Handbook of Biologically Active Peptides

Handbook of Biologically Active Peptides

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Handbook of Biologically Active Peptides

Edited by Abba J. Kastin

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright © 2006, Elsevier Inc. All rights reserved. Except where covered elsewhere. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: [email protected]. You may also complete your request online via the Elsevier homepage (http://elsevier.com), by selecting “Support & Contact” then “Copyright and Permission” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data Application Submitted British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. ISBN 13: 978-0-12-369442-3 ISBN 10: 0-12-369442-6 For information on all Academic Press publications visit our Web site at www.books.elsevier.com Printed in the United States of America 06 07 08 09 10 9 8 7 6 5 4

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Contents Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

Preface: Abba J. Kastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xxxix

Foreword: Andrew V. Schally . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xliii

I: Plant Peptides Section Clarence A. Ryan 1.

4-kDa Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hisashi Hirano

1

2.

AtPep1 Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alisa Huffaker, Yube Yamaguchi, Gregory Pearce, and Clarence A. Ryan

5

3.

CLAVATA3: A Putative Peptide Ligand Controlling Arabidopsis StemCell Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jun Ni and Steven E. Clark

9

4.

DVL Peptides Are Involved in Plant Development . . . . . . . . . . . . . . . . . . . . . . . . . . . Jiangqi Wen and John Walker

17

5.

The POLARIS Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keith Lindsey, Stuart A. Casson, and Paul M. Chilley

23

6.

Phytosulfokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshikatsu Matsubayashi and Youji Sakagami

29

7.

RALF Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel S. Moura, Gregory Pearce, and Clarence A. Ryan

33

8.

ROTUNDIFOLIA4: A Plant-Specific Small Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . Takahiro Yamaguchi and Hirokazu Tsukaya

37

9.

The S-Locus Cysteine-Rich Peptide SCR/SP11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushma Naithani, Daniel Ripoll, and June B. Nasrallah

41

10.

Systemins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory Pearce, Javier Narvaez-Vasquez, and Clarence A. Ryan

49

II: Bacterial/Antibiotic Peptides Section Robert E. W. Hancock 11.

Cationic Antimicrobial Peptides—The Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nikolinka Antcheva, Igor Zelezetsky, and Alessandro Tossi

55

12.

Cathelicidins: Cationic Host Defense and Antimicrobial Peptides . . . . . . . . . . . . . . . Neeloffer Mookherjee, Kelly L. Brown, and Robert E. W. Hancock

67

v

Contents Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

Preface: Abba J. Kastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xxxix

Foreword: Andrew V. Schally . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xliii

I: Plant Peptides Section Clarence A. Ryan 1.

4-kDa Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hisashi Hirano

1

2.

AtPep1 Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alisa Huffaker, Yube Yamaguchi, Gregory Pearce, and Clarence A. Ryan

5

3.

CLAVATA3: A Putative Peptide Ligand Controlling Arabidopsis StemCell Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jun Ni and Steven E. Clark

9

4.

DVL Peptides Are Involved in Plant Development . . . . . . . . . . . . . . . . . . . . . . . . . . . Jiangqi Wen and John Walker

17

5.

The POLARIS Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keith Lindsey, Stuart A. Casson, and Paul M. Chilley

23

6.

Phytosulfokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshikatsu Matsubayashi and Youji Sakagami

29

7.

RALF Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel S. Moura, Gregory Pearce, and Clarence A. Ryan

33

8.

ROTUNDIFOLIA4: A Plant-Specific Small Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . Takahiro Yamaguchi and Hirokazu Tsukaya

37

9.

The S-Locus Cysteine-Rich Peptide SCR/SP11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushma Naithani, Daniel Ripoll, and June B. Nasrallah

41

10.

Systemins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory Pearce, Javier Narvaez-Vasquez, and Clarence A. Ryan

49

II: Bacterial/Antibiotic Peptides Section Robert E. W. Hancock 11.

Cationic Antimicrobial Peptides—The Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nikolinka Antcheva, Igor Zelezetsky, and Alessandro Tossi

55

12.

Cathelicidins: Cationic Host Defense and Antimicrobial Peptides . . . . . . . . . . . . . . . Neeloffer Mookherjee, Kelly L. Brown, and Robert E. W. Hancock

67

v

Contents / vii 31.

Allatostatins in the Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen S. Tobe and William G. Bendena

201

32.

The FXPRLamide (Pyrokinin/PBAN) Peptide Family. . . . . . . . . . . . . . . . . . . . . . . . . Reinhard Predel and R. J. Nachman

207

33.

Insect Pigment Dispersing Factor and Bursicon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inge Mertens, Arnold De Loof, and Peter Verleyen

213

34.

Crustacean Bioactive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Kwok, S. M. Chan, and S. S. Tobe

221

35.

Crustacean Chromatophorotrophins and Hyperglycemic Hormone Peptide Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Kwok, S. M. Chan, F. Martínez-Pérez, S. Zinker, and S. S. Tobe

229

36.

Molluscan Bioactive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anna Di Cosmo and Carlo Di Cristo

235

37.

Molluscan Peptides and Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anna Di Cosmo and Carlo Di Cristo

241

38.

Free-Living Nematode Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. P. Masler

247

39.

Parasitic Nematode Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angela Mousley, Nikki J. Marks, and Aaron G. Maule

255

V: Amphibian Peptides Section J. Michael Conlon 40.

Amphibian Tachykinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cinzia Severini and Giovanna Improta

261

41.

Opioid Peptides from Frog Skin and Bv8-Related Peptides . . . . . . . . . . . . . . . . . . . . Lucia Negri and Pietro Melchiorri

269

42.

Amphibian Bombesin-Like Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eliot R. Spindel

277

43.

Host Defense Peptides from Australian Amphibians: Caerulein and Other Neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John H. Bowie and Michael J. Tyler

283

44.

Bradykinin-Related Peptides from Frog Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Michael Conlon

291

45.

The Dermaseptins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pierre Nicolas and Mohamed Amiche

295

46.

The Temporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Michael Conlon

305

47.

Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maité Montero-Hadjadje, Djida Ait-Ali, Valérie Turquier, Johann Guillemot, Mohammed Boutahricht, Rabia Magoul, Maria M. Malagon, Laurent Yon, Hubert Vaudry, and Youssef Anouar

48.

Sodefrin and Related Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sakae Kikuyama

311

321

viii / Contents 49.

Amphibian Neurohypophysial Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sunny K. Boyd

327

50.

Bombinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maria Luisa Mangoni, Daniela Fiocco, Donatella Barra, and Maurizio Simmaco

333

VI: Venom Peptides Section Jean-Marc Sabatier 51.

Scorpion Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lourival D. Possani and Ricardo C. Rodríguez de la Vega

339

52.

Snake Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alan L. Harvey

355

53.

Sea Anemone Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raymond S. Norton

363

54.

Spider Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graham M. Nicholson

369

55.

Conus Snail Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Baldomero M. Olivera

381

56.

Insect Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mario Sergio Palma

389

57.

Worm Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William R. Kem

397

58.

Targets and Therapeutic Properties of Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . Christine Beeton, George A. Gutman, and K. George Chandy

403

59.

Structure-Function Strategies to Improve the Pharmacological Value of Animal Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michel De Waard and Jean-Marc Sabatier

415

VII: Cancer/Anticancer Peptides Section Terry Moody 60.

Analogs of Luteinizing Hormone-Releasing Hormone (LHRH) in Cancer . . . . . . . Andrew V. Schally and Jorg Engel

61.

Bombesin-Related Peptides and Neurotensin: Effects on Cancer Growth/Proliferation and Cellular Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . Robert T. Jensen and Terry W. Moody

421

429

62.

Somatostatin and NPY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Meike Körner and Jean Claude Reubi

435

63.

Bradykinin and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John M. Stewart and LaJos Gera

443

64.

Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katherine Grant, Marilena Loizidou, and Irving Taylor

447

65.

Adrenomedullin: An Esoteric Juggernaut of Human Cancers . . . . . . . . . . . . . . . . . . Frank Cuttitta, Sergio Portal-Núñez, Christie Falco, Merche Garayoa, Rubén Pío, Luis M. Montuenga, Miguel Julián, Alfredo Martínez, and Enrique Zudaire

453

66.

Angiotensin Peptides and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Ann Tallant, Jyotsana Menon, David R. Soto-Pantoja, and Patricia E. Gallagher

459

Contents / 67.

Gastrin and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jens F. Rehfeld

467

68.

VIP and PACAP as Autocrine Growth Factors in Breast and Lung Cancer . . . . . . . . Terry W. Moody and Robert T. Jensen

473

69.

Oxytocin and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paola Cassoni and Gianni Bussolati

479

70.

Antagonists of Growth Hormone–Releasing Hormone (GHRH) in Cancer . . . . . . . Jozsef L. Varga and Andrew V. Schally

483

VIII: Vaccine Peptides Section Pravin T. P. Kaumaya 71.

Cancer Immunotherapy with Rationally Designed Synthetic Peptides . . . . . . . . . . . Joan T. Steele, Stephanie D. Allen, and Pravin T. P. Kaumaya

491

72.

Peptide Vaccines for Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wolfgang M. Wagner and Mary L. Disis

499

73.

Antiadhesin Synthetic Peptide Consensus Sequence Vaccine and Antibody Therapeutic for Pseudomonas Aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel J. Kao and Robert S. Hodges

507

74.

Peptide Vaccines for Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . José Manuel Lozano, Adriana Bermúdez, and Manuel Elkin Patarroyo

515

75.

Peptide Vaccine for Otitis Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lauren O. Bakaletz

527

76.

Peptide Vaccine for Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beka Solomon

535

77.

Peptide Dendrimers as Immunogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . James P. Tam

541

IX: Immunological and Inflammatory Peptides Section Roland Martin and Joost J. Oppenheim 78.

Chemotactic Peptide Ligands for Formylpeptide Receptors Influencing Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ji Ming Wang and Keqiang Chen

547

79.

Complement-Derived Inflammatory Peptides: Anaphylatoxins . . . . . . . . . . . . . . . . . De Yang

553

80.

Chemokines: A New Peptide Family of Neuromodulators . . . . . . . . . . . . . . . . . . . . . Patrick Kitabgi, Stéphane Mélik-Parsadaniantz, and William Rostène

559

81.

Immune Peptides Related to Dipeptidyl Aminopeptidase IV/CD26 . . . . . . . . . . . . . Siegfried Ansorge and Dirk Reinhold

567

82.

RGD-Peptides and Some Immunological Problems . . . . . . . . . . . . . . . . . . . . . . . . . . Ignacy Z. Siemion, Alicja Kluczyk, and Marek Cebrat

573

83.

Neuropeptides That Regulate Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . Ning Zhang and Joost J. Oppenheim

579

84.

Peptides as Targets of T Cell-Mediated Immune Responses . . . . . . . . . . . . . . . . . . . . Roland Martin

585

ix

x / Contents 85.

The Use of Positional Scanning Synthetic Peptide Combinatorial Libraries to Identify Immunological Relevant Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mireia Sospedra and Clemencia Pinilla

595

86.

Copolymer 1 and Related Peptides as Immunomodulating Agents . . . . . . . . . . . . . Ruth Arnon

603

87.

CLIP—A Multifunctional MHC Class II–Associated Self-Peptide . . . . . . . . . . . . . . . . Anne Vogt and Harald Kropshofer

611

X: Brain Peptides Section Hubert Vaudry 88.

Vasopressin and Oxytocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John F. Morris

621

89.

Thyrotrophin-Releasing Hormone: New Functions for an Ancient Peptide . . . . . . . Albert Eugene Pekary

629

90.

Gonadotrophin Releasing Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. P. Millar

635

91.

Brain Somatostatin-Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jacques Epelbaum and Rephaelle Winoky-Sommerer

645

92.

Corticotrophin-Releasing Hormone (CRH) Peptide Family . . . . . . . . . . . . . . . . . . . David A. Lovejoy

655

93.

Growth Hormone-Releasing Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. M. Malagón, R. Vázquez-Martínez, A. J. Martínez-Fuentes, F. Gracia-Navarro, and J. P. Castaño

663

94.

PACAP/VIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Min Li, Tomoya Nakamachi, and Akira Arimura

673

95.

Neuropeptide Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Remi Quirion and Yvan Dumont

683

96.

Melanocortins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sylvie Jégou, Roger D. Cone, Portland, OR, Alex N. Eberlé, and Hubert Vaudry

689

97.

Cocaine- and Amphetamine-Regulated Transcript (CART) . . . . . . . . . . . . . . . . . . . . Csaba Fekete and Ronald M. Lechan

697

98.

The Melanin-Concentrating Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jean-Louis Nahon

705

99.

CCK/Gastrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Margery C. Beinfeld

715

100.

The Hypocretins (Orexins) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luis De Lecea and J. Gregor Sutcliffe

721

101.

Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Masayasu Kojima and Kenji Kangawa

731

102. Neurotensin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paul R. Dobner and Robert E. Carraway

737

Neuromedin U (NMU): Brain Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preeti H. Jethwa, Caroline J. Small, and Stephen R. Bloom

745

103.

Contents / 104.

Galanin and GALP Systems in Brain—Molecular Pharmacology, Anatomy, and Putative Roles in Physiology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrew L. Gundlach and Sebastian R.-F. Jungnickel

753

105.

Brain Tachykinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nigel M. Page

763

106.

CGRP and Adrenomedullin in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanda Tilakaratne and Patrick M. Sexton

771

107.

The RFamide-Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas Chartrel, Kazuyoshi Tsutsui, Jean Costentin, and Hubert Vaudry

779

108.

Apelin: Discovery, Distribution, and Physiological Role . . . . . . . . . . . . . . . . . . . . . . . Xavier Iturrioz, Annabelle Reaux-le Goazigo, and Catherine Llorens-Cortes

787

109.

Urotensin II and Urotensin II–Related Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isabelle Lihrmann, Howard Bern, and Hubert Vaudry

795

110.

Brain/B-Type Natriuretic Peptide (BNP) and C-Type Natriuretic Peptide (CNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshio Takei

805

111.

Endozepines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marie-Christine Tonon, Jérôme Leprince, Pierrick Gandolfo, Vincent Compère, Georges Pelletier, María M. Malagón, and Hubert Vaudry

813

112.

KiSS-1/Metastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manuel Tena-Sempere

821

XI: Endocrine Peptides Section Gastone Giovanni Nussdorfer 113.

Role of Opioid Peptides in the Local Regulation of Endocrine Glands . . . . . . . . . . Kazuhiro Takekoshi

114.

Role of Tachykinin-Gene-Related Peptides in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ludwik K. Malendowicz

829

833

115.

Neuropeptide Y and the Regulation of Endocrine Function . . . . . . . . . . . . . . . . . . . J. P. Hinson

839

116.

Effects of PACAP in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . David Vaudry, Aurélia Ravni, Olivier Wurtz, Magalie Bénard, Béatrice Botia, Valérie Jolivel, Alain Fournier, Bruno Gonzalez, and Hubert Vaudry

847

117.

Endothelins in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . . . . . Gian Paolo Rossi and Domenico Montemurro

855

118.

Adrenomedullin and Related Peptides in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfredo Martínez

861

119.

Ghrelin in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . . . . . . . . . Oreste Gualillo, Francisca Lago, Rosalia Gallego, Tomas Garcia-Caballero, Jose Ramon Gonzalez-Juanatey, Juan J. Gomez-Reino, Felipe F. Casanueva, and Carlos Dieguez

869

120.

Atrial Natriuretic Peptide in Local Regulation of Endocrine Glands . . . . . . . . . . . . Jolanta Gutkowska

877

xi

xii / Contents 121.

Galanin, Neurotensin, and Neuromedins in the Local Regulation of Endocrine Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giuseppina Mazzocchi, Raffaella Spinazzi, and Gastone G. Nussdorfer

883

XII: Ingestive Peptides Section David York 122. Neuropeptide Y: A Conductor of the Appetite-Regulating Orchestra in the Hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Satya P. Kalra and Pushpa S. Kalra

889

123. Hypothalamic Galanin and Ingestive Behavior: Relation to Dietary Fat, Alcohol, and Circulating Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sarah F. Leibowitz

895

124. Effects of Melanocortins on Ingestive Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patricia Rene and Roger D. Cone

903

125. CART Peptide and Ingestive Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kelly B. Philpot and Michael J. Kuhar

913

126. Orexins and Opioids in Feeding Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catherine M. Kotz, Charles J. Billington, and Allen S. Levine

919

127. Melanin-Concentrating Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eleftheria Maratos-Flier

929

128. Corticotrophin-Releasing Hormone (CRH) and Ingestive Behavior . . . . . . . . . . . . . Mary Ann Pelleymounter

937

129. Peptide YY (PYY) and Neuromedin U (NMU): Effects on Ingestive Behavior . . . . . Caroline J. Small, Preeti H. Jethwa, and Stephen R. Bloom

945

130. Ghrelin and Ingestive Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diego Pérez-Tilve, Rubén Nogueiras, and Matthias Tschöp

953

131. Cholecystokinin and Satiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Timothy H. Moran, Jie Chen, and Sheng Bi

961

132. Enterostatin, a Peptide Regulator of Dietary Fat Ingestion . . . . . . . . . . . . . . . . . . . . David A. York and Miejung Park

969

133. Regulation of Feeding Behavior by Glucagonlike Peptide 1 (GLP-1) . . . . . . . . . . . . Patricia M. Vuguin and Maureen J. Charron

975

134. Role of Amylin and Calcitonin-Gene-Related Peptide (CGRP) in the Control of Food Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas A. Lutz

981

135.

Leptin and the Regulation of Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Martin G. Myers, Jr.

987

136.

Ingestive Peptides: Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen C. Woods

993

XIII: Gastrointestinal Peptides Section Yvette Taché 137.

Adrenomedullin in Gastrointestinal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitchell L. Schubert

999

138.

Calcitonin Gene-Related Peptide and Gastrointestinal Function . . . . . . . . . . . . . . . . Vicente Martinez and Yvette Taché

1005

Contents / 139.

Peripheral Cholecystokinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joseph R. Reeve, Jr., David A. Keire, and Gary M. Green

140.

Corticotrophin-Releasing Hormone (CRH) Family in the Gastrointestinal System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lixin Wang, Pu-Qing Yuan, and Mulugeta Million

1023

Paneth Cell α-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andre J. Ouellette

1029

142. Galanin in the Gastrointestinal Tract: Distribution and Function . . . . . . . . . . . . . . . Laura Anselmi, Ilaria Cavalli, and Catia Sternini

1037

143. Gastrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. J. Dockray

1043

144. Gastrin-Releasing Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. S. Lehmann and C. Beglinger

1047

141.

145. Glucagonlike Peptides 1 and 2, Enteroglucagon, Glicentin, and Oxyntomodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jens Juul Holst

1013

1057

146.

Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theo L. Peeters

1065

147.

Leptin and the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sandra Guilmeau, Robert Ducroc, and André Bado

1071

148.

Motilin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pierre Poitras

1077

149. Neurotensin in Regulation of Gastrointestinal Functions . . . . . . . . . . . . . . . . . . . . . . Dezheng Zhao and Charalabos Pothoulakis

1085

150. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) . . . . . . . . . . . . . . . . . . . Patrizia M. Germano and Joseph R. Pisegna

1091

151. Pancreatic Polypeptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiaoying Deng and David C. Whitcomb

1097

152. Peptide YY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guillermo Gomez, Guiyun Wang, Ella W. Englander, and George H. Greeley, Jr.

1109

Secretin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William Y. Chey and Ta-Min Chang

1115

154. Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathias Gugger and Jean Claude Reubi

1123

155.

Somatostatin Analogs in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . Alan G. Harris, Adrian F. Daly, Maria Tichomirova, Albert Beckers, and Steven W. Lamberts

1131

156.

Substance P and Related Tachykinins in the Gastrointestinal Tract . . . . . . . . . . . . . Peter Holzer

1139

157.

TFF (Trefoil Factor Family) Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Werner Hoffmann

1147

158.

Signaling by Vasoactive Intestinal Peptide in Gastrointestinal Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karnam S. Murthy and Satish C. Rattan

153.

1155

xiii

xiv / Contents XIV: Cardiovascular Peptides Section Naoto Minamino 159. Adrenomedullin and Its Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kazuo Kitamura and Johji Kato

1163

160. Angiotensin II and Its Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yasukatsu Izumi and Hiroshi Iwao

1169

161. Bradykinin and Its Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duncan John Campbell

1175

162. Calcitonin Gene-Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Christina W. L. Tam and Susan D. Brain

1181

163. Endothelins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katsutoshi Goto

1187

164.

Ghrelin: Its Therapeutic Potential in Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . Noritoshi Nagaya and Kenji Kangawa

1193

165.

Natriuretic Peptides in the Cardiovascular System . . . . . . . . . . . . . . . . . . . . . . . . . . . Naoto Minamino, Takeshi Horio, and Toshio Nishikimi

1199

166.

Urotensin and Its Related Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kazuhiro Takahashi and Kazuhito Totsune

1209

167.

Vasoactive Intestinal Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Darrell R. Sawmiller and Robert J. Henning

1215

168.

Cardiovascular Peptides: Vasopressin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natalie N. Rizk, Noreen F. Rossi, and Joseph C. Dunbar

1223

XV: Renal Peptides Section Willis K. Samson 169.

Renal Effects of Neurohypophyseal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joseph G. Verbalis

1227

170.

Renal Renin-Angiotensin System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. Gabriel Navar, Minolfa C. Prieto-Carrasquero, and Hiroyuki Kobori

1235

171.

Renal Natriuretic Peptide System and Actions of Urodilatin . . . . . . . . . . . . . . . . . . . Markus Meyer, Jochen R. Hirsch, and Wolf-Georg Forssmann

1243

172.

ANP and Its Role in the Regulation of Renal Tubular Transport Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jochen R. Hirsch, Markus Meyer, and Wolf-Georg Forssmann

1251

173.

Adrenomedullin as a Renal Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michihisa Jougasaki

1257

174.

Adrenomedullin 2/Intermedin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshio Takei

1263

175.

Renal Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Michael Williams and David M. Pollock

1269

176.

Prolactin and Kidney Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Willis K. Samson

1277

Contents / XVI: Respiratory Peptides Section Sami Said 177. Therapeutic Potential of Adrenomedullin for Pulmonary Hypertension . . . . . . . . . Noritoshi Nagaya

1283

178. Endothelin in the Airways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bengt W. Granström and Lars Edvinsson

1289

179. PACAP’s Role in Pulmonary Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Christiane Otto and Juan Carlos Prieto

1293

180. Tachykinins and Their Receptors in the Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stefania Meini and Alessandro Lecci

1301

181. Vasoactive Intestinal Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sami I. Said

1307

XVII: Opioid Peptides Section Fred Nyberg 182.

Proenkephalin-Derived Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patricia J. McLaughlin

1313

183.

Prodynorphin-Derived Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Santi Spampinato

1319

184.

POMC Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Margaret E. Smith

1325

185.

Endomorphins as Endogenous Peptides for μ-Opioid Receptor: Their Differences in the Pharmacological and Physiological Characters . . . . . . . . . . . . . . Shinobu Sakurada and Tsukasa Sakurada

1333

Casomorphins and Hemorphins—Opioid Active Peptides Released by Partial Hydrolysis of Structural Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fred Nyberg

1339

186.

187.

Anti-Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John Q. Wang, Eugene E. Fibuch, Shinobu Sakurada, and Ji-Sheng Han

1345

188.

Nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jean-Claude Meunier and Brice Bes

1351

189. Role of Tachykinins in Spinal Nociceptive Mechanisms and Their Interactions with Opioids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiao-Jun Xu and Zsuzsanna Wiesenfeld-Hallin

1359

190. Exorphin-Opioid Active Peptides of Exogenous Origin . . . . . . . . . . . . . . . . . . . . . . . Masaaki Yoshikawa

1365

191. Opioid-Substance P Chimeric Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrzej W. Lipkowski, Daniel B. Carr, Iwona Bonney, and Piotr Kosson

1373

XVIII: Neurotrophic Peptides Section Illana Gozes 192.

VIP- and PACAP-Related Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Illana Gozes

1379

xv

xvi / Contents 193.

Insulin-Like Growth Factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Itay Bentov and Haim Werner

194. Erythropoietin—A Hematopoietic Hormone with Emerging Diverse Activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sara Prutchi-Sagiv, Moshe Mittelman, and Drorit Neumann

1385

1393

Neuregulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kim B. Seroogy and Lixin Zhang

1401

196. The Neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ljubica Ivanisevic and H. Uri Saragovi

1407

195.

XIX: Blood-Brain Barrier Peptides Section Weihong Pan 197. Amino Acid Transport Across the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . Quentin R. Smith, Haritha Mandula, and Jagan M. R. Parepally

1415

198. Oligopeptide Transport at the Blood–Brain and Blood–CSF Barriers . . . . . . . . . . . . Richard F. Keep, Ann Arbor, MI, and David E. Smith

1423

199. Opiate Peptides and the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard D. Egleton, Ken A. Witt, and Thomas P. Davis

1429

200. Permeability of the Blood–Brain Barrier to Neurotrophic Peptides . . . . . . . . . . . . . Weihong Pan

1435

201. Transport of Basic Peptides at the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . Yoshiharu Deguchi and Tetsuya Terasaki

1443

202. Fibroblast Growth Factor and the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . Conrad E. Johanson, John E. Donahue, Edward G. Stopa, and Andrew Baird

1449

203. Ingestive Peptides and the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William A. Banks, Ryota Nakaoke, Akihiko Urayama, and Thanh Q. Vo

1455

204. Functional Aspects of Vasoactive Peptides at the Blood–Brain Barrier . . . . . . . . . . . Arvind K. Chappa, Kelly E. Desino, Susan M. Lunte, and Kenneth L. Audus

1461

205. Hypothalamic Neuropeptides and the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . David J. Begley

1469

206. Diseases Mediated by the BBB: From Alzheimer’s to Obesity . . . . . . . . . . . . . . . . . . William A. Banks

1475

XX: Other Peptide Topics Abba J. Kastin 207. Prebiotic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernd M. Rode and Kristof Plankensteiner

1481

208. Mixture-Based Combinatorial Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. A. Houghten, C. T. Dooley, and J. R. Appel

1487

209. Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jörg Votteler, Karsten Bruns, Peter Henklein, Victor Wray, and Ulrich Schubert 210. Pheromone Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miriam Altstein

1495 1505

Contents / Fish Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kazuhiro Takahashi

1515

212. Peptides and Sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Steiger

1521

213. Peptide Chronomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Franz Halberg, Germaine Cornélissen, Eugene Kanabrocki, Robert B. Sothern, Erhard Haus, Samuel Zinker, Rita Jozsa, Weihong Pan, Roberto Tarquini, Federico Perfetto, Cristina Maggioni, and Earl E. Bakken

1529

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1565

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Contributors Michael E. Adams (163) Dept. of Cell Biology & Neuroscience University of California Riverside, CA 92521

J. R. Appel (1487) Torrey Pines Institute for Molecular Studies San Diego, CA 92121 Akira Arimura (673) USJBRL Tulane University F. Edward Hebert Research Center Belle Chasse, LA 70037

Djida Ait-Ali (311) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France

Ruth Arnon (603) Pharmaceutical Research F. Hoffman La Roche Ltd. CH-4070 Basel, Switzerland

Stephanie D. Allen (491) Tzagournis Medical Research Facility Ohio State University Columbus, OH 43210

Kenneth L. Audus (1461) School of Pharmacy University of Kansas Lawrence, KS 66045

Miriam Altstein (1505) Dept. of Entomology Agricultural Research Organization Volcani Center Bet Dagan, 50250, Israel

Andre Bado (1071) Faculte de Medecine Xavier Bichat INSERM Unite 683 75018 Paris, France

Mohamed Amiche (295) FRE 2852 CNRS-Universite Paris—6 Institut Jacques Monod 75251 Paris Cedex 05, France

Andrew Baird (1449) Molecular Neuroscience Group, School of Medicine University of Birmingham Edgbaston, UK

Youssef Anouar (311) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France

Lauren O. Bakaletz (527) Center for Microbial Pathogenesis Columbus Children’s Research Institute Dept. of Pediatrics Ohio State University College of Medicine & Public Health Columbus, OH 43205-2696

Laura Anselmi (1037) CURE, Digestive Diseases Research Center Los Angeles, CA 90073 Siegfried Ansorge (567) Zenit Building IMTM GmbH D-39120 Magdeburg, Germany

Earl E. Bakken (1529)

Nikolinka Antcheva (55) Dept. of Biochemistry University of Trieste Trieste, Italy 34127

William A. Banks (1455, 1475) VAMC St. Louis University School of Medicine St. Louis, MO 63106

xix

xx / Contributors Donatella Barra (333) Dipartimento di Scienze Biochimiche “A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari Universita” La Sapienza Istituto Pasteur-Fondazione Cenci Bolognetti 00185 Rome, Italy Albert Beckers (1131) Dept. of Endocrinology CHU de Liège 4000 Liège, Belgium Christine Beeton (403) Dept. of Physiology & Biophysics, Medical School University of California Irvine, CA 92697 David J. Begley (1469) Dept. of Physiology King’s College, Strand London WC2R 2LS, England United Kingdom C. Beglinger (1047) Professor of Medicine Head Division of Gastroenterology University Hospital CH-4031 Basel, Switzerland Margery C. Beinfeld (715) Dept. of Pharmacology and Experimental Therapeutics Tufts University School of Medicine Boston, MA 02111 Magalie Bénard (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France William G. Bendena (201) Dept. of Biology Queen’s University Kingston Ontario K7L3W6, Canada

University of California Berkeley, CA 94720-3140 Brice Bes (1351) CNRS-UMR 5089 Institut de Pharmacologie et de Biologie Structurale Cedex 04, France Sheng Bi (961) Dept. of Psychiatry and Behavioral Science Johns Hopkins School of Medicine Baltimore, MD 21205-2196 Charles J. Billington (919) VA Medical Center Minneapolis, MN 55417-2399 Stephen R. Bloom (745, 945) Faculty of Medicine, Investigative Sciences Commonwealth Building Royal Postgraduate Medical School London W12 0NN, England, United Kingdom Raquel Regina Bonelli (97) Institut für Medizinische Mikrobiologie und Immunologie Universität Bonn 53115 Bonn, Germany Iwona Bonney (1373) Tufts-New England Medical Center Boston, MA 02111 Béatrice Botia (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France Mohammed Boutahricht (311) Laboratory of Animal Physiology University Sidi Mohamed Ben Abdellah Fès-Atlas, Morocco

Itay Bentov (1385) Dept. of Human Molecular Genetics & Biochemistry Sackler School of Medicine Tel Aviv University Tel Aviv 69978, Israel

John H. Bowie (283) Dept. of Chemistry University of Adelaide South Australia 5005, Australia

Adriana Bermúdez (515) Fundación Instituto de Inmunología de ColombiaFIDIC Bogotá, Colombia

Sunny K. Boyd (327) Biological Sciences University of Notre Dame Notre Dame, IN 46556

Howard A. Bern (795) Dept. of Integrative Biology & Cancer Research Lab

Susan D. Brain (1181) Cardiovascular Division

Contributors / King’s College London London, SE1 1UL England Dag Anders Brede (107) Dept. of Chemistry, Biotechnology, and Food Science Norwegian University of Life Sciences N-1432 As, Norway

J. P. Castaño (663) Dept. of Cell Biology Campus Universitario de Rabanales 14014 Cordoba, Spain Ilaria Cavalli (1037) CURE, Digestive Diseases Research Center Los Angeles, CA 90073

Kelly L. Brown (67) Centre for Microbial Diseases and Immunity University of British Columbia Vancouver, British Columbia, Canada V6T 1Z4

Marek Cebrat (573) Faculty of Chemistry University of Wroclaw 50-383 Wroclaw, Poland

Karsten Bruns (1495) Dept. of Structural Biology German Research Centre for Biotechnology Braunschweig, Germany

S. M. Chan (221, 229) University of Hong Kong Hong Kong, SAR, People’s Republic of China

Gianni Bussolati (479) Dept. of Biomedical Sciences and Human Oncology, University of Torino 10126 Torino, Italy Duncan John Campbell (1175) St. Vincent’s Institute of Medical Research Dept. of Medicine St. Vincent’s Hospital University of Melbourne Fitzroy, Victoria 3065, Australia

K. George Chandy (403) Dept. of Physiology & Biophysics Medical School University of California Irvine, CA 92697 Ta-Min Chang (1115) Rochester Institute for Digestive Diseases & Sciences Rochester, NY 14607

Daniel B. Carr (1373) Tufts-New England Medical Center Boston, MA 02111

Arvind K. Chappa (1461) Dept. of Pharmaceutical Chemistry University of Kansas Lawrence, KS 66045

Robert E. Carraway (737) Dept. of Molecular Genetics and Microbiology Program in Neuroscience University of Massachusetts Medical School Worcester, MA 01655

Maureen J. Charron (975) Dept. of Biochemistry Co-Director, Institute for Obesity Research Albert Einstein College of Medicine Bronx, NY 10461

Felipe F. Casanueva (869) Dept. of Medicine Molecular Endocrinology Section University of Santiago de Compostela–School of Medicine 15782 Santiago de Compostela, Spain

Nicolas Chartrel (771) Laboratory of Cellular and Molecular Neuroendocrinology INSERM U413, UA CNRSEuropean Institute for Peptide Research (IFRMP 23) University of Rouen 76821 Mont-Saint-Aignan Cedex, France

Stuart A. Casson (23) Integrative Cell Biology Laboratory Durham University Durham DH1 3LE, United Kingdom

Jie Chen (961) Dept. of Gastroenterology Children’s Hospital Zhejiang University School of Medicine Hangzhou, China

Paola Cassoni (479) Dept. of Biomedical Sciences and Human Oncology, University of Torino 10126 Torino, Italy

Keqiang Chen (547) LMI, CCR, NCI-Frederick Frederick, MD 21702

xxi

xxii / Contributors William Y. Chey (1115) Rochester Institute for Digestive Diseases & Sciences Rochester, NY 14607 Paul M. Chilley (23) Integrative Cell Biology Laboratory Durham University Durham DH1 3LE, United Kingdom K. T. Chu (125) Dept. of Biochemisty, Faculty of Medicine Chinese University of Hong Kong Shatin, Hong Kong, China Paulos B. Chumala (151) Dept. of Chemistry University of Saskatchewan Saskatoon, SK S7N 5C9, Canada Steven E. Clark (9) Dept. of Molecular, Cellular and Developmental Biology University of Michigan Ann Arbor, MI 48109-1048 Geoffrey M. Coast (157) School of Biological and Chemical Sciences Birkbeck University of London London WC1E 7HX, United Kingdom Vincent Compère (813) Roger D. Cone (689, 903) Vollum Institute Oregon Health and Science University Portland, OR 97201-3011 J. Michael Conlon (291, 305) Dept. of Biochemistry United Arab Emirates University 17666 Al-Ain, United Arab Emirates Germaine Cornélissen (1529) Halberg Chronobiology Center University of Minnesota Minneapolis Campus 420 Delaware Street SE Minneapolis, MN 55455 Jean Costentin (779) Unite de Neuropsychopharmacologie Experimentale Faculte de Medecine et de Pharmacie Rouen, France William A. Cramer (115) Dept. of Biological Sciences Purdue University West Lafayette, IN 47907-2054

Frank Cuttitta (453) Cell and Cancer Biology Branch National Cancer Institute, Center for Cancer Research Bethesda, MD 20892 Adrian F. Daly (1131) Dept. of Endocrinology CHU de Liège 4000 Liège, Belgium Thomas P. Davis (1429) Dept. of Medical Pharmacology University of Arizona College of Medicine Tucson, AZ 85724-5050 Yoshiharu Deguchi (1443) Dept. of Drug Disposition & Pharmacokinetics School of Pharmaceutical Sciences Teikyo University Tsukui-gun, Kanagawa 199-0195, Japan Luis De Lecea (721) Depts. of Molecular Biology and Neuropharmacology, MB-10 Scripps Research Institute La Jolla, CA 92037 Arnold De Loof (213) Genomics and Proteomics K. U. Leuven Zoological Institute Laboratory of Development Physiology Leuven, Belgium Xaioying Deng (1097) Dept. of Pathology State University of New York Syracuse, NY 13210 Kelly E. Desino (1461) Dept. of Pharmaceutical Chemistry University of Kansas Lawrence, KS 66045 Michel De Waard (415) CEA, DRDC Laboratoire Canaux Caliques, Fonctions et Pathologies Inserm U607 38054 Grenoble Cedex 09, France Anna Di Cosmo (235, 241) Dept. of Biological and Environmental Sciences University of Sannio 82100 Benevento, Italy Carlo Di Cristo (235, 241) Dept. of Biological and Environmental Sciences

Contributors / University of Sannio 82100 Benevento, Italy

S-22185 Lund Sweden

Carlos Dieguez (869) Dept. of Physiology University of Santiago de Compostela–School of Medicine 15782 Santiago de Compostela, Spain

Richard D. Egleton (1429) Dept. of Medical Pharmacology University of Arizona College of Medicine Tucson, AZ 85724-5050

Mary L. (Nora) Disis (499) Center for Translational Medicine in Women’s Health Tumor Vaccine Group University of Washington Seattle, WA 98195-8050 Paul R. Dobner (737) Dept. of Molecular Genetics and Microbiology Program in Neuroscience University of Massachusetts Medical School Worcester, MA 01655

Jorg Engel (421) VA Medical Center Miami, FL Ella W. Englander (1109) Dept. of Surgery Shriners Hospital for Children Galveston, TX 77555 Jacques Epelbaum (645) UMR.549 INSERM-Université Paris 5 75014 Paris, France

Graham J. Dockray (1043) Physiological Laboratory School of Biomedical Sciences University of Liverpool Liverpool, L69 3BX, United Kingdom

Christie Falco (453) Vascular Biology Faculty National Cancer Institute Center for Cancer Research Bethesda, MD 20892

John E. Donahue (1449)

Csaba Fekete (697) Dept. of Endocrine Neurobiology Institute of Experimental Medicine Hunagrian Academy of Sciences Budapest H-1083, Hungary

C. T. Dooley (1487) Robert Ducroc (1071) Faculte de Medecine Xavier Bichat INSERM Unite 683 75018 Paris, France Yvan Dumont (683) Douglas Hospital Research Centre Montreal, QC, Canada H4H 1R3 Joseph C. Dunbar (1223) Dept. of Physiology 5374 Scott Hall Wayne State Univ School of Medicine 540 E. Canfield Detroit, MI 48201 Alex N. Eberlé (689) Dept. of Research University Hospital and University Children’s Hospital Basel, Switzerland Lars Edvinsson (1289) Dept. of Internal Medicine University Hospital of Lund

Eugene E. Fibuch (1345) Dept. of Anesthesiology University of Missouri–Kansas City School of Medicine Kansas City, MO 64108-2792 Daniela Fiocco (333) Dipartimento di Scienze Biochimiche “A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari Universita” “La Sapienza” Istituto Pasteur-Fondazione Cenci Bolognetti 00185 Rome, Italy Wolf-Georg Forssmann (1243, 1251) Institute of Medicine Hochschule Hannover Feofor Lynen Strasse Hannover, 30625 Germany Alain Fournier (847) INRS-Institut Armand Frappier University of Quebec Pointe-Claire, Canada H9R 1G6

xxiii

xxiv / Contributors Gerd Gäde (189) Zoology Dept. University of Cape Town Rondebosch 7701, South Africa Patricia E. Gallagher (459) Hypertension and Vascular Disease Center Wake Forest University School of Medicine Winston-Salem, NC 27157 Rosalia Gallego (869) Dept. of Morphological Sciences University of Santiago de Compostela–School of Medicine 15782 Santiago de Compostela, Spain Pierrick Gandolfo (813) Laboratory of Cellular and Molecular Neuroendocrinology 76821 Mont-Saint-Aigan, France Merche Garayoa (453) Cellular & Pathology Dept. University of Navarra Pamplona, Spain Tomas Garcia-Caballero (869) Dept. of Morphological Sciences University of Santiago de Compostela–School of Medicine 15782 Santiago de Compostela, Spain Lajos Gera (443) Patrizia M. Germano (1091) CURE Digestive Diseases Research Center VA Greater Los Angeles Healthcare System David Geffen School of Medicine at UCLA Los Angeles, CA 90073

Santiago University Clinical Hospital 15706 Santiago de Compostela, Spain Katsutoshi Goto (1187) Dept. of Pharmacology Institute of Basic Medical Sciences University of Tsukuba Tsukuba, Japan Illana Gozes (1379) Laboratory for Molecular Neuroendocrinology Sackler Faculty of Medicine Tel Aviv University Tel Aviv 69978, Israel F. Gracia-Navarro (663) Dept. of Cell Biology Campus Universitario de Rabanales 14014 Cordoba, Spain Bengt W. Granström (1289) Dept. of Medicine Lund University Lund, Sweden Katherine Grant (447) Dept. of Surgery Royal Free and University College London Medical School London, United Kingdom George H. Greeley, Jr. (1109) Dept. of Surgery University of Texas Medical Branch Galveston, TX 77555-0725 Gary M. Green (1013) Dept. of Physiology University of Texas Health Science Ctr. San Antonio, TX 78229

Guillermo Gomez (1109) Juan J. Gomez-Reino (869) NEIRID Research Lab 4 Santiago University Clinical Hospital 15706 Santiago de Compostela, Spain Bruno Gonzalez (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France Jose Ramon Gonzalez-Juanatey (869) Research Area Molecular and Celullar Cardiology Lab 1

Jan Grünerwald (89) FB Chemie/Biochemie Philipps University of Marburg D-35032 Marburg, Germany Oreste Gualillo (869) NEIRID Research Lab 4 Santiago University Clinical Hospital 15706 Santiago de Compostela, Spain Mathias Gugger (1123) Division of Cell Biology and Experimental Cancer Research Institute of Pathology University of Bern CH-3010 Bern, Switzerland

Contributors / Johann Guillemot (311) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France Sandra Guilmeau (1071) Faculte de Medecine Xavier Bichat INSERM Unite 683 75018 Paris, France Andrew L. Gundlach (753) Howard Florey Institute University of Melbourne Victoria 3010, Australia Jolanta Gutkowska (877) Research Ceneter—Hotel-Dieu Pav. Masson Centre Hospitalier de l’Universite de Montreal (CHUM) Montreal Quebec, Canada H2W 1T8 George A. Gutman (403) Dept. of Physiology & Biophysics, Medical School University of California Irvine, CA 92697 Franz Halberg (1529) Halberg Chronobiology Center University of Minnesota Minneapolis Campus 420 Delaware Street SE Minneapolis, MN 55455 Ji-Sheng Han (1345) Neuroscience Research Institute Peking University Beijing 100083, People’s Republic of China Robert E. W. Hancock (67) Director, Centre for Microbial Diseases and Immunity University of British Columbia Vancouver, British Columbia, Canada V6T 1Z4 Alan G. Harris (1131) Dept. of Endocrinology CHU de Liège 4000 Liège, Belgium Alan L. Harvey (355) Strathclyde Institute for Drug Research University of Strathclyde Glasgow, G4 0NR, United Kingdom

Erhard Haus (1529) Dept. of Laboratory Medicine & Pathology University of Minnesota St. Paul, MN 55101 Nicholas C. K. Heng (75) Dept. of Microbiology Otago School of Medical Sciences Dunedin, New Zealand Peter Henklein (1495) Institute of Biochemistry Humboldt University Berlin, Germany Robert J. Henning (1215) College of Medicine James A. Haley Hospital University of South Florida Tampa, FL 33612 J. P. Hinson (839) Centre for Endocrinology WHRI Barts and the London Queen Mary School of Medicine and Dentistry London EC1M 6BQ, United Kingdom Hisashi Hirano (1) International Graduate School of Arts and Sciences Yokohama City University Totsuka Yokohama, 244-0813, Japan Jochen R. Hirsch (1243, 1251) Bildung on demand GmbH Karlsruhe, Germany Robert S. Hodges (507) Dept. of Biochemistry and Molecular Genetics University of Colorado at Denver Health Sciences Center Aurora, CO 80045 Werner Hoffmann (1147) Institute of Molecular Biology and Medical Chemistry Otto-von-Guericke-University D-39120 Magdeburg, Germany Helge Holo (107) Melik-Parsadomanz (Kitabgi) Philpot (Kuhar) Yoonoong Park (Michael Adams) P. Rene (R. Cone) Peter Holzer (1139) Dept. of Experimental and Clinical Pharmacology

xxv

xxvi / Contributors Medical University of Graz A-8010 Graz, Austria Takeshi Horio (1199) Division of Hypertension and Nephrology Dept. of Medicine National Cardiovascular Center Osaka, 565-8565, Japan R. A. Houghten (1487) Torrey Pines Institute for Molecular Studies 3550 General Atomics Court San Diego, California, 92121 Alisa Huffaker (5) Institute of Biological Chemistry Washington State University Pullman, WA 99164-6340 Giovanna Improta (261) Dept. of Human Physiology & Pharmacology “Vittorio Erspamer” University La Sapienza 00185 Rome, Italy Xavier Iturrioz (787) Unite 691 College de France Institute National de la Sante et de la Recherche Medicale 75231 Paris, France Ljubica Ivanisevic (1407) Lady Davis Institute Jewish General Hospital McGill University Montreal, Quebec, Canada H3T 1E2 Hiroshi Iwao (1169) Dept. of Pharmacology Osaka City University Medical School Osaka, 545-8585, Japan Yasukatsu Izumi (1169) Dept. of Pharmacology Osaka City University Medical School Osaka, 545-8585, Japan

Building 10, Room 9C 103 10 Center Drive MSC 1804 Bethesda MD 20892-1804 Preeti H. Jethwa (745, 945) Dept. of Metabolic Medicine Imperial College London London, W12 ONN, United Kingdom Conrad E. Johanson (1449) CSF Research Laboratories Brown University Rhode Island Hospital 593 Eddy Street Providence RI 02903-0001 Valérie Jolivel (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France Michihisa Jougasaki (1257) NHO Kyushu Cardiovascular Center Kagoshima, 892-0853, Japan Rita Jozsa (1529) Dept. of Anatomy Poes University Medical Faculty and Neurohumocal Regulations Research Group of the Hungarian Academy of Sciences Szigeti v 12, Hungary Miguel Julián (453) Dept. of Chemistry University of San Pablo CEU Madrid, Spain Sebastian R.-F. Jungnickel (753) Max-Planck Institute for Experimental Endocrinology Hanover, D-30625, Germany

Ralph W. Jack (75) Dept. of Microbiology Otago School of Medical Sciences Dunedin, New Zealand

Jens Juul Holst (1057) Dept. of Medical Physiology The Panum Institute University of Copenhagen Blegdamsvej 3 DK 2200 Copenhagen Denmark

Sylvie Jégou (689) European Institute for Peptide Research University of Rouen 76821 Mont-Saint-Aignan, France

Pushpa S. Kalra (889) Dept. of Physiology & Functional Genomics University of Florida McKnight Brain Institute Gainesville, FL 32610-0244

Robert T. Jensen (429, 473) NIH/NIDDK/DDB

Satya P. Kalra (889) Dept. of Neuroscience

Contributors / University of Florida College of Medicine Gainesville, FL 32610-0244

Miyazaki Medical College Kiyotake, Miyazaki, 889-1692, Japan

Eugene Kanabrocki (1529)

Alicja Kluczyk (573) Faculty of Chemistry University of Wroclaw 50-383 Wroclaw, Poland

Kenji Kanagawa (731, 1193) Dept. of Biochemistry National Cardiovascular Center Research Institute Osaka, 565-8565, Japan Daniel J. Kao (507) Dept. of Biochemistry and Molecular Genetics University of Colorado at Denver Health Sciences Center Aurora, CO 80045 Johji Kato (1163) First Dept. of Internal Medicine Miyazaki Medical College Kiyotake, Miyazaki, 889-1692, Japan Pravin T. P. Kaumaya (491) Tzagournis Medical Research Facility Ohio State University Columbus, OH 43210 Richard F. Keep (1423) Dept. of Neurosurgery University of Michigan Ann Arbor, MI 48109 David A. Keire (1013) VA GLAHS CURE: Digestive Diseases Research Center Los Angeles, CA 90073 William R. Kem (497) Dept. of Pharmacology and Therapeutics University of Florida College of Medicine Gainesville, FL 32610-0267 Sakae Kikuyama (321) Dept. of Biology School of Education Waseda University Tokyo 169-8050, Japan Young-Joon Kim (163) Dept. of Periodontics and Dental Science Research Institute Chonnam National University Kwang-Ju, Korea

Hiroyuki Kobori (1235) Dept. of Physiology Tulane University Health Sciences Center New Orleans, LA 70112 Masayasu Kojima (731) Institute of Life Science Kurume University Fukuoka, 839-0864, Japan Meike Körner (435) Division of Cell Biology and Experimental Cancer Research Institute of Pathology University of Bern CH-3010 Bern, Switzerland Piotr Kosson (1373) Medical Research Centre Polish Academy of Sciences 02106 Warsaw, Poland Catherine M. Kotz (919) VA Medical Center Minneapolis, MN 55417-2399 Harald Kropshofer (611) Pharmaceutical Research F. Hoffman La Roche Ltd. CH-4070 Basel, Switzerland Michael J. Kuhar (913) Yerkes Primate Center Emory University 954 Gatewood NE, Atlanta, GA 30329 R. Kwok (221, 229) Dept. of Zoology University of Toronto Toronto M5S 3G5, Canada

Patrick Kitabgi (559) Hospital St-Antoine 75571 Paris, France

Francisca Lago (869) Research Area Molecular and Celullar Cardiology Lab 1 Santiago University Clinical Hospital 15706 Santiago de Compostela, Spain

Kazuo Kitamura (1163) First Dept. of Internal Medicine

Steven W. Lamberts (1131) Dept. of Internal Medicine

xxvii

xxviii / Contributors Erasmus University Medical Center Rotterdam, Netherlands Angela B. Lange (177, 193) Dept. of Biology University of Toronto at Mississauga Mississauga, Ontario, Canada L5L 1C6 Alessandro Lecci (1301) Dept. of Clinical Pharmacology Menarini Research 50131 Florence, Italy Ronald M. Lechan (697) Div. of Endocrinology Diabetes and Metabolism Tupper Research Institute and Dept. of Medicine Boston, MA 02111 F. S. Lehmann (1047) Division of Gastroenterology University Hospital CH-4031 Basel, Switzerland Sarah F. Leibowitz (895) Rockefeller University New York, NY 10021-6399 Jérôme Leprince (813) Laboratory of Cellular and Molecular Neuroendocrinology 76821 Mont-Saint-Aigan, France Allen S. Levine (919) VA Medical Center Minneapolis, MN 55417-2399 Min Li (673) USJBRL Tulane University F. Edward Hebert Research Center Belle Chasse, LA 70037

Andrzej W. Lipkowski (1373) Medical Research Centre Polish Academy of Sciences 02106 Warsaw, Poland Catherine Llorens-Cortes (787) Unite 691 College de France Institute National de la Sante et de la Recherche Medicale 75231 Paris, France Marilena Loizidou (447) Dept. of Surgery Royal Free and University College London Medical School London, United Kingdom David A. Lovejoy (655) Dept. of Zoology University of Toronto Toronto Ontario, Canada L4A 1K6 José Manuel Lozano (515) Fundación Instituto de Inmunología de Colombia– FIDIC Bogotá, Colombia Susan M. Lunte (1461) Dept. of Pharmaceutical Chemistry University of Kansas Lawrence, KS 66045 Thomas A. Lutz (981) Institute of Veterinary Physiology Vetsuisse Faculty University of Zurich 8057 Zurich, Switzerland Cristina Maggioni (1529) Rabia Magoul (311) Laboratory of Animal Physiology University Sidi Mohamed Ben Abdellah Fès-Atlas, Morocco

Isabelle Lihrmann (795) Laboratory of Cellular and Molecular Neuroendocrinology INSERM U413, UA CNRS European Institute for Peptide Research (IFRMP 23) University of Rouen 76821 Mont-Saint-Aignan Cedex, France

Maria M. Malagón (311, 663, 813) Dept. of Cell Biology, Physiology & Immunology Campus Universitario de Rabanales University of Cordoba 14014 Cordoba, Spain

Keith Lindsey (23) Integrative Cell Biology Laboratory Durham University Durham DH1 3LE, United Kingdom

Ludwik K. Malendowicz (833) School of Medicine Dept. of Histology and Embryology PL-60-781 Poznan, Poland

Contributors Haritha Mandula (1415) Dept. of Pharmaceutical Sciences Texas Tech University Health Sciences Center Amarillo, TX 79106 Maria Luisa Mangoni (333) Dipartimento di Scienze Biochimiche “A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari Universita” “La Sapienza” Istituto Pasteur-Fondazione Cenci Bolognetti 00185 Rome, Italy Mohamed A. Marahiel (89) FB Chemie/Biochemie Philipps University of Marburg D-35032 Marburg, Germany Eleftheria Maratos-Flier (929) Dept. of Medicine Division of Endocrinology Beth Israel Deaconess Medical Center Boston, MA 02215 Heather G. Marco (189) Zoology Dept. University of Cape Town Rondebosch 7701, South Africa Nikki J. Marks (255) Parasitology Research Group Queen’s University Belfast Belfast BT9 7BL, Northern Ireland, United Kingdom Inge Martens (213) Genomics and Proteomics K. U. Leuven Zoological Institute Laboratory of Development Physiology Leuven, Belgium Roland Martin (585) Institució Catalana de Recerca i Estudis Avançats Fundacio per a la Recerca Hospital Unitat de Neuroimmunologia Clinica, Hospital Universitari Vall D’Hebron Barcelona, 08035, Spain Alfredo Martínez (453, 861) Dept. of Neuroanatomy & Cell Biology Institute Cajal CSIC Madrid, Spain Vicente Martínez (1005) Integrative Pharmacology—GI Biology AstraZeneca R&D Mölndal SE-43183 Mölndal, Sweden A. J. Martínez-Fuentes (663) Dept. of Cell Biology

Campus Universitario de Rabanales 14014 Cordoba, Spain F. Martínez-Pérez (229) Departamento de Genetica y Biologia Molecular CINVESTAV Mexico D. F. 07000, Mexico Edward P. Masler (247) Nematology Laboratory United States Dept. of Agriculture Plant Sciences Institute Beltsville, MD 20705 Yoshikatsu Matsubayashi (29) Graduate School of Bio-Agricultural Sciences Nagoya University Chikusa, Nagoya 464-8601, Japan Aaron G. Maule (255) Parasitology Research Group Queen’s University Belfast Belfast BT9 7BL, Northern Ireland United Kingdom Giuseppina Mazzocchi (883) Dept. of Human Anatomy and Physiology (Section of Anatomy) University of Padua I-35121 Padua, Italy Patricia J. McLaughlin (1313) Graduate Program in Anatomy Dept. of Neural & Behavioral Sciences H-109, Room C3727 Penn State Univ. College of Medicine Hershey, PA 17033-0850 Stefania Meini (1301) Dept. of Pharmacology Menarini Research 50131 Florence, Italy Pietro Melchiorri (269) Dept. of Human Physiology & Pharmacology “Vittorio Erspamer” University La Sapienza 00185 Rome, Italy Stéphane Mélik-Parsadaniantz (559) Jyotsana Menon (459) Hypertension and Vascular Disease Center Wake Forest University School of Medicine Winston-Salem, NC 27157

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Contributors Haritha Mandula (1415) Dept. of Pharmaceutical Sciences Texas Tech University Health Sciences Center Amarillo, TX 79106 Maria Luisa Mangoni (333) Dipartimento di Scienze Biochimiche “A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari Universita” “La Sapienza” Istituto Pasteur-Fondazione Cenci Bolognetti 00185 Rome, Italy Mohamed A. Marahiel (89) FB Chemie/Biochemie Philipps University of Marburg D-35032 Marburg, Germany Eleftheria Maratos-Flier (929) Dept. of Medicine Division of Endocrinology Beth Israel Deaconess Medical Center Boston, MA 02215 Heather G. Marco (189) Zoology Dept. University of Cape Town Rondebosch 7701, South Africa Nikki J. Marks (255) Parasitology Research Group Queen’s University Belfast Belfast BT9 7BL, Northern Ireland, United Kingdom Inge Martens (213) Genomics and Proteomics K. U. Leuven Zoological Institute Laboratory of Development Physiology Leuven, Belgium Roland Martin (585) Institució Catalana de Recerca i Estudis Avançats Fundacio per a la Recerca Hospital Unitat de Neuroimmunologia Clinica, Hospital Universitari Vall D’Hebron Barcelona, 08035, Spain Alfredo Martínez (453, 861) Dept. of Neuroanatomy & Cell Biology Institute Cajal CSIC Madrid, Spain Vicente Martínez (1005) Integrative Pharmacology—GI Biology AstraZeneca R&D Mölndal SE-43183 Mölndal, Sweden A. J. Martínez-Fuentes (663) Dept. of Cell Biology

Campus Universitario de Rabanales 14014 Cordoba, Spain F. Martínez-Pérez (229) Departamento de Genetica y Biologia Molecular CINVESTAV Mexico D. F. 07000, Mexico Edward P. Masler (247) Nematology Laboratory United States Dept. of Agriculture Plant Sciences Institute Beltsville, MD 20705 Yoshikatsu Matsubayashi (29) Graduate School of Bio-Agricultural Sciences Nagoya University Chikusa, Nagoya 464-8601, Japan Aaron G. Maule (255) Parasitology Research Group Queen’s University Belfast Belfast BT9 7BL, Northern Ireland United Kingdom Giuseppina Mazzocchi (883) Dept. of Human Anatomy and Physiology (Section of Anatomy) University of Padua I-35121 Padua, Italy Patricia J. McLaughlin (1313) Graduate Program in Anatomy Dept. of Neural & Behavioral Sciences H-109, Room C3727 Penn State Univ. College of Medicine Hershey, PA 17033-0850 Stefania Meini (1301) Dept. of Pharmacology Menarini Research 50131 Florence, Italy Pietro Melchiorri (269) Dept. of Human Physiology & Pharmacology “Vittorio Erspamer” University La Sapienza 00185 Rome, Italy Stéphane Mélik-Parsadaniantz (559) Jyotsana Menon (459) Hypertension and Vascular Disease Center Wake Forest University School of Medicine Winston-Salem, NC 27157

/ xxix

Contributors Tomoya Nakamachi (673) USJBRL Tulane University F. Edward Hebert Research Center Belle Chasse, LA 70037 Ryota Nakaoke (1455) VAMC St. Louis University School of Medicine St. Louis, MO 63106 Javier Narvaez-Vasquez (49) Dept. of Botany and Plant Science University of California Riverside, CA 92921-0001 June B. Nasrallah (41) Dept. of Plant Biology Cornell University Ithaca, New York 14853 Dick R. Nässel (171) Dept. of Zoology Stockholm University Svante Arrhenius vag 14 S-10691 Stockholm, Sweden L. Gabriel Navar (1235) Dept. of Physiology Tulane University Health Sciences Center New Orleans, LA 70112 Lucia Negri (269) Dept. of Human Physiology & Pharmacology “Vittorio Erspamer” University La Sapienza 00185 Rome, Italy Ingolf F. Nes (107) Dept. of Chemistry and Biotechnology Laboratory of Microbial Gene Technology Agricultural University of Norway 1432 Aas, Norway Drorit Neumann (1393) Dept. of Cell & Developmental Biology Sackler School of Medicine Tel Aviv University Tel Aviv 69978, Israel

University of Michigan Ann Arbor, MI 48109-1048 Graham M. Nicholson (369) Neurotoxin Research Group Dept. of Health Sciences University of Technology, Sydney Broadway NSW 2007 Sydney, Australia Pierre Nicolas (295) FRE 2852 CNRS-Universite Paris—6 Institut Jacques Monod 75251 Paris Cedex 05, France Toshio Nishikimi (1199) Dept. of Hypertension and Cardiorenal Medicine Dokkyo University School of Medicine Tochigi, 321-0293, Japan Rubén Nogueiras (953) Dept. of Psychiatry University of Cincinnati Cincinnati, OH 45237 Raymond S. Norton (363) Structural Biology Division Walter and Eliza Hall Institute of Medical Research Parkville 3052, Australia Gastone G. Nussdorfer (883) Dept. of Human Anatomy and Physiology (Section of Anatomy) University of Padua I-35121 Padua, Italy Fred Nyberg (1339) Dept. of Pharmaceutical Biosciences Division of Biological Research on Drug Dependence Uppsala University SE-751 24 Uppsala, Sweden Baldomero M. Olivera (381) Dept. of Biology University of Utah Salt Lake City, Utah 84112

Tzi Bun Ng (125, 131, 137, 145) Dept. of Biochemisty, Faculty of Medicine Chinese University of Hong Kong Shatin, Hong Kong, China

Joost J. Oppenheim (579) Laboratory of Molecular Immunoregulation Center for Cancer Research National Cancer Institute at Frederick Frederick, MD 21702-1201

Jun Ni (9) Dept. of Molecular, Cellular and Developmental Biology

Ian Orchard (177, 193) University of Toronto at Mississauga Mississauga, Ontario, Canada L5L 1C6

/ xxxi

xxxii / Contributors Christiane Otto (1293) CRBA Gynecology and Andrology Schering AG Female Health Care 13342 Berlin, Germany Andre J. Ouellette (1029) Dept. of Pathology & Laboratory Medicine University of California Irvine, CA 92697-4800 Nigel M. Page (763) School of Life Sciences Kingston University Whiteknights, Reading RG6 6AJ, United Kingdom Mario Sergio Palma (389) Laboratory of Structural Biology & Zoochemistry Center of Study of Social Insects (CEIS) Dept. of Biology Institute of Biosciences São Paulo State University (UNESP) Rio Claro, SP-13506-900, Brazil Weihong Pan (1435, 1529) Pennington Biomedical Research Center Blood Brain Barrier Group Baton Rouge, LA 70808 Jagen M. R. Parepally (1415) Dept. of Pharmaceutical Sciences Texas Tech University Health Sciences Center Amarillo, TX 79106

Centre for Gastroenterological Research B-3000 Leuven, Belgium Albert Eugene Pekary (629) VA Greater Los Angeles Healthcare System Bldg.114, Rm. 229 Los Angeles, CA 90073 Georges Pelletier (813) Dept. of Molecular Endocrinology Le Centre Hospital de l’Univ Laval 2705 Blvd Laurier Quebec City PQ G1V 4G2 Canada Mary Anne Pelleymounter (937) Dept. of Metabolic Research Bristol-Myers Squibb Pennington, NJ 08534 Diego Pérez-Tilve (953) Dept. of Psychiatry University of Cincinnati Genome Research Institute Cincinnati, OH 45237 Federico Perfetto (1529) Facolta di Medicina e Chirorgia University of Florence 50139 Florence, Italy Kelly B. Philpot (913)

Miejung Park (969) Pennington Biomedical Research Center Baton Rouge, LA 70808

Clemencia Pinilla (595) Torrey Pines Institute for Molecular Studies San Diego, CA 92121

Yoonseong Park (163)

Rubén Pío (453) Cellular & Pathology Dept. University of Navarra Pamplona, Spain

Manuel Elkin Patarroyo (515) Fundación Instituto de Inmunología de Colombia–FIDIC Bogotá, Colombia Gregory Pearce (5, 33, 49) Institute of Biological Chemistry Washington State University Pullman, WA 99164-6340 M. Soledade C. Pedras (151) Canada Research Chair in Bioorganic and Agricultural Chemistry Thorvaldson Dept. of Chemistry University of Saskatchewan Saskatoon, SK S7N 5C9, Canada Theo L. Peeters (1065) Gasthuisberg, O&N

Joseph R. Pisegna (1091) Division of Gastroenterology and Hepatology VA Greater Los Angeles Healthcare System David Geffen School of Medicine at UCLA Los Angeles, CA 90073 Kristof Plankensteiner (1481) Division of Theoretical Chemistry Institute for General, Inorganic & Theoretical Chemistry University of Innsbruck A-6020 Innsbruck, Austria Pierrre Poitras (1077) Hospital St Luc Montreal, Canada H2X-3J4

Contributors / xxxiii David M. Pollock (1269) Vascular Biology Center Medical College of Georgia Augusta, GA 30912-2500 Sergio Portal-Núñez (453) Vascular Biology Faculty National Cancer Institute, Center for Cancer Research Bethesda, MD 20892 Lourival D. Possani (339) Dept. of Molecular Medicine & Bioprocesses Institute of Biotechnology National Autonomous University of Mexico Cuernavaca Morelos 62210, Mexico Charalabos Pothoulakis (1085) Beth Israel Deaconess Medical Center Division of Gastroenterology Dana 601 330 Brookline Ave. Boston, MA 02215 Reinhard Predel (207) Institut fuer Allegemeine Zoologie Friedrich Schiller Universitaet 07743 Jena, Germany

European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France Annabelle Reaux-Le Goazigo (787) Unite 691 College de France Institut National de la Sante et de la Recherche Medicale 75231 Paris, France Joseph R. Reeve, Jr. (1013) CURE: Digestive Diseases Research Center VA Greater Los Angeles Healthcare System 11301 Wilshire Blvd. Bldg. 115, Rm. 115 Los Angeles, CA 90073, USA Jens F. Rehfeld (467) Dept. of Clinical Biochemistry (KB-3014) Rigshospitalet DK-2100 Copenhagen, Denmark Dirk Reinhold (567) Institute of Immunology Otto-von-Guericke-University Magdeburg D-39120 Magdeburg, Germany Patricia Rene (903)

Juan Carlos Prieto (1293) Departamento de Bioquimica y Biologia Molecular Universidad de Alcalá Modrid, Spain

Jean Claude Reubi (435, 1123) Division of Cell Biology and Experimental Cancer Research Institute of Pathology University of Bern CH-3010 Bern, Switzerland

Minolfa C. Prieto-Carrasquero (1235) Dept. of Physiology Research Building Ponce School of Medicine Ponce, PR 00716-2348

Daniel Ripoll (41) Computational Biology Service Unit Cornell University, Cornell Theory Center Ithaca, NY 14853

Sara Prutchi-Sagiv (1393) Dept. of Cell & Developmental Biology Sackler School of Medicine Tel Aviv University Tel Aviv 69978, Israel Remi Quirion (683) Douglas Hospital Research Centre Montreal, QC, Canada H4H 1R3 Satish C. Rattan (1155) Jefferson Medical College Thomas Jefferson University 1025 Walnut Street, Room # 901 College Philadelphia, PA 19107, USA Aurélia Ravni (847) Laboratory of Cellular and Molecular Neuroendocrinology

Natalie N. Rizk (1223) Dept. of Physiology Wayne State University School of Medicine Detroit, MI 48201-1928 Bernd M. Rode (1481) Institute for General, Inorganic & Theoretical Chemistry University of Innsbruck A-6020 Innsbruck, Austria Ricardo C. Rodríguez de la Vega (339) Dept. of Molecular Medicine & Bioprocesses Institute of Biotechnology National Autonomous University of Mexico Cuernavaca Morelos 62210, Mexico Gian Paolo Rossi (855) DMCS Internal Medicine 4

xxxiv / Contributors University Hospital 35126 Padua, Italy Noreen F. Rossi (1223) Dept. of Medicine and Physiology Wayne State University School of Medicine Detroit, MI 48201 William Rostène (559) Hospital Saint-Antoine 75012 Paris, France Clarence A. Ryan (5, 33, 49) Institute of Biological Chemistry Washington State University Pullman, WA 99164-6340 Jean-Marc Sabatier (415) Laboratoire ERT-62 “Ingénierie des Peptides à Visée Thérapeutique,” Université de la Méditérranée & Ambrilia Biopharma Inc., Faculté de Médecine Nord Boulevard Pierre Dramard, 13916—Marseille Cedex 20 France Hans-Georg Sahl (97) Institut für Medizinische Mikrobiologie und Immunologie Universität Bonn 53115 Bonn, Germany Sami I. Said (1307) Dept. of Medicine Pulomonary Critical Care Medicine Stonybrook, NY 11794-8172

Darrell R. Sawmiller (1215) College of Medicine University of South Florida Tampa, FL 33612 Andrew V. Schally (421, 483) VA Medical Center Miami, FL Lilliane Schoofs (183) Laboratory for Developmental Physiology, Genomics & Proteomics K. U. Leuven B-3000 Leuven, Belgium Mitchell L. Schubert (999) Gastroenterology Division McGuire VAMC; code 111N Richmonnd, VA 23249 Ulrich Schubert (1495) Institute of Clinical & Molecular Virology Univeristy of Erlangen-Nurnberg D-91054 Erlangen, Germany Kim B. Seroogy (1401) Neuroscience Graduate Program University of Cincinnati College of Medicine Vontz Center for Molecular Studies Rm 2320, 3125 Eden Ave. Cincinnati, OH 45267-0536 Cinzia Severini (261) Institute of Neurobiology & Molecular Medicine CNR 00143 Rome, Italy

Youji Sakagami (29) Shinobu Sakurada (1333, 1345) Dept. of Physiology & Anatomy Tohoku Pharmaceutical University Sendai, Japan

Patrick M. Sexton (771) Howard Florey Institute Level 2 Alan Gilbert Building University of Melbourne Carlton South, 3053 Victoria, Australia

Tsukasa Sakurada (1333) Dept. of Biochemistry Daiichi College of Pharmaceutical Sciences Fukuoka, 815-8511, Japan

O. Sharma (115) Dept. of Biological Sciences Purdue University West Lafayette, IN 47907-2054

Willis K. Samson (1277) Pharmacological and Physiological Science Saint Louis University School of Medicine St. Louis, MO 63104-1004

Ignacy Z. Siemion (573) Dept. Chemistry University of Wroclaw 50-383 Wroclaw, Poland

H. Uri Saragovi (1407) Lady Davis Institute–Jewish General Hospital McGill University Montreal, Quebec, Canada H3T 1E2

Maurizio Simmaco (333) Dipartimento di Scienze Biochimiche “A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari Universita” “La Sapienza”

Contributors Istituto Pasteur-Fondazione Cenci Bolognetti 00185 Rome, Italy Caroline J. Small (745, 945) Dept. of Metabolic Medicine Imperial College London London, W12 ONN, United Kingdom

Joan T. Steele (491) Tzagournis Medical Research Facility Ohio State University Columbus, OH 43210 A. Steiger (1521) Max Planck Institute of Psychiatry 80804 Munich, Germany

David E. Smith (1423) Dept. of Pharmaceutical Sciences University of Michigan Ann Arbor, MI 48109

Catia Sternini (1037) CURE, Digestive Diseases Research Center Los Angeles, CA 90073

Margaret E. Smith (1325) Dept. of Physiology Div. of Medical Sciences University of Birmingham Birmingham, B152TT, United Kingdom

John M. Stewart (443) Dept. of Biochemistry University of Colorado Health Sciences Center Denver, CO 80262

Quentin R. Smith (1415) Dept. of Pharmaceutical Sciences Texas Tech University Health Sciences Center Amarillo, TX 79106

Edward G. Stopa (1449) Dept. Pathology Brown Medical School, Rhode Island Hospital Providence, RI 02903

Beka Solomon (535) Dept. of Molecular Microbiology & Biotechnology Tel-Aviv University Ramat Aviv, Tel Aviv, Israel 69978 Mireia Sospedra (595) Unitat de Neuroimmunologia Clinica Hospital Universitari Vall D’Hebron Barcelona, 08035, Spain

J. Gregor Sutcliffe (721) Dept. of Molecular Biology Scripps Research Institute La Jolla, CA 92037

Robert B. Sothern (1529)

Yvette Taché (1005) Center for Neurovisceral Sciences & Women’s Health VA Greater Los Angeles Healthcare System Los Angeles, CA 90073

David R. Soto-Pantoja (459) Hypertension and Vascular Disease Center Wake Forest University School of Medicine Winston-Salem, NC 27157

Kazuhiro Takahashi (1209, 1515) Dept. of Analytical Medical Technology Tohoku University School of Health Sciences Sendai, 980-8575, Japan

Santi Spampinato (1319) Dept. of Pharmacology University of Bologna 40126 Bologna, Italy

Yoshio Takei (805, 1263) Laboratory of Physiology, Dept. of Marine Bioscience Ocean Research Institute University of Tokyo Tokyo, 164-8639, Japan

Raffaella Spinazzi (883) Dept. of Human Anatomy and Physiology (Section of Anatomy) University of Padua I-35121 Padua, Italy Eliot R. Spindel (277) Division of Neuroscience Oregon National Primate Research Center Oregon Health & Science University Beaverton, OR 97006

Kazuhiro Takekoshi (829) Institute of Clinical Medicine Dept. of Clinical Pathology University of Tsukuba Tsukuba 305-8575, Japan E. Ann Tallant (459) Hypertension and Vascular Disease Center Wake Forest University School of Medicine Winston-Salem, NC 27157

/ xxxv

xxxvi / Contributors Christina W. L. Tam (1181) Cardiovascular Division King’s College London London, SE1 1UL, England James P. Tam (541) Dept. of Medical Sciences Scripps Florida Jupiter, FL 33458 Roberto Tarquini (1529) Irving Taylor (447) Dept. of Surgery Royal Free and University College London Medical School London, United Kingdom Manuel Tena-Sempere (821) Physiology Section Dept. of Cell Biology, Physiology and Immunology Faculty of Medicine University of Cordoba 14014 Cordoba, Spain Tetsuya Terasaki (1443) Membrane Transport & Drug Targeting Lab Gradutae School of Pharmaceutical Sciences Tohoku University Aobaku, Sendai 980-8578, Japan Maria Tichomirova (1131) Dept. of Endocrinology CHU de Liège 4000 Liège, Belgium Nanda Tilakaratne (771) Howard Florey Institute Level 2 Alan Gilbert Building University of Melbourne Carlton South, 3053 Victoria, Australia Stephen S. Tobe (201, 221, 229) Dept. of Zoology University of Toronto Toronto M5S 3G5, Canada Marie-Christine Tonon (813) Laboratory of Cellular and Molecular Neuroendocrinology 76821 Mont-Saint-Aigan, France Alessandro Tossi (55) Dept. of Biochemistry University of Trieste Trieste, Italy 34127

Kazuhito Totsune (1209) Dept. of Clinical Pharmacology and Therapeutics Tohoku University Graduate School of Pharmaceutical Sciences and Medicine Miyagi, 980-8575, Japan Matthias Tschöp (953) Medical Sciences Building Dept. of Psychiatry University of Cincinnati Cincinnati, OH 45267 Hirokazu Tsukaya (37) National Institute for Basic Biology Okazaki Institute for Integrative Bioscience Okazaki, Aichi 444-8585, Japan Kazuyoshi Tsutsui (779) Laboratory of Brain Sciences Hiroshima University Higashi-Hiroshima 739-8521, Japan Valérie Turquier (311) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France Michael J. Tyler (283) Environmental Biology School of Earth & Environmental Sciences University of Adelaide South Australia 5005, Australia Akihiko Urayama (1455) VAMC St. Louis University School of Medicine St. Louis, MO 63106 Jozsef L. Varga (483) VA Medical Center Miami, FL R. Vásquez-Martínez (663) Dept. of Cell Biology Campus Universitario de Rabanales 14014 Cordoba, Spain David Vaudry (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) Mont-Saint-Aigan, France Hubert Vaudry (311, 689, 779, 795, 813, 847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France

Contributors / xxxvii Joseph G. Verbalis (1227) Dept. of Medicine Georgetown University Medical Center Washington, DC 20007 Peter Verleyen (213) Juan Carlos Prieto Villapun Dept. of Biochemistry and Molecular Biology University of Alcala Madrid España, Spain Thanh Q. Vo (1455) VAMC St. Louis University School of Medicine St. Louis, MO 63106 Anne Vogt (611) Pharmaceutical Research F. Hoffman La Roche Ltd. CH-4070 Basel, Switzerland Jörg Votteler (1495) Institute of Clinical & Molecular Virology Univeristy of Erlangen-Nurnberg D-91054 Erlangen, Germany Patricia M. Vuguin (975) Dept. of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 Wolfgang M. Wagner (499) Center for Translational Medicine in Women’s Health Tumor Vaccine Group University of Washington Seattle, WA 98195-8050 John Walker (17) Division of Biological Sciences University of Missouri Columbia, MO 65211-7400 B. A. Wallace (83) Dept. of Crystallography Birkbeck College University of London London WC1E 7HX, United Kingdom

Ji Ming Wang (547) LMI, CCR, NCI-Frederick Frederick, MD 21702 John Q. Wang (1345) Depts. of Anesthesiology & Basic Medical Science University of Missouri–Kansas City School of Medicine Kansas City, MO 64108-2792 Lixin Wang (1023) CURE/UCLA Los Angeles, CA 90073 Jiangqi Wen (17) Division of Plant Biology The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore, OK 73402 Haim Werner (1385) Dept. of Human Molecular Genetics & Biochemistry Sackler School of Medicine, Tel Aviv University Tel Aviv 69978, Israel David C. Whitcomb (1097) Division of Gastroenterology, Hepatology and Nutrition University of Pittsburgh and University of Pittsburgh Medical School Mezzanine Level, C-Wing 200 Lothrop Street Pittsburgh, PA 15213 L. Whitmore (83) Dept. of Crystallography Birkbeck College University of London London WC1E 7HX, United Kingdom Imke Wiedermann (97) Institut für Medizinische Mikrobiologie und Immunologie Universität Bonn 53115 Bonn, Germany

Guiyun Wang (1109) Dept. of Surgery The University of Texas Medical Branch Galveston, TX 77555-0725

Zsuzsanna Wiesenfeld-Hallin (1359) Division of Clinical Neurophysiology Huddinge University Hospital SE-141 86 Huddinge Sweden

H. X. Wang (125, 137) Dept. of Microbiology China Agricultural University Beijing, China

Jan Michael Williams (1269) Vascular Biology Center Medical College of Georgia Augusta, GA 30912-2500

xxxviii / Contributors Raphaelle Winsky-Sommerer (645) Institute of Pharmacology and Toxicology University of Zurich CH-8057 Zurich, Switzerland Ken A. Witt (1429) Dept. of Medical Pharmacology University of Arizona College of Medicine Tucson, AZ 85724-5050 Jack H. Wong (131) Dept. of Biochemisty, Faculty of Medicine Chinese University of Hong Kong Shatin, Hong Kong, China Stephen G. Woods (993) Dept. of Psychiatry University of Cincinnati Cincinnati, OH 45267 Victor Wray (1495) Dept. of Structural Biology German Research Centre for Biotechnology Braunschweig, Germany Olivier Wurtz (847) Laboratory of Cellular and Molecular Neuroendocrinology European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France Xiao-Jun Xu (1359) Dept. of Materials Science and Engineering University of Pennsylvania Philadelphia, PA 19104 Takahiro Yamaguchi (37) National Institute for Basic Biology Okazaki Institute for Integrative Bioscience Okazaki, Aichi 444-8585, Japan Yube Yamaguchi (5) Institute of Biological Chemistry Washington State University Pullman, WA 99164-6340 De Yang (553) SAIC-Frederick, Inc. National Cancer Institute at Frederick NIH Frederick, MD 21702-1201 Laurent Yon (311) Laboratory of Cellular and Molecular Neuroendocrinology

European Institute for Peptide Research (IFRMP 23) 76821 Mont-Saint-Aigan, France David A. York (969) Pennington Biomedical Research Center Baton Rouge, LA 70808 Masaaki Yoshikawa (1365) Div. of Food Bioscience & Technology Graduate School of Agriculture Kyoto University Gokasho Uji, Kyoto 611-0011 Japan Pu-Qing Yuan (1023) CURE/UCLA Los Angeles, CA 90073 S. D. Zakharov (115) Dept. of Biological Sciences Purdue University West Lafayette, IN 47907-2054 Igor Zelezetsky (55) Dept. of Biochemistry University of Trieste Trieste, Italy 34127 Lixin Zhang (1401) Ning Zhang (579) Laboratory of Molecular Immunoregulation Center for Cancer Research National Cancer Institute at Frederick Frederick, MD 21702-1201 Dezheng Zhao (1085) Division of Gastroenterology Beth Israel Deaconess Medical Center Harward Medical School Boston, MA 02215 Samuel Zinker (229, 1529) Departamento de Genetica y Biologia Molecular CINVESTAV Mexico D. F. 07000, Mexico Dusan Zitnan (163) Enrique Zudaire (453) Vascular Biology Faculty National Cancer Institute Center for Cancer Research Bethesda, MD 20892

Preface

When Bernard Westerop, the skilled publishing editor of the journal Peptides, suggested that I edit a book on peptides, my initial reaction was the same as it had been when I was asked many years ago to be editor of the first peptide journal: I’d better wait until I stop doing basic research [1]. However, it didn’t take me long to realize that if the book was properly organized, I could greatly accelerate my main goal as a journal editor: bringing together all aspects of biologically active peptides. Investigators in selected areas of research frequently are oblivious to work in related areas. In the peptide field, for example, many investigators working with mammalian peptides are unaware of the large number of peptides from other forms of life, such as the hundreds of venom peptides from scorpions, snakes, sea anemones, spiders, insects, and worms. They also usually overlook the applicability of many pertinent observations made with different types of peptides, such as those in bacteria. This point is emphasized by a statement in the chapter by Nicolas and Amiche: “The discovery of the considerable extent of sequence identity between the neuropeptide and antimicrobial peptide precursors is unprecedented”; I suspect that it is also little known. and who would have thought that plants make peptides (the first being identified in 1991 by Clarence A. (Bud) Ryan, editor of the Plant Peptides Section) and that they can function as hormones. Senior Publishing Editor, Tari Paschall, and Director of Development, Jasna Markovac, were very helpful and encouraged the publication of this amalgamation in a book large in length, width, and depth. One of the first decisions I had to make was whether the book should contain a few large chapters or many small ones. Since my goal was to assemble the disparate aspects of the field in one place for the first time, I chose to try to include as many types of peptides as possible, discussed by experts for each category. After careful consideration, I decided to organize this vast field into 20 sections, with some peptides intentionally discussed in more than one section. I then carefully chose the editors for these 20 sections. Thereafter, I was largely guided by their choices of chapters and authors. Although we tried to include articles for as many important peptides as possible, there were obviously some omissions, including at least one of my own favorite peptides, but we were more interested in showing the breadth of the peptide field. The next big problem arose in enforcing the page restriction for each chapter. Understandably, there was the tendency for the authors to include everything. Although we tried to impose uniformity in design of the chapters, we were flexible in allowing authors to choose a balance between the amount of text and number of references within the space limitation. Reviews were cited wherever possible to save space. Even so, most chapters were initially too long to allow publication of all the material in a single volume. There was one figure, however, suggested by Hubert Vaudry, that was incorporated into all chapters of his Brain Peptides section. This was a template of a sagittal section of the brain depicting the distribution of the peptide (cell bodies and fibers) or the peptide precursor mRNA. It serves to unify his large section of the Handbook, and his outline provided homogeneity to several other sections, including those involving Plant, Bacteria, Invertebrate, and Amphibian peptides: 1. Discovery 2. Structure of the Precursor mRNA/Gene 3. Distribution of the mRNA

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xl / Preface 4. 5. 6. 7. 8.

Processing of the Precursor Receptors and Signaling Cascades Information on Active and/or Solution Conformations (if available) Biological Actions Specific to the Organ or System Pathophysiological Implications

Since the first few of these items could be discussed in several sections of the book, it was decided that the general background sections common to several chapters dealing with mammalian peptides would be discussed primarily in the Brain Peptides Section. For other sections, such as GI Peptides, this same outline is followed except that the focus is specific for the GI tract. Variations of this outline are used in other sections. For example, for the Ingestive Peptides Section, David York recommended this outline: 1. Description of the effects on feeding behavior 2. Studies from genetic manipulations or mutations—for example, knockouts, transgenics, siRNA experiments 3. Functional response of the peptide/gene to differing metabolic and feeding states 4. Sites of action and neural networks affected 5. Interactions with other peptidergic/aminergic systems 6. Physiological and pathophysiological implications Other section editors recommended further variations. The Vaccine Peptides Section included the disease target as one of the categories. Naoto Minamino, discoverer of several peptides, recommended use of the Brain Peptides Section format but also noted that cardiovascular peptides are known to participate in the pathogenesis of cardiovascular diseases, and their plasma concentrations are often used as markers for their diagnosis, prognosis, and therapy. Therefore, he instructed authors in his Cardiovascular Peptides Section to focus on the peptides’ endogenous form and concentration in plasma as well as their alteration under normal (physiological) and diseased (pathological) conditions. The book tries to keep the abbreviations consistent. However, for some—for example, LHRH/ GnRH and orexin/hypocretin—we allowed differences to reflect the authors’ previous work already in the literature, even though I was an author of the paper showing that LHRH released both gonadotropins [4] and even though my one paper [2] on orexin/hypocretin used only the term orexin. Therefore, while there are a large number of chapters written by different authors, there is more uniformity in organization than would be expected from so many contributors. Discussion of some of the same peptides in different sections emphasizes the system involved. Conversely, some peptides discussed in only one section have actions that could fit into several sections (e.g., IGF-1). Many of the chapters are written by the investigator who made the initial discovery of the peptide being discussed. For those mammalian peptides included in more than one section of the book, the emphasis is different in each section. Some degree of overlap was necessary, but it was kept to a minimum by the early transmission of the chapters in the Brain Peptides Section to the authors of chapters dealing with the same peptides in other sections. This was accomplished by the imposition of a deadline for the Brain Peptides chapters that was two months sooner than that for the other chapters. Moreover, each author was provided with the e-mail addresses of all authors and encouraged to communicate directly, especially after receiving drafts of related chapters from me. It is very satisfying for me to see the tremendous body of knowledge in the field of biologically active peptides brought together in a single volume. I am especially gratified that so many chapters inherently reflect some of the concepts with which I have been personally involved: Peripheral peptides can affect the brain, endogenous antiopiate peptides exist, peptides can exert multiple effects, peptides can cross the blood–brain barrier directly, and others [3]. It is now almost inconceivable that they were once so controversial. Despite the restrictions inherent in this large undertaking, the section editors and contributors were extremely cooperative, and I thank all of them. Their enthusiastic encouragement should

Preface / ensure that this book will not only serve as an important reference source but will also widen the scope of the important field of biologically active peptides. Abba J. Kastin Pennington Biomedical Research Center Baton Rouge, LA 70808–4124 [email protected] After the publisher received all manuscripts, including this Preface, they decided that the formidable task of restricting the length of each chapter had been so successful that all materials designated for the supplemental disk could now be incorporated into this book.

References [1] Kastin, A. J. Editorial: Twenty-five years of peptides. Peptides 25:315–317; 2004. [2] Kastin, A. J.; Akerstrom, V. Orexin A but not orexin B rapidly enters brain from blood by simple diffusion. J Pharmacol Exp Ther 289:219–223; 1999. [3] Kastin, A. J.; Zadina, J. E.; Banks, W. A.; Graf, M. V. Misleading concepts in the field of brain peptides. Peptides 5:249– 253; 1984. [4] Schally, A. V.; Arimura, A.; Kastin, A. J. Hypothalamic regulating hormones. Science 179:341–350; 1973.

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Foreword

It is a great privilege to write a Foreword for this important handbook of peptides, to which my coworkers and I have also contributed a couple of chapters. The Handbook of Biologically Active Peptides, with more than 200 chapters, presents for the first time all biologically active peptides in one text. The chapters are organized in sections on Plant, Bacterial/Antibiotic, Fungal/Antifungal, Invertebrate, Amphibian, Venom, Cancer/Anticancer, Vaccine, Inflammatory and Immunological, Brain, Endocrine, Ingestive, Gastrointestinal, Cardiovascular, Respiratory, Renal, Opiate, Neurotrophic, Blood–Brain Barrier, and other peptide topics. All the chapters are edited by expert section editors. This handbook brings together some of the world’s most distinguished scientists who have made major contributions to their particular fields of hormone peptides. The contributors represent some of the most sophisticated investigators working in basic as well as clinical areas. Each contributor has been involved in research in his or her particular area of expertise. Each chapter is well documented with suitable references. Abba Kastin, because of his magnetic personality, wide circle of acquaintances, and international reputation as a scientist, has been able to bring together this group of distinguished individuals. Indeed, we are indebted to him for this book, its excellent planning, systematic organization, and the coverage of so many areas. The book identifies the current state of knowledge in the field, and some chapters include the currently available therapies and future treatments. Thus, the book will be most useful and practical not only for all investigators working with biologically active peptides, but also for other scientists and even clinicians who work to continue and update their scientific and medical education. A famous Spanish endocrinologist, Gregorio Maranon, once said, “Many books are written, and they come and go and are forgotten if they are not significant.” This book by Dr. Kastin, however, will be the gold standard of our time. This book is a seminal contribution to science and medicine, and should become a classic. I hope readers enjoy it as much as I do. Andrew V. Schally Nobel Prize in Physiology or Medicine

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C

H

A

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1 4-kDa Peptide HISASHI HIRANO

STRUCTURE OF THE PRECURSOR mRNA/GENE

ABSTRACT The 4-kDa disulfide-rich peptide (4k-P) is a ligand for the 43-kDa basic glycoprotein (43k-P) in legumes. 4k-P stimulates protein kinase activity of the β-subunit of 43k-P by binding to the α-subunit of 43k-P, and regulates growth, differentiation, and cell proliferation of plant callus. 4k-P consists of 37 residues and assumes a T-knot scaffold containing three β-strands. 43k-P shows some structural and functional similarity to animal hormone receptors. Both 4k-P and 43k-P locate around the plasma membranes and cell walls, showing that 4kP is present in a site suitable for interaction with 43k-P and signal transduction. 4k-P and 43k-P may function as a hormone peptide and a hormone receptor protein, respectively.

A comparison of the amino acid sequence deduced from the cDNA sequence with the actual sequence determined showed that the cDNA contained open reading frames for a putative signal peptide followed by sequences for 4k-P, linker peptide, and 6-kDa peptide. 4k-P has a molecular mass of 3,920. The complete amino acid sequence of 4k-P was determined in soybean and other legume species [12]. 4k-P consisting of 37 amino acid residues contains six half-cystines in three disulfide bridges. The sequences were highly conserved between these species (Fig. 1), suggesting the functional importance of 4k-P. Two mass signals with 3863.9 and 3920.5 were detected in the mass spectrometry of 4k-P. This mass difference, 56.6, corresponds to the mass of the Cterminal Gly [11], but not the mass of the amide (58 Da). Therefore, the penultimate Thr of 4k-P is not amidated. After S-aminoethylation, 4k-P was digested with lysylendopeptidase. Mass spectrometry of the digests detected two types of the C-terminal peptides with and without the C-terminal Gly. The processing of the C-terminal may be related to the activation of the peptide.

DISCOVERY The involvement of hormone peptides in regulatory metabolic mechanisms in plants and microorganisms has yet to be fully understood. In the late 1980s, a 43kDa glycoprotein (43k-P) was isolated from soybean seeds, which showed similarity to animal hormone receptors in protein structure, subcellular localization, and protein kinase activity [11]. 43k-P would appear to be a hormone receptor protein. The presence of a hormone receptorlike protein suggests that the hormonal peptides that are capable of binding to 43k-P may be present in plants. Based on this speculation, many efforts have been made to isolate and characterize peptides that were capable of binding 43k-P. As a result, a 4-kDa biologically active peptide (4k-P) was isolated from the soybean germinated radicles [11]. Handbook of Biologically Active Peptides

DISTRIBUTION OF THE mRNA Western blotting and DNA cloning experiments have revealed that proteins structurally similar to 43k-P are distributed in a number of legume species such as azuki bean, cowpea, French bean, lupin, mung bean, and winged bean, and nonlegume species such as carrot,

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2 / Chapter 1 S–S Soybean Azuki bean Mung bean G.soja Pea Lupin

S–S

S–S

ADCNGACSPFEVPPCRSRDCRCVPIGLFV-GFCIHPTG ADCNGACSPFEMPPCGSTDCRCVPIALVG-GFCIHPTG ADCNGACSPFQMPPCGSTDCLCIPAGLLFVGYCTYPSG ADCNGACSPFEVPPCRSRDCRCVPIGLFV-GFCIHPTG ASCNGVCSPFEMPPCGSSACRCIPVGLVV-GYCRHPSG XXXXXXXXXXXIPPSRSSDCRCVPITLIV-GFCIHPTG * *** **** *** * * * * * * * * *

FIGURE 1. Amino acid sequences of 4k-P superfamilies. The sequences for soybean [11]; Glycine soja (wild Glycine sp.) [10]; pea [4]; lupin [5] 4k-P are shown. Underlined Ser, possibly sequencing error; X, not determined.

rice, and Arabidopsis. However, 4k-P was found in the legume species, but not in carrot, rice, and Arabidopsis, suggesting that no peptides similar in sequence to 4k-P may be present in carrot, rice, or Arabidopsis. Immunocytochemistry has shown that 4k-P is localized around the plasma membranes and cell walls. The subcellular localization of 4k-P is similar to that of 43k-P [9], showing that 4k-P is present in the site suitable for interaction with 43k-P.

tive transmembrane domain in the β subunit. Both 43kP and IR are synthesized as a precursor polypeptide that is posttranslationally cleaved on the N-terminal side of Ser residue to generate α and β subunits. A consensus sequence of ATP-binding site indispensable for protein phosphorylation activity is present in both 43k-P and IR. The soybean 43k-P was found to have protein kinase activity about two-thirds that of tyrosine kinase in the rat IR [8], and the carrot 43k-P to have the activity equivalent to the rat IR [5]. Immunocytochemistry has indicated that, like IR, 43k-P locates in the plasma membranes and in the middle lamellae of cell walls that are unique to plants [9]. Comprehensive posttranslational modification analysis of the carrot 43k-P has shown that this protein contains six disulfide bonds, four glycosylation sites with Asn-linked glycan, and N-terminal modification (pyroglutamic acid) [14]. The binding activity of the deglycosylated 43k-P for 4k-P is about one-third that of the native 43k-P, and the reduced and S-carboxymethylated 43k-P has no binding activity for 4k-P, suggesting that the glycosylation and disulfide bonds play important roles in binding 4k-P [13].

PROCESSING The structure of the precursor mRNA indicates that 4k-P is synthesized as a precursor polypeptide containing a putative signal peptide, 4k-P, linker peptide, and 6-kDa peptide in ribosome bound to the endoplasmic reticulum, and then the putative signal peptide is cotranslationally removed. Finally the precursor polypeptide is posttranslationally cleaved to isolate 4k-P, and intradisulfide bonding is formed to generate the mature 4k-P [11]. The 6-kDa peptide was found to localize in the protein body [4], suggesting that although 4k-P and the 6-kDa peptide are generated from the same precursor polypeptide, they are sorted to different sites.

RECEPTORS The receptorlike protein, 43k-P, has a high isoelectric point (9.05–9.26) and is made of four pairs of α (27-kDa) and β (16-kDa) subunits linked together by a disulfide bond. 43k-P has no sequence similarity with the animal hormone receptors except that there is limited sequence homology between 43k-P and the human insulinlike growth factor binding protein. However, 43k-P shows some structural and functional similarities to animal hormone receptors such as the insulin receptor (IR). For example, like IR, 43k-P has a disulfide-bound α and β subunit structure, a Cys-rich domain in the N-terminal side of α subunit, and a puta-

ACTIVE AND/OR SOLUTION CONFORMATION 4k-P assumes a T-knot scaffold containing three βstrands (Fig. 2)[7, 12]. Two adjacent β-strands connected by a distorted type-I β-turn around Gly26-Val29 make up a two-stranded antiparallel β-sheet that is stacked by a long N-terminal loop, two type-I β-turns around Ser8Glu11 and Ser17-Cys20, and a cis Pro at position 13. The stacked structure is stabilized by three disulfide bridges formed between the β-sheet and the N-terminal loop. It has been reported that the T-knot scaffold is shared by several small disulfide-rich proteins with diverse functions, such as carboxypeptidase A inhibitor from potato and the calcium channel blockers ω-conotoxin of the cone snail and ω-agatoxin of the funnel web spider. These are discussed in chapters in the Venome Peptides section of this book. A structure similar to that of 4k-P was reported for the pea analog, PA1b [6].

BIOLOGICAL ACTIONS 4k-P can be purified from the soybean radicles by affinity chromatography using a Sepharose CL-4B column immobilized with 43k-P as a ligand. Ligand blotting experiments using the radioiodinated 4k-P and surface plasmon resonance spectroscopy has confirmed that the peptide is capable of binding to the α-subunit of 43k-P [14].

4-kDa Peptide /

a

b

FIGURE 2. A. The best-fit superposition of the backbone (N, Ca, and ′C) atoms of the 15NMR-derived structures of 4k-P. The structures are superimposed against the energy-minimized average structure using the backbone coordinates of residues 3–35 (r.m.s.d. of 0.62 ± 0.14 Å for backbone atoms and 1.16 ± 0.14 Å for all heavy atoms). B. Ribbon drawing of the energy-minimized average structure of the 4k-P. The disulfide bridges are shown as ball-and-stick models.

18

Incorporated (cpm × 10–2)

4k-P

32P

4k-P can stimulate the protein kinase activity of 43kP [11]. The maximum stimulatory effect was observed at relatively low 4k-P concentration (1nM)(Fig. 3), indicating the possible involvement of 4k-P and 43k-P in the cellular signal transduction mechanism in a manner similar to that of insulin and IR. To know the biological functions of 4k-P, 4k-P was expressed transiently in the cultured carrot and bird’sfoot trefoil cells transfected with the pBI121 plasmid containing the 4k-P gene and the β-glucuronidase (GUS) gene as a reporter using the Agrobacterium transformation system [12]. The obtained transgenic plants were confirmed to carry the 4k-P gene by Southern blotting and to transcribe the 4k-P mRNA by Northern blotting. GUS activity was constitutively detected in the roots and leaves of the transgenic plants. The transgenic callus rapidly grew compared with the wild callus at the early developmental stage. However, the phenotype of the intact transgenic plants regenerated from these calli was not noticeably different from that of the wild plant. 43k-P has been detected in the wild carrot cells, but not in 4k-P. This result suggests that the 4k-P that is synthesized by the transfected gene can stimulate protein kinase activity of the carrot 43k-P and the signal transduction pathway can be activated for the regulation of growth of the callus. On the other hand, the effect of 4k-P on the growth, differentiation, and cell proliferation of in vitro

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12

Reduced 4k-P 10

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100

Amount (nM) of the 43k-P

FIGURE 3. Stimulation of 43k-P phosphorylation activity with 4k-P. Phosphorylation activity was measured in a reaction mixture containing [γ-32P]ATP. 43k-P and different concentrations of 4k-P, reduced 4k-P or atrial natriuretic peptide (ANP). Results are the mean of three experiments.

3

4 / Chapter 1 cultured carrot callus was investigated [12]. The results suggest that 4k-P is also involved in the regulation of the growth and differentiation of the callus and cell proliferation. Disruption of the disulfide bridges leads to a complete loss of the stimulatory effect of 4k-P on the phosphorylation activity of 43k-P [11], indicating that the disulfide bridges play an important role in maintaining the correct three-dimensional structure of 4k-P required for its function. The amino acid sequences are highly homologous among legume species; in particular, the sites of Cys residues are completely conserved. This also shows the significance of intradisulfide bonds for 4k-P to function. The three-dimensional structure of 4k-P determined by NMR spectroscopy suggests that the specific binding activity of 4k-P to 43k-P and its stimulatory effect on protein phosphorylation could be attributed to the spatial arrangements of the hydrophobic residues at the solvent-exposed surface of the twostranded β-sheet. A structure-activity relationship study using site-directed mutagenesis and surface plasmon resonance spectroscopy of 4k-P confirmed that the substitution of the hydrophobic residues at the solventexposed surface of the two-stranded β-sheet made a significant change in its binding activity to 43k-P [2, 3]. Recently, the pea 4k-P (PA1b) was found to have defensive actions against cereal weevils Sitophilus spp. [1]. The mechanism of action of PA1b and the toxic effect of 4k-P of other legume species on cereal weevils are not completely understood.

References [1] Gressent F, Rahioui I, Rahbe Y. Characterization of a highaffinity binding site for the pea albumin 1b enterotoxin in the weevil Sitophilus. Eur J Biochem 2003; 270: 2429–35.

[2] Hanada K, Hirano H. Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean. Biochemistry 2004; 43: 12105–12. [3] Hanada K, Nishiuchi Y, Hirano H. Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein. Eur J Biochem 2003; 270: 2583–92. [4] Higgins TJ, Chandler PM, Randall PJ, Spencer D, Beach LR, Blagrove RJ, et al. Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds. J Biol Chem 1986; 261: 11124–30. [5] Ilgoutz SC, Knittel N, Lin JM, Sterle S, Gayler KR. Transcription of genes for conglutin γ and a leginsulin-like protein in narrowleafed lupin. Plant Mol Biol 1997; 34: 613–27. [6] Jouvensal L, Quilien L, Ferasson E, Rahbe Y, Gueguen J, Volvelle F. PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H-2-D NMR study and molecular modeling. Biochemistry 2003; 42: 11915–23. [7] Katoh E, Hirano H, Nishiuchi Y, Yamazaki T. The solution structure of leginsulin, a plant hormone with insulin-like activity, by nuclear magnetic resonance spectroscopy. In: Shioiri T ed. Peptide Science, Japan Peptide Soc; 2000, 97–100. [8] Komatsu S, Koshio O, Hirano H. Protein kinase activity and insulin-binding activity in plant basic 7S globulin. Biosci Biotech Biochem 1994; 58: 1705–6. [9] Nishizawa NK, Mori S, Watanabe Y, Hirano H. Ultrastructural localization of the basic 7S globulin in soybean (Glycine max) cotyledons. Plant Cell Physiol 1994; 35: 1079–85. [10] Tan J, Lou C, Hirano H. Analysis of leginsulin gene in soybean cultivar (Glycine max) and wild species (Glycine soja). Chin. J Appl Environ Biol 1999; 5: 259–63. [11] Watanabe Y, Barbashov SF, Komatsu S, Hemming AM, Miyagi M, Tsunasawa S, et al. A peptide that stimulates phosphorylation of the plant insulin-binding protein: Isolation, primary structure and cDNA cloning. Eur J Biochem 1994; 224: 167–72. [12] Yamazaki T, Takaoka M, Katoh E, Hanada K, Sakita M, Sakata K, et al. A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes. Eur J Biochem 2003; 270: 1269–76. [13] Shang C, Sassa H, Hirano H. The role of glycoprotein in the function of a 48-kDa glycoprotein from carrot. Biochem Biophys Res Commun 2005; 328: 144–9. [14] Shang C, Shibahara T, Hanada K, Iwafune Y, Hirano H. Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein. Biochemistry 2004; 43: 6281– 92.

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2 At Pep1 Peptides ALISA HUFFAKER, YUBE YAMAGUCHI, GREGORY PEARCE, AND CLARENCE A. RYAN

had full biological activity, compared with the native peptide.

ABSTRACT AtPep1 is a 23-amino-acid defense-related peptide isolated from leaves of Arabidopsis thaliana that is derived from the carboxyl terminal of a 92-amino-acid precursor polypeptide called proAtPep1. The gene encoding proAtPep1 is expressed in leaves in response to several defense-related stimuli, including wounding, methyl jasmonate (MeJA), and ethylene. AtPep1 regulates the expression of plant defense genes against pathogens, including those encoding the antifungal proteins PDF1.2 (defensin) and several pathogen-related (PR) proteins that are known to be regulated through the jasmonate/ethylene defense signaling pathway. Constitutive overexpression of the proAtPep1 gene in Arabidopsis plants causes a constitutive expression of defense genes and enhances plant resistance to infection by an oomycete pathogen.

STRUCTURE OF AtPep1 AND THE GENE ENCODING THE PRECURSOR PROTEIN ProAtPep1 AtPep1 is derived from the 23 carboxyl terminal residues of a 92-amino-acid precursor protein polypeptide, proAtPep1 [2], deduced from an EST encoding the gene At5g64900 (Fig. 1). Seven proAtPep paralogs are encoded in the Arabidopsis genome share a 10-amino-acid motif, at the carboxyl terminus of each deduced protein where the putative AtPep peptides would be found, is SSG(K/R)xGxxN. Analyses of the biological activities of alanine-substituted analogs, or of AtPep1 with deletions at its N-terminus, revealed that the 10 C-terminal residues are necessary for activity, and modifications within the core motif above reduced the activity of the peptide severalfold [10]. Substituting Gly-17 in the conserved SSG sequence of the motif with Ala totally abolished activity, but the analog was not a competitor of native AtPep1 in the alkalinization assay [10]. The amino-terminal regions of the AtPep peptides deduced from all of the paralogs are cationic in nature. AtPep1 has a predicted pI of 11.2 with five lysines and two arginines concentrated in the amino-terminal half of the peptide [2]. The proAtPep1 precursor gene contains two exons separated by a single intron [1]. Five of the seven proAtPep paralogs present in Arabidopsis share these features, whereas two paralogs have no introns. All of the proAtPep precursor proteins have negatively charged glutamate/aspartate repeats (EKE motifs [9]), interrupted by single lysine/arginine residues. The proAtPep1 protein, as well as the deduced proteins of other family members, lack signal sequences that would target

DISCOVERY AtPep1 peptide was isolated from Arabidopsis thaliana (mouse-ear cress) [2] utilizing an activity assay using suspension cultured cells derived from Arabidopsis thalania [6]. This assay is derived from early signal transduction events, triggered by plant peptidereceptor interactions [6–9] that cause a rapid and transient alkalinization of the extracellular medium surrounding the cells [1, 4, 10]. With this assay, fractions separated from Arabidopsis leaf extracts were found to contain a bioactive peptide, called AtPep1, that was purified to homogeneity [2]. Purified AtPep1 was active in the suspension culture assay at nanomolar concentrations, but the peptide had no effect on suspensioncultured cells from other plant species [2]. The peptide is composed of 23 amino acids, and it is not posttranslationally modified. A synthetic 23-amino-acid AtPep1 Handbook of Biologically Active Peptides

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6 / Chapter 2 1 10 20 30 40 50 MEKSDRRSEE SHLWIPLQCL DQTLRAILKC LGLFHQDSPT TSSPGTSKQP60 70 80 90 92 KEEKEDVTME KEEVVVTSR ATKVKAKQRGKEKVSSGRPGQHN 1 AtPcp1 23

FIGURE 1. Active AtPep1 is a 23-amino-acid peptide derived from the carboxyl terminus of a 92-amino-acid precursor polypeptide, proAtPep1.

Paralogs ProAtPepl At5g64890 At5g64905 At5g09980 At5g09990 Unannot. At2g22000

Precursor Length 1 23 69-ATKVKAKQRGKEKVSSGRPGQHN* 92 74-DNKAKSKKRDKEKPSSGRPGQTNSVPNAAIQVYKED* 109 73-EIKARGKNKTKPTPSSGKGGKHN* 96 55-GLPGKKNVLKKSRESSGKPGGTNKKPF* 81 59-SLNVMRKGIRKQPVSSGKRGGVNDYDM* 86 53-VSGNVAARKGKQQTSSGKGGGTN* 75 81-ITAVLRRRPRPPPYSSGRPGQNN* 104

Orthologs Canola 74-VARLTRRRPRPP-YSSGQPGQIN* Potato 93-PTERRGRPPSRPKVGSGPPPQNN* Poplar 94-DAAVSALARRTPPVSRGGGGQTNTTTS* Medicago 87-LSSMGRGGPRRTPLTQGPPPQHN* Soybean 93-ASLMATRGSRGSKISDGSGPQHN* Rivel 131-ARLRPKPPGNPREGSGGNGGHHH* Rice2 65-DDSKPTRPGAPAEGSGGNGGAIHTAASS*

95 116 121 111 115 154 93

FIGURE 2. The C-terminal regions of proAtPep orthologs and paralogs that contain putative AtPep sequences. A. Amino acid sequence alignment of the predicted protein sequence of Arabidopsis proAtPep family members as found by BLAST searching the Arabidopsis genome. Subfamily I members are proAtPep1 (At5g64900), At5g64890, and At5g64905. Subfamily II includes At5g09990, At5g09980, and the unannotated gene. At2g22000 is the lone member of Subfamily III. B. Sequence alignment of proAtPep1 orthologs from other plant species.

them through the secretory pathway, and therefore appear to be synthesized on cytosolic ribosomes. The Arabidopsis proAtPep gene family can be classified into three subfamilies based on chromosomal locations [2]. Six of the seven paralogs are tandemly arrayed on chromosome V in two groups [NCBI Arabidopsis Genome Database]. In Subfamily I, At5g64890, At5g64900 [proAtPep1], and At5g64905 are sequentially positioned in a 5.5-kilobase region on chromosome V, whereas Subfamily II, At5g09980, At5g09990, and a nonannotated family member are positioned together in a 3.8-kilobase region of chromosome V. One gene, At2g22000, is the lone member of Subfamily III and is located on chromosome II. ProAtPep orthologs have been identified in species of several plant families, including important crop plants such as canola, potato, rice, soybean, Medicago, and poplar [2]. All of these genes encode putative precursor proteins of 93 to 154 amino acids in length, and all lack signal sequences, contain EKE motif regions, and have predicted amphipathic helical regions. The

putative proAtPep regions of these orthologs are Lys/ Arg rich. The only absolutely conserved residue in this region among all paralogs and orthologs is the glycine residue that when substituted with alanine in AtPep1 abolished activity.

DISTRIBUTION OF ProAtPep1 mRNA The gene encoding proAtPep1 is expressed at low basal levels in all Arabidopsis tissues from newly germinated seedlings to mature, seed-bearing plants [2]. ProAtPep1 gene expression is not specific to any particular plant organ and is expressed in roots, leaves, stems, and flowers, with basal levels in flowers and roots being somewhat higher than in other tissues. The proAtPep1 gene in leaves is inducible by wounding, MeJA, and ethylene. The cell-specific localization of both mRNA and protein are not known. Other gene family members are expressed in a wide distribution of tissues in response to MeJA, with most genes showing higher basal levels of

AtPep1 Peptides / 7 expression in the flowers and roots. For one family member, the unannotated open reading frame from Subfamily II, expression was not detectable, indicating that only six of the seven In addition to being jasmonate-inducible, all of the Subfamily I gene family members that are tandemly arrayed on chromosome V (including proAtPep1) are inducible by the AtPep1 peptide, but the Subfamily II and Subfamily III genes, located at other chromosomal loci, are not responsive to the peptide [2]. Thus, a possible mechanism for differential regulation of the subfamilies may be related to chromosomal associations.

PROCESSING OF ProAtPep1 The precursors of all proAtPep family members lack signal sequences that would target them through the secretory pathway and therefore are predicted to be cytosolic proteins. However, AtPep1 interacts with a cell surface receptor [9] to initiate defense signaling, indicating that it, or its precursor, is exported from the cell. Currently, how AtPep1 arrives at the cell surface is not known. The EKE charged motifs and amphipathic helix motif residing in the precursor protein have both been previously described as protein-protein interaction motifs [9]. Thus, these regions of proAtPep1 and the other precursors may interact with some as yet unknown protein[s] such as chaperones or processing enzymes to facilitate processing and/or export.

AtPep1 RECEPTOR The AtPep1-binding protein was identified by photoaffinity labeling and purified. Radioiodinated azido-labeled AtPep1 bound reversibly to a 170 kD Arabidopsis cell surface protein, called AtPepR1, being completed with unlabeled AtPep1 but not with tomato systemin [14]. Binding was inhibited by suramin, a heterocyclic, polysulfonated inhibitor of peptide hormonereceptor interactions in animal and plant cells [13]. Kinetic analyses of AtPepR1-binding to Arabidopsis suspension cultured cells using radiolabeled AtPep1 showed that AtPep1 binds to the cell surface bindingprotein within a minute, with a Kd of 0.25 nM, a binding constant typical of hormone-receptor interactions [14]. The photoaffinity-labeled protein was purified to homogeneity from the Arabidopsis suspension cultured cells. Tryptic digests, analyzed by mass spectroscopy, provided sequence data that was used to identify AtPepR1 as At5g73080 [9]. AtPepR1 consists of 1123 amino acids and has typical receptorlike kinase motifs including 27 leucine-rich repeats, a transmembrane region, and an intracellular Ser/Thr protein kinase domain. AtPepR1

belongs to the subfamily LRR XI, based on the phylogenetic analysis previously reported [12].

SOLUTION CONFORMATION No information regarding the conformation of the AtPep1 peptide has been obtained.

BIOLOGICAL ACTIONS OF AtPep1 AtPep1 is a component of signal transduction processes to initiate plant defense against pathogens. Gene expression analysis of excised leaves supplied through their cut petioles with a solution of the peptide reveals many genes induced by AtPep1 (Table 1). Arabidopsis plants constitutively expressing the proAtPep1 gene express the same defense genes that are induced by MeJA and ethylene. These transgenic plants were more resistant to the oomycete root pathogen Pythium irregulare than were wild-type plants [2]. As seen in Fig. 3, transgenic plants inoculated with the pathogen have larger leaf rosettes and more well-developed and robust root architectures than pathogen-treated wild-type plants. AtPep1 is the first peptide defense signal to be found in Arabidopsis and has provided a new understanding of pathogen defense in this plant. The presence of multiple orthologs of AtPep1 in Arabidopsis suggests that they may have important roles in plant defense, perhaps in a tissue-specific manner. AtPep orthologs throughout the plant kingdom suggest that they should be investigated for their possible roles in plant defense within the ecological niches in which they evolved. The elucidation of the roles of proAtPep paralogs and orthologs should not only contribute to our fundamental knowledge [3]. The plant wound hormone systemin binds with the N-terminal part to its receptor but needs the C-terminal part to activate it.

TABLE 1. Genes induced in excised Arabidopsis leaves by AtPep1. Leaves were excised and supplied with 10 nM AtPep1 for 2 h, and the expression of each gene was analyzed using semiquantitative RT PCR. Genes proAtPep1 At5g64890 At5g64905 PDF1.2 TAT3 PR-1 PR-3 PR-4

Relative Induction +++ +++ +++ +++ ++ ++ +/− +/−

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FIGURE 3. The increased resistance of Arabidopsis plants overexpressing the proAtPep1 gene to infection of the oomycete Pythium irregulare. Upper panel: Transgenic plants constitutively expressing proAtPep1 (far left) and wild-type plants (far right). The two center plants are transgenic, and wildtype plants infected with Pythium irregulare strain 110305 2.5 weeks earlier. Lower panel: Roots of plants shown above, 3.5 weeks after inoculation with either water (far right and far left plants) or Pythium (middle two plants).

Acknowledgments Research reported as unpublished data was supported by National Science Foundation Grant IBN 0090766 and the Charlotte Y. Martin Foundation.

References [1] Felix G, Boller T (1995) Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells. Plant Journal, 7:381–89.

[2] Huffaker A, Pearce G, Ryan CA (in Review) AtPep1: A plant peptide that regulates pathogen defense in Arabidopsis thaliana via the jasmonate/ethylene signaling pathway. [3] Meindl T, Boller T, Felix G (1998) The plant wound hormone systemin binds with the N-terminal part to its receptor, but needs the C-terminal part to activate it. Plant Cell, 10:1561– 70. [4] Moyen C, Johannes E (1996) Systemin transiently depolarizes the tomato mesophyll cell membrane and antagonizes fusicoccin induced extracellular acidification of mesophyll tissue. Plant Cell Environ., 19:464–70. [5] Pearce G, Huffaker A, Ryan CA (in Review) Structure-function relationships of AtPep1, a novel defense signaling peptide in Arabidopsis thaliana. [6] Pearce G, Moura DS, Stratmann J, Ryan CA (2001) RALF, a 5-Kd ubiquitous polypeptide in plants arrests root growth and development. Proc. Natl. Acad. Sci. USA, 98:12843–47. [7] Pearce G, Moura DS, Stratmann J, Ryan CA (2001b) Production of multiple plant hormones from a single polyprotein precursor. Nature, 411:817–20. [8] Pearce G, Ryan CA (2003) Systemic signaling in tomato plants for defense against herbivores. Isolation and characterization of three novel defense signaling glycopeptide hormones coded in a single precursor gene. Journal of Biological Chemistry, 278: 30044–50. [9] Scheer JM, Pearce G, Ryan CA (2005) LeRALF, a plant peptide that regulates root growth and development, specifically binds to 25 and 120 kDa cell surface membrane proteins of Lycopersicon peruvianum. Planta 221:667–74. [10] Schaller A, Oecking C (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell, 11:263–72. [11] Realini C, Rogers SW, Rechsteiner M (1994) KEKE motifs: Proposed roles in protein-protein association and presentation of peptides by MHC Class I receptors. FEBS Letters, 348:109– 13. [12] Shiu S-H, Bleecker AB (2001) Receptor-like kinases from Arabidopsis form a monphyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. USA 98:10763– 68. [13] Stratmann J, Scheer JM, Ryan CA (2000) Suramin inhibits initiation of defense signaling by systemin, chitosan, and a β-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells. Proc. Natl. Acad. Sci. USA 97:8862–67. [14] Yamaguchi Y, Pearce G, Ryan CA (in Review) Isolation and properties of the AtPep1 receptor, AtPepR1, a LRR receptor kinase.

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3 CLAVATA3: A Putative Peptide Ligand Controlling Arabidopsis Stem Cell Specification JUN NI AND STEVEN E. CLARK

both anticlinally and periclinally on the meristem periphery, giving rise to the bulk of organ tissue. The inner L3 layer divides in various planes to give rise to the central tissues of organs as well as the vascular system [25]. At the center of the SM are the stem cells (Fig. 2). Maintenance of these cells in an undifferentiated state is essential for future organogenesis. Stem cell daughters on the flanks of the meristem make a switch toward differentiation, where an auxin signal is important to organize distinct organ primordia among these differentiating cells [21].

ABSTRACT CLAVATA3 (CLV3) is a putative peptide ligand with a well-characterized developmental role. CLV3 and its potential receptor complex, CLAVATA1 (CLV1)/ CLV2, comprise a putative ligand-receptor pair in Arabidopsis. CLV3 is a small-secreted peptide expressed primarily within the stem cells of shoot and flower meristems. Genetic studies indicate CLV genes function in the same pathway to regulate stem cell specification at the Arabidopsis shoot and flower meristems. CLV3 is a founding member of a gene family named CLE (CLVATA3/ESR-related) found throughout land plant species.

Discovery of the CLAVATA3 Locus Mutants that affect SM development have been discovered in various genetic screens in Arabidopsis and other species [4, 10, 16, 22, 26, 32, 33]. Within Arabidopsis, mutations in three loci, CLAVATA1 (CLV1) [24, 26], CLV2 [20], and CLV3 [16, 37], were among the first meristem mutants described [16]. Mutations in all three genes result in similar phenotypes at both the shoot and flower meristems. The SM of clv mutants is larger than the wild-type SM as early as three days after germination [16]. The size of the shoot apex continues to expand as stem cells accumulate in eventually massive populations [5, 16, 19, 37]. Similarly in the flower primordia, clv mutants accumulate stem cells, leading to the eventual generation of supernumerary floral organs and additional whorls of organs. The net result is a club-shaped gynoecium, from whence the gene name arises. The disruption of shoot and flower meristem structure that occurs in clv mutants leads to a number of indirect effects including stem fasciation, altered phyllotaxy, and alterations in floral organ number.

DISCOVERY Organization of the Shoot Apical Meristem In contrast to most animals, plant embryos only establish a simple axis with few organs. The vast majority of organogenesis and developmental patterning occurs postembryonically. Higher plants reiteratively form lateral organs from a group of specialized stem cells at the shoot and root apices, at structures termed the shoot meristem (SM) and the root meristem (RM). SMs are the source for the above-ground portion of the plant (Fig. 1). Based on data from cell lineage analysis [31, 36], the SM consists of three clonally distinct cell layers: the epidermal layer (L1), the subepidermal layer (L2), and corpus (L3). The outer L1 layer divides anticlinally to give rise to the plant epidermis. In the meristem center, the L2 layer also divides anticlinally, but the L2 divides Handbook of Biologically Active Peptides

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10 / Chapter 3 STRUCTURE OF THE PRECURSOR mRNA/GENE The CLV3 Gene and Protein

FIGURE 1. Meristems in plant development. A. A diagram of an Arabidopsis embryo. The locations of the shoot and root meristems are indicated. B. A diagram of an adult Arabidopsis plant, with major organs indicated. Shoot meristems are located at the tip of each active shoot, as well as in the axils of each leaf, indicated by arrows. C. A mature Arabidopsis flower, with floral organs labeled. (See color plate.)

The CLV3 gene (Fig. 3A) was cloned by taking advantage of two insertion alleles: a T-DNA insertion in the weak clv3-3 allele and an En-1 transposon insertion in the strong clv3-7 [12]. CLV3 consists of three exons and two introns. There is potential enhancer element downstream CLV3 as the T-DNA insertion in clv3-3 is located 175 base pairs downstream of the polyadenylate site. In addition to clv3-3, other mutant lesions have also been characterized [12]. The En-1 element in clv3-7 inserted in the second intron, close to the intron-exon 3 boundary. The only missense mutations are found in the intermediate clv3-1 and clv3-5 alleles, both of which have a Gly75 to Arg substitution. The strong γ rayinduced clv3-2 allele and x-ray-induced clv3-4 allele both have chromosomal rearrangement breakpoints between the Mfe I and Dra I restriction sites flanking exon 3. In strong EMS-induced clv3-6 allele, the third exon splice acceptor site is mutated. The CLV3 gene encodes a small 96-amino acid protein (Fig. 3B). The N-terminus contains a putative hydrophobic signal peptide, and no signal anchor was detected, indicating an extracellular localization of the protein. CLV3 has multiple mono- or di-basic residues, which are characterized recognition sites for convertases such as Kex2 and Furin in yeast and animal systems [29]. Evidence for processing of CLV3 is described below.

FIGURE 2. Arabidopsis shoot meristem organization. A. A scanning electron micrograph of an Arabidopsis shoot meristem. Stem cells and the surrounded differentiating daughter cells are falsely colored. Organ primordia are indicated by stars. Scale bar = 50 μm. B. A diagram of a longitudinal section through the shoot meristem in A, showing the organization of the meristem into cell layers (L1, L2, L3). The RNA expression regions for CLV3, CLV1, and WUS are labeled in color. (See color plate.)

A M

clv3-7 En clv3-6

G R clv3-1 clv3-5 STOP

Mfe I

Poly(A)

clv3-3 T-DNA

Dra I

B

MDSKSFVLLLLLFCFLFLHDASDLTQAHAHVQGLS

35

NRKMMMMKMESEWVGANGEAEKAKTKGLGLHEELR

70

TVPSGPDPLHHHVNPPRQPRNNFQLP

96

C AtCLV3 1 AtCLE1 1 AtCLE2 1 AtCLE3 1 AtCLE4 1 AtCLE5 1 AtCLE6 1 AtCLE7 1 AtCLE8 1 AtCLE9 1 AtCLE10 1 AtCLE11 1 AtCLE12 1 AtCLE13 1 AtCLE14 1 AtCLE16 1 AtCLE17 1 AtCLE18 1 AtCLE19 1 AtCLE20 1 AtCLE21 1 AtCLE22 1 AtCLE25 1 AtCLE26 1 AtCLE27 1 AtCLE40 1 ZmESR2g11 1 HgCLE

–––––––––––––––––MDSKSFVLLLLLFCFLFLHDASDLTQ––AHA––HVQGLS–NRKMMMMKMESEW–––––––VGANGE––––––––––––––––––––––––––AEKA––––––– –––––––––––––MANL––KFLLCLFLICV––SL–SRSSASRP––MFPNADGI–KRGRMMIEAE–––––––EVLK––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MAK–LSFTFCFLLFLLLS–SI–A––AGSRP––LEGA–––––––––––––––RVGVKVRGLSP––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MASI––KLWVCLVLLLVL–EI–TSVHECRP––LVAEERFS–GSSRLKKIRR–––––––ELFE––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MASF––KLWVCLILLLLE––––FSVHQCRP––LVAEESPS–DSGNIRKIMR–––––––ELLK––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MATL––ILKQTLIILLIIFSLQTLSSQARI––LRSYRAVSMGNMDSQVLLHELGFDLSKFKG––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MASF––KLWVCLILLLLE––––FSVHQCRP––LVAEESPS–DSGNIRKIMR–––––––ELLK––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––MASKALLLFVMLTFLLVI–EM–E––GRILR––VNSK–––T–KDGESNDLLKRLGYNVSELKR––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––MKTNRNRPINILIVFFLLTTARAA––––––––––––TRNWTNR–––––––––THRTVP–KV–QHAYYAYPHRSCESFSRPYARS–MCIELERIHRSSRQ–PLF ––––––––––––––––MTHLNRLILISLLFVSLLLKSSTASST––VVDEGNRTSRNFRYR–––––––––THRFVP–RFNHHPYHVTPHRSCDSFIRPYARS–MCIELQRIHRSSRKQPLL –––––––––––––––––MKTNRNRPINILIVFFLLTTARAA––––––––––––TRNWTNR–––––––––THRTVP–KV–QHAYYAYPHRSCESFSRPYARS–MCIELERIHRSSRQ–PLF –––––––––MTKQPKPCSFLFHISLLSALFVFLLISFAFTTSY––KL––––KSGINSL––––––––––GHKRILASNFDFTPFL–––––––––––––––––––––––KNKDRTQRQRQSP –––MLRISSSSSMALKFSQILFIVLWLSLFFLLLHHLYS-LNF––RRLYSLNAVEPSLLKQHYRSYRLVSRKVLSDRFDETPFH––––––––––––––––––––––SRDNSRHNHRSGEQ –––––––––––MATTRVSHVLGFLLWISLLIFVSIGLFGNFSS––KPI–––NPFPSPVITLPALYYRPGRRALAVKTFDFTPFL–––––––––––––––––––––––KDLRRSNHRKALP ––––––––––––––––MKVWSQRLSFLIVMIFILAGLHSSSAG–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––RKLP MEACSRKRRRRRAYTTSTTGYAAVFFCGIFVFAQFGISSSALF––AP–DHYPSLPRKAGH–––––––––FHEMAS––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––MTMCFFIFFFVFYVSFQIVLSSSAS––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––VGYSRLHL––––––VA –––––––––––––MHLL––KGGVVLIITLILFLITSSIVAIRE––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ––––––––––MKIKG––––––LMILASSLLILAFIHQSES–AS––MRSLLMNNGSY–EEEEQVLKYD––––––––––––––––––––––––––––––––––––––––––––––––SMGTI –––––––––––MKNKNMNPSRPRLLCLIVFLFLVIVLSKASRI––HV–ERRRFSSKPSGE–––––––––NREFLP––––––––––––––––––––––––––––––––––––––––––––– –––MLILSSRYAMKRDV––––LIIVIFTVLVLIIISRSSSIQA––GRFMTTGRNRN–LSVARSLYYKNHHKVVITEMSNFNKV––––––––––––––––––––––––––RRRSSRRFRKT –––MGNYYSRRKSRKHITTVALIILLLLLFLFLYAKASSSSPN––IHH––HSTHGS–LKKSGNLDPKLHD–––––––LDSNAA––––––––––––––––––––––––––SSRGSKYTNYE –––––––MGGNGIRAIVGVIASLGLIVFLLVGILA–––––––––––––NSAPSVPS––––S––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ––––MRNNHSLRLQLWFRTLFTVGVVTLLMID–––AFVLQNNK––EDDKTKEITTAVNMNNSDAKEIQQ––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––MTHAREWRSSLTTTLLMVILLSYMLHLFCVYSRV––GAIRIFPETPA––––SGKRQEEDLMKKYFG–A––G–––––––––––––––––––––––––––––––––––––––– –––––––MAAMKYKGSV––––FIILVILLLSSSLLAHSSSTKS––FFW––LGETQD–T––––––––––––––––––––––––––––––––––––––––––––––––––––KAMKKEKKID ––––––––––––––––MASRMGMVAIVSLFVCALVASTSVNANVWQTDEDAFYSTNKLGVNGNMEMAQQQSGFIG––––––––––––––––––––––––––––––––––––––––––––– ––––––––––––––––MPNIFKILLIVLLAVV–––SFRLSAST––GDKKTANDG–––SGNNSSAGIGTKIKRIVT–A––GLLFTSLATGGAEAIGRSNAQGGNAAGLVPSHLTNRS–––M

AtCLV3 59 AtCLE1 48 AtCLE2 41 AtCLE3 49 AtCLE4 47 AtCLE5 59 AtCLE6 47 AtCLE7 53 AtCLE8 79 AtCLE9 92 AtCLE10 79 AtCLE11 73 AtCLE12 93 AtCLE13 82 AtCLE14 32 AtCLE16 64 AtCLE17 36 AtCLE18 29 AtCLE19 53 AtCLE20 53 AtCLE21 85 AtCLE22 80 AtCLE25 38 AtCLE26 61 AtCLE27 63 AtCLE40 53 ZmESR2g1 60 91 HgCLE

––––––––––––––––––––––––––K–TKGLGLHEELRTVPSGPDPLHHHVNPPRQPRNNFQLP–––––––––––––––––––––––––––––––––––––– –––––––––––––––ASME––––––––ELMERGFNESMRLSPGGPDPRHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––SIEATSPTVEDQAAG–SHGKSPERLSPGGPDPQHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––RLKEMKGRSEGEETILGNTLDSKRLSPGGPDPRHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––RSEELKVRSKDGQTVLG–TLLSKRLSPGGPDPRHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––HNE––––––––––––RRFLVSSDRVSPGGPDPQHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––RSEELKVRSKDGQTVLG–TLLSKRLSPGGPDPRHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––IGRELSV–––––––––––QNEVDRFSPGGPDPQHHSYPLSSKPRI––––––––––––––––––––––––––––––––––––––––––– SPPPPP––––––––––––––––––––T–EIDQRYGVEKRLVPSGPNPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– SPPPP–––––––––––––––––––––––EIDPRYGVDKRLVPSGPNPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– SPPPPP––––––––––––––––––––T–EIDQRYGVEKRLVPSGPNPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– S––––––––––––––––––––––LTVK–ENGFWYNLEERVVPSGPNPLHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––YDGD–EIDPRYGVEKRRVPSGPNPLHH––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––AGGS–EIDPRYGVEKRLVPSGPNPLHH––––––––––––––––––––––––––––––––––––––––––––––––––––– SMTTTEEFQRL–SFDGKRILSEVTADK–KYDRIYGASARLVPKGPNPLHNK–––––––––––––––––––––––––––––––––––––––––––––––––––– FQAPKAT––––VSFTGQR–––––REEE–NRDEVYKIDKRLVHTGPNPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– SPPPPPPPKALRYSTAPF–––––RGPL–SRDDIYGDDKRVVHTGPNPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– ––––––––––––––––––––––––––––––DPSLIGVDRQIPTGPDPLHNPPQPSPKHHHWIGVEENNIDRSWNYVDYESHHAHSPIHNSPEPAPLYRHLIGV –––––––––––––––––––––––––––––ANSSALDSKRVIPTGPNPLHNR–––––––––––––––––––––––––––––––––––––––––––––––––––– SQPTFPV––––V–––––––––––––––––DAGEILPDKRKVKTGSNPLHNKR––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––DGDEEEEEKRSIPTGPNPLHNK–––––––––––––––––––––––––––––––––––––––––––––––––––– ––––––––––––––––––––––––––G–GGEDVFEDGKRRVFTGPNPLHNR–––––––––––––––––––––––––––––––––––––––––––––––––––– ENVKTLRFSGK––––––––––––––––––DVNLFHVSKRKVPNGPDPIHNRFLSLLSRIFNLLLLLL–––––––––––––––––––––––––––––––––––– ––––––––––––––––––––ELEDGSR–NDDLSYVASKRKVPRGPDPIHNRFLLLSRFILSLLTNPYPYLHICVLDVSV–––––––––––––––––––––––– KFPPVDSFVGK–––––––––––––––––––––GISESKRIVPSCPDPLHN––––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––GGTANEVEERQVPTGSDPLHHKHIPFTP–––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––HRPRLASFNRASKQLDSEKRPVPSGPDPIHHSIPSHAPQHPPSYGKAPYEDDRSRASPGLSNPIGPPPFLDRY––––––––––– APPPPPAQFEKGAATRVEKMRAQLREL–AEKMTDKDPKRLSPSGPDPHHH–––––––––––––––––––––––––––––––––––––––––––––––––––––

FIGURE 3. CLV3 gene and protein structure. A. The CLV3 genomic region (GenBank AF126009) is shown. Exons are represented by boxes. The translational start (M) and Poly A tail sites are indicated. Lesions found in clv3 mutant alleles are labeled. The two restriction sites (MfeI and DraI) flanking the exon3 are shown. B. The predicted CLV3 amino acid sequence. N-terminal signal peptide is in red. C-terminal CLE domain is underlined. The glycine amino acid altered in both clv3-1 and clv3-5 alleles is in bold type. Arrows indicate intron positions. C. Alignment of CLV3 and Arabidopsis CLE sequences. Also included in the alignment are ESR2g1 sequence from maize (GenBank CAA67125) and the esophageal gland cell polypeptide from Heterodera glycines (GenBank AAG21331), referred to as HgCLE in the alignment.

12 / Chapter 3 CLV3/ESR-Related Gene Family CLV3 is the founding member of the plant specific CLAVATA3/ESR (CLE) protein family. This gene family contains at least 25 genes in Arabidopsis as well as genes from other plant species [6, 7]. Embryo-surrounding region (ESR) is one example from maize (Zea mays), which is expressed in a specific zone of the developing endosperm [28]. Almost all of the Arabidopsis CLEs (AtCLEs) consist of small intronless open reading frames. A notable exception is AtCLE40, which contains 3 exons and 2 introns similar to the CLV3 genomic structure [6, 14]. AtCLEs encode peptides and polypeptides of 80–120 residues. They share several common structural features including an amino terminal signal peptide or membrane anchor and a conserved 14 amino acid carboxyl terminal domain that defines the signature of this gene family, termed the CLE domain (Fig. 3C) [6, 7]. This region has been suggested to be crucial for CLV3 function because the missense mutations in both clv3-1 and clv3-5 mutants occur in this region and because it is conserved within the family. Interestingly, CLE domain-containing proteins HgCLE and GrSYV46 have been identified from nematodes that parasitize soybean and potato, respectively [27, 39a]. It is suggested that this is an example of adaptive mimicry, with the nematode CLE proteins activating plant receptors to facilitate the formation of feeding structures. Consistent with this hypothesis, GrSYV46 can replace CLV3 function in vivo [39a]. The lack of any significant sequence similarity outside of the CLE domain among these proteins suggests that this domain is critical, perhaps sufficient for function. Indeed, extensive functional conservation among various CLE proteins can be attributed to the CLE domain. Furthermore, the CLV3 CLE domain can completely replace CLV3 function when placed in a chimeric protein with an unrelated signal sequence [26a].

CLV3 Expression and Function CLV3 mRNA is first expressed in heart stage embryos, within the nascent shoot apical meristem [12]. CLV3 continues to be expressed at the center of shoot and flower meristems throughout development (Fig. 2). It is detected mainly in L1 and L2 cell layers and in a few underlying L3 cells, with no expression detected on the meristem flanks or within organ primordia. In the floral meristem, the CLV3 signal appears in the center of the flower at stage 2 and persists until flower meristem cells are consumed for carpel initiation at stage 6. Throughout development, CLV3 mRNA is always restricted in a few putative stem cells [12]. Indeed, CLV3 has been the best available marker for stem cell identity, although recent evidence has revealed that CLV3 expression and stem cell identity can be uncoupled [13].

Many of the CLE genes are transcribed in one or more tissues during development. Based on RT-PCR analysis, many are widely expressed; some are restricted to one or a few tissue types [35]. Due to the small size of the CLE genes, they are often unannotated in the database. There are also underrepresented in EST and microarray databases, where usually cDNAs with sizes larger than 400 bp are selected for analysis. Among all the CLEs, CLE19 has been reported to show a root consumption phenotype in overexpression lines [3, 11]. CLE40 is able to rescue clv3-2 phenotype under CLV3 promoter [14].

RECEPTORS Three lines of evidence that the CLE domain is proteolytically released from CLV3. The first is the sufficiency of the CLE domain of CLV3, that only the CLE domain is conserved across plant species [26a] (Fig. 3C). Secondly, a peptide containing only CLE domain from CLV3 is active on Arabidopsis roots. Addition of the CLE peptide to roots drives differentiation and is dependent on CLV2 for function [11a]. Finally, protein extracts from Arabidopsis and cauliflower cleave CLV3 releasing the conserved CLE domain [26a]. Mutations at CLV1 and CLV2 result in phenotypes very similar to clv3 mutants. CLV1 encodes a leucinrich repeat (LRR) kinase. The N-terminus contains a potential secretion signal peptide, followed by 21 putative extracellular complete LRRs with 15 consensus sites (N-X-S/T) for N-linked glycosylation. Flanking the LRR region are pairs of conservatively spaced cysteines, which is common in many LRR-containing proteins. There is a transmembrane domain following the LRR domain. The putative intracellular domain is a functional serine/threonine protein kinase [5, 38, 40]. Similar to CLV1, CLV2 also encodes an LRRcontaining putative transmembrane protein. However, CLV2 lacks the cytoplasmic kinase domain. CLV2 DNA and protein sequences are highly polymorphic among various Arabidopsis ecotypes [15, 19]. CLV2 is similar in structure to disease-resistant proteins of the Cf family [9, 18]. CLV1 mRNA is expressed in a specific fashion within the shoot and flower meristems [5]. CLV1 is expressed in a broad central region, largely within the L3 and more basal cells (Fig. 2). Interestingly, CLV1 and CLV3 are expressed in adjacent, largely nonoverlapping cell populations, suggesting these proteins may act to mediate signals between two different, adjacent tissues. CLV2, by contrast, is more broadly expressed throughout the plant. CLV2 was detected in apices, opened flowers, siliques, four-day-old seedlings, and rosette leaves

CLAVATA3: A Putative Peptide Ligand Controlling Arabidopsis Stem Cell Specification / by RNA gel blot analysis [15]. The wider expression pattern of CLV2 is consistent with its being involved in a number of different developmental processes [19].

BIOLOGICAL ACTIONS CLV Signaling Genetic analysis of mutations at CLV1, CLV2, and CLV3 indicate they act in a common pathway [5, 37]. clv3 null mutants have the most severe phenotypes and are epistatic at the meristem to mutations in clv1 and clv2 [15, 37]. Based on the genetic interactions and the protein structures, CLV3 was proposed to be a secreted peptide ligand for a presumed CLV1/CLV2 receptor complex. The facts that the fate of the SAM is sensitive to CLV3 dosage [1] and that phenotype of CLV3 overexpression requires functional CLV1 and CLV2 support this hypothesis [1]. Consistent with a prediction of secretion, CLV3 was detected in the extracellular space when transiently expressed in leek epidermal cells [30]. CLV3 was also revealed in the apoplast in transgenic plant roots by immunoelectron microscopy. Furthermore, it has been demonstrated that the extracellular localization is necessary for CLV3 to function properly [30]. CLV3 has been reported to be able to move from its producing cells to neighboring cells and act over a distance of several cell diameters as an intercellular signal. CLV1, on the other hand, might function to limit CLV3 diffusion through ligand sequestration, consistent with the idea it being the receptor for CLV3 [23]. CLV1 is present in two protein complexes in vivo as detected by gel chromatography [39]. Around 35% of CLV1 was associated with a ∼185 KD complex, and about 65% was found in a ∼450 KD complex. In both complexes, CLV1 was likely linked to other protein(s) through disulfide bonds [39]. Interesting enough, a large number of plant LRR-receptor like proteins contain a pair of conservatively spaced cysteine residues immediately before and after the LRRs [17]. CLV3 is required for assembly of the larger-mass CLV1 complex, as it is absent in clv3 mutant plants. All evidence to date is consistent with CLV3 acting as the ligand for CLV1; however, direct binding of CLV3 to CLV1 is an unresolved issue, further complicated by the likely processing of CLV3 that occurs (see above). Even if CLV3 acts through CLV1, it must not be an exclusive partnership. While the most severe clv1 alleles exhibit phenotypes similar to clv3 null alleles, recent work has shown that these strong clv1 alleles are dominant-negative and that clv1 null alleles exhibit weak phenotypes [8]. Thus, CLV3 still has a role in limiting stem cells in a clv1-null mutant background, presumably by activating redundant receptors.

13

Downstream Target(s) Current results suggest that CLV signaling functions to repress the homeodomain-containing transcription factor WUSCHEL (WUS) at the level of transcription. WUS is required for specifying stem cell identity, as wus mutants lack stem cells at both the shoot and flower meristems [22]. wus is epistatic to clv1 and clv3 [34]. In wild-type plants, WUS is expressed in a small cell group immediately below the three layers of stem cells, in what has been termed the meristem organizing center [34] (Fig. 2). Several studies indicate that in clv1 or clv3 mutant backgrounds WUS expression expands to the L3 stem cell layer, recruiting additional cells to maintain stem cell identity [1, 34]. However, recent results have indicated that in strong clv mutant inflorescence shoot meristems, WUS expression is lost, yet the meristems remain undifferentiated [13]. Ectopically expressing a WUS transgene within the meristem causes stem cell accumulation, similar to the clv phenotype. This observation indicates that the altered WUS expression pattern in clv meristems is the cause of the phenotypic defect [34].

Regulation of CLV3 Interestingly, while CLV3 acts to restrict WUS expression, WUS activates CLV3 expression [1]. wus-1 embryos fail to express PCLV3:GUS, indicating that WUS function is required for the early activation of CLV3 expression in the embryo. It has also been reported that WUS is sufficient to promote CLV3 expression in the shoot apical meristem but not in other tissues, such as leaves [2]. Another homeobox gene SHOOT MERISTEMLESS (STM), which functions independently of WUS, is required at later stages of the maintenance of stem cell population. In stm mutant embryo and seedling, PCLV3: GUS is weaker but detectable, suggesting CLV3 expression does not absolutely require STM function. When both WUS and STM are ectopically expressed, PCLV3:GUS signal could be detected in nonmeristematic tissues, indicating both WUS and STM function are sufficient to activate CLV3 expression in nonmeristematic cells.

Model for CLV3 Function Current models put the CLV/WUS pathway at the heart of stem cell maintenance (Fig. 4). WUS is necessary and sufficient to promote stem cell identity, while CLV signaling, initiated by CLV3, acts to restrict WUS expression, thus restricting the stem cell population. In the absence of WUS activity, a failure to specify stem cells occurs, while in the absence of CLV signaling, WUS expression expands and recruits additional stem cells. A balance between these two activities is obviously critical for maintaining a stable stem cell population over

14 / Chapter 3

[9] CLV3 CLV1 CLV2

[10]

[11] WUS Stem Cells

[11a]

[12]

FIGURE 4. A feedback loop between CLV signaling and WUS is proposed to maintain a stable stem cell population.

[13]

[14]

time. This balance appears to be achieved by a feedback loop between CLV signaling and WUS, as CLV3 initiates the repression of WUS, while WUS promotes the expression of CLV3. This should continually drive the meristem toward an equilibrium with a stable stem cell population. Transient expansion of the stem cell population should result in extra CLV3 expression and more differentiation, while transient reduction in the stem cell population should lead to reduced CLV3 expression and reduced differentiation. Indeed, one might speculate that alterations in the parameters of this signaling pathway might lead to increases/decreases in the stable number of stem cells in plants.

[15]

[16] [17] [18]

[19]

[20]

References [1] Brand, U., et al., Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science, 2000. 289(5479): p. 617–9. [2] Brand, U., et al., Regulation of CLV3 expression by two homeobox genes in Arabidopsis. Plant Physiol, 2002. 129(2): p. 565–75. [3] Casamitjana-Martinez, E., et al., Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance. Curr Biol, 2003. 13(16): p. 1435–41. [4] Clark, S.E., et al., The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. Development, 1996. 122(5): p. 1567–75. [5] Clark, S.E., Williams, R.W. and Meyerowitz, E.M., The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell, 1997. 89(4): p. 575–85. [6] Cock, J.M. and McCormick, S., A large family of genes that share homology with CLAVATA3. Plant Physiol, 2001. 126(3): p. 939– 42. [7] DeYoung, B.J. and Clark, S.E., Signaling through the CLAVATA1 receptor complex. Plant Mol Biol, 2001. 46(5): p. 505–13. [8] Dievart, A., et al., CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate

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CLAVATA3: A Putative Peptide Ligand Controlling Arabidopsis Stem Cell Specification / [28] Opsahl-Ferstad, H.G., et al., ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo. Plant J, 1997. 12(1): p. 235–46. [29] Rockwell, N.C., et al., Precursor processing by kex2/furin proteases. Chem Rev, 2002. 102(12): p. 4525–48. [30] Rojo, E., et al., CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway. Plant Cell, 2002. 14(5): p. 969–77. [31] Satina, S., Blakeslee, A.F. and Avery, A.G., Demonstration of the three germ layers in the shoot apex of Datura by means of induced polyploidy in periclinal chimeras. American Journal of Botany, 1940. 27: p. 895–905. [32] Satoh, N., et al., Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes. Development, 1999. 126(16): p. 3629–36. [33] Satoh, N., Itoh, J. and Nagato, Y., The SHOOTLESS2 and SHOOTLESS1 genes are involved in both initiation and maintenance of the shoot apical meristem through regulating the number of indeterminate cells. Genetics, 2003. 164(1): p. 335–46. [34] Schoof, H., et al., The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell, 2000. 100(6): p. 635–44.

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[35] Sharma, V.K., Ramirez, J. and Fletcher, J.C., The Arabidopsis CLV3-like (CLE) genes are expressed in diverse tissues and encode secreted proteins. Plant Mol Biol, 2003. 51(3): p. 415–25. [36] Steeves, T.A. and Sussex, I.A., Patterns in Plant Development, 2nd ed. 1989. [37] Steven, E., Clark, M.P.R., Meyerowitz, E.M., CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1. Development, 1995(1212057– 2067). [38] Stone, J.M., et al., Control of meristem development by CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions. Plant Physiol, 1998. 117(4): p. 1217–25. [39] Trotochaud, A.E., et al., The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein. Plant Cell, 1999. 11(3): p. 393– 406. [39a] Wang, X., et al., A parasitism gene from a plant nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana. Mol Plant, 2005. 6(2): p. 187–191. [40] Williams, R.W., Wilson, J.M. and Meyerowitz, E.M., A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. Proc Natl Acad Sci USA, 1997. 94(19): p. 10467–72.

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4 DVL Peptides Are Involved in Plant Development JIANGQI WEN AND JOHN WALKER

Activation tagging screens involve generating random genomic insertions of a transgene that contains strong transcriptional enhancers that are capable of increasing the expression of nearby genes [19]. In a gain-offunction genetic screen for genes that affect fruit growth and development in Arabidopsis, we generated a medium-sized activation tagging pool in er-116, a weak allele of ERECTA [5]. After screening the transgenic pool, a dominant transgenic line that exhibited a dwarfed stature, rounder leaves, shortened petioles, clustered inflorescences, shortened pedicels and siliques, and moderately horned fruit tips was collected. Segregation of the er-116 locus by backcrossing to wild-type (ecotype Columbia, Col-0) did not alter the pleiotropic phenotypic changes (Fig. 1, left), suggesting the phenotypic alterations were not dependent on the ERECTA signaling pathway. The activation-tagged line in Col-0 background was named dvl1-1D (devil 1–1 Dominant) for the horned fruit tip phenotype (Fig. 1, right). Compared with wild-type plants (Col-0), dvl1-1D plants had smaller rosettes and shorter flower buds [20]. The sepals and petals in dvl1-1D plants were also shorter than those in wild-type plants. The shortened sepals and petals resulted in the protrusion of the pistils in dvl1-1D flower buds prior to the opening of flowers. However, the filaments and stamens were able to extend to reach the stigma when flowers were opening and dvl1-1D plants were fully fertile. The numbers of petals, sepals, stamens, and carpels were not altered in dvl1-1D plants. Leaves of dvl1-1D plants were slightly shorter but wider than those of wild-type plants, resulting in a rounder leaf appearance and a smaller rosette (Fig. 1, left panel). Mature dvl1-1D plants were about 70% of the height of wild-type plants (Fig. 1, left). The lengths of pedicels and siliques in dvl1-1D plants were shorter than those of Col-0 plants. The number of seeds in the silique of dvl1-1D plants was not significantly different from that of Col-0 plants. Interestingly, the two valves

ABSTRACT In a gain-of-function genetic screen for genes that influence fruit development in Arabidopsis, we identified a novel gene, DVL1 (DEVIL1), that encodes a small protein. Overexpression of DVL1 results in pleiotropic phenotypes featured by shortened stature, rounder rosette leaves, clustered inflorescences, shortened pedicles, and siliques with pronged tips. DVL1 cDNA has a 153-nucleotide open reading frame encoding a 51amino-acid peptide that shares no significant similarity to previously identified proteins. Sequence alignment shows that DVL1 belongs to a family of related genes that are limited to plants. The DVL family is a novel class of peptides, and the overexpression phenotypes suggest DVL peptides may function in plant development.

DISCOVERY Plant development is an intricate process requiring cell-to-cell communication to coordinate growth and differentiation [12]. A growing body of evidence indicates that small peptides are critical elements of cellular communication in plants [10]. Since the characterization of tomato systemin, the first plant peptide hormone discovered [11, 14], several additional groups of small signaling peptides have been identified in plants [2, 17]. Among these peptides, some were identified by biochemical purification, such as tomato systemins [11, 14], tobacco systemins (Tob Sys I and Sys II) [17], rapid alkalinization factor (RALF) [16], and phytosulfokines (PSKs) [9]. Some were identified and characterized by genetic loss-of-function analysis, such as CLAVATA3 [3], POLARIS [2], and inflorescence deficient in abscission (IDA) [1]. We used an activation-tagging screen approach and identified the plant-specific DEVIL (DVL) family of peptides [20]. Handbook of Biologically Active Peptides

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FIGURE 1. Phenotypes of dvl1-1D plants. Left panels: comparison of wild-type (WT, Col-0) and dvl1-1D plants at rosette stage (three-week-old) and flowering stage (six-week-old). Right panel shows a close-up SEM view of the dvl1-1D fruit tips. Bar = 1 mm. (See color plate.)

of the silique in dvl1-1D plants did not taper apically as observed in siliques of wild-type plants. Instead, the ends of the valves were broadened and led to the hornlike protrusions at the fruit tip (Fig. 1, right). When grown on the MS medium, no significant phenotype was observed in dvl1-1D roots. Overall, compared to wild-type plants, dvl1-1D plants exhibited pleiotropic phenotypic changes affecting all aerial organs.

STRUCTURE OF THE PRECURSOR mRNA/GENE Southern blot analysis and the observation of genetic segregation ratios indicated that there was a single activation-tagging T-DNA insertion in dvl1-1D plants. To determine the genetic basis of the phenotypes observed in the dvl1-1D plants, inverse polymerase chain reaction (IPCR) and plasmid rescue [6, 7], followed by DNA sequencing, demonstrated that the activation-tagging T-DNA was inserted 1482 base pairs (bp) downstream of the stop codon of the gene At5g16020 with the enhancer elements toward the gene At5g16020 [20]. There are three annotated genes close to the activationtagging T-DNA insertional site (At5g16010, At5g16020, and At5g16030). Unexpectedly, the enhancers in the inserted T-DNA activated none of these three annotated genes, as demonstrated by Northern blot analyses [20]. However, when the 1.5 kb genomic fragment between the enhancers on the right border of the T-DNA and the stop codon of the gene At5g16020 was used to probe the same Northern blot, elevated expression of a ∼600 nucleotide (nt) transcript was observed in dvl1-1D plants, while there was no detectable signal in Col-0. When the 4.5 kb fragment between the left border of

the T-DNA and the start codon of the gene At5g16030 was used as a probe, there were no noticeable signals in both Col-0 and dvl1-1D plants. These observations implied that the expression of a small nonannotated gene in the 1.5 kb genomic fragment was increased by the enhancers in the inserted activation-tagging T-DNA. After screening a cDNA library constructed from dvl11D poly (A)+RNA, a 581 basepair (bp) cDNA clone was retrieved and designated DVL1 (DEVIL1). DVL1 contains a 153 nt open reading frame (ORF) encoding a putative 51-amino-acid peptide. Alignment of DVL1 cDNA and genomic sequences demonstrates that there are no introns in the DVL1 gene. The deduced DVL1 peptide has a molecular mass of 6209 Daltons from 51 amino acids and has an isoelectric point of 10.52. DVL1 has no predicted signal peptide at the N-terminus of the peptide and shares no sequence similarity with other previously identified proteins.

DISTRIBUTION OF THE DVL1 mRNA The expression of DVL1 in wild-type plants is extremely low (not detectable by Northern blot analysis) [20]. But when the expression of DVL1 is elevated by the transcription enhancers in the activation-tagging T-DNA (as in dvl1-1D plants), DVL1 transcripts were detected in all the tissues tested (roots, rosette leaves, cauline leaves, inflorescence stems, flower buds, and young fruits) by Northern blot analysis. Reverse transcription PCR (RT-PCR) results also indicated that DVL1 is expressed in roots, rosette leaves, inflorescence stems, and flowers with relatively higher levels in the rosette leaves. When a DVL1 promoter-GUS fusion construct was transformed into wild-type plants, no GUS activity

DVL Peptides Are Involved in Plant Development / was detected in transgenic seedlings. Similarly, when the promoter with the four transcription enhancers was fused with GUS reporter gene, strong GUS staining was observed in all the tissues.

PROCESSING The deduced DVL1 peptide contains 51 amino acid residues and has no predicted signal peptide at its Nterminus. Database searching revealed that DVL1 belongs to a gene family with 21 members in the Arabidopsis genome. Among the 21 members, except for four that have recently been annotated (At1g13245 for DVL4, At2g39705 for DVL11, At2g29125 for DVL13, and At5g59515 for DVL18), most are nonannotated genes. Members of the DVL gene family share homology primarily at their C-termini and the closely related family members are clustered in a single clade (Fig. 2). Homologous genes of DVL1 are also found in other angiosperm plants and also show sequence similarity at the C-termini. For example, in rice there are at least 24 members in the DVL gene family. Although we still lack direct evidence of the processing of the DVL peptides, some indirect clues suggest that DVL peptides are processed to a shorter biologi-

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cally active form. First, in all of the 21 DVL family members, monobasic or dibasic residues were found just before the conserved C-terminus. These mono or dibasic residues are thought to be potential proteolytic cleavage sites [6]. Moreover, DVL homologs have very diverse N-terminal sequences but share high homology at their C-termini. Overexpression of the DVL genes results in similar pleiotropic phenotypes, indicating that the conserved C-terminal sequences of the DVL peptides likely cause the phenotypic changes. Y37, I38, C42, and L46 are absolutely conserved amino acid residues across all of the 21 members in the Arabidopsis DVL gene family. Single mutations at any of these conserved residues abolished DVL1 overexpression phenotypes, suggesting that the conserved DVL C-terminus is essential for DVL overexpression phenotypes. We hypothesize that the DVL1 peptide may be processed to a shorter peptide consisting of its conserved C-terminal portion beginning with the dibasic residues (R23-R24). Transgenic plants overexpressing DVL1 with a mutation at one of the dibasic residues (R23E) failed to exhibit pleiotropic phenotypes (data not shown), implying that the dibasic residues in DVL1 are essential for DVL1 function, perhaps involved in proteolytic processing. We made several DVL1-GFP fusion constructs to test the possible processing event. sGFP denotes secreted

MEMKRVMMSSAERSKEKKRSISRRLGKYMKEQKGRIYIIRRCMVMLLCSHD MES---IMS-LKR-KEKK-SQSRRLGKYLKEQKGRIYIIRRCVMMLLCSHD ------MKG-----TKKKTPCNKKLGGYLKEQKGRLYIIRRCVVMLICWHD MK-----MG----GSKRRVSS-KGLGAVLKEQRAKLYIIRRCVVMLLCWHD MKTTGSSVG----GTKRKMWS-RGVGGVVREQKAKLYIIRRCVVMLLCWHD -------MG----VLKRRVSSSRGLGGVLREQRAKLYIIKRCVVMLLCWQD

DVL1 DVL2 DVL3 DVL4 DVL5 DVL6 DVL1 DVL2 DVL3 DVL5 DVL6 DVL4 DVL12 DVL14 DVL21 DVL8 DVL11 DVL19 DVL16 DVL20 DVL9 DVL13 DVL15 DVL18 DVL10 DVL7 DVL17

FIGURE 2. DVL1 belongs to a gene family. Top panel: Alignment of six Arabidopsis genes that are most closely related to DVL1. Bottom panel: A neighbor-joining tree of the DVL gene family in Arabidopsis thaliana constructed using ClustalW [18].

20 / Chapter 4 GFP, which contains a signal peptide at the N-terminus of the GFP protein; eGFP denotes endoplasmic reticulum (ER)-localized GFP, which contains the N-terminal signal peptide and the C-terminal ER retention sequence; cGFP denotes cytoplasmic GFP, which contains the soluble GFP protein only. These differently localized GFP proteins were translationally fused to the DVL1 peptide at its N-terminus (sGFP-DVL1, eGFPDVL1, and cGFP-DVL1) or C-terminus (DVL1-sGFP, DVL1-eGFP, and DVL1-cGFP). All the constructs were driven by the cauliflower mosaic virus 35S promoter and transformed into wild-type plants. Transgenic plants carrying all the DVL1 C-terminus fused GFP constructs, and those carrying sGFP-DVL1 and eGFP-DVL1 constructs did not show any DVL1-overexpression phenotypes, whereas transgenic plants carrying cGFP-DVL1 construct exhibited similar phenotypes as observed when only DVL1 was overexpressed. These observations may imply that DVL1 was processed to a smaller biologically active form, and the processing event took place in the cytoplasm. If the DVL1 protein was targeted to the ER or secreted to outside the plasma membrane, it could not be processed to the functional form, and therefore transgenic plants overexpressing sGFP-DVL1 or eGFP-DVL1 showed no phenotypes. Even though the transgenic plants overexpressing DVL1-cGFP did not exhibit DVL1 overexpression phenotypes, the DVL1-cGFP fusion protein may be still processed, but the processed DVL1 C-terminus was linked with a big “tail”-GFP. This big tail was thought to interfere with the function of DVL1. If the tail was small (such as FLAG tag), it would not affect the DVL1 function. Our results of DVL1-cGFP overexpression were contrary to the observation by Narita et al. in ROT4GFP overexpression [13]. Narita et al. overexpressed ROT4 (DVL16)-GFP and observed similar phenotypes as overexpression of ROT4 alone. Based on their findings, we then tried to test if overexpression of other DVL family members fused with GFP would show the similar phenotypes as the DVL members alone did. The results, however, indicated that none of the DVL2 through DVL5 fused with cGFP showed the DVL overexpression phenotypes. This difference may be due to the unique properties of DVL16 (ROT4) or may be caused by some other difference in experimental design. Although DVL1 does not contain a signal peptide at its amino terminus, DVL1 may still be processed and secreted outside the cell. In plants, there is a precedent for a small peptide that is processed from a larger precursor having no signal peptide but nevertheless is found outside the cell. Systemin (18 amino acids) is proteolytically processed from its precursor, prosystemin (200 amino acids), which does not have a signal peptide at its N-terminus [17]. However, systemin is

known to circulate through plants and bind a cell surface receptor [15, 17].

RECEPTORS Although we think DVL peptides probably function with other partners (receptors?), we have not yet identified the partners (receptors). However, progress has been made in this aspect. Using EMS (ethyl methanesulfonate) as a mutagen, approximately 20,000 dvl1-1D seeds were mutagenized, and 15 potential suppressors were collected after screening about 300,000 M2 plants. Mapping and characterization of these suppressors may provide evidence or clues of the DVL receptors.

BIOLOGICAL ACTION DVL1 was first identified in an activation-tagging screen for genes that affect fruit development. Overexpression of DVL1 in the activation-tagged line dvl1-1D not only resulted in the misshaped fruits/siliques but also caused rounder leaves and more clustered inflorescences (Fig. 1). There are 21 homologous DVL genes in the Arabidopsis genome. When the cDNAs of DVL1 through DVL5, DVL8, DVL9, DVL15, DVL16, DVL17, and DVL20 were individually overexpressed and driven by the cauliflower mosaic virus (CaMV) 35S promoter with a dual enhancer, transgenic plants of each gene construct (except for DVL15) showed similar phenotypes in rosettes, inflorescences, and siliques. These results were similar to those found with the genomic DVL1 recapitulation studies. However, a wide range of phenotypic variation, especially in siliques, was observed in the transgenic lines overexpressing the 35S:DVL cDNAs relative to the activation tagged DVL1 genomic DNA. For example, overexpression of DVL15 showed very weak phenotypes in rosette leaves and fruits. In addition, overexpression of DVL9 displayed an additional phenotype: a sharp turn in the pedicels (Fig. 3). Further analysis of these DVL overexpressing plants revealed that some of the leaf epidermal cells form a stalklike protrusion of the epidermal layer that terminates with a trichome (Fig. 3). The stalklike protrusions are composed of mesophyll cells and appear green. This abnormal phenotype is limited to the epidermis of organs that produce trichomes (leaves, stems, sepals) of all transgenic plants overexpressing all of the DVL genes we have tested. The overexpression of each DVL gene was confirmed by Northern blot analyses. Although the five DVL genes are closely related, there is no cross-hybridization among different DVL genes. These results support the conclusion that all of the DVL genes

DVL Peptides Are Involved in Plant Development /

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FIGURE 3. Leaf epidermal phenotypes wild-type (Col-0) and DVLoverexpressing transgenic plants. Left panels: Scanning electron microscope images of the adaxial surface of a leaf epidermis. Right panels: DVL9overexpressing transgenic plants exhibit a sharp turn in the pedicels. (See color plate.)

tested may have similar functional activity. However, some members, such as DVL9, may have additional functions. Since DVL members affect fruit development, we examined other genes involved in fruit development to see if there is a connection with DVLs. For example, the MADS-box gene family encodes transcriptional regulators involved in many aspects of plant development [8]. One of the MADS-box genes, FRUITFUL (FUL/AGL8), is required for fruit valve differentiation during fruit development [4]. Because overexpression of DVL genes causes dramatic phenotypic changes in the valve formation and fruit shape (Fig. 2), expression of the FUL/ AGL8 gene was examined in the DVL-overexpressing lines. RT-PCR results showed that the level of FUL/AGL8 mRNA was reduced in both dvl1-1D and 35S:DVLoverexpressing plants. After normalization, it was observed that the expression of FUL/AGL8 was decreased by two- to fivefold in the 35S:DVL-overexpressing plants. Moreover, when the expression level of DVL1 was decreased by overexpressing antisense DVL1, FUL/ AGL8 expression level was restored to nearly that of wild-type. These results suggest that the DVL peptides may affect the FUL/AGL8 regulatory pathway in control-

ling fruit development. Further insights into DVL mechanism of action will likely be afforded by molecular identification of DVL genetic modifiers. These modifiers will enable us to better understand this fascinating family of plant peptides.

References [1] Butenko MA, Patterson SE, Grini PE, Stenvik GE, Amundsen SS, Mandal A, Aalen RB. Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants. Plant Cell 2003; 15:2296–307. [2] Casson SA, Chilley PM, Topping JF, Evans IM, Souter MA, Lindsey K. The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning. Plant Cell 2002;14:1705–21. [3] Fletcher LC, Brand U, Running MP, Simon R, Meyerowitz EM. Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 1999; 283:1911–4. [4] Gu Q, Ferrandiz C, Yanofsky MF, Martienssen R. The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 1998; 125:1509–17. [5] Lease KA, Lau NY, Schuster RA, Torii KU, Walker JC. Receptor serine/threonine protein kinases in signaling: analysis of the erecta receptor-like kinase of Arabidopsis thaliana. New Phytologist 2000; 151:133–43.

22 / Chapter 4 [6] Li J, Lease KA, Tax FE, Walker JC. BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana. Proc Natl Acad Sci USA 2001; 98:5916–21. [7] Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 2002; 110:213–22. [8] Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF. SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 2000; 404:766–70. [9] Matsubayashi Y, Sakagami Y. Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 1996; 93:7623– 27. [10] Matsubayashi Y, Yang H, Sakagami Y. Peptide signals and their receptors in higher plants. Trends Plant Sci 2001; 6:573–7. [11] McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA. Structure, expression, and antisense inhibition of the systemin precursor gene. Science 1992; 255:1570–3. [12] Meyerowitz EM. Plants compared to animals: the broadest comparative study of development. Science 2002; 295:1482–5. [13] Narita N, Moore S, Horiguchi G, Kubo M, Demura T, Fukuda H. Overexpression of a novel small peptide ROTUNDIFOLIA4

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decreases cell proliferation and alters leaf shape in Arabidopsis thaliana. Plant J 2004; 38:699–713. Pearce G, Strydom D, Johnson S, Ryan CA. A polypeptide from tomato leaves induces wound-inducible inhibitor proteins. Science 1991; 253:895–8. Pearce G, Moura DS, Stratamann J, Ryan CA. Production of multiple plant hormones from a single polyprotein precursor. Nature 2001; 411:817–20. Pearce G, Moura DS, Stratamann J, Ryan CA. RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development. Proc Natl Acad Sci USA 2001; 98:12843–47. Ryan CA, Pearce G, Scheer J, Moura DS. Polypeptide hormones. Plant Cell 2002; 14 (suppl.):S251–64. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 1994; 22:4673–80. Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, Fankhauser C, et al. Activation tagging in Arabidopsis. Plant Physiol 2000; 122:1003–13. Wen J, Lease KA, Walker JC. DVL, a novel class of small polypeptides: Overexpression alters Arabidopsis development. Plant J. 2004; 37:668–77.

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5 The POLARIS Peptide KEITH LINDSEY, STUART A. CASSON, AND PAUL M. CHILLEY

The promoter trap strategy we developed involved transforming populations of plants with a promoterless gusA gene engineered into a T-DNA vector, allowing agrobacterium-mediated gene transfer. This vector, designated pΔGUSBin19 [21], is illustrated in Fig. 1. It is activated at high frequency in transgenic populations of several species, and GUS activity can be localized histochemically to allow the screening for insertion into genes that are expressed in tissue-specific patterns [9]. An area of interest to us has been the identification and functional analysis of genes expressed in embryos and roots. The basal region of the embryo represents the cellular origin of the primary root [3]. We exploited the promoter trapping approach to screen transgenic Arabidopsis containing the vector pΔGUSBin19 for lines exhibiting GUS activity in embryos [19]. One line of interest, designated AtEM101, showed a pattern of GUS activity that was polar in the developing embryo, and hence the tagged gene was named POLARIS (PLS). GUS activity is detectable from the early heart stage of embryogenesis, a stage at which apical-basal polarity is clearly visible in morphological terms, with cotyledonary primordia flanking the incipient shoot apical meristem at one end and the developing root apical meristem adjacent to the suspensor at the other end, which is the region of attachment to the maternal tissue (Fig. 2). AtEM101 plants homozygous for the PLS promoter trap insertion event exhibited a short root phenotype [2]. Using the promoter trap T-DNA as a molecular anchor point, we used RACE-PCR to clone the PLS cDNA. Northern blot analysis using the PLS cDNA as a probe showed that wild-type seedlings accumulate a single low-abundance transcript of ca. 500 nucleotides (Fig. 3A), and Southern analysis indicated a single gene copy per haploid genome [2]. Subsequent analysis of the Arabidopsis genome sequence has supported the view that PLS is a single copy gene.

ABSTRACT The POLARIS peptide was identified in the plant species Arabidopsis thaliana by promoter trapping. The gene encodes an mRNA of ca. 600 bases, at the 3′ end of which is a 36-amino-acid ORF. Translation of the ORF is required for biological activity. The transcriptional start of the gene overlaps with the 3′ UTR of an upstream gene. Expression of the POLARIS gene is strongest in root tips but is also detectable in young leaves and is induced by auxin. Mutation of POLARIS leads to several developmental defects, including a short root phenotype and reduced vascular complexity in leaves.

DISCOVERY Arabidopsis thaliana has proved to be an enormously valuable model organism for the study of plant growth and development, not least because of its ease of transformation and small diploid genome, which has allowed the use of insertional mutagenesis for gene tagging and functional analysis. Several years ago, we adopted a promoter trapping strategy to allow gene identification, based on the transcriptional activation of a promoterless gusA gene following random insertion into the genomes of plants, including Arabidopsis. A major advantage of this approach over conventional mutagenic strategies is that it not only allows gene detection by the identification of mutant phenotypes but simultaneously can provide information on the expression pattern of the tagged gene. It also facilitates gene isolation by virtue of the fact that the insertion element provides a molecular tag on the genomic sequence in which the gene of interest is located. This can subsequently be cloned by one of a number of PCRbased techniques [8]. Handbook of Biologically Active Peptides

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FIGURE 1. Diagrammatic representation of the promoter trap binary vector pΔGUSBin19, used to identify the POLARIS gene. At the T-DNA left border (LB) is a promoterless gusA gene, which can be activated following insertion into a transcription unit. At the T-DNA right border (RB) is a selectable marker, npt-II, conferring kanamycin resistance on transgenic plants. N = nos terminator.

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FIGURE 2. POLARIS-GUS expression in the basal region of a heart-stage embryo (A) and in the tips of emerging lateral roots of seedlings (B, C). (See color plate.)

A

B Transcript Start Sites

1

2

POLARIS Gene

mRNA 427 - 606 nt

500 nt

PLS ORF 401-511 nt FIGURE 3. The PLS gene encodes a low abundance ca. 500 nt mRNA revealed in Northern blots (A). Transcription initiated has been mapped by RNAse protection assays and 5′ RACE PCR to either of two start sites (1, 2), with the proximal site (2) used more frequently. The PLS ORF is located towards the 3′ end of the gene, at positions 401–511 bp (including the translational stop codon) downstream of transcription start site 1.

The POLARIS Peptide /

STRUCTURE OF THE PRECURSOR mRNA/GENE The genomic sequence at the PLS locus, which is on chromosome 4, reveals that the promoter trap T-DNA had inserted into a small ORF of 108 bp, located in a 755 bp region between two larger genes. One gene, which is not annotated and that we designated Gene X, is 450 bp upstream of the promoter trap T-DNA and encodes a predicted 92-amino-acid peptide of unknown function. The second gene encodes the brassinosteroid receptor BRI1 [7]. The identification of the ca. 500nucleotide PLS transcript suggested that PLS encodes a distinct gene, and the ORF is not an exon of either of the flanking genes. This was confirmed by both 5′ RACE PCR and RNAse protection assays, which demonstrated that the transcription of the PLS ORF initiates within the 3′ untranslated region of Gene X. Specifically, we identified two transcript initiation sites, 95 bp apart and one of which is used more frequently than the other (Fig. 3). The more distal start site (Start Site 1) occurs ca. 117 bp upstream of the Gene X polyadenylation sequence and has the sequence ATCCGTAT, with the underlined G representing the initiation site. The more proximal start site (Start Site 2), which is more frequently used, is ca. 23 bp upstream of the polyadenylation site of Gene X, with the sequence CCACTTAATA. It was not possible to resolve which of the three underlined bases is the start point for transcription. Start Site 2 has a predicted TATA sequence (TATATAA) at positions −32 to −26, and Start Site 1 has a poorer TATA-like sequence at −35 to −29 (AATAATA). Sequence analysis of PLS 3′ RACE products revealed a variable length of polyadenylation, such that the length of the PLS transcript is between 427 and 606 nucleotides, depending on which transcriptional start site is used and the length of the poly(A) tail. Sequences 5′ to the Start 2 transcription initiation site were cloned upstream of the gusA reporter gene, and transformed Arabidopsis plants were generated for functional analysis. A relatively short (−370 bp) sequence was sufficient to drive GUS activity in the root tip of transgenics. A longer 5′ flanking sequence (−1190 bp upstream of Start Site 2) promoted stronger GUS expression than the shorter sequence [2]. These results provided further support for transcription of the PLS gene initiating within the upstream Gene X sequence. While apparently unusual in eukaryotes, an overlapping organization has similarly been described for the EhMCM3 and EhPAK genes of Entamoeba histolytica [4] and for the OTC and AUL1 genes of Arabidopsis [15]. Mouse and human examples have also been reported [5, 17]. The PLS gene was sequenced from two ecotypes of Arabidopsis—namely, C24 (the original promoter trap

25

background) and Columbia (Col-0)—and was found to be identical. The PLS ORF, disrupted by the promoter trap T-DNA, encodes a 36-amino-acid peptide of predicted 4.6 kD. The PLS ORF is located towards the 3′ end of the PLS transcript (Fig. 3B) and is preceded by an in-frame 27 bp ORF (encoding a predicted 9-aminoacid peptide) with a stop codon immediately preceding the PLS ATG and an 8-amino-acid peptide that overlaps with the PLS ORF. These upstream ORFs may play a role in regulating the PLS transcript stability and/or PLS translation [e.g., 10, 23]. Proof for a functional role for the PLS ORF comes from transgenic complementation experiments in which the short root phenotype of the mutant is complemented by transformation with a partial cDNA containing the wild-type PLS ORF sequence. Mutation of the PLS ORF ATG prevented complementation, demonstrating that translation of the PLS peptide is required for biological activity [2].

DISTRIBUTION OF THE mRNA Northern blot, RT-PCR, and cloned promoter analysis have confirmed the distribution of PLS expression throughout different organs, as indicated by the histochemical localization of GUS activity in the original promoter trap line. The PLS mRNA is a very lowabundance transcript but is detectable most readily in the distal (apical) regions of roots and also in young leaves as well as embryos. This is reflected also in promoter-GUS activity in transgenics. In the root, short (5 min) histochemical staining times reveal GUS activity in the root columella initials and lateral root cap, similar to the auxin-responsive DR5-GUS reporter [16]. Indeed, PLS transcription is strongly auxininducible, as determined by either promoter-GUS histochemistry, Northern analysis, or competitor RT-PCR [2]. This induction, which occurs within 30 min of auxin application, is only seen with functional auxins and not with nonfunctional structural analogs such as 2-naphthylacetic acid or 2,3-dichlorophenoxyacetic acid. The PLS promoter contains potential TGTCTClike auxin response elements [22]. Further support for a dependence of PLS expression on correct auxin signaling comes from studies in the gnom/emb30 mutant background. The GNOM protein is an ARF-GEF required for vesicle trafficking and the correct polar localization of PIN proteins, themselves components of the auxin efflux transport system [18]. In a severe gn genetic background, PLS expression in embryos and seedlings is depolarized, consistent with the depolarized auxin distribution [20]. The expression of PLS in young leaves is also consistent with their being a site of

26 / Chapter 5 auxin biosynthesis and relatively high concentration, as for roots [11, 12]. PLS expression is therefore a good marker of auxin responses and auxin localization.

PROCESSING The N-terminal 24 amino acids of the PLS peptide are predicted to form two β-sheets, and the C-terminal 12 amino acids are predicted to form an α-helical domain. There is no obvious targeting sequence in the PLS sequence, suggesting it is not secreted but remains cytosolic. There is a possibility that the N-terminal region may be processed, as the presence of three basic arginine residues at positions 10–12 may form a turn region and represent a cleavage site. The second βsheet contains a repeated SIS sequence that, due to the proximity to the arginine residues, may represent a cAMP-/cGMP-dependent protein kinase phosphorylation site, though this has not been determined experimentally. The C-terminal α-helical domain contains the motif KLFKLFK, which has potential to act as a leucine zipper for protein-protein interactions. We have evidence for interactions in yeast-two hybrid studies for the C-terminal but not N-terminal regions of PLS, and candidate interactors are being characterized further.

BIOLOGICAL ACTIONS Our effort to investigate the function of the PLS peptide has focused on an analysis of the pls mutant phenotype. Although PLS expression is detectable in the developing embryo, there is no obvious mutant phenotype at this stage of the life cycle. However, the first evidence of a role in development is seen at the seedling stage, with defects apparent in root architecture and leaf venation [2]. Seedlings homozygous for the pls mutation show a reduced length of the primary root, to about 50% that of wild-type over the first 14 days postgermination. Cells of the root are shorter and wider (more isodiametric) than wild-type, and they divide less frequently to account for the reduced root length. pls also produces fewer lateral roots (unpublished) but may produce more anchor roots, indicative of a failure of auxin transport to the root tip. Seedlings hemizygous for the pls mutation show a primary root length that is intermediate between wild-type and homozygous mutants, indicating that the mutation is semidominant. The overall stature of pls flowering plants is reduced compared to wild-type and more bushy, indicative of a reduced apical dominance (unpublished). Microscopic analysis of leaves of the rosette at 12 days postgermina-

tion revealed a reduced venation compared with wildtype and in particular a reduced number of higher order veins branching from the primary strands [2]. Taken together, these results suggest a requirement for PLS in regulating cell shape and division in the root, vascular patterning in the leaf, and shoot architecture. One hypothesis we can consider is that PLS is required for correct auxin or cytokinin signaling, both of which are known to affect the development of the root and shoot. Auxin in particular has been implicated in vascular patterning [13], lateral root development [1], and the control of apical dominance [6]. To investigate auxin and cytokinin responses of the pls mutant, seedlings were grown in vitro in the presence of exogenous auxin or cytokinin over a range of concentrations. pls seedlings showed reduced responses to auxins, seen as a reduced inhibition of primary root growth in the presence of exogenously supplied 2,4-D or 1-NAA. For example, in the presence of 10 nm 1NAA, wild-type primary roots were inhibited to 84% the length of untreated roots, whereas pls roots were 106% the length of untreated, thus showing a slight increase rather than inhibition in growth [2]. This suggests that pls seedlings may be defective in auxin transport, responses, or accumulation. The expression of at least some auxin-regulated genes is reduced in pls, shown as the reduced steady-state level of the IAA1 gene transcript, supporting the view that pls has reduced auxin responses. In the presence of exogenous cytokinins, pls primary roots exhibited an enhanced response compared with wild-type. For example, in the presence of 10 nm BA, the wild-type root is not significantly shorter than untreated. However, at the same concentration, the pls root growth was significantly inhibited to 71% the length of the untreated root [2]. Transgenic plants overexpressing the PLS transcript show reduced responses to cytokinins and increased leaf vascularization, an auxin-regulated process, compared with the mutant. This antagonistic relationship between auxin and cytokinin responses in pls may reflect the antagonistic interplay of auxin and cytokinin biosynthesis [14] and suggests the requirement for PLS in regulating this interaction, either directly or indirectly. This hypothesis is currently being tested, and results to date confirm that pls has a reduced free IAA content compared with wild-type (unpublished). In summary, the POLARIS peptide represents a new component required for cross-talk between more conventional plant signaling pathways, and modulation of its expression plays a significant role in the modulation of growth. Key biological questions being addressed in this laboratory include the elucidation of its mode of action (site of action, identity of interacting partners, downstream target pathways); the regulation of its activ-

The POLARIS Peptide / ity; and the extent of its distribution in the plant kingdom.

References [1] Casimiro, I., Beeckman, T., Graham, N., Bhalerao, R., Zhang, H., Casero, P. J., Sandberg, G. and Bennett, M. J. (2003). Dissecting Arabidopsis lateral root development. Trends Plant. Sci. 8, 165–171. [2] Casson, S. A., Chilley, P. M., Topping, J. F., Evans, I. M., Souter, M. A. and Lindsey, K. (2002). The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root rowth and leaf vascular patterning. Plant Cell 14, 1705–1721. [3] Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and Scheres, B. (1993). Cellular organisation of the Arabidopsis thaliana root. Development 119, 71–84. [4] Gangopadhyay, S. S., Ray, S. S., Sinha, P. and Lohia, A. (1997). Unusual genome organisation in Entamoeba histolytica leads to two overlapping transcripts. Mol. Biochem. Parasitol. 89, 73–83. [5] Koskimies, P., Spiess, A.-N., Lahti, P., Huhtaniemi, I. and Ivell, R. (1997). The mouse relaxin-like factor gene and its promoter are located within the 3′ region of the JAK3 genomic sequence. FEBS Lett. 419, 186–190. [6] Leyser, O. (2003). Regulation of shoot branching by auxin. Trends Plant Sci. 8, 541–545. [7] Li, J. and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, 929–938. [8] Lindsey, K. and Topping, J. F. (1996). Insertional mutagenesis by T-DNA. In Plant Gene Isolation (ed. G. D. Foster and D. Twell), pp. 275–305. Chichester: John Wiley & Sons. [9] Lindsey, K., Wei, W., Clarke, M. C., McArdle, H. F., Rooke, L. M. and Topping, J. F. (1993). Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Research 2, 33–47. [10] Linz, B., Koloteva, N., Vasilescu, S. and McCarthy, J. E. G. (1997). Disruption of ribosomal scanning on the 5′-untranslated region, and not restriction of translational initiation per se, modulates the stability of nonaberrant mRNAs in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 272, 9131–9140. [11] Ljung, K., Bhalerao, R. P. and Sandberg, G. (2001). Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative development. Plant J. 28, 465–474.

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[12] Ljung, K., Hull, A. K., Celenza, J., Yamada, M., Estelle, M., Normanly, J. and Sandberg, G. (2005). Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17, 1090– 1104. [13] Mattsson, J., Sung, Z. R. and Berleth, T. (1999). Responses of plant vascular systems to auxin transport inhibition. Development 126, 2979–2991. [14] Nordström, A., Tarkowski, P., Tarkowska, D., Norbaek, R., Astot, C., Dolezal, K. and Sandberg, G. (2004). Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development. Proc. Natl. Acad. Sci. USA 101, 8039–8044. [15] Quesada, V., Ponce, M. R. and Micol, J. L. (1999). OTC and AUL1, two convergent and overlapping genes in the nuclear genome of Arabidopsis thaliana. FEBS Lett. 461, 101–106. [16] Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T., Malamy, J., Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P. et al. (1999). An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99, 463–472. [17] Speek, M., Barry, F. and Miller, W. L. (1996). Alternate promoters and alternate splicing of human tenascin-X, a gene with 5′ and 3′ ends buried in other genes. Human Mol. Gen. 5, 1749– 1759. [18] Steinmann, T., Geldner, N., Grebe, M., Mangold, S., Jackson, C. L., Paris, S., Gälweiler, L., Palme, K. and Jürgens, G. (1999). Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286, 316–318. [19] Topping, J. F., Agyeman, F., Henricot, B. and Lindsey, K. (1994). Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping. Plant Journal 5, 895–903. [20] Topping, J. F. and Lindsey, K. (1997). Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis. Plant Cell 9, 1713–1725. [21] Topping, J. F., Wei, W. and Lindsey, K. (1991). Functional tagging of regulatory elements in the plant genome. Development 112, 1009–1019. [22] Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J. (1997). Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971. [23] Wang, L. and Wessler, S. R. (1998). Inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize R gene. Plant Cell 10, 1733– 1745.

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6 Phytosulfokine YOSHIKATSU MATSUBAYASHI AND YOUJI SAKAGAMI

requirement is greater for more mature explanted cells. Dilution of dispersed mature mesophyll cells in excess culture medium significantly prevents callus formation, even if otherwise sufficient amounts of growth regulators and nutrients are supplied [9]. A similar phenomenon has also been observed for transdifferentiation of mesophyll cells into tracheary elements (TEs) [12]. If dispersed Zinnia mesophyll cells are cultured below a certain cell density, TE formation is greatly suppressed, indicating that cell-to-cell communication is involved in transdifferentiation. Similarly, somatic embryo formation from a suspension of carrot cells is dependent on cell density [4]. Interestingly, this population dependence is lessened by addition of conditioned medium in which cells have previously been grown, indicating that such cell-to-cell communication is mediated by chemical signal(s) produced by cells [4, 9, 12].

ABSTRACT Almost all plant cells, even when fully differentiated, can dedifferentiate and proliferate in vitro to form a callus, in which they can then differentiate to form various organs. These sequential processes can be promoted by exposing the cells to conditioned medium in which cells have previously been grown, indicating the involvement of cell-to-cell communication mediated by chemical signals. Phytosulfokine (PSK), a 5-amino-acid sulfated peptide that has been detected in conditioned medium, is the primary signal responsible for these phenomena. Addition of synthetic PSK to culture medium, even at nanomolar concentrations, significantly promotes cellular proliferation and/or differentiation of cells. PSK is synthesized from ≈80-amino-acid precursor peptides, and it binds to PSKR1, which is a membranelocalized leucine-rich repeat receptor-like kinase.

DISCOVERY AND BIOLOGICAL ACTIONS INTRODUCTION Phytosulfokine (PSK), a 5-amino-acid sulfated peptide that has been detected in conditioned medium of plant cell cultures, is the primary signal molecule responsible for the preceding examples of cell-to-cell communication (Fig. 1A) [1, 4, 9, 12]. PSK was first purified from conditioned medium derived from an asparagus suspension culture, based on its ability to promote cell division of asparagus mesophyll cells incubated at low cell density [9]. Addition of chemically synthesized PSK to culture medium, even at nanomolar concentrations, significantly improves the efficiency of cellular proliferation (Fig. 1B) [9, 13] and/or differentiation (Fig. 1C, 1D) [1, 3, 4, 12] of individual cells, even when the initial cell population is far below the critical density. PSK also promotes adventitious root formation of hypocotyls [15] and improves genetic transformation efficiency by promoting proliferation of surviving cells on selective

Plants, due to their sessile nature, have developed a greater ability to adapt to dynamic environmental conditions than have animals. This plasticity allows plants to flexibly alter their developmental program and metabolism according to the environment. Particularly important aspects of this adaptation are the potentials to initiate cell division (called callus formation) from almost any tissue of the plant irrespective of their age and to regenerate a new plant irrespective of the type of original tissue. With most plant species, stem cells can be created de novo from a single differentiated cell detached from a parent plant, and those stem cells can develop into a new plant. Lines of evidence suggest that the population of living cells are required to support callus formation from explants and successive proliferation in vitro. This Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

30 / Chapter 6 A

Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln

B

–PSK

media [6]. PSK has been detected at nanomolar levels in culture media of various plant cell lines including asparagus [9], rice [13], maize [13], Zinnia [12], carrots [1], and Arabidopsis [18]. This suggests that among higher plants, PSK is widely distributed with strict sequence conservation. Structure-activity analysis indicates that the N-terminal tripeptide fragment Tyr (SO3H)-Ile-Tyr(SO3H) is the active core of PSK [7].

+PSK

STRUCTURE OF THE PRECURSOR mRNA/GENE

C

–PSK

+PSK

–PSK

+PSK

D

FIGURE 1. Structure and biological activities of PSK. PSK is a 5-amino-acid peptide containing 2 sulfated tyrosines (A). PSK promotes cell division of asparagus mesophyll cells (B), tracheary element differentiation of Zinnia mesophyll cells (C), and somatic embryogenesis of carrot suspension cells (D). PSK was added to each culture at final concentration of 10 nM.

AtPSK1 AtPSK2 AtPSK3 AtPSK4 AtPSK5

PSK is generated by enzymatic processing of an ≈80amino-acid precursor peptide that has a secretion signal at its N-terminal (Fig. 2) [16–18]. Di-basic amino acid residues are located immediately upstream from the PSK domain. It is generally believed that in animal prohormone precursor proteins, the primary processing recognition sequence for endoproteolysis is a pair of basic amino acid residues that bracket the peptide hormone. PSK precursor genes are redundantly distributed throughout the genome (5 genes in Arabidopsis) and are found in a variety of angiosperm [5] and gymnosperm [3] plant species. In Arabidopsis, expression of PSK genes is not limited to tissues in which cells actively divide and differentiate and has been detected in most plant parts including leaves, stems, roots, and calluses ([18] and unpublished observation). This suggests that PSK is not a simple mitogen or differentiation initiator and is consistent with the finding that overexpression of the PSK precursor gene causes no apparent changes in plant growth or development (unpublished observation). Another important observation is that rapidly proliferating suspension cells and immature cells from young tissues often grow in the absence of PSK even

MKTKSEVLIFFFTLVLLLSMASSVILRE---DGFAP------PKPSPTTHEKASTK-G-M---ANVSALL-TIALLL--CSTLMCT----ARPEPAISISITTAADPCNMEKKIE-GKL M---KQSLCLA-VLFLILSTSSSAIRRGKEDQEINPLV----SATSVEEDSVNKLM-G-M---GKFTTIF-IMALLL--CSTLTYA----ARLTPTT----TTALSRENSVKEIE-G-M---VKFTTFLCIIALLL--CSTLTHAS---ARLNP------TSVYPEENSFKKLEQG-* : : : *:* .*: * . . . *

48 49 49 43 44

Signal sequence

AtPSK1 AtPSK2 AtPSK3 AtPSK4 AtPSK5

-DRDGV---ECKNSDSEEEC-LVKKTVA-AHTDYIYTQDLNLSP DDMHMVD-ENC-GAD-DEDC-LMRRTLV-AHTDYIYTQKKKH-P -----ME--YC-GEG-DEEC-LRRRMMTESHLDYIYTQHHK--H -DK--VEEESCNGIG-EEEC-LIRRSLV-LHTDYIYTQNHK--P -E---V---ICEGVG-EEECFLIRRTLV-AHTDYIYTQNHN--P : * . . :*:* * :: :. * ******. :

86 87 81 79 77

PSK domain

FIGURE 2. Alignment of deduced amino acid sequences of PSK precursor proteins in Arabidopsis. The 5-amino-acid PSK domain is boxed, and predicted amino terminal signal sequences are underlined. Identical amino acid residues are indicated by an asterisk, and similar amino acid residues are indicated by a colon.

Phytosulfokine / when they are diluted with excess medium. These observations sharply contrast with the finding that PSK is necessary for initiation of proliferation and/or transdifferentiation of fully differentiated mature cells. Thus, the available evidence suggests that cells gradually lose the potential to restart proliferation and/or differentiation during cellular maturation and that PSK reactivates this potential, which is necessary for initiation of in vitro culture.

TYROSINE SULFATION PSK is the first tyrosine-sulfated peptide to have been detected in higher plants. Elimination of the sulfate esters of PSK results in complete loss of its biological activities [7], indicating that a tyrosine sulfation pathway is a key step in PSK biosynthesis in plants. Although the general role of tyrosine sulfation in protein function is not well understood, it is the most frequent posttranslational modification of many secretory and membrane proteins of various eukaryotes [14]. In mammalian cells, tyrosine sulfation is mediated by Golgi-localized tyrosylprotein sulfotransferase (TPST), which catalyzes transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to tyrosine residues within acidic motifs of polypeptides. With use of a PSK precursor peptide fragment as substrate, it has been demonstrated that plant TPST activity is localized in Golgi membranes and that one of the most important determinants of sulfation of PSK precursor is the aspartic acid adjacent to the tyrosine residue [2]. However, in sequence similarity searches, no ortholog of animal TPST has been found in Arabidopsis, suggesting that plant TPST is structurally distinct from animal TPST.

RECEPTORS Specific binding sites for [35S]PSK or [3H]PSK have been detected on the surface of suspension-cultured cells and in plasma-membrane-enriched fractions of various plants [10, 13]. Binding of PSK to those sites is reversible and saturable, and only biologically active PSK analogs can effectively displace the radioligand. Scatchard analysis using [3H]PSK has revealed the existence of a high-affinity PSK binding site in microsomal fractions derived from carrot cells: approximately 150 fmol per mg microsomal proteins, with a dissociation constant (Kd) of 4.2 nM [8]. This PSK receptor protein has been visualized by photoaffinity labeling of carrot plasma membrane fractions using the photoactivatable 125I-labeled PSK analog [N ε-(4-azidosalicyl)Lys5] PSK [8, 11]. SDS-PAGE analysis of the labeled proteins indicates that a 120-kD protein and a minor 150-kD protein specifically interact with PSK.

31

A SP

LRRs

ID

TM

KD

B

WT

PSKR1ox

FIGURE 3. Structure of PSK receptor PSKR1. A. The diagram shows the signal peptide (SP), extracellular leucine-rich repeats (LRRs), a 36-amino-acid island domain (ID), a transmembrane domain (TM), and a cytoplasmic kinase domain (KD). B. Transgenic carrot cells overexpressing PSKR1 show accelerated growth, compared with wild-type cells.

The PSK-binding protein was purified from microsomal fractions of carrot cells by detergent solubilization and specific ligand-based affinity chromatography using a PSK-Sepharose column [8]. Several independent purifications were performed, yielding 50 μg of the protein with 96,000-fold purification from 4800 mg of microsomal proteins (corresponding to 24 L of suspension-cultured cells), with an overall recovery rate of 40%. Based on the internal sequence of the PSKbinding protein, the 120-kD and 150-kD proteins have been identified as leucine-rich repeat receptor-like kinases (LRR-RLK) derived from a single gene (Fig. 3A) [8]. It is likely that the difference between their molecular sizes is due to differences in posttranslational modification such as glycosylation and/or truncation of part of the peptide backbone. Transgenic carrot cells overexpressing the PSK-binding LRR-RLK showed a significant increase in PSK binding sites in the membrane fractions and accelerated growth in response to PSK, compared with control cells (Fig. 3B). These findings indicate that the PSK-binding receptor kinase is a component of a functional PSK receptor that directly interacts with PSK. The PSK-binding LRR-RLK has been named PSKR1. Expression of PSKR1 has been detected throughout tissues of the leaves, apical meristem, hypocotyl, and root of carrot seedlings, although much higher expression has been detected in cultured carrot cells. PSKR1 knockdown calluses can survive and proliferate, but their growth stops within a short time after inoculation, resulting in formation of a smaller callus than the wildtype. However, PSKR1 knockdown calluses can still regenerate morphologically normal shoots and roots.

CONCLUSION The studies that have revealed the in vitro function of PSK and the molecular basis of ligand-receptor interaction in PSK signaling have paved the way for research

32 / Chapter 6 aimed at characterization of the in vivo role of PSK and its downstream signaling pathway in plants. The carrot PSK receptor, PSKR1, exhibits a high percentage of amino acid identity with several LRR-RLKs found in Arabidopsis. The in vivo function of PSK is currently being researched in studies of knockout mutants of the genes for those LRR-RLKs. Differentiated plant cells retain features characteristic of totipotent stem cells. That is, even fully differentiated single cells have the potential to dedifferentiate, proliferate, and give rise to all the organs of a new plant. However, little is known about the mechanisms responsible for such extreme cellular plasticity. The roles that PSK and PSK receptors play in these cellular events at the molecular level may have profound implications for the basis of cellular plasticity and also may reveal general mechanisms of ligand-receptor interactions in plants.

Acknowledgments The author thanks Dr. H. Hanai (Akita Prefectural University) for providing photographs of carrot somatic embryos.

References [1] Hanai H, Matsuno T, Yamamoto M, Matsubayashi Y, Kobayashi T, Kamada H, et al. A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. Plant Cell Physiol 2000;41:27–32. [2] Hanai H, Nakayama D, Yang H, Matsubayashi Y, Hirota Y, Sakagami Y. Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro. FEBS Lett 2000;470:97– 101. [3] Igasaki T, Akashi N, Ujino-Ihara T, Matsubayashi Y, Sakagami Y, Shinohara K. Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica. Plant Cell Physiol 2003;44:1412–6. [4] Kobayashi T, Eun C, Hanai H, Matsubayashi Y, Sakagami Y, Kamada H. Phytosulphokine-α, a peptidyl plant growth factor, stimulates somatic embryogenesis in carrot. J Exp Bot 1999;50: 1123–8.

[5] Lorbiecke R, Sauter M. Comparative analysis of PSK peptide growth factor precursor homologs. Plant Sci 2002;163:321–32. [6] Matsubayashi Y, Goto T, Sakagami Y. Chemical nursing: phytosulfokine improves genetic transformation efficiency by promoting the proliferation of surviving cells on selective media. Plant Cell Rep 2004;23:155–8. [7] Matsubayashi Y, Hanai H, Hara O, Sakagami Y. Active fragments and analogs of the plant growth factor, phytosulfokine: structure-activity relationships. Biochem Biophys Res Commun 1996;225:209–14. [8] Matsubayashi Y, Ogawa M, Morita A, Sakagami Y. An LRR receptor kinase involved in perception of a peptide plant hormone, phytosulfokine. Science 2002;296:1470–2. [9] Matsubayashi Y, Sakagami Y. Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 1996;93:7623–7. [10] Matsubayashi Y, Sakagami Y. Characterization of specific binding sites for a mitogenic sulfated peptide, phytosulfokine-α, in the plasma-membrane fraction derived from Oryza sativa L. Eur J Biochem 1999;262:666–71. [11] Matsubayashi Y, Sakagami Y. 120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine-α, in rice plasma membranes. J Biol Chem 2000;275:15520–5. [12] Matsubayashi Y, Takagi L, Omura N, Morita A, Sakagami Y. The endogenous sulfated pentapeptide phytosulfokine-α stimulates tracheary element differentiation of isolated mesophyll cells of zinnia. Plant Physiol 1999;120:1043–8. [13] Matsubayashi Y, Takagi L, Sakagami Y. Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites. Proc Natl Acad Sci USA 1997;94:13357–62. [14] Moore KL. The biology and enzymology of protein tyrosine Osulfation. J Biol Chem 2003;278:24243–6. [15] Yamakawa S, Sakuta C, Matsubayashi Y, Sakagami Y, Kamada H, Satoh S. The promotive effects of a peptidyl plant growth factor, phytosulfokine-α, on the formation of adventitious roots and expression of a gene for a root-specific cystatin in cucumber hypocotyls. J Plant Res 1998;111:453–8. [16] Yang H, Matsubayashi Y, Hanai H, Nakamura K, Sakagami Y. Molecular cloning and characterization of OsPSK, a gene encoding a precursor for phytosulfokine-α, required for rice cell proliferation. Plant Mol Biol 2000;44:635–47. [17] Yang H, Matsubayashi Y, Nakamura K, Sakagami Y. Oryza sativa PSK gene encodes a precursor of phytosulfokine-α, a sulfated peptide growth factor found in plants. Proc Natl Acad Sci USA 1999;96:13560–5. [18] Yang H, Matsubayashi Y, Nakamura K, Sakagami Y. Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor. Plant Physiol 2001;127:842–51.

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7 RALF Peptides DANIEL S. MOURA, GREGORY PEARCE, AND CLARENCE A. RYAN

9, 10]. The alkalinization is a result of intercellular signaling, initiated by ligand-receptor interactions [6, 9–14] that alters the activity of a membrane proton pump and is an essential component of the signal transduction pathway for defense gene activation [12]. As peptides in leaf extracts elute from HPLC columns, small aliquots from each fraction (1–10 μL) are added to 1 mL of suspension-cultured cells that are growing at a pH near 5.0. The fractions containing bioactive peptides are identified by the increase in pH of the cell medium that takes place within a few minutes after their addition to the cell cultures [8, 9]. A peptide identified in the assay that caused an exceptionally rapid alkalinization response was called RALF and was isolated and characterized [8]. Homologous RALF peptides from tomato and alfalfa leaf extracts have been similarly identified and purified. None of the peptides was posttranslationally modified, and all have equivalent activities when assayed with tomato or tobacco cells. Chemically synthesized tomato RALF is as active as the native tomato RALF in the alkalinization assay [8].

ABSTRACT RALF (Rapid Alkalinization Factor) peptides are composed of 49 to 52 amino acids and are ubiquitous in plants. When added to suspension-cultured plant cells at low nM concentrations, the peptides cause a rapid activation of a MAP kinase and the blockage of a membrane-bound ATP-dependent proton pump that results in the rapid alkalinization of the culture medium. When added to the growth medium of germinating seeds, RALF causes an immediate arrest of root growth and elongation that can be reversed by transferring the seeds to a medium lacking the peptide. RALF peptides are cleaved from larger precursor proteins that are synthesized through the Golgi and secretory pathway and sequestered in the apoplast. RALF precursor genes are members of gene families that exhibit tissue-specific expression in roots, stems, leaves, and flowers. RALF interacts strongly and reversibly with a cell surface receptor complex composed of 120 kD and 25 kD RALFbinding proteins. In Arabidopsis roots, an endogenous RALF gene is expressed at high levels in slowly dividing cells and at low levels in rapidly dividing cells, suggesting that RALF peptides may play important tissuespecific roles in regulating cell division and cell expansion during growth and development.

STRUCTURE OF THE RALF PEPTIDE AND ITS PRECURSOR cDNA AND GENE A partial N-terminal sequence analysis of the pure tobacco RALF peptide, determined by Edman degradation, was +ATKKYISYGALQKNSVP—-The molecular weight of the purified peptide, determined by MALDI mass spectroscopy, is 5332.7 mass units [8]. A full-length tobacco RALF cDNA from a tobacco leaf cDNA library codes for a preproRALF polypeptide of 115 amino acids with the RALF sequence of 49 amino acids harbored within the C-terminal region of the protein (Fig. 2) [8]. The RALF sequence extends from a putative cleavage site of –Leu66-Ala67– to the C-terminal Ser115 of the

DISCOVERY The 49-amino-acid peptide called RALF (Fig. 1) was discovered in leaf extracts of tobacco during a search for signaling peptides. The name RALF is derived from the “alkalinization” assay that was employed to identify and purify the peptides. When added to suspensioncultured cells, RALF causes a receptor-mediated alkalinization of the cell medium of about one pH unit, a characteristic response of plant peptide hormones [6, Handbook of Biologically Active Peptides

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34 / Chapter 7 MGVPSGLILC VLIGAFFISM AAAGDSGAYD WVMPARSGGG 40 CKGSIGECIA EEEEFELDSE SNRRILATKK YISYGALQKN 80 SVPCSRRGAS YYNCKPGAQA NPYSRGCSAI TRCRS 115

FIGURE 1. The primary structure of the deduced preproRALF protein, deduced from a cDNA identified in tobacco leaves. The RALF peptide sequence is underlined.

A T K K Y I SY G A L QKN S V PC SR R G s A s S S C G R S Y P NA Q A G P K C NY Y A s I T s RCR S

FIGURE 2. The primary structure of tobacco RALF peptide, with the disulfide placements.

protein. The molecular mass, calculated from the deduced RALF sequence, coincides exactly with the molecular weight determined by MALDI mass spectroscopy. The preproRALF protein contains six cysteines, with four in the active peptide region as disulfide bonds at Cys84-Cys94 and Cys107-Cys113 [8]. Chemically synthesized tomato RALF requires folding under oxidizing conditions to achieve the correct disulfide linkages found in the native, biologically active, peptide (Fig. 2). Biological assays of correctly folded synthetic RALF peptides of various lengths indicate that loss of 16 amino acids from the N-terminal region of the peptide totally abolishes activity (G. Pearce and C. A. Ryan, unpublished). Tomato and alfalfa RALF peptides have molecular masses of 5346.8 and 5375.9, respectively [8]. The amino acid sequence of the tomato RALF precursor, deduced from an EST sequence (cf. Fig. 1), indicated that a single residue difference is present between the mature tobacco and tomato RALF peptides (a Ser in tobacco is a Thr in tomato at residue 38). Alfalfa RALF, from the more distantly related Leguminosae family, differed in only three positions from tobacco RALF, again implying an important conservation of structure and function in distantly related species. The amino acid sequence of the tomato RALF precursor exhibits three domains: a leader sequence of 23 amino acids (suggesting that the peptide is synthesized through the Golgi and secretory pathway); an upstream sequence of 43 amino acids, rich in acidic residues, between the leader sequence and the RALF sequence; and the C-terminal RALF sequence, containing four conserved half cystines [8].

DISTRIBUTION OF THE mRNA RALF is unique among plant peptide hormones, having highly conserved homologs throughout the plant kingdom. RALF genes have now been identified in various tissues and organs from over 20 species of plants representing 9 families [4, 5, 7, 8]. Among the numerous plant species from which RALF-like ESTs have been derived are Arabidopsis thaliana, Solanum chacoense, and Populus trichocarpa X Populus deltoides. In Arabidopsis, a large family of 34 preproRALF paralogs (AtRALFs) are present with C-terminal coding regions of various degrees of sequence identity with tobacco leaf preproRALF [7]. The expression patterns of many of the genes exhibit different tissue specificities. Five preproRALF cDNAs are present in ovary tissues of Solanum chacoense, and the deduced peptides are called ScRALFs. The five genes coding for ScRALFs [4] are differentially expressed in flower tissues and in other parts of the plants, including roots, stems, and leaves. Five RALF peptides were purified from hybrid poplar leaves, as well as two unique cDNA clones from a poplar cDNA library [5]. Both cDNAs were differentially expressed in tissues of poplar saplings, supporting a developmental role for the peptides. A RALF ortholog in tomato plants is found in pollen, associated with pollen elongation (P. A. Bedinger, personal communication).

PROCESSING An Arg63-Arg64 motif is present just two residues upstream from the N-terminus of the RALF peptide, suggesting that, as found in many animal peptide prohormones, RALF may be processed by a convertase-like enzyme. This dibasic site is highly conserved among different plant species and may be the initial cleavage site for processing, which suggests that the internal cleavage is followed by trimming. Transforming Arabidopsis plants with a gene with the Arg-Arg motif mutated to Arg-Ala residues blocked the processing of proRALF to RALF, suggesting that the dibasic amino acids at this site are important for processing (D. S. Moura, G. Pearce, and C. A. Ryan, in preparation).

RECEPTOR RALF-binding proteins of 25 kD and 120 kD were identified on the external surface of suspensioncultured tomato cells by use of a photoaffinity analog of RALF [14]. Both the 25 kD and 120 kD proteins are associated with the cell membranes. Reagents known to solubilize peripheral proteins from membranes did not

RALF Peptides / solubilize either protein. Kinetic analyses indicate that the binding of RALF involves high-affinity sites that are saturated at 2 nM RALF. Unlabeled RALF strongly competes with labeling with the fully active photoaffinity analog of RALF at a concentration as low as 2 nM and is completely blocked at 200 nM, while two biologically inactive RALF analogs do not compete [14]. Suramin, an inhibitor of peptide ligand receptor interactions in animals and plants, was also a potent competitor of labeling. The properties of the two labeled proteins suggest that both are integral membrane components of the RALF receptor. While the RALF-receptor interactions result in the alkalinization response and the activation of a MAP kinase, the mechanism of signal transduction remains obscure.

SOLUTION CONFORMATION OF RALF The solution conformation of the RALF peptide or its precursor protein have not been investigated. The peptide does require that the two disulfide linkages be correctly cross-linked for activity. This implies that specific conformations are maintained by the disulfide linkages that are involved in receptor interactions. Investigations of the solution conformations of RALF and its active and inactive analogs should provide insights into the regions of the RALF structure that may be important for its interactions with its receptor.

BIOLOGICAL ACTION OF RALF Tomato RALF at a 10 μM concentration causes a striking arrest of growth of developing roots emerging from tomato and Arabidopsis seeds (Fig. 3) [8]. Reduction of the disulfide bonds of RALF and alkylation of the free sulfhydryl groups severely reduced its rootarresting activity and its activity in the alkalinization assay [8]. The tobacco RALF precursor gene is synthesized initially through the secretory pathway and translocated to the extracellular matrix [3], where it is processed in response to an unknown cue and where the RALF peptide interacts with its receptor (14). An analysis of one of the orthologs, AtRALF-1 (At1g02900) in six cell types of growing and elongating roots, from the tip through the elongating zone [1, 2], revealed that the gene is highly expressed in the stele, endodermis, and endodermis plus cortex but not in the epidermis or lateral root cap. Where cell division was high, endogenous AtRALF-1 expression was found to be low, and where endogenous cell division was low, AtRALF-1 was highly expressed. RALF appears to be associated somehow in limiting cell division and elongation. This hypothesis is supported by the effect of transforming

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FIGURE 3. Root inhibition of germinating seedlings of tomato caused by the addition of 10 μM RALF peptide to the medium. Seedlings germinated with water (left pair of plantlets) or an inactive alkylated RALF (alkRALF) peptide (right pair of plantlets) are controls.

Arabidopsis with a constitutively overexpressed AtRALF11 gene under control of the 35S-CaMV promoter, which resulted in a semidwarf phenotype having reduced root and hypocotyl lengths (D. Moura and C. A. Ryan, unpublished). Incorporation of 3H-thymidine into root cells in the presence and absence of RALF indicated that RALF was blocking cell division (incorporation of label). The label in the absence of RALF was identified in the root tips, mainly in the region between the root tip initials and the elongation zone primarily in cells of the vascular bundles, endodermis, and cortex, while no label was found in the initial cells, root epidermis cells, or lateral root cap cells. The labeling was strongly inhibited by treating the root tips with RALF. The biological roles of RALF peptides have only just begun to be understood. The powerful effects of the peptide on cell division and elongation in roots is only a preview of the roles the various gene family members play in their differential gene expression in different tissues of the plant. As more knowledge accumulates concerning the physical, biochemical, and biological properties of RALF family members, their roles will emerge within the broader context of plant growth and development.

Acknowledgment Research reported as unpublished data was supported by National Science Foundation Grant IBN 0090766 and the Charlotte Y. Martin Foundation. D. S. Moura is a recipient of a Young Researcher Grant from FAPESP, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo.

36 / Chapter 7 References [1] Birnbaum K, Shasa DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN. A gene expression map of the Arabidopsis root. Science 2003;302:1956–60. [2] Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B. Cellular organisation of the Arabidopsis thaliana root. Development 1993;119:71–84. [3] Escobar NM, Haupt S, Thow G, Boevink P, Chapman S, Oparka K. High throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata. Plant Cell 2003;15:1507–10. [4] Germain H, Chevalier E, Caron S, Matton DP. Characterization of five RALF like genes from Solanum chacoense provides support for a developmental role in plants. Planta 2005;220:447–54. [5] Haruta M, Constabel CP. Rapid alkalinization factors in poplar cell cultures. Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells. Plant Physiol 2003;131:814–23. [6] Meindl T, Boller T, Felix G. The plant wound hormone systemin binds with the N-terminal part to its receptor but needs the Cterminal part to activate it. Plant Cell 1998;10:1561–70. [7] Olsen AN, Mundy J, Skriver K. Peptomics, identification of novel cationic Arabidopsis peptides with conserved sequence motifs. In Silico Biol 2002;2:441–51.

[8] Pearce G, Moura DS, Stratmann J, Ryan CA. RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development. Proc Natl Acad Sci USA 2001;98: 12843–47. [9] Pearce G, Moura DS, Stratmann J, Ryan CA Production of multiple plant hormones from a single polyprotein precursor. Nature 2001;411:817–820. [10] Pearce G, Ryan CA. Systemic signaling in tomato plants for defense against herbivores. Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene. J Biol Chem 2003;278: 30044–50. [11] Ryan CA, Pearce G. Systemins: A functionally defined family of peptide signals that regulate defensive genes in Solanaceae species. Proc Natl Acad Sci USA 2003;100:14577–80. [12] Schaller A, Oecking C. Modulation of plasma membrane H+ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 1999;11: 263–72. [13] Scheer JM, Ryan CA. The systemin receptor SR160 from Lycopersicon esculentum is a member of the LRR receptor kinase family. Proc Natl Acad Sci USA 2002;99:9585–90. [14] Scheer JM, Pearce G, Ryan CA. LeRALF, a plant peptide that regulates root growth and development, specifically binds to 25 and 120 kDa cell surface membrane proteins of Lycopersicon peruvianum. Planta 2005;221:667–74.

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8 ROTUNDIFOLIA4: A Plant-Specific Small Peptide TAKAHIRO YAMAGUCHI AND HIROKAZU TSUKAYA

tion along the two major leaf axes: leaf-length and leaf-width. For example, ROTUNDIFOLIA3 and ANGUSTIFOLIA regulate leaf cell expansion along the leaflength and leaf-width axes, respectively [2, 3, 7]. In rot4-1D mutants, the lengths of the leaf blades and leaf petioles are severely shortened, while their widths are only slightly affected (Fig. 1). Closer observation shows that cell number, but not cell size, is greatly reduced along the leaf-length axis in rot4-1D leaves [4]. These observations suggest that ROT4 negatively regulates specific cell proliferation along the leaf-length axis when overexpressed. Genomic sequence analysis around the T-DNA insertion shows a small potential open reading frame (ORF) of 53 amino acids, which was overlooked by the gene annotation program. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that the mRNA corresponding to the small ORF accumulates at a higher level in the rot4-1D mutant than in the wild type. Moreover, overexpression of the full coding region of the ORF under the constitutive 35S promoter results in the rot4-1D phenotype in Arabidopsis. These results strongly suggest that the ORF is attributable to the ROT4 gene [4].

ABSTRACT Leaf shape is controlled by cell expansion and cell proliferation along the leaf axes. In Arabidopsis thaliana, a dominant mutation in the ROTUNDIFOLIA4 (ROT4) gene results in a short-leaf phenotype, with decreased cell proliferation along the leaf-length axis. ROT4 encodes a small peptide of 53 amino acids, which belongs to a plant-specific protein family of small peptides with 22 members in Arabidopsis. These members share a highly conserved 29-amino-acid domain (RTF domain) in the C-terminus, and the RTF domain alone, when overexpressed, produces a short-leaf phenotype. We review the identification of the ROT4 peptide and examine its putative roles in plant development.

DISCOVERY Peptides are thought to play important roles in the regulation of developmental and/or defense processes in both plants and animals. However, very few species of peptides have been identified in plants, primarily because it is difficult to isolate them and identify their genes using biochemical purification methods and conventional mutational analyses. ROT4 was discovered using a gain-of-function genetic screen for genes affecting leaf development in Arabidopsis [4]. In an activation-tagging pool constructed in the laboratory of Dr. Justin Goodrich (Institute for Cellular and Molecular Biology, Edinburgh, UK), one family displaying short, rounded leaves; floral organs; and relatively short stems was isolated. Segregation analysis in the T2 family indicated a single dominant mutation, which was linked to the enhancer-containing T-DNA insertion. Thus, the mutation was named rot4-1D [4]. In Arabidopsis, several genes have been identified in the polar control of cell expansion and cell proliferaHandbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The longest isolated ROT4 cDNA sequence is 430 bp and contains a 159-nt ORF encoding a putative 53amino-acid peptide [4]. There is no intron in the ROT4 gene, and the deduced ROT4 peptide has a molecular mass of 6.2 kDa. A database analysis has revealed 22 other putative Arabidopsis proteins homologous to the ROT4 peptide and designated ROT FOUR LIKE1-22 (RTFL1-22). One of the RTFL genes in Arabidopsis (DVL1, corresponding to RTFL18) produces a

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38 / Chapter 8

FIGURE 2. The RTF domain is conserved among RTFL members. A. Schematic diagram of the ROT4 peptide. Solid and shaded rectangles indicate protein-coding regions and RTF domains in ROT4, respectively. B. Alignment of the RTF domains from various angiosperms. ROT4 from Arabidopsis thaliana; OsRTFL1 from Oryza sativa. The rest are RTFL from Tm, Triticum monococcum; Sb, Sorghum bicolor; Hv, Hordeum vulgare; Zm, Zea mays; Pb, Populus balsamifera; Me, Manihot esculenta; Gm, Glycine max; Lj, Lotus japonicus; and Mt, Medicago truncatula. FIGURE 1. Overexpression of ROT4 specifically affects leaf length in Arabidopsis. A. Seedlings of the wild type (left) and rot4-1D (right) at 25 days. Scale bars = 10 mm. B. Leaves of the wild type (top) and rot4-1D (bottom) at 33 days. Left to right: a cotyledon pair; the first through the seventh foliage leaves; and a cauline leaf. Scale bars = 10 mm. Reproduced from [4] with permission.

phenotype similar to the rot4-1D mutant when it is overexpressed [8], suggesting that RTFL proteins share similar functions in Arabidopsis. RTFL genes are also found in other seed plants, such as Oryza sativa (rice), Glycine max (soybean), and Populus balsamifera (poplar). RTFL genes appear to form a multigene family in the genomes of these plant species. For example, 26 RTFL genes (OsRTFL1-26) have been identified in the O. sativa genome (Yamaguchi and Tsukaya, unpublished data), but no such genes have been detected in animals or fungal genomes, which suggests that RTFL genes are specific to plants. All RTFL proteins share a highly conserved 29-aminoacid region in the C-terminus called the RTF domain (Fig. 2; [4]). The RTF domain is rich in basic amino acids and lacks similarities to other proteins of known functions. The N-terminal region is less conserved among RTFL members, and no predictable signal peptide has been identified.

DISTRIBUTION OF THE mRNA RT-PCR analyses show that ROT4 mRNA is weakly expressed in flowers, shoots, and roots [4]. ROT4 is

expressed in leaves during the early developmental stages, but ROT4 transcripts are not found in mature leaves. More precise localization of ROT4 mRNA remains to be determined because clear signals cannot be obtained using in situ hybridization analyses in wildtype Arabidopsis plants. Preliminary analysis of a reporter β-glucuronidase (GUS) expression driven by the 1.2-kb 5′-region of the ROT4 translation start site shows strong GUS localization in the leaf petioles of young leaf primordia. GUS expression becomes weaker as the leaves develop (Narita and Tsukaya, unpublished data), indicating that the ROT4 gene functions in young leaf primordia, the site of active cell proliferation.

PROCESSING It has not been shown whether the ROT4 peptide is subject to protein processing. When the RTF domain alone is overexpressed in Arabidopsis, a phenotype similar to that of the rot4-1D mutant is produced, characterized by the presence of short leaves and floral organs [4]. This suggests that overexpression of the RTF domain alone is sufficient to cause the rot4-1D phenotype in Arabidopsis. Thus, it is plausible that the ROT4 protein is subjected to processing to produce a shorter peptide. In animals, dibasic residues are known to serve as a part of the recognition site for processing peptides from their precursors [1]. Highly conserved single or dibasic residues are found in all RTFL protein domains. Whether these residues are potential process-

ROTUNDIFOLIA4: A Plant-Specific Small Peptide / 39 ing sites cannot be determined without further studies to determine other factors, such as the molecular mass of the RTFL peptides in plants.

RECEPTORS Most small peptides are thought to function as peptide hormones, which are secreted into extracellular spaces and interact with specific receptors. However, the ROT4 protein does not have any predicted signal sequences at the N-terminus, suggesting that ROT4 is synthesized in the cytosol on free ribosomes and does not enter the secretory pathway. The same condition, which has not been found in animal peptide hormone precursors, occurs in those of tomato systemin and legume ENOD40s, in which no signal sequence is known to exist [reviewed in 6]. However, it has been demonstrated that tomato systemin is transported to the extracellular spaces by wounding stimuli and can interact with its receptor on cell surfaces. In contrast, ENOD40 acts as an intracellular modulator of the enzyme involved in sucrose synthesis. The ROT4 protein has been shown to intracellularly localize to the plasma membrane (Fig. 3; [4]), indicating that ROT4 is a membrane protein or a part of a protein complex localizing

FIGURE 3. ROT4-GFP localized to the plasma membrane. A. Localization of the ROT4-GFP fusion protein in onion epidermal cells. B. Localization of the ROT4-GFP fusion protein in cells treated with 0.8 M mannitol, showing the plasma membrane detached from the cell wall (plasmolysis). Fluorescent images (left) and images integrated with Nomarski images (right) are shown in each panel. Scale bars = 100 μm.

to the plasma membrane. The possibility that ROT4 is transported to the extracellular spaces and interacts with a receptor cannot be excluded. Further studies on in situ localization of the ROT4 mRNA and ROT4 peptide are needed to determine the molecular functions of ROT4 proteins.

BIOLOGICAL ACTIONS Loss-of-function phenotypes of RTFL members are unknown; a single loss-of-function mutant did not produce any detectable phenotype in either Arabidopsis or O. sativa [4]. These results strongly suggest a functional redundancy among RTFL members, as would be expected by the more than 20 members with similar protein sequences in each species. Further studies on the loss-of-function phenotypes of multiple RTFL genes are needed to better understand the role they play in wild-type leaf development. In contrast, ROT4 inhibits cell proliferation along the leaf-length axis when it is overexpressed in Arabidopsis. Similar protein activity would be conserved among RTFL members in seed plants because overexpression of several O. sativa RTFL genes in Arabidopsis results in the short-leaf phenotype of the rot4-1D mutant (Fig. 4; Yamaguchi and Tsukaya, unpublished data). It would be interesting to see if overexpression of the RTFL genes in O. sativa results in a similar short-leaf phenotype as in Arabidopsis, because the formation of grass leaves depends largely on cell proliferation in the leaf-length direction. How does ROT4 negatively regulate cell proliferation in leaf primordia? ROT4 is expressed in young leaf primordia but is not expressed in mature leaves. ROT4 mRNA is also detected in flowers, shoots, and roots. These regions, as well as young leaf primordia, are rich in tissues of actively dividing cells. Thus, it has been suggested that ROT4 may function to either trigger cell

FIGURE 4. Rice OsRTFL1 has the same effect on leaf length in Arabidopsis. A. A wild-type seedling. B. A 35S: OsRTFL1 seedling. Each plant was 14 days old. Scale bars = 10 mm.

40 / Chapter 8 cycle arrest or negatively regulate the progression of the cell cycle in actively dividing cells. Interestingly, the effect of ROT4 is most apparent in the lateral organs, including the petals and carpels, which are believed to be modified leaves; however, effects are relatively mild in stems and nonexistent in roots, even when constitutively expressed. Lateral organs show determinate development, while stems or roots show indeterminate growth, indicating that ROT4 is more strongly associated with determinate organ development. It is noteworthy that a signal that restricts cell proliferation is transmitted as a wave from the tip to the base of leaves [5]. If the ROT4 peptide is involved in such a signal transduction, ectopic ROT4 function may result in decreased cell numbers along the leaf-length axis. However, because it is localized to the plasma membrane, ROT4 may participate in the indirect control of the cell cycle through signals transmitted from a membrane complex in which it is involved. Because the RTFL family is specific to plants, this signal transduction may have evolved to control cell proliferation in a plant-specific manner. Understanding the molecular function of the RTFL peptide in controlling the cell cycle will elucidate the evolutionary mechanisms of multicellular organs in plants.

References [1] Harris RB. Processing of pro-hormone precursor proteins. Arch Biochem Biophys 1989; 275:315–333. [2] Kim GT, Shoda K, Tsuge T, Cho KH, Uchimiya H, Yokoyama R, Nishitani K, Tsukaya H. The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation. EMBO J 2002; 21:1267– 1279. [3] Kim GT, Tsukaya H, Uchimiya H. The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells. Genes Dev 1998; 12:2381–2391. [4] Narita NN, Moore S, Horiguchi G, Kubo M, Demura T, Fukuda H, Goodrich J, Tsukaya H. Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana. Plant J 2004; 38:669–713. [5] Nath U, Crawford BC, Carpenter R, Coen E. Genetic control of surface curvature. Science 2003; 299:1404–1407. [6] Ryan CA, Pearce G, Scheer J, Moura DS. Polypeptide hormones. Plant Cell 2002; Suppl:S251–S264. [7] Tsuge T, Tsukaya H, Uchimiya H. Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 1996; 122:1589– 1600. [8] Wen J, Lease KA, Walker JC. DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development. Plant J 2004; 37:668–677.

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9 The S-Locus Cysteine-Rich Peptide SCR/SP11 SUSHMA NAITHANI, DANIEL RIPOLL, AND JUNE B. NASRALLAH

common feature—namely the inhibition of selfpollination. The information provided here relates specifically to the SI system that operates in the Brassicaceae family, and SCR homologs are not expected to be present in families that use a different SI system [18]. The SI system of Brassicaceae is controlled by a single locus, the S (Sterility) locus (see [10, 18, 34] for recent reviews). In self-incompatible members of the Brassicaceae [such as Brassica oleracea, B. rapa (syn. campestris), B. napus, Raphanus sativus, and Arabidopsis lyrata], pollinations between plants that express the same S-locus variant are prevented by arrest of pollen tube development at the surface of the epidermal cells of the stigma, which are located at the tip of the female reproductive apparatus. Previous analysis of SI had indicated that the SI specificity-determining S locus, although behaving genetically as a single Mendelian locus, was in fact a complex locus consisting of at least two genes, one encoding the stigma specificity determinant and the other encoding the pollen specificity determinant. Molecular analysis of the S haplotype had already identified the highly polymorphic S-locus receptor kinase (SRK) gene as the determinant of SI specificity in the stigma [31, 33], and detailed analyses of DNA flanking the SRK gene in various S haplotypes were initiated through long-range physical mapping coupled with transcriptional and sequence analyses [3, 5, 26, 32]. In 1999, Schopfer et al. provided the first experimental proof through loss- and gain-of-function experiments that SCR was required for SI and that it indeed represented the pollen determinant of SI specificity [26]. A self-compatible γ-ray-induced mutant of B. oleracea that exhibited a pollen-specific defect in the SI response was found to lack SCR transcripts [26]. In addition, transformation of an S2S2 plant with an SCR6 allele conferred S6 specificity on the pollen of transgenic plants [26]. Subsequent verification of these results was provided by additional transgenic experiments [29], as well as

ABSTRACT The S-locus cysteine-rich protein SCR, also known as SP11, is a basic protein of approximately 6 kDa, which was identified in members of the Brassicaceae that exhibit self-incompatibility (SI). SI is a genetic system that prevents self-pollination by disrupting the early events of the interaction between pollen grains and the epidermal cells of the stigma, a specialized structure that caps the female reproductive apparatus. SCR is genetically and physically tightly linked to the gene for its receptor, the S-locus receptor kinase (SRK) gene, which encodes a single-pass transmembrane serine/ threonine kinase. Together, these two genes comprise the SI specificity-determining S-locus haplotype, with the pollen coat-localized SCR and the stigma epidermisexpressed SRK functioning as the pollen and stigma determinants of SI specificity, respectively. Like its receptor SRK, SCR is highly polymorphic, but SCR variants adopt a conserved overall three-dimensional structure consisting of an α helix and a triple-stranded antiparallel β sheet stabilized by four intramolecular disulfide bonds. SCR and SRK exhibit allele-specific interactions. Upon self-pollination, the binding of SCR to its cognate SRK (encoded within the same S haplotype) triggers a signal transduction cascade within the stigma epidermal cell that leads to inhibition of pollen hydration, germination, and tube growth.

DISCOVERY OF SCR The S-locus cysteine-rich protein SCR/SP11 (herein designated SCR) was discovered in a search for the pollen determinant of self-incompatibility in the Brassicaceae (crucifer) family. Self-incompatibility (SI) is a generic term that encompasses several evolutionarily and mechanistically divergent systems that share one Handbook of Biologically Active Peptides

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42 / Chapter 9 pollination bioassays in which SCR protein, either chemically synthesized or expressed in E. coli, was applied to stigmas and found to activate the SI response in an allele-specific manner [11, 36]. And most recently, transformation of at least one accession of the selffertile Arabidopsis thaliana with A. lyrata SRKb and SCRb genes demonstrated that these two genes are sufficient to confer self-incompatibility in a self-compatible species [20, 21].

SCR Sequence Polymorphism SCR alleles were first cloned from Brassica species (B. rapa and B. oleracea). DNA sequence is now available for 17 B. oleracea, 23 B. rapa, 2 B. napus, and 7 Raphanus sativus SCR variants (http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=PubMed). Additionally, two SCR alleles have been cloned from Arabidopsis lyrata [12], a self-incompatible close relative of the self-fertile model plant A. thaliana, which diverged from Brassica ∼15 to 20 million years ago [40]. The total number of SCR alleles within a particular species is predicted to be much higher, since each S haplotype must contain an

SCR variant and S haplotypes can number as many as 100 in one species. Consistent with their SI recognition functions, both SCR and its receptor SRK are highly polymorphic genes that must coevolve to maintain their allelespecific interaction. The diversification of Brassica S haplotypes is believed to have taken place over 20 to 40 million years ago [37], before speciation in the crucifer family, and S haplotypes have been grouped into two classes on the basis of sequence similarity of their SRK alleles. SCR alleles exhibit a particularly high level of intraspecific sequence divergence as illustrated in Fig. 1 for a representative sample of SCR variants from Brassica species and A. lyrata. In Brassica class-I S haplotypes, the mature SCR proteins share between 20 and 76% amino acid similarity, while their predicted signal sequences are more highly conserved [26, 39]. In contrast, Brassica class-II SCR proteins exhibit a relatively high degree of similarity (63 to 94%) in the sequence of both their predicted signal peptide and mature forms [27]. When all available SCR sequences are aligned, it is clear that only eight cysteine residues (designated C1 to C8, the glycine in GxC2, and an aro-

Brassica Class-I Bo S3(CAC19880.1) Bo S6(AF195625_1) Br S8(AF195627_1) Bo S13 (AF195626_1) Br S21 (BAA96392.1) Br S32 (BAA96394.1) Br S45 (BAA96401.1) Br S52 (BAA92247.1) Bn S910(CAC80853.1)

mrkatiyall-cflfivssrgqevea-------NVMKNCPIQFNLG----GQCGNSGGDA-CVEEYNRKKKKKKIF--CSCGGVRVGQ------CCCIV mksa-iyal--cfiflvsshgqevea-------NLKKNCVGKTRLP----GPCGDSGASS-CRDLYNQTEKTMPVS—-CRCVPTG----R----CFCSL--CK mksav-yall-cfifivsghiqelea-------NLMKRCTRGFRKL----GKCTTLEE-EKCKTLYPRGQ--------CTCSDSKMNTHS----CDCKS--C mksav-yall—cfifivsghiqevea-------NLMMPCGSFMF------GNCRNIGARE-CEKLNSPGKRKPSH---CKCTDTQMGTYS----CDCKL--C -----------ifi-iisshfq-vea----------KPCADTFP------GDCRNGGN-ERCAISFSSYKKRKASN--CQCRP-YDDKKRL---CDCE---C -----------ifv--vsihvqgvea-------NPTKLCRGSVT-SR---GVCDNSGV-QRCVTEFS--KKIDKDPRLCSCICRHHEGRRF---CPCECK-C m-----------fi-i-sshgqgvea-------DLRKECNGYTQLS----GPCPNLGG-EACANRYEIRAKKR--PRSCNCNDVDEVG-F----CHCSL--CK mksvl-yall-cfifivsshaqdvea-------NLMNRCTRELPFP----GKCGSSEDGG-CIKLYSSEKKLHPS-R-CECEPRYKA--RF---CRCKI--C mkspi-ytllwviliiv-shfq-vea-------NTMKRCNQTFK------GDCQNNGNQL-CKDSYWNVLKKRAYN—-CTCE-RFDGKQRV---CKCNLREC

Class II Bo S2(BAB86354.1) Bo S5(BAB86355.1) Br S29 (BAB86356.1) Br S40 (BAB86357.1) Br S44 (BAB86358.1) Br S60 (BAB86353.1)

mryatsiynfltkihylcfifwtltyvq(al)dvgpLECPEGVAKSGPIRGSCLNSTSAA-CQKHFGQNVT----N-—CLCFAFSKHNRGRIN-CYC----CKVKS mryatyifffltkihylcfifltltyvq(al)dvgaWKCPGAVAKADNITGTCVNSVSED-CQRYVGQNVN----N-—CLCLNFSKHNRGRIT-CYC----CKVKS mryatsifffltkihylcfifltltsvq(al)dvgaRNCPEGIAKSNDVIGTCLNTKSRD-CQKHFGPNVT----N—-CLCYPFSTHNRVRIT-CYC----CKVKS mryatyiyffltkihylcfifltltyvq(al)dvgaWKCPGAVAKADNITGTCVNSVSED-CQRFVGPNVN----N—-CLCLKFSEHNRGRIT-CYC----CKVKS mryatsiynfltkihylcfifwtltyvq(al)DVGPLECPKGVAESGPIRGSCLNSTSAA-CKTHFRQNVT----N—-CLCINFSNHNRGRIN-CYC----CKVKS mryatsiytfltnihylcfifliltyvq(al)dvgaWKCPEGIVYPSPISGRCINSRSTE-CKKHYEVEGQNVT-N—-CRCDTYSMQNPARIT-CYC----CKVKS

Arabidopsis Al Sa(BAB40984.1) Al Sb(BAB40985.1)

MRCSVLFVVSYVIMSLLISHVQG------MEDQKWKKVCNLEGNFP----GRCVGNGDEQ-CKRDLTEDGNNP-SK--CRCRFRAGRRH-----CRCIYEVFGM MRNATFFIVFYVFISLVLSNVQDVTA------QKNK--CMRSEMFPT---GPCGNNGEET-CKKDFKNIYRTPIQ---CKCLDKYDFARL----CDCRF--C C1

GxC2

C3xxxY/F

C4 C5

C6 C7

FIGURE 1. Multiple sequence alignment of the predicted amino-acid sequences of SCR variants from various S haplotypes of different Brassicaceae species. The signal peptide sequence for each SCR variant is shown in lowercase letters. The SCRs from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn) are arranged into class-I and class-II alleles, and two variants from Arabidopsis lyrata are grouped together. Residues that are conserved in most SCR variants within a group are shown in bold. Residues that are conserved among SCRs in the various groupings are underlined, and the consensus sequence motifs associated with some of these residues are shown below the alignment. In the class-II variants, arrows point to the predicted signal peptide cleavage sites in the proteins predicted from two alternative Brassica class-II SCR transcripts [27]. One form encodes an open reading frame of 95 amino acids that is predicted to produce a mature SCR protein of 66 amino acids after removal of the signal peptide (left arrow). The second form encodes a 93-amino-acid protein that lacks 2 amino acids (“AL” shown in parentheses), which would produce a mature SCR protein of 61 amino acids after cleavage of the signal peptide at an alternative cleavage site (right arrow).

C8

The S -Locus Cysteine-Rich Peptide SCR/SP11 matic residue in C3xxxY/F are conserved in most SCR variants (Fig. 1).

Criteria for Identification of SCR Alleles Because of the extensive polymorphism of SCR variants and the existence of SCR-like genes in the genomes of Brassicaceae [38], identification of a cloned sequence as an SCR allele is not straightforward. Linkage to the S locus and SRK is the major and essential criterion for identifying an SCR-like sequence as a bona fide new SCR allele. Accordingly, the isolation of a candidate SCR sequence must be verified by genetic analysis of plant populations that segregate for different S haplotypes or by physical linkage to SRK. When SCR alleles are sought from a new Brassicaceae species, the isolation of SCR alleles requires the cloning of the S locus from large-insert genomic libraries with subsequent “chromosome walking” from SRK to the SCR gene as was performed for Arabidopsis lyrata [12]. This already laborious process is made even more complicated by the fact that S haplotypes within one species exhibit extensive structural heteromorphism, a feature that is thought to contribute to the apparent reduced recombination in the S-locus region and the maintenance of the tight linkage of the SRK-SCR gene pair [1]. Both the orientation of SCR relative to SRK and its physical distance from SRK can vary among S haplotypes [1, 12, 17]. In Brassica, S haplotypes range from ∼10 kb to >200 kb in size, and they also differ in their complement of retroelements and haplotype-specific intergenic sequences [1, 17]. Additionally, some S haplotypes contain partial or complete duplications of the SI-specificity genes. For example, two identical copies of SCR are found in the Sb haplotype of A. lyrata [12], and three nonidentical copies were reported in the S15 haplotype of B. rapa [28].

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of SCR [21]. It is currently not known if any intact SCR genes exist in A. thaliana.

STRUCTURE OF THE PRECURSOR mRNA/GENE Comparison of SCR genomic and cDNA sequences indicates that the SCR gene consists of two exons separated by an intron. The first exon contains a predicted signal sequence of 29–32 amino acids, and the second exon contains an open reading frame of 50–66 amino acids that corresponds to the mature SCR protein [14, 26, 28, 35]. The size of the intron varies among SCR alleles from 0.3 kb in B. rapa SCR12 to 4.1 kb in SCR8 [26, 35]. A single ∼450-bp processed SCR transcript is observed for some S haplotypes that contain either one copy of the SCR gene [26, 32] or two identical SCR genes (e.g., the A. lyrata Sb haplotype [12]). However, more than one SCR transcript has been reported for the S15 haplotype of B. oleracea, which contains three nonidentical tandem copies of SCR (designated S15-SP11a, S15-SP11b, and S15SP11b′) [28]. Only the first two of these genes are transcribed to produce transcripts of two different sizes, a 0.65-kb transcript resulting from cotranscription of S15SP11a and exon 1 of S15-SP11b and a less abundant 1.4-kb transcript containing the full-length S15-SP11a and S15SP11b sequences and exon1 of S15-SP11b′ [28]. Additionally, two SCR transcripts generated by alternative splicing have been detected for some class-II SCR alleles (e.g., S29, S40, S60 of B. rapa) [27], and these transcripts are predicted to encode two mature protein forms differing by five amino acids (Fig. 1). The biological significance, if any, of these alternative transcripts is not known.

DISTRIBUTION OF THE mRNA SCR Pseudogenes in Self-Fertile Brassicaceae Not all strains within a predominantly selfincompatible species and not all species in the Brassicaceae are expected to have functional SCR genes because the loss of SI that underlies the switch to selffertility in this family is often accompanied by inactivation of S-locus genes. Therefore, the S loci of self-fertile species may contain only nonfunctional remnants of these genes. For example, the reference Col-0 accession of the self-fertile model plant A. thaliana contains only three truncated SCR sequences and an SRK pseudogene [12]. Furthermore, additional and presumably nonfunctional SCR variants might exist in A. thaliana because a significant number of other A. thaliana geographical isolates do not hybridize with Col-0 SCR, suggesting that they carry either deletions or highly diverged versions

All SCR alleles exhibit anther-specific expression, although the details of this expression vary with the allele being studied. In situ hybridization and reporter gene analyses have shown that all SCR alleles examined to date are expressed in the tapetum [12, 25, 35], and a subset of SCR alleles, such as the Brassica class-I SCRs and the A. lyrata SCRb allele, are also expressed in microspores [12, 25, 27, 35]. SCR expression in the tapetum is likely to be the biologically relevant site of expression: It is clearly sufficient for SI, and it is consistent with the localization of SCR protein in the pollen coat and with the genetic control of pollen SI phenotype in the Brassicaceae. The tapetum is a layer of nurse cells that lines the anther cavities in which pollen grains develop, and it serves as a source of many components of the pollen coat. The tapetum degenerates at late

44 / Chapter 9 stages of pollen maturation, and as a result, tapetumexpressed SCR transcripts are lost at late stages of anther development. Furthermore, the tapetum is derived from sporophytic (i.e., 2N) cells of the anther. Expression of SCR in the tapetum thus explains the sporophytic control of SI in the Brassicaceae, in which the SI phenotype of a pollen grain is controlled by the diploid genotype of its parental plant rather than by its own haploid genotype [18]. Thus, the pollen grains produced by an S1S2 plant will be phenotypically S1S2 (assuming codominance of S1 and S2) because SCR1 and SCR2 are expressed in the tapetum and the SCR1 and SCR2 proteins will become incorporated in the pollen coat of all pollen grains, whether they carry the SCR1 or SCR2 allele. A peculiarity of the SCR gene is that its expression can be influenced by other SCR alleles in heterozygous anthers [14, 27]. Some SCR alleles exhibit genetic interactions of dominance-recessiveness [18]. For example, in Brassica, class-II SCR alleles are generally recessive to class-I SCR alleles, and in A. lyrata SCRa is recessive to SCRb. In situ hybridization has shown that the recessive SCR allele is silenced in the presence of the dominant SCR allele, leading to the absence of recessive SCR transcripts and protein in heterozygous anthers [14, 27]. The mechanism of this allele-specific silencing is not known.

PROCESSING OF THE SCR PROTEIN SCR variants differ in both length and amino acid composition (Fig. 1). Different SCR alleles predict open reading frames that range from 71 to 95 amino acids with a predicted signal peptide at their N-terminus. For example, the mRNA sequence of the B. rapa SCR8 allele predicts a 74-amino-acid protein of ∼8 kDa, but the mature SCR protein extracted from the pollen coat was determined by mass spectrometric analysis to be 5.716 kDa, which corresponds to 50 amino acids [36]. The primary SCR translational product is therefore processed by removal of the signal sequence that targets the protein for secretion. Although little biochemical characterization has been performed, the SCR protein is not apparently further processed or modified because SCR variants expressed in E. coli [11] or synthetic SCR [36] exhibit biological activity in pollination bioassays.

THE SCR RECEPTOR The receptor for SCR, SRK (S-locus receptor kinase), is encoded by a gene located within the S locus in tight genetic and physical linkage to SCR. SRK mRNA levels attain their highest levels in stigmas just before flower

opening, which correlates with the onset of SI in the developing stigma [31]. The full-length SRK transcript encodes a protein that is expressed exclusively in the stigma epidermal cells and is an integral plasma membrane protein [30]. SRK is composed of a ligand-binding region located extracellularly [15], followed by a single transmembrane domain and a cytoplasmic serine/threonine kinase. Although the structure of SRK has not been solved, sequence-based searches indicate that the SRK extracellular ligand-binding domain has a modular structure (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi). Additionally, statistical analyses of multiple sequence alignments of the SRK ectodomain have identified three hyper-variable regions and a less variable C-terminal region [13, 22], which are thought to be targets for positive selection and therefore to contain residues involved in ligand recognition. However, the functional role of these hypervariable regions and of the various modules in the SRK ectodomain remains to be tested. SRK variants produce alternative mRNA transcripts [31] that produce a truncated protein equivalent to the SRK ectodomain (eSRK) in addition to the full-length SRK [7]. The physiological significance of the soluble eSRK, which is predicted to be secreted into the wall of stigma epidermal cells, is not known. Interestingly, in Brassica, most S haplotypes contain a third gene, the S-locus glycoprotein (SLG) gene, which shares a high degree of sequence similarity with the SRK ectodomain sequence and is coordinately regulated with SRK [19, 31]. The abundant SLG is expressed specifically in the stigma epidermis and is therefore similar to the eSRK. While SRK is clearly the sole determinant of SI specificity in the stigma, SLG is required for the accumulation of some SRKs to physiologically relevant levels [6], and it can enhance the SI response in SRK transformants [33]. The eSRKs might have a similar role to that of SLG.

STRUCTURE The structure of a synthetic version of the SCR variant from the S8 haplotype, S8 -SP11, has been determined [16], and modeling of the three-dimensional structures of several SCR variants [4] predicts that all SCRs adopt a similar overall structure despite their extensive sequence divergence (Fig. 2). The NMR structure of S8 -SP11 [16] suggests that the protein folds into a compact α/β barrel structure made of a twisted threestranded β-sheet backed by another layer formed by an α-helix with flanking loop regions. Four intramolecular disulfide bonds stabilize the structure. The disulfide bridges that link C2 to C5, C3 to C6, and C4 to C7 form the hydrophobic core of the molecule, while the C1–C8

The S -Locus Cysteine-Rich Peptide SCR/SP11

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FIGURE 2. Predicted conservation of secondary structure in SCR variants. A. Structure-based sequence alignment of SCR variants from B. rapa (Br) S52, B. oleracea (Bo) S2 and S5, and A. lyrata (Al) Sa and Sb. The NMR structure (model 1) of the B. rapa S8 variant [16] (PDB code: 1UGL) was used as a template. The secondary structure of the experimental model was computed using the method of Kabsch and Sanders [9]. The rectangles depict the α-helices, and the arrows show the β-strands. Hydrophilic residues are depicted in dark gray and hydrophobic residues in light gray. The conserved C1–C8 residues are shown above the alignment. B. Three-dimensional (3D) models of the SCR variants encoded by the Brassica S52 and S2 and the A. lyrata Sa, and Sb haplotypes. These predicted models were generated with the program MODELLER [23] based on the S8 SCR NMR structure (PDB code: 1UGL) determined by Mishima et al. [16] and the alignments given in panel A. The 3D models show the N/C-termini, the location of the conserved cysteine residues (C1–C8) with disulphide linkages and the glycine (G) in the GxC2 motif. The α-helix is highlighted and β-sheets depicted by are arrows. The aromatic residue in the C3xxxY/F motif found in most SCRs is shown (stick representation) for SCR variants of S52 (Y28), S2 (F30), and Sb (F28). SCRa contains a lysine (L27) at the corresponding position. (See color plate.)

bridge tethers the N- and C-termini. This structure is similar to that of plant defensins, with the difference being that the edges of the loop region between the α-helix and β2 strand are extensively stabilized by disulfide bonds and hydrophobic packing in S8 -SP11 [16]. The aromatic (Y/F) residue that is conserved in many, but not all, SCRs (Fig. 1) appears to have a key role in stabilizing this loop, in part through its hydrophobic contact with C7. The conserved glycine in the GxC2

motif apparently facilitates formation of a stable type-II β-turn in the flanking segment, connecting the β1 strand to the α-helix.

Structure-Function Studies of SCR Structure-function studies of SCR variants have two goals: (1) to determine which residues, especially those conserved in most SCRs, are important for SCR

46 / Chapter 9 function; and (2) to identify sequences that determine recognition specificity. Working with proteins encoded by the S6 and S13 haplotypes of B. oleracea, SCR sequence variants were generated by site-directed mutagenesis of individual residues in SCR6 or by swapping one or more regions between SCR6 and SCR13 [4]. The mutated or chimeric SCRs were expressed in E. coli and tested in vitro for binding to the ectodomains of SRK6 and SRK13 and in vivo for activation of the SI response on stigmas [4]. These studies showed that SCR function was abrogated by mutations of the conserved cysteine residues and the tyrosine residue in the C3xxxY/F motif, probably because these mutations disrupt SCR structure. Surprisingly, mutation of the glycine residue in the GxC2 motif was tolerated. Analysis of SCR6-SCR13 chimeras showed that SCR13 specificity resides in four contiguous amino acids within the C5–C6 loop [4]. However, the specificitydetermining residues of SCR6 could not be identified by this approach, possibly because noncontiguous residues in this variant determine specificity. These results indicate that residues imparting specificity to SCR can be arranged differently in different SCR variants. This conclusion is supported by in vivo studies using transgenic plants, which suggested that the C2–C3 and C5– C6 regions harbor the specificity determinants of the SCR variant encoded by the S52 haplotype of B. rapa, with some contribution from other regions [24]. Taken together, these studies point to the C5–C6 loop region, rather than the C3–C4 region suggested previously by Mishima et al. [16], as an important region for recognition specificity, in at least two SCR variants.

BIOLOGICAL ACTIONS SCR is the pollen determinant of specificity in the SI response, and it functions as a ligand for the stigma SRK receptor. Although the SCR protein is located in the pollen coat, the outermost surface of the pollen grain, which makes initial contact with the stigma, the site of SCR activity is at the plasma membrane of the stigma epidermis. Immunoelectron microscopic images show that, upon pollen-stigma contact, the SCR peptide is translocated from the pollen coat to the stigma epidermal cell surface and subsequently diffuses across the cell wall [8]. It then presumably reaches the plasma membrane, where its specific interaction with its SRK receptor determines the fate of a pollen grain. In selfpollination, the binding of SCR to its cognate “self” SRK results in receptor phosphorylation [2, 36] and activation of a poorly understood signal transduction pathway within the stigma epidermal cell, resulting in inhibition of pollen tube growth. In cross-pollination, SCR cannot bind or activate “non-self” SRK (i.e., an SRK encoded

in an S haplotype different from that which encodes SCR), the signal transduction pathway is not activated, and pollen tube growth proceeds. Thus, SCR may be thought of as the molecular tag on pollen that allows the stigma epidermal cell to perceive, through its SRK, a pollen grain as being a “self” grain that must be arrested and prevented from hydrating, elaborating a pollen tube, and effecting fertilization.

References [1] Boyes, D. C., Nasrallah, M. E., Vrebalov, J., Nasrallah, J. B. The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin. Plant Cell. 1997;9:237–47. [2] Cabrillac, D., Cock, J. M., Dumas, C., Gaude, T. The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature. 2001;410:220–23. [3] Casselman, A. L., Vrebalov, J., Conner, J. A., Singhal, A., Giovannoni, J., Nasrallah, M. E., Nasrallah, J. B. Determining the physical limits of the Brassica S locus by recombinational analysis. Plant Cell. 2000;12:23–33. [4] Chookajorn, T., Kachroo, A., Ripoll, D. R., Clark, A. G., Nasrallah, J. B. Specificity determinants and diversification of the Brassica self-incompatibility pollen ligand. Proc Natl Acad Sci U S A. 2004;101:911–7. [5] Cui, Y., Brugiere, N., Jackman, L., Bi, Y. M., Rothstein, S. J. Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines. Plant Cell. 1999;11:2217–31. [6] Dixit, R., Nasrallah, M. E., Nasrallah, J. B. Post-transcriptional maturation of the S receptor kinase of Brassica correlates with co-expression of the S-locus glycoprotein in the stigmas of two Brassica strains and in transgenic tobacco plants. Plant Physiol. 2000;124:297–311. [7] Giranton, J. L., Ariza, M. J., Dumas, C., Cock, J. M., Gaude, T. The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea. Plant J. 1995;8:827–34. [8] Iwano, M., Shiba, H., Funato, M., Shimosato, H., Takayama, S., Isogai, A. Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa. Plant Cell Physiol. 2003;44:428–36. [9] Kabsch, W., Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22:2577–637. [10] Kachroo, A., Nasrallah, M. E., Nasrallah, J. B. Self-incompatibility in the Brassicaceae: receptor-ligand signaling and cell-to-cell communication. Plant Cell. 2002;14 Suppl:S227–38. [11] Kachroo, A., Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Allele-specific receptor-ligand interactions in Brassica selfincompatibility. Science. 2001;293:1824–6. [12] Kusaba, M., Dwyer, K., Hendershot, J., Vrebalov, J., Nasrallah, J. B., Nasrallah, M. E. Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. Plant Cell. 2001;13:627–43. [13] Kusaba, M., Matsushita, M., Okazaki, K., Satta, Y., Nishio, T. Sequence and structural diversity of the S locus genes from different lines with the same self-recognition specificities in Brassica oleracea. Genetics. 2000;154:413–20. [14] Kusaba, M., Tung, C. W., Nasrallah, M. E., Nasrallah, J. B. Monoallelic expression and dominance interactions in anthers of self-incompatible Arabidopsis lyrata. Plant Physiol. 2002;128: 17–20.

The S -Locus Cysteine-Rich Peptide SCR/SP11 [15] Letham, D. L. D., Blissard, G. W., Nasrallah, J. B. Production and characterization of the Brassica oleracea self-incompatibility locus glycoprotein and receptor kinase in a baculovirus infected insect cell culture system. Sex Plant Reprod. 1999;12:179–87. [16] Mishima, M., Takayama, S., Sasaki, K., Jee, J. G., Kojima, C., Isogai, A., Shirakawa, M. Structure of the male determinant factor for Brassica self-incompatibility. J Biol Chem. 2003;278: 36389–95. [17] Nasrallah, J. B. Cell-cell signaling in the self-incompatibility response. Curr Opin Plant Biol. 2000;3:368–73. [18] Nasrallah, J. B. Recognition and rejection of self in plant selfincompatibility: Comparisons to animal histocompatibility. Trends Immunol. 2005;In press. [19] Nasrallah, J. B., Kao, T.-H., Goldberg, M. L., Nasrallah, M. E., A cDNA clone encoding an S-locus-specific glycoprotein from Brassica oleracea. Nature. 1985;318:263–67. [20] Nasrallah, M. E., Liu, P., Nasrallah, J. B. Generation of selfincompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata. Science. 2002;297:247–9. [21] Nasrallah, M. E., Liu, P., Sherman-Broyles, S., Boggs, N. A., Nasrallah, J. B. Natural variation in expression of selfincompatibility in Arabidopsis thaliana: implications for the evolution of selfing. Proc Natl Acad Sci U S A. 2004;101:16070–4. [22] Sainudiin, R., Wong, W. S., Yogeeswaran, K., Nasrallah, J. B., Yang, Z., Nielsen, R. Detecting site-specific physicochemical selective pressures: applications to the Class I HLA of the human major histocompatibility complex and the SRK of the plant sporophytic self-incompatibility system. J Mol Evol. 2005;60:315–26. [23] Sali, A., Potterton, L., Yuan, F., van Vlijmen, H., Karplus, M. Evaluation of comparative protein modeling by MODELLER. Proteins. 1995;23:318–26. [24] Sato, Y., Okamoto, S., Nishio, T. Diversification and alteration of recognition specificity of the pollen ligand SP11/SCR in selfincompatibility of Brassica and Raphanus. Plant Cell. 2004;16: 3230–41. [25] Schopfer, C. R., Nasrallah, J. B. Self-incompatibility. Prospects for a novel putative peptide-signaling molecule. Plant Physiol. 2000;124:935–40. [26] Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. The male determinant of self-incompatibility in Brassica. Science. 1999; 286:1697–700. [27] Shiba, H., Iwano, M., Entani, T., Ishimoto, K., Shimosato, H., Che, F. S., Satta, Y., Ito, A., Takada, Y., Watanabe, M., Isogai, A., Takayama, S. The dominance of alleles controlling selfincompatibility in Brassica pollen is regulated at the RNA level. Plant Cell. 2002;14:491–504. [28] Shiba, H., Park, J. I., Suzuki, G., Matsushita, M., Nou, I. S., Isogai, A., Takayama, S., Watanabe, M. Duplicated SP11 genes produce alternative transcripts in the S15 haplotype of Brassica oleracea. Genes Genet Syst. 2004;79:87–93.

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[29] Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F. S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A. A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol. 2001;125:2095–103. [30] Stein, J. C., Dixit, R., Nasrallah, M. E., Nasrallah, J. B. SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. Plant Cell. 1996;8:429–45. [31] Stein, J. C., Howlett, B., Boyes, D. C., Nasrallah, M. E., Nasrallah, J. B. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci U S A. 1991;88:8816–20. [32] Suzuki, G., Kai, N., Hirose, T., Fukui, K., Nishio, T., Takayama, S., Isogai, A., Watanabe, M., Hinata, K. Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa). Genetics. 1999;153:391–400. [33] Takasaki, T., Hatakeyama, K., Suzuki, G., Watanabe, M., Isogai, A., Hinata, K. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature. 2000;403:913–6. [34] Takayama, S., Isogai, A. Self-incompatibility in plants. Annu Rev Plant Biol. 2005;56:467–89. [35] Takayama, S., Shiba, H., Iwano, M., Shimosato, H., Che, F. S., Kai, N., Watanabe, M., Suzuki, G., Hinata, K., Isogai, A. The pollen determinant of self-incompatibility in Brassica campestris. Proc Natl Acad Sci U S A. 2000;97:1920–5. [36] Takayama, S., Shimosato, H., Shiba, H., Funato, M., Che, F. S., Watanabe, M., Iwano, M., Isogai, A. Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature. 2001;413:534–8. [37] Uyenoyama, M. K. A generalized least-squares estimate for the origin of sporophytic self-incompatibility. Genetics. 1995;139: 975–92. [38] Vanoosthuyse, V., Miege, C., Dumas, C., Cock, J. M. Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response. Plant Mol Biol. 2001;46:17–34. [39] Watanabe, M., Ito, A., Takada, Y., Ninomiya, C., Kakizaki, T., Takahata, Y., Hatakeyama, K., Hinata, K., Suzuki, G., Takasaki, T., Satta, Y., Shiba, H., Takayama, S., Isogai, A. Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L. FEBS Lett. 2000;473:139–44. [40] Yang, Y.-W., Lai, K.-N., Tai, P.-Y., Ma, D.-P., Li, W.-H. Molecular phylogenetic studies of Brassica, Rorippa, Arabidopsis and allied genera based on the internal transcribed spacer region of 18S–25S rDNA. Mol. Phylogenet. Evol. 1999;13: 455–62.

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10 Systemins GREGORY PEARCE, JAVIER NARVAEZ-VASQUEZ, AND CLARENCE A. RYAN

peptide was isolated from approximately 60 pounds of tomato leaves and identified as an 18-amino-acid peptide called systemin [21]. LeSys is the first peptide hormone to be identified and isolated from a plant tissue. It was chemically synthesized and, when supplied to young tomato plants through their cut stems, was half maximally active at about 40 fmol/plant [21]. 14C-labeled LeSys, when applied to wounds on leaves of tomato plants, was translocated through the vascular system [21]. That the peptide was a primary wound signal for the systemic induction of defense genes in tomato plants was established with antisense technology, which demonstrated that inhibition of the synthesis of the precursor of the peptide also blocked systemic wound signaling and defense against herbivore attacks [7, 15]. A more recent discovery in tobacco and tomato of several small hydroxyproline-rich glyocopeptides (HypSys peptides) that powerfully activate defensive genes in leaves has added a new level of complexity to the understanding of wound signaling [19, 20]. The peptides were discovered as a consequence of earlier observations that Le Sys was not found in leaves of tobacco plants, although leaves of young tobacco plants do exhibit a systemic wound-inducible synthesis of a tobacco trypsin inhibitor (TTI) [18]. TTIs are members of the potato tuber inhibitor II family of proteinase inhibitors [2, 10, 18] that are wound-inducible in tomato and potato leaves. Thus, the defense-signaling pathway for defensive genes that exists in tobacco bears many similarities to the signaling pathway found in tomato and potato, even though the peptide signal LeSys is missing. These observations led to a search for a systemic signal in leaves of young tobacco plants and to the discovery of the HypSys peptide signals [19]. The discovery of the tobacco peptide signals was facilitated by the development of a biological assay using suspension cultured cells that was much simpler and less

ABSTRACT Systemins are a family of functionally related peptide signals in plants of 15 to 20 amino acids in length, derived from precursors that activate defensive genes in response to herbivore and pathogen attacks [25]. Two subfamilies of nonhomologous peptides are included in the systemin superfamily, based on several structural and functional properties. Members of one subfamily are homologous to tomato systemin [21], now called LeSys (the name derived from Lycopersicon esculentum Systemin), with members in potato, pepper, and nightshade [3]. The precursors of this subfamily lack leader sequences and are synthesized in the cytosol and are not posttranslationally modified. The second subfamily members are hydroxyproline-rich glycopeptides (called HypSys peptides) that are derived from precursor proteins that have homologs in tobacco [19], tomato [20], petunia, nightshade, and potato [G. Pearce and C. A. Ryan, unpublished data). Members of each subfamily exhibit the following characteristics: They are all small peptides of about 15–20 amino acids each, are derived from larger precursor proteins, contain multiple proline and/or hydroxyproline residues, are active at low nM concentrations, appear to be receptor mediated, and the precursor genes are all activated by methyl jasmonate.

DISCOVERY OF SYSTEMINS The first identified member of the systemin family, now called LeSys, was discovered during a search for an unknown systemic wound signal that is generated in tomato and potato leaves in response to herbivore attacks or other mechanical wounding [4]. After nearly two decades of intense efforts to identify the “proteinase inhibitor inducing factor,” approximately 1 μg of the Handbook of Biologically Active Peptides

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50 / Chapter 10 time consuming than the assay with whole plants that was previously employed for the isolation of LeSys [21]. The new assay is based on an observation that the addition of LeSys to suspension-cultured tomato cells causes a rapid increase in medium pH [9, 26] (up to 1 pH unit/10 min) that is receptor mediated [27, 28]. The pH response is among the earliest responses to the interaction of LeSys with its receptor, resulting in the blockage of an ATPase-driven proton pump in the plasma membranes [26]. Addition of a few μL of a crude peptide fraction from tobacco leaves caused a strong alkalinization response, suggesting that tobacco leaf extracts contained a signal that mimicked the response of LeSys and might possibly be a peptide. By assaying fractions eluting from HPLC or other columns, two 18amino-acid glycopeptides composed of 18 amino acids each that contained multiple hydroxyproline residues were identified and characterized [19]. Both peptides cause a rapid activation of a 48 kD MAP kinase, similar to the activation of the 48 kD MAP kinase activated by LeSys in tomato cells [30], and both peptides are active at nM concentrations in inducing the synthesis of trypsin inhibitors when supplied to young excised tobacco plants through their cut stems [19]. Neither peptide exhibited homology with LeSys, but because of their similarities to tomato systemin in size, hydroxyproline contents (derived from prolines), and their signaling characteristics, they were called Tobacco Systemin I and II, here designated Nicotiana tabacum Hydroxyprolinerich Systemins, or NtHypSys I and II. The isolation of the HypSys peptides from tobacco systemins led to a search for similar peptides in tomato plants. Three peptides in addition to LeSys were isolated from extracts of tomato leaves [20]. All three are hydroxyproline-rich glycopeptides that exhibit limited sequence homology to the NtHypSys peptides. The tomato peptides, LeHypSys I, II, and III, respectively, are powerful inducers of protease inhibitor synthesis in tomato leaves [20]. The three peptides are composed of 18 (LeHypSys I), 15 (LeHypSys) II), and 20 (LeHypSys III) amino acids, and each contains regions with continuous sequences of from 5 to 11 amino acids that are composed of hydroxyprolines, prolines, serines, and threonines, flanked by charged amino acids.

STRUCTURES OF SYSTEMINS AND OF THEIR PRECURSOR cDNAs The LeSys Subfamily The systemin peptide LeSys peptide is not posttranslationally modified and has the following amino acid sequence: +AVQSKPPSKRDPPKMQTD−. Substitution of the penultimate threonine residue at position #17 of

LeSys LeproSys (200 aa) 1 18 LeSys AVQSKPPSKRD PPKMQTD

FIGURE 1. Box diagram of the LeproSys precursor of tomato leaf LeSys. The amino acid sequence of LeSys is shown below.

LeSys with an alanine totally abolishes activity while generating a powerful competitive inhibitor [17]. LeSys is derived from the C-terminus of a precursor protein of 200 amino acids [8], called LeproSys (Fig. 1). The localization of LeproSys in the cytoplasm was demonstrated using electron microscopy [14]. The 200 amino acids that comprise LeproSys are rich in charged residues, with 10% being aspartic acid, 17% glutamic acid, and 15% lysine, and a few hydrophobic residues. Regions rich in lysine and glutamic residues resemble KEK motifs [23] that are thought to be involved in protein-protein interactions. The precursor is localized in the cytosol and nucleus of phloem parenchyma cells of minor veins and midribs of leaves and in the bicollateral phloem bundles of petioles and stems of tomato plants [14]. The KEK-like motifs may have a role in anchoring the precursor protein in the cytosol. The LeproSys precursor gene, which is wound inducible, has 10 introns and 11 exons [8]. The first ten exons are organized in pairs, with the remaining exon encoding the LeSys peptide. The five exon pairs are homologous with each other, indicating that the gene is a product of several gene duplication-elongation events from a much smaller ancestral gene. The gene is wound-inducible in leaves, and its mRNA can be detected within 30 min after wounding [24], maximizing at about 3 h. It is considered an “early” gene, together with several signal pathway genes. This is in contrast to several defense-related genes, such as protease inhibitor genes, called “late genes,” whose synthesis begins at about 1–2 h after wounding and maximizes after about 8 h [1].

The LeHypSys Subfamily NtHypSys I and II peptides originate from a single preproprotein of 165 amino acids in length, including a signal sequence, with the NtHypSys I sequence near the N-terminus and the NtHypSys II sequence near the C-terminus [19]. Figure 2 shows a simplified box diagram of the NtproHypSys protein and the locations of the two NtHypSys peptides within the sequence. The primary amino acid sequences of the two peptides are shown, with the putative locations of the carbohydrate decorations. Neither peptide exhibits homology with LeSys, but the –OOS– motifs (O = hydroxyproline resi-

Systemins / I

NtpreproHypSys (165 aa)

II

Pentoses 1 18 NtHypSys I RGANLPOOSOASSOOSKE

9

1 18 NtHypSys II NRKPLSOOSOKPADGQRP

6

FIGURE 2. Box diagram of tobacco (Nicotiana tabacum) NtpreproHypSys, the precursor of the two NtHypSys I and II peptide defense signals. The amino acid sequences of the peptides are shown below. The number of pentose units of each peptide is shown in the right column.

dues that are posttranslational modifications of proline) are derived from the motif –PPS– that is found in LeSys [21]. The two new peptides have multiple regions that are contiguous hydroxyproline, serine, and threonine residues. These residues, flanked by charged amino acids, comprise over 50% of each peptide. Mass spectroscopy of the two peptides revealed that the attached carbohydrate moieties consist of pentose residues: nine in NtHypSys I and six in NtHypSys II [19]. The structural properties of NtHypSys I and II, including their leader sequences, hydroxyproline residues, and their carbohydrate decorations indicated that they are synthesized through the Golgi and secretory pathway. The presence of multiple signaling peptides contained in a single precursor is a characteristic of many animal peptide hormones, but the tobacco HypSys precursor protein provides the first example in plants. LeHySys peptides, like the tobacco peptides, are decorated with various numbers of pentose residues, but their identities and locations on the peptides have not been determined. The three tomato HypSys peptides are derived from a single precursor (Fig. 3). The amino

LepreproHypSys (146 aa)

I

III

II

1 18 RTOYKTOOOOTSSSOTHQ 1 20 LeHypSys II GRHDYVASOOOOKPQDEQRQ 1 15 LeHypSys III GRHDSVLPOOSOKTDOO LeHypSys I

Pentoses Variable 8-17 Variable 12-16 10

FIGURE 3. Box diagram of tomato (Lycopersicon esculentum) LepreproHypSys precursor of tomato HypSys I, II, and III peptide signals. The amino acid sequences of the peptides are shown below. Hydroxyproline (O), threonine (T), and serine (S) residues are in orange; charged amino acids are in blue; and neutral amino acids are in black. The number of pentose units of each peptide is shown in the right column.

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acid sequences of the LeHypSys peptides share limited amino acid sequence identities, indicating that they have resulted from gene duplication-elongation events during evolution from a common ancestral precursor. A comparison of the nucleotide and amino acid sequences of LeproHypSys with those of NtproHypSys indicate that they have evolved from a common ancestral protein [19, 20]. A 10-amino-acid sequence is present in the N-terminal region of each protein that is identical at eight positions. The nucleotide sequence similarities in this region were 90%. Multiple HypSys peptides have been isolated from petunia, nightshade, and potato [G. Pearce and C. A. Ryan, unpublished data] that were shown in each case to be derived from the single precursor proteins, homologous to the tomato and tobacco precursors. The peptides are active in low nM concentrations in the alkalinization assay, which was used to monitor the peptides during purification.

DISTRIBUTION OF SYSTEMIN SUBFAMILY mRNAs LeproSys has no paralogs, but orthologs are found in potato, nightshade, and pepper, members of the Solanoideae subfamily of the Solanaceae family [3]. The gene has not been detected in tobacco or petunia, members of the Cestroideae subfamily, or in species of any other plant families. The amino acid sequences of Sys peptides from tomato, potato [Solanum tuberosum (St)], nightshade [Datura stromonium (Ds)], and pepper [Capsicum annum (Ca)] are very similar, differing in only a few residues among them. The presence of a proSys gene in a species outside of the Solanaceae family has not been reported.

PROCESSING OF THE SYSTEMIN FAMILY PRECURSOR PROTEINS The processing enzymes that produce LeSys from LeproSys have not been identified, nor is it known if or how LeSys or LeproSys is transported to the apoplast from the cytoplasm [28]. Wound-inducible proteases have been identified in leaves [1], and they may have a role in the processing of LeproSys to LeSys and of LeproHypSys to LeHypSys I, II, and III. LeproHypSys is sequestered in the cell wall matrix [13] and is likely processed there in response to wounding. This suggests that either cytoplasmic proteases are mixed with the cell walls and cleave to the precursor or that specific proteases are synthesized in response to wounding that process the peptides from their precursors. Identification of the processing proteases and their locations will be important to resolving

52 / Chapter 10 whether LeproSys or LeSys is transported to the apoplast. The identification of the processing enzymes in tomato leaves may provide information that will be applicable to other peptide hormone precursors in plants as well.

THE LeSys RECEPTOR Le Sys interacts with a cell-surface receptor, called SR160, to initiate the defense signaling pathway in tomato plants [28]. SR160 was identified by photoaffinity labeling and purified to homogeneity. Binding of radiolabeled LeSys to the receptor was competed by nonradiolabeled LeSys, as well as by an inactive competitive analog and by suramin, a heterocyclic chemical that inhibits ligand-receptor interactions in animals [30]. SR160 is a 160 kDa, single span, receptor kinase, containing 21 leucine-rich repeats (LRR) in its extracellular domain [28]. The receptor binds to LeSys with a Kd of 0.17 nM and a Hill coefficient of 0.92. Treating cell cultures with methyl jasmonate for 24 h causes a threefold increase in binding activity, suggesting that the SR160 gene is up-regulated with other defenserelated genes that are activated in planta by jasmonates. The receptor is identical to the tomato BRI1 receptor that interacts with brassinolides (BL), which are general regulators of plant growth and development [11, 27]. A loss of function mutation (loss of BR signaling) in the tomato BRI1 gene is also severely limited in LeSys perception [27]. Transformation of tobacco plants with a constitutively expressed LeBRI1/SR160 gene resulted in the recovery of a tobacco plant with a gain in function of LeSys reception [27]. Although the HypSys peptides activate defense signaling as though they are receptor-mediated, a receptor for the HypSys subfamily peptides has not been identified.

SOLUTION CONFORMATIONS OF SYSTEMIN SUBFAMILY MEMBERS Structural features of all Systemin family members include charged amino acids flanking two tandem proline motifs (cf. Figs. 1–3). The tandem prolines are thought to cause the weak poly(L-proline) II, 31 helical conformation PP II (close) found in LeSys [31]. PPII conformations tend to form left-handed helices and are known to be involved in recognition events in animals [22]. All of the systemin family members have multiple proline or hydroxyproline motifs that are flanked by charged residues, providing compelling evidence that the PPII conformation may play an important role in receptor recognition.

BIOLOGICAL ACTIONS OF SYSTEMIN FAMILY MEMBERS Intracellular signaling in response to LeSys involves rapid early events including ion transport, the activation of MAP kinase, and phospholipase activities [9, 12, 26, 30], followed by the release of linolenic acid from chloroplast membranes and its conversion to jasmonates through the octadecanoid pathway [29]. Also involved in the defense signaling pathway are ethylene, abscisic acid, and hydrogen peroxide [6, 16, 24, 29]. Le proSys is synthesized in phloem parenchyma cells [14], while systemin signals the synthesis of jasmonic acid in nearby phloem companion cells [5, 29]. This indicates that systemin is behaving as a hormone in the classical sense. A primary role for systemin in plant defense against herbivory was established with transgenic tomato plants that constitutively express an antisense LeproSys gene, regulated by the CaMV 35S promoter. The antisense plants do not synthesize Le proSys or LeSys and are deficient in long-distance wound signaling. The plants cannot defend themselves against attacking insects in (or by) contrast to wild-type plants [15]. Tomato plants transformed with the LeproSys gene in its sense orientation overexpressed its own gene as well as 20 signaling and defense-related “early” and “late” genes [1]. This phenotype is thought to result from the synthesis of LeproSys in cells throughout the plants where it is abnormally processed to LeSys in the absence of wounding or herbivore attacks. The plants exhibit an increased resistance toward herbivory. The three LeHypSys peptides do not serve as systemic signals, since Le proSys antisense tomato plants are incapable of systemic signaling in response to wounding [27], but may be localized signals that amplify the jasmonate signal. Le proSys antisense plants exhibit a localized wound response, and the HypSys peptides may be part of the localized signaling. How the combined roles of LeSys and LeHypSys are coordinated during wounding is not fully understood, but the production of multiple defense signaling peptides at wound sites may have an important role in providing an early, strong synthesis of jasmonic acid. If the enzymes that process the peptide precursors were among the proteases that are inducible by wounding, then jasmonates would induce the production of both the precursors and the peptide signals as it moves along the vascular bundles, amplifying the levels of jasmonates. Both LeproSys and Le preproHypSys are localized in phloem parenchyma cells adjacent to companion cells [5, 14, 29] where synthesis of jasmonic acid takes place, and a cooperative interaction between these cells may be important in tomato plants to achieve a

Systemins / strong defense response [29]. Tobacco, which has a much weaker systemic response than tomato, does not synthesize systemin but may amplify jasmonic acid synthesis by the release of the HypSys peptides as part of an amplification mechanism for jasmonates. Understanding the complex interactions between Sys, HypSys, jasmonic acid, ethylene, and H2O2, will be a major challenge for future research on wound signaling.

Acknowledgments Research from the authors’ laboratory was supported by the National Science Foundation and by the Charlotte Y. Martin Foundation.

References [1] Bergey D, Howe G, Ryan CA (1996) Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc Natl Acad Sci USA 93:12053–12058. [2] Bryant J, Green T, Gurusaddaiah T, Ryan CA (1976) Proteinase inhibitor II from potatoes: Isolation and characterization of the isoinhibitor subunits. Biochemistry 15:3418–3423. [3] Constabel CP, Bergey DR, Ryan CA (1998) Prosystemin from potato, black nightshade, and bell pepper: Primary structure and biological activity. Plant Mol Biol 36:55–62. [4] Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175:776–777. [5] Hause B, Hause G, Kutter C, Miersch O, Wasternack C (2002) Enzymes of jasmonate biosynthesis occur in tomato sieve elements. Plant Cell Physiol 44:643–648. [6] Leon J, Roo E, Sanchez-Serrano JJ (2001) Wound signaling in plants. J Exp Bot 52:1–9. [7] McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Structure, expression and antisense inhibition of the systemin precursor gene. Science 255:1570–1573. [8] McGurl B, Ryan CA (1992) The organization of the prosystemin gene. Plant Mol Biol 20:405–409. [9] Meindl T, Boller T, Felix G (1998) The plant wound hormone systemin binds with the N-terminal part to its receptor but needs the C-terminal part to activate it. Plant Cell 10:1561–1570. [10] Miller EA, Lee MC, Atkinson AH, Anderson MA (2000) Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata. Plant Mol Biol 42:329–333. [11] Montoya T, Nomura T, Farrar K, Kaneta T, Yokata T, Bishop GJ (2002) Cloning the tomato curl3 gene highlights the putative dual role of the leucine-rich receptor kinase tBRI1/S160 in plant steroid hormone and peptide hormone signaling. Plant Cell 14:3163–3176. [12] Narváez-Vásquez J, Florin-Christensen J, Ryan CA (1999) Positional specificity of a phospholipase A2 activity induced by wounding, systemin and oligosaccharide elicitors in tomato leaves. Plant Cell 11:1–13. [13] Narváez-Vásquez J, Pearce G, Ryan CA (2005) The plant cell wall matrix harbors a precursor of defense signaling peptides. Proc Natl Acad Sci USA 102:12974–12977.

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[14] Narvaez-Vasquez J, Ryan CA (2004) The cellular localization of prosystemin: A functional role for phloem parenchyma in systemin signaling. Planta 218:360–369. [15] Orozco-Cardenas M, McGurl B, Ryan CA (1993) Transformation of tomato plants with an antisense prosystemin gene decreases resistance toward Manduca sexta larvae. Proc Natl Acad Sci US 90:8273–8276. [16] Orozco-Cardenas M, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide behaves as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin and methyl jasmonate. Plant Cell 13:179–191. [17] Pearce G, Johnson S, Ryan CA (1993) Structure-activity of deleted and substituted systemin, an eighteen amino acid polypeptide inducer of plant defensive genes. J Biol Chem 268:212– 216. [18] Pearce G, Johnson S, Ryan CA (1993) Purification and characterization from tobacco leaves of six small, wound inducible, proteinase iso-inhibitors of the potato inhibitor II family. Plant Physiol 102:639–644. [19] Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411:817–820. [20] Pearce G, Ryan CA (2003) Systemic signaling in tomato plants for defense against herbivores: Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene. J Biol Chem 278:30044– 30050. [21] Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253:895–897. [22] Rath A, Davidson AR, Deber CM (2005) The structure of “unstructured” regions in peptides and proteins: Role of the polyproline II helix in protein folding and recognition. Biopolymers 80:179–185. [23] Realini C, Rogers SW, Rechsteiner M (1994) KEK motifs: Proposed roles in protein-protein association and presentation of peptides by MHC Class I receptors. FEBS Lett 348:109–113. [24] Ryan CA (2000) The systemin signaling pathway: Differential activation of plant defensive genes. Biochem Biophys Acta 1477:112–121. [25] Ryan CA, Pearce G (2003) Systemins—A functionally defined family of peptide signals that regulate defensive genes in Solanaceae species. Proc Natl Acad Sci USA 100:14573–14577. [26] Schaller A, Oecking C (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11:263–272. [27] Scheer JM, Pearce G, Ryan CA (2003) Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene. Proc Natl Acad Sci USA 100:10144–10147. [28] Scheer JM, Ryan CA (2002) The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family. Proc Natl Acad Sci USA 99:9585–9590. [29] Schilmiller AL, Howe GA (2005) Systemic signaling in the wound response. Curr Opin Plant Biol 8:369–377. [30] Stratmann JW, Ryan CA (1997) Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors. Proc Natl Acad Sci (USA) 94:11085–11089. [31] Toumadje A, Johnson WC Jr. (1995) Systemin has the characteristics of a poly(L-proline) II type helix. J Am Chem Soc 117:7023–7024.

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11 Cationic Antimicrobial Peptides—The Defensins NIKOLINKA ANTCHEVA, IGOR ZELEZETSKY, AND ALESSANDRO TOSSI

the “thionins”—were known in plants since the early fifties [2], but it was only in the mid-nineties that the γ-thionins, based on their resemblance to the insect and mammalian defensins, were defined as “plant defensins” [11, 15]. A structurally quite different type of circularized peptide more recently isolated in monkeys was found to derive from the ligation of two truncated αdefensins and assigned the name “θ-defensin,” with reference to its cyclic structure [16, 24]. Finally, the recent discovery of a defensin-like peptide, plectasin, in a mold [13], with potent antimicrobial activity, underlines the antiquity of this peptide superfamily. The term defensin thus defines peptide families based on functional (host defense) and structural (compact β-structure) similarities, but does this also imply a phylogenetic relationship? There is evidence that mold, invertebrate animal, and plant defensins form an evolutionarily related group [5, 7, 21], and all the vertebrate defensins form another [31]. Whether these two groups also connect into a phylogenetically unified whole is controversial [31], although a relationship has been suggested between insect and β-defensins [12].

ABSTRACT The term defensin relates different families of host defense peptides (HDPs) in vertebrates, invertebrates, plants, and molds that display structural similarities based on a cystine stabilized antiparallel β-sheet core, with an N-terminal α-helical stretch in many members. Despite structural and functional similarities, invertebrate/plant and vertebrate defensins belong to two distinct phylogenetic groups, and whether a unified relationship exists is controversial. Most defensins show a direct, salt- and medium-sensitive antimicrobial activity in vitro, with varying spectra, which requires interaction with the microbial membrane, although the mode of action differs markedly for defensins both within and from different families. A regulatory role in innate and adaptive immunity has also been observed for mammalian defensins.

DISCOVERY Defensins were among the first HDPs to be discovered [2, 9, 16]. α-defensins were first identified in the early eighties as cationic, cysteine-rich components of mammalian neutrophil phagocytes, by Lehrer et al., who coined the name “defensin.” Analogous molecules termed “cryptdins” were later found in specialized host defense cells of the intestinal crypts [10, 16]. The ability to inhibit adrenocortical steroidogenesis also led to the alternative definition “corticostatin” [33]. In the early nineties, a new type, termed “β-defensin,” was identified in mammalian epithelia and neutrophils, as well as in avian leukocytes [2, 10, 16, 24, 25]. In the late eighties, isolation of the inducible peptides from insect hemolymph [1, 5] with a significant similarity to the mammalian peptides prompted the name “insect defensin.” Cysteine-rich peptides with antimicrobial properties— Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE All defensins are gene encoded, ribosomally synthesized molecules. Analysis of their gene organization in numerous species indicates that they occur in multigene families, as the result of numerous duplication and diversification events. They are all synthesized as prepropeptides (see Fig. 1) and processed to various degrees, depending on the context. The mammalian α- and β-defensins are products of distinct gene families likely deriving from an ancestral β-defensin gene, as only this type is found in avian and reptilian species [19, 25]. The θ-defensin precursors are

55

Copyright © 2006 Elsevier

56 / Chapter 11 In humans, a gene cluster for both α- and β-defensins was mapped to chromosomal region 8p21-p23 [17, 23], and there is evidence for a similar arrangement in syntenic chromosomal locations in other mammals, confirming the homology between these gene families. Clusters of β-defensin-like peptides of unknown function map to other chromosomal locations in humans and mice [23]. In avian species, only β-defensin genes have been identified, and 13 genes are clustered densely on chicken chromosome 3q3.5-q3.7 [31]. Mammalian defensin genes have at least two exons, the first encoding for the signal and the propiece, and the second for the HDP (see Fig. 1), separated by an intron of variable size, just before the mature peptide coding region. Myeloid α-defensins have a third exon in the 5′-UTR [24]. Invertebrate defensins show a more varied gene organization (see Fig. 1). In addition to presenting diverse coding regions, the presence, number, and location of introns also varies [7, 21]. Defensin genes from related species, or even the same species, can show significant homology in the mature HDP region but less in that coding for the leader and propeptides. Plant defensins show two types of gene organizations (Fig. 1) [15], the most common consisting of only signal peptide and the mature defensin domain, and the other found in solanaceous species, presenting a C-terminal propiece. Only one intron is present. The propiece in plant, invertebrate, and vertebrate defensins is often anionic, in contrast to the cationic mature HDP. This may be required for folding, subcellular trafficking, or processing, or it may serve to render the HDP nontoxic until needed, protecting the producer cell [10, 15]. For α-defensins this charge complementarity may be an evolutionarily selected trait [12]. FIGURE 1. Gene organization of different defensin families. The coding region for the mature peptide is in black. The signal or leader sequences are hashed. The proregions are in gray. Introns are indicated by gray triangles. Gene region relative sizes are only indicative. For θ-defensin, two different genes can provide precursors (hashed circles) that are spliced into a single cyclic molecule. For α-defensin, the broken rectangle indicates the 5′-UTR. The dotted part of the bee defensin mature peptide region represents a C-terminal extension found only in heminopteran defensins. Adapted from [7, 8, 15, 21, 24, 32] and Lay and Anderson, personal communication.

truncated α-defensin paralogs from which fragments are excised and spliced to give the mature, backbonecyclized peptide (Fig.2) [16, 24]. Their genes are active in Old World monkeys and orangutans, not present in New World monkeys or prosimians, and present only as pseudogenes in chimpanzees and man [18].

DISTRIBUTION AND EXPRESSION OF THE mRNA Defensins are expressed at sites where the producer organism encounters pathogens or needs to control the natural microbial flora—namely, in phagocytic cells and at epithelial surfaces. Mammalian α-defensins were originally isolated from the cytoplasmic granules of polymorphonuclear phagocytes, where they are most concentrated (>10 mg/ml) [9, 10, 24]. Humans and mouse Paneth cells also store α-defensins in secretory granules for release into intestinal crypts, where a concentration of 10 mg/ml can be reached. In both cases, expression seems to be constitutive. β-defensins have a wider tissue distribution [9, 10, 24], involving barrier and secretory epithelial cells in particular, and can also be produced at quite high concentrations. Expression is in some cases constitutive and in others induced by

Cationic Antimicrobial Peptides—The Defensins /

α-defensins

57

• •• • • (x0/6)CxCRxxxCxxxExxxGxCxxxgxxxxLCCr(x0/2) k f k

β-defensins • • • GxCxxxxCpxxx rqiGtCxxxxxkCC r(x )[ ] (x4/10)Cxxxga k 0/3 * kiq

θ-defensins

v CRCICx Gz R lRxCICRCzG v l

invertebrate defensins •••• • ••• x1/2tCdlls(x0/8)xxxaCaxhCxxxgxxxGGyCxx(x2/3)xxvCvCr(x0/13)

plant defensins

• •••• ••• ••• •••• r • x1/7 C x e kxxsxx xGxCxxx(x0/5)CxxxC(x2/3)E(x4/6)GxCx(x4/6) kCxCxxxCx

FIGURE 2. Schematic representation of the primary structure, topology, disulfide connectivities, and residue variation in defensin families. Highly conserved residues are in uppercase, conserved residues are in lowercase, x indicates variable residues, and stretches of variable size are defined by the numbers in parentheses. The degree of residue variation is indicated by the bars below the sequence, where the increasing number of filled squares indicates increasing degrees of variation. Dots above the sequence indicate positions of possible residue deletions. For β-defensins, * indicates a long C-terminal extension present in some members (e.g., epididymis hBD25–29). For θ-defensins, z indicates variable but hydrophobic residues. For plant defensins, indicates aromatic residues. Gray arrows represent β-strands. Cylinders represent α-helical segments. Hashed segments are present in some family members but not others.

bacteria or their components (e.g., LPS, LTA) or by proinflammatory cytokines (interferons-γ and -1β, TNF) and is often mediated by Toll-like receptors, induction being NF-κB dependent [10, 24]. The distribution pattern in both granulocytes and epithelial cells varies considerably among vertebrate species, as well as within each species [10, 24]. α-defensins have been isolated from the leukocytes of primates and various rodents, but not mice. Pig granulocytes lack defensins, whereas ungulate ones express numerous βdefensins. In humans, expression of defensins has been reported also in monocytes, NK, and T-cells, but only

rabbits have appreciable amounts in alveolar macrophages. Only certain primate species express θ-defensins in monocytes and neutrophils [16, 24]. Only the more ancient β-defensins are expressed in avian species, in both heterophils and epithelia [25]. Invertebrate defensins have mainly been isolated from the blood of insects, arachnids, and mollusks [1, 3, 5, 14]. In insects, they are induced by bacterial components, in the fat body for release into the hemolymph, in surface epithelia, and in the midgut. In the latter case they may serve both to prevent infection of blood-sucking insects by blood-borne pathogens and

58 / Chapter 11 parasites and to protect the stored bloodmeal from microbial attack. Curiously, a similar role has been proposed for the avian β-defensin spheniscin, isolated from the preserved stomach contents of the king penguin [25]. Insect defensin-like peptides have also been isolated from scorpion venom [14]. The distribution of plant defensins also is consistent with their defensive role [11, 15], as they are mainly found in vulnerable tissues or at entry points for pathogens, where they are constitutively produced in a tissue or organ specific manner, or induced in peripheral cell layers, and in cells that line cavities such as stomata. They are abundant in seed and present in leaves, tubers, roots, flowers, pods, and fruit, with a wide distribution in species throughout the plant kingdom [15]. A common feature of defensins in both animals and plants is that they can in many cases be induced by pathogen-associated molecular patterns (PAMPs) [6, 15, 24]. The regulatory mechanisms show a remarkable analogy, although it is uncertain whether they represent an ancient evolutionary relationship or a case of convergent evolution. Invertebrate defensins are principally selective for gram-positive bacteria and fungi, and activation occurs via NF-κ B-like signaling pathways initiated by Toll receptors, which respond to cytokines produced after activation of pattern recognition receptors for different types of PAMPs [6]. Induction of mammalian defensins can be mediated by Toll-like receptors (TLRs), which directly recognize PAMPs, or by receptors for chemokines induced in bacterial components [10, 24] and is often under NF-κ B control. Plants have also acquired the ability to recognize PAMPs via receptors that resemble animal TLRs, and several components of the resulting signalling cascades leading to the transcriptional activation of immune response genes are shared among the two kingdoms [20, 29].

in granules, in the mature form in mice, after processing by the matrix metalloproteinase 7 (matrylisin), and in the proform in humans, with processing by Paneth cell trypsin during or after release. β-defensin precursors are simpler, having a shorter propiece or lacking it altogether, and their biosynthesis and intracellular trafficking seems quite different from that of the α-defensins [10, 24]. Human β-defensin 1 (hBD1) is constitutively produced in many epithelia and is present in plasma and urine, where several Nterminal truncated forms indicate continued processing. hBD2 lacks a propiece and is processed to the mature form by signal peptidases in the ER, ready for secretion [10]. The biosynthesis of ovine neutrophil β-defensins is completed early in myelopoiesis and the peptides are stored in the dense granules in the mature form. θ-defensins are derived from the processing of αdefensin isoforms in which a premature stop-codon interrupts translation of the mature peptide after 12 residues. Although the intermediate processing steps are unknown, in monkey PMN two of these truncated peptides are then trimmed and spliced to give the mature, 18-residue cyclic peptide [16, 24]. Invertebrate defensins are also synthesized as precursors composed of a hydrophobic signal peptide and a relatively long prosequence, joined to the C-terminal mature HDP by a dibasic processing site [5]. This overall organization resembles that of vertebrate and plant defensins. The prodomain, like the vertebrate αdefensins, shows a high content of anionic residues and likely has similar chaperone or protective functions. In contrast to animal systems, there is little information about proteolytic processing of defensins in plants.

RECEPTORS PROCESSING All defensins are synthesized as precursors with an N-terminal signal or leader sequence necessary for secretion or storage, and usually also with a prosequence, which can be either N- or C-terminal to the mature peptide region (Fig. 1). The posttranslational processing steps are best understood for myeloid and enteric α-defensins, which are transferred into the ER by the signal peptide, which is then removed, leaving a relatively long anionic propiece. This may serve as an intramolecular chaperone for folding and/or to neutralize the activity of the mature peptide region, preventing cell damage [10, 24]. They are then stored as the mature, active form on removal of the propiece during granulogenesis. Enteric defensins are also stored

The antimicrobial activity of mammalian defensins derives from both direct and indirect mechanisms. In the first case, they act as effectors of immunity via a direct interaction with the microbial membrane that leads to disruption or lesion formation, or their translocation into the cytoplasm, where they can reach intramolecular targets. This is not a receptor-mediated process, although the ability to recognize specific membrane components is in some cases important. The antifungal activity of insect and plant defensins requires either direct interaction with specific sphingolipids and related glucosylceramides [15, 26] in lipid rafts or with phosphatidylinositol-anchored proteins associated with these rafts. The antiviral activity of θ-defensins has been related to their capacity to act as lectins and bind viral glycoproteins or glycolipids [16, 18, 30].

Cationic Antimicrobial Peptides—The Defensins / Some mammalian defensins have a regulatory role, acting as signaling molecules to recruit or activate cellular components of innate or adaptive immunity. They have variously been shown to chemoattract leukocytes, antigen presenting cells, and lymphocytes [32]. CCR6 mediates chemotaxis of immature dentritic cells (iDC) and CD4+ T-cells by β-defensin, whereas the receptormediating chemotaxis of monocytes is unknown. Certain α-defensins selectively chemoattract T-cells and iDC by as yet undefined G-protein–coupled receptors, as this activity is pertussis toxin sensitive. Mouse βdefensin 2 has the unique ability to stimulate iDC maturation, acting through TLR4. The capacity of certain α-defensins to interfere with the production of immunosuppressive glucocorticoids, possibly by blocking ACTH receptors, was also recognized very early [33].

ACTIVE CONFORMATION Defensins have quite variable primary structures, not only when comparing members of different families but also for members within each family (see Fig. 2 and Fig. 3). Effectively only the cysteine residues involved in disulfide bonding, and very few other residues are conserved, while the remainder show different degrees of variability, as does the number of residues separating the cysteines (Fig. 2). The connectivities are conserved within each family, although they differ between families, being C1-C6, C2-C4, C3-C5 for αdefensins; C1-C5, C2-C4, C3-C6 for β-defensins; C1-C4, C2-C5, C3-C6 for invertebrate defensins; and C1-C8, C2-C5, C3-C6, C4-C7 for plant defensins. Some insect defensin-like peptides, such as heliomicin and drosocin, also show an added C0-C7 disulfide-bridge [5, 15, 27, 28]. Despite the differences in sequence and connectivities, molecules from the different families fold into quite similar tertiary structures (see Fig. 4). This involves a core formed by a twisted antiparallel β-sheet, formed by two or three strands linked to each other and to the rest of the molecule by the disulfide bridges. In insect and plant defensins, the presence of an Nterminal helix gives rise to the cysteine-stabilized α/β fold (CSαβ), which defines a super family in the SCOP classification that also includes scorpion and spider toxins and protease and amylase inhibitors. Vertebrate α- and β-defensins share an overall quite similar fold, although only some beta-defensins have a C-terminal α-helical segment. While this segment, when present, makes the fold similar to the CSαβ fold, they have been placed in a separate “defensin-like” SCOP superfamily [27]. Thus, defensins have in common a conserved scaffold that is insensitive to sequence variations. The

59

distribution of cationic and hydrophobic residues on this scaffold in any case favors interaction with microbial membranes, which are rich in anionic components but could also accommodate more specific receptor/based interactions. The lack of sequence constraints and the fact that these peptides are involved in host:pathogen interactions has ensured their rapid evolution, which explains the heterogeneity of their sequences.

BIOLOGICAL ACTIONS Defensins have an undeniably important role in host defense, both as effectors and regulators [1, 2, 4, 5, 9– 11, 15, 16, 22, 24, 25]. As effectors, most defensins show a direct activity, in vitro, against bacteria and fungi, which is however quite medium and salt sensitive. α- and θ-defensins tend to have a broader spectrum of activity than β-defensins, while the canonical insect defensins tend to be more active against gram-positive bacteria, and the four cysteine insect defensin-like peptides and plant defensins tend to be principally active against fungi. The precise mechanisms of action of these peptides, regarding this direct microbicidal activity, is still undefined. For many of them, interaction with the microbial membrane seems a crucial step, although the mode of action can vary remarkably. Membrane disruption would lead to loss of the transmembrane potential gradient and release of cytoplasmic contents that could be a principal factor in the microbicidal activity. For the vertebrate defensins and the canonical three cysteine invertebrate defensins, membrane interaction is proposed to occur principally with anionic phospholipids such as phosphatidylglycerol or cardiolipin [1, 2, 5, 10, 24, 27, 28]. For the four cysteine antifungal insect and plant defensin, a common membrane target has been identified in fungal sphingolipids or related glucosylceramides (see above) [15, 26]. In any case, membrane interaction and compromised permeability would also favor translocation of the peptides into the cytoplasm, where they could interact with internal targets and disrupt vital functions. For mammalian defensins in particular, roles as regulators of innate and adaptive immune cells have been amply demonstrated [32]. These variously include the capacity to chemoattract monocytes, neutrophils, immature dendritic cells, mast cells, and T-cells; to stimulate maturation of antigen presenting cells; to induce or suppress proinflammatory mediators; to activate phagocytes; to regulate the function of complement components; to inhibit the production of immunosuppressive adrenal glucocorticoids; and to act as adjuvants for antigen presenting cells.

60 / Chapter 11 ALPHA DEFENSINS

Homo sapiens

DEF1 DEF2 DEF3 DEF4 DEF5 DEF6

-----acycripaciagerrygtciyqgrlwafcc------macycripaciagerrygtciyqgrlwafcc------mdcycripaciagerrygtciyqgrlwafcc-------vcscrlvfcrrtelrvgncliggvsftycctrv -qaratcycrtgrcatreslsgvceisgrlyrlccr-straftchcrr-scysteysygtctvmginhrfccl--

Macaca mulatta

DEF1 DEF2A DEF2B DEF3 DEF4 DEF5 DEF6 DEF7 DEF8

-----acycripaclagerrygtcfylgrvwafcc-------acycripaclagerrygtcfymgrvwafcc--sqarrtcrcrfgrcfrresysgscningrifslccr------acycripaclagerrygtcfyrrrvwafcc------rtcrcrfgrcfrresysgscningrifslccr----rrtcrcrfgrcfrresysgscningrifslccr----rrtcrcrfgrcfrresysgscningrisslccr-----rtcrcrfgrcfrresysgscningrisslccr------acycripaclagerrygtcfylrrvwafcc--

Mus musculus

DEF1 DEF2 DEF3 DEF4 DEF5 DEF6 DEF7 DEF8 DEF9 DEF10 DEF11 DEF13 DEF14 DEF15 DEF16 DEF17

-lrdlvcycrsrgckgrermngtcrkghllytlccr--lrdlvcycrtrgckrrermngtcrkghlmytlccr--lrdlvcycrkrgckrrermngtcrkghlmytlccr--lrgllcycrkghckrgervrgtc--g-irflyccprr -skklicycrirgckrrervfgtcrnlfltfvfccs--lrdlvcycrargckgrermngtcrkghllymlccr--lrdlvcycrtrgckrrehmngtcrkghlmytlccr--lrdlvcycrkrgckrrehmngtcrkghlmytlccrmw -lrdlvcycrkrgckrrehmngtcrkghllymlccr--lrdlvcycrkrgckgrermngtcrkghllytmccr--lrdlvcycrsrgckgrermngtcrkghllymlccr--lrdlvcycrkrgckrrehmngtcrrghlmytlccr--lrdlvcycrtrgckrrermngtcrkghlmhtlccr--lrdlvcycrkrgckrrehingtcrkghllymlccr--lrdlvcycrsrgckgrermngtcrkghlmytlccr--lrdlvcycrkrgckrrehmngtcrkghllytlccr--

Rattus norvegicus

DEF1 DEF2 DEF3 DEF4 DEF5

----vtcycrrtrcgfrerlsgacgyrgriyrlccr-----vtcycrstrcgfrerlsgacgyrgriyrlccr-----vtcscrtsscrfgerlsgacrlngriyrlcc-------acycrigacvsgerltgacglngriyrlccr-vlrdlkcfcrrkscnwgegimgickkrygspilccr--

Mesocricetus auratus

DEF1 DEF2 DEF3 DEF4

----vtcfcrrrgcasrerhigycrfgntiyrlccrr------cfckrpvcdsgetqigycrlgntfyrlccrq----vtcfcrrrgcasrerligycrfgntiyglccrr----vtcfckrpvcdsgetqigycrlgntfyrlccrq-

Oryctolagus cuniculus

DEF1 DEF2 DEF3 DEF4 DEF5 DEF6 DEF7 RK1

----gicacrrrfcpnserfsgycrvngaryvrccsrr ----grcvcrkqlcsyrerrigdckirgvrfpfccpr----vvcacrr-lclprerragfcrirgrihplccrr----vvcacrralclplerragfcrirgrihplccrr----vsctcrrfscgfgerasgsctvngvrhtlccrr----vfctcrgflcgsgerasgsctingvrhtlccrr----gicacrrrfclnfeqfsgycrvngaryvrccsrr ----mpcsckk-ycdpwevidgscglfnskyi-ccrek

Cavia porcellus

DEF

----rrcicttrtcrfpyrrlgtcifqnrvytfcc---

FIGURE 3. Sequences of known members of different defensin families. Sequences are aligned and gaps inserted so as to maintain a constant cysteine residue spacing. Residues or types of residues that are significantly conserved, as indicated in Figure 2, are shaded. Nomenclature follows that in the AMSDb database (http://www.bbcm.units.it/~tossi/amsdb. html), unless sequences are not yet entered. Peptides present only as fragments in the database are not listed.

BETA DEFENSINS

Homo sapiens

BD01 BD02 BD03 BD04

---dhyn---cvssggqclysacpiftkiqgtcyrgkakcck---------------gigdpvtclksgaichpvfcprrykqigtcglpgtkcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------efeldricgygtarc-rkkcrsqeyrigrc-pntyacclrkwdesllnrtkp

Gorilla gorilla

BD01 BD02 BD03 BD01 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD02 BD03 BD01 BD03

---dhyn---cvssggqclysacpiftkiqgtcyggkakcck---------------gigdpvtclksgaichpvfcprrykqigtcglpgtkcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhym---cvknkgiclysacplytkiigtcyggkakccg---------------dhyn---cvssggqclysacpiftkiqgtcyggkakcck---------------gisdpvtclksgaichpvfcprrykqigtcglpgtkcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvssggqclysacpiftkiqgtcyrgkakcck---------------gigdpvtclksgaichpvfcprrykqigtcglpgtkcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyqgkakcck---------------dirnpvtclksgaichpvfcprrykqigtcglpgtkccrkp----------glmntlqkyycrvrggwcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyqgkakcck---------------dirnpvtclksgaichpvfcprrykqigtcglpgtkccrkp----------giintlqkyycrvrggrcavlsclpkeeqigkcpmrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyqgkakcck---------------dhyn---cvrsggqclysacpiytriqgtcyhgkakcck---------------dirnpvtcvrsgaiclpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytriqgtcyhgkakcck---------------dirnpvtcvrsgaiclpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytriqgtcyhgkakcck---------------dirnpvtcvrsgaiclpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpitclksgaichpgfcpgrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcallsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpvtclksgaichpvfcprrykqigtcglpgtkccrkp----------giintlqkyycrvrggrcaLlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpitclksgaichpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcaLlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpitclksgaichpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyi---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpitclksgaichpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvrsggqclysacpiytkiqgtcyhgkakcck---------------dirnpvtclrsgaichpgfcprrykhigvcgvsaikcckkp----------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk------------dhyn---cvkgggqclysacpiytkvqgtcyggkakcck------------giintlqkyycrvrggrcavlsclpkeeqigkcstrgrkccrrkk----------

Mus musculus

BD01 BD01P BD02 BD03 BD04 BD05 BD06 BD07 BD08 BD40

---dqyk---clqhggfclrsscpsntklqgtckpdkpnccks--------------vrik---cmpkmtavfgdncsfyssmgdlcnntksvccmvpvrmdni-------e-aeldhchtnggycvraicppsarrpgscfpeknpcckymk-----------kkinnpvsclrkggrcwn-rcigntrqigscgvpflkcckrk------------qiinnpitcmtngaicwg-pcptafrqigncghfkvrcckir------------ktinnpvsccmiggicry-lckgnilqngncgvtslncckrk------------qlinspvtcmsyggscqr-scnggfrlgghcghpkirccrrk------------qdinskracyreggeclq-rciglfhkigtcnfrf-kcckfqipekktkil---qkinepvscirnggicqy-rciglrhkigtcgspf-kcck---------------dtik---clqgnnnchiqkcpwfllqvstcykgkgrccqkrrwfarshvyhv

Rattus norvegicus

BD01 BD02 BIN1

---dqyr---clqnggfclrsscpshtklqgtckpdkpnccrs-------------qsinnpitcltkggvcwg-pctggfrqigtcglprvrcckkk-------------girntv-cfmqrghcrlfmcrsgerkgdicsdpwnrccvsssiknr------

Bos Taurus

BD01 BD02 BD03 BD04 BD05 BD06 BD07 BD08 BD09

------dfaschtnggiclpnrcpghmiqigicfrprvkccrsw--------------vrnhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw------------qgvrnhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw------------qrvrnpqscrwnmgvcipflcrvgmrqigtcfgprvpccrr-------------qvvrnpqscrwnmgvcipiscpgnmrqigtcfgprvpccrrw------------qgvrnhvtcriyggfcvpircpgrtrqigtcfgrpvkccrrw------------qgvrnfvtcrinrgfcvpircpghrrqigtclgprikccr----------------vrnfvtcrinrgfcvpircpghrrqigtclgpqikccr--------------qgvrnfvtcrinrgfcvpircpghrrqigtclapqikccr-------------

Galagoides demidoff Pan troglodytes Pongo pygmaeus Hylobates concolor Hylobates lar Hylobates moloch Macaca mulatta Macaca fascicularis Papio anubis Presbytis cristata Presbytis melalophos Presbytis obscurus Cercopithecus aethiops Cercopithecus erythrogaster Cercopithecus preussi Saguinus oedipus

FIGURE 3.

(Continued)

62 / Chapter 11

BD10 BD11 BD12 BD13 EAP LAP TAP BD01 BD02 BD01 BD02 BD01

--qgvrsylscwgnrgicllnrcpgrmrqigtclaprvkccr------------------gplscrrnggvcipircpgpmrqigtcfgrpvkccrsw----------------gplscgrnggvcipircpvpmrqigtcfgrpvkccrsw----------------gplscgrnggvcipircpvpmrqigtcfgrpvkccrsw----------------nplscrlnrgicvpircpgnlrqigtcftpsvkccrwr-------------gvrnsqscrrnkgicvpircpgsmrqigtclgaqvkccrrk----------------npvscvrnkgicvpircpgsmkqigtcvgravkccrkk------------qgvrnrlschrnkgvcvpsrcprhmrqigtcrgppvkccrkk------------hgvtdslscrwkkgicvltrcpgtmrqigtcfgppvkccrlk----------------srrschrnkgvcaltrcprnmrqigtcfgppvkccrkk------------qgiinhrscyrnkgvcaparcprnmrqigtchgppvkccrkk-------------nignsvsclrnkgvcmpgkcapkmkqigtcgmpqvkcckrk-----------

Meleagris gallopavo

BD01 BD02

------krekclrrngfcaflkcptlsvisgtc-srfqvccktllg----------------lfc--krgtchfgrcpshlikvgsc-fgfrscckwpwda--------

Gallus gallus

BD01 BD01A BD02 BD03

-----grksdcfrksgfcaflkcpsltlisgkc-srfylcckriwg-------------grksdcfrkngfcaflkcpyltlisgkc-srfhlcckriw-----------------lfc--kggschfggcpshlikvgsc-fgfrscckwpwna-------------tatqcrirggfcrvgscrfphiaigkc-atfisccgray

Aptenodytes patagonicus

BD01 BD02

------sfglcrlrrgfcahgrcrfpsipigrc-srfvqccrrvw---------------sfglcrlrrgfcargrcrfpsipigrc-srfvqccrrvw----------

Ovis aries Capra hircus Sus scrofa

THETA DEFENSINS

Pongo pygmaeus

Hylobates syndactylus Pigtailed macaque Macaca mulatta

Kikuyu colobus

FIGURE 3.

(Continued)

DEFT-1,2,4 DEFT-3 DEFA-1 DEFA-2 DEFT-1 DEFT-1 TDEF-1B TDEF-1A TDEF-3 TDEF-4 DEFT-1

rcicrrgvc rcicgrgvc rcicrrgic rcicrrgvc rcicgrgvc rcicrrgvc rclcrrgvc rcictrgfc rcicvlgic rcictrgvc rcvctrgfc

rll rll rfl rll rll qll qll rll rll qll hll

Cationic Antimicrobial Peptides—The Defensins / INVERTEBRATE DEFENSINS

Acalolepta luxuriosa Aedes aegypti

DEF1 DEFA DEFB DEFC DEF Aedes albopictus DEFD DEFI Aeschna cyanea DEFI Allomyrina dichotoma Androctonus australis hector DEF4 DEFA Anomala cuprea DEFB DEF1 Anopheles gambiae DEF2 DEFI Apis mellifera DEFI Bombus pascuorum Bombus ignitus DEF1 DEF1 Dermacentor variabilis DEFI Drosophila melanogaster DMYC Eristalis tenax DEF DEFN Heliothis virescens Leiurus quinquestriatus DEF DEFA Mamestra brassicae DEFI Mytilus galloprovincialis DEF Oryctes rhinoceros DEFI Palomena prasina SCRP Pandinus imperator DEFI Phlebotomus duboscqi Phormia terranovae DEFI DEFI Pyrrhocoris apterus DEFA Rhodnius prolixus DEFC SAPB Sarcophaga peregrine SAPC SAPE Stomoxys calcitrans DEF1 DEF1A DEF2 DEF2A DEFI Tenebrio molitor DEFA Zophobas atratus

rftcdvlsveakgvklnhaacgihclfrrrt-ggycnkk--rvcicr-------------atcdlls----gfgvgdsacaahciarrnr-ggycnak—-kvcvcrn------------atcdlls----gfgvgdsacaahciargnr-ggycnsq—-kvcvcrn------------atcdlls----gfgvgdsacaahciargnr-ggycnsk—-kvcvcrn------------atcdlls----gfgvgdsacaahciarrnr-ggycnak—-tvcvc--------------atcdlls----gfgvgdsacaahciargnr-ggycnsk—-kvcvcpi-----------gfgcpl----------dqmqchrhcqtitgrsggycsgplkltctcyr------------vtcdllsfeakgfaanhslcaahclaigrr-ggscer---gvcicrr-----------gfgcpf----------nqgachrhcrsirrr-ggycaglfkqtctcyr------------vtcdllsfeakgfaanhsicaahclaigrk-ggscqn---gvcvcrn------------vtcdllsfeakgfaanhsicaahclvigrk-ggacqn---gvcvcrn------------atcdlas----gfgvgsslcaahciarryr-ggycnsk-—avcvcrn------------atcdlas----gfgvgnnlcaahciarryr-ggycnsk-—avcvcrn------------vtcdllsf---kgqvndsacaanclslgka-gghcek---gvcicrktsfkdlwdkyf-vtcdllsi---kgvaehsacaanclsmgka-ggrcen---giclcrkttfkelwdkrf-vtcdllsi---kgvaehsacaanclsmgka-ggrcen---gvclcrktnfkdlwdkrfg gfgcpl----------nqgachnhcrsirrr-ggycsgiikqtctcyrn----------ratcdlls----kwnwnhtacaghciakgfk-ggycndk-—avcvcrn------------adclsgrykgpcavwdnetcrrvckeegrs-sghcsps—-lkcwcegc-----------atcdlls----flnvnhaacaahclskgyr-ggycdgk-—kvcncr------------igscvw------gavnytsdcngeckrrgyk-gghcgsfanvncwcet-----------gfgcpl----------nqgachrhcrsirrr-ggycagffkqtctcyrn----------pascylldgyaagrddgrahcia---prnr--rlycas-—yqvcvcry-----------gfgcp-----------nnyqchrhcksipgrcggycggxhrlrctcyrcg---------rltcdllsfeakgfaanhslcaahclaigrk-ggacq---ngvcvcrr------------atcdalsfsskwltvnhsacaihcltkgyk-ggrcvn---ticncrn-----------efqcmanm-------dmlgncekhcqtsgek—-gych---gtkckcgtplsy--------atcdlls----afgvghaacaahcighgyr-ggycnsk--avctcrr-----------ratcdlls----gtginhsacaahcllrgnr-ggycngk—-gvcvcrn------------atcdilsfqsqwvtpnhagcalhcvikgyk-ggqcki—--tvchcrr------------atcdlfsfrskwvtpnhaacaahcllrgnr-ggrck---gtichcrk------------atcdllsltskwftpnhagcaahciflgnr-ggrcv---gtvchcrk------------ltcei----------drslcllhcrlkgyl-raycsqq—-kvcrcvq------------atcdlls----gigvqhsacalhcvfrgnr-ggyctgk—-gicvcrn------------atcdlls----gtginhsacaahcllrgnr-ggycngk-—avcvcrn-----------gitcdlls----lwkvghaacaahclvlgdv-ggyctke—-glcvcke------------itcdlls----lwkvghaacaahclvlgnv-ggyctke—-glcvcke-----------ratcdlls----mwnvnhsacaahclllgks-ggrcndd—-avcvcrk-----------ratcdlls----mwnvnhsacaahclllgks-ggrcndd--avcvcrk -vtcdilsveakgvklndaacaahclfrgrs-ggycngk--rvcvcr-------------ftcdvlgfeiagtklnsaacgahclalgrr-ggycnsk-—svcvcr------------MOLD DEFENSIN

Pseudoplectania nisella Plectasin

FIGURE 3.

(Continued)

gfgcngpwd------eddmqchnhcksikgykggycak-ggfvckcy

63

PLANT DEFENSINS

Aesculus hippocastanum PDEF1 PDEF1 Arabidopsis thaliana PDEF2 PDEF3 PDEF4 PDEF11 Beta vulgaris PDEF2 BSD1 Brassica campestris PDEF3 Brassica napus PDEF1 PDEF3 Brassica rapa PDEF1 Cajanus cajan PDEF1 Capsicum annuum PDEF2 Capsicum chinense CCD1 Cassia fistula CFD1 CFD2 PDEF1 Clitoria ternatea Dahlia merckii PDEF1 Elaeis guineensis EGAD1 SE60 Glycine max Hardenbergia violacea HVAMP1 PDEFA Helianthus annuus PDEF1 Heuchera sanguinea PDEF Hordeum vulgare Lycopersicon esculentum TPP TGAS118

------lcnerpsqtwsgncgntah---cdkqcqdwe--kashgachkrenhwkcfcyfnc----qklc-erpsgtwsgvcgnsna---cknqcinle—-karhgscnyvfpahkcicyfpc----aelc-kresetwsgrcvndyq---crdhcinnd—-rgndgycaggypyrscfcffsc----artc-esqshrfkgtcvsasn---canvchn-e--gfvggncrg—f-rrrcfctrhc----artc-asqsqrfkgkcvsdtn---cenvchn-e--gfpggdcrg—f-rrrcfctrnc-----aic-kkpskffkgacgrdadv--cekacdq-e--nwpggvc-vpf--lrcecqrsc-----atc-rkpsmyfsgacfsdtn---cqkacnr-e--dwpngkclvgf---kcecqrpc----qrsc-krqpnsgskncmkdse---crevciyae--kamratcdytfprrrcfchfpcq ---eaklc-erssgtwsgvcgnnna---cknqcirle—-gaqhgscnyvfpahkcicyfpc----qklc-erpsgtwsgvcgnnna---cknqcinle—-karhgscnyvfpahk----------aqklc-erssgtwsgvcgnnna---cknqrinle—-garhgscnyvfpyhrcicyfpc----aktc-enladkyrgpcfsg-----cdthcttke--havsgrcrddf---rcwctkrc-----kic-ealsgnfkglclssrd---cgnvcrr-e--gftdgscig-f-rlqcfctkpca -----rtc-esqshrfkglcfsksn---cgsvcht-e--gfngghcrg—f-rrrcfctrhc---qnnic-kttskhfkglcfadsk---crkvciq-e-dkfedghcskl--qrkclctknc-----ktc-ekpskffsggcigttgnkqcdylcrrge--gllsgackg---l-kcvctkac-----ktc-evlsgkfggacstiingpkcdktcknqe--hyisgtcksdf---kcwctknc-----nlc-erasltwtgncgntgh---cdtqcrnwe—-sakhgachkrginwkcfcyfdc-----elceekasktwsgncgntgh---cdnqckswe—-gaahgachvrngkhmcfcyfnc-----rtc-esqshkfqgtclresn---canvcqt-e—-gfqggvcrgv--rrrcfctrlc-----rvc-esqshgfhglcnrdhn---calvcrn-e—-gfsggrckg-f-rrrcfctric-----ktc-eslantyrgpcftdgs---cddhcknke—-lislgrcrndv---rcwctrnc-----rtc-esqshkfkgtclsdtn---canvchs-e—-rfsggkcrg-f-rrrcfctthc--dgvklc-dvpsgtwsgvcgsssk---csqqckdreehfayggachyqfpsvkcfckrqc-----ric-rrrsagfkgpcvsnkn---caqvcmq-e--gwgggncdg—-plrrckcmrrc-----qic-kapsqtfpglcfmdss---crkycik-e--kftgghcskl--qrkclctkpc-----rtc-esqshrfkgpcvsekn---casvcet-e—-gfsggdcrg-f-fffcfctrpc-

Medicago sativa Nicotiana alata Nicotiana tabacum

PDEF1 PDEF1 PDEF1 NTS13 NETHIO1 NETHIO2 PDEF1 PDEF1 PDEF1 PDEF1 PDEF2

----artc-enladkyrgpcfs-g----cdthcttk-e--navsgrcrddf---rcwctkrc-----rec-ktesntfpgicitkpp---crkacis-e—-kftdghcski--lrrclctkpc-----rec-ktesntfpgicitkpp---crkacis-e—-kftdghcskl--lrrclctkpc-----rtc-esqshrfkgpcsrdsn---catvclt-e—-gfsggrcpwi--pprcfctspc-----rec-are-i-ftglcitnpq---crkaci-ke—-kftdghcski--lrrclctkpc-----kdc-ktesntfpgicitkpp---crkaci-ke—-kftdghcski--lrrclctkpc----kstc-kaesntfpglcitkpp---crkacls-e—-kftdgkcski--lrrcicykpc----arhc-lsqshrfkgmcvssnn---canvcrt-e—-sfpdgeckshglerkcfckkvc-----rtc-esqshrfhgtcvresn---casvcqt-e--gfiggncra—f-rrrcfctrnc-----atc-kaecptwdsvcinkkp---cvacckka---kfsdghcski--lrrclctkecv -----gtc-kaecptwegicinkap---cvkcckaqpe-kftdghcski—-lrrclctkpca

SPI1 PDEF1 PDEF1 PDEF2 DRR230-C PDEF3 PDEF4 PPDFN1 PDN-1 PDEF1 PDEF2 PDEF3 PDEF4 PDEF1 PDEF2A PDEF2B PDEF1 PDEF1 PDEF2 PDEF3 PDEF2 PDEF1 PDEF2 TAD1 PDEF1 PDEF1 PDEF2 PDEF1 PDEF1 THGC PDEF1 THZ2

-----rtc-ktpsgkfkgvcassnn---cknvcqt-e—-gfpsgscdfhvanrkcycskpcp ----artc-ktpsgkfkgvcassnn---cknvcqt-e--gfpsgscd-hvanrkcycskpcp -----ntc-ehladtyrgvcftnas---cddhcknka—-hlisgtchd-w---kcfctqnc-----ntc-enlagsykgvcfgg-----cdrhcrtqe--gaisgrcrddf---rcwctknc-----ntc-ehladtyrgvcftdas---cddhcknka—-hlisgtchn-f---kcfctqnc-----ktc-ehladtyrgvcftnas---cddhcknka—-hlisgtchn—w---kcfctqnc-----ktc-enlsgtfkgpcipdgn---cnkhcrnne-—hllsgrcrddf---rcwctnrc-----rtc-esqsnrfkgtcvstsn---casvcqt-e—-gfpgghcrg-f-rrrcfctkhc-----rtc-eaasgkfkgmcfssnn---cantcar-e—-kfdggkckg-f-rrrcmctkkc----qklc-erpsgtwsgvcgnnna---cknqcinle—-karhgscnyvfpahkcicyfpc----qklc-qrpsgtwsgvcgnnna---cknqcirle—-karhgscnyvfpahkcicyfpc-----klc-erssgtwsgvcgnnna---cknqcirle--gaqhgscnyvfpahkcicyfpc----qklc-erpsgtwsgvcgnnna---cknqcinle--garhgscnyifpyhrcicyfpc----qklc-erpsgtwsgvcgnnna---cknqcinle—-karhgscnyvfpahkcicyfpc----qklc-qrpsgtwsgvcgnnna---crnqcinle—-karhgscnyvfpahkcicyfpc----qklcarpsgtwssgncrnnna---crnfcikle—-ksrhgscnipfpsnkcicyfpc-----rhc-eslshrfkgpctrdsn---casvcet-e--rfsggnchg-f-rrrcfctkpc-----rvc-mgksqhhsfpcisdrl---csnecvk-eeggwtagych----lrycrcqkac-----rvc-mgksagfkglcmrdqn---caqvclq-e-—gwgggncdgv—-mrqckcirqc-----rvc-rrrsagfkglcmsdhn---caqvclq-e—-gwgggncdgv—-irqckcirqcgifssrkc-ktpsktfkgictrdsn---cdtscry-e--gypagdckgi—-rrrcmcskpc-----kic-rrrsagfkgpcmsnkn---caqvcqq-e—-gwgggncdg—-pfrrckcirqc-----kvc-rqrsagfkgpcvsdkn---caqvclq-e--gwgggncdg—-pfrrckcirqc-----rtc-lsqshkfkgtclsnsn---caavcrt-en--fpdgecnthlverkcyckrtc---aqklc-ekssgtwsgvcgnnna---cknqcinle—-garhgscnyifpyhrcicyfpc---llgrc-kvksnrfhgpcltdth---cstvcrg-e--gykggdchgl--rrrcmc-l-c---llgrc-kvksnrfngpcltdth---cstvcrg-e--gykggdchgl--rrrcmc-l-c----artc-mikkegw-gkclidtt---cahscknr---gyiggnckgm—-trtcyclvnc-----ktc-enlvdtyrgpcfttgs---cddhcknke--hllsgrcrddv---rcwctrnc-----rvc-esqshgfkgactgdhn---calvcrn-e—-gfsggncrg-f-rrrcfcltkc-----rvc-rrrsagfkgvcmsdhn---caqvclq-e--gygggncdgi—-mrqckcirqc-----rvc-mgksqhhsfpcisdrl---csnecvk-edggwtagych----lrycrcqkac-

Nicotiana excelsior Nicotiana paniculata Oryza sativa Petunia inflata Petunia hybrida Picea abies Picea glauca Pisum sativum

Prunus persica Pyrus pyrifolia Raphanus sativus

Sinapis alba Solanum tuberosum Sorghum bicolor Spinacia oleracea Triticum aestivum Wasabia japonica Vicia faba Vigna radiata Vigna unguiculata Zea mays

FIGURE 3.

(Continued)

Cationic Antimicrobial Peptides—The Defensins /

HNP-3 (human)

RK-1 (rabbit)

Crp-4 (mouse)

hBD-1 (human)

hBD-2 (human)

hBD- 3 (human)

65

bBD12 (bovine)

α-defensins

RTD-1 (rhesus)

crotamine (snake venom)

θ-defensins

mBD-7 (mouse)

spheniscin (avian)

DLP-2 (platypus venom)

β-defensins and β-defensin-like peptides

insect def A (blowfly) sapecin (fleshfly)

Mgd-1 (mussel)

mBD-8 (mouse)

heliomicin (moth)

drosomycin (fruit fly)

termicin (termite)

charybdotoxin (scorpion)

invertebrate-defensins and defensin-like peptides

RS-AFP1 (Raphanus sativus)

AH-AFP1 (horse chestnut)

NaD1 (Nicotiana alata)

Psd1 (Pisum sativum)

PhD1 (Petunia hybrida)

plant-defensins

FIGURE 4. Comparison of structurally characterized defensins and defensin-like peptides from different families. Structures were prepared with the DSViewer Pro from Accelrys, using coordinates deposited in the PDB databank (HNP3, 1DFN; RK-1, 1EWS; cryptdin-4, 1TV0; hBD-1, 1IJV; hBD-2, 1FD3; hBD-3, 1KJ6; bBD12, 1BNB; crotamine, 1H5O; mBD8, 1E4R; mBD-7, 1E4T; spheniscin, 1UT3; DLP-2, 1D6B; RTD-1, 1HVZ; insect def A, 1ICA; sapecin 1L4V; heliomicin, 1I2U; termicin, 1MMO; Mgd-1, 1FJN; drosomycin, 1MYN; charybdotoxin, 2CRD; RS-AFP1, 1AYJ; AH-AFP1, 1BK8; Psd1, 1JKZ; NaD1, 1MR4; PhD1, 1N4N).

Acknowledgments N.A. is supported by an Italian Ministry of Research and Universities PRIN grant. I.Z. is supported by a fellowship from the Consortium for International Development of the University of Trieste.

References [1] Bulet P, Hetru C, Dimarcq JL, Hoffmann D. Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 1999;23:329–44. [2] Castro MS, Fontes W. Plant defense and antimicrobial peptides. Protein Pept Lett 2005;12:13–8. [3] Charlet M, Chernysh S, Philippe H, Hetru C, Hoffmann JA, Bulet P. Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis. J Biol Chem 1996;271:21808–13.

[4] Crovella S, Antcheva N, Zelezetsky I, Boniotto M, Pacor S, Verga Falzacappa MV, et al. Primate beta-defensins—structure, function and evolution. Curr Protein Pept Sci 2005;6:7–21. [5] Dimarcq JL, Bulet P, Hetru C, Hoffmann J. Cysteine-rich antimicrobial peptides in invertebrates. Biopolymers 1998;47:465– 77. [6] Ferrandon D, Imler JL, Hoffmann JA. Sensing infection in Drosophila: Toll and beyond. Semin Immunol 2004;16:43–53. [7] Froy O, Gurevitz M. Arthropod and mollusk defensins—evolution by exon-shuffling. Trends Genet 2003;19:684–7. [8] Froy O, Gurevitz M. Membrane potential modulators: a thread of scarlet from plants to humans. Faseb J 1998;12:1793–6. [9] Ganz T. Defensins and other antimicrobial peptides: a historical perspective and an update. Comb Chem High Throughput Screen 2005;8:209–17. [10] Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710–20. [11] Garcia-Olmedo F, Molina A, Alamillo JM, Rodriguez-Palenzuela P. Plant defense peptides. Biopolymers 1998;47:479–91.

66 / Chapter 11 [12] Hughes AL. Evolutionary diversification of the mammalian defensins. Cell Mol Life Sci 1999;56:94–103. [13] Kristensen H-H, Yaver D. Antimicrobial peptides. Innovations in Pharmaceutical technology 2004:77–82. [14] Kuhn-Nentwig L. Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci 2003;60:2651–68. [15] Lay FT, Anderson MA. Defensins—components of the innate immune system in plants. Curr Protein Pept Sci 2005;6:85–101. [16] Lehrer RI. Primate defensins. Nat Rev Microbiol 2004;2:727–38. [17] Liu L, Zhao C, Heng HH, Ganz T. The human beta-defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry. Genomics 1997;43:316–20. [18] Nguyen TX, Cole AM, Lehrer RI. Evolution of primate thetadefensins: a serpentine path to a sweet tooth. Peptides 2003;24:1647–54. [19] Nicastro G, Franzoni L, de Chiara C, Mancin AC, Giglio JR, Spisni A. Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur J Biochem 2003;270:1969–79. [20] Nurnberger T, Brunner F, Kemmerling B, Piater L. Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 2004;198:249–66. [21] Rodriguez de la Vega RC, Possani LD. On the evolution of invertebrate defensins. Trends Genet 2005;21:330–2. [22] Sahl HG, Pag U, Bonness S, Wagner S, Antcheva N, Tossi A. Mammalian defensins: structures and mechanism of antibiotic activity. J Leukoc Biol 2005;77:466–75. [23] Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh MJ, et al. Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proc Natl Acad Sci USA 2002;99:2129–33.

[24] Selsted ME, Ouellette AJ. Mammalian defensins in the antimicrobial immune response. Nat Immunol 2005;6:551–7. [25] Sugiarto H, Yu PL. Avian antimicrobial peptides: the defense role of beta-defensins. Biochem Biophys Res Commun 2004; 323:721–7. [26] Thomma BP, Cammue BP, Thevissen K. Mode of action of plant defensins suggests therapeutic potential. Curr Drug Targets Infect Disord 2003;3:1–8. [27] Torres AM, Kuchel PW. The beta-defensin-fold family of polypeptides. Toxicon 2004;44:581–8. [28] Tossi A, Sandri L. Molecular diversity in gene-encoded, cationic antimicrobial polypeptides. Curr Pharm Des 2002;8: 743–61. [29] Veronese P, Ruiz MT, Coca MA, Hernandez-Lopez A, Lee H, Ibeas JI, et al. In defense against pathogens. Both plant sentinels and foot soldiers need to know the enemy. Plant Physiol 2003;131:1580–90. [30] Wang W, Cole AM, Hong T, Waring AJ, Lehrer RI. Retrocyclin, an antiretroviral theta-defensin, is a lectin. J Immunol 2003; 170:4708–16. [31] Xiao Y, Hughes AL, Ando J, Matsuda Y, Cheng JF, SkinnerNoble D, et al. A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genomics 2004;5:56. [32] Yang D, Biragyn A, Hoover DM, Lubkowski J, Oppenheim JJ. Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Annu Rev Immunol 2004;22:181–215. [33] Zhu Q, Bateman A, Singh A, Solomon S. Isolation and biological activity of corticostatic peptides (anti-ACTH). Endocr Res 1989;15:129–49.

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12 Cathelicidins: Cationic Host Defense and Antimicrobial Peptides NEELOFFER MOOKHERJEE, KELLY L. BROWN, AND ROBERT E.W. HANCOCK

a part of the precursor protein, that is highly homologous, followed by a heterogeneous C-terminal mature peptide (Fig. 1). Cathelicidins were originally described as antimicrobial peptides [53, 55] and include proteins such as pig protegrins and cattle indolicidins that served as templates for some of the most clinically advanced antimicrobial peptide drugs [56]. However, it is becoming clear that while virtually all cathelicidins have some direct antimicrobial activity in dilute media, there is a broad range of potencies, and in some cases it has been difficult to demonstrate antimicrobial activity at the concentrations and salt conditions found physiologically [3]. Thus, given recent findings that they possess a broad variety of immunomodulatory and antiinflammatory activities [3, 12, 22, 51] that are relevant to innate immunity, they are increasingly being referred to as host defense peptides. Ongoing research to further understand the complex myriad of biological functions of cathelicidins has captured the interest of researchers, particularly in the health sciences.

ABSTRACT Cathelicidins are members of a family of protective, anti-infective peptides, also known as host defense peptides, and are defined by the high degree of conservation of their “pre-pro” precursor sequences and exceptional diversity of sequence and structure in the mature peptide. These endogenous cationic peptides play a key role in innate immune responses and are widely distributed in mammalian species, as well as being recently observed in nonmammalian vertebrates including hagfish, trout, and chickens. They exhibit a variety of functions ranging from direct antimicrobial activity against bacteria, fungi, eukaryotic parasites, and viruses through to immunomodulatory and antiinflammatory activities.

DISCOVERY All cathelicidins are small cationic, amphiphilic peptides composed of 12 to 97 amino acids. This family of peptides was first discovered in bovine neutrophils by Zanetti and collaborators [53] in the early 1990s. The term cathelicidin was coined in 1995 to describe this family of cationic, bipartite peptides that shared a conserved DNA and pre-pro protein sequence at the Nterminus that is highly homologous to that upstream of the coding sequence for the protein cathelin, a 96amino-acid, cathepsin L protease inhibitor from porcine neutrophils [54]. Thus, cathelicidins are paradoxically named based on this homology in the part of the precursor protein involved in secretion and processing despite the fact that this family has become better known for its anti-infective properties of the mature (processed) protein, and potential protease inhibitory roles of cathelicidins are rarely investigated. Thus, all cathelicidins have a prosequence at their N-terminus as Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/STRUCTURAL GENE The cathelicidin gene family can be traced back 300 to 500 million years [49], demonstrating the early evolutionary origin of these peptides. Like the defensin family [Chapter 11], these peptides are encoded by a single gene that belongs to a highly homologous gene family. Thus, cathelicidin encoding genes have a highly homologous 5′ region that characterizes this family of peptides and encodes for the conserved cathelin domain [48, 54]. The presence of this homologous 5′ region has enabled the discovery of many new cathelicidins. Cathelicidin-encoding genes contain four exons [17, 57]. Exons 1 through 3 encode the conserved signal

67

Copyright © 2006 Elsevier

68 / Chapter 12

FIGURE 1.

Gene structure and processing of cathelicidins (modified from [51]).

peptide (29–30 amino acids) and the cathelin-like domain (95–114 amino acids), whereas exon 4 is hypervariable [44], encoding the extremely heterogenous mature peptide (12–97 amino acids) in addition to the processing site and 3′ UTR (Fig. 1). Interestingly, the 5′ flanking sequences upstream of the coding sequence have several potential consensus sequences for transcription factors involved in hematopoiesis and inflammation, indicating that the cathelicidin gene expression maybe regulated and/or coordinated along with the expression of other entities of innate immunity [30, 48, 51, 57].

DISTRIBUTION OF CATHELICIDINS Cathelicidin mRNAs and precursor or mature proteins are widely distributed in different mammalian species (Table 1), as well as in a variety of tissue types and cellular lineages. Remarkably a cathelicidin-related peptide was recently reported in the Atlantic hagfish, which is known to be devoid of adaptive immune responses [49]. In addition, new cathelicidins have been described in hagfish, trout, and chickens [48], indicating that this family of peptides is even more widely distributed than at first assumed and indeed plays an important role in innate immunity. In mammals, cathelicidins are widely distributed among various tissue types, including the skin, intestine, oral cavity, cervix, lungs, mucosa, and so on, and are found largely in their unprocessed (pre-pro) form in the granules of neutrophils as well as in myeloid precursors, epithelial cells, mast cells, keratinocytes, and lymphocytes [10, 19, 31, 48]. They are found in their processed forms in a number of bodily fluids, including gastric juices, saliva, semen, sweat, plasma, airway surface liquid, and breast milk. The expression of cathelicidin mRNA appears to be differentially regulated depending on the lineage and/or differentiation state of particular cell types [20, 24], and expression

can be constitutive or inducible. For example, the levels of expression of both human LL-37 and mouse CRAMP are affected by infection (or the inflammation accompanying infection) and/or injury in skin keratinocytes [10]. Similarly, the expression of LL-37 mRNA and protein can be up-regulated by 10- to 50-fold in response to inflammatory stimuli but not conserved bacterial products like LPS [31]. Conversely, infection of the intestinal epithelium with particular pathogens, like Shigella, leads to down-regulation of the expression of cathelicidin [24]. Interestingly, the promoter regions of some cathelicidin genes contain consensus binding sites for transcription factors such as NFκB, and NF-IL6, acute phase response factor, and IFNresponse elements [17, 48, 51, 57], all of which are activated by products associated with inflammation. Therefore, cathelicidin mRNA expression may be coregulated with (or by) inflammatory responses [57], consistent with their functional roles.

PROCESSING Cathelicidins need to be proteolytically processed to be biologically active [49]. Initially, they are synthesized as precursor peptides containing both a leader sequence (for secretion) and a prodomain—namely, highly homologous N-terminal cathelin domain (Fig. 1). In neutrophils, these inactive pre-propeptides are stored at high concentrations in granules and upon neutrophil activation to undergo processing by appropriate proteases to release the mature peptide that is found extracellularly. However, the exact details of where and when the processing occurs is still uncertain as the unprocessed form of the propeptide can be found extracellularly as can a variety of processing forms. The active human cathelicidin LL-37 is cleaved from its precursor protein hCAP18 by elastase or proteinase 3 [17, 45], whereas in cattle and pigs cathelici-

Cathelicidins: Cationic Host Defense and Antimicrobial Peptides / TABLE 1.

69

Distribution of cathelicidins in vertebrates.

Peptide

Structure

Sequence

Origin

LL-37 RL-37 mCRAMP rCRAMP CAP18 CAP11 BMAP-27 BMAP-28 BMAP-34 SMAP-29 SMAP-34 PMAP-23 PMAP-36 PMAP-37 eCATH-1 eCATH-2 eCATH-3 Dog CATH HFIAP-1 HFIAP-3 Bac4 Bac5 Bac7

α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical α-helical Extended Extended Extended

Human Rhesus monkey Mouse Rat Rabbit Guinea pig Cow Cow Cow Sheep Sheep Pig Pig Pig Horse Horse Horse Dog Hagfish Hagfish Cow Cow Cow

OaBac5 OaBac6

Extended Extended

OaBac7.5

Extended

OaBac11

Extended

Trout CATH ChBac5 PR-39 Prophenin-1

Extended Extended Extended Extended

Prophenin-2

Extended

Indolicidin Dodecapeptide OaDode Protegrin-1 Protegrin-2 Protegrin-3 Protegrin-4 Protegrin-5 CHICATH

Extended Cyclic β-turn Cyclic β-turn β-hairpin β-hairpin β-hairpin β-hairpin β-hairpin Unknown

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTAS GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE GLVRKGGEKFGEKLRKIGQKIKEFFQKLALEIEQ GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI GRFKRFRKKFKKLFKKLSPVIPLLHL GGLRSLGRKILRAWKKYGPIIVPIIRI GLFRRLRDSIRRGQQKILEKARRIGERIKDIFR RGLRRLGRKIAHGVKKYGPTVLRIIRIA GLFGRLRDSLQRGGQKILEKAERIWCKIKDIFR RIIDLLWRVRRPQKPKFVTVWVR GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGC GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQS KRFGRLAKSFLRMRILLPRRKILLAS KRRHWFPLSFQEFLEQLRRFRDQLPFP KRFHSVGSLIQRHQQMIRDKSEATRHGIRIITRPKLLLAS KKIDRLKELITTGGQKIGEKIRRIGQRIKDFFKNLQPREEKS GFKKAWRKVKHAGRRVLDTAKGVGRHYVNNWLNRYR GWFKKAWRKVKNAGRRVLKGVGIHYGVGLI RRLHPQHQRFPRERPWPKPLSLPLPRPGPRPWPKPL RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP RRIRPRPPRLPRPRPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPLPF PRPGPRPIPRPL RFRPPIRRPPIRPPFRPPFRPPVRPPIRPPFRPPFRPPIGPFP RRLRPRHQHFPSERPWPKPLPLPLPRPGPRPWPKPLPLPLPRPGLR PWPKPL RRLRPRRPRLPRPRPRPRPRPRSLPLPRPQPRRIPRPILLPWRPPRP IPRPQIQPIPRWL RRLRPRRPRLPRPRPRPRPRPRSLPLPRPKPRPIPRPLPLPRPRPKP IPRPLPLPRPRPRRIPRPLPLPRPRPRPIPRPLPLPQPQPSPIPRPL unknown RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFP RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP AFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPRFPPPN FPGPPFPPPIFPGPWFPPPPPFRP PPFGPPRFP AFPPPNVPGPRFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPN FPGPPFPPPIFPGPWFPPPPPFRPPPFGPPRFP ILPWKWPWWPWWRR RLCRIVVIRVCR RYCRIIFLRVCR RGGRLCYCRRRFCVCVGR RGGRLCYCRRRFCICV RGGGLCYCRRRFCVCVGR RGGRLCYCRGWICFCVGR RGGRLCYCRPRFCVCVGR RVKRVWPLVIRTVIAGYNLYRAIKKK

dins are exclusively processed by elastase during neutrophil degranulation [42]. It has been suggested that the cathelin-like domain exhibits antimicrobial and protease inhibitory activities, although these have not been proven to be relevant in vivo. However, the cationic peptide domain shows a broad range of an antimicrobial and immunomodulatory activities.

Sheep Sheep Sheep Sheep Rainbow trout Goat Pig Pig Pig Cow Cow Sheep Pig Pig Pig Pig Pig Chicken

RECEPTORS The ligand-receptor interactions of cathelicidins are not well understood, with LL-37 representing the most studied peptide in this regard. Overall LL-37 has been shown to have a variety of receptors according to the function being studied and the cell type. For example,

70 / Chapter 12

Proposed Mechanism for Cellular Activation by LL-37 EGFR ligand

Pro-EGFRligand

EGFR

MP (ADAM?)

LL-37

AG1478

PD98059 U0126

MEK1/2 GM6001 ERK1/2

gene transcription

FIGURE 2. Proposed mechanism for cellular activation by LL-37 [47]. LL-37 results in activation of a metalloproteinase, which subsequently cleaves membrane-anchored EGFR ligands that activate the EGFR resulting in ERK1/2 activation and gene transcription.

LL-37 directly mediates chemotaxis of human peripheral blood neutrophils, monocytes, and T cells through formyl peptide receptor-like 1 (FPRL1) [52], whereas two alternative (as yet uncharacterized) receptors are involved in chemotaxis of mast cells [32], and production of IL-8 in epithelial cells in response to LL-37 is also independent of FPRL1 [27]. LL-37 has been shown to mediate IL-1β secretion from human monocytes by activating P2X7 receptor but conversely porcine protegrins mediate IL-1β secretion independent of P2X7 receptors [11]. In addition, it is now known that LL-37 can activate mitogen-activated protein kinases (MAP kinases) in epithelial cells and monocytes [5, 47] and that activation of epithelial cells but not monocytes requires the transactivation of epidermal growth factor (see Fig. 2 for model). LL-37-induced phosphorylation and activation of the MAP kinases p38 and Erk1/2 in human peripheral blood–derived monocytes is independent of FPRL1 [5] but required for chemokine induction by LL-37. In addition, LL-37 can traffic into monocytic and epithelial cells, and this process is also required for IL-8 induction. One hypothesis to explain these results is that there are multiple receptors for LL37 that mediate different events in particular cells, but the issue of cathelicidin receptor interactions and their function in mediating the biological activity is clearly not yet resolved.

ACTIVE AND/OR SOLUTION CONFORMATION Peptides of the cathelicidin family are representative of all of the known secondary structure groups into which antimicrobial peptides are classified. Indeed, they exhibit a remarkable variety of sizes, sequences, and structures, making it unlikely that they have a common origin. They typically have a net positive charge at neutral pH of +2 to +7 due to excess lysine and arginine basic residues and contain around 50% hydrophobic amino acids. This permits them to fold into amphipathic or amphiphilic structures (often upon membrane contact), which is important in their mechanisms of action [18]. Mature (processed, biologically active) cathelicidins fold into one of four structural classes: (i) amphipathic α-helices (that form after interaction with membranes), (ii) β-hairpin molecules with two β-strands interconnected by intramolecular disulfide bonds, (iii) extended structures (upon membrane interaction) including peptides enriched in one or two amino acids such as proline and arginine, or tryptophan, and (iv) the peptide bactenecin (dodecapeptide) that is cyclized by a single disulfide bond and contains a β-turn structure (Fig. 3, Table 1). Interestingly, in recent years there has been several studies that indicate that the amino acid sequence of cathelicidins can be

Cathelicidins: Cationic Host Defense and Antimicrobial Peptides / modified to obtain potent antimicrobial peptides with reduced undesired properties, such as hemolytic and cytotoxic activity. The combinatorial chemistry approach based on sequences of natural cathelicidins has lead to the discovery of biologically active de novo peptides with potential therapeutic use [22, 44, 56].

Indolicidin

71

BIOLOGICAL FUNCTIONS Cathelicidins can function as potent microbicidal agents and/or immunomodulators in the innate immune system [12, 22, 51]. The most studied cathelicidin peptides—human LL-37, porcine PR-39, and bovine BMAP-28—have been demonstrated to control bacterial load and prevent sepsis and mortality in rats and mice after bacterial challenge (Table 2). In humans with atopic dermatitis, low expression of LL-37 (and possibly other peptides) in skin lesions from patients coincides with their enhanced susceptibility to skin infections [37], while individuals with morbus Kostmann suffer from frequent oral bacterial infections and severe periodontal disease that correlates with a deficiency in LL-37 [38]. Together, these observations demonstrate, in vivo, a significant role for cathelicidins in host defense. The protective effects of the peptides have in the past been attributed to their direct antimicrobial killing properties [55]. Indeed, some peptides have exceptionally good antimicrobial activity (e.g., pig protegrin 1 [46]), and ex vivo studies [42] favor the hypothesis that this is responsible for protection. However, most cathelicidins are not nearly as potent as protegrin, and some are only active in very dilute media. A recent wave of reports have established that in addition to being antimicrobial, host defense peptides stimulate a broad range of biological effects in innate immune cells (neutrophils and epithelial cells) and

Protegrin

CRAMP

FIGURE 3. Ribbon structures of cathelicidin mature peptides bovine indolicidin (extended structure), porcine protegrin 1 (β-hairpin structure), and murine CRAMP (amphipathic α-helical structure).

TABLE 2. Functions of selected cathelicidins. Reference

Cathelicidin Functions Anti-infective effects observed in vivo Protects against bacterial challenge and sepsis Controls bacterial load Selectively chemotactic Reduces amounts of pro-inflammatory mediators Enhances adaptive immune response Immunomodulatory effects observed in vitro Induces and influences signal transduction Induces gene expression and enhances protein secretion Suppresses inflammatory stimuli-induced gene expression and protein secretion Influences cell proliferation, differentiation, and migration Promotes angiogenesis and/or wound healing Cooperates with immune mediators Influences adaptive immune response

Human LL-37

Murine CRAMP

3, 13, 38 3 35 35, 40 2

34 23, 39 26

5, 27, 33, 40, 47 5, 6, 9, 11, 27, 33, 40, 47

26

21, 25, 41 3, 4, 9 9

Porcine PR-39

16

16 26

3

3, 8, 30 1, 9, 20, 32, 36, 41, 52

Bovine BMAP-28, Bac2A, or Indolicidin

7, 15, 43 14, 28

4 26

4

50 14, 28

72 / Chapter 12 in cells that are involved in and bridge the innate and adaptive immune systems (monocytes/macrophages and dendritic cells). Cathelicidins promote chemotaxis of effector cells, induce the transcription and secretion of chemokines, and induce mast cell degranulation, resulting in enhanced vascular permeabilization and leukocyte infiltration (Table 2). In this sense, cathelicidins might be considered pro-inflammatory mediators. However, they also protect the host against detrimental, potentially lethal, effects resulting from an excessive inflammatory response to microbial invasion. Cathelicidins suppress gene transcription and the release of proinflammatory mediators induced by LPS and other bacterial products (Table 2). In addition, cathelicidins prevent the release of toxic components that cause excess tissue damage and inflammation (e.g., PR-39 inhibits the production of reactive oxygen species, while BMAP-28 induces apoptosis of activated (infected) lymphocytes) and actively promote tissue regeneration. The neutralizing and resolving effect of cathelicidins provides a balance between the protective and destructive components of inflammation, protecting against lethal conditions, while promoting responses to eradicate the infectious agent. These neutralizing activities also implicate cathelicidins in maintaining homeostasis, particularly in regions of the gut rich in commensals that contain the same bacterial molecules that activate the innate immune response through TLRs. There is also some indication that cathelicidins can act at the interface of innate and adaptive immunity [51], since LL-37 modulates dendritic cell function [9], while both LL-37 and CRAMP can act as adjuvants [2, 26]. Cathelicidins may exert these effects through multiple mechanisms that may involve direct binding to LPS or putative surface receptors or intracellular signaling molecules. It has been reported that cathelicidins activate components of the MAPK signal transduction pathways [5, 26, 27, 47], induce Ca2+ mobilization [26, 33], bind to SH3-domain containing proteins [7, 43], and inhibit NFκB translocation [15, 29]. The biological effects exerted by cathelicidins at a given time and place are likely determined by the physiological setting, including the concentration of the peptide, cellular environment, and soluble components of the extracellular milieu. It has been suggested that at physiological salt and serum conditions, LL-37 is not antimicrobial at the concentrations normally found in adults at mucosal surfaces [3, 4], but it does exhibit immunomodulatory functions under these conditions [3, 25, 40, 47]. Further, LL-37 has been shown to be synergistic with other inflammatory/immune mediators [3, 4, 9]. The antimicrobial and immunomodulatory functions are localized to different regions of the peptide. Further structure-function studies will fuel the synthesis of peptides with specific and potent activities.

In summary, cathelicidins actively participate in the regulation of host defense, exerting antimicrobial activity through direct killing and/or stimulation of biological functions in immune effector cells during the inflammatory and immune response. The functional redundancy between species, the antiseptic activities, and adjuvant properties of cathelicidins makes them attractive antibacterial therapeutic agents. The ability of cathelicidins to reduce inflammatory mediators, induce chemotaxis, and enhance the cell-mediated and humoral immune response in vivo validates these as key functions of cathelicidins in protection against bacterial challenge. However, the vast number of reports under varying conditions illuminates the need for experiments to confirm that in vitro activity is physiologically relevant in vivo.

Acknowledgments We gratefully acknowledge the support of Genome Canada and the Canadian Institutes of Health research for our Peptide research. REWH was supported by a Canada Research Chair Award.

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C

H

A

P

T

E

R

13 Microcins NICHOLAS C. K. HENG AND RALPH W. JACK

have been steady reports of low-molecular-weight “antibiotics” produced by gram-negative bacteria since at least the early 1970s, and they are now divided into two subgroups: class I microcins undergo posttranslational modification during their biosynthesis/processing (see Processing), and class II microcins remain unmodified [35]. More recently, observations concerning the structure and biological activity of microcins, such as those discussed here, have prompted speculation that microcins may represent novel lead structures for the development of new-generation antitumor and anti-infective chemotherapeutic compounds [10–12, 21–23, 27, 28, 35].

ABSTRACT Microcins are peptide antibiotics (<10 kDa) from gram-negative bacteria that share structural and biosynthetic similarities with a number of other bacteriocins from a variety of sources. They range from linear, unmodified peptides to structures that undergo extensive posttranslational modification to yield considerable chemical diversity—for example, peptides with novel heterocyclic backbone structure, carrying novel nucleotide modifications or modifications with catechol-like, iron-binding structures. Their biological activities, like their chemical structures, are widely diverse; some microcins act at the cytoplasmic membrane, instigating depolarization and halting microbial energetics, while others inhibit intracellular functions such as DNA gyrase, RNA polymerase, or mRNA translation. Partly because of their chemical and biological diversity, a number of microcins may warrant further development as novel lead structures in the search for pharmacophores with antitumor and/or antimicrobial activities.

STRUCTURE OF THE PRECURSOR mRNA/STRUCTURAL GENE The structural genes encoding microcins that have been described encode polypeptides with several conserved features and are in many ways very similar to the lantibiotics or other bacteriocins of gram-positive bacteria described in other chapters in this section of the book. Microcins are translated from their respective mRNA as precursor peptides (Fig. 1), composed of a leader peptide and a propeptide portion that in some cases may undergo further posttranslational modification to yield the biologically active molecule [12, 35]. The exception to this rule appears to be MccC7, which does not appear to possess a leader peptide [12, 17, 18]. Proteolytic processing of the leader sequence from a number of microcins appears to occur at a conserved Gly(-2)—Gly/Ala(-1) site, typical of a number of other antibacterial peptides of bacterial origin that are transported by ABC-transport systems incorporating an intrinsic protease domain [20].

DISCOVERY The initial observation that gram-negative bacteria, particularly members of the Enterobacteriaceae family such as Escherichia coli, can produce antibacterial substances that inhibit the growth of other bacteria (a phenomenon known as bacterial antagonism) may date back as far as Louis Pasteur (see [14]) and has been a driving force in antibiotic discovery. Microcins (abbreviated Mcc) are peptide antibiotics (<10 kDa) from gramnegative bacteria and are distinguished by their size from their larger cousins, the colicins (>20 kDa), which are protein antibiotics (see Chapter 18) [2, 24]. There Handbook of Biologically Active Peptides

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MccL:

MREITLNEMNNVSGA - GDVNWVDVGKTVATNGAGVIGGAFGAGLCGPVCAGAFAVGSSAAVAALYDAAGNSNSAKQKPEGLPPEAWNYAEGRMCNWSPNNLSDVCL

MccV:

MRTLTLNELDSVSGG - ASGRDIAMAIGTLSGQFVAGGIGAAAGGVAGGAIYDYASTHKPNPAMSPSGLGGTIKQKPEGIPSEAWNYAAGRLCNWSPNNLSDVCL

MccE492: Mcc24: MccH47: MccB17:

MELRMREISQKDLNLAFGA - GETDPNTQLLNDLGNNMAWGAALGAPGGLGSAALGAAGGALQTVGQGLIDHGPVNVPIPVLIGPSWNGSGSGYNSATSSSGSGS MYMRELDREELNCVGG - AGDPLADPNSQIVRQIMSNAAWGPPLVPERFRGMAVGAAGGVTQTVLQGAAAHMPVNVPIPKVPMGPSWNGSKG MREITESQLRYISGA – GGAPATSANAAGAAAIVGALAGIPGGPLGVVVGAVSAGLTTAIGSTVGSGSASSSAGGGS MELKASEFGVVLSVDALKLSRQSPLG - VGIGGGGGGGGGGSCGGQGGGCGGCSNGCSGGNGGSGGSGSHI

MccJ25: MIKHFHFNKLSSGKKNNVPSPAKGVIQIKKSASQLTK - GGAGHVPEYFVGIGTPISFYG MccC7:

MRTGNAN

FIGURE 1. The amino acid sequences of the precursor peptides of several typical microcins, including microcins B17 (MccB17 [24]), J25 (MccJ25 [43]), C7 (MccC7 [18]), L (MccL [34]), V (MccV [GenBank P22522]), E492 (MccE492 [25]), 24 (Mcc24 [GenBank Q46971]), and H7 (MccH7 [39]). Sequence positions (given above) are relative to the processing site at which the leader peptide is removed (indicated by –). Note that MccC7 does not have a leader peptide.

FIGURE 2. The arrangement and transcriptional orientation of the genetic elements responsible for the production, transport, and immunity to the representative microcins: MccL [34], MccJ25 [43], MccB17 [27], and MccE492 [26]. The functions of the various gene products encoded within the clusters are given in the text.

DISTRIBUTION OF THE mRNA The genetic determinants for microcin biosynthesis occur in a variety of gram-negative bacteria, mainly those belonging to the Enterobacteriaceae. Generally, the structural genes (which may be either plasmid or chromosomally located) are found within gene clusters encoding additional proteins necessary for the maturation of the mature, biologically active microcin (Fig. 2). In the simplest of cases (nonpostranslationally modified microcins—for example, MccL), these gene clusters encode proteins responsible for the precursor peptide (mclC) export out of the cell (mclAB), removal of the leader peptide (unknown) and for producer selfprotection (immunity; mclI) [34]. Surprisingly, biosynthesis of the modified microcin MccJ25 also requires only four gene products, consisting of a structural gene (mcjA), two gene products involved in the novel lactam formation and cleavage of the leader peptide (mcjBC), and an export system (mcjD) [43]. As might be expected, the gene clusters directing the symphony of biosynthetic processes required for the production of highly

modified microcins encode additional proteins (see following) responsible for the posttranslational modification of the propeptide portion of the prepeptide [12]. For example, the MccB17 gene cluster encodes seven distinct products [12, 22, 23]: the MccB17 precursor (mcbA), and the proteins involved in posttranslational modification (mcbBCD), export from the cell (mcbEF), and immunity to microcin B17 (mcbG); the cleavage of the leader peptide is carried out by the instrinsic cellular product of pmbA [39]. Likewise, the catecholatemodified MccE492 gene cluster encodes at least 10 genes encoding the precursor peptide (mceA), an immunity-related peptide (mceB), five products involved in maturation (mceCDE and mceIJ), a protease (mceF), and an export system (mceGH) [25, 26, 44].

PROCESSING In general, microcins are synthesized ribosomally as a precursor peptide from which a leader peptide is cleaved. For example, MccB17 export requires the

Microcins / ABC-transport protein McbEF for export out of the producing cell and the concomitant assistance of PmbA that cleaves the leader peptide and yields the active, mature MccB17 [39]. Interestingly, the transport protein McbEF, in conjunction with a protein of unknown function (McbG), is also required for producer self-protection against the effects of MccB17, suggesting that immunity may involve an efflux system [12, 16]. Other microcins, such as MccE492 contain a dedicated protease and transport system believed to be involved in processing of the leader peptide and transport from the cell, while others (e.g., MccV, Fig. 3) utilize ABC-transport systems incorporating a proteolytic domain for leader peptide removal during export [20]. With respect to posttranslational modification, the biosynthetic processes involved in MccB17 production are undoubtedly the best studied overall (Fig. 3), and it is the first modified peptide antibiotic to be produced by in vitro reconstitution of an enzyme complex

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capable of modifying an exogenously supplied precursor peptide [10, 12, 21–23, 27, 28]. Mature MccB17 contains two residues each of 2-aminomethyloxazole-4carboxylic acid (oxazole) and 2-aminomethylthiazole4-carboxylic acid (thiazole) and one residue each of 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4carboxylic acid [3, 4, 45], that originate from the modification of Gly-Ser, Gly-Cys, Gly-Ser-Cys and Gly-Cys-Ser di- and tri-peptide tracts in the precursor peptide, respectively (Fig. 1). The formation of these hetrocyclic structures is mediated by the microcin B17 synthetase enzyme complex composed of McbB, -C, and -D (Fig. 2) and proceeds from the cyclization of either the serinyl alcohol or cysteinyl thiol with the carbonyl of the preceding glycine residue, followed by dehydration and dehydrogenation to yield the respective oxazole or thiazole; formation of bis-heterocycles follows the same procedure but involves two rounds each of cyclization, dehydration, and dehydrogenation [27]. The

FIGURE 3. Schematic representation for the proposed mechanism of formation of mono- and bis-heterocyclic oxazole and thiazole rings in the backbone of MccB17 A. [27] and the primary structures of MccB17 B. [3, 4], MccV C. [24], and MccC7 D. [18, 19].

78 / Chapter 13 components of the synthetase complex have been studied in detail: McbB is a zinc-binding protein involved in the initial cyclodehydration step [48], McbC has a flavin cofactor and probably carries out the dehydrogenation [31], and McbD is an ATPase/GTPase that acts as the site of initial recognition for the enzyme-substrate complex [30]. In addition, the leader peptide has been shown to be essential for directing posttranslational modification of the propeptide portion [29], and the synthetase complex shows extraordinary regioselectivity and directionality [6, 41]. Although the mechanism(s) by which they occur are not nearly so well characterized, a number of other posttranslationally modified microcins have also been identified (Fig. 3). For example, despite first being reported as an unmodified peptide [36], posttranslational processing of MccE492 is now known to involve the action of a number of additional gene products (Fig. 2) and results in the O-glycosidic modification of the C-terminal residue (Ser84; Fig. 1) with a β-d-glucose, which is itself modified with a trimer of N-(2,3-dihydroxybenzoyl)-L-serine attached via C-glycosidic linkage [44]. Overall, this modification mimics a catechol-like siderophore and is thought to be essential for uptake and targeting of the microcin in susceptible cells. In contrast, the heptapeptide precursor of MccC7 (Fig. 3) is N-terminally formylated, and the C-terminus is modified with the nucleotide adenosine monophosphate aminopropanol [19]. As with other microcins, the MccC7 gene cluster encodes a variety of protein products potentially involved in this novel posttranslational modification or in export or immunity. However, unlike other microcins discussed here, MccC7 does not possess a leader sequence (Fig. 1) and is therefore not further processed during/after export [17, 18].

RECEPTORS Like the colicins (see Chapter 18), a number of microcins interact with specific receptors. The best studied are those microcins that are actively taken up into sensitive cells in an energy-dependent manner via the TonB system [9]. For example, the catecholmodified, ion-channel-forming microcin MccE492 is taken up in a TonB-dependent manner via (at least) three catechol siderophore uptake systems present in sensitive cells [9, 13, 44]. Not surprisingly perhaps, MccH47, which shares significant sequence similarity with MccE492 (Fig. 1), has also been linked to catecholate siderophore production [1]. Similar studies with the RNA polymerase inhibitor MccJ25 have shown that its uptake is also TonB-dependent [9, 12]. Uptake via a specific receptor system appears to confer both specificity and to allow translocation of the respective microcin

across the outer membrane of the gram-negative bacterial target cell.

ACTIVE AND/OR SOLUTION CONFORMATION Analysis of the solution structure of the MccJ25 has revealed some startling features (Fig. 3). Although MccJ25 was initially thought to be 21-amino-acid macrocycle linked head to tail [8], recent NMR and MS analyses have shown that it actually adopts a structure variously referred to as a lassoed tail, a threaded side chain to backbone ring, or a 21-residue lariat protoknot [5, 40, 47]. Overall, the maturation of MccJ25 results in the formation of an internal lactam between the αamino of the N-terminal glycine and the γ-carboxyl of Glu-8. The “tail” of the polypeptide (residues 9–21) is fed through the octa-peptide loop formed by the side chain to backbone cyclization, creating a nooselike structure and is held in place by the bulky, hydrophobic side chains of the aromatic residues Phe19 and Tyr20 that straddle each side of the loop. Surprisingly the noncovalent interaction of these aromatic side chains with the peptide backbone is sufficiently strong to stop the tail from slipping out of the “knot,” even after digestion of the MccJ25 with thermolysin. Moreover, this unique structure appears essential to the biological activity of the MccJ25, and it is interesting to note that thermolysin-cleaved MccJ25 retains biological activity [7]. Analysis of the solution structure of MccB17 (Fig. 3) proved critical in determining the nature and extent of the posttranslational modification in this microcin [3, 4, 28] and the structure of MccB17 has also been confirmed by complete de novo synthesis [45]. Because the heterocyclic nature of MccB17 is reminiscent of a number of antitumor and antibacterial chemotherapeutic agents, a number of synthetic and biosynthetic analogs of MccB17 have been created in order to determine structure function relationships and to define minimum scaffolds for further drug development [42, 49]. From these studies, it has been shown that both the number and position of the rings is critical for antibiotic activity but that the bis-heterocycle had a greater influence, suggesting they may form the basis of useful pharmacophores. Similarly, NMR analysis of MccC7 (Fig. 3) was critical in determining the nature and extent of modification [19]. Interestingly, the peptide unit alone (i.e., without further modification) appears to be sufficient to inhibit translation in vitro, suggesting that the C-terminal modification at the C-terminus is necessary for either transport of MccC7 into the susceptible cell or for targeting of the peptide to the translation machinery.

Microcins /

BIOLOGICAL ACTIONS Different microcins display different biological activities. Currently microcins have been identified acting either as inhibitors of microbial energetics [35], nucleic acid biosynthesis at the level of RNA polymerase, or nucleic acid biosynthesis at the level of DNA gyrase or as inhibitors of translation [12]. For example, a number of microcins, including MccV, MccE492, MccH47, MccL, and Mcc24, have been shown to share numerous characteristics with the unmodified bacteriocins of gram-positive bacteria (see Chapter 17) and are thought to act at the cytoplasmic membrane of the sensitive bacterium to form ion-permeable channels that result in disruption of the proton-motive force and hence ATP production [35]. The molecular mechanism of action of MccB17 has been the subject of considerable research and is therefore probably the best understood of all microcins [12, 22, 23, 27]. The peptide targets DNA gyrase, a topoisomerase that is essential for ATP-dependent negative supercoiling of DNA. Treatment with MccB17 results in trapping of gyrase-DNA complexes in a cleavage complex form, leading to the accumulation of doublestranded DNA breaks and subsequent cell death from the inhibition of DNA replication [46]. While this mode of action is reminiscent of the quinolone antibiotics (e.g., ciprofloxacin), recent evidence suggests that the molecular mechanism is different and intimately involves DNA strand passage, trapping a transiently present intermediate state of the gyrase reaction [34]. This difference in molecular mechanism is one of the forces driving the development of novel anti-infectives based on the unusual bis-heterocyclic structure of MccB17 [10, 22, 27]. The molecular basis for MccJ25 activity appears less clear. Like MccB17, the unusual cyclic peptide MccJ25 also induces SOS-responses and causes cell filamentation. However, it has been shown to interact with, and interfere in the action of, RNA-polymerase in order to exert its antimicrobial activity [11, 12]. At the same time studies have also shown that MccJ25 may be involved in the inhibition of bacterial energetics [37, 38], although this may be due to its application at high concentration, well above that which is necessary for the inhibition of RNA polymerase [12]. Recent analysis of the effects of MccJ25 on mitochondria have shown it can insert into the lipid bilayer membrane dissipating proton motive force in a manner reminiscent of the type A lantibiotics (see Chapter 16) as well as interacting with cytochrome c reductase (complex III), inhibiting its respiratory activity [32]. Taken together, these results have also been one of the drivers behind investigation of the use of such microcins as antitumor and antimicrobial agents [11, 32].

79

Alternatively, treatment of susceptible cells with MccC7 has been shown to markedly reduce protein synthesis without having an effect on RNA synthesis or amino acid uptake. It has therefore been suggested that MccC7 acts as an inhibitor of translation [15]. While it has been shown in vitro that the peptide component is the biologically active portion of the molecule, the molecular mechanism by which translation is effected remains to be elucidated [19].

Acknowledgments The authors wish to thank John Tagg for helpful comments. RWJ wishes to dedicate this publication to his friend, mentor, and the discoverer of the novel heterocyclic nature of microcin B17, Professor Dr. Günther Jung, on the occasion of his 68th birthday.

References Due to space limitations, references have been kept to a minimum and reviews have been cited wherever possible; no sleight to authors of original data is implied or intended. [1] Azpiroz MF, Laviña M. Involvement of enterobactin synthesis pathway in production of microcin H47. Antimicrob. Agents Chemother. 2004;48:1235–1241. [2] Baquero F, Moreno F. The microcins. FEMS Microbiol. Lett. 1984;23:117–124. [3] Bayer A, Freund S, Jung G. Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor. Eur. J. Biochem. 1995;234: 414–426. [4] Bayer A, Freund S, Nicholson G, Jung G. Posttranslational backbone modifications in the ribosomal biosynthesis of the glycinerich antibiotic microcin B17. Agnew. Chem. Intl. Ed. Engl. 1993;32:1336–1339. [5] Bayro MJ, Mukhopadhyay J, Swapna GV, Huang JY, Ma LC, Sineva E, Dawson PE, Montelione GT, Ebright RH. Structure of antibacterial peptide microcin J25: A 21-residue lariat protoknot. J. Am. Chem. Soc. 2003;125:12382–12383. [6] Belshaw PJ, Roy RS, Kelleher NL, Walsh CT. Kinetics and regioselectivity of peptide-to-heterocycle conversions by microcin B17 synthetase. Chem. Biol. 1998;5:373–384. [7] Blond A, Cheminant M, Destoumieux-Garzon D, Ségalas-Milazzo I, Péduzzi J, Goulard C, Rebuffat S. Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity. Eur. J. Biochem. 2002;269:6212–6222. [8] Blond A, Péduzzi J, Goulard C, Chiuchiolo MJ, Barthélémy M, Prigent Y, Salomón RA, Farias RN, Moreno F, Rebuffat S. The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli. Eur. J. Biochem. 1999;259: 747–755. [9] Braun V, Patzer SI, Hantke K. Ton-dependent colicins and microcins: Modular design and evolution. Biochemie. 2002; 84:365–380. [10] Couturier M, Bahassi EM, van Melderen L. Bacterial death by DNA gyrase poisoning. Trends Microbiol. 1998;6: 269–275.

80 / Chapter 13 [11] Darst SA. New inhibitors targeting bacterial RNA polymerase. Trends Biochem. Sci. 2004;29:159–160. [12] Destoumieux-Garzon D, Péduzzi J, Rebuffat S. Focus on modified microcins: structural features and mechanisms of action. Biochimie. 2002;84:511–519. [13] Destoumieux-Garzon D, Thomas X, Santamaria M, Goulard C, Barthélémy M, Boscher B, Bessin Y, Molle G, Pons AM, Letellier L, Péduzzi J, Rebuffat S. Microcin E492 antibacterial activity: Evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli. Mol. Microbiol. 2003;49: 1031–1041. [14] Florey HW. Historical introduction. In: Florey HW, Chain E, Heatley NG, Jennings MA, Sanders AG, Abraham EP, Florey ME. Antibiotics. Oxford University Press. 1949, pp. 4–5. [15] Garcia-Bustos JF, Pezzi N, Mendez E. Structure and mode of action of microcin 7, an antibacterial peptide produced by Escherichia coli. Antimicrob. Agents Chemother. 1985;27:791– 797. [16] Garrido MC, Herrero M, Kolter R, Moreno F. The export of the DNA replication inhibitor Microcin B17 provides immunity for the host cell. EMBO J. 1988;7:1853–1862. [17] González-Pastor JE, San Millán JL, Moreno F. The smallest known gene. Nature. 1994;369:281. [18] González-Pastor JE, San Millán JL, Castilla MA, Moreno F. Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7. J. Bacteriol. 1995;177:7131– 7140. [19] Guijarro JI, González-Pastor JE, Baleux F, San Millan JL, Castilla MA, Rico M, Moreno F, Delepierre M. Chemical structure and translation inhibition studies of the antibiotic microcin C7. J. Biol. Chem. 1995;270:23520–23532. [20] Håvarstein LS, Diep DB, Nes IF. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol. Microbiol. 1995;16:229– 240. [21] Heddle JG, Blance SJ, Zamble DB, Hollfelder F, Miller DA, Wentzell LM, Walsh CT, Maxwell A. The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition. J. Mol. Biol. 2001;307:1223–1234. [22] Jack RW, Bierbaum, G, Sahl H-G. Lantibiotics and related peptides. Springer, Heidelberg, 1998. [23] Jack RW, Jung G. Lantibiotics and microcins: polypeptides with unusual chemical diversity. Curr. Opin. Chem. Biol. 2000;4:310– 317. [24] Kolter R, Moreno F. Genetics of ribosomally-synthesized peptide antibiotics. Ann. Rev. Microbiol. 1992;46:141–163. [25] Lagos R, Villanueva JE, Monasterio O. Identification and properties of the genes encoding microcin E492 and its immunity protein. J. Bacteriol. 1999;181:212–217. [26] Lagos R, Baeza M, Corsini G, Hetz C, Strahsburger E, Castillo JA, Vergara C, Monasterio O. Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin. Mol. Microbiol. 2001;42:229–243. [27] Li YM, Milne JC, Madison LL, Kolter R, Walsh CT. From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase. Science. 1996; 274:1188–1193. [28] Liu J. Microcin B17: posttranslational modifications and their biological implications. Proc. Natl. Acad. Sci. USA. 1994;91:4618– 4620. [29] Madison LL, Vivas EI, Li YM, Walsh CT, Kolter R. The leader peptide is essential for the post-translational modification of the DNA-gyrase inhibitor microcin B17. Mol. Microbiol. 1997;23:161– 168.

[30] Milne JC, Eliot AC, Kelleher NL, Walsh CT. ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17. Biochemistry. 1998;37:13250– 13261. [31] Milne JC, Roy RS, Eliot AC, Kelleher NL, Wokhlu A, Nickels B, Walsh CT. Cofactor requirements and reconstitution of microcin B17 synthetase: A multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17. Biochemistry. 1999;38:4768–4781. [32] Niklison Chirou MV, Minahk CJ, Morero RD. Antimitochondrial activity displayed by the antimicrobial peptide microcin J25. Biochem. Biophys. Res. Comm. 2004;317:882– 886. [33] Pierrat OA, Maxwell A. Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase. Biochemistry. 2005;44:4204–4215. [34] Pons AM, Delalande F, Duarte M, Benoit S, Lanneluc I, Sable S, van Dorsselaer A, Cottenceau G. Genetic analysis and complete primary structure of microcin L. Antimicrob. Agents Chemother. 2004;48:505–513. [35] Pons A-M, Lanneluc I, Cottenceau G, Sable S. New developments in non-post translationally modified microcins. Biochimie. 2002;84:531–537. [36] Pons AM, Zorn N, Vignon D, Delalande F, Van Dorsselaer A, Cottenceau G. Microcin E492 is an unmodified peptide related in structure to colicin V. Antimicrob. Agents Chemother. 2002;46:229–230. [37] Rintoul MR, de Arcuri BF, Morero RD. Effects of the antibiotic peptide microcin J25 on liposomes: Role of acyl chain length and negatively charged phospholipid. Biochim. Biophys. Acta. 2000;1509:65–72. [38] Rintoul MR, de Arcuri BF, Salomón RA, Farias RN, Morero RD. The antibacterial action of microcin J25: Evidence for disruption of cytoplasmic membrane energization in Salmonella newport. FEMS Microbiol. Lett. 2001;204:265–270. [39] Rodriguez-Sainz MC, Hernandez-Chico C, Moreno F. Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17. Mol. Microbiol. 1990;4:1921–1932. [40] Rosengren KJ, Clark RJ, Daly NL, Goransson U, Jones A, Craik DJ. Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. J. Am. Chem. Soc. 2003;125:12464–12474. [41] Sinha Roy R, Belshaw PJ, Walsh CT. Mutational analysis of posttranslational heterocycle biosynthesis in the gyrase inhibitor microcin B17: Distance dependence from propeptide and tolerance for substitution in a GSCG cyclizable sequence. Biochemistry. 1998;37:4125–4136. [42] Sinha Roy R, Kelleher NL, Milne JC, Walsh CT. In vivo processing and antibiotic activity of microcin B17 analogs with varying ring content and altered bisheterocyclic sites. Chem. Biol. 1999;6:305–318. [43] Solbiati JO, Ciaccio M, Farias RN, González-Pastor JE, Moreno F, Salomon RA. Sequence analysis of the four plasmid genes required to produce the circular peptide antibiotic microcin J25. J. Bacteriol. 199;181:2659–2662. [44] Thomas X, Destoumieux-Garzon D, Péduzzi J, Afonso C, Blond A, Birlirakis N, Goulard C, Dubost L, Thai R, Tabet JC, Rebuffat S. Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity. J. Biol. Chem. 2004;279:28233–28242. [45] Videnov GI, Kaiser D, Brooks M, Jung G. Synthesis of the DNA gyrase inhibitor microcin B17, a 43 peptide antibiotic with eight aromatic heterocycles in its backbone. Agnew. Chem. Int. Ed. Engl. 1996;35:1506–1508.

Microcins / [46] Vizan JL, Hernandez-Chico C, del Castillo I, Moreno F. The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase. EMBO J. 1991;10:467–476. [47] Wilson KA, Kalkum M, Ottesen J, Yuzenkova J, Chait BT, Landick R, Muir T, Severinov K, Darst SA. Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. J. Am. Chem. Soc. 2003;125:12475–12483.

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[48] Zamble DB, McClure CP, Penner-Hahn JE, Walsh CT. The McbB component of microcin B17 synthetase is a zinc metalloprotein. Biochemistry. 2000;39:16190–16199. [49] Zamble DB, Miller DA, Heddle JG, Maxwell A, Walsh CT, Hollfelder F. In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites. Proc. Natl. Acad. Sci. USA. 2001;98:7712–7717.

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14 Peptaibols L. WHITMORE AND B. A. WALLACE

considerable sequence identity between the peptaibols, especially between peptides isolated from the same biological source. It is possible to group the peptaibol sequences from a range of biological sources into subfamilies according to their sequences. At present more than 300 peptaibol sequences have been identified; however, very few high-resolution structures of peptaibols have been reported. All of the currently available structures are primarily helical, as would be expected, since Aib is a strongly helix-promoting residue. The helical nature of these peptides is key to their biological activity, which appears to be primarily to form helical bundles that facilitate ion transport across membranes. This activity is harmful to other organisms and can cause cell death, which has led to the speculation that fungi produce peptaibols as a passive defense mechanism. As a result, many peptaibols are effective antibiotics, with differential potencies toward different target organisms. An online searchable database of natural peptaibol sequences, structures, nomenclature, and literature references [56, 57] may be found at http:// peptaibol.cryst.bbk.ac.uk.

ABSTRACT The peptaibols are a large family (>300 members have been identified thus far) of homologous peptides isolated from soil fungi, ranging in length from 5 to 20 amino acids. They are characterized by having a high content of alpha-aminoisobutyric acid (Aib) residues and generally have both N-terminal modifications (mainly acetyl groups) and C-terminal modifications in the form of amino alcohol groups rather than amino acids. Peptaibols are nonribosomally synthesized on large protein complexes that permit the incorporation of nonstandard amino acids. During the process of peptide chain elongation, some of the synthetase modules are not absolutely specific for amino acid type, resulting in a diversity of isoforms being produced by a single organism. Most members of the family exhibit antibacterial or antifungal activity, which has been attributed to their ability to induce membrane permeability by forming multimeric helical bundle ion channels.

INTRODUCTION CHARACTERISTIC FEATURES OF THE PEPTAIBOLS

The peptaibols are a family of short peptides isolated from soil fungi. Their name is derived from a combination of the words PEPTide, AIB and alcohOLs, the characteristic features of the molecules. They are mainly distinguished by compositions consisting of a large number of “nonstandard” amino acid residues, with Aib accounting for a high proportion of all residues in each of the sequences (for examples of some peptaibol sequences, see Fig. 1). Peptaibols are synthesized on large protein synthetase complexes rather than ribosomally, which permit the inclusion of nonstandard, noncoded residues. As would be expected for a family of peptides with a single dominant residue, there is Handbook of Biologically Active Peptides

An important feature of peptaibol sequences is the presence of a high proportion of alpha, alpha disubstituted amino acids, primarily Aib. The Aib residue, otherwise known as alpha-methylalanine, has two methyl groups attached to its alpha carbon atom, making it more hydrophobic and conformationally constrained than the standard (ribosomally coded) alanine (see Fig. 2). Another disubstituted residue found in peptaibols is the related isovaline, which is also known as methylethylalanine. The percentages and locations of Aib residues in peptaibol sequences vary among members of

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84 / Chapter 14 a) Ac Phe b) Ac Aib c) Ac Aib Pro d) Ac Leu e)

Aib Aib Aib Iva Gly Leu Aib Gly Aib Leu Aib Glu Aib Aib Aib Ala Aib Ala Gln Aib Val Ac Aib Aib Leu Aib Phe OH

Aib Aib Aib Asn

Hyp Ala Gly Ile

Gln Aib Leu Ile

Iva Aib Aib Aib

Pro Pro Pro Pro

Aib Leu Val Leu

Pro Aib Aib Leu

Phe Iva Aib Aib

OH Gln Val OH Gln Gln Phe OH Pro Ile OH

FIGURE 1. Examples of sequences of peptaibols of different lengths. a) Antiamoebin II, b) Trichotoxin A_40, c) Alamethicin F_50, d) Trichorovin TV_XIIa, and e) Peptaibolin. Alphaaminoisobutyric acid (Aib) residues are shaded in light gray, and proline (Pro) residues are shaded in dark gray.

H3C CH3

CH3 H2C

H3C C HO

C

C HO

O Aib

FIGURE 2.

NH2

NH2

C O Iva

Chemical structures of Aib and Iva residues.

the family [58]. However, no peptaibols have been discovered that feature a C-terminal hydroxylated Aib residue, and in the longer sequences, Aib has not been found at positions 18, 19, or 20. On average, 38% of peptaibol residues in the known peptaibols are Aib, and 3% are isovaline; the upper and lower ranges for Aib composition of individual sequences are 56% and 14%, respectively. There are Aib content trends within groups of peptaibols, such as the shorter peptaibols containing greater proportions of Aib than the longer ones. The distribution of Aib residues and their neighbors is not random, as certain pairs of residues occur more frequently than expected statistically. A prime example of this is that virtually all proline residues in peptaibols (98%) have Aib as their N-terminal neighbor. The AibPro peptide bond forms the characteristic kink, which is often seen in the peptaibol structures. Furthermore, 100% of hydroxyproline residues are preceded by disubstituted amino acids (75% Aib and 25% isovaline). While Aib is the most common noncoded residue to occur in peptaibols, it is not the only one. The imino acid hydroxyproline comprises 2% of all residues, while etnorvaline (alpha-amino-alpha-ethyl-n-pentanoic acid) and 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMO) are also found but in fewer than 10 examples across all known peptaibols. As well as containing these nonstandard amino acids, peptaibol sequences also have a distinct lack of many of the regular charged and polar amino acids [58]. While the functionally important glutamine is abundant in peptaibol sequences, constituting almost 11% of the

residues and featuring in 86% of the sequences, there are as yet no known peptaibols that contained any arginine, asparagine, cystine, histidine, lysine, or methionine residues, and there is only one example of a tyrosine-containing peptaibol. Glutamines (or occasionally glutamic acids) are found at specific locations, primarily at position 6 or near the C-terminus; these appear to be crucial for functional activity. Prolines are also frequently found and are often located near the middle of the sequences. These residues tend to break helical structures because of the absence of the amino protons needed for forming intramolecular hydrogen bonds. It has been proposed that they may be responsible for kinks or bends in the molecules that may be related to their ion channel gating properties [46]. Aromatic residues also tend to feature prominently in peptaibol sequences, especially at the termini, where it is thought that they may act to stabilize the transmembrane orientations of the peptide chains, much as has been proposed for the transmembrane segments of membrane proteins [55]. A further sequence feature of peptaibols is that both their N- and C-termini tend to be modified. The Nterminal amino acids are typically attached to an acyl group, although other aliphatic groups such as octyl groups in the trikoningins [4], and decyl groups in LP237 F7 [52], have been reported. The C-terminal residues generally have their carbonyl groups reduced to hydroxyl groups (hence they are amino alcohols rather than amino acids), although there are some variations on this. Clonostachin is a peptaibol that has an ester-linked sugar alcohol terminal group [16]. Sequence length is often considered to be a defining feature of peptaibols. The shortest known natural peptaibol, peptaibolin, is five residues long [28], and the longest ones are 20 residues long. Of all known peptaibols, the greatest number (>30% of all sequences) have 20 residues, and the distribution of sizes shows a decreasing trend with decreasing size (Fig. 3). Even-numbered members predominate over odd-numbered ones, with the exception of the highly populated 11-residue group (which could be found more frequently because it corresponds to a half-bilayer thickness) [58].

Length of Sequence

Peptaibols / 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

10

20

30

40

50

60

70

80

90

100

Number of Examples

FIGURE 3. [58]).

Distribution of peptaibol sequence lengths (after

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are indications that other factors may modify the peptaibol sequence: Raap et al. [40] recently showed that zervamicins produced in a medium containing labeled isovaline will contain both labeled isovaline and labeled Aib residues. Some evidence hints at other aspects (perhaps postsynthetic modification) of the peptaibol synthesis process. Sharman et al. [48] have shown that XR586, a peptaibol-like peptide from Acremonium persicinum, is sequentially very similar to the zervamicins, although it terminates in the sequence Phe-Gly (i.e., with an extra Gly), rather than the Phe residue common to the zervamicins. This might suggest that the final stage of synthesis of some peptaibols could include a cleavage enzyme that is not present in all organisms.

PEPTAIBOL STRUCTURES BIOLOGICAL ORIGINS AND BIOSYNTHESIS OF PEPTAIBOLS All known peptaibols have been isolated from soil fungi or molds. Many of the peptaibols have been isolated from various Trichoderma species, but others have come from Hypocrea peltata [38], Hypocrea muroiana [6], Clonostachys [16], Emericellopsis donezkii [7], Emericellopsis salmosynnemata [33], Apiocrea chrysosperma [20], Sepedonium ampullosporum [45], Acremonium tubakii [47], Mycogone cervina [60], Mycogone rosea [27], and Verticimonosporium ellipticum [36]. Some sequences are known to be produced by different organisms. Antiamoebins, for example, have been isolated from at least three species: sequences I to V are from Emericellopsis poonensis, Emericellopsis synnematicola, and Cephalosporium pimprina [45], while sequences VI to XVI are obtained from Stilbella erythrocephal, Stilbella fimetaria, and Gliocladium catenulatum [31]. Peptaibols are not formed ribosomally but are enzymatically synthesized on large nonribosomal peptide synthetase (NRPS) complexes [59]. The peptaibols are assembled by these NRPSs from various precursor units, which may be either standard or nonstandard amino acids or their derivatives. The synthetases have a modular structure, with different modules independently responsible for attaching specific consecutive residues. Synthetase complexes have been cloned and expressed and, depending on the modules present, used to produce defined peptaibol molecules [59]. Some modules appear to be specific for particular residues, while others have two or three specificities and will attach residues according to their availability within the growth medium [59]. This variable specificity leads to the micro-heterogeneity of the peptaibols extracted from a given organism. Also, there

Various methods have been employed to group peptaibols according to their sequences. The oldest classification method was based on sequence length and terminal groups. The groupings according to this classification system are “long,” “short,” and “lipopeptaibol.” The long peptaibols are characterized by a sequence length of 18–20 residues, such as the saturnisporins [41]. Short peptaibols have sequence lengths in the range of 11–16 residues and include the trichorovins [53] (11 residues) and heptaibin [30] (15 residues). It should be noted that this classification was made before the identification of 17 residue peptaibols, such as cephaibols P and Q [47]. Lipopeptaibols are 11 residues or shorter and have fatty acids such as octanoic acid at their N-termini, as exemplified by trichogin A IV [3]. A classification method based on sequence homology was devised by Chugh and Wallace in 2001 [18]. Homology grouping into subfamilies, with special attention to functionally important residues, had the advantage of being more sequence length-independent, and tended to cluster family members with similar functional properties. A total of nine distinct subfamilies with sequence identities of greater than 50% were identified. The advantage of this classification was that it made clear that the large number of closely related sequences provides a naturally occurring pool of “mutants” with slightly modified functional properties that can be used to facilitate structure/function studies. In contrast to the wealth of information available about peptaibol sequences, there are relatively few structures of peptaibols, and even among them not all of the coordinates are publicly available. The latter is probably because peptaibols fall in the intermediate

86 / Chapter 14 size category, smaller than the macromolecular structures traditionally found in the Protein Data Bank [8] and larger than the small molecule structures deposited in the Cambridge Structural Data Bank [1]. To date there are only a limited number of x-ray structures [13, 17, 19, 24, 33–35, 50, 51] and even fewer deposited NMR structures [2, 5, 23, 25, 26, 49]. For one of the peptaibols, antiamoebin I, there are two crystal structures (in different solvents) [34, 50] and an NMR structure [26]. For zervamicin IIb, there are two NMR structures in different environments [5, 49]—one from an isotropic solvent and one in micelles—as well as a crystal structure for a modified version of this peptaibol [33]. The availability of structures in different environments (solution and crystal, different solvents) means that the flexibility and dependence of the structures on their environments can be examined. The dearth of structures (there are structures for less than 3% of the known sequences) may reflect the inherent difficulties of crystallizing membrane-active peptides and solving intermediate-sized crystal structures [54] and of determining structures of peptides with high redundancy of residue types from NMR data [26]. Structural information (albeit in less detail) under various solvent conditions and/or in lipid bilayers or micelles has been frequently obtained using circular dichroism (CD) spectroscopy. CD information is available for hypelcins [38], trichorzianines [9, 42], alamethicin [14, 32], paracelsin [11], trichogins [3], trikoningins [4], trichotoxin A-40 [12], chrysospermins [20], trichosporins [29], saturnisporins [41], harzianins [43], trichologins [44], antiamoebin [50], and XR586, a peptaibol-like antibiotic molecule [48]. They all have common spectral shapes, featuring negative peaks at ∼205–209 nm and 221–226 nm, and positive peaks ∼192–198 nm (the ranges reflect the total span of peak positions quoted in the literature), which are characteristic of helical structures. Because the structures are highly constrained by extensive hydrogenbonded networks, the structures tend to show little solvent-dependence, although their spectra (especially the peak positions) do exhibit a significant dependence on solvent permanent dipole (dielectric constant) [15]. All of the peptaibol structures determined to date include helical conformations, although they exhibit considerable structural diversity in the type of helix (310 or alpha) present and in whether they are straight or bent [50]. The bending tends to be a result of the number and position of the proline or hydroxyproline residues present (although not all imino acid-containing peptaibols are bent—see the structure of trichotoxin, which has a central proline, but is essentially a straight helix) [17].

FIGURE 4. End-on view of the hexameric bundle pore structure of trichotoxin, after [21].

FUNCTIONS AND ACTIVITIES OF PEPTAIBOLS Functionally, most peptaibols exhibit antibacterial and antifungal properties against different target organisms. For example, chrysospermins display activity against a range of bacteria, including gram-negative Escherichia coli [20], while trikoningins inhibit the grampositive bacterium Staphylococcus aureus [4]. Antifungal behavior includes that of hypelcin A, which prevents the growth of Lentinus edodes, a Japanese edible mushroom and other fungi [38]. The physical basis of their anitibiotic properties appears to lie in the abilities of peptaibols to permeabilize lipid bilayers. They can either form transmembrane pores or carriers [50] or modify the membrane surface in a surfactant-like mode. When pores are formed in membranes, they generally take the form of voltage gated ion-channels [21, 22, 24, 46]. Voltagedependent activity is usually associated with the longer peptaibols, which can directly span a bilayer, but it has been observed in peptaibols as short as 14 residues [43]. The channels themselves are thought to be formed by several peptaibol molecules coming together to form a bundle [10, 21, 46], which inserts into the membrane, creating a hydrophilic channel through its center [14] (Fig. 4). The longer peptaibols (16 to 20 residues), when in a helical conformation, are of appropriate lengths to span a lipid bilayer [54]. The number of peptide chain in the bundle differs depending on the peptaibol type, ranging from 6 to more than 12. Somewhat surprisingly, the membrane activity of peptaibols is as pronounced in some short peptides as it is in longer ones. Auvin-Guette et al. [4] reported that in the trikoningins, the 11-residue lipopeptaibols KB I and KB II show similar antibacterial activity against Staphylococus aureus, as does the 19-residue KA V. Similarly, the 11-residue trichogin A IV was shown to have significant membrane-modifying properties in

Peptaibols / experiments designed to measure carboxyfluorescein leakage from egg phosphatidycholine unilamellar vesicles [3]. Clearly, the shorter peptaibols cannot span a membrane in the same way that the longer ones can. Two possible methods of action could occur: either the shorter peptaibols form two end-to-end bundles within the bilayer, thus effectively doubling up their length perpendicular to the bilayer, or they simply associate with the membrane surfaces and alter the permeability of the bilayer. Finally, while membrane permeabilization is clearly a feature common to the peptaibols, it is not certain that these activities represent their true biological function within the native organism. One could speculate that they could act as a defense mechanism in vivo against other organisms with different membrane characteristics to their own, especially as many of them exhibit differential activity towards different membrane lipid types [21, 22]. In summary, the peptaibols are a naturally occurring family of hydrophobic nonribosomally produced peptides that appear to act primarily in altering membrane permeabilities. They exhibit considerable sequence diversity [18] and thus represent an excellent natural resource of “mutants” or homologs, which can be used to elucidate structure/function roles of specific residues in ion channel formation and activity [39].

Acknowledgments This work was supported, in part, by grants from the UK Biotechnology and Biological Sciences Research Council to BAW. The original idea of creating a Peptaibol Database was suggested by Dr. Chris Snook when he was a student in the Wallace Laboratory. The database was designed and is curated by Dr. Whitmore. Some updates of the database and curation were the work of Dr. Jasveen Chugh. We thank both Drs. Chugh and Snook for helpful discussions, and Dr. Chugh for Fig. 4.

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15 Nonribosomally Synthesized Microbial Macrocyclic Peptides JAN GRÜNEWALD AND MOHAMED A. MARAHIEL

are synthesized by heterocyclization of threonine residues [14]. Subsequent oxidation of the oxazoline ring leads to oxazole, as found in the telomerase inhibitor telomestatin 2 [28]. This remarkable peptide product contains not only the constraint imposed by heterocyclization but also a second cyclization constraint that is based on macrocyclization. Although structurally diverse, many nonribosomal-produced peptides share this structural key feature. Macrocyclization can be realized by “head to tail” lactam formation as in the case of the antibiotic tyrocidine A 3 [19]. Branchedcyclic structures are also common as seen for the antibiotic bacitracin 4, which is composed of a heptapeptide lactam ring derived from cyclization of the L-lysine side chain onto the C-terminal carboxyl group [6]. In addition to macrolactamization, diversity is also increased by various macrolactonization strategies. In the case of the lipodepsipeptide surfactin A 5, the branch point is a β-hydroxylated fatty acid moiety [36]. In contrast, the daptomycin 6 and fengycin 7 lipopeptide antibiotics [11, 32] contain hydroxylated amino acid side chains as branch points. The scope of macrocyclic lactones and lactames can be further broadened by cyclo-oligomerization strategies. The macrolactam gramicidin S 8, for example, is composed of two identical pentapeptides bridged head to tail [18], whereas the siderophore bacillibactin 9 has been cyclo-trimerized to give an iron-chelating macrolactone [24]. Finally, an alternate biosynthetic strategy to constrain the conformation of nonribosomal peptides is exemplified by the glycopeptide antibiotic vancomycin 10 [10]. Oxidative crosslinks that occur between electron-rich aryl side chains convert this acyclic, floppy heptapeptide into rigid, cup-shaped scaffolds. This constraint structure sequesters D-Ala-DAla termini of bacterial peptidoglycan strands with five hydrogen bonds and inhibits the transpeptidation reaction [37].

ABSTRACT Nonribosomally synthesized microbial peptides constitute a large set of highly diverse natural products that show a great structural diversity. Peptide synthesis takes place on large multienzyme complexes, the nonribosomal peptide synthetases (NRPS), which simultaneously represent template and biosynthetic machinery. In contrast to the ribosomal machinery, which relies on the 20 proteinogenic amino acids, the small NRPS assembled peptides may contain hundreds of different building blocks, including, for example, D-amino acids, heterocyclic elements, glycosylated, and N-methylated residues. The sphere of activity of these complex natural products reaches from antibiotic to immunosuppressive, cytostatic to toxic, a fact that makes them attractive targets for the development of new drugs with improved or altered activities.

STRUCTURAL DIVERSITY OF NRPS SYNTHESIZED PEPTIDES A common feature of many nonribosomally produced peptides is their constraint structure, which ensures the presentation of the proper functionality in the precise orientation required for interaction with a dedicated molecular target [12, 19, 30]. This structural rigidity is achieved by either cyclization or oxidative cross-linking of side chains, which constrain a nonribosomal peptide to a biologically active conformation. Selected structures of some nonribosomally produced bacterial peptides are shown in Fig. 1. Heterocyclization is one important structural feature of these secondary metabolites that mediates selective interaction with proteins, RNA, and DNA targets [37]. For example, the iron-chelating siderophore vibriobactin 1 contains two oxazoline rings that Handbook of Biologically Active Peptides

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FIGURE 1. A selection of nonribosomally synthesized peptides. Ester or amide linkages of peptidic macrolactones/macrolactames are highlighted by shading.

MODULAR STRUCTURE OF NRPSs NRPSs are large multifunctional enzymes of remarkable size, which represent at the same time template and biosynthetic machinery. The calcium-dependent antibiotic (CDA) synthetase from S. coelicolor, for instance, consists of three subunits—CDA1 (799 kDa), CDA2 (395 kDa), and CDA3 (259 kDa)—which are encoded on an 82 kb region of the genome [3]. The subunits can be further subdivided into 11 modules, which specifically incorporate 11 dedicated amino acids

into the peptide backbone of CDA 11 (Fig. 2) [9]. These modules contain domains necessary to catalyze the individual steps of nonribosomal peptide synthesis. Specifically recognized amino acids are first activated by the adenylation domain (A domain, about 550 amino acids). By analogy to aminoacyl-tRNA synthetases, this process consumes ATP and generates a highly reactive aminoacyl adenylate [5]. The second step of the nonribosomal machinery is the transfer of the activated amino acid to the free thiol group at the terminus of the cofactor phosphopantetheine, which is

FIGURE 2. The nonribosomal CDA synthetase consists of 11 modules (gray and black), which specifically encode 11 amino acids. The modules are further subdivided into 37 catalytically independent domains responsible for substrate recognition/activation (A domain), binding (T domain), elongation (C domain), epimerization (E domain), and release by cyclization (TE domain) HPG, 4-hydroxyphenylglycine.

92 / Chapter 15 posttranslationally attached to an invariant serine residue in the thiolation domain (T domain, or peptidyl carrier protein (PCP), about 80 amino acids) [33, 38]. The amino acid bound to the downstream T domain and the activated thioester of the upstream T domain-bound intermediate serve as substrates of the condensation domain (C domain, about 450 amino acids), which catalyzes peptide bond formation [4]. The N-terminal sequence of CDA1 (about 495 amino acids) presumably constitutes a condensation-like domain (C′) that is involved in the transfer of a putative fatty acid CoA moiety to the serine residue attached to the T domain of the initiation (first) module [9]. In addition to these essential A, T, and C domains, which constitute an elongation module, extra enzyme activities (optional domains) can be found within some modules of the CDA synthetase. For example, the epimerization domain (E domain, about 400 amino acids), which is found in modules 3, 6, and 9, catalyzes racemization (equilibration between L and D isomers) of the respective T domain bound amino acids [23]. To ensure selective incorporation of the D-amino acids into the CDA peptide backbone, the corresponding Cterminally adjacent C domains are D-specific for the incoming cofactor-bound peptide chains [4]. In contrast to modifications, which are catalyzed by optional domains, modifications of the peptide backbone can also be introduced by NRPS-associated tailoring enzymes. The genes encoding these enzymes are typically associated with the NRPS genes in the same biosynthetic gene cluster. In the case of CDA, a putative asparagine oxygenase (AsnO) might be required for the β-oxidation of D-Asn at position 9 [9]. This is consistent with the existence of the gene asnO in the CDA biosynthetic gene cluster, which exhibits high similarity with clavaminate synthase (Cas) 1 and 2, that catalyzes β-hydroxylation of the arginine of a β-lactam precursor of clavaminic acid [40].

MACROCYCLIZATION The C-terminal module of NRPSs, also referred to as a termination module, typically contains a thioesterase domain (TE domain, about 250 amino acids) to cleave the fully assembled peptide from the multienzyme. Chain release takes place by transferring the cofactorbound peptide chain from the T domain to an active site serine residue of the adjacent TE domain to generate an acyl-O-TE intermediate [27]. Following this acylation, the TE domain is deacylated either by an intramolecular reaction with an internal nucleophile to release a macrocyclic peptide or by hydrolysis to give a linear peptide acid. The latter strategy is exemplified by the glycopeptide antibiotics of the vancomycin and

teicoplanin class, whose peptide backbones are constrained by oxidative crosslinks that occur between electron-rich aryl side chains [10]. However, in most cases the structural rigidity of nonribosomal peptides is achieved by macrocyclization. For example, the conformation of CDA 11 is constrained by a decapeptide lactone ring derived from TE-mediated cyclization of an L-threonine side chain onto the C-terminus [9]. In this case, the acyl-O-TE intermediate is kinetically sequestered from water, while a specific conformer of the acyl chain is populated to allow chemoselective capture of the acyl chain carbonyl by the L-threonine side chain at position 2 as shown in Fig. 2. To date, many cyclization strategies have been uncovered, which give rise to a large and diverse set of cyclic or cyclic branched molecules with distinct biological activities as just described. This great versatility in TEcatalyzed cyclization reactions is reflected by the low overall identity among TE domains, which is in the range of 10–15% [26]. Moreover, these peptide cyclization catalysts regioselectively choose only one specific residue of the acyl-O-TE intermediate from a large source of nucleophilic residues for cyclization [19].

STRUCTURE OF THE SURFACTIN THIOESTERASE DOMAIN The structure of the excised macrocyclizing TE domain from surfactin synthetase (Srf TE) responsible for surfactin A biosynthesis has been determined by Bruner et al. [2]. In agreement with the previously observed homology with lipases and serine hydrolases, nonribosomal TE domains belong to the superfamily of α/β hydrolases. In common with other α/β hydrolases, there is a conventional catalytic triad composed of serine, histidine, and aspartic acid. Further structural studies revealed a possible docking site for the T domain, suggesting that the phosphopantetheine prosthetic group with the bound peptidyl chain is directed through a cleft into the active site of the TE domain (Fig. 3). The serine residue of the catalytic triad is then in position to nucleophilically attack the acyl-thioester of the upstream holo-T domain. The resulting negative charge of the tetrahedral enzyme-linked intermediate is stabilized by an oxyanion hole. Further studies with a cocrystallized boronic acid inhibitor revealed well-defined binding pockets for the two C-terminal leucine residues of surfactin, while the rest of the peptide seemed to be less ordered [36]. In addition, the requirement of these aliphatic residues for efficient macrocyclization has been confirmed by biochemical studies. Peptide cyclization may be passively directed through the bowl-shaped hydrophobic active site that accommodates the acylO-TE intermediate [30]. Acyl-O-TE breakdown then

Nonribosomally Synthesized Microbial Macrocyclic Peptides /

FIGURE 3. Accessible surface representation of Srf TE. A modeled phosphopantetheine arm carrying a five-amino acid peptide points into the active site cavity. The putative T-domain interaction site is indicated in yellow. Structural details are described by Bruner et al. [2]. (See color plate.)

occurs by intramolecular nucleophilic attack of the fatty acid β-hydroxyl group on the acyl-enzyme ester bond to exclusively release the macrolactone. This high chemoselectivity for cyclization may be triggered by a rigid proline residue, which is involved in the formation of the oxyanion hole [36]. Sequence alignment studies confirmed the conservation of this residue among TE domains, whereas the lipases, which exclusively hydrolyze their substrates, contain a conserved glycine residue in this region of the active site. The flexible glycine residue may increase the conformational freedom of the oxyanion hole and creates more access for water to capture the acyl-enzyme intermediate. In fact, mutation of proline to glycine in the Srf TE resulted in a 12-fold change in the product partition ratio in favor of intermolecular hydrolysis [36]. This residue may therefore represent a switch between cyclic or linear product outcome among α/β hydrolases.

BIOCHEMICAL CHARACTERIZATION OF EXCISED THIOESTERASE DOMAINS NRPS cyclases are stereo- and regioselective peptide cyclization catalysts. From the synthetic point of view, disconnected TE domains could be highly valuable for the production of novel cyclic compounds in vitro [1]. Since synthetic chemistry faces many difficulties in the reliable production of cyclic peptides, recent work has aimed to combine solid-phase peptide chemistry with peptide cyclization mediated by excised NRPS cyclases. This chemoenzymatic approach was first demonstrated by Trauger et al., who disconnected the TE domain from the tyrocidine synthetase (Tyc TE) from Bacillus brevis [34]. After overexpression and purification, the

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excised TE domain was incubated with a chemically synthesized decapeptide thioester (Fig. 4). Specifically, N-acetylcysteamine (SNAC) was used as a short mimic of the T domain-bound cofactor phosphopantetheine. The authors found specific cyclization activity, which resulted in the formation of tyrocidine A 3. However, hydrolysis of the decapeptide thioester was also observed to a lesser extent. Since the tyrocidine cyclase is usually embedded in a hydrophobic multienzyme complex, its production as an excised TE domain may enhance the exposure of the active site to water [30]. Recently, it was shown that the addition of nonionic detergent significantly improves the cyclic to hydrolyzed product ratio of the recombinant Tyc TE [39]. In this case, detergent micelles may provide a hydrophobic environment that prevents capture of the acyl-O-TE intermediate by water molecules. Detailed biochemical studies have shown that only the substitution of amino acids near the end of the decapeptide thioester significantly decreased the efficiency of the catalyzed macrolactamization [34]. Moreover, it was shown that the excised Tyc TE catalyzes cyclization to form 6–14 residue cyclic peptides [18]. Synthesis of a modified substrate in which the N-terminal amino group of the tyrocidine decapeptide was replaced with a hydroxyl group revealed that this cyclase can also be used as a macrolactonization catalyst [35]. Finally, systematic alteration of the peptide backbone either by the replacement of amino acid blocks with flexible spacers or by replacement of individual amide bonds with ester bonds has been used to probe the role of backbone hydrogen bonds in peptide cyclization. The results suggested that at least two backbone-to-backbone hydrogen bonds may contribute to preorganization of the peptidic substrate for Tyc TE-mediated cyclization. Based on these studies, a model of a minimal cyclization substrate for this cyclase was postulated [35]. To explore the generality of cyclization catalyzed by excised TE domains, additional NRPS cyclases were disconnected from their respective multienzymes and assessed for their ability to catalyze peptide cyclization in vitro. The Srf TE from the surfactin synthetase assembly line catalyzes macrolactonization of an Nacylated lipoheptapeptide. The corresponding excised TE domain retains macrocyclization activity with a 3hydroxybutyryl-heptapeptidyl SNAC substrate [29]. However, in contrast to the recombinant Tyc TE, this peptide cyclase shows much less substrate tolerance. For example, insertion of two additional residues into the peptide thioester completely abolished cyclization yields [36]. Moreover, a change in the cyclization nucleophile from a hydroxyl to an amine group was not tolerated. These observations may be explained by a lower β-sheet content of the heptapeptide surfactin compared to the decapeptide tyrocidine. This lower β-sheet content may

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FIGURE 4. Chemoenzymatic cyclization mediated by excised Tyc TE is shown. The active site serine residue of Tyc TE is acylated by the reactive peptidyl thioester. The generated acyl-O-TE intermediate is then captured either by the N-terminus to generate a decapeptide lactam or by water to release the linear peptide acid. Recognition of the artificial substrate by the cyclase is ensured by the phosphopantetheine cofactor mimic SNAC (highlighted by shading).

hamper substrate preorganization by backbone-tobackbone hydrogen bonds and may therefore contribute to the lower substrate tolerance of Srf TE. Alternatively, substrate presentation by the SNAC leaving group might have been insufficient for efficient peptide cyclization catalysis [30]. Surprisingly, substitution of SNAC with thiophenol resulted in a 15-fold higher catalytic cyclization efficiency for a linear surfactin heptapeptide thioester based on kcat/KM values [31]. Although thiophenol is not a cofactor mimic, it has excellent leaving group properties due to delocalization of the thiolate electrons throughout the aromatic ring system. Therefore, this result emphasizes that chemical reactivity is more important for enzyme acylation than structural similarity to the phosphopantetheine cofactor. Remarkably, activity-based cyclase acylation with peptidyl thiophenol substrates led to the formation of macrocyclic products of the nonribosomal fengycin (Fen), mycosubtilin (Myc), and syringomycin (Syr) TE domains, which displayed no cyclization activities for the corresponding peptidyl SNAC substrates [31]. An alternative approach to characterize excised TE domains that show no activity with conventional peptidyl SNAC substrates was developed by Sieber et al. [32].

To overcome the limitation of this short cofactor mimic, the respective TE domain was excised with the preceding apo-T domain. The recombinant didomain was then loaded in vitro with peptidyl CoA by using the promiscuous phosphopantetheine transferase Sfp from B. subtilis. The cofactor bound peptide chain of the holo-T-TE didomain mimics the natural substrate presentation in the NRPS multienzyme. Using this strategy, the branched-chain cyclization catalyzed by Fen cyclase was characterized. However, the single turnover nature of the reaction has proved to be a limitation. After product release by the TE domain, the phosphopantetheine cofactor remains attached to the didomain, which blocks further Sfp mediated transfer of additional peptidyl-CoA onto the cofactor.

CHEMOENZYMATIC APPROACHES TOWARD NOVEL CYCLOPEPTIDES Excised TE domains can also catalyze macrocyclization of peptide substrates that are dramatically different from their native linear precursors. In the case of Tyc TE, the broad substrate tolerance of this cyclase was

Nonribosomally Synthesized Microbial Macrocyclic Peptides / used to design cyclopeptides with different therapeutic potential. Incorporation of an Arg-Gly-Asp (RGD) sequence motif into the linear peptide precursor led to the formation of macrolactames that confer nanomolar potency in the inhibition of ligand binding by integrin receptors [17]. The versatility of Tyc TE to cyclize diverse linear peptides was further utilized in an approach for production of cyclic hybrid peptide/ polyketides [16]. Specifically, it was demonstrated that ε-amino acid building blocks can be used to introduce polyketide epitopes into linear peptide structures, which are subsequently macrocyclized upon reaction with Tyc TE. This chemoenzymatic route could be used to further optimize macrocyclic peptide/polyketide natural products, such as the immunosuppressant rapamycin and the anticancer agent epothilone [13]. The chemoenzymatic potential of Tyc TE was also used to generate carbohydrate-modified cyclic peptide antibiotics. Using this cyclase, various macrocyclized tyrocidine decapeptide derivatives were synthesized with unnatural propargylglycine residues incorporated at position 3 to 8 [22]. The peptide backbones containing these alkyne amino acids allowed subsequent postsynthetic modification to selectively introduce azido-functionalized sugar residues by copper(I)mediated [2 + 3] cycloaddition reactions. Using this “click chemistry,” glycopeptides with more than sixfold improved therapeutic indexes (MHC/MIC) over the native tyrocidine A 3 were obtained while maintaining the antibacterial potency of this nonribosomally produced antibiotic. Later, Walsh and coworkers developed an alternative method to prepare glycosylated tyrocidine analogs. This approach is based on the chemical incorporation of glycosylated amino acids into linear peptide precursors to act as substrates for Tyc TE [21]. Extension of the glycosylation strategies to other nonribosomal cyclopeptide scaffolds may provide a useful way to increase aqueous solubility and to reduce hemolytic activity of these natural compounds. Another important structural feature of nonribosomal cyclopeptides is lipidation as seen for the acidic lipopeptide antibiotics CDA 11, A54145 12, friulimicins, amphomycins, and daptomycin 6 [25]. The last lipopeptide, under the trade name Cubicin, has gained approval for the treatment of infections caused by gram-positive bacteria [15]. Recently, the thioesterase domain of CDA was excised as a free-standing enzyme [8]. To determine the scope of substitution permitted in linear peptide precursors by this cyclase, a daptomycin-like tridecapeptide thioester was synthesized. Although six residues of the cognate CDA peptide backbone were altered simultaneously, efficient total synthesis of an analog differing from daptomycin by substitution of Glu for 3-methylglutamate (3-MeGlu) at position 12 has been achieved [7]. However, it is very likely that CDA TE also

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tolerates this nonproteinogenic amino acid for catalytic activity due to its occurrence in four natural variants of cognate CDA at the equivalent position.

THE COMBINATORIAL POTENTIAL OF TE DOMAINS TO SYNTHESIZE CYCLIC PEPTIDE LIBRARIES To evaluate the potential utility of excised TE domains for generating large cyclic molecule libraries that can be screened for improved therapeutic activity, a combinatorial approach was developed by Kohli et al. [20]. In a biomimetic synthetic strategy, solid-phase peptide synthesis was used to construct the desired linear peptide tethered to a linker, which mimics the natural phosphopantetheine prosthetic group. When the decapeptide sequence of tyrocidine was synthesized on the resin and incubated with Tyc TE, the cyclase could efficiently catalyze peptide release by cyclization. This enzymatic on-resin cyclization was carried out with more than 300 linear tyrocidine derivatives bound to a solid support. The library of cyclopeptides revealed that replacement of D-Phe4 in tyrocidine by a positively charged D-amino acid resulted in a 30-fold improved therapeutic index (MHC/MIC). This combinatorial method may be applicable to other nonribosomal thioesterase domains, thereby providing a powerful tool for the generation of novel drug leads by large cyclic library screens.

Acknowledgments J. G. gratefully acknowledges a fellowship from the Fonds der Chemischen Industrie (FCI). M. A. M. receives support from the German Research Foundation (DFG), the European Union (EU), and the FCI.

References [1] Bordusa F. Enzymes for peptide cyclization. Chembiochem 2001;2:405–9. [2] Bruner SD, Weber T, Kohli RM, Schwarzer D, Marahiel MA, Walsh CT, Stubbs MT. Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE. Structure (Camb) 2002;10:301–10. [3] Chong PP, Podmore SM, Kieser HM, Redenbach M, Turgay K, Marahiel MA, Hopwood DA, Smith CP. Physical identification of a chromosomal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2). Microbiology 1998;144:193–9. [4] Clugston SL, Sieber SA, Marahiel MA, Walsh CT. Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst. Biochemistry 2003;42:12095–104. [5] Dieckmann R, Lee YO, van Liempt H, von Dohren H, Kleinkauf H. Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases. FEBS Lett 1995;357:212–6.

96 / Chapter 15 [6] Eppelmann K, Doekel S, Marahiel MA. Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis. J Biol Chem 2001;276:34824–31. [7] Grünewald J, Sieber SA, Mahlert C, Linne U, Marahiel MA. Synthesis and derivatization of daptomycin: a chemoenzymatic route to acidic lipopeptide antibiotics. J Am Chem Soc 2004; 126:17025–31. [8] Grünewald J, Sieber SA, Marahiel MA. Chemo- and regioselective peptide cyclization triggered by the N-terminal fatty acid chain length: the recombinant cyclase of the calciumdependent antibiotic from Streptomyces coelicolor. Biochemistry 2004;43:2915–25. [9] Hojati Z, Milne C, Harvey B, Gordon L, Borg M, Flett F, Wilkinson B, Sidebottom PJ, Rudd BA, Hayes MA, Smith CP, Micklefield J. Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor. Chem Biol 2002;9:1175–87. [10] Hubbard BK, Walsh CT. Vancomycin assembly: nature’s way. Agnew Chem Int Ed Engl 2003;42:730–65. [11] Jung D, Rozek A, Okon M, Hancock RE. Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin. Chem Biol 2004;11:949–57. [12] Kahne D, Leimkuhler C, Lu W, Walsh CT. Glycopeptide and lipoglycopeptide antibiotics. Chem Rev 2005;105: 425–48. [13] Kealey JT. Creating polyketide diversity through genetic engineering. Front Biosci 2003;8:c1–13. [14] Keating TA, Marshall CG, Walsh CT. Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH. Biochemistry 2000;39:15522– 30. [15] Kirkpatrick P, Raja A, LaBonte J, Lebbos J. Daptomycin. Nat Rev Drug Discov 2003;2:943–4. [16] Kohli RM, Burke MD, Tao J, Walsh CT. Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules. J Am Chem Soc 2003;125:7160–61. [17] Kohli RM, Takagi J, Walsh CT. The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides. Proc Natl Acad Sci USA 2002;99: 1247–52. [18] Kohli RM, Trauger JW, Schwarzer D, Marahiel MA, Walsh CT. Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases. Biochemistry 2001;40:7099–108. [19] Kohli RM, Walsh CT. Enzymology of acyl chain macrocyclization in natural product biosynthesis. Chem Commun (Camb) 2003: 297–307. [20] Kohli RM, Walsh CT, Burkart MD. Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature 2002;418: 658–61. [21] Lin H, Thayer DA, Wong CH, Walsh CT. Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase. Chem Biol 2004;11:1635–42. [22] Lin H, Walsh CT. A chemoenzymatic approach to glycopeptide antibiotics. J Am Chem Soc 2004;126:13998–14003. [23] Linne U, Doekel S, Marahiel MA. Portability of epimerization domain and role of peptidyl carrier protein on epimerization activity in nonribosomal peptide synthetases. Biochemistry 2001;40:15824–34.

[24] May JJ, Wendrich TM, Marahiel MA. The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J Biol Chem 2001;276:7209–17. [25] Micklefield J. Daptomycin structure and mechanism of action revealed. Chem Biol 2004;11:887–8. [26] Schwarzer D, Finking R, Marahiel MA. Nonribosomal peptides: from genes to products. Nat Prod Rep 2003;20:275–87. [27] Shaw-Reid CA, Kelleher NL, Losey HC, Gehring AM, Berg C, Walsh CT. Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization. Chem Biol 1999;6:385–400. [28] Shin-ya K, Wierzba K, Matsuo K, Ohtani T, Yamada Y, Furihata K, Hayakawa Y, Seto H. Telomestatin, a novel telomerase inhibitor from Streptomyces anulatus. J Am Chem Soc 2001; 123:1262–63. [29] Sieber SA, Marahiel MA. Learning from nature’s drug factories: nonribosomal synthesis of macrocyclic peptides. J Bacteriol 2003;185:7036–43. [30] Sieber SA, Marahiel MA. Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem Rev 2005;105:715–38. [31] Sieber SA, Tao J, Walsh CT, Marahiel MA. Peptidyl thiophenols as substrates for nonribosomal peptide cyclases. Agnew Chem Int Ed Engl 2004;43:493–8. [32] Sieber SA, Walsh CT, Marahiel MA. Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains. J Am Chem Soc 2003;125:10862–6. [33] Stachelhaus T, Huser A, Marahiel MA. Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases. Chem Biol 1996;3: 913–21. [34] Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 2000;407:215–18. [35] Trauger JW, Kohli RM, Walsh CT. Cyclization of backbonesubstituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase. Biochemistry 2001;40:7092–8. [36] Tseng CC, Bruner SD, Kohli RM, Marahiel MA, Walsh CT, Sieber SA. Characterization of the surfactin synthetase Cterminal thioesterase domain as a cyclic depsipeptide synthase. Biochemistry 2002;41:13350–59. [37] Walsh CT. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 2004;303:1805–10. [38] Weber T, Baumgartner R, Renner C, Marahiel MA, Holak TA. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases. Structure Fold Des 2000;8:407–18. [39] Yeh E, Lin H, Clugston SL, Kohli RM, Walsh CT. Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent. Chem Biol 2004; 11:1573–82. [40] Zhang Z, Ren J, Stammers DK, Baldwin JE, Harlos K, Schofield CJ. Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat Struct Biol 2000;7: 127–33.

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16 Lantibiotics RAQUEL REGINA BONELLI, IMKE WIEDEMANN, AND HANS-GEORG SAHL

STRUCTURE OF THE PRECURSOR mRNA/GENE

ABSTRACT Lanthionine-containing antibiotic peptides (lantibiotics) have been known for more than 60 years and the prototype peptide nisin has been used safely in food preservation for half a century. Lantibiotics derive from ribosomally synthesized prepeptides through unique posttranslational modifications involving serine, threonine, and cysteine residues. Approximately 50 lantibiotics are known to date, and almost 20 gene clusters, containing the determinants for prepeptides, modification, processing, export, and producer self-protection have been sequenced. Recent progress in in vitro synthesis and insights into multiple antibiotic mechanisms combined in a single molecule make lantibiotics attractive model compounds for design of novel antiinfective drugs.

Lantibiotics are expressed as prepeptides, with an N-terminal leader sequence and a C-terminal propeptide part that is posttranslationally modified (Fig. 1). Biosynthesis starts with enzymatic dehydration of serine and/or threonine residues in the propeptide part yielding the unusual amino acids 2,3-dehydroalanine (Dha) and 2,3-dehydrobutyrine (Dhb). To some of these dehydroamino acids, the thiol group of a neighboring cysteine residue is added resulting in the characteristic lanthionine (from Dha) and methyllanthionine (from Dhb) residues. The stable, thioether-based intramolecular rings determine the three-dimensional structure of the peptides and are essential for biological activity.

DISTRIBUTION OF THE mRNA DISCOVERY Genetic determinants for lantibiotic synthesis are exclusively found among gram-positive bacteria and can be located either on chromosomes or on mobile elements such as plasmids and transposons. The prepeptide structural genes (lanA) are part of gene clusters that also contain the genes responsible for modification (lanB, lanC, lanM, and lanD), proteolytic processing (lanP and lanT), transport (lanT), immunity (lanI, lanFEG), and biosynthesis regulation (lanK, lanR, lanQ) (Fig. 2) (reviewed in [14, 26, 30]). Two different biosynthetic machineries have been identified [30]. The first requires two separate enzymes (LanB and LanC) to synthesize lanthionines and is found among the entire group of lantibiotics that contain the nisin-like lipid II binding motif and among the Pep5 group (Fig. 3). In the second machinery, only one enzyme, LanM, is necessary to form a thioether

The most prominent lantibiotic nisin was first described as a group-N streptococci inhibiting substance in the 1920s and identified as a lanthioninecontaining peptide by Berridge in 1949 [2]. Its structure was elucidated by Gross and Morell [13]. The designation “lantibiotics” was introduced to emphasize the occurrence of lanthionine and the antibiotic nature of these peptides (lanthionine-containing antibiotics) [33]. Particularly between 1985 and 1995, a significant number of new lantibiotics were discovered and their genetic determinants and biosynthesis pathways characterized [31]. A number of natural variants of lantibiotics have been identified since, and “prototype” peptides, such as nisin, mersacidin, lacticin 481, Pep 5, and lacticin 3147, were defined for various subgroups [14]. Handbook of Biologically Active Peptides

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98 / Chapter 16 bridge. This group of lantibiotics comprises the mersacidin-like lipid II binding motif and various others. Whether these two groups of lantibiotics reflect, in evolutionary terms, two independent “inventions” of a thioether-producing machinery, remains to be studied.

PROCESSING After posttranslational modification, lantibiotics become activated by removal of the leader peptide segment. Genetic determinants for dedicated proteases were found in all the gene clusters except for subtilin, which appears to be processed by one of the numerous extracellular proteases of Bacillus subtilis [9]. LanP proteases are associated with the LanBC modification system (nisin, epidermin, mutacin III, mutacin I, Pep 5, epilancin K7, and lactocin S) and have sequence similarity with subtilisin-like serine proteases (reviewed in [26, 30]). Peptides that are processed by such LanP proteases have leader sequences with a number of charged residues and relatively conserved motifs at position −15 and −20 (FNLD) and at the cleavage site (Fig. 1). NisP and EpiP are synthesized as preproproteins—that is, with a signal peptide for export and a prosequence that possibly functions as an intracellular chaperone. NisP possess, in addition, a C-terminal extension that might act as a membrane anchor to attach the enzyme to the cytoplasmic membrane; EpiP lacks the C-terminal anchor. No details are known for MutP proteins (from mutacin I and III) that were reported to have strong similarity to NisP and EpiP [29]. PepP, ElkP, and LasP appear to be located in the cytoplasm. Peptides resulting from LanM biosynthesis machineries, such as mersacidin, lacticin 481 (and analogs), and lacticin 3147 A1/A2, are processed concomitantly with export by bifunctional ABC-transporters LanT [26, 30]. Such LanT transporters possess an N-terminal extension that contains a cysteine protease domain. The cleavage site of these peptides is usually preceded by a “double-Gly” motif, referring to conserved GG (GA or GS) residues in positions −1 and −2 (Fig. 1); bifunctional transporters are also found with many nonlantibiotic bacteriocins [16].

RECEPTORS The antimicrobial activity of lantibiotics does not involve receptors sensu strictu, although specific targets have been identified (see Biological Actions). However, lantibiotic biosynthesis may be autoregulated through a quorum sensing process, in which the respective lantibiotic acts as an upregulating signal. In this case, the lantibiotic binds to a receptor that is the histidine kinase of a dedicated two-component regulatory system (Fig. 2) [24].

ACTIVE AND/OR SOLUTION CONFORMATION The antibiotic activity is strictly associated with the fully modified lantibiotic—that is, lanthionine ring structures and dehydro-residues provide structural features that are essential for activity. Based on their overall structure, two groups, type A and type B lantibiotics, have been proposed [23]. Meanwhile, because many lantibiotics with intermediate features have been described, categorization has become more difficult. Typical type A lantibiotics such as nisin, subtilin, gallidermin, and Pep5 are elongated and flexible in solution. Defined conformations can only be found within or close to the rings. In membrane-mimicking solvents, these peptides adopt rod-shaped structures, with pronounced amphiphilicity [36]. The type B lantibiotics are also strong amphiphiles; however, they possess much less conformational freedom than the type A peptides. Cinnamycin, duramycins, and ancovenin have an intertwined bridging pattern including a head-to-tail thioether. Studies on the conformation of cinnamycin and duramycins in water and water : acetonitrile (1 : 1) have shown that these peptides present a U-shaped topology, with the hydrophobic residues clustered in the U-bend area of the molecule and the hydrophilic side chains located in the terminal segments [36]. The solution conformations of the type B lantibiotics mersacidin and actagardine were determined in methanol and acetonitrile : water (7 : 3), respectively [28, 43]. Both peptides adopt an overall globular structure with neutral side chains pointing outward. The hydrophilic

FIGURE 1. Prepeptide sequences of lantibiotics described up to April 2005. Peptides were aligned manually and separately for leader and propeptide segments; the proteolytic processing site is indicated by an arrow. For some lantibiotics (*) information for the leader peptides is missing. Lantibiotics were grouped according to structural similarities. Peptides that can be considered natural variants were listed with normal line spacing; groups of natural variants that still share important structural features were separated by enlarged line spacing and marked additionally with a cleavage site arrow. The ring-forming residues in conserved binding motifs for lipid II in the nisin group (rings A and B) and the mersacidin group (six-membered ring with a conserved E/D residue) are highlighted. References are only given when not cited in reviews [10, 14, 17, 26, 30, 31].

Lantibiotics / Nisin A

MST–KD----FNLDLVSVSKK---DSGASPR ITSISLCTPGC--KTGALMGCNMKTATCHCSIHVS-K

Nisin Z

MST–KD----FNLDLVSVSKK---DSGASPR ITSISLCTPGC--KTGALMGCNMKTATCNCSIHVS-K

Nisin Q [42]

MST–KD----FNLDLVSVSKT---DSGASTR ITSISLCTPGC--KTGVLMGCNLKTATCNCSVHVS-K

Subtilin

MSKFDD----FDLDVVKVSKQ---DSKITPQ WKSESLCTPGC--VTGALQTCFLQTLTCNCKI--S-K

Ericin S

MSKFDD----FDLDVVKVSKQ---DSKITPQ WKSESVCTPGC--VTGVLQTCFLQTITCNCHI--S-K

Ericin A

MSKFDD----FDLDVVKVSKQ---DSKITPQ VLSKSLCTPGC--ITGPLQTCYLCFPTFAKC

Epidermin

MEAVKEKNDLFNLD-VKVNAKESNDSGAEPR IASKFICTPGC--AKTGSFNSYCC

Gallidermin

MEAVKEKNELFDLD-VKVNAKESNDSGAEPR IASKFLCTPGC--AKTGSFNSYCC

V1L6Epidermin

* VASKFLCTPGC--AKTGSFNSYCC

Mutacin 1140/III Mutacin-B-Ny266

MSNTQLLEVLGTETFDVQEDLFAFDTTDTTIVASNDDPDTR FKSWSLCTPGC--ARTGSFNSYCC * FKSWSFCTPGC--AKTGSFNSYCC MSNTQLLEVLGTETFDVQEDLFAFDTTDTTIVASNDDPDTR FSSLSLCSLGCTGVKNPSFNSYCC

Mutacin I

MNNTIKD----FDLDL-KTNKK---DT-ATPY VGSRYLCTPGSCWKLVCFTTTVK

Streptin Duramycin

* CKQSCSFGPFTFVCDGNTK

Duramycin B

* CRQSCSFGPLTFVCDGNTK

Duramycin C

* CANSCSYGPLTWSCDGNTK

Ancovenin Cinnamycin

* CVQSCSFGPLTWSCDGNTK MTASILQQSVVDADFRAALLENPAAFGASAAALPTPVEAQDQASLDFWTKDIAATEAFA CRQSCSFGPFTFVCDGNTK

Mersacidin

MEQEAIIRS-WKDPFSRENSTQNPAGNPFSELKEAQMDKLVG--AGD---ME-AA CTFTLPGGGGVC--------TLTSECIC

Actagardine Salivaricin A Sal A1 Lacticin 481 Mutacin II SA-FF22 Streptococcin A-M49 [22] Variacin Butyrivibricin OR79A Nukacin ISK-1 Ruminococcin A Bovicin HJ50 Plantaricin C Lacticin 3147 A1

* SSGWVC--------------TLTIECGTVICAC MKNSKDILNNAIEEVSEKELMEVAGG KRGSGWIA------------TITDDCPNSVFVCC MKNSKDILTNAIEEVSEKELMEVAGG KKGSGWFA------------TITDDCPNSVFVCC M-KEQ-NSFNLLQEVTESELDLILGA –KGG-SGVIH----------TISHECNMNSWQFVFTCCS MNKLNSNAVVSLNEVSDSELDTILGG NRWWQGGVVP----------TVSYECRMNSWQHVFTCC MEKNN-EVINSIQEVSLEELDQIIGA ---GKNGVFK----------TISHECHLNTWAFLATCCS MTKEH-EIINSIQEVSLEELDQIIGA ---GKNGVFK----------TISHECHLNTWAFLATCCS M---T-NAFQALDEVTDAELDAILGG ---G-SGVIP----------TISHECHMNSFQFVFTCCS MNKEL-NA—LTNPIDEKELEQILGG ---G-NGVIK----------TISHECHMNTWQFIFTCCS MENSKVMKVM-KD-IEVANLLEEVQEDELNEVLGA -KK—KSGVIP----------TVSHDCHMNSFQFVFTCCS M-RNDVLTLTNPME--EKELEQILGG ---G-NGVLK----------TISHECNMNTWQFLFTCC MMNATENQIFVETVSDQELEMLIGG –AD-R-GWIK----------TLTKDCPNVISSICAGTIITACKNCA * KKTKKNSSDGIC--------TLTSECDHLATWVCC MNKNEIETQP-VTWLEEVSDQNFDEDVFGA -CSTNTFSLSDYWGNNGAWCTLTHECMAWCK

Staphylococcin C55α C55

MKSSFLEKDIEEQ--VTWFEEVSEQEFDDDIFGA -CSTNTFSLSDYWGNKGNWCTATHECMSWCK

Plantaricin Wα W [18]

MKISKIEAQARKDFFKKIDTNSNLLNVNGA -KCK-WWNISCDLGNNGHVCTLSHECQVSCN

Smb B [41]

MKEIQKAGLQEELSILMDDANNLEQLIAGI GTTVVNSTFSIVLGNKGYICTVTVECMRNCSK

Lacticin 3147 A2

MKEKNMKKNDTIELQLGKYLEDDMIELAEGDESHGG TTPATPAISILSAYI---STNTCPTTKCTRAC

Plantaricin Wβ [18]

MTKTSRRKNAIANYLEPVDEKSINESFGAGDPEAR SGIPCTIGAAVAA-----SIAVCPTTKCSKRCGKRKK

Staphylococcin C55β

MKNELGKFLEENELELGKFSESDMLEITDDEVYAA GTPLALLGGAATGVIGYISNQTCPTTACTRAC

Smb A [41]

MKSNLLKINNVTEMEKNMVTLIKDEDMLAG STPAC-AIGVVGIAVTGI—-----STACTSRCINK

Pep5

MKNNKNLFDLEIKKETSQNTDELEPQ TAG---PAIRASVKQCQKTLKATRLFTVSCKGKNGCK

Epicidin 280

MENKKDLFDLEIKKDNMENNNELEAQ SLG---PAIKATRQVCP---KATRFVTVSCKKSDCQ

Epilancin K7

MNN—-SLFDLNLNKGVETQKSDLSPQ SASVLKTSIKVSKKYCK-------GVTLTCGCNITGGK

Epilancin 15X [12] Lactocin S

* SASIVKTTIKASKKLCR-------GFTLTCGCHFTGKK MKTEKKVLDELSLHASAKMGARDVESSMNADST PVLASVAVSMELLPTASVLYSDVAGCFKYSAKHHC

Cytolysin A1

MENLSVV-PSFEELSVEEMEAIQSG GDVQAETTPVCAVAATAAASSAACGWVGGGIFTGVTVVVSLKHC

Cytolysin A2

VLNKENQENYYSNKLELVGPSFEELSLEEMEAIQSG GDVQAETTPACFTI----------GLGVGALF-------SAKFC

Sublancin SapB [25]

MEKLFKEVKLEELENQKGSG LGKAQCAALWLQCASGGTIGCGGGAVACQNYRQFCR MNLFDLQSMETPKEEAMGDVE TGSRASLLLCGDSSLSITTCN

99

100 / Chapter 16

FIGURE 2. Lantibiotic biosynthesis gene clusters sequenced up to April 2005. lanA genes encode the lantibiotic prepeptides; lanB, lanC, lanM, and lanD, modification enzymes; lanP, proteolytic enzymes; lanT, transporters; lanI, lanE, lanF, and lanG, immunity determinants; lanR, lanK, and lanQ, regulators of biosynthesis. (*) Gene nomenclature has been unified for clarity and is not consistent with the original publications.

residue Glu, which belongs to a highly conserved motif in mersacidin and actagardine, is directed to the center of the molecule. Mersacidin is the only lantibiotic for which a crystal structure is available [32]. The plantaricin C solution structure shares features from type A and B lantibiotics. The first six N-terminal residues, four of which are lysines, are structurally undefined; the remaining part of the molecule is globular because of a bridge connecting residues 7 and 27 and the presence of three internal rings (Fig. 3) [35].

BIOLOGICAL ACTIONS Lantibiotics are active against gram-positive bacteria and exert multiple activities at the cytoplasmic mem-

brane [17]. Resistance of gram-negative bacteria results from the protective effect of the outer membrane that is impenetrable to these peptides. Numerous studies performed with intact bacterial cells, membrane vesicles, and artificial liposomes have shown that nisin and other type-A lantibiotics in μM concentrations are able to destabilize membranes by forming nonselective, transient pores, which leads to dissipation of the membrane potential, rapid efflux of small metabolites, and cessation of cellular biosynthetic processes (reviewed in [17]). Structural analysis of nisin in the presence of micelles [37] and 31P-NMR studies [11] supported a “wedge model” [11], which predicts that the peptides remain surface bound during pore formation rather than inserted into the hydrophobic core.

FIGURE 3. Structure of mature lantibiotics described up to April 2005. Peptides in bold type are prototype peptides; natural variants, subsequently described, are given in regular type. A. Lantibiotics of the nisin group. B. Lantibiotics of the mersacidin group. Some of the structures have been proposed (*), or suggested based on primary sequence similarity (**); experimental proof is missing.

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FIGURE 3. (Continued ) C. Lantibiotics of the cinnamycin group. D. Miscellaneous lantibiotics with only few variants and unknown molecular target. Ala-S-Ala, lanthionine; Abu-S-Ala, 3-methyllanthionine; Abu, 2-aminobutyric acid; Dha, α,β-didehydroalanine; Dhb, α,β-didehydrobutyric acid; Me2A, twofold methylated alanine; aI, allo isoleucine; A*, alanine in the D-configuration. N-terminal modification given in Fig 3D occur spontaneously from Dha and Dhb after proteolytic cleavage. (_) residues conserved with respect to the prototype peptide; (-) missing residue.

Lantibiotics More recently, the membrane-bound peptidoglycan precursor lipid II was identified as a specific target for nisin and mersacidin [7, 8]. Binding of lipid II blocks the precursor from incorporation into the bacterial cell wall. While this appears to be the only bactericidal activity of mersacidin, the nisin-type lantibiotics combine this activity with a unique, target-mediated pore formation [6, 38]. Such a combination of two killing mechanisms may explain the potent in vivo activity (minimal inhibitory concentration in the nM range) of nisin and related lantibiotics. The molecular basis of the interaction between nisin and lipid II was intensively investigated. Genetically engineered nisin variants [38] and NMR spectroscopy [20] identified structural features of nisin that are important for its biological activities. The two first rings in the N-terminal part of the peptide are responsible for interaction with lipid II, whereas the C-terminal and the central flexible hinge region are essential for pore formation. Obviously, the pyrophosphate moiety of the lipid II molecule contributes significantly to binding nisin [3, 21]. The N-terminus of nisin was found to form a cage-like structure, which allows the formation of five intermolecular hydrogen bonds between the backbone amides of nisin and the pyrophosphate linkage group of lipid II [21]. These structural elements were also found in a complex of nisin and undecaprenylpyrophosphate; however, for target-mediated pore formation, nisin appears to need a second docking site on the Mur-NAc-pentapeptide moiety present in lipid I and lipid II [3]. Nisin and lipid II are both intrinsic parts of the pore [5, 15] although the unique pore assembly process and the pore architecture need to be studied further. In black lipid bilayers doped with lipid II, nisin pores have an average lifetime of 6 seconds and a diameter of 2 nm, whereas in the absence of lipid II lifetimes were in the millisecond range with diameters up to 1 nm [39]. Mersacidin and actagardine also bind to lipid II and thus inhibit the cell wall biosynthesis at the level of transglycosylation. It was shown that the binding site of these peptides differs from that of the glycopeptide antibiotic vancomycin [7]. NMR studies of the interaction of lipid II and mersacidin identified a hinge region between residues 12 and 13, which serves in opening and closing of the adjacent rings during lipid II binding [19]. Moreover Glu17 is indispensable for antibiotic activity [34]. The two-peptide lantibiotics lacticin 3147 and its relatives appear in an interesting case in which the two functions, lipid II binding and pore formation, are attributed to two individual peptides rather than being combined into one molecule [27]. It is interesting to note that in both cases, nisin and mersacidin, the structural elements that are predicted to be directly involved in the interaction with lipid II

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(in other words, the first two rings in nisin and the intertwined rings in mersacidin with the conserved negative charge), are also found in many other peptides. In Fig. 3 lantibiotics were aligned according to their ring patterns. It seems likely that peptides that possess these conserved structural features exert the same mechanism of action as the respective prototype lantibiotics, at least regarding their interactions with lipid II. In addition to pore formation and inhibition of the cell wall biosynthesis, further mechanisms of action have been reported for lantibiotics (reviewed in [17]). Nisin and the related cationic lantibiotic Pep5 have been shown to induce autolysis of susceptible staphylococcal cells. Due to high affinity for teichoic and lipoteichoic acid, these lantibiotics displace, and thus activate, cationic autolytic enzymes, which are kept inactive when bound to the anionic cell wall polymers. Furthermore, nisin and subtilin inhibit the germination of bacterial spores. This activity depends on the presence of Dha residues in position 5 of both peptides. The cinnamycin-like type-B lantibiotics display antibactericidal activity against a few bacterial strains, in particular Bacillus strains. The basis for the antibiotic activity appears to be specific binding of phosphoethanolamine, which causes increased membrane permeability, impaired ATP-dependent protein translocation and calcium uptake, and phospholipase inhibition [17]. It should not be left unmentioned that lanthioninecontaining peptides may have nonantibiotic functions. SapB does not display antibacterial properties but functions as biological surfactant. It is important for aerial mycelium formation by the filamentous bacterium Streptomyces coelicolor [25, 40]. Also, cytolysin, another twopeptide lantibiotic produced by some enterococci, has cytotoxic activity and has been shown to represent a significant virulence factor in clinical cases of enterococcal infections [4].

Acknowledgments R. R. B. gratefully acknowledges a CAPES stipendium. I. W. and H. G. S. receive support from the German Research Foundation (DFG, various projects).

References Space restrictions required citation of the following reviews [10, 14, 17, 26, 30, 31] wherever possible. [1] Aso Y, Sashihara T, Nagao J, Kanemasa Y, Koga H, Hashimoto T, Higuchi T, Adachi A, Nomiyama H, Ishizaki A, Nakayama J, Sonomoto K. Characterization of a gene cluster of Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1. Biosci Biotechnol Biochem 2004; 68:1663–71.

104 / Chapter 16 [2] Berridge NJ. Preparation of the antibiotic nisin. Biochem J 1949;45:486–93. [3] Bonev BB, Breukink E, Swiezewska E, de Kruijff B, Watts A. Targeting extracellular pyrophosphates underpins the high selectivity of nisin. FASEB J 2004;18:1862–9. [4] Booth MC, Bogie CP, Sahl HG, Siezen RJ, Hatter KL, Gilmore MS. Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic. Mol Microbiol 1996;21: 1175–84. [5] Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S, Heck AJ, de Kruijff B. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 2003;278:19898–903. [6] Breukink E, Wiedemann I, van Kraaij C, Kuipers OP, Sahl HG, de Kruijff B. Use of the cell wall precursor lipid II by a poreforming peptide antibiotic. Science 1999;286:2361–4. [7] Brötz H, Bierbaum G, Leopold K, Reynolds PE, Sahl HG. The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II. Antimicrob Agents Chemother 1998;42: 154–60. [8] Brötz H, Josten M, Wiedemann I, Schneider U, Götz F, Bierbaum G, Sahl HG. Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol Microbiol 1998;30:317–27. [9] Corvey C, Stein T, Dusterhus S, Karas M, Entian KD. Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA, and Vpr. Biochem Biophys Res Commun 2003;304:48–54. [10] Cotter PD, Hill C, Ross PR. Bacterial lantibiotics: strategies to improve therapeutic potential. Curr Protein Pept Sci 2005;6: 61–75. [11] Driessen AJ, van den Hooven HW, Kuiper W, van de Kamp M, Sahl HG, Konings RN, Konings WN. Mechanistic studies of lantibiotic-induced permeabilization of phospholipid vesicles. Biochemistry 1995;34:1606–14. [12] Ekkelenkamp MB, Hanssen M, Shang-Te DS, de Jong A, Milatovic D, Verhoef J, van Nuland NAJ. Isolation and Structural characterization of epilancin 15X, a novel lantibiotic from clinical strain of Staphylococcus epidermidis. FEBS Lett 2005; 1917–22. [13] Gross E, Morell JL. The structure of nisin. J Am Chem Soc 1971;93:4634–5. [14] Guder A, Wiedemann I, Sahl HG. Posttranslationally modified bacteriocins—the lantibiotics. Biopolymers 2000;55:62–73. [15] Hasper HE, de Kruijff B, Breukink E. Assembly and stability of nisin-lipid II pores. Biochemistry 2004;43:11567–75. [16] Havarstein LS, Diep DB, Nes IF. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol Microbiol 1995;16: 229–40. [17] Héchard Y, Sahl HG. Mode of action of modified and unmodified bacteriocins from Gram-positive bacteria. Biochimie 2002;84:545–57. [18] Holo H, Jeknic Z, Daeschel, M, Stevanovic S, Nes IF. Plantaricin W from Lactobacillus plantarum belongs to a new family of twopeptide lantibiotics. Microbiology 2001;147:643–51. [19] Hsu ST, Breukink E, Bierbaum G, Sahl HG, de Kruijff B, Kaptein R, van Nuland NA, Bonvin AM. NMR study of mersacidin and lipid II interaction in dodecylphosphocholine micelles. Conformational changes are a key to antimicrobial activity. J Biol Chem 2003;278:13110–17. [20] Hsu ST, Breukink E, de Kruijff B, Kaptein R, Bonvin AMJJ, van Nuland NAJ. Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles. Biochemistry 2002;41:7670–6.

[21] Hsu ST, Breukink E, Tischenko E, Lutters MA, de Kruijff B, Kaptein R, Bonvin AM, van Nuland NA. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nat Struct Mol Biol 2004;11: 963–7. [22] Hynes WL, Friend VL, Ferretti JJ. Duplication of the lantibiotic structural gene in M-type 49 group A streptococcus strains producing Streptococcin A-M49 Appl Environ Microbiol 1994; 60:4207–9. [23] Jung G. Lantibiotics: a survey. In: Jung G, Sahl HG, editors. Nisin and Novel Lantibiotics, Leiden: Escom; 1991, p. 1–34. [24] Kleerebezem M. Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis. Peptides 2004;25:1405–14. [25] Kodani S, Hudson ME, Durrant MC, Buttner MJ, Nodwell JR, Willey JM. The SapB morphogen is a lantibiotic-like peptide derived from the product of the developmental gene ramS in Streptomyces coelicolor. Proc Natl Acad Sci USA 2004;101:11448– 53. [26] McAuliffe O, Ross RP, Hill C. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol Rev 2001;25:285–308. [27] Morgan SM, O’Connor PM, Cotter PD, Ross RP, Hill C. Sequential actions or the two-component peptides of the lantibiotic lacticin 3147 explain its antimicrobial activity at nanomolar concentrations. Antimicrob Agents Chemother 2005;49:2606–11. [28] Prasch T, Naumann T, Markert RL, Sattler M, Schubert W, Schaal S, Bauch M, Kogler H, Griesinger C. Constitution and solution conformation of the antibiotic mersacidin determined by NMR and molecular dynamics. Eur J Biochem 1997; 244:501–12. [29] Qi F, Chen P, Caufield PW. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes. Appl Environ Microbiol 1999;65:3880–7. [30] Sahl HG, Bierbaum G. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. Annu Rev Microbiol 1998;52:41–79. [31] Sahl HG, Jack RW, Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem 1995;230:827–53. [32] Schneider TR, Karcher J, Pohl E, Lubini P, Sheldrick GM. Ab initio structure determination of the lantibiotic mersacidin. Acta Crystallogr D Biol Crystallogr 2000;56(Pt 6):705–13. [33] Schnell N, Entian KD, Schneider U, Götz F, Zahner H, Kellner R, Jung G. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 1988; 333:276–8. [34] Szekat C, Jack RW, Skutlarek D, Farber H, Bierbaum G. Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin. Appl Environ Microbiol 2003;69:3777–83. [35] Turner DL, Brennan L, Meyer HE, Lohaus C, Siethoff C, Costa HS, Gonzalez B, Santos H, Suárez JE. Solution structure of plantaricin C, a novel lantibiotic. Eur J Biochem 1999;264: 833–9. [36] van de Ven FJ, Jung G. Structures of lantibiotics studied by NMR. Antonie Van Leeuwenhoek 1996;69:99–107. [37] van den Hooven, HW, Spronk CA, van de Kamp M, Konings RN, Hilbers CW, van de Van FJ. Surface location and orientation of the lantibiotic nisin bound to membrane-mimicking micelles of dodecylphosphocholine and of sodium dodecylsulphate. Eur J Biochem 1996;235:394–403. [38] Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, de Kruijff B, Sahl HG. Specific binding of nisin to the

Lantibiotics peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J Biol Chem 2001;276:1772–9. [39] Wiedemann I, Benz R, Sahl HG. Lipid II-mediated pore formation by the peptide antibiotic nisin: a black lipid membrane study. J Bacteriol 2004;186:3259–61. [40] Willey, J, Santamaria R, Guijarro J, Geistlich M, Losick R. Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor. Cell 1991;65:641–50.

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[41] Yonezawa H, Kuramitsu, HK. Genetic analysis of a unique bacteriocin, Smb, produced by Streptococcus mutans GS5. Antimicrob Agents Chemother 2005;49:541–8. [42] Zendo T, Fukao M, Ueda K, Higuchi T, Nakayama J, Sonomoto, K. Identification of the lantibiotic nisin Q, a new natural variant produced by Lactococcus lactis 61-14 isolated from a river in Japan. Biosci Biotechnol Biochem 2003;67:1616–19. [43] Zimmermann N, Jung G. The three-dimensional solution structure of the lantibiotic murein-biosynthesis-inhibitor actagardine determined by NMR. Eur J Biochem 1997;246:809–19.

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C

H

A

P

T

E

R

17 The Nonlantibiotic Heat-Stable Bacteriocins in Gram-Positive Bacteria INGOLF F. NES, DAG ANDERS BREDE, AND HELGE HOLO

includes gram-positive Class II bacteriocins that have been characterized chemically and genetically as unique bacteriocins. This is not a complete list, and many bacteriocins are not included because they have only been partly characterized. Separating Class II bacteriocins into different subgroups has not been very successful because of their great diversity and the lack of unifying criteria for defining subgroups. In our opinion, only two well-characterized subgroups of Class II bacteriocins exist: the strong antilisterial pediocin-like bacteriocins, which share some unifying structural motifs, and the two-peptide bacteriocins. In addition, some bacteriocins are circular (cyclic) peptides, and only a few have been characterized, but they also seem to belong to a well-defined group. It has been suggested that the circular bacteriocins should constitute a separate group [25], since the genetic apparatus needed for synthesis of circular bacteriocins is different from that of most other Class II bacteriocins [8], but we have included them in this review as a subgroup of Class II. Circular proteins are also found in gram-negative bacteria and among the nonribosomally synthesized antimicrobial peptides. Recent years have also seen the isolation of antimicrobial bacterial peptides that are derived from larger proteins through processing both at the N-terminus and/or C-terminus. Such peptides are observed also in eukaryotes where histones can be specifically degraded to form antimicrobial peptides [32]. The gram-positive antibacterial peptides derived from proteins by processing are also included in this review as bacteriocins (Table 1).

ABSTRACT Gram-positive bacteria produce a range of antimicrobial peptides, and most of them are classified as bacteriocins. Most of these bacteriocins are found in the lantibiotic (Class I) and in the heat stable, nonlantibiotic group (Class II). Class II comprises by far the most numerous and diverse group with respect to peptide sequence, production, and range of target specificity. The pediocin-like bacteriocins (Class IIA) are the largest unifying subgroup and the most studied. This review summarizes the diversity of Class II bacteriocins, by reference to the most thoroughly biochemically and/or genetically characterized bacteriocins.

DISCOVERY Ribosomally synthesized antibacterial peptides produced by gram-positive bacteria were first identified with the discovery of nisin. Such peptides are most frequently referred to as bacteriocins and are now grouped into three classes: Class I—lantibiotics, Class II—nonlantibiotics, and Class III—heat-labile antimicrobial proteins. This chapter focuses on the peptide bacteriocins that belong to Class II. The Class II bacteriocins have been identified most commonly in the lactic acid bacteria (LAB), although it is not known if this is because such bacteriocins are more frequently found among LAB or because these bacteria are the most studied. For years it was believed that the antimicrobial activity of LAB was due solely to the production of organic acids (largely lactic acid). In 1989–1991 the lactococcins were chemically and genetically identified as the first peptide bacteriocins since the initial discovery of nisin [20, 42]. Since then, we have witnessed an overwhelming increase in the isolation and characterization of new bacteriocins. Table 1 Handbook of Biologically Active Peptides

SYNTHESIS The genetic organization for production of most bacteriocins in gram-positive bacteria is quite simple.

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Copyright © 2006 Elsevier

108 / Chapter 17 TABLE 1.

Overview of the most studied Class II bacteriocins.

Class II Subgroups

Number of Amino Acid Residues

Pedocin-Like Bacteriocins Enterocin A Divericin V41 Coagulin Pediocin PA1, Pediocin AcH Sakacin P, Sakacin 674 Listeriocin 743A Piscicolin 126, Piscicocin V1a Carnobacteriocin BM1, Piscicocin V1b Sakacin 5X Leucocin A-TA33a, Leucocin A-UAL 187 Leucocin C Mesenterocin Y105 Lactococcin MMFII Plantaricin 423 Sakacin A, Curvacin A Enterocin P Bacteriocin 31 Carnobacteriocin B2 Bavaricin MN Bifidocin B Piscicocin CS526 Acidocin A Sakacin G Mundticin KS, Mundticin ATO6, Enterocin CRL35 Divergicin M35 Plantaricin C19

47 43 44 44 43 43 44 43 43 37 43 37 37 37 41 44 43 48 42 36 43 58 37 43 43 >36

Two-Peptide Bacteriocins Lactococcin G (LcnGα and LcnG . β) Lactococcin M (LcnM and LcnN) Lactacin F (LafA and LaX) Thermophilin 13 (ThmA and ThmB) Plantaricin S (PlsA and PlsB) Plantaricin EF (PlnE and PlnF) Plantaricin JK (PlnJ and PlnK) Enterocin 1071 (A and B) Plantaricin NC8 (α and β) Brochocin C (A and B)

39, 48, 57, 48, 27, 33, 25, 39, 28, 59,

Sakacin T (α and β) Acidocin J1132 (α and β) Gassericin T (GatT and GatX) Acidocin LF221 (A and B) ABP-118 (Abp118a and Abp118b)

Inhibition Spectrum (*) A A A A A A A A A A A A A A A A A A A A A A A A A A

35 29 47 43 26 34 32 34 35 43

Lactococcus Lactococcus Lactobacillus, Enterococcus LAB, Bacillus, Clostridium, Listeria Lactobacillus Lactobacillus Lactobacillus Enterococcus spp., Listeria, Lactobacillus salivarius Lactobacillus Brochothrix thermosphacta, Lactobacillus, Listeria, Clostridium

51, 43 58, 48 53, 48 45, 46

Cyclic Bacteriocins Gassericin A

58

Enterocin AS-48, enterocin 4

70

Circularin A

69

Butyrivibriocin AR10 Subtilosin

51 32

Lactobacillus Lactobacillus LAB, Bacillus, Clostridium Bacillus, Listeria, Enterococcus, Staphylococcus Lactobacillus, Listeria, Bacillus cereus, Staphylococcus aureus Enterococcus, Lactobacillus, Listeria, Corynebacterium Clostridium, Lactobacillus, Lactococcus, Enterococcus Butyrivibrio Listeria

The Nonlantibiotic Heat-Stable Bacteriocins in Gram-Positive Bacteria / 109 TABLE 1. (Continued)

Class II Subgroups

Number of Amino Acid Residues

Other Class II Bacteriocins Lactococcin A Lactococcin B Lactobin A, Amylovorin L471 Bovicin 255

50 79

Closticin 574 Enterocin B Piscicolin 61 Divergicin A Propionicin T1 Acidocin B Piscicolin 61, Carnobacteriocin A Acidocin (Gassericin) LF221A

82 53 53 46 65 59 53 53

Acidocin (Gassericin) LF221B, Gassericin K7 B

47

Lsb A LsbB

44 30

Leaderless Bacteriocins Enterocin Q Enterocin L50 (A and B), Enterocin EJ97

34 43, 42

Enterocin RJ-11 Enterocin EJ97

44 44

Aureocin A70 (AurA, AutrB, AurC, AurD) Aureocin A53

31, 30, 31, 31 51

Bacteriocins Produced From Larger Proteins PAMP Closticin 574 Propionicin F

65 82 43

54

Inhibition Spectrum (*) Lactococcus Lactococcus Lactobacillus, Enterococcus LAB, Listeria, Bacillus, Clostridium, Ruminococcus, Butyrivibrio Clostridium, Lactobacillus Enterococcus, Lactobacillus Carnobacterium, Enterococcus, Listeria Carnobacterium Propionibacterium Listeria, Clostridium, Brochothrix, Lactobacillus Carnobacterium, Lactobacillus, Enterococcus Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus Lactococcus Lactococcus Enterococcus LAB, Listeria, Bacillus, Clostridium, Staphylococcus LAB, Listeria, Bacillus, Staphylococcus Enterococcus, Bacillus, Listeria, Staphylococcus aureus LAB, Listeria Staphylococcus, Micrococcus, Listeria, Corynebacterium Propionibacterium, Lactobacillus Lactobacillus, Clostridium Propionibacterium freudenreichii

(*) Abbreviations: A: Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Carnobacterium, Listeria. LAB: lactic acid bacteria.

The host possesses a structural gene of the bacteriocin, which usually encodes a prepeptide with an N-terminal leader sequence. The most common leader sequence is the so-called double-glycine leader, but in some cases it can be a sec-dependent leader. However, bacteriocins without leader sequences are also found. An immunity gene is found in conjunction with the bacteriocin gene, and the highly specific immunity protein protects the producer bacterium against self-destruction. The immunity gene is almost exclusively found adjacent to its corresponding bacteriocin gene and in the same operon, although exceptions exist. Each Class II bacteriocin has its own unique immunity gene, including the two-peptide bacteriocins (encoded by two separate

genes next to each other) that have only a single immunity gene that is involved in the self-protection. Bacteriocins with double-glycine leaders are secreted by dedicated ABC transporters that contain N-terminal proteolytic domains that remove the leader during export [19]. Some bacteriocins are exported by the secdependent system [26, 44]. It has been suggested that bacteriocins without leader sequences are secreted by ABC transporters [5, 6, 17]. Over the past decades it has been shown that many bacteria produce multiple bacteriocins. The bacteriocin genes—for example, the plantaricins in Lactobacillus plantarum C11 [10, 36]—can be found in a coregulated cluster of genes with the peptides sharing the same

110 / Chapter 17 transport system. On the other hand, it is quite common for multiple bacteriocins to be independently expressed in a bacterium, either by being discretely located on the bacterial chromosome or by being plasmid encoded. The biosynthesis of some bacteriocins is regulated by a quorum sensing system made up of three components: (1) a peptide pheromone (induction peptide), (2) a receptor (Histidine Protein Kinase), and (3) a response regulator (DNA binding) protein [11, 29]. While the quorum sensing mechanism is fairly well understood, other mechanisms of regulation also seem to exist. For example, it has been shown that temperature may directly or indirectly affect the expression or level of expression of some bacteriocins. Enterococcus faecium L50 is a strain that produces at least three bacteriocins. Enterocin L50 activity is found only when the producer is grown between 16 and 25°C—that is, no activity is found at 37°C, while maximum activity of enterocin Q and enterocin P is observed at growth temperatures of 37°C and 47°C, respectively. Conversely, growth temperature affects sakacin A production through its quorum-sensing mechanism [9]. The quorum sensing is active only in the narrow temperature range between 33 and 35°C. Growth below 33°C leads to constitutive production of sakacin A, while no activity is found in the medium when the bacteria are grown above 35°C.

BIOLOGICAL ACTIONS AND RECEPTORS Bacteriocins are either bactericidal—killing bacteria—or bacteriostatic—inhibiting their growth. These different effects are probably determined by growth conditions and stage of growth of the target strain as well as the choice of target strain and bacteriocin concentration used in the assay. However, more interesting is the observed wide diversity in the antimicrobial inhibitory spectrum. Most of the Class II bacteriocins have restricted inhibitory spectra, and none can compare with the broad spectrum of inhibition of nisin. Some Class II bacteriocins such as lactococcin A have an extremely narrow spectrum of inhibition, being able to inhibit only members of the same genus as the producing strain. Others have an intermediate spectrum of inhibition, being restricted to inhibiting a few genera related to the producer. Many bacteriocins from grampositive bacteria have a much broader range of targets than the gram-negative bacteriocins, but susceptibility can vary greatly between bacteria not only within the same genera but also within the same species. Table 1 indicates the variation of target specificity among Class II bacteriocins. The Class IIA bacteriocins (the pediocin family) are characterized by a more or less common inhibitory spec-

trum in addition to sequence homology in the Nterminal region [11]. These bacteriocins inhibit lactic acid bacteria, with the exception of lactococci and streptococci, and have a strong effect on Listeria [23]. All Class IIA bacteriocins studied to date exert their activity on the cytoplasmic membrane of the target cell. These bacteriocins show inhibitory effects at nanomolar concentrations. At such concentrations only the cytoplasmic membranes of sensitive cells are permeabilized, but at extremely high concentrations, even liposomes have been found to be affected by pediocin PA-1 [4]. The physicochemical properties of Class II bacteriocins, which are cationic and amphiphilic, may strongly influence the results of in vitro mechanistic studies. An environment having high ionic strength or containing amphiphilic or hydrophobic compounds will have detrimental effect on the antimicrobial activity [1]. Despite such issues, several studies have suggested similar mechanisms of action. Many studies have observed that bacteriocins can cause efflux of potassium ions from susceptible bacteria. Such leakage will lead to the depolarization of the bacterial cytoplasmic membrane and excess ATP consumption, which in many cases can be defined as secondary effects. One of the very early studies of mode of action of Class II bacteriocins was carried out with lactococcin A, using whole cells, vesicles, and liposomes. Liposomes derived from the lipids of susceptible lactococci were not affected by lactococcin A, while vesicles from susceptible cells were permeabilized by lactococcin A and at a concentration that correlated with those used in vivo [43]. These results strongly indicate that a receptor might be involved in the action of lactococcin A. By comparison, it has been determined that the activity of nisin involves a receptor, which is more frequently referred to as docking molecule (see lantibiotic chapter). Among the Class II bacteriocins, the pediocin-like bacteriocins probably recognize susceptible cells through a receptor-like molecule. Several published studies have convincingly demonstrated that mannose PTS system of susceptible cells is involved in killing by pediocin-like bacteriocins [7, 18, 33, 34]. It was observed that the highly sensitive Listeria monocytogenes become resistant to pediocin-like bacteriocins at a high frequency [35], and such resistance to pediocins was linked to the mannose phosphotransferase system in L. monocytogenes, as well as Enterococcus faecalis. In the resistant L. monocytogenes the IIAB subunit of the mannose PTS is not detectable. Furthermore, a σ54-dependent promoter regulates the expression of the mptACD operon that encodes this PTS system. Construction of knockout mutants of the mptACD operon or the σ54 encoding gene conferred resistance [7]. Moreover, expression of the mptC gene in the leucocin A resistant

The Nonlantibiotic Heat-Stable Bacteriocins in Gram-Positive Bacteria / 111 L. lactis rendered this bacterium sensitive to the bacteriocin [33]. These investigations thus strongly indicate that receptors or docking molecules are frequently involved in the host specificity of such peptides. However, as observed with nisin, additional mechanisms may also explain the large differences observed for such compounds in the minimal inhibitory concentrations against individual bacteria. For example, it was observed for pediocin-like bacteriocins that killing of Listeria monocytogenes requires a >100-fold range of bacteriocin concentrations depending on the strain [23]. Bacteriocins have either a strong positive net charge, with a high pI, or at least a strong localized positive charge, making them very sensitive to anionic environments. The hydrophobicity/amphilicity property of bacteriocins makes them vulnerable to antagonism by hydrophobic compounds in the environment including target organisms. Changes in the cell wall such as increased D-alanylation of teichoic acid and lipoteichoic acid introduce additional positive charges that may render the target bacteria less susceptible to bacteriocins. Likewise, it has been suggested that lysinylation of the cell-membrane phospholipids may give the same effect [41]. Several studies have investigated the fatty acid composition of membranes of sensitive and resis-

tant target organisms and an increase in membraneassociated saturated fatty acids is correlated with the resistance of pediocin-like bacteriocins [27, 37].

STRUCTURE Several structural studies of pediocin-like bacteriocins have been published [16, 38, 40]. NMR as well as x-ray analysis have been published on their immunity proteins [21, 39]. Such studies have contributed significantly to our understanding of how such antimicrobial peptides and their dedicated immunity proteins work. It has been demonstrated convincingly that the pediocin-like bacteriocins consist of two domains that are separated by a flexible hinge [12, 16, 40]. Studies of hybrid pediocin-like bacteriocins have shown that the positively charged (and most conserved) N-terminal domain binds to the target cell surface [3, 24], while the more hydrophobic (and more diverse) C-terminal domain interacts with the cell membrane [12, 13, 28]. The N-terminal domain comprises a β-strand, while the hydrophobic C-terminal domain starts with an α-helical structure and ends with short hairpin structure (Fig. 1).

Structure of sakacin P

Structure of leucocin A

FIGURE 1. NMR structure of (A) sakacin P [40] and (B) leucocin A [16]. The graphical presentation of the three-dimensional structure is performed by Entrez’ Molecular Modeling Database [2].

112 / Chapter 17 In the construction of hybrid bacteriocins it was demonstrated how important it was to choose the recombination point between the two domains as precisely as possible in order to make a functional hybrid bacteriocin. It was also shown that the C-terminal half of the immunity protein contains a region that directly or indirectly recognizes the membrane-penetrating Cterminal hairpin domain of pediocin-like bacteriocins [22]. These results are consistent with the proposal that pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants, possibly a receptor. It has been suggested that the pediocin-like bacteriocin (Class IIA) can be even further divided into at least three subgroups based on the diversity of their Cterminal domains [15]. While all pediocin-like bacteriocins have an N-terminal cysteine disulfide bridge (Fig. 1) [11, 30], which is required for antimicrobial activity, some pediocin-like bacteriocins have a second disulfide bridge located in the C-terminal domain. The pediocinlike bacteriocins with two disulfide bridges exert a much higher specific antimicrobial activity compared with those containing only an N-terminal bridge. Introduction of a disulfide bridge in the C-terminal part of sakacin P, which normally contains only one N-terminal disulfide bridge, increased its specific activity about 20fold against certain indicator strains and rendered the bacteriocin activity less temperature-dependent [14]. Wild type sakacin P is not active at 37°C, while the activity of modified sakacin P was insensitive to this temperature, probably due to the stabilizing effect of the second disulphide bridge.

FUTURE TRENDS The most challenging aspect of bacteriocin research is to understand their mechanisms of action. In particular it is important to understand if and how receptors are involved, how immunity proteins act, and the mechanisms leading to resistance. Despite the large number of Class II bacteriocins identified, we may have seen just the tip of the iceberg. Bacterial genome sequences have revealed many potential new bacteriocin genes providing a route to identifying new, potent antibacterial peptides [31]. The traditional search for new antimicrobial peptides suffers from the lack of a general functional assay. Often bacteriocins are not expressed during standard growth/screening conditions, and consequently many antimicrobial activities will not be detected. Genome sequencing and analysis will be a powerful tool in identifying new antimicrobial peptides, and hopefully the future will find application for such compounds in the war against pathogens.

Acknowledgments The authors have been supported by the Norwegian Research Council (NRC). Dr. D. A. Brede is a post doc fellow supported by NRC.

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membranes in a voltage-independent, protein-mediated manner. J. Bacteriology. 173(24):7934–41. [44] Worobo, R. W., M. J. Van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1995. A signal peptide secretiondependent bacteriocin from Carnobacterium divergens. J. Bacteriology. 177(11):3143–9.

C

H

A

P

T

E

R

18 Colicins: Bacterial/Antibiotic Peptides O. SHARMA, S. D. ZAKHAROV, AND W. A. CRAMER

tor specific for that type of colicin. Indeed, as was confirmed later, colicins enter cells by parasitizing receptors in the outer membrane whose physiological purpose is the binding and import of metabolites (e.g., vitamins, sugars, metals such as iron). Subsequently, several colicins differing in their mode of action (discussed in [34]) and the receptors they parasitize to gain entry into target cells, have been identified. Ribbon diagrams of those colicins (Ia, N, B, which are pore formers and E3, an rRNase), whose 3-D structure has been solved, are shown in Fig. 1. Colicins have been classified according to the translocation system that they appropriate to enter the bacteria. Group A colicins utilize the bacterial Tol-dependent translocation system consisting of the proteins TolA, TolB, TolQ, TolR, and Pal (Table 1). The name of these proteins is derived from the fact that their mutants are “tolerant” to colicin action. Group B colicins utilize the Ton-dependent system, consisting of TonB and ExbB-ExbD (Table 2). “Ton” mutants are resistant to the phage T1—hence the name “Ton.” As can be seen from the tables, the lethal action of most colicins is exerted either by cellular de-energization [22, 26] that results from the formation of a highly conducting pore [29] in the cytoplasmic membrane [12], or by an enzymatic nuclease digestion mechanism [1, 23, 24].

ABSTRACT The functions of receptor binding, cytotoxicity, and translocation across the cell envelope, necessary for colicins to be imported into the cell and to exert their lethal effect on the cell, are encoded in defined peptide domains of the colicin polypeptide. The major lethal effects of the colicins are exerted through pore formation and intracellular nuclease activity. High-resolution x-ray structures of four colicins and x-ray/NMR structures of four C-terminal domains encoding cytotoxic function are known. Cells producing colicin are protected against autocytotoxicity by a ∼10 kDa immunity protein, which is a tightly bound soluble component of the nuclease colicins or a membrane-bound peptide present in low abundance in cells producing poreforming colicins. Import into the cell is guided by a transenvelope network of “tol” or “ton” proteins.

DISCOVERY AND CLASSIFICATION [28] Colicins are plasmid-encoded bacteriocins, produced by Escherichia coli under stress conditions, which are cytotoxic to closely related strains that contain the required outer membrane receptor(s) but do not produce the cognate immunity protein. Colicins were first discovered in 1925 by Gratia who showed that one strain of E. coli (“V” for virulent) released a substance found in the filtrate that was toxic to a sensitive strain. Gratia also showed that colicin-resistant mutants could be derived from sensitive strains. Fredericq later suggested that resistance to colicins involved the loss of a surface recepHandbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE Colicin-producing cells are protected from its cytotoxic action by coordinate synthesis of an immunity

115

Copyright © 2006 Elsevier

116 / Chapter 18 TABLE 1. Colicins that are imported via the Tol system. Although these colicins have a common theme of import into target cells, the receptors and translocators utilized by these colicins are different. They also differ in their cytotoxic activities. Colicin A2 E1 N E2 E7 E9 E3 E6

Activity Protein1

Immunity Protein1

Receptor

Translocation

Cytotoxic Activity

592 522 387 581 566 582 551 551

178 113 131 86 87 86 85 85

BtuB BtuB OmpF BtuB BtuB BtuB BtuB BtuB

OmpF, TolQRAB TolC, TolQRAB TolQRA OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB

Pore forming Pore forming Pore forming DNase DNase DNase 16SrRNase 16SrRNase

1

Length in amino acids. Sequence of Colicin A from Citrobacter freundii. Tol-dependent colicins E4, E5, E8, K, U, 28b, and DF13 have not been included in the table. Modified from [20]. 2

TABLE 2. Colicins that are imported via the Ton system. Although the mechanism of import of these colicins into the target cells are similar, the cytotoxic activity differs. A common feature of these colicins is the presence of a TonB box that interacts with the TonB protein which transduces energy from the inner membrane to these import processes [27]. Activity Protein1

Immunity Protein1

Receptor protein

B D

510 697

175 87

FepA FepA

Ia Ib

626 626

111 115

Cir Cir

Colicin

Activity Channel Protein synthesis2 Channel Channel

1

Length in amino acids. Inhibits protein synthesis by cleavage of arginine tRNAs. Ton-dependent colicins Js, M, 5 and 10 have not been included in the table. Modified from [6]. 2

protein that acts specifically against that colicin. Immunity proteins are small (MW ∼10 kDa) polypeptides that protect the colicin-producing cells from both endogenous and exogenous colicins [5, 15]. In the case of pore forming colicins, a small number of immunity molecules (∼102–103) in the cytoplasmic membrane are able to provide immunity against a functional and lethal channel in that membrane [45]. Regions of homology between the immunity proteins of pore-forming colicins A, B and N have suggested that extramembrane loops L1 and L3 of the 3–4 TM helix protein interact with the respective colicins. Thus, mutations in these regions affect the immunity activity of these proteins [11]. A lysis protein localized in the periplasmic space functions to release colicin from producing cells to the extracellular medium, both for colicin-immunity

protein complex of enzymatically active colicins and for pore-forming colicins. In pore-forming colicins, the Imm protein remains in the cytoplasmic membrane [3]. The gene encoding colicin, its cognate immunity protein, and the lysis protein form an operon that is regulated by the SOS (DNA damage-induced) promoter [8]. In the absence of induction, the immunity gene is expressed by a constitutive promoter that provides basal levels of immunity protein for protection against exogenous colicins.

THE DOMAIN CONCEPT There are three defined steps in the killing of a sensitive E. coli by most colicins, each of which is carried

Colicins: Bacterial/Antibiotic Peptides / 117

FIGURE 1. (A) Generic domain structure of colicin polypeptides (top): T, translocation; R, receptor binding; C, catalytic or activity containing domain (B–E). Ribbon diagrams of structures of pore-forming colicins, Ia (B), B (E), and N (D), and of rRNase colicin E3 (C). Note the prominent long (210 Å and 100 Å in colicins Ia and E3) coiled-coil structure of the R-domain. Structures, predominantly α-helical, have also been obtained of the C-domains of (i) colicins A and E1 by x-ray diffraction, and (ii) of E7 and E9 by solution NMR. The structures of E3 and E7 contain bound immunity protein, as is characteristic of the nuclease colicins. (See color plate.)

out by a separate domain of the colicin that occupies approximately one-third of the polypeptide (Fig. 1A; [24]). The first step involves the central “R” (receptor) domain, which mediates the binding of colicin to its outer membrane receptor. The N-terminal “T” (translocation) domain mediates the second step of translocation from the outer membrane receptor to the colicin target in the cell. Finally, the carboxy-terminal “C” (catalytic) domain functions in cytotoxicity.

SEQUENCE COMPARISONS (Fig. 2) Groups A and B have subgroups with extended regions of high homology. In group A, colicins with endonuclease activity (E2–E9) have a high degree of homology in the T and R domains (residues 1–449) based on 85% similarity (70% for E7). The subgroup of pore-forming colicins (E1, A, and N) have low homol-

ogy between thvemselves and with colicins E2–9 (∼13% between any two pore-forming group A colicins) in the T and R domains. However, the homology is higher between C-domains of pore-forming colicins (∼30%), which correlates with a common motif consisting of a 10-helix globular structure including a hydrophobic helical hairpin. The subgroup of endonuclease colicins has high homology in the C-domain between 16sRNase colicins E3 and E6 (90%), and between DNase colicins E2, E7 and E9 (80%). A conserved pentapeptide sequence “TolB box” is present in most of the Tol-dependent colicins. This TolB box has been shown to interact with the TolB protein [4, 30], defining a step in the translocation process. A site of proteolytic cleavage between TR- and C-domains (R447) has been defined in E7 [32]. In group B, the pore-forming colicins Ia and Ib use the CirA iron transport receptor for recognition [6, 7]. They are identical in the T and R domains including

Group A 1 I

E7: E3: E6: E2: E9: E1: A: N:

21 I

161 I

181 I

241 I

261 I

321 I

281 I

341 I

361 I

VKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAELNQANKDVARNQERQAKAVQVYNSRKSELDAAN------------VKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAAN------------KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN------------KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN------------KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN------------AKYKELDELVKKLSPRANDPLQNRPFFEATRRR-VGAGKADITQIQKAISQVSNNRNAGIARVHEAE------------QRVQETLKFI-------NDPIRSRIHFNMRSGL-IRAQHSQLSQANNILQNARNEKSAADAALSAATAQRLQAEAALRAA --IKGTEVYT----------------FHTRKGQYVKVTVWKGPKYNNKLVK----RFVSQFLLFRKE------------381 I

E7: E3: E6: E2: E9: E1:

201 I

TPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYVSVTDVLTPAQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNNSTPEVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNDSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP AQSE--------------VVKMDGEIKTLNSRLSSSI--HARDAEMKTLAGKRNELAQAIREEKQKQVTASETRINRINS DAGKRVEAAQAA------INTAQLNVNNLSGAVSAAN--QVITQKQAEMTPLKNELAAANVDTKQNEINAAVANRDALNN KPGR--------------YISSNPEYSLLAKLIDAES----------------------P------DSNIDKMRVDYVN301 I

E7: E3: E6: E2: E9: E1: A: N:

121 I

LPSEIAKDDPNMMSKIVTSLPAETVTNVQVSTLPLDQATVSVTKRV----TDVVKDTRQHIAVVAGVPMSVPVVNAKPTR LPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRV----VDDVKDERQNISVVSGVPMSVPVVDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV----VDDVKDERQNISVVSGVPMSVPVVDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV----VDDVKDERQNISVVSGVPMSVPVVDAKPTE GGLAVSISASELSAAIAGILPADDITESPVSSLPLDKATVNVNVRV----VDDVKDERQNISVVSGVPMSVPVVDAKPTE HNASRTPSATELAHANNAAEKAFQEAEQRRKEIEREKAETERQLKL-------AEAEEKRLAALSEEAKAVEIAQKKLSA SPAGQNGGKSPVQTAVENYGNERTWTVKVPREVPQLTASYNEGMRIRQEAADRARAEANARALAEEEARAIASGKSKAEF NSGNRGNNGDGASAKVGEI------------------------------------------------------------S 221 I

E7: E3: E6: E2: E9: E1: A: N:

101 I

HGNGGGNSNSGGGS-----NSSVAAPMAFG--FPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSAWGIALYGI HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGV HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA RGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA KKTQAEQAARAKAAAEAQAKAKANRDALTQRLKDIVNEALMQAEDE-RLRLAK--------------AEEKARKEAEAAR IINAAGQPTMNGTVMTADNSSMVPYGRGFTRVLNSLVNNPLMVQSG-NLPPGYWLSNGKVMTEV---REERTSGGGGKNV PHKNDGFHSDG------------SYHITFHGDNNSKPKPGTITPDN---------------------------------G 141 I

E7: E3: E6: E2: E9: E1: A: N:

61 I

MSGGDGRGHNSGAHNTGGNI-----NGGPTGLGGNGGASDGSGWSSENNPWGGGS--------------GSGVHWGGGSG MSGGDGRGHNTGAHSTSGNI-----NGGPTGLGVGGGASDGSGWSSENNPWGGGS--------------GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI-----NGGPTGLGVGGGASDGSGWSSENNPWGGGS--------------GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI-----NGGPTGLGVGGGASDGSGWSSENNPWGGGS--------------GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI-----NGGPTGIGVSGGASDGSGWSSENNPWGGGS--------------GSGIHWGGGSG METAVAYYKDGVPYDDKGQVIITLLNGTPDGSGSGGGGGKGGSKSESSAAI------------------HATAKWSTAQL -MPGFNYGGKG--------------DGTGWSSERGSGPEPGGGSHGNSGGHDRGDSSNVGNESVTVMKPGDSYNTPWGKV ------MGSNG--------------ADNAHNNAFGGGKNPGIGNTSGAGSNGSASSNR-----------GNSNGWSWSNK 81 I

E7: E3: E6: E2: E9: E1: A: N:

41 I

401 I

----------------------KTLADAKAEI--KQFERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAF-DAAAKE ----------------------KTLADAIAEI--KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ----------------------KTLADAIAEI--KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ----------------------KTLADAIAEI--KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ----------------------KTLADAIAEI--KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ----------------------ENLKKAQNNLLNSQIKDAVDATVSFYQTLTEKYGEKYSKMAQELADKSK---GKKIGN

FIGURE 2. Amino acid residues of colicins E3 and Ia are numbered in groups A and B, respectively. Residues considered to be similar are: D and E; K and R; N and Q; S and T; I, L, and V. Coloring: red (bold)—similar residues in all or most (except one) of sequences. Blue (bold)—similar residues in poreforming colicins (E1, A, N) of group A. Orange and green—used to show similarity within subgroups of DNase and RNase colicins of group A. Green and blue—used to show similarity within subgroups of CirA- and FepAdependent colicins of group B. Alignment was done using the MUSCLE program.

Colicins: Bacterial/Antibiotic Peptides / 119 A: N:

AEAAEKARQRQAEEAERQRQAMEVAEKAKDER--ELLEKTSELIAGMGDKIGEHLGDKYKAIAKDIADNIKNFQGKTIRS -------------------------EKEKNEK--EALLKASELVSGMGDKLGEYLGVKYKNVAKEVANDIKNFHGRNIRS 421 I

E7: E3: E6: E2: E9: E1: A: N:

441 I

461 I

KSDADVALSSALERRKQKENK-EKDAKAKLDKESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKS KSDADAALSSAMESRKKKEDK-KRSAENNLNDEKNKPRKG-------------FKDYGHDY-HPAP-------KTENIKG KSDADAALSSAMESRKKKEDK-KRSAENKLNEEKNKPRKG-------------VKDYGHDY-HPDP-------KTEDIKG KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKN KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKS VNEALAAFEKYKDVLNKKFSKADRDAIFNALASVKYDDWAKH-----------LDQFAKYLKITGHVSFGYDVVSDILKI FDDAMASLNKITANPAMKINKADRDALVNAWKHVDAQDMANK-----------LGNLSKAFKVADVVMKVEKVREKSIEG YNEAMASLNKVLANPKMKVNKSDKDAIVNAWKQVNAKDMANK-----------IGNLGKAFKVADLAIKVEKIREKSIEG 481 I

E7: E3: E6: E2: E9: E1: A: N:

501 I

521 I

F--DDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKA----PKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDNISVVT L--GDLKPGI-------PKTPKQNGGGKRKRWT---------------GDKGRKIYEWDSQHGELEGYRASDGQHLGSFD L--GELKEGK-------PKTPKQGGGGKRARWY---------------GDKGRKIYEWDSQHGELEGYRASDGQHLGSFE F--DDFRKKFWEEVSKDPDLSKQFKGSNKTNIQKGKA----PFARKKDQVGGRERFELHHDKPISQDGGVYDMNNIRVTT F--DDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYS----PFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTT KDTGDWKPLF-------LTLEKKAADAGVSYVVALLFS----------LLAGTTLGIWGIAIVTGILCSYIDKNKLNTIN YETGNWGPLM-------LEVESWVLSGIASSVALGIFSATLGAYALSLGVPAIAVGIAGI-LLAAVVGALIDDKFADALN YNTGNWGPLL-------LEVESWIIGGVVAGVAISLFGAVLSFLPIS-GLAVTALGVIGI-MTISYLSSFIDANRVSNIN 541 I

E7: E3: E6: E2: E9: E1: A: N:

PKRHIDIHRGK--------PKTGNQLKGPDPKRNIKKYL PKTGNQLKGPDPKRNIKKYL PKRHIDIHRGK--------PKRHIDIHRGK--------EVLGI--------------NEIIRPAH-----------NIISSVIR------------

Group B 1 I

Ia: Ib: B: D:

21 I

101 I

181 I

141 I

201 I

221 I

LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQNADKKAA--DMLAEYERRKGILDTRLSELEKNGGAALAVLD LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQNADKKAA--DMLAEYERRKGILDTRLSELEKNGGAALAVLD ------ARLEPASGNEQKI----IRLMVTQQLEQVTDIPASQLPAAGNNVPVKY---------RLTDLMQNGTQYMAIIG ------ARLEPASGNEQKI----IRLMVTQQLEQVTDIPASQLPAAGNNVPVKY---------RLMDLMQNGTQYMAIIG 241 I

Ia: Ib: B: D:

121 I

EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EEGDWSGWSVSVHSPWGNEKVSAARTVLE------------NGLRGGLPEPSRPAAVSF--------------------EEGDWSGWSVSVHSPWGNEKVSAARTVLE------------NGLRGGLPEPSRPAAVSF--------------------161 I

Ia: Ib: B: D:

61 I

MSD-----PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIWGGNEWVDDSPTRSDIEKRDK MSD-----PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIWGGNEWVDDSPTRSDIEKRDK MSDNEGSVPTEGIDYGDTMVVWPSTGR-IPGGDVKPGGSS–GLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL MSDYEGSGPTEGIDYGHSMVVWPSTGL-ISGGDVKPGGSS–GIAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL 81 I

Ia: Ib: B: D:

41 I

261 I

281 I

301 I

AQQARLLGQQTRNDRAISEARNKLSSVTESLNTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIVVSGDPR AQQARLLGQQTRNDRAISEARNKLSSVTESLKTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIVVSGDPR GIPMTV-------------------PVVDAVPVPDR--SRPGTNIKDVYSAPVSPNL--PDLVLSVGQMNTPVRSNPEIQ GIPMTV-------------------PVVDAVPVPDR--SRPGTNIKDVYSAPVSPNL--PDLVLSVGQMNTPVLSNPEIQ 321

FIGURE 2.

(Continued)

341

361

381

120 / Chapter 18 I

Ia: Ib: B: D:

I

401 I

Ia: Ib: B: D:

I

421 I

441 I

AVNSARNNLSARTNE---QKHANDALNALLKEK-------------ENIRNQLSGINQKIAEEKRKQDELKATKDAI-NF AINSARNNVSARTNE---QKHANDALNALLKEK-------------ENIRSQLADINQKIAEEKRKRDEINMVKDAI-KL AENNAKDDFRVKKEQ------ENDEKTVL----------------------------------TKTSEVIISVGDKVGEY AENKAKDDFRVKKEEAVARAEAEKAKAELFSKAGVNQPPVYTQEMMERANSVMNEQGALVLNNTASSVQLAMTGTGVWTA 461 I

Ia: Ib: B: D:

481 I

501 I

TTEFLKSVSEKYGAKAEQLAREMAG-----------------QAKGKKI---RNVEEALKTYEKYRADIN---------TSDFYRTIYDEFGKQASELAKELAS-----------------VSQGKQI---KSVDDALNAFDKFRNNLN---------LGDKYKALSREIAENINNF-------------------------QGKTI---RSYDDAMSSINKLMANPS---------AGDIAGNISKFFSNALEKVTIPEVSPLLMRISLGALWFHSEEAGAGSDIVPGRNLEAMFSLSAQMLAGQGVVIEPGATSV 521 I

Ia: Ib: B: D:

I

FAGTIKITTSAVIDNRANLNYLLSHSGLDYKRNILNDRNPVVT-EDVEGDKKIYNAEVAEWDKLRQRLLDARNKITSAES FAGTIKITTSAVIDNRANLNYLLTHSGLDYKRNILNDRNPVVT-EDVEGDKKIYNAEVAEWDKLRQRLLDARNKITSAES EDGVISET-----GNYVEAGYTMSS----------NNHDVIVRFPEGSGVSPLYISAVE--------ILDS-NSLSQRQE EEGVIAET-----GNYVEAGYTMSS----------NNHDVIVRFPEGSDVSPLYISTVE--------ILDS-NGLSQRQE

541 I

561 I

-------------------KKINAKDRAAIAAALESVKLSDISSNLNRFSRGLGYAGKFTSLADWITEFGKAVRTENWRP -------------------KKYNIQDRMAISKALEAINQVHMAENFKLFSKAFGFTGKVIERYDVAVELQKAVKTDNWRP -------------------LKINATDKEAIVNAWKAFNAEDMGNKFAALGKTFKAADYAIKANNIREKSIEGYQTGNWGP NLPVRGQLINSNGQLALDLLKTGNESIPAAVPVLNAVRDTATGLDKITLPAVVGAPSRTILVNPVPQPSVPT-DTGNHQP 581 I

Ia: Ib: B: D:

LFV-------KTETII------------------------AGNAATALVALVFSILTGSA-------------------FFV-------KLESLA------------------------AGRAASAVTAWAFSVMLGTP-------------------LML-------EVES----------------------WV--ISGMASAVALSLFSLTLGSALIA-------------FGLS VPVTPVHTGTEVKSVEMPVTTITPVSDVGGLRDFIYWRPDAAGTGVEAVYVMLNDPLDSGRFSRKQLDKKYKHAGDFGIS 601 I

Ia: Ib: B: D:

621 I

------LGIIGY-----------------------------------GLLMAVTGALIDESLV--EKANKFWG-----I ------VGILGF-----------------------------------AIIMAAVSALVNDKFI--EQVNKLIG-----I ------ATVVGF---------------------------------VGVVIAGAIGAFIDDKFV--DELNHKII-----K DTKKNRETLTKFRDAIEEHLSDKDTVEKGTYRREKGSKVYFNPNTMNVVIIKSNGEFLSGWKINPDADNGRIYLETGEL

FIGURE 2.

(Continued)

most of the coiled-coil structure (residues 1–426), and have a 60% similarity in the C-domain. Colicins B and D containing 510 and 627 residues, respectively, act through pore-forming and DNase activities, and use the ferro-enterobactin receptor FepA for recognition [6]. They have a high degree of homology (95%) in the Nterminal segment (res. 1–310). The homology of the aligned segment of central R- and C-terminal domains, excluding that unique to colicin D, is smaller (∼30%). Homology between any three of the four colicins of group B aligned in Fig. 2 is 25%. A common feature of the Ton-dependent colicins is the presence of a “TonB box” pentapeptide sequence in a position proximal to the N-terminus [31]. This sequence is present not only in the Ton-dependent colicins, but also in the outer membrane receptor proteins whose function is dependent on the Ton system.

X-RAY STRUCTURES OF COLICINS (Figs. 1B–E) (A) The four complete or incomplete structures of colicins that have been solved by x-ray diffraction of three-dimensional crystals and their major properties are summarized: (i) The structure of residues 23–39 and 83–624 of the 626 residue pore-forming colicin Ia (“B-type,” translocated by the Ton system) has been solved to 3.0 Å resolution (Fig. 1B; [40]). (ii) The structure encompassing residues 84–551 of the 551 residue “A-type” (translocated by the Tol system) endoribonuclease colicin E3 has been solved to 3.0 Å (Fig. 1C; [35]). The x-ray structure of a complex of the BtuB receptor with bound coiled-coil R-domain of colicin E3 [18], which is relevant to the mechanisms of cellular import, is discussed below. (iii) 292 residues, 93–385,

Colicins: Bacterial/Antibiotic Peptides / 121 of a 321 residue chymotryptic fragment of the 387 residue pore-forming colicin N, has provided a 3.1 Å structure that contains the globular channel forming domain (C, in blue, Fig. 1D) but is missing a substantial part of the T- (and possibly of the R-) domain [38]. (iv) The structure of most of the 511 residue poreforming colicin B (Ton-dependent) (Fig. 1E) was solved at 2.5 Å resolution [14]. The x-ray and NMR structures of the C-domains of pore-forming colicins A [25] and E1 [10] show a bundle of 10 α-helices with a hydrophobic helical hairpin anchor at the core. Structures of the endonuclease domain of colicins E3, E7, E9 (Toldependent), and Ton-dependent colicin D in complex with Imm or nucleic acids have also been solved [13, 17, 36]. The T-, R-, and C-domains in colicins E3 (RNase; Tol-dependent) and Ia (pore-forming; Ton-dependent) are structurally defined (Figs. 1B, C) and connected by long α-helices that form coiled-coils, whereas in colicins B (pore-forming; Ton-dependent; Fig. 1E), and probably in N (pore-forming; Tol-dependent; Fig. 1D), the T- and R-domain functions are located in a globular domain which is separated from the C-domain by a single long helix, forming the shape of a dumbbell. These two distinct shapes are not related to either the import pathway (Tol- or Ton-dependent), or to the mechanism of cytotoxicity (pore-formation or endonuclease). It has been proposed that the coiled-coil structure in colicin E3 is necessary for delivery of T- and C-domains of a receptor-bound colicin to an outer membrane porin through which the C-domain, at least, is translocated to the periplasm [18]. However, involvement of outer membrane porins in the import of Tondependent colicin Ia has not yet been implied. (B) T-domains. The N-terminal T-domain of colicin Ia contains a “TonB box” (E23–V27) that is necessary for translocation across the periplasm as part of an antiparallel helix bundle. This secondary structure of the T domain of colicin Ia contrasts with the β-sheet “jelly roll” that forms most of the colicin E3 T domain. The N-terminal region of the colicin E3 T domain contains a DGSGW (D35-W39) “TolB box” needed for translocation [4] and is glycine-rich (34 of 79 residues), accounting for the disorder in this region of the structure. A possible reason for the difference in the Tdomain structures of colicins Ia and E3 is that the immunity protein is tightly bound to the T domain in the latter. The N-terminal domain of colicin B is composed of 17 β-strands, 2 α-helices, and long loops. The central region (residues 130–291) of its sequence shares some similarity with the T domain of colicin E3, but the folding pattern does not fit to a jellyroll motif. In both colicins Ia and B, the N-terminal segments, containing the TonB box (residues 23–27 and 17–21 in Ia and B, respectively) are folded back and interact with antipar-

allel helices of the coiled-coil and T-domain in colicin Ia, and with the N-terminal lobe (TR-domains) of colicin B. (C) R-domains. The 104 residue region at the tip of the 160 Å coiled-coil, consisting of an amphipathic twostranded β-hairpin folded around the tip-terminal helix of the coiled-coil was proposed to bind to the colicin Ia Cir receptor [40]. The R-domain of colicin N was proposed to consist of a 65 residue (residues 97–161) sixstranded β-strand wrapped around the C-terminal end of a 45 residue extended helix (65 Å) that would be part of the pore-forming domain [38]. The receptorbinding function of colicin B was attributed to two antiparallel β-strands (res. 262–282) of the same N-terminal lobe that contains the T-domain. Comparison of the structure of colicin Ia (Fig. 1B) with genetic and functional analysis of the pore-forming colicins implies that the entire coiled-coil might be a good fit to the receptor binding R-domain. Subsequent studies of the minimum receptor binding domain in the A type colicins E9 and E3 that inhibit intracellular protein synthesis have indicated that a large part of the coiled-coil, but perhaps only its distal half, might be necessary for recognition and effective binding to the receptor [18]. The coiled-coil nature of the receptor domain in the structure of intact colicins Ia and E3 implies that this is a necessary structure motif for interaction with the receptor that initially sequesters the colicin. The markedly smaller length of the extended helices of colicin N and B is suggestive of a mechanism that is different from those used by colicins Ia and E3 for receptor binding and translocation across the outer membrane and periplasmic space. (D) Cytotoxicity domains. The isolated soluble poreforming 20 kDa domain of colicins A [25] and E1 [10], along with this domain in the intact colicins B, Ia, and N, have a similar 10-helix globular structure with tightly packed apolar core formed by hydrophobic helical hairpin consisting of helices VIII and IX. The hydrophobic core is isolated from the polar environment by 7–8 hydrophilic helices, part of which have amphipathic patterns. The average length of these helices is 12–13 residues, shorter than required (ca. 20 residues) to span the membrane bilayer. Beyond the implied role of the buried helical hydrophobic hairpin as a potential membrane anchor, the structure of the soluble channel domain provides few clues about the mechanism of pore forming insertion into the membrane. Rather, the structure implies that the soluble C-domain must undergo large conformational changes in order to accomplish the insertion [21, 43]. The function of the hydrophobic helical hairpin is to serve as an anchor during pore-forming domain insertion into membrane bilayer. This interaction

122 / Chapter 18 requires the presence of an optimum content of anionic lipids—that is, an optimum membrane surface potential [44]. Electrostatic interaction with the negatively charged membrane surface (i) guides binding of the C-terminal pore-forming domain with the membrane surface and (ii) induces unfolding that results in the formation of a closed pre-pore state [21, 43]. The ability of pore-forming colicins to form ion channels in planar bilayer membranes was demonstrated for the first time by Schein et al. [29]. The transition of the colicin from a closed-to-open state requires the presence of a transmembrane potential, negative on the side of the membrane opposite to the side of protein binding and insertion. The electrical field across the cytoplasmic membrane (in vivo) created by the electrochemical proton gradient favors poreformation. The structure of the open channel state is not known. The channel is formed by a single colicin molecule [reviewed in [19]]. In the open state of the channel, a significant part of the C-domain (68 residues, helices II–V of colicin Ia) becomes accessible to ligand binding from the trans side of membrane [16, 33]. The dependence of channel-formation by colicins on the lipid content of membrane points to the involvement of lipids in the formation and structure of ion channel that has been characterized as a “toroidal pore” [42]. Cytotoxic activity of endonuclease colicins also resides in the C-terminal domain. The size of the Cterminal cytotoxic domain of these colicins ranges from 96 to 135 residues, respectively. The immunity proteins bind these domains with very high affinity (Kd < 10−13 M; [39]) efficiently blocking the interaction of colicins with their substrates, DNA or RNA. Extensive, highly complementary interaction surfaces in endonucleaseImm complexes that involve hydrophobic and polar side chains of both proteins, account for the highaffinity nature of Imm binding. Active sites in both DNase and rRNase colicins involve residues C terminal to the residues involved in Imm binding. This can allow Imm binding to an incompletely translated colicin in order to afford protection to host cells during the toxin synthesis.

MECHANISM OF IMPORT ACROSS OUTER MEMBRANE Colicins parasitize receptors that have a metabolic function in the binding and transport of vitamins, metals, and sugars. The best characterized colicin receptor system is that of vitamin B12 receptor (BtuB) utilized by colicin E3 (Table 1). The colicin binds tightly (Kd ≤ 10−10 M) to BtuB. A 2.75 Å structure of BtuB-R-domain has been obtained [18]. Translocation of colicin E3

across the outer membrane appears to require a second outer membrane protein, the OmpF porin [2], to which the T-domain can bind, forming a two-receptor translocon for colicin import across the outer membrane. The coupled and concomitant events required for this import are (i) high-affinity binding to BtuB [37], (ii) unfolding of the colicin and extracellular release of immunity protein, (iii) insertion of disordered Nterminal region of the T-domain into the OmpF channel [41], and (iv) presumed cleavage of C-domain at an exposed R-C junction [9, 32].

AREAS OF RESEARCH ENCOMPASSED BY COLICIN STUDIES As just described, colicin studies span a range of general interests that include mechanisms of protein secretion, protein import, protein-receptor interactions, high-affinity protein-protein interactions, ion channel structures and function, and intracellular nuclease activities.

Acknowledgment Studies of the authors reported here were supported by a grant (GM-18457) from the NIH.

References [1] Almendinger R, Hager LP. Studies on the mechanism of action of colicin E2, 107–33. In “Chemistry and function of colicins” (Hager LP, ed.). Academic Press, New York. [2] Bénédetti H, Frenette M, Baty D, Lloublès R, Geli V, Lazdunski C. Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coli. J Gen Microbiol 1989;135:3413–20. [3] Bishop LJ, Bjes ES, Davidson VL, Cramer WA. Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1. J Bacteriol 1985;164:237–44. [4] Bouveret E, Rigal A, Lazdunski C, Bénédetti H. The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step. Mol Microbiol 1997;23:909–20. [5] Bowman CM, Sidikaro J, Nomura M. Specific inactivation of ribosomes by colicin E3 in vitro and mechanism of immunity in colicinogenic cells. Nat New Biol 1971;234:133–7. [6] Braun V, Patzer SI, Hantke K. Ton-dependent colicins and microcins: modular design and evolution. Biochimie 2002;84: 365–80. [7] Cardelli J, Konisky J. Isolation and characterization of an Escherichia coli mutant tolerant to colicins Ia and Ib. J Bacteriol 1974;119:379–85. [8] Cavard D, Oudega B. (1992). General introduction to the secretion of bacteriocins, 297–305. In “Bacteriocins, Microcins, and Lantibiotics” (James R, Lazdunski C, Pattus F, eds.). SpringerVerlag, Berlin. [9] de Zamaroczy M, Mora L, Lecuyer A, Geli V, Buckingham RH. Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Mol Cell 2001;8:159–68.

Colicins: Bacterial/Antibiotic Peptides / 123 [10] Elkins P, Bunker A, Cramer WA, Stauffacher CV. A mechanism for protein insertion into the membranes is suggested by the crystal structure of the channel-forming domain of colicin E1. Structure 1997;5:443–58. [11] Géli V, Baty D, Pattus F, Lazdunski C. Topology and function of the integral membrane protein conferring immunity to colicin A. Mol Microbiol 1989;3:679–87. [12] Gould JM, Cramer WA. Studies on the depolarization of the Escherichia coli cell membrane by colicin E1. J Biol Chem 1977;252:5491–97. [13] Graille M, Mora L, Buckingham RH, van Tilbeurgh H, de Zamaroczy M. Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein. EMBO J 2004;23: 1474–82. [14] Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Kang C. Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution. Mol Microbiol 2004;51:711–20. [15] Jakes K, Zinder ND. Highly purified colicin E3 contains immunity protein. Proc Natl Acad Sci USA 1974;71:3380–84. [16] Kienker PK, Jakes K, Blaustein RO, Miller C, Finkelstein A. Sizing the protein translocation pathway of colicin Ia channels. J Gen Physiol 2003;122:161–76. [17] Kühlmann UC, Pommer AJ, Moore GR, James R, Kleanthous C. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes. J Mol Bio 2000;301:1163–78. [18] Kurisu G, Zakharov SD, Zhalnina MV, Bano S, Eroukova VY, Rokitskaya TI, Antonenko YN, Wiener MC, Cramer WA. The structure of BtuB with bound colicin E3 R-domain implies a translocon. Nat Struct Biol 2003;10:948–54. [19] Lakey JH, Slatin SL. Pore-forming colicins and their relatives. Curr Top Microbiol Immunol 2001;257:131–61. [20] Lazzaroni J-C, Dubuisson J-F, Vianney A. The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins. Biochimie 2002;84:391–97. [21] Lindeberg M, Zakharov SD, Cramer WA. Unfolding pathway of the colicin E1 channel protein on a membrane surface. J Mol Biol 2000;295:679–92. [22] Luria SE. On the mechanisms of action of colicins. Annales de L’Institut Pasteur 1964;107:67–73. [23] Nomura M. Mechanism of action of colicins. Proc Natl Acad Sci USA 1964;52:1514–21. [24] Ohno-Iwashita Y, Imahori K. Assignment of the functional loci in colicin E2 and E3 molecules by the characterization of their proteolytic fragments. Biochemistry 1980;19:652–59. [25] Parker MW, Postma JPM, Pattus F, Tucker AD, Tsernoglou D. Refined structure of the pore-forming domain of colicin A at 2.4 Å resolution. J Mol Biol 1992;224:639–57. [26] Phillips SK, Cramer WA. Properties of the fluorescence probe response associated with the transmission mechanism of colicin E1. Biochemistry 1973;12:1170–76. [27] Postle K, Kadner RJ. Touch and go: tying TonB to transport. Mol Microbiol 2003;49:869–82. [28] Reeves P, ed. (1972). “The Bacteriocins,” Chapter 1. SpringerVerlag, New York.

[29] Schein SJ, Kagan BL, Finkelstein A. Colicin K acts by forming voltage-dependent channels in phospholipid bilayer membranes. Nature 1978;276:159–63. [30] Schneider CG, Penfold CN, Moore GR, Kleanthous C, James R. Identification of residues in the putative TolA box which are essential for the toxicity of the endonuclease toxin colicin E9. Microbiology 1997;143:2931–38. [31] Schramm E, Mende J, Braun V, Kamp RM. Nucleotide sequence of the colicin B activity gene cba: Consensus pentapeptide among TonB-dependent colicins and receptors. J Bacteriol 1987;169:3350–57. [32] Shi Z, Chak KF, Yuan HS. Identification of an essential cleavage site in ColE7 required for import and killing of cells. J Biol Chem 2005;280:24663–8. [33] Slatin SL, Qiu X-Q, Jakes KS, Finkelstein A. Identification of a translocated protein segment in a voltage-dependent channel. Nature 1994;371:158–61. [34] Šmarda J. (1978). “The effects of colicins,” p. 213. J. E. Purkyne University Brno. [35] Soelaiman S, Jakes K, Wu N, Li CM, Shoham M. Crystal structure of colicin E3: Implications for cell entry and ribosome inactivation. Mol Cell 2001;8:1053–62. [36] Sui MJ, Tsai LC, Hsia KC, Doudeva LG, Ku WY, Han GW, Yuan HS. Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of Cole7/Im7 in complex with a phosphate ion and different divalent metal ions. Prot Sci 2002;11:2947–57. [37] Taylor R, Burgner JW, Clifton J, Cramer WA. Purification and characterization of monomeric Escherichia coli vitamin B12 receptor with high affinity for colicin E3. J Biol Chem 1998;273:31113–18. [38] Vetter IR, Parker MW, Tucker AD, Lakey JH, Pattus F, Tsernoglou D. Crystal structure of a colicin N fragment suggests a model for toxicity. Structure 1998;6:863–74. [39] Walker D, Moore GR, James R, Kleanthous C. Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3. Biochemistry 2003;42:4161–71. [40] Wiener M, Freymann D, Ghosh P, Stroud RM. Crystal structure of colicin Ia. Nature 1997;385:461–64. [41] Zakharov SD, Eroukova VY, Rokitskaya TI, Zhalnina MV, Sharma O, Loll PJ, Zgurskaya HI, Antonenko YN, Cramer WA. Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import. Biophys J 2004;87:3901–11. [42] Zakharov SD, Kotova EA, Antonenko YN, Cramer WA. On the role of lipid in colicin pore formation. Biochim Biophys Acta 2004;1666:239–49. [43] Zakharov SD, Lindeberg M, Griko YV, Salamon Z, Tollin G, Prendergast FG, Cramer WA. Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc Natl Acad Sci USA 1998;95:4282–87. [44] Zakharov SD, Rokitskaya TI, Shapovalov VL, Antonenko YN, Cramer WA. Tuning the membrane surface potential for efficient toxin import. Proc Natl Acad Sci USA 2002;99:8654–59. [45] Zhang YL, Cramer WA. Intramembrane helix-helix interactions as the basis of inhibition of the colicin E1 ion channel by its immunity protein. J Biol Chem 1993;268:10176–84.

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C

H

A

P

T

E

R

19 Fungal Peptides with Antifungal Activity K. T. CHU, H. X. WANG, AND T. B. NG

proteins [11, 17, 18, 24, 33, 34, 36] have been isolated from the fruiting bodies of various mushroom species. The antifungal peptides and proteins are in general unadsorbed on DEAE-cellulose and adsorbed on Affigel blue gel and CM-cellulose/S-Sepharose/Mono S. A 7-kDa peptide, which suppresses mycelial growth in the fungi Fusarium oxysporum, Mycosphaerella arachidicola, and Physalospora piricola, has been purified from the oyster mushroom Pleurotus ostreatus [4]. A 9-kDa antifungal peptide has been isolated from the mushroom Agrocybe cylindracea [26]. A 10-kDa antifungal peptide from the mushroom Pleurotus eryngii, designated as eryngin, inhibits mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola [35]. Isarfelin, a cyclic peptide with inhibitory activity on mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum and insecticidal activity toward Leucania separata, was isolated from the mycelia of Isaria felina [12]. The cyclic peptides pseudodestruxins A and B from cultures of Nigrosabulum globosum possess both antifungal and antibacterial activities. Samarosporin, a neutral cyclopeptide from a Samarospora species, exerts antimicrobial activity [13]. Clavariopsin A and B, cyclic depsipeptide antibiotics from the fermentation broth of Clavariopsis aquatica, inhibit the growth of Aspergillus fumigatus, Aspergillus niger, and Candida albicans [14, 32]. Burmeister et al. [1] described a process for production of a cyclodepsipeptide complex from autoclaved white corn grits fermented with Fusarium tricinctum. Conspicuous swelling of Penicillium digitatum conidia and hyphal tips incubated in a medium supplemented with the CDPC was observed. The presence of one major and two minor cyclodepsipeptides in CDPC was disclosed. The latter differs from the former only in the substitution of either isoleucine or valine for leucine in the threonyl-alanylalanyl-glutaminyl-tyrosyl-leucine peptide portion of the molecule.

ABSTRACT Antifungal peptides and proteins with different Nterminal sequences have been isolated from basidiomycete fungi (mushrooms). Most of them inhibit mycelial growth with IC50 values in micromolar concentrations. Other activities may include translation-inhibitory activity, mitogenic/antimitogenic activity toward spleen cells, antiproliferative activity toward tumor cells, and HIV-1 reverse transcriptase inhibitory activity. Other fungi produce short pentaibol peptides with an alcohol amino acid at the C-terminus and nonstandard amino acids such as aminoisobutyric acid in the sequence. Antifungal lipopeptides are produced by Cryptosporiopsis and Mycogone spp. Aspergillus giganteus and A. niger elaborate antifungal peptides. Cyclic peptides with antifungal and insecticidal activity are produced by Isaria felina.

DISCOVERY Fungal attack of agricultural crops leads to a serious decline in the quality and yield of crops and brings about massive economic losses. Research on antifungal proteins may provide solutions to the problem. Antifungal proteins are a heterogeneous assembly of structurally diverse proteins produced by a wide range of organisms encompassing mammals, insects, flowering plants, gymnosperms, fungi, and bacteria. Plant antifungal proteins can be classified, in accordance with their structure or function, into different types [23]. Despite the existence of numerous fungal species, antifungal proteins have been isolated from only a few species, including those shown in Table 1. The intent of the present review that covers antifungal peptides from fungi is to compare their characteristics with antifungal peptides and proteins from flowering plants (amgiosperms). Antifungal peptides [4, 25, 26, 35] and Handbook of Biologically Active Peptides

125

Copyright © 2006 Elsevier

Agrocybin Molecular mass (kD) Monomeric (M)/ Homodimeric (H) Chromatographic behavior (i) DEAE-cellulose (ii) Affi-gel Blue gel (iii) CM-Sepharose/CMcellulose (iv) Mono S/S-Sepharose (v) Red-Sepharose (vi) Heparin-Sepharose (vii) Mono Q Antifungal activity (IC50) against (i) Botrytis cinerea (ii) Coprinus comatus (iii) Colleotrichum gossypii (iv) Fusarium oxysporum (v) Mycosphaerella arachidicola (vi) Physalospora piricola (vii) Rhizoctonia solani Mitogenic activity Antimitogenic activity on splenocytes Antiproliferative activity (IC50) against (i) L1210 cells (ii) Hep G2 cells Cell-free translation inhibitory activity HIV-1 reverse transcriptase inhibitory activity

Alveolarin

Eryngin

Ganodermin

Hypsin

Lyophyllum antifungal protein Lyophyllin

Lentin

Pleurostrin

Pleurotus sajor-caju RNase

Trichogin

9 M

28 H

10 M

15 M

14 M

14 M

20 M

27.5 M

7 M

12 M

27 M

Unadsorbed Adsorbed —

Unadsorbed Adsorbed —

Unadsorbed Absorbed —

Unadsorbed Adsorbed Adsorbed

— Adsorbed Adsorbed

— Adsorbed Adsorbed

— Adsorbed Adsorbed

Unadsorbed Adsorbed —

Unadsorbed Adsorbed —

— — Adsorbed

Unadsorbed Adsorbed Adsorbed

Adsorbed — — —

— — — —

Adsorbed — — —

— — — —

Unadsorbed

Adsorbed — — —

Adsorbed — — —

— — — —

— — — —

— Adsorbed Adsorbed —

— — — —

— — —

— — —

— — —

15.2 μM — —

60 nM — —

— Inactive Inactive

— Active Inactive

Active — —

— — —

— — —

— — —

—

Active

1.35 μM

12.4 μM

14.2 μM

—

—

—

>15.6 μM

95 μM

Present

125 μM

Active

3.5 μM

—

2.7 μM

Active

Inactive

17.5 μM

 15.6 μM

72 μM

3.8 μM

—

—

—

18.1 μM

2.5 μM

70 nM

2.5 μM

Active

<15.6 μM

—

Present

— Present

— — —

— — —

— — —

— — 10 μM

— — Absent

Inactive — Present

— — —

— — —

— — 65 nM

— —

— Inactive —

— — —

— — —

— — —

5 μM 30 μM 7 nM

— — 70 μM

— — 1 nM

— 0.2 μM 1.5 μM

15 μM 41 μM 28 μM

0.1 μM 0.22 μM 158 nM

— — 83 nM

60 μM

—

—

—

8 μM

5.2 nM

7.9 nM

—

—

—

Information in table from refs [4, 17, 18, 24–26, 34–36].

126 / Chapter 19

TABLE 1. Comparison of characteristics of mushroom antifungal peptides and proteins.

Fungal Peptides with Antifungal Activity / 127 Tricholongins B1 and B2, peptaibol peptides the fungus Trichoderma longibrachiatum, display antifungal and antibacterial activities [19, 28]. Peptaibol antibiotics, designated as chrysospermins, have been isolated from the mycelium of Apiocrea chrysosperma. These nonadecapeptides exert antibacterial and antifungal activities, and induce pigment formation by the fungus Phoma destructive [6]. Helioferins A and B, aminolipopeptides in cultures of Mycogone rosea DSM 8822, demonstrate antimicrobial activity when tested against Candida albicans and grampositive bacteria including Mycobacterium spp. [8].

STRUCTURE Agrocybin, the antifungal peptide isolated from Agrocybe cylindracea, exhibits remarkable homology to RP13, a cysteine-rich protein expressed during fruiting initiation in Agrocybe chaxingu. Agrocybin is also very similar to grape (Vitis vinifera) antifungal peptide in N-terminal sequence [26]. The results suggest that the antifungal function of agrocybin is important during fruiting initiation for protecting the fruiting bodies. The Nterminal sequence of agrocybin is also similar to a small portion of the sequence in an endochitinase from Populus trichocarpa and a lipid transfer protein from Psyscomitrella patens that manifests antifungal activity. Thus, it appears that the N-terminal sequence of agrocybin is related to the antifungal activity of the peptide (Table 2). The N-terminal sequence of eryngin demonstrates some similarity to the antifungal protein from the mushroom Lyophyllum shimeiji [35]. Although there is a certain degree of similarity between the N-terminal sequence of lentin (Table 2) and a portion of the sequences of some endoglucanases near the C-terminal, those endoglucanases are much larger in molecular size than lentin. Nevertheless, this sequence similarity may contribute to the antifungal activity of lentin because TABLE 2.

glucanases demonstrate antifungal activity [24]. Grenier et al. [9] have demonstrated that some fungi express β-1,3-glucanases similar to thaumatin-like proteins. Some angiosperms produce glucanases or glucanaselike proteins with antifungal activity. The sequence resemblance between lentin and heat shock protein may indicate that lentin may be produced under the stress of attack by fungi [24]. The solution structure of Aspergillus giganteus antifungal protein has been determined. Its folding is composed of five antiparallel β-strands connected in a −1. −1, topology and highly twisted, defining a small and compact β barrel stabilized by four internal disulfide bridges. A cationic site constituted by up to three lysine side chains next to a hydrophobic stretch, both at the protein surface, may form a potential phospholipid binding site that would furnish the basis of its antifungal activity [11]. The antifungal peptide isolated from the culture medium of Aspergillus niger exhibits sequence homology to cysteine-rich antifungal peptides from the seeds of Sinapsis alba and Arabidopsis thaliana or the extracellular media of Aspergillus giganteus and Penicillium chrysogenumsome. Potent inhibitory activity against yeast and filamentous algae has been observed [10]. Crytocandin is a 1.1-kDa lipopeptide from the endophytic fungus Cryptosporiopsis cf. quercina. It is inhibitory toward Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum, Sclerotina sclerotiorum, and Botrytis cinerea [31]. Some fungi produce a special kind of small peptide, known as peptaibol antibiotics that exhibit fungicidal and also antibacterial activities by forming pores in the lipid bilayer and inducing leakage of cytoplasm [15]. The peptides are characterized by different degrees of nonstandard amino acid residues such as α-aminoisobutyric acid, α-methylalanine, isovaleric acid, and the imino acids hydroxyproline and proline. Another characteristic is the presence of an alcohol amino acid at the C-terminus (perhaps the unexpected Ser-octanoyl in one form of mammalian ghrelin should

Comparison of N-terminal sequences of mushroom antifungal peptides and proteins.

Agrocybin Alveolarin Eryngin Ganodermin Hypsin Lyophyllin Lyophyllum antifungal protein Lentin Pleurostrin Pleurotus sajor-caju RNase

ANDPQCLYGNVAAKF GVCDMADLA ATRVVYCNRRSGSVVGGDDTVYYEG AGETHTVMINHAGRGAPKLVVGGKKLS ITFQGDLDARQQVITNADTRRKRDVRAA ITFQGASPARQTVITNAITRARADVRAA AGTEIVTCYNAGTKVPRGPSAXGGAIDFFN CQRAFNNPRDDAIRW VRPYLVAFYESH DNGEAGRAAR

Sequences from refs [4, 17, 18, 24–26, 34–36].

128 / Chapter 19 not have been such a surprise). Nine antifungal trichorzianines A that vary mainly at positions 5, 14, 16, and 19 have been isolated from the mold Trichoderma harzianum. The antifungal peptides, classified into peptaibol, contain 19-amino-acid residues with an acetylated residue at the N-terminus and tryptophanol or phenylalaninol at the C-terminus [1]. Tricholongins are highly hydrophobic nonadecapeptides with 19-amino-acid residues in which the N-terminus is acetylated and the C-terminus is leucinol. The full sequence of tricholongin B1 is Aib-Gly-Phe-Aib-Aib-Gln-Aib-Aib-Aib-Ser-LeuAib-Pro-Val-Aib-Aib-Gln-Gln-Leuol. Tricholongin B2 differs from B1 in residue 16 [27]. A peptaibol peptide designated ampullosporin with antifungal and antibacterial activities has been isolated from the fungus Sepedonium ampullosporum. Its full amino acid sequence is Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Gln-LeuAib-Gln-Leuol [28]. Six longibrachins, natural Aibcontaining with a C-terminal phenylalaninol, have been purified from Trichoderma longibrachiatum. Longibrachins demonstrate significant bactericidal activity against mycoplasmas and also perturb the permeability of membrane bilayers [19]. Boletusin, a new peptaibol, was isolated from the fruiting body of Boletus spp. with one acetylated N-terminus residue, phenylalanine, and a C-terminal amino alcohol, tryptophanol. This peptide exhibits antimicrobial activity against several grampositive bacteria [20]. Two aminolipopeptides, helioferins A and B from the fungus Mycogone rosea, inhibit the growth of Candida albicans and gram-positive bacteria [8]. From the fungus Cordyceps heteropoda, cicadapeptins I and II were isolated. Cicadapeptin I has the sequence Hyp-Hyp-Val-Aib-Gln-Aib-Leu. Cicadapeptin II has Ile instead of Leu. Both peptides have antibacterial activity and limited antifungal activity [14].

BIOLOGICAL ACTION Most of the mushroom antifungal peptides and proteins reported to date resemble, in their broad spectra of activity against different fungal species, many angiosperm antifungal proteins. It is noteworthy that some antifungal proteins have a specificity of action against only certain fungal species [23]. Agrocybin differs from Lyophyllum antifungal protein and P. sajor-caju RNase in that the former exhibits mitogenic, whereas the latter two demonstrate antimitogenic, activity [17, 25, 26]. The HIV-1 reverse transcriptase inhibitory activity of agrocybin is in line with similar reports on angiosperm antifungal proteins [23]. The potency of pleurostrin, Lyophyllum antifungal protein, lentin, and P. sajor-caju RNase in inhibiting cell-free translation in the rabbit reticulocyte lysate system are in line with previous reports on angiosperm antifungal proteins

[23]. The antiproliferative activity of pleurostrin, lentin, P. sajor-caju RNase, and Lyophyllum antifungal protein is also consistent with findings on antifungal protein from chive [23]. However, agrocybin is devoid of activity toward Hep G2 cells [26]. Some angiosperm antifungal proteins have hemagglutinating (lectin), deoxyribonuclease, and protease inhibitory activities [23]. The mushroom antifungal peptides and proteins reported to date do not have any of these activities. The antifungal peptide from Aspergillus giganteus demonstrates strong antifungal activity against Magnaporthe grisea, Fusarium moniliforme, and Phytophthora infestans. It does not inhibit the growth of Aspergillus giganteus, yeast, or bacteria [16, 23, 33, 38]. Heterologous expression of the gene encoding this antifungal protein reduces the formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondite f. sp. tritici) in transgenic wheat [27]. Trichorzianine A, produced by Trichoderma harzianum when cultured with the cell wall of the fungus Botrytis cinerea, exhibits synergistic antifungal effect together with chitobiohydrolase, endochitinase, or beta-1,3-glucanase on spore germination and hyphal growth of fungi Botrytis cinerea and Fusarium oxysporum [30]. The synergistic action of an antifungal protein and a ribosome inactivating protein from the mushroom Lyophyllum shimeiji has also been noted [17].

References [1] Burmeister HR, Vesonder RF, Peterson RE, Costello CE. Production and purification of a peptide of Fusarium tricinctum that causes conidia of Penicillium to swell. Mycopathologia 1985; 91: 53–6. [2] Campos-Olivas R, Bruix M, Santoro J, Lacadena J, Martinez del Pazo A, Gavilanes JG, Rico M. NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism. Biochem 1995; 34: 3009–21. [3] Che Y, Swenson DC, Gloer JB, Koster B, Malloch D. Pseudodestruxins A and B: new cyclic depsipeptides from the coprophilous fungus Nigrosabulum globosum. J Nat Prod 2001; 64: 555–8. [4] Chu KT, Xia L, Ng TB. Pleurostrin, an antifungal peptide from the oyster mushroom. Peptides 2005; 26: 2098–103. [5] Chugh JK, Wallace BA. Peptaibols: models for ion channels. Biochem Soc Trans 2001; 29: 565–70. [6] Dornberger K, Ihn W, Ritzau M, Grafe U, Schlegel B, Fleck WF, Metzger JW. Chrysospermins, new peptaibol antibiotics from Apiocrea chrysosperma Ap101. J Antibiot (Tokyo). 1995; 48: 977– 89. [7] el Hajji M, Rebuffat S, Lecommandeur D, Bodo B. Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum. Int J Pept Protein Res 1987; 29: 207–15. [8] Grafe U, Ihn W, Ritzau M, Schade W, Stengel C, Schlegel B, Fleck WF, Kunkel W, Hartl A, Gutsche W. Helioferins; novel antifungal lipopeptides from Mycogone rosea: screening, isolation, structures and biological properties. J Antibiot (Tokyo) 1995; 48: 126–33.

Fungal Peptides with Antifungal Activity / 129 [9] Grenier J, Potvin C, Asselin A. Some fungi express β-1,3glucanases similar to thaumatin-like proteins. Mycologia 2000; 92: 841–8. [10] Gun Lee D, Shin SY, Maeng CY, Jin ZZ, Kim KL, Halm KS. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem Biophys Res. Commun 1999; 263: 646–51. [11] Guo Y, Wang H, Ng TB. Isolation of trichogin, an antifungal protein from fresh fruiting bodies of the edible mushroom Tricholoma giganteum. Peptides 2005; 26: 575–80. [12] Guo YX, Liu QH, Ng TB, Wang HX. Isarfelin, a peptide with antifungal and insecticidal activities from Isaria felina. Peptides 2005; 26: 284–91. [13] Inoue N, Inoue A, Furukawa M, Kanda N. Samarosporin, a new peptide antibiotic. I. Fermentation, isolation and characterization. J Antibiot (Tokyo) 1976; 29: 618–22. [14] Kaida K, Fudou R, Kameyama T, Tubaki K, Suzuki Y, Ojika M, Sakagami Y. New cyclic depsipeptide antibiotics, clavariopsins A and B, produced by an aquatic hyphomycetes, Clavariopsis aquatica. 1. Taxonomy, fermentation, isolation, and biological properties. J Antibiot (Tokyo) 2001; 54: 17–21. [15] Krasnoff SB, Reategui RF, Wagenaar MM, Gloer JB, Gibson DM. Cicadapeptins I and II: new Aib-containing peptides from the entomopathogenic fungus Cordyceps heteropoda. J Nat Prod 2005; 68: 50–5. [16] Lacadena J, Martinez del Pozo A, Gasset M, Patino B, CamposOlvias R, Vazquez C, Martinez-Ruis A, Mancheno JM, Onaderra M, Gavilanes JG. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch Biochem Biophys 1992; 324: 273–81. [17] Lam SK, Ng TB. First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence for synergism of their antifungal effects. Arch Biochem Biophys 2001; 393(2): 271–80. [18] Lam SK, Ng TB. Hypsin, a novel thermostable ribosomeinactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem Biophys Res Commun 2001; 285(4): 1071–5. [19] Leclerc G, Goulard C, Prigent Y, Bodo B, Wroblewski H, Rebuffat S. Sequences and antimycoplasmic properties of longibrachins LGB II and LGB III, two novel 20-residue peptaibols from Trichoderma longibrachiatum. J Nat Prod 2001; 64: 164–70. [20] Lee SJ, Yeo WH, Yun BS, Yoo ID. Isolation and sequence analysis of new peptaibol, boletusin, from Boletus spp. J Pept Sci 1999; 5: 374–8. [21] Marx F, Haas H, Reindl M, Stoffler G, Lottspeich F, Redl B. Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity. Gene 1995; 167: 167– 71. [22] Nakaya K, Omata K, Okahashi I, Nakamura Y, Kolckenbrock H, Ulbrich N. Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus. Eur J Biochem 1990; 193: 31–8.

[23] Ng TB. Antifungal proteins of leguminous and non-leguminous origins. Peptides 2004; 25: 1215–22. [24] Ngai PH, Ng TB. Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells. Life Sci 2003; 73(26): 3363–74. [25] Ngai PHK, Ng TB. A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju. Peptides 2005; 25: 11–17. [26] Ngai PH, Zhao Z, Ng TB. Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea. Peptides 2005; 26(2): 191–6. [27] Oldach KH, Becker D, Lorz H. Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat. Mol Plant Microbe Interact 2001; 14: 832–8. [28] Rebuffat S, Prigent Y, Auvin-Guette C, Bodo B. Tricholongins BI and BII, 19-residue peptaibols from Trichoderma longibrachiatum. Solution structure from two-dimensional NMR spectroscopy. Eur J Biochem 1991; 201: 661–74. [29] Ritzau M, Heinze S, Dornberger K, Berg A, Fleck W, Schlegel B, Hartel A, Grafe U. Ampullosporim, a new peptaibol-type antibiotic from Sepedonium ampullosporum HK1-0053 with neuroleptic activity in mice. J Antibiotic (Tokyo) 1997; 50: 722–8. [30] Schirmbock M, Lorito M, Wang YL, Hayes CK, Arisan-Atac I, Scala F, Harman GE, Kubicek CP. Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl Environ Microbiol 1994; 60: 4364–70. [31] Strobel GA, Miller RV, Martinez-Miller C, Condron MM, Teplow DB, Hess WM. Cryptocandin, a potent antimycotic from the endophytic fungus Cryptospoiopsis cf, quercina. Microbiol 1999; 145: 1919–26. [32] Suzuki Y, Ojika M, Sakagami Y, Kaida K, Fudou R, Kameyama T. New cyclic depsipeptide antibiotics, clavariopsins A and B, produced by an aquatic hyphomycetes, Clavariopsis aquatica. 2. Structure analysis. J Antibiot (Tokyo) 2001; 54(1): 22–8. [33] Vila L, Lacadena V, Fontanet P, Martinez de Pozo A, San Segundo B. A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens. Mol Plant Microbe Interact 2001; 14: 1327–31. [34] Wang H, Ng TB, Liu Q. Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris. Peptides 2004; 25: 693–6. [35] Wang H, Ng TB. Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii. Peptides 2004; 25: 1–5. [36] Wang HX, Ng TB. Ganodermin, an antifungal protein from fruiting bodies of the medicinal mushroom Ganoderma lucidum. Peptides 2005; 27: 27–30. [37] Whitmore L, Wallace BA. The Peptaibol Database: a database for sequences and structures of naturally occurring peptaibols. Nucleic Acids Res 2004; 32: D593–4. [38] Wnendt S, Ulbrich N and Stahl U. Molecular cloning, sequence analysis and expression of the gene encoding an antifungal protein from Aspergillus giganteus. Curr Genet 1994; 25: 519– 23.

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20 Toxins from Basidiomycete Fungi (Mushroom): Amatoxins, Phallotoxins, and Virotoxins JACK H. WONG AND T. B. NG

ABSTRACT

DISCOVERY OF MUSHROOM TOXINS

Toxic mushrooms (basidiomycete mushrooms), including Amanita, Galerina, and Lepiota spp., produce proteinaceous toxins like phallolysin, peptidic toxins including amatoxins, phallotoxins, and virotoxins, and nonpeptidic toxins such as orellanine, gyromitrin, and coprine. Amatoxins are bicyclic octapeptides, phallotoxins are bicyclic heptapeptides, and virotoxins are monocyclic peptides. Amatoxins inhibit DNAdependent RNA polymerase II, whereas both phallotoxins and virotoxins stimulate the polymerization of G-actin and stabilization of F-actin filaments. Chromatographic methods are available for the separation and determination of these toxic peptides. Symptoms of poisoning due to these peptides and its treatment have been described. Phalloidin derivatives can be used as contraction modifiers for a comparative study of cardiac and skeletal muscle.

Toxins in mushroom can in general be divided into two groups: toxic compounds and toxic peptides/proteins. There are three kinds of common toxic compounds: orellanine, gyromitrin, and coprine [5, 12, 14]. Orellanine (3,3′,4,4′-tetrahydroxy-2,2′-bipyridine-1, 1′-dioxide) found in Cortinarius mushrooms causes tubulointerstitial nephritis and renal failure. Gyromitra mushrooms contain gyromitrin (N-methyl-N-formylhydrazone). It decomposes in the stomach to produce, a toxin that causes gastroenteritis, hemolysis, methemoglobinemia, hepatorenal failure, seizures, and coma. Coprinus mushrooms contain coprine (N5-1-hydroxycyclopropyl-L-glutamine), which is metabolized to 1aminocyclopropanol, an inhibitor of the enzyme aldehyde dehydrogenase, producing a clinical syndrome similar to disulfiram (Antabuse) alcohol reaction. Toxic proteins include phallolysin (about 30 kDa), and toxic peptides include amatoxins, phallotoxins, and virotoxins [19, 23]. The first documented experiment designed to elucidate the toxicity of Amanita toxins was conducted by William W. Ford in collaboration with Hermann Schlesinger. A partially purified fraction of the crude extract of Amanita virosa killed a guinea pig (250–400 g) after a subcutaneous dose of 0.4 mg. The name “amanitatoxin” was given to the thermostable toxin. In 1937, Foedor Lynen and Ulrich Wieland obtained the first crystalline toxic component named phalloidin [16]. Intraperitoneal injection of phalloidin brought about the death of a white mouse within two to five hours. In 1941, Rudolf Hallermayer and Heinrich Wieland described the crystallization of “amanitin” [20]. Later on, with new methods of paper chromatography and electrophoresis, it was found that a crystalline

INTRODUCTION Mushrooms (basidiomycete mushrooms) are popular in the diet. Recently, there is increased interest in the use of mushrooms or mushroom extracts as dietary supplements based on theories that they boost immune function and promote health. However, people are not satisfied with the existing mushroom species. The hunting and eating of wild higher fungi has become increasingly popular all over the world. Collectors may confuse edible with toxic species, due to misidentification based on superficial characteristics. Even very knowledgeable wild mushroom gatherers are sometimes poisoned. Mushroom poisoning is a relatively common medical emergency. Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

132 / Chapter 20 preparation of Amanita toxin consisted of two components: a neutral one designated as alpha-amanitin and an acid one called beta-amanitin. In the following years, scientists isolated gamma- and delta-amanitins [25] and the nonpoisonous component amanullin from Amanita phalloides and amaninamide from Amanita virosa [4]. The family of amatoxins comprises these

amanitins (Fig. 1). The family of phallotoxins includes phalloin [24], phalloidin, phallisin, phallacidin, phallacin, and phallisacin [22] (Fig. 2). The virotoxins (Fig. 3) are clearly separated from phallotoxins present in A. virosa by chromatography on Suphadex LH-20. In their mode of action, virotoxins are closed related to the phallotoxins.

R1 HO

CH

H3 C

CH

HN

CH

CH

NH

CO

H

CH

C H2

CO NH

OS

N

HO

NH

CO

H2C

CO

CH2

CO

CH

NH

CO

CH

N H

R2 NH

CO

C H2

CH3

CH

CH

CO

C2 H5

NH

C H2 CO

R3

Peptide -Amanitin -Amanitin -Amanitin -Amanitin Amanin FIGURE 1.

R1 CH 2OH CH 2OH CH 3 CH 3 CH2OH

R2 OH OH OH OH H

R3 NH2 OH NH2 OH OH

Structures of amatoxins.

R3 H3C

CH

CO

CH

NH

CO

NH

CH

CO

H2C

NH CO

NH CH

S C H2 N

CO

CH

N H

RR1 1

CO NH

CO

CH

NH

H

R2

OH

Peptide

R1

R2

R3

Phalloin

CH3

CH(OH)CH3

CH2(CH3)2OH

Phalloidin

CH3

CH(OH)CH3

CH2(C2H5OH)OH

Phallisin

CH3

CH(OH)CH3

CH2(CH2OH)2OH

Phallacin

CH(CH3)2

CH(OH)CO2H

CH2(CH3)2OH

Phallacidin

CH(CH3)2

CH(OH)CO2H

CH2(C2H5OH)OH

Phallisacin

CH(CH3)2

CH(OH)CO2H

CH2(CH2OH)2OH

FIGURE 2.

Structures of phallotoxins.

Toxins from Basidiomycete Fungi (Mushroom): Amatoxins, Phallotoxins, and Virotoxins / 133 R2 H 2 C OH

C

OH

CH2 H3C

CH

CO

CH

NH

CO

CO

CO

NH CH

R1 R1

N H

HO N

CO

OH

FIGURE 3.

CH

H2C

NH

Peptide Viroidin Desoxoviroidin Ala-viroidin Viroisin

NH

R1 SO2 SO SO2 SO2

CH

R3

CO NH

CO

HO CH 2

CH

NH

CHOH C H3

R2 CH3 CH3 CH3 CH2OH

R3 CH(CH3)2 CH(CH3)2 CH3 CH(CH3)2

Structures of virotoxins.

An antitoxic cyclic decapeptide called antamanide from A. phalloides, when injected at a dosage of one milligram per kg body weight prior to a lethal dose of phalloidin, could prevent death in mice [23]. Phallotoxins and amatoxins are also present in Conocybe lactea and C. filaris, respectively [11]. Malthies and Laatsch [17] refuted the existence of toxic fluorescent cyclic decapeptides named cortinarins in the toxic mushroom Cortinarius speiossimus and presented evidence that the fluorescence is partly caused by photodecomposition products of orellanine. Amaniamide found in A. virosa differs from alpha-amanitin in that it lacks the 6′-OH groups of Trp and forms amanin by presence of a carboxamide group instead of carboxylic acid [4].

STRUCTURES OF MUSHROOM TOXINS All amatoxin-containing fungi are found within the families Amanitaceae (genus Amanita), Agaricaceae (genus Lepiota), and Cortinariaceae (genus Galerina). Amatoxins are bicyclic octapeptides (Fig. 1). The amatoxins consist exclusively of L-amino acids. Uncommon building blocks are the crosslinking 6-hydroxytryptathionine-(R)-sulfoxide. In addition, the nonpoisonous components amanullin from Amanita phalloides and amaninamide from Amanita virosa have been isolated. The phallotoxins are bicylic heptapeptides (Fig. 2). The solid state and solution conformation of [Ala7]phallodin, a synthetic analog, has been reported [27]. Virotoxins, monocyclic heptapeptides (Fig. 3) of the white mushroom Amanita virosa, produce the same toxicological symptoms as phallotoxins. They are prob-

ably derived from phallotoxins or from a common precursor molecule. Virotoxins differ from phallotoxins in that the former are monocyclic peptides and contain D-serine instead of L-cysteine. In addition, two unusual amino acids are detected in virotoxins: 2,3trans-3,4-dihydroxy-L-proline and 2′-(methylsulfony)-Ltryptophan. Two-dimensional NMR spectroscopy and restrained molecular dynamics simulations revealed that viroisin has a well-ordered conformation in solution. The functional groups important for toxicity are oriented in the same direction for binding target proteins [1]. The toxins can be separated from one another by chromatography such as HPLC and LC-MS [18].

TOXIC ACTIONS OF MUSHROOM TOXINS Amatoxins Toxicity of amatoxin. Amatoxins are powerful thermostable poisons. Ingestion of a full-grown A. phalloides mushroom (20–25 g) would mean exposure to 5–8 mg of amatoxins, which may be fatal. The molecular mechanism of action of amatoxins is inhibition of DNAdependent RNA polymerases II, with 50% inhibition at a concentration as low as 10−9 M. Depletion of mRNA will lead to diminished protein synthesis. Ultimately, the affected cells will undergo necrosis or apoptosis. Symptoms of amatoxin poisoning. After the mushroom meal there is a latency period of about 12 h prior to the onset of violent gastrointestinal symptoms, including intense diarrhea, abdominal pain, and vomiting. If not treated properly, the patient may become dehydrated

134 / Chapter 20 and hypovolemic with subsequent circulatory instability, oliguria, functional renal insufficiency, hypoglycemia, electrolyte disturbances, and metabolic acidosis. The hypoglycemia may be due to a cytotoxic effect on beta cells and induction of insulin release [6]. After 36–48 h, liver damage as indicated by increase of liver transaminases, lactate dehydrogenase, and bilirubin in serum is detectable. Clinical symptoms, including jaundice and a slightly enlarged and tender liver, may appear. Acute hepatic failure may result with hyperbilirubinemia, coagulation disorders and bleeding, hyperammonemia, metabolic acidosis, hypoglycemia, and encephalopathy. In some cases liver injury can be reversed. In fatal cases patients succumb after 6 to 16 days. During the initial gastrointestinal phase the fluid loss may lead to functional renal impairment, which is reversible upon rehydration. However, subsequently another type of renal dysfunction may ensue, caused either by the failing hepatic function (hepatorenal syndrome) or a direct toxic renal damage. Signs and symptoms of this recurring kidney dysfunction appear three to five days postingestion and indicate a poor prognosis, reflecting a heavy toxic exposure [15]. Treatments. The current treatment methods include basic medical care, detoxification, and liver transplantation. Careful monitoring of the patients is necessary. Based on the toxicokinetic study, large amounts of amatoxins (60–80%) are filtered through the glomeruli, and urinary amatoxin concentrations are much higher than those of serum, so monitoring the urinary amatoxin concentration would yield positive results within the first 8 hours and for 3–4 days after mushroom ingestion. Diarrhea and emesis can produce hypovolemic shock. Stabilization of the patient with the correction of hypoglycemia and electrolyte balance is necessary. After 36–48 h or sooner, biochemical signs of liver damage appear with increase of serum transaminases, lactate dehydrogenase, and serum bilirubin. If the hepatic condition deteriorates, a liver transplant should be considered. For detoxication, gastric lavage is conducted only if the patient is admitted in the asymptomatic phase and not beyond 6 h postingestion. Multidose activated charcoal is administered during three days postingestion to prevent reabsorption of toxin excreted to the gut, primarily through the bile. Despite the unavailability of specific amatoxin antidote, therapeutic agents such as penicillin G, silibinin, thioctic acid, and antioxidant drugs are used to minimize liver damage. Penicillin G (benzylpenicillin) inhibits hepatic amatoxin uptake. It has been suggested that penicillin G could displace amanitin from its binding site on serum protein. Unfortunately, penicillin G commonly causes allergic drug reactions with an incidence of 1–10%. Silibinin may reduce amatoxin uptake in the liver and acts as a free radical scavenger. The mechanism of

detoxication of thioctic acid has not been clearly established due to lack of unanimity of opinions. Silibinin and N-acetylcysteine are the most effective therapeutic agents [7].

Phallotoxins The LD50 of amatoxins is 0.3–0.7 mg/kg after intraperitoneal injection to mice compared with the value of 1.5–3 mg/kg for phallotoxins. Oral administration of phalloidin to the same species does not produce toxic effects. Phalloidin, one of the phallotoxins, causes swelling of the liver and vacuolization of the hepatic parenchyma cells. In the isolated perfused rat liver, potassium efflux was observed a few minutes after application of the phalloidin. Phalloidin can form a tight complex with filament actin from liver cells and muscle. As a consequence, the actin filaments become strongly stabilized against various chemical and physical stresses like 0.6 M KI, heat, proteolytic enzymes, ultrasonic ruptures, and pH changes [21]. The binding site of actin for phallotoxins has been determined by covalent labeling of rabbit muscle F-actin with affinity-labelled phallotoxins. Phalloidin possesses high affinity to actin filaments. The apparent dissociation constant (Kd) of this peptide from rabbit muscle actin is as low as 3.6 × 10−8 M. Toxin also binds to nonmuscle actin with a comparable affinity. The high affinity remains almost unaltered after toxin derivatization at the side chain of dihydroxyleucine, even after an aminomethyldithiolano group has been introduced. It is high enough to still permit specific labeling of actin filaments in cells. Fluorescent phallotoxin is an excellent tool for studying actin structures in eukaryotic cells [26]. Phalloidin enters hepatocytes by a carrier-mediated mechanism. Phalloidin may share a common uptake mechanism with organic anions like bile salts. It is noteworthy that the hepatocellular bile acid transporter Ntcp facilitates hepatic uptake of alpha-amanitin [9]. Because the bile-salt-binding polypeptides of hepatocyte membranes have been identified by photoaffinity labeling and the toxic effect of phalloidin in hepatocytes is prevented by bile salts and by antamanide, the hypothesis that the transport systems for these substance are identical was examined with photolabile derivatives of these compounds. In a study on human transporters the uptake of phalloidin into transfected HEK293 cells stably expressing the recombinant hepatocyte-specific organic anion uptake transporters OATP2 (also termed OATP1B1, OATP-C, LST1, symbol SLC21A6) or OATP8 (OATP1B3 or SLC21A8) was analyzed. Time-dependent uptake of phalloidin is observed with SLC21A6-expressing cells and is inhibited by typical

Toxins from Basidiomycete Fungi (Mushroom): Amatoxins, Phallotoxins, and Virotoxins / 135 substrates of SLC21A6 such as bromosulfophthalein or cholyltaurine [9]. Secophalloidin and phalloidin-(S)-sulfoxide can be used as contraction modifiers for comparative study of skeletal and candiac muscle [3].

Virotoxins The biological activity of viroisin, the main toxin in A. virosa, is comparable to that of the phallotoxins: for example, with 2.5 mg of viroisin per kg (white mouse), 50% of the animals die within two to five hours due to hepatic hemorrhagia. At the molecular level, the virotoxins behave like the phallotoxins. Thus, viroisin binds to rabbit muscle actin with an apparent equilibrium dissociation constant of approximately 2 × 10−8 M, very similar to that of phalloidin. However, the flexibility of the monocyclic structure and the presence of two additional hydroxy groups in the virotoxins suggest a different mode of interaction with actin. While there is proof that the bicyclic phallotoxins possess a rigid binding site, the virotoxins may adopt the biological active conformation by an induced-fit mechanism upon contact with actin [2, 8, 13, 15, 19].

Acknowledgments We thank Ms. Fion Yung for excellent secretarial assistance.

References [1] Bhaskarun R, Yu C. NMR spectra and restrained molecular dynamics of the mushroom toxin virosin. Int J Pept Protein Res 1994; 43: 393–401. [2] Bonnet MS, Basson PW. The toxiciology of Amanita virosa: the destroying angel. Homeopathy 2004; 93: 216–20. [3] Bukatina AE, Kirkpatrick RD, Campbell KB. Secophalloidin and phalloidin-(S)-sulfoxide as contraction modifiers for comparative study of skeletal and candiac muscle. Tsitologia 2000; 42: 37–41. [4] Buku A, Wieland T, Bodenmuller H, Faulstich H. Amaniamide, a new toxin of Amanita virosa mushrooms. Experientia 1980; 36: 33–4. [5] Coulet M, Guillot J. Poisoning by Gyromitra: a possible mechanism. Med Hypotheses 1982; 8: 325–34. [6] De Carlo E, Milanesi A, Martini C, Maffei P, Tamagno G, Parnigotto PP, Scandellari C, Sicolo N. Effects of Amanita phalloides toxins on insulin release: in vivo and in vitro studies. Arch Toxciol 2003; 77: 441–5. [7] Enjalbert F, Rapior S, Nouguier-Soule J, Guillon S, Amouroux N, Cabot C. Treatment of amatoxin poisoning: 20-year retrospective analysis. Toxicol Clin Toxicol 2002; 40: 715–57.

[8] Faulstich H, Buku A, Bodenmuller H, Wieland T. Virotoxins: actin-binding cyclic peptides of Amanita virosa mushrooms. Biochemistry 1980; 19: 334–43. [9] Fehrenbach T, Cui Y, Faulstich H, Keppler D. Characterization of the transport of the bicyclic peptide phalloidin by human hepatic transport proteins. Naunyn Schmiedebergs Arch Pharmacol 2003; 368: 415–20. [10] Gundala S, Wells LD, Milliano MT, Talkad V, Luxon BA, Neuschwander-Tetri BA. The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin alpha-amanitin. Arch Toxicol 2004; 78: 68–73. [11] Hallen HE, Watling R, Adams GC. Taxonomy and toxicity of Conocybe lactea and related species. Mycol Res 2003; 107: 969–79. [12] Holmdahl J, Ahlmem J, Bergek S, Lundberg S, Persson SA. Isolation and nephrotoxic studies of orellanine from the mushroom Cortinarius speciosissimus. Toxicon 1987; 25: 195–9. [13] Karlson-Stiber C, Persson H. Cytotoxic fungi—an overview. Toxicon 2003; 42: 339–49. [14] Lindberg P, Bergman R, Wickberg B. Isolation and structure of coprine, the in vivo aldehyde dehydrogenase inhibitor in Coprinus atramentarius. Syntheses of coprine and related cyclopropa4one derivatives. J Chem Soc [Perkin 1] 1977; 6: 684–91. [15] Loranger A, Tuchweber B, Gicquaud C, St-Pierres, Cote MG. Toxicity of peptides of Amanita virosa mushrooms in mice. Fundam Appl Toxicol 1985; 5: 1144–52. [16] Lynen F, Wieland U. Toxins of Amanita species (IV). Liebigs Ann Chem 1938; 533: 93–117. [17] Matthies L, Laatsch H. Cortinarins in Cortinarins speciossimus. A critical revision. Experientia 1991; 47: 634–40. [18] Mauer HH, Schmitt CJ, Weber AA, Kraemer T. Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins alpha- and betaamanitin in urine after immunoaffinity extraction. J. Chromatogr B Biomed Sci Appl 2000; 748: 125–35. [19] Vetter J. Toxins of Amanita phalloides. Toxicon 1998; 36: 13–24. [20] Wieland H, Hallermayer R, Zilg W. Amanita toxins (VI) amanitine, poison of the mushroom. Liebigs Ann Chem 1941; 548: 1–18. [21] Wieland T. Modification of actins by phallotoxins. Naturwissenschaften 1977; 64: 303–9. [22] Wieland T. The toxic peptides from Amanita mushrooms. Int J Pept Protein Res 1983; 22: 257–76. [23] Wieland T, Faulstich H. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Re-Biochem 1978; 5: 185– 260. [24] Wieland T, Mannes K. Amanita toxins (XIII) phaleosin, another toxin. Angew Chem 1957; 69: 389–90. [25] Wieland T, Rempel D, Gebert U, Buku A, Boehringer H. Components of immature Amanita phalloides XXXII. Chromatographic separation of the total toxin and isolation of the new secondary toxins amanine and phallisine and the non-toxic amanalline. Liebigs Ann Chem 1967; 704: 226–36. [26] Wulf F, Deboben A, Bautz FA, Faulstich H, Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci USA 1979; 76: 4498–502. [27] Zanotti G, Falcigno L, Javiano M, D’Auria G, Bruno BM, Campanile T, Paolillo L. Solid state and solution conformation of [Ala7]-phalloidin: a synthetic phallotoxin analogue. Chemistry 2001; 7: 1479–85.

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21 Fungal Peptides with Ribonuclease Activity T. B. NG AND H. X. WANG

to mushroom proteins with ribonuclease activity. Fungal RNases belonging to the RNase T1 family, and Aspergillus RNases, including α-sarcin, restrictocin, and mitogillin, have been reviewed previously [1, 3, 4, 15] and will not be discussed in detail here. Peptides with ribonuclease activity and a ubiquitinlike N-terminal sequence have been reported from the fruiting bodies of several mushroom species [6, 8]. The peptides are in general unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S (Table 1). Ribonucleolytic peptides [6, 7, 9, 16] and proteins [5, 10–14] with N-terminal sequences different from ubiquitin-like peptides can also be isolated from fruiting bodies of various mushroom species by using a similar procedure (Tables 2–4). Fungal species from which RNase T1 family RNases (G-specific, ca 11 kDa) have been sequenced [3] include Aspergillus clavatus, A. oryzae, A. pallidus, Fusarium lacteritium, F. moniliforme, Neurospora crassa, Penicillium brevi-compactum, P. chrysogenum, Pleurotus ostreatus, and Trichoderma harzianum. Alpha-sarcin, mitogillin, restrictocin are from Aspergillus spp. [15].

ABSTRACT Basidiomycete fungi (mushrooms) produce ribonucleolytic peptides with sequence homology to ubiquitin and also other peptides and proteins with ribonuclease (RNase) activity. Fungi including Aspergillus, Fusarium, Neurospora, Penicillium, Trichoderma, and Ustilago species produce RNase T1 family RNases, which have about 100 amino acid residues. Aspergillus spp. also produce 17kDa RNases comprising α-sarcin, restrictocin, and mitogillin. The basidiomycete RNases and ubiquitin-like RNases exhibit a variety of pH and temperature optima and polyhomoribonucleotide specificities. Some of the basidiomycete ubiquitin-like peptides and ribonucleases have been shown to have antiproliferative activity toward tumor cells and mitogenic/antimitogenic activity toward splenocytes. Fungal ribonucleases may degrade foreign RNA and thus play a defensive role in fungi.

DISCOVERY Ribonucleases (RNases) belong to an important family of proteins that have been studied intensively. Several Nobel Prizes have been awarded to eminent researchers who contributed to the early studies on RNases. RNases isolated from different mammalian tissues, such as brain, liver, and pancreas, may have different structures. Seminal and milk RNases have also been characterized. Other isolated RNases consist of those from submammalian vertebrates, invertebrates, plants, fungi, and bacteria. A spectrum of activities in RNases has been enumerated, including antiviral, immunomodulatory, antitumor, angiogenic, and antifungal activities. This review covers fungal peptides with ribonuclease activity, including mushroom ubiquitinlike peptides and small mushroom ribonucleases. For the sake of completeness, some reference is also made Handbook of Biologically Active Peptides

STRUCTURE Ubiquitin, as its name implies, is found in a variety of organisms. The amino acid sequences of ubiquitins from phylogenetically distant organisms can be almost invariant. The mushroom ubiquitin-like peptides isolated display N-terminal sequences either similar (MQIFVKTLLG for Pleurotus ostreatus and MQIFVKSSTQKCIIIFFE for Termitomyces globulus) or identical (MQIFVKTLTG for Cantharellus cibarius, Calvatia caelata, and Pleurotus sajor-caju) to human ubiquitin (Table 1). The molecular masses of these mushroom ubiquitin-like peptides are similar to each other and to human ubiquitin (Table 1).

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TABLE 1.

Comparison of characteristics of mushroom ubiquitin-like peptides with ribonuclease activity. Agrocybe cylindracea

Molecular mass (kDa) Chromatographic behavior on (i) DEAE-cellulose (ii) Affi-gel Blue gel (iii) CM-Sepharose/CM-cellulose (iv) Mono S (v) Q Sepharose Specific RNase activity (U/mg) Optimal pH for RNase activity Optimal temperature for RNase activity Activity toward polyhomoribonucleotides Anti-mitogenic activity toward splenocytes Antiproliferative activity toward tumor cells (IC50) (i) M1 cells (ii) HepG2 cells (iii) breast cancer cells Macrophage-stimulatory activity Translation-inhibiting activity (IC50)

Pleurotus ostreatus

Cantharellus cibarius

Pleurotus sajor-caju

Calvatia caelata

Termitomyces globulus

7

9

8

8

8

13

Unadsorbed Adsorbed — Adsorbed — 4200 pH 6 0–60°C

Adsorbed — Adsorbed — Adsorbed 199 pH 7 70°C

Unadsorbed Adsorbed — — — 14 — —

Unadsorbed Adsorbed — — — 450 — —

Unadsorbed Adsorbed — — — 1 — —

Unadsorbed Adsorbed Adsorbed — — 217 pH 5–8 70°C

poly C > poly U > poly A > poly G

poly C > poly A > poly U > poly G

—

—

—

poly A ⬄ poly C

—

—

—

—

100 nM

—

10 μM 100 μM Present — —

— — — — —

— — — —

— — — — 30 nM

— — 100 μM — —

— — — — —

Information in table derived from refs [6, 8].

Fungal Peptides with Ribonuclease Activity / 139 TABLE 2. N-terminal sequences of ribonucleases from Clitocybe maxima (CM), Dictyophora indusiata (DI), Ganoderma lucidum (GL), Irpex lacteus (IL), Lentinus edodes (LE), black oyster mushroom (BPO), Pleurotus ostreatus (PO), Pleurotus pulmonarius (PP), Pleurotus sajor-caju (PS), Pleurotus tuber-regium (PT), Russula virescens (RV), and Volvariella volvacea (VV). CM: DI: GL: IL: LE: BPO: PO: PP: PS: PT: RV: VV:

ETAHTHAGIQYSTVDVNNSIMKAVGGGAGN GQPRQPQPQLLV HLPBVPSFAYGSIKVYIN VNSGCGTSGAESCSNSDDGTCCFEAPGGLL ISSGCGTTGALSCSSNAKGTCCFEAPGGLI AISAANNRKA ETGVRSCNCAG ⋅ R ⋅ SFTGTDVTNAIRSARAGGSGN AISANNERKGVNQQSVQNTYQENDV DNGEAGRAAR ALTAQDNRVRVGNRIVGNNFNFAAVQAAYY TDHTLDTMMTHTLRD APYVQLFRPLIQPQVLATFAIANNMAQY

Sequences are from refs [5, 7, 9, 10, 11–14].

Unlike mushroom ubiquitin-like peptides, mushroom ribonucleases may differ markedly from one another in an amino acid sequence (Table 2). In general they have a larger molecular mass than mushroom ubiquitin-like peptides (Tables 3 and 4). These characteristics suggest that mushroom ribonucleases and mushroom ubiquitin-like peptides belong to two different groups of ribonucleases. The antiproliferative and antimitogenic activities of mushroom ubiquitin-like peptides [8], however, are also observed in ribonucleases of nonmushroom origins. The N-terminal sequence of Pleurotus sajor-caju RNase bears resemblance to the C-terminal sequence of a bacteriocin peptide from Lactobacillus sp. and to a portion of the sequence in two enzymes involved in RNA metabolism: a streptomyces RNA polymerase and a Tetracodon RNA-specific editase. This structural feature of Pleurotus sajor-caju RNase may be related to its antibacterial and RNase activities [7]. Similarity of the N-terminal sequence of P. pulmonarius RNase to the sequence of Bacillus subtilis transcriptional regulator (197–215) may also account for the translation-inhibiting activity of the RNase. It is also interesting to observe the similarity between the N-terminal sequence of P. pulmonarius RNase and a portion of the sequence of the ribosome inactivating protein abrin d precursor (204–217). It is well documented that ribosome inactivating proteins potently inhibit translation. Hence this sequence resemblance may also explain the high translation-inhibiting potency of P. pulmonarius RNase [6]. Black oyster mushroom RNase [12] closely resembles P. pulmonarius RNase in N-terminal sequences. It also deserves mention that different cultivars of the oyster mushroom P. ostreatus produce RNases with different N-terminal sequences and polyhomoribonucleotide specificities (Tables 2 and

3). This observation is reminiscent of the different Nterminal sequences exhibited by antifungal proteins from the green and gold varieties of kiwi fruit. It is noteworthy that, despite the lack of structural homology among ribonucleases of various Pleurotus species, sequence similarity is discernible between ribonucleases from Pleurotus ostreatus (Family Pleurotaceae) [9] and Clitocybe maxima (Family Tricholomataceae) [13]. Nevertheless, the homology observed is less marked than that between Lentinus edodes [5] and Irpex lacteus [14]. Of the various Pleurotus species examined, Pleurotus ostreatus ribonuclease is the only one that manifests substantial sequence relationship to Clitocybe maxima ribonuclease. Normally it would be expected that proteins from the same genus bear a more pronounced structural relationship than proteins from different genera.

BIOLOGICAL ACTION Ubiquitin-conjugated proteins including cell cycle regulatory proteins, p 53 tumor suppressor, the transcriptional regulator NF-kB and its inhibitor, many transcription factors, and the mos protooncogene, are targeted for degradation by the 26S proteasome. The ubiquitin-mediated pathway regulates cell-cycle progression, signal transcription regulation, receptor down-regulation, endocytosis, immune response, development, and apoptosis. Defects in ubiquitin-mediated events have been implicated in the development of pathological conditions, including malignant transformation. Whether ubiquitins have similar significance in the life of mushrooms remain to be elucidated, but it is likely in view of the conserved sequence exhibited by

140 / Chapter 21

TABLE 3.

Pleurotus eryngii Molecular mass (kDa) Chromatographic behavior on (i) DEAE-cellulose (ii) Affi-gel Blue gel (iii) CM-Sepharose/CMcellulose (iv) Mono S/S-Sepharose (v) Red-Sepharose (vi) Heparin-Sepharose Specific RNase activity Optimum pH Optimum temp Activity toward polyhomoribonucleotides

Cell-free translation inhibitory activity (IC50) Antiproliferative activity Antimitogenic activity

16

Comparison of characteristics of ribonucleases from various Pleurotus mushrooms.

Pleurotus ostreatus 10.76

Pleurotus ostreatus cv. “Cream Oyster Mushroom”

Pleurotus ostreatus cv. “Cream Oyster Mushroom”

Pleurotus ostreatus cv. “Black Oyster Mushroom”

Pleurotus ostreatus cv. “Brown Oyster Mushroom”

Pleurotus pulmonarius

Pleurotus sajor-caju

Pleurotus tuber-regium

9

12

14

9

14.4

12

29

Unadsorbed Adsorbed —

— — Adsorbed

— — Adsorbed

Unadsorbed Adsorbed Adsorbed

— Adsorbed Adsorbed

— Adsorbed Adsorbed

— — Adsorbed

Unadsorbed Adsorbed Adsorbed

Adsorbed — —

— — — 4,539 pH 7.0 30–60°C Co-specific for poly C and poly U

— — — 941 pH 7.0–8.0 50–70°C poly A > poly C > poly G >>>> poly U

Adsorbed Adsorbed Adsorbed 25,114 pH 7.0 55°C poly C > poly A > poly G >>> poly U

— — — 34,621 pH 5.0–7.0

— — — 39,002 pH 6.5

poly U >>> poly A > poly G > poly C

Poly G

—

—

6.33 nM

158 nM

0.09 nM

— —

— —

— —

— 65 nM

— —

pH 7.0 65°C poly A > poly G > poly U > poly C —

pH 8.0 — poly G

Adsorbed — — 650 — — —

—

15 nM

Adsorbed — — 11,490 pH 7.0 40°C poly U > poly A ⬄ poly C >>> poly G 240 nM

— —

— —

— —

— —

Information in table derived from references cited in footnote to Table 2.

TABLE 4.

Comparison of characteristics of ribonucleases from various mushroom excluding those from Pleurotus species. Clitocybe maxima

Ganoderma lucidum

Russulus virescens

Volvariella volvacea

17.5

28

43

28

42.5

Unadsorbed Adsorbed Adsorbed — 1173 pH 6.5–7.0 70°C poly A > poly G > poly U > poly C

Adsorbed — Adsorbed Adsorbed

Adsorbed — Unadsorbed Adsorbed 180.6 pH 4.0 60°C poly U > poly A > poly C > poly G

Adsorbed — Adsorbed Adsorbed 216.9 pH 4.5 60°C poly C > poly A > poly U > poly G

Unadsorbed Adsorbed Adsorbed — — pH 6.5–7.5 — poly U

4.0–4.5 60°C poly U > poly A > poly G > poly C

Information in table derived from references cited in footnote to Table 2.

Fungal Peptides with Ribonuclease Activity / 141

Molecular mass (kDa) Chromatographic behavior on (i) DEAE-cellulose (ii) Affi-gel Blue gel (iii) CM-Sepharose (iv) Q-Sepharose Specific RNase activity Optimum pH Optimum temperature Activity toward polyhomoribonucleotides

Dictyophora indusiata

142 / Chapter 21 ubiquitins [2]. The inhibitory effect of mushroom ubiquitin-like peptides on cell-free translation [8] is consistent with the role of the ubiquitin-proteasome pathway in degradation of transient and regulatory proteins involved in various cellular processes. The ubiquitin-like peptide from A. cylindracea shows antiproliferative activity on leukemia cell line M1 and hepatoma cell line HepG2, and enhances nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide [8]. Wang and Ng [6] reported a ubiquitin-like peptide with RNase, cell-free translation-inhibitory and HIV-1 reverse transcriptase-inhibitory activities from the oyster mushroom Pleurotus ostreatus. Subsequently, another ubiquitin-like peptide that possesses antimitogenic activity toward mouse splenocytes and antiproliferative activity toward tumor cell lines in addition to RNase and cellfree translation-inhibitory activities was isolated from the mosaic puffball mushroom Calvatia caelata. Ng et al. [7] isolated a ubiquitin-like peptide from Pleurotus sajorcaju cv hsiu tseng with higher translation-inhibitory and RNase activities than the former two ubiquitin-like peptides. The ubiquitin-like peptide from C. caelata reduces viability of breast cancer cells with an IC50 of 100 nM [6]. A. cylindracea ubiquitin-like peptide similarly inhibits proliferation of HepG2 and L1210 cells. However, its immunoenhancing effect on peritoneal macrophages as reflected in stimulation of nitric oxide production is in contrast to the antimitogenic activity of Calvatia caelata ubiquitin-like peptide on splenocytes. This finding is reminiscent of the observation of antimitogenic activity in mung bean cyclophilin-like antifungal protein but mitogenic activity in chickpea cyclophilin-like antifungal protein. Some lectins have mitogenic activity, while other lectins display antimitogenic activity. The stimulatory activity of A. cylindracea ubiquitin-like peptide toward macrophages [8] apparently contradicts the inhibitory activity of C. caelata ubiquitin-like peptide on splenocytes [6]. This discrepancy awaits elucidation. A. cylindracea ubiquitin-like peptide possesses an RNase activity that is much higher than those of other mushroom ubiquitin-like peptides reported to date. What accounts for this large variation in RNase activity among the ubiquitin-like peptides from different mushroom species remains to be elucidated. It may not be due to a difference in molecular size in view of the large discrepancy in RNase activity between peptides from Calvatia caelata and Agrocybe cylindracea despite their similar sizes. Unlike the activity of RNase from the mushroom Pleurotus tuber-regium, which is resistant to heating at 100°C for 30 minutes, the RNase activity of A. cylindracea ubiquitin-like peptide declines when the temperature is raised above 60°C. A pH of 6.0 is required for optimal RNase activity. Its RNase activity is stable

over the temperature range of 0–60°C. It exerts ribonucleolytic activity preferentially on poly C, much lower activity on poly U, and negligible activity on poly A and poly G [8]. The various mushroom ubiquitin-like peptides exhibit different patterns of polyhomoribonucleotide specificity (Table 1). The same applies to the mushroom RNases (Tables 3 and 4). Russula virescens RNase [6] shows cospecificity toward poly A and poly C. The RNases previously isolated from Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus tuber-regium [6], and Lentinus edodes [5] demonstrate specificity toward poly G, poly C, poly G, and poly A, respectively. P. tuber-regium RNase is much more robust compared with R. virescens RNase [6]. The activity of the former RNase is preserved after exposure to 100°C for 30 minutes. An abrupt drop in activity of R. virescens RNase is observed when the ambient temperature is elevated above 70°C. Its optimal temperature is 60°C, close to that for P. pulmonarius RNase, which also undergoes rapid inactivation when the temperature rises above 60°C. A pH of 4.5 is optimal for the activity of R. virescens RNase. In contradistinction to the marked thermostability of Pleurotus tuber-regium RNase, the RNase activity of P. sajor-caju RNase declines rapidly when it is exposed to a temperature at or above 80°C for one hour. The RNase activity of P. sajor-caju RNase is at or near its maximum over pH range 5–7 and is essentially poly Uspecific [7]. The optimal pH is found to be 6.5–7.0 for Clitocybe maxima ribonuclease. Values for ribonucleases from Pleurotus tuber-regium. Volvariella volvacea, Pleurotus pulmonarius, and Pleurotus ostreatus are 6.5, 6.5–7.6, 7.0, and 8.0, respectively [6]. Clitocybe maxima ribonuclease [13] exhibits maximal activity at 70°C, a relatively high temperature, compared with the value of 55°C for Pleurotus pulmonarius ribonuclease, indicating its relative thermostability. However, its activity undergoes a decline when the temperature is raised above or lowered from 70°C. The mushroom ubiquitin-like peptides have very little RNase activity left at 100°C. Their optimum pH is in the range of 6–7. The effects of salts on the enzymatic activity of Pleurotus tuber-regium RNase [6] and Pleurotus ostreatus RNase [9] have been examined. It appears that divalent metal ions exert an inhibitory action on the activity of P. sajor-caju RNase [7]. The ability of P. sajor-caju RNase and ubiquitinlike peptide and other mushroom RNases to inhibit translation in a cell-free rabbit reticulocyte lysate system is probably attributable to its ribonucleolytic activity. However, the translation-inhibiting potency of P. sajorcaju RNase is lower, while its ribonucleolytic activity is on the high side compared with its counterparts from other mushrooms.

Fungal Peptides with Ribonuclease Activity / 143

References [1] D’Alessio G, Riordan JF. Ribonucleases. Structures and Functions. Academic Press, 1997. [2] Hershko A. The ubiquitin system. In Ubiquitin and the Biology of the Cell (Peter A, ed.), Plenum Press, New York, 1998, pp. 1– 17. [3] Irie M. RNase T1/RNase T2 Family RNases. In Ribonuclease. Structures and Functions (D’Alessio G, Riordan JF, eds.), Academic Press, New York, 1997, pp. 101–130. [4] Kao R, Davies J. Fungal ribotoxins: a family of naturally engineered targeted toxins? Biochem Cell Biol 1995; 73: 1151–9. [5] Kobayashi H, Inokuchi N, Koyama T, Watanabe H, Iwami M, Ohgi K, Irie M. Primary structure of a base nonspecific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes. Biosci Biotechnol Biochem 1992; 55: 2003–10. [6] Ng TB. Peptides and proteins from fungi. Peptides 2002; 23: 1361–6. [7] Ngai PHK, Ng TB. A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju. Peptides 2004; 25: 11–7. [8] Ngai PHK, Wang HX, Ng TB. Purification and characterization of a ubiquitin-like peptide with macrophage stimulating, anti-

[9]

[10] [11] [12] [13]

[14]

[15]

[16]

proliferative and ribonuclease activities from the mushroom Agrocybe cylindracea. Peptides 2003; 24: 639–45. Nomura N, Inokuchi N, Kobayashi H, Koyama T, Iwama M, Ohgi K, Irie M. Purification and primary structure of a new guanylic acid-specific ribonuclease from Pleurotus ostreatus. J Biochem (Tokyo) 1994; 116: 26–33. Wang HX, Ng TB. A novel ribonuclease from fruiting bodies of the mushroom Pleurotus eryngii. Peptides 2004; 25: 1365–8. Wang HX, Ng TB. A novel ribonuclease from the veiled lady mushroom Dictyophora indusiata. Biochem Cell Biol 2003; 81: 373–7. Wang HX, Ng TB. A new ribonuclease from the black oyster mushroom Pleurotus ostreatus. Peptides 2004; 25: 685–7. Wang HX, Ng TB. Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima. Peptides 2004; 25: 935–9. Watanabe H, Hamid F, Iwami M, Onda T, Ohgi K, Irie M. Primary structure of RNase from Irpex lacteus. Biosci Biotechnol Biochem 1995; 59: 2092–103. Wool IG. Structure and mechanism of action of the cyototoxic ribonuclease α-sarcin. In Ribonucleases. Structures and Functions (D’Alessio G, Riordan JF, eds.), Academic Press, New York, 1997, pp. 131–62. Xia LX, Chu KT, Ng TB. A low-molecular-mass ribonuclease from fruiting bodies of the brown oyster mushroom. J Peptide Res 2005; 66: 1–8.

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22 Fungal Ribosome Inactivating Proteins T. B. NG

ties that RIPs possess, RIPs have received the attention of many investigators. In contrast to the abundant literature pertaining to angiosperm RIPs, mushroom RIPs have been isolated from only a handful of species including Volvariella volvacea [31], Flammulina velutipes [18, 25, 27], Lyophyllum shimeiji [7], Calvatia caelata [14], Hypsizigus marmoreus [8], and Pleurotus tuber-regium [21]. The chromatographic behavior of most mushroom RIPs [7, 8, 14, 25, 26, 27] on cationic exchangers and anionic exchangers and the affinity chromatographic media Affi-gel blue gel is similar to that of angiosperm RIPs [1, 9, 13]. They are unadsorbed on DEAE-ion exchangers and adsorbed on CM- or SP-ion exchangers and Affi-gel blue gel. It is an interesting finding that the mushroom F. velutipes produces a multiplicity of RIPs: flammin (molecular mass 30 kDa), velin (molecular mass 19 kDa), flammulin (40 kDa), and velutin (13.8 kDa) [25, 27]. The observation of multiple RIPs from the mushroom F. velutipes is reminiscent of the finding of three momorcharins from bitter gourd seeds [24]. The momorcharins are structurally related and similar in molecular size [5, 24]. To the contrary, the four F. velutipes RIPs are structurally disparate and show large differences in molecular mass. Gigantin, purified from the culture medium of the fungus Aspergillus giganteus IFO 5818, is a ribonuclease with a molecular mass of 17 kDa. It exhibits differences in only 9 amino acid residues from α-sarcin, a ribosomeinactivating protein from A. giganteus MDH 18894 [23, 30]. Clavin, also known as c-sarcin [6], is a 17-kDa type 1 ribosome inactivating protein from Aspergillus clavatus [3]. Restrictocin and mitogillin are other well-known ribosome inactivating proteins, also referred to as ribotoxins. Structurally related ribotoxins have been demonstrated in other species [12]. Tricholin is a 14-kDa ribosome inactivating protein elaborated by soil-borne Trichoderma viride [11].

ABSTRACT Ribosome inactivating proteins (RIPs) have been isolated from a number of fungi including Aspergillus and Trichoderma spp. and several mushrooms. Ribosome inactivating peptides found in Cucurbitaceous seeds have not been detected in fungi. The smallest fungal RIP isolated to date, velutin—from the mushroom (basidiomycete fungus) Flammulina velutipes—has a molecular mass of about 14 kDa. Small RIPs with a molecular mass of approximately 20 kDa, and RIPs about 30 kDa in molecular mass, are found in both fungal and plant RIPs. The Aspergillus RIPs are ribonucleases with highsequence homology. In contrast, most mushroom RIPs do not show structural resemblance to each other and have little or no ribonuclease activity. All fungal RIPs demonstrate potent inhibitory activity in the cell-free translation system. Other activities of fungal RIPs include mitogenic/antimitogenic activity toward splenocytes and antiproliferative activity toward tumor cells. RIPs probably serve a role of defense in fungi.

DISCOVERY Ribosome inactivating proteins (RIPs), characterized by the ability to arrest protein synthesis, have been isolated from a great variety of angiosperm species [1, 13] and several mushroom [7, 8, 14, 25, 26, 27, 31] and other fungal [6, 11, 30] species. RIPs manifest an array of biological activities including translation-inhibitory [1, 9], N-glycosidase [10, 13, 14], antimitogenic, immunomodulatory [13, 28], antiproliferative [13], and antifungal [20, 22] activities. RIP-based immunotoxins have been constructed and used in cancer therapy [2]. Transgenic plants carrying the RIP gene are less susceptible to viral infections [10]. RIPs have been cloned and expressed [5]. Due to the potentially exploitable activiHandbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

146 / Chapter 22 TABLE 1. Comparison of N-terminal sequences of various mushroom ribosome inactivating proteins (RIPs) and Aspergillus RIPs (α-sarcin, restrictocin, and mitogillin). Flammin Velin Flammulin Velutin Hypsin Lyophyllin Pleuturegin Calcaelin α-sarcin Restrictocin Mitogillin

SPVIPANTFVAFRLYEVGFUPA SGSPLTQAQAEALLKPQGLAYSSGGNT APSHFSHPGVLADRAQIDFIXGKVNEGAEPWXSAYN XHPDLFXXRPDNTASPKFEDPRLNP ITFQGDLDARQQVITNADTRRKRDVRAA ITFQGASPARQTVITNAITRARADVRAA ARTQPGNIAPVGDFTLYPNAPRQGHIVA ANPIYNIDAFRV AVTWTCLNDQKWPKTNKYETKRLLYNQNKAESNSHHAPLS A-TWTCINQQLWPKTNKWEDKRLLYSQAKAESNSHHAPLS A-TWTCINQQLWPKTNKWEDKRLLYSQAKAESNSHHAPLS

The sequences are from refs [7, 8, 14, 18, 25–27].

STRUCTURE With the exception of hypsin from H. marmoreus [7] and lyophyllin from L. shimeiji [6], which show related N-terminal sequences, the majority of reported mushroom RIPs are structurally dissimilar when their Nterminal sequences are compared (Table 1). The sequence similarity of calcaelin at the N-terminal region to American ginseng RIP is readily discernible [14]. By comparison, resemblance with the fungal proteins αsarcin and restrictocin is less conspicuous. The Nterminal sequences of flammin and velin show some likeness to plant RIPs [18]. The Aspergillus RIPs are highly homologous in their sequences. Flammin and velin demonstrate a molecular mass of 30 kDa and 19 kDa, respectively, well within the range of molecular masses shown by angiosperm RIPs including α- and β-momorcharins (circa 30 kDa) [24] and pisavins (circa 20 kDa) [9]. Flammulin [25] and velutin [27] from the same mushroom exhibit a molecular mass of 40 kDa and 13.8 kDa, respectively. A wide range of molecular masses from 13.8 kDa to 40 kDa is demonstrated by the quadruplet of RIPs from F. velutipes fruiting bodies. The production of calcaelin by the mushroom Calvatia caelata, possessing two subunits with a molecular mass close to 20 kDa and the same N-terminal sequence, and exhibiting translation-inhibiting and ribonuclease activities, is reminiscent of the finding in the pea Pisum sativum var. macrocarpon [8]. The pea RIPs demonstrate identical N-terminal sequences and only a difference of 1.8 kDa in molecular mass. An analogous phenomenon has also been described for α- and β-kirilowins, RIPs from Trichosanthes kirilowii seeds. However, in the case of the pea and Trichosanthes kirilowii seeds, two singlechained RIPs with similar N-terminal sequences are observed, whereas calcaelin is composed of two subunits with similar N-terminal sequences. The molecular masses of calcaelin (39 kDa) and pluturegin (38 kDa)

are higher than the range of 25–32 kDa reported for most plant type 1 RIPs [1] and the value of 17 kDa for the fungal RIP α-sarcin. The RIP volvarin from the straw mushroom has a molecular mass (30 kDa) within the range. The molecular mass of lyophyllin and hypsin is similar to those of α- and β-pisavins, ribosome inactivating proteins from the pea Pisum sativum [9] but lower than the range of 25–32 kDa reported for the majority of plant ribosome inactivating proteins [13]. In addition to type 1 ribosome inactivating proteins with molecular masses in the range of 25 to 32 kDa [1], the seeds of flowering plants produce peptides and small proteins with translation-inhibiting activity. Examples are hispin from the hairy gourd [15], α- and β-benincasins (11 kDa) from the wax gourd Benincasa hispida [17], moschin (12 kDa) from the brown pumpkin Cucurbita moschata [16], charantin (9.7 kDa) from the bitter gourd Momordica charantia [21], luffacylin (7.8 kDa) from the sponge gourd Luffa cylindrica [20], luffangulin (5.6 kDa) from the ridge gourd Luffa acutangula [29], and α- and β-pisavins (about 20 kDa) from the garden pea Pisum sativum [5]. Hence, velin [18], velutin [27], hypsin [8], and lyophyllin [7] are the mushroom counterparts of the foregoing angiosperm peptides and small proteins with ribosome inactivating activity. Most of these angiosperm peptides and small proteins possess N-terminal sequences distinct from those of angiosperm type 1 ribosome inactivating proteins. The N-terminal sequences of these angiosperm peptides and small proteins are characterized by an abundance of arginine and glutamate or glutamine residues. However, this structural feature is not obvious in their mushroom counterparts including velin [18], velutin [27], hypsin [8], and lyophyllin [7].

BIOLOGICAL ACTIONS Tricholin acts on reticulocyte ribosomal RNA to produce an α-sarcin RNA fragment. Interestingly,

TABLE 2. Comparison of characteristics of various mushroom ribosome activating proteins and α-sarcin from Aspergillus sp. Calcaelin

Flammulin

Hypsin

Lyophyllin

Pluturegin

Velin

Velutin

Volvarin

α-Sarcin

39 D

30 S

40 S

20 S

20 S

38 S

19 S

13.8 S

30 S

17 S

Unadsorbed Adsorbed — Adsorbed 4 nM

Unadsorbed Adsorbed Adsorbed — 1.4 nM

Unadsorbed Adsorbed Adsorbed — 0.24 nM

— Adsorbed Adsorbed — 7 nM

— Adsorbed Adsorbed Adsorbed 1 nM

Adsorbed — — Unadsorbed 0.5 nM

Unadsorbed Adsorbed Adsorbed — 2.5 nM

Unadsorbed Adsorbed Adsorbed — 0.29 nM

Unadsorbed — — — 0.5 nM

Present

6 μM

—

—

10 μM

Present

—

—

—

—

—

2.3 μM Absent Absent —

— — — —

— — — —

— — Present 8 μM

— — Present 7.9 nM

— — — —

— — — —

— — — —

— — — —

— Present Absent Absent —

— = not tested Information in table derived from refs [7, 8, 14, 18, 25–27, 31].

Fungal Ribosome Inactivating Proteins / 147

Molecular mass (kDa) Single-chain (S)/double-chain (D) Chromatographic behavior (i) DEAE-cellulose (ii) Affi-gel Blue gel (iii) CM-Sepharose/CM-cellulose (iv) Mono S/SP-Sepharose Cell-free translation inhibitory activity (IC50) Antimitogenic activity (IC50) on splenocytes Antiproliferative activity against (IC50) breast cancer cells Antibacterial activity Antifungal activity HIV-1 reverse transcriptase inhibitory activity (IC50)

Flammin

148 / Chapter 22 anti-α-sarcin antibodies show a strong cross-reaction with tricholin but antitricholin antibodies cross-react only weakly with α-sarcin [11]. Gigantin exhibits preference for poly C and poly U, while α-sarcin shows preference for poly A and poly I. Gigantin and α-sarcin are immunologically distinct. Gigantin demonstrates a pH optimum around 7.0 and a temperature optimum at 45–55°C. Flammin, velin, flammulin [25], and velutin [27] from F. velutipes are devoid of RNase and protease activities. RNases and proteases may interfere in the assay for cell-free translationinhibitory activity and produce an apparent inhibition because RNA important for protein synthesis can be degraded by RNases and the newly synthesized proteins can be broken down by proteases. The cell-free translation-inhibitory activity of flammin, velin, flammulin, and velutin is thus ascribed to ribosome inactivation and not to hydrolysis of RNA and proteins. They inhibit translation in rabbit reticulocyte lysate with a potency analogous to those of other mushroom RIPs [7, 8, 14, 25–28] and angiosperm RIPs [1, 9, 13]. They demonstrate N-glycosidase activity typical of RIPs. The translation-inhibiting potency of mushroom ribosome inactivating proteins are 7 nM, 1 nM, 0.29 nM, 0.25 nM, 0.5 nM, and 4 nM for hypsin, lyophyllin, velutin, flammulin, pluturegin, and calcaelin, respectively [7, 8, 14, 25–28], within the range of values for plant RIPs [1]. The ability to inhibit translation in a cell-free rabbit reticulocyte lysate system is a characteristic of RIPs. The oligonucleotide generated by the action of the fungal RIP α-sarcin on rat liver ribosomes is derived from the 3′ end of 28S rRNA and has a size of 488 bases, while the oligonucleotide similarly produced by plant RIP (e.g., ricin A-chain) has a size of 553 bases. When analyzed in agarose gel electrophoresis, the oligonucleotide band produced by α-sarcin on ribosomal RNA shows only a slightly faster mobility than the mobility of the band generated by the small plant RIP luffin-S without aniline treatment. Similarly, the action of calcaelin on ribosomal RNA produces a band with a mobility slightly faster and thus a size slightly smaller than the Endo’s band produced by the plant RIP trichosanthin [14]. The ribonuclease activity in calcaelin [14] is substantially lower than those of ribonucleases from mushrooms like Pleurotus tuber-regium ribonuclease [26]. The fungal RIP α-sarcin also expresses ribonuclease activity. Calcaelin lacks antifungal activity. On the other hand, lyophyllin suppresses fungal growth, in line with the findings in case of plant ribosome inactivating proteins [20]. The antifungal action of lyophyllin is speciesspecific: It is effective toward Physalospora piricola and Coprinus comatus but not toward Rhizoctonia solani or Mycosphaerella arachidicola. Calcaelin does not manifest

antibacterial action. In fact, plant type 1 RIPs [5] and the fungal RIP α-sarcin have been cloned in E. coli and expressed. α-Sarcin does not display antimicrobial or antifungal activity. Both calcaelin and lyophyllin have an inhibitory effect on the mitogenic response of murine splenocytes. Plant type 1 RIPs inhibit the Con A-stimulated mitogenic response of splenocytes [13] without significant alterations in splenocyte viability, indicating that the antimitogenic effect is not a consequence of lymphotoxicity. The mechanism of the antimitogenic effect of the plant ribosome inactivating protein β-momorcharin involves inhibition of protein synthesis. It is likely that calcaelin has a similar mechanism of action. Calcaelin displays a cytotoxic activity on human breast carcinoma cells. It is known that plant type 1 RIPs exert cytotoxic actions on tumor cell lines. The cytotoxic activity of calcaelin may be related to its translation-inhibiting activity because RIPs inhibit protein synthesis in tumor cells [14]. Immunotoxins based on clavin [3] and restrictocin [19] have been constructed. Histidine 137 is located at the active site of α-sarcin, since replacement by glutamine abolishes the catalytic activity of α-sarcin. The antimitogenic and N-glycosidase activities of lyophyllin are in accord with findings regarding angiosperm ribosome inactivating proteins [13, 28].

Acknowledgments The excellent secretarial assistance of Miss Fion Yung and the award of a grant (CUHK 4272/04M) from Research Grants Council.

References [1] Barbieri WK, Batteli HG, Stirpe F. Ribosome inactivating proteins from plants. Biochim Biophys Acta 1993; 1154: 237–82. [2] Battelli MG, Polito L, Bolognesi A, Lafleur L, Fadat Y, Stirpe F. Toxicity of ribosome-inactivating protein-containing immunotoxins to a human carcinoma cell line. Int J Cancer 1996; 65: 485–500. [3] D’Alatri L, Di Massimo AM, Anastasi AM, Pacilli A, Novelli S, Saccinto MP, De Santis R, Mele A, Parente D. Production and characterization of a recombinant single-chain anti ErB2-clavin immunotoxin. Anticancer Res 1998; 18: 3369–73. [4] Endo Y, Tsurugi K. N-Glycosidase activity of ricin A chain. Mechanism of action of toxic lectin ricin on eukaryotic ribosomes. J Biol Chem 1987; 262: 8128–30. [5] Ho WKK, Liu SC, Shaw PC, Yeung HW, Ng TB, Chan WY. Cloning of the cDNA of α-momorcharin: a ribosome inactivating protein. Biochim Biophys Acta 1991; 1088: 311–4. [6] Huang KC, Huang YY, Hwu L, Lin A. Characterization of a new ribotoxin gene (c-Sar) from Aspergillus clavatus. Toxicon 1997; 350: 383–92. [7] Lam SK, Ng TB. First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeiji) together with evidence for synergism of their antifungal effects. Arch Biochem Biophys 2001; 393: 271–80.

Fungal Ribosome Inactivating Proteins / 149 [8] Lam SK, Ng TB. Hypsin, a novel thermostable ribosomeinactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem Biophys Res Commun 2001; 285: 1071–5. [9] Lam SS, Wang HX, Ng TB. Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem Biophys Res Commun 1998; 253: 135–42. [10] Lam YH, Wong YS, Wang B, Wong RNS, Yeung HW, Shaw PC. Use of trichosanthin to reduce infection by turnip mosaic virus. Plant Sci 1996; 114: 111–7. [11] Liu A, Chen KD, Chen YJ. Molecular action of tricholin, a ribosome-inactivating protein isolated from Trichoderma viride. Mol Microbiol 1991; 5: 3007–13. [12] Martinez-Ruiz A, Kao R, Davies J, Martinez del Pozo A. Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed. Toxicon 1999; 37: 1549–63. [13] Ng TB, Chan WY, Yeung HW. Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants. Gen Pharmacol 1992; 23: 575–90. [14] Ng TB, Lam YW, Wang HX. Calcaelin, a new protein with translation-inhibiting, antiproliferative and antimitogenic activities from the mosaic puffball mushroom Calvatia caelata. Planta Med 2003; 69: 212–7. [15] Ng TB, Parkash A. Hispin, a novel ribosome inactivating protein with antifungal activity from hairy melon seeds. Protein Expr Purif 2002; 26: 211–7. [16] Ng TB, Parkash A, Tso WW. Purification and characterization of moschins, arginine-glutamate-rich proteins with translationinhibiting activity from brown pumpkin (Cucurbita moschata) seeds. Protein Express Purif 2002; 26: 9–13. [17] Ng TB, Parkash A, Tso WW. Purification and characterization of alpha- and beta-benincasins, arginine/glutamate-rich peptides with translation-inhibiting activity from wax gourd seeds. Peptides 2003; 24: 11–16. [18] Ng TB, Wang HX. Flammin and velin: new ribosome inactivating polypeptides from the mushroom Flammulina velutipes. Peptides 2004; 25: 929–33. [19] Orlandi R, Canevari S, Conde FP, Leoni F, Mezzanzanica D, Ripamonti M, Colnaghi MI. Immunoconjugate generation between the ribosome inactivating protein restrictocin and an

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

anti-human breast carcinoma MAB. Cancer Immunol Immunother 1988; 26: 114–20. Parkash A, Ng TB, Tso WW. Isolation and characterization of luffacylin, a ribosome inactivating peptide with antifungal activity from sponge gourd (Luffa cylindrica) seeds. Peptides 2002; 23: 1019–24. Parkash A, Ng TB, Tso WW. Purification and characterization of alpha- and napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds. Peptides 2002; 59: 197–202. Roberts WK, Selitrennikoff CP. Isolation and partial characterization of two antifungal proteins from barley. Biochem Biophys Acta 1986; 880: 161–70. Salvarelli S, Munoz S, Conde FP. Purification and characterization of a ribosome inactivating protein from Aspergillus giganteus IF 5818, the gigantin. Immunological and enzymic comparison with α-sarcin. Eur J Biochem 1994; 225: 243–51. Tse PMF, Ng TB, Fong WP, Yeung HW, Wong RNS, Mak NK. New ribosome inactivating proteins from fruits and seeds of the bitter gourd Momordica charantia. Int J Biochem Cell Biol 2000; 31: 895–902. Wang HX, Ng TB. Flammulin: a novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes. Biochem Cell Biol 2000; 78: 699–702. Wang HX, Ng TB. Isolation of pleuturegin, a novel ribosomeinactivating protein from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Biochem Biophys Res Commun 2001; 288: 718–21. Wang HX, Ng TB. Isolation and characterization of velutin, a novel low-molecular-weight ribosome-inactivating protein from winter mushroom (Flammulina velutipes) fruiting bodies. Life Sci 2001; 68: 2151–8. Wang HX, Ng TB. Studies on the anti-mitogenic, anti-phage and hypotensive effects of several ribosome inactivating proteins. Comp Biochem Physiol Part C 2001; 128: 359–66. Wang HX, Ng TB. Luffangulin, a novel ribosome inactivating peptide from ridge gourd (Luffa acutangula) seeds. Life Sci 2002; 70: 890–900. Wool IG. Structure and mechanism of action of the cytotoxic ribonuclease α-sarcin. In: Ribonucleases (D’Alessio G, Riordan J F, eds.), Academic Press. New York, 1997; pp. 131–62. Yao QZ, Yu MM, Ooi LS, Ng TB, Chang ST, Sun SS, Ooi VE. Isolation and characterization of a ribosome-inactivating protein from fruiting bodies of the edible mushroom (Volvariella volvacea). J Agric Food Chem 1998; 46: 788–92.

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23 Peptides and Depsipeptides from Plant Pathogenic Fungi M. SOLEDADE C. PEDRAS AND PAULOS B. CHUMALA

cation of current knowledge to agriculture and food production.

ABSTRACT This chapter describes representative examples of peptides and depsipeptides produced by plant pathogenic fungi, their biological and ecological roles, as well as potential application of current knowledge to agriculture and food production. Fungi are known to produce an enormous array of secondary metabolites, many of which have unknown function and bioactivity. Despite their incredible structural diversity, according to their basic biosynthetic precursors—their “building blocks”—these metabolites can be classified into four main groups: (1) acetyl coenzyme A and malonyl coenzyme A, which lead to the formation of polyketides; (2) mevalonic acid, which leads to synthesis of terpenoids and steroids; (3) shikimic acid, which leads to formation of shikimates and derivatives of aromatic amino acids, including some alkaloids; and (4) nonaromatic amino acids, including unusual amino acids, which lead to formation of alkaloids [4]. The last two categories comprise depsipeptides and peptides. Common investigation of secondary metabolites starts with their isolation, purification, and chemical structure elucidation. However, in many cases the low availability of metabolites—no longer an impediment to chemical structure determination—still poses constraints to an integrated understanding of fungal secondary metabolites. Nonetheless, it is well known that secondary metabolites have a variety of roles in fungi, including mediation of the interactions between fungi and plants, fungi and animals, and fungi and microorganisms. Although secondary metabolites of extremely diverse chemical structures have been isolated from fungi, this chapter describes only representative examples of peptides and depsipeptides produced by plant pathogenic fungi, their biological and ecological roles, and potential appliHandbook of Biologically Active Peptides

PHYTOTOXINS Since plant fungal diseases cause enormous economic losses, most of the current studies on fungal metabolites are directed to plant-fungal interactions that cause disease. Many phytopathogenic fungi produce phytotoxins in culture and in their host plants. Phytotoxins are broadly defined as secondary metabolites, which have deleterious effects on plants and are produced by bacterial or fungal phytopathogens. Host-selective toxins (HSTs) damage selectively tissues of plants that host the pathogen, whereas nonselective toxins (NSTs) cause necrosis and cell death of both host and nonhost plants [1]. The isolation of phytotoxins from plant pathogenic fungi requires a bioassay-directed isolation and elucidation of the complete threedimensional chemical structure of the pure compound. Assignment of each particular chemical structure is usually based on a thorough analysis of spectroscopic data (nuclear magnetic resonance, mass, infrared, and ultraviolet spectroscopy, x-ray crystallography) and is often confirmed by chemical synthesis. HSTs mimic the pathogenicity range of the toxin-producing pathogen, reproducing the disease symptoms in the absence of the pathogen. They can determine the virulence and specificity of pathogenic fungi. Knowledge of the mechanism of action of HSTs and their targets in the host cells contributes to the understanding of the molecular basis of fungal diseases [3, 10]. Several studies have been carried out to understand the role of HSTs in the development of the disease. Of more than 20 HSTs, and a much larger number of NSTs known to date, a few of them, such as destruxins, AM-toxins I-III, victorin C,

151

Copyright © 2006 Elsevier

152 / Chapter 23 HC-toxin, BZR co-toxins I-III, sirodesmins, phomalirazine, phomalide, and depsilairdin, are of peptidic or depsipeptidic nature.

planta as well, causing necrotic and chlorotic symptoms in different species, suggesting that destruxin B is a host-selective phytotoxin. Homodestruxin B also appears to have a role in facilitating colonization of plant tissue; it is produced in planta as well. Evidence that destruxin B and homodestruxin B play important roles in the development of Alternaria blackspot disease was obtained from metabolic studies in plants [8, 9]. Plant detoxification of destruxin B and homodestruxin B was observed both in resistant and susceptible plants but at faster rates in resistant than in susceptible plant species. Feeding 14C-labeled destruxin B to leaves of blackspotresistant (Sinapis alba, Camelina sativa, Capsella bursa-pastoris, and Eruca sativa) and susceptible plants (Brassica napus, B. juncea, and B. rapa) showed that destruxin B was transformed sequentially to hydroxydestruxin B, β-Dglucosyl hydroxydestruxin B and then to (6′-Omalonyl)hydroxydestruxin B β-D-glucopyranoside (Fig. 2). The structures of hydroxydestruxin B and β-Dglucosyl hydroxydestruxin B were determined by spectroscopic data and confirmed by total synthesis. Interestingly, hydroxylation was the rate-limiting step in susceptible plants, whereas glucosylation was the ratelimiting step in the resistant species. Moreover, the detoxification product, hydroxydestruxin B, induced production of phytoalexins in resistant plants, which was not observed in susceptible plants. Similarly, a detoxification reaction converted homodestruxin B to hydroxyhomodestruxin B at a faster rate in resistant plants as compared with susceptible plants [9].

DEPSIPEPTIDES AND PEPTIDES Depsipeptides are alkaloids derived from amino acids and hydroxy acids, which contain both amide and ester bonds, whereas peptides are derived from amino acids and contain only amide bonds. Most of the peptidic or depsipeptidic phytotoxins have cyclic structures containing various amino/hydroxy acids (Figs. 1–5). Cyclic dipeptides are called dioxopiperazines, also known as diketopiperazines, and comprise a great variety of alkaloids with phytotoxic activity (Fig. 5).

Host-Selective Toxins from the Genus Alternaria Alternaria brassicae Destruxin B and homodestruxin B are cyclodepsipeptides characterized as HSTs produced by Alternaria brassica, the causative agent of blackspot disease of Brassica species. The structures and absolute stereochemistry of destruxin B and homodestruxin B were established by chemical and spectroscopic methods and later on through total synthesis [9]. Destruxin B is produced in

Alternaria alternata f. sp. mali Alternaria blotch of apple caused by A. alternata f. sp. mali is a disease mediated by host-selective toxins. Multiple host-selective toxins—AM-toxin I, II, and III—have been isolated from the culture filtrates of A. alternata f. sp. mali, and the chemical structures were determined by spectroscopic methods and total synthesis [3, 15, 16]. AM-toxins are cyclic tetradepsipeptides consisting of Lhydroxyisovaleric acid, L-alanine, α-amino acrylic acid,

FIGURE 1. Depsipeptide and peptide phytotoxins from the genus Alternaria.

O HO HO HO HO

O

HO HO

O

O

O

HO

b-D-glucosyl hydroxydestruxin B

FIGURE 2. Detoxification of the phytotoxin destruxin B in crucifer plants [8].

*

Hydroxydestruxin B

O

*

*

* = 14C

HO2C

(6'-O-malonyl)hydroxydestruxin B b-D-glucopyranoside

Peptides and Depsipeptides from Plant Pathogenic Fungi / 153

FIGURE 3. Depsipeptide and peptide phytotoxins from the genus Cochliobolus.

FIGURE 4. Depsipeptide and peptide phytotoxins from Bipolaris zeicola race 3.

OH OH

OH

O N

N

O

Sirodesmins n=1,2,3,4

N O O

O

HO

Depsilairdin

H

Phomalide Phomalirazine H N

H

O N H Polanrazine A

FIGURE 5. lingam).

O H N H

MeS

O

H N

O SMe

HO

N H

N H Polanrazine B

O

H N

O SMe N H

N H Polanrazine C

MeS

O

H N

O OH N H

N H Polanrazine D

HO

O

H N

O OR N H

N H Polanrazine E R=CH3 Polanrazine F R=H

Depsipeptide and dioxopiperazine phytotoxins from Leptosphaeria maculans (Phoma

and derivatives of L-α-amino-δ-phenylvaleric acid (Fig. 1). The major toxin of the three derivatives, AM-toxin I is described as [cyclo(α-hydroxyisovaleryl-α-amino-pmethoxyphenylvaleryl-α-aminoacryl-alanyl-lactone)]. These AM-toxins showed extremely potent biological activity—for instance, AM-toxin I at a concentration of 10−8 M caused an increase in electrolyte loss and necro-

sis in susceptible apple tissue. Moreover, AM-toxins exhibit host-selectivity that corresponds to the pathogenicity range of A. alternata f. sp. mali, indicating that they play an important role in host recognition during the early stages of infection. Further, mutants of A. alternata apple pathotype that do not produce AM-toxin were unable to cause disease symptoms on susceptible

154 / Chapter 23 apple cultivars. Thus, AM-toxins appeared to be primary determinants of virulence and specificity in the A. alternata apple pathotype-apple interaction [2].

Host-Selective Toxins from the Genus Cochliobolus Pathogenic species of the fungal genus Cochliobolus are known to cause toxin-mediated plant diseases [3, 10, 17]. Of these diseases, Victoria blight of oats and maize leaf spot, which are caused by C. victoriae and C. carbonum, respectively, are among the most studied diseases in regard to the biochemistry of disease development and disease resistance. These pathogenic species of Cochliobolus produce peptidic and depsipeptidic hostspecific toxins that act as host recognition and virulence factors. C. victoriae The pathogenic fungus C. victoriae, the causative agent of Victoria blight of oats, produces in culture several host selective toxins, of which victorin C is the major host selective toxin [18]. Victorin C is constituted by glyoxylic acid, 5,5-dichloroleucine, erythro-βhydroxyleucine, victalanine, threo-β-hydroxylysine, and α-amino-β-chloroacrylic acid (Fig. 3). The biological activity of victorin C is quite remarkable—for instance, growth of susceptible oat (Avena sativa) roots was inhibited at a concentration of 0.1 ng/mL. Additionally, victorin C affects selectively susceptible oats (Avena sativa L.), reproducing disease symptoms caused by the pathogenic fungus. The studies on the structure-activity relationships of victorin C revealed that the glyoxylic acid residue particularly the hydrated aldehyde group is essential for biological activity. For instance, victorin C tested for its effect on dark CO2 fixation in susceptible oat leaf slices showed inhibition of dark CO2 fixation, whereas a victorin C derivative without the glyoxylic acid residue had no effect on dark CO2 fixation. Moreover, several studies have shown that specificity in toxicity of victorin C is correlated with its specificity in binding to victorin C receptor, which is only available in susceptible oat (Avena sativa L.) [5]. Importantly, those mutants that did not produce victorin C were found to be nonpathogenic. Consequently, victorin C appears to determine host-specificity and virulence of C. victoriae. C. carbonum Race 1 Northern leaf spot of maize caused by C. carbonum race 1 is a toxin-mediated disease [10]. The fungus C. carbonum produces in culture HC-toxin a host-selective toxin having a cyclic peptide structure composed of four amino acid residues (Fig. 3; cyclo-[(L-2-amino-

9,10-epoxy-8-oxodecanoyl)-D-prolyl-L-alanyl-L-alanyl]) [17]. HC-toxin caused necrosis and inhibited growth of seedlings of susceptible maize, whereas it had no effect on resistant maize. HC-toxin was essential for C. carbonum race 1 colonization of susceptible maize [17]. Nonpathogenic C. carbonum and C. victoriae, which were unable to colonize C. carbonum race 1 susceptible maize, were found to successfully colonize C. carbonum race 1-susceptible maize in the presence of HC-toxin. Thus, genetic variants of the pathogen unable to biosynthesize HC-toxin were not pathogenic and lacked the genes required for the biosynthesis of HC-toxin and pathogenicity. Studies of structure-activity relationships of HC-toxin showed that reduction of the keto group of 2-amino-8-oxo-9,10-epoxydecanoic acid moiety of the HC-toxin to the corresponding alcohol resulted in a HC-toxin derivative that showed no toxicity to maize sensitive to HC-toxin. Similarly, HC-toxin lost its biological activity by converting the epoxide of the 2-amino-8-oxo-9,10-epoxydecanoic acid moiety to the corresponding dihydroxy derivative [17]. Additionally, HC-toxin treated with 2-mercaptoethanol, which modified the keto group of the 2-amino-8-oxo-9,10epoxydecanoic acid moiety, was found to lose reversibly the inhibitory activities on histone deacetylases (HD1-A, HD1-B, and HD2) of C. carbonum race 1 susceptible maize in vitro. As these studies indicated, both epoxy and keto groups are essential for the biological activity of HC-toxin. Most importantly, HC-toxin was found to be detoxified in maize resistant to C. carbonum race 1 by enzymatic conversion of the 8-keto group of 2-amino8-oxo-9,10-epoxydecanoic acid moiety to the 8-hydroxy derivative of HC-toxin [17]. Occurrence of the detoxifying enzyme, NADPH dependent HC-toxin reductase, in the resistant maize cultivar (genotype Hm/Hm or Hm/hm) was attributed to be the biochemical basis of resistance to infection by C. carbonum race 1 [17].

Host-Selective Toxins from Bipolaris zeicola Race 3 Bipolaris zeicola race 3, the causative agent of leaf spot disease in maize, produces host-selective toxins—namely, BZR-cotoxins I-IV (Fig. 4)—in liquid culture and in spore-germination fluids. Chemically BZR-cotoxins are cyclic depsipeptides, whose structures were established by both spectroscopic and chemical methods [11–14]. BZR-cotoxins are biologically less active when assayed separately, but in combination they exhibit potent phytotoxicity and host-selectivity. Their host-selectivity correlated with the pathogenicity range of B. zeicola race 3, and caused disease symptoms on susceptible rice and maize plants. Interestingly, a nonpathogenic fungus like Bipolaris victoriae that could not penetrate into maize tissues was found penetrating and colonizing maize

Peptides and Depsipeptides from Plant Pathogenic Fungi / 155 tissues in the presence of BZR-cotoxins. On the other hand, mutants not producing BZR-cotoxins were also unable to infect host plants compared to wild B. zeicola race 3. During infection, the germinating spores release BZR-cotoxins to facilitate colonization of host tissues of the toxin producing fungus. These facts indicate that BZR-cotoxins are the determinant factors of virulence and host selectivity of B. zeicola race 3.

Host-Selective Toxins from Leptosphaeria maculans Although blackleg is a devastating disease of economically important cruciferous crops caused by the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not. (asexual stage Phoma lingam (Tode ex Fr.) Desm), not as much work has been done regarding the biochemistry and molecular genetics of disease development and disease resistance, as compared with plant diseases caused by the fungal genera Cochliobolus and Alternaria. However, blackleg appears to be one of the toxin-mediated diseases, since the causative agent, L. maculans (P. lingam), like the species and pathotypes of Cochliobolus and Alternaria, is a host-selective pathogen. So far, two host-selective toxins with a depsipeptide structure—phomalide and depsilairdin (Fig. 5)—were reported from this fungal pathogen (L. maculans/P. lingam). Phomalide was isolated from 24–60-h-old cultures of virulent isolates and its structure was elucidated by combination of both spectroscopic methods and chemical degradation [6]. Chemically, phomalide is a 15-membered cyclic depsipeptide composed of dehydrothreonine (DhThr), (S)-valine [(S)-Val], (R)-leucine [(R)-Leu], (S)-3-phenyllactic acid [(S)-O-Phe], and (S)2-hydroxyisovaleric acid [(S)-O-Leu], in a sequence of cyclo(DhThr-Val-Leu-O-Leu-O-Phe). The chemical structure was confirmed by total synthesis [6]. Phomalide was also identified to be produced in planta by virulent isolates of L. maculans and cause disease symptoms on susceptible plants similar to the pathogen [6]. The hostselectivity of phomalide correlated with the pathogenicity range of the pathogen; both phomalide and the pathogen cause damage on leaves of canola (Brassica napus, B. rapa) and other susceptible species. From the structure-activity relationships studies of phomalide, the (E)-double bond configuration of phomalide appears to be essential for the observed host-selective phytotoxicity. Therefore, phomalide appears to be essential for host-selectivity and virulence of the pathogenic fungus. However, much work remains to be done to establish the role of phomalide in the disease development [9]. Currently, the metabolism of phomalide by resistant and susceptible plant tissues is being studied to establish if and how disease resistance correlates with phomalide detoxification.

Depsilairdin was recently isolated from a new group of blackleg fungal isolates able to cause disease on brown mustard (B. juncea) but not on canola [7]. The leaf lesions observed on blackleg-infected mustard suggested that colonization by these “new” blackleg isolates (Laird 2 and Mayfair 2) would be mediated by a rather potent toxin. A comprehensive metabolite search for new phytotoxic compounds led to the discovery of depsilairdin, a highly selective phytotoxic metabolite. The remarkable selectivity of depsilairdin to brown mustard was observed over a broad concentration range from μM to mM. Depsilairdin could be an excellent probe to detect, for example, genes and/ or receptors targeted by the pathogen to defeat the plant.

Dioxopiperazines from Leptosphaeria maculans Dioxopiperazines containing epipolysulfide bridges were isolated from extracts of many plant pathogenic fungal species [6]. Sirodesmin PL is the major component of phytotoxic extracts (50–70%, w/w) produced by the fungal pathogen L. maculans, whereas phomalirazine is produced in very small amounts [6]. Bioassays conducted with mesophyll protoplasts of a number of cruciferous species (B. carinata, B. juncea, B. napus, and S. alba L.) showed extreme sensitivity to sirodesmin PL at concentrations ≥ 1 × 10−6 M; 95% of the protoplasts were killed within 24 hours. When using lower concentrations of sirodesmin PL (1 × 10−10 − 1 × 10−7 M) the protoplast viability ranged from 45 to 80% relative to the controls. Interestingly, B. juncea microspores were more sensitive to phomalirazine (LD50 = 1.9 × 10−6 M) than to sirodesmin PL (LD50 = 3.0 × 10−6 M). By contrast, microspores of B. napus were as sensitive to phomalirazine (LD50 = 1.0 × 10−6 M) as to sirodesmin PL (LD50 = 0.8 × 10−6 M). The dioxopiperazines polanrazines A–E were isolated from a group of fungal isolates of L. maculans (P. lingam) from Poland, virulent on brown mustard but not on canola (Fig. 5). Phytotoxicity assays of polanrazines A–E on plants resistant and susceptible to this pathogen indicated that polanrazines C and E had moderate but selective toxicity, causing necrotic and chlorotic lesions on brown mustard leaves, whereas no damage was observed on canola or white mustard leaves. Nonetheless, it remains to be established if the virulence of the Polish fungal isolates is related with the production of polanrazines A–E [6].

CONCLUSION Many studies have shown that depsipeptides and peptides are host-selective toxins essential for virulence

156 / Chapter 23 of phytopathogenic fungi [3]. The pathogenic fungus releases host-selective toxins at an infection site during the early stages of the infection process. These hostselective toxins disrupt the normal cell function and facilitate the successful infection of host tissues. Toxindeficient mutants (Tox−) of pathogenic fungi are usually unable to cause infection and have been shown to elicit successful infection of host tissues when inoculated with host-selective toxins of the pathogen. Thus, secretion of host-selective toxins is critical for the virulence of the fungus. The factors involved in plant-fungal interactions, which are decisive for the pathogenicity of fungus on one hand and disease resistance in plants on the other hand, determine plant disease development. Factors such as phytotoxin detoxifying enzymes, which are involved in plant resistance/defense reactions, can be crucial for disease resistance to some fungal pathogens. Two examples of detoxification of a peptide (HCtoxin) and a depsipeptide (destruxin B) by plants indicate that it is possible to use these HSTs to determine mechanisms of disease resistance in plants. For example, the destruxin B detoxification pathway present in S. alba is also present in three wild species—Camelina sativa, Capsella bursa-pastoris, and Eruca sativa—suggesting a conservation of this pathway across crucifers. Considering that C. sativa and C. bursa-pastoris detoxify destruxin B and produce the phytoalexins camalexins that are highly inhibitory to A. brassicae, these wild crucifers appear to represent unique and perhaps useful sources of resistance to A. brassicae in strategic plant breeding.

References [1] Graniti A. Phytotoxins and their involvement in plant diseases. Introduction. Experientia 1991;47:51–55. [2] Johnson LJ, Johnson RD, Akamatsu H, Salamiah A, Otani H, Kohmoto K, Kodama M. Spontaneous loss of a conditionally dispensable chromosome from the Alternaria alternata apple pathotype leads to loss of toxin production and pathogenicity. Curr Genet 2001;40:65–72.

[3] Kohmoto K, Otani H. Host recognition by toxigenic plant pathogens. Experientia 1991;47:755–764. [4] Mann J. Chemical aspects of biosynthesis. 1994. Oxford University Press, Oxford, UK, p 2–4. [5] Navarre DA, Wolpert TJ. Victorin induction of an apoptotic/ senescence-like response in oats. The Plant Cell 1999;11:237– 249. [6] Pedras MSC. Phytotoxins from fungi causing blackleg disease on crucifers: isolation, structure determination, detection, and phytotoxic activity. Recent Res Devel Phytochem 2001;5:109– 117. [7] Pedras MSC, Chumala PB, Quail JW. Chemical mediators: the remarkable structure and host-selectivity of depsilairdin, a sesquiterpenic depsipeptide containing a new amino acid. Organic Letters 2004;6:4615–4617. [8] Pedras MSC, Montaut S, Zaharia IL, Gai Y, Ward DE. Biotransformation of the host-selective toxin destruxin B by wild crucifers: probing a detoxification pathway. Phytochemistry 2003;64:957–963. [9] Pedras MSC, Zaharia IL, Ward DE. The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry 2002;59:579–596. [10] Scheffer RP, Livingston RS. Host-selective toxins and their role in plant diseases. Science 1984;223:17–21. [11] Ueda K, Xiao JZ, Doke N, Nakatsuka S. Structure of BZR-cotoxin II produced by Bipolaris zeicola race 3, the cause of leaf spot disease in corn. Tetrahedron Lett 1992;33:5377–5380. [12] Ueda K, Xiao JZ, Doke N, Nakatsuka S. Structure of BZR-cotoxin I produced by Bipolaris zeicola race 3, the cause of leaf spot disease in corn. Tetrahedron Lett 1994;35:7033–7036. [13] Ueda K, Xiao JZ, Doke N, Nakatsuka S. Isolation and structure of BZR-cotoxin IV produced by Bipolaris zeicola race 3, the cause of leaf spot disease in corn. Tetrahedron Lett 1995a;36:741– 744. [14] Ueda K, Xiao JZ, Doke N, Nakatsuka S. Structure of BZR-cotoxin III produced by Bipolaris zeicola race 3, the cause of leaf spot disease in corn. Nat Pro Lett 1995b;6:43–48. [15] Ueno T, Hayashi Y, Nakashima T, Fukami H, Nishimura S, Kohmoto K, Sekiguchi A. Isolation of AM-toxin I, a new phytotoxic metabolite from Alternaria mali. Phytopathology 1975a;65: 82–83. [16] Ueno T, Nakashima T, Hayashi Y, Fukami H. Structures of AMtoxin I and II, host specific phytotoxic metabolites produced by Alternaria mali. Agr Biol Chem 1975b;39:1115–1122. [17] Walton JD. Host-selective toxins: agents of compatibility. The Plant Cell 1996;8:1723–1733. [18] Wolpert TJ, Macko V, Acklin W, Jaun B, Arigoni D. Structure of minor host-selective toxins from Cochliobolus victoriae. Experientia 1986;42:1296.

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24 Insect Diuretic and Antidiuretic Hormones GEOFFREY M. COAST

punctata [13]. Dippu-DH31 (Fig. 1) has low-sequence identity to vertebrate CT but shares the unusual Cterminal Gly-Pro-NH2. CT-like DH are encoded in the genomes of the fruit fly (Drosophila melanogaster), the malaria mosquito (Anopheles gambiae), the honeybee (Apis mellifera), and the silk moth (Bombyx mori). Kinins: Kinins are known from 10 species of insect, and frequently (but not always) multiple isoforms are present. The first (Leuma-Ks; Fig. 1) were identified from the Madeira cockroach (Leucophaea maderae) by their myostimulatory action on the hindgut [15] but were later shown to be potent diuretics. CAP2b: Manse-CAP2b (Fig. 1) was first identified as a cardioacceleratory peptide (CAP) [16] but was later shown to stimulate secretion by D. melanogaster MT. Other CAP2b peptides have been identified by mass analysis of tissue extracts and by mining genomic databases. Antidiuretic factors (ADFs): Tenmo-ADFa and TenmoADFb (Fig. 1) were identified from head extracts of T. molitor pupae on the basis of their ability to increase cGMP production by MT from adult beetles [9, 10]. Both are extremely potent in reducing secretion by the free portion of larval MT with an EC50 value of 10 fM for Tenmo-ADFa. To date, they have not been identified from any other insect. Ion Transport Peptide (ITP): Schgr-ITP (Fig. 1) is the only identified ADH that stimulates fluid reabsorption from the insect hindgut and was isolated from the locust Schistocerca gregaria on the basis of its ability to increase electrogenic Cl− transport across the ileum [1]. A cDNA encoding the prepropeptide was cloned and the Cterminal amidated peptide has 39–42% sequence identity to CHH from the shore crab, Carcinus maenas [24]. A cDNA encoding a putative lepidopteran ITP was obtained from B. mori [11] and a homologous peptide is encoded in the fruit fly genome. An alternative spliced form of Schgr-ITP encodes a Schgr-ITP-like peptide (ITP-L) with a nonamidated C-terminus (Fig. 1).

ABSTRACT A diverse group of peptides are implicated in the control of Malpighian tubule and hindgut function in insects. They are the products of neurosecretory cells in the brain and ventral nerve cord ganglia and are present in identified neurohemal structures from where they can be released into the circulation to function as diuretic and/or antidiuretic hormones. Receptors are known for some, and they couple to cAMP, cGMP, or Ca2+ signaling pathways to regulate a variety of transport processes.

DISCOVERY In the excretory process of insects, Malpighian (renal) tubules (MT) generate a flow of primary urine that is subsequently modified by reabsorptive and secretory processes in the hindgut (ileum and rectum). Diuretic hormones (DH) accelerate primary urine production, whereas antidiuretic hormones generally stimulate fluid reabsorption in the hindgut, although some reduce MT secretion. CRF-like peptides: To date, 18 CRF-related DHs are known from 14 species of insects representing 7 Orders. They comprise paralogous subfamilies of “long” and “short” peptides as exemplified by two CRF-related DH from the tobacco hornworm Manduca sexta, Manse-DH [20] and Manse-DPII [3] (Fig. 1), both of which act via cAMP to increase MT secretion. Manse-DH has 41% sequence identity with sauvagine, but somewhat less with CRF, and has only 9 residues identical to ManseDPII. “Short” paralogs are present only in lepidopteran and coleopteran insects. The CRF-related DH from the beetle Tenebrio molitor (Tenmo-DH47 and Tenmo-DH37) are unique in being nonamidated [31]. Calcitonin (CT)-like peptides: The first CT-like DH was identified in the Pacific beetle cockroach, Diploptera Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

158 / Chapter 24 CRF-related DH Manse-DH Manse-DP2

RMPSLSIDLPMSVLRQKLSLEKERKVHALRAAANRNFLNDI-NH2 ---SFSVNPAVDILQH-------RYMEKVAQ-NNRNNLNRV-NH2

CT-like DH Dippu-DH31

GLDLGLSRGFSGSQAAKHLMGLAAANYAGGP-NH2

Kinin Leuma-K-1

DPAFNSWG-NH2

CAP2b Manse-CAP2b

pQLYAFPRV-NH2

Tenmo-ADFa Tenmo-ADFb

VVNTPGHAVSYHVY-COOH YDDGSYKPHIYGF-COOH

ADFs

ITP Schgr-ITP

SFFDIQCKGVYDKSIFARLDRICEDCYNLFREPQLHSLC RSDCFKSPYFKGCLQALLLIDEEEKFNQMVEIL-NH2 Schgr-ITP-L SFFDIQCKGVYDKSIFARLDRICEDCYNLFREPQLHSLC RKDCFTSDYFKGCIDVLLLQDDMDKIQSWIKQIHGAEPGV-COOH

KR

FIGURE 1. Sequences of representative diuretic and antidiuretic peptides. In each case, the sequence shown is that of the first representative of the family to be described.

GKR

D ro m e -D H 4 4

356 r e s id u e s

KR

GRR

D ro m e -D H 3 1

117 r e s id u e s

s ig n al p e p tid e KR

GRR

D ro m e -K

160 r e s id u e s

RR GR

KK GR KR

flan k in g s e q u e n ce

GKR

D ro m e -C AP 2 b

m atu r e p e p tid e

151 r e s id u e s cap a-1 KR

cap a-2 C 40

D ro m e -ITP D ro m e -ITP-L

cap a-3 GRK 108 r e s id u e s

119 r e s id u e s

FIGURE 2. Schematic representations (approximately to scale) of precursor structures and processing sites for diuretic and antidiuretic peptides that are encoded by the D. melanogaster genome.

STRUCTURE OF THE PRECURSOR mRNA/GENE CRF-related DH: The CRF-DH gene (Dh) of D. melanogaster (CG8348) is located in region 85E on chromosome 3R. It is composed of 4 exons, with the

prepropeptide (Fig. 2) encoded by exons 2, 3, and 4 [4]. The sequence C-terminal to the cleavage and amidation signal of Drome-DH44 resembles no known peptide. The prepropeptide of Manse-DH [7] has a similar structure, but is incomplete since Manse-DPII is encoded on the ˇ itn same gene (D. Z ˇ an, personal communication).

Insect Diuretic and Antidiuretic Hormones / 159 CT-like DH: The CT-like DH gene (Dh31) of D. melanogaster (CG13094) is located in region 29D2–3 on chromosome 2L and is composed of 5 exons. The prepropeptide (Fig. 2) is encoded by exons 3, 4, and 5. The flanking sequences have no similarity to other known peptides. Kinins: The kinin gene (pp; leucokinin) of D. melanogaster (CG 13480) is located in region 70E2 of chromosome 3L. The prepropeptide (Fig. 2) is encoded by a single exon and contains one copy of Drome-K. In contrast, three kinins (Aedae-K-1, -2, and -3) are encoded on the corresponding gene of Ae. aegypti [33]. CAP2b: The capability (capa) gene of D. melanogaster (CG15520) is located in region 99C6 of chromosome 3R. The prepropeptide (Fig. 2) is encoded by 3 exons, with exon 2 encoding two CAP2b peptides (capa-1 and -2), and exon 3 encoding a peptide of the pyrokinin family. ADFs: No gene sequence is available for these peptides. ITP: The gene ion transport peptide (itp) of D. melanogaster (CG13586) maps to region 60D4–D5 on chromosome 2R. The N- and C-termini of ITP are encoded by exons 9 and 11, which are spliced together at the equivalent of Cys40 in the mature peptide (Fig. 2) [28]. The same splice site (equivalent to Cys39) is used in S. gregaria to produce either ITP or ITP-L (Fig. 1).

to the CC [26] but is not found in MNCs of locusts, houseflies, and honeybees. Kinin-ir is, however, consistently present in posterior-lateral NSCs of abdominal ganglia 1–7 and associated neurohemal areas [5]. These NSCs colocalize CRF-DH-ir. CAP2b: The D. melanogaster CAP2b prepropeptide is present in three pairs of ventral NSCs in abdominal neuromeres, with neurohemal release sites on abdominal median nerves of larvae and on the dorsal surface of the thoracico-abdominal ganglion of adults [21]. It is also present in a pair of NSCs in the labial neuromere with axons projecting to the brain and retrocerebral complex. ADFs: Nothing is known of the distribution of TenmoADFa, but Tenmo-ADFb-ir is present in two pairs of lateral NSCs in the protocerebrum and in another pair located anterior and lateral to these. Axons from these cells project to ADFb-ir varicosities that might form neurohemal release sites [10]. ITP: Schgr-ITP mRNA is expressed in the brain and CC of adult locusts [24], and ITP-like-ir material is present in the PI and CC of S. gregaria [31]. There are also ITP-ir NSCs in each thoracic and abdominal PVO. Similarly, a number of cells in the frontal area of fifth instar B. mori brain express Bommo-ITP [11].

PEPTIDE PROCESSING DISTRIBUTION OF mRNA AND PEPTIDES CRF-related peptides: In D. melanogaster, the Dh transcript is found in three bilateral pairs of cells in the pars intercerebralis (PI) of the larval brain [4]. Drome-DH44 immunoreactivity (ir) is present in the cells and in their axons projecting to the retrocerebral complex. In other insects, CRF-DH-ir material is present in median neurosecretory cells (MNCs) of the brain, posterior-lateral neurosecretory cells (NSCs) of abdominal ganglia, and associated neurohemal organs, corpora cardiaca (CC), perivisceral organs (PVO), and abdominal nerves [5]. CRF-DH-ir material is also present in endocrine cells of the midgut and MT ampullae. CT-like peptides: CT-like-ir material is present in lateral NSCs of the brain in R. prolixus and in axons projecting to the CC and dorsal vessel (heart) [32]. In the mesothoracic ganglion mass, CT-like-ir is present in bilaterally paired NSCs and in five dorsal unpaired medial (DUM) neurons, which also contain serotonin, an identified DH. Axons from CT-like-ir cells extend into abdominal and trunk nerves that form neurohemal release sites. Kinins: Kinin-like-ir material is widely distributed in the CNS in both interneurons and NSCs. Kinin-ir is present in MNCs of the PI in L. maderae, and in axons

DH and ITP prepropeptides contain an N-terminal signal sequence of about 20 amino acid residues followed by active and inactive peptide sequences separated by some combination of two basic amino acids (Fig. 2). However, to obtain Aedae-K-2 and Drome-CAP2b peptides, the propeptide must be cleaved at single Arg residues by a mono-arginyl convertase. In vertebrates, PC1/PC3 convertase, which normally cleaves at Lys-Arg, can in an appropriate context cleave at single Arg residues. With the exception of the T. molitor CRF-like DH and ADFs, the mature peptides are amidated and the C-terminal of the propeptide ends with a cleavage and amidation signal -Gly-Xxx-Xxx (Xxx represents Arg or Lys) or -Gly-Arg for Drome-CAP2b peptides (Fig. 2). A propeptide convertase first cleaves off the C-terminal basic amino acids to expose the C-terminal glycine. Peptidylglycine α-amidating monooxygenase then converts the peptide to the des-Gly peptide amide.

RECEPTORS CRF-related peptides: Receptors for CRF-related DH belong to the secretin-like (Family B) family of G protein-coupled receptors (GPCRs). Two orthologs are present in D. melanogaster (CG8422 and CG12370), and

160 / Chapter 24 CG8422 encodes a functional receptor (Drome-DH44R1) for Drome-DH44 [18]. However, Drome-DH44-R1-ir material is not present in MT principal cells, which suggests CG12370 encodes a receptor that mediates the diuretic activity of Drome-DH44 [19]. Indeed, only the CG12370 transcript is enriched in MT compared with whole bodies [34]. A separate receptor probably exists for the short paralogs, but there are no data on this. Interestingly, a CRF-binding protein is also present in insects, providing further evidence for the long evolutionary history of the CRF system [17]. CT-like peptides: The D. melanogaster genome encodes a Family B GPCR (CG17415) related to the calcitonin receptor-like receptor (CLR) family [19]. CG17415 signals in response to Drome-DH31, but in common with other CLR this requires coexpression with accessory proteins, either mammalian or D. melanogaster receptor component protein or with mammalian receptor activity modifying proteins. In MT, Drome-DH31-R1 is present only in principal cells, and the gene transcript is enriched 17-fold compared with whole bodies [34]. Drome-DH31R1 is present also in corazonin-expressing neurons within the CNS, which also express Drome-DH44-R1. Drome-DH31 and Drome-DH44 may therefore control corazonin release, possibly at the time of ecdysis. Kinins: The kinin receptors of D. melanogaster [14] and Ae. aegypti [29] belong to Group III-B of Family A GPCRs, and appear to be paralogs of the true neurokinin receptors. Drome-K-R is present only in stellate cells of MT, and the gene (Lkr) transcript is enriched threefold compared with whole bodies [34]. The receptor is present throughout the larval and adult CNS, notably on MNCs of the PI that express Drome-DH44. Outside the CNS, the receptor is present in testes and ovary, and these tissues respond to Drome-K with an increase in intracellular Ca2+. CAP2b: CAP2b peptides have a similar C-terminal motif (PRX-NH2) to neuromedin U (NMU) and arginine vasopressin (AVP), and receptors related to those for NMU and AVP are encoded in the D. melanogaster genome. One of these (CG14575; capaR) responds functionally to Drome-CAP2b peptides ending with PRVNH2 but not to other PRX-NH2 peptides [27]. This receptor most likely mediates the diuretic activity of CAP2b, and the gene transcript is enriched 11-fold in MT compared with whole bodies [34]. Nothing is known of the receptors for ADFs or for ITP.

STRUCTURE-ACTIVITY AND ACTIVE CONFORMATION CRF-related peptides: The N-terminus is required for receptor activation, while the C-terminal two-thirds of

the peptide is needed for receptor binding [31]. The C-terminal amide is also important for high-affinity binding, although the nonamidated Tenmo-DH37 is a potent stimulant of T. molitor MT [12]. Manse-DH and Manse-DPII may adopt a folded helix-loop-helix conformation, bringing the C-terminal amide close to the N-terminus, and the constrained analogue [Cys3,40]Manse-DH has similar efficacy and potency to Manse-DH [31]. Kinins: The C-terminal pentapeptide is highly conserved and is the minimum sequence required for activity, but residues outside of this “active core” may be required for high-affinity binding [31]. Within the active core, Phe1 and Trp4 are invariant and essential for activity. The active core adopts a type VI β-turn when bound to receptors [25], bringing the side chains of Phe1 and Trp4 together on one face of the molecule where they can interact with the receptor. CAP2b: The minimum sequence for full activity in M. domestica MT is the C-terminal hexapeptide (YAFPRVNH2), within which the critical residues appear to be Arg and Val (G. M. Coast and R. J. Nachman, unpublished observations). ITP : At the N-terminus of Schgr-ITP, Ala substitutions at positions 1, 4, 5, and 6 have no effect on activity, but Phe2 and Phe3 are essential for receptor activation [28]. The C-terminal Leu-NH2 is also essential as are the six Cys residues, and disulfide bridges probably fix the distance between essential sites at the N- and C-termini [22]. There have been no structure/activity studies with ADFs.

BIOLOGICAL ACTIONS MT fluid secretion is coupled to ion transport, and DH stimulate KCl/NaCl secretion into the lumen. This is driven by active proton transport across the principal cell apical membrane by a V-type ATPase, with secreted protons returning to the cell via cation/H+ antiporters. Drome-DH44 and Drome-DH31 act via cAMP to stimulate V-ATPase activity, possibly by increasing recruitment of the V-ATPase to the apical membrane or by promoting association of the catalytic (V1) and proton-translocating (V0) domains. The Na+ : K+ concentration ratio of the secreted urine is probably determined by their uptake across the basolateral membrane, and the natriuretic response of mosquito MT to Anoga-DH31 appears to result from the opening of a cAMP-dependent Na+ channel [2, 6]. Kinins open a Cl−-selective conductance pathway making more Cl− available for KCl/NaCl secretion into the MT lumen [2]. The binding of Drome-K to DromeK-R on stellate cells activates phospholipase Cβ, promoting the inositol trisphosphate (IP3)-mediated release

Insect Diuretic and Antidiuretic Hormones / 161 of Ca2+ from intracellular stores [8]. Secretion is stimulated by the opening of Ca2+-activated “maxi” Cl− channels. In contrast, kinin activity in Ae. aegypti may be mediated by principal cells and results in the Ca2+dependent opening of a paracellular Cl− conductance through septate junctions [2]. Drome-CAP2b also uses IP3 as a second messenger, but acts on principal cells not stellate cells [8]. The emptying of IP3-sensitive Ca2+ stores opens storeoperated channels and the resultant influx of extracellular Ca2+ activates a Ca2+/calmodulin-sensitive nitric oxide (NO) synthase expressed only in principal cells [8]. The NO generated activates a soluble guanylate cyclase to elevate levels of cGMP, which increase VATPase activity. However, CAP2b activity in housefly MT is independent of NO and cGMP, and leads to the opening of a Cl− conductance pathway [31]. CAP2b acts via cGMP to reduce secretion by MT from R. prolixus and T. molitor larvae. It antagonizes the diuretic activity of serotonin (R. prolixus) [30] and Tenmo-DH37 (T. molitor) [35], which act via cAMPdependent mechanisms. CAP2b may therefore activate a cGMP-dependent cAMP phosphodiesterase, which will lower cAMP levels and hence reduce fluid secretion [30]. There is no evidence of NO involvement, and CAP2b may activate a membrane-bound guanylate cyclase. Tenmo-ADFa and Tenmo-ADFb act via cGMP to reduce secretion by T. molitor MT and probably activate a cAMP-specific phosphodiesterase, since ADFa antagonize the actions of Tenmo-DH37 [31]. Tenmo-ADFa also reduces secretion by A. aegypti MT but has no effect on tubule electrophysiology or on the concentrations of Na+, K+, and Cl− in the secreted urine [23]. This is consistent with reduced levels of cAMP and the down-regulation of a cAMP-dependent electroneutral transporter. Fluid uptake from the locust ileum is coupled to active salt (NaCl/KCl) transport. Schgr-ITP acts via cAMP to increase transepithelial NaCl/KCl transport by stimulating an apical membrane electrogenic Cl− pump [28]. Na+ and K+ cross the apical membrane through separate channels, both of which are activated by cAMP. In addition, Schgr-ITP inhibits active proton secretion into the lumen via a cAMP-independent mechanism [28].

CONCLUSION Insect DH and ADH constitute a diverse group of peptides that have in common their role in controlling fluid and ion loss via the excretory system. Their effects on epithelial transport processes in MT and hindgut are reasonably well understood, but there is still much to

be learned about the contribution these peptides make singly and collectively to the control of ion and fluid homeostasis in vivo. It is also clear that they may have additional roles within the CNS, as is evident from the presence of kinin receptors on NSCs expressing CRFrelated DH and of receptors for CRF-related and CTlike DH on corazonin-containing neurons.

References [1] Audsley, N.; McIntosh, C.; Phillips, J. E. Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport. J. Exp. Biol. 1992;173:261–274. [2] Beyenbach, K. W. Transport mechanisms of diuresis in Malpighian tubules of insects. J. Exp. Biol. 2003;206:3845– 3856. [3] Blackburn, M. B.; Ma, M. C. Diuretic activity of Mas-DP II, an identified neuropeptide from Manduca sexta: an in vivo and in vitro examination in the adult moth. Arch. Insect Biochem. Physiol. 1994;27:3–10. [4] Cabrero, P.; Radford, J. C.; Broderick, K. E.; Costes, L.; Veenstra, J. A.; Spana, E. P.; Davies, S. A.; Dow, J. A. T. The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP. J. Exp. Biol. 2002;205:3799–3807. [5] Coast, G. M.; Orchard, I.; Phillips, J. E.; Schooley, D. A. Insect diuretic and antidiuretic hormones. In: Evans, P. D., Ed. Adv. Insect Physiol. London: Academic Press; 2002:279–409. [6] Coast, G. M.; Garside, C. S.; Webster, S. G.; Schegg, K. M.; Schooley, D. A. Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles). J. Exp. Biol. 2005; in press. [7] Digan, M. E.; Roberts, D. N.; Enderlin, F. E.; Woodworth, A. R.; Kramer, S. J. Characterization of the precursor for Manduca sexta diuretic hormone Mas-DH. Proc. Natl. Acad. Sci. USA 1992;89:11074–11078. [8] Dow, J. A. T.; Davies, S. A. Integrative physiology and functional genomics of epithelial function in a genetic model organism. Physiol. Rev. 2003;83:687–729. [9] Eigenheer, R. A.; Nicolson, S. W.; Schegg, K. M.; Hull, J. J.; Schooley, D. A. Identification of a potent antidiuretic factor acting on beetle Malpighian tubules. Proc. Natl. Acad. Sci. USA 2002;99:84–89. [10] Eigenheer, R. A.; Wiehart, U. M.; Nicolson, S. W.; Schoofs, L.; Schegg, K. M.; Hull, J. J.; Schooley, D. A. Isolation, identification and localization of a second beetle antidiuretic peptide. Peptides 2003;24:27–34. [11] Endo, H.; Nagasawa, H.; Watanabe, T. Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori. Insect Biochem. Mol Biol. 2000;30:355–361. [12] Furuya, K.; Schegg, K. M.; Wang, H.; King, D. S.; Schooley, D. A. Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor. Proc. Natl. Acad. Sci. USA 1995; 92:12323–12327. [13] Furuya, K.; Milchak, R. J.; Schegg, K. M.; Zhang, J.; Tobe, S. S.; Coast, G. M.; Schooley, D. A. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc. Natl. Acad. Sci. USA 2000;97:6469–6474. [14] Hewes, R. S.; Taghert, P. H. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res. 2001;11:1126–1142. [15] Holman, G. M.; Cook, B. J.; Nachman, R. J. Isolation, primary structure and synthesis of two neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins. Comp. Biochem. Physiol. C 1986;84:205–211.

162 / Chapter 24 [16] Huesmann, G. R.; Cheung, C. C.; Loi, P. K.; Lee, T. D.; Swiderek, K. M.; Tublitz, N. J. Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta. FEBS Lett. 1995;371:311–314. [17] Huising, M. O.; Flik, G. The remarkable conservation of corticotropin-releasing hormone (CRH)-binding protein in the honeybee (Apis mellifera) dates the CRH system to a common ancestor of insects and vertebrates. Endocrinology 2005; 146:2165–2170. [18] Johnson, E. C.; Bohn, L. M.; Taghert, P. H. Drosophila CG8422 encodes a functional diuretic hormone receptor. J. Exp. Biol. 2004;207:743–748. [19] Johnson, E. C.; Shafer, O. T.; Trigg, J. S.; Park, J.; Schooley, D. A.; Dow, J. A.; Taghert, P. H. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J. Exp. Biol. 2005;208:1239–1246. [20] Kataoka, H.; Troetschler, R. G.; Li, J. P.; Kramer, S. J.; Carney, R. L.; Schooley, D. A. Isolation and identification of a diuretic hormone from the tobacco hornworm, Manduca sexta. Proc. Natl. Acad. Sci. USA 1989;86:2976–2980. [21] Kean, L.; Cazenave, W.; Costes, L.; Broderick, K. E.; Graham, S.; Pollock, V. P.; Davies, S. A.; Veenstra, J. A.; Dow, J. A. T. Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster. Am J Physiol Regul Integr Comp Physiol 2002;282:R1297–1307. [22] King, D. S.; Meredith, J.; Wang, Y. J.; Phillips, J. E. Biological actions of synthetic locust ion transport peptide (ITP). Insect Biochem. Mol. Biol. 1999;29:11–18. [23] Massaro, R. C.; Lee, L. W.; Patel, A. B.; Wu, D. S.; Yu, M.-J.; Scott, B. N.; Schooley, D. A.; Schegg, K. M.; Beyenbach, K. W. The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti. J. Exp. Biol. 2004;207:2877– 2888. [24] Meredith, J.; Ring, M.; Macins, A.; Marschall, J.; Cheng, N. N.; Theilmann, D.; Brock, H. W.; Phillips, J. E. Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system. J. Exp. Biol. 1996;199:1053–1061. [25] Nachman, R. J.; Zabrocki, J.; Olczak, J.; Williams, H. J.; Moyna, G.; Ian Scott, A.; Coast, G. M. cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation. Peptides 2002;23:709–716.

[26] Nässel, D. R.; Cantera, R.; Karlsson, A. Neurons in the cockroach nervous system reacting with the antisera to the neuropeptide leucokinin I. J. Comp. Neurol. 1992;322:45–67. [27] Park, Y.; Kim, Y. J.; Adams, M. E. Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligandreceptor coevolution. Proc. Natl. Acad. Sci. USA 2002;99:11423– 11428. [28] Phillips, J.; Meredith, J.; Wang, Y.; Zhao, Y.; Brock, H. Ion Transport Peptide (ITP): structure, function, evolution. In: Goos, H. J.; Rastogi, R. K.; Vaudry, H.; Pierantoni, R., Eds. Perspective in Comparative Endocrinology: Unity and Diversity. Bologna: MEDIMOND Inc.; 2001:745–752. [29] Pietrantonio, P. V.; Jagge, C.; Taneja-Bageshwar, S.; Nachman, R. J.; Barhoumi, R. The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins. Insect Mol. Biol. 2005;14:55–67. [30] Quinlan, M. C.; Tublitz, N. J.; O’Donnell, M. J. Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: the peptide CAP2b and cyclic GMP inhibit Malpighian tubule fluid secretion. J. Exp. Biol. 1997;200:2363–2367. [31] Schooley, D. A.; Horodyski, F. M.; Coast, G. M. Hormones controlling homeostasis in insects: Endocrinology. In: Gilbert, L. I.; Iatrou, K.; Gill, S. S., Eds. Comprehensive Molecular Insect Science: Elsevier; 2005:493–550. [32] Te Brugge, V. A.; Lombardi, V. C.; Schooley, D. A.; Orchard, I. Presence and activity of a Dippu-DH31-like peptide in the bloodfeeding bug, Rhodnius prolixus. Peptides 2005;26:29–42. [33] Veenstra, J. A.; Pattillo, J. M.; Petzel, D. H. A single cDNA encodes all three Aedes leucokinins, which stimulate both fluid secretion by the Malpighian tubules and hindgut contractions. J. Biol. Chem. 1997;272:10402–10407. [34] Wang, J.; Kean, L.; Yang, J. L.; Allan, A. K.; Davies, S. A.; Herzyk, P.; Dow, J. A. T. Function-informed transcriptome analysis of Drosophila renal tubule. Genome Biol. 2004;5:art. no.-R69. [35] Wiehart, U. I. M.; Nicolson, S. W.; Eigenheer, R. A.; Schooley, D. A. Antagonistic control of fluid secretion by the Malpighian tubules of Tenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers. J. Exp. Biol. 2002;205:493– 501.

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25 Developmental Peptides: ETH, Corazonin, and PTTH MICHAEL E. ADAMS, YOUNG-JOON KIM, YOONSEONG PARK, AND DUSAN ZITNAN

premature ecdysis when injected into pharate M. sexta larvae. The first isolation of an ETH was accomplished by reversed-phase HPLC using an extract of ∼40 M. sexta epitracheal glands [36]. The peptide was pure following a two-step purification guided by ability of fractions to trigger premature ecdysis behavior in pharate M. sexta larvae upon injection into the hemocoel. ETHs of M. sexta (MasETH) and B. mori (BomETH) are linear peptides with a conserved C-terminal sequence motif of –NIPRMamide [2, 36]. Pre-ecdysis triggering hormone (PETH), discovered through precursor analysis and biological activity (see following), is identical in the two moth species [37]. The Drosophila peptides DrmETH1 and DrmETH2 were deduced using BLAST analysis of the fly genome database using the moth precursor sequences and confirmed by RT-PCR [22]. Inka cells and associated ETH-like immunoreactivity occur in all insect Orders thus far examined, including Thysanura (silverfish), Orthoptera (crickets, grasshoppers, cockroaches, walking sticks), Plecoptera (stoneflies), Hemiptera and Homoptera (bugs), Megaloptera (alderflies), Neuroptera (antlions), Coleoptera (beetles), Lepidoptera (moths), Diptera (flies, mosquitos, crane flies), and Hymenoptera (bees, wasps, ants). Extracts from epitracheal glands or trachea containing Inka cells from many of these species induce ecdysis behavior in B. mori [39], suggesting that the ETH signaling system is widespread and conserved throughout the Insecta.

ABSTRACT Ecdysis triggering hormones (ETHs) are small, linear, C-terminally amidated peptides produced and released by endocrine Inka cells under the control of ecdysteroids and other peptides. Released into the blood at the end of each molt, they act directly on the central nervous system to trigger a sequence of physiological and behavioral events culminating in ecdysis, or shedding of the old cuticle. Corazonin is an Nand C-terminally blocked neuropeptide produced by neurosecretory cells in the brain and ventral nerve cord. It has a number of physiological actions, including cardioacceleration, melanization, and ecdysis initiation. Prothoracicotropic hormones (PTTHs) is a large (22 kDa) homodimeric peptide hormone produced by two pairs of lateral neurosecretory cells in the brain. It acts on the prothoracic glands to promote synthesis and release of ecdysteroids. Precursors of all these peptide hormones contain associated peptides of unknown function.

DISCOVERY Ecdysis Triggering Hormones Ecdysis triggering hormones (ETHs) were first isolated and sequenced from epitracheal glands of the tobacco hawkmoth, Manduca sexta, and the silkmoth, Bombyx mori [2, 36]. Epitracheal glands consist of a large, peptidergic Inka cell, a smaller putative endocrine cell, an exocrine cell, and a canal cell [16]. Zitnan observed strong immunoreactivity with antiserum to a neuropeptide FMRFamide in Inka cells on the surface of tracheal tubes immediately adjacent to each spiracle in the waxmoth Galleria mellonella and noticed that they lost staining after ecdysis [34]. A critical experiment demonstrated that extracts of the epitracheal glands trigger Handbook of Biologically Active Peptides

Corazonin Corazonin, an undecapeptide blocked at both Nand C-termini, originally was isolated from corpora cardiaca of the cockroach, Periplaneta americana [29]. The name—literally “heart” in Spanish—derives from its signature cardioacceleratory activity in the cockroach Periplaneta americana. The identical peptide subsequently

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Copyright © 2006 Elsevier

164 / Chapter 25 PETH (B. mori, M. sexta) ETH (B. mori) ETH (M. sexta)

SFIKPN.NVPRVa SNEA...FDEDVMGYVIKSNKNIPRMa SNEAISPFDQGMMGYVIKTNKNIPRMa

ETH1 (D. melanogaster) ETH2 (D. melanogaster) ETH1 (A. gambiae) ETH2 (A. gambiae)

DDSSPGFFLKITKNVPRLa GENFAIKNLKTIPRIa SESPGFFIKLSKSVPRIa GDLENFFLKQSKSVPRIa

ETH-AP (B. mori) ETH-AP (M. sexta)

NYDSGNHFDIPKVYSLPFEFYGDNEKSLNNDDAEE...YYAKKMGSM-OH NYDSENRFDIPKLYPWRAENTELYEDDAQPTNGEEINGFYGKQRENM-OH

ETH-AP1 (D. melanogaster) SE.H...SSVTPLLAW..LWDLD.TSPS-OH AGGFH..MAAVAPQEAHGSSW.FERYMKTA-OH ETH-AP1 (A. gambiae)

Corazonin His7-corazonin

pQTFQYSRGWTNa pQTFQYSHGWTNa

was identified in other cockroaches, crickets, and moths. A variant of corazonin, His7-corazonin, was isolated from the grasshopper Schistocerca americana [31] and subsequently from a number of other orthopterans, including S. gregaria, Locusta migratoria, and the stick insect Carousius morosus [28]. The amino acid sequence of corazonin is somewhat related to adipokinetic hormone and red pigment concentrating hormone of crustaceans, suggesting a common ancestry (Fig. 1).

Prothoracicotropic Hormone (PTTH) Stefan Kopec’s demonstration of a brain hormone that initiates molting in insects was an propitious event that launched the field of insect endocrinology [17, 18]. The brain hormone constituted the first definitive example of hormonal signaling in insects, yet it took more than 65 years for its molecular structure to be defined as PTTH. The hormone’s molecular size was estimated initially to be ∼25 kDa [15], and PTTH-like bioactivity was located in two pairs of lateral protocerebral neurosecretory cells projecting to release sites in the corpora allata [3, 4]. Ultimate determination of molecular identity came through a combination of direct peptide sequencing and cDNA precursor analysis. Sequencing of the peptide was made possible by tissue preparation on a heroic scale—first, 500,000 silkmoth heads to obtain the N-terminal 13 amino acids, and subsequently by processing of 1.8 million Bombyx heads to yield virtually the entire peptide sequence and the realization that the hormone is a dimer [11, 12]. For elucidation of the precursor sequence, polyclonal antibodies directed against the N-terminus were used to screen a cDNA expression library. The amino acid sequence of the predicted precursor was in complete agreement with that obtained by Kataoka and colleagues [13] (Genbank accession number D90082). Bombyx PTTH was thus defined as a homodimer consisting of

FIGURE 1. Amino acid sequence alignments of ETHs and corazonins.

identical 109-amino-acid subunits. Nucleotide probes based on the Bombyx sequence were used to determine the primary structures of PTTH in Samia cynthia, Antheraea pernyi, and Hyalophora cecropia. Related peptide sequences have been detected in silico in Drosophila and Anopheles genome databases.

STRUCTURE OF THE PRECURSOR mRNA/ GENE AND PEPTIDE PROCESSING Ecdysis Triggering Hormones Manduca sexta and Bombyx mori The MasETH and BomETH precursors contain a 23-amino-acid signal sequence and one copy each of PETH, ETH, and ETH-AP [35, 37]. Dibasic proteolytic cleavage sites are depicted in Fig. 2. Direct isolation of peptides from M. sexta epitracheal glands yielded, in addition to PETH, ETH, and ETH-AP, significant amounts of precursor peptides. These include the unprocessed precursor minus the signal sequence PETH-ETH-ETH-AP (PEA), PETH-ETH (PE), and ETH-ETH-AP (EA) [37]. Analysis of peptide content in Inka cells and circulating peptides in the hemolymph during ecdysis behavior reveals a 30% excess of PETH over ETH, despite the 1 : 1 stoichiometry of these peptides in the precursor. Lower concentrations of ETH result from incomplete processing of the EA precursor. Ecdysteroid pulses that initiate the molt trigger increased synthesis of ETH peptides in Inka cells. This is especially evident during the fifth (last) larval instar of M. sexta, where the pupal steroid commitment pulse is followed by a much larger second pulse that programs the pupal stage. Levels of ETHs and precursors rise in synchrony with the leading edges of each of these two steroid peaks [40], indicating that expression

Developmental Peptides: ETH, Corazonin, and PTTH /

165

FIGURE 2. Schematic diagrams of precursors and processing sites for ETHs [22, 37], corazonins [6, 8, 30], and PTTHs [13].

of the ETH gene and precursor processing are steroid-regulated.

closely related to DrmETH1 and DrmETH2 are evident in the mosquito precursor, as well as associated peptides downstream of ETH2.

Drosophila melanogaster and Anopheles gambaie The ETH precursor in Drosophila is encoded by the gene CG18105, which occurs on the right arm of the second chromosome at position 60E1-2. The preprohormone consists of a 21-amino-acid signal sequence and one copy each of DrmETH1 and DrmETH2, both of which have ecdysis-triggering biological activity [22]. Assignment of the ETH1 N-terminus was not obvious from inspection of the precursor sequence. However, synthesis and bioassay of the two most likely peptides (N-terminus beginning with either AIS– or DDS–) revealed that the DDS– sequence was significantly more bioactive in inducing adult ecdysis [22]. Three additional putative peptides occurring downstream of ETH2 have not been tested for biological activity. These include a 21-amino-acid peptide unblocked at the Cterminus (DrmETH-AP1), a 63-amino-acid peptide with a blocked C-terminus (DrmETH-AP2), and a 54-aminoacid peptide unblocked at the C-terminus [22]. Processing sites for the precursor are depicted in Fig. 2. An ETH precursor has been deduced from the mosquito Anopheles gambiae genome database [39]. Peptides

Corazonin The corazonin gene has been characterized in Drosophila melanogaster (CG3302) and other Drosophila species, including D. virilis, D. simulans, and D. erecta [6, 30]. The cDNA encoding the precursor also has been characterized in the waxmoth Galleria mellonella [8]. The precursor consists of a signal sequence, a single copy of the 11-mer corazonin peptide, and a corazonin-associated peptide (CRZ-AP or CAP) of ∼120 amino acids in flies and 80 amino acids in the waxmoth (Fig. 2). The genomic sequence indicates the presence of an intron in the region corresponding to the D. melanogaster CAP coding region. A second intron occurs in the 5′ UTR region of D. virilis. While the CRZ sequence is highly conserved in all species examined, the CAP sequence is highly variable and hence appears to be rapidly evolving.

PTTH The PTTH gene of B. mori consists of five exons that encode a 224-amino-acid precursor protein with three

166 / Chapter 25 proteolytic cleavage sites [1, 13]. The C-terminal 109amino-acid fragment of the precursor is processed to form the biologically relevant monomeric subunit, which contains 7 cysteines (Fig. 2). Homodimerization of these subunits via a single disulfide bond formed between Cys15 of each monomer yields the active hormone [9]. Disulfide bonds within each monomer are formed according to the following pattern: Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98. Interestingly, the 109-amino-acid monomer is about 50% as active as the native dimeric hormone. Glycosylation accounts for the disparity between the predicted (13 kDa) and observed (17 kDa) molecular weights of the monomer and also contributes to full activity of the native hormone, since the expression product from bacteria without glycosylation has lower biological activity than the native hormone. Two putative peptides of 2 kDa and 6 kDa predicted from sequences upstream of the 109-mer fragment are flanked by canonical proteolytic cleavage sites. No function has been associated with these peptides as yet. PTTHs cloned and characterized from other moth species (Samia, Hyalophora, Antheraea, Manduca) and nonlepidopteran species (Drosophila, Anopheles) have been summarized recently [24].

DISTRIBUTION OF THE mRNA AND PEPTIDES Ecdysis Triggering Hormones The ETH gene is expressed only in Inka cells. Northern blots were used to detect ETH-encoding mRNA in M. sexta epitracheal glands; the signal was absent in the CNS, epidermis, muscle, and trachea [14]. An antiserum specific for the MasETH N-terminus stained exclusively Inka cells of M. sexta and B. mori. No other cells or tissues were stained. Cell-specific expression also was demonstrated using the ETH promoter to drive expression of EGFP in transgenic flies. A nucleotide sequence encoding the following fusion protein was constructed: the ETH gene upstream promoter region (∼300 bp), the ORF encoding part of the ETH precursor, and a nucleotide sequence encoding EGFP. Transformed fly lines showed EGFP-associated fluorescence only in Inka cells [19].

a DUM cell of the subesophageal ganglion, an interneuron in the optic lobes, and additional cells at base of optic lobes, as well as in the tritocerebrum and deutocerebrum [32]. In the last larval instar of the waxmoth G. mellonella, in situ hybridization detected mRNA in four pairs of brain lateral neurosecretory neurons [8]. These same cells showed CRZ-IR, verifying the presence of both mRNA and peptide in the same cells. No other cells in the CNS were observed. Roller et al. [23] surveyed six Orders of insects for CRZ-IR and found central neurons, both in the brain and ventral nerve cord of most insects, except for Coleoptera and the albino strain of the grasshopper Locusta migratoria. Extracts of ganglia displaying CRZ-IR induced melanization in the albino grasshopper, providing further evidence for the widespread presence of the peptide in the CNS. In Drosophila, in situ hybridization demonstrated a pattern of corazonin expression in central neurons that changes during the life history of the animal. During larval life, mRNA is present in one pair of brain dorsomedial neurons and three pairs of dorsolateral neurons. In addition, eight bilateral pairs are observed in the ventral nerve cord [6]. In the adult, hybridization signals occurred in six to eight pairs of brain dorsolateral neurons and widely on the anterior surface of the optic lobe medulla but none in the dorsomedial region or in the ventral nerve cord corresponding to cells previously observed in larvae. Despite the presence of mRNA in optic lobe neurons, CRZ-IR is absent. It has been suggested that the absence of immunoreactivity may be due either to rapid turnover of peptides or translational silencing [6].

PTTH Expression and synthesis of PTTH are confined to two pairs of lateral neurosecretory cells in the brain. This was demonstrated originally in M. sexta by singlecell dissection and bioassay [4] and was substantiated in many species of moth (B. mori, A. polyphemus, M. sexta) by in situ hybridization and immunohistochemical staining. Possibly homologous cells were demonstrated in the Drosophila brain [38] and in the subesophageal and ventral ganglia of Drosophila, but these have not been confirmed by in situ hybridization or bioassay.

Corazonin Corazonin mRNA and the peptide itself are confined to the central nervous system in all insects examined. The pattern of cellular expression depends on the particular insect and is stage-dependent. In the cockroach, P. americana, corazonin-like immunoreactivity (CRZ-IR) is found in ten lateral neurosecretory cells of the brain, which project ipsilaterally to release sites in the corpus cardiacum. CRZ-IR also occurs in bilateral pairs of neurons in the thoracic and abdominal neuromeres, in

RECEPTORS Ecdysis Triggering Hormone Receptors ETH receptors identified in D. melanogaster are G protein-coupled receptors [10, 21]. A single gene (CG5911) containing five exons encodes two ETH receptor subtypes through alternative splicing of the two 3′ exons to generate CG5911a and CG5911b. Phylogenetic analysis shows that CG5911 is most closely

Developmental Peptides: ETH, Corazonin, and PTTH / related to the vertebrate thyrotropin-releasing hormone and neuromedin receptors, as well as to Drosophila receptors for PBAN-like pyrokinin receptors, including HUGγ, Drm-PK2, and CAP2B peptides. Pharmacological profiles of the ETH receptors were determined by heterologous expression in mammalian CHO cells using aequorin as an intracellular calcium reporter. ETH receptors show high sensitivity and selectivity to fly and moth ETHs. Remarkably, CG5911b shows considerably higher sensitivity to ETH1 and ETH2 as compared to CG5911a. Both DrmETH1 and DrmETH2 activate CG5911b with EC50 values of ∼1 nM. In contrast, ETH1 and ETH2 activate CG5911a with EC50 values of ∼400 nM and ∼4 μM, respectively [21]. Thus, while CG5911b is more sensitive to the cognate ETH ligands than CG5911a, the latter is nevertheless more discriminating between ETH1 and ETH2.

Corazonin Receptors The corazonin receptor (CRZR) in D. melanogaster is a GPCR encoded by CG10698 [5, 20]. Phylogenetic analysis groups this receptor with that of adipokinetic hormone (AKH) and vasopressin receptors. Expressed either in Xenopus oocytes or mammalian CHO cells, CG10698 is highly selective for corazonin, whose EC50 is in the low nanomolar range. Other ligands tested at up to 10 μM concentration (including AKH) were inactive [20]. The M. sexta corazonin receptor is involved in ecdysis initiation [14]. Northern blots show expression in the CNS and epitracheal glands but not in muscle, epidermis, and trachea. The receptor is highly sensitive and selective for corazonin when expressed in Xenopus oocytes (EC50 = 200 pM) or CHO cells (EC50 = 75 pM). Inka cells respond to corazonin (≥10 pM) by releasing ETH. Interestingly, high concentrations of corazonin (>1 nM) suppress ETH release from Inka cells, indicating the likelihood that receptor desensitization plays a role in cellular responses. Such densitization could place a limit on the amount of ETH release, ensuring that only low levels are released in the initial stages of the ecdysis behavioral sequence.

PTTH Receptor No receptor for PTTH has been identified at the time of this writing.

ACTIVE AND/OR SOLUTION CONFORMATION Three-dimensional structures for peptides described in this review were not available at the time of this writing.

167

BIOLOGICAL ACTIONS Ecdysis Triggering Hormones ETHs released into the hemocoel by Inka cells act directly on the CNS to trigger a behavioral sequence culminating in ecdysis of the old cuticle. It is remarkable that ETH signaling mediates ecdysis in insects with widely divergent body plans. Even though the specific motor patterns may be different as a result, the signaling system and the behaviors appear to be conserved over millions of years. Injection of MasETH into pharate M. sexta larvae initiates pre-ecdysis contractions within minutes. The animal switches to ecdysis contractions after about one hour [36]. The peptide also elicits patterned motor output from the isolated CNS, the parameters of which (e.g., burst frequency, burst duration) correspond precisely to those governing contractions in the whole animal. These observations indicate that the peptide acts directly on the CNS to elicit centrally patterned pre-ecdysis and ecdysis behaviors. Injection of PETH into pharate Manduca larvae helped to define two pre-ecdysis behaviors: Pre-ecdysis I and pre-ecdysis II. Injection of PETH alone triggers pre-ecdysis I only; thereafter the animal ceases all contractions. Subsequent injection of ETH triggers pre-ecdysis II followed by ecdysis behavior. It also has been observed that injection of a naive pharate larva with MasETH triggers simultaneously both pre-ecdysis I and pre-ecdysis II. Observations of natural behavior reveal that the animal begins the behavioral sequence with pre-ecdysis and 20 minutes later begins pre-ecdysis II contractions. Both behaviors continue in parallel for the next 40 minutes, after which a transition to ecdysis behavior occurs. Therefore, the action of PETH dominates during the first 20 minutes of the ecdysis behavioral sequence. Injection of DrmETH1 into Drosophila larvae elicits the entire ecdysis behavioral sequence in Drosophila larvae [7, 19]. These steps include tracheal collapse and inflation, pre-ecdysis behaviors consisting of anterior-posterior contractions and squeezing waves, and peristaltic ecdysis contractions. On the other hand, ETH2 injection triggers tracheal collapse and inflation and ecdysis but fails to elicit pre-ecdysis behaviors. Deletion of the gene encoding Drosophila ETHs causes failure of successful ecdysis and lethality [19]. In pharate adults, injection of ETH1 (>1 fmol) or ETH2 (>10 fmol) trigger the entire eclosion behavioral sequence, although ETH2 is less potent than ETH1. The steps in the behavior include head inflation (pre-eclosion), forward head thrusts (onset of ecdysis), bilateral, alternating contractions of the thorax, and strong peristaltic abdominal contractions pushing the body forward [22].

168 / Chapter 25 Corazonin Corazonin is a potent cardioaccelerator in P. americana but not in other insects. His7-corazonin of locusts triggers melanization and hence is also known as a “pigmentotropin” [28, 31]. The melanization activity of His7-corazonin is confined to locusts. Corazonin is involved in events related to molting, ecdysis, and adult development. For example, the peptide prolongs spinning behavior in fifth instar B. mori larvae and delays adult development during the pupal stage in the same species [27]. As just mentioned, corazonin injection into moth larvae elicits ecdysis behaviors. The peptide was detected in the hemolymph of Manduca larvae just prior to onset of pre-ecdysis behavior. Exposure of isolated Inka cells to corazonin leads to a low level release of ETHs. It thus seems likely that, in Manduca, corazonin is involved in initiating the early stages of the ecdysis behavioral sequence by causing ETH release from Inka cells [14]. Finally, corazonin is implicated in circadian rhythms and diapause. The peptide is colocalized with the PER gene product in lateral neurosecretory 1a1 cells of Manduca [33], and these cells are implicated in the regulation of circadian rhythms [25]. Ablation of these cells in Manduca suppresses diapause during short days, indicating their possible role in measuring day length [26].

PTTH The sole tissue target of PTTH is the prothoracic gland, which responds to the hormone at low to subnanomolar concentrations by elevating synthesis of ecdysteroids. Consistent with this function, hemolymph levels of PTTH rise immediately prior to ecdysteroid pulses, which initiate molting at each stage of development [24]. Although the PTTH receptor has yet to be defined, it is hypothesized to be a GPCR coupled to a number of signal transduction pathways, including influx of extracellular calcium, phosphoinositide turnover, and cAMP elevation.

References [1] Adachi-Yamada T, Iwami M, Kataoka H, Suzuki A, Ishizaki H. Structure and expression of the gene for the prothoracicotropic hormone of the silkmoth Bombyx mori. Eur J Biochem 1994;220:633–43. [2] Adams ME, Zitnan D. Identification of ecdysis-triggering hormone in the silkworm Bombyx mori. Biochemical and Biophysical Research Communications 1997;230:188–91. [3] Agui N, Bollenbacher WE, Granger NA, Gilbert LI. Corpus allatum is release site for insect prothoracicotropic hormone. Nature 1980;285. [4] Agui N, Granger NA, Gilbert LI, Bollenbacher WE. Cellular localization of the insect prothoracicotropic hormone: In vitro

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assay of a single neurosecretory cell. Proc Natl Acad Sci USA 1979;76:5694–8. Cazzamali G, Saxild N, Grimmelikhuijzen C. Molecular cloning and functional expression of a Drosophila corazonin receptor. Biochem Biophys Res Commun 2002;298:31–6. Choi YJ, Lee G, Hall JC, Park JH. Comparative analysis of Corazonin-encoding genes (Crz’s) in Drosophila species and functional insights into Crz-expressing neurons. J Comp Neurol 2005;482:372–85. Clark AC, del Campo ML, Ewer J. Neuroendocrine control of larval ecdysis behavior in Drosophila: complex regulation by partially redundant neuropeptides. J Neurosci 2004;24:4283– 92. Hansen IA, Sehnal F, Meyer SR, Scheller K. Corazonin gene expression in the waxmoth Galleria mellonella. Insect Mol Biol 2001;10:341–6. Ishibashi J, Kataoka H, Isogai A, Kawakami A, Saegusa H, Yagi Y, et al. Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide. Biochemistry 1994;33:5912–19. Iversen A, Cazzamali G, Williamson M, Hauser F, Grimmelikhuijzen CJ. Molecular identification of the first insect ecdysis triggering hormone receptors. Biochem Biophys Res Commun 2002;299:924–31. Kataoka H, Nagasawa H, Isogai A, Ishizaki H, Suzuki A. Prothoracicotropic hormone of the silkworm, Bombyx mori: amino acid sequence and dimeric structure. Agric Biol Chem 1991; 55:73–86. Kataoka H, Troetschler RG, Kramer SJ, Cesarin BJ, Schooley DA. Isolation and primary structure of the eclosion hormone of the tobacco hornworm, Manduca sexta. Biochem Biophys Res Commun 1987;146:746–50. Kawakami A, Kataoka H, Oka T, Mizoguchi A, Kimura-Kawakami M, Adachi T, et al. Molecular cloning of the Bombyx mori prothoracicotropic hormone. Science 1990;247:1333–5. Kim YJ, Spalovska-Valachova I, Cho K-H, Zitnanova I, Park Y, Adams ME, et al. Corazonin receptor signaling in ecdysis initiation. Proc Natl Acad Sci USA 2004;101:6704–9. Kingan TG. Purification of the prothoracicotropic hormone from the tobacco hornworm Manduca sexta. Life Sci 1981;28: 2585–94. Klein C, Kallenborn HG, Radlicki C. The “Inka cell” and its associated cells: ultrastructure of the epitracheal glands in the gypsy moth, Lymantria dispar. J Insect Physiol 1999;45: 65–73. Kopec S. Experiments on metamorphosis of insects. Bull Int Acad Cracovie B 1917:57–60. Kopec S. Studies on the necessity of the brain for the inception of insect metamorphosis. Biol Bull 1922;42:322–42. Park Y, Filippov V, Gill SS, Adams ME. Deletion of the ecdysistriggering hormone gene leads to lethal ecdysis deficiency. Development 2002a;129:493–503. Park Y, Kim YJ, Adams ME. Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc Natl Acad Sci USA 2002b;99:11423–8. Park Y, Kim YJ, Dupriez V, Adams ME. Two subtypes of ecdysistriggering hormone receptor in Drosophila melanogaster. J Biol Chem 2003;278:17710–15. Park Y, Zitnan D, Gill SS, Adams ME. Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster. FEBS Letters 1999;463:133–8. Roller L, Tanaka Y, Tanaka S. Corazonin and corazoninlike substances in the central nervous system of the Pterygote and Apterygote insects. Cell Tissue Res 2003;312: 393–406.

Developmental Peptides: ETH, Corazonin, and PTTH / [24] Rybczynski R. Prothoracicotropic Hormone. In: Gilbert LI, Iatrou, K., and Gill, S. S., editor. Comprehensive Molecular Insect Science. Oxford: Elsevier; 2005, p. 61–123. [25] Sauman I, Reppert SM. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of Period protein regulation. Neuron 1996;17:889–900. [26] Shiga S, Davis NT, Hildebrand JG. Role of neurosecretory cells in the photoperiodic induction of pupal diapause of the tobacco hornworm Manduca sexta. J Comp Neurol 2003;462:275–85. [27] Tanaka Y, Hua Y, Roller L, Tanaka S. Corazonin reduces the spinning rate in the silkworm, Bombyx mori. J Insect Physiol 2002;48:707–14. [28] Tawfik AI, Tanaka S, De Loof A, Schoofs L, Baggerman G, Waelkens E, et al. Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant. Proc Natl Acad Sci USA 1999;96:7083–7. [29] Veenstra JA. Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Lett 1989;250: 231–4. [30] Veenstra JA. Isolation and structure of the Drosophila corazonin gene. Biochem Biophys Res Commun 1994;204:292–6. [31] Veenstra JA. Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana. Peptides 1991;12:1285–9. [32] Veenstra JA, Davis NT. Localization of corazonin in the nervous system of the cockroach Periplaneta americana. Cell Tissue Res 1993;274:57–64.

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[33] Wise S, Davis NT, Tyndale E, Noveral J, Folwell MG, Bedian V, et al. Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta. J Comp Neurol 2002;447: 366–80. [34] Zitnan D. Regulatory peptides and peptidergic organs in insects [Ph.D Dissertation]. Bratislava: Slovak Academy of Sciences; 1989. [35] Zitnan D, Hollar L, Spalovska I, Takac P, Zitnanova I, Gill SS, et al. Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori. J Exp Biol 2002;205: 3459–73. [36] Zitnan D, Kingan TG, Hermesman J, Adams ME. Identification of ecdysis-triggering hormone from an epitracheal endocrine system. Science 1996;271:88–91. [37] Zitnan D, Ross LS, Zitnanova I, Hermesman JL, Gill SS, Adams ME. Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence. Neuron 1999;23:523–35. [38] Zitnan D, Sehnal F, Bryant PJ. Neurons producing specific neuropeptides in the central nervous system of normal and pupariation-delayed Drosophila. Dev Biol 1993;156:117–35. [39] Zitnan D, Zitnanova I, Spalovska I, Takac P, Park Y, Adams ME. Conservation of ecdysis-triggering hormone signalling in insects. J Exp Biol 2003;206:1275–89. [40] Zitnanova I, Adams ME, Zitnan D. Dual ecdysteroid action on the epitracheal glands and central nervous system preceding ecdysis of Manduca sexta. J Exp Biol 2001;204:3483–95.

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26 Tachykinins and Tachykinin-Related Peptides in Invertebrates DICK R. NÄSSEL

from the locust Locusta migratoria. The amino acid sequences of these two locust nonapeptides, designated locustatachykinin-I and -II, differ in a major way from vertebrate tachykinins and eledoisin by having a Cterminal pentapeptide FXGVRamide. Subsequently, it became apparent that nervous system- and intestinederived tachykinins of insects and several other invertebrates share this structural feature (Table 1) [3, 4, 9]. Thus, these invertebrate peptides are not referred to as tachykinins but as tachykinin-related peptides (TKRPs). Interestingly it has been found that vertebrate type of FXGLMamide–containing tachykinins are present in salivary glands of a mosquito and two cephalopod species but not in the CNS of any invertebrate [9]. The salivary gland-derived tachykinins are henceforth referred to as invertebrate tachykinins (invTKs) as suggested by Satake et al. [9]. The first insect TKRPs were identified by their myostimulatory actions in the cockroach hindgut muscle contraction bioassay, an assay used for isolation of a large number of novel peptides from HPLC-purified tissue extracts. Most invertebrate TKRPs have been identified either by the hindgut bioassay, a combination of this and an immunoassay with antiserum to locust TKRP, or by mining existing genome data bases [3, 4, 9]. Additional TKRPs have been identified by classical homology cloning technique from the Honeybee and the cockroaches Leucophaea maderae and Periplaneta americana. Thus, sequence data are now available for TKRPs from molluscs, an annelid-like worm Urechis unicinctus, many insects, and some decapod crustaceans (see Table 1). The invTKs have been identified in the salivary glands of two cephalopods and a mosquito, Aedes aegypti. In one of the cephalopods, Octopus vulgaris, typical TKRPs have in addition been identified in the CNS [9], suggesting that TKRPs are neuronal peptides and invTKs nonneuronal.

ABSTRACT Peptides related to vertebrate tachykinins have been identified in many invertebrates, including a nematode, an annelid-like worm, mollusks, crustaceans, and insects. Two major types can be distinguished: tachykininrelated peptides (TKRPs) with an FXGXRamide Cterminus and invertebrate tachykinins (invTKs) with FXGLMamide. The TKRPs are present in the nervous system and intestine, whereas the invTKs have been found only in salivary glands in a few species. The TKRPs have a wide neuronal distribution and a broad spectrum of biological activities, including myostimulatory, diuretic, and neuromodulatory actions. Receptors for TKRPs, likely to be ancestrally related to vertebrate neurokinin receptors, have been identified in a few species. In heterologous expression systems these receptors couple to calcium and cyclicAMP signaling. Several enzyme activities have been identified that may be responsible for TKRP inactivation.

DISCOVERY Tachykinins constitute a large and structurally varied family of multifunctional peptides present in the nervous system, gastrointestinal tract, and many other tissues. Members of this family have been identified in a nematode, an annelid, molluscs, arthropods, as well as in vertebrates. Already in 1931 the first tachykinin, substance P, was isolated from horse brain. Substance P was followed by a large number of related peptides isolated from various vertebrates, all characterized by a common C-terminal sequence FXGLMamide [7]. The first tachykinin to be identified from an invertebrate was eledoisin, isolated from the salivary glands of the cephalopod Eledone moschata. In 1990 the first insect peptides with similarities to tachykinins were identified Handbook of Biologically Active Peptides

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172 / Chapter 26 TABLE 1.

Sequences of representative members of the extended tachykinin family.

Species Decapod crustaceans L. maderae

Drosophila

Stomoxys calcitrans Anodonta cygnea Eledone moschata Aedes aegypti C. elegans Mammals

Designation

Sequence

TKRPs (FXGXRamide) CabTRP-I APSGFLGMRa LemTRP-1 APSGFLGVRa LemTRP-2 APEESPKRAPSGFLGVRa LemTRP-3 NGERAPGSKKAPSGFLGTRa APSGFLGTRa LemTRP-311–19 DTK-1 APTSSFIGMRa DTK-2 APLAFVGLRa DTK-3 APTGFTGMRa DTK-4 APVNSFVGMRa DTK-5 APNGFLGMRa DTK-6 AALSDSYDLRGKQQRFADFNSKFVAVRa StcTK-I APTGFFAVRa AncTK pQYGFHAVRa InvTKs and substance P (FXGLMamide) Eledoisin Sialokinin I nlp-81 Substance P

pEPSKDAFIGLMa SGNTGDKFYGLMa SFDRMGGTEFGLMa RPKPQQFFGLMa

The dibasic cleavage sites in LemTRPs are indicated in bold; the G to A substitution in some TKRPs is indicated by underlining. 1 The C. elegans invTK has no designation, but is derived from the nlp-8 gene.

STRUCTURE OF THE PRECURSOR mRNA/GENE The three principal mammalian tachykinins—substance P, neurokinin A, and neurokinin B—are processed from two precursors—preprotachykinin A and B [7]. More recently additional tachykinins, the hemokinins, were identified on a third precursor gene, preprotachykinin C, expressed in hematopoietic cells [7]. The first invertebrate cDNA to be identified for a TKRP precursor was isolated from U. unicinctus [9]. This precursor consists of 243 amino acid residues, from which 7 different TKRPs can be liberated. Next, a gene (tk; CG14734) coding for multiple TKRPs was identified by searching the Drosophila genome database. The Drosophila precursor consists of 289 amino acids derived from an 870 bp open reading frame. There are six predicted TKRPs (DTK-1 to -6) in the Drosophila precursor, five of which could be confirmed in the brain by mass spectrometry. Through data mining and classical cloning techniques we now know the sequences of several additional TKRP precursors from insects, crustaceans, and a cephalopod. Thus, there are 7 TKRPs on the precursor from the Honeybee Apis mellifera, 3 in the mosquito Anopheles gambiae, 13 in each of the cockroaches P. americana and L. maderae, 7 in Octopus vulgaris, and 7 each in the crayfish Procambarus clarkii and spiny lobster Panulirus interruptus [4, 8, 9].

The crayfish and lobster precursors, which are very similar to each other, each contain 7 identical TKRPs (CabTRP-I) and no further peptides. This is unique, so far, among the TRP precursors. In all other organisms, multiple, closely related TKRP isoforms are found on the precursors. The crustacean precursors may provide insights into the evolution of tachykinin isoforms. Possibly precursors, like the crustacean ones, with several identical copies of a peptide are more ancestral, and such a precursor could evolve by diversification into one with multiple closely related peptides. An interesting feature of the cockroach TKRPs is that two N-terminally extended TKRPs exist that have internal dibasic cleavage sites (Table 1). These sites appear more likely to be utilized by processing enzymes in the brain than in the intestine. As a consequence, the nonapeptides LemTKRP-1 and LemTKRP-311–19 are much more abundant in the brain than LemTKRP-2 (17mer) and LemTKRP-3 (19mer), whereas the extended forms are prominent in the intestine [8]. Thus, there might be a differential posttranslational processing of these peptides in the brain and the gut. In total the L. maderae precursor can yield up to 15 TKRP isoforms, as confirmed by MALDI-TOF mass spectrometry. Two of these are produced posttranslationally. Judging from cDNAs the sequences of the TKRP precursors of different invertebrates are conserved mainly in the peptide progenitor regions. The sequences providing spacing between peptide progenitors vary in

Tachykinins and Tachykinin-Related Peptides in Invertebrates / 173 length between species: from being just the dibasic cleavage sites and amidation signals (Urechis) to substantial spacer sequences (several of the insects). In cockroaches there is a mix between the two spacer lengths. The genomic organization of TKRP precursor genes has been published for Drosophila [4]. Three exons separated by introns are seen in the Drosophila Tk gene. The DTK-1–4 peptides are located on exon 2 and DTK5 and -6 on exon 3. No evidence for alternative splicing of the precursor mRNA is available. The precursor of the invTK of Aedes is small (96 amino acids, 406 bp cDNA) and codes for a single peptide, Sialokinin. The Sialokinin gene consists of three exons. Also the Octopus invTK precursor, OctTK-I is small (87 residues, 489 bp cDNA) and predicts one peptide [9]. In Octopus a second, nearly identical, invTK precursor (OctTK-II) was identified. The two Octopus precursors may be the result of a polymorphism. The C. elegans gene nlp-8 predicts three peptides, one of which has the C-terminal tetrapeptide identical to that of substance P (Table 1) [5]. Since the other two peptides of the precursor are clearly unrelated, it is not clear whether nlp-8 is ancestrally related to genes coding for tachykinins and TKRPs. Thus, maybe the C. elegans peptide should not be grouped with the invTKs.

DISTRIBUTION OF mRNA AND PEPTIDES The localization of TKRP precursor transcript and/ or peptides is known for some insects and crustaceans. In Drosophila in situ hybridization has revealed a distribution of Tk transcript in interneurons of the CNS and endocrine cells of the intestine. Immunocytochemistry confirmed the presence of TKRPs (DTKs) in the same set of neurons. The number of neurons is relatively small in late third instar larvae (about 40–50 neurons in the total CNS, 30–36 in brain) and somewhat higher (about 200 neurons in the CNS, about 150 in brain) in adult Drosophila [4]. Immunocytochemistry revealed neuronal arborizations in specific brain neuropils, such as optic lobes, antennal lobes, central body, and pars intercerebralis (area of branches of protocerebral neurosecretory cells). No neurosecretory cells were detected. The principal distribution of TKRP-containing neuron processes follows a basic pattern in the insects studied (Fig. 1), but there is some variation between species in some other parts of the brain [3, 4]. In all studied insects the same neuropils as listed for Drosophila are supplied by TKRP immunoreactive branches and most insects seem to lack TKRPs in brain neurosecretory

FIGURE 1. Distribution of TKRP immunoreactive neurons in the brain of the cockroach L. maderae (frontal compressed views). Most of the cell bodies are located in bilateral clusters. A large number of cell bodies are associated with the antennal lobes (AL), the protocerebral bridge (PB), and the central body (CB). Neuropils rich in immunoreactive processes are highlighted, AL, PB, CB, as well as tritocerebral glomeruli (TN) and mushroom body calyces (LCa and MCa). Not shown here are the processes in the pars intercerebralis (PI), and optic lobes are not depicted. Other abbreviations: AN—antennal nerve; OP—optic peduncle; aL and bL— alpha- and beta-lobes of mushroom bodies; P—protocerebrum, D—deutocerebrum; T— tritocerebrum. Altered from Muren JE, Lundquist CT, Nässel DR. Phil Trans R Soc Lond B 1995;348:423–444.

174 / Chapter 26 cells. In honeybees there is additional expression of the TKRP gene transcript in intrinsic neurons (Kenyon cells) of the mushroom bodies. Also, TKRP immunoreactivity has been detected in Kenyon cells of the bee. The mushroom bodies are higher olfactory centers but also important for olfactory learning and memory. Thus, TKRPs may have a role in higher olfactory processing or even in olfactory learning in honeybees. In the brain of decapod crustaceans the distribution of TKRP immunoreactive neurons is similar to that of insects: in olfactory interneurons, optic lobe interneurons, and in the central body complex [3]. The neuronal distribution of TKRPs has also been studied in the snails Helix pomatia and Lymnaea stagnalis [3]. Numerous TKRP-containing neurons were found especially in cerebral ganglia, and neuronal processes arborize in every ganglion. There is no report on the distribution of TKRPs in U. unicinctus, but mass spectrometry analysis has indicated that the TKRPs are expressed in the CNS and not in the intestine. The invTK precursor distribution has been mapped by in situ hybridization in Aedes and Octopus. In the mosquito the transcript is present only in adult female salivary glands, and in the cephalopod it was found in a portion of the salivary glands only. The distribution of the C. elegans “invTK” encoded by nlp-8 was seen in sets of identified neurons throughout the animal and processes on the gut [1].

PEPTIDE PROCESSING The TKRPs are likely to be liberated from their precursor proteins by common endoproteolytic enzymes such as prohormone convertases [10]. The enzymatic cleavage of some extended TKRPs from the precursor of cockroaches might follow a tissue specific pattern as described in a previous section. Enzymatic inactivation is an important means of terminating neuropeptide action. Several enzyme activities are known to cleave TKRPs and thus render them inactive: dipeptidyl peptidase IV (DPP IV), angiotensin converting enzyme (ACE), and nephrilysin (NEP) [4, 10]. Prolyl endoprotease activity of the DPP IV type has been identified in the cockroach brain and localized histochemically to specific brain regions also containing TKRPs. Enzymes of this kind inactivate neuropeptides with a Pro in position 2, like most of the TKRPs. In Drosophila the gene CG5355 predicts a prolyl endopeptidase. The ACE metalloproteases remove dipeptides from the C-terminus. A family of ANCE/ACER genes encoding enzymes with ACE activity has been identified in Drosophila. Nephrilysin (enkephalinase) is a key enzyme in neuropeptide metabolism. Nephrilysin activity was purified from L. maderae and found to efficiently degrade cockroach TKRPs and its localization in

the brain was revealed by enzyme histochemistry. At least 16 genes of the NEP family have been identified in Drosophila.

RECEPTORS OF TKRPs In mammals three types of G-protein-coupled receptors (GPCRs) for tachykinins have been identified [7], as has been discussed in the tachykinin chapter of the Brain Peptides section of this book. These are designated NK1-3 and display preferential affinities for substance P, neurokinin A, and neurokinin B, respectively. The hemokinins seem to associate with the NK1 receptor. In invertebrates GPCRs for the TKRPs have been identified in several species of insect and in the worm U. unicinctus [4, 9, 10]. Two main types of GPCRs with TKRPs as ligands have been distinguished in Drosophila among a set of six GPCRs that fall in the class of neurokinin-like receptors [10]. The first to be identified, designated DTKR, has recently been fully characterized with endogenous Drosophila TKRPs (DTK-1 to -6). GPCRs closely related to DTKR have been identified in the mosquito Anopheles, the stable fly Stomoxys calcitrans, and the worm U. unicinctus. The second type of TKRP receptor is represented by NKD in Drosophila and its ortholog in Anopheles. The DTKR type of tachykinin receptors display sequence similarities to mammalian tachykinin receptors, and the gene organization is similar in terms of location of intron-exon boarders [9]. Thus, it is likely that the invertebrate and mammalian receptors are ancestrally related. The Stomoxys receptor STKR has been extensively investigated in different cellular expression systems. It responds to its native ligand, StcTK, of Stomoxys but also to other insect TKRPs. In heterologous expression systems the STKR can couple both to calcium and cyclicAMP signaling systems. The Drosophila receptor DTKR when expressed in HEK-293 cells could be activated equally well with the six native DTKs (DTK-1 to -6), and it coupled both to calcium and cyclicAMP signaling. Upon prolonged exposure of DTKR to DTKs β-arrestin translocation is initiated and the receptor internalizes. Attempts to characterize NKD with Drosophila DTKs in calcium and cyclic AMP signaling assays were not successful, but β-arrestin translocation was initiated upon DTK activation and the receptor internalized. Originally NKD, when expressed in mammalian cells, could be activated by a heterologous TKRP—namely, LomTK-II from the locust—and was shown to couple to phospholipase c. Antisera to a portion of DTKR were used for immunocytochemical mapping of the receptor protein in the CNS and intestine of Drosophila. The distribution of DTKR immunoreactivity in the CNS matches that of the

Tachykinins and Tachykinin-Related Peptides in Invertebrates / 175 DTK peptide distribution. High density of DTKR is seen in the antennal lobes, central body, and pars intercerebralis. Peripheral sites of DTKR expression appear to be the intestine and Malpighian tubules, based on immunocytochemistry and western blots. Both the intestine and the tubules are known to respond to TKRPs in several insect species. The protein of the other TKRP receptor, NKD, has not yet been mapped in Drosophila, but antiserum to NKD was used for immunocytochemistry in a locust and a fleshfly. In situ hybridization, however, did reveal a CNS localization of NKD in the Drosophila embryo. In C. elegans eight GPCRs have been identified that are related to the vertebrate neurokinin receptors [1]. The ligands of these receptors have not yet been identified, but the behavioral role of one of the GPCRs (AC7.1) has been analyzed after RNA interference. The phenotype was described as “sick” or as a slight, statistically insignificant, slowing of locomotion [1].

STRUCTURE-ACTIVITY AND ACTIVE CONFORMATION The insect and U. unicinctus TKRPs have been investigated with respect to structure activity relations. At least the C-terminal hexapeptide is required for full biological activity of insect TKRPs in a few bioassays [3], whereas in cells transfected with STKR, a pentapeptide seems sufficient for activation. Several amino acid substitutions are possible in this C-terminus, but in principle FX1GX2Ramide is required. It has been shown, however, that a substitution of the G for an A in this sequence changes the bioactivity only slightly. Natural isoforms with an FX1AX2Ramide do exist in Stomoxys (Stc-TK) and Drosophila (DTK-6), as well as in a mollusk (Table 1) [9]. The Ramide is very critical for biological activity: Substitution of Ramide to Mamide renders the invertebrate TKRPs inactive on invertebrate receptors such as STKR and UTKR (U. unicinctus) [9]. Conversely, if the Mamide of a mammalian tachykinin is changed to a Ramide, the peptide can activate the invertebrate receptors. A comparison was made of the aqueous solution conformations of a conformationally restricted bioactive analog of an insect TKRP and an analogous substance P conformer [2]. NMR spectra and molecular dynamics calculations showed that the insect and mammalian peptides adopt similar active conformations.

BIOLOGICAL ACTIONS A range of biological actions of TKRPs has been established in insects, using in vitro bioassays: modulation of different types of muscle, weak diuretic activity, neuro-

modulatory activity, and induction of adipokinetic hormone release [3]. TKRPs have been shown to increase spontaneous muscle contractions in cockroach foregut, Drosophila midgut, cockroach hindgut, locust and cockroach oviduct, and heart (dorsal vessel) of beetles, and to modulate contractile properties of hindleg muscles in a locust. A neuromodulatory action was demonstrated on locust dorsal unpaired median neurons. These neurons respond to TKRPs by depolarization, possibly mediated by activation of adenylate cyclase. TKRPs are probably involved in olfactory processing, since insects and crayfishes express this type of peptide in olfactory interneurons. The TKRPs are colocalized with the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in olfactory interneurons (at least in cockroaches and Drosophila). Thus, the peptide may act as cotransmitter and/or neuromodulator in GABA signaling in antennal lobes. The TKRPs may also act as neuromodulators in several other circuits of the insect CNS (e.g., the central body and motor centers of thoracic ganglia). In crustaceans the neuromodulatory action of TKRPs has been studied more extensively. In the lamina of the crayfish visual system amacrine neurons contain colocalized TKRP and GABA and it was shown that application of these two substances to retinal photoreceptors produces dose-dependent hyperpolarization [4]. This action can follow two time courses: a short latency hyperpolarization in the order of seconds and another evolving over minutes. The TKRP action involves a reduction of the photoreceptor potential and the light-elicited current and a potentiation of the presynaptic inhibitory action of GABA. The GABA and TRP actions may subserve lateral inhibition or feedback inhibition of the primary visual synapse (over seconds) and possibly light adaptation of photoreceptors (over minutes). In another crustacean, the crab Cancer borealis, a TKRP has been identified in neurons of the stomatogastric ganglion, STG [6]. Different overlapping subsets of STG neurons form networks that generate rhythms controlling chewing and filtering behaviors of the foregut. An endogenous TKRP localized in one of the neurons innervating the STG was found to trigger a specific pyloric motor pattern. In this neuron the TRP is colocalized with proctolin and GABA, and the three substances act together to elicit the appropriate response.

CONCLUSION Tachykinins, invTKs, and TKRPs constitute a large family of multifunctional neuropeptides whose signaling mechanisms seem to be partially conserved through evolution [3, 4, 9]. Although the vertebrate tachykinins and invertebrate TKRPs and their precursors display only limited sequence identities, their G-protein-

176 / Chapter 26 coupled receptors (GPCRs) display more striking similarities, suggesting ancestral relationships [9]. The invTKs, which display strong similarities to vertebrate tachykinins, seem to have evolved separately in the salivary glands of some invertebrates that use saliva in their interactions with vertebrate prey organisms. Thus, these invTKs may have evolved by mimicry to cause vasodilation or other actions in the prey vertebrates. The gene coding for the C. elegans invTK appears to have diverged from the others early in evolution (or may be unrelated). In invertebrates the function of TKRPs is far from clear. Characteristically, the first invertebrate peptides of the TKRP family were isolated by a very general muscle contraction assay and no single major in vivo function has been determined. It is more likely that the TKRPs have multiple functions distributed in different parts of the CNS, intestine, and maybe other tissues.

References [1] Keating CD, Kriek N, Daniels M, Ashcroft NR, Hopper NA, Siney EJ, Holden-Dye L, Burke JF. Whole-genome analysis of 60 G protein-coupled receptors in Caenorhabditis elegans by gene knockout with RNAi. Curr Biol 2003;13:1715–1720.

[2] Nachman RJ, Moyna G, Williams HJ, Zabrocki J, Zadina JE, Coast GM, Vanden Broeck J. Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/ Tyr-W-MIF neuropeptide family pairs. Ann NY Acad Sci 1999; 897:388–400. [3] Nässel DR. Tachykinin-related peptides in invertebrates: a review. Peptides 1999;20:141–158. [4] Nässel DR. Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 2002;68:1–84. [5] Nathoo AN, Moeller RA, Westlund BA, Hart AC. Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc Natl Acad Sci USA 2001;98:14000– 14005. [6] Nusbaum MP, Blitz DM, Swensen AM, Wood D, Marder E. The roles of co-transmission in neural network modulation. Trends Neurosci 2001;24:146–154. [7] Pennefather JN, Lecci A, Candenas ML, Patak E, Pinto FM, Maggi CA. Tachykinins and tachykinin receptors: a growing family. Life Sci 2004;74:1445–1463. [8] Predel R, Neupert S, Roth S, Derst C, Nässel DR. Tachykininrelated peptide precursors in two cockroach species: molecular cloning and peptide expression in gut and brain neurons. FEBS 2005;272:3365–75. [9] Satake H, Kawada T, Nomoto, K, Minakata H. Insight into tachykinin-related peptides, their receptors and invertebrate tachykinins: a review. Zool Sci 2003;20:533–549. [10] Taghert PH, Veenstra JA. Drosophila neuropeptide signaling. Adv Genet 2003;49:1–65.

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27 Proctolin in Insects ANGELA B. LANGE AND IAN ORCHARD

It appears to have an identical amino acid sequence in all arthropods studied. There is one report, however, of the presence of proctolin along with two possible alternative sequences (AYLPT, XYLPT) in methanolic head extracts of Leptinotarsa decemlineata [16].

ABSTRACT The neuropeptide proctolin (RYLPT) was discovered in the cockroach Periplaneta americana, where it was considered a neurotransmitter associated with hindgut visceral muscle. Proctolin was the first insect neuropeptide to be sequenced and is now known to be widely distributed in insects and other arthropods and to be extremely active on a variety of arthropod visceral and skeletal muscle preparations. The gene for proctolin has been cloned in Drosophila, as too has the gene for its receptor. Structure–activity studies have identified the critical positions in the proctolin structure that enable binding to the receptor and biological activity, and agonists and antagonists have been designed. Many biological processes appear to be influenced by proctolin, which is involved in regulating muscles associated with posture, the digestive system, the reproductive system, and the circulatory system. Proctolin may also act as a releasing factor for hormones and/or act as a neurohormone.

STRUCTURE OF THE PRECURSOR mRNA/GENE Proctolin is a small peptide, and early searches of the Drosophila genome databases failed to identify the proctolin gene (see [19]). Recently, however, several expressed sequenced tags (ESTs) were recognized in the Drosophila melanogaster genome database that encode predicted genes containing the sequence RYLPT. One of these (CG7105) is likely to encode the Drosophila proctolin precursor [19]. The predicted protein sequence for this gene has potential peptide cleavage sites flanking the RYLPT sequence. The gene has three exons, and the pre-proprotein is 140 amino acids in length. This conceptual pre-proproctolin contains a secretion signal peptide (amino acids 1–38) followed by a single copy of the proctolin sequence, which is C-terminally flanked by a 94-amino-acid peptide. This protein structure is conserved in Drosophila pseudoobscura [5]. A search of the Anopheles gambiae and the Apis mellifora genome databases fails to reveal a gene with homology to the Drosophila proctolin gene (see [5]).

DISCOVERY The pentapeptide proctolin (RYLPT) was the first insect neuropeptide to be characterized and sequenced [17]. The active material, monitored by its ability to stimulate hindgut contractions in Periplaneta americana, was extracted from 125,000 cockroaches. This extraction and subsequent purification provided 180 μg of peptide for sequencing by Edman degradation. Proctolin is present in a number of insect orders, identified more recently by mass spectrometry, and is extensively distributed throughout the nervous systems of insects and other arthropods. Unlike many biologically active peptides, proctolin is nonamidated and is not considered to be a member of an extended family of peptides. Handbook of Biologically Active Peptides

DISTRIBUTION OF mRNA In insects, immunohistochemical studies have indicated the presence of proctolin-like immunoreactive neurons distributed throughout the nervous system, but unlike many other insect neuropeptides, no

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178 / Chapter 27 proctolin-like immunoreactive endocrine cells are associated with the midgut. Proctolin-like immunoreactivity is found in a variety of neuronal cell types, including interneurons, identified motoneurons projecting to visceral and skeletal muscles, and neurosecretory cells projecting to neurohemal areas [2, 11, 14]. With the identification of the putative Drosophila proctolin gene, the power of in situ hybridization has now been combined with immunohistochemistry to provide a complete map of proctolinergic neurons in D. melanogaster. In situ hybridization, using a probe against the Drosophila CG7105 transcript, results in staining of cell bodies in the brain and ventral nerve cords of third instar larvae of D. melanogaster [19]. The pattern of labeled cell bodies is very similar to that reported for proctolin-like immunoreactivity in D. melanogaster (see [13, 19]). Neither immunohistochemistry nor in situ hybridization reveals any endocrine-like cells in the midgut. With regard to the ventral nerve cord, in situ hybridization reveals both lateral and medial cell body clusters, with both clusters showing stronger signals in the abdominal neuromeres than in the thoracic neuromeres. In the brain, there are hybridization signals in two to three lateral cell bodies in the protocerebrum and a set of three cell bodies lying ventrally within the tritocerebrum near the esophageal foramen. Interestingly, proctolin-like immunoreactive staining reveals that the two to three lateral cells of the protocerebrum are neurosecretory cells with axons projecting to the corpora cardiaca (CC) portion of the ring gland. In the CC, there are numerous varicose terminals displaying proctolin-like immunoreactivity [19]. Immunohistochemistry with antisera generated against the proctolin precursor [19] reveals similar neurons to those labeled by in situ hybridization and with proctolin-like immunoreactivity.

PROCESSING The biochemistry of proctolin biosynthesis and processing from its precursor in Drosophila has recently been reviewed [5, 19]. A signal peptidase is most likely to cleave Gly38-Arg39 and Gly31-Arg32 peptide bonds of the preproproctolin in D. melanogaster and D. pseudoobscura, respectively. This exposes the N-terminal Arg of proctolin without the need for a proprotein convertase (PC)-like endoprotease. It has been proposed that a second cleavage occurs at Arg44-Ser45 by a PC, since two of the three D. melanogaster PCs (FUR1 and FUR2) recognize and cleave between Arg-Ser of prodynorphin. This cleavage in Drosophila generates an Arg-extended proctolin intermediate, which would need to be converted to proctolin in secretory granules by the action of a carboxypeptidase D.

In order for a neuroactive chemical to function effectively as an intercellular messenger, there must be some means of inactivating, removing, or otherwise terminating its action. Proctolin, injected into the hemolymph of P. americana, has a half-life of approximately 4 minutes (see [14]). By following the fate of radioactively labeled proctolin it was suggested that the initial inactivation step is cleavage of the Arg-Tyr bond followed by cleavage of the Tyr-Leu bond, indicating an aminopeptidase as the primary inactivation mechanism. A number of in vitro studies have further indicated peptidases as responsible for the metabolic inactivation of proctolin (see [5, 14]). By use of a variety of tissue homogenates from P. americana (hindgut, leg muscle, brain, and terminal abdominal ganglion), a soluble aminopeptidase was identified as a key proctolin inactivating enzyme. These tissues also contained another peptidase that removed the N-terminal dipeptide of proctolin by cleavage of the Tyr-Leu peptide bond. The Tyr-Leu cleavage site appears to be more important in tissues that are richer in proctolin, leading to the suggestion of the existence of a proctolinase. Hemolymph also contains the soluble aminopeptidase activity, but the second peptidase seems mainly responsible for proctolin degradation in cockroach hemolymph in vitro at neutral pH. Similar peptidases are found in homogenates of brain and thoracic ganglia of Schistocerca gregaria (see [5]). The enzyme responsible for cleaving the N-terminal dipeptide is mainly cytosolic, whereas the aminopeptidase is enriched in locust synaptic membranes and might therefore be considered a neuronal proctolinase. This proctolinase has a Km of 23 μM and is inhibited by amastatin. A similar amastatin-sensitive aminopeptidase is also considered to be the major proctolin degrading enzyme associated with locust oviduct and hindgut membranes. The enzyme responsible for removing the N-terminal dipeptide of proctolin has activity resembling that of mammalian dipeptidylaminopeptidase III (EC 3.4.14.4, DPP-III), which is an enzyme associated with the metabolism of enkephalin and angiotensin. Two proteins with relative molecular masses of 80K and 76K have recently been isolated from foregut membranes of the cockroach, Blaberus craniifer, and identified as insect orthologs of mammalian DPP-III (see [12]). The purified cockroach enzyme hydrolyzes proctolin with a Km of about 4 μM. The cockroach DPP-III is a metalloenzyme, inhibited by EDTA and by tynorphin, a peptide inhibitor of mammalian DPP-III. Subsequent analysis of the D. melanogaster DPP-III gene (CG7415) and cDNA sequences predicts the expression of two insect variants (89,194 Da and 81,937 Da) resulting from different translational start sites [12]. When the smaller cDNA is expressed in S2 cells, there is the generation of the 82 kDa DPP-III but also the generation of an 86 kDa protein, suggesting that some DPP-III

Proctolin in Insects / 179 undergoes posttranslational modification that influences protein mobility in SDS-PAGE. The majority of the recombinant DPP-III is located in the soluble fraction of transfected S2 cells, but a significant amount is also detected in washed membranes. A search of the genome databases of D. pseudoobscura and A. gambiae reveals DPP-III proteins that, like D. melanogaster, have similar amino acid sequences to the mammalian DPPIII with complete conservation of key catalytic and zinccoordinating residues (see [5]).

tochemistry reveals the receptor is present in several neuropil regions and is associated with neurosecretory cell bodies in the adult and larval brain, but there is little evidence of proctolin receptor expression in thoracic and abdominal ganglia. Strong immunostaining is obtained in muscle layers of the Drosophila adult and larval hindgut, the aorta, and on neuronal endings in adult heart (see [5]). Screening of the A. gambiae genome database fails to reveal any potential A. gambiae proctolin receptor [3, 5].

RECEPTORS Early attempts at isolating proctolin receptors (see [5, 9, 12]) made use of tritiated proctolin to bind to crude membrane preparations from locust hindgut and oviduct (two tissues highly responsive to proctolin). Proctolin binding was found to be time-dependent, proportional to the concentration of membrane protein, saturable, specific, and reversible. Further work isolated proctolin-binding fractions from hindgut and from oviduct tissues using isoelectric focussing, anion exchange, HPLC, and size exclusion HPLC. The specific binding was detected in fractions with an elution volume equivalent to a molecular weight of 50,000. More recently, the orphan G-protein coupled receptor (GPCR) CG6986 from the Drosophila genome database has been identified as a putative proctolin receptor [3, 7] and verified by use of G-protein coupled receptorsβ-arrestin2 interactions [6]. This Drosophila proctolin receptor is a typical GPCR with seven transmembrane regions. The unmodified protein has a mass of 62 kDa and shows only weak identity with other known GPCRs. The D. pseudoobscura genome possesses a highly homologous gene. The cloned DNA of the D. melanogaster proctolin receptor gene has been expressed in mammalian cells, and screening with neuropeptides reveals that the expressed receptor is specific for proctolin with an EC50 of 0.3 nM or 0.6 nM [3, 7]. Membrane preparations of mammalian cells transfected with the proctolin receptor have been used in competition studies. The putative proctolin receptor binds proctolin with high affinity (IC50, 4 nM) [7]. Microarray analyses reveal the receptor transcript in head mRNA of different genotypes and under different environmental conditions. Northern blots indicate that the proctolin receptor is only weakly expressed in embryos, larvae, pupae, and in the thorax and abdomen of adult flies but is strongly expressed in the heads of adult flies [3]. Immunohistochemistry using antibodies against the receptor reveals immunoreactive signals in the hindgut, heart, and in distinct neuronal populations within the CNS, and Western blots reveal a band in tissue homogenates similar to the predicted size of the protein. Immunohis-

STRUCTURE—ACTIVITY AND ACTIVE CONFORMATION Numerous analogs have been synthesized in an attempt to gain insight into the structure–activity requirements of proctolin and its receptor. These experiments have focussed on the contractions of foregut, hindgut, oviduct, and cardiac muscle in a variety of insect species. Radioactively labeled proctolin has also been employed to study the binding characteristics of receptors in membranes prepared from target tissues. These results have been well reviewed by a variety of authors (see [9, 15]). In early studies (see [18]), it was found that removal of the C-terminal Thr, or its replacement with Ser, resulted in a loss of 99% of the peptide’s activity. Replacement of any of the amino acids with its D isomer or with Ala illustrated that each of them are necessary for full activity with activities for the analogs ranging from 5 to 16% of the activity of equimolar proctolin. A number of di-, tri-, and tetrapeptide fragments were also inactive. One analog with significant activity, three times as potent as proctolin, was [Phe(p-OMe)2]-proctolin. Other super analogs have also been made by modifying the second residue, and indeed, some antagonists have also been generated (see [8, 9]). These latter observations, combined with the observation that Phe2-proctolin has only 15% of proctolin’s potency, leads to the conclusion that the aromatic ring in the second position is important in receptor binding. Other studies have also concluded the importance of the aromatic ring in the second position and further evidence for this has been derived from a theoretical analysis [1]. Thus, the total energy for the five most probable conformations of proctolin indicates the preferred conformation is a quasi-cyclic structure with the tyrosyl side chain pointing outwards. This would place it in an ideal position to interact with active sites on the receptor. The majority of the published results concerns the exchange of one or more of the five amino acids of proctolin with other natural or nonnatural α-amino acids. In addition, some analogs have been generated

180 / Chapter 27 that modify the peptide backbone. As summarized in [9], of 100 synthetic proctolin analogs produced, 45 have myotropic activity on various insect visceral muscles. Of these, 40 are agonists and 5 are antagonists, with [α-MeTyr2]-proctolin being the most potent antagonist. The agonistic property is dependent on the para substitution at position 2 and on the presence of the peptide bond hydrogen atom between Arg1 and Tyr2and between Tyr2 and Leu3. Additional substitution at the meta position of the Tyr phenyl ring facilitates interaction with the receptor. The Arg1, Leu3, and Pro4 residues are essential for myotropic activity. These classical structure–activity studies have certainly provided insight into the possible interaction between the proctolin molecule and its receptor, and the importance of the individual amino acid residues in these interactions. They have also been important for identifying super analogs and antagonists of the proctolin receptor for applied purposes. The cloning of the proctolin receptor in Drosophila will now allow a much greater insight into these interactions and allow the power of molecular biology, including mutagenesis, to be brought to bear on this important area of research.

BIOLOGICAL ACTIONS Proctolin was originally isolated from P. americana based on its myostimulatory action on the hindgut and subsequently shown to be a potent stimulator of contraction of a variety of insect visceral and skeletal muscle (see [14]). Much of the research on proctolin has focused on its effects on muscle contraction, prompted in part because of proctolin’s presence within identified motoneurons projecting to various muscles. However, proctolin’s relevance extends to other physiological and behavioral processes. In addition to the cockroach hindgut, proctolin stimulates contractions of foregut in S. gregaria and midgut of Diploptera punctata and L. migratoria, and therefore might be regarded as a major neuromuscular modulator in the gut of insects (see [9, 15]). Proctolin is also a potent modulator of reproductive tissue, stimulating contractions of the oviducts in P. americana, Leucophaea maderae, L. migratoria, and spermathecae in L. migratoria and Rhodnius prolixus. In addition, proctolin influences egg-laying activity of A. mellifora queens and promotes the onset of vitellogenesis in B. craniifer (see [5, 9]). Proctolin also appears to be a cardioacceleratory peptide, increasing the rate and amplitude of contractions of heart and antennal heart of several insects, although interestingly the hearts of Manduca sexta and Stomoxys calcitrans are not responsive to proctolin. In addition, proctolin enhances contractions of skeletal

muscle, such as the extensor tibiae in L. migratoria, the coxal depressor in P. americana, the ventral protractor muscle, and the ovipositor opener muscle of L. migratoria. In vivo application of antiproctolin antiserum in Gryllus bimaculatus indicates that proctolin, which is present in antennal motoneurons, stabilizes the prolonged antennal forward position during flight [4]. Proctolin appears to be necessary to induce a large enough muscle tension to hold the antenna in a forward position during flight. This matches the observation that proctolin is found in slow motoneurons that control skeletal muscle involved in maintaining posture (see [13]). Within the skeletal and visceral muscle preparations just described, it is accepted that proctolin is a cotransmitter that acts alongside a more classical neurotransmitter, such as glutamate. Immunohistochemistry and in situ hybridization indicates that proctolin is present within interneurons within the CNS. Dorsal unpaired median neurons can be activated by proctolin and proctolin influences ovipositional digging behaviors when applied to abdominal ganglion VIII, and it alters the rhythmic bursting of centrally generated action potentials recorded from the oviductal nerve in L. migratoria (see [10, 11, 13]). However, the functional roles of proctolin centrally are not well understood in insects (see [13]), although more is known about proctolin’s central actions in crustacea. For example, proctolin activates the rhythm generating networks in the crayfish swimmeret system and the crab stomatogastric and cardiac ganglion. Proctolin is often colocalized along with other neuroactive chemicals that act in different combinations as neuromodulators in these crustacean preparations (see [13]). As mentioned earlier, immunoreactivity and in situ hybridization for proctolin reveals its presence in insect neurosecretory cells. This, coupled to the detection of proctolin in the hemolymph, is consistent with proctolin acting as a neurohormone (see [2, 5, 11]). A number of tissues that are not innervated (e.g., Malpighian tubules and upper lateral oviducts of L. migratoria, and heart of P. americana and Tenebrio molitor) are sensitive to proctolin, so proctolin may be acting upon these tissues in its capacity as a neurohormone. This concept has been criticized, however, because of the short half-life of proctolin when injected into the hemolymph. It has been pointed out that the concentration of any active peptide in the vicinity of a receptor is determined by a number of factors, including the rate of release, hemolymph flow, and distance from the release site to the receptor, and is therefore not only a function of the rate of metabolism [5]. These points, along with the fact that responsive tissues are activated very quickly by proctolin in low concentrations, suggest that a half-life of four minutes does not

Proctolin in Insects / 181 argue against proctolin as a neurohormone. The role of proctolin in lateral neurosecretory cells projecting to the CC/CA in L. migratoria and to the CC in D. melanogaster has recently been investigated. In L. migratoria, proctolin may well be a neurohormone (see [2]) but may also be a releasing factor for other hormones associated with the CC/CA. Thus, proctolin stimulates the release of adipokinetic hormones from the glandular lobe of the CC and also stimulates the production and release of juvenile hormone from the CA in L. migratoria [2]. In D. melanogaster, proctolin is colocalized with corazonin in the lateral neurosecretory cells in the brain. Corazonin has different actions in different insect species and seems to be involved in the control of heart, morph-specific color changes, silk gland activity, larval ecdysis behavior, and the circadian clock system. Ectopic expression of the proctolin gene in the CNS and midgut of D. melanogaster has been used to overexpress proctolin in pupae. Within these pupae, there is a 14% increase in heart rate, suggesting a cardioacceleratory endocrine function for proctolin [19]. Thus, there can be no doubt as to the importance of proctolin as a neuroactive chemical in insects, where it functions in quite diverse roles, roles that far exceed the original discovery of it being a cockroach hindgut neurotransmitter.

References [1] Betins JR, Nikiforovich GV. 1979. Theoretical conformational analysis of proctolin molecule. Bioorganicheskaya Khimya 5:1581–1583. [2] Clark L, Zhang JR, Tobe S, Lange AB. 2006. Proctolin: a possible releasing factor in the corpus cardiacum/corpus allatum of the locust. Peptides vol. 27 issue 3, 559–566. [3] Egerod K, Reynisson E, Hauser F, Williamson M, Cazzamali G, Grimmelikhuijzen CJP. 2003. Molecular identification of the first insect proctolin receptor. Biochem Biophys Res Comm 306:437–442. [4] Gebhardt M. 2004. In vivo application of anti-proctolinantiserum affects antennal flight posture in crickets. J Comp Physiol A 190:359–364.

[5] Isaac RE, Taylor CA, Hamasaka Y, Nässel DR, Shirras AD. 2004. Proctolin in the post-genomic era: new insights and challenges. Invert Neurosci 5:51–64. [6] Johnson EC, Bohn LM, Barak LS, Birse RT, Nässel DR, Caron MG, Taghert PH. 2003. Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-β-Arrestin2 interactions. J Biol Chem 278:52172–52178. [7] Johnson EC, Garczynski SF, Park D, Crim JW, Nässel DR, Taghert PH. 2003. Identification and characterization of a G proteincoupled receptor for the neuropeptide proctolin in Drosophila melanogaster. PNAS 100:6198–6203. [8] Konopin´ska D. 1997. Insect neuropeptide proctolin and its analogues: an overview of the present literature. J Peptide Res 49:457–466. [9] Konopin´ska D, Rosin´ski G. 1999. Proctolin, an insect neuropeptide. J Peptide Sci 5:533–546. [10] Kwok R, Orchard I. 2002. Central effects of the peptides, SchistoFLRFamide and proctolin, on locust oviduct contraction. Peptides 23:1925–1932. [11] Lange AB. 2002. A review of the involvement of proctolin as a cotransmitter and local neurohormone in the oviduct of the locust, Locusta migratoria. Peptides 23:2063–2070. [12] Mazzocco C, Fukasawa KM, Auguste P, Puiroux J. 2003. Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster. Eur J Biochem 270: 3074–3082. [13] Nässel DR. 2002. Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 68:1–84. [14] Orchard I, Belanger JH, Lange AB. 1989. Proctolin: a review with emphasis on insects. J Neurobiol 20:470–496. [15] Osborne RH. 1996. Insect neurotransmission: neurotransmitters and their receptors. Pharmacol Ther 69:117–142. [16] Spittaeles K, Vankeerberghen A, Torrekens S, Devreese B, Grauwels L, Van Leuven F, Hunt D, Shabanowitz J, Schoofs L, Van Beeumene J, De Loof A. 1995. Isolation of Ala1-proctolin, the first natural analogue of proctolin, from the brain of the Colorado potato beetle. Mol Cell Endocrinol 110:119–124. [17] Starratt AN, Brown BE. 1975. Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects. Life Sci 17:1253–1256. [18] Starratt AN, Brown BE. 1979. Analogs of the insect myotropic peptide proctolin: synthesis and structure-activity studies. Biochem Biophys Res Comm 90:1125–1130. [19] Taylor CAM, Winther AME, Siviter RJ, Shirras AD, Isaac RE, Nässel DR. 2004. Identification of a proctolin preprohormone gene (Proct) of Drosophila melanogaster: expression and predicted prohormone processing. J Neurobiol 58:379–391.

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28 Sulfakinins LILIANE SCHOOFS AND RONALD J. NACHMAN

fakinin was isolated from an extract containing 9000 central nervous systems (CNSs) of the locust Locusta migratoria [24, 25]. Initially only one sulfakinin was biochemically isolated and fully sequenced from dipteran species—that is, Calliphora vomitoria (12,000 heads) and Lucilia cuprina [5]. A second longer sulfakinin could be partially identified in the flesh fly Neobellieria bullata (42,000 heads) [7]. Peptidomic experiments have reduced the time required to isolate and sequence peptides from years to days, when sufficient (genomic) sequence information is available. For instance, mass spectrometric techniques have revealed the complete structure of (dro)sulfakinin I and II starting with only 50 larval Drosophila brains [2, 3], as well as the locustasulfakinin sequence from a single locust corpus cardiacum [1, 4]. Extracts of neurohaemal release sites, the corpora cardiaca, were the source for the isolation of various posttranslationally modified sulfakinins from Periplaneta americana by conventional HPLC methods in conjunction with either a hindgut [29] or in situ heart [23] bioassay. Sulfakinins have also been identified in crustaceans [9, 28]. Interestingly, one of the isolated forms in Litopenaus vannamei is disulfated [28]. Such a disulfotyrosyl sequence is also found in cionin, a CCK-gastrin-like neuropeptide found in the protochordate Ciona intestinalis.

ABSTRACT Sulfakinins constitute a family of arthropod neuropeptides that typically contain the C-terminal hexapeptide Y(SO3H)GHMRFamide, the active core sequence. They display structural and functional similarities to the vertebrate gastrin-cholecystokinin family of peptides. Sulfakinins are synthesized by a limited number of neurosecretory cells and exhibit several effects, including myotropic activities, inhibition of food intake, and stimulation of digestive enzyme release. The sulfate group is a requirement for biological activity. The gastrin/CCK and sulfakinin signaling system is a good example of the coevolution of neuropeptides and their receptors.

DISCOVERY Sulfakinins are arthropod neuropeptides that typically contain a sulfated tyrosine residue—hence their name. They are characterized by the C-terminal hexapeptide Y(SO3H)GHMRFamide and display structural and physiological similarities to gastrin and cholecystokinin (CCK), their sulfated neuropeptide counterparts in vertebrates [19, 20]. The initial evidence indicating that gastrin/CCK-like peptides are present in invertebrates came from immunocytochemical studies using gastrin and CCK antibodies on the insect neuroendocrine system [10] and on various tissue extracts of 26 other species of invertebrates, including the phyla of Mollusca, Annelida, Arthropoda, and Ectoprocta [12]. The first sulfakinins were isolated from insect sources by successive chromatographic processing of extracts of 3000 heads of the cockroach Leucophaea maderae [19, 20]. The cockroach hindgut assay was used to monitor the isolation process. With the same assay, a locustasulHandbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE Thus far, genes encoding sulfakinins have been identified in Drosophila melanogaster and C. vomitoria [5, 22]. The first sulfakinin gene to be reported was identified by screening a D. melanogaster genomic library with oligonucleotides designed for leukosulfakinins. The

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184 / Chapter 28 TABLE 1. Sequence comparison of arthropod sulfakinins and some gastrin/CCK-related peptides. Amino acids that are conserved throughout the sulfakinin family are in boldface. pQ indicates a pyroglutamic acid residue. X indicates an undetermined amino acid. Subphylum (Phylum) Crustacea (Arthropoda)

Species

Name

Sequence

Litopenaeus vannamei

Pev-SK 1 Pev-SK 2 Pem SKI Pem SKII PemSKIII CavSKI CavSKII LucSKI LucSKII DrmSKI DrmSKII NebSKI NebSKII LemSKI LemSKII PeaSKI PeaSKII LomSK Cionin Gastrin II CCK-8

AGGSGGVGGEY(SO3H)DDY(SO3H)GH(L/I)RF-NH2 pQFDEY(SO3H)GHMRF-NH2 pQFDEY(SO3H)GHMRF-NH2 AGGSGGVGGEYDDY(SO3H)GHLRF-NH2 VGGEYDDY(SO3H)GHLRF-NH2 FDDY(SO3H)GHMRF-NH2 GGEEQFDDY(SO3H)GHMRF-NH2 FDDY(SO3H)GHMRF-NH2 GGEEQFDDY(SO3H)GHMRF-NH2 FDDY(SO3H)GHMRF-NH2 GGDDQFDDY(SO3H)GHMRF-NH2 FDDY(SO3H)GHMRF-NH2 XXEEQFDDY(SO3H)GHMRF-NH2 EQFEDY(SO3H)CHMRF-NH2 pQSDDY(SO3H)GHMRF-NH2 EQFDDY(SO3H)GHMRF-NH2 pQSDDY(SO3H)GHMRF-NH2 pQLASDDY(SO3H)GHMRF-NH2 NY(SO3H)Y(SO3H)GWMDF-NH2 pQGPWLEEEEEAY(SO3H)GWMDF-NH2 DY(SO3H)MGWMDF-NH2

Penaeus monodon

Hexapoda (Arthropoda)

Calliphora vomitoria Lucilia cuprina Drosophila melanogaster Neobellieria bullata Leucophaea maderae Periplaneta americana

Urochordata Craniata (Chordata)

Locusta migratoria Ciona intestinalis Homo sapiens Ovis aries

128-amino-acid translation product contains an N-terminal signal peptide and three neuropeptide sequences, flanked by conventional dibasic cleavage sites, including two sulfakinin sequences termed Drm-SK I and II. Another putative peptide that is unrelated to the sulfakinins (NQKTMSFamide, termed Dsk-0), is also encoded. The only significant region of sequence similarity between the sulfakinin precursors of both flies is that encoding the sulfakinin peptides.

TISSUE DISTRIBUTION mRNA Level In situ hybridization studies with a digoxigeninlabeled sulfakinin gene probe (from the blowfly L. cuprina) revealed only four pairs of neurons in the brain of C. vomitoria [5].

Protein Level PROCESSING After cleavage from their inactive propeptide sulfakinin precursor (most likely by subtilisin-like proprotein convertases), sulfakinins undergo further posttranslational modification [23]. The tyrosine residue can be sulfated. Both nonsulfated and sulfated forms have been biochemically isolated, although it is not clear whether this is due to loss of the unstable sulfate group during isolation procedures. The Nterminal glutamate can be modified into a pyroglutamate residue. N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in cockroach neurohemal release sites. Third, the presence of a naturally occurring perisulfkinin with O -methylated glutamic acid has also been demonstrated. Finally, all sulfakinins are amidated at their C-terminus.

Immunocytochemical studies on the holometabolous blowfly C. vomitoria with antisera against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster also revealed only four pairs of sulfakinin neurones in the entire nervous system [5]. Despite the small number of sulfakinin-immunoreactive cells in the CNS, extensive arborization of dendrites exhibiting sulfakinin-like immunoreactivity occurs within the neuropile of the brain, thoracic, and abdominal ganglia of all investigated species, suggesting a neurotransmitter neuromodulator function for the sulfakinins. Such a neurotransmitter function for sulfakinins appears to be a general characteristic across insect orders. In hemimetabolous insects such as locusts and cockroaches immunocytochemical studies suggest an additional hormonal role for sulfakinins [6, 23]. In the

Sulfakinins / 185 cockroach, sulfakinin-like material is produced in 10 pairs of anterior cells in the pars intercerebralis, as well as two pairs of medial and one major pair of lateral posterior brain cells. In addition, sulfakinin-like immunoreactivity is present along axons to the foregut tissue, and a plexus of retrocerebral nerves is likely to serve as a neurohemal release site. It is also postulated that neurohemal release occurs from the corpora cardiaca, which contains sulfakinins, into the dorsal aorta. The hormonal role of sulfakinins in dipterans is questioned because no evidence for a release site has been found [5]. Using specific Drm-DSK antisera characterized by affinity chromatography, Nichols et al. examined the temporal and spatial sulfakinin distribution of sulfakinins in Drosophila and found that they are expressed in the CNS of embryos, larvae, pupae, and adults [21]. In larvae, sulfakinin immunoreactivity is not only found in the protocerebral brain but also in thoracic ganglia and the most abdominal ganglion (Figs. 1 and 2). In pupae and adults, additional neurons are stained.

RECEPTORS Shortly after the publication of the Drosophila genome sequence, Hewes and Taghert suggested that the Drosophila G-protein coupled receptors (GPCRs) CG6857 and CG6881 probably diverged from a common ancestor of the CCK and gastrin family of receptors. The same authors speculated that the gastrin/CCK-like peptide homologs in Drosophila, the sulfakinins, may interact with either or both Drosophila GPCRs [14]. This was later confirmed by the cloning and functional expression of one of these GPCRs [11]. When expressed in mammalian cellular expression systems, this receptor (DSK-R1) was activated by a sulfated Met7-Leu7 substituted analog of drosulfakinin I. The Met-Leu substitution was carried out to improve chemical stability and did not influence functional activity. Unsulfated sulfakinin was 3000 times less potent than the sulfated form, indicative of the importance of the sulfate moiety for receptor activation. The observed calcium signaling was PTX insensitive in HEK-293 cells, expressing the sulfakinin receptor, indicating that the receptor couples exclusively to Gq and or G11 proteins. This is in agreement with mammalian CCK receptors where CCK B activates PLC, triggering an increase in IP3 production. Human CCK-8 and gastrin II were unable to activate the sulfakinin receptor, which is consistent with structure-activity studies performed on tissue preparations [17]. The Anopheles genome encodes a putative ortholog of the DSK receptor, which is situated on the genomic scaffold AAAB01008811 of the Anopheles genome project [14].

FIGURE 1. DSK I/II immunoreactivity in larval neural tissue at 96 h. Immunoreactive material is expressed in SP1, SP2, SP3, LP1, MP1, SE2, Tv1–3, T2dm, and A8 cells. An immunoreactive fiber projects away from the central nervous system in an abdominal ganglion (filled arrow). Bar: 50 μm. From R. Nichols and I.A. Lim, Spatial and temporal immunocytochemical analysis of drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system. Cell Tissue Res (1996) 283: 107–116, with kind permission of Springer Science and Business Media.

STRUCTURE/CONFORMATIONACTIVITY RELATIONSHIPS In a cockroach hindgut myotropic bioassay, the minimum sequence (active core) capable of eliciting a response is reported to be the C-terminal hexapeptide sequence. Within the active core region (Tyr(SO3H)Gly-His Met-Arg-Phe-NH2), the His (or alternate aromatic residue), Arg (or alternate basic residue), and Phe residues are critical for myotropic activity. Indeed, replacement of the Asp residue with Arg in both gastrin and CCK can confer activity in the insect bioassay to these otherwise inactive mammalian hormones [16, 17]. The sulfate residue is a strict requirement for myotropic activity, although its position within the sequence is not critical. A shift in the location (“gastrin” position)

186 / Chapter 28 group in sulfakinins by stable, acidic α-aminosuberic acid, which lacks a phenyl ring, is accompanied by retention of cockroach myotropic and locust food intake-inhibition activity [18]. Replacement of the basic Arg in the sulfakinins and acidic Asp in the analogous position of CCK with neutral Pro is accompanied by significant retention of bioactivity [17], leading to an argument that these residues do not directly interact with the receptor but may stabilize the active conformation. This is consistent with an aqueous solution conformation study of a sulfakinin, which revealed a relatively rigid α-helical loop structure stabilized by alternating salt bridges between the Arg and the two acidic residues in the N-terminal region and between His and the Tyr(SO3H) (Nachman RJ, and Williams H, unpublished data).

BIOLOGICAL ACTIONS

FIGURE 2. DSK I/II immunoreactivity in larval protocerebrum and subesophageal ganglion at 96 h. Immunoreactive material is expressed in SP1, SP2, SP3, LP1, MP1, Sv, and SE2 cells. Immunoreactive fibers project from the MP1 cells—one extends through the subesophageal ganglion into the ventral ganglion, while another crosses the midline, turns and extends through the subesophageal ganglion into the ventral ganglion. Bar : 50 μm. From R. Nichols and I.A. Lim, Spatial and temporal immunocytochemical analysis of drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system. Cell Tissue Res (1996) 283: 107–116, with kind permission of Springer Science and Business Media.

Sulfakinins affect several biological processes. They increase the frequency and amplitude of hindgut contractions [19, 20, 23, 25] and increase the frequency of heartbeat in the cockroach [29]. Sulfakinins also reduce food intake in locusts [24, 29] and in cockroaches [13]. In addition, they have been shown to stimulate the release of the digestive enzyme amylase from the insect digestive tract [8, 15]. Apparently, the sulfakinins not only display structural but also physiological similarities to the vertebrate peptides gastrin and CCK. The gastrin/CCK and sulfakinin signaling system represents a good example of coevolution of neuropeptides and their receptors. So far, sulfakinin peptides have only been isolated from arthropods, but gastrin-CCK-related peptides have been detected by immunological methods in annelids [27], nematodes [26], and snails [4]. In addition, putative sulfakinin receptors occur in the C. elegans genome [11, 14].

References of the Tyr(SO3H) in sulfakinins by one position toward the N-terminus (“CCK” position) leads to retention of nearly 40% of myotropic activity. A shift of the sulfate group by two to five positions toward the N-terminus or one position toward the C-terminus leads to reduced, but significant, activity [18]. These results indicate that the sulfakinin myotropic receptor site demonstrates a degree of flexibility with respect to interaction with the sulfate group and suggests that movement of this group could occur during the molecular evolutionary process. The sulfate group is also a requirement for food intake inhibition activity in locusts and cockroaches [13, 18, 30]. Replacement of the acidic, unstable Tyr(SO3H)

[1] Baggerman G, Clynen E, Huybrechts J, Verleyen P, Clerens S, De Loof A, Schoofs L. Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry. Peptides 2003;24:1475–85. [2] Baggerman G, Boonen K, Verleyen P, De Loof A, Schoofs L. Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry. J Mass Spectrom 2005;40:250–60. [3] Baggerman G, Cerstiaens A, De Loof A, Schoofs L. Peptidomics of the larval Drosophila melanogaster central nervous system. J Biol Chem 2002;277:40368–74. [4] Clynen E, Baggerman G, Veelaert D, Cerstiaens A, Van der Horst D, Harthoorn L, Derua R, Waelkens E, De Loof A, Schoofs L. Peptidomics of the pars intercerebralis-corpus cardiacum

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[6]

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[8]

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[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

complex of the migratory locust, Locusta migratoria. Euro J Biochem 2001;268:1929–39. Duve H, Thorpe A, Scott AG, Johnsen AH, Rehfeld JF, Hines E, East PD. The sulfakinins of the blowfly Calliphora vomitoria— Peptide isolation, gene cloning and expression studies. Euro J Biochem 1995;232:633–40. East PD, Hales DF, Cooper PD. Distribution of sulfakinin-like peptides in the central and sympathetic nervous system of the American cockroach, Periplaneta americana (L) and the field cricket, Teleogryllus commodus (Walker). Tissue & Cell 1997;29: 347–54. Fonagy A, Schoofs A, Proost P, Van Damme J, De Loof A. Isolation and primary structure of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata. Comp Biochem Physiol CPharmacol Toxicol & Endocrin 1992;103:135–42. Harshini S, Nachman RJ, Sreekumar S. In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.). Peptides 2002;23:1759–63. Johnsen AH, Duve H, Davey M, Hall M, Thorpe A. Sulfakinin neuropeptides in a crustacean—Isolation, identification and tissue localization in the tiger prawn Penaeus monodon. Euro J Biochem 2000;267:1153–60. Kramer KJ, Speirs RD, Childs CN. Immunochemical evidence for a gastrin-like peptide in insect neuroendocrine system. Gen Comp Endocrin 1977;32:423–26. Kubiak TM, Larsen MJ, Burton KJ, Bannow CA, Maritn RA, Zantello MR, et al. Cloning and functional expression of the first Drosophila melanogaster sulfakinin receptor DSK-R1. Biochem Biophys Res Commun 2002;291:313–20. Larson BA, Vigna SR. Species and tissue distribution of cholecystokinin gastrin-like substances in some invertebrates. Gen Comp Endocrin 1983;50:469–75. Maestro JL, Aguilar R, Pascual N, Valero ML, Piulachs MD, Andreu D, Navarro I, Belles X. Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach—Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins? Euro J Biochem 2001;268:5824–30. Meeusen T, Mertens I, De Loof A, Schoofs L. G protein-coupled receptors in invertebrates: A state of the art. Internat Rev Cytol—Survey Cell Biol 2003;230:189+. Nachman RJ, Giard W, Favrel P, Suresh T, Sreekumar S, Holman GM. Insect myosuppressins and sulfakinins stimulate release of the digestive enzyme α-amylase in two invertebrates: the scallop Pecten maximus and insect Rhynchophorus ferrugineus. Ann NY Acad Sci 1997;814:335–8. Nachman RJ, Holman GM, Haddon WF, Vensel WH. Effect of sulfate position on myotropic activity of the gastrin/CCKlike insect leucosulfakinins. Internat J Peptide Prot Res 1989; 33:223–9. Nachman RJ, Holman GM, Haddon WF. Structural aspects of the gastrin-cholecystokinin-like insect sulfakinins. Peptide Res 1989:2:171–7.

[18] Nachman RJ, Vercammen T, Williams H, Kaczmarek K, Zabrocki J, Schoofs L. Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects. Peptides 2005; 26:115–20. [19] Nachman RJ, Holman GM, Haddon WF, Ling N. Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin. Science 1986;234:71–3. [20] Nachman RJ, Holman GM, Cook BJ, Haddon WF, Ling N. Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin. Biochem Biophys Res Commun 1986;140:357–64. [21] Nichols R, Lim IA. Spatial and temporal immunocytochemical analysis of drosulfakinin (DSK) gene products in the Drosophila melanogaster central nervous system. Cell Tissue Res 1996;283: 107–16. [22] Nichols R, Schneuwly SA, Dixon JE. Identification and characterization of a Drosophila homolog to the vertebrate neuropeptide cholecystokinin. J Biol Chem 1988;263:12167– 70. [23] Predel R, Brandt W, Kellner R, Rapus J, Nachman RJ, Gade G. Post-translational modifications of the insect sulfakinins—Sulfation, pyroglutamate-formation and O-methylation of glutamic acid. Euro J Biochem 1999;263:552–60. [24] Schoofs L, Clynen E, Cerstiaens A, Baggerman G, Wei Z, Vercammen T, Nachman R, De Loof A, Tanaka S. Newly discovered functions for some myotropic neuropeptides in locust. Peptides 2001;22:219–27. [25] Schoofs L, Holman GM, Hayes TK, De Loof A. Isolation and identification of a sulfakinin-like peptide with sequence homology to vertebrate gastrin and cholecystokinin, from the brain of Locusta migratoria. In: McCaffery A, Wilson I, editors, Chromatography and isolation of insect hormones and pheromones, 1990;231–41. New York, Plenum Press. [26] Sithigorngul P, Cowden C, Stretton AOW. Heterogeneity of cholecystokinin/gastrin-like immunoreactivity in the nervous system of the nematode Ascaris suum. J Comp Neurol 1996; 370:427–42. [27] Smiri Y, Bulet P, Andries JC. Molecular heterogeneity of gastrin cholecystokinin-like immunoreactive peptides in Nereis diversicolor (Annelida, Polychaeta). Comp Biochem Physiol CPharmacol Toxicol Endocrin 1992;101:71–3. [28] Torfs P, Baggerman G, Meeusen Nieto TJ, Nachman RJ, Calderon J, De Loof A, Schoofs L. Isolation, identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropod, the white shrimp Litopenaeus vannamei. Biochem Biophys Res Commun 2002;299:312–20. [29] Veenstra JA. Isolation and structure of two gastrin/CCK-like neuropeptides from the American cockroach homologous to the leucosulfakinins. Neuropeptides 1989;14:145–49. [30] Wei Z, Baggerman G, Nachman RJ, Goldsworthy G, Verhaert P, De Loof A, Schoofs L. Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria. J Insect Physiol 2000;46: 1259–65.

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29 The Invertebrate AKH/RPCH Family GERD GÄDE AND HEATHER G. MARCO

In insects, extracts of the corpus cardiacum (CC) from either the American cockroach or the migratory (Locusta migratoria) and desert locust were shown, during the 1960s, to have metabolic effects: elevation of the blood sugar trehalose (hypertrehalosemic effect) or of the blood lipids (adipokinetic or hyperlipemic effect). In 1976, the complete primary sequence of one of the locust’s adipokinetic neuropeptides from the CC, today denoted as Locmi-AKH-I (Table 1), was obtained by using enzymatic cleavage and mass spectrometry following purification by size-exclusion gel chromatography on controlled-pore glass and thin-layer chromatography on silica gel [11]. The sequence of the decapeptide Locmi-AKH-I showed close structural similarities with Panbo-RPCH; both peptides are cross-reactive in the reciprocal biological system. Today, almost 40 members of this AKH/RPCH family of peptides are known by primary sequence from the two arthropod sister groups, Crustacea and Insecta. Interestingly, Crustacea have only one AKH member, and that is the highly conserved Panbo-RPCH (Table 1), whereas Insecta contain up to three AKH peptides in an individual and show a high degree of variability in the sequence. There is, however, group-specificity in the insect AKHs and moreover some insects synthesize the crustacean form Panbo-RPCH (see [4, 7]) (Table 1).

ABSTRACT Color change in the integument is the obvious action of the red pigment-concentrating hormone (RPCH), which is produced in neurosecretory cells of the Xorgan of crustaceans. Mobilization of various fuels used during energy-demanding processes is the classical regulatory role of adipokinetic/hypertrehalosemic/hyperprolinemic hormones (AKHs), which are produced in neurosecretory cells of the insect corpus cardiacum. These neuropeptide hormones have common structural features, such as a chain length of 8 to 10 amino acids, blocked N-(pyroglutamate) and C-termini (carboxyamide), and aromatic amino acids at positions 4 (phenylalanine or tyrosine) and 8 (tryptophan). The precursor is always organized as signal peptide, AKH/ RPCH peptide, amidation and processing site, followed by another peptide of diverse length and unknown function.

DISCOVERY Already in the 1920s to 1940s, extracts from certain glands of crustaceans and insects caused a change of integumental color from dark to light when injected into shrimps (see [8]). Complete purification of the active material from the eyestalks of the prawn Pandalus borealis (Panbo) was achieved mainly by gel chromatography on Sephadex LH-20 material in water/butanol mixtures, and the structure of the active principle was elucidated as a blocked octapeptide using quantitative amino acid analysis, proteolytic cleavage, Edman-dansyl sequencing, and high-resolution mass spectrometry. This resulted, in 1972, in the first complete primary structure of an invertebrate neuropeptide [2], which was termed red pigment-concentrating hormone (RPCH), and today denoted as Panbo-RPCH (Table 1). Handbook of Biologically Active Peptides

PEPTIDE AND PRECURSOR STRUCTURE AND PROCESSING Commonly, AKH peptides are characterized by a chain length of 8 to 10 amino acids, a blocked Nterminus (pGlu), a blocked C-terminus (carboxyamide), and aromatic amino acids at position 4 (mostly Phe but sometimes also Tyr) and 8 (Trp). In addition to the modified termini, other posttranslational modifications encompass an unusual C-glycosylation (at

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190 / Chapter 29 TABLE 1. Peptide Code Name

Primary structures of selected peptides of the AKH/RPCH family. Primary Structure

Comments

Panbo-RPCH

pGlu-Leu-Asn-Phe-Ser-Pro-Gly-TrpNH2

Locmi-AKH-I

pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2

Locmi-AKH-II Locmi-AKH-III Phymo-AKH-I

pGlu-Leu-Asn-Phe-Ser-Ala-Gly-TrpNH2 pGlu-Leu-Asn-Phe-Thr-Pro-Trp-TrpNH2 pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-SerNH2

Schgr-AKH-II Phymo-AKH-III Emppe-AKH

pGlu-Leu-Asn-Phe-Ser-Thr-Gly-TrpNH2 pGlu-Ile-Asn-Phe-Thr-Pro-Trp-TrpNH2 pGlu-Val-Asn-Phe-Thr-Pro-Asn-TrpNH2

Manto-CC

pGlu-Val-Asn-Phe-Ser-Pro-Gly-TrpNH2

Trp residue) in a stick insect AKH, an as yet unknown modification in AKHs of various cicadas (see [8]) and recently, a unique peptide of the AKH family was isolated from the CC of a cetoniid beetle and found to have a phosphorylated Thr residue at position 6 [9]. Direct protein chemical methods, as well as molecular cloning techniques have been used to demonstrate in detail the biosynthesis of up to three AKHs in locusts, including the characterization of the precursor (see [8]). Similar studies have been done on AKHs of the tobacco hornworm moth (Manduca sexta), a blaberid cockroach, a fruit fly, and on RPCH of two crab species. Apparently, a distinct mRNA encodes each AKH or RPCH precursor, which is translated into a preprohormone with the following general organization: signal peptide, AKH or RPCH peptide, Gly residue for amidation, dibasic processing site, and C-terminally a so-called “tail” or “precursor-related peptide” of variable length of 28 to 46 residues in insects and more than 70 residues in the two crustacean species (see [3]). For the latter peptides, specific functions have not yet been annotated. In the case of the migratory locust, two independently translated monomers of the pro-Locmi-AKH-I are oxidized to a unique precursor dimer by the formation of a disulfide bond at the “tail peptide.” Processing thus results in two monomeric Gly-Lys-Arg-extended Locmi-AKH-I decapeptides in addition to the dimeric molecule of the “tail peptide.” Firstly, the two basic amino acids are enzymatically removed by carboxypeptidase, and the resulting Gly-extended Locmi-AKH-I is modified to the amidated form by a peptidylglycine-αamidating monooxygenase (see [8]). This processing scheme has not been verified for any RPCH, and it is not even known whether dimers of other AKHs are formed.

Color change hormone of Pandalus borealis and adipokinetic hormone of stinkbug Nezara viridula Adipokinetic hormones of Locusta migratoria; three isoforms known

Adipokinetic hormones of the grasshopper Phymateus morbillosus; three isoforms known

Adipokinetic peptide of the mantid Empusa pennata Adipokinetic peptide of a Namibian member of the new order Mantophasmatodea

DISTRIBUTION The results from several immunocytochemical and in situ hybridization studies have shown that AKHs are localized and synthesized in the glandular lobe of the insect CC (see [8]). Interestingly, in the case of the migratory locust, mRNAs encoding the three LocmiAKHs were shown to colocalize in neurosecretory cells of the locust CC (see [8]), suggesting that the three AKH peptides are synthesized and released from the same cells. Although some have also reported immunoreactivity in neurons of the brain and other parts of the central nervous system of insects, this non-CC immunoreactivity was demonstrated to be a nonspecific crossreaction to other unidentified peptides [1]. Such cross-reactions were clearly abolished by the use of antisera preabsorbed with RPCH, whereas the specific immunoreactions in neurons of the CC remained unaffected by preabsorption [1]. Immunocytochemistry in crustaceans have localized RPCH in neurons of the X-organ and, to a lesser extent, in the medulla terminalis (in the eyestalks of decapods). RPCH immunoreactivity was also distributed in neurons of abdominal ganglia in a crayfish and in neurons that projected to the neuropil of the stomatogastric ganglion of a crab and a spiny lobster (see [8]). This distribution pattern ties in well with the functional activity of RPCH in crustaceans. In situ hybridization studies have confirmed that RPCH is synthesized in neurons of the eyestalks of crustaceans [10].

SOLUTION CONFORMATION OF AKH AND RECEPTORS FOR AKH Studies on the solution conformation of a few selected AKH peptides (mostly Locmi-AKHs and Emppe-AKH

The Invertebrate AKH/RPCH Family / 191 but not Panbo-RPCH) have been performed using either circular dichroism or nuclear magnetic resonance techniques and applying constrained molecular dynamics. All studies are in favor of a β-turn between residues 4 to 8, but they are at variance with each other about the N-terminus where β-sheet, “extended structure” and “extended PII conformation” have been proposed. Receptor studies for AKH peptides are also scarce. The first report on an AKH receptor protein in insects mentioned specific binding studies with tritiated Manse-AKH on membrane fractions purified from the fat body of adult M. sexta (see [5]). In crustaceans, it has only been shown that unidentified membrane proteins from various neuronal tissues are able to bind Panbo-RPCH. Recently, however, with the help of molecular biological methods, receptors for the AKH peptides of the fruit fly Drosophila melanogaster and the silkworm Bombyx mori have been cloned and characterized (see [5]). Interestingly, the G protein-coupled receptor with 7 membrane-spanning domains are structurally related to receptors of the vertebrate gonadotropin-releasing hormone. Receptor specificity in heterologous systems has been tested by functionally expressing the receptor of the fruit fly in Chinese hamster ovary cells or in frog oocytes and assuring that Drome-AKH had the lowest EC50 value of all peptides tested, including other AKH peptides (see [5]). For a number of insect species and AKH peptides, we have a good knowledge of what happens after receptor binding—that is, the mode of action pathways; this is the topic of a very recent review [6]. In brief, for the activation of glycogen phosphorylase that ultimately results in the increase of trehalose in the hemolymph (see following: biological actions), a Gq protein is activated, phospholipase is stimulated, the second messenger inositol trisphosphate is produced, calcium ions from internal stores are released, but, apparently, entry of extracellular calcium into the cell is promoted as well. For the activation of triacylglycerol lipase that leads to the increase of diacylglycerol in hemolymph, a Gs protein is activated, adenylate cyclase is stimulated, the second messenger cyclic AMP is increased, and intracellular calcium also plays a role. Much less is known of the mode of action of PanboRPCH in crustaceans, but it appears to be clear that for both the aggregation of the integumentary pigment and the retraction of distal retinal pigment in the compound eye, internal and external calcium ions are necessary and that phospholipase and inositol trisphosphate may be involved in the signaling processes (see [8]).

BIOLOGICAL ACTIONS In crustaceans, the major biological function of Panbo-RPCH is surely the aggregation of pigment gran-

ules in the epithelial chromatophores. Thereby, one or more different types of chromatophores (and, thus, pigment colors) can be affected. Next, the peptide is also responsible for the retraction (aggregation) of the distal retinal pigment in the ommatidia. Extrapigmentary effects of Panbo-RPCH are known as well. For example, it is implicated in the stimulation of the release of methyl farnesoate from mandibular organs. Other effects are neuromodulatory, which affect the rhythms of certain parts of the stomatogastric and swimmeret system. Moreover, modulation of photoreceptor cells has been reported (see [8]). However, no true metabolic effects of RPCH on energy metabolism (as is known for AKH peptides in insects—see following) have been demonstrated to occur in crustaceans. In insects, the basic physiological action of AKH peptides is the increase of those metabolites (irrespective of the nature of the metabolites—see following) that are used by the muscles (and other tissues) as a source of energy especially during exercise, such as flight and running. The AKH peptides target the fat body tissue in order to convert its stored triacylglycerols or glycogen to diacylglycerol or trehalose, respectively, or to synthesize the amino acid proline from alanine and fatty acids. Diacylglycerols, trehalose, or proline are released from the fat body, and their concentrations are consequently elevated in the hemolymph. There is not a specific AKH peptide that is responsible to elicit the increase of diacylglycerols in the hemolymph of locusts, for example, and another one that elevates the concentration of hemolymph trehalose in cockroaches or the level of proline in the tsetse fly. The activation of the respective pathways for lipid-, carbohydrate-, or proline mobilization is inherent in the enzymatic machinery of the fat body of the respective species and correlates with its main fuel for intensive locomotory activities— for example, flight activity (see [5]). Other actions of AKH peptides are on the hemolymph level to increase the concentration of lipophorins—that is, proteins that elevate the capacity of shuttling more lipids from fat bodies to muscles or on the flight muscle level where AKH peptides are proposed to stimulate the oxidation of lipids. Other stimulatory actions are the acceleration of the rate of the heartbeat that is interpreted as a mechanism to facilitate faster distribution of the metabolites and the increase in the oxidative capacity of mitochondria. Inhibitory actions of AKH peptides are also known of the biosynthesis of fatty acids (which is a logical effect if the main effect is to stimulate lipid degradation), RNA, and protein. The latter effect is mainly discussed under the physiological aspect of reproduction where AKH peptides and the repression of the synthesis of the egg yolk precursor protein (vitellogenin) have been analyzed. It appears that AKH peptides are somehow intricately linked with certain actions

192 / Chapter 29 of Juvenile hormone in its inhibitory effect on vitellogenin production (see [5]). In certain insects (firebug, cricket) it has been shown that AKH peptides have an effect on the locomotory activity. The peptides are also implicated in the immune response of locusts (see [5]). In summary, although the mobilization of substrates for high-energetic phases is the major and most well-known function of AKH peptides in insects, these peptides are truly multifunctional and exert pleiotropic action, as is also known from the majority of other hormones in invertebrates and vertebrates.

Acknowledgments Own research was financially supported in part by the National Research Foundation (Pretoria, South Africa; latest grant number: 2053806) and by the University of Cape Town.

References [1] Eckert M, Gabriel J, Birkenbeil H, Greiner G, Rapus J, Gäde G. A comparative immunocytochemical study using an antiserum against a synthetic analogue of the corpora cardiaca peptide Pea-CAH-I (MI, neurohormone D) of Periplaneta americana. Cell Tiss Res 1996;284:401–13. [2] Fernlund P, Josefsson L. Crustacean color change hormone: amino acid sequence and chemical synthesis. Science 1972;177:173–75.

[3] Gäde G. The revolution in insect neuropeptides illustrated by the adipokinetic hormone/red pigment-concentrating hormone family of peptides. Z Naturforsch 1996;51c: 607–17. [4] Gäde G. The explosion of structural information on insect neuropeptides. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm C, editors. Progress in the Chemistry of Organic Natural Products, Vol. 71. Wien: Springer Verlag; 1997. p. 1–128. [5] Gäde G. Regulation of intermediary metabolism and water balance of insects by neuropeptides. Ann Rev Entomol 2004; 49:93–113. [6] Gäde G, Auerswald L. Mode of action of neuropeptides from the adipokinetic hormone family. Gen Comp Endocrinol 2003; 132:10–20. [7] Gäde G, Auerswald L, Simek P, Marco HG, Kodrik D. Red pigment-concentrating hormone is not limited to crustaceans. Biochem Biophys Res Commun 2003;309:967–73. [8] Gäde G, Marco HG. Structure, function and mode of action of select arthropod neuropeptides: In: Atta-Ur-Rahmann, editor. Studies in Natural Product Chemistry. Bioactive Natural Products (Part M), Vol. 33. Amsterdam: Elsevier; 2006; p. 69– 139. [9] Gäde G, Simek P, Clark KD, Auerswald L. Unique translational modification of an invertebrate neuropeptide: A phosphorylated member of the adipokinetic hormone peptide family. Biochem J. 2006;393:705–13. [10] Linck B, Klein JM, Mangerich S, Keller R, Weidemann WM. Molecular cloning of crustacean red pigment concentrating hormone precursor. Biochem Biophys Res Commun 1993;195: 807–13. [11] Stone JV, Mordue W, Batley KE, Morris, HR. Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature 1976;263:207–11.

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30 Insect Myosuppressins/FMRFamides and FL/IRFamides/NPFs IAN ORCHARD AND ANGELA B. LANGE

Famide (where X1 is pQ, P, T, or A; X4 is D, G, or V; and X6 is V or S). Extended versions of this common sequence have been isolated but only from the midgut of parasitized Manduca sexta.

ABSTRACT Since the discovery of the molluscan cardioacceleratory peptide FMRFamide [8], a variety of neuropeptides that share the C-terminal RFamide have been characterized from both invertebrates and vertebrates. In insects these include the myosuppressins, the Nterminally extended FMRFamides (found only in dipterans) and FL/IRFamides, the neuropeptide Fs (NPFs), and the sulfakinins (discussed in Chapter 28). Although often referred to collectively as FMRFamide-related peptides (FaRPs), it is now clear that these peptide families are distinct and not related to one another. The extended RFamides are found throughout the central nervous system (CNS) in a variety of neuronal types, the stomatogastric nervous system, and within endocrine cells of the midgut. The genes for the peptides and their receptors have been cloned, and structure–activity relationships established in some cases. Many biological processes appear to be influenced by these peptides, including reproduction, circulation, ecdysis, and development. However, these peptides seem to be particularly involved in aspects of feeding, digestion, and/or food transport.

Extended FMRFamides and Extended FL/IRFamides N-terminally extended FMRFamides (see [6]) have only been identified in the dipterans, by sequencing or prediction from the gene. In Drosophila species, genes encoding 13 extended FMRFamides have been characterized and a number of the predicted peptides sequenced. Similarly, 13 extended FMRFamides have been sequenced from thoracic ganglia of the blowfly, Calliphora vomitoria, and are located on a single gene. The dipteran genes also code for some extended FIRFamides. N-terminally extended FL/IRFamides have been sequenced in other insects (see Table 1).

Neuropeptide F-like Peptides and Head Peptides The first member of the invertebrate neuropeptide F (NPF) family was identified in a tapeworm and shown to be related to the neuropeptide Y (NPY) family of vertebrate peptides. Subsequently, NPFs were identified in molluscs. In insects (see refs. in [1]) several sequenced peptides have been referred to as NPF/NPYlike peptides. These peptides (Table 1) are divided into long NPFs (the true NPFs) and short NPFs (also referred to as head peptides). The short peptides were isolated (often from the head) using an RIA with an antiserum against the tapeworm NPF or FMRFamide. They are occasionally referred to as FaRPs or as NPFs or as head peptides. The sequence RXRFamide is shared with the insect long NPFs, which typically contain 36 amino acids.

DISCOVERY Myosuppressins The first N-terminally extended RFamide to be sequenced in insects was leucomyosuppressin (LMS), which inhibits spontaneous contractions of Leucophaea maderae hindgut (see [6]). Other members of this socalled “myosuppressin” family have been sequenced (see refs. in [6]) some of which are shown in Table 1. These peptides share the sequence X1DVX4HX6FLRHandbook of Biologically Active Peptides

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194 / Chapter 30 TABLE 1. Sequences of representative members of the extended RFamide peptides in insects. Peptide Sequence

Acronym

Myosuppressins pQDVDHVFLRFamide PDVDHVFLRFamide TDVDHVFLRFamide pQDVVHSFLRFamide

Leucomyosuppressin SchistoFLRFamide Dromyosuppressin ManducaFLRFamide

Extended FMRFamides SVKQDFMHFamide DPKQDFMRFamide TPAEDFMRFamide SDNFMRFamide SPKQDFMRFamide PDNFMRFamide SAPQDFVRSamide MDSNFIRFamide

Drosophila Drosophila Drosophila Drosophila Drosophila Drosophila Drosophila Drosophila

FMRFamide FMRFamide FMRFamide FMRFamide FMRFamide FMRFamide FMRFamide FMRFamide

Species Leucophaea maderae Schistocerca gregaria Drosophila melanogaster Manduca sexta 1 2 7 9 10 11 12 13

Drosophila melanogaster ″ ″ ″ ″ ″ ″ ″

Extended FL/IRFamides GNSFLRFamide DPSFLRFamide AFIRFamide GQERNFLRFamide GGRSNDNFIRFamide DRSDNFIRFamide ARPDNFIRFamide

Pea-FMRFa-4 Pea-FMRFa-7 Pea-FMRFa-11

Manduca sexta ″ Locusta migratoria ″ Periplaneta americana ″ ″

Neuropeptide F (Long) LVAARPQDSDAASVAAAIRYLQELETKHAQHARPRFamide SFTDARPQDDPTSVAEAIRLLQELETKHAQHARPRFamide SNSRPPRKNDVNTMADAYKFLQDLDTYYGDRARVRFamide

Ang-NPF Aea-NPF Drm-NPF

Anopheles gambiae Aedes aegypti Drosophila melanogaster

Neuropeptide F (Short) AQRSPSLRLRFamide SPSLRLRFamide ARGPQLRLRFamide APSLRLRFamide pQRPHypSLKTRFamide TRFamide ANRSPSLRLRFamide

Drosophila sNPF1 Drosophila sNPF2 Led-NPF1 Led-NPF2 Aedes head peptide 1 Aedes head peptide 2 Periplaneta head peptide

Drosophila melanogaster ″ Leptinotarsa decemlineata ″ Aedes aegypti ″ Periplaneta americana

STRUCTURE OF THE PRECURSOR mRNA/GENE Myosuppressins The myosuppressin precursor has been determined through cloned DNA from Diploptera punctata, Blattella germanica, Pseudaletia unipuncta, and M. sexta and predicted from the genomes of Drosophila melanogaster and Anopheles gambiae (see refs. in [1, 14]). In all species studied, only a single decapeptide myosuppressin is released from the C-terminus of the precursor upon cleavage at dibasic and tribasic endoproteolytic processing sites and amidation. In D. punctata, the myosuppessin gene is divided into three exons. There is no evidence to suggest alternative splicing in the expression of the myosuppressin gene, although there may be unusual processing in the midgut of parasitized M. sexta

MasFLRFamide II MasFLRFamide III

larvae. The organization of the D. melanogaster myosuppressin gene is similar to the myosuppressin genes in other insect species. Analysis of the nucleotide sequence shows that a 100 amino acid translation product contains a signal peptide of 24 amino acid residues. A phylogenetic analysis has been described [14].

Extended FMRFamides and Extended FL/IRFamides The D. melanogaster FMRFamide gene was identified through oligonucleotide isolated DNA clones, using the sequence DPKQDFMRFamide (see [5]). The gene contains two exons and one intron. The precursor is 347 amino acids in length and contains a predicted signal sequence of 20 residues. This is a single copy gene that encodes five copies of DPKQDFMRFamide, two of TPAEDFMRFamide, and one each of SDNFMRFamide,

Insect Myosuppressins/FMRFamides and FL/IRFamides/NPFs / 195 SPKQDFMRFamide, and PDNFMRFamide, all bounded by conventional proteolytic sites. Precursors containing N-terminally extended FMRFamides have been identified in the dipterans D. melanogaster, Drosophila virilis, C. vomitoria, and Lucilia cuprina (see [6]). Precursors in C. vomitoria and L. cuprina specify 16 potential extended FMRFamides and 2 potential extended FIRFamides. There is no evidence for alternative splicing of the mRNA transcript; however, expression data indicates that the intron of D. melanogaster may contain regulatory enhancer sequences. Using tandem mass spectrometry, extended RFamides were structurally elucidated in Periplaneta americana and sequence information used for subsequent cloning of the gene [7]. This precursor gene encodes for 24 putative peptides, 23 of which were detected by mass spectrometric methods. Many of these are N-terminally extended FIRFamides, although the gene is referred to as the cockroach FMRFamide gene. The cDNA encodes for a protein of 505 amino acids that has several features typical of the D. melanogaster FMRFamide cDNA, including a 22-amino-acid signal peptide, N- and C-terminal sequences not coding for FMRFamides, and a central region with 24 putative peptide sequences. Although clearly similar in sequence to the extended dipteran FMRFamides, none of the encoded peptides contains the C-terminal FMRFamide. Instead, 10 FIRFamides, 3 FIRLamides, 2 FVRFamides, as well as peptides with an individual C-terminus of FVRLamide, LVRIamide, TLRVamide, SVRLamide, and LMRFamide are found. The remaining four putative peptides are nonamidated peptides.

Neuropeptide F-like Peptides and Head Peptides The prepropeptide encoded by the D. melanogaster NPF gene has 96 amino acids, of which the first 26 constitute a putative signal peptide (see [1]). Removal of the signal peptide, posttranslational processing, and amidation produces the 36 amino acid D. melanogaster NPF. This peptide shares sequence similarity with mammalian NPY and related peptides, although it is more closely related to invertebrate NPFs. The D. melanogaster NPF gene is present as a single copy in the genome. Similarly, the genomes of A. gambiae and Aedes aegypti contain sequences encoding an NPF each. The A. gambiae NPF gene is similar to the D. melanogaster NPF gene. The gene for A. aegypti head peptides encodes a protein containing three copies of bioactive peptides, whereas the short NPF genes of A. gambiae and D. melanogaster encode five and four peptides, respectively, with differing, but related sequences (see [1]). The short NPF gene has been cloned in D. melanogaster [4]. The cDNA encodes a 281-amino-acid protein that contains a 30-amino-acid signal peptide and 2 putative extended RLRF peptides (11 amino

acids and 19 amino acids) flanked by pairs of basic residues. The Drosophila genome database contains the two predicted transcripts for the short NPF gene. The coding region of the transcript has four exons. The amino acid sequences of the short NPFs are quite different from vertebrate NPY, in contrast to the long NPF that has 31% homology with NPY. Thus, the long NPFs (not the short NPFs) are considered as homologs of the vertebrate NPY.

DISTRIBUTION OF mRNA In insects, immunohistochemical studies have indicated the presence of FMRFamide-like immunoreactivity distributed throughout the CNS and PNS and within endocrine cells of the midgut. Most of these studies used antisera raised against the C-terminal RFamide and potentially indicate the distribution of multiple members of the extended RFamide families. The specific localization of mRNA by Northern blot and/or in situ hybridization is described here.

Myosuppressins In situ hybridization [6] shows that expression of the D. punctata myosuppressin gene is most abundant in the brain and optic lobes and in the frontal and subesophageal ganglia. In the brain, expression is found in cells of the pars intercerebralis, a region known to contain the medial neurosecretory cells. In P. unipuncta, 57% of the cells producing the myosuppressin gene transcript are found in the optic lobe. Within the digestive tract, D. punctata myosuppressin mRNA-positive endocrine cells are found in the anterior portion of the midgut, as they are in P. unipuncta. In situ hybridization also reveals a variety of cell types within the CNS, a peripheral neurosecretory cell, and midgut endocrine cells in M. sexta that express the myosuppressin gene.

Extended FMRFamides and Extended FL/IRFamides In situ hybridization reveals that the D. melanogaster FMRFamide gene is expressed in approximately 44 neurons of the 10,000 in the larval CNS (see [6]). In P. americana a nested reverse-transcription PCR analysis of the “FMRFamide” cDNA (that codes for extended FIRFamides) shows expression in the brain, and in thoracic and abdominal ganglia [7].

Neuropeptide F-like Peptides and Head Peptides In situ hybridization for D. melanogaster NPFs reveals medial and lateral cell pairs in the larval and adult

196 / Chapter 30 brains, although the lateral cells are only weakly stained in the adult (see refs. in [1]). There are about 15 endocrine cells in larval midgut, and 75–150 endocrine cells in the adult midgut. The transcript expression of A. gambiae NPF shows that the PCR product is present in eggs and in head, thorax, and abdomen of fourth instar larvae, and in pupae. It is present in the head and abdomen of adult males and females. Northern blot analysis has also revealed the presence of A. gambiae NPF transcript in the brain and midgut of adult female mosquitoes. The A. aegypti cDNA is expressed in brain and midgut (see [1]). Expression of the D. melanogaster short NPF mRNA has been studied by Northern blot analysis and in situ hybridization. Similar amounts of the transcript are expressed throughout all developmental stages of embryos and larvae. In situ hybridization reveals expression of the short NPF transcript in the nervous system from stage 14 embryos onwards and its presence in neurons of the brain and ventral ganglia. No expression is apparent in the midgut. The A. aegypti head peptide gene is expressed predominantly in the brain, terminal ganglion, and midgut (see [4]).

PROCESSING The genes for enzymes that process or degrade regulatory peptides have been identified in D. melanogaster and A. gambiae (see [9, 13]). Peptides are synthesized as a larger precursor that is subsequently processed into the active substances. After cleavage of the signal peptide, further proteolytic processing occurs predominantly at basic amino acid residues. Some rules have been proposed that allow a prediction of which putative proteolytic sites are actually used in insects (see [13]). The great majority of known or predicted insect peptides, including the extended RFamides, are α-amidated, so the significance of amidating mechanisms for peptide sequencing is great. Peptide signaling is terminated by a combination of mechanisms that include receptor desensitization or internalization, peptide diffusion, and enzymatic degradation (see [13]). A number of peptidases responsible for neuropeptide inactivation have been identified in insects (see [9]), but little work has examined the extended RFamides.

Myosuppressins Endoproteolytic and amidation enzymes liberate a single myosuppressin neuropeptide from the precursor protein. Alternative processing might exist in parasitized M. sexta larvae, since two extended myosuppressins have been identified in the midgut.

Extended FMRFamides and Extended FL/IRFamides There is considerable conservation of gene organization for the extended FMRFamides in dipterans. The single-copy gene encodes a precursor containing multiple copies of N-terminally extended FMRFamides liberated by endoproteolytic and amidation enzymes. In D. melanogaster there is one monofunctional PHM gene and two monofunctional PAL genes, which are required to produce α-amidated bioactive products (see [2]). The DNA regulatory sequences controlling D. melanogaster FMRFamide neuropeptide gene expression is a model for insect neuropeptide gene expression (see [6]). There is some debate over cell-specific processing of the D. melanogaster FMRFamide gene. Immunohistochemistry using antisera generated against three of the D. melanogaster FMRFamides demonstrates a unique, nonoverlapping cellular expression pattern indicative of differential processing of the precursor. In contrast, others have concluded that there is a single predominant pattern of expression, and it is unlikely that there are widespread cell-specific differences in precursor processing (see [5, 6]). The P. americana FMRFamide gene [7] liberates 24 putative peptides with appropriate endoproteolytic and amidation processing.

Neuropeptide F-like Peptides and Head Peptides The long NPFs in dipterans are encoded in a single gene that contains one copy of the neuropeptide following endoproteolytic and amidation enzyme processing. The short NPF gene in dipterans is also a single gene that contains multiple copies of short NPFs that would be liberated by endoproteolytic and amidation enzymes.

RECEPTORS Publication of the genomes for a variety of insect species enables analysis of putative G-protein coupled receptors (GPCRs) with “reverse pharmacology” identifying the natural ligand of functionally expressed orphan receptors.

Myosuppressins Two D. melanogaster GPCRs have been cloned and characterized that are specific for the D. melanogaster myosuppressin and that do not react with other D. melanogaster neuropeptides (refs. in [10]). The corrected genes from the database (coding for receptor 1 and 2) have multiple exons and introns. The cDNA codes for a protein of 478 amino acid residues (receptor 1) and

Insect Myosuppressins/FMRFamides and FL/IRFamides/NPFs / 197 488 amino acids (receptor 2), which each contains 7 transmembrane domains. The extracellular N-terminus contains potential N-glycosylation sites. Northern blot analysis illustrates that receptor 1 is strongly expressed in the adult head (essentially absent in the thorax, and abdomen), with receptor 2 present in the adult head, thorax, and abdomen. Similarly, a GPCR closely related to the D. melanogaster myosuppressin receptor has also been identified in the A. gambiae Genome Project database and subsequently cloned and characterized [10]. Anopheles gambiae appears to have only one myosuppressin receptor, in contrast to the two sequenced from D. melanogaster. An in vitro binding assay has been used to characterize putative receptors for Locusta migratoria myosuppressin associated with the oviduct and CNS of L. migratoria [6]. The specific binding for oviducts suggests the presence of two receptor types of high and low affinity.

Extended FMRFamides and Extended FL/IRFamides A receptor for the extended FMRFamides has been cloned, sequenced, and characterized in D. melanogaster (refs. in [10]) and predicted in A. gambiae. The cDNA sequence codes for a protein of 549 amino acid residues, which contains 7 transmembrane domains and potential N-glycosylation sites. Reverse pharmacology using the receptor in Chinese hamster ovary (CHO) cells reveals that the receptor responds to the D. melanogaster extended FMRFamides, the most potent of which was PDNFMRFamide. Interestingly, other extended FMRFamides were much less effective. These results imply that there are other receptors for the remaining extended FMRFamides. This receptor can also be activated by some other RFamide peptides, including the D. melanogaster short NPF and the D. melanogaster myosuppressin, which leads to some debate on the true physiological relevance of this FMRFamide receptor. Northern blot analysis reveals that the Drosophila receptor is expressed in all stages but mostly in larvae and adults. Expression analysis by RT-PCR illustrates that the receptor is present in trachea, which might therefore contaminate the positive results for CNS, fat body, intestine, and Malpighian tubules.

Neuropeptide F-like Peptides and Head Peptides The Drosophila genome has a genomic sequence with similarity to NPY receptors (refs. in [1]). This gene for D. melanogaster NPF receptor 1 has been cloned and sequenced. The deduced protein is 481 amino acids long and is predicted to have 7 transmembrane domains. This receptor resembles the vertebrate Y2 receptors and is most closely related to the Lymnaea NPY receptor.

CHO-K1 cells expressing the RNA of the NPF receptor 1 have been used in radioreceptor binding assays. The NPF receptor 1 also inhibits forskolin-stimulated adenylyl cyclase activity in transfected CHO cells. In situ hybridization indicates that the transcripts for this receptor are found in many cells of the larval CNS and midgut. The A. gambiae genome contains sequences encoding for an NPF receptor, and the cDNA has been cloned and sequenced [1]. The deduced protein is 424 amino acids and is predicted to have 7 transmembrane domains. This receptor has also been stably expressed for radioligand binding analysis and exhibits high affinity for A. gambiae NPF. RT-PCR analysis reveals that the receptor transcript is present in all life stages. A phylogenetic analysis of NPY/NPF receptors has been made [1]. A gene from the Drosophila Genome Project database has been cloned and represents a seven transmembrane GPCR that shows some structural similarity to vertebrate NPY2 receptors and is activated by the short NPFs of D. melanogaster (refs. in [1]). This short NPF receptor, expressed in CHO-K1 cells, is not activated by other peptides. Functional expression of the short NPF receptor illustrates that the activated receptor produces inward rectifying potassium currents by a pertussis toxin-sensitive G-protein mediated pathway. The D. melanogaster short NPFs are more potent than long NPFs (refs. in [1]).

STRUCTURE–ACTIVITY AND ACTIVE CONFORMATION In vitro bioassays, receptor binding, and molecular cloning/functional expression of receptors have been used to define ligands and structure–activity relationships of the extended RFamides and active conformation examined for the myosuppressin family.

Myosuppressins The structure–activity relationships of the myosuppressins have been examined in L. maderae hindgut and L. migratoria oviduct (see [6]). In hindgut, VFLRFamide appears to be the active core for inhibitory biological activity. In oviduct, the amide is critical for both binding with the receptor and inhibitory biological activity. Interestingly, VFLRFamide is the minimum sequence required for receptor binding, whereas HVFLRFamide is the minimum sequence for comparable inhibitory biological activity. The nonpeptide benzethonium chloride (Bztc) shares several chemical features with the sequence VFLRFamide, and Bztc mimics the biological activity of myosuppressins in a variety of target tissues (see [6]). A molecular dynamic/modeling study identified plausible structural explanations for this activity.

198 / Chapter 30 Myosuppressin analogs containing restricted conformational components have also been developed and shown to antagonize the myosuppressin receptors (refs. in [6]).

ecdysis, and development (see [1, 6]). The extended RFamides appear to act as neurotransmitters, neuromodulators, or neurohormones.

Myosuppressins Extended FMRFamides and Extended FL/IRFamides There have been few studies examining the structure–activity of the dipteran-extended FMRFamides. The question of functional redundancy has been addressed in D. melanogaster and C. vomitoria, where multiple extended FMRFamides are encoded on the same gene (see [5, 6]). By examining twitch tension of body wall muscles, it was concluded that the D. melanogaster FMRFamides are functionally redundant. In contrast, by use of a heart rate or crop assay, some of the D. melanogaster FMRFamides were found to be functionally distinct, with some showing activity and others not. The D. melanogaster FMRFamide receptor expressed in CHO cells shows differential potency to the extended FMRFamides (see [10]). A similar functional distinctiveness has been observed in C. vomitoria, where the semi-isolated heart and salivary glands respond to some of the C. vomitoria FMRFamides but not to others (see [6]).

Neuropeptide F-like Peptides and Head Peptides Structure–activity studies have not been reported in any detail for the NPFs. Functional expression of the receptors was used to identify the ligands for the receptor, so the NPF receptors are, by definition, more responsive to NPFs than to other extended RFamides. Using functional expression and radio-ligand binding analysis, the A. gambiae NPF receptor exhibits highaffinity binding for A. gambiae NPF but lower affinity for the NPFs from A. aegypti and D. melanogaster [1]. The functionally expressed D. melanogaster NPF receptor is more responsive to short NPFs than long NPFs (refs. in [1]).

BIOLOGICAL ACTION A range of biological actions have been established for N-terminally extended RFamides by use of in vitro bioassays, injection, and molecular techniques. It is often difficult to correlate these actions with the particular families, because of the likelihood of the higher doses of peptides cross-reacting with receptors for a different family. However, many target tissues for extended RFamides have been identified, and many biological processes appear to be influenced by these peptides, including reproduction, feeding, circulation,

Myosuppressins, as their name implies, reduce the frequency of spontaneous contractions of visceral and cardiac muscle (see [5, 6]). In some insect species, they have also been shown to enhance the force of neurally evoked contractions of skeletal muscle. Myosuppressins also stimulate enzyme secretion and inhibit short-circuit current in midgut and inhibit the release of adipokinetic hormones. Injection of the cockroach myosuppressin into B. germanica inhibits food intake, with food accumulating in the foregut [14].

Extended FMRFamides and Extended FL/IRFamides The extended FMRFamides influence heart rate, gut mobility, and skeletal muscle activity in D. melanogaster, with the various extended FMRFamides having differing effectiveness (see [5, 6]). In C. vomitoria a similar functional distinctiveness is suggested by the fact that only two of the six C. vomitoria extended FMRFamides tested are active on the semi-isolated heart and only three of the six tested on salivary gland are active sialagogues (see [6]). The extended FL/IRFamides also influence oviduct contractions in L. migratoria, contractions of the antennal heart in P. americana, and reduce spike frequency of dorsal unpaired median neurons in P. americana (see [6, 7]).

Neuropeptide F-like Peptides and Head Peptides The NPY signaling pathway is implicated in the stimulation of food uptake in the vertebrates and in the regulation of food conditioned foraging behaviors in Caenorhabditis elegans. The NPF neuronal network in D. melanogaster has been described and its food-dependent modifications examined. Gustatory stimulation by sugar is sufficient to trigger long-term changes in the NPF neuronal circuit [11]. Expression of D. melanogaster NPF is high in the brain of larvae attracted to food, and its down-regulation coincides with food aversion, hypermobility, and cooperative burrowing in the older larvae. Young transgenic larvae that have lost their NPF signaling prematurely display the behavioral phenotype associated with the older larvae. Overexpression of NPF in older larvae prolongs feeding and suppresses hypermobility and cooperative burrowing behaviors [15]. The hemolymph titer of A. aegypti NPF changes in females shortly after a blood meal and during the course of the reproductive cycle [1]. In D. melanogaster, short NPF

Insect Myosuppressins/FMRFamides and FL/IRFamides/NPFs / 199 mutants, in which the pathway is overexpressed, have elevated food intake, whereas mutants in which the pathway is suppressed have reduced food intake [4]. Injection of A. aegypti head peptide inhibits host-seeking by females, and its hemolymph titer is low prior to a blood meal but peaks at 36 to 48 hours post–blood meal (see [12]). Blood feeding also results in an increased titer of RFamide peptides in the hemolymph of R. prolixus that peaks at 3, 6, and 10 hours postfeeding (see [6]). A short NPF in Leptinotarsa decemlineata is a potent gonadostimulin in L. migratoria, stimulating oocyte length following injection (see [3]). Within L. decemlineata itself, the two short NPFs are absent from the brains of insects in diapause [3].

References [1] Garczynski SF, Crim JW, Brown MR. Characterization of neuropeptide F and its receptor from the African malaria mosquito, Anopheles gambiae. Peptides 2005;26:99–107. [2] Han M, Park D, Vanderzalm PJ, Mains RE, Eipper BA, Taghert PH. Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2. J Neurochem 2004;90:129–141. [3] Huybrechts J, De Loof A, Schoofs L. Diapausing colorada potato beetles are devoid of short neuropeptides FI and FII. Biochem Biophys Res Commun 2004;317:909–916. [4] Lee KS, You KH, Choo JK, Han YM, Yu K. Drosophila short neuropeptide F regulates food intake and body size. J Biol Chem 2004;279:50781–50789.

[5] Nichols R. Signaling pathways and physiological functions of Drosophila melanogaster FMRFamide-related peptides. Annu Rev Entomol 2003;48:485–503. [6] Orchard I, Lange AB, Bendena WG. FMRFamide-related peptides: A multifunctional family of structurally related neuropeptides in insects. Adv Insect Physiol 2001;28:267–329. [7] Predel R, Neupert S, Wicher D, Gundel M, Roth S, Derst C. Unique accumulation of neuropeptides in an insect: FMRFamiderelated peptides in the cockroach, Periplaneta americana. European J Neurosci 2004;20:1499–1513. [8] Price DA, Greenberg EJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977;197:670–671. [9] Riehle MA, Garczynski SF, Crim JW, Hill CA, Brown MR. Neuropeptides and peptide hormones in Anopheles gambiae. Science 2002;298:172–175. [10] Schöller S, Belmont M, Cazzamali G, Hauser F, Williamson M, Grimmelikhuijzen CJP. Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae. Biochem Biophys Res Commun 2005;327:29–34. [11] Shen P, Cai HN. Drosophila neuropeptide F mediates integration of chemosensory stimulation and conditioning of the nervous system by food. J Neurobiol 2001;47:16–25. [12] Stanek DM, Pohl J, Crim JW, Brown MR. Neuropeptide F and its expression in the yellow fever mosquito, Aedes aegypti. Peptides 2002;23:1367–1378. [13] Taghert PH, Veenstra JA. Drosophila neuropeptide signaling. Adv Genet 2003;49:1–65. [14] Vilaplana L, Castresana J, Bellés X. The cDNA for leucomyosuppressin in Blatella germanica and molecular evolution of insect myosuppressins. Peptides 2004;25:1883–1889. [15] Wu Q, Wen T, Lee G, Park JH, Cai HN, Shen P. Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system. Neuron 2003;39:147–161.

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31 Allatostatins in the Insects STEPHEN S. TOBE AND WILLIAM G. BENDENA

the family) [20]. Two years later, the first member of the PISCF family was identified in the moth Manduca sexta [8]. In both instances, the ability of the peptides to inhibit the production of JH was the principal assay used for identification, following HPLC separation of brain extracts. The FGLa family has numerous other biological actions, including modulation of myotropic activity and gut enzyme activity, and it also can act as a neurotransmitter. The FGLa family of ASTs is now known to consist of at least 70 different peptides, and members are found in many invertebrate phyla, including all arthropods examined to date, as well as in nematodes and trematodes (Table 1). However, only in cockroaches, crickets, and termites do they function as inhibitors of JH production. The PISCF family is less widely distributed, occurring principally in Lepidoptera and Diptera. Peptides of this family do inhibit JH biosynthesis in some members of these orders. A third family of peptides was subsequently discovered in crickets and is characterized by the consensus sequence W(X)6Wa [18]. This family also occurs in stick insects, although the ability to inhibit JH production appears restricted to the crickets. Recently, mining of insect genome databases has revealed additional ASTs of all three families. However, the FGLa family is the most widely distributed and variable. For example, FGLamide-like peptides in Diptera were purified from Drosophila by use of a “reverse physiology” approach to identify cognate ligands for orphan receptors [2].

ABSTRACT There are three families of allatostatins (ASTs), peptides discovered on the basis of their ability to inhibit juvenile hormone (JH) production in insects: the cockroach type representing the FGLa family, the cricket type representing the W(X)6Wa family, and the Manduca type representing the PISCF family. Each of these families performs multiple functions, including the modulation of myotropic activity and neurotransmission. These effects are species- and order-specific, and each family inhibits JH production in only a limited number of related orders, suggesting that their original functions were probably not the inhibition of JH production. This review summarizes the expression and distribution of these peptides, their interaction with receptors, and their biological activities.

DISCOVERY JH produced by the insect corpora allata (CA) is the regulator of metamorphic and reproductive functions in insects. The production of these compounds is regulated by other compounds originating in the central nervous system. Measurement of production of JH has revealed large changes in the rates of biosynthesis, which are also believed to be reflected in changes in the titers in the hemolymph. ASTs, inhibitors of production of JH, were hypothesized to exist in 1980 [15], following from demonstrations by Scharrer and colleagues [11] that presumed peptidergic factors in the brains of cockroaches modulate the volume of the CA, which in turn was believed to translate into changes in hormone production. The first ASTs were discovered in the cockroach Diploptera punctata and are members of the FGLa family (the common C-terminal sequence for all members of Handbook of Biologically Active Peptides

STRUCTURE OF ALLATOSTATIN PRECURSORS The first preproallatostatin protein was characterized from a gene sequence obtained from the cockroach Diploptera punctata [1, 3]. The Diploptera AST precursor

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202 / Chapter 31 TABLE 1.

Sequences of representative members of the three allatostatin families.

Species Diploptera punctata

Drosophila melanogaster Bombyx mori C. elegans

Gryllus bimaculatus

Manduca sexta Drosophila melanogaster Anopheles gambiae

Designation Cockroach FGLa family Dippu-AST 1 Dippu-AST 2 Dippu-AST 5 Drome-AST 3 Bommo-AST 5 nlp-51 nlp-61

Sequence LYDFGLa AYSYVSEYKRLPVYNFGLa DRLYSFGLa SRPYSFGLa ARMYSFGLa ALSTFDSLGGMGLa MAAPKQMVFGFa YKPRSFAMGFa AAMRSFNMGFa LIMGLa

Cricket W(X)6Wa family Grybi-AST 1 Grybi-AST 5

GWQDLNGGWa AWDQLRPGWa

Manduca PISCF family Manse-PISCF AST Drome-PISCF AST Anoga-PISCF AST

pQVRFRQCYFNPISCF-OH pQVRYRQCYFNPISCF-OH pQIRYRQCYFNPISCF-OH

One dibasic cleavage site in Dippu-AST 2 is shown in bold. Substitutions in the PISCF family are underlined. 1C. elegans AST-like peptides have no designation but are derived from the nlp5 and nlp6 genes.

has 370 amino acid residues and contains 13 unique ASTs that range from 6 to 18 amino acids and when processed share a COOH-terminal core sequence Y/ FXFGL/I-amide. Each AST in the precursor is followed by glycine and a dibasic KR or RK endoproteolytic cleavage site. After cleavage, the terminal glycine functions as the substrate for peptidyl-glycine α-amidating monooxygenase. Purified ASTs are C-terminally amidated and require amidation for biological activity, confirming that these signals are utilized in vivo. The ASTs are clustered in groups within the precursor, with each group separated by spacer regions containing acidic amino acids that effectively neutralize the basic charge contribution of the ASTs and their processing sites. The structure and organization of the Diploptera precursor serves as a prototype for all AST precursors identified thus far. A variation between different orders is the number of ASTs present in their respective precursor. For example, Lepidoptera AST precursors contain 8 to 10 peptides, Dipterans contain 4 to 5, and nematode AST precursors contain 7 (see Chapter 26). In crickets, the W(X)6Wamide precursor is structurally similar to the Dippu AST precursor. The preproAST polypeptide sequence for the W(X)6Wa cricket family of ASTs has been identified from cloned DNA in Drosophila [18] and partially characterized in the cricket G. bimaculatus [18]. The Drosophila W(X)6Wa precursor

has sequences for five unique W(X)6Wamide peptides that are followed by GKR, indicating COOH-terminal amidation and cleavage. The Gryllus precursor contains sequence for six cricket ASTs, four of which are unique in sequence. The sequence of the preproAST polypeptide of Manduca PISCF was deduced from the DNA sequence in the moth Pseudaletia unipuncta [1] and in Drosophila [1, 9]. A single 15-amino-acid AST peptide flanked by dibasic endoproteolytic cleavage sites is released as a nonamidated peptide from the carboxyl terminus of the 125-amino-acid precursor. The PISCF AST differs between Manduca and Drosophila by one conservative amino acid substitution (Y4 for F4) and an additional substitution in Anopheles (I2 for V2) (see Table 1).

DISTRIBUTION OF mRNA AND PEPTIDES The distribution and expression of ASTs have been examined with antibodies specific to AST peptides and through tissue in situ hybridization whereby specific nucleic acid probes detect cells expressing AST mRNA. The following describes the extensive expression of ASTs noted in D. punctata. Four medial cells of the pars intercerebralis express ASTs and extend axons that arborize and terminate adjacent to 25–30 lateral neurosecretory cell bodies of the protocerebrum. These lateral cells project AST-immunoreactive nerve fibers in

Allatostatins in the Insects / 203 the nervi corporis cardiaci (NCC) II and arborize within the corpus cardiacum and CA. Extensively arborizing AST-immunoreactive neurons are also detected in the optic, central, and antennal brain lobes, as well as the pulsatile organ muscle [14]. Clearly, the brain and associated structures contain large amounts of FGLa ASTs, but we do not know the relative percentage of this material that projects to the CC and CA for inhibition of JH biosynthesis. However, these observations do suggest that the FGLa peptides play other major roles in the brain. AST-immunoreactive neurons innervate the rectal dilator muscles, muscles of the rectum, anterior hindgut, midgut [14], and the lateral and common oviducts [5]. AST-immunoreactive cells are also detected in opentype midgut endocrine cells. These cells extend from the basal lamina to lumen of the midgut and are sites of AST mRNA synthesis. AST expression and production are also found in a population of circulating hemocytes that comprise approximately 5% of the total hemocytes [13, 17]. In other insects—such as flies, moths, and earwigs— ASTs are detected in cells of the brain and optic lobe but generally are not detected in nerve fibers extending within the CA. Immunoreactive nerve fibers are rarely found in the midgut. However, expression in endocrine cells of the midgut is common. Little data is available for expression of the two FGLa receptors in Drosophila. However, expression studies suggest that one receptor subtype may predominate in brain and the other in the gut [2, 10].

PEPTIDE PROCESSING The occurrence of multiple ASTs in the preproallatostatins, with associated cleavage and amidation signals, suggests that all of the peptides are likely to be cleaved and present at the same time in any given tissue. Prohormone convertases are likely to be responsible for the liberation of the various AST peptides. There is no evidence for selective processing of the precursor or of tissue-specific processing. Degradation of the FGLa ASTs to terminate their biological actions occurs in several different ways and is dependent on the precise sequence of the peptides and the tissues in which degradation is studied. For example, [4] concluded that endopeptidases as well as aminoand carboxypeptidases were the principal proteolytic enzymes for degradation of the ASTs. In tissues, the FGLamide peptides are cleaved by carboxypeptidases to yield the corresponding nonamidated FG-OH peptides (removal of L-amide), resulting in inactive peptides. Hemolymph of D. punctata does not contain carboxypeptidases but rather aminopeptidases, which produce

peptides with intact core sequence, and hence continued biological activity. Interestingly, changes in sequence can also alter the half-life of the AST peptides. For example, Dippu-AST 5 has a much longer half-life than other ASTs in hemolymph. To date, the peptidases responsible for the cleavage of the ASTs have not been identified or cloned.

ALLATOSTATIN RECEPTORS The sequencing of the Drosophila genome has led to the identification of at least 32 neuropeptide-encoding genes. These neuropeptides interact with G-protein coupled receptors (GPCRs), which are characterized as seven-transmembrane domain receptors. Drosophila has approximately 160 GPCRs, of which 44 have characteristics of receptors for peptide ligands [6]. As assigning a ligand to a specific receptor(s) is dependent on an appropriate assay, only half of these peptide GPCRs have been functionally characterized. The presence of multiple cockroach-like or AST receptors was first suggested by ligand-membrane binding studies in the cockroach, D. punctata [14]. Two Drosophila genes encode GPCRs functionally specific for AST peptides. Both GPCRs belong to the rhodopsinlike GPCR family and are most closely related to the vertebrate galanin receptors. The first, gene CG2872, is located at chromosomal locus 3D6-E1 and encodes a GPCR (AlstR, DAR-1) of 394 amino acids that responds to ASTs [2]. AlstR/DAR-1 mRNA is not expressed in embryos but appears in larvae, pupae, and adults. Expression is found in both heads and bodies of adults. The second Drosophila gene CG10001 (DAR-2) is located a chromosomal locus 98DE and encodes a GPCR of 357 amino acids [10]. DAR-2 is likely a paralog of AlstR/ DAR1 as DAR-2 shares 47% overall sequence identity and 60% identity in the transmembrane regions. DAR2 mRNA expression begins in 8–16 hour embryos and is found in all later stages of development. In third instar larvae and adults, mRNA expression in the gut/ abdomen is greater than that in isolated brain/head [10]. AST receptor orthologs have been sequenced in Periplaneta americana and Bombyx mori. Bombyx fifth instar larvae express GPCR mRNA in foregut, midgut, hindgut, and brain. Additional AST receptor orthologs have been identified through sequencing projects to include invertebrates such as Apis mellifera, Anopheles gambiae, and Caenorhabditis elegans. The Drosophila AST-B receptor gene (CG14484 = CG30106) was identified through a functional assay [7] to be a member of the bombesin family of peptide GPCRs. The gene is localized on the second chromosome, locus 54D3. A possible paralog (CG14593) was

204 / Chapter 31

Cockroach-type AST NH2

Manduca-type AST

Y/FXFGL-NH2

NH2

CYFNPISCF

CNS Galanin-like GPCRs

?

Somatostatin-like GPCRs

GUT Myoinhibitory Peptides?

NH2 W(X)6W-NH2

Bombesin-like GPCRs

?

Cricket-type AST FIGURE 1. The cockroach-type ASTs interact with two galanin-like G-protein coupled receptors (GPCRs). One type of receptor may be more prevalent in the CNS and the other more abundant in the gut tissue. Manduca-type ASTs with oxidized cysteines C7–C14 bind with two somatostatin-like receptors. In Drosophila, these receptors appear localized to the pars intercerebralis of the brain as well as the optic lobe. Somatostatin-like receptors have not been found to be expressed in the gut, although Manduca-type AST immunoreactivity is present in gut tissues. Cricket-type ASTs interact with Bombesin-like GPCRs, but expression data is not yet available. The cricket-type ASTs share sequence similarity with myoinhibitory peptides (MIPs) and galanin. As the N-terminus of galanin is required for receptor binding, cricket-type ASTs and myoinhibitory peptides could hypothetically interact with the galanin-like GPCRs.

identified in the Drosophila sequence database but has not yet been functionally tested. Two Drosophila PISCF AST receptor genes (CG7285 and CG13702) were identified in two separate functional assays [7, 9]. CG7285 and CG13702 encode GPCRs of 483 amino acids (Drostar1) and 549 amino acids (Drostar2), respectively. Both receptors share 60% overall identity and 76% identity in the transmembrane regions. These genes are paralogs located at cytological locus 75D1 and 75D2 and are separated by 80 kb. Drostar 1 and 2 are orthologs of the vertebrate somatostatin receptors sharing 42% amino acid identity in the transmembrane region. This identity is concentrated on those parts of the transmembrane regions facing the cytosol where receptors couple with their heterotrimeric G-protein. In situ hybridization has localized the Drostar genes to cells in the lamina region of the optic lobes and to cells of the pars intercerebralis of the brain of adult flies [9]. As with AST GPCRs, invertebrate genome projects are rapidly uncovering sequences that identify orthologs to W(X)6Wamide ASTs and PISCF-GPCRs.

STRUCTURE–ACTIVITY AND ACTIVE CONFORMATIONS Structure–activity studies have demonstrated that the core sequence for biological activity in the FGLa family is the C-terminal pentapeptide Y/FXFGL/Iamide. Amidation is also essential for biological activity. The consensus sequence for the cricket family appears to be W(X)6Wamide and amidation is similarly necessary. The minimal biologically active region for the PISCF family is unknown although the oxidation of the two cysteine residues is critical for receptor binding. Receptor binding activity was not dependent on the presence of a blocked N terminus [9]. The core region of the FGLa family appears to possess secondary structural elements which are probably essential for interaction with the receptors. In particular, this region probably contains a type II β turn, with the most critical residues being F and L, at least in selected ASTs. Incorporation of moieties promoting a β turn in the core region permitted the synthesis of analogs of these peptides that show high biological activity [12, 14].

Allatostatins in the Insects / 205

BIOLOGICAL ACTIONS Based on immunocytochemical distribution, isolation, and sequencing, or mining of genomic databases, it has become clear that the AST families are ubiquitous throughout the Class Insecta and many other invertebrate phyla, and regulate many other processes in addition to JH production [16]. Other actions of ASTs have been largely defined in individual species, and therefore, it is unknown at present if such actions can be inferred across the insects. In the case of the FGLamide family, such actions include (1) the inhibition of vitellogenin production by fat body in cockroaches; (2) the inhibition of myotropic activity, particularly in gut tissue of Dictyoptera and Orthoptera; (3) regulation of release of digestive enzymes in the midgut of cockroaches; and (4) neurotransmission in crab stomatogastric ganglion and possibly cockroach CNS [17]. Treatment of cultured mammalian neurons expressing DAR-1 receptor with the FGLa ligand is being used to selectively silence these neurons. Biological actions of the W(X)6Wamide family include (1) inhibition of ecdysteroid biosynthesis (prothoracicostatic activity) by prothoracic glands in silk moth and ovaries of crickets, (2) inhibition of locust oviduct muscle, and (3) inhibition of spontaneous muscle contraction in foregut of the cockroach. The PISCF family displays biological effects similar to the other two AST families, dramatically inhibiting contraction in larval Drosophila heart muscle, for example (hence, its name flatline), and its localization in gut suggests similar functions in this tissue. This peptide family may also play a role in the regulation of migratory flight in Lepidoptera. Although the PISCF family appeared to inhibit JH production only in members of the Lepidoptera, the discovery of a conservatively modified PISCF peptide in the Anopheles gambiae genome database and its apparent ability to inhibit JH production in adults of another mosquito, Aedes aegypti [11], suggests that this peptide family may be important as an allatoregulator in Diptera as well. The modes of action of these three peptide families remains largely unexplored, although studies with the FGLa family indicate that second messengers cAMP, cGMP, and calcium are all effectors. With our present knowledge of second-messenger cascades, the time is appropriate for a reexploration of this area.

CONCLUSION As is the case with many invertebrate peptide families, the allatostatin families are multifunctional peptides. The large number of peptides in the FGLa family,

with significant variation in length but conservation of the core C-terminal pentapeptide, indicates that this family serves important functions across the invertebrates. However, the relatively limited number of orders in which they act as inhibitors of JH biosynthesis [16] also suggests that this function appeared secondarily; thus, the FGLa family was coopted as inhibitors by cockroaches, crickets, and termites early in evolution. In these instances, specificity of function resides with the tissues and the cognate allatostatin receptors. Because the CA of these orders are heavily invested by FGLa-containing nerves, with multiple release sites, the local concentration of the peptides is likely to be very high. It is therefore conceivable that differing concentrations of the peptides or differing receptor affinity could confer multiple functionality. Receptor affinity in both the CA and other target tissues such as gut and oviduct muscle no doubt dictate responsiveness. The occurrence of at least two receptor types in insects and their similarity to the galanin GPCRs suggests an ancient origin. The same ideas can be applied to the evolutionary necessity of varying insects to adopt alternative peptides as ASTs. A clue may come from the cricket ASTs whereby the most potent molecule resembles the mammalian galanins and hence may interact with the galanin-like receptor as does the cockroach type of AST. Subtle changes in the receptor “binding pocket” may have provided the “evolutionary pressure” to solicit other peptide molecules to perform the “allatostatic” function. The Manduca type of AST is further removed in sequence but has the same functional quality of the cockroach type of ASTs in that a major role is inhibition of muscle contraction. As muscle contraction is the likely ancestral function, a reasonable hypothesis would be that all ASTs were coopted for allatostatic regulation somewhat later in evolutionary time.

References [1] Bendena WG, Donly BC, Tobe SS. Allatostatins: a growing family of neuropeptides with structural, and functional diversity. Ann NY Acad Sci 1999;897:311–29. [2] Birgul N, Weise C, Krienkamp H-J, Richter D. Reverse physiology in Drosophila: Identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. EMBO J 1999;18:5892–900. [3] Donly BC, Ding Q, Tobe SS, Bendena WG. Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata. Proc Natl Acad Sci USA 1993;90:8807–11. [4] Garside CS, Hayes TK, Tobe SS. Degradation of Dip-allatostatins by haemolymph from the cockroach, Diploptera punctata. Peptides 1997;18:17–25. [5] Garside CS, Koladich PM, Bendena WG, Tobe SS. Expression of allatostatin in the oviducts of the cockroach Diploptera punctata. Insect Biochem Mol Biol 2002;32:1089–99.

206 / Chapter 31 [6] Hewes R, Taghert PH. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 2001;11:1126–42. [7] Johnson EC, Bohn LM, Barak LS, Birse RT, Nassel DR, Caron MG, Taghert PH, Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions. J Biol Chem 2003;278:52172–78. [8] Kramer SJ, Toschi A, Miller CA, Kataoka H, Quistad GB, Li GP, Carney RL, Schooley DA. Identification of an allatostatin from the tobacco hornworm Manduca sexta. Proc Natl Acad Sci USA 1991;88:9458–62. [9] Kreienkamp H-J, Larusson HJ, Witte I, Roeder T, Birgul N, Honck H-H, Harder S, Ellinghausen G, Buck F, Richter D. Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology. J Biol Chem 2002;277:39937–43. [10] Lenz C, Williamson M, Hansen GN, Grimmelikhuijzen CJ. Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2. Biochem Biophys Res Commun 2001;286:1117–22. [11] Li Y, Hernandez-Martinez S, Noriega FG. Inhibition of juvenile hormone biosynthesis in mosquitoes: Effect of allatostatic head factors, PISCF- and YXFGL-amide-allatostatins. Regul Pept 2004;118:175–82. [12] Nachman RJ, Moyna G, Williams HJ, Tobe SS, Scot AI. Synthesis, biological activity and conformational studies of insect allatostatin neuropeptide analogues incorporating turn-promoting moieties. Biorg Med Chem 1998;6:1379–88.

[13] Stay B. A review of the role of neurosecretion in the control of juvenile hormone synthesis: A tribute to Berta Scharrer. Insect Biochem Mol Biol 2000;30:653–62. [14] Stay B, Tobe SS, Bendena WG. Allatostatins: Identification, primary structures, functions and distribution. Adv Insect Physiol 1994;25:267–338. [15] Tobe SS. Regulation of corpora allata in adult female insects. In Insect Biology in the Future: VBW 80, Academic Press, NY. 1980, pp. 345–67. [16] Tobe SS, Zhang JR, Bowser PRF, Donly BC, Bendena WG. Biological activities of the allatostatin family of peptides in the cockroach Diploptera punctata and potential interaction with receptors. J Insect Physiol 2000;46:231–42. [17] Tobe SS, Stay B. Neuropeptide regulators of juvenile hormone production. Encyclopedia of Hormones. Elsevier Science, 2003, San Diego, CA. pp. 19–29. [18] Wang J, Meyering-Vos M, Hoffmann KH. Cloning and tissuespecific localization of cricket-type allatostatins from Gryllus bimaculatus. Mol Cell Endocrinol 2004;227:41–51. [19] Williamson M, Lenz C, Winther ME, Nassel DR, Grimmelikhuijzen CJP. Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster. BBRC 2001;281: 544–50. [20] Woodhead AP, Stay B, Seidel SL, Khan MA, Tobe SS. Primary structure of four allatostatins: Neuropeptide inhibitors of juvenile hormone synthesis. Proc Natl Acad Sci USA 1989;86:5997– 6001.

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32 The FXPRLamide (Pyrokinin/PBAN) Peptide Family REINHARD PREDEL AND RONALD J. NACHMAN

terminally extended peptides from various species of moths. The functional diversity of these moth peptides is reflected by various designations such as pheromone biosynthesis activating neuropeptides (PBAN; [35]), melanization and reddish coloration hormone (MRCH; [19]), and diapause hormone (DH; [14, 19]). Since truncated peptides, containing only the common Cterminal pentapeptide, show cross-reactivity in the different physiological assays, we refer here to this family of peptides as FXPRLamides. Another group of FXPRLamides is typical of neurosecretory cells of the abdominal ventral nerve cord and was first identified from the American cockroach [29]. These FXPRLamides, which always contain a Trp preceding the C-terminal pentapeptide, are products of capa-gene related precursors [16] and occur as only a single isoform in all insects studied so far. Members of the FXPRLamide family have also been identified from a crustacean [43], whereas a related peptide (FXPRGamide) was found in the nervous system of the Freshwater Hydrozoan Hydra [41]. A significant degree of N-terminal sequence similarity exists with the neuromedin-U peptide family of vertebrates (see Table 1 and neuromedin-U chapters by Bloom’s group elsewhere in this book).

ABSTRACT FXPRLamides are pleiotropic neuropeptides that are mainly associated with the neuroendocrine system. FXPRLamides mediate such diverse functions as pheromone biosynthesis, hindgut/oviduct contraction, melanization, pupariation, and diapause in insects. At least two genes encoding for FXPRLamides are known in insects. A single pyrokinin is always encoded on the capa-gene. Homologs are found in many insects and are typical of neurohemal release sites from the abdominal ventral nerve cord. Other FXPRLamides are synthesized in neurosecretory cells of the subesophageal ganglion and transported into the retrocerebral complex. Products of the PBAN (pheromone biosynthesis activating neuropeptide) gene are particularly well studied. The vertebrate neuromedin U not only shares Cterminal sequence similarity with insect FXPRLamides but its receptor exhibits a high degree of similarity with invertebrate FXPRLamide receptors.

DISCOVERY FXPRLamides are neuropeptides that exhibit highly diverse physiological functions in different insect groups. The first member of the peptide family was identified in 1986 from head extracts of the cockroach Leucophaea maderae and named leucopyrokinin because of its N-terminal pyroglutamate residue and its myotropic action in the hindgut assay [12]. By use of similar bioassays to monitor bioactivity during the purification procedure, multiple isoforms of pyrokinins were subsequently purified from the central nervous system or retrocerebral complex of different hemimetabolous insects—mainly Locusta migratoria [39] and the American cockroach, Periplaneta americana [30]. Completely different bioassays were used to identify sequence-related but NHandbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The first FXPRLamide gene was cloned from the silkworm Bombyx mori [15, 37]. Diapause hormone, pheromone biosynthesis activating neuropeptide (DHPBAN), and three shorter FXPRLamides (α-, β-, and γ-subesophageal ganglion neuropeptides, SGNPs) have been shown to be a product of a polyprotein precursor, which is encoded by a single mRNA. Molecular characterization of the cDNA revealed that the mRNA encodes an open reading frame consisting of 192 amino acid

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208 / Chapter 32 TABLE 1.

Sequences of selected FXPRLamides from insects and related peptides from other organisms.

Species Leucophaea maderae Periplaneta americana D. melanogaster Bombyx mori

Penaeus vannamei Hydra magnipapillata Homo sapiens

Abbreviation

Sequence

Lem-PK Pea-PK-5 Pea-PK-2 Drm-PK-1 Drm-PK-2 Bom-PBAN Bom-DH Bom α-SGNP Bom β-SGNP Bom γ-SGNP Pev-PK Hym-355 NmU-25

pQTSFTPRLa GGGGSGETSGMWFGPRLa SPPFAPRLa TGPSASSGLWFGPRLa SVPFKPRLa LSEDMPATPADQEMYQPDPEEMESRTRYFSPRLa TDMKDESDRGAHSERGALWFGPRLa IIFTPKLa SVAKPQTHESLEFIPRLa TMSFSPRLa DFAFSPRLa FPQSFLPRGa FRVDEEFQSPFASQSRGYFLFRPRNa

residues. Similar DH-PBAN cDNAs, always encoding five FXPRLamides, have subsequently been sequenced from many other moth species [2, 46]. In Drosophila melanogaster, two FXPRLamide encoding genes were identified; specifically the capa-gene (CG15520; see [16]) and the hugin-gene (CG6371; see [20]). The translation of the mRNA of the hugin-gene yields a precursor that consists of 191 amino acids derived from a 573 bp coding sequence. Only a single FXPRLamide (Drm-PK2) can be predicted from this precursor, although another related peptide that lacks a Phe in the C-terminus (hug γ) can be deduced from the gene [20]. The expression of Drm-PK-2 could be confirmed by mass spectrometry [3, 33]. The capa-gene of D. melanogaster contains two introns; the expression of the single FXPRLamide (Drm-PK-1), which is located on exon 3, was also confirmed by mass spectrometry [33]. A homolog gene was sequenced from Manduca sexta [17]. The DH/PBAN precursor sequences usually contain the cleavage sites and amidation signals between the deduced peptides, without providing additional spacer sequences. Only the first FXPRLamide of the precursor (DH) is always separated from the second FXPRLamide by an extensive spacer sequence. In contrast, peptide precursors that result from the hugin- and capa-genes include longer spacer sequences between the deduced peptide sequences.

DISTRIBUTION OF mRNA AND PEPTIDES The distribution of FXPRLamides in the CNS of insects is known for many species. Neurosecretory cells in the subesophageal ganglion (SEG) and in the abdominal ganglia are the main source of FXPRLamides, which are likely released into the hemolymph. By in situ hybridization, expression of the PBAN/DH gene was

observed in a number of median cells in the SEG of Bombyx mori [38]. These cells belong to the mandibular (4 cells), maxillar (6 cells), and labial (2 cells) neuromeres of the SEG and subsequent studies using many other moth species always revealed a similar expression pattern; only the number of neurons demonstrated slight variability between species. Backfill experiments and immunostainings with antisera against FXPRLamides revealed putative release sites of PBAN/DH gene products in moths. Thus, neurits of neurosecretory cells of the mandibular and maxillar segments join each other in the dorsal hemispheres of the SEG and separate from the ganglion via the maxillar nerves (see [11]). A smaller nerve (NCC-V), which originates from the maxillar nerve, probably serves as a neurohemal release site rather than the retrocerebral complex. Neurons of the labial segment send neurits via the circumesophageal connectives to the brain, where they separate from the CNS and reach the retrocerebral complex via the NCC-3. Although PBAN/DHs are only known from Lepidoptera, homolog cells in the SEG were immunostained in other insects as well. As revealed by mass spectrometric screening of nervous tissues, shorter FXPRLamides are responsible for these immunostainings [30]. Studies on locusts [5] and cockroaches [30] have also shown that FXPRLamide immunoreactive cells have a peripheral projection very similar to that of the aforementioned SEG cells of moths. In hemimetabolous insects, the corpora allata often contain large amounts of FXPRLamides [9, 30]. This unique accumulation, which is not accompanied by other abundant peptides, primarily results from FXPRLamide containing fibers of the NCA2, which run over the CA into the periphery. FXPRLamide producing cells of the labial neuromere may have neurits that project into the ventral nerve cord and leave the abdominal ganglia via the segmental (locusts;

The FXPRLamide (Pyrokinin/PBAN) Peptide Family / 209 [4]) or median/transverse nerves (cockroaches) to innervate targets as far as the dorsal vessel. The FXPRLamide of the capa-gene is expressed in median neurosecretory cells of the abdominal ganglia. Although the number of cells strongly differs between species, the neurits always project via the median nerves into abdominal perisympathetic organs. In D. melanogaster and M. sexta, the FXPRLamide containing precursors of the capa-gene are also expressed in cells of the labial neuromere of the SEG [16, 17], likely together with FXPRLamides of the PBAN/DH gene (M. sexta) or hugin gene (D. melanogaster). In P. americana, the ortholog FXPRLamide (Pea-pyrokinin-5) is not expressed in neurosecretory cells of the SEG. Instead, two sequence-related FXPRLamides were found in these cells [30]. Altogether, the localization of FXPRLamides in neurosecretory cells of the CNS of insects seems to be highly conserved. An obvious exception is the expression of a subset of FXPRLamides in the tritocerebrum of hemimetabolous insects [30], which does not occur in the holometabolous insects studied so far.

PEPTIDE PROCESSING No specific data are available on endoproteolytic enzymes that cleave the predicted FXPRLamides from their precursor proteins. However, mass spectrometry was performed to confirm sequences of peptides, which are cleaved from the precursors as well as to reveal a

PSO

NCC-1

brain

The tritocerebral neurons are neurosecretory as well and project via the NCC-1 into the retrocerebral complex [42]. Neurosecretory neurons in the SEG and abdominal ganglia synthesize the different FXPRLamides in the late embryo/early larval instars, and the immunoreactivity persists throughout development [17, 31, 40]. In contrast, the limited number of interneurons from the brain/SEG/thoracic ganglia that produce the FXPRLamide of the capa-gene may decrease during development, at least in holometabolous insects [17]. A summary of the distribution of FXPRLamides in an insect is given in Fig. 1.

CC/CA PAN

transverse nerve

NCA-2 NCC-3

median nerve

TC

Md

SEG

L Mx

sagittal view

abdominal ganglion

dorsal view

FIGURE 1. Simplified schematic drawing of the distribution of FXPRLamides in the cockroach CNS. The expression in the different neuromers of the SEG is typical of all insects investigated so far. In moths, homolog neurons produce PBAN/DH. The neurosecretory cells in the TC are only known from hemimetabolous insects. In the abdominal ganglion, those neurons are included that first express FXPRLamides during the embryogenesis. Whereas in hemimetabolous insects more FXPRLamide expressing neurons appear during development and project into the PSOs, the expression in holometabolous insects is restricted to homologs of these two cells throughout development. In all insects, however, these cells contain a single FXPRLamide likely cleaved from a capa-gene-related precursor. In cockroaches, labial cells of the SEG send neurites (see arrows) into the ventral nerve cord with side branches emerging in every abdominal ganglion that leave the respective ganglia via the median nerve. CC/CA, corpus cardiacum/corpus allatum. Md/Mx/L, mandibular/ maxillar/labial neuromer of the SEG. NCC, nervus corporis cardiaci. PAN, postallatal nerve. PSO, perisympathetic organ. SEG, subesophageal ganglion. TC, tritocerebrum.

210 / Chapter 32 possible tissue specific distribution of peptides originating from a common precursor. For D. melanogaster, it was found that the predicted Drm-PK-2 from the hugingene is indeed expressed in cells of the SEG, no truncated or extended form was observed in the SEG neurons and/or the respective neurohemal release sites [33]. Identical results were obtained with Drm-PK-1 from the capa-gene that was found in neurosecretory cells of the abdominal ventral nerve cord. Surprisingly, a truncated form of Drm-PK-1 (Drm-PK-12–15) was detected in neurosecretory cells of the SEG and the retrocerebral complex [33]. This truncated form was not adjoined by Drm-PK-1 or other products of the capa-gene (Cap2bs/periviscerokinins), which are always coexpressed in the respective abdominal neurosecretory cells of insects. In the precursor, Drm-PK-1 is flanked by dibasic cleavage sites, whereas the other two peptides encoded on the capa-gene have upstream dibasic cleavage sites but monobasiic downstream signals [16]. Expression of different convertases in the cells of the SEG and abdominal ganglia might be responsible for the tissue specific distribution but does not explain the N-terminal truncation of Drm-PK-1 observed in the labial neurosecretory cells of the SEG. Although the PBAN/DH hormone precursor is known from many moth species, a limited knowledge is available about the peptides that result from precursor processing. So far, only the occurrence of Hez-PBAN gene products has been studied by MALDI-TOF mass spectrometry [18], which revealed that the processing of the PBAN-gene might be more complicated than originally anticipated. A differential distribution of products from the PBAN gene was postulated for neurons of the SEG, and is not limited to only truncated and extended forms of the predicted FXPRLamides. Cell-type specific sorting mechanisms of FXPRLamides were detected in neurosecretory cells and interneurons of P. americana [28]. In interneurons of the brain, Pea-pyrokinin-5 is packed into dense core vesicles, which are separate from the periviscerokinins that are cleaved from the same precursor. In median neurosecretory cells of the abdominal ganglia, however, the different dense core vesicles, containing the putative capa-gene products, fuse with each other in the cytoplasm in a highly regulated heterotypic fusion process and become translucent on their way to the axon hillock. Consequently, Pea-pyrokinin-5 cannot be released separately into the hemolymph, but rather together with periviscerokinins.

RECEPTORS OF IXPRLamides In D. melanogaster, two structurally related FXPRLamide receptors were identified that can be activated

by low concentrations of Drm-pyrokinin-2 [36]. The cDNAs of the annotated receptor genes CG8784 and CG8795 were cloned and expressed in Chinese hamster ovary cells. The cDNAs code for proteins of 658 (CG8784) and 595 amino acid residues (CG8795); both contain seven transmembrane helices, suggesting that they are G protein-coupled receptors (GPCRs). The second D. melanogaster FXPRLamide (Drm-PK-1) demonstrated a weak interaction with these receptors but activates another GPCR (receptor gene CG9918) that needed much higher concentrations of Drm-pyrokinin2 to be activated [6]. Related PBAN GPCRs were cloned and characterized from two moth species—specifically Helicoverpa zea [7] and Bombyx mori [13]. In both cases, the gene was identified based on hypothesized sequence similarity to neuromedin U receptors in vertebrates [see 27]. Tissue distribution analyses revealed that the receptor transcript is specific to the pheromone glands where it undergoes up-regulation the day preceding eclosion. Expressed PBAN receptors respond to PBAN and related peptides by mobilizing extracellular calcium. Thus, FXPRLamides act directly on pheromone glands by using calcium as a second messenger. The B. mori PBAN receptor is structurally and functionally distinct from that of H. zea due to a 67aa C-terminal extension, which mediates internalization of the receptor after PBAN binding [13].

STRUCTURE–ACTIVITY RELATIONSHIPS AND ACTIVE CONFORMATION The C-terminal pentapeptide amide fragment common to the FXPRLamides represents the active core for biological activities such as pheromone biosynthesis, hindgut/oviduct contraction, melanization, and diapause induction [2, 23]; longer sequences are required for a full response. In contrast, the core sequence required to elicit pupariation acceleration in the flesh fly is the C-terminal tripeptide sequence PRLamide [25], although more than trace activity requires longer sequences. The Pro, Arg, and Leu have been shown to be the most critical core residues for pheromonotropic and pupariation activity [25, 26]. Evaluation of a rigid, conformationally restricted, cyclic pyrokinin analog indicates that the active conformation is a transPro, type I β-turn over core residues XPRL for pheromone biosynthesis, hindgut/oviduct contraction, diapause induction, and pupariation acceleration in insects [21, 22, 23, 25]. Subsequent solution conformation studies on more flexible, linear pyrokinins also provide evidence for a turn in the core region [9, 45]. Cyclic, sidechain-to-backbone analogs containing a DPhe in the variable X position of the core have demon-

The FXPRLamide (Pyrokinin/PBAN) Peptide Family / 211 strated antagonism of the pyrokinin PBAN in pheromonotropic assays [2]. Amphiphilic analogs of the pyrokinin core containing various hydrophobic components attached to the N-terminus elicit prolonged biosynthesis of high titers of pheromone in the moth Heliothis virescens following either topical and/or oral delivery [1, 24].

BIOLOGICAL ACTIONS PBAN regulates pheromone biosynthesis in female moths by directly activating receptors (see above) on the pheromone gland cells (see [34]). The signal transduction cascade is initiated by an influx of calcium [10], which leads to production of species-specific sex pheromones via PBAN-induced fatty acid synthesis. Release of DHs can cause embryonic diapause in Bombyx mori, but recent results have shown that the same peptide may be involved in breaking pupal diapause in Helicoverpa armigera [47]. FXPRLamides are also known to accelerate melanin biosynthesis in moths, to influence puparation behavior in flies [44], and to stimulate visceral muscle contractions in locusts and cockroaches (see [2]). In contrast to pheromone gland cells, which usually do not clearly discriminate between different FXPRLamides, the efficacy of the different cockroach pyrokinins differs dramatically in visceral muscle assays [32]. Gene silencing of Drosophila FXPRLamide receptors, as well as ubiquitous ectopic expression, leads to lethal consequences in larvae [20, 36].

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[9] Clark BA, Prestwich, GD. Evidence for a C-terminal turn in PBAN. An NMR and distance geometry study. Int J Pept Protein Res 1996;47:361–8. [10] Clynen E, Baggerman G, Huybrechts J, Vanden Bosch L, De Loof A, Schoofs L. Peptidomics of the locust corpora allata: Identification of novel pyrokinins (FXPRLamides). Peptides 2003;24:1493–500. [11] Davis NT, Homberg U, Teal PEA, Altstein M, Agricola HJ, Hildebrand JG. Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: immunoreactivities to PBAN and other neuropeptides. Microsc Res Technique 1996;35:201–29. [12] Holman GM, Cook BJ, Nachman RJ. Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae. Comp Biochem Physiol C 1986;85:219–24. [13] Hull JJ, Ohnishi A, Moto K, Kawasaki Y, Kurata R, Suzuki MG, Matsumoto S. Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori: Significance of the carboxyl terminus in receptor internalization. J Biol Chem 2004;279:51500–7. [14] Imai K, Konno T, Nakazawa Y, Koiya T, Isobe M, Koga K, Goto T, Yaginua T, Sakakibara K, Hasegawa K, Yamashita O. Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proc Jap Acad 1991;67:98–101. [15] Kawano T, Kataoka H, Nagasawa H, Isogai A, Suzuki A. cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori. Biochem Biophys Res Commun 1992;189:221–6. [16] Kean L, Cazenave W, Costes L, Broderick KE, Graham S, Pollock VP, Davies SA, Veenstra JA, Dow JAT. Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster. Am J Physiol 2002;282:R1297–1307. [17] Loi PK, Tublitz NJ. Sequence and expression of the CAPA/ CAP2b gene in the tobacco hawkmoth, Manduca sexta. J Exp Biol 2004;207:3681–91. [18] Ma PW, Garden RW, Niermann JT, O’Connor M, Sweedler JV. Characterizing the Hez-PBAN gene products in neuronal clusters with immunocytochemistry and MALDI MS. J Insect Physiol 2000;46:221–30. [19] Matsumotu S, Yamashita O, Fonagy A, Kurihara M, Uchiumi K, Nagamine T, Mitsui T. Functional diversity of a pheromonotropic neuropeptide: induction of cuticular melanization and embryonic diapause in lepidopteran insects by Pseudaletia pheromonotropin. J Insect Physiol 1992;38:847–51. [20] Meng X, Wahlström G, Immonen T, Kolmer M, Tirronen M, Predel R, Kalkkinen N, Heino TI, Sariola H, Roos C. The Drosophila hugin gene codes for myostimulatory and ecdysis modifying neuropeptides. Mech Developm 2002;117: 5–13. [21] Nachman RJ, Roberts VA, Dyson HJ, Holman GM, Tainer JA. Active conformation of an insect neuropeptide family. Proc Natl Acad Sci USA 1991;88:4518–22. [22] Nachman RJ, Kuniyoshi H, Roberts VA, Holman GM, Suzuki A. Active conformation of the pyrokinin/PBAN neuropeptide family for pheromone biosynthesis in the silkworm. Biochem Biophys Res Commun 1993;193:661–6. [23] Nachman RJ, Radel PA, Abernathy R, Teal PEA, Holman GM, Yamashita O. Mimetic analog development for the insect pyrokinin/PBAN/diapause induction (FXPRLa) neuropeptide family. In: Suzuki A, Kataoka H, Matsumoto S (Eds.). Molecular Mechanisms of Insect Metamorphosis and Diapause, Industrial Publishing and Consulting, Inc., Tokyo. 1995;97–106. [24] Nachman RJ, Teal PEA, Strey A. Enhanced oral availability/ pheromonotropic activity of peptidase-resistant topical

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secreted from the suboesophageal ganglion of the silkworm Bombyx mori: characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. Proc Natl Acad Sci USA 1993;90:3251–5. Sato Y, Ikeda M, Yamashita O. Neurosecretory cells expressing gene for common precursor for diapause hormone and pheromone biosynthesis-activating neuropeptide in the suboesophageal ganglion of the silkworm, Bombyx mori. Gen Comp Endocrinol 1994;96:27–36. Schoofs L, Veelaert D, Vanden Broeck J, De Loof A. Peptides in the locusts, Locusta migratoria and Schistocerca gregaria. Peptides 1997;18:145–56. Sun JS, Zhang QR, Zhang TY, Zhu ZL, Zhang HM, Teng MK, Niu LW, Xu WH. Developmental expression of FXPRLamide neuropeptides in peptidergic neurosecretory cells of diapauseand nondiapause-destined individuals of the cotton bollworm, Helicoverpa armigera. Gen Comp Endocrinol 2005;141:48–57. Takahashi T, Muneoka Y, Lohmann J, Lopez de Haro MS, Solleder G, Bosch TC, David CN, Bode HR, Koizumi O, Shimizu H, Hatta M, Fujisawa T, Sugiyama T. Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families. Proc Natl Acad Sci USA 1997; 94:1241–6. Tips A, Schoofs L, Paemen L, Ma M, Blackburn M, Raina A, De Loof A. Co-localization of locustamyotropin- and pheromone biosynthesis activating neuropeptide-like immunoreactivity in the central nervous system of five insect species. Comp Biochem Physiol 1993;106A:195–207. Torfs P, Nieto J, Cerstiaens A, Boon D, Baggerman G, Poulos C, Waelkens E, Derua R, Calderon J, De Loof A, Schoofs L. Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei. Eur J Biochem 2001;268:149–54. Verleyen P, Clynen E, Huybrechts J, Van Lommel A, VandenBosch L, De Loof A, Zdarek J, Schoofs L. Fraenkel’s pupariation factor identified. Dev Biol 273:38–47. Wang YS, Kempe TG, Raina AK, Mazzochi PH. Conformation of a biologically active C-terminal hexapeptide analog of the pheromone biosynthesis activating neuropeptide by NMR spectroscopy. Int J Peptide Protein Res 1994;43:277–83. Xu W-H, Denlinger DL. Identification of a cDNA encoding DH, PBAN and other FXPRL neuropeptides from the tobacco hornworm, Manduca sexta, and expression associated with pupal diapause. Peptides 2004;25:1099–106. Zhang T-Y, Sun J-S, Zhang L-B, Shen J-L, Xu W-H. Cloning and expression of the cDNA encoding the FXPRLamide family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera. J Insect Physiol 2004; 50:25–33.

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33 Insect Pigment Dispersing Factor and Bursicon INGE MERTENS, ARNOLD DE LOOF, AND PETER VERLEYEN

pigment migration, since they do not possess epithelial chromophores.

ABSTRACT Insect pigment dispersing factors (PDFs) are neuropeptides that are closely related to the crustacean PDH peptide family. In Drosophila, PDF is an important neurochemical that carries circadian timing information from the central pacemaker’s neurons, the ventrolateral neurons (LNv). PDF mutants display strong arrhythmic locomotor activity, suggesting that PDF is the key outgoing signal for the entrainment of circadian rhythms. PDF mutants also display negative geotaxis behavior and arrhythmic eclosion. The neuropeptide activates a G-protein coupled receptor that belongs to the family of calcitonin and CGRP receptors. Bursicon is responsible for the crucial completion of each molt: It induces hardening and darkening of the new cuticle and wing expansion after the final molt of an insect’s metamorphosis. Bursicon is a heterodimer that activates the Drosophila leucine-rich repeats containing G proteincoupled receptor 2 (DLGR2).

Structure of the Precursor mRNA/Gene The Drosophila PDF-encoding gene is intronless and encodes the 102 amino acid pre-pro-PDF. This precursor contains a 16-amino-acid signal peptide, followed by a sequence of 63 amino acids termed PAP (PDF-associated peptide), followed by the mature 18-amino-acid PDF (Fig. 1). The PAP region contains another cleavage site that could result in peptides of 18 and 43 amino acids [24]. The PAP region is highly diverged from those of the crustacean precursors, whereas the primary structure of the mature PDF is highly conserved among other members of the pigment-dispersing hormone family (Table 1).

Processing The processing sites of the fly’s pre-pro-PDF appear to be different from those of crustacean PDHprecursors. The proteolytic cleavage site preceding the mature PDF consists of triple basic amino acids (RKR), in contrast to the dibasic crustacean cleavage site (KR). Also, the α-amidation signal for crustacean PDHs (GKK) is different from that of the fly’s PDF (GK).

INSECT PIGMENT DISPERSING FACTOR Discovery Pigment dispersing hormones (PDH) were first identified in the shrimp Pandalus borealis [8]. They are secreted from the sinus gland of the eyestalk and are responsible for the daily color change in crustaceans by dispersing the epithelial chromatophoral pigment. They can also function as natural sunglasses by translocating the retinal distal pigment. Related peptides have been identified in insects such as cockroaches, grasshoppers, flies, bugs, moths, and crickets (Table 1). Because their physiological role in insects was not clear, they were named pigment dispersing factors (PDFs). In insects, it is unlikely that PDFs are involved in cuticular Handbook of Biologically Active Peptides

Distribution of the mRNA Drosophila pdf is expressed in a cluster of four neurons in each larval brain hemisphere and these neurons persist during metamorphosis, developing into the small ventrolateral neurons (s-LNv) in the adult brain (Fig. 2). Nerve fibers stemming from these cells project into the superior protocerebrum near mushroom body calyxes, and these neuronal processes are essential for normal rhythmicity. In the vicinity of s-LNv perikarya, another cluster of four pdf-expressing neurons with

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Copyright © 2006 Elsevier

214 / Chapter 33 TABLE 1. Comparison of the primary structure of the mature crustacean pigment dispersing hormones (PDHs) and insect pigment dispersing factors (PDFs). Species

Peptide Sequence

Crustacean PDH Uca pugilator Cancer magister Callinectes sapidus Orconectus limosus Penaeus vannamei Pandalus borealis Insect PDF Gryllus bimaculatus Acheta domesticus Romalea microptera Meimuna opalifera Drosophila melanogaster Phormia regina Musca domestica Bombyx mori

NSELINSILGLP-KVMNDAamide NSELINSILGLP-KVMNDAamide NSELINSILGLP-KVMNDAamide NSELINSILGLP-KVMNEAamide NSELINSLLGIP-KVMNDAamide NSGMINSILGIP-RVMTEAamide NSEIINSLLGLP-KVLNDAamide NSEIINSLLGLP-KVLNDAamide NSEIINSLLGLP-KLLNDAamide NSEIINSLLGLP-KVLNDAamide NSELINSLLSLP-KNMNDAamide NSELINSLLSLP-KNMNDAamide NSELINSLLSLP-KSMNDAamide NADLINSLLALP-KDMNDAamide

SP

PAP

16AA

63AA

RKR

PDF 18AA

NH2

FIGURE 1. Schematic diagram of the Drm-PDF precursor structure (SP = signal peptide, PAP = PDF-associated peptide, PDF = pigment dispersing factor).

larger somata (the large ventrolateral neurons—l-LNv) emerge during metamorphosis. These cells project ipsilaterally and contralaterally into the medulla cortex (Fig. 2) [22]. pdf mRNA levels and PDF immunoreactivities are severely reduced or eliminated in arrhythmic cyc0 or dClkJrk mutant brains, respectively, suggesting that pdf expression is positively regulated by CYCLE and CLOCK. Indeed, the dCLK:CYC heterodimer is a functional unit that activates transcription of target genes via binding to the consensus 6-bp E-box sequence present in these target gene promoters. However, despite the presence of an E-box sequence in the pdf gene promoter, it is definitely not involved in pdf transcriptional regulation, suggesting an indirect regulation by dCLK and CYC [22]. pdf is also regulated posttranslationally by different clock genes such as period and timeless, that are proposed to regulate the PDF-release from the nerve terminals [11, 23]. As in flies and other insect species, PDF-immunoreactive neurons are also found in the accessory medulla of the cockroach Leucophaea maderae [12].

Receptors The Drosophila PDF-receptor is a secretin-related Gprotein coupled receptor (GPCR) that is specifically

responsive to PDF and is most similar to the calcitonin and CGRP receptors in mammals [19].

Biological Actions Circadian clocks are ticking in organisms from bacteria to humans and allow biological events within the organisms to anticipate changing environmental conditions [1, 6, 21]. To impose its rhythm on behaviors such as activity, sleep, and feeding, the oscillating molecules that make up the clock must communicate through some kind of outgoing signals to the brain areas that drive those behaviors [3]. In the fruit fly Drosophila melanogaster, the neuropeptide PDF is the key outgoing signal from the LNv, the primary pacemakers. The Drosophila mutant for PDF was discovered resident among laboratory stocks of long standing [25]. These animals contain a nonsense mutation in the signal sequence of pre-pro-PDF and are protein nulls. The mutant animals appear and behave normally in most respects. PDF neurons are present and appear fully differentiated in the mutant background. However, the circadian clock-regulated behavior of the pdf mutants is highly abnormal: while they entrain to light signals, a large majority of the population display a gradual loss of rhythmicity under constant darkness (DD) during the first three days, becoming completely arrhythmic. pdf mutants

Insect Pigment Dispersing Factor and Bursicon / 215

FIGURE 3. Neobellieria (Sarcophaga) bullata, decapitated shortly after eclosion, sealed with paraffin, and immersed in ethanol four hours later. Flies showed no (left), intermediate or complete (right) darkening of the cuticle. Injection of hemolymph from unmolested flies 30 min after eclosion or CNS extracts of eclosing flies provoke complete darkening in decapitated or neck-ligated flies. (See color plate.)

FIGURE 2. In situ expression of the pdf gene in wholemount CNS. (A–C) PDF immunohistochemistry. (A) Wildtype third-instar larval CNS. Normally, PDF is expressed in four lateral neurons (LN) in each brain lobe, and four to six neurons in the abdominal ganglion (Ab). (B) dClkJrk/+ heterozygous third-instar larval CNS. These PDF-immunosignals are indistinguishable from those of wild-type. (C) PDF-immunoreactive neurons in the adult brain from dClkJrk/+ heterozygotes. These immunostaining patterns are also identical to those of wild-type adult brains. Two clusters of neurons (sLNv and l-LNv) are clearly visible in the anterior view (left). In the posterior view (right), dorsal projection from s-LNv is indicated by an arrowhead. (D, E) pdf promoter-driven lacZ reporter expression detected by X-gal histochemistry of thirdinstar larval CNS. (D) Control. The lac-Z gene expression patterns are identical to those observed by Jrk PDF-immunohisJrk tochemistry shown in A. (E) dClk /dClk homozygotes. Consistent with previous pdf in situ hybridization and immunohistochemistry results reported elsewhere [23], pdf promoter-mediated lac-Z expression is absented only from the lateral neurons. At least five brain specimens were processed and examined for each panel. Reproduced with kind permission from Humana Press Inc. from Park, J. H. (2002), Downloading central clock information in Drosophila, Mol. Neurobiol. 26, 217–233. (See color plate.)

also display a strong negative geotaxis behavior [26]. Ablation of the pdf neurons in an otherwise wild-type background produced a behavioral phenotype that was in all respects comparable to that produced by pdf mutant flies [25]. PDF also seems necessary for the clock of the prothoracic gland (PG), a tissue required for fly development, and eclosion rhythms [20]. It is possible that the role of the LNvs is to provide, via PDF, a signal to the PG clock. For instance, in per0 and tim0 flies, PDF is no longer released in a rhythmic way from the dorsal LN projections, and eclosion is arrhythmic as well [23]. Also in pdf mutant flies, not only locomotor activity but also eclosion is arrhythmic, which indicates that the endogenous clock inside the PG cannot function without PDF in the fly. However, there may also be other inputs to the PG, such as light.

BURSICON Discovery The biological action of bursicon was simultaneously described by two different groups in 1962 [4, 9]. It was demonstrated that a neck ligation of flies within minutes after eclosion prevented tanning and sclerotization of the cuticle. Injection of such flies with either extracts from the central nervous system (CNS) or hemolymph from a tanning fly could still induce tanning and sclerotization (Fig. 3). The presumed principle causing this effect was later given the name “bursicon,” which is derived from the Greek bursikos and means “pertaining to tanning” [10]. Finally, after 43 years and countless unsuccessful attempts, again two groups simultaneously revealed the structure of this hormone [17, 18]. This finding was preceded by many large-scale purifications of bursicon from the CNS of different insect species, each time predicting an approximate MW of ±30 kDa. The purification attempts accumulated in the retrieval of five partial

216 / Chapter 33 amino acid sequences. These sequences were derived by trypsin digestion of a bioactive spot, obtained after high-performance liquid chromatography followed by two-dimensional gel electrophoresis of homogenates of 2850 nerve cords of the cockroach Periplaneta americana [13]. Three of the five sequences could be aligned with the Drosophila melanogaster gene CG13419 (Fig. 4) [5]. This CG13419 encodes a polypeptide of 15 kDa and belongs to the cystine knot family, which includes vertebrate glycoprotein hormones. The fact that mutations of CG13419 caused defects in cuticle sclerotization and wing expansion sustained the hypothesis of bursicon being a homodimer of this gene product. The finding

that cells transfected with CG13419 cDNA were devoid of activity in the bursicon bioassay and failed to stimulate the putative bursicon receptor DLGR2 motivated two research groups to further investigate this enigmatic neurohormone [17, 18]. One group discovered that the CG13419 ortholog of the honey bee Apis mellifera was fused with a comparable cystine knot protein in a single open reading frame. This second cystine knot protein appeared to be the ortholog of the Drosophila CG15284 gene product [18]. The other group actively searched a dimerization partner on Genbank for CG13419, since most glycoproteins are heterodimeric, and found the same CG15284 [17]. Interestingly, the two remaining

bm ag dm am dm ag bm

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MSVLNTFLVIVALILCYVNDFPVTG----------------------HEVQLPPGTKFFC MKSTFLVVLELAFFLLPGRVLYA------------------------QKDSEDGGSHYSS MLRHLLRHENNKVFVLILLYCVLVSILKLCTA-----------QPDSSVAATDNDITHLG MKENFSIMFIHSIFLILIIFIYSNETIA-------------------------------MHVQELLFVAAILVPQCLRA------------------------------------LRYS MCNSVRTALAASNCCSIVLCCVLLLTLTLTVAVTA------------------------MNIMITKIFFLVQLFYIVVSKSSA------------------------------------

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Bursα -----------------------------------------------------------Burs TKCEGFCNSQVQPSVASTTGFSKECYCCRESYLKERHITLHHCYDADGIKLMNEENGVME Bursβ NKCEGLCNSQVQPSVITPTGFLKECYCCRESFLKEKVITLTHCYDPDGTRLTSPEMGSMD Bursβ NKCEGKCNSQVQPSVITATGFLKECYCCRESFLRERQLQLTHCYDPDGVRMTDHESATME Bursβ NKCEGMCNSQVHPSISSPTGFQKECFCCREKFLRERLVTLTHCYDPDGIRFEDEENALME Burs ESFLR LTQEGQASME

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PQEVAGYSEEGPLYN--HFRKS--L PQELTSFADEGTLTGY--FQKSHYKSIE PQEIAGYSDEGPLNN--HFRRIA-LQ PQEIAGFADEGPFTTSAHFRRSSDLQ

126 122 134 118

160 163 173 269

FIGURE 4. Alignments of insect bursicon proteins. CG13419 and CG15284 orthologs (called Bursα and Bursβ, respectively) from mosquito (ag: Anopheles gambiae), silkworm (bm: Bombyx mori), and fruit fly (dm: Drosophila melanogaster) were aligned along the bee (am: Apis mellifera) single-chain multidomain bursicon. The tryptic peptides identified in bursicon preparations from the cockroach (pa: Periplaneta americana), a heterometabolic insect, are also aligned. Conserved sequences are shaded in gray, respectively. Residues identical among three, four, or five species are dark shaded, while residues identical between two species are pale shaded. The putative signal peptides are shown at the left side on the top of the figure. GenBank accession: Silkworm Bursα (BN000691), Bursβ (BN000690); mosquito Bursα (AY735442), Bursβ (BN000689); and bee singlechain bursicon (BN000692). Reproduced with kind permission from FEBS Letters from F. M. Mendive et al. (2005), Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2, FEBS Lett. 579, 2171–2176.

Insect Pigment Dispersing Factor and Bursicon / 217 partial peptide sequences, obtained from cockroach nerve cords, could be aligned with the second cystine knot protein (Fig. 4). Moreover, cells cotransfected with both genes (named burs and pburs or Bursα and Bursβ) produced convincing activity in the bursicon bioassay and stimulated the DLGR2 receptor. In addition, the transcript levels for CG13419, CG15284, and DLGR2 showed a similar pattern of regulation, peaking shortly before eclosion. Thus, ample data confirmed that bioactive bursicon is the heterodimer of Drosophila CG13419 and CG15284 [17, 18].

Structure of the Precursor mRNA/Gene Drosophila CG13419 and CG15284 consist of three and two exons, respectively, separated by rather short introns. The orthologs of CG15284 in Anopheles, Bombyx, or Apis also consist of two or three exons. Bombyx exon 1 and 2 are fused in Drosophila, and Bombyx exon 2 and

3 are fused in Anopheles [17]. Interestingly, the Apis orthologs of CG15284 and CG13419 are found in a single gene, whereas both are clearly encoded in distinct loci in the other available insects [18].

Distribution of the mRNA Transcript levels for CG13419 and CG15284 are both low in the first two larval stages of Drosophila and gradually increase in the pupal stages. Their highest levels are reached before eclosion, only to drop again in the adult stage [17]. Long before the structure of bursicon was known, it was demonstrated that certain CCAPimmunoreactive cells possessed bursicon activity [16]. The identification of CG13419 as (a part of) bursicon allowed in situ hybridization. CG13419 appeared to be present in almost all CCAP-immunoreactive bilateral neurons of the Drosophila third instar CNS (Fig. 5A–C) [5]. Unexpectedly, the mRNA of CG15284 is only

FIGURE 5. Localization of bursicon mRNA (CG13419 or CG15284) in CCAP neurons of the CNS of third instar larval D. melanogaster. (A) In situ labeling of bursicon mRNA (CG13419). Bursicon is found in distinct bilateral neurons in the ventral nervous system (VNS). (B) Corresponding pattern of CCAP immunoreactivity in the same preparation. (C) Superimposition of (A) and (B) showing that bursicon RNA is expressed in all CCAP-immunoreactive neurons except for two pairs in the brain (Br) and two pairs in the first thoracic neuromere (arrow and arrowhead, respectively). (D and E) Detection of bursicon mRNA (CG13419) in the CNS of control (D) and CCAP knockout (E) larvae. (G and H) D. melanogaster in situ labeling of bursicon mRNA (CG15284) in four bilateral neurons in the VNS (G). These signals were absent in the ventral nervous system of flies bearing targeted ablations of CCAP neurons (H). Scale bars 75 μm (A–E) and 100 μm (G–H). Reproduced (G–H) from Luo, C. W. et al. (2005), Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2, PNAS 102, 2820–2825, with permission (Copyright 2005 National Academy of Sciences, USA), and (A–E) from Dewey, E. M. et al. (2004), Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading, Curr. Biol. 14, 1208–1213, with kind permission from Elsevier. (See color plate.)

218 / Chapter 33 present in no more than four bilateral neurons, which express CG13419 (Fig. 5G) [17]. These results were confirmed by the absence of bursicon mRNAs in transgenic Drosophila bearing targeted ablations of CCAP neurons (Fig. 5E, H) [5, 17].

sustaining the heterodimeric bioactive bursicon. The presence of posttranslational modifications such as phosphorylations cannot be excluded yet [17].

Receptors Surprisingly, the bursicon receptor was identified a few years before its ligand upon investigation of Drosophila flies that are mutant for the rk locus [2]. These mutations affect the rickets gene, resulting in a viable phenotype with folded wings, weak postecdysial sclerotization and melanization, and kinked femurs (hence the name of these mutants). The rickets gene encodes

Processing The two Drosophila bursicon precursors have a signal peptide followed by the mature protein. Both contain 11 cysteine residues, which form intramolecular cystines, resulting in the typical cystine knot structure (Fig. 6B) and, most probably, intermolecular cystines

A

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FIGURE 6. Predicted sequences, gene structure, and phylogenetic analyses of pburs from different insects. (A) Sequence alignment of pburs (CG15284) from different insects and burs (CG13419) from D. melanogaster (D.m.). Conserved residues are shaded, and cysteine residues are numbered. A.g., Anopheles gambiae; A.m., Apis mellifera; B.m., Bombyx mori. A stretch of four residues (boxed) in D.m. pburs is identical to that of a fragment purified from protease-treated bursicon from P. americana. (B) Predicted cystine knot structure of pburs. (C) Intron–exon arrangement of pburs genes in different insects. Exons are represented by boxes. The numbers of amino acid residues for each exon and nucleotides for each intron are shown. (D) Phylogenetic tree of D.m. pburs and burs together with seven human BMP antagonists. Reproduced from Luo, C. W. et al. (2005), Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2, PNAS 102, 2820–2825, with permission (Copyright 2005 National Academy of Sciences, USA).

Insect Pigment Dispersing Factor and Bursicon / 219 the glycoprotein hormone receptor DLGR2. This Drosophila leucine-rich repeats-containing G-proteincoupled receptor 2 (DLGR2) was detected and cloned based on homology with DLGR1 [7]. Both show striking structural homology with members of the vertebrate glycoprotein hormone receptor family, like a seven transmembrane region and a very large extracellular amino terminus. The phenotype of the rickets mutants is very similar to the phenotype induced by neck-ligation immediately after eclosion. The mutants have bioactive bursicon in their hemolymph 15 min after eclosion, whereas their phenotype cannot be rescued by injection of bursicon extracts or of an analog of cyclic AMP, the suggested second messenger for bursicon. From these results it was concluded that DLGR2 is the bursicon receptor [2].

Biological Actions Bursicon has been mainly studied in flies, where it is released into the hemolymph shortly after eclosion. However, the presence of bursicon was shown in many insects, including those without a complete metamorphosis. Bursicon, for example, appears in the hemolymph of the cockroach Periplaneta americana after each molt [9]. Drosophila and blowflies are interesting models in which to study the biological role of bursicon because of the delay between the emergence of an adult and the subsequent wing expansion and cuticular tanning. Based on these models, bursicon is thought to be involved in both the transient plasticization, allowing the cuticle and wings to expand, and the following hardening and darkening of the expanded cuticle. In addition, there is strong evidence that bursicon triggers the programmed death of epidermal wing cells during wing spreading [15]. Finally, the process of sclerotization or tanning of the new cuticle itself is mediated by diphenolic compounds derived from tyrosine. These compounds permeate the cuticle and serve as precursors for quinonoid derivatives that both sclerotize and pigment the exoskeleton [14].

References [1] Allada R, Emery P, Takahashi JS, Rosbash M. Stopping time: The genetics of fly and mouse circadian clocks. Ann Rev Neurosci 2001;24:1091–1119. [2] Baker JD, Truman JW. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J Exp Biol 2002;205:2555–2565. [3] Barinaga M. Circadian rhythms: Possible clock messenger identified. Science 2004;286:2434–2436. [4] Cottrell CB. The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties. J Exp Biol 1962;39:413–430.

[5] Dewey EM, McNabb SL, Ewer J, Kuo GR, Takanishi CL, Truman JW, et al. Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading. Curr Biol 2004;14:1208–1213. [6] Dunlap JC. Molecular bases for circadian clocks. Cell 1999; 96:271–290. [7] Eriksen KK, Hauser F, Schiott M, Pedersen KM, Sondergaard L, Grimmelikhuijzen CJ. Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster. Genome Res 2000;10: 924–938. [8] Fernlund P. Structure of a light-adapting hormone from shrimp, Pandalus borealis. Biochim Biophys Acta 1976;439: 17–25. [9] Fraenkel G, Hsiao C. Hormonal and nervous control of tanning in the fly. Science 1962;138:27–29. [10] Fraenkel G, Hsiao C. Bursicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J Insect Physiol 1965;11:513–556. [11] Helfrich-Förster C, Täuber MP, Mühlig-Versen M, Schneuwly S, Hofbauer A. Ectopic expression of the neuropeptide pigment dispersing factor alters behavioral rhythms in Drosophila melanogaster. J Neurosci 2004;20:3339–3353. [12] Homberg U, Reischig T, Stengl M. Neural organization of the circadian system of the cockroach Leucophaea maderae. Chronobiol Int 2003;20:577–591. [13] Honegger HW, Market D, Pierce LA, Dewey EM, Kostron B, Wilson M, et al. Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone. J Comp Neurol 2002;452: 163–177. [14] Hopkins TL, Kramer KJ. Insect cuticle sclerotization. Ann Rev Entomol 1992;37:273–302. [15] Kimura K, Kodama A, Hayasaka Y, Ohta T. Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster. Dev 2004;131:1597–1606. [16] Kostron B, Kaltenhauser U, Seibel B, Braunig P, Honegger HW. Localization of bursicon in CCAP-immunoreactive cells in the thoracic ganglia of the cricket Gryllus bimaculatus. J Exp Biol 1996;199:367–377. [17] Luo CW, Dewey EM, Sudo S, Ewer J, Hsu SY, Honegger HW, et al. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G proteincoupled receptor LGR2. Proc Natl Acad Sci USA 2005;102: 2820–2825. [18] Mendive FM, Van Loy T, Claeysen S, Poels J, Williamson M, Hauser F, et al. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2. FEBS Lett 2005;579:2171–2176. [19] Mertens I, Vandingenen A, Johnson EC, Shafer OT, Li W, Trigg JS, et al. PDF receptor signalling in Drosophila contributes to both circadian and geotaxic behaviors. Neuron 2005;48:213–219. [20] Myers EM, Yu JJ, Sehgal A. Circadian control of eclosion: Interaction between a central and peripheral clock in Drosophila melanogaster. Curr Biol 2003;13:526–533. [21] Panda S, Hogenesch JB, Kay SA. Circadian rhythms from flies to human. Nature 2002;417:329–335. [22] Park JH. Downloading central clock information in Drosophila. Mol Neurobiol 2002;26:217–233. [23] Park JH, Helfrich-Förster C, Lui L, Rosbash M, Hall J. Differential regulation of circadian pacemaker output by separate clock genes in Drosophila melanogaster. Proc Natl Acad Sci USA 2000;97:3608–3613.

220 / Chapter 33 [24] Park JH, Hall JC. Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster. J Biol Rhyth 1998;13:219–228. [25] Renn SC, Park JH, Rosbash M, Hall J, Taghert PH. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause

severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 1999;99:791–802. [26] Toma DP, White KP, Hirsch J, Greenspan RJ. Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nature Gen 2002;31:349–353.

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34 Crustacean Bioactive Peptides R. KWOK, S. M. CHAN, AND S. S. TOBE

model systems. For this reason they are covered in a separate chapter.

ABSTRACT The crustacean subphyla is made up of over 30 orders, though research has concentrated mainly on the decapods and the terrestrial isopods. A growing number of peptides have been sequenced from these two orders, the majority from decapods. As is typical of bioactive peptides in other taxa, crustacean peptides regulate a range of physiological functions, including color change, metabolic function, metamorphosis, development, and reproduction.

PROCTOLIN Discovery The first neuropeptide sequenced in insects was the peptide proctolin (Arg-Tyr-Leu-Pro-Thr) [6]. In crustaceans, the presence of proctolin was confirmed in the lobster, Homarus americanus and later in the crab, Carcinus maenas (see citation in Table 1).

INTRODUCTION Biological Actions As observed in other arthropod subphyla, the sequencing of several insect genomes and the refinement of sensitive mass spectrometric techniques has led to a rapid increase in the number of identified peptide sequences in crustacean species. Yet, of the numerous orders that compose the subphylum Crustacea, bioactive peptides have only been isolated/identified from species belonging to two: the Decapoda and Isopoda (see Table 1). The vast majority of identified sequences are from decapod species, with the terrestrial isopod, Armadillidium vulgare being the only nondecapod crustacean. This is a clear reflection of past and present research interests, but it underscores our lack of knowledge of bioactive peptides from other crustacean orders and in crustaceans in general. The first sequenced crustacean neuropeptides were the chromatophorotrophic hormones, pigment dispersing hormone (PDH) and red pigment concentrating hormone (RPCH). These two peptide families, along with the crustacean hyperglycemic hormone family, have been studied extensively in crustacean Handbook of Biologically Active Peptides

Although the name proctolin was derived from its potent myogenic effects on the hindgut (proctodeum), proctolin also has myogenic effects on insect cardiac, skeletal, and visceral muscle contraction [6, 8—includes review of structure-activity relationship]. In crustaceans, proctolin has been identified as one of many neuropeptides present in the stomatogastric nervous system (STNS), where it is involved in modulating neural pattern generation [6].

CRUSTACEAN CARDIOACTIVE PEPTIDE Discovery Crustacean cardioactive peptide (CCAP; ProPhe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2), initially isolated from C. maenas, is a cyclic nonapeptide that contains a disulfide bond between two cysteine residues in positions three and nine [2]. In crustaceans, CCAPimmunoreactive material has been localized within the

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Neuropeptide sequences in crustacean species identified by various purification and sequencing methodologies or from cloned pre-propeptides. Taxonomy refers to infraorder in all cases except the Penaeoidea, which is classified as a superfamily.

Family

Subfamily

PDH/PDF

PDH

Common Sequence

Species (# of Sequences)

N-S-E-X-I-N-S-X-L-X-XLitopenaeus vannamei (1) P-L-X-X-X-X-A -NH2 Marsupenaeus japonicus (2) Penaeus aztecus (1) Pacifastacus leniusculus (1) Procambarus clarkii (1) Orconectes immunis (1) Orconectes limosus (1) Cancer magister (1) Callinectes sapidus (2)

Taxonomy (Infraorder) Penaeoidea

Astacidea

Brachyura

Carcinus maenas (1)

RPCH/AKH

RPCH

Proctolin

Proctolin

Opioid CHH

Enkephalin CHH—subtype

MIH—subtype

pQ-X-X-F-X-X-X-W -NH2

R-Y-L-P-T Y-G-G-F-M/L subtype A and B

subtype A and B (includes MOIH, GIH, VIH)

Uca pugilator (1) Pandalus borealis (1) Pandalus jordani (1) Armadillidium vulgare (1) Orconectes limosus (1) Cancer magister (1) Carcinus maenas (1) Pandalus borealis (1) Homarus americanus (1) Carcinus maenas (1) Carcinus maenas (2) Litopenaeus vannamei (1) Metapenaeus ensis (>10) Marsupenaeus japonicus (6) Penaeus monodon (>5) Penaeus vannamei (2) Procambarus clarkii (5) Orconectes limosus (1) Homarus americanus (2) Cancer pagurus (>6) Carcinus maenas (>6) Libinia emarginata (2) Macrobrachium rosenbergii (4) Armadillidium vulgare (1) Metapenaeus ensis (2) Marsupenaeus japonicus (1) Homarus americanus (1) Nephrops norvegicus (1) Charybdis feriatus (2) Cancer pagurus (3) Callinectes sapidus (1) Macrobrachium rosenbergii (2) Armadillidium vulgare (1)

Caridea Oniscidea Astacidea Brachyura Caridea Astacidea Brachyura Brachyura Penaeoidea

Astacidea

Reference Desmoucelles-Carette et al., 1996, Biochem. Biophys. Res. Commun. 25;221(3):739–43. Ohira et al., 2002, Mar. Biotechnol. 4:463–470. Phillips et al., 1988, Soc. Neurosci. Abstr. 14:534. Rao and Rhiem, 1993, Ann. New York Acad. Sci. 680:78–88. McCallum et al., 1991, Pigment Cell Res. 4:201–208. Rao and Rhiem, 1993, Ann. New York Acad. Sci. 680:78–88. De Kleijn et al., 1993, FEBS Lett. 321:251–255. Kleinholz et al., 1986, Biol. Bull. (Woods Hole) 170:135–143. Klein et al., 1994, Biochem. Biophys. Res. Commun. 205:410–416. Klein et al., 1992, Biochem. Biophys. Res. Commun. 189:1509–1514. Rao and Riehm, 1989, Biol. Bull. 177:225–229. Fernlund, 1976, Biochem. Biophys. Acta 439:17–25. Rao et al., 1985, PNAS USA 82:5319–5322. Knowles, 1992, Master thesis. U. of West Florida. Gauss et al., 1990, J. Comp. Physiol. B. 160:373–379.

Fernlund and Josefsson, 1972, Science 177:173–175. Schwarz et al., 1984, J. Neurosci. 4:1300–1311. Stangier et al., 1986, Peptides 7:67–72. Luschen et al., 1991, PNAS USA 88:8671–8675. Note: incomplete list for the CHH/MIH/GIH peptide family. These examples show the distribution of known CHHs in different infraorders. For refs. and a description of the subtypes see Chan et al., 2003; Gen. Comp. Endocrin. 134:214:219; for accession numbers see Chen et al., 2005 Marine Biotech. First examples of CHH/MIH/ GIH and MOIH cited below.

Brachyura First CHH (C. maenas): Kegel et al., 1989, FEBS Lett. 255(1):10–4. Caridea Oniscidea Penaeoidea Astacidea Brachyura

Caridea Oniscidea

First MIH and GIH (H. americanus): Chang et al., 1990, Biochem. Biophys. Res. Commun. 171(2):818–826; Soyez et al., 1991, Neuropeptides 20(1):25–32. First MOIH (C. pagurus): Wainwright et al., 1996, J. Biol. Chem. 271(22):12749–12754.

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TABLE 1.

CCAP

CCAP

Orcokinin

Orcokinin

P-F-C-N-A-F-T-G-C– -NH2 Orconectes limosus (1) Homarus americanus (1) Carcinus maenas (1) N-F-D-E-I-D-R-X-X-F-G-F-X Procambarus clarkii (1) Orconectes limosus (2) Cherax destructor (5) Cancer borealis (3)

Orcomyotropin I Orcomyotropin II

Carcinus maenas (3) F-D-A-F-T-T-G-F -NH2 Orconectes limosus (1) F-D-A-F-T-T-G-F-G-H-S Procambarus clarkii (1) Cherax destructor (1) Cancer borealis (1)

RFamide

FLRFamide

F/X-L-R-F -NH2 Penaeus monodon (6) Procambarus clarkii (2) Homarus americanus (2) Cancer borealis (6) Callinectes sapidus (1) Macrobrachium rosenbergii (8)

Sulfakinin NPF

G-R-F -NH2 Callinectes sapidus (2) G-Y-R-K-P-P-F-N-G-S-I-F -NH2 Penaeus monodon (1) Procambarus clarkii (1) Cancer borealis (1)

Tachykinins

Tachykinin

Allatostatins

Allatostatins

Kinins

Kinins

Pyrokinin/ PBAN Corazonin

Pyrokinin Corazonin

F-X-G-X-R -NH2 Penaeus monodon (1) Procambarus clarkii (1) Cancer borealis (2) Y/F-X-F-G-L/I -NH2 Penaeus monodon (41) Orconectes limosus (3) Cancer borealis (9) Carcinus maenas (20) F-X-X-W-G -NH2 Litopenaeus vannamei (2) Cancer borealis (2) F-X-P-R-L -NH2 Litopenaeus vannamei (2) pQ-T-F-Q-Y-S-R-G-W-T-N -NH2 Cancer borealis (1)

Brachyura Astacidea

Brachyura

Astacidea

Brachyura Penaeoidea Astacidea Brachyura

Caridea Penaeoidea Penaeoidea Brachyura Brachyura Penaeoidea

Stangier and Keller, 1990, see citation in Dircksen, 1998. Schwiemann, 1999, see citation in Dircksen, 1998. Stangier et al., 1987, PNAS USA 84(2):575–579. Yasuda-Kamatani and Yasuda, 2000, Gen. Comp. Endocrinol. 118:161–172. Stangier et al., 1992, Peptides 13:859–864; Burdzik et al., 1993. Skiebe et al., 2002, J. Comp. Neurol. 444:245–259. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Bungart et al., 1995, Peptides 16:67–72. Dircksen et al., 2000, J. Exp. Biol. 203(18):2807–2818. Yasuda-Kamatani and Yasuda (2000), Gen. Comp. Endocrinol. 118:161–172. Skiebe et al., 2002, J. Comp. Neurol. 444:245–259. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Sithigorngul et al., 2002, Comp. Biochem. Physiol. B. 131:325–337. Mercier et al., 1993, Peptides 14:137–143. Trimmer et al., 1987, J. Comp. Neurol. 266(1):1–15. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Krajniak 1991, Peptides 12:1295–1302. Sithigorngul et al., 1998, Comp. Biochem. Physiol. B. 120:587–595; Sithigorngul et al., 2001, Peptides 191–197. Torfs et al., 2003, Biochem. Biophys. Res. Commun. 299:312–320. Johnsen et al., 2000, Eur. J. Biochem. 267:1153–1160. Sithigorngul et al., 2002, Peptides 23:1895–1906. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Yasuda, 1993, Comp. Biochem. Physiol. B. 104(2):235–40.

Penaeoidea Brachyura Penaeoidea

Sithigorngul et al., 2002, Comp. Biochem. Physiol. B. 131:325–337. Yasuda et al., 2004, Gen. Comp. Endocrinol. 135(3):391–400. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Nieto et al., 1998, Biochem. Biophys. Res. Commun. 248(2):406–411. Yasuda-Kamatani, 2004, Eur. J. Biochem. 271:1546–1556. Christie et al., 1997, J. Exp. Biol. 200(17):2279–2294. Duve et al., 2002, Peptides 23:1039–1051. Dircksen et al., 1999, Peptides 20:695–712. Huybrechts et al., 2003, Biochem. Biophys. Res. Commun. 308:535–544. Duve et al., 1997, Eur. J. Biochem. 250:727–734. Nieto et al., 1998, Biochem. Biophys. Res. Commun. 248(2):406–411. Li et al., 2003, J. Neurochem. 87:642–656. Torfs et al., 2001, Eur. J. Biochem. 268(1):149–154.

Brachyura

Li et al., 2003, J. Neurochem. 87:642–656.

Astacidea Brachyura Penaeoidea Astacidea Brachyura Penaeoidea Astacidea Brachyura

Crustacean Bioactive Peptides / 223

SIFamide

Antho-RFamide-like SIFamide

Y(So(3)H)-G-H-M-R-F -NH2 Litopenaeus vannamei (2) Penaeus monodon (3) R-P-R-F -NH2 Penaeus monodon (4) Cancer borealis (1)

Astacidea

224 / Chapter 34 CNS and hemolymph in a number of decapods [2]. CCAP or peptides with similar structure are also present in insects and molluscs [24].

in vivo has been difficult to determine. A range of actions, including effects on dark adaptation, on skeletal muscles, and on aspects of the circulatory and digestive systems, have previously been reviewed [11].

Biological Actions The peptide CCAP, named for its excitatory activity on the crustacean heart, has been shown to have excitatory effects on other invertebrate muscles [2]. It is released into the hemolymph in large amounts during ecdysis, when it is likely involved in coordinating a number of physiological and behavioral processes, including the regulation of hemolymph pressure and flow, the potentiation of muscular force, and postecdysial ventilatory behavior to increase hemolymph PO2 [15]. Within the nervous system, CCAP is involved in the modulation of the stomatogastric and swimmerette pattern generating circuits, as well as direct modulatory effects on motor neurons [24].

RFamide PEPTIDES There are many peptides with the C-terminus Arg-Phe-NH2 (RFamide), and these may or may not be evolutionarily related to the original tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) isolated from the mollusc, Macrocallista nimbosa [16]. At present there is no general consensus as to which peptides compose a family of FMRFamide-related peptides (FaRPs) or FMRFamide-like peptides (FLPs), as members continue to be identified by both antigen cross-reactivity, as well as consensus sequence. This section will include those crustacean peptides possessing a C-terminal Arg-PheNH2 (RFamide). Discussion of the ancestral relatedness of these peptides is beyond the scope of this chapter.

FLRFamide Discovery The majority of isolated crustacean FLPs share the C-terminal sequence Phe/Xxx-Lys-Arg-Phe-amide (FLRFamide). Two FLRFamides isolated from H. americanus were the first identified crustacean RFamides, and other FLRFamides have since been identified in the crayfish Procambarus clarkii, the shrimp Penaeus monodon and Macrobrachium rosenbergii, and the crabs Cancer borealis and Callinectes sapidus (see refs. in [11]).

SULFAKININS Members of the sulfakinin peptides have been identified from two tropical shrimp species, Litopenaeus vannamei and P. monodon (see [20]). The sulfakinins, first discovered in the cockroach Leucophea maderae, share the common C-terminal sequence Asp-Tyr-GlyHis-Met-Arg-Phe-amide, in which the Tyr residue is sulfated [12]. One of the sulfakinin homologs from L. vannamei has two sulfated tyrosyl residues [20].

NEUROPEPTIDE F Pancreatic polypeptide (PP) and the related peptides, neuropeptide Y (NPY) and peptide YY (PYY), are distributed throughout the vertebrates. The first invertebrate NPY/PYY-like peptide was isolated from a flatworm and designated neuropeptide F (NPF). NPF homologs have since been identified in gastropods and insects. All consist of about 35 residues and are similar in length to the vertebrate NPY/PYYs (see refs. in [19]). The first crustacean NPF-like peptide was isolated from P. monodon by use of an antiserum raised against the C-terminal hexapeptide of PP [19]. These NPF-like peptides (designated Pem-PYFs) are shorter, having only 6–10 residues, but share a high sequence identity with the C-terminus of the longer NPFs. Other short NPFs (designated sNPF in [23]) have been identified from a cephalopod and an insect [19]. Furthermore, in Drosophila melanogaster, a putative precursor has been identified by database search that contains four sNPFs [23]. Although sharing a high sequence identity with the long NPFs, the sNPFs may represent a separate group of peptides, since they do not appear to be recognized by the long NPF receptors [3].

ANTHO-RFamide-LIKE Two short peptides isolated from the crab C. sapidus end in Gly-Arg-Phe-amide. One sequence is identical to a coelenterate neuropeptide Antho-RFamide [26].

OPIOID–ENKEPHALIN

Biological Actions

Discovery

Numerous effects have been demonstrated for FLRFamides in vitro in crustaceans, but whether they occur

Despite numerous reports of opioid immunoreactivity in invertebrate tissues and accounts of opioid effects in

Crustacean Bioactive Peptides / 225 invertebrate assays, enkephalins (Tyr-Gly-Gly-Phe-Met/ Leu) have been isolated from only three invertebrates: a mollusc, an annelid, and a crustacean (C. maenas) (see Table 1; [7]). Both [Leu]-enkephalin and [Met]enkephalin, identical to the vertebrate sequences, were isolated from extracts of thoracic ganglia of C. maenas.

Receptors Three receptor types resembling the vertebrate δand κ-type opioid receptors have been characterized pharmacologically in nervous tissues (eyestalk and thoracic ganglia) of C. maenas [4]. These receptors bind both [Leu]- and [Met]-enkephalins, which are competitively displaced by a number of opioids and antagonists.

Biological Actions A number of effects have been reported for the enkephalins in crustaceans, which include the stimulation of release of chromatophorotrophins, the slowing of ovarian maturation, and the regulation of carbohydrate metabolism (see refs. in [7]). The effects of the enkephalins on hemolymph glucose concentration appear to be species dependent, as the enkephalins have been reported to cause both hypoglycemia (e.g., P. clarkii, Uca pugilator) and hyperglycemia (e.g., Oziotelphusa senex senex, Penaeus indictus, Metapenaeus monocerus). These glycemic effects appear to be regulated through inhibition or stimulation of the release of CHHs from the sinus gland [7].

ORCOKININ AND ORCOMYOTROPIN Discovery The orcokinins are a family of peptides that until recently had only been identified in crustaceans ([14] and references therein). The first orcokinin was isolated from ventral nerve cord extracts of the crayfish, Orconectes limosus, and subsequently, several other homologs have been isolated from O. limosus hindgut extracts, as well as from a number of other crustaceans (C. maenas, Cherax destructor, P. clarkii, and C. borealis) [14]. Recently, the first insect orcokinin has been identified in the cockroach Blattella germanica [14]. The same authors also reported putative homologs of the crayfish pre-pro-orcokinin identified by database searches in the mosquito Anopheles gambiae and in the nematode Caenorhabditis elegans. A functionally related peptide named orcomyotropin, of which there are two homologs, shares slight sequence similarity with the orcokinins (see refs. in

[14]). A copy of orcomyotropin is present on the each of two genes coding for P. clarkii pre-pro-orcokinins [29].

Structure of the Precursor mRNA/Gene Two genes code for the orcokinin precursor proteins in P. clarkii [29]. Preproorcokinin A contains seven copies of orcokinin, two copies of [Val13]orcokinin, single copies of two other homologs, and a single copy of orcomyotropin. Preproorcokinin B has the same structure as preproorcokinin A but contains an additional copy of orcokinin.

Biological Actions The myotropic effects of the orcokinins are most pronounced on O. limosus hindgut, although stimulatory activity is also seen on the O. limosus heart and C. borealis hindgut [14]. Orcomyotropin is more potent than the orcokinins on the O. limosus hindgut contraction assay. Orcokinin-like immunoreactivity has been localized in the CNS, STNS, neurohemal structures, and in the hemolymph, suggesting both neural and hormonal roles. Exogenous application of orcokinins to the STNS has been shown to affect pyloric rhythm in H. americanus [14].

ALLATOSTATIN Discovery The term allatostatin (AST) describes a peptide that has an inhibitory effect on the insect corpora allata and thus encompasses a number of insect peptides grouped into three families determined by sequence consensus [1]. The first identified insect ASTs were isolated from the cockroach Diploptera punctata and share the consensus sequence Tyr/Phe-Xxx-Phe-Gly-Leu/Ileamide. Over 60 FGLamide-type ASTs have been identified from just 3 species of crustacean: 20 from the crab C. maenas, 3 from the crayfish O. limosus, and over 40 from the shrimp P. monodon (see ref. [9]). In addition, ASTs continue to be identified in other species by mass spectrometric techniques (e.g., [5, 10]).

Biological Actions In crustaceans, FGLamide-AST immunoreactivity (AST-IR) has been localized throughout the CNS and STNS, in neurohemal structures, and in the hemolymph, which suggests both neural and hormonal functions [1, 9]. Through the STNS, the ASTs have modulatory effects on pattern-generating circuits as

226 / Chapter 34 well as pre- and postsynaptic effects at the neural muscular junction [1]. The FGLamide ASTs have been shown to stimulate the release of methyl farnesoate (MF) from the mandibular organ of P. clarkii [9]. MF is widely regarded to be the crustacean equivalent of insect juvenile hormone, which regulates processes such as molting and reproduction.

TACHYKININ-RELATED PEPTIDES Discovery Tachykinin-related peptides (TRPs; Phe-Xxx-GlyXxx-Arg-NH2) that have been isolated from insects, molluscs, and crustaceans constitute a large group of invertebrate peptides that share a sequence identity with the vertebrate tachykinins [17]. Two TRPs have been isolated from C. borealis (Cab-TRP 1a and 1b), and one TRP with the same sequence as Cab-TRP 1a has been isolated from L. vannamei [17]. Cab-TRP 1a has also been shown to be present in the crayfish P. clarkii and the spiny lobster Panulirus interruptus by cloning of the pre-pro-TRPs from both species [28].

Structure and Distribution of the Precursor mRNA/Gene The pre-pro-TRPs cloned from cDNA libraries of P. clarkii and P. interruptus are highly conserved, and each contains seven copies of the Cab-TRP 1a peptide [28]. In P. clarkii pre-pro-TRP mRNA was expressed in the nervous tissue (ES and CNS) but was absent from midgut and hindgut tissue [28].

Biological Actions The TRPs are present within the crustacean STNS, where they have been shown to stimulate pyloric motor patterns in the crab C. borealis [17]. In the crayfish Pacifastacus leniusculus TRP may act in combination with GABA to exert inhibitory effects within the lamina ganglionaris [17].

KININS Discovery The only known crustacean members of the insect kinin peptide family, Pev-kinin 1 through 6, were isolated from L. vannamei [22]. The original kinins were isolated from the cockroach L. maderae by monitoring their stimulatory effect on hindgut muscle contraction, the same in vitro assay used in the isolation of the

Pev-kinins [22]. The insect kinins share the same Cterminal sequence Phe-Xxx-Xxx-Trp-Gly-NH2 as Pevkinin 1, 3, and 4, whereas the remaining Pev-kinins have an Ala residue substituted for the C-terminal Gly [22]. Pev-kinin 2 and 4 have also been identified in C. borealis by mass spectrometry [10].

Biological Actions The insect kinins have both myomodulatory and diuretic effects, but in crustaceans no endogenous roles have yet been identified [22]. The Pev-kinins were able to stimulate fluid secretion and muscle contraction in insect tissues, yet they either have no effect or only a slight effect on crustacean hindgut muscle contraction assays [22].

PYROKININ/PBAN Discovery The only noninsect members of the Pyrokinin/PBAN family of peptides have been isolated and sequenced from L. vannamei [21]. The two peptides, Pev-PK 1 and 2, possess the C-terminal consensus sequence Phe-XxxPro-Arg-Leu-NH2 characteristic of the insect Pyrokinin/ PBAN family.

Biological Actions Both Pev-PKs are able to stimulate contractions in cockroach and crayfish hindgut in vitro, but the in vivo roles for the pyrokinin/PBAN peptides within crustaceans is presently unknown.

SIFAMIDE Discovery The first member of the SIFamide peptide family in crustaceans was isolated along with several RFamides from P. monodon eyestalk extracts by use of a mixture of three monoclonal antibodies raised against three different RFamides [18]. For this reason it was named PemFLP 7 (FMRFamide-like peptide). At the same time, mining of the A. gambiae genome revealed an identical peptide, Angam-SIFamide, which was cited as evidence for a new family whose original member was NebLFamide, previously isolated from the gray flesh fly Neobellieria bullata [25 and references therein]. Two more crustacean SIFamides with a sequence identical to Angam-SIFamide have been identified from C. borealis and P. clarkii [5, 27]. The name crustaceanSIFamide has been proposed for the three identical

Crustacean Bioactive Peptides / 227 crustacean sequences (Gly-Tyr-Arg-Lys-Pro-Pro-Phe-AsnGly-Ser-Ile-Phe-amide).

Structure and Distribution of the Precursor mRNA/Gene A crustacean-SIFamide precursor containing one copy of the peptide was cloned from a cDNA library of brain and SOG of P. clarkii [27]. In P. clarkii, the SIFamide precursor mRNA was expressed in all nervous tissues tested but was absent from heart, hepatopancreas, and skeletal muscle [27].

[3]

[4]

[5]

[6]

[7]

Biological Actions The endogenous roles of the SIFamides in both insects and crustaceans have yet to be determined, although there is some indication of myomodulatory effects [27].

MISCELLANEOUS This brief survey of identified neuropeptides in crustaceans has omitted some neuropeptides from a list that is rapidly expanding. For example, the insect peptide corazonin has recently been identified by spectrometry in the nervous tissues of C. maenas (listed in Table 1; [10]). It is certain that other as yet unidentified neuropeptides and neuropeptide families will soon be added to the list of crustacean neuropeptides. Androgenic gland hormone (AGH) has not been included in this review, but is covered in more detail elsewhere in this Handbook. Although in the literature, AGH is occasionally refered to as a “protein,” it is more accurately described as a heterodimer composed of two peptide chains, one of which is glycosylated [13]. AGH, characterized from A. vulgare, is involved in regulation of sexual differentiation in crustaceans [13]. Last, other peptides that could be considered to be bioactive peptides have been omitted due to space limitations. These include the antimicrobial peptides, about which there is a growing body of literature, and the pheromonotropic peptides, of which there is little known in crustaceans at present. Both of these topics, especially antimicrobial peptides, are covered elsewhere in this book.

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17]

[18]

[19]

References [1] Bendena WG, Donly BC, Tobe SS. Allatostatins: a growing family of neuropeptides with structural and functional diversity. Ann N Y Acad Sci 1999;897:311–29. [2] Dircksen H. (1998). Conserved crustacean cardioactive peptide (CCAP) neuronal networks and functions in arthropod evolution. In Recent Advances in Arthropod Endocrinology, vol. 65 (ed.

[20]

G. M. Coast and S. G. Webster), pp. 302–333. Cambridge, UK: Cambridge University Press. Garczynski SF, Crim JW, Brown MR. Characterization of neuropeptide F and its receptor from the African malaria mosquito, Anopheles gambiae. Peptides 2005;26(1):99–107. Hanke J, Willig A, Yinon U, Jaros PP. Delta and kappa opioid receptors in eyestalk ganglia of a crustacean. Brain Res 1997;744(2):279–84. Huybrechts J, Nusbaum MP, Vanden Bosch L, Baggerman G, De Loof A, Schoofs L. Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab Cancer borealis. Biochem Biophys Res Commun 2003;308:535–544. Isaac RE, Taylor CA, Hamasaka Y, Nassel DR, Shirras AD. Proctolin in the post-genomic era: new insights and challenges. Invert Neurosci 2004;5(2):51–64. Kishori B, Premasheela B, Ramamurthi R, Reddy PS. Evidence for a hyperglycemic effect of methionine-enkephalin in the prawns Penaeus indicus and Metapenaeus monocerus. Gen Comp Endocrinol 2001;123(1):90–9. Konopinska D, Rosinski G. Proctolin, an insect neuropeptide. J Pept Sci 1999;5(12):533–46. Kwok R, Rui Zhang J, Tobe SS. Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: A possible role for allatostatins. J Insect Physiol 2005;51(4):367–78. Li L, Kelley WP, Billimoria CP, Christie AE, Pulver SR, Sweedler JV, Marder E. Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis. J Neurochem 2003;87(3):642–56. Mercier AJ, Friedrich R, Boldt M. Physiological functions of FMRFamide-like peptides (FLPs) in crustaceans. Microsc Res Tech 2003;60(3):313–24. Nachman RJ, Holman GM, Haddon WF, Ling N. Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin, and cholecystokinin. Science 1986; 234:71–3. Okuno A, Hasegawa Y, Ohira T, Katakura Y, Nagasawa H. Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare. Biochem Biophys Res Commun 1999;264(2):419–23. Pascual N, Castresana J, Valero ML, Andreu D, Belles X. Orcokinins in insects and other invertebrates. Insect Biochem Mol Biol 2004;34(11):1141–6. Phlippen MK, Webster SG, Chung JS, Dircksen H. Ecdysis of decapod crustaceans is associated with a dramatic release of crustacean cardioactive peptide into the haemolymph. J Exp Biol 2000;203(Pt 3):521–36. Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977;197(4304):670–1. Satake H, Kawada T, Nomoto K, Minakata H. Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review. Zoolog Sci 2003;20(5):533–49. Sithigorngul P, Pupuem J, Krungkasem C, Longyant S, Chaivisuthangkura P, Sithigorngul W, Petsom A. Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon. Comp Biochem Physiol B Biochem Mol Biol 2002;131(3):325–37. Sithigorngul P, Pupuem J, Krungkasem C, Longyant S, Panchan N, Chaivisuthangkura P, Sithigorngul W, Petsom A. Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon. Peptides 2002;23(11):1895–906. Torfs P, Baggerman G, Meeusen T, Nieto J, Nachman RJ, Calderon J, De Loof A, Schoofs L. Isolation, identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei. Biochem Biophys Res Commun 2002;299(2):312–20.

228 / Chapter 34 [21] Torfs P, Nieto J, Cerstiaens A, Boon D, Baggerman G, Poulos C, Waelkens E, Derua R, Calderon J, De Loof A, Schoofs L. Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei. Eur J Biochem 2001;268(1):149–54. [22] Torfs P, Nieto J, Veelaert D, Boon D, van de Water G, Waelkens E, Derua R, Calderon J, de Loof A, Schoofs L. The kinin peptide family in invertebrates. Ann N Y Acad Sci 1999;897:361–73. [23] Vanden Broeck J. Neuropeptides and their precursors in the fruitfly, Drosophila melanogaster. Peptides 2001;22(2):241–54. [24] Vehovszky A, Agricola HJ, Elliott CJ, Ohtani M, Karpati L, Hernadi L. Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis. Neurosci Lett 2005;373(3):200–5. [25] Verleyen P, Huybrechts J, Baggerman G, Van Lommel A, De Loof A, Schoofs L. SIFamide is a highly conserved neuropep-

[26]

[27]

[28]

[29]

tide: a comparative study in different insect species. Biochem Biophys Res Commun 2004;320(2):334–41. Yasuda A, Naya Y, Nakanishi K. Isolation of Antho-RFamide related peptides from the eyestalks of blue crab. Comp Biochem Physiol B 1993;104(2):235–40. Yasuda A, Yasuda-Kamatani Y, Nozaki M, Nakajima T. Identification of YRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis. Gen Comp Endocrinol 2004;135(3):391–400. Yasuda-Kamatani Y, Yasuda A. APSGFLGMRamide is a unique tachykinin-related peptide in crustaceans. Eur J Biochem 2004;271(8):1546–56. Yasuda-Kamatani Y, Yasuda A. Identification of orcokinin generelated peptides in the brain of the crayfish Procambarus clarkii by the combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectrometries and molecular cloning. Gen Comp Endocrinol 2000;118(1):161–72.

C

H

A

P

T

E

R

35 Crustacean Chromatophorotrophins and Hyperglycemic Hormone Peptide Families R. KWOK, S. M. CHAN, F. MARTÍNEZ-PÉREZ, S. ZINKER, AND S. S. TOBE

ion transport peptide (ITP) family for the CHHs [12, 22, 23].

ABSTRACT The chromatophorotrophic hormones, pigment dispersing hormone (PDH) and red pigment concentrating hormone (RPCH), were the first crustacean peptides to be isolated and sequenced. These peptide families, along with the crustacean hyperglycemic hormone (CHH) peptide family, have been extensively studied in crustaceans and have been found to regulate aspects of color change, metabolism, development, and reproduction. All three peptide families have insect homologs as well as some homologs in other taxa.

CHROMATOPHOROTROPHIC HORMONES The chromatophores are cells that concentrate or disperse pigments, changing the color of the bearer (see review [11]). The chromatophorotrophic hormones, RPCH, and PDH, were the first crustacean peptides to be purified (from the northern shrimp Pandalus borealis) and chemically synthesized [17]. The octapeptide, PDH, and the octadecapeptide, RPCH (see Tables 1 and 2), are produced by the X organ-sinus gland complex located in the optic peduncle of crustaceans. Both peptides and their homologs have been found in several other species of decapods, and PDH has been isolated from the terrestrial isopod A. vulgare [9]. An RPCH homolog has been identified in one insect species, the stink bug, Nezara viridula [22]; it contains Ile as an amino acid at position 2 and functions as a lipid mobilizing hormone. RPCH is structurally related to its insect ortholog, adipokinetic hormone (AKH), and likewise PDH and its insect ortholog, pigment dispersing factor (PDF), are considered to be a peptide family [12, 22].

INTRODUCTION Although many peptide families have been identified in the crustaceans, the chromatophorotrophic hormones, PDH, and RPCH, as well as the CHH family, are special in that much of the characterization of these peptides has been done with crustacean model systems. As a contrasting example, the cyclic peptide, crustacean cardioactive peptide (CCAP), was originally discovered in a crustacean and has since been characterized almost exclusively in insects, in which it has also been found (see previous chapter). Many of the other crustacean peptides belong to families that were first identified in insects and characterized mainly using insect model systems. Considering that the vast majority of crustacean peptides have been isolated from decapod species, it is interesting to note that isoforms of PDH and CHH have also been identified in the terrestrial isopod Armadillidium vulgare [6, 9]. In addition, all three peptide families have insect homologs: the pigment dispersing factor (PDF) family for crustacean PDH, the adipokinetic hormones (AKH) for crustacean RPCH, and the Handbook of Biologically Active Peptides

Structure of the Precursor mRNA/Gene The structure of a genomic 646 bp DNA fragment from the blue crab Callinectes sapidus that codes for the ORF and an intervening sequence of 272 bp of RPCH are known. The classical intron is located between two inframe codons (phase 0 intron), similar to that present in AKH II [22]. In addition, sequences of three cDNAs from crabs Carcinus maenas and C. sapidus and from the

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230 / Chapter 35 crayfish Cherax quadricarinatus are known. The genes for RPCH, AKH, and APGWamide from mollusks may have a common origin [22], and their target receptors most probably evolved independently. Genes PDH 1 and PDH 2 have been cloned from the shrimp Marsupenaeus japonicus. PDH 1 has two introns and three exons, whereas PDH 2 has one intron and two exons. Expression of the RPCH mRNA [22] in the crayfish C. quadricarinatus and pigment migration controlled by beta-PDH in the crab Chasmagnathus granulata [13] are regulated by the circadian cycle. (GeneBank accesión nos: RPCH—C. maenas AAB28133, C. quadricarinatus AY642684, C. sapidus L36824; PDH—M. japonicus I AB073367 and II AB073368, C. sapidus II L36717, Litopenaeus vannamei I Y11722 and II Y11723, C. sapidus I L36716, Orconectes limosus S59496, C. maenas L08635, P. sp. JC4756).

AKH injected into crustaceans induces pigment concentration.

CRUSTACEAN HYPERGLYCEMIC HORMONES Discovery The X-organ sinus gland complex (XOSG) of the crustacean eyestalk produces hyperglycemic hormone (CHH), molt inhibiting hormone (MIH), and gonad inhibiting hormone (GIH) [5]. These neuropeptides are composed of 72–81 amino acid residues with 6 conserved cysteine residues implicated in the formation of disulfide bonds that determine the tertiary structure. CHH was first isolated from the crab C. maenas, MIH and GIH from the lobster Homarus americanus, MOIH from the crab Cancer pagurus, and subsequently from several other decapod species [2, 5, 6]. Because of their structural similarity, these hormones are grouped together as members of the CHH/MIH/GIH family. However, based on their biological, biochemical, and molecular properties, the CHH/MIH/GIH family can be divided into two major subtypes—the CHH (also known as type I) and MIH/GIH (type II) peptides— with a further division into two forms within each subtype [5]. Apart from decapods, CHH has also been identified in the isopod A. vulgare [6]. In insects, the ion transport peptide (ITP) family are the homologs of the crustacean CHH/MIH/GIH family [5, 23].

Biological Action RPCH and PDH elicit aggregation (triggered by dopamine) or dispersion (triggered by 5-hydroxytryptamine and norepinephrine) of pigment granules in tegumentary chromatophores and retinal cells, respectively. PDH also is released by a neural reflex triggered by light acting on extraretinal photoreceptors [22]. RPCH, which is structurally related to AKH, induces lipid mobilization following injection into insects.

TABLE 1. Consensus for RPCH/AKH. 1

2

23

pGlu

3

pGlu

5

6

12

17

21 2

S T 1 A

19

6

F T

12

6

11

3

po

a

F

po

np

L V 2 I, F 1 Y np

a

4

N T

7

P T 1 A, S

8

11

D G 2 S 1 V, W any

24

Gene Organization

9 24

W

The gene structure/organization of the CHH/MIH/ GIH family was first reported for the shrimp Metapenaeus ensis through screening of a genomic DNA library and by genomic Southern blot analysis [15]. It was estimated that the shrimp genome contains >20 copies of CHH-type neuropeptide genes, with a greater number of the CHH-A subtype relative to the CHH-B subtype. The CHH-A gene comprises several isoforms of the mature peptide, which share a high overall sequence identity (>97%). However, the gene sequences that

G

8

W

G

a

a

Superscript = times that amino acid is present in all species studied. Last line: ainvariable amino acid; np = nonpolar amino acid; po = polar amino acid; bold = amino acid in RPCH; bold and underlined = amino acid in APGWamide.

TABLE 2. Consensus for PDH/PDF. 1 29

N

2

3

4

5

28

28

23

29

S

A N

a

S

a

E D E

a

L

I

6 29

N

7 29

S

5

I 1 M np

8

9

20

29

L

L

I I

a

N

a

S

a

np

L

a

10

11

12

13

23

21

28

27

20

23

5

7

1

3

6

G S 1 A po/np

L

I 1 A np

P

S

L A

1

P

a

L

a

14 V N 2L 1 F, S, D, T np / po / nc

15 M L

np

16

17

18

19

26

22

29

29

N T 1 I po 2

D E 1 N nc

A

G

6

Superscript = times that amino acid is present in all species studied. Last line: ainvariable amino acid; np = nonpolar amino acid; po = polar amino acid; nc = noncharged amino acid.

A

a

G

a

Crustacean Chromatophorotrophins and Hyperglycemic Hormone Peptide Families / 231 encode the signal peptide and CHH-precursor-related peptides (CPRP) exhibit far more variability. There is some confusion in the naming of the different CHHs among different decapods. For example, in H. americanus and Procambarus clarkii, two forms of CHH have been named CHH-A and CHH-B, yet they share >98% amino acid sequence identity and should therefore be considered the same subtype. The CHH gene consists of three relatively small exons separated by two relatively large introns (Fig. 1).

For CHH genes, exon 1 encodes a portion of the signal peptide, and exon 2 encodes the remaining signal peptide plus the CPRP and the N-terminal portion of the mature peptide. Exon 3 encodes the coding sequence of the remaining mature peptide. Four-exon variants of the CHH gene have been reported in C. pagurus [10], P. clarkii (GenBank #AF474409), and the shrimps—Macrobrachium rosenbergii (GenBank #AY466494) and L. vannamei (GenBank #AY167044). In C. pagurus, the four-exon variant is also expressed in the

CHH-A/CHH-B gene clusters in M. ensis

A CHH-B

CHH-A

CHH-A

CHH-A

CHH-A

CHH-A CHH-A

CHH-A

CHH-A CHH-A CHH-B

CHH-A

CHH-A

Cluster 1

CHH-A

Cluster 2

CHH-A

CHH-A

CHH-A

B Gene duplication and mutation

CHH-A1…N Gene duplication and mutation

CHHA12,..N CHH-B Extensive mutation

CHH-A1…N CHH-B

CHH-A1…N CHH-B

3-exons (major)

MIH1,2,3.

CHH-like

4-exons (minor)

MIH GIH MOIH.

3-exons (major)

FIGURE 1. (A) Gene clustering of the M. ensis CHH neuropeptide and (B) proposed evolution of the CHH neuropeptide gene (A) Two clusters of CHH subtype neuropeptides have been identified by genomic lambda clones: cluster 1 consists of 8 CHH-A genes (gray boxes) and 1 CHH-B gene (black box); cluster 2 consists of 7 CHH-A gene and 1 CHH-B gene; (B) proposed view on the evolution of crustacean CHH family neuropeptide. Gene duplication of CHH-A gene produced many CHH-A isoforms, a few of the CHH-A genes will mutate to produce the CHH-B gene. Formation of the 4-exon gene is the result of enlargement of intron 2 followed by the formation of exon 3 and intron 3. Formation of the MIH gene is the result of extensive mutation of the CHH-A gene.

232 / Chapter 35 pericardial organ. These four-exon CHH genes may have evolved by the insertion of the additional exon. However, the coding sequence of exon 4 is not translated because of the presence of a stop codon in exon 3. Alternative splicing that removes exon 3 will produce the commonly reported CHHs identified in the eyestalk from other decapods. Based on the results from phylogenetic analysis, the four-exon CHH gene has most likely evolved recently from one of the CHH genes (Fig 1). Interestingly, in the locust the long form of ITP is formed by alternative splicing of the four-exon gene. The gene organization of the MIH/GIH-type peptides is basically identical to the three exon CHH-type genes except that CPRP is absent from exon 2 [14]. There are only a few reports of MIH/GIH gene organization, including genes from M. ensis, Charybdis feriatus, and the MOIH gene in C. pagurus [5]. Although the total number of MIH/GIH peptides in each species has yet to be confirmed, this subtype has fewer isoforms within a single species than CHH-type peptides. It is premature to speculate if MOIH belongs to a third subtype of the MIH/GIH-type peptides, because GIH has not been identified in the species from which MOIH was characterized [20]. However, based on phylogenetic tree analysis of MIH/GIH subtypes in the shrimp, it is likely that three major forms of the MIH/GIH neuropeptides exist in the Penaeidae (see following). The CHH/MIH/GIH genes may form clusters along the loci on the chromosome. For example, in shrimp, >14 CHH-A and 2 CHH-B genes arranged in two clusters have been identified [15]. It is not known whether these two clusters of CHH-like genes are located at the same locus of the chromosome. But analysis of the sequences of these CHH genes indicates that the major gene structure of M. ensis comprises three rather than four exons. Since examples of the four-exon gene variant are fewer in number and the first 41 amino acid residues in the splice variant of the CHH peptide are conserved, we suggest that the four-exon CHH gene is a recently evolved gene organization (Fig. 1). The organization of the MIH/GIH gene is most likely derived from the extensive mutation of a CHH gene, involving (1) the elimination of the CPRP and (2) the addition of more coding sequence (i.e., forming additional glycine residues and extending the C-terminus). In C. pagurus, MOIH and MIH genes are also clustered on a locus of the chromosome [20]. Whether all the CHH/MIH/GIH genes are located on the same chromosome remains to be determined. Despite the clustering of multiple genes, the mechanism controlling the expression of these forms is not known. There are only a few reports on the characterization of the gene regulatory region [4]. In conclusion, the study of CHH/MIH/GIH neuropeptide gene organization has been complicated by the use of different species, incomplete knowledge of the

total number of genes, and incomplete cloning of the major forms, making any comparison difficult until all CHH neuropeptide sequences from a single species are known. However, we speculate that there are three CHH subtypes and three MIH/GIH subtypes in decapods. An extensive gene duplication event may have occurred to produce multiple CHH-A isoforms, which may show tissue-specific expression. CHH-B may be a result of CHH-A mutation, and further mutation of the CHH gene may have produced the MIH/GIH subtypes.

Gene Expression There have been comprehensive studies on the expression of the CHH neuropeptide family in M. ensis, as members of the four major CHH-subtypes have been cloned and identified (for review, see [5]). Although CHH-A, CHH-B, MIH, and GIH are expressed in the eyestalk, transcripts of both the CHH-B and GIH of M. ensis can be detected in other neuronal tissues such as brain, thoracic ganglion, and ventral nerve cord. A similar expression profile occurs in another shrimp, M. japonicus [1]. The presence of multiple forms of CHHA suggests that the neuropeptide may have a wide tissue distribution; expression studies indicate the presence of CHH-like peptides in other nonneuronal tissues. The CHH gene transcript is expressed at relatively high levels and seems to have no regular pattern for different developmental stages. However, there are conflicting results for the expression of the MIH gene transcripts. For example, in M. ensis and C. sapidus, MIH transcripts were low during the premolt and high during the intermolt [14, 19], whereas a recent study reported that both the CHH and MIH mRNA levels were increased during premolt in C. maenas [7]. GIH expression in M. ensis and H. americanus decreased in the early phase of gonadal maturation and increased to a relatively high level at the middle to late stage of gonadal maturation [8, 16].

Receptors To date, no receptors for the CHH/MIH/GIH neuropeptides have been cloned. However, in characterizing the receptor for MIH in C. maenas, Chung and Webster reported that there were no changes in receptor number or receptor affinity during the molt cycle (except for the possible recruitment of MIH receptors to the organ during postmolt). They suggest that this is an important mechanism involved in control of the molt cycle and relates to intracellular signaling pathways within the Y-organ [7]. The receptor for the MIH of M. japonicus has been characterized by radioligand binding assay using recombinant protein for MIH and shown to be 70 kDa in size [1].

Crustacean Chromatophorotrophins and Hyperglycemic Hormone Peptide Families / 233

Processing The primary structure of the CHH-type preprohormone is composed of the CPRP, the signal peptide, and the mature peptide [5]. All CHH-type peptides are amidated at the C-terminus. Although there is conservation of the six cysteine residues in the mature CHH and MIH/GIH subtypes, the CHH subtype lacks the Gly residue at position 12 (relative to the mature peptide, of MIH). The primary structure of the MIH/GIH preprohormone lacks the CPRP. The prohormone is composed of only the signal peptide and the mature peptide [5].

Biological Actions The biological actions of the CHH/MIH/GIH peptides have often been difficult to resolve as a result of the structural similarities of the peptides, the use of unpurified extracts, and the use of heterologous assays to characterize biological activity. Recently, a number of studies have employed purified, synthesized, or recombinant CHH/MIH/GIH peptides to more accurately assess the activities in vitro and in vivo. Even with the use of the appropriate peptide in homologous assays, there is still overlap in biological activities, probably as a result of the structural similarities [2]. The primary function of CHH, as the name suggests, is the regulation of carbohydrate metabolism through elevation of sugar in the hemolymph. Recombinant CHH can mimic the effects of purified CHH or eyestalk extract causing a rapid increase in hemolymph glucose levels following injection into the crustacean [18]. Structure–activity studies have determined some critical residues, such as the 6 Cys residues that determine the 3-D structure of CHH, which is required for hyperglycemic activity [24]; that insertion of a Gly residue into CHH at position 12, an invariable residue in MIH sequences, results in a decrease in hyperglycemic activity by one order of magnitude [18]. Other recorded activities for the CHH peptides are effects on lipid mobilization, osmoregulation, secretagogue activity on the hepatopancreas, and a possible role for CHH-B in stimulating oocyte growth (see reviews [2, 26]). MIH suppresses molting by inhibiting the synthesis of the molting hormone (ecdysteroids) produced by the Y-organ [2]. Injection of recombinant MIH in M. japonicus resulted in a prolonged molt interval and a decrease in hemolymph ecdysteroid levels, mimicking the results obtained using purified MIH ([25] and reference therein). GIH (also known as vitellogenin inhibiting hormone, VIH) in vitro suppresses total protein synthesis in the ovary [8]. Neither the mode of action nor the function of these peptides in vivo is presently clear. Peptides with

GIH activity (in homologous assays) have exclusively had MIH-like sequences, except for one example in Penaeids where a CHH from one species was found to inhibit protein synthesis in ovaries of another Penaeid species, whereas the one MIH tested had no effect (see [2]). No doubt the recently cloned vitellogenin gene will be an invaluable tool in resolving the identities and modes of action of the GIHs. MOIHs have been shown to inhibit the activity of the mandibular organ in vitro, and although their effects in vivo have not been clearly demonstrated, they are assumed to regulate the activity of the MO, likely in combination with other peptidergic factors [21]. The MO produces and releases the sequiterpenoid compounds methyl farnesoate (MF) and its immediate precursor farnesoic acid (FA). MF is regarded as the crustacean version of the insect juvenile hormone, whereas FA has recently been shown to stimulate vitellogenin gene expression [3, 21]. Last, it is of interest that the CPRP has been shown to be coreleased with CHH from sinus glands of potassium-evoked eyestalk preparations in vitro [27]. CPRP is detectable in the hemolymph following stress-evoked in vivo release and has a longer half life in vivo than CHH [27]. There are as yet no known functions associated with the CPRPs.

References [1] Asazuma H, Nagata S, Katayama H, Ohira T, Nagasawa H. Characterization of a Molt-Inhibiting Hormone (MIH) Receptor in the Y-Organ of the Kuruma Prawn, Marsupenaeus japonicus. Ann N Y Acad Sci 2005;1040:215–18. [2] Bocking D, Dircksen H, Keller R. (2001) “The crustacean neuropeptides of the CHH/MIH/GIH family: structures and biological activities.” In: Wiese K. eds., The Crustacean Nervous System, Springer Verlag, New York, pp. 84–97. [3] Borst DW, Wainwright G, Rees HH. In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus. Proc Biol Sci 2002;269(1490):483–90. [4] Chan S-M, Chen X-G, Gu P-L. PCR cloning and expression of the molt-inhibiting hormone gene for the crab Charybdis feriatus. Gene 1998;224:23–33. [5] Chan SM, Gu PL, Chu KH, Tobe SS. Crustacean neuropeptide genes of the CHH/MIH/GIH family: implications from molecular studies. Gen Comp Endocrinol 2003;134(3): 214–19. [6] Chen SH, Lin CY, Kuo CM. In silico analysis of crustacean hyperglycemic hormone family. Mar Biotechnol (NY). 2005; 7(3):193–206. [7] Chung JS, Webster SG. Molt cycle-related changes in biological activity of moult-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) in the crab Carcinus maenas: from target to transcript. Eur J Biochem 2003;270: 3280–8. [8] de Kleijn DP, Janssen KP, Waddy SL, Hegeman R, Lai WY, Martens GJ, Van Herp F. Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus. J Endocrinol 1998;156(2):291–8.

234 / Chapter 35 [9] Desmouccelles-Carette C, Sellos D, Van Wormhoudt A. Molecular cloning of the precursors of pigment dispersing hormone in crustaceans. Biochem Biophys Res Comm 1996;221:739–43. [10] Dircksen H, Bocking D, Heyn U, Mandel C, Chung JS, Baggerman G, Verhaert P, Daufeldt S, Plosch T, Jaros PP, Waelkens E, Keller R, Webster SG. Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes. Biochem J 2001;356:159–70. [11] Finguerman M. Chromatophores. Physiol Rev 1965;45:296– 339. [12] Gade G. The revolution in insect neuropeptides illustrated by the adipokinetic hormone/red pigment-concentrating hormone family of peptides. Z Naturforsch [C] 1996;9–10:607–17. [13] Granato FC, Tironi TS, Maciel FE, Rosa CE, Vargas MA, Nery LE. Circadian rhythm of pigment migration induced by chromatophorotropins in melanophores of the crab Chasmagnathus granulata. Comp Biochem Physiol A Mol Integr Physiol 2004;138:313–19. [14] Gu P-L, Chan S-M. Molecular cloning of the shrimp (Metapenaeus ensis) eyestalk cDNA encoding the putative molt inhibiting hormone. Mar Mole Biol and Biotech 1998;7:214–20. [15] Gu P-L, Chan S-M. The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of gene arranged in a cluster. FEBS Letters 1998;441:397–403. [16] Gu PL, Yu KL, Chan S-M. Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins. FEBS Letters 2000;472:122–8. [17] Joseffson L. Invertebrate neuropeptide hormones. Int J Pept Protein Res 1983;21:459–70. [18] Katayama H, Nagasawa H. Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity. Zoolog Sci. 2004;21(11):1121–4.

[19] Lee KJ, Watson RD, Roer RD. Molt-inhibiting hormone mRNA levels and ecdysteroid titer during a molt cycle of the blue crab, Callinectes sapidus. Biochem Biophys Res Commun 1998;249:624– 7. [20] Lu W, Wainwright G, Webster SG, Rees HH, Turner PC. Clustering of mandibular organ-inhibiting hormone and moultinhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression. Gene 2000;253:197–207. [21] Mak AS, Choi CL, Tiu SH, Hui JH, He JG, Tobe SS, Chan SM. Vitellogenesis in the red crab Charybdis feriatus: Hepatopancreas-specific expression and farnesoic acid stimulation of vitellogenin gene expression. Mol Reprod Dev 2005;70(3):288–300. [22] Martinez-Perez F, Zinker S, Aguilar G, Valdes J, Arechiga H. Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus. Peptides 2005; DOI:10.1016/j.peptides.2005.05.018. In press. [23] Meredith J, Ring M, Macins A, Marschall J, Cheng NN, Theilmann D, Brock HW, Phillips JE. Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system. J Exp Biol 1996;199:1053–61. [24] Mettulio R, Giulianini PG, Ferrero EA, Lorenzon S, Edomi P. Functional analysis of crustacean hyperglycemic hormone by in vivo assay with wild-type and mutant recombinant proteins. Regul Pept 2004;119(3):189–97. [25] Okumura T, Ohira T, Katayama H, Nagasawa H. In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. Zoolog Sci 2005;22(3):317–20. [26] Tensen CP, Janssen KPC, Herp R. Isolation, characterizaation and physiological specificity of the crustacean hyperglycemic factors from the sinus gland of the lobster Homarus americanus (Milne-Edwards). Invert Reprod Dev 1989;16:155–64. [27] Wilcockson DC, Chung SJ, Webster SG. Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs? Cell Tissue Res 2002;307(1):129–38.

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36 Molluscan Bioactive Peptides ANNA DI COSMO AND CARLO DI CRISTO

Structure of the Precursor mRNA/Gene

ABSTRACT

RFamide peptides are characterized by sharing the sequence Arg-Phe-NH2 (RFamide) at the C-terminus (see also chapter on Insect Myosuppressins/ FMRFamides/NPFs by Orchard and Lange in this section of the book). These peptides are translated from specific genes as propeptides. These contain multiple copies of either the same or different peptides, the latter sharing in almost all cases only the C-terminal RFamide. Each peptide contained in the propeptide is proteolytically cleaved and amidated during posttranslational processing. The gene coding for FMRFamide has been identified in the gastropods Lymnaea stagnalis [47], Aplysia californica [49], and Helix aspersa [42]; in the bivalve Mytilus edulis [13]; and in the cephalopods Loligo pelaeii and Loligo opalescens [53], Sepia officinalis [32], and Octopus vulgaris [11]. The FMRFamide locus of Lymnaea is one of the best characterized (see [47] for references). This gene consists of five exons (I–V) punctuated by four introns. Two differentially spliced transcripts have been identified: one that consists of exons I and II (precursor 1) and another that comprises exons I, III, IV, and V (precursor 2). Exon II encodes a battery of tetra- and pentapeptide species. The sequence of exon III contains the heptapeptides and pentapeptides. Exon V encodes the two hexapeptides, while the sequence of the heptapeptide SKPYMRFamide is formed at the splicing junction between exons II and IV. Posttranslational processing of the two Lymnaea FaRP precursors allows the formation of two long non-FaRP peptides: the 22aa peptide, called SEEPLY, encoded in precursor 1 and the 35aa peptide, named acidic peptide, present in precursor 2 [47].

In this review we give a detailed overview of the studies reporting gene organization, mapping, receptors, and sites of action of peptides in the phylum Mollusca. Peptides or peptide families have been collected in groups according to molecular structure. However, when possible, structurally different peptides sharing a common biological action were described in separate subsections. According to these criteria, the RFamide peptides, as well as tachykinins, have been discussed as members of one peptide family. Other peptides are presented according to their involvement in energy flow and growth, feeding behavior, and water and ion balance.

FMRFamide, FMRFamide-RELATED PEPTIDES (FaRPs) Discovery The tetrapeptide Phe-Met-Phe-Arg-NH2 (FMFRamide), discovered in the ganglia of the clam Macrocallista [41], was the first member of the extended family of the FMRFamide-related peptides (FaRPs, also named RFamide peptides). They are known as molluscan cardioexcitatory peptides [37] but also as neurotransmitters and neuromodulators in the central nervous system [47]. Biochemical and molecular approaches showed that the sequences vary among members of the RFamide family in the N-terminal region [12]. Other RFamide peptides have been described: the Fusinus ferrugineus and Aplysia californica FRFamide family (see reference in [6]), the Achatina fulica cardioexcitatory peptide [15], the Lymnaea stagnalis cardioexcitatory peptide (LyCEP, [56]), and the recent Lymnaea stagnalis family of LFRFamide peptides [21], which is up-regulated during parasitization. Handbook of Biologically Active Peptides

Distribution of mRNA In Lymnaea the alternate mRNA expression of the FMRFamide gene is specific to single identified neurons.

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236 / Chapter 36 The two different transcripts are expressed in a mutually exclusive manner in different neurons of adult CNS [47]. By in situ hybridization and immunocytochemistry with peptide-specific antibodies, it was revealed that all neurons that express precursor 1 mRNA contain SEEPLY-immunoreactivity and, similarly, all precursor 2 mRNA-expressing neurons contain the acidic peptide [47].

Receptors A G-protein-coupled receptor (GPRC) was cloned from the mollusc Lymnaea stagnalis [56]. The ligand for this receptor was purified and identified as an RFamide peptide, the Lymnaea cardioexcitatory peptide (LyCEP), which displays significant similarity to the Achatina cardioexcitatory peptide (ACEP-1). In the Lymnaea brain, the cells that produce the egg-laying hormone represent the predominant site of RFamide receptor gene expression.

Biological Actions The cardioacceleratory action of RFamide peptides has been documented in numerous gastropods (reviewed by [37]). The well-characterized central network controlling cardiorespiration in Lymnaea represents a good system to study the physiological consequences of alternative splicing of this neuropeptide gene. Most of the effects of the peptides from both precursors are inhibitory, and it has been suggested that the global inhibitory action of a released mixture of peptides may be more effective than the specific action of each peptide [47]. FMRFamide also affects the feeding in Lymnaea, Aplysia, Limax, and Helisoma (see reference in [12]). Moreover, FaRPs appear to be involved in osmoregulation in Helisoma trivolvis, Helisoma duryi, and Melampus bidentatus (see [26] for reference). In Aplysia FMRFamide inhibits the 30-min period of firing of the bag cell neurons of Aplysia californica (afterdischarge), which causes the animal to undergo a long-lasting sequence of stereotypical reproductive behaviors [14] and presynaptically inhibits the sensory neurons of the gill- and siphon-withdrawal reflex of Aplysia [34]. In cephalopods, FMRFamide exerts a positive contractile action on muscles of both the male and female reproductive systems [10, 20]. In the central nervous system this tetrapeptide seems to be involved in the inhibition of both the secretory activity of the endocrine optic gland [9] and the screeningpigment migration in combination with dopamine [11]. Finally, several FaRPs are potent excitators of the chromatophore muscles, causing chromatophore expansion [31].

PEPTIDES AND METABOLISM: ENERGY FLOW AND GROWTH Insulin-Related Peptides and NPY: Discovery The neuroendocrine light green cells (LGCs) in the cerebral ganglia of Lymnaea stagnalis have been suggested to be involved in the control of growth [45]. In these neuroendocrine cells molluscan insulin-related peptides (MIPs) have been found. To date, many members of the Lymnaea MIP family have been identified and characterized [52]. Six insulin-related peptides from pedal ganglions of Anodonta cygnea have also been isolated [50]. Recently, differential screening of cDNA libraries of the CNS of parasitized and nonparasitized snails also revealed specific alterations in neuropeptide encoding gene expression [7]. Among these, one of the significantly upregulated genes encodes for a Lymnaea neuropeptide Y homolog (LyNPY). Moreover, peptides with striking similarity to NPY/peptideYY (PYY)/polypeptideY (PY)/pancreatic polypeptide (PP) have also been isolated in other mollusks (for reference, see [23]).

Structure of the Precursor mRNA/Gene Seven MIP genes have been identified in Lymnaea stagnalis (see reference [52] for a complete list) and one in Aplysia californica (Accession number: AAL99711). Five of the Lymnaea MIPs (I, II, III, V, and VII) are expressed in the light green cells and the canopy cells [52]. The MIP-IV and MIP-VI genes are pseudogenes [52]. All the MIPs cDNAs encode a preprohormone resembling the organization of pre-proinsulin [52]. The invertebrate counterpart of NPY peptides resembles more neuropeptide F (NPF), which shares structural similarity to members of the vertebrate NPY/ PYY/PY/PP superfamily [23]. NPY genes have been characterized in Lymnaea and Aplysia [7, 43].

Distribution of mRNA In Lymnaea LGCs express genes encoding the molluscan MIPs (for complete references, see [52]): type-A cells express all MIP genes, whereas type-B cells do not express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Uniquely, the molluscan MIP VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea. In situ hybridization and specific immunoreactivity for NPY have been reported in Lymnaea and Aplysia. In Lymnaea, LyNPY-positive cells contact in a nonsynaptic way the neuroendocrine centers involved in the regulation of reproduction, ovulation and egg laying, and growth

Molluscan Bioactive Peptides / 237 [7]. In Aplysia, bag cell neurons are positive for NPY, and Northern blot analysis indicates that the peptide is abundantly expressed in the CNS [43].

Receptors Receptors for both MIP and NPY have been found in the nervous system of Lymnaea. Typical insulinreceptor features, including a cysteine-rich domain, a single transmembrane domain, and a tyrosine-kinase domain, are conserved in the cloned putative MIP receptor [44]. The Lymnaea cloned NPY receptor [55] is a typical G-protein coupled receptor (GPCR).

Biological Actions In contrast to vertebrate insulins, the MIPs are produced by neuroendocrine cells of the CNS (see [52]). The MIPs of LGCs are involved in the regulation of growth of both the soft body parts and the shell, as well as for protein and carbohydrate metabolism [18]. MIP VII may also be involved as a neuromodulator in the feeding circuitry [52]. In Lymnaea, female reproductive effort was decreased early on by NPY action on caudodorsal cells. Growth was also suppressed almost immediately by the action of NPY on the MIPsproducing LGCs. By contrast, after treatment with NPY, food consumption was unchanged, suggesting a different role for this peptide in invertebrates [8].

PEPTIDES AND FEEDING BEHAVIOR Discovery In gastropods, three families of peptides are directly involved in the control of the accessory radula closer (ARC) muscle in Aplysia: myomodulins, buccalins, and small cardioactive peptides (SCPs) (see [2, 5]). All these peptides are localized in the motoneurons B15 and B16 of the buccal ganglia of Aplysia (see [5]). Myomodulin is also present in several other mollusks [28], and three myomodulin-related peptides have been purified and sequenced from extracts of the snails Helix aspersa [19]. Small cardioactive peptides (SCPA, SCPB) have been isolated from a wide range of molluscan groups [3, 30, 42]. Recently in Aplysia other peptide families have been added to the list of feeding behaviorcontrolling peptides: the enterins [16], the feeding circuit-activating peptides (FCAPs) [54], cerebrin [29], and the pentapeptide PRQFVamide [17].

Structure of the Precursor mRNA/Gene Myomodulins (A-I) have been characterized from the motor neuron B16 and ARC muscle in Aplysia

californica (see [2, 5]). The complete sequence of this cDNA encodes a 42-kD pre-propeptide. The myomodulin-related peptides have been also characterized in Aplysia [2]. In the snail Lymnaea, a single gene encodes five structurally similar forms of myomodulin [25]. Buccalin cDNA from Aplysia contains 19 distinct buccalinrelated peptides, several of which are present in multiple copies [36]. The small cardioactive peptide (SCP) gene of Aplysia encodes a precursor protein that contains two peptides related in structure and other unrelated peptides [35]. In the snail Lymnaea, the SCP gene encodes a pre-propeptide containing two structurally related SCPs [40]. Enterin cDNA codes for a total of 35 copies of 20 distinct but related amidated peptides named enterin A (ENa) through enterin T (ENt) [16]. Analysis of the precursor structure encoded in the FCAPs cDNA predicts eight different structurally related peptides, named FCAPa through FCAPh [54]. Cerebrin cDNA codes for a precursor containing a single copy of the peptide and one additional peptide. The C-terminal glycine indicates that the peptide is amidated at the C-terminus [29]. PRQFVamide cDNA codes for a total of 33 copies of PRQFVamide and four related pentapeptides [17].

Distribution of mRNA The myomodulin and buccalin mRNA are localized in specific neurons in each ganglia of the Aplysia CNS, mainly in the motoneurons B16 [33]. Buccalin transcript is also localized in the motor neurons B15 [36]. In situ hybridization analysis indicates that in Lymnaea the myomodulin gene is widely and abundantly expressed in all ganglia of the brain [25]. In Aplysia and Lymnaea, SCPs are present mainly in the buccal ganglion [30, 40]. In Lymnaea, SCP and myomodulin genes are expressed within some large motoneurons (B1–B4) (see [39]). Both the enterin and FCAP mRNA are unevenly distributed throughout the central ganglia of Aplysia [16, 54]. Cerebrin RNA distribution studies performed on Aplysia showed detectable levels only in the cerebral ganglia [29], while the PRQFVamide precursor is abundant in the abdominal ganglion [17].

Biological Actions In Aplysia several of these peptides act in concert to enhance acetylcholine-induced contractions in a specific muscle of the buccal mass (see [2]). Myomodulins induce potentiation, depression of the amplitude of the contractions, and acceleration of the relaxation rate of ARC muscle [2], while buccalins decrease the size of contractions of the ARC muscle (see [5]). SCPa and SCPb are capable of acting postsynaptically on the muscle to potentiate the action of the primary

238 / Chapter 36 neurotransmitter, acetylcholine [22]. The concerted actions of SCPs and myomodulin on the feeding behavior and the innervation of the foregut have also been demonstrated in Lymnaea [46]. Enterins are active in both the gut and the CNS [16]. They play an inhibitory role on the contractions of the gut, inducing the switch from egestive to ingestive behaviors. Conversely, FCAPs are capable of initiating rhythmic feeding motor programs—mainly ingestive [54]. Also, cerebrin has a profound effect on the feeding motor pattern, dramatically shortening the duration of the radula protraction in a concentration-dependent manner [29]. Finally, PRQFVamide was found to decrease the excitability of some but not all neurons of the buccal feeding circuit [17].

PEPTIDES AND RENAL FUNCTION Discovery Apart from the evidence that FaRPs are involved in osmoregulation (see above), other peptides have been found to be involved in maintaining the water balance in mollusks. A bradykinin peptide is present in the left upper quadrant (LUQ) cells in Aplysia californica, which extensively innervate the kidney [58]. Moreover, peptides contained in the neuron R15 present in the abdominal ganglion of Aplysia have been shown to increase the water content in this gastropod [57]. In Lymnaea the peptide lymnokinin PSFHSWSamide, which belongs to the leucokinin family of peptides [4] (see also Coast chapter in this section of the book) and the sodiuminflux-stimulating peptide [1], were characterized.

Structure of the Precursor mRNA/Gene A bradykinin clone encodes a putative 16.3-kD precursor peptide, which contains potential proteolytic cleavage sites that could generate smaller mature peptides [58]. A cDNA cloned from R15 neurons encoded for a propeptide contains three major peptides that were named R15α, R15β, and R15γ peptides [57]. R15 mRNA undergoes alternative RNA splicing to yield two different mature mRNA species, R15-1 and R15-2. Lymnokinin was only purified with classical HPLC/MALDI approaches [4], while a cDNA clone, encoding the prohormone of the sodium-influx-stimulating peptide of the freshwater snail Lymnaea stagnalis, was isolated and characterized [51].

Distribution of mRNA In Aplysia the expression of bradykinin RNA was revealed only in neuron L5 among the LUQ cells [58],

while R15 peptide was mainly localized in that neuron [57]. In situ hybridization showed that the Lymnaea sodium-influx-stimulating peptide gene is expressed by the neuroendocrine Yellow Cells of the central nervous system [51].

Receptors The cloned GPCR receptor GRL104 from Lymnaea represents the leucokinin-like peptide receptors, and only lymnokinin is able to activate GRL104 at the nanomolar level [4].

Biological Actions In Aplysia bradykinin is supposed to regulate some renal functions [58]. On the contrary, substantial evidence indicates that neuron R15 is involved in the regulation of extracellular water volume or ionic composition [27]. Unfortunately, there is a paucity of data concerning the role of leucokinin-like peptides (lymnokinin) in mollusks. Indeed, the Lymnaea sodium-influxstimulating peptide controls the activity of sodium pumps in the integument, pericardium, ureter, and nephridial gland [51].

TACHYKININS Discovery Tachykinins (TKs) constitute the largest vertebrate neuropeptide family with multifunctions in central and peripheral tissues. In several invertebrate species, two types of structurally related peptides, tachykinin-related peptides (TKRPs) and invertebrate tachykinins (invTKs), have been identified [48] (see Chapter 26, in this section of the book). The first tachykinin to be sequenced, named eledoisin, was isolated from the posterior salivary glands of cephalopods Eledone moschata and Eledone aldrovandi (see [48]). Two novel tachykinins (OctTK-I and OctTK-II) were recently isolated from the posterior salivary glands of the Octopus vulgaris. Finally, a type of TKRP was isolated from the bivalve Anodonta cygnea (see [48]).

Structure of the Precursor mRNA/Gene The cDNA of preproOctTK-I and preproOctTK-II are very similar [24]. OctTKs were included in the sequences flanked by a typical dibasic signal at the Ntermini and an amidation signal and a single basic residue at the C-termini.

Molluscan Bioactive Peptides / 239

Distribution of mRNA The mRNA of preproOctTK-I was expressed in some mucus-containing cells of type A epithelium of the salivary glands of Octopus vulgaris [24].

Biological Actions inv-TKs have been shown to be present exclusively in the salivary glands of several species [24]. Furthermore, inv-TKs have not been found to exhibit any action on invertebrate tissues at physiological concentrations. Therefore, despite high sequence similarity to vertebrate TKs, inv-TKs are not anticipated to be the functional counterparts of vertebrate TKs [48]. The undecapeptide eledoisin that shows vasodilatory and smooth muscle stimulatory actions on mammals has an obscure role and significance in the octopus salivary glands; it may have no function in the nervous system of cephalopods [38]. OctTK-I and -II showed immediate contractile activities when applied on the longitudinal muscle of the guinea-ileum [24]. The OctTKs, in particular OctTK-I, are secreted in mucous saliva as a venomous substance acting on vertebrates such as fishes that are prey and/or natural enemies of the octopus.

References [1] Boer, HH, Montagne-Wajer, C, van Minnen, J, Ramkema, M, and de Boer, P. Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail, Lymnaea stagnalis, studied by in situ hybridization and immunocytochemistry. Cell Tissue Res, 1992. 268: 559–66. [2] Brezina, V, Bank, B, Cropper, EC, Rosen, S, Vilim, FS, Kupfermann, I, et al. Nine members of the myomodulin family of peptide cotransmitters at the B16-ARC neuromuscular junction of Aplysia. J Neurophysiol, 1995. 74: 54–72. [3] Candelario-Martinez, A, Reed, DM, Pritchard, SJ, Doble, KE, Lee, TD, Lesser, W, et al. SCP-related peptides from bivalve mollusks: identification tissue distributions and actions. Biol Bull, 1993. 185: 428–39. [4] Cox, KJ, Tensen, CP, Van der Schors, RC, Li, KW, van Heerikhuizen, H, Vreugdenhil, E, et al. Cloning, characterization, and expression of a G-protein-coupled receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide, PSFHSWSamide, as its endogenous ligand. J Neurosci, 1997. 17: 1197–205. [5] Cropper, EC, Vilim, FS, Alevizos, A, Tenenbaum, R, Kolks, MA, Rosen, S, et al. Structure, bioactivity, and cellular localization of myomodulin B: a novel Aplysia peptide. Peptides, 1991. 12: 683–90. [6] Cropper, EC, Brezina, V, Vilim, FS, Harish, O, Price, DA, Rosen, S, et al. FRF peptides in the ARC neuromuscular system of Aplysia: purification and physiological actions. J Neurophysiol, 1994. 72: 2181–95. [7] De Jong-Brink, M, Reid, CN, Tensen, CP, and Ter Maat, A. Parasites flicking the NPY gene on the host’s switchboard: why NPY? Faseb J, 1999. 13: 1972–84. [8] de Jong-Brink, M, ter Maat, A, and Tensen, CP. NPY in invertebrates: molecular answers to altered functions during evolution. Peptides, 2001. 22: 309–15.

[9] Di Cosmo, A, and Di Cristo, C. Neuropeptidergic control of the optic gland of Octopus vulgaris: FMRF-amide and GnRH immunoreactivity. J Comp Neurol, 1998. 398: 1–12. [10] Di Cristo, C, Paolucci, M, Iglesias, J, Sanchez, J, and Di Cosmo, A. Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH). J Exp Zool, 2002. 292: 267–76. [11] Di Cristo, C, Delli Bovi, P, and Di Cosmo, A. Role of FMRFamide in the reproduction of Octopus vulgaris: molecular analysis and effect on visual input. Peptides, 2003. 24: 1525–32. [12] Dockray, GJ. The expanding family of -RFamide peptides and their effects on feeding behaviour. Exp Physiol, 2004. 89: 229– 35. [13] Favrel, P, Lelong, C, and Mathieu, M. Structure of the cDNA encoding the precursor for the neuropeptide FMRFamide in the bivalve mollusc Mytilus edulis. Neuroreport, 1998. 9: 2961– 5. [14] Fisher, T, Lin, CH, and Kaczmarek, LK. The peptide FMRFa terminates a discharge in Aplysia bag cell neurons by modulating calcium, potassium, and chloride conductances. 1993. 69: 2164–73. [15] Fujimoto, K, Ohta, N, Yoshida, M, Kubota, I, Muneoka, Y, and Kobayashi, M. A novel cardio-excitatory peptide isolated from the atria of the African giant snail, Achatina fulica. 1990. 167: 777–83. [16] Furukawa, Y, Nakamaru, K, Wakayama, H, Fujisawa, Y, Minakata, H, Ohta, S, et al. The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia. J Neurosci, 2001. 21: 8247–61. [17] Furukawa, Y, Nakamaru, K, Sasaki, K, Fujisawa, Y, Minakata, H, Ohta, S, et al. PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia. J Neurophysiol, 2003. 89: 3114–27. [18] Geraerts, PM, Smit, AB, Li, KW, Vreugdenhil, E, and van Heerikhuizen, H. Neuropeptide gene families that control reproductive behaviour and growth in molluscs, In: O. NN, Editor Current Aspects of the Neurosciences. London: McMillan Press, 1991. pp. 255–304. [19] Greenberg, MJ, Doble, KE, Lesser, W, Lee, TD, Pennell, NA, Morgan, CG, et al. Characterization of myomodulin-related peptides from the pulmonate snail Helix aspersa. 1997. 18: 1099–106. [20] Henry, J, Zatylny, C, and Boucaud-Camou, E. Peptidergic control of egg-laying in the cephalopod Sepia officinalis: involvement of FMRFamide and FMRFamide-related peptides. 1999. 20: 1061–70. [21] Hoek, RM, Li, KW, van Minnen, J, Lodder, JC, de Jong-Brink, M, Smit, AB, et al. LFRFamides: a novel family of parasitationinduced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis. J Neurochem, 2005. 92: 1073–80. [22] Hooper, SL, Probst, WC, Cropper, EC, Kupfermann, I, and Weiss, KR. SCP application or B15 stimulation activates cAPK in the ARC muscle of Aplysia. Brain Res, 1994. 657: 337–41. [23] Hoyle, CH. Neuropeptide families: evolutionary perspectives. Regul Pept, 1998. 73: 1–33. [24] Kanda, A, Iwakoshi-Ukena, E, Takuwa-Kuroda, K, and Minakata, H. Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris. Peptides, 2003. 24: 35–43. [25] Kellett, E, Perry, SJ, Santama, N, Worster, BM, Benjamin, PR, and Burke, JF. Myomodulin gene of Lymnaea: structure, expression, and analysis of neuropeptides. 1996. 16: 4949–57. [26] Khan, HR, Price, DA, Doble, KE, Greenberg, MJ, and Saleuddin, AS. Osmoregulation and FMRFamide-related peptides in the

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[43] Rajpara, SM, Garcia, PD, Roberts, R, Eliassen, JC, Owens, DF, Maltby, D, et al. Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons. Neuron, 1992. 9: 505–13. [44] Roovers, E, Vincent, ME, van Kesteren, E, Geraerts, WP, Planta, RJ, Vreugdenhil, E, et al. Characterization of a putative molluscan insulin-related peptide receptor. Gene, 1995. 162: 181– 8. [45] Roubos, EW, Geraerts, WP, Boerrigter, GH, and van Kampen, GP. Control of the activities of the neurosecretory Light Green and Caudo-Dorsal Cells and of the endocrine Dorsal Bodies by the lateral lobes in the freshwater snail Lymnaea stagnalis (L.). Gen Comp Endocrinol, 1980. 40: 446–54. [46] Santama, N, Wheeler, CH, Burke, JF, and Benjamin, PR. Neuropeptides myomodulin, small cardioactive peptide, and buccalin in the central nervous system of Lymnaea stagnalis: purification, immunoreactivity, and artifacts. 1994. 342: 335– 51. [47] Santama, N, and Benjamin, PR. Gene expression and function of FMRFamide-related neuropeptides in the snail Lymnaea. 2000. 49: 547–56. [48] Satake, H, Kawada, T, Nomoto, K, and Minakata, H. Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review. Zoolog Sci, 2003. 20: 533–49. [49] Schaefer, M, Picciotto, MR, Kreiner, T, Kaldany, RR, Taussig, R, and Scheller, RH. Aplysia neurons express a gene encoding multiple FMRFamide neuropeptides. 1985. 41: 457–67. [50] Shipilov, VN, Shpakov, AO, and Rusakov, YI. Pleiotropic action of insulin-like peptides of mollusk, Anodonta cygnea. Ann N Y Acad Sci, 2005. 1040: 464–5. [51] Smit, AB, Thijsen, SF, and Geraerts, WP. cDNA cloning of the sodium-influx-stimulating peptide in the mollusc, Lymnaea stagnalis. Eur J Biochem, 1993. 215: 397–400. [52] Smit, AB, Spijker, S, Van Minnen, J, Burke, JF, De Winter, F, Van Elk, R, et al. Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis. Neuroscience, 1996. 70: 589–96. [53] Sweedler, JV, Li, L, Floyd, P, and Gilly, W. Mass spectrometric survey of peptides in cephalopods with an emphasis on the FMRFamide-related peptides. J Exp Biol, 2000. 203: 3565–73. [54] Sweedler, JV, Li, L, Rubakhin, SS, Alexeeva, V, Dembrow, NC, Dowling, O, et al. Identification and characterization of the feeding circuit-activating peptides, a novel neuropeptide family of aplysia. J Neurosci, 2002. 22: 7797–808. [55] Tensen, CP, Cox, KJ, Burke, JF, Leurs, R, van der Schors, RC, Geraerts, WP, et al. Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor. Eur J Neurosci, 1998. 10: 3409–16. [56] Tensen, CP, Cox, KJ, Smit, AB, van der Schors, RC, Meyerhof, W, Richter, D, et al. The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family. J Neurosci, 1998. 18: 9812–21. [57] Weiss, KR, Bayley, H, Lloyd, PE, Tenenbaum, R, Kolks, MA, Buck, L, et al. Purification and sequencing of neuropeptides contained in neuron R15 of Aplysia californica. Proc Natl Acad Sci USA, 1989. 86: 2913–7. [58] Wickham, L, and Desgroseillers, L. A bradykinin-like neuropeptide precursor gene is expressed in neuron L5 of Aplysia californica. DNA Cell Biol, 1991. 10: 249–58.

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released during this activity, including the egg-laying hormone (ELH; [1]), αBCP, βBCP, δBCP, and γBCP [30]. Two additional peptides—the acidic peptide (AP; [35]) and εBCP (see reference in [30])—have been chemically characterized. Peptides A and B, as well as three peptide complexes, have been isolated from the atrial gland in the reproductive tract of Aplysia (see reference in [34]). The two peptides, and the complexes as well, are able to induce egg-laying behavior. Three other biologically active peptides—califin A, B, and C—were isolated and characterized from the atrial gland [34].

ABSTRACT Many aspects of molluscan reproduction rely on peptides. Egg laying, secretory activity, sexual behavioral repertoires, and chemical attraction are some of the key actions of sexual behavior controlled by peptides in mollusks. In this chapter a detailed analysis of peptides involved in molluscan reproduction is reported. In particular, the discovery, the structure of the gene, and the distribution and biological actions of egg laying hormones, GnRH, APGWamide, vasopressin/oxytocin, and pheromones are discussed. Only those peptides for which the primary structure is known are reported in this chapter.

Structure of the Precursor mRNA/Gene In Lymnaea the CDCs express three different caudodorsal cell hormone (CDCH) genes: CDCH-1, -2, and -3 (see reference in [19]). The CDCH-1 gene gives rise to nine different peptides (see reference in [19]), including the caudodorsal cell hormone-1, the εpeptide, the calfluxin, the α-caudodorsal cell peptide, the δ-peptide, β1- and β2-peptides, and the carboxyl terminally located peptide, from the cerebral commissure. The CDCH-2 gene encodes the same peptides, with the exception of caudodorsal cell hormone-2, which is different from the ovulation hormone (see reference in [19]). At least five distinct genes for ELH exist in Aplysia (see references in [35]). Transcription of this small multigene family results in the expression of at least five distinct RNA transcripts encoding ELH. The pattern of transcripts differs strikingly in different tissues: Bag cells express three distinct mRNA species, whereas the atrial gland expresses two distinct mRNAs. All cDNAs contain an exon that spans the coding region (exon III) and one or two additional exons. Genomic clones encoding peptides A and B have been isolated and subjected to nucleotide sequence analysis [35].

PEPTIDES AND REPRODUCTION Egg-laying Hormones (ELHs): Discovery The caudodorsal cell (CDC) system of Lymnaea and the bag cells of Aplysia function as a peptidergic command center that initiates and coordinates ovulation and egg mass production with a fixed sequence of overt behaviors: cessation of locomotion, turning, modulation of feeding movements, oviposition, and inspection [3, 25]. In Lymnaea egg laying and correlated processes are initiated and coordinated by CDC, neuroendocrine cells located in the cerebral ganglia near the intercerebral commissure (COM), the neurohemal area of the CDC. Around the time of ovulation, all CDC display a synchronous spiking activity (discharge), which allows peptide release [25]. In a similar way, the bag cells of Aplysia, two clusters of neuroendocrine cells located in the abdominal ganglion, show a prolonged and synchronous burst of electrical activity (afterdischarge) that precedes egg-laying behavior [1]. At least nine peptides are Handbook of Biologically Active Peptides

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242 / Chapter 37 Distribution of mRNA In Lymnaea, in situ hybridization experiments demonstrated that CDCH genes appeared to be expressed in the CDC, while a group of small neurons in the cerebral ganglia express the CDCH-I gene only [43]. A widespread expression of the CDCH genes has also been demonstrated in peripheral tissues [44]. Finally, sensory neurons in the head skin and mantle edge were found to express the CDCH-I gene [43]. In Aplysia it has been shown that greater than 90% of the ELH genes’ family expression occurs in the bag cells, but these genes are expressed also in the atrial gland and in an extensive system of neurons distributed in four of the five ganglia of the central nervous system [2].

Biological Actions In Lymnaea stagnalis, during discharges, CDCH-I is released into the blood, where it acts as a hormone that stimulates ovulation and egg mass formation as well as synthesis of secretory products in the female accessory sex glands and affects neurons in the neuronal circuits controlling locomotion and feeding (see references in [22]). During the discharge, calfluxin stimulates the release and/or synthesis of perivitelline fluid and, in addition, affects feeding movements, and α-CDCP elicits electrical discharge activity in all CDCs, causing the maximal release of hormone (see reference in [19]) and induces the transition from cessation of locomotion to the turning phase of egg-laying behavior (see reference in [19]). Egg laying in Aplysia californica is controlled by the bag cells, which have similar morphological and electrophysiological characteristics to the CDCs. They release a battery of peptides, including ELH and bag cell peptides αBCP, βBCP, and γBCP [1, 33]. ELH induces ovulation and alters the electrical activities of several neurons in the abdominal, buccal, and pedal ganglia (see references in [28]). αBCP mimics bag cellinduced inhibition of several identified and unidentified neurons (see references in [28]). Two additional peptides released by bag cells—the acidic peptide [35] and εBCP [29]—have no known biological or pharmacological activities.

GnRH: DISCOVERY Gonadotrophin-releasing hormone (GnRH) (discovered by Schally, an author of this book; also see GnRH chapter in the brain peptides section of this book) is a decapeptide neurohormone crucial for the regulation of reproductive and neural functions in vertebrates. Multiple reports indicated the presence of bioactive

molecules that immunologically and biochemically resemble vertebrate GnRH in gastropods [15, 39, 46, 49], bivalves [31], and cephalopods [9, 10, 20].

Structure of the Precursor mRNA/Gene A GnRH precursor cDNA has been cloned from the cephalopod Octopus vulgaris [20]. The precursor protein contains 87 amino acids. The octopus GnRH-like peptide contains two additional amino acids at positions 2 and 3, but the general motif of a pyroglutamyl residue at the N-terminus and amidated glycine residues at the C-terminus is conserved. A peptide similar to the chicken I form of GnRH (cGnRH-I) has been found and partially purified in the same species of octopus [10]. It is likely that the recently isolated Octopus GnRH-like peptide [20] is different from the peptide described by Di Cristo et al. [10], since it is unlikely that these two peptides share identical immunological and biochemical properties given the marked structural differences.

Distribution of mRNA In situ hybridization analysis is only reported in Octopus vulgaris [21]. Oct-GnRH mRNA-expressing cell bodies were located in the supraesophageal and subesophageal parts of the central nervous system (CNS). However, the authors did not provide any new aspects on the localization of GnRH in Octopus that was already completed [9, 10]. GnRH neurons are mainly present in the posterior olfactory lobule. These neurons send axons to the secretory cells of the optic gland. GnRH is also present in the subpedunculate area that controls the same gland. GnRH neurons have also been localized in both the subesophageal and supraesophageal masses, as well as in the plexiform layer of the deep retina in the optic lobe.

Biological Actions In Helisoma trivolvis, CNS extract was capable of stimulating gonadotrophin release from dispersed goldfish pituitary cells [15, 46]. In addition, the application of mammalian GnRH to Helisoma CNS elicited diverse electrophysiological and morphological effects on individual neurons [15]. Application of vertebrate GnRH to bag cells of Aplysia californica inhibited the electrical response [49], a finding supported by a previous study on Lymnaea stagnalis [46]. The reproductive and neural involvement of the molluscan GnRH was further corroborated by studies on the bivalves and cephalopods. These studies demonstrated a strong mitogenic action of GnRH on bivalve gonadal cells [31] and a spatial distribution of GnRH-IR in the CNS that is highly sug-

Molluscan Peptides and Reproduction / 243 gestive of reproductive regulation [9]. In Octopus, GnRH was proposed to act on the secretory cells of the gonadotropic optic gland. Interestingly, GnRH neurons in Octopus are located in the olfactory lobe consistently with the known relationship between GnRH and chemosensory system observed in vertebrates [9]. These results are reinforced by the presence of GnRH in the osphradium of Aplysia californica [39]. It has also been proposed that GnRH in Octopus is involved in the control of reproductive ducts [10] and the contractions of the heart [21].

DISCOVERY Ala-Pro-Gly-Trp-NH2 (APGWamide) was isolated from ganglia of the prosobranch mollusk Fusinus ferrugineus [26]. In Lymnaea stagnalis [7] and Helix aspersa [16], APGWamide is primarily localized in neurons of the asymmetrical right lobe of the cerebral ganglion. APGWamide-related peptides have also been found in bivalves [14]. In the cephalopod Sepia officinalis, the dipeptide GWamide has been purified from the optic lobe [17], and immunoreactivity has been found recently in the nervous system as well as in the oviducal gland of Octopus vulgaris APGWamide [11].

Structure of the Precursor mRNA/Gene The cDNA encoding for APGWamide has been cloned from Lymnaea stagnalis [36] and Aplysia californica [12], whereas an APGW-related peptide-containing cDNA has been found in Mytilus edulis [14]. In Lymnaea stagnalis, the primary structure of the APGW preprohormone deduced from the cDNA sequence predicts that the prohormone can be endoproteolytically cleaved to give rise to 10 copies of the sequence APGWG and a C-terminal peptide of 40 residues, called CALP (Cterminal anterior lobe peptide) [36]. Both the APGWG peptides and CALP are posttranslationally amidated. In Aplysia, sequence analysis of the cDNA clone demonstrated that it contained a 209-amino-acid APGWamide precursor [12]. In Mytilus, the nucleotide sequence of the APGWamide related-peptide cDNA clone encodes a 196-amino-acid pre-proprotein [14]. The full processing of the Mytilus precursor has the potential to generate five RPGWamide peptides, one KPGWamide, one TPGWamide, and a C-terminal peptide, but no copies of APGWamide.

Distribution of mRNA The gene encoding the precursor of APGWamide is mainly expressed in the neurons of the right anterior lobe of the cerebral ganglia of Lymnaea stagnalis [36].

In addition, gene expression was also detected in the left cerebral ganglion and in several neurons in other parts of the CNS [36]. In Aplysia, Northern blot analysis of total RNA indicated that a major transcript was expressed predominantly in the right cerebral and right pedal ganglia [12].

Biological Actions In Lymnaea, it has been shown that APGWamide inhibits contraction of the penis retractor muscle [6, 7] and functions as a neurotransmitter within the CNS, inhibiting the activity of certain neuroendocrine cells and coordinating the activity of different populations of penial motoneurons [6]. APGWamide and related peptides have also been suggested to play a key role in the control of male copulation behavior in other gastropods such as Helix aspersa [8] and to function as an inhibitory neurotransmitter in the central nervous system of both Helix aspersa and Achatina fulica [27]. The APGWamide-related peptides of Mytilus show similar potency on contraction of both the pedal retractor and anterior byssus retractor bivalve muscles [18]. In the cephalopod Sepia officinalis, the dipeptide GWamide purified from the optic lobe shows a strong inhibiting action on the motility of the mature oviduct [17]. APGWamide in O. vulgaris is strictly linked and associated to those systems and lobes that directly process close and distant chemical signals [11]. These results open new insights into the possible effects that both distant and close chemical stimuli can exert on neuropeptidergic circuitries, which may affect the reproductive behavior of cephalopods [11].

VP/OT: DISCOVERY The members of the vasopressin (VP) and oxytocin (OT) superfamily are widely distributed throughout the animal kingdom. In the snail Lymnaea stagnalis, Lysconopressin is the only member present [40], while cephalotocin (CT) and octopressin (OP) were found in Octopus vulgaris [32, 38].

Structure of the Precursor mRNA/Gene In Lymnaea stagnalis, conopressin cDNA encodes for a precursor that is organized very similarly to the prohormones of the vasopressin/oxytocin superfamily with a hydrophobic signal peptide, a nominal peptide, a neurophysin domain, and a C-terminal copeptin homologous extension of the neurophysin domain [41]. In Octopus vulgaris, the OP and CT precursor cDNAs predict a polypeptide of 145 and 152 amino acids, respectively [38]. The two genes of the oxytocin/vaso-

244 / Chapter 37 pressin superfamily in Octopus vulgaris lack introns and consist of a single exon in their protein-coding regions, unlike most vertebrates and Lys-conopressin precursors [23]. The N-terminal sequences of the OP precursor and the CT precursor are different from each other [38]. Both of the remaining parts are identical except for two nucleotide bases.

Distribution of mRNA In situ hybridization on Lymnaea CNS showed that the conopressin gene is expressed in a few neurons in both pedal ganglia [41]. In Octopus in situ hybridization using CT- and OP-specific cRNA antisense probes on adjacent sections was performed [38]. The expression of CT mRNA was primarily limited within the ventral median vasomotor lobe of the subesophageal brain. OP mRNA was expressed in the superior buccal, anterior basal, median basal, dorsal basal, vertical, subvertical, posterior brachial, and palliovisceral lobes [38].

Receptor A receptor for vasopressin-related Lys-conopressin has been cloned in Lymnaea stagnalis (LSCPR1; see [42]). Expression of this receptor mRNA was detected in central neurons and peripheral muscles associated with reproduction. In addition, a novel Lys-conopressin receptor subtype, named LSCPR2, was found [42]. In Octopus brain, a G-protein-coupled receptor (GPCR) specific to CT (CTR1) was cloned (see [24]). This cDNA coded for an orphan receptor that was activated by CT. An additional CT receptor, CTR2, and a novel OP receptor, OPR, were later found in the CNS of Octopus vulgaris [24]. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues [24].

Biological Actions Very little is known about the functions of these peptides in mollusks. It has been demonstrated that in Lymnaea Lys-conopressin excites the vas deferens in vitro in a dose-dependent fashion (see in [45]). Upon application, Lys-conopressin also excites pacemaker neurons in the anterior lobe of the right cerebral ganglion, acting on the inward current (see in [45]).

PHEROMONES: DISCOVERY The possibility that mollusks use peptide or protein pheromones to communicate with one another has been explored in a number of species of gastropods, as

well as in cephalopods. Their role in a large number of species, including bacteria, are summarized by Altstein in the last section of this book. The structural and behavioral evidence for peptide pheromonal communication is most complete in Aplysia (see reference in [5]). However, molluscan peptide pheromones include (1) a family of water-borne peptide pheromonal attractants named attractin characterized from five Aplysia species (see for reference [5]) and synthesized by the albumen gland; (2) the tetrapeptide (Ile–Leu–Met–Glu; ILME) characterized in the cephalopod Sepia officinalis [47]; and (3) a Sepia sperm-attracting peptide (SepSAP; PIDPGVamide) synthesized in oocytes [48].

Structure of the Precursor mRNA/Gene The sequence of attractin cDNA from Aplysia californica encodes a 76-amino-acid precursor [13], which contains a single copy of a 58-residue peptide. Northern blot analysis demonstrated the presence of two transcripts in the albumen gland of Aplysia [13]. The nuclear magnetic resonance spectra of a solution of recombinant A. californica attractin has shown that attractin contains two antiparallel alpha-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in attractin function (see in [37]).

Biological Actions Pheromones modulate many aspects of Aplysia social behavior, which is associated with sexual maturity. At least three stimuli lead to the release of pheromones regulating behavior: (1) the presence of Aplysia, (2) the presence of mating animals, and (3) the presence of egg cordons [37]. During the reproductive season, Aplysia moves into breeding aggregations, mates, and lays eggs. The aggregations usually occur where egg cordons are laid. Attractin elutes from the cordons into the surrounding water acting as long-distance signaling [37]. Experimental effects of attractin are (1) attraction, (2) an increase in respiratory pumping, and (3) a reduced latency to mating and an increase in the number of animals mating as hermaphrodites [37]. Some behavioral effects of attractin are species-specific, whereas others are not [37]. This lack of speciesspecificity makes attractin the first nonspecies-specific peptide pheromone in invertebrates and vertebrates. In the last two years, cDNAs encoding novel proteins, enticin, temptin, and seductin, which seem to act synergistically with attractin, have been characterized (see [4] for references). In the cephalopod Sepia officinalis, in vitro bioassays suggests that ILME specifically targets the genital tract and the nidamental glands, which are involved in the

Molluscan Peptides and Reproduction / 245 transport of the oocytes during egg laying and the mechanical secretion of egg capsules, respectively [47]. Whether ILME stimulates overt behaviors in intact male or female cuttlefish is an important issue that remains to be addressed. Moreover, in Sepia PIDPGVamide attracts freshly dissected spermatozoa at low concentrations [48].

References [1] Chiu, AY, Hunkapiller, MW, Heller, E, Stuart, DK, Hood, LE, and Strumwasser, F. Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica. Proc Natl Acad Sci USA, 1979. 76: 6656–60. [2] Chiu, AY and Strumwasser, F. An immunohistochemical study of the neuropeptidergic bag cells of Aplysia. J Neurosci, 1981. 1: 812–26. [3] Cobbs, JS and Pinsker, HM. Role of bag cells in egg deposition of Aplysia brasiliana. I. Comparison of normal and elicited behaviors. J Comp Physiol [A], 1982. 147: 523–35. [4] Cummins, SF, Nichols, AE, Warso, CJ, and Nagle, GT. Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction. Peptides, 2005. 26: 351–9. [5] Cummins, SF, Schein, CH, Xu, Y, Braun, W, and Nagle, GT. Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness. Peptides, 2005. 26: 121–9. [6] De Boer, PA, Ter Maat, A, Pieneman, AW, Croll, RP, Kurokawa, M, and Jansen, RF. Functional role of peptidergic anterior lobe neurons in male sexual behavior of the snail Lymnaea stagnalis. J Neurophysiol, 1997. 78: 2823–33. [7] De Lange, RP, Joosse, J, and Van Minnen, J. Multi-messenger innervation of the male sexual system of Lymnaea stagnalis. J Comp Neurol, 1998. 390: 564–77. [8] de Lange, RP and van Minnen, J. Localization of the neuropeptide APGWamide in gastropod molluscs by in situ hybridization and immunocytochemistry. Gen Comp Endocrinol, 1998. 109: 166–74. [9] Di Cosmo, A and Di Cristo, C. Neuropeptidergic control of the optic gland of Octopus vulgaris: FMRF-amide and GnRH immunoreactivity. J Comp Neurol, 1998. 398: 1–12. [10] Di Cristo, C, Paolucci, M, Iglesias, J, Sanchez, J, and Di Cosmo, A. Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH). J Exp Zool, 2002. 292: 267–76. [11] Di Cristo, C, Van Minnen, J, and Di Cosmo, A. The presence of APGWamide in Octopus vulgaris: A possible role in the reproductive behavior. Peptides, 2005. 26: 53–62. [12] Fan, X, Croll, RP, Wu, B, Fang, L, Shen, Q, Painter, SD, et al. Molecular cloning of a cDNA encoding the neuropeptides APGWamide and cerebral peptide 1: Localization of APGWamidelike immunoreactivity in the central nervous system and male reproductive organs of Aplysia. 1997. 387: 53–62. [13] Fan, X, Wu, B, Nagle, GT, and Painter, SD. Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying. 1997. 48: 167–70. [14] Favrel, P and Mathieu, M. Molecular cloning of a cDNA encoding the precursor of Ala-Pro-Gly-Trp amide-related neuropeptides from the bivalve mollusc Mytilus edulis. Neurosci Lett, 1996. 205: 210–4. [15] Goldberg, JI, Garofalo, R, Price, CJ, and Chang, JP. Presence and biological activity of a GnRH-like factor in the

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nervous system of Helisoma trivolvis. J Comp Neurol, 1993. 336: 571–82. Griffond, B, Van Minnen, J, and Colard, C. Distribution of APGWa-immunoreactive substances in the central nervous system and reproductive apparatus of Helix aspersa. Zoolog Sci, 1992. 9: 533–9. Henry, J, Favrel, P, and Boucaud-Camou, E. Isolation and identification of a novel Ala-Pro-Gly-Trp-amide-related peptide inhibiting the motility of the mature oviduct in the cuttlefish, Sepia officinalis. Peptides, 1997. 18: 1469–74. Henry, J, Zatylny, C, and Favrel, P. HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: Comparative activities on Mytilus muscles. Brain Res, 2000. 862: 162–70. Hermann, PM, de Lange, RP, Pieneman, AW, ter Maat, A, and Jansen, RF. Role of neuropeptides encoded on CDCH-1 gene in the organization of egg-laying behavior in the pond snail, Lymnaea stagnalis. J Neurophysiol, 1997. 78: 2859–69. Iwakoshi, E, Takuwa-Kuroda, K, Fujisawa, Y, Hisada, M, Ukena, K, Tsutsui, K, et al. Isolation and characterization of a GnRHlike peptide from Octopus vulgaris. Biochem Biophys Res Commun, 2002. 291: 1187–93. Iwakoshi-Ukena, E, Ukena, K, Takuwa-Kuroda, K, Kanda, A, Tsutsui, K, and Minakata, H. Expression and distribution of octopus gonadotropin-releasing hormone in the central nervous system and peripheral organs of the octopus (Octopus vulgaris) by in situ hybridization and immunohistochemistry. J Comp Neurol, 2004. 477: 310–23. Jansen, RF and ter Maat, A. Ring neuron control of columellar motor neurons during egg-laying behavior in the pond snail. J Neurobiol, 1985. 16: 1–14. Kanda, A, Takuwa-Kuroda, K, Iwakoshi-Ukena, E, and Minakata, H. Single exon structures of the oxytocin/vasopressin superfamily peptides of octopus. Biochem Biophys Res Commun, 2003. 309: 743–8. Kanda, A, Satake, H, Kawada, T, and Minakata, H. Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris). Biochem J, 2005. 387: 85–91. Kits, KS. States of excitability in ovulation hormone producing neuroendocrine cells of Lymnaea stagnalis (gastropoda) and their relation to the egg-laying cycle. J Neurobiol, 1980. 11: 397–410. Kuroki, Y, Kanda, T, Kubota, I, Fujisawa, Y, Ikeda, T, Miura, A, et al. A molluscan neuropeptide related to the crustacean hormone, RPCH. Biochem Biophys Res Commun, 1990. 167: 273–9. Liu, GJ, Santos, DE, Takeuchi, H, Kamatani, Y, Minakata, H, Nomoto, K, et al. APGW-amide as an inhibitory neurotransmitter of Achatina fulica Ferussac. 1991. 177: 27–33. Mayeri, E, Rothman, BS, Brownell, PH, Branton, WD, and Padgett, L. Nonsynaptic characteristics of neurotransmission mediated by egg-laying hormone in the abdominal ganglion of Aplysia. J Neurosci, 1985. 5: 2060–77. Nagle, GT, Painter, SD, Blankenship, JE, and Kurosky, A. Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity. 1988. 263: 9223–37. Nagle, GT, De Jong-Brink, M, Painter, SD, Bergamin-Sassen, MM, Blankenship, JE, and Kurosky, A. Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity. 1990. 265: 22329–35. Pazos, AJ and Mathieu, M. Effects of five natural gonadotropinreleasing hormones on cell suspensions of marine bivalve gonad: stimulation of gonial DNA synthesis. Gen Comp Endocrinol, 1999. 113: 112–20.

246 / Chapter 37 [32] Reich, G. A new peptide of the oxytocin/vasopressin family isolated from nerves of the cephalopod Octopus vulgaris. Neurosci Lett, 1992. 134: 191–4. [33] Rothman, BS, Weir, G, and Dudek, FE. Egg-laying hormone: Direct action on the ovotestis of Aplysia. Gen Comp Endocrinol, 1983. 52: 134–41. [34] Rothman, BS, Hawke, DH, Brown, RO, Lee, TD, Dehghan, AA, Shively, JE, et al. Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica. J Biol Chem, 1986. 261: 1616–23. [35] Scheller, RH, Jackson, JF, McAllister, LB, Rothman, BS, Mayeri, E, and Axel, R. A single gene encodes multiple neuropeptides mediating a stereotyped behavior. Cell, 1983. 32: 7–22. [36] Smit, AB, Jimenez, CR, Dirks, RW, Croll, RP, and Geraerts, WP. Characterization of a cDNA clone encoding multiple copies of the neuropeptide APGWamide in the mollusk Lymnaea stagnalis. J Neurosci, 1992. 12: 1709–15. [37] Susswein, AJ and Nagle, GT. Peptide and protein pheromones in molluscs. Peptides, 2004. 25: 1523–30. [38] Takuwa-Kuroda, K, Iwakoshi-Ukena, E, Kanda, A, and Minakata, H. Octopus, which owns the most advanced brain in invertebrates, has two members of vasopressin/oxytocin superfamily as in vertebrates. Regul Pept, 2003. 115: 139–49. [39] Tsai, PS, Maldonado, TA, and Lunden, JB. Localization of gonadotropin-releasing hormone in the central nervous system and a peripheral chemosensory organ of Aplysia californica. Gen Comp Endocrinol, 2003. 130: 20–8. [40] van Kesteren, RE, Smit, AB, de With, ND, van Minnen, J, Dirks, RW, van der Schors, RC, et al. A vasopressin-related peptide in the mollusc Lymnaea stagnalis: Peptide structure, prohormone organization, evolutionary and functional aspects of Lymnaea conopressin. Prog Brain Res, 1992. 92: 47–57. [41] van Kesteren, RE, Smit, AB, Dirks, RW, de With, ND, Geraerts, WP, and Joosse, J. Evolution of the vasopressin/oxytocin super-

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38 Free-Living Nematode Peptides E. P. MASLER

A discussion of peptides in free-living nematodes necessarily focuses on Caenorhabditis elegans, which contains the first completely sequenced animal genome and has become an important organism for molecular genetic and cellular biology research. Other free-living species have been studied, including the related C. briggsae and Panagrellus redivivus, but the bulk of peptiderelated information in free-living nematodes is from C. elegans. Studies on biologically active peptides in nematodes are extensive, but the number of different peptide families discovered thus far is small compared with higher invertebrates and vertebrates. However, complexity within each family can be quite high, and genomic analysis suggests that the actual number of families is substantial. Here we consider four families of peptides in free-living nematodes, one of which has been the most extensively studied.

ABSTRACT Nematodes depend on peptide messengers to regulate diverse behavioral, developmental, and metabolic events. In free-living nematodes, sequencing of the first complete animal genome facilitated detection of biologically active peptides, including neuropeptides. This has complemented previous and ongoing efforts in biochemistry, pharmacology, and physiology, revealing that nematodes possess some peptide families that are exceedingly complex. Complexity can result from large numbers of genes encoding related amino acid sequences, as in the case of the FMRFamide-like peptides or from intricate variations of molecular architectures, as seen among members of the insulin-like peptides family. These and two other peptide families are described, along with their established and proposed functions.

FMRFamide-LIKE PEPTIDES (FLPs)

INTRODUCTION

Discovery and Structure

Nematodes are second only to insects in number of species, breadth of distribution, and amount of biomass. They occupy nearly every ecological niche and include both parasitic and nonparasitic species. Most are microscopic, although some animal-parasitic species are quite large. Nematodes are unsegmented roundworms having a longitudinal nerve cord and nerve ring. The nervous system is accompanied by a rather simple anatomy, including muscular mouthparts with a pharyngeal pump to facilitate feeding, an intestine that serves as a metabolic organ somewhat analogous to the mammalian liver, gonads, sexually dimorphic features such as the uterus, a muscular body wall, and cuticle. All nematodes have an egg stage, four larval stages, and one or more adult forms. Parasitic species are covered in Chapter 39. Handbook of Biologically Active Peptides

By far the most comprehensively studied family of peptides in nematodes consists of the FMRFamide-like peptides (FLPs). They are hormones affecting neuromuscular, behavioral, reproductive, and other activities. FLPs have been comprehensively reviewed and cataloged elsewhere [12, 14]. Free-living nematode FLPs are neuropeptides of 4 to ∼15 amino acids, characterized by an amidated C-terminus with an RFNH2 motif. This is a common posttranslational modification of biologically active peptides and requires a glycine residue at the C-terminus of the precursor. Thus, the “RFG” pattern has been used to scan precursor peptides for the presence of potential FLPs. Beyond this signature, there is sequence variation toward the N-terminus. Typically, there is more than one flp-gene, and multiple

247

248 / Chapter 38 sequences of unique FLPs are present, providing a broad diversity. This is best illustrated in C. elegans, where 23 flp-genes have been characterized, encoding precursor peptides predicting 61 unique FLP sequences (Table 1). All predicted FLPs have been confirmed except the product of flp-23, and many of the predicted peptides have been isolated. Four of the C. elegans flpgenes each encodes a single unique FLP (Category I,

Table I), and six genes (flp-6, flp-8, flp-9, flp-14, flp-20, flp-22) encode multiple copies of single sequences. The majority of flp-genes encode multiple unique sequences (Categories III and IV). Genes in Category IV not only for multiple sequences but multiple copies as well. As many as nine unique sequences are produced from a single gene (flp-3). Most of the genes that encode multiple sequences produce peptides in which the last four

TABLE 1. Arbitrary C. elegans flp gene categories. Gene Category I flp-10 flp-12 flp-21 flp-23 Category II flp-6 flp-8 flp-9 flp-14 flp-20 flp-22 Category III flp-1

WormBase ID (T06C10.4) (C05E11.8) (C26F1.10) (F22B7.2)

QPKARSGYIRFG RNKFEFIRFGC CLGPRPLRFG TKFQDFLRFG

(F07D3.2) (F31F6.4a) (C36H8.3) (Y37D8A.15) (E01H11.3) (F39H2.1)

KSAYMRFGC,P KNEFIRFGC KPSFVRFGC KHEYLRFGC,P AMMRFG SPSAKWMRFG

(F23B2.5a)

SADPNFLRFGC,P SQPNFLRFGC ASGDPNFLRFGC SDPNFLRFGC,P AAADPNFLRFGC KPNFLRFGC AGSDPNFLRFG SPREPIRFG LRGEPIRFG SPLGTMRFG TPLGTMRFG EAEEPLGTMRFG ASEDALFGTMRFG NPLGTMRFG EDGNAPFGTMRFG SAEPFGTMRFG SADDSAPFGTMRFG NPENDTPFGTMRFG PTFIRFG ASPSFIRFG GAKFIRFG AGAKFIRFG APKPKFIRFG AMRNALVRFG NGAPQPFVRFG ASGGMRNALVRFG GGPQGPLRFG RGPSGPLRFG WANQVRFG ASWASSVRFG

flp-2

(W07E11.3a)

flp-3

(W07E11.2)

flp-4

(C18D1.3)

flp-5

(C03G5.7)

flp-11

(K02G10.4a)

flp-15

(ZK525.1)

flp-19

(M79.4)

Category IV flp-7

Peptide

(F49E10.3)

SPMQRSSMVRFG TPMQRSSMVRFG SPMERSAMVRFG SPMDRSKMVRFG

Copy Number

Receptor ID

(C39E6.6)

[x6] [x3] [x2] [x4] [x2] [x3]

(T19F4.1) (″)

(C26F1.6)

(C10C6.2) (″)

[x3] [x2]

(C26F1.6)

Free-Living Nematode Peptides / 249 TABLE 1. (Continued) Gene flp-13

WormBase ID (F33D4.3)

flp-16

(F15D4.8)

flp-17

(C52D10.11)

flp-18

(Y48D7A.2)

Peptide SDRPTRAMDSPLIRFG AADGAPLIRFGC APEASPFIRFGC ASPSAPLIRFG SPSAVPLIRFG SAAAPLIRFG ASSAPLIRFG AQTFVRFG GQTFVRFG KSAFVRFG KSQYIRFG DFDGAMPGVLRFG EMPGVLRFG SVPGVLRFGC EIPGVLRFG SEVPGVLRFG DVPGVLRFG

Copy Number

Receptor ID

[x2] [x2]

[x2] [x2]

(C39E6.6) [x3]

Number in parentheses following a gene is the WormBase sequence identifier. All sequences confirmed by cDNA isolation or EST identification except for flp-23 where peptide is predicted. The C-terminal glycine predicts amidation of the mature peptide. Number in brackets following a peptide sequence is copy number per gene. Number in parentheses following peptide sequence is confirmed receptor. CAmidated product isolated from C. elegans, Pamidated product isolated from P. redivivus. Categories used: I-single sequence, single copy; II-single sequence, multiple copies; III-multiple sequences, single copy; IV-mixed multiple and single copies. Data from WormBase, GenBank, and references cited in text.

residues are identical. Exceptions are flp-11, flp-13, flp17, and flp-19. All FLPs have neuromuscular effects, but not all of the effects are the same, leading to the hypothesis that a variety of FLP receptors exists. This argument is supported by the large number of unique sequences identified. Among the many FLPs identified, KHEYLRF (flp-14) is present in all nematode species examined and is the most abundant FLP in C. elegans and P. redivivus. In C. elegans, flp-14 encodes four copies of the peptide. KSAYMRF (flp-6) is also present in all species examined, and six copies are encoded by C. elegans flp-6. Also, SADPNFLRF and SDPNFLRF (flp-1) are widely distributed. The abundance and/or distribution of these FLPs indicates a fundamental importance.

flps are expressed in neurons, some are also expressed in nonneuronal cells, including head muscle (flp-2, flp11), pharyngeal muscle (flp-5, flp-15), sheath cells (flp11, flp-15), vulval cells (flp-10), and uterine cells (flp-2, flp-11). flp-15 is also detected in late embryos. flp-4 and flp-8 expression increases in the dauer stage. When the worm exits dauer diapause and resumes development, expression of these two genes ceases in a specific set of neurons. The large number and variety of unique FLPs can indicate redundancy to ensure that critical physiological and developmental events occur or enable flexible and specific regulation of such events. Receptors, for example, may use multiple ligands to modulate responses to various similar, but distinct, messages [8].

Receptors and Function Expression Nineteen of the 23 flps are expressed throughout larval and adult stages in C. elegans, including both hermaphrodite and male adult forms [8, 12, 22]. Expression is distributed among various sets of neurons and is conservatively estimated to be present in ∼160 of the 300 total neurons in the worm. A number of flps are expressed in male-specific neurons associated with mating, as well as in other neurons. While all detected

Five receptors have been characterized from C. elegans. All are G-protein coupled, 7-transmembrane members of the rhodopsin family. NPR-1 (gene C39E6.6) [10, 22] is a 457aa protein that specifically binds ligand products of flp-18 (EMPGVLRF) and flp-21 (GLGPRPLRF). Two naturally occurring versions of the receptor differ by a single residue at position 215 (NPR1-215F, NPR-1-215V), with NPR-1-215F coded by the ancestral gene. C. elegans strains exhibit either social or

250 / Chapter 38 solitary feeding behaviors, and NPR-1-215V suppresses social feeding and is functionally dominant over NPR1-215F. Each variant, expressed in Xenopus laevis oocytes, binds FLP-21 equally, while only NPR-1-215V binds FLP-18 (EMPGVLRF). In contrast, both receptors expressed in C. elegans pharyngeal muscle bind each ligand equally. This suggests that the receptors may respond differently to the cellular environment. Overexpression of flp-21 or injection of FLP-21 suppresses social feeding in NPR-1-215F animals, while flp-21 deletion promotes social feeding in animals expressing either receptor variant. A homolog is predicted by C. briggsae gene CBG14540. FLP-15 peptides GGPQGPLRF and RGPSGPLRF are ligands for receptor NPR-3, encoded by C10C6.2 [11]. The 376aa receptor protein was expressed in Chinese hamster ovary cells and exhibited low ligand binding at 37°C. Only after a shift of the culture to 28°C was robust binding obtained, as was also observed with NPR-1 [11]. Gene knockout using RNAi produced phenotypes with greatly impaired locomotion and paralysis [7]. A homolog is predicted by C. briggsae gene CBG06153. The gene C26F1.6 coding for receptor VRFa-1 was cloned and expressed in a human kidney cell line [16]. The expressed protein (360aa) was selectively activated by flp-7 product TPMQRSSMVRF and AMRNALVRF from flp-11 at similar ligand doses (1–1.3 μM). The receptor specificity was determined by the entire length of the ligand, indicating that variations toward the Nterminus are functionally important. RNAi gene knockout in C. elegans produced an increase in egg laying and progeny. A homolog is encoded by C. briggsae gene CBG20584. FLPs SPREPIRF and LRGEPIRF (flp-2) were confirmed as ligands for T19F4.1 [15]. Two alternative splice variants of the receptor were isolated (T19F4.1a, T19F4.1b). The T19F4.1b protein (432aa) and T19F4.1a protein (402aa) differ primarily in the C-terminal region, suggesting different specificities in G-protein coupling. SPREPERFNH2 activated both receptor isoforms at ∼50 nM EC50, whereas EC50 for LRGEPIRFNH2 was >6 μM for each variant. In contrast to other C. elegans FLP receptors expressed in mammalian systems, T19F4.1 is active at 37°C. A homolog is encoded by C. briggsae CBG19036.

directly, however, a rigorous examination of the C. elegans genome was made for neuropeptide-like protein encoding genes (nlp-genes), using a combination of structural and alignment searches [18] and evaluation of related ESTs in other species. The search actively eliminated any genes encoding peptides ending in RFG—for example, flps. Thirty-two C. elegans nlp genes encode 151 total and 134 unique putative neuropeptides. The peptides sort into 11 families based on conserved motifs and similar ESTs and neuropeptides in other species. The authors [18] list the families in decreasing order of confidence that neuropeptides are encoded: (1) homology to orcokinin myotropic neuropeptide: nlp-14 and nlp-15 contain a C-terminal GFxGF, nlp-8 and nlp-15 encode peptides structurally related to those of nlp-14 and -15; (2) homology to myomodulin peptides: nlp-2, nlp-22, nlp-23 contain FRPG, with the glycine predicting amidation; (3) homology to buccalin neuropeptide neurotransmitter: nlp-1, nlp-7, nlp-13 contain C-terminal MSFG and related motifs; (4) homology to insect allatostatin, buccalin: nlp-5 and nlp-6 contain C-terminal MGLG and MGFG; (5) nlp-24, nlp25, and nlp-27 through nlp-32 encode YGGWG and YGGYG C-terminal motifs: similar encoded peptides in other nematode and insect ESTs; (6) nlp-9 and nlp-21 encode GGARAF: similar motifs in other nematodes; (7) nlp-18 and nlp-20 code for FAFA and related motifs, similar to motifs predicted in other nematode ESTs; nlp-20 (F45E4.8) may reside in an operon with an endopeptidase (F45E4.7); (8–11) nlp-4, nlp-10 through nlp12, nlp-17, and nlp-19 encode peptides sorted into four groups based on C-terminal motifs and have hallmarks of neuropeptides but are not clearly related to established neuropeptides.

Expression and Function Postembryonic expression in C. elegans was detected for 13 nlps. Neurons in the ventral nerve cord express nlp-7, -9, -14, -15, and -21. Pharyngeal neurons express nlp-3, -8, -13, -18, -19, -20, -21, and -24. Two nlps, nlp-21 and nlp-31, are expressed in embryos. In addition to the potential functions inferred from homologous neuropeptides, two NLPs (NLP-29, B0213.4 and NLP-31, B0213.6) are proposed to function in the immune response (see “Expression and Function” under “Antimicrobial Peptides”).

NEUROPEPTIDE-LIKE PEPTIDES (NLPs) Discovery and Structure Not all nematode neuropeptides are FLPs. Immunochemical screens using antisera against vertebrate and invertebrate antigens provide circumstantial evidence for numerous other neuropeptides in nematodes. More

INSULINS (INSs) Discovery and Structure Thirty-eight insulin-like peptides (INSs) are predicted from the C. elegans genome. They range in length

Free-Living Nematode Peptides / 251 from 67 to 218 residues and exhibit remarkably little sequence homology. Multiple alignments of INS-1 through INS-38 (WormBase) show that only two sequences have as much as 60% homology (INS-7, INS8), two are 47% homologous (INS-25, INS-29), and two pairs are 40% homologous (INS-22, INS-23; INS-25, INS-29). All other alignments reveal less than 39% homology, with the majority less than 30%. Clearly, this family of peptides is not characterized by sequence identity. Only through the combined use of an extensive array of multiple sequence search tools, expression searches, and molecular modeling were the INS sequences identified [20]. Caenorhabditis elegans insulin genes predict three basic peptide precursor structures [19]. All structures contain a signal sequence, and the B-chain and A-chain peptides characteristic of vertebrate insulin. INS-1 and INS-18 precursors have an intervening C-peptide between the A- and B-chain peptides similar to human insulin precursor. INSs 2–9 have an F-peptide between the signal sequence and B-chain peptide but no C-peptide. The A- and B-chain peptides are contiguous. Notable here is a dauer-associated gene, daf-28 (Y116F11B.1), encoding an insulin-like peptide with INSs 2–9 precursor structure [13]. All remaining INSs have only the signal sequence and contiguous A- and B-peptides. *

30

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In addition to signal sequences and the A-chain and B-chain peptides shared with vertebrate insulins, all C. elegans INSs have highly conserved cysteines essential to formation of inter- and intrachain disulfide bonds [19, 20]. Based on the architectures of disulfide bond formation and the comparison of these architectures with that of human insulin C. elegans INSs have been sorted into three structural groups: α, β, and γ [20]. These are illustrated in Fig. 1, using representative C. elegans INS sequences and human insulin as reference. Since sequence homologies are so low, only those portions of the molecules containing the important A-chain peptide and B-chain peptide insulin signatures are compared. α- and β-group members form three interchain bonds (B1-A2, B2-A4, B3-A5) and one intrachain bond (A1A3). The disulfide intrachain bond (A1-A3) is absent from the α-group, but it is suggested that substitute residues (italics, Fig. 1) form hydrophobic interactions to stabilize the structure [19]. The γ-group has two interchain bonds and one intrachain bond, and in this respect is most similar to vertebrate insulins. All C. elegans INS sequences have residues predicted to enable formation of α-helices (Fig. 1, underlined). Despite little primary structural homology among their group and to vertebrate insulin, all C. elegans INSs are capable of forming stable tertiary structures recognized by *

90

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100

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110

*

Hu : --------------FVNQHLC---GSHLVEALYLVC------GERGFF-----

-------------KRGIVEQCCTS--ICSLYQLENY-CN-----------

a-type 24 : -------MGLIRANQGPQKAC---GRSMMMKVQKLCA-----GGCTIQNDD-26 : -------IGNHHHGTKAGLTC---GMNIIERVDKLCN-----GQCTRNYDA-30 : -------REPVVAAQGAKKTC---GRSLLIKIQQLCH-----GICTVHADD-36 : ------IRKRHPEGKLVIRDC---KRYLIMYSRTICK-----EKCEKFD----

----------------LTIKSCST--GYTDAGFISA-CCPSGFVF--------------------LVIKSCHR--GVSDMEFMVA-CCPTMKLFIH------------------LHETACMK--GLTDSQLINS-CCPPIPQTPFVF-----------FIFTDLLVEGCHSNQTLSNERTREL-CCPNAGSN-----

b-type 1 : ----------------SIRLC---GSRLTTTLLAVCRN----QLCTGLTAFKR 2 : ---------------VQKRLC---GRRLILFMLATC------GECDTD----3 : --------------GDKVKIC---GTKVLKMVMVMCG-----GECSS-----4 : ------------VPAGEVRAC---GRRLLLFVWSTC-----GEPCTPQ----6 : -----------VPAPGETRAC---GRKLISLVMAVC-----GDLCNPQ----7 : -----------VPDEKKIYRC---GRRIHSYVFAVCG-----KACESN----8 : -----------VP-EQKNKLC---GKQVLSYVMALCE-----KACDSN----9 : -----------TLETEKIYRC---GRKLYTDVLSACN-----GPCEPG-----

------------KRGGIATECCEK--RCSFAYLKTF-CCNQDDN-----------------SSEDLSHICCIK--QCDVQDIIRV-CCPNSFRK----------------TNENIATECCEK--MCTMEDITTK-CCPSR-------------------EDMDIATVCCTT--QCTPSYIKQA-CCPEK-------------------EGKDIATECCGN--QCSDDYIRSA-CCP---------------------TEVNIASKCCRE--ECTDDFIRKQ-CCP---------------------TKVDIATKCCRD--ACSDEFIRHQ-CCP---------------------TEQDLSKLCCGN--QCTFVEIRKA-CCADKL-------

g-type 17 : ---------------GSLKLCPPGGASFLDAFNLICP-----MRRRRR----19 : YIIDSSESYEVLMLFGYKRTC---GRRLMNRINRVCVK-------DID----1 2 3

SVSENYNDGGGSLLGRTMNMCCET--GCEFTDIFAI-CNPFG--------------PADIDPKIKLSEHCCIK--GCTDGWIKKHICSEEVLNFGFFEN 12 3 45

B-peptide

A-peptide

FIGURE 1. Multiple alignment (ClustalW) amino acid sequences encompassing the B-peptide and A-peptide portions of human insulin precursor (Hu; SwissProt P01308) and selected insulin-like peptides from Caenorhabditis elegans; INS-1 (F13B12.5), INS-2 (ZK75.2), INS-3 (ZK75.3), INS-4 (ZK75.1), INS-6 (ZK84.6), INS-7 (ZK1251.2), INS-8 (ZK1251.11), INS-9 (C06E2.8), INS-17 (F56F3.6), INS-18 (T28B8.2), INS-24 (ZC334.3), INS-26 (ZC334.1), INS-30 (ZC334.2), INS-36 (Y53H1A.4); gene identifiers from WormBase. Numbers at the top are position numbers for human insulin. Numbers at the bottom indicate cysteines (shaded) participating in disulfide bonds. Dark shading indicates additional cysteines not found in human or γ-group sequences. Underline indicates residues predicted to enable formation of α-helices. Residues that substitute for cysteines in intrachain bonding of type α-group insulins are italicized.

252 / Chapter 38 insulin receptors. In fact, INS-6 (ZK84.6) can bind to the human insulin receptor [4].

Expression Insulin genes are widely expressed throughout development and in a number of tissues [20]. Expression patterns for 14 ins genes (ins-1 through ins-9, ins-11, ins18, ins-21, ins-22, and ins-23) reveal that all except ins-9 are expressed in the embryo, all are expressed in larval stages, and all but ins-2 are expressed in the adult. All ins genes are expressed to varying degrees in the nerve ring and sensory neurons. All except ins-9 are expressed in nonsensory neurons. Six tested genes (ins-1, ins-2, ins-4, ins-5, ins-8, ins-18) are also expressed in nonneuronal tissues including the vulva, intestine, pharynx, and hypodermis.

Receptors and Function A single insulin receptor (daf-2; Y55D5A.5) has been described in C. elegans, although additional receptors are proposed [2]. DAF-2 is a transmembrane receptor tyrosine kinase homolog of the mammalian insulin/IGF receptor family [20]. It is a central member of a complex pathway regulating a number of basic events, including nematode development, longevity, stress response, and dauer formation. Caenorhabditis elegans larvae respond to environmental stress, such as crowding or lack of food, by entering a diapause or arrested state (dauer stage). Upon the return of favorable conditions, larvae resume development. Stimulation of DAF-2 promotes increased metabolism, growth, and aging. Downregulation of the receptor can induce larval dauer formation or extended adult lifespan [9, 13, 19, 20, 23]. Insulins are proposed to regulate metabolism as ligands of DAF-2. INS-1 (F13B12.5) is an antagonist of DAF-2, enhancing dauer arrest [20], while INS-18 (T28B8.2) activates DAF-2 and regulates lifespan [13]. Another insulin-like peptide (DAF-28; Y116F11B.1) also may be a DAF-2 agonist and prevent dauer arrest. Expression of ins-genes in sensory neurons and their interaction with DAF-2 support their roles as regulators of response to external and internal cues. Including insulins, over 93 genes encoding at least 233 unique neuropeptides have so far been reported in free-living nematodes.

specific antigens and render long-lasting immunity, as in the higher vertebrates. Innate immunity is a more basic, rapid response to infection characterized by phagocytosis and production of antimicrobial peptides. Both types of immunity are present in higher vertebrates, while lower animals, including nematodes, depend solely on innate immunity as defense against pathogens. In nematodes, the production of antibacterial peptide factors (ABFs) in response to bacterial challenge was demonstrated in the animal parasite Ascaris suum [21], in which a family of antibacterial factor-like peptides (ASABFs) has been reported [5, 21]. The screening of sequence databases with the ASABF amino acid sequence revealed homologies to only two peptides, both in the C. elegans database [6]. No other significant homologies were detected in any other organism. Genes for the C. elegans homologs (abf-1 and abf-2) were isolated and characterized [6]. They are organized sequentially, produce a polycistronic mRNA, and are thought to form an operon. The product peptides ABF1 and ABF-2 possess structural properties generally found in antimicrobial peptides, including conserved cysteine residues necessary to stabilize the molecule and preserve activity [5]. Antimicrobial peptides are a rapidly evolving group and sequence comparisons usually reveal limited homologies with no significant sequence homology outside of the nematodes [24]. A BLAST search done for this report using C. elegans ABF-1 (BAA89489) and ABF-2 (AAK68258) detected no significant homologs outside of C. elegans, A. suum, and C. briggsae. The highest scoring C. briggsae sequence, hypothetical protein CAE66644 (CbHyP), C. elegans ABF-1 and -2, and A. suum ASABF (BAA11943) are compared in Fig. 2. Sequence homology of ABF-1 is low (35–36% to the other three peptides), while homology of ASABF is 52 and 54%, respectively, to ABF-2 and CbHyP. ABF-2 has antibacterial activity [6] and is 72% homologous to CbHyP. Signal peptides are present in ASABF and both C. elegans ABFs (Fig. 2, underlined, italicized), and a signal sequence is suggested for the C. briggsae hypothetical peptide (underlined only). All four sequences share a number of identical residues in 13 locations (Fig. 2, shaded boxes), including the conserved cysteines (eight total).

Expression and Function ANTIMICROBIAL PEPTIDES Discovery and Structure Animals respond to pathogen challenge through the mobilization of complex molecular defenses in the forms of adaptive and/or innate mechanisms. Adaptive systems are characterized by the ability to remember

ABF-2 was detected immunologically only in the pharyngeal region of larval and adult hermaphrodite stages of C. elegans [6]. abf-1 and abf-2 transcripts were detected in adult hermaphrodites and eggs. Protein expression was detected in larval and adult hermaphrodite stages but not in prehatch embryos. ABF-2 was observed in the pharynx, pharyngeal neurons, and excretory cells, whereas ABF-1 was detected in the pharynx and body

Free-Living Nematode Peptides / 253 ASABF ABF-1 ABF-2 CbHyP

MKTAIIVVLLVIFASTNAAVDFSSCARMDVPGLSKVAQGLCISSCKFQNCGTGHCEKRGGRPTCVCDRCGRGGGEWPSVPMPKGRSSRGRRHS MLYFCLLLVLLLPNNGVSSEAS--CARMDVPVMQRIAQGLCTSSCTAQKCMTGICKKVDSHPTCFCGGCSN-ANDVSLDTLIS----QLPHNMFVRSLFLALLLATIVAADIDFSTCARMDVPILKKAAQGLCITSCSMQNCGTGSCKKRSGRPTCVCYRCANGGGDIPLGALIK----RG---MLFRFLLFTFLCVSHISAGIDFSTCARMDVPVLDKAARGLCITSCSMQNCGTGYCEKRRGRPTCVCSRCANGGGNIPLDSLIK----RGKH--

FIGURE 2. Antibacterial peptides. Multiple alignment (ClustalW) of full-length amino acid sequences of antibacterial peptides from Ascaris suum (ASABF, BAA11943), Caenorhabditis elegans (ABF-1, BAA89489; ABF-2, AAK68258), and C. briggsae (CbHyP, CAE66644). Identical positions are shaded, and include eight conserved cysteines; signal peptides are underlined and italicized. Predicted signal sequence is underlined only. Accession numbers are from GenBank.

wall muscle [6]. Only in A. suum has in vivo bacterial challenge been shown to induce production of nematode antibacterial peptides [21], but in vitro experiments and structural analyses of candidate peptides argue strongly that C. elegans ABFs are true antibacterial peptides [6, 24]. It is interesting that expression of two neuropeptide-like proteins (NLP-29 and NLP-31) was induced in C. elegans exposed to fungal or bacterial pathogens [1] and that synthetic NLP-31 has both potent antifungal and moderate antibacterial activities. NLP-29 and NLP-31 each have less than 22% homology to any of the peptides in Fig. 2, and lack the conserved cysteines. Also, the DAF-2 signaling pathway appears to function in pathogen resistance [3, 17].

Acknowledgment My thanks to Dr. Aaron Maule, Queen’s University, Belfast, for helpful discussions and for providing access to a manuscript prior to publication.

References [1] Couillault C, Pujol N, Reboul J, Sabatier L, Guichou J-F, Kohara Y, et al. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM. Nat Immunol 2004;5:488–494. [2] Dlakic M. A new family of putative insulin receptor-like proteins in C. elegans. Curr Biol 2002;12:155–157. [3] Garsin DA, Villanueva JM, Begun J, Kim DH, Sifri CD, Calderwood SB, et al. Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Science 2003;300:1921. [4] Hua Q-x, Nakagawa SH, Wilken J, Ramos RR, Jia W, et al. A divergent INS protein in Caenorhabditis elegans structurally resembles human insulin and activates the human insulin receptor. Genes Develop 2003;17:826–831. [5] Kato Y, Komatsu S. ASABF, a novel cysteine-rich antibacterial peptide isolated from the nematode Ascaris suum. J Biol Chem 1996;271:30493–30498. [6] Kato Y, Aizawa T, Hoshino H, Kawano K, Nitta K, Zhang H. abf1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans. Biochem J 2002;361:221–230. [7] Keating CD, Kriek N, Daniels M, Ashcroft NR, Hopper NA, Siney EJ, et al. Whole-genome analysis of 60 G protein-coupled receptors in Caenorhabditis elegans by gene knockout with RNAi. Curr Biol 2003;13:1715–1720. [8] Kim K, Li C. Expression and regulation of an FMRFamiderelated neuropeptide gene family in Caenorhabditis elegans. J Comp Neurol 2004;475:540–550.

[9] Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G. daf-2 an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 1997;277:942–946. [10] Kubiak TM, Larsen MJ, Nulf SC, Zantello MR, Burton KJ, Bowman JW, et al. Differential activation of “social” and “solitary” variants of the Caenorhabditis elegans G-protein-coupled receptor NPR-1 by its cognate ligand AF9. J Biol Chem 2003a;278:33724–33729. [11] Kubiak TM, Larsen MJ, Zantello MR, Bowman JW, Nulf SC, Lowery DE. Functional annotation of the putative orphan Caenorhabditis elegans G-protein-coupled receptor C10C6.2 as a FLP-15 peptide receptor. J Biol Chem 2003b;278:42115–42120. [12] Li C, Kim K, Nelson L. FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. Brain Res 1999;848:26–34. [13] Li W, Kennedy SG, Ruvkun G. daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 pathway. Genes Devel 2003;17:844–858. [14] Maule AG, Mousley A, Marks NJ, Day TA, Thompson DP, Geary TG, Halton DW. Neuropeptide signaling systems—potential drug targets for parasite and pest control. Curr Topics Med Chem 2002;2:733–758. [15] Mertens I, Meeusen T, Janssen T, Nachman R, Schoofs L. Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans. Biochem Biophys Res Comm 2005;330:967–974. [16] Mertens I, Vandingenen A, Meeusen T, Janssen T, Luyten W, Nachman RJ, et al. Functional characterization of the putative orphan neuropeptide G-protein coupled receptor C26F1.6 in Caenorhabditis elegans. FEBS Lett 2004;573:55–60. [17] Millet ACM, Ewbank J. Immunity in Caenorhabditis elegans. Curr Opinion Immunol 2004;16:4–9. [18] Nathoo AN, Moeller RA, Westlund BA, Hart AC. Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. PNAS 2001;98:14000–14005. [19] Nelson DW, Padgett RW. Insulin worms its way into the spotlight. Genes Develop 2003;17:813–818. [20] Pierce SB, Costa M, Wisotzkey R, Devadhar S, Homburger SA, Buchman AR, et al. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Devel 2001;15:672– 686. [21] Pillai A, Ueno S, Zhang H, Kato Y. Induction of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of ASABF in the nematode Ascaris suum. Biochem J 2003;371:663–668. [22] Rogers C, Reale V, Kim K, Chatwin H, Li C, Evans P, et al. Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1. Nat Neurosci 2003;6:1178–1185. [23] Tatar M, Bartke A, Antebi A. The endocrine regulation of aging by insulin-like signals. Science 2003;299:1346–1351. [24] Zhang H, Kato Y. Common properties specifically found in the CSαβ-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Devel Comp Immunol 2003;27:499–503.

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39 Parasitic Nematode Peptides ANGELA MOUSLEY, NIKKI J. MARKS, AND AARON G. MAULE

ABSTRACT

FMRFamide-LIKE PEPTIDES (FLPs)

Only a small number of peptides have been characterized from parasitic nematodes, and most of these belong to the FMRFamide-like neuropeptide (FLP) family. All of the available results on peptide structure and gene sequence have been derived from work on two gastrointestinal parasites—the pig parasite Ascaris suum and the sheep parasite Haemonchus contortus—and the potato plant parasite Globodera pallida. FLPs are widely expressed in the nervous systems of parasitic nematodes and play a wide variety of roles in the modulation of motor function. The A. suum peptide, TKQELE has been proposed to represent a novel peptide family.

Discovery The vast majority of peptides known from parasitic nematodes are FMRFamide-like peptides (FLPs). These are neuropeptides that encompass an RF.NH2 Cterminus that is preceded by an aromatic-hydrophobic, an aromatic-variable, or a variable-hydrophobic dipeptide. The complete amino acid sequences of 18 distinct FLPs (and partial sequences of another two) have been determined from A. suum, but one of these has a Cterminal FMHF.NH2, such that it lacks the penultimate R residue and is therefore only tentatively assigned. Two of these peptides have also been structurally characterized from the sheep intestinal parasite Haemonchus contortus. A. suum FLPs have been isolated from acid-extracts of head (which contain the anterior nerve ring or brain) and tail (which contains the perianal nerve ring) regions of A. suum and were designated Ascaris FLP 1 (AF1) to AF20 upon discovery (see Table 1; note that ENEKKAVPGVLRF.NH2, an alternative cleavage product for AF3, has been identified by mass spectrometry) [4–7, 34]. AF2 has also since been isolated from acidethanol extracts of third-stage larvae of H. contortus [13, 18]; AF2 is the most abundant nematode FLP detected in these parasitic nematodes. AF8 has also been characterized from acid-ethanol extracts of H. contortus thirdstage larvae [18]; this peptide was also designated PF3 following its isolation from a free-living nematode [19]. There is a preliminary report of the characterization of LQPNFLRF.NH2 from H. contortus [12]. Several of these FLPs (AF1, AF2, AF8, AF9, and AF15) have also been identified in free-living nematodes [19]. A recent mass spectrometry study has identified peptides that match predicted masses of A. suum ESTs [34]; the authors also report the de novo sequencing using tandem mass

INTRODUCTION To date, knowledge on peptides in parasitic nematodes is confined to one family of neuropeptides (FMRFamide-like peptides or FLPs), several antibacterial peptides, and a putative peptide fragment of chromogranin. The peptides described in this chapter are restricted to those that have been structurally characterized or for which the encoding gene has been described. Almost all of the known peptides have been isolated from the large gastrointestinal pig parasite, Ascaris suum. However, it should be noted that there is now a multitude of expressed sequence tag (EST) information for a range of parasitic nematodes, and a recent analysis of the FLP complements across the nematode phylum revealed a high level of conservation of FLP structures, such that homologs of the peptides described in the previous chapter [19] are also present in parasitic nematodes. The peptides described in this chapter are subdivided into the FLPs and TKQELE; antibacterial peptides are covered elsewhere. Handbook of Biologically Active Peptides

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256 / Chapter 39 TABLE 1. Parasitic Nematode Ascaris suum4–7,9

Parasitic nematode FMRFamide-like peptides.

Peptide Code* AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10 AF11 AF12 AF13 AF14 AF15 AF16 AF17 AF18 AF19 AF20

Globodera pallida14

Haemonchus contortus12,13,18

Encoding Gene

afp-1 (= As-flp-18) afp-1 (= As-flp-18)

afp-1 (= As-flp-18)

afp-1 (= As-flp-18) afp-1 (= As-flp-18)

afp-1 (= As-flp-18) Gp-flp-1 Gp-flp-1 Gp-flp-1 Gp-flp-1 Gp-flp-1 Gp-flp-6 Gp-flp-12 Gp-flp-14 Gp-flp-18 Gp-flp-18 Gp-flp-18 Gp-flp-18 Gp-flp-18

AF2 AF8

Amino Acid Sequence KNEFIRF.NH2 KHEYLRF.NH2 AVPGVLRF.NH2 GDVPGVLRF.NH2 SGKPTFIRF.NH2 FIRF.NH2 AGPRFIRF.NH2 KSAYMRF.NH2 GLGPRPLRF.NH2 GFGDEMSMPGVLRF.NH2 SDIGISEPNFLRF.NH2 XXXPNFLRF.NH2 SDMPGVLRF.NH2 SMPGVLRF.NH2 AQTFVRF.NH2 ILMRF.NH2 FDRDFMHF.NH2 XXXXPNFLRF.NH2 AEGLSSPLIRF.NH2 GMPGVLRF.NH2 TSSNFLRF.NH2 SSASMSTSSEPNFLRF.NH2 GGVDPTFLRF.NH2 ANNNFLRF.NH2 QANPNFLRF.NH2 KSAYMRF.NH2 KNKFEFIRF.NH2 KHEYLRF.NH2 DEFVAPGVLRF.NH2 MPGVLRF.NH2 AVPGVLRF.NH2 AEVPGVLRF.NH2 MPQVLRF.NH2 KHEYLRF.NH2 KSAYMRF.NH2 LQPNFLRF.NH2

*Note that peptide codes have only been assigned to peptides that have been structurally characterized.

spectrometry of AQDPNFL/IRF.NH2, ATDPNFL/IRF. NH2, and APKPNFL/IRF.NH2.

Structure of the Precursor mRNA/Gene FMRFamide-like peptides are encoded on FMRFamide-like peptide (flp) genes. Despite the identification of 27 flp genes in the free-living nematode Caenorhabditis elegans [22], little is known about flp genes in parasitic nematodes. Indeed, although EST databases provide us with insight into the flp gene complements in parasitic nematode species (see [22]), flp-encoding genes have been confirmed in only two nematode parasites: A. suum and Globodera pallida. The first known flp gene from A. suum was designated Ascaris FMRFamide-

like Precursor protein 1 (afp-1) [9]; afp-1 encodes six structurally related FLPs with characteristic PGVLRF. NH2 C-termini, all of which have been structurally characterized (AF3, AF4, AF10, AF13, AF14, and AF20) [5, 7]. This gene is a homolog of the C. elegans flp-18, and we propose redesignation as As-flp-18. More recently, five flp-encoding genes (Gp-flp-1, Gp-flp-6, Gp-flp-12, Gpflp-14, and Gp-flp-18) have been characterized from the potato cyst nematode, G. pallida; these encode 13 FLPs [14; see Table 1].

Distribution of Peptide/mRNA Overviews of the expression of FLPs in nematodes have been provided by immunocytochemical studies

Parasitic Nematode Peptides / 257 that employ anti-FLP antisera. Unfortunately, most of these studies do not reveal the expression of individual FLPs or flp mRNAs and simply provide evidence for the widespread expression of flp genes in parasitic nematodes. Three monoclonal antibodies have been raised against AF1, AF2, and AF3/4; only the anti-AF1 serum was highly specific and stained a small subset of neurons in the brain and pharynx as well as single neurons in the dorsal and ventral nerve cords that synapse with muscle and inhibitory motorneurons (see [7]). Up to 75% of the neurons in A. suum have been reported to display FLP-immunoreactivity. Only one study reports the expression of FLP-encoding mRNAs in a parasitic nematode [15]. In this study, in situ hybridization (ISH) was used to explore the expression of the five flp genes known from the potato cyst nematode, G. pallida. Four of the flp genes (Gp-flp-1, Gp-flp6, Gp-flp-12, and Gp-flp-14) are expressed in neurons in the J2 infective stage of G. pallida. Gp-flp-1 (encodes a number of FLRF.NH2’s) expression is restricted to VA/VD and DA/DB-like motorneurons as well as RIF-, RIG-, AVG-, and SAB-like interneurons. Gp-flp-6 (encodes KSAYMRF.NH2) is expressed in neurons associated with the anterior nerve ring, PHA- and PHB-like sensory neurons and lumbar ganglion interneurons. Expression of Gp-flp-12 (encodes KNKFEFIRF.NH2) is localized to the retrovesicular ganglion D/V-like motorneurons, pre-anal ganglion motorneurons, lumbar ganglia interneurons that are associated with body wall and vulval motorneurons, the BDU-like interneurons that are associated with the HSN vulval motorneuron, and an ADE-like sensory neuron. Gp-flp-14 (encodes KHEYLRF.NH2) is expressed in the anterior ganglion and several paired cells posterior to the circumpharyngeal nerve ring; specifically in RMED-, RME-, and RMG-like cells that are associated with head motorneurons and ADA-like interneurons. The expression of Gp-flp-18 was not reported. A recent study employed peptidomic techniques to reveal the expression of selected neuropeptides in distinct brain regions of A. suum using mass spectrometry [34].

Receptors To date, no parasitic nematode FLP receptor has been characterized. However, several Ascaris FLPs have been found to be ligands to a C. elegans G-protein coupled FLP receptor. AF9 and PGVLRFamides encoded on As-flp-18 (afp-1) were matched as ligands for the NPR-1 receptor (Wormbase accession number, C39E6.6) [17, 25, 28]. In addition, studies on A. suum somatic muscle membranes revealed that the AF2 receptor in A. suum is G-protein coupled [16].

Biological Actions Effect on Muscle Ascaris suum body wall Most of the accumulated data on FLP activities in nematodes have been obtained using A. suum somatic body wall muscle strips. Parasitic nematode-derived FLPs with structurally distinct C-terminal motifs have been shown to induce a diverse array of both pre- and postsynaptic inhibitory (slow and prolonged [body wall response type 1, bwRT1] or fast and transient [bwRT2]), excitatory (sustained contractility [bwRT3]), and biphasic (transient relaxation/sustained contraction [bwRT4]) activities, such that four body wall response types (bwRTs) have been described and designated bwRT1– bwRT4. AF1 and AF2 abolish localized locomotory waves when injected directly into the A. suum pseudocoel [4, 6]. AF1 (≥1 nM) and AF2 (≥1 pM) were also shown to induce qualitatively similar biphasic activities on somatic muscle that comprise a transient, rapid relaxation followed by a prolonged phase of increased contractility, indicative of bwRT4 [1, 4, 6, 20, 24, 30]; AF2 is approximately 1000-fold more potent than AF1. The excitatory phase of the biphasic response is abolished upon denervation of the muscle strips [1, 20], indicating that different receptors are involved in the two phases of the AF1/AF2-induced response: a muscle-based receptor mediating the inhibitory phase, and a neuronal receptor conveying the excitatory response. AF2 has also been found to enhance membrane potential responses to acetylcholine (ACh) [31]. Structure-activity studies on AF1 have utilized alanine scan series and N-terminally truncated analogs to show that elimination of functional amino groups or amino acids at either end of the peptide are deleterious to biological activity [1]. Substitution of alanine for any amino acid in AF1 has profound effects on activity; Ala1, Ala2, and Ala7 analogs display no activity; Ala3 and Ala5 substituted peptides have only inhibitory responses; and Ala4AF1 displays strictly excitatory effects. Despite identical AF1/AF2-induced physiology on the body wall muscle, Ala6AF1 completely inhibits the AF1 effect while having no effect on the AF2 response. In addition, AF1/AF2 hybrids (KHEFIRF. NH2, KNEYIRF.NH2, and KNEFLRF.NH2) induce either similar or less potent effects than AF1 such that none was as active as AF2 [1]. In contrast to AF1, all alanine-substituted AF2 derivatives are active on A. suum muscle strip preparations, although with reduced potencies; Ala1, Ala2, Ala4, Ala5, Ala6, and Ala7 analogs display ≥10,000fold potency decreases. In addition, Ala6AF2 does not display the antagonistic activities that were noted for Ala6AF1 [30].

258 / Chapter 39 AF3 and AF4 induce concentration-dependent contractions of dorsal somatic muscle strips indicative of bwRT3 [5, 33]. Four additional Ascaris FLPs (AF10, AF13, AF14, AF20), which share C-terminal homology with AF3 and AF4, also display excitatory activities (bwRT3) as do AF5, AF9, and AF17 [5, 7]. AF11 induces a slow and prolonged inhibition of the body wall muscle indicative of bwRT1 [7]; body wall muscle is also relaxed by AF6 and AF19. AF7 and AF16 exert little or no effect on body wall muscle [7]. The ease in preparation of dorsal, ventral, and denervated muscle strips has enabled further delineation of FLP activities on somatic muscle. For example, AF8 displays a unique differential activity of nervecord dependent excitatory effects on ventral (≥0.1 μM) and inhibitory effects on dorsal (≥1 μM) muscle strip preparations [7, 20, 21]. To date this is the only peptide found to display differential activity on dorsal and ventral body wall muscle strips of a nematode. The effects of AF10, AF12, AF13, AF14, and AF15 on the somatic body wall muscle of A. suum remain unpublished. Ascaridia galli body wall The effects of two Ascaris FLPs have been examined on dorsal muscle strip preparations from the domestic fowl parasite, A. galli; AF3 and AF4 contract A. galli somatic muscle [33]. In addition, AF3 and AF4 elicit an increase in the propagation of phasic spontaneous activity in A. galli. Further examination of the mechanism underlying the excitatory effects of AF3 revealed that its actions were independent of the cholinergic system [33]. Haemonchus contortus body wall The effects of two Ascaris FLPs (AF2 and AF8; also present in H. contortus) have been characterized on the body wall muscle of the sheep nematode H. contortus. While injection of AF2 (≥1 μM) into the pseudocoelomic cavity has no significant effect on spontaneous contractions, ACh-induced contractions are significantly inhibited [18]. Injections of AF8 (≥10 nM) enhance both spontaneous and AChinduced contractions. Further insight into the relationship between the cholinergic and FLPergic systems in H. contortus was facilitated by use of two different Haemonchus isolates; an isolate that was susceptible to ACh and the cholinergic anthelmintic, levamisole (MH isolate), and a resistant isolate (Lawes isolate) that had reduced sensitivity to cholinergic drugs. Although Lawes isolates are significantly less sensitive to AF8 than MH isolates, AF1 effects are identical in both isolates [18], suggesting that AF8 modulates cholinergic transmission. Ascaris suum ovijector The effects displayed by nematode FLPs are diverse and can be delineated into five response types (ovijector response type ovRT1, inhibitory; ovRT2, excitatory; ovRT3, transient contraction; ovRT4, transient contraction/paralysis; ovRT5, contraction frequency increase). With respect to the FLPs from parasitic nematodes, AF2 (≥100 nM) and

AF8 (≥10 nM) induce inhibitory activities on the ovijector comprising a cessation/reduction of contractile activity and/or flaccid paralysis [ovRT1] [11]. AF3 (≥1 nM), AF4 (≥3 nM), and AF9 (≥1 μM) induce excitatory activities comprising an increase in muscle tension and contractile frequency [ovRT2] [10, 23]. AF1 (≥10 nM) induces a transient contraction/paralysis activity comprising an increase in muscle tension followed by inactivity [ovRT4] [11]. Ascaris suum pharynx The effects of six parasitic nematode FLPs on pharyngeal pumping behavior were monitored using a pressure transducer system. Serotonin-induced pumping is significantly inhibited by two nematode FLPs, AF1 (EC50, 168 nM) and AF8 (EC50, 188 nM) [2, 3]. Several nematode FLPs (AF2, AF3, AF4, AF6, and AF16) have no effect on serotonininduced pumping [3].

Effect on Behavior and Body Length Ascaris Suum The effects of injection of five parasitic nematodederived FLPs have been examined on locomotion and body waveforms in adult female Ascaris [26]. Injection of AF1 or AF2 (10 μM) inhibits the propagation of locomotory waves, reduces the number of waveforms, and decreases body length [26]. In addition, worms injected with AF1 show a more linear body posture and a marked decrease in locomotion [8]. The inhibition of locomotory waveforms reported for AF2 appear to be inconsistent with the general increase in motility reported for the same peptide [8]. Injection of AF5 induces a more linear body posture and decreases locomotion [8]. AF8 causes ventral coiling [26], abolishes motility, and induces head tremor activity [8, 26]. Injection of AF9 reduces motility, causes an anterior linear posture, posterior coils, and reduces head searching activity [8, 26]. AF10 increases the number of body waveforms (not propagated) and decreases the body length. AF17 was found to increase body length and inhibit worm movement [26].

Effect on Nerve Ascaris Suum Initial electrophysiology studies [6] revealed that AF1 exerts inhibitory effects on the electrical properties of both the ventral inhibitory (VI) and dorsal inhibitory (DI) motorneurons and decreases their input resistance. The effects of 18 Ascaris FLPs on the membrane potential and input resistance of dorsal excitatory 2 (DE2) and DI motorneurons have since been reported. Endogenous Ascaris FLPs induce five major response types in motorneurons thought to be attributable to distinct FLP receptor subtypes [8].

Parasitic Nematode Peptides / 259 The first response type is triggered by the addition of the -FIRFamide peptides (AF1, AF5, AF6, and AF7), all of which have qualitatively similar depolarizing effects on DE2 motorneurons and hyperpolarizing effects on DI motorneurons. AF1 produced strong and consistent DE2 neuron depolarizations; occasionally, superfusion of AF1 produces weak early transient hyperpolarizations. In addition, AF1 produced the strongest decrease in input resistance seen in DE2 motorneurons. AF1 superfusions also resulted in weak DI neuron hyperpolarizations that occurred early, transiently, and inconsistently. AF5 also induced strong and constant DE2 neuron depolarizations that were consistent with decreases in input resistance. In addition, AF5 superfusions resulted in weak DI neuron hyperpolarizations. In contrast to the effects of AF1 and AF5, superfusion of AF6 and AF7 produced weak DE2 neuron depolarizations. AF6 and AF7 also produced weak DI neuron hyperpolarizations. The second proposed response type is induced by superfusion of AF2 and is based on its unique biphasic effect on the input resistance of the DE2 motorneurons; in contrast to the decrease in input resistance noted for AF1, AF2 induced an early, transient increase followed by a later and sustained decrease in input resistance. This peptide also produced the strongest depolarization of DE2 neurons that was noted for any of the 18 Ascaris FLPs examined. It has no significant effect on DI neurons. AF9-induced DE2 neuron depolarization represents the third response type with superfusion of AF9 producing a weak, transient DE2 neuron hyperpolarization followed by a sustained depolarization. AF9 had no significant effect on the input resistance of the DE2 motorneurons. An early transient, weak DI neuron hyperpolarization was also noted for AF9. The fourth response type is based on the strength of the AF8-induced depolarization of DI motorneurons. Superfusion with AF8 also produced a small hyperpolarization of DE2 neurons. The final response was observed on superfusion of either AF17 or AF19, both of which induced strong DE2 and DI neuron hyperpolarizations. AF19 induced the largest DE2 neuron hyperpolarization that was noted for all 18 Ascaris FLPs.

Effect on cAMP There is very little information on second messenger systems involved in FLP signaling in parasitic nematodes, such that the precise mechanism of action of individual FLPs remains unknown. The only published effects on signaling molecules relate to cyclic adenosine monophosphate (cAMP), which is generated by adenylate cyclase following G-protein activation. cAMP levels

increase following the treatment of A. suum muscle strips with either AF1 or AF2 [12, 27, 30] or following whole worm AF1/AF2 injections [26]. Moreover, similar increases in cAMP levels are seen upon exposure of denervated muscle preparations to AF2, such that the increase in cAMP is thought to be attributable to the inhibitory phase of AF2-induced, biphasic response [30]. In contrast, AF3 and AF17, which cause distinct excitatory effects in A. suum somatic muscle, decrease cAMP levels in A. suum; AF3 also decreases cAMP in A. galli [26, 32]. Together these studies signify a link between increases in cAMP levels and muscle relaxation; decreases in cAMP levels appear to be associated with muscle contraction. The effects of a further 10 FLPs (in addition to AF1 and AF2) on cAMP levels in Ascaris were also examined and, following comparisons with changes in worm length, an inverse relationship between increase/decrease in cAMP levels and muscle contraction/relaxation was not confirmed [26]; several nematode FLPs decreased body length (suggestive of muscle contraction) and cAMP levels. AF10 and AF8 have no effect on intracellular cAMP [26, 30].

TKQELE TKQELE was structurally characterized from the gonoduct of A. suum [29]. This peptide was detected using an antiserum against KGQELE, which flanks the C-terminus of the pancreastatin sequence in rat chromogranin A. Although positive staining was identified throughout the nervous system using an anti-KGQELE antiserum, there is limited evidence to indicate that this is a true fragment of chromogranin, and it was reported as a novel class of peptide.

References [1] Bowman JW, Friedman AR, Thompson DP, Ichhpurani AK, Kellmann MF, Marks N, et al. Structure-activity relationships of KNEFIRFamide (AF1), a nematode FMRFamide-related peptide, on Ascaris suum muscle. Peptides 1996;17:381–7. [2] Brownlee DJA, Holden-Dye L, Fairweather I, Walker RJ. The action of serotonin and the nematode neuropeptide KSAYMRFamide on the pharyngeal muscle of the parasitic nematode, Ascaris suum. Parasitology 1995;111:379–84. [3] Brownlee DJA, Walker RJ. Actions of nematode FMRFamiderelated peptides on the pharyngeal muscle of the parasitic nematode, Ascaris suum. Ann NY Acad Sci 1999;897:228–38. [4] Cowden C, Stretton AOW. AF2, an Ascaris neuropeptide: Isolation, sequence, and bioactivity. Peptides 1993;14:423–30. [5] Cowden C, Stretton AOW. Eight novel FMRFamide-like neuropeptides isolated from the nematode Ascaris suum. Peptides 1995;16:491–500. [6] Cowden C, Stretton AOW, Davis RE. AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum. Neuron 1989;2:1465–73.

260 / Chapter 39 [7] Davis RE, Stretton AOW. The motornervous system of Ascaris: electrophysiology and anatomy of the neurons and their control by neuromodulators. Parasitology 1996;114:S97–S117. [8] Davis RE, Stretton AOW. Structure-activity relationships of 18 endogenous neuropeptides on the motornervous system of the nematode Ascaris suum. Peptides 2001;22:7–23. [9] Edison AS, Messinger LA, Stretton AOW. afp-1: a gene encoding multiple transcripts of a new class of FMRFamide-like neuropeptides in the nematode Ascaris suum. Peptides 1997;18:929–35. [10] Fellowes RA, Maule AG, Marks, NJ, Geary TG, Thompson DP, Halton DW. Nematode neuropeptide modulation of the vagina vera of Ascaris suum: In vitro effects of PF1, PF2, PF4, AF3 and AF4. Parasitology 2000;120:79–89. [11] Fellowes RA, Maule AG, Marks NJ, Geary TG, Thompson DP, Shaw C, et al. Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRFamide-related peptides. Parasitology 1998;116:277–87. [12] Geary TG, Marks NJ, Maule AG, Bowman JW, AlexanderBowman SJ, Day TA, et al. Pharmacology of FMRFamide-related peptides in helminths. Ann NY Acad Sci 1999;897:212–27. [13] Keating C, Thorndyke MC, Holden-Dye L, Williams RG, Walker RJ. The isolation of a FMRFamide-like peptide from the nematode Haemonchus contortus. Parasitology 1995;111:515–21. [14] Kimber MJ, Fleming CC, Bjourson A, Halton DW, Maule AG. FMRFamide-related peptides in potato cyst nematodes. Mol Biochem Parasitol 2001;116:199–208. [15] Kimber MJ, Fleming CC, Prior A, Jones JT, Halton DW, Maule, AG. Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridization. Int J Parasitol 2002;32:1095–105. [16] Kubiak TM, Larsen MJ, Davis JP, Zantello MR, Bowman JW. AF2 interaction with Ascaris suum body wall muscle membranes involves G-protein activation. Biochem Biophys Res Commun 2003;301:456–9. [17] Kubiak TM, Larsen MJ, Nulf SC, Zantello MR, Burton KJ, Bowman JW, et al. Differential activation of “social” and “solitary” variants of the Caenorhabditis elegans G protein-coupled receptor NPR-1 by its cognate ligand AF9. J Biol Chem 2003;278:33724–9. [18] Marks NJ, Sangster NC, Maule AG, Halton DW, Thompson DP, Geary TG, et al. Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus. Mol Biochem Parasitol 1999;100:85– 194. [19] Masler EP. Free-living Nematode Peptides. In: Kastin AJ, editor. The Handbook of Peptides. Elsevier. In press. [20] Maule AG, Geary TG, Bowman JW, Marks NJ, Blair KL, Halton DW, et al. Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips from Ascaris suum. Invertebr Neurosci 1995;1:255–65. [21] Maule AG, Shaw C, Bowman JW, Halton DW, Thompson DP, Geary TG, et al. KSAYMRFamide: A novel FMRFamide-related

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heptapeptide from the free-living nematode, Panagrellus redivivus, which is myoactive in the parasitic nematode, Ascaris suum. Biochem Biophys Res Commun 1994;200:973–80. McVeigh P, Leech S, Mair G, Marks NJ, Geary TG, Maule AG. Analysis of FMRFamide-like peptide (FLP) diversity in phylum Nematoda. Int J Parasitol 2005;35:1043–60. Moffett CL, Beckett AM, Mousley A, Geary TG, Marks NJ, Halton DW, et al. The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamiderelated peptides. Int J Parasitol 2001;33:859–76. Pang FY, Mason J, Holden-Dye L, Franks CJ, Williams RG, Walker RJ. The effects of the nematode peptide, KHEYLRFamide (AF2), on the somatic musculature of the parasitic nematode Ascaris suum. Parasitology 1995;110:353–62. Potter CJ, Luo L. Food for thought: an orphan receptor finds its ligands. Nat Neurosci 2003;1:1119–20. Reinitz CA, Herfel HG, Messinger LA, Stretton AOW. Changes in locomotory behavior and cAMP produced in Ascaris suum by neuropeptides from Ascaris suum or Caenorhabditis elegans. Mol Biochem Parasitol 2000;111:185–97. Rex E, Harmych S, Puckett T, Komuniecki R. Regulation of carbohydrate metabolism in Ascaris suum body wall muscle: a role for the FMRFamide AF2, not serotonin. Mol Biochem Parasitol 2004;133:311–3. Rogers C, Reale V, Kim K, Chatwin H, Li C, Evans P, et al. Inhibition of Caenorhabditis elegans social feeding by FMRFamiderelated peptide activation of NPR-1. Nat Neurosci 2003; 6:1178–85. Smart D, Shaw C, Curry WJ, Johnston CF, Thim L, Halton DW, et al. The primary structure of TE-6: a novel neuropeptide from the nematode Ascaris suum. Biochem Biophys Res Commun 1992;187:1323–9. Thompson DP, Davis JP, Larsen MJ, Coscarelli EM, Zinser EW, Bowman JW, et al. Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle. Int J Parasitol 2003;33:199–208. Trailovic SM, Clark CL, Robertson, AP, Martin RJ. Brief application of AF2 produces long lasting potentiation of nAChR responses in Ascaris suum. Mol Biochem Parasitol 2005;139:51– 64. Trim N, Boorman JE, Holden-Dye L, Walker RJ. The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascarida galli. Mol Biochem Parasitol 1998;93:263–71. Trim N, Holden-Dye L, Ruddell R, Walker RJ. The effects of the peptides AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) on the somatic muscle of the parasitic nematodes Ascaris suum and Ascaridia galli. Parasitology 1997;115:213–22. Yew JY, Kutz KK, Dikler S, Messinger L, Li L, Stretton AO. Mass spectrometric map of neuropeptide expression in Ascaris suum. J Comp Neurol 2005;8:396–413.

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40 Amphibian Tachykinins CINZIA SEVERINI AND GIOVANNA IMPROTA

evident that the short peptides of nonmammalian origin, named TKs by Vittorio Erspamer, represented the phylogenetic precursors of SP and of the other mammalian TKs (neurokinin-A/NKA and neurokininB/NKB). During the following years, a systematic screening of the peptide contents in the skin of as many as 600 amphibian species from all over the world resulted in the discovery and isolation of numerous families of neuropeptides, including that of TKs [2]. Despite the large amount of TKs isolated from extracts of frog skin, there are relatively few studies regarding TKs isolated from amphibian brain or gut. Like all vertebrate TKs, the great majority of amphibian TKs, although with some exceptions, has the classical C-terminal pentapeptide sequence Phe-X-Gly-Leu-Met-NH2, essential but not sufficient for their biological activity. The minimum chain length required for activity is six residues. In addition, the Phe residue at position 5 from the C-terminus and the amidation at the C-terminus are crucial for their biological activity. On the basis of their chemical structure and receptor affinities, both amphibian skin TKs and amphibian brain/gut TKs can be grouped into two subfamilies: “Aromatic TKs” (with the C-terminal pentapeptide Phe-Tyr(Phe)-Gly-Leu-Met-NH2) and “aliphatic TKs” (with the C-terminal pentapeptide PheVal(Ile)-Gly-Leu-Met-NH2) [8]. Parallel bioassays in a number of isolated and in situ test systems, using the natural peptides and selective synthetic analogs alone or in the presence of selective antagonists and, in addition, radioligand binding studies, allowed the receptor selectivity of amphibian TKs to be functionally characterized, largely in mammals and to a lesser degree in their species of origin. The main monoreceptor systems used are the rabbit vena cava for NK1-, endothelium-deprived rabbit pulmonary artery and hamster urinary bladder for NK2-, and rat portal vein for NK3-receptors [7]. “Aromatic TKs”

ABSTRACT As many as 19 tachykinins (TKs) have been isolated from amphibian skin and 11 from amphibian gut or brain. Amphibian TKs exert their biological actions in mammals through activation of the three classical TK receptors and in their species of origin through activation of receptors, which resemble the structure of the mammalian TK receptors, thus suggesting a high degree of conservation of the receptor sequence among different vertebrate species. As amphibian TK potency is comparable to, or can exceed, the potency of the mammalian counterpart, amphibian TKs have represented a widely used tool in the characterization of TK functional roles.

DISCOVERY The history of nonmammalian TKs began in 1947 together with that of amphibian TKs. In that year Vittorio Erspamer, while investigating the occurrence of biogenic amines and, especially, serotonin in the extracts of the posterior salivary glands of a Mediterranean octopod (Eledone moschata), discovered an unidentified substance that lowered blood pressure in rabbits and dogs, stimulated isolated intestinal smooth muscle, and caused profuse salivation in dogs and rats. The new substance, called eledoisin, was structurally characterized in 1962. In the same year, from the skin of the South American leptodactylid frog Physalaemus biligonigerus (formerly fuscumaculatus), a new peptide, with eledoisin-like activity called physalaemin (PHYS), was extracted and isolated. The structural characterization of this first amphibian TK in 1964 led to the prediction of the primary structure of substance P (SP), already identified by bioassay in 1931 but sequenced only 40 years later, in 1971. Therefore, it became Handbook of Biologically Active Peptides

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262 / Chapter 40 TABLE 1.

Amino acid sequence of amphibian skin TKs.

Source/Peptide Aromatic TKs Physalaemus biligonigerus (fuscumaculatus) PHYSALAEMIN (PHYS) Uperoleia rugosa [Lys5,Thr6]PHYS Uperoleia marmorata UPEROLEIN (UP) Uperoleia inundata UPERIN Kassina (Hylambates) maculata HYLAMBATIN (HYL) Rana margaratae RANAMARGARIN Pseudophryne güntheri PG-SPI PG-SPII Agalychnis callidryas AC-AR1 AC-AR2 AC-AR3 AC-AR4 Aliphatic TKs Kassina senegalensis KASSININ (KASS) Kassina (Hylambates) maculata [Glu2,Pro5]KASSININ Phyllomedusa bicolor PHYLLOMEDUSIN (PHYLL) Pseudophryne güntheri PG-KI PG-KII PG-KIII Agalychnis callidryas AC-AL

preferentially interact with the mammalian NK1 receptor, whereas “aliphatic TKs” mainly interact with the mammalian NK2/NK3 receptor. Table 1 summarizes the sequence and source of the 19 TKs extracted from amphibian skin. All these peptides have a nonneuronal origin, being synthesized in the syncytial cells lining the wall of the granular glands. These cells are capable of cosynthesizing, costoring, and cosecreting not only peptides belonging to different families but also amines and alkaloids of various classes and families. The occurrence of such a variety of substances and neuropeptides in the amphibian skin may perhaps be explained by the common embryogenic origin of the integument and the nervous system from the primitive ectoderm. The list of the 11 TKs extracted from frog gastrointestinal or nervous systems is reported in Table 2 and should be completed by authentic NKB found only in the brain of Rana esculenta.

Primary Structure

pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Ala-Asp-Pro-Lys-Thr-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Pro-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Ala-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2 Asp-Pro-Pro-Asp-Pro-Asn-Arg-Phe-Tyr-Gly-Met-Met-NH2 Asp-Asp-Ala-Ser-Asp-Arg-Ala-Lys-Lys-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Tyr-Gly-Leu-Met-NH2 pGlu-Pro-Asn-Pro-Asn-Glu-Phe-Tyr-Gly-Leu-Met-NH2 Gly-Pro-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Pro-Gly-Met-NH2 Gly-Pro-Pro-Asp-Pro-Asp-Lys-Phe-Tyr-Pro-Gly-Met-NH2 pGlu-Pro-Asp-Pro-Asp-Lys-Phe-Tyr-Pro-Gly-Met-NH2 Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Tyr-Pro-Val-Met-NH2

Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH2 Asp-Glu-Pro-Lys-Pro-Asp-Gln-Phe-Val-Gly-Leu-Met-NH2 pGlu-Asn-Pro-Asn-Arg-Phe-Ile-Gly-Leu-Met-NH2 pGlu-Pro-His-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2 pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2 pGlu-Pro-His-Pro-Asn-Glu-Phe-Val-Gly-Leu-Met-NH2 Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2

STRUCTURE OF THE PRECURSOR mRNA/GENE In spite of the large number of TKs isolated from amphibians, to date no information exists about the genes for these peptides. However, the high percent of sequence homology among TKs from amphibians and mammals, and the perfect identity of the C-terminal region in different species, crucial for TK biological activity, suggest that TK genes are phylogenetically ancient genes that have been highly conserved throughout evolution. Therefore, the structure of the TK precursor mRNA/gene and its processing are discussed in Chapter 105 by N. M. Page about mammalian TKs in the brain peptides section of this book. In this regard, summarizing, we may state that mammalian TKs derive from three different genes: preprotachykinin (TAC)-1, TAC-2, and TAC-4. TAC-1 encodes the sequences for SP and NKA (and, obviously, the two elongated forms of

Amphibian Tachykinins / 263 TABLE 2.

Amino acid sequence of amphibian brain/gut TKs.

Source/Peptide Aromatic TKs Rana catesbeiana RANATACHYKININS (RTK) RTKA RTKB RTKD Rana ridibunda RANAKININ (RNK) Bufo marinus BUFOKININ (BK) Amphiuma tridactylus Xenopus laevis XENOPUS SP Aliphatic TKS Rana ridibunda RNK Rana catesbeiana RTKC Amphiuma tridactylus Xenopus laevis XENOPUS NKA

Primary Structure

Brain,Gut Brain Gut

Lys-Pro-Ser-Pro-Asp-Arg-Phe-Tyr-Gly-Leu-Met-NH2 Tyr-Lys-Ser-Asp-Ser-Phe-Tyr-Gly-Leu-Met-NH2 Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Ala-Pro-Met-NH2

Brain

Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2

Gut Gut

Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met-NH2 Asp-Asn-Pro-Ser-Val-Gly-Gln-Phe-Tyr-Gly-Leu-Met-NH2

Gut

Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met-NH2

Gut

His-Lys-Leu-Asp-Ser-Phe-Ile-Gly-Leu-Met-NH2

Gut

His-Asn-Pro-Ala-Ser-Phe-Ile-Gly-Leu-Met-NH2

Gut

His-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2

Gut

Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met-NH2

NKA, neuropeptide k and neuropeptide γ) and is alternately split to yield three different mRNAs in a tissuespecific manner so that the relative amounts of SP and NKA vary in different tissues. TAC-2 encodes only the sequence for NKB. Recently, a third gene (TAC-4), which encodes for novel TK peptides (hemokinin1 and endokinins A-D), has been cloned.

AMPHIBIAN TK DISTRIBUTION Studies on TK-mRNA distribution in nonmammalian vertebrates are very few, but the available information about final peptide localization could be a predictor of the corresponding mRNA expression sites. Specific evidence of the presence of amphibian TKs in amphibian skin, brain, and gut was obtained through techniques of isolation and structural characterization of the original peptides from extracts of these amphibian tissues. On the other hand, the use of radioimmunological and immunohistochemical techniques revealed the presence of TK-like substances in different amphibian tissues and organs. As in mammals and other vertebrates, including amphibians, TK-like immunoreactivity (TK-LI) is highly distributed throughout both the central and peripheral amphibian nervous systems. In Rana pipiens, SP-LI was found in the brainstem with a distribution similar to that detected in amniotes, thus emphasizing the phylogenetic conservation of

large amounts of the structure and possibly of functional activities in the old reticular formation. TK-LI was also identified in the hypothalamic-hypophysary system of Rana esculenta, as well as in the frog (Rana pipiens and Xenopus laevis) visual system, especially in the retina (amacrine cells) and the optic tectum, with a distribution of SP-related peptides remarkably similar to that seen in the homologous regions of mammals. In Rana esculenta, Rana catesbeiana, and Xenopus spinal cord a dense TK-LI was observed in the dorsal horn where the TK functional organization seems to be similar to that of mammals, as well as the colocalization of TK-LI with calcitonin gene-related peptide (CGRP) immunoreactivity restricted to primary afferent fibers. In the peripheral nervous system of mammals, the main source of TKs is represented by the enteric nervous system. Also in the amphibian gut, TKs may be assumed to have a distribution similar to that occurring in mammals, as demonstrated by the dense SP-LI and Bufokinin-LI (BFK-LI) in the myenteric plexus and in the fibers innervating submucosal and mucosal structures and, conversely, by the moderate immunoreactivity detected in the circular muscle of the toad intestine [1]. TK-LI was also found in the nerve fibers innervating the frog palate, the carotid labyrinth, the respiratory system (pharynx and lung of Rana catesbeiana in colocalization with CGRP), and the vasculature. Recently, BFK-LI was mapped in varicose fibers in the wall of

264 / Chapter 40 many peripheral blood vessels in the toad. In Rana ridibunda, TK-LI (SP-, NKA-, and ranakinin/RNK-LI) was observed in peptidergic fibers innervating the adrenal gland, supporting functional data available on TK control of corticosteroid release in amphibians. In addition to neuronal distribution, TKs are also localized in nonneuronal structures. In amphibians, the most complex and richest nonneuronal localization is certainly represented by the skin, which may be considered a huge workshop producing bioactive peptides and a true endocrine system.

RECEPTORS The biological activity of vertebrate TKs depends on their interaction with three main types of G protein-coupled receptors—NK1, NK2, and NK3—which mediate their functional roles by modulating inositolphosphate/calcium second messengers. Whereas mammalian TK receptors have received great attention (for detailed information, see Chapter 105), the nonmammalian counterparts of these receptors have been poorly studied. Because mammalian and nonmammlian TKs have broadly similar actions in both mammalian and nonmammalian species, it is likely that the sequences of the TK receptors have been conserved across different vertebrate species. An SP receptor complementary DNA has been cloned from the sympathetic ganglion of Rana catesbeiana and characterized [9]. It was found to have a protein sequence with 69% identity to the mammalian NK1 receptor, with the largest degree of conservation seen in the transmembrane and intracellular loop region. The endogenous ligands of this receptor, the ranatachykinins (RTKs), act as full agonists at this site, inhibiting a potassium current that causes alterations in neuronal excitability. Recently, three TK NK1-like receptor isoforms (bNK1-A, bNK1-B, and bNK1-C), differing only at the intracellular COOH terminus, were cloned from cane toad brain and intestine by Liu and coworkers, suggesting that the alternative splicing of the NK1 receptor is a mechanism that has been physiologically conserved across different species and that each isoform could play a distinct physiological role. Two of these BFK receptors (bNK1-A and bNK1-B) are similar to the truncated and full-length forms of the mammalian NK1 receptor, while bNK1-C does not correspond to any previously described receptor. The three isoform transcripts are strongly expressed in gut, lung, and skeletal muscle, as well as in spinal cord and brain [5]. BFK receptors were pharmacologically characterized in the Bufo marinus gastrointestinal tract, showing functional similarities to the NK1 mammalian receptor, although

displaying very low affinity for the mammalian NK1 receptor selective agonists and antagonists [4, 6] In addition, in the Xenopus gastrointestinal tract, two different TK receptors were functionally characterized, one a NK1-preferring form and the other a NK2preferring receptor. Both may exist in the stomach, whereas the NK1-like predominates in longitudinal muscle in the small intestine of Xenopus [3]. However, no pharmacological or molecular evidence exists for the presence of both TK NK2 and NK3 receptors in amphibians. Moreover, as classical TK antagonists appear to interact differently with these amphibian receptors in comparison with mammalian receptors, the sequence of the “antagonist binding domain” of amphibian TK receptors is probably not well conserved.

ACTIVE AND/OR SOLUTION CONFORMATION Conformational analysis of amphibian TKs was performed using nuclear magnetic resonance and spectroscopic techniques. PHYS, like SP, exists in solution in a mixture of conformational states. In water, preferentially PHYS exists in an extended chain structure, while in methanol it exists as a mixture of beta-turn conformations in dynamic equilibrium. Unlike SP, in PHYS the midsegment of the peptide may be constrained by formation of a salt bridge. In addition, KASS in a water solution prefers an extended chain conformation, whereas, exhibits in the presence of dodecylphosphocholine micelles, exhibits a helical conformation in the central core and in the C-terminal region. The conformation adopted by KASS in this membrane model system is consistent with the structural motif typical of NK2 selective agonists. The same studies were performed for the RTK peptides (RTKA, RTKB, and RTKC). Also, these amphibian TKs adopted a helical form from the midregion to the C-terminus, which is essential to receptor activation, showing great flexibility in the Nterminal TK sequence.

BIOLOGICAL ACTIONS Amphibian TKs were available in pure form several years before the structure of mammalian TKs was elucidated and, in addition, have been shown to have a counterpart in mammalian tissues and to exert wide ranging biological actions mediated through the activation of the three classical TK receptors. Consequently, the earliest studies on the biological actions of TKs, which mainly referred to the effects of amphibian TKs such as PHYS and KASS, have substantially contributed to the explosive progress of the knowledge of the func-

Amphibian Tachykinins / 265 tional roles of the TK system in mammalian species. Whereas the results obtained with KASS, a NK2/NK3 TK receptor agonist, do not exactly mimic results obtained with either NKA or NKB, the results obtained with PHYS, a selective NK1 agonist, are identical, with negligible quantitative differences, to those obtained later with SP, the mammalian selective NK1 receptor agonist. The availability, in 1990, of PG-SPI and PG-KII, two highly selective and potent NK1 and NK3 TK receptor agonists, provided a further instrument to investigate the role of the TK system in mammals, in which amphibian TKs show a spectrum of biological actions that is usually identical with that of mammalian TKs. Conversely, whereas it has been largely demonstrated that mammalian TKs are able to produce effects in nonmammalian species, the extent to which amphibian TKs mirror these effects in their species of origin is not clear, although some studies exist that indicate a number of similarities. In the present chapter, we therefore summarize the main in vitro and in vivo actions of some amphibian TKs on different systems both in their species of origin and in mammals.

Actions of Amphibian TKs in Their Species of Origin PHYS exerts a strong excitatory action on motoneurons of bullfrog isolated spinal cord. On a molar basis PHYS was 1500 times more active than L-glutamate in depolarizing the motoneurons, suggesting that TKs are excitatory transmitters of primary sensory neurons in this animal species. PHYS and the other SP-like amphibian neuropeptides, RTKs and BFK, produce a potent and long acting hypotensive effect without any reflex tachycardia in the anesthetized cane toad Bufo marinus. The blood pressure depressant action of RTK is left unmodified by a nonpeptidic NK1 antagonist, indicating that the amphibian NK1 receptor mediating this effect differs from its mammalian counterpart. The occurrence of BFK-LI in varicose fibers of blood vessels of the toad suggests that the fall in systemic blood pressure is the consequence of a decrease in peripheral vascular resistance in this species. Perhaps BFK may be released by antidromic stimulation of sensory nerves and may play a role in hemodynamic regulation in the toad. PHYS, KASS, RNK, and BFK are very potent and long acting spasmogens in the toad intestine and the presence of BFK-immunoreactive nerves in the myenteric plexus and muscularis externa supports this finding. The absence of BFK-LI in the intestinal mucosa suggests a minor role in regulating toad intestinal secretion. Among the Xenopus TK-like peptides, both the SP-like (structurally identical to BFK) and the NKA-like peptides induce an equipotent myotropic effect on isolated

strips of circular smooth muscle from Xenopus stomach, whereas only the SP-like peptide acts as a spasmogen on Xenopus intestinal musculature. The maximum response to Xenopus SP is lower than that to the NKAlike peptide, suggesting a more effective interaction of the NKA peptide with the TK receptors in Xenopus stomach. These findings confirm the presence of both NK1 and NK2 receptors in stomach, and only of the NK1 subtype in Xenopus intestine [2]. Administration of PH YS in the Rana porosa porosa had a positive effect on protein and mucous granule secretion from the lingual gland as detected by transmission electron microscopy. This reaction is very different from that observed in mammals, in which TKs stimulate only water secretions. In Rana esculenta skin, TKs stimulate ion transport (Na+ absorption and Cl− secretion, measured by short-circuit currents) by interacting with NK1-like receptors and by involving prostanoid synthesis. The rank order of T K potency is: PG-KI > UP > HYL > KASS > PH YLL > RTKA > PH YS > RNK. The skin of this frog does not produce TKs, and it is probably a target for TKs produced in other tissues that, by promoting fluid secretion, may regulate pain response. Amphibian TKs, which are detected in frog adrenal gland extracts, stimulate corticosteroid secretion by frog adrenal slices through a prostaglandin-dependent mechanism. The rank order of their potency in comparison with mammalian TKs is NKA > RNK > SP > KASS > PHYS > NKB, suggesting that TKs released by nerve fibers control neuroendocrine secretions in amphibians. RNK, the most potent amphibian TK in stimulating steroid secretion, causes mobilization of calcium from adrenochromaffin cells through activation of a NK1-like receptor linked to phospholipase C and pertussis toxin-sensitive G protein, which is different from the mammalian NK1 subtype.

Actions of Amphibian TKs on Mammalian Animal Species Cardiovascular System. TKs can relax or contract in vitro vascular preparations, the relaxation being due to the activation of NK1 receptors (by PHYS) located in the endothelium, while the contraction is caused by the activation of NK2 receptors (by KASS) located on the arterial smooth muscle. Likewise, in the rat portal vein (with intact endothelium) contraction elicited by KASS is brought about by NK3 receptors, presumably located on the smooth muscle membrane. In in vivo studies, the response of the vasculature to TKs is complex, depending on the animal species, density in the smooth muscle cells, and density in the endothelium of the various receptor subtypes, as well as on the TK administered or released. The NK1 receptor preferring TK, PHYS, causes a marked dose-dependent

266 / Chapter 40 hypotension in dogs and rabbits, whereas the NK2/NK3 agonist KASS is 10 to 20 times less potent. Conversely, PHYS produces moderate hypotension with tachyphylaxis, hypotension/hypertension, or frank hypertension in cats, sheep, rats, and pigeons. PHYS is a very potent vasodilator of the dog coronary, skeletal muscle, and hepatic vascular bed, and, in the form of an intradermal injection, increases rat, guinea pig, and human capillary permeability. These findings suggest that the striking amphibian TK hypotensive effect is the consequence of intense vasodilatation in several peripheral vascular beds due to direct TK action on the blood vessel wall promoting the release of endogenous relaxing factors from the endothelium. Gastrointestinal Tract (GI) Motor Effects. TKs, socalled because they evoke a fast contraction of intestinal smooth muscle, represent one of the most active substances inducing a clear-cut stimulation of motility in all sections of the gut (from esophagus to the rectum and in all muscle layers, including longitudinal muscle, circular muscle, and muscularis mucosae) of all examined mammalian and submammalian vertebrates. The actions of mammalian TKs in the gut have been the object of an extensive and thorough investigation (for detailed information, see Chapter 156 by P. Holzer in the gastrointestinal section of this book). As far as amphibian TK GI effects are concerned, we can summarize by saying that all these peptides, with few exceptions, provoke a contractile response in in vitro gut preparations, although the intensity and reproducibility of the response may be considerably different, depending on animal species, gut section, and agonist receptor selectivity. Moreover, in vivo studies have demonstrated that peripherally administered amphibian TKs induce complex, mainly stimulatory, but also inhibitory effects in the GI propulsion of several mammalian species (dog, cat, guinea pig, rat, sheep). GI Secretory Effects. Amphibian TKs exert a potent stimulant effect on salivary secretion, which was recognized several years before the sialagogic factor in a bovine hypothalamic extract was identified as SP. The more sensitive animal species are dog, ferret, rat, and guinea pig. The rank order of potency (PH YS = UP > SP = PG-SPI > PG-KI I = KASS >> NK A >>> NKB) in causing salivary secretion in rats after intravenous injection indicates that this TK effect is preferentially mediated by the NK1 receptor. The greater potency of PHYS and UP in comparison with SP may be explained by the considerably higher resistance to enzyme attack offered in the two first peptides by their N-terminal pGlu residue. PHYS is involved in vomiting, diarrhea, and profuse salivation in dogs. Exogenous TKs appear to cause secretion of fluid and electrolytes from the intestinal mucosa, and it has been suggested that endogenous TKs also may play a messenger role in the

intestinal secretory pathway. Also an increased permeability of the capillaries could contribute to enhance the secretions. Moreover, there is increasing evidence that TKs participate in hypersecretory, vascular, and immunological disorders associated with infection and inflammatory bowel disease. PHYS displays a moderate, short-lasting stimulatory action on exocrine pancreatic secretion in the dog. In dispersed acinar cells from guinea-pig pancreas, both NK1 (PH YS) and NK3 (PG -KII) TK receptor agonists increase amylase release, whereas in rat acinar cells only the NK1 agonist is active. These functional findings indicate a species-related difference in the TK regulation of exocrine pancreatic secretion, which has been confirmed by immunocytochemical studies. PHYS is a potent stimulant of lachrymal secretion in the dog, while the rat is much less sensitive. Airways System. In guinea pigs, KASS increases insufflation pressure, while PHYS, by activating receptors localized in the endothelial cells, increases the microvascular permeability to proteins, indicating that both NK2 and NK1 receptors, respectively, are involved in the guinea pig tracheobronchial tree response to TKs. Also in rats, KASS elicits a bronchoconstrictor action that is much more potent than SP. These findings are discussed in more detail in the pulmonary peptides section of the book. Urogenital Tract. TKs stimulate smooth muscle preparations of the urogenital tract, especially the urinary bladder, and display differences in their agonistic potency, not only depending on the different animal species but also on the particular segment of the urinary tract, probably due to the involvement of different receptor types. Intravenous injection of TKs induces a phasic contraction of the urinary bladder (increase of internal pressure) and an activation of rhythmic contractions, with KASS being the most potent peptide. These different responses appear to be mediated through different receptors and to be brought about both by a direct effect on the bladder’s smooth muscle and by an effect on intramural sensory nervous pathways (“micturition reflex”). Central Nervous System. In the mammalian CNS, TKs occur in large amounts, particularly in areas involved in the central control of peripheral autonomic functions (blood pressure, respiration, micturition, gastrointestinal motility and secretions), of essential functions (e.g., drinking behavior), of affective and emotional life (stereotyped behavior, motility, anxiety, aggressivity, pain), as well as of higher cerebral functions (learning, memory). Therefore, systematic studies using the intracerebroventricular administration of amphibian TKs that are selective for different receptor types allowed the roles of these neuropeptides to be evaluated in the CNS control of several peripheral func-

Amphibian Tachykinins / 267 tions in rats in order to identify the TK preferring receptor types involved, as it is briefly reported here: 1. The NK2 receptor preferring agonists (KASS) induce a dose-related delay in the gastric emptying of a liquid meal, whereas activation of NK3 receptors by the highly selective NK3 agonist PGKII mediates a colonic antipropulsive effect. 2. The NK3 receptor activation (by KASS and PG -KII) mediates an inhibitory action on gastric acid secretion in pylorus-ligated rats. 3. The NK2 receptors participate in the protection against gastric ulcers. 4. Via NK3 receptor activation, amphibian TKs ( PGKII) exert an antidipsogenic effect versus angiotensin II-induced drinking behavior, an antidiuretic effect due to release of vasopressin, and a potent and long-lasting decrease in salt intake. 5. The NK3 receptor preferring agonists (PG -KII) decrease ethanol intake in genetically alcoholpreferring rats. 6. The NK1 and NK3 TK receptor systems are involved in supraspinal analgesia in rats. 7. PHYS and other NK1 agonists do not affect the response of rat cerebellar granule neurons to the cytotoxic action of glutamate, whereas the NK2/ NK3 receptor preferring amphibian TKs (KASS, RKA, PG-KII) significantly increase glutamate excitotoxicity.

PATHOLOGICAL IMPLICATIONS Advances made during the past decade indicate that TKs, besides their physiological roles both in mammals and in amphibians, may also play a significant role under pathological conditions. An imbalance in the functioning of the mammalian TK system may contribute to several diseases, and, therefore, TK receptors could represent attractive targets for novel agonist and antagonist therapeutic agents. In this regard, although results obtained with amphibian TKs are rather scanty, some representative studies both in experimental models and in human disorders, discussed below, show that these peptides, being more selective and more resistant to enzymatic degradation than mammalian TKs, may help to better clarify TK pathological implications. PHYS administered by eye drops increases lachrymal secretion and ameliorates Sjögren’s syndrome and other forms of keratoconjunctivitis sicca due to deficit of lachrymal secretion. Recently, in a screening study of new sialogogic agents, which might be of use in the treatment of dry mouth in humans, PHYS was the most potent substance tested.

Central injections of PG -SPI (NK1 selective agonist) and PG-KII (NK3 selective agonist) in rats stimulate IL1β production and increase granulocyte infiltration into the colon worsening experimental models of colitis, which are reminiscent of human inflammatory bowel disease (Crohn’s disease and ulcerative colitis), thus providing further evidence of the central role of TKs in this gut pathology and of the potential usefulness of NK1 and NK3 TK receptor antagonists as therapeutic agents. In addition, it has been recently reported that the fragment 25 to 35 of the β-amyloid peptide, the primary constituent of senile plaques and cerebrovascular deposits in Alzheimer’s disease and Down syndrome, shows important sequence homology with TKs, being highest in the C-terminal region. Interestingly, the neurotoxic response to β-amyloid (1–40) is completely reversed by SP and PHYS and, partially, by NKB and KASS, raising the possibility that β-amyloid may interact with TK receptors. Furthermore, in neuroblastoma cells the toxic effect induced by βamyloid (1–40) is inhibited by the concurrent treatment of the cells with PHYS. In turn, β-amyloid (1–40), which alone weakly activates the TK receptors, in the presence of glutamate strongly activates both TK receptors (especially the NK1 receptors) and phosphatidylinositol turnover. Taken together, all studies on amphibian TKs should not merely be considered of academic interest, as they have substantially contributed to the progress of research in the field of mammalian TKs. These studies have led the way to, first, the discovery of new TKs and elucidation of their structures, then to providing easily available models for studying structure–activity relationships, and finally, to identification of the pathophysiological implications of TKs in mammals, including humans.

References [1] Conlon JM, Warner FJ, Burcher E. Bufokinin: a substance Prelated peptide from the gut of the toad, Bufo marinus with high binding affinity but low selectivity for mammalian tachykinin receptors. J Pept Res. 1998;51:210–15. [2] Erspamer V. Bioactive secretions of the integument. In: Heatwole H, Barthalmus GT, Eds. Amphibian biology. I. The integuments, Surrey Beatty & Sons: Chipping Norton NSW (Australia); 1994: 178–350. [3] Johansson A, Liu L, Holmgren S, Burcher E. Characterization of receptors for two Xenopus gastrointestinal tachykinin peptides in their species of origin. Naunyn-Schmiedebergs Arch Pharmacol. 2004;370:35–45. [4] Liu L, Warner FJ, Conlon JM, Burcher E. Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinn, in its species of origin. Naunyn-Schmiedebergs Arch Pharmacol. 1999;360:187–95. [5] Liu L, Markus I, Vandeberg RJ, Neilan BA, Murray M, Burcher E. Molecular identification and characterization of three isoforms of

268 / Chapter 40 tachykinin NK 1-like receptors in the cane toad. Bufo marinus. Am J Physiol Regul Integr Comp Physiol. 2004;287:575–85. [6] Liu L, Murray M, Conlon JM, Burcher E. Quantitative structureactivity analyses of bufokinin and other tachykinins at bufokinin (bNK1) receptors of the small intestine of the cane toad. Bufo marinus. Biochem Pharmacol. 2005;69:329–38. [7] Regoli D, Nguyen QT, Jukic D. Neurokinin receptor subtypes characterized by biological assays. Life Sci. 1994;54:2035–47.

[8] Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V. The tachykinin peptide family. Pharmacol Rev. 2002;54:285–322. [9] Simmons MA, Brodbeck RM, Karpitskiy VV, Schneider CR, Neff DP, Krause JE. Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana. Neuroscience 1997;79: 1219–29.

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41 Opioid Peptides from Frog Skin and Bv8-Related Peptides LUCIA NEGRI AND PIETRO MELCHIORRI

subfamily Phyllomedusinae (Phyllomedusa, Agalychnis and Pachymedusa species). They are small peptides codified by different genes and secreted by the syncytial cells that form the luminal wall of serous holocrine glands in the frog integument.

ABSTRACT Amphibian opioids are small peptides found in the skin of South American hylid frogs Phyllomedusinae. Two subfamilies of opioid peptides have been distinguished according to their selectivity for the μ- and δopioid receptors: the dermorphin and deltorphin families. The distinctive feature of opioid peptides is the presence of a D-amino acid in the N-terminal sequence, Tyr-D-Xaa-Phe, that restricts peptide conformation and confers resistance against enzyme degradation. Bv8 is a small protein isolated from the skin secretion of the frog Bombina variegata. Ortologs of Bv8 have been found in snakes (MIT) and in mammals (Prokineticins). Striking characteristics of these proteins are the identical amino terminal sequence AVITG and the presence of 10 cysteines with identical spacing. Bv8/Prokineticin proteins have been shown to modulate complex behaviors, such as pain perception, feeding, drinking, and circadian rhythms. They also are involved in hypothalamic hormone release, neuronal survival, and angiogenesis.

A BRIEF HISTORY The first to be discovered were two dermorphins (1 and 2 in Table 1) identified in the skin of Ph. sauvagei, Ph. rohdei, and Ph. burmeisteri. The very high dermorphin content in the skin of these Phyllomedusinae, about 50–80 μg/g of fresh skin, made it easy to purify and sequence the peptides. Until the discovery of mammalian endomorphins, these peptides represented the most potent and selective μ-opiate receptor agonists identified in living organisms. The screening of a cDNA library prepared from the skin of Ph. sauvagei established the amino acid sequence of several dermorphin precursors. The sequence of one of these cDNAs indicated the existence of another peptide that contained methionine as the second amino acid. This peptide (8 in Table 1) was subsequently isolated from the skin of Ph. sauvagei and proved to have higher affinity and selectivity for δ-opiate receptors than any other known natural compound. It has been given various names: deltorphin, D-Met-deltorphin, dermenkephalin, and deltorphin A. Subsequently, two additional peptides with even higher affinity for the δ-opiate receptor were isolated from the skin of Ph. bicolor. Like dermorphin, these peptides contain D-alanine as the second amino acid. They have been termed D-Aladeltorphin-I and D-Ala-deltorphin-II (9 and 10 in Table 1). Screening of cDNA libraries from the skin of Ph. bicolor revealed the sequence of four precursors for DAla-deltorphin-I and -II. These precursors contained one copy of D-Ala-deltorphin-II and either one, three

OPIOID PEPTIDES Although Vittorio Erspamer and his colleagues discovered the opioid peptides in Amazonian frogs only in the1980s [2], the Matses tribe of the upper Amazonian basin unveiled the pharmacological properties of amphibian skin opiates long ago. For centuries they had habitually applied the dried skin secretions of Phyllomedusa bicolor, called sapo (the Spanish word for “toad”), to cuts in their skin during shamanistic hunting rituals. The abundance of the opioid petides in sapo probably caused the hunters analgesia and behavioral excitation. Amphibian opiate peptides have been found only in the skin of South American hylid frogs belonging to the Handbook of Biologically Active Peptides

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270 / Chapter 41 TABLE 1.

1 2 3 4 5 6 7

Naturally occurring Amphibian opioid peptides and their origin.

Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys-OH Tyr-D-Ala-Phe-Trp-Tyr-Pro-Asn-OH Tyr-D-Ala-Phe-Trp-Asn-OH Dermorphin-Gly-Glu-Ala-OH Dermorphin-Gly-Glu-Ala-Lys-Lys-Ile-OH

8 9 10 11

Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 Tyr-D-Leu-Phe-Ala-Asp-Val-Ala-Ser-ThrIle-Gly-Asp-Phe-Phe-His-Ser-Ile-NH2 12 Tyr-D-Ile-Phe-His-Leu-Met-Asp-NH2

Dermorphins dermorphin [Hyp6]der [Lys7-OH]-der [Trp4,Asn7-OH]-der [Trp4,Asn5-OH]-der Y10A Y131 Deltorphins D-Met-deltorphin D-Ala-deltorphin-I D-Ala-deltorphin-II D-Leu-deltorphin-17 D-Ile-deltorphin

or no copies of D-Ala-deltorphin-I, respectively. The DAla-deltorphin precursors also contained three additional sequences related to dermorphins. As predicted by the absence of glycine at the carboxyl terminus of these peptides, the end products extracted from the skin contain a free α-carboxyl group at the C-terminus. Alternative cleavage sites in the sequence of dermorphin can generate C-terminally elongated dermorphins: Two of these dermorphin have been isolated from Ph. sauvagei skin extracts (6 and 7 in Table 1). In the Brazilian frog Ph. burmeisteri, a linear peptide of 17 amino acids with a D-Leu residue at position 2 has been identified and termed D-Leu-deltorphin-l7 (11 in Table 1). During screening of cDNA libraries prepared from skin of two additional species of Phyllomedusinae, the Pachymedusa dacnicolor and Agalychnis annae, using sequence information from cDNAs encoding dermorphin and deltorphin precursors from Ph. sauvagei and Ph. bicolor, we identified, in addition to four copies of dermorphin, a sequence that contains the genetic information for a novel peptide: Tyr-D-Ile-Phe-His-Leu-Met-Asp-NH2 (12 in Table 1). This peptide, an analog of D-Metdeltorphin, contains a D-Ile at the second position.

STRUCTURE AND CONFORMATION All amphibian opioid peptides contain the Nterminal sequence Tyr-D-Xaa-Phe, where the amino acids Tyrl and Phe3 are of L-configuration and D-Xaa2 is a D-amino acid. The D-amino acid containing opioid peptides arises from precursors showing a common preproregion (22-residue signal peptide and a 18- to 25-residue acidic prosequence) with precursors of the peptide antibiotics dermaseptins (24- to 34-residue polycationic and α-helical amphipathic peptides). The D-enantiomer is encoded, however, by the codon for the L-isomer in the precursor cDNA. Thus, L-Xaa2 must

Ph. sauvagei, burmeisteri, rohdei, hypohchondrialis, tarsius, Agalychnis Ph. bicolor

Ph. sauvagei

Ph. sauvagei, burmeisteri, rohdei, tarsius Ph. bicolor Ph. burmeisteri Pachymedusa dacnicolor, Agalychnis annae

be converted to D-Xaa2 by an unusual posttranslational reaction that presumably takes place in the precursor itself. Because [L-Xaa2]-containing peptides have never been found in amphibian skin extracts, the epimerization mechanism probably involves a quantitative inversion of the chirality of the α-carbon of the amino acid residue, rather than a racemization, which would yield an equimolar mixture of L- and D-isomers. Enzymes catalyzing the formation of D-amino acids are so far known only in yeast. From Bombina skin secretions, Kreil [3] recently purified a 52 kDa glycoprotein, which catalyzes the reaction Ile-Ile-Gly to Ile-D-allo-Ile-Gly. The partial conversion of Ile to D-allo-Ile in peptide linkage proceeds without the addition of cofactors. Similar to mammalian prohormones, all opioid peptides in amphibian skin precursors are flanked by paired dibasic amino acids (Lys-Arg). Moreover, the precursor sequence contains an additional Gly residue at its carboxyl terminus; this extra residue is required for the carboxamidation of the mature heptapeptide. Despite the common N-terminal tripeptide (Tyr-DXaa-Phe), the two families of amphibian opioids differ widely in receptor selectivity but bind to their own receptors with similar affinity. The N-terminal domain contains the minimum sequence essential for opioid receptor binding, whereas the C-terminal domain contains the address requisites for receptor selectivity. The presence within the putative transmembrane domains II and III of the mu-receptor protein of negative charged amino acid residues (Asp [114], Asp [147]) that contribute to ligand binding, may explain why positively charged dermorphins have high μ-opioid receptor selectivity and why amidation of their terminal carboxyl group increases their affinity and potency. Similarly, the negatively charged C-terminal tetrapeptide of deltorphins enhances deltorphin selectivity for the δ-opioid receptor by electrostatic attraction to the positively charged binding site of the delta receptor

Opioid Peptides from Frog Skin and Bv8-Related Peptides / 271 (Arg 292) and electrostatic repulsion from the negatively charged μ-receptor site. Unlike the positively charged C-terminus of dermorphins, the negatively charged C-terminal tail of deltorphins (especially in DAla2-deltorphin-II) comes into close contact with the positively charged N-terminal tripeptide of the molecule and folds the backbone, thus placing the Tyr1 and Phe3 aromatic rings in specific orientations best suited for delta-receptor docking.

a hydrophilic group (a basic amino acid or a glycosyl residue) at the C-terminal end of the molecule enter the CNS at a 7 to 10 times higher rate than dermorphin, suggesting facilitated transport across the blood brain barrier by a carrier or endocytosis. The amphibian opioid with the highest analgesic potency and efficacy is [Lys7]dermorphin (AD50: 30 pmol/rat, by intracerebroventricular route; 0.22 μmol/kg, by subcutaneous route). Its antinociceptive effect lasts more than 3 hours and is never accompanied by catalepsy. In contrast, [Trp4,Asn7]dermorphin induces catalepsy at i.c.v. doses that do not modify the reaction time in the tail-flick test: The ratio of antinociceptive to cataleptic ED50 (AD50/ CD50) is 40, a value 500 times higher than that calculated for [Lys7]dermorphin (AD50/CD50 = 0.073). The ratio of antinociceptive to respiratory-depressant ED50 doses is 17 times lower for [Lys7]dermorphin than for morphine. Indeed, [Lys7]dermorphin, in the range of the analgesic doses (36–120 pmol, icv; 0.12–4.7 μmol/ kg, sc) significantly increases respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. The early onset ventilatory stimulation produced by [Lys7]dermorphin, and ascribed to the intact peptide, is mediated by serotonergic descending excitatory pathways that stimulate neurons of the brainstem respiratory network [19]. In rats and mice, central or peripheral administration of the dermorphin-like peptides induces a significantly slower development of tolerance to the antinociceptive effect than morphine. Withdrawal symptoms precipitated by naloxone are less intense in peptide-dependent than in morphine-dependent rats.

BIOLOGICAL ACTIONS The pharmacology of amphibian opiates has been extensively described [2, 14, 16, 19, 20]. Dermorphin has high affinity and selectivity for μopioid receptors: its opioid potency in guinea pig ileum preparations (GPI) is 100 times higher than that of morphine. The amidated analogs of Ph. bicolor dermorphins display similar or slightly higher μ-opioid receptor affinity but significantly higher (15–20 times) κ-opioid receptor affinity than dermorphin (Table 2). Like μopiate agonists, dermorphins produce antinociception but also catalepsy, respiratory depression, constipation, tolerance, and dependence, although at a lower degree than morphine. Dermorphin-induced antinociception takes place at both the supraspinal and spinal level. The analgesic potency of dermorphin is about 280 times higher than that of morphine after i.c.v. injection but comparable to that of morphine after s.c. injection because of its low CNS permeability and bioavailability. [Lys7]dermorphin and some synthetic analogs bearing

TABLE 2. Affinity and selectivity for μ-, δ-, and κ-opioid receptors; biological activities on guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations; and analgesic potency of dermorphins and deltorphins. IC50, nM (mean ± S.E.)

Ki, nM (mean ± S.E.) Compounds Morphine Dermorphin dermorphin [Hyp6]der [Lys7]der [Lys7-OH]der [Trp4,Asn5]der [Trp4,Asn5-OH]der [Trp4,Asn7]der [Trp4,Asn7-OH]der Deltorphin D-Ala2-delt I D-Ala2-delt II D-Met2-delt D-Leu2-delt D-Ile2-delt D-aIle2-delt

μ

δ

11 ± 1.5 0.6 0.7 0.09 5.7 0.9 4.44 0.32 2.9

± ± ± ± ± ± ± ±

0.02 0.03 0.008 0.51 0.052 0.39 0.026 0.2

1985 ± 224 2222 ± 233 693 ± 37 >10,000 1021 ± 57 452 ± 68

κ

GPI

500 ± 48 41 131 185 172 45 73 57 69

8162 ± — 617 ± — 177 ± 2396 ± 427 ± 905 ±

0.78 ± 0.08 1.03 ± 1.09 1.18 ± 0.21 >10,000 24 ± 3 54 ± 65

>10,000 >10,000 >10,000 >10,000 >10,000 >10,000

929 1200 1105 1150 480 715 690 865

± ± ± ± ± ± ± ±

150 ± 18.5 979 66 12 301 47 61

MVD

Analgesia AD50, nmol/rat

1215 ± 115

8.7 ± 1.1

± ± ± ± ± ± ± ±

0.11 0.12 0.13 0.45 0.52 1.2 0.06 0.2

16.5 18.1 13.6 56.3 73.7 205 6.6 10.4

± ± ± ± ± ± ± ±

1.3 2.9 1.5 7.8 9.1 25 0.9 1.3

0.035 ± 0.01 — 0.026 ± 0.009 — 0.43 ± 0.04 — 2.86 ± 0.31 —

1239 ± 2500 ± 1476 ± >5000 4200 ± 3200 ±

203 170 185

0.18 0.37 0.97 2480 7.0 70

± ± ± ± ± ±

0.02 0.03 0.05 378 0.9 8.2

15 ± 3.0 54 ± 6.0 20 ± 5.0 — 6.7 ± 1.0 9.8 ± 1.2

1.29 1.6 1.15 3.82 5.00 13.1 0.58 1.3

475 307

272 / Chapter 41 Like morphine, dermorphin inhibits gastric acid secretion, delays gastric emptying and slows intestinal and colonic propulsion. Compared with dermorphin, [Lys7]dermorphin is slightly (3–5 times) more potent both in reducing acid gastric secretion and gastric emptying. By contrast [Trp4,Asn7]dermorphin is 20–50 times less potent than dermorphin in inhibiting gastric acid secretion but is 100–1000 times less potent in delaying gastric emptying. In spite of comparable μ-opioid receptor affinity, the two dermorphins from Ph. bicolor, [Lys7]dermorphin and [Trp4,Asn7]dermorphin, differ in biological effects, underlying distinct functional roles of the μ-opioid receptors. The discovery of deltorphins provided the tools for the functional characterization of the delta opiate system. While D-Ala2-deltorphins have delta-binding affinity similar to D-Met2-deltorphin, they consistently have the highest delta-opioid selectivity (Table 2). The rank order of selectivity (Kiδ/Kiμ) is D-Ala2-deltorphinI = D-Ala2-deltorphin-II > D-Met2-deltorphin >> D-Ile2deltorphin >> D-Leu2-deltorphin heptadecapeptide or its N-terminal decapeptide fragment. Both D-Met2deltorphin and D-Ala2-deltorphins are highly resistant to enzyme degradation. D-Ala2-deltorphin-I and -II cross the blood-brain barrier in vivo and in vitro: D-Ala2deltorphin-II was identified as a transport substrate of organic anion transporting polypeptides (Oatp/OATP), a family of polyspecific membrane transporters, strongly expressed in rat and human blood-brain barrier. In mice, D-Ala2-deltorphin II, by the i.c.v. route (EC50 = 2.1 nmol/mouse) is half as potent as morphine and the analgesic effect is antagonized by the δ-selective antagonist, naltrindole. Repeated injection of D-Ala2deltorphin-II induces tolerance to the antinociceptive effect. Isobolographic analysis shows that the supraspinal antinociception induced by the delta opioid agonist DPDPE and the spinal antinociception induced by DAla2-deltorphin-II are synergistic in many nociceptive tests. In rats, intrathecal injections of D-Ala2-deltorphinII (from 0.6 nmol/rat) produce a naltrindole-reversible inhibition of the tail-flick response by inhibiting the nociceptive neurons in the superficial and deeper dorsal horn of the medulla. Conversely, when injected i.c.v. in rats, D-Ala2-deltorphin-II was a weak partial agonist (>30 nmol/rat) and induces fleeting naloxone-sensitive antinociception. Both D-Ala2-deltorphin-I and D-Met2deltorphin at doses between 6.5 to 52 nmol/rat induce a naloxone-sensitive analgesia. Results suggest that the δ-agonists play a predominantly modulatory role in antinociception rather than a primary role. In mice and in rats the intensity of δopioid analgesia depends on coactivation of μ-opiate receptors by endogenous or exogenous opioids. Accordingly, deltorphin-induced analgesia is weaker in homo-

zygotic mice with a disrupted μ-opiate receptor gene than in wild-type mice. Injections of the deltorphins into the rat brain ventricles, ventral tegmental area, and nucleus accumbens at doses 10–100 times lower than those inducing analgesia (0.06 to 3.8 nmol/rat) invariably increase locomotor activity and induce stereotyped behavior. Deltophin-induced motor activity is antagonized by the δ-selective antagonist, naltrindole. Repeated i.c.v. injections of D-Ala2-deltorphin-II in naive rats induced tolerance to the stimulant effects. High doses (10– 50 nmol/rat) of all the D-Met2-deltorphin analogs and His4-substituted D-Ala2-deltorphins induce nonopioid motor disfunction that is completely blocked by the noncompetitive NMDA antagonist, dextrorphan. Deltorphin improves memory consolidation in a passive avoidance test in mice. D-Ala2-deltorphin-I, at low doses, stimulates respiratory activity in fetal lambs. D-Aladeltorphin-II inhibits diarrhea induced by castor oil and colonic bead expulsion, but it leaves the rate of transit through the small intestine unchanged. By the s.c. route, D-Ala-deltorphin-I inhibits acidified alcoholinduced gastric mucosal lesions. It is involved in the control of ingestive behavior, stimulating food intake and angiotensin-mediated water consumption in rats. The discovery of the amphibian opioid peptides confirms that the amphibian skin and its secretions offer an inexhaustible supply of biologically active peptides. The amphibian peptides isolated from methanol extracts of amphibian skin are of small or relatively small molecular mass (700 to 4600 Da). Proteins, externally secreted by syncytial cells forming the wall of the integument glands, can be obtained upon electrical stimulation of the skin of the living frog. By repetitive electrical stimulation at weekly intervals, several milligrams of bioactive proteins can be collected from a single frog, yielding enough biological active proteins for pharmacological research.

Bv8/PROKINETICIN FAMILY (SWISS-PROT: Q9PW66) A basic small protein, containing 77 amino acids, 10 of which are cysteine, has been isolated from the skin secretion of discoglossid frogs Bombina variegata and Bombina bombina [15] and named Bv8, to indicate its origin (Bombina variegata) and its molecular mass (about 8 kDa). The amino acid sequence was established by automated Edman degradation and analysis of proteolytic fragments as well as by cDNA cloning. Homologs of Bv8 have been predicted (but not yet isolated) in skin secretions of Rana temporaria and Rana esculenta and have been demonstrated in the skin secretion of Bombina orientalis (Bo8) and Bombina maxima (Bm8a). These pro-

Opioid Peptides from Frog Skin and Bv8-Related Peptides / 273 Bv8

AVITGACDKDVQCGSGTCCAASAWSRNIRFCIPLGNSGEDCHPASHK VPYDGKRLSSL-CPCKSGLTCSKSGE-KFKC-S

Bo8

AVITGACDRDVQCGSGTCCAASAWSRNIRFCVPLGNSGEECHPASHK VPYDGKRLSSL-CPCKSGLTCSKSGA-KFQC-S

Bm8a

AVITGVCDRDAQCGSGTCCAASAFSRNIRFCVPLGNNGEECHPASHK VPYNGKRLSSL-CPCNTGLTCSKSGE-KFQC-S

MIT

AVITGACERDLQCGKGTCCAVSLWIKSVRVCTPVGTSGEDCHPASHK IPFSGQRKMHHTCPCAPNLACVQTSPKKFKCLSK

PK1

AVITGACERDVQCGAGTCCAISLWLRGLRMCTPLGREGEECHPGSHK VPFFR-R-KHHTCPCLPNLLCS-FPDGRYRCSMDLKNINF

PK2

AVITGACDKDSQCGGGMCCAVSIWVKSIRICTPMGKLGDSCHPLTRK VPFFG-R-MHHTCPCLPGLACL-TSFNRFICLAQK

PK2L

AVITGACDKDSQCGGGMCCAVSIWVKSIRICTPMGKLGDSCHPLTRK VPFFG-R-MHHTCPCLPGLACL-TSFNRFICLAQK NNFGNGRQERRKRKRSKRKKE

FIGURE 1. Aminoacid sequences of the frog skin secretion proteins (Bv8, Bo8, and Bm8a) compared with the snake venom protein (MIT) and the human prokineticis: PK1, PK2, and the long form of PK2 (PK2L) with the 21-amino-acid insert.

teins are derived from simple precursors composed of a putative 19-residue signal peptide and the mature protein. Bo8 and Bm8a showed 96% and 92% sequence identity with Bv8. Moreover the putative signal peptides in all three proproteins were 100% identical. The primary sequence of Bv8 is similar to MIT (mamba intestinal toxin), a constituent of the venom of the snake black Mamba. The amphibian and snake proteins share the same pattern of cystein distribution and their overall identity is 58%. cDNA cloning showed the existence of mouse (mBv8) and human (hBv8) homologs of amphibian Bv8 [21]. Striking characteristics of these proteins are the identical amino terminal sequence, AVITG, and the presence of 10 cysteines with identical spacing. A similar Cys-motif is also present in mammalian co-lipase and in the carboxy-terminal region of members of the Dickkopf family of extracellular signaling proteins. However, the frog protein does not stimulate the activity of pancreatic lipase, and it is also inactive in an assay for Dickkopf functions [4]. Starting from the 3D structure of mammalian co-lipase, we could build a model for frog Bv8 in which the hydrophobic amino terminal sequence AVITG forms sort of a “beak” exposed at the surface of the tightly folded rest of the protein. Two forms of mBv8 and hBv8 have been characterized in the mouse and man testis. These forms differ in an exon coding for 21 amino acids, the majority of which are basic. The predominant site of Bv8 expression in both humans and mice is primary spermatocytes in the seminiferus tubules of the testis. The genomic structure of these murine and human Bv8 genes has been determined, and the chromosomal localization was identified near a synteny breakpoint of mouse chromosome 6 and human 3p21. Searching the EST database using the predicted coding of Bv8, Zhou [10] identified two human EST sequences, one encoding the human protein already described [21] and the other encoding a slightly different Bv8-like protein. The two

proteins were named prokineticin 2 (PK2) and prokineticin 1 (PK1). The name prokineticin refers to the ability of these peptides to contract guinea pig ileum, a property shared with amphibian Bv8. Screening a library of human secreted proteins for the ability to induce proliferation in capillary endothelial cells, Ferrara [7, 8] identified a protein that induced proliferation, migration, and fenestration in the endothelial cells of steroid synthesizing glands (ovary, testis, adrenals) and named it endocrine-gland-derived vascular endothelial growth factor (EG-VEGF). EG-VEGF and PK1 are the same protein and have an overall identity of 58% and homology of 76% with human PK2 and murine Bv8 and 43% identity with amphibian Bv8. PK1/EG-VEGF has been isolated and sequenced also from bovine milk. Rat and human mRNAs for PK1 and PK2 have been cloned and their expression patterns reported in peripheral tissues and the central nervous system [10]. The distribution of murine Bv8-like proteins, PK1 and PK2, and their mRNAs has been reported in brain (olfactory bulb, cerebral cortex, hippocampus, some thalamic and hypothalamic nuclei, Purkinje cells of cerebellum, many nuclei of brain stem), spinal cord, gastrointestinal tract, endocrine glands, and other peripheral organs [13]. PK2 mRNA expression pattern in the suprachiasmatic nucleus (SCN) of mice [1] and of rats (submitted data) is rhythmic (being lowest in the dark phase) and is severely blunted in mutant mice deficient in Clock or cryptochrome genes. Receptors for PK1 and PK2 have been identified in humans, mice, and rats [11, 12]. These receptors, named prokineticin receptor 1 and 2 (PKR1 and PKR2), belong to the family of G-protein-coupled receptors, share approximately 85% amino acid identity, and are about 80% identical to the previously described mouse orphan receptor gpr73. In a study aimed to map the expression of PKR1 and PKR2 in rat and mouse nervous system, we detected PKR2 mRNA in the brain [olfactory

274 / Chapter 41 bulbs, SCN, hippocampus, paraventricular thalamic and hypothalamic nuclei, nucleus arcuatus, subfornical organ (SFO), amygdala, caudate putamen and cortex] and in dorsal root ganglia (DRG). PKR1 is mainly expressed outside the mammalian central nervous system, with the highest density in the neurons of DRG. PKR1 is also expressed by murine macrophages. Mice lacking the PKR2 do not survive; mice lacking PKR1 are severely deficient in their responses to noxious thermal and chemical (capsaicin, protons) stimuli. The biology of Bv8 and its mammalian orthologs remains largely unexplored. Luckily, the isolation of an adequate amount of the amphibian Bv8 protein has provided the opportunity to study the pharmacology of Bv8 by topical and systemic administration. Originally identified as potent agents that contract smooth muscle of gastrointestinal tract [10, 15], Bv8 and its mammalian orthologs have been shown to modulate complex behaviors, such as pain perception, feeding, drinking [17], and circadian rhythms [1] and have been involved in hypothalamic hormone release [6], neuronal survival [13], and angiogenesis [7, 8]. On cultured cerebellar granular cells, Bv8 reduced the extent of apoptotic death induced by switching the concentration of K+ in the growing medium from 25 to 5 mM [13]. In a cell-based assay, Bv8 and EG-VEGF/PK1 induced proliferation, migration, and survival of primary adrenal cortical capillary endothelial (ACE) cells. Delivery of Bv8 or EG-VEGF to the mouse testis resulted in a potent angiogenic response due to activation of PKRs on vascular endothelial cells within the interstitial space [7, 8]. Injections of Bv8 (from 0.25 to 60 pmol) into the lateral ventricles of the rat brain suppressed diurnal, nocturnal, deprivation-induced, and neuropeptide Ystimulated feeding. I.c.v. injected Bv8 also stimulated drinking in a dose-dependent manner. We demonstrated that Bv8 stimulates drinking through its receptors in the SFO and inhibits feeding after injection into the arcuate nucleus. Rat sensitivity to the Bv8-induced anorexogenic response increased significantly during the dark phase of the circadian cycle [17]. Cheng and coworkers [1] showed that PK2 delivered into the rat lateral brain ventricles suppressed the high level of wheel running behavior associated with the dark phase of the circadian cycle and hypothesized that PK2 controls the behavioral (locomotor) circadian rhythm from the SCN. In rats, i.c.v. and s.c. injections of Bv8 induced antidiuresis dependent on vasopressin release. S.c. injections of Bv8 also induced release of oxytocin and corticosterone [6]. Very low doses of Bv8 administered to rats and mice by systemical (intravenous or subcutaneous, from 6 to 25 pmol/kg), intrathecal (50 fmol/rat), or topical

(50 fmol/paw) injection produced an intense and long-lasting decrease in the threshold to thermal and mechanical nociceptive stimuli. We demonstrated that PKR1 and PKR2 expressed in primary sensitive neurons of DRG bind Bv8 with high affinity and, when activated by Bv8, produce [Ca++]i transients and PKCε translocation in cultured dorsal root ganglion neurons—hence sensitizing nociceptors to mechanical and thermal stimuli [18]. We have demonstrated that Bv8 activates prokineticin receptors on macrophages to produce chemotaxis and release inflammatory cytokines (Martucci et al., 2004, submitted). Moreover the mammalian Bv8/PK2 is highly expressed in circulating and inflammatory leukocytes [5, 9]. Systemic in vivo exposure to Bv8 or PK1/EGVEGF resulted in significant increases in total leukocyte, neutrophil, and monocyte counts [9], and PK1 induces a distinct monocyte derived cell population, which is primed for release of proinflammatory cytokines. We would like to propose that Bv8/prokineticins and their receptors are part of a novel signaling pathway that triggers the development of inflammatory and neuropathic pain. To develop PKR antagonists as novel analgesic and antinflammatory drugs, structural determinants required for the biological activity of Bv8 must be identified. The highly conserved amino terminal sequence (AVITGA) of all members of the Bv8/PK family is important for their biological activity: deletions and substitutions in the conserved N-terminal sequence of Bv8 and of PK1 yielded inactive and, sometimes, antagonist molecules. We have demonstrated that an N-terminally shortened form of Bv8, desAlaVal-Bv8, succeeded in antagonizing Bv8-induced hyperalgesia.

References [1] Cheng MJ, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J, Weaver DR, Leslie FM, and Zhou QY. (2002). Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature, 417, 405–10. [2] Erspamer V. (1994). Bioactive secretions of the Amphibian integument. In: Heatwole H (ed). Amphibian Biology. Surrey Beatty & Sons Publ. pp. 178–350. [3] Jilek A, Mollay C, Tippelt C, Grassi J, Mignogna G, Mullegger J, Sander V, Fehrer C, Barra D, and Kreil G. (2005). Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions. Proc. Natl. Acad. Sci. USA, 102, 4235–9. [4] Kaser A, Winklmayr M, Lepperdinger G, and Kreil G. (2003). The AVIT protein family. Secreted cysteine-rich vertebrate proteins with diverse functions. EMBO Rep., 4, 469–73. [5] Lattanzi R, Giannini E, Melchiorri P, and Negri L. (2001a). Pharmacology of Bv8: A new peptide from amphibian skin. Br. J. Pharmacol., 133, 45P. [6] Lattanzi R, Giannini E, and Negri L. (2001b). Bv8, a small protein from frog skin induces antidiuresis in rats. Pharmacological Res., 43(A), 30. [7] LeCouter J and Ferrara N. (2003). EG-VEGF and Bv8: A novel family of tissue-selective mediators of angiogenesis, endothelial phenotype and function. TMC, 13, 276–82.

Opioid Peptides from Frog Skin and Bv8-Related Peptides / 275 [8] LeCouter J, Kowalski J, Foster J, Hass P, Zhang Z, Dillard-Telm L, Frantz G, Rangell L, Deguzman L, Keller Ga, Peale F, Gurney A, Hillan KJ, and Ferrara N. (2001). Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature, 412, 876–84. [9] LeCouter J, Zlot C, Tejada M, Peale F, and Ferrara N. (2004). Bv8 and endocrine gland-derived vascular endothelial growth factor stimulate hematopoiesis and hematopoietic cell mobilization. PNAS, 101, 16813–18. [10] Li M, Bullock CM, Knauer DJ, Ehlert FJ, and Zhou QY. (2001). Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle. Mol. Pharm., 59, 692–98. [11] Lin DCH, Bullock CM, Ehlert FJ, Chen JL, Thian H, and Zhou QY. (2002). Identification and molecular characterization of two closely related G-protein coupled receptors activated by prokineticins/EG-VEGF. J. Biol. Chem., 277, 19276–80. [12] Masuda Y, Takatsu Y, Terao Y, Kumano S, Ishibashi Y, Suenaga M, Abe M, Fukusumi S, Watanabe T, Shintani Y, Yamada T, Hinuma S, Inatomi N, Ohtaki T, Onda H, and Fujino M. (2002). Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors. Biochem. Biophys. Res. Comm., 293, 396–402. [13] Melchiorri D, Bruno V, Besong G, Ngomba Rt, Cuomo L, Deblasi A, Copani A, Moschella C, Storto M, Nicoletti F, Lepperdinger G, and Passarelli F. (2001). The mammalian homologue of the novel peptide Bv8 is expressed in the central nervous system and supports neuronal survival by activating the

[14] [15]

[16]

[17]

[18]

[19]

[20] [21]

MAP kinase/PI-3-kinase pathways. Eur. J. Neuroscience, 13, 1694–1702. Melchiorri P and Negri L. (1996). The dermorphin peptide family. Gen. Pharmacol., 7, 1099–1107. Mollay C, Wechselberger C, Mignogna G, Negri L, Melchiorri P, Barra D, and Kreil G. (1999). Bv8, a small protein from frog skin and its homolog from snake venom induce hyperalgesia in rats. Eur. J. Pharmacol., 374, 189–96. Negri L and Giannini E. (2003). Deltorphins In: Porreca F, Woods JH, Chang KJ (eds). The delta receptors, molecule and effects of delta opioid compounds Marcel Dekker Book Chapters pp. 175–89. Negri L, Lattanzi R, Giannini E, De Felice M, Colucci A, and Melchiorri P. (2004). Bv8, the amphibian homologue of the mammalian prokineticins, modulates ingestive behaviour in rats Br. J. Pharmacol., 142, 141–51. Negri L, Lattanzi R, Giannini E, Metere A, Colucci M, Barra D, Kreil G, and Melchiorri P. (2002). Nociceptive sensitisation by the secretory protein Bv8. Br. J. Pharmacol., 137, 1147–54. Negri L, Lattanzi R, Tabacco F, and Melchiorri P. (1998). Respiratory and cardiovascular effects of the mu-opioid receptor agonist [Lys7]dermorphin in awake rats. Br. J. Pharmacol., 124, 345–55. Negri L, Melchiorri P, and Lattanzi R. (2000). Pharmacology of amphibian opiate peptides. Peptides 21: 1639–47. Wechselberger C, Puglisi R, Engel E, Lepperdinger G, Boitani C, and Kreil G. (1999). The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes. FEBS Letters, 462, 177–81.

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C

H

A

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T

E

R

42 Amphibian Bombesin-like Peptides ELIOT R. SPINDEL

massive secretion of the contents of the glands [2, 4]. Typically levels of peptides found in frog skin are 100- to 1000-fold higher than levels in mammalian tissues. Peptides are secreted from frog skin as a defensive function or potentially as a means of communication. The exact function of bombesin-like peptides in frog skin remains unknown. The mammalian bombesin-like peptides are gastrinreleasing peptide (GRP) and neuromedin B (NMB). Phylogenetic analysis of the prohormones encoding the bombesin-like peptides suggests there are three distinct branches of bombesin-like peptides: the GRP branch, the NMB branch, and a branch that contains the multiple bombesin-like peptides found in amphibian skin. This raises two interesting questions: Do frogs have bombesin-like peptides that fall in the NMB phylogenetic branch, and do mammals have bombesin-like peptides that fall in the skin peptide branch?

ABSTRACT The bombesin-like peptides were originally isolated from frog skin, but the discovery of extensive effects in mammals led to the search for and discovery of mammalian homologs. In frogs, four groupings of bombesin-like peptides have been described: the bombesins, the ranatensins, and the phyllolitorins, which are present in skin, and gastrin-releasing peptide (GRP), which is not present in skin. In mammals, two bombesin-like peptides have been described: gastrin-releasing peptide (GRP) and neuromedin B (NMB). Whether mammals have additional bombesin-like peptides related to the frog skin bombesin-like peptides remains to be determined. The receptors for amphibian bombesin-like peptides are 7-transmembrane G-protein coupled receptors linked to generation of IP3. Despite investigation, the exact role of bombesin-like peptides in frog skin remains unknown.

AMPHIBIAN BOMBESINS INTRODUCTION As just stated, bombesin was originally isolated from the skin of the fire-bellied toad, Bombina bombina, by Anastasi, Erspamer, and coworkers [1]. Bombesin is a tetradecapeptide with the structure shown in Fig. 2. Bombesin is also found in the skin of other Bombina species, including Bombina variegata and Bombina orientalis. The original form of bombesin as isolated by Anastasi et al. [1] is characterized by a Leu as its penultimate residue. This penultimate residue was initially thought to differentiate the bombesin-like peptides isolated from Bombina species from the bombesin-like peptides isolated from Rana species that contain a Phe residue as the penultimate residue. This is not the case, however, as many frog species contain multiple forms of bombesin-like peptides in their skin with both Leu or Phe as penultimate residues. This is illustrated by the

The bombesin-like peptides are one of the classic examples of peptides first discovered in frog skin and then later found to be widely distributed in mammals. The prototypical bombesin-like peptide, bombesin was isolated from the skin of the frog Bombina bombina by Anastasi et al. [1] in 1971. The bombesin-related peptides ranatensin and Leu-8 phyllolitorin were subsequently isolated from the skin of Rana and Phyllomedusa species by Nakajima et al. [17] and by Yasuhara et al. [21]. As discussed following, different genuses of frogs contain different, characteristic, bombesin-like peptides. In frog skin the bombesin-like peptides are located in cutaneous granular glands (Fig. 1). These are myoepithelial glands under adrenergic regulation such that subcutaneous injection of norepinephrine causes Handbook of Biologically Active Peptides

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278 / Chapter 42

FIGURE 1. Bombesin-like peptide mRNA expression in frog skin. A. Low power view showing ranatensin mRNA expression in cutaneous granular glands in the skin of Rana pipiens (100x). B. Higher power view showing bombesin expression in the skin of Bombina orientalis (200x). E = epidermis, D = Dermis, Mu = mucus gland, arrows point to peptide containing glands.

FIGURE 2. Structures of amphibian bombesin-like peptides found in the skin of Bombina orientalis. Residues that differ from Leu-13 bombesin are boxed. A. Peptides classically designated as bombesin-like. B. Peptides classically designated as phyllolitorin-like. C. Peptides classically designated as ranatensin-like.

presence of three forms of bombesin in the skin of Bombina orientalis: Leu13 bombesin, Phe13 bombesin, and Ser3-Arg10-Phe13 bombesin (SAP bombesin) (Fig. 2), as reported by Nagalla et al. [14]. Analysis of genomic DNA shows that these three forms of bombesin are derived from separate genes. As well as being expressed in skin at high levels, lower levels of these peptides are

also expressed in brain, lung, and gut [14]. In the bombesin prohormones, the sequence of bombesin follows a small N-terminal extension peptide that follows the signal peptide. A Gln residue provides the signal for the C-terminal pyroglutamate, a Gly residue provides the nitrogen for the N-terminal amide followed by two basic amino acids to signal the C-terminal cleavage. Fol-

Amphibian Bombesin-like Peptides / 279 lowing the two basic amino acids is a C-terminal extension peptide of varying length [14]. Frog skin is rich in enzymes similar to the mammalian prohormone convertases, so processing of frog skin bombesin is likely to be highly homologous to processing of neuropeptides in mammals. The receptor for Phe-13 bombesin was cloned by Spindel and coworkers [13]. It has 377 amino acids and is a 7-transmembrane, G-protein coupled-receptor linked to increased IP3 formation. The amphibian Phe13 bombesin receptor is clearly homologous to the mammalian receptors for bombesin-like peptides. In Bombina orientalis, the Phe-13 bombesin receptor is expressed primarily in brain. The affinity of the Phe-13 bombesin receptor is 0.2 nM for Phe-13 bombesin, 0.9 nM for Leu-13 bombesin, and 100 nM for SAP bombesin [13]. These differing affinities suggest that distinct receptors for the other forms of amphibian bombesin will also be present in the frog. Consistent with this, partial cDNA sequences of receptors homologous to the Phe-13 bombesin receptor have been identified in frog brain by PCR [13]. In terms of the standardized nomenclature for bombesin receptors, the receptor for Phe-13 bombesin has been designated as the BB4 receptor. Interestingly, the Phe-13 bombesin receptor is most homologous to the BRS-3 receptor, raising the possibility that the BRS-3 ligand in mammals when characterized may fall in the same phylogenetic branch as the frog skin bombesin-like peptides. This is discussed more in the “Phylogenic Considerations and Implications” section. While the function of bombesin in frogs is unknown, bombesin produces diverse physiologic effects in mammals in brain, GI tract, and lung. Within the GI tract, bombesin stimulates the secretion of many GI hormones, including gastrin, somatostatin, VIP, GIP, and glucagons, as well as the release of pancreatic exocrine enzymes [10]. Within the CNS, bombesin is a potent regulator of central homeostatic mechanisms, including regulation of appetite, blood sugar, cardiac function, and gastric acidity [19], and in lung, bombesin stimulates lung cell growth [7]. These effects of bombesin in mammals are mediated by the interaction of bombesin with the mammalian GRP and NMB receptors as discussed in other chapters of this book. Since the multiple forms of bombesin occur in frog brain together with their receptors, it is safe to conclude that bombesin-like peptides also function as neurotransmitters in frog brain.

AMPHIBIAN PHYLLOLITORINS The phyllolitorins were initially characterized by Yasuhara et al. [21] from the skin of Phyllomedusa sau-

vagei. They form a distinct class of bombesin-like peptides characterized by the Ser residue at the third position from the C-terminus as opposed to the His residue that characterizes all other bombesin-like peptides (Fig. 2). Just like bombesin, the phyllolitorins exist in multiple forms distinguished by their penultimate amino acid, a Phe-8 form resembling ranatensin and a Leu-8 form resembling bombesin. Generally, the Leu-8 form shows more bioactivity [9]. The phyllolitorins have distinct pharmacologic profiles that differentiate them from the other bombesin-like peptides, and, despite the fact that their binding to the known bombesin receptors is lower than for other related bombesinlike peptides, they produce similar effects as bombesin on the induction of scratching and grooming behavior and effects on hypothermia [9]. Peripherally, phyllolitorins lower systemic blood pressure [18] in dogs and monkeys in contrast to the hypertensive effects of bombesin and ranatensin. In an unusual mechanism, the two forms of phyllolitorin are produced by RNA editing of a single gene that encodes Phe-8 phyllolitorin [15]. The single primary transcript of the Phe-8 phyllolitorin gene is either processed unedited to the Phe-8 phyllolitorin mRNA or the uracil (U) residue in the codon encoding the Phe-8 is edited in the RNA to a cytosine (C) residue to create the codon encoding the Leu residue in Leu-8 phyllolitorin. Such U to C RNA editing, while less common than C to U RNA editing, has been reported in other systems, including insect sodium channels [8]. The phyllolitorins like bombesin are found in highest levels in skin in the cutaneous granular glands. Lower levels of phyllolitorin mRNA are also present in brain and GI tract [15]. The receptors for phyllolitorins have not been characterized but almost certainly are 7-transmembrane receptors that will be highly homologous to the receptor for Phe-13 bombesin. As for bombesin, the function of phyllolitorin in frog skin remains unknown.

RANATENSIN-LIKE PEPTIDES At about the same time that Anastasi et al. [1] described the isolation of bombesin from the skin of Bombina bombina, Nakajima et al. [17] independently described the isolation of the related peptide ranatensin from the skin of Rana pipiens. As shown in Fig. 2, bombesin and ranatensin are highly homologous, the most critical difference being the penultimate residue that is Leu in bombesin and Phe in ranatensin. Different Rana and Litora species have slightly different but highly related ranatensin-like sequences. In mammals, ranatensin-like bombesin has a variety of effects. Ranatensin has strong effects on smooth muscle and blood

280 / Chapter 42 pressure [3] and also replicates many of bombesin’s effects on GI and CNS function but with less potency [12]. The ranatensin cDNA is similar in structure to the cDNAs for the other bombesin-like peptides. As in the bombesin-prohormone, the sequence of ranatensin follows a short N-terminal extension peptide that immediately follows the signal peptide. If ranatensin is not cleaved from its N-terminal extension, it gives a peptide highly similar to ranatensin-R (Fig. 2B) and to NMB-32, the larger form of mammalian neuromedin B (NMB). In Rana pipiens highest levels of ranatensin mRNA are found in the skin, but the ranatensin mRNA is also clearly detectable in brain. Highly homologous RNAs are also found in other Rana species, including Rana catesbeiana and Rana esculenta. A full-length cDNA for the ranatensin receptor has not been characterized, but sequence analysis of cDNA fragments amplified from RNA prepared from R. pipiens brain suggests that the ranatensin receptor will be highly homologous to the Phe-13 bombesin receptor.

GASTRIN-RELEASING PEPTIDE Shortly after amphibian bombesin was isolated, its ability to stimulate release of gastrin was used by McDonald and coworkers [11] to isolate a homologous peptide from porcine stomach that was named gastrin-releasing peptide (GRP). For many years GRP was considered the mammalian homolog of amphibian bombesin until Kim et al. showed that Xenopus laevis expressed a 29amino-acid peptide in stomach highly homologous to mammalian GRP [5] and Nagalla et al. [16] cloned cDNAs encoding GRP from Bombina orientalis to demonstrate that the same frog expressed different mRNAs to encode both bombesin and GRP. Phylogenetic analysis showed that frog GRP was far more closely related to mammalian GRP than to frog bombesin (Fig. 3). The prohormone structure of amphibian GRP is also highly similar to the prohormone structure of mammalian GRP, with the sequence for GRP immediately following the signal peptide. As for mammalian GRP, there is an Arg residue in frog GRP that allows processing of the

29-amino-acid large form of GRP to the C-terminal decapeptide of GRP (GRP-10). The distribution of frog GRP is also quite distinct from that of bombesin in that frog GRP is not present in skin but rather is present in highest quantities in gut and brain. In situ hybridization analysis of GRP expression shows expression of the frog GRP mRNA in cells in the basal layers of the gastric pits [16]. Kim et al. [5] have also shown that in Xenopus laevis GRP effects contractility of stomach muscle. The localization and function of GRP in stomach suggests that amphibian GRP likely has similar functions in the GI tract of frogs as mammalian GRP does in mammals. Sequence analysis of partial cDNAs isolated from Bombina orientalis suggests that the amphibian GRP receptor is highly homologous to the mammalian GRP receptor.

PHYLOGENETIC CONSIDERATIONS AND IMPLICATIONS FOR ADDITIONAL MAMMALIAN BOMBESIN-LIKE PEPTIDES Despite the 35 years of study of bombesin-like peptides in both frogs and mammals, there are a number of fundamental unanswered questions about the peptide family. In mammals there are three receptors: the GRP receptor, the NMB receptor, and the BRS-3 receptor. The ligand for the BRS-3 receptor is unknown, and phylogenetic analysis shows that the Phe-13 bombesin receptor is closest to the BRS-3 receptor. Thus, it is tempting to speculate that the ligand for the BRS-3 receptor will be the true mammalian bombesin and phylogenetically more closely related to amphibian bombesin than to GRP or NMB. If this is the case, this suggests that one or more mammalian bombesin-like peptides closely related to the frog skin bombesins remain to be characterized. This would be similar to the characterization of the urocortins [6], the mammalian homologs of amphibian sauvagine. Given that frog species tend to have several forms of bombesin, this would suggest that the true mammalian bombesin, like the urocortins, will exist in multiple forms. Another possibility is that frogs also have a BRS-3 receptor, in which case frogs would also have a BRS-3 ligand be

ranatensin phyllolitorin bombesin (X. laevis) bombesin (B. orientalis) NMB (human) NMB (rat) GRP (human) GRP (B. orientalis) 400

300

200

100

0

FIGURE 3. Phylogenetic tree derived from multiple comparison of the known bombesinlike prohormones using MegAlign by DNAstar.

Amphibian Bombesin-like Peptides / 281 distinct from the skin bombesin-like peptides. This would be consistent with the fact that none of the known amphibian bombesin-like peptides have high affinity for the mammalian BRS-3 receptor. Another question is whether frogs have a neuromedin-B-like peptide distinct from the skin bombesin-like peptides in that an amphibian homolog of neuromedin B has yet to be definitively characterized—though Xenopus have a peptide in brain that resembles neuromedin B but is phylogenetically more closely related to Bombina orientalis bombesin [20]. However, since frogs have Phe-form bombesin-like peptides present in brain (Phe-13 bombesin, Phe-8 phyllolitorin), it is possible that these peptides take the place of neuromedin B in amphibians.

References [1] Anastasi A, Erspamer V, Bucci M. Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphibians Bombina and Alytes. Experientia 1971;27:166–67. [2] Chen T, Farragher S, Bjourson AJ, Orr DF, Rao P, Shaw C. Granular gland transcriptomes in stimulated amphibian skin secretions. Biochem J 2003;371:125–30. [3] Clineschmidt BV, Geller RG, Govier WC, Pisano JJ, Tanimura T. Effects of ranatensin, a polypeptide from frog skin on isolated smooth muscle. Br J Pharmacol 1971;41:622–28. [4] Dockray GJ, Hopkins CR. Caerulein secretion by dermal glands. J Cell Biol 1975;64:724–33. [5] Kim JB, Johansson A, Holmgren S, Conlon JM. Gastrin-releasing peptides from Xenopus laevis: purification, characterization, and myotropic activity. Am J Physiol Regul Integr Comp Physiol 2001;281:R902–8. [6] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, Bilezikjian L, Rivier J, Sawchenko PE, Vale WW. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001;98:7570–5. [7] Li K, Nagalla SR, Spindel ER. A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth. J Clin Invest 1994;94:1605–15. [8] Liu Z, Song W, Dong K. Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. Proc Natl Acad Sci USA 2004;101:11862–7.

[9] Masui A, Kato N, Itoshima T, Tsunashima K, Nakajima T, Yanaihara N. Scratching behavior induced by bombesin-related peptides. Comparison of bombesin, gastrin-releasing peptide and phyllolitorins. Eur J Pharmacol 1993;238:297–301. [10] McDonald TJ, Ghatei MA, Bloom SR, Adrian TE, Mochizuki T, Yanaihara C, Yanaihara N. Dose-response comparisons of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide. Regul Pept 1983;5:125–37. [11] McDonald TJ, Jornvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 1979;90:227–33. [12] Mukai H, Kawai K, Suzuki Y, Yamashita K, Munekata E. Stimulation of dog gastropancreatic hormone release by neuromedin B and its analogues. Am J Physiol 1987;252:E765–61. [13] Nagalla SR, Barry BJ, Creswick KC, Eden P, Taylor JT, Spindel ER. Cloning of a receptor for amphibian [Phe13] bombesin distinct from the receptor for gastrin-releasing peptide: Identification of a fourth bombesin receptor subtype (BB4). Proc Natl Acad Sci USA 1995;92:6205–9. [14] Nagalla SR, Barry BJ, Falick AM, Gibson BW, Taylor JE, Dong JZ, Spindel ER. There are three distinct forms of bombesin: Identification of [Leu13] bombesin, [Phe13] bombesin and [Ser3, Arg10, Phe13] bombesin in the frog Bombina orientalis. J Biol Chem 1996;271:7731–7. [15] Nagalla SR, Barry BJ, Spindel ER. Cloning of cDNAs encoding the amphibian bombesin-like peptides Phe8 and Leu8 phyllolitorin from Phyllomedusa sauvagei: Potential role of U to C RNA editing in generating neuropeptide diversity. Mol Endocrinol 1994;8:943–51. [16] Nagalla SR, Gibson BW, Tang D, Reeve JR, Jr., Spindel ER. Gastrin-releasing peptide (GRP) is not mammalian bombesin: Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis. J Biol Chem 1992;267:6916–22. [17] Nakajima T, Tanimura T, Pisano JJ. Isolation and structure of a new vasoactive polypeptide. Fed Proced 1970;29:282. [18] Negri L, Improta G, Broccardo M, Melchiorri P. Phyllolitorins: a new family of bombesin-like peptides. Ann NY Acad Sci 1988;547:415–28. [19] Tache Y, Brown M. On the role of bombesin in homeostasis. Trends Neurosci 1982;5:431–3. [20] Wechselberger C, Kreil G, Richter K. Isolation and sequence of a cDNA encoding the precursor of a bombesin-like peptide from brain and early embryos of Xenopus laevis. Proc Natl Acad Sci USA 1992;89:9819–22. [21] Yasuhara T, Nakajima T, Nokihara K, Yanaihara C, Yanaihara N, Erspamer V, Erspamer GF. Two new frog skin peptides, phyllolitorins, of the bombesin-ranatensin family from Phyllomedusa sauvagei. Biomed Res 1983;4:407–12.

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43 Host Defense Peptides from Australian Amphibians: Caerulein and Other Neuropeptides JOHN H. BOWIE AND MICHAEL J. TYLER

ration of the components of one secretion from one animal. The sequence of each peptide component is determined using a combination of mass spectrometry and Edman sequencing [6]. The amino acid sequences of the bioactive peptides produced by the parotoid and rostral glands of the Magnificent Tree Frog (Litoria splendida) are listed in Table 1 as an illustration. These peptides include caerulein (a neuropeptide; the major topic of this report), caerin 1.1 (a broad spectrum antimicrobial peptide), caerin 2.1 [inhibits the production of nitric oxide from neuronal nitric oxide synthase (nNOS)], caerin 4.1 (narrow spectrum antibiotic, active against gram negative Escherichia coli), and splendipherin (the aquatic male sex pheromone of L. splendida) [48]. Amphibian host-defense peptides often have multifaceted activity. As an illustration, the membrane active peptide caerin 1.1 is active against most gram-positive and some gram-negative organisms (MIC 1–100 μg/ mL), shows IC50 in the 10−6 M range against all the major human cancer types, is active against some viruses (e.g., HIV, MIC 20 μg/mL), is a fungicide (e.g., against the chytrid fungus Batrachochytrium dendrobates, IC50, 10−6 M), kills nematodes (at concentrations of 10−6 M), and inhibits the formation of NO from nNOS (IC50 37 μM). Caerin 1.1 lyses red blood cells at concentrations greater than 250 μg/mL, a concentration higher than those just noted. Another amphibian peptide that shows multifaceted activity is the neuropeptide caerulein.

ABSTRACT Amphibians generally have at least one potent neuropeptide in their dorsal skin secretions. Caerulein [pEQDY(SO3)TGWMDF-NH2] is common in a number of frog genera and has multifaceted activity at nanomolar concentrations. It causes smooth muscle contraction, reduces blood pressure, has gastrin-like activity, and modifies satiety, sedation, and thermoregulation. A number of modified caeruleins occur in Australian frogs of the genus Litoria. Uperoleia species of toadlets produce a number of tachykinin- and bombesin-type neuropeptides, including uperolein [pEPDPNAFYGLMNH2], while froglets of the genus Crinia produce a variety of disulfide-containing neuropeptides that cause smooth muscle contraction at nanomolar concentrations—for example, signiferin 1 [RLCIPYIIPCOH].

INTRODUCTION Amphibians have rich chemical arsenals that form an integral part of their defense systems and also assist with the regulation of dermal physiological action. In response to a variety of stimuli, host defense compounds including peptides are secreted from specialized glands onto the dorsal surface and into the gut. The peptides exhibit a range of bioactivities, including antimicrobial and smooth muscle activity (e.g., see [7, 9, 20, 24]). We have isolated and identified peptides from the secretions of skin glands of some 35 species of Australian frogs and toads of Litoria, Uperoleia, Limnodynastes, Cyclorana, and Crinia [6]. Skin secretions are obtained by electrical stimulation of the glands on the dorsal skin [44]. This process may be repeated at monthly intervals and does not harm the animals. Normally, the major bioactive peptides may be separated using HPLC sepaHandbook of Biologically Active Peptides

THE CAERULEIN PEPTIDES Caerulein was first isolated from dried skins of the Australian Green Tree Frog (Litoria caerulea) by Erspamer and colleagues in 1968 [1] and subsequently from the African Clawed Frog (Xenopus laevis) and Leptodac-

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284 / Chapter 43 TABLE 1.

Host-defense skin peptides of Litoria splendida [48].

Peptide Caerulein* Caerin 1.1* Caerin 1.6 Caerin 1.10 Caerin 2.1 Splendipherin Caerin 3.1 Caeridin 1.1

Amino Acid Sequence

Activity

pEQDY(SO3)TGWMDF-NH2 GLLSVLGSVAKHVLPHVVOVIAEHL-NH2 GLFSVLGAVAKHVLPHVVPVIAEKL-NH2 GLLSVLGSVAKHVLPHVVOVIAEKL-NH2 GLVSSIGRALGGLLADVVKSKGQPA-OH GLVSSIGKALGGLLADVVKSKGQPA-OH GLWQKIKDKASELVSGIVEGLK-NH2 GLLDGLLGTGL-NH2

Neuropeptide Antibiotic Antibiotic Antibiotic nNOS active Sex pheromone Antibiotic Inactive

*Caerulein and caerin 1.1 are the major peptides in the secretion. TABLE 2.

The caerulein family of neuropeptides and some mammalian analogues.

Name

Sequence

MW

Species various Litoria Xenopus laevis Lept. labyrinthicus Phyll. sauvagei Kassina maculata Nyctimystes disrupta Rana erythraea Litoria splendida L. citropa; L. subglandulosa L.c; L.sub L.c; L.sub L.c; L.sub L.c; L.sub L.c; L.sub L.c; L.sub

Caerulein

pEQDY(SO3)TGWMDF-NH2

1351

Phyllocaerulein [Asn2,Leu5] caer. [Glu(OMe)2] caer. [Leu3] Caerulein (3–10) Caerulein 1.2

pEEY(SO3)TGWMDF-NH2 pENDY(SO3)LGWMDF-NH2 pEE(OMe)DY(SO3)TGWMDF-NH2 DY(SO3)LGWMDF-NH2 pEQDY(SO3)TGWFDF-NH2

1352 1352 1366 1124 1367

Caerulein Caerulein Caerulein Caerulein Caerulein Caerulein

pEQDY(SO3)TGAHMDF-NH2 pEQDY(SO3)TGAHFDF-NH2 pEQDY(SO3)GTGWMDF-NH2 pEQDY(SO3)GTGWFDF-NH2 pEQDY(SO3)TGSHMDF-NH2 pEQDY(SO3)TGSHFDF-NH2

1373 1389 1408 1424 1389 1405

2.1 2.2 3.1 3.2 4.1 4.2

Mammalian analogues Cholecystokinin 8 Hexagastrin Eugenin

of caerulein DY(SO3)MGWMDF-NH2 Y(SO3)GWMDF-NH2 pEQDY(SO3)VFMHPF-NH2

tylus labrinthicus [3]. More recently, caerulein has been isolated from live animals of the genus Litoria (using the electrical stimulation method [44]) as follows: L. splendida [43], L. gilleni [50], L. xanthomera [42], L. genimaculata [38], L. citropa [47], L. aurea and L. raniformis [10], L. dahlii [51], L. lesueuri [17], L. subglandulosa [13], L. rothii [10], and L. gracilenta [25]. Caerulein is stored in the glands as inactive procaerulein, which has been sequenced using mRNA and cDNA cloning methods [36, 37, 49]. The procaerulein is converted enzymically to the active caerulein at the time of secretion. Erspamer’s early neuropeptide survey also led to the isolation of phyllocaerulein from the South American hylid frog Phyllomedusa sauvagei [2], Asn2 Leu5 caerulein from the South African hyperoliid frog Hylambates maculata [29], [Glu(OMe)2] caerulein from Nyctimystes disrupta [20, p. 214], and Leu3 caerulein 3–10 from the

1142 896 1372

Philippine ranid frog Rana erythraea [52]. It was later shown that Litoria splendida contains caerulein together with the caerulein analogue caerulein 1.2 [48], while L. citropa [47] and L. subglandulosa [13] produce the caeruleins 1.2 to 4.2 (see Table 2). Caerulein is a primary host-defense peptide, and it also regulates dermal physiological action in the frog. It exhibits a spectrum of activity similar to those of the mammalian intestinal hormones gastrin and cholechystokinin. Caerulein is similar in sequence to the mammalian neuropeptides cholecystokinin 8 (CCK-8) [16] and hexagastrin [26], and the immunomodulator eugenin from the Tammar wallaby Macropus eugenii [8]. The sequences of these mammalian peptides are listed in Table 2. CCK-8 and caerulein have similar spectra of physiological activities. There is an extensive literature concerning the pharmacology of these peptides: For a

Host Defense Peptides from Australian Amphibians: Caerulein and Other Neuropeptides / 285 detailed record, see the comprehensive and definitive review of Erspamer [20]. Both CCK-8 and caerulein contain a tyrosine sulfate residue; the bioactivity is significantly diminished if the sulfate group is hydrolysed. Caerulein induces potent smooth muscle contraction at better than the nanomolar concentration; has gastrinlike activity; modifies satiety, sedation, and thermoregulation; and reduces blood pressure at less than an ng per kg body weight. Caerulein is an analgesic 100 to 7000 times more potent than morphine: The particular figure depends on which biological system is being tested. Caerulein has been used clinically prior to gall bladder surgery [20]. CCK-8 and caerulein bind to CCK receptors [45]. There are two types of CCK receptor: CCK1 and CCK2— differing in anatomical locations and actions [15]. CCK1 receptors are present on smooth muscle and, when activated, contract smooth muscle directly. In contrast, CCK2 receptors act indirectly, causing release of acetylcholine from cholinergic nerves on the myenteric plexus; the acetylcholine then activates muscarinic receptors on smooth muscle. The sequences of the CCK receptors are known [33], and models of their 3D structures have been reported [33, 34]. Both NMR and other experimental data have been used to determine where the partial helix CCK-8 binds to these receptors [21, 22, 33, 34], but similar data are not available for caerulein. Caerulein and CCK-8 are able to act via both CCK1 and CCK2 receptors. During a 3-year survey of the peptide components of the glandular secretion of Litoria splendida, it was shown that the neuropeptide components of the secretions change seasonally, whereas the ratio of antimicrobial peptides was essentially unchanged over the period of the study [48]. Caerulein is the only neuropeptide present in the reproductive season of the frog [December to March (southern hemisphere summer)]. During the inactive winter season, some 50% of caerulein is converted into its less active desulfated analog, and a new peptide, caerin 1.2 (see Table 2), is produced. The difference between caerulein and caerulein 1.2 is that Met8 (in caerulein) is replaced by Phe8 (in caerulein 1.2). The relative ratios of caerulein, desulfated caerulein, and caerulein 1.2 are 1 : 1 : 2 in midwinter. Desulfated caerulein is not an artefact of the isolation and purification processes. Litoria splendida must have an enzyme that is responsible for this hydrolysis. We do not know why the neuropeptide content of the glandular secretion is seasonally variable. However, it is known that caerulein may act at both CCK1 and CCK2 receptors, whereas desulfated caerulein and caerulein 1.2 act only indirectly through the CCK2 receptor—that is, through the central nervous system [20, 31]. Desulfated caerulein is still active but generally at much lower concentrations than caerulein [often activity is reduced

from the nanomolar to micromolar (or below) concentrations]. Caerulein 1.2 and caerulein show almost identical smooth muscle activity. Similar but more complex scenarios are observed for the two closely related species Litoria citropa [47] and Litoria subglandulosa [13]. Both species produce caerulein, caerulein 2.1, 3.1, and 4.1 (all containing Met8) in the reproductive season (see Table 2 for sequences). During the inactive season, caerulein, together with the caeruleins 2.1, 3.1, and 4.1, are partially hydrolyzed to the desulfated forms, and caeruleins 1.2, 2.2, 3.2, and 4.2 (all containing Phe8) are formed. The caeruleins 2, 3, and 4 are all likely to be potent smooth muscle active peptides, but as yet, they have not been tested for bioactivity. It is not clear why these animals produce such a complex range of caerulein neuropeptides.

PEPTIDES RELATED TO CAERULEIN There have been a number of peptides of the tachykinin and bombesin families of neuropeptides isolated from Australian anurans. These are listed in Table 3. Among the tachykinin family are uperolein [4], uperin 1 [11], and the related peptides PGK1-111 [39]. The bombesin family of peptides is represented by litorin [18], [Glu(OMe)2] litorin [5], [Glu (OEt)2] litorin [30], and PC litorin [39]. The physiological activities of the tachykinin and bombesin peptides have been studied extensively and have been comprehensively reviewed [20]. The tachykinins are potent stimulants of gastric secretion. They also stimulate smooth muscle and reduce blood pressure. Their potent vasodilator activity is a result of their interaction with the endothelium that in turn releases factors that reduce the tone of arterial smooth muscle [20]. While Australian Litoria generally produce caeruleins as their primary neuropeptide defense agents, Australian toadlets of the genus Uperoleia produce uperolein-type neuropeptides. An example is uperin 1, the major neuropeptide defense agent of Uperoleia inundata, which shows similar activity to uperolein [4]. Uperin 1 shows smooth muscle activity (guinea pig ileum at 0.4 ng/mL) and reduces rabbit blood pressure at 5 ng/kg body weight [11]. Bombesins, like the tachykinins, show potent pharmacological action in the peripheral and central nervous systems. They stimulate gastric acid secretion and are smooth muscle active at nanomolar concentrations. It is interesting that tachykinin and bombesin peptides have mammalian analogues. For example, litorin has analogs in mammalian tissue, including the gastric releasing peptide GRP 18-27 [18] and neuromedin B [27, 28] (for sequences, see Table 3).

286 / Chapter 43 TABLE 3.

Other pyroglutamate containing neuropeptides from Australian amphibians.

Tachykinins Uperolein Uperin 1 PGK1 PGK11 PGK111

pEPDPNAFYGLM-NH2 pEADPNAFYGLM-NH2 pEPHPDEFVGLM-NH2 pEPNPDEFVGLM-NH2 pEPHPNEFVGLM-NH2

1232 1208 1250 1227 1247

Uperoleia rugosa Uperoleia inundata Pseudophyrne güntheri Pseudophyrne güntheri Pseudophyrne güntheri

Bombesins Litorin* Glu(OMe)2 litorin* Glu(OEt)2 litorin PG litorin

pEQWAVGHM-NH2 pEE(OMe)WAVGHM-NH2 pEE(OEt)WAVGHM-NH2 pEGGGPQWAVGHFM-NH

1084 1100 1115 1352

Litoria aurea Litoria aurea Uperoleia rugosa Pseudophyrne güntheri

Mammalian analogues of litorin GRP 18–27 GNHWAVGHLM-NH2 Neuromedin B GNLWATGHFM-NH2

1119 1131

*Electrical stimulation of the dorsal skin of Litoria aurea gives a glandular secretion that contains caerulein as the major neuropeptide. Litorin is a minor constituent. No Glu(OMe)2 litorin was detected [10].

TRYPTOPHYLLIN PEPTIDES There are two related species of the genus Litoria that have host-defense chemistries quite different from those of any congeners. Most Litoria species have a potent mix of neuropeptides, antibiotic peptides, and peptides that inhibit the operation of the nitric oxide synthase isoforms. The Red Tree Frog Litoria rubella (which is found throughout northern and central Australia) and the Buzzing Tree Frog Litoria electrica (found only in a specific region below the Gulf of Carpentaria in northern Australia) secrete neither neuropeptides analogous to caerulein or uperolein nor antimicrobial peptides (rubella [40, 41]; electrica [46]). How, then, do they protect themselves from predators? These two species secrete large amounts of small peptides that are very similar in sequence to the tryptophyllin peptides isolated by members of the Erspamer group from Phyllomedusa rohdei [19, 23, 30]. The sequences of the tryptophyllins are listed in Table 4. Erspamer has found that neither his tryptophyllins nor selected samples of our tryptophyllins show smooth muscle activity below the micromolar concentration. In addition, we have determined that the tryptophyllins— FPWL-NH2, FPWP-NH2, and pEFPWL-NH2—show no NOS activity. One of Erspamer’s tryptophyllins (FPPWMNH2) induces sedation and behavioral sleep in birds and is immunoreactive to a set of cells in the rat adenohypophysis [35]. Recently, a tryptophyllin-like peptide (LPHypAWVP-NH2) from the Mexican leaf frog Pachymedusa dacnicolor has been shown to contract smooth muscle at the nanomolar concentration [14]. Some tryptophyllins have sequence similarity to the human brain endomorphin YPWF-NH2 [53]. The role of the

TABLE 4. Tryptophyllin peptides from the genera Phyllomedusa [19, 23, 30] and Litoria [40, 41, 46]. pEPWV-NH2 pEAWM-OH pEPWM-NH2 pEPWM-OH pEFPWL-NH2 pEFPWF-NH2 pEIPWFHR-NH2 LPWY-NH2 IPWL-NH2 FPWP-NH2

510 517 542 543 672 706 965 577 527 544

FPWP-OH FPPW-OH PWL-NH2 FPWL-NH2

545 545 414 560

FPPWV-NH2 FPPWL-NH2 FPPWL-OH FPPWM-NH2 FLPWY-NH2 VPPLGWM-OH FPFPWL-NH2 KPHypSWP-NH2 KPPPWVP-OH KPPPWIPV-OH PEEKPYWPPPIYPM-OH PEEKPFYPPPIYPV-OH PEDKPFWPPPIYPV-OH

643 657 658 675 724 798 805 838 918 932 1441 1556 1565

Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Litoria rubella Litoria rubella Litoria rubella Litoria rubella Phyllomedusa sp Litoria rubella Litoria electrica Phyllomedusa sp Litoria rubella Litoria rubella Litoria rubella Litoria rubella Litoria electrica Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Litoria rubella Phyllomedusa sp Litoria rubella Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp Phyllomedusa sp

Host Defense Peptides from Australian Amphibians: Caerulein and Other Neuropeptides / 287 tryptophyllins in Litoria and Phyllomedusa species still remains to be determined.

A O H3N+

O–

HN

DISULFIDE NEUROPEPTIDES FROM THE GENUS CRINIA

NH

1187 1158

B

Acknowledgments We acknowledge ongoing funding from the Australian Research Council for our research on bioactive peptides and permits from State and Territory authorities to collect frogs. This article is dedicated to the many graduate students, postdoctoral fellows, and senior colleagues who have worked on the projects

NH NH2 NH +NH

Crinia signifera Crinia riparia

We are currently working on a series of these peptides: the sequences of two representative examples are shown above. There are peptides isolated from European Rana species that have disulfide bridges similar to those of the Crinia peptides—for example, brevinin 1E [(FLPLLAGLAANFLPKIFCKITRKC-OH] (see review [7]). These peptides differ from the Crinia disulfides in that the N-terminal side chains contain from 15 to 30 residues. Brevinins and related peptides show potent antimicrobial activity. In complete contrast, the Crinia disulfide-containing peptides show neither antimicrobial activity nor do they cause inhibition of NOS enzymes. The secondary structures of the two peptides have been determined by 2D NMR studies; these are shown in Fig. 1 for comparison. The peptide from signifera contracts guinea pig ileum at a nanomolar concentration acting indirectly on smooth muscle via CCK2 receptors. Surprisingly, the peptide from riparia does not contract smooth muscle below 10−6 M. Thus, it seems unlikely that smooth muscle activity is the primary function of these peptides on amphibian skin. In conclusion, Australian anurans produce some of the most potent neuropeptides yet identified in the amphibian integument, including the caeruleins (from Litoria) and the uperoleins (from Uperoleia). There are outstanding questions still to be answered including the roles of the tryptophyllins and the Crinia disulfides.

S

HO

Crinia species follow the general trend of other frogs as far as the type of host defense peptides produced in their glandular skin secretion, in that they contain neuropeptides, antimicrobial peptides, and at least one peptide that inhibits the operation of the NOS isoforms. However, their neuropeptides are unique among Australian frogs in that they contain a disulfide link [31]. RLCIPYIIPC-OH RLCIPVIFPC-OH

S

NH2

3

O– O S

S

FIGURE 1. A. Disulfide neuropeptide from Crinia signifera. B. Disulfide neuropeptide from Crinia riparia.

involving Australian animals. Their names appear in the reference section.

References [1] Anastasi A, Erspamer V, Endean R. Isolation and amino acid sequence of caerulein, the active peptide of Hyla caerulea. Arch Biochem Biophys 1968;125:57–68. [2] Anastasi A, Bertaccini G, Cei JM, DeCaro G, Erspamer V, Impicciatore M. Structure and pharmacological actions of phyllocaerulein, a caerulein like nonapeptide: its occurrence in the extracts of the skin of Phyllomedusa sauvagii and related Phyllomedusa species. Br J Pharmacol 1969;37:198–206. [3] Anastasi A, Bertaccini G, Cei JM, DeCaro G, Erspamer V, Impicciatore M. Presence of caerulein in extracts of the skin of Leptodactylus labyrinthicus labyrinthicus and Xenopus laevis. Br J Pharmacol 1970;38:221–8. [4] Anastasi A, Erspamer V, Endean R. Structure of uperolein a physalaemin-like endecapeptide occurring in the skin of Uperoleia rugosa and Uperoleia marmorata. Experientia 1975;31:394– 7. [5] Anastasi A, Erspamer V, Montecucchi P, Angelucci F, Endean R. Glu(OMe)2-litorin the second bombesin-like peptide occurring

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in methanol extracts of the skin of the Australian frog Litoria aurea. Experientia 1977;33:1289–93. Apponyi MA, Pukala TL, Brinkworth CS, Maselli VM, Bowie JH, Tyler MJ, Booker GW, Wallace JC, Carver JA, Separovic F, Doyle JR, Llewellyn LE. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 2004;25:1035–54. Barra D, Simmaco M. Amphibian skin: a promising resource for antimicrobial peptides. TIBTECH 1995;13:205–7. Baudinette RV, Boontheung P, Musgrave IF, Wabnitz PA, Maselli VM, Skinner J, Alewood PF, Brinkworth CS, Bowie JH. Eugenin. An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii. FEBS J 2005;272:433–43. Bevins CL, Zasloff M. Peptides from frog skin. Annu Rev Biochem 1990;59:395–414. Bowie JH, Tyler MJ; unpublished observations. Bradford AM, Raftery MJ, Bowie JH, Tyler MJ, Wallace JC, Adams GW, Severini C. Novel uperin peptides from the dorsal glands of the Australian flood plain toadlet Uperoleia inundata. Aust J Chem 1996;49:475–84. Brinkworth CS, Bowie JH, Tyler MJ. Unpublished observations. Brinkworth CS, Pukala TL, Bowie JH, Doyle JR, Tyler MJ, Llewellyn LL, Wallace JC. Host defence peptides from the skin glands of Australian amphibians. Caerulein neuropeptides and antimicrobial, anticancer and nNOS inhibiting citropins from the Glandular Frog Litoria subglandulosa. Aust J Chem 2002; 55:605–10. Chen T, Orr DF, O’Rourke M, McLynn C, Bjourson AJ, McClean S, Hirst D, Rao P, Shaw C. Pachymedusa dacnicolor tryptophyllin-1. Structural characterisation, pharmacological activity and cloning of cDNA. Reg Peptides 2004;117:25–32. Cuq P, Gross A, Terraza A, Fourmy D, Clerk P, Dornand J, Magous R. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurgat lymphoblastoid cells. Life Sci 1997;61:543–55. Dockray GJ. Immunological evidence of CCK-like peptides in brain. Nature 1976;264:568–70. Doyle JR, Llewellyn LE, Brinkworth CS, Bowie JH, Wegener KL, Rozek T, Wabnitz PA, Wallace JC, Tyler MJ. Amphibian peptides that inhibit nNOS: the isolation of lesueurin from the skin secretion of the Australian Stony Creek frog Litoria lesueuri, Eur J Biochem 2002;269:100–9. Erspamer V, Negri L, Falconieri Erspamer G, Endean R. Uperolein and other active peptides in the skin of the Australian leptodactylic frogs Uperoleia and Taudactylus. NaunynSchniedeberg’s Arch Pharmacol 1975;289:42–54. Erspamer V, Melchiorri P, Falconeiri Erpamer G, Montecucchi PC, de Castiglione R. Phyllomedusa skin: a huge factory and store house of a variety of active peptides. Peptides 1985; 6:7–12. Erspamer V. Bioactive secretions of the amphibian integument. In: Heatwole H, Barthalmus GT, editors. Amphibian biology: the integument. Vol. 1, Norton-Chipping, NSW; Surrey Beatty and Sons; 1994. pp. 178–350. Giragossian C, Mierke DF. Intermolecular interactions of CCK8 and the third extracellular loop of CCKA receptor. Biochemistry 2001;40:3804–9. Giragossian C, Mierke DF. Intramolecular interactions between CCK-8 and the third extracellular loop of CCKB. Biochemistry 2002;41:4560–6. Gozzini L, Montecucchi PC, Erspamer V, Melchiorri P. Tryptophyllins from extracts of Phyllomedusa rhodei skin: new tetra-, penta-, and hexapeptides. Int J Peptide Protein Res 1985;25:323– 9. Lazarus LH, Attila M. The toad, ugly and venomous wears yet a precious jewel in his skin. Prog Neurobiol 1993;41:473–507.

[25] Maclean MJ, Brinkworth CS, Bilusich D, Bowie JH, Doyle JR, Llewellyn LE, Tyler MJ. New caerin antimicrobial peptides from the skin glands of the Dainty Green Tree Frog Litoria gracilenta. Sequence determination using positive and negative ion electrospray mass spectrometry. Toxicon 2006; in press. [26] Matsumoto M, Park J, Sugano K, Yamada T. Biological activity of progastrin post-translational processing intermediates. Am J Physiol 1987;252:G315–19. [27] Minamino N, Kangawa K, Fukuda A, Matsuo H. A novel bombesin-like peptide identifies in porcine spinal cord. Biochem Biophys Res Commun 1983;114:541–8. [28] Minamino N, Sudoh T, Kangawa K, Matsuo H. Neuromedins: novel smooth muscle stimulant peptides identified in porcine spinal cord. Peptides 1985;6(Suppl. 3):245–8. [29] Montecucchi PC, Falconieri Erspamer G, Visser J. Occurrence of Asn2 Leu5 caerulein in the skin of the African frog Hylambates maculatus. Experientia 1977;33:1138–9. [30] Montecucchi PC, Gozzini L, Erspamer V, Melchiorri P. The primary structure of tryptophan containing peptides from skin extracts of Phyllomedusa rhodei. Int J Peptide Protein Res 1984;24:276–85. [31] Musgrave IF, Maselli VM, Pukala TL, Bowie JH. Unpublished observations. [32] Nakajima P, Yasuhara T, Erspamer V, Falconieri Erspamer G, Negri L. Physalaemin- and bombesin-like peptides in the skin of the Australian leptodactylic frog Uperoleia rugosa. Chem Pharm Bull (Japan) 1980;28:689–95. [33] Noble F, Wank SA, Crawley JN, Bradwejn J, Seroogy KB, Hamon M, Roques BJ. Structure distribution and functions of cholecystokinin receptors. Pharmacol Rev 1999;51:745–81. [34] Pellegrini M, Mierke DF. The complex of CCK-8 and the Nterminus of CCKA receptor by NMR spectroscopy. Biochemistry 1999;38:14775–83. [35] Renda S, D’Este L, Buffa R, Usellini L, Capella C, Vaccaro R. Tryptophyllin-like immunoreactivity in rat adenohypophysis. Peptides 1985;6:197–202. [36] Richter K, Aschauer H, Kreil G. Biosynthesis of peptides from the skin of Xenopus laevis. Isolation of novel peptides from the skin of Xenopus laevis. Isolation of novel peptides predicted from the sequence of cloned cDNAs, Peptides 1985;6(Suppl 3):17–21. [37] Richter K, Egger R, Kreil G. Sequence of preprocaerulein from cDNAs cloned from the skin of Xenopus laevis: a small family of precursors containing one, three and four copied of the final product. J Biol Chem 1986;261:3676–80. [38] Rozek T, Waugh RJ, Steinborner ST, Bowie JH, Tyler MJ, Wallace JC. The maculatin peptides from the skin glands of the tree frog Litoria genimaculata—a comparison of the antibacterial activities of maculatin 1.1 and caerin 1.1. J Peptide Sci 1998;4:111–15. [39] Simmaco M, Severini C, DeBiase D, Barra D, Bossa F, Roberts J, Melchiorri P, Erspamer V. Six novel tachykinin- and bombesinrelated peptides from the skin of the Australian frog Pseudophryne güntheri. Peptides 1990;11:299–304. [40] Steinborner ST, Gao CW, Raftery MJ, Waugh RJ, Blumenthal T, Bowie JH, Wallace JC, Tyler MJ. The structure of four tryptophyllin and three rubellidin peptides from the Australian Red Tree Frog Litoria rubella. Aust J Chem 1994;47:2099–108. [41] Steinborner ST, Wabnitz PA, Waugh RJ, Bowie JH, Gao CW, Tyler MJ, Wallace JC. The structures of new peptides from the Australian Red Tree Frog Litoria rubella—the skin peptide profile as a probe for the evolutionary trends in amphibians. Aust J Chem 1996;49:955–63. [42] Steinborner ST, Waugh RJ, Bowie JH, Wallace JC, Tyler MJ, Ramsey SL. New caerin peptides from the skin glands of the Australian tree frog Litoria xanthomera. J Peptide Sci 1997;3: 181–5.

Host Defense Peptides from Australian Amphibians: Caerulein and Other Neuropeptides / 289 [43] Stone DJM, Waugh RJ, Bowie JH, Wallace JC, Tyler MJ. Peptides from Australian frogs. Structures of the caerins and caeridin 1 from Litoria splendida. J Chem Soc Perkin Trans 1 1992;3173–8. [44] Tyler MJ, Stone DJM, Bowie JH. A novel method for the release and collection of dermal, glandular secretions from the skin of frogs. J Pharm Toxicol Methods 1992;28:199–200. [45] Varga G, Balint A, Burghardt B, D’Amato M. Involvement of endhenous CCK and CCK1 receptors in colonic motor function. Br J Pharmacol 2004;141:1275–84. [46] Wabnitz PA, Bowie JH, Wallace JC, Tyler MJ. Peptides from the skin glands of the Buzzing Tree Frog Litoria electrica. Comparison with the skin peptides of the Red Tree Frog Litoria rubella. Aust J Chem 1999;52:639–45. [47] Wabnitz PA, Bowie JH, Tyler MJ. Caerulein like peptides from the skin glands of the Australian Blue Mountains Tree Frog Litoria citropa. 1. Sequence determination using electrospray mass spectrometry. Rapid Commun Mass Spectrom 1999;13:2498–502. [48] Wabnitz PA, Bowie JH, Tyler MJ, Wallace JC, Smith BP. Differences in skin peptides of the male and female Australian Magnificent Tree Frog Litoria splendida—a three-year monitoring

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survey: the discovery of the aquatic male sex pheromone splendipherin, together with Phe8 caerulein and the antibiotic caerin 1.10. Eur J Biochem 2000;267:269–75. Wakabayashi T, Kato H, Tachibana S. An unusual repetitive structure of caerulein mRNA in the skin of Xenopus laevis. Gene 1984;31:255–9. Waugh RJ, Stone DJM, Bowie JH, Wallace JC, Tyler MJ. Peptides from Australian frogs. The structures of the caerins and caeridins from Litoria gilleni. J Chem Res (S) (M) 1993;139:937–61. Wegener KL, Brinkworth CS, Bowie JH, Wallace JC, Tyler MJ. Bioactive dahlein peptides from the skin secretions of the Australian aquatic frog Litoria dahlii: sequence determination by electrospray mass spectrometry. Rapid Commun Mass Spectrom 2001;15:1726–34. Yasuhara T, Nakajimi T, Erspamer V, Falconieri Erspamer G, Tukamoto GY, More M. Isolation and sequential analysis of peptides in Rana erythraea. Peptide Chemistry 1985. Kis Y ed. Protein Research Foundation, Osaka, 1986; pp. 363–8. Zadina JE, Hackler L, Ge L, Kastin AJ. A potent and selective endogenous agonist for the m-opiate receptor. Nature 1997; 386:499–502.

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44 Bradykinin-Related Peptides from Frog Skin J. MICHAEL CONLON

sequence was identical to that of mammalian BK [2]. The occurrence of BK and related peptides among the different families of frogs, and even among species belonging to the same genus, is sporadic. For example, the skins of the European frog R. temporaria and the African frog R. galamensis contain BK equivalent to >150 μg/g tissue, but BK was undetectable in the skins of R. dalmatina, R. graeca, and R. latastei [19, 20]. Similarly, among the North American ranids, the skin secretions R. muscosa contain high concentrations of BK, whereas the peptide is undetectable in secretions from those of its sister taxa R. aurora and R. draytoni [18]. As well as from frogs belonging to the family Ranidae, BK and/or BK-related peptides have been isolated from species belonging to the families Ascaphidae [9], Bombinatatoridae [4–6, 12–14], Hylidae [1], and Leptodactylidae [17]. The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Consequently, the identification of BK in skin secretions from this species demonstrates that dermal BK production arose early in anuran evolution.

ABSTRACT The skins of a wide range of frogs, particularly those belonging to the families Ascaphidae, Bombinatatoridae, and Ranidae, synthesize bradykinin (BK)-related peptides. BK identical in structure to the mammalian peptide, larger molecular forms of BK extended from either the N- or C-terminus by additional amino acids, and molecular variants of BK containing amino acid substitutions, have been identified. The frog skin BK peptides are not generated by activation of the kallikrein-kinin system as in mammals but by the action of processing enzymes cleaving at the site of single arginyl residues in precursors that generally contain multiple tandem copies of a BK-containing domain. The frog skin BK-related peptides show a wide range of potencies in eliciting contractile or relaxant responses in mammalian smooth muscle preparations, and peptides derived from probradykinins with weak antagonistic and BK-potentiating activities have also been identified.

DISCOVERY AND DISTRIBUTION The skins of amphibians have proved to be a remarkably rich storehouse of myotropic peptides, including kinins. A BK-like ability to elicit a hypotensive response and to contract rat uterine and gastrointestinal smooth muscle has been demonstrated in extracts of the skins of a wide range of frogs from Africa [19], North and South America [10], Australia and Papua New Guinea [11], and Europe [20] by Erspamer and coworkers. In particular, the skins of certain frogs from the genus Rana are associated with very high concentrations of such BK-like bioactivity, and it was shown that the skin of the European common frog Rana temporaria contained a peptide whose amino acid Handbook of Biologically Active Peptides

BIOSYNTHESIS OF BRADYKININ AND RELATED PEPTIDES The pathways of biosynthesis of BK-related peptides in frog skin are quite different from those in the blood and tissues of mammals. The kallikrein-kinin system in mammals involves the sequential action of several wellcharacterized proteolytic enzymes and is illustrated schematically in Fig. 1 (see also D. J. Campbell and J. M. Stewart, this volume). Activation of Factor XII (Hageman factor) in blood results in the activation of plasma prekallikrein and subsequent generation of BK

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292 / Chapter 44

Factor XII

Glandular prokallikrein

Factor XIIa

Plasma prekallikrein

Plasma kallikrein

High Mrkininogen

Glandular kallikrein

[Lys/ Arg0]bradykinin

Low Mrkininogen

Bradykinin

Bradykinin

Des[Arg9]bradykinin

Des[Arg9]bradykinin

Blood

Tissue

FIGURE 1. Generation of bradykinin in the plasma and tissues of mammals by activation of the kallikrein-kinin system.

BK

BK

BK

BK

BK

BK

HV

Repeated domain: LRDLPKINRKGPRPPGFSPFRGKFHSQS

FIGURE 2. A schematic representation of the biosynthetic precursor of bradykinin deduced from the nucleotide sequence of a cloned cDNA from the skin of Bombina maxima. The probradykinin contains six identical repeated copies of the domain shown. The bradykinin sequence is underlined.

by the cleavage of high-molecular mass kininogen. This protein contains only a single copy of the BK sequence. Alternative splicing of the primary transcript of the kininogen gene gives rise to a second mRNA that directs the synthesis of low-molecular mass kininogen. This protein is a substrate for glandular or tissue kallikrein, a serine-protease that is localized predominantly in the kidney, pancreas, and pituitary, that generates either [Lys0]-BK in the human or [Arg0]-BK in the rat. BKrelated peptides have been generated in the plasma of several species of reptile and fish by incubation with porcine pancreatic kallikrein and/or trypsin, but similar attempts to generate BK in the plasma of a range of frog species have been unsuccessful [7]. The primary structure of the biosynthetic precursor of a frog skin BK was first determined for the Chinese red-bellied toad Bombina maxima [12]. As shown schematically in Fig. 2, the probradykinin comprises six copies of the 28-amino-acid-residue repeated domain LRDLPKINRKGPRPPGFSPFRGKFHSQS. Thus, generation of BK (sequence underlined) involves cleavage at the site of single arginyl residues by an as yet uncharacterized processing enzyme rather than by the action of the kallikrein-kinin system as in mammals. As well as BK, posttranslational processing of B. maxima probradykinin generates the N-terminally extended peptide,

bombinakinin M (DLPKINRKGPRPPGFSPFR). Subsequent work has shown that structural organization of probradykinin in the skins of the piebald odorous frog Rana schmakeri [15] and the fire-bellied toad Bombina orientalis [6] is similar to that of the B. maxima precursor, comprising seven and four BK-containing tandem repeats, repectively. More recently, it has been shown that several preprobradykinin genes are expressed in the skin of B. maxima [4, 13, 14], and a cDNA encoding a bombinakinin M variant (DLSKMSFLHGIPPGFSPFR) that does not contain the full sequence of BK has been identified. It has been suggested that environmental factors may influence which particular bombinakinin M gene is expressed [14].

MOLECULAR VARIANTS OF BK The different pathways of posttranslational processing of probradykinin in frog skin give rise to peptides in which the BK sequence is extended from either the Nor the C-terminus by additional amino acid residues. As shown in Fig. 3, a peptide comprising BK extended from its N-terminus by -Gly-Val-Ile-Pro-Leu-Leu and three peptides comprising BK extended from its C-terminus by -Ile-Ala, -Ile-Ala-Pro-Ala-Ser-Thr-Leu, and -Ile-Ala-Pro-

Bradykinin-Related Peptides from Frog Skin / 293 Ascaphus truei Rana temporaria

RPPGFSPFRVD LLPIVGRPPGFSPFR RPPGFSPFRIA RPPGFSPFRIAPASTL RPPGFSPFRIAPASIL

Rana nigromaculata

RPPGFSPFRVAPAS

Phyllomedusa rohdei

RPPGFSPFRIY(SO3H)

Bombina orientalis

RPPGFSPFRGKFH

Bombina maxima

DLPKINRKGPRPPGFSPFR

FIGURE 3. The primary structures of frog skin bradykininrelated peptides that contain the full sequence of bradykinin (residues underlined) extended from either the N- or the Cterminus by additional amino acid residues.

Ascaphus truei

APVPGLSPFR APVPGLSPFRVV

Rana rugosa

RPPGFTPFR RPPGFTPFRIAPEIV

Rana nigromaculata

VPPGFTPFR

Rana palustris

Bombina variegata

IRRPPGFTPLR IRRPPGFTPLRIA AGIRRPPGFTPLRIA AGIRRPPGFTPLRIA RPAGFTPFR VPTGFTPFR

FIGURE 4. Molecular variants of bradykinin isolated from skin extracts or skin secretions of frogs. Those amino acid residues that differ from bradykinin are underlined.

Ala-Ser-Ile-Leu were purified from R. temporaria [8]. Related studies in other species have led to the isolation of BK-related peptides extended from the C-terminus by -Ile-Tyr(SO3) (phyllokinin from Phyllomedusa rohdei) [1], by -Gly-Lys-Phe-His (bombinakinin O from Bombina orientalis) [22], by -Val-Ala-Pro-Ala-Ser (ranakinin N from R. nigromaculata) [16], by -Ile-Ala-Pro-Glu-Ile-Val (ranakinin R from R. rugosa) [23], and by Val-Asp (peptide RD-11 from A. truei) [9]. Metabolites of BK, identified as the (1–7) and (1–8) fragments, have been isolated in high yield from the skins of R. temporaria and R. esculenta and [hydroxyprolyl3]-BK has been purified from R. temporaria and from the South African leptodactylid frog Heleophryne purcelli (reviewed in [7]). In general, the primary structure of BK has been poorly conserved in vertebrates with only two amino acid residues (Gly4 and Arg9) remaining invariant during the evolution of the peptide [7]. As shown in Fig. 4, BK-related peptides whose amino acid sequences are not the same as mammalian BK include [Thr6]-BK from Rana rugosa [23] and Bombina orientalis [21], [Val1,Thr6]-

BK from R. nigromaculata [16], N- and C-terminally extended forms of [Leu8]-BK from R. palustris [3], [Ala3,Thr6]-BK and [Val1,Thr3,Thr6]-BK from B. variegata [5], and [Ala0,Pro1,Val2, Leu5]-BK from Ascaphus truei [9].

BIOLOGICAL ACTIVITIES The myotropic activities of several of the naturally occurring larger molecular forms of BK have been investigated. Phyllokinin [BK-Ile-Tyr(S03H)] was two to three times more potent and longer acting than BK in producing a hypotensive response in the anesthetized dog, but the peptide was less potent than BK in contracting a range of extravascular smooth muscle preparations. Removal of the sulfate group from phyllokinin resulted in an appreciable loss in potency [1]. In contrast, removal of C-terminal extension to the BK sequence in ranakinin N with trypsin results in a 20-fold increase in potency in smooth muscle bioassays [21]. BK-Val-Asp from A. truei was a full agonist at the B2 receptor in mouse trachea, but the BK-related peptide AR-10 (APVPGLSPFR) and its C-terminally extended form AV-12 (APVPGLSPFRVV) functioned as low potency, partial agonists [9]. Bombinakinin M from B. maxima is approximately fourfold less potent than BK in eliciting contraction of the guinea pig ileum. In contrast, the bombinakinin M variant containing the substitution Arg1 → Ile in the BK domain was itself inactive in this preparation but potentiated the action of BK up to twofold [14]. The predicted amino acid sequence of one of the multiple biosynthetic precursors of bombinakinin M contains at its extreme C-terminus the domain Arg-Gln-Ile-ProGly-Leu-Gly-Pro-Arg-Gly. A peptide derived from this domain (pGlu-Ile-Pro-Gly-Leu-Gly-Pro-Arg.NH2) has been termed kinestatin and acts as a weak antagonist at the B2 receptor in the rat tail artery [4]. Similarly, the peptide Asp-Tyr-Thr-Ile-Arg-Thr-Arg-Leu-His.NH2 encoded by a different B. maxima probradykinin gene also weakly inhibited the contractile activity of bombinakinin M at the guinea pig ileum [14]. As well as the bombinakinin M precursor that contains six tandem repeats [12], cDNA cloning has identified a larger precursor that contains eight identical copies of the bombinakinin M sequence plus one copy of a 28-aminoacid-residue peptide, termed bombinakinin M gene associated peptide [13]. This component shows limited structural similarity to mammalian cocaine- and amphetamine-regulated transcript (CART) and intracerebroventricular administration of the peptide induced a significant decrease in food intake in rats. The biological function of myotropic peptides, such as BK, in the skins of amphibia is unclear, but it is

294 / Chapter 44 reasonable to speculate that they may represent an important component of the organism’s survival strategy, protecting the animal against ingestion by predators. Release of large amounts of BK, or a related peptide, into the lumen of the stomach of a predator will stimulate gastric and esophageal motility that may lead to activation of the vomiting reflex and ejection of the prey unharmed.

[12]

[13]

[14]

References [1] Anastasi A, Bertaccini G, Erspamer V. Pharmacological data on phyllokinin (bradykinyl-isoleucyl-tyrosine O-sulphate) and bradykinyl-isoleucyl-tyrosine. Br J Pharmacol 1966;27:479–85. [2] Anastasi A, Erspamer V, Bertaccini G. Occurrence of bradykinin in the skin of Rana temporaria. Comp Biochem Physiol 1965;14: 43–52. [3] Basir YJ, Knoop FC, Dulka J, Conlon JM. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from the skin secretions of the North American pickerel frog, Rana palustris. Biochim Biophys Acta 200;1543:95– 105. [4] Chen T, O’Rourke M, Orr DF, Coulter DJ, Hirst DG, Rao P, et al. Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima. Regul Pept 2003;116:147–54. [5] Chen T, Orr DF, Bjourson AJ, McClean S, O’Rourke M, Hirst DG, et al. Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin. Eur J Biochem 2002;269:4693–700. [6] Chen T, Orr DF, Bjourson AJ, McClean S, O’Rourke M, Hirst DG, et al. Bradykinins and their precursor cDNAs from the skin of the fire-bellied toad (Bombina orientalis). Peptides 2002;23:1547–55. [7] Conlon JM. Bradykinin and its receptors in non-mammalian vertebrates. Regul Pept 1999;79:71–81. [8] Conlon JM, Aronsson U. Multiple bradykinin-related peptides in the skin of the frog, Rana temporaria. Peptides 1997;18:361–5. [9] Conlon JM, Jouenne T, Cosette P, Cosquer D, Vaudry H, Taylor CK, et al. Bradykinin-related peptides and tryptophyllins in the skin secretions of the most primitive extant frog, Ascaphus truei. Gen Comp Endocrinol 2005;143:193–9. [10] Erspamer V, Falconieri Erspamer G, Cei JM. Active peptides in the skins of two hundred and thirty American amphibian species. Comp Biochem Physiol 1986;85C:125–37. [11] Erspamer V, Falconieri Erspamer G, Mazzanti G, Endean R. Active peptides in the skins of one hundred amphibian species

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

from Australia and Papua New Guinea. Comp Biochem Physiol 1984;77C:99–108. Lai R, Liu H, Hui Lee W, Zhang Y. A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product. Biochem Biophys Res Commun 2001;286:259–63. Lai R, Liu H, Lee WH, Zhang Y. Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima. Peptides 2003;24:199–204. Lee WH, Liu SB, Shen JH, Jin Y, Zhang Y. Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide. Regul Pept 2005;127:207–15. Li L, Bjourson AJ, He J, Cai G, Rao P, Shaw C. Bradykinins and their cDNA from piebald odorous frog, Odorrana schmackeri, skin. Peptides 2003;24:863–72. Nakajima T. New vasoactive peptides of nonmammalian origin In: Pisano JJ, Austen KF, editors. The Chemistry and Biology of the Kallikrein-kinin System in Health and Disease. Bethesda: Fogarty International Center Proceedings 1974:165–8. Nakajima T, Yasuhara T, Falconieri Erspamer G, Visser J. Occurrence of Hyp3-bradykinin in methanol extracts of the skin of the South African leptodactylid frog Heleophryne purcelli. Experientia 1979;35:1133–4. Rollins-Smith LA, Woodhams DC, Reinert LK, Vredenburg VT, Buggs CJ, Nielsen PF, Conlon JM. Antimicrobal peptide defenses of the Mountain Yellow-Lagged Frog (Rana muscosa). Dev Comp Immunol 2006; in press. Roseghini R, Falconieri Erspamer G, Severini C. Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than Bufonids. Comp Biochem Physiol 1988;91C:281–86. Roseghini R, Falconieri Erspamer G, Severini C, Simmaco M. Biogenic amines and active peptides in extracts of the skin of thirty-two European amphibian species. Comp Biochem Physiol 1989;94C:455–60. Yanaihara N, Yanaihara C, Sakagami M, Nakajima T, Nakayama T. Synthesis of bradykininyl-Val-Ala-Pro-Ala-Ser-OH and its biological properties. Chem Pharm Bull (Tokyo) 1973;21: 616–21. Yashuhara T, Hira M, Nakajima T, Yanaihara N, Yanaihara C. Active peptides on smooth muscle in the skin of Bombina orientalis Boulenger and characterization of a new bradykinin analog. Chem Pharm Bull (Tokyo) 1973;21:1388–91. Yashuhara T, Ishikawa O, Nakajima T, Araki K, Tachibana S. The studies on the active peptide on smooth muscle in the skin of Rana rugosa, Thr6-bradykinin and its analogous peptide, ranakinin R. Chem Pharm Bull (Tokyo) 1979;27:486–91.

C

H

A

P

T

E

R

45 The Dermaseptins PIERRE NICOLAS AND MOHAMED AMICHE

resemble any members of the other peptide families. To date, these families include (1) the dermaseptins sensu stricto, from Phyllomedusa sauvagei, P. bicolor, P. oreades, P. distincta, Pachymedusa dacnicolor, Agalychnis annae, and A. callidryas (subfamily: Phyllomedusinae), which share a signature pattern consisting of a conserved Trp residue at position 3 and an AG(A)KAAL(V/ G)G(N/K)AV(A) consensus motif in the midregion; (2) the Gly-Leu-rich peptides, from Phyllomedusa sauvagei, P. bicolor, Pachymedusa dacnicolor, Agalychnis annae, and A. callidryas, which are rich in Gly and Leu residues arranged in regular 3-mer motifs GXL (where X is any amino acid residue) that are repeated two to five times; (3) the dermatoxins, from Phyllomedusa sauvagei, P. bicolor, Pachymedusa dacnicolor, and Agalychnis annae; (4) the phylloxins, from Phyllomedusa sauvagei and P. bicolor; (5) the phylloseptins, from Phyllomedusa bicolor, P. oreades, and P. hypochondrialis; (6) the cationic hydrophobic peptides, which have only been described in Phyllomedusa sauvagei; and (7) the orphan peptides, from Pachymedusa dacnicolor and Agalychnis annae, which do not resemble any members of the other peptide families. All these peptide families show such a considerable structural variety that their evolutionary relationships would never have been apparent without the strong conservation of their biosynthetic precursor preproregions. This conserved preproregion is the hallmark of the dermaseptin superfamily. The first member of the dermaseptin superfamily, named dermaseptin S1, was isolated from an extract of dried pieces of skin of Phyllomedusa sauvagei in the early 1990s [21]. This 34-residue peptide has lytic activity against Gram-positive and Gram-negative bacteria, yeast, and protozoa, without harmful effects against mammalian cells [16]. It was the first gene-encoded eukaryotic peptide to show lethal effect against filamentous fungi that are responsible for opportunistic infections that result from the immunodeficiency syndrome

ABSTRACT The dermaseptins are a superfamily of gene-encoded host defense peptides that are produced by the skin of frogs belonging to the family Hylidae. These peptides are genetically related, with a remarkable identity in signal sequences and acidic propieces of their preproforms but have clearly diverged to yield several families of microbicidal cationic peptides that are structurally distinct. These include amphipathic α-helical peptides—for example, the dermaseptins sensu stricto, the Gly-Leu-rich peptides, the phylloxins, the dermatoxins, the phylloseptins, the caerins, and the aureins, as well as α-helical hydrophobic peptides such as dermaseptin S9. Most of these peptides are lethal against Grampositive and Gram-negative bacteria, yeasts, protozoan, fungi, and enveloped viruses, without harmful effect on mammalian cells. Their antimicrobial activity very likely results from their capacity to bind to the outer leaflet of bacterial bilayers and, once bound, to insert into the membrane, thereby triggering transient wormhole formation and membrane disruption.

THE DERMASEPTIN SUPERFAMILY The dermaseptins represent a large family of peptides involved in host defense that are made in the skin of Hylidae frogs. These peptides are derived from precursors that all show a highly conserved N-terminal preprosequence and markedly varied carboxyterminal domains corresponding to very different antimicrobial peptides. These peptides have all been given individual names and grouped to form distinct families on the basis of their structural characteristics (Table 1). Whereas members of a peptide family are structurally related and differ by amino acid substitutions and deletions, peptides belonging to a given family do not Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

296 / Chapter 45 TABLE 1. Amino acid sequences of peptides belonging to the dermaseptin superfamily of antimicrobial peptides present in the skin secretions of South American hylid frogs. Gaps (-) have been introduced to maximize sequence similarities. Identical amino acids have shaded backgrounds. a, amide. The peptides are categorized into families based on structural similarities (see text). DRS = dermaseptin (appended with S, O, DD, or B/PB to indicate that the sequences were identified from Phyllomedusa sauvagei, P. oreades, P. distincta, and P. bicolor, respectively). DRP = dermaseptin-related peptide (appended with PB, PD, AA or AC to indicate that the sequences were identified from Phyllomedusa bicolor, Pachymedusa dacnicolor, agalychnis annae, and A. callidryas, respectively). PS = phylloseptin (appended with O or H to indicate that the sequences were identified from Phyllomedusa oreades and P. hypochondrialis, respectively). Dermaseptins DRS S1 DRS S2 DRS S3 DRS S4 DRS S5 DRS S6 DRS S7 DRS S8 DRS S11a,b DRS S12a,b DRS S13a,b DRS O1 DRS DDL DRS DDK DRS B1 DRS B2d DRS B3 DRS B4 DRS B5 DRS B6 DRS Bg3a DRP-PD-2-2a DRP-PD-3-3a DRP-AA-3-1a DRP-AA-3-6a DRP-AA-3-3a DRP-AC-3a

ALWKTMLKKLG-TMALH----AGKAALGAAADTISQGTQ-ALWFTMLKKLG-TMALH----AGKAALGAAANTISQGTQ-ALWKNMLKGIGK--------LAGKAALGAVKKLVGAES--ALWMTLLKKVLK--------AAAKAALNAV--LVGANA--GLW-SKIKTAGKSVAK----AAAKAAVKAVTNAV------GLW-SKIKTAGKEAAKAAAKAAGKAALNAVSEAI------a GLWKSLLKNVGK--------AAGKAALNAVTDMVNQ----a ALWKTMLKKLG-TVALH----AGKAALGAAADTISQ----a ALWKTLLKGAGKVFGHVA-KQFLG----------SQGQPES GLW-SKIKEAAKT--------AGKMAMGWVNDMV------ac GL-RSKIKEAAKT--------AGKMALGWVNDMA------ac GLW-STIKQKGKEAAIAAAKAAGQAALGAL----------a ALWKTLLKNVGK--------AAGKAALNAVTDMVNQ----GLW-SKIKAAGKEAAKAAAKAAGKAALNAVSEAV------AMWKDVLKKIGTVALH-----AGKAALGAVADTISQ----a GLW-SKIKEVGKEAAKAAAKAAGKAALGAVSEAV------a ALWKNMLKGIGK--------LAGQAALGAVKTLVGA----ALWKDILKNVGK--------AAGKAVLNTVTDMVNQ----a GLW-NKIKEAAKS--------AGKAALGFVNEMV------a ALWKDILKN------------AGKAALNEINQLVNQ----ALWKTIIKGAGKMIGS-----LAKNLLG------SQAQPES ALWKTLLKKVGK--------VAGKAVLNAVTNMANQNEQ-GMW-SKIKNAGKAAAKASKKAAGKAALGAVSEAL------ac SLW-SKIKEMAAT--------AGKAALNAVTGMVNQ----ac GMW-STIRNVGKSAAKAANLPA-KAALGAISEAV------ac GMFTNMLKGIGK--------LAGQAALGAVKTLA------ac SVL-STITDMAK--------AAGRAALNAITGLVNQ----ac

[21, 8] [25] [25] [25] [25] [8] [8] [8]

[4] [4] [4] [6, 22] [6, 11] [6] [6] [6] [6] [15] [35] [35] [35] [35] [35] [33]

Gly-Leu-rich peptides GLVSDLLSTVTGLLGNLGG--GGLKKI-DRS S10a,b DRP-PBN2 GLVTSLIKGAGKLLGGLF---GSVTGGQS DRP-PD 3-6 GVVTDLLNTAGGLLGNLV---GSLSGG---a GLVSGLLNTAGGLLGDLLGSLGSLSGG---ac DRP-AA 2-5a a GLLSGILNTAGGLLGNLI---GSLSNG---a DRP-AC-1 GLLSGILNSAGGLLGNLI---GSLSNGEac DRP-AC2a

[33, 34] [35, 34] [35] [33] [33]

Dermatoxins Dermatoxin sa,e Dermatoxin B DRP-AA-1-1a DRP-PD-1-5a

ALGTLLKGVGSAVATVGKMVADQFGKLLQAGQG SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQG SLGSFMKGVGKGLATVGKIVADQFGKLLEAGQG SLGSFMKGVGKGLATVGKIVADQFGKLLEAGKG

[2] [35] [35]

Phylloxins Phylloxin sa,e Phylloxin B

GWMSKIASGIGTFLSGVQQa GWMSKIASGIGTFLSGIQQa

[27]

The Dermaseptins / 297 TABLE 1. (Continued) Phylloseptins DRP-PBN1a PS-1 H PS-2 H PS-3 H PS-4 O PS-5 O PS-6 H

FLSLIPHIVSGVAALAKHL-ac FLSLIPHAINAVSAIAKHN-a FLSLIPHAINAVSTLVHHF-a FLSLIPHAINAVSALANHG-a FLSLIPHAINAVSTLVHHSGa FLSLIPHAINAVSAIAKHS-a --SLIPHAINAVSAIAKHF-a

[33] [17] [17] [17] [17] [17] [17]

Cationic hydrophobic peptides GLRSKIWLWVLLMIWQESNKFKKM DRS S9a,b Orphan peptides GMWGSLLKGVATVVKHVLPHALSSQQS DRP-AA-3-4a LLGDLLGQTSKLVNDLTDTVGSIV DRP-PD-3-7a

[35] [35]

a

Sequences predicted from cDNA clones; bunpublished results; ccarboxa-midation predicted from the amino acid sequence of the polypeptide precursor; d first isolated by Daly et al. from P. bicolor and named adenoregulin [11]; eChen, T., Walker, B., Zhou, M. and Shaw, C. unpublished results (accession numbers: Dermatoxin s: AJ865345.1; phylloxin s: AJ865344.1).

or the use of immunosuppressive agents. This discovery was followed by the isolation of adenoregulin (also named dermaseptin B2) from P. bicolor skin, a peptide that interacts with the adenosine receptor [11], and dermaseptin B1 [22]. These two peptides were thought to be unrelated until attempts to clone their precursor polypeptides revealed the presence of a common preproregion and 5′- and 3′-UTRs [1]. Since then, additional members of the dermaseptin superfamily were rapidly identified in various South American and Australian hylid species (see Table 1 for references). As shown in Table 1, different species of hylid frogs are equipped with different sets of peptides belonging to different families. Orthologous peptides, as well as paralogous peptides, have different amino acid sequences in different species, and most often display differential activity against a range of microorganisms (Table 1). This impressive divergence between and within species is such that no single peptide from one species has been found with an identical amino acid sequence in another.

THE PREPRODERMASEPTINS Antimicrobial peptides are synthesized in the multinucleated cells of the granular glands of the skin as prepropeptides that are processed by the removal of the signal peptide to the proform and then stored in the large granules of the glands. Glands may release their content onto the skin surface by a holocrine mechanism involving rupture of the plasma membrane and extrusion of the granules through a duct opening to the surface.

For most of the peptides just described, the cDNAencoding precursors are known to code for a single copy of the antimicrobial peptide at the C-terminus of the predicted precursor sequence (Table 2). A comparison of the precursor sequences revealed that they form a single family, the preprodermaseptin, with unique features. Precursors belonging to this family have a common N-terminal preprosequence of approximately 50 residues that is remarkably well conserved both within and between species, while the C-terminal sequence corresponding to antimicrobial peptides varies markedly. The conserved region comprises a 22-residue signal peptide and an acidic intervening sequence that ends in a typical prohormone processing signal Lys-Arg. This conservation also extends into the 5′- and 3′-untranslated regions of the corresponding mRNAs. Preprodermaseptin mRNAs thus show a unique pattern of very high sequence conservation surrounding a region of very high sequence variability. The contrast between the strikingly conserved preproregion and the hyperdivergent mature peptides is one of the most extreme examples observed to date for homologous gene products within a single order of organisms. Moreover, the pattern of conserved and variable regions in skin antimicrobial peptide precursors is the opposite of that of conventional secreted peptides, suggesting that the conserved preproregion is important for the biology of the expressing cell. Australian frogs belonging to the Pelodryadinae subfamily of the family Hylidae also produce in their skins numerous antimicrobial peptides (caerins, aureins, maculatins) that have no structural similarity with South American hylid peptides (see Tyler et al.), but the signal peptide and acidic propiece of the precursors of the

298 / Chapter 45 TABLE 2. Conserved preproregions and hypervariable antimicrobial domains of preprodermaseptins. A. Diagram of preprodermaseptin cDNAs. The coding region, including the signal peptide, the acidic propiece, and the antimicrobial progenitor sequence is drawn as a rectangle. The predicted hydrophobic signal peptide includes the first 22 amino acid residues, while the acidic propiece comprises 21–24 residues. B. Alignment of the predicted amino acid sequences (single-letter code) of the preproregions of preprodermaseptins obtained from South American hylid frogs. Gaps (-) have been introduced to maximize sequence similarities. Identical (black background) amino acid residues are highlighted. DRS = dermaseptin (appended with S or B/PB to indicate that the sequences were identified from Phyllomedusa sauvagei and P. bicolor, respectively). DRP = dermaseptin-related peptide (appended with AA, AC or PD to indicate that the sequences were identified from Agalychnis annae, A. callidryas, and Pachymedusa dacnicolor, respectively). Amino acids in italics are removed during processing of proforms to expose the extra Gly residue (lower case), which serves as an amide donor for the C-terminal residue of mature peptides. See Table 1 for references. ′ ′ DRS SI DRS S6 DRS S7 DRS S8 DRS S9 DRS S10 DRS S11 DRS S12 DRS S13 DRS B1 DRS B2 DRS B3 DRS B4 DRS B6 DRS Bg3 DRP PBN1 DRP PBN2 Dermatoxin s Dermatoxin B Phylloxin s Phylloxin B DRP-AA-1-1 DRP-AA-2-5 DRP-AA-3-1 DRP-AA-3-3 DRP-AA-3-4 DRP-AA-3-6 DRP-PD-1-5 DRP-PD-2-2 DRP-PD-3-3 DRP-PD-3-6 DRP-PD-3-7 DRP-AC1 DRP-AC2 DRP-AC3

C Signal peptide

KR Acidic propiece

Antimicrobial Peptide Progenitor Sequence

MDILKKSLFLVLFLGLVSLSICEEEKRENEDEEK-QEDDEQSEMKRALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ MDILKKSLFFILFLGLVSLSISEEEKRENEDEED-QEDDEQSEEKRGLWSKIKTAGKEAAKAAAKAAGKAALNAVSEAIgEQ MDILKKSLFLVLFLGLISLSFCEEEKRENEDEEE-QEDDEQSEEKRGLWKSLLKNVGKAAGKAALNAVTDMVNQgEQ MDILKKSLFLVLFLGLVSLSICEEEKRENEDEEK-QEDDEQSEMKRALWKTMLKKLGTVALHAGKAALGAAADTISQgAQ MAFLKKSLFLVLFLGLVSLSICDEEKRENEDEEN-QEDDEQSEMRRGLRSKIWLWVLLMIWQESNKFKKM MAFLKKSLFLVLFLALVPLSICEEEKREGENEKE-QEDDNQSEEKRGLVSDLLSTVTGLLGNLGGGGLKKI MAFLKKSLFLVLFLGMVSLSICEEEKRENEDEEE-QEDDEQSEEKRALWKTLLKGAGKVFGHVAKQFLGSQGQPES MASLKKSLFLVLFLGLVSLSICEEEKRENEDEEN-QEDDEQSEMRRGLWSKIKEAAKTAGKMAMGWVNDMVgEQ MAFLKKSLFLVLFLGLVSLSICDEEKRENEDEEN-QEDDEQSEMRRGLRSKIKEAAKTAGKMALGWVNDMAgE MDILKKSLFLVLFLGLVSLSICEEEKRENEDEEK-Q-DDEQSEMKRAMWKDVLKKIGTVALHAGKAALGAVADTISQgEQ MAFLKKSLFLVLFLGLVSLSICEEEKRENEDEEE-QEDDEQSEMKRGLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVgEQ MAFLKKSVFLVLFLGLVSLSICEEEKREEENEEK-QEDDEQSEEKRALWKNMLKGIGKLAGQAALGAVKTLVGAE MAFLKKSLFLVLFLGLVSLSICEEEKRENKDEIE-QEDDEQSEEKRALWKDILKNVGKAAGKAVLNTVTDMVNQgEQ MAFLKKSLFLVLFLGLVSLSVCEEEKRENEDEME-QEDDEQSEEKRALWKDILKNAGKAALNEINQLVNQgEL MAFLKKSLFLVLFLGLVSLSVCEEEKRENEDEEE-QEDDEQSEEKRALWKTIIKGAGKMIGSLAKNLLGQAPES MAFLKKSLFLVLFLGLVSLSICEEEKRETEEKEYDQGEDDKSEEKRFLSLIPHIVSGVAALAKHLG MAFLKKSLFLVLFLALVPLSICEE-KKSEEENEEKQEDD-QSEEKRGLVTSLIKGAGKLLGGLFGSVTGGQS MAFLKKSLFLILFLGLVPLSFCENDKREGENEEE-QDDD-QSEEKRALGTLLKGVGSAVATVGKMVADQFGKLLQAGQG MAFLKKSLFLVLFLGLVPLSLCESEKREGENEEE-QEDD-QSEEKRSLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQG MVFLKKSLLLVLFVGLVSLSICEENKREEHEEVE--ENAEKAEEKRGWMSKIASGIGTFLSGVQQg MVFLKKSLLLVLFVGLVSLSICEENKREEHEEIE--ENKE-AEEKRGWMSKIASGIGTFLSGMQQg MAFLKKSLFLVLFLGLVPLFLCENEKREGENEKE--ENDDQSEEKRSLGSFMKGVGKGLATVGKIVADQFGKLLEAGQG MAFLKKSLFLVLFLAIVPLSICEEEKREEENEEK-QEDDDQS--KRGLVSGLLNTAGGLLGDLLGSLGSLSGgES MAFLKKSLFLVLLLGLISLSICEEEKRENEVEEE-QEDDEQSELRRSLWSKIKEMAATAGKAALNAVTGMVNQgEQ MAFLKKSLFLVLFLGMVSLSICEEEKREEENE---QEDDEQSEEKRGMFTNMLKGIGKLAGQAALGAVKTLAgEQ MAFLKKSLFLVLFLGLVSLSICDEEKRENEDEEE-QEDDEQSEEKRGMWGSLLKGVATVVKHVLPHALSSQQS MAFLKKSLFLVLFLGLVSLSICEEEKRENEDEEE-QEDDEQSEMKRGMWSTIRNVGKSAAKAANLPAKAALGAISEAVgEQ MAFLKKSLFLVLFLGLVPLFLCENEKREGENEKE--ENDDQSEEKRSLGSFMKGVGKGLATVGKIVADQFGKLLEAGKG MALVKKSLFLVLFLGLVSLSICE-EKRENEDEEE-QEDDEQSEEKRALWKTLLKKVGKVAGKAVLNAVTNMANQNEQ MAFLKKSLFLVLFLGLVSLSICE-EKRENEDEEE-QEDDEQSEEKRGMWSKIKNAGKAAAKASKKAAGKAALGAVSEALgEQ MAFLKKSLFLVLFLALVPLSICEAEKREEENEEK-QEDDDESEKKRGVVTDLLNTAGGLLGNLVGSLSGgER MSFMKKSLFLVLFLGVVSLSNCEEEKGE-ENEEDH-EEHH--EEKRLLGDLLGQTSKLVNDLTDTVGSIV MAFLKKSLLLVLFLGLVSLSICEEEKRENEDEEK-QEDDDQSENKRGLLSGILNTAGGLLGNLIGSLSNgES MAFLKKSLLLVLFLALVPLSICEEEKREEEDEEK-QEDDDQSENKRGLLSGILNSAGGLLGNLIGSLSNgES MAFLKKSLLLVLFLGLVSLSICEEEKRENEDEEE-QEDDEQSEMRRSVLSTITDMAKAAGRAALNAITGLVNQgEQ

aureins and caerins are highly similar to the corresponding regions of the precursors of the South American hylid antimicrobial peptides [10, 33] (see Table 3 in the supplemental disk). Similarly, several disulfidecontaining antimicrobial peptides from Eurasian and North American ranid frogs have precursors whose preproregions strikingly resemble those of preprodermaseptins [33]. The remarkable similarity of preproregions

of precursors that give rise to very different end products in distantly related frogs suggests that the corresponding genes all came from a common ancestor of South American and Australian hylids and ranids before their divergence in the Mesozoic age 150 million years ago [33]. A combination of phylogenetic reconstructions, analysis of mutation rates, and geophysical models for the sequence of fragmentation of Gondwana suggests

SUPPLEMENTAL DISK TABLE 3. A comparison of the predicted amino acid sequence of the dermaseptin B2 precursor obtained from P. bicolor with the predicted sequences of A. caerin and aurein precursors obtained from Litoria caerulea and L. aurea, respectively; B. dermorphin and [Thr 6]-phyllokinin precursors obtained from Phyllomedusa sauvagei, the deltorphin precursor obtained from Phyllomedusa bicolor, and Tryptophyllin-1 precursor obtained from Pachymedusa dacnicolor. Gaps (-) have been introduced to maximize sequence similarities. Identical (black background) amino acid residues are highlighted. The sequences of mature peptides are underlined. A DRS B2 Caerin Caerin Caerin Caerin Caerin Caerin Aurein Aurein Aurein Aurein Aurein

1.1 1.11 1.12 1.13 1.14 1.15 2-2 2-3 2-5 3-1 5-3

MAFLKKSLFLVLFLGLVSLSICEEEKRENEDEEEQEDD--EQ----S-EMKRGLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVGEQ MSFLKKSLLLILFLGLVSLSVCKEEKRETEEENENEE-NHEEG---S-EMKRYAFGYPSG... MSFLKKSLLLVLFLGLVSHSVCKEEKRETEEENENEEENHEVG---S-EMKRYAFWYPNG... MNFLKKSLFLVLFLGFVSISFCDEEKRQDDDEGNEREEKKEIQEDGNQEERRDKPPAWVP MDILKKSLFLVLFLGLVSFSICEEEKRDTEEEENDDEIEEE-----SEEKKREAPERPPGFTPFRIY

[33]

[10]

[29] [13] [9] [7]

The Dermaseptins / 299

B DRS B2 Dermorphin Deltorphin Tryptophyllin-1 [Thr6]-Phyllokinin

MAFLKKSLFLVLFLGLVSLSICEEEKR-----ENEDEEEQEDDEQ---SEMKRGLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVGEQ MASLKKSLFLVLLLGFVSVSICEEEKRQ----EDEDEHEEEGESQEEGSEEKRGLLSVLGSVAKHVLPHVVPVIAEHLG MASLKKSLFLVLFLGFVSVSICEEEKRQ----EDEDEHEEEGENQEEGSEEKRGLFSVLGSVAKHVVPRVVPVIAEHLG MAFLKKSLFLVLFLGLVSLSICEEEKRQ----EDEDEHEEEGENQEEGSEEKRGLFGILGSVAKHVLPHVVPVIAEHSG MASLKKSLFLVLFLGFVSVSICEEEKRQ----EDEDENEEEGENQEEGSEEKRGLLSVLGSLKLIVPHVVPLIAEHLG MASLKKSLFLVLFLGFVSVSICEEEKRQ----EDEDENEEEGENQEEGSEEKRSVLGKSVAKHLPHVVPVIAEKTG MASLKKSLFLVLFLGFVSVSICEEEKRQ----EDEDENEEEGESQEEGSEEKRGLFGLAKGSVAKPHVVPVISQLVG MAFLKKSLFLVLFLGLVSLSICEKEKRQNE--EDEDENEAA--NHEEGSEEKRGLFDIVKKVVGALGSLGKRNDLE MAFLKKSLFLVLFLGLVSLSICEKEKRQNG--EDEDENEAA--NHEEGSEEKRGLFDIVKKVVGAIGSLGKRNDVE MAFLKKSLFLVLFLGLVSLSICEKEKRQNE--EDEDENEAA--NHEEGSEEKRGLFDIVKKVVGAFGSLGKRNDLE MAFLKKSLFLVLFLGLVSLSICEKEKRQNE--EDEDENEAA--NHEEGSEEKRGLFDIVKKIAGHIAGSIGKKR MAFLKKSLFLVLFLGLVSLSICEQEKREEENQEEDEENEAA-------SEEKRGLMSSIGKALGGLIVNVLKPKTPAS

300 / Chapter 45 that the impressive diversity of these peptides and the number of peptides per species reflect the combination of multiple duplications of the ancestral gene before and during radiation of these species and within individual species, focal hypermutations of the mature peptide domain, and subsequent actions of diversifying (positive) selection [33]. Frog skin secretions are also a rich source of biologically active neuropeptides and hormones that are very similar to mammalian peptides of the central and peripheral nervous system and the gastrointestinal tract. Most interesting, preprodermaseptins-encoding peptides also include the D-amino acid containing opioid peptides, dermorphin (YdAFGYPSamide) and deltorphins (YdAFDVVGamide) from P. sauvagei and P. bicolor, respectively [13, 29], the bradykinin-related peptide [Thr 6]phyllokinin (RPPGFTPFRIY) from P. sauvagei [7], as well as tryptophyllin-1 (KPPAWVP), a myoactive peptide from Pachymedusa dacnicolor [9] relaxing mammalian arterial smooth muscles and contracting small intestinal smooth muscles (see Table 3 in the supplemental disk). The discovery of the considerable extent of sequence identity between the neuropeptide and antimicrobial peptide precursors is unprecedented. Thus, although the two groups of peptides are genetically related and belong to the same gene superfamily, they have strongly diverged to yield families of peptides that are both structurally and functionally distinct.

ACTIVITIES OF THE PREPRODERMASEPTINDERIVED PEPTIDES Peptides of the dermaseptin superfamily have a very broad range of antimicrobial activity. The dermaseptins sensu stricto are cidal against a wide spectrum of microorganisms, including mollicutes, bacteria, fungi, protozoa, yeast, and enveloped viruses. All the peptides but dermaseptin S4 [14] are devoid of toxic effect on mammalian cells. Despite high sequence similarities, the dermaseptins differ in their potency for killing the various agents (see the supplemental disk). It is noteworthy that the antimicrobial potencies are essentially independent of the bacterial envelope structure. The dermaseptins S exhibit synergy of action upon combination, resulting in some cases in a 100-fold increase in antibiotic activity of the mixture over the activity of the peptides separately [23]. Shortening the peptide chain of dermaseptin S3 to dermaseptin S3-[1–16]–NH2 does not affect the potency of the peptide. Further reduction of the chain length yield derivatives gradually showing reduced activity. However, analogs as short as 10–12 residues in length remain fully active against several bacterial strains [24]. Dermaseptins S derivatives with improved toxicity profiles: A 28-residue [K4, K20]-

dermaseptin S4 and two shorter versions [K4]dermaseptin S4-[1–16] and [K4, K20]-dermaseptin S4-[1–13] have been investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with Pseudomonas aeruginosa [26]. Naive mice exhibited 75% mortality, compared with 18% and 36% mortality in mice that received a single i.p. injection (4.5 mg/kg) of [K4]-dermaseptin S4-[1–16] and [K4, K20]dermaseptin S4-[1–13], respectively. Dermaseptins S1– S5 exhibit cidal activity against Leishmania mexicana in its promastigote form at μM doses [16]. Within 5 min of incubation in the presence of the peptide, the flagellated parasites lost their motility. Immunocytochemical, freeze fracture, and label fracture microscopic observations show that the peptide generates major perturbations of the lipid bilayer, leading to death of the parasites (see Fig. 1 in the supplemental disk). Dermaseptin S3 and derivatives of dermaseptin S4 selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell [12]. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocitic parasite. Dermaseptins O1, DDL, and DDK show antiprotozoan activity against Trypanosoma cruzi in its tryptomastigote and epimastigote forms cultivated in both cell culture and blood media, without toxicity against mouse erythrocytes and white blood cells [4]. Dermaseptins S1–S5 display antiviral activity against herpes simplex virus type I and HIV-1 virus at μM doses [19]. The most potent peptide, dermaseptin S4, inhibits cell-free and cell-associated HIV-1 infection of D4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. The Gly-Leu-rich peptide orthologs all have very similar amino acid sequences, hydrophobicities, and amphipathicities, but differ markedly in their activity spectra [34]. Whereas cationic peptides, such as DRP-PBN2, are hemolytic and very potent against Gram-positive and Gram-negative bacteria and yeasts, Gly-Leu-rich peptides with no net charge have only hemolytic activity. The phylloseptins demonstrate a strong effect against Gram-positive and Gram-negative bacteria without showing significant hemolytic activity toward mammalian cells [17]. Among these peptides, PS4 and PS5 show antiprotozoan activity with IC50 about 5 μM for Trypanosoma cruzi. A single member of the cationic hydrophobic peptide family, dermaseptin S9, has been identified to date in Phyllomedusa sauvagei (unpublished results). Although its amino acid sequence

The Dermaseptins / 301

A/

B/

Figure 1. Electron microscopic observations of Leishmania mexicana promastigotes A. before and B. after 5 min treatment by 5 μM dermaseptin S1. After treatment, the plasma membrane is peeled off (inset), and the microtubular network is the unique cell surface structure that still maintains the shape and integrity of the promastigote ghost.

and physicochemical properties are considerably different than those of all the other peptides of the dermaseptin superfamily, dermaseptin S9 is cidal against Gram-positive and Gram-negative bacteria, with potencies that are equal to or often higher than those of dermaseptins B and S (Table 4). It has been speculated that the presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides [33]. Most of the peptides belonging to the dermaseptin superfamily have been identified primarily by their antimicrobial activity. However, additional biological functions have been recognized that may or may not be directly associated with pathogen clearance. For instance, adenoregulin (dermaseptin B2) was first isolated by Daly and coworkers as a peptide able to stimulate binding of agonists to A1 adenosine receptors [11]. Adenoregulin was further shown to enhance the binding of agonists to several G-protein coupled receptors in rat brain membranes [32]. It was proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein. On the other hand, dermaseptin B4 significantly stimulates insulin release by 1.5- to 2.5-fold in acute incubations with glucose-

responsive BRIN-BD 11 cells [20]. Although it is tempting to speculate about the importance of these extra-antimicrobial activities, their physiological significance is unclear. Dermaseptin S1 stimulates microbicidal activities of polymorphonuclear leukocytes [3]. Treatment of polymorphonuclear leukocytes with dermaseptin S1 (10–100 nM) stimulates production of reactive oxygen species and release of myeloperoxidase. In addition, low peptide concentrations (1–10 nM) prime the stimulation of respiratory burst induced by zymosan particles. The induced burst is inhibited by selective protein kinase inhibitors and is associated with early signaling events such as a rapid and transient elevation of cytosolic-free calcium concentration and phospholipase D activity. These data provide evidence of stimulating and priming properties of dermaseptin S1 on microbicidal activities of neutrophils, suggesting a potential role in modulating host-defense mechanisms.

STRUCTURAL FEATURES AND MECHANISMS OF MICROBICIDAL ACTIVITY The mode of antimicrobial action of most of the preprodermaseptin-derived peptides from hylid frogs is believed to be the permeation/disruption of the lipid plasma membrane of the target cells through a “carpet” mechanism [30]. Fluorescence-based studies using liposomes and surface plasmon resonance analysis of the

302 / Chapter 45 SUPPLEMENTAL DISK TABLE 4. Activity of peptides of the dermaseptin superfamily against a selected panel of microorganisms.a Microorganisms

DRS B2

DRS B3

DRS B4

DRS S1

3.1 1.5

3.1 6.2

25 25

ndc nd

Gram-negative bacteria Escherichia coli Pseudomonas aeruginosa Salmonella typhimurium

1.5 12.5 3.1

1.5 3.1 3.1

6.2 Rb 25

Gram-positive bacteria Staphylococcus aureus Enterobacter faecalis Bacillus megaterium

12.5 50 0.4

3.1 12.5 0.4

Yeasts Candida albicans Saccaromyces cerevisiae

5 5.5

Mollicutes Acholeplasma laidlawii Spiroplasma melliferum

Fungi Aspergillus fumigatus Aspergillus niger Microsporum canis Hemolysisd

DRS S3

DRS S4

DRS S5

DRP-PBN2

DRS S9

nd nd

nd nd

nd nd

nd nd

1 30 nd

2.5 nd nd

4 nd nd

35 nd nd

1.5 3.1 3.1

1.5 3.1 1.5

25 50 0.8

5 5 nd

10 10 nd

10 20 nd

2 40 nd

3.1 12.5 0.8

1.5 nd 3.1

R 12.5

nd 50

10 5

10 5

20 20

10 5

50 3.1

nd nd

20 40 3

nd nd nd

nd nd nd

30 30 15

20 10 15

20 30 15

>70 30 15

nd nd nd

nd nd nd

>100

200

200

>70

80

1

>90

>100

>100

nd nd

a

The antimicrobial activity is expressed as MIC (μM), the minimal peptide concentration required for total inhibition of cell growth in liquid medium. bStrains were considered resistant (R) when their growth was not inhibited by peptide concentrations up to 100 μM. cNot determined. dDose producing 100% hemolysis after 1 hour incubation with the peptide.

interaction of the peptides with immobilized bilayers demonstrated that cationic peptides bind to negatively charged membranes with association affinity constants in the range of 106–107 M−1. CD, FTIR spectroscopy, and 2-D NMR have shown that dermaseptins S, B, and O, phylloxins, and dermatoxins, as well as Gly-Leu-rich peptides, form an amphipathic α-helix in the presence of apolar solvents, or in the presence of SDS micelles or phospholipid liposomes [2, 5, 18, 27, 31, 34]. The resulting spatial segregation of polar and apolar amino acids on opposing faces along the long axis of the helix permits insertion of a well-defined hydrophobic sector into the lipid bilayer, leading to disruption of the membrane once a critical peptide concentration is reached. The carpet model was proposed for the first time to describe the mode of action of dermaseptin S1 [28]. According to this model [30], cationic peptides initially bind onto the negatively charged surface of the target membrane and cover it in a carpet-like manner. The high content of anionic lipids in prokaryotic membranes and their absence from the neutral matrix of erythrocytes account for the preferential binding of cationic peptides to the outer leaflet of bacterial bilayers through nonspecific long-range electrostatic interactions. The four steps proposed to be involved in this model are (1) binding of the cationic peptide to the

phospholipid head groups, with subsequent formation of an amphipathic peptide helix; (2) alignment of the helical peptides on the surface of the membrane so that their hydrophilic surface is facing the phospholipid head groups; (3) rotation of the helical peptides leading to reorientation of the hydrophobic residues toward the hydrophobic core of the membrane (note that the peptides do not become inserted deeply into the acyl core of the membrane because of the snorkeling of the regularly spaced lysine side chains that grip it to the membrane surface); and (4) disintegration of the peptide-coated membrane by disrupting the bilayer curvature. As peptides cover the cell surface, transient holes are formed. They may enable the passage of low molecular weight molecules prior to membrane lysis. It is generally acknowledged that amphipathicity plays a crucial role in mediating the binding of cationic peptide to the outer leaflet of bacterial bilayers and, once bound, to insert into the membrane interior. Dermaseptin S9 represents departures from the rule with its nonamphipathic hydrophobic core sequence flanked at both termini by cationic and polar residues. CD and NMR spectroscopy analysis combined with membrane permeabilization assays and SPR analysis of the interaction of dermaseptin S9 with model bilayers showed that this cationic hydrophobic peptide is helical in mem-

The Dermaseptins / 303 brane mimetic environments and does bind to and permeate bacterial membranes (unpublished results). This demonstrates that an amphipathic character is not a sine qua non for antimicrobial activity of cationic αhelical peptides. It is proposed that membrane-bound dermaseptin S9 adopts a curved, “banana-like” helical structure with both positively charged termini anchored in the water-membrane interface and the helical hydrophobic core immersed in the hydrocarbon region of the membrane.

References [1] Amiche M, Ducancel F, Mor A, Boulain JC, Menez A, Nicolas P. Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins. J Biol Chem 1994;269:17847–52. [2] Amiche M, Seon AA, Wroblewski H, Nicolas P. Isolation of dermatoxin from frog skin, an anti-bacterial peptide encoded by a novel member of the dermaseptin genes family. Eur J Biochem 2000;267:4583–92. [3] Ammar B, Perianin A, Mor A, Sarfati G, Tissot M, Nicolas P, Giroud JP, Roch-Arveiller M. Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes. Biochem Biophys Res Commun 1998;247:870–5. [4] Brand GD, Leite JR, Silva LP, Albuquerque S, Prates MV, Azevedo RB, Carregaro V, Silva JS, Sa VC, Brandao RA, Bloch C Jr. Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-trypanosoma cruzi activity without cytotoxicity to mammalian cells. J Biol Chem 2002;277:49332–40. [5] Castiglione-Morelli MA, Cristinziano P, Pepe A, Temussi PA. Conformation-activity relationship of a novel peptide antibiotic: Structural characterization of dermaseptin DS 01 in media that mimic the membrane environment. Biopolymers. 2005 Feb 2. [6] Charpentier S, Amiche M, Mester J, Vouille V, Le Caer JP, Nicolas P, Delfour A. Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics. J Biol Chem 1998;273:14690–7. [7] Chen T, Shaw C. Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif. Peptides 2003;24:1123–30. [8] Chen T, Tang L, Shaw C. Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI–sVIII) by cloning from a skin secretion-derived cDNA library. Regul Pept 2003;116:139–46. [9] Chen T, Orr DF, O’Rourke M, McLynn C, Bjourson AJ, McClean S, Hirst D, Rao P, Shaw C. Pachymedusa dacnicolor tryptophyllin-1: structural characterization, pharmacological activity and cloning of precursor cDNA. Regul Pept 2004;117:25–32. [10] Chen T, Scott C, Tang L, Zhou M, Shaw C. The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea. Regul Pept 2005;128:75–83. [11] Daly JW, Caceres J, Moni RW, Gusovsky F, Moos M Jr, Seamon KB, Milton K, Myers CW. Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor. Proc Natl Acad Sci USA 1992; 89:10960–3. [12] Efron L, Dagan A, Gaidukov L, Ginsburg H, Mor A. Direct interaction of dermaseptin S4 aminoheptanoyl derivative with intraerythrocytic malaria parasite leading to increased specific antiparasitic activity in culture. J Biol Chem 2002;277: 24067–72.

[13] Erspamer V, Melchiorri P, Falconieri-Erspamer G, Negri L, Corsi R, Severini C, Barra D, Simmaco M, Kreil G. Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites. Proc Natl Acad Sci USA 1989;86:5188–92. [14] Feder R, Dagan A, Mor A. Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity. J Biol Chem 2000;275:4230–8. [15] Fleury Y, Vouille V, Beven L, Amiche M, Wroblewski H, Delfour A, Nicolas P. Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family. Biochim Biophys Acta 1998;1396:228–36. [16] Hernandez C, Mor A, Dagger F, Nicolas P, Hernandez A, Benedetti EL, Dunia I. Functional and structural damage in Leishmania mexicana exposed to the cationic peptide dermaseptin. Eur J Cell Biol 1992;59:414–24. [17] Leite JR, Silva LP, Rodrigues MI, Prates MV, Brand GD, Lacava BM, Azevedo RB, Bocca AL, Albuquerque S, Bloch C Jr. Phylloseptins: a novel class of anti-bacterial and anti-protozoan peptides from the Phyllomedusa genus. Peptides 2005;26: 565–73. [18] Lequin O, Bruston F, Convert O, Chassaing G, Nicolas P. Helical structure of dermaseptin B2 in a membrane-mimetic environment. Biochemistry 2003;42:10311–23. [19] Lorin C, Saidi H, Belaid A, Zairi A, Baleux F, Hocini H, Belec L, Hani K, Tangy F. The anti-microbial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro. Virology 2005;334:264–75. [20] Marenah L, McClean S, Flatt PR, Orr DF, Shaw C, Abdel-Wahab YH. Novel insulin-releasing peptides in the skin of Phyllomedusa trinitatis frog include 28 amino acid peptide from dermaseptin BIV precursor. Pancreas 2004;29:110–5. [21] Mor A, Nguyen VH, Delfour A, Migliore-Samour D, Nicolas P. Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin. Biochemistry 1991;30:8824–30. [22] Mor A, Amiche M, Nicolas P. Structure, synthesis, and activity of dermaseptin b, a novel vertebrate defensive peptide from frog skin: relationship with adenoregulin. Biochemistry 1994;33:6642–50. [23] Mor A, Hani K, Nicolas P. The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms. J Biol Chem 1994;269:31635–41. [24] Mor A, Nicolas P. The NH2-terminal alpha-helical domain 1–18 of dermaseptin is responsible for antimicrobial activity. J Biol Chem 1994;269:1934–9. [25] Mor A, Nicolas P. Isolation and structure of novel defensive peptides from frog skin. Eur J Biochem 1994;219:145–54. [26] Navon-Venezia S, Feder R, Gaidukov L, Carmeli Y, Mor A. Antibacterial properties of dermaseptin S4 derivatives with in vivo activity. Antimicrob Agents Chemother 2002;46:689–94. [27] Pierre TN, Seon AA, Amiche M, Nicolas P. Phylloxin, a novel peptide antibiotic of the dermaseptin family of antimicrobial/opioid peptide precursors. Eur J Biochem 2000;267: 370–8. [28] Pouny Y, Rapaport D, Mor A, Nicolas P, Shai Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 1992;31: 12416–23. [29] Richter K, Egger R, Kreil G. D-alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor. Science 1987;238:200–2. [30] Shai Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1999;1462:55–70.

304 / Chapter 45 [31] Shalev DE, Mor A, Kustanovich I. Structural consequences of carboxyamidation of dermaseptin S3. Biochemistry 2002;41: 7312–7. [32] Shin Y, Moni RW, Lueders JE, Daly JW. Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx. Cell Mol Neurobiol 1994;14: 133–57. [33] Vanhoye D, Bruston F, Nicolas P, Amiche M. Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-

year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain. Eur J Biochem 2003; 270:2068–81. [34] Vanhoye D, Bruston F, El Amri S, Ladram A, Amiche M, Nicolas P. Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions. Biochemistry 2004;43:8391–409. [35] Wechselberger C. Cloning of cDNAs encoding new peptides of the dermaseptin-family. Biochim Biophys Acta 1998;1388: 279–83.

C

H

A

P

T

E

R

46 The Temporins J. MICHAEL CONLON

acid residues, they are among the smallest antimicrobial peptides to be found in nature. Temporins were first identified in the skin of the Asian frog R. erythraea [22] and the European hybrid frog R. esculenta [18] on the basis of their hemolytic activity. These hydrophobic, C-terminally α-amidated peptides were originally described as “Vespa-like” because of their structural similarity to the short (13–14 amino acid residues) peptides with chemotactic and histamine-releasing properties isolated from the venom of wasps of the genus Vespa. The term “temporin” was first used by Simmaco and coworkers to describe a family of 10 structurally related peptides (temporins A–L) with antibacterial and antifungal properties identified in electrically stimulated skin secretions of the European common frog R. temporaria [19]. Subsequent work has demonstrated that the temporin family is widely, but not universally, distributed in ranid frogs of both North American and Eurasian origin (Figs. 1 and 2). Adopting the classification of new world Rana proposed by Hillis and Wilcox [8], temporins have been isolated from the skin of species belonging to the Aquarana (R. catesbeiana group), Amerana (R. boylii group), Nenirana (R. aerolata group), and Stertirana (R. pipiens group) but not in those representatives of the Scurrilirana (R. berlandieri and R. sphenocephala) and Torrentirana (R. tarahumarae) studied to date (reviewed in [4, 5]). R. sylvatica is considered to be a sister-group to the Aquarana, but temporins were not identified in this species. The classification system of Eurasian ranids is less well developed, but temporins have been identified in the skin of the East Asian brown frogs (R. ornativentris, R. pirica, R. tagoi, and R. japonica) but not in the Ryukyu brown frog R. okinavana or in the pond frogs R. brevipoda porosa and R. nigromaculata. Although the European agile frog R. dalmatina is considered to be closely related to R. temporaria, temporins were not identified in the skin of this species.

ABSTRACT The temporins are a family of short (10–14 amino acid residues), hydrophobic, C-terminally α-amidated peptides with antibacterial and antifungal properties that are synthesized in the skin of a wide range of North American and Eurasian frogs of the genus Rana. Not all temporins are cationic, but a positive charge is an important determinant of antimicrobial potency. The temporins show potential for development into therapeutically valuable anti-infective agents, particularly for use against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis, against anaerobic pathogens such as Clostridium difficile, and against the protozoan Leishmania spp. However, the clinical usefulness of certain temporins is restricted by their high hemolytic activity. As well as cidal activity, individual temporins have been shown to stimulate chemotaxis of neutrophils and monocytes, modulate the activity of secretory phospholipase A2, and relax vascular smooth muscle.

DISCOVERY Frogs of the genus Rana synthesize in granular glands in the skin a remarkably diverse range of antimicrobial peptides that are released into skin secretions, often in very high concentrations, in a holocrine manner upon stress or injury as a result of contraction of myocytes surrounding the glands [5]. These peptides represent a component of the animal’s system of innate immunity and serve to protect the frog against invasion by a variety of pathogenic microorganisms (see also Nicolas and Amiche, and Simmaco; this volume). Included in this arsenal of defensive peptides are members of the temporin family. Composed of between 10 and 14 amino Handbook of Biologically Active Peptides

305

Copyright © 2006 Elsevier

306 / Chapter 46 Aquarana

Amerana

Nenirana

Stertirana

R. catesbeiana FLP*IASLLGKYL FISAIASMLGKFL FLSAIASMLGKFL FISAIASFLGKFL FLFPLITSFLSKVL

R. boylii FLPIIAKVLSGLL

R. aerolata FLPIVGRLISGLL

R.pipiens FLPIVGKLLSGLL

Rana muscosaR. FLPIVGKLLSGLL

palustris FLPLVGKILSGLI

Rana grylio SILPTIVSFLSKVF SILPTIVSFLSKFL SILPTIVSFLTKFL FILPLIASFLSKFL

Rana aurora FLPIIGQLLSGLL Rana draytoni HFLGTLVNLAKKIL NFLGTLVNLAKKIL

Rana clamitans FLPFLAKILTGVL FLPLFASLIGKLL FLPFLASLLTKVL FLPFLASLLSKVL FLPFLATLLSKVL

Rana luteiventris VLPLISMALGKLL NFLGTLINLAKKIM FLPILINLIHKGLL

FIGURE 1. Distribution and primary structures of the temporin peptides from North American frogs of the genus Rana. All peptides are C-terminally α-amidated. The classification of the ranids is according to Hillis and Wilcox [8]. *Denotes deletion of an amino acid residue.

Rana septentrionalis FLSAITSILGKFF FLSAITSLLGKLL FLSAITSILGKLF Rana virgatipes FLSSIGKILGNLL FLSIIAKVLGSLF FLPLVTMLLGKLF

R. temporaria FLPLIGRVLSGIL LLPIVGNLLKSLL LLPILGNLLNGLL LLPIVGNLLNSLL VLPIIGNLLNSLL FLPLIGKVLSGIL FFPVIGRILNGIL LSP***NLLKSLL LLP***NLLKSLL FVQWFSKFLGRIL

R. ornativentris FLPLLASLFSRLL FLPLIGKILGTIL FLPLLASLFSRLF FLPLLASLFSGLF

R. japonica ILPLVGNLLNDLL R.pirica LPILGNLLNGLL LPILGNLLNSLL

R. tagoi FLPILGKLLSGIL R. erythraea FLPILGKILGGLL

R. esculenta FLPAIAGIKSQLF FLPLIAGLLGKLF FLPVIAGLLSKLF

BIOSYNTHESIS OF THE TEMPORINS Screening of a cDNA library prepared from the skin of R. temporaria with an oligonucleotide probe derived from the signal peptide region of preproesculentin-1 from R. esculenta led to the identification and characterization of the biosynthetic precursors of temporin B (LLPIVGNLLKSL L.NH2), temporin G (FFPVIGRILNG IL.NH2), and temporin H (LSPNLLKSLL.NH2) [19]. The structural organization of the three preprotemporins is the same and is illustrated schematically for preprotemporin B in Fig. 3. As discussed in detail by Nicolas and Amiche (this volume), the proteins conform to the general rule among the frog skin anti-

FIGURE 2. Distribution and primary structures of the temporin peptides from Eurasian frogs of the genus Rana. *Denotes deletion of an amino acid residue.

microbial peptides that the signal peptide and acidic propeptide regions are very strongly conserved, even in the case of frogs belonging to different families, whereas the antimicrobial peptide domain at the Cterminus of the precursor is hypervariable. Thus, the Cys22 residue represents the site of cleavage of the signal peptide in the preprotemporins as well as in the preprodermaseptins and a range of other precursors. The mature temporin is generated by cleavage at a typical Lys-Arg prohormone processing site, and the penultimate Gly residue in the propeptide acts as a substrate for peptidyl-glycine α-amidating monooxygenase to produce a C-terminal α-amidated residue in the secreted peptide.

The Temporins / 307 C

Signal Peptide 1

K

R

Acidic Propeptide 22

G

K

Temporin 44

61

FIGURE 3. A schematic representation of the biosynthetic precursor of temporin B deduced from the nucleotide sequence of a cloned cDNA from the skin of Rana temporaria. Cys22 is the probable site of cleavage of the signal peptide, Lys45Arg46 is the site of cleavage of a prohormone convertase, and Gly60 acts as nitrogen donor for C-terminal α-amide formation.

MOLECULAR HETEROGENEITY OF THE TEMPORINS As shown in Figs. 1 and 2, more than 50 members of the temporin family have been identified in the skins of Eurasian and North American ranids. Analysis of several of these amino acid sequences by Wade and coworkers [21] led to formulation of the consensus sequence: FLPLIASLLSKLL.NH2 for the temporins and FLPIIGKLLGG LL.NH2 for the wasp venom peptides. However, the temporins are among the most highly variable of all antimicrobial peptides. Deviations from the consensus sequence are considerable and no single residue in the peptide is invariant. Most temporins contain a single basic residue (generally Lys) giving a charge of +2 at physiological pH but temporin-C, -D, and -E from R. temporaria, peptide A1 from R. esculenta, temporin-1Od from R. ornativentris, temporin-1Ja from R. japonica, temporin-1AUa from R. aurora, and temporin-1PRa and -1PRb from R. pirica lack this feature. Conversely, temporin L from R. temporaria, temporin1Lb from R. luteiventris, and temporins -1Da and -1Db from R. draytoni contain two basic residues. All temporins isolated to date contain a preponderance of hydrophobic amino acids and are C-terminally α-amidated. In common with the majority of frog skin antimicrobial peptides, the temporins exist in a random coil conformation in aqueous solution but have the propensity to form a stable amphiphathic α-helix in a membranemimetic solvent, such as 50% trifluoroethanol/water [20]. The all-D enantiomer of temporin A (FLPLIGRVLSGIL.NH2) is equipotent with the native peptide against bacteria, indicating that effects on the cell membrane are mediated through nonchiral interactions [20]. Studies involving the use of liposomes of different composition have indicated that the lytic activities of the temporins are not greatly influenced by composition or surface charge of the lipid membrane, suggesting that hydrophobic rather than ionic interactions are of primary importance [11]. It is proposed that the temporins produce bacterial cell death by formation of

transmembrane pores (“toroid pore” mechanism) rather than by causing a detergent-like disruption of the cell membrane into peptide-coated vesicles (“carpet” mechanism) [15] (see also contributions in the bacterial/antibiotic peptide section of this volume). Structure-activity studies with temporin A have indicated that a hydrophobic N-terminal residue and bulky hydrophobic residues at positions 5 and 12 are important determinants of antibacterial activity [20]. Replacement of the Ile5 and Ile12 residues by leucine resulted in an increase in antimicrobial potency but replacement by Ala abolished activity. The positively charged amino acid is usually located at position 7 or 11 in order to maintain the amphipathic character of the α-helix that is important for activity. Most temporins that lack a basic amino acid residue show very weak or absent antimicrobial properties, the exception being temporin-1Od (FLPLLASLFSGLF.NH2) from R. ornativentris, which is active against S. aureus [10]

BIOLOGICAL ACTIVITIES OF THE TEMPORINS In general, the temporins show greater potencies against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Streptococcus spp. than against Gram-negative bacteria [4, 7, 11, 20]. The exception is temporin L (FVQWFSKFLGRIL.NH2) from R. temporaria, which bears a net positive charge of +3 and is active against clinically relevant Gram-negative species such as Escherichia coli and Pseudomonas aeruginosa and against the opportunistic yeast pathogen Candida albicans [16]. The therapeutic potential of many of the temporins is limited by their toxicities against mammalian cells, and temporin L is strongly hemolytic against human erythrocytes and also cytolytic toward several human tumor cell lines. Temporin A and B, however, have relatively weak hemolytic activity but have been shown to severely damage the plasma membrane of the human parasitic protozoan Leishmania donovani, leading to cell death [12]. Temporin A also showed antiviral activity by reducing the infectivity of channel catfish virus and frog virus 3 [2], and the peptide inhibited the growth of the chytrid fungus Batrachochytrium dendrobatidis that has been implicated in declines in amphibian populations worldwide [17]. The biological properties of the temporins are not confined to cidal activities. Temporins B and L enhanced the ability of secretory phospholipase A2 from bee venom and in human lacrimal fluid to hydrolyze anionic liposomes [23]. In common with the structurally related peptides from wasp venom, temporin A and ranatuerin6 (FISAIASMLGKFL.NH2) from R. catesbeiana induced

308 / Chapter 46 migration of monocytes, neutrophils, and macrophages, both in vitro and in vivo, by a mechanism that involves interaction with the formyl peptide receptor-like 1 [1]. Temporin-1Gb (SILPTIVSFLSKFL.NH2) and temporin1Gd (FILPLIASFLSKFL.NH2), isolated from R. grylio skin, elicited concentration-dependent relaxations of preconstricted vascular rings from the rat thoracic aorta with EC50 values of approximately 2 μM, but the physiological significance, if any, of this observation is unclear [9].

POTENTIAL CLINICAL AND COMMERCIAL APPLICATIONS The emergence of strains of pathogenic microorganisms with resistance to commonly used antibiotics constitutes a serious threat to public health that has necessitated a search for new types of antimicrobial agents. The temporins are promising candidates for development into clinically useful anti-infectives, particularly with regard to topical applications, such as treatment of infected foot ulcers in diabetic patients [3]. Several peptides show high potency in vitro against nosocomial Gram-positive pathogens such as MRSA [4] and antibiotic-resistant strains of E. faecalis [7]. The all D-isomer of temporin B was active against a range of anaerobic bacteria and was stable in a fecal milieu, suggesting the possibility of its use in the treatment of Clostridium difficile–associated diarrhea [13]. Combination therapy with a conventional antibiotic, such as rifampicin, appears promising, as the temporin may increase the permeability of the bacterial plasma membrane facilitating entry of the second bactericidal agent [11]. Infection of indwelling medical devices, such as central venous, urinary, and dialysis catheters and prosthetic heart valves, contributes significantly to mortality rates following surgery. In a subcutaneous pouch model in which rats were infected with drug-resistant clinical isolates of S. epidermidis, presoaking of a Dacron vascular graft in temporin A solution, either alone or in combination with vancomycin, markedly reduced the number of bacteria adhering to the graft [6]. Thus, temporins have potential as prophylactic agents in preventing vascular prosthetic graft infections. The possibility of expressing genes that encode for an antimicrobial peptide to protect commercially important crops from bacterial and fungal diseases has excited interest within the agricultural biotechnology industry. Genetically engineered potatoes expressing the gene for temporin A have been grown successfully and are resistant to Phytophthora infestans, responsible for late blight, and to Phytophthora erythroseptica, responsible for pink rot. The transgenic tubers remained disease-free during prolonged storage [13].

References [1] Chen Q, Wade D, Kurosaka K, Wang ZY, Oppenheim JJ, Yang D. Temporin A and related frog antimicrobial peptides use formyl peptide receptor-like 1 as a receptor to chemoattract phagocytes. J Immunol 2004;173:2652–9. [2] Chinchar VG, Bryan L, Silphadaung U, Noga E, Wade D, RollinsSmith L. Inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides. Virology 2004;323:268–75. [3] Conlon JM. The therapeutic potential of antimicrobial peptides from frog skin. Rev Med Micro 2004;15:17–25. [4] Conlon JM, Abraham B, Sonnevend A, Jouenne T, Cosette P, Leprince J, Vaudry H, Bevier CR. Purification and characterization of antimicrobial peptides from the skin secretions of the carpenter frog Rana virgatipes (Ranidae, Aquarana). Regul Pept 2005;131:38–45. [5] Conlon JM, Kolodziejek J, Nowotny N. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim Biophys Acta 2004;1696:1–14. [6] Ghiselli R, Giacometti A, Cirioni O, Mocchegiani F, Orlando F, Kamysz W, et al. Temporin A as a prophylactic agent against methicillin sodium-susceptible and methicillin sodium-resistant Staphylococcus epidermidis vascular graft infection. J Vasc Surg 2002;36:1027–30. [7] Giacometti A, Cirioni O, Kamysz W, D’Amato G, Silvestri C, Del Prete MS, et al. In vitro activity and killing effect of temporin A on nosocomial isolates of Enterococcus faecalis and interactions with clinically used antibiotics. J Antimicrob Chemother 2005; 55:272–4. [8] Hillis DM, Wilcox TP. Phylogeny of the new world true frogs (Rana). Mol Phylogenet Evol 2005;34:299–314. [9] Kim JB, Halverson T, Basir YJ, Dulka J, Knoop FC, Abel PW, Conlon JM. Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio. Regul Pept 2000; 90:53–60. [10] Kim JB, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. J Peptide Res 2001;58:349–56. [11] Mangoni ML, Rinaldi AC, Di Giulio A, Mignogna G, Bozzi A, Barra D, et al. Structure-function relationships of temporins, small antimicrobial peptides from amphibian skin. Eur J Biochem 2000;267:1447–54. [12] Mangoni ML, Saugar JM, Dellisanti M, Barra D, Simmaco M, Rivas L. Temporins, small antimicrobial peptides with leishmanicidal activity. J Biol Chem 2005;280:984–90. [13] Oh H, Hedberg M, Wade D, Edlund C. Activities of synthetic hybrid peptides against anaerobic bacteria: aspects of methodology and stability. Antimicrob Agents Chemother 2000;44:68– 72. [14] Osusky M, Osuska L, Hancock RE, Kay WW, Misra S. Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot. Transgenic Res 2004;13:181–90. [15] Rinaldi AC, Di Giulio A, Liberi M, Gualtieri G, Oratore A, Bozzi A, et al. Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles. J Pept Res 2001;58:213– 20. [16] Rinaldi AC, Mangoni ML, Rufo A, Luzi C, Barra D, Zhao H, et al. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles. Biochem J 2002;368:91–100. [17] Rollins-Smith LA, Conlon JM. Antimicrobial peptide defenses against chytridomycosis, an emerging infectious disease of amphibian populations. Dev Comp Immunol 2005;29:589–98.

The Temporins / 309 [18] Simmaco M, De Biase D, Severini C, Aita M, Falconieri Erspamer G, Barra D, et al. Purification and characterization of bioactive peptides from skin extracts of Rana esculenta, Biochim Biophys Acta 1990;1033:318–23. [19] Simmaco M, Mignogna G, Canofeni S, Miele R, Magoni ML, Barra D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur J Biochem 1996;242:788–92. [20] Wade D, Silberring J, Soliymani R, Heikkinen S, Kilpeläinen I, Lankinen P, et al. Antibacterial activities of temporin A analogs, FEBS Lett. 2000;479:6–9.

[21] Wade D, Silveira A, Silberring J, Kuusela P, Lankinen H. Temporin antibiotic peptides: a review and derivation of a consensus sequence. Protein Peptide Lett 2000;7:349–57. [22] Yashuhara T, Nakajima T, Erspamer V, Falconieri-Erspamer G, Tukamoto Y, Mori M. Isolation and sequential analysis of peptides in Rana erythraea skin. In: Kiso Y, editor. Peptide Chemistry 1985. Osaka: Protein Research Foundation; 1986. pp. 363–8. [23] Zhao H, Kinnunen PK. Modulation of the activity of secretory phospholipase A2 by antimicrobial peptides. Antimicrob Agents Chemother 2003;47:965–71.

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47 Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model MAITÉ MONTERO-HADJADJE, DJIDA AIT-ALI, VALÉRIE TURQUIER, JOHANN GUILLEMOT, MOHAMMED BOUTAHRICHT, RABIA MAGOUL, MARIA M. MALAGON, LAURENT YON, HUBERT VAUDRY, AND YOUSSEF ANOUAR

are stored and released with hormones, peptides, and neurotransmitters in response to a variety of stimuli. Chromogranin B (CgB) was the second member of this family that was also identified in chromaffin cells, while the third chromogranin-like protein was first characterized in anterior pituitary cells and initially named chromogranin C. However, this protein is now known as secretogranin II (SgII), since it exhibits no sequence homologies with CgA and CgB that are structurally related to each other [35]. This class of acidic, secreted proteins present in secretory granules is currently known as the granin family and has expanded to include other more recently described members such as secretogranins III to VI also called 1B1075, HISL-19, 7B2, and NESP55, respectively [30].

ABSTRACT Chromogranins/secretogranins (Cg/Sg) are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Cloning and characterization of chromogranin A, chromogranin B, and secretogranin II revealed that selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved between amphibian and mammalian species. Two peptides, named secretoneurin and vasostatin, which occur in these regions, have been shown to exert various biological activities. Cg/Sg may also be involved in the formation of secretory granules in amphibians as in mammals. Recent results obtained on the function of Cg/Sg as propeptide precursors and as granulogenic factors in different vertebrate species are discussed.

STRUCTURE OF THE PRECURSOR mRNA/GENE

DISCOVERY

An important breakthrough in the field of the granins was the elucidation of their primary structures that were deduced from the cloning of the corresponding cDNAs in the mid-eighties [35]. It was noticed that CgA encompasses in its sequence numerous pairs of basic amino acids that are potential cleavage sites for prohormone convertases (Table 1). In addition, sequence comparisons showed that CgA may represent the precursor of a peptide named pancreastatin (PST) that has been previously isolated from the porcine pancreas and shown to inhibit glucose-induced insulin secretion [30]. These observations suggested that, akin to the precursors of hormones and neuropeptides, CgA could be a prohormone precursor that is processed to give rise to smaller bioactive peptides such as PST [35]. It was subsequently found that CgB, SgII, and all the granins identified to date contain multiple dibasic sites, further supporting the notion that granins may serve as precursors of biologically active peptides. Molecular

The chromogranin story started with the biochemical studies performed in the mid-sixties on chromaffin granules that were aimed at understanding the physiology and the pharmacology of catecholamines. Thus, analysis of the secretion of catecholamines from chromaffin cells revealed the existence of a granule-specific, high molecular weight protein coreleased with the hormones upon splanchnic nerve stimulation [4]. The term chromogranin A (CgA) was coined for this abundant secretory granule protein of chromaffin cells. Subsequently, hormone- and neurotransmitter-containing organelles have been characterized in other tissues, and CgA was generally found in these organelles [35]. In fact, a large family of chromogranins corresponding to acidic, heat-stable, and soluble proteins has been characterized in the secretory granules of endocrine, neuroendocrine, and neuronal cells, where these proteins Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

312 / Chapter 47 TABLE 1. Comparison of the physicochemical properties of human and frog granins. CgA

Residues Precursor Mature protein Molecular mass (kD)a Calculated Apparent Acidic residues (% E + D) Isoelectric point (pHi) Multibasic sitesb Disulfide bonded loop

CgB

SgII

Human

Frog

Human

Frog

Human

457 439

399 381

677 657

599 580

617 590

601 574

76 120 18 + 6 4.83 15 16–37

70 ND 21 + 7 4.95 20 16–37

68 86 13 + 7 4.47 9 No

67 98 12 + 8 4.57 11 No

49 74 21 + 4 4.37 10 17–38

44 64 21 + 9 4.31 11 17–38

Frog

CgA, chromogranin A; CgB, chromogranin B; SgII, secretogranin II; ND, not determined. a The molecular mass was deduced from the primary structure. The apparent molecular mass was determined by SDS-polyacrylamide gel electrophoresis. b Multibasic sites refer to sites with two or more consecutive R or K residues.

cloning of granins in mammals also revealed the high content of acidic amino acids, which explains the acidic nature of these proteins (Table 1). This characteristic underlies the propensity of granins to aggregate in high calcium and acidic pH conditions that are found in the intragranular milieu. It was thus proposed that granins may participate in the sorting and packaging of hormones and neuropeptide precursors into the secretory granules [6]. Interest in granins in nonmammalian species started when a 33-amino-acid peptide that derives from the endoproteolytic cleavage of SgII was isolated from the frog brain [33]. The characterization of the first graninderived peptide in a nonmammalian vertebrate, whose sequence has been highly conserved between frog and mammals, reinforced the idea that granins could be considered as propeptide precursors. A homologous peptide was subsequently characterized in mammalian species and named secretoneurin (SN), based on its abundance in the brain and its effects on neuronal activity [10]. It was then hypothesized that the molecular characterization of granins in nonmammalian species would allow delineation of the sequences that have been preserved during evolution and that may represent the functional determinants of these proteins. The cloning of the first nonmammalian granins CgA, CgB, and SgII was undertaken in the frog Rana ridibunda. Amphibians, which are the first vertebrates that have evolved to a terrestrial life, are a group of particular interest in which to investigate the neurochemical aspects of evolution. Because there was the possibility that the sequences of granins could considerably vary in frog, cloning of short DNA fragments corresponding

to SN or the C-terminal regions of CgA and CgB that are strongly conserved in mammals was performed. These short sequences were amplified from frog endocrine tissues such as the pituitary or the adrenal and used as probes to isolate the full-length sequences encoding granins from a pituitary cDNA library. The deduced frog CgA, CgB, and SgII amino acid sequences all contain signal peptides that would be cleaved to generate mature proteins comprising 381, 580, and 574 amino acids, respectively [1, 2, 31]. As their mammalian homologs, frog granins are characterized by a high content of acidic amino acids and the occurrence of multiple dibasic cleavage sites, as well as the existence of two cysteine residues at the N-terminal part of CgA and CgB that have been shown to form a disulfide loop in mammals (Table 1, see supplemental disk). The abundance of acidic residues in frog granins largely accounts for the high apparent molecular weight observed for these proteins on SDS-polyacrylamide gel electrophoresis, as previously reported for their mammalian homologs [30]. However, the overall sequence identity for each of these proteins with the corresponding mammalian ortholog does not exceed 40–50% (Fig. 1, see supplemental disk). This is even more striking if specific regions of these proteins are compared between mammals and frog. Thus, while certain discrete regions of frog granins display strong sequence conservation with the mammalian counterparts, the majority of the sequences of these proteins exhibit a rather loose homology between the two vertebrate groups. Interestingly, the sequences with high identity between amphibians and mammals are bounded by dibasic cleavage signals (Fig. 1, see supplemental disk), suggesting that

Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model / 313 A KR

KR

KK RR

KR

KR

KR

Frog 46%

44% 40% 82% 68% RK

KR

KR

30% 70%

40%

KR KR

KR

KR

Human

SN EM66

B KK

KR KR

KR

RK

RR

Frog 40%

71% 32% KK

14%

71% 17% 80% KR KR

KR

KK

RR

Human

VS-1

PST

C

WE14 CST EL35

K R K R KR KK RR KRKK KK

KK KR

Frog 35%

61% 20%

RK

RK

16%

24%

61%

K KR KK KR KR RK KK

Human PE-11 BAM-1745 CCB Secretolytin

these regions can be processed to generate peptides that may occur ubiquitously, at least in vertebrate species. In fact, the most highly conserved region in SgII is that encompassing SN and a flanking peptide (Fig. 1A, see supplemental disk) that was characterized in various mammalian species and named EM66 in consideration of its N- and C-terminal residues, and its size of 66 amino acids [25]. Cloning of SgII from another amphibian species, Xenopus laevis, from goldfish, from chicken, and from lizard [18, 32] confirmed the sequence conservation of these peptides. Within CgA and CgB, the most highly conserved regions occur at the N and C termini of the proteins which show up to 80% sequence identity, whereas the

FIGURE 1. Comparison of the primary structures and the putative processing signals of human and frog SgII (A), CgA (B), and CgB (C). The shaded regions represent the highly conserved peptides that can be potentially generated by proteolytic cleavage at the dibasic sites. The percentages of identity between the amino acid sequences of frog and human granins are indicated. Conserved peptides are indicated in bold.

rest of the two granins markedly diverge; the middle part of these proteins exhibit approximately 20% identity (Fig. 1B and C, see supplemental disk). It has been shown that CgA is processed in a tissue-specific manner to yield several peptides, some of which are endowed with biological activities (see following). These include vasostatins (VS-1 and VS-2), which exert various effects in different model systems [14] (see following), catestatin (CST), which has been shown to inhibit nicotineevoked catecholamine release from chromaffin cells [19] and WE14 [7], a peptide with no known function. As depicted in Fig. 1B (see supplemental disk), the sequences of VS, WE14, and an additional peptide named EL35 [24] were preserved during evolution,

314 / Chapter 47 whereas the region encoding PST in mammals has no homologous sequence in the frog protein and that encoding catestatin is not well conserved in the amphibian species. Several peptides that derive from the C terminus of CgB have also been isolated and characterized (Fig. 1C, see supplemental disk). However, apart from a 13-amino-acid peptide purified from the adrenal medulla that exerts an antibacterial effect and named secretolytin [23], no biological activity was attributed to these peptides. It should be noted that sequence comparisons confirmed that CgA and CgB have probably evolved from an ancestral common precursor since the N- and Cterminal regions of these proteins display 36% and 45% sequence identity [1], respectively, as previously reported for the mammalian proteins [35].

DISTRIBUTION OF THE mRNA The tissue-distribution of granins has been extensively studied in several mammalian species. The majority of endocrine and neuroendocrine cells express at least one member of this family of acidic proteins. Pituitary cells, C cells of the thyroid, parathyroid cells, chromaffin cells of the adrenal medulla, and neuroendocrine cells of the gastrointestinal and pulmonary tracts produce and store granins in their secretory granules. In the central and peripheral nervous systems, granins are widely expressed with highest concentrations occurring in certain brain structures such as the hypothalamus, hippocampus, amygdala, and cerebellum [35]. The relative abundance of granins varies in a tissue- and species-specific manner. For instance, SgII is highly expressed in gonadotrophs compared with other pituitary cells or in glucagon-producing alpha cells compared with the other pancreatic endocrine cell types. Human CgA is predominantly expressed in the adrenal medulla, followed by the pituitary and the gastrointestinal tract [35]. Although granins or granin-like molecules have been detected by immunocytochemistry in various nonmammalian species including birds, fish, flies, and even a unicellular eukaryotic organism, the paramecium, a precise mapping of the sites of synthesis of these proteins has awaited their cloning in frog [32]. Thus, Northern blot, reverse transcription-polymerase chain reaction and in situ hybridization analyses have been performed to provide the first overall mapping of granins in a nonmammalian species [1, 2, 31]. These studies showed that granins are also widely expressed in endocrine, neuroendocrine, and neuronal cells in a nonmammalian vertebrate. Equivalent high levels of SgII mRNA are found in the frog brain, pituitary, and spinal cord, and moderate levels are detected in the

adrenal gland. No expression could be seen in the pancreas, stomach, intestine, and kidney using Northern blot analysis. The CgA gene is more strongly expressed in the frog pituitary than in the brain, spinal cord, or adrenal. Finally, the CgB transcript is abundantly expressed in various organs such as the brain, spinal cord, pituitary, adrenal, pancreas, and heart. In the frog brain, all three granins exhibit a widespread distribution (Fig. 2). In situ hybridization has shown that SgII expression is prominent in the pallium, the amygdaloid complex, and the hypothalamus. CgA mRNA is intensely expressed in thalamic and hypothalamic regions, whereas strong expression of CgB is observed in the lateral pallium, the dorsal hypothalamus, and the anteroventral tegmental area (Fig. 2A and B). Overall, the tissue-distribution studies in frog revealed that SgII is more highly expressed in neuronal tissues, that CgA is strongly expressed in the pituitary compared with all other tissues, and that CgB exhibits a more diffuse expression. Within the frog pituitary, CgA and SgII are present in both the distal and the neurointermediate lobes, whereas CgB only occurs in the pars distalis (Fig. 2B). In amphibians, the intermediate lobe of the pituitary is responsible for the synthesis of melanotrophic peptides that induce skin darkening, allowing the animals to adapt to the color of the environment and therefore to hide from predators. Melanotrophs of the pars intermedia represent a valuable model in which the neuroendocrine regulation of hormone synthesis and release can be studied in physiological conditions. Thus, the biosynthesis of melanotrophic peptides in these cells is tightly regulated through the control of the transcriptional activity of the pro-opiomelanocortin (POMC) gene, mainly by hypothalamic input [34]. Both SgII and CgA mRNA levels [32] are up-regulated in parallel with those of POMC in the intermediate lobe following adaptation to a dark background, and, conversely, the levels of these transcripts are down-regulated when the animals are placed on a white background. We have recently observed that CgA and SgII are differentially expressed and processed in frog melanotrophs depending on the status of the secretory activity of these cells: although both SgII and CgA are elevated in vivo in dark background-adapted animals, the two proteins could play rather different roles in the regulated secretory pathway in melanotrophs. Thus, CgA expression and processing may be related to hormone storage in secretory granules, whereas SgII production and processing could be part of the active process of hormone release from melanotrophs. Further studies using this model are warranted in order to better understand the coordinate regulation of the SgII and CgA genes with the POMC gene during the neuroendocrine reflex of skin color adaptation in amphibians.

Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model / 315

A SgII

CgB Lpd

Lpd

Lpv

P

CNT

CNT

TP

TP

TP NPv DH

TP NPv DH

VH

VH

B

Lpv

P

CgA

CgB OT

OT

TS

TS

RIS AD

AD AV

AV RIS

III

III PN PI

PN PI

Pdis Pdis

FIGURE 2. In situ hybridization histochemistry of granin mRNA in the brain and the pituitary of the frog Rana ridibunda. The left hemisections show autoradiograms obtained from frontal frog brain slices at the level of the diencephalon (A) and mesencephalon (B), hybridized with the CgA, CgB, and SgII antisense probes. The right hemisections designate the anatomical structures. Abbreviations: AD, anterodorsal tegmental nucleus; AV, anteroventral tegmental nucleus; CNT, central thalamic nucleus; DH, dorsal hypothalamic nucleus; Lpd, lateral thalamic nucleus, posterodorsal division; Lpv, lateral thalamic nucleus, posteroventral division; III, oculomotor and trochlear nuclei; NPv, nucleus of the periventricular organ; OT, optic tectum; P, posterior thalamic nucleus; Pdis, pars distalis; PI, pars intermedia; PN, pars nervosa; RIS, nucleus reticularis isthmi; TP, posterior tuberculum; TS, torus semicircularis; VH, ventral hypothalamic nucleus.

316 / Chapter 47 BIOLOGICAL ACTIONS As just mentioned, the search for the roles of granins in mammals has focused on their intracellular implication in the packaging of hormones and the formation of secretory granules, and on their extracellular activities as precursors of several peptides that could be released to exert hormonal effects in autocrine, paracrine, and endocrine manners [10, 30].

Intracellular Functions Because granins are localized in secretory granules of virtually all neuroendocrine cells and because several studies have shown that these proteins aggregate in intravesicular conditions of acidic pH and high calcium, it has been proposed that they may play a fundamental role in granulogenesis [6]. Direct evidence in support of this hypothesis was lacking for several years until the demonstration by Kim et al. [16] of a pivotal role of CgA in the biogenesis of secretory vesicles in neuroendocrine cells. Inhibition of the expression of CgA in PC12 cells leads to a marked decrease in secretory granule formation and to impairment of the release of a transfected hormone. Subsequent studies revealed that CgB [15] and SgII [3] are also efficient in inducing the formation of secretory granule-like structures in nonneuroendocrine cells that are normally devoid of a regulated pathway of secretion, suggesting that granins may play a crucial role in the generation of secretory vesicles. In addition, stored SgII could be released in a regulated manner from transfected, constitutively secreting cells [3]. Although there is currently some controversy as to whether the vesicles generated through granin transfection are or not bona fide secretory granules and whether granin members are master switches or simple cargo proteins for granulogenesis [9, 20], the intracellular function of granins has received more interest recently and the preliminary results obtained should stimulate further investigations in this direction. Interestingly, expression of frog CgA also induced secretory granule-like structures in nonneuroendocrine cells (Fig. 3; Montero-Hadjadje et al., unpublished observations; see supplemental disk), suggesting that CgA could play an intracellular role in secretory granule formation in different vertebrates. In addition, this observation indicates that the high content of acidic amino acids in frog CgA, alone or in combination with the conserved N- and C-terminal regions, is probably sufficient to confer aggregating properties to this granin. Because all granins contain a high proportion of acidic amino acids in all species studied to date, this structural feature may well represent a critical if not the sole requirement for the granulogenic effect of these proteins.

FIGURE 3. Vesicular distribution of frog CgA in transfected COS7 cells. Cells transfected with frog CgA cDNA were incubated with a rabbit antiserum directed against frog CgA (CgA107–234) and processed for immunofluorescence labeling.

Several studies have proposed additional intracellular roles for certain members of the granin family. For instance, it has been shown that CgA and CgB are involved in intracellular calcium (Ca2+) mobilization, since these granins bind to the inositol 1,4,5-trisphosphate (IP3) receptor that is present on the membranes of secretory granules and facilitate Ca2+ release in response to IP3 [38]. It has also been shown that a fraction of immunoreactive CgB in PC12 cells can localize to the nucleus and that the granin is able to stimulate or repress the transcription of different genes, including transcription factors, when overexpressed in neuroblastoma cells [39]. Using antibodies directed against specific peptides derived from CgA, we have observed immunostaining of the nucleus in PC12 cells (MonteroHadjadje et al., unpublished observation). The physiological significance of these findings remains unknown. Finally, a clear intracellular function has been established for the granin-like protein 7B2 that binds and activates PC2 through two domains that have been highly conserved in various species, including the toad Xenopus laevis [22]. Most of the functional characteristics of 7B2 have been deduced from initial studies performed in Xenopus melanotrope cells in which this protein is also regulated in concert with POMC during background color adaptation [21]. More recently, the introduction of 7B2-knockout mice models have allowed to confirm and extend these early studies [26]. Knockout models are currently lacking for granins like CgA, CgB and SgII in order to firmly demonstrate their involvement in intracellular events associated with hormone and neurotransmitter release.

Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model / 317

Extracellular Functions The notion that granins may serve as precursors to biologically active peptides has received much attention since the first demonstration that the CgA-derived peptide PST exerts a potent inhibitory effect on insulin release from pancreatic cells [29]. Proteolytic processing of CgA, CgB, and SgII at both monobasic and dibasic residues gives rise to a wide spectrum of peptides, some of which have been shown to exert biological actions, in particular inhibitory effects on hormone secretion from different endocrine glands [14, 30]. In the context of this review, we will focus our attention only on peptides whose sequence has been conserved between frog and mammals (Table 2, see supplemental disk), which could be relevant to amphibian physiology. Readers are referred to a recent book on chromogranins [14] that compiles several reviews on various granin-derived peptides that have been isolated so far and shown to exert biological activities. Among the peptides that could act in amphibians based on the remarkable preservation of their sequence, VS has been shown to inhibit vasoconstriction of arteries in mammalian species [14]. Interestingly, VS and VS-derived peptides may exert cardioregulatory actions in frog [8]. VS-derived peptides have also been shown to promote fibroblast adhesion [27], to affect human colonic motility in vitro [12], to trigger neuronal apoptosis [30], and to be involved in nociceptive responses in rat [13]. Finally,

TABLE 2.

the N-terminal peptides of CgA also exert potent antibacterial and antifungal activities [23]. Besides, a large polypeptide that derives from the highly conserved C-terminus of CgA has been previously isolated and named parastatin in consideration of its effect on parathyroid hormone secretion from parathyroid chief cells [35]. However, no results are currently available on the occurrence of this peptide and its actions in amphibian species. Nevertheless, a C-terminal peptide named EL35 is present in both frog and mammalian CgA and is produced in rat and human tissues [24, 31], suggesting that the processing of the C-terminal region of this granin may give rise to a ubiquitous peptide that may exert biological activities. Although peptides derived from the N and C termini of CgB have also been identified, there are few functional studies using these peptides. In particular, several C-terminal peptides have been isolated from various tissues such as GAWK and CCB which have been purified from the human pituitary gland, a pyroglutamyl peptide named BAM-1745 that has been identified in bovine adrenochromaffin cells, and an 11-amino acid peptide named PE-11 that has been characterized in the rat and human brain, but the activity of all these peptides remains unknown [35]. One exception is the peptide secretolytin, which exerts antibacterial activity [23]. The sequence of the majority of these peptides is highly conserved in the frog protein but their occurrence as free entities and their function in this species have not been studied.

Functions of granin-derived peptides conserved between frog and mammals. Biological Activity of the Peptide

Chromogranin A-Derived Peptides Vasostatin I (bCgA1–76) Inhibits vasoconstriction; inhibits cardiac performance; promotes fibroblast adhesion; inhibits parathyroid hormone secretion; triggers microglial-cell-mediated neuronal apoptosis; exerts bacteriolytic and antifungal effects. Inhibits parathyroid hormone secretion from parathyroid chief Parastatin (pCgA347–419) cells. Chromogranin B-Derived Peptides Exerts bacteriolytic effects. Secretolytin (bCgB614–626) Secretogranin II-Derived Peptides Stimulates dopamine release from central striatal neurons; Secretoneurin (rSgII154–186) promotes chemotactic attraction of monocytes, eosinophils, and fibroblasts; stimulates proliferation and migration of vascular smooth-muscle; stimulates migration and inhibits proliferation of endothelial cells; stimulates transendothelial migration of monocytes; activates endothelial cells for neutrophil adherence; promotes neurite outgrowth in cerebellar granule cells. bCgA, bovine chromogranin A; pCgA, porcine chromogranin A; rSgII, rat secretogranin II; bCgB, bovine chromogranin B.

318 / Chapter 47 The SgII-derived peptide SN, initially isolated from the frog brain [33], has been extensively studied during the last decade [10]. SN has emerged mainly as a peptide that links the nervous and the immune systems. Thus, SN attracts monocytes, eosinophils, and endothelial cells at sites of injury and acts as an angiogenic factor [17, 10]. In addition, SN stimulates dopamine release from rat striatal slices [10] and influences neurite outgrowth in cerebellar granule cells [11]. Signaling mechanisms involved in the effects of SN have been proposed and include activation of phosphoinositide 3-kinase and protein kinase C [10]. Specific binding sites for SN have been found in human monocytes and may represent the putative functional plasma membrane receptor for SN which has not been identified so far [28]. SN exhibits 82% sequence identity between frog and human and is the most highly conserved region in SgII, indicating that SN may represent an authentic neuropeptide in various species. Flanking SN at its C-terminus, the peptide EM66 that occurs in all species studied to date, including the frog Rana ridibunda [25, 37, and unpublished observations], is also of potential interest. It has recently been shown that the concentration of EM66 is modulated by food deprivation in the jerboa hypothalamus, suggesting that this peptide may be involved in the control of food intake and/or the stress associated with fasting [5]. Finally, a peptide named manserin that also derives from the processing of SgII has been identified in the rat, but no function has been attributed to this 40amino-acid peptide [36]. The function of the neuroendocrine proteins chromogranins and secretogranins has been intensely investigated during the last 40 years, at least for CgA and CgB, which were the first members of this family that were discovered. Although their physicochemical properties—localization, processing, and regulation—are now well known, their function still remains incompletely understood. An intracellular role for these proteins has received strong support during the last years, and some fundamental notions are now emerging that will probably enhance our understanding of the intracellular intervention of these proteins in hormone storage and release. Besides, the role of granins as precursors to neuropeptides and hormones is an attractive hypothesis that still needs to be substantiated. Several questions related to the mode of action of the derived peptides often remain unanswered. SN and VS, whose sequences have been preserved during evolution, have proved to be highly potent at regulating different systems but the issues of the concentrations used, the existence of specific receptors and the effects of these peptides in vivo should be clarified. Other biologically active peptides, such as PST and CST, may be relevant only in mammals, since their sequences are either

lacking or poorly conserved in certain vertebrates such as frog, suggesting that these peptides may have emerged late during evolution and/or their sequence has evolved rapidly. Thus, elucidation of the sequences and the properties of granins in frog and the study of their expression in this model have provided insights to help determine the physiological role of these proteins and to decipher the structure–activity relationships that may govern their function in all species.

Acknowledgments The work performed by the authors has been supported by INSERM (U413), The European Institute for Peptide Research (IFRMP 23), the Regional Platform for Cell Imaging, the Conseil Régional de Haute-Normandie, Ministerio de Educacion y Ciencia (BFI 2001–2007) and an INSERM-CNRST (Morocco) exchange program.

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48 Sodefrin and Related Pheromones SAKAE KIKUYAMA

attracting activity. The minimum effective amount of extract in a sponge block that attracted a female placed in a container filled with 3000 ml of water, was the equivalent of 0.1% of the abdominal gland contents. When the water-soluble fraction of the abdominal gland was subjected to gel-filtration column chromatography, the female-attracting activity emerged in a fraction that had a relative molecular mass below 5000. This fraction was incubated with pronase. The pronase digested sample lost its female-attracting activity, indicating that the active substance is a peptide. To isolate the active peptide from the crude fraction, two purification cycles of reversed-phase HPLC were employed. The final product was subjected to direct sequencing using a pulsed gas-liquid phase protein sequencer, and the active substance was identified as a decapeptide with the amino acid sequence SIPSKDALLK. Furthermore, COOH-terminal analysis by carboxypeptidase-P digestion showed that a free (nonamidated) Lys residue was present at the COOH-terminus. The relative molecular mass of 1071.2 estimated from fast atom bombardment mass spectrometry corresponded with that calculated from the amino acid sequence. Ten nanograms of the final product absorbed in a block of sponge was enough to attract female newts placed in a container filled with 3000 ml of water. A synthetic replicate exhibited female-attracting activity similar to that of the natural material. The peptide showed no sequence similarity with any known peptide. Thus, the novel peptide was designated sodefrin (derived from the ancient Japanese word sodefri, meaning “soliciting”) [7]. To study the localization of sodefrin in the abdominal gland and to develop a specific radioimmunoassay system for the measurement of the peptide, an antiserum against sodefrin was generated. Frozen sections of abdominal glands stained with the antiserum against sodefrin showed that the apical region of the epithelial cells was positive for sodefrin (Fig. 1). An immunoelectron microscopic study of the abdominal gland revealed

ABSTRACT This article describes the current state of understanding of sodefrin, a peptide pheromone from the newt Cynops pyrrhogaster, and [Leu3, Gln8] sodefrin (silefrin) from the congeneric species, C. ensicauda. Both pheromones are composed of 10-amino-acid residues and were isolated from the abdominal glands of the male newts. They exhibit a potent female-attracting activity only to the conspecific females, suggesting a contribution of the pheromones to maintaining the reproductive isolation of the species. Both pheromones are generated from 20 kDa precursor molecules. These pheromones are emitted through the cloaca of the male and are directed toward the female partner’s snout during courtship to act primarily on the lateral nasal sinus cells. Hormone dependency of secretion of and response to the pheromones has been demonstrated.

DISCOVERY It has been well recognized that courting male redbellied newts, Cynops pyrrhogaster, attract their partners by sending water from around the cloaca toward the female’s snout by vibrating their tail and that during courtship they project numerous minute tubules that are connected to the abdominal gland, from the cloaca. This suggests that the male newts emit female-attracting pheromones through the cloaca. In fact, we have found that the water in which sexually active male newts were kept attracted conspecific females. The attractant was believed to be secreted by the abdominal gland of the cloaca because the water in which abdominal glandablated males had been kept did not attract females [13]. Thus, an attempt was made to isolate and characterize the active component. Female-attracting activity was monitored by a preference test [13]. An aqueous extract of the abdominal glands of sexually developed male red-bellied newts exhibited femaleHandbook of Biologically Active Peptides

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322 / Chapter 48

FIGURE 1. Immunofluorescence micrograph showing the localization of sodefrin in the abdominal gland of a red-bellied newt. The sodefrin antibody labels the epithelial cells red with rhodamine. Nuclear DNA is stained blue with 4′ 6-diamino2-phenylindole. Bar represents 50 μm. From Toyoda and Kikuyama Zool Sci 2000; 17: 561–76. Copyright 2000 by the Zoological Society of Japan. (See color plate.)

FIGURE 2. Immunoelectron micrograph showing the localization of sodefrin in the epithelial cells of the abdominal gland of a red-bellied newt. Immunogold particles indicating the presence of sodefrin are observed within the secretory granules. Bar represents 500 nm. From Toyoda and Kikuyama Zool Sci 2000; 17: 561–76. Copyright 2000 by the Zoological Society of Japan.

that the sodefrin-immunoreactive substance was localized mainly in the secretory granules (Fig. 2). This clearly indicates that sodefrin is secreted by the epithelial cells of the abdominal gland. In order to ascertain whether the abdominal gland of a congeneric species, C. ensicauda (the sword-tailed newt), synthesizes molecules that immunoreact with the sodefrin antiserum, an aqueous extract of the abdominal gland was subjected to a sodefrin radioimmunoassay (RIA). This extract showed no cross-reactivity in this RIA system, whereas a water extract from the abdominal gland of C. pyrrhogaster showed an inhibition curve parallel to the sodefrin standard [17]. On the assumption that C. ensicauda abdominal glands contain a molecule possessing a female-attracting activity that is different from sodefrin, purification of putative C. ensicauda pheromone was carried out. The pheromone was isolated from the abdominal glands by gel-filtration chromatography and reversed-phase HPLC. The final product was composed of 10-amino-acid residues with the sequence SILSKDAQLK and a molecular mass of 1102.3 as esti-

mated by electrospray mass spectrometry. This peptide was designated silefrin, a combination of the first three N-terminal amino acids, SIL and -efrin, derived from sodefrin [18]. The sequence of this peptide differed from that of sodefrin by two-amino-acid residues, with substitutions of Leu for Pro and Gln for Leu at positions 3 and 8, respectively. Both native and synthetic silefrin were equipotent in attracting conspecific females, 10 ng being the minimum effective amount. Localization of silefrin in the abdominal gland of C. ensicauda was examined by immunohistochemistry using a specific antiserum. An intense immunofluorescence was observed on the apical side of the epithelial cells in the cryosections incubated with the antiserum.

STRUCTURE OF THE PRECURSOR mRNA/GENE A sodefrin precursor cDNA, isolated from a cDNA library constructed from C. pyrrhogaster abdominal gland

Sodefrin and Related Pheromones / 323 mRNA, contained 1364 bp with an open reading frame of 567 bp and encoded a sodefrin precursor protein of 189 amino acid residues. The precursor protein included a predicted signal peptide and, in a region close to the C-terminus, the sodefrin molecule. The molecular mass of the sodefrin precursor deduced from its sequence was approximately 20 kDa (Fig. 3A). A cDNA clone encoding a silefrin precursor protein was isolated from a C. ensicauda abdominal gland cDNA library. This clone consisted of 1339 bp and contained a predicted open reading frame of 576 bp encoding a precursor protein of 192 amino acid residues, including a domain rich in hydrophobic amino acids that was presumed to be a signal peptide. Its nucleotide sequence showed 82% sequence identity with preprosodefrin from C. pyrrhogaster (Fig. 3B).

DISTRIBUTION OF THE mRNA Northern blot analysis of sodefrin mRNA in the abdominal gland indicated a size of about 1.5 kb and that sodefrin mRNA was expressed in the abdominal gland but not in other organs such as tail, liver, kidney, testis, and brain. Similarly, the expression of the silefrin precursor mRNA of C. ensicauda was also analyzed with silefrin precursor cDNA as a probe. A single band showing positive hybridization corresponding to approximately 1.5 kb was detected exclusively in the abdominal gland [2]. Localization of mRNA was also studied by in situ hybridization using 35S-labelled silefrin precursor cDNA as a probe. Intense specific signals were seen in the epithelial cells of the abdominal gland [4] (Fig. 4).

PROCESSING OF THE PRECURSORS Sequence analysis of the cDNAs encoding sodefrin and silefrin revealed that both peptides are generated from precursor proteins. Both molecules are sandwiched between monobasic amino acid residues (Arg and Lys). It is well known that most of the peptide hormones are derived from precursor proteins as a result of posttranslational processing. The peptide hormone sequence is flanked by pairs of dibasic amino acids that are subjected to cleavage by enzymes such as the prohormone convertases PC1 and PC2. The absence of such dibasic amino acids flanking the sodefrin and silefrin molecules suggests that the mode of processing of these peptide pheromones are different from that of most peptide hormones. Synthetic sodefrin C-terminally extended by IleSer-Ala (the C-terminal portion of sodefrin precursor consisting of 13-amino-acid residues) does not attract

the females, indicating that these three amino residues must be removed from the C-terminus of this molecule for acquisition of biological activity. In order to demonstrate the presence in the abdominal gland of enzymes that cleave the amino acids at the C- and N-termini of sodefrin and silefrin, an assay for enzyme activity was developed using t-butoxycarbonyl (Boc)-LGR-methylcoumaryl-7-amide (MCA), Boc-LLKMCA, and Boc-QLK-MCA as synthetic substrates. These substrates were designed on the basis of the predicted amino acid sequence of the C-terminal region of the sodefrin precursor protein (. . . LGRSIPSKDALLKISA) and the silefrin precursor protein (. . . LGRSILSKDQLKISA). A C. pyrrhogaster abdominal gland extract hydrolyzed both Boc-LGR-MCA and Boc-LLK-MCA to liberate 7-amino-methyl-4-courain (AMC). Similarly, a C. ensicauda abdominal gland extract was shown to hydrolyze both Boc-LGR-MCA and Boc-QLK-MCA. Both enzyme preparations lost activity when they were boiled or when proteinase inhibitors were present during incubation [1].

RECEPTORS At the present time, no receptors for either sodefrin or silefrin have been identified. Bilateral, but not unilateral, nostril plugging in females with a cotton ball soaked in melted vaseline completely abolished the female attracting activity of sodefrin. This strongly suggests that the action of sodefrin is mediated through the olfactory organ. This result concurs with the results of an electrophysiological study demonstrating that sodefrin evokes a marked electro-olfactogram (EOG) response when applied to the lateral nasal sinus. The lateral nasal sinus region is considered to be a primitive vomeronasal organ. The EOG response to sodefrin was found to be markedly enhanced in the females when they were treated with prolactin and gonadotrophin or estrogen. In the male, however, the effects of the similar hormonal treatments on the EOG response to sodefrin were not apparent. Thus, it was concluded that there is a sex difference in the responsiveness of lateral nasal sinus to the hormones and/or the pheromone [14]. Sodefrin was tested for its ability to elevate intracellular Ca2+ ([Ca2+]i) levels in dissociated lateral nasal sinus cells. Sodefrin, but not silefrin, increased [Ca2+]i in dissociated lateral nasal sinus cells from the female C. pyrrhogaster. Species specificity in terms of the responsiveness to the pheromone was confirmed at the cellular level. The number of cells that responded to sodefrin increased concentration dependently. Relatively few cells in the lateral nasal cells from female newts in the nonbreeding season or from male newts responded to sodefrin compared with cells from reproductive females. A

324 / Chapter 48 A

TGGCAGGTGAACAGGTGCAGAGACTCCATCACCCTATTCCTTACTCTCCTAGCACC ATGAGGGCCATCCTTGCAGCTGTCGTCCTGCTCCAGGCACTGATAACTGGAGATTGCCTATTATGCGAGCAGTGT M R A I L A A V V L L Q A L I T G D C L L C E Q C TTCGCTCTCCAAACCAGCAGCTGCTCGGGTATCTTCACGCAGTGCTCTCCTGACGTCACTCACTGCGTCGCAGGC F A L Q T S S C S G I F T Q C S P D V T H C V A G CTAGAGAACTGCACACTGGGGACTCATGTTATTCTAACTGCGTTCAAGGACTGTCTGGATCCTTCCGAAAAAGCA L E N C T L G T H V I L T A F K D C L D P S E K A GCCTGCGGTAGAGAGGTCTCCTTCACAGCTCCAGCGGCCTCTTTATGGACAAGCAGGACGTGCTGTGACTCTGAT A C G R E V S F T A P A A S L W T S R T C C D S D TTCTGCAACGGTGGGGATGTGCAGGTGCCTCCTCCAGACGACACTCCAGIGGTTGTGGCAGTGACCAGCCCTGC F C N G G D V Q V P P P D D T P S G C G S D Q P C ACAGCGCCAGAACACCTAAGGGAAACAGTGCACTCTACAACATCGATTAGAGAGAAGAGAAGAAAGCGATTTTTT T A P E H L R E T V H S T T S I R E K R R K R F F TGGICATATTTTCCGATCAGAAGAACGCATGTGGCACCATCTATGGAACTGCCTCCAGGCCGGCTAAGACTGGGG W S Y F P I R R T H V A P S M E L P P G R L R L G AGGAGTATACCTTCAAAGGATGCACTACTCAAGATTTCTGCATAGCTGGAATTTTCCACATGGCGGGGATGCAAG R S I P S K D A L L K I S A * CCTACGATTATTATGTTTTAAAGTGTTCCCCTGCCCTAAAAGTTTGAGACTTTTGTTCATACCCCATAGGCACTC CTACTCTAGCTTAGTAGTTGTCTGTAGAAACATTCATAAAGCGCTACAAGTATGTGGAATGCAGTGICTGATCTT GTGATGAGGAAGCATATGAACTCATGTGAGCCTCTCTGAGACACAGTGTACAGGTGGCCAATGTGCTTAGTACAA TCTAGGCCGGCATGCTGTTTAACCACTGTCTTCTCTATTCAGCCATCTTAAGCGCCTGGGCATCTCAGAGGGTTA TCTTGGATTCATGCATCGAGTGATCCAAGCACAGGCCAAGCAATCATGCAATGATGCTGTCTTATGGTTGTAGAA GGTGCTTCTCCTGATGTGCTACTAATGCTGACTTCATGAGTAGCCATGAACAGCCATTCCTGCTTTTCTTCTGCT TTTTGGTTGAATACCTCTTCTAACATAAAGTAATTGAGAATATCTGGCGCAGTTGTATTGATGCTGTCAAATATA AGAGGACAGGGTTATTGGTTCATATCTCCAATATGAATGTGCCTTTTAATCCAGCAATAAGCATGCTTTGTGCCA CAGATATAACCCAAAATAGAACAAATATGTAGACCCGCTTTGTACTGCACATTGAAAAAATGAATAAACATTAAT TTACACTGCTGCAAAAAAAAAAAAAAAAAAAAA

B TAGTTGAACAGGTGCAGAGACTCCATCACCCTACTCCTTACTCTCCTAGCACC ATGAGGGCCATCATTGCAGCTGTCGTCCTGCTCCAGGCACTGATAACTGGAGATTGTCTATTATGCGAGCAGTGT M R A I I A A V V L L Q A L I T G D C L L C E Q C TTCGCTCTCCACACCAGCAGCTGCTCGGGTATCTTCACGCAGTGCTCTCCTGACGTCACTCACTGCGCCGCAGCT F A L H T S S C S G I F T Q C S P D V T H C A A A AAGAAGAACAACACAGCGGGGACTCATGTTATTCTAACTGCGTTCAAGGACTGTCTGGATCCTTCCCAAAAAGCA K K N N T A G T H V I L T A F K D C L D P S Q K A GCCTGCGGTAGAGAGGTCTCCTTCACAGCTCCAGCGGCCTCTTTATGGACAAGCAGGACGTGCTGTGACTCTGAT A C G R E V S F T A P A A S L W T S R T C C D S D TTCTGCAACGGCGGGGATGTGCAGGTGCCTCCTCCAGACGACACTCCCAATGGTTGTGGCAGTGACCAGTCCGCG F C N G G D V Q V P P P D D T P N G C G S D Q S A AACGCCTGCACAGCGCCAGGACACCTGAGGGTAACAGTGCGCTCTACAACATCGATTAGAGAGAAGAGAAGAGAG N A C T A P G H L R V T V R S T T S I R E K R R E CGACTGAATGTGTTCAGTGTTCTGGCAAGCAGAAGGCGTGTGGCACCTTCTAAGGAACTGCCTCTAGGCTTGATA R L N V F S V L A S R R R V A P S K E L P L G V I AAACTGGGGAGGAGTATACTTTCAAAGGATGCACAACTCAAGATTTCTGCATAGATGGAATTTTCCACATGGCGG K L G R S I L S K D A Q L K I S A * GGACGCAAGCCTACAATTATGTTTTAAAGTGTTCCCCTGCCCTGAAAGTTTGAGACTTTTGTTCATACCCCATAG GCACTCCTACTCTAGCTTAGTATTGICTGTAGAAACATTCCTAAAGCTCTACAAGTATGTGGAATGCAGTGICTG ATCTTGTGATGAGGAAGCATATGAACTCATGTCAGCCTCTCTGAGACAAAGTGTACAGGTGGCCAATGTGCTCAG TACAATCCAGGCCGGCATGCTGTTTAACCACTGTCTTCTCTATTCAGCCATCTTAAGCGCCTGGGCATCTCAGAG GGTTATCTTGGATTCATGCATTGAGTGATCCGAGCACAGTCCAAGCAATCACCCAATGATGCTGTCTTATGGTTG TAGAAGGTGCTTCTCCTGATGTGCTACTAATGCTGACTTCATGAGTAGCCATGAACAGCCATTCCTGCTTTTCTT CTACTTTTTGGTTGAATACCTCTTCTAACATAAAGTAATTGAGAATATCTGGCGCAGTTGTGTTGATGCTTTCAA ATATAAGAGGACAGGGTTATTGGTTCATATCTCCAATATGAATGTGCCTTTTAATCCAGCAATAAGCATGCTTTG TGCTACAGCTATAACCCAAAATAAAAAAATATATAGACCCGCTTTGTACTGCACATTGAAAAAATGAATAAACAT TAATTTACACA

FIGURE 3. Nucleotide and deduced amino acid sequences of the cDNA clone encoding sodefrin (A) and silefrin (B). The predicted amino acids are shown below the nucleotide sequence. The amino acids comprising sodefrin and silefrin molecules are circled. The asterisk indicates the termination codon, and the polyadenylation signal (AATAAA) is underlined. From Toyoda and Kikuyama Zool Sci 2000; 17: 561–76. Copyright 2000 by the Zoological Society of Japan.

Sodefrin and Related Pheromones / 325 that the sequence Ser4-Lys5 in the sodefrin molecule is indispensable for mediating the female-attracting activity. From an extract of the abdominal glands of male red-bellied newts captured five months prior to the onset of the breeding season, a COOH-terminally extended form of sodefrin (SIPSKDALLKISA) was obtained. This peptide was biologically inactive, suggesting that activation of a protease that cleaves at the Lys-Ile bond to generate the active pheromone must occur by the time of onset of reproductive behavior. Another biologically inactive peptide isolated was a COOH-terminally extended form of [Asn10] sodefrin (SIPSKDALLNISA). The presence of this sodefrin variant raised the possibility of the expression of multiple genes encoding preprosodefrin. Analyses of PCR products derived from total RNAs from the abdominal gland of individual newts collected from three different regions (Chiba, Niigata, and Nara) of Japan were performed. The results confirmed the existence of multiple genes encoding sodefrin and its variants. Their expressions varied according to the individuals and the regions. Among them, mRNA encoding the sodefrin variant, SIPSKDAVLKICA ([Val8] sodefrin. ICA) was expressed exclusively in the animals from Nara, whereas genes encoding sodefrin were expressed in all the specimens sampled [6]. Recently, [Val8] sodefrin was isolated from the abdominal gland of the male newts inhabiting the Nara region. Synthetic [Val8] sodefrin had femaleattracting activity only toward the Nara females [11]. FIGURE 4. Micrographs of the signals for silefrin precursor mRNA in the abdominal gland of the newt, C. ensicauda. In situ hybridization was performed with a specific 35S-labeled probe A and with a 35S-labeled probe containing an excess of nonlabeled silefrin precursor cDNA B. Bars represent 50 μm. From Ref. [4]. Copyright 2000 the Society for the Study of Reproduction, Inc. Zoological Society of Japan.

pharmacological study revealed that there are at least two cell types in the lateral nasal cells that respond to sodefrin by elevating [Ca2+]i. One increases [Ca2+]i through the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate pathway and the other increases [Ca2+]i through the PLCdiacylglycerol-phosphokinase C pathway [5].

STRUCTURE–ACTIVITY RELATIONSHIPS In addition to sodefrin, several peptides related to sodefrin (SIPSKDALLK) have been identified. Minute amounts of peptides such as SKDLLK and DALLK were obtained during purification of sodefrin. These peptides, possibly degradation products of sodefrin, were subjected to the preference test. SKDALLK retained the ability to attract sexually developed female newts, whereas DALLK exhibited no activity. This indicates

BIOLOGICAL ACTIONS The biological test for female-attracting activity is performed according to the method established by Toyoda et al. [13]. This preference test revealed that 10 ng of both native and synthetic sodefrin adsorbed by a sponge block is enough to attract female newts and that the minimum effective concentration lies within the range 0.1–1.0 pM [7]. Both native and synthetic silefrin attract C. ensicauda females, the minimum effective amount absorbed in a sponge block being 10 ng as in the case of sodefrin. Silefrin attracts only C. ensicauda females but not C. pyrrhogaster females. Likewise, sodefrin attracts only C. pyrrhogaster females. A combination of prolactin and androgen produces the structural development of various male organs related to reproduction, including the abdominal gland [8]. The combination of both hormones elevates sodefrin precursor mRNA levels [3] and increases the content of immunoassayable sodefrin [17]. Likewise, it was confirmed that silefrin precursor mRNA as well as silefrin content in the abdominal gland of C. ensicauda were increased by treatment with prolactin and

326 / Chapter 48 androgen [4]. The existence of both prolactin and androgen receptors in the epithelial cells of the abdominal gland has been demonstrated [9, 10]. Arginine vasotocin (AVT) is considered to be a candidate hormone for regulating discharge of the pheromone. The water in which AVT-treated C. pyrrhogaster males had been kept showed considerable femaleattracting activity as compared with the water in which saline-injected males had been kept. In addition, the sodefrin content in the abdominal gland was decreased by the hormonal treatment. The effect of another neurohypophyseal hormone, mesotocin, was less pronounced in this respect. AVT induced contraction of the excised gland concentration dependently. This AVT-induced contraction was blocked completely by a V1a receptor blocker. Thus, AVT may serve as a hormone to induce the release of sodefrin, acting on a contractile structure of the abdominal gland through V1a receptors [15].

PHYSIOLOGICAL IMPLICATION The existence of pheromones in the class Amphibia has long been postulated. Sodefrin is the first amphibian pheromone to be identified and the first peptide pheromone to be identified in a vertebrate. Given the fact that sexually mature newts lead an aquatic life, it is quite reasonable to expect a nonvolatile but watersoluble peptide as a pheromone in this animal. A protein pheromone has been identified in a urodele Plethodon jordani [12]. In this species courtship takes place on land, whereas courtship by Cynops is performed in water. In an anuran Litoria splendida, a female-attracting pheromone was shown to be a peptide consisting of 25-amino-acid residues that is secreted by the male skin gland [16]. In this species mating takes place in water. However, until the chemical nature of sex pheromones from various species of amphibians performing the reproductive behavior in various environments has been elucidated, it may be premature to conclude that peptides are the dominant form of sex pheromone for amphibians in which courtship or mating takes place in water. Nevertheless, water-soluble peptide pheromones might be considered to be the ideal forms of species-specific reproductive pheromones for amphibians performing courtship or mating in aquatic environments where other species are liable to exist, since variant forms that may exert species-specific pheromonal activity can be generated by modification of the nucleotide sequence of the pheromone gene.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17]

References [1] Ishizuka Y, Iwata T, Conlon JM, Toyoda F, Nakada T, Yamamoto K, Kikuyama S. Possible existence of processing enzymes generating sodefrin molecule in the abdominal gland. In: Oishi T, Tsutsui K, Tanaka S, Kikuyama S, editors. Trends in Comparative

[18]

Endocrinology. Nara: The Asia Oceania Society for Comparative Endocrinology; 2004. pp. 426–8. Iwata T, Umezawa K, Toyoda F, Takahashi N, Matsukawa H, Yamamoto K, Miura S, Hayashi H, Kikuyama S. Molecular cloning of newt sex pheromone precursor cDNAs: evidence for the existence of species-specific forms of pheromones. FEBS Letters 1999;457:400–4. Iwata T, Toyoda F, Yamamoto K, Kikuyama S. Hormonal control of urodele reproductive behavior. Comp Biochem Physiol 2000;B126:221–9. Iwata T, Yada T, Shioda S, Toyoda F, Kikuyama S. Effect of prolactin and androgen on the expression of the female-attracting pheromone silefrin in the abdominal gland of the newt, Cynops ensicauda. Biol Reprod 2000;63:1867–72. Iwata T, Kawahara G, Yamamoto K, Zhou CJ, Nakajo S, Shioda S, Kikuyama S. Signal-transduction pathway mediating the action of a newt pheromone, sodefrin. Abstracts 14th International Congress of Comparative Endocrinology, Sorrento; 2001. p. 68. Iwata T, Conlon JM, Nakada T, Toyoda F, Yamamoto K, Kikuyama S. Processing of multiple forms of preprosodefrin in the abdominal gland of the red-bellied newt Cynops pyrrhogaster: regional and individual differences in preprosodefrin gene expression. Peptides 2004;25:1537–43. Kikuyama S, Toyoda F, Ohmiya Y, Matsuda K, Tanaka S, Hayashi H. Sodefrin: a female-attracting peptide pheromone in newt cloacal glands. Science 1995;267:1643–45. Kikuyama S, Yazawa T, Abe S, Yamamoto K, Iwata T, Hoshi K, Hasunuma I, Mosconi G, Polzonetti-Magni AM. Newt prolactin and its involvement in reproduction. Can J Physiol Pharmacol 2000;78:984–93. Matsukawa H, Hasunuma I, Kato T, Yamamoto K, Miura S, Fujita T, Kikuyama S. Expression of prolactin receptor mRNA in the abdominal gland of the newt Cynops ensicauda. Comp Biochem Physiol A Mol Integr Physiol 2004;138:79–88. Matsumoto A, Arai Y, Toyoda F, Kikuyama S, Prins GS. Immunohistochemical analysis of androgen receptor in the abdominal glands of the cloaca of male red-bellied newts, Cynops pyrrhogaster. Zool Sci 1996;13:429–33. Nakada T, Toyoda F, Iwata T, Ishizuka Y, Conlon JM, Kato T, Kikuyama S. Isolation and bioactivity of a sodefrin variant from the abdominal gland of the newt, Cynops pyrrhogaster. Abstracts 15th International Congress of Comparative Endocrinology, Boston; 2005. p. 102. Rollmann SM, Houek LD, Feldhoff RC. Proteinaceous pheromone affecting female receptivity in a terrestrial salamander. Science 1999;285:1907–9. Toyoda F, Tanaka S, Matsuda K, Kikuyama S. Hormonal control of response to and secretion of sex attractants in Japanese newts. Physiol Behav 1994;55:569–76. Toyoda F, Hayakawa Y, Ichikawa M, Kikuyama S. Olfactory responses to a female-attracting pheromone in the newt, Cynops pyrrhogaster. In: Johnston RE, Müller-Schwärz D, Sorensen PW. editors. Advances in Chemical Signals in Vertebrates. New York: Kluwer Academic/Plenum Publishers; 1999. pp. 607–15. Toyoda F, Yamamoto K, Ito Y, Tanaka S, Yamashita M, Kikuyama S. Involvement of arginine vasotocin in reproductive events in the male newt, Cynops pyrrhogaster. Horm Behav 2003;44:346–53. Wabnitz PA, Bowie JH, Tyler MJ, Wallace JC, Smith BP. Aquatic sex pheromone from a male tree frog. Nature 1999;401:444–5. Yamamoto K, Toyoda F, Tanaka S, Hayashi H, Kikuyama S. Radioimmunoassay of a newt sex pheromone, sodefrin, and the influence of hormone on its level in the abdominal gland. Gen Comp Endocrinol 1996;104:356–63. Yamamoto K, Kawai Y, Hayashi T, Ohe Y, Hayashi H, Kawahara G, Toyoda F, Iwata T, Kikuyama S. Silefrin, a sodefrin-like pheromone in the abdominal gland of the sword-tailed newt, Cynops ensicauda. FEBS Letters 2000;472:267–70.

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49 Amphibian Neurohypophysial Peptides SUNNY K. BOYD

35]. In all amphibians investigated, AVT and MT have been found, with the exception of Bufo regularis in which [Ser5,Ile8]-oxytocin replaces mesotocin (seritocin; Table 1 [22]). Finally, anuran amphibians also possess peptides called hydrins.

ABSTRACT The amphibian neurohypophysis releases primarily two peptides: arginine vasotocin (AVT) and mesotocin (MT). Both are synthesized in multiple brain areas. Three specific receptor subtypes have been identified. The VT1R is specific for AVT, found in brain and kidney, and is coupled to a PKC-mediated pathway. The VT2R has a high affinity for AVT and hydrins and is found primarily in the skin and bladder, where it utilizes the cAMP signaling pathway. The MTR is specific for MT, couples to a PKC-linked pathway, and is found in brain and peripheral tissues. Biological actions of AVT include control of osmoregulation, behavior, reproductive tract contractions, and the cardiovascular system. The actions of MT are poorly understood. Finally, the neurohypophysis produces hydrins that are extended forms of AVT with specific effects on skin and urinary bladder.

STRUCTURE OF THE PRECURSOR mRNA/GENE The nonapeptides, AVT and MT, are part of a conserved family. All vertebrates examined to date possess AVT, and it is thus considered the precursor peptide in this family [5]. AVT is replaced by arginine vasopressin (AVP) in most mammals. MT is found not only in amphibians but also in birds and reptiles. Isotocin replaces MT in most fish, and oxytocin (OT) is the mammalian homolog. All have a nine-amino-acid peptide backbone with a disulfide bridge between conserved cysteine residues at positions 1 and 6. All possess an NH2-blocked C-terminal glycine. The primary structure of the mRNA for AVT and MT has been reported for only two amphibians: the toad Bufo japonicus [55] and the caecilian Typhlonectes natans (GenBank accession nos. AAF76848, AAF76847). Following a hydrophobic signal sequence, the amino acids of AVT or MT are represented. Next, a tripeptide is found that may facilitate processing or become incorporated into hydrin. This tripeptide is followed by a 93-amino-acid neurophysin domain and a 36-amino-acid glycoprotein domain. Although these domains result in the production of two peptides in mammals, this is not the case in amphibians, where a single large neurophysin is produced [48]. In the MT precursor, a 93- or 94-amino-acid peptide neurophysin also follows, but a glycoprotein is not present.

DISCOVERY The class Amphibia contains three orders: the Anurans (frogs and toads), the Urodeles (salamanders and newts), and the Gymnophionans (caecilians). In the early 1900s, it was already recognized that the neurohypophysis (posterior pituitary) of frogs contained “active principles” that altered blood pressure [33]. The structure of these principles was first elucidated in an anuran amphibian for AVT (Table 1) in 1960 [3] and for MT (Table 1) in 1964 [4]. AVT was characterized in a caecilian in 1998 [34] but the structure of MT was only recently determined with the cloning of the gene (2000, GenBank accession no. AAF76848). The structure of urodele peptides has not been reported although it is presumed that urodeles possess AVT and MT [21, Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

328 / Chapter 49 DISTRIBUTION OF AVT AND MT Amphibian neurohypophysial peptides are synthesized in a variety of brain nuclei [35, 47, 50, 65]. In all three amphibian orders, the most prominent cell group is the magnocellular preoptic area. It is likely that these cells are the primary source of AVT and MT released from the neurohypophysis. AVT cell bodies can also be found in the bed nucleus of the stria terminalis, preoptic area, ventral thalamus, suprachiasmatic nucleus, hypothalamus, and midbrain tegmentum. On the other hand, MT cells are restricted primarily to diencephalic regions. Fibers are widespread and these peptides likely function as neuromodulators. AVT concentrations in some brain areas are sexually dimorphic and steroid-sensitive [14, 15, 17].

RECEPTOR STRUCTURE AND DISTRIBUTION The MT receptor (MTR) and two subtypes of an AVT receptor have been cloned in anuran amphibians. All three belong to the G-protein coupled receptor family, with typical seven transmembrane domains. One AVT receptor subtype closely resembles the mammalian AVP receptor subtype V1a [2]. The cDNA for this receptor (VT1R) encodes a protein of 419 amino acids. The potency of ligands for this receptor is AVT > OT > AVP ≈ MT. The VT1R preferentially couples to the PKCmediated signaling pathway. The second AVT receptor subtype (VT2R) is more similar to the mammalian V2R [43]. The receptor protein is 363 amino acids. The potency of ligands for this receptor is AVT = hydrin 1 > hydrin 2 = AVP > MT = OT > isotocin. The VT2R likely utilizes the adenylate cyclase-cAMP signaling pathway. Finally, the MTR mRNA codes for a receptor of 384–389 amino acids that shares significant homology with the OT receptor [2, 8, 43]. The sensitivity of the MTR to ligands is MT > OT > AVT > AVP [2] or MT > AVT = OT > AVP > hydrin 1, isotocin, hydrin 2 [8], depending on the species. The frog MTR likely couples exclusively to the PKC-linked signaling pathway [2, 8]. The VT1R mRNA is abundant in brain, anterior pituitary, heart, adrenal gland, kidney, and oviduct [2]. In brain, the distribution of binding sites detected with 3H-AVP autoradiography is very similar to the distribution of the mRNA [2, 13, 18, 68]. Putative receptors are abundant in accumbens, pallium, striatum, lateral amygdala, preoptic area, magnocellular nucleus, hypothalamus, thalamus, and optic tectum. In the pituitary, VT1R mRNA is found in the anterior pituitary and the intermediate lobe but not in the neurohypophysis [2]. Binding studies with 3H-AVP show receptors in the anterior pituitary but, in contrast, also show receptors

in the neurohypophysis. This may reflect the presence of a third subtype of AVT receptor in amphibians, not detected by the VT1R probe. The presence of a VT1R subtype receptor in amphibian kidney is also supported by autoradiographic evidence [9, 11, 42]. Binding sites for 3H-AVP or 125I-OVTA are found specifically located over the glomeruli but not renal tubules. These sites have a ligand specificity more similar to mammalian V1 receptors than V2 receptors. The mRNA for the VT2R is expressed in brain, heart, kidney, urinary bladder, and pelvic skin patch [30, 43]. MTR mRNA is abundant in the brain of anurans [2, 8, 43]. In situ hybridization shows highest levels in pallium, amygdala, ventral striatum, anterior preoptic area, nucleus of the periventricular organ, posterior tuberculum, ventral hypothalamic nucleus, thalamic nuclei, and optic tectum [2]. The MTR mRNA, like that for VT1R, is found in the anterior pituitary and intermediate lobe but not in the neurohypophysis. The neural function of MT is unknown. The MTR mRNA is also found in several peripheral tissues, including heart, adrenal gland, kidney, urinary bladder, skeletal muscle, fat body, and testis [2, 8, 43]. Importantly, the receptor was not found in the oviduct [2]. This suggests that, despite the structural similarities with the OT receptor, the MTR does not play a homologous role in reproductive tract smooth muscle contraction.

MAJOR BIOLOGICAL ACTIONS OF AVT AND MT Osmoregulatory Effects AVT control of osmoregulation in amphibians is complex and involves several different target organs: the kidneys, the urinary bladder, and the skin. At the renal level, AVT causes antidiuresis first by constricting preglomerular arteries and causing a reduction in glomerular filtration rate by as much as 85% [54, 57, 70]. The glomerular action is supported by the location of receptors over the glomeruli and the presence of VT1R mRNA. Whether AVT alters tubule reabsoption in amphibians is more controversial. AVT can cause antidiuresis by acting directly on tubules in a few species [25, 57, 69, 70]. Binding sites for radiolabeled AVT analogs have not been located over tubules, however, and it is clear in some species that AVT has no tubular effects [9, 11, 42, 57]. Mesotocin increases glomerular filtration rate and is diuretic in amphibians [24, 28, 56, 70]. The anuran urinary bladder served as the archetypal tissue for research on the water permeability of membranes for decades. Anurans can store fluid equivalent to 50% of body weight in the bladder [10]. AVT

Amphibian Neurohypophysial Peptides / 329 stimulates movement of water from the bladder back into the vasculature [70]. This likely occurs via the VT2R and it is accompanied by an increase in cAMP and the expression of aquaporins [1, 38, 43]. The anuran bladder also possesses AVT-modulated sodium channels [40]. The permeable skin of amphibians allows the unique movement of water across that surface. AVT increases water permeability of ventral pelvic skin and facilitates rehydration of anurans and urodeles [19, 38, 70]. VT2 receptor mRNA and protein is expressed in the pelvic skin and AVT regulates expression of water channel aquaporins in this tissue [29]. Mesotocin has been reported to increase cutaneous water permeability, but MTR mRNA has not been found here [2, 8, 19, 43]. Neither AVT nor MT alter water permeability of the skin of a caecilian amphibian [66]. Finally, active sodium transport across skin is also facilitated by AVT in some but not all species [70].

Behavioral Effects In recent decades, the behavioral effects of neurohypophysial peptides in amphibians have received the most attention. In many anurans, AVT modulates the display of vocal behavior [18, 51, 71]. The peptide facilitates the advertisement call used to attract mates, while it inhibits the release call given by unreceptive females. In addition, AVT stimulates the attraction of female anurans to the calls of males and alters locomotor behaviors [12, 16]. AVT also alters reproductive behaviors of urodeles. AVT stimulates amplectic clasping, pheromone release, and egg-laying behavior [52, 67]. Effects of AVT are likely occurring via action in the central nervous system [12, 49, 59, 67]. Androgens and corticosterone interact with AVT to control amphibian behaviors [18, 51, 52, 59, 71].

Reproductive Tract Effects As in other vertebrate classes, the amphibian oviduct shows robust smooth muscle contractions following exposure to neurohypophysial peptides [44]. Unexpectedly, however, the oxytocin homolog, MT is not the most potent peptide. Instead, AVT causes oviduct contractions [26, 31, 63]. Sex steroids increase the responsiveness of the oviduct to AVT [26]. In an analogous fashion, AVT stimulates contractions of the male urodele Wolffian duct [72].

Cardiovascular Effects It has been known since 1904, that pituitary extracts alter the circulatory system of the frog [32], but this has received little attention. AVT is vasopressor and causes

constriction of vessels in vitro [20, 23]. Although AVT causes bradycardia in vivo, when autonomic receptors are blocked, AVT increases heart rate and contractile force [20, 64]. MT acts as a vasodepressor in amphibians [20]. All three receptor subtypes (VT1R, VT2R, and MTR) are found in the heart, but it is unknown which receptors are present in the vasculature [2, 8, 43]. It has been proposed that the cardiovascular effects of AVT are the most primitive and that these gave rise to the osmotic effects of the peptide in amphibians and other tetrapods [58].

Other Effects In amphibians, the adrenal gland homolog is the interrenal gland, where chromaffin cells are intermingled with steroidogenic cells. AVT-immunoreactivity is found in virtually all chromaffin granules [45]. AVT causes a rapid increase in corticosterone and aldosterone output [27, 41, 45]. The rank order potency of ligands suggests a VT2R is responsible [46]. However, the effect is via phosphoinositide-specific phospholipase C, and VT2R mRNA has not been found in the interrenal gland [2, 8, 43, 46]. AVT stimulates hepatic glycogenolysis and increases glycogen phosphorylase a activity in representative anuran and urodele amphibians [7, 36]. This effect differs significantly from the stimulation of glycogenolysis in mammalian liver by AVP, however, because it appears to work via a VT2 subtype receptor and coupling to adenylate cyclase [37, 39]. VT2R mRNA has not been reported in the liver but the liver receptor subtype may differ from the bladder VT2R [30, 43].

STRUCTURE AND FUNCTION OF HYDRINS Neurohypophysial extracts of anurans were first reported in 1961 to contain a third class of peptides [53]. These peptides were sequenced and named “hydrins” in 1989 (Table 1; [60]). Hydrin 2 is broadly distributed across anuran families. In Xenopus laevis, two variants of hydrin (termed hydrin 1 and 1′) are found instead. Hydrins have not been detected in any urodele or in any other vertebrate. Hydrins likely result from differential processing of the AVT preprohormone [61]. The result is an extended peptide that is not amidated. Hydrins are as active as vasotocin in the anuran bladder, more active on anuran skin but inactive in the kidney [6, 62]. Given the high affinity of the VT2R for hydrins, it is likely that a receptor of this subtype is responsible for the biological actions of hydrins. Differences in physiological effects of AVT and hydrins on target tissues, however, suggest that two subtypes of VT2R may exist [62].

330 / Chapter 49 TABLE 1.

Vasotocin Mesotocin Seritocin Hydrin 1 Hydrin 1′ Hydrin 2

Amphibian neurohypophysial peptides.

1

2

3

4

5

6

7

Cys— — — — —

Tyr— — — — —

Ile— — — — —

Gln— — — — —

Asn— Ser— — —

Cys— — — — —

Pro— — — — —

8 ArgIleIle— — —

9 Gly— — — — —

(NH2) (NH2) (NH2) Gly-Lys-Arg(OH) Gly-Lys(OH) Gly(OH)

Dashes indicate residues identical with those of vasotocin.

References [1] Abrami L, Capurro C, Ibarra C, Parisi M, Buhler JM, Ripoche P. Distribution of messenger RNA encoding the FA-CHIP water channel in amphibian tissues—effects of salt adaptation. J Membr Biol 1995;143:199–205. [2] Acharjee S, Do-Rego JL, Oh DY, Moon JS, Ahn RS, Lee K, et al. Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors. J Mol Endocrinol 2004;33:293–313. [3] Acher R, Chauvet J, Lenci MT, Morel F, Maetz J. Presence d’une vasotocine dans la neurohypophyse de la grenouille (Rana esculenta L.). Biochim Biophys Acta 1960;42:379–80. [4] Acher R, Chauvet J, Chauvet MT, Crepy D. Phylogenie des peptides neurohypophysaires: Isolement de la mesotocine (ileu8-ocytocine) de la grenouille, intermediaire entre la ser4ile8-ocytocine des poissons osseux et l’ocytocine des mammiferes. Biochim Biophys Acta 1964;90:611–3. [5] Acher R. Neurohypophyseal peptide systems—processing machinery, hydroosmotic regulation, adaptation and evolution. Regul Pept 1993;45:1–13. [6] Acher R, Chauvet J, Rouille Y. Adaptive evolution of water homeostasis regulation in amphibians: Vasotocin and hydrins. Biol Cell 1997;89:283–91. [7] Ade T, Segner H, Hanke W. Hormonal response of primary hepatocytes of the clawed toad, Xenopus laevis. Exp Clin Endocrinol Diabet 1995;103:21–7. [8] Akhundova A, Getmanova E, Gorbulev V, Carnazzi E, Eggena P, Fahrenholz F. Cloning and functional characterization of the amphibian mesotocin receptor, a member of the oxytocin/ vasopressin receptor superfamily. Eur J Biochem 1996;237: 759–67. [9] Ammar A, Rajerison RM, Roseau S, Blochfaure M, Butlen D. Frog glomerular vasotocin receptors resemble mammalian V-1b receptors. Am J Physiol-Regulat Integr Compar Physiol 1994;36: R1198–208. [10] Bentley PJ. The physiology of the urinary bladder of amphibia. Bio Rev Cambridge Philos Soc 1966;42:275–316. [11] Boyd SK, Moore FL. Autoradiographic localization of putative arginine vasotocin receptors in the kidney of a urodele amphibian. Gen Comp Endocrinol 1990;78:344–50. [12] Boyd SK. Effect of vasotocin on locomotor activity in bullfrogs varies with developmental stage and sex. Horm Behav 1991; 25:57–69. [13] Boyd SK, Moore FL. Gonadectomy reduces the concentrations of putative receptors for arginine vasotocin in the brain of an amphibian. Brain Res 1991;541:193–7. [14] Boyd SK, Moore FL. Sexually dimorphic concentrations of arginine vasotocin in sensory regions of the amphibian brain. Brain Res 1992;588:304–6.

[15] Boyd SK, Tyler CJ, Devries GJ. Sexual dimorphism in the vasotocin system of the bullfrog (Rana catesbeiana). J Comp Neurol 1992;325:313–25. [16] Boyd SK. Arginine vasotocin facilitation of advertisement calling and call phonotaxis in bullfrogs. Horm Behav 1994;28:232–40. [17] Boyd SK. Gonadal steroid modulation of the vasotocin concentrations in the bullfrog brain. Neuroendocrinology 1994;60: 150–6. [18] Boyd SK. Brain vasotocin pathways and the control of sexual behaviors in the bullfrog. Brain Res Bull 1997;44:345–50. [19] Brown SC, Brown PS. Water balance in the California newt, Taricha torosa. Am J Physiol 1980;238:R113–R8. [20] Chan DKO. Comparative physiology of the vasomotor effects of neurohypophysial peptides in the vertebrates. Amer Zool 1977;17:751–61. [21] Chauvet J, Rouille Y, Michel G, Ouedraogo Y, Acher R. Adaptative differential processing of neurohypophyseal provasotocin in amphibians—occurrence of hydrin-2 (vasotocinyl-glycine) in anura but not in urodela. Comptes Rendus Acad Sci Ser III-Sci Vie-Life Sci 1991;313:353–8. [22] Chauvet J, Michel G, Ouedraogo Y, Chou J, Chait BT, Acher R. A new neurohypophyseal peptide, seritocin (ser(5),ile(8) -oxytocin), identified in a dryness resistant African toad, Bufo regularis. Int J Pept Protein Res 1995;45:482–7. [23] Chiu KW, Lee YC, Pang PKT. The cardiovascular and renal effects of hydrins and arginine vasotocin in frogs. Gen Comp Endocrinol 1993;91:1–7. [24] Galligallardo SM, Pang PKT, Oguro C. Renal responses of the Chilean toad, Calyptocephalella caudiverbera, and the mud puppy, Necturus maculosus, to mesotocin. Gen Comp Endocrinol 1979;37:134–6. [25] Garland HO, Henderson IW, Brown JA. Micropuncture study of renal responses of urodele amphibian Necturus maculosus to injections of arginine vasotocin and an anti-aldosterone compound. J Exp Biol 1975;63:249–64. [26] Guillette LJ, Norris DO, Norman MF. Response of amphibian (Ambystoma tigrinum) oviduct to arginine vasotocin and acetylcholine in vitro—influence of steroid-hormone pretreatment in vivo. Comp Biochem Physiol C-Pharmacol Toxicol Endocrinol 1985;80:151–4. [27] Gupta OP, Hanke W. Regulation of interrenal secretion in the axolotl, Ambystoma mexicanum. Exp Clin Endocrinol 1994;102: 299–306. [28] Hartenstein HR, Stiffler DF. Renal responses to mesotocin in adult Ambystoma tigrinum and Notophthalmus viridescens. Exp Biol 1990;48:373–7. [29] Hasegawa T, Tanii H, Suzuki M, Tanaka S. Regulation of water absorption in the frog skins by two vasotocin-dependent water channel aquaporins, AQP-h2 and AQP-h3. Endocrinology 2003;144:4087–96.

Amphibian Neurohypophysial Peptides / 331 [30] Hasegawa T, Sugawara Y, Suzuki M, Tanaka S. Spatial and temporal expression of the ventral pelvic skin aquaporins during metamorphosis of the tree frog, Hyla japonica. J Membr Biol 2004;199:119–26. [31] Heller H, Ferreri E, Leathers DHG. The effect of neurohypophysial hormones on the amphibian oviduct in vitro, with some remarks on the histology of this organ. J Endocrinol 1970;47:495– 509. [32] Herring PT. The action of pituitary extracts on the heart and circulation of the frog. J Physiol (Lond) 1904;31:429–37. [33] Herring PT. Further observations upon the comparative anatomy and physiology of the pituitary body. Quart J Exp Physiol 1913; 6:73–108. [34] Hilscher-Conklin C, Conlon JM, Boyd SK. Identification and localization of neurohypophysial peptides in the brain of a caecilian amphibian, Typhlonectes natans (Amphibia : Gymnophiona). J Comp Neurol 1998;394:139–51. [35] Hollis DM, Chu J, Walthers EA, Heppner BL, Searcy BT, Moore FL. Neuroanatomical distribution of vasotocin and mesotocin in two urodele amphibians (Plethodon shermani and Taricha granulosa) based on in situ hybridization histochemistry. Brain Res 2005;1035:1–12. [36] Janssens PA, Kleineke J, Caine AG. Calcium-independent stimulation of glycogenolysis by arginine vasotocin and catecholamines in liver of the axolotl (Ambystoma mexicanum) in vitro. J Endocrinol 1986;109:75–84. [37] Janssens PA, Grigg JA. Hormonal-regulation of hepatic glycogenolysis in the toad, Xenopus laevis, is mediated by cyclic-AMP and not Ca-2+. Gen Comp Endocrinol 1987;67:227–33. [38] Jo I, Harris HW. Molecular mechanisms for the regulation of water transport in amphibian epithelia by antidiuretic hormone. Kidney Int 1995;48:1088–96. [39] Kleineke JW, Janssens PA. Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes—extracellular origin and control by cAMP. Am J Physiol 1993;265:C1281–C8. [40] Kleyman TR, Smith PR, Benos DJ. Characterization and localization of epithelial Na+ channels in toad urinary bladder. Am J Physiol 1994;266:C1105–C11. [41] Kloas W, Hanke W. Neurohypophyseal hormones and steroidogenesis in the interrenals of Xenopus laevis. Gen Comp Endocrinol 1990;80:321–30. [42] Kloas W, Hanke W. Localization and quantification of nonapeptide binding sites in the kidney of Xenopus laevis—evidence for the existence of 2 different nonapeptide receptors. Gen Comp Endocrinol 1992;85:71–8. [43] Kohno S, Kamishima Y, Iguchi T. Molecular cloning of an anuran V-2 type arg(8) vasotocin receptor and mesotocin receptor: Functional characterization and tissue expression in the Japanese tree frog (Hyla japonica). Gen Comp Endocrinol 2003;132:485–98. [44] La Pointe J. Comparative physiology of neurohypophysial hormone action on the vertebrate oviduct-uterus. Amer Zool 1977;17:763–73. [45] Larcher A, Delarue C, Idres S, Lefebvre H, Feuilloley M, Vandesande F, et al. Identification of vasotocin-like immunoreactivity in chromaffin cells of the frog adrenal-gland—effect of vasotocin on corticosteroid secretion. Endocrinol 1989;125:2691– 700. [46] Larcher A, Delarue C, Homodelarche F, Kikuyama S, Kupryszewski G, Vaudry H. Pharmacological characterization of vasotocin stimulation of phosphoinositide turnover in frog adrenal gland. Endocrinology 1992;130:475–83. [47] Lowry CA, Richardson CF, Zoeller TR, Miller LJ, Muske LE, Moore FL. Neuroanatomical distribution of vasotocin in a urodele amphibian (Taricha granulosa) revealed by immunohis-

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332 / Chapter 49 [67] Toyoda F, Yamamoto K, Ito Y, Tanaka S, Yamashita M, Kikuyama S. Involvement of arginine vasotocin in reproductive events in the male newt Cynops pyrrhogaster. Horm Behav 2003;44: 346–53. [68] Tripp SK, Moore FL. Autoradiographic characterization of binding sites labeled with vasopressin in the brain of a urodele amphibian. Neuroendocrinology 1988;48:87–92. [69] Uchiyama M. Sites of action of arginine vasotocin in the nephron of the bullfrog kidney. Gen Comp Endocrinol 1994;94:366–73.

[70] Warburg MR. Hormonal effect on the osmotic, electrolyte and nitrogen-balance in terrestrial amphibia. Zool Sci 1995; 12:1–11. [71] Wilczyniski W, Lynch KS, O’Bryant EL. Current research in amphibians: Studies integrating endocrinology, behavior, and neurobiology. Horm Behav 2005; in press. [72] Zoeller RT, Lais LT, Moore FL. Contractions of amphibian Wolffian duct in response to acetylcholine, norepinephrine, and arginine vasotocin. J Exp Zool 1983;226:53–7.

C

H

A

P

T

E

R

50 Bombinins MARIA LUISA MANGONI, DANIELA FIOCCO, DONATELLA BARRA, AND MAURIZIO SIMMACO

The crude secretion of B. variegata was known to be strongly hemolytic [2], whereas the bombinin-related peptides were antimicrobial but not cytolytic. From the inspection of the sequences of the cDNAs encoding the bombinin precursors, the presence of a second putative 20-residue peptide was noticed, giving rise to a set of novel hydrophobic peptides slightly different from each other [3, 13]. These peptides were subsequently isolated by reversed-phase HPLC from skin secretions of B. variegata and proved to be highly hemolytic [11]. Therefore, they were termed bombinins H, as they are hydrophobic and show antibacterial and hemolytic activities (Table 1, H1–H5). Differences among the various bombinins H were observed at three positions, but the most surprising result was the presence of a Dalloisoleucine in the second position of the sequence in the late HPLC-eluting peaks. A change of configuration around the α-carbon of the gene-encoded isoleucine was hypothesized, leading to the formation of D-alloisoleucine as the result of a posttranslational modification of unknown origin. The enzyme responsible of such modification was purified from skin secretion of B. variegata, partially sequenced and cloned. Its functional characterization as well as sequence comparison with homologs from other vertebrates was recently reported [5]. From B. orientalis secretions, two shorter (17 residues), more hydrophobic versions of the previously isolated bombinins H were found [3, 12], containing an L- or D-leucine as the second residue and lacking the lysine pair at the C-terminus (Table 1, H6 and H7). From the sequences of two bombinin genes [9, 10], two additional peptides related to bombinins H were predicted (Table 1, GH-1 and GH-2), but they were never isolated from B. orientalis skin secretion. Finally, from B. maxima a series of bombinin/ bombinin H-related peptides have been described, the maximins, but the presence of a D-amino acid was

ABSTRACT The bombinin family of antimicrobial peptides is constituted by a number of molecules isolated from skin secretions of frogs belonging to the genus Bombina. These peptides can be grouped into two distinct subfamilies: the bombinin and the bombinin H. The former comprises 27-amino-acid residue peptides with antimicrobial activity against gram-negative and gram-positive bacteria and fungi; the latter includes 20- or 17-residue hemolytic peptides, some of which contain a D-amino acid in the second position of the sequence. The presence of this posttranslational modification confers the two isoforms’ distinctive biological properties, the D-amino acid–containing peptide being always more active than the corresponding L-isomer.

DISCOVERY The term bombinins refers to a family of antimicrobial peptides isolated from skin secretions of frogs belonging to the genus Bombina (B. variegata, B. bombina, B. orientalis, B. maxima). In the seventies, the sequence of a 24-residue peptide from B. variegata, named bombinin, was reported [2]. Later, using peptide sequencing and cDNA cloning, a family of antimicrobial peptides related to bombinin was isolated from both B. variegata [13] and B. orientalis [3]. These peptides differed from each other by only one or a few amino acids, showing a variable amino-terminal sequence and an identical carboxy-terminal region (Table 1). They all have an amidated C-terminus and mostly contain 27amino-acid residues, 3 more than bombinin; it is likely that the original bombinin sequence, probably obtained from a mixture of similar peptides, contained some sequencing errors. Handbook of Biologically Active Peptides

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334 / Chapter 50 TABLE 1. Sequences of bombinin and bombinin H-related peptides. Bombinin-like peptides (BLP) Bombinin Clone 7* 9* 10* 13* 14* 33* 42* BLP-1 BLP-2 BLP-3§ BLP-4 BLP-5 BLP-6 BLP-7§ Maximin 1 Maximin 2 Maximin 3 Maximin 4 Maximin 5 Bombinins H H1 H2 H3 H4# H5# H6 H7# GH-1§ GH-2§ Maximin H1 Maximin H2 Maximin H3 Maximin H4 Maximin H5*

GIG-A-LSA-KGALKGLAKGLAEHFAN-NH2 GIGGALLSAGKSALKGLAKGLAEHFAN-NH2 GIGGALLSAAKVGLKGLAKGLAEHFAN-NH2 GIGASILSAAKVGLKGLAKGLAEHFAN-NH2 GIGGALISAGKSALKGLAKGLAEHFAN-NH2 GIGGALLSDAKVGLKGLAKGLAEHFAN-NH2 GIGASILSAGKSALKGFAKGLAEHFAN-NH2 GIGGALLSAAKVGLKGLAKGLAEHFAN-NH2 GIGASILSAGKSALKGLAKGLAEHFAN-NH2 GIGSAILSAGKSALKGLAKGLAEHFAN-NH2 GIGAAILSAGKSALKGLAKGLAEHF---NH2 GIGAAILSAGKSIIKGLANGLAEHF---NH2 GIGATILSVGKSALKGLAK----HF---NH2 GIGAALLSAGKSALKGLAKGLAEHFAN-NH2 GIGGALLSAGKSALKGLAKGLAEHFAN-NH2 GIGTKILGGVKTALKGALKELASTYAN-NH2 GIGTKILGGVKTALKGALKELASTYVN-NH2 GIGGKILSGLKTALKGAAKELASTYLH GIGGVLLSAGKAALKGLAKVLAEKYAN-NH2 SIGAKILGGVKTFFKGALKELASTYLQ IIGPVLGMVGSALGGLLKKI-NH2 IIGPVLGLVGSALGGLLKKI-NH2 IIGPVLGMVGSALGGLLKKI-NH2 IIGPVLGLVGSALGGLLKKI-NH2 LIGPVLGLVGSALGGLLKKI-NH2 IIGPILGLVSNALGGLL----NH2 ILGPILGLVSNALGGLL----NH2 IIGPVLGLVGKPLESLLE ILGPVLDLVGRALRGLLKKI-NH2 ILGPVISTIGGVLGGLLKNL-NH2 ILGPVLSMVGSALGGLIKKI-NH2 ILGPVLGLVGNALGGLIKKI-NH2 ILGPVISKIGGVLGGLLKNL-NH2 ILGPVLGLVSDTLDDVLGIL-NH2

*Sequences deduced from cDNA. §Sequences deduced from BLP-3 [9] and BLP-7 [10] genes, respectively. #D-amino acids in position 2 are italicized.

not investigated [6]. Their sequences are included in Table 1. The obvious comparison with the other amphibian peptides containing a D-amino acid—that is, the opioid peptides dermorphin and deltorphins (see Chapter 41), allows some considerations to be made: (1) in the case of the opioid peptides, the L- to D-amino acid conversion is complete, as no trace of the L-amino acidcontaining mature peptides has ever been found. In contrast, appreciable amounts of the two types of bombinins H are present in the skin secretion, although the D-amino acid–containing form is always prevalent; (2) for both dermorphin and deltorphins the L-amino

acid–containing isoform is completely inactive, whereas biological activity, although different in specificity and potency toward microorganisms, can be attributed to both bombinin H isoforms (see following); (3) for the Bombina peptides, the presence of the D-amino acid is probably related to increased stability: In fact, bombinins H3 to -5 are completely stable under conditions where bombinins H1 and -2 are partly digested by aminopeptidases [11]. However, in all cases, the presence of the D-amino acid influences the peptide conformation in solution. For bombinins H, this causes a different interaction with bacterial membranes; for the opiates this is essential for the interaction with the receptor.

Bombinins / 335 2 × 106

θ

1 × 106

0

–1 × 106 190

200

210

220 230 nm

240

250

260

FIGURE 1. CD spectra of bombinin (clone 42, Table 1) in water (dotted line) and 30% trifluoroethanol (solid line).

SOLUTION CONFORMATION Both bombinins and bombinins H are able to adopt α-helical conformations in hydrophobic environments. However, in bombinins the helical conformation is mostly amphipathic, as they contain polar residues properly spaced in the sequence and have a net charge of +4–+5. In contrast, bombinins H are highly hydrophobic with a net charge varying from +3 to +1 and positive charges confined in the C-terminal region (H1–H5) and/or at the N-terminus (H6–H7). CD spectra in water or in 70% trifluoroethanol show a change for both bombinin types from a random to a α-helical conformation (see Fig. 1).

like peptide (BLP-3) and one copy of a bombinin Hrelated peptide (GH-1, see Table 1), whereas BLP-7 contains a single copy of each peptide (BLP-7 and GH2, see Table 1). These findings rule out a speciesspecific variation of exon 2, showing in B. orientalis the coexistence in the genome of the same type of construct as that found in B. variegata. The nucleotide sequences of exon 1 and the acidic and spacer peptides in the two genes are very similar. Canonical Lys-Arg sequences are flanking the putative peptides for the correct processing, with the Gly residue necessary for the C-terminal amidation. Also the 3′-non coding regions are highly conserved in the two genes. These considerations allow one to rule out an alternative splicing mechanism in the expression of the bombinin genes, as already observed for other antimicrobial peptide genes from different sources. Moreover, walking chromosome analysis has indicated that this gene family is not densely clustered [10]. In the promoter region, both genes contain NF-κB and NF-IL6 putative binding sites: although their relative location is inverted, the preservation of their distance from the translation initiation site and the proximity of the binding sites to each other are probably critical for synergistic activation. It can be postulated that in amphibians a common regulation of the expression of antimicrobial peptide genes occurs, involving cooperation of a multiplicity of transcription factors, such as those acting in the activation of the mammalian acute-phase response.

BIOLOGICAL ACTIVITY STRUCTURE OF THE PRECURSOR mRNA/GENE The cDNA sequence of several clones from B. variegata presented the same structural organization: the sequences of two putative peptides, a bombinin-like peptide (see Table 1) and a bombinin H-related peptide, separated by spacer peptides [13, 14]. By contrast, in B. orientalis the available cDNA sequence showed the presence of three putative peptides—that is, two identical copies of a bombinin peptide and one copy of a bombinin H-type peptide [3]. Two genes coding for peptides BLP-3 and BLP-7 from B. orientalis were sequenced, revealing a different organization [9, 10]. Both genes contain two exons separated by a large intron of 2045 and 1337 bp, respectively. Exon 1 codes for the signal peptide; exon 2 includes the remaining coding region comprising the sequences of the mature peptides as well as acidic and spacer peptides. The main difference between the two genes is that BLP-3 codes for two copies of a bombinin-

The antimicrobial activity of bombinins and bombinins H was tested by use of the inhibition zone assay on agarose plates [4]. The lethal concentration (LC)— that is, the lower peptide concentration inhibiting bacterial growth—is calculated from the diameter of the zones obtained in serial dilutions of the test substance. As shown in Table 2, the LC values of BLP-1 and -3 on gram-negative strains are similar to those on grampositive bacteria and in the same range as those reported for the antimicrobial peptide cecropin P1 [1]. A stronger effect was displayed against the yeast Candida albicans, and this trend in activity strongly reflects what was found for the maximins [6]. With regard to bombinins H, the data shown in Table 2 clearly indicate that only H2 and H4 are active toward the selected microorganism, with the exception of Aeromonas hydrophila Bo-3N, a member of the B. orientalis natural flora, resistant to most of the antimicrobial peptides from frog skin. In all cases, the D-amino acidcontaining H4 was more potent than its L isomer H2, displaying a 1.4–3 fold lower LC value.

336 / Chapter 50 TABLE 2. Antimicrobial activity of bombinin/bombinin H-related peptides. Peptide

BLP-1

BLP-3

H2

H4

H6

H7

GH-1D

GH-1L

Cecropin P1

Lethal concentration (mM) Gram-negative bacteria Escherichia coli D21 Escherichia coli D22 Yersinia pseudotuberculosis YPIII Pseudomonas aeruginosa ATCC 15692

1.7 1.5 0.8 6.3

3.0 0.9 0.5 9.2

21.4 4.4 7.3 NA

4.7 3.1 2.0 NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

0.6 ND 0.5 ND

FROG ISOLATES Areomonas hydrophila Bo-3N Enterobacter agglomerans Bo-1S

ND ND

NA 1.9

NA 30

NA 11.3

NA NA

NA NA

NA NA

NA NA

2.6 0.6

PLANT PATHOGEN Pseudomonas syringae pv tobaci

ND

ND

32

8.2

NA

NA

NA

NA

ND

Gram-positive bacteria Bacillus megaterium Bm11 Staphylococcus aureus Cowan I

0.3 3.4

0.8 1.7

1.4 4.7

0.8 3.0

NA NA

25 NA

NA NA

NA NA

0.3 ND

Yeast Candida albicans ATCC 10231

0.4

0.4

3.1

1.6

NA

NA

NA

NA

ND

ND, not determined; NA, not active.

MODE OF ACTION STUDIES The results described above reveal that the more positively charged bombinins H2 and H4 are highly active compared with the shorter ones. The finding that almost all bombinins H have some hemolytic activity suggests that they have to interact with the cell membrane and increase its permeation. To investigate whether their target on bacteria is the cytoplasmic membrane, appropriate studies were conducted. One

80

Hemolysis (% of control)

The antifungal activity against Phytophthora nicotianae spores was also investigated, and a minimal fungistatic and fungicidal concentration, corresponding to 18 or 10 μM, was recorded for H2 and H4, respectively. In addition, bombinin H2, at 18 μM, gave rise to a 50% inhibition of P. nicotianae hyphae growth [15]. To evaluate the cytotoxic effect of the peptides on mammalian cells, human red cell lysis was studied in liquid medium at different peptide concentrations. Bombinin H6 displayed the greatest activity, causing 60% hemolysis at 4 μM, whereas a hemolytic capacity below 35% was shown by its diastereomer H7 even at 30 μM (Fig. 2). With respect to H2/H4, the higher extent of hemolysis was obtained with the D-amino acid–containing H4, whereas no cytotoxic effect was recorded, at all concentrations used, for GH-1D and GH-1L, the BLP-3 gene-deduced bombinin H-like peptide, chemically synthesized in an all-L version or with a D-amino acid in position 2. In contrast, significant lysis was detected for the maximins H [6].

70 60

H2 H4 H6 H7 GH-1D GH-1L

50 40 30 20 10 0 4

8

15

30

Peptide concentration (μM) FIGURE 2. Percentage hemolysis of human erythrocytes as a function of bombinin concentration. Erythrocytes were incubated in 0.9% NaCl for 30 min. The absorbance in the supernatant was recorded at 415 nm. Complete lysis was measured by suspending erythrocytes in distilled water.

of the criteria used to reveal a membrane target is the kinetic of action of the peptides. Moreover, to determine whether the microbicidal effect is dependent on salt concentration, rate of killing experiments were performed at high ionic strength, such as in phosphate buffered saline. According to the LC values reported in Table 2, H2 and H4 result to be more potent against the grampositive bacterial strains than the gram-negative ones. In fact, a 90 to 100% colony-forming unit (CFU) reduction in 30 min was noted for Bacillus megaterium and Staphylococcus aureus, whereas a 10-fold higher peptide concen-

Bombinins / 337 tration (25 μM) was necessary to kill Escherichia coli D22 and Yersinia pseudotuberculosis YPIII cells [8]. In all cases, the D-amino acid–containing H4 shows a killing kinetic faster than the corresponding L-isomer. A possible explanation is that the D-amino acid–containing peptide is less aggregated in solution compared with the L-form, and, therefore, it might be easier for it to penetrate through the cell wall and reach its target. Concerning the other two pairs of bombinins H (H6/H7 and GH-1D/GH-1L), H6 lacks microbicidal activity; in contrast, 300 μM of H7 and 200 μM of GH1D/GH-1L are required to cause, after 15 min, a 99% CFU reduction of A. hydrophila Bo-3N, a potential natural target of these peptides. The discrepancy in activity observed between agar and liquid assays may be related to the difference in solubility and diffusion of the peptides in the two media. In order to remove the single positive charge of H6 and H7 and to understand its role in the antimicrobial activity of the molecule, dansylated derivatives were produced. No variation in the killing curves was detected compared with the parent peptides, thus indicating that the single positive charge is not an essential requirement for the antibacterial activity of the molecule. Indeed, in support of this notion is the finding that the anionic maximin H5 also displays antimicrobial properties [7]. To assess the capacity of the bombinins H to alter the bacterial inner membrane permeability, the βgalactosidase activity in the supernatant of E. coli D22 cell culture, after peptide incubation, was measured by use of the chromogenic substrate ONPG. Only H2 and H4 are able to permeate the inner membrane; in fact, the enzymatic activity was absent in the extracellular medium after treatment with the other bombinins H. It is known that ONPG can permeate through pores the outer membrane, whereas the inner membrane represents the main barrier and its influx into the cells cannot occur in the absence of protein transporters such as the lac or melibiose permeases. To shed light into the capacity of H6 and H7 to alter the inner membrane permeability, the β-galactosidase activity in the whole bacterial culture was followed as a function of time. The increasing enzymatic activity that was recorded at a constant H7 concentration reflects the permeabilization of the inner membrane to ONPG [8]. Overall, these results suggest that bombinins H can cause either large damage at the membrane level (H2/ H4) allowing for protein leakage, or local disruption (H7) allowing diffusion of small molecules. Recently, lethal activity against the insect and the mammalian stage of the Leishmania parasite was evidenced for H2 and H4 (Mangoni et al., unpublished results), suggesting bombinins H as potential templates

for the development of new drugs with a new mode of action against Leishmania.

Acknowledgments This work was supported in part by grants from Università La Sapienza and the Italian Ministero dell’Università e della Ricerca Scientifica e Tecnologica.

References [1] Barra D, Simmaco M, Boman HG. Gene-encoded peptide antibiotics and innate immunity. Do “animalcules” have defence budgets? FEBS Lett 1998;430:130–4. [2] Csordas A, Michl H. Monatsh Chem 1970;101:182–9. [3] Gibson BW, Tang D, Mandrell R, Kelly M, Spindel ER. Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis. J Biol Chem 1991;266:23103–11. [4] Hultmark D, Engström A, Bennich H, Kapur R, Boman HG. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem 1982;127:207–17. [5] Jilek A, Mollay C, Tippelt C, Grassi J, Mignogna G, Müllegger J, Sander V, Fehrer C, Barra D, Kreil G. Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions. Proc Natl Acad Sci USA 2005;102:4235–9. [6] Lai R, Zheng Y-T, Shen J-H, Liu G-J, Liu H, Lee W-H, Tang S-Z, Zhang Y. Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 2002;23:427–35. [7] Lai R, Liu H, Lee W-H, Zhang Y. An anionic antimicrobial peptide from toad Bombina maxima. Biochem Biophys Res Commun 2002;295:796–9. [8] Mangoni ML, Grovale N, Giorgi A, Mignogna G, Simmaco M, Barra D. Structure-function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions. Peptides 2000;21:1673–9. [9] Miele R, Ponti D, Boman HG, Barra D, Simmaco M. Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-κB and NF-IL6 binding sites in its promoter. FEBS Lett 1998;431:23–8. [10] Miele R, Borro M, Fiocco D, Barra D, Simmaco M. Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7. Peptides 2000;21:1681–6. [11] Mignogna G, Simmaco M, Kreil G, Barra D. Antibacterial and hemolytic peptides containing D-alloisoleucine from the skin of Bombina variegata. EMBO J 1993;12:4829–32. [12] Mignogna G, Simmaco M, Barra D. Defence peptides in the amphibian immune system. In: Jollès P, Editor. D-Amino Acids in Sequences of Secreted Peptides of Multicellular Organisms; Basel, Birkhauser Verlag; 1998, pp. 29–36. [13] Simmaco M, Barra D, Chiarini F, Noviello L, Melchiorri P, Kreil G, Richter K. A family of bombinin-related peptides from the skin of Bombina variegata. Eur J Biochem 1991;199:217–22. [14] Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from Amphibian skin: What do they tell us? Biopolymers 1998; 47:435–50. [15] Simmaco M, Mangoni ML, Miele R, Borro M, Fiocco D, Barra D. Defence peptides in the amphibian immune system. In: Ascenzi P, Polticelli F, Visca P, editors. Bacterial, plant and animal toxins, Kerala, India, Research Signpost; 2003, pp. 155–167.

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51 Scorpion Venom Peptides LOURIVAL D. POSSANI AND RICARDO C. RODRÍGUEZ DE LA VEGA

nents are toxins specific for K+ or Na+ channels (see following), although several enzymatic activities, like hyaluronidases and phospholipases, have also been reported [19, 32]. Many components for which a precise biological function, in the context of the venom, is not known, have also been recently reported (e.g., see reviews [22, 47]). The primary structure and the threedimensional folding of several scorpion venom components, mainly toxins specific for Na+ channels (NaScTxs) or K+ channels (KTxs), have been determined. The molecular bases defining the specific pharmacological action of these peptides are now under intensive investigation, whereas gene cloning and evolutionary analyses of scorpion venom peptides are generating relevant information on how their function and specificity arose (see following). In this chapter, we outline some of these findings and discuss the state-of-the-art knowledge available on the subject, highlighting the main problems that need further analysis.

INTRODUCTION Scorpion venoms have many different peptides whose structures and functions have been studied. For many of them the exact three-dimensional structure and the receptors to which they bind are known. At least 350 distinct peptides were already isolated and partially characterized. The molecular bases of action are currently being improved, and a better understanding of their function is shaping up. The better known peptides recognize ion-channels (mainly K+ and Na+) and were shown to play an important role in the pharmacological and structural characterization of their receptors. From the wide diversity of scorpion species there is still much to do, and probably other interesting targets will be found and described in the future. Scorpion venoms are complex mixtures of components, where the peptides and proteins play a fundamental role, providing the animals with the tools to defend themselves from predators or subdue their preys. Most of the scorpion venom peptides are composed of 20 to 75 amino acid residues, whereas the few known proteins (enzymes) are over 120 up to 370 residues [13, 32, 49]. Proteomic analyses show that single scorpion venoms might contain more than 100 peptidic components [2, 12, 31]. The neurotoxicity caused by scorpion stings (whole venom) can be reproduced in experimental animals or by in vitro assays, using homogeneous or partially purified components of the venom [49]. Thus, it is not surprising to find that most work reported was conducted with isolated toxic components. From more than 1500 different species of scorpions known to exist in the world [14], only about 50 species were used for individual peptides or protein characterization [36, 37]. No more than 350 different scorpion venom peptides or proteins are known, from a biodiversity expected to be over 100,000 distinct components [34]. Among the best-known venom compoHandbook of Biologically Active Peptides

DISCOVERY AND DOCUMENTED BIODIVERSITY The first evidences regarding biological functions of scorpion venom components were obtained by electrophysiological experiments in the squid giant axon [21]. After these seminal works, it became clear that scorpion venoms contain two main kinds of neurotoxins affecting either Na+ or K+ channel permeability [28, 33]. The laborious work of several groups has completed the amino acid sequence of hundreds of peptides purified from scorpion venoms. Based on the length of the sequences, two groups of peptides were identified: the “short-chain” peptides containing 20--43 amino acids, most of which recognize K+ channels, and the “longchain” peptides with 58--76 amino acids, usually specific for Na+ channels. There is a common signature for the disulfide bridge arrangements of these peptides (see

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340 / Chapter 51 below). Two freely available databases of scorpion toxins can be found on the World Wide Web: SCORPION [41, 43] and Tox-Prot [19]. In the supplementary material accompanying this chapter, an extended list of the available amino acid sequences are presented in FASTA format. Fig. 1A shows a multiple sequence alignment with some of the most extensively characterized scorpion toxins, including their disulfide bonds.

THREE-DIMENSIONAL FOLDING Most scorpion venom peptides characterized until now contain a high proportion of basic amino acids and are rich in Cys residues, tightening the structure with three or four disulfide bridges. The most common Cys pairings bridge the consensus sequence Cysi..Cysi+4, folded into an α-helix segment, with the pair Cysj..Cysj+2

A ChTx 2crd 1sxm NTx AgiTx2 1agt MauTx 1txm Tc1 1jlz Tc32 BeKm1 1lgl CnErg1 1px9 Aah2 1aho LqhαIT 1lqh BmKM1 1djt Kurtx Cn2 1cn2 Css4 Tsγ 1npi Cn11 LqhIT2 BjxtrIT 1bcg Birtoxin KAaH1 Phaitx

PDB

Alignment ------------ZFTNVSCTT--------SKECWSVCQRL--HNTSRG-KCMN-------KKCRCYS---------------------------------------TIINVKCTS--------PKQCSKPCKEL--YGSSAGAKCMN-------GKCKCYNN-------------------------------------GVPINVSCTG--------SPQCIKPCKDA---GMRFG-KCMN-------RKCHCTPK------------------------------------------VSCTG--------SKDCYAPCRKQ--TGCPNA-KCIN-------KSCKCYGC-------------------------------------------AC-----------GSCRKKCK-------GSG-KCIN-------GRCKCY----------------------------------------TGPQTTCQ---------AAMCEAGCKGL-GKSME---SCQG-------DTCKCKA------------------------------------RPTD---IKCSE--------SYQCFPVCKSRFG-KTNG--RCVN----G---FCDCF--------------------------------DRDSCVDKS----RCAKYG-----YYQECQDCCKNAGH--NGG--TCMFF-------KCKCA------------------------------VKDGYIVDD-VNCTYFCGR--------NAYCNEECTKLKGES----GYCQWASPYG--NACYCYKLPD-----HVRTK---GPGRCHGR-----VRDAYIAKN-YNCVYECFR--------DAYCNELCTKNGASS----GYCQWAGKYG--NACWCYALPD-----NVPIR---VPGKCRK------VRDAYIAKP-HNCVYECAR--------NEYCNDLCTKNGAKS----GYCQWVGKYG--NGCWCIELPD-----NVPIR---VPGKCHR------KIDGYPVDY-WNCKRICW-----YN--NKYCNDLCKGLKADS----GYCWGWT-----LSCYCQGLPD-----NARIK---RSGRCRA-------KEGYLVDKNTGCKYECLK----LGD-NDYCLRECKQQYGKG--AGGYC-YAFA------CWCTHLYEQAI--VWPLP----NKRCS--------KEGYLVNSYTGCKFECFK----LGD-NDYCLRECRQQYGKG--SGGYC-YAFG------CWCTHLYEQAV--VWPLP----NKTCN---------EGYLMDH-EGCKLSCFI----RP--SGYCGRECGIK--KG--SSGYC-AWPA------CYCYGLPNWVK--VWDRA----TNKCGKK-----ARDGYPVD-EKGCKLSCLIN-------DKWCNSAC-HSRGGK---YGYCYTGGL-----ACYCEAVPDNV--KVWTY----ETNTC----------DGYIKR-RDGCKVACLIG-------NEGCDKEC-KAYGGS---YGYCWTWGL-----ACWCEGLPDD---KTWKS----ETNTCG-------KKNGYPLDR-NGKTTECSGVNAIAPH---YCNSECTKVYYAE---SGYCCWGA-------CYCFGLEDDKPIGPMKDI---TKKYCDVQIIPS ADVPGNYPLDK-DGNTYKCF----LLGG-NEECLNVC-KLHGVQ---YGYCYASK-------CWCEYLEDD------KD----SV---------ADVPGNYPLDS-SDDTYLCA----PLGE-NPFCIKIC-RKHGVK---YGYCYAFQ-------CWCEYLED-------KNV---KI--------------KFIRHK-DESFYECGQ----LIGYQQYCVDACQAHGSKEK---GYCKGMAPFGLPGGCYCPKLPSN----RVKMCFGALESKCA------

B N C N

KTxs

NaScTxs C

FIGURE 1. General structure of scorpion venom peptides. A. Multiple sequence alignment of selected scorpion peptides. The access codes from Swiss-Prot and PDB are given for references. Vertical lines indicate the pharmacological groups (KTxs and NaScTxs). The conserved disulfide pairing is indicated by horizontal lines at the bottom. The fourth possible disulfide bridge is indicated by underlined cysteines, within the sequences. B. Superimposed three dimensional models of the structures of KTxs (left side): Charybdotoxin (purple), Ergtoxin-1 (blue), and Tc1 (green), and NaScTxs (right side): Aah2 (brown), Cn2 (blue), and BjxtrIT (orange). The disulfide pairings are indicated in yellow (see panel A for PDB identifiers). (See color plate.)

KTxs

Name

NaScTxs

Acc# P13487 P08815 P46111 P80719 P83243 P60211 Q9BKB7 Q86QT3 P01484 P17728 P45697 P58910 P01495 P60266 P15226 P58296 Q26292 P56637 P58752 n.d.e. P84207

Scorpion Venom Peptides / 341 of an extended β-strand structure. This structural arrangement is known as the Cysteine Stabilized αβ motif (CSαβ-motif) [5, 20]. The minimum core of this structural motif is constituted by an αββ topology but might contain extra strands or helixes (reviewed in [29, 42]). This is the general folding found for both the KTxs and the NaScTxs, despite the fact that the KTxs usually are shorter peptides stabilized by three or four disulfides, whereas the NaScTxs are longer-chain peptides mostly packed by four disulfides. Interestingly, the CSαβmotif is also adopted by functionally diverse peptides from other distantly related biological sources (reviewed in [15, 42]). Less represented topological folds were found (e.g., toxins affecting Ryanodine receptors and κ-KTxs [29]). The three-dimensional characterization was determined by x-ray crystallography or by nuclear magnetic resonance. Thus far, there are ∼60 different structures determined (see PDB accession codes in Supplementary Material). Fig. 1B shows the general folding of the CSαβ-motif containing scorpion toxins. For this figure (left-hand side) we superimposed the structure of Charybdotoxin, Ergtoxin 1, and toxin Tc1, and for NaScTxs the three-dimensional structures of Cn2, AaH2, and BjxtrIT toxins (right-hand side).

that contain several transmembrane segments and one reentrant loop that forms the selectivity filter of the channel (reviewed in [27, 46]). The K+ channels are tetrameric structures, whereas the Na+ and the Ca2+ channels are monomeric structures containing four homologous repeats equivalent to the monomers of K+ channels. The short-chain scorpion toxins recognize K+ channels. These toxins bind to the vestibule of the ionconducting pathway, blocking the passage of the ion through the pore of the channel [18, 44]. The longchain scorpion toxins usually modulate Na+ channel function by affecting the gating mechanism of the channel. There are two proposed distinct sites where these toxins bind for modulating the activity. One situated in the segment S3-S4 of domain II (the β-NaScTx type) and the other in the segment S3-S4 of domain IV (the α-NaScTx type). The α- and β-NaScTx types were originally defined based on binding and displacement experiments, as well as using electrophysiological measurements (reviewed in [11, 26, 45]). However, there are several other scorpion venom bioactive peptides that do not strictly correspond to these generalizations, whose receptor sites are not identified yet or are poorly characterized (reviewed in [22, 36, 37, 47]).

SCORPION TOXIN PRECURSORS It is assumed that peptides isolated from scorpion venoms are only produced in the venomous glands, although this needs confirmation. These peptides are translated as pre-peptides, with a characteristic lowcomplexity signal peptides (∼20 amino acid residues long), followed by a highly variable mature region. Most of the scorpion venom peptides are encoded by a single class of precursor, characterized by a conserved architecture including a single intron (phase I, in which the codon is split after the first base) within the region encoding the signal peptide (see [3, 16]). This topological conservation suggests that all scorpion toxins should be genetically related by duplication and divergence events, constituting a multigene family [16]. However, this also needs to be confirmed by genomewide analyses. The messengers encoding scorpion toxins often include a short sequence just before the stop codon, which is postranslationally processed to give C-terminal amidated toxins (e.g., [3]). The information about precursors of other venom components is still limited, although it seems that nondisulfide bridged peptide precursors are more diverse [23].

RECEPTOR SITES Most receptors recognized by scorpion toxins are ion-channels. These are integral membrane proteins,

MOLECULAR BASIS OF THEIR ACTIVITY The solution of the three-dimensional structure of several toxins, as well as that of some K+ channels (reviewed in [24]), the cloning of the genes coding for the Na+ and Ca2+ channels, and the discovery of their homologies [1] has paved the way to elucidate the molecular basis of their interactions. The technique of thermodynamic double mutant analysis applied to both the toxins and the corresponding channels have provided the tools for identification of the interacting surfaces of KTxs and K+ channels (reviewed in [18, 36, 44], but also see [30, 35]). The difficulties to obtain recombinant NaScTxs and Na+ channels mutants have hampered the fine examination of their interacting surfaces. Fortunately, recent success in the recombinant production of both molecules (e.g., [9, 10, 17, 38, 39, 48]) promises significant advances in the near future. Fig. 2A shows the state-of-the-art knowledge for the surface contact points between KTxs and various sub-types of K+ channels. Fig. 2B shows a cartoon of the proposed interactions of NaScTxs with Na+ channels. In the case of the KTxs toxins, at least three distinct surface interacting sides were identified (Fig. 2A, reviewed in [30, 35]), whereas for the Na+ channel-specific toxins, two models were proposed (reviewed in [4, 7]). One of them can be interpreted in the light of the “stop model” of the α-NaScTxs affecting the inactivation of the

342 / Chapter 51

FIGURE 2. Working models for the interaction of scorpion toxins with ion-channels. A. Three distinct forms of interaction of KTxs blocking K+-channels: Agtx 2 with Shaker (left), BmP05 with KCa2.1 (middle), and BeKm-1 with erg channel (right). The figure is modified from [35]. B. Cartoon representation of the interaction of α-NaScTxs (α, left-hand side) and β-NaScTxs (β, right-hand side) with voltage-gated Na+ channels. The effect of α-NaScTx’s is drawn according the “stop-model” as described in [6]. The action of β-NaScTxs follows the “voltage-sensor trapping” mechanism as proposed in [8, 9, 25]. Arrows represent channel movements implicated in gating. Toxin binding does not allow movement of the voltage sensors (arrows with crosses). Cartoons of both K+ and Na+ channels are based on three dimensional structures of KcsA and KvAP, respectively. Only two opposite monomers (K+ channels) or domains (Na+ channels) are depicted for the sake of clarity.

channels (Fig. 2B, [6]), and the other one in the light of the “voltage-sensor trapping” model for the βNaScTxs affecting the mechanism of activation (Fig. 2B, [8, 9, 10]). It is worth mentioning that not all the possible “pharmacophores” of the scorpion toxins are known. Various bona fide K+ channels blockers do not fit any of the models currently defined (reviewed in [29,

30, 35, 36]). Similarly, not all long-chain toxins seem to interact by the two mechanisms just outlined (reviewed in [37]). Moreover, recent publications suggest that the binding of some β-ScTxs to excitable membranes does not require the presence of a specific receptor protein but depends on the very nature of the lipid bilayer [40].

Scorpion Venom Peptides / 343

CONCLUSION It is conceivable that each peptide present in the venom has been selected for a specific target, either in the aggressor or in the prey from which the scorpions feed on. This means that the particular specificity of action of the various peptides has evolved according to specific needs. Our knowledge in this regard is still quite limited. Although we do have convincing examples of a perfect match of the pair toxin-receptor (mainly for the case of ion-channels) the exact fitness for many other peptides are ill defined. The affinity is low and the possible relevance for the toxicity found is not clear. It is expected that each one of the great structurally diverse (in terms of primary sequence) scorpion peptides should correspond to a given subtype of receptor, but this needs additional effort to be clarified. It is necessary to use more specific assays that can provide more information on the fitness of action (the more putative receptors assayed, the better). The whole venom of a given species is a complex mixture of components, which have evolved for a concerted biological effect (“cabals”; see chapter 54 on “conus snail venom peptides” in the Venom Peptides section”.). The pending research task is to uncover the intricate coordination of action of these various components for obtaining a precise objective (to prey or to defend themselves). The future work is huge, since most species of scorpion have not been studied yet (mainly the nonbuthid group); different types of components with distinct structures and functions are expected to be found. Finally, the potential use of these peptides, or improved variants, for the treatment of human channelopathies (see chapters by J. M. Sabatier and K. G. Chandy) is also a major driving force that promotes the continuous research on scorpion venom peptides.

Acknowledgments This work was partially supported by grants 40251-Q from CONACyT (Mexican Government), IN206003-3 from DGAPA-UNAM, and by the company Instituto Bioclón S.A. de C.V. The participation of many collaborators of the laboratory is greatly acknowledged, especially Dr. Georgina Gurrola, Dr. Fernando Zamudio, Dr. Baltazar Becerril, Dr. Cesar V.F. Batista, M.Sc. Timoteo Olamendi, Biol. Cipriano Balderas and Mr. Fredy Coronas.

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Scorpion Venom Peptides / 345 >P13487 (2crd) |Charybdotoxin (aKTx1.1) FTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS >P45628 (1lir) |Lq2 (aKTx1.2) QFTQESCTASNQCWSICKRLHNTNRGKCMNKKCRCYS >P24663 |Iberiotoxin (aKTx1.3) QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ >n.d.e. |Limbatotoxin (aKTx1.4) VFIDVSCSVSKECWAPCKAAVGTDRGKCMGKKCRCYX >Q9NII6 (1big) |BmTx1 (aKTx1.5) QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS >Q9NII5 (2bmt) |BmTx2 (aKTx1.6) QFTNVSCSASSQCWPVCKKLFGTYRGKCMNSKCRCYS >P45660 |Lqh15_1 (aKTx1.7) GLIDVRCYDSRQCWIACKTGSTQGKCQNKQCRCY >P59848 |HgTx2 (aKTx1.9) KFIDVKCTTSKECWPPCKAATGKAAGKCMNKKCKCQX >P83112 |PbTx3 (aKTx1.10) EVDMRCKSSKECLVKCKQATGRPNGKCMNRKCKCYPR >n.d.e. |Slotoxin (aKTx1.11) TFIDVDCTVSKECWAPCKAAFGVDRGKCMGKKCKCYV >P08815 (1sxm) |Noxiustoxin (aKTx2.1) TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN >P40755 (1mtx) |Margatoxin (aKTx2.2) TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH >P45629 |CllTx1 (aKTx2.3) ITINVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP >AAB50864 |Ntx2 (aKTx2.4) TIINEKCFATSQCWTPCKKAIGSLQSKCMNGKCKCYNG >P59847 (1hly) |HgTx1 (aKTx2.5) TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCKCYPH >P45630 |CllTx2 (aKTx2.7) TVIDVKCTSPKQCLPPCKEIYGRHAGAKCMNGKCKC >n.d.e. |Ce1 (aKTx2.8) TVINVKCTSPKQCLKPCKDLYGPHAGAKCMNGKCKCYNN >n.d.e. |Ce2 (aKTx2.9) TIINVKCTSPKQCLKPCKDLYGPHAGAKCMNGKCKCYNN >n.d.e. |Ce3 (aKTx2.10) IFINVKCSLPQQCLRPCKDRFGQHAGGKCINGKCKCYP >n.d.e. |Ce4 (aKTx2.11) TIINVKCTSPKQCLLPCKEIYGIHAGAKCMNGKCKCYKI >n.d.e. |Ce5 (aKTx2.12) TIINVKCTSPKQCLPPCKEIYGRHAGAKCMNGKCHCSKI >P24662 (2ktx) |Kaliotoxin (aKTx3.1) GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTP >P46111 (1agt) |Agitoxin2 (aKTx3.2) GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK >P46112 |Agitoxin3 (aKTx3.3) GVPINVPCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK >P46110 |Agitoxin1 (aKTx3.4) GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK >P45696 |KTx2 (aKTx3.5) VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK >Q9NII7 (1bkt) |BmKTX (aKTx3.6) VGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCTPK >P55896 (1sco) |OsK1 (aKTx3.7) GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK >P59886 |Bs6 (aKTx3.8) GVPINVKCRGSPQCIQPCRDAGMRFGKCMNGKCHCTPQ >P59290 |KTx3 (aKTx3.9) VGIPVSCKHSGQCIKPCKDAGMRFGKCMNRKCDCTPK >P46114 (1hp2) |TsTXKalpha (aKTx4.1) INAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP >P56219 (1tsk) |Tskappa (aKTx4.2) VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC >P59925 |TdK1 (aKTx4.3) VFINVKCTGSKQCLPACKAAVGKAAGKCMNGKCKCYT

346 / Chapter 51 >P60210 |Tc30 (aKTx4.4) VFINVKCRGSKECLPKCKAAVGKAAGKCMNGKCKCYP >AAW72455 |TcoKTx (aKTx4.5) VFINVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP >P16341 (1scy) |Scyllatoxin (aKTx5.1) AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH >P31719 (1pnh) |PO5 (aKTx5.2) TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH >Q9TVX3 |BmPO5 (aKTx5.3) AVCNLKRCQLSCRSLGLLGKCIGDKCECVKH >P59869 |Tamapin (aKTx5.4) AFCNLRRCELSCRSLGLLGKCIGEECKCVPY >P59870 |Tamapin2 (aKTx5.5) AFCNLRRCELSCRSLGLLGKCIGEECKCVPH >Q10726 |Pi1 (aKTx6.1) VKCRGTSDCGRPCQQQTGCPNSKCINRMCKCYGC >P80719 (1txm) |Maurotoxin (aKTx6.2) VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC >P59867 (1quz) |HsTx1 (aKTx6.3) ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC >P58498 (1n8m) |Pi4 (aKTx6.4) IEAIRCGGSRDCYRPCQKRTGCPNAKCINKTCKCYGCS >P58490 (1qky) |Pi7 (aKTx6.5) DEAIRCTGTKDCYIPCRYITGCFNSRCINKSCKCYGCT >AAP73817 |OcKTx1 (aKTx6.6) AEVIKCRTPKDCAGPCRKQTGCPHGKCMNRTCRCNRC >AAP73818 |OcKTx2 (aKTx6.7) AEVIKCRTPKDCADPCRKQTGCPHGKCMNRTCRCNRC >AAP73819 |OcKTx3 (aKTx6.8) AEVIKCRTPKDCAGPCRKQTGCPHAKCMNKTCRCHRC >AAP73820 |OcKTx4 (aKTx6.9) AEIIRCSGTRECYAPCQKLTGCLNAKCMNKACKCYGCV >AAP73821 |OcKTx5 (aKTx6.10) AEVIRCSGSKQCYGPCKQQTGCTNSKCMNCKCYGC > (1wmt) |IsTx (aKTx6.11) VHTNIPCRGTSDCYEPCEKKTNCARAKCMNRHCNCYNNCPW >n.d.e. |Anuroctoxin (aKTx6.12) QKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK > (1v56) |Spinoxin (aKTx6.13) IRCSGSRDCYSPCMKQTGCPNAKCINKSCKCYGCX >P55927 (2pta) |Pi2 (aKTx7.1) TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR >P55928 (1c49) |Pi3 (aKTx7.2) TISCTNEKQCYPHCKKETGYPNAKCMNRKCKCFGR >P56215 (1acw) |P01 (aKTx8.1) VSCEDCPEHCSTQKAQAKCDNDKCVCEPI >Q9U8D2 |BmP01 (aKTx8.2) ATCEDCPEHCATQNARAKCDNDKCVCEPK >P80670 |LpII (aKTx8.3) VSCEDCPDHCSTQKARAKCDNDKCVCEPI >P80671 |LpIII (aKTx8.4) VSCEDCPDHCSTQKARAKCDNDKCVCEPK >Q9NJP7 (1du9) |Bmp02 (aKTx9.1) VGCEECPMHCKGKNAKPTCDDGVCNCNV >Q9U8D1 |Bmp03 (aKTx9.2) VGCEECPMHCKGKNANPTCDDGVCNCNV >P80669 |LpI (aKTx9.3) VGCEECPMHCKGKNAKPTCDNGVCNCNV >P60209 |BTK-2 (aKTx9.4) VGCAECPMHCKGKMAKPTCENEVCKCNIG >n.d.e. |Kbot1 (aKTx9.5) VGCEECPMHCKGKHAVPTCDNGVCNCNV >O46028 (1pjv) |Cobatoxin1 (aKTx10.1) AVCVYRTCDKDCKRRGYRSGKCINNACKCYPY >P58504 |Cobatoxin2 (aKTx10.2)

Scorpion Venom Peptides / 347 VACVYRTCDKDCTSRKYRSGKCINNACKCYPY >P60164 |PBTx1 (aKTx11.1) DEEPKESCSDEMCVIYCKGEEYSTGVCDGPQKCKCSD >P60165 |PBTx2 (aKTx11.2) DEEPKETCSDEMCVIYCKGEEFSTGVCDGPQKCKCSD >AAQ23195 |PBTx10 (aKTx11.3) DEEPKETCSDDMCVIYCKGEEYSTGVCDGPQKCKCS >P59936 (1c55) |Butantoxin (aKTx12.1) WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYT >P83243 (1jlz) |Tc1 (aKTx13.1) ACGSCRKKCKGSGKCINGRCKCY >P83244 |OsK2 (aKTx13.2) ACGPGCSGSCRQKGDRIKCINGSCHCYP >Q967F9 |Bmkk1 (aKTx14.1) TPFAIKCATDADCSRKCPGNPSCRNGFCACT >Q95NK7 (1pvz) |BmKK2 (aKTx14.2) TPFAIKCATDADCSRKCPGNPPCRNGFCACT >Q9BJX2 |BmKK3 (aKTx14.3) TPFEVRCATDADCSRKCPGNPPCRNGFCACT >AAK31986 |SKTx1 (aKTx14.4) TPFAIKCATNADCSRKCPGNPPCRNGFCACT >P60233 |Aa1 (aKTx15.1) QNETNKKCQGGSCASVCRRVIGVAAGKCINGRCVCYP >Q8I0L5 |BmTx3A (aKTx15.2) QVETNVKCQGGSCASVCRKAIGVAAGKCINGRCVCYP >P60208 |AmmTX3 (aKTx15.3) QIETNKKCQGGSCASVCRRVIGVAAGKCINGRCVCYP >Q867F4 |AaTx1 (aKTx15.4) QIETNKKCQGGSCASVCRRVIGVAAGKCINGRCVCYP >Q86SD8 |AaTx2 (aKTx15.5) QVETNKKCQGGSCASVCRRVIGVAAGKCINGRCVCYP >n.d.e. |Discrepin (aKTx15.6) QIDTNVKCSGSSKCVKICIDRYNTRGAKCINGRCTCYP >n.d.e. |TmTX (aKTx16.1) DLIDVKCISSQECWIACKTGRFEGKCQNRQCRCY >Q9NBG9 (1m2s) |Martentoxin (aKTx16.2) FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY >Q8MQL0 |Kchtx1 (aKTx16.3) FGLIDVKCFASSECWIACKTGSVQGKCQNNQCRCY >Q95NJ8 (1klh) |BmKK4 (aKTx17.1) QTQCQSVRDCQQYCLTPDRCSYGTCYCKTT >P60211 |Tc32 (aKTx18.1) TGPQTTCQAAMCEAGCKGLGKSMESCQGDTCKCKA >P83407 (1q2k) |BmBKTx1 (aKTx19.1) AACYSSDCRVKCVAMGFSSGKCINSKCKCYK >Q9BN12 |Tamulustoxin1 (a?) RCHFVVCTTDCRRNSPGTYGECVKKEKGKECVCKS >Q9BN11 |Tamulustoxin2 (a?) RCHFVICTTDCRRNSPGTYGECVKKEKGKECVCKS >AAM94410 |BmK38 (a?) KTATFCTQSICQESCKRQNKNGRCVIEAEGSLIYHLCKCY >AAW72454 |Tco_pKTx (a?) KVETGQYVKCKYDICAKSCQEEKGKRTGFCSNPECICSKD >P45639 (1chl) |ClTx_lqq MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR >P55966 |Lqh8/6 RCSPCFTTDQQMTKKCYDCCGGKGKGKCYGPQCICAPY >P15229 |Bs8 RCSPCFTTDQQMTKKCYDCCGGKGKGKCYGPQCICAPY >P15220 |ITI1 MCMPCFTTRPDMAQQCRACCKGRGKCFGPQCLCGYD >P15222 (1sis) |I5A MCMPCFTTDPNMAKKCRDCCGGNGKCFGPQCLCNR >P81761 |ButaIT RCGPCFTTDPQTQAKCSECCGRKGGVCKGPQCICGIQ

348 / Chapter 51 >P60268 |BeI3 MCMPCFTTDHQTARRCRDCCGGRGRKCFGQCLCGYD >P60269 |BeI4 MCMPCFTTDHNMAKKCRDCCGGNGKCFGPQCLCNR >P60270 |BeI5 MCMPCFTTDPNMANKCRDCCGGGKKCFGPQCLCNR >P69940 |TsTXKb (b1) KLVALIPNDQLRSILKAVVHKVAKTQFGCPAYEGYCNDHCNDIERKDGECHGFKCKCAKD >P69939 |AaTXKb (b2) KLVKYAVPVGTLRTILQTVVHKVGKTQFGCPAYQGYCDDHCQDIKKEEGFCHGFKCKCGIPMGF >AAF31480 |BmTXKb (b3) KNIKEKLTEVKDKMKHSWNKLTSMSEYACPVIEKWCEDHCAAKKAIGKCEDTECKCLKLRK >AAF31479 |BmTXKb2(b4) KLVKYAVPEGTLRTIIQTAVHKLGKTQFGCPAYQGYCDDHCQDIKKEEGFCHGFKCKCGIPMGF >P56972 |Scorpine (b?) GWINEEKIQKKIDERMGNTVLGGMAKAIVHKMAKNEFQCMANMDMLGNCEKHCQTSGEKGYCHGTKCKCGTPLSY >AAQ94352 |Opiscorpine 1 (b?) KWFNEKSIQNKIDEKIGKNFLGGMAKAVVHKLAKNEFMCVANVDMTKSCDTHCQKASGEKGYCHGTKCKCGVPLSY >AAW72464 |TcoScorpine-like (b?) KLVALIPNDQLRSILKAVVHKVAKTQFGCPAYEGYCNNHCQDIERKDGECHGFKCKCAKD >Q86QT3 (1ne5) |CnErg1 (gKTx1.1 ) DRDSCVDKSRCAKYGYYQECQDCCKNAGHNGGTCMFFKCKCA >Q86QV6 |CeErg1 (gKTx1.2 ) DRDSCVDKSRCAKYGYYQECTDCCKKYGHNGGTCMFFKCKCA >Q86QV3 |CgErg1 (gKTx1.3 ) DRDSCVDKSRCAKYGHYQECTDCCKKYGHNGGTCMFFKCKCA >Q86QU6 |CsErg1 (gKTx1.4 ) DRDSCVDKSRCAKYGYYQECQDCCKKAGHNGGTCMFFKCKCA >Q86QV0 |CllErg1 (gKTx1.5 ) DRDSCVDKSRCSKYGYYQECQDCCKKAGHNGGTCMFFKCKCA >Q86QU1 |CexErg1 (gKTx1.6 ) DRDSCVDKSRCAKYGYYQECQDCCKKAGHSGGTCMFFKCKCA >Q9BKB7 (1lgl) |BeKm-1 (gKTx2.1 ) RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF >P59939 |CnErg2 (gKTx3.1 ) GRDSCVNKSRCAKYGYYSQCEVCCKKAGHKGGTCDFFKCKCKV >Q86QV5 |CeErg2 (gKTx3.2 ) DRDSCVDKSRCAKYGYYQQCEICCKKAGHRGGTCEFFKCKCKV >Q86QU5 |CsErg2 (gKTx3.3 ) DRDSCVDKSRCAKYGYYGQCEVCCKKAGHRGGTCDFFKCKCKV >Q86QV2 |CgErg2 (gKTx3.4 ) DRDSCVDKSRCQKYGNYAQCTACCKKAGHNKGTCDFFKCKCT >Q86QU9 |CllErg2 (gKTx4.1 ) DRDSCVDKSKCSKYGYYGQCDECCKKAGDRAGNCVYFKCKCNP >Q86QV7 |CnErg5 (gKTx4.2 ) DRDSCVDKSKCGKYGYYQECQDCCKNAGHNGGTCVYYKCKCNP >Q86QU0 |CexErg2 (gKTx4.3 ) DRDSCVDKSKCGKYGYYGQCDECCKKAGDRAGICEYYKCKCNP >Q86QT9 |CexErg3 (gKTx4.4 ) DRDSCVDKSKCAKYGYYYQCDECCKKAGDRAGTCEYFKCKCNP >Q86QT8 |CexErg4 (gKTx4.5 ) DRDSCVDKSQCAKYGYYYQCDECCKKAGDRAGTCEYFKCKCNP >Q86QV1 |CgErg3 (gKTx5.2 ) DRDSCVDKSRCQKYGPYGQCTDCCKKAGHTGGTCIYFKCKCGAESGR >Q86QU8 |CllErg3 (gKTx4.6 ) DRDSCVDKSKCSKYGYYGQCDKCCKKAGDRAGNCVYFKCKCNQ >Q86QU7 |CllErg4 (gKTx4.7 ) DRDSCVDKSKCAKYGYYGQCDECCKKAGDRAGNCVYLKCKCNQ >Q86QV4 |CeErg3 (gKTx4.8 ) DRDSCVDKSKCGKYGYYHQCDECCKKAGDRAGNCVYYKCKCNP >Q86QU4 |CsErg3 (gKTx4.9 ) DRDSCVDKSRCGKYGYYGQCDDCCKKAGDRAGTCVYYKCKCNP >Q86QU3 |CsErg4 (gKTx4.10) DRDSCVDKSRCGKYGYYGQCDECCKKAGDRAGTCVYYKCKCNP >Q86QV8 |CnErg4 (gKTx4.11)

Scorpion Venom Peptides / 349 DRDSCVDKSQCGKYGYYGQCDECCKKAGERVGTCVYYKCKCNP >P59940 |CsEKerg1 (gKTx4.12) ERDSCVEKSKCGKYGYYGQCDECCKKAGDRAGTCVYYKCKCNP >Q86QV9 |CnErg3 (gKTx4.13) DRDSCVDKSKCGKYGYYGQCDECCKKAGDRAGTCVYYKCKCNP >Q86QU2 |CsErg5 (gKTx5.1 ) DRDSCVDKSRCAKYGYYGQCEVCCKKAGHNGGTCMFFKCMCVNSKMN >Q86SE1 |Aam1 RDGYIVYPHNCVYHCIPSCDGLCKENGATSGSCGYIIKVGIACWCKDLPENVPIYDRSYKCYR >Q86SE0 |Aam2 RDGYIADAGNCGYTCVANDYCNTECTKNGAESGYCQWFGRYGNACWCIKLPDKVPIKVPGKCNGR >Q86SD9 |Aam3 RDGYIAQPNNCVYHCIPLSPGCDKLCRENGATSGKCSFLAGSGLACWCVALPDNVPIKIIGQKCTR >P01479 |Aah1 KRDGYIVYPNNCVYHCVPPCDGLCKKNGGSSGSCSFLVPSGLACWCKDLPDNVPIKDTSRKCT >P01479 |Aah1b KRDGYIVYPNNCVYHCIPPCDGLCKKNGGSSGSCSFLVPSGLACWCKDLPDNVPIKDTSRKCT >P01484 (1ptx) |Aah2 VKDGYIVDDVNCTYFCGRNAYCNEECTKLKGESGYCQWASPYGNACYCYKLPDHVRTKGPGRCHGR >P01480 |Aah3 VRDGYIVDSKNCVYHCVPPCDGLCKKNGAKSGSCGFLIPSGLACWCVALPDNVPIKDPSYKCHSR >P45658 |Aah4 GRDGYIVDSKNCVYHCYPPCDGLCKKNGAKSGSCGFLVPSGLACWCNDLPENVPIKDPSDDCHKR >P56743 |Aah6 GRDGYVVKNGTNCKYSCEIGSEYEYCGPLCKRKNAKTGYCYAFACWCIDVPDDVKLYGDDGTYCSS >P01497 |AahIT1 KKNGYAVDSSGKAPECLLSNYCNNECTKVHYADKGYCCLLSCYCFGLNDDKKVLEISDTRKSYCDTTIIN >P15147 |AahIT2 KKNGYAVDSSGKAPECLLSNYCYNECTKVHYADKGYCCLLSCYCFGLNDDKKVLEISDTRKSYCDTPIIN >P21150 |AahIT4 EHGYLLNKYTGCKVWCVINNEECGYLCNKRRGGYYGYCYFWKLACYCQGARKSELWNYKTNKCDL >P81504 |AahIT5 DGYIKRHDGCKVTCLINDNYCDTECKREGGSYGYCYSVGFACWCEGLPDDKAWKSETNTCD >P80950 |Aahstr1 ARDGYIVHDGTNCKYSCEFGSEYKYCGPLCEKKKAKTGYCYLFACWCIEVPDEVRVWGEDGFMCWS >n.d.e. |KAaH1 ADVPGNYPLDSSDDTYLCAPLGENPFCIKICRKHGVKYGYCYAFQCWCEYLEDKNVKI >n.d.e. |KAaH2 ADVPGNYPLDSSDDTYLCAPLGENPSCIQICRKHGVKYGYCYAFQCWCEYLEDKNVKI >n.d.e. |ABTxL1 ADVPGNYPLDRSGKKYPCTITWKKNPSCIQICKKHGVKYGYCFDFQCWCEIFGRLKTFKI >Q9BLM3 |Aahp985 ARDAYIAKNDNCVYECFQDSYCNDLCTKNGAKSGTCDWIGTYGDACLCYALPDNVPIKLSGECHR >Q9BLM4 |Aahp1005 KKDGYIVDDKNCTFFCGRNAYCNDECKKKGAESGYCQWASPYGNACYCYKLPDRVSTKKKGGCNGR >P01482 |Amm5 LKDGYIIDDLNCTFFCGRNAYCDDECKKKGGESGYCQWASPYGNACWCYKLPDRVSIKEKGRCN >Q7YXD3 |Amm8 KDGYIVNDINCTYFCGRNAYCNELCIKLKGESGYCQWASPYGNSCYCYKLPDHVRTKGPGRCND >P84207 |Phaidotx KFIRHKDESFYECGQLIGYQQYCVDACQAHGSKEKGYCKGMAPFGLPGGCYCPKLPSNRVKMCFGALESKCA >P80962 |BarIT2 DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG >P13488 |Bom3 GRDGYIAQPENCVYHCFPGSSGCDTLCKEKGATSGHCGFLPGSGVACWCDNLPNKVPIVVGGEKCH >P59354 |Bom4 GRDAYIAQPENCVYECAKNSYCNDLCTKNGAKSGYCQWLGKYGNACWCEDLPDNVPIRIPGKCHF >P59896 |BomPI GRDAYIAQPENCVYECAKSSYCNDLCTKNGAKSGYCQWLGRWGNACYCIDLPDKVPIRIEGKCHFA >P60255 |Boma6a VRDAYIAQNYNCVYDCARDAYCNDLCTKNGAKSGYCEWFGPHGDACWCIDLPNNVPIKVEGKCHRK >P60256 |Boma6b VRDAYIAQNYNCVYDCARDAYCNELCTKNGAKSGHCEWFGPHGDACWCIDLPNNVPIKVEGKCHRK >P60257 |Boma6c VRDAYIAQNYNCVYTCFKDAHCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRIPGKCHRK >P60258 |Boma6d

350 / Chapter 51 VRDAYIAQNYNCVYHCGRDAYCNELCSKNGAKSRTRGGYCHWFGPHGDACWCIDLPNNVPIKVEGKCHRK >P60259 |Boma6e VRDAYIAQNYNCVYACARDAYCNDLCTKNGARSGLFATFGPHGDACWCIALPNNVPLKVQGKCHRK >P01488 |Bot1 GRDAYIAQPENCVYECAQNSYCNDLCTKNGATSGYCQWLGKYGNACWCKDLPDNVPIRIPGKCHF >P01485 |Bot3 VKDGYIVDDRNCTYFCGRNAYCNEECTKLKGESGYCQWASPYGNACYCYKVPDHVRTKGPGRCN >P01486 |Bot9 LKDGYIVDDRNCTYFCGTNAYCNEECVKLKGESGYCQWVGRYGNACWCYKLPDHVRTVQAGRCRS >Q17254 |Bot14 VRDGYIAQPHNCAYHCLKISSGCDTLCKENGATSGHCGHKSGHGSACWCKDLPDKVGIIVHGEKCHR >P55902 |BotIT1 VRDAYIAQNYNCVYFCMKDDYCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRIPGKCHS >P59863 |BotIT2 DGYIKGYKGCKITCVINDDYCDTECKAEGGTYGYCWKWGLACWCEDLPDEKRWKSETNTC >P55903 |BotIT4 DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG >P55904 |BotIT5 DGYIRKRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG >P59864 |BotIT6 DGYPKQKDGCKYDCIINNKWCNGCIKMHGGYYGYCWGWGLACWCEGLPEDKKWWYETNKCGR >AAT97992 |Cex1 KEGYLVSKSTGCKYECFWLGKNEGCDKECKAPNQGGGYGYCHAFACWCENLPESTPTYPIPGKSCGKK >AAT97993 |Cex2 KEGYLVSKSTGCKYECFWLGKNEGCDKECKAPNQGGGYGYCHAFACWCENLPESTPTYPIPGKSCGKK >AAT97994 |Cex3 KDGYLVNKSTGCKYECFWLGKNEFCDKECKAKNQGGSYGYCYSFACWCEGLPESTSTYPLPNKSCGRK >AAT97995 |Cex4 KEGYLVNKSTGCKYECFWLGKNEFCDKECKAKNQGGSYGYCYSFACWCEGLPESTSTYPLPNKSCGRK >AAT97996 |Cex5 KDGYLVSKSTGCKYECFWLGKNEGCDKECKAPNQGGGYGYCHAFACWCENLPESTPTYPIPGKSCGKK >AAT97997 |Cex6 REGYLVNKSTGCKYECFWLGKNEFCDKECKAKNQGGSYGYCYSFACWCEGLPESTSTYPLPNKSCGRK >AAT97998 |Cex7 REGYLVSKSTGCKYECFWLGKNEGCDKECKAPNQGGGYGYCHAFACWCENLPESTPTYPIPGKSCGKK >AAT97999 |Cex8 KEGYLVNIYTGCKYSCWLLGENEYCIAECKEIGAGYGYCHGFGCWCEQFPENKPSYPYPEKSCGRK >AAT98000 |Cex9 KDGYPVEVTGCKKSCYKLGENKFCNRECKMKHRGGSYGYCYFFGCYCEGLAESTPTWPLPNKSCGKK >AAT98001 |Cex10 KDGYLVEVTGCKKSCYKLGENKFCNRECKMKHRGGSYGYCYFFGCYCEGLAESTPTWPLPNKSCGKK >AAT98002 |Cex11 KEGYPVNIYTGCKYSCWLLGENEYCIAECKEIGAGYGYCHGFGCWCEQFPENKPSYPYPEKSCGRK >AAT98003 |Cex12 NDGYLFDKRKRCTLECIDKTGDKNCDRNCKKEGGSFGKCSYSACWCKGLPGITPISRTPGKTCRK >AAT98004 |Cex13 KDGYLVIIKTGCKYNCYILGKNKYCNSECKEVGAGYGYCYAFGCWCEGLPESIPTWPLPDKTCGTK >P59897 |Cii1 KEGYLVNHSTGCKYECYKLGDNDYCLRECKQQYGKGAGGYCYAFGCWCTHLYEQAVVWPLPKKTCN >P45667 |Cll1 KEGYLVNKSTGCKYGCFWLGKNENCDKECKAKNQGGSYGYCYSFACWCEGLPESTPTYPLPNKSCS >P45666 |Cll1m KEGYIVNLSTGCKYECYKLGDNDYCLRECKQQYGKGAGGYCYAFGCWCTHLYEQAVVWPLPKKTCT >P59898 |Cll2 KEGYLVNHSTGCKYECFKLGDNDYCLRECKQQYGKGAGGYCYAFGCWCNHLYEQAVVWPLPKKTCN >P59899 |Cll2b KEGYLVNHSTGCKYECYKLGDNDYCLRECKQQYGKGAGGYCYAFGCWCTHLYEQAVVWPLPKKTCN >Q7Z1K9 |Cll3 KEGYIVNYYDGCKYACLKLGENDYCLRECKARYYKSAGGYCYAFACWCTHLYEQAVVWPLPNKTCYGK >Q7Z1K8 |Cll4 KEGYIVNYHDGCKYECYKLGDNDYCLRECKLRYGKGAGGYCYAFGCWCTHLYEQAVVWPLPKKRCNGK >Q7Z1K7 |Cll5b KEGYLVNKSTGCKYGCFWLGKNENCDKECKAKNQGGSYGYCYSFACWCEGLPDSTPTYPLPNKSCSKK >Q7Z1K6 |Cll5c EGYLVNKSTGCKYGCFWLGKNENCDMECKAKNQGGSYGYCYSFACWCEGLPDSTPTYPLPNKSCSKK >Q7YT61 |Cll5c

Scorpion Venom Peptides / 351 KEGYLVNKSTGCKYGCFWLGKNENCDMECKAKNQGGSYGYCYSFACWCEGLPDSTPTYPLPNKSCSKK >Q7Z1K5 |Cll6 KEGYLVNMKTGCKYGCYELGDNGYCDRKCKAESGNYGYCYTVGCWCEGLPNSKPTWPLPGKSCSGK >P59865 |Cll7 KEGYLVNTYTGCKYICWKLGENKYCIDECKEIGAGYGYCYGFGCYCEGFPENKPTWPLPNKTCGRK >Q7Z1K4 |Cll8 KEGYLVKKSNGCKYECFKLGENEHCDTECKAPNQGGSYGYCDTFECWCEGLPESTPTWPLPNKSCGKK >Q8WRY4 |Cll9 EDGYLFDKRKRCTLACIDKTGDKNCDRNCKKEGGSFGHCSYSACWCKGLPGSTPISRTPGKTCKK >P18926 |Clt1 KEGYLVNHSTGCKYECFKLGDNDYCLRECRQQYGKGAGGYCYAFGCWCTHLYEQAVVWPLPNKTCS >P15223 |Cn1 KDGYLVDAKGCKKNCYKLGKNDYCNRECRMKHRGGSYGYCYGFGCYCEGLSDSTPTWPLPNKTCS >P01495 (1cn2) |Cn2 KEGYLVDKNTGCKYECLKLGDNDYCLRECKQQYGKGAGGYCYAFACWCTHLYEQAIVWPLPNKRCS >P80076 |Cn3 KEGYLVELGTGCKYECFKLGDNDYCLRECKARYGKGAGGYCYAFGCWCTQLYEQAVVWPLKNKTCR >P45662 |Cn4 KEGYLVNSYTGCKYECFKLGDNDYCLRECKQQYGKGAGGYCYAFGCWCTHLYEQAVVWPLKNKTCN >P45663 |Cn5 KEGYLVNKSTGCKYGCLLLGKNEGCDKECKAKNQGGSYGYCYAFGCWCEGLPESTPTYPLPNKSCSKK >Q94435 |Cn10 KEGYLVNKSTGCKYNCLILGENKNCDMECKAKNQGGSYGYCYKLACWCEGLPESTPTYPIPGKTCRTK >P58296 |Cn11 ARDGYPVDEKGCKLSCLINDKWCNSACHSRGGKYGYCYTGGLACYCEAVPDNVKVWTYETNTC >P63019 (1pe4) |Cn12 RDGYPLASNGCKFGCSGLGENNPTCNHVCEKKAGSDYGYCYAWTCYCEHVAEGTVLWGDSGTGPCRS >P45664 |CnGTIII KEGYLVNKSTGCKYGCFWLGKNEGCDKECKAKNQGGSYGYCYAFGCWCEGLPESTPTYPLPNKTCSKK >P45665 |CnGTIV KDGYLVDVKGCKKNCYKLGENDYCNRECKMKHRGGSYGYCYGFGCYCEGLSDSTPTWPLPNKRCGGK >P01491 (1b3c) |CsE1 KDGYLVEKTGCKKTCYKLGENDFCNRECKWKHIGGSYGYCYGFGCYCEGLPDSTQTWPLPNKTC >Q95WD2 |CsE3 KEGYIVNYHTGCKYECFKLGDNDYCLRECKLRHGKGSGGYCYAFGCWCTHLYEQAVVWPLPKKKCN >P56646 |CsEM1 KEGYLVNSYTGCKYECLKLGDNDYCLRECRQQYGKSGGYCYAFACWCTHLYEQAVVWPLPNKTCN >P46066 (1nra) |CsE5 KKDGYPVDSGNCKYECLKDDYCNDLCLERKADKGYCYWGKVSCYCYGLPDNSPTKTSGKCNPA >Q95WD1 |CsE8 EKGYLVHEDTGCRYKCTFSGENSYCDKECKSQGGDSGICQSKACYCQGLPEDTKTWPLIGKLCGRK >Q95WC9 |CsE9 EDGYLFDKRKRCTLECIDKTGDKNCDRNCKNEGGSFGKCSYFACWCKGLPGITPISRTPGKTCKI >P01492 (1vnb) |CsEv1 KEGYLVKKSDGCKYDCFWLGKNEHCDTECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNKSCGKK >P01493 (1jza) |CsEv2a KEGYLVNKSTGCKYGCLKLGENEGCDKECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNKSCSRK >P01494 (2sn3) |CsEv3b KEGYLVNKSTGCKYGCLKLGENEGCDKECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNKSCGKK >P58778 |CsEv4 KEGYMVNKSTGCSYSCPKTGESVYCDKECKAKNQGGSYGFCQYSNCWCEGLPESTPTWPLDDKPCD >P58779 (1nh5) |CsEv5 KDGYPVDSKGCKLSCVANNYCDNQCKMKKASGGHCYAMSCYCEGLPENAKVSDSATNIC >P08900 |Css2 KEGYLVSKSTGCKYECLKLGDNDYCLRECKQQYGKSSGGYCYAFACWCTHLYEQAVVWPLPNKTCN >P60266 |Css4 KEGYLVNSYTGCKFECFKLGDNDYCLRECRQQYGKGSGGYCYAFGCWCTHLYEQAVVWPLPNKTCN >P60267 |Css6 KEGYLVNSYTGCKFECFKLGDNDYCKRECKQQYGKSSGGYCYAFGCWCTHLYEQAVVWPLPNKTCN >P24336 |BjIT2 DGYIRKKDGCKVSCIIGNEGCRKECVAHGGSFGYCWTWGLACWCENLPDAVTWKSSTNTCGRKK >P56637 (1bcg) |BjxtrIT KKNGYPLDRNGKTTECSGVNAIAPHYCNSECTKVYYAESGYCCWGACYCFGLEDDKPIGPMKDITKKYCDVQIIPS >AAT52203 |Bj-alphaIT GRDAYIADNLNCAYTCGSNSyCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACRGR >n.d.e. |Isom

352 / Chapter 51 KKNGYAVDSSGKAPECLLSNYCNNECTKVHYADKGYCCLLSCYCFGLSDDKKVLEISDTRKKYCDYTIIN >n.d.e. |Isom2 KKNGYAVDSSGKAPECLLSNYCNNECTKVHYADKGYCCLLSCYCFGLSDDKKVLDISDTRKKYCDYTIIN >P59355 |Lqh2 IKDGYIVDDVNCTYFCGRNAYCNEECTKLKGESGYCQWASPYGNACYCYKLPDHVRTKGPGRCR >P56678 (1fh3) |Lqh3 VRDGYIAQPENCVYHCFPGSSGCDTLCKEKGGTSGHCGFKVGHGLACWCNALPDNVGIIVEGEKCHS >P83644 |Lqh4 GVRDAYIADDKNCVYTCGANSYCNTECTKNGAESGYCQWFGKYGNACWCIKLPDKVPIRIPGKCR >P59356 |Lqh6 VRDGYIAQPENCVYHCIPDCDTLCKDNGGTGGHCGFKLGHGIACWCNALPDNVGIIVDGVKCHK >P59357 |Lqh7 VRDGYIAKPENCAHHCFPGSSGCDTLCKENGGTGGHCGFKVGHGTACWCNALPDKVGIIVDGVKCH >P17728 (1lqh) |Lqh-alphaIT VRDAYIAKNYNCVYECFRDAYCNELCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGKCRK >n.d.e. |Lqhb1 DNGYLLNKATGCKVWCVINNASCNSECKLRRGNYGYCYFWKLACYCEGAPKSELWAYATNKCNGKL >Q26292 |LqhIT2 DGYIKRRDGCKVACLIGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG >n.d.e. |LqhIT3a DGYIRGGDGCKVSCVINHVFCDNECKAAGGSYGYCWAWGLACWCEGLPADREWKYETNTCG >P81240 |LqhIT5 DGYIRGGDGCKVSCVIDHVFCDNECKAAGGSYGYCWGWGLACWCEGLPADREWKYETNTCG >P01487 (1lqq) |Lqq3 VRDAYIAKNYNCVYECFRDSYCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGKCH >P01489 |Lqq4 VRDAYIADDKNCVYTCGSNSYCNTECTKNGAESGYCQWLGKYGNACWCIKLPDKVPIRIPGKCR >P01481 |Lqq5 LKDGYIVDDKNCTFFCGRNAYCNDECKKKGGESGYCQWASPYGNACWCYKLPDRVSIKEKGRCN >P19856 |LqqIT1 KKNGYAVDSSGKAPECLLSNYCYNECTKVHYADKGYCCLLSCYCVGLSDDKKVLEISDARKKYCDFVTIN >P19855 |LqqIT2 ADGYIRKRDGCKLSCLFGNEGCNKECKSYGGSYGYCWTWGLACWCEGLPDEKTWKSETNTCG >P09981 |BeM9 ARDAYIAKPHNCVYECYNPKGSYCNDLCTENGAESGYCQILGKYGNACWCIQLPDNVPIRIPGKCH >P01490 |BeM10 VRDGYIADDKDCAYFCGRNAYCDEECKKGAESGKCWYAGQYGNACWCYKLPDWVPIKQKVSGKCN >P09982 |BeM14 ARDAYIADDRNCVYTCALNPYCDSECKKNGADGSYCQWLGRFGNACWCKNLPDDVPIRKIPGEECR >P15221 |BeI2 ADGYVKGKSGCKISCFLDNDLCNADCKYYGGKLNSWCIPDKSGYCWCPNKGWNSIKSETNTC >P45697 (1sn1) |BmKM1 VRDAYIAKPHNCVYECARNEYCNDLCTKNGAKSGYCQWVGKYGNGCWCIELPDNVPIRVPGKCHR >P58488 (1chz) |BmKM2 VRDAYIAKPHNCVYECARNEYCNNLCTKNGAKSGYCQWSGKYGNGCWCIELPDNVPIRVPGKCH >P15227 |BmKM3 VRDAYIAKPENCVYECATNEYCNKLCTDNGAESGYCQWVGRYGNACXCIKLPDRVPIRVWGKCHG >P58328 (1sn4) |BmKM4 VRDAYIAKPENCVYHCAGNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDDVPIRVPGKCH >P59854 (1kv0) |BmKM7 VRDGYIALPHNCAYGCLNNEYCNNLCTKDGAKIGYCNIVGKYGNACWCIQLPDNVPIRVPGRCHPA >P54135 (1snb) |BmKM8 GRDAYIADSENCTYFCGSNPYCNDVCTENGAKSGYCQWAGRYGNACYCIDLPASERIKEPGKCG >P45698 |BmKM9 VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDRVPIRVPGKCHR >O61705 |BmKM10 VRDAYIAKPENCVYECGITQDCNKLCTENGAESGYCQWGGKYGNACWCIKLPDSVPIRVPGKCQ >Q9N682 |BmKM11 VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGKYGNACWCIKLPDDVPIRVPGKCHR >Q9GQW3 (1omy) |BmKaIT1 VRDAYIAQNYNCVYHCARDAYCNELCTKNGAKSGSCPYLGEHKFACYCKDLPDNVPIRVPGKCHRR >Q9GYX2 |BmKa1 VRDGYIADDKNCPYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG >Q9NJC7 |BmKa2 VKDGYIADDRNCPYFCGRNAYCDGECKKNRAESGYCQWASKYGNACWCYKLPDDARIMKPGRCNGG >Q9GUA7 |BmKa3

Scorpion Venom Peptides / 353 VRDGYIADDKNCAYFCGRNAYCDDECKKKGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG >Q9NJC5 |BmKaTX10 VRDGYIALPHNCAYGCLLNEFCNDLCTKNGAKIGYCNIQGKYGNACWCIELPDNVPIRVPGRCHPS >Q9NJC8 |BmKaTX13 VRDAYIAKPENCVYHCAGNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDDVPIRVPGKCHR >Q9GNG8 |BmKaTX15 VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG >Q9GQV6 |BmKaTX16 VRDAYIAKPHNCVYECARNEYCNDLCTKNGAKSGYCQWVGKYGNGCWCKELPDNVPIRVPGKCHR >Q9NJC4 |BmKaTX17 GRDAYIAKNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGNACWCINLPDDKPIRIPGKCHRR >P82815 |Bukatx VRDGYIADDKNCAYFCGRNAYCDEECIINGAESGYCQQAGVYGNACWCYKLPDKVPIRVSGECQQ >P56569 |MakatxI GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR >Q86BW9 |MakatxII GRDAYIADSENCTYFCGSNPYCNDLCTENGAKSGYCQWAGRYGNACWCIDLPDKVPIRIPGPCRGR >O61668 |BmKIT1 KKNGYAVDSSGKVSECLLNNYCNNICTKVYYATSGYCCLLSCYCFGLDDDKAVLKIKDATKSYCDVQII >P68727 |BmKIT2? DGYIKGKSGCRVACLIGNQGCLKDCRAYGASYGYCWTWGLACWCEGLPDNKTWKSESNTCG >Q17231 |BmKIT3 DGYIRGSNGCKVSCLWGNEGCNKECRAYGASYGYCWTWGLACWCEGLPDDKTWKSESNTCGRKK >Q17230 |BmKIT4 DGYIRGSNGCKISCLWGNEGCNKECKGFGAYYGYCWTWGLACWCEGLPDDKTWKSESNTCGRKK >Q9XY87 |BmKITa DGYIRGSNGCKVSCLWGNEGCNKECRAYGASYGYCWTWGLACWCQGLPDDKTWKSESNTCGGKK >Q95WX6 |BmKITb DGYIRGSNGCKVSCLWGNEGCNKECKAFGAYYGYCWTWGLACWCQGLPDDKTWKSESNTCGGKK >Q9Y1U3 |BmKITc DGYIRGSDGCKVSCLWGNDFCDKVCKKSGGSYGYCWTWGLACWCEGLPDNEKWKYESNTCGSKK >O77091 |BmKITAP KKNGYAVDSSGKVAECLFNNYCNNECTKVYYADKGYCCLLKCYCFGLADDKPVLDIWDSTKNYCDVQIIDLS >Q9UAC9 |BmKAS DNGYLLDKYTGCKVWCVINNESCNSECKIRGGYYGYCYFWKLACFCQGARKSELWNYNTNKCNGKL >Q9UAC8 |BmKAS1 DNGYLLNKYTGCKIWCVINNESCNSECKLRRGNYGYCYFWKLACYCEGAPKSELWAYETNKCNGKM >Q9NBW2 |BmKabT KKSGYPTDHEGCKNWCVLNHSCGILCEGYGGSGYCYFWKLACWCDDIHNWVPTWSRATNKCRA >P15228 |BmKAEP DGYIRGSNGCKVSCLLGNEGCNKECRAYGASYGYCWTWKLACWCQGLPDDKTWKSESNTCG >Q86M31 |BmKAEP2 DGYIRGSNGCKVSCLWGNEGCNKECKAFGAYYGYCWTWGLACWCEGLPDDKTWKSESNTCGGKK >Q9BKJ1 |BmKANEP2 DGYIRGSNGCKVSCLWGNDGCNKECRAYGASYGYCWTWGLACWCEGLPDDKTWKSESNTCGGKK >Q9BKJ0 |BmKANEP3 DGYIRGSNGCKISCLWGNEGCNKECKGFGAYYGYCWTWGLACWCEGLPDDKTWKSESNTCGGKK >P60277 (1dq7) |scx1MESTA GEDGYIADGDNCTYICTFNNYCHALCTDKKGDSGACDWWVPYGVVCWCEDLPTPVPIRGSGKCR >P82811 |BsIT1 DGYILMRNGCKIPCLFGNDGCNKECKAYGGSYGYCWTYGLACACEGQPEDKKHLNYHKKTC >P82812 |BsIT2 DGYIKKSKGCKVSCVINNVYCNSMCKSLGGSYGYCWTYGLACWCEGLPNAKRWKYETKTCK >P82813 |BsIT3 DGYILNSKGCKVSCVVSIVYCNSMCKSSGGSYGYCWTWGLACWCEGLPNSKRWTSSKNKCN >P82814 |BsIT4 DGYIKGNKGCKVSCVINNVFCNSMCKSSGGSYGYCWSWGLACWCEGLPAAKKWLYAATNTCG >P15224 |Os1 ERDGYIVQLHNCVYHCGLNPYCNGLCTKNGATSGSYCQWMTKWGNACYCYALPDKVPIKWLDPKCY >P15225 |Os3 VRDGYIAQPHNCVYHCFPGSGGCDTLCKENGATQGSSCFILGRGTACWCKDLPDRVGVIVDGEKCH >O76963 |OsI1 DGYPKQKDGCKYSCTINHKFCNSVCKSNGGDYGYCWFWGLACWCEGLPDNKMWKYETNTCG >n.d.e. |PgKL1 KIDGYPVDNWNCKRICWYNNKYCYDLCKGLKADSGYCWGWTLSCYCEGLPDNARIKRGGRCN >n.d.e. |PgKL2

354 / Chapter 51 KIDGYPVDNWNCKRICWYNNKYCNDLCKGLKADSGYCWGWTLSCYCEGLPDNARIKRGGRCN >P58910 (1t1t) |Kurtoxin KIDGYPVDYWNCKRICWYNNKYCNDLCKGLKADSGYCWGWTLSCYCQGLPDNARIKRSGRCRA >P58752 |Birtoxin ADVPGNYPLDKDGNTYKCFLLGGNEECLNVCKLHGVQYGYCYASKCWCEYLEDDKDSV >n.d.e. |Ikitoxin ADVPGNYPLDKDGNTYKCFLLGENEECLNVCKLHGVQYGYCYASKCWCEYLEDDKDSV >n.d.e. |Altitoxin ADVPGNYPLDKDGNTYTCLELGENKDCQKVCKLHGVQYGYCYAFFCWCKELDDKDVSV >n.d.e. |Bestoxin ADVPGNYPLDKDGNTYTCLELGENKDCQKVCKLHGVQYGYCYAFSCWCKEYLDDKDSV >n.d.e. |Dorsotoxin ADVPGNYPLDKDGNTYTCLKLGENKDCQKVCKLHGVQYGYCYAFECWCKEYLDDKDSV >P56611 |Tb? KEGYLMDHEGCKLSCFIRPSGYCGSECKIKKGSSGYCAWPACYCYGLPNWVKVWDRATNKCGKK >P56609 |TbIII8 KEGYAMDHEGCKFSCFPRPAGFCDGYCKTHLKASSGYCAWPACYCYGVPSNIKVWDYATNKC >P56608 |TbIV5 KKDGYPVEADNCAFVCFGYDNAYCDKLCGDKKADSGYCYWVHILCYCYGLPDNEPTKTNGKC >P60276 |Tb2II KEGYAMDHEGCKFSCFIRPSGFCDGYCKTHLKASSGYCAWPACYCYGVPSNIKVWDYATNKC >P60275 |TbIT1 GKEGYPVDSRGCKVTCFFTGAGYCDKECKLKKASSGYCAWPACYCYGLPDSVPVYDNASNKCB >n.d.e. |Tc48a NKDGYLMEGDGCKMGCLTRKASYCVDQCKEVGGKNGYCYAWLSCYCYNMPDSVEIWDSKNNKCGK >n.d.e. |Tc48b KDGYLVGNDGCKYNCLTRPGHYCANECARVKGKDGYCYAWNACYCYSMPDWVKGKTWSRSTNRCGR >P60214 |Tc49b KKEGYLVGNDGCKYGCITRPHQYCVHECELKKGTDGYCAYWLACYCYNMPDWVKTWSSATNKCK >AAW72463 |Tcontxp KEGYPADSKGCKVTCFLTAAGYCNTECKLQKASSGYCAWPACYCYGLPDSASVWDSATNKCGKK >AAW72453 |Tcogamma KEGYAMDHEGCKLSCFIRPSGYCGRECGYKKGSSGYCAWPACYCYGLPNWVKVWERATNRCGKK >n.d.e. |Ardiscretin KNGYIIEPKGCKYSCDWGSSTWCNRECKFKKGSSGYCAWPACWCYGLPDNVKFIDYYNNKC >P83435 |Tf4 GKEGYPADSKGCKVTCFFTGVGYCDTECKLKKASSGYCAWPACYCYGLPDSASVWDSATNKC >n.d.e. |Tpa2 KKEGYLVGNDGCKYSCFTRPAQYCVHECELRKGTDGYCYAWLACYCYNMPDHVRTWSRATNRCGS >P15226 (1npi) |Tsgamma EGYLMDHEGCKLSCFIRPSGYCGRECGIKKGSSGYCAWPACYCYGLPNWVKVWDRATNKCGKK >P68410 |Ts2 KEGYAMDHEGCKFSCFIRPAGFCDGYCKTHLKASSGYCAWPACYCYGVPDHIKVWDYATNKC >P01496 |Ts3 KKDGYPVEYDNCAYICWNYDNAYCDKLCKDKKADSGYCYWVHILCYCYGLPDSEPTKTNGKCKS >P45669 |Ts4 GREGYPADSKGCKITCFLTAAGYCNTECTLKKGSSGYCAWPACYCYGLPESVKIWTSETNKC >P46115 |TsV KKDGYPVEGDNCAFACFGYDNAYCDKLCKDKKADDGYCVWSPDCYCYGLPEHILKEPTKTSGRC >O77463 |TsNTxP REGYPADSKGCKITCFLTAAGYCNTECTLKKGSSGYCAWPACYCYGLPDSVKIWTSETNKCGKK >P56612 |Tstgamma KEGYLMDHEGCKLSCFIRPSGYCGRECTLKKGSSGYCAWPACYCYGLPNWVKVWDRATNKC >P68411 |Tst2 KEGYAMDHEGCKFSCFIRPAGFCDGYCKTHLKASSGYCAWPACYCYGVPDHIKVWDYATNKC >n.d.e. |Tz1 KDGYLVGNDGCKYSCFTRPGTYCANECSRVKGKDGYCYAWMACYCYSMPNWVKTWDRATNRCGR LEGEND FOR THE SEQUENCES All amino acid sequences available in databases of toxins from scorpion venom, either specific for K+ or Na+ ion channels are listed in a FASTA format. The first 14 positions of the headings are the access codes from SWISS-PROT or GenBank. The PDB accession numbers are between parentheses. After the vertical line (|) is the known trivial name of the corresponding toxin.

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52 Snake Venom Peptides ALAN L. HARVEY

ABSTRACT

THREE-FINGER TOXINS

Snake venoms are complex mixtures of small molecules, peptides, and proteins. Most of the biologically active toxins are peptides or enzymes. This chapter covers nonenzymatically active peptides and toxins. The peptides belong to several structural classes, and they have many different biological actions. The best characterized are the so-called three-finger toxins that have three peptide loops stabilized by four disulfide bridges. Despite their common 3D shape, these peptides can interfere selectively with different biological targets, including nicotinic and muscarinic acetylcholine receptors, acetylcholinesterase, ion channels, and cell membranes. Other small peptides can block K+ or Ca2+ channels and are based on Kunitz serine proteinase inhibitors, while other toxins have different molecular scaffolds and different pharmacological activities.

The first members of this group (“α-neurotoxins”) were isolated from venoms of cobras and sea snakes because of their potent muscle paralyzing activity. They have 60–74 amino acid residues in a single chain crosslinked by four or five disulfide bonds. They contain a central core stabilized by four S-S bridges and three protruding peptide loops (like three fingers of a hand). It was long apparent that another group of cobra venom toxins with a completely different biological activity (the “cardiotoxins” or “cytotoxins”) had similar molecular architecture as the α-neurotoxins. Since then, nearly 300 different examples of snake peptides with the three-finger motif have been described, and they are known to have several quite different pharmacological actions. This makes them extremely interesting in terms of structure-activity relationships and for an understanding of molecular evolution (see, for example, [4]).

INTRODUCTION a-Neurotoxins

Snake venoms have been long known to have potent biologically acting molecules that are proteins or peptides. While much early work focused on enzymes, the fashion changed in the mid-1960s with the advent of gel filtration and ion-exchange chromatography to look at the chemical and biological properties of smaller peptides. This research has been incredibly productive in terms of understanding how peptide structure contributes to biological activity and also in revealing molecules with highly selective pharmacological actions. More recent research on venom peptides has been aided by application of molecular biology techniques and proteomics. New classes of peptides with novel pharmacological effects are still being discovered. This chapter concentrates on nonenzymatically active peptides. Handbook of Biologically Active Peptides

Discovery These are generally found in venoms of snakes of the Elapidae (cobras, kraits, mambas, etc.) and Hydrophidae (sea snakes) families, although similar molecules occur in venoms of some Colubridae. The α-neurotoxins can be subdivided into “short” (60–62 residues; four disulfides) and “long” (66–74 residues; five disulfides), including the kappa-neurotoxins (66 residues; five disufhides) and “weak” neurotoxins and other homologs (63–66 residues; five disulfides). Structure of the Precursor mRNA/Gene The nucleotide sequence of a cDNA encoding erabutoxin a was derived from material extracted from venom glands, and subsequently the relevant genes encoding erabutoxins were cloned from a genomic library prepared from the sea snake Laticauda semifasciata. The gene for

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356 / Chapter 52 cobrotoxins from Naja naja atra had a similar organization to those for the erabutoxins, with three exons separated by two introns. For a review on work on the gene structures of α-neurotoxins, see [23]. Clearly, the toxins are found in venom that is synthesized and stored in the venom glands of the snake. The control of toxin synthesis is not yet understood, although transcriptional regulation of toxin genes may be involved. Receptors The α-neurotoxins bind to nicotinic acetylcholine receptors. Most early work concentrated on the receptors at the neuromuscular junction or on electroplaques of electric fish. These “muscle-type” receptors are pentameric proteins with two α subunits and one each of β, γ, and ε (or δ), and such receptors are powerfully blocked by the high-affinity binding of both short and long α-neurotoxins. Venoms of Bungarus multicinctus and other kraits also contain κ-bungarotoxins that have little affinity for muscle-type nicotinic receptors but that bind to some neuronal isoforms (particularly the α3β2 subtype). More recently, long α-neurotoxins were found to bind not just to muscle-type nicotinic receptors but also to homo-oligomeric nicotinic receptors (comprising five α subunits) that are found on neurons.The so-called “weak” toxins can also bind with relatively high affinity to such neuronal nicotinic receptors. Conformation The three-dimensional structures of the short chain toxin erabutoxin b, the long-chain αbungarotoxin, and κ-bungarotoxin have been described. The overall shape is formed by a central core that is stabilized by four S-S bonds and three peptide loops protruding like fingers from a hand. Biological Activity The short and long αneurotoxins paralyze skeletal muscle by blocking the ability of the neurotransmitter acetylcholine to bind to and activate the nicotinic receptors at the neuromuscular junction. These α-neurotoxins are among the most potent of venom constituents and presumably evolved to help elapid and hydrophid snakes catch their prey. The biological activities of the neurotoxins that bind to neuronal acetylcholine receptors are not clearly understood. A structurally homologous toxin named γbungarotoxin was isolated from venom of Bungarus multicinctus. It is lethal on injection, presumably because it binds weakly to nicotinic cholinoceptors. γ-Bungarotoxin also inhibits platelet aggregation.

Muscarinic Toxins Discovery Venom of mamba snakes had been reported to contain acetylcholine or a cholinomimetic compound. However, competition binding studies with synaptosomal membranes led to the discovery of threefinger type peptides that bound to some central musca-

rinic receptors (see [8] for a review). The first peptides to be characterized were named muscarinic toxin 1 (MTx1) and 2, although their toxicity has not been established. These toxins are relatively selective for the m1 subtype of muscarinic receptor, and other toxins from mamba venoms have different selectivities. More recently, homologous peptides have been isolated from cobra venoms, and a larger peptide BM14 (82 amino acid residues including 10 cysteines) was found in Bungarus multicinctus venom that bound to m2 muscarinic receptors. γ-Bungarotoxin also binds to m2 receptors. Structure of the Precursor mRNA/Gene The cDNA encoding muscarinic toxin 2 was cloned and that of m1-toxin 1 also. Although not a classical muscarinic toxin, the muscarinic receptor binding peptide BM14 from Bungarus multicinctus venom is coded by a gene that has a similar organization as α-neurotoxin and cardiotoxin genes. Receptors Different muscarinic toxins have different binding affinities for different subtypes of muscarinic receptors (see [1] for a review). From green mamba (Dendroaspis angusticeps) venom, MTx1 is relatively selective for the m1 subtype; MTx3 is highly selective for the m4 subtype; and MTx7 (or m1-toxin) is extremely selective for the m1 subtype. Conformation The solution structure of MTx2 was solved by NMR and shown to be three fingerlike. Biological Activity MTx1 and -2 from green mamba venoms appear to be very unusual for toxins in that they cause an allosteric activation of their receptor target rather than a block. When injected into the hippocampus of mice, these peptides can enhance learning and memory processes, whereas MTx3 (which blocks m4 muscarinic receptors) is inhibitory. MTx7 (or m1-toxin) blocks m1 muscarinic receptors virtually irreversibly. The contribution of the muscarinic toxins to the action of mamba venoms is unknown.

Anticholinesterases Discovery The anticholinesterase activity of green mamba venom was revealed in pharmacological experiments by Osman et al. in 1973 and predicted from the muscle fasciculations caused by injection of venom fractions into mice. The isolated peptides with powerful inhibitory effects on acetylcholinesterase were called “fasciculins.” Structure of the Precursor mRNA/Gene cDNA encoding fasciculin 1 has been cloned. The cDNA is organized similarly to that of α-neurotoxins, although the signal peptide sequence for fasciculin 1 lacks a cysteine residue generally encoded in structurally related toxin genes.

Snake Venom Peptides / 357 Receptors Fasciculins bind tightly to a peripheral anionic site on acetylcholinesterases from most species, but not from birds. They also have very little affinity for butyrylcholinesterases. The interaction of fasciculin with enzyme has been studied in great detail and the complex of enzyme and inhibitor has been studied. Conformation The structure of fasciculin 1 is similar to that of other three-finger toxins. Biological Activity Fasciculins produce a longlasting inhibition of acetylcholinesterase activity, thereby prolonging the action of the neurotransmitter acetylcholine. In vivo, this can lead to visible trembling (“fasciculations”) of muscles under the skin. When combined with other toxins that are present in mamba venoms, the fasciculins can cause muscle paralysis by inducing a depolarization-type blockade due to prolonged activation of acetylcholine receptors at the neuromuscular junction.

Three-finger, Disintegrin-like Peptides from Mamba Venoms Discovery Many components of snake venoms have been found to inhibit platelet aggregation. Most are enzymes, but some peptides that are in the three-finger structural class have also been found, first in mamba venoms (mambin) and then in krait venom (γbungarotoxin). Structure of the Precursor mRNA/Gene There have been no reports on the cloning of these antiplatelet peptides from venoms of mambas, but gene organization for γ-bungarotoxin was shown to be similar those of α-neurotoxin and cardio toxin genes. Receptors Mambin (also known as dendroaspin) and γ-bungarotoxin contain the sequence RGD that is common to proteins that bind to the glycoprotein receptor IIb-IIIa on platelets. Homologous sequences were noted in thrombostatin and peptide S5C1, which are also from mamba venoms and assumed to be platelet inhibitors. Conformation The conformation of γ-bungarotoxin and mambin is similar to other three-finger toxins in being predominantly β-sheet. Biological Activity These peptides prevent aggregation of blood platelets.

Ca2+ Channel Blockers Discovery From screening of components of black mamba venom for activity on isolated smooth muscle, a 60-residue peptide was isolated and named calciseptine. Earlier, a similar molecule named FS2 had been isolated, although its biological activity was not known. They are in the general three-finger class of toxins.

Structure of the Precursor mRNA/Gene The genes for these peptides do not appear to have been cloned. Receptors The peptides bind to L-type Ca2+ channels, probably via the recognition site for 1, 4-dihydropyridines. Calciseptine does not affect N-type or T-type Ca2+ channels. Conformation The solution structure of FS2 by NMR experiments is similar to those of other threefinger toxins. Biological Activity Both calciseptine and FS2 block Ca2+ currents through L-type Ca2+ channels in cardiac and smooth muscle cells, although calciseptine was found to act as an agonist on L-type channels in skeletal muscles [5]. Both in vivo and in vitro, calciseptine and FS2 inhibit contractions of smooth muscle; because of this, the peptides have hypotensive effects.

Cardiotoxins Discovery Cobra venoms were long known to have cardiovascular effects in vivo and to cause direct damage to isolated cardiac preparations. Protein fractions showing such activity were called “cardiotoxins.” Such fractions could lyse cells, particularly erythrocytes, and they also became known as “cytotoxins.” Purified cardiotoxins were shown to be homologous in size and structure to the short chain α-neurotoxins. Structure of the Precursor mRNA/Gene Genomic DNAs encoding several cardiotoxins were isolated from the liver of the Taiwan cobra Naja naja atra. Similarities to the genes for α-neurotoxins were noted. Several cDNAs for cardiotoxins have also been obtained by from RNA isolated from cobra venom glands. Receptors The mechanism of action of cardiotoxins remains obscure despite decades of research. They are generally thought to disrupt cell membranes, but whether it is through binding to a specific receptor is unknown. Cardiotoxins are very basic peptides and they have been suggested to bind to negatively charged lipids on membranes. Certainly, Lys residues on the peptides are important for activity, but cardiotoxins affect some cells much more than others, implying some selective binding. More recently, evidence was found for sulfo-containing receptors on membranes of neuronal and cardiac cells [19]. Conformation The structure of a cardiotoxin has been determined by X-ray crystallography and by NMR spectroscopy. The differences in a projection from the tip of the third loop may correlate with activity. Biological Activity Cardiotoxins depolarize electrically excitable cells (neurones and muscles) in an irreversible manner, leading to sustained contractures of muscles. Some cardiotoxins have hemolytic activity, although this is difficult to ascribe directly to the cardiotoxins because

358 / Chapter 52 they act in a highly synergistic manner with very low amounts of venom phospholipase A2.

Synergistic Toxins Discovery Fractions from mamba venoms were noted to have little lethality in mice until combined, leading to the isolation of several peptides with a threefinger structure that were named as “synergistic toxins.” Some of these were subsequently shown to have anticholinesterase properties, but others act by still unknown mechanisms. Structure of the Precursor mRNA/Gene The cDNA coding for a likely synergistic toxin was cloned from RNA from venom glands of a green mamba Dendroaspis angusticeps. Receptors The binding sites are unknown. Conformation It is assumed that the synergistic toxins have a conformation broadly similar to other members of the three-finger class. Biological Activity Little is known about the biological activity, other than in vivo toxicity.

Homologs of Unknown Activity Protein isolation and cDNA cloning have provided examples of several peptides that are homologous to three-finger toxins but whose activities are not yet known. These so-called “orphan toxins” have been studied from a phylogenetic perspective [4], but this does not point to pharmacological classifications. Further work is required.

PROTEASE INHIBITOR HOMOLOGS Enzyme Inhibitors Discovery Proteinase inhibitors are very widespread throughout nature, so it is not surprising that they have been isolated from various snake venoms, including viperids and elapids. Most are in the Kunitz class of serine proteinase inhibitors. Serum of the snake Trimeresurus mucrosquamatus has a novel glycoprotein that inhibits the activity of zinc-dependent metalloproteinases but not serine proteases. Structure of the Precursor mRNA/Gene cDNA for trypsin and chymotrypsin inhibitors has been cloned from venom glands of Vipera ammodytes and Naja naja atra. There is a shared gene structure across a wide range of homologs, including some with markedly different biological activities (see next section). Receptors The peptides bind to serine proteinases, particularly to trypsin and chymotrypsin. However, their physiological targets are not known.

Conformation The peptides adopt the same conformation as bovine pancreatic trypsin inhibitor. Biological Activity The peptides inhibit the activity of various proteinases, but their purpose in venoms is obscure, although they may interfere with blood clotting by inhibiting enzymes involved in the clotting cascade.

Dendrotoxins Discovery Venom from the Eastern green mamba Dendroaspis angusticeps was shown to increase acetylcholine release from motor nerve endings, an effect subsequently correlated with block of some neuronal K+ channels [6]. The peptides responsible (dendrotoxins) had been isolated and sequenced before their biological activity was known. Structure of the Precursor mRNA/Gene The gene for dendrotoxin K was cloned from a cDNA library made from venom glands of black mamba Dendroaspis polylepis. Most dendrotoxins have a pyroglutamic acid residue at their N-terminus. Receptors α-Dendrotoxin from green mamba and dendrotoxin I from the black mamba block some cloned voltage-dependent K+ channels (Kv1.1, Kv1.2, and Kv1.6) in the low nanomolar range; dendrotoxin K from the black mamba selectively blocks Kv1.1 channels at picomolar concentrations [6]. Interactions with channel proteins have been studied. Conformation The solution structures of dendrotoxins I and K were determined by NMR and the X-ray crystal structure of α-dendrotoxin was also solved. All are similar to bovine pancreatic trypsin inhibitor. Biological Activity The first biological activity of the dendrotoxins to be noticed was the augmentation of twitch responses of skeletal muscle preparations in response to nerve stimulation. Although not very toxic on peripheral injection, dendrotoxins are potent toxins when injected centrally in mice, causing convulsions and death. These effects are due to the peptides blocking some of the voltage-activated K+ channels in neurons, leading to increased neuronal firing and excitability [57].

Ca2+ Channel Blockers Discovery A BPTI homolog with blocking activity at L-type Ca2+ channels was isolated from green mamba venom and named calcicludine. Structure of the Precursor mRNA/Gene The gene for calcicludine has not been characterized. Receptors Calcicludine blocks L-type Ca2+ channels in cerebellar granular cells and cardiac myocytes. The block of the α1C form of the L-type channels is

Snake Venom Peptides / 359 partial but irreversible, while calcicludine can increase the activity of the α1A, -B and -E subtypes [17]. Conformation The NMR structure of calcicludine is similar to that of dendrotoxins. Biological Activity Calcicludine can reduce contractions of cardiac muscle.

Conformation The solution structure of a sarafotoxin has been solved using NMR techniques. The molecule is largely α-helical, with a very flexible C-terminal region. Biological Activity The principal activity is vasoconstriction mediated by actions on endothelin receptors. In vivo, death is by cardiac arrest.

CROTAMINE-LIKE MYOTOXINS CRISP FAMILY TOXINS Discovery Rattlesnake venoms are known to cause damage to skeletal muscle, and several small peptides (42 residues with three disulfides) associated with that activity have been isolated. These include myotoxin A from the prairie rattlesnake Crotalus viridis viridis and crotamine from the South American rattlesnake C. durissus terrificus. Structure of the Precursor mRNA/Gene A 1.8 kb gene Crt-p1 coding for crotamine was found in specimens of C. durissus terrificus. Receptors Crotamine and homologous myotoxins can bind to Na+ channels on skeletal muscle membranes and also to lipid membranes. Conformation The NMR-derived structure of crotamine has a short N-terminal α-helical region and a small anti-parallel triple stranded β-sheet area; overall, the folding has a resemblance to that of βdefensins. Biological Activity The crotamine-like myotoxins can depolarize skeletal muscles and cause myotoxic damage. Crotamine has also been shown to have analgesic activity that is reversed by naloxone. More recently, crotamine was found to penetrate into dividing cells and localize in the nucleus [9].

SARAFOTOXINS Discovery The venom of the burrowing asp Atractaspis engaddensis causes cardiac arrest. A family of toxins, the sarafotoxins, has been characterized. They have 21 amino acid residues in a single chain stabilized by two disulfide bonds [3]. A similar toxin, bibrotoxin, was found in venom of Atractaspis bibroni. Longer homologs, with 24 residues, were discovered in the venom of Atractaspis microlepidota microlepidota. All are homologs of the mammalian endothelins. Structure of the Precursor mRNA/Gene The gene encoding sarafotoxins from the burrowing asp is unusual in coding for five different isotoxins in a “rosary-type” organization. The mechanisms for processing the mature toxin peptides are unknown. Receptors The sarafotoxins bind to endothelin receptors. There may be a different, as yet uncharacterized, subtype that recognizes the “long” sarafotoxins.

Discovery Cysteine-rich secretory proteins (CRISPs) are found in mammalian epididymis and the immune system, though their functions are unknown. They have been found to be widely distributed in snake venoms, with examples being known from colubrid, elapid, and viperid snakes [22]. Pseudechetoxin was discovered by screening a wide variety of venoms for activity on a particular ion channel. Structure of the Precursor mRNA/Gene cDNAs encoding ablomin, catrin, latisemin, ophanin, piscivorin, tigrin, and triflin have been cloned. They code for mature proteins of 221 residues including 16 cysteines [22]. Similar molecules with different pharmacological actions were cloned from Australian elapids: pseudechetoxin from Pseudechis australis and pseudecin from Pseudechis porphyriacus [21]. These gene products have 238 amino acid residues, including a 19-residue signal peptide. A further eight residues are thought to be cleaved enzymatically to give the mature peptide. Receptors It is suggested that ablomin, catrin, latisemin, ophanin, piscivorin, and triflin may be L-type Ca2+ channel blockers (they reduce contractions of arterial smooth muscle stimulated by K+-induced depolarization with no effect on contractions stimulated by release of intracellular Ca2+ by caffeine) [22]. Pseudechetoxin and pseudecin block channels in olfactory and retinal cells that are gated by cyclic nucleotides [21]. Conformation A crystal structure of triflin has been obtained. It has a large globular portion linked to a rodlike cysteine-rich domain containing five disulfide bridges. Biological Activity The function of the CRISP proteins in snake venoms is unknown. Since they are so widely distributed in snakes of different families and in different geographical regions, perhaps they serve a common purpose such as aiding the spread of venom after a bite.

NATRIURETIC PEPTIDES Discovery A vasorelaxant peptide of 38 amino acid residues and with homology to atrial natriuretic peptide (ANP) was isolated from green mamba venom and

360 / Chapter 52 named dendroaspis natriuretic peptide, DNP. Homologous peptides have been isolated from the venom of the inland taipan Oxyuranus microlepidotus. Structure of the Precursor mRNA/Gene DNP is expressed in venom glands, but it is not known if it plays a signalling role elsewhere in the snake. DNP has been found in human tissues [10]. Several Asian and South American snakes contain genes for C-type natriuretic peptides. Receptors DNP activates ANP-A receptors rather than ANP-B receptors. Conformation The overall conformation of DNP has not been described. It is assumed to have a disulphide bridge between residues 7 and 23. Biological Activity DNP acts on ANP-A receptors to activate guanylate cyclase and increase levels of cyclic GMP, which cause vascular relaxation. DNP also causes natriuresis. It has been suggested that DNP may have a use in treating patients with congestive heart failure.

DISINTEGRINS Discovery Many components of snake venoms interfere with blood coagulation through actions on platelets (see review [2]). Disintegrins have a variety of peptide structures (including some three-finger–shaped molecules described in section 2.4) but generally share a three residue section of RGD. They have been divided into five groups according to size and disulphide bonds. The largest also have enzymatic activity. Structure of the Precursor mRNA/Gene Several cDNA sequences have been cloned and found to be quite variable in length, although a common ancestor is likely [2]. Receptors The peptides bind to various integrins, including αIIbβ3, α2β3, and α5β1. Conformation A crystal structure of schistatin from the saw-scaled viper Echis carinatus has been solved. This is a homomeric dimer formed by two disulfide bonds between monomers. Each monomer has three sets of anti-parallel β-strands and four intramolecular disulphide bonds. The conformation of albolabrin has been defined by NMR measurements. Biological Activity The disintegrins prevent platelet aggregation.

C-TYPE LECTINS AND LECTIN-LIKE PROTEINS Discovery Lectins are complex proteins that bind to various sugars and have a variety of biological effects. C-type lectin-like proteins lack carbohydrate binding activity, and have only been found in various snake

venoms. Over 80 are known [15]. They affect platelet functions and/or coagulation factors, and therefore disturb blood coagulation. Structure of the Precursor mRNA/Gene Phylogenetic analysis of the A and B chains of the C-type lectins and the lectin-like proteins indicate that the C-type lectinlike proteins evolved separately from the lectins [15]. Receptors Different C-type lectin-like proteins seem to bind to different receptors on platelets, including the integrins α2β1 and αIIβ3, glycoproteins in the leucine-rich repeat family GPIb-IX-V and the immunoglobulin superfamily GPIV, causing either inhibition or activation [11, 20]. They can also bind to von Willebrand factor, collagen, and fibrinogen. Other C-type lectin-like proteins have anticoagulant activity through binding to different coagulation factors in blood [14, 15]. Conformation The C-type lectin-like proteins are made up of various combinations of homologous subunits (A and B chains, each of about 14 kDa). The structures and structure-activity relationships are being actively studied [11, 12, 14, 15]. Biological Activity Many of the proteins can both inhibit and activate platelets [11]. Overall, most seem to produce thrombocytopenia. Others have anticoagulant properties (e.g., see [14]).

WAGLERINS Discovery Waglerins are small peptides from venom of Wagler’s pit viper, Tropidolaemus (Trimeresurus) wagleri that are lethal to mice. Structure of the Precursor mRNA/Gene The peptides have 22 or 24 amino acid residues, including seven proline residues and one disulfide bond. The genes for waglerins do not appear to have been cloned, but the peptides can be readily synthesized. Receptors Waglerins bind to muscle-type nicotinic acetylcholine receptors containing the ε- but not the γ-subunit [13]. Waglerin-1 also binds to sites on different forms of GABAA receptors. Conformation The 3D conformation of waglerin-1 has been resolved by NMR studies. There is a short, rigid loop formed by the S-S bond and two extended and flexible tails. Biological Activity When injected into mice, waglerins cause respiratory failure because of blockade of acetylcholine receptors at the neuromuscular junction. Rats are insensitive to the toxins.

BRADYKININ-POTENTIATING PEPTIDES Discovery Venom of the Brazilian snake Bothrops jararaca was found to potentiate the effects of bradyki-

Snake Venom Peptides / 361 nin in vitro and in vivo [7]. Several small peptides of 5–14 residues with that activity were isolated, and other examples have been found in other venoms of Bothrops and Crotalus snakes. Since these act by inhibiting the activity of angiotensin-converting enzyme (ACE), they led the way to the discovery and development of clinically useful ACE inhibitor drugs [7]. Structure of the Precursor mRNA/Gene Several genes have been cloned from a cDNA library of venom glands of Bothrops jararaca that code for the precursor of bradykinin potentiating peptides along with C-type natriuretic peptide. Similar genes were found in venom glands of three Asian snakes: Trimeresurus flavoviridis, Trimeresurus gramineus, and Agkistrodon halys blomhoffi. The control of expression and the modification to give the pyroglutamic residue found in the active peptides is not known. However, similar mRNA was detected in various brain regions in the snakes, suggesting that the peptides may have a signaling role within the snake. Receptors Bradykinin potentiating peptides interact with the active sites on ACE. A homologous peptide, blomhotin from Agkistrodon halys blomhoffii, causes contractions of rat stomach preparations in a manner consistent with their being specific receptors for the peptide. These have not been identified. Conformation The conformation of bradykinin potentiating peptide F from Agkistrodon piscivorus piscivorus was studied by NMR. Two major forms were found (stretched and folded), and it was speculated that one was associated with activity on ACE and the other with potentiating responses to bradykinin. Biological Activity The peptides enhance the contractile activity of bradykinin on smooth muscle, although potency does not necessarily correlate with ability to inhibit ACE activity [7].

AVIT PEPTIDES Discovery Among several peptides isolated from black mamba venom and then sequenced were protein A and A’ that had 81 amino acid residues, including 10 cysteines, and that were not homologous to any known peptide group. A very similar peptide was later found to cause contractions of intestinal smooth muscle preparations and was called “mamba intestinal toxin” (MIT1). Structure of the Precursor mRNA/Gene Although MIT1 does not appear to have been cloned, mammalian homologs have been found. These are known as prokineticins, and they are expressed in the gastrointestinal tract, brain, and other tissues. Receptors Schweitz et al. noted that radiolabeled MIT1 bound to ileal and brain membranes with very

high affinity, and there was pharmacological evidence that prokineticins act on G-protein coupled receptors. Two such receptors have been cloned. Conformation MIT1 is unusual in the arrangement of its five disulfide bonds, and, from NMR experiments, it was found to have an overall homology with colipase. As well as the mammalian prokineticins, there are homologs in amphibian skin. All share a conserved Nterminal sequence of AVIT, hence the proposed name of “AVIT protein family”. Biological Activity MIT1 powerfully contracts smooth muscle in sections of the gastrointestinal tract. The peptide also can sensitize pain receptors. However, its contribution to the overall effects of mamba venom is not known.

MISCELLANEOUS PEPTIDES In the late 1960s, the advances in protein purification and analysis techniques gave rise to information about the structures of many venom components whose activity was unknown. Often, it took many years before a biological activity could be ascribed to such peptides. Recently, a similar phenomenon is appearing because of the use of proteomics to reveal the presence of more and more peptides in venoms. Many of these peptides may have biological activity, but that remains to be determined. Other peptides have been ascribed a biological activity because of similarities to other better characterized molecules, but their significance in snake venom is still unknown. A few current examples follow. Vascular endothelial growth factors are proteins that stimulate angiogenesis. Several examples have been found in snake venoms (e.g., Bothrops and Vipera species), and it is suggested that they may act to enhance distribution of venom after a bite by increasing vascular permeability. By fractionating venom and using mass spectrometry to detect peptides with masses different from known classes of toxin, a novel 51-amino acid residue peptide was isolated from the venom of the cobra Naja nigricollis [18]. This is homologous to whey acidic proteins, including the elastase inhibitor elafin. The cobra-derived peptide was named nawaprin. It does not inhibit elastase, and its biological activity is unknown. A small protein of 107 residues was isolated from the venom of the king cobra (Ophiophagus hannah) and named ohanin [16]. It is different from other snake venom proteins, but it has homology with SPRY domain proteins. It is not lethal in mice, but it decreases locomotor activity and sensitizes responses to painful stimuli. Its mechanism of action is unknown.

362 / Chapter 52 References [1] Bradley KN. Muscarinic toxins from the green mamba. Pharmacol Ther 2000;85:87–109. [2] Calvete JJ, Marcinkiewicz C, Monleon D, Esteve V, Celda B, Juarez P, Sanz L. Snake venom disintegrins: Evolution of structure and function. Toxicon 2005;45:1063–74. [3] Ducancel F. The sarafotoxins. Toxicon 2002;40:1541–5. [4] Fry BG, Wuster W, Kini RM, Brusic V, Khan A, Venkataraman D, Rooney AP. Molecular evolution and phylogeny of elapid snake venom three-finger toxins. J Mol Evol 2003;57:110–29. [5] Garcia MC, Hernandez-Gallegos Z, Escamilla J, Sanchez JA. Calciseptine, a Ca2+ channel blocker, has agonist actions on L-type Ca2+ currents of frog and mammalian skeletal muscle. J Membr Biol 2001;184:121–9. [6] Harvey AL, Robertson B. Dendrotoxins: structure-activity relationships and effects on potassium ion channels. Curr Med Chem 2004;11:3065–72. [7] Hayashi MA, Camargo AC. The bradykinin-potentiating peptides from venom gland and brain of Bothrops jararaca contain highly site specific inhibitors of the somatic angiotensinconverting enzyme. Toxicon 2005;45:1163–70. [8] Jerusalinsky D, Harvey AL. Toxins from mamba venoms: Small proteins with selectivities for different subtypes of muscarinic acetylcholine receptors. TIPS 1994;15:424–30. [9] Kerkis A, Kerkis I, Radis-Baptista G, Oliveira EB, ViannaMorgante AM, Pereira LV, Yamane T. Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. FASEB J 2004;18:1407–9. [10] Lee S, Park SK, Kang KP, Kang SK, Kim SK, Kim W. Relationship of plasma Dendroaspis natriuretic peptide-like immunoreactivity and echocardiographic parameters in chronic haemodialysis patients. Nephrology 2004;9:171–5. [11] Lu Q, Navdaev A, Clemetson JM, Clemetson KJ. Snake venom C-type lectins interacting with platelet receptors. Structurefunction relationships and effects on haemostatis. Toxicon 2005;45:1089–98. [12] Matsui T, Hamako J. Structure and function of snake venom toxins interacting with human von Willebrand factor. Toxicon 2005;45:1075–87.

[13] Molles BE, Tsigelny I, Nguyen PD, Gao SX, Sine SM, Taylor P. Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site. Biochemistry 2002;41: 7895–906. [14] Morita T. Structures and functions of snake venom CLPs (C-type lectin-like proteins) with antocoagulant-, procoagulant-, and platelet-modulating activities. Toxicon 2005;45:1099–114. [15] Ogawa T, Chijiaw T, Oda-Ueda N, Ohno M. Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom. Toxicon 2005;45:1–14. [16] Pung YF, Wong PT, Kumar PP, Hodgson WC, Kini RM. Ohanin, a novel protein from king cobra venom, induces hypolocomotion and hyperalgesia in mice. J Biol Chem 2005;280:13137– 47. [17] Stotz SC, Spaetgens RL, Zamponi GW. Block of voltagedependent calcium channel by the green mamba toxin calcicludine. J Memb Biol 2000;174:157–65. [18] Torres AM, Wong HY, Desai M, Moochhala S, Kuchel PW, Kini RM. Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom. J Biol Chem 2003;278:40097–104. [19] Wang CH, Monette R, Lee SC, Morley P, Wu WG. Cobra cardiotoxin-induced cell death in fetal rat cardiomyocytes and cortical neurons: Different pathway but similar cell surface target. Toxicon 2005, in press. [20] Wijeyewickrema LC, Berndt MC, Andrews RK. Snake venom probes of platelet adhesion receptors and their ligands. Toxicon 2005;45:1051–61. [21] Yamazaki Y, Brown RL, Morita T. Purification and cloning of toxins from elapid venoms that target cyclic nucleotide-gated ion channels. Biochemistry 2002;41:11331–7. [22] Yamazaki Y, Hyodo F, Morita T. Wide distribution of cysteinerich secretory proteins in snake venoms: Isolation and cloning of novel snake venom cysteine-rich secretory proteins. Arch Biochem Biophys 2003;412:133–41. [23] Yee JSP, Nanling G, Afifiyan F, Donghui M, Lay PS, Armugam A, Jeyaseelan K. Snake postsynaptic neurotoxins: Gene structure, phylogeny and applications in research and therapy. Biochimie 2004;86:37–149.

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53 Sea Anemone Venom Peptides RAYMOND S. NORTON

characterized classes of sea anemone polypeptides are the 5-kDa toxins that act by binding to the voltagegated sodium channel [31] and the 16–20 kDa cytolysins, known as actinoporins [1]. More recently, new classes of potassium channel blockers have also been characterized. Here I summarize the salient features of these and other less abundant sea anemone peptides. In some cases it has been established that these molecules are localized to nematocysts and are genuine venom constituents, but in others this remains to be confirmed.

ABSTRACT The two most thoroughly characterized classes of peptides and polypeptides from sea anemones are the 5 kDa toxins that act by binding to voltage-gated sodium channels and the 20 kDa cytolysins, known as actinoporins. More recently, new classes of potassium channel blockers have also been characterized. This article summarizes the key features of these and other sea anemone peptides. In some cases it has been established that these peptides are localized to nematocysts and are genuine venom constituents, but in others this remains to be confirmed. A number of these peptides are of interest as new therapeutic leads.

ION CHANNEL BLOCKERS AND MODULATORS Sodium Channel Toxins

INTRODUCTION

The first representatives of the Na+-channel binding proteins were isolated in the early and mid-1970s by Beress at the University of Kiel [3] and by Norton (no relation to the author) at the University of Hawaii [31]. It is now recognized that this group of toxins consists of at least three classes of peptides, two made up of molecules containing around 45–50 amino acid residues and one of shorter peptides containing 27–32 residues. “Long” peptides from the genera Anthopleura and Anemonia (family Actiniidae) have been classified as Type 1 [25], and those from the Indopacific genera Heteractis (formerly Radianthus) and Stichodactyla (formerly Stoichactis), members of the family Stichodactylidae, as Type 2. These two classes of peptides are similar with respect to the locations of the six halfcystines (which form three disulfide bonds), as well as several other residues thought to play a role in biological activity or maintenance of the tertiary structure. However, while there is extensive sequence homology (≥60%) within each class, there is only about 30% homology between the two classes (Fig. 1, where

Sea anemones, in common with other members of the phylum Cnidaria (Coelenterata), possess numerous tentacles containing specialized stinging cells or cnidocytes. These stinging cells are equipped with organelles known as nematocysts that contain small threads which are forcefully everted when stimulated mechanically or chemically. Anemones use this venom apparatus in the capture of prey (crustaceans, small fish), as well as for defense against predators and in intraspecific aggression. Accordingly, they contain a variety of potent and interesting biologically active compounds, including many peptide toxins. Peptides and proteins characterized to date from sea anemones range in molecular mass from 3 to 300 kDa [3]. Even smaller peptides have been identified, such as the tetrapeptide Antho-RF amide and pentapeptides Antho-RW amide I and II from Anthopleura elegantissima, but they have potent biological activities as neurotransmitters or neuromodulators [16] and are unlikely to play a role in venom action. The two most thoroughly Handbook of Biologically Active Peptides

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364 / Chapter 53 amino acid sequences of most peptides described in this article are documented; see http://www.wehi.edu. au/resources/norton). These toxins bind to site 3 on voltage-gated sodium channels (VGSC), one of at least seven toxin binding sites identified on vertebrate and insect channels [7]. The pore-forming α-subunit of each VGSC consists of four homologous domains, each containing six putative transmembrane helices S1–S6, with the Na+ channel thought to be formed by the S5–S6 loops from all four domains (these S5–S6 linkers are further subdivided into the S5-P, P, and P-S6 loops, with the P loop containing the SS1 and SS2 segments). Fujiyoshi and coworkers [40] have determined a low-resolution structure for the eel VGSC by means of cryoelectron microscopy; the channel has a bell-shaped outer surface, with several inner cavities that are connected to four small holes and eight orifices close to the extracellular and cytoplasmic membrane surfaces. Site 3, which the anemone toxins share with scorpion α-toxins, involves extracellular loops of domains I and IV of the VGSC [37, 38], but the structure is not sufficiently well resolved to allow this (or other) toxin binding site to be localized. The effect of site 3 toxin binding is to delay channel inactivation such that the channel remains open for a longer period and the action potential is prolonged. They may achieve this by interfering with conformational changes in the S3–S4 loop upon translocation of the S4 segment, which is an important part of the channel’s voltage sensor [37]. The structure of a Type 1 toxin, anthopleurin-A (APA) [34], is shown in Fig. 1. It consists of a four-stranded, antiparallel β-sheet linked by three loops, the first of which, spanning residues 8–16, is the largest and least well defined in solution, although it contains several residues essential for activity. The Type 2 toxin ShI [43] has a very similar structure (Fig. 1). Calitoxin, from the anemone Calliactis parasitica, is a 46-residue toxin with three disulfide bonds, but a sequence showing several significant differences from those of Type 1 and Type 2 toxins [5] (Fig. S1). It nonetheless exerts similar presynaptic effects on crustacean nerve muscle preparations to these toxins, implying that it may represent yet a third type of long Na+-channel toxins. Gene sequencing subsequently identified an analogue with a Glu6 → Lys substitution [42]. Two shorter sea anemone toxins, ATX III (27 residues, three disulfide bonds) and PaTX (31 residues, four disulfides), also interact with the sodium channel, probably at site 3 [31]. There is clear sequence similarity between them, but very little with the longer toxins (Fig. S2). Recently, three crab toxins of 30–32 residues were isolated from the anemones Dofleinia armata and Entacmaea ramsayi, which were homologous to each other and also to PaTX, indicating that this is a distinct

family of toxins [22]. ATX III has a well-defined structure in solution [28], consisting of four reverse turns and two other chain reversals, but no regular α-helix or β-sheet (Fig. 1). APETx2 is unrelated in sequence to the other Na+channel toxins described here but is closely related to the K+-channel toxins APETx1 and BDS (see below). It inhibits the acid-sensing ion channel ASIC3, which is a proton-gated Na+ channel that has been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues [12]. Despite the lack of sequence similarity, its structure [8] is similar to those of AP-A and ShI.

Potassium Channel Toxins A unique family of potassium channel blockers has been identified in anemones. The first representative

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Equinatoxin II (EqTII) from Actinia equina

FIGURE 1. Structures of various classes of sea anemone toxins. All of these structures were determined for the peptide in solution using nuclear magnetic resonance data. Category, toxin name, RCSB accession code and references are as follows: Long Type 1 toxin, anthopleurin-A (AP-A), 1AHL [34]; Long Type 2 toxin, ShI, 1SHI [43]; Short Na+-channel toxin, ATX III, 1ANS [28]; K+-channel blocking toxin ShK, 1ROO [39]; actinoporin equinatoxin II (EqTII), 1KD6 [2] (note that a crystal structure has also been determined for EqTII, as described in RCSB entry 1IAZ). Structures are shown approximately to scale. Note the cluster of hydrophobic residues at the base of the ATX III structure.

Sea Anemone Venom Peptides / 365 to be isolated and characterized thoroughly was ShK, from Stichodactyla helianthus [6, 35]. A number of others have since been isolated, as illustrated in Fig. S3. The known sequences fall into two groups, with ShK and HmK lacking the basic four-residue insert found in the N-terminal half of AeK, AsKS, and BgK but having two single-residue inserts in the C-terminal half. The solution structure of ShK toxin [39] represents a novel fold consisting of two short α-helices encompassing residues 14–19 and 21–24 and an N-terminus with an extended conformation up to residue 8 followed by a pair of interlocking turns that resembles a 310-helix (Fig. 1). It contains no β-sheet. Its structure is thus quite distinct from the α/β fold found in scorpion K+ channel blockers, such as charybdotoxin, but is similar to that determined subsequently for BgK toxin [9]. Another family of peptides from anemones also acts on K+ channels (Fig. S4); the first members to be characterized were BDS-I and -II, which were isolated some time ago before their target was defined, but have been shown recently to block Kv3.4 [14]. The related toxin APETx1 specifically inhibits human ether-a-go-gorelated gene (HERG, Kv11.1) channels and shares 54% homology with BDS-I [13], although they show no sequence homology with other K+ channel toxins from sea anemones. Recently, Honma et al. [21] described three new crab toxins from Antheopsis maculata, one of which, Am II, also belongs to this family. The precursor proteins of these three toxins consist of a signal peptide, a pro region concluding with a pair of basic residues (Lys-Arg) and the mature peptide; the Am I precursor protein contains as many as six copies of Am I. The structures of the BDS and APETx toxins are similar to those of the Na+-channel toxins such as AP-A (Fig. 1) but quite different from the ShK-BgK family of K+-channel toxins. Anemones are clearly capable of using a common structural scaffold to create blockers of distinct targets (AP-A, APETx1, and APETx2 act on VGSC, HERG, and ASIC channels, respectively), while also using different scaffolds (all-β in APETx1 vs all-α in ShK) to block similar channels. The targets of these toxins are K+ channels, in particular the six-transmembrane channels, which include the voltage-gated K+ channels (Kv) and the smallconductance and intermediate-conductance Ca2+activated K+ channels (KCa). In these channels the region between the fifth and sixth transmembrane segments (the pore loop) forms the ion conduction pathway, and four subunits come together to form a functional channel tetramer. The Kv channels are voltage-activated, opening in response to membrane depolarization. The surface of ShK involved in binding to Kv channels has been probed using alanine scanning and selected toxin analogs [36]. Two residues, Lys22 and Tyr23, are crucial for activity, as also found

subsequently for BgK toxin [9]; other residues also contribute to the K+ channel-binding surfaces. ShK toxin blocks K+ channels by binding to a shallow vestibule at the outer entrance to the ion conduction pathway and occluding the entrance to the pore [24]. It appears that Lys22 and Tyr23 in the toxins represent a conserved dyad of residues that is essential for K+channel blockade by a range of structurally unrelated peptide toxins from scorpions, snakes, and cone snails [9, 33]. Gasparini et al. [15] proposed that this motif be more broadly defined as a lysine and a neighboring hydrophobic residue. ShK toxin blocks the voltage-gated channel Kv1.3 at low picomolar concentrations. As Kv1.3 is crucial for the activation of terminally differentiated effector memory T cells, it is regarded as a promising target for the treatment of T-cell–mediated autoimmune diseases such as multiple sclerosis and the prevention of chronic transplant rejection. ShK and its analogs are currently undergoing evaluation as leads in the development of new biopharmaceuticals for the treatment of multiple sclerosis and other T-cell–mediated autoimmune disorders [33].

CYTOLYSINS The actinoporins are a family of sea anemone toxins that function by forming pores in cell membranes [1]. These highly basic proteins, of mass 16–20 kDa (Fig. S5), display permeabilizing activity in model lipid and cell membranes that is markedly enhanced by the presence of sphingomyelin (SM). The actinoporins differ from bacterial pore-forming toxins in several respects: They are more potent, the pore they form does not have a stable structure and has not yet been visualized directly, and they are of smaller size and very stable toward proteolysis. In common with many pore-forming toxins, the actinoporins are highly water soluble, stable proteins, and yet their only known activity is the formation of oligomeric pores in membranes, consisting of three or, more likely, four monomers. The resulting pores have a radius of about 1 nm and are permeable to small molecules and solutes, with the resulting osmotic imbalance promoting cell lysis [1]. SM plays a key role in the lytic activity of the actinoporins. The hemolytic activity of a cytolysin from Stichodactyla helianthus was inhibited by preincubation with SM, and treatment of erythrocyte membranes with sphingomyelinase rendered them resistant to lysis. The hemolytic activity of equinatoxin II (EqTII) was also inhibited by preincubation with SM. More recent studies with sticholysins I and II on model membranes confirmed that SM enhances lytic activity and suggested that cholesterol may have a minor role [11]. The interaction of EqTII with large unilamellar vesicles contain-

366 / Chapter 53 ing phosphatidylcholine (PC) was reversible and did not involve major conformational changes [4], but the presence of SM enabled irreversible insertion and pore formation, which were associated with major conformational changes. Some EqTII-induced leakage was observed from large unilamellar vesicles containing only PC and cholesterol, again suggesting a minor contribution of this sterol lipid to actinoporin cytolytic activity. Fluorescence studies of EqTII binding to lipid vesicles showed that association was markedly enhanced by the presence of SM [1]. Three-dimensional structures have been reported for EqTII [19] and sticholysin [27]. EqTII consists of two short helices packed against opposite faces of a βsandwich structure formed by two five-stranded β-sheets (Fig. 1). A number of studies have been carried out in an effort to elucidate the molecular mechanism of actinoporin pore formation. By site-directed mutagenesis, it was shown that at least two regions of EqTII became embedded in lipid membranes, the N-terminal region (residues 10–28) and the surface aromatic cluster including tryptophans 112 and 116 [1]. The current model of pore formation proposes that EqTII binds to the membrane initially via the aromatic rich region, then the N-terminal region is transferred to the lipid membrane, and, finally, across the bilayer to form a final functional pore [2, 20]. The structural details of the final oligomeric assembly are not known. The Okinawan sea anemone Phyllodiscus semoni, which causes severe stinging, contains PsTX-60A and PsTX-60B as the major toxins from the isolated nematocysts [29]. These 60-kDa proteins are toxic to shrimp and hemolytic on sheep red blood cells, but they show no sequence similarity to previously reported proteins and thus represent a novel class of cytolysins.

OTHER BIOACTIVE PEPTIDES Honma et al. [23] have described an epidermal growth factor (EGF)-like toxin (gigantoxin I) from Stichodactyla gigantea. Its precursor protein is composed of a signal peptide, a pro region, and the mature peptide, similar to those of gigantoxins II and III. These proteins are found in nematocysts, implying that they do indeed function as toxins. Various protease inhibitors have been isolated from anemones, including several Kunitz inhibitors [3, 10] and, more recently, cysteine proteinase inhibitors such as equistatin [26]. Sea anemones also contain phospholipases A2, which are definitely present in nematocysts and likely to play a role in defense and aggression [17, 30]. Several other classes of peptides and proteins have been identified in sea anemones, although a role in the

venom of these species has not always been established [41]. In contrast to cone shells, which possess potent toxins against a range of voltage- and ligand-gated ion channels [32], sea anemones have not yet yielded many well-characterized blockers of other channels, although recent results suggest that their repertoire is by no means exhausted. With increasing interest in the prospects of toxic peptides from venoms as drug leads, we can look forward to the isolation and characterization of new classes of peptides from sea anemones and related cnidarians.

Acknowledgments I am very grateful to Ronelle Welton for assistance with sequence analyses and Jennifer Sabo for help with the figure. Supplementary material referred to in the text is available at http://www.wehi.edu/resources/ norton. I also wish to pay tribute to my colleagues Laszlo Béress and Ted Norton, whose enthusiasm for sea anemone toxins helped stimulate my enduring interest in this field.

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[26] Lenarcic B, Ritonja A, Strukelj B, Turk B, Turk V. Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain. J Biol Chem 1997;272:13899–903. [27] Mancheño JM, Martin-Benito J, Martinez-Ripoll M, Gavilanes JG, Hermoso JA. Crystal and electron microscopy structures of sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation. Structure (Camb.) 2003;11:1319–28. [28] Manoleras N, Norton RS. Three-dimensional structure in solution of neurotoxin III from the sea anemone Anemonia sulcata. Biochemistry 1994;33:11051–61. [29] Nagai H, Oshiro N, Takuwa-Kuroda K, Iwanaga S, Nozaki M, Nakajima T. Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski. Biochem Biophys Res Commun 2002;294:760–3. [30] Nevalainen TJ, Peuravuori HJ, Quinn RJ, Llewellyn LE, Benzie JA, Fenner PJ, Winkel KD. Phospholipase A2 in cnidaria. Comp Biochem Physiol B Biochem Mol Biol 2004; 139:731–5. [31] Norton RS. Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel. Toxicon 1991;29:1051–84. [32] Norton RS, Olivera BM. Conotoxins down under. Toxicon 2006; in press. [33] Norton RS, Pennington MW, Wulff H. Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases. Curr Med Chem 2004;11:3041–52. [34] Pallaghy PK, Scanlon MJ, Monks SA, Norton RS. Threedimensional structure in solution of the polypeptide cardiac stimulant anthopleurin-A. Biochemistry 1995;34:3782–94. [35] Pennington MW, Byrnes ME, Zaydenberg I, Khaytin I, de Chastonay J, Krafte DS, et al. Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone. Int J Pept Protein Res 1995;46:354–8. [36] Pennington MW, Mahnir VM, Khaytin I, Zaydenberg I, Byrnes ME, Kem WR. An essential binding surface for ShK toxin interaction with rat brain potassium channels. Biochemistry 1996;35:16407–11. [37] Rogers JC, Qu Y, Tanada TN, Scheuer T, Catterall WA. Molecular determinants of high affinity binding of α-scorpion toxin and sea anemone toxin in the S3–S4 extracellular loop in domain IV of the Na+ channel α subunit. J Biol Chem 1996; 271:15950–62. [38] Thomsen WJ, Catterall WA. Localization of the receptor site for α-scorpion toxins by antibody mapping: implications for sodium channel topology. Proc Natl Acad Sci U S A 1989;86:10161–5. [39] Tudor JE, Pallaghy PK, Pennington MW, Norton RS. Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone. Nat Struct Biol 1996;3:317–20. [40] Sato C, Ueno Y, Asai K, Takahashi K, Sato M, Engel A, Fujiyoshi Y. The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature 2001;409:1047–51. [41] Sher D, Knebel A, Bsor T, Nesher N, Tal T, Morgenstern D, et al. Toxic polypeptides of the hydra-a bioinformatic approach to cnidarian allomones. Toxicon 2005;45:865–79. [42] Spagnuolo A, Zanetti L, Cariello L, Piccoli R. Isolation and characterization of two genes encoding calitoxins, neurotoxic peptides from Calliactis parasitica (Cnidaria). Gene 1994;138: 187–91. [43] Wilcox GR, Fogh RH, Norton RS. Refinement of the solution structure of the sea anemone neurotoxin ShI. J Biol Chem 1993;268:24707–19.

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for the identification and characterization of ion channel subtypes and the process of neurotransmitter release, respectively [1, 20]. Yet, others, such as GsAFII, have already been patented as antiarrhythmic and analgesics [9, 10]. The focus of this review is the discovery, processing, structure, and function of spider venom peptides. In particular, it will detail the site and mechanism of their action, detail the molecular determinants for their pharmacology, and discuss the application of these peptides in the fields of pharmacology and neuroscience and the development of novel insecticides.

ABSTRACT Spider peptide and protein toxins are recognized as highly potent and specific molecular tools that modulate neurotransmission via interaction with a variety of ion channels, receptors, and transporters in vertebrates and invertebrates. The recent discovery of the diversity of these peptides means that spider venoms are only now being considered as combinatorial peptide libraries useful for probing synaptic neurotransmission, for validating novel insecticide targets, and for aiding in the design of therapeutics and biopesticides. These studies are being greatly assisted by the determination of the pharmacophore of these toxins. This review details the discovery, structure, and function of these spider venom peptide toxins.

METHODOLOGICAL APPROACHES IN THE ISOLATION OF NOVEL SPIDER VENOM PEPTIDES Several different methodological approaches have led to the isolation of novel spider peptide toxins (for a more extensive overview see [5]). The most commonly used approach has been the use of insect and mammalian bioassays to drive venom fractionation using acute toxicity testing via injection assays or isolated organ bath screening. The weakness with this approach is that numerous toxins remain uncharacterized in terms of target and mode of action (e.g., lasiotoxins 1 and 2 [LpTx1 and 2], Eurypelma toxins [ESTx1 and 2], and covalitoxin II; see [5]). Also the potential array of targets for a novel toxin is too vast to be comprehensively screened. A more recent approach has been to study all components of a single venom, or venoms of closely related species, and characterize their mode of action in target-oriented bioassays. This has been the approach taken with the atracotoxins (ACTXs) and huwentoxins (HWTXs) where several families of toxins that modulate voltage-gated Na+ (Nav), Ca2+ (Cav), and Ca2+-activated K+ channels have been described. The limitation of this approach is that it does

INTRODUCTION Spiders have, during their evolution, developed a complex preoptimized combinatorial peptide library of enzymes, neurotoxins, and antimicrobial and cytolytic peptides in their venom glands. The role of this venom is to paralyze and/or kill prey or predators as rapidly as possible. Therefore, their venoms are particularly rich in neurotoxins that rapidly modify ion conductance (ion channel toxins) and to a lesser extent affect neurotransmitter exocytosis (presynaptic toxins) and interfere with the binding of neurotransmitters (postsynaptic toxins). Many of these peptides are selectively insecticidal or modulate the activity of various targets in vertebrates, including humans. In particular, insect-selective atracotoxins are now being investigated for their possible use as bioinsecticidal agents for the control of phytophagous pests or insect vectors of new or reemerging disease [19]. Other toxins, such as ω-agatoxin IVA and α-latrotoxin, have been invaluable molecular tools Handbook of Biologically Active Peptides

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370 / Chapter 54 not always yield the most active toxin for a specific target and again many toxins remain uncharacterized (e.g., MIT-like ACTX-Hvf17 [22] and HWTX-II, III, V–IX [11]). Currently a number of groups are using a systematic approach employing screening assays using cloned ion channels expressed in Xenopus oocytes or HEK293 cells. Such methods have provided highaffinity and often highly selective ligands that block or modulate ion channel subtypes for which there were no previous pharmacological tools (e.g., PcTx1, SNX482; see following). Unfortunately, this approach is limited if a potential therapeutic or insecticidal target has not been previously validated or if the target has not yet been cloned. This limits discovery of ligands to only those with affinity for known target subtypes. Accordingly, methodologies centered around a combination of the last two approaches are likely to be the most successful.

STRUCTURE OF THE PRECURSOR SPIDER VENOM PEPTIDE AND POSTTRANSLATIONAL PROCESSING The expression of spider venom peptides, like those from marine cone snails and sea anemones, are translated mostly as pre-propeptides that undergo posttranslational modification to yield the mature toxin [18]. These pre-propeptide precursors typically are composed of an N-terminal signal peptide that precedes a propeptide region rich in acidic residues and of highly variable length, which is followed by the mature toxin sequence (Fig. 1A and B). It appears that during evolution families of toxins within spider venoms underwent hypermutation in the mature peptide region while conserving the basic disulfide framework. This cystine framework appears to be associated with a specific signal sequence. However, the signal peptide was conserved, since its role was, most likely, to direct the precursor to a specific secretory pathway, thereby to ensure correct peptide folding. The specific role(s) of the propeptide region, however, is still not understood but most likely involves proteolytic processing and posttranslational modifications such as N-terminal pyroglutamine formation and C-terminal trimming and amidation. In the case of the MIT-like ACTXs [22] and latrotoxins [20], this N-terminal propeptide is absent (Fig. 1A and C).

STRUCTURAL ORGANIZATION OF SPIDER VENOM PEPTIDES: VARIATIONS ON AN ANCESTRAL FOLD A large number of spider venom peptides are compact globular proteins possessing several disulfide

bridges and modified N- and/or C-termini that increase their in vivo stability. These peptides, predominantly targeting voltage-gated ion channels, often contain a “disulfide pseudo-knot,” which places them in a class of toxins and inhibitory polypeptides with an “inhibitory cystine-knot” (ICK) motif [16]. This structural motif is normally exemplified by a triple-stranded, antiparallel β-sheet stabilized by disulfide bridges. Since not all ICK peptides exhibit the N-terminal β-sheet (β1 in Fig. 2C), a modified definition composed of “an antiparallel βhairpin stabilized by a cystine-knot” without a mandatory third β-sheet has been proposed [19]. The consensus sequence for the ICK motif is currently –CIX3–7–CIIX3–8–CIIIX0–7–CIVX1–6–CVX3–13–CVI– where X is any amino acid. The three disulfide bridges and intervening backbone form a pseudo-knot consisting of a ring (CI—CIV, CII—CV) penetrated by a third disulfide bridge (CIII—CVI) (see Fig. 2C). However within this fold-class, the biological activities of spider ICK toxins are quite diverse with activity at Nav, Cav, voltage-gated potassium, protongated, and mechanosensitive channels (see following) and hemagglutination on red blood cells (ShL-I; [11]). This highlights the observation that different biological functions are often grafted onto the same, or similar, structural scaffolds. Recently another structural fold has been defined for spider toxins. The disulfide-directed β-hairpin (DDH) fold lacks the disulfide knot and is composed of a double-stranded antiparallel β-hairpin stabilized by two mandatory disulfide bridges with a current consensus sequence of –CX4–19–CX2[G or P]X2–CX4–19–C– (see Fig. 2A, B). The ICK motif appears to have evolved from this simpler canonical ancestral fold [21]. This DDH fold has been observed in the MIT-like ACTX [22], HWTX-II, ESTx1 and -2, and LpTx1 and -2 [5]. Specific differences in the DDH and ICK structural folds, determined by the spacing between cysteine residues and their connectivity, are critical for the presentation of key functional residues to the target, thus generating a vast array of peptides acting on distinct biological systems. This together with their protease resistance and compact nature provides an effective scaffold for the design of molecular tools and therapeutics [16].

SPIDER PEPTIDE TOXINS MODULATING Nav CHANNEL FUNCTION: BLOCKERS AND GATING MODIFIERS Nav channels are responsible for the initiation and propagation of the action potential in excitable cells. To date, nine mammalian Nav channels (Nav1.1–1.9) have been cloned, functionally expressed, and characterized [7]. Interestingly, much of their structure and

FIGURE 1. Structural models of the precursor architecture for spider toxins including (A) atracotoxin families, (B) other peptide and protein toxins, and (C) latrotoxins. All toxins display a pre-propeptide paradigm except the MIT-like atracotoxins and latrotoxins. In panel (A), values in parentheses are the lowest interspecies identity observed for each region when multiple sequences (∼20) are compared from Atrax and Hadronyche spp. (B) ω-Aga 1A is a heterodimer consisting of a 66-residue major chain that is coupled via a disulfide bond to a 3-residue minor chain. (C) Domains I and IV represent the signal peptide and the C-terminal sequence, respectively. Domain III is composed of ankyrin-like repeats (R1, R2, etc.).

372 / Chapter 54

FIGURE 2. Evolution of DDH-related folds (A, B) into the ICK structural motif (C). Left-hand columns in each panel (Aa, Ba, Ca) show schematic representations of the structural motifs depicting the formation of the cystine-knot and possible addition of the third β-sheet. β-Sheets are shown as gray arrows and disulfide bridges connecting cysteine residues are shown as dark gray lines with roman numerals. The dark arrow (β1) in panel Cc represents the additional β-sheet not always present in ICK spider toxins. Right-hand columns in each panel (Ab, Bb, Cb) show a schematic view of the 3D structures of typical representatives of the two structural motifs. (Aa) MIT-like ACTX-Hvf17 from Hadronyche versuta modelled on the structure of MIT1 from the black mamba snake Dendroaspis p. polylepis [22], (Ba) HWTX-II (PDB 1I25), and (Cb) SGTx1 (PDB 1LA4). (Cc) Stereo view of the pseudo-knot of SGTx1.

function has been elucidated by the use of various plant and animal toxins. These molecular probes bind with seven identified targets, referred to as neurotoxin receptor sites 1–7 [3]. Four sites that bind peptide toxins exist and, according to their functional characteristics on Nav channels, these toxins can be classified as inducing either a depressant or excitatory phenotype. Site 1, located on the extracellular surface of the pore, binds the marine cone snail peptide μ-conotoxins and the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin [3]. These toxins physically occlude the conduction pathway resulting in a depressant phenotype. Site 3 toxins, including the classical scorpion α-toxins, slow channel inactivation while site 4 toxins include the scorpion β-toxins that facilitate channel activation [3]. Site 3 and 4 toxins are thus classified as “gatingmodifiers” with an excitatory phenotype. Finally, site 6

binds the δ-conotoxins, which slow channel inactivation but show different allosteric modulation to site 3 toxins [3]. Spider venoms are becoming an interesting source of peptide toxins that modulate Nav channel conductance and/or gating. For a full overview of the structure and function of spider toxins targeting sodium channels, readers are directed to [15], a summary of which is presented following. Several spider toxins have been found to block Na+ conductance such as HWTX-IV from the venom of Ornithoctonus huwena and the structurally related hainantoxins (HNTX) III–V from O. hainana. This action most likely occurs via an interaction with site 1 of the Nav channel. Interestingly, an ortholog, HNTX-I, also has a similar action but displays a 15-fold increase in selectivity for insect versus mammalian Nav channels. Therefore, these peptides represent the first family of spider

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FIGURE 3. Structural comparison of spider toxins. (A–E) Schematic view of NMR solution structures showing the location of β-strands (black arrows) and α-helices (light and dark gray helices). Disulfide bridges are shown as light gray tubes. Surface representations are shown to the right of each panel indicating the suspected pharmacophore. Residues are coded: black, positively charged; light gray, negatively charged or polar; dark gray, aliphatic or aromatic. (A) HNTX-IV (PDB 1NIY), (B) δ-ACTX-Hv1a (PDB 1VTX), (C) δ-PaluIT2 (PDB 1V91), (D) κ-ACTXHv1c (PDB 1DLO), (E) ω-ACTX-Hv1a (PDB 1AXH). (F) X-ray crystal structure SMase I from Loxosceles laeta (PDB 1XX1) showing the overall structure (left panel) and key residues involved in the Mg2+ ion (gray sphere) coordination and the catalytic site (right panel).

toxins to selectively block Na+ conductance and, in the case of HNTX-I, the first insect-selective toxin to block the Nav channel. The NMR structures of several of these toxins have also been determined and, by synthesis of various alanine mutants, it has been determined that the pharmacophore responsible for the affinity of HNTX-IV for the Nav channel (Fig. 3A) appear to be conserved in HWTX-IV. Interestingly His28 is substituted by the negatively charged Asp26 in HNTX-I providing a possible molecular basis for the selectivity of HNTX-I for the insect Nav channel. Many spider toxins acting on Nav channels bind at site 3 [3]. Spider δ-toxins from funnel-web (Hadronyche and Atrax spp.) and eastern mouse spiders (Missulena bradleyi) are lethal toxins, responsible for the major symptoms of human envenomation. This family of 42residue peptides, the δ-atracotoxins (δ-ACTXs) and δMSTX-Mb1a, have been shown to target the Nav channel (for a complete overview see [14]). Electrophysiologi-

cal studies have identified that δ-ACTXs alter neuronal excitability in both insect and mammalian neurons by causing a prolongation of action potential duration resulting in the appearance of plateau potentials, accompanied by spontaneous repetitive firing. These actions are due to a slowing of TTX-sensitive Nav channel inactivation and a modest hyperpolarizing shift in the voltage dependence of activation. Following radiolabeled binding experiments this was found to result from an interaction with site 3 on both mammalian and insect Nav channels in a comparable manner to scorpion α-toxins. Similar actions to slow Nav channel inactivation have been noted with PnTx2-6 from the venom of the South American “armed” spider Phoneutria nigriventer [6]. A number of residues in spider δ-toxins are believed to provide a complementary surface to the residues identified in the IVS3-S4 loop or key residues forming the likely pharmacophores of scorpion αtoxins. Additional comparisons with the homolog

374 / Chapter 54 δ-ACTX-Hv1b, which lacks insecticidal activity, reveals that a number of charged amino acids at the Nterminus are likely to be important for determining phyla specificity (Fig. 3B). Two other spider toxins represent a growing family of excitatory insect-selective spider neurotoxins targeting site 3 on the Nav channel. Magi 2, a neurotoxin from Macrothele gigas, has been shown to displace the insect site 3 ligand 125I-LqhαIT from cockroach synaptosomes, while another novel toxin, Tx4(6-1) from P. nigriventer, selectively prolongs insect action potential duration via a slowing of Nav channel inactivation and competes with the scorpion α-like toxin Bom IV for site 3 on insect Nav channels [6]. Other toxins are able to discriminate between different Nav channel isoforms. Jingzhaotoxin I (JZTX-I) from the venom of the Chinese tarantula Chilobrachys jingzhao has been shown to slow inactivation of TTXresistant Nav1.5 channels of rat cardiac myocytes, most likely via an interaction with site 3. Interestingly, the toxin, like the related peptide JZSTX-III, failed to modify TTX-resistant Nav channels in rat sensory neurons thus discriminating cardiac from neuronal isoforms. Other excitatory toxins interact with Nav channel site 4 by acting as gating modifiers of activation. δ-Palutoxins (δ-PaluITs) from the spider Paracoelotes luctuosus are a family of four insect-selective peptides that act by slowing insect Nav channel inactivation, similar to site 3 neurotoxins such as LqhαIT. Despite this they have been shown to displace the site 4 excitatory scorpion β-toxin, Bj-xtrIT, from cockroach neuronal membranes and fail to displace LqhαIT binding. Thus, δ-PaluITs represent the first spider toxins that definitively bind to site 4 on insect sodium channels but modulate Nav channel inactivation. The NMR solution structures of δ-PaluIT1 and -IT2 have been recently determined and shown to contain double- and triple-stranded antiparallel β-sheet ICK folds, respectively. Alanine scanning mutagenesis experiments reveal that the putative insectophore of δ-PaluIT2 (Fig. 3C) shares similarity with the bipartite bioactive surface of Bj-xtrIT despite different protein scaffolds. These toxins therefore reveal that modulation of Nav channel inactivation can be achieved by binding to a site thought to be associated with effects on channel activation. In support the μagatoxins from the venom of the American funnel-web spider Agelenopsis aperta and the structurally related curtatoxins (I–III) from the related agelenid spider Hololena curta also appear to share this activity [1]. μAgatoxins are insect-selective neurotoxins that cause irreversible paralysis and repetitive firing in presynaptic terminals of houseflies, and evoke massive release of neurotransmitters by increasing Na+ influx through TTX-sensitive Nav channels, shifting the activation curve

to more negative potentials [1]. This action is analogous to that reported for scorpion β-toxins and therefore it is likely that this family targets site 4. However, μ-agatoxins also slow Nav channel inactivation in insect motoneurons [1], an action shared by δ-PaluIT toxins with whom they share considerable sequence homology. This provides further support for the hypothesis that insect site 4 is a macrosite, which may be allosterically linked to channel inactivation. Magi 5, from the venom of the spider M. gigas, is also believed to interact with site 4 of the Nav channel. It has been shown to be lethal to mice and produce a transient paralysis in insects. Competitive binding assays have revealed that Magi 5 competes with a scorpion βtoxin active on mammals, 125I-CssIV, for binding to site 4 on the Nav channel. Interestingly, Magi 5 shows an intermediate potency to displace LqhαIT binding to insect site 3 on cockroach synaptosomes. The precise mechanism of action therefore awaits analysis by electrophysiological methods. Finally, there are also toxins that interact with unidentified sites on Nav channels. These include ACTX-Hi:OB4219 from the funnel-web spider Hadronyche infensa Orchid Beach and DTX9.2 from the weaving spider Diguentia canities (see [15]).

SPIDER PEPTIDE TOXINS MODULATING VOLTAGE-GATED POTASSIUM CHANNELS Voltage-gated K+ channels are the most abundant ion channels and play important roles in excitable cells such as controlling neuronal excitability, resting membrane potential, and neurotransmitter release. While there are many voltage-gated K+ channel families, two are of particular importance to this discussion. First, the outward-rectifying K+ (Kv) channels classified into four groups Kv1-Kv4, and second, Ca2+-activated K+ (KCa) channels [7]. Spider toxins have been instrumental in the structural and pharmacological characterization of the Kv2 and Kv4 channels particularly those isolated from “tarantula” spider venoms (for a complete overview see [5]). Heteropodatoxins (HpTX1–HpTX3) from the huntsman spider Heteropoda venatoria are a family of toxins that possess a significant degree of homology with the hanatoxins. Heteropodatoxins prolong the duration of the cardiac action potential via a voltage-dependent block of the Shal-related Kv4.2 but not Shaker-related Kv1.4 channels. Moreover, the toxins slowed the time course of channel activation and inactivation and caused a depolarizing shift in the voltagedependence of inactivation. Thus, the heteropodatoxins represent new pharmacological tools with which to study transient outward Kv currents in cardiac tissue. Hanatoxins (HaTx1 and 2) from the Chilean tarantula

Spider Venom Peptides / 375 Grammostola spatulata potently block Kv4.2 as well as the Shab-related Kv2.1 (drk1) channel. Recent studies using alanine-scanning mutagenesis indicate that HaTx interact with the S3–S4 linker of the Kv channel. Binding and electrophysiological studies demonstrate that the channel can still open with HaTx bound and support the hypothesis that HaTx does not physically occlude ion channel conductance but rather modifies channel gating by shifting the voltage dependency of channel activation to more depolarized potentials. Other Kv channel blockers targeting Kv4 channels include phrixotoxins 1 and 2 (PaTx1 and -2) from the venom of Phrixotrichus auratus that potently block Kv4.3 and Kv4.2 channels by alteration of channel gating. Also, stromatoxin (ScTx1), isolated from the venom of the African tarantula Stromatopelma calceata, blocks Kv4.2, but in addition inhibits Kv2.2, and to a lesser extent, Kv2.1 and Kv2.1/Kv9.3 subtypes. Heteroscodratoxins 1 and 2 (HmTx1 and -2), from the tarantula spider Heteroscodra maculata venom, bind to the voltage sensor and inhibit Kv channels. HmTx2 seems to be specific for Kv2 channels, whereas HmTx1 also inhibits the Kv4 subtype with the same potency. HmTx1 is the first peptide to be effective on Kv4.1 (see [5]). Finally, the P. nigriventer peptide toxin 3-1 (PnTx3-1), selectively inhibits A-type Kv channels in GH3 cells without blocking delayedrectifier Kv currents [6]. Of great interest has been the recent discovery of a family of insect-selective neurotoxins from the Australian funnel-web spider H. versuta. κ-ACTXs, formerly Janus-faced ACTXs, target KCa channels in cockroach dorsal unpaired median neurons. The most remarkable feature of their 3D structure is a vicinal disulfide bridge between the sidechains of Cys13 and Cys14. Vicinal disulfide bridges are exceptionally rare in proteins. Using alanine-scanning mutagenesis, the vicinal disulfide, as well as Arg8, Pro9, and Tyr31, were determined to form the insectophore (Fig. 3D). Intriguingly, the functionally critical Arg8 and Tyr31 residues in κ-ACTX-Hv1c align extremely well with the Lys–Phe/Tyr dyad that is conserved across structurally dissimilar KCa channel blockers such as BgK, agitoxin2, and charybdotoxin [19].

SPIDER PEPTIDE TOXINS MODULATING ON Cav CHANNELS A variety of Cav channels are classified into lowvoltage-activated (LVA) channels that inactivate rapidly, and high-voltage-activated (HVA) channels that show no inactivation. HVA Cav channels are further subdivided according to their pharmacological and biophysical characteristics into L-type (Cav1.x), N-, P-, Q-, and

R-type (Cav2.x). LVA Cav channels are only of the T-type (Cav3.x) [7]. Undoubtedly the best characterized Cav channel blockers are the ω-agatoxins from A. aperta spider venom that inhibit release of a variety of neurotransmitters (for a complete overview see [1]). ωAgatoxins vary from 5–10 kDa in length and are classified according to their sequence homology and spectrum of activity against insect and vertebrate Cav channels. Type I ω-agatoxins (ω-Aga IA, IB and IC) and Type II ωagatoxins (ω-Aga IIA and IIB) are selective blockers of insect Cav channels. Type III ω-agatoxins such as ω-Aga IIIA are inactive in insects but block the binding of the N-type Cav channel ligand [125I] ω-conotoxin GVIA to vertebrate membranes, as do toxins from group I. ωAga IIIA binds in a voltage-dependent manner to all HVA but not LVA, Cav channels in mammalian central neurons but only produces a partial block of current. This appears to arise from a reduction in the unitary conductance of single channels. Type IV ω-agatoxins (ω-Aga IVA) are not toxic at the fly neuromuscular junction but do block Cav channels in insect central neurons and block P-type Cav channels in mammals. This results from a depolarizing shift in the voltage-dependence of activation and high affinity of the toxin for the resting, but not the open, state of the channel. P. nigriventer venom also contains several toxins targeting a variety of Cav channel subtypes (for an overview of P. nigriventer toxins see [6]). PnTx3-3 inhibits neurotransmitter release via a block of P/Q- and R-type channels. ω-phonetoxin IIA (PnTx3-4), which shows high-sequence homology with the ω-agatoxin III family, not unexpectedly blocks N-type Cav channels. In addition, it also blocks L-type Cav channels, as do Tx3-2 and Tx3-5. PnTx3-6 also abolishes Ca2+-dependent glutamate release by inactivating intrasynaptosomal P/Qtype Cav channels. Other toxins have also been isolated and characterized from mygalomorph (ω-grammotoxin SIA from G. spatulata and SNX482 from Hysterocrates gigas) and araneomorph (SNX325 from Segestria florentina; DW13.3 from Filistata hibernalis and PLTX-II from Plectreurys tristis) spiders (for a complete review see [17]). Recently a number of insect-selective Cav channel blockers have been discovered in the venom of funnelweb spiders (for a extensive overview see [19]). These peptides are lethal neurotoxins with ω-ACTX-Hv1a one of the most potent insecticidal peptide toxins discovered to date. ω-ACTX-Hv1a binds to orthopteran Cav channels at low nanomolar concentrations, whereas it has no effect on cloned human Cav2.1, Cav2.2, or Cav1.2 channels. Alanine-scanning mutagenesis revealed that the insectophore of the ω-ACTX-1 family is most likely restricted to a small number of conserved residues (Fig. 3E) that forms a single contiguous patch on the

376 / Chapter 54 molecular surface of the toxin. Topological restriction of the insectophore to a single face of the toxin should increase the probability of designing functional small-molecule analogs. More recently, ω-ACTX-Hv2a, a 45-residue insect-selective toxin has been found to inhibit insect Cav channels but with a vastly different 3D structure.

Nav channels by causing a depolarizing shift in the voltage-dependence of activation. However, these toxins also potently block Cav and Kv channels. This promiscuous activity of certain spider toxins on multiple ion channels may arise due to common structural elements shared between ion channels, particularly Nav and Cav. These toxins may recognize a common domain or motif present in voltage-gated ion channels that has a highly conserved tertiary structure.

SPIDER PEPTIDE TOXINS ACTING ON ACID-SENSING ION CHANNELS An exciting development has been the discovery of a spider toxin targeting novel acid-sensitive ion channels (ASIC). These proton-gated sodium channels are associated with nociception, taste transduction, and perception of extracellular pH changes in the brain and sensory ganglia, and may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), isolated from the venom of the tarantula Psalmopoeus cambridgei, has been found to selectively inhibit homomultimeric ASIC1a channel currents. As it lacks significant toxicity, its role in prey capture and the importance of ASIC1a channels as targets of venom components remain unclear. The mechanism of ASIC1a inhibition by PcTx1 is not yet understood, however, given that gating of ASIC channels by protons is likely to be different from that of the voltagegated channel family, this may be via a novel mechanism. The 3D NMR structure of PcTx1 has recently been determined and should provide insights into the pharmacophore of this novel toxin [4]. So far, this is the only selective ligand with which to study the functional role and pathophysiology of ASIC channels.

SPIDER PEPTIDE TOXINS WITH NONSELECTIVE ACTIONS ON VOLTAGE-GATED ION CHANNELS: PROMISCUOUS TOXINS It has been previously noted that peptide toxins can exert their actions both within, and across, voltagegated ion channel families (for an overview see [15]). For example, SNX482 from the venom of the Cameroon red baboon tarantula Hysterocrates gigas is a known blocker of R- and P/Q-type Cav channels. This toxin has also been shown to delay Nav channel inactivation and partially block INa in bovine chromaffin cells at similar concentrations to those that block ICa. This dual activity on Nav and Cav channels has also been reported for the P/Q-type blocker ω-agatoxin IVA. In addition, protoxin I and II from the venom of the green velvet tarantula Thrixopelma pruriens act as gating modifiers to inhibit

SPIDER PEPTIDE TOXINS ACTING ON MECHANOSENSITIVE ION CHANNELS Mechanosensitive ion channels (MSCs) potentially transduce many external (sound, vibration, and touch) and internal stimuli (local blood flow control, cell volume regulation, and dilation-induced heart rate changes). Several spider toxins, which block cationselective MSCs with no action on voltage-gated ion channels, have been isolated from G. spatulata venom. GsAFI and II have been patented as antiarrhythmic and analgesic peptides [9, 10]. GsMTx4 also blocks MSC in ventricular myocytes, suggesting it could also be a new lead compound for the development of antiarrhythmic drugs [2].

SPIDER TOXINS ACTING ON GLUTAMATE RECEPTORS OR TRANSPORTERS Arguably the most important excitatory neurotransmitter in the mammalian CNS and insect peripheral nervous system is L-glutamate. Excessive activation of glutamate receptors is thought to be involved in brain damage following a stroke, ischemic injury, or epilepsy and also in neurodegenerative chronic diseases such as Huntington’s, Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. Glutamate receptor antagonists are therefore being investigated for the treatment of these diseases. While there have been a wide range of glutamate receptor antagonists found in spider venoms, these are mostly acylpolyamines. The only peptide toxin from spider venom known to interact with glutamate receptors is PnTx4(5-5) from P. nigriventer, an insecticidal toxin that also reversibly inhibits ionic currents mediated through NMDA receptors in rat hippocampal neurons (for a review see [6]). However, PnTx 4(3-7) from the same venom also inhibits glutamate uptake in rat cortical synaptosomes. PnTx3-4 has been reported to decrease glutamate release from synaptosomes via a block of glutamate transporters in synaptosomes. These toxins therefore provide P. nigriventer spiders with the means to disrupt specific aspects of insect glutamate neurotransmission.

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SPIDER TOXINS ACTING ON NEUROTRANSMITTER RELEASE (PRESYNAPTIC TOXINS) Arguably the most clinically significant group of spiders is the widow spiders belonging to the genus Latrodectus. For an overview of the clinical pathophysiology see [13]. To date, isolation of the lethal neurotoxin has only been reported from the European widow spider L. tredecimguttatus. At present, seven different latrotoxins have been isolated from the venom of L. tredecimguttatus. Firstly, α-latrotoxin (α-LTx) is a 120kDa protein that is responsible for the effects seen on vertebrate synapses. Two additional types of latrotoxins with molecular weights in excess of 100 kDa have also been purified and cloned from L. tredecimguttatus venom. These act on invertebrates including five insect-selective latroinsectotoxins (α-, β-, γ-, δ-, and εlatroinsectotoxins; LITs) and a crustacean-selective αlatrocrustotoxin (α-LCT). For a recent and extensive overview of latrotoxin structure and function, see [20]. Latrotoxins are synthesized as larger inactive precursor proteins that undergo posttranslational modification. Unlike all other spider toxins, the N-termini of the mature latrotoxin proteins (Domain II and III; Fig. 1C) are not preceded by a classical signal peptide, but instead by a short hydrophilic sequence ending with a series of basic residues (Domain I; Fig. 1C). This sequence resembles the recognition site of the enzyme furin, a ubiquitous subtilisin-like proprotein convertase, involved in the processing of many protein precursors. Thus, during maturation, activation of the toxins results from furin-like proteolytic hydrolysis of the latrotoxin precursors. This is also believed to occur for the C-terminus of each precursor (Domain IV; Fig. 1C). In support, expression of the truncated proteins produces fully active polypeptides. A key feature of all mature latrotoxins is the presence of 15–22 ankyrin repeats (Domain III; Fig. 1C). These ∼33 residue repeats are thought to be involved in intraand intermolecular interactions and oligomerization. Three defined domains make up the tertiary structure of the molecule: the wing, the body, and the head (Fig. 1C). α-LTx was shown by cryoelectron microscopy to insert into the phospholipid bilayers, where its monomers/dimers aggregate and form a tetramer. The tetrameric complex consists of four monomers symmetrically arranged around a central axis, resembling a four-blade propeller with a diameter of 250 Å and a thickness of 100 Å. The central channel forms a constantly open channel-like pore, of 10–36 Å diameter, permeable to Ca2+, that can trigger neurotransmitter exocytosis as well as mediate massive leakage of the transmitter pool present in the cytosol. Under normal conditions, when

Ca2+ is present, the major effect of α-LTx is based on the pore-mediated Ca2+ influx. Furthermore, Ca2+ strongly facilitates tetramerization of α-LTx and, consequently, its pore formation. The overall action of α-LTx is to cause massive vesicle exocytosis from the presynaptic terminal in a wide range of amphibian, molluscan, mammalian, and insect preparations. Despite being phyla-selective, all latrotoxins target only neuronal cells due to a neuron-specific receptor that is distinct in different classes of organisms. These membrane receptors appear to be localized close to the active zone on the presynaptic plasma membrane but belong to distinct families of proteins. These are the Ca2+-dependent neurexin Ia (NRX), a neuronal protein with a single transmembrane domain, and latrophilin 1 (LPH 1), a heptahelical transmembrane protein that belongs to the secretin/calcitonin family of G protein-coupled receptors. Recently, a third receptor, protein tyrosine phosphatases (PTPs), has been described that binds toxin in a Ca2+-independent manner. However, PTPs appear to represent a minor receptor component [20]. It would appear that multiple mechanisms underlie the actions of α-LTx to increase neurotransmitter release. These include Ca2+ influx through the pore formed by the toxin tetramer after its binding to either LPH or NRX receptors and a direct nonvesicular efflux of neurotransmitters through the same pores, again following its binding to either LPH or NRX receptors. However, two further mechanisms reliant on intracellular signaling pathways may also exist. First, a Ca2+-dependent activation of LPH, a G proteincoupled receptor linked to Gaq/11. The downstream effector of Gaq/11 is phospholipase C (PLC). Activated PLC, causes increases in the cytosolic concentration of the phosphoinositide IP3, which in turn induces release of Ca2+ from intraterminal stores. This rise of [Ca2+]i, similar to presynaptic residual Ca2+, may increase the probability of spontaneous exocytosis. In addition, there is a hypothetical Ca2+-independent direct interaction of α-LTx with the exocytotic machinery after its binding to either LPH or NRX receptors [20].

SPHINGOMYELINASES D TOXINS FROM SPIDER VENOMS Loxoscelism is the clinical condition produced by envenomation by spiders belonging to the genus Loxosceles, which can be observed as two clinical conditions: cutaneous loxoscelism and systemic or viscerocutaneous loxoscelism (for an overview of the clinical pathophysiology see [13]). Sphingomyelinases (SMases) D from Loxosceles spider venoms such as L.

378 / Chapter 54 laeta and L. intermedia are the principal toxins responsible for the local and systemic pathological effects of spider envenomation. These include local dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. In the presence of Mg2+, these 31–35 kDa proteins catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. Loxosceles SMase toxins assist activation of the alternative pathway of complement on human erythrocytes by cleavage of glycophorins as a result of the activation of membrane-bound metalloproteinase and activation of the classical pathway of complement. More recently the x-ray crystal structure of SMase D from Loxosceles laeta (SMase I) was determined at 1.75-Å resolution [12]. SMase I folds as an (α/β)8 β-barrel with the interfacial and catalytic sites encompassing hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu32, Asp34, Asp91, and solvent molecules. In the proposed acid base catalytic mechanism, His12 and His47 also play key roles in hydrolyzing sphingomyelin and lysophosphatidylcholine via acid base catalysis (Fig. 3F).

ANTIMICROBIAL AND CYTOLYTIC PEPTIDE TOXINS FROM SPIDER VENOM To date, several families of cytolytic peptides with antimicrobial activity have been discovered in araneomorph spider venoms, especially from members of the superfamily Lycosidoidea. This includes two lycotoxins from the wolf spider Hogna (also described as Lycosa) carolinensis, four cupiennins (formerly CSTX-3 to -6) from the neotropical ctenid spider Cupiennius salei, and two families of toxins known as oxypinins from the lynx spider Oxyopes takobius (formerly kitabensis). For a recent review of the structure and biological activities of cytolytic peptides from spider venom arthropods see [8]. These 27–48 residue cytolytic peptides are linear, highly cationic, and amphipathic peptides lacking cysteine residues and have isoelectric points above pH 10.2. They are characterized by a high total charge of +6 to +10 because of the high proportion of lysine residues. In addition, they often possess an amidated C-terminus. In the case of the cupiennin 1 family, their structure is characterized by a hydrophobic N-terminal region, a lysine repeat segment, and a hydrophilic polar C-terminal region. In the case of lycotoxin II and the oxypinin family this N-terminal region is absent. The

central segment of these peptides consists of either a (i) sixfold repeat of four residues (cupiennins), (ii) four- to fivefold repeat of four to five residues (lycotoxins), or (iii) six- to ninefold repeat of three to four residues (oxypinins). In each case there is a lysine residue at every first position in the repeat, apart from oxypinins, where the lysine is sometimes conservatively substituted by an arginine. In the presence of membranes, the secondary structures of these peptides display a predicted 62–92% α-helix character typical of antimicrobial pore-forming peptides. In support, circular dichroism studies have shown that the secondary structure of the cupiennins is essentially α-helical. The amphipathic motif is characterized by a right-handed ribbon of positively charged lysine and polar residues winding around the α-helix. All these peptides are highly active against gramnegative and gram-positive bacteria with minimal inhibitory concentrations in the low micromolar range. Furthermore, lycotoxins display fungicidal activity (against Candida glabrata). The insecticidal effect of cupiennins on Drosophila melanogaster flies has been reported but is negligible, with very high LD50 values (5–8 nmol/g). However, coinjection of the neurotoxin oxytoxin with oxyopinin 1 into insect larvae resulted in a significant increase in the paralytic and lethal activity of the neurotoxin. Electrophysiological studies using insect Sf9 cells showed that oxypinins reduce cell membrane resistance by opening nonselective ion channels. Unfortunately, these peptides also exhibit a hemolytic effect on human erythrocytes dependent on the content of phosphatidylcholine. Synthetic N- and C-terminal truncates of cupiennin 1d showed that the residues involved in the cytolytic activity are located in the hydrophobic N-terminal chain segment. Removal of the first six N-terminal residues totally abrogated cytolytic activity, while the polar Cterminal region appears to modulate the peptide association at negatively charged cell surfaces to reduce activity. It is believed that the highly cationic lysine ribbons form electrostatic interactions with the negatively charged bacterial cell membrane. This would allow insertion of the hydrophobic N-terminal helix domain into the hydrophobic core of the lipid matrix of erythrocytes leading to subsequent membrane disruption. Several groups have speculated as to the role of these antimicrobial and cytolytic peptides in the venom of various spiders. They may play a dual role: first functioning in a cooperative fashion with neurotoxins in the prey capture strategy, and second to protect the spider from potentially infectious organisms arising from prey ingestion. Thus spider venoms represent a potential source of novel antimicrobial agents with important

Spider Venom Peptides / 379 medical implications. Unfortunately the antimicrobial peptides discovered so far are not well suited for the design of novel antibiotic drugs since they disrupt eukaryotic cells. If the precise mechanism of action of these peptides could be determined, then perhaps in the future a more selective agent could be developed. Presently, other more selective peptides contained in the hemolymph of other arthropods are more promising lead compounds (e.g., the antifungal ETD-151). Nevertheless, the synergistic actions that these peptides have with insect-selective neurotoxins could enhance the activity of recombinant insect-specific baculoviruses being trialed as biopesticides.

CONCLUSION Using several molecular mechanisms, spiders have developed a combinatorial peptide library strategy to diversify their toxin pool [18]. New pharmacologies have been produced by hypermutation of the mature toxin sequence resulting in a rich diversity of neurotoxins with high potency and selectivity for multiple cellular targets. This confers an evolutionary advantage for the spider, enabling them to efficiently immobilize a wide variety of prey. As pharmacologists, we can take advantage of this preoptimized peptide library as a diversified source of molecular probes for identifying subtype differences in specific targets and as leads for novel therapeutics such as analgesics, antiarrhythmics, neuroprotectants, and antimicrobials. Given that spiders, particularly mygalomorphs, rely upon their venom to immobilize or kill their prey it is not surprising that they contain a wide variety of insectselective toxins that may provide the basis for the development of biopesticides. These neurotoxins often target specific insect prey or more precisely subtle differences in the prey’s nervous system. Interestingly, a large number of these phyla-specific toxins target voltagegated ion channels. These represent suitable targets for the future development of insecticides, since they are ubiquitous among insects. However, despite their significance and potential for application in insect-pest control, the molecular basis for the selectivity of these toxins for insect over mammalian ion channels is still largely unknown. The structural basis for these selective interactions requires elucidation of the contact surfaces (i.e., insectophore) between the various toxins and their receptor binding sites on ion channel subtypes. This may lead to the development of more efficacious and more selective insecticidal toxins capable of being employed in a recombinant baculovirus model or used to design nonpeptide mimetics that could be used in foliar sprays.

References [1] Adams ME. Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta. Toxicon 2004;43:509–25. [2] Bode F, Sachs F, Franz MR. Tarantula peptide inhibits atrial fibrillation. Nature 2001;409:35–6. [3] Cestèle S, Catterall WA. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 2000;82:883–92. [4] Escoubas P, Bernard C, Lambeau G, Lazdunski M, Darbon H. Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton-gated cation channels. Protein Sci 2003;12:1332–43. [5] Escoubas P, Rash L. Tarantulas: eight-legged pharmacists and combinatorial chemists. Toxicon 2004;43:555–74. [6] Gomez MV, Kalapothakis E, Guatimosim C, Prado MA. Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels. Cell Mol Neurobiol 2002;22:579–88. [7] Hille B. Ion channels of excitable membranes. Sunderland: Sinauer Associates, Inc.; 2001. [8] Kuhn-Nentwig L. Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci 2003;60:2651–68. [9] Lampe RA. Analgesic peptides from the venom of Grammostola spatulata and use thereof. 1999;US Patent 5,877,026. [10] Lampe RA, Sachs F. Antiarrhythmic peptide from the venom of the spider Grammostola spatulata. 1999;US Patent 5,968,838. [11] Liang S. An overview of peptide toxins from the venom of the Chinese bird spider Selenocosmia huwena Wang [= Ornithoctonus huwena (Wang)]. Toxicon 2004;43:575–85. [12] Murakami MT, Fernandes-Pedrosa MF, Tambourgi DV, Arni RK. Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases D. J Biol Chem 2005;280:13658– 64. [13] Nicholson GM, Graudins A. Antivenoms for the treatment of spider envenomation. J Toxicol-Toxin Rev 2003;23:81–106. [14] Nicholson GM, Little MJ, Birinyi-Strachan LC. Structure and function of delta-atracotoxins: lethal neurotoxins targeting the voltage-gated sodium channel. Toxicon 2004;43:587–99. [15] Nicholson GM, Little MJ. Spider neurotoxins targeting voltage-gated sodium channels. J Toxicol-Toxin Rev 2005;24: 315–45. [16] Norton RS, Pallaghy PK. The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 1998;36:1573– 83. [17] Rash LD, Hodgson WC. Pharmacology and biochemistry of spider venoms. Toxicon 2002;40:225–54. [18] Sollod BL, Wilson D, Zhaxybayeva O, Gogarten JP, Drinkwater R, King GF. Were arachnids the first to use combinatorial peptide libraries? Peptides 2005;26:131–9. [19] Tedford HW, Sollod BL, Maggio F, King GF. Australian funnelweb spiders: master insecticide chemists. Toxicon 2004;43:601– 18. [20] Ushkaryov YA, Volynski KE, Ashton AC. The multiple actions of black widow spider toxins and their selective use in neurosecretion studies. Toxicon 2004;43:527–42. [21] Wang X-H, Connor M, Smith R, Maciejewski MW, Howden MEH, Nicholson GM, et al. Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge. Nature Struct Biol 2000;7:505–13. [22] Wen SP, Wilson DTR, Kuruppu S, Korsinczky MLJ, Hedrick J, Szeto TH, et al. Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins. Peptides 2005;26:2412–26.

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55 Conus Snail Venom Peptides BALDOMERO M. OLIVERA

ebrid (the last two groups have shells with easily recognizable morphology). The toxins characterized from toxoglossate venoms are almost exclusively from cone snails. As a consequence, this review focuses on conotoxins, the peptides from Conus venoms. Present estimates suggest that there are 500 to 700 Conus species, >300 terebrid species, and very likely >10,000 turrids (broadly defined). Recent work from Bouchet and coworkers in New Caledonia found only 51 Conus species but over 1500 “turrid” species, most not yet named [1]. If this were the true ratio for locations in the tropics, since both terebrids and cone snails are largely tropical groups (only turrids are adapted to cold-water environments), the estimate of >10,000 turrid species may very well be a conservative one. Thus, the currently available information about peptides from molluscan venoms is highly skewed and not at all aligned to the actual biodiversity of venomous snails. The turrids, though presently the least characterized of the three groups with respect to their toxins and venoms, probably have the greatest actual molecular diversity of venom peptides. Thus, although the conotoxins that have been characterized (and are reviewed here) are a large, well-defined class of peptides, the peptide toxins from molluscan venoms, in toto, are largely an unexplored frontier.

ABSTRACT Cone snails are a large genus (500–700 species) of venomous predators. They comprise only a minor fraction of the total biodiversity of molluscs; the overwhelming majority of peptides from molluscan venoms are uncharacterized. Peptides from Conus venoms are generally small (10–30 amino acids) and disulfide-rich, often with unusual posttranslationally modified amino acids (i.e., γ-carboxyglutamate, 6-bromotryptophan, dphenylalanine, etc.). Most Conus peptides target ligandgated or voltage-gated ion channels, or G-protein-coupled receptors. Conotoxins are widely used for basic neuroscience research; a few have reached human clinical trials, and one is an approved drug for intractable pain.

INTRODUCTION: THE BIODIVERSITY OF VENOMOUS MOLLUSCS Molluscs are not the first animals that come to mind when venoms are discussed. However, the total number of venomous molluscs is, in fact, surprisingly large. A major gastropod lineage, the toxoglossate “poison tongue” snails (suborder Toxoglossa or superfamily Conoidea), are venomous predators. Toxoglossate snails are believed to have originated in the Cretaceous period; by the Cretaceous Extinction, fossils of several toxoglossate stem groups can be confidently identified. Several major radiations of toxoglossate snails took place in the Cenozoic, giving rise to the three major living conoidean groups: the cone snails (Conus), augers (family Terebridae), and turrids (family Turridae, sensu lato). Of the three groups, the turrids are the least characterized and most heterogeneous, and in some respects, a turrid is defined by exclusion—that is, a toxoglossate snail that is neither a cone snail nor a terHandbook of Biologically Active Peptides

DISCOVERY OF CONOTOXINS The first conotoxins were purified and characterized from the venom of Conus geographus, a dangerous species of Conus that has caused human fatality. The first peptides investigated caused paralysis in either fish or mice and were shown to target ion channels important for neuromuscular transmission [9]. The discovery by Craig Clark, then a 19-year-old student at the University of Utah, that different components of Conus venoms

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382 / Chapter 55 caused different behavioral symptomatologies when injected into the central nervous system of mice led to the characterization of a larger group of Conus peptides, mostly from fish-hunting cone snails. These had biological activity on mammalian neurons [10], and some were subsequently developed for direct therapeutic application. One peptide from Conus magus, ωconotoxin MVIIA, was purified because it caused tremors in mice; it was approved as a drug for severe pain in December 2004 (commercial name Prialt) [8, 17]. Unusual posttranslationally modified amino acids were initially identified in the first group of conotoxins characterized [3]. As detailed following, it is highly likely that there are well over 50,000 different peptides in the venoms of living cone snails; the discovery of novel peptides has been accelerating in recent years. At this time (mid2005), there are probably amino acid sequences of >2000 Conus peptides known, but of these, only a small minority have been biochemically characterized, and the mechanism of action of an even smaller fraction has been clarified. The vast majority of peptides from Conus venoms remain completely uncharacterized. Initially, the discovery of each new conotoxin required biochemical purification from venom. As more cDNA clones encoding conotoxins were elucidated, a molecular cloning/PCR approach to identifying novel conotoxins became feasible [5]. Compared with the large number of venom ducts required for biochemical purification from venom, the molecular analysis requires, in principle, a single specimen harvested from the wild. However, a problem with this approach is that many conotoxins are posttranslationally modified, and knowing the encoding DNA sequence does not generally permit an accurate prediction of which amino acids are posttranslationally modified.

PRECURSOR STRUCTURE, EXPRESSION, AND PROCESSING: CONOTOXIN SUPERFAMILIES The Conus toxins characterized over the last two decades provide an overview framework for Conus venoms. The great majority of biologically active Conus venom components are peptides, initially synthesized through ribosomal translation as polypeptide precursors, and posttranslationally processed to yield the mature, biologically active peptide toxins. The total number of different Conus venom peptides is probably very large. Conus venoms are unusually complex; every Conus species can express its own repertoire of between 100 to 200 different toxins in the venom duct. Since there is virtually no molecular overlap between peptides found in the 500–700 different Conus species, there are

probably >50,000 different peptides in living cone snail venoms. The molecular genetic work done on Conus peptides provides a simplifying framework for Conus peptide toxins: The majority are likely to be encoded by a small number of gene superfamilies, with all peptides that belong to the same superfamily having a number of shared characteristics (for a review, see [16]). The genes encoding Conus peptide toxins are expressed in the epithelial cells of cone snail venom ducts and initially translated as pre-propeptide precursors, with an N-terminal signal sequence, an intervening “pro” region and at the C-terminal end, the mature toxin, always in single copy. All members of a particular Conus peptide superfamily share a remarkable degree of sequence identity in their signal sequences. This is highly anomalous, since in most gene families that encode secreted proteins the signal sequences are the least conserved elements. Like most toxins in animal venoms, Conus peptides are generally disulfide-rich. However, two characteristics differentiate conotoxins from other venom polypeptides. The mature toxins from Conus venoms are unusually small, typically 10–30 amino acids. The fully processed gene products of some major Conus peptide gene superfamilies are among the smallest biologically active gene products known; although many thousands of peptides in Conus venoms are likely to be between 10 and 20 amino acids, the great majority of these will be conformationally rigid because of the multiple disulfide crosslinks. A second unusual feature is the presence, sometimes at high density, of posttranslationally modified amino acids in many of the peptides from Conus. Some of the Conus posttranslational modifications are found in many other peptides. Conus peptides are first translated as longer precursors, and proteolytic processing and the formation of disulfide linkages are required steps in maturation. Other posttranslational events that may occur to generate the ends of a mature Conus peptide toxin are C-terminal amidation and the cyclization of glutamine to pyroglutamate at the Nterminus, well-characterized modifications that occur in many other peptides. Many Conus peptides have nonstandard amino acids that are derived from a standard amino acid by the action of a modification enzyme. Shown in Table 1 is the diverse set of posttranslationally modified amino acids discovered in Conus peptides so far; the presence of these unusual amino acids is one hallmark of peptide toxins from this group of venomous animals. The posttranslational modification of conotoxins was reviewed by Craig et al. [3] and more recently by Buczek et al. (2005, submitted to Cellular and Molecular Life Sciences). The reader is referred to these more comprehensive reviews for a discussion of mechanisms of posttransla-

TABLE 1. Posttranslationally modified amino acids in Conus peptides. Type of Modification Hydroxylation

Enzyme

4-trans-hydroxyproline

Proline hydroxylase Hydroxylase with DAA specificity Lysyl hydroxylase γ-Glutamyl carboxylase Bromo peroxidase Epimerase Epimerase Epimerase Epimerase Glutaminyl cyclase Tyrosyl sulfotransferase Polypeptide HexNAc Polypeptide HexNAc

D-γ-hydroxyvaline

Carboxylation Bromination Epimerization

Cyclization O-sulfation O-glycosylation

5-hydroxylysine γ-carboxyglutamate 6-bromotryptophan D-tryptophana D-leucine D-phenylalanine D-valine Pyroglutamate O-sulfated Tyr O-glycosylated Ser O-glycosylated Thr

Conus peptide μ-GIIIA gld-V*

RDCCTOOKKCKDRQCKOQRCCA* AOANS(D-Hyv)WS

de13a Conantokin-G

DCOTSCOTTCANGWECC(Hyk)GYOCVN(Hyk)ACSGCTH* GEggLQVNQgLIRgKSN*

r7a/Light sleeper Contryphan-R Leu-contryphan-P r11a mus-V Bromoheptapeptide α-EpI

WFGHγγCTYWLGPCγVDDTCCSASCγSKFCGLW GCO(D-W)EPWC* GCV(D-L)LPWC GOSFCKADEKOCEYHADCCNCCLSGICAOSTNWILPGCSTSSF(D-F)KI SOANS(D-V)WS ZCGQAWC* GCCSDPRCNMNNP(Y-SO3H)C*

κA-SIVA Contulakin-G

ZKSLVPS+VITTCCGYD(Hyp)GTMCOOCRCTNSC* ZSEEGGSNAT+KKPYIL

*, C-terminal amidation; O, 4-trans-hydroxyprolline; D-Hyv, D-γ-hydroxyvaline; W, 6-bromotryptophan; Hyk, 5-hydroxylysine; γ, γ-carboxyglutamate; D-X, D-amino acid; Z, pyroglutamate; Y-SO3H, sulfotyrosine; +, O-glycosylation.

Conus Snail Venom Peptides / 383

Modified AA

384 / Chapter 55 tional modification, and the probable function of individual posttranslationally modified amino acids. Another characteristic of conotoxins is that a few characteristic arrangements of cysteine residues in the primary sequence of the mature peptide toxin (“Cys patterns”) are repeatedly found. All peptides belonging to the same conotoxin superfamily will typically have one major “Cys pattern”; additional minor Cys patterns may be observed in some superfamilies. Examples of two different superfamilies, the O- and the A-conotoxin gene superfamilies, are shown in Table 2. All peptides that belong to the A-superfamily have a highly conserved signal sequence different from that of the peptides of the O-superfamily. The arrangement of Cys residues differs between the two superfamilies: In the O-superfamily, only one major Cys pattern (—C—C— CC—C—C—) is found. A different major Cys pattern (—CC—C—C—) occurs in most peptides of the Asuperfamily, but a minor one (—CC—C—C—C—C—) is also found in a minority of the A-superfamily peptides [12]. The Cys patterns of the different conotoxin superfamilies were reviewed by Terlau and Olivera [16].

TABLE 2.

There may be multiple peptides (>20) that belong to the Conus peptide superfamily expressed in a single venom duct. The major conotoxin gene superfamilies are widely distributed over the 500 to 700 different species in the genus. Some conotoxin superfamilies have been intensively investigated by molecular methods; thus, >500 different sequences are known that belong to the A-conotoxin superfamily at the present time, even though only a small minority of Conus species have been examined. A table of conotoxin sequences that have been published, with references to the primary literature, is provided with the supplementary material for this chapter.

DEFINITION OF CONOTOXIN FAMILIES, RECEPTOR TARGETS, STRUCTURES Peptides from a given conotoxin superfamily with the same Cys pattern are believed, to a first approximation, to have generally similar three-dimensional struc-

Conotoxins targeted to ligand-gated ion channels.

Nicotinic acetylcholine receptor; neuromuscular subtypes α-Conotoxin family (A–superfamily) α3/5 Subfamily α-conotoxin GI ECCNPACGRHYSC* α-conotoxin MI GRCCHPACGKNYSC* α4/7 Subfamily α-conotoxin EI RDOCCYHPTCNMSNPQIC* αA-Conotoxin family (A-superfamily) Long αA-subfamily αA-conotoxin PIVA GCCGSYONAACHOCSCKDROSYCGQ* αA-conotoxin EIVA GCCGPYONAACHOCGCKVGROOYCDROSGG* Short αA-subfamily CCGVONAACPOCVCNKTC* αA-conotoxin OIVB1 CCGIONAACHOCVCTGKC αA-conotoxin PeIVB1 ψ-Conotoxin family (M-superfamily) ψ-conotoxin PIIIE HOOCCLYGKCRRYOGCSSASCCQR* Nicotinic acetylcholine receptor; neuronal subtypes α-Conotoxin family α4/7 Subfamily (A-superfamily) α-conotoxin MII2 α-conotoxin PIA3 Other α Subfamilies α-conotoxin ImII4 α-conotoxin BuIA5 5HT3-receptor σ-Conotoxin family (S-superfamily) σ-conotoxin GVIIIA

GCCSNPVCHLEHSNLC* RDPCCSNPVCTVHNPQIC* ACCSDRRCRWRC* GCCSTPPCAVLYC*

GCTRTCGGOKCTGTCTCTNSSKCGCRYNVHPSGBGCGCACS*

*, amidated C-terminus; B, 6-bromotryptophan; 1preferentially targets the fetal subtype; 2preferred subtypes are α3β2- and α6β2-containing receptors; 3preferred subtypes are α6β2-containing receptors; 4preferred subtypes are the α7 and α9/α10 receptors; 5very slowly dissociating from β4-containing neuronal subtypes.

Conus Snail Venom Peptides / 385 tures. Disulfide crosslinks are a dominant determinant of how polypeptide backbones will fold; consequently, peptides sharing a Cys pattern will generally have similar polypeptide folds. Thus, members of a conotoxin superfamily are likely to have similar overall conformations. A number of conotoxin structures have been published [4]. A table of conotoxin structures that have been solved by NMR or x-ray crystallography is provided with the supplementary material for this chapter. Conus peptide superfamilies are subdivided into discrete families. The salient feature that distinguishes the different conotoxin families belonging to the same superfamily is pharmacological specificity. In general, all of the peptides in a particular family have the same class of receptor targets; peptides in different families,

TABLE 3. Sodium Channels Antagonists μ-Conotoxin family (M-superfamily) μ-conotoxin GIIIA μ-conotoxin PIIIA μ-conotoxin KIIIA μO-Conotoxin family (O-superfamily) μO-conotoxin MrVIB Inactivation inhibitors δ-Conotoxin family (O-superfamily) δ-conotoxin PVIA δ-conotoxin SVIE Calcium Channels Nor-P-type Ca channels ω-Conotoxin family (O-superfamily) ω-conotoxin GVIA ω-conotoxin MVIIA ω-conotoxin MVIIC L-type Ca channels ω-Conotoxin family (O-superfamily) ω-conotoxin TxVIIA Contryphan family Glacontryphan-M Potassium Channels Antagonists κ-Conotoxin family (O-superfamily) κ-conotoxin PVIIA κM-Conotoxin family (M-superfamily) κM-conotoxin RIIIK Conkunitzin family conkunitzin-S1 (Family unnamed—I superfamily) ViTx Modulation (Family unnamed—I superfamily) BeTx

but in the same superfamily, will have divergent targeting specificity (even though they may be similar structurally). Thus, in the M-superfamily, ψ-conotoxins inhibit nicotinic acetylcholine receptors, μ-conotoxins block voltage-gated sodium channels, and κMconotoxins are voltage-gated potassium channel antagonists (see Tables 3 and 4). Two different conotoxin families may target the same class of receptors; these invariably belong to different gene superfamilies. Thus, both the ψ-conotoxin and the α-conotoxin families are nicotinic acetylcholine receptor antagonists [7] but belong to the M- and A-conotoxin superfamilies, respectively, and are structurally unrelated (in this particular case, peptides from the two families probably bind to different sites on the nicotinic acetylcholine receptor complex).

Conotoxins targeted to voltage-gated ion channels.

RDCCTOOKKCKDRQCKOQRCCA* ZRLCCGFOKSCRSRQCKOHRCC* CCNCSSKWCRDHSRCC* ACSKKWEYCIVPILGFVYCCPGLICGPFVCV*

EACYAOGTFCGIKOGLCCSEFCLPGVCFG* DGCSSGGTFCGIHOGLCCSEFCFLWCITFID

CKSOGSSCSOTSYNCCRSCNOYTKRCY* CKGKGAKCSRLMYDCCTGSCRSGKC* DECYPOGTFCGIKOGLCCSAICLSFVCISFDF

NγSγCPWHPWC*

CRIONQKCFQHLDDCCSRKCNRFNKCV LOSCCSLNLRLCOVOACKRNOCCT* KDRPSLCDLPADSGSGTKAEKRIYYNSARKQCLRFDYTGQGGNEN NFRRTYDCQRTCLYT SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK

CRAEGTYCENDSQCCLNECCWGGCGHPCRHPGKRSKLQEFFRQR

*, amidated C-terminus; γ, gamma-carboxyglutamate; W, D-tryptophan.

386 / Chapter 55 TABLE 4.

Miscellaneous receptor targets.

Glutamate Receptor Superfamily NMDA receptor Conantokin family conantokin-G conantokin-L G-Protein-Coupled Receptors Agonists ρ-Conotoxin family (A-superfamily) ρ-conotoxin TIA Antagonists Conopressin family conopressin-G conopressin-S (Unnamed family) contulakin-G Conorfamide family conorfamide Transporter χ-Conotoxin family (T-superfamily) χ-conotoxin MrIA

GEγγLQγNQγLIRγKSN* GEγγVAKMAAγLARγDAVN*

FNWRCCLIPACRRNHKKFC*

CFIRNCPKG* CIIRNCPRG* ZSEEGGSNA†KKPYIL GPMGWVPVFYRF*

NGVCCGYKLCHOC

*, amidated C-terminus; γ, γ-carboxyglutamate; †, O-glycosylated Thr.

Some conotoxin families are widely distributed over the entire genus Conus; examples of these are the αconotoxins that target nicotinic acetylcholine receptors and the δ-conotoxins that target voltage-gated Na channels. In contrast, other conotoxin families appear to be very restricted in their phylogenetic distribution within Conus and are found only in a small group of closely related Conus species; this appears to be particularly true of those conotoxin families that target voltagegated K channels. Based on their major prey, the 500–700 species of cone snails are usually divided into fish-hunting, wormhunting, and snail-hunting species [11]. However, it has become apparent that these are not monophyletic groups, with each derived from a single common ancestor. Rather, there are distinct groups of fish-hunting cone snails and an even greater number of divergent groups of worm-hunting Conus species. The subgeneric group to which a cone snail species belongs is strongly predictive of the conotoxins to be found in its venom. For example, different groups of fish-hunting cone snails appear to have different specialized toxin families to target the neuromuscular nicotinic acetylcholine receptor. Although predicting which conotoxins will be found in the venom of a particular Conus species is far from a precise science, the assignment of a species to a particular taxonomic subdivision within the genus Conus has proven to be the most reliable indicator of the peptide toxin families likely to be present. However, individual species often

have evolved idiosyncratic venom components not found at all in very closely related Conus species. This is why purification and characterization of actual venom components should continue to productively yield novel pharmacological agents from Conus venoms.

BIOLOGICAL MECHANISMS, THERAPEUTIC APPLICATIONS The biological effects of a particular Conus venom are a function of the physiological targets of the individual conotoxins expressed in that venom. The known receptor targets of Conus peptides fall into three broad classes: (1) receptors belonging to the ligand-gated ion channel superfamily; (2) voltage-gated ion channels; and (3) targets other than those that belong to the two large superfamilies above. We discuss conotoxins specific for these target classes, and examples are provided of individual conotoxins with receptor targets in each of the three classes. A more comprehensive list of conotoxins is given in a table in the supplementary material. A large number of conotoxins that inhibit members of the ligand-gated ion channel superfamily have been identified; the vast majority of these are nicotinic acetylcholine receptor antagonists. A surprisingly wide range of structural variation is seen for conotoxins that inhibit nicotinic receptors. One large and well-

Conus Snail Venom Peptides / 387 characterized family is the α-conotoxins [12], most of which are competitive antagonists of various nicotinic acetylcholine receptor subtypes. Other classes of toxins that antagonize nicotinic receptors are the αA-conotoxins that have a different disulfide bonding framework (but also belong to the A-superfamily), and the αSconotoxins, which are large, genetically unrelated toxins [14]. The last are more closely related to the σconotoxins, which target another class of ligand-gated ion channels, the serotonin (5HT3) receptor. Some examples of these classes of Conus peptides are shown in Table 2. Conotoxins that target various voltage-gated ion channels are shown in Table 3. The most widely used conotoxins for basic neurobiological research are the ω-conotoxins that block specific voltage-gated Ca channel subtypes. Conotoxins that target Na channels either inhibit conductance (such as the μ- and μOconotoxins) or inhibit channel inactivation (the δconotoxins). Recently, toxins with novel specificity for Na channel subtypes, including some that inhibit tetrodotoxin-resistant sodium channels have been described [2]. Conotoxins that target voltage-gated K channels are particularly diverse: It was recently shown that the three different groups of fish-hunting cone snails have different conotoxin families, all targeted to a single subfamily of K channels, the Shaker (or Kv1) channels (Imperial et al., Proceedings of the American Philosophical Society, submitted). Although most established targets of conotoxins are either in the voltage-gated or ligand-gated ion channel superfamilies, a spectrum of additional targets has been identified; examples are given in Table 4. Several Conus peptides are known to affect the function of G-proteincoupled receptors (GPCRs); however, no major family of Conus peptides that targets diverse GPCRs has been identified so far. The Conus peptides shown to target GPCRs are from a diverse set of families and subfamilies. One peptide family, the conantokins, targets NMDA receptors, a class of ion channels belonging to the glutamate receptor superfamily. The structure of these unusual peptides is stabilized by a posttranslationally modified amino acid, γ-carboxyglutamate, rather than by disulfide crosslinks (which are absent). The presence of the γ-carboxyglutamate side chains every three or four amino acids confers a stable helical structure to these peptides in the presence of Ca++. Additionally, a conotoxin targeted to the norepinephrine transporter has been described [13]. Mechanisms of individual conotoxins provide the most reductionist insights required to understand the physiological activity of the whole venom. It has become clear that several peptide toxins in the venom act together in combination; thus, cone snails have evolved a pharmacological strategy similar to the combination

drug therapy favored by the modern pharmaceutical industry for treating AIDS or cancer. In order to paralyze prey and interfere with neuromuscular transmission, cone snails simultaneously target presynaptic calcium channels, postsynaptic nicotinic receptors, and voltage-gated sodium channels on the muscle membrane of the prey. These combinations of Conus peptides have been termed “toxin cabals”; the combination interfering with neuromuscular transmission is called the “motor cabal.” A different toxin combination causes an extremely rapid excitotoxic immobilization of fish prey [15], resulting in tetanic paralysis (the “lightningstrike cabal”). The conotoxins that belong to a toxin cabal typically have high potency and specificity for their individual receptor targets. It is notable that an increasing number of conotoxins are finding both diagnostic and therapeutic application [6]. Thus, peptides that might be used to facilitate a deadly purpose such as killing prey in the natural context may be benign and useful when applied to an entirely different biological context.

Acknowledgments The work of the author included in this review has been supported by grant GM 48677 from the Natural Institute of General Medical Sciences.

References [1] Bouchet P, Lozouet P, Maestrat P, Heros V. Assessing the magnitude of species richness in tropical marine environments: High numbers of molluscs at a New Caledonia site. Biol J Linnean Soc 2002; 75:421–36. [2] Bulaj G, West PJ, Garrett JE, Marsh M, Zhang M-M, Norton RS, et al. Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels. Biochemistry 2005; 44:7259–65. [3] Craig AG, Bandyopadhyay P, Olivera BM. Post-translationally modified peptides from Conus venoms. Eur J Biochem 1999; 264:271–5. [4] Grant MA, Morelli XJ, Rigby AC. Conotoxins and structural biology: A prospective paradigm for drug discovery. Curr Protein Peptide Sci 2004; 5:235–48. [5] Hillyard DR, Monje VD, Mintz IM, Bean BP, Nadasdi L, Ramachandran J, et al. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 1992; 9:69–77. [6] Livett BG, Gayler KR, Khalil Z. Drugs from the sea: Conopeptides as potential therapeutics. Curr Med Chem 2004; 11:1715– 23. [7] McIntosh JM, Santos AD, Olivera BM. Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. Annu Rev Biochem 1999; 68:59–88. [8] Miljanich GP. Ziconotide: Neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem 2004; 11:3029– 40. [9] Olivera BM, Gray WR, Zeikus R, McIntosh JM, Varga J, Rivier J, et al. Peptide neurotoxins from fish-hunting cone snails. Science 1985; 230:1338–43.

388 / Chapter 55 [10] Olivera BM, Rivier J, Clark C, Ramilo CA, Corpuz GP, Abogadie FC, et al. Diversity of Conus neuropeptides. Science 1990; 249: 257–63. [11] Röckel D, Korn W, Kohn AJ. Book Manual of the Living Conidae. Wiesbaden, Germany: Verlag Christa Hemmen; 1995. [12] Santos AD, McIntosh JM, Hillyard DR, Cruz LJ, Olivera BM. The A-superfamily of conotoxins: Structural and functional divergence. J Biol Chem 2004; 279:17596–606. [13] Sharpe IA, Gehrmann J, Loughnan ML, Thomas L, Adams DA, Atkins A, et al. Two new classes of conopeptides inhibit the α1-adrenoceptor and noradrenaline transporter. Nat Neurosci 2001; 4:902–7.

[14] Teichert RW, Rivier J, Torres J, Dykert J, Miller C, Olivera BM. A uniquely selective inhibitor of the mammalian fetal neuromuscular nicotinic acetylcholine receptor. J Neurosci 2005; 25:732–6. [15] Terlau H, Shon K, Grilley M, Stocker M, Stühmer W, Olivera BM. Strategy for rapid immobilization of prey by a fish-hunting cone snail. Nature 1996; 381:148–51. [16] Terlau H, Olivera BM. Conus venoms: A rich source of novel ion channel-targeted peptides. Physiol Rev 2004; 84:41– 68. [17] Webster LR, Fakata KL. Ziconotide for chronic severe pain. Pract Pain Mgmt 2005; 5:5–6, 47–51.

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56 Insect Venom Peptides MARIO SERGIO PALMA

ABSTRACT

to the extraordinary diversity of their chemical defense mechanisms [51]. In addition to the glandular defensive secretions, some arthropods developed sophisticated offensive/defensive chemical weaponry. In this regard, the development of venoms and its injection apparatus among the Insecta represented evolutionary attributes that contributed to adaptation of the insects to the many different terrestrial environments [51]. Thus, different orders of the Insecta developed their self-chemical weaponry, particularly the Hymenoptera (bees, wasps, and ants) that evolved into their venoms and stinging apparatuses according to their biology and behavior. The species with a solitary life history evolved their venoms to be used as paralytic tools in order to keep their prey alive for feeding and reproduction. The many wasp species taking this evolutionary way include the solitary aculeate wasps belonging to the superfamilies Bethyloidea, Scolioidea, Pompiloidea, Sphecoidea, and Vespoidea. This last superfamily is considered as a single family, the Vespinae, and contains the solitary families Massarinae and Eumeninae as well as the social Vespinae [37]. Members of this group of solitary wasps are seasonal, spending the cooler periods as diapausing larvae in nests provided by the mother wasp. In most cases, the food provided consists of arthropod prey paralyzed by injection of venom into their bodies. The immobilized prey is then carried to the nest, where the eggs are layed on the prey and the larval development takes place [37]. The constituents of these venoms are low-molecular-mass neurotoxins, such as polyamines, a cocktail of neurotransmitters, and a few peptides [26], which are discussed later in this chapter. Another group of solitary wasps, the Terebrant, which include the superfamilies Ichneumoidea, Cynipoidea, and Chalcicoidea evolved in the direction of parasitic behavior—that is, their venom evolved to promote short/ long-lasting transient paralysis of the prey in order to permit egg-laying on/within the prey’s body. In this case, after the egg laying, the prey recovers from the paralysis

The insects of the order Hymenoptera (bees, wasps, and ants) are classified in two groups, based on their life history: social and solitary. The venoms of the social Hymenoptera evolved to be used as defensive tools to protect the colonies of these insects from the attacks of predators. Generally they do not cause lethal effects but cause mainly inflammatory and/or immunological reactions in the victims of their stings. However, sometimes it is also possible to observe the occurrence of systemic effects like respiratory and/or kidney failure. Meanwhile, the venoms of solitary Hymenoptera evolved mainly to cause paralysis of the preys in order to permit egg laying on/within the prey’s body; thus, some components of these venoms cause permanent/transient paralysis in the preys, while other components seem to act preventing infections of the food and future progenies. The peptide components of venoms from Hymenoptera are spread over the molar mass range of 1400 to 7000 Da and together comprise up to 70% of the weight of freeze-dried venoms. Most of these toxins are linear polycationic amphipatic peptides with a high content of α-helices in their secondary structures. These peptides generally account for cell lysis, hemolysis, antibiosis, and sometimes promote the delivery of cellular activators/mediators through interaction with the Gprotein receptor, and perhaps some of them are even immunogenic components. In addition to these peptides, the Hymenopteran venoms also may contain a few neurotoxins that target Na+ and/or Ca+2 channels or even the nicotinic ACh receptor. This review summarizes current knowledge of the biologically active Hymenoptera venoms.

INTRODUCTION The remarkable dominance of insects and other arthropods on land can be attributed, at least partially, Handbook of Biologically Active Peptides

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390 / Chapter 56 and carries the eggs of the parasitic wasps [37]. In this situation, the venom of these solitary wasps evolved to cause prey paralysis and seem to be constituted of highmolecular-mass proteins and low-molecular-mass compounds, generally presenting neurotoxicity. No peptide component is presently known for these venoms so that no further discussion about these components will be considered in the present chapter. Meanwhile, those species that evolved in the direction of social behavior developed the formation of castes and established the hierarchic relationship among nestmates of different castes. Generally the social species built large nests containing many workers and larvae, in addition to storage of a reasonable amount of pollen, honey, or nectar dew, attracting many different types of predators to their nests [45]. These species evolved their venom to prevent the presence of predators, keeping them far from the colonies; the venom of these species is not used to promote lethal actions but to produce mneumonical actions on the victims of their stinging due to the uncomfortable effects of venoms such as pain, local burning, edema, swelling, bradycardia, tachycardia, headache, and, sometimes systemic effects like respiratory and/or kidney failure [32]. The venom of the social Hymenoptera consists of a complex mixture of proteins, peptides, and low molecular mass compounds. The enzymes are responsible for the injury caused to tissues and frequently are immunogenic and therefore related to the allergy caused by these venoms in the victims of wasp and/or bee stings [13, 53]. Most abundant components of the venoms from social wasps and bees consist of peptide toxins. Ants contain some groups of species that may be considered as the most specialized among the social insects, as well as contain species that may be considered as the least specialized ones. Some species form small and secretive cryptic colonies, preying only on a limited group of organisms, while other species live almost anywhere [5]. The ant venoms may be broadly classified as either predominantly consisting of protein and peptides or as a complex mixture of low-molecularmass compounds. The ant species from the subfamilies Ponerinae, Myrmiciinae, Pseudomyrmecinae, and Ecitoninae generally contain venom rich in proteins and peptides. This chapter summarizes the current knowledge concerning the biochemical and pharmacological properties of the most representative peptide toxins from insects of the order Hymenoptera, which are now thoroughly characterized. It is not easy to comprehensively review the extensive literature on all the major components of these venoms in the available space. Therefore, for the interest of readers the focus will be on the most relevant groups of biologically peptides from Hyme-

noptera venoms and the references mentioned will enable a more extensive search of the literature.

PEPTIDES FROM THE VENOMS OF SOCIAL HYMENOPTERA The peptide components of venoms from social Hymenoptera are spread over the molar mass range of 1400 to 7000 kDa and together comprise up to 70% of the weight of freeze-dried Hymenoptera venoms [36]. Most of these peptides have polycationic amphipatic components, presenting a high content of α-helices in their secondary structures; these peptides generally account for cell lysis, hemolysis, antibiosis, and sometimes promote the delivery of cellular activators/mediators. In addition to this, the Hymenopteran venoms also may contain a few neurotoxic peptides.

Peptide Toxins from Honeybee Venom Honeybee (Apis mellifera) venom contains well known peptides such as: melittin, apamin, tertiapin, secapin, and MCD-peptide. Some of these peptides present a detergent-like action on plasma membranes [8], causing cell lysis, while others are neurotoxins. Some aspects of the primary sequences, secondary structures, and the biological actions will be emphasized. Melittin Melittin is the major component of honeybee venom, representing about 50% of the total honeybee venom. It consists of 26 amino acid residues, mostly with hydrophobic or at least uncharged side chains, except for the C-terminal region. Melittin can aggregate into a tetrameric form into the venom reservoir, being apparently inactive under this condition [4]. This peptide lowers the surface tension of water at the level of the plasma membrane, acting mainly by its natural detergent-like effect on the plasma membrane, causing cell lysis (especially of mast cells), followed by histamine delivery. Due to this lytic effect on cell membranes, this peptide may be considered as a venom diffusion factor, facilitating the entry of venom into the bloodstream of the stung victims. X-ray crystallography of mellitin indicated that residues 1 to 10 and residues 13 to 26 form α-helixes aligned about 120° to each other, while the proline in position 14 was suggested to be the cause of a bend in the middle of the rod structure [11]. Thus, a single polypeptide chain has the conformation of a bent alpha-helical cylinder [8]. When packed, the tetramer does so in a double planar layer. In order to achieve tight hydrophobic interactions, the hydrophobic residues of all four chains extend toward the center of the

Insect Venom Peptides / 391 tetramer. Due to its surfactant properties melittin is considered a direct hemolytic factor, acting synergistically with phospholipase A2, activating this enzyme [4].

Apamin The honeybee venom also contains apamin, a peptide of 18 amino acid residues. The CD spectroscopy study of apamin in solution is consistent with a peptide presenting as an alpha-helix and demonstrating a high degree of stability over a wide range of pH values [4]. This stability is a consequence of the rigidity imposed by two disulfide bridges. H1-NMR studies of apamin in solution indicates a rigid, folded structure stabilized not only by the two disulfide bridges but by at least seven intra chain hydrogen bonds. The structure of apamin has been studied by the use of a number of spectroscopic techniques, such as H1-NMR, CD, Raman, FT-IR, and structure prediction algorithms of energy minimization using NMR and CD data. At first, the most suitable interpretation of the experimental results was the proposal of the coexistence of both an alpha-helix and beta-turns [16]. However, until now, the best structure for apamin remains controversial, with several available structural models. Apamin is permeable to the blood-brain barrier, causing its effects on the CNS by several routes of administration. It causes neurotoxic effects in the spinal cord of mammals, producing hyperactivity and convulsions in rats [15]. When peripherically applied, apamin seems to selectively and potently affect the potassium permeabilities of certain membranes, such as the smooth muscle of the gut. At very low levels, it appears that apamin treatment can convert the normal hyperpolarizing response to epinephrine into a calciumdependent depolarization [9]. The peripheral effects of apamin suggest that the central action may be also due to decreased potassium fluxes, since this would broadly reduce inhibitory tone and increase the excitability. Mast Cell Degranulating Peptide (MCD) The MCD peptide is a 22-residue-long toxin. Its secondary structure was studied by CD and 1H-NMR [14, 50], which indicated a close resemblance to αhelical peptides from positions 13 to 19. It is stable over the pH range 2 to 8, stabilized by two disulfide bridges and six intrachain hydrogen bonds, which form a 28atom ring structure, also observed in apamin [4]. It was suggested that this peptide may present two closely related conformations in solution, possibly differing by cys-trans isomerism involving the residues Pro12His13 [49].

It produces cytolysis, thus being considered a potent mast cell degranulator, causing the release of histamine [4]. This peptide is a facilitator of the response of honeybee venom, responsible for the reddening, swelling, and locally occurring pain at the site of honeybee stinging, typical of histamine-mediated responses. Other Peptides Secapin represents about 0.5% of total honey bee venom. It is a 25-amino-acid residue peptide, containing a single disulfide bridge and high proline content. Apparently, this peptide has no known physiological and/or pharmacological activity, probably because it was not sufficiently tested so far [4]. Tertiapin, which comprises about 0.1% of total honey bee venom, is a 21-amino-acid residue peptide, containing a single disulfide bridge and a C-terminal residue in an amidated form. This peptide is not particularly toxic on intravenous injection; it degranulates mast cells with low potency. It has been compared pharmacologically with prostaglandins under the same conditions [31]. Tertiapin has been proposed to have both alpha-helical and beta-sheet tendencies along the length of its sequence, raising the question of which conformational state the molecule is likely to be in. Considering the positions of both disulfide bridge as well as its CD spectrum, which has no indication of a beta-sheet, the structure of tertiapin is probably analogous to that of the MCD peptide [4].

Peptide Toxins from Bumblebee Venoms Bombolitins The bombolitins are a family of heptadecapeptides isolated from the venom of the bumblebee Megabombus pennsyluanicus [3]. These peptides are able to form amphiphilic α-helical structures either by self-association or by interaction with amphiphilic matrices, such as micelles or vesicles. The biological activity of these peptides seems then to be directly correlated to their capability to form an amphiphilic order structure (αhelix) that allows them to associate and penetrate cellular membranes. They are capable of lysing erythrocytes and liposomes, in addition to increasing the activity of the enzyme phospholipase A2. In more general terms the bombolitins share common biological properties with other “toxin” peptides such as melittin, crabrolin, and mastoparans.

PEPTIDE TOXINS FROM SOCIAL WASPS The venoms of social wasps contain a series of polycationic amphipathic peptides, such as mastoparans,

392 / Chapter 56 chemotactic peptides, and wasp kinins, presenting a series of multifunctional pharmacological actions, which generally contribute to the occurrence of intense inflammatory processes.

enhancing the inflammatory effect of wasp stings. A novel type of chemotactic peptide was recently reported in which the acetylation of the N-terminal residue seems to play part in the modulation of polymorphonuclear leukocyte attraction [44].

Mastoparans The most abundant peptide component from Vespide venoms is represented by the mastoparans, which involves a histamine-releasing principle, acting as the MCD-peptide from honeybee venom. Mastoparans generally are polycationic, linear tetradecapeptide amides, rich in hydrophobic residues such as leucine, isoleucine, and alanine. The most representative molecules of species endemic from regions with a cold climate show characteristic lysine residues in positions 4, 11, and 12 [32], but novel peptide sequences showing a different pattern of lysine distribution have been identified recently in wasp species endemic from the tropical regions [29, 48]. Mastoparans are randomly coiled in water, while in methanolic solution and/or in the presence of lipid vesicles they are generally alpha helical peptides, causing their effects by interacting with plasma membranes due to their amphipaticity. According to their mode of action, these peptides may be classified into two groups: (1) those acting by cell lysis due to pore formation on cell membranes [1, 28, 32] and (2) those acting by interaction with G-protein coupled receptors, leading to the activation of degranulation mechanisms and resulting in many different types of secretions, depending on the type of target cell [18, 30]. In any situation the final action will be histamine delivered from mast cells, serotonin from platelets, or even insulin from pancreatic β-cells. In addition to the interaction with membrane components, mastoparans also activate phospholipases A [1]. Recently, antibacterial activities have been described for these peptides [27, 28].

Chemotactic Peptides The second most important group of peptides among the Vespid are the chemotactic peptides, which generally are trideca-polycationic peptides, with primary sequences resembling the mastoparans in regards to the richness of hydrophobic amino acid residues and the amidation of the C-terminal residue [32]. These peptides induce chemotaxis in polymorphonuclear leukocytes and macrophages [52] and sometimes also release histamine from mast cells [32]. The result of such chemotactic activity is the development of a mild edema, accompanied by an inflammatory exudate around the site of stinging, containing mainly polymorphonuclear leukocytes. Thus, the chemotactic peptides do not cause pain directly but play an important role

Kinin-Related Peptides (Wasp Kinins) A third group of peptides biologically important among the Vespid toxins are the wasp kinins that generally are known as pain-producing peptides. These peptides consist of bradykinin molecules elongated at the N-terminus with some extra amino acid residues. Sometimes there is also an elongation from the original bradykinin C-terminal residue [32]. The general pharmacological properties are similar to those presented by human bradykinin; however, the effects of Vespidae kinins are much more pronounced and long lasting. These effects may be summarized as follows: When injected intravenously, they cause hypertension in rats, dogs, rabbits, and cats; hyppotension in chickens; bronchoconstriction in guinea pigs; contraction of most isolated smooth muscle preparations; and relaxation of rat duodenum [42].

Sylverin A fourth group of amphipathic polycationic peptides from Vespide venoms has been described, but the few characterized up to now are the sylverins [10]. These peptides contain from 21 to 25 amino acid residues in their primary sequences, presenting a single disulfide bridge, constituting the first example of this type of structure among the Vespid peptide toxins. These peptides are potent mast cell degranulators without known hemolytic activity.

Crabrolin This is a 13-amino-acid residue polycationic peptide present in the venom of the hornet Vespa crabro, presenting a helical conformation [27]. This peptide produces potent hemolytic and antibacterial activity; however, it is a weak mast cell degranulator, with less than 20% the activity of mastoparan. Crabrolin is reported to be four times less active than mastoparan in guinea pig erythrocytes [2] and showed no effect in rat erythrocytes.

PEPTIDES FROM THE VENOMS OF SOLITARY WASPS Contrary to social wasps, solitary wasps offensively use their venoms for prey capture; they inject their venoms into prey insects or spiders to paralyze and use

Insect Venom Peptides / 393 them to feed their larvae. Therefore, the solitary wasp venoms should contain some neurotoxins acting on nervous systems [37] and antimicrobial peptides to prevent their preys from being colonized by pathogenic microorganisms [25]. Recent investigations indicate that solitary wasp venoms may contain a variety of bioactive substances—in particular, amphipathic peptides with antibiotic and inflammatory actions besides neurotoxins [21–26].

Anoplin Anoplin is a linear polycationic decapeptide presenting an α-helical amphipathic conformation, isolated from the solitary wasp Anoplius samariensi. It is highly conserved in relation to crabrolin and mastoparan-X; therefore, it is also a mast cell degranulating component. This peptide shows antimicrobial activity, both against gram-positive and gram-negative bacteria, and is considered the smallest among the linear antimicrobial peptides yet found in nature [24].

Bradykinin-Related Peptides Neurotoxic kinins have been reported in the venom of solitary wasps, such as threonine-bradykinin (Thr6BK) and megascoliakinin (Thr6-BK-Lys-Ala) isolated from the venom of the European scolid wasp Megascolia flavifrons [38, 39]; Thr6-BK was also reported in the venom of the wasp Colpa interrupta [40, 41]. An analytical survey with the venom extracts of 27 species of solitary wasps from the families Pompilidae, Sphecidae, Eumenidae, and Scoliidae, using MALDI-TOF MS as the experimental tool, demonstrated that BK-related peptides were present in four of these species, with Thr6-BK as the major component in the venom of Megacampsomeris prismatica [26]. The wasp kinins are known to block the synaptic transmission of the nicotinic acetylcholine receptor in the insect central nervous system [19, 38–40].

Pompilidotoxins (PMTXs) The pompilidotoxins (PMTXs) constitute a family of toxins consisting of 13 amino acid residues, isolated from the venoms of the pompilid wasps Anoplius samariensis and Batozonellus maculifrons [22]. PMTXs affect both the vertebrate and invertebrate nervous systems by blockade of sodium channel inactivation [46]. Most notably, this toxin discriminates between neuronal and cardiac sodium channels [21].

Eumenine Mastoparan-AF (EMP-AF) EMP-AF is a tetradeca-polycationic peptide isolated from the solitary wasp Anterhychium flavomargimatum micado, with general characterisics of the mastoparans from social wasp venoms; however, the location of lysine residues in EMP-AF is a little bit different from the mastoparans, being present at positions 5, 8, and 12 instead of positions 4, 11, and 12 [6, 23]. NMR study of this peptide suggested that it consists of an amphipathic α-helix conformation stabilized by the C-terminal residue in the amidated form [47]. EMP-AF is a mast cell degranulating peptide, which also affects the neuromuscular transmission in the lobster walking leg preparation [23].

PEPTIDES FROM ANT VENOMS Most of the ants only present traces of proteins/ peptides in their venoms; however, in those species producing peptides as components of their venoms, these toxins follow the same general pattern already observed for peptides from wasp and bee venoms—that is, short and linear polycationic peptides, presenting a high content of α-helices, which are responsible for cell lysis, hemolysis, histamine release from mast cells, and antimicrobial actions. A few neurotoxins are also known.

“Myr p” Peptides and Pilosulin The venom of the Australian ant Myrmecia pilosula seems to contain a complex mixture of allergenic peptides, some of them forming heterodimers, apparently maintained by disulfide bridges. From this complex was identified Myr p 1, the major expressed allergenic product being a 56-residue peptide, while Myr p 2 is a 27-residue peptide; these peptides may dissociate or are cleaved to minor fragments, such as those previously identified as pilosulin-1 and -2, characterized as the fragments 57–112 from Myr p 1 and 49–75 from Myr p 2 [7]. Pilosulin-1 was characterized as an α-helical peptide with potent and broad spectrum antimicrobial activity, both against standard and multidrug-resistant gram-positive and gram-negative bacteria, and Candida albicans [54]. Other pilosulins have been identified and characterized as hemolysins and histamine release peptides [20].

Poneratoxins Poneratoxin is a neuropeptide found in the venom of the ant Paraponera clavata. It is a peptide containing 25 amino acid residues, which affects the voltagedependent sodium channels and blocks the synaptic transmission in the insect central nervous system in a concentration-dependent manner. The NMR structure shows the peptide in the form of two α-helices

394 / Chapter 56 connected by a β-turn; the helices have quite different characteristics from each other: One of them is apolar, whereas the second contains polar and charged amino acids. This will result in different interactions with cell membranes. The extremely hydrophobic N-terminal helix may interact with uncharged lipid bilayers, while the C-terminal helix, slightly positively charged and terminating with arginine, will be able to attach to negatively charged cell surfaces as previously found for other membrane interacting peptides. Such a toxin can thus use two different complementary modes of interaction to attain its target, cellular membranes [41].

Ponericins Ponericins constitute a group of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, exhibiting hemolysis, insecticidal activity against cricket larvae, and antimicrobial action against gram-positive and gram-negative bacteria. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic αhelical structure in polar environments, such as cell membranes [34, 35].

Ectatomin This peptide is a neurotoxin isolated from the venom of the Ectatomma tuberculatum ant and contains two highly homologous peptide chains (consisting of 37 and 34 amino acid residues) linked to each other by disulfide bonds [43]. Each chain consists of two alphahelices and a hinge region of four residues; this forms a hairpin structure that is stabilized by disulfide bridges; the hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-alpha-helical bundle structure [33]. Ectatomin is a potent inhibitor of calcium currents after a latency of a few seconds in rat ventricular myocites [43].

Aknowledgments The work of the author of this review has been supported by grant of the project “Institute of Immunological Investigations” (iii)/MCT/CNPq and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

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[34] Orivel, J., Redeker, V., Le Caer, J.P., Krier, F., Revol-Junelles, A.M., Longeon, A., Chaffotte, A., Dejean, A., Rossier, J. Ponericins, new antibacterial and insecticidal peptides from the venom of the ant Pachycondyla goeldii. J. Biol. Chem. 2001; 276: 17823– 17829. [35] Orivel, J., Dejean, A. Comparative effect of the venoms of ants of the genus Pachycondyla (Hymenoptera Ponerinae). Toxicon. 2001; 39: 195–201. [36] Palma, M.S., Brochetto-Braga, M.R. Biochemical variability between venoms from different honeybee (Apis mellifera) races. Comparat. Biochem. Physiol. 1993; 106: 423–427. [37] Piek, T., Spanjer, W. Chemistry and Pharmacology of Solitary Wasp Venoms. In: Piek, T., Editor; Venoms of Hymenoptera, pp. 161–327. Biochemical, Pharmacological and Behavioral Aspects, Academic Press, London, UK, 1986, p. 570. [38] Piek T., Hue B., Pelhate M., Mony, L. The venom of the wasp Campsomeris sexmaculata (F.) block synaptic transmission in insect CNS. Comp. Biochem. Physiol. 1987; 87C: 283–286. [39] Piek, T., Hue, B., Mony, L., Nakajima, T., Pelhate, M., Yasuhara, T. Block of synaptic transmission in insect CNS by toxins from insect CNS by toxins from the venom of the wasp Megascolia flavifrons (FAB.). Comp. Biochem. Physiol. 1987; 87C: 287– 295. [40] Piek, T. Neurotoxic kinins from wasp and ant venoms. Toxicon 1991a; 29: 139–149. [41] Piek, T., Duval, A., Hue, B., Karst, H., Lapied, B., Mantel, P., Nakajima, T., Pelhate, M., Schmidt, J.O. Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata. Comp. Biochem. Physiol. C. 1991; 99: 487–495. [42] Pisano, J.J. Kinins in Nature. Hand. Exp. Pharmacol. Suppl. 1979; 25, 273–285. [43] Pluzhnikov, K., Nosyreva, E., Shevchenko, L., Kokoz, Y., Schmalz, D., Hucho, F. Grishin, E. Analysis of ectatomin action on cell membranes. Eur. J. Biochem. 1999; 262: 501–506. [44] Ribeiro, S.P., Mendes, M.A., Santos, L.D., Souza, B.M., Marques, M.R., Azevedo Jr., W.F., Palma, M.S. Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista. Peptides 2004; 25: 2069–2078. [45] Smith, J. Chemistry, Pharmacology and Chemical Ecology of Ant Venoms. In: Piek, T. Ed.; Venoms of Hymenoptera, Biochemical, Pharmacological and Behavioral Aspects, Academic Press, London, UK, 1986, pp. 423–508. [46] Sahara, Y., Gotoh, M., Konno, K., Miwa, A., Tsubokawa, H., Robinson, H.P.C., Kawai, N. A new class of neurotoxin from wasp venom slows inactivation of sodium current. Eur. J. Neurosci. 2000; 12: 1961–1970. [47] Sforça, M.L., Oyama Jr., S., Canduri, F., Lorenzi, C.C.B., Pertinhez, T., Konno. K., Souza, B.M., Palma, M.S., Ruggiero-Neto, J., Azevedo Jr, W.F., Spisni, A. How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the Eumenine solitary wasp. Biochemistry 2004; 43: 5608–5617. [48] Souza, B.M., Marques, M.R., Tomazela, D.M, Eberlin, M.N., Mendes, M.A., Palma, M.S. Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista. Rapid. Commun. Mass Spectrom. 2004; 18: 1095–1102. [49] Walde, P., Jäckle, H., Luisi, P.L., Dempsey, C.E., Banks, B.E.C. Spectroscopic investigation of peptide 401 from bee venom. Biopolymers. 1981; 20: 371–385. [50] Wemmer, D., Kallembach, N.R. Assignments and structure of apamin and related peptides in bee venom. Biochemistry 1982; 22: 191–1906. [51] Whitman, D.W., Blum, M.B., Alsop, D.W. Allomones: Chemicals for Defense. In: Evans, D.L, Smith, J., Eds, Insect Defenses; State University of New York Press, Albany, USA; 1990, pp. 289–351.

396 / Chapter 56 [52] Yasuhara, T., Nakajima, T., Fukuda, K., Tsukamoto, K., Mori, M. Kitada. M., Fujino, M. In: Munekata, E. Ed.; Peptide Chemistry: Structure and activity of chemotactic peptide from the venom sac of Vespinae; Protein Res. Found. Editorial., Osaka, Japan, 1983, pp. 185–190. [53] Yee, C.J., Palma, M.S., Malaspina, O., Morato-Castro, F.M., Azevedo-Neto, R.S., Manso, E.C., Croce, J. Acquired immunity

to African honeybee (Apis mellifera) venom in Brazilian beekeepers. J. Invest. Allergol. Clin. Imunol. 1997; 7: 583–587. [54] Zelezetskya, I., Pagb, U., Antchevaa, N., Sahlb, H.G., Tossia, A. Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin. Arch. Biochem. Biophys. 2005; 434: 358–364.

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57 Worm Venom Peptides WILLIAM R. KEM

ertines, possessing a proboscis stylet capable of puncturing the skin of a prey animal, and the so-called anoplans (paleonemertines and heteronemertines), which apparently lack a means of injecting a venom into the prey. Whereas hoplonemertine toxins are used for offensive as well as defensive purposes, it is thought that the paleo- and heteronemertines utilize their toxins largely to repel predators. In 1936 a Belgian pharmacologist reported the serendipitous discovery of two different types of toxic activities in nemertines [1]. An aqueous homogenate of the hoplonemertine Amphiporus lactifloreus potently contracted isolated frog skeletal muscle and stimulated the cat cervical autonomic ganglion in a manner similar to the neurotransmitter acetylcholine (ACh). However, since this activity was stable in highly alkaline solution, it could not be due to ACh. In extracts of other marine species, Bacq also found a neurotoxic activity lacking nicotinic receptor effects, which he referred to as “la nemertine.” Both “amphiporine” and “la nemertine” caused convulsions, paralysis, and death when injected into crabs. Relative to “amphiporine” activity, “la nemertine” activity only slowly passed across a dialysis membrane. Thirty years elapsed before nemertine toxins were investigated again. Fortunately, during the ensuing decades many new isolation and analytical methods were developed, which made it possible to successfully isolate even small quantities of these natural products. These included classical column, thin-layer, and later high-pressure liquid chromatographic isolation techniques, sensitive amino acid analysis, and Edman sequencing methods for analysis of peptides. As a graduate student, the author isolated the hoplonemertine alkaloid anabaseine, a nicotinoid compound possessing a biological and chemical profile similar to Bacq’s “amphiporine” [22]. Related compounds were found in other hoplonemertines [16, 19]. More recently, the

ABSTRACT Approximately half of the recognized animal phyla are worms. Many of these animal groups receive little scientific attention except as they influence human affairs. Nevertheless, investigation of the substances that they use to attack or defend against other organisms likely will provide useful molecular probes or models for understanding complex physiological properties such as membrane permeability and vulnerability to exogenous agents such as toxins, infectious viruses, microbes, and various parasites. Nemertines are a phylum of carnivorous marine worms that produce a plethora of peptidic as well as alkaloidal toxins. While just a few species have been investigated to date, many toxins used for chemical defense and offense have already been discovered. Heteronemertines, which lack an armed proboscis, secrete small basic peptide neurotoxins and larger peptide cytolysins. The neurotoxins target ion channels involved in generating action potentials. Certain predatory marine annelids possess large protein toxins that stimulate neurotransmitter release at certain synapses by targeting particular calcium channels. Finally, certain parasitic worms (helminths and nematodes) utilize cytolytic peptides to aid in the digestion of host cells and tissues. This article summarizes current knowledge of these worm peptides.

INTRODUCTION Nemertines are a phylum of marine worms that utilize a large, muscular proboscis to capture their prey [12]. Approximately 1000 species have been described. Most species are relatively small and inconspicuous inhabitants of coastal waters. It is likely that many more species will be described in the future. The phylum has been subdivided into two main groups: the hoplonemHandbook of Biologically Active Peptides

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398 / Chapter 57 pharmacological properties of anabaseine and a variety of anabaseine derivatives have been assessed [25]. Several of these derivatives, because they selectively stimulate a nicotinic ACh receptor involved in brain cognitive function, are in clinical tests [21, 26]. Heteronemertines, though lacking alkaloid toxins, were found to possess peptide neurotoxins resembling the activity profile of Bacq’s “la nemertine” [16, 17]. This chapter summarizes current knowledge concerning the biochemical and pharmacological properties of the neurotoxic or cytolytic nemertine peptide toxins. In addition, the toxic properties of certain other peptide toxins found in other worm phyla are reported.

NEMERTINE PEPTIDE NEUROTOXINS Biochemistry Many heteronemertines have been shown to possess peptide neurotoxins as assayed by their ability to rapidly paralyze crabs [16]. However, the only neurotoxins that have been isolated to date belong to a large (>1 meter) Atlantic coast species, Cerebratulus lacteus. The genus Cerebratulus is widely distributed in the Pacific and other oceans, and it is likely that at least some of these other species contain similar toxins. The Cerebratulus neurotoxins have molecular sizes of approximately 6000 daltons and are cross-linked by three disulfide bonds [18]. These were designated as B toxins because during G50 Sephadex gel chromatography they eluted after a cytolytic peptide A fraction, which will be discussed later. Only the sequences of the two most abundant and active isotoxins, B-II and B-IV, are known [5, 8]. Both are very basic peptides and contain a single residue of hydroxyproline at position 10. Unlike the scorpion and sea anemone peptide sodium channel neurotoxins, whose secondary structures are mostly composed of antiparallel B-strands, the B toxins are devoid of B-sheet structure but rich in alpha-helix. The secondary structure of B-IV calculated using circular dichroism measurements was similar to that predicted using Chou-Fasman and other methods [24]. Later, the secondary and tertiary structure of B-IV was determined by Norton’s lab using NMR methods [13]. It was observed that there are two long stretches of helix, represented by positions 11–23 and 34–49. The two helices are connected by a loop consisting of two inverse gamma-turns and a beta-turn. This sequence, residues 11–49, thus constitutes a rather unique helical hairpin structure [2]. Considerable data is available implicating some amino acid side chains in the toxic action of toxin B-IV. The initial studies utilized a chemical modification approach and focused on the few aromatic residues (2

tyrosyls, 2 tryptophanyls). By manipulating the conditions of the reactions, it was possible to differentially label the two Tyr and two Trp residues, the former by nitration [6] and the latter by alkylation [4]. By bioassay of the toxin samples at different degrees of modification it was deduced that Tyr9 and Trp30 are probably involved in receptor binding, since their modification did not affect secondary structure as measured by CD spectroscopy. Blumenthal’s lab has utilized molecular biological methods to express B-IV in E. coli and to obtain mutants for SAR studies [14, 15]. Initial experiments showed that replacement of the hydroxyproline at position 10 with Pro did not affect crayfish paralytic activity, nor did replacement of Ala residues at either position 3 or 8 with serine. In fact, the toxicity of B-IV was enhanced by simultaneous substitution of serine at positions 3 and 8. Arg17 has been implicated in receptor binding, as toxicity was undetectable but the CD spectrum remained unchanged when glutamine, Ala, or Lys was substituted. Replacement of Arg25 with Lys reduced crayfish toxicity 400-fold [31]. Some of the side chains implicated in toxicity of B-IV include the guanidinyl side chains of Arg 17, 25, and 34, and the aromatic side chains of Trp 30, and Tyr 9. It was rather surprising that the implicated residues are found along the entire length of one surface of the toxin. This implies that the toxin binds to an extensive portion of receptor surface. Alternately, modification of certain residues such as Tyr9 and Trp30 may have altered the folded structure sufficiently to deleteriously affect activity without affecting the CD spectrum. Experiments with other toxin mutants, coupled with more intensive NMR structural analyses of tertiary structure should provide further insights regarding the binding surface of this toxin.

Pharmacology Toxin B-IV was not toxic when injected intravenously into mice, insects, and snails. However, it was extremely toxic to crustaceans, especially crayfish [18]. Initially the crustacean displays tremors and flipping of the tail but then convulses in a massive contracture of the limbs and tail. In a few minutes the contractural paralysis is replaced by flaccid paralysis and eventually death. Toxicity of the Cerebratulus B toxins seems to be mediated through its action on the crustacean nerves. Although the limb contracture can be observed on isolated, perfused crayfish cheliped preparations, the motor nerve terminals seem to be the site of action in this preparation, as tetrodotoxin effectively blocks the action of toxin B-IV. At relatively high (μM) concentrations, toxin B-IV also causes some repetitive spiking in isolated crab walking leg nerves. It is likely that this toxin activates a small population of sodium channels, which generates

Worm Venom Peptides / 399 repetititive spiking and a massive release of excitatory glutamate neurotransmitter at the neuromuscular synapse (Kem, unpublished results). Lieberman and Blumenthal [29] measured the binding of iodinated toxin B-IV to membranes prepared from lobster (Homarus vulgaris) nerves. The specific binding could not be displaced by scorpion alpha-toxin. The radiolabeled toxin was cross-linked to the lobster membranes and SDS gel electrophoresis of the solubilized membrane proteins revealed a 40,000 dalton band, of smaller molecular size than would be predicted for the sodium channel alpha-subunit. This suggests that the toxin either binds predominantly to a smaller (B?) subunit of the sodium channel or that it interacts with some other membrane protein, perhaps another ion channel. All species of the heteronemertine genus Lineus so far examined have been found to be quite toxic [16]. These toxins are also low molecular weight (3000–6000 daltons) peptides [17]. While they paralyze crustaceans in a manner indistinguishable from the Cerebratulus toxins, the Lineus toxins primarily prolong action potential duration in crustacean neurons, whereas Cerebratulus toxin B-IV causes repetitive spiking without significantly delaying repolarization of each action potential (Kem, unpublished results).

NEMERTINE PEPTIDE CYTOLYSINS Lytic toxins are practically ubiquitous among all living organisms, including bacteria, plants, and animals. Almost all animal venoms include some lytic substances that enhance the penetration of the other toxins into the circulation of the affected organism and possibly potentiate the actions of certain toxins. So far, cytolytic proteins have only been found in the anoplan class of nemertines, which includes both heteronemertines (as described following) and paleonemertines (Kem, unpublished results).

Biochemistry Cerebratulus contains at least four homologous integumentary protein lysins (approximately 10,000 dalton molecular size) called A toxins [20, 23]. The most abundant isotoxin, A-III, was sequenced and shown to contain three disulfide bonds [7]. CD and Raman spectroscopic analyses of this toxin revealed the presence of approximately 60% alpha-helix and 10% B-sheet (Kem et al., in preparation). The C-terminal portion that is not crosslinked by a disulfide bond is thought to exist as a helical hairpin structure. Because of its amphipathic nature this portion of toxin sequence may interact with membrane lipids and be of critical importance in pore formation.

Recently homologous cytolysins have been isolated from the gigantic Antarctic heteronemertine Parbolasia corrugatus. This species feeds on dead organic materials as well as live animals, and the only known predator is a jellyfish. Despite many attempts with a variety of chromatography supports, it was not possible to resolve the very similar isotoxins from each other. Nevertheless, Edman sequencing revealed a sequence similar to that of the Cerebratulus A toxins [3]. A circular dichroism analysis also revealed a very similar secondary structure.

Pharmacology The Cerebratulus A toxins are relatively potent cytolysins on a variety of cells. Hemolysis is a useful assay system although these toxins presumably attack other sites under natural conditions, such as gill apparatus and enteral system of potential predators. Cerebratulus toxin A-III blocks squid axon sodium channels and also affects the kinetics of opening and closing of voltagegated potassium channels. It also increases the resting membrane ion permeability, which probably reflects a loss of membrane integrity [20]. A-III inhibits brain phospholipid sensitive Ca2+-dependent protein kinase in vitro, as do other peptide toxins that possess a detergent-like action [27]. In maritime Canada Cerebratulus lacteus is a nuisance in aquaculture beds of bivalve mollusks, where it is a dominant predator [10]. Although its proboscis is unarmed, it may paralyze its molluscan prey by inserting the proboscis between the two shells and secreting its peptide toxins within this enclosed space.

ANNELID (GLYCERA) NEUROTOXIN Biochemistry and Pharmacology Bloodworms belonging to the genus Glycera are active intertidal predators that attack and paralyze their prey by means of a muscular pharynx that possesses four venom-injecting fangs, each attached to its own venom gland. Only very minute amounts of the venom peptide toxins have been partially purified [9, 30]. The major neurotoxin possesses a molecular size of approximately 320,000 daltons by SDS-PAGE. This toxin causes a massive release of neurotransmitter at certain synapses. This action has been primarily studied electrophysiologically at amphibian neuromuscular synapses, as the toxin is not active at most or all mammalian neuromuscular synapses. The reason for this selectivity is that glycerotoxin targets a particular voltage-gated calcium channel, the so-called N-type, which is present at amphibian but not mammalian neuromuscular synapses [30].

400 / Chapter 57 PEPTIDE CYTOLYSINS FROM OTHER WORM PHYLA Cytolysins are probably the most widely distributed peptide toxins in nature and are produced by organisms as diverse as bacteria, plants, and animals. These toxins cause cell death and breakdown by a variety of mechanisms. One commonly observed mechanism is that of pore formation, usually by aggregation of several monomers. An additional mechanism resembles that of detergents, which disrupt lipid-lipid as well as lipidprotein interactions. In the former mechanism the cell membrane of the dead cell may be largely intact but perforated by macromolecular complexes of the lytic peptide or protein. In the latter case the cell membrane is solubilized by the interfacial activity of the detergentlike peptide or protein. Many pore-forming peptides probably can solubilize the cell membrane at even higher concentrations than are required for cell death. Two recent publications report the occurrence of cytolytic peptides in parasitic helminth worms (liver fluke belonging to phylum Platyhelminthes) and in hookworms (phylum Nematoda). These pore-forming peptides are thought to be important for the initial breakdown of the host cells on which the parasite feeds, thus facilitating digestion. The helminth peptide clonorin (molecular weight estimated as approximately 9000) possesses a sequence with six half-cystines located at essentially the same positions as in amoebapore, thus suggesting that these peptides may be homologous, even though the overall sequence similarity with amoebapore is quite low [28]. The hookworm hemolytic peptide was not completely purified but appeared to have a size of 60,000–65,000 daltons [11].

CONCLUSION It is predicted that systematic investigations of other worm phyla will reveal a plethora of peptide toxins. Many invertebrate toxins primarily target invertebrate receptors, as they have evolved over time to deal with potential invertebrate predators or prey. An understanding of their actions upon invertebrate nervous system receptors may ultimately lead the way toward the design of more selective pesticides and antiparasitic drugs. It is also expected that toxins targeting homologous receptors occurring in the mammalian nervous system will also be found. For instance, although the narrow phylogenetic activity of the Cerebratulus B neurotoxins presently limits their utility as molecular probes to crustacean nervous systems, it seems likely that natural or synthetic variants of these toxins will eventually be found that act upon homologous ion channels in vertebrates.

References [1] Bacq ZM. Les poisons des Nemertiens. Bull Acad Roy Belg Cl Sci 1936;22 (Ser. 5):1072–9. [2] Barnham KJ, Dyke TR, Kem WR, Norton RS. Structure of neurotoxin B-IV from the marine worm Cerebratulus lacteus: a helical hairpin cross-linked by disulphide bonding. J Mol Biol 1997;268:886–902. [3] Berne S, Sepcic K, Krizaj I, Kem WR, McClintock JB, Turk T. Isolation and characterization of a cytolytic protein from mucus secretions of the Antarctic heteronemertine Parborlasia corrugatus. Toxicon 2003;41:483–91. [4] Blumenthal KM. Inactivation of Cerebratulus lacteus toxin B-IV concomitant with tryptophan alkylation. Arch Biochem Biophys 1980;203:822–6. [5] Blumenthal KM, Kem WR. Primary structure of Cerebratulus lacteus toxin B-IV. J Biol Chem 1976;251:6025–9. [6] Blumenthal KM, Kem WR. Inactivation of Cerebratulus lacteus toxin B-IV by tyrosine nitration. Arch Biochem Biophys 1980;203:816–21. [7] Blumenthal KM, Kem WR. Primary structure of Cerebratulus lacteus toxin A-III. J Biol Chem 1980;255:8266–72. [8] Blumenthal KM, Keim PS, Heinrikson RL, Kem WR. Structure and action of heteronemertine polypeptide toxins. Amino acid sequence of Cerebratulus lacteus toxin B-II and revised structure of toxin B-IV. J Biol Chem 1981;256:9063–7. [9] Bon C, Saliou B, Thieffry M, Manaranche R. Partial purification of alpha-glycerotoxin, a presynaptic neurotoxin from the venom glands of the polychaete annelid Glycera convoluta. Neurochem Int 1985;7:63–75. [10] Bourque D, Miron G, Landry T. Predation on soft-shell clams (Mya arenaria) by the nermertean Cerebratulus lacteus in Atlantic Canada: Implications for control measures. Hydrobiol. 2001;456: 33–44. [11] Don TA, Jones MK, Smyth D, O’Donoghue, Hotez P, Loukas A. A pore-forming haemolysin from the hookworm, Ancylostoma caninum. Intern. J. Parasit. 2004;34:1029–35. [12] Gibson R. Nemerteans. London: Hutchinson University Library; 1972. [13] Hansen PE, Kem WR, Bieber AL, Norton RS. 1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold. Eur J Biochem 1992;210:231–40. [14] Howell ML, Blumenthal KM. Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV. J Biol Chem 1989;264:15268–73. [15] Howell ML, Blumenthal KM. Mutagenesis of Cerebratulus lacteus neurotoxin B-IV identifies NH2-terminal sequences important for biological activity. J Biol Chem 1991;266:12884–8. [16] Kem WR. A study of the occurrence of anabaseine in Paranemertes and other nemertines. Toxicon 1971;9:23–32. [17] Kem WR. Biochemistry of Nemertine Toxins. In: Martin DF and Padilla GM, editors. Marine Pharmacognosy: Marine Biotoxins as Probes of Cellular Function, New York: Academic Press; 1973, pp. 37–84. [18] Kem WR. Purification and characterization of a new family of polypeptide neurotoxins from the heteronemertine Cerebratulus lacteus (Leidy). J Biol Chem 1976;251:4184–92. [19] Kem WR. Pyridine alkaloid distribution in the hoplonemertinea. Hydrobiol 1988;156:145–53. [20] Kem WR. Structure and membrane actions of a marine worm cytolysin, Cerebratulus toxin A-III. Toxicol 1994;87:189–203. [21] Kem WR. The brain alpha7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer’s disease: Studies with DMXBA (GTS-21). Behav Brain Res 2000; 113:169– 183.

Worm Venom Peptides / 401 [22] Kem WR, Abbott BC, Coates RM. Isolation and structure of a hoplonemertine toxin. Toxicon 1971;9:15–22. [23] Kem WR, Blumenthal KM. Purification and characterization of the cytolytic Cerebratulus A toxins. J Biol Chem 1978;253:5752–7. [24] Kem WR, Tu C-K, Williams RW, Toumadje A, Johnson WC, Jr. Circular dichroism and laser Raman spectroscopic analysis of the secondary structure of Cerebratulus lacteus toxin B-IV. J Prot Chem 1990;9:433–43. [25] Kem WR, Mahnir VM, Papke R, Lingle C. Anabaseine is a potent agonist upon muscle and neuronal alpha-bungarotoxin sensitive nicotinic receptors. J Pharmacol Exper Therap 1997;283:979–92. [26] Kem WR, Mahnir VM, Prokai L, Papke RM, Cao XF, LeFrancois S, Wildeboer K, Porter-Papke J, Prokai-Tatrai K, Soti F. Hydroxy metabolites of the Alzheimer’s drug candidate DMXBA (GTS21): Their interactions with brain nicotinic receptors, and brain penetration. Mol Pharmacol 2004;65:56–67. [27] Kuo JF, Raynor RL, Mazzei GJ, Schatzman RC, Turner RS, Kem WR. Cobra polypeptide cytotoxin I and marine worm polypep-

[28]

[29]

[30]

[31]

tide cytotoxin A-IV are potent and selective inhibitors of phospholipid sensitive Ca2+-dependent protein kinase. FEBS Lett 1983;153:183–6. Lee J-Y, Cho P-Y, Kim TY, Kang S-Y, Song K-Y, Hong S-J. Hemolytic activity and developmental expression of pore-forming peptide, clonorin. Biochem Biophys. Res Commun 2002;296: 1238–44. Lieberman DL, Blumenthal KM. Structure and action of heteronemertine polypeptide toxins. Specific cross-linking of Cerebratulus lacteus toxin B-IV to lobster axon memebrane vesicles. Biochim Biophys Acta 1986;855:41–48. Meunier FA, Feng Z-P, Molgo J, Zamponi GW, Schiavo G. Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity. EMBO J 2002;21: 6733–43. Wen PH, Blumenthal KM. Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus. J Biol Chem 1996; 271:29752–8.

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58 Targets and Therapeutic Properties of Venom Peptides CHRISTINE BEETON, GEORGE A. GUTMAN, AND K. GEORGE CHANDY

Ancient Egyptians also revered scorpion goddesses. In spite of all this bad press, venomous animals have also been associated with healing, and in early Babylon the symbol of two intertwined snakes represented fertility, wisdom, and healing. Asclepius, the Greek god of medicine, carried a staff with a sacred snake coiled around it, which has become the Caduceus, the symbol of Western medical science. As in ancient times, modern medicine uses venom as a rich source of compounds with a wide range of useful pharmacological activities. In this review, we discuss recent scientific advances in the exploitation of venom, an extensive and largely untapped pharmacopoeia, for the development of therapeutics. The structures of selected compounds are shown in Supplementary Fig. 1.

ABSTRACT Venoms contain numerous peptides with a large array of biological activities. In this chapter we examine the potential therapeutic application of venom-derived peptides. Toads, snakes, and other venomous animals are wellknown ingredients of witch potions, as extolled by Shakespeare in Macbeth, Act IV, Scene 1. The three witches . . . Round about the caldron go; In the poison’d entrails throw— Toad, that under cold stone, Days and nights has thirty-one Swelter’d venom sleeping got, Boil thou first i’ the charmed pot! Double, double, toil and trouble; Fire, burn; and caldron, bubble. Fillet of a fenny snake, In the caldron boil and bake; Eye of newt, and toe of frog, Wool of bat, and tongue of dog, Adder’s fork, and blind-worm’s sting, Lizard’s leg, and howlet’s wing— For a charm of powerful trouble, Like a hell-broth boil and bubble.

ANTICOAGULANTS AND THROMBOLYTIC AGENTS Venomous creatures are an abundant source of anticoagulants, and thrombolytic agents that include disintegrins, direct thrombin inhibitors, fibrinolytic compounds, and plasminogen activators [51]. Integrilin (barbourin, eptifibatide), a cyclic heptapeptide from the venom of the Pygmy rattlesnake (Sistrurus miliarus barbouri), is a disintegrin that inhibits platelet aggregation by binding with high affinity to the fibrinogen receptor (integrin αIIbβ3) via a Lys-Gly-Asp recognition sequence. It was approved in 1998 by the U.S. Food and Drug Administration for anticoagulation in patients with acute coronary syndrome and for patients undergoing angioplasty. Other disintegrins from snake venom (Table 1, Fig. 1) use the more common ArgGly-Asp recognition sequence to interact with their target receptor. Aggrastat® (tirofiban), a mimetic of echistatin, obtained FDA approval for anticoagulant use in 1998. ViprinexTM is being evaluated as a late-

The ability of these animals to kill with tiny amounts of powerful venom has inspired both fascination and fear in humans around the world. Serpents were worshipped as deities in many ancient religions in Egypt, Greece, India, and Mesoamerica. In China, too, the snake, in the form of a dragon, is a traditional divinity. In the Garden of Eden, the snake tempted Eve into tasting the forbidden fruit, forcing her and Adam into exile in an impermanent world. The Aztec deity Quetzalcoatl was a feathered snake, and in Greek mythology, Medusa’s head of writhing snakes inspired terror. Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

404 / Chapter 58 Salmosin (disintegrin)

A

Rhodostomin (disintegrin)

B

Dendroaspin (disintegrin)

D

F

OSK1 (blocker of potassium channels)

H

Chlorotoxin (blocker of chloride channels)

J

BgK (blocker of potassium channels)

I

Crotamin (analgesic)

w -conotoxin MVIIA (blocker of calcium channels)

K

Cobrotoxin (analgesic)

Magainin-2 (antimicrobial)

GsMtx-4 (blocker of cardiac stretch-activated channels)

Melittin (antimicrobial and inhibitor of COX-2)

M

C

E

G

Echistatin (disintegrin)

L

Psalmotoxin (inhibitor of acid-sensing ion channels)

N

Exendin-4 (stimulator of insulin secretion)

O

FIGURE 1. Structures of selected peptides with potential therapeutic applications. Peptide backbones are shown as ribbon diagrams, and disulfide bonds are shown in yellow. (See color plate.)

stage therapy for the management of acute ischemic stroke. Hirudin, a polypeptide of 65 amino acids from the saliva of the medicinal leech, exemplifies antithrombin anticoagulants. Hirudin forms a biomolecular complex with thrombin via its acidic C-terminus and thereby prevents thrombin’s activity. Desirudin, a recombinant form of hirudin, is effective in the treatment of heparininduced thrombocytopenia. A number of derivatives of hirudin are available, including hirugen, a synthetic C-terminal peptide fragment of hirudin; hirulog

(bivalirudin), a derivative of hirugen; and Argatroban (heterocyclic peptidomimetic) [24]. Fibrolase from the Southern copperhead snake is a fibrinolytic enzyme that cleaves fibrin and belongs to the class of nonglycosylated metalloproteinases known as metzincins or alpha-fibrinogenases [48]. Alfimeprase, a recombinant fibrinolytic enzyme derived from fibrolase, is in phase II clinical trials for the treatment of peripheral arterial occlusions [22]. Desmoteplase, a salivary plasminogen activator from the vampire bat Desmodus rotundus (DSPA alpha 1), exhibits 85%

TABLE 1. Peptides. Name

Source

Size

Target

Use

Cardiovascular diseases Integrilin (barbourin, Snake Sistrurus eptifibatide) miliarus barbouri

73 amino acids

Disintegrin/Fibrinogen receptor (integrin αIIbβ3) antagonist

Salmosin

73 amino acids

Disintegrin

Anticoagulant in acute coronary syndrome (unstable angina and non-Q-wave myocardial infarction) and angioplasty Anticoagulant

68 amino acids

Disintegrin

Anticoagulant

Disintegrin

Dendroaspin (mambin) Crotavirin Hirudin and desirudin (recombinant hirudin) Fibrolase

Rhodostomin (kistrin) Ancrod (ViprinexTM)

Approved by the US FDA in 1998

49 amino acids

Disintegrin

Anticoagulant for acute ischemic stroke Anticoagulant

Snake Dendroaspis jamesoni kaimose Snake Crotalus viridis

59 amino acids

Disintegrin

Anticoagulant

Disintegrin

Medicinal leech

65 amino acids

Anticoagulant for infectious endocarditis Heparin-induced thrombocytopenia

Snake Agkistrodon contortrix contortrix

203 amino acids Fibrinolytic enzyme

Peripheral arterial occlusions

Desmoteplase (DSPA α1) Nonapeptide, SQ 20,881 or teprotide

Bat Desmodus rotundus Snake Bothrops jararaca jararacussa

441 amino acids plasminogen activator

Acute ischemic stroke

9 amino acids

Small molecule mimetics captopril, enalapril, and lisinopril

Margaratensin Ranatensin

Frog Frogs of the genus Rana Spider Grammostola spatulata Snake Oxyuranus microlepidotus

Hypertension, congestive heart failure, and renal syndromes such as diabetic nephropathy and scleroderma Hypertension Hypertension Suppression of atrial fibrillation

May have use in therapy of atrial fibrillation

GsMtx-4

Inhibitors of angiotensinconverting enzyme (ACE) Acts like neurotensin Acts like neurotensin

35 amino acids 35–39 amino acids

Cardiac stretch-activated ion channels Act like ANF

Congestive heart failure

Analogs hirugen, hirulog, (bivalirudin) and Argatroban Recombinant form (Alfimeprase) in phase II clinical trial Phase II clinical trials

/ 405

Natriuretic-like peptides TNP-a, TNP-b, and TNP-c

Thrombin

Aggrastat (tirofiban, mimetic of echistatin) approved by the US FDA in 1998

Targets and Therapeutic Properties of Venom Peptides

Echistatin

Snake Agkistrodon halys brevicaudus Snake Calloselasma rhodostoma Snake Calloselasma rhodostoma Snake Echis carinatus

Comments

Name

Source

Size

Target

Use

Infection Tigerinins

Frog Rana tigerina

Japonicin-2

Frog Rana japonica

11–12 amino acids 21 amino acids

Esculentin-2

Frog Rana esculenta

37 amino acids

Brevinin-1 and -2

Frog Rana brevipoda porsa and Rana esculenta Frog Rana temporaria

24 and 33 amino acids

Frog Rana palustris Frog Rana japonica Frogs Rana grylio and Rana clamitans Frogs Rana esculenta, Rana palustris, and Rana areolata

48 amino acids 14 amino acids 20 amino acids 46 amino acids

Gram-positive and -negative bacteria and Candida Albicans

Broad-spectrum antimicrobial

Frog Xenopus laevis Frogs Phyllomedusa sauvagii and Phyllomedusa bisolor Ant Myrmecia pilosula

23 amino acids 28–34 amino acids

Gram-negative bacteria Bacteria, yeast, and filamentous molds

Broad-spectrum antimicrobial Antimicrobial

27–56 amino acids

Gram-positive and -negative bacteria and fungi

Broad-spectrum antimicrobial

Scorpion Opistophtalmus carinatus Scorpion Parabuthus schlechteri Scorpion Pandinus imperator Scorpion Pandinus imperator Scorpion Hadrurus aztecus Scorpion Opisthacanthus madagascariensis Spider Lycosa carolinensis

44 amino acids

Gram-negative bacteria and fungi Gram-negative bacteria and fungi Gram-positive bacteria

Antimicrobial

Temporin L

Palustrin-3 Japonicin-1 Ranalexin Esculentin-1

Magainins Dermaseptins

Pilosulins

Opistoporin-1 Parabutoporin Pandinin-1 Pandinin-2 Hadrurin IsCT

Lycotoxins I and II

13 amino acids

45 amino acids 44 amino acids 26 amino acids 41 amino acids 13 amino acids

25 amino acids

Gram-positive and -negative bacteria Gram-positive and -negative bacteria Gram-positive and -negative bacteria Gram-positive and -negative bacteria and Candida Albicans Gram-positive and -negative bacteria and Candida Albicans Escherichia Coli Escherichia Coli Cryptosporidium parvum

Gram-positive bacteria and Candida albicans Gram-positive and -negative bacteria Gram-positive and -negative bacteria E. coli and Candida glabrata

Comments

Broad-spectrum antibacterial Broad-spectrum antibacterial Broad-spectrum antibacterial Broad-spectrum antimicrobial

Broad-spectrum antimicrobial

Antibacterial Antibacterial Antiparasite Analog [Leu28] esculentin-1 as potent as the native peptide without hemolytic activity

Analog of pilosulin-1 with increased antimicrobial activity and reduced hemolytic activity

Antimicrobial Antibacterial Antimicrobial Broad-spectrum antimicrobial Antibacterial

Antimicrobial

Analog [K7,P8,K11]-IsCT with higher potency

406 / Chapter 58

TABLE 1. (Continued)

Oxyopinins Melittin Anoplin Protonectin and Agelaia Cabrolin Cecropins

Immunomodulators Myrmexins OSK1 (α-KTx3.7)

Margatoxin Kaliotoxin Agitoxin-2

Hongotoxin Noxiustoxin Pi1

48 amino acids

Bacteria

26 amino acids

Bacteria, fungi, viruses, protozoa 10 amino acids Gram-positive and -negative bacteria 12 and 16 amino Gram-positive and acids -negative bacteria 13 amino acids Bacteria 30–35 amino Gram-positive and acids -negative bacteria

Antimicrobial Broad-spectrum antimicrobial Broad-spectrum antibacterial Antibacterial Antimicrobial Antibacterial

Bacteria Bacteria, endotoxins

Antimicrobial Antibacterial Antiendotoxin

Tunicate Halocynthia aurantium

2 subunits of 18 and 15 amino acids

Methicillin-resisitant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa

Antibacterial

Ant Pseudomyrmex triplarinus Scorpion Orthochirus scrobiculosus

62 amino acids

Unknown

Antiinflammatory

38 amino acids

Lymphocyte Kv1.3 channels

Immunosuppressant for autoimmune diseases

Scorpion Centruroides margaritatus Scorpion Androctonus mauretanicus Scorpion Leiurus quinquestriatus var. hebraeus Scorpion Centruroides limbatus Scorpion Centruroides noxius Scorpion Pandinus imperator

39 amino acids

Lymphocyte Kv1.3 channels Lymphocyte Kv1.3 channels Lymphocyte Kv1.3 channels

Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases

Lymphocyte Kv1.3 channels Lymphocyte Kv1.3 channels Lymphocyte Kv1.3 channels

Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases

38 amino acids

39 amino acids 39 amino acids 35 amino acids

Analog OSK1-K16D20 with high affinity and specificity for Kv1.3

/ 407

19 amino acids 13 amino acids

38 amino acids

Sequence similarities with frog dermaseptins

Targets and Therapeutic Properties of Venom Peptides

Drosocin Phospholipase A2-derived peptides Halocidin

Spider Oxyopes kitabensis Bees Apis florea and Apis mellifera Wasp Anoplius samariensis Wasp Agelaia pallipes pallipes Hornet Vespa crabo Silk moth Hyalophora cecropia and Aedes aegypti, and worms Ascaris suum, Ascaris lumbricoides, and Toxocara canis Drosophila Snake Bothrops asper

Name Anuroctoxin

Source

Size

Target

Scorpion Anuroctonus phaiodactylus Scorpion Scorpio maurus Scorpion Leiurus quinquestriatus var. hebraeus Sea anemone Stichodactyla helianthus Sea anemone Bunadosoma granulifera Snake

35 amino acids

Bees Apis florea and Apis mellifera Frog Rana margaratae

26 amino acids

Cyclooxygenase COX-2

Rheumatoid arthritis

14 amino acids

Tachykinin

Antiinflammatory

Scorpion Leiurus quinquestriatus

36 amino acids

Chloride channels on glioma cells

Anticancerous

Bombesin and analogs

Frogs of the Bombina genus

14 amino acids

Bombesin receptors on tumor cells

Anticancerous

Contortrostatin

Snake Agkistrodon contortrix

Adhesion molecules

Prevention of metastasis

Jerdonin

Snake Trimeresurus jerdonii Mollusk Elysia rufescens

2 subunits of 65 amino acids 71 amino acids

Maurotoxin Charybdotoxin

ShK

BgK

Cobratoxin-derivative CAM-NTX Melittin Ranamargarin Oncology Chlorotoxin

Kahalalides

Analgesia ω-conopeptide MVIIA

Marine cone snail Conus magus

38 amino acids

Kv1.3 and KCa3.1 channels

Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases Immunosuppressant for autoimmune diseases

35 amino acids

Lymphocyte Kv1.3 channels

Immunosuppressant for autoimmune diseases

37 amino acids

Lymphocyte Kv1.3 channels

Immunosuppressant for autoimmune diseases

34 amino acids

Lymphocyte Kv1.3 channels KCa3.1 channel

Use

Comments

Analog ShK(L5) with higher selectivity

Prevented a guinea pig model of multiple sclerosis

I-TM-601 in clinical trials of targeted radiotherapy Directed killing when conjugated to camptothecin

Anticancerous

Up to 13 amino acids

25 amino acids

131

Prostate cancer

N-type voltage-sensitive calcium channels in mammalian painsensing neurons

Antinociceptive for severe chronic pain

Synthetic Ziconotide (Prialt®) approved by the US FDA in 2004

408 / Chapter 58

TABLE 1. (Continued)

Scorpion Buthus martensis Karsh

31 amino acids

Frog Hyla caerula

16 amino acids

Ceruletide

Frog Litoria citropa

Crotamine

Snake Crotalus durissus terrificus Snake Ophiophagus hannah Snake Naja naja atra

Hannalgesin (Oh9-1) Cobrotoxin

Psalmotoxin

Liver disease ?

Diabetes mellitus Exendin-3 and -4

Frog skin insulinotropic peptide (FSIP)

72 amino acids

Neural diseases such as apoplexy, epilepsy, paralysis, and analgesia Antinociceptive, sedation, inhibition of water intake, anticonvulsive Antinociceptive, sedation, inhibition of water intake, anticonvulsive Analgesic, 30-fold more potent than morphine Analgesic

235 amino acids

Analgesic

C-terminus similar to cholecystokinin

42 amino acids

Spider Psalmopoeus Cambridgei

40 amino acids

Snake Agkistrodon halys pallas

?

Increased bile flow, improved hepatic microcirculation, altered expression of bile salt transporter, fibrinolytic, and antithrombotic

Lizard Heloderma suspectus

39 amino acids

Type-2 diabetes

Frog Agalychnis litodryas

Acid-sensing ion channel

Caerulin analog

Synthetic analog not beneficial in patients with adrenomyeloneuropathy

Acid-induced pain

Type-2 diabetes

Synthetic exenatide approved by the U.S. FDA in 2005

Targets and Therapeutic Properties of Venom Peptides

BmK dIT-AP, BmK dIT-AP3, and BmK AS1 Caerulein

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410 / Chapter 58 sequence similarity with human tissue plasminogen activator. It is in phase II clinical trials for use in acute ischemic stroke [45, 47].

ANTIHYPERTENSIVE AGENTS The Brazilian arrowhead viper (Bothrops jararaca) produces peptide inhibitors (e.g., nonapeptide SQ 20,881 or teprotide) of angiotensin-converting enzyme (ACE) that generates angiotensin II, a hormone that causes vasoconstriction and increased blood pressure. These peptide inhibitors bind to the active site of ACE in the same manner as natural substrates and reduce blood pressure [11]. Captopril, enalapril, and lisinopril are peptidomimetics used widely to treat hypertension, congestive heart failure, and diabetic nephropathy. Neurotensin-like peptides identified from frog skin (e.g., margaratensin and ranatensin) are hypotensive in rats and have the potential to be developed into novel therapeutics.

ANTIARRHYTHMIC AGENTS Venom from the South American tarantula (Grammostola spatulata) contains GsMtx-4, a small peptide belonging to the “cysteine-knot” family that blocks cardiac stretch-activated ion channels and suppresses atrial fibrillation in rabbits [4]. Efforts are under way to develop therapeutics for atrial fibrillation based on GsMtx-4.

CONGESTIVE HEART FAILURE Natriuretic peptides are used in the management of congestive heart failure. Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) have been isolated from the venom of the Inland Taipan snake (Oxyuranus microlepidotus), and TNP-c is equipotent to atrial natriuretic peptide and may have use in the treatment of congestive heart failure [12].

ANTIMICROBIAL PEPTIDES The increasing resistance of pathogens to antibiotics is a major health concern. Venom from frogs of the genus Rana are a rich source of peptides (tigerinin, japonicin-2, esculentin-2, brevinin-1, brevinin-2, temporin L) that target both gram-positive and gramnegative bacteria and even pathogenic fungi such as Candida albicans. These peptides have mild to potent hemolytic activities that preclude their use in humans

[8]. An analog of esculentin-1 ([Leu28] esculentin-1) lacks hemolytic activity and is very potent against S. aureus, P. aeruginosa, E. coli, and C. albicans. Broadspectrum antibacterial and antifungal helical amphiphilic peptides called Magainins have been isolated from the skin of the African clawed frog (Xenopus laevis) [54]. These 23-amino-acid peptides are nonhemolytic at their effective antimicrobial concentration and improve survival in a rat model of gram-negative pathogen septic shock. MSI-78 (pexiganin acetate) did not receive FDA approval [28]. Dermaseptins (lysine-rich peptides 28–34 amino acids) from the skin of the South American clawed frogs Phyllomedusa sauvagii and Phyllomedusa bicolor are active against bacteria, yeast, and filamentous molds [1, 37, 38]. Analogs improve survival in a peritonitis model in mice infected with Pseudomonas aeruginosa and prevent staphylococcal infections in a rat skin-graft model. The jumper ant Myrmecia pilosula produces broad-spectrum antibacterial and antifungal peptides called pilosulins and modification of pilosulin-1 increased antimicrobial activity while reducing hemolytic activity [17, 55]. South African scorpions Opistophtalmus carinatus and Parabuthus schlechteri each produce an antimicrobial peptide, opistoporin-1 and parabutoporin, respectively [35]. Both potently inhibit growth of gram-negative bacteria and fungi. They exhibit little or no hemolytic activity and are thus of potential use as new antimicrobial drugs. Antimicrobial peptides called pandinin-1 and pandinin2 have been isolated from the African scorpion Pandinus imperator and show activity against gram-positive bacteria [9]. Hadrurin, an antimicrobial peptide from the Mexican scorpion Hadrurus aztecus, displays broad antimicrobial activity [50] and may serve as templates for the development of novel therapeutics. Peptide lycotoxins I and II with activity against E. coli and Candida glabrata have been identified from the venom of the wolf spider (Lycosa carolinensis) [52]. Antimicrobial peptides called oxyopinins (48-amino-acid residues) were isolated from the another species of wolf spider called Oxyopes kitabensis and share sequence similarity with the frog dermaseptins [10]. A 10-residue antimicrobial peptide called Anoplin from the venom of the solitary wasp Anoplius samariensis [16, 25] exhibits broad-spectrum antimicrobial activity. Protonectin and Agelaia, two peptides from the neotropical social wasp Agelaia pallipes pallipes, also exhibit potent antimicrobial activity [33], while Crabrolin, a 13-residue peptide present in the venom of the hornet Vespa crabro, has antibacterial activity but is hemolytic [26]. Melittin, a 26-residue peptide from bee venom, also has antibacterial, antifungal, antiviral, and antiprotozoal properties [8]. Antimicrobial peptides called cecropins (30–35 amino acids) have been isolated from the silk moth

Targets and Therapeutic Properties of Venom Peptides [15], mosquitos (Aedes aegypti), worms (Ascaris suum, Ascaris lumbricoides, Toxocara canis), and silkworms [5, 43]. Shiva-11, a synthetic cecropin derivative, is effective against ocular pathogens. Antimicrobial peptides called attacins have been identified in the moth [15]. Other antimicrobial peptides from insects include drosocin, diptericin, MPAC, drosomycin, and metchnikowin. Peptides derived from snake venom phospholipase A2 are broad-spectrum bactericidal agents and also antiendotoxic compounds [46]. Halocidin, a heterodimer antimicrobial peptide from the sea squirt Halocynthia aurantium, is effective against methicillin resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa [18, 19].

IMMUNOMODULATORY PEPTIDES Venom-derived peptides from a number of sources are being evaluated as immunosuppressants for the treatment of autoimmune diseases and the prevention of graft rejection. Venom from the tropical ant Pseudomyrmex triplarinus contains heterodimeric peptides called myrmexins that relieve pain and inflammation in patients with rheumatoid arthritis and inhibit inflammatory carragenin-induced edema in mice, but their mode of action still remains to be determined [40, 58]. The venom of scorpions and sea anemone have been a rich source of peptides that block the Kv1.3 and KCa3.1 channel in human T cells that are therapeutic targets for autoimmune diseases. The most potent Kv1.3 inhibitors include OSK1, ShK, margatoxin, kaliotoxin, agitoxin-2, hongotoxin, noxiustoxin, HsK1, Pi1, and Anuroctoxin [3, 6]. Kaliotoxin inhibited immune responses in rats mediated by terminally differentiated effector memory T cells including delayed type hypersensitivity, adoptive experimental autoimmune encephalomyelitis (a model for multiple sclerosis), and bone resorption in experimental periodontal disease [3]. Margatoxin was effective in inhibiting the delayed type hypersensitivity response in minipigs [3]. ShK(L5), an analog of ShK, suppression is effective in vivo in preventing and treating disease in an animal model of multiple sclerosis and exhibits a safety index of 100 in rodents [3]. Maurotoxin is the most potent inhibitor of the KCa3.1 channel [6]. A derivative of cobratoxin from the Thailand cobra called CAM-NTX prevents disease in a guinea pig model of multiple sclerosis by reducing lymphocytic infiltration into the brain [13]. Apitherapy (the use of bee products) is used in patients with arthritis. Bee venom is often injected at a traditional acupuncture point (Zusanli), and its analgesic and anti-inflammatory actions have been demonstrated both in animal models and human trials of

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autoimmune arthritis [29, 41, 44]. The antiarthritic effects of bee venom are at least in part mediated by melittin through the inhibition of cyclooxygenase COX2 and of the production of superoxide by neutrophils, both major players in the maintenance and amplification of inflammation in the joints during rheumatoid arthritis [41, 58]. However, mellitin also exhibits hemolytic and cytotoxic activities against mammalian cells and would have to be modified before it can be safely developed into a drug. Bombolitins from the bumblebee (Megabombus pennsylvanicus) degranulate mast cells and induce the release of histamine and other bioactive compounds and may have therapeutic use [2]. Tachykinins and their receptors are recognized as therapeutic targets for inflammatory diseases. Ranamargarin is a 14-residue tachykinin isolated from frog skin and could serve as a template for a novel antiinflammatory [49].

ANTITUMOR PEPTIDES Venomous creatures are an abundant source of antiproliferative peptides, some of which are in clinical trials as chemotherapeutic agents for cancer therapy. Chlorotoxin, a 36-amino-acid basic peptide from the venom of the scorpion Leiurus quinquestriatus, blocks chloride channels expressed on glioma cells but not by normal glial cells [30]. 131I-labeled chlorotoxin (131I-TM601) has been effective in targeted radiotherapy of gliomas in mice and is being evaluated in clinical trials as a therapeutic for gliomas. Bombesin, a tetradecapeptide neurohormone first isolated from skin of firebellied toad (Bombina bombina), binds to Bombesin receptors that are frequently expressed at high levels by a variety of tumors (breast, colon, lung, and prostate). Bombesin conjugated to camptothecin, a cytotoxic compound, has been used to selectively kill human lung cancer cells in vitro [36]. Disintegrins from snake venom may have therapeutic use in suppressing tumor growth by inhibiting angiogenesis [31, 53]. Contortrostatin from the Broad Banded copperhead snake (Agkistrodon contortrix) is a homodimeric disintegrin, each subunit having a molecular mass of 6750. Contortrostatin has been reported to inhibit β1-integrin-mediated human melanoma cell adhesion and block experimental metastasis of these tumors and breast cancers. Jerdonin, another disintegrin from the venom of the Oriental pit viper (Trimeresurus jerdonii), improves the survival time of B16 tumor-bearing mice [57]. Kahalalides are peptides isolated from the Hawaiian mollusk (Elysia rufescens) [20, 21]. Seven of these are cyclic depsipeptides (kahalalides A–F and O), and three are linear (kahalalides G, H, and J) [20, 21]. Kahalide F is in phase I trials in patients with advanced androgen

412 / Chapter 58 refractory prostate cancer [20, 21]. Cyclic depsipeptides called didemnins isolated from the Caribbean tunicate Trididemnum solidum [14] are in clinical trials for the treatment of solid tumors and lymphomas [27]. Dolastatin 10, an antimitotic peptide originally identified in the sea hare and recently shown to derive from cyanobacterial metabolism, is in phase II clinical trials as an anticancer therapeutic [42].

ANALGESIA Marine cone snails use neurotoxins to paralyze their preys. One of these toxins, ω-conopeptide MVIIA from Conus magus and its synthetic equivalent ziconotide, blocks N-type voltage-sensitive calcium channels in mammalian pain-sensing neurons and is therefore potently anti-nociceptive in conditions where morphine is poorly or not active [34]. Ziconotide (Prialt®) was approved for the treatment of severe chronic pain by intrathecal infusion by the U.S. Food and Drug Administration in 2004 [44]. Extracts from the scorpion Buthus martensis Karsh have long been used in China to treat neurological diseases such as apoplexy, epilepsy, and paralysis [44]. These effects have been partly attributed to the peptides BmK dIT-AP, BmK dIT-AP3, and BmK AS1 that exhibit analgesic effects in mice and rats [44]. These are short 31-amino-acid peptides held together by three disulfide bonds, but their tertiary fold differs from that of other scorpion toxins. Caerulein, a decapeptide from the skin of the Tree frog Hyla caerula [56], shares sequence similarity to the C-terminal octapeptide of cholecystokinin. Peptides related to Caerulein have been isolated from the skin of the Australian Blue Mountain tree frog Litoria citropa [56]. Caerulin and its analogs exhibit antinociceptive activity and cause sedation, inhibition of water intake, and anticonvulsive effects [56]. Ceruletide, an analog of caerulin, is effective as an analgesic in biliary colic. Snake toxins have antinociceptive effects in rodents [44]. Crotamine (molecular mass ∼5 kDa) derived from the venom of the Northeastern Brazilian rattlesnake (Crotalus durissus terrificus) is about 30-fold more potent than morphine as an analgesic [44]. Hannalgesin (a.k.a. Oh9-1) from the venom of the King cobra (Ophiophagus hannah) and Cobrotoxin from the venom of the Chinese cobra (Naja naja atra) are potent analgesics that do not cause neurological or muscular deficits. However, in a clinical trial, a modified version of cobrotoxin with oral bioavailability did not produce significant pain relief to patients with Adrenomyeloneuropathy [39]. Psalmotoxin, a 40-amino-acid peptide from the South American tarantula (Psalmopoeus Cambridgei), inhibits the acid-sensing ion channel and may have use in reducing acid-induced pain [7].

DIABETES MELLITUS Diabetes mellitus, a disease in which the body is unable to sufficiently produce or properly use insulin, wreaks a deadly toll on the populace. It is the seventh leading cause of death in the United States, the leading cause of blindness, the leading cause of end-stage kidney disease, and causes nerve damage in about 60–70% of patients. Newer therapeutic modalities for this devastating disease are urgently needed. The venom of the gila monster (Heloderma suspectus) holds the key to the cure of type-2 diabetes [23]. It produces exendin-3 and exendin-4, two peptides that stimulate insulin secretion in response to increases in glycemia and modulate gastric emptying to slow the entry of ingested sugars in the blood. Exendin-4 has been developed into a drug, Exenatide, approved for the treatment of type-2 diabetes by the U.S. Food and Drug Administration in 2005. Peptides with insulin-releasing activity have been isolated from the skin secretions of the frog Agalychnis litodryas (e.g., FSIP) and may serve as templates for a novel class of insulin secretagogues [32]. In conclusion, venom from insects, snakes, arachnids, amphibians, marine cones, and sea anemones are widely recognized sources of peptides with potential medical applications; several have already proven effective in curing several human diseases and many more are on the way to be developed into drugs. Most certainly there are useful hidden venoms waiting to be discovered.

Acknowledgments We thank Professor James Hall for critiquing this manuscript. We also acknowledge grants from the NIH (NS048252), American Heart Association, and the American Diabetes Association that have allowed us to prepare this review.

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59 Structure-Function Strategies to Improve the Pharmacological Value of Animal Toxins MICHEL DE WAARD AND JEAN-MARC SABATIER

production. Also, the high yields of peptide productions are assets for detailed structural analyses and functional characterizations of the peptides. Finally, the production of peptides for therapeutic purposes is less cumbersome when a chemical approach is chosen over a genetic one. Indeed, safety, reproducibility, and lack of cell contaminants clearly argue in favor of chemical synthesis. In fact, the vast majority of studies relating structure-function analyses are based on peptides produced by chemical synthesis.

ABSTRACT Animal venoms are rich sources of bioactive compounds that possess obvious pharmacological, therapeutic, and/or biotechnological values. A majority of these compounds are peptides that mainly target enzymes, membrane receptors, or ion channels. These peptides are most often in a size range that allows their production in vitro by chemical synthesis or genetic engineering. Unfortunately, they rarely display the required characteristics in terms of selectivity, affinity, stability, and targeting with regard to the desired application. In recent years, a number of structural approaches or strategies have been developed to improve the intrinsic potential of venom peptides. They are reviewed herein for their effectiveness.

PEPTIDE IMPROVEMENT For valuable strategies of peptide improvement, one needs to know the peptide target(s), 3-D structure, functional effect(s), and specific application(s) pursued. With uncharacterized venom peptides, identification of actual target(s) is generally a difficult task. Pharmacological profiling using well-defined screening approaches often reveals that peptides recognize several targets (e.g., different ion channel types) or may be derived for multiple applications. From pharmacological studies, useful information can be gained that includes peptide affinity for its target(s), selectivity profile, and mode of action. Gathering structural data is an essential step in defining the interacting surfaces of both the peptide and its target(s). In most cases, venom peptides are readily amenable to 1H-NMR-based 3D structure determination. Alternatively, computer-assisted molecular modeling is an interesting route that can be used for peptide structural analysis provided that appropriate 3D structures of related compounds are available from databases to serve as templates. The accurate structural determination of the peptide’s target(s) or, at least, of its binding site(s) is a considerably more difficult challenge. Although this condition is not always met satisfactorily, it is clearly beneficial for the design of novel

CHEMICAL SYNTHESIS VERSUS GENETIC ENGINEERING In animal venoms, peptide sizes are mainly in the range of 0.4 to 8.0 kDa. This would correspond to peptides of approximately 4 to 70 amino acid residues. Because of the intrinsic structural complexities of these peptides (different types of folds and 1–5 disulfide bridges), this size range implicates that only a fraction of them (<50-mer peptides) can easily be produced by chemical synthesis [17, 22]. Longer peptides are more difficult to produce chemically and have generally required the use of molecular biology techniques for their productions. Basically, chemical synthesis has several advantages over genetic engineering, making the former approach more appropriate to improve the structural and functional properties of peptides of interest. In particular, the possibility to incorporate nonnatural amino acid derivatives or to form nonamide pseudo-peptide bonds is a definite benefit over genetic Handbook of Biologically Active Peptides

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416 / Chapter 59 and more potent peptide analogs. Structural information, from both peptide and target, allow more sophisticated analyses that include in silico molecular docking simulations. From the most energetically favorable docking solutions, relevant hypotheses can be drawn on the characteristics of the interacting surfaces involved. These can then be verified experimentally by using complementary mutagenesis of the peptide and its target(s), leading to a precise view of the peptide’s structural and functional properties [11].

substitutions based on natural amino acid residues only. Owing to the conformational change of the peptide that is potentially induced by residue substitution, it is wise to gain insight of the structural features of each analog designed, produced, and tested for bioactivity (e.g., 3D solution structure determination by 1H-NMR, circular dichroism analysis, molecular modeling). Combined with docking simulation experiments of these analogs over their targets, all the data gathered should be of value for a still more rational design of novel more potent and/or selective peptide analogs by an iterative feedback information process.

DESIGN OF NOVEL PEPTIDE ANALOGS The design of novel peptide analogs by selective residue substitution(s) is largely employed to obtain novel analogs with unique pharmacological properties or to pursue detailed structure-function studies. It represents the method of choice to address whether certain amino acid residues from the peptide are involved in target recognition and binding. It also allows a precise mapping of the peptide interacting surface, provided that the substituted residues do not alter the global peptide conformation. Generally, residue substitutions are made on a systematic basis, using the alaninescanning approach. By this method, the contribution of scorpion toxin functional dyads to K+ channel blockage was experimentally investigated [4, 18]. Mutation of amino acid residues also represents a powerful strategy to improve the pharmacological value and therapeutic potential of a particular peptide. Two general strategies can be envisioned. First is substituting amino acid residues within the peptide active site. This strategy can be used to increase the affinity of the peptide toward one specific target or for improving its selectivity profile (similar or increased affinity for one target but decreased affinity toward one or several other targets) [23] or both [1]. Second is substituting amino acid residues outside the peptide active site. This strategy may generate more subtle variations in peptide pharmacological properties, although in a more unpredictable manner. Indeed, it is expected that amino acid residue substitutions outside the active site should slightly alter the peptide’s interacting surface. Stronger conformational changes are expected from substitutions that target residues key for a proper peptide folding. For instance, it has been shown that single residue mutations in scorpion maurotoxin can affect its pattern of disulfide bridging and, hence, its conformation and pharmacology [9]. Apart from acting on peptide affinity and selectivity by substituting natural amino acid residues by others, one can also act on peptide stability (e.g., sensitivity to proteases) by using nonnatural residues for the substitutions instead. It is worth mentioning, however, that a similar protease resistance can also be acquired by selective

BENEFITS OF SIZE REDUCTION IN PEPTIDES Since many large peptides (>50-mer) are not readily amenable to chemical production, it may appear attractive to reduce their size without compromising on their pharmacological value. Unfortunately, this strategy has seldom been used, and too little information is available from the literature to reasonably draw a conclusion about their effectiveness. However, some remarks may be pertinent. Size reduction of peptides may be used for delineation of their pharmacophore(s). Similarly, it may prove useful for restricting the peptide selectivity profiles. This approach would certainly be beneficial in terms of production costs and should bypass the complex use of a genetic production strategy. In the case of venom peptides, one would expect that such a strategy should severely decrease the pharmacological potencies of the resulting low-mass analogs, since the integrity of the various toxin folds characterized so far, that would presumably be affected by peptide size reduction, is required for the correct spatial distribution of functionally key amino acid residues [17]. However, this strategy might be particularly valuable for peptides presenting small pharmacophores and thus less susceptible to alteration of the active site conformation by peptide size trimming.

RELEVANCE OF CHIMERA AND LABELING APPROACHES Structure-activity relationship studies have demonstrated that a given venom peptide can be active on several targets (large selectivity profile) through different interacting surfaces. The existence of a variety of folds in venom peptides [17] allows the occurrence of distinct relative localizations of the active interacting surfaces [21]. For instance, it has been documented that scorpion toxins generally fold according to the α/β architectural motif (an N-terminal helical structure

Structure-Function Strategies to Improve the Pharmacological Value of Animal Toxins / 417 connected by disulfide bridges to a C-terminal antiparallel, two- or three-stranded, β-sheet structure) [20]. Many of these scorpion venom peptides act on voltagegated K+ (Kv) channels through interacting surfaces primarily involving their β-sheet structures, whereas they may interact with small conductance Ca2+-activated K+ (SK) channels through opposite interacting surfaces that implicate their helical structures. Given the fact that interacting surfaces of some venom peptides (with enlarged target specificity) are associated with residues of specific secondary structures, it may be worth using a chimera approach to produce peptide analogs with either enlarged or restricted pharmacological profiles. A representative example of such an approach was provided with a chimera made by the N-terminal helical region of maurotoxin and the C-terminal β-sheet region of HsTx1 [19]. In this case, a “gain of function” was obtained, since the chimera acquires the activity of maurotoxin on SK channels while preserving the pharmacological profile of HsTx1 on Kv channels. Conversely, a “loss of function” can be attributed to this chimera, since the activity of maurotoxin on a specific Kv channel subtype is lost. The cited approaches relate to change in peptide selectivity (enlargement or restriction). Of note, the chimera approach can tentatively be used to modulate peptide affinity toward a particular target without altering the initial pharmacological profile. This was successfully experienced using a chimera of butantoxin and maurotoxin in which the addition of the 9 N-terminal residues of butantoxin to the 31 C-terminal residues of maurotoxin provides some additional molecular contacts for the interaction with Kv1.2 channel (pharmacophore enlargement), thereby enhancing peptide affinity for this channel subtype [15]. Other technical applications are worth mentioning. First, peptides can be modified for tracking purposes. Tracking can be useful for the determination of peptide tissue distribution, subcellular receptor localization, or classical binding experiments. For example, such an “add-on” function has proven valuable for demonstrating the cell-penetration ability of maurocalcine, a venom peptide acting on intracellular ryanodinesensitive Ca2+ channels [7]. Site-directed chemical modifications of peptides, by labeling with fluorescent derivatives or radioactive materials (e.g., 125I), or biotinylation, are routinely used for binding experiments. Second, venom peptides can be derived to become multifunctional. Chemical coupling to other molecules, such as antibodies or independent functional domains, may represent an interesting strategy for novel types of “gain of function,” including cell targeting, cell toxicity, cell labeling, cell delivery of compounds, and so on. Unfortunately, these research avenues are still largely unexploited and their add-on values underevaluated.

STRATEGIES IMPLYING A CHANGE IN THE PATTERN OF HALF-CYSTINE PAIRS Venom peptides are often highly reticulated structures. It has been evidenced that disulfide bridges contribute to the acquisition and stability of the various peptide folds. Secondary structures are frequently connected to each other by at least one disulfide bridge. The pattern of a half-cystine connection is also crucial for the correct spatial distribution of the functionally key amino acid residues from the peptide interacting surfaces. Strategies based on the pattern of half-cystine pairs can be classified into three categories. The first one aims at increasing the number of disulfide bridges. The extra half-cystine pair might decrease peptide flexibility and, possibly, that of the interacting surfaces, provided that the additional disulfide bridge is inserted in an appropriate location within the peptide amino acid sequence. In turn, a reduced flexibility may lessen peptide adaptability to their target(s), and one may expect a greater pharmacological selectivity for the analogs designed this way. However, an experimental validation of this approach is still needed. In contrast, the second strategy relies on a reduction in the number of peptide half-cystine pairs instead; a strategy that may conceivably increase peptide flexibility, thereby potentially affecting its pharmacological behavior. In both cases, adding or removing a disulfide bridge may have consequences for the pattern of other half-cystine pairs, therefore highlighting the need for a careful structural characterization of the analogs. The third strategy relies on a change in the pattern of disulfide bridges within the peptide (with or without variation in the number of half-cystine pairs). Enforcing new disulfide bridge arrangements in a peptide is an effective way to alter the conformation of its pharmacophore(s) and, hence, its pharmacology. This strategy has been applied twice for the scorpion maurotoxin. By replacing two halfcystine residues belonging to two different disulfide bridges, a novel three-disulfide-bridged maurotoxin analog was produced in which the third half-cystine pair was unique. This analog still folded along the common α/β architectural motif of scorpion toxins while presenting a new selectivity toward Kv channels [8]. Also, by using an innovative strategy of chemical synthesis, a four-disulfide-bridged maurotoxin variant was produced in which the two last bridges were differently arranged as compared with native maurotoxin (although halfcystine pairings are similar to those found in other toxins from the same structural family). Again, for this analog, the common architectural motif was maintained, but the change in disulfide bridge pattern was accompanied by changes in pharmacology [16]. It is worth mentioning that removal of all half-cystine pairs from a reticulated venom peptide—in the cases

418 / Chapter 59 where it has been studied—abolishes peptide bioactivity, suggesting that the presence of all disulfide bridges are required for the maintenance of peptide fold. This property has been taken into consideration for peptidebased immunizations during vaccination programs, with the aim of neutralizing the toxicity of properly folded and reticulated toxins [10, 24].

WHAT ABOUT THE DIPOLE MOMENT OF VENOM PEPTIDES? Many venom peptides exhibit a marked electric charge anisotropy. A formal representation of this anisotropy is given by the peptide dipole moment. It has been proposed that the dipole moment guides and orients the peptide towards its target(s), thereby being key for peptide specificity [3]. Although the concept is attractive by itself, it remains to be supported experimentally. In fact, the main flaw of this concept is that it is somehow incompatible with the existence of several interacting surfaces, which are sometimes located on opposite faces of the peptide (e.g., activity of maurotoxin on both SK and Kv channels involving opposite faces). Of note is the noticeable charge anisotropy of venom peptide maurocalcine, which possesses a highly basic face and is likely to contribute to its singular ability to translocate into cells where the peptide acts [7]. This property is shared with other cell-penetrating, structurally unrelated, peptides such as Tat and penetratin [13].

FUNCTIONAL DERIVATION OF VENOM PEPTIDES Venom peptides exhibit a great variety of folds. In the case of animal toxins acting on ion channels, up to 14 different types of folds were identified [17]. Along with other favorable characteristics, such as small size and high stability, diversity of fold can advantageously be used for a functional derivation of venom peptides. Indeed, active sites of venom peptides can be substituted by functionally unrelated pharmacophores of biological value to benefit of the gainful peptide characteristics. For example, the CD4 binding surface for the HIV-1 gp120 envelope glycoprotein has been successfully transferred into the structure of a scorpion toxin possessing the α/β scaffold [12]. This approach leads to the production of interesting anti-HIV compounds with demonstrated efficacies in vitro. Venom peptides with novel pharmacophores represent new tools for biological, biotechnological, and medical applications. A therapeutic development may also be envisioned in case the novel compounds possess the appropriate drug properties.

Other types of functional derivation can be achieved that are not necessarily based on the replacement of the peptide pharmacophore by an exogenous one. In that case, different properties of the peptide are derived, such as cell targeting, receptor recognition, membrane translocation, nucleic acid binding, and so on. It is worth noting that, in some cases, it is mandatory to eliminate the original pharmacological activity of the peptide to prevent some unwanted side effects. For instance, the pharmacological activity of maurocalcine on intracellular ryanodine-sensitive calcium channels can be suppressed [6] in order to use only its cellpenetration property for the cell-entry of larger nonmembrane permeable compounds, including whole proteins [7]. In other cases, structures of venom peptides can be modified to properly target toxins to a given cell type in vivo. Such a strategy has been developed in the case of immunotoxins, in which a toxin is coupled to a cell-targeting ligand or antibody. The main focus of the immunotoxin approach is targeted cancer therapy [14]. So far, bacterial and plant toxins have been used, but the technique could prove valuable with animal toxins.

IMPROVING PEPTIDE STABILITY Since the ever-increasing interest in studying animal venom peptides is related to their potential use in therapeutics, it appears suitable to optimize their stability in vivo. Several methods can be used to improve peptide stability. Among them, one can mention (1) amino acid residue substitution(s) aimed at protecting the peptide from proteolytic cleavage. Residue replacement(s), which should not alter the pharmacophore properties, can be achieved using L-, D-, or nonnatural amino acid residues (including residues modified on their sidechains). (2) A selective chemical modification of the peptide (e.g., changing the nature of the N-(acetylation) [5] and/or C-terminal extremities (amidation, addition of a specific group such as a fatty acid). Such a strategy may also be valuable for improving the pharmacological characteristics of a given peptide, as shown in the case of ShK sea anemone toxin in which grafting of a phosphotyrosine moiety at the N-terminus enhances its selectivity profile towards the Kv1.3 potassium channel [2]. (3) The replacement of natural peptide bonds by pseudo-peptide bonds during chemical synthesis (e.g., CO—NH by NH—CO, or CH2—CH2, or CO—CH2, etc). (4) Cysteine-based cyclization of the peptide. These modifications, which might by themselves be detrimental to peptide biological properties, should be attempted only to overcome specific problems, such as short peptide half-life, poor peptide stability and/or solubility, and inadequate tissue distribution.

Structure-Function Strategies to Improve the Pharmacological Value of Animal Toxins / 419 Of note, although peptide modifications have been used in the development of several therapeutically useful peptides, it has seldom been put into application for venom peptides. Apart from chemical modifications of the peptides, it is also possible to act on peptide stability by using specific delivery systems (liposomes, nanoparticles, glycan polymers, hydrogels). The latter do not belong to structure-function strategies and thus will not be developed further.

CONCLUSION Despite the richness in biologically active venom peptides, they rarely come with the desired set of characteristics. A rationale structure-function strategy is therefore a key step to obtain a valuable compound for any given application. Strategies are now numerous and have proven to be efficient to reach these goals. The choice of a particular strategy actually depends on the peptide biological properties that one seeks to reinforce, create, or suppress. Not all strategies are equivalent, and sometimes a combination thereof should be used. An iterative feedback information process is often required, which combines the benefits of several approaches, to generate compounds with the appropriate set of requisite characteristics. Combined strategies will prove valuable for the future development of venom peptide-derived drugs in the treatment of specific human diseases.

References [1] Alessandri-Haber N, Lecoq A, Gasparini S, Grangier-Macmath G, Jacquet G, Harvey AL, et al. Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity. J Biol Chem 1999;274:35653–61. [2] Beeton C, Pennington MW, Wulff H, Singh S, Nugent D, Crossley G, et al. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Mol Pharmacol 2005;67:1369–81. [3] Blanc E, Sabatier JM, Kharrat R, Meunier S, El Ayeb M, Van Rietschoten J, et al. Solution structure of maurotoxin, a scorpion toxin from scorpio maurus, with high affinity for voltagegated potassium channels. Proteins 1997;29:321–33. [4] Dauplais M, Lecoq A, Song J, Cotton J, Jamin N, Gilquin B, et al. On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J Biol Chem 1997;272:4302– 9. [5] de Haan EC, Wauben MH, Wagenaar-Hilbers JP, GrosfeldStulemeyer MC, Rijkers DT, Moret EE, et al. Stabilization of peptide guinea pig myelin basic protein 72-85 by N-terminal acetylation—Implications for immunological studies. Mol Immunol 2004;40:943–8. [6] Estève E, Smida-Rezgui S, Sarkozi S, Szegedi C, Regaya I, Chen L, et al. Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of maurocalcine in skeletal muscle cells. J Biol Chem 2003;278:37822–31.

[7] Estève E, Mabrouk K, Dupuis A, Smida-Rezgui S, Altafaj X, Grunwald D, et al. Transduction of the scorpion toxin maurocalcine into cells—Evidence that the toxin crosses the plasma membrane. J Biol Chem 2005;280:12833–9. [8] Fajloun Z, Ferrat G, Carlier E, Fathallah M, Lecomte C, Sandoz G, et al. Synthesis, 1H NMR structure, and activity of a threedisulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins. J Biol Chem 2000;275:13605– 12. [9] Fajloun Z, Mosbah A, Carlier E, Mansuelle P, Sandoz G, Fathallah M, et al. Maurotoxin Versus Pi1/HsTx1 toxins: Toward new insights in the understanding of their distinct disulfide bridge patterns. J Biol Chem 2000;275:39394–402. [10] Gazarian KG, Gazarian T, Hernández R, Possani LD. Immunology of scorpion toxins and perspectives for generation of antivenom vaccines. Vaccine 2005;23:3357–68. [11] Gilquin B, Racape J, Wrisch A, Visan V, Lecoq A, Grissmer S, et al. Structure of the BgK-Kv1.1 complex based on distance restraints identified by double mutant cycles. Molecular basis for convergent evolution of Kv1 channel blockers. J Biol Chem 2002;277:37406–13. [12] Huang CC, Stricher F, Martin L, Decker JM, Majeed S, Barthe P, et al. Scorpion-toxin mimics of CD4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth. Structure 2005;13:755– 68. [13] Lindgren M, Hallbrink M, Prochiantz A, Langel U. Cellpenetrating peptides. Trends Pharmacol Sci 2000;21:99–103. [14] MacDonald GC, Glover N. Effective tumor targeting: strategies for the delivery of armed antibodies. Curr Opin Drug Discov Devel 2005;8:177–83. [15] M’Barek S, Chagot B, Andreotti N, Visan V, Mansuelle P, Grissmer S, et al. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity. Proteins 2005;60:401–11. [16] M’Barek S, Lopez-Gonzalez I, di Luccio E, Visan V, Grissmer S, Judge S, et al. A maurotoxin with constrained standard disulfide bridging—Innovative strategy of chemical synthesis, pharmacology, and docking on K+ channels. J Biol Chem 2003;278:31095– 104. [17] Mouhat S, Jouirou B, Mosbah A, De Waard M, Sabatier JM. Diversity of folds in animal toxins acting on ion channels. Biochem J 2004;378:717–26. [18] Mouhat S, De Waard M, Sabatier JM. Contribution of the functional dyad of animal toxins acting on voltage-gated Kv1-type channels. J Pep Sci 2005;11:65–8. [19] Regaya I, Beeton C, Ferrat G, Andreotti N, Darbon H, De Waard M, et al. Evidence for domain-specific recognition of SK and Kv channels by MTX and HsTx1 scorpion toxins. J Biol Chem 2004;279:55690–6. [20] Rodríguez de la Vega RC, Merino E, Becerril B, Possani LD. Novel interactions between K+ channels and scorpion toxins. Trends Pharmacol Sci 2003;24:222–7. [21] Rodríguez de la Vega RC, Possani LD. Current views on scorpion toxins specific for K+-channels. Toxicon 2004;43:865–75. [22] Sabatier JM. Chemical synthesis and characterization of small proteins: example of scorpion toxins. In: Rochat H, Martin-Eauclaire MF, editors. Animal toxins. Basel, Switzerland: Birkhaüser Verlag; 2000. pp. 196–216. [23] Shakkottai VG, Regaya I, Wulff H, Fajloun Z, Tomita H, Fathallah M, et al. Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SKCa2. J Biol Chem 2001;276:43145–51. [24] Zenouaki I, Kharrat R, Sabatier JM, Devaux C, Karoui H, Van Rietschoten J, et al. In vivo protection against Androctonus australis hector scorpion toxin and venom by immunization with a synthetic analog of toxin II. Vaccine 1997;15:187–94.

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60 Analogs of Luteinizing Hormone-Releasing Hormone (LHRH) in Cancer ANDREW V. SCHALLY AND JORG ENGEL

known as gonadotrophin-releasing hormone (Gn-RH) [40]. However, for reasons of historical continuity, the abbreviation LHRH is recommended for naming its analogs [40]. Moreover, the abbreviation Gn-RH is confusing because it is too similar to GH-RH (growth hormone-releasing hormone), for which many agonistic and antagonistic analogs already exist [40]. LHRH is the primary link between the brain and the pituitary in the regulation of gonadal function and plays a pivotal role in vertebrate reproduction [40]. LHRH-II (Gn-RHII) and other isoforms of decapeptide LHRH have been also isolated from chicken brain, bony fish, and mammals, including human [31]. LHRH-II has been likewise found in tumors such as breast carcinomas [4, 31], but the presence of a functional receptor for LHRH-II in humans remains controversial [16, 31]. Since the oncological importance of LHRH-II is not established, it is not covered in this chapter. The discovery of LHRH has had a major impact on medicine and has led to a variety of clinical uses of LHRH analogs in oncology and gynecology [40, 42]. The interest in possible medical applications of LHRH stimulated various groups including ours to synthesize more than 3000 LHRH analogs in the past 34 years. The aims were to develop analogs with prolonged biological activity so that they would be more useful therapeutically than LHRH itself in the field of reproduction and its control [22, 40, 42]. Thus, several LHRH analogs substituted in positions 6, 10, or both are much more potent than LHRH and also possess prolonged activity [22, 40, 42]. Of these, the most important are [D-Trp6]LHRH (Decapeptyl, Triptorelin), Leuprolide or Lupron [dLeu6,Pro9-NHET]LHRH, Buserelin [d-Ser(But)6,Pro9NHET]LHRH, and Zoladex or Goserelin [d-Ser(But)6, Aza-Gly10]LHRH [22, 40, 42], which are 50–100 times more active than LHRH. This greater biologic activity of the analogs is due both to increased resistance to enzymatic degradation and an enhancement in affinity to

ABSTRACT The development of agonists and antagonists of luteinizing hormone-releasing hormone (LHRH), also known as GnRH, and their clinical uses are reviewed. Clinical applications of LHRH agonists are based on gradual downregulation of pituitary receptors for LHRH and inhibition of the pituitary and gonads. LHRH antagonists produce an immediate suppression of the secretion of gonadotrophins and sex steroids and thus achieve rapid therapeutic effects. LHRH agonists and antagonists can be used for the treatment of endometriosis, uterine leiomyomas, and in assisted reproduction programs. The preferred primary treatment of advanced androgendependent prostate cancer is based on the periodic administration of depot preparations of LHRH agonists. LHRH agonists can be likewise applied for treatment of breast cancer. LHRH antagonists have been successfully tried for therapy of prostate cancer and benign prostatic hypertrophy. Cytotoxic analogs of LHRH AN-152 and AN-207 containing doxorubicin or 2-pyrrolinodoxorubicin that can be targeted to LHRH receptors on tumors have been synthesized and successfully tested in in vivo experimental cancer models of human prostate, breast, ovarian, and endometrial cancers, and renal cell carcinomas, melanomas, and lymphomas.

INTRODUCTION More than 34 years have passed since the laboratory of one of us (AVS) clearly edged out other groups in the race for the isolation, structure, and synthesis of hypothalamic luteinizing hormone-releasing hormone (LHRH) [29, 30, 41] as well as in its clinical testing [45]. Since LHRH is likewise accepted now as the main FSH-releasing hormone, the decapeptide pGlu1-His2Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly10-NH2 is also Handbook of Biologically Active Peptides

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422 / Chapter 60 the LHRH receptors. The substitution of Gly6 by damino acids renders the analog more resistant to degradation by endopeptidases, which cleave LHRH at this position and are widely distributed in mammalian tissues [22, 40]. An acute injection of superactive agonists of LHRH induces a marked and sustained release of LH and FSH. However, a continuous stimulation of the pituitary by chronic administration of LHRH agonists produces inhibition of hypophysealgonadal axis through a process of “downregulation” (that is a reduction in the number) of pituitary receptors for LHRH, decrease in expression of LHRH receptor gene, desensitization of the pituitary gonadotrophs, and a suppression of circulating levels of, LH, FSH, and sex steroids [10, 22, 40, 42, 43]. The phenomena of downregulation of pituitary receptors for LHRH and inhibition of sex steroid levels by LHRH agonists are being used for treatment of precocious puberty, endometriosis, uterine leiomyomas, and other conditions [10, 42]. Chronic administration of LHRH agonists is being utilized to induce the regression of hormonedependent malignant neoplasms, especially prostate cancer, breast cancer, and endometrial carcinoma [10, 42]. Initially, agonists of LHRH were given daily by the subcutaneous or intranasal route. Subsequently, we developed a long-acting delivery system for [DTrp6]LHRH in microcapsules of a biodegradable polymer, poly (DL-lactide-co-glycolide), designed to release the peptide over a 30-day period [40]. Microcapsules or implants of peptide analogs that can be administered once every 30–90 days make the treatment of patients much more convenient and ensure compliance [40, 42]. LHRH agonists are also being used for in vitro fertilization and embryo transfer (IVF-ET) [40] in procedures known as the controlled ovarian stimulationassisted reproductive technology (COS-ART).

ANTAGONISTS OF LHRH LHRH antagonists represent another class of compounds that may be useful for treatment of sex-hormone dependent cancers and various gynecological applications. Potent antagonistic analogs of LHRH such as Cetrorelix, Ganirelix, azaline-B, degarelix, and abarelix have been synthesized in recent years and some are now available as depot preparation [2, 19, 33, 39]. Modern antagonists like Cetrorelix [Ac-d-Nal(2)1,d-Phe(4Cl)2,dPal(3)3,d-Cit6,d-Ala10]LHRH possess structural modifications in positions 1, 2, 3, 6, and 10 and other antagonists, also in positions 5 and 8 [2, 19, 33, 39]. LHRH antagonists exhibit no intrinsic activity but compete with native LHRH for the binding sites and cause an immediate inhibition of the release of gonadotrophins and sex steroids [40, 42, 43]. The principal

mechanism of action of LHRH antagonists was thought to be due only to their competitive blockade of LHRH receptors, but recently we demonstrated that at adequate doses of Cetrorelix, receptor downregulation also occurs [40, 43]. At low doses, however, LHRH antagonists suppress the pituitary gonadal axis only due to a competitive receptor blockade and additional downregulation of LHRH receptors was not observed [18]. Clinical studies with Cetrorelix in patients with prostate cancer and BPH demonstrated a marked clinical improvement and lowering of serum testosterone levels to castration values [15]. LHRH antagonists are used for IVF-ET and are in phase III clinical trials in patients with BPH, endometriosis, and myomas [40, 42, 43].

RECEPTORS FOR LHRH ON TUMORS Specific receptors for LHRH were first detected on human breast, prostatic, ovarian, endometrial, and pancreatic cancers [7, 10, 12, 17, 40, 42, 43]. Thus, highaffinity binding sites for LHRH were found on 52% of human breast cancer specimens and also in various human breast cancer cell lines [7, 12, 40, 43]. LHRH receptors and expression of mRNA for LHRH receptors were also demonstrated in about 86% of human prostate cancer samples and in several human prostate cancer lines [17]. Similarly receptors for LHRH were detected in 78% of human epithelial ovarian cancer specimens and ovarian cancer cell lines [10] as well as in 80% of human endometrial carcinomas and many endometrial cancer lines [10] and more recently in melanomas [34], non-Hodgkin’s lymphomas, and renal cell carcinomas. The evidence for the production of an LHRH-like peptide and/or the expression of mRNA for LHRH was also demonstrated in human breast, prostatic, ovarian, and endometrial cancer cell lines (for a review see 9, 44). These findings suggest that locally produced LHRH may be involved in the growth of these tumors, forming an autocrine regulatory loop. This system would consist of locally produced LHRH-like peptides and tumoral LHRH receptors. This concept is supported by an inhibitory action of LHRH agonists and antagonists in vitro on human mammary, prostatic, ovarian, and endometrial cancer cell lines through specific LHRH receptors on tumor cells [9, 44.] However, classical LHRH receptor signal transduction mechanisms for tumors appear to be different from those for the pituitary [9]. Other investigators [6, 8] postulate that this LHRH produced by tumor cells might have an inhibitory function. However, inhibitory effects of LHRH agonists can be best explained by receptor downregulation. Additional investigations are necessary to clarify the role and action of endogenous LHRH-like peptides produced by various tumors.

Analogs of Luteinizing Hormone-Releasing Hormone (LHRH) in Cancer / 423

CYTOTOXIC LHRH ANALOGS Targeted chemotherapy represents a modern oncological strategy designed to improve the effectiveness of cytotoxic drugs and decrease peripheral toxicity. The first concept of targeted therapy—so-called “Magic Bullets”—was proposed by Paul Ehrich more than 100 years ago [46]. However, this approach remained unexplored for many decades until the discovery of receptors. Thus, the receptors for peptide hormones on tumor cells can serve as targets for peptide ligands that can be linked to various cytotoxic agents [46–48]. Consequently, on the basis of the presence of specific receptors for LHRH on tumor cells, we developed a new class of targeted antitumor agents by linking cytotoxic radicals to analogs of LHRH [35, 46–48]. The first series of cytotoxic LHRH hybrid molecules was synthesized in our institute in the late 1980s [46]. By the middle of the 1990s, we developed a novel class of targeted cytotoxic LHRH analogs containing doxorubicin (DOX) or its intensely potent derivative 2pyrrolino-DOX (AN-201), which is 500 times more active in vitro than its parent compound [35, 47, 48]. AN-201 can also be characterized as a noncardiotoxic and noncross-resistant derivative of doxorubicin. One of our conjugates, AN-152, consists of [D-Lys6] LHRH linked to DOX-14-O-hemiglutarate, while AN-207 contains 2-pyrrolino-DOX also conjugated to d-Lys6LHRH through glutaric acid linker [35]. Cytotoxic analogs AN-152 and AN-207 have been tested extensively in preclinical studies. Both analogs show high-affinity binding to LHRH receptors on various tumors and retain the powerful cytotoxic activities of their respective cytotoxic radicals [35, 47, 48]. Clinical phase I trials with AN-152 are in progress in Germany.

EFFECTS OF ANALOGS OF LHRH ON TUMORS Prostate Cancer The greatest therapeutic impact of LHRH analogs came about in the field of prostate cancer. Carcinoma of the prostate is the most common noncutaneous malignancy in men [40, 42]. About 70% of human prostate cancers are testosterone-dependent and the endocrine therapy of advanced prostate cancer with agonists of LHRH is based on androgen deprivation [40, 42]. The principles of this approach were established by experimental studies in rats in our laboratory [37]. The first clinical study with LHRH agonists in men with advanced prostate cancer was carried out in collaboration with Tolis in 1980–1981 [49]. This and other clinical trials [40, 42] demonstrated a fall in

testosterone levels and marked improvement in patients with stage C or D prostate carcinoma after treatment with agonistic analogs Triptorelin, Leuprolide, or Zoladex. Long-term administration of depot preparation of LHRH agonists with or without antiandrogens is currently the preferred primary palliative treatment for men with advanced prostate cancer [40, 42]. Various recent studies also indicate that adjuvant treatment with LHRH agonists, after radiation or prostatectomy, is associated with a survival benefit in patients with localized or locally advanced prostate cancer [5]. Side effects caused by chronic administration of LHRH agonists are due to androgen deficiency and include impotence, loss of libido, and hot flashes. An occasional flareup during the first week of treatment can be prevented by initial coadministration of antiandrogens [40, 42]. The use of LHRH antagonists would avoid the temporary flareup of the disease that can occur in 10–20% of the patients with prostate cancer when LHRHagonists are given as single agents [40, 42]. We were the first to investigate inhibitory effects of the LHRH antagonist Cetrorelix on experimental androgen-dependent Dunning R-3327-H and human PC-82 prostate cancers in rats and nude mice [38]. Clinical trials showed that a decrease in testosterone and prostate-specific antigen (PSA) levels and prostate size measured by ultrasonography can be achieved in patients with advanced prostate cancer treated with Cetrorelix [15]. Patients who were paraplegic due to metastatic invasion of spinal cord showed neurologic improvement during therapy with Cetrorelix [14]. Cetrorelix may be indicated for patients with prostate cancer and metastases to the spinal cord, bone marrow, and other sites in whom the LHRH agonists cannot be used as single drugs because of the possibility of flareup. Recent studies performed with a depot formulation of the LHRH antagonist abarelix demonstrated that its administration leads to a faster decline of the testosterone levels than leuprolide with or without antiandrogens [50]. Cetrorelix is also suitable for the therapy of BPH and extensive clinical phase 3 trials are in progress [42]. In conclusion, it has been documented in thousands of patients with advanced prostate cancer that LHRH agonists provide an effective palliative therapy. However, all hormonal therapies, including LHRH agonists or antagonists, can provide only a remission with a limited duration, and most patients relapse in two to three years [42].

CYTOTOXIC ANALOGS OF LHRH It is possible that recently developed cytotoxic analogs of LHRH could be used in the management of relapsed prostate cancer. In view of ongoing clinical trials on cytotoxic analog AN-152, we investigated its effects on

424 / Chapter 60 the growth of LNCaP androgen sensitive prostate cancer in castrated nude mice [28]. Treatment with AN-152 markedly enhanced the effect of androgen ablation and produced a 67% tumor volume inhibition as compared with the effect of castration alone. The cytotoxic analog AN-152 also strongly suppressed growth of androgen sensitive MDA-PCa-2b prostate cancers and was more effective than equimolar doses of DOX [28]. In nude mice bearing androgen-independent intraosseous C4-2 prostate cancers, treatment with AN-152 decreased serum PSA levels, while DOX had no effect. All these three lines were LHRH receptor positive. The results indicate that the cytotoxic analog AN-152 should be effective in patients with prostate cancer who relapse following androgen deprivation therapy. After initial tests with AN-207 in rats with Dunning prostate cancers and in nude mice with human PC-82 tumors, we showed that cytotoxic LHRH analog AN-207 inhibited growth of MDA-PCa-2B prostate cancers xenografted s.c. into nude mice [36] and lowered PSA levels. AN-207 also inhibited growth of DU-145 tumors and LuCaP-35 human prostate cancers and was less toxic than AN-201. Our work supports the concept that targeted chemotherapy based on cytotoxic LHRH analogs should be more efficacious and less toxic than the current systemic chemotherapeutic regimens and should permit an escalation of doses. Targeted cytotoxic analogs of LHRH could be used for the treatment of advanced prostate cancer after the relapse. Cytotoxic analogs of LHRH might be also indicated for primary therapy of patients with advanced prostate cancer, thus extending the oncological uses of LHRH analogs from the current palliation toward an eventual cure.

Breast Cancer Breast cancer is the most common malignancy in women and a leading cause of cancer-related deaths [40, 42]. Women with breast cancer that express estrogen or progesterone receptors can be treated by hormonal manipulations such as the antiestrogens Tamoxifen and raloxifene or by oophorectomy [40, 42]. Agonists of LHRH may also be used for treatment of estrogen-dependent breast cancer in premenopausal and perimenopausal women. Experimental studies in rat and mouse models of breast cancer showed that chronic administration of LHRH agonists decreased tumor weight and volume [40, 42] and suggested that LHRH agonists are suitable as a novel endocrine treatment modality for estrogen sensitive mammary cancer. Clinical trials in premenopausal patients with advanced or metastatic breast cancer demonstrated 41–53% of objective responses after treatment with LHRH agonists such as buserelin or goserelin [3]. It was also concluded that combined estrogen blockade with an LHRH agonist

and tamoxifen is superior to either treatment modality alone [27]. Recently, large multicenter trials in premenopausal women with estrogen receptor positive breast cancer [23] demonstrated that adjuvant treatment with Zoladex for two to three years is as effective as chemotherapy and burdened with fewer side effects. Cetrorelix might also be an effective treatment for premenopausal breast cancers, but this was shown so far only in experimental models of breast cancer [40]. Estrogen independent tumors have a poor prognosis. The treatment options available are not satisfactory and new therapeutic modalities must be explored [40, 42]. Chemotherapy is a major modality for treatment of patients with breast cancer, but chemotherapeutic drugs have toxic side effects [40, 42, 46–48]. Targeted cytotoxic analogs of LHRH bind with high-affinity to LHRH receptors on human breast cancers and inhibit tumor growth [46–48]. Thus, analog AN-152 powerfully inhibited growth of MX-1 hormone independent doxorubicin-resistant human breast cancers in nude mice, while doxorubicin had no effect [1]. In another study, a single administration of AN-207 caused a complete regression of MX-1 hormone-independent doxorubicin-resistant human breast cancers in nude mice [21]. AN-207 also inhibited the growth of MDAMB-231 and MDA-MB-435 estrogen independent human breast cancer in nude mice and prevented metastatic spread. The results suggest that targeted cytotoxic LHRH analogs could be considered for treatment of human mammary cancers expressing receptors for LHRH.

Epithelial Ovarian Cancer Epithelial ovarian cancer is the fourth most frequent cause of cancer-related deaths in women [40, 42]. The treatment based on surgery or chemotherapy is not very effective, and new approaches are needed. In a multicenter trial in women with advanced ovarian carcinoma no beneficial effects of therapy with [d-Trp6]LHRH could be found [40, 42]. Since LHRH receptors are present on about 80% of ovarian tumors, we tried an approach based on targeted cytotoxic LHRH analogs. In the first study [32], we showed that the cytotoxic analog AN-152 inhibited the growth of LHRH receptorpositive OV-1063 human ovarian tumors in nude mice for at least four weeks. AN-152 had no effect on receptor-negative SK-OV-3 human ovarian cancers. In two subsequent studies, we demonstrated that the growth of OV-1063 and ES-2 human ovarian cancers could be suppressed by administration of cytotoxic LHRH analog AN-207 in doses 100 times smaller than those of AN-152 [40, 42, 43, 46–48]. Thus, targeted chemotherapy based on analogs such as AN-152 and AN-207 may improve the management of ovarian cancer and clinical trials are in progress.

Analogs of Luteinizing Hormone-Releasing Hormone (LHRH) in Cancer / 425

Endometrial Cancer Endometrial cancer is a common gynecologic malignancy in the Western world [10, 40, 42, 43, 47]. Surgery and radiotherapy are successful in 75% of cases, but new methods are needed for advanced or relapsed cancers. In limited clinical studies carried out so far, administration of depot preparations of LH-RH agonists produced a partial or complete remission in 35% of patients with recurrent endometrial cancer [13]. In view of the presence of LHRH receptors on nearly 80% of endometrial cancers, targeted cytotoxic analogs have been investigated. Thus, AN-152 was found to be more efficient and less toxic than DOX in the treatment of HEC-1B human endometrial cancers in nude mice. AN152 likewise strongly inhibited the growth of HEC-1A endometrial tumors [11]. AN-207 also significantly suppressed the proliferation in vivo of HEC-1A and RL95-2 endometrial cancers. Cytotoxic radicals DOX and AN-201 had no effect [11]. These experimental findings have to be confirmed in clinical trials.

Renal Cell Carcinoma (RCC) Each year about 36,000 patients in the United States are diagnosed with renal cancer, and an estimated 12,000 die from it [40, 42, 43]. The prognosis for patients with metastatic renal cell carcinoma (RCC) is poor because of its resistance to both chemotherapy and radiotherapy. The present methods of treatment of RCC must be improved. Therapy with the LH-RH antagonist Cetrorelix inhibited the growth of the CAKII RCC line xenografted into nude mice [20], but clinical trials with Cetrorelix in patients with advanced RCC were inconclusive. The expression of LHRH receptors was found in 28 of 28 surgically removed specimens of human renal cell carcinoma (RCC) by immunohistochemistry and in three human RCC cell lines A-498, ACHN, and 786-0 by Western immunoblotting and RT PCR [25]. The cytotoxic analog AN-207 significantly inhibited the growth of these three human RCC lines xenografted into nude mice [25]. Our findings indicate that LHRH receptors expressed in human RCC specimens can be used for targeted chemotherapy with cytotoxic LHRH analogs.

Non-Hodgkin’s Lymphoma Non-Hodgkin’s lymphoma (NHL) is the most frequently diagnosed hematological malignancy and the sixth most common cancer in the United States with an estimated incidence of about 56,000 new cases and 21,000 deaths in 2005 [26]. Patients with advanced disease face a dismal prognosis with a five-year survival rate of only 26%, emphasizing the need for novel

therapeutic approaches [26]. LHRH receptors were detected in 92% of the surgically removed human NHL specimens and in RL and HT NHL cell lines. AN-207 significantly inhibited the growth of RL HT tumors, while the nontargeted AN-201 had no effects [26]. Thus, LHRH receptors found in human NHL can be therapeutic targets for the cytotoxic LHRH analog AN-207.

Melanoma In the past four decades, the incidence of malignant cutaneous melanoma has steadily increased [24]. When detected early, cutaneous melanoma can be cured by surgical excision. However, once melanoma metastasizes to distant sites, the prognosis is very poor [24], and new effective therapies must be developed. The first demonstration of LHRH receptors in melanomas was made by Moretti et al. [34]. We also found LHRH receptor expression by immunohistochemistry in 19 of 19 human melanoma specimens derived from primary tumors or metastases [24]. The mRNA for LHRH receptors and LHRH receptor protein were similarly detected in transplantable MRI-H255 and MRI-H187 melanoma tumor lines [24]. We then showed that AN-207 significantly inhibited the growth of MRI-H255 and MRI-H187 tumors xenografted into nude mice [24]. Thus, LHRH receptors expressed in human malignant melanoma can be used for targeted chemotherapy with the cytotoxic LHRH analog AN-207.

Other Cancers Receptors for LH-RH are present in hamster and human pancreatic cancers and human colorectal cancers, but their function is not known. Various clinical trials with LH-RH agonists in patients with pancreatic carcinoma were unsuccessful [40, 42, 43].

SIDE EFFECTS OF CYTOTOXIC LHRH ANALOGS AN-207 can cause selective damage to the rat gonadotroph cells after an IV injection, but the pituitary function completely recovers after one week [46–48]. Because receptors for LHRH are not expressed in high concentrations in most normal tissues, side effects related to targeting, other than myelotoxicity, are not expected after treatment with AN-207 or AN-152. In all the experimental studies, targeted cytotoxic LHRH analogs AN-152 and AN-207 were much more effective and significantly less toxic than their chemotherapeutic radical doxorubicin or 2-pyrrolino doxorubicin [46– 48].

426 / Chapter 60 CONCLUSION Agonists of LHRH have found important applications in cancer therapy. LHRH antagonists are also being developed for oncological uses. Novel therapeutic modalities for breast, prostate, ovarian, and endometrial cancers, RCC, melanomas, and lymphomas could consist of the use of targeted cytotoxic analogs of LHRH containing doxorubicin (DOX) or 2-pyrrolino-DOX. The continued development of these three classes of LHRH analogs should lead to a more effective treatment for various cancers.

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61 Bombesin-Related Peptides and Neurotensin: Effects on Cancer Growth/Proliferation and Cellular Signaling in Cancer ROBERT T. JENSEN AND TERRY W. MOODY

derived from a 148-amino-acid precursor, whereas NMB is a 10-amino-acid peptide derived from a 32-amino-acid precursor that originated from a larger 76-amino-acid precursor [6]. The cellular actions of these peptides are mediated by two G protein-coupled receptors (GPCR): a GRP-R (384 amino acids) and NMB-R (399 amino acids) [7]. A third GPCR, termed bombesin receptor subtype 3 (BRS-3) (399 amino acids) [13], which shows 46–49% amino acid identities with GRP-R/NMB-R, has been cloned, but the endogenous ligand for it is unknown. All three of these mammalian BN receptors are coupled to phospholipase C (PLC), as well as tyrosine phosphorylation and MAP kinase cascades [2, 32, 43, 58].

ABSTRACT Mammalian bombesin-related peptides and neurotensin are regulatory peptides that have important roles in a number of physiological and pathological processes. In this chapter their effects on human cancer are briefly reviewed, including evidence that these peptides and/or their receptors occur in cancer, stimulate growth, are involved in cellular signaling cascades, and have potential roles in cancer treatment.

INTRODUCTION Bombesin (BN)-related peptides [Gastrin-releasing peptide (GRP) and neuromedin B (NMB)] as well as neurotensin (NT) are regulatory peptides widely distributed in the central nervous system and gastrointestinal (GI) tract, where they primarily function as neurotransmitters/neuromodulators. It has been proposed that these peptides are important in a number of physiological/pathological processes [6, 66]. In this chapter we will briefly review the evidence for their possible role in tumor growth, the mechanisms involved, and their possible role in treatment of human cancer.

Expression of BN-Peptides and Their Receptors in Cancer Many studies have demonstrated that a wide range of cancers synthesize BN-related peptides, including GRP and NMB fully processed and processing intermediates, and many possess GRP-R, NMB-R, and BRS-3 [20, 43, 53, 56, 81]. In human cancer cell lines and human cancers, either BN receptors or receptor mRNA was detected frequently in small cell lung cancer (SCLC) (85–100% GRP-R, 55% NMB-R, 25% BRS-3); nonsmall cell lung cancer (74–78% GRP-R, 67% NMB-R, 8% BRS3); breast cancer (38–72% GRP-R, 0% NMB-R, BRS-3); pancreatic cancer cell lines (75% GRP-R, 100% NMBR); pancreatic cancer (10% GRP-R); prostate cancer (62–100% GRP-R, 0% NMB-R, BRS-3); head/neck squamous cell cancer (100% GRP-R); glioblastomas (85% GRP-R); intestinal carcinoids (46% NMB-R, 0%, BRS-3,GRP-R); bronchial carcinoids (35% BRS-3, 4% NMB-R, 0% GRP-R); and neuroblastomas (72% GRP-R) [1, 8, 15, 30, 43, 56, 64, 67, 81].

BN-RELATED PEPTIDES (GRP, NMB) General Only a few general points will be made here in regard to the structure, pharmacology, and cellular action of these peptides and their receptors because they are covered in other sections in this book (Spindel and Beglinger). GRP is a 27-amino-acid peptide, which is Handbook of Biologically Active Peptides

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430 / Chapter 61 Proliferative Effect of BN-Receptor Activation on Cancer BN-like peptides have been shown to be critical to SCLC cell growth [8]. SCLC cells not only produce BNlike peptides and overexpress BN-receptors (especially GRP-R), but inhibition of the secreted BN-like peptide with antibody results in interference with the growth of the SCLC cells demonstrating an autocrine growth loop in these cells [8, 42, 43]. A similar autocrine role of BN-related peptides has been proposed in a number of other cancers [34, 47, 51, 64, 73, 76]. NMB-R or GRP-R expression in rat-1 or BALB/3T3 cells promotes proliferation [14, 33]. In some tumors, such as prostate cancer [40], BN-receptor (GRP-R) overexpression has been shown to correlate directly with neoplastic transformation. BN-related peptides have been shown to stimulate growth of a wide range of cancers [53, 61, 78]. Using either BN-receptor agonists or antagonists [27] BN-related peptides are reported to stimulate growth of cancers of the pancreas [23, 61], lung [43, 45], head/ neck [37], glioblastomas [50, 64], renal cell cancer [49], prostate cancer [51, 61], breast cancer [77], colorectal cancers [26, 53, 61], ovarian cancer [61], and gastric cancers [61]. Furthermore, BN-related peptides stimulate invasive growth by a number of tumors, including intestinal adenocarcinomas [23], colonic cancer [60], and prostate cancer [23]. BN-related peptides also function as proangiogenic factors in various tumors [28, 35, 41].

Mechanism of BN-Related Peptide-Induced Growth Effects on Cancer Cells Activation of all mammalian BN-Rs (i.e., GRP-R, NMB-R, BRS-3) stimulates phospholipase C (PLC) activity and causes activation of tyrosine and MAP kinase cascades [2, 32, 57, 58]. Only the growth stimulatory signaling cascades of the GRP-R have been investigated in detail. Activation of the GRP-R results in activation of Gq; PLC with generation of IP3 (1,4,5) and diacylglycerol; mobilization of cellular calcium; activation of classical and novel PKC isoforms; stimulation of MAP kinases, protein kinase D (PKD), phospholipase A2, and D, Src, p21rho; and stimulation of tyrosine and serine phosphorylation [p125 focal adhesion kinase (p125FAK), paxillin, p130CAS] [22, 57]. In some tumors the growth stimulatory effects appear to be mediated directly by the preceding GRP-R activated cascades, whereas in other cases it is carried out through transactivation of the EGF receptor (EGF-R), and in still other cases by stimulation of the release of growth factors [35, 37, 57]. In head/neck squamous cancer cells, activation of the GRP-R results in rapid phosphorylation of the EFG-R, which is mediated by stimulation of matrix metallopro-

teinases stimulating TGF-α and amphiregulin release by an Src-dependent mechanism [37, 80]. In these cells, activation of c-SRC and EGF-R transactivation are essential for GRP-R to stimulate proliferation [80]. The role of GRP and/or GRP-R overexpression in tumor growth/ differentiation may differ in different tumors. In contrast to head/neck squamous tumors and a number of other tumors, in recent studies in colon cancer [26], GRP/GRP-R co-expression was seen equally in Stage A and D cancers, rarely detected in metastases, and there was no correlation of survival with the level of GRP/ GRP-R expression. In fact, GRP/GRP-R expression was found primarily in well-differentiated cancers. From these studies it was proposed that GRP/GRP-R are functioning primarily as morphogens or differentiating factors and not as a growth factor in colon cancer [26]. More recent studies [17] suggest that the morphogenic properties are mediated by activation of p125FAK, which inhibits invasion/metastases by enhancing cell attachment to the cell matrix.

Role of BN-Related Peptides in Treatment of Human Tumors BN-related peptides/receptors currently play potentially important roles in four areas of treatment of human cancers: as prognostic factors, as targets to inhibit tumor growth, as targets to image tumor extent/ location, and as targets to deliver BN-R mediated cytotoxicity through radiolabeled compounds and/or cytotoxic agents coupled to BN-R ligands [46, 61, 68, 78, 81]. In some studies, plasma levels of GRP precursors (i.e., such as pro-GRP) [48, 65] or assessment of GRP expression in lung cancer [21] gives important prognostic information. Second, attempting to inhibit the autocrine effect of BN-like peptides on tumor growth by monoclonal antibodies to GRP/GRP-R, antisense constructs, antagonists, or other inhibitors are being studied [81]. In one study [29] of 13 patients with SCLC treated with a monoclonal antibody (A211) to GRP, 1 out of 12 (8%) had complete resolution radiologically of the tumor. Third, because tumors frequently overexpress BN-Rs, radiolabeled analogs of GRP with enhanced stability have been developed and are being evaluated as agents to image these tumors to assess tumor location/extent [4, 68]. Furthermore, cytotoxic GRP conjugates and radiolabeled analogs to target antitumor treatment to these tumors is receiving considerable attention [4, 46, 61]. Cytotoxic GRP analogs formed by coupling GRP-related peptides to chemotherapeutic agents (doxorubicin)(AN-215) have been shown to inhibit growth of pancreatic, lung, prostate, and gastric cancers [61, 62] and GRP analogs coupled to camptothecin to inhibit lung cancer growth [46]. At present the usefulness of BN-like peptides/receptors in any of

Bombesin-Related Peptides and Neurotensin /

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the four areas of investigation for tumor treatment just listed is not established.

NTR1, and NTR3 were present in all cancer cell lines, whereas NTR2 was not present in any cancer cell line [9].

NEUROTENSIN (NT)

Proliferative Effect of NT in Cancer

General

NT stimulates growth of normal gastric antrum, small bowel, colon, and pancreas [73]. NT stimulates proliferation of colon cancer cell lines [38, 79] both in vitro and in vivo when the tumor cells are xenografted into nude mice. NT also promoted colonic cancer carcinogenesis in rats treated with azoxymethane [71]. NT stimulates the proliferation of pancreatic cancer cell lines both in vitro and in vivo [70], promotes gastric carcinogenesis induced by MNNG in rats [72], and also stimulates growth of SCLC cells [44] and prostate cancer cells [63].

NT is a 13-amino-acid peptide derived from a 170amino-acid precursor, which contains both NT and neuromedin N (NmN), a hexapeptide that shares a Cterminal sequence with NT. Both of these peptides are structurally related to Xenopsin [66, 74]. Three subtypes of NT receptors have been cloned: NTR1, NTR2, and NTR3 [31, 74]. NTR1 (421 amino acids) and NTR2 (410 amino acids) are both G protein-coupled receptors, whereas NTR3 has a single transmembrane domain and belongs to a family of sorting receptors with an identical structure to the sorting protein, sortilin [31, 74]. NTR1 is coupled to PLC activation via Gq, through Gi/o, to inhibition of adenylate cyclase and production of arachidonic acid as well as stimulation of MAP kinase activation [31, 74]. NTR2 causes activation of PLC and MAP kinase and stimulates accumulation of arachidonic acid; however, it shows species- and expression-dependent pharmacologic properties wherein NT behaves as an NTR2 antagonist in some cells suggesting that the endogenous activator of this receptor has yet to be described [16, 31].

Expression of NT and NT Receptors in Cancer Cells Pancreatic ductal cancers, but not pancreatic endocrine cancers, possess NT receptors in 75–100% of cases [11, 54, 75] by binding studies or mRNA expression. In one study [75], NTR1 levels were increased a mean of 4.4-fold in pancreatic ductal cancer, and higher expression was found in advanced (GradeIII/1V) than early tumor stages (Stages I/II, P < 0.05). NT expression was detected in 47% of human colon cancers by binding studies but not in normal colonic mucosa [39]. Using autoradiographic binding studies, NT receptors were found in 11 out of 17 (65%) Ewing’s sarcomas, 21 out of 40 (52%) meningiomas, 10 out of 23 (43%) astrocytomas, 5 out of 12 (38%) medulloblastomas, 7 out of 24 (29%) medullary thyroid cancers, 2 out of 8 (25%) SCLC, and is rare or absent in non-SCLC cells, and cancers of the breast, prostate, ovary, kidney, or liver [55]. In the preceding study [55], all NT receptors were of the NTR1 subtype. A number of human colon cancer cell lines express proNT mRNA and produce NT [12] as well as cancers of the pancreas, prostate, and SCLC cells [31]. In a recent study of prostate (n = 2), colon (n = 6), and pancreatic cell lines (n = 1), using RT-PCR,

Mechanism of NT-Induced Effect on Tumor Cell Growth NT has been proposed to have an autocrine action in pancreatic cancer cells, colon cancer, lung cancer, and prostate cancer [44, 63, 73]. NTR1 receptor antagonists inhibited the growth of SCLC cells [44] and pancreatic cancer cells [24]. In human colon cancer cells and CHO-transfected cells with the NTR1, NT stimulates activation of both p44 and p42 and the activation was inhibited by the NTR1 antagonist, SR-48692 [11]. The activation of MAP kinases by NT is partially dependent on pertussis-toxin sensitive G proteins as well as on PKC activation [52]. In pancreatic cancer cells, NT also stimulates activation of MAP kinase as well as PKD, and the increase in DNA synthesis is dependent on PKC activation [18, 19, 59]. In lung cancer cells NT stimulates tyrosine phosphorylation of p125FAK, which is inhibited by the NTR1 antagonist, SR-48692 [36]. However, it has not been established that p125FAK activation is involved in mediating the NT growth effect in these cells. NT in breast cancer cells has an antiapoptotic effect [69] that is associated with an increase in Bcl-2 protein and mRNA levels resulting in Bcl-2 transcriptional activation, which in turn is dependent on NT-stimulation of the MAP kinase pathway [69]. In some cancer cells NT may stimulate growth by an indirect mechanism because the NTR1 antagonist SR-48692 inhibited the trophic effect in vivo of NT on LoVo colon cancer cells; however, these cells did not have NTR1 receptors [25].

Role of NT Peptides/Receptors (NTR) in Treatment of Human Cancer The possibility of using NTR overexpression either to localize tumor extent or to allow NT receptor-directed

432 / Chapter 61 cytotoxicity using radiolabeled analogs has received increasing attention [3, 5, 10]. NT analogs with improved stability have been synthesized, coupled to 111 In, using either a DTPA or DOTA linker or to 99MTc, and shown to localize to tumors (colorectal cancer) as well as be internalized by the tumor cells [3, 5, 10]. In one in vivo study in four patients with ductal pancreatic cancer, a 99mTc-labeled NT analog (NT-XI) localized the tumor in one of four patients and in one of the two patients whose tumor possessed NT receptors [5].

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[47] Murphy LO, Abdel-Wahab YH, Wang QJ, Knezetic JA, Permnert J, Larsson J, Hollingsworth AM, Adrian TE: Receptors and ligands for autocrine growth pathways are up-regulated when pancreatic cancer cells are adapted to serum-free culture. Pancreas 2001;22:293–8. [48] Okusaka T, Eguchi K, Kasai T, Kurata T, Yamamoto N, Ohe Y, Tamura T, Shinkai T, Saijo N: Serum levels of pro-gastrinreleasing peptide for follow-up of patients with small cell lung cancer. Clin Cancer Res 1997;3:123–7. [49] Pansky A, Peng F, Eberhard M, Baselgia L, Siegrist W, Baumann JB, Eberle AN, Beglinger C, Hildebrand P: Identification of functional GRP-preferring bombesin receptors on human melanoma cells. Eur J Clin Invest 1997;27(1):69–76. [50] Pinski J, Schally AV, Halmos G, Szepeshazi K, Groot K: Somatostatin analogues and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo. Cancer Res 1994;54:5895–901. [51] Plonowski A, Nagy A, Schally AV, Sun B, Groot K, Halmos G: In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215. Int J Cancer 2000;88:652–7. [52] Point-Chazel C, Portier M, Bouaboula M, Vita N, Pecceu F, Gully D, Monroe JG, Maffrand JP, Le Fur G, Casellas P: Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24. Biochem J 1996;320:145–51. [53] Preston SR, Miller GV, Primrose JN: Bombesin-like peptides and cancer. Crit Rev Oncol Hematol 1996;23:225–38. [54] Reubi J-C, Waser B, Friess H, Buchler M, Laissue J: Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma. Gut 1998;42:546–50. [55] Reubi J-C, Waser B, Schaer JC, Laissue JA: Neurotensin receptors in human neoplasms: high incidence in Ewing’s sarcomas. Int J Cancer 1999;82:213–8. [56] Reubi JC, Wenger S, Schumuckli-Maurer J, Schaer JC, Gugger M: Bombesin receptor subtypes in human cancers: detection with the universal radoligand (125)I-[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin(6–14). Clin Cancer Res 2002; 8:1139–46. [57] Rozengurt E: Signal transduction pathways in the mitogenic response to G protein-coupled neuropeptide receptor agonists. J Cell Physiol 1998;177:507–17. [58] Ryan RR, Weber HC, Hou W, Sainz E, Mantey SA, Battey JF, Coy DH, Jensen RT: Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. J Biol Chem 1998;273: 13613–24. [59] Ryder NM, Guha A, Hines OJ, Reber HA, Rozengurt E: G protein-coupled receptor signaling in human ductal pancreatic cancer cells: Neurotensin responsiveness and mitogenic stimulation. J Cell Physiol 2001;186:53–64. [60] Saurin JC, Fallavier M, Sordat B, Gevrey JC, Chayvaille JA, Abello J: Bombesin stimulates invasion and migration of Isrecol colon carcinoma cells in a Rho-dependent manner. Cancer Research 2002;62:4829–35. [61] Schally AV, Comaru-Schally AM, Nagy A, Kovacs M, Szepeshazi K, Plonowski A, Varga JL, Halmos G: Hypothalamic hormones and cancer. Front Neuroendocrinol 2001;22:248–91. [62] Schally AV, Nagy A: Cancer chemotherapy based on targeting of cytotoxic peptide conjugates to their receptors on tumors. Eur J Endocrinol 1999;141:1–14. [63] Sehgal I, Powers S, Huntley B, Powis G, Pittelkow M, Maihle NJ: Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer. Proc Natl Acad Sci U S A 1994;91:4673–7.

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62 Somatostatin and NPY MEIKE KÖRNER AND JEAN CLAUDE REUBI

other systems of the human body, where it has mainly inhibitory regulative functions [17]. These inhibitory actions of somatostatin are not restricted to physiologic processes, but also play a role in neoplasia, for which mainly three mechanisms have to be considered: the inhibition of tumoral hormone secretion, the direct and indirect inhibition of tumor cell proliferation, and the inhibition of tumoral angioneogenesis. These aspects are of current investigational interest because they represent the molecular basis for the clinical use of somatostatin in tumor therapy.

ABSTRACT Somatostatin and NPY are two different peptide hormones with multiple functions in neoplasia. Somatostatin exerts inhibitory effects on tumoral hormone secretion, tumor cell proliferation, and tumoral angioneogenesis, which represent the basis for the clinical use of somatostatin in tumor therapy, whereas emerging data show effects of NPY on tumor growth and angioneogenesis. Moreover, many tumors overexpress somatostatin receptors that are targeted for diagnostic and therapeutic purposes with radioactive and cytotoxic somatostatin analogs. Similarly, an increasing number of tumors are found to overexpress NPY receptors that may also be targeted in vivo with adequate NPY analogs.

Inhibition of Hormone Release from Tumors by Somatostatin Somatostatin is a strong inhibitor of hormone secretion from normal endocrine cells and their neoplastic counterparts. Thus, somatostatin and somatostatin analogs are highly effective in reducing hormonal symptoms caused by hypersecretory endocrine tumors and therefore now form an integral part of the routine therapy of several such tumors. For somatostatin therapy, metabolically stable and/or long-acting somatostatin analogs have been developed, such as octreotide acetate (Sandostatin®), lanreotide, or their respective long-lasting release (LAR) formulations, as native somatostatin has a very short half-life in the circulation. These somatostatin analogs are generally well tolerated and cause only mild side effects. They potently inhibit tumoral growth hormone (GH) secretion and reduce GH-related symptoms in patients suffering from GH-producing pituitary adenomas [17]. They are also the drugs of first choice to control hormone-related symptoms in several functionally active gastroenteropancreatic neuroendocrine tumors (see also chapter by Gugger and Reubi). For instance, in patients with carcinoid syndrome, somatostatin analogs achieve symptomatic control in 73% and a significant improvement of the quality of life [6].

INTRODUCTION The two peptides discussed in this chapter do not have much in common at first glance. Physiologically, they control and regulate different biological systems in the body. Historically, somatostatin was already discovered in 1973 [2], while NPY was first reported in 1982 [24]. This one decade implies that much more information is available on the somatostatin than on the NPY systems, not only in general biology but also regarding clinical applications. In particular, the relation between somatostatin and cancer has been known for a relatively long time, whereas NPY has only very recently been linked to neoplasia. Nevertheless, there are numerous converging features appearing that suggest that the very young NPY cancer field may learn and strongly be influenced by the decades’ experience with the somatostatin cancer field.

SOMATOSTATIN Somatostatin and Cancer Somatostatin is a peptide hormone with a wide distribution in nervous, endocrine, gastrointestinal, and Handbook of Biologically Active Peptides

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436 / Chapter 62 Direct and Indirect Antiproliferative Effects of Somatostatin in Tumors More than 20 years ago, Schally and coworkers performed seminal studies in various animal tumor models showing that somatostatin and somatostatin analogs inhibit tumor cell proliferation [22]. This effect is probably achieved via several independent mechanisms [18]. Somatostatin may indirectly reduce tumor cell proliferation via inhibition of various physiologically occurring growth stimulating hormones, such as inhibition of pituitary GH and prolactin release, inhibition of the GH/insulin-like growth factor (IGF)-I axis, and suppression of release or action of other gut hormones. Moreover, somatostatin may directly act on tumor cells by binding to tumoral somatostatin receptors and interfering with intracellular signaling cascades of cell cycle and apoptosis [12, 22]. The tumor growth inhibitory effect of somatostatin has been documented in many experimental models. In animals bearing pancreatic, breast, and prostate carcinoma, as well as chondro- and osteosarcoma, somatostatin analogs were shown to reduce tumor size and to prolong survival [18, 22]. In human tumor cell lines, including pancreatic, breast, and small cell lung cancer (SCLC) cell lines, the antiproliferative effect of somatostatin analogs could also be reproduced. Worth mentioning are the elegant studies by Susini and collaborators, showing that the introduction of the somatostatin receptor subtype 2 (sst2) in pancreatic tumor cell lines originally devoid of sst2 lead to a marked inhibition of tumor growth, indicating the pivotal role of sst2 [3]. In patients, reproducible inhibitory effects of somatostatin analogs on tumor growth were observed primarily in GH-producing pituitary adenomas: In most patients, tumor growth control was reported, and overall in 42% of patients a mean tumor size reduction of 50% could be achieved. Unfortunately, however, results of clinical trials on other tumors were disappointing. While a reduction of tumor size under somatostatin therapy was only very rarely observed in single patients with advanced gastroenteropancreatic neuroendocrine tumors [6, 22], evidence of limited tumor growth control by somatostatin analogs was provided by Arnold and coworkers who documented a stabilization of tumor size over 2 to 60 months in 36–70% of patients with malignant neuroendocrine tumors [1]. In summary, however, the current clinical data are not sufficiently convincing to propose the use of nonradioactive or noncytotoxic somatostatin analogs for a therapy of nonneuroendocrine tumors [6]. The reasons for the marked differences in somatostatin analog efficacy between animal studies and clinical trials remain presently unclear.

Antiangiogenic Effects of Somatostatin Induction of new blood vessels is an important feature of malignant tumors for growth and metastasis, and inhibition of tumoral angiogenesis is a promising therapeutic approach. Somatostatin is antiangiogenic by inhibiting endothelial cell proliferation. It reduces the new formation of both normal and tumorassociated blood vessels in several experimental animal and human models. In particular, octreotide was shown to reduce tumor vascularization in vivo in a human breast carcinoma model [25]. Of note, vascular somatostatin receptors are locally upregulated in the vicinity of tumors [4]. This is important for a somatostatin-based antiangiogenic tumor therapy, as it may allow specific targeting of tumorassociated vessels while sparing normal vessels. Although the present in vitro and animal tumor model data look promising, no clinical experience exists on an antiangiogenic effect of somatostatin analogs in tumor patients so far. Molecular Signaling of Somatostatin Receptors The multiple somatostatin effects are mediated by six different somatostatin receptor (sst) subtypes, named sst1, sst2A, sst2B, sst3, sst4, and sst5. The molecular signaling of sst is complex, as sst couple to various different pertussis toxin-sensitive and -insensitive Gproteins, which in turn modulate multiple interdependent intracellular signal transduction pathways [12]. Correspondingly, the effect of somatostatin on normal and tumor cell proliferation can be mediated by multiple mechanisms. Cell cycle arrest is mediated by various sst subtypes, leading for instance to activation of tyrosine phosphatases, interference with the MAP kinase ERK pathway, induction of cyclin-dependent kinase inhibitors, and accumulation of hypophosphorylated Rb protein. Moreover, the antiproliferative effect of somatostatin may also be mediated indirectly by induction of apoptosis via several sst. Endothelial cell proliferation is regulated by sst3 mediated inhibition of endothelial nitric oxide synthase activity [12]. Only recently, it was recognized that sst not only are activated by somatostatin agonist binding to a single sst entity but also may undergo complex and yet poorly understood regulating mechanisms of homo- and heterooligomerization [12]. Sst oligomerization not only affects intracellular signal transduction but also alters sst binding affinity and internalization. For instance, sst3 is completely inactivated by heterodimerization with sst2A, and sst1 internalization depends on heterodimerization with sst5. Of note, these effects have been observed only in transfected cells and have not been established in native tissues so far—in particular not in sst expressing tumors. However, as tumors often

Somatostatin and NPY / 437 coexpress multiple sst subtypes [19], sst heterodimerization may significantly affect binding and internalization properties of the various tumoral sst.

Somatostatin Receptors in Cancer The discovery that sst are expressed in many malignant human tumors has led to further important clinical applications, which are based on sst properties independent of the regulation of hormone release, proliferation, and angiogenesis [19]. Indeed, tumoral sst overexpression allows a highly effective in vivo tumor targeting for diagnostic and therapeutic purposes based on the binding and internalization of radiolabeled and cytotoxic somatostatin analogs to sst [11, 23]. Somatostatin Receptor Expression in Human Tumors Sst are expressed on the tumor cell surface in a broad spectrum of human cancers. As expected from the sst distribution in nonneoplastic tissues, sst expression is very high in neuroendocrine and brain tumors, particularly in GH-producing pituitary adenomas, gastroenteropancreatic and bronchial neuroendocrine tumors, pheochromocytomas, paragangliomas, neuroblastomas, medulloblastomas, meningiomas, medullary thyroid carcinomas, and SCLC. In comparison, sst are expressed in lower incidence and density in many common epithelial tumors (breast carcinomas, prostatic carcinomas, renal cell carcinomas, gastrointestinal adenocarcinomas, hepatocellular carcinomas, ovarian carcinomas), lymphomas, and mesenchymal tumors [19]. Sst are also overexpressed in tumor-associated blood vessels—in particular in peritumoral veins and, more rarely, in intratumoral blood vessels, as compared with normal blood vessels in corresponding nonneoplastic tissues [4]. The degree of vascular sst expression varies according to the tumor type. Tumor-associated vascular sst expression is important because it may also render those tumors responsive to in vivo sst targeting (see following) that do not express sst on tumor cells [25]. The six sst subtypes are differentially and often concomitantly expressed in tumors. However, sst2A is by far the most common and most predominant subtype both on tumor cells and in tumor-associated blood vessels [19, 25]. This is of great clinical advantage because sst2A, compared with other sst subtypes, has a high affinity for the commercially available somatostatin analogs. Somatostatin Receptor Targeted Tumor Imaging Sst overexpressing tumors can be specifically visualized with scintigraphy using radiolabeled somatostatin

analogs such as 111In-DTPA–octreotide (OctreoScan®). This method is very effective in detecting tumors with high and homogeneous sst expression and therefore became the gold standard method for localizing and staging gastroenteropancreatic neuroendocrine tumors. Indeed, compared with computed tomography and ultrasound, OctreoScan® often shows more lesions and thus modifies tumor staging and changes surgical strategy in a substantial number of cases. For the detection of gastrinomas, OctreoScan® is even superior to all other imaging techniques [5]. OctreoScan® is also very reliable in imaging paragangliomas as well as pituitary adenomas, meningiomas, and medulloblastomas; however, for the latter three, its value is limited because other radiographic methods are already established. Conversely, the sensitivity of OctreoScan® is lower compared with other imaging techniques for tumors with inhomogeneous sst expression, such as breast carcinomas [19]. Moreover, OctreoScan® has a relatively low sensitivity for tumors that lie next to tracer accumulating organs like the kidney, such as primary renal cell carcinomas, limiting the usefulness of OctreoScan® to detection of metastatic spread of these tumors. Somatostatin Receptor Targeted Tumor Therapy Radiolabeled and cytotoxic somatostatin analogs may be used to specifically target tumoral sst. They will be internalized into the cells and develop their radiotoxic and cytotoxic effects in tumor cells and tumor vessels. Such an sst targeting represents a very promising approach in cancer therapy, with the advantage of a more favorable benefit-toxicity profile compared with conventional radio- and cytotoxic therapy [11, 23]. For sst targeted radiotherapy, somatostatin analogs coupled with a radioisotope like 111In, 90Y, or 177Lu have been used [11]. For instance, 90Y-DOTATOC achieved in animals complete tumor destruction dependent on tumor size. Moreover, preliminary clinical trials on patients with advanced malignant gastroenteropancreatic neuroendocrine tumors treated with 90Y-DOTATOC yielded tumor shrinkage in 10–30% as well as clinical improvement in over half of the patients. It is very likely that sst targeted radiotherapy will be clinically effective also in other human tumors with high sst expression. A relative limitation of sst targeted radiotherapy is the renal toxicity of the radionuclide compounds, a problem that has been given full consideration for instance by routinely performing renal protection by infusing amino acid solutions together with the radiotracer. Sst targeted cytotoxic therapy is an alternative approach unimpaired by this renal side effect. The most active compound developed for this purpose is AN-238, a doxorubicin derivative coupled to a somatostatin analog. AN-238 has so far been tested only in

438 / Chapter 62 animal models—in particular in human cancer cell lines xenografted in nude mice [23]. It achieved significant growth inhibition of a wide variety of therapeutically challenging tumors, including breast carcinomas, ovarian adenocarcinomas, renal cell carcinomas, prostate carcinomas, SCLC and NSCLC, glioblastomas, gastroenteropancreatic carcinomas, and hepatocellular carcinomas. Importantly, intrinsic tumor resistance to conventional chemotherapy may be overcome if chemotherapeutics are delivered directly into the tumor cell via sst. Indeed, AN-238 was shown to be effective in an anthracycline resistant renal cell carcinoma [23]. The first clinical trials on sst targeted cytotoxic tumor therapy in humans are foreseen in the near future [23].

NPY Neuropeptide Y (NPY) was first described and characterized in 1982 by Tatemoto and coworkers [24]. It forms together with the structurally related peptide YY (PYY) and pancreatic polypeptide (PP) the NPY family of peptides. NPY is primarily a neurotransmitter with a wide distribution in the central, peripheral, and vegetative nervous system, where it regulates many diverse functions—for instance, feeding and anxiety behavior, reproduction, and blood pressure. While these functions have been extensively investigated and are discussed elsewhere in this book, evidence of a role of NPY in cell proliferation, angioneogenesis, and, more generally, neoplasia has only recently been emerging. This is the subject of the present review.

NPY Effects on Normal and Tumor Cell Proliferation An effect of NPY on cell proliferation has been observed in normal and tumor cells. In normal cells, NPY generally stimulates cell proliferation, which so far has been demonstrated only in animal cell cultures, including rat olfactory neuronal precursor cells, guinea pig retinal glial cells, mouse pancreatic β-cells, and rat vascular smooth muscle cells. The NPY receptor subtype Y1 seems to be mainly involved. Of note, Y1 can mediate both a proliferative and an antiproliferative effect, depending on the experimental conditions: Low concentrations of NPY inhibited DNA synthesis, whereas high concentrations stimulated it in various cell cultures. In contrast to the predominantly growth-promoting effects of NPY on normal cells, the results in tumor cells are more variable and depend on several parameters. An inhibition of tumor cell growth was reproducibly

observed in the Y1 expressing human neuroepithelioma cell line SK-N-MC [7, 21]. Similar results were also obtained in an NPY receptor subtype Y5 expressing human cholangiocarcinoma cell line. Conversely, a mitogenic and/or growth stimulating effect of NPY was reported in the NPY receptor subtype Y2 expressing human neuroblastoma cell line SK-N-BE(2) [7], in NPY receptor-expressing human and hamster pancreatic cancer cell lines, as well as in the SK-N-MC cell line. Therefore, NPY may have different effects on tumor cell proliferation, depending on the tumor cell type and on the NPY receptor subtypes.

NPY Effects on Angioneogenesis There is increasing data suggesting that NPY induces angioneogenesis. It was demonstrated in multiple in vitro and in vivo models that NPY can induce the formation of new vessels in normal and ischemic tissues, probably via complex activation of several NPY receptors, including Y2 and Y1. Furthermore, NPY may also play a role in tumoral angioneogenesis: Tumor cell lines xenografted in mice showed increased tumor vascularity after treatment with NPY [7].

NPY Receptors: Subtypes, Molecular Signaling The actions of NPY are mediated by at least five different NPY receptor subtypes that form the NPY receptor family [15]. In humans, the subtypes Y1, Y2, Y4, and Y5 are expressed, whereas a physiological correlate of y6 has not been described. The existence of the putative Y3 receptor remains to be proven. All NPY receptor subtypes are members of the G-protein-coupled receptor superfamily. The NPY receptors almost universally act via pertussis toxin-sensitive Gi- and Go-proteins and inhibit adenylyl cyclase; in a restricted number of cell types they modulate calcium and potassium channels. The intracellular signaling pathways downstream to Gprotein-coupling are very incompletely characterized and may vary depending on NPY receptor subtypes, cell lines, tissues, and species [16]. Regarding NPY mediated cell proliferation, there is increasing data indicating involvement of the MAP kinase ERK. ERK phosphorylation was shown to be effected either via a Gi- and phosphatidylinositol 3-kinase (PI3K)-dependent pathway in Y1, Y2, Y4, and Y5 transfected CHO cell lines and in endogeneously Y1 expressing human and animal cell lines or via a Go- and protein kinase C (PKC)dependent pathway in a Y5 transfected CHO cell line and in various endogeneously Y1 expressing animal cell cultures. Conversely, tumor growth reduction was shown to be related to Y1-mediated activation of proapoptotic caspases-3/7.

Somatostatin and NPY / 439

NPY Receptors and Human Tumors Only very recently was it recognized that NPY receptors are expressed not only in normal tissues and in tumor cell lines but also in human tumor tissues. These findings may be of considerable importance in view of the potential clinical applications of NPY analogs.

NPY Receptor Expression in Human Tumors NPY receptors have been described in various human tumors. Like somatostatin receptors, they were found to be expressed predominantly in (neuro-)endocrine and epithelial tumors, and also in embryonal and rare mesenchymal tumors. Regarding endocrine active tumors, NPY receptors were highly expressed in adrenal cortical tumors, pheochromocytomas, paragangliomas [8], and ovarian granulosa cell tumors and SertoliLeydig cell tumors [9]. In epithelial malignancies, NPY receptors were found in breast cancer, renal cell carcinomas, and ovarian adenocarcinomas [9, 10, 21]. Breast TABLE 1.

carcinomas are particularly noteworthy because of their extremely high NPY receptor expression. Moreover, NPY receptors were also found in high incidence in several embryonal tumors—namely neuroblastomas and nephroblastomas [8, 10], in accordance with NPY receptor expression during human fetal development. Conversely, in gastrointestinal stromal tumors (GIST), NPY receptors were identified in a relatively small number of cases. It is of particular interest to mention that NPY receptors were identified not only on tumor cells but also often in the muscular wall of intra- and peritumoral blood vessels [8–10]. Regarding NPY receptor subtypes, tumor cells expressed predominantly Y1 and/or Y2, whereas tumor blood vessels expressed only Y1. Expression of significant amounts of Y4 or Y5 could be ruled out with pharmacological displacement experiments [8–10, 21]. Some tumors expressed the same NPY receptor subtype like their precursors, as was the case for adrenal cortical tumors and nonneoplastic adrenal cortex, and renal cell carcinomas and nonneoplastic kidney tubules [8,

Somatostatin, NPY, their respective receptors, and cancer. Somatostatin

Peptide structure Peptide distribution

Receptors Cancers with peptide production Cancers with receptor expression

Peptide action on tumors

Clinical applications in oncology

Tools

14 and 28 amino acids central nervous system, gastrointestinal tract, endocrine systems sst1, sst2A, sst2B, sst3, sst4, sst5 somatostatinoma, neuroblastoma, pheochromocytoma gastroenteropancreatic, lung and thyroid neuroendocrine tumors, pituitary adenomas, pheochromocytomas, paragangliomas, neuroblastomas, medulloblastomas, meningiomas, breast carcinomas, prostatic carcinomas, renal cell carcinomas, gastric adenocarcinomas, lymphomas ↓ hormone secretion ↓ cell proliferation ↓ angioneogenesis • symptomatic therapy of hormone secreting neuroendocrine tumors (S) • diagnostic tumor visualization (D) • radiotherapeutic tumor targeting (R) • cytotoxic tumor therapy (C) • octreotide (Sandostatin®), lanreotide (S) • 111In-octreotide (OctreoScan®) (D) • 90Y-DOTATOC (R) • AN-238 (C)**

*Depending on tissue and NPY receptor subtype. **Preclinical use only.

NPY 36 amino acids central and peripheral nervous system, adrenal gland Y1, Y2, Y4, Y5, y6 paraganglioma, pheochromocytoma, neuroblastoma breast carcinomas, ovarian adenocarcinomas, ovarian granulosa cell tumors, ovarian Sertoli-Leydig cell tumors, adrenal cortical tumors, pheochromocytomas, paragangliomas, neuroblastomas, renal cell carcinomas, nephroblastomas, GIST ↓/↑ cell proliferation* ↑ angioneogenesis none identified yet

• daunorubicin-coupled Y1 analog** • 99mTc-labeled Y2 analog**

440 / Chapter 62 10]. Conversely, some tumors expressed an NPY receptor subtype other than their nonneoplastic counterparts. For instance, normal breast ducts and lobules expressed Y2, whereas ductal and lobular breast carcinomas expressed predominantly Y1 [21], suggesting a switch in NPY receptor subtype expression during neoplastic transformation. The in vivo significance of the NPY receptors expressed by tumor cells and tumor blood vessels is unknown up to now. A functional role is however suggested by the presence of endogeneous NPY in close proximity to tumoral NPY receptors. Indeed, in kidney tumors, NPY-containing nerve fibers were observed next to tumor cells and tumor blood vessels, suggestive of an interaction of the peripheral nervous system with renal tumors via NPY [10]. Moreover, concomitant NPY peptide production and NPY receptor expression was observed in tumor cells in pheochromocytomas, paragangliomas, and neuroblastomas [8], as well as in a human neuroblastoma cell line [7], suggesting an autocrine effect of NPY on tumor cells and a paracrine effect on tumor blood vessels. Interaction of NPY with tumoral NPY receptors could possibly affect tumor cell proliferation or apoptosis, tumoral blood supply, or tumoral angioneogenesis. NPY Receptors and Tumor Targeting In analogy to somatostatin receptor targeting of tumors, it has been proposed to use NPY analogs to target NPY receptors for tumor therapy. As the effects of NPY itself on tumor proliferation are not yet fully elucidated, an NPY receptor-targeted radiotherapy or cytotoxic tumor therapy with radiolabeled or cytotoxic NPY analogs is more timely and promising. NPY analogs suitable for this purpose have indeed already been developed, such as a Y1-selective, daunorubicin-coupled cytotoxic NPY analog [13] and a Y2-selective, 99mTclabeled radioactive NPY analog [14], which would be ready for pilot studies in tumors. Breast tumors with their high receptor density would represent first choice candidate tumors. The same general principles as for somatostatin receptor targeting could be applied. Advantages that should be put forward are a more favorable benefit-toxicity profile compared with conventional radio- or chemotherapy and the rarity of side effects. The targeting of tumor blood vessels alone or together with NPY receptor expressing tumor cells may also represent an attractive strategy for therapy. Finally, as many of the NPY receptor expressing tumors can express multiple peptide receptors concomitantly, NPY receptors may be suitable for a multireceptor targeting with a cocktail containing NPY and other therapeutic peptide analogs directed against various peptide hormone receptors. For such a multireceptor approach,

good candidate tumors seem to be breast tumors targeted with NPY and bombesin analogs.

References [1] Arnold R, Simon B, Wied M. Treatment of neuroendocrine GEP tumours with somatostatin analogues: a review. Digestion 2000;62:84–91. [2] Burgus R, Ling N, Butcher M, Guillemin R. Primary structure of somatostatin, a hypothalamic peptide that inhibits the secretion of pituitary growth hormone. Proc Natl Acad Sci USA 1973;70:684–8. [3] Delesque N, Buscail L, Esteve JP, Saint-Laurent N, Muller C, Weckbecker G, Bruns C, Vaysse N, Susini C. sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells. Cancer Res 1997;57:956–62. [4] Denzler B, Reubi JC. Expression of somatostatin receptors in peritumoral veins of human tumors. Cancer 1999;85: 188–98. [5] Gibril F, Reynolds JC, Doppman JL, Chen CC, Venzon DJ, Termanini B, Weber HC, Stewart CA, Jensen RT. Somatostatin receptor scintigraphy: its sensitivity compared with that of other imaging methods in detecting primary and metastatic gastrinomas. Ann Intern Med 1996;125:26–34. [6] Hejna M, Schmidinger M, Raderer M. The clinical role of somatostatin analogues as antineoplastic agents: much ado about nothing? Ann Oncol 2002;13:653–68. [7] Kitlinska J, Abe K, Kuo L, Pons J, Yu M, Li L, Tilan J, Everhart L, Lee EW, Zukowska Z, Toretsky JA. Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors. Cancer Res 2005;65:1719–28. [8] Körner M, Waser B, Reubi JC. High expression of NPY receptors in tumors of the human adrenal gland and extraadrenal paraganglia. Clin Cancer Res 2004;10:8426–33. [9] Körner M, Waser B, Reubi JC. Neuropeptide Y receptor expression in human primary ovarian neoplasms. Lab Invest 2004;84:71–80. [10] Körner M, Waser B, Reubi JC. Neuropeptide Y receptors in renal cell carcinomas and nephroblastomas. Int J Cancer 2005;115:734–41. [11] Kwekkeboom DJ, Mueller-Brand J, Paganelli G, Anthony LB, Pauwels S, Kvols LK, O’Dorisio TM, Valkema R, Bodei L, Chinol M, Maecke HR, Krenning EP. Overview of results of peptide receptor radionuclide therapy with 3 radiolabeled somatostatin analogs. J Nucl Med 2005;46:62S–6S. [12] Lahlou H, Guillermet J, Hortala M, Vernejoul F, Pyronnet S, Bousquet C, Susini C. Molecular signaling of somatostatin receptors. Ann N Y Acad Sci 2004;1014:121–31. [13] Langer M, Kratz F, Rothen-Rutishauser B, Wunderli-Allenspach H, Beck-Sickinger AG. Novel peptide conjugates for tumorspecific chemotherapy. J Med Chem 2001;44:1341–8. [14] Langer M, La Bella R, Garcia-Garayoa E, Beck-Sickinger AG. 99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents. Bioconjug Chem 2001;12:1028–34. [15] Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T. XVI. International union of pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 1998;50:143–50. [16] Mullins DE, Zhang X, Hawes BE. Activation of extracellular signal regulated protein kinase by neuropeptide Y and pancreatic polypeptide in CHO cells expressing the NPY Y(1), Y(2), Y(4) and Y(5) receptor subtypes. Regul Pept 2002;105: 65–73. [17] Reichlin S. Somatostatin. N Engl J Med 1983;309:1556–63.

Somatostatin and NPY / 441 [18] Reubi JC. A somatostatin analogue inhibits chondrosarcoma and insulinoma tumour growth. Acta Endocrinol (Copenh) 1985;109:108–14. [19] Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 2003;24:389–427. [20] Reubi JC, Gugger M, Waser B. Coexpressed peptide receptors in breast cancers as molecular basis for in vivo multireceptor tumor targeting. Eur J Nucl Med 2002;29:855–62. [21] Reubi JC, Gugger M, Waser B, Schaer JC. Y1-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer Res 2001;61:4636–41.

[22] Schally AV. Oncological applications of somatostatin analogues. Cancer Res 1988;48:6977–85. [23] Schally AV, Nagy A. Chemotherapy targeted to cancers through tumoral hormone receptors. Trends Endocrinol Metab 2004;15:300–10. [24] Tatemoto K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA 1982;79: 5485–9. [25] Woltering EA. Development of targeted somatostatin-based antiangiogenic therapy: a review and future perspectives. Cancer Biother Radiopharm 2003;18:601–9.

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63 Bradykinin and Cancer JOHN M. STEWART AND LAJOS GERA

ABSTRACT

KININ CHEMISTRY AND BIOLOGY

Bradykinin is a pluripotent factor for cancer growth. It stimulates cancer cell growth directly and also stimulates angiogenesis by stimulating release of vascular endothelial growth factor as well as facilitating tumor invasion and migration by stimulating action of matrix metalloproteases. Bradykinin antagonists, both peptides and nonpeptide mimetics, are able to block all three aspects of bradykinin function in tumors. These antagonists offer great promise as new anticancer agents.

Two kinin precursor proteins, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK), are synthesized in the liver and circulate normally in the bloodstream [4]. Kinin peptides are processed from these precursors by two trypsin-like enzymes, plasma kallikrein, and tissue (glandular) kallikrein. Plasma kallikrein normally circulates as an inactive precursor but is rapidly activated concomitantly with initiation of blood clotting by contact with a negatively charged surface such as basement membranes (as occurs in wounds,) or by lowered pH (as occurs in hypoxia or infection). It then acts on HMWK to produce BK. Tissue kallikrein can be released from a variety of organs such as pancreas, salivary glands, and kidney. It acts mainly on LMWK (but also on HMWK) to produce kallidin. In the circulation BK is rapidly metabolized by several enzymes. Most important is angiotensin I converting enzyme (ACE), localized primarily to the pulmonary vascular wall, where it normally cleaves more than 90% of circulating BK on a single passage. ACE removes Cterminal dipeptides from the kinins, totally inactivating them. C-terminal arginine residues are removed from the kinins by plasma carboxypeptidases such as carboxypeptidase N (CPN). The products lacking the Arg residue are inactive on kinin B2 receptors, which require the C-terminal arginine residue, but are the normal agonists for kinin B1 receptors. In some inflammatory conditions ACE is inactivated or lost, and most circulating kinins are then processed by carboxypeptidases to yield kinin B1 agonists. Neutral endopeptidase (NEP) is also membrane-bound; it cleaves kinins at the internal phenylalanine residue (Phe5 in BK). Aminopeptidases also cleave kinins. With this battery of degrading enzymes present, it is not surprising that BK has a circulating lifetime of only a few seconds.

INTRODUCTION The kinin peptides bradykinin (Arg-Pro-Pro-GlyPhe-Ser-Pro-Phe-Arg; BK) and its homolog kallidin (Lys-BK) have been studied mainly for their roles in inflammation, where they are primary mediators and stimulate the release of many other agents. Among these inflammatory agents whose release is stimulated by BK are growth factors critical to such inflammatory events as wound repair. Many tumor types have mutated to stimulate increased production of BK and expression of BK receptors. These activities work together for autocrine stimulation of tumor growth. In tumors, BK stimulates growth directly and also stimulates neovascularization of tumors by stimulating release of vascular endothelial growth factor (VEGF). BK also facilitates tumor migration and invasion by stimulating release of matrix metalloproteases (MMPs). Thus, BK is a pluripotent agent for stimulating tumor growth and invasion. BK antagonists, both peptides and peptidomimetics, are under investigation as potential new anticancer drugs. They offer the unique potential of triple combination cancer chemotherapy with a single agent. Handbook of Biologically Active Peptides

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444 / Chapter 63 Biological actions of kinins are mediated by two G protein-coupled receptors, designated B1 and B2 [4, 11]. B2 receptors are constitutively expressed in a wide variety of tissues; they require the full kinin chain for agonist action. In contrast, B1 receptors are normally not present in most tissues, but their expression is rapidly induced in inflammation. They are thus particularly important in chronic inflammation. Kinin B1 receptors are not activated by intact kinins, but their preferred ligands are the kinin metabolites lacking the C-terminal arginine residue, produced by carboxypeptidases. Both receptor types are functional in advanced prostate cancers [3]. In various tissues, kinin receptors are linked to a wide variety of second messenger systems. Activation of phospholipase C leads to elevated intracellular free calcium concentration, stimulating cells for a wide variety of activities. Inositol trisphosphate is also liberated in this pathway and leads to growth factor stimulation. Particularly important for cancer growth is stimulation of the JUN kinase-MAP kinase pathway, which causes cell proliferation. Also important for cancer is the link to phospholipase A2 leading to prostaglandin production; prostaglandins are tumor growth factors.

BRADYKININ ANTAGONISTS Antagonists for kinin B1 receptors were discovered by Regoli in 1977 [14]. The key change in the BK(1-8) molecule to produce antagonists was replacement of the C-terminal phenylalanine residue with an aliphatic residue, such as leucine. The first antagonists for B2 receptors were reported by Vavrek and Stewart in 1985 [22]. In that case the critical change was replacement of the 7-proline residue with an aromatic amino acid of the D-configuration. The resulting DPro7-BK was a very weak partial antagonist, but additional changes gave

TABLE 1. Bradykinin: (B2 agonist) Kallidin: (B2 agonist) Modern potent agonist 1st Generation antagonist 2nd Generation antagonist 3rd Generation antagonist Modern B1-B2 antagonist Modern B1 antagonist Modern B1 antagonist Anti-cancer dimer

large increases in receptor affinity and gave the first useful B2 antagonist, known as NPC-349. (See Table 1 for structures of peptides.) In NPC-349 the D-phenylalanine residue at position seven blocks cleavage by ACE, and the N-terminal D-arginine residue blocks aminopeptidase action, but this peptide was still cleaved by CPN and NEP. As a result it has an effective plasma halflife in rats of only a few minutes. A major improvement in potency and lifetime of B2 antagonists was made by investigators at Hoechst, who introduced HOE-140, which has a D-tetrahydroisoquinoline-3-carboxylic acid (D-Tic) residue at position seven and an octahydroindole-2-carboxylic acid (Oic) residue at position eight [10]. HOE-140 is not cleaved by CPN and has been used in clinical trials for asthma and angioedema [2]. In contemporary B2 antagonists such as B-9430 the final enzymatic cleavage site, that by NEP at the 5-6 bond, is blocked by incorporation of an indanylglycine (Igl) residue at position five [20]. B-9430 is extremely potent and is not degraded by incubation with plasma or by lung or kidney homogenates. In rats or guinea pigs a single subcutaneous injection of 100 μg/kg blocks systemic BK action for more than two days. In rats B9430 shows about 10% oral bioavailability [18]. Perhaps most remarkable is that B-9430 also shows good antagonism at B1 receptors, even though it has the C-terminal arginine residue that normally blocks B1 action. A related antagonist, B-9340, has been used in humans [23]. There has also been progress in improving B1 antagonists. Modifications to block enzymic degradation similar to those made in B2 antagonists have yielded excellent, long-lasting compounds. For example, B9958 (see Table 1) is extremely potent, and its action is limited only by slow aminopeptidase action [9]. Changing the N-terminal lysine residue to the D-configuration yielded a B1 antagonist with very long persistence of action (B-10352) [12].

Peptide ligands for kinin receptors.

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg DArg-Arg-Pro-Hyp-Gly-Igl-Ser-Oic -Igl-Arg DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DIgl-Oic-Arg DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg Lys-Lys-Arg-Pro-Hyp-Gly-CpG-Ser-DTic-CpG DLys-Lys-Arg-Pro-Hyp-Gly-CpG-Ser-DTic-CpG SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg)2

B-9972 NPC-349 HOE-140 B-9340 B-9430 B-9958 B-10352 B-9870

Abbreviations: CpG: cyclopentylglycine; Hyp: 4-hydroxyproline; Igl: α-(2-indanyl)glycine; Oic: octahydroindole-2-carboxylic acid; Tic: tetrahydroisoquinoline-3-carboxylic acid; Thi: β-(2-thienyl)alanine; SUIM: suberimidyl crosslinker at N-terminus.

Bradykinin and Cancer Incorporation of enzyme-resistant amino acids into the native BK structure has yielded modern agonists such as B-9972, which is resistant to degradation and shows remarkable persistence of action in vivo [21]. Discovery of nonpeptide antagonists of peptide hormones has long been a major goal of investigators. Discovery of nonpeptide BK B2 antagonists was first announced by investigators at Sterling-Winthrop [16] and subsequently also from Fujisawa [1], Sanofi [13], and Novartis [8]. Development of BK nonpeptide antagonists in the Stewart laboratory has followed the Sterling-Winthrop lead, has been directed at anticancer agents, and has led to some highly effective compounds such as BKM-296 and BKM-570 [19]. Nonpeptide antagonists have also been discovered for B1 receptors [15].

BRADYKININ AND CANCER The presence of receptors for BK and other neuropeptides in lung cancer was reported in 1989 [24]. Since that time, evidence has been presented for the presence of BK receptors on a wide variety of cancers, notably small cell (SCLC) and nonsmall cell (NSCLC) lung cancer [5] and various prostate cancer (PC) cell lines [17]. Some clinical work was done with substance P antagonists in lung cancer, but the potency of the antagonists tried was low, and the results were not encouraging. BK antagonists seem to hold much more promise. The potent and enzyme-resistant BK antagonist B9430 was found to inhibit BK-evoked increase in intraTABLE 2. Compound Number B-9430 B-9870 B-10324 B-10396 BKM-296 BKM-570 BKM-638

a

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cellular calcium concentration in SCLC cells in culture, but it did not inhibit cell growth. However, when B-9430 was crosslinked with a suberimide linker at the amino terminus, the resulting dimer, B-9870 (also known as CU-201), was found to be a very potent stimulus for apoptosis in cultured SCLC, and it inhibited tumor growth of SCLC xenografts in athymic nude mice [7] (see Table 2). This peptide is currently in development at the U.S. National Cancer Institute. It shows activity in vitro against a broad range of cancer types. In xenografts it is much more potent against SCLC than standard anticancer agents such as cisplatin, and does not produce toxic side effects. This antagonist stimulates apoptosis in SCLC cells by an unusual “biased agonist” mechanism; it inhibits the Gq,11-mediated pathway that leads to increased intracellular calcium concentration but stimulates the G12,13-mediated pathway linked to JUN-kinase that stimulates growth factor action [6]. Although B-9430 did not have significant anticancer activity, certain derivatives of single chain B2 and B1 antagonists have been found to be active in vivo against prostate cancer (Table 2). In search of more druglike BK antagonists as potential anticancer agents, work in the Stewart laboratory has concentrated on a series of nonpeptides. These are generally derivatives of acylated tyrosine amides (Table 2) [19]. Lead compound BKM-570 is much less potent a BK antagonist on isolated guinea pig ileum than is the peptide B-9430, and in vitro it is less potent against SCLC than is the peptide dimer B-9870, but it is a very potent inhibitor of tumor growth of several cancers in vivo in nude mouse xenografts. This impressive activity in vivo is probably related to its potent inhibition of

Inhibition of tumor growth by bradykinin antagonists. Biological Activity

Structure

GPIa

SHP-77b

PC3b

MM2b,c

D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg SUIM-(B9430)2 F5c-Lys-Lys-Arg-Pro-Hyp-Gly-CpG-Ser-DTic-CpG F5c-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-PFF-Arg Pya-Bip-Atmp F5c-OC2Y-Atmp DDD-(D-Arg-Igl-Arg-Matp)2 Cisplatin Taxotere

8.2 8.4 — 6.2 — 5.6 <4.0

15% 65 86 — — 91 78d 60 49

— 78% 43 — 57 65 52d 39 42

— 33% — 53 — 46 57d — —

pA2 for bradykinin antagonist activity on isolated guinea pig ileum, for reference. Percent inhibition of growth of xenografts in nude mice. Compounds were injected i.p. at 5 mg/kg/day; except for BKM-638, which was given at 1.0 mg/kg/day. Cisplatin and Taxotere were given at the maximum tolerated doses. c PC3 strain MM2. d Injected at 1 mg/kg/day; the compound was toxic at 5 mg/kg/day. Abbreviations: Atmp 4-amino-2,2,6,6-tetramethylpiperidine; Bip, 3-(4-biphenylyl)alanine; DDD, dodecandioyl; F5c, 2,3,4,5,6pentafluorocinnamoyl; Matp, 4-(methylamino)-2,2,6,6-tetramethylpiperidine; OC2Y, O-(2,6-dichlorobenzyl)tyrosine; Pya, 3-(3pyridyl)acryloyl; PFF, p-fluorophenylalanine. For other abbreviations, see Table 1. b

446 / Chapter 63 angiogenesis and of MMP action in treated tumors in addition to its direct inhibition of cell growth. Tunel staining of sections of treated tumors shows markedly increased cell death, and staining for CD-31, a vascular endothelial marker, shows greatly diminished vascularity [19]. Extracts of treated tumors show much less MMP activity than do extracts of control tumors. The low water solubility of BKM-570 is a disadvantage, as some DMSO has been used in solutions for injection. This disadvantage has been overcome in BKM-296, which has similar potency and is water-soluble. This remarkable combination of activities in these compounds provides in effect triple combination cancer therapy with one compound and suggests that they have great potential for drug development.

References [1] Abe Y, Kayakiri H, Satoh S, Inoue T, Sawada Y, Inamura N, et al. A novel class of orally active non-peptide bradykinin B2 antagonists. J Med Chem 1998;41:4053–61. [2] Austin CE, Foreman JC, Scadding GK. Reduction by HOE-140, the B-2 kinin receptor antagonist, of antigen-induced nasal blockage. Br J Pharmacol 1994;111:969–71. [3] Barki-Harrington L, Daaka Y. Kinin-regulated growth of androgen-insensitive prostate cancer PC3 cells requires direct interaction between bradykinin 1 and bradykinin 2 receptors. J Urol 2001;165:2121–25. [4] Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 1992;44:1– 80. [5] Bunn PA Jr, Chan DC, Dienhart DG, Tolly R, Togawa M, Jewett PB. Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity. Cancer Res 1991;52:24–31. [6] Chan D, Gera L, Stewart J, Helfrich B, Verella-Garcia M, Johnson G, et al. Bradykinin antagonist dimer CU-201 inhibits the growth of human lung cancer cells by a novel “biased agonist” mechanism. Proc Natl Acad Sci USA 2002;99:4608–13. [7] Chan D, Gera L, Stewart J, Helfrich B, Zhao TL, Feng WY, et al. Bradykinin antagonist dimer CU-201 inhibits the growth of human lung cancer cell lines in vitro and in vivo and produces synergistic growth inhibition in combination with other antitumor agents. Clin Cancer Res 2002;8:1280–87. [8] Dziadulewicz EK, Ritchie TJ, Hallett A, Snell CR, Ko SY, Wrigglesworth R, et al. 1-(2-nitrophenyl)thiosemicarbazides: a novel class of potent, orally active non-peptide antagonist for the bradykinin B2 receptor. J Med Chem 2000;43:769–71.

[9] Larrivee JF, Gera L, Houle S, Bouthillier J, Bachvarov DR, Stewart JM, et al. Non-competitive pharmacological antagonism at the rabbit B1 receptor. Brit J Pharmacol 2000;131:885–92. [10] Lembeck F, Griesbacher T, Eckhardt M, Menke S, Breipohl G, Knolle J. New, long-acting, potent bradykinin antagonists. Br J Pharmacol 1991;102:297–304. [11] Marceau F, Hess JF, Bachvarov DR. The B1 receptors for kinins. Pharmacol Rev 1998;50:357–86. [12] Marceau F. Personal communication. [13] Pruneau D, Paquet JL, Luccarini JM, Defrêne E, Fouchet C, Franck, et al. Pharmacological profile of LF 16-0687, a new potent non-peptide bradykinin antagonist. Immunopharmacol 1999;43:187–94. [14] Regoli D, Barabe J, Park WK. Receptors for bradykinin in rabbit aorta. Can J Physiol Pharmacol 1977;55:855–67. [15] Ritchie TJ, Dzaidulewicz EK, Culshaw AJ, Muller W, Burgess GM, Bloomfield GC, et al. Potent and orally bioavailable nonpeptide antagonists at the human bradykinin B1 receptor. J Med Chem 2004;47:4642–44; Gougat J, Ferrari BSL, Planchenault C, Poncelet M, Mauani J, Alonso R, et al. J Pharmacol Exp Therap 2004;309:661–69. [16] Salvino JM, Seoane PR, Douty BD, Awad MMA, Dolle RE, Houck WT, et al. Design of potent, non-peptide antagonists of the human bradykinin B2 receptor. J Med Chem 1993;36:2583–84. [17] Stewart JM. Bradykinin antagonists as anti-cancer agents. Curr Pharmaceut Design 2003;9:2036–42. [18] Stewart JM, Gera L, Chan DC, Whalley ET, Hanson WL, Zuzack JS. Potent, long-acting, orally active bradykinin antagonists for a wide variety of applications. Immunopharmacol 1997;36: 167–72. [19] Stewart JM, Gera L, Chan DC, York EJ, Simkeviciene V, Bunn PA Jr, et al. Combination cancer chemotherapy with one compound: pluripotent bradykinin antagonists. Peptides 2005;26:1288–91. [20] Stewart JM, Gera L, Hanson W, Zuzack JS, Burkard M, McCullough R, et al. A new generation of bradykinin antagonists. Immunopharmacol 1996;33:51–60. [21] Taraseviciene-Stewart L, Scerbavicius R, Stewart JM, Gera L, Demura Y, Cool C, et al. Treatment of severe pulmonary hypertension: a bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy. Peptides 2005;26:1292–300. [22] Vavrek RJ, Stewart JM. Competitive antagonists of bradykinin. Peptides 1985;6:161–64. [23] Witherow FN, Helmy A, Webb DJ, Fox KAA, Newby DE. Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure. Circulation 2001;104:2177–81. [24] Woll PJ, Rozengurt E. Multiple neuropeptides mobilize calcium in small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin, galanin and neurotensin. Biochem Biophys Res Commun 1989;164:66–73.

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64 Endothelin KATHERINE GRANT, MARILENA LOIZIDOU, AND IRVING TAYLOR

ABSTRACT

ET-1 ASSOCIATED SIGNAL TRANSDUCTION PATHWAYS

Endothelin-1 is a small vasoactive peptide that was first identified in 1988. It is one of a family of three endothelins that exert their action through two G-protein coupled receptors known as ETA and ETB. This chapter reviews the evidence implicating ET-1 in tumorigenesis. In particular, we concentrate on the role of ET-1 in mitogenesis, apoptosis, angiogenesis, tumor invasion, and metastasis. Evidence relating to downstream effectors is discussed, and trials relating to the potential for endothelin-system modulation as an adjuvant therapeutic strategy are reviewed.

The consequences of ET-1 induced receptor stimulation are complex in that many intracellular pathways can be activated (Fig. 1). Docking of ET-1 on its receptor results in dissociation of the α and βγ subunits of one of several possible associated G-proteins [27]. The activated G-protein may then phosphorylate one of several upstream pathway initiators, including phospholipase C and D, phospholipase A2, adenylate cyclase, and guanylate cyclase [5, 43, 47, 50]. The major downstream mitogenic pathways converge on mitogen-activated protein kinase (MAPK), and ultimately transcription of early response genes such as fos and jun [24]. The MAPK cascade may be activated by phosphatidylinositol 3-kinase (PI3-K) mediated stimulation of Ras [20] or by protein kinase C (PKC), either by direct targeted activation of Raf-1, or by Ca2+-mediated activation of Ras [9]. MAPK may also be activated secondary to tyrosine kinase receptor (TKR) transactivation by GPCRs. Epidermal growth factor receptor (EGFr) transactivation by ET-1 was first demonstrated in rat-1 fibroblasts [13] and has more recently been shown in human ovarian cancer [46]. ET-1 also potentiates the effect of platelet-derived growth factor (PDGF) on human smooth muscle cells, suggesting interactions with other TKRs [49].

ENDOTHELIN-1 The potent vasoconstrictor peptide endothelin 1 (ET-1) is one of a family of three multifunctional peptides (ET-1, -2, and -3). Of these isoforms, ET-1 has been the most extensively studied to date and has been most implicated in tumorigenesis. The ET physiological effect is exerted via ETA and ETB, which are G-protein coupled transmembrane receptors (GPCR) found in both vascular and nonvascular tissues. The ETA receptor has varying affinities for each isoform (ET-1 > ET-2 > ET-3), whereas the ETB receptor shows no selective affinity for any of the ET subtypes [39]. The endothelins have been implicated in numerous pathological conditions including hypertension and cardiac failure. Interest in the role of ET-1 in cancer has grown over the last decade, and currently there is evidence that ET-1 can modulate mitogenesis, apoptosis, angiogenesis, tumor invasion, and development of metastases. Handbook of Biologically Active Peptides

ENDOTHELIN EXPRESSION IN CANCER Elevated plasma levels of ET-1 have been detected in patients with various solid tumors, including gastric and prostate cancer, where levels are greatest in patients with metastastic, hormone refractory disease [17, 31].

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Copyright © 2006 Elsevier

448 / Chapter 64 Growth Factor

Na+ H+

ET1 TKR

PLA2

ETA

PLC

Na/H+ Porter

PLD

AS

GC

G IP3

Arachidonic acid

Cyclo-oxygenase products

PG+TX

DAG

Ca

Ras CAMP

CGMP

PKA

PKG

Raf

PKC

Lipo-oxygenase products

LT

PA

2+

MAPKinase Phosphorylation Event Physiological Effect

Contraction Relaxation

Mitosis/Differentiation (via fos/Jun)

Inflammation

FIGURE 1. Signal transduction pathways associated with stimulation of endothelin receptors. Upstream transduction molecules activated by G-proteins are shown in italics and include phospholipase A2 (PLA2), phospholipase C and D (PLC, PLD), tyrosine kinase receptors (TKR), adenylate cyclase (AS), and guanylate cyclase (GC). Downstream effectors include diacylglycerol (DAG), phospholipase A (PA), phosphatidyl inositol 3 kinase (IP3), cyclic AMP (CAMP), cyclic GMP (CGMP), protein kinase C, A and G (PKC, PKA, PKG), prostaglandins and thromboxanes (PG, TX), and leukotrienes (LT).

In colorectal cancer, elevated plasma levels of ET-1 have been demonstrated in patients with primary tumors with and without liver metastases [41]. Plasma levels of Big ET-1 have also been found to be significantly raised in colorectal cancer patients compared with controls [44]. This study also found that both preoperative and intraoperative portal plasma levels were significantly higher in metastatic disease. Many human cancer cell lines have been shown to synthesize ET-1, including colonic, breast, stomach, prostate, and glioblastoma cells [3, 25]. Similarly, in vivo, increased tissue immunoreactivity for ET-1 has been demonstrated in several cancer types, including ovarian, breast and colorectal tumors [1, 7]. Furthermore, expression of endothelin system components can be altered in pre-malignant tissues. In colorectal adenomas, increased expression of pre-pro ET-1 and ECE mRNA has been shown compared with normal colon [16]. ET-1 immunoreactivity in breast

ductal carcinoma in situ (DCIS) specimens is also significantly higher (P < 0.005) than that of normal breast tissue [1], suggesting that modulation of the endothelin system may be an early phenomenon in tumorigenesis.

ENDOTHELIN RECEPTOR EXPRESSION IN CANCER Increased ETA receptor expression in malignant tissue has been demonstrated using immunohistochemistry and/or autoradiography in several cancer types, including colorectal, ovarian, and prostate tumors [2, 7, 31]. In prostate tumors, levels of receptor expression have been found to correlate with the presence of metastases [19]. Of note, in normal tissue from these sites the ETB receptor predominates, whereas the ETA receptor becomes prevalent in both primary tumors and metas-

Endothelin / 449 tases. Interestingly, relative hypermethylation of the ETB gene has been demonstrated in several prostate, bladder, and colon cancer cell lines. Furthermore, this has also been found to correlate with transcriptional downregulation [33], providing a plausible mechanism for reduced ETB receptor expression in malignant tissue.

ENDOTHELIN AS A MITOGEN ET-1 has been shown to stimulate growth of several human cancer cell lines in vitro including colorectal, ovarian, and prostate cancer, Kaposi’s sarcoma, and melanoma cells. Several groups have demonstrated that this mitogenic effect is mediated via the ETA receptor [3, 6, 7, 23, 31, 31]. The growth of nonepithelial tumors, however, does not appear to be ETA dependent. Studies on human melanoma cells have shown that the mitogenic effect of ET-1 is purely ETB receptor dependent [23], whereas antagonism of either receptor partially inhibits in vitro growth of Kaposi’s sarcoma cells [6]. This has also been demonstrated in vivo, where the specific ETB antagonist (BQ788) was shown to significantly slow melanoma tumor growth in nude mice [26]. The role of ET-1 as an autocrine growth factor has been demonstrated in human ovarian and colon cancer cell lines [2]. A paracrine role for ET-1 has also been elucidated in ovarian cancer, where ET-1 production by human ovarian cancer cells stimulated growth of carcinoma associated fibroblasts in coculture, an effect that was partially inhibited by both ETA and ETB antagonism [30]. However, ET-1 had no effect on human colonic subepithelial myofibroblast proliferation, but contraction and migration were stimulated through ET receptor mediated myosin phosphorylation [22].

ENDOTHELIN AND APOPTOSIS In addition to its mitogenic effect, there is evidence that ET-1 may contribute to tumor growth by acting as an antiapoptotic agent. Peduto-Eberl and colleagues have demonstrated that ET-1 is a survival factor for rat colon carcinoma cells against FasL mediated apoptosis [36]. The same group has also shown that the dual endothelin receptor antagonist bosentan sensitizes resistant human colorectal cancer cells to FasL-induced, caspase mediated apoptosis [35]. In ovarian cancer, ET-1 protects cells from both serum-deprivation and paclitaxel-induced apoptosis, in part via suppression of Bcl-2 phosphorylation and PI3K mediated Akt activation [14]. In both melanocytes and melanoma cell lines, ET-1 has been shown to decrease basic apoptotic rates [15].

Like the proliferative action of ET-1 in these cells, the effect on apoptosis is ETB dependent.

ENDOTHELIN AND ANGIOGENESIS Endothelin-1 may also facilitate tumor growth through promotion of angiogenesis. Human umbilical vein endothelial cells (Huvecs) actively produce and secrete ET-1, while simultaneously expressing the ETB receptor, suggesting autocrine activity [18]. ET-1 is a potent mitogen for both endothelial cells and vascular smooth muscle cells (VSMC) [24] and can stimulate migration of endothelial cells via the ETB receptor [29]. ET-1 may also indirectly enhance endothelial cell proliferation through stimulation of vascular endothelial growth factor (VEGF) production by other cell types [34]. The reverse situation has also been demonstrated in endothelial cells, where VEGF enhanced ET-1 mRNA expression and ET-1 secretion [28]. Furthermore, ET-1 potentiates the effect of several proangiogenic factors in vitro, including PDGF and VEGF [34, 49]. In ovarian cancer cells, up-regulation of VEGF production has been shown, in part due to ET-1 induced prostaglandin signaling, particularly PGE2. COX-1 and COX-2 expression in human ovarian cancer cells is increased significantly by exogenous ET-1. This occurs via ETA receptor activation and stimulation of several MAPK-dependent signaling pathways, including p38 MAPK and p42/22 MAPK[45]. ET-1 also stimulates invasion and morphological differentiation of Huvecs in matrigel, and this may be facilitated via ET-1-induced production of matrix metalloproteinase-2 (MMP-2) by endothelial cells [40]. In addition to an effect on tumor cells, ET-1 is known to protect human endothelial cells and VSMCs from serum deprivation-induced apoptosis in vitro [42]. This suggests that ET-1 could have a proangiogenic effect by acting as a survival factor for newly formed blood vessels. In vivo data is conflicting concerning the putative role of ET-1 in angiogenesis. Early studies demonstrated that ET-1 was unable to stimulate angiogenesis in both a rat sponge model and the chick embryo choreoallentoic membrane (CAM) model. However, when combined with VEGF, ET-1 has been shown to stimulate angiogenesis in subcutaneously implanted matrigel plugs in mice [40]. ET-1 has also been shown to stimulate angiogenesis in a rat corneal model with a similar efficacy to VEGF [8]. Finally, ET-1 was shown to have an angiogenic effect in CAM using ET-1-secreting Chinese hamster ovary cells. In both these models the ET-1 angiogenic effect was inhibited by ETA or dual antagonists but not by ETB antagonism.

450 / Chapter 64 ENDOTHELIN-1 AND TUMOR PROGRESSION/METASTASES Further to its proangiogenic role, ET-1 may also influence tumor invasion and metastases by stimulating cancer cells to secrete matrix remodeling proteins. Specifically, ET-1 has stimulated expression of several MMPs, particularly MMP-2 and MMP-9, and downregulated tissue inhibitors of matrix metalloproteinases, TIMP1 and -2, in human ovarian carcinoma cells [38]. Similar upregulation of MMPs has also been shown in Kaposi’s sarcoma cells [37]. Furthermore, ET-1 can contribute to the creation of a “reactive” tumor stroma by stimulating myofibroblast induction (via ETA), and the expression of matrix remodeling genes by these cells [48]. In prostate cancer ET-1 may contribute to the growth of bony metastases. In vitro, ET-1 production by prostate cancer cells is enhanced by bone contact, which in turn blocks osteoclastic bone reabsorption [12]. Similarly, in an in vivo osteoblastic tumor model, tumors transfected to overexpress ET-1 produced significantly more bone growth in nude mice compared with vector only controls [32]. Furthermore, we have demonstrated increased ET-1 immunoreactivity in endothelial cells within colorectal liver metastases compared with surrounding vessels [41], suggesting that ET-1 may be involved in modulation of tumor blood flow.

ENDOTHELIN ANTAGONISM IN VIVO Several in vivo models have been used to assess the role of endothelin antagonism in tumorigenesis. Work originating from our department using intraportally injected syngeneic MC28 cells in rats demonstrated that ETA antagonism with BQ123 significantly reduced hepatic tumor load compared with controls [4]. The effect of bosentan, a dual receptor antagonist, on growth of peritoneal tumors derived from a syngeneic rat colonic adenocarcinoma cell line has been investigated [36]. Although bosentan was not able to control tumor progression, tumors were generally of lower grade, and there were fewer spontaneous deaths in the treated versus the untreated groups. Egidy and colleagues used the same tumor model to assess histological differences between tumors of bosentan treated animals and controls [16]. They demonstrated that tumor cells were less densely packed, and there was less collagen matrix around tumor nodules in the treated compared to the untreated group. Finally, using an osteoblastic tumor model in nude mice Nelson and colleagues have shown that ETA antagonism with A127722 significantly reduced the growth

of new bone compared with vehicle treated controls [32]. Although in vivo results have not so far yielded dramatic results, they are encouraging and warrant further investigation.

CLINICAL TRIALS Three phase I trials of the ETA receptor antagonist atrasentan have been undertaken in patients with refractory adenocarcinomas (reviewed in [21]). Side effects due to the physiological consequences of ETA blockade included headache, hypotension, and peripheral edema, which were generally tolerated up to a maximum dose of 60 mg. A phase II randomized, placebo-controlled trial in 288 patients with asymptomatic hormone-resistant metastatic prostate cancer evaluated three groups: placebo, 2.5 mg, or 10 mg atrasentan [11]. Delayed time to progression (TTP) was observed in the 10 mg group and stabilization of biochemical markers, including prostate specific antigen and lactate dehydrogenase compared with controls. A phase III trial in a similar population of 809 prostate cancer patients has shown a nonsignificant delay in TTP compared with controls [10]. However, secondary endpoint analysis has demonstrated significantly delayed progression of bone acid phosphatase levels and preserved prostate cancerspecific quality of life, particularly in terms of painrelated symptoms. As ET-1 appears to influence metastatic spread in prostate cancer, a further phase III clinical trial is ongoing to evaluate the role of atrasentan in delaying metastatic spread in hormone refractory nonmetastatic disease. Other ETA antagonists in phase II clinical trials include YM598 and AZD4054. The endothelin axis is altered in cancer, aiding tumor growth and progression. It seems that selective ETA antagonism provides the most effective method of endothelin system inhibition in cancer. With generally acceptable side effects, and suggested antitumor activity, further clinical evaluation of these agents is warranted to determine possible therapeutic potential as an adjuvant anticancer strategy.

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65 Adrenomedullin: An Esoteric Juggernaut of Human Cancers FRANK CUTTITTA, SERGIO PORTAL-NÚÑEZ, CHRISTIE FALCO, MERCHE GARAYOA, RUBÉN PÍO, LUIS M. MONTUENGA, MIGUEL JULIÁN, ALFREDO MARTÍNEZ, AND ENRIQUE ZUDAIRE

AM is up-regulated in a variety of human malignancies of both neural and epithelial origins, being produced by cancers of the brain, lung, colon, breast, ovary, uterus, prostate, skin, kidney, and eye [5, 50]. It has been shown that a patient’s serum AM level begins to rise with the onset of lung and gastrointestinal tumor progression [6]. In addition, elevated AM levels in tumor tissue or patient serum indicate a poor clinical prognosis for individuals having prostate or ovarian cancer [11, 38]. Hypoxia and chronic inflammation have been identified as major biological anomalies leading to neoplastic conversion and are also known to be key regulatory pathways responsible for the induction of AM expression [3, 8, 32, 39, 42]. Hence, there appears to be several pieces of anecdotal evidence linking AM with the tumor promotion process. In this chapter we summarize our cumulative knowledge of AM in human cancers, reveal molecular mechanisms modulating its expression, identify biological functions of the cancer cell it regulates, and define new intervention approaches to malignant disease based on disrupting AM/AM receptor interaction.

ABSTRACT Recent discoveries have linked adrenomedullin (AM) with fetal development, wound repair, and cancer, underlying its importance in cellular proliferative events. In this chapter we examine the role of AM in human carcinogenesis, focusing on its ability to modulate cell growth, induce angiogenesis, suppress apoptosis, control cell migration/invasion, and regulate immune response mechanisms. We identify regulatory pathways that affect AM mRNA/protein expression and function as it relates to malignant disease and propose alternative therapeutic strategies for the clinical management of human cancers targeting this peptide.

INTRODUCTION Adrenomedullin (AM) was discovered over a decade ago by Kitamura et al., who have written a chapter for the Cardiovascular Peptides section of this book [18]. AM was initially isolated from a human pheochromocytoma and shown to be a 52-amino-acid peptide amide with hypotensive activity [18]. AM has now been established as a pluripotent peptide with diverse biological actions in normal physiology and disease states [20]. It is expressed in both vertebrates and invertebrates, tracing its evolutionary ancestry back over 500 million years to the echinoderms (starfish) and playing a critical role in species survival [4, 5, 21, 41]. Recent observations have demonstrated a crucial involvement of AM in proliferative pathways such as embryogenesis, wound repair (tissue remodeling), and carcinogenesis [4, 5, 10, 28, 41, 45, 50]. Handbook of Biologically Active Peptides

AM EXPRESSION As previously mentioned, AM was first isolated and characterized from an adrenal gland tumor and subsequently found in a multitude of human cancers [5, 18, 50]. To begin to understand why AM expression is selectively enhanced in tumor tissue as compared with normal anatomical counterparts, it is important to review the known regulatory pathways controlling AM transcriptional and translational product formation. Hypoxia and the resulting increase of hypoxia-

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454 / Chapter 65 inducible factor-1 (HIF-1) transcription factor have been implicated as one of the underlying pathways leading to AM overexpression in human tumors [8, 34]. Initial studies by Nakayama et al. gave the first definitive data to support the existence of a hypoxia induced AM relationship in human colorectal carcinoma cells [30]. These investigators found that colon cancer cells exposed to reduced oxygen tension (1%) or cobalt chloride (hypoxia mimetic) would develop a timedependent increase in AM message expression and show an accompanying increase in immunoreactive AM released into the surrounding culture fluid. Follow-up studies performed by Garayoa et al. confirmed Nakayama’s initial findings and demonstrated that HIF-1 binding to distinct hypoxia response elements (HRE) in the AM gene would trigger message expression in a variety of human cancer cell lines [8]. This HIF-1/AM linkage was not only dependent on peptide message induction but also included a transcript stabilization element that increased the biological half-life of the AM message [8]. Since the surrounding microenvironment of human tumors tends to be oxygen depleted [3], superimposing the existence of the HIF-1/AM linkage on that background would suggest that cancer cells have a built-in predisposition to overexpress AM. Chronic inflammation is an alternative mechanism by which tumor cells can selectively elevate their AM levels. Initial studies by Sugo and colleagues demonstrated that proinflammatory cytokines interleukin-1 (IL-l) and tumor necrosis factor-alpha (TNF-α), as well as lipopolysaccaride (LPS), could augment AM expression in vascular smooth muscle cells [43]. Intravenous injection of LPS in the rat resulted in a dramatic elevation of AM serum content and stimulated AM message expression in a variety of internal organs including liver, heart, intestine, and kidney [42]. There are several recent reports that now clearly demonstrate the bioactive products of inflammatory cell infiltrates (macrophage and mast cell) as being a driving force in tumor development [32, 39]. Both of these hematopoietic cell types are not only known to be producers of AM, but also to release IL-1 during immune activation [31, 46, 51]. Hence, tumor infiltrated cells can add to the overall AM concentration of their surrounding microenvironment by either autocrine production of the peptide or through an indirect mechanism via IL-1 induction of AM. Compounding the complexity of AM production in human tumors, recent scientific evidence suggests there may be cross-communication between the hypoxia and inflammatory pathways in ovarian cancer cells, which allows for HIF-1 protein to increase intracellularly under normoxic conditions, hence driving AM expression in tumors [7]. Interestingly, IL-1 was shown to increase HIF-1 nuclear accumulation in ovarian cancer

cells exposed to normal atmospheric conditions and also amplified HIF-1 activation under reduced oxygen tension. These results suggest that cooperative interaction between the hypoxia response mechanism and the inflammatory pathway could synergistically enhance AM production in human cancer cells. Thus, the hypoxia/inflammation/AM axis could ultimately lead to a more aggressive tumor type, which will be discussed in the remaining sections of this text.

GROWTH REGULATION AM has been shown to be a universal growth factor for several anatomically distinct tumor cell types and suppression of peptide/receptor interaction via neutralizing antibodies or peptide antagonists was demonstrated to inhibit cancer cell growth in vitro and in vivo [15, 27, 35]. Miller et al. first revealed the existence of an AM autocrine/paracrine growth relationship in tumor cell populations of lung, colon, breast, ovary, and prostate cancers [27]. They not only showed that a variety of human tumor cell lines had the ability to produce AM but that this endogenous peptide could stimulate cell proliferation, an event that could be blocked with the anti-AM monoclonal antibody MoAb-G6 [27]. A similar AM regulated growth mechanism has been shown to exist for glioblastoma, pancreatic, and endometrial cancer cells [15, 33, 35].

ANTIAPOPTOSIS Seminal papers from Hirata’s group at Tokyo Medical and Dental University Graduate School were the first reported documentation that AM could function as an autocrine/paracrine suppressor of programmed cell death in rat endothelial cells [16, 40]. This AMmediated phenomenon was shown to be independent of cAMP but involved a Max bHLH driven mechanism [16, 40]. Recently published findings from this same group have shown that the c-Myc protein (dimeric partner of Max) has a positive/negative regulatory effect on AM expression depending on the intracellular c-Myc levels achieved [36]. Using stable transfection technology to overexpress AM in human breast cancer cell lines, Martínez et al. (see his chapter in the Endocrine Peptide section of this book) demonstrated that such cells were highly resistant to apoptosis induced by serum deprivation compared to empty plasma control cells [24]. This resistant phenotype to serum starvation was abolished by exposure to the neutralizing anti-AM monoclonal antibody MoAb-G6, confirming the AMmediated dependency of this culture phenomenon [24]. In addition, protein/molecular analysis of the AM

Adrenomedullin: An Esoteric Juggernaut of Human Cancers / 455 overexpressing breast cancer cells revealed that the antiapoptotic molecule Stat3 was elevated in these stable transfectants while proapoptotic proteins (i.e., Bax, Bid, caspase 6, caspase 7, caspase 8, MEKK3, and TRADD) were lower when compared with empty plasmid control cells [24]. Similar growth protective effects of AM overexpression have been seen with hypoxia-induced cell death in endometrial cancer cells [34]. Hence it appears that tumor cells overexpressing AM have a selective growth advantage for surviving harsh environmental conditions like serum deprivation or hypoxic insult.

MIGRATION/INVASION AM was first reported as an inhibitor of serum- or platelet-derived growth factor-induced migration of rat vascular smooth muscle cell [12] and later as an inhibitor of angiotensin II induced migration of human coronary artery smooth muscle cells [19]. Recently, studies by Huang et al. have shown that AM can block seruminduced retinal pigmented epithelial cell migration [13]. Conversely, AM appears to be a direct stimulator of cell migration for human ovarian cancer ECV cell [24]. The ability of normal cells (tissue remodeling and trophoblast implantation) or malignant cells (metastasis) to penetrate tissue substroma (invasion) is dependent on a variety of proteolytic enzymes that digest basement membrane structures and allow for the invasion process to proceed [2]. Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, has long been recognized as a major contributor to the degradation of the extracellular matrix during wound repair, implantation, and metastasis [2]. Montuenga and colleagues were first to demonstrate that mouse/rat trophoblasts expressed high levels of AM during the implantation process of the early placenta [29], a fact that was further validated by the work of Yotsumoto et al. [47]. It has now been shown that AM can induce MMP-2 expression in rat aortic adventitial fibroblasts implicating a similar relationship may occur during trophoblast implantation [44]. Recent findings by Zhang and coworkers have in fact confirmed that AM enhances the ability of trophoblasts to invade the substroma by up-regulating gelatinase activity and suppressing plasminogen activator inhibitor-1 mRNA expression [48]. Interestingly, it has also been shown that MMP-2 can rapidly degrade AM and that complement factor H or AM-binding protein-1 (AMBP-1) can selectively protect AM from this proteolytic degradation [23, 37]. Given the biological connection between AM, AMBP-1, MMP-2, and implantation/invasion, it is not too surprising to find that both the amnion and tumor cells are high produc-

ers of all of these components [1, 2, 5, 17, 47]. Thus, correct anatomical placement of these components could effectively drive implantation events during normal fetal development or heighten tumor aggression during the metastatic process.

ANGIOGENESIS The paradigm setting paper by Zhao et al. convincingly demonstrated that AM was a direct mitogen for endothelial cells and could stimulate neovascularization in vivo when analyzed by the CAM assay [49]. Multiple follow-up studies have now clearly shown the importance of AM in tumor angiogenesis [9, 15, 24, 33]. Although there are many angiogenic growth factors known to exist that stimulate tumor cell growth, the most well known is vascular endothelial growth factor (VEGF) [32, 39]. Interestingly, a recent study by Iimuro et al. has demonstrated that AM can induce VEGF expression in human aortic endothelial cells [14]. These same investigators have shown that heterozygotic AM KO mice (AM+/−) exposed to sarcoma 180 cells generate slower growing tumors with reduced capillary density than did wild-type mice (AM+/+), and treatment of AM+/+ mice with the peptide antagonist AM22–52 caused the same type of diminished tumor growth due to neovascular suppression [14]. Finally, a new AM gene-related angiogenic growth factor has now been identified, proadrenomedullin N-terminal 20 peptide (PAMP), which is a million times more potent, on a molar basis, than VEGF, bFGF, or AM in stimulating capillary growth in vivo [26]. Expanding on their initial discovery, Martínez and colleagues have shown that the peptide antagonist PAMP12–20 is a powerful suppressor of tumor cell growth when applied to the mouse xenograft model [26].

IMMUNE REGULATION AM has been previously demonstrated to be both a positive and a negative regulator of macrophage function, stimulating IL-1β, IL-6, and MIF secretion in LPStreated NR8383 cells, while suppressing TNF-α release in these same cells [46]. Pío et al. have shown that the AM/AMBP-1 complex can stimulate the proteolytic cleavage of C3b, an event that could ultimately lead to the suppression of complement lysis [37]. This same investigator has recently found that human lung tumor cell lines that produce AMBP-1 are selectively resistant to the lytic effects of complement fixation; hence cells producing both AM and AMBP-1 should acquire an augmented protective state [1]. Mast cells (MC) have been shown to enhance tumor cell growth by releasing

456 / Chapter 65 Wound Repair (Tissue Remodeling)

Ischemic Insult

Embryogenesis

Cancer

Bacterial Sepsis

Involved In Cellular Proliferative Events

Inflammation

Hypoxia ADRENOMEDULLIN

Exposure To Environmental Toxins?

Growth

Migration

Exposure To Parasitic Infection?

Regulator Of Biological Functions

Invasion

Antiapoptosis

Angiogenesis Immune (Lymphangiogenesis?) Response

FIGURE 1. Summary of adrenomedullin’s role in major proliferative events and the biological functions it regulates.

a variety of angiogenic factors when activated [31]. Recent studies by Zudaire and coworkers have revealed that AM at picomolar concentrations performs as a chemotactic factor for MCs, while at nanomolar levels it stimulates the production of VEGF, bFGF, and MCP-1 in MCs, and at micromolar amounts it initiates MC degranulation [51]. Hence, these pivotal studies have identified a novel tumor/MC axis that may underlie the ability of chronic inflammation to drive neoplastic growth and metastasis.

THERAPEUTIC STRATEGIES AND CONCLUSION Given AM’s ability to function as a major regulator of human carcinogenesis via its influence on direct growth, antiapoptotic, migration/invasion, angiogenesis, and immune suppression (summarized in Fig. 1), it therefore naturally begs the question: Could AM be used as a biological target for the clinical management of malignant disease? We have already presented results from several laboratories that utilize neutralizing antiAM antibodies or AM peptide antagonists to suppress tumor cell growth in vitro and in vivo [15, 17, 27, 35], and these reagents represent a logical approach to therapeutic intervention of cancer. More recently, small molecule inhibitors of AM have been identified that disrupt multiple biological functions of AM and, in

theory, could also be used in the treatment of human cancers [22, 25]. Just how and when such innovative strategies will be evaluated in clinical trials depends on accrued pharmaceutical support and the investigative fortitude of physicians/scientists.

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GRP blocker 77427 inhibits tumor growth in vitro and in vivo. Oncogene 2005;24:4106–13. Martínez A, Zudaire E, Portal-Núñez S, Guédez L, Libutti SK, Stetler-Stevenson W, et al. Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, its inhibition results in reduction of tumor growth. Cancer Res 2004;64:6489–94. Miller MJ, Martínez A, Unsworth EJ, Thiele CJ, Moody TW, Elsasser T, Cuttitta F. Adrenomedullin expression in human tumor cell lines. Its potential role as an autocrine growth factor. J Biol Chem 1996;271:23345–51. Miyashita K, Itoh H, Sawada N, Fukunaga Y, Sone M, Yamakara K, et al. Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo. FEBS Letters 2003;544:86–92. Montuenga LM, Martínez A, Miller MJ, Unsworth EJ, Cuttitta F. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 1997;138:440–51. Nakayama N, Takahashi K, Murakami O, Shirato K, Shibahara S. Induction of adrenomedullin by hypoxia and cobalt chloride in human colorectal carcinoma cells. Biochem Biophys Res Commun 1998;243:514–7. Norrby K. Mast cells and angiogenesis. APMIS 2002;110: 355–71. O’Byrne KJ, Dalgleish AG. Chronic immune activation and inflammation as the cause of malignancy. Br J Cancer 2001;85: 473–83. Oehler MK, Hague S, Rees MCP, Bicknell R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene 2002;21:2815–21. Oehler MK, Norbury C, Hague S, Rees MC, Bicknell R. Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells. A possible promotion mechanism for tumour growth. Oncogene 2001;20:2937–45. Ouafik LH, Sauze S, Boudouresque F, Chinot O, Delfino C, Fina L, et al. Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell line in vitro and suppresses tumor xenograft growth in vivo. Am J Path 2002;160:1279– 92. Ozawa N, Shichiri M, Fukai N, Yoshimoto T, Hirata Y. Regulation of adrenomedullin gene transcription and degradation by the c-myc gene. Endocrinology 2004;145:4244–50. Pío R, Martínez A, Unsworth EJ, Kowalak JA, Bengoechea JA, Zipfel PF, et al. Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J Biol Chem 2001;276:12292– 300. Rocchi P, Boudouresque F, Zamora AJ, Murocciole X, Lechevallier E, Martin PM, et al. Expression of adrenomedullin and peptide amidation activity in human prostate cancer and human prostate cell lines. Cancer Res 2001;61:1196–206. Shacter E, Weitzman SA. Chronic inflammation and cancer. Oncology (Huntingt) 2002;16:217–26, discussion 230–2. Shichiri M, Kato H, Doi M, Marumo F, Hirata Y. Induction of Max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol Endocrinol 1999;13: 1353–63. Shindo T, Kurihara Y, Nishimatsu H, Moriyama N, Kakoki M, Wang Y, et al. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circulation 2001;104:1964–71. Shoji H, Minamino N, Kangawa K, Matsuo H. Endotoxin markedly elevates plasma concentration of gene transcript of adrenomedullin in rat. Biochem Biophys Res Commun 1995; 215:531–7.

458 / Chapter 65 [43] Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H. Interleukin-1, tumor necrosis factor and lipopolysaccharide activity stimulates production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun 1995;207:25–32. [44] Tsuruda T, Kato J, Cao YN, Hatakeyama K, Masuyama H, Imamura T, et al. Adrenomedullin induces matrix metalloproteinase-2 activity in rat aortic adventitial fibroblasts. Biochem Biophys Res Commun 2004;325:80–84. [45] Wang H, Tomikawa M, Jones MK, Pai R, Sarfeh J, Tarnawski AS. Sequential expression of adrenomedullin and its receptor during gastric ulcer healing in rats. Dig Dis Sci 2000;45: 591–8. [46] Wong LYF, Chang BMY, Li YY, Tang F. Adrenomedullin is both proinflammatory and anti-inflammatory: its effects on gene expression and secretion of cytokines and macrophage migration inhibitory factor in NR8383 macrophage cell line. Endocrinology 2005;146:1321–7.

[47] Yotsumoto S, Shimada T, Cui CY, Nakashima H, Fujiwara H, Ko MSH. Expression of adrenomedullin, a hypotensive peptide, in the trophoblast giant cells at the embryo implantation site in mouse. Devel Biol 1998;203:264–75. [48] Zhang X, Green KE, Yallampalli C, Dong YL. Adrenomedullin enhances invasion by trophoblast cell lines. Biol Reprod 2005;Published Online, PMID: 15917349. [49] Zhao Y, Hague S, Manek S, Zhang L, Bicknell R, Rees MCP. PCR display identifies tamozifen induction of a novel angiogenic factor adrenomedullin by a nonestrogenic mechanism in the human endometrium. Oncogene 1998;16:409– 15. [50] Zudaire E, Martínez A, Cuttitta F. Adrenomedullin and cancer. Reg Peptides 2003;112:175–83. [51] Zudaire E, Martínez A, Garayoa M, Pío R, Kaur G, Woolhiser MR, et al. Adrenomedullin serves as a cross-talk molecule that regulates tumor and mast cell function during human carcinogenesis. Amer J Path 2006;168:280–91.

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66 Angiotensin Peptides and Cancer E. ANN TALLANT, JYOTSANA MENON, DAVID R. SOTO-PANTOJA, AND PATRICIA E. GALLAGHER

Ang II are mediated by two subtypes of receptors, classified as subtype 1 (AT1) and subtype 2 (AT2) receptor. These receptors are typical 7-transmembrane spanning, G protein-coupled receptors with limited homology that are separate gene products. In rodents, there are two AT1 receptors (AT1a and AT1b) that have similar functions but have differential localization, while humans have a single AT1 receptor. The majority of physiological effects associated with Ang II (vasoconstriction, natriuresis, diuresis) are mediated by the AT1 receptor and antagonists selective for blockade of AT1 receptor function (angiotensin receptor blockers or ARBs) are also commonly used in the treatment of hypertension [42]. In addition, the AT1 receptor mediates the mitogenic effects of Ang II in cardiac myocytes and fibroblasts and inhibition of either the production of Ang II by ACE inhibitors or the function of Ang II by ARBs is used to reduce cardiac hypertrophy. The AT2 receptor has a more limited tissue distribution but is present in increased amounts prenatally and following tissue injury, such as myocardial infarction. Its proposed functions include stimulation of apoptosis and inhibition of cell growth. Although Ang-(1-7) and Ang-(2-8) [or Ang III] were initially considered degradation productions of Ang II catabolism, both heptapeptides have physiological functions. The majority of responses to Ang III mimic those of Ang II and are mediated by AT1 receptors. In contrast, Ang-(1-7) opposes the effects of Ang II, with vasodilatory, and antinatriuretic and -diuretic effects and inhibits the growth of vascular and cardiac cells [6, 7, 41]. Most of the responses to Ang-(1-7) are mediated by a selective non-AT1, non-AT2 receptor and the orphan G protein-coupled receptor mas was recently identified as an Ang-(1-7) receptor [36, 43]. Ang-(1-7) is produced from both Ang I and Ang II, by endopeptidases and the recently discovered homolog of ACE, angiotensin converting enzyme 2 (ACE2), respectively [6]. In

ABSTRACT The angiotensin peptides angiotensin II and angiotensin-(1-7) have opposing effects on the growth of cancer cells. Angiotensin II stimulates cell growth, and blockade of either its synthesis using angiotensin converting enzyme inhibitors or its effects using angiotensin receptor blockers reduces tumor cell growth and angiogenesis. Since angiotensin converting enzyme inhibitors and angiotensin receptor blockers are in clinical use for the control of hypertension, they may represent novel therapeutics in the treatment of cancer. In contrast, angiotensin-(1-7) reduces the growth of human lung and breast cancer cells and inhibits angiogenesis. This suggests that treatment with angiotensin-(1-7) or drugs that elevate its concentration may serve as potential therapeutic modalities for cancer treatment.

INTRODUCTION The renin-angiotensin system plays a significant role in the control of blood pressure and the regulation of both cardiac and renal function, as reviewed in this book by Izumi and Iwao and by Navar. The parent compound of this system is angiotensinogen, predominantly produced in the liver and secreted into the circulation, where it is degraded by circulating renin to the decapeptide angiotensin I (Ang I), as shown in Fig. 1. Ang I is subsequently cleaved into a number of physiologically relevant peptides, including angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)]. Angiotensin converting enzyme (ACE) catalyzes the conversion of Ang I to Ang II and attenuation of its actions by selective inhibitors (enalopril, captopril, perindopril, etc.) is among the therapeutic modalities commonly used to treat patients with high blood pressure. The effects of Handbook of Biologically Active Peptides

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460 / Chapter 66 Angiotensinogen Renin ACE Chymase

Ang II

AT1R Losartan

Ang I ACE2 PE

AT2R PD 123319

Neprilysin PE

by similar studies of hypertensive patients treated with ARBs (such as the recently completed LIFE or RENAAL studies) to eliminate conflicting effects due to increased bradykinin and Ang-(1-7) caused by treatment with ACE inhibitors.

Ang-(1-7)

mas

ACE AND AT1 RECEPTOR POLYMORPHISMS AND CANCER

D-[Ala7]-Ang-(1-7)

FIGURE 1. Pathways for the formation and activities of angiotensin peptides. ACE, angiotensin converting enzyme; ACE2, angiotensin converting enzyme 2; PE, prolyl endopeptidase; Ang I, angiotensin I; Ang II, angiotensin II; Ang-(1-7), angiotensin-(1-7); AT1R, angiotensin subtype 1 receptor; AT2R, angiotensin subtype 2 receptor; and [D-Ala7]Ang-(1-7), [D-Alanine7]-angiotensin-(1-7).

addition, Ang-(1-7) is degraded by ACE, contributing to an increase in its concentration following treatment with ACE inhibitors. Treatment with ACE inhibitors results in both a reduction in Ang II and an increase in Ang-(1-7). In addition, bradykinin, which also has antiproliferative effects, is degraded by ACE to inactive peptides and treatment with ACE inhibitors increases bradykinin. Thus, treatment with ACE inhibitors results in a decrease in Ang II and a concomitant increase in both Ang-(1-7) and bradykinin. A role for angiotensin peptides in cancer growth was suggested by several recent epidemiological studies. Patients receiving ACE inhibitors were included as a control group in two studies assessing the increased risk of cancer in hypertensive patients taking calcium channel blockers. In both of these studies, the relative risk of cancer in ACE inhibitor-treated patients was 0.73 and 0.79 [19, 32]. While these reductions in risk were not statistically significant, the number of participants was small and the data suggested a correlation between relative risk of cancer and ACE inhibitor treatment. In a retrospective study of 5207 patients in Scotland, the relative risks of incident and fatal cancer among the 1559 patients treated with ACE inhibitors were significantly reduced, to 0.72 and 0.65, respectively [22]. The relative risk was lowest in patients with lung, colon, or sex-specific cancer, as compared with other sites including skin cancer and lymphoma leukemias. These results suggest that drugs that inhibit ACE may attenuate tumor cell growth. In contrast, several epidemiology studies found no correlation between relative risk of cancer and treatment with ACE inhibitors [10, 27, 28]. It is clear that additional studies are needed to resolve this conflicting information. However, more direct assessment of the effect of Ang II on cancer may be obtained

Koh et al. [20] conducted a case control study of women without and with diagnosed breast cancer within the Singapore Chinese Health Study Cohort to determine whether there was a correlation between known ACE polymorphisms and breast cancer incidence. Singapore Chinese women with the I/D or A-240T genotypes, polymorphisms associated with decreased ACE activity, showed a significant reduction in risk compared with subjects with DD or TT alleles, respectively, following adjustment for breast cancer risks. In a second report, this group found that three polymorphisms in the promoter of the AT1 receptor, resulting in reduced receptor expression, correlated significantly with decreased breast cancer incidence [21]. Further, women with combined low risk genotypes for both ACE and the AT1 receptor had an odds ratio of 0.35 compared with subjects with high activity ACE alleles and genotypes without altered AT1 receptor expression. These results suggest that pharmacological agents that decrease Ang II production, such as ACE inhibitors, or block Ang II action, such as ARBs, may provide effective breast cancer treatment. In conflicting reports, Freitas-Silva et al. [9] found that normotensive women with the I/D or I/I genotype were associated with early age onset of endometrial cancer. In contrast, Haiman et al. [16] showed no correlation between ACE genotypes I/D or A-240T and breast cancer risk among African-American, Japanese, Latina, and non-Hispanic white women in the Multiethnic Cohort Study, while a modest association was observed in women with the I/I genotype. These conflicting results indicate that further studies are warranted to determine whether polymorphisms in the genes encoding the enzymes that regulate the levels of angiotensin peptides or the receptors that mediate their actions are associated with cancer incidence.

EFFECTS OF ACE INHIBITORS AND ARBs ON CANCER CELL GROWTH AND TUMORIGENESIS The use of ACE inhibitors to block the production of Ang II or ARBs to block responses to Ang II was used

Angiotensin Peptides and Cancer / 461 to demonstrate a role for Ang II in the regulation of cancer cell growth and tumorigenesis. The effect of inhibition of ACE on cancer cell growth was studied in three different types of human cancer cells and in a number of animal carcinoma cell lines. In human neuroblastoma cells of the SH-SY5Y cell line [3], human breast cancer cells of the T-47D and Hs578T cell lines [37], human SN12K-1 renal cell carcinoma [17], and murine pancreatic duct carcinoma [34], inhibition of ACE activity using three different inhibitors (captopril, enalaprilat, and lisinopril) reduced cell proliferation. However, the antiproliferative effects of ACE inhibition were only observed at millimolar concentrations of the drug, which are much higher than their concentration in patients treated with ACE inhibitors. Additionally, the ACE inhibitors used in these studies were often restricted to compounds with a sulfhydryl binding site and their antiproliferative effects may be related to inhibition of metalloprotease activity [24]. Inhibition of tumor growth in vivo by ACE inhibitors was also evaluated by a number of investigators. Hii et al. [17] showed a significant reduction in renal cell carcinoma following captopril treatment of SCID mice transplanted with human SN12K-1 cells. Captopril also slowed tumor development of chemically induced foci of preneoplastic liver cells and experimental fibrosarcoma, in agreement with a reduction in angiogenesis [46]. Perindopril suppressed the tumor development in a murine hepatocellular carcinoma model, using BNL-HCC murine cells transplanted into BALB/c mice [49]. Although these studies suggest that reduced production of Ang II by treatment with ACE inhibitors attenuates malignant cell growth and tumor formation, more definite conclusions can be drawn from studies where the effects of Ang II are selectively blocked by ARBs, in conjunction with ablation studies using AT1 receptor knockout mice. The ARB candesartan (TCV-116, CV11974) was used in a number of studies to show that blockade of AT1 receptor activity inhibits tumorigenesis. The growth of engrafted B16-F1 melanoma, sarcoma-180 (S-180) and NFSA fibrosarcoma cells, metastatic lung tumors of renal cell carcinoma (RCC) cells, SKOV-3 ovarian cancer cells, and DU145 prostate cancer cells was significantly reduced by treatment with candesartan [5, 11, 12, 29, 40, 45]. In agreement with these studies, the rate of tumor growth was slower in mice with an ablated AT1 receptor and treatment with ARBs was ineffective in AT1 knockout mice, further demonstrating a role for the AT1 receptor in stimulating tumorigenesis [5, 11]. The AT1 receptor antagonist losartan also caused a significant reduction in the volume of C6 rat glioma, in agreement with a reduction in cellular proliferation in cultured glioma cells [35]. These studies suggest that treatment with ARBs may provide a novel and

effective strategy for the treatment of malignant tumors.

MECHANISMS OF ACTION OF ANG II Cell Signaling Ang II receptors of both the AT1 and AT2 subtype are present on many different types of cells and are upregulated on some types of malignant cells and tumors. AT1 receptors are present on C6 glioma cells as well as human astrocytoma cells [1, 18], human lung cancer cells [33], both invasive and borderline ovarian adenocarcinoma [40], human prostate cancer [45], and human breast carcinoma [4]. The expression of the AT1 receptor is elevated in ovarian carcinoma as compared with benign cystadenomas [40], in human prostate cancer as compared with normal prostate tissues [45], as well as in hyperplastic breast tissue and ductal carcinoma in situ (DCIS) but not in invasive carcinoma of the breast [4]. The AT1 receptor is a typical G-protein coupled receptor and causes an increase in the intracellular concentration of calcium in human lung adenocarcinoma cells (A549 cells) [2] and primary cultures of normal and cancerous breast cancer cells [14]. Ang II also increases mitogen-activated protein (MAP) kinase activity in breast and prostate cancer cells [15, 30, 45], expression of transcription factors (c-fos and STAT3, respectively), and activation of protein kinase C [15]. In addition, AT1 receptors and receptors for epidermal growth factor (EGF) are transactivated in both breast cancer and prostate cancer cells [15, 45]. These results indicate that signaling pathways activated by AT1 receptors in malignant cells are similar to those which regulate growth via AT1-receptor mediated pathways in normal cells and tissues.

Stimulation of Angiogenesis by Ang II Ang II increased the growth of vascular cells in vitro and stimulated angiogenesis in several in vivo models of angiogenesis [25, 31]. In contrast, ACE inhibitors that block endogenous Ang II production reduce angiogenesis. Perindopril inhibition of tumor growth in the murine hepatocellular carcinoma model in BNL-HCC allograft mice was associated with a reduction in vascular endothelial growth factor (VEGF) production and a decrease in angiogenesis [49]. In vitro studies showed a reduction in endothelial cell tubule formation by the prodrug of perindopril, perindoprilat, and a reduction in VEGF mRNA in cultured BNL-HCC cells. Captopril also inhibited endothelial cell migration, an in vitro assessment of angiogenesis, and blocked neovascularization in the rat cornea [46]. In agreement with the

462 / Chapter 66 ACE inhibitor-mediated reduction in angiogenesis, administration of ARBs to tumor-bearing mice also resulted in a significant reduction in angiogenesis. Vascular density in B16-F1 melanoma xenografts was markedly reduced by treatment with the AT1 receptor antagonist candesartan as well as in AT1a−/− mice, in agreement with a reduction in the number of infiltrating macrophages [5]. Angiogenesis and increased VEGF following implantation of S-180 sarcoma cells was blocked by an AT1 receptor antagonist and was reduced in AT1a receptor null mice; the AT1a receptor and VEGF were predominantly expressed in tumor stroma, suggesting that stromal Ang II receptors and their upregulation of VEGF are critical in regulating tumor angiogenesis [11]. In a previous study by the same group, AT1 receptors were also expressed on the neovascularized endothelial cells in S-180 tumor xenografts and candesartan treatment significantly reduced angiogenesis, suggesting a more direct effect by blockade of endothelial cell growth. ARB blockade also inhibited angiogenesis in mice implanted with human ovarian cancer cells (SKOV-3) or human prostate cancer cells (DU145) [40, 45]. These results demonstrate that Ang II receptors stimulate angiogenesis to increase tumor cell growth. Further, AT1 receptors on vascular endothelial cells, infiltrating macrophages, tumor stromal cells, as well as the tumor cells themselves, may contribute to the angiogenic effects of Ang II. Further studies are necessary to clarify the molecular mechanisms that participate in Ang II-stimulated angiogenesis in cancer cells.

Stimulation of Apoptosis by Ang II A series of studies in human and rat alveolar epithelial cells (AEC), as well as the AEC-derived carcinoma cell line A549, showed that Ang II stimulated apoptosis. This effect was blocked by losartan, indicating that it was mediated by Ang II activation of the AT1 receptor [33, 48]. In addition, apoptosis in A549 cells in response to either Fas ligand or bleomycin was associated with synthesis of angiotensinogen and its conversion to Ang II and was blocked by antisense oligonucleotides to angiotensinogen, ACE inhibitors, or ARBs [23, 47]. Although these results suggest that ACE inhibitors or ARBs may be useful in the treatment of lung fibrogenesis and subsequent lung injury, a role for blockade of Ang II-mediated apoptosis in treatment of cancer cell growth is not clear. In contrast, treatment of C6 rat gliomas or cultured C6 glioma cells with the ARB losartan resulted in an increase in the rate of apoptosis, suggesting that Ang II inhibits apoptosis through activation of an AT1 receptor or stimulates apoptosis at the AT2 when AT1 receptors are blocked by ARBs [1]. It is clear that further investigation of the regulation of

apoptosis by Ang II in cancer cells and the subtype of receptor that mediates this response is warranted.

INHIBITION OF HUMAN CANCER CELL GROWTH BY ANG-(1-7) Results from our laboratory showed that Ang-(1-7) reduced mitogen-stimulated growth of vascular smooth muscle cells (VSMCs) in vitro and prevented neointimal formation in vivo [8, 39, 41]. Since treatment of patients with ACE inhibitors reduced the risk of cancer, especially lung cancer [22], and ACE inhibitors elevate plasma levels of Ang-(1-7) [6], we hypothesized that Ang-(1-7) would reduce the growth of lung cancer cells. Recently, we reported that Ang-(1-7) inhibited serum-stimulated growth and DNA synthesis in three different human lung cancer cells: A549, SK-MES-1, and SK-LU-1 [13]. The attenuation of cell growth was dose-dependent with EC50s in the subnanomolar range, similar to the concentration of Ang-(1-7) measured after infusion of the heptapeptide [39] or treatment of rats with the ACE inhibitor lisinopril [6]. Inhibition of the serum-stimulated growth of SK-LU-1 cells by Ang(1-7) was blocked by the Ang-(1-7) selective antagonist [D-Ala7]-Ang-(1-7), while neither AT1 nor AT2 angiotensin receptor antagonists were effective, suggesting that the antiproliferative effect of Ang-(1-7) in lung cancer cells is mediated by a novel AT(1-7) receptor. Since all three of these cell lines contain the AT(1-7) receptor mas, it is likely that mas mediates the antiproliferative response to Ang-(1-7) in lung cancer cells. Neither Ang I, Ang III, Ang-(3-8), Ang IV, Ang-(3-7), nor Ang II mimics the growth inhibitor effects of Ang(1-7). These results suggest that the antiproliferative effect of Ang-(1-7) is mediated by a novel Ang-(1-7) receptor and may represent a new therapeutic treatment for these cancers. Similarly, mitogen-stimulated proliferation and DNA synthesis were inhibited markedly in ZR-75–1 and MCF7 cells, human ductal breast carcinoma cell lines, following treatment with Ang-(1-7) [38]. The attenuation of DNA replication was dependent on the dose of Ang(1-7) with a maximal reduction of approximately 40% of serum-stimulated 3H-thymidine incorporation and IC50s in the subnanomolar range. These effects correlated with a time-dependent reduction in mitogenstimulated hyperphosphorylation of retinoblastoma that causes cell cycle arrest and inhibition of cell growth. Taken together, these studies suggest that Ang(1-7) or agents which increase endogenous Ang-(1-7) may be an effective chemotherapeutic or chemopreventive agent in lung or breast cancer. These results are in agreement with epidemiological studies showing that hypertensive patients treated with ACE inhibitors

Angiotensin Peptides and Cancer / 463 have a reduced risk of cancer, particularly gender-specific and lung cancers. Since ACE inhibitors cause a significant elevation in both tissue and circulating Ang(1-7), our results suggest that the reduced cancer risk observed in patients after administration of ACE inhibitors may be due, at least in part, to the elevated levels of Ang-(1-7).

ment of hypertension, they could easily and quickly be used in the treatment of malignant cell growth. In contrast, more recent evidence suggests that the angiotensin heptapeptide Ang-(1-7) inhibits the growth of both lung and breast cancer cells as well as reduces angiogenesis. This suggests that Ang-(1-7) or a drug that would increase endogenous synthesis of Ang-(1-7) may represent a novel treatment for cancer.

Ang-(1-7) and the Inhibition of Angiogenesis Ang-(1-7) inhibited angiogenesis in a murine sponge model, a technique representative of the formation of new blood vessels from preexisting blood vessels during wound healing [25]. In this model, a cannulated sponge disc was implanted subcutaneously in the dorsa of mice to induce a wound repair response. Infusion of Ang-(17) reduced hemoglobin content, blood flow, and proliferative activity in the disc, as compared with a disc containing vehicle. The antiangiogenic effect of Ang(1-7) in this model was regulated by a [D-Ala7]-Ang(1-7)-sensitive Ang-(1-7) receptor [26], in agreement with our identification of an Ang-(1-7) receptor on endothelial cells from canine coronary artery and bovine aortic endothelial cells [7, 44]. Further, the antiangiogenic response to Ang-(1-7) was blocked by preincubation with either aminoguanidine or N G-nitrol-arginine methyl ester (l-NAME), indicating that the response is mediated by nitric oxide (NO) [26]. Ang(1-7) stimulates the release of NO from bovine aortic endothelial cells and causes the vasodilation of blood vessels through an endothelial release of NO [6]. While this is a model of angiogenesis during wound healing, it suggests that Ang-(1-7) may inhibit the angiogenesis that occurs during tumor formation to supply necessary nutrients for tumor growth.

CONCLUSION Although the renin-angiotensin system is one of the most well-characterized peptidergic systems, a role for angiotensin peptides in the regulation of cancer cell growth and strategies to regulate their concentrations and activities to control tumorigenesis is in its infancy. Both Ang II and Ang-(1-7) play important roles in the regulation of cardiovascular function and growth. However, recent studies using antagonists for the AT1 subtype Ang II receptor and mice in which the AT1a receptor is ablated provide strong evidence of a role for Ang II in the growth of cancer of the lung, breast, brain, ovary, and prostate. Since the molecular mechanism of the inhibition of tumor cell growth by Ang II includes a reduction in angiogenesis, ARBs may be used in the treatment of a broad range of malignant cell growth. Further, since ARBs are already prescribed for the treat-

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464 / Chapter 66 [13] Gallagher, P. E.; Tallant, E. A. Inhibition of lung cancer cell growth by angiotensin-(1-7). Carcinogenesis 2004;25:2045– 2052. [14] Greco, S.; Elia, M. G.; Muscella, A.; Storelli, C.; Marsigliante, S. AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture. Cell Calcium 2002;32:1–10. [15] Greco, S.; Muscella, A.; Elia, M. G.; Salvatore, P.; Storelli, C.; Mazzotta, A.; Manca, C.; Marsigliante, S. Ang II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor in breast cancer cells. J Cell Physiol 2003;196:370–377. [16] Haiman, C. A.; Henderson, S. O.; Bretsky, P.; Kolonel, L. N.; Henderson, B. E. Genetic variation in angiotensin I-converting enzyme (ACE) and breast cancer risk: the multiethnic cohort. Cancer Res 2003;63:6984–6987. [17] Hii, S. I.; Nicol, D. L.; Gotley, D. C.; Thompson, L. C.; Green, M. K.; Jonsson, J. R. Captopril inhibits tumour growth in a xenograft model of human renal cell carcinoma. Br J Cancer 1998;77:880–883. [18] Jaiswal, N.; Tallant, E. A.; Diz, D. I.; Khosla, M. C.; Ferrario, C. M. Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes. Hypertension 1991;17:1115– 1120. [19] Jick, H.; Jick, S.; Derby, L. E.; Vasilakis, C.; Myers, M. W.; Meier, C. R. Calcium-channel blockers and risk of cancer. Lancet 1997;349:525–528. [20] Koh, W.-P.; Yuan, J.-M.; Sun, C.-L.; van den Berg, D.; Seow, A.; Lee, H.-P.; Yu, M.C. Angiotensin I-converting enzyme (ACE) gene polymorphism and breast cancer. Risk among Chinese women in Singapore. Cancer Res 2003;63:573–578. [21] Koh, W.-P.; Yuan, J.-M.; van den Berg, D.; Lee, H.-P.; Yu, M. C. Polymorphisms in angiotensin II type 1 receptor and angiotensin I-converting enzyme genes and breast cancer risks among Chinese women in Singapore. Carcinogenesis 2005;26:459– 464. [22] Lever, A. F.; Hole, D. J.; Gillis, C. R.; McCallum, I. R. M. G. T.; MacKinnon, P. L.; Meredith, P. A.; Murray, L. S.; Reid, J. L.; Robertson, M. J. Do inhibitors of angiotensin-I-converting enzyme protect against risk of cancer? The Lancet 1998;352: 179–184. [23] Li, X.; Zhang, H.; Soledad-Conrad, V.; Zhuang, J.; Uhal, B. D. Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo. Am J Physiol Cell Physiol 2003;284:L501–L507. [24] Lindberg, H.; Nielsen, D.; Jensen, B. V.; Eriksen, J.; Skovsgaard, T. Angiotensin converting enzyme inhibitors for cancer treatment? Acta Oncol 2004;43(2):142–152. [25] Machado, R. D.; Santos, R. A.; Andrade, S. P. Opposing actions of angiotensins on angiogenesis. Life Sci 2000;66(1):67–76. [26] Machado, R. D. P.; Santos, R. A. S.; Andrade, S. P. Mechanisms of angiotensin-(1-7)-induced inhibition of angiogenesis. Am J Physiol Regulatory Integrative Comp Physiol 2001;280:994– 1000. [27] Meier, C. R.; Derby, L. E.; Jick, S. S.; Jick, H. Angiotensinconverting enzyme inhibitors, calcium channel blockers, and breast cancer. Arch Intern Med 2000 Feb;160(3):349–353. [28] Michels, K. B.; Rosner, B. A.; Walker, A. M.; Stampfer, M. J.; Manson, J. E.; Colditz, G. A.; Hennekens, C. H.; Willett, W. C. Calcium channel blockers, cancer incidence, and cancer mortality in a cohort of U.S. women: the nurses’ health study. Cancer 1998 Nov;83(9):2003–2007. [29] Miyajima, A.; Kosaka, T.; Asano, T.; Asano, K.; Seta, K.; Kawai, T.; Hayakawa, M. Angiotensin II type I antagonist prevents pulmonary metastasis of murine renal cancer by inhibiting tumor angiogensis. Cancer Res 2002;62:4176–4179.

[30] Muscella, A.; Greco, S.; Elia, M. G.; Storelli, C.; Marsigliante, S. PKC-z is required for angiotensin II-induced activation of ERK and synthesis of C-FOS in MCF-7 cells. J Cell Physiol 2003;197:61–68. [31] Nadal, J. A.; Scicli, G. M.; Carbini, L. A.; Scicli, G. Angiotensin II stimulates migration of retinal microvascular pericytes: involvement of TGF-β and PDGF-BB. Am J Physiol Heart Circ Physiol 2002;282:H738–H748. [32] Pahor, M.; Guralnik, J. M.; Salive, M. E.; Corti, M.-C.; Carbonin, P.; Havlik, R. J. Do calcium channel blockers increase the risk of cancer? Am J Hypertens 1996;9:695–699. [33] Papp, M.; Li, X.; Zhuang, J.; Wang, R.; Uhal, B. D. Angiotensin receptor subtype AT1 mediates alveolar epithelial cell apoptosis in response to ANG II. Am J Physiol Lung Cell Mol Physiol 2002;282:L713–L718. [34] Reddy, M. K.; Baskaran, K.; Molteni, A. Inhibitors of angiotensin-converting enzyme modulate mitosis and gene expression in pancreatic cancer cells. Proc Soc Exp Biol Med 1995 Dec;210(3):221–226. [35] Rivera, E.; Arrieta, O.; Guevara, P.; Duarte-Rojo, A.; Sotelo, J. AT1 receptor is present in glioma cells; its blockage reduces the growth of rat glioma. Br J Cancer 2001 Nov;85(9):1396– 1399. [36] Santos, R. A. S.; Simoes, E.; Silva, A. C.; Maric, C.; Silva, D. M. R.; Machado, R. D.; DuBuhr, I.; Heringer-Walther, S.; Pinheiro, S. V.; Lopes, M. T.; Bader, M.; Mendes, E. P.; Lemos, V. S.; Campagnole-Santos, M. J.; Schultheiss, H.-P.; Speth, R.; Walther, T. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor mas. Proc Natl Acad Sci USA 2003;100:8258–8263. [37] Small, W., Jr.; Molteni, A.; Kim, Y. T.; Taylor, J. M.; Chen, Z.; Ward, W. F. Captopril modulates hormone receptor concentration and inhibits proliferation of human mammary ductal carcinoma cells in culture. Breast Cancer Res Treat 1997 Aug;44(3):217–224. [38] Soto-Pantoja, D.; Gallagher, P. E.; Tallant, E. A. Inhibition of human breast cancer cell growth by angiotensin-(1-7). Proceedings of the American Association for Cancer Research 2004;45. [39] Strawn, W. B.; Ferrario, C. M.; Tallant, E. A. Angiotensin-(1-7) reduces smooth muscle growth after vascular injury. Hypertension 1999;33[part II]:207–211. [40] Suganuma, T.; Ino, K.; Shibata, K.; Kajiyama, H.; Nagasaka, T.; Mizutani, S.; Kikkawa, F. Functional expression of the angiotensin II type 1 receptor in human ovarian carcinoma cells and its blockade therapy resulting in suppression of tumor invasion, angiogenesis, and peritoneal dissemination. Clin Cancer Res 2005;11:2686–2694. [41] Tallant, E. A.; Diz, D. I.; Ferrario, C. M. Antiproliferative actions of angiotensin-(1-7) in vascular smooth muscle. Hypertension 1999;34(part 2):950–957. [42] Tallant, E. A.; Ferrario, C. M. Biology of angiotensin II receptor inhibition with a focus on losartan: a new drug for the treatment of hypertension. Exp Opin Invest Drugs 1996;5:1201– 1214. [43] Tallant, E. A.; Ferrario, C. M.; Gallagher, P. E. Angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor. Am J Physiol Heart Circ Physiol 2005;289: H1560–66. [44] Tallant, E. A.; Lu, X.; Weiss, R. B.; Chappell, M. C.; Ferrario, C. M. Bovine aortic endothelial cells contain an angiotensin-(1-7) receptor. Hypertension 1997;29[part 2]:388– 393. [45] Uemura, H.; Ishiguro, H.; Nakaigawa, N.; Nagashima, Y.; Miyoshi, Y.; Fujinami, K.; Sakaguchi, A.; Kubota, Y. Angiotensin II receptor blocker shows antiproliferative activity in prostate

Angiotensin Peptides and Cancer / 465 cancer cells: A possibility of tyrosine kinase inhibitor of growth factor. Molecular Cancer Therapeutics 2003;2:1139– 1147. [46] Volpert, O. V.; Ward, W. F.; Lingen, M. W.; Chesler, L.; Solt, D. B.; Johnson, M. D.; Molteni, A.; Polverini, P. J.; Bouck, N. P. Captopril inhibits angiogenesis and slows the growth of experimental tumors in rats. J Clin Invest 1996;98:671–679. [47] Wang, R.; Zagariya, A.; Ang, E.; Ibarra-Sunga, O.; Uhal, B. D. Fas-induced apoptosis of alveolar epithelial cells requires ANG II generation and receptor internalization. Am J Physiol Lung Cell Mol Physiol 1999;277:L1245–L1250.

[48] Wang, R.; Zagariya, A.; Ibarra-Sunga, O.; Gidea, C.; Ang, E.; Deshmukh, S.; Chaudhary, G.; Baraboutis, J.; Filippatos, G.; Uhal, B. D. Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. Am J Physiol 1999 May;276(5 Pt 1): L885–L889. [49] Yoshiji, H.; Kuriyama, S.; Kawata, M.; Yoshii, J.; Ikenaka, Y.; Noguchi, R.; Nakatani, T.; Tsujinoue, H.; Fukui, H. The angiotensin-I-converting enzyme inhibitor perindopril suppresses tumor growth and angiogenesis: possible role of the vascular endothelial growth factor. Clin Cancer Research 2001;7: 1073–1078.

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67 Gastrin and Cancer JENS F. REHFELD

Growth factors are often involved in cancer. But the oncogenic role of gastrin is not yet clear. A large number of studies, however, implicate gastrin peptides and their receptors in cancer development. This review attempts to discuss the carcinogenetic role of gastrin critically.

ABSTRACT The gastrin and gastrin receptor genes are frequently expressed at the protein level in common carcinomas of the esophagus, stomach, pancreas, colorectal mucosa, bronchial mucosa, ovary, and thyroid gland. In addition, rare neuroendocrine tumors also express gastrin and its receptor. The expression is often modified at the posttranscriptional and posttranslational levels. Thus, as a result of incomplete processing, progastrin and gastrin-processing intermediates often accumulate and are released from cancer cells. Moreover, the receptor gene may be expressed as splice variants and in larger or truncated forms due to abnormal posttranslational modifications. The growth promoting effect of gastrin peptides suggests that the gastrin system (ligands and receptor) may have broad carcinogenic significance. Consequently, attempts to target different components of the gastrin system in cancer by receptor antagonists or vaccination are under way.

EXPRESSION OF GASTRINS IN NORMAL TISSUE In the normal organism by far most gastrin is produced in antroduodenal G-cells (for reviews, see [11, 44]). Consequently, gastrin biosynthesis studies have focused on antral tissue [8, 19, 26, 65] and shown that gastrin synthesis is quite complex. As a result of the elaborate biosynthetic pathway, antral G-cells release a mixture of progastrin products from the secretory granules. A small percentage are nonamidated processing-intermediates, of which glycine-extended gastrins have attracted considerable interest [19, 59, 65]. But in adult man more than 98% are α-amidated bioactive gastrins, of which nearly 85% are sulfated and nonsulfated gastrin-17, 5–10% are gastrin-34, and the rest is a mixture of gastrin-71, -52, -14, and -6 [17, 20, 26, 49]. Due to gross differences of metabolic clearance rates, the larger gastrins with long half-lives predominate over gastrin-17 and shorter gastrins in plasma [26]. In achlorhydria and gastrinomas the translational activity in the gastrin producing cells increases considerably, and apparently the progastrin processing enzymes cannot keep up with such synthesis. Consequently, the fraction of immature precursors and processing intermediates increases markedly in tissue and plasma [2, 3, 26]. The gastrin gene is expressed also in extraantral cells, that normally, however, contribute only little to the gastrin measured in blood. So far we have encoun-

INTRODUCTION Gastrin was discovered in extracts of the gastric antrum in 1905 [12] and identified as a pair of heptadecapeptides in 1964 [14, 15]. Until the 1970s, gastrin was believed to be produced only in antroduodenal cells and its function to be regulation of gastric acid secretion. Now, the structure of the gastrin and gastrin receptor genes as well as their widespread expression also outside the stomach are well known [5, 7, 29–32, 54, 58, 72, 74, 76; for review, see 55]. Moreover, it has been shown that gastrin is also a growth factor. Thus, gastrin controls the growth of the fundic mucosa cells, and seems to influence also the growth of epithelial mucosal cells elsewhere [27, 28, 37, 60; for review, see 56]. Handbook of Biologically Active Peptides

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468 / Chapter 67 tered extraantral gastrin expression in the small intestine [30], in the colorectal mucosa [35], in pancreatic islet cells [5, 32], in pituitary corticotrophs and melanotrophs [31, 42], in hypothalamo-pituitary [42, 51], cerebellar [43] and vagal neurons [68], in bronchial mucosa [48], and in spermatogenic cells [58]. The significance of extraantral gastrin synthesis is largely unknown, but it may have carcinogenetic relevance. Thus, gastrin producing cancers seem to originate from cells that normally express the gastrin gene at low level at some stage of the development (for review, see [47]).

THE GASTRIN RECEPTOR(S) The gastrin receptor was cloned and identified in 1992 by Kopin et al. [29]. It is a protein of 453 amino acids residues, and it belongs to the superfamily of G-protein coupled receptors with seven transmembrane α-helical domains. The gastrin receptor binds acid stimulatory peptides containing the C-terminal sequence –Gly-Trp-Met-Asp-Phe• NH2. Hence, the receptor binds all carboxyamidated gastrins and the homologous cholecystokinin (CCK) peptides. The gastrin and CCK peptides are bound with similar affinity irrespective of length and degree of tyrosyl-sulfation. But in terms of hormonal regulation the CCK peptides are of marginal significance as gastrin receptor ligands because they circulate in 10- to 20-fold lower concentrations than the gastrins in plasma [45]. The gastrin receptor is structurally closely related to the CCK receptor also identified in 1992 but by Wank et al. [72]. The amino acid sequences of the two receptors display nearly 50% homology [71]. But the CCK receptor is more specific as it binds only tyrosyl-sulfated CCK peptides with high affinity. The naming of the gastrin and CCK receptors has been confusing because the gastrin receptor also binds CCK peptides. It has therefore been proposed [40] that the CCK receptor should be named the “CCK-A or CCK1” receptor, and the gastrin receptor the “CCK-B or CCK2” receptor. Since further variants later have been found (vide infra), this nomenclature has become inconsistent. Consequently, the original names as gastrin and CCK receptors are used in the following. The gastrin receptor in normal mammals is expressed primarily on ECL and parietal cells in the stomach and on neurons in most regions of the brain. In the central nervous system CCK-8 is the principal ligand, whereas gastrin-17 and -34 are major ligands in the periphery. The gastrin receptor is promiscuous in the sense that it is expressed—although with some species differences— at a lower level on other cells and in other tissues during

ontogenetic development [57] and during malignant transformation (for reviews, see [47 and 55]). In recent years a number of gastrin receptor variants have been discovered. One isoform is the result of a splicing variation of the fourth exon, and it encodes a receptor with five additional amino acid residues near the third intracellular loop [24, 39, 64]. Another form is truncated without the N-terminal extracellular domain [38]. Moreover, both high (74 KDa) and low (40 KDa) molecular weight forms have been described [9, 36]. The differences are assumed to reflect different degrees of glycosylation and other posttranslational modifications [34, 66]. Finally, a gastrin receptor variant of particular oncogenetic relevance has been found in pancreatic [62] and colorectal carcinomas [18]. This variant has retained the fourth intron of the receptor gene and encodes accordingly an extra sequence of 69 amino acid residues in the third intracellular loop [18, 62]. The prolongation changes the function of G-proteins associated with calcium signaling [61]. The splicing variation is caused by reduced activity of the U2AF35 ribonucleoprotein particle splicing factor [10]. The intron-4 containing splice variant is of major interest for pancreatic and colorectal carcinogenesis [63]. The interest has led to the suggestion that it deserves the name of a “CCK-C” receptor [63]. At our present state of knowledge, and considering that other variants exist, such a name is, however, somewhat premature.

EXPRESSION OF GASTRIN IN TUMORS In 1955 Zollinger and Ellison described a syndrome consisting of severe duodenal ulcer disease, gastric acid hypersecretion, and endocrine tumors in the pancreas [77]. The syndrome was subsequently shown to be due to hypersecretion of gastrin from pancreatic G-cell tumors, gastrinomas [16]. Although many gastrinomas originate in the pancreas, small gastrinomas have also been found with increasing frequency in the duodenum, and in the ovaries as well as a few other locations [33, 41, 67, 69]. Some pancreatic gastrinomas are mixed and also contain cells that produce other pancreatic hormones. The gastrinoma syndrome occurs with a frequency of one new case per million inhabitants per year [25]. It is important to diagnose and localize gastrinomas at an early stage, because gastrinomas in spite of slow growth are malignant [3]. The gastrin gene is, however, much more frequently expressed at the peptide level in some brain tumors [50], pituitary adenomas [6], pheochromocytomas [4], and bronchogenic [48], pancreatic [13, 62, 63], and colorectal [18, 21, 70] carcinomas. The level of expression is often so low that gastrins from these tumors do

Gastrin and Cancer not contribute significantly to the concentration of gastrin in plasma. Consequently, these neoplasias do not elicit symptoms associated with the gastrinoma syndrome. Often only a minor fraction of the progastrin is processed to bioactive gastrins although the processing varies considerably in each individual tumor. However, even though the secretion of gastrin from brain, lung, pancreatic, and colonic tumors is too small to increase the concentration in plasma, the expression of gastrin may have carcinogenic significance. Local secretion may stimulate growth of tumor cells equipped with receptors for gastrin, variants or not [21–23, 73, 75]. Moreover, it is possible that the release of progastrin processing intermediates may contribute to the precursor concentrations in plasma. Hence progastrin may serve as a tumor marker [3, 48, 70]. Progastrin in plasma may even be an indicator of the degree of malignancy [3].

GASTRIN AS A TUMOR GROWTH FACTOR Gastrin has growth-promoting effects on the normal gastric and colorectal mucosa [27, 28, 60], and several normal gastric, pancreatic, and colorectal epithelial cells are equipped with gastrin receptors (for reviews, see [47, 53, 55]). Moreover, in countless studies of cancer cell lines gastrin peptides have been shown to stimulate growth. It is consequently likely that in vivo growth of human bronchial, gastric, colorectal, ovarian, esophageal, and pancreatic carcinomas is stimulated either by autocrine, endocrine, neurocrine, or paracrine release of gastrin. There is, however, confusion about the molecular nature of the growth-promoting gastrins. It is generally agreed that carboxyamidated and acid-stimulatory gastrins (mainly gastrin-17 and gastrin-34) can stimulate growth of normal and transformed epithelial cells that express the gastrin receptor. But the observation that the progastrin products in colorectal carcinomas are incompletely processed and not amidated has led to the suggestion that also processing intermediates such as glycine-extended gastrins via specific receptors stimulate the growth of carcinoma cells [59]. The suggestion is interesting, but two essential pieces of evidence are missing. First, 12 years after the initial suggestion [59] and in spite of subsequent intensive search since 1994 in many laboratories, the proposed receptor or receptor mechanism has still not materialized. Second, half of all known bioactive peptide systems are carboxyamidated and often affect, as does gastrin, growth of their target cells. Since the glycine residue is the obligatory amide donor for carboxyamidated peptides, glycineextended precursors for all amidated bioactive peptides

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are necessarily produced during hormone and neuropeptide biosynthesis. Nevertheless, it is striking that independent activity via specific receptors of glycineextended intermediates has been proposed only for the gastrin system. Taken together, the hypothesis about a growth-promoting carcinogenetic role of glycineextended gastrins is therefore still short of definitive evidence. Another confusing issue is progastrin. Several authors have suggested that the progastrin molecule of 80 amino acid residues in itself stimulates growth (for reviews, see [1 and 53]). Since the concentration of progastrin in normal, adult tissue and plasma is negligible [3, 46], progastrin as such is not available for promotion of growth of normal cells. But in bronchogenic, colorectal, and pancreatic cancer cells, there is an accumulation of progastrin [13, 48, 70]. Hence, the cellular concentration of progastrin and cellular growth may correlate in certain carcinomas. Such correlation or co-occurrence constitutes, however, no proof. Moreover, in most reports on this issue progastrin is rarely or poorly defined [1, 53]. The expression often refers to immunoreactivity without precise epitope or sequence definition. Consequently, the studies may have dealt with undefined progastrin products and not with the progastrin molecule itself. Again, as for glycine-extended gastrins, progastrin as such is not a ligand for the gastrin receptor, and therefore a separate progastrin-receptor remains to be found if the progastrin theory shall survive. Finally, specific bioactivity of the primary precursor (i.e., the proper prohormone) has so far not been observed for other prohormones. In view of the pronounced molecular heterogeneity of carboxyamidated gastrins and the many processing intermediates produced along the biosynthetic pathway in cells that express the gastrin gene, there is an urgent need for precise definition and naming of the progastrin products that are studied. Such nomenclature was recently proposed [52] and should be followed. Hopefully systematic definitions will reduce the confusion about gastrins as tumor growth factors especially because of the many variants of the gastrin receptor (which also needs further characterization and definition).

CONCLUSION The evidence for a role of gastrin in the carcinogenesis of common cancer forms within and outside the gastrointestinal tract has grown markedly in the last decades. The poor prognosis and limited success in treatment of, for instance, ovarian, pancreatic, colorectal, and lung carcinomas so far have brought gastrin

470 / Chapter 67 peptides and their receptors (in their multiple molecular variants) into focus as possible targets for new therapeutic approaches. These include specific receptor antagonists and programs for vaccination against gastrin peptides (for review, see [53]). Rational and effective therapy, however, requires precise molecular understanding and definition of the system as well as an unequivocal naming of the targets. The literature published so far abounds with inconsistencies in these respects. When the necessary ambiguity has been resolved, the gastrin system may open new avenues for rational attacks on widespread cancers.

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68 VIP and PACAP as Autocrine Growth Factors in Breast and Lung Cancer TERRY W. MOODY AND ROBERT T. JENSEN

patients as well as lung cancer patients. The five-year survival of lung cancer patients is less than 20%. VIP, which elevates cAMP in lung cancer cell lines, increases secretion of growth factors such as bombesinlike peptides [24]. Also, VIP increases expression of angiogenic factors such as vascular endothelial cell growth factor (VEGF) [6]. In particular, VIP increases expression of VEGF isoforms containing 121, 165, and 189 amino acids in a protein kinase A-dependent manner [7]. In preclinical studies, VIPhyb decreases the breast and lung cancer tumor growth [35, 57]. Thus, blockade of VIP receptors is a therapeutic strategy for treatment of breast and lung cancer patients.

ABSTRACT VIP and PACAP are synthesized by several human breast and lung cancer cell lines. These peptides bind to cell surface receptors and stimulate adenylylcyclase activity. This leads to increased expression of nuclear oncogenes such as c-fos and c-jun, ultimately leading to cellular proliferation. The growth of breast and lung cancer cell lines is inhibited by VIP receptor antagonists and VIP-chemotherapeutic conjugates.

INTRODUCTION VIP/PACAP PEPTIDES

Other chapters in this book (see Arimura, Gozes, Henning, Li, Murthy, Pisegna, Said, Vallapus, Vaudry) have shown that vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are biologically active in the brain, gastrointestinal tract, heart, lung, and pituitary. Normally, VIP and PACAP are released from neurons and function in a paracrine manner, activating receptors in adjacent cells. In human breast and lung cancer, VIP and PACAP are expressed in the cancer cells, released from the cancer cells, and bind to receptors on the cancer cell surface [33]. Thus, VIP and PACAP function in an autocrine manner in breast and lung cancer cells to stimulate proliferation [32]. Breast cancer causes the death of 40,000 women in the United States annually, whereas lung cancer causes the deaths of 160,000 U.S. citizens annually [8]. Traditionally, breast cancer and lung cancer are treated with chemotherapeutic agents as well as radiation therapy. In addition, breast cancer, which is estrogen receptor positive, is treated with selective estrogen receptor modifiers, such as tamoxifen. New therapeutic approaches are needed for estrogen receptor negative breast cancer Handbook of Biologically Active Peptides

VIP is synthesized as a 170-amino-acid precursor protein (pre-pro-VIP) and metabolized to 148-aminoacid (pro-VIP) and 28-amino-acid forms (VIP) [4]. In contrast, PACAP is synthesized from a different precursor protein (176 amino acids) and metabolized to 38 (PACAP-38) and 27 (PACAP-27) amino acid forms [50]. Lung cancer cells have mRNA for pre-pro-VIP and prepro-PACAP [14]. Also, VIP and pro-VIP immunoreactivity are present in lung cancer cells [28]. PACAP mRNA and immunoreactivity is present in breast cancer biopsy specimens. Pre-pro-PACAP (19.9 kDa) but not PACAP38 was present in peritumoral and tumoral breast tissues; as well as alveolar epithelial cells and leukocytes in connective tissue [11]. The results indicate that breast and lung cancer tumors can synthesize VIP, PACAP, and their precursors. Both the fully processed peptides and their precursors have biological activity [9]. VIP and PACAP have sequence homologies at the C-terminal [2, 47]. VIP binds with high affinity to VPAC1 and VPAC2 but not PAC1 receptors, resulting in increased

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474 / Chapter 68 adenylyl cyclase activity. PACAP-27 as well as PACAP-38, which consists of PACAP-27 plus a C-terminal extension of 11 amino acids, bind with high affinity to VPAC1, VPAC2, and PAC1 receptors. Activated PAC1 receptors increase intracellular cAMP as well as cause phosphatidylinositol turnover. Selective agonists for the VPAC1 (Lys15, Arg16, Leu27)VIP1–7GRF8–27, and VPAC2 [R0251553] receptors have been identified [3, 17, 18]. Maxidilan is a 63-amino-acid peptide, which is a selective agonist for PAC1 receptors [52]. Deletion of the N-terminal nonapeptide of VIP and N-terminal pentapeptide of PACAP-38 results in VIP10–28 and PACAP6–38, which function as antagonists for VPAC1 and PAC1 receptors, respectively [10, 26, 27]. Subsequently, chimeric peptides such as neurotensin(6– 11)VIP(7–28) (VIPhyb) were developed [15]. VIPhyb is a broad-spectrum antagonist that blocks VPAC1, VPAC2, and PAC1 receptors. (N-Stearyl, Nle17)VIPhyb (SN)VIPhyb strongly inhibits the growth of human breast, lung, and pancreatic cancer cells [31, 58]. Selective peptide antagonists for VPAC1 and VPAC2 receptors include (N-Ac-Tyr1, D-Phe2)GRF(1–29)NH2, and PG99– 465, respectively [16, 36]. Nonpeptide antagonists for the VPAC1, VPAC2, and PAC1 receptors have not yet been developed.

VIP RECEPTORS The actions of VIP and PACAP are mediated by Gprotein coupled receptors. The human VPAC1 receptor is a 457-amino-acid glycoprotein that crosses the plasma membrane seven times [23]. It has 49% homology with the human VPAC2 receptor, which also contains 457 amino acids [21]. The PAC1 receptor basic form contains 467 amino acids, but splice variants have been identified that contain 28 or 56 amino acid inserts [40, 51]. Almost all breast and lung cancer cells and tumors contain VPAC1 receptors [13, 48, 54]. We have found that all breast tumors examined have VPAC1 receptor mRNA. In contrast, few breast tumors or cells have mRNA for VPAC2 receptors, whereas approximately half of the cell lines have PAC1 receptor mRNA. Using autoradiographic techniques, VPAC1 receptors are abundant in bladder, breast, colon, liver, lung, prostate, stomach, thyroid, and uterine cancer tumors [43]. VPAC2 receptors are abundant in stomach leiomyoma cancer, whereas PAC1 receptors predominate in glioblastoma, neuroblastoma, adrenal, and pituitary cancer [44, 45, 46]. VPAC1 and VPAC2 receptor immunoreactivity has been detected in lung cancer biopsy specimens [5]. VPAC1, VPAC2, and PAC1 immunoreactivity has been detected in breast cancer biopsy specimens [11].

Several amino acids have been identified in the VPAC1 receptor, which are essential for biological activity. A large protein groove may be present in the VPAC1 receptor and VPAC1 receptor acidic amino acids (Glu36 and Asp68) interact electrostatically with basic VIP amino acids (Arg14, Lys15, and Lys21) [37]. Substitution of Ala for VIP amino acids at positions 1, 6, 12, 14, or 23 reduced binding affinity to VPAC1 receptors by over two orders of magnitude. Substitution of Ala for VIP amino acids at positions 3, 5, 7, 10, 15, 20, or 21 reduced binding affinity to VPAC1 receptors by over one order of magnitude. The analog (Ala2,8,9,11,19,24,25,27,28)VIP was synthesized and found to be a potent VPAC1 receptor agonist that is resistant to degradation [22].

SECOND MESSENGERS Upon binding VIP, the VPAC1 receptor interacts with a stimulatory guanine nucleotide binding protein (Gs), which activates adenylyl cyclase [25, 49]. The addition of 10 nM VIP increases the cAMP 10–30 fold within minutes after addition to human breast or lung cancer cells. The elevated cAMP may activate protein kinase A, resulting in increased phosphorylation of protein substrates such as CREB [56]. When CREB is phosphorylated in the nucleus, it alters gene expression. Nanomolar concentrations of VIP caused increased expression of c-fos, c-jun, and c-myc oncogenes, one hour after addition to lung or breast cancer cells [57]. After translation, the c-fos and c-jun proteins may form heterodimers and activate AP-1 sites on the 5′ regulatory region of growth factor genes such as VEGF. In this regard, 10 nM VIP increased VEGF expression in lung cancer cells after 4 hours [6]. The VEGF may be secreted from cancer cells and bind to receptors on endothelial cells, facilitating tumor angiogenesis. (SN)VIPhyb blocks the increase in second messenger production caused by VIP. One μM (SN)VIPhyb antagonized the increase in cAMP caused by addition of 10 nM VIP to breast or lung cancer cells. One μM (SN)VIPhyb reversed the increase in c-fos mRNA caused by VIP addition to lung cancer cells. Preliminary data (T. Moody, unpublished) indicate that SN(VIPhyb) inhibited VEGF mRNA and tumor angiogenesis. VIP causes neuroendocrine differentiation of LNCaP prostate cancer cells [20]. Further VIP increased Ras-GTP in LNCaP cells [19]. VIP caused neurite remodeling in human neuroblastoma SH-SY5Y cells in a Cdcdependent manner [1]. This suggests that Rho family GTPases are important in neurite remodeling induced by VIP. VPAC2 receptors have a signal transduction mechanism similar to that of VPAC1 receptors. PAC1 receptors, however, can cause phosphatidyl inositol (PI) turnover

VIP and PACAP as Autocrine Growth Factors in Breast and Lung Cancer / 475 in addition to adenylylcyclase activation. One nM, PACAP-27, caused increased cAMP regardless of the PAC1 SV used. PACAP-27 (100 nM) strongly increased PI turnover in cells containing PAC1 SV-2 (hop) receptors, moderately increased PI turnover in cells transfected with SV-3 or the null receptors, but only weakly increased PI turnover when PAC1 receptor SV-1 was present [41]. Because the SVs are present in the third intracellular loop of the PAC1 receptor, this region may be important in interacting with G proteins such as Gq. These results suggest that VPAC1 and VPAC2 receptors interact with Gs leading to increased adenylyl cyclase activity, whereas the PAC1 receptor interacts with Gs as well as Gq, leading to increased intracellular cAMP and PI turnover.

PROLIFERATION Ten nM concentrations of VIP or PACAP-27 stimulate the clonal growth of breast and lung cancer cells. The increase in growth caused by VIP or PACAP-27 was reversed by μM concentrations of (SN)VIPhyb. Also, it was found that (SN)VIPhyb inhibited the basal proliferation of 51 of 56 human cancer cell lines tested, including leukemia, melanoma, breast, colon, lung, and prostate cancer [28]. Further (SN)VIPhyb potentiated the cytotoxicity of chemotherapeutic agents such as taxol in breast cancer. Taxol, which interacts with microtubules, blocks cancer cells in the M-phase of growth, whereas VIP receptor antagonists prevent G1 to S transitions. Thus, using taxol with VIP receptor antagonists causes a double cell cycle block. Using the MTT assay and MCF-7 breast cancer cells, 3 μM VIPhyb shifted significantly the taxol dose response curve (IC50 values) from 1 to 0.3 μg/ml and the doxorubicin dose response curve from 0.4 to 0.2 μg/ml [30]. Similar results were observed using vinorelbine (antimicrotubule agent), gemcitabine (antimetabolite), irinotecan (topoisomerase I inhibitor), and cisplatin (alkylating agent) [12]. Thus VIP receptor antagonists are synergistic with chemotherapeutic agents in inhibiting lung and breast cancer growth. It remains to be determined if VIPhyb will potentiate the action of chemotherapeutic drugs in cancer patients. An alternative approach is to use VPAC1 receptors to deliver VIP-chemotherapeutic conjugates to breast and lung cancer cells. VIP-LALA was conjugated to ellipticine (E), a topoisomerase II inhibitor. VIP-LALA-E bound with moderate affinity to breast cancer cells and was internalized [29]. VIP-LALA-E functioned as an agonist in that it elevated cAMP and increased c-fos mRNA. The VIP-LALA-E was metabolized by lysosomal enzymes and cytotoxic E released into the cytosol. E accumulated in the nucleus and prevented the unwind-

ing of DNA, ultimately leading to cancer cell apoptosis. Thus, VPAC1 receptors can be utilized to deliver chemotherapeutic drugs into cancer cells. Recently VIP receptors were used in a preclinical study to deliver an 131I-antisense oligonucleotide to nude mice bearing HT29 colon cancer tumors [38]. The antisense oligonucleotide for the c-myc oncogene, which was conjugated to VIP-polylysine, decreased the tumor growth rate by 9.7-fold. Thus VPAC1 receptors were utilized to deliver radioisotopes, which killed the cancer cells.

TUMOR IMAGING Due to the high densities of VPAC1 receptors in cancer cells (100,000/cell), they may be useful in imaging tumors. Initially 123I-VIP was used to localize primary tumors in colon cancer patients [53]. Subsequently 123I-VIP localized 84% of the primary tumors in carcinoid patients and 85% of liver metastases [42]. 99m Tc labeled VIP (TP-3645) has localized primary tumors in breast cancer patients [39, 55]. An 18F derivative of (Arg15, Arg21)VIP was used to localize mammary cancer tumors in animal models of breast cancer [34]. Currently, lung cancer is detected by chest x-ray when it has undergone metastasis to lymph nodes [8]. Breast cancer is detected by mammography, and frequently it has already undergone metastasis to lymph nodes. It remains to be determined if a radiolabeled VIP analog will be useful for early detection of lung and breast cancer.

CONCLUSION VIP and PACAP are peptide growth factors synthesized in breast and lung cancer cells. After secretion, they bind to cell surface VPAC1 receptors, leading to increased cellular proliferation. Because VIP and PACAP function in an autocrine manner, VPAC1 receptor antagonists such as SN(VIPhyb) decrease breast and lung cancer cellular growth. It will be important to develop nonpeptide VPAC1 receptor antagonists that bind with high affinity but are slowly degraded. A unique factor of breast and lung cancer cells is the high density of VPAC1 receptors. VIP-chemotherapeutic conjugates such as VIP-LALA-ellipticine have been developed whereby the VPAC1 receptor can be used to deliver chemotherapeutic drugs as a result of receptormediated endocytosis. Thus, the VPAC1 receptor can be utilized as a molecular target to deliver cytotoxic drugs resulting in the apoptosis of cancer cells. New VIPchemotherapeutic conjugates need to be developed

476 / Chapter 68 that are specific for the VPAC1 receptor and are more resistant to degradation by blood proteases. It remains to be determined if the VPAC1 receptor can be utilized for detection of breast and lung cancer. Initially, 123I-VIP was utilized to image tumors in carcinoid patients and subsequently 99mTc labeled VIP (TP3645) was used in breast cancer patients. New agents need to be developed for early detection of cancer in patients.

References [1] Alleaume C, Eychene A, Hnarnois T, Bourmeyster N, Constantin B, Caigneaux E, Muller JM, Philippe M. Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway. Exp. Cell Res. 2004; 299:511–24. [2] Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): Discovery and current status of research. Regul. Peptides 1992; 37:287–303. [3] Bolin DR, Michelewsky J, Wasserman MA, O’Donnell, M. Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma. Biopolymers 1995; 37:57–66. [4] Bodner M, Fridkin M, Gozes I. Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome. Proc. Natl. Acad. Sci. USA 1985; 82:3548–51. [5] Busto R, Prieto JC, Bodega G, Zapatero J, Fogue L, Carrero I. VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: A study in biopsy specimens. Peptides 2003; 24:429–36. [6] Casibang MC, Purdom S, Zia R, Jakowlew S, Neckers L, Ben-Av P, Hla T, You L, Jablons D, Moody TW. Prostaglandin E2 and VIP increase VEGF mRNAs in lung cancer cells. Lung Cancer 2001; 31:203–12. [7] Collado B, Gutierrez-Canas I, Rodriguez-Hence N, Prieto JC, Carmena MJ. Vasoactive intestinal peptide increases vascular endothelial growth factor expression and neuroendocrine differentiation in human prostate cancer LNCaP cells. Reg. Peptides 2004; 119:69–75. [8] DeVita VT, Hellman S, Rosenberg, SA. Cancer Principles and Practice of Oncology. 6th Edition, Lippincott, Williams and Wilkins, Philadelphia, 2001. [9] Fahrenkrug J. Glycine-extended processing intermediate of proVIP: A new bioactive form of VIP in the rat. Biomedical Res. 1992; 13 Sup. 2, 19–23. [10] Fishbein JA, Coy DH, Hocart SJ, Jiang NY, Mrozinski JE Jr, Mantey SA, Jensen RT. A chimeric VIP-PACAP analogue but not VIP pseudopeptides function as VIP receptor antagonists. Peptides 1994; 15:95–100. [11] Garcia-Fernandez MO, Bodega G, Ruiz-Villaespesa A, Cortes J, Prieto JC, Carmena, MJ. PACAP expression and distribution in human breast cancer and healthy tissue. Cancer Let. 2004; 205:189–95. [12] Gelber E, Granoth R, Fridkin M, Dreznik Z, Brenneman D, Moody TW, Gozes I. A lipophilic vaoactive intestinal peptide analog enhances the antiproliferative effect of chemotherapeutic agents on cancer cell lines. Cancer 2001; 92:2172–80. [13] Gespach C, Bawab W, Decremoux P, Calvo F. Pharmacology, molecular identification and functional characteristics of vasoactive intestinal peptide receptors in human breast cancer cells. Cancer Res. 1988; 48:5079–83.

[14] Gozes I, Davidson A, Draoui M, Moody TW. The VIP gene is expressed in non small cell lung cancer cell lines. Biomed. Res. 1993; 13:37–40. [15] Gozes I, McCune SK, Jacobson L, Warren D, Moody TW, Fridkin M, Brenneman DE. A hybrid antagonist to vasoactive intestinal peptide: Effects on cellular function in the central nervous system, J. Pharm Expt. Therapeutics 1991; 257:959–66. [16] Gourlet P, De Neef P, Cnuddle J, Waelbroeck M, Robberecht P. In vitro properties of a high affinity selective antagonists of the VIP1 receptor. Peptides 1997; 18:1555–60. [17] Gourlet P, Vandermeers A, Rathe J, DeNeef P, Cnuddle J, Robberecht P, Waelbroeck M. Vasoactive intestinal peptide modification at position 22 allows discrimination between receptor subtypes. Eur. J. Pharmacol. 1998; 354:105–11. [18] Gourlet P, Vandermeers A, Vertongen P, Rathe J, DeNeef P, Cnuddle J, Waelbroeck M, Robberecht, P. Development of high affinity selective VIP1 receptor agonists. Peptides 1997; 18: 1539–45. [19] Guttierrez-Canas I, Juarranz MG, Collado B, Rodriguez-Hence N, Chiloeches A, Prieto JC, Carmena MJ. Vasoactive intestinal peptide induces neuroendocrine differentiation in the LNCaP prostate cancer cell line through PKA, ERK and PI3K. The Prostate 2005; 63:44–55. [20] Gutierrez-Canas I, Rodriguez-Hence N, Bolanos O, Carmena MJ, Prieto JC, Juarranz MG. VIP and PACAP are autocrine factors that protect the androgen-independent prostate cancer cell line PC-3 from apoptosis induced by serum withdrawal. Br. J. Pharmacol. 2003; 139:1050–8. [21] Harmar T, Lutz E. Multiple receptors for PACAP and VIP. Trends in Pharmacological Sciences 1993; 15:97–9. [22] Igarishi H, Ito T, Hou W, Mantey SA, Pradhan TK, Ulrich II C, Hocart SJ, Coy DH, Jensen, RT. Elucidation of vasoactive intestinal peptide pharmacophore for VPAC1 receptors in human, rat and guinea pig. J. Pharm. Expt. Ther. 2002; 301:37–50. [23] Ishihara T, Shigemoto R, Mori K, Takahashi K, Nagata S. Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 1992; 8:811–19. [24] Korman LY, Carney DN, Citron ML, Moody TW. Secretin/VIP stimulated secretion of bombesin-like peptides from human small cell lung cancer. Cancer Res. 1986; 46:1214–18. [25] Lee M, Jensen RT, Huang SC, Bepler G, Korman L, Moody TW. Vasoactive intestinal polypeptide binds with high affinity to nonsmall cell lung cancer cells and elevates cyclic AMP levels. Peptides 1990; 11:1205–9. [26] Leyton J, Coelho T, Hida T, Jakowlew S, Birrer M, Fridkin M, Gozes I, Wank SA, Pisegna JR, Moody TW. PACAP hybrid antagonizes PACAP receptor splice variants. Life Sci. 1997; 61: 631–9. [27] Leyton J, Gozes Y, Pisegna J, Coy D, Purdom S, Casibang M, Zia F, Moody TW. PACAP(6-38) is a PACAP receptor antagonist for breast cancer cells. Breast Cancer Research and Treatment 1999; 56:177–86. [28] Moody TW, Chan D, Fahrenkrug J, Jensen RT. Neuropeptides as autocrine growth factors in cancer cells. Current Pharmaceutical Design 2003; 9:495–509. [29] Moody TW, Czerwinski G, Tarasova NI, Michejda CJ. VIP ellipticine derivatives inhibit the growth of breast cancer cells. Life Sci. 2001; 19:1005–14. [30] Moody TW, Leyton J, Chan D, Brenneman DC, Fridkin M, Gelber E, Levy A, Gozes I. VIP receptor antagonists and chemotherapeutic agents inhibit the growth of breast cancer cells. Breast Cancer Research and Treatment 2001; 68:55–64. [31] Moody TW, Leyton J, Coelho T, Knight M, Takahashi K, Koh M, Fridkin M, Gozes I. (Stearyl, Norleucine17)VIP hybrid is a potent non-small cell lung cancer VIP receptor antagonist. Life Sci. 1997; 17:1657–66.

VIP and PACAP as Autocrine Growth Factors in Breast and Lung Cancer / 477 [32] Moody TW, Leyton J, Gozes I, Lang L, Eckelman WC. VIP and breast cancer. Ann. N.Y. Acad. Sci. 1998; 865:290–6. [33] Moody TW, Walters J, Casibang M, Zia F, Gozes Y. VPAC1 receptor in lung cancer. Ann. N.Y. Acad. Sci. 2000; 921:26–32. [34] Moody TW, Leyton J, Unsworth E, John C, Lang L, Eckelman W. (Arg15, Arg21)VIP: A potent VIP agonist for localizing breast cancer tumors. Peptides 1998; 19:585–92. [35] Moody TW, Zia F, Brenneman D, Fridkin M, Davidson A, Gozes, I. A VIP antagonist inhibits the growth of non-small cell lung cancer. Proc. Natl. Acad. Sci. USA. 1993; 90:4345–9. [36] Moreno D, Gourlet P, DeNeef J, Cnuddle M, Waelbroeck M, Robberecht P. Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC2 receptor. Peptides 2000; 21:1543–9. [37] Nicole P, Lins L, Rouyer-Fessard C, Drouot C, Fulcrand P, Thomas A, Couvineau, A, Martinez J, Brasseur R, LaBurthe M. Identification of key residues for interaction of vasoactive intestinal peptide with human VPAC1 and VPAC2 receptors and development of a highly selective VPAC1 receptor agonist. Alanine scanning and molecular modeling of the peptide. J. Biol. Chem. 2000; 275:24003–12. [38] Ou X, Tan T, He L, Li Y, Li J, Kuang A. Antitumor effects of radioiodinated antisense oligonuclide mediated by VIP receptor. Cancer Gene Therapy 2005; 12:313–20. [39] Pallela VR, Thakur ML, Chakder S, Rattan S. 99mTc-labeled vasoactive intestinal peptide receptor agonist. Functional studies. J. Nuclear Medicine 1999; 40:357–60. [40] Pisegna J, Wank SA. Molecular cloning and functional expression of the pituitary adenylate cyclase activating polypeptide type I receptor. Proc. Natl. Acad. Sci. USA 1993; 90: 6345–9. [41] Pisegna JR, Wank SA. Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C. J. Biol. Chem. 1996; 271:17267–74. [42] Radener M, Kurtanan A, Leimer M, Angelberger P, Niederle B, Vierhapper H, Voreck F, Hejna MHL, Schneithauer W, Pidrich J, Virgolini I. Value of peptide receptor scintigraphy using 123Ivasoactive intestinal peptide and 111In-DPTA-D-Phe1-octreotide in 194 carcinoid patients: Vienna University experience, 1993– 1998. J. Clinical Oncology 2000; 18:1331–6. [43] Reubi JC. In vitro identification of vasoactive intestinal peptide receptors in human tumors: Implications for tumor imaging. J. Nucl. Med. 1995; 36:1846–53. [44] Reubi JC. In vitro evaluation of VIP/PACAP receptors in healthy and diseased human tissues. Clinical implications. Ann. N.Y. Acads. Sci 2000; 921:1–25. [45] Robberecht P, Vertongen P, Velkeniers B, De Neef P, Vergani P, Raftopoulos C, Brotchi JH, Hooghe-Peters EL, Christophe J. Receptors for pituitary adenylate cyclase activating peptides in

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69 Oxytocin and Cancer PAOLA CASSONI AND GIANNI BUSSOLATI

ABSTRACT

MOVING FROM PHYSIOLOGY TO NEOPLASTIC PATHOLOGY

For a long time, the hypothalamic nonapeptide oxytocin (OT) has been known to play a crucial role in many reproductive and behavioral functions. In recent years an additional new biological role of OT has been identified in neoplastic pathology. Through the activation of a specific G-coupled transmembrane receptor (the OT receptor, OTR), OT may act as a growth regulator in various tumors. In vitro, OT inhibits proliferation of neoplastic cells of either epithelial (mammary, endometrial, prostatic), nervous, or bone origin, all expressing OTR. However, in neoplastic cells derived from two additional OT target tissues, trophoblast and endothelium, OT was found to promote cell proliferation. Similarly, the proliferation of small cell lung carcinomas has been reported to be stimulated by OT in vitro. The signal transduction pathways of OT can be different. The effect of OT on growth inhibition or growth enhancement may depend on the membrane localization of the OTR itself. The OT inhibiting effect could apparently be mediated by the activation of the cAMP-PKA pathway, a nonconventional OT signaling pathway. The mitogenic effect is coupled to the increase of intracellular calcium and tyrosine phosphorylation, both known as “classical” OT transducers. The OTR localization in lipid rafts enriched in caveolin-1 leads to a proliferative response, eliciting different patterns of EGFR/MAPK activation. This unexpected role of OT (and OT analogs) in regulating neoplastic cell proliferation, as well as the wide distribution of OTR in neoplastic tissues of different origin, opens new perspectives on the biological role of the OT-OTR system in the control of cancer growth. Handbook of Biologically Active Peptides

OTR are G-protein coupled receptors with a typical seven transmembrane domain structure [24]. These receptors mediate many different biological functions of OT, mostly related to reproduction and behavior [22, 25, 38]. OTR are therefore expressed in different target tissues such as endometrium [35], myometrium [18], trophoblast [36], and ovary [23], where they are involved in various functions, mainly related to parturition or ovulation; in the breast, where OTR are necessary to mediate the myoepithelial cell contraction required for milk ejection [29]; and in the central nervous system, where OTR activation participates in the control of various behavioral functions [22]. In recent years OTR have also been described in other, different tissues, which usually were not considered conventional targets for the peptide, such as the vasculature [21, 37], prostate [39], and bone [15]. In the preceding tissues, OTR presence and activation is related to physiological or paraphysiological functions. In the last 10 years, the presence of OTR in tumors arising from some of these OT target tissues (physiologically expressing OTR), as well as their possible role, has been investigated. In breast carcinomas, OTR were demonstrated in the large majority (80%) of primary breast lesions both at the mRNA level (by RT-PCR) and the protein level (by immunohistochemistry) by our group and others [2, 20]. The presence of OTR has also been described in breast cancer cell lines, regardless of their estrogen dependency or independency [4]. Recently, it has been reported that

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480 / Chapter 69 estradiol and progesterone may regulate OTR expression and binding in breast carcinoma cell lines. 1OT seems to modulate the expression of ER in the same breast carcinoma cell lines, causing a reduction of the number of ER alpha binding sites, which is balanced by the increase in both the receptor binding affinity and transcriptional activity [10]. These observations suggest the existence of an additional, indirect role for steroids in breast neoplastic pathology, via the OTOTR system, as well as the existence of an unexpected OT-dependent modulation of ER in breast cancer. OTR are present also in primary adenocarcinomas of the endometrium [8], a tissue that is known to express OTR under physiological conditions, as well as myometrium. Because of the wide distribution of OTR in the normal uterus, either in its myometrial or endometrial component, the effective site of localization of the OTR mRNA in endometrial carcinomas needed to be verified by a technique other than RT-PCR, such as in situ hybridization (ISH). It was shown by ISH that neoplastic cells of endometrial origin had OTR mRNA. Therefore, the RT-PCR positive results were not dependent on the presence of a myometrial component in the extracted tissue [8]. In all the primary endometrial carcinomas examined the ISH positivity was intense and diffuse. Interestingly, the pattern of protein expression as revealed by immunohistochemistry (IHC) varied according to the degree of tumor differentiation [8]. More precisely, poorly differentiated carcinomas had diffuse OTR immunoreactivity, whereas well-differentiated tumors presented a clonal pattern of OTR immunoreactivity [8]. This different level of protein expression was independent from ISH positivity, which was equally present in all tumors examined, regardless of their degree of differentiation. It still remains to be clarified if the overexpressed OTR in poorly differentiated endometrial cancers have biological activity or if they are defective, mutated, inactive receptors. Within the female “reproductive area,” OTR are also present in a human choriocarcinoma cell line, as well as in two normal trophoblast cell lines [9]. In the male reproductive system, OTR are diffusely present within prostate carcinomas [12]. The significance of OTR in the normal prostate is still debated. OTR expression in neoformed, neoplastic glands has been demonstrated to be unrelated to the androgen-receptor status of the cancer cells [12]. It remains to be determined if patients with hormone-independent prostate tumors, who usually have poor outcome, would benefit from hormonal therapy. Among tumors of epithelial origin, OTR have been described in lung small cell carcinoma cell lines [31]. The OTR expression in neoplastic cells of endocrine origin, such as small cell lung carcinoma, synthesize OT

itself, as was reported by North and coworkers, suggesting the possible existence of an autocrine effect of OT in these tumors [26, 27]. The possible autocrine role of OT in endocrine tumors warrants further investigation [16]. Finally, OTR are present in tumors of neural and glial origin of both the central and peripheral nervous system. Primary glioblastomas and neuroblastomas, as well as human cell lines originating from these tumors, express OTR both at the mRNA and protein level [7]. In analogy to these tissues, it has been reported that normal bone cells also express OTR [15]. Accordingly to what has previously been observed in all the neoplasm derived from tissues expressing OTR under physiological conditions, human osteosarcoma cell lines express OTR mRNA as well, and synthesize the receptor at protein level [28].

OT AS A MODULATOR OF CELL GROWTH IN OTR-EXPRESSING TUMORS Beside the well-known roles of OT in regulating reproductive and behavioral functions under physiological conditions, some other roles and biological activities have become more and more evident in the neoplastic area. In vitro, we demonstrated a unique effect of inhibition in the proliferation of breast [4], endometrial [8], prostatic [12], glial and neural [7], and bone [28] neoplastic cells. Such growth inhibition ranges from 30 to 50% for all the different cell lines and was evident within the first 48 hours of OT treatment. The lowest concentration of the peptide that could significantly reduce cell proliferation was 10 nM or 100 nM, depending on the cell line studied. The inhibition of breast tumor growth was also confirmed by in vivo experiments on Balb-c mice injected with the mammary TS/A tumor and treated by subcutaneous pulsatile administration of the peptide [5]. Although the inhibiting effect of OT on cell proliferation was reproducible in all the previously described cells, in the BeWo human choriocarcinoma cell line OT stimulated cell proliferation [9]. In fact, 10 nM to 1 μM OT treatment significantly increased the cell number from the earliest time of administration (48 hours). Furthermore, a selective OT antagonist (OTA) reversed the mitogenic effect of OT. OTA alone significantly inhibited cell proliferation [9]. Similarly, OT stimulated cell proliferation in an endothelial-derived sarcomatous cell line, KS-IMM, derived from a Kaposi’s sarcoma [11]. Interestingly, this biological effect was identical to that observed in normal, nonneoplastic, endothelial cells in culture [37]. Also, the mitogenic effect of OT was shown in small cell lung carcinoma cell lines in vitro and, since OT is directly produced by the cells themselves, the existence of an effective autocrine-paracrine loop

Oxytocin and Cancer participating in the regulation of cell growth is hypothesized [31]. In previous studies, we reported that these opposite effects of OT on cell proliferation (inhibition vs. stimulation) are coupled to the activation of different signaling pathways. The cAMP-PKA signaling pathway was involved in the antiproliferative effect in breast, endometrial, bone, and nervous system tumors. Specifically, intracellular cAMP levels were increased following OT treatment, and a selective inhibitor of the PKA eliminated the antiproliferative effect of OT [5, 6]. The activation of OTR involves intracellular Ca2+ increase and the inositol phosphate pathway. Some authors hypothesize that the activation of the cAMPPKA pathway could depend on the binding of OT to the vasopressin receptor type 2 and not to OTR [17]. In choriocarcinoma cells, as well as on the Kaposi’s sarcoma derived cell line, the mitogenic effect of OT was coupled to intracellular Ca2+ increase [9, 11]. The use of OTA (already defined) blocked the Ca2+ increase after vasopressin infusion and the Ca2+ increase induced by the OT agonist, Thr4-OT. In the choriocarcinoma cell line, the proliferative effect was also associated to the tyrosine phosphorylation of three major proteins of 125, 60, and 45 kDa [9]. Similarly, in the small cell carcinoma cell lines the mitogenic effect of OT is coupled to an increase in cytosolic Ca2+, and followed by the activation of the MAP-kinase pathway [32]. Recently, Guzzi and coworkers [19] demonstrated that a different localization of OTR within the cell membrane may play a critical role in determining the peptide stimulating or inhibiting effect on cell growth. They proposed two allosteric states of OTR (active and inactive) [13]. Recent results on the membrane localization of OTR demonstrate that when the vast majority of OTR are excluded from lipid rafts enriched in caveolin-1, OT inhibits cell proliferation, while, when OTR are targeted to lipid rafts, the hormone has a strong mitogenic effect [19]. The crucial role of lipid raft in endocytosis as well as in membrane dynamics explains the spatial segregation of certain signaling pathways emanating from the plasma membrane. Lipid rafts are highly ordered islands constituted of lipids and cholesterol that could be involved in regulating the membrane-receptor mediated processes [30]. Therefore, OTR stimulation leads to different patterns of EGFR and MAPK activation, depending on its localization. When OTR is located outside lipid rafts, it inhibits cell growth, whereas when OTR is located in lipid rafts, it stimulates cell growth. If the OTR is localized outside lipid rafts, it causes activation of p21WAF1/CIP1, a cycle inhibitor [34]. Interestingly, an OT antagonist (atosiban) that binds to OTR but does not induce any contractile response in typical target cells, such as myometrial

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cells, acts on OTR in neoplastic cells, inducing inhibition of cell growth [33]. This may suggest a possible therapeutical use of such antagonists in OTR-positive tumors.

THE OT/OTR SYSTEM AND CANCER The distribution of the OTR within neoplastic tissues of even very different origin is wider than expected. Through these specific binding sites, OT may play a regulatory role in the tumor growth, inhibiting or stimulating cell proliferation. Further studies would clarify if the different biological action of OT may depend on the different receptor locations within the cell membrane. Alternatively, different, still unknown, OTR subtypes may be coupled to different signaling pathways. The expression of OTR may have future applications in the radioimaging of different neoplasms as well as in their therapy, using OT analogs conserving a high OTR affinity as either radiotracers or chemotherapeutics vector [3, 14].

References [1] Amico JA, Rauk PN, Cai HM. Estradiol and progesterone regulate OT receptor binding and expression in human breast cancer cell lines. Endocrine 2002;18:79–84. [2] Bussolati G, Cassoni P, Ghisolfi G, Negro F, Sapino A. Immunolocalization and gene expression of OT receptors in carcinoma and non-neoplastic tissue of the breast. Am J Pathol 1996; 148:1895–903. [3] Bussolati G, Chinol M, Chini B, Nacca A, Cassoni P, Paganelli G. 111In-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′tetraacetic acid-lys(8)-vasotocin: a new powerful radioligand for OT receptor-expressing tumors. Cancer Res 2001;61:4393–7. [4] Cassoni P, Sapino A, Negro F, Bussolati G. OT inhibits proliferation of human breast cancer cell lines. Virchows Archiv 1994;425:467–72. [5] Cassoni P, Sapino A, Papotti M, Bussolati G. OT and OTanalogue F314 inhibit cell proliferation and tumour growth of rat and mammary carcinomas. Int J Cancer 1996;66:817–20. [6] Cassoni P, Sapino A, Fortunati N, Munaron L, Chini B, Bussolati G. OT inhibits the proliferation of MDA-MB231 human breastcancer cells via cyclic adenosine monophosphate and protein kinase A. Int J Cancer 1997;72:340–4. [7] Cassoni P, Sapino A, Fortunati N, Bussolati G. Presence and significance of OT receptors in human neuroblastomas and glial tumors. Int J Cancer 1998;77:695–700. [8] Cassoni P, Fulcheri E, Carcangiu ML, Stella A, Deaglio S, Bussolati G. OT receptors in human adenocarcinomas of the endometrium: presence and biological significance. J Pathol 2000;190:470–7. [9] Cassoni P, Sapino A, Munaron L, Deaglio S, Chini B, Graziani A, Ahmed A, Bussolati G. Activation of functional OT receptors stimulates cell proliferation in human trophoblast and choriocarcinoma cell lines. Endocrinology 2001;142:1130–6. [10] Cassoni P, Catalano MG, Sapino A, Marrocco T, Fazzari A, Bussolati G, Fortunati N. OT modulates estrogen receptor alpha expression and function in MCF7 human breast cancer cells. Int J Oncol 2002;21:375–8.

482 / Chapter 69 [11] Cassoni P, Sapino A, Deaglio S, Bussolati B, Volante M, Munaron L, Albini A, Torrisi A, Bussolati G. OT is a growth factor for Kaposi’s sarcoma cells: evidence of endocrine-immunological cross-talk. Cancer Res 2002;62:2406–13. [12] Cassoni P, Marrocco T, Sapino A, Allia E, Bussolati G. Evidence of OT/OT receptor interplay in human prostate gland and carcinomas. Int J Oncol 2004;25:899–904. [13] Chini B, Fanelli F. Molecular basis of ligand binding and receptor activation in the OT and vasopressin receptor family. Exp Physiol 2000;85:59S–66S. Review. [14] Chini B, Chinol M, Cassoni P, Papi S, Reversi A, Areces L, Marrocco T, Paganelli G, Manning M, Bussolati G. Improved radiotracing of OT receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue. Br J Cancer 2003;89:930–6. [15] Copland JA, Ives KL, Simmons DJ, Soloff MS. Functional OT receptors discovered in human osteoblasts. Functional OT receptors discovered in human osteoblasts. Endocrinology 1999;140:4371–4. [16] Emmer T, Volante M, Pagani A, Allia E, Crafa P, Bussolati G. Potential applications of molecular biology in neuroendocrine tumors. Endocr Pathol 2003;14:319–28. Review. [17] Fay MJ, Du J, Longo KA, North WG. OT does not induce a rise in intracellular free calcium in human breast cancer cells. Res Commun Mol Pathol Pharmacol 1999;103:115–28. [18] Fuchs AR, Fuchs F, Soloff MS. OT receptors in nonpregnant human uterus. J Clin Endocrinol Metab 1985;60:37–41. [19] Guzzi F, Zanchetta D, Cassoni P, Guzzi V, Francolini M, Parenti M, Chini B. Localization of the human OT receptor in caveolin1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response. Oncogene 2002; 21:1658–67. [20] Ito Y, Kobayashi T, Kimura T, Matsuura N, Wakasugi E, Takeda T, Shimano T, Kubota Y, Nobunaga T, Makino Y, Azuma C, Saji F, Monden M. Investigation of the OT receptor expression in human breast cancer tissue using newly established monoclonal antibodies. Endocrinology 1996;137:773–9. [21] Jankowski M, Wang D, Hajjar F, Mukaddam-Daher S, McCann SM, Gutkowska J. OT and its receptors are synthesized in the rat vasculature. PNAS 2000;11:6207–11. [22] Kendrick KM. OT, motherhood and bonding. Exp Physiol Mar 2000; 85 Spec No:111S–124S. [23] Khan-Dawood FS. OT in intercellular communication in the corpus luteum. Semin Reprod Endocrinol 1997;15:395–407. [24] Kimura T, Tanizawa O, Mori K, Brownstein MJ, Okayama H. Structure and expression of a human OT receptor. Nature (London) 1992;356:526–9. [25] Kimura T, Takemura M, Nomura S, Nobunaga T, Kubota Y, Inoue T, Hashimoto K, Kumazawa I, Ito Y, Ohashi K, Koyama M, Azuma C, Kitamura Y, Saji F. Expression of OT receptor in human pregnant myometrium. Endocrinology 1996;137: 780–5.

[26] North WG. Neuropeptide production by small cell carcinoma: vasopressin and OT as plasma markers of disease. J Clin Endocrinol Metab 1991;73:1316–20. [27] North WG, Friedmann AS, Yu X. Tumor biosynthesis of vasopressin and OT. Ann N Y Acad Sci 1993;689:107–21. [28] Novak JF, Judkins MB, Chernin MI, Cassoni P, Bussolati G, Nitche JA, Nishimoto SK. A plasmin-derived hexapeptide from the carboxyl end of osteocalcin conteracts OT-mediated growth inhibition of osteosarcoma cells. Cancer Res 2000;60: 3470–6. [29] Olins GM, Bremel RD. OT-stimulated myosin phosphorylation in mammary myoepithelial cells: roles of calcium ions and cyclic nucleotides. Endocrinology 1984;114:1617–25. [30] Parton RG, Richards AA. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 2003;4:724–38. [31] Pequeux C, Breton C, Hendrick JC, Hagelstein MT, Martens H, Winkler R, Geenen V, Legros JJ. OT synthesis and OT receptor expression by cell lines of human small cell carcinoma of the lung stimulate tumor growth through autocrine/paracrine signaling. Cancer Res 2002;62:4623–9. [32] Pequeux C, Keegan BP, Hagelstein MT, Geenen V, Legros JJ, North WG. OT- and vasopressin-induced growth of human small-cell lung cancer is mediated by the mitogen-activated protein kinase pathway. Endocr Relat Cancer 2004;11:871–85. [33] Reversi A, Rimoldi V, Marrocco T, Cassoni P, Bussolati G, Parenti M, Chini B. The OT receptor antagonist atosiban inhibits cell growth via a “biased agonist” mechanism. J Biol Chem 2005;280:16311–18. [34] Rimoldi V, Reversi A, Taverna E, Rosa P, Francolini M, Cassoni P, Parenti M, Chini B. OT receptor elicits different EGFR/ MAPK activation patterns depending on its localization in caveolin-1 enriched domains. Oncogene 2003;22:6054–60. [35] Takemura M, Nomura S, Kimura T, Inoue T, Onoue H, Azuma C, Saji F, Kitamura Y, Tanizawa O. Expression and localization of OT receptor gene in human uterine endometrium in relation to the menstrual cycle. Endocrinology 1993;132:1830–5. [36] Takemura M, Kimura T, Nomura S, Makino Y, Inoue T, Kikuchi T, Kubota Y, Tokugawa Y, Nobunaga T, Kamiura S, et al. Expression and localization of human OT receptor mRNA and its protein in chorion and decidua during parturition. J Clin Invest 1994;93:2319–23. [37] Thibonnier M, Conarty DM, Preston JA, Plesnicher CL, Dweik RA, Erzurum SC. Human vascular endothelial cells express OT receptors. Endocrinology 1999;140:1301–9. [38] Wathes DC, Borwick SC, Timmons PM, Leung ST, Thornton S. OT receptor expression in human term and preterm gestational tissues prior to and following the onset of labour. J Endocrinol 1999;161:143–51. [39] Whittington K, Assinder S, Gould M, Nicholson H. OT, OTassociated neurophysin and the OT receptor in the human prostate. Cell Tissue Res 2004;318:375–82.

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70 Antagonists of Growth Hormone–Releasing Hormone (GHRH) in Cancer JOZSEF L. VARGA AND ANDREW V. SCHALLY

information on VIP and PACAP, see the chapters by Moody and Arimura). The main site of production of GHRH is the hypothalamus, but various peripheral organs, including placenta, ovary, mammary, testis, prostate, and the gastrointestinal tract, also synthesize GHRH [1, 14, 22, 29]. GHRH is produced, sometimes in large quantities, by human malignant tissues of diverse origin [29, 33]. In fact, the identification of GHRH was facilitated by the demonstration of ectopic production of GHRH by carcinoid and pancreatic islet tumors that caused acromegaly [9]. The 44- and 40amino-acid forms of GHRH were first isolated and sequenced from human pancreatic tumors and only subsequently identified in human hypothalamus. Hypothalamic GHRH, acting through specific GHRH receptors on the pituitary, stimulates the synthesis and the secretion of hypophyseal GH (see chapter by Malagon). The functions of GHRH in extrapituitary tissues are less clear. However, there is increasing evidence for the role of GHRH as an autocrine/paracrine growth factor in various cancers [4, 24, 26, 33]. Splice variants (SV) of receptors for GHRH, different from those expressed in the pituitary, have been found in a wide range of human cancers and in some normal tissues [13, 14, 31, 40]. Recently, expression of the pituitary type of GHRH receptor was also detected in a number of human tumors, including lymphomas and lung cancers [14]. GHRH has been shown to stimulate the proliferation of cancer cells and transfected cell lines, which express the pituitary type, as well as the SV1 isoform of GHRH receptor [23, 25]. Thus, the actions of tumoral autocrine/paracrine GHRH could be exerted on both types of these receptors. In addition, receptors of VIP and other as yet unidentified receptors of this family could be targets of local GHRH [33, 34].

ABSTRACT In the past 10 years potent antagonistic analogs of growth hormone-releasing hormone (GH-RH) have been synthesized. These GH-RH antagonists bind to pituitary receptors for GH-RH and inhibit the release of GH in vitro and in vivo. The presence of GH-RH ligand was demonstrated in various cancers, suggesting that GH-RH could be an autocrine growth factor. GHRH antagonists inhibit the growth of various human cancer lines xenografted into nude mice, including breast cancers, prostate cancers, small cell lung carcinomas (SCLC) and non-SCLC, malignant gliomas, renal cell carcinomas, pancreatic cancers, colorectal carcinomas, and lymphomas. These effects of GHRH antagonists could be exerted in part indirectly through inhibition of the secretion of pituitary GH and the resulting reduction in the levels of hepatic IGF-I. However, the principal effect of GH-RH antagonists appears to be the direct suppression of action of the autocrine GHRH and the secretion of IGF-I and IGF-II in tumors, as well as expression of the genes encoding them. These direct effects of GHRH antagonists are exerted on GHRH receptors and their splice variants on tumors. Further development of GH-RH antagonists should lead to potential therapeutic agents for various cancers.

INTRODUCTION Growth hormone-releasing hormone (GHRH) belongs to the secretin/glucagon family of neuroendocrine and gastrointestinal peptides that also includes vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), and others. (For Handbook of Biologically Active Peptides

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484 / Chapter 70 THERAPEUTIC INDICATIONS OF GHRH ANTAGONISTS In view of the role of GHRH as a hypothalamic regulator of GH release and as an autocrine/paracrine growth factor in cancers, therapeutic strategies based on the use of GHRH analogs have been developed for the treatment of various pathological conditions. The GHRH antagonists could be tried in conditions such as acromegaly, diabetic retinopathy, and diabetic nephropathy (glomerulosclerosis), where a reduction of serum GH levels is desirable. Nevertheless the main applications of GHRH antagonists would be in the field of cancer [33, 34]. By blocking the pituitary GHRH receptors, GHRH antagonists inhibit the synthesis and release of pituitary GH, leading to a decrease of production of IGF-I in the liver and other organs [34]. IGF-I is an established mitogen for various cancers; thus, a decrease of serum IGF-I levels after treatment with GHRH antagonists is likely to contribute to the experimentally observed antitumor effects of GHRH antagonists [34]. However, GHRH antagonists also bind with high affinity to the SV and pituitary isoforms of GHRH receptors expressed on tumor membranes and exert direct antiproliferative effects on the cancer cells [13, 14, 24, 31, 33]. These effects are independent of their indirect effects mediated by the endocrine pituitary GH-hepatic IGF-I axis. Recent evidence indicates that the direct inhibitory effects of GHRH antagonists on tumoral GHRH receptors and consequently the antiproliferative properties of these analogs are not always correlated with their antagonistic effects on the pituitary GHRH receptors [20, 41]. Thus, for a more complete characterization of their properties and therapeutic indications, antagonistic analogs of GHRH are tested both in endocrine assays for the inhibition of GH release and in oncological assays for the inhibition of tumor proliferation.

ANTAGONISTIC ANALOGS OF GHRH The full intrinsic biological activity of human GHRH is retained by the amino-terminal 29-amino-acid sequence [hGHRH(1–29)NH2]. Consequently, most agonistic and antagonistic analogs of GHRH were synthesized based on the structure of this peptide. Early studies revealed that replacement of Ala2 in GHRH by D-Ala2 or N-methyl-D-Ala2 leads to superpotent agonists, while the replacement by D-Arg2 produces antagonists [32, 34]. The earliest GHRH antagonist, defined as “standard antagonist” (see Table 1 for structure) [32], has been tested clinically. Large doses of this antagonist (400 μg/kg) inhibited the response to GHRH and reduced GH levels in a patient with acromegaly caused

by GHRH-secreting carcinoid tumor [15]. However, for clinical use, much more potent antagonists of GHRH are required. Synthetic work in our laboratory has been aimed at developing GHRH antagonists with increased binding affinities to the pituitary and tumoral GHRH receptors, enhanced enzymatic and chemical stabilities, greater and more protracted GH inhibitory effects in vitro and in vivo, and increased direct antitumor activities [33, 34, 41]. Initial work yielded the potent GHRH antagonists MZ-4-71 and MZ-5-156, containing hydrophobic and helix stabilizing amino acid substituents, combined with a hydrophobic N-terminal acyl moiety and enzymatically resistant agmatine at the C-terminus (see Table 1) [34]. Subsequent work revealed that incorporation of positively charged arginine or homoarginine (Har) residues in position 9 and a positively charged, enzymatically resistant D-Arg28-Har29-NH2 sequence at the C-terminus, produces antagonists with further increased activities in vivo, such as JV-1-36 and JV-1-38 (Table 1) [34]. Later we synthesized antagonistic analogs of hGHRH(1–29)NH2 with other substituents at positions 8, 9, and 10 [41]. Representative analogs of this series are JV-1-63, JV-1-65, and JV-1-68, shown in Table 1. JV-1-63 is one of the most potent endocrine antagonists of GHRH reported to date [41]. In the rat pituitary superfusion system, 30 nM JV-1-63 causes a nearly total blockade of GHRH-evoked GH release that lasts for more than two hours. In vivo, JV-1-63 injected intravenously at 80 μg/kg inhibits the GH release in rats evoked by 3 μg/kg hGHRH(1–29)NH2 for at least one hour. The binding affinity of JV-1-63 peptide for the rat pituitary GHRH receptors is about 80 times higher than that of the standard antagonist [41]. In contrast, GHRH antagonist JV-1-65 exhibits only a weak and short inhibitory action on GH release in vivo [41]. Nevertheless, antagonist JV-1-65 was more potent than JV-1-63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice [17, 41]. This indicated that oncological activity of GHRH antagonists is not primarily correlated with the endocrine inhibitory effects on GH release but is based on several mechanisms. Subsequent work revealed that incorporation of fatty acids into the N-terminal position of GHRH antagonists, such as octanoic acid in MZ-J-7-114 or dodecanedicarboxylic acid in MZ-J-7-110, leads to a further increase in antitumor potency [19] (Table 1). Furthermore, we found that Arg and Lys residues, prone to enzymatic attack by trypsin-like proteases, can be replaced by His and ornithine (Orn) residues to yield GHRH antagonists with increased antitumor activities. Representative antagonists of this series include JV-1-80, MZ-J-7-132, MZ-J-7-118, and MZ-J-7-138 (Table 1). These antagonists exhibit increased antitumor effects in vivo on human experimental prostatic and

Antagonists of Growth Hormone–Releasing Hormone (GHRH) in Cancer / 485 endometrial cancers, and lymphomas but weaker endocrine effects on the inhibition of hepatic IGF-I in serum, as compared to earlier antagonists such as MZ-4-71 or MZ-5-156 [7, 20, 35, 36].

GHRH AND GHRH RECEPTORS IN HUMAN CANCERS Evidence accumulated mainly since 1999 indicates that GHRH is an autocrine/paracrine growth factor for many cancers and that its effects are mediated by specific tumoral receptors for GHRH [13, 14, 26, 31, 33]. The proliferation of many cancer cell lines cultured in vitro, including those of the lung, prostate, breast, endometrium, ovary, stomach, pancreas, colon, and bone, is stimulated by exogenous GHRH and, conversely, inhibited by GHRH antagonists or antisera to GHRH [3, 4, 11, 23, 24, 26, 33, 34, 38]. The presence of mRNA for GHRH or GHRH peptide has been demonstrated in surgical specimens of human pancreatic, lung, prostatic, breast, ovarian, endometrial, adrenal, and pituitary tumors [1, 5, 8, 9, 12, 16, 22, 29, 33, 39]. In addition, various human cancer lines including lung, breast, endometrial, ovarian, pancreatic, gastric, colorectal, prostatic, brain, bone sarcomas, and lymphomas express mRNA or show GHRH peptide [3, 4, 6, 7, 11, 14, 16, 17, 20, 21, 26, 30, 38]. The overexpression of the GHRH gene in pituitary tumors has been associated with neoplastic progression and aggressive behavior [39]. A significantly higher expression of mRNA for GHRH, or increased secretion of GHRH peptide, was also found in specimens of human ovarian and breast cancers [1, 22] and in cell lines of immune cell-derived tumors [21], as compared with normal tissues and cell lines. These observations suggest that a deregulation of GHRH gene transcription may contribute to the pathogenesis of some of these malignancies. Initial attempts to detect the tumoral receptors that mediate the direct effects of GHRH and its antagonists were unsuccessful, since the pituitary-type GHRH receptor (pGHRH-R) is not expressed in a number of human prostatic, pancreatic, renal, breast, and ovarian cancers, and SCLC models [13, 31, 33, 34]. However, we found that these and other tumors, including primary human prostate cancers, express the mRNAs for four truncated splice variants (SVs) of GHRH-R [12, 13, 31]. Radioligand binding studies using GHRH antagonist JV-1-42 (Table 1) as a special ligand revealed the presence of tumoral binding sites for GHRH with characteristics different from the pituitary receptors [12, 13, 31]. Of the four SVs of GHRH-R, SV1 has the greatest structural similarity to the pGHRH-R and is probably the main splice variant that mediates the effects of

GHRH and its antagonists on tumors [23, 25, 31]. The deduced protein sequence of SV1 differs from the pGHRH-R only in the N-terminal extracellular domain, the first 89 amino acids of the pGHRH-R being replaced by a different, 25-aa sequence [31]. Thus, SV1 would encode a functional G-protein coupled receptor with 7 transmembrane domains. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain [31]. To confirm that mRNA for SV1 is translated into a corresponding GHRH-R isoform protein, we used antisera generated against the N-terminal 25-amino-acid sequence, which is present in SV1 but not in the pGHRH-R [40]. In these studies, SV-specific immunostaining or Western blot signals consistent with the expected molecular mass of ∼40 kDa of SV1 protein were detected in cell lines of human endometrial, lung and pancreatic cancers, lymphomas, and glioblastomas, as well as in surgical specimens of endometrial, breast, and lung cancers [5, 14, 23, 40]. The functionality of the GHRH-R isoform encoded by SV1 was tested in experiments involving the ectopic expression of this protein into 3T3 fibroblasts or antisense RNA-mediated ablation of its expression in HEC1A human endometrial cancer cells [23, 25]. We showed that the expression of SV1 is correlated with binding of GHRH analogs, immunostaining with the specific antiserum, and that SV1 mediates the mitogenic effect of GHRH and antimitogenic effects of GHRH antagonists [23, 25]. In contrast to the pGHRH-R, SV1 possesses relatively high-intrinsic, ligand-independent activity [23]. The expression of SV1, but not of the pGHRH-R, stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH [23]. Therefore, SV1 exerts significant baseline mitogenic activity, but maximal activation of the receptor-mediated cell proliferation is only reached after binding to GHRH. Recently, we reinvestigated whether tumors can express the pGHRH-R. Using real-time PCR, Western blots, and radioligand binding assays with antibodies and ligands specific for the pGHRH-R, we showed that the pituitary-type receptor is detectable in human lymphoma, glioblastoma and SCLC cell lines, and in surgical specimens of human lung cancers [14]. Since various tumors can express not only the SVs, but also the pGHRH-R, both types of GHRH receptors should be considered as potential targets for anticancer therapy with GHRH antagonists. So far, the expression of GHRH receptors has been described in primary human prostatic, lung, endometrial, breast, and adrenal carcinomas [5, 8, 10, 12, 14, 23] and in cell lines of virtually all major types of malignancies, including prostatic, lung (SCLC and nonSCLC), breast, ovarian, endometrial, gastric, pancreatic,

486 / Chapter 70 colorectal, renal, glioblastomas, osteogenic and Ewing’s sarcomas, and lymphomas [3, 4, 6, 7, 11, 13, 14, 17, 20, 23, 27, 30, 31, 36, 38, 40].

MECHANISMS OF TUMOR INHIBITION BY GHRH ANTAGONISTS The antitumorigenic actions of GHRH antagonists are exerted directly on the cancer cells by blocking the tumoral isoforms of GHRH-R and preventing the effects of tumoral autocrine/paracrine GHRH, as well as indirectly at the pituitary level with the resulting suppression of the pituitary GH/hepatic IGF-I axis [33]. In addition, GHRH antagonists inhibit the production of tumoral IGF-I and IGF-II and possibly several other autocrine/paracrine and endocrine growth factors and their receptors that are known to stimulate the growth of neoplastic cells [33, 34]. The relative importance of these mechanisms could vary in different tumors. In studies with nude mice, the tumor inhibitory effects of high doses (40–80 μg/day) of early antagonists such as MZ-4-71 and MZ-5-156 on the growth of osteogenic sarcomas, prostatic, renal and lung cancers, and lymphomas were associated with a significant inhibition of serum GH and IGF-I levels [2, 20, 33, 34]. However, lower doses of these antagonists, or more recently developed analogs with decreased endocrine inhibitory effects such as JV-1-65, MZ-J-7-118, and MZ-J7-138, inhibit the growth of pancreatic, colorectal, prostatic, breast, ovarian, endometrial, lung cancers, and lymphomas in the absence of any significant effects on serum IGF-I [7, 19, 20, 24, 26, 28, 33, 35, 38]. This indicates that the direct inhibitory effects of GHRH antagonists on cancer cells are in general more important for tumor inhibition than their endocrine activities on suppression of GH release and serum IGF-I. The tissue levels of IGF-I and/or IGF-II in tumors, and the expression of their mRNAs, were decreased by GHRH antagonists in pancreatic, colorectal, renal, prostatic, lung, ovarian, breast, bone tumors, and glioblastomas [2, 17, 24, 27, 33, 34, 37, 38]. This is presumably due to the direct effects of antagonists on tumor tissue. However, in H69 SCLC, MDA-MB-435 breast, LNCaP prostatic, and in HEC-1A endometrial tumors, tumoral IGF-I and -II levels were not reduced indicating that GHRH antagonists can inhibit tumor growth without the participation of the tumoral IGF system [7, 24, 27, 33]. In MXT mouse mammary cancers, tumoral GH and GH receptor levels were strongly decreased after treatment with GHRH antagonists [37]. The concentration and mRNA levels of tumoral angiogenic growth factors, VEGF, VEGF receptors, and/or bFGF,

were reduced in human prostatic cancer, SCLC and non-SCLC, and lymphoma models [20, 27, 35]. Recently we also found that GHRH antagonists decrease the protein and mRNA expression of EGF receptors and related HER-2, -3, and -4 receptors in human SCLC, non-SCLC, and prostate cancer models [18, 35]. Intracellularly, GHRH antagonists inhibit some of the signaling mechanisms involved in cell proliferation, survival, and metastasis, and activate pro-apoptotic signaling mechanisms. Thus, we found that GHRH antagonists inhibit the PKC-MAPK and PI3K-Akt signaling pathways, decrease the expression of c-jun and c-fos oncogenes, and mutant p53 protein levels in human SCLC, non-SCLC, and prostate cancer models, and reduce the telomerase activity in glioblastomas and other cancers [17, 19, 24]. In addition, GHRH antagonists decrease antiapoptotic Bcl-2 and increase proapoptotic Bax protein levels in non-SCLC models, and in the prostate cancer line LNCaP they trigger a Ca2+dependent apoptotic mechanism.

INHIBITORY EFFECTS OF GHRH ANTAGONISTS ON HUMAN EXPERIMENTAL CANCERS IN VIVO The antitumor effects and mechanisms of GHRH antagonists were investigated extensively in human cancer cell lines grown in nude mice. These studies helped elucidate the potential therapeutic benefits of GHRH antagonists and provided a basis for planning future clinical trials with cancer patients.

Prostate Cancer The growth of s.c. xenografts of human androgenindependent PC-3 and DU-145 prostate cancers in nude mice was inhibited by high doses (20 μg b.i.d.) of early GHRH antagonists MZ-4-71 and MZ-5-156 or much lower doses (2.5–5 μg/day) of new and more potent antagonists MZ-J-7-118 and MZ-J-7-138 [33–35]. GHRH antagonists also inhibited the orthotopic growth and metastatic spread, as well as the intraosseous growth of PC-3 tumors [36]. Given alone, GHRH antagonists JV-1-38 (20 μg/day) or MZ-J-7-118 (5 μg/day) were ineffective for the treatment of human androgen-sensitive prostate cancers LNCaP and MDA-PCa-2b, but they greatly enhanced the inhibitory effects of androgen deprivation therapies such as surgical castration and LHRH agonists or antagonists on the growth of s.c. and orthotopic prostate tumors [27, 28, 33]. Therapy with GHRH antagonists should be considered for the management of both androgen-dependent and -independent prostate cancers.

Antagonists of Growth Hormone–Releasing Hormone (GHRH) in Cancer / 487

Lung Cancer Early GHRH antagonists MZ-4-71 and MZ-5-156 (20 μg b.i.d. each) significantly inhibited growth of human H69 SCLC and H157 non-SCLC tumors; GHRH antagonist JV-1-38 (10 μg b.i.d.) also suppressed the growth of H838 human non-SCLC xenografted s.c. into nude mice [33, 34, 38]. Newer and more potent antagonists including JV-1-65, JV-1-68, MZ-J-7-114, MZ-J-7-118, MZ-J-7-138, and MZ-J-7-132 inhibit the s.c. or orthotopic growth of H69 and DMS-153 SCLC, as well as H-460 and A-549 non-SCLC tumors in nude mice at doses of 5–10 μg/day [17, 19].

Gynecologic Cancers In nude mice bearing s.c. xenografts of human estrogen-independent MDA-MB-468 breast cancers, treatment with GHRH antagonists MZ-5-156 or JV-1-36 (20 μg/day) induced regression in 30–40% of the tumors, and arrested the growth of other tumors [33]. GHRH antagonist JV-1-36 (20 μg/day) also inhibited the growth of orthotopic implants of MDA-MB-435 human estrogen-independent breast cancers and nullified their metastatic potential [24]. GHRH antagonists JV-1-36 and JV-1-38 (20 μg/day) also suppressed growth of MXT estrogen-independent mouse mammary cancers [37]. GHRH antagonists MZ-5-156, JV-1-36 (20 μg/day each), and MZ-J-7-138 (5 μg/day) inhibit the proliferation of s.c. OV-1063 human ovarian carcinomas in nude mice [24, 33]. GHRH antagonist JV-1-36 (20 μg/day) also suppressed growth of LHRH receptor negative UCI-107 human ovarian cancer xenografts and exposure to this antagonist in vitro decreased the tumorigenicity of UCI-107 cells in nude mice [24, 33]. GHRH antagonist MZ-J-7-118 (10 μg/day and 20 μg b.i.d.) dosedependently inhibited the growth of s.c. xenografts of HEC-1A human endometrial cancers in nude mice, and at the higher dose it extended the tumor volume doubling time by more than 100% [7].

Pancreatic and Colorectal Cancers GHRH antagonists MZ-4-71 and MZ-5-156 (10–40 μg/ day) hindered the growth of nitrosamine-induced pancreatic cancers in hamsters and SW-1990 human pancreatic cancers xenografted s.c. into nude mice [33]. GHRH antagonists including MZ-5-156 and JV-1-36 (20 μg/day each) also inhibited the tumor growth of s.c. xenografts of HT-29 human colon cancers [33].

Renal Cell Carcinoma Treatment with GHRH antagonist MZ-4-71 (20 μg b.i.d.) strongly suppressed the proliferation of s.c.

tumors of Caki-I human renal adenocarcinoma in nude mice and extended tumor doubling time [33, 34]. GHRH antagonist JV-1-38 (20 μg/day) also reduced the orthotopic growth of Caki-I tumors and reduced the development of metastases to lung and lymph nodes [13].

Brain Tumors GHRH antagonists MZ-5-156 and JV-1-36 (20 μg/day) inhibited the proliferation of U-87MG human glioblastomas xenografted s.c. into nude mice and extended the survival of animals implanted orthotopically [24, 33]. Exposure in vitro to GHRH antagonists decreased the tumorigenicity of U-87MG cells in nude mice and extended the latency period for the development of tumors [24].

Bone Tumors GHRH antagonists MZ-4-71 (40 μg/day or 25 μg b.i.d.) and JV-1-38 (20 μg b.i.d.) significantly reduced the tumor volumes and tumor weights of human MNNG/HOS osteosarcomas and SK-ES-1 Ewing’s sarcomas xenografted s.c. into nude mice [2, 33, 34].

Lymphomas Nude mice bearing s.c. xenografts of human nonHodgkin’s lymphoma cell lines RL and HT were treated with GHRH antagonists MZ-5-156 and MZ-J-7-138 at a dose of 40 μg b.i.d. [20]. Both antagonists strongly inhibited the growth of RL and HT tumors; the tumor volume doubling time of HT tumors was more than tripled by antagonist MZ-J-7-138.

CONCLUSION GHRH antagonists inhibit the growth, tumorigenicity, and metastases of a wide range of human experimental malignancies. This could be linked to their multiple mechanisms of action. GHRH antagonists may offer distinctive advantages over other classes of antitumor agents. Thus, they lack the toxic side effects typically associated with cytotoxic therapies. Since they operate on different pharmacological principles, GHRH antagonists could be combined with standard chemotherapeutic agents for an enhanced antitumor effect. Because GHRH antagonists inhibit IGF-II-dependent tumors, they should be superior to GH antagonists, as the synthesis of IGF-II is not controlled by GH [33, 34]. GHRH antagonists could be also used for suppression of tumors that do not express somatostatin receptors,

488 / Chapter 70 such as osteogenic sarcomas or those that contain only low levels of SST receptors [33, 34]. Our studies support the merit of development of GHRH antagonists for the clinical therapy of various cancers.

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71 Cancer Immunotherapy with Rationally Designed Synthetic Peptides JOAN T. STEELE, STEPHANIE D. ALLEN, AND PRAVIN T. P. KAUMAYA

who noted the spontaneous regression of tumors was often preceded by infectious episodes. Within the past 20 years, there have been significant advances in the development of diverse therapeutic approaches against cancer. However, to date there have been no cancer vaccines approved or licensed for clinical use, presumably because cancer vaccine approaches are mostly therapeutic vaccines administered after the onset of disease to treat patients who are immunosuppressed and with high tumor burden. Thus, this approach has not been clinically very useful as compared with the successful prophylactic vaccine approaches used for infectious agents to prevent disease. Despite the progress made, the treatment and/or prevention of cancer remains a considerable challenge because of cancer cells’ ability to evade the immune system. This is a consequence of tumor-associated antigens (TAAs), in many cases being expressed in normal tissues. As a result, many of the lymphocytes reactive with epitopes derived from these TAAs may have been eliminated through mechanisms involved in immune tolerance. An argument against active immunotherapy is that because of tolerance to self, TAAs are not antigenic due to the fact that they are self-antigens. The conventional strategies to treat cancer include chemotherapy, radiotherapy, and surgical excision. These approaches lack tumor specificity, resulting in toxicity. Chemotherapy and radiotherapy target other rapidly dividing cells, including hair follicle cells, gastrointestinal epithelium, and leukocytes, resulting in adverse side effects. There are several potential advantages associated with active specific immunotherapy in which the host immune system is activated to elicit an effective tumor-specific response against cancer cells. Active specific immunotherapy against TAAs offers

ABSTRACT Cancer immunotherapy involves exploiting the impressive powers of the immune system for the treatment of cancer without causing adverse effects on healthy tissues. Immunotherapeutic approaches to cancer offer advantages over chemotherapy and radiation therapy that kill normal cells as well as tumors cells, resulting in severe side effects. Immunotherapy specifically targets only tumor cells expressing the antigen/ oncogene of interest, thereby developing specific immunity against neoplastic but not normal cells. There are several active immunotherapeutic strategies currently in development that include protein-based vaccines, DNA-based vaccines, cell-based vaccines, and peptidebased vaccines, as well as passive treatment with monoclonal antibodies. The use of synthetic peptide vaccines as an immunotherapeutic strategy offers the advantages of being safe, easy to produce, devoid of oncogenic potential, and can be chemically engineered into defined conformational epitopes. Thus, the chemical synthesis of peptides allows for a rationally designed vaccine to elicit a focused immune response. In this article, we review many of the promising vaccine strategies with special emphasis on the control of tumor cells by enhancing humoral immunity and in particular the design of B-cell epitopes for eliciting high-affinity antibodies.

CANCER IMMUNOTHERAPY The concept of active immunotherapy, that the immune system can recognize and respond to tumors, was first popularized by Coley in the nineteenth century, Handbook of Biologically Active Peptides

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492 / Chapter 71 the advantage of generating immunological memory, leading to a sustained and long-lasting response against the TAA. Immunotherapy offers tumor-specific rejection with relatively fewer side effects compared with chemotherapy and radiotherapy. Both specific TAA antibodies and CD8+ cytotoxic T cells (CTLs) have been found in cancer patients. Generally, most cancer immunotherapeutic strategies have focused on eliciting CD8+ CTL responses that kill tumors. More recently the importance of CD4+ helper T cells (TH) in the treatment of certain cancers as a therapeutic modality that can aid in tumor killing has been realized. In this review we summarize the main strategies for cancer immunotherapy and emphasize our own approach to the design of the next generation of cancer vaccines focusing on the development of antibodies and activation of the humoral immune system rather than activation of cellular immunity.

CURRENT VACCINATION STRATEGIES Passive versus Active Immunotherapy Passive immunotherapy involves enhancing or stimulating the immune system using exogenous cytokines, antibodies, immune cells such as tumor-specific CTLs, or growth factors. Passive immunotherapy is highly specific; it leads to tumor cell death. However, there is a limited duration associated with monoclonal antibodies (mAbs); one study found at a dosage of 4 mg/kg the mean half-life of Herceptin, an mAb used against the TAA HER-2/neu, to be 5.8 days. Thus, there is a need for subsequent treatments. In addition there is possible toxicity and immunogenicity associated with passive immunotherapy. Active immunotherapy involves a specific TAA eliciting an endogenous immune or antitumor response. Weak but detectable levels of HER-2 (human epidermal growth factor receptor-2), specific Abs (antibodies) and T cells have been found in early stage breast cancer patients that over-express HER-2/neu [13]. Thus, active immunotherapy could augment an established immunogenic response in those patients, since the preexisting immunity has failed to control or eradicate the tumor. In addition, immunologic memory is elicited with an active approach; the longevity of the immune response could prevent the reemergence of the tumor. Hence, active immunotherapy is an attractive strategy for developing a prophylactic vaccine. We review here various examples of passive and active immunization strategies followed by specific aspects of peptide vaccine formulations with an emphasis on B-cell epitopes and the generation of specific antitumor immune responses.

Passive Immunotherapy with Monoclonal Antibodies The effectiveness of mAbs as a treatment for cancer and other diseases was recognized soon after the development of hybridoma technology in 1975. Antibodies have a high affinity for antigen and discriminating specificity, so mAbs have the ability to bind small quantities of antigen and neutralize their function. In addition, Abs bound to antigen can trigger effector functions via the Fc region. The Fc region can recruit lymphocytes or the complement system that subsequently results in cell death of the antibody-bound cell. Nearly all mAbs used clinically today are engineered to be chimeric or fully humanized because of the rapid induction of human antimouse antibody (HAMA) responses. There has been a major effort to identify proteins that are present or overexpressed in cancerous cells but not normal cells. The HER-2/neu (or c-erbB-2) protooncogene was first identified from a cancerous neuro/ glioblastoma cell line [35]. HER-2/neu is a transmembrane receptor protein of 185 kDa that is present in normal tissues but overexpressed in a significant number of breast and ovarian cancers [36]. This TAA is an attractive target for both passive and active immunization strategies. The murine mAb 4D5 has been shown both in in vitro assays and in animal models to inhibit the proliferation of human tumor cells that overexpress HER-2/neu [21]. Herceptin (trastuzumab), a recombinant DNA-derived humanized mAb derived from 4D5, was one of the first mAbs approved by the FDA for the treatment of cancer in 1998. Herceptin is available for patients with HER-2 overexpressing metastatic breast cancer. Clinical trials have shown that breast cancer patients receiving chemotherapy in combination with Herceptin gave a higher rate of objective response, improved one-year survival, and increased longer overall survival [37]. The crystal structure of the Herceptin Fab fragment bound to HER-2 reveals that it binds at the C-terminal portion of subdomain IV of the HER-2 extracellular domain (ECD) [7]. The structure of HER-2 bound to Herceptin facilitates the design of new therapeutics to target HER-2. In particular, antibodies raised against a peptide that could closely mimic the native structure of the pocketlike Herceptin-binding region of HER-2 may likely be able to modulate the behavior of the receptor and result in beneficial therapeutic properties. An emerging target for cancer therapy has been vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen involved in angiogenesis. The interaction of VEGF with its receptors (Flt-1 and KDR) leads to endothelial cell proliferation and new blood

Cancer Immunotherapy with Rationally Designed Synthetic Peptides / 493 cell proliferation in in vitro models of angiogenesis. Avastin (bevacizumab) is a recombinant humanized IgG1 mAb that binds VEGF and prevents the interaction of VEGF with its receptors on the surface of endothelial cells. This mAb blocks the growth of several tumor cell lines in nude mice [17]. In 2004 Avastin was approved by the FDA as a first-line treatment for patients with metastatic colon cancer in combination with 5-FU (5fluorouracil)-based chemotherapy. In addition active immunotherapy approaches against VEGF and its receptors have been pursued. Animal model studies have shown promising results by DCs pulsed with KDR and a DNA vaccination with VEGF [32].

ACTIVE IMMUNOTHERAPEUTIC STRATEGIES USING PEPTIDES A number of active immunotherapy strategies have been investigated including DNA, whole cell, dendritic cell (DC), and peptide vaccines. Sundaram et al. have discussed the advantages and pitfalls associated with these vaccine formulations [39]. Here we will focus on peptide vaccines that can be broadly characterized into two groups: cellular (T cell) and humoral (B cell) vaccines. Most cancer studies have focused on CD8+ T cell epitopes due to the fact that when presented in the context of MHC (major histocompatibility complex) Class I molecules, the resulting activated cytotoxic T cells can become activated and lyse transformed tumor or infected cells. This response is thought to be the most prominent mechanism utilized by the immune system to control viral replication and kill transformed tumor cells. There is a widespread consensus that CD4+ TH cells are paramount in helping CTLs mount an effective immune response. A drawback of the CTL approach for clinical use is the effectiveness is limited to patients who express the appropriate HLA haplotype. However, it has been proposed that this issue can be circumvented using subdominant epitopes with a synthetic peptide vaccine [14]. Alternatively, the natural sequence of an epitope can be modified at amino acid residues that contact the MHC molecules to increase affinity and prevent tolerance (see Martin’s chapter on MHC peptides in this book). Additionally, it is now recognized that optimum T cell responses require a second signal provided by costimulatory molecules. This priming of an effective antitumor T cell response is provided by the receptors on T cell and antigen presenting cells (APC) such as CD28-B7, CD40-CD40L, and others. However, there is also compelling evidence that the humoral immune responses (antibodies) may also play a critical role in control-

ling tumor growth. Antibodies are efficient at inhibiting metastasis by killing tumor cells.

T Cell Epitopes There are two main types of T cells: cytotoxic CD8+ T cells and helper CD4+ T cells. Cancer researchers have heavily relied on strategies designed to elicit cytotoxic T cell responses, known to lyse target tumor or virally infected cells. CTLs express CD8 on their surface with the T cell receptor (TCR), which interacts with MHC Class I bound to an 8- to 11-residue peptide on the target cell. The target cell can be any nucleated cell. These peptides are processed intracellularly by the proteosome, loaded on MHC Class I molecule, and targeted to the cellular surface of target cells. TH cells express the coreceptor CD4 on their surface with the TCR, which interacts with MHC Class II bound to a 12to 20-residue peptide on the surface of APCs. These peptides are derived from exogenous antigens that are phagocytosed and degraded in lysosome vesicles. TH cells act to enhance the reactivity of CTLs, therefore indicating a peptide construct that can activate both subsets would have optimal effectivity [25]. One of the most well-studied peptide vaccine target systems is melanoma. Melanoma AntiGEn-3 (MAGE-3) has been tested in multiple clinical trials. Other melanoma epitopes tested include gp100, Melan-A/MART-1, and tyrosinase [25]. Studies with Plasmodium falciparum and Trypanosoma cruzi have utilized T cell epitopes from circumsporozoite protein and Trypomastigote surface antigen, respectively, in peptide vaccines [33]. Tax as a T cell epitope for human T cell lymphotropic virus type I (HTLV-1) and Env, Gag, reverse transcriptase, and Tat as T cell epitopes for human immunodeficiency virus (HIV) have also been studied.

B Cell Epitopes B cell epitope vaccines are designed to induce a protective humoral response. This response includes creation of antibody-producing plasma cells as well as immunologic memory. B cell epitopes have been used in a variety of vaccines. Oomen et al. designed a peptide mimic of the immunodominant β-turn of PorA from Neisseria meningitidis to protect against meningococcal meningitis [28]. A malaria vaccine against Plasmodium falciparum is made up of multiple tandem repeats of NANP, which is the immunodominant B cell epitope of the circumsporozoite protein [3]. United Biochemical, Inc. has developed a B cell epitope vaccine for HIV targeting residues 39–66 on the CD4 T cell molecule. Resulting antibodies are expected to cause steric hindrance, preventing viral infection of CD4+ T cells [43].

494 / Chapter 71 IDENTIFICATION OF T AND B CELL EPITOPES A fundamental step in the development of peptide vaccines for treating cancer is the identification of epitopes that are recognized by B cells to stimulate a neutralizing humoral response and T cells to induce a cellular response. Identification of T cell epitopes is discussed and has been reviewed previously [12]. There are numerous Web-based predictive algorithms for identifying peptides that can bind a variety of MHC Class I molecules (SYFPEITHI: http://www.syfpeithi.de; MHCBN: http:// www.imtech.res.in/raghava/mhcbn; MHCPEP: http:// wehih.wehi.edu.au/mhcpep). A summary of related links including other databases and servers can be accessed at http://www.imtech.res.in/raghava/ mmbpred/link.html. In the past several years, there has been a wealth of publications demonstrating the effectiveness of epitope enhancement in the design of CTL peptide vaccines [27, 34, 42]; this strategy involves altering the anchor residues of Class I epitopes to increase the affinity of peptide binding to MHC molecule or increase the affinity of the peptide-MHC complex for the TCR [2]. The use of heteroclitic analogs (epitope enhanced peptides) has been shown to be a promising methodology to increase the affinity and potency of vaccine-induced CTL responses. Epitope enhancement operates on the premise that there is a direct correlation between peptide-MHC I affinity and the potency of the CTL responses. By optimizing the anchor residues of an antigenic peptide, stable peptide-MHC I complexes are more likely to form promoting signaling cascades necessary for activation and differentiation of naïve CTLs into effectors. B cell epitopes can be classified as either continuous or discontinuous, depending on whether or not the residues involved in the epitope are contiguous in the polypeptide chain. Discontinuous (also called conformational) epitopes are made up of residues that are not continuous in sequence but are assembled at the surface of the protein either by constraints imposed by the three-dimensional structure of the protein or by the folding of the polypeptide chain. Identification of continuous epitopes can be determined from the primary structure of the protein, while determination of conformational epitopes usually entails analysis of the x-ray crystal structure of the protein. Crystallographic studies allow detailed information about antigen-antibody information. However, crystallographic studies are not amenable to widespread use, given the time necessary to acquire and interpret such results and the necessity that the protein antigen must be isolated in pure crystalline form. Thus, other methods have been developed to identify B cell epitopes.

Various theoretical and experimental methods have been developed to determine potential antigenic sequences of a protein. Antibody:antigen interfaces have been assumed to generally be hydrophilic and transiently accessible to solvent surrounding the antigen. This is the basis for the prediction of antigenic sites in the seminal work by Hopp and Woods [22] in which hydrophilic stretches in a protein’s linear sequence are identified. In addition, one can predict a protein’s flexibility, mobility, solvent accessibility, and amphiphilicity having the primary sequence of the protein in hand. Kaumaya et al. provide detailed analysis of computational methods available to identify B cell epitopes [23] by using a variety of algorithms that locate peptide accessibility, flexibility, hydrophilicity, and protrusion. A number of various antigenic B cell epitope predictions are available on the Web at http://www.peptideresource.com/software.html. However, further testing on individual epitopes identified from computational methods need to be performed to confirm that the sequence is immunogenic and possesses biological relevance. This is usually achieved by immunization of animals with the peptide by use of an adjuvant and performing a variety of in vitro testing and functional assays.

Epitope Mapping Epitope mapping, or determination of the antigenbinding site to an antibody, is a useful tool in identifying B cell epitopes of a protein. This procedure necessitates having a panel of monoclonal or polyclonal antibodies available against the protein. One experimental method to map protein epitopes involves the synthesis of peptides, which represent overlapping segments of the antigen sequence [19]. However, this procedure can be costly and time prohibitive, even when multiple peptides are screened simultaneously with a series of antibodies using the PEPSCAN approach. Several procedures have been developed that use mass spectrometry to identify B cell epitopes [15]. MALDI mass spectrometry is used to identify nonbinding peptides through a direct comparison of the spectra of a mixture of proteolytic peptide fragments before and after reaction with a mAb. Peptides that are part of the epitope are determined based on the absence of their ions in the spectrum of the antibody reaction mixture versus unreacted control spectrum. A comprehensive database is available at http://www.jenner.ac.uk/antijen.

B Cell Mimotopes The term mimotope is defined as “a peptide capable of binding to the paratope of an antibody but unrelated in sequence to the protein antigen used to elicit the

Cancer Immunotherapy with Rationally Designed Synthetic Peptides / 495 antibody” [18]. These peptides are usually identified through synthetic peptide libraries or recombinant peptide libraries (mostly phage display libraries) [26]. To be a true mimotope, the peptide should be able to elicit antibodies that recognize the epitope being mimicked, in addition to binding the antibody. This phenomenon is explained by the fact that dissimilar amino acid residues in the two cross-reactive peptides nevertheless contain similar atomic groups that are able to interact with atoms of the CDR regions of the antibody. Mimotopes to Herceptin, a mAb against HER-2/neu, have been identified using phage libraries. Antibodies raised against one of these peptides recognized HER2/neu and caused internalization of the receptor from the cell surface in a similar manner as Herceptin. A phage/algorithm approach has been described in which mAbs specific to HIV-1 are used to select specific phages from a combinatorial phage-display library that is then used as an epitope-defining database. This database can then be applied via a computer algorithm to analyze the crystalline structure of the original antigen [16], and epitopes on the protein that bind to the mAbs can be predicted.

RATIONAL DESIGN OF PEPTIDE VACCINES Peptides have the potential to be safe, noninfective, well-defined, and stable vaccines. However, many barriers remain in the rational design of peptide-based vaccine in spite of a mounting knowledge of the molecular recognition and stimulation of the immune system. Several factors need to be considered in formulating an effective peptide vaccine; these factors include inclusion of a universal T helper epitope and the necessity to mimic the structure of the parent antigen to generate high-affinity Abs.

Peptide Immunogenicity In the design of a synthetic peptide vaccine, it is necessary that the B cell epitope be presented along with an appropriate helper T cell epitope. The interaction between B cells and T cells recognizing the same antigen is essential for B cell proliferation and differentiation and the generation of high-affinity antibodies that are of the IgG isotype. For sequences shorter than 15 residues, peptides are traditionally coupled to a larger carrier protein such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). This approach can be problematic for several reasons including epitope suppression by the carrier protein and poorly defined constructs due to lack of control during the chemical coupling reaction. Several alternatives have been described to produce conformationally stable

peptides. One approach involves the use of branched oligo-lysines as a template for the attachment of antigenic peptides known as multiple antigenic peptides (MAP) [40]. This approach has been found effective in a number of cases; however, it’s possible that due to the density of the peptides in the construct, peptides assume a conformation that in some cases does not mimic the structure of the peptide monomer or the parent protein [41]. Another alternative involves B and T cell epitopes constructed in a colinear fashion. This has limited usage when the T helper epitope is recognized by one or a few MHC Class II alleles. Thus, the identification of “universal” or “promiscuous” T helper epitopes that bind multiple MHC haplotypes is necessary in the design of an optimum vaccine. Universal T helper epitopes from tetanus toxoid (TT) and measles virus fusion protein (MVF) have been identified [29, 31]. Eight predicted B cell epitopes derived from the HER-2/neu oncoprotein were synthesized collinearly with a a 288– 302 from the MVF protein. All eight of the peptides investigated generated high-titered antibodies in outbred rabbits and importantly produced undetectable levels of antibodies against the MVF TH epitope [8, 10], thus proving the utility of a chimeric vaccine construct incorporating B and TH epitopes in a collinear fashion.

Molecular Mimicry to the Cognate Antigen The introduction of functional humoral immunity with unstructured peptide vaccines may be difficult. Even if the protein contains a continuous epitope, peptides containing this sequence may induce antibodies that lack cross-reactivity with the parent protein. To induce functional cross-reactive immune responses, it appears essential that peptide design take into account conformational correctness. One approach to achieve molecular mimicry to the parent protein is through constricting the peptide by cyclization. Linear peptides are highly flexible and can adopt a variety of conformations in solution. However only a few of these conformations are responsible for their immunoreactivity [20]. Cyclic peptides can cause preferred spatial arrangements that duplicate the bioactive conformation resulting in improved binding and immunological properties. Davies has written an extensive review on the cyclization of peptides [11]. In addition Annis et al. provide a review on the methods of disulfide bond formation for synthetic peptides [1]. Constraining peptides by cyclization has been investigated as a diagnostic tool in identifying the human papillomavirus type 16 (HPV-16) oncoprotein in cervical cancer patients. With a hydrazone link approach to mimic the helix secondary structure of HPV-16, it was found that a conformationally restricted α-helix peptide showed strong positive

496 / Chapter 71 reaction with sera from women having invasive cervical carcinoma whereas the linear version showed little reaction to the sera [5]. The utility of peptide cyclization with the incorporation of the two native disulfide bonds of the 628–647 sequence of HER-2/neu has been shown [9]. Antibodies raised against the disulfidepaired peptide showed enhanced cross-reactivity to tumor cells overexpressing HER-2/neu and demonstrated superior antitumor responses in the context of ADCC and IFN-γ induction. In addition, mice vaccinated with the disulfide-bonded epitope showed a significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T cell epitope (MVF) control peptide.

has strong inflammatory properties, causes degranulation of neutrophils, and recombinant IL-12 has a relatively long half-life in the body compared to other cytokines [38]. A multiepitope HER-2/neu vaccine in combination with IL-12 caused a significant reduction in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2/neu [10]. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been studied extensively as a vaccine adjuvant because of its ability to recruit antigen presenting cells at the site of vaccination. There have been a number of studies demonstrating the adjuvant effects of GM-CSF using a range of cancer vaccine methods [6].

Multi-Epitope Vaccines ANTITUMOR IMMUNITY WITH PEPTIDE VACCINES To date, most peptide cancer vaccine strategies seek to induce a cellular antigen-specific T-cell response, and there are numerous reviews on this subject [4, 24, 44]. However, the humoral arm could play a critical role in the generation of an antitumor response. The successful clinical usage of passively infused mAbs points to the effectiveness of the humoral arm of the immune system. A recent study found that the induction of anti-HER2/neu antibodies are both necessary and sufficient in protecting mice transgenic for the HER-2/neu oncogene and that have been depleted of CD4+ and CD8+ cells from developing tumors [30]. Thus, an effective peptide vaccine should have components that activate both the cellular and humoral arms of the immune system. The vaccine should be composed of appropriate tumor antigen B-cell epitopes, CTL epitopes, and a universally immunogenic T-helper epitope. In addition, there have been several recent advances in peptide vaccine formulations; the utility of cytokines and the use of multiple epitopes in vaccine are discussed below.

Cytokines as Vaccine Adjuvants Considerable attention has been directed to the use of immune adjuvants to overcome immunologic tolerance of tumor antigens. To this end, the use of recombinant cytokines has been exploited as an adjuvant for tumor vaccines. Cytokines lead to either a Th1 or Th2 response [39]. The production of interleukin-2 (IL-2), IL-12, and interferon-gamma (IFN-γ) allows the selective enhancement of a Th1-type cellular response, while production of IL-4, IL-5, or IL-10 directs a Th2-type humoral immunity. IL-12 is a promising cytokine with the possibility of clinical usage as a vaccine adjuvant. It

Antibodies against TAAs can mediate diverse effects. There are antibodies that result in stimulation of cells overexpressing HER-2/neu [45]. Therefore, it is crucial to identify epitopes on TAAs that result either in stimulation or inhibition of tumor cell growth to develop an effective peptide vaccine that creates an antibody response for therapeutic benefit. The enhanced efficacy of a combination vaccine containing two HER-2/ neu B cell epitopes as compared with a single epitope vaccine has been shown; a phase 1b human clinical trial with the multiepitope HER-2/neu vaccine was conducted based on the results [10]. Ideally, an optimum cancer vaccine should include not only a combination of epitopes from a single TAA but epitopes from various TAAs [4]. This is a fundamental requirement to overcome the problems imposed by the antigenic heterogeneity of each tumor type and prevent the reemergence of tumors.

CONCLUSION Although the rational design of synthetic peptides as cancer vaccines remains a major challenge, general rules governing immunogenicity with regards to vaccine optimization are beginning to be understood, such as identification of the biologically relevant epitopes, understanding epitope structure, devising strategies to engineer conformationally dependent B cell epitopes, adopting ways to increase the immunogenicity in an outbred population, delivering the immunogen in a safe and efficacious vehicle, and using novel adjuvants for eliciting immune responses and enhancing biological activity and efficacy by modifying MHC class I epitopes. The immune system’s exquisite specificity offers one of the simplest and most effective ways to prevent and control disease. Recent findings have implications not only for understanding of immune response to self

Cancer Immunotherapy with Rationally Designed Synthetic Peptides / 497 peptides in normal and pathologic conditions but also for the development of immunotherapies for cancer. Novel vaccines designed to stimulate both antibody and T cell responses against human tumors are being developed. Passive immunotherapy against TAAs offers the advantage of immediately targeting tumor cells and has been used clinically since 1998. Active specific immunotherapy offers several advantages over passive, particularly when the application is primary or secondary cancer prevention, and several vaccine approaches are under investigation. Active immunotherapy provides a prolonged antitumor immune response; there are several active immunotherapy formulations including DNA, whole cell, DC, and peptide vaccines. Peptide vaccines are a particularly viable option since peptides may be able to circumvent immune tolerance of TAAs using subdominant B cell epitopes. For peptide vaccines to become a practical reality, rationally designed synthetic constructs must incorporate enough antigenic determinants to elicit all three arms of the immune system—namely B cell, TH, and CTL epitopes. Synthetic peptide immunogens offer a safe, stable, and costeffective alternative to other vaccine preparations under investigation, and peptide vaccine formulations can be manipulated in many different ways to achieve optimal and protective immune responses.

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[37] Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–92. [38] Spadaro M, Lanzardo S, Curcio C, Forni G, Cavallo F. Immunological inhibition of carcinogenesis. Cancer Immunol Immunother 2004;53:204–16. [39] Sundaram R, Dakappagari NK, Kaumaya PT. Synthetic peptides as cancer vaccines. Biopolymers 2002;66:200–16. [40] Tam JP. Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system. Proc Natl Acad Sci USA 1988;85:5409–13. [41] Van Regenmortel MHV, Muller S. Synthetic peptides as antigens. Amsterdam; New York: Elsevier, 1999. [42] Vertuani S, Sette A, Sidney J, Southwood S, Fikes J, Keogh E, et al. Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues. J Immunol 2004;172: 3501–8. [43] Wang CY, Walfield AM. Site-specific peptide vaccines for immunotherapy and immunization against chronic diseases, cancer, infectious diseases, and for veterinary applications. Vaccine 2005;23:2049–56. [44] Weber J. Peptide vaccines for cancer. Cancer Invest 2002;20: 208–21. [45] Yip YL, Ward RL. Anti-ErbB-2 monoclonal antibodies and ErbB2-directed vaccines. Cancer Immunol Immunother 2002;50: 569–87.

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72 Peptide Vaccines for Cancer Treatment WOLFGANG M. WAGNER AND MARY L. DISIS

bed and thus can penetrate a mass of tumor cells at any location. Finally, the clonal expansion of tumor specific T cells will occur as long as antigen is available for recognition. In this way, the tumor itself serves as the growth stimulus for immune cells capable of its destruction. It is still debated whether a natural protective tumor immunity exists, as suggested by the concept of tumor immunosurveillance. Immunocompromised individuals, such as transplant recipients and AIDS patients, develop cancer at a much higher incidence than do individuals with intact immune systems. Most of these tumors are induced by viruses, but retrospective longterm studies revealed an increased risk for transplant recipients to develop tumors with no viral etiology [12]. Indeed, human tumors can elicit immune responses in cancer patients. In the case of paraneoplastic neurologic disorders, the tumor expresses an antigen that is normally only expressed in the immune-privileged nervous system. T cells will recognize this antigen and mount an immune response that leads to damage of the nervous system resulting in paraneoplastic syndromes. Of note, such neurologic disorders usually precede the diagnosis of cancer and the tumor may be found only months or even years after the development of neurologic symptoms [4]. The immune system does play an important role in the therapy of cancer as illustrated by the role of the T cell in eradication of leukemia. Patients who develop graft-versus-host disease (GvHD) after allogeneic transplantation have lower relapse rates, although transplant-related mortality is higher, a phenomenon called graft-versus-leukemia effect (GvL). T cells in the graft recognize and attack leukemia cells in the recipient and are essential for the success of the therapy. This effect can be further exploited by giving donorlymphocyte infusions when the leukemia relapses after allogeneic transplant.

ABSTRACT Tumors express a multitude of antigens that distinguish them from normal tissue and can be specifically recognized by T cells. However, the tumor microenvironment favors the induction of tolerance to the tumor and limits the development of efficient T cell immunity. Tumor vaccines aim at reversing tumor-induced immunosuppression by eliciting high-avidity T cells directed against subdominant epitopes of tumor antigens. Peptide-based cancer vaccines are particularly well suited for use in the clinic because they are known to be safe, specifically target a definite epitope, and allow for precise monitoring of the induced immune response.

USING THE IMMUNE SYSTEM TO TREAT CANCER Cancer is the leading cause of death for persons younger than 85 in the United States, and the lifetime probability of developing some form of malignancy is currently 46% for men and 38% for women [16]. Standard cancer treatments, mainly surgery, radiotherapy, and chemotherapy can reduce the tumor load even in advanced stage disease but fail in many cases to eliminate all tumor cells. The surviving malignant cells will ultimately result in disease relapse. The failure of standard cancer therapies is due, in large part, to the inability of such treatments to elicit total tumor eradication. Harnessing the immune system has many potential advantages over standard therapy. First, immunologic memory can be generated so that cancer relapse, even when occurring at a distant time point, can be eliminated. Second, T cells have the ability to migrate out of the vasculature to the tumor Handbook of Biologically Active Peptides

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500 / Chapter 72 IDENTIFICATION OF TUMOR ANTIGENS AND PEPTIDES USING ENDOGENOUS IMMUNITY PRESENT IN CANCER PATIENTS Defining Antigens and Immunogenic Peptides: Endogenous Immune Approach There are two major ways to identify novel tumor antigens. One uses tumor-specific T cells or immune sera to screen cDNA libraries for antigens. The other uses “reverse immunology” by selecting potential tumor antigens based on theoretical reasons—for example, tumor-specific expression and then verifying the candidate’s immunogenicity in vitro and in vivo [33]. Tumor-specific T cell lines can be generated by in vitro stimulation of a cancer patient’s PBMC or tumor infiltrating lymphocytes with autologous tumor cells. Such T cell lines can further be cloned and used as epitope-specific probes to screen DNA expression libraries of tumor cells to identify the tumor antigen recognized by the T cells [34]. For the identification of the actual epitope, overlapping peptides spanning the identified antigen are synthesized and tested for recognition by the T cell clone. Another less commonly used approach is to elute peptides from MHC molecules of tumor cell lines, fractionate them by chromatography, and load these peptide fractions onto HLA-matched antigen presenting cells followed by screening with tumor-specific T cell clones. The peptides can then be identified by mass spectrometry. This latter method has the advantage of being able to identify posttranslationally modified epitopes [30]. Tumor antigens that elicit antibody responses can be identified by serological recombinant expression cloning (SEREX). In this technique, cDNA expression libraries of tumor cells are screened for expressed proteins binding to serum IgG of cancer patients. Since production of IgG in most cases requires the presence of T helper cells specific for the same antigen, proteins identified by SEREX are also putative T cell antigens. Several tumor antigens have been discovered by this approach—for example, NY-ESO-1 that also contains CTL epitopes [15].

Defining Antigens and Immunogenic Peptides: Theoretical Approach Some of the proteins expressed by tumors are potential tumor antigens based on theoretical considerations—for example, proteins known to be overexpressed in a specific tumor. The challenge in this case is to find peptide epitopes that are naturally presented as well as the MHC allele by which they are presented.

One method is to synthesize peptides, assess their binding affinity to MHC molecules, sensitize the T cells against higher affinity binders in vitro and, finally, use these peptide-specific T cells as probes to test whether the peptide is actually naturally presented by tumor cells [26]. For MHC class I molecules, algorithms are available that screen protein sequences for binding motifs, based on anchor positions of MHC molecules and predict potential T cell epitopes with good probability [27]. Additional software programs predicting proteasomal cleavage sites within protein sequences may be used to narrow down potential peptide epitopes to a number amenable to experimental analysis. Several software programs are available to predict epitopes for MHC class II molecules, but they are somewhat less reliable. In any case, the immunogenicity and natural presentation of such predicted peptide epitopes has to be verified experimentally, such as by screening blood samples of cancer patients with peptide-MHC tetramers containing the predicted peptides.

COMMON TUMOR ANTIGENS AND ANTIGENIC PEPTIDES RECOGNIZED BY T CELLS In 1991, using T cell clones, MAGE-1 (melanoma antigen-1) was identified by Thierry Boon and colleagues [34]. Since then a large number of human tumor-associated antigens (TAA) in a variety of cancers has been described [23], and tumor antigens are commonly grouped into five categories. Common class I and class II peptides derived from tumor antigens are shown in Tables 1 and 2, respectively.

Cancer-Testis Antigens MAGE-1 is naturally expressed in the testis and placenta and belongs to a group of tumor antigens that are normally only expressed at early differentiation stages or at immunoprivileged sites. Proteins of the MAGE family are not only expressed in melanoma but also in a variety of carcinomas and certain sarcomas. Aside from the MAGE family, several other tumor antigens have been identified that belong into this group, such as BAGE, GAGE, PRAME, NY-ESO-1, and LAGE-1.

Tissue-Specific Differentiation Antigens Tissue-specific differentiation antigens are shared between the tumor and the tissue from which the tumor

Peptide Vaccines for Cancer Treatment / 501 TABLE 1. Category Cancer-testis antigens Differentiation antigens

Tumor-specific antigens Overexpressed proteins

Viral antigens

Gene

Epitope Sequence

HLA

MAGE-A1 NY-ESO-1 Melan-A/MART-1 gp100 tyrosinase Mammaglobin-A p21ras HER-2/neu MUC-1 Survivin WT-1 HPV16 E7

SLFRAVITK SLLMWITQC EAAGIGILTV YLEPGPVTA YMDGTMSQV PLLENVISK VVVGAVGVG KIFGSLAFL STAPPVHNV ELTLGEFLKL CMTWNQMNL TLGIVCPI

A3 A2 A2 A2 A2 A3 B35 A2 A2 A2 A24 A2

TABLE 2. Category Cancer-testis antigens Differentiation antigens Tumor-specific antigens Overexpressed proteins

Tumor antigens recognized by MHC class I restricted T cells. Tissue Expression Melanoma, carcinomas, normal germline cells Tissue-specific expression in melanocytes and melanoma Mammary gland/breast cancer Pancreatic and colon carcinoma Various carcinomas, such as breast cancer, ovarian cancer Various carcinomas, melanoma Various leukemias, carcinomas Cervix carcinoma

Tumor antigens recognized by MHC class II restricted T cells.

Gene

Epitope Sequence

HLA-Restriction

MAGE-A1, A2, A3 NY-ESO-1 GP 100 Tyrosinase CDC27 Annexin II HER-2/neu

LLKYRAREPVTKAE AADHRQLQLSISSCLQQL WNRQLYPEWTEAQRLD QNILLSNAPLGPQFP FSWAMDLDPKGA DVPKWISIMTERSVPH KVPIKWMALESILRRRF

DRβ1*1301, 1302 DRβ4*0101-0103 DRβ1*0401 DRβ1*0401 DRβ1*0401 DRβ1*0401 DR1, DR4, DR7

Tissue Expression Melanoma, carcinomas, normal germline cells Melanocytes and melanoma Melanoma Melanoma Various carcinomas

arose. The first tumor antigen that was discovered in this group was tyrosinase, a protein enzyme involved in melanin synthesis and almost exclusively expressed in melanocytes and melanomas. Other examples are the melanoma antigens gp100/Pmel17 and MelanA/ MART-1.

proteins. The best example is human papilloma virus, which is implicated in cervix cancer. The viral proteins E6 and E7 that are required for the malignant phenotype contain peptide epitopes that can be recognized by CTL.

Overexpressed Antigens

Tumor-Specific Antigens Resulting from Genetic Alterations

Some proteins are expressed at higher levels in tumor cells than in normal cells. As a consequence subdominant or cryptic epitopes are presented at higher levels by MHC molecules and allow the recognition by T cells that have not been eliminated in the thymus in the course of negative thymic selection. One example is HER-2/neu, a growth factor receptor that is overexpressed in breast cancer as well as several other carcinomas [8].

Viral Proteins Tumors that are induced by viruses contain the provirus integrated in the genomic DNA and express viral

Tumors acquire and accumulate mutations during their development, which give rise to novel tumorspecific antigens that can be recognized by the immune system. The vast majority of these mutations are specific for every cancer patient or even every tumor cell clone. Mutations in genes that are involved in the malignant transformation of the cell can be shared among many or almost all patients with the same tumor—for example, the fusion gene bcr/abl is found in all patients with chronic myelogenous leukemia. Another class of tumor-specific antigens represent products of defective transcription caused by the usage of cryptic promoters or frame shift mutations. T cells

502 / Chapter 72 specific for products of pseudogenes or antisense DNA have been described.

FEATURES OF TUMOR-SPECIFIC T CELLS The T cell repertoire of healthy individuals contains naive T cells capable of recognizing tumor antigens, although usually at levels too low to be detected directly ex vivo. Relatively few studies have tried to determine actual tumor specific precursor frequencies in patients. In one report, the frequency of T cells specific for MAGE-3 epitopes in individuals without cancer was estimated to be in the range of approximately 1 × 107 of all peripheral blood CD8+ T cells [2] and potentially this value could apply to the majority of T cell epitopes. A notable exception is the large T cell pool directed against the self-antigen Melan-A/MART-1 in HLA-A2 nontumor bearing individuals, which consists of largely cross-reactive subsets of naive T cells displaying multiple specificities [13]. The situation is different in cancer patients, where some T cells specific for the tumor may have already encountered antigen and exist as memory cells at elevated levels. Cancer patients mount spontaneous T cell and antibody immune responses to their tumor, even though these are obviously not able to control the disease. For example, 5 of 45 (11%) patients with stage III or IV, HER-2/neu overexpressing breast or ovarian cancer have a significant HER-2/neu specific T cell response as defined by a stimulation index > or = 2.0 (range 2.0–7.9) upon restimulation with HER-2/neu protein [10]. Spontaneous tumor specific T cell responses against a single antigen are usually of very low magnitude and difficult to quantify and thus are often only detected in a minority of patients. However, since patients develop tumor-specific T cells of multiple specificities, the combined multiantigenic response amounts to a more substantial proportion of the T cell pool. A recent study found antitumor cytotoxic T lymphocyte precursors at a frequency of 6 × 105 to 2 × 103 of all peripheral blood CD8+ T cells in melanoma patients [14]. However, it is important to note that tumorspecific T cells in cancer patients are often found to be nonfunctional in vivo. Most TAA are nonmutated self-proteins, which are expressed in normal tissues including the thymus. Highavidity T cells against the immunodominant epitopes of such TAAs are deleted in the thymus, while T cells against cryptic epitopes, on the other hand, pass negative thymic selection and may be targeted in tumor vaccines. Cryptic epitopes are often poor MHC binders, a problem that can be circumvented by the use of heteroclitic peptides with stronger binding affinity to the MHC molecule but retained T cell receptor specificity.

For example, substitution of methionine for threonine at position 2 in the HLA-A*0201 restricted gp100(209– 217) epitope increases its immunogenicity in vitro and in vivo by enhancing the stability of the peptide/MHC complex [1]. The clinical translation of what has been learned about antigen specific T cell epitopes to clinical trials of cancer vaccines focuses on these issues.

CLINICAL TRIALS OF PEPTIDE BASED CANCER VACCINES By active immunization targeting tumor antigens, precursor frequencies of tumor-specific T cells can be increased in cancer patients. Tumor vaccines based on peptide epitopes, proteins, naked DNA, viral constructs, whole tumor cells, or antigen-loaded dendritic cells have been tested in phase I/II clinical trials [20], and tumor-specific T cells mostly of low magnitude could be elicited in many, but not all, patients. Clinical trials of cancer vaccines are unfortunately notoriously difficult to compare due to the heterogeneity of vaccine formulations and patient populations. Most of the clinical trials have been rather small in size and showed low response rates, which, together with wide interpatient variability, impairs the ability to draw strong conclusions. An association between vaccine-induced increase in specific T cell frequencies in peripheral blood and clinical responses to antigen-specific immunotherapy has not yet been established. Peptide vaccines are particularly promising because they target primarily only a small set of T cells and induced T cell responses can thus be readily monitored even at relatively low magnitudes. Peptides are safe to administer, can be manufactured under rigorous standards at comparatively low cost, allow easy reproducibility of vaccinations, and may elicit immunity to self-proteins in cases where whole protein vaccines fail [9]. Although the potential use of peptide vaccines is limited to patients who express both the antigen and the HLA-allele, the use of multipeptide vaccines circumvents this problem [28]. Peptide-based vaccinations are usually monitored by the analysis of peptide-specific T cells in the peripheral blood of patients. Assays based on soluble tetrameric MHC-peptide complexes (commonly known as “tetramers”) allow direct ex vivo quantification and isolation of peptide-specific T lymphocytes but yield no functional data on these cells. Functional analyses on the other hand require in vitro antigenic stimulation. Conventional proliferation and cytotoxicity assays do not allow for direct quantification, are imprecise, and are being replaced by enzyme-linked immunospot assays (Elispot) and cytokine flow cytometry. Elispot assays have the lowest limit of detection, while cytokine flow

Peptide Vaccines for Cancer Treatment / 503 cytometry offers the advantage of identifying subsets of reactive cells [35]. These quantitative assays allow an assessment of the potency of the peptide based vaccine approach. The T cell response elicited in patients by a peptide vaccine is not restricted solely to the peptide contained in the vaccine. Through epitope spreading, T cell populations arise after vaccination that recognize additional epitopes derived from the TAA in the vaccine or even a different TAA. Epitope spreading has been frequently observed after peptide based cancer immunotherapies and is possibly associated with a clinical antitumor response. After vaccination of patients with HER-2/ neu-overexpressing breast, ovarian, or non-small-cell lung cancers, with peptides derived from the HER-2/ neu extracellular domain (ECD) or intracellular domain (ICD) mixed with GM-CSF as an adjuvant, epitope spreading was observed in 84% of patient’s T cell responses to portions of the HER-2/neu protein not included in the immunizing vaccine [7]. Moreover, of 35 patients immunized with HER-2/neu peptide based vaccines, 60 percent developed HER-2/neu IgG specific antibody responses to at least one peptide included in their vaccine, and humoral intramolecular epitopespreading within the HER-2/neu protein occurred in 49% of immunized patients [6]. Similarly, in melanoma patients vaccinated with MAGE MHC class I peptides, antivaccine CTL accounted for only a small portion of the induced increase in antitumor CTL response. This effect was even more pronounced in tumor invaded lymph nodes and metastases [19]. The development of robust and long-term CD8 T cell immunity requires help by CD4 T cells at the time of priming, which can be accomplished by the inclusion of CD4 T cell epitopes in the vaccine, such as tetanus helper peptide or KLH. In a phase I clinical trial using putative HER-2/neu helper peptides encompassing HLA-A2 binding motifs CD4+ and CD8+ T cell responses developed in the majority of patients. Frequencies of HLA-A2 restricted CD8+ to HER-2/neu, which were not detected in most patients before vaccination, increased to levels of approximately 100/106 PBMC. T cell precursor frequencies increased after vaccination and remained elevated for more than one year after the final immunization [18]. In contrast, patients who received an immunodominant HER-2/neu-derived HLA-A2 peptide alone in the absence of exogenous help developed only short-lived peptide-specific immunity that was not detectable five months after the last vaccination [17]. Frequencies of vaccine-induced peptide-specific T cells remain, however, usually relatively low, even after multiple vaccinations and using peptides with enhanced immunogenicity. Smith et al. report a clinical trial with HLA-A2-positive melanoma patients that received a modified gp100 with improved HLA-A2 binding affinity

up to 13 times in the course of six months. Increase of peptide-specific CD8+ T cells was observed in 28 of 29 patients with a median frequency of 0.02% before and 0.34% after vaccination [31]. Advanced stage cancer patients are sometimes immunosuppressed, and thus it is necessary to include suitable controls into clinical trials that ensure an assessment of immunocompetence. Frequently used control antigens include influenza or CMV peptide epitopes to measure recall responses. Tumor-induced immunosuppression is a major hurdle in the development of successful cancer vaccines [37]. Serum levels of immunosuppressive cytokines such as IL-10 and TGF-β are often elevated in cancer patients. These cytokines are produced by tumors themselves as well as tumor-infiltrating leukocytes and can inhibit T cell function directly or indirectly via modulating APC. There is often a lack of correlation between the presence of vaccine-induced circulating T cells and tumor regression. In some cases, it is uncertain whether the peptide epitopes are naturally presented in vivo by tumors [24]. In other cases, vaccine-induced T cells display a nonactivated, “quiescent” phenotype, a state in which they lack direct ex vivo effector function [21]. Moreover, in the course of immunoselection, tumor variants may develop that have lost or down-regulated the expression of either MHC molecules or tumor antigen and thus evade immune attack by T cells.

STRATEGIES FOR ENHANCING THE EFFICACY OF PEPTIDE-BASED VACCINES In order to make peptide-based vaccines more immunogenic, current research focuses on optimizing the other major component of a vaccine, the adjuvant. To date, there are only a few adjuvants licensed for use in humans [25]. Adjuvants have two functions: They optimize antigen delivery to professional APC—for example, by slowing down diffusion of vaccine, and they stimulate the innate immune system to provide the necessary milieu required for a robust and effective adaptive immune response. The use of dendritic cells as “natural adjuvants” in cancer vaccines has shown some promising clinical results [22]; however, the levels of vaccine-specific T cell expansion achieved by DC-based vaccines are not higher than those observed with peptide vaccines. Protocols used so far for cultivating and loadings cells vary considerably, which makes comparison of various trials almost impossible. Another approach is to recruit dendritic cells in vivo to the vaccination site by adding adjuvants such as Flt3 ligand that induce an increase in peripheral DC and skin [11]. GM-CSF has emerged as the adjuvant of choice for tumor vaccines and

504 / Chapter 72 is especially effective in recruiting antigen-presenting cells when given intradermally [5]. However, GM-CSF does not by itself activate dendritic cells and high-dose GM-CSF may even impair immune responses through recruitment of myeloid suppressor cells. Thus, in order to induce efficient T cell responses, it is essential to include stimuli that induce the activation and maturation of DC in the vaccine, such as ligands for Toll-like receptors (TLR), a family of molecules that recognize microbial components, and mediates activation of DC in the course of natural infection. The addition of a CpG oligonucleotide, a TLR9 ligand, to a Melan-A/ MART-1 peptide vaccine increased the number of induced vaccine-specific CTL by one order of magnitude in melanoma patients, compared with a control group that received the vaccine without adjuvant [32]. Regulatory T cells (T[reg]) play a crucial role in maintaining self-tolerance, but they also contribute to tumor growth by suppressing tumor-specific T cell immunity. T(reg) cells preferentially move to and accumulate in tumors and ascites and blocking T(reg) function by specific antibodies helps to defeat cancer in animal models. A complementary cancer vaccine strategy aims at breaking the tolerance to tumor antigens, such as in vivo CTLA-4 blockade or B7-H1/PD-1 blockade [3]. Initial clinical trials have resulted in significant autoimmunity being generated in treated patients; thus, the line between the induction of antitumor immunity and autoimmunity is blurred [29]. Alternatively, the coadministration of danger signals such as Toll-like receptor ligands can bypass T(reg) mediated tolerance in the setting of established tolerance [36]. Thus, combination therapies matching peptide-based vaccines with approaches that will modulate the immune microenvironment may improve immunologic and potentially therapeutic efficacy.

CONCLUSION Over the last decade, with the identification of a multitude of tumor antigens and associated immunogenic peptide epitopes, peptide vaccines have become one of the most common immunotherapeutic approaches in cancer treatment. Computer modeling has facilitated the identification of peptides suitable for vaccination. Advances in immunologic monitoring have allowed the assessment of the immunologic potency of the peptide approach in a more precise manner. Finally, new agents that can modulate the dampening effects of the tumor environment, to be used in combination with vaccines, may improve immunogenicity.

Acknowledgments This work has been supported by NIH NCI K24 CA85218 and a gift by Athena Water.

References [1] Borbulevych, O. Y.; Baxter, T. K.; Yu, Z.; Restifo, N. P.; Baker, B. M. Increased immunogenicity of an anchor-modified tumorassociated antigen is due to the enhanced stability of the peptide/MHC complex: implications for vaccine design. J Immunol. 174:4812–4820; 2005. [2] Chaux, P.; Vantomme, V.; Coulie, P.; Boon, T.; van der Bruggen, P. Estimation of the frequencies of anti-MAGE-3 cytolytic T-lymphocyte precursors in blood from individuals without cancer. Int. J Cancer. 77:538–542; 1998. [3] Curiel, T. J.; Wei, S.; Dong, H.; Alvarez, X.; Cheng, P.; Mottram, P. et al. Blockade of B7-H1 improves myeloid dendritic cellmediated antitumor immunity. Nat. Med. 9:562–567; 2003. [4] Darnell, R. B.; Posner, J. B. Paraneoplastic syndromes involving the nervous system. N. Engl. J Med. 349:1543–1554; 2003. [5] Disis, M. L.; Bernhard, H.; Shiota, F. M.; Hand, S. L.; Gralow, J. R.; Huseby, E. S. et al. Granulocyte-macrophage colonystimulating factor: an effective adjuvant for protein and peptidebased vaccines. Blood. 88:202–210; 1996. [6] Disis, M. L.; Goodell, V.; Schiffman, K.; Knutson, K. L. Humoral epitope-spreading following immunization with a HER-2/neu peptide based vaccine in cancer patients. J Clin. Immunol. 24:571–578; 2004. [7] Disis, M. L.; Gooley, T. A.; Rinn, K.; Davis, D.; Piepkorn, M.; Cheever, M. A. et al. Generation of T-cell immunity to the HER2/neu protein after active immunization with HER-2/neu peptide-based vaccines. J. Clin. Oncol. 20:2624–2632; 2002. [8] Disis, M. L.; Grabstein, K. H.; Sleath, P. R.; Cheever, M. A. Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin. Cancer Res. 5:1289–1297; 1999. [9] Disis, M. L.; Gralow, J. R.; Bernhard, H.; Hand, S. L.; Rubin, W. D.; Cheever, M. A. Peptide-based, but not whole protein, vaccines elicit immunity to HER-2/neu, oncogenic self-protein. J Immunol. 156:3151–3158; 1996. [10] Disis, M. L.; Knutson, K. L.; Schiffman, K.; Rinn, K.; McNeel, D. G. Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer. Breast Cancer Res. Treat. 62:245–252; 2000. [11] Disis, M. L.; Rinn, K.; Knutson, K. L.; Davis, D.; Caron, D.; Dela, R. C. et al. Flt3 ligand as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines in patients with HER-2/neuoverexpressing cancers. Blood. 99:2845–2850; 2002. [12] Dunn, G. P.; Bruce, A. T.; Ikeda, H.; Old, L. J.; Schreiber, R. D. Cancer immunoediting: from immunosurveillance to tumor escape. Nat. Immunol. 3:991–998; 2002. [13] Dutoit, V.; Rubio-Godoy, V.; Pittet, M. J.; Zippelius, A.; Dietrich, P. Y.; Legal, F. A. et al. Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-a peptide multimer(+) CD8(+) T cells in humans. J Exp. Med. 196:207–216; 2002. [14] Germeau, C.; Ma, W.; Schiavetti, F.; Lurquin, C.; Henry, E.; Vigneron, N. et al. High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens. J Exp. Med. 201:241–248; 2005. [15] Jager, E.; Chen, Y. T.; Drijfhout, J. W.; Karbach, J.; Ringhoffer, M.; Jager, D. et al. Simultaneous humoral and cellular immune

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[27] Rammensee, H.; Bachmann, J.; Emmerich, N. P.; Bachor, O. A.; Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 50:213–219; 1999. [28] Salazar, L. G.; Fikes, J.; Southwood, S.; Ishioka, G.; Knutson, K. L.; Gooley, T. A. et al. Immunization of cancer patients with HER-2/neu-derived peptides demonstrating high-affinity binding to multiple class II alleles. Clin. Cancer Res. 9:5559– 5565; 2003. [29] Sanderson, K.; Scotland, R.; Lee, P.; Liu, D.; Groshen, S.; Snively, J. et al. Autoimmunity in a phase I trial of a fully human anticytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin. Oncol. 23:741– 750; 2005. [30] Skipper, J. C.; Hendrickson, R. C.; Gulden, P. H.; Brichard, V.; Van Pel, A.; Chen, Y. et al. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp. Med. 183:527–534; 1996. [31] Smith, J. W.; Walker, E. B.; Fox, B. A.; Haley, D.; Wisner, K. P.; Doran, T. et al. Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses. J Clin. Oncol. 21:1562– 1573; 2003. [32] Speiser, D. E.; Lienard, D.; Rufer, N.; Rubio-Godoy, V.; Rimoldi, D.; Lejeune, F. et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin. Invest. 115:739–746; 2005. [33] Stevanovic, S. Identification of tumour-associated T-cell epitopes for vaccine development. Nat. Rev. Cancer. 2:514–520; 2002. [34] van der Bruggen, P.; Traversari, C.; Chomez, P.; Lurquin, C.; De Plaen, E.; Van den, E. B. et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 254:1643–1647; 1991. [35] Walker, E. B.; Disis, M. L. Monitoring immune responses in cancer patients receiving tumor vaccines. Int. Rev. Immunol. 22:283–319; 2003. [36] Yang, Y.; Huang, C. T.; Huang, X.; Pardoll, D. M. Persistent Tolllike receptor signals are required for reversal of regulatory T cell-mediated CD8 tolerance. Nat. Immunol. 5:508–515; 2004. [37] Zou, W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer. 5:263– 274; 2005.

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73 Antiadhesin Synthetic Peptide Consensus Sequence Vaccine and Antibody Therapeutic for Pseudomonas Aeruginosa DANIEL J. KAO AND ROBERT S. HODGES

from the normal flora of healthy humans. Pa rarely causes disease in healthy hosts.

ABSTRACT A synthetic peptide vaccine against Pseudomonas aeruginosa (Pa) has been designed. This vaccine targets the epithelial cell binding domain of the type IV pilus, a key adhesin of Pa. This vaccine uses a novel approach, where a consensus sequence has been developed that generates antibodies that are cross-reactive among pili from multiple strains of Pa. Structural studies have been used to understand the way in which the neutralizing epitope interacts with its host receptor. We describe the use of synthetic peptides to map the adhesintope of the type IV pilus and the use of synthetic peptides in the design of the consensus sequence. We also describe the use of the consensus sequence immunogen in the development of a monoclonal antibody therapeutic for use as a passive vaccine.

BACKGROUND AND AVAILABLE THERAPIES Microbiology Pa is an aerobic, Gram-negative bacillus that can be isolated from soil, water, plants, and animals. It is a frequent contaminant of hospitals and medical equipment. Pa has minimal nutritional requirements, allowing it to grow in distilled water and on relatively inert surfaces, such as stainless steel. Pa is a versatile pathogen that has the potential to express a wide array of virulence-associated factors. These factors can be divided into two groups: constitutive factors and exoproducts. The constitutive factors are important for bacterial adhesion, transport of molecules across the inner and outer membranes, and enzymatic processes important for Pa metabolism. These include pili, flagella, alginate, lectins, outer membrane proteins, endotoxins, and lipopolysaccharide, among others. The exoproducts are important for invasive properties of Pa. These include proteases, elastases, exotoxin A, siderophores, and β-lactamases, among others. The pathogenesis of Pa infection involves the coordinated expression of these virulence-associated factors, which depends on the site of infection and the host response. A brief review of the pathogenesis of respiratory infection by Pa can serve to illustrate how the organism uses these virulence-associated factors and can give us insight into how one may treat these infections. Initially, bacteria are inhaled or come into contact with

DISEASE TARGET Infections Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of nosocomial infection as well as infection in hosts with compromised defenses. It is the second leading cause of hospital-acquired pneumonia and accounts for 10–20% of all nosocomial infections [20]. The most common Pa-related infections are Pa pneumonia, followed by bacteremia and skin and soft tissue infections. Pa is also a major cause of serious infection in patients with burn wounds, those who are immunocompromised, and those with cystic fibrosis. Pa is ubiquitous in the environment and can be isolated Handbook of Biologically Active Peptides

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508 / Chapter 73 the respiratory epithelium by other means, such as through contact with an object that has been colonized by Pa. In the absence of normal mucociliary clearance mechanisms or innate immune responses, the organisms adhere to the epithelium through pili and flagella. Once adhered to the surface, exoproducts such as elastase, alkaline protease, and pyocyanin are produced that facilitate invasion of the host tissue. As the individual enters the chronic stage of infection, there is a transition of colonizing Pa to a mucoid phenotype, characterized by increased production of alginate. There is also selective pressure for nonpiliated and nonflagellar mutants. Pa biofilms form as cell densities increase, and this serves to protect the bacteria from phagocytic cells and the actions of most antibiotics. Once infections reach this stage, they become increasingly difficult to treat. In our view, it would be preferable to focus treatment on prevention of the initial stages of infection before damaging exoproducts and resistant biofilms have come into play. Following this reasoning, we have developed an antiadhesin vaccine that inhibits pilus-mediated adhesion to eukaryotic epithelia.

Immunotherapy

Conventional Treatment

WHY PEPTIDES

Because of the large number of virulence-associated factors, it is impractical to treat Pa infections through neutralization of specific factors. Instead, conventional treatment targets cell wall synthesis and other metabolic processes that are necessary for bacterial growth. Pa infection is conventionally treated by combined antimicrobial therapy. There are a number of currently available antibiotics with antipseudomonal activity, including certain penicillins, third-generation cephalosporins, carbapenems, monobactams, quinolones, and aminoglycosides. Specific agents are selected based on the site of infection, status of the host immune system, and local observations of antimicrobial susceptibility. A significant impediment to the effective treatment of Pa infection is the organism’s ability to rapidly develop antimicrobial resistance [2]. Pa evades the actions of antibiotics by enzymatic modification of the drugs, extrusion of the drugs to the extracellular space by efflux pumps, and mutation of antimicrobial targets. Hypermutable strains of Pa have been observed that may represent another mechanism that contributes to this organism’s ability to develop de novo antibiotic resistance [23]. The selective pressures created by antibiotic use make the development of antibiotic resistance a near inevitability. As a result, the effective lifetime of any individual antibiotic is expected to be limited. This illustrates the need for other forms of therapy for the prevention and treatment of Pa infection [25].

Vaccines have been effective means of controlling diseases caused by a number of bacterial and viral pathogens. Attempts to control Pa infection by vaccination have been ongoing since the 1960s. Approaches have targeted nearly every known Pa virulence factor [15]. Many approaches have resulted in increased survival of hosts in animal models of infection; however, few vaccines have advanced to clinical trials. Vaccines targeting lipopolysaccharide (LPS), a toxic component of the outer membrane of Pa, generally have unacceptable side effects due to the nature of the vaccine constituents. Immunization with inactivated exotoxins increases survival by neutralizing toxic effects of Pa, but these approaches do not necessarily prevent colonization by the organism and may require additional antimicrobial treatment to kill the bacteria. Antiadhesin vaccines tend to not be toxic to the host and have resulted in increased survival in animal models. An obstacle to the implementation of adhesin-based vaccines is the antigenic variability of adhesins among Pa strains.

Type IV Pilus The type IV pili found on Pa are filamentous, unipolar adhesins with an average length of 2.5 μm and diameter of 5.2 nm [24]. Pili bind tracheal, buccal, and corneal epithelial cells. Pili have also been shown to bind DNA [30], stainless steel [13], and other inert surfaces [28, 31]. Because they are the longest extensions from the cell surface, pili are proposed to mediate initial binding of the bacterium to the epithelial surface. Type IV pili are also responsible for the twitching motility observed in Pa [21]. The importance of pili in the binding of Pa to epithelial cell surfaces has been shown, where nonpiliated strains of Pa show a 90% decrease in their ability to bind human A549 type II pneumocytes [10, 12]. In another study using a neonatal mouse model of Pa pneumonia, nonpiliated strains caused 28–96% fewer cases of pneumonia compared with piliated control strains [29]. These results show that pili are important, although not essential for establishing infection by Pa. These results also point to the role of other adhesins such as flagella and alginate in adhesion. Pili are multimeric, nonbranching filaments consisting of thousands of pilA protein monomers arranged in a helical array (Fig. 1) [11]. Five pilin monomers form each turn of the helix. Pili are dynamic structures that can extend and retract through the assembly and disassembly of pilin monomers from the base of the

Antiadhesin Synthetic Peptide Consensus Sequence Vaccine / 509 with 14-residue loops, four residues are identical (C129, D134, P139, C142). Two residues are identical in all but one strain (K140, G141). Two other positions show high conservation (positions 131 [S,T] and 137 [F,Y]). The remaining six positions within the loop are variable (positions 130, 132, 133, 135, 136 and 138, denoted X1 to X6, Fig. 2). Pili from all Pa strains share the same eukaryotic receptor. Pa pili bind a GalNAcβ(1–4)Gal carbohydrate moiety that is found on the surfaces of a number of epithelial cell types [16, 27]. This moiety exists in the glycolipids asialo-GM1 and asialo-GM2, which have been suggested to be physiologically important host cell receptors for pilus-mediated adhesion of Pa. These surface glycolipids appear to be conserved host cell receptors among a number of respiratory pathogens, including Candida albicans [19], Bordetella pertussis [4], Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae [16], and Moraxella catarrhalis [1]. The observation that this is a conserved receptor among pathogenic bacteria points to its promise as a therapeutic target. The C-terminal disulfide loop of the type IV pilus presented itself as an excellent vaccine candidate. Purified pili have been used as immunogens to generate neutralizing antibodies that block pilus-mediated adhesion, thereby reducing the rate of infection. Purified pili do not generally make effective vaccines because while pili do generate neutralizing antibodies specific for the C-terminal receptor binding domain, the majority of the antibodies that are generated against pili are specific for other regions of the protein, namely the major immunological region which spans residues 70 to 110 [6]. This major immunological region does not contain neutralizing epitopes and varies significantly in amino acid sequence among strains. To replace pili as an immunogen and improve the preceding situation, a peptide vaccine is an attractive approach for the following reasons. First, the receptor binding domain is

pilus in a complex and coordinated fashion [21]. The functional domains of the pilin monomer have been elucidated. An N-terminal α-helix mediates multimerization [14], and a model has been proposed for the quaternary structure of the pilus fiber [11]. It has been hypothesized that this helix is buried upon pilus formation. The domain that has been the focus of our work is the 14- to 19-residue disulfide-bridged loop at the C-terminus of the pilin monomer [18]. It has been shown that this loop region mediates binding to eukaryotic epithelial cells. This well-structured loop contains one type I β-turn and one type II β-turn [22], both of which are conserved among known strains of Pa pilin. The epithelial cell receptor binding domain is only exposed on the five pilin monomers at the tip of each pilus (Fig. 1) [17]. The amino acid sequence of the pilin receptor binding domain is semiconserved among Pa strains. Pili serotypes can be divided into two groups based on the length of the receptor binding domain loop: those with a 14-residue C-terminal disulfide loop and those with a 19-residue loop (Fig. 2). Among the pili serogroups

FIGURE 1. Schematic diagram showing the architecture of the type IV pilus. Type IV pili are polar appendages extending from the surface of the bacterium (left), which are composed of pilin protein monomers arranged in a helical array (center) with fivefold symmetry (right).

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FIGURE 2. Amino acid sequences of the receptor binding domains from five strains of Pa. Alignment of amino acid sequences of the receptor binding domain from Pa strains PAK, PAO, KB7, and P1. Boxed residues show identical positions. Shaded residues indicate positions with conservative substitutions.

510 / Chapter 73 contained within a continuous epitope constrained by a disulfide bridge, so the design of the peptide immunogen is greatly simplified. Second, peptide sequences of native strains of the receptor binding domain can compete with purified pili and whole bacteria for binding to host epithelia, which indicate that these native peptides adopt similar conformations to those found in intact pili. Third, in the setting of a peptide vaccine, antibodies are only generated against the neutralizing epitope, which enhances its potential to develop a protective response, rather than against the major immunological region.

CONTRIBUTIONS The ideal vaccine would provide protection against infection from challenge by multiple Pa strains. This implies that antibodies raised against the vaccine would have to be cross-reactive among pili from multiple Pa strains. We hypothesized that it would be possible to achieve a cross-protective immune response because of the semiconserved amino acid sequence of the receptor binding domain (Fig. 2) and the observation that all strains bind the same host receptor [27]. Furthermore, we hypothesized that if we were able to generate a crossreactive immune response, it would be unlikely that escape mutants would evolve that retain their ability to bind the eukaryotic host receptor, but would not be recognized by antibodies from the vaccine. Development of a cross-protective vaccine has been complicated by the observation that antibodies raised against homologous strain pili are not protective against challenge by heterologous Pa strains [26]. A conventional approach to avoid this problem would be to actively immunize with pili from multiple Pa strains (a cocktail vaccine) with the intent of inducing specific immunity to each constituent. Previous studies of such multivalent vaccines, however, revealed that antigenic competition among the pili strains caused antibody production against one pili strain to dominate the immune response [6]. The weaker responses to other serogroups were not expected to be adequate to confer protection; therefore, a multivalent approach is not an option. In order to avoid complications caused by antigenic competition, we have pursued the development of a consensus sequence immunogen designed to generate antibodies that are cross-reactive among different Pa strain pili. We have undertaken a novel approach, using biochemical and structural techniques, to understand components of the receptor binding domain that contribute to strain-specificity and cross-reactivity. In conjunction with the development of the consensus sequence vaccine, we are developing a monoclonal antibody therapeutic which will have optimal cross-

reactivity among strains. This antibody therapeutic can be administered to individuals who require immediate protection or who have a weakened or compromised immune system (e.g., burn wound patients). Structural studies of the receptor binding domain have been performed with the goal of identifying structural similarities and differences between strains of Pa that may be used to design a vaccine that is crossprotective among strains. The three-dimensional X-ray crystal structures of pilin proteins from two strains, PAK [14] and K122-4 [3], have been determined. The NMR solution structures of peptide analogs of the receptor binding domains from strains PAK [22], PAO, KB7 [8], and P1 [9] have also been determined. Among the most notable conserved structural features for both the pilin proteins and peptides were the two conserved β-turns, which are key features for immunogenicity. In addition, there were only subtle differences in the conformation of the receptor binding domains in PAK and K122-4 pilins (Fig. 3). The NMR solution structures of peptides of the receptor binding domains from strains PAK, PAO, and KB7 showed more dramatic differences (Fig. 3). In these peptides, the orientations of the β-turns with respect to each other differ among the peptides from the three strains. These differences may be accounted for by the arrangement of hydrophobic side chains in each of the peptides. Residues F137 and P139 form hydrophobic cores in both the PAK and PAO peptides. I138 also contributes to the hydrophobic core of the PAO. In contrast, the KB7 peptide forms a hydrophobic face comprised of V133 and F137. The differ-

FIGURE 3. A. Backbone conformations of the PAK and K122-4 receptor binding domains from X-ray crystal structures of the monomeric pilin proteins [3, 14]. The white strand corresponds to the PAK receptor binding domain (129–144), and the gray strand corresponds to the K122-4 receptor binding domain (129–144). β-turns are highlighted by the hatched regions in both strands. The two structures are aligned along the type 1 β-turn. The disulfide bonds are shown in black. B. NMR solution structures of PAK, PAO, and KB7 synthetic peptides of the receptor binding domains [8, 22]. The white strand corresponds to the PAK peptide (129– 144), the gray strand corresponds to the PAO peptide (129– 144), and the hatched strand corresponds to the KB7 peptide (129–144). The three peptide structures are aligned along the type 1 β-turn. The disulfide bonds are shown in black.

Antiadhesin Synthetic Peptide Consensus Sequence Vaccine / 511 ences in the hydrophobic pockets in the three analogs are the most likely cause of the observed differences in backbone conformation surrounding the β-turns. Because the crystal structures of the pilin protein monomers are not known for the PAO and KB7 strains, the significance of these differences within the receptor binding domains is unclear. Structural comparison of the receptor binding domain seen in the monomeric pilin proteins to those seen in the synthetic peptides suggests that the pilin protein and the peptides may present conformationally different epitopes to the immune system (Fig. 3). The receptor binding domain in the pilin monomer makes a number of intrachain contacts with other regions of the protein (Fig. 4), which serve to define a specific conformation of the receptor binding domain and are expected to make the receptor binding domain more conformationally rigid in comparison to the synthetic peptides of the receptor binding domain. Furthermore, the receptor binding domain in the peptide is completely accessible and only partially accessible in the pilin protein (Fig. 4). In the development of an active vaccine, the additional flexibility of the peptide immunogen could be advantageous in enhancing antibody cross-reactivity among different strains of Pa. In the development of a monoclonal antibody therapeutic, however, the benefits of using a peptide immunogen are not as clear. In this case, it is critical to maximize its affinity for the epitope in the context in which the epitope is presented by the pathogen, in this case the pilin protein. As the structural results suggest, this is not necessarily the same as the epitope presented by a peptide immunogen. We needed to determine whether there is a sacrifice in the affinity of antibodies raised against a peptide immunogen compared to a protein immunogen. We hypothesized that a peptide immunogen may generate antibodies of higher cross-reactivity but lower affinity for the pilin protein receptor binding domain because

the immunogen has a different conformation and is more conformationally flexible. Our second objective was to determine whether there was a difference in cross-reactivity among Pa strains with the use of a peptide immunogen in comparison with use of a protein immunogen. Our findings will help us determine the best way to produce a monoclonal antibody with the desired properties of high affinity and cross-reactivity. In addition, these findings will have implications on the peptide vaccines in general. To test this hypothesis, two synthetic peptide immunogens and two recombinant pilin protein immunogens were used to generate polyclonal and monoclonal antibodies. For each type of immunogen, one was based on the PAK strain sequence and the other was based on the consensus sequence [6]. Preliminary data from the polyclonal antibodies shows that the peptide and protein immunogens generate antibodies of comparable affinity for Pa antigens. The cross-reactivity of the peptide-specific antisera also appears to be greater than the antisera raised against the protein immunogens. Further analysis, including affinity purification of receptor binding domain specific antibodies from the polyclonal pools, is required to determine which immunogen generates antibodies of optimal affinity and cross-reactivity. In order to understand the interactions that occur between the receptor binding domain and the host receptor, the “adhesintopes” of the PAK and KB7 strains were mapped by use of single alanine substitution peptide analogs of the receptor binding domains from each strain [32]. These peptide analogs were used as competitors for binding of PAK pili to cultured A549 human pneumocyte cells. The IC50 of each peptide analog was determined in order to determine which positions were critical for receptor binding. Analogs that increased the IC50 by at least threefold were considered critical residues. The PAK analogs revealed that residues S131, Q136, I138, P139, G141, and K144 were

FIGURE 4. A. Comparison of the conformation of the PAK receptor binding domain (128–144) as part of the pilin protein (dark chain) [14] and as a peptide (light chain) [8]. B and C. Crystal structure of PAK monomeric pilin (29–144), showing the receptor binding domain as a stick model and the remainder of the protein as a molecular surface. White patches on the surface represent contacts between the receptor binding domain and other regions of the protein.

512 / Chapter 73 critical for receptor binding. The KB7 analogs showed that A130, T131, T132, V133, D134, A135, K136, R138, and P139 were critical for receptor binding. These residues were considered to compose the adhesintope of each strain. Comparsion of these findings with the NMR solution structures of the PAK and KB7 peptide analogs suggested that certain residues of the adhesintopes were important for imposing conformational constraints on the peptides. Other residues appeared to be important for interactions with the host receptor. The findings suggested that there are more potential interactions between the peptides and the host receptor than any one peptide can occupy. Each peptide occupies a subset of the total possible interactions, but the subsets are different for the PAK versus the KB7 strains. These findings gave insight into the types of interactions that occur between the pilus and the host receptor, but the findings did not point to a single group of interactions that was shared among strains. The feasibility of designing a consensus sequence immunogen has been demonstrated. Polyclonal antibodies were raised against the native strain PAK peptide analog as well as three single substitution analogs of the PAK sequence (E135A, I138A, and E135P) [5]. These experiments showed that the native PAK sequence generated high-affinity antibodies for the PAK strain, with much lower affinity for the PAO, KB7, and P1 strains. By use of single alanine substitution analogs of the PAK sequence, it was shown that certain side chains were critical for antibody recognition of the PAK sequence, while other residues appeared to be nonessential for PAK recognition. It was hypothesized that the immunogens that generated antibodies of increased crossreactivity among strains could be designed by making substitutions at nonessential positions, but maintaining the identities of residues that were critical for antibody binding to PAK. To demonstrate this, antibodies raised against the E135A analog had a higher apparent affinity

for the PAK strain and greatly increased cross-reactivity among the PAO, KB7, and P1 strains. It was also shown that the cross-reactivity determined in the binding assay correlated to cross-protection in an animal model of Pa infection. These experiments demonstrated that it was feasible to design immunogens that generate antibodies with increased cross-reactivity, while retaining a high affinity. The next step in the design of a consensus sequence immunogen that generated more cross-reactive antibodies was brought about through the hypothesis that making substitutions of the PAO sequence into the PAK sequence would further increase cross-reactivity. These immunogens became true consensus sequences, combining elements from more than one strain in individual immunogens. A series of peptide immunogens with one, two, or three substitutions of the PAO sequence into the PAK sequence were designed. One promising PAK analog with substitutions from the PAO sequence at positions 130 and 135 was identified in this screen and has been pursued as a putative consensus sequence, called Cs1 [6]. In the process of vaccine development, we have developed a novel approach for the measurement of antibody affinity and cross-reactivity using surface plasmon resonance (Fig. 5) [7]. Compared with the competitive ELISA assays that we were using previously to measure relative antibody affinity, this new technique has the advantage of consuming smaller quantities of synthetic peptide antigens and allows for kinetic analysis of antibody-antigen interactions. This technique is based on a novel peptide delivery and capture system using a stable heterodimeric α-helical coiled-coil interaction. Two peptides, denoted E-coil and K-coil, are used in the experiment to form the coiled-coil. The peptides have been designed such that at neutral pH, electrostatic interactions prevent formation of E/E or K/K homodimers, while favoring formation of E/K IgG

PA Antigen

K-coil Immobilization

Loading of E-coil ligand

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IgG Association

PA Antigen

IgG Disssociation

Regeneration of K-coil surface

FIGURE 5. Synthetic K-coil peptide is covalently immobilized on the sensor chip surface, followed by delivery and capture of the ligand-E-coil conjugate. The antibody solution is injected over the sensor chip surface, and the binding and subsequent dissociation are measured by the SPR instrument. Finally, the K-coil surface is regenerated to prepare for the next cycle.

Antiadhesin Synthetic Peptide Consensus Sequence Vaccine / 513 heterodimer formation. The affinity of this interaction has been measured at 0.47 to 0.50 nM, with a relatively slow dissociation rate (kd) of 2 × 10−4 s−1. Peptide ligands are prepared by iodoacetylation specifically at the Nα-amino group. These peptides are then conjugated to E-coil peptides that have been designed with an N-terminal cysteine residue. During the experiment (Fig. 5), the K-coil peptide is covalently immobilized on the sensor chip surface. The ligand-E-coil conjugate is then delivered to the surface and captured by the K-coil peptide. A low surface density is used to minimize the effects of avidity. An antibody solution is then injected over the surface, and the association and dissociation phases are monitored by the Biacore. Finally, the E/K-coil heterodimer is dissociated from the surface, regenerating the K-coil surface. This surface can be reloaded with the same ligand-E-coil conjugate for further analysis or loaded with another conjugate to analyze a different antibody-antigen interaction. In this way, a single surface can be used for up to 150 cycles, with multiple ligands. The utility of this system has been demonstrated with one polyclonal antibody raised against the PAK strain monomeric pilin and one polyclonal antibody raised against the synthetic PAK peptide. The antibody raised against the PAK monomeric pilin was PAK strain specific and did not bind epitopes from heterologous strains. This polyclonal antibody had an apparent KD of 1.03 × 10−8 M and 2.80 × 10−7 M for the PAK pilin and synthetic PAK peptide, respectively (Table 1). On the other hand, the antibody raised against the synthetic PAK peptide bound all four strains that were examined: PAK, PAO, KB7, and P1 peptides. The apparent Kd of this antibody for the synthetic peptide ligands ranged from 2.89 × 10−6 M to 7.67 × 10−8 M. These results show that the polyclonal antibody raised against the synthetic peptide immunogen was more cross-reactive and had an affinity that was comparable to an antibody raised against the monomeric pilin. TABLE 1. Dissociation constants (KD) determined using surface plasmon resonance peptide capture and delivery technique for binding of antibodies raised against PAK monomeric pilin and PAK synthetic peptide (129–144) to Pa type IV pilus antigens (PAK, PAO, KB7, P1, PAK pilin). Polyclonal antibody Antigen PAK PAO KB7 P1 PAK pilin

PAK monomeric pilin KD (M) 1.03 × 10−8 — — — 1.69 × 10−7

PAK peptide KD (M) 2.80 4.67 2.89 7.67 2.24

× × × × ×

10−7 10−6 10−6 10−8 10−7

FUTURE OUTLOOK An alternative approach to the design of a consensus sequence is being pursued using a synthetic combinatorial peptide library. Rather than rational design of a consensus sequence immunogen, a combinatorial peptide library is being used to identify a peptide ligand with optimal binding to the eukaryotic epithelial cell receptor. In this case, a competitive binding assay is being used to assess relative affinities of library pools for the eukaryotic host receptor. Briefly, peptide ligands are incubated with cultured A549 human pneumocytes and compete for binding with biotinylated PAO bacteria. The remaining biotinylated bacteria are then quantitated. A peptide library has been constructed based on the PAO strain receptor binding domain, where the identities of the conserved positions were maintained and the variable positions were library positions (Fig. 2). This library is currently being deconvoluted by use of an iterative approach by selection of library pools that show optimal binding. After screening 20 pools of 3.2 million peptides per pool, the amino acid residue at position X1 is defined. A second library was prepared where 20 pools of 160,000 peptides per pool was screened to define position X2. After deconvolution of the first two positions of the library, the affinity was increased 1700 times bringing the apparent Ki from 12 μM for the native PAO peptide to 6.9 nM for the library pool. After deconvolution of all six positions, we anticipate having a peptide ligand with sub-nanomolar affinity for the host receptor. This peptide can then be used as an immunogen, with the idea that it will give an optimal cross-protective response.

CONCLUSION We have demonstrated that the development of synthetic peptide vaccine against Pa infection is feasible and that it can be effective. Using biochemical and structural techniques, we have characterized the neutralizing epitope of the type IV pilus. This information has guided the design of a consensus sequence immunogen that can generate antibodies of increased cross-reactivity among strains. The major obstacle to the development of this vaccine has been how to design a consensus sequence that is protective against all strains of Pa, rather than being specific for a single strain or a few strains. Understanding the molecular determinants of strain-specificity and crossreactivity should be applicable to vaccine design in general.

514 / Chapter 73 References [1] Ahmed, K., Y. Suzuki, D. Miyamoto, and T. Nagatake, Asialo-GM1 and asialo-GM2 are putative adhesion molecules for Moraxella catarrhalis., Med Microbiol Immunol (Berl), 2002. 191(1): p. 5–10. [2] Alonso, A., E. Campanario, and J. L. Martinez, Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa, Microbiology, 1999. 145(Pt 10): p. 2857–62. [3] Audette, G. F., R. T. Irvin, and B. Hazes, Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture, Biochemistry, 2004. 43(36): p. 11427–35. [4] Brennan, M., J. H. Hannah, and E. Leininger, Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids., J Biol Chem, 1991. 266(28): p. 18827–31. [5] Cachia, P. J., L. M. Glasier, R. R. Hodgins, W. Y. Wong, R. T. Irvin, and R. S. Hodges, The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa, J Pept Res, 1998. 52(4): p. 289–99. [6] Cachia, P. J. and R. S. Hodges, Synthetic peptide vaccine and antibody therapeutic development: Prevention and treatment of Pseudomonas aeruginosa, Biopolymers, 2003. 71(2): p. 141–68. [7] Cachia, P. J., D. J. Kao, and R. S. Hodges, Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and crossreactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy, J Mol Recognit, 2004. 17(6): p. 540–57. [8] Campbell, A. P., C. McInnes, R. S. Hodges, and B. D. Sykes, Comparison of NMR solution structures of the receptor binding domains of Pseudomonas aeruginosa pili strains PAO, KB7, and PAK: implications for receptor binding and synthetic vaccine design, Biochemistry, 1995. 34(50): p. 16255–68. [9] Campbell, A. P., H. Sheth, R. S. Hodges, and B. D. Sykes, NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn, Int J Pept Protein Res, 1996. 48(6): p. 539–52. [10] Chi, E., T. Mehl, D. Nunn, and S. Lory, Interaction of Pseudomonas aeruginosa with A549 pneumocyte cells, Infection and Immunity, 1991. 59(3): p. 822–28. [11] Craig, L., R. K. Taylor, M. E. Pique, B. D. Adair, A. S. Arvai, M. Singh, S. J. Lloyd, D. S. Shin, E. D. Getzoff, M. Yeager, K. T. Forest, and J. A. Tainer, Type IV pilin structure and assembly. X-ray and EM analyses of Vibrio cholerae toxin-coregulated Pilus and Pseudomonas aeruginosa PAK pilin, Mol Cell, 2003. 11(5): p. 1139–50. [12] Farinha, M. A., B. D. Conway, L. M. Glasier, N. W. Ellert, R. T. Irvin, R. Sherburne, and W. Paranchych, Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice, Infect Immun, 1994. 62(10): p. 4118–23. [13] Giltner, C. L., E. J. van Schaik, D. J. Kao, and R. S. Hodges, The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for binding to both biotic and abiotic surfaces, Mol Microbio, 2005. [14] Hazes, B., P. A. Sastry, K. Hayakawa, R. J. Read, and R. T. Irvin, Crystal structure of Pseudomonas aeruginosa PAK pilin suggests a main-chain-dominated mode of receptor binding, J Mol Biol, 2000. 299(4): p. 1005–17. [15] Holder, I. A., Pseudomonas vaccination and immunotherapy: an overview, J Burn Care Rehabil, 2001. 22(5): p. 311–20. [16] Krivan, H. C., D. D. Roberts, and V. Ginsburg, Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence

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74 Peptide Vaccines for Malaria JOSÉ MANUEL LOZANO, ADRIANA BERMÚDEZ, AND MANUEL ELKIN PATARROYO

Due to this parasite’s increasing resistance to antimalarial agents and that of the mosquito to insecticides, the search for different methods for developing vaccines has become an urgent need, especially the development of multiantigen, subunit-based, synthetic vaccines, thus becoming one of the most exiting conceptual challenges for vaccine research today. The first multiantigen, multistage, synthetic vaccine (named SPf66) was developed by our institute 18 years ago in the search for this goal; it provided complete protection for a limited number of Aotus monkeys [63] and humans during experimental challenge [64]. Large-scale phase II trials, involving thousands of individuals older than one year of age, showed that this synthetic vaccine, formulated with aluminum hydroxide as sole adjuvant, was safe and immunogenic, showing a protective efficacy of about 40% (31–60%) in endemic areas [4, 61, 81, 92] for up to two years. This multiantigen, multistage, synthetic malarial vaccine was not effective in children aged less than one year [3], and a batch produced elsewhere was not protection-inducing in a field trial performed using a different ethnic group [60]. The large-scale vaccine trials also revealed that the immune response against SPf66 was controlled by the major histocompatibility complex (MHC), since most nonresponder individuals typed HLA-DRβ1*04 and those that did not produced antibodies preferentially using T-cell receptor (TCR) Vβ3 and Vβ10 family members [58]. These seminal results presented by SPf66 provided the basis for further developing a logical and rational methodology for a synthetic, subunitbased, multistage, antimalarial vaccine. Microbes and the immune system have coevolved, both trying to protect themselves against mutual aggression. It is thus not strange that microbial high activity binding peptides (so-called HABPs), binding to the cells that they will infect and being critical for their

ABSTRACT Obtaining a highly effective malaria vaccine is a worldwide priority. The first approach aimed at obtaining a malarial vaccine using synthetic peptides was a polymeric chimeric molecule named SPf66, which conferred limited protective efficacy in Aotus monkeys and in large human field-trials. Our efforts then became focused on obtaining a second generation malarial vaccine based on the rational selection of conserved high activity binding peptides (HABPs) whose critical binding residues were to be systematically replaced by others precisely selected. An alternative approach has consisted of replacing peptide bonds involving these HABPs’ critical binding residues; this has also returned promising results to date. Our overall results have suggested a correlation between modified HABPs’ three-dimensional structure, HLA-DR β1* binding preferences, and their protection-inducing capacity in monkeys. Basic knowledge of a parasite’s functionally active peptides, their 3D structure, and their interaction for forming the MHC II- peptide-TCR complex will thus contribute toward designing fully effective multicomponent, multistage, subunit-based malarial vaccines.

INTRODUCTION Plasmodium falciparum is the causative agent for the most lethal form of malaria in humans, being responsible for more than 2 million deaths per year [6, 14, 53]. Around 25% of all cause mortality in areas of stable endemic transmission in Africa among children under five years old has been directly attributed to malaria. Besides the direct demographic consequences of this alarming level of mortality, there are also significant negative economic effects [78]. Handbook of Biologically Active Peptides

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516 / Chapter 74 survival, have evolved so as to remain undetected or silent to avoid immune system pressure. These HABPs are normally derived from malaria parasite conserved binding regions when used in our studies [90]. It is also not unexpected that microbes have selected certain of the host’s genetic characteristics to survive in order to induce inappropriate immune responses in them, such as immunological silence, high nonprotectioninducing, or short-lived antibody levels, apoptosis, immunosuppression, or autoimmunity. Therefore, site-directed chemical modifications in specific HABPs’ critical binding residues can render them immunogenic and protection-inducing or solely immunogenic. A multicomponent subunit-based malarial vaccine is therefore feasible if the appropriate fitting into HLA-DRβ1* molecules can be achieved and the appropriate MHC-II peptide-TCR complex formed. One of the strategies for obtaining structural and immunomodulated antigens (useful for malarial vaccine development) consists of site-directed modification of peptide bonds from selected HABPs by obtaining pseu-

dopeptides in which peptide bond isosters are elements for antigen backbone modification, such as reduced amide [45, 46] or partial d-substitutions for stereochemical alteration [48]; both of these have proven to be potential components of a subunit malarial vaccine formulation. This review summarizes the relevance of antigenmodified malaria sequences and their structural and immunological impact when assessed as protectioninducing agents in Aotus monkeys as novel malarial vaccine components.

MALARIAL VACCINES Historically, the need for a malarial vaccine has led to immunization trials with x-ray-irradiated P. falciparum sporozoites inoculated via infected Anopheles mosquito bites [32] (Fig. 1A). Human volunteers immunized with such attenuated malaria parasites have developed a protective immune response against experimental malarial infections [20]. However, disadvantages associated with

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AMA-1 PFNBP-1 PFRBP-2-HaHb RAP1,2 MSP-1

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MSP-2 MSP-4 MSP-5

ABRA

MSP-6

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FIGURE 1. Adapted from Miller [52] and Bannister [7]. A. The Plasmodium falciparum lifecycle, showing the malaria parasite’s distinct blood and liver stages. B. Merozoite representation describing the main protein ligands involved in malarial parasite invasion of RBCs. (See color plate.)

Peptide Vaccines for Malaria / 517 x-ray attenuated vaccines (such as producing large amounts of irradiated material) have led to the search for protective antigens derived from the malaria parasite’s different developmental stages. Several strategies have been explored related to developing an antimalarial vaccine. Such a vaccine should ideally function as follows: (1) block sporozoites during invasion or development within hepatocytes (anti-infection vaccine), (2) block merozoite invasion of RBCs or inhibit their intracellular development (antidisease vaccine), (3) block cytokine-pathologyinducing effects or parasite sequestration (diseasemodifying vaccine), or (4) block human-mosquito transmission by immunization against the parasite’s sexual stages (transmission-blocking vaccines), thereby preventing the spread of disease within a population, having no direct impact in terms of preventing sideeffects among vaccinated individuals. An effective antimalarial vaccine should advantageously follow different strategies by simulating protective immune responses against several stages of the lifecycle at the same time. Many candidate antigens for P. falciparum malaria subunit vaccines have been studied and identified during the last three decades of malarial research. Efficacy has been measured on the basis of protection studies in animal models (murine and primate) and on in vitro antisera and monoclonal antibody activities blocking host cell invasion and/or in vitro parasite development. Many of these strategies have reached clinical trials, but only the synthetic SPf66 malarial vaccine has provided protective efficacy in different field trials in different ethnic groups and epidemiological setups. The efficacy of a recombinant product comprising residues 83 to 383 from the Plasmodium falciparum circumsporozoite protein (CSP), named RTS,S/AS02A, formulated with a powerful adjuvant mixture, has been recently reported to have decreased infection by 29% among young African children [5]. However, its protective efficacy did not last more than three months and was only directed against the first episode [83], confirming previous results concerning semi-immune adults (34% protective efficacy for only 2 months) [12].

MAIN MALARIAL ANTIGENS INVOLVED IN TARGET CELL BINDING The recognition and invasion of human erythrocytes by the malaria parasite depends on multiple interactions. A specific assay has been developed [90] for identifying any malaria protein’s amino acid sequences involved in RBC binding; several such different antigens have been suggested as targets for protective

immunity (Fig. 1B). Therefore, thousands of peptides (all derived from the main malarial protein ligands) have been synthesized by using both standard t-Boc and Fmoc solid-phase peptide synthesis (SPPS) [37, 52] with the aim of determining those peptide ligands involved in specific binding to RBCs, as well as identifying their critical binding residues as determined by glycine analog scanning binding competition experiments against the native 125I radio-labeled peptide. Once critical binding residues have been identified in each case, these residues are systematically replaced by other amino acids having similar sidechain mass but different polarity for immunological assessment in in vitro as well as in vivo experiments. The Plasmodium falciparum merozoite surface protein1 (MSP-1) is one of this parasite’s most abundant merozoite surface membrane molecules and has thereby been regarded as an antimalarial vaccine candidate. This molecule is proteolytically processed into 83 kDa, 30 kDa, 38 kDa, and 42 kDa fragments forming a noncovalent complex on the merozoite surface, together with the MSP-6 and MSP-7 molecules that have been implicated in merozoite invasion [11, 36]. Urquiza [90] has identified seven MSP-1 HABPs and their corresponding critical RBC binding residues; four of them were derived from nonpolymorphic Plasmodium falciparum MSP-1 regions. Another P. falciparum antigen ligand involved in merozoite invasion of RBCs is membrane surface protein-2 (MSP-2). Two erythrocyte binding peptides (HABPs) have been identified from MSP-2 by use of the previously reported strategy [62]; one of them was variable, while the other was conserved and thus chosen for further research and its critical RBC binding residues identified. Members of the erythrocyte-binding-like (ebl) family, including erythrocyte binding antigen-175 (EBA-175), are responsible for high-affinity binding to glycoproteins on RBC surface [31]. Six conserved HABPs having high specific RBC binding were found for EBA-175 by using the same strategy, representing clear evidence of these sequences’ role in the invasion process. Critical binding residues were also identified in the same work [77]. The serine repeat antigen (SERA) protein, also known as pf140 [69], serine repeat protein (SERP) [39] p126 or pl13 [24], is a 111-kDa and 989-amino acid antigen, including a 35-serine residue repeat region [15]. Although SERA’s function is not known, it has been shown to contain a domain having significant amino acid sequence homology with the active site of cysteine proteinases [57]; however, protease activity has not yet been reported for SERA. The 140-kDa/130-kDa/110-kDa rhoptry protein complex (RHOPH) has been found to be associated

518 / Chapter 74 with a 120-kDa SERA protein, to bind directly to human erythrocyte membranes, inside-out vesicles, and intact mouse erythrocytes [68]; it therefore leads to the formation of a soluble macromolecular complex deposited on RBC membrane during merozoite invasion. Six HABPs have been identified in SERA; some of them have inhibited in vitro merozoite invasion of erythrocyte and also the parasite’s intraerythrocyte development [71]. Ring-infected erythrocyte surface antigen (RESA) is a P. falciparum protein located in dense granules but also found in culture supernatants. The role of RESA during merozoite invasion is not clear; however, specific anti-RESA antibodies inhibit merozoite invasion [82, 95]. The presence of anti-RESA antibodies in individuals from P. falciparum-holoendemic areas has been correlated with acquiring clinical immunity against malaria [16]. One of these HABPs contains a B-cell epitope recognized by antibodies elicited by natural exposure to P. falciparum that are able to efficiently inhibit in vitro merozoite invasion of RBC not only by Pf155/RESA+ parasites but also by Pf155/RESA-deficient parasites, suggesting cross-reactivity with other as yet unrecognized molecules. Moreover, this region contains T-cell epitopes recognized by humans living in holoendemic areas [40]. The apical membrane antigen-1 (AMA-1) is a highly conserved minor surface antigen from the malaria parasite’s apical complex. The 83-kDa precursor PfAMA-1 is a Type I integral membrane protein that is proteolytically processed to 66-kDa and then to 48- and 44-kDa products (released as soluble protein in supernatant culture) located on merozoite surface around the time of merozoite release from the infected RBC [38]. Four HABPs belonging to this protein domain I, two from domain II, and one from domain III have been identified [91]; as four of them are conserved, their critical binding residues have been necessarily identified. Three histidine-rich proteins (HRP) synthesized by the parasite are present on the RBC membrane in P. falciparum-infected RBC. One of these proteins is the knob-associated histidine-rich protein (KAHRP-I), phenotypically associated with the expression of knoblike protuberances on the surface of infected erythrocytes which have been involved in erythrocyte agglutination, or rosetting [42], and therefore indirectly in severe malaria. A novel P. falciparum membrane-associated histidine-rich protein located in Maurer’s clefts (MAHRP-1) has been described recently. KAHRP-I has a molecular mass varying between 80 and 110 kDa. It is a histidine-lysine-rich protein presenting three different domains: an N-terminal histidine-rich domain (Region I), a central lysine-rich domain (Region II),

and a C-terminal decapeptide repeat domain (Region III) [74]. No high-activity binding peptides were found in region I (residues 41–300) or in region III (residues 481–660) but three HABPs have been identified in region II (residues 301–480) [44]; one of them is conserved, leading to its critical binding residues being identified by glycine scanning. The Plasmodium falciparum acidic-basic repeat antigen (ABRA) is a highly conserved 101-kDa protein, located on the merozoite surface and in the parasitophorous vacuole within infected erythrocytes [13]. ABRA is recognized by antibodies eluted from immune cluster of merozoites (IMC) [51]. It has been shown that ABRA binds to erythrocytes and to MSP-142; the interaction with RBC seems to be mediated by band 3, since ABRA showed dose-dependent and saturable binding to band 3 protein. This interaction is mediated by the highly conserved N-terminus [41]. Five HABPs have been found in this region [23]; four of them are conserved, thereby leading to their critical binding residues being identified.

VACCINE DESIGN BASED ON A STRUCTURAL-FUNCTION RELATIONSHIP 3D structural models obtained from 1H-NMR data gathered from hundreds of HABPs derived from selected P. falciparum merozoite proteins involved in RBC invasion (MSP-1, MSP-2, EBA-175, SERA, RESA, AMA-1, HRP-II, and ABRA) have allowed us to find that modifications performed on non-immunogenic, nonprotection-inducing peptides have generated a large panel of peptide analogs all having different immunological behavior as a consequence of adopting a specific bioactive structural conformation. 1 H-NMR experiments have revealed that most native HABPs have a compact α-helical structure [21, 22], different types of β-turn [18], or a totally random tendency [9, 17, 79, 88]; these results were always confirmed by spectroscopic CD analysis. NMR structural studies have chronologically shown different patterns in structural-immunological activity relationship when correlated with protection against experimental malaria induced by either native or chemically modified HABPs in vaccinated Aotus monkeys. A significant shortening of the formerly high α-helical portion in the native HABPs has been associated with induced protective immunity against malaria [18, 21, 22]. When a second set of formerly random native HABPs were modified, they acquired a small α-helical region associated with immune protection, while others adopted a distorted β-turn structure, and some others induced displacement of previously present structured regions [33]. Some modified HABPs (having slightly

Peptide Vaccines for Malaria / 519 different modifications) induced different immune mechanisms associated with their 3D structures (this will be discussed further). Some other modified HABPs induced short-lived antibodies that were not associated with protection against malaria [65]; very few modified HABP analogs induced protection without inducing antibody production but did induce high cytokine liberation, suggesting that an immune cellular mechanism

had been activated during the protection process [66]. Another group of modified HABPs induced highly nonprotective, long-lasting antibody levels [67], while a large group of modified HABPs effectively induced a protective humoral immune response against malaria [22, 26, 79], as can be observed in Table 1. Our 1H-NMR structural data has a remarkably high structural homology in regions where the most relevant

TABLE 1. Immunological and structural characteristics for both native and modified HABPs. Conserved HAPB amino acid sequences and those of their analogues used for immunizing Aotus monkeys. These peptides were aligned according to the HLA-DRB1 molecule’s Pocket 1, 4, 6, and 9 (shadowed). PI, II10, II15, and III15 are the days when monkeys were bled and antibody titres determined by IFA (shown in brackets). The prefix corresponds to the total number of Aotus presenting these antibody titres. Prot = corresponds to the total number of Aotus protected against experimental challenge with the 100% infective Aotus adapted P. falciparum FVO strain. Ref = referenced papers. Sequence Protein

Peptide

MSP-1

1585 15484 19992 19994 13450 1783 12860 12912 14014 15492 22814 1815 24292 6746 21742 20466 23230 1522 15474 15476 17898 22904 13446 1779 14012 19750 24080 22812 6737 14096 24308 22834 6671 10000 17964 22826 22792 17962 23778 13494 13492

EBA-175

EBA-175 SERA

MSP-1

EBA-175

SERA

RESA

P1 P4 P6 P9

EVLYLKPLAGVYRSLKKQLE EVLYHMPLAGVYRALKKQLE VLYHKPLAGVYRALKKQLG VIYHKPLAGVYRALKKQIG EVLYLLDLAGVYRSLKKQLE HRNKKNDKLYRDEWWKVIKK LFNKKNDKLYRDEYWKDIKK FFNKKNDKLYRDEYWKDIKK LFNKMNDKLYRDEYWKDIKK HRNKMNDKLYRDEYWKTIKK NDKLYRMEYWKTIKKDVW YTNQNINISQERDLQKHGFH LTNQNINIDQEFNLMKHGFH DQGNCDTSWIFASKYHLETI DQGNSDTSYIFASKYHH DQGNSDTSWNFAAKYLLETI GNSITAWIRASKYLLET QIPYNLKIRANELDVLKKLV QIPYNLKIFAIMLDTHKMLV QIPYNLKIFAIMLDTHKMLT QIPYNLKIFANMLDTHKMG QIPYNLKIFANMLDVHKMLV QIPYNLKIRANMLDVDKKLV NIDRIYDKNLLMIKEHILAI NNPRIYDKNHLKIKMHILAI NIQRIYNKNHLMIKMHILAI GNNIRIYDKNLLMIKEHIL NNDRIYDMNHLMIKMHILAI DNILVKMFKTNENNDKSELI DNIHVKMRKVIMNNDKSELI DNIHVKMFKTPMNNDKSELI DNIHVKMFKVIENNDKSELI MTDVNRYRYSNNYEAIPHIS MTDVNRYRYSNNYERPHIS VIRYRYSNDYDANDHIS MTDVIRYRYSNNYESSDK MTDVIRYRYSNIYEASDK VIRYRYSNNYEANDHIS MTDVIRFRYSNNYMSNPH MTDVIRYRYSNNYEAESHIS MTDVIRYRYSNNYEASDHIS

PI

II10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 3 (320) 0 0 1 (5120) 0 1 (320) 0 0 0

II15 1 1 1 1

1 3 4 2 1

0 0 0 0 0 3 (1280) 0 0 0

0 0 0 2 (320) 0 0 0 1 (160) 2 (320) 2 (640)

2 (5120)

2 3 1 6 2 3 4 1

3 1 1 3 1 1

4 1

0 (320) (320) (320) (2560) 0 0 (640) (320) (640) (5120) 0 (640) 0 (320) (320) (320) 0 (320) (320) (320) (320) (160) 0 0 (320) (320) (2560) 0 (640) (640) (2560) 0 0 0 0 0 0 0 (5120) (1280)

1

1

2 3 1 1

III15

Prot

Ref

0 0 0 0 (5120) 0 0 0 0 0 ND 0 ND 0 0 0 ND 0 0 0 0 ND ND 0 (320) 0 ND ND 0 0 ND ND 0 0 0 0 (640) (640) (320) (320) ND

0/5 0/5 0/6 0/7 2/4 0/5 0/5 0/5 0/4 0/7 2/10 0/5 1/9 0/5 0/8 0/5 1/9 0/5 0/4 0/3 0/6 0/7 1/5 0/5 0/4 0/7 0/7 1/9 0/5 0/6 0/8 2/9 0/5 0/4 0/8 0/10 0/10 0/5 0/7 0/2 1/6

[26]

[17]

[33] [1]

[22]

[9]

[21]

[2]

520 / Chapter 74 TABLE 1. (Continued) Sequence Protein

Peptide

MSP-2

4044 13458 14502 14504 9942 10010 24180 24112 4325 20032 14522 17930 20034 4313 13766 14022 22914 10022 4337 14048 9200 14044 6800 11880 11882 24230 5501 23754 13466 13980 13984 24148 2150 24296 23394 25854 25852 24922 1818 23390 24166

AMA-1

AMA-1

AMA-1

HRP-2

MSP-1

ABRA

EBA-175

P1 P4 P6 P9

KNESKYSNTFINNAYNMSIR KLMSKYSNTFEVNAYNMSIR KIMSKWGNTFNINAYNMSNF KIMSKWVNTFNINAYIMSNF KNESKYSNTFQMNAYNMSIR KNESKYSNTFQVNAYNMSIR KNMSMYSNAFDINAYNMANRR SKYSNTFNINAYNMVIRRSM MIKSAFLPTGAFKADRYKSH MIKAAFLPTGAFKADRYKSH MIKVGFLPTGAFKSPRYKSH KASFDVTGAFKAPRYKS MIKAAFLPTGAFMADRYKSH DAEVAGTQYRLPSGKCPVF DAEVAGTQWFDPSGKSPVFG DAEVAGTQWFTPSGKTPVFG EDAEVAGTQWFTPSGKSGC DAEVAGTQYFHPSGKSPVFG WGEEKRASHTTPVLMEKPYY YSEMKRASLTTPVLKEMPWY WGEGKRASHTTPVLMEKPYY YSEMKRASLTTPVLKEKPYY YNNSAFNNNLCSKNAQGLNLN NNSAFNNNLSSKNAMGLVLN NVSAFNNNLSSKNAMGLVLN SAFDDNLTAANAMGLILNKR MLNISQHQCVKKQCPQNSY MHNISQLQVVKKMVPQK MLNISQMQSVKKQSDQNS MHNISQHQSVMKMSDQNS MHNISQHQSVMMMSDQNS MLNISMLQTVMMMTPQK KMNMLKENVDYIQKNQNLFK KMNMHMENVAWIMKNQNLFK KKMNMHMENVIYIMKNQNLFK KMNMNLEHVPWIMKNQNLFK KMNMHLEHVPWIMKNQNLFK KMNMHLENVPWIMNKQNLFK NNNFNNIPSRYNLYDKKLDL NNIPSRYNLYDKMLDLDDL FNNIPSRYNLYDKMLPLDD

HABPs reported by our group are located when comparing it to recently published work by other groups in which the native 3D structure of important malarial proteins such as EBA-175 (87), AMA-1 (28), and MSP-1 (70) has been reported.

DESIGN BASED ON THE MHC-PEPTIDE-TCR COMPLEX CONFORMATION The primary immunological function of major histocompatibility complex (MHC) molecules is to bind and “present” peptides on the surface of specialized cells for

PI

II10

II15

III15

Prot

Ref

0

0

0

0

0/5

[18]

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 (320) 2 (320)

0 0 0 0 1 (2560)

0 0 1 (320) 1 (320) ND ND 0 1 (320) 1 (640) 2 (320) 1 (160) 0 2 (1280) 1 (320) 1 (320) 1 (5120) 0 2 (320) 1 (320) 1 (320) 0 0 0 ND 0 ND 0 1 (640) 2 (320) ND 0 ND ND ND ND ND 0 9 (320) 2 (640)

0/3 0/3 0/5 0/5 0/9 1/8 0/6 0/8 0/3 0/7 2/8 0 0/5 0/5 0/9 1/5 0/5 0/5 0/5 1/5 0/5 0/5 0/5 1/7 0/5 0/8 0/6 0/4 0/5 3/16 0/5 0/9 0/9 0/8 0/6 2/7 0 0/8 2/15

1 (5120) 0 0 0 0 0

1 2 2 2 3

1 1 1 (320) 1 (320)

1 1 1

0 2 (2560)

1 3

3 (2560)

3 1 3 2 1 1 2 1

0 (160) (640) (320) (320) 0 (640) 0 0 0 0 0 (320) (160) 0 (640) (640) (320) 0 (320) (1280) 0 0 (2560) 0 (1280) (1280) (320) (320) (320) 0 (320) (320)

[21]

[72]

[79]

New

[88]

[80]

[19]

antigen presentation [or Antigen Presenting Cells (APC)] and recognition (binding) by lymphocytes’ antigen-specific T-cell receptors (TCRs). MHC class II molecules bind peptide fragments derived from exogenously internalized and proteolytically processed proteins, including macrophages, dendritic cells, and B cells. The resulting peptide fragments are compartmentalized in the endosome where they are associated with MHC class II molecules before being routed to the cell surface for helper TH lymphocyte recognition. The three-dimensional structure of molecules from MHC class I (10), class II (84), T-lymphocyte receptor

Peptide Vaccines for Malaria / 521 (TCR) [34], and macromolecular complexes formed by these MHC molecules, antigenic peptides, and TCR (the MHC-peptide-TCR) (29, 75) have been determined by different molecular techniques by other groups, thereby constituting a useful tool for complementing our particular work. Recent studies have concluded that MHC class-II molecules present a molecular groove or peptidebinding region (PBR) having four pockets named Pockets 1, 4, 6, and 9 [84] into which a peptide’s aminoacid side-chains fit properly [73]. This fitting is stabilized by 11 or 12 hydrogen bonds established between MHC II molecules’ α and β chain amino-acid side chain atoms and the peptide backbone [25, 30]. Based on the primary structure of the HABPs studied so far at FIDIC and knowing the HLA-DR isolated molecules to which they bind to plus the previously reported specific HLA allele binding motifs [73] we have found that if different modified HABPs are synthesized, all having relevant amino acid changes, these changes would induce 3D structural changes that would subsequently allow them to fit flawlessly into the MHC-II molecule, inherent in a given immune response. The HLADRB1*0401 human molecule crystal structure was used for modeling the docking contact for protectioninducing peptide 24112 (from the MSP-2 group shown in Table 1) (Fig. 2). The modified HABPs so obtained were assayed in Aotus monkeys to determine their ability to induce antibodies recognizing the individual peptides by ELISA, native denatured protein by Western Blot, antiparasite antibodies by immunofluorescence (IFA), as well as their protection-inducing ability against an experimental challenge with the highly infective Aotus-adapted P. falciparum FVO strain.

DESIGN BASED ON STRUCTURALLY MODIFIED ANTIGEN BINDING TO HLADRb1* MOLECULES Since controlling an immune response against malaria is associated with the MHC, particularly class II molecules (human HLA-DR) [58], association was thus sought between these peptides’ immunological characteristics (immunogenic, protection-inducing, or not), their three-dimensional structure and these molecules’ HLA-DRβ1* purified molecules’ binding capacity [94]. It is well known that HLA-DRβ1-genetic-region-encoded molecules have 16 allelic forms of expression denominated HLA-DRβ1*01 to HLA-DRβ1*16, with more than 250 variants. These alleles have been serologically, molecularly, and phylogenetically grouped into five large groups or haplotypes, including several of these alleles. These haplotypes are HLA-DR1 (including alleles HLA-DRβ1*01, 10 and 103), HLA-DR51 (includ-

FIGURE 2. Modified immunogenic and protection-inducing HABP docking into HLA-DRβ1*0401 binding groove. HLADR α chain Connolly surfaces are shown in pink and HLADR β chains in pale blue (upper panel). Van der Waals surface for protection-inducing MSP-2 peptide 24112 (lower panel). Color code for amino acids fitting into these Class-II molecules: fuchsia (Pocket 1), red (P2), pale blue (P3), dark blue (Pocket 4), rose (P5), orange (Pocket 6), gray (P7), yellow (P8), green (Pocket 9). Note the buried amino acids corresponding to residues fitting into pockets P1, P4, P5, P6, P7, and P9, as well as putative TCR P2, P3, and P8 contact residues pointing upward. (See color plate.)

ing HLA-DRβ1*15 and 16), HLA-DR52 (including HLA-DRβ1*03, 11, 12, 13, 14; 1403 and 1404), HLADR8 (exclusively represented by HLA-DRβ1*08), and HLA-DR53 (including alleles HLA-DRβ1*04, 07, and 09) [89]. It should be kept in mind that sequencing studies regarding genes encoding these Aotus class II molecules (HLA-DRβ1*-like) carried out on more than 110 Aotus monkeys (a New World primate highly susceptible to developing human malaria) has shown homology ranging from 100–88% with human HLA-DRβ1*04, 03,

522 / Chapter 74

TCR MHCII

HLA-DRb1*0301

TCR

HLA-DRb1*1101

HLA-DR52

08, 11, 13, 14, 10, 07, and 01 molecules [89] and ≥88% homology with human TCR molecules [54] and other immune system molecules [35], making this primate an excellent model for human vaccine development. Therefore, the results obtained with these peptides in these monkeys could be extrapolated (with minimal modifications) to humans (Fig. 3).

TCR MHCII

HLA-DR1

HLA-DRb1*0101

TCR MHCII

HLA-DRb1*0701

TCR MHCII

HLA-DRb1*0401

HLA-DR53

TCR MHCII

HLA-DRb1*0801

HLA-DR8

MHCII

FIGURE 3. Modified (protection-inducing) HABP backbone structures. The left-hand side shows the front views of backbone structures for different protection-inducing HABPs binding to HLA haplotypes, as determined by proton NMR. The right-hand side shows colorgrams representing the same for both TCR and MHC-II peptide contacting residues. (See color plate.)

It should also be remarked that all data regarding HABP binding ability to a particular HLA-DRβ1* allele is based on in vitro purified HLA-DRβ1 molecule binding activity (measured as the capacity to displace ≥50% of control peptide) by identifying their characteristic allele binding motifs and by recognizing the register for their binding to the corresponding allele.

DESIGN BASED ON MALARIAL ANTIGENS’ STRUCTURAL MODIFICATION BY INTRODUCING PEPTIDE BOND ISOSTERS Approaches used for designing synthetic peptidebased vaccines have included synthesizing cyclic peptides [43, 93] and template-bound peptides [56]. Most efforts aimed at developing synthetic peptide-based malaria vaccines have been focused on using short, linear peptides or multipeptide constructs [64, 76]. As we have demonstrated in previously published works, selected HABPs’ 3D structure has to be modified to render them immunogenic and protection-inducing against malaria as a powerful approach in overcoming this problem [26]. It has been demonstrated that MSP-142–61 (1513 HABP) possesses the 48K-KMV52 high binding motif for specific receptors on RBC [90], but this peptide is poorly immunogenic and nonprotection-inducing against malaria itself [27]. Five 1513 peptide analogs were synthesized. Each contained a ψ[CH2-NH] isoster bond on the K-KMV RBC binding motif. “ψ” nomenclature for pseudopeptides has been proposed by Arno Spatola [85]. Monoclonal antibodies induced by such methylene amine surrogates have also been obtained, some of them possessing neutralizing properties regarding in vitro malaria infection. In a subsequent study, an all-L amino acid made peptide MSP-142–61 (1513 peptide) and a set of pseudopeptide analogs, including one all-D peptide (a peptide made up with only D residues), seven partially substituted D-peptides (as a sequence scan), and reduced amide ψ[CH2-NH] surrogates (a peptide having a methylene group instead of an amide peptide bond carbonyl group), one Retro (a peptide made up with only D residues but having a totally inverted backbone) and one Retro-inverso analog (a peptide made up only with L-amino acid residues but having a totally inverted backbone) were obtained. In vivo tests have demonstrated that most Aotus monkeys immunized with either of the 1513-derived reduced amide pseudopeptides have induced P. falciparum merozoite antibody titers. Remarkably, three out of eight monkeys immunized with the K48-ψ[CH2-NH]-E49 pseudopeptide became fully protected against malaria after experimental challenge; two out of eight animals from the group immu-

Peptide Vaccines for Malaria / 523 Also modified pseudopeptide HABPs derived from MSP-1282–1301 (all-L 1585 peptide) have demonstrated that specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with ψ-[CH2-NH] isoster bonds, making this molecule become a highly specific HLAβ1*1101 allele binder associated with partial protection against malaria in experimentally performed challenge [47, 49] (Fig. 4B–F). As can be deduced from this approach aimed at ascertaining malaria vaccine subunit candidates, a single reduced amide isoster bond, as well as peptide α-carbon chiral transformation, are efficient antigen structure-modulating elements of a given entire peptide conformation, mimicking parasite ligand’s transient-states due to internal backbone mobility.

nized with the K50-ψ[CH2-NH]-M51 delayed parasitaemia when compared to monkeys immunized with nonmodified 1513 HABP and the control group. These findings have revealed that receptors on RBC for the MSP-142–61 epitope are fairly selective for Lnatural conformations on amino acid ligands which can be mimicked by specific ψ-[CH2–NH] modifications; so inducing modulated molecular ψ[CH2–NH] surrogate antibodies can differentiate topochemistry but not chirality. The rigidity and right-handed properties of the all-L 1513 nonmodified molecule will potentially prevent the molecule inducing a protective immune response against malaria (Fig. 4A). Concomitantly, pseudopeptides derived from the MSP-138–58 have proved to be efficient human T-cell clone stimulators [8].

A

C

All-L 1513 Ψ- 437 (V52-L53) Ψ- 439 (M51-V52)

All-L-1585

B

All-L 1585 Ψ-9473 (K6-P7) Ψ-9475 (P7-L8)

D 25799 (D-K6)

26.16 Å

15.35 Å

E

F

9475 (LP7-ψ[CH2-NH]-LL8)

25807 (D-L8) 19.84 Å

26.09 Å

FIGURE 4. Conformational effects induced by D-substitutions and ψ[CH2-NH] isoster bonds on MSP-1 derived pseudopeptide 3D structure (49) A. Ribbon representation of overlapped consensus 3D structures for MSP-142–61 (1513 HABP) and two of its most representative reduced amide pseudopeptides. B. Overlapped consensus 3D structures for MSP-11282–1301 (1585 HABP) and two reduced amide pseudopeptides. The most representative structure for nonmodified all-L 1585 peptide in C and its D-partial substituted analogues and a reduced amide analogue are analyzed, respecting their 3D structure in D, E, and F. Molecular distance between Y4 and Y12 is shown in Å.G. (See color plate.)

524 / Chapter 74 Taken together, these results support the notion of including nonnatural peptide analogs in the next generation of synthetic peptide-based, multiepitope, antimalarial vaccines.

Colombian entity responsible for the advance of science and technology) and is being wholly performed by FIDIC’s functional groups.

References CONCLUSION The results summarized in this review show that chemically synthesized, subunit-based, multicomponent, multistage, malaria vaccines are feasible. A deep knowledge of immunogenetics, immunogenic protection-inducing modified peptides’ three-dimensional structure, and class II-peptide-TCR complex formation are clearly needed for their development. Some of the subunits for such vaccines and some of the rules dictating their immunogenicity and protection-inducing activity are described in those papers referenced at the end of this chapter, as they display some of the principles governing the development of any chemically synthesized, multicomponent, multistage, subunit-based vaccine. As a result of our findings, and many other works, it can also be suggested that immunization employing large recombinant molecules [12, 86], DNA-encoded ones [55], or synthetic [59] nonmodified molecules does not induce or improve protection-inducing immune response but could rather have a negative or adverse effect. Also, pseudopeptides are becoming a viable alternative tool for vaccine design and use with MHC, but a great deal of work still remains to be done [50]. However, predicting any particular peptide’s affinity for an MHC molecule is currently beyond our capacities. In this review we have presented some evidence that peptide side chains, as well as peptide backbone, make a critical contribution toward TCR recognition and T-cell stimulation as a consequence of appropriate antigen presentation within the context of MHC-II molecules. Pseudopeptide chemistry, just like our first mentioned approach, offers the possibility of mimicking certain peptide conformations or antigenic protein transient stages by inducing both local and global structural constraints. These molecules thus represent structural probes having broad molecular interaction possibilities allowing them increasing biological half-time, protease blocking effects, modulated human HLA class II molecule binding activity, and efficient antigen human HLADR and -DP restricted T-cell clone stimulation.

Acknowledgments The research described in this review is being supported by the president of Colombia’s office and the Ministry of Social Protection and Colciencias (the

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75 Peptide Vaccine for Otitis Media LAUREN O. BAKALETZ

complication of OM [6, 22] with behavioral, educational, and language development delays being additional consequences of early onset OM with effusion or OME [23, 46]. The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the United States alone [1, 11, 25, 45].

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important causative agent of bacterial otitis media (OM). Of interest for vaccine development are several conserved or semiconserved NTHI surface proteins. We’ve focused our efforts on a structure expressed by 100% of clinical NTHI isolates recovered from children with chronic OM [5] that is composed of a 36.4 kDa protein subunit, which is highly homologous to outer membrane protein (OMP) P5 of H. influenzae type b [31, 32]. This “P5-homologous adhesin” (also called OMP P5 or P5-fimbrin) mediates bacterial binding to epithelial cells [24, 44] and mucin [30, 40, 41]. Due to its surface location and important biological functions, we targeted this adhesin for vaccine development, generating a highly efficacious, chimeric peptide that derives from a specific predicted surface-exposed epitope of interest [4, 20, 21, 26, 28, 34–36, 39].

BACKGROUND OF AVAILABLE THERAPIES To date, antibiotic use, both therapeutically and prophylactically, has been largely relied upon for medical management of the spectrum of clinical entities known collectively as OM [45]. Widespread use of antimicrobials for OM has met with controversy, however [45, 50], and the emergence of multiple-antibiotic resistant microorganisms is a sobering consequence of this wellestablished practice [13, 19, 29, 33]. Surgical management of OM involves the insertion of tympanostomy tubes through the tympanic membrane (or eardrum) while a child is anesthetized. While this procedure is commonplace [10] and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and the incumbent risks of putting a child under general anesthesia [8, 10, 37, 45]. Clearly, there is a tremendous need to develop more effective and accepted approaches to the management and, preferably, the prevention of OM. Vaccine development holds the greatest promise and would be the most cost-effective method to accomplish this goal [15, 18]. However, progress in terms of vaccine development for NTHI, the Gram-negative pathogen that predominates both in chronic OM with effusion [7], as well as being a significant etiologic agent of acute OM [27], is hampered by our incomplete understanding of the

DISEASE TARGET Otitis media, both acute and chronic, are highly prevalent pediatric diseases worldwide. The most recently available statistics indicate that 24.5 million physician office visits were made for OM in the United States alone in 1990, representing a greater than 200% increase over those reported in the 1980s [9, 37]. OM is the most frequently diagnosed illness in children less than 15 years of age and is the primary cause for emergency room visits [12]. It is estimated that 83% of all children will experience at least one episode of acute OM (AOM) by age three and that greater than 40% of children will experience three or more episodes of AOM by this age [47]. While only very rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is the most common Handbook of Biologically Active Peptides

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528 / Chapter 75 pathogenesis and immunobiology of OM, a polymicrobial disease caused by one or more of three predominant bacterial pathogens, whose ability to invade the tympanum is facilitated by virtually any upper-respiratory tract (URT) virus [2]. In addition, due to the fact that NTHI are an extremely heterogeneous group of microorganisms, identification of appropriate and conserved targets for vaccine development has proved to be quite challenging. Because adherence and colonization are of primary importance in the disease course of OM, we’ve focused our vaccine development efforts to date on a candidate derived from one of several known NTHI adhesins [17].

WHY PEPTIDES? In 1988, we made the observation that 100% of the clinical NTHI isolates tested that were recovered from children with chronic OM expressed the filamentous OMP P5-homologous adhesin [5]. To begin to characterize the biological function(s) of this structure, we first isolated, cloned, and sequenced the gene that encodes the adhesin subunit protein in NTHI strain 1128. The nucleotide sequence of the P5-fimbrin gene contained an ORF of 1077 bp, which would encode a mature protein of 338 amino acids with a calculated molecular mass of 36.4 kDa. To directly test whether these structures were involved in pathogenesis, the gene was disrupted, and the biological consequences of disruption were determined. The P5-fimbrin mutant neither expressed the filamentous appendage nor was labelled with specific antisera directed against isolated fimbrial protein. Importantly, the P5-fimbrin mutant showed reduced adherence to human oropharyngeal cells in vitro, was cleared more rapidly from the middle ears of animals post-transbullar inoculation and was significantly less able to induce OM post-intranasal inoculation in a chinchilla model when compared with the fimbriated parent strain [44]. After showing that the P5-fimbrin was indeed an adhesin and a virulence factor for OM in chinchilla models, we attempted to use isolated native P5-fimbrin as an immunogen. Whereas this immunization strategy was effective against challenge with the homologous isolate [3, 4, 44], there was no (or limited) protection conferred against heterologous challenge, as had been

shown with many other NTHI OMPs when used in their entirety as an immunogen. To overcome this obstacle, we subjected the translated AA sequence of P5-fimbrin to multiple algorithmic analyses and determined that there were two predominant putative B-cell epitopes of interest that resided in two of four predicted surfaceexposed regions of P5-fimbrin (regions 3 and 4) [16, 49]. Thereby, we synthesized two 40-mer peptides, called LB1 and LB2, that were composed of these B cell epitopes colinearly synthesized with a “promiscuous” T-cell epitope derived from the fusion protein of measles virus (MVF) [4] (Fig. 1). Sera obtained from immunized rabbits and chinchillas demonstrated significant reciprocal titers against both the homologous peptide and isolated P5-fimbrin. These antisera also immunolabeled native fimbriae expressed by whole unfixed NTHI. Immunization of chinchillas with either isolated P5fimbrin or LB1 (but not LB2) also resulted in elimination of NTHI from the chinchilla nasopharynx two to three weeks earlier, respectively, than was observed in sham-immunized controls. Whereas these were encouraging results, we were concerned that due to the known heterogeneity of NTHI, there may be significant diversity within surfaceexposed region 3, from which the peptide immunogen LB1 had been derived, thus potentially compromising the breadth of coverage this immunogen might provide. To determine the relative conservation of AA sequence within this region, 99 clinical NTHI isolates were subjected to PCR amplification and nucleotide sequencing of the 19-mer putative B-cell epitope of mature P5fimbrin contained in LB1 [this region was called LB1(f)] and grouped according to analysis of the deduced AA sequence (Table 1) [3]. After alignment of sequences (each 13 to 22 AAs long; at approximate positions 110 to 140), NTHI strains from both the United States and Europe segregated into three major groups, using the criterion that assignment to a particular group required at least 75% identity to the consensus sequence of that group. Group 2 was further divided into subgroups 2a and 2b based on limited but consistent sequence differences. Approximately 75% of these 99 isolates belonged to group 1, 16% belonged to group 2, and 8% belonged to group 3 [3]. Thus, whereby based on these data, LB1 could be predicted to induce the formation of antibodies that would potentially cross-react with ∼75% of circulating NTHI isolates (since the AA sequence of the B

117

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LB1 H2N-Arg-Ser-Asp-Tyr-Lys-Phe-Tyr-Glu-Asp-Ala-Asn-Gly-Thr-Arg-Asp-His-Lys-Lys-Gly-ProSer-Leu-Lys-Leu-Leu-Ser-Leu-lle-Lys-Gly-Val-lle-Val-His-Arg-Leu-Glu-Gly-Val-Glu-COOH 163

180

LB2 H2N-Tyr-Gln-Trp-Leu-Thr-Arg-Val-Gly-Lys-Tyr-Arg-Pro-Gln-Asp-Lys-Pro-Asn-Thr-Gly-ProSer-Leu-Lys-Leu-Leu-Ser-Leu-lle-Lys-Gly-Val-lle-Val-His-Arg-Leu-Glu-Gly-Val-Glu-COOH

FIGURE 1.

Synthetic chimeric peptide immunogens LB1 and LB2.

Peptide Vaccine for Otitis Media / 529 TABLE 1. NTHI strain 1128 1715 183 1729

Aligned NTHI group 1, 2, and 3 LB1(f) peptide sequences.

Group

Consensus sequence

No. of amino acids

No. of U.S. strains (% of total)a

No. of European strains (% of total)a

1 2a 2b 3

RSDYKFYEDANGTRDHKKG---RSDYKLYNKNSSSNSTLKNLGE RSDYKLYNKNSS------TLKDLGE RSDYKFYDN------------KRID------

19 22 19 13

53 (76) 7 (10) 3 (4) 7 (10)

22 (76) 4 (14) 2 (7) 1 (3)

a

Of 99 U.S. and European strains tested. Reprinted from Infection and Immunity [3] with permission of the publisher.

b

TABLE 2. Amino acid sequences of peptides representing the four predicted surfaceexposed regions of P5-fimbrin. Peptide Region Region Region Region

Amino acid sequence 1 2 3a 4

SFHDGINNNGAIKKGLSSSNYGYRRNTF GRAKLREAGKPKAKHTNHG LVRSDYKFYEDANGTRDHKKGRHT TRVGKYRPQDKPNTAINYNPWIG

a

Identical in sequence to LB1(1) peptide noted in text and Table 3. Reprinted from Infection and Immunity [36] with permission of the publisher.

b

A B C

D E F G H I J

359 359

1 22 38 65 region 1

136 136

93 111 region 2

136 159 188 210 region region 3 4

147 142

148

136 136 136

159 153

153

159 162 160

FIGURE 2. Schematic diagram of synthetic peptides. A. P5-fimbrin with leader peptide. B. 8- to 10-mer peptides representing the mature protein. C. Predicted surface-exposed region peptides. D. Region 3 peptide LB1(1). E to G. Partial sequence peptides LB1ps1 to 3. H to J. Group peptides LB1(2a), LB1(2b), and LB1(3), respectively. (Reprinted from Infection and Immunity [36] with permission of the publisher.)

cell epitope in LB1 was indeed derived from a gp. 1 isolate), we were interested in determining if perhaps LB1 might also protect against NTHI strains belonging to group 2 or 3 as well. In addition to identifying protective, and perhaps shared, epitopes within P5-fimbrin, particularly within surface-exposed region 3, in a parallel effort, we also wanted to identify the adhesin-binding domain(s) of this bacterial protein. Toward accomplishing both of these goals, we synthesized three sets of peptides based on the deduced AA sequence of P5-fimbrin of NTHI strain 1128 [44] and used these to map epitopes of this adhesin [36]. These peptides are depicted schematically in Fig. 2. The first series of 34 peptides represented

mature P5-fimbrin sequentially in 10-residue segments, except the C-terminal peptide 34, which was 8 residues long. The second series of four “region” peptides represented each of the predicted major surface-exposed regions of this adhesin [16, 49] (Fig. 2 and Table 2). A final series of seven peptides was designed to specifically map the third predicted surface-exposed region from which LB1 had been derived (Fig. 2 and Table 3). Within this region [called LB1(f)], three major groups and one subgroup had been identified as described above, and thus a peptide representing each was created. These are designated the “group” peptides: LB1(1), LB1(2a), LB1(2b), and LB1(3). In addition, a set of three short, overlapping “partial sequence” peptides

530 / Chapter 75 p.01209 TABLE 3.

Amino acid sequences of peptides designed to map LB1(f).

Peptide

Amino acid sequencea

LB1 LB1(1)b LB1ps1 LB1ps2 LB1ps3 LB1(2a) LB1(2b) LB1(3)

RSKYKFYEDANGTRDHKKG LVRSDYKFYEDANDTRDHKKGRHT LVRSDYKFYEDA KFYEDANGTRDH NGTRDHKKGRHT LVRSDYKLYNKNSSSNSTLKNLGEHHR LVRSDYKLYNKNSS—NTLKDLGEHHR LVRSDYKFYDN-------------KRID-------SHR

a

Dashes were inserted to align sequences. Identical in sequence to region 3 peptide noted in text and in Table 2. c Reprinted from Infection and Immunity [36] with permission of the publisher.

Percent inhibition of adherence

b

30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0

**

1728MEE Group 3 1885MEE Group 2a

*

86-028NP Group 1

Region 1

Region 2

Region 3

(LB1ps1, LB1ps2, and LB1ps3) were synthesized to more precisely map the consensus sequence of group 1 NTHI strains. We then assayed the ability of the 10-mer peptides to inhibit adherence of three NTHI strains representing groups 1, 2a, and 3 to human OP cells and showed a group-specific inhibitory effect (data not shown). Substantial inhibition of adherence against the group 1 isolate (strain #86-028L) was shown by peptides representing the N-terminal portion of this adhesin (peptides 1 to 19). These peptides were based on the sequence from a different group 1 NTHI strain. Less activity was shown against the group 2a strain (#1885MEE), and minimal activity was demonstrated against the group 3 strain (#1728MEE). For the group 1 strain 86-028L, of the four “region” peptides, those that spanned region 3 (peptides 12 to 15) showed the greatest relative ability to inhibit adherence of this isolate to human OP cells, suggesting the presence of an adhesin-binding domain in predicted surfaceexposed region 3 of P5-fimbrin. Longer peptides representing each of the four predicted surface exposed regions of P5-fimbrin confirmed

Region 4

FIGURE 3. Percent inhibition of adherence of NTHI strains to human OP cells by synthetic peptides representing the four predicted surface exposed regions of P5-fimbrin. *, significant difference from strain 1728MEE; **, significant difference from strains 1728MEE and 1885MEE (P ≤ 0.05). (Reprinted from Infection and Immunity [36] with permission of the publisher.)

the group-specific result obtained with the 10-mer peptides (Fig. 3). Peptides representing regions 1 and 2 did not inhibit adherence above a mean value of 10% for either the group 1, 2a, or 3 NTHI isolate. The region 3 peptide was significantly more inhibitory to adherence of a group 1 isolate (P ≤ 0.05) than to the group 3 isolate, with greatest interassay variability shown against the group 2a strain. These results further indicated the presence of a second potential adhesin-binding domain within region 4 that had not been detected with the 10-mer peptides. As had been shown with the region 3 peptide, the region 4 peptide significantly inhibited the adherence of a group 1, but not a group 2a or 3, isolate to human OP cells (P ≤ 0.05). Collectively, our data suggested the presence of a group-specific adhesinbinding domain within surface-exposed regions 3 and 4. Thereby, antibodies directed against these domains would be predicted to inhibit bacterial binding, or colonization of a mucosal surface, which we later showed was indeed the case. We have also conducted assays to determine potential immunodominant and/or protective domains among the four predicted surface-exposed regions of

Peptide Vaccine for Otitis Media / 531 P5-fimbrin. To briefly summarize our findings to date, antibodies directed against LB1 reacted most strongly with peptides derived from surface-exposed region 3 when assayed by biosensor and not to regions 1, 2, or 4, as could be expected [36]. Moreover, these antibodies were most reactive with a synthetic peptide derived from a group 1 NTHI isolate with regard to sequence diversity within surface-exposed region 3, also as could be expected. However, antibodies directed against LB1 also demonstrated reactivity against peptides derived from region 3 of NTHI strains belonging to minority groups 2b and 3, with minimal reactivity to a peptide derived from minority group 2a. These results suggested that immunization with LB1 might also confer partial protection against challenge by heterologous isolates of NTHI.

OUR CONTRIBUTIONS TO THE AREA As mentioned briefly earlier, active immunization with either isolated native P5-fimbrin or LB1 induces significant protection in chinchilla models against homologous challenge, but to assay for potentially greater breadth of protective efficacy, we conducted a passive protection study in which antiserum directed against the peptide immunogen LB1 was now assayed for ability to confer protection against heterologous challenge using NTHI strains representing majority group 1 but also minority groups 2 and 3 isolates as well [26]. We found that passive immunization of chinchillas

with serum specific for LB1 prior to challenge with heterologous NTHI isolates significantly inhibited the signs and incidence of OM (P ≤ 0.01) induced by all three challenge isolates (Fig. 4). These results suggested that the approximately nine-residue N-terminal portion of the 19-mer sequence that is shared among all three NTHI groups (Table 1) and is incorporated into LB1 [RSDYFK(L)YE(N,D)D(K,N) is potentially an immunodominant region of this epitope or that it perhaps comprises a key protective epitope [26]. In addition to noting the ability of anti-LB1 antiserum to provide significant cross-protection relevant to induction of culture-positive OM, in this study we also observed that the ability of this antiserum pool to induce total eradication of NTHI from the nasopharynx was not equivalent among challenged cohorts. Importantly, these results thus suggested that while early, complete eradication of NTHI from the nasopharynx (as seen in the homologously challenged cohort) was highly protective against OM, reduction of the bacterial load to below a critical threshold level (as seen in the heterologously challenged cohorts) appeared to be similarly effective. This observation has very important clinical implications, in that induction of sterilizing immunity did not appear to be necessary for protection against ascending OM, and, instead, by immunizing in a manner that simply reduced the load of bacteria present in the NP, one could have a similar protective effect against disease. Given the serotype conversion issues that are currently confounding the efficacy of multivalent pneumococcal capsular-conjugate-based vaccines [42, 48],

FIGURE 4. Cross-strain protection against ascending OM afforded by passive transfer of antiserum directed against the peptide immunogen LB1. (Reprinted from The Journal of Immunology [34] with permission of the publisher. Copyright 2003. The American Association of Immunologists, Inc.) (See color plate.)

532 / Chapter 75 this was a valuable piece of information to have and keep in mind as we moved forward with our vaccine development efforts. Having now shown that LB1 could induce the formation of antibodies that were protective against both homologous and heterologous challenge in chinchilla models after parenteral immunization, variants of this vaccine candidate were then blindly evaluated in a rat model of mucosal immunization [28]. For this study, the 18-mer B-cell epitope contained within LB1 was conjugated to either keyhole limpet hemocyanin or bovine serum albumin and delivered to rats via direct immunization of the Peyer’s patches followed 14 days later by an intratracheal boost. All immunogens tested induced a high-titered and specific response in the rat. Clearance of NTHI, measured four hours after challenge, occurred significantly earlier from the lungs of rats immunized with a combination of three LB1 peptides (representing each of the groups, see Table 1) than from sham-immunized controls. Clearance of NTHI from the middle ears of rats challenged intrabullarly also occurred significantly earlier for those rats administered the combination of three LB1-derived peptides. The efficacy shown for LB1 in a second, independent rodent model lent strong support to the merits of its continued development and evaluation as a component of a vaccine directed against NTHI-induced OM. Given the significant protective efficacy of LB1, delivered parenterally in chinchilla models and mucosally in rat models of OM, we now wanted to examine the response induced when LB1 was delivered intranasally to chinchillas. We also wanted to determine the optimal prime-boost regimen for this immunogen when admixed with the adjuvant monophosphoryl lipid A (MPL). Thereby, we immunized cohorts of chinchillas with either 30 μg of LB1 plus MPL, 10 μg LB1 plus MPL, or MPL alone. Formulations were administered as either three intranasal (IN) doses via micro-aerosolized spray, or as one subcutaneous (SQ) dose followed by two IN doses, all at biweekly intervals. Chinchillas were then challenged IN with first adenovirus, then NTHI to better mimic the polymicrobial nature of natural OM where children typically develop OM approximately 1 week after having a viral URT infection [20, 21, 35]. Clearance of NTHI from the NP occurred most rapidly in the cohort immunized mucosally with 10 μg LB1/dose. In the directly challenged middle ear, significantly earlier clearance of NTHI (p < 0.01) occurred in cohorts immunized with either 10 μg LB1 delivered IN/ IN/IN or with 30 μg LB1 delivered SQ/IN/IN. Immunization via either regimen induced LB1-specific serum Igs in all cohorts except those that received MPL only. We also detected immunogen-specific immunoglobulins in effusions recovered from the middle ears of chinchillas after NTHI challenge, with greatest levels

occurring in those that cleared bacteria most rapidly. Whereas total serum Ig was greatest in cohorts receiving 30 μg LB1/dose, regardless of the immunization regime, based upon induced augmented clearance observed among cohorts, LB1-specific immunoglobulins available locally appeared to have also played a key role in mediating bacterial clearance from both the NP and middle ears. Collectively, our results have shown that a protective immune response could be elicited in two unique animal models following delivery of LB1, or one of its derivatives, via either an exclusively mucosal regime, an exclusively parenteral strategy, or one of parenteral priming followed by mucosal boosting. Whether or not the efficacy shown for LB1, or its derivatives, in animal models will translate into similar protection in children awaits clinical evaluation.

FUTURE OUTLOOK Despite their limitations, peptide-based vaccines have the potential to direct the immune response against prespecified immunodominant or protective epitopes of native proteins [14]. Recent reports from several laboratories, including ours, have shown that the inclusion of both B- and T-cell epitopes in synthetic peptide immunogens can induce antibodies of higher affinity for the incorporated B-cell epitopes [38, 43]. The orientation, number, and secondary character of peptide sequences included in these immunogens have been found to influence antigen processing and presentation to T cells, thus affecting the specificity and affinity of the antibodies produced for native proteins [14, 38, 43]. Using this epitope-targeted, peptide-based vaccine strategy to develop the immunogen LB1 [24, 30, 40, 41, 44], we have shown in multiple preclinical trials that this strategy is highly successful.

References [1] Alsarraf R, Jung CJ, Perkins J, Crowley C, Alsarraf NW, Gates GA. Measuring the indirect and direct costs of acute otitis media. Arch Otolaryngol Head Neck Surg 1999;125:12–8. [2] Bakaletz LO. 14. Otitis Media, In Brogden KA, Guthmiller JM, editors. Polymicrobial Diseases. ASM Press, Washington, D.C. 2002;259–95. [3] Bakaletz LO, Kennedy BJ, Novotny LA, Duquesne G, Cohen J, Lobet Y. Protection against development of otitis media induced by nontypeable Haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection. Infect Immun 1999;67:2746–62. [4] Bakaletz LO, Leake ER, Billy JM, Kaumaya PT. Relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeable Haemophilus influenzae in the chinchilla. Vaccine 1997;15:955–61.

Peptide Vaccine for Otitis Media / 533 [5] Bakaletz LO, Tallan BM, Hoepf T, DeMaria TF, Birck HG, Lim DJ. Frequency of fimbriation of nontypable Haemophilus influenzae and its ability to adhere to chinchilla and human respiratory epithelium. Infect Immun 1988;56:331–5. [6] Baldwin RL. Effects of otitis media on child development. Am J Otol 1993;14:601–4. [7] Barenkamp SJ, Kurono Y, Ogra PL, Leiberman A, Bakaletz LO, Murphy TF, Chonmaitree T, Patel JA, Heikkinen T, Sih TM, Hurst DS, St Geme JW, 3rd, Kawauchi H, Stenfors LE. Recent advances in otitis media. 5. Microbiology and immunology. Ann Otol Rhinol Laryngol Suppl 2005;194:60–85. [8] Berman S, Roark R, Luckey D. Theoretical cost effectiveness of management options for children with persisting middle ear effusions. Pediatrics 1994;93:353–63. [9] Bluestone CD. Pathogenesis of otitis media: role of Eustachian tube. Pediatr Infect Dis J 1996;15:281–91. [10] Bright RA, Moore RM, Jr., Jeng LL, Sharkness CM, Hamburger SE, Hamilton PM. The prevalence of tympanostomy tubes in children in the United States, 1988. Am J Public Health 1993;83: 1026–8. [11] Cassell GH. New and Reemerging Infectious Diseases: A Global Crisis and Immediate Threat to the Nation’s Health: The Role of Research. American Society for Microbiology, Washington, D.C. 1997;1–11. [12] Cassell GH, Archer GL, Beam TR, Gilchrist MJ, Goldmann D, Hooper DC, Jones RN, Kleven SH, Lederberg J, Levy SB, Lein DH, Moellering RC, O’Brien TF, Osburn B, Osterholm M, Shlaes DM, Terry M, Tolin SA, Tomasz A. Report of the ASM Task Force on Antibiotic Resistance. American Society for Microbiology, Washington, D.C. 1994. [13] Cohen R, Bingen E, Varon E, de La Rocque F, Brahimi N, Levy C, Boucherat M, Langue J, Geslin P. Change in nasopharyngeal carriage of Streptococcus pneumoniae resulting from antibiotic therapy for acute otitis media in children. Pediatr Infect Dis J 1997;16:555–60. [14] Craig L, Sanschagrin PC, Rozek A, Lackie S, Kuhn LA, Scott JK. The role of structure in antibody cross-reactivity between peptides and folded proteins. J Mol Biol 1998;281:183–201. [15] Cripps AW, Otczyk DC, Kyd JM. Bacterial otitis media: a vaccine preventable disease? Vaccine 2005;23:2304–10. [16] Duim B, Bowler LD, Eijk PP, Jansen HM, Dankert J, van Alphen L. Molecular variation in the major outer membrane protein P5 gene of nonencapsulated Haemophilus influenzae during chronic infections. Infect Immun 1997;65:1351–6. [17] Foxwell AR, Kyd JM, Cripps AW. Nontypeable Haemophilus influenzae: pathogenesis and prevention. Microbiol Mol Biol Rev 1998;62:294–308. [18] Giebink GS, Kurono Y, Bakaletz LO, Kyd JM, Barenkamp SJ, Murphy TF, Green B, Ogra PL, Gu XX, Patel JA, Heikkinen T, Pelton SI, Hotomi M, Karma P. Recent advances in otitis media. 6. Vaccine. Ann Otol Rhinol Laryngol Suppl 2005;194:86– 103. [19] Green M, Wald ER. Emerging resistance to antibiotics: impact on respiratory infections in the outpatient setting. Ann Allergy Asthma Immunol 1996;77:167–73. [20] Hill SR, Novotny LA, Bakaletz LO. Characterization of the immune response induced by a parenteral prime-mucosal boost immunization strategy vs. a mucosal priming and boosting regimen using the vaccine candidate LB1 against nontypeable Haemophilus influenzae-induced otitis media. Abst. 5th Extraordinary International Symposium on Recent Advances in Otitis Media 2005;148. [21] Hill SR, Novotny LA, Bakaletz LO. Characterization of the protective response induced by a mucosal immunization strategy vs. one of parenteral priming and mucosal boosting using an adhesin-based vaccine candidate against otitis media caused by

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534 / Chapter 75 [39] Poolman JT, Bakaletz L, Cripps A, Denoel PA, Forsgren A, Kyd J, Lobet Y. Developing a nontypeable Haemophilus influenzae (NTHi) vaccine. Vaccine 2000;19 Suppl 1:S108–15. [40] Reddy MS, Bernstein JM, Murphy TF, Faden HS. Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyngeal mucin. Infect Immun 1996;64:1477–9. [41] Reddy MS, Murphy TF, Faden HS, Bernstein JM. Middle ear mucin glycoprotein: purification and interaction with nontypable Haemophilus influenzae and Moraxella catarrhalis. Otolaryngol Head Neck Surg 1997;116:175–80. [42] Reinert RR. Pneumococcal conjugate vaccines—a European perspective. Int J Med Microbiol 2004;294:277–94. [43] Shaw DM, Stanley CM, Partidos CD, Steward MW. Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization. Mol Immunol 1993;30:961–8. [44] Sirakova T, Kolattukudy PE, Murwin D, Billy J, Leake E, Lim D, DeMaria T, Bakaletz L. Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A. Infect Immun 1994;62:2002–20. [45] Stool SE, Berg AO, Berman S, Carney CJ, Cooley JR, Culpepper L, Eavey RD, Feagans LV, Finitzo T, Friedman EM, Goertz JA, Goldstein AJ, Grundfast KM, Long DG, Macconi LL, Melton L, Roberts JE, Sherrod JL, Sisk JE. Otitis Media with Effusion in

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76 Peptide Vaccine for Alzheimer’s Disease BEKA SOLOMON

Various theories have suggested that besides amyloid plaques, circulating amyloid, vascular amyloid and, more recently, soluble oligomeric species of amyloid may be responsible for the cognitive impairment and neural degeneration in AD [14, 16, 31, 32]. The most studied hypothesis, amyloid cascade [16], states that overproduction of AβP, or failure to clear this peptide, leads to AD primarily through amyloid deposition which is presumed to be involved in neurofibrillary tangles formation [2, 15]. These lesions are then associated with cell death, which is reflected in memory impairment, the hallmark of this dementia [14, 15, 10].

ABSTRACT The pathology of Alzheimer’s disease (AD) shows a significant correlation between β-amyloid peptide (AβP) deposition and severity of dementia. We have shown that antibodies toward the N-terminal region of β-amyloid peptide suppress in vitro and in vivo formation of toxic β-amyloid (Aβ). The epitope of these antiaggregating antibodies displayed on phage proved to be a potent antigen that induces antibodies against the whole AβP. In Alzheimer’s diseased transgenic mice, these antibodies are delivered from the periphery to the CNS, preventing β-amyloid formation and/or dissolving such aggregates. Performance of such antigens offers possibilities for development of an efficient, longlasting immunization for AD treatment.

IN VITRO MODULATION OF b-AMYLOID FORMATION Many investigators have studied the propensity of beta-amyloid peptide or its fragments to assemble into insoluble aggregates [19, 24]. Beta-amyloid peptide can exist in two alternative conformations, depending on the secondary structure adopted by the N-terminal domain [17, 38] under various environmental conditions [1]. The N-terminal domain contains sequences that permit the existence of a dynamic equilibrium between the alpha-helix and the beta-strand conformations [38] (Fig. 1). The perturbations of the equilibrium of various conformational states of the betaamyloid peptide can be caused by local pH changes, alterations of environmental hydrophobicity, or binding of other proteins [20, 38]. The involvement of the N-terminal region in conformational transformations of beta-amyloid peptide was confirmed by studies using synthetic peptides bearing the beta-amyloid peptide sequence in various solvents and in studies with synthetic beta-amyloid peptides containing single amino acid substitutions [19]. The polar

INTRODUCTION The pathology of AD is characterized primarily by extracellular senile plaques and intracellular neurofibrillary tangles [31, 32]. The presence of amyloid in senile plaques and in cerebral blood vessels (amyloid angiopathy) has long been pathologically recognized. In 1984, Glenner and Wong were the first to sequence the deposits, showing them to be β-pleated sheets of amyloid (Aβ) [11]. Soluble amyloid beta-peptide is a normal metabolite of ∼4-kDa that is produced by processing a large transmembrane glycoprotein called amyloid beta-protein precursor (APP) [13, 33]. The pathological conditions and mechanisms that transform soluble beta-amyloid peptide into the fibrillary, toxic, beta-sheet form found in the plaques and vessels of AD patients is not yet completely understood, but it is clear that the same amino acid sequence of betaamyloid peptide can have both a fibrillar and soluble conformation. Handbook of Biologically Active Peptides

535

Copyright © 2006 Elsevier

536 / Chapter 76 N-terminal

Beta 1-42

597

C-terminal

638

D1-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-V-V-I-A42

BETA 1-28 Alpha helix

Random coil

BETA 29-42 Beta sheet

Beta sheet

Random coil

domain of beta-amyloid peptide (1–28) is capable of stable self-association to form fibrils at acid pH, even in the absence of the stabilizing influence of the hydrophobic carboxyl terminus [20]. The importance of the N-terminal region in maintaining the solubility of the beta-peptide has been confirmed by studies demonstrating that amino-terminal deletions within the (1–12) and (1–17) peptides enhance the aggregation of the beta-amyloid peptide in vitro in parallel with the neurotoxicity effect [28]. As beta-amyloid peptide might be critical for inducing the pathology seen in AD, its accumulation may result in a cascade effect, thereby allowing for intervention at multiple different points to slow disease progression. The treatment may be directed toward decreasing beta-amyloid peptide production, increasing its removal, and decreasing beta-amyloid aggregation [3]. We demonstrated for the first time that antibodies raised against the N-terminal region of the AβP peptide prevent amyloid formation and bind to preformed Aβ fibrils, leading to their disaggregation and inhibition of their neurotoxic activity [35, 36]. Antibodies interact at strategic sites where protein aggregation is initiated and are able to stabilize the antigen by preventing aggregation and resolubilizing already formed protein aggregates. For such an active role, the mAbs require a high binding constant to the “strategic” positions on the antigen molecule, and to be noninhibitors of the biological activity of the respective antigen [37]. The ability of these antibodies to modulate βA formation in vitro and in vivo was found to be epitope-dependent. We localized the epitope of anti-aggregating antibodies in the N-terminal region of beta-amyloid peptide, which modulates amyloid formation [5, 6], using the phage libraries. The construction and application of several epitope libraries in which different peptides are expressed on the surface of filamentous phage (fd) has been described [25, 30]. The minimal epitope for

FIGURE 1. Amino acid sequence of β-amyloid peptide—C-terminal domain of AβP between 29 and 42 amino acids develops a β-strand structure in aqueous solutions independent of pH or temperature conditions, while the N-terminal region, 1–28 amino acids, can exhibit different conformations and solubility properties depending on environmental conditions. EFRH epitope of antiaggregating antibodies located at positions 3–6 is in bold.

these mAbs was determined employing combinatorial phage-displayed 6 and/or 15 peptide libraries. The immunopositive peptide-presenting phages were selected by AβP anti-aggregating antibody according to the higher affinity binding of about 10-9 M (half maximal binding). A consensus sequence of four residues—namely EFRH (Glu-Phe-Arg-His)—was found and this motif is sequentially located at positions 3–6 of the beta-amyloid molecule. The fact that the EFRH epitope is available for mAbs both when beta-amyloid peptide is either in solution or is an aggregate suggests that the N-terminal is exposed for antibody binding in both forms [5, 6]. We found that the epitope EFRH, which is located at the soluble tail of the N-terminal region, is involved in the aggregation process and acts as a regulatory site controlling both the solubilization and the disaggregation process of the beta-amyloid molecule. Locking of this epitope by antibodies modulates the dynamics of aggregation as well as resolubilization of already formed aggregates. If so-called pathological chaperones, like ApoE and heparan sulfate, increase the extent of β-amyloid formation, we propose the use of site-directed monoclonal antibodies against β-amyloid peptides, which decrease β-amyloid formation as therapeutic chaperones. Finding the “aggregating epitopes” as sequences that are related to the physiological protein regions where conformational changes are initiated, and preparation of antibodies against them, are the bases of the immunological approach in treatment of conformational diseases, such as Alzheimer’s disease.

EFRH PHAGE ELICITS ANTIBODIES AGAINST b-AMYLOID PEPTIDE We developed an immunization procedure for the production of effective anti-aggregating AβP antibodies based on filamentous phages displaying on their surface

Peptide Vaccine for Alzheimer’s Disease / 537 the EFRH (Glu-Phe-Arg-His) peptide as the antigen [7, 8] that was found to be the main regulatory site for amyloid modulation [6]. Small synthetic peptides, consisting of antibody epitopes, are generally poor antigens and need to be coupled to a large carrier, but even then they may induce only a low-affinity immune response. The generation of antisera to define peptide sequences usually involves the chemical synthesis of a peptide and its conjugation to a carrier molecule. Many efforts have been made to circumvent the low affinity response with limited success. A novel turn has been accomplished by use of filamentous bacteriophage as peptide carrier. Filamentous bacteriophages offer an obvious advantage over other immunocarriers [42]. Filamentous bacteriophages have been used extensively in recent years for the “display” on their surface of large repertoires of peptides generated by cloning random oligonucleotides at the 5′-end of the genes coding for the phage coat proteins [25, 30]. Filamentous phages are long thread-like single-stranded DNA phages that infect bacteria via sex pili. The best known, the Ff phages, are a group of three phages (M13, fd, and fl). The phage protein that is responsible for binding to the pilus tip, pIII, is present in four copies, encoded by gene 3. If foreign DNA that encodes a peptide or protein is inserted downstream of the leader sequence of gene 3, it will be translated and exposed at the N terminus of the mature pIII without compromising the ability of pIII to mediate infection via the F pilus. Although pIII is the protein most used for phage display, the major coat protein, pVIII, has also been used. The main difference between pIII and pVIII is the copy number of the displayed protein; while pIII is present in four copies pVIII is found in 2700 copies. Parenteral administration of filamentous phages in animals induced a strong immunological response to the phage proteins in all animals tested [42]. Mimotopes displayed on filamentous phages could be used as immunogens, and form the basis for developing a wide range of vaccines. Immunization with the EFRH-phage of BALB/c mice may, in a short period of time (a few weeks), raise the high concentration of high affinity (IgG) antiaggregation antibodies. Seven days after each injection of 1011 phages displaying the epitope without the adjuvant, the mice were bled and their sera were tested by ELISA for antibody IgG reactivity against wild type (wt) phage (not bearing the peptide EFRH on its surface) and against whole beta-amyloid peptide. Highly specific antibodies (IgG) against the beta-amyloid peptide were achieved after the first injection and the titers increased with the second and the third injections till 1 : 50000 [7]. Moreover, it was found that specific IgA antibodies against the beta-amyloid peptide anti-aggregation

epitope EFRH were obtained seven days after only one boost through intranasal administration [7]. The level of antibody in the sera was found to be related to the number of copies per phage. The high immunogenicity of filamentous phages enables the raising of antibodies against self-peptides or antigens. Immunization of guinea pigs with EFRH-phage as antigen, in which the beta-amyloid peptide sequence is similar to that in humans, resulted in the production of antibodies against self-peptides. While in mice there is a switch of one amino acid (glycine instead of arginine in position 5-EFGH), in guinea pigs the amino acid sequences are identical to those in humans [8]. Highly specific antibodies (IgG) against the AβP were achieved after the fourth injection effective in concentrations as dilute as 1 : 5000. Similar results were 11 obtained after immunization with 10 phage per intranasal injection without the adjuvant with an additional administration of antigen. The antibodies resulting from EFRH phage immunization are similar regarding their immunological properties to monoclonal antibodies previously studied, and to the antibodies raised by direct injection with fibrillar toxic beta-amyloid [7].

PEPTIDES AS VACCINES FOR PREVENTION AND/OR REDUCTION OF AMYLOID PLAQUES IN AD TRANSGENIC MICE The abundant evidence that AβP aggregation is an essential early event in AD pathogenesis has prompted an intensive search for therapeutics that target amyloid β-peptide, especially after the development of animal models of the disease. Several labs have bred AD diseased models of transgenic mice that produce human AβP, which develop plaques and neuron damage in their brains, as reviewed [40]. Although they do not develop the widespread neuronal death and severe dementia seen in the human disease, they are used as models for the study of AD and putative treatments. With the development of transgenic mice (Tg), the immunological concept in the treatment of conformational diseases became immunization approaches being pursued in order to stimulate clearance of brain Aβ plaques [18, 37]. They include both active and passive immunization techniques. Active immunization approaches employ AβP epitopes and/or immunogenic AβP conjugates, as well as various routes of administration and types of adjuvants. Passive immunization approaches include monoclonal antibodies or specific antibody fractions (Fabs) directed against specific AβP epitopes. The idea of eliciting an immune response to exogenously administered Aβ peptide in humans was

538 / Chapter 76 originally described in a 1990 U.S. patent by a physician and an experimental immunologist [21]. They proposed to parenterally administer low amounts (up to 10 μg/day) of Aβ peptide to slow the development of amyloid plaques. Later, Schenk et al. [29] demonstrated that immunization with human synthetic fibrillar Aβ 42 in transgenic mice (PDAPP) harboring a mutant version of human APP770 (V717F) produced high serum antibody titers against Aβ 42 (1 : 10,000) and inhibited the formation of amyloid plaques and associated histopathologic lesions. The group, headed by Thomas Wisniewski at New York University, believes that Aβ 42 immunization in humans may be unsafe because this peptide may cross the blood–brain barrier and form toxic fibrils [34]. In addition, administration of human Aβ 42 in AD patients may induce autoimmunity reactions. In order to reduce these risks, this group developed antigens displaying peptide sequences homologous to AβP and devoid of fibrillogenic properties. In particular, they prepared peptides by adding poly-lysine (K6) or polyaspartate (D6) to the N- or C-terminus of AβP. Scientists at Northwestern University in Evanston have developed a vaccine against AβP 42 oligomers [22]. Praecis Pharmaceuticals developed small peptidic ligands with subnanomolar affinity for AβP as inhibitors of Aβ polymerization and fibril formation [4]. Using natural peptide ligands that bind to Aβ plaques, they have created fusion proteins between these ligands and sequences from proteins of the immune system. These fusion proteins bind to Aβ plaques with high specificity and affinity. Scientists at Pasteur University started their work based on the knowledge that monoclonal antibodies, raised against the sequence AβP 1–16 of the amyloid protein, are able to dissolve in vitro Aβ 42 fibrils [27]. They also noted that palmitoylation of specific peptide sequences of an antigen may elicit a strong immune response against the entire protein. Recombinant fusion proteins that link the nontoxic B subunit of verotoxin with specific Aβ peptide sequences are being developed by Neurochem as strong AβP antigens. These fusion proteins should target AβP antigens directly to dendritic cells, the cells of the immune system that direct and control antibody production and cellular immunity [18]. In an attempt to increase the immune response to AβP, Chiesi Farmaceutici developed fusion proteins formed by the covalent bonding of Aβ peptide, or its fragments, and heat shock proteins (Hsp), released by cells in response to thermal shock and other types of injury (hypoxemia, chemical, etc.) of various molecular weights [10].

ACTIVE IMMUNIZATION OF hAPP TRANSGENIC MICE WITH EFRH-PHAGE AS PEPTIDE VACCINE We developed a series of antigens displaying the EFRH epitope on filamentous phages, which enabled modulating the immune response against AβP, according to the number of EFRH copies [23]. APP[V717I] transgenic mice (16 months old) were immunized with the EFRH-phage and analyzed at age 21 months [9]. Amyloid burden in the brain was significantly reduced in the immunized APP[V717I] transgenic mice that developed anti-AβP titers of at least 1 : 100, indicating that a relatively low antibody-titer may be enough to reduce reduce brain amyloid load [9]. Another set of experiments was performed on transgenic mice that express human APP751 regulated by the neuronal murine Thy1 promoter. The hAPP gene carries both the London (717) and the Swedish (670/671) mutations, resulting in an age-dependent increase in Aβ. Overexpression of the mutated human APP resulted in the development of typical AβP depositions as amyloid plaques in the neocortex and hippocampus. The mice were provided by JSW Research Co., Austria [23]. hAPP transgenic mice were immunized with phages displaying EFRH sequences by intraperitoneal administration and analyzed at age 15 months. At this age the amyloid plaque pathology is maximally and stably established. Titer levels, although moderate, were positively correlated to the copy number of the EFRH epitopes displayed by the phage antigens. Titers obtained from sera of nontransgenic mice or mice that were injected only with PBS were at background levels. The amyloid burden in the brain was reduced in the immunized hAPP transgenic mice that developed anti-AβP titers of at least 1 : 100, similar to previous experiments [7], showing a dose-response relationship between antibody-titer and reduced amyloid load. A considerable difference in plaque load content exists between treated and untreated mice. The reduction in total amyloid burden is related to the level of antibodies. We showed that a low but continuous titer of anti-EFRH is sufficient to reduce the amyloid burden [23]. The development of AβP anti-aggregating antibodies via immunization with phage-EFRH of AD models of transgenic mice showed not only a considerable reduction of amyloid in the brains of the affected animals but also, as recently reported, a considerable improvement in cognitive functions of the treated mice [23]. The advantages of phage-EFRH antigen in raising anti-aggregating β-amyloid antibodies versus whole βamyloid can be summarized as follows:

Peptide Vaccine for Alzheimer’s Disease / 539 1. The high immunogenicity of the phage enables production of high titers of IgG antibodies in a short period of weeks without need for adjuvant administration. Self-expression of the antigen led to long-lasting immunization. 2. The key role of the EFRH epitope in β-amyloid formation and its high immunogenicity leads to anti-aggregating antibodies that recognize whole βamyloid peptide and can substitute for whole toxic β-amyloid fibrils.

FUTURE OUTLOOK In spite of uncertainty regarding the efficacy of AβP immunization in humans, the very promising results with APP Tg mice generated by several laboratories led ELAN/AHP to start human clinical trials after two and a half years of preclinical animal studies by injection of fibrillar Aβ 42 into participants in the clinical trials. Patients with mild to moderate AD were immunized with AN1792 (fibrillar Aβ 1-42 plus adjuvant) in singleand multiple-dose Phase 1 safety studies, which were well tolerated and demonstrated no significant safety concerns. Elan suspended inoculations in mid-January 2002 after some volunteers fell ill with what it called “clinical signs consistent with inflammation in the central nervous system.” Clinical trials showed stabilized cognition and slowed progression of dementia, but there was neuroinflammation in some patients. Meningoencephalitis is not a feature of AD pathology and is likely to be a consequence of the immunotherapy. Vaccination appeared to have both beneficial and harmful effects, suggesting that the vaccine can have potent clinical utility if an appropriate immunological response can be generated. An inflammatory reaction was probably triggered by T-lymphocyte activation after immune system stimulation with fibrillar Aβ 1-42. The adverse events observed in patients with AN-1792 could reflect an autoimmune inflammatory response [26] due to T-cell epitopes to AβP 1-42 that reside in the mid-to-carboxy-terminal region of the beta-peptide [12]. Improved immunotherapeutic strategies may be used to obtain a beneficial effect without untoward side effects. It is possible , to induce AβP antibodies with no AβP T-cell response as the N-terminal of AβP (1-15) contains the dominant B-cell epitopes that bind to Aβ plaques with no T-cell epitopes [26, 34], or with only small sequences from this region [23], as described below. On the other hand, humans may develop selfantibodies when immunized with whole or fragments of AβP. These antibodies are capable of binding to a

variety of Aβ species in the brain. Thus immunization could have contradictory effects: the desired inhibition of amyloid fibril formation versus Fc microglial overactivation leading to neuroinflammation [39]. How many antibodies are required for an effective immunization? No data are available on the quantitative decrease of AβP concentration necessary to reduce Aβ deposition. An increase of ∼30% of Aβ concentration shifts the disease onset earlier by several decades. These assumptions may suggest that a low level of antibodies is recommended for optimum effect of vaccination, similar to the level of antibodies found in older but healthy people [41]. Taking these arguments into consideration, the proposed EFRH sequence, devoid of T-cell epitopes, displayed on phage as antigens, able to provide a relatively low titer of AβP anti-aggregating antibodies, may become a real alternative for AD vaccination. Despite the setbacks in the halted phase II Elan/ Wyeth vaccination trial, amyloid immunotherapy strategies remain a novel and promising approach for the treatment and/or prevention of AD. To move forward it will take considerable effort to determine whether immunotherapy will work in humans.

Acknowledgments The author wishes to thank Rachel Cohen-Kupiec for phage EFRH construction, Vered Lavie and Maria Becker for neuropathology staining, Rela Koppel for technical assistance, and Faybia Margolin for editing.

References [1] Barrow, C.J. and Zagorski, M.G., Solution structures of β-peptide and its constituent fragments: Relation to amyloid deposition, Science 253 (1991), pp. 179–182. [2] Busciglio, J., Lorenzo, A., Yeh, J. and Yankner, B.A., β-amyloid fibrils induce tau phosphorylation and loss of microtubule binding, Neuron 14 (1995), pp. 879–888. [3] Drachman, D.A., Preventing and treating Alzheimer’s disease: Strategies and prospects, Expert Rev. Neurotherapeutics 3 (2003), pp. 565–569. [4] Findeis, M.A., Lee, J.J., Kelley, M., Wakefield, J.D., Zhang, M.H., Chin J. et al., Characterization of cholyl-leu-val-phe-phe-ala-OH as an inhibitor of amyloid β-peptide polymerization, Amyloid: Int J Exp Clin Invest 8 (2001), pp. 231–241. [5] Frenkel, D., Balass, M. and Solomon, B., N-terminal EFRH sequence of Alzheimer’s β-amyloid peptide represents the epitope of its anti-aggregating antibodies, J Neuroimmunol 88 (1998), pp. 85–90. [6] Frenkel, D., Balass, M., Kachalsky-Katzir, E. and Solomon, B., High affinity binding of monoclonal antibodies to the sequential epitope EFRH of β-amyloid peptide is essential for modulation of fibrillar aggregation. J Neuroimmunol 95 (1999), pp. 136–142. [7] Frenkel, D., Katz, O. and Solomon, B., Immunization against Alzheimer’s β-amyloid plaques via EFRH phage administration. Proc Natl Acad Sci USA 97 (2000), pp. 11455–11459.

540 / Chapter 76 [8] Frenkel, D., Kariv, N. and Solomon, B., Generation of autoantibodies towards Alzheimer’s disease vaccination. Vaccine 19 (2001), pp. 2615–2619. [9] Frenkel, D., Dewachter, Van Leuven, I.F. and Solomon, B., Reduction of beta-amyloid plaques in brain of transgenic mouse model of Alzheimer’s disease by EFRH-phage immunization. Vaccine 21 (2003), pp. 1060–1065. [10] Ghirardi S., Armani, E., Amari, G., Puccini, P., Imbimbo, B.P. and Villetti, G., Fusion proteins as immunization treatments of Alzheimer’s disease, PCT/EP01/12242, October 23 (2001). [11] Glenner, G.G. and Wong, C.W., Alzheimer’s disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem Biophys Res Com 120(3) (1984), pp. 885–890. [12] Grubeck-Loebenstein B., Blasko, I., Marx, F.K. and Trieb, I., Immunization with beta-amyloid: Could T-cell activation have a harmful effect? Trends Neurosci 23 (2000), p. 114. [13] Haass, C., Schlossmacher, M.G., Hung, A.Y. et al., Amyloid βpeptide is produced by cultured cells during normal metabolism, Nature 359 (1992), pp. 322–324. [14] Hardy, J. and Allsop, D., Amyloid deposition as the central event in the aetiology of Alzheimer’s disease, Trends Pharmacol. Sci 12 (1991), pp. 383–388. [15] Hardy, J., Duff, K., Hardy, K.G., Perez-Tur, J. and Hutton, M., Genetic dissection of Alzheimer’s disease and related dementias: Amyloid and its relationship to tau. Nat Neurosci 1 (1998), pp. 355–358. [16] Hardy, J. and Selkoe, D.J., The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics, Science 297 (2002), pp. 353–356. [17] Hollosi, M., Otvos, L. Kaijtar, J., Percell, A. and Lee, V.M.Y., Is amyloid deposition in Alzheimer’s disease preceded by an environment induced double conformational transition? Pept Res 2 (1989), pp. 109–113. [18] Imbimbo, B.P., β-Amyloid immunization approaches for Alzheimer’s disease. Drug Dev Res 56 (2002), pp. 150–162. [19] Kirschner, D.A., Inouye, H., Duffy, L.K., Sinclair, A., Lind, M. and Selkoe, D.J., Synthetic peptide homologous to β-protein from Alzheimer disease forms amyloid-like fibres in vitro. Proc Natl Acad Sci USA 84 (1987), pp. 6953–6957. [20] Kirshenbaum, K. and Daggett, V., PH-Dependent conformations of the amyloid β (1-28) peptide fragment explored using molecular dynamics. Biochemistry 34 (1995), pp. 7629– 7639. [21] Kline, E.L. and McMichael, J., Method and composition for treatment of central nervous systems disease states associated with abnormal amyloid β protein. WO 91/16819 (1991). [22] Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos., M., et al., Diffusible, nonfibrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins, Proc Natl Acad Sci USA 95 (1998), pp. 6448–6453. [23] Lavie, V., Becker, M., Cohen-Kupiec, R., Yacoby, I., Koppel, R., Wedenig, M. et al., EFRH—phage immunization of Alzheimer’s disease animal model improves behavioral performance in Morris Water Maze trials, J Molec Neurosci 24(1) (2004), pp. 105–113. [24] Maggio, J.E. and Mantyh, P.W., Brain amyloid—A physicochemical perspective, Brain Pathol 6 (1996), pp. 147–162.

[25] Medynski, D., Phage display: All dressed up and ready to role, Biol Technol 12 (1994), pp. 1134–1136. [26] Monsonego, A., Immunogenic aspects of amyloid beta peptide: Implications for pathogenesis and treatment of Alzheimer’s disease, Neurobiol Aging 23 (2002), S112. [27] Nicolau, C., Greferath, R., Balaban, T.S., Lazarte, J.E. and Hopkins, R.J., A liposome-based therapeutic vaccine against βamyloid plaques on the pancreas of transgenic NORBA mice, Proc Natl Acad Sci USA 99:2 (2002), pp. 332–2337. [28] Pike, C.J., Overman, M.J. and Cotman, C.W., Amino-terminal deletions enhance aggregation of β-amyloid peptides in vitro. J Biol Chem 270 (1995), pp. 23895–23898. [29] Schenk, D., Barbour, R., Dunn W. et al., Immunization with amyloid-β attenuates Alzheimer’disease-like pathology in the PDAPP mouse. Nature 400:1 (1999), pp. 73–177. [30] Scott, J.K. and Smith, G.P., Searching for peptide ligands with an epitope library, Science 249 (1990), pp. 386–390. [31]Selkoe, D.J., Amyloid β protein and the genetics of Alzheimer’s disease, J Biol Chem 271 (1996), pp. 18295–18298. [32] Selkoe, D.J., Toward a comprehensive theory for Alzheimer’s disease. Hypothesis: Alzheimer’s disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein, Ann NY Acad Sci 924 (2000), pp. 17–24. [33] Seubert, P., Vigo-Pelfrey C., Esch, F. et al., Isolation and quantification of soluble Alzheimer’s β-peptide from biological fluids, Nature 359 (1992), pp. 324–325. [34] Sigurdsson, E.M., Scholtzova, H., Mehta, P.D., Frangione, B. and Wisniewski, T., Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer’s diseaseassociated pathology in transgenic mice, Am J Pathol 159 (2001), pp. 439–447. [35] Solomon, B., Koppel, R., Hanan E. and Katzav, T., Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer’s β-amyloid peptide, Proc Natl Acad Sci USA 93(1) (1996), pp. 452–455. [36] Solomon, B., Koppel, R., Frankel, D. and Hanan-Aharon, E., Disaggregation of Alzheimer β-amyloid by site-directed mAb. Proc Natl Acad Sci USA 94 (1997), pp. 4109–4112. [37] Solomon, B., Alzheimer’s disease and immunotherapy, Curr Alz Res 1 (2004), pp. 149–163. [38] Soto, C., Castano, E.M., Frangione, B. and Inestrosa, N.C., The α-helical to β-strand transition in the amino-terminal fragment of the amyloid β-peptide modulates amyloid formation, J Biol Chem 270 (1995), pp. 3063–3067. [39] Ulvestad, E., Williams, K., Matre, R. et al., Fc receptors for IgG on cultured human microglia mediate cytotoxicity and phagocytosis of antibody-coated targets, J Neuropathol Exp Neurol 53 (1994), pp. 27–36. [40] Van Leuven, F., Single and multiple transgenic mice as models for Alzheimer’s disease, Prog Neurobiol 61(3) (2000), pp. 305– 312. [41] Weksler, M.E., Relkin, N., Turkenich, R., LaRusse, S., Zhou, L. and Szabo, P., Patients with Alzheimer disease have lower levels of serum anti-amyloid peptide antibodies than healthy elderly individuals, Exp Gerontol 37 (2002), pp. 943–948. [42] Willis, E.A., Perham, N.R. and Wraaith, D., Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage, Gene 128 (1993), pp. 79–83.

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77 Peptide Dendrimers as Immunogens JAMES P. TAM

peptide dendrimers reported to date are in the range of 6 to 20 kDa and are often homomeric with generations of 8 branching units or less.

ABSTRACT Peptide dendrimers are radically branched macromolecules that are designed to mimic proteins. Their multimeric nature, unambiguous composition, design flexibility, and ease of production render them popular for various immunological and biochemical applications. Applications include uses as immunogens, synthetic vaccines, biomedical diagnostic reagents, protein mimetics, anticancer and antiviral agents, and delivery vehicles for drugs. This chapter focuses on the design, properties and preparation of peptide dendrimers as immunogens, commonly known as multiple antigen peptides (MAPs).

BACKGROUND AND DISCOVERY An impetus for developing peptide dendrimer by our laboratory in the 1980s was to mimic forms and functions of proteins [9, 14, 19]. Our work was inspired by polyamino-acid dendrimers that employed trifunctional amino acids such as Lys as repeating branch units to form spherical polymers. These spherical dendrimers consist of many generations of branching units of amino acids radiating from a central core. Early examples of polyamino-acid dendrimers were the highly branched polylysines of 64 Lys and 128 amino branches of unequal lengths. These molecules are found to be monodisperse, nondraining spheres which could find applications in drug delivery and encapsulation. Thus far, only a few polyamino-acid dendrimers based on repeating amino acids have been described, but there is an explosion of research activity on chemical dendrimers consisting of nonamino acids over the past two decades [2, 8]. Since protein-protein interactions often involve oligomers, we emphasized the polyvalency effects of peptide dendrimers to validate biological goals, particularly as immunogens. The first example of peptide dendrimer intended for producing antipeptide antibodies is the MAPs [9, 14]. Common designs for MAPs (Fig. 2) consist of a core built up of a low generation of 2 or 16 Lys residues (n < 4) and a surface of peptide chains attached to the core matrix. MAPs have been used successfully to produce both polyclonal and monoclonal antibodies that specifically recognize native proteins. Comparative studies also show that they often produce sera that have a significantly higher antibody titer than sera with antibodies against the same peptides conjugated to a carrier protein [9, 14, 19].

INTRODUCTION Multiple antigen peptides (MAPs) belong to the family of peptide dendrimers that are branched, multichain, cascade-shaped polymers [9, 14, 19]. MAPs and peptide dendrimers often contain three components: a core, branching units, and surface peptides (Fig. 1). Generally, 2 to 16 peptides of the same or different sequences are found in a peptide dendrimer. Unlike most polymers, peptide dendrimers are synthesized under controlled conditions that produce macromolecules with defined molecular weights, structures, and multiple N- or C-terminal ends. Such molecular architectures are different from conventional polypeptides or proteins that are single chains. Peptide dendrimers are also different from chemical dendrimers [2, 8] that often consist of branches >16 small organic monomers and no tethering peptide chains. The sizes of peptide dendrimers, varying widely from 3 to >100 kDa, are strongly influenced by the number and lengths of tethering peptide chains. In turn, they account for the bulk of their molecular mass. Most Handbook of Biologically Active Peptides

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DESIGN OF PEPTIDE DENDRIMERS AS IMMUNOGENS The design of peptide dendrimers or MAPs contains Lys, Orn or their homologs, often as polyamino-acid core and branching units. Lysine with Nα- and Nε-amino groups as reactive ends is a suitable trifunctional amino acid to form the Bn-type core by a limited sequential propagation of lysine. These low-molecular-weight cores with 2n reactive amino ends can then serve as attachment sites for peptides. Furthermore, they are amenable to divergent stepwise synthesis by solid-phase methods through successive branching of a di-protected Lys. By virtue of their trivalency, reactivity, and synthetic expedience, a K2K (Lys2Lys) unit containing four surface amino groups and one carboxylic group is a popular design for a dendrimer core [9, 14, 19]. The K2K-core consists of three lysines to give a tetravalent

peptide dendrimer (Fig. 2). Further divergence of the K2K unit to one or two additional levels will generate di-K2K and tetra-K2K core for MAPs with reactive ends of 8 and 16 amino groups, respectively, to which peptides can be attached. These cores can be further functionalized with electrophiles and thiol nucleophiles for convergent ligation of peptides. The K2K-core is small when compared with the bulk of peptides layered around the surface. As an example, an octabranched core and eight copies of the peptide, each containing 14 amino acid residues, would have an average molecular weight of 13 to 14 kDa. Only 7% of the mass is due to a lysine-based core; 93% of the mass is due to peptide sequences. The core is oligomeric and contains noncationic peptidyl and isopeptidyl lysine amide bonds. Several variations of K2K cores have evolved. A symmetrical version with a β-alanine as a spacer at the α-amine of Lys provides the core unit that has sym-

Peptide Dendrimers as Immunogens / 543 metrical branchings [19]. To limit the flexibility of the branched lysyl core, ornithine, their lower homologs as well as constrained diamino acids have been used. Nonbranched polylysine form of MAPs that are linear, helical, and cyclic peptides have also been developed to impart different forms of dendrimeric architectures.

IMMUNOLOGICAL PROPERTY OF MAPs Comparative studies have identified what has now become a general trend in the properties of antisera elicited by MAPs [15, 19]. Sera obtained from animals inoculated with MAPs is site-specific, of a higher titer, and produced more rapidly than those obtained with protein conjugate peptides. The MAP format allows investigators to overcome the immunodominance conferred by linker and carrier proteins and elicit antibody responses to weakly immunogenic subdominant peptide sequences. To further increase the immunogenicity of MAPs, T-helper epitope(s) are often added [11, 12, 17]. There are many possible arrangements of linking B- and Thelper epitopes on MAPs. A convenient approach is to link the epitopes in tandem. The main drawback of this method is that new epitopes may be formed within the tandem peptide, and the MAP is not processed and presented as intended. One solution is to insert a longer T epitope to preserve the diversity of T-cell recognition. Alternatively, convergent strategies may be employed where the dendrimer contains copies of each peptide sequence on separate “arms,” thereby decreasing the possibility of neo-epitope formation and increasing the response to the desired epitopes. Tandem or di-epitope MAP constructs have been extensively studied in the malaria synthetic vaccine model. Ten MAP constructs were synthesized, each containing a combination of described B- and T-cell epitopes; the constructs were designed to evaluate the relevance of the number of copies, stoichiometry, and orientation of T and B sequences in a di-epitope model [11, 12, 15, 17]. In this case it was concluded that there was no advantage in using octameric instead of tetrameric MAPs and that having the B-cell epitope at the N-terminal produced a more efficient immunogen. The degree of protection against challenge of immunized mice with 2000 Plasmodium berghei sporozoites correlated with the antibody levels obtained by the immunization protocol. Tetra and octameric MAPs have been utilized in most studies to date. The number of branches required tends to depend largely on the amino acid residues: with peptides <15 amino acids we have found that there is no real advantage in using more than four branches. Similar observation was found when guinea

pigs were immunized with constructs containing one, two, four, or eight copies of a foot-and-mouth disease virus peptide. It was determined that in this case presentation in a tetrameric structure was sufficient for antibody response. Di-epitope MAPs as synthetic vaccine model for hepatitis B uses a different chemical approach [15]. B-cell epitope and T-helper epitope were used in mono- and di-epitope configurations; in the latter the two peptides were connected in alternating forms on the lysine core instead of in tandem. Only when the B-cell epitope was presented in the MAP form with the T-helper epitope was it immunogenic in rabbits. Therefore, these results confirmed that a di-epitope MAP format may overcome the poor immunogenicity of a linear peptide. Mono- and di-epitope MAP configurations were further analyzed in a synthetic model for the human immunodeficiency virus type 1 (HIV-1) [3, 7]. Monoepitope MAPs consisting of four copies of a major neutralizing epitope of HIV-1 were synthesized along with di-epitope MAPs that also contained a known T-helper epitope at the C-terminus of the B-cell epitopes. The di-epitopes were immunogenic, whereas the mono-epitope MAPs elicited species-specific responses. The use of universal or promiscuous Thelper epitopes, which are recognized by three or more strains of mice, are particularly useful for di-epitope constructs and have been used to enhance the immunogenicity of B-cell epitopes in a number of MAPs [11, 12, 17, 19].

CELL-MEDIATED RESPONSES INDUCED BY LIPIDATED MAPs Induction of a cell-mediated response relies on the delivery of CTL epitopes to an intracellular endosomal pathway that results in the presentation of peptides in association with class I MHC molecules. Others have found the value of lipidated peptide in eliciting cellular responses because of the increased efficiency of delivery of peptides to the intracellular compartment. Longchain lipidic amino acids have been developed in combination with detergent-solubilized liposome as a delivery system [1, 3, 7, 21]. This approach was applied to the MAP format in successful vaccine models against HIV-1 [1, 3, 7]. A cytotoxic response was induced by a lipidated MAP containing a peptide sequence from gp120 envelope protein of HIV-1, III-B isolate, which overlaps a B-cell epitope, a T-helper epitope, and a CTL epitope. This response was detected after a single inoculation without extraneous adjuvant and was superior to the response induced by a full cycle of inoculations with the nonlipidated MAP in complete Freund’s adjuvant and

544 / Chapter 77 was still detectable seven months later. The lipidated MAP was found to produce systemic antibody and cellular responses regardless of the route of inoculation employed [7]. The ability of MAPs containing the lipid moiety tripalmitoyl-S-glyceryl cysteine (PC) to induce mucosal antibody response via oral administration adds a new dimension to applications of the MAP constructs, and it may be particularly useful in preventing transmission of pathogens, such as HIV, through mucosal surfaces.

REASONS FOR INCREASED IMMUNOGENICITY The multimeric property of MAP has been shown to increase the immunogenicity of weakly immunogenic monomeric peptides. The multimeric nature of the MAPs of closely packed epitopes could lead to greater B-cell surface cross-linking through surface immunoglobulins resulting in increased activation and antibody production. MAPs attaining an unnatural polymeric structure compared to linear peptides may provide resistance to proteolytic degradation leading to a longer immunogen half-life. Persistence of MAPs as immunogens in the systemic circulation possibly induces longer lasting immune responses. The increase in immunogenicity could also be due to new T-helper epitopes being generated within the MAP format. Even though the lysine core itself is immunologically silent, the contribution of one or two residues to the attached peptide sequences could result in the production of new epitopes. This would provide a plausible explanation for the fact that the antibody response to the repetitive NANP peptide from Plasmodium falciparum CS protein is restricted to a single mouse strain when administered as a linear peptide, but administration of the peptide in a MAP format results in antibody production in a further five strains that differ in MHC haplotype. An alternative view is that the MAP construct allows the desired epitopes to be processed and presented by APCs in a more efficient manner. For instance, all residues in a linear peptide may be accessible to degrading enzymes, whereas the MAP configuration may actually protect certain residues from enzymatic cleavage, resulting in the presentation of different epitopes. In many cases the more immunogenic di-epitope MAP constructs are synthesized with the desired T-helper epitope in close proximity to the branching lysine core. The design of a MAP provides a scaffold for close packing of peptide sequences that may allow the stabilization of the secondary structure and the reverse turns of peptides. Furthermore, the distal end of the peptide away from the core of MAPs is more exposed and flex-

ible than the proximal end. For these reasons, we may expect the immunogenicity of the B-cell epitope to be greater if the epitope is located at the distal N-terminal site.

SYNTHESIS Synthesis of peptide dendrimers embraces a broad range of chemistry from conventional solid-phase peptide synthesis schemes in organic solvents to the formation of regiospecific amide or nonamide bonds in aqueous solutions. Strategies for preparing peptide dendrimers can generally be divided into two categories—namely the divergent and convergent approaches [4, 9, 10, 14, 18, 19]. The divergent strategy is a direct approach by which the dendrimer is built stepwise and diverges outward (Fig. 3). Stepwise solid-phase synthesis has been used to prepare the dendrimer in a continuous operation on a solid support. However, the convergent strategy is an indirect, modular approach by which peptidyl surface functional groups and the branching units are prepared separately. The purified components are then linked together and peptide sequences converge to the branching unit as a dendrimer. Speed and efficiency are advantages of the divergent stepwise strategy particularly by solid-phase methods because intermediates are neither purified nor characterized. This strategy is suitable for validation of concepts and for small peptide dendrimers that may be separated from their by-products by powerful systems. In many cases stepwise synthesis is not practical, either because of the nature of the branching unit preventing integration into a stepwise strategy or because the resulting dendrimer is too large (e.g., >15 kDa) to be separated from its by-products with confidence. An advantage of the convergent strategy is chemical unambiguity because the protected or unprotected peptide segments and the branching unit used in coupling reactions are purified prior to reaction, thereby limiting the range of by-products and facilitating purification. A disadvantage lies in the operational complexity: The convergent strategy requires more steps than stepwise synthesis, including additional purification and characterization steps for intermediates. The convergent strategy could also pose significant problems with regard to the solubility of peptide intermediates when protected peptide segments are being used. Thus, a recent trend has developed in which aqueous, soluble, unprotected peptides are used that are convenient to handle, purify, and characterize. Several laboratories have made extensive efforts to develop new methodologies of ligation chemistry that involve the fewest steps possible. These chemistries are

Peptide Dendrimers as Immunogens / 545 Abu-Wang

βAla-MBHA resin

resin

1. Fmoc -Lys(Fmoc)/BOP/DIEA 2. Piperidine 3. repeat 1, 2 twice di-K2K-Abu-Wang

resin

1. Fmoc-amino acid 2. n cycles 3. TFA

1. [Boc -Lys(Boc)]2O 2. TFA 3. repeat 1, 2 twice di-K2K-βAla-MBHA resin

1. Boc -amino acid 2. HF

(Peptide)8-di-K2K-Abu-OH

(Peptide)8-di-K2K-βAla-OH

Fmoc

Boc

based on chemoselectivity of unprotected intermediates for assembling peptide dendrimers [4, 10, 18]. An advantage of the convergent strategy is chemical unambiguity because the purified protected or unprotected peptide segments and the template or cores are used in coupling reactions that limit the range of side products and facilitate the purification of the desired products. A disadvantage is its operational complexity because it requires more steps than stepwise synthesis, including additional purification and characterization steps for intermediates. The convergent strategy could pose significant problems in regard to the solubility of peptide intermediates when protected peptide segments are being used. Thus, a recent trend is to prepare peptide dendrimers by the convergent approach using aqueous soluble unprotected peptides that are convenient to handle, purify, and characterize. Recently, several laboratories have made extensive efforts to develop new methodologies of ligation chemistry based on chemoselectivity of unprotected intermediates for assembling peptide dendrimers [4, 10, 18]. Methods for purification and characterization of peptide dendrimers are similar to those of peptides and proteins. Crude synthetic peptide dendrimers derived from the divergent strategy often require multiple methods that may include sequential steps of dialysis or gel-filtration chromatography, followed by RP-HPLC or high-performance ion-exchange chromatography. Even by these steps it may still not be possible to remove byproducts with a single modification or deletion of an amino acid from the desired product. Synthetic products assembled using a convergent strategy can be refined by most chromatographic methods resulting in a homomeric product. Characterization methods for peptide dendrimers include the usual panel of techniques: amino acid analysis, SDS-PAGE, capillary zone electrophoresis, and enzymatic digestion. Mass spectro-

FIGURE 3. A schematic comparison of divergent synthesis methods by Boc and Fmoc chemistries.

metric analysis has now become an indispensable tool for determining the molecular weights indicating product homogeneity of these complex molecules.

CONTRIBUTION AND FUTURE OUTLOOK Our laboratory introduced peptide dendrimers in 1988 [9, 14]. The original proposed forms of MAPs are still widely popular for current use and the original synthetic methods by stepwise solid-phase synthesis commonly employed for their preparation. The multivalency of peptide dendrimers is advantageous to increase binding avidity of antigens to antibodies in various immunological assays and diagnostic tests [5, 13, 16]. Examples in the literature have reported that the MAP format increases the binding avidity and sensitivity—in some cases >100,000-fold [13]. In addition, MAPs and peptide dendrimers have found application in areas such as inhibitors, artificial proteins, affinity purifications, and intracellular transport, as well as in drug discovery. Currently, the majority of vaccines approved for human use consist of either heat-killed or liveattenuated infectious agents. Despite the successes of such vaccines, development by conventional methods is limited by several factors—namely (1) hazardous production; (2) cold chain required for storage; (3) presence of contaminating materials; (4) risk of reversion to infectious state; and (5) side effects of vaccination. Subunit vaccines based on MAPs design are appealing alternatives because they are selective and chemically defined. MAPs have additional advantages in that desired sequences may be synthesized that only contain the epitope of interest and deleterious epitopes may be omitted. Large quantities of chemically purified peptide vaccines can be prepared with automated methods and

546 / Chapter 77 peptide-based immunogens are more likely to be resistant to denaturation, and they can be easily stored and transported without refrigeration [6, 20]. Our laboratory is optimistic about developing lipidated MAPs entrapped in liposomes for vaccine development [3, 7, 21]. The use of a mixture of MAPs anchored on lipid vesicles to mimic viral surface proteins may hold promise for future vaccine design. Immunogenicity could be broadened and enhanced by the inclusion of a noncovalent mixture of B-, T-helper, and CTL epitopes from various proteins. At the same time, the combination of adjuvant effects of liposome and the built-in lipid anchor may replace the need for additional adjuvant.

References [1] Defoort JP, Nardelli B, Huang W, Ho DD, Tam JP. Macromolecular assemblage in the design of a synthetic AIDS vaccine. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 3879–83. [2] Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: From biomimicry to drug delivery and biomedical applications. Drug Discov. Today 2001; 6: 427–36. [3] Huang W, Nardelli B, Tam JP. Lipophilic multiple antigen peptide system for peptide immunogen and synthetic vaccine. Mol. Immunol. 1994; 31: 1191–9. [4] Lu YA, Clavijo P, Galantino M, Shen ZY, Liu W, Tam JP. Chemically unambiguous peptide immunogen: Preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system. Mol. Immunol. 1991; 28: 623– 30. [5] Marsden HS, Owsianka AM, Graham S, McLean GW, Robertson CA, Subak-Sharpe JH. Advantages of branched peptides in serodiagnosis. Detection of HIV-specific antibodies and the use of glycine spacers to increase sensitivity. J. Immunol. Methods. 1992; 147: 65–72. [6] Moreno CA, Rodriguez R, Oliveira GA, Ferreira V, Nussenzweig RS, Moya Castro ZR. Calvo-Calle JM, Nardin E. Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice. Vaccine 1999; 18: 89–99. [7] Nardelli B, Haser PB, Tam JP. Oral administration of an antigenic synthetic lipopeptide (MAP-P3C) evokes salivary antibodies and systemic humoral and cellular responses. Vaccine 1994; 12: 1335–9.

[8] Newkome GR, Lin X, Weis CD. Polytryptophane terminated dendritic macromolecules. Tetrahedron-Asymmetry 1991; 2: 957–60. [9] Posnett DN, McGrath H, Tam JP. A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain. J. Biol. Chem. 1988; 263: 1719–25. [10] Rao C, Tam JP. Synthesis of peptide dendrimer. J. Am. Chem. Soc. 1994; 116: 6975–6. [11] Romero PJ, Tam JP, Schlesinger D, Clavijo P, Gibson H, Barr PJ, Nussenzweig RS, Nussenzweig V, Zavala F. Multiple T helper cell epitopes of the circumsporozoite protein of Plasmodium berghei. Eur. J. Immunol. 1988; 18: 1951–7. [12] Schott ME, Wells DT, Schlom J, Abrams SI. Comparison of linear and branched peptide forms (MAPs) in the induction of T helper responses to point-mutated ras immunogens. Cell Immunol. 1996; 174: 199–209. [13] Shin SY, Lee MK, Kim SY, Jang SY, Hahm KS. The use of multiple antigenic peptide (MAP) in the immunodiagnosis of human immunodeficiency virus infection. Biochem. Mol. Biol. Int. 1997; 43: 713–21. [14] Tam JP. Synthetic peptide vaccine design—synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 5409–13. [15] Tam JP, Lu Y-A. Vaccine engineering—enhancement of immunogenicity of synthetic peptide vaccines related to hepatitis in chemically defined models consisting of T-cell and B-cell epitopes. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 9084–8. [16] Tam JP, Zavala F. Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solidphase immunoassays. J. Immunol. Methods 1989; 124: 53–61. [17] Tam JP, Clavijo P, Lu Y-A, Nussenzweig V, Nussenzweig R, Zavala F. Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria. J. Exp. Med. 1990; 171: 299–306. [18] Tam JP, Lu Y-A, Liu C-F, Shao J. Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 12485–9. [19] Tam JP. Recent advances in multiple antigen peptides. J. Immunol. Methods 1996; 196: 17–32. [20] Wang CY, Looney DJ, Li ML, Walfield AM, Ye J, Hosein B, Tam JP, Wong-Staal F. Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen. Science 1991; 254: 285–8. [21] Zhong G, Toth I, Reid R, Brunham RC. Immunogenicity evaluation of a lipidic amino acid-based synthetic peptide vaccine for Chlamydia trachomatis. J. Immunol. 1993; 151: 3728–36.

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78 Chemotactic Peptide Ligands for Formylpeptide Receptors Influencing Inflammation JI MING WANG AND KEQIANG CHEN

methionyl-leucyl-phenylalanine (fMLF) activate at least two 7-transmembrane (STM), G protein-coupled receptors, the high-affinity FPR and its low-affinity variant FPR-like 1 (FPRL1), in human myeloid cells [17, 19, 25, 27]. In human cells, there is a third formylpeptide receptor—namely FPR-like-2 (FPRL2)—that is not activated by fMLF but interacts with a peptide fragment of hemebinding protein released by damaged cells [24]. When activated by agonist peptides, formylpeptide receptors in phagocytic leukocytes transduce a typical pertussis toxin sensitive, G protein-mediated, signaling cascade, which increases cell migration, phagocytosis, and release of proinflammatory mediators [17, 19, 25, 27]. Formylpeptide receptors were identified and cloned in the early 1990s. However, their biological role was poorly understood until recently. Mice depleted of the gene encoding the counterpart of FPR do not show significant alterations in their phenotype, but such mice are more susceptible to bacterial infection [9], indicating that FPR is involved in innate antibacterial host defense by recognition of bacterium-derived agonists. During the past few years, a wide variety of novel peptide agonists that activate one or more of the formylpeptide receptors have been identified. These include additional bacterial peptides, peptide fragments of HIV-1 envelope proteins, small synthetic peptides obtained from random peptide libraries, and peptide agonists produced by mammalian cells [17, 18, 19]. Most of the newly identified peptide agonists are not formylated at the N-termini and are associated with inflammatory responses and antimicrobial host defense.

ABSTRACT Bacterial peptide fMet-Leu-Phe (fMLF) is one of the first identified chemoattractants for phagocytic leukocytes by interacting with two seven transmembrane, G protein-coupled formylpeptide receptors, FPR, and its variant FPRL1. Recently a number of novel and hostderived formylpeptide receptor agonist peptides have been identified. These agonists selectively activate one or more formylpeptide receptors including another FPR variant FPRL2. Activation of formylpeptide receptors by peptide agonists results in increased cell chemotaxis, phagocytosis, and release of proinflammatory mediators by leukocytes. Injection of peptide agonists in mice also promotes antibody responses. Therefore, formylpeptide receptor agonists may play important roles in the pathogenesis of inflammatory and immunological diseases.

BACKGROUND Leukocytes accumulate at the sites of inflammation and infection by responding to pathogen or host-derived chemoattractant peptides. Such mediators are categorized in general into two families, the classical chemoattractants [18] and chemokines [21] (see chapter by Ansorge and Reinhold). Members of the classical chemoattractants include proinflammatory anaphylotoxins and neuropeptides (see chapters by Yang as well as Zhang and Oppenheim, respectively, in this same section) and N-formylpeptides as cleavage products of bacterial [22] and mitochondrial proteins [1], all of which induce potent directional migration of mammalian phagocytic leukocytes. This chapter will review those peptides that interact with members of the formylpeptide receptor (FPR) family. Although these peptides are structurally unrelated, they have similar functional effects. The bacterial and synthetic analogs of formylHandbook of Biologically Active Peptides

FORMYLPEPTIDE RECEPTORS In order to discuss the peptide ligands that share the members of the formylpeptide receptor family, we will describe the receptors expressed by humans and mice. In humans, there are three genes encoding functional

547

548 / Chapter 78 formylpeptide receptors, FPR, FPRL1, and FPRL2 [25, 27]. All three genes cluster on chromosome 19q13.3. FPR and FPRL1 share 69% identity at the amino acid level, while FPRL2 has 56% amino acid sequence identity to human FPR and 83% to FPRL1. Both FPR and FPRL1 are expressed at high levels in peripheral blood monocytes and neutrophils, whereas FPRL2 is preferentially expressed in monocytes and immature dendritic cells (DC) [38]. In addition to phagocytic leukocytes, some nonhematopoietic cells, including hepatocytes [23], fibroblasts [36], and neuroblastoma [41] and glioblastoma cells [42], have recently been found to also express one or more functional formylpeptide receptors. In different mammalian species, the number of genes coding for the formylpeptide receptor family varies. The mouse FPR gene family has at least six members, as opposed to only three in humans. mFPR1, encoded by Fpr1, is the mouse ortholog of human FPR, whereas both Fpr-rs2 and Fpr-rs1 encode receptors that

TABLE 1.

are structurally and functionally most similar to human FPRL1 [10]. The Fpr-rs2 product mFPR2 is an fMLF receptor, whereas the product of murine Fpr-rs1 has been reported to function as a receptor for the lipid mediator lipoxin A4 (LXA4) [34]. The other four mouse formylpeptide receptor genes are expressed in leukocytes but encode putative receptor proteins without characterized agonists. The high sequence divergence between species orthologs (∼30% between human and mouse) is indicative of the complex evolution of the FPR gene family, which is a common feature for immunoregulatory proteins.

AGONIST PEPTIDES OF FPRs The peptide agonists described thus far for FPRs are derived from four major sources—that is, microbial pathogens, random peptide libraries, mammalian cells, and amphibian antibacterial peptides (Table 1).

Formylpeptide receptor agonists.

Peptides

Origin

Human receptors

References

Bacterial peptides fMLF and analogues Hp(2-20)

E. Coli, S. Aureus Helicobacter pylori

FPR, FPRL1 FPRL1, FPRL2

[22] [5]

HIV-1 env peptides T20/DP178 T21/DP107 N36 F peptide V3 peptide

HIV-1LAV gp41 (aa643-678) HIV-1LAV gp41 (aa558-595) HIV-1LAV gp41 (aa546-581) HIV-1Bru gp120 (aa414-434) HIV-1MN gp120 (V3 loop)

FPR FPR, FPRL1 FPRL1 FPRL1 FPRL1

[31] [30] [16] [6] [29]

Peptide library derived agonists W peptide (WKYMVm) WKYMVM MMK-1

Random peptide library Random peptide library Random peptide library

FPR, FPRL1, FPRL2 FPRL1, FPRL2 FPRL1

[3, 14] [12]

FPRL1 FPRL1 FPR

[39] [13] [33]

FPR, FPRL1, FPRL2 FPRL1 FPRL1 FPRL1 FPRL1 FPRL1

[37, 26, 8] [28] [32] [15] [20] [41]

sCKβ8-1

hCAP18 (aa1-37) Mouse LL-37 orthologue Neutrophil granule Annexin I (aa1-26) Annexin I (aa9-25) Annexin I (aa1-25) uPAR (aa88-274) Acute phase protein APP (aa1-42) Prion (aa106-126) Neuronal tissue N-terminus of heme-binding protein Truncated CCL23/MIPIF-1

FPRL2, FPRL1 FPRL1

[24] [7]

Amphibian peptides Temporin A Rana-6

Frog skin Frog skin

FPRL1 FPRL1

[2]

Mammalian peptides LL-37 CRAMP Cathepsin G Ac1-26 Ac9-25 Ac1-25 D2D388-274 SAA Aβ42 PrP106-126 Humanin F2L

Chemotactic Peptide Ligands for Formylpeptide Receptors Influencing Inflammation

Microbial Peptides In addition to the bacterial fMLF, a Helicobacter pylori peptide Hp(2-20) activates two FPRs, FPRL1 and FPRL2, and may contribute to the development of pyloritis by recruitment of monocytes and basophils to the gastric mucosa in response to bacterial infection [5]. Moreover, HIV-1 envelope proteins contain domains capable of interacting with either or both FPR and FPRL1, including at least three domains in gp41, as well as two sequences from gp120. While T20/DP178 from gp41 specifically activates human FPR in vitro [31] and the murine FPR homologue mFPR1 in vivo [11], T21/DP107 from gp41 uses both FPR and FPRL1 with higher efficacy on FPRL1 [30] and N36 from gp41, which partially overlaps with T21/DP107 and solely signals through FPRL1 [16]. Two peptide domains in HIV-1 gp120 are potent chemoattractants and activators for FPRL1, but not for FPR, in human phagocytic leukocytes [6]. One peptide domain, F peptide, consists of 20 amino acid residues and is located in the C4-V4 region of gp120 of the HIV-1 LAI strain. Another peptide of 33 amino acids (V3 peptide) was derived from a linear sequence of the V3 region of the HIV-1 MN strain [29]. Despite the existence of peptide domains that interact with formylpeptide receptors in HIV-1 envelope proteins, it remains unclear whether such domains are released by enzymatic cleavage of the envelope proteins in vivo during HIV-1 infection.

Agonists from Peptide Libraries Random small peptide libraries have been a rich source of formylpeptide receptor agonists. They have yielded a number of potent chemotactic stimulants for leukocytes. For instance, WKYMVm, a hexapeptide representing a modified sequence isolated from a random peptide library, was initially reported to be an efficacious stimulant of human B lymphocytes and monocytic cell lines, as well as peripheral blood neutrophils. It was subsequently found that WKYMVm uses both FPR and FPRL1, with a markedly higher potency for FPRL1, to chemoattract and activate human phagocytic cells [14]. The WKYMVm analog produced by converting the Damino acid methionine at the C-terminus into an Lamino acid, becomes a more selective agonist of FPRL1 and also a weaker activator of FPRL2 [3]. Another peptide, MMK-1, which is also derived from a random peptide library, is a potent and very selective chemotactic agonist for FPRL1 [12].

MAMMALIAN PEPTIDES Peptides Associated with Amyloidogenic Diseases At least three amyloidogenic polypeptides associated with chronic inflammation and amyloidosis have been

/ 549

identified as agonists for FPRL1. Serum amyloid A (SAA), an acute phase protein that increases its serum concentration by 1000-fold during inflammation, is the first mammalian cell-derived chemotactic peptide ligand identified for FPRL1 [32]. Subsequently, the 42amino-acid form of Aβ42, which is a cleavage product of the amyloid precursor protein in the brain and a causative factor in Alzheimer’s disease [15], was also found to activate FPRL1. An additional amyloidogenic diseaseassociated FPRL1 agonist is a prion protein fragment PrP106-126, which is produced in human brains with prion disease [20]. FPRL1 mediates the migration and activation of monocytic phagocytes, including macrophages and brain microglia, induced by these agonists. Moreover, FPRL1 promotes the endocytosis of the agonists by macrophages and microglia in the form of receptor and ligand complexes. If the exposure of macrophages to Aβ42 is transient, the internalized Aβ42 is degraded and FPRL1 is rapidly recycled back to the cell surface. However, prolonged exposure results in accumulation of Aβ42 and FPRL1 complex in macrophages, which culminates in progressive fibrillary aggregation of Aβ42 and the macrophage death [40]. These observations suggest that FPRL1 not only mediates the proinflammatory activity of the peptide agonists associated with amyloidogenic diseases, it may also play an important role in the fibrillary deposition of the peptides, a typical pathologic feature of the diseases that causes tissue and organ destruction [4]. In addition, FPRL1 may exert a neuroprotective effect by recognizing a small peptide, humanin [41], which is derived from a cDNA cloned from tissues of a relatively healthy region of an Alzheimer’s disease brain. Humanin is chemotactic for monocytes through the use of FPRL1, and by blocking the receptor abrogates the intracellular fibrillary aggregation of Aβ42. In neuronal cells, humanin protects the cells from apoptosis induced by Aβ42. The blockade of Aβ42 aggregation in monocytes and its neurotoxicity by humanin was apparently through its competitive occupation of FPRL1 in both cell types. Thus, FPRL1 may transduce life and death signals in neuronal cells, depending on the nature of the agonists it encounters, and may determine the outcome of Alzheimer’s disease.

Peptides Associated with Inflammatory and Antibacterial Responses Urokinase-type plasminogen activator (uPA) contains a peptide domain that is a chemotactic agonist of FPRL1. uPA as a serine protease is best known for its ability to regulate fibrinolysis, but it also participates in tissue remodeling and tumor invasion. uPA is required for leukocyte trafficking to sites of inflammation in vivo and it indirectly activates FPRL1 through the liberation

550 / Chapter 78 of a chemotactic peptide D2D388-274 from uPA receptor (uPAR) (CD87) [28]. Consistent with this, the presence of both uPAR and FPRL1 on the cell surface is required for the chemotactic activity of uPA, whereas FPRL1 alone is sufficient for the effect of D2D388-274. Thus, uPAR may facilitate fibrinolysis as well as serve as a source of chemotactic proinflammatory peptides necessary for host defense. Formylpeptide receptors interact with several bactericidal peptides contained in human neutrophil granules. LL-37, an enzymatic cleavage fragment of the neutrophil granule-derived cathelicidin [39] and its mouse homolog CRAMP [13] are agonists for FPRL1. Another antibacterial granule protein, cathepsin G, which is a serine protease and participates in wound healing, is a specific agonist for FPR [33]. The capacity of antimicrobial neutrophil granule peptides, by interacting with formylpeptide receptors, may aid in the recruitment of additional phagocytic leukocytes to sites of infection and thus accelerate the killing and clearance of the invading bacteria. It is intriguing that some formylpeptide receptor agonists have dual roles in inflammatory host responses. Annexin I (lipocortin I) is a glucocorticoid-regulated protein possessing both pro- and anti-inflammatory activity that may be mediated in part by activation of FPR [37]. Expressed in a variety of cell types, annexin I is particularly abundant in neutrophils, where it is expressed on the outer cell surface and serves to inhibit transendothelial migration. Although both annexin I holoprotein and its N-terminal peptides (Ac1-26 and Ac9-25) are FPR agonists, at low concentrations they elicit Ca2+ transients through FPR without fully activating the MAP kinase pathway. This is associated with neutrophil desensitization and inhibition of transendothelial migration induced by other chemoattractants such as the chemokine IL-8. In contrast, at high concentrations, annexin I peptides fully activate neutrophils in vitro and become potent proinflammatory stimulants. The antimigratory activity of exogenous and endogenous annexin I has been shown in both acute and chronic models of inflammation. FPR knockout mice [26] exhibit normal neutrophil accumulation during thioglycolate-elicited peritonitis. However, a significant reduction in intraperitoneal neutrophil infiltration observed in annexin I-treated wild-type mice was abolished in FPR-/- mice. More recent studies revealed that annexin peptides may also use FPRL1 and, to a lesser extent, FPRL2 [8], suggesting a complex role for formylpeptide receptors in annexin-mediated regulation of host responses. FPRL1 may also interact with a chemokine variant that potently activates phagocytic leukocytes. A chemokine CKbeta8-1 (CCL23/MPIF-1) uses a typical G-

protein coupled receptor CCR1 for its leukocyte chemotactic activity. However, an N-terminally truncated form of the CKbeta8 splice variant CKbeta8-1 (22-137 aa) activates myeloid cells and FPRL1 transfected cell lines at a low nanomolar concentration range and thus is considered one of the most potent FPRL1 agonists identified so far [7]. It remains unclear from which cell sources and under what circumstances this FPRL1 agonist would be released in vivo. It is relevant that the identity of a potential and highly efficacious agonist peptide for FPRL2 has recently been isolated from spleen extracts. The agonist, termed F2L, is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers typical G-protein-mediated intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of ERK 1/2 MAP kinases [24]. F2L also chemoattracts and activates monocytederived DCs. Thus, F2L appears to be a novel and unique natural chemoattractant peptide for FPRL2 in DCs and monocytes, in agreement with the selective expression of FPRL2 in these cells [38]. Therefore, the host-derived FPRL2 agonist peptide may play a role in linking innate and adaptive immune responses by activating antigen-presenting FPRL2+ DCs, which express reduced levels of FPR and FPRL1.

Frog Skin Peptides Temporin A (TA) is a frog-derived antimicrobial peptide which was found to induce the migration of human monocytes, neutrophils, and macrophages [2] (see chapter on temporins in the amphibian peptides section). Characterization of the signaling characteristics of TA in monocytes and the use of a receptor transfected HEK293 epithelial cell line revealed that this peptide uses FPRL1 as a receptor. TA is also chemotactic in vivo since it elicited infiltration of neutrophils and monocytes into the injection site in mice. Another temporin peptide Rana-6 was chemotactic for human phagocytes by using FPRL1. Thus, frog-derived temporins have the capacity to chemoattract phagocytes through human FPRL1 and the mouse homolog mFPR2, suggesting the participation of amphibian antimicrobial peptides in host antimicrobial innate immunity. Since the expression of formylpeptide receptor homolog(s) has not been reported in species other than mammals, it will be interesting to clarify whether such a receptor exists in amphibians and other species, such as insects that normally rely on the secretion of antimicrobial peptides as a natural host defense.

Chemotactic Peptide Ligands for Formylpeptide Receptors Influencing Inflammation

PERSPECTIVES The identification of novel agonist peptides from various sources has promoted considerable progress in the understanding of the biological roles of formylpeptide receptors. The original hypothesis that the formylpeptide receptors may be involved in host antimicrobial defense was supported by increased susceptibility of FPR gene knockout mice to bacterial infection. This was further supported by the observations that many antibacterial peptides, derived from mammalians and frogs, are able to chemoattract and activate phagocytic leukocytes, presumably to promote host antibacterial responses. The use of FPRL1 by peptides associated with amyloidogenic diseases suggests that this receptor may not only contribute to the proinflammatory aspects of these diseases but also affect the process of peptide aggregation and amyloid deposition. In addition, some formylpeptide receptor agonists when injected in mice enhanced specific antibody response suggesting that these agonists may potentially act as adjuvants of adaptive immunity [2, 35]. The newly identified agonists for formylpeptide receptors do not share substantial amino acid sequence homology between one another, but they seem to possess “molecular patterns” recognized by common receptors. While a full understanding of the role of the formylpeptide receptors and their agonist peptides in disease states requires further investigation, these molecules constitute a group of pharmacological targets for the development of therapeutic agents for diseases, which may benefit from either the enhancement or suppression of the ligand-receptor interaction.

Acknowledgments

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

The authors thank Wanghua Gong and Nancy M. Dunlop for technical support, and C. Fogle and C. Nolan for secretarial assistance. [14]

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552 / Chapter 78 [18] Le, Y., Oppenheim, J. J., and Wang, J. M. (2001). Pleiotropic roles of formyl peptide receptors. Cytokine Growth Factor Rev. 12: 91–105. [19] Le, Y., Yang, Y., Cui, Y., Yazawa, H., Gong, W., Qiu, C., and Wang, J. M. (2002). Receptors for chemotactic formyl peptides as pharmacological targets. Intl. Immunopharm. 2: 1–13. [20] Le, Y., Yazawa, H., Gong, W., Yu, Z., Ferrans, V. J., Murphy, P. M., and Wang, J. M. (2001). The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1. J. Immunol. 166: 1448–1451. [21] Le, Y., Zhou, Y., Iribarren, P., and Wang, J. M. (2004). Chemokines and chemokine receptors: their manifold roles in homeostasis and disease. Cell. Mol. Immunol. 1: 95–104. [22] Marasco, W. A., Phan, S. H., Krutzsch, H., Showell, H. J., Feltner, D. E., Nairn, R., Becker, E. L., and Ward, P. A. (1984). Purification and identification of formyl-methionyl-leucylphenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J. Biol. Chem. 259: 5430– 5439. [23] McCoy, R., Haviland, D. L., Molmenti, E. P., Ziambaras, T., Wetsel, R. A., and Perlmutter, D. H. (1995). N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation. J. Exp. Med. 182: 207–217. [24] Migeotte, I., Riboldi, E., Franssen, J. D., Gregoire, F., Loison, C., Wittamer, V., Detheux, M., Robberecht, P., Costagliola, S., Vassart, G., Sozzani, S., Parmentier, M., and Communi, D. (2005). Identification and characterization of an endogenous chemotactic ligand specific for FPRL2. J. Exp. Med. 201: 83–93. [25] Murphy, P. M. (1996). The N-formyl peptide chemotactic receptors. In: Horuk R, editor. Chemoattractant ligands and their receptors. CRC Press, Inc., Boca Raton, FL, p. 269–299. [26] Perretti, M., Getting, S. J., Solito, E., Murphy, P. M., and Gao, J. L. (2001). Involvement of the receptor for formylated peptides in the in vivo anti-migratory actions of annexin 1 and its mimetics. Am. J. Pathol. 158, 1969–1973. [27] Prossnitz, E. R., and Ye, R. D. (1997). The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function. Pharmacol. Ther. 74: 73–102. [28] Resnati, M., Pallavicini, I., Wang, J. M., Oppenheim, J., Serhan, C. N., Romano, M., and Blasi, F. (2002). The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc. Natl. Acad, Sci. (USA). 99: 1359– 1364. [29] Shen, W., Proost, P., Li, B., Gong, W., Le, Y., Sargeant, R., Murphy, P. M., Van Damme, J., and Wang, J. M. (2000). Activation of the chemotactic peptide receptor FPRL1 in monocytes phosphorylates the chemokine receptor CCR5 and attenuates cell responses to selected chemokines. Biochem. Biophys. Res. Commun. 272: 276–283. [30] Su, S. B., Gao, J-L., Gong, W., Dunlop, N. M., Murphy, P. M., Oppenheim, J. J., and Wang, J. M. (1999). T21/DP107, A synthetic leucine zipper-like domain of the HIV-1 envelope gp41, attracts and activates human phagocytes by using Gprotein-coupled formyl peptide receptors. J. Immunol 162: 5924– 5930.

[31] Su, S. B., Gong, W. H., Gao, J. L., Shen, W. P., Grimm, M. C., Murphy, P. M., Oppenheim, J. J., and Wang, J. M. (1999). T20/ DP178, an ectodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte N-formyl peptide receptor. Blood 93: 3885–3892. [32] Su, S. B., Gong, W., Gao, J-L., Shen, W., Murphy, P. M., Oppenehim, J. J., and Wang, J. M. (1999). A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells. J. Exp. Med. 189: 395–402. [33] Sun, R., Iribarren, P., Zhang N., Zhou, Y., Gong, W., Cho, E. H., Lockett, S., Chertov, O., Rogers, T. J., Oppenheim, J. J., and Wang, J. M. (2004). Identification of neutrophil granule protein cathepsin G as a novel chemotactic agonist for the G proteincoupled formyl peptide receptor. J. Immunol. 173: 428–436. [34] Takano, T., Fiore, S., Maddox, J. F., Brady, H. R., Petasis, N. A., and Serhan, C. N. (1997). Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors. J. Exp. Med. 185: 1693–1704. [35] Tani, K., Murphy, W. J., Chertov, O., Oppenheim, J. J., and Wang, J. M. (2001). The neutrophil granule protein cathepsin G activates murine T lymphocytes and upregulates antigenspecific IG production in mice. Biochem. Biophys. Res. Commun. 282: 971–976. [36] VanCompernolle, S. E., Clark, K. L., Rummel, K. A., and Todd, S. C. (2003). Expression and function of formyl peptide receptors on human fibroblast cells. J. Immunol. 171: 2050–2056. [37] Walther, A., Riehemann, K., and Gerke, V. (2000). A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell 5: 831–840. [38] Yang, D., Chen, Q., Gertz, B., He, R., Phulsuksombati, M., Ye, R. D., and Oppenheim, J. J. (2002). Human dendritic cells express functional formyl peptide receptor-like2 (FPRL2) throughout maturation. J. Leukoc. biol. 72: 598–607. [39] Yang, D., Chen, Q., Schmidt, A. P., Anderson, G. M., Wang, J. M., Wooters, J., Oppenheim, J. J., and Chertov, O. (2000). LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192: 1069–1074. [40] Yazawa, H., Yu, Z-X., Takeda, K., Le, Y., Gong, W., Ferrans, V. J., Oppenheim, J. J., Li, C. C., and Wang, J. M. (2001). Beta amyloid peptide (Abeta42) is internalized via the G-protein-coupled receptor FPRL1 and forms fibrillar aggregates in macrophages. FASEB J. 15: 2454–2642. [41] Ying, G., Cui, Y., Zhou, Y., Gong, W., Zhang, N., Iribarren, P., Yu, Z., Le, Y., and Wang, J. M. (2004). Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. J. Immunol. 172: 7078–7085. [42] Zhou, Y., Bian, X., Le, Y. Y., Gong, W., Zhang, X., Wang, L., Iribarren, P., Salcedo, R., Howard, O. M. Z., Farrar, W., and Wang, J. M. (2005). Formylpeptide receptor FPR and the rapid growth of malignant human gliomas. J. Nat. Cancer. Inst. 97: 823–835.

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79 Complement-Derived Inflammatory Peptides: Anaphylatoxins DE YANG

olysis. At the beginning of the twentieth century, anaphylatoxin was first described by Friedberger, who demonstrated that treatment of fresh serum with immune precipitates resulted in the formation of a substance that elicits anaphylactic shock in animals and speculated that the substance was derived from complement [13]. In the first half of the twentieth century, it was established that complement contained more than one proteinaceous component. The 1960s and 1970s witnessed the purification and characterization of the individual components of the complement system including the identification of anaphylatoxic peptides and the establishment of the classic and alternative pathways of the complement activation cascade, as well as the biochemical characterization of the receptors used by complement components including the anaphylatoxins [21, 25]. The genes for the complement components, anaphylatoxin, and their receptors were cloned in the 1980s and 1990s [28]. We will cover only C3a and C5a as anaphylatoxins. Although C4a is also generally considered an anaphylatoxin [21, 25], C4a has only been shown to bind to the C3a receptor (C3aR) with low affinity, and no C4aspecific receptor has been identified. In addition, human C4a has no agonistic effect on human C3aR despite showing anaphylactic activity in guinea pigs [23].

ABSTRACT Anaphylatoxins are complement-derived, 74∼77 aa residue-long, cationic, inflammatory peptides that can cause anaphylactic reaction. This chapter covers various aspects of anaphylatoxins, with a particular focus on their genes, expression and generation, structure, and receptors, as well as or the biological actions responsible for their inflammatory activities and implications in diseases.

HISTORICAL PERSPECTIVE AND SCOPE Anaphylatoxin is a substance derived from complement activation that causes smooth muscle contraction, capillary leakage, and even anaphylactic shock. Historically, the development of our knowledge of anaphylatoxin is closely linked with the advancement in the understanding of complement system, which parallels the history of immunology. In the late nineteenth century, two theories on the mechanisms involved in protecting the host from microbial attack emerged: the “cellular theory,” based on the existence of blood cells capable of ingesting bacteria, and the “humoral theory,” based on the capacity of cell-free serum to cause bacteriolysis. Toward the end of the nineteenth century, Bordet, working at the Pasteur Institute, found that serum from guinea pigs immunized with cholera organisms lost its bacteriolytic activity after heating but that the activity could be fully restored by the addition of nonimmune serum [4]. Bordet hypothesized that two humoral factors were involved, one of which was heatliable and the other a thermostable substance present in immune serum. Later, the thermostable factor present only in immune serum was identified as antibody and the heat-labile factor present in both nonimmune and immune sera was termed complement, since it “complemented” the activity of antibody in bacteriHandbook of Biologically Active Peptides

PRECURSOR mRNA/GENE STRUCTURE, EXPRESSION, AND REGULATION OF ANAPHYLATOXINS The genome organization, transcription, translation, and processing of human C3 and C5 are schematically illustrated in Fig. 1. Human C3 and C5 structural genes are located on chromosome 19p13.3-p13.2 and 9q33, respectively [30, 31]. Although C3 (41 kb) and C5 (79 kb) genes are quite different in size, their

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554 / Chapter 79 fore, C3 is considered one of the prominent acute response proteins. The expression of C3 is regulated at both transcriptional and posttranscriptional levels. In contrast, proinflammatory cytokines and LPS do not modulate the hepatic expression of C5.

GENERATION OF C3a AND C5a ANAPHYLATOXINS

FIGURE 1. Schematic illustration of the genome, synthesis, and processing of the precursors of human anaphylatoxins. The vertical bars in the genome represent exons.

intron-exon organization is very similar, with each consisting of 41 exons [7, 11]. Transcription of the genes leads to the generation of C3 and C5 mRNAs of approximately 5.0∼5.4 kb, which contain C3- and C5-coding sequences of 4992 and 5031 bases (including three bases of the stop codon), respectively [10, 17]. The major site for both C3 and C5 synthesis is hepatocytes, however, many other cell types, such as monocytes/macrophages and alveolar type II epithelial cells, can also produce C3 and C5 [2, 12, 25, 28]. Translation of C3 mRNA gives rise to a single-chain C3 precursor of 1663 amino acid (aa) residues consisting of a 22-aa leader peptide, a 645-aa β chain peptide, a 4-aa (ArgArg-Arg-Arg) linker sequence, and a 992-aa α chain peptide [10]. Similarly, C5 mRNA is translated into a single-chain precursor of 1676 aa including an 18-aa leader peptide, a 655-aa β chain peptide, a 4-aa (ArgPro-Arg-Arg) linker sequence, and a 999-aa α chain peptide [17]. Both the leader peptide and the linker sequence are removed during intracellular processing, leading to the generation of mature C3 or C5, that possesses a similar two-chain structure held together by one interchain disulfide bond and noncovalent interactions [10, 17, 25, 28]. Mature C3 and C5 are secreted into the extracellular fluids. In the serum, C3 is the most abundant complement component with a concentration ranging from 1 to 1.5 mg/ml, whereas C5 is present at approximately 70∼80 μg/ml [12, 25, 28]. The expression of C3 by many types of cells can be up-regulated by inflammatory stimuli such as proinflammatory cytokines (e.g., TNFα, IL-1, IL-6, and IFNγ) and microbial products (e.g., LPS) [28]. There-

C3a and C5a are generated in the course of complement activation [12, 21, 25, 28]. In order to understand how C3a and C5a are generated, we will briefly describe the complement activation cascade. The complement system is activated through three pathways termed classic, lectin, and alternative pathways, as illustrated in Fig. 2A (reviewed in detail [28]). Although initiated uniquely and differing in the generation of C3 and C5 convertases, the three activation pathways converge on the activation of C5. The classic pathway is initiated by the activation of C1, which is achieved primarily by the binding of C1 to the CH2 region of certain antibodies (IgG1∼3 and IgM) that have complexed with antigens. In contrast, the lectin pathway is initiated by the activation of mannose-binding lectin-associated serine proteases, which is triggered by the binding of mannose-binding lectin to mannose-containing carbohydrate moieties on the microbial cell surfaces. Activated C1 and MASP sequentially cleave C4 and C2, leading to the generation of C4b2a C3 convertase. The alternative pathway is initiated by many microbial cell wall components, such as zymosan, LPS, and teichoic acid. The exact mechanism of initiation of the alternative pathway is not completely understood; however, the most likely explanation is the “tickover” hypothesis. This hypothesis is based on the continuous low-rate hydrolysis of circulating C3 to produce a molecule, C3(H2O), which structurally and functionally resembles C3b but is extremely unstable unless it binds to the surface of a microbial cell membrane. Subsequently, C3(H2O), in the presence of factor B, D, and properdin, converts to C3bBbP, the C3 convertase of the alternative complement activation pathway. Once C3 is cleaved by C3 convertases, C3b binds with C3 convertase to form a complex termed C5 convertase [either C4b2a3b or C(3b)nBbP], which, in turn, cleaves C5. C5b continues the complement activation cascade by binding to C6∼8 and multiple C9 to ultimately form a cylindrical transmembrane channel, C5b678(9)n, which is termed the membrane attack complex, for the destruction of target cell membrane. In the complement activation cascade, cleavage of either C3 or C5 by C3 or C5 convertases occurs at a site close to the N-termini of their α chains [12, 21, 25, 28], leading to the generation of a big fragment, C3b or C5b, and a small peptide fragment, C3a or C5a (Fig.

Complement-Derived Inflammatory Peptides: Anaphylatoxins / 555

FIGURE 3. Structure of anaphylatoxins. A. Primary sequences of human C3a (NCBI accession No. = P01024) and C5a (NCBI accession No. = P01032) with the C-terminal arginine residue colored. B. The tertiary structure of human C5a. Green: α-helices; Blue: N-terminus, the three loops connecting the α-helices, and the C-terminal tail. Brown: disulfide bonds. (See color plate.)

STRUCTURE OF ANAPHYLATOXINS

FIGURE 2. Generation of anaphylatoxins. A. Outline of the complement activation cascade. Overlined components represent activated enzymes. Abbreviations used: AAC, antigen-antibody complex; MBL, mannose-binding lectin; MASP, MBL-associated serine protease. B. Cleavage of the α chain of C3 or C5 by respective convertases leads to the release of C3a or C5a anaphylatoxin.

2B). C3b and C5b continue the complement activation cascade on the surface of microbial or cell surfaces, whereas C3a and C5a are released into the fluid phase to act as anaphylatoxins. The life span of C3a and C5a in vivo is limited by carboxypeptidase N, an enzyme in the plasma that cleaves off the C-terminal Arg residue from both C3a and C5a, leading to the formation of desArg derivatives, C3adesArg and C5adesArg. Although C3adesArg is inactive, C5adesArg retains about 1% of C5a’s anaphylactic activity.

Anaphylatoxins purified from many vertebrate species are 74∼77-aa residue peptides, highly cationic (with pI’s of 8.6~9.6), and characteristically contain six cysteine residues [21, 25, 28]. The primary sequences of human C3a (77 aa) and C5a (74 aa) are shown in Fig. 3A. Elucidation of the tertiary structures of C3a and C5a by x-ray crystallography and nuclear magnetic resonance spectroscopy has revealed that both C3a and C5a adopt a compact globular folding, which consists of four antiparallel α-helices connected by three peptide loops [27, 33], as illustrated by the structure of human C5a (Fig. 3B). The globular core of four antiparallel α-helices is stabilized by three intrachain disulfide bonds, whereas the C-terminal tail is relatively disordered and flexible (Fig. 3B). For both C3a and C5a, the C-terminal tail, particularly the C-terminal pentapeptide (LGLAR for C3a and MQLGR for C5a), plays an important role in mediating both binding and activation of their receptors [16, 20, 21, 24, 25, 33]. However, other regions of the surface of the globular core, such as the first and second loops, also participate in receptor binding [8, 16, 20, 24, 27, 33].

556 / Chapter 79 RECEPTORS FOR ANAPHYLATOXINS C3a and C5a exert their biological actions by binding to their receptors. To date, one C3a receptor (C3aR) and two C5a receptors (C5aR, also known as CD88, and C5L2) have been cloned and characterized [3, 5, 6, 9, 15]. As illustrated by Fig. 4, all three receptors belong to the family of G-protein coupled receptors (GPCRs), which share a common structural motif of seven transmembrane domains joined together by three extracellular and three intracellular hydrophilic loops. These receptors also have an extracellular N-terminal portion that contains an N-glycosylation site, and an intracellular C-terminal tail that harbors multiple Ser/Thr phosphorylation sites. C3aR contains a large second extracellular loop. C3aR is expressed by many tissues including those of the central nervous system and by myeloid leukocytes such as neutrophils, eosinophils, basophils, mast cells, and monocytes/macrophages [3, 9, 21, 25, 28]. C5aR is expressed by many leukocytes such as neutrophils, eosinophils, basophils, mast cells, monocytes/macrophages, dendritic cells, and a certain subset of lymphocytes, as well as nonleukocytes such as endothelial, epithelial, glial, and blood vessel smooth muscle cells [5, 15, 20, 21, 25, 28, 32]. The N-terminal part, the second extracellular loops, and some residues of the transmembrane domains of the receptors contribute to the binding of their respective ligands [6, 8, 20, 28]. Binding of ligands to C3aR and C5aR, in particular through interacting with the boxed residues (Fig. 4), triggers the conformational change of the receptor, which leads to the activation of heterotrimeric G proteins. Subsequently, multiple intra-

cellular kinases (e.g., PLCs, PI-3Ks, PKC, PKB, MAPKs) and small G proteins (e.g., Ras, Rac, Rho, Cdc42) are activated, which ultimately lead to diverse biological actions. Although it can bind with both C5a and C5adesArg with high affinity, C5L2 does not elicit the biological actions that C5aR does [6, 14], raising the possibility that C5L2 may act as a decoy receptor for C5a.

BIOLOGICAL ACTIONS OF ANAPHYLATOXINS Binding and activation of C3aR and C5aR by their respective ligands has diverse effects on various target cells, which play important roles in inflammation and immunity.

Anaphylatoxic Effect C3a and C5a can cause smooth muscle contraction, an increase in capillary permeability, vasodilation, and anaphylactic shock if systemically generated or applied. This anaphylatoxic effect is predominantly due to the activation of anaphylatoxin receptors on mast cells and basophils, which leads to their degranulation and release of histamine and other vasoactive or inflammatory mediators such as arachidonic acid metabolites (prostaglandins, leukotrienes, lipoxins), lipid mediators (e.g., platelet-activating factor), and the nucleoside adenosine [12, 21, 25, 28]. Together with the direct action of anaphylatoxins on smooth muscle and vessel endothelial cells, these mediators elicit constriction of bronchial, intestinal, and uterine smooth muscles,

FIGURE 4. Sequence alignment human C3aR (NCBI accession no. = Q16581), C5aR (NCBI accession no. = P21730), and C5L2 (NCBI accession no. = NP060955). The seven transmembrane domains are underlined. Residues involved in ligand-induced receptor activation in C5aR and conserved in C3aR and C5L2 are boxed. The “DRY” motif residues are both outlined and boxed. The intracellular loop 3 Ser/Thr-containing motif is shown as boxed shadow residues. The extra residues of large extracellular loop of C3aR are unbolded and budded out in order for appropriate alignment.

Complement-Derived Inflammatory Peptides: Anaphylatoxins / 557 promote local vasodilatation, augment capillary permeability, and induce mucous secretion from bronchial, gastric, and intestinal epithelial cells.

Chemotactic Effect Anaphylatoxins are potent chemoattractants and can induce the migration of leukocytes expressing their receptors [8, 12, 15, 20, 21, 25, 28, 32]. C5a is more potent than C3a, and is actually one of the most potent peptide chemoattractants that are active at concentrations as low as 0.1 nM. Both C3a and C5a are chemotactic for granulocytes (neutrophils, eosinophils, and basophils), monocytes/macrophages, and mast cells. In addition, C5a is also chemotactic for dendritic cells, germinal center B cells, and T cells [22, 26, 32]. Thus, anaphalatoxins participate in the recruitment of various leukocytes into sites of inflammation during infection and tissue injury.

Phagocyte-Activating Effect Anaphylatoxins act as strong activators for neutrophils and monocytes/macrophages [12, 21, 28]. Activation of neutrophils by anaphylatoxins increases their adhesiveness, causes neutrophil aggregation, triggers the release of antimicrobial substances such as lysozyme, defensins, and cathelicins, and remarkably stimulates neutrophil oxidative metabolism and the production of toxic reactive oxygen species. Activation of monocytes/macrophages by anaphylatoxins augments their cytotoxic activity and stimulates their production of inflammatory mediators such as cytokines and chemokines.

and an impaired inflammatory response in experimental reverse Arthus reactions [18, 19]. Anaphylatoxins are associated with nearly all types of inflammation [14, 21, 25, 28, 29]. Their inflammatory effects, particularly when excessive, are harmful and play a role in the pathogenesis of many diseases such as anaphylaxis, adult respiratory distress syndrome, acute transplant ejection, ischemia reperfusion, sepsis, brain inflammation, and many autoimmune diseases like psoriasis, rheumatoid arthritis, and systemic lupus erythematosus [28, 29]. Blockade of anaphylatoxins by neutralizing antibody or antagonists ameliorates the pathogenesis and symptoms of these diseases in experimental animal models. Therefore, great effort has been made for the development of effective antagonists for anaphylatoxins [1, 29]. Thus, anaphylatoxic peptides have moved from gross observation to the understanding of their genomic complexity, regulation at the molecular level of their expression and action, as well as roles in inflammation and immunity in the twentieth century, and finally culminating in the twenty-first century with the development of molecularly targeted antagonists for the potential treatment of various inflammatory diseases.

Acknowledgment I thank Drs. Joost J. Oppenheim and O. M. Zack Howard (National Cancer Institute, National Institute of Health, USA) for critical reading of the manuscript. This publication has been funded in whole or in part with federal funds from the National Cancer Institute, National Institute of Health under DHHS#NO1-CO-12400.

References PATHOPHYSIOLOGICAL IMPLICATIONS OF ANAPHYLATOXINS Based on their multiple biological actions, it is clear that the physiological function of anaphylatoxins is participation in immune defense and inflammation of the host [21, 25, 28]. During infection, anaphylatoxins contribute to the establishment of inflammatory responses with the ultimate goal of elimination of invading pathogens by facilitating bacterial killing or the induction of adaptive immunity. Therefore, it is not unexpected that individuals genetically deficient in either C3 or C5 display severe recurrent bacterial infection, likely due to the lack of both terminal membrane attack complex and anaphylatoxins. The critical role of C5a in host antimicrobial immune defense and inflammation have been clearly demonstrated by the use of C5aR knockout mice that, in comparison with wild-type mice, exhibit elevated susceptibility to mucosal bacterial challenge

[1] Allegretti M, Moriconi A, Beccari AR, Di Bitondo R, Bizzarri C, Bertini R, and Colotta F. Targeting C5a: recent advances in drug discovery. Curr Med Chem 2005;12:217–36. [2] Alper CA, Johnson AM, Birtch AG, and Moore FD. Human C′3: evidence for the liver as the primary site of synthesis. Science 1969;163:286–8. [3] Ames RS, Li Y, Sarau HM, Nuthulaganti P, Foley JJ, Ellis C, Zeng Z, Su K, Jurewicz AJ, Hertzberg RP, Bergsma DJ, and Kumar C. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J Biol Chem 1996;271:20231–4. [4] Bordet J and Gengou O. Sur l’existence de substances sensibilisatrices dans la plupart des serum antimicrobiens. Ann Inst Pasteur (Paris) 1901;15:289–302. [5] Boulay F, Mery L, Tardif M, Brouchon L, and Vignais P. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry 1991;30:2993–9. [6] Cain SA and Monk PN. The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). J Biol Chem 2002;277:7165–9. [7] Carney DF, Haviland DL, Noack D, Wetsel RA, Vik DP, and Tack BF. Structural aspects of the human C5 gene: intron/exon organization, 5′-flanking region features, and characterization of two truncated cDNA clones. J Biol Chem 1991;266:18786–91.

558 / Chapter 79 [8] Chao TH, Ember JA, Wang M, Bayon Y, Hugli TE, and Ye RD. Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function. J Biol Chem 1999; 274:9721–8. [9] Crass T, Raffetseder U, Martin U, Grove M, Klos A, Kohl J, and Bautsch W. Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells. Eur J Immunol 1996;26:1944–50. [10] de Bruijn MH and Fey GH. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci USA 1985;82:708–12. [11] Fong KY, Botto M, Walport MJ, and So AK. Genomic organization of human complement component C3. Genomics 1990; 7:579–86. [12] Frank MM. Complememt & Kinin. In: Medical Immunology (ninth ed.), edited by Stites DP, Terr AI, and Parslow TG. Stamford: Appleton & Lange, 1997, pp. 169–81. [13] Friedberger E. Weitere Untersuchungen uber Eiweissanaphylaxie. Z Immunforsch Exp Ther 1910;4:636–89. [14] Gao H, Neff TA, Guo RF, Speyer CL, Sarma JV, Tomlins S, Man Y, Riedemann NC, Hoesel LM, Younkin E, Zetoune FS, and Ward PA. Evidence for a functional role of the second C5a receptor C5L2. Faseb J 2005;19:1003–5. [15] Gerard NP and Gerard C. The chemotactic receptor for human C5a anaphylatoxin. Nature 1991;349:614–17. [16] Gerardy-Schahn R, Ambrosius D, Saunders D, Casaretto M, Mittler C, Karwarth G, Gorgen S, and Bitter-Suermann D. Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes. Eur J Immunol 1989;19:1095–102. [17] Haviland DL, Haviland JC, Fleischer DT, Hunt A, and Wetsel RA. Complete cDNA sequence of human complement pro-C5: evidence of truncated transcripts derived from a single copy gene. J Immunol 1991;146:362–8. [18] Hopken UE, Lu B, Gerard NP, and Gerard C. The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 1996;383:86–9. [19] Hopken UE, Lu B, Gerard NP, and Gerard C. Impaired inflammatory responses in the reverse Arthus reaction through genetic deletion of the C5a receptor. J Exp Med 1997;186:749–56. [20] Huber-Lang MS, Sarma JV, McGuire SR, Lu KT, Padgaonkar VA, Younkin EM, Guo RF, Weber CH, Zuiderweg ER, Zetoune FS, and Ward PA. Structure-function relationships of human C5a and C5aR. J Immunol 2003;170:6115–24.

[21] Hugli TE. Biochemistry and biology of anaphylatoxins. Complement 1986;3:111–27. [22] Kupp LI, Kosco MH, Schenkein HA, and Tew JG. Chemotaxis of germinal center B cells in response to C5a. Eur J Immunol 1991;21:2697–701. [23] Lienenklaus S, Ames RS, Tornetta MA, Sarau HM, Foley JJ, Crass T, Sohns B, Raffetseder U, Grove M, Holzer A, Klos A, Kohl J, and Bautsch W. Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor. J Immunol 1998;161:2089–93. [24] Lu ZX, Fok KF, Erickson BW, and Hugli TE. Conformational analysis of COOH-terminal segments of human C3a: evidence of ordered conformation in an active 21-residue peptide. J Biol Chem 1984;259:7367–70. [25] Muller-Eberhard HJ. Molecular organization and function of the complement system. Annu Rev Biochem 1988;57:321–47. [26] Nataf S, Davoust N, Ames RS, and Barnum SR. Human T cells express the C5a receptor and are chemoattracted to C5a. J Immunol 1999;162:4018–23. [27] Nettesheim DG, Edalji RP, Mollison KW, Greer J, and Zuiderweg ER. Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations. Proc Natl Acad Sci USA 1988;85:5036–40. [28] Volanakis JE and Frank MM. Human complement system in health and disease. New York, Marcel Dekker, 1998. [29] Ward PA. The dark side of C5a in sepsis. Nat Rev Immunol 2004;4:133–42. [30] Wetsel RA, Lemons RS, Le Beau MM, Barnum SR, Noack D, and Tack BF. Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9. Biochemistry 1988;27:1474–82. [31] Whitehead AS, Solomon E, Chambers S, Bodmer WF, Povey S, and Fey G. Assignment of the structural gene for the third component of human complement to chromosome 19. Proc Natl Acad Sci USA 1982;79:5021–5. [32] Yang D, Chen Q, Stoll S, Chen X, Howard OM, and Oppenheim JJ. Differential regulation of responsiveness to fMLP and C5a upon dendritic cell maturation: correlation with receptor expression. J Immunol 2000;165:2694–702. [33] Zuiderweg ER, Nettesheim DG, Mollison KW, and Carter GW. Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data. Biochemistry 1989;28:172–85.

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80 Chemokines: A New Peptide Family of Neuromodulators PATRICK KITABGI, STÉPHANE MÉLIK-PARSADANIANTZ, AND WILLIAM ROSTÈNE

ABSTRACT

unique ligand-receptor pairs exist. To date, more than 50 chemokines and about 20 chemokine receptors have been identified. Their classification and nomenclature can be found in several reviews [5, 7, 25]. Interestingly, some chemokine receptors have been shown to serve as coreceptors for HIV entry into cells. Clearly, a comprehensive review of all the preceding aspects for such a large family of mediators would be largely beyond the scope of this paper. The interested reader is referred to excellent recent reviews that cover these topics [2, 5, 7, 32]. It has become apparent over the past years that chemokines are involved in virtually all pathologies that present an inflammatory component. This includes nervous system pathologies, such as neurodegenerative diseases like Alzheimer’s disease; autoimmune diseases, such as sclerosis; and pathologic conditions, such as brain injury, stroke, and ischemia. Neuroinflammation induces the expression of a number of chemokines and chemokine receptors in activated astrocytes and microglia, thus suggesting their involvement in the activation of the central nervous system defense mechanisms. These aspects of chemokine action have been comprehensively reviewed elsewhere [5, 7, 29]. In addition, it has been recently recognized that some chemokines and their receptors play a role in brain development and may function as neuromodulators in the normal adult brain, acting in this respect somehow like the more classical neuropeptides discussed in this series of papers. It is this emergent concept that we would like to illustrate by reviewing recent results pertaining to the brain functions of the chemokine SDF-1 and its receptor CXCR4.

Chemokines are a family of small, inducible, secreted proteins that chemoattract and activate immune and nonimmune cells by interacting with G protein-coupled receptors on their target cells. They thus mediate immune and inflammatory responses in a wide range of pathologies, including neuroimmune and neuroinflammatory conditions. Also, some chemokines play major roles in tissue/organ development both during embryogenesis and throughout life. In particular, they take part in a number of aspects concerning brain development like neuronal migration, axonal guidance, and process formation. Recently, evidence was provided that chemokines and chemokine receptors are constitutively expressed in a highly regionalized fashion by neurons in the central nervous system where they can directly modulate neuronal activity and neurotransmitter release. It is these aspects of chemokine function that we address in this paper, taking as an example the chemokine SDF-1 and its receptor CXCR4. Chemokines are a family of peptides (60–100 aa) that have been discovered for their roles in immune and inflammatory responses. In particular, they all have the capability to chemoattract leukocytes to sites of injury or infection. They also share other effects involved in tissue repair such as hematopoiesis, angiogenesis, and tissue growth regulation. All chemokines signal through G protein-coupled receptors (GPCR). In general, several chemokines can bind to the same receptor, and, conversely, a given chemokine may recognize more than one receptor. However, there are exceptions where Handbook of Biologically Active Peptides

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560 / Chapter 80 DISCOVERY

SDF-1alpha

SDF-1 was originally cloned from a murine bone marrow stromal cell line for its ability to partially replace the need of stromal cells for the in vivo generation of B cells—hence its original name “stromal cell-derived factor” [27]. In addition to being required for B cell maturation, SDF-1 is a potent chemoattractant for T cell lymphocytes. It also directs the traffic of other cell types involved in embryogenesis and tissue regeneration and plays a role in metastasis by attracting cancer cells [19]. The more recent chemokine nomenclature attributes the name CXCL12 to this chemokine, but we will retain the more descriptive acronym SDF-1 throughout this review.

human mouse rat

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STRUCTURE OF THE PRECURSOR mRNA/GENE Two SDF-1 protein isoforms, SDF-1α and SDF-1β, that arise from alternative mRNA splicing were originally characterized in the process of cloning the chemokine [27]. Their amino acid sequence is shown in Fig. 1. SDF-1α consists of a 67-residue protein that is preceded by a 21-residue signal peptide at its Nterminus. SDF-1β is identical to SDF-1α except for a five-residue extension at the C-terminus. Recently, a third mRNA variant, termed SDF-1γ, was described in rat brain [15], but the corresponding protein has yet to be characterized. Should it be translated, SDF-1γ would be identical to SDF-1α with a 31-residue C-terminal extension (Fig. 1). Most functional data on SDF-1 were obtained with the α variant.

DISTRIBUTION OF SDF-1 mRNA AND PROTEIN SDF-1 mRNA is differentially expressed in the developing and mature CNS. At embryonic day 15, SDF-1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex [34]. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF-1 in situ hybridization signal that decreased progressively within the next two weeks. In other regions such as cortex, thalamus, and hippocampus, SDF-1 transcripts progressively increased from birth to later postnatal stages. The presence of SDF-1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF-1 mRNA signal

rat

119 ---ALNGR EEKVGKKKE KIGKKKRQKK RKAAQKKKN

FIGURE 1. Amino acid sequences of human, mouse, and rat SDF-1. The 21-aa signal peptide is highlighted in green, and the 67-aa mature SDF-1α in yellow. Species-variation in the sequences are highlighted in magenta. Note the very high degree of sequence conservation between species. The five-residue extension of mouse and rat SDF-1β is shown highlighted in blue, and the putative 31-residue extension of rat SDF-1γ in italic and gray. The first two Cys residues in the SDF-1 sequence in positions 9 and 11, separated by only one residue, are shown in red. This CXC motif is common to one of the two major subfamilies of chemokines, hence the designation CXCLi for members of this subfamily and CXCRi for their receptors. (See color plate.)

increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life [34]. More recently, we established by immunohistochemistry that SDF-1 was constitutively expressed in adult rat brain neurons in a highly regionalized fashion [9]. The chemokine was mainly found in the cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, and oculomotor nuclei. Moreover, we provided evidence that SDF-1 was constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in the substantia nigra pars compacta and the ventral tegmental area. SDF-1 was also detected in vasopressin-expressing magnocellular neurons of the supraoptic and paraventricular hypothalamic nuclei and in melanin-concentrating hormone (MCH)expressing neurons of the lateral hypothalamus [8]. These results are recapitulated in Fig. 2A.

Chemokines: A New Peptide Family of Neuromodulators / 561

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FIGURE 2. Schematic representation of the neuronal distribution (nerve cell bodies and fibers) of A. SDF-1 and B. CXCR4 proteins in the adult rat brain, as determined by immunohistochemistry. Note that in a number of brain regions, the distribution of both proteins overlaps. •, cell bodies; —, fibers.

LRN

562 / Chapter 80 PROCESSING OF THE PRECURSOR In all cells, precursors of secreted proteins such as chemokines or neuropeptides are usually targeted to early compartments of the secretory pathway—that is, the endoplasmic reticulum (ER)—by a signal peptide that is then removed by a signal peptidase while the protein is being translocated to the ER. In addition, virtually all classical neuropeptides and peptide hormones, such as those reviewed in the present series of articles, are synthesized in inactive form as part of a larger protein in which they are flanked at one or both ends by cluster(s) of basic residues. Cleavage at the basic site(s) is effected in late compartments of the regulated secretory pathway of neuroendocrine cells by proteases known as prohormone convertases. The active peptides are then stored in dense core secretory vesicles that are a landmark of neuroendocrine cells. In contrast, chemokine precursors do not undergo further cleavage events after removal of the signal peptide until they are released from the cells. Furthermore, they do not appear to be stored in the cells that produce them. Instead, they seem to be constitutively secreted at a rate that is governed by their rate of synthesis. This is consistent with the fact that chemokines are usually inducible proteins whose expression is triggered in the course of inflammation and other pathological conditions. However, the recent recognition that some chemokines like SDF-1 are expressed by neurons in the normal brain and exert neuromodulatory actions (see following) suggests that they might be stored and released by neurons in a regulated manner. This interesting question, which has yet to be investigated, clearly deserves some attention.

RECEPTORS AND SIGNALING CASCADES The sole receptor for SDF-1, CXCR4, was originally cloned from bovine Locus Coeruleus and named LCR1 for Locus Coeruleus Receptor 1 and wrongly classified as a neuropeptide Y receptor [30]. It was then cloned by others from leukocytes (thus named LESTR for Leukocyte-Derived Seven Transmembrane Receptor) [21]. It was subsequently rediscovered as a co-receptor for HIV fusion and entry into target cells and was thus also named Fusin [10, 14]. Finally, the recent nomenclature attributed the name CXCR4 to this receptor [25]. The only selective CXCR4 antagonist reported to date is a bicyclam compound, AMD3100, developed by AnorMED [17]. This compound has a relatively low affinity for CXCR4 (around 100 nM, compared with the sub-nM affinity of SDF-1). It has nonetheless proven

useful to probe the functions associated with SDF-1 and CXCR4. CXCR4 is a GPCR that preferentially couples to the Gi/Go family of G proteins to initiate adenylate cyclase inhibition and phospholipase C activation [31]. CXCR4 may also activate signaling cascades further distal from these early responses such as the ERK and JAK/STAT pathways [4, 36].

RECEPTOR EXPRESSION AND DISTRIBUTION The onset of CXCR4 mRNA expression in rats was approximately embryonic on day 9 and was high in proliferative areas of the brain during development [18, 24]. CXCR4 mRNA expression was prominent in olfactory bulbs, hypothalamus, thalamus, hippocampus, cerebellum, pituitary gland, and the spinal cord, with levels peaking before birth. Similarly, it was reported that CXCR4 mRNA was expressed in the developing mouse brain, from embryonic day 9.5 to maturity at postnatal day 21 [35]. At all stages, CXCR4 was prominently transcribed in ventricular zones of neuronal proliferation, from the spinal cord to the forebrain. Moreover, a time-dependent regulation was observed, as the expression of CXCR4 mRNA in the forebrain was delayed, relative to posterior parts of the CNS, at early stages. In macaque brain, CXCR4 was expressed in early stages of CNS ontogenesis, its expression increasing significantly from birth to 9 months of age [37]. Recently, we showed by immunohistochemistry that CXCR4 was constitutively expressed by neurons in the rat brain [6]. Neuronal expression of CXCR4 was highly regionalized and predominated in the cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus, and substantia nigra. Furthermore, we demonstrated the presence of CXCR4 in cholinergic neurons of the caudate putamen and substantia innominata, as well as in dopaminergic neurons of the substantia nigra pars compacta. CXCR4 expression was also demonstrated in vasopressin-expressing magnocellular neurons of the supraoptic and paraventricular hypothalamic nuclei [6] and in MCH-expressing neurons of the lateral hypothalamus [16]. These results are summarized in Fig. 2B and can be compared with those shown in Fig. 2A for SDF-1 localization in rat brain neurons. Interestingly, the distribution of both the chemokine and its receptor can be seen to overlap in a number of regions, and, in particular, to coincide in septal cholinergic, nigral dopaminergic, and hypothalamic peptidergic neurons.

Chemokines: A New Peptide Family of Neuromodulators / 563

CONFORMATION

BIOLOGICAL ACTIONS

The conformation of SDF-1 was solved by NMR and is depicted in Fig. 3. Its structure comprises three distinct domains that each constitutes about one third of the molecule: a highly flexible N-terminal domain that is anchored to the middle portion by two disulfide bridges, a middle portion made of three antiparallel β-pleated sheets, and a C-terminal α helix that runs across the β sheets [13]. Structure-activity studies revealed that the N-terminal region is important for receptor binding and activation [13]. Interestingly, all chemokine structures solved to date more or less conform to this three-dimensional pattern even though they may bear little homology in their primary amino acid sequence [12].

Brain Development It has been reported that SDF-1/CXCL12 and CXCR4 knockout mice die perinatally and present anomalies in cerebellar development because of abnormal inward migration of external granule cells [23, 26, 40]. Further examination of these mouse lines indicated additional defects in other brain regions, such as the hippocampus and the neocortex [22, 33]. The reported alterations in the brain architecture suggested dysfunctions in cell migration, axonal guidance, and/or process formation. Actually, all of these cellular events were found to be regulated by SDF-1. Thus, SDF-1 was shown to be involved in migration of hippocampal, cerebellar and cortical neurons [3, 33, 39], axon guidance [11, 38], and axon elongation [1]. More recently, we presented evidence that SDF-1 also regulates axonal patterning in hippocampal neurons at an early stage of neuronal development [28]. Thus, SDF-1 clearly appears as a major factor in the setting up of neuronal circuits during brain development.

Neuromodulation

FIGURE 3. Solution conformation of SDF-1 as determined by NMR. The N-terminal random coil region is represented in blue, the middle β strand portion in red, and the C-terminal α helix in green. The two disulfide bonds are depicted in yellow. (Adapted from [13].) (See color plate.)

As reported some years ago, SDF-1 may indirectly modulate neuronal activity. Thus, SDF-1-induced glutamate release from astrocytes generated Ca2+ transients in cerebellar granule cells and evoked a slow inward current in neurons recorded from cerebellar slices [20]. However, the observation that SDF-1 and CXCR4 are both expressed by discrete populations of neurons in the brain (see preceding) suggests that the chemokine could also directly modulate neuronal activity. In particular, the coexpression of both ligand and receptor in cholinergic, dopaminergic, vasopressinergic, and MCH-expressing neurons raises the question as to whether SDF-1 could modulate the release of these neuromediators. In a recent study, we provided evidence that low concentrations of SDF-1, acting via CXCR4, could depress action potential discharge of MCH-expressing neurons in the lateral hypothalamus [16]. This does not prove but strongly suggests that SDF-1 might directly modulate MCH release from hypothalamic neurons. We also obtained evidence that SDF-1 modulates the electrical activity of magnocellular vasopressin neurons in the supraoptic and paraventricular nuclei of the hypothalamus [8]. Furthermore, SDF-1 inhibited secretagog-evoked vasopressin release both in vitro and in vivo [8]. Finally we recently observed that low concentrations of SDF-1 directly modulated Ca++ currents in DA neurons of the substantia nigra and potentiated potassium-evoked DA release

564 / Chapter 80 from these neurons (Guyon A, Skrzydelski D, Banisadr G, Rovere C, Apartis E, Rostene W, Kitabgi P, MelikParsadaniantz S, Nahon JL, submitted). All the preceding effects were reversed by the CXCR4 antagonist, AMD3100. These results provide strong support to the hypothesis that chemokines might represent a new class of neuromodulators.

Note: While this chapter was submitted, the identification of the orphan receptor. RDC1 as a SDF-1 receptor in T lymphocytes was reported (Balabanian et al., J Biol Chem 2005;280:35760–35766). It is not known as yet whether this receptor is involved in SDF-1 signaling in the brain.

References PATHOPHYSIOLOGICAL IMPLICATIONS As mentioned in the introduction, there is growing evidence that chemokines are implicated in the neuroinflammatory process that accompanies a number of nervous system pathologies such as Alzheimer’s disease, multiple sclerosis, brain ischemia, and stroke. However, almost all these studies have focused on chemokine and chemokine receptor expression and activation in endothelial, astrocytic, and microglial cells. The recent realization that these mediators and their receptors are also expressed by neurons has unraveled new brain functions for chemokines. Thus, as illustrated here for SDF-1, it is clear that chemokines play key roles in brain development. Furthermore, they also appear to act as modulators of neuronal function in restricted brain areas. It is interesting that SDF-1 modulates the activity of the hypothalamic neurons that express vasopressin and MCH, two neuropeptides involved in the control of drinking and eating behaviors, respectively. These behaviors are often altered in pathological conditions and it might be speculated that SDF-1-mediated signaling in hypothalamic neurons could contribute to such alterations. Also of great interest is the observation that SDF-1 and CXCR4 are expressed by septal cholinergic and nigral dopaminergic neurons, two neuronal populations that degenerate in Alzheimer’s and Parkinson’s diseases, respectively. Both pathologies are marked by neuroinflammatory conditions in degenerating brain areas. There is evidence that some chemokines and their receptors may contribute to plaque-associated inflammation and neurodegeneration in Alzheimer’s disease [7]. Although there is no indication at present that SDF-1 and CXCR4 may contribute to these pathologies, it would certainly be worthwhile to investigate this possibility. In conclusion, the diversity of functions exerted by chemokines makes this recently identified family of peptide mediators fascinating. Exploration of chemokine actions in the brain is an emerging field which promises to yield a wealth of data pertaining to important aspects of normal brain functioning, such as development, neuro-glial communication, and neuroendocrine modulation, as well as to neurodegenerative and neuroinflammatory brain pathologies.

[1] Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemotp-Kimura S, Kimura K, Nozaki K, Hashimoto N, Narumiya S. Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons. J Cell Biol 2003; 161:381–391. [2] Baggiolini M, Loetscher P. Chemokines in inflammation and immunity. Immunol Today 2000; 21:418–420. [3] Bagri A, Gurney T, He X, Zou YR, Littman DR, Tessier-Lavigne M, Pleasure SJ. The chemokine SDF-1 regulates migration of dentate granule cells. Development 2002; 129:4249–4260. [4] Bajetto A, Barbero S, Bonavia R, Piccioli P, Pirani P, Florio T, Schettini G. Stromal cell-derived factor-1alpha induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway. J Neurochem 2001; 77:1226–1236. [5] Bajetto A, Bonavia R, Barbero S, Schettini G. Characterization of chemokines and their receptors in the central nervous system: physiopathological implications. J Neurochem 2002; 82:1311–1329. [6] Banisadr G, Fontanges P, Haour F, Kitabgi P, Rostene W, Melik Parsadaniantz S. Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons. Eur J Neurosci 2002; 16:1661–1671. [7] Banisadr G, Rostene W, Kitabgi P, Melik-Parsadaniantz S. Chemokines and brain functions. Curr Drug Targets—Inflammation & Allergy 2005; 4:387–399. [8] Banisadr G, Skrzydelski D, Callewaere C, Desarmenien M, Kitabgi P, Rostene W, Melik-Parsadaniantz S. Chemokines and chemokine receptors in the brain: possible implications in neuromodulation and neuroendocrine regulation. Am Soc for Neurosci 2004; abstract 660.6. [9] Banisadr G, Skrzydelski D, Kitabgi P, Rostene W, Parsadaniantz SM. Highly regionalized distribution of stromal cell-derived factor-1/CXCL12 in adult rat brain: constitutive expression in cholinergic, dopaminergic and vasopressinergic neurons. Eur J Neurosci 2003; 18:1593–1606. [10] Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 1996; 382:829–833. [11] Chalasani SH, Sabelko KA, Sunshine MJ, Littman DR, Raper JA. A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axonal pathfinding. J Neurosci 2003; 23:1360–1371. [12] Clore GM, Gronenborn AM. Three-dimensional structures of alpha and beta chemokines. FASEB J 1995; 9:57–62. [13] Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, Virelizier JL, Baggiolini M, Sykes BD, Clark-Lewis I. Solution structure and basis for functional activity of stromal cell-derived factor 1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. Embo J 1997; 16:6996–7007. [14] Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996; 272:872–877. [15] Gleichmann M, Gillen C, Czardybon M, Bosse F, Greiner-Petter R, Auer J, Muller HW. Cloning and characterization of SDF-

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1gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system. Eur J Neurosci 2000; 12:1857–1866. Guyon A, Banisadr G, Rovere C, Cervantes A, Kitabgi P, MelikParsadaniantz S, Nahon JL. Complex effects of stromal cellderived factor-1alpha on melanin-concentrating hormone neuron excitability. Eur J Neurosci 2005; 21:701–710. Horuk R, Ng HP. Chemokine receptor antagonists. Med Res Rev 2000; 20:155–168. Jazin EE, Soderstrom S, Ebendal T, Larhammar D. Embryonic expression of the mRNA for the rat homologue of the fusin/ CXCR-4 HIV-1 co-receptor. J Neuroimmunol 1997; 79:148– 154. Kucia M, Jankowski K, Reca R, Wysoczynski M, Bandura L, Allendorf DJ, Zhang J, Ratajczak J, Ratajczac MZ. CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion. J Mol Histol 2004; 35:233–245. Limatola C, Giovannelli A, Maggi L, Ragozzino D, Castellani L, Ciotti MT, Vacca F, Mercanti D, Santoni A, Eusebi F. SDF-1alphamediated modulation of synaptic transmission in rat cerebellum. Eur J Neurosci 2000; 12:2497–2504. Loetscher M, Geiser T, O’Reilly T, Zwahlen R, Baggiolini M, Moser B. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem 1994; 269:232–237. Lu M, Grove EA, Miller RJ. Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor. Proc Natl Acad Sci U S A 2002; 99:7090–7095. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA. Impaired Blymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 1998; 95:9448–9453. McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev Biol 1999; 213(2):442–456. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 2000; 52:145–176. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T. Defects of Bcell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996; 382: 635–638.

[27] Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc Natl Acad Sci U S A 1994; 91:2305–2309. [28] Pujol F, Kitabgi P, Boudin H. The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons. J Cell Sci 2005; 118:1071–1080. [29] Ragozzino D. CXC chemokine receptors in the central nervous system: Role in cerebellar neuromodulation and development. J Neurovirol 2002; 8:559–572. [30] Rimland J, Xin W, Sweetnam P, Saijoh K, Nestler EJ, Duman RS. Sequence and expression of a neuropeptide Y receptor cDNA. Mol Pharmacol 1991; 40:869–875. [31] Roland J, Murphy BJ, Ahr B, Robert-Hebmann V, Delauzun V, Nye KE, Devaux C, Biard-Piechaczyk M. Role of the intracellular domains of CXCR4 in SDF-1-mediated signaling. Blood 2003; 101:339–406. [32] Schols D. HIV co-receptors as targets for antiviral therapy. Curr Top Med Chem 2004; 4:883–893. [33] Stumm RK, Zhou C, Ara T, Lazarini F, Dubois-Dalcq M, Nagasawa T, Hollt V, Schultz S. CXCR4 regulates interneuron migration in the developing neocortex. J Neurosci 2003; 23:5123–5130. [34] Tham TN, Lazarini F, Franceschini IA, Lachapelle F, Amara A, Dubois-Dalcq M. Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system. Eur J Neurosci 2001; 13:845–856. [35] Tissir F, Wang CE, Goffinet AM. Expression of the chemokine receptor CXCR4 mRNA during mouse brain development. Brain Res Dev Brain Res 2004; 149:63–71. [36] Vila-Coro AJ, Rodriguez-Frade JM, Martin De Ana A, Moreno-Ortiz MC, Martinez AC, Mellado M. The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J 1999; 13:1699–1710. [37] Westmoreland SV, Alvarez X, deBakker C, Aye P, Wilson ML, Williams KC, Lackner AA. Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain. J Neuroimmunol 2002; 122:146–158. [38] Xiang Y, Li Y, Zhang Z, Cui K, Wang S, Yuan XB, Wu CP, Poo MM, Duan S. Nerve growth cone-guidance mediated by G protein-coupled receptor. Nat Neurosci 2002; 5:843–848. [39] Zhu Y, Yu T, Zhang XC, Nagasawa T, Wu JY, Rao Y. Role of the chemokine SDF-1 as the meningeal attractant for embryonic cerebellar neurons. Nat Neurosci 2002; 5:719–720. [40] Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998; 393:595–599.

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81 Immune Peptides Related to Dipeptidyl Aminopeptidase IV/CD26 SIEGFRIED ANSORGE AND DIRK REINHOLD

ABSTRACT

substrate specificity makes DP IV/CD26 and the related peptidases relatively unique among other proteases. The crystal structure of DP IV homodimers has been published recently (for review, see [11]). Each subunit consists of two domains, an α/β-hydrolase domain and an eight-bladed ß-propeller domain. The cavity of approximately 30–45 Å between the two domains is accessible via two openings. DP IV is ubiquitously distributed and highly expressed in intestine, kidney, and liver, but lower expression is detectable in nearly every tissue. Functionally relevant also is its localization on endothelial cells of all organs examined and on intestinal epithelial cells. The unique substrate specificity of DP IV results in the peptidase playing a key role in the catabolism of a number of neuropeptides, immunopeptides, and peptide hormones, discussed elsewhere in this book, containing the X-Pro or X-Ala amino terminal sequences, such as substance P, neuropeptide Y, peptide YY, enterostatin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) [8]. Recently, the emphasis of the research related to DP IV has been concentrated on its ability to cleave a dipeptide from the N-terminus of the GLP-1 (see chapters in ingestive peptides and gastrointestinal peptides sections). This incretin appears to play an important role in management of glucose levels. Therefore, GLP-1based therapies are of potential use in treatment of diabetes Type II.

Many biologically active peptides of the immune system like cytokines and chemokines are generated or inactivated by a concerted action of proteolytic enzymes. One of the most prominent peptidases is the dipeptidyl aminopeptidase IV (DP IV, CD26, EC 3.4.14.5), which is expressed on all lymphocytes and that plays a crucial role in regulation of the immune response. In this chapter, we describe the activities of biologically active peptides that are regulated by dipeptidyl aminopeptidase IV (DP IV) and that themselves may specifically inhibit this peptidase and thereby modulate the immune response.

INTRODUCTION The ectoenzyme dipeptidyl peptidase IV (DP IV, E. C. 3.4.14.5) is identical with the leukocyte surface antigen CD26 and belongs to the group of postproline dipeptidyl aminopeptidases, which consists of the gene family of DP IV, FAP (fibroblast activation protein), DP8 and DP9, and DPII (E.C.3.4.14.2) (for review see [11, 31]). DP IV is a homodimeric Type II transmembranic glycoprotein with a total molecular mass of 220–240 kD. It appears that DP IV is associated with glycolipid- and cholesterol-rich micro domains known as “rafts” [28]. FAP (Seprase) is also a dimeric peptidase expressed in the plasma membrane whereas DP8 and DP9 are soluble, monomeric cytoplasmic proteins [11]. DP IV/CD26 is a serine exopeptidase catalyzing the release of N-terminal dipeptides from oligo- and polypeptides preferentially with proline, hydroxyproline, and, with less efficiency, alanine or serine in the penultimate position (Fig. 1). The postproline cleaving Handbook of Biologically Active Peptides

DP IV/CD26 IN THE IMMUNE SYSTEM Within the immune system, DP IV/CD26 is expressed on the surface of resting and activated T cells, activated B cells, and activated NK cells [5, 11, 14, 21]. DP IV/

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568 / Chapter 81

DP IV +

H3N

FIGURE 1.

P2

P1

P1‘

Gl Gly Ala Val Leu Ile Pro ...

Pro Ala ... Hyp Ser Gly Val Leu

Pro Hyp

Pn‘

Substrate specificity of DP IV.

CD26 expression is upregulated after stimulation of T cells with mitogen, antigen, anti-CD3 antibodies, or IL-2 after B-cell stimulation with staphylococcal proteins and IL-2 stimulation of NK cells. Thus, activated antigenspecific CD4+ T-cell clones express high levels of DP IV/CD26 [27]. In 1985, Schön and coworkers [29] showed for the first time that N-Ala-Pro-O-(nitrobenzoyl)-hydroxylamine, which irreversibly inhibits DP IV, is capable of suppressing the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated with mitogens. This was the first evidence that DP IV plays a critical role in the regulation of DNA synthesis of immune cells and that the enzymatic activity of DP IV is involved in this process. In the meantime, a multitude of biochemically distinct synthetic compounds inhibiting DP IV and/or DP IV-like enzymes have been studied under different stimulation conditions with a variety of target cells. The most frequent reversible inhibitors used were Lys[Z(NO2)]-thiazolidide, -piperidide, and -pyrrolidide, which cause a 50% inhibition of DP IV in the range of 2–3 μM. The inhibitory effects of these inhibitors on leukocyte proliferation and cytokine production were confirmed in different mitogen- or antigen-stimulated T cells and T-cell clones from human and mouse [27, 30, 32]. Besides their influence on DNA synthesis, inhibitors of DP IV enzymatic activity also exhibit strong suppressive effects on the production of various cytokines. It was shown that Lys[Z(NO2)]-thiazolidide, -piperidide, and -pyrrolidide inhibit the production of IL-2, IL-10, IL-12, and IFN-γ of PWM (pokeweed mitogen)-stimulated PBMC and purified T cells (cf. [27]) as well as the production of IL-2, IL-6, and IL-10 of PHA (phytohemagglutinin)-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes (cf. [27]). These inhibitors also reduce in a dose-dependent manner IFN-γ, IL-4, and TNF-α production of myelin basic protein (MBP)-stimulated T cell clones from MS patients [27]. DP IV inhibitors, on the other hand, elicit the enhanced production and secretion of latent trans-

forming growth factor-ß1 (TGF-ß1), which partially explain the suppressive effects on DNA and cytokine synthesis ([14], cf. [27]). In vivo, the administration of DP IV inhibitors suppresses antibody production in mice after stimulation with bovine serum albumin [16], as well as hind paw swelling in an arthritis model in rats [41, 42], acute rejection in a rat cardiac transplantation model [15], and experimental allergic encephalomyelitis in mice [32]. In all, results convincingly demonstrate the involvement of DP IV or DP IV-like proteolytical activity in immune functions. It should be mentioned that in addition to DP IV, lymphocytes do express DP8 and DP9 which have a similar substrate specificity as DP IV. Both are also inhibited by a number of typical DP IV inhibitors [44]. This means that many DP IV inhibitors are selective for the DP IV family rather than DP IV itself [30]. Although little is known about the functional relevance of DP8 and DP9, it cannot be excluded that the functional effects of DP IV inhibitors described may be related, at least in part, also to their effect on these members of the DP IV family. Recent results show that combined inhibition of the DP IV and alanyl-aminopeptidases have much stronger synergistic anti-inflammatory effects compared with the DP IV inhibition alone [1].

DP IV EFFECTOR PEPTIDES Although the detailed molecular mechanism of DP IV action in the immune system is not known yet, the most probable explanation for its role lies in the processing of immunorelevant bioactive oligo- and polypeptides and/or the inhibitory effects of DP IV-binding peptides—that is, peptides with N-terminal X-Pro- or X-Ala-motifs, on the enzymatic activity of DP IV itself. During the last few years, a number of bioactive peptides were experimentally identified as substrates of DP IV. Most of them belong to the group of chemokines and endocrine factors (Table 1). However, the group of potential DP IV effector peptides is much larger. A number of bioactive polypeptides like cytokines have the N-terminal X-Pro or X-Ala sequence required for cleavage by DP IV but are not processed by soluble DP IV (Table 2). Probably their N-terminus is not susceptible under in vitro conditions. The N-terminal peptides of some, if not all, of them are, however, susceptible to DP IV cleavage after endoproteolytical processing of the native peptide. Moreover, potential DP IV substrates can be also generated from any proteins or peptides which express the -X-Pro sequence within the peptide chain, namely via endo- or exoproteolytical processing. One example is the endogenous peptide hemorphin-7 belonging to the family of atypical opioid peptides

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TABLE 1. The in vitro truncation of natural immunorelevant DP IV peptide substrates (primary DP IV effector peptides). Peptide

N-terminus

CXCL12 (SDF-1α) CCL22 (MDC) CXCL11 (I-TAC) CXCL10 (P-10) CXCL9 (Mig) CCL11 (Eotaxin) CCL5 (RANTES) CCL3L1 (LD78β) Peptide YY GRP Substance P Neuropeptide Y PACAP38 VIP PACAP27 Hemorphin 7 PCT

KP↓VSLS GP↓YGAN FP↓MFKR VP↓LSRT TP↓VVRK GP↓ASVP SP↓YSSD AP↓LAAD YP↓IKPE VP↓LPAG RP↓KPQE YP↓SKPD HS↓DGIF DA↓VFTD HS↓DGIF YP↓WTQR AP↓FRSA

TABLE 2. Secondary immunorelevant peptide substrates of DP IV. Peptide

N-terminus

Residues

IL-1β IL-1Ra IL-2 IL-3 IL-5 IL-8 IL-10 IL-11 IL-13 G-CSF GM-CSF LIF TNF-β Thrombopoietin MCP-1 (CCL2) MCP-2 MCP-3 MIP-1α (CCL3) MIP-1β (CCL4) IP-10 MIF VEGF CNTF aFGF bFGF NT-3 OSM PF-4 EGF IFN-β NGF

AP↓VRSL RP↓SGRK AP↓TSSS AP↓MTQT IP↓TEIP SA↓KELR SP↓GQGT AP↓GPPG GP↓VPPS TP↓LGPA AP↓ARSP SP↓LPIT LP↓GVGL SP↓APPA QP↓DAIN QP↓DSVS QP↓DSVS AS↓LAAD AP↓MGSD VP↓LSRT MP↓MFIV AP↓MAEG MA↓FTEH MA↓EGEI MA↓AGSI YA↓EHKS AA↓IGSC EA↓EEDG NS↓DSEC MS↓YNLL SS↓SHPI

153 152 133 133 115 72 160 179 112 177 127 180 171 332 76 76 76 70 69 77 115 189 200 155 155 119 196 70 53 166 120

Residues 68 69 73 77 103 74 68 70 36 27 11 36 38 28 27 7 116

kcat/Km (106 M−1 s−1)

References

5 4 1.2 0.5 >0.4 0.08 0.04 0.003 1.9 1.8 0.91 0.76 0.024 0.012 0.0042 0.137 n.d.

18 18 18 18 18 18 18 18 20 17 22 17, 18 17 17 17 2 37

released from hemoglobin B-chain sequence 35–41 [2]. From this, one can conclude that the DP IV substrate scenario is characterized by a vast number of oligo- and polypeptides, which potentially can act either as substrates or as competitive inhibitors.

PRIMARY DP IV EFFECTOR PEPTIDES In vitro studies on processing of immunorelevant peptides by soluble DP IV have identified a number of candidates where DP IV is playing a critical role in regulating their bioactivity (Table 1). In vivo, bioactive peptides are acting in nano- or picomolar concentration ranges. The physiological relevance of in vitro inactivation of such peptides is defined by the physiological efficiency or selectivity constant kcat/Km. Substances with higher kcat/Km values are better substrates than those with lower values. Chemokines are playing a fundamental role in the immune system, particularly in traffic and homing of lymphocytes and other immune cells. They bind to specific receptors on the surface of endothelial or immune cells or to the extracellular matrix. Many chemokines exhibit a DP IV susceptible N-terminal sequence, and some of them have been found in a DP IV truncated form in blood and tissues. Therefore, it is highly probable that DP IV-mediated truncation is an important mechanism of regulation chemokinesis. One of the most rapidly DP IV-processed chemokines is CXCL12 (stromal derived factor -1a and -1ß). The CXCL12 form found in blood is predominantly the

570 / Chapter 81 truncated form generated by DP IV cleavage [3]. CXCL12 is also a ligand of the HIV coreceptor CXCR4 and inhibits infection of macrophages by special HIV strains (for reviews, see [6, 11, 19]). CCL11/Eotaxin is a significant factor for the recruitment of eosinophils to inflammatory sites, particularly in allergic reactions. DP IV-mediated processing of CCL11 results in a clear loss of its biological activity [6]. Coincubation of CCL11 with human T cells show a processing of full-length CCL11 to CCL11 (3–74), reflecting the specific cleavage of CCL11 by DP IV. Intravenous administration of CCL11 in wild-type Fischer 344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DP IV-deficient Fischer 344 mutants. This result clearly underscores the role of DP IV in controlling eosinophilia [10]. Neuropeptide Y (NPY) and peptide YY (PYY) belong to the best natural substrate of DP IV. Several lines of evidence suggest that NPY exerts regulatory effects in local inflammatory response. Lymphocytes express NPY Y1 receptor. It was shown that NPY, PYY, and an NPY Y5 receptor agonists potentiated concanavalin A-induced paw edema in the rat. The DP IV inhibitor Ilethiazolidide exerted synergistic and potentiating effects in vivo. This clearly shows an involvement of DP IV in NPY- and PYY-mediated immune response [7]. Hemorphins are endogenous peptides that belongs to the family of atypical opioid peptides released from sequentially hydrolyzed hemoglobin. Hemorphin-7 (heptapeptide [35–41] of human hemoglobin) has been shown to be capable of inhibiting the inflammatory response in acute, but not chronic, injury conditions. Inhibition kinetic studies conducted with purified DP IV demonstrated that hemorphin-7 constitutes a good substrate for this enzyme but could also act as a selective competitive inhibitor by substrate binding site competition. Therefore, this group of blood peptides could represent endogenous regulators of DP IV activity [2]. Substance P (SP) is an undecapeptide that is secreted by nerves and inflammatory cells such as macrophages, lymphocytes, and dendritic cells. It has proinflammatory effects in immune and epithelial cells and plays a crucial role in inflammatory diseases [24]. Besides the neutral endopeptidase (NEP) and the angiotensin converting enzyme (ACE), DP IV is one of the most important peptidases involved in the degradation of SP. In many studies, it has been reported that vasoactive intestinal peptide (VIP) could play an important role in modulation of the immunological response. VIP can be produced by immunological cells, and also the receptors for this neuropeptide are present in many of these cells. Recent work has demonstrated that VIP is pro-

duced by Th2 cells and that through specific receptors it exerts immunological functions typically ascribed to Th2 cytokines in nervous and immune system. VIP strongly reduces the inflammatory response under different conditions. Thus, it was added to the list of endogenous anti-inflammatory agents [25]. The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the VIP/secretin glucagon family of peptides produced by immune cells, which exerts a wide spectrum of immunological functions controlling the homeostasis of the immune system through different receptors expressed on various immunocompetent cells. Like VIP, PACAP has been identified as a potent anti-inflammatory factor that exerts its function by regulating the production of both anti- and proinflammatory mediators [4]. DP IV inhibitors are presumed to protect both PACAP and VIP from inactivation and thereby to favor the antiinflammatory status. The tetradecapeptide gastrin-releasing peptide (GRP) is the mammalian homolog of bombesin and has been shown to affect cytokine production and release from various immune cells. It is present in joint fluids in rheumatoid arthritis and correlates clearly with proinflammatory cytokines [12]. Procalcitonin (PCT), a protein of 116 amino acids, was discovered as a prohormone of calcitonin produced by C cells of the thyroid gland. Circulating levels of PCT in healthy subjects are below the detection limit. Blood concentration of PCT is increased in systemic inflammation, especially when this is caused by bacterial infection, like sepsis [9]. Recently it was found that not PCT (1–116) but only the truncated form, lacking the dipeptide Ala-Pro, PCT (3–116), is present in the serum of patients with bacterial sepsis and that this DP IV catalyzed processing of PCT can be imitated also in vitro [37].

SECONDARY IMMUNORELEVANT DP IV PEPTIDE SUBSTRATES A number of cytokines, chemokines, endocrine factors, and other bioactive peptides exhibit X-Pro or X-Ala sequences at the N-terminus (Table 2) but were not processed by soluble DP IV under in vitro conditions [13, 14, 21]. A number of candidates have to be studied yet with respect to their DP IV susceptibility in vitro. In the context of DP IV action, it is of interest that the biological activity is changed strongly by short deletions and mutations at the N-terminal part of the bioactive peptide (cf. [13]). Using IL-2 oligopeptides with different chain lengths, it was shown that the DP IV catalyzed hydrolysis

Immune Peptides Related to Dipeptidyl Aminopeptidase IV/CD26 rate was negatively correlated to their chain length [13]. Similar effects were observed with IL-6 and other peptides [13]. Obviously, the free and flexible Nterminus of the peptide is required. Probably binding of the bioactive peptide to the receptor could change conformation and susceptibility. Nevertheless, DP IVresistant bioactive peptides can be processed by endoproteases in vivo to suitable substrates for DP IV, which then are capable of acting as competitive inhibitors and immune suppressors. The great number of such peptides makes them a powerful natural tool in regulation of immune response via DP IV in vivo.

PEPTIDE INHIBITORS OF DP IV In contrast to the increasing number of non-peptide inhibitors of DP IV [8, 36], the examples of natural peptide inhibitors already studied are limited (Table 3). One should remember, however, that the previous peptide substrates of DP IV can principally act also as competitive inhibitors of this peptidase. This means that the number of potential DP IV, inhibitors with proline in the second position is much larger. A special feature of DP IV inhibition is the finding that peptides with proline in the third position can also act as potent inhibitors of DP IV, particularly if tryptophan is present in the second position of the N-terminus (Table 3). Examples are Tat protein and its derivatives of the human immunodeficiency virus 1 [38]. Peptides described in this chapter probably represent only a small portion of the total number of peptide candidates related to DP IV. Future work will show that the number of immunorelevant peptides which are actively (DP IV inhibitors as suppressor peptides) or passively (cytokines and chemokines as instructur peptides) involved in DP IV-mediated immune regulation is much larger.

TABLE 3.

DP IV peptide inhibitors.

Peptide name

Peptide sequence

Ki value [μM]

Reference

Diprotin A Diprotin B Hemorphin 7 HIV-Tat (1-86) Tat (1-9) Trp2-Tat (1-9) TXA2-R (1-9) Met-IL-2 (1-6)

IPI VPV YPWTQR MDPVDP MDPVDP MWPVDP MWPNGS MAPTSS

2.2 7.6 n.d. n.d. 267 2.12 5.06 100

35 35 2 35 38 38 38 13

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572 / Chapter 81 [19] Lambeir AM, Durinx C, Scharpe S, De Meester I. Dipeptidylpeptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev Clin Lab Sci 2003; 40:209–94. [20] Mentlein R, Dahms P, Grandt D, Krüger R. Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul Pept 1993; 49:133–44. [21] Mentlein R. Cell-surface peptidases. Int Rev Cytol 2004; 235: 165–213. [22] Nausch I, Heymann E. Substance P in human plasma is degraded by dipeptidyl peptidase IV, not by cholinesterase. J Neurochem 1985; 44:1354–7. [23] Nausch I, Mentlein R, Heymann E. The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta. Biol Chem Hoppe Seyler 1990; 371:1113–8. [24] O’Connor TM, O’Connell J, O’Brien DI, Goode T, Bredin CP, Shanahan F. The role of substance P in inflammatory disease. J Cell Physiol 2004; 201:167–80. [25] Pozo D, Delgado M. The many faces of VIP in neuroimmunology: a cytokine rather a neuropeptide? FASEB J 2004; 18: 1325–34. [26] Qi SY, Riviere PJ, Trojnar J, Junien JL, Akinsanya KO. Cloning and characterization of dipeptidyl peptidase 10, a new member of an emerging subgroup of serine proteases. Biochem J 2003; 373:179–89. [27] Reinhold D, Hemmer B, Gran B, Born I, Faust J, Neubert K, McFarland HF, et al. Inhibitors of dipeptidyl peptidase IV/CD26 suppress activation of human MBP-specific CD4+ T cell clones. J Neuroimmunol 1998; 87:203–9. [28] Riemann D, Hansen GH, Niels-Christiansen L, Thorsen E, Immerdal L, Santos AN, et al. Caveolae/lipid rafts in fibroblastlike synoviocytes: ectopeptidase-rich membrane microdomains. Biochem J 2001; 354:47–55. [29] Schön E, Mansfeld HW, Demuth HU, Barth A, Ansorge S. The dipeptidyl peptidase IV, a membrane enzyme involved in the

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82 RGD-Peptides and Some Immunological Problems IGNACY Z. SIEMION, ALICJA KLUCZYK, AND MAREK CEBRAT

ABSTRACT

In the search for biologically active peptides, the sequential analogs of signal sequences present in the proteins are also of interest. This review is concerned with the RGD signal sequence, which may be considered as the sequential analog of thymopentin. The RGD tripeptide fragment appears in many adhesive proteins and plays a role in their main adhesive site. The cellular adhesion processes could be divided into two categories: the cell-cell and the cell-intracellular matrix protein adhesion. In both cases, a major role is played by the cellular integrin receptors for cell adhesion. The integrin glycoproteins appear as dimers composed of α- and β-subunits. The sequencing of the human genome has identified as many as 24 α- and 9 β-subunits and 24 dimeric integrins known to exist in mammals. The history of integrin recognition was recently presented [12]. In addition to their role in mediating cell adhesion, integrins are responsible for the transmembrane connections to the actin cytoskeleton and activation of the intracellular signaling pathways. Integrin receptors may interact specifically with such matrix proteins as fibronectin, vitronectin, and laminin, with serum fibrinogen and von Willebrand factor, and with different kinds of collagen, recognizing mainly the RGD signaling sequence. The adhesive protein signal sequences are collected—for example, in the review of Yamada [46]. The cellular adhesion processes are important for many fundamental biological processes, such as cell differentiation and growth, tumor metastasis, several immune functions and tissue repair. They regulate cell movement and migrations, wound healing, and the inflammatory phenomena, as well as embryonal development.

The application of adhesive RGD-peptides and their molecular mimetics as the inhibitors of several cardiovascular and immunological processes as well as the development of studies on the inhibition of platelet aggregation is reviewed. The role of RGDpeptides in fibronectin-integrin receptors interaction is also discussed.

DISCOVERY OF THE ANTIADHESIVE RGD-PEPTIDES A very useful approach in the search for biologically active peptides consists of the utilization of particular sequences of definite proteins. The classic example of such a procedure is the application of antigenic epitopes of proteins to obtain the selective antibodies and vaccines (for review, see [27]). Another possibility is the utilization of protein fragments that form the sites of interaction of regulatory proteins. Peptide inhibitors of the processes regulated by these proteins could be obtained this way. From this point of view especially interesting are the “universal signal sequences” of proteins. The “universal signal sequences” can be recognized by their broad distribution among different regulatory proteins of various living species. Tuftsin and thymopentin are examples of sequences that could be considered as “signals.” Thymopentin (RKDVY) is an active pentapeptide fragment of thymopoietin, a polypeptide immunoregulator produced by epithelial cells of thymus. Tuftsin (TKPR), the natural phagocytosis stimulating factor, is a part of the molecules of leucokinins (leucophilic portion of IgG proteins). Handbook of Biologically Active Peptides

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574 / Chapter 82 The crucial role of adhesive interactions of cells with other cells and with matrix proteins within the immune system was reviewed by Springer [36]. In 1982 the complete amino acid sequence of the cell attachment domain of human fibronectin was determined by Pierschbacher. Pierschbacher and Ruoslahti also reported that the tetrapeptide RGDS of this domain plays a role of the adhesive site of fibronectin. The Arg (R) and Asp (D) residues present in the tetrapeptide were shown to be of major importance for this activity [26]. The peptides containing the RGD sequence compete with fibronectin for cellular receptors and may act as antiadhesive substances [30].

In other RGD mimetics, the guanidine group was replaced by the p-anisidinophenyl moiety. It opened a way to the RGD peptide mimetics of the benzodiazepine series (structure A):

HOOC

H N

COOH

O O N

HN NH

O

O OH

O

HN

B A

NH

NH2

RGD-PEPTIDES IN PLATELET AGGREGATION Because of the role of the RGD-peptides in the pathophysiology of thromboses, the interactions of adhesive proteins with platelets have gained the attention of many laboratories. Platelet aggregation consists of the cross-linking of fibrinogen or von Willebrand factor to the GPIIb/IIIa (αIIbβ3) platelet integrin receptor. It takes place during the development of such cardiovascular diseases as myocardial infarction, transient ischemic attacks, strokes, and so on. Each of the Aα peptide chains of fibrinogen contains two adhesive sites (sequences RGDF and RGDS) and the HHLGGAKQAGDV fragment responsible for receptor recognition. Six binding sites of a dimeric form of fibrinogen participate in binding to the integrin receptors, thus mediating platelet aggregation. Disintegrins, very potent inhibitors of the αIIbβ3 receptors, are polypeptides containing the exposed RGD motif obtained from the venom of Viperidae snakes and leaches. Echistatin, omatins, kistrin, and decorsin belong to this family. In barbourin and ussuristatin only the RGD motif is changed into the KGD sequence [31]. Cyclic RGD-containing peptides represent another very potent class of αIIbβ3 inhibitors. The search for new antithrombotic agents was also oriented at the acyclic semipeptides. As an example of such structures, the compound containing a nonpeptidic 8-guanidinooctanoyl residue could be presented here:

COOH

O NH

H2N NH

NH Y

NH O

COOH

Y= phenyl, 1-naphthyl, indolyl

These investigations clearly showed that the distance between the guanidine and carboxyl groups is crucial for the activiy of the antithrombotic agents. It should be similar to the respective distance in the RGDS peptide in the extended conformation. Assuming that this distance should be about 10–20 Å, Egbertson et al. [7] proposed a substituted N-benzyloxycarbonyl tyrosin as a new lead compound in the search for antithrombotic drugs (structure B). It was found that the RGD sequence is the absolute minimum for cell attachment activity. The L-Arg residue could be substituted by D-Arg, while replacing Gly by D-Ala, or L-Asp by D-Asp results in inactive compounds. An RGD amide is sufficient for platelet aggregation inhibition at a high concentration. The Ser residue in RGDS could by replaced by other amino acids, especially hydrophobic ones. Substitution of the Asp β-carboxyl group by a tetrazole moiety leads to a completely inactive compound. The peptides bearing a primary amine instead of the guanidine group (i.e., with Arg/ Lys substitution) exhibited enhanced platelet aggregation potency and αIIbβ3 selectivity. The introduction of Har (homoarginine) instead of Arg into the cyclic RGD peptides also greatly increases the platelet aggregation inhibitory potency. In the search for peptides binding to specific integrin receptors, the phage display library method was used [20]. In order to get additional information on the RGD binding domains of the platelet integrins, a series of (G)n-RGDF peptides with a varying number of N-terminal Gly residues was immobilized on polyacrylonitrile beads. The platelet agglutinating effects of these beads in relation to the number of Gly residues and the presence of platelets agonists and inhibitors were examined [2]. A novel chimeric protein was engineered by insertion of the GRGDS motif within the loop of the epidermal growth factor-like molecule [44].

RGD-Peptides and Some Immunological Problems / 575

BIOLOGICALLY ACTIVE CONFORMATION OF RGD-PEPTIDES

1

Scarborough et al. [31] have pointed out that various conformations, proposed as the receptor-bound conformations of RGD ligands, have led to a number of independent approaches to the design of RGD-peptide mimetics. NMR, CD, and molecular modeling studies were applied in the conformational investigations, with attention given to semirigid, cyclic RGD-peptide analogs, showing increased selectivity for a definite type of the integrin receptor [3]. It follows from the work of Kostidis et al. [21] that the pseudo-dihedral angle between the Arg Cβ and Asp Cβ atoms can be used as the criterion for evaluation of the structure–activity relationship of RGD-containing peptides. The value of this angle in the active compounds is between +45° and −45°. Activity increases with an increase of the distance between the charged centers and diminishes with the increase of ionic interactions between Arg and Asp. Miyashita et al. [22] found that the optimal distance between the central carbon atoms of each basic and acidic group involved in the interaction with the receptor should be about 15.3 Å. At somewhat lower value of about 8 Å was given by Stavrakoudis et al. [37] after the studies performed with the series of rigid peptides containing the Cys-Asp-Cys sequence. Recently the crystal structure of the extracellular part of the αvβ3 integrin receptor alone and complexed with the RGD-ligand (cyclic pentapeptide: cyclo-(ArgGly-Asp-D-Phe-(N-Me)Val) was resolved by Xiong et al. [45]. The ligand is located at the major interface between the αv and β3 subunits, binding them together. The guanidine group of Arg forms a bidendate salt bridge to Asp218 and Asp150 of this receptor, and the carboxyl group of Asp participates in a hydrogen bond with the amide carbonyls of Tyr122 and Asn215. The distance between the carbon atoms of these two groups equals 13.72 Å, and the Arg and Asp side chains are extended in opposite directions. These results enabled a more precise analysis of the interaction of adhesive peptides with integrins, presented in the paper of Arnaout et al. [1].

RGD PEPTIDES IN IMMUNOLOGICAL PHENOMENA A very important role of RGD peptides in immunological phenomena results from the principal role of adhesion in the immune cell interactions. It plays a key role in antigen presentation, in antibody response, T cell help and suppression, aninflammatory processes, as well as in cytotoxic killing. Inflammation consists of

the local accumulation of blood lymphocytes and plasma proteins resulting from the antigen stimulation. The bacterial and other pathogen invasion into the cells is also mediated by adhesive reactions in which the integrin receptors and matrix proteins participate. The possibility of an influence of the RGD peptides on such processes is, of course, evident. The homotypic adhesion of T cells is mediated by intercellular adhesion molecules (ICAM-1), which interact with the leukocyte integrin receptors LFA-1 (leukocyte-function associated antigen-1) and MAC-1 expressed on macrophage cells. MAC-1 mediates the adhesion of granulocytes and monocytes to endothelial cells. LFA-1 contributes to the binding of killer T cells to target cells. Natural killer cells constitute a conserved T lymphocyte subpopulation that regulates many types of the immune response by a rapid secretion of such cytokines as IL-4 and IFN-γ. ICAM-1 belongs to the group of most intensively studied adhesion molecules of the immune system. Its crystal structure was reported by Casasnovas et al. [4]. ICAM-1 is also the cell surface receptor for human rhinoviruses and red blood cells infected by Plasmodium falciparum. Blocking of the cell adhesion signal by inhibition of the ICAM-1–LFA-1 interactions suppresses the allograft rejection and may also induce the apoptosis of antigen-specific T cells. The blocking could be achieved by means of linear and cyclic peptides derived from the ICAM-1 sequence [17, 29]. The interacting site of ICAM-1 consists of the PSKVILPRGGSVLVTG sequence. The PRGGS sequence closely resembles the recognition sequence of fibronectin (GRGDS). The solution conformations of two potent LFA-1–ICAM-1 interaction inhibitors, cyclic peptides closed by disulfide bridges: cyclo(1,12)-Pen-Pro-ArgGly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH and cyclo(1,12)Pen-Pro-Ser-Lys-Val-Ile-Leu-Pro-Arg-Gly-Gly-Cys-OH (Pen-L-penicillamine (ß,ß-dimethyl-L-cysteine)) were studied by Gürsoy et al. [8] and Jois et al. [16], respectively. The sequential analog of thymopentin with the RGDVY sequence was found in HLA-DQ, the molecule of human leukocyte antigen class II, located on the surface of macrophages. The antigens of this class present the peptide fragments of foreign proteins to the T cells. They are composed of two peptide chains (α and β). The RGDVY sequence occupies the 167–171 fragment of the β2-extracellular domain of the β-chain of HLA and is located within the exposed loop of this domain. The synthetic linear peptides containing this sequence suppress the cellular immune response (DTH test), while thymopentin was found to be inactive in this test. The effect of the peptides on the humoral response (PFC test) depends on the length of the peptide chain—for example, for the elongated QRGDVY

576 / Chapter 82 sequence, stimulation of the immune response is observed—but the further elongation of the peptide chain results in suppression of the response. The presence of the adhesive RGD sequence within the HLA-DQ suggests that the molecule may interact with some integrin receptors, enhancing its binding to the CD4 cell receptor. The synthetic peptides related to the indicated HLA-DQ fragment inhibit the adhesion of platelets to fibrinogen, as well as the adhesion of tumor K562 cells to fibronectin. A disulfide bridged, semirigid CRGDVYC peptide showed a significant increase in inhibitory potency as compared with the RGDVY linear pentapeptide [38]. It seems interesting to note that the RGDVF sequence, very similar to the RGDVY motif, appears in the molecule of an adhesive protein, vitronectin. In the molecules of HLA-DP and HLA-DR subclasses of HLA II, the RGDVY sequence of HLA-DQ is changed into QGDVY and SGEVY, respectively. It is also of interest that a similar sequence, ILDVP, without the basic Arg residues, was identified as the second adhesive site on fibronectin [46]. Therefore, it seems that the indicated mutational changes do not destroy the adhesive properties of the respective HLA-DP and HLA-DQ fragments. The peptides which correspond to these sequences showed a distinct immunosuppressory activity in both the cellular and humoral immune response in mice [41]. The immunological properties of the peptides derived from the HLA-DQ protein were also discussed in several other papers by the same research group. Cyclo-(Suc-TPQRGDVK) (Suc—succinyl) was synthesized as a model of the RGD-containing loop of the HLA molecule [40].

RGD-PEPTIDES AND PATHOGEN INVASIONS In the process of infection and internalization of various pathogens into the cells, the adhesion of pathogens to the host cells is a crucial phenomenon. Many microbial proteins contain the RGD motif, which enables binding to human integrins. Several microbial pathogens bind the host matrix proteins, like fibronectin, to facilitate entry into the host cells. A short review of the role of cell adhesion molecules in the pathogenesis and host defense against microbial pathogens including protozoa, fungi, bacteria, and viruses was presented by Kerr [18]. In particular, the entry of Mycobacteria into the leukocyte cells is mediated by formation of the complex between the bacterial antigen Ag85 and fibronectin [24]. Mycobacteria can invade the cells only in the form of this complex interacting with the fibronectin-specific integrin receptor.

It was shown by Siemion and Wieczorek that the GRGDS fibronectin peptide, as well as the RGDVY HLADQ peptide and their analogs, inhibit the entry of Mycobacterium kansasii into leukocyte cells [34]. The effect consists of the competition of fibronectin and the investigated peptide for the same integrin receptor. Thymopentin (RKDVY) was also found to be a potent inhibitor of Mycobacteria phagocytosis. The exchange of Asp by Glu in splenopentin (RKEVY) results in a decrease of activity. The fragments of such proteins as cecropine, systemine, and BRCT proteins, containing the modified RGD sequences, were also examined for inhibitory activity. In the series of peptides of the general formula GRGnDV, the best fit of the spatial conditions needed for a peptide–integrin receptor interaction was found for the compound with a triglycine linker between the Arg and Asp residues (RG3DV) [32]. The ω-guanidino acids (H2N-C(=N)-NH-(CH2)nCOOH) were also used as mimetics of RGD peptides in the Mycobacteria phagocytosis inhibition. Inhibitory activity appears for amidinohexanoic acid. The compounds with a shortened hydrocarbon chain are practically devoid of any activity, with the exception of the guanidinoacetic acid [33]. Staphylococcus aureus, Streptococcus pyrogens, and several other bacteria also use the binding to fibronectin as a tool for host cell invasion [14]. However, the RGDVY peptide is inactive in S. aureus phagocytosis inhibition [34]. The role of fibronectin binding in bacterial adherence and entry into mammalian cells was reviewed by Joh et al. [15]. Candida albicans yeasts exploit the vitronectin matrix protein as a tool for the cell adhesion. Their binding to vitronectin is strongly inhibited by RGD, but not RGE-peptides [35]. The synthetic peptidomimetics of RGD were also used for the inhibition of binding of adenoviruses to the integrin receptors. The human adenovirus contains the RGD sequence in the penton base protein [9]. The γ-herpesviruses also use integrin receptors for entry into the cells via the RGD sequence [43]. It follows from the results presented above that the RGD peptides and their mimetics could serve as potential drugs not only in cardiovascular, but also in various infectious diseases.

RGD-PEPTIDES IN OTHER PATHOLOGICAL PHENOMENA The possibility of use of the α4β1 and α4β1 integrin receptor inhibitors in the treatment of autoimmune disorders was discussed by Jackson [13] and the relevance of the RGD peptides and their orally active mimetics in treatment of renal disease by Horton [10].

RGD-Peptides and Some Immunological Problems / 577 Humphries et al. [11] showed that the GRGDS pentapeptide may inhibit the experimental metastasis of murine melanoma cells. Reinmuth et al. [28] found that RGD-mimetics decrease colon cancer metastasis and angiogenesis, and improve survival in mice. The inhibitory effects of the RGD peptides on tumor cell migration through tissue and on restriction of metastatic dissemination of tumor cells injected into the circulation were also noted earlier by Thomas et al. [42] and Ouaissi et al. [25].

[5] [6]

[7]

[8]

RGD RETROSEQUENCES IN PROTEINS In some proteins, retro RGD sequences are present. Such a situation appears—for example, in the human CD23 molecule (low-affinity receptor for IgE)— expressed on a variety of hematopoietic cell types. The (GCY)CPDPDGRLPTPSAPLHS peptide derived of this protein was found to bind to MHC class II molecules, but not to CD23 [19]. The reversed RGD sequence appears also in ubiquitin [39]. A series of linear peptides, derived from ubiquitin, was synthesized and investigated for immunomodulatory properties. It was shown that all of them act as strong immunosuppressors in PFC and DTH tests. A model of the 48–59 loop of ubiquitin, a cyclic peptide cyclo-(Glt-QLEDGRTLSDK)-NH2 (Glt—glutaryl), with the bridge between Glt and the ε-amino group of Lys, was also synthesized. These experiments showed that the retro-RGD sequence is able to interact with immune cells. In this context it is also worth noting that the ubiquitin system is also involved in processing MHC class I antigens [5]. Ubiquitin also augments the production of TNFα in murine macrophages and may be involved in immune response modulation [23]. The retro-inverso RGD peptides showed a two- to threefold decrease in potency or a total loss of activity in platelet aggregation [6]. The presented material strongly suggests that beside the well-known RGD-peptides in cardiovascular processes, they will gain the increasing importance in various immunological phenomena.

[9]

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[11]

[12] [13] [14]

[15]

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[20]

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intercellular adhesion molecule-1. Proc Natl Acad Sci U S A. 1998;95:4134–9. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 1998;17:7151–60. Dal Pozzo A, Fagnoni M, Bergonzi R, Vanini L, de Castiglione R, Aglio C, Colli S. Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides. J Pept Res. 2000;55: 447–54. Egbertson MS, Chang CT, Duggan ME, Gould RJ, Halczenko W, Hartman GD, et al. Non-peptide fibrinogen receptor antagonists. 2. Optimization of a tyrosine template as a mimic for Arg-Gly-Asp. J Med Chem. 1994;37:2537–51. Gürsoy RN, Jois DS, Siahaan TJ. Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-ValLeu-Val-Thr-Gly-Cys-OH. J Pept Res. 1999;53:422–31. Hippenmeyer PJ, Ruminski PG, Rico JG, Lu HS, Griggs DW. Adenovirus inhibition by peptidomimetic integrin antagonists. Antiviral Res. 2002;55:169–78. Horton MA. Arg-Gly-Asp (RGD) peptides and peptidomimetics as therapeutics: relevance for renal diseases. Exp Nephrol. 1999;7:178–84. Humphries MJ, Olden K, Yamada KM. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science. 1986;233:467–70. Hynes RO. The emergence of integrins: a personal and historical perspective. Matrix Biol. 2004;23:333–40. Jackson DY. Alpha 4 integrin antagonists. Curr Pharm Des. 2002; 8:1229–53. Joh D, Speziale P, Gurusiddappa S, Manor J, Hook M. Multiple specificities of the staphylococcal and streptococcal fibronectin-binding microbial surface components recognizing adhesive matrix molecules. Eur J Biochem. 1998;258:897– 905. Joh D, Wann ER, Kreikemeyer B, Speziale P, Höök M. Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol. 1999;18:211–23. Jois DS, Pal D, Tibbetts SA, Chan MA, Benedict SH, Siahaan TJ. Inhibition of homotypic adhesion of T-cells: secondary structure of an ICAM-1-derived cyclic peptide. J Pept Res. 1997; 49:517–26. Jois SD, Tibbetts SA, Chan MA, Benedict SH, Siahaan TJ. A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion. J Pept Res. 1999;53:18–29. Kerr JR. Cell adhesion molecules in the pathogenesis of and host defence against microbial infection. Mol Pathol. 1999; 52:220–30. Kijimoto-Ochiai S, Noguchi A. Two peptides from CD23, including the inverse RGD sequence and its related peptide, interact with the MHC class II molecule. Biochem Biophys Res Commun. 2000;267:686–91. Koivunen E, Gay DA, Ruoslahti E. Selection of peptides binding to the α5β1 integrin from phage display library. J Biol Chem. 1993;268:20205–10. Kostidis S, Stavrakoudis A, Biris N, Tsoukatos D, Sakarellos C, Tsikaris V. The relative orientation of the Arg and Asp side chains defined by a pseudodihedral angle as a key criterion for evaluating the structure-activity relationship of RGD peptides. J Pept Sci. 2004;10:494–509. Miyashita M, Akamatsu M, Hayashi Y, Ueno T. Three-dimensional quantitative structure-activity relationship analyses of RGD mimetics as fibrinogen receptor antagonists. Bioorg Med Chem Lett. 2000;10:859–63. Nabika T, Terashima M, Momose I, Hosokawa Y, Nagasue N, Tanigawa Y. Synergistic effect of ubiquitin on lipopolysac-

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83 Neuropeptides That Regulate Immune Responses NING ZHANG AND JOOST J. OPPENHEIM

ABSTRACT

Neuropeptides influence host immune defenses based on two mechanisms. First, these peptides indirectly regulate the immune response by activating the different pathways of the central nervous system. For example, Met-enkephalin, an endogenous opioid, stimulates the CNS-mediated production of corticosterone, a potent immunosuppressive hormone [35]. Opioids also activate the sympathetic nervous system, resulting in an increase in the level of circulating epinephrine from the adrenal medulla and secretion of the norepinephrine by sympathetic nerve terminals. Increased catecholamine levels have been linked to suppression of natural killer cell and lymphocyte function. Second, all the peptides listed in Table 1 are able to directly regulate the functions of receptor expressing leukocytes, independent of neuronal activities. Studies using RT-PCR or Western blotting analysis reveal the expression of a wide variety of neuropeptide receptors on various types of leukocytes. For example, all three subtypes of opioid receptors, μ-, δ-, and κ- receptors, have been detected on T cell, B cells, monocytes, neutrophils, and natural killer cells [40]. Stimulation of these neuropeptide receptors can modulate leukocyte production of proinflammatory cytokines. Most neuropeptide receptors are seven-transmembrane receptors which exert their function by activating heterotrimeric G protein. Neuropeptides are mainly expressed and active in the central and peripheral nervous system. However, these peptides can also be produced and are functional in other peripheral tissues. For example, adrenocorticotrophic hormone/corticotrophin (ACTH) is not only produced by pituitary cells, but also to a limited extent by leukocytes [7]. Endogenous opioids are expressed by leukocytes and play an important role in peripheral

Increasing evidence has shown that many neuropeptides modulate immune responses in addition to their well-documented function in the central and peripheral nervous systems. RT-PCR, Western blotting, and functional assays reveal the production of these neuropeptides by the immune system and the expression of their receptors on the leukocyte surface. This chapter provides a brief summary of the immunological activities of neuropeptides. The nervous and immune systems are tightly integrated and orchestrated to serve complicated tasks of host sensation and response. A disturbance in one system often has a profound effect on the other. For example, activation of hypothalamo-pituitary-adrenal (HPA) axis by long-term stress often elicits suppression of both innate and adaptive immunity [17, 23, 61]. Conversely, inflammatory immune responses activate central nervous system sensory pain pathways [53, 58, 65, 66]. Increasing evidence suggests that a family of immunoactive neuropeptides provides key signals between these two systems. Beyond their well-characterized effects in the nervous system, these neuropeptides also interact with their receptors on leukocytes and thus modulate the function of these pivotal mediators of host defense. Furthermore, leukocytes are also capable of producing many of these peptides in the peripheral immune system. The structure, properties, and roles of neuropeptides in the central and peripheral nervous systems is discussed in other chapters of this book. This section briefly describes the immunological effects of these neuropeptides, as summarized in Table 1. Other actions of these peptides are discussed elsewhere in this book. Handbook of Biologically Active Peptides

579

580 / Chapter 83 TABLE 1.

Neuropeptides

Size

Immunological effects of neuropeptides.

Receptors in immune system

CRH

41aa

Neutrophils, eosinophils, thymus, spleen [3, 48, 49]

Somatostatin

14aa or 28aa

T cells, monocytes [33, 60]

Vasopressin (AVP) 9aa

T cells, B cells, monocytes [8, 29, 37]

Gastrin-releasing peptides (GRP)

27aa

Macrophage, T cells, natural killer cells [20, 21, 22]

Met-enkephalin and endorphins

5aa

ACTH

39aa

αMSH (melanocortin)

13aa

T cell, B cells, monocytes, neutrophils, natural killer cells [40] T helper cells, B cells, macrophages [30] Monocytes, macrophages, natural killer cells, T helper cells, and dendritic cells [1, 39]

Neuropeptide Y

36aa

VIP

28aa

Immune effects

Nonimmune effects

Enhances production of immunosuppressive glucocorticoids through HPA axis, [23, 57] enhances peripheral inflammation, ([2, 34] Urocortin (Ucn), UcnI, and UcnII share similar properties Inhibits the secretion of proinflammatory cytokines and antibodies by immune cells [33]; inhibits lymphocyte proliferation [60] Enhances production of immunosuppressive glucocorticoids through HPA axis [61], enhances secretion of IFN-γ by T-cells [31] Stimulates phagocytic activities of macrophages [20], inhibits Con-A induced proliferation of lymphocytes [22], Bombesin and neuromedin C share the similar properties Induces heterologous desensitization of chemokine receptors, inhibits natural killer cell activity, suppresses IL2 and IFNγ production [40, 44]

Endocrine-signaling peptide involved in the stress response [17]

Enhances production of immunosuppressive glucocorticoids through HPA axis [41]

Endocrine-signaling peptides involved in the response to stress [17]

Inhibits secretion of numerous growth hormones [25]

Potentiates CRH effects, respond to osmotic and hemodynamic stress [54, 62] Activates the sympathetic nervous system to modulate stress, fear, and anxiety responses [42] Analgesic peptides, induces the secretion of corticosterone [10, 50]

Down-regulates the production of Regulation of energy proinflammatory cytokines, downhomeostasis [18] regulates expression of costimulatory molecules (CD86, CD40, ICAM-1) on antigenpresenting dendritic cells, upregulates IL-10, inhibits cutaneous inflammation [16, 39] Monocytes, T cells, Elicits neurogenic inflammation by Regulates energy balance, B cells, natural inducing the secretion of substance food intake, and killer cells [4, 5] p, contributes to colitis, potentiates antihyperalgesia, paw edema, enhances T-cell antimicrobial peptide, affects adhesion to fibronectin, increases skin color, antipyretic effects release of oxidative reagents by [43, 46, 59] neutrophils, suppresses NK cell activity, enhances phagocytosis by monocyte [4, 24, 36, 43] T cells, B cells, Inhibits leukocyte recruitment, Neurotransmitter, mast cells, cytokine production; polarizes secretagogue, macrophages, TH2 responses, induces neuroprotective, [28, 32, 47] T lymphocyte adhesion and neurotrophic, and chemotaxis [28, 32, 47] PACAP vasodilator [26, 12] possesses similar anti-inflammatory activities

Neuropeptides That Regulate Immune Responses / 581 TABLE 1. (Continued)

Neuropeptides

Size

Receptors in immune system

Substance P (neurokinin1, tachykinin 1)

11aa

T cells, macrophages, dendritic cells [45]

CGRP

37aa

Bradykinin 9aa

9aa

T cells, B cells, dendritic cells [55, 15, 27] Monocytes, neutrophils, eosinophils, T cells [9]

Immune effects Contributes to neurogenic inflammation, involved in various respiratory diseases and pancreatitis [45], substance K (neurokinin A) possesses similar proinflammatory activities Contributes to neurogenic inflammation, involved in various aspiratory diseases [55] Induces chemotaxis of neutrophils, stimulates the secretion of superoxide radicals, exacerbates infection, contributes to ischaemiareperfusion, involved in respiratory diseases, [9, 13, 19]

Nonimmune effects Induces hyperalgesia [52]

Induces vasodilatation [11]

Induces hyperalgesia [53, 58]

CRH: Corticotrophin-releasing hormone; αMSH: alpha-melanocyte stimulating hormone; ACTH: adrenocorticotrophic hormone/corticotrophin; CGRP: Calcitonin gene-related peptide; VIP: Vasoactive intestinal polypeptide.

analgesic effects [56]. In addition to being produced in the pituitary gland, α-melanocyte-stimulating hormones (αMSH) is also highly expressed in skin and GI tract cells [38]. Substance P mRNA and protein has been detected in the thymic medulla and purified CD5+ thymocytes [51]. The neuropeptides of the HPA axis play a profound role in inflammation. Stressful stimuli activate the HPA axis through release of a sequential series of neuropeptide hormones, beginning with production of hypothalamic corticotrophin-releasing hormone (CRH) and followed by pituitary derived ACTH, culminating in secretion of glucocorticoids, potent anti-inflammatory hormones produced by the adrenal cortex [17]. Vasopressin (AVP) potentiates the capacity of CRH to activate the HPA-axis [61]. The resultant elevated glucocorticoid levels in the circulation contribute to stressinduced immunosuppression [23]. Surprisingly, CRH and AVP are also produced by leukocytes, and their receptors are expressed by leukocytes. While hypothalamic CRH and AVP have indirect anti-inflammatory effects through stimulation of glucocorticoids, these peptides appear to have direct proinflammatory effects in the peripheral tissues. AVP stimulates secretion of interferon-γ by T-lymphocytes [31]. CRH, a peptide of 41 amino acids, elicits production of IL-1 by human monocytes and enhances mouse NK cell activity [14, 64]. CRH and its family members, Urocortin (Ucn), Ucn I, and Ucn II have been shown to exacerbate symptoms of experimental autoimmune encephalomyelitis

and toxin A-induced inflammatory diarrhea [2, 6]. Their antagonists have been tested as therapeutics for arthritis and preliminary results have been encouraging [63]. Inflammation induces hyperalgesia. This is in part attributable to chemokine receptor-induced desensitization of analgesic opioid receptors and concomitant sensitization of algesic Vanilloid receptors (TRPV1) on neurons [65, 66]. Conversely, opioid receptors on leukocytes have immunosuppressive effects. Metenkephalin, an endogenous opioid, is an analgesic peptide of 5 amino acids [10]. It exerts immunosuppressive effects by heterologous desensitization of chemokine receptors and enhancing the secretion of corticosteroids [40, 45, 50]. Painful stimuli activate peripheral neurons to release several immunostimulatory peptides, including substance P and calcitonin gene-related peptide (CGRP). These peptides have proinflammatory effects and are associated with neurogenic inflammation. Substance P, a hyperalgesic peptide, belongs to the tachykinin family. It stimulates the influx of neutrophils and eosinophils into the human dermis, induces secretion of TNFα by macrophages, and enhances leukocyte adhesion to endothelial cells, which may contribute to its proinflammatory effects in various respiratory diseases, pancreatitis, and inflammatory bowel disease [45]. Substance K, another peptide in the tachykinin family, possesses similar proinflammatory effects. CGRP is also involved in neurogenic respiratory diseases such as ashma,

582 / Chapter 83 chronic obstructive pulmonary disease or chronic cough [55]. Neuropeptide Y (NPY), a key peptide regulating energy balance and food intake in CNS, also induces the secretion of substance P by peripheral neurons, contributing to colitis and paw edema in animal models of inflammation [24, 43, 46]. Furthermore, NPY directly enhances T-cell adhesion to fibronectin [36]. Bradykinin, another hyperalgesic peptide, facilitates septic shock, exacerbates ischaemiareperfusion, and enhances airway inflammation based on its vasodilatory effects [13, 19]. There are a number of other neuropeptides that down-regulate immune responses. Vasoactive intestinal polypeptide (VIP) has immunosuppressive effects based on its capacity to inhibit leukocyte recruitment to inflammatory sites [28, 40]. VIP also induces polarization to TH2 type of antibody-based allergic responses [47]. αMSH down-regulates the production of proinflammatory cytokines, inhibits the expression of costimulatory molecules (CD86, CD40, and ICAM-1) on dendritic cells, and up-regulates IL-10, resulting in immunosuppressive effects [16, 39]. Somatostatin inhibits secretion of IFN-γ and CSF, enhances the secretion of IL-10 and IL-4, down-regulates the secretion of antibodies by B lymphocytes, and impairs lymphocyte proliferation [33, 60]. Gastrin-releasing peptide (GRP) and neuromedin C belong to the bombesin peptide family. They stimulate phagocytic activities of macrophages but inhibit concanavalin-A induced proliferation of lymphocytes [20, 22]. In summary, increasing evidence has confirmed the production of neuropeptides and the expression of their receptors by cells of the immune system. Furthermore, neuropeptides have been found to directly contribute to a wide range of diseases, such as stress-induced immunosuppresion, asthma, and inflammatory bowel disease. However, many more studies are needed to connect in vitro observation, based on the use of isolated leukocytes to the in vivo systemic effects of neuropeptides in disease. Furthermore, it is critical to understand that the anatomical and temporal distribution of these peptides may determine their overall effects on immune responses. For example, CRH acts as an anti-inflammatory agent when it exerts its effects in the CNS to stimulate the production of glucocorticoids. But in peripheral tissues, CRH is proinflammatory and its antagonists can be used to treat arthritis in animal models. In summary, a broad range of neuropeptides is capable of influencing immune responses. They may serve to integrate a host’s immune and nervous system response to injurious environmental stimuli. The physiological relevance of immunoregulatory activity of these neuropeptides needs further investigation.

Acknowledgment We thank Dr. Esther M. Sternberg for reviewing our manuscript and constructive discussion. This research was supported [in part or whole] by the Intramural Research Program of the NIH, National Cancer Institute.

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[60] ten Bokum, AM, Hofland, LJ, and van Hagen, PM. Somatostatin and somatostatin receptors in the immune system: a review. Eur. Cytokine Netro. 2000;11:161–76. [61] Turnbull, AV, Lee, S, and Rivier, C. Mechanisms of hypothalamic-pituitary-adrenal axis stimulation by immune signals in the adult rat. Ann. N.Y. Acad. Sci. 1998;840:434–43. [62] Volpi, S, Rabadan-Diehl, C, and Aguilera, G. Vasopressinergic regulation of the hypothalamic pituitary adrenal axis and stress adaptation. Stress. 2004;7:75–83. [63] Webster, EL, Barrientos, RM, Contoreggi, C, Isaac, MG, Ligier, S, Gabry, KE, Chrousos, GP, McCarthy, EF, Rice, KC, Gold, PW, and Sternberg, EM. Corticotropin releasing hormone (CRH) antagonist attenuates adjuvant induced arthritis: role of CRH in peripheral inflammation. J. Rheumatol. 2002;29:1252–61. [64] Woloski, BM, Smith, EM, Meyer, WJ, III, Fuller, GM, and Blalock, JE. Corticotropin-releasing activity of monokines. Science. 1985;230:1035–7. [65] Zhang, N, Inan, S, Cowan, A, Sun, R, Wang, JM, Rogers, TJ, Caterina, M, and Oppenheim, JJ. A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1. Proc. Natl. Acad. Sci. USA. 2005;102:4536–41. [66] Zhang, N, Rogers, TJ, Caterina, M, and Oppenheim, JJ. Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons. J. Immunol. 2004;173:594–9.

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84 Peptides as Targets of T Cell-Mediated Immune Responses ROLAND MARTIN

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cells (APC) such as dendritic cells (DC), B cells, or macrophages. This central observation by Zinkernagel and Doherty [85], Bevan [4], and others is now referred to as MHC/HLA-restriction of T cells. There are many subtypes of T cells that are characterized by specific surface molecules such as differentiation markers or by their functional phenotype such as the secretion of cytokines or chemokines, and the list is steadily growing. For the purpose of this chapter, only the two main populations will be mentioned, and these are defined by the expression of either one of the two coreceptors, the cluster of differentiation (CD) markers CD4 [41] or CD8 [84]. CD4+ T cells are also referred to as T helper cells (Th cells), while CD8+ T cells are often referred to as cytotoxic or effector T cells [49]. The subdivision into these two main categories of T cells is important not only because of their differing functions but also because they recognize antigen in the context of different MHC/HLA (from here on these abbreviations will be used interchangeably) molecules. CD8+ T cells are restricted by MHC-class I molecules, and CD4+ by MHC-class II molecules with very few exceptions to these rules [20]. Both CD4+ and CD8+ T cells express on their surface a clonotypic T cell receptor (TCR), a heterodimeric transmembrane receptor composed of an alpha (TCR-α) and beta-chain (TCR-β) with remarkable homologies to immunoglobulin molecules with respect to genetic organization of the genes that form the TCR-α/β chains and their molecular structure [50, 51]. The site-specific recombination of the variable (V-), diversity (D-), and joining (J-) gene segments during thymic maturation of T cells results in the random reassortment of a limited number of germ-line encoded complementary determining region (CDR) 1 and 2 loops of their TCR together with a large number of somatically encoded CDR3 loops [35, 39, 44, 53, 70].

Adaptive immune responses are mediated by either soluble immunoglobulin molecules or thymusdependent T lymphocytes. T cells are the basis for cellmediated, antigen-specific immune recognition, and orchestrate almost every aspect of physiological as well as pathological immune responses including the destruction of virus-infected cells, the differentiation of antibody-forming B cells, the lysis of tumor cells, but also allergic responses, autoimmune diseases, transplant rejection, and graft-versus-host disease. During the last three decades, a large proportion of research in immunology has been devoted to understanding T cell specificity. Following the description that T cells recognize antigen in the context of self-major histocompatibility complex (MHC) molecules, which was coined “self-MHC restriction of T cells,” the nature of the antigen was studied in detail. As is briefly described in this chapter, T cells respond with their clonotypic T cell antigen receptor to short peptides that are generated by the antigen processing machinery of antigenpresenting cells and displayed on the surface of these in the context of self-MHC molecules.

INTRODUCTION Different from antibodies that are specific largely for conformational determinants of macromolecules and can interact with these antigens in solution, T cells do not recognize antigen derived from, for example, a foreign pathogen itself and in solution but only in the context of self-major histocompatibility complex (MHC; in humans also referred to as human leukocyte antigen; HLA) molecules on the surface of antigen-presenting Handbook of Biologically Active Peptides

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586 / Chapter 84 Thus, generation of T cells in the thymus results in a T cell repertoire expressing a diverse set of clonally distributed TCR. The theoretical number of different TCR molecules has been estimated to be in the range of 1014, but thymic negative and positive selection processes eliminate the majority of TCR with the propensity to recognize self antigens in the context of self-MHC, and the resulting peripheral T cell repertoire is much smaller, in the order of 107 or even less different specificities [1]. Following the observation that cytotoxic, virus-specific T cells recognize short overlapping peptides of viral proteins [76, 77], a combination of cell biology-, cellular immunology-, biochemical-, and later structural biology techniques led to the discovery that CD8+ cytotoxic T cells recognize with their TCR nonamer peptides embedded in the antigen binding groove of MHC-class I molecules, while CD4+ T cells recognize longer, usually 12–20-mer, peptides in the binding groove of MHC-class II molecules [14, 15, 32, 34, 61, 67, 68, 79]. The restriction of CD8+ T cells by HLA-class I molecules and vice versa CD4+ T cells by HLA-class II results from the fact that the coreceptors CD4 and CD8 interact with HLA-class II and I, respectively, independent of the interaction of the TCR with the HLA-peptide complex. It is impossible to summarize here the many studies and reviews on the general subject of T cell antigen recognition, details how exogenous proteins or endogenously synthesized antigens are processed, loaded onto nascent MHC molecules, and later displayed at the surface, or details on the structural interactions between TCR and MHC-peptide complex. Instead this chapter highlights a few aspects that are important for understanding the overall context and the most important molecules, particularly the MHC molecules and peptides that are involved in T cell recognition. Some important original articles and reviews will be mentioned for the interested reader.

MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I AND CLASS II MOLECULES The human HLA complex is encoded on the short arm of chromosome 6 (6p. 21), which harbors over 150 genes [6, 22, 46]. HLA-class I molecules are heterodimeric transmembrane molecules composed of the invariant β2-microglobulin together with either of the HLA-class I heavy chains encoded by the polymorphic HLA-A, -B, or -C genes [22]. Over 1000 allelic HLAclass I gene variants have been described so far [46]. HLA-class II molecules are also heterodimers consisting of either a nonpolymorphic (DR-α) or a polymorphic (DQ-α, DP-α) alpha chain that pairs with a polymorphic beta chain encoded by the HLA-DR-β,

-DQ-β, and -DP-β genes. If an HLA-class I or -class II molecule is mentioned following, the respective heterodimer is meant (e.g., DR15 as the dimer of DRα and the beta chain encoded by DRB1*1501); if a specific allele is referred to, the gene designation will be mentioned in parentheses (e.g., DRB1*1501 as the DR-β1 chain gene number 1501) [7]. To give an impression of the complexity of HLA-class II allelic polymorphisms, there are currently 61 different HLA-DRB1*04 alleles listed by the American Society of Histocompatibility and Immunogenetics (ASHI), and there are hundreds more polymorphic DR alleles. It is important to note that most HLA-class II haplotypes encode for two DR heterodimers, one encoded by DRα together with the DRB1* gene product and the other by DRα paired with the DRB3*-, -B4* or -B5* gene product [46, 79]. Hence, in humans each chromosome six codes for a set of three different class I molecules (HLA-A, -B, -C), and up to four different HLA-class II molecules (two different DR-, one DQ-, and one DP molecule). If the individual is heterozygous, full HLA-typing will therefore show six HLA-class I and eight class II molecules. This enormous complexity explains the difficulties in finding an unrelated organ transplant donor who fits in all HLA loci. For details on HLA-typing and nomenclature the reader is referred to the periodic updates of the International HLA Workshops or the ASHI website (http://www.ashi-hla.org). The main functional role of MHC/HLA molecules is to display antigenic peptides to T cells. Under physiological conditions the vast majority of surface HLA molecules on APC are occupied by self peptides [34]. Self-peptides in the context of the immune system refers to peptides derived from proteins of the host organism in distinction from peptides that are derived from foreign agents such as viruses, bacteria, fungi, or proteins from other organisms—for example, proteins from meat or vegetables that we ingest. The repertoire of self peptides that is continuously presented to T cells serves two major roles. During T cell development in the thymus, recognition of complexes of self-MHC that display a broad selection of self peptides [11] ensures that the emerging T cell repertoire is tolerant to self proteins. This is achieved by processes referred to as positive and negative selection [2]. T cells expressing TCR with high affinity for self-MHC/self peptide complexes would pose the risk of T cell reactivity against the body’s own tissue and organs, and hence they are deleted by activation-induced cell death. T cells with very low affinity for self-MHC/self peptide complexes fail to be sufficiently stimulated in the thymus to undergo further maturation—that is, they die by neglect. Only T cells recognizing self-MHC/self peptide complexes within a range of intermediate affinities are positively selected [2], mature, and leave the thymus to

Peptides as Targets of T Cell-Mediated Immune Responses / 587

T cell receptor

Antigenic Peptide

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HLA DR Molecule TCR Comparison of the crystal structures of DR2a with MBP (86105) (A) and DR2b with MBP (85-99) (E)

HLA-DR

FIGURE 1. A schematic lateral view of the interaction between T cell receptor, antigenic peptide, and HLADR molecule (top), and a top view (from above) onto the structures of two HLA-DR molecules with the embedded MBP peptide (bottom). On the right side of the graph, a schematic interaction between T cell receptor embedded in a T cell membrane, and the antigenic peptide that is presented in the context of an HLA-DR molecule on the surface of an antigen-presenting cell, is shown. From Li, Y. et al., J. Mol. Biol. 304: 177–188 (2000).

populate the peripheral lymphoid compartments. While these rules generally apply, there are peptides from, for example, splice variants of certain proteins such as proteolipid protein (PLP), a major myelin component, that are not expressed in the thymus [37]. As a result, relatively higher numbers of T cells with specificity for such peptides can be found in the periphery, and these T cells also express higher-affinity TCR. Beyond thymic T cell development, the constant exposure of peripheral T cells to self-MHC/self-peptide complexes guarantees a degree of low-level stimulation that is important for their homeostasis [38, 63, 74, 75]. Therefore, the recognition of self-MHC/self-peptide complexes in the thymus will select for a broad set of T cells that do not cause harm to the host’s own tissues in the periphery. However, these T cells are ready to be activated as soon as complexes of self-MHC with peptides derived from an infectious agent—that is, nonselfpeptides—are recognized with higher affinity during,

for example, a viral infection. Transplantation of histoincompatible or allogeneic cells or tissue—cells that express a different set of MHC molecules—also leads to strong activation of T cells and consequently organ rejection, since thymic selection processes take place in the context of the autologous MHC molecules, and T cells with high-affinity TCR for complexes of foreign MHC and self-peptides are found at high frequencies in the peripheral repertoire [13].

CHARACTERISTICS OF PEPTIDES PRESENTED BY MHC-CLASS I Due to their important role as cytotoxic effector cells during viral infections, CD8+ MHC-class I-restricted T cells have probably received greater attention and at earlier times. MHC restriction was discovered by examining virus-specific CD8+ T cells with specificity for

588 / Chapter 84 lymphocytic choriomeningitis virus [85], and, as already mentioned, studies of influenza virus-specific cytotoxic T cells led to the observation that T cells recognize short overlapping stretches of viral peptides rather than whole proteins or conformational structures of infectious agents [75, 76]. Subsequently, synthetic peptides rather than whole proteins or transfected genes were employed for in vitro and in vivo immune studies in order to understand the nature of the peptides that are recognized by T cells in the context of MHC-class I. The observation of the periodic occurrence of amino acids with certain physicochemical characteristics initially led to the assumption that antigenic peptides assume a helical conformation, when they associate with MHC molecules [45, 65, 66]. However, soon thereafter the x-ray crystallographic study of MHC-class I molecules and bound peptide showed that antigenic peptides are embedded in an extended linear conformation in a peptide binding groove that is formed by a beta-pleated sheet at the bottom of the groove and two flanking alpha helices that are formed by the α1 and α2 domains of the MHC-class I heavy chain [69, 73]. A number of different cell biology-, molecular biology-, biochemical-, and protein chemistry techniques have been applied to identify the characteristics of the class-I–associated peptides including direct sequencing of pools of self-peptides eluted from either class I or class II molecules, the sequencing of individual self-peptides eluted from HLA molecules, the use of truncated or L-ala-substituted peptides that were known to bind to a given allele [15, 34, 67, 68, 79, 82] but also peptide libraries either generated recombinantly in phage or synthetically [9, 16, 26, 27]. The following characteristics of peptides that are presented by MHC-class I molecules emerged from these studies. Crystal structure analysis of MHC-class I molecules showed that the class I binding groove is closed at both ends [69, 73], and the groove itself accommodates peptides that are usually octamers or nonamers, sometimes decamers, and less often longer peptides are found that do not fit completely into the groove but extrude in part outside [61]. Peptides as short as pentamers have been shown to be efficient antigens [62]. Depending on the amino acids that line the groove in individual MHC-class I molecules, certain amino acids with charge, hydrophobicity, or size preferences are accommodated preferentially in separate binding pockets, which are numbered from N- to C-terminus of the peptide as P1 through P9. In almost every class I molecule, one dominant anchor is located at the Cterminus (P9 in nonamer peptides) [61]. The second dominant anchor residue is found at varying positions but most frequently at P2 of the peptide. Peptides that have only dominant anchor residues in common can

differ in their binding affinity for the same MHC molecule by four orders of magnitude [12, 57, 58]. Amino acid residues present at other positions in the peptide can have significant positive or negative effects on peptide binding. Nondominant amino acids with positive effects are termed auxiliary anchor positions. Using the preceding strategies, the binding motifs and most important anchor amino acids have now been identified for many different MHC-class I alleles in mice, rats, and humans. These anchor motifs have been used to devise prediction algorithms that allow to scan any protein and to predict the presence of areas that bind well to an MHC-class I allele of interest—for example, in the context of looking for peptide vaccine candidates [25, 58]. One very useful and publicly available database that lists peptide binding motifs for class I and class II alleles and includes a prediction tool is SYFPEITHI, at the Department of Immunology, University of Tübingen (www.syfpeithi.de) [61]. Peptides that are presented in the context of MHC-class I to CD8+ T cells are usually generated from cytosolic proteins, proteolytically degraded by the proteasome involving a number of distinct proteases, and subsequently transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they are loaded onto nascent class I molecules and subsequently shuttled to the cell surface. This pathway is referred to as the endogenous processing pathway, and it is most important for mounting T cell responses against infections with intracellular pathogens such as enveloped viruses [17, 19].

CHARACTERISTICS OF PEPTIDES PRESENTED BY MHC-CLASS II CD4+ T helper cells recognize peptides that are bound by MHC-class II molecules (see Fig. 1). The same investigational approaches that have been mentioned above for MHC-class I have also been employed to study class II–bound peptides, and a number of distinct characteristics were found. Different from the MHC-class I binding groove, the one of class II molecules is open at either end and hence accommodates peptides that are often substantially longer than those bound by class I [15, 73, 79, 82]. Most peptides that have been eluted from MHC-class II molecules were 12–20 amino acids long, but even trimers were found to bind to class II and stimulate T cells [28]. At the other extreme, it was shown that entire proteins such as myelin basic protein (MBP), a polypeptide of 170 amino acids that may be present in unfolded form in solution, can bind to surface MHC-class II [78]. The MHC-class II binding groove is formed by the polymorphic stretches encoded

Peptides as Targets of T Cell-Mediated Immune Responses / 589 by exon 2 of the class II α- and β-chain genes and typically depicted from the top with the alpha helical parts of the DR/DQ/DP α-chain on top, and the β-chain at the bottom. As for class I, the binding groove accommodates usually 9 amino acids, and the numbering of the putative pockets follows the same order from P1-P9, and different MHC-class II molecules favor distinct anchor amino acids of the peptide in the various pockets, although there is considerable sharing of the requirements at least in some of the main anchors such as P1 and P4 between different alleles. Certain other features lead to additional characteristics such as the amino acid in position 86 of exon 2 in the different HLA-DR alleles, which determines the size of pocket P1 [54]. All alleles have either a valine or glycine in this position, and accordingly the respective allele can either accommodate a large aromatic residue in P1 of the peptide if the DR allele expresses G86 or a substantially smaller residue, if a valine flanks P1 [54]. Due to the fact that class II binding grooves are open at both ends, peptides are not necessarily fixed in one position but can bind in multiple different registers—that is, either shifted to the left or right depending on whether additional favored anchor motifs are present. Since the interface of the MHC-peptide complex to the TCR is affected by such a register shift, it may negatively or positively influence T cell recognition. Furthermore, one needs to take into account not only amino acids in anchor positions that interact with the specificity pockets but also those that contribute negative influences—that is, by the size of the side chain, length of the molecule, proline or charge of amino acids in other positions [26, 27]. Antigen processing of peptides that are presented by MHC-class II molecules usually follows the so-called exogenous pathway [20], which differs from the above class I–associated endogenous processing of antigens. Peptides that are later loaded onto MHC-class II originate from endocytosed or phagocytosed proteins—for example, from a bacterial protein—that are proteolytically degraded in the acidic environment of endo-lysosomes, where they are loaded onto newly synthesized MHC-class II molecules. Upon entering the processing compartment as heterotrimers of class II α- and β-chain complexed with the class II–associated invariant chain (Ii) [19], the Ii-derived CLIP peptide is removed from the antigen presenting groove under the influence of HLA-DM and -DO [40, 80], that serve as peptide editors, and replaced by peptides that are derived from proteolysis of the exogenous protein. Similarly to the main class I–associated processing pathway, the majority of class II–bound peptides are generated through the above exogenous pathway, although different loading compartments and processing paths have been described.

BIOLOGICAL ROLES OF MHC-BOUND PEPTIDES Defense against Foreign Agents/Vaccines As just outlined, our T cell repertoire constantly probes the internal environment, which is formed by cells expressing self-MHC molecules loaded with self peptides. As long as this environment does not change substantially in the quality of MHC-peptide complexes or the relative amounts of antigenic peptides displayed, T cells stay alive but are not fully activated. Upon infection with, for example, an enveloped virus such as influenza virus, epithelial cells are infected and viral proteins are produced at relatively large quantities and loaded onto MHC-class I molecules of these virus-infected cells. The display of viral peptides in the context of self-MHC class I is recognized by CD8+ T cells with TCR that are specific for these MHC-peptide complexes, which become rapidly activated, then lyze the virus-infected cells and interrupt viral spread from one infected cell to the next [76]. In the context of other infectious agents, for example, those that replicate extracellulary such as many bacteria, the infectious agent itself or protein breakdown products are taken up by phagocytes and shuttled into the class II–associated presentation pathway. Eventually, peptides derived from the infectious organism are presented on the surface of APC in the context of MHC-class II molecules to CD4+ helper T cells, which, according to their functional phenotype, produce either proinflammatory cytokines such as interferon-gamma or immunomodulatory cytokines such as interleukin-4, which is central for B cell development. The dynamics of the infection, the site of inflammation, the tropism and other characteristics of the infectious organism, the amount of antigen that is produced, the cell types that are infected or take up antigen, and many other factors determine the relative contribution and role of CD4+ versus CD8+ T cells. In most instances, efficient immune responses will require both potent activation of effector cells but also T cell help, including the production of growth factors such as interleukin-2, which is required for the expansion of both CD4+ and CD8+ cells but also helper function to B cells in order to mount efficient antibody responses and others. Since the presentation of peptide antigens from infectious organisms in the context of self-MHC plays such a central role for effective immune responses, many of the above strategies that have been used for deciphering MHC-peptide interactions and for understanding the specificity of T cells are also of interest for vaccine developments. Due to the complexity of some infectious organisms and the fact that numerous

590 / Chapter 84 antigenic sites of viruses, bacteria, and parasites are rapidly mutated under the pressure of the host’s cellular immune system, vaccine design aims at combining multiple immunodominant peptides and variants thereof in vaccines—for example, for HIV or malaria— although many questions as to how to render these effective vaccines remain open.

Transplantation The existence and importance of histocompatibility molecules was discovered in the context of transplant rejection [10]. Up to this date the rejection of organ and cell transplants by the host’s cellular immune system or graft versus host responses—for example, after hematopoietic stem cell transplants after leukemia treatment, remain major obstacles to transplantation medicine or cell reconstitutive approaches in oncology or any field, where autologous cell sources are not available. The reason for these usually very strong and often lethal allospecific immune responses has already been mentioned briefly. T cell development involves “education” of T cells on self-MHC/self-peptide complexes, and T cells expressing high-affinity TCRs for these complexes are eliminated, thus creating a TCR repertoire that is broad and reactive with high affinity to complexes that are different from self-MHC/self-peptide. If an allogeneic organ transplant is introduced into the host, all nucleated cells of this transplant express nonself MHC-class I. Furthermore, since the binding grooves of these molecules bind different self peptides than the autologous MHC molecules, the T cell repertoire of the host is exposed to large numbers of complexes of nonself MHC-class I together with different self peptides than the ones that it was “educated” on in the thymus. As a result, the frequencies of cells with alloreactivity are usually high in the peripheral blood, and allo-specific immune responses are fulminant [13]. Consequently, matching donor and recipient carefully for the entire HLA-class I and -class II haplotype is necessary, although many other aspects need to be taken into account as well.

AUTOIMMUNE DISEASES T cells not only mediate physiological immune responses against infectious organisms, but also may cause pathological immune reactions [86]. If organs or tissues of the host are affected, the resulting diseases are referred to as autoimmune diseases, and many of these are due to damage by T cells themselves or by T cell-dependent immune mechanisms. The best-known examples are rheumatoid arthritis (RA) [56], insulindependent or type 1 diabetes mellitus (IDDM) [3], and

multiple sclerosis (MS) [72], among numerous others. One piece of evidence that strongly suggests a role of CD4+ T cells in these diseases is the observation that HLA-DR or -DQ genes are the strongest susceptibilityconferring genetic factors in each of them [24]. To give one example, MS in Caucasians, the ethnic group with the highest prevalence of MS, is associated with the DR15 haplotype that includes the two DR-β alleles DRB1*1501 and DRB5*0101 and the DQ molecules DQA1*0102 and DQB1*0602. The relative risk to develop MS that is contributed by these class II alleles has been estimated to be between 10 and 50% of all the genetic susceptibility factors [24]. It is currently not known how the presence of one HLA-class II allele or several that are part of one haplotype confer risk at the molecular level. The following major possibilities have been considered and are in part supported by experimental data. (1) It has been speculated that diseaseassociated HLA-class II molecules preferentially present certain autoantigenic peptides from a tissue, in the case of MS—for example, myelin protein-derived peptides resulting in T cell responses against these peptides. (2) As a variation of possibility 1, it has been shown that certain disease-associated MHC-class II molecules present an overall narrower range of peptides with the result that negative selection for autoreactive T cells is less complete in the thymus, and relatively more autoreactive T cells escape to the periphery. (3) MHC-class II gene expression could be regulated differentially in certain tissues, resulting in overexpression of certain MHC-class II alleles in the target organ. Such a mechanism would be particularly attractive in the brain, where most cells do not express any MHC-class II [72]. There is, however, currently no evidence for this possibility. Trying to understand which self-peptides are the most important targets for autoreactive T cells in an autoimmune disease and which foreign agents might trigger their initial activation is a central goal of research in this area. T cell responses against some autoantigenic peptides such as myelin basic protein (MBP) (83–99) [47, 55, 60] are probably among the best examined of all T cell responses. MBP (83–99) has been analyzed systematically with respect to peptide binding to the MS-associated HLA-DR alleles—that is, DRB1*1501 [82] and DRB5*0101 [80], and later also cocrystallized with them [42, 71]. While the crystal structure analysis yielded important new insights, many conclusions that had previously been derived from T cell studies and binding experiments were confirmed. Recently, the two preceding MBP (83–99)/DR complexes were cocrystallized with two different TCR derived from MS patients, one restricted by DRB1*1501 [23], the other by DRB5*0101 [43]. Different from the few foreign antigen-specific class II–restricted TCR that had been cocrystallized before, these autoreactive TCR showed

Peptides as Targets of T Cell-Mediated Immune Responses / 591 either an unusual binding topology on the MHC-class II/peptide complex [23] or a remarkable absence of any salt bridges or hydrogen bonds between TCR and MHC-bound peptide explaining the low affinity of this autoreactive TCR [43]. Despite this remarkable progress, many questions related to T cell reactivity to autoantigens remain open, and there is currently no single autoantigen, which can be assumed a general target in all patients suffering from one particular autoimmune disease. Regarding the foreign agents that may trigger autoreactive T cells, a mechanism referred to as molecular mimicry appears particularly attractive. Molecular mimicry in the context of T cell responses means that a peptide derived from a foreign agent looks similar to a self peptide. Seminal studies by Fujinami and Oldstone [18], who proposed this mechanism, showed that molecular mimicry requires sequence identity over a stretch of 6 or more amino acids—for example, between a hepatitis B virus-derived peptide and a peptide from MBP. They demonstrated that injection of the viral peptide into rabbits causes an immune-mediated encephalomyelitis and cross-reactivity of immune cells against the brain protein MBP [18]. The subsequent dissection of the molecular recognition of peptides by T cells led to a better understanding of the rules of T cell cross-reactivity. It became clear that some amino acids in an antigenic peptide are particularly relevant for binding to the MHC molecule and others for contacting the TCR. Based on this knowledge searches for molecular mimics concentrated on the presence of these contact amino acids or motifs rather than complete sequence homology between an autoantigen and a peptide from a virus or bacterium [83]. Based on theoretical considerations that the T cell repertoire in a mammalian host is orders of magnitude smaller than the potential spectrum of antigenic peptides that can be encountered, it has been proposed that single T cells must be able to recognize up to 106 different peptides [48], and these speculations are supported by data that was mainly generated by probing the specificity repertoire of single T cell clones with systematically arranged peptide mutations [29, 30] or combinatorial peptide libraries [31]. These tools are currently being used to better understand the range of molecular mimicry, the relative affinity of single clones for peptides, and other related questions.

THERAPEUTIC USE OF PEPTIDES Not surprisingly, many investigators quickly began to apply the knowledge about the central role of peptides for T cell recognition during infections, tumor control and autoimmune diseases also for therapeutic purposes.

The potential relevance of peptides or pools of peptides for vaccines has already been mentioned above, and the author anticipates that this will be one interesting strategy to counter the occurrence of rapidly mutating immunodominant epitopes and immune evasion that is observed in some infectious diseases, such as HIV and malaria [33]. Tumor immunologists have identified the specific target peptides for tumor-infiltrating lymphocyte CD4+ and CD8+ populations, and in numerous cases, these were “neoantigens”—that is, peptides that emerged through mutations in the tumor [81]. There are numerous clinical trials ongoing, in which either dendritic cells pulsed with the tumor peptide, peptide alone, or the former in combination with in vitro expanded tumor-infiltrating lymphocytes are used for tumor-specific immunotherapies [64]. Some of these approaches have already shown remarkable success in patients with large tumor burdens and widespread metastases [64]. In the case of infectious diseases and tumor immunology, the use of peptides or peptidespecific T cells aims at enhancing the respective immune responses. In contrast, under conditions where immune responses are damaging—such as allergic responses or autoimmune diseases—investigators have tried to modulate the phenotype and reactivity of T cells by modifying autoantigenic peptides or peptides derived from an allergen or administered the peptide in a way that causes immunomodulation. Such therapeutic approaches were spurred by the discovery that systematic amino acid modifications of a peptide that serves as an agonist to a T cell clone—that is, it stimulates a full immune response including proliferation, cytokine release, and cytotoxicity—can result in partial agonists or even TCR antagonists [21]. In summary, such peptides are described as altered peptide ligands (APL). Partial agonism results from modifications of peptides that still stimulate some functions in the responding T cell but not others, such as proliferation in the absence of cytokine release. TCR antagonists are peptides that have been modified in a way that they inhibit a T cell response even if the agonist peptide is present at the same time by altering the TCR signaling machinery [21]. Each of these effects could be shown in vitro or in vivo in a number of different experimental systems, and APL peptides were also successfully used as therapeutic agents in experimental animal models of MS [8, 52]. One particularly attractive mechanism of action that emerged from these studies is APL-induced bystander suppression [52]. This mechanism refers to the generation of T cells upon vaccination with the APL that are specific for the APL peptide and express immunomodulatory cytokines that attenuate the proinflammatory autoimmune process. The induced APL-specific T cells cross-react with the native autoantigenic peptide, and hence, whenever tissue is damaged and autoantigen

592 / Chapter 84 released, the APL-specific T cell population is stimulated to secrete “beneficial” immune mediators. Despite the elegant in vitro and in vivo findings, the use of APL in human autoimmune diseases remains problematic. A phase II trial of an APL derived from MBP (83–99) in MS resulted in disease exacerbation of some patients upon injection of high doses [5] and a trend toward benefit at lower doses [36]. These observations showed, however, that autoantigens that had been discovered in animal or human in vitro studies are indeed relevant targets in the human autoimmune diseases. Future research will have to address how such peptides should be applied—that is, by what route, at which dose, and what other factors need to be considered.

[10]

[11]

[12]

[13]

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CONCLUSION The investigation of T cell specificity for antigenic peptides under physiological and pathological conditions remains a focus of basic research in immunology. Beyond that, the use of antigenic peptides for therapeutic or vaccine purposes rapidly gains in importance in clinical medicine in infectious diseases, tumor immunology, for allergic diseases, and in the field of T cellmediated autoimmune diseases.

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85 The Use of Positional Scanning Synthetic Peptide Combinatorial Libraries to Identify Immunological Relevant Peptides MIREIA SOSPEDRA AND CLEMENCIA PINILLA

ABSTRACT

described method, the iterative approach [11], uses a selection and enhancement process to identify individual compounds. Following the identification of active mixtures having defined positions, the remaining positions are then defined, one diversity position at a time, through a synthesis and selection process, until individual compounds are identified. The second deconvolution approach, the positional scanning method [18], simultaneously addresses each position of the library by testing at the same time individual sublibraries in which each diversity position is defined with a single aa, while the remaining positions are composed of mixtures of aa. The assay data derived from each positional sublibrary provides information about the most important aa for every diversity position of the Positional Scanning (PS)-SCL. An illustration of the PS-SCL concept using a tripeptide combinatorial library is shown in Fig. 1. Four different amino acids are incorporated at each of the three diversity positions resulting in a diversity of 64 individual peptides. When the same diversity is arranged as a PS-SCL, only 12 peptide mixtures (4 amino acids × 3 positions) need to be synthesized. Each of the three positional sublibraries—namely OXX, XOX, and XXO—contains the same diversity of peptides, but differ only in the location of the defined aa position. The O positions represent one of the four amino acids, while the remaining two diversity positions are mixtures (X) of the same four amino acids. Shown below each mixture are the 16 peptides (42) that make up that mixture. Assuming that ART is the only active tripeptide in this library that is recognized, the ART tripeptide (outlined below each sublibrary in Fig. 1) is present in all three positional sublibraries. Thus, the only mixtures with activity are AXX, XRX, and XXT because the ART

Many receptor-ligand interactions in biology involve the binding of peptidic molecules to complex surface receptors. Among these, the immune system is a particularly interesting example, since peptides are the main targets for T lymphocytes and can also be recognized by antibodies. The systematic identification of ligands for biologically relevant receptors has been greatly facilitated by the introduction of combinatorial peptide chemistry. Here, we introduce the underlying principles of the use of positional scanning synthetic combinatorial peptide libraries and provide a few informative examples how they have been employed in the recent past.

DEFINITION OF POSITIONAL SCANNING SYNTHETIC COMBINATORIAL PEPTIDE LIBRARIES (PS-SCL) Synthetic combinatorial libraries (SCLs) made up of mixtures having one position defined with a given amino acid (aa), while other positions have amino acids incorporated as mixtures. These libraries represent a very large number of compounds—in this case synthetic peptides. SCLs are generated by use of the multiple solid phase synthesis method known as the “tea bag approach” [10]. Then they are cleaved from the solid support and assayed in solution; this allows each peptide within each mixture to freely interact with a given receptor and thus these libraries can be used in virtually all existing assay systems. Two different deconvolution methods can be used to identify individual active compounds from these mixture-based SCLs. The first Handbook of Biologically Active Peptides

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596 / Chapter 85

Synthetic combinatorial Library (SCL) Tripeptide library 4 amino acids in each position A = Alanine

T = Threonine

R = Arginine

W = Tryptophan

Individual peptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

XXX 43 = 64

DIVERSITY = 64 individual peptides

A A A A A A A A A A A A A A A A

A A A A R R R R T T T T W W W W

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

A R T W A R T W A R T W A R T W

R R R R R R R R R R R R R R R R

A A A A R R R R T T T T W W W W

A R T W A R T W A R T W A R T W

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

T T T T T T T T T T T T T T T T

A A A A R R R R T T T T W W W W

A R T W A R T W A R T W A R T W

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

W W W W W W W W W W W W W W W W

A A A A R R R R T T T T W W W W

A R T W A R T W A R T W A R T W

Positional Scanning Synthetic combinatorial Library (PS-SCL) 0 A R T W

Position 1 1 2 3 4 A A A A A A A A A A A A A A A A A

X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

R R R R R R R R R R R R R R R R R

FIGURE 1.

X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

T T T T T T T T T T T T T T T T T

X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

X X X X X W W W W W W W W W W W W W W W W W

X X X X X X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

5 6 7 8 X A A A A R R R R T T T T W W W W

A A A A A A A A A A A A A A A A A

X A R T W A R T W A R T W A R T W

X A A A A R R R R T T T T W W W W

R R R R R R R R R R R R R R R R R

X A R T W A R T W A R T W A R T W

X X X X X

X 0 X A X R X T X W

Position 2

X A A A A R R R R T T T T W W W W

T T T T T T T T T T T T T T T T T

X A R T W A R T W A R T W A R T W

X A A A A R R R R T T T T W W W W

W W W W W W W W W W W W W W W W W

X A R T W A R T W A R T W A R T W

X X X X X

Position 3 9 10 11 12 X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

A A A A A A A A A A A A A A A A A

X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

R R R R R R R R R R R R R R R R R

X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

T T T T T T T T T T T T T T T T T

X 0 X A X R X T X W X A A A A R R R R T T T T W W W W

X A R T W A R T W A R T W A R T W

W W W W W W W W W W W W W W W W W

DIVERSITY 12 peptide mixtures

Conceptual illustration of tripeptide PS-SCL.

tripeptide is present only in those mixtures. The combination of these aa in their respective positions yields the tripeptide ART, which would then be synthesized and tested for its activity against the receptor. It should be noted that the activity observed for each of the three mixtures (AXX, XRX, and XXT) is due to the presence of the tripeptide ART within each mixture, and not due to the individual amino acids (A, R, and T) that occupy the defined positions. In more complex libraries, more than one mixture is often found to have activity at each position. Selection of the aa for the synthesis of individual peptides is based first on activity and then on differences in the chemical character. Although the above example is a simple representation of the arrangement and use of a PS-SCL, the same concept applies to all types of mixture-based libraries having defined and mixture positions. To give an estimate of the complexity of PS-SCL, a hexapeptide library using 20 amino acids represents a total diversity of approximately 50 million (196) (Cys in not included in the mixture positions) individual peptides, while a decapeptide library using the same number of aa represents a total diversity of

approximately 6 trillion (1910) individual peptides, a number of peptides that could clearly not be synthesized or tested individually. A hexamer PS-SCL can be formatted into 120 mixtures (20 aa × 6 positions) and a decamer PS-SCL into 200 mixtures (20 aa × 10 positions). PS-SCLs provide an invaluable resource for characterizing the molecular interactions that underlie many biological processes and for identifying novel sequences that have a desired biological activity. In this chapter we describe the use of PS-SCL in identifying immunological relevant peptides.

USE OF PS-SCL TO DETERMINE ANTIBODY LIGANDS Antibodies are glycoproteins synthesized and secreted by B cells that mediate humoral immunity. Monoclonal antibodies (mAb) are identical antibodies produced by one type of B cell that recognize antigens with high affinity and specificity. The remarkable specificity of

The Use of Positional Scanning Synthetic Peptide Combinatorial Libraries / 597 antibodies makes them very valuable for protection against disease organisms, but also attractive candidates to target other types of molecules found in the body and promising agents for human therapy. Furthermore, antibodies have an important medical application in diagnosis of infectious diseases and autoimmune disorders. In this context the identification of antibody ligands is of great interest. Despite the high specificity that characterizes antibodies, it is important to notice that cross-recognition of peptides by single antibodies also exists. The identification of synthetic mimics recognized by an antibody is also very interesting in the development of more effective immunodiagnostics and synthetic vaccines (see chapter by Kaumaya in the previous section of this book). The epitopes recognized by antibodies can be classified as either continuous or discontinuous. It is believed that antibodies recognize mainly native discontinuous or conformational epitopes and therefore rarely peptides. However, it has been described that antibodies that recognize discontinuous epitopes can cross-react with linear peptide fragments, indicating that peptides can be used effectively to determine antibody specificity. The classical approach used to identify antibody ligands involves the synthesis and testing of overlapping peptides. Although this methodology allows the identification of ligands, it requires substantial time and resources. Several different peptide combinatorial library approaches, including synthetic and phage display, have emerged as more straightforward and useful approaches for mapping antigen-antibody interactions. Among these approaches PS-SCL offer an important advantage since no prior information regarding the specificity of the antibody is needed. The first use of PS-SCL in immunology was for the identification of high-affinity peptides recognized by monoclonal antibodies (Mabs) of known specificity. In 1992 Pinilla et al. [18] demonstrated that the antigenic determinants recognized with high affinity by two antibodies could be correctly identified upon a single screening using hexamer PS-SCL. In 1994 Pinilla et al. [19] extended the practical range to decamer libraries. With a decapeptide PS-SCL the known six-residue antigenic determinant sequence recognized by an antibody was found, with the most specific residues appearing to “walk through” the 10 positions of the peptide library. Interestingly, the antibody recognition in this system was stronger when the antigenic determinant was located at the C-terminus of the decapeptide library. Individual decapeptides corresponding to sequences derived from the most active peptide mixtures at each position were synthesized to confirm the results of the screening. Four peptides were found to be 5–10 times more active than the known control peptides. Additional studies were performed using hexa- and decamer

PS-SCL to identify antigen determinants of antibodies (reviewed in [12, 21]). One of the main advantages of the use of PS-SCL for the study of antibody specificity is that no prior specificity information is required. In 1996, two studies reported the use of PS-SCL to determine the epitope determinant for antibodies for which the target protein was known, but not the epitope determinant. The first study [1] reported the identification of high affinity, crossreactive peptides recognized by a Mab (MAb 12) known to recognize the surface antigen of hepatitis B virus (HBsAg). The binding specificity of MAb 12 was studied with a hexapeptide PS-SCL that was screened by competitive enzyme-linked immunosorbent assay (ELISA). This led to the identification of the peptides STTSMM (IC50 = 170 nM) and SVGPPH (IC50 = 165 nM), which specifically inhibited the interaction between HBsAg and MAb 12. Since motifs from these two hexapeptides, -STTS- and -GP-, were located in the primary sequence of the protein (residues 114–120), overlapping hexapeptides of this region were used to identify the most active hexapeptide, TTSTGP (IC50 = 2.3 μM), which was 10-fold less active than either hexapeptide found from the library. In the second study [4], an epitope for a monoclonal antibody 201/9, raised against betafactor XIIa was determined with octapeptide PS-SCL libraries. One important observation was that highly active peptide sequences having little or no resemblance with the known immunogen were identified for some antibodies. Park et al. [17] using a hexapeptide PS-SCL identified peptide sequences that inhibit the binding of IgG antibodies specific of thyroid-stimulating-hormone receptor (TSHR) obtained from patients with Graves’ disease. Interestingly, these peptides do not resemble the linear sequence of TSHR and thus may mimic a spatial arrangement of the key antigenic residues. The structural basis for the molecular mimicry shown by monoclonal antibodies remains unclear. It has been suggested that the flexible hypervariable loops of antibodies and the fact that they recognize functional conformation rather than identical structures can facilitate cross-recognition. It is often assumed that cross-reactive antigens share some structural similarity that is specifically recognized by a monoclonal antibody. Antibody cross-reactivity was examined by Pinilla et al. [20] screening hexamer PS-SCL, consisting of all-L or all-D aa, for inhibition of the monoclonal antibody HGAC 39.G3 which binds to N-acetyl-d-glucosamine (GlcNAc) on a polyrhamnose backbone. Inhibitory activity by mixtures from the all-D hexapeptide library was greater than the activity from the all-L libraries. The sequence Ac-yryygl-NH2 was specifically recognized by mAb HGAC 39.G3 with a relative affinity of 300 nM when measured in a competitive binding assay. This peptide

598 / Chapter 85 shared little homology with the carbohydrate antigen, suggesting that the peptide might interact differently with the Mab.

USE OF PS-SCL FOR T CELL EPITOPE MAPPING T lymphocytes play a central role in controlling the acquired immune response orchestrating both the “cellular” and “humoral” (helping B cells) arms of acquired/ specific immunity. Like B lymphocytes, T cells express a clonal antigen-specific receptor that recognizes fragmented, linear antigenic peptides when these are bound to proteins encoded by major histocompatibility complex (MHC) genes (see chapter by Martin in this section of the book). There are two major subsets of T lymphocytes that differ in effector function and MHC restriction. CD8+ T cells recognize peptides bound to MHC class I molecules and act as effector cells during viral infections through antigen-specific cytotoxic activity. CD4+ T cells recognize peptides bound to MHC class II molecules and act as inducers of T and B cell functions producing cytokines. While T cell recognition was previously considered exquisitely specific for single peptides or close homologs with similar sequence, it is now clear that T cell recognition is very flexible and cross-reactivity is a common phenomenon. T lymphocytes play important roles in infectious diseases, autoimmunity, and also immune responses against tumors. For many of these disorders, the relevant target antigens are not known. Designing effective methods that allow the search for T cell epitopes is therefore an important goal, and several approaches have been applied with variable success. Epitope mapping with series of overlapping peptides can be applied only when the antigenic protein is known. Biochemical analysis of pools of peptides eluted from MHC molecules is another approach but requires sophisticated instrumentation. Molecular biology approaches using expression libraries or the search of epitopes using MHC-binding motifs are biased since they require the previous selection of an organism or tissue of interest. Several studies have demonstrated the efficacy of using PS-SCL for identifying target antigens (Ags) and highly active peptide mimics for both CD4+ and CD8+ T cells of known and unknown specificity. Initially CD8+ T cells received more attention not only because they often show higher antigen affinities but also because the class I binding groove accommodates shorter peptides (8–10 aa) and less complex peptide libraries can be used to determine their specificity. In 1996, Gundlach et al. [5] presented a new approach to T cell epitope determination for cytotoxic T cells using

PS-SCL. Sequences for potential T cell epitopes were deduced from scan profiles by use of combinations of the active amino acids. An octamer PS-SCL was successfully used by this group to identify self-MHC-restricted antigenic peptides cross-recognized by an allo-MHCspecific CTL clone [31] and also to study the specificity and degeneracy of H-3-specific, H-2K(b)-restricted CTL clones [6]. The identification of defined human tumor antigens recognized by autologous CTLs opened new opportunities for the development of antigen-specific cancer vaccines (see chapter by Wagner and Disis in the previous section of this book). In this context to elucidate the nature of antigens recognized by tumor-reactive T cells is a prerequisite for designing such vaccines. In 2000, Linnemann et al. [15] used a nonamer PS-SCL in a standard chromium release assay to identify a synthetic epitope for the My-La CTL specific for cutaneous T cell lymphoma. The response toward this epitope was comparable to the response toward their natural target My-La. The identification of synthetic epitopes for tumor-specific CTL clones that are recognized in an MHC-restricted manner can be used for the development of vaccines for immune therapies of cancer. PS-SCL also can be used to improve vaccines, for example, enhancing the activity of peptide ligands in order to reduce the amount of peptide vaccine needed to obtain a clinical CTL response in an immunization strategy. In this context is important the study by La Rosa et al. [14] in which the authors using a nonamer PS-SCL identified a series of peptide analogs for the cytomegalovirus (CMV)-specific T Cell Clone, 3-3F4, that were recognized better than the native peptide sequence. The identification of the specificity of CD4+ class II restricted T cell clones appeared more difficult; since the affinity of these cells is lower than described for CD8+ T cells, and peptides bound to class II molecules are longer (typically 12–15 amino acids). CD4+ class IIrestricted T cells specific for self antigens are thought to be involved in the pathogenesis of most human autoimmune diseases, and molecular mimicry between foreign and self ligands has been implicated as a possible mechanism for their activation. The identification of peptide mimics for these autoreactive CD4+ T cells is crucial to understand the autoimmune process and to develop future therapies. In 1997, Hemmer et al. [7] demonstrated that an 11-mer PS-SCL could be used to identify cross-reactive artificial peptides for a CD4+ T cell clone (TCC), TL5G7, specific for the myelin basic protein peptide (MBP) (86–96) and derived from the peripheral blood of a patient with multiple sclerosis. Interestingly these artificial peptides induced proliferative responses at much lower concentrations than the original peptide MBP (86–96) used to generate the

The Use of Positional Scanning Synthetic Peptide Combinatorial Libraries / 599 TCC. In addition stimulatory ligands derived from protein sequences of self and microbial proteins were identified, some of them more potent agonists than MBP (86–96). A more extensive study was carried out with decamer PS-SCL to study the specificity of two additional MBP (86–96)-specific TCC, also established from peripheral blood lymphocytes of MS patients: TL5F6 that is DR2b restricted, and TL3A6 that is DR2a restricted [9]. The use of PS-SCL allowed the identification of TCR motifs and stimulatory antigenic peptides. In this study the authors suggested for the first time that each amino acid residue contributes to the overall potency of the antigenic peptide ligand. The decapeptide PS-SCL was also successfully used to identify peptide mimics for CD4+ T cell clones of known specificity in two additional studies. The first study published in 1999 [33] demonstrated the used of 10-mer PS-SCL to identify peptide epitopes more effective than the native peptide in stimulating proliferative responses in a CD4+ T cell clone derived from transgenic mice. This TCC expresses a TCR specific for the 88–104 peptide fragment of pigeon cytochrome c and the Ek class II molecule. A second study demonstrating the power of PS-SCL in identifying epitopes for CD4+ T cell clones was published in 2001 [13]. Nonobese diabetic (NOD) mice spontaneously develop destruction of the insulinproducing pancreatic islet beta cells similar to type 1 diabetes mellitus in humans. The diabetogenic NOD CD4+ T cell clone, BDC2.5, adoptively transfer disease after being activated in vitro with syngenic islets cells. The identity of the islet cell Ag responsible for pathogenicity is not known. PS-SCL was used to identify decapeptides able to stimulate BDC2.5 cells and transfer disease to NOD-scid mice. Surprisingly, some of these peptides include sequences similar to those found within the 528–539 fragment of glutamic acid decarboxylase 65, a candidate autoantigen in diabetes. An enormous step forward was achieved in the use of PS-SCL to identify T cell epitopes when Hemmer et al. [8] incorporated biometrical analysis to this approach. PS-SCL-based biometrical analysis developed as a strategy that systematically compares the data derived from a PS-SCL with all peptide fragments present in protein databases. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries consisting of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by

means of molecular mimicry to tissue autoantigens. Using this strategy the authors effectively identified both microbial epitopes, from Borrelia burgdorferi, and candidate autoantigens, from the host, recognized by a TCC, CSF-3, of unknown specificity that was established from the cerebrospinal fluid of a patient with chronic neuroborreliosis using Borrelia burgdorferi lysate. The PSSCL-based biometrical analysis was analyzed in detail by Zhao et al. [34], and its predictive power was demonstrated with a CD4+ TCC, GP5F11, that was established from peripheral blood of a patient with multiple sclerosis using influenza virus hemagglutinin (HA) peptide 309–318 as antigen. The experimental data from the screening of a PS-SCL was used to generate a matrix, which was used to score all the decapeptides in all the viral proteins in the Genpept database. The information obtained was a list of peptides ranked by their score, and the native peptide ranked 6th. Selected peptides were synthesized and tested and their stimulatory capacity for GP5F11 confirmed. Afterward, the effectiveness of PS-SCL-based biometrical analysis has also been demonstrated with tumor antigen-specific CD8+ clones of known (Melan-A [22, 26], and Tyrosinase [27]) and unknown [25] specificity; these studies as well as an overview of the PS-SCL-based biometrical analysis of the study of T cell specificity and cross-reactivity were recently reviewed [16].

USE OF PS-SCL TO IDENTIFY MHC BINDING MOTIFS CD8+ and CD4+ T lymphocytes recognize peptides bound to class I or class II MHC molecules, respectively. These complexes are assembled intracellularly during the biosynthesis and trafficking of MHC molecules. MHC class I and II molecules have different peptide binding sites. MHC class I molecules have a closed groove that only allows short peptides of 8–10 amino acids in length. In contrast MHC class II molecules bind peptides that are 10–25 amino acids in length. Considerable interest has focused on understanding how MHC specificity is generated with the ultimate goal of predicting peptide binding. PS-SCL has emerged as a highly efficient, universal, and unbiased approach to address MHC specificity. In the first report in 1995, Udaka et al. [30] identified patterns of amino acid preferences from testing an octapeptide PS-SCL against the mouse MHC-I molecule H2Kb using stabilization experiments. A year later, Stryhn et al. [28] used PS-SCL and competitive binding assays to describe peptide binding motifs for class I molecules, including primary and secondary anchor residues. The quantification of the effect of any amino acid in each position also allowed the identification of disfavored

600 / Chapter 85 residues very important in shaping MHC class I specificity. The same year a second study used PS-SCL to determine the impact of the amino acids in every sequence position of octapeptides using stabilization experiments for the mouse MHC-I molecule H2-Ld [24]. Later, Udaka et al. [32] also used this approach to determine the specificities of three mouse major MHC class I molecules, Kb, Db, and Ld. The result of this study was used to create a scoring program to predict MHC-binding peptides in proteins. However, it is important to note that the authors found frequently MHC-binding peptides with incomplete or no anchor amino acid(s) suggesting a substantial bias that is introduced by natural antigen processing in peptide selection by MHC class I molecules. PS-SCL have also been employed to study the binding characteristics of MHC class II molecules. In 1996, Fleckenstein et al. [3] used an undecapeptide PS-SCL to estimate the influence of every amino acid on the binding to human MHC-DRB1*0101 molecules (HLADR1) and to identify new peptide ligands. The amino acids favorable or unfavorable for DR1-binding at every sequence position were defined and individual peptides were identified. Recently, a nonapeptide PS-SCL has been used in a new study [2] to purify RT1.Da and to determine its binding characteristics.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

CONCLUSION This chapter presents a summary of the use of PSSCLs to elucidate the specificity of a number of immunologically relevant receptor-peptide interactions. These receptors include antibody, T cell receptors, and MHC molecules. It is important to note that peptide PS-SCL libraries have also been successfully used for the identification of the substrates of several proteases relevant in immunology such as human caspases and granzyme B [29]. Furthermore, nonpeptidic and small molecule PS-SCLs represent a source of diversity for the identification of protein-protein, or protein receptor interactions in which inhibitors or modulators of non-peptidic composition would be favorable. We and others have successfully used these libraries for the identification of highly active agonists and antagonists of G-protein coupled receptors, antimicrobials, enzyme inhibitors, as well as other biological targets (reviewed [12, 23]).

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that inhibit the stimulation of thyrotropin receptor by Graves’ immunoglobulin G from peptide libraries. Endocrinology 138:617–626. Pinilla, C., J. R. Appel, P. Blanc, and R. A. Houghten. 1992. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. Biotechniques 13:901–905. Pinilla, C., J. R. Appel, and R. A. Houghten. 1994. Investigation of antigen-antibody interactions using a soluble nonsupportbound synthetic decapeptide library composed of four trillion sequences. Biochem. J. 301:847–853. Pinilla, C., J. R. Appel, G. D. Campbell, J. Buencamino, N. Benkirane, S. Muller, and N. Greenspan. 1998. All-D peptides recognized by an anti-carbohydrate antibody identified from a positional scanning library. J. Mol. Biol. 283:1013–1025. Pinilla, C., R. Martin, B. Gran, J. R. Appel, C. Boggiano, D. B. Wilson, and R. A. Houghten. 1999. Exploring immunological specificity using synthetic peptide combinatorial libraries. Curr. Opin. Immunol. 11:193–202. Pinilla, C., V. Rubio-Godoy, V. Dutoit, P. Guillaume, R. Simon, Y. Zhao, R. A. Houghten, J.-C. Cerottini, P. Romero, and D. Valmori. 2001. Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor reactive cytolytic T lymphocytes. Cancer Res. 61:5153–5160. Pinilla, C., J. R. Appel, E. Borras, and R. A. Houghten. 2003. Advances in the use of synthetic combinatorial chemistry: Mixture-based libraries. Nat. Med. 9:118–122. Pridzun, L., K.-H. Wiesmüller, S. Kienle, G. Jung, and P. Walden. 1996. Amino acid preferences in the octapeptide subunit of the major histocompatibility complex class I heterodimer H-2Ld. Eur. J. Biochem. 236:249–253. Rubio-Godoy, V., M. Ayyoub, V. Dutoit, C. Servis, A. Schink, D. Rimoldi, P. Romero, J.-C. Cerottini, R. Simon, Y. Zhao, R. A. Houghten, C. Pinilla, and D. Valmori. 2002. Combinatorial peptide library based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity. Eur. J. Immunol. 32:2292–2299. Rubio-Godoy, V., V. Dutoit, Y. Zhao, R. Simon, P. Guillaume, R. Houghten, P. Romero, J. C. Cerottini, C. Pinilla, and D. Valmori. 2002. Positional scanning-synthetic peptide librarybased analysis of self- and pathogen-derived peptide crossreactivity with tumor-reactive Melan-A-specific CTL. J. Immunol. 169:5696–5707.

[27] Rubio-Godoy, V., C. Pinilla, V. Dutoit, E. Borras, R. Simon, Y. Zhao, J.-C. Cerottini, P. Romero, R. A. Houghten, and D. Valmori. 2002. Towards synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ tumor-reactive T-cell ligands: A comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ CTL. Cancer Res. 62:2058–2063. [28] Stryhn, A., L. O. Pedersen, T. Romme, C. H. Holm, A. Holm, and S. Buus. 1996. Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: Quantitation by peptide libraries and improved prediction of binding. Eur. J. Immunol. 26:1911–1918. [29] Thornberry, N. A., T. A. Ranon, E. P. Pieterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom, S. Roy, J. P. Vaillancourt, K. T. Chapman, and D. W. Nicholson. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B—Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272: 17907–17911. [30] Udaka, K., K.-H. Wiesmüller, S. Kienle, G. Jung, and P. Walden. 1995. Tolerance to amino acid variations in peptides binding to the major histocompatibility complex class I protein H-2Kb. J. Biol. Chem. 270:24130–24134. [31] Udaka, K., K.-H. Wiesmüller, S. Kienle, G. Jung, and P. Walden. 1996. Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone. J. Immunol. 157:670–678. [32] Udaka, K., K.-H. Wiesmüller, S. Kienle, G. Jung, H. Tanamura, H. Yamagishi, K. Okamura, P. Walden, T. Suto, and T. Kawasaki. 2000. An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries. Immunogenetics. 51:816–828. [33] Wilson, D. B., C. Pinilla, D. H. Wilson, K. Schroder, C. Boggiano, V. Judkowski, J. Kaye, B. Hemmer, R. Martin, and R. A. Houghten. 1999. Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands. J. Immunol. 163:6424–6434. [34] Zhao, Y., B. Gran, C. Pinilla, S. Markovic-Plese, B. Hemmer, A. Tzou, L. W. Whitney, W. E. Biddison, R. Martin, and R. Simon. 2001. Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions. J. Immunol. 167:2130–2141.

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86 Copolymer 1 and Related Peptides as Immunomodulating Agents RUTH ARNON

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immunogenic proteins [27, 58]. The system that had been studied most extensively is experimental autoimmune encephalomyelitis (EAE), which served as the animal model for multiple sclerosis (MS). MS is a chronic, inflammatory disease of the CNS, usually diagnosed in young adults and is characterized by localized myelin destruction and axonal damage or loss [22, 31]. Although the etiology and pathogenesis of MS remain largely unknown, there are indications that the disease is of autoimmune nature. Results obtained both from MS patients and from the animal model point to the involvement of T cell–mediated immune response towards several myelin antigens, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) [15, 32, 43]. However, in addition to the involvement of activated lymphocytes, myelin-specific antibodies may also be implicated in the pathogenesis of MS [53]. In view of the autoimmune nature of MS, the drugs recommended for its treatment, particularly for the most common type of the disease, relapsingremitting MS, were designed for reduction of the autoimmune responses. However, the limited success and low tolerability of the general immunosuppressants led to the introduction of general immunomodulators as therapeutic agents—namely several forms of recombinant interferon β—as well as the more specific immunomodulator copolymer 1 (Cop-1) also known as glatiramer acetate (GA). These two nomenclatures are used interchangeably in this chapter. Cop-1 is a random polymer consisting of the amino acids l-alanine, l-lysine, l-glutamic acid, and l-tyrosine, in a molar ratio of 4.2 : 3.4 : 1.4 : 1.0 [10]. It was designed to simulate the myelin protein MBP, and was shown to suppress very efficiently experimental autoimmune encephalomyelitis (EAE) in several species

Copolymer 1 (Cop-1) is a synthetic random polypeptide composed of l-alanine, l-lysine, l-glutamic acid, and l-tyrosine, also known as glatiramer acetate. It is highly effective in the suppression of experimental autoimmune encephalitis (EAE), the animal model for multiple sclerosis (MS), in various species including primates. Following several clinical trials it was approved as a drug for the treatment of MS, under the name Copaxone®. Its mode of action is by initial strong binding to the major histocompatibility (MHC) class II molecules and competition with myelin proteins for such binding and presentation to T cells, followed by the induction of specific suppressor T cells of the Th2 type. Copolymer 1 and related polypeptides exert the suppressive effect primarily by immunomodulation and have recently demonstrated ameliorating effects in a few additional autoimmune disorders, as well as in graft rejection. This is the first drug based on an antigen-specific intervention in an autoimmune disease.

INTRODUCTION Peptides and polypeptides have demonstrated reactivity in various manifestations of the immune response, including autoimmunity. In autoimmune conditions, T cells reactive to self antigens escape elimination in the thymus and are activated in the periphery, where they can provoke damage in specific organs. Restoration of self-tolerance without suppressing the immune system nonspecifically is a major challenge and attempts towards it have been made in the case of several autoimmune disorders, with either proteins that had been rendered tolerogenic or peptides derived from the Handbook of Biologically Active Peptides

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604 / Chapter 86 including primates [13]. Following several clinical trials, it was approved as a drug named Copaxone® for the treatment of MS patients. Cop-1 is cross-reactive with MBP, and its beneficial effect in EAE may be explained in terms of this cross-reactivity. However, the suppressive effect of GA, which stems from its immunomodulatory capacity, is not limited to EAE and MS and can be demonstrated in the case of several other autoimmune disorders. This chapter includes a brief description of the studies on the suppression of EAE by Cop 1 in experimental animal models, and the subsequent clinical trials that led to its approval as a novel drug, followed by a discussion on its mode of action. The main focus, however, will be on our more recent findings on the immunomodulatory and immunoprotective activity of GA in the target organ of MS/EAE—namely the CNS, and on some new applications of GA—in other autoimmune disorders and in the suppression of graft rejection that are based on its mechanism of activity.

SUPPRESSION OF EAE BY GA GA was designed to simulate MBP, one of the major myelin-derived autoantigens that induces EAE and that has been implicated in the pathogenesis of MS. Indeed, GA was demonstrated to suppress EAE induced by MBP in a variety of species: guinea pigs, rabbits, mice, and two species of primates—rhesus monkeys and baboons. In contrast to rodents where GA inhibits the onset of the disease, in primates it suppressed an ongoing disease. Furthermore, GA was effective in suppressing the chronic relapsing EAE, a disease that shows a closer resemblance to MS, which can be induced in young guinea pigs by MBP, or in mice by encephalitogenic peptides derived from PLP or MOG. The effectivity of GA in suppression was demonstrated in all three models [13, 49]. Thus, the suppressive effect of GA in EAE is a general phenomenon and is not restricted to a particular species, disease type, or the encephalitogen used for EAE induction. More recent studies have demonstrated that in addition to the parenteral route of administration used in all the studies described so far, oral administration of GA is also effective in suppressing EAE in rats, mice, and in primates. Furthermore, oral GA was more effective than oral MBP in suppressing the disease [46, 57]. The suppressive effect of GA in EAE is a specific one, since GA lacked any suppressive effect on the immune response in several systems—humoral and cellular immune responses to a variety of antigens and vaccination against various induced infections [47]. Neither did it suppress some other experimental autoimmune diseases, including myasthenia gravis, thyroiditis, diabe-

tes, and systemic lupus erythematosus. However, more recently GA has been reported to inhibit another autoimmune disorder—namely experimental uveoretinitis [59]—a disease interrelated with MBP and EAE and, as will be discussed below, GA is also effective in the case of experimental colitis. The specific effect of GA in EAE may be explicable in terms of immunological specificity. Indeed, marked cross-reactivity was demonstrated between GA and MBP, at both the cellular and the humoral levels of the immune response. Thus, using monoclonal antibodies, we could demonstrate clearly that several monoclonal anti-MBP antibodies reacted with GA and vice versa [44]. At the cellular level, cross-reaction was observed both in vitro and in vivo [55] and was correlated with the suppressive effect on EAE [56].

OTHER PEPTIDES RELATED TO MS Besides Cop1, which comprises a synthetic polymer of amino acids cross-reactive with the myelin protein, several short synthetic peptides have demonstrated capacity to inhibit EAE, and possibly MS. These peptides may be categorized into several groups: (1) Peptides of myelin basic protein, in either their native form or modified, that bind to MHC class II molecules and thereby compete for the binding of the encephalitogenic molecules. This is illustrated by the immunodominant epitope Ac1-11 of MBP which not only inhibits EAE by competition with disease-inducing peptides [54], but its analog leads to the reversal of acute EAE by forming long-lived peptide-MHC complexes [37]. An additional MBP epitope with a similar effect is the region 69–89, which was recently shown to form stable complexes with two-domain MHC class II molecules that suppress EAE very efficiently [17]. Then (2) there are altered peptide ligands (APL) that act as T cell receptor antagonists and thus induce bystander suppression for modulating EAE. A representative example is the region 139–151 of the PLP of myelin. This peptide as such is encephalitogenic, but its analog L144/R147 serves as a TCR antagonist and blocks the activation of encephalitogenic Th1 helper cells and thus inhibits EAE induced by multiple antigens [35]. Another example ameliorating EAE is the encephalitogenic peptide 87–99 of MBP, altered peptide ligands in which the lysine in position 91 or the arginine in position 97 were substituted by alanine [21]. (3) Finally, there are the peptides representing TCR determinants, since immunoregulation directed at them may lead to inhibition of the development of autopathogenic Th1 cells reactive with tissue-specific antigens. This is exemplified in the case of EAE by the effect of vaccination with the BV8S2 proteins of T cells, which

Copolymer 1 and Related Peptides as Immunomodulating Agents / 605 resulted in selective inhibition of MBP-specific Th1 cells and protection against EAE [36]. These findings led to a clinical trial using TCR peptides for the treatment of MS.

nor is it associated with the influenza-like syndrome reported in patients treated with interferon β. Based on the preceding, it was concluded that GA is a valuable first-line treatment option for MS patients [42].

CLINICAL TRIALS WITH COPOLYMER 1

MECHANISM OF ACTION OF GLATIRAMER ACETATE

The clinical trials that led to the approval of copolymer 1 as a drug for the treatment of MS were described in three comprehensive reviews [40, 42, 45], and hence only a very brief description is given in the following. Two phase I trials were conducted in Israel and the USA, respectively. They led to a phase II, two-year double-blind pilot trial in relapsing-remitting (RRMS) patients conducted in the USA [16], which demonstrated highly significant efficacy of copolymer 1 over placebo-treated patients. This was manifested in threefold reduction in the cumulative number of exacerbations and higher proportion of relapse-free patients over the two years. Both the phase I and this pilot phase II trial indicated that Cop 1 was well tolerated, with no toxicity noted and no systemic adverse side effects. The phase III trials for evaluation of the therapeutic efficacy included two trials, in the United States [23] and Europe/Canada [18]. In the U.S. trial, which lasted two years, the mean relapse rate in the GA receiving patients was 29% lower than in the placebo-treated group. Concerning the progression to sustained disability, as measured by the Kurtzke Expanded Disability Scale (EDSS), the patients receiving GA were significantly more likely to experience improvement in their disability status, and placebo recipients were more likely to experience worsening disability. The beneficial effect of GA persisted far beyond the duration of the trials, as shown in the extension of the U.S. trial up to 35 months [24]. Furthermore, the annualized relapse rate for patients who had received a GA throughout the six-year active-treatment extension phase [25] was 72% less than the annualized relapse rate at study entry (p = 0.0001). Patients receiving GA for eight years [26] had an annualized relapse rate for the eighth year of 0.16 compared with a baseline annualized rate of 1.49. The European/Canadian trial lasted nine months and its primary end point was the number of enhancing lesions in MRI. GA was found to decrease significantly (p < 0.002) the burden of disease compared with patients in the placebo group [18]. As for its safety profile—from all these clinical trials, it emerges that GA is well tolerated. The most commonly reported treatment-related adverse events are localized injection-site reactions and transient postinjection systemic reactions that are mild and self-limited [52]. GA does not induce neutralizing antibodies [48],

The mechanism by which GA induces its beneficial effect in animals and in patients was extensively investigated during the years by us and by others. These studies demonstrated that GA exerts its therapeutic activity by immunomodulating various levels of the immune response, which differ in their degree of specificity. The prerequisite step is the binding of GA to class II major histocompatibility complex (MHC) molecules. GA exhibited a very rapid, high, and efficient binding to various MHC class II molecules on murine and human antigen presenting cells, and even displaced peptides from the MHC binding site [20]. This modulation on the level of antigen presenting cells is the least specific step. In MS and EAE, however, in addition to MHC blocking, GA was shown to inhibit the response to the immunodominant epitope of MBP peptide 82–100 in a strictly antigen-specific manner by acting as a T cell receptor antagonist [9] and is further involved in T cell responses. For example, it was recently demonstrated that GA promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells [51]. In early studies we demonstrated that GA-treated animals develop GA-specific T cells in the peripheral immune system. These cells can adoptively transfer protection against EAE [30]. Furthermore, T cell lines and hybridomas could be isolated from spleens of mice rendered unresponsive to EAE by GA [8]. Both cell types act as regulatory suppressor cells, as they inhibited in vitro the response of MBP-specific effector cells and inhibited in vivo EAE induced by different CNS antigens. These GA-induced cells were indeed characterized as Th2/3 cells secreting high amounts of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β but not Th1 cytokines, in response to both GA and MBP [11]. It is noteworthy that other myelin antigens such as PLP and MOG could not activate the GAinduced cells to secrete by Th2 cytokines, but the disease induced by PLP and MOG can be suppressed by GA as well as by GA-induced cells, probably by “bystander suppression mechanisms” [10]. A shift from Th1-biased cytokine profile toward a Th2-biased profile was also observed in GA-treated MS patients [19, 33, 34], indicating that such GA-specific cells are involved in the therapeutic effect brought about by GA in MS. In all these studies, GA-induced Th2 regulatory cells were

606 / Chapter 86 demonstrated only in the periphery (spleens and lymph nodes of experimental animals or peripheral blood mononuclear cells in human) and not in the organ in which the pathological processes of EAE and MS occur. Our more recent studies demonstrate that the mechanism of activity of GA includes actual immunomodulation in the brain.

IN SITU IMMUNOMODULATION AND NEUROGENRATION IN THE CNS More recently we demonstrated the ability of GAspecific T cells induced in the periphery, either by injection or by oral treatment with GA, to pass the blood–brain barrier and accumulate in the CNS. This was manifested by GA-specific proliferation and by Th2 cytokine secretion, in whole lymphocyte population obtained from brains of EAE-induced mice treated by GA parenterally [11] or orally [5]. As was also demonstrated in these studies, highly reactive GA-specific T-cell lines that secrete in vitro IL-4, IL-5, IL-10, and TGF-β in response to GA and cross-react with MBP at the level of Th2 cytokine secretion could be obtained from both brains and spinal cords of GA-treated mice but not from untreated controls. Moreover, adoptively transferred fluorescently labeled GA-specific cells were found in the brain, indicating that the GA cells induced in the periphery penetrate and persist in the CNS. Using a double labeling approach in which fluorescently prelabeled specific T cells were adoptively transferred and subsequently detected immunohistologicaly, we demonstrated in the brain the expression of the two antiinflammatory cytokines, IL-10 and TGF-β (but no trace of the inflammatory cytokine IFN-γ), not only by the GA-labeled cells themselves but also by surrounding unlabeled cells [4, 14]. Such Th2/3 spreading suggests a bystander therapeutic effect of GA on the CNS resident cells, since IL-10 is a potent regulatory cytokine in autoimmunity that inhibits Th1 cells and macrophage activation and hence may contribute to the therapeutic activity of GA. It is of special importance that in addition to Th2/3 cytokines we demonstrated that GA-specific cells express in situ the potent brain-derived neurotrophic factor BDNF [2, 14]. BDNF is a key regulator of neuronal development that supports neuronal survival and regulates neurotransmitter release and dendritic growth. Activated GA-specific cells have been shown to secrete significant amounts of BDNF [28]. The in situ intense staining for BDNF, IL-10, and TGF-β and not for antiIFN-γ was observed only in brains of mice that had been adoptively transferred with GA-specific cells but not in control mice. The elevated expression of BDNF can be demonstrated not only after the adoptive transfer of

cells but also as a result of active treatment of EAEinflicted mice with Copolymer 1 (in preparation). This augmented expression of these alleviating factors in the CNS draws a direct linkage between the therapeutic activity of GA in MS/EAE and its in situ immunomodulatory effect as part of its mode of action. Very recently, we have demonstrated that the effect of GA in EAE-induced mice is manifested also by actual neurogenesis [1]. The results showed that GA treatment in various stages of the disease led to sustained reduction in the neuronal/axonal damage that is typical to the neurodegenerative disease course. Moreover, three processes characteristic of neurogenesis—namely cell proliferation, migration, and differentiation—were augmented and extended by GA treatment. The newborn neuroprogenitors migrated into the injury sites in the brain and differentiated to mature neuronal phenotype (Fig. 1). Some studies from other laboratories have revealed an additional activity of GA, demonstrating that active immunization with GA, as well as adoptive transfer of T cells reactive to GA, can inhibit the progression of secondary degeneration after crush injury of the rat optic

FIGURE 1. Pyramidal neuron in the cortex born during the concurrent injections of the proliferation marker BrdU and GA to an EAE-induced transgenic mouse that selectively express YFP on its neuronal population. One month after completion of GA treatment, neurons coexpressing BrdU (red) and YFP (green) with apical dendrites and axons are seen, indicative of mature functional neurons. (See color plate.)

Copolymer 1 and Related Peptides as Immunomodulating Agents / 607 nerve [29]. Furthermore, vaccination with GA protected neurons against glutamate cytotoxicity, while immunity to MBP and MOG that provides effective neuroprotection after axonal injury did not protect the neurons from toxicity caused by glutamate [36]. It is of interest that in these experiments, immunization with GA protected also retinal ganglion cells from death induced by ocular hypertension in rats.

POTENTIAL OF GA FOR THE SUPPRESSION OF OTHER AUTOIMMUNE DISORDERS— STUDIES ON INFLAMMATORY BOWEL DISEASES The capacity of Cop 1 to induce specific Th2 suppressor cells as a major path in its mechanism of action prompted us to explore the potential of its use for the suppression of other autoimmune diseases. A case in point is that of inflammatory bowel diseases (IBD). IBD are severe gastrointestinal disorders, characterized by detrimental immune reactivity in the gut that also involve mainly CD4+ Th1 cells, and an imbalance between proinflammatory and anti-inflammatory reactivity [41]. Current medical treatments for IBD rely on the use of nonspecific anti-inflammatory, as well as immunosuppressive drugs, which ameliorate the symptoms but also induce severe side effects that limit their use. In view of the immunopathological “autoimmunelike” nature of IBD and the proposed mechanism for GA, we tested the effect of GA on two relevant animal models—the trinitrobenzene sulfonic acid (TNBS)induced colitis, a murine model that resembles human Crohn’s disease (CD), and a dextran sulfate-induced colitis (DSS). The results obtained in three strains of mice clearly indicate that GA, administered either parenterally or orally, significantly ameliorated the various pathological manifestations of TNBS-induced colitis [3]. Thus, the macroscopic colonic damage characteristic of the disease were all drastically reduced by GA treatment and histological analysis confirmed the prevention of colonic damage. The beneficial effect of GA was also manifested by less weight loss and resulted in improved long-term survival. GA treatment resulted in significant reduction in the overall secretion of the proinflammatory cytokine TNF-α, and in elevation in the secretion of the beneficial Th3 cytokine TGF-β (3), suggesting that GA can modulate the detrimental immune response involved in the pathogenesis of experimental colitis, which resembles Crohn’s disease. A similar beneficial effect of GA was recently demonstrated in another experimental model of IBD, namely dextran sulfateinduced colitis (DSS), concerning both the acute and the chronic diseases, manifested in reduced weight loss,

increased survival, and reduction of the clinical score of the mice (unpublished results).

EFFECT OF COPOLYMER 1 ON GRAFT REJECTION The pathological process of immune rejection is mediated by T cells that recognize alloantigens presented on self MHC molecules as nonself. In view of the strong capacity of GA to bind promiscuously to MHC II [20], taken together with its immunomodulating activities and the immunopatholoical nature of graft rejection, the ability of GA to alleviate this detrimental reactivity was investigated. Indeed, in our early studies we showed that GA inhibited mixed lymphocyte reaction (MLR) in vitro [38], as well as the onset and severity of graft versus host disease (GVHD) [6]. We investigated the ability of GA by itself, as well as in combination with these two immunosuppressive drugs, cyclosporin A (CyA) and FK506 (tacrolimus), to suppress the rejection of strongly mismatched allografts. We demonstrated that Cop 1 treatment prolonged significantly skin graft survival and inhibited the functional deterioration of thyroid grafts, in various strain combinations, across minor and major histocompatibility barriers [7]. The effect was similar to that of a low to medium dose of the potent immunosuppressive drugs CyA and FK506. Concerning the mechanism by which GA alleviates the immune rejection, our results show that Cop 1 treatment inhibited the Th1 response to graft and induced a Th2 response, leading to improved survival and function of the transplanted grafts. This is in accord with the mechanism prevailing in the GA-induced suppression of EAE/MS. In an attempt to reduce the dosage and toxicity of the current immunosuppressive regimens and to improve their efficacy, we investigated the ability of GA to inhibit graft rejection in a combined treatment with suboptimal and less toxic doses of these suppressants [12]. We found that combined treatments of GA with various doses of CyA or FK 506 significantly extended skin graft survival compared with the effect of each drug alone and led to a synergistic effect, even suggesting that blood supply and engraftment have taken place. The combined treatment led also to efficient inhibition of the functional deterioration of thyroid grafts in mice, manifested by 2- to 20-fold increase in iodine absorbance by the transplanted thyroids, as compared with each drug alone. The most significant effect of the combination therapy was demonstrated in the system of heterotrophic heart transplantation in rats [12]. In this system, GA by itself was not effective in prolonging the graft survival, and the two immunosuppressive drugs were

608 / Chapter 86 effective only at medium and high doses. In contrast, cardiac allograft (BN→ Lewis) survival following the combined treatment with GA and low dose of CyA was longer than the survival obtained with a fourfold higher dose of CyA alone. Similarly, with FK506, the combination with GA resulted in longer graft survival than that obtained with higher, toxic, doses of the drug alone. Hence, the application of GA, which is a well-tolerated approved drug, may lead to reduction of the adverse effect and even improve the efficacy of the current immunosuppressive regimens in organ transplantation.

[6]

[7]

[8]

[9]

CONCLUSION [10]

This chapter summarizes the data available on the therapeutic activity and immunomodulatory properties of copolymer-1, also known under the trade name Copaxone®. GA is the only noninterferon novel drug for the treatment of multiple sclerosis. It is a synthetic random polymer of amino acid and has a specific effect on the autoimmune process involved in both EAE and MS. It is thus the only known polymeric (polypeptide) material that is responsible for a successful treatment of a disease. Its mode of action includes a prerequisite stage of binding to the MHC II molecules, thus competing with the binding of the myelin proteins that induce the neurological damage. But primarily GA is an immunomodulator that affects different levels of the immune response, as an MHC blocker, T-cell receptor antagonist, and as a potent inducer of regulatory T cells. These immunomodulatory activities as well as the high safety profile of GA, support its application for various pathological autoimmune disorders such as multiple sclerosis and inflammatory bowel disease, as well as other immune-related pathology such as graft rejection.

References [1] Aharoni R. Arnon R. Eilam R. Neurogenesis and neuroprotection induced by peripheral immunomodulatory treatment of experimental autoimmune encephalomyelitis. J. Neuroscience 2005; 25 (6): 8217–8228. [2] Aharoni R. Eilam R. Sela M. Arnon R. Glatiramer acetate specific T-cells in the brain express TH2/3 cytokines and brainderived neurotrophic factor in situ. PNAS. 2003;Vol 100. 24: 14157–14162. [3] Aharoni R. Kayhan B. Arnon R. Therapeutic effect of the immunomodulator glatiramer acetate on trinitrobenzene sulfonic acid-induced experimental colitis. Bowel 2005; 48: 47 106 –115. [4] Aharoni R. Kayhan B. Eilam R. Sela M. Arnon R. Glatiramer acetate specific T-cells in the brain express TH2/3 cytokines and brain-derived neurotrophic factor in situ. PNAS. 2003; 100: 24, 14157–14162. [5] Aharoni R. Meshorer A. Sela M. Arnon R. Oral treatment of mice with copolymer 1 (glatiramer acetate) results in accumula-

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[35] Nicholson LB. Murtaza A. Hafter BP. Sette A. Kuchroo VK. A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple antigens. Proc. Nat. Acad. Sci. USA 1997; 94: 9279–9284. [36] Offner H. Adlard K. Bepo BF. Jr. Schuster J. Burrows GG. Buenafe AC. Vandenbark AA. Vaccination with BV8S2 protein amplifies TCR-specific regulation and protection against experimrental autoimmune encephalomyelitis in TCR BV8S2 transgenic mice. J. Immunol. 1998; 161: 2178–2186. [37] Samson MF. Smilek DE. Reversal of acute experimental autoimmune encephalomyelitis and prevention of relapses by treatment with a myelin basic protein peptide analogue modified to form long-lived peptide-MHC complexes. J. Immunol. 1995; 155: 2737–2746. [38] Schlegel PG. Aharoni R. Chen Y. Chen J. Teitelbaum D. Arnon R. Sela M. Chao NJ. A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T cell proliferative responses to major and minor histo-compatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo. Proc. Natl. Acad. Sci. USA 1996; 93: 5061–5066. [39] Schori H. Kipnis J. Yoles E. Schwartz M. Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma. Proc. Natl Acad. Sci. USA 2001; 298: 3398–3403. [40] Sela M. Teitelbaum D. Glatiramer acetate in the treatment of multiple sclerosis. Exp. Opin. Pharmacother. 2001; 2: 1149– 1165. [41] Shanahan F. Inflammatory bowel disease: Immunodiagnostic and ecotherapeutics immunotherapeutics. Gastroenterology 2001; 120 (3): 622–635. [42] Simpson D. Noble S. Perry C. Glatiramer acetate—a review of its use in relapsing-remitting multiple sclerosis. CNS Drugs 2002; 16: 826–850. [43] Steinman L. Weisman A. Altman D. Major T cell responses in multiple sclerosis. Mol. Med. Today 1995; 1: 79–83. [44] Teitelbaum D. Aharoni R. Sela M. Arnon R. Cross-reactions and specificities of monoclonal antibodies against myelin basic protein and against the synthetic copolymer 1. Proc. Natl. Acad. Sci. USA 1991; 88: 9528–9532. [45] Teitelbaum D. Arnon R. Sela M. Case history of Copaxone®— from the bench to the bedside. 2005; Medicinal Chemistry (in press). [46] Teitelbaum D. Arnon R. Sela M. Immunomodulation of autoimmune encephalomyelitis by oral administration of copolymer 1. Proc. Natl. Acad. Sci. USA 1999; 96: 3842–3847. [47] Teitelbaum D. Ben-Yedidia T. Tarrab-Hazdai R. Orr N. KarpovRoikhel O. Arnon. R. Specificity of the suppressive effect of copolymer 1. J. Neurol. 1996; 243: (Suppl. 2): S85. [48] Teitelbaum D. Brenner T. Abramsky O. Aharoni R. Sela M. Arnon R. Antibodies to glatiramer acetate do not interfere with its biological functions and therapeutic activity. Multiple Sclerosis 2003; 9: 592–599. [49] Teitelbaum D. Fridkis-Hareli M. Arnon R. Sela M. Copolymer 1 inhibits chronic relapsing allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice and interferes with PLP specific T cell responses. J. Neuroimmunol. 1996; 64: 209–217. [50] Teitelbaum D. Meshorer M. Hirshfeld T. Sela M. Arnon R. Suppression of experimental allergic encephalomyelitis by a synthetic basic copolymer. Eur. J. Immunol. 1971; 1: 242– 248. [51] Vieira PL. Heyslek HC. Wormmester J, Wierenga E. Kaspenberg ML. Glatiramer acetate (copolymer-1, Copaxone) promotes

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87 CLIP—A Multifunctional MHC Class II–Associated Self-Peptide ANNE VOGT AND HARALD KROPSHOFER

ABSTRACT

murine equivalent of CLIP had already been described before as one of the prominent self-peptides in normal murine B cells [47]. Likewise, a large variety of length variants of human CLIP was found in several EpsteinBarr virus-transformed human B cells and later on also in other types of antigen presenting cells (APC) in vitro and ex vivo [13]. Upon gaining insight that it is the CLIP region of Ii that is mainly responsible for the interaction of intact Ii with the peptide binding groove of class II MHC molecules [56, 61], it became obvious why CLIP is the best example of a peptide ligand that binds promiscuously to class II MHC proteins [51]. Furthermore, as Ii and class II MHC molecules are widely coexpressed in all types of professional and nonprofessional APC, CLIP is a key representative in the vast majority of MHC II– associated self-peptide repertoires and thus is prone to play a key role in the induction and maintenance of self-tolerance. Most recently, evidence was provided that self-peptides on the surface of dendritic cells contribute to the regulation of helper T cell responses against foreign antigens, with CLIP being a key player in this scenario.

A key step in induction of a cellular immune response involves recognition of antigenic peptides bound to MHC class II molecules via T cell receptors expressed on effector CD4+ T cells. Here, we summarize the function of a principal player in MHC class II–associated antigen processing and presentation, the invariant chain derived self-peptide CLIP (class II–associated invariant chain peptide). CLIP has unique features, enabling it to serve as a promiscuously binding selfpeptide and to be replaced by cognate antigenic peptide. Most recently, CLIP has been shown to be abundantly expressed on the cell surface of mature antigen presenting cells, where it contributes to shaping the immune response as a part of the immunological synapse.

INTRODUCTION In 1992, Peter Cresswell and colleagues analyzed human TxB hybrid cell lines which were defective in major histocompatibility complex (MHC) class II– restricted antigen processing and thereby identified a set of naturally processed peptides derived from the MHC class II–associated chaperone invariant chain (Ii) [45]. This set of CLass II–associated Ii Peptides was named “CLIP” and became the most frequently cited natural peptide in the MHC literature. The striking observation with this particular type of mutants in those days was that in the context of most MHC class II alleles, human CLIP covered 50–100% of the associated selfpeptide repertoire [45, 50]. Interestingly, the shorter Handbook of Biologically Active Peptides

THE CLIP RECEPTORS: MHC CLASS II MOLECULES As yet, CLIP has only been described as a ligand of MHC class II molecules. The latter are expressed on the surface of APC and in the majority of cases carry a peptide ligand bound in their peptide binding cleft [55]. Only a small percentage of surface MHC II molecules are thought to be “empty,” which means they do

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612 / Chapter 87 not carry a ligand. The primary function of MHC II peptide complexes is to stimulate CD4+ T helper cells. T cell receptors on the surface of these helper T cells are selected to interact with both self-structures at the rim of the MHC class II peptide binding groove and amino acids side chains of the peptide ligand. The peptide determinants that are specifically recognized by T cell receptors in an MHC-restricted fashion are called “T cell epitopes.” Thus, in contrast to “B cell epitopes,” which are linear or conformational structures on the surface of an antigen to be recognized by soluble or membrane-bound immunoglobulins of B cells, T cell epitopes are generated by proteolytic cleavage of antigenic proteins and presented as linear structures in the context of MHC II.

The Structure of MHC Class II Peptide Complexes MHC II molecules are membrane bound heterodimers consisting of one α and one β chain. The aminoterminal α1 and β1 domains constitute a peptide binding groove—consisting of two flanking α-helices on a bottom of eight β-sheet strands—and both are linked to an Ig-like domain, a transmembrane region, and a short cytoplasmic tail [55]. For reasons of stability, MHC II αβ dimers have to accommodate a third subunit: either a peptide ligand of at least 12 amino acids length or a chaperone, such as Ii. As shown by x-ray crystallography, about half of the peptide binding enthalpy

arises from interactions of the peptide backbone with side chains of the MHC II binding groove, while the other half is due to interactions of amino acid side chains of the peptide with residues of the so-called specificity pockets of the MHC II peptide binding groove [55]. Peptide residues fitting into the pockets of the peptide binding groove are denoted as “anchors.” Importantly, and in contrast to MHC I, the peptide groove of MHC II is open at both ends [8] so that peptide or polypeptide ligands of variable lengths can be accommodated [11, 49]. The advantage of this feature is that unfolded polypeptides carrying an epitope can bind first and then be trimmed by proteases afterwards, thereby rescuing these precursors from quantitative proteolysis in protease-enriched endosomal-lysosomal compartments where antigenic peptide loading of MHC class II molecules takes place (see following). In the case of CLIP bound to human MHC class II molecules, CLIP residues Val-88 to Ala-101 are buried in the binding groove, as originally shown with crystals of CLIP-HLA-DR3 complexes (Fig. 1; [22]). However, the N-terminal tail of CLIP covering residues Leu-81 to Pro-87 is apparently protruding from the groove. Only human CLIP carries this 7-mer extension, whereas murine CLIP is shortened. This may be due to the fact that the N-terminal tail of human CLIP contains 4 Pro residues, which are known to slow down endo- and exoproteases, whereas murine CLIP contains only two Pro residues.

FIGURE 1. X-ray crystal structure of CLIP bound to the antigen binding groove of HLA-DR3. Only CLIP residues between Pro-87 and Ala-101 could be resolved. Black dotted lines between CLIP and HLA-DR3 residues indicate the formation of 17 hydrogen bonds (from [22]). (See color plate.)

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Impact of CLIP on the MHC II Peptide Binding Groove There are good reasons to believe that the CLIP region of Ii has decisively impacted the structural evolution of the peptide binding cleft of MHC class II molecules with regard to both its openness and polymorphism: First, Ii binding to MHC II via the CLIP region is fundamental to the chaperone role of Ii during the first hours of the life cycle of MHC II molecules and before HLA-DM is taking over the chaperone task in endosomes and lysosomes [67]. Accommodation of CLIP in the peptide binding groove as a part of the Ii polypeptide is vital for most class Ii dimers, as they otherwise would dissociate and/or unfold. As a consequence, fixation of N- and C-termini of a peptide ligand, with the distance between both termini not exceeding the length of about 25Å of the peptide binding groove, as with MHC class I molecules, could no longer have been an option during evolution of the MHC class II processing pathway. Second, CLIP appears to have been acting as a powerful factor shaping the polymorphism of MHC II molecules that is restricted to residues constituting the binding groove [20]. On the one hand, the increase in polymorphism increases the capacity to cope with the high degree of variability among foreign invaders. On the other hand, mutations in hypervariable regions of MHC II alleles that prevent CLIP from binding or interfere with CLIP exchange for cognate antigenic peptides are necessarily subject to negative selection during evolution. In accordance with this view, the CLIP core region encompassing residues Met-91 to Met-99 is highly conserved among all species investigated and

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capable of anchoring CLIP to any class II groove, with Met-91, Ala-94, Pro-96, and Met-99 serving as the universal P1, P4, P6, and P9 anchor, respectively [22]. The unique sequence of CLIP enables this peptide to serve as a ligand for nearly all different MHC class II alleles, albeit with variable binding affinity ranging from high affinity to low affinity [51].

The Parent Protein of CLIP: The Invariant Chain MHC II molecules assemble in the endoplasmic reticulum (ER) involving the concerted activities of the chaperones calnexin and Ii. While calnexin serves several types of antigen receptors [40], such as immunoglobulins and MHC class I, Ii is dedicated to MHC II dimers only, despite the high degree of allelic variability in the MHC class II molecules [17]. Ii is a type II transmembrane protein bearing a single transmembrane region close to the N-terminus (Fig. 2). The cytosolic tail contains a sorting signal which directs Ii and associated class II molecules to endosomal-lysosomal organelles. In the C-terminal half, Ii carries a trimerization region (residues 118–192 in human Ii). This region together with the transmembrane domain contributes to homotrimerization of Ii (Fig. 3). The sequential binding of calnexin and Ii to newly synthesized individual α and β chains facilitates the assembly of αβIi heterotrimers followed (after dissociation of calnexin) by the formation of a nonameric (αβIi)3 complex. Ii protects αβ dimers from aggregation in a chaperone-like fashion. Equally importantly, the formation of (αβIi)3 complexes, with the CLIP region of Ii occupying the peptide binding groove, prevents premature binding of peptides and polypeptides

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LPKPPKPVS KMRMATPLLMQALPM Internalization signal (residue 7-17) Flanking region (residue 71-81; 104-128)

Transmembrane region (residue 30-56) CLIP effector site (residue 81-90)

Trimerization region (residue 118-192) CLIP core region (residue 90-104)

FIGURE 2. Functional regions of human invariant chain. Schematic of the p31 form of human Ii. Colored boxes indicate the regions that are responsible for targeting (internalization signal), membrane anchoring (transmembrane region), trimerization (trimerization region) and binding to MHC class II molecules (CLIP core region, CLIP effector site, and CLIP flanking region). Letters beneath the CLIP box indicate the CLIP amino acid sequence. Black numbers refer to the p31 variant of human Ii and indicate the borders of the functional domains. (See color plate.)

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FIGURE 4. Invariant chain proteolysis and the formation of CLIP. Schematic representation of the proteolytic steps in an endosomal compartment (in grey) within antigen presenting cells leading to the generation of CLIP. The p31 variant of Ii (in green), bound to a class II MHC molecule (in black) enters the compartment and, upon cleavage by the asparagin endoproteinase (AEP) or another protease, gives rise to the p22 Ii intermediate. Ii p22 is transformed to Ii p10 and finally to CLIP via cleavage by the indicated cathepsins. (See color plate.)

FIGURE 3. Model of nonameric MHC class II–invariant chain complexes. Ii trimers (in green) form a scaffold, with trimerization being accomplished through the transmembrane regions and regions close to the C-terminus. Heterodimeric class II MHC molecules (in black) binding to Ii trimers, mainly via the CLIP region, give rise to nonameric (αβ)3(Ii)3 complexes. (See color plate.)

in the ER and during transport to endosomal-lysosomal loading compartments [9]. The cytosolic tail of Ii harbors two sorting signals that target the nonameric (αβIi)3 complex from the trans-Golgi network to endocytic compartments.

PROTEOLYSIS OF II AND GENERATION OF CLIP During transit from endosomal to lysosomal compartments within APCs, stepwise proteolysis, denoted as “processing,” of Ii takes place—hence Ii processing and antigen processing mainly colocalize [64]. By using specific protease inhibitors and genetic knockouts of lysosomal proteases, Ii processing was shown to be a staged process, proceeding from the lumenal C-terminus to the membrane-proximal N-terminus (Fig. 4). Inhibition of cysteine proteases by leupeptin results in accumulation of a 22 kD Ii fragment (p22) in human cells

and in accumulation of a 10 kD fragment (p10) in murine cells [4]. Here, the leupeptin-insensitive cysteine protease AEP (asparagin endo-proteinase) is initiating Ii (p31) processing by targeting two Asn residues close to the C-terminal trimerization region, resulting in generation of the p22 fragment of Ii [39]. Further processing is observed in the case of murine Ii because of additional Asn residues that are located closer to the N-terminus. Besides AEP, the cysteine protease Cat S is key in Ii processing, with at least two subsequent Cat S cleavage steps finally giving rise to CLIP [60]. CLIP keeps on occupying the peptide binding groove, thereby preventing αβ disassembly. Proteolytic cleavage generating the N-terminus of CLIP at the same time releases αβ dimers from their trimeric oligomerization state previously maintained by Ii trimers kept together via trimerized transmembrane regions (Fig. 2; [60]). Cat S k.o. mice are healthy and normal in most respects, but they show a decrease in Ii degradation in B cells and DCs and an accumulation of CLIP in the self-peptide repertoire of APC, underlining the critical role of Cat S in the generation of CLIP [44]. The moderate effects seen in macrophages of Cat Sdeficient may be due to Cat F taking over Ii processing functions [52]. In murine thymic cortical epithelial cells, the related enzyme Cat L processes Ii upon deletion of the Cat S gene [43]. The importance of Cat S in Ii processing of APCs translates also into impaired antigen presentation in APC lacking Cat S activity.

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FLANKING RESIDUES AND SELF-RELEASE OF CLIP A closer look at the naturally occurring CLIP length variants in the human system reveals that there are two principal variants: CLIP(long), represented by 21to 26-mers, and CLIP(short), represented by 14 - to 19-mers [13, 14]. The x-ray structural analysis of the HLA-DR3-CLIP crystal shows that CLIP(short) contains the residues critical for interacting with the peptide binding groove (Fig. 1; 22). According to this crystal structure, the N-terminal flanking residues of CLIP, positions 81–89, have no defined folding, as they remained invisible. However, CLIP(long) dissociated rapidly from HLA-DR molecules at endocytic pH, whereas CLIP(short) displayed a lower off-rate [35, 59]. These findings suggested that the N-terminal 9 residues of CLIP, which are positioned outside the 9mer region occupying the binding cleft, have their own function. The critical impact of flanking residues is reminiscent of naturally occurring hen-egg-lysozyme (HEL) derived epitopes: C-terminal Trp residues enhance the immunogenicity of HEL(52–63) resulting in altered T cell receptor variable region usage [10]. Likewise, the N-terminal flanking region of CLIP augments the immunogenic potential of the cryptic “self” epitope from the C-erb oncogene (Her2/neu) leading to protective antitumor immunity of the chimeric tumor antigen [27]. Although the majority of class II allelic products are dependent on the chaperone HLA-DM with respect to CLIP removal and processing of foreign protein antigens (see following), HLA-DM or the murine counterpart H2-DM is not essential for peptide loading of DRB1*0401, I-Ak, or I-Ad [3, 7]. Likewise, loading with

conventional self-peptides other than CLIP has been demonstrated with several HLA-DM-defective mutants [36, 45]. The rationale is that at endosomal pH, the N-terminal residues 81–89 facilitate rapid release of CLIP(long) [36]. Furthermore, CLIP(81–89) catalyzes the release of CLIP(short) and a subset of other selfpeptides. Flanking residues of CLIP, in particular Lys83, Lys-86, and Pro-87 are thought to interact with an effector site outside the binding-cleft (Fig. 5). This would be consistent with an allosteric mode of action [36]. Interestingly, a lateral HLA-DM-interacting surface on HLA-DR that includes acidic and hydrophobic HLADR residues near the N-terminus of the peptide has been mapped [19]. Hence, from these parallels one could envisage that class II molecules bear a binding site outside the groove close to the P1 pocket that allows induction of conformational changes in and around the P1 pocket, thereby initiating the release of bound peptides. According to this model, the chaperone HLADM has evolved to exploit the allosteric effector site: HLA-DM appears to be an optimized effector molecule that has release capacities superior to the original effector, the N-terminal flank of CLIP.

A CHAPERONE FACILITATING CLIP RELEASE: HLA-DM/H2-DM CLIP needs to be removed from the MHC II peptide binding groove before cognate self-peptide or foreign peptides can have access. In the context of most MHC II allelic products, efficient exchange of CLIP against cognate peptide requires an accessory molecule, designated HLA-DM in humans and H2-DM in mice. Only in B cells, peptide loading is modulated by HLA-DO (H2-DO in mice) [1].

acidic pH FIGURE 5. Model of the self-release activity of CLIP. The N-terminal residues of CLIP (green circles) bind to an effector site outside the antigen binding groove, most probably located in the α1 domain. Binding to the effector site at acidic pH leads to accelerated dissociation of the CLIP core region, mediated via conformational changes in and around the P1 pocket occupied by the universal CLIP anchor residue Met-91. The critical residues in the N-terminal tail of CLIP are marked in red. (See color plate.)

616 / Chapter 87 HLA-DM (short: DM) is a non-classical MHC II molecule [15, 33]. It cannot bind peptide ligands [42] but colocalizes with classical MHC class II molecules in endosomal/lysosomal loading compartments [48]. Here, DM catalyzes one of the rate-limiting steps in MHC II antigen processing: the removal of CLIP (Fig. 6; [53]). Mutant APCs devoid of DM, therefore, have a defect in presentation of protein antigens, originally leading to the discovery of DM [1]. The kinetics of DM-mediated exchange of CLIP for cognate peptide follows the rules of Michaelis-Menten derived from enzyme catalysis [62]. Consistent with a catalytic pH optimum around pH 4.5 in vitro, DM : DR rations of about 1 : 5 and acidic conditions characterized by pH 4.5–5.5 were found in typical loading compartments where DM resides [37]. After the release of CLIP, the binding cleft is “empty,” which renders αβ dimers susceptible to denaturation and aggregation, in particular at the low pH of endosomal/lysosomal compartments. DM has been shown to bind to peptidereceptive empty MHC II molecules and prevent them from unfolding [37]. This property is regarded as the chaperone function of DM [37]. In B cells, about 20– 25% of HLA-DR molecules were shown to engage in HLA-DR-DM complexes, thereby constituting a pool of antigen-receptive HLA-DR molecules that may respond promptly upon being confronted with an antigenic challenge (Fig. 6; [37]).

MEMBRANE MICRODOMAINS

minority in relation to the multitude of endogenous self-peptides, such as CLIP, the question emerges how T cells manage to track down the few copies of cognate peptide. At least 200–300 class II-peptide complexes are estimated to be required to fully activate a naive CD4+ T cell so that it can acquire effector functions in immune defense [18, 25]. However, it remained open whether abundant self-peptides are to be excluded from the contact zone where cognate T cell receptors recognize cognate antigenic peptide. Until recently, it was believed that supra-molecular assemblies of monospecific T cell receptors on the T cell surface are inducing cluster formation of cognate class II MHC-peptide complexes on APC [5]. Current evidence suggests that professional APC actively organize the lateral distribution of class II MHC-peptide complexes on their own—before they engage in immunological synapses with T cells—by segregation of class II molecules into membrane microdomains [63]. B cells and some other APC concentrate class II–peptide complexes in detergent-resistant cholesterol- and glycosphingolipid-enriched microdomains at the cell surface, denoted as “lipid rafts” [2, 28]. This type of microdomains can accumulate in synapses and thereby favor antigen presentation at low doses of antigen, but lipid rafts do not seem to be equally important in all types of APC [29, 38]. Microdomains formed by socalled tetraspanins seem to be more broadly distributed across leukocytes [26].

Tetraspan Microdomains

The density of MHC class II on the surface of APC is a critical parameter in activating helper T cells. The fact that cognate antigenic peptides can only be a

Tetraspanins are a large superfamily of more than 150 members [57]. They are characterized by four transmembrane domains and short cytoplasmic regions.

MHC class II-peptide peptide (high-stability)

5.

4.

MHC class II-peptide peptide (low-stability)

3. Antigen CLIP

MHC class IICLIP

DM

1.

2.

FIGURE 6. CLIP removal and antigen processing mediated by HLA-DM. The following steps are facilitated by the nonclassical MHC II molecule HLA-DM (DM): (1) DM binds to MHC II-CLIP complexes and releases CLIP; (2) DM remains bound to empty MHC II molecules and prevents their denaturation (chaperone activity); (3) DM catalyzes loading with antigenic peptides (catalytic activity); (4) DM exchanges low-stability ligands against high-stability ligands (peptide editing); (5) MHC II-peptide complexes migrating to the cell surface are long-lived and bear a conformation imposed by DM (conformational editing). (See color plate.)

CLIP—A Multifunctional MHC Class II–Associated Self-Peptide / 617 They form homo- or heterodimers through their stalk subdomain, while their head subdomain appears to serve as an interface to contact other membrane proteins, such as integrins, signaling molecules or MHC molecules [34, 63]. Tetraspanins are expressed in a wide variety of cell types and contribute to diverse physiological processes, such as cellular adhesion, motility, activation, and tumor invasion. APC express almost 20 different tetraspanins, with the core family members being denoted as CD9, CD37, CD53, CD63, CD81, and CD82 [57]. Together with their homologs, they are thought to constitute a novel type of membrane microdomain, also named “tetraspan web,” which may act as a kind of membrane skeleton that forms the glue between molecules destined to form a functional unit.

CLIP in Tetraspan Microdomains A critical characteristic of tetraspan microdomains, found with human B cells and DCs, may further improve the capacity to trigger T cells: Tetraspan microdomains carry a selected set of peptides rather than the complex repertoire representative for whole APCs [6, 63]. This is most likely accomplished by recruitment of the peptide editor HLA-DM, which associates to CD82 even in the absence of classical MHC II molecules [24, 38]. The presence of HLA-DM obviously leads to accumulation of peptides that bind with moderate stability to MHC II molecules, whereas high-stability MHC IIpeptide complexes are excluded [38, 63]. Together, tetraspan microdomains preferentially form around MHC II-peptide complexes of moderate stability thereby compensating by a gain in avidity for the usually lower copy number of those complexes as compared with those attaining higher stability. Strikingly, CLIP occupies a majority of the MHC II binding sites in these tetraspan microdomains on the surface of mature monocyte-derived DCs [46]. As 9– 12% of the HLA-DR molecules on mature DCs reside in tetraspan microdomains [38], CLIP covers 5–10% of the total population of HLA-DR molecules, which equals to about 4–8 × 105 molecules [46]. A similar enrichment of MHC II-CLIP complexes was found ex vivo in tetraspan microdomains on APC of human tonsils [46]. Confocal microscopy studies revealed that HLA-DR-CLIP complexes can localize to both the central and peripheral molecular activation cluster apparently relying on the exogenous antigen to be recognized by the T cell receptor. This is consistent with the observation that tetraspan microdomains segregate to those areas where APC get in contact with T cells [46] and that the tetraspanin CD81 accumulates at the central zone of the immune synapse formed by B and T lymphocytes [41]. The

conclusion is that a network of tetraspan molecules populates the APC side of the immune synapse, thereby stabilizing clusters of MHC molecules loaded with cognate peptide and CLIP. Attempts to define the physiological relevance of elevated MHC II-CLIP complexes in tetraspan domains on DC revealed that CLIP contributes to the regulation of whether T helper cells differentiate toward the type 0, type 1 or type 2 of polarization upon interaction with DC ([46]; see following).

CLIP in the Pseudodimer Model The monoclonal antibody FN1, which has been instrumental in the identification of tetraspan microdomains, recognizes superdimers of HLA class II molecules, embedded in a network of tetraspanins [21, 63]. Hence, two HLA class II molecules in close proximity surrounded by tetraspan molecules constitute the socalled CDw78 determinant. CLIP, as a major resident of tetraspan microdomains on the surface of DC and as a constituent of immune synapses, appears to be a candidate self-peptide that may facilitate di- and oligomerization of T cell receptors (TCRs) recognizing cognate foreign antigen. The concept that self- and foreign peptides collaborate in initiating the formation of TcR clusters has been suggested in the “pseudodimer” model pioneered by M. Davis and colleagues [30, 32]. Pseudodimers have been defined as heterologous superdimers of MHC II molecules carrying a self-peptide and an agonistic peptide. In particular in the initial phase of TCR docking, pseudodimers localizing to the synapse may be essential in overcoming limitations in TCR triggering caused by too low local density of agonistic MHC II-peptide complexes [30, 32, 65]. The prerequisite of recognizing both MHC class II molecules carrying agonistic peptide or CLIP may apply, in particular, to those TCR that have been positively selected on MHC II-CLIP complexes in the thymic cortex, as those TCR are thought to recognize CLIP with moderate affinity (Fig. 7). CLIP is actually one of the most abundant self-peptides on APC of the thymic cortex. Later on, at the stage when the TCR begins to transmit signals, CLIP is supposed to reduce the local density of agonistic ligands in the synapse, as compared with the situation when only agonistic ligands are engaging the TCR. This would mirror the situation in which very low doses of agonist or low-affinity agonists give rise to low-avidity T cell interactions, thereby favoring TH2 polarization [16].

CLIP AS AN ANTAGONIST OF TH1 POLARIZATION Many investigators have proposed that endogenous self-peptides may be critical for the extraordinary

618 / Chapter 87

FIGURE 7. A role for CLIP in T cell receptor recognition of cognate peptide. MHC II-CLIP complexes segregate into tetraspan microdomains on the surface of APC. Together with cognate antigenic peptide, CLIP may form dimers of MHC-peptide complexes, also denoted as pseudodimers. According to the pseudodimer model, these heterodimers may facilitate dimerization of T cell receptors at the initial stage of T cell/APC interactions when the local density of cognate peptide on the APC surface is too low. (See color plate.)

sensitivity of T cells for foreign antigen [31, 54, 66]. This view is supported by the fact that T cells rely on the weak interaction with MHC-self-peptide complexes in the thymus to progress to full maturity [23] and in the periphery to guarantee for survival [54]. In addition, along with agonistic ligands, large quantities of class II MHC-self-peptide complexes, attaining up to 20% relative abundance, accumulate in the immunological synapse [30, 66]. Until most recently, it remained open whether each self-peptide can influence T cell responsiveness in a similar manner or whether distinct self-peptides modulate T cell activity in a distinct way. CLIP is the first defined self-peptide that could be demonstrated to have an impact on the quality of T cell activation [12, 46, 58]. During maturation, DCs strongly up-regulate class II-CLIP complexes on the surface, apparently driven by down-regulation of the catalytic activity of HLA-DM. Class II-CLIP complexes attain an abundance of up to 10% of the total class II-self-peptide pool on the surface of mature DC [46]. CLIP and cognate antigen cosegregate in immunological synapses of DCs and naive CD4+ T cells, which is in accordance with class II-CLIP complexes being constituents of tetraspan microdomains. The increase in class II-CLIP complexes on mature DC led to a significant shift in the polarization of naive T cells [46]: CLIP reduced the number of TH1 cells secreting IFN-γ and promoted the secretion of the TH2 cytokine IL-4. Likewise, APCs from H2-DM k.o. mice favored polarization of TH2 cells, whereas APCs from wild-type mice gave rise to more TH1 cells. Mechanistically, CLIP may interfere with signalling relevant for TH1 polarization. This would be equivalent to the situation when low doses of agonist or low-affinity agonists give rise to low-avidity APC-T cell interactions, thereby favoring TH2 polarization.

Class II MHC alleles linked to autoimmune diseases, such as rheumatoid arthritis, juvenile dermatomyositis, autoimmune hepatitis, and Graves disease, form unstable class II-CLIP complexes [46]. As most autoimmune diseases rely on TH1 cells, a lack of CLIP may be accomplished by a low capacity to counterbalance TH1 polarization [58]. Thus, the CLIPlow phenotype may increase the risk for autoimmunity, whereas the CLIPhigh phenotype may confer protection. This mechanism may under normal conditions prevent the organism from mounting excessively strong TH1 responses but maintain balance in the challenging task to combat the high variety of foreign invaders without losing tolerance against the plethora of potential self-antigens.

CONCLUSION A decade ago, CLIP was viewed as an Ii-derived remnant peptide that needs to be exchanged for cognate ligands before helper T cells can successfully mount an immune response against foreign invaders. In the meantime, there is accumulating evidence that CLIP is a key ligand of the self-peptide repertoire and presented on the surface of most antigen presenting cells throughout the body. Colocalizing with cognate antigenic peptides on dendritic cells engaging in immune synapses, CLIP seems to play a role in regulating the polarization of naïve T helper cells and, thus, is likely to promote the diversification of adaptive immune responses to pathogens. CLIP appears to be predisposed to this task due to its ubiquitous expression: CLIP-MHC II complexes positively select a repertoire of T cells in the thymus, maintain them through recurrent contact in secondary lymphatic organs in the

CLIP—A Multifunctional MHC Class II–Associated Self-Peptide / 619 periphery, and may facilitate their recruitment during early stages of infectious processes when foreign antigenic peptides are scarcely presented on antigen presenting cells. Hence, CLIP supports the current concept that self-recognition can augment immunity rather than constraining it.

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88 Vasopressin and Oxytocin JOHN F MORRIS

ABSTRACT

used interchangeably today. Ott and Scott discovered the mammary effects of OT, Gaines separated milkejection from milk secretion, and Dale demonstrated its uterotonic properties. At that time synthesis of the hormonal activities was thought to reside in the NL and it was the studies of the Scharrers and Bargmann which defined their hypothalamic origin in magnocellular neurons (MCN) and the whole concept of neurosecretion into the systemic circulation was born. The structure of the hormonal peptides was elucidated by DuVigneaud in 1953 (Fig. 1). The studies of Acher on neurophysin (NP) and Sachs and Takabatake on precursor processing showed that the hormones were produced from larger precursors. The molecular era of understanding opened in 1984 when Ivell and Richter described the VP and OT genes, and shortly after that the gene defect giving rise to diabetes insipidus in the Brattleboro rat was identified. The development of radioactively labeled hormones in the late 1950s ushered in the study of the receptors for the hormones and gradually the VP receptor subtypes were identified (see Jard in [18]) by the use of hormonal analogs and the elucidation of subcellular mechanisms of action. At the same time, the use of immunocytochemistry was demonstrating that not all VP and OT axons originate from MCN in the SON or PVN and project to the neural lobe; there also are substantial projections from smaller PCN in the PVN and other nuclei to many parts of the central nervous system (CNS). Studies on the CNS receptors seek to link them with neural actions. Recently, the dendrites of MCN have been shown to secrete the hormones independently of the axons adding a further sophistication to the mechanisms and their control (see Morris

Vasopressin (VP) and oxytocin (OT) are archetypal brain peptides intimately linked with the discovery of neurosecretion and, more recently, central actions of neuropeptides. Magnocellular neurons (MCN) responsible for the systemic secretion of the hormones from the neural lobe (NL) have been robust models because of their large size, grouping in the paraventricular nucleus (PVN) and supraoptic nucleus (SON), and projecting to the NL where huge amounts of the peptides are stored. Smaller parvocellular neurons (PCN) project widely within the CNS but their functions are only recently becoming clearer compared with the long established systemic actions of the hormones. Vasopressin is crucial for body water/osmolality control and involved in cardiovascular regulation; oxytocin’s functions are associated with reproductive processes and viviparity.

DISCOVERY [e.g., 8] Vasopressin (VP) or antidiuretic hormone (ADH) and oxytocin (OT) have pioneered endocrine research and the peptides themselves have a long evolutionary history. The discovery, between 1895 and 1915 of the hormonal activities of VP and OT is well documented. Oliver and Schäfer demonstrated vasopressor activity in the pituitary that Howell showed came from its neural lobe (NL); similarly, Schäfer demonstrated antidiuretic activity and von den Velden showed its NL source. Originally it was thought that there were two hormones— antidiuretic and vasopressor—hence the two names Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

622 / Chapter 88 and Ludwig in [7]). It seems likely that the same is true for the PCN. Note: Most references are to recent volumes with collected reviews [6, 7, 10, 11, 12, 13, 15, 18] or specific reviews, with author reference where appropriate.

STRUCTURE OF THE PRECURSOR mRNA/GENE (Fig. 2) [2, 17] The VP and OT genes are situated on chromosome 20, tail to tail and transcribed in opposite directions, reflecting duplication during evolution. The intergenic region contains a linear repeat (LINE) sequence and enhancers for both the VP and OT genes, some of which are reversed with respect to the prohormonecoding sequences. The close linkage of the VP and OT genes may be important as there is evidence for regulatory communication between components of the two transcription units. The full extent of the controlling promoter regions remains to be determined (Gainer in [18]); one directs trafficking of the mRNA (Mohr and Richter in [7]). Each gene consists of 3 exons (ca. 2 kb). Exon A codes for the signal, the hormone, a Gly-Lys-Arg cleavage sequence, and the nine N-terminal amino acids

Vasopressin Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 Oxytocin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

FIGURE 1. Amino acid structure of arginine vasopressin (Mr 1084.38) and oxytocin (Mr1007.35). The Arg in vasopressin is replaced by Lys in pigs and some marsupials; the Arg (or Lys) is critical for vasopressin action. Oxytocin has the same structure in all mammals; the Ile is critical for oxytocin action. (In birds, reptiles, amphibia, and lungfishes, vasotocin and mesotocin are the VP and Oxy equivalents; in bony fishes, vasotocin and isotocin.)

(aa) of NP. Exon B codes for the middle part of NP (aa 10-76), which is very highly conserved, reflecting a role of NP both as a chaperone and binding the hormone within the large dense-cored vesicles (LDCV). Interestingly, there is evidence for ancestral duplication within exon B. Exon C codes for the remaining aa (77-93/95) of the NP, a single Arg and, for VP only, a 39 aa GP. Occasional hybrid VP/OTNP and OT/VP mRNAs and peptides can be found in both wild-type and Brattleboro (di/di) rats. The VP/OTNP hybrid peptides accumulate in the ER of the neurons and are not packaged into LDVC or secreted (Morris et al. in [6]). Transcription of the VP and OT genes in MCN is controlled by activity of the cells (and therefore their neuronal inputs) in ways that are still unclear. This reflects both the size and amount of repetitive elements in the genome and the difficulty of studying gene regulation in a small group of neurons. In both VP and OT neurons gene transcription, synthesis, and secretion increase along with c-fos induction, and c-fos induction was thought to link MCN activation and transcription. However, c-fos is induced in osmotically stimulated aged rats where there is no increase in VP mRNA transcription. An inducible glucocorticoid receptor (GR) in VP neurons appears to act on a GR site in the VP gene promoter reflecting the secretion of VP in stress. Osmotic stimulation also induces a doubling of the poly(A) tail length of the VP and OT mRNAs which may alter mRNA stability. However, modelling studies by Fitzsimmons (in [8]) indicate that control of transcription is much more important than mRNA stability. Rather little is known about the promoters. Cyclic AMP response elements (CRE) are present in human, rat, and bovine VP genes; they can be blocked by CRE binding proteins (CREB). Glucocorticoids influence VP transcription in both MCN and PCN but the mechanism of this is not clear. Diurnal variation in VP transcription may be influenced by CLOCK and BMAL1 via a conserved G box. Sex steroids exert important effects on OT secretion in most species and ERβ but not ERα is expressed in VP and OT MCN. The promoter of the

vasopressin CRE

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Vasopressin and Oxytocin / 623 OT but not the VP gene contains a number of AGGTCA motifs (in particular at −172 to −148) where ER and other members of the nuclear receptor family (e.g., THR, RAR) including orphans (e.g., COUP-TF1 which inhibits induction by estradiol -E2—and triiodothyronine -T3) could interact. Sex steroids could also influence transcription via nongenomic mechanisms (they have rapid effects on electrical activity, axonal, and dendritic secretion) and/or via E2-responsive neurons afferent to the MCN such as those of the olfactory lobes.

DISTRIBUTION OF THE mRNA (Figs. 3, 4; see [1]) Magnocellular neurons: The SON consists almost entirely of MCN: the OT cells are situated dorsally/ rostrally, the VP cells ventrally/caudally. In the PVN OT MCN are situated more rostrally, VP cells more caudally. In both nuclei there is some mixing. Other MCN are located in the preoptic area (POA), lateral hypothalamic and perifornical areas, near the anterior commissure, and in the nucleus circularis. The MCN project almost exclusively to the neural lobe but have collateral axonal branches within the hypothalamus. Nucleus circularis VP neurons also act on the anterior hypothalamus V1a receptors which have a role in aggression

linked to a 5-HT input (see Ferris in [10]) MCN contain very large amounts of message (up to 1.5 × 106) in each VP or OT neuron. The VP message has a long life (1.9d days). Although it was thought that MCN produce either VP or OT, it now clear that most MCN in unstimulated animals produce small amounts of the other peptide, but in lactation <17% of OT cells produce substantial amounts of both peptide. MCN also produce, and probably copackage, many other neuropeptides at low levels, which suggest local actions [1]. Of the copackaged peptides, CCK in OT cells can stimulate both OT and VP secretion, while dynorphin selectively inhibits OT secretion. The role of the copackaged peptides in other CNS axonal terminals is unexplored. Parvocellular neurons: The PVN contains more mixed cell types than the SON; PCN predominate in its medial and caudal parts. In the medial part, CRH PCN can coexpress VP; many of these project to the median eminence to control the anterior pituitary. Other VPsecreting PCN in the PVN project to other parts of the forebrain, brainstem, and spinal cord to affect autonomic function. One group of small VP neurons in the shell of the suprachiasmatic nucleus (SChN) is involved in clock function, displays rhythmic clock gene expression that is reduced in aging, and projects dorsocaudally to the zone between the SChN and PVN and into

FIGURE 3. Distribution of VP cells and fibers. Magnocellular neurons ( ; —). Parvocellular neurons: (a) projecting to median eminence (䉫) fibers not shown; (b) circadian rhythmic neurons (䉱; - - - -); (c) sex dimorphic neurons (䉬; –·–·–); (d) autonomic group (䉫; ·····). Abbreviations in Figs. 3 and 4 not in main list: A aminergic neurons in ventral medulla; C cingulate; F frontal; IML intermediolateral column autonomic neurons; LS lateral septum; NC nucleus circularis; POA preoptic area; PV periventricular group; VH ventral hippocampus.

624 / Chapter 88

FIGURE 4. Distribution of OT cells and fibers. Magnocellular neurons ( ; —). Parvocellular neurons: projecting to forebrain (䉬; - - - -); projecting to brainstem and cord (䉬; ·····). For additional abbreviations, see Fig. 3.

the DMH, also along the third ventricle into the medial preoptic and PVN, and caudally to the retrochiasmatic area and VMH. A fourth sexually dimorphic group in the BNST and medial amygdala projects widely in the lateral septum, diagonal band, ventral hippocampus, and periaqueductal gray (e.g., Buijs and Kalsbeek in [13]; DeVries and Miller in [15]). Hypothalamic parvocellular OT neurons are located in the mediobasal hypothalamus, the median preoptic area, the periventricular area, and in the BNST. OT projections and receptors are more widespread than those for VP, being found in the hypothalamus, olfactory system, association cortex, limbic system, nucleus accumbens, central gray, and reticular nucleus, with especially large amounts in parts of the olfactory system and insular cortex. In addition to these central neuronal sources, VP mRNA has been detected in choroid plexus epithelium, in a subset of pituicytes in the NL; and OT mRNA and peptide are produced in ovarian follicles, corpora lutea, endometrium, testicular interstitial cells, and other peripheral tissues implicated in reproductive functions.

PROCESSING OF THE PRECURSOR (Fig. 5) [1] The genes on chromosome 20 are transcribed in opposite directions to yield heteronuclear RNA, which

is then spliced to give mature RNA which leaves the nucleus and is translated on the rough endoplasmic reticulum (rER) to give the preprohormones, preprovasopressin or preprooxyphysin. In the rER the signal is cleaved, S-S bonds and folding are established to produce the prohormones, and proximal glycosylation of the GP moiety of provasopressin occurs. This is then passed to the Golgi where terminal glycosylation and cleavage of the VP-associated GP occurs and the mature prohormone is sequestered into LDCV together with processing enzymes (the neurophysin moiety is essential for this). During subsequent transport into the axons or dendrites, the prohormone is cleaved and the C-terminal glycine of the hormone is amidated. The acidic intragranular pH is optimal for the cleavage enzymes and also for binding of the cleaved hormones to their neurophysin which promotes LDCV stability. VP neurons express prohormone convertases (PC) 1 and 3, but PC5 predominates in OT neurons. These cleave at the Gly-Lys-Arg which separates the hormone from the neurophysin and then carboxypeptide H/E trims the remaining C-terminal aa to reveal the glycine, which is then amidated by peptidylglycine α-amidating monoxygenase. Interestingly production of active VP occurs in fetal life, but mature OT is only produced postnatally. In stimulated cells processing appears to be upregulated to match increased transcription. The LDCV are either released from the axons or dendrites by what appears to be classical exocytosis or they can be

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FIGURE 5. Processing of vasopressin (left) and oxytocin (right). The signal peptidase (SP) removes the signal peptide from the preprohormone. Prohormone convertases first remove GP in provasopressin, then cleave between hormone and neurophysin within the large dense-cored vesicles.

destroyed by lysosomal action in NL Herring bodies if not required.

RECEPTORS [1, 5, 9] The receptors for VP and OT are all members of the G-protein associated 7-transmembrane group, the hormones binding to both extracellular and transmembrane domains. As with the hormones there is a high degree of homology of the receptors across species, especially in the extracellular loops and transmembrane helices. Around 25% of their aa are invariant across all VP and OT receptors. Three subtypes of VP receptor (VR) have been identified (V1R or V1a; V2R; V3R or V1b); but apparently only a single type of OT receptor (OTR) exists. The three VP receptors are relatively specific for VP (Ki ∼ 1 nM) compared with OT (Ki 64–1782 nM) whereas the OT receptor is relatively nonspecific (Ki 1 nM for OT, 16 nM for VP) (Thibonnier et al. in [15]). The V1R acts via Gq/11 to regulate the β-isoform of phospholipase C (PLC) and stimulates PLC, D and A2. It is expressed in liver, vascular smooth muscle, and most peripheral tissues that express VR, and is probably the most common VR in brain, including expression on VP MCN. The V2R preferentially couples to the αsubunit of Gs, simulating adenyl cyclase and the cAMP cascade. It is expressed only in the kidney of adult rats, but also in the brain of neonates. The V3R also acts via Gq/11 to stimulated PLCs; it was first identified in pituitary corticotrophs but is now known to occur in many brain regions including the VP MCNs, and in kidney,

heart, lung, spleen, thymus, uterus, and breast [9]. The OTR is also coupled via Gq to PLC-β. It is expressed in the myometrium, breast, and other reproductive structures, including many parts of the brain with functions allied to reproduction. OTR are abundant in parts of the olfactory system (anterior olfactory nucleus and tubercle—dependent on sex-steroids); the limbic system, basal ganglia (neostriatum), and spinal cord (superficial dorsal horn). In the ventral hippocampus and BNST, OTR and OT fibers coincide, but in the amygdala there is marked OT binding and OTR mRNA but few OT fibers. There is little information on the regulation of VPR expression, but OTR are strongly regulated by E2 (though they do not disappear entirely after gonadectomy) and adrenal steroids. In nonexpressing tissues OTR expression is constitutively repressed, apparently by hypermethylation. Regulatory elements that control tissue specific expression are present in the 5′ sequences but have not yet been elucidated. Insel (in [12]) has evidence that these could provide a molecular basis for pair-bonding behavior (see following) and therefore the evolution of monogamy. On ligand binding the receptors are rapidly (5–10 min) internalized; V1R but not OTR or V2R are recycled to the membrane. Recent studies indicate that homo and heterodimerization of the receptors occurs and can control the amount of receptor on the cell surface by varying dissociation in endosomes after ligand binding (see Devost and Zingg in [7]). The possibility of a direct progesterone effect on OTR is controversial; the brain is known to produce progesterone derivatives but whether these affect OTR is not known.

626 / Chapter 88 ACTIVE AND/OR SOLUTION CONFORMATION [5] There is little information because solubilization of OTR causes a loss of high-affinity binding which can be restored by addition of cholesterol and Mg2+ as would be present in the cell.

BIOLOGICAL ACTIONS WITHIN THE BRAIN AND PITUITARY Both VP and OT have well-known systemic effects in the kidney, cardiovascular, and reproductive systems, discovered long before their effects in the brain, but there is no evidence for actions of either the neurophysins or VP-associated GP. Systemic VP is the main controller of body water via V2R (all mammals control water to regulate osmolality) and a minor player in pressurevolume regulation via V1R (see [2] and the sections in this book on renal peptides and cardiovascular peptides for these actions). OT stimulates milk-ejection from the breast (though OTR expression is greater in glandular epithelium than myoepithelial cells) and myometrial contraction (OTR increase 200-fold at parturition), which may be controlled by circulating oxytocinase during pregnancy. OT and OTR are also produced in many reproductive tissues where OT exerts local effects. In the fetal membranes OT stimulates the production of prostaglandins near term; OT mRNA also appears in the endometrium of some species. In the ovary, OT is involved in follicular steroidogenesis and is also produced in the corpus luteum where it is luteotrophic. In the male, OT and OTR are present in testicular Leydig cells and OT may influence tubule contractility; OT also affects the prostate and epididymis (see [4]; Soloff et al. in [10]). In the heart OT is produced and can reduce the rate and force of contraction. OT also acts in the pancreas via glucagon, in the thymus to influence T cell differentiation, in adipocytes where it has insulin-like actions, and in the adrenal. In the CNS, a good correlation between hormonecontaining terminals and receptors (present on both neurons and astrocytes) would be expected, but this is often not the case. The peptides act both as a result of axonal secretion at distant targets and in an autocrine and paracrine manner by dendritic secretion. In the SON and PVN, VP inhibits VP neurons but OT facilitates OT neuronal activity (separation of VP and OT MCN and bundling of OT dendrites that occurs in parturition and lactation may compensate for the nonselectivity of the OTR). VP apparently has fewer CNS projections and actions than OT and the distribution of their receptors and

their action differ markedly. In the SChN and its projections (see preceding) VP is involved in rhythm generation via V1a and V1b receptors. In the anterior hypothalamus VP and V1aR and OT are involved in aggression (see Ferris in [10]). In the limbic system VP reinforces learning and memory related to aversion, whereas OT attenuates it (it reduces NMDA receptor activity); both OT and VP are released by noxious or aversive stimuli. VP and OT and their receptors are located in centers involved in osmotic and cardiovascular regulation (OVLT, median preoptic nucleus, area postrema, dorsal motor nucleus of vagus). Brain OT and OTR are involved primarily in reproductive and related social behaviors such as recognition memory, pair bonding/grooming, and care of offspring, so not surprisingly there is considerable variation among species, during development, with steroid status, and between male and female (see [12]; Choleris et al. in [7]; Insel et al. in [18]). Most results come from rats, mice, voles, and sheep. Large amounts of OT are released in parturition and lactation, and in humans plasma OT peaks at orgasm. In males and females OT is secreted centrally by stress, activates stress-associated grooming, and has anxiolytic properties that reduce stress-activated behaviors that might impair maternal care and pair bonding. In males, OT causes penile erection via axons that end on the sexually dimorphic nucleus in the sacral cord. In females OT appears to be critical for the initiation and development of maternal behavior (see Kendrick in [10]) and also influences receptive (lordosis) behavior. In the PVN, OT acts as short term satiety hormone. In the pituitary VP and OT are involved in the control of the secretion of both ACTH and prolactin. VP released at the median eminence from CRH parvocellular PVN neurons is certainly involved in stress responses and there is now evidence [3] both VP and OT from magnocellular system also affect the hypothalamo-pituitary stress axis. In the pituitary VP amplifies CRH-stimulated ACTH secretion via V1bR on corticotrophs (Aguilera et al. [10]). Toward the end of gestation OT acts to stimulate PRL release via E2-induced OTR.

PATHOPHYSIOLOGICAL IMPLICATIONS (see Swaab in [15]; sections on cardiovascular and renal peptides) Because VP is primarily involved in control of water/ osmolality, the most prominent VP-associated pathological conditions are diabetes insipidus (DI) and the syndrome of inappropriate ADH secretion (SIADH). In the Brattleboro rat, homozygous deletion of a deoxyguanosine in Exon B causes a frameshift which destroys

Vasopressin and Oxytocin / 627 5 of the 14 codons for cysteines involved in S-S bonds, the glycosylation site, and the stop codon so that a poly(Lys) tail of up to 70 residues is formed. The mRNA is not efficiently translated and does not reach the LDCV. Human central DI differs and may be partial or complete. Mutations in the signal, VP, and NP parts of the gene cause autosomal dominant familial neurohypophysial DI and may cause progressive loss of VP MCN neurons. Unlike in Brattleboro rats, one human mutant allele causes DI, interferes with synthesis of the normal VP precursor, and cause aggregates of misfolded protein to accumulate in the rER. X-linked mutations in the human V2R cause nephrogenic DI by affecting the folding of the receptor. In the nephrogenic (di/di) mouse the cause is quite different—an overactive phosphodiesterase which prevents the action of V2R. Interestingly solitary MCNs in aged Brattleboro rats do express VP, NP, and GP due to a further loss of a GA pair apparently due to “molecular misreading” [16], a concept that has been extended to Alzheimer’s disease. In SIADH, plasma VP is inappropriately high for plasma osmolality. It is often caused by lung or breast carcinoma, the cells of which also express all three VP receptors [see North in [10]), and by a variety of CNS disorders with no obvious common link. Serotonin reuptake inhibitors can cause SIADH particularly in the elderly. Neither DI nor SIADH are associated with obvious behavioral disturbances. In Brattleboro rats a diurnal rhythm persists despite the lack of VP. In mice, knockout of OT prevents milk ejection but not parturition, but there are no well-defined pathologies associated with loss or excess oxytocin in humans. It has been suggested, however, that autism is associated with reduced central OTR caused by homologous downregulation at birth and a resultant defect in social bonding; that high CNS OT is associated with obsessivecompulsive disorder in which “grooming” behavior is excessive; and that OT might also be involved in eating disorders such as anorexia and Prader-Willi obesity. Pharmaceutical agonists and antagonists of VP and OT have been created both for research and for treatment (see Chan et al. in [10]). Long-acting VP analogs are used to treat central DI; VP antagonists to treat SIADH; OT is used to induce parturition and OT antagonists are being developed to treat premature labor.

References [1] Burbach JPH, Luckman SM, Murphy D, Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system. Phys Revs 2001;81:1197–1267. [2] Coote JH. A role for the paraventricular nucleus of the hypothalamus in the autonomic control of heart and kidney. Exp Physiol 2005;90:169–173. [3] Engelmann M, Landgraf R, Wotjak CT. The hypothalamicneurohypophysial system regulates the hypothalamicpituitary-adrenal axis under stress: an old concept revisited. Front Neuroendocrinol 2004;25:132–149. [4] Filippi S, Vignozzi L, Vannelli GB, Ledda F, Forti G, Maggi M. Role of oxytocin in the ejaculatory process. J Endocrinol Invest 2003;26:82–86. [5] Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function and regulation. Phys Revs 2001;81:629–684. [6] Ivell R, Russell JA, editors. Oxytocin. Cellular and molecular approaches in medicine and research. Adv Exp Med Biol 1995;395:p.1–673. [7] Kawata M, Leng G, editors. Neurohypophysial hormones. J. Neuroendocrinol 2004;16:293–408. [8] Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, editors. Williams textbook of endocrinology. Ed. 10. Philadelphia, Saunders. 2003. [9] Lolait SJ, O’Carroll AM, Brownstein MJ. Molecular biology of vasopressin receptors. Ann NY Acad Sci 1995;771:273–292. [10] Oxytocin and vasopressin—from molecules to function. Exp Physiol 2000;85S:1S–272S. [11] Poulain D, Oliet S, Theodosis D, editors. Vasopressin and oxytocin: from genes to clinical applications. Prog Brain Res 2002;139:1–376. [12] Russell JA, Douglas AJ, Windle RJ, Ingrm CD, editors. The maternal brain. Neurobiological and neuroendocrine adaptation and disorders in pregnancy and post partum. Prog Brain Res 2001;133:1–365. [13] Saito T, Kukawa K, Yoshida S, editors. Neurohypophysis. Recent Progress of Vasopressin and Oxytocin Research. Amsterdam, Elsevier; 1995. p.1–689. [14] Saper CB, Lu J, Chou TC, Gooley J. The hypothalamic integrator for circadian rhythms. Trends Neurosci 2005;28: 152–157. [15] Urban IJA, Burbach JPH, DeWied D, editors. Advances in brain vasopressin. Prog Brain Res 1998;119:p.1–655. [16] Van Leeuwen, FW, Burbach JP, Hol EM. Mutations in RNA: a first example of molecular misreading in Alzheimer’s disease. Trends Neurosci 1998;21:331–335. [17] Young WS 3rd, Gainer H. Transgenesis and the study of expression, cellular targeting and function of oxytocin, vasopressin and their receptors. Neuroendocrinology 2003; 78:185–203. [18] Zingg HH, Bourque CW, Bichet DG. Vasopressin and oxytocin. Molecular, cellular, and clinical advances. Adv Exp Med Biol 1998;449:p.1–483.

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89 Thyrotrophin-Releasing Hormone: New Functions for an Ancient Peptide ALBERT EUGENE PEKARY

ABSTRACT

that the substance being isolated contained only three amino acid residues, Glu, His, and Pro. During synthesis of all possible tripeptides containing these amino acids, Glu-His-Pro was found to have a low, but unmistakable, activity in the dispersed anterior pituitary bioassay being used to detect TSH-releasing activity in chromatographic fractions of hypothalamic extracts. Given that all of the other hypophysiotropic hormones are much larger than TRH [34] and almost always require amidation of the C-terminal residue, complete characterization of these other releasing hormones using the technology and funding available at the time would have been nearly impossible. Acceptance of neuroendocrinology as a legitimate research discipline could have been long delayed but for this extraordinary confluence of chance, rivalry, and scientific skepticism.

Thyrotrophin-releasing hormone (TRH) was the first of the hypothalamic releasing factors to be fully characterized. It consists of the tripeptide pGlu-HisPro-NH2 that is derived from a precursor protein with multiple copies of the precursor sequence Lys-Arg-GlnHis-Pro-Gly-(Lys/Arg)-Arg. Pre-proTRH is distributed throughout the animal kingdom, occurring in species lacking a pituitary. In addition to its neuroendocrine release from the hypothalamus of mammals, resulting in secretion of TSH and PRL from the anterior pituitary, it functions as a neurotransmitter, neuromodulator, and neuroprotective agent in the central and peripheral nervous systems. Unlike hypothalamic preproTRH mRNA levels and TRH content that are subject to thyroid hormone negative feedback inhibition, extrahypothalamic brain TRH biosynthesis and release are unresponsive to thyroid hormone status. TRH suppresses glycogen synthase kinase-3β (GSK-3β) expression, a process that may lead to treatments for Alzheimer’s disease, depression, bipolar disorder, and diabetes.

STRUCTURE OF THE PRECURSOR mRNA/GENE Genes for frog, rat, mouse, and human pre-proTRH contain three exons and two introns [5, 20, 22, 32, 39]. The first exon codes for the 5′-untranslated region; the second exon contains the signal sequence and most of the N-terminus. The remainder of pre-proTRH is coded by the third exon (Fig. 1). Two closely related preproTRH genes have been identified in frog, with sequence differences occurring primarily within the 5′flanking regions [5, 20]. This may provide additional opportunities for tissue and cell-specific regulation of TRH synthesis in this species which produces extraordinarily high levels of TRH in its skin [17]. Each preproTRH mRNA contains multiple TRH precursor

DISCOVERY OF TRH The nearly simultaneous reporting of the structure of the first hypothalamic releasing factor, thyrotrophinreleasing hormone (TRH), as pGlu-His-Pro-NH2, by Roger Guillemin and Andrew Schally, was the culmination of a five-decade effort to validate the neuroendocrine hypothesis [11]. This story contains elements of intense rivalry between competing research groups, serendipitous combinations of events and sheer luck Handbook of Biologically Active Peptides

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630 / Chapter 89 Putative signal peptide 5′-UT

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FIGURE 1. Structure of rat pre-proTRH gene: schematic diagram of the messenger RNA (mRNA) (top line) and genomic DNA (bottom line). The 5′-untranslated region (5′UT) and 3′-untranslated regon (3′-UT) are shown by the solid line (top) and open boxes (bottom). The signal peptide is indicated by a stippled box. The striped boxes indicate connecting and terminal peptides. The solid boxes indicate the TRH-coding regions. (F.L. Strand, Neuropeptides, 1999, with permission from MIT Press).

sequences: 7 in frog [5, 20], 6 in human [39], and 5 in rat and mouse [22, 32]. Peptide sequences connecting some of the TRH precursors in rat pre-proTRH have been shown to have biological activity [26, 31], but these are not strongly conserved between species [20].

DISTRIBUTION OF PRE-PROTRH mRNA AND PRE-PRO-TRH-DERIVED PEPTIDES Pre-proTRH mRNA is expressed at very high levels in mouse hypothalamus and testis [32]. Immunohistochemical staining with antibodies to intervening sequences of rat pre-proTRH showed staining in neuronal perikarya and processes in all regions of the brain previously demonstrated to immunostain for TRH, including dense innervation of the external zone of the median eminence. Immunocytochemical and in situ hybridization studies localize TRH-producing perikarya in extrahypothalamic brain areas such as the olfactory system, reticular thalamic nucleus, and amygdaloid complex (Fig. 2). Rat pre-proTRH(160–169) is readily measured in hippocampus, pyriform cortex, amygdala, striatum, and anterior cortex [26]. High levels of TRH immunoreactivity are also found in the posterior pituitary, retina, pineal gland, and spinal cord [34].

PROCESSING OF THE TRH PRECURSOR Processing of pre-proTRH begins with removal of the signal peptide during its passage into the lumen of the rough endoplasmic reticulum [25]. The dibasic amino acid residues flanking the N-terminus and Cterminus of the repeated TRH precursor sequence GlnHis-Pro-Gly are cleaved by serine proteases, PC1 and PC2, that are constituent membrane proteins of the large dense core vesicles (LDCV) into which the preproTRH is packaged following synthesis in the endoplasmic reticulum. The C-terminal basic residues are removed by the zinc-dependent carboxypeptidase H. The remaining C-terminal Gly is cleaved by the copper, ascorbate, and oxygen-requiring peptidylglycine alpha amidating monooxygenase (PAM). The N-terminal Gln is cyclized to pyroglutamate with the aid of an N-glutaminyl cyclase [25, 34].

DISTRIBUTION OF TRH RECEPTORS AND THE TRH DEGRADING ENZYME Three different receptors for TRH have been reported to occur in frog, two in rat, mouse and a teleost, and one in chicken and man [3, 6, 12, 16, 23, 35, 39]. The distribution of the rat mRNA for the TRH

Thyrotrophin-Releasing Hormone: New Functions for an Ancient Peptide / 631

FIGURE 2. Pre-proTRH mRNA, expressed protein, and derived peptides are widely distributed in rat brain [15, 21, 25, 26, 29]. However, the extent of processing of the pre-proTRH precursor protein to form tripeptide TRH, prior to its extracellular release, is limited in some brain areas but is nearly complete in the hypothalamus.

receptor 1 (TRH-R1) and receptor 2 (TRH-R2) have been studied in detail [13]. TRH-R2 mRNA is widely distributed throughout brain with highest cDNA levels throughout the thalamus, cerebral and cerebellar cortex, medial habenulae, medial geniculate nucleus, pontine nuclei, and the entire reticular formation. Consistent with the endocrine function of TRH, TRH-R1 predominates in hypothalamic nuclei, the anterior pituitary thyrotrophs, mammotrophs (PRL-secreting cells), and to a lesser extent, somatotrophs (GH-secreting cells) [34]. TRH-R1 in brainstem regions and spinal cord motoneurons is involved with autonomic and somatomotor control [8, 11]. An overlapping distribution of the TRH degrading enzyme (TRH-DE) mRNA [1] with TRH-R2 mRNA is consistent with the involvement of TRH in higher cognitive functions as well as effects on arousal, locomotor activity, and pain perception [4, 7, 14, 33, 34].

G11 [9], pertussis toxin-insensitive G proteins that activate phosphoinositide-specific phospholipase C-β (PPIPLC) [33]. PPI-PLC is the enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Inositol trisphosphate binds to the inositol trisphosphate receptor, a calcium channel on the endoplasmic reticulum, resulting in an increase in cytoplasmic calcium levels [38]. Diacylglycerol, along with calcium, activates protein kinase C (PKC) resulting in increased phosphorylation of Ser and Thr on a variety of second messenger enzymes [9]. Altered phosphorylation of ion channels, cytoplasmic and plasma membrane proteins, and nuclear transcription factors modify gene expression in the cell nucleus thereby transducing the TRH receptor binding signal [33].

BIOLOGICAL ACTIONS OF TRH WITHIN THE BRAIN AND PITUITARY TRH RECEPTOR SIGNALING CASCADES TRH-R1 and TRH-R2 are typical G-protein-coupled receptors that bind TRH and N-methyl-TRH with high affinity and specificity [14]. TRH-R3 from frog binds TRH, and other TRH-like peptides weakly, suggesting that it is an orphan receptor (Hinkle and Pekary, unpublished results; [23]). TRH-R1 couples to Gq and

TRH is the principal regulator of the “set point” for thyrotrophin (TSH) synthesis and secretion by the anterior pituitary thyrotrophs [28]. TSH stimulates the thyroid gland to produce T4 and T3, which provide a negative feedback suppression of hypothalamic TRH and pituitary TSH synthesis and release [25]. Knock out of the pre-proTRH gene results in hypothyroidism and

632 / Chapter 89 diabetes mellitus [40]. Hypothalamic TRH release is altered in many conditions including thyroid disease, cold stress, the onset of sleep, and changes in hormone and cytokine levels resulting from physical or emotional stress or severe illness [33]. It has long been assumed that TRH has other functions besides the control of TSH given its wide distribution throughout the brain and peripheral tissues, its

occurrence in vertebrate and invertebrate species lacking a pituitary, and the unresponsiveness of TRH mRNA and peptide levels of the limbic system to changes in thyroid status [28] (see Fig. 3). It is generally accepted that TRH has neurotransmitter and neuromodulatory functions [34]. It is colocalized and cosecreted with other neurotransmitters in a variety of different nerve cell types located in both CNS and peripheral tissues

FIGURE 3. Effect of progressive, PTU-induced, hypothyroidism on HPLC profile of TRH and TRH-like peptides in amygdala of male Sprague-Dawley rats. Note that the TRH-like peptides (X-TRH, pGlu-X-Pro-NH2) decreased, on average, 71 and 79% on days 2 and 3 of daily ip injections of 3 mg 6-N-propylthiourea (PTU) while the TRH level was completely unaffected (p < 0.001) [30].

Thyrotrophin-Releasing Hormone: New Functions for an Ancient Peptide / 633 Proposed Model for TRH Regulation of Tau Phosphorylation in Hippocampal Neurons

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[34]. CNS regulation of autonomic functions, including blood flow, heart rate, blood pressure, breathing, thirst, hunger, pain, liver blood flow, exocrine, endocrine pancreas functions, gastrointestinal motility and gastric acid secretion are mediated, at least in part, by TRH release within the CNS [8, 11]. Interestingly, high levels of TRH also occur within the β-cells of the pancreatic islets [40] and Leydig cells of the testis [27] of all mammals so far tested.

PATHOPHYSIOLOGICAL IMPLICATIONS TRH has an essential neuroprotective function that is mediated by its suppression of glycogen synthase kinase-3β (GSK-3β) expression in neurons [24]. This enzyme is responsible for the hyperphosphorylation of tau that results in neurofibrillary tangles that are associated with the neuropathologies of Alzheimer’s and other dementias and regulates microtubule assembly in nerve axons and dendrites [41]. A signal transduction pathway for TRH downregulation of GSK-3β has been proposed [24] (Fig. 4). This ability of TRH to suppress GSK-3β has implications not only for Alzheimer’s disease but also severe depression, bipolar disorder, diabetes mellitus, stroke, prostate cancer, and aging [2, 10, 18, 36, 37, 42]. Antidepressants and the major mood stabilizers for bipolar disorder, lithium and valproate, also inhibit GSK-3β and can reverse the associated neurologic damage to glia and neurons in brain regions associated with mood regulation [7]. TRH and TRH-like peptides can be viewed as endogenous antidepressants, anticonvulsants, analeptics, and neuroprotectants [14, 29]. Because TRH is rapidly degraded by TRH-DE [1, 19], develop-

FIGURE 4. A scheme to describe the signal transduction pathways of TRH on Tau phosphorylation in hippocampal neurons. Binding its receptor, TRH dissociates the α unit from αβγ complex. The βγ unit then activates Src and PKA independently. Src could inhibit GSK-3β directly or through inhibiting PKC activation to reduce GSK-3β activation. In addition, the βγ unit could activate PKA, which has been reported to directly inhibit Tau phosphorylation as indicated by the arrow in the scheme. However, PI3K may affect GSK-3β during Tau phosphorylation, but further studies are necessary to confirm this. (Reprinted from J. Alzheimer’s Dis., Vol. 6, L Luo and E Stopa, Thyrotrophin releasing hormone inhibits tau phosphorylation by dual signaling pathways in hippocampal neurons, pp. 527–536, 2004, with permission from IOS Press.)

ment of TRH-DE-resistant TRH analogs [8], use of naturally occurring TRH-like peptides with antidepressant properties [14, 29], or development of nontoxic, selective PAPII inhibitors [19] will be required for practical treatments.

References [1] Bauer K. TRH-degrading aminopeptidase: A regulator and terminator of TRH action? In: Aminopeptidases in Biology and Disease, Leudeckel U, Hooper N, Eds., Kluwer Plenum Publishers, New York, 2004, pp. 127–143. [2] Bhat RV, Budd Haeberlein SL, Avila J. Glycogen synthase kinase 3: A drug target for CNS therapies. J. Neurochem. 2004;89: 1313–1317. [3] Bidaud I, Lory P, Nicolas P, Bulant M, Ladram A. Characterization and functional expression of cDNAs encoding thyrotrophin-releasing hormone receptor from Xenopus laevis. Identification of a novel subtype of thyrotrophin-releasing hormone receptor. Eur J. Biochem. 2002;269:4566–4576. [4] Boschi G, Desiles M, Reny V, Rips R, Wrigglesworth S. Antinociceptive properties of thyrotrophin releasing in mice: Comparison with morphine. Br. J. Pharmacol. 1983;79:85–92. [5] Bulant M, Richter K, Kuchler K, Kreil G. A cDNA from brain of Xenopus-laevis coding for a new precursor of thyrotrophinreleasing hormone. F.E.B.S. Lett. 1992;296:292–296. [6] Cao J, O’Donnell D, Vu H, Payza K, Pou C, Godbout C, Jakob A, Pelletier M, Lembo P, Ahmad S, Walker P. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotrophin-releasing hormone receptor. J. Biol. Chem. 1998;273:32281–32287. [7] Charney DS, Nestler EJ, Eds., Neurobiology of Mental Illness, 2nd Edition, Oxford University Press, 2004. [8] Gary KA, Sevarino KA, Yarbrough GG, Prang AJ Jr, Winokur A. The thyrotrophin-releasing hormone (TRH) hypothesis of homeostatic regulation: Implications for TRH-based therapeutics. J. Pharmacol. Exp. Ther. 2003;305:410–416. [9] Gershengorn MC, Osman R. Molecular and cellular biology of thyrotrophin-releasing hormone receptors. Physiol. Rev. 1996; 76:175–191.

634 / Chapter 89 [10] Gould TD, Einat H, Bhat R, Manji HK. AR-014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int. J. Neuropsychopharmacol. 2004;7:387–390. [11] Guillemin R. Hypothalamic hormones a.k.a. hypothalamic releasing factors. J. Endocrinol. 2005;184:11–28. [12] Harder S, Dammann O, Buck F, Zwiers H, Lederis K, Richter D, Bruhn TO. Cloning of two thyrotrophin-releasing hormone receptor subtypes from a lower vertebrate (Cotostomus commersoni): Functional expression, gene structure, and evolution. Gen. Comp. Endocrinol. 2001;124:236–245. [13] Heuer H, Schafer MK, O’Donnell D, Walker P, Bauer K. Expression of thyrotrophin-releasing hormone 2 (TRH-R2) in the central nervous system of rats. J. Comp. Neurol. 2000;428:319– 336. [14] Hinkle PM, Pekary AE, Senayake S, Sattin A. Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides. Brain Res. 2002;935:59–64. [15] Ishikawa K, Inoue K, Tosaka H, Shimada O, Suzuki M. Immunohistochemical characterization of thyrotrophin-releasing hormone-containing neurons in rat septum. Neuroendocrinology 1984;39:448–452. [16] Itadani H, Nakamura T, Itoh J, Iwaasa H, Kanatani A, Borkowski J, Ihara M, Ohta M. Cloning and characterization of a new subtype of thyrotrophin-releasing hormone receptors. Biochem. Biophys. Res. Commun. 1998;250:68–71. [17] Jackson IMD, Reichlin S. Thyrotrophin-releasing hormone: abundance in the skin of the frog, Rana pipiens. Science 1977; 198:414–415. [18] Kelley S, Zhao H, Hua Sun G, Cheng D, Qiao Y, Luo J, Martin K, Steinbeck GK, Harrison SD, Yenari MA. Glycogen synthase kinase 3beta inhibitor Chir025 reduces neuronal death resulting from oxygen-glucose deprivation, glutamate excitotoxicity, and cerebral ischemia. Exp. Neurol. 2004;188:378–386. [19] Kelley JA, Slator GR, Titpton KF, Williams CH, Bauer K. Kinetic investigation of the specificity of porcine brain thyrotrophinreleasing hormone-degrading ectoenzyme for thyrotrophinreleasing hormone-like peptides. J. Biol. Chem. 2000;275: 16746–16751. [20] Kuchler K, Richter K, Trnovsky J, Egger R, Kreil G. Two precursors of thyrotrophin-releasing hormone from skin of Xenopus laevis. Each contains seven copies of the end product. J. Biol. Chem. 1990;265:11731–11733. [21] Lechan RM, Wu P, Jackson IMD. Immunocytochemical distribution in rat brain of putative peptides derived from thyrotrophin-releasing hormone prohormone. Endocrinology 1987;121:1879–1891. [22] Lee SL, Stewart K, Goodman RH. Structure of the gene encoding rat thyrotrophin releasing hormone. J. Biol. Chem. 1988;263:16604–16609. [23] Lu X, Bidaud I, Ladram A, Gershengorn MC. Pharmacological studies of thyrotrophin-releasing hormone (TRH) receptors from Xenopus laevis: Is xTRHR3 a TRH receptor? Endocrinology 2003;144:1842–1846. [24] Luo L-G, Stopa EG. Thyrotrophin releasing hormone inhibits tau phosphorylation by dual signaling pathways in hippocampal neurons. J. Alzheimer’s Dis. 2004;6:527–536.

[25] Nillni EA, Severino KA. The biology of pro-thyrotrophin-releasing hormone-derived peptides. Endocr. Rev. 1999;20:599–648. [26] Pekary AE. Is Ps4 (prepro-TRH[160–169]) more than an enhancer for thyrotrophin-releasing hormone? Thyroid 1998;8: 963–968. [27] Pekary AE, Meyer NV, Vaillant C, Hershman JM. Thyrotrophinreleasing hormone and a homologous peptide in the male rat reproductive system. Biochem. Biophys. Res. Commun. 1980;95:993–1000. [28] Pekary AE, Sattin A. Regulation of TRH and TRH-related peptides in rat brain by thyroid and steroid hormones. Peptides 2001;22:1161–1173. [29] Pekary AE, Sattin A, Meyerhoff JL, Chiligar M. Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain. Peptides 2004;25:458–647. [30] Pekary AE, Sattin A, Stevens SA. Rapid modulation of TRH-like peptides in rat brain by thyroid hormones. Peptides 2006 (in press). [31] Redei E, Rittenhouse PA, Revskoy S, McGivern RF, Aird F. A novel endogenous corticotropin release inhibiting factor. Ann. N.Y. Acad. Sci. 1998;840:456–469. [32] Satoh T, Yamada M, Monden T, Iizuka M, Mori M. Cloning of the mouse hypothalamic preprothyrotrophin-releasing hormone (TRH) cDNA and tissue distribution of its mRNA. Brain Res. Mol. Brain Res. 1992;14:131–135. [33] Schatzberg AF, Nemeroff CB, Eds. Textbook of Psychopharmacology. The American Psychiatric Publishing, Inc., Washington, DC, 2004. [34] Strand FL. Neuropeptides. Regulators of Physiological Processes. MIT Press, Cambridge, 1999. [35] Sun YM, Millar RP, Ho H, Gershengorn MC, Illing N. Cloning and characterization of the chicken thyrotrophin-releasing hormone receptor. Endocrinology 1998;139:3390–3398. [36] Wagman AS, Johnson KW, Bussiere DE. Discovery and development of GSK3 inhibitors for the treatment of type 2 diabetes. Curr. Pharm. Des. 2004;10:1105–1137. [37] Wang L, Lin HK, Hu YC, Xie X, Yang L, Chang C. Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells. J. Biol. Chem. 2004;279:32444–324452. [38] Waring P. Redox active calcium ion channels and cell death. Arch. Biochem. Biophys. 2005;33–42. [39] Yamada M, Radovick S, Wondisford FE, Nakayama Y, Weintraub BD, Wilber JF. Cloning and structure of human genomic DNA and hypothalamic cDNA encoding human preprothyrotrophinreleasing hormone. Mol. Endocrinol. 1990;4:551–556. [40] Yamada M, Saga Y, Shibusawa N, Hirato J, Murakami M, Iwasaki T, Hashimoto K, Satoh T, Wakabayashi K, Taketo MM, Mori M. Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotrophin-releasing hormone gene. Proc. Natl. Acad. Sci. USA 1997;94:10861–10867. [41] Zhou F-Q, Snider WD. GSK-3β and microtubule assembly in axons. Science 2005;308:211–214. [42] Zmijewski JW, Jope RS. Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblasts. Aging Cell 2004;3:309–317.

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and was first isolated from mammalian hypothalami as the decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGly.NH2) [1]. GnRH is processed in specialized neurons of the hypothalamus from a precursor polypeptide by enzymatic processing and packaged in storage granules that are transported down axons to the external zone of the median eminence. GnRH is released as synchronized pulses from the nerve endings of about 1000 neurons into the hypophyseal portal system every 30– 120 mins to stimulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs [4]. Each GnRH pulse stimulates a pulse of LH, but FSH pulses are less clear. The frequency of pulses is highest at the ovulatory LH surge and lowest during the luteal phase of the ovarian cycle. The asynchronous patterns of LH and FSH release result from changes in GnRH pulse frequency, modulating effects of gonadal steroid and peptide hormones on FSH and LH responses to GnRH, and differences in the half-lives of the two hormones. Moreover, while LH is stored and largely dependent on GnRH for secretion, FSH tends to be constitutively secreted and more dependent on biosynthesis for secretion.

ABSTRACT GnRH is the central hypothalamic hormone regulating reproduction. Over 20 forms of the decapeptide have been identified in vertebrates and invertebrates. The NH2- and COOH-terminal sequences, which are essential for receptor binding and activation, are conserved. In mammals, there are two forms: GnRH I, which regulates gonadotrophin, and GnRH II, which appears to be a neuromodulator and stimulates sexual behavior. GnRHs and receptors are also expressed in reproductive and peripheral tissues and immune cells, as well as tumors in which a paracrine/autocrine role is postulated. GnRH agonists and antagonists are now extensively used to treat hormone-dependent diseases, in assisted conception, and have promise as novel contraceptives. Nonpeptide orally active GnRH antagonists have been recently developed and may increase the flexibility and range of utility. As with GnRH, GnRH receptors have undergone coordinated gene duplications such that cognate receptor subtypes for respective ligands exist in most vertebrates. Interestingly, in man and some other mammals (e.g., chimp, sheep, and cow) the Type II GnRH receptor has been silenced. However, GnRH I and GnRH II still appear to have distinct roles in signaling differentially through the Type I receptor (ligand-selective-signaling) to have different downstream effects. The ligand-receptor interactions and receptor conformational changes involved in receptor activation have been partly delineated. These advances are setting the scene for the generation of novel selective GnRH analogs with potential for wider and more specific applications in a range of diseases.

STRUCTURE OF GnRHS AND THEIR PRECURSORS Primary Structures Mammalian hypothalamic GnRH was thought to be a unique structure with a primary role in regulating LH and FSH and conserved among vertebrates. However, it became apparent that diverse forms exist in vertebrates (see [12]). This led to the structural identification of 13 different forms in vertebrates and another 9 in protochordates (Fig. 1) [3]. It also became apparent that GnRHs are distributed in a wide range of tissues in vertebrates, where they apparently have a diversity of func-

DISCOVERY AND BACKGROUND Gonadotrophin-releasing hormone (GnRH) is the central initiator of the reproductive hormonal cascade Handbook of Biologically Active Peptides

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636 / Chapter 90

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FIGURE 1. Primary amino acid sequences of naturally occurring GnRH structural variants spanning approximately 600 million years of evolution. The boxed regions show the conserved NH2 and COOH-terminal residues, which interact with the receptors. Nonconserved residues are either unimportant or convey ligand-selectivity for a particular GnRH receptor. Note that the GnRHs are named according to the species in which they were first discovered and they may be represented in more than one species. For example, mammalian GnRH is widely present in amphibians and primitive bony fish, and chicken GnRH II is present in most vertebrate species, including man.

tions in addition to their primary role in gonadotrophin secretion. These include other neuroendocrine funtions, paracrine and autocrine functions in GnRH neurons, gonads, immune cells, breast and prostatic cancer cells, and neurotransmitter/neuromodulatory roles in the central and peripheral nervous system. Although a single GnRH is theoretically capable of serving these roles, it is evident that at least two and

usually three forms of GnRH are differentially expressed in these tissues as regulators. The most ubiquitous GnRH in vertebrates is chicken GnRH II (first isolated from chicken brain), which is totally conserved from bony fish to man [6] and is probably the earliest evolved form. This form has been designated GnRH II while the hypophysiotropic form is designated GnRH I. In many vertebrate species a third form of GnRH occurs (local-

Gonadotrophin Releasing Hormone / 637 ized to the forebrain in fish) and is designated GnRH III. Analysis of the genes encoding the GnRHs supports this general classification [16].

Three-Dimensional Structures The amino acid sequences of the 24 GnRHs from vertebrate and protochordate species reveal (Fig. 1) that there has been conservation in peptide length (10 amino acids) and in the NH2 terminus (pGluHis-Trp) and the COOH terminus (Pro-Gly.NH2), over 600 million years of evolution, indicating that these features are critically important for receptor binding and activation (Fig. 2). This is borne out by structure-activity data from thousands of analogs that were developed largely on an empirical basis (see [5, 20]). Position eight is the most variable amino acid (Arg, Gln, Ser, Asn, Leu, Try, Lys, Ala, Trp, Thr, Met), suggesting that virtually any residue is tolerated in this position. However, this is not the case for the mammalian pituitary type I receptor, which requires Arg in position eight [20], and recent work on cloned nonmammalian receptors also indicates certain specificities for the amino acid in this position. Thus, this residue seems to play an important role in ligandselectivity of the different GnRH receptors and in modulating intracellular signalling at the same receptor (see [12]). The conserved NH2- and COOH-terminal domains of GnRH are closely apposed when mammalian GnRH binds its receptor, resulting from a β-II-type turn involving residues 5–8 (Fig. 2 [5, 12, 20]). This is partly due to intramolecular interactions with the side-chain of Arg8, Trp fluorescence. Computer simulations using the technique of conformational memories and nuclear magnetic resonance (NMR) studies have shown that substitution of Arg8 results in a more extended structure, with a loss of predominance of

the folded conformers and a low biological activity ([3] and see [12, 20] for review). Yet, these extended forms (e.g., Gln8 GnRH) have high activity in many nonmammalian GnRH receptors, in spite of their low activity at the mammalian receptor. The β-II-type turn conformation of GnRH also appears to be induced in part by the interaction of Arg8 with an acidic residue (D302) in extracellular loop 3 (EC3) of the mammalian receptor (Fig. 5). Substitution of a d-amino acid for Gly6 enhances the β-II-type conformation, and increases the activity of GnRH about tenfold, thus creating a super-active agonist [5, 12, 20]. The aminoterminal residues of GnRH are involved in receptor activation, and modification of these residues in GnRH produces analogs with antagonistic properties (Fig. 2).

GnRH DISTRIBUTION The diversity in molecular structure, function, and anatomical distribution of GnRHs raises the question as to whether GnRH neurons expressing the different GnRHs are embryonically derived from a single precursor population, or have multiple origins. The neuroanatomical organization of GnRH neurons in different vertebrates has been reviewed [6, 17]. As discussed earlier at least two different forms of GnRH are expressed in most vertebrate species; GnRH II, and a second/third form that varies in the different species. The GnRH I cell bodies regulating the brain-pituitarygonad axis are located principally in the medial septum and anterior preoptic area in mammals, and give rise to the major GnRH axonal projection through the ventral hypothalamus to the median eminence. GnRH cell bodies are also present in the terminal nerve, associated with the olfactory system and ventral forebrain, and in several regions of the posterior diencephalon

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FIGURE 2. Schematic representation of mammalian gonadotrophin-releasing hormone (GnRH) in the folded conformation in which it is bound to the GnRH pituitary receptor. The molecule is bent around the achiral glycine in position six. Substitution with D-amino acids in this position stabilizes the folded conformation, and increases binding affinity and decreases metabolic clearance. This feature is incorporated in all agonist and antagonist analogs. The NH2 (red) and COOH (green) termini are involved in receptor binding. The NH2 terminus alone is involved in receptor activation, and substitutions in this region produce antagonists. (From [12] with permission.)

638 / Chapter 90 and midbrain. GnRH fibers project to almost all areas of the central nervous system, the vasculature, and the ventricles. A model for GnRH neuronal systems has been proposed by Muske [5]. (a) GnRH neurons have at least two embryonic origins: the olfactory placode, which gives rise to the terminal nerve-septo-preoptic system, and a second, nonplacodal structure, which gives rise to the posterior diencephalon/anterior midbrain system. (b) The two embryonically distinct populations express different molecular forms of GnRH. The posterior neurons express GnRH II, while the terminal nervesepto-preoptic neurons express a different form(s) of GnRH, which varies in different species. Immunocytochemical studies of brain regions reveal many similarities among vertebrate species in their cytoarchitectonic organization, suggesting that like the peptides, the neuronal systems are phylogenetically ancient. The GnRH neuronal system in amphibians is typical of that described in species from other vertebrate classes (Fig. 3). The terminal nerve-septo-preoptic system is the prime regulator of gonadotrophin release in most vertebrates and the neurons originate in the embryonic olfactory placode and migrate centrally during development. The ontogeny of the terminal nerve-septopreoptic system is thought to reflect its evolutionary origin as a peripheral endocrine organ associated with the olfactory system. The second GnRH system, which arises from nonplacodal precursors, comprises cell bodies in periventricular regions of the posterior dienHAB MP

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PROCESSING OF THE GnRH PRECURSOR Initial studies claimed a non-ribosomal biosynthesis of GnRH, but other studies provided evidence for biosynthesis from a precursor of about 5000 molecular weight through tryptic and carboxypeptidase B cleavage of a dibasic site [13]. These researchers proposed that the product was further processed by cyclization of Gln to the N-terminal pyro Glu and amidation of the C-terminal glycine using the adjacent glycine as NH2 donor (Fig. 4). These proposals were supported by the subsequent revelation of the GnRH cDNA sequence [21]. The 56-amino-acid sequence following that of GnRH was thought to affect prolactin and gonadotrophin secretion but this notion is now in doubt.

GnRH RECEPTORS AND SIGNALLING CASCADES

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cephalon and midbrain. Evidence suggests that these cells served as the ancestral brain GnRH system, and are the cellular locus of the early-evolved GnRH II peptide. While GnRH I and GnRH II are both present and have distinct neural distribution patterns in primates, GnRH II has been deleted from the mouse and rat genome [14, 16]. It appears that GnRH I has taken over the role of GnRH II in neurons in which this peptide was previously expressed. In vertebrate species, it is widely established that the different roles of GnRHs are as a releaser of pituitary gonadotrophins, and as a neurotransmitter or neuromodulator in extrahypothalamic areas of the brain.

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ME

FIGURE 3. GnRH-immunoreactive neuronal systems in an amphibian, Taricha granulosa. Mammalian GnRH antiserum labelled cell bodies (red circles) in the anterior forebrain, including terminal nerve, medial septum, and anterior preoptic area, and a restricted set of projections to the medial pallium, habenula, and median eminence (red lines). GnRH II antiserum-labeled cell bodies (blue circles) in the paraventricular organ and posterior tubercle and a diffuse system of fibers to most brain area (blue lines). Abbreviations: HAB, habenula; HYP, hypothalamus; ME, median eminence; MED, medulla; MP, medial pallium; MS, medial septum; OB, olfactory bulb; OT, optic tectum; POA, preoptic area; PT, posterior tubercle; PVO, paraventricular organ; TEG, midbrain tegmentum; TN, terminal nerve. (Adapted from Muske [17].) (See color plate.)

The amino acid sequence of the GnRH receptor was first deduced for the mouse receptor in the αT3 gonadotroph cell line [23], and this provided the basis for the cloning of GnRH pituitary receptors from the rat, human, sheep, cow, and pig, which share over 80% amino acid identity. More recently, GnRH receptors have been cloned from a marsupial (possum), chicken, and a number of fish and amphibian species [12, 16]. A second form of GnRH receptor (type II) was identified in man [10] and subsequently cloned from primates [11, 19]. However, the human homolog appears to be a nonfunctional pseudogene whose function has been taken over by the type I receptor [14, 16]. In some amphibian and fish species, a third GnRH receptor exists [12, 16]. The findings suggest an early evolution of the three GnRH receptor subtypes in vertebrates that parallels that of the GnRH ligands [12]. The conservation of amino acids during evolution from bony fish to mammals is likely to indicate those residues crucial to GnRH receptor function (see Fig. 5).

Gonadotrophin Releasing Hormone / 639 CYCLIZATION TO pGlu

PRE -23

CLEAVAGE & AMIDATION SITE

-1 1

10

Gln His Trp Ser Tyr Gly Leu Arg Pro Gly

11

14

Gly Lys Arg

PUTATIVE CLEAVAGE SITES 27

37

46

50

53

Lys

Arg

Arg Arg Lys

67

68

69

Lys Lys lle

GnRH FIGURE 4. Schematic diagram of the structure of the human GnRH precursor. The precursor consists of a signal sequence (PRE) of 23 amino acids followed immediately by the GnRH decapeptide sequence. Cleavage of the signal peptide reveals an NH2-terminus Gln which cyclizes (enzymatically or spontaneously) to pyro-Glu. The GnRH sequence is followed by a Gly which is the donor for the COOH-terminal amide of GnRH, and Lys-Arg which is a conventional dibasic amino acid cleavage site. This is followed by a 56-aminoacid peptide sequence. The positions of pairs and single basic amino acid residues present in the 14–69 sequence are shown as potential sites of further processing. Glycosylation

Extracellular

14

Q

N Q E P

S

A

M S N A

NH2

197 L N T H C S F 196 200 V R M P G E C A L E Q Q Y G T L T W 205 S S Q L L Q K W P S D 302 T 292 C 114 V H L W 206 S V D S L A F D P F K H I T G 179 102 290 291 101 289 M Q I W V L V A N R I K N Y S R F F W H 211 Y V I I W 98 T L 121 Y 214 F G M N Y 212 T V L G L K Q L L D F F V F F L P F F Y P 215 T F F S Y 282 A G M F L A P M 284 V F F 313 F Y T L C S A S V L L I W 280 S L A P F L L S I P N 53 E T I V C A W 164 C T L I A F 223 F P F 319 131 L M 87 N F T S N D L M L G A V A T 320 F L P L I M V I V F A F S L T Q S LM G Y I L V A L S G I L 82 H V K Y 323 N C R D 138 K M T L K L L A S K S 139 L S F K 328 L L Q L N I L K T F I K S K R 262 A COOH L T R A261 M I W K T 260 PKC 75 R P T 146 147 L T G 240 R q/11 74 I R S Gs Q 73 P L A N Gq/11 V 241 L N K K L 242 E K G KK K Gq/11 PKA H S Q Q D N P H E L Q L

P

PKC

G L N

S C I A N N S I 18 L P Q M

H

N

Intracellular

FIGURE 5. Two-dimensional representation of the human GnRH receptor showing TM domains (boxed) connected by ECs and ICs. Putative ligand binding sites (red) and residues thought to be important in receptor structure or binding pocket formation are shown in green letters. These include disulfide bond formation and glycosylation sites. Residues involved in receptor activation are shown in blue. Residues in squares are the ones highly conserved throughout the rhodopsin family of GPCRs. Residues involved in coupling to G proteins are shown in orange. Protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites are indicated. (From [12] with permission.)

The GnRH receptor has the characteristic features of G protein-coupled receptors (GPCRs) with an Nterminal domain, followed by seven putative α-helical transmembrane (TM) domains that are connected by three extracellular (EC) loop domains and three intracellular (IC) loop domains (Fig. 5). The EC domains and superficial regions of the TMs are usually involved in binding of peptide hormones like GnRH; the TMs are believed to be involved in conformational change associated with signal propagation (receptor activation), while the intracellular domains are

involved in interacting with G proteins for signal transduction. A unique feature of the mammalian GnRH receptor is the absence of a C-terminal tail present in all other GPCRs and in all of the nonmammalian GnRH receptors and that is responsible for rapid desensitization. The structures of the genes encoding the mouse and human GnRH receptors have been elucidated. In both genes the three exons are separated by introns in the coding regions of TM4 and IC3, and there are numerous splice variants whose function may be regulatory, as some truncated recep-

640 / Chapter 90 tor variants affect the expression of the functional receptor. The promoters of the rat and human receptors have been analyzed and the genes are highly regulated by GnRH and its signallers, and by gonadal, steroid, and peptide hormones.

Insights into the Tertiary Structure of the GnRH Receptor A knowledge of the three-dimensional structure of the GnRH receptor are essential for a complete understanding of its molecular functioning. To date it has only been possible to crystallize and obtain x-ray structural information on rhodopsin, the light sensing GPCR. Molecular models of the GnRH receptor have been generated based on the rhodopsin structure and form the basis for experimental studies designed to test hypotheses and refine the model. The positioning and orientation of the TM helices are therefore crucial. In this regard an unusual feature of the GnRH receptor has allowed identification of interhelical interactions. Two residues that are highly conserved in GPCRs, aspartate in TM2 and asparagine in TM7, appear to have undergone reciprocal mutation to Asn87 and Asp319 (see Fig. 5). This suggests that the two residues interact with each other. Mutation of Asn87 in TM2 to aspartate abolished receptor function, but a second mutation of Asp319 to asparagine in TM7, recreating the arrangement found in other GPCRs (Asp87, Asn318), regenerated ligand binding [see 7]. This restoration of binding by reciprocal mutation shows that the side chains of two residues in helixes TM2 and TM7 have complementary roles in maintaining the structure of the receptor and occupy the same microenvironment within the receptor. The 7 TM helical domains are known from physical structure studies in the rhodopsins to be arranged in a tight bundle enclosing a hydrophilic pocket and surrounded by the hydrophobic membrane environment. The evolutionary conservation of residues along a distinct face of the TM domains in the various GnRH receptors is clearly evident in Fig. 5. This suggests that the conserved, more hydrophilic faces are orientated toward the hydrophilic pocket formed by the helical bundle.

GnRH Agonist Binding to Its Receptor In the course of the systematic and targeted mutation of almost one-third of all the amino acid residues of the GnRH receptor, considerable advances in identifying ligand contact sites in the mammalian GnRH receptor have been made (Fig. 5). Four putative ligand interaction sites in the GnRH receptor. These are pGlu1 with N212, His2 with K121 and D98, Trp3

with Y280, Tyr5 with Y290, Arg8 with D302, and Gly.NH210 with N102 [12] (Fig. 5). All of these sites have been conserved in vertebrate pituitary GnRH receptors cloned from bony fish, amphibian, and bird species. For details on GnRH binding, activation, and desensitization of its receptors, the reader is referred to [12].

Intracellular Signalling Pathways The coupling of the GnRH receptor to intracellular-signalling pathways has been intensely investigated and comprehensively reviewed (see [7, 9, 18]). Although a wide range of intracellular signalling pathways have been implicated in GnRH action, there is a general consensus that the primary pathway is the activation of phospholipase Cβ (PLCβ) through the Gq/11 small guanine nucleotide-binding protein (Fig. 6). PLCβ hydrolyzes phosphatidylinositol-4-5 bisphosphate (PIP2) to inositol-1-4-5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to receptors in the endoplasmic reticulum to release Ca2+ transiently from these intracellular stores, and this elicits a rapid spike of LH release. DAG activates protein kinase Cs (PKCs) that phosphorylate proteins involved in the more sustained release of LH and in gonadotrophin biosynthesis such as the mitogen-activated protein kinases (MAPKs) which include ERK, JNK, and P38. A second major effect of GnRH binding to its receptor is the activation of L-type voltage-operated Ca2+ channels (VOCCs), which results in the influx of extracellular Ca2+ required for recharging intracellular stores and the prolonged second phase of LH release (Fig. 6). GnRH stimulates DAG production biphasically in pituitary cells, and at a high molar ratio relative to IP3. This suggests that GnRH stimulates DAG production from a second source independent of PLCβ action on PIP2. Phospholipase D (PLD) was found to be activated in a gonadotroph cell line a few minutes after PLC activation. Since its substrate phosphatidylcholine is present at almost 100 times higher concentrations in the plasma membrane than that of PIP2, GnRH stimulation of PLD may be the main source of sustained DAG production. Another potential signalling pathway of GnRH action is through phospholipase A2 (PLA2), which produces arachidonic acid and its 5-lipoxygenase products, the leukotrienes, in pituitary cells some minutes after the rise in IP3. Although PLA2 might be activated directly by the GnRH receptor, it is likely that activation is by MAPKs, which are known to phosphorylate and activate PLA2. A number of studies have shown that GnRH indirectly activates MAPKs in pituitary cells and gonadotroph cell lines via PKC phosphorylation of Raf-1.

Gonadotrophin Releasing Hormone / 641 GnRH

Ca2+

Gq/11

EGFR/FAP PLA2

PLD

PLCβ

PC

PC

PIP2

AA

DAG

LTs

PKCs

L-type VOCC

ER IP3

Ca2+ Ca2+ Ca2+

Cytosolic Ca2+

MAPKs (ERK, JNK, P38)

Gonadotropin biosynthesis Gonadotropin secretion

FIGURE 6. Signalling pathways of mammalian, gonadotrophin-releasing hormone (GnRH) type I receptor. The primary pathway shown by heavy arrows involves the activation of phospholipase Cβ (PLC) via the Gq/11 G-protein and the hydrolysis of phosphatidylinositol-1-4 bisphosphate (PIP2) to diacyl glycerol (DAG), which activates protein kinase C (PKC), and to inositol-1-4-5trisphosphate (IP3) which stimulates Ca2+ release from the endoplasmic reticulum (ER). A second major effect is the entry of extracellular Ca2+ through voltage-operated Ca2+ channels (VOCCs). The GnRH receptor also activates phospholipase D (PLD), which hydrolyzes phosphatidyl choline (PC) to DAG, and phospholipase A2 (PLA2), which generates arachidonic acid (AA) and leukotrienes (LTs) from PC. PLA2 activation may be mediated via a primary stimulation of mitogenactivated protein kinases (MAPK). DAG activates protein kinase C (PKC), which in turn can activate MAPKs. MAPK isoforms may also be stimulated via focal adhesion proteins (FAPs) and epidermal growth factor receptor (EGFR) and monomeric G-proteins such as ras. The products of the diverse pathways affect gonadotrophin biosynthesis and secretion. Note that many of the signalling events occur in the plasma membrane, where enzymes (e.g., PLCβ, PLD, PLA2) and substrates (e.g., PIP2) are anchored. For simplicity, this is not shown.

MAPKs may also be activated by focal adhesion protein complexes involving integrins and by transactivation of the epidermal growth factor receptor (EGFR). All of the signalling pathways just outlined can result in the activation of PKC isoforms (Fig. 6). However, there are distinct differences in the timing and the PKC isoform activated, such that a sequential, coordinated, and sustained activation of PKCs occurs. The early DAG and Ca2+ generated by PLCβ action might activate Ca2+-dependent PKCs, while late DAG generated by PLD might activate Ca2+-independent PKCs. Similarly, the more slowly generated products

of PLA2, such as arachidonic acid and leukotrienes, are known to activate specific PKC isoforms (see [7, 9, 18]).

GnRH Regulation of Gonadotrophin Gene Expression The distal targets of the signalling pathways, PKC isoforms, Ca2+ calmodulin, MAPKs, and leukotrienes are all variously involved in gonadotrophin gene expression, biosynthesis, and exocytosis (see [7, 18]). The mobilization of Ca2+ and activation of PKCs resulting

642 / Chapter 90 from the primary GnRH signalling pathway have both been reported to increase α-, LHβ-, and FSHβ-subunit mRNA levels and PKC response elements have been identified in the promoters of these subunits. The more protracted activation of PKCs resulting from the products of the PLD and PLA2 pathways presumably contributes to the increased gonadotrophin subunit mRNA levels. GnRH activation of MAPKs also stimulates the expression of subunit mRNA, and since PKCs can activate MAPKs, part or all of the PKCs effects may be via MAPKs. These effects may be mediated via distinct GnRH-response elements in the gene promoters. The relative stimulation of gonadotrophin subunits in the rat pituitary is influenced by GnRH pulse frequency. High-pulse frequencies stimulate LHβ mRNA levels more than FSβ, while the converse is true at lowpulse frequencies. Thus, the combination of alterations in pulse frequency together with different phasing and duration of the various signalling pathways provides the potential for fine regulation of gonadotrophin secretion. Feedback by gonadal steroid and peptide hormones at the gonadotroph provides additional modulation and the means for asynchronous LH and FSH secretion in response to GnRH which is crucial for normal ovarian function.

Desensitization Exposure of the gonadotroph to continuous high doses of GnRH agonists results in the phenomenon of desensitization (see [9, 12]). Gonadotroph desensitization comprises many contributing phenomena, including GnRH receptor down-regulation, receptor uncoupling from cognate G-proteins, additional downstream uncoupling (e.g., protein kinase C, Ca2+ stores, inositol trisphosphate receptor), inhibition of gonadotrophin synthesis, and alterations in the glycosylation of gonadotrophins rendering them biologically inactive or even antagonistic. At the level of the GnRH receptor, agonist activation leads to two potential mechanisms of desensitization. For many GPCRs, agonist stimulation results in the activation of protein kinases, which phosphorylate IC domains, resulting in uncoupling from Gproteins and internalization of the receptor. GnRH receptors have a number of putative phosphorylation sites in the IC domains which may serve this function (Fig. 5). However, the mammalian GnRH receptors lack the carboxy-terminal IC domain, which is the prime target for phosphorylation by G-protein-coupled receptor kinases and protein kinases. Phosphorylation in this region facilitates the docking of arrestins, thus inducing G-protein uncoupling and receptor internalization. Hence, the absence of the carboxy-terminal tail in the mammalian GnRH receptors is associated with a lack of rapid desensitization in inositol phosphate production

and an arrestin-independent slow rate of receptor internalization, sevenfold less than that of the chicken GnRH receptor which has a carboxy-terminal tail. Removal of the carboxy-terminal tail from the chicken receptor greatly reduces the rate of internalization to one similar to that of the human receptor. Addition of the carboxy-terminal tail of a non-mammalian GnRH receptor to a mammalian GnRH receptor conveys both rapid desensitization of inositol phosphate production and a more rapid internalization. It appears that physiological requirements have selected for removal of the carboxy-terminal tail during evolution of the mammalian GnRH receptor, to prevent rapid desensitization and internalization. A possible driving force is the physiological need of a prolonged LH surge for ovulation in mammals. Thus, although pharmacological doses of GnRH can produce desensitization of the gonadotroph, and this phenomenon has extensive clinical application, the GnRH receptor is less susceptible to ligand-mediated desensitization than other GPCRs and may not undergo desensitization under physiological conditions.

GnRH Receptor Genes The structures of the genes encoding GnRH receptors from a number of mammalian, amphibian, bird, and fish have been elucidated [16]. In all genes, there are introns in the coding regions of TM4 and IC3. There are numerous splice variants whose function may be regulatory, as some truncated receptor variants affect the expression of the functional receptor. The promoters of the rat and human receptors have been analyzed, and the genes are highly regulated by GnRH and its signalling molecules, and by gonadal, steroid, and peptide hormones.

PHYSICAL STRUCTURE OF GnRH GnRH is highly flexible in solution and a crystal structure has yet to be accomplished. However, structure–activity relations and molecular modelling had suggested that GnRH interacts with its receptor in a folded conformation incorporating a βII′ bend around Gly6 [5, 12, 20] and section 2. This conformation is supported by NMR studies and recent approaches combining molecular modelling and ion mobility mass spectrometry [3].

BIOLOGICAL ACTIONS GnRH was thought to function exclusively as a stimulator of gonadotrophin release from the vertebrate

Gonadotrophin Releasing Hormone / 643 pituitary, but it soon emerged that the peptide has been recruited for diverse functions in vertebrates and protochordates during evolution. This conclusion arose from revelations that: 1. Multiple forms of GnRH are present in tissues of single species. 2. GnRH is expressed in extrahypothalamic regions of the nervous system (e.g., midbrain, spinal cord, sympathetic ganglia) and in nonneural tissue (e.g., gonads, placenta, breast). 3. GnRH receptors are present in extrapituitary tissues and GnRH affects cell activity in these tissues. 4. GnRH stimulates the release of other pituitary hormones (e.g., growth hormone in fish). 5. GnRH is present in organisms that do not have a pituitary. The isolation of several forms of GnRH in neural tissue of tunicates, which represent chordate progenitors, and their activation of the gonads suggest that direct regulation of the gonads was an early-evolved function, and that the neuroendocrine role in regulating the pituitary was a later evolutionary development. The presence of GnRH and GnRH receptors in the gonads of various vertebrate species may reflect this early function. The stimulation of sexual reproduction in yeast by the α-mating factor, which appears to be structurally related to GnRH, may point to an even more ancient example of the direct effects of GnRH on reproduction. It is plausible, therefore, that GnRH peptides were originally involved in cellular communication in sexual reproduction of simple unicellular and multicellular organisms. Later, they were recruited for expression in nerve cells to translate external and internal signals into activation of reproduction, initially by acting directly on germ cells and subsequently via pituitary gonadotroph activation. While the peptide has been co-opted as a regulator at a number of levels in the reproductive system (hypothalamus, gonad, breast, and placenta), there is apparently considerable plasticity in also recruiting it as a regulator in nonreproductive tissues (e.g., adrenal, extrahypothalamic brain, the immune system, retina, and pancreas). The second form of GnRH identified from chicken brain (chicken GnRH II (Fig. 1) is ubiquitous in vertebrates from primitive bony fish to man. This complete conservation of structure over 500 million years suggests that the type II GnRH has an important function that is mediated through a discriminating receptor that has selected against any structural change in the ligand. The wide distribution of type II GnRH in the central and peripheral nervous systems suggests a neurotrans-

mitter/neuromodulatory role. This has been thoroughly demonstrated by the inhibition of M-currents in the bullfrog sympathetic ganglion, which sensitizes neurons to depolorization. GnRH II is present in amphibian sympathetic ganglia, and the receptors are highly selective for the type II peptide (see [6, 12]). GnRH had been shown to have direct effects on sexual arousal in vertebrates, and GnRH II is localized in brain areas associated with reproductive behavior, suggesting that this may be a role for the peptide. GnRH II and an analog are potent stimulators of reproductive behavior in ring doves and song sparrows (see [6, 12, 20]). Recently, the cognate receptor for GnRH II was cloned from the marmoset [11] and found to be distributed in those areas of primate brain associated with reproductive behaviors. In both the female marmoset [2] and musk shrew [22], central administration of GnRH II is much more effective than GnRH I in stimulating reproductive behavior. However, the type II GnRH receptor is nonfunctional in man [14, 16], and it has emerged that the cellular context can alter the pharmacology of the type I receptor and that GnRH II can induce different signalling from that of GnRH I [8].

PATHOPHYSIOLOGY Clinical applications of GnRH and its agonist and antagonist analogs are summarized in Fig. 7 (also see [15]). Low doses of GnRH (pg/ml) delivered in a pulsatile fashion equivalent to that found in the hypothalamic portal vessels restore fertility in hypogonadal men and women and are also effective in the treatment of delayed puberty. However, high doses of GnRH or agonist analogs desensitize the gonadotroph, with a resultant decrease in LH and FSH and a decline in ovarian and testicular function. This desensitization phenomenon is extensively applied in clinical medicine for the treatment of a wide range of diseases (Fig. 7). GnRH peptide antagonists also inhibit the reproductive system but through competition with endogenous GnRH. The doses required are much higher than the desensitizing agonist doses, and antagonists are currently less extensively employed. However, they have the advantage of inducing an immediate inhibition in contrast to agonists that initially (7–14 days) stimulate gonadotrophin before the desensitization phenomenon sets in. The development of nonpeptide, orally active GnRH antagonists (see [12]) will probably lead to the replacement of agonists, as they avoid the undesirable stimulation that precedes desensitization, dosage can be more easily modified than for injectable peptides, and treatment may be rapidly withdrawn when required.

644 / Chapter 90 Mammary Endometrial Prostate

Antisteroidogenic Antispermatogenic Antipregnancy Luteolytic Antiovulatory Cryptorchidism

Ovarian

Anticancer Infertility (+ gonadotropins) Endometriosis Precocious puberty Polycystic ovarian syndrome

Antifertility Therapeutic

Delayed puberty Steroidogenesis Spermatogenesis Ovulation

Hirsutism Infertility

Diagnostic

FIGURE 7.

Profertility GnRH pulsatile

GnRH agonists and antagonists

Acne Porphyria

Clinical applications of GnRH and GnRH agonists and antagonists. Modified from [15].

References [1] Baba Y, Matsuo H, Schally AV. Structures of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses. Biochem Biophys Res Commun. 1971;44:459:463. [2] Barnett DK, Bunnell TM, Millar RP, Abbott DH. Gonadotrophinreleasing hormone II stimulates female sexual behavior in marmoset monkeys. Endocr. 2005;147:615–623. [3] Barran PE, Roeske RW, Pawson AJ, Sellar R, Bowers MT, Morgan K, Lu Z-L, Tsuda M, Kusakabe T, Millar R. Evolution of constrained GnRH ligand conformation and receptor selectivity. J Biol Chem 2005;280:38569–38575. [4] Fink G. Gonadotrophin secretion and its control. In: The physiology of reproduction. Knobil E, Neill J, eds. New York: Raven Press 1988;1349–1377. [5] Karten MJ, Rivier JE. Gonadotrophin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: Rationale and perspective. Endocr Rev. 1986;7:44–66. [6] King JA, Millar RP. Co-ordinated evolution of GnRHs and their receptors. In: GnRH neurons: Gene to behavior. Parhar IS, Sakuma Y, eds. Tokyo: Brain Shuppan. 1997;51–77. [7] Kraus S, Naor Z, Seger R. Intracellular signaling pathways mediated by the gonadotrophin-releasing hormone (GnRH) receptor. Arch Med Res. 2001;32:499–509. [8] Maudsley S, Davidson L, Pawson AJ, Chan R, de Maturana RL, Millar RP. Gonadotrophin-releasing hormone (GnRH) antagonists promote proapoptotic signaling in peripheral reproductive tumor cells by activating a Gαi-coupling state of the type I GnRH receptor. Cancer Res. 2004;64:7533–7544. [9] McArdle CA, Franklin J, Green L, Hislop JN. Signalling, cycling and desensitization of gonadotrophin-releasing hormone receptors. J Endocrinol. 2002;173:1–11. [10] Millar RP, Conklin D, Lofton-Day C, Hutchinson E, Troskie B, Illing N, Sealfon SC, Hapgood J. A novel human GnRH receptor homolog gene: Abundant and wide tissue distribution of the antisense transcript. J Endocrinol. 1999;162:117–126. [11] Millar RP, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A. A novel mammalian receptor

[12]

[13]

[14] [15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 2001;98:9636–9641. Millar RP, Lu Z, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR. Gonadotrophin-releasing hormone receptors. Endo Rev. 2003;25(2):235–275. Millar RP, Aehnelt C, Rossier G. Higher molecular weight immunoreactive species of luteinizing hormone releasing hormone: Possible precursors of the hormone. Biochem Biophys Res Commun. 1977;79:720–731. Millar RP. GnRH II and type II GnRH receptors. Trends Endocrinol Metab. 2003;14:35–43. Millar RP, King JA, Davison JS, Milton RC. Gonadotrophinreleasing hormone—diversity of functions and clinical applications. S Afr Med J. 1987;72:748–755. Morgan K, Millar RP. Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species. Gen Comp Endocrinol. 2004;139:191–197. Muske LE. Evolution of gonadotrophin-releasing hormone (GnRH) neuronal systems. Brain Behav Evol. 1993:42:215– 230. Naor Z, Bernard O, Seger R. Activation of MAPK cascades by G-protein-coupled receptors: The case of gonadotrophinreleasing hormone receptor. Trends Endocrinol Metab. 2000;11:91–99. Neill JD, Duck LW, Sellers JC, Musgrove LC. A gonadotrophinreleasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun. 2001;282:1012– 1018. Sealfon SC, Weinstein H, Millar RP. Molecular mechanisms of ligand interaction with the gonadotrophin-releasing hormone receptor. Endocr Rev. 1997;18:180–205. Seeburg PH, Mason AJ, Stewart TA, et al. The mammalian GnRH gene and its pivotal role in reproduction. Recent Prog Haorm Res. 1987;43:690–698. Temple JL, Millar RP, Rissman EF. An evolutionarily conserved form of gonadotrophin-releasing hormone co-ordinates energy and reproductive behavior. Endocrinology. 2003;144:13–19. Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC. Cloning and functional expression of a mouse gonadotrophin-releasing hormone receptor. Mol Endocrinol. 1992;6:1163–1169.

C

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91 Brain Somatostatin-Related Peptides JACQUES EPELBAUM AND RAPHAELLE WINSKY-SOMMERER

ABSTRACT

later on in the gut [4] (Fig. 1). SRIF-14 is predominantly produced by neurons and secretory cells in the central and peripheral nervous system and SRIF-28 in the gastrointestinal tract. However, a number of additional expression sites have been described such as the placenta, the kidney, the retina, and in cells of the immune system [1]. Cortistatin (CST) was discovered in 1996 by Luis de Lecea while using directional tag PCR substractive hybridization techniques in order to fish out genes selectively involved in long-term potentiation (LTP), a phenomenon likely to correspond to cellular memory at the electrophysiological level on neurons. Among several candidates, he sequenced a novel 112-aminoacid protein with a striking homology to somatostatin at its distal C-terminal end. The cloning and localization of the gene on human chromosome 1 at 1p36.3p36.2 increased the complexity of the somatostatin signaling system. Sharing 11 amino acids with somatostatin, CST-14 is predicted to occur in a short or 17amino-acid form (CST-17) in rat, mouse, and human, respectively, and a longer one of 29 amino acids (CST29) in rat and human (Fig. 1). In the central nervous system CST mRNA expression is mainly restricted to the cerebral cortex and hippocampus. Only in mouse brain have low levels of pre-pro-CST mRNA been detected in the amygdala and the hypothalamus, which is an important SRIF expression site.

Somatostatin-14 was originally characterized as a hypothalamic neurohormone responsible for the inhibition of pituitary growth hormone secretion. In the mammalian brain, two genes encode for the prosomatostatin-derived peptides, somatostatin-14 and -28, and procortistatin-related ones, respectively. Each peptide binds with similar affinities to the five cloned sst receptors, which belong to the GPCR family and cortistatins bind specifically to the MrgX2 receptor. Besides the initial neuroendocrine role, many studies suggest that somatostatin and cortistatin peptides act as neuromodulators in the CNS, influencing different sensory processes, motor activity, sleep, and cognitive functions, as well as playing a role in brain diseases such as affective disorders, epilepsy, and Alzheimer’s disease.

DISCOVERY Somatostatin (SRIF: somatotropin release inhibiting factor) was discovered in 1972 by Roger Guillemin and his colleagues, who were engaged in the quest to purify and characterize the hypothalamic neurohormones involved in the control of pituitary hormone secretion [5]. They were aiming for a growth hormone (GH)releasing-hormone (GHRH) (cf. M. Malagon chapter) but ended up with a 14-amino-acid peptide which inhibited GH release from pituitary cells in monolayer culture. Rapidly thereafter, SRIF immunoreactivity was localized in the pancreas and gastrointestinal tract as well as in the brain. Two biologically active forms arise from the C-terminal portion of a single propeptide: SRIF-14, originally described in the hypothalamus [5] and the amino-terminally extended SRIF-28, discovered Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The pre-pro-SRIF gene structure and regulation have been extensively reviewed elsewhere. In humans, rats, and mice this gene is respectively located in 3q28, on

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646 / Chapter 91

SOMATOSTATIN RELATED PEPTIDES AND RECEPTORS

Peptides: genes:

Somatostatin gene

precursors:

prepro-somatostatin

active peptides

r,m,hsomatostatin-14

Cortistatin gene prepro-cortistatin

somatostatin-28

r,mcortistatin-14 hcortistatin-17

Ala-Gly-Cys-Lys-Asn-Phe-Phe Trp

Pro-Cys-Lys-Asn-Phe-Phe

Cys-Ser-Thr-Phe -Thr Lys

cortistatin-29 Trp

Lys-Cys-Ser-Ser-Phe-Thr Lys Asp-Arg-Met-Pro-Cys-Arg-Asn-Phe-Phe Trp Lys-Cys-Ser-Ser-Phe-Thr Lys

Receptors: 5 genes: sst1 to sst5

genes: proteins

1 gene: MrgX2

2 subfamilies: S-S

SRIF1 (sst2 A/B, sst3, sst5)

S-S

SRIF2 (sst1, sst4) COOH

COOH

FIGURE 1. Somatostatin-related peptides and receptors. The somatostatin gene generates two bioactive peptides, somatostatin-14 and somatostatin-28, the former being totally conserved in mammals and representing the majority of somatostatin immunoreactivity in brain. In contrast, cortistatin gene putative peptides are different in murine and primate species and, according to remaining levels in somatostatin −/− mouse, represent only a minority of somatostatin immunoreactivity in brain. Somatostatins and putative cortistatins can bind to the six somatostatin receptors, which can be subdivided into two families according to their functional properties: SRIF1 and SRIF2. Cortistatins can also bind to a former orphan receptor MrgX2 highly expressed in the dorsal root ganglia. (See color plate.)

chromosome 11 and 16. The human, mouse, and rat gene organizations are very similar. A single intron interrupts the two exons encoding pre-pro-SRIF. SRIF14 and SRIF-28, located at the carboxy-terminus of the precursor, are generated by posttranslational enzymatic processing (see following). In brain cells, transcription is regulated positively by many factors acting on cisregulatory elements such as bZip proteins and a cAMP response element (CRE), homeodomain proteins, and TAAT elements such as Pdx-1 or by bHLH proteins such as Sim1 and arnt2, and negatively by two proximal silencers which may be active in cerebrocortical but not hippocampal cells. SST gene regulation appears notably different in brain and pancreas since enhancers in the latter (SST-TAAT1, SST-TAAT2, and SST-UE-B) act as silencers in neural cells.

The human and mouse pre-pro-CST gene is located in 1p36.3-1p36.2 and on chromosome 4, respectively. Very few results are available concerning its regulation. While the sequences of SRIF 14 and 28 are identical in all mammals tested so far, cortistatin sequences vary notably within this lineage, indicating a much more rapid rate of evolution for the latter gene.

DISTRIBUTION OF THE mRNAs AND PEPTIDES SRIF immunoreactivity is largely distributed in many neurons in mammalian brain, including the human brain. The highest levels are found in the mediobasal hypothalamus and median eminence, amygdala, preop-

Brain Somatostatin-Related Peptides / 647 tic area, accumbens nucleus, cerebral cortex, striatum olfactory regions, and brain stem. Two main categories of neurons can be identified: those that project to a distance from their soma (long projecting) and short GABAergic interneurons coexpressing the neuropeptide either alone (cortex and hippocampus) or with neuropeptide Y (striatum and a few cells in the cortex). Pre-procortistatin mRNAs are mostly localized in a subset of cortical and hippocampal GABAergic interneurons, which partially colocalize with SRIF-positive neurons. No study has yet reported the distribution of CST peptides in the brain using selective antibodies which do not recognize SRIF moieties. In srif −/− mouse, the level of immunoreactive material remaining in brain, which is likely to correspond to CST, is below the sensitivity of the assay while CST expression does not change. Three long-projecting neuronal somatostatinergic systems can be differentiated according to their localization:

four layers that are parallel to the ventricle wall. Their axons run laterally and turn caudally through the retrochiasmatic area to enter the median eminence where they make contacts with the fenestrated capillary loops of the portal vasculature, from which they reach pituitary somatotrophs directly. SRIF cells in the Pev also project to many limbic (olfactory tubercle, septum, habenula, hippocampus) and mesencephalic (substantia nigra and locus coeruleus) regions. Cocaineamphetamine-regulated transcript (CART) colocalizes in 38% of the somatostatinergic neurons and is the only neuropeptide produced by hypophysiotropic somatostatinergic cells. • Amygdaloid somatostatin neurons connect the different nuclei of the amygdala with the notable exception of central nucleus ones which innervate many limbic regions through the stria terminalis. • Solitary tract neurons, through their projections to the ambiguus nucleus, control esophagean motility and, through their ascending projections to magnocellular hypothalamic neurons, cardiovascular processes. Interestingly these neurons are the only ones in the brain to contain only SRIF 28.

• Hypophysiotropic somatostatin neurons projecting to the median eminence are located within the rostral periventricular nucleus (Pev) and the parvocellular paraventricular nucleus. These cells are located close to the third ventricle, in three or

Cx Hi

CC OB CPut

S

Th PAG

Pev

AcbT

SON

Hpt DMH

PVN DBB

VMH

SN

LC NST

Arc ME

Amy

C

DR PBN

BST

NL IL AP

Amb

LRN

Cortistatin Somatostatin FIGURE 2. Schematic distribution of somatostatin (blue) and cortistatin (red) neurons in mammalian brain. In adults, somatostatin neurons are essentially distributed in the forebrain (OB, BST, Acb, claustrum, all parts of Cx including HI, entopeduncular nucleus, Amy, and Hpt) and the lower brain stem (central gray, para and lateral lemniscal nuclei, reticular formation), next to the prepositus hypoglossal nucleus, suprageniculate nucleus, and NST. Cortistatin neurons are essentially localized in the Cx and Hi. (See color plate.)

648 / Chapter 91 PROCESSING OF THE PRECURSORS AND DEGRADATION OF THE PEPTIDE The convertases Furin and PACE-4 appear to be involved in the constitutive pathway to generate SRIF28. In secretory vesicles, pro-SRIF is a physiological substrate of PC1 and/or PC2 and in vivo generation of SRIF-14 from the precursor requires this last convertase in human brain. These results are in keeping with the study of Furuta et al., which showed that the processing of pro-SRIF is severely impaired in PC2−/− mice pancreatic islets. Furthermore, PC1 and PC2 immunoreactivities are localized in numerous human cortical neurons and their topographic and cellular distribution broadly coincides with that previously reported for SRIFcontaining neurons—that is, mainly in the cortical layers II, III, as well as V and VI. SRIF peptides are degraded primarily by two endopeptidases: EC.3.4.24.15 and EC.3.4.24.16. The former is a soluble enzyme, while the latter is membrane associated. Subsequently exopeptidases, such as aminopeptidases A and M are able to cleave the N-terminal fragments. SRIF appears also to be cleared from brain interstitial space by sequestration within microvessels. No data are available concerning pro-CST processing and degradation in mammalian brain.

RECEPTORS AND SIGNALING CASCADES SRIF mediates its biological functions via at least six receptor subtypes, termed sst1, sst2A, sst2B, sst3, sst4, and sst5, which all belong to the family of seven transmembrane domain G-protein-coupled receptors (for review, see [2, 3]). The natural ligands of the different somatostatin receptor subtypes, SRIF-14 and SRIF-28 and CST, bind to all somatostatin receptor subtypes with high affinity. Nonetheless, it has not been shown that CST mediates sst receptor activation in vivo. Recently a putative CST receptor has been identified as MrgX2, which is highly expressed in dorsal root ganglia. Surprisingly, CST also appears able to interact with the ghrelin receptor while this is not the case for SRIF (for review, see [2]). The identification in the early 1990s of the sst receptor subtypes was a major step forward in elucidating the somatostatin signaling system. In recent years researchers aimed to find correlations between cloned and native receptors and to assign specific functions to individual receptor subtypes. Furthermore, the recent generation of specific antibodies against all somatostatin receptor variants allowed a precise cellular and subcellular localization of somatostatin receptor subtypes in the central nervous system, which also provided important insights into the somatostatin signaling. The

development of specific somatostatin analogues and antagonists and the results of experiments investigating genetically modified mice also contributed to a better understanding of the individual somatostatin receptor characteristics. A number of detailed studies have been conducted, especially in rodent tissues, to examine the pattern of expression of individual sst receptor subtypes mRNAs in the brain. Sst receptor transcripts are widely distributed throughout the CNS and the pituitary, displaying distinct but overlapping patterns of expression. However, mRNA levels do not necessarily reflect receptor protein expression. Today, immunolocalization of the sst1-5 proteins in the mammalian brain and pituitary has been achieved using selective antibodies (Fig. 3). Contrasting with the broad distribution of sst1 mRNAs, the sst1 protein appears essentially restricted to the rostral part of the periventricular hypothalamic nucleus from the organum vasculosum laminae terminalis extending caudally to the rostral part of the median eminence when using a C-terminal antibody. In contrast, a broad distribution of the presumptive deglycosylated form of the sst1 receptor protein in rat brain has been reported using an antibody directed against the N-terminal sequence. Further experiments are needed to resolve this discrepancy. It also remains to be established whether the presumptive nonglycosylated sst1 is functional. At any rate, the localization of the sst1 receptors and functional studies in the basal ganglia and retina are consistent with a role as a presynaptic inhibitory autoreceptor. Using C-terminal antibodies, sst2A receptor immunoreactivity appears widely distributed, in keeping with the regional distribution of both sst2mRNA and SRIF-1 binding sites. Rostrally, numerous strongly immunoreactive nerve cell bodies are observed throughout the pyramidal layer of the olfactory tubercle, extending into the polymorph layer. More sparse and less intensely labeled nerve cell bodies are also found in layer II of the piriform cortex. Diffuse but intense immunoreactive labeling is present in the endopiriform, anterior olfactory, and lateral olfactory tract nuclei. In the cerebral cortex, frontal, parietal, temporal, and occipital lobes exhibit prominent perikarya immunostaining in layers II–III. A few less intensely immunoreactive cell bodies are also evident in layer V. Diffuse but intense sst2A labeling is found in layers V–VI of the cerebral cortex. High densities of immunoreactive perikarya and dendrites are detected in the lateral division of the bed nucleus of the stria terminalis. Low densities of sst2Apositive cells are also found in the vertical limb of the diagonal band of Broca’s area. In the habenula nucleus, immunoreactive cell bodies are detected among a dense network of immunoreactive processes in the medial part while the lateral one appears devoid of any positive

Brain Somatostatin-Related Peptides / 649

Cx Hi CC

CS

epl

CI

OB S

Th

CPut

PAG C

BST

Hpt

Acb

TUO

DR PBN

PVN

DBB

SON Amy BL

DMH Pev VMH Arc ME

LC SN NTS NL IL

Amb LRN

SSTR1 SSTR2 SSTR4 SSTR5 SSTR3 ? FIGURE 3. Schematic representation of somatostatin receptor immunolocalization in mammalian brain. Color intensity is proportional to the levels of SSTR2A receptor. Same abbreviations as in Fig. 2. (See color plate.)

signal. Medium density of immunoreactive nerve cell bodies and proximal dendrites are encountered throughout the ventrolateral and caudal segments of the neostriatum, within the core and the shell divisions of the nucleus accumbens, and in the dorsolateral part of the septum. Diffuse but intense immunoreactive labeling is observed in the claustrum. Within the hippocampus, pyramidal cells including their basal and apical dendrites of CA1-CA2 fields are intensely stained. A few labeled perikarya are also observed in the stratum radiatum. Diffuse but intense immunoreactive signal is present in the stratum lacunosum moleculare and the subiculum and is also apparent, although less prominently, in the molecular layer of the dentate gyrus. The CA3 subfield remains consistently immunonegative. Both high densities of perikarya and diffuse labeling are detected in the amygdaloid complex, the former in the central and the latter in the basolateral nucleus. Medium densities of immunoreactive nerve cell bodies are also encountered in the medial and cortical nuclei. The endopiriform nucleus displays diffuse but intense immunoreactive labeling.

In the hypothalamus, only sparse immunoreactive perikarya are detected, except in the tuberomammillary nucleus in which they form a tight cluster of moderately immunoreactive cells. Diffuse labeling is apparent in a few nuclei including arcuate and paraventricular nuclei. In the midbrain, low densities of labeled cell bodies with long and fine processes are evident in the deep layers of the superior colliculus as well as in periaqueductal gray matter. Additionally, diffuse labeling is found in the dorsal and lateral segment of periaqueductal gray, gray layers of the superior colliculus, pars compacta of the substantia nigra, and ventral tegmental area. In the pons, intensely labeled perikarya and processes are detected in the locus coeruleus. Labeled cell bodies are also apparent in the lateral dorsal tegmental area and parabrachial nucleus. More caudally, moderate densities of immunopositive cell bodies are visible in the dorsal motor nucleus of the vagus and in the lateral reticular nucleus. Diffuse labeling is found in the nucleus tractus solitarius and medial vestibular nucleus. Cerebellar cortex and deep cerebellar nuclei are devoid of immunostaining. In the

650 / Chapter 91 spinal cord, sst2A immunoreactivity is exclusively found in the dorsal horn. A dense network of immunoreactive processes is present in the substantia gelatinosa (Layer II), while immunoreactive cells are apparent in the border zone between layers I and II. Only occasional cell bodies are stained in layer VI. Double labeling immunohistochemistry for pituitary hormones and the sst2A receptor reveals an almost complete colocalization of the sst2A receptor with GH expressing cells. Only a small percentage of prolactin cells (<10%) bear the sst2A receptor, while a substantial overlap of the receptor is found with gonadotrophs (LH/FSH) (40–50%), corticotrophs (<60%), and thyrotrophs (<30%). Sst3 receptor mRNAs are broadly expressed in the mammalian central nervous system. However, SSTR3 is immunolocalized exclusively in neuronal cilia. Sst4 mRNAs are expressed in a relatively restricted number of rat brain areas, in contrast to the rather broad expression pattern of sst1, sst2, and sst3 receptors. Expression of the sst4 receptor mRNA is greatest in the pyramidal cells of the CA1 region of the hippocampus, lower in other hippocampal regions and the cortex, and absent from the striatum, cerebellum, and hypothalamus. Immunoreactivity is essentially restricted to the cerebral cortex and CA1 of the hippocampus. Sst5 receptor immunoreactivity appears only sparsely present in neuronal perikarya and proximal dendrites exclusively in the rostral part of the brain but the protein is clearly present in the pituitary. MgrX2 is moderately expressed in subsets of CA2-4 neurons in the hippocampus, but it is not detected in the cerebral cortex. SRIF/CST interactions with their cognate receptors in the brain may trigger the recruitment of many intracellular effectors (for review see [3]). The five sst receptors can activate through pertussis toxin-sensitive G proteins such as Gail-3 and Ga, the adenylyl cyclasecAMP-protein kinase A pathway. Many potassium channels (inward rectifier, noninactivating voltage-sensitive, ATP-sensitive, large conductance Ca2+-activated) can be opened, either directly or through activation of the phospholipase C-protein kinase C pathway and phospholipase A2-arachidonic acid pathway. In addition, sst1 receptors can also recruit pertussis toxin-insensitive G proteins and modulate the Na+/H+-exchanger. By these mechanisms, somatostatin can hyperpolarize cell membranes therefore reducing [Ca2+]i. It was also proposed to modulate Ca2+ influx either directly through voltage-sensitive Ca2+ channels (L-, N-, P/Q- types) and Gαo proteins or indirectly through the guanylate cyclase-cGMP-dependent protein kinase pathway. Stimulated sst receptors can also activate tyrosine (PTP)and serine/threonine-phosphatases, some of the latter, PP2A, and calcineurin (PP2B) being involved in synap-

tic plasticity processes. Finally, sst receptors also seem to be able to inhibit the mitogen-activated protein kinase pathway, possibly through activation of PTPs. Such mechanisms may contribute to the monitoring of tumor growth by SRIF but this has not been convincingly demonstrated for brain tumors. All the previously mentioned signal transduction pathways seem to be effectively modulated by the cloned receptors (see Table 1 for further details). However, it appears that multiple sst receptor subtypes can be expressed in single neurons, the best examples being hypothalamic neurons in which somatostatin can either enhance (through an sst1 pertussis-toxin-independent mechanism) or decrease (through an sst2 pertussistoxin-dependent mechanism) AMPA/kainate-receptormediated responses to glutamate, depending on their ability to modulate [Ca2+]i. SRIF receptors can be differentiated by their ability to internalize. With the noticeable exception of sst4, all subtypes do internalize in various cellular models following agonist treatment. In the brain, this has only been shown in vivo for the sst2 receptor. In transfected cells, SRIF receptors have also been reported to homoand heterodimerize as well as interact with PDZ-containing proteins. Such receptor-multidomain protein complexes are likely to intervene in the cell- and tissuespecific modulation of SRIF/CST-mediated signalling processes.

INFORMATION ON ACTIVE AND/OR SOLUTION CONFORMATION SRIF and CST are disulfide-bridged peptides and the receptor pharmacophore lies in the 4amino-acid sequence: Phe-Trp-Lys-Thr, totally conserved among peptides. The lysine and d-tryptophan side chains in cyclic hexapeptide analogs exist in an equatorial orientation relative to the backbone of the peptides. Octreotide ([D]-Phe-c[Cys-Phe-(D)-Trp-LysThr-Cys]-Thr-ol, sandostatin) was the first SRIF analogue in clinical use for the treatment of several endocrine and malignant disorders. It binds predominantly to hsstr2 and hsstr5 and also with low affinity to hsstr3. Octreotide exists in solution in two conformational forms, differing mainly by the conformation of the C-terminal tail. The molecule adopts an overall antiparallel β-sheet conformation, with a type II′, β-turn centered at the (D)-Trp8-Lys9 region. In one conformational form, the residues following this β-turn continue the β-sheet structure. Based on elaborate studies of various somatostatin analogs, this form is thought to contain the active conformation(s). In the second conformational form, the residues following the β-turn adopt a 310 helical conformation.

Brain Somatostatin-Related Peptides / 651 TABLE 1.

Characteristics of human somatostatin receptors.

sst1 (SRIF2A)

sst2 (SRIF1A)

sst3 (SRIF1B)

sst4 SRIF2B

sst5 SRIF2C

Adenylyl cyclase Tyrosine phosphatase MAP kinase K+ channels Ca2+ channels Na+/H+ exchanger PLP C/IP3 pathway PLPA2/AA pathway

− + +

− + − + −

− + +/− +

− + + +

− + − +

+

+

+ +

+/−

Gene location Swiss prot. Amino acids Mol Weight (kDa) Transcript (kb) Glycosylation site Phosphorylation site Tissue distribution

14q13 P30872 391 42.7 4.8 3 6 Brain, Pituitary Islets Stomach Liver Kidneys

17q24 P30874 369/356 41.3 8.5 4 8 Brain, Pituitary Islets Stomach

22q13.1 P32745 418 45.9 5.0 2 5 Brain, Pituitary Islets Stomach

20p11.2 P31391 388 41.9 4.0 1 3 Brain

16p13.3 P35346 364 39.2 4.0 3 5

Subtypes

− + +

Kidneys Adrenals

BIOLOGICAL ACTIONS WITHIN THE BRAIN AND PITUITARY The best characterized action of brain SRIF is the inhibition of pituitary hormone secretion, most notably GH, but also TSH and in some cases ACTH and TSH [1]. Moreover, SRIF is a widely distributed neuromodulator in the brain. Experimental depletion of somatostatin by cysteamine disrupts avoidance learning and decreases locomotor activity, while somatostatin agonists induce the opposite effects. CST shares several functional properties with SRIF, such as the depression of neuronal activity. However, some biological functions of CST are unique—for example, the induction of slow-wave sleep and reduction of locomotor activity. Intracerebro ventricular (i.c.v.) administration of CST-14 induces a twofold increase in deep slow wave sleep (SWS) during both light and dark periods; CST mRNA levels depend on the physiological demand for sleep as they oscillate during the light/dark cycle and are induced fourfold upon total sleep deprivation (but not selective REM sleep), returning to basal levels after eight hours of rebound sleep. Interestingly, and also in contrast to SRIF, CST antagonizes the effects of acetylcholine at the cortical level (system related to alertness) to promote cortical synchrony and to control SWS/wakefulness/ SWS transitions. These data suggest that CST is a spe-

Islets Stomach Liver Adrenals Lung Placenta

Pituitary Islets Stomach Adrenals

cific sleep factor, which accumulates during wakefulness to generate sleep pressure, then is released during sleep. In contrast to SRIF, CST produces a reduction of locomotor activity. Also unlike SRIF, expression of CST mRNA is not upregulated in response to kainic acidinduced seizure activity in vivo. Data on the effects of sst1, sst2, and srif gene deletion on brain activity in mice are summarized in Table 2. A change in basal GH release from somatotroph cells has been reported in sst1 −/− mice, while somatostatin inhibition is reduced in sst2 −/− mice. Sexual dimorphism in GH pulsatile secretion and ACTH/corticosterone secretion no longer occur in srif −/− mice. Subtle changes in motor and cognitive behaviors have been also observed in sst2 −/− mice. Studies also show that sst1, sst2, and srif gene deletion modify kainate-induced seizure sensibility. Finally, sst2 −/− mice appear less sensitive to ischemia. Though the mice have been developed, no report on brain activity is available for sst3 -5 −/− mice at the present time.

PATHOPHYSIOLOGICAL IMPLICATIONS Long-acting sst2 preferring agonists, such as octreotide and lanreotide, are used for the treatment of acromegalic patients, gastrointestinal or pancreatic tumors,

652 / Chapter 91 TABLE 2. Summary of the CNS-related phenotypes observed in the different strains invalidated for the somatostatin receptors or peptide genes. sst1 KO versus Controls

sst2 KO versus Controls

SRIF KO versus Controls

−23

=46,49 +46

−27

Motor coordination and learning Spontaneous motor activity Static rod test Wire hang test Beam-walking test Constant speed rotarod test Accelerating rotarod

=6,46 =6,46 =6,46 −6 =46

=27 =27 =27

Open-field exploratory activity Open-field test

=6, −46

=27

Anxiety-related behaviors Dark-light box test Elevated-plus maze test Forced-swim test Reaction to novelty test

−46 +6, −46 −46 −46

=27

Social interaction Resident-intruder test

=46

Neuroendocrinology Pituitary hormones GH secretion ACTH secretion Behavior

Sensorimotor responses Acoustic startle response Prepulse inhibition Learning and memory Radial maze learning Skinner box paradigm Conditioned fear Seizure susceptibility Neurodegeneration after focal ischemia

=27 −27

=27 =27 +16,46 −16,46 +4

−28 +42

=27 +8

Abbrev.: = no change, − decreased effect/responses , + improved effect/responses. Numbers correspond to references.

and other gastrointestinal disorders. Specifically designed analogs are also used or are in development for tumour imaging and radiotherapy. Not surprisingly, the physiopathological implications for neuropsychiatric diseases are less developed. Nevertheless, changes in SRIF and sst receptors have long been known to be associated with dementia, epilepsy, and major affective disorders [1]. Concerning affective disorders, one of the most consistent neuropeptide alterations in depression has been a state-dependent decrease of CSF-SRIF. This decrease does not appear to be specific to depression, as it has been observed in other patient populations where cognition may be impaired, such as schizophrenia, Alzheimer’s dementia, drug refractory epilepsy, and

active multiple sclerosis. The SRIF decrease in affective illness and multiple sclerosis has been considered state dependent, in that levels normalize with recovery from the acute episode or treatment. In contrast, increased SRIF levels have been reported in psychiatric illness where cognition is accelerated, as in mania, as well as in obsessive-compulsive disorder. Temporal lobe epilepsy (TLE) is characterized by hippocampal sclerosis together with profound phenotypic changes of different classes of interneurons. Evidence from human studies suggests that hilar SRIF interneurons undergo extensive degeneration in patients with hippocampal sclerosis. Therefore SRIF receptors may represent potential therapeutic targets for temporal lobe epilepsy. Indeed, SRIF is released

Brain Somatostatin-Related Peptides / 653 under conditions characteristic of seizures; seizures induce changes in the levels of the peptide; SRIF and its analog affect seizures. However, information regarding the precise contribution of each SRIF receptor on the SRIF-induced inhibition of epileptiform activity is still limited. Although the sst2 receptor is the candidate likely to mediate the anticonvulsant effects of SRIF in the rat hippocampus, in the mouse hippocampus recent observations support a central role of sst4 and/or sst1 receptors in mediating SRIF inhibition of epileptiform activity. Finally, in Alzheimer’s disease cortical SRIF deficiency correlates with the dementia score in frontal cortex and with some indices of severity of the illness. Selective assays indicate that somatostatin- but not cortistatin-related peptides are mainly involved in these deficits. On the other hand, overexpression of the human β-amyloid precursor protein downregulates CST mRNA in PDAPP mice. Pierotti et al. observed that the proportion of pro-SRIF is significantly lower in the temporal cortex of Alzheimer’s patients, and SRIF-28 proportions were significantly elevated. These results suggest that total SRIF deficiency may be related to a reduction in the rate of biosynthesis and/or to a change in the proteolytic processing of the precursor form to yield the bioactive peptides SRIF-28 and SRIF-14 but not through PC2 protein convertase. Monitoring brain SRIF levels may be of interest, since the neuropeptide seems to be the only one out of nearly 50 reagents to increase neprylysin activity, thereby promoting the degradation of the amyloid β peptide Aβ 42, which is considered to be the main pathogenic factor in the disease.

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[7] de Lanerolle NC, Kim JH, Robbins RJ, Spencer DD. Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy. Brain Res. 1989; 495:387–95. [8] de Lecea L, Criado JR, Prospero-Garcia O, Gautvik KM, Schweitzer P, Danielson PE, Dunlop CL, Siggins GR, Henriksen SJ, Sutcliffe JG. A cortical neuropeptide with neuronal depressant and sleep-modulating properties. Nature. 1996;381(6579): 242–5. [9] Dournaud P, Delaere P, Hauw JJ, Epelbaum J. Differential correlation between neurochemical deficits, neuropathology, and cognitive status in Alzheimer’s disease. Neurobiol Aging. 1995;16(5):817–23. [10] Dournaud P, Slama A, Beaudet A, Epelbaum J. Somatostatin receptors in peptide receptors. Handbook of Chemical Neuroanatomy, Vol 16, Part 1. Quirion R, Björklund A, Hökfelt T eds. Elsevier, Boston MA, pp. 1–43, 2000. [11] Dutar P, Vaillend C, Viollet C, Billard JM, Potier B, Carlo AS, Ungerer A, Epelbaum J. Spatial learning and synaptic hippocampal plasticity in type 2 somatostatin receptor knock-out mice. Neuroscience. 2002;112(2):455–66. [12] Epelbaum J. Somatostatin in the central nervous system: Physiology and pathological modifications. Prog Neurobiol. 1986; 27(1):63–100. [13] Falb E, Salitra Y, Yechezkel T, Bracha M, Litman P, Olender R, Rosenfeld R, Senderowitz H, Jiang S, Goodman M. A bicyclic and hsst2 selective somatostatin analogue: design, synthesis, conformational analysis and binding. Bioorg Med Chem. 2001; 9(12):3255–64. [14] Frye MA, Pazzaglia PJ, George MS, Luckenbaugh DA, Vanderham E, Davis CL, Rubinow DR, Post RM. Low CSF somatostatin associated with response to nimodipine in patents with affective illness. Biol Psychiatry. 2003;53(2):180–3. [15] Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, Steiner DF. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci U S A. 1997; 94(13):6646–51. [16] Grouselle D, Winsky-Sommerer R, David JP, Delacourte A, Dournaud P, Epelbaum J. Loss of somatostatin-like immunoreactivity in the frontal cortex of Alzheimer patients carrying the apolipoprotein epsilon 4 allele. Neurosci Lett. 1998;255(1):21–4. [17] Handel M, Schulz S, Stanarius A, Schreff M, ErdtmannVourliotis M, Schmidt H, Wolf G, Hollt V. Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience. 1999; 89(3):909–26. [18] Hannon JP, Bruns C, Wesbecker G, Hoyer D. Somatostatin receptor gene family-subtype selectivity for ligand binding. In Somatostatin. Srikant CB ed. Kluwers Academic Publishers, Norwell MA. pp. 81–106, 2004. [19] Kreienkamp HJ, Akgun E, Baumeister H, Meyerhof W, Richter D. Somatostatin receptor subtype 1 modulates basal inhibition of growth hormone release in somatotrophs. FEBS Lett. 1999;462(3):464–6. [20] Kreienkamp HJ, Liew CW, Bächner D, Mamezza MG, Soltau M, Quitsch A, Christenn M, Wente W, Richter D. Physiology of somatostatin receptors: from genetics to molecular analysis. In Somatostatin. Srikant CB ed. Kluwers Academic Publishers, Norwell MA. pp. 185–202, 2004. [21] Larsen PJ, Seier V, Fink-Jensen A, Holst JJ, Warberg J, Vrang N. Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit. J Neuroendocrinol. 2003;15(3):219–26. [22] Leake A, Ferrier IN. Alterations in neuropeptides in aging and disease. Pathophysiology and potential for clinical intervention. Drugs Aging. 1993;3(5):408–27.

654 / Chapter 91 [23] Low MJ, Otero-Corchon V, Parlow AF, Ramirez JL, Kumar U, Patel YC, Rubinstein M. Somatostatin is required for masculinization of growth hormone-regulated hepatic gene expression but not of somatic growth. J Clin Invest. 2001;107(12): 1571–80. [24] Moneta D, Richichi C, Aliprandi M, Dournaud P, Dutar P, Billard JM, Carlo AS, Viollet C, Hannon JP, Fehlmann D, Nunn C, Hoyer D, Epelbaum J, Vezzani A. Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice. Eur J Neurosci. 2002;16(5): 843–9. [25] Olias G, Viollet C, Kusserow H, Epelbaum J, Meyerhof W. Regulation and function of somatostatin receptors. J Neurochem. 2004;89(5):1057–91. [26] Patel YC. Somatostatin and its receptor family. Front Neuroendocrinol. 1999;20(3):157–98. [27] Peineau S, Potier B, Petit F, Dournaud P, Epelbaum J, Gardette R. AMPA-sst2 somatostatin receptor interaction in rat hypothalamus requires activation of NMDA and/or metabotropic glutamate receptors and depends on intracellular calcium. J Physiol. 2003;546(Pt 1):101–17. [28] Pierotti AR, Harmar AJ, Simpson J, Yates CM. High-molecularweight forms of somatostatin are reduced in Alzheimer’s disease and Down’s syndrome. Neurosci Lett. 1986 Jan 16;63(2):141– 6. [29] Piqueras L, Martinez V. Role of somatostatin receptors on gastric acid secretion in wild-type and somatostatin receptor type 2 knockout mice. Naunyn Schmiedebergs Arch Pharmacol. 2004 Dec;370(6):510–20. [30] Pradayrol L, Jornvall H, Mutt V, Ribet A. N-terminally extended somatostatin: the primary structure of somatostatin-28. FEBS Lett. 1980;109(1):55–8. [31] Reichlin S. Somatostatin. N Engl J Med. 1983;309(24):1495– 501. [32] Reichlin S. Somatostatin (second of two parts). N Engl J Med. 1983;309(25):1556–63. [33] Robas N, Mead E, Fidock M. MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. J Biol Chem. 2003;278(45):44400–4. [34] Robbins RJ, Brines ML, Kim JH, Adrian T, de Lanerolle N, Welsh S, Spencer DD. A selective loss of somatostatin in the hippocampus of patients with temporal lobe epilepsy. Ann Neurol 1991;29:325–332. [35] Saito T, Iwata N, Tsubuki S, Takaki Y, Takano J, Huang SM, Suemoto T, Higuchi M, Saido TC. Somatostatin regulates brain amyloid beta peptide Abeta(42) through modulation of proteolytic degradation. Nat Med. 2005;11(4):434–9. [36] Schreff M, Schulz S, Handel M, Keilhoff G, Braun H, Pereira G, Klutzny M, Schmidt H, Wolf G, Hollt V. Distribution, targeting, and internalization of the sst4 somatostatin receptor in rat brain. J Neurosci. 2000;20(10):3785–97. [37] Spier AD, de Lecea L. Cortistatin: a member of the somatostatin neuropeptide family with distinct physiological functions. Brain Res Brain Res Rev. 2000;33(2–3):228–41.

[38] Stroh T, Kreienkamp HJ, Beaudet A. Immunohistochemical distribution of the somatostatin receptor subtype 5 in the adult rat brain: predominant expression in the basal forebrain. J Comp Neurol. 1999;412(1):69–82. [39] Strowski MZ, Kohler M, Chen HY, Trumbauer ME, Li Z, Szalkowski D, Gopal-Truter S, Fisher JK, Schaeffer JM, Blake AD, Zhang BB, Wilkinson HA. Somatostatin receptor subtype 5 regulates insulin secretion and glucose homeostasis. Mol Endocrinol. 2003 Jan;17(1):93 –106. [40] Strowski MZ, Dashkevicz MP, Parmar RM, Wilkinson H, Kohler M, Schaeffer JM, Blake AD. Somatostatin receptor subtypes 2 and 5 inhibit corticotropin-releasing hormone-stimulated adrenocorticotropin secretion from AtT-20 cells. Neuroendocrinology. 2002 Jun;75(6):339–46. [41] Stumm RK, Zhou C, Schulz S, Endres M, Kronenberg G, Allen JP, Tulipano G, Hollt V. Somatostatin receptor 2 is activated in cortical neurons and contributes to neurodegeneration after focal ischemia. J Neurosci. 2004;24(50):11404–15. [42] Tostivint H, Trabucchi M, Vallarino M, Conlon JM, Lihrmann I, Vaudry H. Molecular evolution of somatostatin genes. In Somatostatin. Srikant CB ed. Kluwers Academic Publishers, Norwell MA. pp. 47–64, 2004. [43] Vale W, Brazeau P, Grant G, Nussey A, Burgus R, Rivier J, Ling N, Guillemin R. [Preliminary observations on the mechanism of action of somatostatin, a hypothalamic factor inhibiting the secretion of growth hormone.] C R Acad Sci Hebd Seances Acad Sci D. 1972;275(25):2913–6. [44] Vallejo M. Somatostatin gene structure and regulation. In Somatostatin. Srikant CB ed. Kluwers Academic Publishers, Norwell MA. pp. 1–16, 2004. [45] Vezzani A, Hoyer D. Brain somatostatin: a candidate inhibitory role in seizures and epileptogenesis. Eur J Neurosci. 1999; 11(11):3767–76. [46] Viollet C, Vaillend C, Videau C, Bluet-Pajot MT, Ungerer A, L’Heritier A, Kopp C, Potier B, Billard J, Schaeffer J, Smith RG, Rohrer SP, Wilkinson H, Zheng H, Epelbaum J. Involvement of sst2 somatostatin receptor in locomotor, exploratory activity and emotional reactivity in mice. Eur J Neurosci. 2000;12(10): 3761–70. [47] Winsky-Sommerer R, Grouselle D, Rougeot C, Laurent V, David JP, Delacourte A, Dournaud P, Seidah NG, Lindberg I, Trottier S, Epelbaum J. The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer’s disease. Neuroscience. 2003;122(2):437–47. [48] Winsky-Sommerer R, Spier AD, Fabre V, de Lecea L, Criado JR. Overexpression of the human beta-amyloid precursor protein downregulates cortistatin mRNA in PDAPP mice. Brain Res. 2004;1023(1):157–62. [49] Zheng H, Bailey A, Jiang MH, Honda K, Chen HY, Trumbauer ME, Van der Ploeg LH, Schaeffer JM, Leng G, Smith RG. Somatostatin receptor subtype 2 knockout mice are refractory to growth hormone-negative feedback on arcuate neurons. Mol Endocrinol. 1997;11(11):1709–17.

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92 Corticotrophin-Releasing Hormone (CRH) Peptide Family DAVID A. LOVEJOY

ABSTRACT

that the structure of “the long sought after corticotrophin-releasing factor” may be similar to sauvagine [9]. About the same time, on the other side of the Atlantic, Lederis and his colleagues [15] had isolated and characterized a 41-amino-acid peptide from the caudal neurosecretory system (urophysis) of carp (Cyprinus carpio) called urotensin-I. The structure of CRH was reported by Vale and his associates in 1981 [30] after extracting CRH from 100,000 sheep hypothalami. The triple discoveries of CRH, urotensin-I, and sauvagine within 2–3 years immediately led to speculation as to the evolutionary and physiological reasons for the presence of three similar peptides in different regions of the body. At this time the concept of molecular paralogy and orthology was poorly understood. Over the next 10 years, the discovery of CRH in fishes and amphibians indicated that urotensin-I and sauvagine were likely homologs of CRH [19]. The rat and human orthologs of urotensin-I were cloned and characterized by the Vale and Sawchenko laboratories and were termed urocortin [7, 31]. Urocortin, like sauvagine, was 40 amino acids long, in contrast to the 41-residue sequence that characterized CRH and urotensin-I (Fig. 1). This reason, along with the uncertainty surrounding the phylogenetic relationship of sauvagine, played a role in the peptide being called urocortin instead of urotensin-I. Later, the sequencing of urocortins and urotensin-I in other vertebrates [3, 19] helped establish the orthology of the urotensin I and urocortins. The mRNA encoding sauvagine was eventually cloned from P. sauvageii in 2005 (Fig. 1), and it appeared to be a highly derived urocortin sequence. Neuroanatomical observations [4] and phylogenetic studies [19] suggested that as many as four CRH/urotensin-I-like peptides may be found in chordates. Then

The corticotrophin-releasing hormone (CRH) family of peptides consists of four distinct paralogs including CRH, urocortin (Ucn)/urotensin-I, urocortin 2 (Ucn2), and urocortin 3 (Ucn3). The peptides range between 38 and 41 amino acids in length and exist predominantly as amphiphilic alpha helices. Each is located on separate chromosomes and all possess a similar gene structure consisting of two exons and an intron of a variable length. CRH is responsible for the hypothalamic activation of the hypothalamic-pituitary adrenal axis but all paralogs appear to play roles in the regulation of stress homeostasis. The CRH family of peptides has been linked pathologically to affective and neurodegenerative disorders.

DISCOVERY The quest for the hypothalamic releasing factor ultimately responsible for the adrenal glucocorticoid release began almost immediately after Harris and Green’s landmark studies that finally cemented the foundation for hypothalamic-pituitary interaction [12, 18]. The first evidence of a hypothalamic corticotrophinreleasing substance was reported in 1955 [11, 28], but the isolation of this substance was thwarted by its low concentrations and hydrophobic nature. Ironically, the first family member of the CRH family was found not in the hypothalamus but rather in the skin of the neotropical monkey frog, Phyllomedusa sauvageii by Erspamer and his laboratory [22]. This peptide, called sauvagine, consisted of 40 amino acids and proved to be effective at releasing ACTH from rat pituitary cells (Fig. 1). Writing later in 1981, Erspamer speculated Handbook of Biologically Active Peptides

655

Copyright © 2006 Elsevier

656 / Chapter 92 Corticotropin Releasing Hormone

human Xenopus zebrafish trout

(NM000756) (S50096) (BC085458) (AF296672)

SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-NH2 AEEPPISLDLTFHLLREVLEMARAEQIAQQAHSNRKLMDII-NH2 SEEPPISLDLTFHLLREVLEMARAEQMAQQAHSNRKMMEIF-NH2 SDDPPISLDLTFHMLRQMMEMSRAEQLQQQAHSNRKMMEIF-NH2

Urocortin/Urotensin-I

human Xenopus P. sauvageii dogfish zebrafish trout

(NM003353) (AY596827) (AY943910) (AAB47080) (CAH68941) (AJ005264)

.DNPSLSIDLTFHLLRTLLELARTQSQRERAEQNRIIFDSV-NH2 .EDPPISIDLTFHILRQMIEIAKTQNQKQQAEQNRIIFDSV-NH2 .QGPPISIDLSLELLRKMIEIEKQEKEKQQAANNRLLLDTI-NH2 PAETPNSLDLTFHLLREMIEIAKHENQQMQADSNRRIMDTI-NH2 NDDPPISIDLTFHLLRNMIEMARIENQREQAELNRKYLDEV-NH2 NDDPPISIDLTFHLLRNMIEMARIESQKEQAELNRKYLDEV-NH2

Urocortin 2

human chicken pufferfish

(BC022096) (XP425157) (see legend)

...IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV-NH2 ...VSLSLDVPTHILRILLDLAREKELQAKAAANAELMARI-NH2 ...FALSLDVPTSILSVLIDLAKNQDMRSKAAANAELMARI-NH2

Urocortin 3

human Xenopus pufferfish

(NM053049) (AAT70727) (CAB96535)

...FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2 ...FTLSLDVPTNLMNILFDIAKAKNIRAKAAANAQLMAQI-NH2 ...LTLSLDVPTNIMNVLFDVAKAKNLRAKAAENARLLAHI-NH2

FIGURE 1. Primary sequence of CRH superfamily orthologues and paralogs. Light gray regions indicate amino acid identity within each set of orthologous peptides. Dark gray regions indicate amino acids that have been conserved in all orthologs and paralogs. The accession number is given for each sequence. The pufferfish sequence was obtained as a predicted translated protein product from the pufferfish genome database (accession number: lcl SINFRUP00000059907).

in 2001, the presence of two additional CRH-related peptides were reported by the Vale and Sawchenko laboratories who called the peptides urocortin 2 (Ucn2) and urocortin 3 (Ucn3) [17, 26] and independently by Hsu and Hseuh [13], who named the peptides stresscopin and stresscopin-related peptide on the basis of their anxiolytic effects (Fig. 1). The apparent sequence dissimilarity of Ucns 2 and 3 to CRH and Ucn/urotensin-I suggested, however, that the former were not urocortins, per se, but rather the product of a gene duplication early in CRH ancestry. This is consistent with the closer sequence similarity of the CRH/urotensin-I lineage to the insect diuretic hormones and the Ucn 2 and 3 sequences resembling a recently discovered insect peptide diuretic hormone-31 (DH31) [18]. Further genomic analysis has not supported the existence of additional CRH peptides, although a recently discovered family of neuropeptides, the teneurin C-terminalassociated peptides (TCAP), have about a 20% identity to the CRH family of peptides [32]. However, the evolutionary relationship of the TCAPs to the CRH family is uncertain.

STRUCTURE OF THE PRECURSOR mRNA/GENE The gene structure of the CRHs and the urocortins support a gene duplication early in chordate ancestry. The basic structures of the CRH and urocortin genes

are similar but are located on chromosomes 8q13 and 2p23-p21, respectively, in humans and chromosomes 3 and 13 in mice. There are two exons of unequal sizes where the first encodes for a portion of the 5′ untranslated region (UTR) and exon 2, the remainder of the 5′ UTR, entire preprohormone sequence, and the entire 3′ UTR. The intron length varies considerably among species, although the urocortin intron is much smaller compared with that of CRH (Fig. 2). Whereas the CRH cDNA structure remains consistent among orthologs, there is considerable variability among the urocortins and urotensins-I. The urotensins-I mRNA is 3 to 5 times longer in fishes than the mammal urocortin mRNA. This length difference is due to a much shorter gene-associated peptide in the Ucns and extended 3′ UTR in fish urotensin-I, particularly in rainbow trout (Oncorhynchus mykiss). In this species, multiple polyadenylation sites correspond to the differential mRNA processing that occurs in the urophysis and brain [3]. The gene structure of the Ucns 2 and 3 also reflect a structural relationship to each other, although these genes have undergone considerable change. Both Ucn2 and 3 genes possess two exons similar to the arrangement for urocortin and CRH (Fig. 2). Ucn 2 is found on human chromosome 3p21.3 and on mouse chromosome 9. In both cases, the start of the urocortin gene is only a few hundred bases from the terminal exon of type VII α1 collagen. In the chicken Gallus gallus (Fig. 1) the Ucn2 gene appears to have

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FIGURE 2. Gene and cDNA structure of CRH family members. For each paralog, a schematic of the gene structure (not to scale) is shown along with its position on the relevant human chromosome. Boxes represent exons and lines indicate intronic regions. The number of bases of the 5′ and 3′ untranslated regions, and introns, are shown above the relevant regions. The translated region of the gene is indicated by the human preprohormone sequence corresponding to the light gray box. The mature peptide is shown bracketed by the cleavage and amidation motifs shown in clear boxes.

been assimilated into the chicken ortholog of type VII α1 collagen and is present as the terminal exon of this gene. It is not clear if the chicken Ucn2 peptide is cleaved from the collagen protein and has an independent function. Ucn3 differs from the other CRH

family members by having an extended intron of over 8000 bases. Moreover, the position of the 3′ UTR of the Ucn3 mRNA is unclear. The gene is found on human chromosome 10p15.1 and on mouse chromosome 5.

658 / Chapter 92 DISTRIBUTION AND EXPRESSION OF mRNA In mammals, the regulation of pituitary ACTH by CRH occurs primarily via projections from the parvocellular division of the paraventricular nucleus in the hypothalamus to the fenestrated capillaries of the median eminence. Its role as a hypothalamic releasing hormone for ACTH has been conserved in both sarcopterygian and actinopterygian lineages. Indeed, its presence in the nucleus preopticus and nucleus lateralis tuberalis of elasmobranches indicates that its role as a hypothalamic releasing factor evolved early in vertebrate ancestry [20, 27]. In many teleost species, CRH is also synthesized in the nucleus preopticus and is transported along neurosecretory processes to the neurohypophysis, where it is released near the pituitary corticotropes. However, CRH is expressed widely within the brain of all vertebrates, where it plays neuromodulator and neurotransmitter roles. CRH mRNA expression occurs predominantly in rostral regions of the brain. In the rat forebrain, there are clusters of expression in the bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), septal regions, olfactory lobe, and olfactory tract as well as in isolated expression throughout the cortex. In caudal regions, the adrenergic cell groups, A1 and A5, and the parabranchial nucleus are sites of CRH expression (Fig. 3) [4, 27]. It is difficult to determine how homologous sites of CRH expression in fishes are to mammals, as few in situ hybridization studies in the former have been performed (see Takahashi and Totsune, this volume). Immunohistochemical studies suggest that the general pattern of CRH expression in the rostral regions is essentially retained, although the topology of the teleost forebrain differs considerably from that of mammals. For example, in the tilapia (Oreochromis mossambicus), the greatest concentration of immunoreactive CRH occurs in the lateral part of the ventral telencephalon, where it may be released into the systemic circulation [24]. Many fish species show reduced CRH immunoreactivity in caudal regions. Amphibians have caudal CRH expression patterns similar to those found in mammals. Thus, CRH expression patterns may have evolved differently among actinopterygian and sarcopterygian lineages [20]. In comparison with CRH, Ucn has a much more caudal distribution. The primary site for Ucn mRNA expression in mammals is the Edinger-Westphal nucleus although there are small regions of Ucn expression in the lateral hypothalamic area, and the supraoptic nucleus (SON) of the hypothalamus [31]. Ucn is also expressed in a number of brain stem sites (Fig. 3). Urotensin-I, the piscine ortholog of Ucn is expressed in the nucleus of the medial longitudinal fasciculus in fishes. However, the majority of urotensin-I expression

in fishes actually occurs in the urophysis. In phylogenetically older vertebrates and actinopterygians in general, urotensin-I expression is predominant in the spinal cord and brain stem sites where CRH appears to be not present [20]. In amniotes, however, CRH expression is associated with nuclei that contribute descending autonomic fibers. Ucns 2 and 3 have a restricted pattern of expression in comparison with CRH and urocortin (Fig. 3). In the rat hypothalamus, Ucn2 mRNA has been detected in the PVN, SON, and arcuate nucleus (Arc). In the brain stem, Ucn2 mRNA is present in the locus coeruleus (LC) and in the trigeminal (V), facial (VII), and hypoglossal (VII) motor nuclei [17]. The main region of Ucn3 expression occurs as clusters of cells associated with the columns of the fornix and throughout the rostral hypothalamus. Regions of expression are also detected in the posterior aspects of the BNST and lateral hypothalamic regions, lateral to the PVN, and in dorsal and lateral regions around the dorsomedial hypothalamic nucleus. A few Ucn3-expressing cells have also been detected in the anterodorsal part of the medial amygdaloid nucleus and near the anterior periventricular nucleus and retrochiasmatic area. In the brain stem, the superior paraolivary nucleus is one of the few sites of Ucn3 expression [26].

PROCESSING OF PREPROHORMONE In all CRH paralogs, the prohormone consists of a signal peptide, a gene-associated peptide, and a mature peptide (Fig. 2). The comparative simplicity of the preprohormone and conservation of the general structure among paralogs suggest that the peptide is processed in a similar manner regardless where the gene is expressed. All CRH paralogs generally possess a welldefined signal peptide with a basic residue within a few residues from the initial methionine. Human Ucn2 lacks this basic residue, however. The signal peptides consist of fewer than 30 residues with a strong hydrophobic core. The gene-associated peptide, or cryptic peptide, spanning the region between the signal peptide and the mature peptide is highly variable where there is little consensus among paralogs. CRH and Ucn3 possess the longest gene-associated peptides, and Ucn and Ucn2, the shortest. There may be a relationship between the length of the gene-associated peptide and the physiological constraints on the peptide as CRH and Ucn3 are the most highly conserved whereas the Ucn and Ucn2 paralogs are the least conserved among vertebrates. The mature peptide is flanked by prohormone convertase (PC) cleavage sites (Fig. 2). The organization of the PC cleavage sites vary between the CRH/urocortin

Corticotrophin-Releasing Hormone (CRH) Peptide Family / 659

FIGURE 3. Neuroanatomical localization of CRH family member mRNA in rat brain. Regions of immunoreactivity not confirmed by in situ hybridization histochemistry are not shown. See text for further description. Abbreviations: A1, A5, adrenergic cell groups; Acb, nucleus accumbens; Amb, nucleus ambiguous; Amy, amygdala; AP, anterior pituitary; Arc, arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; Cput, caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; EW, Edinger-Westphal nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of the pituitary; LC, locus coeruleus; LRN, lateral reticular nucleus; LSO, lateral superior olive; ME, median eminence; NL, neural lobe of the pituitary; NST, nucleus of the solitary tract; OB, olfactory bulb; OT, olfactory tract; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular nucleus of the hypothalamus; S, septum; SN, substantia nigra; SON, supraoptic nucleus; Th, thalamus; V, trigeminal motor nucleus; VII, facial motor nucleus; VMH, ventromedial nucleus of the hypothalamus; XII, hypoglossal motor nucleus.

660 / Chapter 92 lineage, and the urocortin 2/3 lineage. CRH and urocortin possess an R-X2-R motif, consistent with recognized PC cleavage motifs of R/K-Xn-R/K where X is any amino acid and typically varies between 2, 4, and 6 residues [29]. Human Ucn2 has the motif R-X4-R, but in mouse and rat there are 7 residues between the two basic residues. The number of residues between the basic residues also varies among species at the Ucn3 cleavage. In most, if not all, cases, the mature peptide is amidated. Human Ucn2 has a peculiar motif of GHC instead of the usual G-R/K-R/K, so it is not clear whether this version of the peptide is amidated, or if it exists as a larger disulfide-bridged cyclic polypeptide [26]. All peptides are amphiphilic and exist predominantly as α helices.

RECEPTORS AND BINDING PROTEINS The first human CRH receptor (CRH1) was isolated by expression cloning of cDNA from a human Cushing’s corticotropic adenoma [5]. This discovery followed the characterization of receptor orthologs in numerous tetrapod and actinopterygian species. The CRH1 receptor also exists in a series of splice variants including a frameshift mutation in the rat, a 29-aminoacid insertion in the human form, a 133 bp deletion in the sheep isoform, and a variant possessing a deletion in the seventh transmembrane domain expressed in human myometrium. A paralogous CRH receptor system (CRH2), discovered shortly after that of the CRH1, exists in at least three different functional splice variants although there are no major pharmacological differences among them [5]. Both CRH1 and CRH2 receptors belong to a family of G-protein-coupled receptors and are structurally related to the calcitonin and the CRH-like insect diuretic hormone receptors [10]. In the brain, stimulatory coupling the CRH receptors to adenylate cyclase appears to be the primary signal transduction system, although a number of other pathways have been associated with CRH signaling systems in nonneural tissues and cells [5]. The CRH1 receptor is predominant in the brain, whereas the CRH2 receptor is more prevalent in peripheral tissues. The mammal CRH1 receptor binds CRH and urocortin with similar affinity but shows little affinity for urocortins 2 and 3. However, there are a number of species differences in the relative affinities among the various CRH paralogs. In addition to the receptors, a high-affinity and selective soluble CRH binding protein is present in the brain and pituitary gland [25]. The function of the CRHbinding protein is not entirely understood. It shows the greatest affinity for urocortin, less affinity for CRH, and little if any affinity for Ucns 2 and 3. This protein may

act as a sequestering mechanism to attenuate abrupt changes in CRH release, to reduce the CRH/ urocortin response after the peptide release, or it may provide ligand specificity in loci where multiple CRH family peptides are released. The basic structure of the CRH binding protein has evolved early in metazoan history as evidenced by the recent cloning of a honeybee (Apis mellifera) CRH-binding protein ortholog [13].

BIOLOGICAL ACTIONS IN THE CENTRAL NERVOUS SYSTEM Generally in rats, the CRH-related peptides elicit behaviors that have elements resembling the behavioral response to stress and environmental novelty: decreased food intake, rearing, and increased grooming. This is achieved by activating both the hypothalamic-pituitaryadrenal and sympathetic branches of the stress response. However, this aspect of CRH physiology appears to be mediated by the CRH1 receptors. In a number of studies, the CRH2 receptor has been associated with anxiolytic effects; thus, Ucns 2 and 3 have been deemed anxiolytic peptides as they appear to bind exclusively to this receptor. CRH and Ucn are generally associated with anxiogenic actions [5]. Currently, it is unclear if all four CRH paralogs act in an integrated manner to regulate stress-associated homeostasis. CRH family peptides have well-described effects on feeding and weight regulation in both mammals and fishes. In rats, Ucn is the most potent peptide at decreasing food intake [5, 20]. In fishes, food intake is also reduced significantly after CRH administration [6]. A recent report indicating a role for the CRH-like diuretic hormones in insect feeding suggests that this mechanism may be phylogenetically quite ancient [2]. The overexpression of the CRH-BP in a mouse also affects body weight [19], presumably by sequestering free CRH, thereby reducing its receptor activation ability in circuits associated with food intake. A number of other neurological actions of CRH family peptides have been described. For example, CRH-related peptides administered centrally can elicit dose-dependent hypothermia in rats [16]. In addition, a CRH-mediated role on locomotor activity was established shortly after the peptides’ discovery and appears to be conserved among vertebrates [8]. In the brain stem of the roughskin newt (Taricha granulosa), discharges of neurons of the raphe region of the midbrain, associated with walking and swimming behaviors, could be modified by injections of CRH into the lateral ventricle [21]. Other actions induced by CRH peptides include vocalization changes, modulation of the reproductive axis, and modulation of circadian-induced locomotor behaviors [5, 8, 20].

Corticotrophin-Releasing Hormone (CRH) Peptide Family / 661

PATHOPHYSIOLOGICAL IMPLICATIONS CRH is a critical mediator of fear conditioning and other forms of emotional memory to both aversive and rewarding stimuli. In humans, the most recent investigations of the etiology of depression and mood disorders implicate a central role for CRH. Over-activation of the hypothalamic-pituitary-adrenal axis occurs in about half of all of patients presenting with clinical depression [23]. The CRH-secreting brain cells receive their primary positive feedback from the amygdala and negative feedback from the hippocampus. The hippocampus and neocortex mediate the cognitive aspects of depression such as memory impairment and feelings of worthlessness, hopelessness, guilt, and suicidal feelings. A number of postmortem studies of suicidal victims have shown increased levels of CRH and cell numbers in the hypothalamus [1]. Generally, injection of CRH into the brain results in decreased appetite, increased heart rate and blood pressure, and an increased incidence of behaviors associated with anxiety. Recent studies implicate a role for the CRH family peptides in neural development and plasticity and thus may be involved in the onset of some neurodegenerative disorders such as Alzheimer’s disease [5, 8, 16, 23].

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18] [19]

References [1] Arato M, Banki CM, Bissette G, Nemeroff CB. Elevated CSF CRF in suicide victims. Biol Psychiatry 1989;25:355–359. [2] Audsley N, Goldsworthy GJ, Coast GM. Circulating levels of locust diuretic hormone: The effect of feeding. Peptides 1997;18:59–65. [3] Barsyte D, Tipping DR, Smart D, Conlon JM, Baker BI, Lovejoy DA. Rainbow trout (Oncorhynchus mykiss) urotensin-I: Structural differences between urotensins-I and urocortins. Gen Comp Endocrinol 1999;115:169–177. [4] Bittencourt JC, Sawchenko PE. Do centrally administered neuropeptides access cognate receptors?: An analysis in the central corticotrophin-releasing factor system. J Neurosci 2000;20:1142– 1156. [5] Dautzenberg FM, Hauger RL. The CRF peptide family and their receptors: Yet more partners discovered. Trends Pharm Sci 2002;23:71–77. [6] De Pedro N, Pinillos ML, Valenciano AI, Alonzo-Bedate M, Delgado MJ. Inhibitory effect of serotonin on feeding behaviour in goldfish: Involvement of CRF. Peptides 1998;19:505–511. [7] Donaldson CJ, Sutton SW, Perrin MP, Corrigan AZ, Lewis KA, Rivier JE, Vaughan JM, Vale WW. Cloning and characterization of human urocortin. Endocrinology 1996;137:2167–2170. [8] Dunn AJ, Berridge CW. Physiological and behavioral responses to corticotrophin-releasing factor administration: Is CRF a mediator of anxiety or stress responses? Brain Res Rev 1990;15:71– 100. [9] Erspamer V, Melchiorri P, Broccardo M, Erspamer GF, Falaschi P, Improta G, Negri L, Renda T. The brain-gut-skin triangle: New peptides. Peptides 1981;2, Suppl. 2:7–16. [10] Fredricksson R, Lagerstrom L, Lundin LG, Schioth H. The G protein coupled receptors in the human genome form five

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main families. Phylogenetic analysis, paralogon groups and fingerprints. Mol Pharmacol 2003;63:1256–1272. Guillemin R, Rosenberg B. Humoral hypothalamic control of anterior pituitary: A study with combined tissue cultures. Endocrinology 1955;57:599–607. Harris GH. Neural control of the pituitary gland. Physiological Rev 1948;28:139–179. Hsu SY, Hseuh AJW. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotrophinreleasing hormone receptor. Nature Medicine 2001;7:605–611. Huising MO, Flik G. The remarkable conservation of corticotrophin-releasing hormone (CRH)-binding protein in the honeybee (Apis mellifera) dates the CRH system to a common ancestor of insects and vertebrates. Endocrinology 2000;146: 2165–2170. Lederis K, Letter A, McMaster D, Ichikawa T, MacCannell KL, Rivier J, Rivier C, Vale W, Fryer J. Isolation, analysis of structure, synthesis, and biological actions of urotensin I neuropeptides. Can J Biochem Cell Biol 1983;61:602–614. LeFeuvre RA, Rothwell NJ, White A. A comparison of the thermogenic effects of CRF, sauvagine and urotensin-I in the rat. Horm Metab Res 1989;21:525–526. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, Bilezikjian L, Sawchenko PE, Vale WW. Identification of urocortin III, an additional member of the corticotrophin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001;98:7570–7575. Lovejoy DA. Neuroendocrinology: An Integrated Approach. John Wiley and Sons. 2005. Lovejoy DA, Aubry J-M, Turnbull A, Sutton S, Potter E, Yehling J, Rivier C, Vale WW. Ectopic expression of the CRFbinding protein: Minor impact on HPA axis regulation but induction of sexually dimorphic weight gain. J Neuroendocrinol 1998. Lovejoy DA, Balment RJ. Evolution and physiology of the corticotrophin-releasing factor (CRF) family of neuropeptides in vertebrates. Gen Comp Endocrinol 1999;155:1–22. Lowry CA, Rose JD, Moore FL. Corticotrophin-releasing hormone factor enhances locomotion and medullary neuronal firing in an amphibian. Horm Behav 1996;30:50–59. Montecucchi PC, Henschen A, Erspamer V. Structure of sauvagine, a vasoactive peptide from the skin of a frog. Hoppe-Seyler’s Z Physiol Chem 1979;360:1178. Nemeroff CB, Owens MJ. Treatment of mood disorders. Nature Neurosci Suppl 2002;1068–1070. Pepels PPLM, Meek J, Wendelaar Bonga SE, Balm PHM. Distribution and quantification of the corticotrophin-releasing hormone (CRH) in the brain of the teleost fish Oreochomis mossambicus (tilapia). J Comp Neurol 2002;453:247–268. Potter E, Behan DP, Fischer WH, Linton EA, Lowry PJ, Vale WW. Cloning and characterization of the cDNAs for human and rat corticotrophin-releasing factor-binding proteins. Nature 1991;349:423–426. Qian X, Barsyte-Lovejoy D, Wang L, Chewpoy B, Gautam N, Al Chawaf A, Lovejoy DA. Cloning and characterization of teneurin C-terminus associated peptide (TCAP)-3 from the hypothalamus of an adult rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 2004;137:205–216. Reyes TM, Lewis K, Vale WW. Urocortin II: A member of the corticotrophin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001;98:2843–2848. Sawchenko PE, Swanson LW. Localization, colocalization, and plasticity of corticotrophin-releasing factor immunoreactivity in rat brain. Fed Proc 1985;44:221–227.

662 / Chapter 92 [29] Schally AV, Saffran M. The release of corticotrophin by anterior pituitary tissue in vitro. Can J Physiol Pharmacol 1955;33: 408–415. [30] Seidah NG, Chretien M. Proprotein and prohormone convertases: A family of subtilases gene diverse bioactive polypeptides. Brain Res 1999;848:45–62. [31] Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotrophin and b-endorphin. Science 1981;213:1394– 1397.

[32] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull A, Lovejoy D, Rivier C, Rivier J, Sawchenko PE, Vale W. Urocortin, a mammalian neuropeptide related to fish urotensin-I and to corticotrophin-releasing factor. Nature 1995;378:287–292. [33] Wang L, Rotzinger S, Al Chawaf A, Elias CF, Barsyte-Lovejoy D, Qian X, Wang N-C, De Cristofaro A, Belsham D, Bittencourt JC, Vaccarino F, Lovejoy DA. Teneurin proteins possess a carboxy terminal sequence with neuromodulatory activity. Mol Brain Res 2005;133:253–265.

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93 Growth Hormone-Releasing Hormone M. M. MALAGÓN, R. VÁZQUEZ-MARTÍNEZ, A. J. MARTÍNEZ-FUENTES, F. GRACIA-NAVARRO, AND J. P. CASTAÑO

ABSTRACT

chordate [45, 57]. GHRH is a member of a growing superfamily of brain-gut peptides, which also includes, among others, pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon, secretin, and vasoactive intestinal polypeptide (VIP) discussed in this and other sections [45, 57].

Growth hormone-releasing hormone (GHRH), a 44amino-acid peptide primarily secreted by hypothalamic neurons of the arcuate nucleus, is the main regulator of pituitary somatotrophs. GHRH binds to its G-proteincoupled receptor (GHRH-R) to activate diverse signaling pathways predominantly involving cAMP and Ca2+ and thereby stimulates GH secretion, GH gene transcription, and somatotroph proliferation. These actions, coupled to hypothalamic GHRH interactions with somatostatin- and ghrelin-producing neurons, underlie pulsatile GH secretion. GHRH production is markedly dependent upon age, sex, and metabolic status and is also involved in sleep and feeding regulation. Defects in GHRH/GHRH-R severely alter somatotroph function and GH production, thereby causing abnormal growth.

STRUCTURE OF THE PRECURSOR mRNA/GENE The human pancreatic tumor samples that enabled isolation of GHRH also provided the first cDNA sequences, whose cloning and characterization revealed that GHRH mRNA codes for a pre-pro-GHRH of 108 amino acids composed of a typical signal peptide, the 44-amino-acid GHRH, and a 31-amino-acid C-terminal peptide (Fig. 1) (reviewed by [23, 43]). A second isoform coding for a 107-amino-acid precursor lacking the Ser-103 in the C-peptide was also found. Subsequently, the cDNA and/or gene was isolated and characterized in rat, mouse, and other mammals, as well as in birds, amphibians, and fish (reviewed by [45, 57]). In nonmammalian species, GHRH is encoded together with PACAP by a common precursor gene [45, 57]. The primary structure of GHRH is highly variable among species, especially at the C-terminus. In fact, rat GHRH, which is 43 amino acids, shares 68% homology with the human peptide, yet the N-terminal two-thirds of the molecule, corresponding to the bioactive core of the peptide (GHRH1–29), are more conserved. In most species but not in rodents, GHRH is amidated at its C-terminus due to the presence of Gly [45, 46, 57]. The human and rat genomes contain a single GHRH gene that maps to chromosomes 20 and 3, respectively.

DISCOVERY OF GHRH The original evidence for the existence of a hypothalamic factor that stimulated secretion of growth hormone (GH) from pituitary somatotrophs [54] was followed by a long, unfruitful search through classic purification and bioassay methods using large amounts of hypothalamic extracts, which ended unexpectedly in 1982 when two independent teams isolated and characterized human GH-releasing hormone (GHRH) from two pancreatic tumors that caused acromegaly [28, 55]. Two of the three GHRH forms found in these tumors, of 44 and 40 amino acids, were found to be identical to the human hypothalamic GHRH forms [39]. Subsequently, GHRH was isolated and identified in a number of vertebrates, from rodents to fish, including a protoHandbook of Biologically Active Peptides

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FIGURE 1. A. Organization of the GHRH gene and posttranslational processing of its protein product by the sequential participation of the proprotein convertases furin and PC1. The 5′ nontranslated region (5′-NT), the signal peptide (SP), GHRH, the C-terminal peptide, and the 3′ nontranslated region (3′-NT) are indicated. B. Alignment of human and rat GHRH precursor sequences. Bold letters illustrate mismatched amino acids in the rat GHRH sequence when compared to the human sequence. Open boxes encompass the amino acid sequence of GHRH. Gray boxes encompass amino acid residues involved in the processing of the precursor protein.

In both species, the GHRH gene spans approximately 10 kb and includes five exons: exon 1 contains the 5′untranslated region and exons 2–4 encode the pre-proGHRH together with exon 5, which also contains the 3′-untranslated region (Fig. 1). The 5′-splice donor site of intron 4 in human and rat genes differ, which explains differences between the C-terminal portion of human and rat GHRH precursors. In the human gene, differential use of two consensus splice-acceptor sites at the 5′-end of exon 5, which are also present in the rat gene sequence, gives rise to the two distinct types of mRNAs coding for the 108- and 107-amino acid precursor forms (reviewed in [43]). The 5′-flanking region of human and rat GHRH genes includes consensus TATA and CAT box sequences. Analysis of the 5′-promoter region of the rat gene has revealed consensus sites for the transcription factor Gsh-1, which seems to be required for both development of GHRH neurons and tissuespecific regulation of the GHRH gene [47], as well as

for the nuclear factor of activated T cells (NFAT), which appears to be involved in the acute regulation of GHRH gene transcription [5].

EXPRESSION OF GHRH IN THE BRAIN Shortly after isolation of GHRH, the distribution of the peptide was reported in the human and primate brain, wherein GHRH immunoreactivity is associated with neuronal cell bodies in the arcuate nucleus (Arc) of the hypothalamus (Hpt) as well as with fibers in the median eminence (ME) [6, 7]. In rat, the majority of GHRH-immunoreactive cell bodies are found in the ventrolateral part of the Arc, which also exhibits the highest GHRH mRNA levels in the brain (Fig. 2) (reviewed by [43, 46]). These cells project their fibers toward the external layer of the ME, where GHRHimmunoreactive terminals are found around the capil-

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FIGURE 2. Schematic parasagittal section of the rat brain illustrating the distribution of GHRH expression. Black triangles represent GHRH mRNA-containing cell bodies. In rat, the highest GHRH mRNA levels in the brain are found in the ventrolateral part of the arcuate nucleus (Arc).

laries of the portal vessels. In addition to the Arc GHRH-containing neurons, GHRH mRNA can be detected in some scattered perikarya in the dorsolateral region of the ventromedial nucleus, basolateral Hpt, and to a lesser extent, in the paraventricular nucleus. This distribution correlates well with that of GHRH immunoreactivity, which also has been localized to a few cells in the dorsal part of the dorsomedial nucleus, ventral premammillary nucleus, as well as in the medial perifornical region, zona incerta, and the hypothalamic portion of the stria terminalis. GHRHimmunoreactive fibers from these cells extend through the periventricular region of the Hpt and adjoining parts of the basal telencephalon. Nevertheless, the presence of the peptide in extrahypothalamic regions is quite limited as only the cortex and brain stem have been reported to contain detectable amounts of GHRH mRNA [42]. In rat, GHRH-mRNA containing neurons in the Arc appear at E16 and acquire the adultlike distribution pattern at E20 [9]. Transcript levels increase gradually over the course of maturation both in males and females, although GHRH expression is greater in the male than the female from 10 days onward [4]. Finally, both GHRH mRNA levels and the number of immunoreactive Arc GHRH neurons decrease in aged rats [15, 34, 35].

PROCESSING OF GHRH PRECURSOR In vitro analysis of GHRH precursor processing in rat hypothalamic neurons demonstrated that, after removal of the signal peptide, pro-GHRH is first processed to an 8.8-kDa intermediate form that is cleaved to yield the 5.2-kDa GHRH and the 3.6-kDa C-peptide [48]. This process involves sequential participation of two members of the subtilisin-like group of proprotein convertases, furin and PC1, which cleave pro-GHRH at two typical recognition sites for these enzymes located at the Nand C-termini of GHRH, respectively (Fig. 1) [16, 52]. Specifically, processing of pro-GHRH is initiated by furin to give rise to the 8.8-kDa intermediate form, which is subsequently cleaved by PC1 to yield mature GHRH and the C-peptide.

GHRH RECEPTOR Earlier studies reported specific GHRH binding sites with high affinity and low capacity in membrane extracts of intact cells from pituitary [26, 44, 46, 50]. However, detailed knowledge of GHRH-R was only achieved after its cloning, first in rat, then in mouse, human, and pig, and more recently in other vertebrate species [26, 44, 50]. GHRH-R belongs to the class II of the superfamily

666 / Chapter 93 of 7 transmembrane domains, G-protein-coupled receptors, which includes receptors for its related PACAP/ VIP/secretin/glucagon family of peptides (Fig. 3) [44]. Contrary to other receptors of this family, GHRH-R is highly selective for its cognate ligand, showing subnanomolar affinity [26]. Specificity is conferred by the N-terminal extracellular region, transmembrane domains and associated extracellular loops, and also C-terminal intracellular tail [26, 30]. The GHRH-R gene is localized to human chromosome 7p14-15 and rat chromosome 4q24. The human GHRH gene spans over 15 kb and is composed of 13 exons separated by introns of variable size, which thereby enable alternative mRNA splice generating receptor isoforms [26, 44]. The main form of GHRH-R in most mammals is a 423 amino acid protein [44]. Additional, less abundant isoforms bearing C-termini with distinct length and structure have been reported

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in the pituitary of several species including human (normal and tumoral), rat, and pig, and, although their precise functional significance is as yet unclear, they show distinct binding and/or cAMP signaling properties and may modulate signaling by the predominant receptor [30, 44]. The N-terminal extracellular domain of GHRH-R contains a site for N-glycosylation, and six Cys, which are conserved in this receptor family, while the third intracellular loop and C-intracellular domain contain several potential phosphorylation sites, which may regulate signaling and receptor internalization [26, 44, 50]. Human and rat GHRH-R gene promoters lack a canonical TATA box motif but contain putative binding sites for a number of transcription factors including (in human) Pit-1, Oct-1, Brn-2, NF-1, cAMP response elements (CRE) and an estrogen-responsive element (ERE) [50]. Of note, studies in dwarf mice and humans

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FIGURE 3. Amino acid sequence and membrane topology of the rat GHRH-R. Arrowheads indicate the six Cys residues in the NH2-terminal domain. Asterisks represent several potential phosphorylation sites and arrows illustrate two N-glycosylation sites.

Growth Hormone-Releasing Hormone / 667 [44, 46]. In contrast, GHRH-R expression is positively regulated by glucocorticoids and thyroid hormone [26, 44, 46].

indicate that GHRH-R expression is strictly and reciprocally dependent upon Pit-1 gene expression [26, 44]. Accordingly, GHRH-R is predominantly expressed in pituitary, in a temporal and spatial pattern corresponding to GH gene expression, and therefore essentially restricted to somatotrophs [38]. In addition, GHRH-R mRNA is present in periventricular, Arc, and ventromedial nuclei, and the anterior hypothalamic area as well as in some peripheral tissues (e.g., placenta, kidney) [44, 46]. Regulation of GHRH-R is exerted at different levels. Rapid desensitization occurs upon ligand binding, through internalization of the GHRH-R [26, 44, 58]. More lasting regulatory effects are exerted on GHRH-R expression by a number of signals including GHRH, which evokes a dual, time-dependent effect: an acute decrease in GHRH-R expression likely mediated via cAMP [26, 40, 44] and a long-term up-regulation of mRNA levels [26, 44]. Acute inhibition of expression is also caused by ghrelin [40]. Consistent with the presence of corresponding binding sites in GHRH-R promoter, and with its sexually dimorphic expression in pituitary, estrogen inhibits GHRH-R transcription

GHRH SIGNALING Upon GHRH binding, GHRH-R activates diverse signaling cascades, which primarily involve two second messengers, cAMP and Ca2+ (Fig. 4) [23, 44, 46, 50]. GHRH increases cAMP levels by stimulating adenylate cyclase (AC) through a Gs protein. Subsequently, increased cAMP levels induce activation of protein kinase A (PKA), which, in turn, activates several proteins by phosphorylation, including ion channels [23, 44, 46, 50]. Likewise, GHRH increases cytosolic free Ca2+ concentration levels in somatotrophs through a mechanism essentially dependent on extracellular Ca2+ influx through voltage-sensitive Ca2+ channels, a process that involves K+ and Na+ channels and PKA-dependent phosphorylation [11, 12]. There is also evidence that GHRH activates and requires additional signaling

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GH release FIGURE 4. Schematic diagram summarizing the main signaling pathways involved in the response of somatotrophs to GHRH. Abbreviations: AC, adenylate cyclase; CREB, cAMP response element (CRE)-binding protein; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogenactivated protein kinase; NO, nitric oxide; NOS, nitric oxide synthase; PKA, protein kinase A; PLC, phospholipase C; RER, rough endoplasmic reticulum; sGC, soluble guanylyl cyclase; VSCC-L, L-type voltage-sensitive Ca2+ channels.

668 / Chapter 93 components in somatotrophs [23, 46]. Thus, GHRHinduced GH release is partially dependent on phospholipase C (PLC)/inositol phosphate (IP) pathway and/or mobilization of Ca2+ from intracellular stores in rat and pig somatotrophs [51, 53]. Increasing evidence also indicates that the signaling cascade involving nitric oxide and cGMP also participates in mediating GHRH-induced GH release, and may represent a subsequent, interlinked step in the AC/cAMP/PKA signaling cascade, via PKA-mediated phosphorylation of soluble guanylyl cyclase [23, 27, 41, 46]. GHRH-R signaling also activates the MAP kinase pathway, which may mediate the effect of the peptide on GH synthesis and somatotroph proliferation [26, 44]. Finally, the signaling properties conveyed by the GHRH-R may be modulated by other G-protein-coupled receptors such as that for ghrelin/GH secretagogues [14].

CONFORMATION OF GHRH AND SYNTHESIS OF ANALOGS The secondary structure of human GHRH in solution consists of a short β-strand and two α-helices spanning between residues 6–13 and 16–29 of the protein [8]. This highly helical structure appears to be critical for effective binding to GHRH-R. Since the discovery of the sequences crucial for GHRH somatotrophic activity, much effort has been invested in generating synthetic analogs with increased potency. Thus, generation of a loop between amino acids 8 and 12 of [Asp8, Asp15]GHRH(1–29)NH2 yielded a GHRH analog exhibiting increased biological activity in vitro [21]. C-terminal PEGylation has also been introduced into GHRH sequence to enhance its activity [10] and to confer further stability to its helical conformation [17]. These analogs have been proven as potent stimulators of GH release and, therefore, very useful to combat human GH deficiency disorders.

BIOLOGICAL ACTIVITY OF GHRH It is now well established that GHRH is the primary hypothalamic stimulus for GH secretion from pituitary somatotrophs [23, 27, 46]. Indeed, the remarkable ability of GHRH to induce GH release specifically, both in vivo and in vitro, in mammals, birds, and amphibians (and to a lesser extent in fish) with a marginal effect on the release of other pituitary hormones, constitutes the major role for this peptide [23, 27, 44–46]. Moreover, GHRH acts as a true somatotrophic factor in that it also increases intracellular GH content by activating GH gene transcription and GH mRNA levels, and stimulates somatotroph proliferation, this latter function being

fundamental for somatotroph development during pituitary organogenesis [24, 46]. Consistent with the well-known pulsatile pattern of GH release in plasma, GHRH has been shown to be released in an episodic manner into portal blood at the ME in various species [27, 46]. In fact, initial studies on male rats based on measurements of GHRH and somatostatin in portal plasma and of GH in peripheral plasma led to a valuable model wherein GH pulses were the result of alternating episodes of GHRH and somatostatin release from Hpt, which would stimulate and suppress, respectively, GH release [27, 46]. Yet, subsequent studies revealed that this model did not satisfactorily explain GH secretory patterns in human and other species in diverse pathophysiological conditions (e.g., GH pulsatility is maintained in the absence of GHRH input due to a mutation in GHRH-R [56]). The available evidence led Veldhuis and coworkers to postulate, in the context of highly complex and elaborated mathematical models, that the generation of a given physiological pattern of pulsatile GH release is the combined result of intricate, time-delimited interactions among GHRH, somatostatin, and GH autofeedback, as well as a new major player, ghrelin, which are exerted at both hypothalamic and pituitary levels [18, 19, 27]. In this scenario, GHRH acts on the pituitary to stimulate GH release and synthesis but would also act by stimulating Arc somatostatin neurons, as suggested by the tight neuroanatomical interplay between these factors. In turn, GHRH secretion from Arc neurons is negatively regulated by hypothalamic somatostatin neurons as well as by GH feedback, whereas ghrelin would act upon GHRH neurons stimulating its release. The components of this cycle are discussed elsewhere in this book. There is ample evidence that GH levels and secretory patterns are markedly affected by development, sex, aging, and diverse pathological conditions both in human and other species, and that, accordingly, hypothalamic GHRH production is strictly and distinctly regulated by these factors [27, 46]. Indeed, the sexually dimorphic pattern of pulsatile GH secretory profiles in both human and rat, which underlies the distinct (higher in males) gender-dependent growth pattern, is likely due to the higher number and mRNA content of hypothalamic GHRH neurons, and presumably, to their distinct neuroanatomical connectivity with other neurons (e.g., somatostatin) in the male compared with the female. Although the precise mechanisms by which gonadal steroids contribute to this sex-dependent differences at the hypothalamic level has not been fully elucidated, a number of studies have shown that the steroid environment, especially during development and around puberty, profoundly affects GHRH producing neurons, their number, responsiveness to steroids

Growth Hormone-Releasing Hormone / 669 in adult age, and even synaptic organization [13]. Similarly, basal and pulsatile GH release is markedly influenced by age, with a decline in GH during life span and aging in human and animal models, which seems to be paralleled by and significantly dependent on a decreased production of hypothalamic GHRH and reduced somatotroph secretory responsiveness to this peptide, which is accompanied by an increased somatostatinergic tone [27, 34, 35, 46]. In addition to controlling somatotroph function, GHRH also possesses extrapituitary central effects. Thus, it has been shown that GHRH promotes sleep and, accordingly, it has been proposed to provide the subcortical mechanism synchronizing sleep and GH secretion (reviewed by [49]). Specifically, non-REM sleep increased in human, rat, and rabbit after GHRH administration, whereas sleep was reduced in rats when GHRH action was inhibited by a specific antagonist or by GHRH antibodies. This sleep-promoting effect of GHRH has been proposed to occur through a direct action of the peptide on GABAergic neurons in the anterior Hpt/medial preoptic region. Additionally, GHRH also stimulates REM sleep, although this action seems to depend on GH. GHRH is also involved in the central regulation of feeding and likely contributes to the circadian pattern of food intake (reviewed by [46]). In support of this, central administration of GHRH stimulates food intake in rat and sheep by a mechanism that was initially proposed to involve GHRH-mediated effects on the suprachiasmatic nucleus/medial preoptic area [20]. Interestingly, hypothalamic GHRH content is positively correlated with the feeding-related neuropeptides αMSH, orexin A, and cocaine amphetamine-regulated transcript (CART) [32].

PATHOPHYSIOLOGICAL IMPLICATIONS Due to the direct involvement of GHRH in somatotroph proliferation, defects in tandem GHRH/GHRHR profoundly alter the morphological and functional characteristics of the pituitary, provoking generalized GH secretion disorders. Thus, excessive GHRH release into the median eminence induces somatotroph hyperplasia and an abnormal enlargement of the pituitary [25]. In rodents, symptomatology of GHRH excess has been extensively studied since Mayo and coworkers established a strain of transgenic mice expressing a mouse metallothionein I/human GHRH fusion gene, an animal model of acromegaly [29]. In these mice, chronic GHRH production leads to development of enormous pituitary glands as a result of uncontrolled proliferation of somatotrophs that secrete large amounts of GH, an effect that is GHRH-specific and not medi-

ated by GH or IGF-I [33]. In humans, GHRHdependent somatotroph hyperplasia is very rare; it is limited to certain cases of extrapituitary GHRH overproduction and sporadic examples of gigantism [31]. Moreover, mutations in the GHRH gene have never been described, contrary to that observed in the GHRHR gene, where a variety of spontaneous punctal mutations affecting GHRH-R activity have been reported. One of these mutations (or gene polymorphism) is produced by a single base change in the GHRH-R gene in human somatotrophinomas that confers hypersensitivity to GHRH binding [1]. On the other hand, deficient GHRH secretion and/ or activity is generally associated with somatotroph hypoplasia, subsequent low GH release, and consequently, development of dwarfism. These observations have been obtained from rodent (i.e., little mouse) or humans with inactivating mutations in the GHRH-R (reviewed by [3]) since, as mentioned earlier, mutations in the GHRH gene have not been reported to date, neither in humans nor in rodents. Despite this, rodent models with GHRH deficiency secondary to deletion of other genes (Gsh-1 [37]; PC1 [59]) or caused by targeted expression of the human GH transgene in the Hpt [22] have been already reported. Certainly, the recent development of GHRH knockout mice [2, 36] will pave the way to unveil the precise role of GHRH in the regulation of normal and pathophysiological somatotroph function.

References [1] Adams EF, Symowski H, Buchfelder M, Poyner DR. A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro. Biochem Biophys Res Commun 2000; 275:33–6. [2] Alba M, Salvatori R. A mouse with targeted ablation of the growth hormone-releasing hormone gene: a new model of isolated growth hormone deficiency. Endocrinology 2004; 145:4134–43. [3] Alba M, Salvatori R. Familial growth hormone deficiency and mutations in the GHRH receptor gene. Vitam Horm 2004; 69:209–20. [4] Argente J, Chowen JA, Zeitler P, Clifton DK, Steiner RA. Sexual dimorphism of growth hormone-releasing hormone and somatostatin gene expression in the hypothalamus of the rat during development. Endocrinology 1991; 128:2369–75. [5] Asai M, Iwasaki Y, Yoshida M, Mutsuga-Nakayama N, Arima H, Ito M, Takano K, Oiso Y. Nuclear factor of activated T cells (NFAT) is involved in the depolarization-induced activation of growth hormone-releasing hormone gene transcription in vitro. Mol Endocrinol 2004; 18:3011–9. [6] Bloch B, Brazeau P, Ling N, Bohlen P, Esch F, Wehrenberg WB, Benoit R, Bloom F, Guillemin R. Immunohistochemical detection of growth hormone-releasing factor in brain. Nature 1983; 301:607–8. [7] Bresson JL, Clavequin MC, Fellmann D, Bugnon C. [Human ontogeny of the hypothalamic neuroglandular system

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[24] Frohman LA, Kineman RD. Growth hormone-releasing hormone and pituitary somatotrope proliferation. Minerva Endocrinol 2002; 27:277–85. [25] Frohman LA, Kineman RD, Kamegai J, Park S, Teixeira LT, Coschigano KT, Kopchic JJ. Secretagogues and the somatotrope: signaling and proliferation. Recent Prog Horm Res 2000; 55:269–90; discussion 290–1. [26] Gaylinn BD. Growth hormone releasing hormone receptor. Receptors Channels 2002; 8:155–62. [27] Giustina A, Veldhuis JD. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocr Rev 1998; 19:717–97. [28] Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982; 218:585–7. [29] Hammer RE, Brinster RL, Rosenfeld MG, Evans RM, Mayo KE. Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 1985; 315:413–6. [30] Hassan HA. Biological activities of two porcine growth hormonereleasing hormone receptor isoforms. Arch Biochem Biophys 2001; 387:20–6. [31] Horvath E, Kovacs K, Scheithauer BW. Pituitary hyperplasia. Pituitary 1999; 1:169–79. [32] Kappeler L, Zizzari P, Alliot J, Epelbaum J, Bluet-Pajot MT. Delayed age-associated decrease in growth hormone pulsatile secretion and increased orexigenic peptide expression in the Lou C/JaLL rat. Neuroendocrinology 2004; 80:273–83. [33] Kineman RD, Teixeira LT, Amargo GV, Coschigano KT, Kopchick JJ, Frohman LA. The effect of GHRH on somatotrope hyperplasia and tumor formation in the presence and absence of GH signaling. Endocrinology 2001; 142:3764–73. [34] Kuwahara S, Kesuma Sari D, Tsukamoto Y, Tanaka S, Sasaki F. Age-related changes in growth hormone (GH)-releasing hormone and somatostatin neurons in the hypothalamus and in GH cells in the anterior pituitary of female mice. Brain Res 2004; 1025:113–22. [35] Kuwahara S, Sari DK, Tsukamoto Y, Tanaka S, Sasaki F. Agerelated changes in growth hormone (GH) cells in the pituitary gland of male mice are mediated by GH-releasing hormone but not by somatostatin in the hypothalamus. Brain Res 2004; 998:164–73. [36] Le Tissier PR, Carmignac DF, Lilley S, Sesay AK, Phelps CJ, Houston P, Mathers K, Magoulas C, Ogden D, Robinson IC. Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene. Mol Endocrinol 2005; 19: 1251–62. [37] Li H, Zeitler PS, Valerius MT, Small K, Potter SS. Gsh-1, an orphan Hox gene, is required for normal pituitary development. Embo J 1996; 15:714–24. [38] Lin C, Lin SC, Chang CP, Rosenfeld MG. Pit-1-dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. Nature 1992; 360:765–8. [39] Ling N, Esch F, Bohlen P, Brazeau P, Wehrenberg WB, Guillemin R. Isolation, primary structure, and synthesis of human hypothalamic somatocrinin: growth hormone-releasing factor. Proc Natl Acad Sci U S A 1984; 81:4302–6. [40] Luque RM, Kineman RD, Park S, Peng XD, Gracia-Navarro F, Castano JP, Malagon MM. Homologous and heterologous regulation of pituitary receptors for ghrelin and growth hormone-releasing hormone. Endocrinology 2004; 145: 3182–9. [41] Luque RM, Rodriguez-Pacheco F, Tena-Sempere M, GraciaNavarro F, Malagon MM, Castano JP. Differential contribution of nitric oxide and cGMP to the stimulatory effects of GHRH

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mitter, and neuroendocrine hormone, activating adenylate cyclase (AC) through G protein-coupled receptors. In 1989, during an attempt to discover a novel hypothalamic hypophysiotropic hormone, PACAP was isolated from ovine hypothalamic tissue because of its ability to stimulate accumulation of cAMP in rat pituitary cell cultures [14]. Two forms of PACAP were isolated, an amidated peptide with 38 amino acids (PACAP38) and another, a C-terminally truncated form with 27 amino acids (PACAP27). PACAP is closely related to VIP (68% sequence homology within the N-terminal 28 residues) and more distantly related to glucagon, glucagon-like peptide 1, peptide histidine isoleucine amide, secretin, and GRF. However, the AC activating potencies of PACAP38 and PACAP27 were 1000–10,000 times greater than VIP. Among its family of peptides, the primary structure of VIP and PACAP has been extremely well conserved in vertebrates including mammals, birds, reptiles, amphibians, and fish [21]. PACAP38 is most abundant in the hypothalamus, with lower levels in other brain regions. PACAP has a distinct distribution in the central and peripheral nervous systems, where it functions as a neurotransmitter, neuromodulator, neurotrophic factor, vasodilator, noncholinergic catecholamine secretagogue, and also an immunomodulator affecting immune cells. PACAP is also present in several peripheral tissues, including the gastrointestinal tract, adrenal gland, and testis, suggesting a variety of functions in respective organs and tissues.

PACAP and VIP are two prominent structurally related neuropeptides with a broad distribution in the body, which exert very important pleiotropic functions in several systems. They share two common G proteincoupled receptors, VPAC1 and VAPC2 receptors, whereas PACAP has an additional specific receptor, PAC1 receptor. PACAP/VIP has played a vital role in the adaptation of living organisms to the ever-changing environmental conditions. This chapter describes the present knowledge regarding aspects of PACAP/VIP, including (1) discovery; (2) structure of the precursor mRNA/gene; (3) distribution of the mRNA; (4) processing of the precursor; (5) receptors and signaling cascades; (6) information on active and/or solution conformation; (7) biological actions within the brain and pituitary; and (8) pathophysiology of PACAP/VIP.

DISCOVERY Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of a superfamily of structurally related peptide hormones that include glucagon, glucagon-like peptide, secretin, and growth hormone-releasing factor (GRF). VIP is a 28-amino-acid amidated peptide isolated from porcine duodenum based on its vasodilator activity in the canine femoral artery [19, 20]. VIP is not only present in gastrointestinal tissues but also in neural tissues, especially of the cerebral cortex. VIP has a number of actions in both central and peripheral tissues, including activities as a neurotransmitter and neurotrophic factors, vascular and nonvascular smooth muscle relaxation, and electrolyte secretion in the gut. It functions as a gastrointestinal hormone, neurotransHandbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The VIP precursor polypeptide (pre-pro-VIP) contains a 170-amino-acid sequence, which includes an

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674 / Chapter 94 additional biologically active peptide, called peptide histidine methionine (PHM) amide in humans or peptide histidine isoleucine (PHI) in sheep, rats, mice, and chickens [23]. PHM/PHI possesses moderate amino acid sequence similarity with VIP. Pre-pro-PACAP contains 176 amino acids in human beings, sheep, and mouse, and 175 amino acids in rat [23]. The sequence of PACAP38 is located in the C-terminal domain of the precursor and is identical in all mammalian species studied so far. The precursor comprises a 24-amino-acid putative signal peptide and also contains a 29-aminoacid peptide, termed PACAP-related peptide (PRP). The gene encoding PACAP has been cloned in human beings and mice. The overall architecture of the two genes is similar and is composed of five exons. Unlike the PACAP gene, the VIP gene has seven exons. The sequences of PHM/PHI and PRP are encoded by exon 4, respectively, and those of VIP and PACAP by exon 5 of the respective gene. Furthermore, the locus of the PACAP gene was found to be in chromosome 18p11, whereas the VIP gene is located in chromosome 6q26q27. The region 18p11 is associated with a hereditary developmental defect of the forebrain in human beings, and PACAP was suggested to be involved in the control of neural tube patterning and neurogenesis. In mammals, the structural organization and the sequence of the PACAP precursor and gene exhibit strong similarities with those of the VIP precursor and gene. Presumably, they arose by a genomic duplication of the exon encoding a common ancestral gene, and they both appeared very early during evolution, prior to the emergence of vertebrates.

DISTRIBUTION OF THE mRNA Distributions of PACAP and VIP mRNAs in the CNS are substantially different. PACAP mRNA is mainly expressed in the hypothalamus, highly contained in the PVN and SON [23]. On the other hand, VIP mRNA is localized in the SCN, paraventrolateral thalamic region, and neocortical region. In the thalamus, a few VIP fibers were found running up the wall of the third ventricle, whereas a dense network of PACAP fibers was observed in the central thalamic nuclei. In the BST, PACAP fibers surround unstained, round-shaped neuronal cell bodies, whereas VIP fibers are homogeneously distributed (Fig. 1). Significant amounts of PACAP38 are also found in extrahypothalamic regions. In situ hybridization revealed the presence of scattered PACAPexpressing cell bodies in the cingulate and frontal cortex. The transcripts of PACAP were also detected in the cerebellum. In the spinal cord, PACAP mRNA is expressed in a subpopulation of sensory neurons of the dorsal root ganglia.

In peripheral organs, PACAP and VIP mRNA are often coexpressed in the same cells. Both PACAP and VIP transcripts were demonstrated in nerve fibers and cell bodies in the human esophageal sphincter and the gut [22]. PACAP mRNA was also detected in most endocrine glands. By immunohistochemistry, PACAP dense fibers were found at the periphery of the posterior lobe in the vicinity of the IL. However the VIP fibers were evenly distributed in the central part of the posterior lobe. The adrenal medulla expresses a high level of PACAP mRNA. In the rat, the highest amounts of PACAP mRNA are found in the testis. In fact, the total amount of PACAP transcript in the testis is greater than in the whole brain and exceeds the concentration of any other known peptide. However, in the male rat, VIP mRNA was exclusively localized to neural fibers derived from the inferior spermatic nerve. Numerous VIP transcript-containing nerve fibers were observed in the cauda epididymis, ductus deferens, accessory glands, and penis. No report indicating the presence of VIP mRNA in the testicular cells has appeared.

PROCESSING OF THE PRECURSOR Many proteins and peptides are initially synthesized as large precursor molecules that require highly specific intracellular proteolysis to generate the biologically active end-products. After the signal peptide is cleaved, the precursor undergoes proteolytic processing, initiated by endoproteolytic cleavage at sites marked by basic amino acids. A novel family of proteases has been identified that represent these processing endoproteases. The proteases found thus far are all related to the yeast dibasic-specific endoprotease kex2, and include prohormone convertase (PC) 2, PC3/PC1, PC4, furin/PACE, PACE4, PC5/PC6, and PC7/PC8/ LPC. The emerging characteristics of these endoproteases, including their tissue-specific expression, subcellular localization, and cleavage site selectivity, suggest that members of this family arose during evolution to process a diverse group of functionally distinct precursors in a highly specific, compartmentalized, and regulated fashion. PACAP is a neuropeptide that is synthesized in neuroendocrine cells. In the regulated processing pathway characteristic of neuroendocrine cell types, precursors are packaged into granules, where they are proteolytically processed and stored for later release in response to the appropriate extracellular signals. In mammals, the primary structure of the PACAP precursor reveals the existence of seven monoor dibasic residues that can be cleaved by various prohormone convertases. Studies with PC2 and PC3/PC1 indicate that these proteases function specifically within this pathway. Both enzymes are expressed only

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FIGURE 1. The distribution of the neuronal systems expressing PACAP (red) and VIP (blue). (•): Cell bodies and (−): Fibers. Acb, nucleus accumbens; Amb, nucleus ambiguous; Amy, amygdala; AP, anterior pituitary; Arc, arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; Cput, caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of the pituitary; LC, locus coeruleus; LRN, lateral reticular nucleus; ME, median eminence; NL, neural lobe of the pituitary; NST, nucleus of the solitary tract; OB, olfactory bulb; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular nucleus of the hypothalamus; S, septum; SC, nucleus suprachiasmaticus; SN, substantia nigra; SON, supraoptic nucleus; Th, thalamus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VMH, ventromedial nucleus of the hypothalamus; VPM, ventroposteromedial thalamic nucleus. (See color plate.)

LRN

676 / Chapter 94 in neuroendocrine tissues, and PC2 has been localized intracellularly to the secretory granules. Cotransfection experiments have confirmed that both PC1 and PC2 can actually process the rat PACAP precursor to generate mature PACAP38 and PACAP27 [13]. On the other hand, the testicular cells do not express either PC1 or PC2. With in situ hybridization, furin and another kex2 homologue, PC4, were found in the germ cells of both mouse and rat testes. In the testis, PC4 processes the PACAP precursor to generate both PACAP38 and PACAP27 [12]. The cleavage at three dibasic sites, Arg79-Arg80, Lys129-Arg130, and Arg170-Arg171, generates a large intermediate precursor of PRP (big PRP) and a glycine-extended form of PACAP38. Cleavage at the single Arg110, followed by hydrolysis of this C-terminal Arg residue by carboxypeptidases E, H, or M, generates PRP. The Gly169 residue is used by peptidyl glycine αamidating monooxygenase for the amidation of the Lys168 residue at the C-terminal amino acid of PACAP38 [3]. Finally, the tripeptide Gly158-Lys159-Arg160 can be cleaved to generate the α-amidated PACAP27 isoform. Prepro-VIP mRNA is transcripted from 7 exon regions on chromosome 6q24 in human beings. The VIP precursor is processed to at least three biologically active peptides: VIP, PHI, and PHV and another three peptides: pre-pro-VIP (22–79), pre-pro-VIP (111–122), and pre-pro-VIP (156–170).

RECEPTORS AND SIGNALING CASCADES Three PACAP/VIP receptors have been cloned so far and officially termed the PAC1, VPAC1, and VPAC2 receptors [9]. The PAC1-R is PACAP-specific, whereas VPAC1-R and VPAC2-R have similar binding affinities for PACAP and VIP. The human PAC1-R cDNA, which codes for a 525-amino-acid G protein-coupled heptahelical receptor with a seven transmembrane domain, exhibits a high degree of sequence similarity with the VIP, GHRH, glucagon, gastric inhibitory peptide, glucagon-like peptide-1, and secretin receptor cDNAs [17]. Unexpectedly, this family also comprises receptors for secretin, GRF, glucagon, glucagon-like peptide, calcitonin, calcitonin gene-related peptide, parathyroid hormone, and corticotropin releasing factor, with sequence identities ranging between 30 and 50%. The three PACAP/VIP receptors are preferentially coupled to Gs (adenylate cyclase activation); the PAC1 and VPAC1 are also coupled to Gq and Go, respectively (calcium mobilization). In human beings, the chromosomal locations for genes of PAC1-R, VPAC1-R, and VPAC2-R are 7p15-p14, 3p22, and 7q36.3, respectively. Alternative splicing of the PAC1-R gene also occurs in the untranslated region and could represent a regulatory mechanism involved in tissue-selective expression

of the gene and/or in mRNA stability. At least ten subtypes of the rat PAC1-R resulting from alternate splicing were cloned. Each subtype was coupled to specific signaling pathways, and their expressions are either tissueor cell-specific. Six variants resulting from alternative splicing in the third intracellular loop have been identified. The splice variants are characterized by the presence of either one or two cassettes of 28 (hip or hop1 variant) or 27 (hop2 variant) amino acids in the third intracellular loop or their absence (short variant, also named the null variant). The presence of the hip cassette impairs AC stimulation and abolishes phospholipase C (PLC) activation, suggesting that various cassettes are involved in second messenger coupling. In the brain and pituitary, the short variant is the most abundant form, whereas the PAC1-R hop1 variant predominates in the testes and adrenal gland. Moreover, two very short splice variants of PAC1-R (PAC1-R-vs1 and PAC1-R-vs2) displayed either a 21- or 57-amino-acid deletion in the N-terminal extracellular domain, revealing similar high binding affinities, and AC- and PLCstimulating potencies for PACAP38 and PACAP27. In comparison, PACAP38 had a substantially higher binding affinity for the PAC1-R short (PAC1-R-s) variant than PACAP27 and was more potent as an activator of PLC. Another PAC1-R variant termed PAC1-R-TM4 has been cloned from the cerebellum. This receptor differs from the PAC1-R-s by discrete sequence substitutions located in transmembrane domains II and IV. Surprisingly, activation of PAC1-R-TM4 has no effect on AC or PLC activity but causes calcium influx through L-type voltage-sensitive calcium channels. A testis-specific splice variant with the insertion of 24 amino acids (exon 3) in the N-terminal extracellular domain was recently cloned and named PAC1-R-3a. The transcripts for PAC1-R-3a are preferentially expressed at the highest levels in Sertoli cells and at lower levels in germ cells, and have a sixfold higher binding affinity for PACAP38 than for PACAP27. Moreover, the PAC1-R-3a, in the membrane preparations from stably transfected HEK293 cells expressing the two receptor isoforms, reveals a sixfold increase in the affinity of the PAC1-R-3a for PACAP38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild-type PAC1-R. Rat and human VPAC1-R (457 amino acids) have been cloned, corresponding to the “classic” VIP binding site [11]. In the cells transfected with VPAC1-R cDNA, PACAP and VIP bind with a similar potency for AC stimulation. Besides AC stimulation, VIP induces mobilization of Ca2+ from intracellular Ca2+ stores in the cells that express VPAC1-R. However, PACAP enhances Ca2+-dependent neurotransmitter release from PC12 cells and cultured cerebellar granule cells without affecting intracellular Ca2+ mobilization. More recently, VPAC2-R (438 amino acids) was

PACAP/VIP cloned from rat, mouse, and human tissues [11]. VPAC2-R has similar affinity in binding and displays a similar potency of AC activation for PACAP and VIP. Rat VPAC2-R has 50 and 51% similarity with rat PAC1-R and VPAC1-R, respectively. In VPAC2-R-expressed Xenopus oocytes, PACAP and VIP induced calcium-activated chloride currents, suggesting that VPAC2-R is coupled to PLC activation. In plasma membranes isolated from dispersed gastric muscle cells, a Ca2+/ calmodulin dependent nitric oxide synthase was activated by VIP and PACAP via a common receptor coupled to pertussis toxin-sensitive Gil-2. PACAP stimulated cell growth in pancreatic AR4-2J cell lines through a pertussis toxin-sensitive G protein. This effect appears to be mediated by phospholipase D and/or tyrosine kinase via VPAC and PAC1 receptors. Furthermore, PACAP and VIP trigger synaptic currents in Drosophila through the coactivation of the cAMP pathway and a signaling pathway involving the small G protein Ras and the subsequent activation of Raf. Cerebellar granule neurons were protected from apoptosis via mitogen-activated protein kinase (MAPK) activation by 100 nM PACAP38 treatment. In neuron/astroglia cocultures, at least two major signaling pathways are involved in the neuroprotection by PACAP: stimulation of adenylate cyclase with increased level of cAMP and stimulation of ERK type MAPK. The former is stimulated by nanomolar PACAP, whereas the latter is stimulated by subpicomolar PACAP. However, even at the femtomolar level of PACAP, a small increase in cAMP levels (4–5 times higher than the baseline) was demonstrated. Both Rap-1 GTPases and B-Raf were required for recruiting the activation of ERK1/2 by VIP or PACAP. In summary, PAC1-R, which binds to PACAP with a high affinity and VIP with 1000 times lower affinity, is coupled to activation of both AC and LPC through G proteins, and then increases Ca2+. One exception is PAC1-R TM4, which is coupled to Ca2+ mobilization through the activation of L-type Ca2+ channels but without activation of AC and PLC. Additional signaling pathways appear to be coupled with PAC1-R, and they involve Ras/Rap-1, Raf, and MAPK signaling cascade. These PACAP/VIP receptors, as well as PAC1-R subtypes, are distributed in discrete tissues, and some tissues express more than one type of PACAP/VIP receptor with a mixed ratio, and the density of each receptor expressed may be variable. The coupling of a receptor to a signaling pathway may depend on such factors as the types of G protein, AC, and PLC expressed in the cells, and the concentration and pattern of the ligand that the cells receive. Thus tissues and cells may display unique and varying effector responses to endogenous and exogenous PACAP. Exogenous VIP stimulates prolactin secretion from the pituitary and catecholamine release from the adrenal medulla only by a pharmacological dose,

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whereas PACAP stimulates release of catecholamine at a physiological concentration.

SOLUTION STRUCTURE COMPARISON Structural studies of PACAP in solution by NMR spectroscopy have shown an initial disordered N-terminus sequence of eight amino acid residues, followed by a region from amino acid residues 9 to 24 that consists of four distinct domains [10]. The first domain, encompassing residues 9 to 12, forms a β-turn-like conformation whereas the three others are composed of distinct helical regions that extend from residues 12 to 14, 15 to 20, and 22 to 24, respectively. The conformation of the C-terminal segment exhibits a short helix attached by a flexible hinge to the 1–27 region. The threedimensional structure of PACAP exhibits substantial similarities with those of other members of the VIP. In particular, both PACAP and VIP possess two helices separated by a disordered region, but the position of the first α-helix of PACAP is shifted by two residues toward the C-terminus, and the conformation of the second helix of PACAP is closer to an α-helix than that of VIP. These minor conformational differences between PACAP and VIP may contribute to the selectivity of the peptides for their receptors.

BIOLOGICAL ACTIONS IN THE BRAIN AND THE PITUITARY PACAP and VIP have well-established roles in central and peripheral nervous system function and development. The neurophysiological effects of VIP overlap significantly with those of PACAP. VIP is a neuropeptide that has a wide distribution in peripheral and central nervous systems and a large spectrum of biological actions in humans. The presence of VIP and VIP receptors in defined pathways in the brain indicates that it plays important roles in CNS function, including regulation of cerebral energy metabolism, cerebral blood flow, neural activity and survival [1]. VIP has also neurotrophic properties during development and repair of the nervous tissues. Subnanomolar VIP protects the spinal cord neurons in spinal cord cultures against tetrodotoxin. The neuroprotective action of VIP is contingent on the presence of astroglia. Astroglia stimulated by VIP releases several neurotrophins, including cytokines, protease nexin I, activity-dependent neurotrophic factor, and activity-dependent neurotrophic protein. In sympathetic neuroblasts, VIP increases neurite sprouting. VIP stimulates neurite outgrowth in several cells such as human neuroblastoma and PC12 (pheochromocytoma) cells. Furthermore, VIP

678 / Chapter 94 was shown to stimulate early brain growth, and VIP blockade during development resulted in severe microcephaly. More recently, depression of hypoxic arousal response in adolescent mice was observed following antenatal VIP blockade. In a model of in vivo excitotoxic brain lesions in the newborn mice that mimic brain lesions observed in preterm and full-term human neonates, VIP and VIP agonists protected the white matter from NMDA-induced damage. The most abundant population of PACAP-containing neurons and the highest density of PACAP binding sites are found in the hypothalamus [6]. In particular, a dense accumulation of PACAP-immunoreactive neurons and PACAP receptors are present in the magnocellular region of the PVN and SON, where the neurosecretory perikarya producing oxytocin and vasopressin are located. Administration of PACAP within the PVN and SON increases the firing rate activity and causes membrane depolarization of magnocellular neurons. Intracerebroventricular and intracisternal injection of PACAP causes a dose-dependent elevation of plasma vasopressin. In the NL, PACAP stimulates the release of oxytocin and vasopressin through activation of the cAMP/protein kinase A signaling pathway. Notably the NL contains a large amount of PACAP, but not VIP. Although the neurohypophysial PACAP could also be released in response to certain stimuli, its physiological role has not been explored. Both PACAP and VIP facilitate neuronal depolarization and excitability, induce second-messenger production, and increase neurotransmitter levels by augmenting biosynthetic enzyme phosphorylation, activity, and mRNA expression in the brain. These two peptides have also been identified in specific sensory and autonomic neurons and implicated in neuron-mediated cardiovascular vasodilation, gastrointestinal smooth-muscle relaxation, and neuroendocrine hormone secretion. In addition to their roles as neurotransmitter and neuromodulator, both PACAP and VIP play important roles as neurotrophic factors. In this chapter, the neurotrophic and neuroprotective activity of PACAP is presented only briefly, since it is also discussed in the neurotrophic peptides section. PACAP regulates both proliferation and differentiation of neurons during development. Expression of PACAP and PAC1-R protein in the neural tube suggests its important role in regulation of neural development. In a gestational day 13 rat embryo, PACAP stimulates growth of precursors of cortical neurons, whereas in gestational day 18, the peptide suppresses cell growth and stimulates differentiation of neurons. The differential regulation of neuronal development by PACAP may be induced by temporal expression of specific subtypes of PAC1-R on developing neurons.

Many studies have shown neuroprotective effects of PACAP, such as significant suppression of neuronal death induced by hypoxia, withdrawal of nerve growth factor or potassium in neural cultures, excitotoxicity, and toxic envelop protein of HIV, gp120, and others. In vivo, neuronal death in the hippocampus resulting from global brain ischemia in rats was dose-dependently suppressed by intracerebroventricular infusion or even a systemic administration of PACAP38. Systemic administration of PACAP38 also reduced infarct size in the brain after a transient focal ischemia induced by occlusion of the middle cerebral artery in rats. This indicates that a therapeutic concentration of the peptide can be reached in the brain parenchyma after systemic intravenous administration of the peptide. Indeed, despite the relatively large size of the peptide, PACAP38 is transported from the blood to the brain across the blood– brain barrier with a high efficacy in a saturable manner, and the efficacy of the transport is six times greater than morphine. In neuron/astroglia cocultures, PACAP completely prevented neuronal death induced by gp120, with a bimodal dose response, peaking at 10−13 M and 10−10 M, respectively. In a similar study, VIP showed a single dose-response curve, with the peak in the nanomolar range. The finding suggests that PACAP interacts with PAC1-R as well as VIP receptors. In neuronal enriched cultures, PACAP shows a single bell-shaped dose-response curve as does VIP, suggesting that glia are involved in the neuroprotective mechanism. Activitydependent neurotrophic protein (ADNP), which was found to be expressed by stimulation of VIP in astrocyte cultures, is also expressed by PACAP38. RANTES and other chemokines and cytokines also appear to play a role in PACAP-mediated neuroprotection in gp120induced neuronal death. Although PACAP was discovered during an attempt to isolate a novel hypothalamic-hypophysiotrophic hormone, the role of PACAP as a hypophysiotrophic hormone has not been fully elucidated. The presence of PACAP in the hypothalamus and innervation of its neuroterminals in both the external and internal zones of the ME, the higher concentration of PACAP in the hypophysical portal blood than the peripheral blood, the presence of its receptors on the pituitary cells, and the ability of PACAP to directly stimulate adenylate cyclase in the pituitary cells indicate that PACAP is indeed a physiological hypophysiotrophic hormone. PACAP stimulates the release of GH, prolactin, ACTH, and LH in vitro under certain conditions, but to lesser magnitudes than the respective classic releasing hormones [18]. Unlike classic releasing hormones, all pituitary cell types and folliculostellate (FS) cells express their receptors. Cytofluorometric studies, conducted on dispersed rat pituitary cells, showed that PACAP

PACAP/VIP induced calcium mobilization in all categories of endocrine cells. The most marked response of the pituitary cells to the PACAP is IL-6 release from FS cells; thus PACAP may function as a trophic hormone, rather than as a releasing hormone. Follicular granular cells extend processes between granular cells and regulate growth and differentiation of these cells by releasing trophic factors, including IL-6. Innervation of VIP containing nerve terminals in the pituitary stalk is less marked than PACAP. Yet, VIP has been suggested as a prolactin releasing factor in the presence of estrogens. In the mammalian pituitary gland, simultaneous inhibition of pituitary VIP mRNA expression and VIP release seems to be required for dopaminergic inhibition of prolactin mRNA expression and prolactin release.

PATHOPHYSIOLOGY OF PACAP/VIP Many biological actions of PACAP and VIP have been described above. However, these effects may not necessarily represent their physiological actions. The true physiological roles in the body may be best revealed when the expression of their genes is interfered with. Several laboratories generated PACAP or PAC1-R-deficient animals and reported their phenotypes. One report on VPAC2-R deficient animals also has appeared [5]. In Drosophila, mutation of a homolog of the PACAP gene, amnestic, produced impaired olfactory-related learning and altered sensitivity to alcohol. Otto et al. [15] generated mice lacking PACAP-specific receptor, PAC1-R. PAC1-R-deficient mice showed a deficit in hippocampus-dependent associative learning, accompanied on the cellular level by an impairment of mossy fiber long-term potentiation (LTP). In mice, the PAC1-R within the hippocampus is localized presynaptically in the mossy fiber synapse and mossy fiber LTP depends on a presynaptic rise of calcium and cAMP. Therefore, the extreme conservation of PACAP and PAC1-R might implicate their involvement in a phylogenetically old learning paradigm—that is, associative learning. Moreover, it was demonstrated that LTP in vivo in the dentate gyrus of PAC1-R-deficient mice and heterozygous PACAP (PACAP+/−) was induced by both suprathreshold and threshold tetanic stimulation. However, the population spike at threshold but not suprathreshold LTP decreased significantly in both PAC1-R-deficient and heterozygous PACAP mice. At threshold, LTP of PACAP+/− was impaired more than that of PAC1-R−/− mice. Thus, both PACAP and PAC1-R could contribute to the establishment of LTP. Furthermore, the surviving adult PACAPdeficient mice displayed remarkable behavioral changes: they exhibited hyperactive and explosive jumping

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behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. On the other hand, PAC1-R-deficient mice also displayed the similar phenotype of altered emotional behavior as PACAP-deficient mice—that is, reduced anxiety and elevated locomotion [16]. These findings suggest that both PACAP and PAC1-R might play a pivotal role in learning and memory function. Light information reaches the suprachiasmatic nucleus through a subpopulation of retinal ganglion cells that contain both glutamate and PACAP. Although the role of glutamate in this pathway has been well studied, the involvement of PACAP and its receptors is only beginning to be understood. To investigate the function of PACAP in vivo, Colwell et al. [2] demonstrated that PACAP-deficient mice exhibited significant impairment in the magnitude of response to brief light exposures, with both light-induced phase delays and advances of the circadian system being affected. This mutation equally impacted phase shifts induced by bright and dim light exposure. Despite these effects on phase shifting, the loss of PACAP had only limited effects on the generation of circadian oscillation, as measured by rhythms in wheel-running activity. Unlike melanopsin-deficient mice, the mice lacking PACAP exhibited no loss of function in the direct light-induced inhibition of locomotor activity—that is, masking. Finally, the PACAP-deficient mice exhibited normal phase shifts in response to exposure to discrete dark treatments. Thus, the loss of PACAP produced selective deficits in the light response of the circadian system. Although phenotypes in PACAP-deficient or PAC1R-deficient mice reported by different laboratories varied considerably, all reported early death of these animals. The adrenal gland is important for homeostatic response to metabolic stress. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Costaining with PACAP antibody and the vesicular acetylcholine transporter revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. PACAP-deficient mice had otherwise normal cholinergic innervations and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia [8]. However, insulin-induced hypoglycemia was more profound and longer lasting in PACAP-deficient mice, and was associated with a dose-related lethality absent in wild type mice. Failure of PACAP-deficient mice to adequately counter-regulate plasma glucose levels could be accounted for by impaired long-term secretion of adrenaline, secondary to a lack of induction of tyrosine

680 / Chapter 94 hydroxylase that normally occurs after insulin hypoglycemia in wild-type mice, and consequent depletion of adrenomedullary adrenaline stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress and PACAP appears to function as an “emergency response” cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under pathophysiological conditions by a neuropeptide. The Sherwood group [7] reported that most of the PACAP-deficient mice died within the first two weeks after birth in a wasted state with lipid droplets in the liver, heart, and skeletal muscle cells. More recently, Gray [8] of the same group reported that the premature death of the PACAP-deficient mice that occurred when the animals were raised at 21°C resulted from impaired adaptive thermogenesis. The premature death of PACAP-deficient mice was prevented when these animals were raised at an environmental temperature of 24°C. Furthermore, when the 7-day-old mice were separated from the mother and placed at 21°C, the PACAP null mice showed a greater loss of core body temperature compared with the wild type mice, indicating an impaired thermal adaptation in the PACAPdeficient mice. Thermogenesis in response to cold stress takes place by two different mechanisms: adaptive thermogenesis that occurs in the brown adipose tissues and shivering thermogenesis in the skeletal muscle. In the neonate and small rodents, adaptive thermogenesis that takes place in the brown adipose tissues plays a major role in the production of heat in a cold environment. Adaptive thermogenesis is mediated by norepinephrine, which is released from sympathetic nerve endings distributed in the brown adipocytes and the blood vessels in the brown adipose tissues. The importance of norepinephrine and epinephrine in thermogenesis is supported by the finding that mice deficient in dopamine β-hydroxylase, the enzyme that catalyzes the conversion of dopamine to norepinephrine, are highly cold sensitive. In short, Gray et al. [8] provided important information about the physiological role of PACAP in the adaptation of the living body to a cold environment, through adaptive thermogenesis by the brown adipose tissues. The adaptive action of the low temperature and the protective action of PACAP to other stresses appear to be mediated by its stimulatory action to sustain the production of catecholamines in the sympathetic nerves and the adrenal medulla. Indeed gene targeting to create null mutations in mice is a powerful new tool in biology that will allow the molecular dissection of complex phenotypes. However, the attempt to interpret the phenotypical changes which arise in null-mutant mice are subject to several caveats [4]. For example, the ability to disrupt

a single gene in a targeted manner might lead one to overlook the effects of other genes. Ignoring the genetic background might lead to misinterpretation of results. Interpretation of the phenotype of gene-deficient mice must be made with these cautions.

References [1] Brenneman DE, Hill JM, Gozes I. Vasoactive intestinal peptide in the central nervous system: the American College of Neuropsychopharmacology, 2000. [2] Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Waschek JA. Selective deficits in the circadian light response in mice lacking PACAP. Am J Physiol Regul Integr Comp Physiol 2004;287: R1194–1201. [3] Eipper BA, Green CB, Campbell TA, Stoffers DA, Keutmann HT, Mains RE, Ouafik L. Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM). J Biol Chem 1992;267: 4008–4015. [4] Gerlai R. Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci 1996;19: 177–181. [5] Goetzl EJ, Voice JK, Shen S, Dorsam G, Kong Y, West KM, Morrison CF, Harmar AJ. Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide. Proc Natl Acad Sci USA 2001;98: 13854–13859. [6] Gottschall PE, Tatsuno I, Miyata A, Arimura A. Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide. Endocrinology 1990;127: 272–277. [7] Gray SL, Cummings KJ, Jirik FR, Sherwood NM. Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism. Mol Endocrinol 2001;15: 1739–1747. [8] Gray SL, Yamaguchi N, Vencova P, Sherwood NM. Temperaturesensitive phenotype in mice lacking pituitary adenylate cyclaseactivating polypeptide. Endocrinology 2002;143: 3946–3954. [9] Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 1998;50: 265–270. [10] Inooka H, Endo S, Kitada C, Mizuta E, Fujino M. Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues. Conformation determined by 1H NMR and CD spectroscopies and distance geometry in 25% methanol solution. Int J Pept Protein Res 1992;40: 456–464. [11] Laburthe M, Couvineau A. Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regul Pept 2002;108: 165–173. [12] Li M, Mbikay M, Arimura A. Pituitary adenylate cyclaseactivating polypeptide precursor is processed solely by prohormone convertase 4 in the gonads. Endocrinology 2000;141: 3723– 3730. [13] Li M, Shuto Y, Somogyvari-Vigh A, Arimura A. Prohormone convertases 1 and 2 process ProPACAP and generate matured, bioactive PACAP38 and PACAP27 in transfected rat pituitary GH4C1 cells. Neuroendocrinology 1999;69: 217–226. [14] Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH. Isolation of a novel 38 residue-

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hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989;164: 567–574. Otto C, Kovalchuk Y, Wolfer DP, Gass P, Martin M, Zuschratter W, Grone HJ, Kellendonk C, Tronche F, Maldonado R, Lipp HP, Konnerth A, Schutz G. Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice. J Neurosci 2001;21: 5520–5527. Otto C, Martin M, Wolfer DP, Lipp HP, Maldonado R, Schutz G. Altered emotional behavior in PACAP-type-I-receptordeficient mice. Brain Res Mol Brain Res 2001;92: 78–84. Pisegna JR, Wank SA. Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor. Proc Natl Acad Sci USA 1993;90: 6345–6349. Rawlings SR, Hezareh M. Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal poly-

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peptide receptors: actions on the anterior pituitary gland. Endocr Rev 1996;17: 4–29. Said SI, Mutt V. Isolation from porcine-intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur J Biochem 1972;28: 199–204. Said SI, Mutt V. Polypeptide with broad biological activity: isolation from small intestine. Science 1970;169: 1217–1218. Sherwood NM, Krueckl SL, McRory JE. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr Rev 2000;21: 619–670. Sundler F, Ekblad E, Absood A, Hakanson R, Koves K, Arimura A. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 1992;46: 439–454. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52: 269–324.

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95 Neuropeptide Y YVAN DUMONT AND REMI QUIRION

ABSTRACT

(NPY) [32]. These two peptides contained a tyrosine residue at their N- and C-termini, 36 amino acids possessing structural homologies with the PPs. All three peptides were thus included in the same family usually designated as the Y, NPY, or PP family. Further information on this peptide family is provided in the ingestive and gastrointestinal peptide sections. Antibodies were developed against NPY, and immunohistochemical and radioimmunoassay techniques revealed the presence of very high amounts of NPY-ir in the mammalian CNS, including brain, and established that the PP-like ir previously observed in brain tissues was in fact due to cross reactivity of the antibody used with NPY [13]. NPY is believed to be exclusively expressed in the central and peripheral nervous systems, while PYY and PPs appear to be mostly restricted to endocrine cells of the intestine.

Neuropeptide Y (NPY) is one of the most abundant peptides found in various brain structures and one of the most evolutionary conserved ones. NPY and its homologs (peptide YY and the pancreatic polypeptides) have been implicated in several biological processes including food intake, anxiety, and depression-related behaviors, neuronal excitability and seizures, circadian rhythms, alcohol consumption, neuroendocrine secretions, nociception, and cardiorespiratory functions. The various effects of NPY in the central nervous system are mediated by the activation of at least five receptor subtypes designated as Y1, Y2, Y4, Y5, and y6, the last one not being functionally expressed in human and rat. We review here some of the unique features of this major peptide family.

DISCOVERY OF NPY AND HOMOLOGS

STRUCTURE OF THE PRECURSOR mRNA/GENE

Three decades ago, Kimmel and collaborators [19] isolated a 36-amino-acid peptide from the chicken pancreas and termed it avian pancreatic polypeptide (aPP). Few years later, a strong PP-immunopositive signal was observed in the central nervous system (CNS) of several species [24]. These results were subsequently confirmed by other investigators, suggesting the presence of a PPlike protein in mammalian brains. However, the isolation of genuine PP proteins from the CNS remained fruitless. The nature of the PP-like immunoreactive (ir) materials detected in mammalian brains was resolved in 1982 with the discovery by Tatemoto and collaborators of two peptides isolated from porcine intestine and brain extracts; the first one was called peptide YY (PYY); while the second became known as neuropeptide Y Handbook of Biologically Active Peptides

The human gene coding for NPY is located on chromosome 7 and nonisotopic in situ hybridization revealed that it is on 7q15.1 in proximity to the HOXA cluster (7q15–q14). It exists as a single-copy gene [6]. Early analysis of the gene sequence revealed that the transcription unit spans approximately 8 kilobase pairs divided in four exons separated by three introns of approximately 965, 4300, and 2300 bp as well as 5′ and 3′-untranslated domains (Fig. 2). The 5′ nontranscribed region of the NPY gene regulates the expression of the NPY mRNA and contains the sequence ATAAA or TATA box, as well as nucleotide sequences for DNA-binding proteins such as CAAT, AP1, and several SP1 [6]. The mRNA transcript includes the

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FIGURE 1. Schematic distribution of NPY-ergic neurons. Levels of expression of NPY are expressed in gray. Dark gray represents the highest amount of expression, while light gray shows the lowest ones.

7p22 7p21

7q21

Exon 2

7p15 7p14 7p13 7p12 7p11+2 7p11+1 7q11+1 7q11+2

7q22

7q31

7q32 7q33 7q34 7q35 7q36

Exon 1

Exon 3

Intron 1 965 bp

Intron 2 4070 bp

188 bp

Exon 4 Intron 3 2083 bp

81 bp

5’ Untranslated domain

FIGURE 2.

3’ Untranslated domain

Schematic representation of the human NPY gene organization.

first exon, which contains the 5′-untranslated domain; the second exon, which codes for the initiating codon and the main part of the mature NPY sequence up to glutamine 34; the third exon, which codes for the last two amino acids, the glycine amine donor site, the dibasic cleavage site, and the main portion of the carboxy terminal peptide of neuropeptide Y (CPON);

and the fourth exon, which includes the end of CPON and the 3′-untranslated region, including the polyadenylation recognition signal AATAAA (Fig. 2). The NPY mRNA consists of 551 bp and translates for a 97amino-acid precursor protein (Fig. 3). Comparable NPY gene organization has been reported in various species [6].

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process and ensures that the translation of the polypeptide coding mRNA occurs at the surface of the rough endoplasmic reticulum. The second function of the signal sequence is to facilitate the passage of the nascent polypeptide through the lipid bilayer of the endoplasmic reticulum into the lumen to be packed into secretory vesicles [6]. The 97-amino-acid pre-pro-NPY is then cleaved by signal peptidases to generate a 69-amino-acid peptide, or pro-NPY, and the signal peptide [6]. The pro-NPY is next processed by enzymatic cleavage to generate a 39-amino-acid peptide that contains NPY and the amidation proteolytic site motif (glycine-lysinearginine) and a 30-amino-acid residue peptide usually referred as CPON [6]. The C-terminal lysine and arginine are removed by a carboxypeptidase from the immature NPY and the glycine residue donates its amino group to generate the amide of the C-terminal tyrosine residue of NPY [6]. NPY and CPON are then stored in vesicles and coreleased upon stimulation. In contrast to NPY, the role of CPON is mostly unknown.

DISTRIBUTION OF NPY mRNA AND PROTEIN

FIGURE 3. Representation of the human NPY mRNA and the pre-pro-NPY protein. Light gray, the signal peptide; Midgray, the mature NPY in bold letters and the amidation proteolytic site (GRK), black; CPON, dark gray. Black boxes represent the 5′ and 3′ untranslated domains.

PROCESSING OF THE NPY PRECURSOR As for most low-molecular-weight peptides, NPY is synthesized as a precursor protein containing a hydrophobic signal peptide (highlighted in light gray), the mature peptide NPY (highlighted in mid-gray), the amidation-proteolytic site (highlighted in black), and the carboxy-terminal extension CPON (highlighted in dark gray) (Fig. 3) [6]. The signal sequence has two main functions. In the early stage of translation, as the first amino acid residue of the nascent polypeptide emerges from the large subunit of the ribosome, this region of the nascent polypeptide interacts with the signal recognition particle, a ribonucleoprotein, resulting in slowing down translation in the cytoplasmic space [6]. The complex (ribosome-nascent chain-signal recognition particle) is then targeted to the endoplasmic reticulum, where it interacts with the signal recognition particle receptor. This event reactivates the translation

Anatomical studies have demonstrated the presence of important quantities of NPY-like ir material and NPY mRNA in the brain of multiple species including the rat, mouse, and human, and a high degree of similarity in the distribution of NPY-ergic neurons is observed between species [13]. In situ hybridization and immunostaining studies revealed that NPY mRNA and immunoreactive materials are distributed very similarly [13]. In mammalian brain, NPY-ergic neurons are found in interneurons, as well as long projection neurons. While interneurons are mostly located in the forebrain, projection neurons are located, for example, in the arcuate nucleus, lateral geniculate nucleus, and various brainstem nuclei [13]. Schematic distribution of NPY-ir is presented in Fig. 1. Enrichment in NPY-ergic neurons varies between different cortical areas, with the highest concentrations found in the cingulate and temporal cortices and the lowest in the occipital lobe. Cortical NPY-positive cells are mostly interneurons (13). In the hippocampus, large quantities of NPY-containing cells are located in the hilus of the dentate gyrus, striatum, and orien layers of the CA2 and CA3 subfields, and near or within the stratum pyramidale of the CA1 subfield [13]. The majority of this innervation is from local projections, but some NPY-like ir terminals may have extrahippocampal origins. In the striatum, the majority of NPYergic neurons are interneurons with the highest densities located in the nucleus accumbens and bed nucleus of the stria terminalis [13]. The anterior

686 / Chapter 95 olfactory nucleus, olfactory tubercle, islands of Calleja, lateral septum, and tenia tecta also contain important amounts of NPY-like ir neurons. The hypothalamus is the structure that contains the highest levels of NPY. NPY-like ir and mRNA-containing cell bodies are mainly found in the arcuate nucleus and lateral hypothalamus [13], while fibers and terminals spread throughout this region. The suprachiasmatic nucleus is innervated by NPY axons originating from the ventral lateral geniculate nucleus [13]. NPY’s innervation of the paraventricular nucleus of the hypothalamus arises from the brainstem with shorter projections originating in the arcuate nucleus [13]. The amygdaloid complex is also an area that contains several NPY-like ir cell bodies and numerous fibers. On the other hand, in most thalamic nuclei and in the cerebellum, no or very low levels of NPY-like material are expressed. The only thalamic nucleus that contains significant amounts of NPY is the lateral geniculate nucleus with fibers projecting to the suprachiasmatic nucleus. Significant amounts of NPY-like ir neurons are also found in the inferior colliculus, central gray area, medulla oblongata, lateral reticular nucleus, raphe nuclei, and locus coeruleus. The dorsal horn of the spinal cord also expresses significant amounts of NPY, especially under pathological conditions.

STRUCTURE OF NPY-LIKE PEPTIDES Few investigators have evaluated the secondary and tertiary structures of NPY and homologs. Using a combination of molecular modelling and structural dynamics, comparison of the possible structure of NPY with that of aPP has shown that structural elements found in aPP were maintained in NPY (2). In the model proposed, N- and C-termini are stabilized by the intramolecular association of the hydrophobic moieties of the polyproline type II helix (residues 1–9) and the amphiphilic α-helix (residues 14 –30), these two structures being connected by a type II β-turn [2]. This confers a hairpin-like structure to the NPY molecule, where the tyrosine residue in position 1 and amino acid residues 30–36 are located in close proximity to each other, a structure often referred to as the PP-fold [2]. Structure–activity studies have also demonstrated that the central segment of NPY, amino acid residues 19–23, is important in conferring the helical conformation and ensuring high affinity for various NPY receptor subtypes [4].

RECEPTORS AND SIGNALING CASCADES Thus far, five NPY receptor subtypes have been cloned and designated as Y1, Y2, Y4, Y5, and y6 [25].

They all belong to the subfamily of seven transmembrane G protein-coupled receptors type 1 [10]. They are expressed in all mammalian brains except for the y6 subtype, which is restricted to only few species such as the mouse, rabbit, and dog [10]. Rather surprisingly, NPY receptors display relatively low sequence homologies between themselves and appear to be among the most divergent receptor families [21]. Some NPY receptor subtypes even have higher homology for other families of G protein-coupled receptors [10]. The detailed CNS distribution of the various NPY receptor subtypes has also been evaluated in various species. Generally, the Y1 and Y2 subtypes are the most abundant NPY receptors expressed in the CNS, while the Y4 and Y5 classes are found in much lower amounts [8, 9]. NPY receptors are usually coupled to Gi/o leading to the inhibition of the production of cAMP. However, coupling to other signal transduction pathways can also occur. In HEK293 cells transfected with the human Y1 receptor cDNA, the receptor couples to a pertussis toxin-sensitive G protein that mediated the inhibition of cAMP accumulation while in CHO cells this same receptor is associated with the elevation of intracellular Ca++ levels [10, 25]. Likewise, transfection of different cell types with the human Y2 receptor cDNA can generate a receptor that can couple to a pertussis toxinsensitive G protein mediating the inhibition of cAMP production and the elevation of intracellular Ca++ levels [10, 25]. Similar findings have been reported for the human and rat Y5 receptor subtype [10, 25].

BIOLOGICAL ACTIONS Numerous studies have addressed the biological functions of NPY and homologs in the CNS and demonstrated a broad range of effects (for most recent data see [1, 3, 7–9, 11, 12, 14, 15, 18, 22, 23, 26, 27, 29–31, 34, 35, 37]). Intracerebroventricular (icv) injections of NPY as well as direct administration of this peptide into specific nuclei such as the paraventricular and perifornical hypothalamic nuclei, have shown that it is the most potent substance known thus far to stimulate feeding behaviors and water consumption in various species including rat, mouse, sheep, pig, rabbit, and pigeon. NPY has also been shown to increase the respiratory quotient indicative of increased carbohydrate utilization as energy substrates, revealing its role in catabolism. It has also been reported that NPY can facilitate learning and memory processes, modulate locomotor activities, produce hypothermia, decrease sexual behavior, shift circadian rhythms, modulate cardiorespiratory parameters, produce antinociceptive effects, and generate anxiolytic-like effects. The secre-

Neuropeptide Y tion of neuroendocrine hormones is also altered by NPY and congeners. For example, NPY modulates the release of luteinizing hormone releasing hormone, and induces the release of corticotrophin releasing hormone. Several of these effects are mediated by a given NPY receptor subtype and appear to be physiologically relevant.

PATHOPHYSIOLOGICAL IMPLICATIONS Based on data obtained using NPY antibodies, receptor antagonists, antisense oligonucleotides, siRNA, and knockout and transgenic animals, it has been suggested that NPY could be implicated in various pathophysiological disorders including feeding and satiety, cardiovascular functions, memory impairment and cognition, anxiety- and depression-related behaviors, pain, epilepsy, alcohol consumption, and drug dependence [1, 3, 8, 9, 11, 12, 14, 23, 27, 34, 37]. For example, neutralizing endogenous NPY effects by the administration of NPY antibody into specific brain regions such as the hippocampus induced amnesia in the rat [30], while NPY antibodies administered in the ventromedial or paraventricular hypothalamic nuclei induced decreases in food intake. Similarly, blocking the synthesis of NPY by icv administration of NPY antisense oligonucleotides resulted in decreased food intake and attenuation of the progesterone-induced LH surge [15]. Injection of an NPY Y1 receptor antisense induced marked anxiogenic-like behavior [12]. Moreover, NPY-deficient mice exhibited mild spontaneous seizures, while kainateinduced seizures resulted in death in these animals, which can be prevented by the exogenous administration of NPY [38]. Furthermore, NPY-deficient mice are alcohol preferring and less sensitive to the sedative effect of alcohol, while transgenic mice overexpressing NPY drank lower amounts of alcohol [34, 35]. Additionally, young and old transgenic rats were shown to be less anxious than wild type rats [5, 12]. Furthermore, NPY Y2 knockout mice were found to possess anxiolytic-like behavior [28] and to drink less alcohol as compared with wild-type mice [33]. Interestingly, similar results were obtained using Y2 receptor antagonists [36]. In humans, Karvonen and collaborators [17] have identified a 1128T-C polymorphism that resulted in substitution of leucine by proline at residue 7 in the signal peptide part of pre-pro-NPY, and its frequency was found to be higher in alcohol-dependent subjects [20]. Finally, Y1 knockout mice display highly aggressive behaviors [16]. Hence, it would appear that NPY and its receptors could be associated with various aberrant CNS behaviors, suggesting their possible relevance in the development of new therapeutic avenues to treat neurological and mental illnesses.

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Acknowledgment Supported by the Canadian Institutes of Health Research (CIHR).

References [1] Baraban, S. C. Neuropeptide Y and epilepsy: recent progress, prospects and controversies. Neuropeptides 2004;38:261– 265. [2] Beck-Sickinger, A. G. The importance of various parts of the NPY molecule for receptor recognition. In: Grundemar, L.; Bloom, S. R., Eds. Neuropeptide Y and Drug Development. London, UK: Academic Press; 1997:107–126. [3] Berglund, M. M.; Hipskind, P. A.; Gehlert, D. R. Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp. Biol. Med. (Maywood.) 2003;228:217–244. [4] Cabrele, C.; Wieland, H. A.; Langer, M.; Stidsen, C. E.; BeckSickinger, A. G. Y-receptor affinity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y5receptor ligands with picomolar affinity. Peptides 2001; 22:365–378. [5] Carvajal, C. C.; Vercauteren, F.; Dumont, Y.; Michalkiewicz, M.; Quirion, R. Aged neuropeptide Y transgenic rats are resistant to acute stress but maintain spatial and non-spatial learning. Behav. Brain Res. 2004;153:471–480. [6] Cerda-Reverter, J. M.; Larhammar, D. Neuropeptide Y family of peptides: structure, anatomical expression, function, and molecular evolution. Biochem. Cell Biol. 2000;78:371–392. [7] Chronwall, B. M.; Zukowska, Z. Neuropeptide Y, ubiquitous and elusive. Peptides 2004;25:359–363. [8] Dumont, Y.; Chabot, J. G.; Quirion, R. Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: Focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides. Peptides 2004;25:365–391. [9] Dumont, Y.; Jacques, D.; St. Pierre, J. A.; Tong, Y.; Parker, R.; Herzog, H.; Quirion, R. Neuropeptide Y, peptide YY and pancreatic polypeptide receptor proteins and mRNAs in mammalian brains. In: Quirion, R.; Bjorklund, A.; Hokfelt, T., Eds. Handbook of Chemical Neuroanatomy, Vol 16 Peptide Receptors, Part 1 Peptide Receptors, Part 1. London, UK: Elsevier; 2000:375–475. [10] Dumont, Y.; Redrobe, J. P.; Quirion, R. Neuropeptide Y receptors. In: Pangalos, M. N.; Davies, C. H., Eds. Understanding G Protein-coupled Receptors and Their Role in the CNS Molecular and Cellular Neurobiology series. Oxford, UK: Oxford University Press; 2002:372–401. [11] Gehlert, D. R. Introduction to the reviews on neuropeptide Y. Neuropeptides 2004;38:135–140. [12] Heilig, M. The NPY system in stress, anxiety and depression. Neuropeptides 2004;38:213–224. [13] Hendry, S. H. Organization of neuropeptide Y neurons in the mammalian central nervous system. In: Colmers, W. F.; Wahlested, C., Eds. The Biology of Neuropeptide Y and Related Peptides. Totawa, NJ: Humana Press, Inc.; 1993:65–156. [14] Kalra, S. P.; Kalra, P. S. NPY and cohorts in regulating appetite, obesity and metabolic syndrome: beneficial effects of gene therapy. Neuropeptides 2004;38:201–211. [15] Kalra, S. P.; Kalra, P. S. NPY—an endearing journey in search of a neurochemical on/off switch for appetite, sex and reproduction. Peptides 2004;25:465–471. [16] Karl, T.; Lin, S.; Schwarzer, C.; Sainsbury, A.; Couzens, M.; Wittmann, W.; Boey, D.; von Horsten, S.; Herzog, H. Y1 recep-

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[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

tors regulate aggressive behavior by modulating serotonin pathways. Proc. Natl. Acad. Sci. USA 2004;101:12742–12747. Karvonen, M. K.; Pesonen, U.; Koulu, M.; Niskanen, L.; Laakso, M.; Rissanen, A.; Dekker, J. M.; Hart, L. M.; Valve, R.; Uusitupa, M. I. Association of a leucine(7)-to-proline(7) polymorphism in the signal peptide of neuropeptide Y with high serum cholesterol and LDL cholesterol levels. Nat. Med. 1998;4:1434–1437. Kask, A.; Harro, J.; von Horsten, S.; Redrobe, J. P.; Dumont, Y.; Quirion, R. The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y. Neurosci. Biobehav. Rev. 2002;26:259–283. Kimmel, J. R.; Hayden, L. J.; Pollock, H. G. Isolation and characterization of a new pancreatic polypeptide hormone. J. Biol. Chem. 1975;250:9369–9376. Lappalainen, J.; Kranzler, H. R.; Malison, R.; Price, L. H.; Van Dyck, C.; Rosenheck, R. A.; Cramer, J.; Southwick, S.; Charney, D.; Krystal, J.; Gelernter, J. A functional neuropeptide Y Leu7Pro polymorphism associated with alcohol dependence in a large population sample from the United States. Arch. Gen. Psychiatry 2002;59:825–831. Larhammar, D. Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide. Regul. Pept. 1996;65:165–174. Leibowitz, S. F.; Wortley, K. E. Hypothalamic control of energy balance: different peptides, different functions. Peptides 2004;25:473–504. Lin, S.; Boey, D.; Herzog, H. NPY and Y receptors: lessons from transgenic and knockout models. Neuropeptides 2004;38: 189–200. Loren, I.; Alumets, J.; Hakanson, R.; Sundler, F. Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: preliminary immunocytochemical observations. Cell Tissue Res. 1979;200:179–186. Michel, M. C.; Beck-Sickinger, A.; Cox, H.; Doods, H. N.; Herzog, H.; Larhammar, D.; Quirion, R.; Schwartz, T.; Westfall, T. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. Rev. 1998;50:143–150. Morris, M. J.; Tortelli, C. F.; Hart, D. P.; Delbridge, L. M. Vascular and brain neuropeptide Y in banded and spontaneously hypertensive rats. Peptides 2004;25:1313–1319.

[27] Pedrazzini, T. Importance of NPY Y1 receptor-mediated pathways: assessment using NPY Y1 receptor knockouts. Neuropeptides 2004;38:267–275. [28] Redrobe, J. P.; Dumont, Y.; Herzog, H.; Quirion, R. Characterization of neuropeptide Y, Y2 receptor knockout mice in two animal models of learning and memory processing. J. Mol. Neurosci. 2004;22:159–166. [29] Redrobe, J. P.; Dumont, Y.; Quirion, R. Neuropeptide Y (NPY) and depression: From animal studies to the human condition. Life Sci. 2002;71:2921–2937. [30] Redrobe, J. P.; Dumont, Y.; St. Pierre, J. A.; Quirion, R. Multiple receptors for neuropeptide Y in the hippocampus: putative roles in seizures and cognition. Brain Res. 1999;848:153– 166. [31] Sajdyk, T. J.; Shekhar, A.; Gehlert, D. R. Interactions between NPY and CRF in the amygdala to regulate emotionality. Neuropeptides 2004;38:225–234. [32] Tatemoto, K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. USA 1982;79: 5485–5489. [33] Thiele, T. E.; Naveilhan, P.; Ernfors, P. Assessment of ethanol consumption and water drinking by NPY Y(2) receptor knockout mice. Peptides 2004;25:975–983. [34] Thiele, T. E.; Sparta, D. R.; Hayes, D. M.; Fee, J. R. A role for neuropeptide Y in neurobiological responses to ethanol and drugs of abuse. Neuropeptides 2004;38:235– 243. [35] Thiele, T. E.; Stewart, R. B.; Badia-Elder, N. E.; Geary, N.; Massi, M.; Leibowitz, S. F.; Hoebel, B. G.; Egli, M. Overlapping peptide control of alcohol self-administration and feeding. Alcohol Clin. Exp. Res. 2004;28:288–294. [36] Thorsell, A.; Rimondini, R.; Heilig, M. Blockade of central neuropeptide Y (NPY) Y2 receptors reduces ethanol self-administration in rats. Neurosci. Lett. 2002;332:1. [37] Vezzani, A.; Sperk, G. Overexpression of NPY and Y2 receptors in epileptic brain tissue: an endogenous neuroprotective mechanism in temporal lobe epilepsy? Neuropeptides 2004;38: 245–252. [38] Vezzani, A.; Sperk, G.; Colmers, W. F. Neuropeptide Y: emerging evidence for a functional role in seizure modulation. Trends Neurosci. 1999;22:25–30.

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96 Melanocortins SYLVIE JÉGOU, ROGER D. CONE, ALEX N. EBERLÉ, AND HUBERT VAUDRY

ABSTRACT

hypothesis was confirmed when Nakanishi et al. [27] reported the nucleotide sequence of a cDNA encoding a protein that encompasses both ACTH and β-LPH. This precursor protein that has the potential to generate the opioid peptide β-endorphin, three MSH sequences (α-, β-, and γ-MSH), and ACTH, was termed proopiomelanocortin (POMC) [25]. The POMC gene is primarily expressed in corticotroph cells of the pars distalis and melanotroph cells of the pars intermedia of the pituitary [11]. Besides, it has long been known that biologically active MSHs are also present in the hypothalamus [38] and that central administration of α-MSH facilitates learning and memory, elicits stretching, yawning, and grooming, and induces antipyretic effects [1, 10, 22], suggesting that melanocortins could also act as authentic neuropeptides. The occurrence of the highly potent opioid peptide β-endorphin within the precursor sequence further supported the notion that POMC could generate various neuroactive peptides. Finally, the cloning of melanocortin receptors (MC-Rs) [26] and the demonstration that some members of the MC-R family are expressed in the brain [13, 31] confirmed the existence of a brain melanocortin system.

Melanocortins are a family of related peptides comprising α-MSH, γ-MSH, and ACTH that all derive from the precursor protein POMC. In the brain, processing of POMC gives rise essentially to α-MSH and γ-MSH. A prominent group of POMC neurons is located in the arcuate nucleus of the hypothalamus, and these neurons project toward multiple brain regions. Melanocortins exert a wide range of biological activities in the brain, including control of feeding, grooming, social and sexual behaviors, central regulation of inflammation, fever, and cardiovascular functions, as well as neurotrophic activities. The central effects of melanocortins are mediated through three types of receptors, namely MC3-R, MC4-R, and MC5-R.

DISCOVERY The term melanocortins refers to a family of structurally related peptides that includes α-melanocytestimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotrophic hormone (ACTH). α-MSH and β-MSH were initially isolated and characterized from the porcine pituitary by virtue of their ability to induce darkening of frog skin [15, 17]. ACTH was first characterized from the ovine pituitary on the basis of its capacity to stimulate adrenocortical activity [23]. Finally, β-lipotropin (β-LPH), whose sequence encompasses βMSH, was isolated as a side fraction during the course of purification of ACTH from the ovine pituitary gland [24]. The observation that the α-MSH sequence corresponds to the first third of the ACTH molecule and that the β-MSH and β-endorphin sequences are part of β-LPH suggested the existence of a precursor-product mode of biosynthesis for this family of peptides. This Handbook of Biologically Active Peptides

STRUCTURE OF THE PROOPIOMELANOCORTIN mRNA/GENE The POMC genes of all species examined so far exhibit essentially the same structural organization with 3 exons separated by 2 large introns [11]. In mammals, the second exon encodes for the signal peptide and the first 18-amino-acid residues, while the third exon encodes for all bioactive POMC-derived peptides (Fig. 1). In mammals and amphibians, POMC encompasses four melanocortins, namely α-MSH, β-

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FIGURE 1. Structural organization of the human POMC gene and processing of the precursor in neurons. : paired basic residue cleavage sites where processing of POMC occurs to yield various peptide products. The numbers of the amino acid residues contributing to the cleavage site are indicated. K = Lys; R = Arg : MSH core sequence : site of N-glycosylation : site of O-glycosylation : site of phosphorylation CLIP: corticotropin-like intermediate lobe peptide JP: joining peptide (See color ground.)

MSH, γ-MSH, and ACTH (Fig. 2). In teleost fish, POMC lacks the γ-MSH region, whereas in elasmobranch fish POMC contains an additional MSH peptide named δ-MSH that is located between the ACTH and β-MSH sequences. The sequence of the POMC cDNA has been preserved in the regions coding for pro-γ-MSH/γ-MSH, ACTH/α-MSH, β-MSH, and βendorphin. The POMC sequence is more variable in the regions corresponding to the joining peptide (JP) and the N-terminal portion of β/γ-LPH. Most of the dibasic motifs that are typical cleavage sites for prohormone convertases are conserved across species, except for the Arg63 and Lys190 of human POMC that are lacking in the rat and mouse POMC sequences, indicating that in rodents γ3-MSH and γ-LPH cannot be further processed to generate γ-MSH and β-MSH, respectively. In contrast to other mammals that have only one POMC gene, the mouse also possesses a pseudogene. Several submammalian vertebrates, including xenopus, salmon, trout, and sturgeon, possess two functional POMC genes.

DISTRIBUTION OF POMC mRNA AND MELANOCORTINS IN THE BRAIN The arcuate nucleus (AN) of the hypothalamus and the nucleus of the solitary tract (NTS) in the medulla are the only two sites of POMC expression in the rodent brain (Fig. 3) [21, 29]. In primates, POMC expression has been demonstrated in the AN but has not yet been reported in the NTS. In rat, the predominant POMC neuronal population is located in the AN. POMC neurons project ventrally to the median eminence, rostrally to the anterior hypothalamic and medial preoptic nuclei, the nucleus of the stria terminalis, and the lateral septum, dorsally to the thalamic periventricular nucleus and the periventricular, paraventricular, dorsomedial, and posterior hypothalamic nuclei, laterally to the amygdala, and caudally to the substantia nigra, the dorsal raphe nucleus, the periaqueductal gray, the parabrachial nucleus, the locus coeruleus, the nucleus ambiguus, the lateral reticular nucleus, and the nucleus of the solitary tract (Fig. 3). NTS POMC neurons project

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NH2-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-ValLys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH

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FIGURE 2. Amino acid sequences of human melanocortins. All melanocortins share the invariant sequence His-Phe-Arg-Trp.

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FIGURE 3. Sagittal section of the rat brain depicting the distribution of POMC and AgRP neuronal systems. Solid lines denote high density and dashed lines denote moderate density of fibers.

within the nucleus solitarius, as well as to the nucleus reticularis gigantocellularis, the A1 group, and all along the spinal cord (Fig. 3).

PROCESSING OF PROOPIOMELANOCORTIN After removal of the signal peptide from the precursor protein, the first steps of POMC maturation consist

of glycosylation of pro-γ-MSH and ACTH, and phosphorylation of ACTH (Fig. 1), two events that apparently do not affect processing or release of the mature peptides [11]. Then, prohormone convertases 1 and 2 (PC1 and PC2) catalyze differential cleavage of POMC at dibasic or tetrabasic sites in a tissue-dependent manner. Thus, in corticotroph cells of the pars distalis, which only express PC1, cleavage occurs at three Lys-Arg sites to generate the large N-terminal fragment pro-γ-MSH, JP,

692 / Chapter 96 ACTH, and the large C-terminal peptide β-LPH. In melanotroph cells of the pars intermedia and in hypothalamic POMC neurons, which both express PC1 and PC2, processing of the precursor gives rise to the formation of shorter peptides including (Lys-)γ3-MSH, ACTH1–17, corticotrophin-like intermediate lobe peptide (CLIP), γ-LPH, and β-endorphin. (Lys-)γ3-MSH can be further processed to yield (Lys-)γ2-MSH. Carboxyterminal basic residues are trimmed from the cleaved peptides par carboxypeptidase E. Then, ACTH1–14 and (Lys-)γ2-MSH are C-terminally amidated by peptidylglycine-α-amidating-monooxygenase (PAM) to form desNα-acetyl α-MSH and (Lys)γ1-MSH, respectively. Finally, Nα-acetylation of des-Nα-acetyl α-MSH and β-endorphin may occur, and this last event profoundly affects the biological activity of the two peptides: mono- and diacetylation of the N-terminal serine residue of desNα-acetyl α-MSH generally enhances the biological potency of the peptide, while acetylation of the Nterminal tyrosine residue of β-endorphin suppresses its analgesic activity. In the rat brain, des-Nα-acetyl α-MSH is the prominent form in the hypothalamus, whereas mono- and diacetylated α-MSH species predominate in projection areas of POMC neurons, suggesting that acetylation of α-MSH happens at a late stage of axonal transport, just before or during exocytosis [19].

CONTROL OF BIOSYNTHESIS AND RELEASE OF BRAIN MELANOCORTINS Arcuate nucleus POMC neurons are innervated by axon terminals containing various aminergic or peptidergic neurotransmitters. Serotonin, acting through 5-HT2C receptors [9, 36], and glutamatergic agonists, via activation of NMDA receptors [41], have been shown to stimulate α-MSH secretion from hypothalamic neurons. Conversely, GABA and benzodiazepines acting through the GABAA receptor complex, and neuropeptide Y inhibit α-MSH release [4, 5], as well as POMC gene transcription [14, 20]. POMC neurons are also the targets of peripheral mediators of feeding behavior and energy homeostasis. Leptin and insulin directly activate POMC neurons and stimulate POMC mRNA expression [28]. Glucocorticoids, which exert a major inhibitory effect on POMC gene expression in pituitary corticotrophs, actually stimulate POMC transcription in hypothalamic neurons [30, 40].

MELANOCORTIN RECEPTOR FAMILY Five subtypes of MC-Rs have been cloned to date and termed MC1-R to MC5-R [35]. All MC-Rs belong to the G protein-coupled receptor superfamily and activate

the adenylyl cyclase-proteine kinase A pathway via coupling to Gs. The MC-R subtypes differ with respect to their anatomical distribution and ligand selectivity (Table 1). Thus, MC1-R is expressed almost exclusively in melanocytes and mediates effects of melanocortins on skin and coat pigmentation. MC2-R is expressed primarily in adrenocortical cells and mediates the effects of ACTH on corticosteroid secretion. MC3-R and MC4-R both occur in the brain (Fig. 4): MC3-R is expressed in discrete regions of the diencephalon and midbrain, notably in the hypothalamus and thalamus, while MC4-R is widely distributed in the CNS including the autonomic centers of the hindbrain and the spinal cord. MC5-R is primarily expressed in exocrine glands and in skeletal muscle but low levels of MC5-R mRNA are also found in a few regions of the rat brain, notably, in the olfactory bulb, cerebral cortex, striatum, and medulla. Two endogenous melanocortin receptor antagonists participate in the control of melanocortin signaling—that is, the agouti protein, expressed in dermal papillae, which confers the yellow color typical of agouti mice, and the agouti-related protein (AgRP), mainly expressed in the hypothalamus and adrenal gland, which acts as an orexigenic peptide [8]. AgRP-containing neurons of the arcuate nucleus project globally to the same hypothalamic and septal regions as POMC-containing neurons (Fig. 3), indicating that AgRP can actually modulate melanocortin neurotransmission.

STRUCTURE–ACTIVITY RELATIONSHIPS FOR MELANOCORTINS α-MSH has been the subject of extensive structureactivity relationship studies [11, 18]. The tetrapeptide His6-Phe7-Arg8-Trp9 is the minimal sequence exhibiting melanotrophic activity, while the N-terminal pentapeptide and the C-terminal tetrapeptide act as modulators of the linear central core peptide. The Met4 residue is particularly important since its oxidation considerably lowers the potency of α-MSH. The reverse β-turn conformation centered at the Phe7 residue plays a critical role for the biological activity of α-MSH. The N-terminal acetyl group protects the peptide from aminopeptidase degradation and enhances the actions of α-MSH on pigmentation, arousal, memory, attention, excessive grooming, and inhibition of food intake. Conversely, desacetyl-α-MSH is more effective than αMSH at blocking opiate analgesia. α-MSH and γ-MSH exhibit structural differences mainly within the Cterminal region. The natural γ-MSH ligands are still the most selective agonists of MC3-R and several studies have shown that the C-terminal portion of MSHs plays an important role in the interaction with MC3-R.

Melanocortins / 693 TABLE 1. Pharmacological properties and distribution of melanocortin receptor subtypes. Ligands Potency/Endogenous Antagonists

Tissue Distribution

MC1-R

α-MSH = ACTH > β-MSH >> γ-MSH/Agouti

MC2-R MC3-R MC4-R MC5-R

ACTH α-MSH = β-MSH = γ-MSH = ACTH/AgRP α-MSH = ACTH > β-MSH >> γ-MSH/Agouti, AgRP α-MSH > ACTH > β-MSH > γ-MSH

Melanocytes, keratinocytes Macrophages, leukocytes Adrenal cortex CNS, Gut, placenta CNS Exocrine glands, muscle, CNS

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MC3-R mRNA MC4-R mRNA FIGURE 4. Comparative distribution of MC3-R and MC4-R mRNA expression in the rat brain.

BIOLOGICAL ACTIONS WITHIN THE BRAIN Energy Homeostasis This topic is covered in the ingestive peptides section.

Stretching-Yawning Syndrome and Grooming One of the first reported behaviors induced by intracerebroventricular (ICV) injection of melanocortins are the stretching-yawning syndrome (SYS) and excessive grooming [12, 16]. The brain regions that respond to local injection of ACTH1–24 and α-MSH with SYS and grooming behavior are the hypothalamic periventricular regions surrounding the third ventricle including the paraventricular nucleus, the dorsomedial nucleus,

the anterior hypothalamic area, and the ventromedial nucleus [3]. Grooming is also elicited by local injection of ACTH1–24 in the periaqueductal gray or the substantia nigra. Excessive grooming is observed when animals are exposed to a novel environment and various studies converge to show the implication of brain endogenous melanocortins in this process. Structure–activity relationships indicate that MC4-R is probably involved in melanocortin-induced SYS and grooming behavior [1, 3].

Sexual and Social Behavior Local injection of α-MSH or ACTH1–24 in the hypothalamic periventricular region induces penile erection in rat [3], and MC4-R has been implicated in the

694 / Chapter 96 modulation of erectile and sexual function [37]. In contrast, ICV injection of ACTH1–24 or ACTH4–10 delays the copulatory behavior in inexperienced male rats, and ACTH1–24 reduces sexual performance. In female rats that exhibit a low level of receptivity, α-MSH stimulates lordosis, whereas in receptive females, α-MSH has the opposite effect. ICV administration of melanocortins generally reduces social interactions and increases aggressive behavior. The same effects are observed when α-MSH or ACTH4–10 is injected in the ventromedial hypothalamic nucleus or the septum, respectively. The MC4-R is involved in social interactions and mediates the anxiogenic effect of brain melanocortins [32]. In rodents, peripheral and central melanocortins appear to influence sexual activity and social interactions in a coordinate manner, since circulating α-MSH stimulates the release of pheromone from sebaceous and preputial glands, which in turn influence sexual behavior, while ACTH stimulates the secretion of glucocorticoids which promote aggressive behavior.

Antipyretic and Anti-inflammatory Activities Central administration of α-MSH provokes marked antipyretic effects in different species [11, 33]. In rabbit, α-MSH inhibits cytokine-induced fever and, reciprocally, fever causes an increase in α-MSH concentrations in the septum. In rat, α-MSH reduces lipopolysaccharide-evoked fever, and this effect is antagonized by an MC4-R antagonist. Enhanced febrile responses to interleukin-1 (IL-1) and to endotoxin have been reported in rabbit and rat after ICV injection of either an α-MSH antiserum or an MC3-R/MC4-R antagonist, indicating that endogenous α-MSH actually exerts antipyretic activity. Melanocortins inhibit different types of inflammatory responses including acute and chronic inflammation and contact hypersensitivity. ICV injection of α-MSH inhibits the response to tumor necrosis factor-α in endotoxin-induced brain inflammation as well as cutaneous inflammation provoked by application of cytokines. α-MSH can only reduce inflammation induced in the mouse hind paw when the spinal cord is intact, indicating that α-MSH probably acts through descending neurogenic pathways capable of modulating inflammation in peripheral tissues. With respect to neuroendocrine functions, α-MSH antagonizes the stimulatory effects of IL-1 on the hypothalamopituitary-adrenal axis in rodents and primates.

Nerve Regeneration Perinatal treatment with melanocortins enhances maturation of the CNS and accelerates maturation of the neuromuscular system. In various peripheral nerve

injury models, melanocortins are effective growth factors improving axonal regeneration, muscle reinnervation and recovery of sensory and motor functions. In addition, melanocortins stimulate neurite outgrowth in neuroblastoma cells and primary cultures of dorsal root ganglia and spinal cord neurons [34]. The stimulatory effect of α-MSH on Neuro 2A cells is blocked by a specific MC4-R antagonist. Since MC4-R is the only receptor subtype found in the spinal cord and in sympathetic ganglia, MC4-R likely mediates the neurotrophic effects of melanocortins. However, the ACTH4–9 analog ORG2766, which fails to activate MC-Rs, also enhances recovery of neuromuscular functions, suggesting that a distinct receptor which does not belong to the MC-Rs family may be involved. Nerve injury provokes an increase in POMC expression and ACTH/MSH immunoreactivity in mouse spinal cord motoneurons. ACTH/MSH levels are also higher in Wobler mice, which exhibit motoneuron disease, and in diabetic neuropathy. An increase in ACTH binding sites also occurs on diabetic, developing, and dystrophic muscle, suggesting that melanocortins may act as paracrine factors involved in the innervation of muscle, probably through MC5-R, which is uniquely expressed in muscle.

Cognitive Functions Melanocortins restore the deficient acquisition of shuttle box avoidance behavior in hypophysectomized rats. ACTH and α-MSH facilitate acquisition and delay extinction of active and passive avoidance responses and of rewarded behavior in intact rats, suggesting that these peptides influence motivation, attention, learning, and memory [10]. While α-MSH and β-MSH have similar positive effects on active and passive avoidance, γ-MSH facilitates extinction of active avoidance and attenuates passive avoidance behavior. The short chain analog ACTH4–7 is the smallest fragment that exhibits full activity on learning and memory, whereas the minimum sequence required to stimulate MC-Rs is ACTH6–9. Indeed, structure–activity studies strongly suggest that the effects of melanocortins on avoidance behavior are not mediated by a receptor of the MC-Rs family [1].

Cardiovascular Effects Melanocortins exert a variety of effects on the cardiovascular system [39]. ICV injection of γ2-MSH or ACTH4–10 induces a slight pressor effect that is not mediated through any of the identified MC-Rs but may involve receptors for RFamide peptides (see chapter on RFamide peptides), which have affinity for peptides that possess a C-terminal Arg-Phe-NH2 sequence like γ1-MSH. Microinjection of γ2-MSH into the caudal part

Melanocortins / 695 of the NTS causes bradycardia and reduces blood pressure. A depressor effect is also observed when α-MSH is injected into the dorsal motor nucleus of the vagus nerve, where MC4-R is abundantly expressed, and the response is blocked by an MC4-R antagonist indicating that the depressor activity of melanocortins is likely mediated via MC4-R. Stimulation of POMC neurons in the AN has a depressor effect that is mediated through the dorsal vagal complex, suggesting that endogenous melanocortins may play a role in the control of cardiovascular functions.

Interactions with Opiates The melanocortin and opiate systems have opposite activities in many tests and may be considered as functional antagonists [2, 33]. Central administration of α-MSH and ACTH causes hyperalgesia in various pain models and reverses the analgesic effect of morphine and β-endorphin. Melanocortins also antagonize morphine tolerance and dependence and even induce opiate withdrawal-like effects in naive animals. Reciprocally, chronic administration of morphine downregulates the expression of MC4-R in the striatum and periaqueductal gray, two regions implicated in drug reward and withdrawal.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS Cloning of MC-Rs, studies with genetic models, and development of selective MC-R ligands have opened new perspectives for the comprehension of the physiological roles of melanocortins. In particular, the role of α-MSH, MC3-R, and MC4-R in feeding and energy homeostasis as well as sexual activity has received a great deal of attention and has provided a framework to explore the melanocortin system for the treatment of obesity or other metabolic disorders, and sexual dysfunctions. Indeed, an MC-R agonist has proven to be effective in treating erectile dysfunction in a clinical trial [42]. The recent development of a selective, nonpeptidic MC4-R antagonist has revealed that MC4-R plays an important role in stress responses and in the regulation of emotional states, suggesting that blockade of MC4-R may be of therapeutic value for the treatment of stress-related disorders such as anxiety and depression [7]. α-MSH acts centrally to inhibit fever and inflammation, and a number of potential therapeutic targets for melanocortins have been proposed based on preclinical investigations in inflammatory disorders, such as acute and chronic inflammation diseases, brain inflammation, neurodegenerative diseases, and systemic host reactions [6].

References [1] Adan RAH. Effects of melanocortins in the nervous system. In Cone RD, editor. The melanocortin receptors. Totowa: Humana Press Inc; 2000. p. 109–41. [2] Alvaro JD, Tatro JB, Duman RS. Melanocortins and opiate addiction. Life Sci 1997;61:1–9. [3] Argiolas A, Melis MR, Murgia S, Schioth HB. ACTH- and alpha-MSH-induced grooming, stretching, yawning and penile erection in male rats: site of action in the brain and role of melanocortin receptors. Brain Res Bull 2000;51: 425–31. [4] Blasquez C, Jégou S, Tranchand Bunel D, Delbende C, Braquet P, Vaudry H. Central-type benzodiazepines inhibit release of alpha-melanocyte-stimulating hormone from the rat hypothalamus. Neuroscience 1991;42:509–16. [5] Blasquez C, Jégou S, Tranchand Bunel D, Fournier A, Vaudry H. Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein. Brain Res 1992;596:163–8. [6] Catania A, Gatti S, Colombo G, Lipton JM.Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev 2004;56:1–29. [7] Chaki S, Okuyama S. Involvement of melanocortin-4 receptor in anxiety and depression. Peptides 2005;26:1952–64. [8] Cone RD. Anatomy and regulation of the central melanocortin system. Nat Neurosci 2005;8:571–8. [9] Cowley MA. Hypothalamic melanocortin neurons integrate signals of energy state. Eur J Pharmacol 2003;480:3–11. [10] De Wied D. Melanotropins as neuropeptides. Ann NY Acad Sci 1993;680:21–8. [11] Eberle AN. The melanotropins: chemistry, physiology and mechanisms of action. Basel: Karger; 1988. [12] Ferrari W. Behavioural changes in animals after intracisternal injection with adrenocorticotrophic hormone and melanocytestimulating hormone. Nature 1958;181:925–6. [13] Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, DelValle J, Yamada T. Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 1993;268:15174–9. [14] Garcia de Yebenes E, Li S, Fournier A, St Pierre S, Pelletier G. Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus. Brain Res 1995;674: 112–6. [15] Geschwind II, Li CH, Barnabi L. Isolation and structure of melanocyte-stimulating hormone from porcine pituitary glands. J Am Chem Soc 1956;78:4494–5. [16] Gispen WH, Wiegant VM, Greven HM, de Wied D. The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structure-activity studies. Life Sci 1975;17:645–52. [17] Harris JI, Lerner AB. Amino-acid sequence of the alphamelanocyte-stimulating hormone. Nature 1957;179:1346–7. [18] Hruby VJ, Han G. The molecular pharmacology of alphamelanocyte stimulating hormone. Structure-activity relationships for melanocortins at melanocortin receptors. In Cone RD, editor. The melanocortin receptors. Totowa: Humana Press Inc; 2000. p. 239–61. [19] Jégou S, Tranchand Bunel D, Delbende C, Blasquez C, Vaudry H. Characterization of α-MSH related peptides released from rat hypothalamic neurons in vitro. Mol Brain Res 1989; 5:219–26. [20] Jégou S, Tong Y, Blasquez C, Pelletier G, Vaudry H. Activation of GABAA-benzodiazepine receptor complex inhibits proopiomelanocortin gene expression in the rat arcuate nucleus. Mol Cell Neurosci 1991;2:440–5.

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[33] Starowicz K, Przewlocka B. The role of melanocortins and their receptors in inflammatory processes, nerve regeneration and nociception. Life Sci 2003;73:823–47. [34] Strand FL, Zuccarelli LA, Williams KA, Lee SJ, Lee TS, Antonawich FJ, Alves SE. Melanotropins as growth factors. Ann NY Acad Sci 1993;680:29–50. [35] Tatro JB. Melanocortins receptor expression and function in the nervous system. In Cone RD, editor. The melanocortin receptors. Totowa: Humana Press Inc; 2000. p. 173–207. [36] Tiligada E, Wilson JF. Regulation of alpha-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus by serotonin and the interaction of serotonin with the dopaminergic system inhibiting peptide release. Brain Res 1989;503: 225–8. [37] Van der Ploeg LH, Martin WJ, Howard AD, Nargund RP, Austin CP, Guan X, Drisko J, Cashen D, Sebhat I, Patchett AA, Figueroa DJ, DiLella AG, Connolly BM, Weinberg DH, Tan CP, Palyha OC, Pong S, MacNeil T, Rosenblum C, Vongs A, Tang R, Yu H, Sailer AW, Fong TM, Huang C, Tota MR, Chang RS, Stearns R, Tamvakopoulos C, Christ G, Drazen DL, Spar BD, Nelson RJ, MacIntyre DE. A role for the melanocortin 4 receptor in sexual function. Proc Natl Acad Sci USA 2002;99: 11381–6. [38] Vaudry H, Tonon MC, Delarue C, Vaillant R, Kraicer J. Biological and radioimmunological evidence for melanocyte stimulating hormones (MSH) of extrapituitary origin in the rat brain. Neuroendocrinology 1978;27:9–24. [39] Versteeg DH, Van Bergen P, Adan RA, De Wildt DJ. Melanocortins and cardiovascular regulation. Eur J Pharmacol 1998; 360:1–14. [40] Wardlaw SL, McCarthy KC, Conwell IM. Glucocorticoid regulation of hypothalamic proopiomelanocortin. Neuroendocrinology 1998;67:51–7. [41] Wayman CP, Pike NV, Wilson JF. N-methyl-D-aspartate (NMDA) stimulates release of alpha-MSH from the rat hypothalamus through release of nitric oxide. Brain Res 1994;666:201–6. [42] Wessels H, Gralnek D, Dorr R, Hruby VJ, Hadley ME, Levin N. Effect of an alpha-melanocyte stimulating hormone analog on penile erection and sexual desire in men with organic erectile dysfunction. Urology 2000;56:641–6.

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97 Cocaine- and Amphetamine-Regulated Transcript (CART) CSABA FEKETE AND RONALD M. LECHAN

ABSTRACT

noncoding region generates two CART mRNAs approximately 700 and 900 bases long [15]. In addition, alternate splicing generates even further mRNA species due to the presence or absence of an in-frame 39 base insert in the coding region [15]. This results in the production of two proCART peptides with 102 or 89 amino acid residues, respectively [15]. Approximately one-third of the CART mRNA content in the rat brain contains the 39 nucleotide insert [15]. Two different CART genes are also present in the goldfish, resulting in the production of two different proCART peptides with 117 and 120 amino acid residues, respectively [52]. In contrast, although multiple polyA sites are also present in the human CART gene, only one site is utilized resulting in an approximately 900 base long CART mRNA [14]. In addition, human CART mRNA does not contain the 39 base long insert observed in the rat [14]. The chromosomal location of the CART gene has been identified in humans on chromosome 5 and in mice on chromosome 13 [2, 14]. Comparison of the cDNA sequence to that of the genomic DNA in different species reveals that in all instances, mature CART mRNA is encoded by three exons [2, 14, 15, 51].

Cocaine- and amphetamine-regulated transcript (CART) peptides are widely expressed in the central nervous system and in the pituitary. ProCART peptides are directed to secretory granules by a signal sequence and further processed by prohormone convertases into smaller biologically active peptides. These peptides have an important role in the regulation of a number of physiological processes in the central nervous system including reward, feeding, neuroendocrine function, pain, cardiovascular function, and gastrointestinal motility.

DISCOVERY Cocaine- and amphetamine-regulated transcript (CART) was discovered by Douglass et al. in 1995 as a striatal transcript induced by psychostimulant administration [15]. As revealed by differential display PCR, the acute administration of either cocaine or amphetamine is capable of inducing an approximately four- to fivefold increase in CART mRNA in the striatum, but not in other regions of the brain [15]. It was later recognized that a somatostatin-like peptide fragment isolated by Spiess et al. in 1981 [47] is identical to the N-terminal portion of CART 55–102 peptide.

PROCESSING OF CART PEPTIDES CART peptides derive from two precursor peptides of 129 and 116 amino acid residues that arise via alternative splicing of a single CART gene in rats and mice [15]. The longer form of the CART precursor contains a 13-amino-acid insert [15]. In contrast to rodents, only the shorter CART precursor exists in humans [14]. Both forms of pre-proCART begin with a 27-amino-acid

STRUCTURE OF THE PRECURSOR mRNA/GENE CART transcript is present in rat tissues as an RNA doublet [15]. Alternate polyA site utilization in the 3′ Handbook of Biologically Active Peptides

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698 / Chapter 97 signal peptide motif, suggesting that CART peptides are targets for posttranslational processing [15]. The resulting prohormone of either 102 or 89 amino acids is further processed by protein convertases [12]. The long form of proCART gives rise to two bioactive peptides, CART 55–102 and CART 62–102, and an intermediate peptide, CART 33–102 [12]. Similarly, the shorter form is also processed into an intermediate residue, CART 10–89, and two bioactive forms, CART 55–102 and CART 62–102 [12]. (The nomenclature of CART peptides is based on the amino acid sequence of the long form of CART precursor; see [30].) The two main enzymes involved in processing of CART are PC1/3 and PC2 protein convertases [12]. PC1/3 is much more potent than PC2 in producing intermediate CART residues. It cleaves the long form of the CART precursor to result in CART 33–102 [12] and is exclusively responsible for the production of CART 10–89 from the short CART precursor [12]. In contrast, PC2 is the primary enzyme responsible for the production of CART 55–102 and is exclusively responsible for generation of CART 62–102 [12]. Both intermediate forms and the two bioactive forms of CART peptides are present in the brain [12, 50]. In contrast, only CART 62–102 is present in the neurointermediate lobe where both PC1/3 and PC2 are present, while CART 55–102 is present in the anterior lobe which expresses only PC1/3 [12, 50].

DISTRIBUTION OF CART mRNA AND PEPTIDES CART mRNA is expressed in discrete neuronal groups in the brain, and it is also present in the anterior lobe of the pituitary gland (Fig. 1) [15]. The highest concentration of the CART mRNA is found in the hypothalamus and, in particular, neurons of the arcuate, paraventricular, supraoptic and dorsomedial nuclei, periventricular region, zona inzerta, and lateral hypothalamus [15]. Lower concentrations of CART mRNA are also present in the ventral and medial premamillary nuclei and in the lateral aspect of the supramamillary nucleus [15]. In the telencephalon, CART mRNA is localized to the olfactory tubercle, nucleus accumbens, primary somatosensory and piriform area of the cortex, bed nucleus of the stria terminalis, dentate gyrus, and amygdaloid complex [15]. The brainstem also contains CART expressing cell groups, including the EdingerWestphal nucleus, locus ceruleus, parabrachial nucleus, nucleus of tractus solitarius, A1/C1 region, and raphe nuclei [15]. The pituitary gland contains as high concentration of CART mRNA as the hypothalamus [8], expressed in 80% of the gonadotrophs but not in GH-, TSH-, ACTH- or prolactin-producing cells [32]. The distribution of CART-immunoreactive (IR) perikarya shows a pattern similar to that observed for CART mRNA-containing cells [8]. However, there are some

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FIGURE 1. The location of main populations of CART synthesizing neurons.

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Cocaine- and Amphetamine-Regulated Transcript (CART) / 699 discrepancies. While in situ hybridization identifies numerous CART expressing neurons in the hypothalamic dorsomedial (DMN) and ventral premamillary nuclei, only a few CART-IR cells can be detected in these regions. The projection fields of CART neurons have been described in detail by Koylu et al. [27, 28]. Of particular importance is the observation that CART fibers densely innervate vegetative centers of the central nervous system, including the PVN, DMN, arcuate nucleus, nucleus tractus solitarius, C1–3 adrenergic cell groups and the intermediolateral column of the spinal cord [16, 27, 28]. Furthermore, a dense CART-IR fiber network is found in the median eminence and in the posterior lobe of the pituitary arising from the CART expressing hypophysiotropic neurons and magnocellular neurosecretory neurons of the PVN and supraoptic nuclei, respectively [28].

RECEPTORS AND SIGNALING CASCADES Until now, little has been known about CART receptors and the intracellular signaling cascades mediating the effects of CART. Despite the fact that CART was discovered 10 years ago, CART receptors have not yet been identified and characterized. However, it is conceivable that CART may act on more than one receptor. Yermolaieva et al. [60] demonstrated that CART inhibits voltage-dependent intracellular Ca(2+) signaling in the hippocampus, likely through the inhibition of L-type voltage-gated Ca(2+) channel activity via a G-protein-dependent pathway. Sarkar et al. [43] found that CART induces phosphorylation of cyclic-AMP response element binding protein (CREB) in corticotropin-releasing hormone (CRH)-synthesizing neurons of the PVN, suggesting that the cAMP-CREB signaling pathway may also be involved in the mediation of CART effects. In addition, in vitro studies from Lakatos et al. [33] showed that CART can rapidly induce ERK phosphorylation in AtT20 cells, mediated through pertussis toxin sensitive G-protein coupled receptors [33].

BIOLOGICAL ACTIONS OF CART PEPTIDES WITHIN THE BRAIN AND PITUITARY CART has been implicated in the central regulation of an increasing number of physiological processes that now include reward, feeding, energy metabolism, neuroendocrine regulation, pain, cardiac function, and gut motility [31]. Each action is briefly summarized herein.

Reward One of the first recognized actions of CART in the CNS is its involvement in reward circuits, based on its expression in the nucleus accumbens, striatum, and ventral tegmental area (VTA), and regulation of CART gene expression by cocaine and amphetamine [15]. CART exerts psychostimulant-like effects when injected into the VTA, while simultaneously increases locomotor activity and promotes conditional place preference [26]. Since these effects of CART can be blocked by dopamine antagonists [26] and CART is present in axon terminals that establish synaptic specializations on both dopaminergic and GABA-ergic neurons of the VTA, it is suggested that CART either influences dopaminergic neurons of the VTA directly or indirectly through disinhibition of GABA-ergic interneurons [13, 26]. Indicative of site specific effects on locomotor activity [24], CART administration into the nucleus accumbens inhibits cocaine induced locomotor activity [23]. Similarly, CART inhibits the dopamine-induced locomoter activity in the nucleus accumbens [23], suggesting that CART interacts with postsynaptic targets of dopaminergic neurons and attenuates the effect of the cocaine-induced increase of dopamine transmission [23].

Feeding and Energy Metabolism The synthesis of CART peptides in specific neuronal groups that have been implicated in the central control of energy homeostasis has suggested an important role for CART in the regulation of feeding an energy metabolism. These neuronal groups include alpha-melanocyte-stimulating hormone (αMSH) producing neurons of the hypothalamic arcuate nucleus, melanin-concentrating hormone (MCH) neurons of the lateral hypothalamus, neurons of the DMN and NTS and adrenaline-synthesizing neurons of the C1–3 regions of the brainstem [16, 20, 27, 29]. Along these lines, an acute injection of CART into the CSF induces c-fos in the abovementioned feeding related regions [55], decreases feeding [29, 34], and inhibits the orexinogenic effects of NPY [29, 34], whereas chronic administration induces significant weight loss [42]. Conversely, the central administration of CART antiserum increases food intake [29, 34]. In the arcuate nucleus, the synthesis of CART is highly regulated by circulating levels of leptin [3]. Exogenous administration of leptin or elevated leptin levels induced by high fat diet-induced obesity increase CART mRNA in the arcuate nucleus [29, 42]. Conversely, CART gene expression is decreased in the arcuate nucleus of leptin

700 / Chapter 97 resistant fa/fa Zucker rats and leptin deficient ob/ob mice [29]. In the later animal model, CART mRNA level can be normalized by leptin replacement [29]. The actions of leptin on CART gene expression is believed to be exerted directly on CART-producing neurons of the arcuate nucleus, as these neurons express leptin receptors and show evidence of SOCS 3 expression following leptin administration [16, 17]. CART-synthesizing arcuate neurons project directly to the PVN [18, 20] and the intermediolateral column of the spinal cord [16], thereby coordinating the effects of CART to simultaneously regulate food intake and energy metabolism [45, 56]. CART also influences food intake through effects on brainstem feeding centers, as administration of CART into the fourth ventricle inhibits food intake [4]. While there is general agreement that icv administration of CART has anorexigenic effects, some discrepancies have been reported. Wang et al. [56] observed inhibition of food intake when CART is injected directly into the PVN, whereas Abbott et al. [1] observed increased food intake after focal injection of CART into either the PVN or arcuate nucleus. Nevertheless, consistent with the role of CART as an anorexic peptide, CART deficient transgenic mice develop obesity when placed on a high-caloric diet [5], and polymorphisms of the 5′ flanking region of the CART promoter are associated with obesity [59].

Neuroendocrine Regulation of Hypothalamic Hypophysiotropic Neurons CART has a number of important actions on hypophysiotropic neurons of the hypothalamic-pituitary axes as indicated by the synaptic association of CARTcontaining axon terminals with CRH-, TRH- and GnRHproducing hypothalamic neurons [18, 36, 43, 54]. The CART-IR innervation of TRH and CRH neurons originates primarily from two sources: adrenergic C1–3 regions of the medulla and the hypothalamic arcuate nucleus [18, 19, 57, 58]. The central administration of CART activates CRH neurons [54], and its stimulatory effect on ACTH and corticosterone secretion can be blocked by the CRH antagonist, Astressin B [46]. CART also exerts stimulatory effects on hypophysiotropic TRH neurons in the PVN, increasing TRH content and secretion from hypothalamic cells [18, 48] and preventing the fasting-induced decline in TRH gene expression in the PVN [18]. While these actions may be involved in the mediation of leptin via projections from CART/αMSH neurons [18, 58] of the arcuate nucleus [18], dense CART-projection fields from the brainstem neurons indicate that CART may also be involved the regulation of the hypophysiotropic TRH neurons by other physiological processes.

CART also seems to play a crucial role in mediating the effects of leptin on the central regulation of gonadal function [40]. LHRH pulse amplitude is similarly increased by both leptin and CART in female rats [40]. Further, immunoneutralization of CART prevents the leptin induced increase of LHRH pulse amplitude in hypothalamic explants [40]. In addition to regulation of hypophysiotropic neurons through afferent inputs, CART is also coexpressed in hypophysiotropic neurons that synthesize TRH, somatostatin, vasopressin, and oxytocin [18, 35, 53], presumably to regulate pituitary function. CART has a direct inhibitory effect on prolactin secretion in vitro [32] and can completely block the stimulatory effects of TRH on prolactin secretion at even low concentrations (10−8 to 10−10 M) [41]. CART has no effect, however, on either basal or stimulated TSH, LH, or FSH secretion [32, 41]. As noted previously, CART has been identified in gonadotropes in the rat anterior pituitary gland [32], suggesting the additional possibility that it may be involved in paracrine regulation of prolactin secretion [32].

Pain CART peptides 55–102 and 62–102, when administered icv or intrathecally, have been reported to produce significant antinociceptive effects as measured by hindpaw withdrawal latency in the hot-plate test [6, 38] and to enhance antinociceptive effects of morphine in the tail flick test [9]. It is presumed that the major analgesic actions of CART are exerted in the spinal cord, consistent with the presence of dense networks of CART-IR fibers in the dorsal horn [38].

Vegetative Functions Intracerebroventricular and intracisternal administration of CART increases mean arterial pressure and heart rate in rats and rabbits [21, 37] and is a potent vasoconstrictor in the cerebral circulation [22]. Since a relatively high dose of CART is necessary to influence cardiovascular function when administered intrathecally [45], the brain may be the primary site of the action for these physiologic effects of CART. At least two different regions of the brainstem have been identified as mediating these responses including the rostral ventrolateral medulla, a critical pressor area [10] where intracisternal CART administration induces cfos expression [21] and the nucleus tractus solitarius, where local administration of CART attenuates phenylephrineinduced bradycardia [44]. However, CART alone is relatively ineffective in influencing the cardiovascular system if administered intrathecally [45]; but even low doses of CART are sufficient

Cocaine- and Amphetamine-Regulated Transcript (CART) / 701 to potentiate the stimulatory effects of intrathecally administered glutamate on cardiovascular function [45]. It is presumed that this effect is exerted through the intermediolateral column of the spinal cord [45], which is heavily innervated by CART-synthesizing vasomotor neurons of the rostral ventrolateral medulla [7]. CART also acts in the CNS to inhibit gastric acid secretion and gastric motility, and stimulate colonic motility [39, 49]. It is presumed that these actions are mediated in the brain by CRH-synthesizing neurons, as the effects of CART on gastrointestinal function can be inhibited with CRH antagonists [39, 49].

PHYSIOPATHOLOGICAL IMPLICATIONS To date little is known about the role of CART in development of pathological conditions. However, a role of CART is suggested in the development of obesity and alcoholism by genetic linkage studies. A polymorphism of the 5′ flanking region of the CART gene (− 156) is significantly associated with increased BMI [59]. Furthermore, a missense mutation at codon 34, in the N terminal region of CART has been found in an obese family, where the mutation cosegregated with the severe obese phenotype [11]. Jung et al. [25] found that a AvaII polymorphism in intron I predisposed to alcoholism but is not associated with depression or schizophrenia [25].

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702 / Chapter 97 [23] Jaworski, J.N., Kozel, M.A., Philpot, K.B. and Kuhar, M.J., Intraaccumbal injection of CART (cocaine-amphetamine regulated transcript) peptide reduces cocaine-induced locomotor activity. J Pharmacol Exp Ther 2003; 307:1038–1044. [24] Jaworski, J.N., Vicentic, A., Hunter, R.G., Kimmel, H.L. and Kuhar, M.J., CART peptides are modulators of mesolimbic dopamine and psychostimulants. Life Sci 2003; 73:741– 747. [25] Jung, S.K., Hong, M.S., Suh, G.J., Jin, S.Y., Lee, H.J., Kim, B.S., Lim, Y.J., Kim, M.K., Park, H.K., Chung, J.H. and Yim, S.V., Association between polymorphism in intron 1 of cocaine- and amphetamine-regulated transcript gene with alcoholism, but not with bipolar disorder and schizophrenia in Korean population. Neurosci Lett 2004; 365:54–57. [26] Kimmel, H.L., Gong, W., Vechia, S.D., Hunter, R.G. and Kuhar, M.J., Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55–102 induces locomotor activity and promotes conditioned place preference. J Pharmacol Exp Ther 2000; 294:784–792. [27] Koylu, E.O., Couceyro, P.R., Lambert, P.D. and Kuhar, M.J., Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol 1998; 391:115 –132. [28] Koylu, E.O., Couceyro, P.R., Lambert, P.D., Ling, N.C., DeSouza, E.B. and Kuhar, M.J., Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J Neuroendocrinol 1997; 9:823–833. [29] Kristensen, P., Judge, M.E., Thim, L., Ribel, U., Christjansen, K.N., Wulff, B.S., Clausen, J.T., Jensen, P.B., Madsen, O.D., Vrang, N., Larsen, P.J. and Hastrup, S., Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 1998; 393:72–76. [30] Kuhar, M.J., Adams, L.D., Hunter, R.G., Vechia, S.D. and Smith, Y., CART peptides. Regul Pept 2000; 89:1–6. [31] Kuhar, M.J., Adams, S., Dominguez, G., Jaworski, J. and Balkan, B., CART peptides. Neuropeptides 2002; 36:1–8. [32] Kuriyama, G., Takekoshi, S., Tojo, K., Nakai, Y., Kuhar, M.J. and Osamura, R.Y., Cocaine- and amphetamine-regulated transcript peptide in the rat anterior pituitary gland is localized in gonadotrophs and suppresses prolactin secretion. Endocrinology 2004; 145:2542–2550. [33] Lakatos, A., Prinster, S., Vicentic, A., Hall, R.A. and Kuhar, M.J., Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors. Neurosci Lett 2005; 384:198–202. [34] Lambert, P.D., Couceyro, P.R., McGirr, K.M., Dall Vechia, S.E., Smith, Y. and Kuhar, M.J., CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 1998; 29:293–298. [35] Larsen, P.J., Seier, V., Fink-Jensen, A., Holst, J.J., Warberg, J. and Vrang, N., Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit. J Neuroendocrinol 2003; 15:219–226. [36] Leslie, R.A., Sanders, S.J., Anderson, S.I., Schuhler, S., Horan, T.L. and Ebling, F.J., Appositions between cocaine and amphetamine-related transcript- and gonadotropin releasing hormoneimmunoreactive neurons in the hypothalamus of the Siberian hamster. Neurosci Lett 2001; 314:111–114. [37] Matsumura, K., Tsuchihashi, T. and Abe, I., Central human cocaine- and amphetamine-regulated transcript peptide 55–102 increases arterial pressure in conscious rabbits. Hypertension 2001; 38:1096–1100. [38] Ohsawa, M., Dun, S.L., Tseng, L.F., Chang, J. and Dun, N.J., Decrease of hindpaw withdrawal latency by cocaine- and

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98 The Melanin-Concentrating Hormone JEAN-LOUIS NAHON

ABSTRACT

by a paling neurohypophysial hormone. The development of in vitro teleost scale bioassays allowed functional characterization and purification of this hormone. Indeed, based on a tilapia scale bioassay, Kawachi and coworkers [19] purified melanin-concentrating hormone (MCH) from pituitary glands of chum salmon (Oncorhynchus keta) and characterized its structure. Sequence analysis revealed that MCH is a peptide of 17 amino acids with one disulfide bond (Fig. 1). This seminal work paved the road for immunocytochemical localization, structure-activity studies, and investigation of MCH function and regulation, apart from its pigmentary action, in fish models [2, 4, 15]. On the other hand, the availability of salmon MCH (sMCH) antisera facilitated the characterization of the MCH counterpart in higher vertebrates. Thanks to the strong expression of MCH-like peptide in the rat hypothalamus and using the sMCH RIA, Vaughan and coworkers [26] isolated the rat MCH and established its sequence. Rat MCH is a cyclic nonadecapeptide that displays strong structural similarity with the fish peptide but an N-terminal extension and four additional substitutions (Fig. 1). The role of MCH in mammalian systems remained elusive until 1996 when MCH mRNA expression studies revealed striking overexpression in obese leptin-deficient (Ob/Ob) mice and after fasting [22]. Intracerebral injections of MCH further revealed an acute orexigenic effect in the rat that was confirmed in several laboratories [25]. The importance of the MCH neuronal system in the regulation of feeding behavior, body weight, and energy homeostasis is by far the most documented. However, MCH is also involved in the control of a broad spectrum of cerebral and peripheral functions [15–17, 21, 23].

The melanin-concentrating hormone (MCH) is a cyclic peptide highly conserved in vertebrates that was originally characterized as a neurohypophysial skin paling factor in teleosts. In fish, MCH may also participate in the control of the stress response. Mammalian MCH is a hypothalamic neuropeptide that displays multiple functions, mostly controlling feeding behavior and energy homeostasis and regulating the stress axis and emotion. The MCH precursor may generate additional putative peptides in the brain and peripheral organs but their roles remain poorly explored. In mammals, alternative splicing and antisense transcription at the MCH gene locus lead to production of novel proteins whereas variant MCH genes were found only in primates. Two G protein–coupled MCH receptors were found in fish and primates, while a single one appears functional in rodents. Transgenic mouse models and pharmacological studies have demonstrated the importance of the MCH signaling pathway as a potential target to treat appetite disorders, obesity, and also anxiety and depression.

DISCOVERY The existence of a paling factor present in the pars tuberalis of amphibia that would regulate skin pigmentation in response to environmental changes in lower vertebrates was first proposed by Hogben and Slome in 1931 [18]. While this concept never received any experimental support in amphibians, melanin-concentrating activity was firmly established in teleosts with the demonstration of pituitary control of teleost melanophores Handbook of Biologically Active Peptides

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FIGURE 1. Structure of salmon and rat/human MCH. Conserved amino acids are boxed and the disulfide bridge between the two cysteines is indicated.

Cloning of the cDNAs encoding the MCH precursor in teleost fishes, then in rat and human, opened the era of molecular characterization of MCH gene structure and expression. Most of this research concentrated on mammalian genes and led to the discovery of additional putative peptides generated from the MCH precursor, a splice variant of the MCH primary transcript, a natural antisense MCH gene, and a family of variant MCH genes that arose lately in the Hominidae lineage [17]. All of these MCH gene–derived products may serve functions, distinct from MCH effects, that are still to be defined. Recently, a long-awaited achievement was the cloning and functional characterization in cellular and animal models of two MCH receptors, both members of the G protein–coupled receptor (GPCR) family [7, 14, 17]. The first MCH receptor, MCH-R1, was isolated by a reverse-pharmacology strategy and was found in all mammalian species while the second, designated here MCH-R2, was identified by computer data mining and appeared absent in rat and mouse but present in higher mammalian species. Since MCH-R1 per se seems to mediate MCH actions in the regulation of food intake and energy expenditure, several pharmaceutical companies have developed a diverse array of MCH-R1 antagonists [8, 20]. Preclinical studies with MCH-R1 antagonists are still limited but provide some evidence that nonpeptidic MCH-R1 antagonists represent attractive molecules for the treatment of obesity, metabolic syndrome, and anxiety in humans.

STRUCTURE OF THE PRECURSOR mRNA/GENE Two MCH genes and/or corresponding mRNAs have been cloned and sequenced from salmonids, tilapia, bonito, eel, and flounder [15]. Both are intronless and display the highest level of substitutions when compared within the same species rather than among other species, in agreement with early tetraploidization during teleost fish evolution (Fig. 2A). The salmonid and tilapia/flounder pre-pro-MCHs are 132 amino acids and 135 amino acids long, respectively, with the MCH peptide located at the C-terminus. In contrast with the

strong conservation in the MCH region between fish species, the remaining part of the precursor displays marked sequence divergence [9]. However, additional putative peptides could be generated from the fish proMCH and named MCH gene–related peptide (Mgrp) in tilapia, neuropeptide-E (glutamic acid)-V (valine) (NEV) in chum salmon, or neuropeptide-A(alanine)-L (leucine) in barfin flounder, but their functional importance, if any, remains elusive [15]. The sequence of the gene encoding the MCH precursor is composed of three exons and two introns and exhibits high conservation in mammalian species (Fig. 2B). In humans, this gene is located on chromosome 12q24 covering about 1.4 kb of genomic DNA. The first exon encodes the 5′ untranslated region of the mRNA and the N-terminal part of pro-MCH including the signal peptide that allows targeting to the secretory pathway. The second exon is composed of the sequence corresponding to putative neuropeptideglycine(G)-glutamic acid(E) (NGE), neuropeptideglutamic acid(E)-isoleucine(I) (NEI), and the first three amino acids of MCH. The last 15 amino acids of MCH and the 3′ untranslated amino acids are localized in exon III. Intron B splits the methionine codon with the nucleotide A on exon II and the bases TG on exon III. This intronic organization is identical in the rat, mouse, and primate MCH genes. This unusual intron position for a neuropeptide encoding gene is of primary importance to generate by alternative splicing a short mRNA encoding a protein named MCH-gene-overprintedpolypeptide (MGOP) [24] (Fig. 2C). Rat/mouse MGOP contains 125 amino acid residues and shares an identical N-terminal part with the MCH precursor (encoded by exon I of the MCH gene) but lacks NGE and NEI (encoded by exon II of the MCH gene) as well as MCH that is replaced by a new C-terminal part (due to a shift in the reading frame within exon III of the MCH gene). Recently, structural analysis of MGOP-IR and distribution in the rat and mouse brain revealed a strict colocalization with MCH in neurons of the zona incerta/lateral hypothalamus and unique expression of an MGOP-like antigen that was characterized as the pro-SRIF1–64, in neurons of the hypothalamic periventricular nucleus and other brain areas [1]. The role of MGOP in the brain remains elusive.

The Melanin-Concentrating Hormone / 707

A

TATA NEV MCH

sMCH1

5'UT

3'UT

ATG

86%

TATA

sMCH2

NEV MCH

5'UT

3'UT

100 bp

ATG

B

HUMAN MOUSE RAT

EXON I 86% EXON I 85% EXON I MCH mRNA

70% 86%

INTRON A

88%

INTRON B 3'UT

100 bp

NGE NEI MCH

( )

NGE NEI MCH

Signal

pre-pro-MCH

EXON III 78% EXON III 87% EXON III

66%

5'UT ATG

C

EXON II 90% EXON II 94% EXON II

( )

EXON I

MCH mRNA

MCH gene EXON I

INTRON A

EXON I

MGOP mRNA pre-MGOP

EXON II

EXON II

EXON III

INTRON B

EXON III

Signal

In addition to the MCH/MGOP gene, two other MCH gene–related transcription units were found in mammalian genomes. First, characterization of high-molecularweight transcripts recognized by MCH cDNA/ oligoprobes in the pheochromocytoma cell line PC12 cells and rat tissues revealed the existence of “natural” antisense RNAs transcribed from the genomic region complementary to the MCH gene. This new gene was named AROM for antisense-RNA-overlapping-MCH [6]. Two major classes of RNAs appear produced from the AROM gene: (1) noncoding unspliced RNAs overlapping exon II/exon III and flanking intronic/regulatory sequences of the MCH gene; and (2) alternatively spliced mRNAs complementary to the 3′ flanking region of the MCH gene and coding for a new family of RNA/

EXON III

AAAA

FIGURE 2. The MCH genes. A. Comparison of the two MCH genes in salmon. TATA, putative TATA box; UT, untranslated region; ATG, first codon of the preproMCH. The putative signal peptide is indicated by a striped box. Arrowheads and black bars indicate the basic residues involved at the cleavage sites to generate MCH and the putative NEV. DNA sequence percent identities are noted. B. Comparison of the human, mouse, and rat MCH genes. Exons and introns are indicated by boxes and lines, respectively. The putative signal peptide and the location of peptides NGE, NEI, and MCH are noted as in A, C. Structure of the MCH and MGOP mRNA and deduced proteins produced by alternative splicing of the MCH primary transcript. Black box indicates the MGOP-specific C terminus due to exon II skipping and ORF frameshift in exon III.

DNA binding proteins. Because of the reciprocal regulation of MCH and AROM gene expression in PC12 cells, it is tempting to speculate that AROM mRNA and/or proteins may control MCH gene expression, but this remains to be directly tested. Recently, we provided evidence for the emergence in higher primates of two chimeric genes [11]. These genes were named PMCHL1 and PMCHL2, genes based on partial homology with the authentic MCH gene, and were found respectively on human chromosome 5p14 and 5q13—that is, located on chromosomal regions clearly distinct from those of the genuine MCH gene. Detailed structural expression and phylogenetic analyses showed that the PMCHL1 gene was created about 25 million years ago (mya) by a complex mechanism of

708 / Chapter 98 exon-shuffling through retroposition of an antisense MCH messenger RNA coupled to de novo creation of splice sites. PMCHL2 arose 5–10 mya by an event of duplication involving a large chromosomal region encompassing the PMCHL1 locus. Interestingly, both sense and antisense transcripts from the PMCHL1 gene are expressed in different areas of the developing human brain, while the PMCHL2 gene is totally silent in the central nervous system. Furthermore, one of the PMCHL1 mRNAs would encode a 72-amino-acid protein carrying a putative nuclear localization signal, suggesting an intranuclear function for this PMCHL1 gene-derived protein in primates. The PMCHL1 gene represents a prototype of the so-called “primatespecific” genes that are presently under intensive investigation because of their major interest in understanding the basis of human evolution and emergence of novel functions.

DISTRIBUTION OF THE mRNA/PEPTIDES The distribution of MCH-IR perikarya and projection fields has been established in most of the vertebrate taxa (reviewed in [3, 15]). In fish, MCH neurons are located in two distinct areas, the nucleus lateralis tuberis (NLT) and in more dorsal nuclei above the lateral ventricular recess (LVR). The magnocellular

MCH neurons of the NLT predominate in lately evolved actinopterigians, including teleosts, and project massively to the posterior neurohypophysis, where MCH is released into the bloodstream to act peripherally as a skin paling hormone. In contrast, the parvocellular MCH neurons of LVR are prominent in early evolved vertebrates, including lampreys, and project widely to different brain areas with the exception of the pituitary, suggesting that LVR-originated MCH likely acts as a neurotransmitter/neuromodulator. Numerous data indicate that both MCH neuronal cell groups function differently under white or black environmental challenge in tilapia, rainbow trout, or barfin flounder [3]. The mapping of MCH-IR in the rat brain revealed two striking features highly conserved in mammals: (1) quite exclusive clustering of MCH neuronal perikarya in the subzona incerta, lateral hypothalamic area (LHA), and posterior hypothalamic area (PHA); and (2) extensive distribution of the projections throughout all brain with the notable exception of cerebellum, external median eminence, and pituitary gland (Fig. 3; [5]). Recently, Fellmann and Risold’s team performed a series of immunocytochemical investigations looking for differential expression of neurokinin 3 (NK3) receptor and cocaine- and amphetamine-regulated transcript (CART) in MCH neurons and projections. In particular, two MCH cell groups were clearly identified, based

FIGURE 3. Schematic representation of MCH-producing neurons (dots) and projections (lines/arrow) in the rat brain (from [5]).

The Melanin-Concentrating Hormone / 709 on coexpression of MCH/CART/NK3 (type B neurons) or only MCH (type A neurons) [12]. Furthermore, they display very different projection pathways; type A neurons have quite exclusively descending inputs to reach the spinal cord, whereas type B neurons project massively to all known telencephalic areas receiving ascending MCH inputs. Intriguingly, at least four MCH neuronal subpopulations can be distinguished according to their times of genesis during rat diencephalon development, two of them overlapping with type A and type B MCH neurons. Minor MCH-expressing neuronal groups are also present in extrahypothalamic areas such as the olfactory tubercle and pontine tegmentum in the adult rat (Fig. 3; [5]). Transient expression of MCH-IR containing neurons was identified throughout rat embryo development in the lateral geniculate nucleus, lateral ZI, PVN, and lateral septum and also in the medial preoptic area and PVN of lactating female rats [15]. The significance of this transient appearance of MCH neurons remains elusive but could be related to similar expression patterns reported for other neuropeptide systems.

PROCESSING OF THE MCH PRECURSOR Very few studies have attempted to investigate the biosynthesis of the pro-MCH-derived peptides in teleost precursor [15]. Large-molecular-weight MCH precursors are produced in the hypothalamus of salmon or trout and are further cleaved to generate intermediate products and mature MCH in the trout pituitary glands. Interestingly, the main intermediate corresponds to the pro-MCH101–132—that is, the NEV-MCH peptide generated after cleavage at a pair of arginine residues at position 99–100 of the trout MCH precursor. In the mammalian brain, most of the pro-MCHderived peptides are mature MCH and amidated NEI peptide (Fig. 4A). Conversely, fully processed MCH and NEI peptides are lacking or weakly produced in rodent and human peripheral organs (spleen, gut, thymus, and testis), while a large MCH-IR product is found. This large product was identified as a pro-MCH intermediate, bearing at least NEI and MCH (Fig. 4B). In this context, the rat pro-MCH131–165 (NEI-MCH peptide) was synthesized and ICV injected to investigate the effect on appetite behavior and energy metabolism. The NEIMCH exerts long-term inductions in feeding and body weight gain, much more potent than MCH itself, likely due to better resistance to peptidase degradation [17]. It is attractive to suggest that a similar kind of pro-MCH intermediate may be either transiently expressed in the brain or produced peripherally under particular physiopathological conditions and cross the blood-brain barrier to exert sustained MCH-like activity.

A

Translated region

-65 +1

mRNA Precursor

5' UT +1

3' UT

( ) +165 NGE NEI MCH

+687 (A) n

signal

Peptides R

MCH NEI

B

+495

V

G

L

M

Y R P

M D F D H C R L C

WQ V OH

H E I G D E E N S A K F P I NH 2

NEI

Brain PC2

MCH

NH2

PC1/3, PC2, PC5/6-A or ?

129-130

145-146

NEI QEKRE

MCH IGRRD

? Periphery

NEI

MCH

FIGURE 4. Rat MCH precursor processing and peptide production. A. Rat MCH mRNA structure (as in Fig. 2B) and peptide sequences. B. Pro-MCH processing by prohormoneconvertases (PCs) in the brain and in peripheral organs. Sequences at the cleavage sites are noted as well as PCs with demonstrated effects on pro-MCH to generate MCH and NEI. The question mark indicates that the PC involved in MCH precursor processing at the periphery is not yet known.

The processing of the mammalian MCH precursor by prohormone-convertases (PCs) was examined using recombinant vaccinia virus-infected cellular systems, PC-transfected PC12 cells, and an in vivo PC2 KO-mouse model (Fig. 4B; [17]). The main finding was that active PC2 is necessary and sufficient to generate NEI in the mouse brain and in PC12 cells. In contrast, several PCs, including PC1/3, PC2, and PC5/6A, may cleave at the appropriate site to produce MCH either in vaccinia expression systems or in the mouse brain. Incidentally, colocalization studies demonstrated simultaneous expression of MCH mRNA and PC2 in all MCH-expressing cell bodies of the lateral hypothalamus, whereas only 15–20% of these perikarya contained PC1. Therefore, PC2 is likely to be the key enzyme that cleaves MCH precursor to generate MCH and NEI in the brain. The counterpart in peripheral organs remains unknown [17].

RECEPTORS AND SIGNALING CASCADES The pioneer attempts to characterize MCH receptors were largely based on binding assays with MCH analogs such as (Phe13, Tyr 19)-MCH and allowed

710 / Chapter 98 identification of MCH binding sites in different mammalian cell types, including melanoma and neuroblastoma [13]. The lack of ligand selectivity in competition experiments, ligand-induced signal transduction pathways associated with these MCH binding sites, and rapid internalization of the radioligand likely through scavenger receptor binding have cast some doubt on the reliability of these putative MCH receptors. However, a functional MCH receptor distinct from the two presently identified MCH receptors (and named MCH-Rpc) could be found in mouse and human melanoma, but its sequence is still unknown [13].

MCH-R1 Structure and Signaling A milestone discovery in the MCH receptor field was the quite simultaneous identification by several laboratories in 1999 of an orphan GPCR called SLC-1 (GPR24) as being a bona fide MCH receptor, named here MCHR1 [7, 20]. This receptor belongs to the class 1 subfamily of GPCRs and displays strong sequence conservation among mammals (91% protein sequence identity between rat and human). The MCH-R1 gene is composed of two exons that encode for a 353-aminoacid-long protein with seven putative transmembrane domains, three N-glycosylation sites in the N-terminal part, that appear necessary for cell surface targeting, and multiple phosphorylation sites in the intracellular loops; some of them, located in its C-terminal tail, are involved in the MCH-dependent internalization of the MCH-R1. MCH-R1 is widely and strongly expressed in rat, mouse, and primate brains with a distribution that fits with the MCH projection fields, suggesting that this receptor may mediate most of MCH actions in mammals [17]. MCH-R1 signaling has been mostly investigated using stably transfected cells, and these studies revealed that this MCH receptor may couple to a variety of G proteins, resulting in activation of multiple signaling pathways. Indeed, in MCH-R1 overexpressing CHO or HEK293 cells, MCH inhibits forskolin-induced cAMP synthesis through a PTX-sensitive mechanism, indicating the coupling to Gαi/Gαo. In the same cellular models, MCH also stimulates IP3 production and transiently increases intracellular Ca2+ via Gαq interaction with MCH-R1. Finally, MCH-stimulated MAP kinase activity in transfected CHO cells appears mediated through both Gαi and Gαq coupled signaling pathways. Intriguingly, the combination of MCH with forskolin potentiates MAP kinase phosphorylation in HEK293 cells stably expressing MCH-R1 and in various regions of the rat brain suggesting that a complex interaction between MCH-R1 and receptors coupled to Gαs could be the rule rather than the exception in the mammalian brain. In contrast to these data, the signal-

ing pathways that mediate MCH action in mammalian cells endogenously expressing MCH-R1 remain poorly defined [13].

MCH-R2 Structure and Signaling The second MCH receptor, named here MCH-R2, was identified in the human genome by use of so-called “in silico data mining,” and its cDNA was subsequently isolated from human brain cDNA libraries [17]. In contrast to the MCH-R1 gene, which is intronless in its coding region, MCH-R2 gene encompasses five coding exons and one noncoding exon and may produce receptor variants by alternative splicing that would lead to a truncated version of MCH-R2. It is noteworthy that human MCH-R1 and MCH-R2 display a low sequence identity (only 36%), which is rather unusual for a family of GPCR that bind an identical ligand, suggesting weak evolutionary constraints. In this context, the MCH-R2 gene was found in teleosts (pufferfish, zebrafish), dog, ferret, and primates (including human) but a functional MCH-R2 gene was selectively lost in the rodent/lagomorph lineage. This proved to be useful for elucidating the physiological importance of the single MCH-R1 in rat and mouse but also hampered functional characterization of MCH-R2 in available animal models. In higher mammals, the overall brain expression of MCH-R2 appears quite similar to that of MCH-R1 but with a pattern more restricted to the limbic regions, including medial and central amygdala, entorhinal and temporal cortex, CA1 of the hippocampus, and at the lowest level in the ventral hypothalamus and claustrum. The differential distribution of the two MCH receptors, particularly in the human brain, may indicate distinct biological roles in mediating the large spectrum of MCH effects. In the periphery, weak MCH-R2 expression was detected in pituitary, adipose tissue, and pancreas, which also expressed MCH-R1, but the precise cellular distribution and functional relevance of MCH-R2 in these organs are presently unknown. The signaling pathways associated with MCH-R2 activation have been studied only in transfected mammalian cells and proved to be much more restricted than those reported for MCH-R1. Indeed, MCH stimulates an increase in intracellular free Ca2+ levels and IP3 production, but it has no effect on basal or forskolininduced cAMP synthesis. This action is not blocked by PTX treatment indicating that MCH-R2 couples mainly to Gαq. The difference in G protein coupling between MCH-R1 and MCH-R2 could be attributed to a marked sequence difference in the second and third intracellular loops, which are essential for specific G protein interaction in the GPCR family.

The Melanin-Concentrating Hormone / 711

ACTIVE STRUCTURE OF THE MCH PEPTIDE AND RECEPTOR CONFORMATION There have been numerous studies characterizing MCH analog for their functional activity initially using fish skin bioassays and more recently cellular models expressing cloned mammalian MCH receptors [7, 10, 15]. All information converges towards the following model for MCH ligand-receptor interactions: (1) The cyclic peptide fragment ring (MCH 5–14 in fishes and MCH 7–16 in mammals; see Fig. 1) is necessary but not sufficient to get maximal activity; indeed, the Cterminal part (mainly the Tyr 15 residue) of the fish peptide and additional amino acids flanking the ring structure (Arg6 and Trp17) in the rat/human MCH are required for agonist potency; (2) only Met 8, Arg 11, and Tyr 13 of the mammalian MCH are essential to elicit full and potent responses with both MCH receptors in binding and functional tests; and (3) changes in Arg6, Leu 9-Gly 10, or Gln18 of the rat/human MCH generate peptides that are less efficient in binding or activating MCH-R2 than MCH-R1. In addition, studies using NMR spectrometry, circular dichroism, or molecular dynamics indicate conformational flexibility of the salmon MCH 5–14 core backbone, whereas mammalian MCH exhibits a rather limited variability in the 4–17 region. Site-directed mutagenesis of the MCH-R1 and comparative molecular dynamics simulations of both MCH receptors in their free and ligand-bound conformation have highlighted the importance of ionic interactions between Arg 11 and Tyr 13 of rat/human MCH and portions of the receptors close to the E/DRY and NPxxY motifs under the active state.

BIOLOGICAL ACTIONS OF PRO-MCHDERIVED PEPTIDES Pigmentation MCH was originally characterized and incidentally named because of its potent action on melanin granule aggregation. However, the role of MCH as a neurohypophysial hormone involved in adaptive color change appears confined to neopterygians (holosteans and teleosts). Accordingly, targeted disruption of either the MCH or the MCH-R1 gene in the mouse did not lead to obvious modifications in coat or skin color. However, one study has indicated both MCH and MCH-R1 receptor expression in human skin and functional antagonism of MCH and αMSH in cultured human melanocytes. Furthermore, autoantibodies directed toward MCH-R1 domains were identified in patients with vitiligo, a common depigmenting skin disorder. While these data

suggest some effects of MCH on human melanocytes, further investigations are needed to recognize an overt role of MCH in skin or hair pigmentation in man [13, 23].

Feeding Behavior and Energy Homeostasis The dominant expression of MCH in the lateral hypothalamus of mammalian brains, a so-called “feeding-drinking center,” has early suggested that this peptide may regulate appetite and/or energy homeostasis. The role of MCH and MCH-R1 signaling in the control of feeding behavior and energy balance is now firmly established in rodents [16, 23, 25]. Most important results are detailed in (should be chapter by MARAD-foldier on MCPE) Chapter 126, Chapter 127 1 in the Ingestive Peptide section of this book. Overall, pharmacological studies and animal models have demonstrated that MCH and MCH-R1 neuronal networks play a major role in the control of energy homeostasis by increasing food intake and mostly regulating energy expenditure and thermogenesis. It remains to decipher the relative importance of central, neuroendocrine, and local influences versus sympathetic-innervated peripheral regulatory pathways involved in mediating the MCH effects on energy balance under normal and pathological conditions.

MCH and Feeding Behavior and Energy Homeostasis First, acute ICV injections of MCH (over 5 μg per animal) transiently stimulates food intake in rats, an orexigenic effect that is not mediated by NPY, galanin, or orexins (also named hypocretins) but could be blocked by aMSH (see corresponding peptide chapters in this book). Intrahypothalamic injection studies and electrophysiological characterization further identified the PVN, VMH, and ARC as major targets of MCH action, with differential effects in small-litter and overnourished rats. Interestingly, MCH also stimulates water consumption independently from its feeding and water osmolality actions, consistent with the strong adipsia observed in LHA-lesioned rats. Second, chronic ICV infusion of MCH in rats or mice induced hyperphagia and body weight gain, especially on a moderate high-fat diet. The development of obesity is accompanied by an increase in liver and fat weights resulting in lipogenic activity stimulation of white adipose tissue (WAT) and concomitant reduction of brown adipose tissue (BAT) functions. Furthermore, plasma glucose, insulin, and leptin levels are also raised in these animals, all these parameters being associated with obesity in man. Third, while acute or chronic ICV injections in rodents of MCH-R1 agonists fully recapitulate the

712 / Chapter 98 feeding and/or obese phenotype observed with MCH itself, central administration of MCH-R1 antagonists leads to a sustained reduction of appetite, body weight, and fat storage without overt alteration of lean mass. Finally, in agreement with the pharmacological data, genetically modified mouse models also demonstrated an important role of MCH and MCH-R1 genes in appetite and, mainly, energy metabolism. Indeed, transgenic mice overexpressing the MCH gene develop hyperphagia, mild obesity, and insulin resistance, when maintened on a high-fat diet. Conversely, MCH gene ablation in mice leads to a leanness phenotype associated with hypophagia and an increase in oxygen consumption, both effects being highly dependent upon the genetic background and food diet. In this regard, the reduction in body fat observed in mice combining MCH and leptin deficiencies, by crossing MCH null mice and ob/ob mice, does not result from feeding behavior changes (the mice remaining hyperphagic) but improvements in energy expenditure regulation (increased locomotor activity and resting metabolic rate). It is worth noting that targeted disruption of the MCH-R1 gene in mice maintained on regular chow also leads to a lean phenotype associated with both hyperphagia on one hand and increased energy metabolism on the other. Consistent with the hyperactive phenotype, MCH-R1 null mice are resistant to high-fat diet-induced obesity.

Stress Response In fish, MCH depresses the stress-induced activity of the hypothalamo-pituitary-adrenal (HPA) axis and repeated moderate stress enhances MCH release in the neurohypophysis and MCH gene expression in the NLT or LVR groups of MCH neurons, depending on the stress paradigm. Early modulation of LVR-MCH neurons might be related to a central inhibitory action on HPA, whereas the late response of the NLT-MCH neurons might be secondary to their regulation on pigmentation. Interestingly, MCH synthesis and release appear enhanced under mild stress, whereas intense stress tends to suppress MCH production, a regulatory balance that is under plasma glucocorticoid control [3, 4]. In mammals, there are conflicting reports regarding the action and regulation of MCH on the HPA axis [16]. Indeed, ICV injections of MCH in rats either increase or do not modify the basal secretion of ACTH in two separate studies. Results of intravenous administration of MCH are consistent with a stimulatory effect on the HPA axis. On the contrary, ICV-injected MCH inhibits ACTH release after mild stress, such as ether or handling; this action can be reversed by the coadminitration of NEI or αMSH. In addition, some chronic stressors, such as footshock stress or dehydration, induce

a reduction in MCH mRNA expression and/or peptide release, while others, such as immobilization or cold stress, do not modify or even stimulate (after fasting, for instance) MCH gene activity. This apparent confusing situation may be solved by considering the diurnal pattern of MCH gene expression (and likely peptide secretion) that appears higher at night and closely follows plasma glucocorticoid levels as well as the heterogeneity of the MCH neuronal population. Both parameters could explain differential responses to distinct stimuli or even opposite regulation in MCH sub-populations under a single stress (for instance, a hypertonic saline regimen increases MCH mRNA levels in ZI and decreases them in LHA).

Other Functions The actions of MCH and/or NEI on a large spectrum of behaviors have been studied in rats. These include reproduction, grooming, rearing, aggressive behavior, locomotion, exploratory behavior, learning, and memory formation. It is beyond the scope of this chapter to enumerate these behavioral studies discussed in exhaustive reviews [3, 15, 21]. Besides these multiple behavioral effects, recent evidence suggests that MCH neurons also play a role in sleep-wake regulation. ICV injection of low doses of MCH (below 5 μg per rat) induces paradoxal sleep (PS also named REM sleep) and slow wave sleep in rats. Furthermore, MCH neuronal activity (monitored by cFos induction) is increased selectively in PS recovery rats. Overall, these results suggest a potential hypnogenic role for MCH. I would also recommend the above-mentioned reviews when considering the local expression and role of MCH and its receptor outside the brain. Indeed, the importance of MCH/MCH-R1 in the digestive tract, adipose and bone tissues, cardiovascular or immune systems, for instance, should be considered in analyzing phenotype alterations in MCH-null or overexpressing transgenic mice.

PATHOPHYSIOLOGICAL IMPLICATIONS Feeding studies and characterization of MCH/MCHR1 transgenic mouse models have boosted interest in MCH as a suitable target for pharmaceutical intervention in the treatment of the obesity syndrome. Most of the efforts focus on the development of small-molecule antagonists for MCH-R1, because of the promising pharmacology of this receptor and the lack of suitable in vivo models for testing MCH-R2-selective antagonists. Initially, a series of peptidic analogs with antagonist activity was generated and proved to counteract the orexigenic effect of MCH agonists but was ineffective

The Melanin-Concentrating Hormone / 713 alone on feeding behavior. This limited effect and the complications inherent in the use of peptidic analogs as therapeutic tools (blood-brain barrier crossing, sensitivity to peptidase degradation) prompted most of the pharmaceutical and biotech companies working in the obesity field to invest in high throughput and targeting screening for discovering new nonpeptide MCH-R1 antagonists. The first orally active MCH antagonist in rats was originally isolated by the Takeda group in 2002 and named T-226296. This compound binds with high affinity to MCH-R1, suppresses the stimulatory effects of MCH on food intake, and significantly decreases feeding and body weight in rats under high-fat diet by reducing meal size but not meal frequency. The T-226296 molecule does not alter locomotor activity or induce malaise in a onehour test period. These results provide encouraging clues that the T-226296 molecule (and derivatives) may be effective in treatment of overeating disorders. The second nonpeptide MCH-R1 antagonist, namely SNAP-7941, was characterized in 2002 by the Synaptic group. This molecule is a competitive antagonist of MCH with high affinity for MCH-R1 and displays potent anorectic effects when injected intraperitoneally (ip) alone or in preventing the orexigenic effect of MCH. Chronic ip administration of SNAP-7941 in diet-induced obese (DIO) rats suppresses feeding and provokes a sustained weight loss that is reversible after the termination of drug treatment. This compound does not have apparent toxic or aversive actions. Strikingly, oral administration of SNAP-7941 molecule leads to unexpected anxiolytic and antidepressant effects as demonstrated in rat forced-swim test, rat social interactions, and guinea pig maternal-separation vocalization tests. The potential antidepressant property of SNAP-7941 was independently replicated at the Institut de Recherche Servier. Interestingly, two new MCH-R1 antagonists synthesized at Taisho Pharmaceutical Company and named ATC0065 and ATC0175 also exhibit antidepressant and anxiolytic effects in rodents after oral administration, without sedation or motor coodination impairment. These results provide further support for an anxiogenic effect of MCH, as found after MCH injection in the medial preoptic area or nucleus accumbens shell in rats and offer novel approaches to treat depression and/or anxiety. However, conflicting data were reported regarding the role of MCH in the regulation of mood as found for the regulation of the HPA axis. Furthermore, the specificity of SNAP-7941 as well as other MCH antagonists needs to be evaluated in a MCH-R1 null mouse model to preclude the possibility that the in vivo activities of these compounds may be mediated by another receptor. Thus, since the pioneer studies using T-226296 or SNAP-7941 as MCH antagonists, a number of compa-

nies have synthesized, tested, and patented a variety of nonpeptide MCH antagonists acting at MCH-R1 (close to 40 patent applications are presently registered [8, 20]). As such, the compounds are claimed to be useful in suppressing appetite and in treating or preventing a wide range of diseases, including diabetes, hyperlipidemia, depression, anxiety and associated somatic symptoms, neurodegenerative disorders, as well as even more distantly related pathologies such as bacterial, fungal, and viral infections. There is no doubt that among this huge number of compounds suitable candidates will be identified and subsequently enter clinical trials. However, major questions remain about the role of MCH and MCH-R1 in the control of energy balance (e.g., phenotype discrepancy between MCH and MCHR1 null mice) and other behaviors (stress response, reproduction, sleep, and learning). Furthermore, the MCH physiological functions mediated by MCH-R2 in the human brain deserve thorough investigations.

References [1] Allaeys I, Bouyer K, Loudes C, Faivre-Bauman A, Petit F, Ortola C, Cardinaud B, Epelbaum J, and Nahon JL. Characterization of MCH-gene overprinted-polypeptide-immunoreactive material in hypothalamus reveals an inhibitory role of prosomatostatin 1-64 on somatostatin secretion. Eur. J. Neurosci. 2004; 19:925–936. [2] Baker B.T. in Melanotropic peptides (Vaudry H, and Eberle A, eds) 1993, Vol. 680, pp. 279–289, New York Academy of Sciences Series, New York. [3] Baker BI. Melanin-concentrating hormone: A general vertebrate neuropeptide. Int Rev Cytol 1991; 126:1–47. [4] Baker BI, and Kawauchi H. in MCH and seizures: neuromolecular and neuroendocrine aspects (Knigge K, Prased A, Preteland S, and Wagner JE, eds) 1997, pp. 1–30, Research Signpost, India. [5] Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W, and Sawchenko PE. The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J. Comp. Neurol. 1992; 319: 218–245. [6] Borsu L, Presse F, and Nahon JL. The AROM gene: spliced mRNAs encoding new DNA/RNA binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals. J. Biol. Chem. 2000; 275: 40576–40587. [7] Boutin JA, Suply T, Audinot V, Rodriguez M, Beauverger P, Nicolas JP, Galizzi J-P, and Fauchère J-L. Melanin-concentrating hormone and its receptors: state of the art. Can. J. Physiol. Pharmacol. 2002; 80:388–395. [8] Browning A. Recent developments in the discovery of melaninconcentrating hormone antagonists: novel antiobesity agents. Expert Opin. Ther. Patents 2004; 14:313–325. [9] Cardinaud B, Darré-Toulemonde F, Duhault J, Boutin JA, and Nahon JL. Comparative analysis of melanin-concentrating hormone structure and activity in fishes and mammals. Peptides 2004; 25:1623–1632. [10] Collins CA, and Kym PR. Prospects for obesity treatment: MCH receptor antagonists. Curr. Opin. Invest. Drugs 2003; 4: 386–394. [11] Courseaux A, and Nahon JL. Birth of two chimeric genes in the Hominidae lineage. Science 2001; 291:1293–1297.

714 / Chapter 98 [12] Cvetkovic V, Brischoux F, Jacquemard C, Fellmann D, Griffond B, and Risold PY. Characterization of subpopulations of neurons producing melanin-concentrating hormone in the rat ventral diencephalon. J. Neurochem. 2004; 91:911–919. [13] Eberle AN, Mild G, Schlumberger SE, Drozdz R, Hintermann E, and Zumsteg U. Expression and characterization of melaninconcentrating hormone receptors on mammalian cell lines. Peptides 2004; 25:1585–1595. [14] Forray C. The MCH-receptor family: feeding brain disorders? Curr. Opin. Pharmacol. 2003; 3:85–89. [15] Griffond B, and Baker BI. Cell and Molecular cell biology of melaninconcentrating hormone. Int. Rev. Cytol. 2002; 213:233–277. [16] Hervieu G. Melanin-concentrating hormone functions in the nervous system: food intake and stress. Expert Opin. Ther. Targets 2003; 7:495–511. [17] Hervieu G, Maulon-Feraille L, Chambers JK, Cluderay JE, Wilson S, Presse F, and Nahon JL. In Handbook of chemical neuroanatomy. Peptide receptors, Part II (Quirion R, Björklund A, and Hökfelt T, eds) Vol. 20; 2003. pp. 31–101, Elsevier Sci. [18] Hogben LT, and Slome D. In Vol. VI, The dual character of endocrine coordination In Amphibian colour change. Ser. B , 931, Vol. 108, pp. 10–16, Proc. Roy. Soc., London. [19] Kawauchi H, Kawazoe I, Tsubokawa M, Kishida M, and Baker BI. Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 1983; 305:321–323.

[20] Kowalski TJ, and McBriar M. Therapeutic potential of melaninconcentrating hormone-1 receptor antagonists for the treatment of obesity. Expert Opin. Investig. Drugs 2004; 13: 1113–1122. [21] Nahon JL. The melanin-concentrating hormone: from the peptide to the gene. Crit. Rev. Neurobiol. 1994; 8: 221–262. [22] Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przupek J, Kanarek R, and MaratosFlier E. A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 1996; 380: 243–247. [23] Shi Y. Beyond skin color: emerging roles of melanin-concentrating hormone in energy homeostasis and other physiological functions. Peptides 2004; 25:1605–1611. [24] Toumaniantz G, Bittencourt JC, and Nahon JL. The rat melaninconcentrating hormone gene encodes an additional putative protein in a different reading frame. Endocrinology 1996; 137:4518–4521. [25] Tritos NA, and Maratos-Flier E. Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides 1999; 33:339–349. [26] Vaughan JM, Fischer WH, Hoeger C, Rivier J, and Vale W. Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 1989; 125:1660–1665.

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99 CCK/Gastrin MARGERY C. BEINFELD

ABSTRACT

CCK and gastrin share the same five carboxyl terminal amino acids and gastrin is thought to have evolved from CCK by gene duplication. CCK has a long evolutionary history [22]. The final processed form of both CCK and gastrin has a sulfated tyrosine residue. CCK is completely sulfated, while the degree of sulfation of gastrin varies.

Cholecystokinin (CCK) is one of the most abundant and widely distributed brain peptides. This review details how CCK was originally discovered in the intestine and later found in the brain. It describes where CCK neurons are located and their projections. The process by which it is made and which enzymes are involved is detailed. The signal transduction pathways that are activated when CCK binds to its two receptors are described. CCK plays a major role of CCK in anxiety, satiety, analgesia, modulation of dopamine neurotransmission, learning and memory, and sexual behavior. Possible pathophysiological implications of these activities are discussed.

STRUCTURE OF THE PRECURSOR mRNA/GENE The CCK cDNA was cloned first using mRNA from a rat thyroid medullary carcinoma [11]. This was followed in rapid succession by the cloning of the cDNAs from many species. CCK’s transcriptional unit spans 7 kb and is interrupted by two introns. The promoter is found within 144 base pairs of the transcription start site [12]. The complex regulation of CCK expression has been studied in detail, reviewed in [18]. The CCK promoter contains cyclic AMP and estrogen response elements. It also contains an E-box element. CCK expression is stimulated synergistically by cAMP and fibroblast growth factor.

DISCOVERY The discovery of the substance that would eventually be called cholecystokinin (CCK) dates to the beginning of the twentieth century. Bayliss and Starling demonstrated that intestinal acidification, which later was shown to cause the release of CCK and other peptides into the circulation caused gallbladder contraction. Administration of CCK was subsequently found to cause gallbladder contraction. The sequencing of porcine intestinal CCK 33 by Mutt and Jorpes in the 1960s was another important milestone [26]. The name CCK/ gastrin probably arose in 1975 with the detection of CCK-like immunoreactivity in the brain with a gastrin antiserum which cross-reacted with CCK [34]. Based on its biological activity, chromatographic profile, and amino acid sequencing, it soon became clear that this material was not gastrin but CCK 8 [13]. Small amounts of gastrin are found in the brain and pituitary of several species. Handbook of Biologically Active Peptides

DISTRIBUTION OF CCK CCK is one of the most abundant and widely distributed peptides in the brain. There are numerous CCK mRNA positive cell bodies in the cerebral cortex (Cx), amygdala (Amy), thalamus (Th), hippocampus (Hi), olfactory bulb (OB), hypothalamus (Hpt), various nuclei in the mesencephalon, ventral tegmental area (VTA), and substantia nigra (SN) [6, 30]. The distribution of CCK cell bodies and their projections is shown in Fig. 1. One fascinating aspect of the anatomy of CCK

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716 / Chapter 99 in the Cx and Hi is the overlap between CCK neurons which also express GABA and cannabinoid CB1 receptors [16].

gastrin in the gut are discussed in other chapters in this book. A strict temporal order of cleavages has been shown for a number of prohormones, including CCK. It is thought that the first site where cleavage occurs is the most accessible. After cleavage of the first site, the remaining prohormone changes structure, making additional sites available for cleavage in a specific order. The order of pro-CCK processing to CCK 22 and CCK 8 is indicated in Fig. 2. It is based on products identified in normal and fat/fat mouse brain and on site-directed mutagenesis studies in endocrine tumor cells [28, 35].

PROCESSING OF THE PRECURSOR The processing of pro-CCK occurs mainly at single Arg or Lys residues (Fig. 2). The sequence and structure of pro-CCK and gastrin are similar and they both display tissue-specific processing. CCK 8 is the major form in brain, while intestine has larger forms. CCK and

III

Signal Peptide Cleavage

CCK 39

CCK 58

QPVVPAETDPVEQRAQEAPRRQLR AVLRTDGEPRARLGALLA RYIQQVR II

CCK 33

CCK 8

CCK 22

The signal sequence of pre-pro-CCK allows it to be inserted into the lumen of the endoplasmic reticulum (ER). The signal sequence is removed by the signalase enzyme located on the luminal surface of the ER membrane.

I

KAPSGRMSVLK NLQSLDPSHRISDR [DYMGWMDF]GRR SAEDYEYPS

FIGURE 1. Sequence of mouse pro-CCK written in the single amino acid code. The endoproteolytic cleavage sites are indicated by spaces and downward arrows, the CCK 8 sequence is underlined. CCK peptides are numbered backward from the carboxyl terminal of CCK 8. The amino terminal end of the major forms of amidated CCK peptides is indicated by the downward arrows. Stars mark the location of the sulfated tyrosine residues. The temporal order of endoproteolytic cleavages for the production of CCK 8 is indicated by the roman numerals.

Tyrosine Sulfation Three out of four tyrosine residues in pro-CCK are sulfated by protein tyrosine sulfotransferase in the transGolgi network (TGN). The tyrosines that are sulfated have adjacent acidic residues that constitute the consensus sequence for sulfation.

Cx

Hi CC

OB CPut

S

Th PAG

AON

C BST

E W PVN

Acb

Hpt VTA DMH

SN NST

DBB

SO N

Arc

VMH ME Amy

NL I L AP

Amb

FIGURE 2. Distribution of CCK neurons and their major projections in the rat brain. CCK cell bodies are indicated by the dots and their projections by the lines.

LRN

CCK/Gastrin Sorting into Secretory Granules In the TGN, sulfated pro-CCK is sorted into secretory granules with the enzymes responsible for its processing. Cleavage 1 on the Carboxyl-Terminal of CCK 8 Gly Arg Arg (GRR) Mutation of the GRR site on the carboxyl-terminal of CCK 8 to Gly Ala Ala blocks production of amidated CCK [35]. Cleavage 2 at RA (Arg Ala) at CCK 58 (Removal of the Pro-Peptide) Mutation of the RA cleavage site to Ala Ala completely prevents subsequent production of amidated CCK 22 or CCK 8. CCK 58 GRR does not accumulate in brain or in cell lines because it is efficiently cleaved to smaller forms [35]. Cleavage 3 at CCK 22 Lys Asn and Arg Asp CCK 8 to Produce CCK 22 GRR and CCK 8 GRR Removal of these cleavage sites by mutation prevents their cleavage but otherwise has a minor impact on any other aspect of the processing [35]. Conversion of CCK 22 G and CCK 8 G to CCK 22 Amide and CCK 8 Amide by the Amidation Enzyme The last step in CCK biosynthesis is the amidation of glycine-extended CCK peptides by peptidylglycine-αamidating monooxygenase [14]. Enzymology of Pro-CCK Processing Tyrosyl Protein Sulfotransferase Two Golgi enzymes (TSP1 and TSP2) have been identified that are thought to be responsible for the sulfation of the three tyrosine residues in pro-CCK. Endoproteases The most likely candidates enzyme(s) responsible for the endoproteolytic cleavages in CCK during its processing are the prohormone convertases (PCs) [32], most notably PC1, PC2, and PC5. They are widely co-localized with CCK in the brain [7, 9] and present in cell lines that express CCK mRNA. A number of studies have demonstrated that they have the correct catalytic activity to cleave pro-CCK. Antisense and RNA studies in cell lines also support a role for these enzymes in CCK processing. PC1 and 2 knockout mice have decreased levels of CCK in specific brain regions, although considerable

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CCK is still detected. This is strong evidence that they are involved in CCK processing. There also appears to be mechanisms that compensate for their loss and continue to provide some CCK. Carboxypeptidases In adult fat/fat mice that lack carboxypeptidase E, the levels of amidated CCK in brain are decreased by 85% showing that this enzyme is required for CCK processing. There is also a large accumulation of glycine and arginine extended CCK 8 [8, 37], the immediate precursor of CCK 8. This also shows that CCK 33 or CCK 22 amide cannot be the immediate precursor of CCK 8 amide in brain. Amidating Enzyme Although C-terminal amidation is a widespread modification, glycine extended CCK is abundant in rodent brain, intestine, and CCKexpressing endocrine cells. This enzyme is dependent on copper and ascorbate, and at least in some cell lines, the quantity of glycine extended CCK is decreased by adding ascorbate [3].

RECEPTORS AND SIGNALING CASCADES Two CCK receptors have been cloned. The CCK 1 subtype is relatively specific for sulfated CCK 8, while unsulfated CCK 8, CCK 4, and gastrin are 2–3 orders of magnitude less potent. For the CCK 2 subtype (identical to the gastrin receptor), this difference in potency is about one order of magnitude. The CCK 2 receptor is more abundant and widely distributed in the brain than the CCK 1 receptor, although distinct populations of the CCK 1 receptor are present. The CCK 1 receptor is present on soma and fibers in the SN, VTA, nucleus accumbens (Acb), and dorsal striatum but not in the prefrontal Cx, Amy, or Hi. The CCK 2 receptor is present on soma and fibers in the SN, VTA, Cx, Abc, dorsal striatum, Hi, and Amy. What happens after CCK activates its receptors has been extensively studied and was reviewed recently [40]. The model that has been studied in the most detail is the rodent pancreatic acinar cell which expresses mainly CCK 1 receptors. The AR42J pancreatic cell line and adrenal chromaffin cells which express the CCK 2 receptor have also been studied. In pancreatic acinar cells, CCK receptor activation recruits the heterotrimeric G proteins Gq and G11 and results in increased levels of inositol triphosphate, increased intracellular calcium, and activation of protein kinase C. CCK at higher concentrations can activate adenylate cyclase, increasing cAMP levels. CCK also activates three mitogen activated protein kinase cascades leading to ERK, JNK, and p38 MAPK. CCK also

718 / Chapter 99 activates the NF-κB pathway and the P13K-PKB-m-TOR pathway, which regulates protein synthesis. Activation of the CCK 2 receptor in gastric cancer cells activates protein kinase D2 [33] and in human adrenal chromaffin cells it increases adenylate cyclase and cAMP [33].

to be particularly evident in neuropathic pain, where increased CCK release as well as changes in expression of CCK receptors has been reported [39].

ACTIVE AND/OR SOLUTION CONFORMATION

CCK is colocalized with dopamine in the VTA [20], and these neurons project to medial and posterior subdivisions (shell) of the Acb, the olfactory tubercle, and pre-frontal Cx [43]. CCK 1 receptors in Acb shell display DA agonist-like activities (increased dopamine release and enhancement of dopamine-mediated behaviors), while in lateral portions of the Acb (core), CCK 2 receptors display DA antagonist-like activities (decreased dopamine release and inhibition of dopamine-mediated behaviors). CCK infusion into Acb shell increases dopamine release, while core infusion decreases dopamine release. Treatment of rats with psychostimulants and stress increases CCK release and augments their locomotor behavior (sensitization). The CCK 1 antagonist blocks expression of psychostimulant sensitization while the CCK 2 antagonist blocks acquisition of this behavior, reviewed in [5]. OLETF rats lacking the CCK 1 receptor display much less behavioral sensitization to repeated cocaine than wild type LETO rats [4]. CCK 2 receptor knockout mice behave as if they have augmented dopamine neurotransmission. They displayed more locomotor activity than the wild-type [10] and more locomotor activity in response to a single high dose of amphetamine [23]. This implies that the CCK 1 receptor is required for full expression of cocaine sensitization, while the CCK 2 receptor is tonically inhibiting dopamine release.

Biophysical measurements of sulfated and nonsulfated CCK 8 demonstrate that these peptides are found mainly in folded forms with beta and gamma turns around the sequence Gly-Trp-Met-Asp and Met-AspPhe-NH2, respectively [15]. NMR spectroscopy and molecular modeling methods have demonstrated that CCK 15 assumes a helical conformation [2].

BIOLOGICAL ACTIONS WITHIN THE BRAIN AND PITUITARY The actions of CCK in the brain are diverse and have been the subject of intense investigation. Progress has been aided by the development of specific agonists and antagonists and discovery or creation of rodent models that lack the receptors or the peptide. Satiety CCK released from the intestine by ingestion of food was found to have a major influence on food intake even before CCK was detected in the brain [17]. A chapter in this volume details the role of CCK in satiety.

CCK Modulation of Dopamine Neurotransmission, Dopamine-Mediated Reward, and Psychostimulant Sensitization

Analgesia CCK acting through the CCK 2 receptor has antiopiate activity. Opiate analgesia is mainly mediated by neurons in the rostral ventromedial medulla (RVM). CCK exerts its inhibitory effect on morphine analgesia by inhibiting activation of pain inhibiting output neurons of the RVM [19]. Acute administration of CCK into the RVM causes acute tactile and thermal hypersensitivity that is antagonized by the CCK 2 receptor antagonist or lesion of the dorsolateral funiculus. Continuous administration of morphine produces sustained tactile and thermal hypersensitivity that is reversed by the CCK 2 antagonist. Continuous morphine treatment resulted in a fivefold increase in basal CCK levels in the RVM relative to controls [42]. Activation of the endogenous CCK system by repeated morphine treatment may be partially responsible for the observed hypersensitivity. The opiate antagonist effect of CCK appears

Learning, Memory, and Sexual Behavior CCK is very abundant in the Cx, Amy, and Hi, so it is not surprising that it plays a role in cognition. CCK acting through the CCK 2 receptor in the Hi is involved in spatial memory [31]. Other memory tasks which CCK facilitates involve a dopaminergic projection from the VTA to the Nac, central Amy, and Hi [41]. CCK improves spatial memory in elderly rats [36] and in rats whose dopamine has been depleted by 6 hydroxydopamine [29]. CCK acting through the CCK 1 receptor is involved in maternal behavior and in infant-maternal recognition in sheep [27] and in appetitive learning in infant rats [38]. CCK released by a meal activates peripheral CCK 1 receptors to initiate these behaviors. CCK interacting with estrogen and endogenous opiates in the Hpt is involved in regulating sexual behavior in female rodents [25].

CCK/Gastrin Anxiety A large literature describes the ability of CCK to trigger panic attacks in human subjects and to produce anxiety-related behaviors in rodents and primates. This is mediated through the CCK 2 receptor.

[4]

[5]

PATHOPHYSIOLOGICAL IMPLICATIONS There is currently no CCK-based drug in clinical practice. CCK and its receptors are widely distributed and have many different physiological functions in the brain and the periphery. Selectively targeting CCK receptors in a single organ will not be easy. Satiety Although CCK does cause short-term inhibition of food intake, this activity has not been utilized therapeutically. Given the anatomical and physiological relationship between cannabinoids, CCK, and leptin, it is possible to speculate that the new CB1 cannabinoid antagonist soon to be approved as a diet drug (Rimonabant) may work in part through stimulating CCK release.

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Analgesia CCK 2 antagonists have been tested in humans where they improve pain management when given with morphine [24]. It is possible that CCK 2 antagonist treatment might be promising particularly in neuropathic pain to increase morphine efficacy and reduce tolerance. Novel ligands incorporating an opiate peptide with a CCK 2 antagonist has been designed, which might be useful in the treatment of pain [21]. Anxiety CCK 2 antagonists have not been shown to be beneficial in the treatment of anxiety, but questions remain about whether these antagonists get into the brain [1].

References [1] Adams JB, Pyke RE, Costa J, Cutler NR, Schweizer E, Wilcox CS, Wisselink PG, Greiner M, Pierce MW, Pande AC. A doubleblind, placebo-controlled study of a CCK-B receptor antagonist, CI-988, in patients with generalized anxiety disorder. J Clin Psychopharmacol 1995;15:428–34. [2] Albrizio S, Carotenuto A, Fattorusso C, Moroder L, Picone D, Temussi PA, D’Ursi A. Environmental mimic of receptor interaction: conformational analysis of CCK-15 in solution. J Med Chem 2002;45:762–9. [3] Beinfeld MC, Perloff MD, Venkatakrishnan K. Identification of glycine-extended CCK peptides in endocrine cells and modula-

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tion of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation. Peptides 1998;19:1393–8. Beinfeld MC, Connolly K, Pierce RC. OLETF (Otsuka LongEvans Tokushima Fatty) rats that lack the CCK 1 (A) receptor develop less behavioral sensitization to repeated cocaine treatment than wild type LETO (Long Evans Tokushima Otsuka) rats. Peptides 2001;22:1285–90. Beinfeld MC. What we know and what we need to know about the role of endogenous CCK in psychostimulant sensitization. Life Sci 2003;73:643–54. Burgunder J-M, Young WS, I. Cortical neurons expressing the cholecystokinin gene in the rat: distribution in the adult brain, ontogeny, and some of their projections. J Comp Neurol 1990;300:26–46. Cain B, Connolly K, Blum A, Vishnuvardhan D, Marchand JE, Beinfeld MC. The distribution and co-localization of cholecystokinin with the prohormone convertase enzymes PC1, PC2 and PC5 in rat brain. J Comp Neurol 2003;467:307–25. Cain BM, Wang WG, Beinfeld MC. Cholecystokinin (CCK) levels are greatly reduced in the brains but not the duodenums of Cpefat/Cpefat mice: A regional difference in the involvement of carboxypeptidase E (Cpe) in pro-CCK processing. Endocrinology 1997;138:4034–7. Cain BM, Connolly K, Blum AC, Vishnuvardhan D, Marchand JE, Zhu X, Steiner DF, Beinfeld MC. Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain. J Neurochem 2004;89:307–13. Dauge V, Sebret A, Beslot F, Matsui T, Roques BP. Behavioral profile of CCK2 receptor-deficient mice. Neuropsychopharmacology 2001;25:690–8. Deschenes RJ, Lorenz LJ, Haun RS, Roos BA, Collier KJ, Dixon JE. Cloning and sequence analysis of a cDNA encoding rat precholecystokinin. Proc Natl Acad Sci USA 1984;81:726–30. Deschenes RJ, Haun RS, Funckes CL, Dixon JE. A gene encoding rat cholecystokinin. J Biol Chem 1985;260:1280–6. Dockray GJ, Gregory RA, Hutchinson JB. Isolation, structure, and biological activity of two cholecystokinin octapeptides from sheep brain. Nature 1978;264:568–70. Eipper BA, Milgram SL, Husten EJ, Yun H-Y, Mains RE. Peptidylglycine alpha-amidating monooxygenase: A multifunctional protein with catalytic, processing and routing domains. Protein Sci 1993;2:489–97. Fournie-Zaluski MC, Belleney J, Lux B, Durieux C, Gerard D, Gacel G, Maigret B, Roques BP. Conformational analysis of cholecystokinin CCK26–33 and related fragments by 1H NMR spectroscopy, fluorescence-transfer measurements, and calculations. Biochemistry 1986;25:3778–87. Freund TF. Interneuron diversity series: Rhythm and mood in perisomatic inhibition. Trends Neurosci 2003;26:489–95. Gibbs J, Young SN, Smith GP. Cholecystokinin elicits satiety in rats with open gastric fistulas. Nature 1973;245:323–5. Hansen TO. Cholecystokinin gene transcription: promoter elements, transcription factors and signaling pathways. Peptides 2001;22:1201–11. Heinricher MM, McGaraughty S, Tortorici V. Circuitry underlying antiopioid actions of cholecystokinin within the rostral ventromedial medulla. J Neurophysiol 2001;85:280–6. Hokfelt T, Skirboll L, Rehfeld JF, Goldstein M, Markey K, Dann O. A subpopulation of mesencephalic dopamine neurons projecting to limbic areas contain a cholecystokinin-like peptide: evidence from immunocytochemistry combined with retrograde tracing. Neurosci 1980;5:2093–124.

720 / Chapter 99 [21] Hruby VJ, Agnes RS, Davis P, Ma SW, Lee YS, Vanderah TW, Lai J, Porreca F. Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain. Life Sci 2003;73:699–704. [22] Johnsen AH. Phylogeny of the cholecystokinin/gastrin family. Front Neuroendocrinol 1998;19:73–99. [23] Koks S, Volke V, Veraksits A, Runkorg K, Sillat T, Abramov U, Bourin M, Huotari M, Mannisto PT, Matsui T, Vasar E. Cholecystokinin2 receptor-deficient mice display altered function of brain dopaminergic system. Psychopharmacology (Berl) 2001;158:198–204. [24] McCleane G. Cholecystokinin antagonists: A new way to improve the analgesia from old analgesics? Curr Pharm Des 2004; 10:303–14. [25] Micevych P, Sinchak K. Estrogen and endogenous opioids regulate CCK in reproductive circuits. Peptides 2001;22:1235–44. [26] Mutt V. Historical perspectives on cholecystokinin research. Ann NY Acad Sci 1994;713:1–10. [27] Nowak R, Breton G, Mellot E. CCK and development of mother preference in sheep: a neonatal time course study. Peptides 2001;22:1309–16. [28] Pratt JS, Blum A, Vishnuvardhan D, Kitagawa K, Beinfeld MC. Cleavage-site mutagenesis alters post-translation processing of pro-CCK in AtT-20 cells. Biochemistry 2004;43:9502–11. [29] Rex A, Fink H. Cholecystokinin tetrapeptide improves water maze performance of neonatally 6-hydroxydopamine-lesioned young rats. Pharmacol Biochem Behav 2004;79:109–17. [30] Schiffmann SN, Vanderhaeghen J-J. Distribution of cells containing mRNA encoding cholecystokinin in the rat central nervous system. J Comp Neurol 1991;304:219–33. [31] Sebret A, Lena I, Crete D, Matsui T, Roques BP, Dauge V. Rat hippocampal neurons are critically involved in physiological improvement of memory processes induced by cholecystokininB receptor stimulation. J Neurosci 1999;19:7230–7. [32] Seidah NG, Day R, Marcinkiewicz M, Chrétien M. Precursor convertases: An evolutionary ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and proteins. Ann NY Acad Sci 1998;839:9–24. [33] Sturany S, Van Lint J, Gilchrist A, Vandenheede JR, Adler G, Seufferlein T. Mechanism of activation of protein kinase

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100 The Hypocretins (Orexins) LUIS DE LECEA AND J. GREGOR SUTCLIFFE

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tractive hybridization to identify messenger RNA species restricted to discrete nuclei within the rat hypothalamus. Among these was a species whose expression, as detected by in situ hybridization, was restricted to a few thousand neurons that were bilaterally distributed within the dorsolateral hypothalamus [15, 22]. A large collaborative study to identify endogenous ligands for orphan G protein-coupled receptors discovered the peptides independently [62]. This group referred to the peptides as orexins because they stimulated acute food intake when administered to rats during the daytime. In this chapter, we will refer to the peptides by their first-used name, the hypocretins, but both names have been used extensively in the large literature that has grown up around the peptides.

The hypocretins (also called the orexins) are two Cterminally amidated neuropeptides of related sequence. They are produced from a common precursor whose expression is restricted to a few thousand neurons of the rat dorsolateral hypothalamus. Two G proteincoupled receptors for the hypocretins have been identified, and these have different distributions within the CNS and differential affinities for the two hypocretins. The hypocretins project to areas within the posterior hypothalamus that are implicated in feeding behaviors and hormone secretion. Hypocretin fibers also project to diverse targets in other brain regions and the spinal cord, including several areas implicated in cardiovascular function and sleep-wake regulation. The peptides are generally neuroexcitatory. Administration of the hypocretins stimulates food intake, affects blood pressure, hormone secretion, and locomotor activity, and increases wakefulness while suppressing rapid eye movement (REM) sleep. Inactivating mutations in the hypocretin receptor 2 gene in dogs result in narcolepsy. Mice whose hypocretin gene has been inactivated exhibit a narcolepsy-like phenotype. Most human patients with narcolepsy have greatly reduced levels of hypocretin peptides in their cerebral spinal fluid and no or barely detectable hypocretin neurons in their hypothalami, suggestive of autoimmune attack.

STRUCTURE OF THE PRECURSOR mRNA/GENE The mouse hypocretin gene, HCRT, is located on chromosome 11, and the human HCRT gene maps to chromosome 17q21–q24. Genes that encode conserved pre-prohypocretins have been detected in pufferfish, zebrafish, frog [21], and chicken species, suggesting that the gene arose early in the chordate lineage. Sequence similarities with various members of the incretin family, especially secretin, suggest that the preprohypocretin gene was formed from the secretin gene by three genetic rearrangements: First, a duplication of the secretin gene; second, deletions of the N-terminal portion of the 5′ duplicate and the C-terminal portion of the 3′ duplicate to yield a secretin with its N- and Ctermini leapfrogged (circularly permuted); and third, a further duplication of the permuted gene, followed

DISCOVERY An open-system search for undiscovered hypothalamic regulatory peptides provided the first glimpse of the hypocretins. Gautvik and colleagues [22] used subHandbook of Biologically Active Peptides

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722 / Chapter 100 by modifications, to form a secretin derivative that encoded two related hypocretin peptides. The human pre-prohypocretin gene consists of two exons and one intron distributed over 1432 base pairs. The 143-base pair first exon includes the 5′-untranslated region and a small part of the coding region that encodes the first seven residues of the secretory signal sequence. The second exon contains the remaining portion of the open reading frame and 3′-untranslated region. The 3.2 kilobase pairs of the 5′-upstream region from a cloned human pre-pro-Hcrt gene promoter is sufficient to direct the expression of the E. coli β-galactosidase (lacZ) gene in transgenic mice to neurons in the lateral hypothalamic area and adjacent regions [63].

hypothalamus. Both mRNA and peptide expression diminish after one year of age. The human lateral hypothalamus contains 50,000–80,000 hypocretin neurons [51]. Hcrt neurons are 20–30 μm in diameter and are multipolar or fusiform in shape, with 2–4 primary dendrites bearing few spines. In addition to rats, mice, and humans, Hcrt neurons with a similar restricted hypothalamic distribution have been detected in monkey, hamster, cat, sheep, pig, chicken, various amphibians, and zebrafish. Glutamate, the excitatory amino acid transporter EAAT3, and the vesicular glutamate transporters VGLUT1 and VGLUT2 are expressed by Hcrt neurons [13, 73]; thus, Hcrt neurons are likely to be glutamatergic. Other proteins detected in Hcrt neurons include dynorphin, GABAA receptor epsilon subunit, 5-HT1A receptor, mu opioid receptor, pancreatic polypeptide Y4 receptor, adenosine A1 receptor, leptin receptor, transcription factor Stat-3, and the neuronal pentraxin Narp, implicated in clustering of ionotropic glutamate receptors. There are several reports of hcrt and hcrt receptor expression in the periphery, including the enteric nervous system [37], pancreas, kidney, stomach, and ileum [52]. There are also several reports of hcrt expression in the human anterior pituitary [6] and adrenal gland [5]. One study showed hcrt-immunoreactivity in amacrine cells in the human retina [67].

DISTRIBUTION OF THE mRNA The rat pre-prohypocretin mRNA migrates in northern blots at ∼700 nucleotides in samples obtained from brain [15]. A few thousand neurons highly positive for Hcrt mRNA and immunoreactivity are located between the rat fornix and the mammillothalamic tracts [15, 56]. These are first detected at embryonic day E18 [69]. Beginning at E20, hypocretin antisera detect a prominent network of axons that project from these cells to other neurons in the perifornical and posterior

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FIGURE 1. Distribution of hypocretin-containing cell bodies and projections. In the rodent brain there are ~3000 neurons that produce hypocretin, all located in the periformical area of the posterior hypothalamus. These neurons send projections throughout the brain (arrows) with strong innervation of aninergic nuclei. For a comprehensive description of the anatomical projections of hypocetin neurons see Peyron et al. [56]. (See color plate.)

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PROCESSING OF THE PRECURSOR Preprohypocretin encodes a 130-residue putative secretory protein with an apparent signal sequence and two additional phylogenically conserved sites for potential proteolytic maturation followed by modification of the carboxy-terminal glycines by peptidylglycine αamidating monooxygenase [15]. These features suggested that the product of this hypothalamic mRNA served as a pre-prohormone for two C-terminally amidated, secreted peptides. One of these, hypocretin 2 (Hcrt2 or OxB), was, on the basis of the putative preprohormone amino-acid sequence, predicted to contain precisely 28 residues. The other, hypocretin 1 (Hcrt1 or OxA), had a defined predicted amidated C terminus, but because of uncertainties as to how the amino terminus might be proteolytically processed, an undefined N-terminal extent [15]. The C-terminal 19 residues of these two putative peptides shared 13 amino acid identities, suggesting that the peptides had related structures and functions. This region of Hcrt2 contained a sevenamino-acid match with secretin. Antisera generated against synthetic peptides corresponding to regions of the deduced prohypocretin sequence and to bacterially expressed preprohypocretin have been generated [15, 56]. The antisera are specific for the hypocretin-related peptides and have been used extensively to characterize the protein in both anatomical and ELISA-type studies. The detection of the two hypocretin peptides within the brain allowed the exact structures of these endogenous peptides to be determined by mass spectroscopy [62]. The sequence of endogenous Hcrt2, RPGPPGLQGRLQRLLQAN GNHAAGILTM-amide, was the same as that predicted from the cDNA sequence. The N-terminus of Hcrt1 was found to correspond to a genetically encoded glutamine that was derivatized as pyroglutamate. Hcrt1 (33 residues: *EPLPDCCRQKTC SCRLYELLHGAGNHAAGILTL-amide) contains two intrachain disulfide bonds. Human Hcrt1 is identical to the rodent peptide, whereas human Hcrt2 differs from rodent Hcrt2 at two residues [62]. The enzyme that cleaves preprohypocretin and yields the mature peptides has not been determined. Anatomical evidence suggests that prohormone convertase 1 colocalizes with hcrt in the lateral hypothalamus [53] and may be involved in the processing of the precursor.

RECEPTORS AND SIGNALING CASCADES Sakurai and collaborators [62] prepared transfected cell lines stably expressing each of 50 orphan G-proteincoupled receptors (GPCRs), and then measured

calcium fluxes in these cell lines in response to fractions from tissue extracts. One of these transfected cell lines responded to a substance in a brain extract. Mass spectroscopy showed that this substance was a peptide whose sequence was later identified as that of endogenous Hcrt1. The initial orphan GPCR, Hcrtr1 (also referred to as OX1R), bound Hcrt1 with high affinity but Hcrt2 with 100- to 1000-fold lower affinity. A related GPCR, Hcrtr2 (OX2R), sharing 64% identity with Hcrtr1, was identified by searching database entries with the Hcrtr1 sequence and had a high affinity for both Hcrt2 and Hcrt1 [62]. These two receptors are highly conserved (95%) across species. Radioligandbinding studies and calcium flux measurements have shown Hcrt1 to have equal affinity for Hcrtr1 and Hcrtr2, whereas Hcrt2 has ∼10-fold greater affinity for Hcrtr2 than Hcrtr1 [74]. The mRNAs that encode the two hypocretin receptors and the receptor proteins themselves, detected by immunohistochemistry, are both enriched in the brain and moderately abundant in the hypothalamus, but have different distributions within the brain [45]. Hcrtr1 mRNA is prominent in the prefrontal Cx, Hi, paraventricular thalamic nucleus, VMH, DR, LC, LDT/ PPT (at higher density than Hcrtr2), pontine raphe, dorsal motor vagal complex, and spinal cord. Additional forebrain regions expressing moderate to high levels of Hcrtr1 mRNA are the tenia tecta, BST, DBB, and medial amygdala. Immunoreactive terminals have been detected in the LH. This receptor has also been detected in the retina [67]. Hcrtr2 mRNA (there are two alternative splice forms, each encoding proteins with slightly different structure and anatomical distribution but no demonstrated functional differences) [12] is detected in the Cx, S, Hi, Th, DR, and various nuclei of the hypothalamus, including the tuberomammillary nucleus (TMN), PVN, and DMH. PAG and midbrain reticular formation are also positive. Hcrtr2 mRNA is prominent is the S, DBB, and central Amy. Both receptors are detected in the dorsal raphe, VTA, and midline thalamic nuclei. In the Cx, Hcrtr1 mRNA is expressed primarily in layers II, III and V, whereas Hcrtr2 mRNA is found at higher density in layers II and VI and more diffusely in other layers. In the hippocampus, Hcrtr1 is expressed mainly in the CA2 region and medial dentate gyrus, while Hcrtr2 was most abundant in CA3 [29]. The distribution of Hcrt receptors is largely consistent with Hcrt axon innervation patterns. The composite distribution of the two Hcrt receptors strongly resembles the distribution of the MCH receptor [35]. In the LC, amygdala, and other brainstem noradrenergic groups, MCH receptor mRNA distribution is similar to that of the Hcrtr1. In regions such as the septum,

724 / Chapter 100 hypothalamus, and much of the brainstem, the distribution of MCH receptor mRNA resembles that of the Hcrtr2 [35]. The Hcrt receptors are not restricted to the central nervous system: In the periphery, they are widely expressed, especially in endocrine tissues. Hcrtr1 or Hcrtr2 have been detected in the pituitary, adrenal gland, testis, gastrointestinal tract, pancreas, and pineal gland. Both Hcrt1 and Hcrt2 evoke increases in Ca2+, as measured by fura-2 imaging, in about one-third of hypothalamic neurons, probably by opening a TRPCtype calcium channel [40]. Responses to hypocretin are completely blocked by the protein kinase C-specific inhibitors and by phospholipase C (PLC) inhibitors, [77] suggesting that the hypocretins work through a family of GTP-binding proteins (Gq) that activate protein kinase C (PKC) and mobilization of intracellular calcium. Gq-activated signaling cascades result in phosphorylation of Ca2+ channels, which can increase Ca2+ conductance and neuronal excitability [68, 75]. The nonamidated forms of the peptides are not electrophysiologically active [68]. In the SN, hypocretin stimulation was sensitive to an inhibitor of protein kinase A, which mediates effects of cAMP, but insensitive to blockers of PKC [39], implicating a role of the Gs system.

INFORMATION ON ACTIVE AND/OR SOLUTION CONFORMATION The structures of both hcrt1 and hcrt 2 in solution have been determined by nuclear magnetic resonance [36, 43]. Consistent with the hypothesis that the hypocretins and secretin are phylogenetically related, portions of their three-dimensional solution structures are similar despite their leapfrogged primary sequence, consisting of two adjacent α-helices (6–7 and 9–14 amino acids long) separated by a short 2- to 3-aminoacid turn [25, 36, 43]. The longer helix corresponds to the region of identity between the two peptides.

BIOLOGICAL ACTIONS WITHIN THE BRAIN Administration of the hypocretins to experimental animals stimulates food intake, affects autonomic and endocrine parameters, and increases arousal. These are discussed in the following in that order, but because the disease of the hypocretin system is the sleep disorder narcolepsy, the arousal aspects are the most important and will be discussed in a later section of the chapter as well as in the sleep peptides chapter of this book.

Feeding and Metabolism Sakurai and colleagues [62] found that intracerebroventricular (ICV) administration of either Hcrt1 or Hcrt2 increased short-term food consumption in rats. Furthermore, rats that had been deprived of food for 48 hours showed increased concentrations of hypocretin mRNA and peptides in the hypothalamus [50, 62]. Feeding responses can be elicited by local administration of Hcrt1 to the paraventricular nucleus, the dorsomedial nucleus, the lateral hypothalamus, or the perifornical area [16]. ICV administration of Hcrt2 also increases food intake in sheep [66] and goldfish [78]. Many observations leave little doubt that the hypocretin system influences and is influenced by primary nutritional homeostasis circuits. For instance, hcrt neurons are sensitive to glucose, leptin, and triglyceride concentrations [9, 82]. Hcrt neurons are contacted by NPYcontaining neurons in the arcuate nucleus [30, 31], and much of the food intake increase elicited by hcrt-1 seems to be mediated by NPY [33, 85]. Other findings suggest that the hypocretins are not critical players in feeding activities but rather play roles in increasing arousal and motivation levels so that feeding can take place. Continuous administration of Hcrt1 for seven days in rats does not significantly alter daily food intake, body weight, blood glucose, total cholesterol, or free fatty acid levels [86], suggesting that many of hypocretin’s effects may be limited to shortterm, immediate stimulation of feeding behavior due to increased wakefulness. If hypocretin has a direct role on food intake, one would expect a lean phenotype in hcrt-deficient mice, as has been described for melanin concentrating hormone [58]. Hcrt knockout mice show very modest [81], if significant, differences in food intake. Hypocretin-ataxin 3 mice, which are genetically depleted of hcrt neurons and would be expected to be lean, show obesity and hypolocomotion [27], and this effect appears to be dependent on diet and genetic background [28]. During fasting, Hcrt1 accumulation in the CSF does not exceed concentrations normal for the waking period [20]. Also, hcrt ataxin-3 mice, unlike wild-type animals, do not show increase in locomotor activity after fasting [84]. All these data suggest that some of the food-uptake effect may result from arousal rather than direct feeding pressure.

Autonomic and Endocrine Effects Hypocretin neurons receive input from brainstem areas that are associated with cardiovascular function, and project to the ventrolateral medulla, the LC, the lateral paragigantocellular nucleus, the nucleus of the solitary tract, the pre-Botzinger region of the ventrolateral medulla, phrenic motoneurons, and other areas

The Hypocretins (Orexins) / 725 that have been implicated in the regulation of blood pressure, heart rate, and breathing. Projections to the arcuate nucleus also suggest a role in the regulation of hormone release. In the ovine hypothalamus, there are hypocretin terminals on the neurons that produce gonadotropin-releasing hormone, suggesting that hypocretin might particularly modulate reproductive endocrinology. In addition, projections to the raphe magnus and subcoeruleus suggested a role for hypocretins in the regulation of body temperature. The dense hypocretinergic projections to the ventrolateral preoptic area, tuberomammillary nucleus (TMN), pontine reticular formation, PPT/LDT area, and LC suggested involvement in states of arousal [56]. Very strong hypocretin-immunoreactive projections have been described in regions of the spinal cord that are related to modulation of pain [76], and hypocretin-like immunoreactivity has also been detected in the intestinal epithelium [37]. In accordance with the wide distribution of hypocretin terminals, ICV administration of the hypocretins affects not only feeding but also several other functions. Both Hcrt1 and Hcrt2 elevate mean arterial blood pressure, heart rate (both suppressed by the hypocretin receptor 1 antagonist SB334867), and oxygen consumption [11, 64]. Hcrt1 increases body temperature independent of peripheral thermogenesis, at least in part via clozapine-sensitive pathways in the cerebral cortex [49, 88]. Hcrt1 also increases water consumption and stimulates gastric acid secretion in the gut [41] and increases locomotor activity and wakefulness, while decreasing slow wave and depressing REM sleep [7, 26, 57]. The peptides also stimulate the secretion of luteinizing hormone in ovarectomized and proestrus female rats (suppressed by central administration of SB334867) and hypothalamic explants of male pituitaries [61]. Consistent with these findings, in humans, LH concentration in serum and pulsatile LH secretion are lower in male narcolepsy patients (who lack hypocretin function; see following) than in unaffected controls [38]. Hcrt concentrations are elevated during proestrus in cycling female rats but not noncycling middle-aged rats and in pregnant rats, and c-fos is increased in Hcrt neurons in lactating mice [17]. In male rats, Hcrt1 increased basal testosterone secretion [2]. Hcrt1 decreases the concentrations of circulating growth hormone and prolactin, while increasing corticosterone, ACTH, and insulin levels [26]. Hypoglycemiainduced elevation of pancreatic nerve firing is antagonized by SB334867 microinjection in the dorsal motor nucleus of the vagus, indicative of a central circuit in this pathway [83]. Hcrt2, but not Hcrt1, increases circulating thyroid-stimulating hormone [34] and has direct effects on the pituitary, adrenal, and pineal glands [48, 59, 65].

Both peptides depolarize CRF neurons in the paraventricular hypothalamic nucleus (PVN) and increase CRF, c-fos, and arginine vasopressin mRNA concentrations in the PVN, thus exerting clear effects on the HPA axis and stress-related physiology; various stress paradigms increase c-fos expression by Hcrt neurons [1]. Hcrt2 is directly excitatory on superficial dorsal horn neurons of the spinal cord [76] and exhibits an analgesic effect in models of pain [4].

Motivation and Addiction Hcrt neurons are highly responsive to morphine and are activated by naltrexone-precipitated withdrawal. However, the response of these neurons is heterogeneous, suggesting that there might be different populations of Hcrt cells [18]. The expression of the Hcrt gene increases only after precipitated withdrawal. The neurons express the μ-opioid receptor; hence their response may be directly related to morphine and naltrexone [23]. These observations might explain why animals self-administer heroin to the LH [14]. Hcrt knockout mice exhibit dramatically attenuated morphine withdrawal symptoms [23]. Hcrt neurons have extensive projections to the mesolimbic dopamine and noradrenergic (LC) pathways, regions well studied for their roles in drug addiction. These neurons also project to and inhibit nucleus accumbens neurons [46]. Rats can be trained to turn a wheel to deliver electrical current to the LH: LH self-stimulation (LHSS). LHSS thresholds measure brain reward systems; lower thresholds represent increased reward. Most drugs of abuse lower LHSS thresholds. LHSS is thought to be rewarding in part because it activates cholinergic neurons in the LDT and the PPT nuclei that consequently activate dopaminergic neurons in the VTA [19]. Hypocretins excite LDT cholinergic neurons both directly and indirectly, acting synergistically with glutamatergic afferents [8] to drive dopamine release in the nucleus accumbens by exciting dopaminergic neurons in the VTA. Thus, the Hcrt system acts as a modulator of brain reward function. Interestingly in this regard, naltrexone injected into the accumbens suppresses the Hcrt1-induced acute feeding response, suggesting that the response is mediated by opioidergic pathways involved in motivation [71].

Pain and Anesthesia In models of pain elicited by noxious stimuli or chronic constriction, Hcrts administered intrathecally or microinjected into the posterior hypothalamus decreased pain parameters [3, 70]. Hypocretin knockout mice (see following) exhibited greater hyperalgesia and less stress-induced analgesia than wild-type mice

726 / Chapter 100 [79]. Hcrt1 decreases barbiturate anesthesia time in rats by 15–40%, an action reversed by SB-334867 [42]. In vitro, barbiturates inhibit Hcrt-induced norepinephrine release, although they do not interact directly with Hcrt receptors. In isoflorane-anesthetized animals, Hcrt1 elicits arousal without cardiovascular activation, in contrast to its effect on awake animals [87]. Pain and stress activate Hcrt neurons, which respond by inhibiting pain pathways.

PATHOPHYSIOLOGICAL IMPLICATIONS The first case of human narcolepsy was reported in 1877 by Westphal, and the sleep disorder acquired its name from Gélineau in 1880. Narcolepsy affects around 1 in 2000 adults, appears between the ages of 15 to 30 years, and shows four characteristic symptoms: (1) excessive daytime sleepiness with irresistible sleep attacks during the day; (2) cataplexy (brief episodes of muscle weakness or paralysis precipitated by strong emotions such as laughter or surprise); (3) sleep paralysis, a symptom considered to be an abnormal episode of REM sleep atonia, in which the patient suddenly finds himself unable to move for a few minutes, most often upon falling asleep or waking up; and (4) hypnagogic hallucinations, or dreamlike images that occur at sleep onset. These latter symptoms have been proposed as pathological equivalents of REM sleep. The disorder is considered to represent a disturbed distribution of sleep states rather than an excessive amount of sleep. Studies with monozygotic twins have shown that narcolepsy is weakly penetrant: In only 25% of cases does the monozygotic twin of an affected individual also develop the disorder. Sporadic narcolepsy (which accounts for 95% of human cases) is highly correlated with particular class II HLA-DR and -DQ histocompatibility haplotypes in about 90% of patients, but most people with these haplotypes are not narcoleptic [47]. Because many autoimmune disorders are HLA-linked and because of the late and variable age of disease onset, narcolepsy has long been considered a likely autoimmune disorder, but the targets of the immune attack were not known.

gene that encodes the hypocretin receptor, Hcrtr2 [44]. The mutation in the Doberman lineage is an insertion of a short interspersed repeat (SINE element) into the third intron of HCRTR2 that causes aberrant splicing of the Hcrtr2 mRNA (exon 4 is skipped) and results in a truncated receptor protein. In cells that have been transfected with the mutant gene, the truncated Hcrtr2 protein does not properly localize to the membrane and, therefore, does not bind its ligands [32]. Analysis of a colony of narcoleptic Labradors revealed that their HCRTR2 gene contained a distinct mutation that resulted in the skipping of exon 6, also leading to a truncated receptor protein. A third family of narcoleptic Dachshunds carries a point mutation in HCRTR2 that results in a receptor protein that reaches the membrane but cannot bind the hypocretins.

Mouse Knockout Mutants Continuous recording of the behavior of knockout mice in which the hypocretin gene was inactivated by homologous recombination in embryonic stem cells revealed periods of ataxia, which were especially frequent during the dark period [10]. EEG recordings showed that these episodes were not related to epilepsy and that the mice suffered from cataplectic attacks, a hallmark of narcolepsy. In addition, the mutant mice spent almost twice as much time in REM sleep during the dark period as did their wild-type littermates, and their EEGs showed episodes of direct transition from wakefulness to REM sleep, another event that is unique to narcolepsy. Similar observations were made in rats in which the hypocretin neurons of the lateral hypothalamus were inactivated by saporin targeting [24], although in this model, cataplexy was not observed. Mice with an inactivated HCRTR2 gene have a milder narcoleptic phenotype than the HCRT knockouts; HCRTR1 knockouts exhibit only a sleep fragmentation phenotype, whereas double HCRTR1 and HCRTR2 mutants recapitulate the full HCRT knockout phenotype [80], suggesting that signaling through both receptors contributes to normal arousal, although the role of HCRTR2 is greater than that of HCRTR1.

Human Narcolepsy Canine Narcolepsy Both sporadic and heritable narcolepsy are observed in dogs, and the symptoms resemble those exhibited by human narcoleptics. The first link between the hypocretins and narcolepsy came from genetic linkage studies in a colony of Doberman pinschers in which narcolepsy was inherited as an autosomal recessive, fully penetrant phenotype. Fine mapping and cloning of the defective canine narcolepsy gene showed it to be the

Nishino and colleagues [54] studied hypocretin concentrations in the cerebral spinal fluid (CSF) of normal controls and patients with narcolepsy by radioimmunoassay. In control CSF, hypocretin concentrations were highly clustered, suggesting that tight regulation of the substance is important. However, of nine patients with narcolepsy, only one had a hypocretin concentration within the normal range. One patient had a greatly elevated concentration, while seven patients had no

The Hypocretins (Orexins) / 727 detectable circulating hypocretin. In an expanded study, hypocretin was undetectable in 37 of 42 narcoleptics and in a few cases of Guillain-Barré syndrome [60]. CSF hypocretin was in the normal range for most neurological diseases but was low, although detectable, in some cases of CNS infections, brain trauma, and brain tumors. Peyron [55], Thannikal [72], and their teams of collaborators found that, in the brains of narcolepsy patients, they could detect few or no hypocretinproducing neurons. Whether the hypocretin neurons are selectively depleted, as is most likely, or only no longer expressing hypocretin is not yet known, although one report showed some indications of gliosis [72]. The codistributed MCH neurons were unaffected. Furthermore, a single patient with a non-HLA-linked narcolepsy carries a mutation within the hypocretin gene itself. The mutation results in a dominant negative amino acid substitution in the secretion signal sequence that sequesters both the mutant and heterozygous wild-type hypocretin nonproductively to the smooth endoplasmic reticulum [55]. These findings leave no doubt as to the central role of the hypocretin system in this sleep disorder. Because most cases are sporadic, mutations in the hypocretin gene or those for its receptors can account for no more than a small subset of the human narcolepsies. The HLA association, loss of neurons with signs of gliosis, and age of disease onset are consistent with autoimmune destruction of the hypocretin neurons accounting for the majority of narcolepsy [44], although a nonimmune-mediated degenerative process has not been ruled out. Whether hypocretin itself or some other protein that is selectively expressed by the hypocretin neurons is the target antigen is yet to be determined. The precipitating factor for the development of the autoimmunity is also unknown, but there must be one because only a small percentage of individuals with the predisposing HLA haplotypes develop the disorder. The narcolepsies as a group are probably a collection of disorders that are caused by defects in the production or secretion of the hypocretins or in their signaling, and these could have numerous genetic, traumatic, viral, and/or autoimmune causes.

References [1] Al-Barazanji, KA, Wilson, S, Baker, J, Jessop, DS, Harbuz, MS. Central orexin-A activates hypothalamic-pituitary-adrenal axis and stimulates hypothalamic corticotropin releasing factor and arginine vasopressin neurones in conscious rats. J Neuroendocrinol. 2001; 13: 421–424. [2] Barreiro, ML, Pineda, R, Navarro, VM, Lopez, M, Suominen, JS, Pinilla, L, Senaris, R, Toppari, J, Aguilar, E, Dieguez, C, TenaSempere, M. Orexin 1 receptor messenger ribonucleic acid expression and stimulation of testosterone secretion by orexinA in rat testis. Endocrinology. 2004; 145: 2297–306.

[3] Bartsch, T, Levy, MJ, Knight, YE, Goadsby, PJ. Differential modulation of nociceptive dural input to [hypocretin] orexin A and B receptor activation in the posterior hypothalamic area. Pain. 2004; 109: 367–78. [4] Bingham, S, Davey, PT, Babbs, AJ, Irving, EA, Sammons, MJ, Wyles, M, Jeffrey, P, Cutler, L, Riba, I, Johns, A, Porter, RA, Upton, N, Hunter, AJ, Parsons, AA. Orexin-A, an hypothalamic peptide with analgesic properties. Pain. 2001; 92: 81–90. [5] Blanco, M, Garcia-Caballero, T, Fraga, M, Gallego, R, Cuevas, J, Forteza, J, Beiras, A, Diéguez, C. Cellular localization of orexin receptors in human adrenal gland, adrenocortical adenomas and pheochromocytomas. Regul Pept. 2002; 104: 161–165. [6] Blanco, M, Lopez, M, Garcia-Caballero, T, Gallego, R, VazquezBoquete, A, Morel, G, Senaris, R, Casanueva, F, Dieguez, C, Beiras, A. Cellular localization of orexin receptors in human pituitary. J Clin Endocrinol Metab. 2001; 86: 1616–9. [7] Bourgin, P, Huitrón-Reséndiz, S, Spier, A, Fabre, V, Morte, B, Criado, J, Sutcliffe, J, Henriksen, S, de Lecea, L. Hypocretin-1 modulates REM sleep through activation of locus coeruleus neurons. J Neurosci. 2000; 20: 7760–5. [8] Burlet, S, Tyler, CJ, Leonard, CS. Direct and indirect excitation of laterodorsal tegmental neurons by hypocretin/orexin peptides: implications for wakefulness and narcolepsy. J Neurosci. 2002; 22: 2862–72. [9] Chang, GQ, Karatayev, O, Davydova, Z, Leibowitz, SF. Circulating triglycerides impact on orexigenic peptides and neuronal activity in hypothalamus. Endocrinology. 2004; 145: 3904–12. [10] Chemelli, RM, Willie, JT, Sinton, CM, Elmquist, JK, Scammell, T, Lee, C, Richardson, JA, Williams, SC, Xiong, Y, Kisanuki, Y, Fitch, TE, Nakazato, M, Hammer, RE, Saper, CB, Yanagisawa, M. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell. 1999; 98: 437–51. [11] Chen, CT, Hwang, LL, Chang, JK, Dun, NJ. Pressor effects of orexins injected intracisternally and to rostral ventrolateral medulla of anesthetized rats. Am J Physiol Regul Integr Comp Physiol. 2000; 278: R692–7. [12] Chen, J, Randeva, HS. Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants. Mol Endocrinol. 2004; 18: 2790–804. [13] Collin, M, Backberg, M, Ovesjo, ML, Fisone, G, Edwards, RH, Fujiyama, F, Meister, B. Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight. Eur J Neurosci. 2003; 18: 1265– 78. [14] Corrigall, WA. Heroin self-administration: effects of antagonist treatment in lateral hypothalamus. Pharmacol Biochem Behav. 1987; 27: 693–700. [15] de Lecea, L, Kilduff, TS, Peyron, C, Gao, X, Foye, PE, Danielson, PE, Fukuhara, C, Battenberg, EL, Gautvik, VT, Bartlett, FS, 2nd, Frankel, WN, van den Pol, AN, Bloom, FE, Gautvik, KM, Sutcliffe, JG. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA. 1998; 95: 322–7. [16] Dube, MG, Kalra, SP, Kalra, PS. Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res. 1999; 842: 473–7. [17] Espana, RA, Berridge, CW, Gammie, SC. Diurnal levels of Fos immunoreactivity are elevated within hypocretin neurons in lactating mice. Peptides. 2004; 25: 1927–34. [18] Fadel, J, Deutch, AY. Anatomical substrates of orexin-dopamine interactions: lateral hypothalamic projections to the ventral tegmental area. Neuroscience. 2002; 111: 379–87. [19] Forster, GL, Blaha, CD. Laterodorsal tegmental stimulation elicits dopamine efflux in the rat nucleus accumbens by activation of acetylcholine and glutamate receptors in the ventral tegmental area. Eur J Neurosci. 2000; 12: 3596–604.

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[68] Smart, D, Jerman, JC, Brough, SJ, Rushton, SL, Murdock, PR, Jewitt, F, Elshourbagy, NA, Ellis, CE, Middlemiss, DN, Brown, F. Characterization of recombinant human orexin receptor pharmacology in a chinese hamster ovary cell-line using FLIPR. Br J Pharmacol. 1999; 128: 1–3. [69] Steininger, TL, Kilduff, TS, Behan, M, Benca, RM, Landry, CF. Comparison of hypocretin/orexin and melaninconcentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain. J Chem Neuroanat. 2004; 27: 165–81. [70] Suyama, H, Kawamoto, M, Shiraishi, S, Gaus, S, Kajiyama, S, Yuge, O. Analgesic effect of intrathecal administration of orexin on neuropathic pain in rats. In Vivo. 2004; 18: 119–23. [71] Sweet, DC, Levine, AS, Kotz, CM. Functional opioid pathways are necessary for hypocretin-1 (orexin-A)-induced feeding. Peptides. 2004; 25: 307–14. [72] Thannickal, TC, Moore, RY, Nienhuis, R, Ramanathan, L, Gulyani, S, Aldrich, M, Cornford, M, Siegel, JM. Reduced number of hypocretin neurons in human narcolepsy. Neuron. 2000; 27: 469–74. [73] Torrealba, F, Yanagisawa, M, Saper, CB. Colocalization of orexin a and glutamate immunoreactivity in axon terminals in the tuberomammillary nucleus in rats. Neuroscience. 2003; 119: 1033–44. [74] Upton, N, In vivo pharmacology of orexin (hypocretin) receptors. In L de Lecea and JG Sutcliffe (Eds.), The hypocretins: integrators of physiological signals, Springer, New York, 2005. [75] Uramura, K, Funahashi, H, Muroya, S, Shioda, S, Takigawa, M, Yada, T. Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area. Neuroreport. 2001; 12: 1885–9. [76] van den Pol, AN. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J Neurosci. 1999; 19: 3171–82. [77] van den Pol, AN, Gao, XB, Obrietan, K, Kilduff, TS, Belousov, AB. Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci. 1998; 18: 7962–71. [78] Volkoff, H, Bjorklund, JM, Peter, RE. Stimulation of feeding behavior and food consumption in the goldfish, Carassius auratus, by orexin-A and orexin-B. Brain Res. 1999; 846: 204–9. [79] Watanabe, S, Kuwaki, T, Yanagisawa, M, Fukuda, Y, Shimoyama, M. Persistent pain and stress activate pain-inhibitory orexin pathways. Neuroreport. 2005; 16: 5–8. [80] Willie, JT, Chemelli, RM, Sinton, CM, Tokita, S, Williams, SC, Kisanuki, YY, Marcus, JN, Lee, C, Elmquist, JK, Kohlmeier, KA, Leonard, CS, Richardson, JA, Hammer, RE, Yanagisawa, M. Distinct narcolepsy syndromes in orexin receptor-2 and orexin null mice: molecular genetic dissection of non-REM and REM sleep regulatory processes. Neuron. 2003; 38: 715–30. [81] Willie, JT, Chemelli, RM, Sinton, CM, Yanagisawa, M. To eat or to sleep? Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci. 2001; 24: 429–58. [82] Wortley, KE, Chang, GQ, Davydova, Z, Leibowitz, SF. Orexin gene expression is increased during states of hypertriglyceridemia. Am J Physiol Regul Integr Comp Physiol. 2003. [83] Wu, X, Gao, J, Yan, J, Owyang, C, Li, Y. Hypothalamus-brain stem circuitry responsible for vagal efferent signaling to the pancreas evoked by hypoglycemia in rat. J Neurophysiol. 2004; 91: 1734–47. [84] Yamanaka, A, Beuckmann, CT, Willie, JT, Hara, J, Tsujino, N, Mieda, M, Tominaga, M, Yagami, K, Sugiyama, F, Goto, K, Yanagisawa, M, Sakurai, T. Hypothalamic orexin neurons regulate arousal according to energy balance in mice. Neuron. 2003; 38: 701–13.

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by a fatty acid: ghrelin is modified at its Ser3 by noctanoic acid, and this modification is essential for ghrelin’s activity [14]. In the rat stomach, a second type of ghrelin peptide has been purified and identified as des-Gln14-ghrelin [9]. Except for the deletion of Gln14, des-Gln14-ghrelin is identical to ghrelin, even retaining the n-octanoic acid modification. Des-Gln14-ghrelin has the same potency of activities as that of ghrelin. The deletion of Gln14 in des-Gln14-ghrelin arises due to the use of a CAG codon to encode Gln, which results in its recognition as a splicing signal. However, des-Gln14-ghrelin is only present in low amounts in the stomach, indicating that ghrelin is the major active form. In the course of purifying human ghrelin from the stomach, several minor forms of ghrelin peptide were isolated [10]. These could be classified into four groups by the type of acylation observed at Ser3: nonacylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg28, and are derived from the same ghrelin precursor through two alternative pathways. As was the case in the rat, the major active form of human ghrelin is a 28-amino-acid peptide with octanoylated Ser3. Synthetic octanoylated and decanoylated ghrelins stimulate the increase of intracellular Ca2+ in GHS-Rexpressing cells and stimulate GH release in rats to a similar degree. Fig. 2 shows a sequence comparison of the identified vertebrate ghrelins [13].

Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary through the GHS-R, a G-protein-coupled receptor. Using a reverse pharmacology method with a stable cell line expressing GHS-R, we purified an endogenous ligand for GHS-R from rat stomach and named it “ghrelin,” after a word root (“ghre”) in Proto-Indo-European languages meaning “grow.” Ghrelin is a peptide hormone in which the third amino acid is modified by a fatty acid; this modification is essential for ghrelin’s activity. Ghrelin plays important roles in maintaining growth hormone release and energy homeostasis in vertebrates.

DISCOVERY Ghrelin was discovered from stomach tissues as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [14]. The name ghrelin is based on ghre, a word root in Proto-Indo-European languages for “grow,” referring to its ability to stimulate growth hormone (GH) release. In 1976, C. Y. Bowers found that some opioid peptide derivatives had weak GH-releasing activity. After this observation, many peptide derivatives and nonpeptide compounds with potent GH-releasing activity were synthesized. These are called growth hormone secretagogues (GHS) [2]. In 1996, GHS-R was identified by expression cloning [11]. After the identification of GHS-R, a search for its endogenous ligand was actively undertaken with the use of the “orphan receptor strategy,” and finally in 1999 the ligand was discovered in the stomach, an unexpected tissue for the GHS-R ligand (Fig. 1) [14]. The structure of ghrelin is unprecedented. Ghrelin is the first case of a bioactive peptide that is modified Handbook of Biologically Active Peptides

STRUCURE OF THE PRECURSOR AND mRNA/GENE The human ghrelin gene, localized on the chromosome 3p25–26 comprises five exons (Fig. 3) [12, 21]. The short first exon contains only 20 bp, which encode

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FIGURE 1. Structures of human and rat ghrelins. Both human and rat ghrelins are 28-amino-acid peptides, in which Ser3 is modified by a lipid, primarily n-octanoic acid. This modification is essential for ghrelin’s activity.

Mammalian Human Rhesus Monkey Mouse Mongolian Gerbil Rat Dog Porcine Sheep Bovine Avian Chicken Duck Emu Goose Turkey Fish Rainbow Trout 1 Rainbow Trout 2 Japanese Eel Goldfish Zebrafish Tilapia Amphibian Bullfrog

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The 28 amino acids of the ghrelin peptide are encoded in exons 2 and 3. In the rat and mouse ghrelin genes, the codon for Gln14 (CAG) is used as an alternative splicing signal to generate two different ghrelin mRNAs. One mRNA encodes the ghrelin precursor, and another encodes a des-Gln14-ghrelin precursor. Complementary DNA analyses indicated that desGln14-ghrelin cDNA also exists in the human stomach (GenBank Accession number: AB035700). However, the number of human des-Gln14-ghrelin cDNA clones is low, and des-Gln14-ghrelin peptides have not yet been isolated from stomach tissue. Moreover, two cDNA clones from Homo sapiens fetus library that code for human des-Gln14-ghrelin are deposited in the NCBI nucleotide database (AI338429 and BY149645). There are two types of porcine ghrelin cDNA, which encode ghrelin and des-Gln14-ghrelin that are present at an approximate ratio of 1 : 1 (GenBank Accession number: AB035703 & AB035704). In the cow, only one ghrelin mRNA exists, and it encodes a 27-amino acid ghrelin.

DISTRIBUTION OF GHRELIN mRNA

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FIGURE 2. Sequence comparison of vertebrate ghrelins. Identical amino acids in each species of mammal, bird, and fish are colored. The asterisks indicate acyl-modified third amino acids. N-terminal cores with acyl-modification sites are well conserved among all vertebrate ghrelins.

part of the 5′-untranslated region. There are two different transcriptional initiation sites in the ghrelin gene; one occurs at −80 and the other at −555 relative to the ATG initiation codon, resulting in two distinct mRNA transcripts (transcript-A and transcript-B).

Ghrelin mRNA is mainly expressed in the stomach [14]. In situ hybridization and immunohistochemical analyses indicated that ghrelin-containing cells are a distinct endocrine cell type found in the submucosal layer of the stomach [7]. These cells, known as X/A-like cells, contain round, compact, electron-dense granules filled with ghrelin. Ghrelin immunoreactive cells are also found in the small and large intestines. The ghrelin content of the central nervous system is low. By means of immunohistochemical analysis, ghrelin-responsive neuronal cells can be demonstrated in a very limited region in the hypothalamic arcuate nucleus [3, 14, 18]. This region is known to control appetite, suggesting that ghrelin might be involved in the regulation of food intake. RT-PCR analyses of ghrelin have shown that the mRNA is present in many tissues: brain, heart, lung, kidney, pancreas, stomach, and small and large intestines.

PROCESSING OF THE GHRELIN PRECURSOR The amino acid sequences of mammalian ghrelin precursors are well conserved (Fig. 4). The rat and human ghrelin precursors are both composed of 117 amino acids. In these precursors, the 28-amino-acid active ghrelin sequence immediately follows the signal peptide. The cleavage site for the signal peptide is the

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GSSFLSPEHQRVQQRKESKKPPAKLQPR n-Octanoyl ghrelin

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FIGURE 3. From the human ghrelin gene to an active peptide. The human ghrelin gene comprises five exons. The first exon encodes the 5′-untranslated region and is very short. cDNA analyses of human ghrelin have revealed that transcript-A, an alternative splicing product from exon 2 to exon 4, is the main form of human ghrelin mRNA in vivo. This mRNA is translated into a 117amino-acid ghrelin precursor (preproghrelin). Protease cleavage and acyl-modification of the ghrelin precursor result in the production of a 28-aminoacid-long active acyl-modified ghrelin peptide. In rat, mouse, and pig, another splicing variant encoding des-Gln14-ghrelin is produced by alternative splicing at the end of intron 2.

ghrelin

Human: Rat: Mouse: Porcine: Bovine: Ovine: Canine:

Human: Rat: Mouse: Porcine: Bovine: Ovine: Canine:

FIGURE 4. Amino-acid sequences of mammalian ghrelin precursors. A sequence comparison between mammalian ghrelin precursors is shown. Identical amino acids are colored. The asterisk shows the position of the acyl-modified Ser3. Note that the amino-acid sequences of mammalian ghrelin precursors are well conserved; in particular, the Nterminal 10 amino acids of all of these active ghrelin peptides, each of which contains an acyl-modified serine in its active core, are identical.

same in all mammalian ghrelins. Although propeptides are usually processed at dibasic amino acid sites by prohormone convertases, the C-terminus of the ghrelin peptide sequence is processed at an uncommon ProArg recognition site. Acyl-modification is an important processing step for ghrelin, because ghrelin needs acyl-modification, in particular n-octanoyl modification, for exerting its full activity. However, an enzyme that catalyzes the acylmodification of ghrelin has not yet been identified. The universal incorporation of n-octanoic acid in mammals, fish, birds, and amphibians suggests that this putative enzyme is rather specific in its selection of mediumchain fatty acid substrates. Recent studies indicate that ingestion of either medium-chain fatty acids (MCFAs) or medium-chain triacylglycerols (MCTs) specifically increases production of acyl-modified ghrelin without changing the total (acyl- and des-acyl-) ghrelin level [16]. When mice ingested either MCFAs or MCTs, the acyl group attached to nascent ghrelin molecules corresponded to that of the ingested MCFAs or MCTs. Moreover, n-heptanoyl (C7:0) ghrelin, an unnatural form of ghrelin, was produced in the stomach of mice following ingestion of

734 / Chapter 101 n-heptanoic acid or glyceryl triheptanoate. These findings indicate that ingested fatty acids are directly utilized for acyl-modification of ghrelin.

RECEPTOR Ghrelin receptor, or GHS-R, is a typical G proteincoupled receptor with seven transmembrane domains (7-TM) [11]. Two distinct ghrelin receptor cDNAs have been isolated. The first, GHS-R type 1a, encodes a 7-TM GPCR with binding and functional properties consistent with its role as ghrelin’s receptor. Another GHS-R cDNA, type 1b, is produced by an alternative splicing mechanism. The GHS-R gene consists of two exons; the first exon encodes TM-1 to 5, and the second exon encodes TM-6 to 7. Type 1b is derived from only the first exon and encodes only five of the seven predicted TM domains. The type 1b receptor is thus a C-terminal truncated form of the type 1a receptor and is pharmacologically inactive. The ghrelin receptor (GHS-R) has several homologs, whose endogenous ligands are gastrointestinal peptides or neuropeptides, like motilin, neuromedin U, and neurotensin [13]. The ghrelin receptor is most homologous to the motilin receptor; the human forms share 52% identical amino acids. Moreover, their ligands, ghrelin and motilin peptides, have similar amino acid sequences. Preliminary studies have shown that motilin can stimulate the ghrelin receptor, albeit at a low level. In contrast, ghrelin does not activate the motilin receptor. The ghrelin receptor is well conserved across all vertebrate species examined, including a number of mammals, chicken, and pufferfish (Fugu). This strict conservation suggests that ghrelin and its receptor serve important physiological functions. Ghrelin receptor mRNA is prominently expressed in the arcuate (ARC) and ventromedial nuclei (VMN) and in the hippocampus [8]. Ghrelin receptor (GHS-R) mRNA is also detected in multiple hypothalamic nuclei and in the pituitary, as well as the dentate gyrus, CA2, and CA3 regions of the hippocampus, the substantia nigra, the ventral tegmental area, and the dorsal and median raphe nuclei. RT-PCR analyses demonstrated ghrelin receptor mRNA expression in many peripheral organs, including heart, lung, liver, kidney, pancreas, stomach, small and large intestines, adipose tissue and immune cells, indicating that ghrelin has multiple functions in these tissues.

Ghrelin specifically stimulates GH release from cultured pituitary cells in a dose-dependent manner (Fig. 5A) [14]. This fact also indicates that ghrelin acts directly on the pituitary. Intravenous injection of ghrelin induces potent GH release both in the rat and in humans [14, 17, 20]. When anesthetized rats were injected intravenously with ghrelin, an increase in GH plasma concentration was observed (Fig. 5B). The release of GH peaks at about 5–15 min after ghrelin injection and returns to basal levels 1 hour later. Single intracerebroventricular administration of ghrelin also increased rat plasma GH concentrations in a dosedependent manner. Together, these in vivo assays confirmed that ghrelin is a specific GH-releasing peptide. Immunohistochemical analyses indicate that ghrelincontaining neural cells are localized to the arcuate nucleus of the hypothalamus, a region involved in the regulation of appetite. This localization suggests that ghrelin might control food intake. When ghrelin was injected into the cerebral ventricles of rats, their food intake was potently stimulated (Fig. 5C) [15, 22]. In fact, ghrelin was found to be the most powerful stimulator of appetite of all known peptides. At least part of this orexigenic effect of ghrelin is evidently exerted by its stimulatory action on the genes encoding the potent appetite stimulators, agouti-related peptide and neuropeptide Y (NPY), located primarily in the arcuate nucleus. Peripherally injected ghrelin stimulates hypothalamic neurons and stimulates food intake. The detection of ghrelin receptors on vagal afferent neurons in the rat nodose ganglion suggests that some of ghrelin signals from the stomach are transmitted to the brain via the vagus nerve. Moreover, the observation that ICV administration of ghrelin induces c-fos in the dorsomotor nucleus of the vagus and stimulates gastric-acid secretion indicates that ghrelin activates the vagal system [6]. In contrast, vagotomy inhibits the ability of ghrelin to stimulate food intake and GH release. However, the basal level of ghrelin concentration is not affected and a decrease of ghrelin levels is not observed after vagotomy. On the other hand, fasting-induced elevation of plasma ghrelin is completely abolished by subdiaphragmatic vagotomy or atropine treatment. These results indicate that the response of ghrelin to fasting is transmitted through vagal afferent transmission.

PATHOPHYSIOLOGICAL IMPLICATIONS BIOLOGICAL ACTIONS WITHIN THE BRAIN Growth-hormone releasing activity and appetite stimulation are the two main functions of ghrelin.

Plasma ghrelin concentration is low in obese people and high in lean people [19]. Related to this fact, plasma ghrelin level is greatly increased in anorexia nervosa (AN) patients [1]. AN is a syndrome often seen

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in young women characterized by a combination of weight loss, amenorrhea, and behavioral changes. Some of these changes are reversible with weight gain. Plasma ghrelin levels in AN patients are high and return to control levels after weight gain by renutrition. AN patients often show markedly elevated GH levels, which may be due to high circulating levels of ghrelin. Moreover, high ghrelin increases ACTH, prolactin, and cortisol levels in humans, which may explain the amenorrhea and behavioral changes observed in AN patients. Patients with gastric bypass lose their weight, and their ghrelin levels decrease [4]. Changes in ghrelin concentration associated with food intake are diminished in these patients, confirming that the stomach is the main site of ghrelin production. Plasma ghrelin concentrations are also decreased in patients with short bowel syndrome, probably due to the loss of ghrelinproducing tissues. High-plasma ghrelin concentrations are observed in Prader-Willi syndrome (PWS) patients [5]. PWS is a complex genetic disorder characterized by mild mental retardation, hyperphagia, short stature, muscular hypotonia, and distinctive behavioral features. Excessive appetite in PWS causes progressive severe obesity, which in turn leads to an increase of cardiovascular morbidity and mortality. The PWS genotype is characterized by a loss of one or more paternal genes in region q11–13 on chromosome 15. It has been suggested that this genetic alteration leads to dysfunction of several hypothalamic areas, including appetite regulatory regions. The mean plasma concentration of ghrelin was higher by three- to fourfold in PWS than in a reference population. Thus, ghrelin may be responsible, at least in part, for the hyperphagia seen in PWS. It is unclear, however, what underlies the increased ghrelin levels in these patients.

References Saline 3

10 50 200 500 1nmol pmol

FIGURE 5. Effects of ghrelin on pituitary hormone secretion and food intake. A. Effects of a high dose (10−6 M) of ghrelin on hormone secretion from rat primary pituitary cells in vitro. ACTH, adrenocorticotropin; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; and TSH, thyroid-stimulating hormone. B. Time course of plasma hormone concentrations after intravenous injection of ghrelin into male rats in vivo. C. Two-hour food intake of free-feeding rats injected with various doses of ghrelin. Control rats were given 0.9% saline.

[1] Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86(10): 4753–8. [2] Bowers CY. Growth hormone-releasing peptide (GHRP). Cell Mol Life Sci 1998;54(12):1316–29. [3] Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003;37(4):649–61. [4] Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002;346(21): 1623–30. [5] Cummings DE, Clement K, Purnell JQ, Vaisse C, Foster KE, Frayo RS et al. Elevated plasma ghrelin levels in Prader Willi syndrome. Nat Med 2002;8(7):643–4.

736 / Chapter 101 [6] Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H et al. The role of the gastric afferent vagal nerve in ghrelininduced feeding and growth hormone secretion in rats. Gastroenterology 2002;123(4):1120–8. [7] Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T et al. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000;141(11):4255–61. [8] Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ et al. Distribution of mRNA encoding the growth hormone secretagog receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997;48(1):23–9. [9] Hosoda H, Kojima M, Matsuo H and Kangawa K. Purification and characterization of rat des-Gln14-Ghrelin, a second endogenous ligand for the growth hormone secretagog receptor. J Biol Chem 2000;275(29):21995–2000. [10] Hosoda H, Kojima M, Mizushima T, Shimizu S and Kangawa K. Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing. J Biol Chem 2003;278(1):64–70. [11] Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996; 273(5277):974–7. [12] Kanamoto N, Akamizu T, Tagami T, Hataya Y, Moriyama K, Takaya K et al. Genomic structure and characterization of the 5′-flanking region of the human ghrelin gene. Endocrinology 2004;145(9):4144–53. [13] Kojima M and Kangawa K. Ghrelin: structure and function. Physiol Rev 2005;85(2):495–522.

[14] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H and Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402(6762):656–60. [15] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K et al. A role for ghrelin in the central regulation of feeding. Nature 2001;409(6817):194–8. [16] Nishi Y, Hiejima H, Hosoda H, Kaiya H, Mori K, Fukue Y et al. Ingested medium-chain fatty acids are directly utilized for the acyl modification of ghrelin. Endocrinology 2005;146(5): 2255–64. [17] Peino R, Baldelli R, Rodriguez-Garcia J, Rodriguez-Segade S, Kojima M, Kangawa K et al. Ghrelin-induced growth hormone secretion in humans. Eur J Endocrinol 2000;143(6): R11–14. [18] Sato T, Fukue Y, Teranishi H, Yoshida Y and Kojima M. Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2-deoxy-D-glucose administration Endocrinology 2005. [19] Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002;87(1):240–4. [20] Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M et al. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab 2000;85(12): 4908–11. [21] Tanaka M, Hayashida Y, Iguchi T, Nakao N, Nakai N and Nakashima K. Organization of the mouse ghrelin gene and promoter: occurrence of a short noncoding first exon. Endocrinology 2001;142(8):3697–700. [22] Tschop M, Smiley DL and Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407(6806):908–13.

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102 Neurotensin PAUL R. DOBNER AND ROBERT E. CARRAWAY

ABSTRACT

C-terminal six residues. For example, NT(8–13) is nearly as potent as NT, and it exhibits full intrinsic biological activity in a number of systems. Consistent with the importance of the C-terminal residues for function, this region is highly conserved in evolution [11] and present in a number of intraspecies variants [14]. NT administration produces diverse biological effects, indicating that endogenous NT likely plays roles in the regulation of digestion, inflammation, pain perception, temperature, and stress that seem to involve complex interactions with monoamine transmitter systems and pituitary hormones. This review focuses on the biosynthesis, distribution, receptor signaling, and potential functions of NT in the CNS.

Neurotensin (NT) and neuromedin N, bioactive peptides of brain and gut origin, are derived from a common precursor by cleavages at KR processing sites. Differential processing occurs in some tissues, yielding large molecular, N-terminally extended forms. Two G protein–linked receptors show preference for NT but bind both peptides. NTR-1 signaling elevates intracellular [Ca2+] and can also increase cGMP, cAMP, and arachidonic acid metabolites. NTR-1/sortilin heterodimers appear to modulate NT signaling and trafficking of internalized receptors, and NT may also signal through sortilin. Intimately associated with dopaminergic systems, NT is an endogenous antipsychotic, and has also been implicated in pain perception, appetite control, stress responses, and reproductive functions.

STRUCTURE OF THE PRECURSOR mRNA/GENE

DISCOVERY OF NEUROTENSIN

The amino acid sequence of the NT precursor was first inferred from DNA sequences of cDNAs isolated from a library prepared from canine intestinal endocrine N cells [25]. The canine probe was subsequently used to isolate the corresponding neuronal precursor cDNA from cow and the rat gene [38]. The predicted prepro form of the precursor ranges from 165 to 170 amino acids in length and is highly conserved from chicken to man. The NT sequence and the sequence of the related hexapeptide neuromedin N are located in tandem near the carboxyl terminus of the precursor, bounded and separated by Lys-Arg processing sites, and for this reason it was named the NT/N precursor. The putative signal peptide sequence (amino acids 1–24 in the canine sequence) was initially predicted based on sequence comparisons and the structure of the rat gene, and direct protein sequencing of processing

In 1967, Susan Leeman discovered that extracts of bovine hypothalami induce vasodilation and anaphylactic shock in rats. Using this assay, her laboratory eventually isolated [12], sequenced [13], and synthesized [10] the active substance, a 13-residue peptide they named neurotensin (NT). Using radioimmunoassay and immunohistochemistry, they and others have since determined that NT is located primarily in brain neurons and intestinal endocrine cells but is also found in various glands and in peripheral nerves in many organs [53]. NT is a member of a family of related peptides that are strikingly similar in their C-terminal regions (Table 1). These peptides share some of the pharmacological properties demonstrated for NT, and structure– function studies indicate a strong dependence on the Handbook of Biologically Active Peptides

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738 / Chapter 102 TABLE 1. Peptides of the neurotensin family. Peptide NT (bovine, canine, human) NT (Gallus) NMN (porcine) LANT-6 (Gallus) XP (Xenopus) XP (canine, turkey)

Sequence
NT, neurotensin; NMN, neuromedin N; LANT-6 [Lys8, Asn9]-neurotensin 8–13; XP, xenopsin.

intermediates isolated from canine intestine indicated that signal peptide cleavage occurs between amino acids 23 and 24 [17]. The analysis of the rat, mouse, and human NT/N genes revealed that the coding region is spread over four exons, separated by three introns spanning just over 10 kb [3, 26, 38]. The gene is transcribed to yield two different transcripts (1.0 and 1.5 kb) that differ only in the extent of the 3′ untranslated region, due most likely to differential utilization of alternative polyadenylation signals [38]. The two mRNAs are expressed in approximately equal amounts in the brain, but the 1.0 kb mRNA greatly predominates in the gastrointestinal tract [38]. Sequences immediately upstream of the transcriptional start site contain a series of response elements, including AP-1, CRE, and GRE sequences, that are important for NT/N gene induction in response to environmental stimuli in PC12 cells [39]. A variety of stimuli regulate NT/N gene expression in a brain region-specific manner.

NT/N mRNA EXPRESSION IN BRAIN Alexander et al. first described the distribution of NT/N mRNA in the rat forebrain [1]. NT/N mRNA is expressed in many forebrain regions, with higher levels in limbic regions, including hippocampus and subiculum, VP, BNST, medial Acb, and Cput. NT/N mRNApositive neurons are located mainly in the dorsomedial and ventral aspects of the Cput more rostrally but are more evenly distributed in more caudal regions. A continuum of positive cells was observed stretching from the S through the preoptic area, including the lateral S, DBB, lateral BNST, and lateral Hpt. Endocrine hormones and neurotransmitters have been implicated in the regulation of NT/N mRNA expression in several brain regions, including the medial preoptic area, Arc, PVN, periventricular nucleus, amygdala, Cput, and Acb. In the brainstem, NT mRNA-positive neurons are most abundant in the PAG and DR but also can be found in adjacent lateral

structures (cuneiform nucleus, microcellular tegmental nucleus), particularly during chronic pain [70].

PROCESSING OF THE PRECURSOR The NT precursor undergoes complex posttranslational processing in a tissue-specific manner. Whereas NT and NMN are the primary products in brain, NT and a large molecular form of NMN are formed in intestine [15] and other large molecular forms are present in the adrenal gland [19]. Since large NT and large NMN are metabolically stable agonists that are secreted into blood, they could produce longer-lasting effects [32]. Pepsin has been used as a tool for the biochemical analysis of these proteins since it excises NT413 and NMN in high yield [18]; however, natural processing is likely performed by prohormone convertases (PCs) of the Kex2 family. When cells expressing the unprocessed NT precursor are transfected with PC2, they display the processing pattern seen in brain, whereas PC1 imparts the intestinal pattern [59]. Although PC2 immunoreactivity colocalizes with NT in rat brain, PC2 knockout animals display only a 15% decrease in brain levels of NT [68]. Since PC1 and PC5 are also present in some NT neurons, it has been suggested that they might compensate for the loss of PC2. Barbero et al. [2] have found that colon cancer cell lines, which often express PC5 but not PC1 or PC2, exhibit a processing pattern resembling that in adrenal, a tissue known to express high levels of PC5. Thus, the available evidence suggests that PC1, PC2, and PC5 are used to generate a variety of NT-related peptides in a tissue-specific manner. The predicted secondary structure for the canine NT precursor shows a high probability of α-helix formation with a strong tendency for β-turns in the vicinity of the major cleavage sites [18]. Helical wheel analyses indicate the presence of three amphipathic regions that could potentially generate three hydrophobic surfaces, each consisting of 7–8 aligned nonpolar residues.

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RECEPTORS AND SIGNALING CASCADES Four NT receptors have been identified (reviewed in [28]), two that are 7-transmembrane G protein–coupled receptors (NTR-1, NTR-2) and two primarily intracellular proteins related to yeast sorting receptors (NTR3/sortilin, NTR-4/SorLA). NTR-1 displays the highest affinity (Kd, 0.1 nM) and appears to mediate NT effects on dopaminergic transmission [40], hot-plate latency, temperature regulation, and smooth muscle contractility [52]. First described by Mazella [44] as a lower affinity receptor (Kd, 3 nM) with a differing specificity, NTR-2 has been implicated in the analgesic effects of NT [29]. Although the role of NTR-3 is less clear, one possibility is that its binding and internalization of NT serves to regulate turnover of the peptide [45]. Recent evidence supports the involvement of NTR-3 in the growth promoting effects of NT [24] and in NT-induced migration of brain microglial cells [43]. Signal transduction studies in transfected cell systems and cancer cell lines indicate that NTR-1 is primarily coupled to Gαq and signals via the activation of phospholipase C and the generation of IP3, causing an elevation in intracellular [Ca2+] (reviewed in [69]). Concomitant generation of DAG leads to activation of PKC in some cells, stimulating MAP kinases and PI3 kinase via complex mechanisms that can involve the transactivation of EGF receptor [35]. In neuroblastoma cells and rat brain slices, NT can elevate cGMP formation, inhibit cAMP synthesis, and stimulate phosphoinositol turnover [33, 50], and recent evidence suggests that the third intracellular loop of NTR-1 is required for interactions with Gq [72], while the carboxy-terminal tail interacts with Gi/o and Gs [47]. NT activation of dopamine neurons appears to involve cAMP signaling [66], and NT potentiates cAMP formation (via NTR-1) in response to Gs-agonists by facilitating the activation of Ca2+-dependent adenylyl cyclases [20]. This might possibly relate to the ability of NT to modulate responses to monoamines in the CNS. Perhaps secondary to its effects on [Ca2+], NT also activates PLA2 and DAG lipase, stimulating arachidonic acid release and the formation of bioactive metabolites via the cyclooxygenase [73] and lipoxygenase pathways [9]. NT is the most potent substance known to stimulate leukotriene formation in rats [16], and the possible relevance of this to its central effects is not well investigated. Signaling mechanisms for NTR-2 and NTR-3 are not yet clear. NTR-2 constitutively activates inositol phosphate production, and this process is stimulated by the NTR-1 antagonist SR48692 but suppressed by NT [55]. NTR-3 is primarily localized to ER-Golgi membranes but ≅10% is on the cell surface. NTR-3 can form heterodimers with NTR1, decreasing the potency of NT to stimulate inositol phosphate formation and to activate

MAP kinase. Thus, NTR-3, which cannot promote either response, modulates these effects of NTR1.

INFORMATION ON ACTIVE OR SOLUTION CONFORMATION 2D solid-state NMR spectroscopy has recently been used to study the conformational changes of NT8-13 upon interaction with an N-terminally truncated form of rat NTR-1 [41, 71]. Upon binding to the receptor, the peptide rearranged to adopt a β-strand conformation, characterized by a ψ(Pro10) dihedral angle of 146 ± 15°. Furthermore, a lactam-bridged analog of NT8-13 with ψ(Pro10) angles restricted to an angle (130°) close to the experimentally determined one exhibited ∼1000fold higher affinity for porcine NTR-1 than the stereoisomer (−130°), providing further evidence for the NMR-derived bioactive conformation [8].

BRAIN FUNCTIONS NT May Be an Endogenous Antipsychotic Considerable evidence suggests that NT mediates at least a subset of responses to certain APDs. NT was first suggested to be a possible endogenous neuroleptic based on similarities between the effects of APDs and centrally administered NT [48]. APDs have also been shown to increase NT expression in both the Cput and Acb and NT microinjection in the Acb was found to suppress amphetamine-stimulated locomotor activity, similar to APDs, suggesting that increased NT signaling could underlie some APD effects (reviewed in [28]). A broad spectrum of APDs increases NT expression in the Acb; however, increased expression in the dorsolateral striatum is associated with typical APDs (e.g., haloperidol), which suggests a possible involvement in the production of extrapyramidal side effects. Several reports indicate that endogenous NT signaling is required for at least some actions of certain APDs in two animal models of schizophrenia, prepulse inhibition (PPI), and latent inhibition (LI). LI measures the suppression of associative learning by preexposure to the conditioned stimulus and APD treatment improves the acquisition of LI. Pretreatment with NT antagonists suppressed acquisition and blocked the ability of haloperidol to improve LI [6, 7] and similarly blocked haloperidol and quetiapine reversal of PPI deficits in isolation-reared rats [6]. Quetiapine treatment restored NT mRNA expression in the Acb of isolation-reared rats, suggesting that increased NT expression may underlie improved PPI [6]. Consistent with these results, NT knockout mice display decreased basal PPI levels and fail to

740 / Chapter 102 respond to both haloperidol and quetiapine, but respond normally to the atypical APD clozapine [37]. Collectively, these results suggest that increased striatal NT expression is required for at least some effects of certain typical and atypical APDs.

responses, there may be physiological correlates of the biphasic response to exogenous NT. NTR-2 has been suspected to play a role in NT-induced analgesia and appears to play a prominent role in NT suppression of irritant-induced writhing; however, NTR-1 is also required for certain somatic pain responses [28, 29].

NT and Amphetamine Sensitization NT microinjection into the VTA results in increased dopamine release in the Acb and increased locomotor activity, and repeated administration sensitizes both NT and amphetamine responses (reviewed in [27]). Psychostimulants also increase NT expression, particularly in the dorsomedial and ventrolateral aspects of the Cput. NT knockout mice and SR 48692 pretreated mice and rats display selective defects in amphetamineelicited c-fos expression in the medial striatum, suggesting that NT is required for neuronal activation in this region [30]. Although NT antagonist pretreatment has been shown to block amphetamine sensitization [23, 51, 57], other evidence suggests that NT may act to limit psychostimulant sensitization. For instance, peripheral administration of the NT agonist NT69L suppresses nicotine sensitization and reportedly also blocks amphetamine and cocaine sensitization [31], NT antagonist administration potentiates rather than blocks supersensitive locomotor responses to l-dopa in dopamine-deficient mice [21], and NT knockout mice display enhanced amphetamine sensitization (P.R.D., unpublished results). The different results obtained using NT antagonists and NT knockout mice could be explained be either adaptive changes in NT knockout animals or unanticipated antagonist effects.

NT and Pain Given centrally, NT enhances nociceptive responses at lower doses but produces a potent analgesic response at higher doses that in most studies does not require μopioid receptor signaling (reviewed in [28]). The latter observation suggested that NT might be involved in μopioid receptor independent stress-induced antinociception (SIAN). NT and NT receptors are expressed in the central descending circuitry involved in pain modulation, including the PAG, the rostroventral medulla, and the dorsal horn of the spinal cord. There is increasing evidence that NT plays a key role in SIAN. Severe stress-induced SIAN is blocked in both NT knockout mice and in rats pretreated with SR 48692 and is instead replaced by a stress-induced hyperalgesia [34]. The observation that cold water swim stress increases NT mRNA levels in certain Hpt regions that project to the PAG suggests that stress-induced increases in NT signaling in the PAG may underlie SIAN [64]. Since disruption of NT signaling also attenuates basal nociceptive

Appetite Control NT has been implicated as a possible satiety factor [42] and mediator of the central suppressive effects of leptin on food consumption [63]. Leptin administration has been shown to suppress food consumption and weight gain possibly through the decreased expression of orexigenic peptides and increased NT expression in the hypothalamus [56, 62]. Similarly, NT expression is decreased in specific Hpt regions in response to a high fat diet that results in obesity [4]. Combined central administration of NT and leptin potentiates both the short-term inhibitory effects of NT on spontaneous food intake and early but not late (24 hr) leptin effects [5]. Finally, NTR-1 knockout mice display increased food consumption (10%) compared to wild type that apparently results in a modest increase in body weight (∼15%) [54]. Collectively, these results indicate that dynamic regulation of NT in specific Hpt nuclei in response to alterations in the levels of circulating leptin and perhaps other factors is involved in appetite suppression and body weight balance.

NT and Stress Responses Stress increases NT expression in several Hpt nuclei, suggesting that NT may modulate stress responses. Centrally administered NT was found to rapidly increase levels of circulating ACTH and corticosterone and these increases were attenuated by corticotropin releasing hormone (CRH) antagonists and bilateral lesions of the PVN [46, 60]. Furthermore, SR 48692 administration in the PVN suppresses stress-induced increases in plasma levels of ACTH and corticosterone and decreases CRH expression in the PVN [49, 61].

NT and Reproductive Functions NT has been implicated in the preovulatory luteinizing hormone (LH) surge and the Hpt control of prolactin (PRL) release, and estrogen has been shown to increase NT expression in several Hpt regions implicated in reproductive functions (reviewed in [58]). NT most likely participates in the preovulatory LH surge through the stimulation of gonadotropin releasing hormone (GnRH) release from rostral medial preoptic nucleus GnRH neurons expressing NTR-1 [67]. NT has also been implicated in both positive and negative regu-

Neurotensin / 741 lation of PRL secretion from pituitary lactotrophs (see endocrine peptides section). The evidence suggests that NT directly activates tuberinfundibular dopamine (TIDA) neurons and mediates feedback inhibition of PRL release through activation of these neurons [36]. Estrogen increases NT expression in TIDA neurons and NT is intensely expressed in virtually all TIDA neurons in nursing rats, but decreases markedly after removal of pups [22]. Increased NT release from TIDA neurons in the median eminence would most likely counteract the inhibitory effects of dopamine on PRL secretion, since NT stimulates pituitary PRL release. NT knockout mice reproduce and successfully rear their young, suggesting that NT is not essential for reproductive functions [26].

PATHOPHYSIOLOGICAL IMPLICATIONS There is now considerable evidence from animal models that endogenous NT signaling is both required for certain APD actions and that decreased NT signaling, particularly in the Acb, may underlie deficits in PPI. Comparison of NT levels in cerebrospinal fluid (CSF) from schizophrenics both prior to and following APD treatment indicated that low CSF NT levels were associated with more severe psychopathology and that clinical improvement was associated with increased CSF NT [65]. These studies suggest that reductions in NT signaling could be involved in the etiology of schizophrenia in at least a subset of patients, and that increased NT signaling may underlie the therapeutic effects of certain APDs. Animal studies have provided convincing evidence that reduced NT signaling results in sensorimotor gating deficits and that NT is required for reversal of these deficits by a subset of APDs [6, 37]. These results suggest that the development of NT agonists could provide an important new class of APDs. In the shorter term, there could be some therapeutic benefit associated with the use of NT-dependent (e.g., quetiapine) and NT-independent (e.g., clozapine) APDs in combination. The NMR structure of receptorbound NT(8–13) could aid in agonist development [41].

References [1] Alexander, MJ, Miller, MA, Dorsa, DM, Bullock, BP, Melloni, RH, Jr., Dobner, PR and Leeman, SE. Distribution of neurotensin/neuromedin N mRNA in rat forebrain: Unexpected abundance in hippocampus and subiculum. Proc Natl Acad Sci USA 1989; 86:5202–5206. [2] Barbero, P, Rovere, C, De Bie, I, Seidah, NG, Beaudet, A and Kitabgi, P. PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5transfected PC12 cells. J Biol Chem 1998; 273:25339–25346.

[3] Bean, AJ, Dagerlind, A, Hokfelt, T and Dobner, PR. Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls. Neurosci 1992; 50:259–268. [4] Beck, B, Burlet, A, Nicolas, J-P and Burlet, C. Opposite influence of carbohydrates and fat on hypothalamic neurotensin in LongEvans rats. Life Sci 1996; 59:349–356. [5] Beck, B, Stricker-Krongrad, A, Richy, S and Burlet, C. Evidence that hypothalamic neurotensin signals leptin effects on feeding behavior in normal and fat-preferring rats. Biochem Biophys Res Comm 1998; 252:634–638. [6] Binder, E, Kinkead, B, Owens, MJ, Kilts, CD and Nemeroff, CB. Enhanced neurotensin neurotransmission is involved in the clinically relevant behavioral effects of antipsychotic drugs: evidence from animal models of sensorimotor gating. J Neurosci 2001; 21:601–608. [7] Binder, EB, Gross, RE, Nemeroff, CB and Kilts, CD. Effects of neurotensin receptor antagonism on latent inhibition in Sprague-Dawley rats. Psychopharmacol 2002; 161:288–295. [8] Bittermann, H, Einsiedel, J, Hubner, H and Gmeiner, P. Evaluation of lactam-bridged neurotensin analogues adjusting ψ(Pro10) close to the experimentally derived bioactive conformation of NT(8–13). J Med Chem 2004; 2004:5587–5590. [9] Canonico, PL, Speciale, C, Sortino, MA and Scapagnini, U. Involvement of arachidonate metabolism in neurotensininduced prolactin release in vitro. Am J Physiol 1985; 249: E257–E263. [10] Carraway, R and Leeman, SE. The synthesis of neurotensin. J Biol Chem 1975; 250:1912–1918. [11] Carraway, R, Ruane, SE and Kim, HR. Distribution and immunochemical character of neurotensin-like material in representative vertebrates and invertebrates: Apparent conservation of the COOH-terminal region during evolution. Peptides 1982; 3:115–123. [12] Carraway, RE and Leeman, SE. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 1973; 248:6854–6861. [13] Carraway, RE and Leeman, SE. The amino acid sequence of a hypothalamic peptide, neurotensin. J Biol Chem 1975; 250:1907–1911. [14] Carraway, RE and Ferris, CF. Isolation, biological and chemical characterization, and synthesis of a neurotensin-related hexapeptide from chicken intestine. J Biol Chem 1983; 258:2475–2479. [15] Carraway, RE and Mitra, SP. Differential processing of neurotensin/neuromedin N precursor(s) in canine brain and intestine. J Biol Chem 1990; 265:8627–8631. [16] Carraway, RE, Cochrane, DE, Salmonsen, R, Muraki, K and Boucher, W. Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats. Peptides 1991; 12:1105–1111. [17] Carraway, RE and Mitra, SP. Purification of large neuromedin N (NMN) from canine intestine and its identification as NMN125. Biochem Biophys Res Comm 1991; 179:301–308. [18] Carraway, RE, Mitra, SP and Spaulding, G. Posttranslational processing of the neurotensin/neuromedin-N precursor. Ann NY Acad Sci 1992; 668:1–16. [19] Carraway, RE, Mitra, SP and Joyce, TJ. Tissue-specific processing of neurotensin/neuromedin-N precursor in cat. Regul Pept 1993; 43:97–106. [20] Carraway, RE and Mitra, SP. Neurotensin enhances agonistinduced cAMP accumulation in PC3 cells via Ca2+-dependent adenylyl cyclase(s). Mol Cell Endocrinol 1998; 144:47–57. [21] Chartoff, EH, Szczypka, MS, Palmiter, RD and Dorsa, DM. Endogenous neurotensin attenuates dopamine-dependent locomotion and stereotypy. Brain Res 2004; 1022:71–80.

742 / Chapter 102 [22] Ciofi, P, Crowley, WR, Pillez, A, Schmued, LL, Tramu, G and Mazzuca, M. Plasticity in expression of immunoreactivity for neuropeptide Y, enkephalins, and neurotens in in the hypothalamic tubero-infundibular dopaminergic system during lactation in mice. J Neuroendocrinol 1993; 5:599–602. [23] Costa, FG, Frussa-Filho, R and Felicio, LF. The neurotensin receptor antagonist, SR48692, attenuates the expression of amphetamine-induced behavioral sensitisation in mice. Eur J Pharmacol 2001; 428:97–103. [24] Dal Farra, C, Sarret, P, Navarro, V, Botto, J-M, Mazella, J and Vincent, J-P. Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 2001; 92:503–509. [25] Dobner, PR, Barber, DL, Villa-Komaroff, L and McKiernan, C. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc Natl Acad Sci USA 1987; 84:3516–3520. [26] Dobner, PR, Fadel, J, Deitemeyer, N, Carraway, RE and Deutch, AY. Neurotensin-deficient mice show altered responses to antipsychotic drugs. Proc Natl Acad Sci USA 2001; 98:8048–8053. [27] Dobner, PR, Deutch, AY and Fadel, J. Neurotensin: Dual roles in psychostimulant and antipsychotic drug responses. Life Sci 2003; 73:801–811. [28] Dobner, PR. Multitasking with neurotensin in the central nervous system. Cell and Mol Life Sci 2005; (in press). [29] Dubuc, I, Sarret, P, Labbe-Jullie, C, Botto, JM, Honore, E, Bourdel, E, Martinez, J, Costentin, J, Vincent, JP, Kitabgi, P and Mazella, J. Identification of the receptor subtype involved in the analgesic effect of neurotensin. J Neurosci 1999; 19: 503–510. [30] Fadel, J, Dobner, PR and Deutch, AY. Amphetamine-elicited striatal Fos expression is attenuated in neurotensin-deficient mice. (submitted). [31] Fredrickson, P, Boules, M, Yerbury, S and Richelson, E. Novel neurotensin analog blocks the initiation and expression of nicotine-induced locomotor sensitization. Brain Res 2003; 979:245–248. [32] Friry, C, Feliciangeli, S, Richard, F, Kitabgi, P and Rovere, C. Production of recombinant large proneurotensin/neuromedin N-derived peptides and characterization of their binding and biological activity. Biochem Biophys Res Comm 2002; 290:1161–1168. [33] Gilbert, JA and Richelson, E. Neurotensin stimulates formation of cyclic GMP in murine neuroblastoma clone N1E-115. Eur J Pharmacol 1984; 99:245–246. [34] Gui, X, Carraway, RE and Dobner, PR. Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats. Neurosci 2004; 126: 1023–1032. [35] Hassan, S, Dobner, PR and Carraway, R. Involvement of MAPkinase, PI3-kinase and EGF-receptor in the stimulatory effect of neurotensin on DNA synthesis in PC3 cells. Regul Pept 2004; 120:155–166. [36] Hentschel, K, Cheung, S, Moore, KE and Lookingland, KJ. Pharmacological evidence that neurotensin mediates prolactininduced activation of tuberoinfundibular dopamine neurons. Neuroendocrinol 1998; 68:71–76. [37] Kinkead, B, Dobner, PR, Egnatashvili, V, Murrary, T, Deitemeyer, N and Nemeroff, CB. Neurotensin-deficient mice have deficits in prepulse inhibition: Restoration by clozapine but not haloperidol, olanzapine or quetiapine. J Pharmacol Exp Ther 2005; (in press). [38] Kislauskis, E, Bullock, B, McNeil, S and Dobner, PR. The rat gene encoding neurotensin and neuromedin N: Structure, tissue-specific expression, and evolution of exon sequences. J Biol Chem 1988; 263:4963–4968.

[39] Kislauskis, E and Dobner, PR. Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells. Neuron 1990; 4:783–795. [40] Leonetti, M, Brun, P, Clerget, M, Steinberg, R, Soubrie, P, Renaud, B and Suaud-Chagny, M-F. Specific involvement of neurotensin type 1 receptor in the neurotensin-mediated in vivo dopamine efflux using knock-out mice. J Neurochem 2004; 89:1–6. [41] Luca, S, White, JF, Sohal, AK, Filippov, DV, van Boom, JH, Grisshammer, R and Baldus, M. The conformation of neurotensin bound to its G protein-coupled receptor. Proc Natl Acad Sci USA 2003; 100:10706–10711. [42] Luttinger, D, King, RA, Sheppard, D, Strupp, J, Nemeroff, CB and Prange, AJ, Jr. The effect of neurotensin on food consumption in the rat. Eur J Pharmacol 1982; 81:499–503. [43] Martin, S, Vincent, J-P and Mazella, J. Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia. J Neurosci 2003; 23:1198–1205. [44] Mazella, J, Poustis, C, Labbe, C, Checler, F, Kitabgi, P, Granier, C, van Rietschoten, J and Vincent, J-P. Monoiodo[Trp11]neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity, and binding properties to rat brain synaptic membranes. J Biol Chem 1983; 258:3476–3481. [45] Mazella, J, Zsurger, N, Navarro, V, Chabry, J, Kaghad, M, Caput, D, Ferrara, P, Vita, N, Gully, D, Maffrand, J-P and Vincent, J-P. The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor. J Biol Chem 1998; 273: 26273–26276. [46] Mussdorfer, GG, Malendowicz, LK, Meneghelli, V and Mazzocchi, G. Neurotensin enhances plasma adrenocorticotropin concentrations by stimulating corticotropin-releasing hormone concentration. Life Sci 1992; 50:639–643. [47] Najimi, M, Gailly, P, Maloteaux, J-M and Hermans, E. Distinct regions of C-terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G-proteins. FEBS Lett 2002; 512:329–333. [48] Nemeroff, CB. Neurotensin: Perchance an endogenous neuroleptic? Biol Psychiatry 1980; 15:283–302. [49] Nicot, A, Bérod, A, Gully, D, Rowe, W, Quirion, R, de Kloet, ER and Rostène, W. Blockade of neurotensin binding in the rat hypothalamus and of the central action of neurotensin on the hypothalamic-pituitary-adrenal axis with non-peptide receptor antagonists. Neuroendocrinol 1994; 59:572–578. [50] Oury-Donat, F, Thurneyssen, O, Gonalons, N, Forgez, P, Gully, D, Le Fur, G and Soubrie, P. Characterization of the effect of SR 48692 on inositol monophosphate, cyclic GMP and cyclic AMP responses linked to neurotensin receptor activation in neuronal and non-neuronal cells. Br J Pharmacol 1995; 116:1899–1905. [51] Panayi, F, Dorso, E, Lambas-Senas, L, Renaud, B, Scarna, H and Berod, A. Chronic blockade of neurotensin receptors strongly reduces sensitized, but not acute, behavioral response to Damphetamine. Neuropsychopharmacol 2002; 26:64–74. [52] Pettibone, DJ, Hess, JF, Hey, PJ, Jacobson, MA, Leviten, M, Lis, EV, Mallorga, PJ, Pascarella, DM, Snyder, MA, Williams, JB and Zeng, Z. The effects of deleting the mouse neurotensin receptor NTR1 on central and peripheral responses to neurotensin. J Pharmacol Exp Ther 2002; 300:305–313. [53] Reinecke, M. Neurotensin: Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone. Progr Histochem Cytochem 1985; 16:1–175. [54] Remaury, A, Vita, N, Gendreau, S, Jung, M, Arnone, M, Poncelet, M, Culouscou, JM, Le Fur, G, Soubrie, P, Caput, D,

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103 Neuromedin U (NMU): Brain Peptide PREETI H. JETHWA, CAROLINE J. SMALL, AND STEPHEN R. BLOOM

icosapentapeptide and has no internal dibasic cleavage site for the generation of a shorter version. The comparison of human and rat cDNA encoding NMU revealed approximately 70% homology between the two species suggesting that NMU is synthesized from a 174-aminoacid precursor containing the NMU peptide within the C-terminus [2, 27]. The precursor contains a hydrophobic signal peptide and several dibasic cleavage sites that give rise to a number of possible secreted peptides, including NMU (Fig. 1). Southern blot analysis of genomic DNA reveals that NMU is a single copy gene [27].

ABSTRACT Neuromedin U (NMU) is one of the most abundant neuropeptides and has been found in significant concentrations in both the gastrointestinal (GI) tract and the central nervous system (CNS). Within the CNS NMU cell bodies are found in the rostrocaudal part of the arcuate nucleus (Arc) of the hypothalamus with more widespread distribution of NMU fibers in the nucleus accumbens, medial thalamus, and brainstem, and in the paraventricular nucleus (PVN), ventromedial nucleus, and dorsomedial nucleus, areas where the NMU2 receptor is expressed. Recently NMU has been shown to be a potent anorexigenic peptide and that alterations of NMU signaling can predispose to obesity.

NMU DISTRIBUTION AND PROCESSING NMU shows remarkable conservation throughout evolution, the C-terminal pentapeptide (Phe-Arg-ProArg-Asn-NH2) is conserved across all species and the heptapeptide (Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) is conserved throughout all mammalian species [27]. This degree of conservation suggests the importance of the carboxyl-terminus sequence for biological activity [16, 36]. NMU is an abundant peptide found at significant concentrations in both the gastrointestinal tract [1, 3, 7] and CNS [3, 7, 12, 18, 20, 22, 37]. It is also found in other tissues such as the pituitary gland [7], the thyroid gland [5], and the urogenital tract [7]. In the rat brain, the highest levels of NMU-like immunoreactivity are found in the nucleus accumbens (Acb), hypothalamus, anterior pituitary, and thalamus [6, 7]. NMU-immunoreactive cell bodies are found in the rostrocaudal part of the Arc with more widespread distribution of immunoreactive fibers in the Acb, medial thalamus, PVN, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), supraoptic nucleus (SON), and brainstem with dense terminals field

NMU DISCOVERY Neuromedin U (NMU) was first isolated from porcine spinal cord in 1985 and named for its potent contractile activity on the uterus [29, 30]. Two molecular forms were purified; an icosapentapeptide (NMU-25) and an octapeptide (NMU-8) identical to the C-terminal of NMU-25 [29, 30]. Both forms are biologically active, stimulating contraction of rat uterus in vitro and causing potent vasoconstriction in rats and dogs [11, 18, 39]. NMU has since been fully sequenced in several species and most of the NMU peptides that have been isolated from different species are icosapentapeptides with the exception of rat NMU, NMU-23 [4].

STRUCTURE OF THE PRECURSOR mRNA/GENE The human cDNA encoding the precursor protein for NMU has been sequenced [2]. The human NMU is an Handbook of Biologically Active Peptides

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FIGURE 1. Neuromedin U-25 precursor peptide [2].

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FIGURE 2. NMU expression in the mouse hypothalamus. Autoradiographs showing NMU mRNA expression in the mouse hypothalamus (ad libitum fed hooded listers). In situ hybridization was conducted using [35]S-labeled NMU antisense RNA probe. Specific expression was observed in the (a) DMH and Arc, (b) VMH, (c) DMH and VMH, and (d) SCN. Scale bar in (d) represents 2 μm [12].

primarily in the nucleus of the solitary tract (NST) and parabrachial nucleus (PBN) [3, 18, 20, 37, 38]. In situ hybridization has shown NMU mRNA to be most abundant in the pituitary pars tuberalis and slightly less in the Arc and median eminence, the caudal brainstem [NST, area postrema (AP), and dorsal motor nucleus of the vagus (DMV)] and the spinal cord [18, 20, 22, 37]. The presence of the NMU mRNA in the median eminence has been questioned, with authors arguing that expression is localized to the par tulberalis [22]. However, more recently Graham et al. [12] showed that expression of NMU mRNA was species dependent (Figs.

2 and 3). In mice NMU mRNA is detected in the Arc, DMH, VMH, and suprachiasmatic nucleus (SCN) in contrast to the expression of NMU mRNA in the rat in which it is absent from the median eminence, the VMH, and the DMH but highly expressed in the pituitary par tuberalis [12].

NMU RECEPTORS In 1998 a G-protein coupled receptor (GPCR) FM-3 was cloned from human and murine cDNA libraries due to its homology with the growth hormone secreta-

Neuromedin U (NMU): Brain Peptide / 747

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FIGURE 3. NMU expression in the rat hypothalamus. Autoradiographs showing NMU mRNA expression in the rat hypothalamus (ad libitum fed hooded listers). NMU mRNA was detected in (a) the Arc (−2.8 to Bregma), (b) pars tuberalis (−3.6 to Bregma), (c) dorsomedial compact zone of the DMH (−3.3 to the Bregma), and (d) in the SCN (−1.4 to Bregma). Scale bar in (d) represents 2 mm [12].

gogue receptor and the neurotensin receptor. Cloning enabled a subsequent “reverse pharmacological” approach for the identification of NMU as an endogenous ligand for the orphan human GPCR, FM-3 [9, 17, 19, 20, 35, 37, 40]. NMU binds to and activates human FM3 with subnanomolar affinity and potency. Extracts from rat tissue were also found to contain endogenous ligand for FM-3, the brain and small intestine extracts having the highest affinity. A 23-amino-acid peptide identical to rat NMU-23 was subsequently chromatographically purified. Screening of potential ligands revealed that only various forms of NMU peptides activated the receptor. Peptides that show some similarity to NMU, such as neuromedin B, C, K, and N, as well as neurotensin, ghrelin, motilin, vasoactive intestinal peptide, and pancreatic polypeptide had no affinity [9, 17, 19, 20, 35, 37, 40]. Simultaneously a second NMU receptor, a GPCR, FM-4 was cloned from the human and rat [19, 20, 35, 37], and more recently the murine receptor [10, 20] was cloned. FM-3 and FM-4 are cognate receptors and have been renamed NMU1R and NMU2R, respectively (nomenclature as recommended by IUPHAR) [21]. Both receptors have significant abilities to distinguish between different forms of NMU, and have species and tissue selectivity in the biological actions of NMU [9, 17, 19, 20, 35, 37, 40].

NMU1R The gene for human NMU1R has been mapped to SHGC-33253, which is localized on chromosome 2q34937 [35] and is encoded by two exons [37]. NMU1R has a molecular weight of 44,979 Da. The mRNA for NMU1R is expressed in a wide variety of tissues but levels are greatest in peripheral tissues, in particular, the small intestines and stomach. However, mRNA for NMU1R is present in relatively high levels in the pancreas, testis, adrenal cortex, liver, heart, lung, trachea, mammary gland, bone marrow, and peripheral blood leukocytes [17, 19, 20, 40]. A similar distribution pattern exists in the rat with highest levels in the small intestine, lung, and bone marrow [9]. NMU1R mRNA expression is also found in approximately 25% of the small/medium diameter neurons within the dorsal root ganglia [42]. There is relatively low expression of NMU1R in human or rodent brain [9, 10, 20].

NMU2R The gene for human NMU2R has been mapped to SHGC-8848, which is localized to chromosome 5q31.1q31.1 [35]. NMU2R has a molecular weight 47,450 Da. In humans, NMU2R mRNA is confined predominantly

748 / Chapter 103 to specific regions within the brain, with the greatest expression observed in the substantia nigra, medulla oblongata, pontine reticular formation, spinal cord, and thalamus. Moderate to high levels are present in the indusium griseum, setophippocampal nucleus, vascular organ of the lamina terminalis, the PVN, CA1 region of the hippocampus, parafascicular thalamic nucleus, dorsal raphe nucleus, and along the wall of the third ventricle in the hypothalamus [20]. More recently a detailed analysis of NMU2R expression was completed in the rat [12]. The NMU2R gene was found in the medial postal region of the Arc adjacent to the third ventricle, isolated neurons in other portions of the Arc [12], and the PVN. NMU2R was found in less abundance in the bed nucleus of the stria terminalis, subfornical organ, dorsal fornix, dorsal tuberomammillary nucleus, raphe obscurus and raphe pallidus nuclei, cunneate nucleus, and paratrigeminal nucleus [20]. Peripherally the NMU2R is expressed in the testes with lower levels present in the GI tract, genitourinary tract, liver, pancreas, adrenal gland, thyroid gland, lung, trachea, spleen, and thymus [20].

BIOLOGICAL ACTIONS OF NMU IN THE BRAIN The Role of NMU in the Regulation of Energy Balance Intracerebroventricular (ICV) administration of NMU reduces nocturnal food intake and decreases body weight [22, 25, 32, 33, 41]. In addition, ICV administration of NMU antiserum increases food intake [25] while fasting reduces levels of NMU mRNA in the ventromedial hypothalamic region [20]. Wren et al. [41] demonstrated a dose-dependent reduction of feeding 1 hour following administration, NMU into the PVN and Arc [20]. In addition to the immediate reduction in food intake observed following NMU administration into the PVN or Arc, NMU has a much delayed inhibitory (4–8 hours post injection) effect following administration into the medial preoptic area (MPOA) of the hypothalamus [41]. The MPOA is involved in reproductive function and c-fos expression and neuropeptide Y (NPY) levels increase after feeding, while αMSH reduces food intake following administration in the MPOA [24]. Thus, NMU may interact with other neuropeptides to mediate the delayed feeding effect [41]. The decrease in food intake following a single ICV injection of NMU is associated with a significant increase in gross locomotor activity, core body temperature, heat production, and oxygen consumption [13, 20, 22, 32, 41]. However, NMU does not cause shivering, suggest-

ing that the effects on body temperature are mediated via changes in sympathetic outflow to brown adipose tissue and skeletal muscle. Ivanov et al. [22] proposed that the effects of NMU on feeding and energy expenditure are independent as they observed an increase in core body temperature in the absence of a decrease in food intake in satiated animals. More recently NMU null (NMU−/−) mice [14] and NMU overexpressing (NMU Tg) mice [26] have been developed. NMU−/− mice show increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese NMU−/− mice develop hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia, and hyperlipidemia [14]. The NMU Tg mice, on the other hand, show decreased body weight and adiposity, hypophagia, and a modest increase in respiratory quotient during food deprivation and refeeding. Unlike the NMU−/− mice, the NMU Tg mice also show improved insulin sensitivity on both normal and high fat diet [26]. In addition, many neuropeptides important to the regulation of feeding are altered in these transgenic animals; in the NMU−/− mice there is a decrease in proopiomelanocortin (POMC), while NPY and agouti-gene-related peptide (AgRP) remain unchanged [14]. However, in the NMU Tg mice, both POMC and NPY are increased, while AgRP remains unchanged [26]. The phenotype of these animals suggests that alterations in the NMU system leads to altered energy metabolism.

The Role of Gastric Acid Secretion and Emptying Intracerebroventricular administration of NMU reduces gastric acid secretion [31]. This CNS effect of NMU appears to be independent of vagal innervation, since NMU significantly suppressed pentagastrin secretion in vagotomized rats. Prostaglandin E2 reduces gastric acid secretion directly by inhibiting parietal cell secretion and indirectly by inhibiting gastric acid release. Peripheral administration of indomethacin, to block prostaglandin synthesis, prevents the inhibitory action of neuropeptides on gastric secretion. However, administration of NMU suppressed acid secretion in indomethacin-treated rats suggesting that NMU acts independently of prostaglandin E2 [31].

The Role of NMU in the Regulation of the HPA Axis and Stress Response Corticotropin releasing hormone (CRH) is another hypothalamic peptide located in the PVN, which decreases food intake, increases energy expenditure via stimulation of the sympathetic nervous system, induces stress responses, and increases locomotor and groom-

Neuromedin U (NMU): Brain Peptide / 749

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FIGURE 4. Distribution of neuromedin U (NMU) cell bodies (•) and immunoreactive fibers (Æ) where NMU mRNA has been found using in situ hybridization.

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FIGURE 5. Distribution of neuromedin U 2 receptor (NMU2R) (䉱). This receptor is found predominantly in the brain, unlike the NMU1 receptor (NMU1R), which is found predominantly in peripheral tissues.

ing behavior [23, 28]. Intracerebroventricular and intra-PVN administration of NMU increases plasma adrenocorticotropic hormone and corticosterone. In addition, the release of CRH and arginine vasopressin is increased from ex vivo hypothalamic explants incu-

bated with NMU [34]. Consistent with these effects, NMU increases c-fos expression in CRH containing neurons in the parvocellular region of the PVN and SON [22, 34] and augments CRH mRNA expression in the PVN [14].

750 / Chapter 103 The mechanism by which hypothalamic NMU alters feeding behavior and energy homeostasis is not clear. However, given that the ICV effects of CRH appear to be similar to those observed following NMU administration, Hanada et al. [15] hypothesized that the changes in feeding behavior and energy homeostasis following NMU may be via CRH. Intracerebroventricular administration of NMU did not alter dark phase food intake or fasting-induced feeding in CRH null (CRH−/−) mice when compared to wild-type [15]. In addition, the increase in oxygen consumption, body temperature, and locomotor activity observed following NMU administration was suppressed in the CRH−/− mice. In further experiments, the effects of NMU were also abolished in wild type mice pre-treated with the CRH receptor antagonist alpha-helical-CRH(9–41) when compared to their controls [13]. Further supporting this, recently Hanada et al. [14] showed that CRH mRNA expression is reduced in the PVN of NMU−/− mice and showed a lower plasma corticosterone level. However, surprisingly, NMU Tg mice showed no changes in CRH mRNA expression [26], but this may be due to the differing method of detection. To further support the hypothesis, NMU-induced inhibition of gastric acid secretion is blocked by pretreatment with anti-CRH IgG, suggesting that NMUinduced acid inhibition is mediated by CRH. CRH inhibits gastric acid secretion by activation of the sympathetic, noradrenergic nervous system and not vagal fibers [8]. This finding supports the possibility that CRH mediates NMU-induced acid inhibition through a vagus-independent pathway. Intracerebroventricular administration of NMU also inhibited gastric emptying in rats, however, peripheral NMU had no effect [31]. These findings suggest that NMU mediates its effects on food intake and energy expenditure via hypothalamic CRH.

PATHOPHYSIOLOGICAL IMPLICATIONS The development of the NMU null (NMU−/−) mice [14] and the NMU overexpressing (NMU Tg) mice [26] further supports the role of NMU as an endogenous anorexigenic peptide. The NMU−/− mice lack the gene encoding NMU and develop an obese phenotype, while NMU Tg mice have ubiquitous expression of the NMU transgene and develop a lean phenotype. These findings suggest NMU is an important regulator of energy balance as perturbation of NMU signaling can result in obesity. It is likely that NMU mediates its effects on body weight via alterations in hypothalamic CRH. While this is an important and interesting hypothalamic circuit, it may not prove a useful antiobesity target. Stimulation would result in weight loss but also an

overactive hypothalamo-pituitary-adrenal axis. Thus, NMU may not be useful as a long-term anti-obesity therapy.

References [1] Augood, S. J.; Keast, J. R.; Emson, P. C. Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine. Regul Pept 1988 Apr; 20(4):281–292. [2] Austin, C.; Lo, G.; Nandha, K. A.; Meleagros, L.; Bloom, S. R. Cloning and characterization of the cDNA encoding the human neuromedin U (NmU) precursor: NmU expression in the human gastrointestinal tract. J Mol Endocrinol 1995 Apr; 14(2):157–169. [3] Ballesta, J.; Carlei, F.; Bishop, A. E.; Steel, J. H.; Gibson, S. J.; Fahey, M.; Hennessey, R.; Domin, J.; Bloom, S. R.; Polak, J. M. Occurrence and developmental pattern of neuromedin Uimmunoreactive nerves in the gastrointestinal tract and brain of the rat. Neuroscience 1988 Jun;25(3):797–816. [4] Conlon, J. M.; Domin, J.; Thim, L.; DiMarzo, V.; Morris, H. R.; Bloom, S. R. Primary structure of neuromedin U from the rat. J Neurochem 1988 Sep;51(3):988–991. [5] Domin, J.; Al Madani, A. M.; Desperbasques, M.; Bishop, A. E.; Polak, J. M.; Bloom, S. R. Neuromedin U-like immunoreactivity in the thyroid gland of the rat. Cell Tissue Res 1990 Apr;260(1):131–135. [6] Domin, J.; Ghatei, M. A.; Chohan, P.; Bloom, S. R. Characterization of neuromedin U like immunoreactivity in rat, porcine, guinea-pig and human tissue extracts using a specific radioimmunoassay. Biochem Biophys Res Commun 1986 Nov; 140(3):1127–1134. [7] Domin, J.; Ghatei, M. A.; Chohan, P.; Bloom, S. R. Neuromedin U—a study of its distribution in the rat. Peptides 1987 Sep;8(5):779–784. [8] Druge, G.; Raedler, A.; Greten, H.; Lenz, H. J. Pathways mediating CRF-induced inhibition of gastric acid secretion in rats. Am J Physiol 1989 Jan;256(1 Pt 1):G214–G219. [9] Fujii, R.; Hosoya, M.; Fukusumi, S.; Kawamata, Y.; Habata, Y.; Hinuma, S.; Onda, H.; Nishimura, O.; Fujino, M. Identification of neuromedin U as the cognate ligand of the orphan G proteincoupled receptor FM-3. J Biol Chem 2000 Jul;275(28):21068– 21074. [10] Funes, S.; Hedrick, J.; Yang, S.; Shan, L.; Bayne, M.; Monsma, F.; Gustafson, E. Cloning and characterization of murine neuromedin U receptors. Peptides 2002 Sep;23(9):1607. [11] Gardiner, S. M.; Bennett, T. Brain neuropeptides: actions on central cardiovascular control mechanisms. Brain Res Brain Res Rev 1989 Jan;14(1):79–116. [12] Graham, E. S.; Turnbull, Y.; Fotheringham, P.; Nilaweera, K.; Mercer, J. G.; Morgan, P. J.; Barrett, P. Neuromedin U and neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status. J Neurochem 2003 Dec;87(5):1165–1173. [13] Hanada, R.; Nakazato, M.; Murakami, N.; Sakihara, S.; Yoshimatsu, H.; Toshinai, K.; Hanada, T.; Suda, T.; Kangawa, K.; Matsukura, S.; Sakata, T. A role for neuromedin U in stress response. Biochem Biophys Res Commun 2001 Nov;289(1):225– 228. [14] Hanada, R.; Teranishi, H.; Pearson, J. T.; Kurokawa, M.; Hosoda, H.; Fukushima, N.; Fukue, Y.; Serino, R.; Fujihara, H.; Ueta, Y.; Ikawa, M.; Okabe, M.; Murakami, N.; Shirai, M.; Yoshimatsu, H.; Kangawa, K.; Kojima, M. Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nat Med 2004 Oct;10(10):1067–1073.

Neuromedin U (NMU): Brain Peptide / 751 [15] Hanada, T.; Date, Y.; Shimbara, T.; Sakihara, S.; Murakami, N.; Hayashi, Y.; Kanai, Y.; Suda, T.; Kangawa, K.; Nakazato, M. Central actions of neuromedin U via corticotropin-releasing hormone. Biochem Biophys Res Commun 2003 Nov;311(4):954– 958. [16] Hashimoto, T.; Masui, H.; Uchida, Y.; Sakura, N.; Okimura, K. Agonistic and antagonistic activities of neuromedin U-8 analogs substituted with glycine or D-amino acid on contractile activity of chicken crop smooth muscle preparations. Chem Pharm Bull (Tokyo) 1991 Sep;39(9):2319–2322. [17] Hedrick, J. A.; Morse, K.; Shan, L.; Qiao, X.; Pang, L.; Wang, S.; Laz, T.; Gustafson, E. L.; Bayne, M.; Monsma, F. J., Jr. Identification of a human gastrointestinal tract and immune system receptor for the peptide neuromedin U. Mol Pharmacol 2000 Oct;58(4):870–875. [18] Honzawa, M.; Sudoh, T.; Minamino, N.; Kangawa, K.; Matsuo, H. Neuromedin U-like immunoreactivity in rat intestine: regional distribution and immunohistochemical study. Neuropeptides 1990 Jan;15(1):1–9. [19] Hosoya, M.; Moriya, T.; Kawamata, Y.; Ohkubo, S.; Fujii, R.; Matsui, H.; Shintani, Y.; Fukusumi, S.; Habata, Y.; Hinuma, S.; Onda, H.; Nishimura, O.; Fujino, M. Identification and functional characterization of a novel subtype of neuromedin U receptor. J Biol Chem 2000 Sep;275(38):29528–29532. [20] Howard, A. D.; Wang, R.; Pong, S. S.; Mellin, T. N.; Strack, A.; Guan, X. M.; Zeng, Z.; Williams, D. L., Jr.; Feighner, S. D.; Nunes, C. N.; Murphy, B.; Stair, J. N.; Yu, H.; Jiang, Q.; Clements, M. K.; Tan, C. P.; McKee, K. K.; Hreniuk, D. L.; McDonald, T. P.; Lynch, K. R.; Evans, J. F.; Austin, C. P.; Caskey, C. T.; Van der Ploeg, L. H.; Liu, Q. Identification of receptors for neuromedin U and its role in feeding. Nature 2000 Jul;406(6791):70–74. [21] IUPHAR RECEPTOR DATABASE. http://www.iuphar-db.org/ iuphar-rd/. 2005. [22] Ivanov, T. R.; Lawrence, C. B.; Stanley, P. J.; Luckman, S. M. Evaluation of neuromedin U actions in energy homeostasis and pituitary function. Endocrinology 2002 Oct;143(10):3813– 3821. [23] Kalra, S. P.; Dube, M. G.; Pu, S.; Xu, B.; Horvath, T. L.; Kalra, P. S. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 1999 Feb;20(1):68– 100. [24] Kim, M. S.; Rossi, M.; Abusnana, S.; Sunter, D.; Morgan, D. G.; Small, C. J.; Edwards, C. M.; Heath, M. M.; Stanley, S. A.; Seal, L. J.; Bhatti, J. R.; Smith, D. M.; Ghatei, M. A.; Bloom, S. R. Hypothalamic localization of the feeding effect of agouti-related peptide and alpha-melanocyte-stimulating hormone. Diabetes 2000 Feb;49(2):177–182. [25] Kojima, M.; Haruno, R.; Nakazato, M.; Date, Y.; Murakami, N.; Hanada, R.; Matsuo, H.; Kangawa, K. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem Biophys Res Commun 2000 Sep;276(2):435–438. [26] Kowalski, T. J.; Spar, B. D.; Markowitz, L.; Maguire, M.; Golovko, A.; Yang, S.; Farley, C.; Cook, J. A.; Tetzloff, G.; Hoos, L.; Del Vecchio, R. A.; Kazdoba, T. M.; McCool, M. F.; Hwa, J. J.; Hyde, L. A.; Davis, H.; Vassileva, G.; Hedrick, J. A.; Gustafson, E. L. Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice. J Endocrinol 2005 Apr;185(1):151– 164. [27] Lo, G.; Legon, S.; Austin, C.; Wallis, S.; Wang, Z.; Bloom, S. R. Characterization of complementary DNA encoding the rat neuromedin U precursor. Mol Endocrinol 1992 Oct;6(10):1538– 1544. [28] Masaki, T.; Yoshimichi, G.; Chiba, S.; Yasuda, T.; Noguchi, H.; Kakuma, T.; Sakata, T.; Yoshimatsu, H. Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding, adi-

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104 Galanin and GALP Systems in Brain—Molecular Pharmacology, Anatomy, and Putative Roles in Physiology and Pathology ANDREW L. GUNDLACH AND SEBASTIAN R.-F. JUNGNICKEL

the central nervous system. The human galanin gene was cloned in 1988 and the equivalent genes in other species were cloned and sequenced soon after. Galaninlike peptide (GALP) was first isolated from porcine intestine in 1999 and GALP genes from pig, rat, and human were cloned [26]. GALP expression was detected in brain, intestine, and testis.

ABSTRACT The galanin peptide family comprises two members: galanin, a widely distributed 29- to 30-amino-acid neuropeptide discovered in 1983, and galanin-like peptide (GALP), a structurally related 60-amino-acid neuropeptide first isolated and cloned in 1999, with a more restricted distribution in mammalian brain. Three G protein–coupled receptors—GalR1, GalR2, and GalR3—bind galanin with high affinity and are expressed in brain of mammalian species, including rat, mouse, and human. Activation of these receptors by galanin and analogs activates conventional signaling cascades associated with cAMP/protein kinase-A and inositol phosphate/Ca2+/phospholipase-C/protein kinase-C; and galanin is known to affect both neuronal K+- and Ca2+-channel activity. GALP also binds to these receptors, but its preferred native receptor has not been identified. Galanin regulation of classical transmitter and neurohormone release is associated with a wide range of putative central actions, including roles in the physiology of metabolism, arousal/sleep-wake cycle, nociception, cognition, and development; and in the pathophysiology of seizures and epilepsy, chronic pain, and neural injury and repair. GALP production in the hypothalamus is strongly regulated by leptin (and insulin) and GALP interacts with other feeding-related circuits and GnRH signaling to regulate the function of the hypothalamic-pituitary-gonadal axis and influence metabolism, sexual behavior, and reproduction.

STRUCTURE AND REGULATION OF THE GALANIN AND GALP GENES Galanin. The human galanin gene spans 6.5 kb and consists of 6 exons: exon 1 is noncoding, exons 2 to 5 encode the preprogalanin peptide, and exon 6 encodes the stop codon and a polyadenylation tail. The gene is located on chromosome 11q13.3-q13.5 with a very similar exon:intron organization in the human and mouse. Galanin gene expression can be regulated by a range of stimulatory/inhibitory transcription factors and cellular signaling molecules—consistent with “plasticity” of galanin levels in multiple systems. In chromaffin cells, human neuroblastomas and other cell types, galanin expression can be altered by cytokines, growth factors, hormones, cAMP, and activation of protein kinase C (phorbol esters) [27]. A phorbol ester responsive element is present in the promoter region about 50 bp upstream of the human galanin gene. In the bovine galanin promoter, CRE-like elements have been identified that initiate galanin transcription by binding other phorbol ester induced proteins, such as Jun, activating transcription factor (ATF, both homo- and heterodimers) and cAMP-response element binding protein (CREB, alone and homodimers) [27]. Leukemia inhibitory factor (LIF) and nerve growth factor (NGF) also activate galanin gene transcription and NRE- and CRE-like elements may be largely responsible

DISCOVERY OF GALANIN AND GALANINLIKE PEPTIDE (GALP) Galanin was first isolated from porcine intestine in 1983 and was soon identified in other tissues including Handbook of Biologically Active Peptides

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754 / Chapter 104 for maintenance of basal expression [27], while under conditions of stress, NGF has been shown to suppress LIF-mediated activation of galanin. GALP. The porcine GALP cDNA is 974 bp with a 360bp open-reading frame encoding the precursor peptide, while the rat and human cDNAs have similar sized open-reading frames and precursor products [26]. The human gene is located on chromosome 19q13.43, while the rat and mouse genes are on chromosome 1q12 and 7A1, respectively. The regulation of the GALP gene has not been described, but in vivo studies suggest that signaling associated with the cytokine, leptin, and the circulating hormone, insulin may be primarily responsible for maintenance of GALP expression. Under fasting conditions that reduce GALP mRNA levels in hypothalamus, leptin treatment increases GALP mRNA levels; and in hypoleptinemic rats and mice (i.e., ob/ob obese) and hypoinsulinemic diabetic rats, in which hypothalamic GALP mRNA levels are markedly reduced, leptin and insulin treatment can restore expression [6]. In addition, thyroidectomy decreases and thyroxine treatment normalizes GALP mRNA expression in rat brain [4]. GALP expression is increased in pituicytes of the posterior pituitary by lactation, osmotic-challenge, and diabetics, by unknown mechanisms [4, 28].

DISTRIBUTION OF GALANIN AND GALP mRNA AND PEPTIDE-IMMUNOREACTIVITY Galanin. Galanin mRNA and galanin-like immunoreactivity (-LI) is distributed throughout the CNS of several species including rat, mouse, and human; and it coexists with classical neurotransmitters (see [12, 23]). Galanin mRNA is most abundant in hypothalamic and brainstem areas of rat [12] (Fig. 1) and mouse, with very high levels in the preoptic-, periventricular-, and dorsomedial-hypothalamic nuclei, bed nucleus of the stria terminalis, medial and lateral amygdala, locus coeruleus, and nucleus of the solitary tract. Low to medium galanin mRNA levels are observed in olfactory bulb, septal nuclei, thalamus, parabrachial nucleus, and the spinal trigeminal tract nucleus. Recently galanin mRNA was detected in the proliferative zones of developing and adult brain—the subventricular zone and subgranular zone of hippocampus, and in oligodendrocyte precursor cells in the corpus callosum [29]. Galanin is coexpressed with different neurotransmitters and neuropeptides in different types of neurons. For example in the rat, 80% of locus coeruleus norepinephrine neurons and 60% of dorsal raphe serotonin neurons contain galanin, as do populations of GABA neurons in the spinal cord, cholinergic neurons in the septum and histamine cells in the tuberomammillary nucleus. Galanin is also coexpressed with vasopressin,

cholecystokinin, and tyrosine hydroxylase in the paraventricular hypothalamic nucleus. Aspects of galanin expression in brain appear to be species-specific. For example, galanin-LI is normally low in hippocampus of the mouse, while it is abundant in this structure in the monkey; and the cell bodies and dense galanin-LI fibers present in the nucleus accumbens of the monkey are not present in the mouse or rat. While rats and mice display a quite similar galanin distribution pattern, galanin mRNA and -LI are readily detected in the dorsal motor nucleus of the vagus in mouse but not rat. Galanin mRNA is observed in inferior olive neurons of mouse but not rat, but in contrast no galanin mRNA is present in mouse cerebellum, yet galanin transcripts are observed in rat cerebellum in a subset of Purkinje cells in the flocculus, paraflocculus, and several lobules, with the number of positive Purkinje cells in lobule 10 in excess of the number of cells in the rest of the adult cerebellum. GALP. In all species studied so far, including rat and mouse, GALP mRNA has a more restricted distribution in the CNS in neurons of the periventricular regions of the arcuate nucleus and median eminence of the hypothalamus, and in pituicytes in the posterior pituitary gland [16, 28]. GALP-immunopositive neurons are present in the arcuate nucleus, particularly the posteriormedial regions; and GALP-immunoreactive fibers are present in the arcuate and paraventricular nuclei, the lateral hypothalamus, medial preoptic area, bed nucleus of the stria terminalis (BST), and lateral septum [31] (Fig. 1). A majority (85%) of arcuate GALP neurons express leptin receptors and smaller numbers express orexin-1 receptors. GALP cells also contain α-melanocyte-stimulating hormone, derived from proopiomelanocortin (POMC). Neuropeptide Y- and orexin-terminals innervate GALP cells in the arcuate and GALP-nerve terminals make contact with orexin- and melaninconcentrating hormone-neurons in lateral hypothalamus. GALP neurons innervate gonadotropin-releasing hormone (GnRH) neurons and fibers in the medial preoptic area and BST of rats [31]. On the basis of this data, two major GALP pathways are apparent: one to the paraventricular nucleus and a second to the medial hypothalamic area, bed nucleus of the stria terminalis and lateral septum.

NATURE AND PROCESSING OF GALANIN AND GALP PRECURSORS Preprogalanin is a 123-amino-acid galanin precursor and is cleaved at two sites to produce galanin and galanin message-associated peptide. In most mammals, galanin is a 29-amino-acid peptide (30 in human), with a highly conserved NH2-terminal sequence (15 amino

Galanin and GALP Systems in Brain / 755

Cx

Hi CC

OB

LS CPu

Th PAG Cb BST

DR

PBN

Hyp

AON

PVN Acb

LC

DMH SN

MPA

VR NST

LH

SpC SON

DBB

VMH

Arc

Amy

ME

Amb

NL IL AP

7

FIGURE 1. Schematic representation of the contrasting distribution of galanin and GALP cells and innervation in rat brain. In a stylized parasagittal brain section, cells that express mRNA (and immunoreactivity) for galanin (red dots) and GALP (blue dots) are illustrated, along with the approximate localization of their immunoreactive nerve fibers/terminals (red and blue stippled lines). Nonneuronal expression in astrocytes (pituicytes) and oligodendrocyte and other progenitors is shown in open symbols. The two major GALP pathways that have been proposed from the arcuate nucleus to the paraventricular nucleus and from arcuate to other hypothalamic and limbic regions are indicated by blue arrows. The relative topography of the many and widespread galanin pathways has been omitted for clarity. (For more details on these distributions, see [12, 16, 23, 26, 28, 29, 31]). Abbreviations: Acb, nucleus accumbens; Amb, nucleus ambiguus; Amy, amygdala; AON, anterior olfactory nucleus; AP, anterior pituitary; Arc, arcuate nucleus of hypothalamus; BST, bed nucleus of stria terminalis; Cb, cerebellum; CC, corpus callosum; CPu, caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of hypothalamus; DR, dorsal raphe nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of pituitary; LC, locus coeruleus; LS, lateral septum; ME, median eminence; MPA, medial preoptic area; NL, neural lobe of the pituitary; NST, nucleus of solitary tract; OB, olfactory bulb; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular nucleus of hypothalamus; SN, substantia nigra; SON, supraoptic nucleus; SpC, spinal trigeminal nucleus; Th, thalamus; VMH, ventromedial nucleus of hypothalamus; VR, ventral raphe nuclei; 7, facial nucleus. (See color plate.)

acids) (see [23]). The GALP precursor is 115 amino acids in rat and mouse (116/120 in human/pig) [26]. The N-terminal 20–22 residues have characteristics of a signal sequence with hydrophobic clusters and small polar residues. In all species, GALP amino acids 9–21 are homologous to the 13 N-terminal amino acids of galanin, which confer its biological activity and are also conserved across species. Notably, the DNA sequences for the 13-shared galanin/GALP residues are more conserved within GALP sequences of different species than galanin and GALP of the same species. GALP residues 1–24 and 41–53 are also conserved across species and are presumably important for receptor interaction or trafficking of the peptide [26].

GALANIN FAMILY RECEPTORS Biological actions of galanin are mediated via G protein–coupled receptors. Following the cloning of the first human receptor (GalR1) from a Bowes melanoma cell line cDNA library in 1994, all other galanin receptors (GalR1, GalR2, and GalR3) have subsequently been cloned from rat, mouse, and human, and their genetic and physicochemical characteristics have been determined, including their chromosomal location and intron-exon organization, their relative degree of homology between each other and across species, their signal transduction pathways, and their pharmacological profile including their relative affinity for [125I]-

756 / Chapter 104 TABLE 1. Characteristics of cloned galanin receptors. GalR1 Human—chromosome 18q23 (349 amino acids) Mouse—chromosome 18E4 Rat—346 amino acids (91% homology with human GalR1) The affinity (KD) of [125I]-galanin (1–29) for GalR1 is 0.12 nM Rank order of peptide binding: galanin (1–29) > galanin (1–16) >> galanin (2–29) >> [D-Trp2] galanin (1–29) > galanin (3–29) GalR2 Human—chromosome 17q25.3 (human, mouse, rat share >90% homology) The affinity (KD) of porcine [125I]-galanin for GalR2 is 0.15–0.6 nM Rat—372 amino acids (38% homology with rat GalR1) Rank order of peptide binding: galanin = M35/M40 > galanin (1–16) = [D-Trp2]-galanin (1–29) >> galanin (3–29) GalR3 Human—chromosome 22q13.1 Rat—370 amino acids (36% and 54% homology with rat GalR1 and GalR2) The affinity (KD) of porcine [125I]-galanin for GalR3 is 0.6 nM Rank order of peptide binding: rat galanin > porcine galanin > M35 > galantide > human galanin > M40 > galanin (1–16) > [D-Trp2] galanin (1–29) > galanin (3–29) (For further details on these receptors, see reference [11]).

galanin(1-29) (KD values 0.1 to 0.6 nM) and their characteristic, relative affinities for a range of truncated galanin analogs (Table 1, supplementary material; [11]). Such studies of cloned receptors have shown that GalR1 is negatively coupled to adenylate cyclase via the inhibitory G protein (Gi) and can influence neuronal Ca2+- and inward-rectifying K+-currents and activate mitogen-activated protein kinase (MAPK). GalR2 has been shown to couple via Go and Gq11 to activation of cellular inositol phosphate production and protein kinase C. GalR3 also produces inhibition of adenylate cyclase, but its physiological roles are largely unexplored (see [1, 11]). Distribution of galanin receptors. GalR1 mRNA is widely expressed in the mammalian CNS. In the mouse and rat, expression is high in olfactory structures and subregions/nuclei of the amygdala, thalamus, hypothalamus, pons, medulla, and spinal cord. GalR2 mRNA is also broadly expressed in the CNS, with high levels present in the hippocampus, particularly in the dentate gyrus and the CA3 field, and in the hypothalamus in the supraoptic, arcuate, and mammillary nuclei. In the hindbrain, GalR2 is abundant in the spinal trigeminal tract and the dorsal vagal complex [25]. GalR3 mRNA is relatively abundant in peripheral tissues, but expression in the CNS is more limited with expression largely confined to the hypothalamus and areas of the mid- and hindbrain [22]. Recent anatomical studies have also identified GalR1 and GalR2 mRNA in cells within the subventricular zone and the rostral migratory stream, regions associated with neurogenesis in adult brain [20, 29].

The autoradiographic distribution of high affinity [125I]-galanin binding sites correlates with that of GalR1 mRNA in rat and mouse brain [12], a finding consistent with a more limited and lower level of GalR2/3 expression and a lower affinity of the radioligand for nonGalR1 receptors [22, 25]. The distribution of galanin receptors (and galanin) in the developing CNS suggests that galanin regulates developmental processes including cell proliferation and survival, neurite growth, and synaptic maturation [19, 20, 29]. GALP receptors. GALP was originally identified as a possible native ligand for GalR2 [26], but GALP binds to GalR1 and may also have high affinity for GalR3 (this activity has not been widely reported). Many in vivo studies have, however, shown differences between the effects of GALP and galanin or galanin receptor-specific analogs on neuronal activity and animal behavior, suggesting that a unique receptor for GALP might exist that still awaits discovery.

CENTRAL ACTIONS OF GALANIN AND GALP IN NORMAL AND PATHOPHYSIOLOGY Galanin—normal physiology. Galanin is thought to regulate numerous physiological actions in the adult mammalian nervous system, including arousal/sleep regulation, metabolism, reproduction [2, 10], nociception [17], and cognition [14]; and recent studies are helping to establish which galanin receptors are involved in these actions, with the availability of subtype-selective agonists and antagonists and transgenic mouse models,

Galanin and GALP Systems in Brain / 757 including galanin [32] and GalR1 and GalR2 genedeletion strains [9, 13]. For example, using a combination of these approaches, GalR2 has been shown to be involved in transmission in spinal cord [17] and hippocampus [20]; to mediate increased neurite outgrowth [5, 19]; and to control neuronal survival and neurogenesis in injured hippocampus [5, 20]. In light of the number of putative physiological actions of galanin, details of the many supporting studies are beyond the scope of this chapter, but this section will provide a brief review of galanin actions in some of these central processes, based on data from rats and normal and transgenic mice. Learning and memory. Many studies implicate galanin in learning and memory. Central injection of galanin impairs the formation but not the retrieval of memory— decreasing the ability of rats to perform learning tasks such as the Morris water maze. The GalR1 antagonist M35, which enhances retention performance, reverses this effect. Galanin also disrupts learning and memory when injected into ventral hippocampus. Galanin coexists with acetylcholine in septal afferents to the hippocampus and lesions of the medial septum attenuate learning and memory in rats in radial and Morris water mazes and passive avoidance tasks, so it has been postulated that galanin inhibits cognition by reducing cholinergic release. Galanin injected into ventral hippocampus can also have a biphasic dose-dependent effect on spatial learning tasks, facilitating and impairing task acquisition at different doses; suggesting additional effects beyond inhibition of cholinergic transmission. The effect of galanin on learning and memory has also been studied in two types of galanin-overexpressing (OE) mice. In one strain the galanin gene is linked to the dopamine-β-hydroxylase (DβH) promoter [30], restricting galanin overexpression to (nor)adrenergic neurons; and in a second strain, the galanin gene is linked to the platelet-derived growth factor-β (PDGF) promoter, producing a wider distribution and higher levels (four- to eightfold) of galanin expression in hippocampus and cortex [15] (see [3]). DβH galanin-OE mice displayed deficits in learning and memory tests such as spatial navigation-, olfactory-, and emotionalmemory associated with attenuated depolarizationinduced glutamate release from hippocampal neurons, while measures of sensory and motor abilities and levels of extracellular norepinephrine or serotonin release in the hippocampus were normal [3, 30]. Notably, DβH galanin-OE mice are also impaired in a trace-fear conditioning paradigm with a time delay between the conditioned and unconditioned stimuli. These mice displayed a significant decrease in freezing behavior [14], reflecting a reduced ability to associate the conditioning tone with the unconditioned foot

shock. It has been proposed that trace-fear conditioning is dependent on the integrity of the amygdala and the hippocampus [14] and the behavior will initiate higher rates of neuronal firing that favor release of neuropeptides. Hence, the decreased performance of galanin-OE mice is thought to result from an increased release of galanin during the complex memory task, and reduced glutamate release. Feeding and metabolism. Galanin and active analogs stimulate food intake upon central injection into different regions of hypothalamus; and several studies have been conducted to explore the associated mechanisms, differences in acute and chronic effects, and effects on macronutrient choice. Notably, galanin KO mice are more sensitive to leptin treatment, but neither GalR1or GalR2-KO mice display any marked phenotype related to differences in body weight, feeding behavior or responses to fasting or leptin, relative to littermates [9, 13]. This topic is reviewed in the ingestive peptides section and will not be further discussed here. Osmotic regulation. Early studies of galanin dynamics in hypothalamic vasopressin neurons and the effects of hypothalamic galanin administration revealed that galanin is involved in osmotic regulation. Vasopressin is centrally involved in osmotic regulation and vasopressin-deficient and salt-loaded rats with increased plasma osmolality have reduced concentrations of galanin in the neurointermediate lobe of the pituitary and in the median eminence, suggesting increased release of galanin. Furthermore, central administration of galanin inhibits osmotically induced increases in vasopressin mRNA in the supraoptic and paraventricular nuclei and inhibits vasopressin release. Infusion of the galanin antagonist, M15 increased vasopressin mRNA in normal rats, which further suggests tonic inhibition by galanin. Galanin-LI in the supraoptic nucleus is altered in pharmacologically induced diabetes mellitus, and salt loading with 2% saline as drinking water increased galanin mRNA and GalR1 mRNA in the paraventricular/supraoptic nuclei of the rat. Water deprivation and salt loading also increased galanin binding and GalR1LI in these neurons (see [10]). These data suggest that salt loading and dehydration increase vasopressin release and increase levels of galanin, which acts as a negative feedback modulator of vasopressin release. Nociception. Galanin is involved in the regulation of nociception with evidence for galanin acting as both an inhibitory (analgesic) and excitatory (hyperalgesic) mediator (see [17]). The degree of pro- or antinociceptive action of galanin appears to depend on the acute or chronic nature of the nociceptive stimulus (see following), whether the stimulus is derived from a thermal, mechanical or chemical source, and on the concentration of galanin available to act on the nociceptive afferent nerves.

758 / Chapter 104 Galanin antagonists induce allodynia in naïve rats and in electrophysiological studies galanin reduces spinal hyperexcitability. Nonanalgesic doses of galanin strongly depress activity-dependent increases in spinal reflex magnitude after repetitive nerve activation. These data strongly suggest that galanin has analgesic properties during acute pain. Transgenic mice provide a model in which to assess the effect of the absence of galanin on nociception; and galanin KO mice have greater sensitivity to acute mechanical and thermal pain, while galanin-OE mice have reduced responses to acute nociceptive heat [32]. Galanin—pathophysiology. In pathological, chronic pain conditions such as neuropathic pain and inflammation, expression of galanin (and several other peptides) is markedly increased in nociceptive pathways in dorsal root ganglia (DRG) and spinal cord. The analgesic effect of galanin is concentration-dependent and high doses of exogenous galanin alleviate neuropathic pain behaviors following peripheral nerve injury, while low doses of galanin or GalR1/GalR2 agonists (ARM1896 or AR-M961) by intrathecal infusion increase pain sensitivity to thermal and mechanical pain [18]. Sensitivity to pain is also increased when galanin is administered chronically by intrathecal osmotic infusion. In chronic pain models, galanin KO mice display a reduced pain response and autotomy [32]. Therefore, galanin may increase the neuropathic pain response through excitatory (GalR2) galanin receptor activation. This would suggest that endogenous galanin released in response to chronic pain influences acute pain responses through activation of excitatory pathways, which differ from acute nociceptive insults in the naïve animal. This idea was supported by electrophysiological findings of increased postdischarge and wind-up in wide dynamic response neurons of rat lumbar spinal cord, after galanin administration in vivo, suggesting a pronociceptive effect. GalR2 may mediate the hyperalgesic response in chronic pain, while GalR1 mediates the analgesic effect. Changes in GalR1–3 expression in DRG neurons are suspected to underlie the shift in galanin activity in neuropathic pain, but further studies are required to elucidate this situation. Galanin is thought to play an important role in other neuropathological conditions, such as Alzheimer’s disease and epilepsy [15, 21], and galanin expression and signaling are also activated during neural injury and repair [32], suggesting trophic actions of the peptide. Seizures and epilepsy. Galanin-LI normally present in nerve fibers in the hippocampus of the rat is dramatically depleted in all hippocampal areas for up to a week after experimental stimulation of the perforant pathdentate gyrus pathway to induce self-sustaining status epilepticus (SSSE; a state of nearly continuous seizure

activity lasting for hours or days) [21]. Galanin-positive fibers reappear at a reduced density in the hippocampus, an effect caused by “release fatigue” induced by overactivation of septal (or locus coeruleus) galanincontaining projections to the hippocampus. Duration of SSSE can be significantly shortened by injection of galanin into the dentate hilus before stimulation of the perforant path, an effect reversible by injection of a GalR1 antagonist, M35. Furthermore, M35 alone promotes the establishment of seizures and prolongs their duration, indicating that galanin can affect the maintenance phase of established SSSE, possibly via GalR1 [21]. Galanin-OE mice display a retarded seizure threshold and duration during hippocampal kindling, presumably due to increased release of galanin from hippocampal mossy fibers, which interacts with presynaptic GalR2 receptors to reduce glutamate release and seizure activity [15]. Galanin-KO mice that totally lack the peptide are more susceptible to perforant-path stimulation-induced SSSE than wild-type mice, suggesting that endogenous galanin modulates the excitability of the perforant path-dentate granule cell complex and hippocampal excitability. Galanin KO mice display a similar increase in susceptibility to seizures induced by pentylenetetrazole, which acts on brainstem and medial thalamic nuclei that also contain galanin fibers and receptors. Galanin KO mice do not have spontaneous seizures [32], while GalR1 KO mice do [13]. Although the reason for the discrepancy is unknown, there are morphological differences between brains of wild-type and GalR1 KO mice, with a decrease in galanin-positive fibers in the hippocampal granule cell layer of GalR1 KO. Potentially galanin activation of GalR1 is anticonvulsant, while GalR2 activation is proconvulsant, which is consistent with spontaneous seizures in GalR1 KO mice. While in the galanin-KO both systems are nonfunctional, so tonic seizures are absent. Irrespective of this particular mechanism, galanin appears to be an anticonvulsant in wild-type mice. Alzheimer’s disease. Alzheimer’s disease is characterized by a progressive loss of cognitive function accompanied by neuronal loss in cerebral cortex, hippocampus, basal forebrain, locus coeruleus, and dorsal raphe. Alzheimer’s brain is characterized by neurofibrillary tangles and neuritic plaques composed of neurites, astrocytes, and glial cells around an amyloid core; and cholinergic cell loss in the nucleus basalis of Meynert, and a reduction in choline acetyltransferase and acetylcholinesterase actively in basal forebrain. In postmortem brain from Alzheimer’s patients, an upregulation of galanin receptor binding sites of up to 200% was observed in several layers of the hippocampal CA1 region, the stratum radiatum of CA3, the hilus of the dentate gyrus, and the substantia nigra. Increased

Galanin and GALP Systems in Brain / 759 galanin receptor expression is also evident in the central nucleus of the amygdala and the cortico-amygdaloid transition area in the early stages of Alzheimer’s disease, but levels decrease toward the end stages. Galaninpositive fibers and terminals are present in a higher density in the basal forebrain and hyperinnervate remaining cholinergic cell bodies. It has been proposed that degeneration of a collateral network induced by Alzheimer’s disease leads to upregulation of galanin production in the remaining, unaffected nerve terminals, similar to neuronal injury. By contrast, in Down syndrome, which also produces cholinergic neuron degeneration, no galanin hyperinnervation occurs. Thus, degeneration per se is not sufficient to induce galanin upregulation, an idea supported by a lack of correlation between galanin fiber hypertrophy and the level of cholinergic cell loss after lesions of the septum in rats. Interestingly, DβH galanin-OE mice display performance deficits in memory tests [30], which are quite analogous to deficits seen in Alzheimer’s disease. Based on these findings, it has been proposed that the inhibitory activity of galanin might inhibit ACh release and worsen symptoms; although more recent findings indicate otherwise. In electrophysiological studies of acutely dissociated rat cholinergic neurons from basal forebrain galanin inhibited K+-currents but not Ca2+- or Na+-currents. Hence, galanin could excite cholinergic neurons and act to augment ACh release from any remaining cells in Alzheimer brain. Thus, it is still unclear whether upregulation of galanin in Alzheimer’s is a contributing factor to the disease, the result of nerve injury, or a compensatory change to maintain cholinergic transmission. Anxiety. Research into the effects of galanin in models of anxiety was prompted by the presence of galanin in brain areas relevant to emotional behavior and initial pharmacological studies revealing that galanin injected into the amygdala and the BST produced an anxiolytic effect but no effect on locomotor activity, analgesia, thirst or appetitive motivation, which may have otherwise influenced the responses seen in the Vogel punished-drinking test [24]. Neuroendocrine function was modulated, however, with a decrease in plasma ACTH observed in the stressed rats. Depressive disorders, recognizable by depressed moods, associated social dysfunction, and increased risk of suicide, have been thought of as involving biogenic amine imbalances in areas such as the locus coeruleus, as monoamine oxidase- and monoamine reuptakeinhibitors have antidepressant actions. Thus injection of yohimbine, a presynaptic α2-adrenoceptor antagonist, has been used as a model of anxiety in mice and rats; and both yohimbine and immobilization have an anxiogenic effect on rats. Notably, central administra-

tion of galanin had an anxiolytic effect, which was blocked by the galanin antagonist, M40. In line with this, DβH galanin-OE mice had no response to the proanxiety effects of yohimbine, presumably due to a decrease in norepinephrine or glutamate release in regions mediating anxiety-like behaviors, such as amygdala and hippocampus. Surprisingly, PDGF-β galanin-OE mice displayed increased hippocampal norepinephrine and serotonin release after a forced swimming stress, which is more consistent with increased anxiety. However, it was argued that any additional upregulation of galanin by stressors in these galanin-OE mice did not reduce serotonin or norepinephrine levels because galanin receptors were already fully occupied due to higher basal galanin levels. Hence, galanin appears to be anxiolytic in the mouse and rat. Neural injury and repair. Galanin expression is strongly activated by neural injury in several models and recent data suggests GalR2 is primarily involved in these regenerative functions in DRG and hippocampus [5, 19]. DRG neurite outgrowth was similar in cells from GalR1 KO and wild-type mice and was not altered by a GalR1 antagonist, indicating that the regenerative capabilities of DRG neurons are GalR1-independent [19]. A selective GalR2 agonist, AR-M1896, restored neurite outgrowth in galanin-KO neurons, with equal potency to galanin [19] and this in vitro data is consistent with an upregulation of GalR2 but not GalR1 mRNA in the facial nucleus after crush injury of the facial nerve. Galanin has also been implicated in control of neuronal survival and neurogenesis in normal/injured brain [5, 20, 29]. GALP—normal physiology. Since its recent discovery there have only been around 40 articles published on GALP biology, but most of these reports, including recent reviews [4, 8, 10], have provided consistent anatomical, physiological, and pharmacological evidence for its potential role in affecting and integrating metabolism and reproduction via actions in the hypothalamus. Initially it was reported that central GALP infusion altered feeding in rats (acute stimulation and subsequent inhibition) and mice (inhibition only). Studies of acute and chronic GALP infusion in leptin-deficient ob/ob obese mice revealed that acute GALP induced a long-lasting (4 days) decrease in food intake and body weight (BW), while chronic GALP produced a sustained decrease in BW and an increase in core body temperature, despite significant recovery of food intake. In a pair-fed model, chronic GALP treatment resulted in a decrease in BW and increased body temperature and thermogenesis in brown adipose tissue, suggesting that leptin’s activation of the sympathetic nervous system and ultimately thermogenesis may be partially mediated by GALP [8]. GALP is also present in the

760 / Chapter 104 gastrointestinal tract [26], and levels of immunoreactive GALP in blood are reportedly reduced by food deprivation. Fasting also decreased a rapid blood-tobrain influx of intact GALP that was increased by glucose treatment. In relation to the reproductive axis, central infusion of GALP (5 nmol), but not galanin, in male rats activated GnRH neurons and increased plasma luteinizing hormone (LH) levels for up to 60 min posttreatment, without altering plasma levels of other pituitary hormones. Furthermore, the LH response was blocked by pretreatment with a GnRH receptor antagonist. GALP also stimulated LH and testosterone secretion in the mouse. In vitro studies demonstrated that GALP induced GnRH release from rat hypothalamic explants and GALP antiserum inhibited leptin-induced GnRH release (see [8]). GALP was also recently shown to increase male sexual behavior, whereas galanin inhibited it; and the effect of GALP was maintained in castrated animals, suggesting effects independent of testosterone secretion [7]. Together these findings indicate that GALP is an important mediator of the physiological effects of leptin on GnRH secretion and the reproductive axis [8]. GALP—pathophysiology. The absence of leptin signaling in obese Zucker rats and hypoleptinemia in streptozotocin-induced diabetic (STZ-DM) rats is associated with decreased hypothalamic expression of GALP. The downregulation of hypothalamic GALP and the upregulation of NPY may act in concert to promote hyperphagia in these animals. Current findings are consistent with a tonic influence of leptin receptor signaling on hypothalamic GALP expression under normal conditions and possible abnormalities in GALP neuronal signaling and their putative targets—TRH and GnRH neurons—under pathological conditions such as diabetes and obesity [8].

References [1] Branchek TA, Smith KE, Gerald C, Walker MW. Galanin receptor subtypes. Trends Pharmacol Sci 2000;21:109–16. [2] Crawley JN. The role of galanin in feeding behavior. Neuropeptides 1999;33:369–75. [3] Crawley JN, Mufson E, Hohmann J, Teklemichael D, Steiner R, Holmberg K, et al. Galanin overexpressing transgenic mice. Neuropeptides 2002;36:145–56. [4] Cunningham MJ, Krasnow SM, Gevers EF, Chen P, Thompson CK, Robinson IC, et al. Regulation of galanin-like peptide gene expression by pituitary hormones and their downstream targets. J Neuroendocrinol 2004;16:10–8. [5] Elliott-Hunt CR, Marsh B, Bacon A, Pope R, Vanderplank P, Wynick D. Galanin acts as a neuroprotective factor to the hippocampus. Proc Natl Acad Sci USA 2004;101:5105–10. [6] Fraley GS, Scarlett JM, Shimada I, Teklemichael DN, Acohido BV, Clifton DK, et al. Effects of diabetes and insulin on the expression of galanin-like peptide in the hypothalamus of the rat. Diabetes 2004;53:1237–42.

[7] Fraley GS, Thomas-Smith SE, Acohido BV, Steiner RA, Clifton DK. Stimulation of sexual behavior in the male rat by galaninlike peptide. Horm Behav 2004;46:551–7. [8] Gottsch ML, Clifton DK, Steiner RA. Galanin-like peptide as a link in the integration of metabolism and reproduction. Trends Endocrinol Metab 2004;15:215–21. [9] Gottsch ML, Zeng H, Hohmann JG, Weinshenker D, Clifton DK, Steiner RA. Phenotypic analysis of mice deficient in the type 2 galanin receptor (GALR2). Mol Cell Biol 2005;25:4808–11. [10] Gundlach AL. Galanin/GALP and galanin receptors: role in central control of feeding, body weight/obesity and reproduction? Eur J Pharmacol 2002;440:255–68. [11] Gundlach AL. GAL1, GAL2, GAL3 galanin receptors. In: Enna SJ, Bylund DB, eds. http://www.xpharm.com/citation?Article ID=200/189/190. New York: Elsevier, 2004. [12] Jacobowitz DM, Kresse A, Skofitsch G. Galanin in the brain: chemoarchitectonics and brain cartography—a historical review. Peptides 2004;25:433–64. [13] Jacoby AS, Hort YJ, Constantinescu G, Shine J, Iismaa TP. Critical role for GALR1 galanin receptor in galanin regulation of neuroendocrine function and seizure activity. Mol Brain Res 2002;107:195–200. [14] Kinney JW, Starosta G, Holmes A, Wrenn CC, Yang RJ, Harris AP, et al. Deficits in trace cued fear conditioning in galanintreated rats and galanin-overexpressing transgenic mice. Learn Mem 2002;9:178–90. [15] Kokaia M, Holmberg K, Nanobashvili A, Xu ZQ, Kokaia Z, Lendahl U, et al. Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin. Proc Natl Acad Sci USA 2001;98:14006–11. [16] Larm JA, Gundlach AL. Galanin-like peptide (GALP) mRNA expression is restricted to arcuate nucleus of hypothalamus in adult male rat brain. Neuroendocrinology 2000;72:67–71. [17] Liu H, Hökfelt T. The participation of galanin in pain processing at the spinal level. Trends Pharmacol Sci 2002;23:468–74. [18] Liu HX, Brumovsky P, Schmidt R, Brown W, Payza K, Hodzic L, et al. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: Selective actions via GalR1 and GalR2 receptors. Proc Natl Acad Sci USA 2001;98: 9960–4. [19] Mahoney SA, Hosking R, Farrant S, Holmes FE, Jacoby AS, Shine J, et al. The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons. J Neurosci 2003;23:416–21. [20] Mazarati A, Lu X, Kilk K, Langel U, Wasterlain C, Bartfai T. Galanin type 2 receptors regulate neuronal survival, susceptibility to seizures and seizure-induced neurogenesis in the dentate gyrus. Eur J Neurosci 2004;19:3235–44. [21] Mazarati AM, Liu H, Soomets U, Sankar R, Shin D, Katsumori H, et al. Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J Neurosci 1998;18:10070–7. [22] Mennicken F, Hoffert C, Pelletier M, Ahmad S, O’Donnell D. Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system. J Chem Neuroanat 2002;24:257–68. [23] Merchenthaler I, Lopez F, Negro-Vilar A. Anatomy and physiology of central galanin-containing pathways. Prog Neurobiol 1993;40:711–69. [24] Möller C, Sommer W, Thorsell A, Heilig M. Anxiogenic-like action of galanin after intra-amygdala administration in the rat. Neuropsychopharmacology 1999;21:507–12. [25] O’Donnell D, Ahmad S, Wahlestedt C, Walker P. Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: Distinct distribution from GALR1. J Comp Neurol 1999;409:469– 81.

Galanin and GALP Systems in Brain / 761 [26] Ohtaki T, Kumano S, Ishibashi Y, Ogi K, Matsui H, Harada M, et al. Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus. J Biol Chem 1999;274:37041– 5. [27] Rökaeus Å, Jiang K, Spyrou G, Waschek JA. Transcriptional control of the galanin gene. Tissue-specific expression and induction by NGF, protein kinase C and estrogen. Ann NY Acad Sci 1998;863:1–13. [28] Shen J, Larm JA, Gundlach AL. Galanin-like peptide mRNA in neural lobe of rat pituitary. Increased expression after osmotic stimulation suggests a role for galanin-like peptide in neuronglial interactions and/or neurosecretion. Neuroendocrinology 2001;73:2–11.

[29] Shen PJ, Larm JA, Gundlach AL. Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression. Eur J Neurosci 2003;18:1362–76. [30] Steiner R, Hohmann J, Holmes A, Wrenn C, Cadd G, Jureus A, et al. Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer’s disease. Proc Natl Acad Sci USA 2001;98:4184–9. [31] Takatsu Y, Matsumoto H, Ohtaki T, Kumano S, Kitada C, Onda H, et al. Distribution of galanin-like peptide in the rat brain. Endocrinology 2001;142:1626–34. [32] Wynick D, Bacon A. Targeted disruption of galanin: new insights from knock-out studies. Neuropeptides 2002;36:132–44.

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Inference was drawn from the detection of immunoreactive tachykinin-like activity in mammalian tissues other than SP. In addition, eledoisin and physalaemin, two tachykinins isolated in the early 1960s from the Mediterranean octopus and the South American frog, respectively, were found to produce greater pharmacological responses on mammalian tissues than SP. In 1983, these suppositions gave way to the confirmation of the existence of two further members, both isolated from the porcine spinal cord and designated neurokinin A (NKA) and NKB. Two biologically active Nterminally extended forms of NKA—neuropeptide K (NPK) and neuropeptide gamma (NPγ)—were also later discovered in the mid-1980s. More recently (2000 onward), additional species-divergent members have been discovered, including hemokinin-1 (HK-1) in mice and rats, endokinin-1 (EK-1) in rabbits, and endokinin A (EKA) and EKB in humans [2]. Furthermore, in 2003–2004, tachykinin gene-related peptides were identified in rabbit, EK-2, and in humans, EKC and EKD.

ABSTRACT The mammalian tachykinins constitute a family of peptides that all share the common C-terminal sequence FXGLM-NH2 and that act as neurotransmitters and neuromodulators in the brain. They mediate their biological actions through interactions with three known tachykinin receptors (NK1, NK2, and NK3) that are located widely and in distinct locations in the brain. They are implicated in the control of several autonomic, affective, and higher cerebral functions and in the pathophysiology of some neurogenerative and psychiatric disorders.

DISCOVERY The history of the tachykinins can be traced back to 1931 with the discovery of substance P (SP) by von Euler and Gaddum. A preparation (and hence the “P” of SP) was purified from equine brain and intestine that was found to cause the vasodilatation and stimulation of gut mobility in rabbits. Numerous attempts to isolate the pure form of SP over the next 40 years were unsuccessful. Nonetheless, in 1967, a peptide named sialogen that stimulated salivation was discovered in the bovine hypothalamus, and in the following year Lembeck and Starke suggested that this peptide might be the same as SP. In 1970, upon purification and comparison of sialogen’s biological properties with those of impure preparations of SP, it was realized that these two peptides were indeed the same. This enabled the chemical structure of SP to be determined as that of an amidated undecapeptide (Table 1), allowing its chemical synthesis and enabling the production of a specific radioimmunoassay. Over the next 10 years, SP was believed to be the only mammalian tachykinin even though there were indications that this might not be entirely the case. Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The mammalian tachykinins are encoded on three different genes, termed preprotachykinin 1 (TAC1), TAC3, and TAC4 (Fig. 1). TAC1, the first to be cloned in 1983 from bovine brain, was initially believed to be two different genes, one containing SP (TAC1) and the other also containing NKA (TAC2). In the following year, it was realized that a discrete genomic segment encoded NKA by alternative RNA splicing of the same gene to yield αTAC1 and βTAC1. The βTAC1 splice transcript was determined to be encoded by seven exons with exon 3 and exon 6 containing SP and NKA, respectively. Exon 6 is lacking from αTAC1. Existence of a third TAC1 precursor was found first in the rat with the

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764 / Chapter 105 TABLE 1. Amino acid sequences of the mammalian tachykinins. Tachykinins SP NKA NPK NPγ NKB EKA EKB EK-1 HK-1

Peptide Sequence RPKPQQFFGLM-NH2 HKTDSFVGLM-NH2 DADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLM-NH2 DAGHGQISHKRHKTDSFVGLM-NH2 DMHDFFVGLM-NH2 DGGEEQTLSTEAETWVIVALEEGAGPSIQLQLQEVKTGKASQFFGLM-NH2 DGGEEQTLSTEAETWEGAGPSIQLQLQEVKTGKASQFFGLM-NH2 GKASQFFGLM-NH2 SRTRQFYGLM-NH2

elucidation of a sequence corresponding to the precise deletion of exon 4 (γTAC1). In addition, a fourth splice variant δTAC1 lacking both exon 4 and exon 6 was identified. In essence, the alternative splicing of exon 6 of TAC1 produces either SP alone (α, δTAC1) or SP along with NKA (β, γTAC1). The differential inclusion or exclusion of exon 4 into β and γTAC1 allows the potential formation of the two extended NKA peptides, either NPK or NPγ, respectively. Marked species dependent differential expression of the TAC1 splice variants has been found, although within a given species no established evidence of tissue-specific alternative splicing has yet been reported. The gene organization for TAC3 encoding NKB, like TAC1, is composed of seven exons with exon 5 encoding the sequence for NKB. In the bovine mRNA two TAC3 transcripts have been reported that differ only at the 5′ extremity of their untranslated regions. In humans, two TAC3 transcripts (α, βTAC3) are found that differ in their C-terminal coding region. Nevertheless, a thorough investigation of TAC3 has concluded that it encodes only the single tachykinin. The gene structures of TAC1 and TAC3 are highly conserved across the mammalian species when compared with the highly species-divergent structure of TAC4. Mouse TAC4 is encoded by four exons, rat by five, of which exon 2 in both encodes HK-1. In the rabbit, alternative splicing produces α and βTAC4, whereas the alternative splicing of exons 3 and 4 in humans produces α-, β-, γ-, and δTAC4. These splicing events allow either a tachykinin (EK-1, EKA, or EKB) to be synthesized alone or in conjunction with one of the tachykinin gene-related peptides (EK-2, EKC, or EKD). The tachykinin gene-related peptides were not predicted by the mouse or rat genome.

DISTRIBUTION OF THE mRNA In the brain, distribution of the tachykinins has been most extensively studied in the rat, where widespread

and distinct regions containing labeled TAC1 and TAC3 neurons are found [1]. In the case of rat TAC1, these are in the anterior olfactory nucleus, olfactory tubercle, the islands of Calleja, the nucleus accumbens, the caudate-putamen, amygdala, hypothalamus, the medial habenular nucleus, nuclei of the pontine tegmentum, raphe nuclei, portions of the reticular formation, and nucleus of the solitary tract. The characterized positions of the SP and NKA containing cell groups and fibers in the rat brain are shown in Fig. 2, where it is generally believed that SP is co-synthesized, colocalized, and co-secreted with NKA. Levels of TAC1 have also been measured during rat brain development from embryonic day 15 to adult, indicating significant changes in region- and time-specific regulation of the TAC1 gene, particularly in the cingulate cortex. In the case of rat TAC3, neurons are located in the cerebral cortex, the hippocampal formation, the amygdaloid complex, the bed nucleus of the stria terminalis, the ventral pallidum, the habenula, the medial preoptic area, the arcuate nucleus, and the lateral mammillary bodies. In human brain, there is a distinct and complementary distribution pattern of TAC1 and TAC3 neurons. TAC3 is the predominant tachykinin in the rostal hypothalamus, whereas TAC1 predominates in the posterior hypothalamus. Numerous TAC3 neurons are identified in the magnocellular basal forebrain, the bed nucleus of stria terminalis, and the anterior hypothalamic area with scattered neurons in the infundibular and paraventricular nuclei, paraolfactory gyrus, posterior hypothalamic area, lateral division of the medial mammillary nucleus, and amygdala. Numerous neurons expressing TAC1 are identified in the premammillary, supramammillary, and medial mammillary nuclei; the posterior hypothalamic area; and the corpus striatum. Scattered SP neurons are also observed in the preoptic area; the infundibular, intermediate, dorsomedial, and ventromedial nuclei; the infundibular stalk; the amygdala; the bed nucleus of stria terminalis; and the paraolfactory gyrus. The expression levels for TAC1

Brain Tachykinins / 765 TAC1 Human

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and TAC3 have been translated into respective amounts of immunoreactive peptides by immunoassay, which have proved largely confirmatory of the mRNA expression sites. In the different regions of rat brain, the amounts of SP are generally greater than those of NKA, which are in turn greater than those for NKB. TAC4 has been found predominantly expressed in the periphery. Nevertheless, analysis of numerous regions of the murine brain (including the brain stem, cerebellum, cortex, caudate, hippocampus, middle brain, hypothalamus, and thalamus) has revealed a similar regional distribution to that for brain TAC1, although it is present at consistently (around 100-fold) lower levels.

PROCESSING OF THE PRECURSOR The tachykinin peptides are synthesized as large precursor proteins that undergo posttranslational proteolytic processing to generate the mature peptide products. It is alternative mRNA splicing that leads to the distinct tachykinin precursor preteins. Each precursor has a strongly hydrophobic signal peptide sequence at its N-terminus, responsible for translocating it to the endoplasmic reticulum (ER). Cleavage of this signal sequence in the ER forms the precursor, which is further cleaved, normally at dibasic (or monobasic) amino acid residues by prohormone convertases to yield the smaller tachykinin peptides. Each tachykinin is typically flanked

upstream (except human EKA and EKB) and downstream by potential dibasic cleavage sites, and all possess an adjacent glycine at their C-terminus for amidation. Typically, following prohormone convertase action, a carboxypeptidase removes the C-terminal dibasic residues and a peptidylglycine α-amidating enzyme converts the exposed glycine into a C-terminal amide. Intermediate products of this process, including SPglycine and SP-glycine-lysine, can be found throughout the rat brain, where levels of these extended products are lowest in the amygdala and highest in the medulla. Such differences are believed to be an indicator of different rates of tachykinin synthesis coupled with utilization within the different brain regions. However, the most significant differences in tachykinin processing are found at their N-termini. In SP, where the Nterminal dibasic cleavage site is adjacent to a proline residue and because of the resistance of arginine-proline bonds to prohormone converting enzymes, an arginine residue from the dibasic cleavage site is exceptionally maintained. In addition, in the case of SP, many studies have shown further processing can occur including the production of SP(1-4) and SP(1-7), which are biologically active SP metabolites. There is also evidence of differential processing at the N-terminal dibasic cleavage site of NKA with such processing responsible for the production of both NPK and NPγ. Indeed, early evidence from the late 1980s noted that, although SP cleavage appeared to be easily handled, cleavage of the different NKA tachykinins might involve multiple

Brain Tachykinins / 767 enzymes, it is indicated by the fact that multiple peptides were derived from the NKA portion of the TAC1 precursor. Likewise, the apparent loss of the N-terminal dibasic processing site of EKA and EKB with a monobasic cleavage site could be due to the evolutionary pressure of differential processing.

RECEPTORS AND SIGNALING CASCADES In the early 1980s, following an intense pharmacological investigation, the existence of three tachykinin receptors was proposed—termed NK1, NK2, and NK3 [3]. Their existence was confirmed in the late 1980s with the cloning of each subtype from a number of different mammalian species. The receptors all constitute members of the G-protein-coupled receptor family and are encoded on five exons. For example, the NK1 receptor contains seven putative hydrophobic α-helical transmembrane domains in which the first two extracellular loops are joined by a disulfide bridge. Its extracellular N-terminus contains two predicted asparagine glycosylation sites, and its intracellular C-terminal tail contains predicted sites of palmitoylation and a series of potential serine and threonine phosphorylation sites. The order of potency of the tachykinin receptors for each tachykinin is as follows: NK1, SP = HK-1 = EKA/B ≥ NKA > NKB; NK2, NKA > NKB > SP = EKA/B and NK3, NKB > NKA > SP = EKA/B. NPK and NPγ preferentially bind to the NK2 receptor. However, there is significant opportunity for the cross-selectivity of each receptor subtype with each different tachykinin, owing to their common activating C-terminal motif, allowing them to act as full agonists at each receptor. Overall, the signaltransduction mechanisms of the three tachykinin receptors have been studied in transfected Chinese hamster ovary (CHO) cells. Here, they all markedly activate phospholipase C-β via a pertussis-toxin-insensitive Gprotein (Gαq and/or Gα11), leading to the hydrolysis of membrane-bound inositol phospholipids. Receptor activation can also activate the arachidonic acid cascade; however, the activation of adenylate cyclase is less efficient and requires approximately one order of magnitude higher concentrations of each tachykinin. In the central nervous system, binding sites for NKA were reported despite the finding that NK2-receptor mRNA transcripts could not be located. This enigma eventually gave way to the realization that alternative conformations of the NK1 receptor existed. In CHO cells overexpressing the NK1 receptor, classic NK1-receptor binding sites were found coupled to adenylate cyclase, whereas septide-sensitive NK1-receptor sites were found coupled to phospholipase D. At classic NK1 binding sites, SP was found to be the only endogenous tachykinin to display high affinity, whereas NKA, NPK, NPγ, and NKB instead

exhibited a high affinity for the septide-sensitive NK1 binding site. However, in vivo, classic NK1-receptor binding sites are associated with phospholipase C and rarely with adenylate cyclase. Consequently, each NK1 receptor subtype represents a different receptor conformation that can be coupled to a distinct transduction system with the capacity to activate different functions. Furthermore, the NK1 receptor also exists as a truncated isoform that is missing its C-terminal tail; this possesses different receptor-binding efficiencies and effector system interactions. The untruncated NK1 isoform is the most prevalent throughout the human brain, whereas in peripheral tissues the truncated isoform is the most represented.

ACTIVE AND SOLUTION CONFORMATION The three-dimensional structures of the tachykinins have been derived by nuclear magnetic resonance and circular dichroism. In water, SP presents, in addition to an aggregation response, a complex conformational equilibrium that represents an extended chain structure that is distinct from the completely unfolded peptide. In contrast, the addition of sodium dodecyl sulfate to the aqueous solution induces a preferred α-helical conformation representing 10–30% of the peptide population. However, in dodecylphosphocholine micelles, an amphiphilic helical conformation is induced in the midregion of SP that is adopted by most of the SP peptide population. This conformation is believed to yield a structural motif typical of many selective NK1 ligands. In water, NKA, like SP, prefers to be in an extended chain formation where, again, a helical conformation is predominantly induced in the central core and the C-terminal region in the presence of dodecylphosphocholine micelles. The N-terminal of NKA, although less defined, also displays some order and a possible turn structure. The extended forms of NKA, NPK, and NPγ also display the same helical conformation induced in the central core and the C-terminal region in the presence of dodecylphosphocholine micelles. In addition, in NPγ a β-turn has been found to precede the helical core, whereas NPK has been found to adopt a well-defined helical structure in its N-terminal domain and possess a relatively disordered C-terminal domain. NKB also forms a helical structure in the presence of dodecylphosphocholine micelles. The overall induction of a helical conformation in the midregion of each of the tachykinins appears to be crucial for tachykinin receptor activation. Selectivity for each receptor has been attributed to changes in the helix length having an effect on the distribution of the hydrophobic and hydrophilic extremes of the tachykinin peptides.

768 / Chapter 105 BIOLOGICAL ACTIONS WITHIN THE BRAIN There is substantial evidence that in the brain the tachykinins play very important roles as neurotransmitter, neuromodulatory, and neurotrophic agents. They occur particularly in large quantities in the areas involved in the control of several autonomic and endocrine functions, of affective and emotional responses, and of higher cerebral functions. They are also often co-localized with other neurotransmitters such as dopamine, endomorphin, and serotonin. The biological actions of the tachykinins have been recorded in a number of different brain regions, and some of the more defined are summarized here. 1. In the cerebral cortex, SP has been found to excite 91% of the spontaneously active neurons tested. 2. In the striatum, tachykinins are released by high potassium concentrations. SP has been reported to increase the firing of some striatal neurons and to evoke the release of dopamine, Met-enkephalin, and acetylcholine. 3. In the globus pallidus, microinjection of tachykinin antagonists into the entopeduncular nucleus has been shown to reduce muscle tone in genetically spastic rats, suggesting that SP-dependent mechanisms can regulate muscle tone. 4. In the substantia nigra, SP is released in a calciumdependent manner in response to depolarizing stimuli and on electrophoretic application to produce long-lasting excitation. SP infusion into the substantia nigra also produces marked increases in stereotyped behaviors such as grooming, sniffing, and rearing. SP has also been suggested to have an excitatory regulation on nigrostriatal dopamine release. NKA is also believed to exert potent excitatory action on nigral dopaminergic and nondopaminergic neurons. 5. In the ventral tegmental area, a close relationship between dopamine and SP has been suggested. SP infusion into the ventral causes a marked increase in locomotion and rearing that can be completely blocked by infusion of dopamine antagonists. An important role for NKA in the ventral tegmental area has also been suggested because it has been found to be 10 times more potent than SP in producing an increase in motor activity. 6. In the nucleus accumbens, where evidence suggests that tachykinins can modulate dopamine release through presynaptic mechanisms, dopamine neurons richly innervate. The activation of this pathway is believed to cause an increase in locomotor activity. In addition, SP injection into this nucleus exerts memory-promoting effects.

7. In the limbic system, the hippocampus and amygdala have been implicated in memory processing, where NKA and NPK administered intracerebroventricularly after footshock avoidance training enhanced memory attention in mice. Localization studies also showed that NPK improved memory when injected directly into the hippocampus and amygdala. The amygdala has also been implicated in the regulation of sexual behavior where higher levels of SP are found in male rather than female rats and where the castration of adult rats reduces amygdala SP staining. It has also been suggested that the medial amygdala is a site of action for a NK3-mediated mechanism to inhibit salt intake. 8. In the hypothalamo-hypophysial system, SP may be secreted directly into the portal circulation to reach the anterior pituitary. In doing so, SP may affect the release of anterior pituitary hormones. Such released SP is believed to inhibit the release of rat growth hormone, whereas SP antagonists increase the concentrations of plasma growth hormone. Also, SP applied to pituitary cells exerts a stimulant effect on prolactin secretion. Furthermore, a number of biological actions of the tachykinins in the brain are demonstrated by direct intracerebroventricular injection. These are summarized next. 1. Cardiovascular responses promoted by SP and NKA typical of a defense reaction that increases blood pressure, heart rate, sympathetic efferent activity, visceral vasoconstriction, and hindlimb vasodilation. These responses are accompanied by behavioral defense reactions including increased locomotion, scratching, skin biting, and grooming. 2. Dose-related inhibition of gastric secretion, gastric emptying, and colonic propulsion with NK3 agonists. 3. Drinking behavior evoked by angiotensin II intracerebroventricular injection, which acts by inhibiting water uptake and causing water deprivation and cell dehydration, affected by the administration of eledoisin. Injection of eledoisin also causes the release of vasopressin with ensuing antidiuresis and independently displays a potent and lasting inhibitory effect on salt uptake. 4. Changes in respiration caused by the dosedependent administration of SP leading to minute ventilation, due to increased volume stimulated respiration. 5. An increase in anxiety with the infusion of NK1 agonists causing long-lasting and pronounced vocalization in guinea pigs.

Brain Tachykinins / 769 6. Behavior affected by the administration of SP and some of its N-terminal-derived peptides. SP(1-7) inhibited not only nociception but also aggressive and grooming behavior, while, like SP, stimulating investigative motor behavior.

PATHOPHYSIOLOGICAL IMPLICATIONS The tachykinins have been implicated in a number of centrally mediated disease states. These have included, among others, the neurodegenerative disorders of Parkinson’s and Alzheimer’s diseases. Parkinson’s disease, a serious motor disorder, is caused by the degeneration of dopamine neurons in the substantia nigra. However, there are lines of evidence indicating a role for the tachykinins in its pathogenesis. First, decreases of TAC1 and SP immunoreactivity are found in the nigral and striatal regions of animal and postmortem Parkinson’s disease patients. Second, the tachykinins have been found to exert neuroprotective effects on neurons. In addition, an abundance of NK1 and NK3 receptors are localized in the dopaminergic neurons of the basal ganglia, indicating these neurons are under the physiological regulation of the tachykinins. Furthermore, in an animal model of Parkinson’s disease, tachykinin receptor agonists were found to modulate motor activity. In the case of Alzheimer’s disease, SP function has been related to memory and learning, where it is highly expressed in several brain areas from the forebrain to hindbrain. In this regard, the basal forebrain, particularly the nucleus of Meynert, has received attention because it is these SP-containing nuclei that have been shown to degenerate in the senile dementia of Alzheimer’s disease. SP has also been found to interact with the cholinergic ascending system of the nucleus of Meynert, and patients with Alzheimer’s disease show a marked loss of cholinergic neurons from the nucleus of Meynert to the cortex and diminished brain SP immunoreactivity. SP has also received particular focus in its role in psychiatric disorders. This rationale has been based on the observation that, like clinically used antidepressant/anxiolytic drugs, SP antagonists suppress anxiety-related behaviors and defensive cardiovascular changes in rodents. In addition, mice with selective deletions of the gene encoding, the NK1 receptor, or SP itself showed decreased anxiety-related behaviors. The mechanism by which SP antagonists bring about their effects is not fully understood, but SP is found in close association with 5-hydroxytryptamine and norepinephrine-containing neurons that are targeted

by currently used antidepressant drugs. The NK1 antagonist MK-869 was recently shown to display antidepressant activity comparable to that of the selective serotonin reuptake inhibitor paroxetine but with a better sideeffect profile. However, further research is needed to ascertain the relevance of NK1 antagonists in depression following the subsequent discontinuation of MK-869 from Phase III trials, after it was shown to be no more effective than placebo. Nevertheless, there still remains an interest in the role of SP in psychiatric illnesses, which also extends to conditions such as panic disorder, obsessive-compulsive disorder, generalized anxiety disorder, and posttraumatic stress disorder. Interestingly, MK-869 has recently received regulatory approval for use as an anti-emetogenic therapy in cancer chemotherapy patients along with a combination of 5-HT3 antagonist and dexamethasone. SP is found in the neurons of the brain stem and is believed to suppress the centrally controlled vomiting reflex. Because the tachykinins are well known for producing marked behavioral changes in animals, they are plausible candidates for playing roles in other pathological behavioral states, including schizophrenia and bipolar mood disorders. Changes in brain levels of SP and SP-binding sites have been implicated in schizophrenia. Significant reductions of TAC1 mRNA expression are detected in the basal, lateral, and accessory basal amygdaloid nuclei in patients with schizophrenic or bipolar disorder. It has been hypothesized that the involvement of the tachykinins in mood disorders and schizophrenia suggests a generalized impairment of the SP system in the amygdala. NK1 antagonists have also been used in the study of acute migraine, where it is believed that SP is a mediator of pain and inflammation through the NK1 receptor. SP binding to postcapillary NK1 receptors in the brain vasculature is believed to produce plasma extravasation, platelet aggregation, and white cell endothelial adhesion. These effects could mediate both the inflammatory process and pain reported in migraine. Furthermore, NK1 antagonists are very effective in animal models of inflammation that are thought to be indicators of activity in acute migraine.

References [1] Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;73:229–308. [2] Page NM. New challenges in the study of the mammalian tachykinins. Peptides 2005;26:1356–1368. [3] Regoli D, Drapeau G, Dion S, D’Orleans-Juste P. Pharmacological receptors for substance P and neurokinins. Life Sci 1987;40:109– 117.

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106 CGRP and Adrenomedullin in the Brain NANDA TILAKARATNE AND PATRICK M. SEXTON

was later named CGRP-α (or CGRP-I) to distinguish it from another related peptide CGRP-β (or CGRP-II). CGRP-α and CGRP-β differ from one another by only a single amino acid in rats and by three residues in humans. CGRP-β is encoded by a gene distinct from the CALCA gene. The following discussion is limited to CGRP-α. In 1993, Kitamura and colleagues discovered a peptide that was able to stimulate cyclic AMP (cAMP) production in blood platelets [12]. The 52-amino-acid peptide was isolated from an extract of a pheochromocytoma—a tumor of the adrenal medulla. Hence, the peptide was named adrenomedullin (AM). However, AM is also produced by healthy adrenal medulla and a wide range of other tissues, including vascular endothelium, vascular smooth muscle, and the central nervous system. Although AM and CGRP share only a 30% sequence homology, the two structures required for biological activity—a C-terminal aromatic amide and a ring structure made up of six amino acids linked by disulfidebonded cysteines—are well conserved between the two peptides. These features are also present in the related peptides, CT, amylin, and intermedin (AM2) and there is a degree of cross-reactivity of the peptides across their target receptors [7, 20].

ABSTRACT Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are two structurally related peptides. They are synthesized and secreted by many mammalian tissues, including the central nervous system. The two peptides, which were initially described as vasodilators, are now considered as multifunctional regulatory agents. Their cellular actions are mediated by pharmacologically distinct receptors, each comprising two subunits, a classic seven-transmembrane-spanning G-protein-coupled receptor (GPCR) and one of three single-transmembrane-spanning accessory receptor activity-modifying proteins (RAMP). Two GPCRs, namely the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR), contribute to CGRP/AM receptor phenotypes. In the brain, CGRP and AM are widely expressed and have been shown to activate, in addition to the vasodilator effect, several autonomic centers involved in the regulation of fluid and electrolyte balance. In addition, they have modulatory effects on the synthesis and release of several other neuropeptides. The potent vasodilator activity of CGRP in cerebral microvasculature is possibly relevant to the pathology of migraine.

DISCOVERY STRUCTURE OF THE PRECURSOR mRNA/GENE

Discovered in 1983, calcitonin gene-related peptide (CGRP) was so named because the peptide is a product of the same gene, CALCA, that encodes calcitonin (CT)—a thyroidal hormone that regulates bone metabolism. Rosenfeld and colleagues found that the CT gene transcript, which yields CT in thyroidal C cells, is alternately spliced in brain cells to produce CGRP [21]. CGRP is a 37-amino-acid peptide that shares 55% amino-acid-sequence homology with CT. This peptide Handbook of Biologically Active Peptides

The CT/CGRP cDNAs have been cloned from several species. In humans, the genomic copy is localized to chromosome 11 (11p15.2-p15.1; Fig. 1). The gene spans a region of approximately 6.3 kilobases (kb) and comprises six exons. As mentioned previously, the primary transcript undergoes tissue-specific alternate splicing. In the brain, the transcript is princi-

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pally spliced to yield CGRP mRNA; it is formed by the splicing of exon I (5′ noncoding) with exons II, III, and V, which form the coding region, and exon VI (3′ noncoding) (Fig. 1). In normal thyroid, the splicing involves exons I, II, III, and IV to form the CT mRNA. The CALCB gene encoding CGRP-β is also localized to chromosome 11 [25, 28]. Human AM is encoded by a gene also localized to chromosome 11 (11p15.4). The gene spans approximately 2.1 kb of DNA and consists of four exons and three introns

(Fig. 1) [9]. The 5′ flanking region contains several elements such as TATA, CAAT, and GC boxes, required for its functional expression. There are also consensus transcription-factor binding sites for activator protein-2 (AP-2) and a cAMP-regulated enhancer element, suggesting that gene expression may be subject to regulation by protein kinase C (PKC) and cAMP [10]. The AM gene codes for two bioactive peptides, AM and pro-adrenomedullin-Nterminal 20 peptide (PAMP), which are released from a common precursor protein (Fig. 1).

CGRP and Adrenomedullin in the Brain / 773 motor nuclei (III, IV, V, VI, VII, XII) in the hindbrain also exhibit CGRP immunoreactivity. CGRP is also detected in cell bodies in the parabrachial nuclei; the ventral tegmental nucleus, superior olive, and nucleus ambiguous; and fibers in the central amygdala, caudal caudate putamen, sensory trigeminal area, and substantia gelatinosa (Fig. 2A). Other areas containing CGRPimmunoreactive fibers include the septal area, bed nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, and median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus of the solitary tract, and confines of clusters of cell bodies (Fig. 2A). The neuroanatomical localization of CGRP is reviewed more comprehensively elsewhere [13, 26, 27]. There is also enrichment of CGRP in the cerebral microvasculature, both in vascular endothelial cells and nerve fibers that are closely associated with blood vessels.

DISTRIBUTION OF THE mRNA/PEPTIDE CGRP and AM are widely expressed in many regions of the mammalian brain. This has been revealed by several studies that detected both mRNA, by in situ hybridization, and the peptide, by immunohistochemistry.

CGRP Neurons and fibers that are positive for CGRP mRNA and immunoreactive to anti-CGRP antibodies are widely but unevenly distributed in the central nervous system (CNS). CGRP-positive cell bodies are observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, and medial forebrain bundle), premammillary nucleus, medial amygdala, hippocampus, dentate gyrus, central gray, and ventromedial nucleus of the thalamus. In the midbrain, a large cluster of CGRP-positive cells is found in the peripeduncular area. The cholinergic

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Adrenomedullin

PROCESSING OF THE PRECURSOR

Although initial studies suggested that AM was poorly expressed in brain, more recent work indicates that it has a widespread distribution and that it is dynamically regulated by events such as ischemia and glucose deprivation. Several autonomic centers, including the hypothalamic paraventricular nucleus (PVN), hypothalamic supraoptic nucleus (SON), locus coerulus, and ventrolateral medulla, express high levels of AM, with low to moderate AM immunoreactivity found in many other regions, including multipolar neurons and pyramidal cells of layers IV–VI of the cerebral cortex, parts of the thalamus, cerebellar Purkinje cells, and cerebellar nuclei (Fig. 2B). Immunoblotting of brain extracts has shown that the fully processed peptide is the predominantly expressed form, with a relatively smaller amount of the pro-peptide. The detailed distribution of AM in the brain is reviewed elsewhere [23, 27]. AM and PAMP are also expressed in the pituitary with particularly high levels in the adenohypophysis and neural lobe, with relatively lower expression in the intermediate pituitary. The role of AM in the pituitary is covered elsewhere [16].

As previously described, the precursor for CGRP is derived from alternately spliced mRNA from the CALCA gene. The CGRP precursor in humans consists of 128 amino acids with a molecular mass of 13.87 kDa. The N-terminal region that is encoded principally by exons II and III incorporates a signal peptide from residues 1–21; it is cleaved at the dibasic motif KR (residues 81–82) to yield the biologically active 37-aa CGRP peptide together with a short C-terminal fragment. The glycine at residue 120 of the precursor provides the amide for amidation of the C-terminal phenylalanine (aa. 119 in the precursor) (Fig. 1). The primary protein product of the human AM mRNA is 185 amino acids in length and is called preproAM. The N-terminal signal sequence of 21 residues is cleaved to yield proAM. Further processing of proAM yields two peptides: PAMP derived from residues 22–41 and AM (a 52-aa peptide) composed of the amino acid residues 95–146 of preproAM. Glycine 147 provides the amide for the amidation of tyrosine 52 (aa. 146 of the precursor) of the mature AM.

CGRP and Adrenomedullin in the Brain / 775 Some investigators have reported the presence of two forms (A and B) of the AM mRNA. Form A is devoid of introns and results in a prohormone containing both AM and PAMP. Form B retains the third intron, which introduces a premature stop codon, producing a shorter prohormone with only PAMP. Treatments that modify AM expression, such as hypoxia, have been shown to change B/A ratio and the relative levels of AM and PAMP, indicating that the splicing mechanism is subject to modification and is physiologically relevant [17].

RECEPTORS AND SIGNALING CASCADES CGRP Receptors CGRP receptors are widely distributed in the brains of many species, and there has been extensive work mapping the distribution of these receptors using 125ICGRP, particularly in rat brain [24, 27, 28]. High densities of CGRP binding sites are present in several regions, including accumbens nucleus; OVLT; ventral caudate putamen; median eminence; much of the hypothalamus, including enrichment in the arcuate nucleus and premammillary area; various amygdaloid nuclei; the superior and inferior colliculi; pontine nuclei; cerebellar cortex; nucleus of the solitary tract; inferior olivary nucleus; hypoglossal complex; and the vestibular and cochlear nuclei, along with lower levels of binding to many other brain regions. CGRP receptors are also present on most of the cerebral vasculature. CGRP receptor distribution has been extensively reviewed elsewhere [24, 27]. However, the sites labeled by 125ICGRP are heterogeneous and there are multiple subtypes of CGRP receptor.

AM Receptors Although membrane-based radioligand binding studies have demonstrated the existence of significant levels of AM receptors in the brain, there is only limited information on the anatomical distribution of AM receptors in this tissue. Using 125I-AM13–52, Juaneda and colleagues demonstrated high levels of AM binding in several regions, including choroid plexus, the linings of the cerebral ventricles, cerebellum, parts of the amygdala, and the neural lobe of the pituitary gland [2, 11]. Elsewhere, only low levels of binding were observed, although this may reflect the sensitivity of the methodology and radioligand probe because there is evidence for more widespread distribution of the constituent components of the AM receptor complexes [19]. AM receptors are also heterogeneous, with two high-affinity receptors characterized.

Molecular Basis of CGRP and AM Receptor Phenotype The molecular basis of CGRP and AM receptor phenotype(s) is complex; the ligand-binding domains of the receptors comprise two subunits, a classic seven transmembrane-spanning G-protein-coupled receptor (GPCR) and one of three single-transmembranespanning accessory receptor activity-modifying proteins (RAMP) [7, 20]. Two GPCRs, namely the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR), contribute to CGRP/AM receptor phenotypes. The CLR/RAMP1 heterodimer is a classic CGRP1 receptor and has a high affinity for CGRPs but a lower affinity for related peptides. CLR/RAMP2 is a high-affinity AM receptor (termed AM1 receptor) and interacts only weakly with CGRP. CLR/RAMP3 is also a high-affinity AM receptor (termed AM2 receptor), but it may respond with moderate potency to CGRP in a system-dependent manner. In addition to CLR-based receptors, a highaffinity CGRP receptor is also formed by heterodimerization of CTR/RAMP1. This receptor, like other CTR/RAMP-based receptors, has a high affinity for the peptide amylin and is termed the AMY1 receptor (previously also known as a C3 receptor). Other CTR/RAMP receptors have only a low affinity for CGRP, and all CTR/RAMP receptors have a very low affinity for AM. Signaling via CLR-based receptors also involves an additional protein, termed receptor component protein (RCP), that is required for efficient coupling of CLR/ RAMP heterodimers to at least Gαs proteins [5]. CLR, RAMPs, and CTR are all expressed within the brain, although with often discrete patterns of distribution. The distribution of the mRNA of the molecular components for CGRP and AM receptors generally correlates well with that of radioligand binding sites, in some regions, including the hypothalamus, caudate putamen, and cerebral vasculature, supporting the heterodimer as the basic unit for these receptors. An additional receptor, termed the CGRP2-subtype, has also been proposed. CGRP2-subtype receptors are defined as having a low affinity for CGRP1 receptor antagonists such as CGRP8–37 and BIBN4096BS, but they can be activated with reasonable potency by linear CGRP analogs [27]. Recent results suggest that most of the pharmacology ascribed to CGRP2 receptors can be attributed to the CTR/RAMP1, CTR/RAMP3, or CLR/ RAMP3 complexes [6, 14]. The possible existence of additional non-RAMPmodulated CGRP receptors has been suggested. This comes principally from expression data in the cerebellum. The cerebellum contains abundant CGRP binding sites that are pharmacologically similar to CGRP1 receptors. However, there is some controversy about their molecular identity because there are some reports

776 / Chapter 106 indicating no expression of CLR or RAMP1 mRNA in this tissue, whereas others suggest that a low expression of these mRNAs does indeed occur. The cerebellum, however, is particularly enriched in RCP, and this may contribute to efficient and stable expression of the CLR and RAMP protein components in a high-affinity complex (reviewed in [7]).

Signaling CGRP and AM receptors are family B GPCRs; these receptors pleiotropically couple to multiple G-proteins, with prominent coupling to Gαs. CGRP and AM receptors strongly activate Gαs, and many of their actions are downstream of cAMP generation. This has been confirmed by a vast number of studies in various tissues, primary cultures, or transfected cells. As previously described, efficient coupling of the CLR-based receptors to Gαs requires the presence of RCP. However, the receptors can also activate pertussin-toxin-sensitive Gproteins and probably also Gαq/11 proteins, the latter leading to Ca2+ mobilization. Only limited functional studies have been performed in nervous tissue, primarily in neuronal tumor cells or primary cultures of astrocytes and glial cells. Rat AM stimulated cAMP production in rat astrocytes and NG108-15 neuroblastoma/glial hybrid cells in a dose-dependent manner. This effect was not blocked by a CGRP1 receptor antagonist, indicating that the activation of selective AM receptors was involved. Similarly, CGRP-induced cAMP production has been demonstrated in human glioblastoma A172 cells and SKNMC neuroblastoma cells. The effects of AM on Ca2+-signaling mechanisms have also been investigated in neuroblastoma and glial tumor-cell lines because endothelial NO has been implicated in AMmediated vasodilation, and there is a well-known link between intracellular Ca2+ and the activation of NO synthase (NOS) [8, 23].

PEPTIDE CONFORMATION Nuclear magnetic resonance and circular dichroism studies have indicated that CGRP can adopt an α-helical secondary structure in a solvent-dependent manner. Organic solvents such as TFE promote formation of the α-helix, and this is thought to more closely resemble the active conformation at the receptor. Under such conditions, NMR studies indicate that an amphipathic α-helix is formed from residues 8–18, two β-turn structures from amino acids 17–21 and amino acids 29–34, and a random coil structure from amino acids 23–29. It is thought that the disulfide-bonded N-terminal region (aas. 1–7) is important in stabilization of the

helix but that residues 19–37 do not contribute significantly (reviewed in [27]). Less is known about the conformation adopted by AM. However, it is thought, by analogy, that AM is likely to adopt a similar secondary structure to CGRP when bound to its receptor(s). The N-terminal 14-amino-acid extension seen in AM, but not in other related peptides, does not appear to be important for biological activity and may not directly interact with the receptor.

BIOLOGICAL ACTIONS WITHIN THE BRAIN CGRP and AM are best known for their hypotensive effects through their vasodilatory action. However, because the peptides and their receptors are widely distributed in the brain, outside the cerebral vasculature, there is interest in their potential roles in other biological actions within the brain. There is evidence that some of the peripheral effects of both these peptides result from their actions within the brain, particularly on neurons in the autonomic centers.

CGRP Consistent with its widespread distribution in brain, CGRP has been implicated in a wide range of centrally mediated functions. As with peripheral vascular beds, CGRP is a potent dilator of cerebral and pial vessels and, via this action, contributes to the pain of migraine headache. Central injection of CGRP elicits multiple effects, including hypothermia, catalepsy, reduced motor activity, and actions on the hypothalamic-pituitary axis, including modulation of growth hormone, luteinizing hormone, and prolactin secretion. CGRP potentiates haloperidol-induced catalepsy and decreases apomorphine-induced hypermotility. Central CGRP can also inhibit thalamic neuronal firing evoked by peripheral noxious mechanical stimuli (at least in rat) and can modulate pain responses at the level of the spinal cord. CGRP can decrease feeding via receptors in the hypothalamus and also in the area postrema (reviewed in more detail elsewhere [15]), but has a more prominent effect on diuresis and natriuresis. It can also decrease gastric acid secretion and gastric motility. In contrast to the hypotensive effect of peripherally administered CGRP, centrally administered CGRP increases the mean arterial pressure and heart rate through increased sympathetic outflow. The coincident distribution of CGRP fibers and receptors in functionally linked nuclei involved in auditory, gustatory, and visual processing suggests that CGRP may also play a role in these systems. The central biological actions of CGRP are reviewed in more detail elsewhere [2, 24, 27, 28].

CGRP and Adrenomedullin in the Brain / 777

Adrenomedullin The most characterized physiological role of AM is in the regulation of circulation and control of fluid and electrolyte homeostasis (these are described in detail in chapters in the Cardiovascular and Renal Peptides sections of this Handbook). This function of AM is mediated via both peripheral and central actions. The effects of AM in the brain have been studied mostly by AM infusion into various brain regions. Action sites of AM in the rat brain have been mapped by c-fos immediateearly gene expression. Intracerebroventricular (ICV) injection in conscious rats results in fos expression in supra-optic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus, and area postrema. This suggests that centrally administered AM causes the activation of a neural network in the hypothalamus and brain stem. In the PVN, mostly the corticotrophinreleasing hormone (CRH) and nitric oxide (NO)– producing cells are activated. The c-fos induction is blocked by pretreatment with the AM receptor antagonist hAM22–52. These observations suggest that AM stimulates activity of the hypothalamic-pituitary axis, the sympathetic nervous system, and the hypothalamic NO system. ICV injection of AM in rats also augments tyrosine hydroxylase gene expression in the locus coeruleus and plasma oxytocin levels. Thus, AM activates the neurosecretory cells of the PVN and SON via selective AM receptors and stimulates oxytocin secretion by activating hypothalamic-OXY-producing cells [22, 23]. Investigations of this type have been extended to specific populations of CNS neurons as in patch-clamp studies on dissociated rat area postrema (AP) neurons. They have revealed AM-concentration-dependent neuronal activity and that the effects were mediated via specific ion channel activity. The results have implications for the cardiovascular actions of AM because the AP is a CNS site implicated in cardiovascular control. AM also has protective effects against ischemic/hypoxic injury to neurons. AM expression is upregulated in cultured neurons subjected to hypoxic conditions. It was recently reported that AM gene transfer via a viral vector could protect experimentally induced ischemic brains of rats from developing cerebral infarcts [29]. The effect was abolished by prior treatment with an AM receptor antagonist. Thus, there is evidence to suggest that AM can act as a neurotransmitter, neuromodulator, or a neurohormone in brain autonomic pathways involved in maintenance of homeostasis and also as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role in the brain microvasculature. AM has actions at each level of the hypothalamopituitary-adrenal axis, suggesting that the peptide plays

a role in the organization of neuroendocrine responses to stress. ICV injection to rat brain causes secretion of corticosterone and prolactin but not growth hormone. The pituitary actions of AM are described in more detail in the chapter by Martinez in the Endocrine Peptides Section of this Handbook [16].

PATHOLOGICAL IMPLICATIONS Both CGRP and AM have been linked to a broad range of clinical conditions, the majority of them being inflammatory conditions of the cardiovascular system. In regard to those associated with the brain, the strongest link has been established for a role for CGRP with the physiopathology of headache, particularly migrainetype headache. The intracranial extracerebral blood vessels (e.g., middle meningeal artery and dural arterioles) are thought to dilate in response to CGRP and, as a consequence, stimulate perivascular sensory nociceptive nerve fibers, producing a pain sensation. Intravenous infusion of CGRP exacerbates headache pain, and CGRP levels are increased in external jugular venous blood proportionally with pain intensity during migraine attacks [3]. Early clinical trials have demonstrated the efficacy of the CGRP1-receptor-specific antagonist BIBN4096BS in the treatment of migraine pain [4]. Studies with CGRP−/− mice have provided evidence that CGRP is important for generation of opiate withdrawal symptoms [18] and may therefore contribute to this behavior in humans. AM has been implicated as having a role in the pathogenesis of several psychiatric disorders such as schizophrenia, bipolar disorder, and depression. It is believed that the effect of AM occurs through its nitric oxide–generating action. There are also suggestions that AM may have a role in autism [30]. However, the best evidence is for a protective role in ischemic episodes, which is also seen in peripheral tissue [1].

Acknowledgments PMS is a Senior Research Fellow of the National Health and Medical Research Council of Australia.

References [1] Bunton DC, Petrie MC, Hillier C, Johnston F, McMurray JJ. The clinical relevance of adrenomedullin: A promising profile? Pharmacol Ther 2004; 103: 179–201. [2] Dumont Y, Chabot J-G, Quirion R. Receptor autoradiography as a mean to explore the possible functional relevance of neuropeptides: Focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides. Peptides 2004; 25: 365–91.

778 / Chapter 106 [3] Edvinsson L. Blockade of CGRP receptors in the intracranial vasculature: a new target in the treatment of headache. Cephalagia 2004; 24: 611–22. [4] Edvinsson L. Clinical data on the CGRP antagonist BIBN4096BS for treatment of migraine attacks. CNS Drug Rev 2005; 11: 69–76. [5] Evans BN, Rosenblatt MI, Mnayer LO, Oliver KR, Dickerson IM. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J Biol Chem 2000; 275: 31438–43. [6] Hay DL, Christopoulos G, Christopoulos A, Poyner DR, Sexton PM. Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol Pharmacol 2005; 67: 1655–65. [7] Hay DL, Poyner DR, Sexton PM. 2005. GPCR modulation by RAMPs. Pharmacol Ther 2006; 109: 173–97. [8] Hayakawa H, Hirata Y, Kakoki M, Suzuki Y, Nishimatsu H, Nagata D, Suzuki E. Role of nitric oxide-cGMP pathway in adrenomedullin-induced vasodilation in the rat. Hypertension 1999; 33: 689–93. [9] Ishimitsu T, Kojima M, Kanagawa K, Hino J, Matsuoka H, Kitamura K, Eto T, Matsuo H. Genomic structure of human adrenomedullin gene. Biochem Biophys Res Commun 1994; 203: 631–39. [10] Ishimitsu T, Ono H, Minami J, Matsuoka H. Pathophysiological implication of adrenomedullin and its therapeutic application to cardiovascular disorders. Pharmacol Ther 2005; (in press). [11] Juaneda C, Dumont Y, Chabot J-G, Quirion R. Autoradiographic distribution of adrenomedullin receptors in rat brain. Eur J Pharmacol 2001; 421: R1–R2. [12] Kitamura K, Kanagawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993; 192: 353–60. [13] Kresse A, Jacobowitz DM, Skofitsch G. Detailed mapping of CGRP mRNA expression in the rat central nervous system: Comparison with previous immunocytochemical findings. Brain Res Bulletin 1995; 36: 267–74. [14] Kuwasako K, Cao YN, Nagoshi Y, Tsuruda T, Kitamura K, Eto T. Characterization of the human calcitonin gene-related peptide receptor subtypes associated with receptor activity-modifying proteins. Mol Pharmacol 2004; 65: 207–13. [15] Lutz TA. Role of amylin and calcitonin gene-related peptide (CGRP) in the control of food-intake. In: Handbook of Biologically Active Peptides. Academic Press, pp. 881–88. [16] Martinez A. Adrenomedullin and related peptides in the local regulation of endocrine glands. In: Handbook of Biologically Active Peptides. Academic Press, pp. 1001–06. [17] Martinez A, Hodge DL, Garayoa M, Young HA, Cuttita F. Alternate splicing of the proadrenomedullin gene results in differential expression of gene products. J Mol Endocrinol 2001; 27: 31–41.

[18] Muff R, Born W, Lutz TA, Fischer JA. Biological importance of the peptides of the calcitonin family as revealed by disruption and transfer of the corresponding genes. Peptides 2004; 25: 2027–38. [19] Oliver KR, Kane SA, Salvatore CA, Mallee JJ, Kinsey AM, Koblan KS, Keyvan-Fouladi N, Heavens RP, Wainwright A, Jacobson M, Dickerson IM, Hill RG. Cloning, characterization and central nervous system distribution of receptor activity modifying proteins in the rat. Eur J Neurosci 2001; 14: 618–28. [20] Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, Foord SM. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 2002; 54: 233–46. [21] Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vi WW, Evans RM. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983; 304: 129–35. [22] Serino R, Ueta Y, Hara Y, Nomura M, Yamamoto Y, Shibuya I, Hattori Y, Kitamura K, Kanagawa K, Russell JA, Yamashita H. Centrally administered adrenomedullin increases plasma oxytocin levels with induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of the rat. Endocrinol 1999; 140: 2334–42. [23] Serrano J, Alonso D, Fernandez AP, Encinas JM, Lopez JC, Castro-Blanco S, Fernandez-Vizarra P, Richart A, Santacana M, Uttenthal LO, Bentura ML, Martinez-Murillo R, Martinez A, Cuttitta F, Rodrigo J. Adrenomedullin in the central nervous system. Micro Res Tech 2002; 57: 76–90. [24] Sexton PM. Central nervous system binding sites for calcitonin and calcitonin gene-related peptide. Mol Neurobiol 1991; 5: 251–73. [25] Sexton PM, Findlay DM, Martin TJ. Calcitonin. Curr Med Chem 1999; 6: 1067–83. [26] Skofitsch G, Jacobowitz DM. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 1985; 6: 6721–45. [27] van Rossum D, Hanisch U-W, Quirion R. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci & Behav Rev 1997; 21: 649–78. [28] Wimalawansa SJ. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: A peptide superfamily. Crit Rev Neurobiol 1997; 11: 167–239. [29] Xia CF, Yin H, Borlongan CV, Chao J, Chao L. Adrenomedullin gene delivery protects against cerebral ischemic injury by promoting astrocyte migration and survival. Hum Gene Therap 2004; 15: 1243–54. [30] Zoroglu SS, Yurekli M, Meram I, Sogut S, Tutkun H, Yetkin O, Sivasli E, Savas H, Yanik M, Herken H, Akyol O. Pathophysiological role of nitric oxide and adrenomedullin in autism. Cell Biochem Funct 2003; 21: 55–60.

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107 The RFamide-Related Peptides NICOLAS CHARTREL, KAZUYOSHI TSUTSUI, JEAN COSTENTIN, AND HUBERT VAUDRY

balance, and neuromodulatory activities. Immunohistochemical results with antibodies directed against FMRFamide had long suggested that RFRPs also exist in vertebrates. Indeed, several RFRPs have been identified in fish [14, 39, 51], amphibians [6, 22, 24, 60], and birds [9, 50, 58]. To date, seven RFRPs encoded by five distinct genes have been characterized in the brain of mammals (Table 1). Neuropeptide FF (NPFF) and neuropeptide AF (NPAF) have been isolated from a bovine brain extract by use of antibodies directed against FMRFamide [62]. Prolactin-releasing peptide (PrRP) has been purified from a bovine hypothalamic extract by reverse pharmacology as the endogenous ligand of the human orphan receptor hGR3/GPR10 [18]. A search for unknown members of the RFamide peptide family in the databases has led to the identification of two human ESTS that encode two RFRPs designated RFamide-related peptide-1 and -3 (RFRP-1 and -3) [19]. A fifth mammalian RFRP, named metastin or kisspeptin, has been isolated from the human placenta on the basis of its affinity for the human orphan receptor GPR54 [25, 40, 44] (see Chapter 111). Finally, the precursor of a novel RFRP initially isolated from a frog brain extract [5], referred to as 26RFa, has been characterized in several mammalian species [5, 16, 20].

ABSTRACT The term RFamide-related peptides (RFRPs) designates a family of regulatory peptides that possess the signature Arg-Phe-NH2 at their C-terminus. So far, the genes encoding seven distinct RFRPs have been characterized in mammals and five receptors for RFRPs have been recently identified. RFRPs show a discrete localization in the central nervous system (CNS) and are frequently co-regionalized. The main populations of RFRP-containing neurons are present in the periventricular region of the hypothalamus, in the nucleus of the solitary tract and the reticular nucleus of the brain stem, and in the dorsal horn of the spinal cord. Consistent with this distribution, RFRPs exert various effects in the CNS such as modulation of nociceptive transmission, control of feeding behavior, and regulation of pituitary hormone secretion.

DISCOVERY OF THE MAMMALIAN RFAMIDERELATED PEPTIDES Bioactive peptides possessing the motif Arg-PheNH2 at their C-terminal extremity are collectively termed RFamide-related peptides (RFRPs). The first member of this family, the tetrapeptide Phe-Met-ArgPhe-NH2, was originally isolated from the ganglia of the venus clam on the basis of its cardiovascular activity [47]. Since then, numerous RFRPs have been characterized in cnidarians, nematodes, annelids, molluscs, and arthropods (for review see [6]). In particular, in Caenorhabditis elegans, 22 genes encoding 59 distinct RFRPs have been identified [33]. Pharmacological studies indicate that in invertebrates RFRPs exert a large array of biological effects including cardioexcitatory activities, modulation of muscle contraction, control of locomotor activity, regulation of hydric Handbook of Biologically Active Peptides

STRUCTURE OF THE RFAMIDE-RELATED PEPTIDE PRECURSORS Molecular cloning of the NPFF cDNA from human, bovine, rat, and mouse has revealed that a single gene encodes both NPFF and NPAF [46, 61]. In all four species, the NPFF sequence is located in the central region, whereas NPAF is found at the C-terminal extremity of the precursor (Fig. 1). The primary structure of NPFF has been fully conserved in mammals, whereas

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Copyright © 2006 Elsevier

780 / Chapter 107 TABLE 1. Primary structure of the various RFamide-related peptides characterized in mammals. Species

Peptide

Amino Acid Sequence

Reference

Bovine Bovine Bovine Bovine Bovine Bovine Human

NPFF NPAF PrRP20 PrRP31 RFRP-1 RFRP-3 Metastin

[62] [62] [18] [18] [15] [64] [25, 40, 44]

Rat Human

26RFa 43RFa (QRFP)

FLFQPQRF-NH2 AGEGLSSPFWSLAAPQRF-NH2 TPDINPAWYAGRGIRPVGRF-NH2 SRAHQHSMEIRTPDINPAWYAGRGIRPVGRF-NH2 SLTFEEVKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH2 AMAHLPLRLGKNREDSLSRWVPNLPQRF-NH2 GTSLSPPPESSGSRQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYN WNSFGLRF-NH2 ASGPLGTLAEELSSYSRRKGGFSFRF-NH2 <EDEGSEATGFLPAAGEKTSGPLGNLAEELNGYSRKKGGFSFRF-NH2

A NPFF precursor

SP 68

A

SP

R PrRP31

22

1

R

78

92

NPFF

1

PrRP precursor

R

113

RRR PrRP20

33

55

57

87

SP 1

KK NPAF

R RFRP precursor

R

R R

94

122 123

RFRP-1

SP 1

196

133

K R Metastin

66 67

SP 1

145

123 124

R 26RFa precursor

R RFRP-3

55

R R Metastin precursor

[5, 16, 20] [16]

K 43RFa

90

R R 26RFa

107

136

FIGURE 1. Schematic representation of the RFamide-related peptide (RFRP) precursors characterized in human. Potential mono/polybasic cleavage sites are indicated by vertical bars. RFRPs generated by each precursor are shown. Amino acids are numbered under each precursor. A, alanine; K, lysine; NPFF, neuropeptide FF; NPAF, neuropeptide AF; PrRP, prolactin-releasing peptide; R, arginine; RFRP-1 and -3, RFamiderelated peptide-1 and -3; SP, signal peptide.

the sequence of NPAF is more divergent [61]. In particular, NPAF encompasses 18 amino acids in human and ox and only 13 amino acids in rat and mouse [61]. The characterization of the cDNA encoding PrRP in the human, cow, and rat has shown that the PrRP sequence is located in the central region of the precursor (Fig. 1) [18]. The primary structure of PrRP has been strongly preserved in mammals—bovine and rat

PrRP show only one amino acid difference with the human sequence [18]. The cDNA encoding RFRP-1 and RFRP-3 has been cloned in human, cow, rat, and mouse [19]. The putative sequences of RFRP-1 and RFRP-3 are located in the central region of the precursor (Fig. 1). The RFRP-1 sequence is identical in rat and mouse and shows two residue substitutions with the human sequence. Bovine RFRP-1 exhibits one amino

The RFamide-Related Peptides / 781 acid substitution with its human counterpart and two substitutions with rat/mouse RFRP-1 [19]. Similarly, the RFRP-3 sequence is fully conserved between rat and mouse and between human and cow. Two residue substitutions occur between rat/mouse and human/cow RFRP-3 [19]. Metastin is the mature product of the KiSS-1 gene, previously characterized as a metastasis suppressor gene [31]. The cDNA encoding the metastin precursor has been cloned in human [25, 40, 44], rat, and mouse [56]. In the three species, the metastin sequence is located in the C-terminal region of the precursor (Fig. 1). Human metastin is a 54-amino-acid peptide [25, 40, 44], whereas rat and mouse metastin encompass only 52 residues [56]. Rat and mouse metastin show an overall identity of 83%, but the percentage of identity between rodent and human metastin is only 52–57%. Interestingly, human metastin is an authentic RFamide peptide, whereas the rat and mouse orthologs exhibit an RY-NH2 motif at their C-terminus [56]. In addition, rat and mouse metastin possess a disulfide bridge in their N-terminal region, whereas the human counterpart is linear [56]. The 26RFa precursor has been characterized in human, cow, rat, and mouse [5, 16, 20]. The 26RFa sequence is located at the C-terminal extremity of the precursor (Fig. 1). The cow, rat, and mouse 26RFa precursors contain only one copy of RFamide peptide, whereas human prepro-26RFa encompasses a second putative RFRP of nine residues upstream of 26RFa [5, 16, 20]. The primary structure of 26RFa has been highly conserved across mammals— the human sequence shares 81% identity with the rat/ mouse sequence and 85% with the bovine sequence [20].

PROCESSING OF THE RFAMIDE-RELATED PEPTIDE PRECURSORS Isolation of NPFF from tissue extracts indicates that prepro-NPFF is efficiently cleaved at an Ala site (at the N-terminus of NPFF). However, an N-elongated form of NPFF, possessing three additional residues, may also be generated from the rodent precursors by cleavage at an Arg-Arg doublet [3]. NPAF has been characterized in ox, indicating that bovine prepro-NPFF is cleaved at a single Arg residue upstream of the NPAF sequence. This Arg residue is also found in the human NPFF precursor but not in the rodent precursors, suggesting that in rat and mouse NPAF is not produced. Indeed, NPAF has never been characterized in rodent tissues, but a shorter form of the peptide of eight amino acids that results from processing at a Trp residue, referred to as NPSF, has been detected in rat and mouse [3]. Two molecular forms of PrRP have been isolated from a bovine hypothalamic extract, namely PrRP20 and its N-

extended form PrRP31 (Table 1) [18]. The PrRP20 sequence is preceded by a single Arg residue that is fully conserved across mammalian PrRP precursors, suggesting that PrRP20 is the common form present in the mammalian brain. In contrast, PrRP31 could be generated by the cleavage of the bovine PrRP precursor at an Ala residue, which is also found in human prepro-PrRP but not in the rat precursor. Biochemical characterization of mature products of the RFRP precursor has shown that RFRP-1 and RFRP-3 produced in vivo exhibit a sequence different from those predicted from the structure of the precursor [15, 59, 64]. Thus, RFRP-1 was predicted to be a 12-amino-acid peptide [19], but the purification of endogenous RFRP-1 in a bovine hypothalamic extract indicates that the mature form is a 35-amino-acid peptide (Table 1) [15]. Similarly, RFRP3 was predicted to possess nine amino acids [19], but the isolation of RFRP-3 from the hypothalamus revealed that the peptide actually consists of 28 amino acids [64] (Table 1). Metastin has been purified from the human placenta as a 54-amino-acid peptide [25, 44]. Two shorter forms of metastin of 14 and 13 residues have also been identified that may correspond to breakdown products generated during the extraction procedure [25]. 26RFa has been originally isolated from a frog brain extract [5]. In the rat, mouse, cow, and human precursors, the 26RFa sequence is preceded by a single Lys residue that is not considered as a conventional processing site. However, recent data indicate that 26RFa is actually generated in the human central nervous system (CNS). In Chinese hamster ovary (CHO) cells transfected with the 26RFa cDNA, an N-extended form of 26RFa designated 43RFa (or QRFP) (Table 1) is generated by cleavage at a single Arg residue [16] that is conserved in all mammalian prepro-26RFa, suggesting that 43RFa is also produced in vivo. Consistent with this notion, the presence of 43RFa has also been detected in human hypothalamic and spinal cord extracts (unpublished data).

DISTRIBUTION OF RFAMIDE-RELATED PEPTIDES IN THE BRAIN Neuroanatomical studies have shown that RFRPexpressing neurons display a discrete localization in the CNS of mammals and that several RFamide-related peptides are frequently co-regionalized. The main population of NPFF-immunoreactive neurons is found between the ventromedial and the dorsomedial hypothalamic nuclei [45] (Fig. 2). Scattered NPFF-containing neurons are also observed in the paraventricular and the suprachiasmatic nuclei [45]. Additional populations of NPFFpositive neurons are present in the nucleus of the solitary tract and in the dorsal horn of the spinal cord

782 / Chapter 107 Brain

A Cx

Hi CC

OB CPut

S

Th PAG C BST

DR PVN

Acb

PBN

Hpt DMH

LC SN

LHA

NST DBB

B

Spinal cord

SON

Rch Sch Amy

DH

LH

VH WM

Cc

VMH Arc ME

NL IL

Rt

AP

NPFF PrRP RFRP-1 and -3 Metastin 26RFa

FIGURE 2. Neuroanatomical localization of RFamide-related peptides (RFRPs) in the central nervous system. A. Schematic parasagittal section depicting the distribution of RFRP-expressing neurons (colored dots) in the rat brain. B. Schematic coronal section depicting the distribution of RFRP-expressing neurons (colored dots) in the rat spinal cord. Abbreviations: Acb, accumbens nucleus; Amy, amygdala; AP, anterior lobe of the pituitary; Arc, arcuate nucleus; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; Cc, central canal; Cput, caudate putamen; Cx, cerebral cortex; DBB, bed nucleus of the diagonal band of Broca; DH, dorsal horn; DMH, dorsomedial hypothalamic nucleus; DR, dorsal raphe nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of the pituitary; LC, locus coeruleus; LH, lateral horn; LHA, lateral hypothalamic area; ME, median eminence; NL, neural lobe of the pituitary; NST, nucleus of the solitary tract; OB, olfactory bulb; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular hypothalamic nucleus; Rch, retrochiasmatic area; Rt, reticular nucleus; S, septum; Sch, suprachiasmatic nucleus; SN, substantia nigra; SON, supraoptic nucleus; Th, thalamus; VH, ventral horn; VMH, ventromedial hypothalamic nucleus; WM, white matter.

[45] (Fig. 2). PrRP-expressing neurons are primarily localized in the posterior aspect of the dorsomedial hypothalamic nucleus [21, 32, 48] (Fig. 2). In the brain stem, a high density of PrRP neurons is found in the medial portion of the nucleus of the solitary tract [32, 48] as well as in the ventrolateral reticular nucleus [48] (Fig. 2). RFRP-1- and -3-expressing neurons have been observed only in a region of the hypothalamus localized between the ventromedial and dorsomedial hypothalamic nuclei, extending to the lateral hypothalamic area [15, 19, 63] (Fig. 2). Metastin is present in three neuronal populations located in the dorsomedial

hypothalamic nucleus and, at the level of the brain stem, in the nucleus of the solitary tract and the caudoventral region of the reticular nucleus [4, 10] (Fig. 2). Scattered metastin-immunoreactive cells have also been detected in the superficial layers of the dorsal horn in the spinal cord [10] (Fig. 2). 26RFa-expressing neurons are located in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the lateral aspect of the arcuate nucleus, and the retrochiasmatic area [5, 16]. 26RFa-containing neurons have also been detected in the dorsal horn of the human spinal cord (unpublished data).

The RFamide-Related Peptides / 783

RFAMIDE-RELATED PEPTIDE RECEPTORS Five RFamide peptide receptors, all belonging to the GPCR family, have already been characterized in mammals. Screening of a library of peptidic neurotransmitters has led to the identification of NPFF and NPAF as cognate ligands of two orphan GPCRs, OT7TO22, and HLWAR77 [2, 12], which have thus been renamed NPFF1 and NPFF2, respectively. However, subsequent studies have shown that RFRP-1 and RFRP-3 also bind with high affinity NPFF1 [19, 34, 64] and NPFF2 [13, 64]. NPFF1 and NPFF2 share 49% amino acid sequence identity [2]. Co-expression of NPFF1 and NPFF2 with chimeric proteins suggests that the two receptors activate Gi/o proteins, leading to an inhibition of the adenylyl cyclase pathway [2, 12]. GPR10 has been found to be a PrRP receptor by use of a reverse pharmacology approach [18]. Contrasting with the low specificity of the two NPFF receptors, GPR10 shows a high affinity for PrRP20 and PrRP31 but a low affinity for NPFF, RFRP1, and RFRP-3 [13]. G-protein coupling of GPR10 depends on the cell system in which it is expressed. Thus, in GPR10-transfected HEK293 cells, PrRP fails to alter basal or forskolin-stimulated levels of intracellular cAMP [26], whereas in both GH3 cells and rat anterior pituitary cells GPR10 appears to be coupled, to a large extent, to a Gi/o protein and possibly also to a Gq protein [23]. Metastin has been identified as the endogenous ligand of the orphan receptor GPR54 [25, 40, 44]. Currently, it is not known whether other mammalian RFRPs can bind to GPR54. In both CHO and HEK293 cells transfected with GPR54, metastin stimulates calcium mobilization [25, 40]. This effect is not affected by pertussis toxin pretreatment and no modification of cAMP accumulation is observed, suggesting that GPR54 is coupled to G-proteins of the Gq family. The strong structural identity of GPR103 (also referred to as SP9155 and AQ27) with the NPFF1 and NPFF2 receptors (49% identity) [20] has suggested that GPR103 could be a RFamide peptide receptor. Binding studies have shown that 26RFa and its N-elongated form, 43RFa, exhibit a high affinity for GPR103 [16, 20]. In contrast, PrRP, RFRP-1, and RFRP-3 are unable to activate GPR103 [8]. In CHO cells transfected with GPR103, 26RFa increases intracellular calcium concentrations and decreases cAMP production, indicating that the receptor probably couples to the Gi/o and Gq signaling pathway [16].

INFORMATION ON SOLUTION CONFORMATION OF RFAMIDERELATED PEPTIDES The three-dimensional structures of NPAF, PrRP20, and 26RFa have been elucidated by use of nuclear mag-

netic resonance and molecular modeling. The structure of NPAF consists of a disordered N-terminal region (residues 1–6) followed by an α-helix in the central region (residues 7–14) and a C-terminal domain that either is unstructured or forms a type I β-turn (residues 15–18), depending on the solvent [38]. The structure of PrRP20, in a cell membrane mimetic medium, exhibits an α-helix within its C-terminal region (residues 11–19) and an N-terminal flexible domain with a weak tendency to form a β-turn between residues 2 and 5 [11]. 26RFa adopts a well-defined conformation in methanol, consisting of an amphipathic α-helix of approximately three turns in its central region, extending from residues 4 to 17, that is flanked by two unstructured N- and C-terminal domains [57]. These structural results indicate that all members of the RFamide peptide family investigated so far possess an α-helix that probably plays an important role in the interaction of RFRPs with their receptors.

BIOLOGICAL ACTIONS OF RFAMIDERELATED PEPTIDES WITHIN THE BRAIN AND PITUITARY The expression pattern of RFRPs and their receptors in the CNS of mammals suggests that members of this family exert important functions. Consistent with this notion, it is now clearly established that NPFF acts as a modulator of the opioid system as intracerebroventricular (icv) administration of the peptide reduces morphine-induced analgesia [62] and produces an abstinence syndrome in naive and morphine-tolerant mice [35] (see Chapter 186). NPFF has also been shown to reduce food intake [41, 55]. Because NPFF inhibits the opioid system and because endogenous opioids stimulate appetite, it has been proposed that the anorexigenic activity of NPFF may be mediated, at least in part, through the modulation of opioid neurotransmission [41]. In support of this hypothesis, it has been recently reported that injection of NPFF in the parabrachial nucleus inhibits the stimulation of food intake induced by the μ-opioid receptor agonist DAMGO [43]. In addition, NPFF binding sites are present in the pituitary [45] and NPFF is able to stimulate prolactin release [1]. PrRP has been initially identified as a prolactin-releasing factor [18], but this effect has not been consistently observed [27]. In contrast, there is now strong evidence that PrRP is involved in the central control of food intake: (1) icv administration of PrRP reduces food intake in both fasted and ad libitum fed rats [28] and (2) GPR10 knockout mice are obese [17]. Several lines of evidence suggest that the anorexigenic action of PrRP is mediated through the modulation of the

784 / Chapter 107 corticotrophin-releasing hormone (CRH) neuronal system. It has notably been observed that PrRP-immunoreactive fibers make synaptic contacts with CRH neurons in the paraventricular nucleus [36]. In addition, icv administration of PrRP increases CRH secretion in the hypothalamus [52], and the anorexigenic action of PrRP is blocked by CRH receptor antagonists [29]. PrRP also acts as a hypophysiotropic neurohormone. In addition to its prolactin-releasing activity [18], PrRP stimulates adrenocorticotropin [52] and gonadotropin release [53]. However, it seems that PrRP exerts its releasing activity at the hypothalamic rather than at the pituitary level [52, 53]. Finally, the presence of both PrRP and GPR10 in brain areas involved in pain modulation [21] suggests that PrRP may affect the transmission of nociceptive stimuli. Consistent with this notion, it has been shown that the microinjection of PrRP20 into the nucleus of the solitary tract induces marked antinociceptive responses [21]. Central administration of RFRP-1 enhances prolactin release [19, 49] and reduces morphine-induced nociception [34]. There is now clear evidence that, in addition to its role as a metastasis suppressor [25, 44], kisspeptin plays a key role in the control of reproductive function (see Chapter 111). In particular, metastin elevates plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels [42] and stimulates hypothalamic GnRH secretion [37]. GPR54 knockout mice fail to undergo sexual maturation [54], and several mutations of the GPR54 gene are associated with hypogonadotropic hypogonadism in human [7, 54]. The expression of both 26RFa and GPR103 in hypothalamic nuclei involved in the control of feeding behavior [5, 16] has raised the possibility that 26RFa may affect energy homeostasis. Indeed, icv injection of 26RFa provokes a dose-dependent stimulation of food consumption in semi-fasted mice [5]. However, by contrast to NPFF and PrRP, nothing is known regarding the neuronal pathways mediating the orexigenic effect of 26RFa. Both 26RFa and GPR103 are expressed in the pituitary [16, 30] and 26RFa stimulates cAMP production by rat pituitary cells [5], indicating that the neuropeptide, like other mammalian RFRPs, may act as a hypophysiotropic neurohormone.

References [1] Aarnisalo AA, Tuominen RK, Nieminen M, Vainio P, Panula P. Evidence for prolactin-releasing activity of neuropeptide FF in rats. Neuroendocrinol Lett 1997;18:191–6. [2] Bonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, et al. Identification and characterization of two G protein-coupled receptors for neuropeptide FF. J Biol Chem 2000;275:39324–31.

[3] Bonnard E, Burlet-Schiltz O, Francés B, Mazarguil H, Monsarrat B, Zajac JM. Identification of NPFF-related peptides in rodent spinal cord. Peptides 2001;22:1085–92. [4] Brailoiu GC, Dun SL, Ohsawa M, Yin D, Yang J, Kan Chang J, et al. KiSS-1 expression and metastin-like immunoreactivity in the rat brain. J Comp Neurol 2004;481:314–29. [5] Chartrel N, Dujardin C, Anouar Y, Leprince J, Decker A, Clerens S, et al. Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc Natl Acad Sci USA 2003;100:15247–52. [6] Chartrel N, Dujardin C, Leprince J, Desrues L, Tonon MC, Cellier E, et al. Isolation, characterization and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the brain of the European green frog Rana esculenta. J Comp Neurol 2002; 448:111–27. [7] de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–6. [8] Dockray GJ. The expanding family of RFamide peptides and their effects on feeding behavior. Exp Physiol 2004;89:229–35. [9] Dockray GJ, Reeve JR, Shively J, Gayton RJ, Barnard CS. A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide. Nature 1983;305:328–30. [10] Dun SL, Brailoiu GC, Parsons A, Yang J, Zeng Q, Chen X, et al. Metastin-like immunoreactivity in the rat medulla and spinal cord. Neurosci Lett 2003;335:197–201. [11] D’Ursi AM, Albrizio S, Di Fenza A, Crescenzi O, Carotenuto A, Picone D, et al. Structural studies on Hgr3 orphan receptor ligand prolactin-releasing peptide. J Med Chem 2002;45:5483–91. [12] Elshourbagy NA, Ames RS, Fitzgerald LR, Foley JJ, Chambers JK, Szekeres PG, et al. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J Biol Chem 2000;275:25965–71. [13] Engström M, Brandt A, Wurster S, Savola JM, Panula P. Prolactin-releasing peptide has high affinity and efficacy at neuropeptide FF2 receptors. J Pharmacol Exp Ther 2003;305:825–32. [14] Fujimoto M, Takeshita K, Wang X, Takabatake I, Fujisawa Y, Teranishi H, et al. Isolation and characterization of a novel bioactive peptide, Carassius RFamide (C-RFa), from the brain of the Japanese crucian carp. Biochem Biophys Res Commun 1998;242:436–40. [15] Fukusumi S, Habata Y, Yoshida H, Iijima N, Kawamata Y, Hosoya M, et al. Characteristics and distribution of endogenous RFamide-related peptide-1. Biochim Biophys Acta 2001; 1540:221–32. [16] Fukusumi S, Yoshida H, Fujii R, Maruyama M, Komatsu H, Habata Y, Shintani Y, et al. A new peptidic ligand and its receptor regulating adrenal function in rats. J Biol Chem 2003;278:46387–495. [17] Gu W, Geddes BJ, Zhang C, Foley KP, Stricker-Krongrad A. The prolactin-releasing peptide receptor (GPR10) regulates body weight homeostasis in mice. J Mol Neurosci 2003;22:93–103. [18] Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S, et al. A prolactin-releasing peptide in the brain. Nature 1998;8393:272–6. [19] Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, et al. New neuropeptides containing carboxyterminal RFamide and their receptor in mammals. Nature Cell Biol 2000;2:703–8. [20] Jiang Y, Luo L, Gustafson EL, Yadav D, Laverty M, Murgolo N, et al. Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155. J Biol Chem 2003;278:27652–7. [21] Kalliomäki ML, Pertovaara A, Brandt A, Wei H, Pietilä P, Kalmari J, et al. Prolactin-releasing peptide affects pain, allodynia and

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786 / Chapter 107 [57] Thuau R, Guilhaudis L, Ségalas-Milazzo I, Chartrel N, Oulyadi H, Boivin S, et al. Structural studies on 26RFa, a novel human RFamide-related peptide with orexigenic activity. Peptides 2005;26:779–89. [58] Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, Ishii S, Sharp PJ. A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 2000;275:661–7. [59] Ukena K, Iwakoshi E, Minakata H, Tsutsui K. A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett 2002;512: 255–8. [60] Ukena K, Koda A, Yamamoto K, Kobayashi T, Iwakoshi-Ukena E, Minakata H, Kikuyama S, Tsutsui K. Novel neuropeptides related to frog growth hormone-releasing peptide: Isolation, sequence, and functional analysis. Endocrinology 2003;144:3879–84.

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108 Apelin: Discovery, Distribution, and Physiological Role XAVIER ITURRIOZ, ANNABELLE REAUX-LE GOAZIGO, AND CATHERINE LLORENS-CORTES

angiotensin system, exerting tonic stimulatory control over arterial blood pressure (BP) [34], triggering arginine vasopressin (AVP) release, and activating magnocellular vasopressinergic neurons in the supraoptic nucleus (SON) [42, 43]. However, these effects in the SON are not mediated by the conventional AT1 or type 2 (AT2) Ang II receptors because no mRNA or binding sites corresponding to AT1 or AT2 receptors have been found in the SON. Ang III--induced AVP stimulation may therefore involve another angiotensin receptor subtype. In an attempt to isolate this receptor, we screened rat SON RNA by reverse transcription and polymerase chain reaction (PCR) amplification, using primers corresponding to the conserved region of the transmembrane domains of the rat angiotensin receptor subtypes. The PCR fragment was then used to screen a rat brain cDNA library, leading to isolation of a cDNA encoding the rat APJ receptor, displaying a high level of amino acid sequence identity (between 90 and 96%) to the corresponding receptors in mice [10] and humans [30]. Two other groups [21, 29] simultaneously isolated the rat APJ receptor sequence. We stably expressed the rat APJ receptor fused to enhanced green fluorescent protein (EGFP) in Chinese hamster ovary (CHO) cells and performed binding experiments and functional assays to determine whether this receptor bound angiotensin fragments. No specific binding of [125I] Ang II, Ang III, or Ang IV was observed, and stimulation of the rat APJ receptor by Ang II or Ang III did not modify cAMP production [8], demonstrating that this receptor was not an angiotensin receptor subtype. It therefore remained an orphan GPCR for which the endogenous ligand had to be isolated. We developed a new process for identifying the endogenous ligand of this orphan GPCR based on direct observation of the ligand-induced internalization of fluorescently tagged receptors expressed in mammalian cells after incubation with prepurified

ABSTRACT The APJ receptor was originally isolated as an orphan seven-transmembrane-domain G-protein-coupled receptor in humans. Interest in this receptor in mammals increased with the identification of its endogenous ligand, apelin, in 1998. Apelin is a peptide generated from a 77-amino-acid precursor, proapelin. Apelin and its receptor are strongly expressed in vasopressinergic neurons in the hypothalamus. The central injection of apelin in lactating rats decreases the phasic electrical activity of vasopressinergic neurons and the systemic secretion of AVP, leading to aqueous diuresis. Apelin is therefore a natural inhibitor of the antidiuretic effect of AVP. Intravenous administration of apelin decreases arterial blood pressure, and apelin also improves cardiac contractility and reduces cardiac loading in vivo. The development of nonpeptide agonists of the apelin receptor could lead to new therapeutic tools for the treatment of the syndrome of inappropriate secretion of AVP, thirst disorders, and heart and kidney failure.

DISCOVERY The apelin story began in 1993 with the cloning of the cDNA for the orphan APJ receptor (putative receptor protein related to the type 1 (AT1) angiotensin receptor) from a human genomic library by O’Dowd et al. [30]. This receptor is 380 amino acids long and was identified as a member of the family of orphan seventransmembrane-domain G-protein-coupled receptors (GPCRs). Its amino acid sequence was found to be 31% identical to that of the human AT1 receptor. We initially searched for another angiotensin receptor subtype specific for angiotensin III (Ang III), because we had previously shown that Ang III, but not angiotensin II (Ang II), was a major effector peptide of the brain reninHandbook of Biologically Active Peptides

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788 / Chapter 108 tissue extracts [22]. We used this procedure to evaluate the ability of reverse-phase high-performance liquid chromatography (HPLC) fractions obtained from purification of frog-brain extracts to induce internalization of the rat APJ receptor fused to EGFP. We were at the third stage in the purification process when Tatemoto et al. [37] isolated the endogenous ligand of the human APJ receptor from bovine stomach tissue extracts, using the Cytosensor microphysiometer to detect the metabolic activation of cells expressing the human APJ receptor. They named this ligand apelin, for endogenous ligand for the APJ receptor.

(2 and 3). This may account for the presence of transcripts of two different sizes (∼3 kb and ∼3.6 kb) in various tissues [21, 29]. The alignment of proapelin amino acid sequences from cattle, humans, rats, and mice has demonstrated strict conservation of the C-terminal 17 amino acids, known as apelin 17 or K17F (Fig. 1B). In vivo, proapelin gives rise to various molecular forms of apelin, probably through the action of prohormone convertases due to the presence of pairs of basic residues in proapelin. In rat brain and plasma, the predominant forms of apelin are the pyroglutamyl form of apelin 13 (pE13F) and, to a lesser extent, K17F (Fig. 1B) [9]. In rat lung, testis, and uterus and bovine colostrum apelin 36 predominates, whereas in the rat mammary gland both apelin 36 and pE13F have been detected [16, 19].

STRUCTURE OF THE PRECURSOR mRNA/GENE AND PROCESSING OF THE PRECURSOR

RECEPTORS AND SIGNALING CASCADES Apelin is a 36-amino-acid peptide (apelin 36) generated from a larger precursor, the 77-amino-acid proapelin (Fig. 1A). This precursor has been isolated from various species [15, 21, 37]. The human proapelin gene is located on chromosome X at locus Xq25--q26.1 and contains three exons, with the coding region spanning exons 1 and 2. The 3′ untranslated region also spans two exons

A

Several groups have shown that rat and human apelin receptors, stably expressed in CHO cells, are negatively coupled to adenylate cyclase activity [8, 15, 27]. The most potent inhibitors of forskolin-induced cAMP production were found to be apelin 36, K17F, apelin 13 (Q13F), and pE13F, whereas apelin fragments apelin 10

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FIGURE 1. A. Amino-acid sequence of the apelin precursor, proapelin, in cattle, humans, rats, and mice. The first amino acid of apelin 36 is indicated by an arrowhead and the apelin 17 sequence is indicated by a box (see [15, 38]). B. Apelin fragments detected in vivo in mammals: apelin 36, apelin 17 (K17F), and the pyroglutamyl form of apelin 13 (pE13F).

Apelin: Discovery, Distribution, and Physiological Role / 789 (R10F) and apelin 5 (G5F) were inactive [8, 11, 27]. Ala scan of pE13F [27] or N- or C-terminal deletions of K17F [11] showed that the arginine residues in positions 2 and 4 or the leucine in position 5 in pE13F played a critical role in binding affinity or in the inhibition of cAMP production. Apelin 36, K17F, and pE13F increase intracellular calcium mobilization in both NTera 2 human teratocarcinoma (NT2N) cells, which differentiate into postmitotic neurons following retinoic acid stimulation, and RBL-2H3 cells derived from rat basophils stably expressing the human APJ receptor [7, 27]. Interestingly, apelin 36, K17F, and pE13F also induce internalization of the rat and human apelin receptors [11, 27, 32, 40], whereas deletion of the C-terminal phenylalanine of K17F abolishes internalization without affecting the adenylate cyclase coupling of the apelin receptor [11]. Masri et al. have also shown that pE13F activates extracellular-signal-regulated kinases (ERKs) via a pertussis toxin-sensitive G-protein and Ras-independent pathway [25]. In addition, ERK activation by apelin is mediated by an unidentified isoform of protein kinase C (PKC) [25]. More recently, pE13F was shown to activate p70S6 kinase in human umbilical vein endothelial cells (HUVEC) or in CHO expressing the mouse apelin receptor via two intracellular cascades, one ERKdependent and the other, PI3 kinase-dependent [26].

DISTRIBUTION OF APELIN AND ITS RECEPTOR IN THE RAT BRAIN The production of a polyclonal antibody with high affinity and selectivity for K17F [9] has made it possible to visualize, for the first time, apelin neurons in the rat central nervous system. The precise central topographical distribution of apelin immunoreactivity shows that apelin-immunoreactive (IR) cell bodies are particularly abundant in the structures of the hypothalamus and medulla oblongata involved in neuroendocrine control, drinking behavior, and the regulation of arterial BP, notably in the hypothalamic SON and the magnocellular part of the paraventricular nucleus (PVN), the arcuate nucleus, the lateral reticular nucleus, and the nucleus ambiguus (Fig. 2) [33]. Conversely, apelin-IR nerve fibers are much more widely distributed in many brain regions than are neuronal apelin cell bodies. The density of IR nerve fibers and apelinergic nerve endings is highest in the inner layer of the median eminence and in the posterior pituitary [4, 32], suggesting that the apelin neurons of the SON and PVN, like the magnocellular AVP and ocytocin neurons, project into the posterior pituitary. Double immunofluorescence staining confirmed this finding, showing that apelin co-localized with AVP [9, 13] and ocytocin [4] in magnocellular hypothalamic neurons.

Apelin-IR nerve fibers also innervate the mesencephalon, the pons, the medulla oblongata, and several circumventricular organs such as the vascular organ of the lamina terminalis (OVLT), the subfornical organ (SFO), the subcommissural organ, and the area postrema [33]. Like apelin, the apelin receptor is widely distributed throughout the rat central nervous system [8, 21, 29]. In situ hybridization has shown that apelin receptor mRNA is present in the piriform and entorhinal cortices, the septum, the hippocampus, and structures containing monoaminergic neuronal cell bodies (pars compacta of the substantia nigra, dorsal raphe nucleus, and locus coeruleus). The apelin receptor is particularly abundant in the apelin-rich hypothalamic nuclei, including the SON, PVN, and arcuate nucleus, and in the pineal gland and the anterior and intermediate lobes of the pituitary gland [8]. Furthermore, double-labeling studies combining munocytochemistry and in situ hybridization have demonstrated that, in the SON and PVN, apelin receptors [28, 32], like type 1A and 1B AVP receptors (V1A and V1B) [17], are synthesized by magnocellular AVP neurons suggesting an interaction between AVP and apelin.

BIOLOGICAL ACTIONS WITHIN THE BRAIN AND PITUITARY GLAND Involvement of Apelin in the Regulation of Food Intake Apelin-IR cell bodies and nerve fibers have been detected in the hypothalamic nuclei involved in the control of food intake, including the PVN, the suprachiasmatic, arcuate, ventromedial, and dorsomedial nuclei [33]. Intravenous injection of apelin 13 does not affect food intake in fed rats or rats fasted for 24 h. Conversely, central injection of apelin 13 or apelin 12 has been reported to reduce [35], to increase [31], or to have no effect [36] on food intake in various animal models including fed and fasted rats. Apelin precursor mRNA and apelin immunoreactivity have also been detected in the periphery in rats, in the stomach and intestine [16, 21, 39]. In a murine intestinal enteroendocrine cell line (STC-1 cells) producing and secreting cholecystokinin, apelin stimulates cholecystokinin secretion, suggesting that apelin may play a physiological role in the gastrointestinal tract [39]. Boucher et al. recently demonstrated that, in experimental animal models of obesity, plasma apelin levels are significantly higher than normal but only in states associated with hyperinsulinemia [3]. Apelin appears to play a role in the peripheral and central

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FIGURE 2. Topographical distribution of apelin-IR cell bodies and nerve fibers on a sagittal section of adult colchicine-treated rat brain. Apelin-IR cell bodies and nerve fibers are shown as dots and lines, respectively. Adapted from [33]. Abbreviations: Acb, nucleus accumbens; Amb, nucleus ambiguus; Amy, amygdala; AP, anterior pituitary; AP, area postrema; Arc, arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; Cput, caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of the pituitary; LC, locus coeruleus; LPO, lateral preoptic area; LRN, lateral reticular nucleus; ME, median eminence; MPO, medial preoptic nucleus; NL, neural lobe of the pituitary; NST, nucleus of the solitary tract; OB, olfactory bulb; OVLT, vascular organ of the lamina terminalis; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVA, paraventricular thalamic nucleus; PVN, paraventricular nucleus of the hypothalamus; Re, reuniens thalamic nucleus; SCN, suprachiasmatic nucleus; SFO, subfornical organ; SN, substantia nigra; SON, supraoptic nucleus; Sp5, spinal trigeminal nucleus; S, septum; Th, thalamus; VMH, ventromedial nucleus of the hypothalamus.

regulation of food intake. However, given the conflicting nature of the data obtained to date, further investigations are required to highlight the mode of action of apelin in the control of this function.

Involvement of Apelin in the Regulation of Water Balance The neurosecretory neurons release AVP, an antidiuretic vasoconstrictor peptide, into the fenestrated capillaries of the posterior pituitary in response to changes in plasma osmolality and volemia [5, 24]. The recent report of colocalization of AVP and apelin in the magnocellular neurons of the hypothalamus and the presence of receptors for AVP and apelin on these same neurons (Fig. 3) suggest a potential apelinergic response to these stimuli. Regarding the involvement of apelin in the regulation of water balance, it is possible that, indepen-

dently of the feedback control exerted by AVP on its own release, apelin may regulate AVP release. This hypothesis has been tested in lactating rats exhibiting a reinforced phasic pattern of AVP neurons during lactation, thereby facilitating systemic AVP release to maintain body water content for optimal milk production. In this model, the intracerebroventricular (icv) injection of K17F inhibits the phasic firing activity of AVP neurons, thereby decreasing AVP release into the bloodstream, leading to aqueous diuresis [9] (Fig. 3). Similarly, a marked decrease in systemic AVP release is observed following the icv injection of K17F or pE13F in mice deprived of water for 24 h [32], a condition known to increase AVP neuron activity. These data suggest that apelin is probably released from the SON and PVN AVP cell bodies and inhibits AVP neuron activity and release by acting directly on the apelin autoreceptors expressed by AVP/apelin-containing neurons. This mechanism probably involves

Apelin: Discovery, Distribution, and Physiological Role / 791 A Apelin receptor mRNA expression

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apelin acting as a natural inhibitor of the antidiuretic effect of AVP. The colocalization and opposite biological actions of these two peptides raises questions concerning how these two peptides are regulated to maintain body fluid homeostasis. For this purpose, the effect of water deprivation on the neuronal content and systemic release of both apelin and AVP were studied. In rats deprived of water for 24 h, a large increase in the hypothalamic apelin content especially in that of the PVN and SON magnocellular neurons [13] is mirrored by a decrease in plasma apelin levels [9], suggest-

FIGURE 3. Apelin, a potent diuretic neuropeptide counteracting the effects of AVP through inhibition of AVP neuron activity and AVP release. A. Colocalization of apelin and its receptor in magnocellular AVP neurons, suggesting an interaction between apelin and AVP. B. Apelin ICV. C. Inhibition of the phasic electrical activity of SON AVP neurons. D. Decreased systemic AVP release and consequently increased aqueous diuresis. Adapted from [9]. (See color plate.)

ing that, under these conditions, apelin accumulates within AVP neurons rather than being released. The apelin response to dehydration is therefore the opposite of that of AVP, which is released faster than it is synthesized [9, 41]. This interpretation implies that apelin and AVP are released differentially by the magnocellular AVP neurons in which they are produced. Consistent with this hypothesis, double-labeling and confocal microscopy studies have demonstrated that AVP and apelin are mainly present in populations of vesicles differing in size and distribution in magnocellular neurons [9, 13] (Fig. 3).

792 / Chapter 108 These opposite regulatory patterns of apelin and AVP suggest that these molecules act in concert to maintain body fluid homeostasis. During dehydration, increases in the somatodendritic release of AVP optimize the phasic activity of AVP neurons [14, 23], facilitating the release of AVP into the bloodstream, whereas apelin accumulates in these neurons rather than being released into the bloodstream and, probably, into the nuclei. Thus, decreases in the local supply of apelin to SON and PVN AVP cell bodies may facilitate the expression by AVP neurons of optimized phasic activity by decreasing the inhibitory effects of apelin on these neurons. This concerted regulation by apelin and AVP has a biological purpose, making it possible to maintain the water balance of the organism by preventing additional water loss via the kidneys. Consistent with a role for apelin in the control of water balance, which depends not only on AVP secretion but also on the regulation of water and salt intake, apelin administered icv clearly and significantly decreases water intake in rats deprived of water for 24 h [32].

CONCLUSION The identification of apelin as the endogenous ligand of the orphan APJ receptor constitutes a major advance, both for fundamental research and, potentially, for clinical practice. It demonstrates the validity of the deorphanization approach to GPCRs for the identification of new bioactive peptides and new therapeutic targets. The experimental data obtained to date demonstrate that apelin, by inhibiting the phasic electrical activity of AVP neurons and the systemic secretion of AVP, induces water diuresis. In the periphery, apelin decreases arterial BP and increases the contractile force of the myocardium. Overall, these data show that this new circulating vasoactive peptide may play a key role in the maintenance of water balance and cardiovascular function. The development of nonpeptide agonists of the apelin receptor, based on knowledge of the structures of apelin and its receptor, could lead to new therapeutic tools for the treatment of the syndrome of inappropriate secretion of AVP, thirst disorders, and heart and kidney failure.

PERIPHERAL CARDIOVASCULAR ACTIONS Apelin also has cardiovascular effects. The mRNAencoding apelin receptors has been detected in endothelial cells of large conduit arteries, coronary vessels, and the endocardium of the right atrium [20, 29]. The injection of apelin into the bloodstream decreases arterial BP [11, 21, 32, 38], via a mechanism dependent on nitric oxide (NO) production [38]. In normotensive or hypertensive rats, apelin increases the contractile force of the myocardium via a positive inotropic effect [2, 6]. Moreover, APJ knockout mice display an enhanced vasopressor response to systemic Ang II, suggesting a counterregulatory action of apelin on Ang II [18]. Acute administration of apelin in vivo results in a vasodilatation-induced decrease in left ventricular preload and afterload and a potent increase in contractility, accompanied by a slight decrease in cardiac output. Conversely, chronic apelin infusion increases cardiac output without causing hypertrophy [1]. Apelin immunoreactivity has been found to increase in the plasma of patients in the early stages of heart failure and then to decrease during later, more severe stages of heart failure [12]. These data suggest that apelin and its receptor could constitute potential therapeutic targets in the treatment of heart failure. Indeed, the administration of apelin or of a nonpeptide agonist of the apelin receptor might improve the contractile performance of the myocardium while reducing cardiac loading and increasing aqueous diuresis in patients with heart failure.

Acknowledgments This work was funded by INSERM, the Société Française d’Hypertension Artérielle, the Fonds de la Recherche en Santé du Québec and the FranceHungary cooperation program BALATON.

References [1] Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res 2005; 65:73--82. [2] Berry MF, Pirolli TJ, Jayasankar V, Burdick J, Morine KJ, Gardner TJ, et al. Apelin has in vivo inotropic effects on normal and failing hearts. Circulation 2004; 110:II187--93. [3] Boucher J, Masri B, Daviaud D, Gesta S, Guigne C, Mazzucotelli A, et al. Apelin, a newly identified adipokine up-regulated by insulin and obesity. Endocrinology 2005; 146:1764--71. [4] Brailoiu GC, Dun SL, Yang J, Ohsawa M, Chang JK, Dun NJ. Apelin-immunoreactivity in the rat hypothalamus and pituitary. Neurosci Lett 2002; 327:193--7. [5] Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and release of posterior pituitary hormones. Science 1980; 207: 373–8. [6] Chen MM, Ashley EA, Deng DX, Tsalenko A, Deng A, Tabibiazar R, et al. Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction. Circulation 2003; 108:1432--9. [7] Choe W, Albright A, Sulcove J, Jaffer S, Hesselgesser J, Lavi E, et al. Functional expression of the seven-transmembrane HIV-1 co-receptor APJ in neural cells. J Neurovirol 2000; 6:S61--9. [8] De Mota N, Lenkei Z, Llorens-Cortes C. Cloning, pharmacological characterization and brain distribution of the rat apelin receptor. Neuroendocrinology 2000; 72:400--7.

Apelin: Discovery, Distribution, and Physiological Role / 793 [9] De Mota N, Reaux-Le Goazigo A, El Messari S, Chartrel N, Roesch D, Dujardin C, et al. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release. Proc Natl Acad Sci USA 2004; 101:10464--9. [10] Devic E, Rizzoti K, Bodin S, Knibiehler B, Audigier Y. Amino acid sequence and embryonic expression of msr/apj, the mouse homolog of Xenopus X-msr and human APJ. Mech Dev 1999; 84:199--203. [11] El Messari S, Iturrioz X, Fassot C, De Mota N, Roesch D, LlorensCortes C. Functional dissociation of apelin receptor signaling and endocytosis: implications for the effects of apelin on arterial blood pressure. J Neurochem 2004; 90:1290--301. [12] Foldes G, Horkay F, Szokodi I, Vuolteenaho O, Ilves M, Lindstedt KA, et al. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure. Biochem Biophys Res Commun 2003; 308:480--5. [13] Goazigo AR, Morinville A, Burlet A, Llorens-Cortes C, Beaudet A. Dehydration-induced cross-regulation of apelin and vasopressin immunoreactivity levels in magnocellular hypothalamic neurons. Endocrinology 2004; 145:4392--400. [14] Gouzenes L, Desarmenien MG, Hussy N, Richard P, Moos FC. Vasopressin regularizes the phasic firing pattern of rat hypothalamic magnocellular vasopressin neurons. J Neurosci 1998; 18:1879--85. [15] Habata Y, Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Hinuma S, et al. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta 1999; 13:25--35. [16] Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma S, et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem 2000; 275:21061--7. [17] Hurbin A, Boissin-Agasse L, Orcel H, Rabie A, Joux N, Desarmenien MG, et al. The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons. Endocrinology 1998; 139:4701--7. [18] Ishida J, Hashimoto T, Hashimoto Y, Nishiwaki S, Iguchi T, Harada S, et al. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo. J Biol Chem 2004; 279:26274–9. [19] Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, et al. Molecular properties of apelin: tissue distribution and receptor binding. Biochim Biophys Acta 2001; 23:2--3. [20] Kleinz MJ, Davenport AP. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells. Regul Pept 2004; 118:119-25. [21] Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, et al. Characterization of apelin, the ligand for the APJ receptor. J Neurochem 2000; 74:34--41. [22] Lenkei Z, Beaudet A, Chartrel N, De Mota N, Irinopoulou T, Braun B, et al. A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts. J Histochem Cytochem 2000; 48:1553--64. [23] Ludwig M. Dendritic release of vasopressin and oxytocin. J Neuroendocrinol 1998; 10:881--95. [24] Manning M, Lowbridge J, Haldar J, Sawyer WH. Design of neurohypophyseal peptides that exhibit selective agonistic and antagonistic properties. Fed Proc 1977; 36:1848--52. [25] Masri B, Lahlou H, Mazarguil H, Knibiehler B, Audigier Y. Apelin (65–77) activates extracellular signal-regulated kinases via a PTX-sensitive G protein. Biochem Biophys Res Commun 2002; 290:539--45.

[26] Masri B, Morin N, Cornu M, Knibiehler B, Audigier Y. Apelin (65--77) activates p70 S6 kinase and is mitogenic for umbilical endothelial cells. Faseb J 2004; 22:22. [27] Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn KY, et al. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 2003; 84:1162--72. [28] O’Carroll AM, Don AL, Lolait SJ. APJ receptor mRNA expression in the rat hypothalamic paraventricular nucleus: regulation by stress and glucocorticoids. J Neuroendocrinol 2003; 15:1095--101. [29] O’Carroll AM, Selby TL, Palkovits M, Lolait SJ. Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues. Biochim Biophys Acta 2000; 21:72--80. [30] O’Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, et al. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene 1993; 136:355--60. [31] O’Shea M, Hansen MJ, Tatemoto K, Morris MJ. Inhibitory effect of apelin-12 on nocturnal food intake in the rat. Nutr Neurosci 2003; 6:163--7. [32] Reaux A, De Mota N, Skultetyova I, Lenkei Z, El Messari S, Gallatz K, et al. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain. J Neurochem 2001; 77:1085--96. [33] Reaux A, Gallatz K, Palkovits M, Llorens-Cortes C. Distribution of apelin-synthesizing neurons in the adult rat brain. Neuroscience 2002; 113:653--62. [34] Reaux A, Fournie-Zaluski MC, David C, Zini S, Roques BP, Corvol P, et al. Aminopeptidase A inhibitors as potential central antihypertensive agents. Proc Natl Acad Sci USA 1999; 96:13415– 20. [35] Sunter D, Hewson AK, Dickson SL. Intracerebroventricular injection of apelin-13 reduces food intake in the rat. Neurosci Lett 2003; 353:1--4. [36] Taheri S, Murphy K, Cohen M, Sujkovic E, Kennedy A, Dhillo W, et al. The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats. Biochem Biophys Res Commun 2002; 291:1208--12. [37] Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 1998; 251:471--6. [38] Tatemoto K, Takayama K, Zou MX, Kumaki I, Zhang W, Kumano K, et al. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism. Regul Pept 2001; 99:87--92. [39] Wang G, Anini Y, Wei W, Qi X, Am OC, Mochizuki T, et al. Apelin, a new enteric peptide: localization in the gastrointestinal tract, ontogeny, and stimulation of gastric cell proliferation and of cholecystokinin secretion. Endocrinology 2004; 145: 1342–8. [40] Zhou N, Fan X, Mukhtar M, Fang J, Patel CA, DuBois GC, et al. Cell-cell fusion and internalization of the CNS-based, HIV-1 coreceptor, APJ. Virology 2003; 307:22--36. [41] Zingg HH, Lefebvre D, Almazan G. Regulation of vasopressin gene expression in rat hypothalamic neurons. Response to osmotic stimulation. J Biol Chem 1986; 261:12956--9. [42] Zini S, Demassey Y, Fournie-Zaluski MC, Bischoff L, Corvol P, Llorens-Cortes C, et al. Inhibition of vasopressinergic neurons by central injection of a specific aminopeptidase A inhibitor. Neuroreport 1998; 9:825--8. [43] Zini S, Fournie-Zaluski MC, Chauvel E, Roques BP, Corvol P, Llorens-Cortes C. Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci USA 1996; 93:11968--73.

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109 Urotensin II and Urotensin II–Related Peptide ISABELLE LIHRMANN, HOWARD A. BERN, AND HUBERT VAUDRY

cells. Consistent with these notions, the cDNAs encoding the UII precursors have been cloned in various mammalian species including mouse [16], rat [16], pig [46], monkey [23], and human [14]. Recently, a paralog of UII named urotensin II–related peptide (URP) has been characterized in the rat brain, and the URP cDNAs have been cloned in mouse, rat, and human [62]. The occurrence of UII, URP, and their receptor in the central nervous system (CNS), together with the variety of their behavioral effects, suggests that these peptides act as neurotransmitters and/or neuromodulators.

ABSTRACT Urotensin II (UII) is a cyclic neuropeptide initially isolated from the urophysis of teleost fish. In the central nervous system (CNS) of tetrapods, UII is almost exclusively produced in motoneurons located in brainstem nuclei and in the ventral horn of the spinal cord. Recently, a paralog of UII called urotensin II–related peptide (URP) has been characterized in mammals. The cyclic region of UII and URP, which has been fully conserved during evolution, plays a crucial role in the biological activity of these peptides. A single receptor for UII and URP, called UT receptor, has been identified so far. In agreement with the widespread distribution of UT receptor in the CNS, it has been shown that UII and URP exert a large array of activities in the brain.

STRUCTURE Gene Structure The gene encoding prepro-UII comprises five exons and maps on human chromosome 1p36. In human, two distinct precursor isoforms with 139 (isoform a) and 124 (isoform b) amino acids have been characterized. These two isoforms differ only in their N-terminal extremity and give rise to the same mature UII peptide (Fig. 2A). In all other mammalian species studied so far (mouse, rat, pig, and monkey), only isoform b has been identified [16, 23, 46]. In fish, two variants of isoform b are expressed as a result of genome duplication. The gene encoding prepro-URP has five exons and maps on human chromosome 3q23. The human URP precursor consists of 119 amino acids and exhibits very low sequence identity with the UII precursor (<20%) (Fig. 2B).

DISCOVERY The caudal neurosecretory system of teleost fish incorporates a neurohemal organ called the urophysis, which contains various regulatory factors including neurotransmitters and neuropeptides (Fig. 1). Urotensin II (UII) is a cyclic neuropeptide that was originally isolated from the goby urophysis on the basis of its ability to induce contractions of the trout hindgut [4, 50]. In all species of fish investigated so far, the UII sequence encompasses 12 amino acids with a fully conserved cyclic region at positions 6–11 that exhibits some structural similarity to somatostatin. It had long been thought that UII was the appanage of the fish urophysis [4]. However, characterization of UII from the brain of the European green frog [13] has revealed that a UIIencoding gene actually exists in certain species of tetrapods and that this gene is not expressed only in the caudal portion of the spinal cord but also in brain nerve Handbook of Biologically Active Peptides

Processing of the Precursors The general organization of the UII and URP precursors is identical, the sequence of each peptide being located at the C-terminal extremity of the protein [14, 16, 23, 46] (Fig. 2). Interestingly, somatostatin-14, which

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796 / Chapter 109 Hypothalamo neurohyposial system

Caudal neurosecretory system

Caudal neurosecretory neurons (Dahlgren cells)

Spinal cord

Central canal

.....

..... .. ..... ... . ..... ....

UROPHYSIS

FIGURE 1. The fish caudal neurosecretory system consists of large UII-immunoreactive neurons located in the caudal portion of the spinal cord in the last (∼5–6) preterminal vertebrae. Large unmyelinated axons from these cells extend lateroventrally and turn caudally to join the spinal-urophysial tract. UII is released in the vicinity of fenestred capillaries in the urophysis and ultimately joins the renal portal system. (See color plate.)

possesses a structural similarity to UII in the biologically important cyclic region, is also located at the C-terminal end of its precursor (see chapter on Somatostatin in this section of the Handbook). Proteolytic cleavage of pro-UII and pro-URP at dibasic (Arg-Lys) or tribasic (Lys-Lys-Arg) sites has the potential to generate mature peptides whose sequences may vary from 8 (URP) to 17 (mouse UII) amino acids in length. In tetrapods, up to now, the structure of UII has only been determined in the frog brain [13] and in the porcine [46] and human [10] spinal cord. In the human brainstem and in the SW-13 adrenocortical carcinoma cell line, additional UII-related peptides have been isolated that may correspond to large processing products at one of the upstream Arg-Lys dibasic sites [10, 64]. The UIIconverting enzyme has been partially characterized in human epicardial mesothelial cells, vascular endothelial cells, and cardiac fibroblasts by the study of the processing of the 25-amino-acid C-terminal fragment of pro-UII [58]. The results show the occurrence of furin-like activity capable of converting this precursor fragment into UII.

Peptide Structure Strong evolutionary pressure has acted to preserve the primary structure of the C-terminal portion of UII, whereas the N-terminus is highly variable (Fig. 3). Thus,

in all species examined so far, the amino acid sequence of the cyclic region of UII and URP (Cys-Phe-Trp-LysTyr-Cys) has been fully conserved. In the UII sequence, the cyclic portion is preceded by an acidic residue (Asp or Glu) and is flanked at its C-terminus by a hydrophobic residue (Val or Ile). In the URP sequence, the cyclic region is flanked by an Ala residue at its N-terminus and a Val residue at its C-terminus (Fig. 3).

LOCALIZATION In teleost fish, UII is primarily produced by neurosecretory cells located in the caudal portion of the spinal cord, called Dalghren cells, that project axons into the urophysis [4]. To date, UII-containing cell bodies have not been detected in the brain of fish [49], although UII has been isolated and sequenced from extracts of trout and skate brains [70]. In the CNS of tetrapods, UII and URP are almost exclusively expressed in motoneurons located in the brainstem and the dorsal horn of the spinal cord [9, 14, 16, 22, 51, 52]. In particular, in the rat brain, a high concentration of UII mRNA has been observed in motoneurons located in the trigeminal, abducens, fascial, ambiguus, and hypoglossal nuclei [16] (Fig. 4). Similarly, intense expression of the UII gene occurs in the ventral horn of the spinal cord both in adult and developing rat [15, 16]. UII mRNA is also

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FIGURE 2. Structure of the human (A) prepro-UII and (B) prepro-URP genes. The prepro-UII gene comprises five exons and encodes two precursor isoforms (variants a and b) that differ only at their N-terminal extremity. The same mature UII peptide is generated from both isoforms a and b following proteolytic cleavage at a tribasic site (KKR). The prepro-URP gene comprises five exons. The URP sequence is localized at the C-terminal extremity of the URP precursor and the mature peptide is generated by proteolytic cleavage at a dibasic site (KR). (See color plate.)

798 / Chapter 109 Species

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Zebrafish UIIα Zebrafish UIIβ Goby UII Trout UII Sucker UIIA Sucker UIIB Carp UIIα Carp UIIβ1 Carp UIIβ2 Carp UIIγ Flounder UII Sturgeon UII Paddlefish UII Skate UII Dogfish UII Lamprey UII

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Primary structure pGlu

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Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

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FIGURE 3. Alignment of UII and URP sequences identified so far. AA, number of amino acids. (See color plate.)

Cx

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[125I]UII binding sites UII neurons FIGURE 4. Schematic drawing showing the distribution of UII neurons (red dots) and [125I]UII binding sites (blue staining) in the rat central nervous system. (See color plate.)

Urotensin II and Urotensin II–Related Peptide / 799 detectable, albeit at a much lower level, in various brain regions, including the caudate nucleus, amygdala, thalamus, hippocampus, hypothalamus, substantia nigra, cerebral peduncle, pons, medulla oblongata, and cerebellum [2, 14, 62, 68]. In the frog brain, long varicose processes coursing along the ventral surface of the midtelencephalon to the medulla oblongata have been described [9]. Globally, the profiles of expression of the UII and URP genes in the CNS of mammals are similar, except in the human spinal cord, where prepro-URP levels are much lower [62]. A recent study has shown that, in the mouse spinal cord, the UII and URP precursors are generally expressed in the same motoneurons [51].

RECEPTORS AND SIGNALING CASCADES The UT Receptor The orphan G-protein-coupled receptor GPR14 (also termed SENR) has been initially characterized by a homology-based approach [43, 65]. GPR14 has been subsequently identified by reversed pharmacology as the UII receptor [2] and then renamed UT receptor. This receptor exhibits the highest sequence identity (∼25%) with somatostatin, opioid, and galanin receptors [65]. In the human, the UT receptor comprises 389 amino acids and is encoded by an intronless gene located on chromosome 17q25.3. The UT receptor possesses two N-glycosylation sites in the C-terminal domain [6] and several putative phosphorylation sites in the second and third intracellular loops and the C-terminal domain. Two methionine residues within the fourth transmembrane domain of the UT receptor interact with the phenylalanine residue present in the cyclic region of UII and URP [6].

Signaling Cascades The biological effects of UII are mediated through activation of a number of transduction pathways. In arterial smooth muscle cells, the UT receptor is positively coupled to phospholipase C (PLC) via the heterotrimeric Gαq protein, thus stimulating protein kinase C (PKC) and causing Ca2+ mobilization from intracellular calcium stores [27, 48, 56]. UII also activates the small GTPase RhoA and its downsteam effector Rhokinase [59]. In rat spinal cord motoneurons, UII stimulates the adenylyl cyclase/protein kinase A (PKA) cascade and provokes Ca2+ influx through N-type Ca2+ channels [24]. In frog motoneuron terminals, the effect of UII on neurotransmitter release is mediated via both the adenylyl cyclase/PKA and the PLC/PKC pathways [7]. UII binds tightly to its receptor [1, 19, 54] and the UII receptor desensitizes slowly [53, 57], two phenom-

ena that probably account for the prolonged duration of the contractile effects of UII [8, 19].

Distribution of the UT Receptor In peripheral organs, the highest concentrations of UT receptor mRNA are found in the heart and the thoracic aorta [2, 23, 26, 39, 44, 65]. UT receptor transcripts are also found, albeit at a much lower level, in the kidney, bladder, pancreas, intestine, skeletal muscle, esophagus, lung, adipose, and endocrine tissues [2, 23, 39, 65, 68]. In the CNS, the UT receptor is widely expressed notably in the piriform cortex, the pyramidal cell layer and the dentate gyrus of the hippocampus, the amygdala, several tegmental nuclei, the pontine nuclei, the medulla oblongata, and the cerebellum [26, 32, 68]. [125I]UII-binding sites have also been visualized in various brain nuclei [12, 32, 42] (Fig. 4), indicating that these receptors are functional.

Structure-Activity Relationships The cyclic region that is common to UII and URP (Fig. 3) plays a pivotal role in the biological activity of these peptides on native and recombinant UII receptors [11, 25, 30, 31, 34, 41]. Thus, disruption of the disulfide bridge or point substitution of individual amino acids within the cyclic region by Ala or the Disomer markedly reduces the potency of UII and URP [11, 25, 33, 34, 41]. Although the Asp/Glu residue flanking the cyclic core of UII on its N-terminus has been fully preserved among vertebrates, in the URP sequence, this residue is replaced by an alanine moiety, indicating that it does not play a crucial role for the activity of the molecule. In contrast, the presence of the conserved residues (Val or Ile) at the C-terminal extremity of UII and URP contributes to the biological potency. Determination of the solution structure of UII [3] and URP [11] by nuclear magnetic resonance (NMR) spectroscopy has shown that the cyclic region adopts a highly ordered compact conformation, whereas the N-terminal segment of UII is disordered. In both UII and URP, the side chains of the Phe, Trp, and Tyr residues of the cyclic region are oriented on the same side of the peptides, suggesting that they may all be involved in docking in the binding pocket of the UII receptor.

BIOLOGICAL ACTIONS WITHIN THE BRAIN Central Regulation of Cardiovascular Functions The genes encoding the UT receptor as well as the UII and URP precursors are all expressed in brainstem

800 / Chapter 109 nuclei associated with regulation of cardiovascular functions [2, 14, 26, 32, 62, 68]. In the conscious rat, intracerebroventricular (ICV) administration of UII causes hypertension and tachycardia [38]. In the anesthesized rat, microinjection of UII into the A1 area of the brain stem provokes bradycardia and a hypotensive response, whereas injection of UII into the paraventricular or arcuate nuclei of the hypothalamus induces tachycardia and hypertension [40]. In freely moving sheep, ICV administration of UII causes chronotropic and inotropic responses [69]. In conscious trout, ICV injection of low doses of UII increases heart rate without modifying blood pressure, whereas a higher dose of UII provokes hypertension [35, 36]. These observations indicate that central administration of UII causes site- and dosedependent cardiovascular responses in both fish and mammals.

Neuroendocrine Actions The occurrence of the UT receptor mRNA in several hypothalamic nuclei involved in the neuroendocrine control of the pituitary, including the paraventricular, supraoptic, and arcuate nuclei [32, 68], suggests that UII may regulate neuroendocrine functions. Indeed, ICV administration of UII provokes an increase in plasma prolactin and thyroid-stimulating hormone (TSH) in the rat [26] and adrenocorticotropic hormone (ACTH) in sheep [69]. Thus, the stimulatory effect of UII on prolactin, TSH, and ACTH release is probably mediated through activation of thyrotrophin-releasing hormone (TRH) and corticotrophin-releasing hormone (CRH) neurons that are both located in the paraventricular nucleus (see chapters on TRH and CRH in this section of the Handbook).

Behavioral Responses Consistent with the widespread distribution of GPR14 mRNA and UII binding sites in the CNS [12, 26, 32, 42], central administration of UII elicits a number of behavioral effects in rodents [17, 26, 45]. In particular, ICV injection of UII increases rearing, grooming, and motor activity in a familiar environment [26], and induces anxiogenic and depressantlike effects [17, 45]. The occurrence of a high density of UII receptors in cholinergic neurons of the mesopontine tegmental area [12, 32] suggests that UII may be involved in the control of functions regulated by cholinergic pathways. In agreement with this notion, it has been recently shown that local administration of UII into the pedunculopontine nucleus modulates rapid-eye-movement sleep through direct activation of brainstem cholinergic neurons [29].

Motor Functions ICV injection of UII increases horizontal and vertical locomotor activity in mice [17, 26] and stimulates motor activity in trout [35]. The expression of the mRNA encoding UII and URP precursors and the UII receptor in motoneurons of the brainstem and spinal cord [14– 16, 22, 32, 39, 47, 51, 52] strongly suggests that UII and URP may act as autocrine modulators of motoneuron activity. Consistent with this hypothesis, it has been shown that UII stimulates neurotransmitter release from frog motor-nerve terminals [7].

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL SIGNIFICANCE In vitro studies have shown a great variability in the contractile efficacity of UII in isolated human arteries and veins [18]. In healthy volunteers, intradermal injection of UII causes local vasoconstriction [37] and intrabrachial artery infusion of UII provokes a dosedependent decrease in forearm blood flow [5]. The expression of UII is enhanced in myocardial tissue from patients with congestive heart failure [20] and an increase in plasma or urine concentrations of UII has been reported in various pathological states including essential hypertension [44], congestive heart failure [21, 55], renal dysfunction [68], cirrhosis [28], and diabetes mellitus [67]. There is also a positive correlation between blood pressure and the concentration of UII in the cerebrospinal fluid of hypertensive patients [66]. Whether these changes in circulating UII concentrations are due to an increase in UII production or a decrease in UII excretion remains to be determined. The fact that UII inhibits glucose-induced insulin secretion [60, 61] suggests that the increase in plasma UII concentration observed in diabetic patients may contribute to the etiology of the disease. In support of this notion, the association of polymorphisms in the UII gene with type 2 diabetes has recently been reported in Chinese and Japanese subjects [63, 71].

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802 / Chapter 109 [34] Labarrère P, Chatenet D, Leprince J, Marionneau C, Loirand G, Tonon MC, Dubessy C, Scalbert E, Pfeiffer B, Renard P, Calas B, Pacaud P and Vaudry H. Structure-activity relationships of human urotensin II and related analogues on rat aorta ring contraction. J Enz Inh Med Chem 2003;18:77–88. [35] Lancien F, Leprince J, Mimassi N, Mabin D, Vaudry H and Le Mével JC. Central effects of native urotensin II on motor activity, ventilatory movements and heart rate in the trout Oncorhynchus mykiss. Brain Res 2004;1023:164–74. [36] Le Mével JC, Olson KR, Conklin D, Waugh D, Smith DD, Vaudry H and Conlon JM. Cardiovascular actions of trout urotensin II in the conscious trout Oncorhynchus mykiss. Am J Physiol 1996;271: R1335–43. [37] Leslie SJ, Denvir M and Webb DJ. Human urotensin II causes vasoconstriction in the human skin microcirculation. Circulation 2000;102(Suppl. II):I–113 (abstract). [38] Lin Y, Tsuchihashi T, Matsumura K, Abe I and Iida M. Central cardiovascular action of urotensin II in conscious rats. J Hypertens 2003;21:159–65. [39] Liu Q, Pong SS, Zeng Z, Zhang Q, Howard AD, Williams DL, Davidoff M, Wang R, Austin CP, McDonald TP, Bai C, George SR, Evans JF and Caskey CT. Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 1999;266:174–8. [40] Lu Y, Zou CJ, Huang DW and Tang CS. Cardiovascular effects of urotensin II in different brain areas. Peptides 2002;23:1631– 5. [41] McMaster D, Kobayashi Y, Rivier J and Lederis K. Characterization of the biologically and antigenically important regions of urotensin II. Proc West Pharmacol Soc 1986;29:205–8. [42] Maguire JJ, Kuc RE and Davenport AP. Orphan-receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin-1. Br J Pharmacol 2000;131:441–6. [43] Marchese A, Heiber M, Nguyen T, Heng HH, Saldivia VR, Cheng R, Murphy PM, Tsui LC, Shi X, Gregor P, George SR, O’Dowd BF and Docherty JM. Cloning and chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8, neuropeptide Y, and somatostatin receptors. Genomics 1995;29:335–44. [44] Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N and Hirata Y. Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 2001;19:2185–90. [45] Matsumoto Y, Abe M, Watanabe T, Adachi Y, Yano T, Takahashi H, Sugo T, Mori M, Kitada C, Kurokawa T and Fujino M. Intracerebroventricular administration of urotensin II promotes anxiogenic-like behaviors in rodents. Neurosci Lett 2004;358: 99–102. [46] Mori M, Sugo T, Abe M, Shimomura Y, Kurihara M, Kitada C, Kikuchi K, Shintani Y, Kurokawa T, Onda H, Nishimura O and Fujino M. Urotensin II is the endogenous ligand of a G-proteincoupled orphan receptor, SENR (GPR14). Biochem Biophys Res Commun 1999;265:123–9. [47] Nothacker HP, Wang Z, McNeill AM, Saito Y, Merten S, O’Dowd B, Duckles SP and Civelli O. Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. Nat Cell Biol 1999;1:383–5. [48] Opgaard OS, Nothacker H, Ehlert FJ and Krause DN. Human urotensin II mediates vasoconstriction via an increase in inositol phosphates. Eur J Pharmacol 2000;406:265–71. [49] Owada K, Kawata M, Akaji K, Takagi A, Moriga M and Kobayashi H. Urotansion II-immunoreactive neurons in the caudal neurosecretory system of freshwater and seawater fish. Cell Tissue Res 1985;239:349–54.

[50] Pearson D, Shively JE, Clark BR, Geschwind II, Barkley M, Nishioka RS and Bern HA. Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc Natl Acad Sci USA 1980;77:5021–4. [51] Pelletier G, Lihrmann I, Dubessy C, Luu-The V, Vaudry H and Labrie F. Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord. Neuroscience 2005;132:689– 96. [52] Pelletier G, Lihrmann I and Vaudry H. Role of androgens in the regulation of urotensin II precursor mRNA expression in the rat brainstem and spinal cord. Neuroscience 2002;115:525– 32. [53] Proulx CD, Simaan M, Escher E, Laporte SA, Guillemette G and Leduc R. Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization. Biochem J 2005;385:115– 23. [54] Qi JS, Minor LK, Smith C, Hu B, Yang J, Andrade-Gordon P and Damiano B. Characterization of functional urotensin II receptors in human skeletal muscle myoblasts: comparison with angiotensin II receptors. Peptides 2005;26:683–90. [55] Richards AM, Nicholls MG, Lainchbury JG, Fisher S and Yandle TG. Plasma urotensin II in heart failure. Lancet 2002;360:545– 6. [56] Rossowski WJ, Cheng BL, Taylor JE, Datta R and Coy DH. Human urotensin II-induced aorta ring contractions are mediated by protein kinase C, tyrosine kinases and Rho-kinase: inhibition by somatostatin receptor antagonists. Eur J Pharmacol 2002;438:159–70. [57] Russell F. Emerging roles of urotensin-II in cardiovascular diseases. Pharmacol Ther 2004;103:223–43. [58] Russell FD, Kearns P, Toth I and Molenaaar P. Urotensin-IIconverting enzyme activity of furin and trypsin in human cells in vitro. J Pharmacol Exp Ther 2004;310:209–14. [59] Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P and Loirand G. Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 2001;88:1102–4. [60] Silvestre RA, Egido EM, Hernandez R, Leprince J, Chatenet D, Tollemer H, Chartrel N, Vaudry H and Marco J. Urotensin-II is present in pancreatic extracts and inhibits insulin release in the perfused rat pancreas. Eur J Endocrinol 2004;151:803–9. [61] Silvestre RA, Rodriguez-Gallardo J, Egido EM and Marco J. Inhibition of insulin release by urotensin II—a study on the perfused rat pancreas. Horm Metab Res 2001;33:379–81. [62] Sugo T, Murakami Y, Shimomura Y, Harada M, Abe M, Ishibashi Y, Kitada C, Miyajima N, Suzuki N, Mori M and Fujino M. Identification of urotensin II-related peptide as the urotensin IIimmunoreactive molecule in the rat brain. Biochem Biophys Res Commun 2003;310:860–8. [63] Sun HX, Du WN, Zuo J, Wu GD, Shi GB, Shen Y, Qiang BQ, Yao ZJ, Hang JM, Wang H, Huang W, Chen Z and Fang FD. The association of two single nucleotide polymorphisms in PRKCZ and UTS2 respectively with type 2 diabetes in Han people of northern China. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2002;24:223–7. [64] Takahashi K, Totsune K, Murakami O and Shibahara S. Expression of urotensin II and urotensin II receptor mRNAs in various human tumor cell lines and secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells. Peptides 2001;22:1175–9. [65] Tal M, Ammar DA, Karpuj M, Krizhanovsky V, Naim M and Thompson DA. A novel putative neuropeptide receptor expressed in neural tissue, including sensory epithelia. Biochem Biophys Res Commun 1995;209:752–9.

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cause potent inotropic and chronotropic actions. Hypertension 2003;42:373–9. [70] Waugh D and Conlon JM. Purification and characterization of urotensin II from the brain of a teleost (trout, Oncorhynchus mykiss) and an elasmobranch (skate, Raja rhina). Gen Comp Endocrinol 1993;92:419–27. [71] Wenyi Z, Suzuki S, Hirai M, Hinokio Y, Tanizawa Y, Matsutani A, Satoh J and Oka Y. Role of urotensin II gene in genetic susceptibility to type 2 diabetes mellitus in Japanese subjects. Diabetologia 2003;46:972–6.

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110 Brain/B-Type Natriuretic Peptide (BNP) and C-Type Natriuretic Peptide (CNP) YOSHIO TAKEI

amphibians, and fishes [36]. In the killifish and eel, it was initially named ANP-like or BNP-like peptide, but C-type NP (CNP) is now generally used in all species [38]. CNP is most abundantly synthesized and stored in the brain. Thus, CNP is the true brain peptide of the NP family in this sense. However, its peripheral actions have attracted more attention than the central actions, as revealed by the CNP gene-deficient mice. CNP has a structural characteristic of lacking C-terminal extension from the intramolecular ring. CNP has been identified in all vertebrate classes ranging from cyclostomes to mammals [15]. Phylogenetic studies of the NP family revealed the evolutionary history of this hormone family in vertebrates [12, 15]. In ray-finned fishes including teleostei, four CNPs have been isolated from the brain in addition to ANP and BNP, and these are named CNP1, 2, 3, and 4 (Fig. 2). Synteny of the neighboring genes on the same chromosome showed that CNP4 is an ortholog of mammalian CNP. CNP1 is a major brain peptide of teleost fish and thus initially isolated from fish brains. Despite the difference in their origin, mammalian CNP(4) and teleost CNP1 are structurally most similar to one another at the mature sequence of 22 amino acid residues (Fig. 1). CNP3 is localized in tandem with ANP and BNP on the same chromosome, showing that ANP and BNP are duplicated from CNP3. A novel NP has been identified in the cardiac ventricle of teleostean and chondrostean fish and named ventricular NP (VNP), in addition to ANP and BNP [14, 38]. Linkage analyses in the rainbow trout show that VNP is also produced from CNP3 by tandem duplication [13]. Only a single CNP has been isolated from the brain and heart of cartilaginous fish (sharks and rays) and cyclostomes (lamprey), whereas a single NP with long C-terminal sequence and amidated C-terminal has been found in the brain and heart of another cyclostome, hagfish, the most primitive extant vertebrate species [15, 16, 37].

ABSTRACT Among the natriuretic peptide (NP) family, brain (B-type) and C-type NP (BNP and CNP) were initially regarded as brain peptides. However, BNP is now shown to be a circulating hormone secreted from the heart. CNP is most abundant in the brain, but its cerebral actions are not fully understood yet. In fact, CNP gene knockout mice exhibit dwarfism because of the lack of its peripheral action on endochondral ossification. Four CNPs (CNP1 through 4) are found in teleost fish, of which CNP4 is an ortholog of mammalian CNP. Phylogenetic and comparative genomic analyses of all NPs thus far obtained show that CNP4 is an ancestral molecule of the NP family and that ANP and BNP are produced by tandem duplication from CNP3 in early bony fish.

DISCOVERY The natriuretic peptides (NPs) have a basic structure consisting of an intramolecular ring of 17 amino acid residues and N-terminal and C-terminal extensions of variable length (Fig. 1). Following the discovery of potent diuretic/natriuretic and hypotensive factor(s) in the rat atria in 1981 and subsequent isolation of its factor, atrial NP (ANP) in 1983 [20, 38], the second member of the NP family, was isolated from the porcine brain in 1988 and named brain NP (BNP) [35]. In contrast to pigs, BNP and its gene transcript were practically undetectable in the brain of rodents and humans, but it is synthesized abundantly in the heart, particularly in the failing ventricle [24]. BNP is also identified in the heart of birds, amphibians, and fishes [12, 38]. Therefore, it is now thought that B of BNP does not stand for “brain” but “B-type.” In 1990, a third member of the NP family was isolated from the brain of mammals, Handbook of Biologically Active Peptides

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806 / Chapter 110 Atrial Natriuretic Peptide (ANP) sturgeon NRGSSGCFGSRIDRIGSMSSMGCGGSRK-NH 2 eel SKSSSPCFGGKLDRIGSYSGLGCNS-RK-NH 2 trout SKAVSGCFGARMDRIGTSSGLGCSPKRRS bullfrog SSDCFGSRIDRIGAQSGMGC-GRRF man SLRRSSCFGGRMDRIGAQSGLGCNSFRY rat SLRRSSCFGGRIDRIGAQSGLGCNSFRY B-type Natriuretic Peptide (BNP) sturgeon KRYSGCFGRRLDRIGSMSALGCNGGSRLSYKRS eel NRYSGCFGRKMDRIGSMSSLGCKTVSKRN trout KRYSGCFGRRLDRIGSMSSLGCTTVGKYNAKTR bullfrog SNCFGRRIDRIDSVSGMGCNGSRNRYP chicken MMRDSGCFGRRIDRIGSLSGMGCNGSRKN man SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH rat NSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF C-type Natriuretic Peptide (CNP) spotted dogfish GPSRGCFGVKLDRIGAMSGLGC sturgeon QQGRGCFGMKLDRIGSMSGLGC eel GWNRGCFGLKLDRIGSLSGLGC trout GWNRGCFGLKLDRIGSMSGLGC bullfrog GYSRGCFGVKLDRIGAFSGLGC chicken GLSRSCFGLKLDRIGSMSGLGC man GLSKGCFGLKLDRIGSMSGLGC Ventricular Natriuretic Peptide sturgeon SMNGCFGNRIERIGSWSSLGCNNSRFGSKKRIF eel KSFNSCFGTRMDRIGSWSGLGCNSLKNGTKKKIFGN trout KSFNSCFGNRIERIGSWSGLGCNNVKTGNKKRIFGN

FIGURE 1. Amino acid sequences of the natriuretic peptide family sequenced to date. Brackets show disulfide bonds. Shaded amino acids show that they are conserved in more than half of the species.

C-type Natriuretic Peptide-1 (CNP1) eel GWNRGCFGLKLDRIGSLSGLGC trout GWNRGCFGLKLDRIGSMSGLGC killifish GWNRGCFGLKLDRIGSMSGLGC medaka GWNRGCFGLKLDRIGSMSGLGC zebrafish GWNRGCFGLKLDRIGSMSGLGC fugu GWNRGCFGLKLDRIGSMSGLGC C-type Natriuretic Peptide-2 (CNP2) medaka VAGGGCFGMKMDRIGSISGLGC zebrafish GVARGCFGMKVDRIGVISGLGC fugu VGGRGCFGMKIDRIGSISGLGC C-type Natriuretic Peptide-3 (CNP3) medaka GGMRSCFGVRLERIGSFSGLGC zebrafish GDLRSCFGVRLERIGSFSGLGC fugu GGLRSCFGVRLARIGSFSGLGC C-type Natriuretic Peptide-4 (CNP4) medaka TSRSGCFGHKMDRIGTISGMGC zebrafish TARSGCFGHKMDRIGTMSGMGCQPSHYHSINNLS fugu SSRSGCFGHKMDRIGTISGMGC trout AARSGCFGHKMDRIGTISGMGC

FIGURE 2. Amino acid sequences of four types of C-type natriuretic peptides in teleost species. Brackets show disulfide bonds. Shaded amino acids show that they are conserved in more than half of the species.

Brain/B-Type Natriuretic Peptide (BNP) and C-Type Natriuretic Peptide (CNP) / 807 Phylogenetic analyses of these NPs from primitive species showed that shark CNP is CNP3 type and lamprey CNP and hagfish NP are CNP4 type (Fig. 3). These results unveiled an evolutionary history of the NP family of molecules. An ancestral molecule of the NP family is CNP4, which is duplicated to produce CNP3 during evolution from agnathans to gnathostomes, whereas CNP4 disappeared in the cartilaginous fish lineage. In the bony fish lineage, four CNPs still exist and ANP, BNP, and VNP are produced by tandem duplication from CNP3. It should be determined whether other NPs identified only in bony fish are also present in mammals and other tetrapods. In amphibians, CNP3 and CNP4 cDNAs have been cloned from the bullfrog.

responsive element, and the GATA sequence have been identified. The cDNAs coding for CNP have been sequenced in several species of mammals, chicken, frogs, several species of teleost fish, sturgeon, and two species of elasmobranchs (Table 1). The CNP gene is expressed also in the venom gland of snakes and duckbill, an egglaying mammal [38]. The genomic DNA of the CNP gene, which is localized on a different chromosome from the ANP/BNP gene, has been sequenced in mammals and teleost fishes (Table 1). A cyclic AMP responsive element-like sequence and GC boxes are localized in the promoter region of the CNP gene.

DISTRIBUTION OF mRNA STRUCTURE OF THE PRECURSOR mRNA AND GENE The cDNAs encoding BNP have been cloned in several species of mammals, chicken, frog, and several species of bony fishes including chondrostei (Table 1) [38]. The precursor sequence of BNP is most versatile among the members of the NP family in mammals, but it is conserved between mammals and other tetrapod species. All BNP mRNA sequences thus far cloned are characterized by the presence of AUUUA repeats in the 3′-untranslated region, which is known to facilitate mRNA degradation. The genomic DNA sequences of the BNP gene, which is localized in tandem with the ANP gene on the same chromosome, are also determined in several species of mammals and teleost fish (Table 1). In the vicinity of the human BNP gene, cisacting elements such as activator protein-1 site, serum

Cyclostomes hagfish hfNP

Cartilaginous fish

Bony fish

elasmobranch holocephalan

teleostei chondrostei

lamprey C4

C4

CNP-4 type

C3

C1 C2 C3 C4

A B V

Although BNP was initially isolated from the brain, its brain concentration is much smaller than in the heart [24]. Furthermore, BNP is much less abundant than ANP in the brain of rodents and humans [1]. Therefore, BNP is not a brain peptide but a cardiac hormone that acts in the periphery for cardiovascular and fluid homeostasis just like ANP. Recently, much attention has been paid to BNP in terms of diagnosis and treatment of cardiac failure (see Minamino, Morio and Nishikimi’s Chapter on Natriuretic peptides (ANP, BNP, CNP) in the Cardovascular Peptides Section). In the pig brain, BNP is localized preferentially in the striatum, medulla-pons region, hypothalamus, and spinal cord when each brain region is measured by specific radioimmunoassay [41]. In situ hybridization histochemistry of the rat brain showed that BNP mRNA was not detectable but that ANP mRNA is expressed in the anterior olfactory nuclei; limbic cortices; dorsal endopiriform nucleus; hippocampal subfield CA1;

Tetrapods

mammal amphibian A

B

C3

C4

FIGURE 3. Diagram showing evolutionary history of the natriuretic peptide family in vertebrates. A, ANP; B, BNP; C1, CNP1; C2, CNP2; C3, CNP3; C4, CNP4; V, VNP.

808 / Chapter 110 TABLE 1. Accession numbers of BNP and CNP cDNA and genomic DNA. BNP Animals Mammals human chimpanzee gorilla orangutan rat mouse hamster guinea pig hopping mouse sheep pig ox dog cat Birds chicken Amphibians Xenopus bullfrog Fishes eel tilapia fugu medaka zebrafish salmonids sturgeon spotted dogfish spiny dogfish

CNP

Genome

cDNA

Genome

cDNA

AB037521 AB037522 AB037523 AB037524 M60266 AB039044 D17314

M25296

D90337

AC013435

M25297 AB03905

D90219 D28873

NM010933

AF193572 AF460241 AF037466 M22477 M31777 AF425738

M64710 M64758 Z48477 AF253495

AF295101

E02222

BU417608

BG346231

BJ069969 D17413 D88022 AB087285

AB087284 AB089934 AB099700 CN023754 CK883047 AB087729

cortical amygdaloid nuclei; medial habenula; anteroventral, periventricular, and arcuate nuclei; periventricular stratum; zona incerta; mammillary nuclei; inferior olive; nucleus ambiguous; and pontine paragigantocellular nuclei [19]. In rodents and humans, ANP appears to play a more significant role in brain function than BNP. In bony fishes, BNP mRNA expression is also sparse in the brain and abundant in the heart. BNP has not been localized in the brain of nonmammals yet. CNP peptide and its mRNA expression are more abundant in the brain than in the periphery in all mammalian species thus far examined [22]. Immunoreactive CNP and its gene transcripts are ubiquitously distributed in the whole brain, with a highest concentration in the olfactory bulb. In the rat and mouse, relatively high expression of CNP gene was detected in the tegumentum, as determined by RNase protection assay [34]. More detailed mapping of CNP mRNA by in situ hybridization revealed that CNP gene transcripts were localized in the olfactory nuclei; limbic cortices;

AB089935 AB081455 AL927379 AB076601 AB087731 AB047081 X59991

dorsal endopiriform nucleus; hippocampal subfields CA1–3; anteroventral, periventricular, and arcuate nuclei; and numerous brain-stem regions including pontine, lateral reticular, solitary tract, prepositus hypoglossal, and spinal trigeminal nuclei [19]. Co-localization of CNP and ANP in the same brain nucleus suggests their cooperative function. In the hypothalamus, both CNP and ANP gene transcripts are abundantly expressed in the anteroventral preoptic area and arcuate nucleus, which is consistent with their role in the regulation of neuroendocrine function [10]. Only CNP mRNA was detectable in the vasopressin-producing magnocellular paraventricular nucleus and supraoptic nucleus. Interestingly, water deprivation decreased and salt loading (2% NaCl for drinking water) increased CNP gene expression in the olfactory region of the rat brain, as shown by Northern blot and in situ hybridization [6]. CNP is also detected in the pineal gland of the dog. From a comparative viewpoint, all CNPs thus far obtained are expressed abundantly in the brain in all

Brain/B-Type Natriuretic Peptide (BNP) and C-Type Natriuretic Peptide (CNP) / 809 species examined to date including lampreys, elasmobranchs, holocephalan, bony fishes, amphibians, and birds, although CNP is also expressed abundantly in the heart of lampreys, elasmobranchs, and holocephalans where CNP is the only peptide of the NP family. Four CNP genes identified in teleostei and chondrostei are all expressed briskly in the fish brain. CNP3, from which cardiac ANP, BNP, and VNP are derived, was expressed also in the heart of the medaka, Oryzias latipes [12]. A detailed localization of CNP or its transcripts in the brain by immunohistochemistry or in situ hybridization has not been examined in nonmammalian species yet.

PROCESSING OF PRECURSOR The BNP prohormone is cleaved by a processing enzyme, furin, to produce mature BNP in the brain [32]. Furin cleaves it after a consensus sequence, ArgX-X-Arg, so that mature human and porcine BNP consist of 32 amino acid residues (BNP-32) and that of rats and mice contain 45 amino acid residues (BNP-45) depending on the location of the consensus sequence. In the porcine brain, BNP is also processed to BNP-26, which was initially isolated from brain [41]. BNP-32 circulates in the blood of the human and pig and acts as a mature hormone. Only BNP prohormone is detected in the porcine atria, but BNP-32 is a major form stored in the human atria [24]. BNP-45 is a major form circulating in the rat blood, and both prohormone and BNP-45 are stored in the rat atria. Although BNP exists in several nonmammalian species, their mature circulating forms have not been determined yet. There is a furin processing signal in the prosegment of chicken BNP, but the signal is absent in most other species. Instead, two consecutive basic amino acids for cleavage by typical proprotein convertase exist in the prosegment of nonmammalian BNP. BNP has been identified in all species of bony fishes, amphibians, reptiles, birds, and mammals thus far examined, whereas ANP and its cDNA could not be detected in some species of birds, reptiles, and bony fishes. Therefore, BNP, rather than ANP, may be a basic circulating NP secreted from the heart of vertebrates. All BNPs have two consecutive basic amino acids at positions 4 and 5 of the intramolecular ring. Two molecular forms of CNP have been isolated from the porcine brain, CNP-22 and CNP-53 [22]. CNP22 is processed after dibasic amino acid residues, whereas CNP-53 is cleaved by furin. Both forms have similar biological activities and have been identified in the brain of all mammalian species. Among four teleost CNPs, CNP4 is an ortholog of mammalian CNP [12]. Interestingly, CNP4 is the only teleost CNP that has a furin processing signal. CNP1 is a major teleost CNP in

the brain and is most similar to mammalian CNP-22, but it lacks a furin processing signal. Elasmobranch CNP is an ortholog of teleost CNP3, whose mature 22amino-acid form is highly similar to mammalian CNP (∼90%) but lacks a furin processing signal. Thus, mature forms of mammalian CNP (CNP4), teleost CNP (CNP1), and elasmobranch CNP (CNP3) have undergone convergent evolution, probably because of the binding to their specific receptor. Because cardiac NPs—ANP, BNP, and VNP—are produced by tandem duplication from CNP3, it is relevant that elasmobranch CNP3 is synthesized abundantly in both the brain and heart. Medaka CNP3 is also synthesized in both the brain and heart, but medaka CNP1 is found exclusively in the brain [12]. Lamprey CNP is an ortholog of teleost CNP4, and it has a furin processing signal in the prohormone sequence to form mature peptide [15].

RECEPTORS AND SIGNALING CASCADE BNP, and ANP also, binds with high affinity to the A-type NP receptor (NPR-A or GC-A) that possesses guanylyl cyclase and kinase-like domains intracellularly in its molecule [27]. CNP, on the other hand, binds specifically to cognate B-type NP receptor (NPR-B or GC-B). After ligand binding to the receptors, guanylyl cyclase is activated with the help of ATP to produce cGMP. Increased cGMP acts as a second messenger to activate G-kinase, resulting in phosphorylation of various target proteins. In addition, both BNP and CNP bind with high affinity to a C-type NP receptor (NPR-C) that has only a short intracellular domain and exists ubiquitously in various tissues. Judging from the rapid internalization after ligand binding, NPR-C was initially thought to be a clearance-type receptor that regulates NP concentration in plasma and locally in various tissues. However, new physiological functions have been suggested for this receptor through an interaction with Gi proteins [23]. In situ hybridization histochemistry showed that significant levels of NPR-A mRNA expression was observed in the restricted cell populations of the rat brain that also contain high levels of ANP [9]. These include the olfactory bulb and the circumventricular organs such as the subfornical organ and area postrema. In contrast, NPR-B mRNA was widely expressed throughout the brain, which coincides well with the wide distribution of CNP in the brain. Autoradiography using 125I-labeled ANP, CNP, or cANF-(4–23) that binds only to NPR-C showed that NPRA is localized in the olfactory bulb, nucleus tractus solitarius, and circumventricular organs such as the subfornical organ and area postrema, as well as the choroid plexus, in the rat brain [5, 28]. However, the autoradiographic study could not detect CNP-specific NPR-B.

810 / Chapter 110 In addition to NPR-A, -B, and -C, another NP receptor devoid of the guanylyl cyclase domain was identified in the eel and named NPR-D [38]. The relative abundance of NPR-D in the brain suggests some biological function by an intracellular messenger other than cGMP, as suggested with NPR-C. Two types of NPR-A and NPR-B have been cloned in the medaka fish [18]. In other nonmammalian species, NPR-A has been cloned in the bullfrog [33] and NPR-B in the dogfish, where only CNP3 exists in the brain and heart [2]. It is interesting to note that medaka CNP1, 2, and 4 bind with high affinity to NPR-B but that medaka CNP3, from which ANP, BNP, and VNP originated, binds to a medaka homolog of NPR-A [12]. Our preliminary immunohistochemical study using eel-specific NPR antisera showed that immunoreactive NPR-A is detected in the area postrema, glossopharyngeal vagal motor complex, reticular formation, and commissural nucleus of Cajal (equivalent to the nucleus tractus solitarius) in the eel brain, whereas immunoreactive NPR-B is distributed widely in the vascular smooth muscle cells of whole brain (Tsukada T and Takei Y, unpublished data).

BIOLOGICAL ACTIONS IN THE BRAIN As already mentioned, BNP is detected in the brain of pigs, but is undetectable in the rodent brain by radioimmunoassay, immunohistochemistry, or in situ hybridization. The BNP concentration is only one-third that of ANP in the human brain. In the rat brain, ANP is much more abundant than BNP, and it exists as ANP(4–28) or -(5–28). The biological action of ANP in the brain has been well characterized in the rat. Because ANP and BNP bind a common receptor, NPR-A, the biological actions of BNP may be similar to those of ANP. ANP is implicated in the central regulation of body fluid homeostasis in the rat. It has been shown that water deprivation reduces ANP content in several discrete regions of the brain such as the neural lobe of pituitary, organum vasculosum lamina terminalis, and supraoptic and suprachiasmatic nuclei but not in other regions [29]. In fact, infusion of rat ANP-(4–28) that is predominant in the brain into the third ventricle inhibits drinking induced by water deprivation or by central infusion of angiotensin II in normally hydrated rats [3]. Interestingly, intravenous infusion of ANP at similar doses equally inhibited drinking induced by these dipsogenic treatments, suggesting that ANP acts on the central site devoid of a blood–brain barrier such as the circumventricular organs. Similarly, central infusion of ANP causes dose-dependent and long-lasting inhibition of sodium appetite in sodium-depleted rats [11].

In nonmammalian species, eel ANP infused into the cerebral ventricle inhibits drinking in a dosedependent manner in seawater-adapted eel [17]. We also have preliminary results showing that direct topical injection of eel ANP to the area postrema inhibits drinking in seawater eels (Tsukada T. and Takei Y., unpublished data). Peripheral injection of ANP also inhibits drinking, but lesioning of the area postrema abolishes the antidipsogenic action of ANP. Thus, ANP may act on this circumventricular structure. Central ANP also influences hypothalamic and pituitary hormone secretion in mammals. The hormones that are regulated include vasopressin, GnRH, and CRH secreted from the neural tissues and prolactin, growth hormone, ACTH, and luteinizing hormone secreted from the anterior pituitary [20]. Established among those is vasopressin, whose enhanced secretion by angiotensin II or dehydration is inhibited by ANP in vivo or administered directly into the supraoptic or paraventricular nuclei in vitro. ANP also inhibits sympathetic tone through its action on the nucleus tractus solitarius and inhibits the release of catecholamines from the sympathetic nerve endings. Noradrenergic projections from the nucleus tractus solitarius to the anterior hypothalamus play pivotal roles in the central cardiovascular control in mammals. ANP is shown to act on both sites and decrease arterial pressure via the inhibition of noradrenalin release at the anterior hypothalamic area [26]. In contrast to the many studies on the central action of ANP, studies on the CNP effects have been rather scarce. Initially, potent central actions of CNP were expected because it is a true brain peptide of the NP family. However, CNP effects were not so prominent as those of ANP and generally less potent than ANP. For instance, neuroendocrine actions of CNP such as inhibition of vasopressin and luteinizing hormone secretion were not as prominent as those of ANP and BNP [20]. With respect to body fluid regulation, CNP weakly stimulates, rather than inhibits, water drinking in the rat when administered into the cerebral ventricle [31]. There has been no report on the effect of CNP on sodium appetite. Similar opposing actions of CNP and ANP are suggested on prolactin secretion: stimulation by CNP and inhibition by ANP [30]. In the tilapia, Oreochromis mossambicus, fish CNP stimulated growth hormone secretion from the isolated pituitary, but ANP had no effect [8]. Both CNP and ANP had no effects on prolactin secretion.

PATHOPHYSIOLOGICAL IMPLICATIONS As already mentioned, BNP is a circulating hormone secreted from the heart, particularly in the case of con-

Brain/B-Type Natriuretic Peptide (BNP) and C-Type Natriuretic Peptide (CNP) / 811 gestive heart failure and acute myocardial infarction [24]. In fact, increased plasma BNP concentration in these patients is correlated with the enlargement, decreased contractility, and increased stiffness of the left ventricle. Thus, BNP is thought to be involved in ventricular remodeling. Indeed, mice with targeted disruption of the BNP gene showed cardiac fibrosis defined as a proliferation of interstitial fibroblasts and biosynthesis of extracellular matrices in the cardiac ventricle [40]. Knockout of the gene coding for the BNP receptor, NPR-A, also produced mice with ventricular hypertrophy and fibrosis [25]. Because the mice with targeted disruption of the ANP gene are free from cardiac fibrosis [21], BNP and ANP play different roles in the physiological regulation of cardiovascular homeostasis through NPR-A. CNP is a phylogenetically ancient peptide and CNP4, an ortholog of mammalian CNP, is the ancestral molecule of the NP family [12]. Thus, CNP and its receptor, NPR-B, genes are expressed early in ontogeny. Although CNP is principally a brain peptide of the NP family, its physiological action seems to be related more closely with the periphery than with the brain. Mice lacking the CNP gene showed severe dwarfism as a result of impaired endochondral ossification [7]. Furthermore, most of them died early during postnatal development. Targeted expression of the CNP gene in the growth-plate chondrocyte rescued the skeletal defect of the CNP-deficient mice and prolonged their survival. Thus, their short life is due to the skeletal abnormalities. Disruption of NPRB appears to be lethal during intrauterine life. In the human, autosomal recessive skeletal dysplasia is known as a genetic disease. Analyses of the causative gene mapped it in the NPR-B gene [4]. In fact, obligate carriers of NPR-B gene mutations have skeletal heights that are below the mean for matched controls. These results show that the major role of CNP/NPR-B is in the regulation of skeletal growth, although the peptides are abundantly expressed in the brain.

[5]

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C

H

A

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111 Endozepines MARIE-CHRISTINE TONON, JÉRÔME LEPRINCE, PIERRICK GANDOLFO, VINCENT COMPÀRE, GEORGES PELLETIER, MARÍA M. MALAGÓN, AND HUBERT VAUDRY

STRUCTURE OF THE PRECURSOR mRNA/GENE

ABSTRACT The existence of specific binding sites for benzodiazepines has led to the discovery of endogenous ligands for benzodiazepine receptors that are collectively designated by the term endozepines. In the brain and in peripheral nervous tissues, endozepines are exclusively expressed by glial cells. The release of endozepines from cultured astrocytes is finely regulated by neurotransmitters and neuropeptides. Endozepines exert their effects either through classical central- and peripheraltype benzodiazepine receptors or through G-proteincoupled receptors. Intracerebroventricular injection of endozepines causes marked effects on anxiety, sleep, and food consumption. The concentration of endozepines in the brain and cerebrospinal fluid is affected in various diseases, including depression and hepatic encephalopathy.

In humans, the DBI gene is located on chromosome 2 and is composed of four exons [22] (Fig. 2). The mammalian DBI promoter displays consensus recognition sites for various known transcription factors, including AP-1/2, SP-1, ETF, Y-box-binding protein, CTF/NF-1, C/EBF, HNF-3, SRE-like sequence, and GREs (Fig. 2). Although the DBI gene exhibits several typical features of housekeeping genes (e.g., lack of TATA and CAAT boxes, high GC content, multiple transcription initiation sites, and consensus sequence for binding of SP-1), its expression is clearly regulated by insulin [42] and androgens [12].

DISTRIBUTION OF DBI mRNA AND ENDOZEPINE IMMUNOREACTIVITY

DISCOVERY

In situ hybridization histochemical labeling has shown that DBI mRNA is widely distributed in the rat brain, the highest concentrations being found in the cerebellum, the ependyma, and the area prostrema [1, 4]. In the cerebellum, the DBI gene is strongly expressed in Bergman (astrocyte-like) cells [1, 47]. High concentrations of DBI mRNA are also present in other glial cell populations, for example, ependymocytes bordering the third ventricle and tanycytes in the median eminence [1, 47] (Fig. 3). In the pituitary, DBI mRNA occurs in the neural lobe in adult rat [1, 47], but is expressed only in the anterior and intermediate lobes during embryonic development [4].

Benzodiazepines (BZD) are synthetic artificial compounds that are widely used for their sedative, anxiolytic, anticonvulsant, and myorelaxant activities. The search for endogenous ligands for BZD receptors has led to the discovery of diazepam-binding inhibitor (DBI), a 10-kDa peptide originally identified from the rat brain for its ability to displace diazepam from its binding sites [19]. Subsequently, the purification or the molecular cloning of DBI in different species has shown that the sequence of the protein has been relatively well conserved during evolution from yeast to mammals (Fig. 1). Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

814 / Chapter 111 Species

Sequence identity %

DBI sequences

Human

10 20 30 40 50 60 70 80 SQAEFEKAAE EVRHLKTKPS DEEMLFIYGH YKQATVGDIN TERPGMLDFT GKAKWDAWNE LKGTSKEDAM KAYINKVEEL KKKYGI

100

Rat

•••D•DK••• ••KR•K•Q•T ••••••••S• FK••••••V• •DR••LL•LK •K•K••S••K •K•••K•N•• KT•VEK•••• KKK•••

78

Mouse

•••••DK••• ••KR•K•K•T ••••••••S• FK••••••V• •DR••LL•LK •K•K••S••K •K•••K•S•• KT•VEK•D•• KKK•••

79

Pig

••••••K••• ••KN•K•K•A •D••••••S• •K•••••••• ••R•••••LK •K•K•••••G •K•••K•••• K••••K•••• KKK•••

90

Beef

•••••DK••• ••K••K•K•A ••••••••S• •K•••••••• ••R••••••K •K•K•••••• •K•••K•••• K•••DK•••• KKK•••

93

Dog

•••••DK••• D•K••K•K•A •••••Y••S• •K•••••••• ••R••L••LR •K•K•••••Q •K•••K•••• K••V•K••D• KKK•••

84

Armadillo

••••••K••• ••KN•K•K•A •D••••••S• •K•••••••• ••R••••••K •K•K•••••Q •K•••K•••• KS••DK•••• KKK•••

90

Rabbit

••••••K••• ••KN•K•K•A •A••••••S• •K••••••V• ••R•••••LK •K•K•••••• •K•••K•S•• R••VDK•••• KQK•••

86

T•••••••••• ••KK•K•R•T •••LKEL••F •K•••••••• I•C•••••LK •K•K•E•••L KK•I•K•••• N•••SKAKTM VEK•••

64

•E•A••K••• ••KE•KSE•T •••••DV•S• •K•••••••• •DR••••••K •K•K•••••S •K•I•K•••• K••VAK•••• KGK•••

79

Frog

SP••D•DK••G D•KK•K•K•T •D•LKEL••L •K•S•••••• I•C•••••LK •K•K•••••L KK•L•K•••• S••VSKAH•• IEK••L

66

Carp

•E•••QK••• ••KQ•KAK•T •A•••EI•SL •K••••E•V• •AR••••••• •K•K••••EA KK•M•KD••• K•••AK•••• KGK•••

77

Zebrafish

•E•••QK••• ••KQ•KAK•T •A•••E••SL •K••••••V• •AR••••••• •K•K••••DA KK•••K•••V K•••AK•••• KGK•••

79

Turtle

•••••DK••• ••KQ•KSQ•T •••••Y••S• FK•••••••• ••R••F•••K •K•K••••DA •K•MAK•E•• K•••AK•••• KGK•••

79

Duck Chicken

Fly (Drosophila)

VSEQ•NA••• K•KS•TKR•• •D•F•QL•AL FK••S•••ND •AK••L••LK •K•K•E•••K QK•K•S•A•Q QE••TF••G• VAK•A

49

Mosquito

•LDQQ•NEA•• K•KTFTKR•• •Q•L•EL•AL FK•S••••NT ••K•••F•LK •K•K•Q••SD KK•I•QDA•K E••VKF•••• SAKCL

50

Silkworm

SL•EKFDQ••A N•KN•KAL•T •AQL•NLYA• FK••••••ADPANR•••••LK •K•KF•••HK •A•••KD••Q K•••IEIV•G LIASIGLKE

49

Tobacco hornworm

SL•EQ•DQ••S N•RN•KSL•• •NDL•EL•AL FK••SA••ADPANR••L••LK •K•KF•••HK KA•L•K•••Q K•••AK••S• IASL•LQ

51

Nematode A.t.

53

TLSFDDA••A T•KT•K•S•• ND•L•KL•AL FK•G••••NT •DK•••F•LK •K•K•S••D• KK•LAKD••Q K••VAL•••• IAK••A

45

GLKEE•EEH•• K•NT•TEL•• N•DLLIL•GL •K••KF•PVD •SR•••FSMK ER•K••••KA VE•K•S•E•• ND••TK•KQ• LEVAASKAST

S.c.

48

VSQL•EEK•K A•NE•P•K•• TD•L•E••AL •K••••••ND K•K••IFNMK DRYKWE••EN LK•K•Q•••E KE••AL•DQ• IAK•SS

FIGURE 1. Comparison of the amino acid sequences of DBI from yeast to human. Dots denote identical amino acids to the human sequence. Basic amino acids are in bold letters. A.t. Aradbidopsis thaliana; S.c. Saccharomyces cerevisiae.

- 1600

- 60

PPRE

- 1525

HNF-3 C/EBF AP-1

- 824

- 791

- 747

SP-1

- 484

AP-2

- 320

CTF/NF-1

- 143 100 bp

DBI 12

41

62

86

FIGURE 2. Genomic organization of the rat DBI gene. Recognition sites for transcription factors (PPRE, HNF-3,C/EBF, AP-1, SP-1, AP-2 CTF/NF-1) are indicated by black boxes. The transcribed regions are boxed and grey boxes denote the coding regions. The shaded box represents the DBI sequence.

Immunohistochemical labeling has confirmed that in the brain and pituitary of tetrapods including frog [27], rat [31, 49], monkey [44], and human [2], endozepine immunoreactivity is observed exclusively in glial cells, that is, astrocytes, Gomori-positive astrocytes (a subset of brain astrocytes most prominent in the

arcuate nucleus of the hypothalamus), Bergman cells, ependymocytes, tanycytes, and pituicytes. Surprisingly, in the fish brain, endozepines are synthesized in a subpopulation of hypothalamic neurons [30]. DBI mRNA and DBI-like immunoreactivity are also present in numerous peripheral organs, including the

Endozepines / 815

Cx Hi CC OB CPut

S

Th PAG C DR PBN

BST PVN Acb

Hpt DMH

DBB

SN

NST

Arc

SON

LC

VMH ME Amy

NL IL AP

Amb LRN

FIGURE 3. Schematic parasagittal section through the rat brain depicting the distribution of endozepineimmunoreactive cells. The density of dots (•) is meant to be proportional to the density of endozepine-positive cells. Acb, nucleus accumbens; Amb, nucleus ambiguous; Amy, amygdala; AP, anterior pituitary; Arc, arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; Cput, caudate putamen; Cx, cerebral cortex; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of the pituitary; LC, locus coeruleus; LRN, lateral reticular nucleus; ME, median eminence; NL, neural lobe of the pituitary; NST, nucleus of the solitary tract; OB, olfactory bulb; PAG, periaqueductal gray; PBN, parabrachial nucleus; PVN, paraventricular nucleus of the hypothalamus; S, septum; SN, substantia nigra; SON, supraoptic nucleus; Th, thalamus; VMH, ventromedial nucleus of the hypothalamus.

adrenal, testis, heart, kidney, liver, and intestine [4, 40, 45]. In these organs, endozepines are selectively expressed in specific cell types, notably in steroidsecreting cells [11, 43] and peripheral glial cells [49].

PROCESSING OF DBI Endoproteolytic cleavage of DBI generates several biologically active peptides, including the triakontatetraneuropeptide TTN (DBI17–50) and the octadecaneuropeptide ODN (DBI33–50), which are present in various proportions in the different brain regions [31]. The DBI sequence is devoid of dibasic motifs that are typical cleavage sites for prohormone convertases, but it contains multiple individual lysine residues (Fig. 1), three of which, located at positions 16, 32, and 50, delimit the sequences of TTN and ODN (Fig. 4). However, to date, the enzymes responsible for the processing of DBI have not yet been characterized.

RECEPTORS AND SIGNALING CASCADES Endozepines have been originally characterized as inverse agonists of the central-type benzodiazepine receptor (CBR) that is an intrinsic part of the GABAA receptor–chloride channel complex. Electrophysiological studies have confirmed that DBI and ODN attenuate GABA-induced Cl− efflux in neurons and endocrine cells [3, 29], indicating that endozepines act as negative allosteric modulators of the GABAA receptor. DBI and TTN can also interact with the peripheral-type benzodiazepine receptor (PBR), a five-transmembranedomain mitochondrial protein of 18 kDa. In addition, endozepines can activate PBR located at the plasma membrane, leading to an influx of Ca2+ through voltagedependent calcium channels in glial cells [14]. Finally, recent studies have shown that endozepines can interact with metabotropic membrane receptors. In particular, in rat astrocytes, ODN stimulates phospholipase C through a Gαi/0 protein-coupled receptor [25, 38],

816 / Chapter 111 1

K16

K32

K50

86

DBI 17

50

TTN 33

50

ODN FIGURE 4. Schematic representation of DBI and two of its processing products, TTN and ODN. Cleavage sites, corresponding to individual lysine (K), that delimit the sequences of TTN and ODN are indicated.

OH

whereas, in frog adrenocortical cells, TTN activates adenylyl cyclase, which in turn causes Ca2+ influx through T-type calcium channels [26].

Y84 L15

D21

K52

CONTROL OF BIOSYNTHESIS AND RELEASE OF ENDOZEPINES The neuronal mechanisms regulating the secretion of endozepines have been studied in cultured rat astrocytes. The release of endozepines is stimulated by pituitary adenylyl cyclase–activating polypeptide (PACAP), acting through the PACAP-specific receptor PAC1-R [48] (see also Chapter 92 on PACAP/VIP in the Brain Peptides section) positively coupled to adenylyl cyclase and phospholipse C [33] and by β-amyloid peptides acting through formyl peptide receptors [46]. Endozepine release is inhibited by GABA, acting through GABAB receptors [37] and by somatostatin acting through sst1, sst2, and sst4 receptors [34], all four receptors being negatively coupled to adenylyl cyclase (see Chapter 89 on Somatostatin/Cortistatin, in the Brain Peptides section). These factors also regulate DBI gene expression in a coordinated manner. In particular, β-amyloid peptides increase while somatostatin reduces DBI mRNA levels in cultured astrocytes. Altogether, these observations indicate that the biosynthesis and release of endozepines are finely regulated by various neuronal factors, including classical neurotransmitters and neuropeptides.

INFORMATION ON SOLUTION CONFORMATION OF ENDOZEPINES Molecular modeling of bovine DBI under nuclear magnetic resonance (NMR) constrains has shown that the peptide exhibits a characteristic four-helix bundle structure, stabilized by three identified hydrophobic minicores (Fig. 5) [21]. The α-helix H1 forms strong

H3

K62

H4 H1

H2

V36

A3 S65

H FIGURE 5. Three-dimensional NMR structure of bovine DBI. Helixes are referred to as H1, H2, H3, and H4. Residues limiting α-helixes are indicated (adapted from [21]).

interface contacts with α-helix H4, made up of the conserved hydrophobic residues Phe5, Ala9, Val12, and Leu15 in H1 and of Tyr73, Val77, and Leu80 in H4. TTN, which is a selective ligand of PBR, contains the hydrophobic portion corresponding to H2, whereas ODN, which preferentially binds CBR, fails to adopt an α-helical configuration. A three-dimensional quantitative structureactivity relationship study performed over 130 PBR ligands supports the hypothesis that two lipophilic regions are essential for interaction with PBR [5]. The three-dimensional structures of DBI and TTN, derived from NMR data, reveal the existence of a triad of lipophilic amino acids located in the H2 helix (i.e., Met46, Leu47, and Phe49) as likely residues underlying PBR binding [5]. Structure-activity relationship studies have shown that the C-terminal octapeptide of ODN (OP, ODN11–18)

Endozepines / 817

Leu7

Asp6

Lys8 Leu5

β-turn Arg1 Leu4 Gly3

Pro2

g -turn FIGURE 6. Lowest energy conformer of cyclo1–8OP from simulated annealing. The dotted lines indicate hydrogen bonds consistent with NMR data (from [25] with permission). (See color plate.)

is the minimum sequence retaining full activity on metabotropic PLC-coupled receptors in astrocytes [24]. The cyclic OP analog cyclo1–8OP is three times more potent than OP, whereas the D-Leu5–substituted analog cyclo1–8[D-Leu5]OP is a full antagonist of this receptor [25]. In water, cyclo1–8OP adopts a single conformation encompassing a γ- and a β-turn (Fig. 6).

BIOLOGICAL ACTIONS OF ENDOZEPINES WITHIN THE BRAIN Some of the biological effects of endozepines are mediated through CBR or PBR, and others can be accounted for by activation of metabotropic receptors. Intracerebroventricular (ICV) administration of DBI, TTN, or ODN increases aggressive interactions [20] and induces anxiety and proconflict behavior [8] in rodents. These effects are prevented either by the specific CBR antagonist flumazenil or by the PBR antagonist PK11195. DBI and ODN, acting through CBR, also shorten pentobarbital-induced sleeping time in mice [10]. ICV injection of ODN markedly reduces drinking and increases the aversive response to 0.9% NaCl in mice, and these effects are mediated through CBR [32]. ODN, acting via CBR, also inhibits apomorphine-induced yawning in rat, suggesting that endozepines can modulate dopaminergic and cholinergic

neurotransmission involved in yawning [17]. Incubation of cultured rat astrocytes with picomolar concentrations of endozepines causes a robust increase in DNA synthesis through activation of CBR and PBR [15, 16], indicating that endozepines act as autocrine/paracrine factors modulating the proliferation of astroglial cells. On isolated mitochondria from a glioma cell line, endozepines activate the formation of the steroid hormone precursor pregnenolone [36], suggesting that endozepines also act as intracrine factors stimulating the synthesis of neurosteroids in astrocytes. Because several neuroactive steroids are potent allosteric modulators of the GABAA receptor, it appears that endozepines can indirectly regulate the activity of the GABAA–benzodiazepine receptor complex through their ability to control the production of neurosteroids. A single ICV injection of very low doses of ODN (30–100 ng) provokes a dose-dependent reduction of food intake in rodents [9]. Chronic ICV infusion of ODN for 15 days inhibits food consumption and causes a significant reduction in body weight [9]. Although CBR agonists are known to stimulate food intake, the anorexigenic effect of ODN is not mediated through CBR or PBR, but probably via metabotropic endozepine receptors. ICV injection of ODN increases proopiomelanocortin (POMC) mRNA level and decreases neuropeptide Y (NPY) mRNA level in the arcuate nucleus of the hypothalamus [6], suggesting that the effects of ODN on food consumption can be accounted for by stimulation of the production of the anorexigenic peptide α-MSH and inhibition of the production of the orexigenic peptide NPY (see Chapter 93 on NPY and Chapter 94 on Melanocortins in the Brain Peptides and Ingestive Peptides sections). Endozepines also regulate the expression of the genes encoding the hypothalamic hypophysiotropic neurohormones CRH and GnRH [6, 7] and the pituitary adrenocorticotrophic hormone precursor POMC [18]. Some of these effects are mediated through activation of metabotropic endozepine receptors [7].

PATHOPHYSIOLOGICAL IMPLICATIONS GABA is the most important inhibitory neurotransmitter in the central nervous system, and endozepines, by affecting GABA-mediated inhibition of neurotransmission, may be involved in various neurological diseases (e.g., epilepsy) and psychiatric disorders (e.g., anxiety, mood impairment, and psychotic syndromes). Indeed, patients with major depression exhibit significantly higher concentrations of DBI in their cerebrospinal fluid (CSF) as compared with normal volunteers [41]. In chronic morphine-treated rats, an increase in DBI mRNA level is observed in the the CA1 region of

818 / Chapter 111 the hippocampus, the ventral tegmental area, the nucleus accumbens, the prefrontal cortex, and the amygdala, which play a crucial role in addiction, suggesting that endozepines may also be involved in psychological and physical dependence to opiates [28] and possibly other drugs, including nicotine and alcohol. An increased level of endozepines in human CSF is also observed in other neurological diseases such as hepatic encephalopathy [39] and Alzheimer’s disease [13]. In these patients, the high concentration of endozepines is usually associated with an increase in PBR density [23] or neurosteroid biosynthesis [35], suggesting that an alteration of DBI gene expression could be implicated in various neuronal diseases. In rat brain, intense expression of the DBI gene and high concentrations of endozepines occur during late gestation [4, 31], a period of active neurogenesis. Endozepines are also actively expressed in various human brain tumors and glioblastomas [2]. These latter observations support the view that endozepines could play a role in neuronal cell proliferation and tumorigenesis.

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potent endozepine antagonist. Eur J Biochem 2001;268: 6045–57. Lesouhaitier O, Kodjo MK, Cartier F, Contesse V, Yon L, Delarue C, Vaudry H. The effect of the endozepine triakontatetraneuropeptide on corticosteroid secretion by the frog adrenal gland is mediated by activation of adenylyl cyclase and calcium influx through T-type calcium channels. Endocrinology 2000;141: 197–207. Lihrmann I, Plaquevent JC, Tostivint H, Raijmakers R, Tonon MC, Conlon JM, Vaudry H. Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning, and expression in the brain. Proc Natl Acad Sci USA 1994;91:6899–903. Liu X, Li Y, Zhou L, Chen H, Su Z, Hao W. Conditioned place preference associates with the mRNA expression of diazepam binding inhibitor in brain regions of the addicted rat during withdrawal. Mol Brain Res 2005;137:47–54. Louiset E, Vaudry H, Cazin L. Allosteric modulation of the GABA-induced chloride current in frog melanotrophs. Ann NY Acad Sci 1993;680:564–6. Malagon M, Vallarino M, Tonon MC, Vaudry H. Localization and characterization of diazepam-binding inhibitor (DBI)-like peptides in the brain and pituitary of the trout (Salmo gairdneri). Brain Res 1992;576:208–14. Malagon M, Vaudry H, Van Strien F, Pelletier G, Gracia-Navarro F, Tonon MC. Ontogeny of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain. Neuroscience 1993; 57:777–86. Manabe Y, Toyoda T, Kuroda K, Imaizumi M, Yamamoto T, Fushiki T. Effect of diazepam binding inhibitor (DBI) on the fluid intake, preference and the taste reactivity in mice. Behav Brain Res 2001;126:197–204. Masmoudi O, Gandolfo P, Leprince J, Vaudry D, Fournier A, Patte-Mensah C, Vaudry H, Tonon MC. Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA-dependent mechanism. FASEB J 2003;17:17–27. Masmoudi O, Gandolfo P, Tokay T, Leprince J, Ravni A, Vaudry H, Tonon MC. Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes. J Neurochem 2005;34:561–71. Papadopoulos V. Peripheral benzodiazepine receptor: structure and function in health and disease. Ann Pharm Fr 2003;61:30– 50. Papadopoulos V, Guarneri P, Kreuger KE, Guidotti A, Costa E. Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor. Proc Natl Acad Sci USA 1992;89:5113–7.

[37] Patte C, Gandolfo P, Leprince J, Thoumas JL, Fontaine M, Vaudry H, Tonon MC. GABA inhibits endozepine release from cultured rat astrocytes. Glia 1999;25:404–11. [38] Patte C, Vaudry H, Desrues L, Gandolfo P, Strijdveen I, Lamacz M, Tonon MC. The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes. FEBS Lett 1995;362: 106–10. [39] Rothstein JD, McKhann G, Guarneri P, Barbaccia ML, Guidotti A, Costa E. Cerebrospinal fluid content of diazepam binding inhibitor in chronic hepatic encephalopathy. Ann Neurol 1989;26:57–62. [40] Rouet-Smih F, Tonon MC, Pelletier G, Vaudry H. Characterization of endozepine-related peptides in the central nervous system and in peripheral tissues of the rat. Peptides 1992;13:1219–25. [41] Roy A. Cerebrospinal fluid diazepam binding inhibitor in depressed patients and normal controls. Neuropharmacology 1991;30:1441–4. [42] Sandberg MB, Bloksgaard M, Duran-Sandoval D, Duval C, Staels B, Mandrup S. The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor a in hepatocytes. J Biol Chem 2005;280:5258–66. [43] Schultz R, Pelto-Huikko M, Alho H. Expression of diazepam binding inhibitor-like immunoreactivity in rat testis is dependent on pituitary hormones. Endocrinology 1992;130:3200–6. [44] Slobodyansky E, Kurriger G, Kultas-Ilinsky K. Diazepam binding inhibitor processing in the rhesus monkey brain: an immunocytochemical study. J Chem Neuroanat 1992;5:169–80. [45] Steyaert H, Tonon MC, Tong Y, Smih Rouet F, Testart J, Pelletier G, Vaudry H. Distribution and characterization of endogenous benzodiazepine receptor ligand (endozepine)-like peptides in the rat gastrointestinal tract. Endocrinology 1991;129:2101–9. [46] Tokay T, Masmoudi O, Gandolfo P, Leprince J, Pelletier G, Vaudry H, Tonon MC. Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes. J Neurochem 2005;94:607–16. [47] Tong Y, Toranzo D, Pelletier G. Localization of diazepambinding inhibitor (DBI) mRNA in the rat brain by high resolution in situ hybridization. Neuropeptides 1991;20:33–40. [48] Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52:269– 324. [49] Yanase H, Shimizu H, Yamada K, Iwanaga T. Cellular localization of the diazepam binding inhibitor in glial cells with special reference to its coexistence with brain-type fatty acid binding protein. Arch Histol Cytol 2002;65:27–36.

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nosis factor for tumor progression and metastasis in esophageal squamous cell carcinoma, gastric carcinoma, and bladder cancer, among others [2]. In addition to its role in tumor progression, fragmentary information already showed some years ago additional roles for KiSS-1–derived peptides in the endocrine/paracrine control of specific biological functions. Notably, expression of the KiSS-1 gene in normal human tissues was reported to be maximal in the placenta, which probably accounts for the dramatic increase in serum metastin levels during pregnancy, whose endocrine relevance is yet to be determined [2]. In terms of paracrine regulation, placental KiSS-1 peptides have been suggested to play a role in the physiological regulation of trophoblast invasion. In addition, some preliminary evidence pointed out that KiSS-1 may participate in the regulation of specific neuroendocrine systems, such as oxytocin release [5]. Indeed, the expression of KiSS-1 and its putative receptor was demonstrated in a variety of normal tissues, including not only placenta but also different brain areas, spinal cord, pituitary, pancreas, testis, small intestine, and human plasma [2], which strongly suggested additional, as yet unknown, physiological roles of this newly discovered system. In this context, the neuroendocrine facet of the KiSS-1 system was rediscovered by the striking observation that some forms of idiopathic hypogonadotropic hypogonadism are associated with inactivating mutations of the gene encoding the putative receptor for KiSS-1 peptides (GPR54) [17, 18]. Such a hypogonadal phenotype was reproduced in mouse models where the GPR54 gene had been knocked out [2, 18]. These clinical and experimental findings boosted an extraordinary interest among reproductive physiologists, who endeavored to characterize the physiological role of the KiSS-1 system in the neuroendocrine control of the gonadotrophic axis.

ABSTRACT KiSS-1 was originally identified as a metastasis suppressor gene that encodes a number of related peptides, metastin, and other kisspeptins and that, acting through the G-protein-coupled receptor GPR54, are able to inhibit tumor progression. The neuroendocrine dimension of the KiSS-1/GPR54 system was disclosed by the observation (reported in late 2003) that loss-offunction mutations of the GPR54 gene are associated with the absence of puberty and hypogonadotropic hypogonadism. This seminal finding underscored a pivotal role of the KiSS-1 system in control of reproduction and elicited intense research in an attempt to characterize the neuroendocrine physiology of kisspeptins. Indeed, in the last months, KiSS-1 has emerged as a major physiological gatekeeper of GnRH neurons and, hence, of the reproductive axis. The major features of the KiSS-1 system are comprehensively described here.

DISCOVERY OF KiSS-1 SYSTEM Identification of the KiSS-1 gene was accomplished in late 1996 in the context of tumor biology. By the use of subtractive hybridization in melanoma cell lines with different metastatic capacity, the KiSS-1 gene was originally reported to be selectively overexpressed in metastasis-suppressed tumor cells [7]. This original finding was further elaborated in the following years by assessing KiSS-1 gene expression and biological actions in additional tumor specimens and cell lines. The gathered data led to the conclusion that KiSS-1 is provided with potent antimetastasis activity in several tumors, such as papillary thyroid carcinoma, breast carcinoma, melanoma, and pancreatic cancer cells, whereas loss of KiSS-1 gene expression appeared as a bad progHandbook of Biologically Active Peptides

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822 / Chapter 112 STRUCTURE OF THE KiSS-1 GENE In the human genome, the gene encoding KiSS-1 has been mapped to chromosome 1q32 [24]. The human KiSS-1 gene (ENSG00000170498 at www.ensembl.org) is composed of three exons. The first exon contains 111 bases and it is not translated, whereas exon II is composed of 141 bases with the start codon at position 39. Finally, exon III consists of 332 translated bases, followed by the translational stop codon and the polyadenylation sequence [24]. The deduced sequence of the precursor KiSS-1 protein is composed of 145 amino acids. The mouse KiSS-1 gene has been mapped to chromosome 1, whereas the rat KiSS-1 gene is located in chromosome 13q13. The cDNA sequences and the (predicted) genomic organization of the rat and mouse KiSS-1 genes are highly homologous. There is, however, some uncertainty regarding the precise structure of rodent genes. According to GeneBank database, rat and mouse KiSS-1 genes are composed of two exons, and their cDNA sequences show 90% identity (rat: NM 181692; mouse: NM 178260). These two exons are highly homologous to the corresponding exons II and III of the human sequence, with analogous splicing sites and a similar spacing intron of approximately 2 kb. However, a comparison of human and mouse genomic sequences at www.ensembl.org (gene NM_026680; transcript NM_178260) reveals a structure of the mouse gene that is strikingly similar to the human, with three exons: a noncoding exon I of 102 bases, an exon II with start codon at base 41, and a final exon III harboring the stop codon and polyadenylation site. In the rat gene, only two exons, corresponding to mouse and human exons II and III, have been annotated (see www. ensembl.org). However, a blast search using human and mouse exon I sequences detects a highly similar sequence in the rat genome, located ∼7500 bp upstream from the corresponding KiSS-1 gene, that conserves all putative splicing signals. Assuming that such a sequence actually corresponds to exon I of the rat KiSS-1, the structure of human, mouse, and rat genes would be essentially identical. Moreover, in human and rodent species, there is a TATA-box-like sequence located 15–18 bp upstream from the predicted 5′-end of the putatively common exon I (H. Leffers, personal communication).

PROCESSING OF KiSS-1 PRECURSOR: THE KISSPEPTINS In the human, the KiSS-1 gene encodes a 145-aminoacid precursor, which contains a putative 19-amino-acid signal sequence, two potential dibasic cleavage sites (at

amino acids 57 and 67), and one putative site for terminal cleavage and amidation (at amino acids 121–124) [2, 10, 15]. These structural features predicted the generation of secretory product(s), in spite of the original proposal of KiSS-1 as a major intracellular mediator. Indeed, through several proteolytic steps, the full-length KiSS-1 precursor was later proven to give rise to a number of structurally related secreted peptides, globally termed kisspeptins [2, 5]. Among these, the major product of the KiSS-1 gene appears to be a 54-aminoacid peptide, largely secreted by the placenta, named metastin or kisspeptin-54 [15]. In addition, other derivatives of the KiSS-1 precursor, such as kisspeptin-14, kisspeptin-13, and kisspeptin-10, were also identified. Notably, the shorter 10-amino-acid fragment at the Cterminus (kisspeptin-10) retains maximal activity at the receptor level. All kisspeptins share the C-terminal region of the metastin molecule and harbor an ArgPhe-NH2 motif distinctive of the RF-amide peptide family. Other prominent members of this family have been recently involved in relevant neuroendocrine functions (Chapter 107 RFRPs-PrRP in this volume). In rodent species, the overall structure of the KiSS-1 precursor and derived kisspeptins is roughly conserved. Nonetheless, rat and mouse metastin is composed of 52 amino acids, where the terminal RF-amide signature is substituted by an Arg-Tyr-NH2 motif.

DISTRIBUTION OF KiSS-1 mRNA/PROTEIN WITHIN THE BRAIN On identification of kisspeptins as endogenous ligands for the previously orphan GPR54, expression analyses were conducted to characterize the pattern of KiSS-1 (as well as GPR54) gene expression in normal tissues. Although they did not totally overlap, these screening analyses showed prominent expression of the KiSS-1 gene in human placenta and, at lower levels, in testis and small intestine. Expression of KiSS-1 mRNA was also detected in the human brain, with scattered distribution at moderate relative levels being detected throughout the central nervous system (CNS), including the basal ganglia and the hypothalamus [10, 15]. These initial observations were recently completed by a similar screening in rodent tissues. In the rat, the highest levels of KiSS-1 mRNA were found in the colon and cecum, with moderate to low expression levels in different peripheral tissues including placenta [22]. In addition, KiSS-1 gene expression was demonstrated in rat brain (and specifically in the hypothalamic area) [11, 22]. These expression data at the CNS were further extended by in situ hybridization assays that revealed KiSS-1 expressing neurons at different levels along the rostral-caudal extent of the hypothalamus in the mouse,

KiSS-1/Metastin / 823 including the arcuate nucleus (Arc) and the periventricular nucleus (PeN). In addition, KiSS-1 mRNAexpressing cells were also detected in the anterodorsal preoptic nucleus, the medial amygdala (Amy), and the bed nucleus of the stria terminalis (BST) (Fig. 1) [3]. Likewise, expression of KiSS-1 mRNA has been recently detected in the Arc of pubertal primates [19]. At the peptide level, an exhaustive immunohistochemical analysis of the distribution of kisspeptins within the CNS has been recently reported. In this study, metastin-like immunoreactivity (irMT) was widely detected throughout the CNS. In the brain, the dorsomedial hypothalamic nucleus, ventromedial nucleus, and Arc within the hypothalamus; the nucleus of the solitary tract (NST); and the caudal ventrolateral medulla were found clearly positive for irMT. In addition, fibers with varying intensity of irMT were observed

in different areas of the telencephalon (amygdala, BST, septal nuclei, nucleus accumbens, and basal ganglia), diencephalon (several hypothalamic regions, thalamus, and zona incerta), mesencephalon (periaqueductal grey and raphe nuclei), metencephalon, and myelencephalon (lateral parabrachial nuclei, locus coeruleus, spinal trigeminal tract, and medullary reticular nucleus) (Fig. 1) [1]. Although it has to be noted that the immunohistochemical analyses were conducted using a polyclonal antiserum against kisspeptin-10, which might cross-react with other peptides showing the terminal RF-amide motif, such a scattered anatomical distribution within the CNS strongly suggests potential roles of KiSS-1 peptides in the central control of diverse biological systems, including nociception, visceral regulation, and, relevant here, different neuroendocrine axes.

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FIGURE 1. Anatomical distribution of KiSS-1 mRNA (blue dots), KiSS-1 immunoreactivity –irMT- (red dots), and GPR54 mRNA (black dots) in different brain areas of rodent brain. In the mouse, KiSS-1 mRNA signals are detected in the hypothalamus, mostly in the arcuate nucleus (Arc) and periventricular nucleus (PeN), as well as in the preoptic area (POA), amygdala (amy), and bed nucleus of stria terminalis (BST); taken from [3]. In addition, expression of KiSS-1 gene has been detected in basal ganglia in the human (denoted by light blue dots); taken from [10]. In rat brain, irMT has been identified mainly in neurons at the Arc, ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei, as well as in the nucleus of solitary tract (NST); taken from [1]. In addition, multiple fibers containing irMT have been reported throughout the rat brain; for sake of simplicity, these are not specifically depicted in the scheme. Finally, in the rat, GPR54 mRNA has been detected in different forebrain areas, including diagonal band of Broca (DBB), septum, POA, anterior and lateral hypothalamus (LH), and DMH. Likewise, prominent expression of GPR54 has been observed in locus coeruleus (LC), amy, and periaqueductal grey (PAG), among others; taken from [4, 6]. In addition, in the human brain, scattered expression of GPR54 mRNA has been reported, including basal ganglia –Cput-, hippocampus –Hi-, substantia nigra –SN-, and, at low levels, thalamus –Th-, cerebellum –C-, and corpus callosum –CC-; taken from [5, 10]. These are denoted by light grey dots. (See color plate.)

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824 / Chapter 112 GPR54 AS RECEPTOR FOR KISSPEPTINS: STRUCTURE AND SIGNALING CASCADES Using a degenerate polymerase chain reaction (PCR) strategy and a rat brain cDNA library, Lee and coworkers published in 1999 the identification of an orphan G-protein-coupled receptor, with a typical heptahelical conformation and significant identity (>40%) with the transmembrane regions of galanin receptors, which was termed GPR54 [6]. The human ortholog of GPR54 was subsequently cloned and named AXOR12 or hOT7T175 [2]. In 2001, this receptor was deorphanized when three groups independently reported that KiSS-1–derived peptides are the natural ligands of GPR54 [5, 10, 15]. As was the case for the ligand, expression analyses were conducted to screen the tissue distribution of GPR54 gene. These showed a rather scattered pattern of expression, with maximal mRNA levels in placenta, pancreas, pituitary, spinal cord, and various brain areas (including hypothalamus, basal ganglia, amygdala, substantia nigra, and hippocampus). These observations further suggested the potential involvement of the KiSS-1-GPR54 system in the control of diverse biological systems, including different CNS functions. Within the hypothalamus, GPR54 mRNA was proven to be maximally expressed in the arcuate nucleus, the dorsomedial nucleus, and the lateral hypothalamic area (Fig. 1) [4, 6]. Interestingly, expression of GPR54 mRNA has been detected in gonadotropinreleasing hormone (GnRH) neurons in the rat (in different forebrain regions such as diagonal band of Broca, medial septum, medial and lateral preoptic areas, median preoptic nucleus, anterior hypothalamus, and lateral hypothalamus) and fish species [4, 16]. These observations are in good agreement with the proposed role of the KiSS-1 system as a major gatekeeper of the reproductive axis. Analyses of the intracellular signaling systems recruited by GPR54 to convey the biological actions of kisspeptins have revealed that, on ligand-receptor interaction, the activation of phospholipase C and an increase in PIP2 hydrolysis take place, followed by the accumulation of inositol-(1,4,5)-triphosphate, Ca2+ mobilization, arachidonic acid release, and phosphorylation of ERK1/2 and p38 MAP kinases [5]. Although extraordinarily illustrative, it has to be noted that such signaling studies were conducted in a heterologous system (Chinese hamster ovary K1 cells stably expressing GPR54). Indeed, although metastin has been proven to activate extracellular-signal-regulated kinase (ERK) and p38 kinase in thyroid and pancreatic cancer cells, the signaling cascades used by GPR54 in normal tissues (such as the hypothalamus and other brain areas), where this receptor is expressed and where biological

effects of kisspeptins have been reported, remain mostly unexplored.

BIOLOGICAL ACTIONS OF KiSSPEPTINS: BRAIN AND PITUITARY EFFECTS As indicated in previous sections, the KiSS-1 gene was initially labeled as metastasis suppressor and its major product, metastin, named after its proven ability to inhibit tumor metastasis and progression [2]. However, the possibility of additional physiological functions of the KiSS-1 system was strongly suggested by the reported widespread pattern of expression of KiSS-1 and GPR54 genes in different peripheral and central tissues. Indeed, based on its biological properties on cell migration and prominent expression in placenta, locally produced KiSS-1 was recently proposed as playing a major role in the physiological regulation of trophoblast invasion. At the CNS, the scattered distribution of KiSS-1 and GPR54 mRNAs, as well as of irMT, allowed the speculation of potential roles of kisspeptins in different brain functions, including neuroendocrine regulation. However, up to late 2003, such biological functions remained largely unexplored. In 2003, two groups independently reported that a number of point mutations and deletions of the GPR54 gene are found in patients suffering familial (or even sporadic) forms of idiopathic hypogonadotropic hypogonadism, a clinical syndrome that was reproduced in mouse models carrying null mutations of the GPR54 gene [2, 17, 18]. These findings provided conclusive evidence for a major, previously unsuspected role of KiSS-1 system in the regulation of the development and function of the hypothalamic-pituitary-gonadal axis. Moreover, these observations underpinned the need for a complete characterization of essential aspects of KiSS-1 physiology in the neuroendocrine control of reproduction, including its key biological effects, primary site(s) of action, major signaling pathways, and main regulatory mechanisms. Such an assessment has been initiated in the last few months. Studies conducted in laboratory animals (rodents and primates) have now demonstrated that kisspeptins (metastin and kisspeptin-10) are extraordinarily potent elicitors of luteinizing hormone (LH) (and to a lesser extent follicle-stimulating hormone, FSH) secretion. This effect is observed both after central (intracerebroventricular) and systemic (intravenous, intraperitoneal, and subcutaneous) administration of the peptides [3, 4, 8, 11–14, 19, 23]. The sensitivity of the gonadotropic system to the stimulatory effect of KiSS-1 is illustrated by the fact that doses as low as 1.3 ng (equivalent to 1 pmol) injected centrally and 0.1 μg (equivalent to

KiSS-1/Metastin / 825 75 pmol) injected systemically are able to significantly increase serum LH levels (personal observations). Notably, FSH release in vivo appeared to be approximately 100-fold less sensitive to the stimulatory effect of kisspeptin than LH (Fig. 2). Nevertheless, comparative meta-analyses with previously published data on the LH releasing activity of other neuropeptides and neurotransmitters, such as glutamate and galanin-like peptide, show that the KiSS-1 system is probably the most potent elicitor of the GnRH-gonadotropin axis known so far. Based on the proven expression of KiSS-1 and GPR54 genes at the hypothalamus (the major center for neuroendocrine integration), a primary site of action of kisspeptins at this level was already suggested in the original publications underscoring its essential role in the control of the reproduction [2]. Hence, direct regulatory actions of KiSS-1 on the hypothalamic GnRH system, as the pivotal element driving the gonadotropic axis, were proposed. However, the fact that hypothalamic GnRH content was preserved in GPR54-null mice strongly suggested that KiSS-1 is not critically involved in the process of GnRH neuron migration from the olfactory placode that takes place during development but rather plays a role in the control of GnRH secretion. Such a contention was indirectly supported by the initial observation that the potent LH-releasing effect of kisspeptin is blunted by pretreatment with a GnRH antagonist [3, 8, 11]. Thereafter, a demonstration of GPR54 expression in GnRH neurons has been provided, and

the direct stimulatory effects of kisspeptin-10 on GnRH release have been demonstrated using in vitro (rat) and in vivo (sheep) settings [9, 23]. Moreover, metastin was recently reported to induce c-fos expression (an early sign of activation) in hypothalamic GnRH neurons after its central injection [4]. Altogether, these data conclusively demonstrate that GnRH neurons are direct targets for the KiSS-1 system. In addition to functional tests, expression analyses indirectly also supported a prominent role of the KiSS1 system in key aspects of the maturation and function of the reproductive axis. For instance, hypothalamic expression of KiSS-1 and GPR54 genes appeared developmentally regulated, with maximum levels around puberty, both in the rat and primates [11, 19]. This observation suggests a pivotal function of KiSS-1 system in the timing of puberty onset in mammals, in keeping with the original data from human and mouse models of inactivating mutations of GPR54. Indeed, this contention has been corroborated by functional studies, including a demonstration of the ability of kisspeptin-10 to induce precocious activation of the reproductive axis in immature female rats [14]. Similarly, expression analyses strongly suggested that the KiSS-1 system may play a key role in mediating the classical negative feedback effect of sex steroids on gonadotropin secretion because hypothalamic expression of the KiSS-1 (and to a lesser extent the GPR54) gene significantly increased after gonadectomy, whereas testosterone and estradiol replacement fully prevented such a response in male

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Kisspeptin-10 FIGURE 2. Analysis of the effects of central administration of KiSS-1 peptide upon serum LH and FSH levels. A range of doses of KiSS-1 (5 nmol, 1 nmol, 500 pmol, 100 pmol, 10 pmol, 1 pmol, and 100 fmol) were tested (ICV injection). Calculation of the mean effective dose (ED50), as determined by nonlinear regression, allowed us to estimate that LH secretion is approximately 100-fold more sensitive to the stimulatory effect of kisspeptin than FSH. Taken from [13], with modifications.

826 / Chapter 112 and female rats, respectively [11]. Interestingly, in situ hybridization assays recently demonstrated that such a negative regulation of hypothalamic KiSS-1 gene expression appears to be restricted to the Arc (an area classically recognized as pivotal for negative feedback of sex steroids), whereas at the anteroventral periventricular nucleus (AVPN) KiSS-1 mRNA decreased after gonadectomy and increased after sex steroid replacement [20]. Considering that the AVPN, which is a sexually dimorphic nucleus with far more KiSS-1–expressing neurons in the female, has been involved in mediating the positive feedback effect of estrogen on GnRH and LH surges, it is tempting to propose that the KiSS-1 neurons (at the AVPN) might be involved also in the generation of the preovulatory gonadotropin surge via positive regulation of GnRH secretion. Finally, hypothalamic expression of the KiSS-1 gene also appeared sensitive to the organizing effects of estrogen during critical stages of brain sex differentiation [11]. This finding might contribute to the sexually dimorphic content of KiSS-1 neurons within the hypothalamus and opens up the possibility that decreased expression of KiSS-1 at the hypothalamus (as observed following neonatal estrogenization) may be causative for the plethora of developmental and functional defects of the gonadotropic axis following early exposure to pharmacological doses of estrogenic compounds. In contrast to the hypothalamus, assessment of direct biological effects of kisspeptins at the pituitary level in the control of gonadotropin secretion has received less attention. In principle, the possibility of direct stimulatory actions of kisspeptins upon LH and FSH release is supported by the observation of prominent expression of GPR54 gene at the pituitary. Moreover, peripheral administration of kisspeptin has been proven to potently elicit gonadotropin release, which is compatible with direct effects at the pituitary. However, it is also possible that the releasing effects of systemically delivered kisspeptins may derive from its ability to elicit GnRH secretion by GnRH neuron nerve terminals located at the median eminence–arcuate nucleus complex, the median eminence lying outside the blood–brain barrier. To date, the direct effects of kisspeptins on gonadotropin secretion have been evaluated by means of in vitro settings, involving cultures of dispersed anterior pituitary cells or static incubations of pituitary tissue. Results from these analyses are somewhat controversial because either no effect or a moderate stimulatory action of kisspeptin-10 (which was at least four times less potent than that of GnRH) on LH secretion in vitro has been reported [8, 12, 23]. Although differences in the in vitro settings (cell culture vs. static incubation) and developmental stage (adult vs. pubertal) tested may account for part of the indicated divergences, the available data strongly suggest that the pituitary is not the

major primary site of action for the potent gonadotropin-releasing effect of kisspeptins. Nonetheless, the possibility that GPR54 signaling at the pituitary might play a physiological role in the modulation of gonadotropin secretion or might be involved in additional endocrine functions cannot be excluded and warrants additional studies. Considering that the reproductive facet of KiSS-1 was proposed only recently, it is striking how our knowledge of the neuroendocrine role of this novel system has rapidly expanded, leading to the hypothesis that it represents an essential gatekeeper for GnRH neurons and, hence, the gonadotropic axis. However, note also that there are still key aspects of KiSS-1 physiology that remain to be elucidated. For instance, although the ability of kisspeptin to elicit GnRH release is now undisputed, the hierarchy of KiSS-1 neurons within the hypothalamic network governing GnRH secretion is yet to be fully established. Nonetheless, pharmacological studies, involving testing of kisspeptin effects after blockade of endogenous excitatory amino acid and nitric oxide pathways (i.e., relevant neurotransmitters in the neuroendocrine control of LH secretion) strongly suggested that KiSS-1 is acting downstream of other neuroendocrine regulators to elicit GnRH secretion [12, 13]. Likewise, the major intracellular signals recruited by kisspeptins on binding to GPR54 in GnRH neurons await elucidation and it remains to be characterized whether desensitization of its gonadotropinreleasing effects takes place after repeated administration of kisspeptin. Finally, the functionality of the KiSS-1 system in the control of the gonadotropic axis in situations of negative energy balance, as well as in different reproductive or developmental stages, has not been explored to date.

PATHOPHYSIOLOGICAL IMPLICATIONS As indicated in previous sections, the identification of the KiSS-1/GPR54 system as the pivotal element in the neuroendocrine network controlling the reproductive axis is probably one of the best recent examples of transfer of knowledge from bedside to bench research. Accordingly, the disturbance of this system poses evident pathophysiological implications in terms of reproductive function. To date, several inactivating mutations of the GPR54 gene have been reported to lead to a lack of onset of puberty and hypogonadism of central (hypothalamic) origin. These have included the homozygous deletion of 155 nucleotides around the intron IV–exon V junction, the homozygous L148S mutation, and different compound heterozygous (R331X and X399R; C223R and R297L) mutations [17, 18]. Notably, not a single KiSS-1 gene mutation causing hypogonadotro-

KiSS-1/Metastin / 827 phic hypogonadism has been published so far. However, given the short time that has elapsed since the first reports on GPR54 mutations, it is premature to speculate about whether this condition might be a causative entity for clinical forms of reproductive insufficiency. In this sense, the generation of KiSS-1 knock-out mice will be extremely instrumental in defining the expected (reproductive and nonreproductive) phenotype of humans carrying null mutations of the KiSS-1 gene. From an epidemiological standpoint, initial analyses already have suggested that the frequency of GPR54 null mutations as a cause for hypogonadotropic hypogonadism is (probably) not high; yet, the picture might be incomplete as additional studies are certainly required to determine the actual frequency GPR54 mutations in different human populations. Nonetheless, besides the obvious physiological implications, identification of inactivating mutations of GPR54 as a causative factor for reproductive failure has helped us to expand our knowledge on the molecular basis of some forms of infertility. In this sense, GPR54 enlarges the set of genes (such as KAL1, steroidogenic factor 1, DAX-1, GnRH, and leptin) whose inactivation had been previously linked to hypogonadotropic hypogonadism. In addition, considering GPR54 physiology and the situation with other G-protein-coupled receptors with key roles in reproduction, such as the LH receptor, for which both inactivating and activating mutations have been reported [21], it is predictable that constitutive activation of GPR54 may lead to central precocious puberty, an etiological entity that is yet to be identified. Moreover, taking into account the widespread pattern of expression of GPR54 in the central and peripheral tissues and its proposed biological roles (e.g., control of tumor progression and metastasis), it will be interesting to evaluate in detail whether patients suffering mutations of this system display additional nonreproductive phenotypes. Overall, complete characterization of the panel of GPR54 (and eventual KiSS-1) gene mutations inducing clinical alterations of sexual development and function will help to elucidate not only the molecular basis of some forms of reproductive pathology but also the whole array of physiological roles and physiopathological implications of this newly discovered system.

Acknowledgments The author is indebted with V. M. Navarro, R. Fernandez-Fernandez, J. M. Castellano, C. Dieguez, E. Aguilar, L. Pinilla, and H. Leffers for helpful discussions during preparation of this manuscript. Experimental work conducted in the author’s laboratory, was supported by grants BFI 2000-0419-CO3-03 and BFI 200200176 from DGESIC (Ministerio de Ciencia y Tecnología,

Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082), and EU research contract EDEN QLK4-CT-2002-00603.

References [1] Brailoiu GC, Dun SL, Ohsawa M, Yin D, Yang J, Chang JK, Brailoiu E, Dun NJ. KiSS-1 expression and metastin-like immunoreactivity in the rat brain. J Comp Neurol 2005;481:314– 429. [2] Colledge WH. GPR54 and puberty. Trends Endocrinol Metab 2004;15:448–453. [3] Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004;145:4073–4077. [4] Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, et al. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004;80:264–272. [5] Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001;276:34631–34636. [6] Lee DK, Nguyen T, O’Neill GP, Cheng R, Liu Y, Howard AD, et al. Discovery of a receptor related to galanin receptors, FEBS Lett 1999;446:103–107. [7] Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Natl Cancer Inst 1996;88:1731–1737. [8] Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004;320:383–388. [9] Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, et al. Kisspeptin directly stimulates gonadotropin-releasing hormone secretion via G protein-coupled receptor 54. Proc Natl Acad Sci USA 2005;102:1761–1766. [10] Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, et al. AXOR12, a novel human G proteincoupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001;276:28969–28975. [11] Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor GPR54 in rat hypothalamus and potent LH releasing activity of KiSS-1 peptide. Endocrinology 2004;145:4565–4574. [12] Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, et al. Characterization of the potent LH releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146:156–163. [13] Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, et al. Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 2005;146:1689–1697. [14] Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, et al. Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 2004;561:379–386. [15] Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G protein-coupled receptor. Nature 2001;411:613–617.

828 / Chapter 112 [16] Parhar IS, Ogawa S, Sakuma Y. Laser captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel G protein-coupled receptor (GPR54) during maturation in cichlid fish. Endocrinology 2004;145:3613–3618. [17] Roux N de, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–10976. [18] Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, et al. The GPR54 gene as a regulator of puberty. New Engl J Med 2003;349:1614–1627. [19] Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005;102:2129–2134. [20] Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, et al. Differential regulation of KiSS-1 mRNA expression by

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sex steroids in the brain of the male mouse. Endocrinology 2005;146:2976–2984. Tena-Sempere M, Huhtaniemi I. Gonadotropins and gonadotropin receptors. In: Fauser BCJM, editor. Reproductive Medicine—Molecular, Cellular and Genetic Fundamentals. New York: Parthenon Publishing; 2003, p. 225–244. Terao Y, Kumano S, Takatsu Y, Hattori M, Nishimura A, Ohtaki T, et al. Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. Biochim Biophys Acta 2004;1678:102–110. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, et al. Central and peripheral administration of kisspeptin-10 stimulates the hypothalamo-pituitary-gonadal axis. J Neuroendocrinol 2004;16:850–858. West A, Vojta PJ, Welch DR, Weissman BE. Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene (KISS1). Genomics 1998;54:145–148.

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113 Role of Opioid Peptides in the Local Regulation of Endocrine Glands KAZUHIRO TAKEKOSHI

a direct effect on the thyroid and parathyroid glands. Therefore, this review focuses on the pituitary gland, pancreatic islets, and adrenal gland.

ABSTRACT The classic opioids (i.e., enkephalins, endorphins, and dynorphins) and their cognate receptors (δ, μ, and κ) are known to be expressed in several endocrine glands. Thus, it can be suggested that they have regulatory roles, especially in terms of local regulation of endocrine function, such as hormone secretion and synthesis. However, there is insufficient evidence to establish whether opioids have a direct effect on the thyroid and parathyroid glands. Therefore, this review focuses on the pituitary gland, pancreatic islets, and adrenal gland. The review emphasizes the diverse role of the opioid peptides and their cognate receptors in endocrine function.

PITUITARY GLAND Opioid-receptor agonists have a broad effect on hormone release from the anterior pituitary (AP), such as increasing the release of prolactin (PRL), growth hormone (GH), and pro-opiomelanocortin (POMC), while decreasing the release of luteinizing hormone (LH). Consistent with these findings, endogenous opioid peptides [15, 21, 25] as well as opioid receptors have been detected in the pituitary gland [36]. It is traditionally believed that opioids mainly affect the hypothalamus without significantly affecting the pituitary gland. During stress, opioids are released, which tonically inhibits corticotrophin-releasing hormone (CRH) release from the hypothalamus without any direct effect on either the pituitary or adrenal glands [33]. Indeed, naloxone, a μ-opioid receptor antagonist, appears to have a direct stimulatory effect on paraventricular nuclear (PVN) CRH neurons, resulting in elevated levels of plasma adrenocorticotrophic hormone (ACTH) and cortisol [6, 14, 16, 28, 39]. However, more recent studies show that opioids directly inhibit gonadotrophin-releasing hormone (GnRH)-stimulated and basal LH release [19] in the pituitary [3]. Furthermore, tonic regulation of LH release is modulated by opioids. Indeed, in vitro studies show that β-endorphin stimulates LH release from the anterior pituitary gland tissue in the presence of a high level of estrogen, but this is inhibited by addition of progesterone [19]. Thus, it would appear that β-endorphin acts to potentiate the production of the LH surge when estrogen levels are high. Furthermore, β-endorphin may play an important role in terminating the mid-cycle surge in concert with

INTRODUCTION Classic opioids (i.e., enkephalins, endorphins, and dynorphins) are known to exert significant effects on divergent systems via a number of different receptors The opioid receptors (δ, μ, and κ) are members of the seven-transmembrane G-protein-coupled group of receptors and have unique pharmacological properties. These receptors, which are distributed differently within the central nervous system (CNS), are implicated in a broad range of behaviors and functions, including the regulation of pain, reinforcement, and reward (reviewed in the Opioid Peptides Section of this book) [21]. Opioids also have regulatory roles, especially in terms of the local regulation of endocrine function, such as hormone secretion and synthesis. It is now firmly established that normal adrenal chromaffin cells as well as transformed cells, such as pheochromocytoma cells, express opioids and their receptors, which are involved in pathophysiological functions. However, there is insufficient evidence to establish whether opioids have Handbook of Biologically Active Peptides

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830 / Chapter 113 progesterone. Opioids were reported to differentially modify the release of LH, depending on the opioid receptor subtype that was activated. Spontaneous LH release is inhibited by opioids acting via the μ- and κreceptors, and GnRH-stimulated LH release is inhibited by the μ- and δ-receptors. Finally, opioids have been shown to decrease the number of GnRH receptors [2]. Opioids are known to be involved in pathophysiological conditions, such as polycystic ovary syndrome (PCOS). Abnormalities in LH-secretory patterns and their regulation have been observed in PCOS. Abnormally rapid GnRH pulse generation is thought to underlie the atypical LH secretion in PCOS. Abnormalities in other neuroendocrine modulators, such as the endogenous opioids, dopamine, and leptin, have also been proposed as determinants of gonadotrophin secretion in PCOS. An excess of endogenous opioid may sensitize the gonadotroph to GnRH, particularly in association with hyperinsulinemia [23, 24].

PANCREATIC ISLETS Endogeneous opioid peptides are reported to be expressed in the endocrine pancreas [4, 17, 22]. Thus, it appears that islet cell secretion of these peptides could influence β-cell function. Consistent with these findings, a number of in vitro and in vivo studies have demonstrated the direct effect of endogenous and selective opioid receptor agonists on insulin release [1, 8]. However, conflicting results with marked variations among species have been reported [17]. For example, dynorphin-A was reported to increase glucose-induced insulin release [9, 10], whereas conflicting results have been reported with Met-enkephalin [8, 12]. Also βendorphin was reported to inhibit glucose-induced insulin secretion [34], whereas a stimulatory effect was demonstrated after administration of β-endorphin as an intravenous (IV) bolus [32]. Interestingly, the analgesic tramadol, which acts through activation of opioid μ-receptors, can increase the use of glucose and decrease hepatic gluconeogenesis to lower plasma glucose in STZ-induced diabetic rats lacking insulin [5].

ADRENAL GLAND Growing evidence suggests that endogenous opioid peptides and their cognate receptors are expressed in the endocrine adrenal cortex, especially in the medulla [29]. Thus, paracrine interaction between the cortex and medulla may play an important role in regulating their physiological function.

The effect of opioids on the secretory activity of the adrenal cortex has been the subject of much debate. Some reports concluded that enkephalins have no effect on aldosterone secretion [11, 31]. In contrast, it has been demonstrated that Met-enkephalins increase both basal and agonist (ACTH or AT-II)-stimulated aldosterone secretion and that these effects are mediated by μ-opioid receptors [13]. Enkephalins are also reported to affect glucocorticoid secretion, although other studies contradict these findings. Similarly, there are conflicting reports concerning the regulatory effect of endorphins and dynorphins during in vitro studies of adrenocortical cells. The specific reasons for these discrepancies have not been established, although differences in the species or experimental conditions (e.g., hormone concentration) are probably contributing factors. Although the precise role of opioids in chromaffin cells is unclear, mounting evidence suggests that opioids exert their action through the inhibition of catecholamine release in a paracrine or autocrine manner. Normal adrenal chromaffin cells produce only δ-opioid peptides such as enkephalin, and they inhibit basal and nicotine-induced catecholamine secretion [7, 27, 35]. This property of normal chromaffin cells may be changed as a result of the transformation to pheochromocytomas. Indeed, human pheochromocytomas produce κ-opioid peptides such as dynorphin in addition to enkephalin and have a κ-opioid binding site [18, 40, 41]. Furthermore, Margioris et al. demonstrated that PC12 cells, a rat pheochromocytoma cell line and an appropriate model to investigate pheochromocytomas, contain and secrete dynorphin in addition to enkephalin [20, 26]. Moreover, it was demonstrated in several studies that a κ-opioid agonist, but not a δ or μ agonist, significantly inhibited nicotine-induced dopamine release in PC12 cells [30, 38]. These findings indicate that κ-opioids are the most potent inhibitors of catecholamine release from PC12 cells. Recently, we have shown that the anticholinergic actions of κ-opioids are mediated partially by inhibition of thyroid hormone (TH) enzyme activity and TH synthesis through suppression of the cAMP/protein kinase A (PKA) pathway. The inhibitory effect of this pathway is mediated, at least in part, by the pertussis toxin-sensitive G-protein [37]. Kampa et al. recently demonstrated that the κ sites were the dominant opioid binding sites in human pheochromocytomas [18]. Our findings presented here combined with previous studies suggest that κ-opioids may inhibit catecholamine biosynthesis and catecholamine release in human pheochromocytomas (Fig. 1). In addition, differences in the tissue contents of κopioid peptide may contribute to clinical manifestations of pheochromocytomas through the inhibition of catecholamine biosynthesis [40, 41].

Role of Opioid Peptides in the Local Regulation of Endocrine Glands / 831 Ach

K -receptor VDCC

Na+

+

Gi

Ca+

AC

TH-enzyme

cAMP

Opioid peptide

catecholamine

TH-gene Chromaffin cell

FIGURE 1. Proposed inhibitory role for κ opioid peptide in the regulation of catecholamine synthesis. We have shown that the anticholinergic actions of κ opioids are mediated partially by inhibition of TH-enzyme activity and TH-synthesis through suppression of the cAMP/PKA pathway. The inhibitory effect of this pathway is mediated, at least in part, by the pertussis toxin-sensitive G-protein. Modified from [3].

CONCLUSION We have reviewed the role of opioid peptides in the local regulation of endocrine glands, focusing mainly on the pituitary gland, pancreatic islets, and adrenal gland. The review emphasizes the diverse role of the opioid peptides and their cognate receptors in endocrine function. The discovery of novel potent and selective agonists/antagonists of opioid peptides will open new frontiers in our knowledge of endocrine function and will inevitably lead to the development of new therapeutic agents.

References [1] Ahren B. Effects of beta-endorphin, met-enkephalin, and dynorphin. A on basal and stimulated insulin secretion in the mouse. Int J Pancreatol, 1989;5:165–178. [2] Barkan A, Reigiani S, Duncan J, Papavasiliou S, Marshall JC. Opioids modulate pituitary receptors for gonadotropinreleasing hormone. Endocrinology, 1983;112:387–389. [3] Blank MS, Fabbri A, Catt KJ, Dufau ML. Inhibition of luteinizing hormone release by morphine and endogenous opiates in cultured pituitary cells. Endocrinology, 1986;118:2097–2101. [4] Cetin Y. Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas. Cell Tissue Res, 1990;259:313–319. [5] Cheng JT, Liu IM, Chi TC, Tzeng TF, Lu FH, Chang CJ. Plasma glucose–lowering effect of tramadol in streptozotocin-induced diabetic rats. Diabetes, 2001;50:2815–2821. [6] Estienne MJ, Kesner JS, Barb CR, Kraeling RR, Rampacek GB. On the site of action of naloxone-stimulated cortisol secretion in gilts. Life Sci, 1988;43:3161–3166. [7] Franklin SO, Yoburn BC, Zhu YS, Branch AD, Robertson HD. Preproenkepharin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster: Comparison with central nervous system tissues. Brain Res Mol Brain Res, 1991;10:241–250.

[8] Giugliano D, Torella R, Lefébvre PJ, D’Onofrio F. Opioid peptides and metabolic regulation. Diabetologia, 1988;31:3– 15. [9] Green C, Perrin D, Penman E, Yaseen A, Bay K, Howell SL. Effect of dynorphin on insulin and somatostatin secretion, calcium uptake, and c-AMP levels in isolated rat islets of Langerhans. Diabetes, 1983;32:685–690. [10] Green C, Tadayyon M. Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro. Life Sci, 1988;42:2123–2130. [11] Guaza C, Borrell J. The Met-enkephalin analog D-Ala2Met-enkephalinamide decreases the adrenocortical response to ACTH in dispersed rat adrenal cells. Peptide, 1984;5:895–898. [12] Hermansen K. Enkephalins and the secretion of pancreatic somatostatin and insulin in the dog: studies in vitro. Endocrinology, 1983;113:1149–1154. [13] Hinson JP, Kapas S. Effects of sodium depletion on the response of rat adrenal zona glomerulosa cells to stimulation by neuropeptides: Actions of vasoactive intestinal peptide, enkephalin, substance P, neuropeptide Y and corticotrophin-releasing hormone. J Endocrinol, 1995;146:209–214. [14] Hockings GI, Grice JE, Walters MM, Crosbie GV, Torpy DJ, Jackson RV. A synergistic adrenocorticotropin response to naloxone and vasopressin in normal human: evidence that naloxone stimulates endogenous corticotropin-releasing hormone. Neuroendocrinology, 1995:61:198–206. [15] Imura H, Kato Y, Nakai Y, Nakao K, Tanaka I, Jingami H, Koh T, Yoshimasa T, Tsukada T, Suda M. Endogenous opioids and related peptides: From molecular biology to clinical medicine. J Endocrinol, 1985;107:147–157. [16] Jackson RV, Grice JE, Hockings GI, Torpy DJ. Naloxone-induced ACTH release: Mechanism of action in humans. Clin Endocrinol, 1995;43:423–424. [17] Josefsen K, Buschard K, Sorensen LR, Wollike M, Ekman R, Birkenbach M. Glucose stimulation of pancreatic β-cell lines induces expression and secretion of dynorphin. Endocrinology, 1998;39:4329–4336. [18] Kampa M, Margioris AN, Hatzoglou A, Dermitzaki I, Denizot A, Henley JFm Oliver C, Gravanis A, Castanas E. K1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line. Eur J Pharmacol, 1999; 364:255–262. [19] Kandeel FR, Swerdloff RS. The interaction between βendorphin and gonadal steroids in regulation of luteinizing hormone (LH) secretion and sex steroid regulation of LH and proopiomelanocortin peptide secretion by individual pituitary cells. Endocrinology, 1997;138:649–656. [20] Karl M, Saviolakis GA, Gravanis A, Chrousos GP, Margioris AN. The PC12 rat pheochromocytoma cell line express the prodynorphin gene and secretes the 8kDa dynorphin product. Regul Peptides, 1996;61:99–104. [21] Khachaturian H, Lewis ME, Schäfer MKH, Watson SJ. Anatomy of the CNS opioid systems. Trends in Neurosciences, 1985;8:111– 119. [22] Khawaja XZ, Green IC, Thorpe JR, Titheradge MA. The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Biochem J, 1990; 267:233–240. [23] Lanzone A, Apa R, Fulghesu AM, Cutillo G, Caruso A, Mancuso S. Long-term naltrexone treatment normalizes the pituitary response to gonadotropin-releasing hormone in polycystic ovarian syndrome. Fertil Steril, 1993;59:734–737. [24] Lanzone A, Fulghesu AM, Cucinelli F, Ciampelli M, Caruso A, Mancuso S. Evidence of a distinct derangement of opioid tone in hyperinsulinemic patients with polycystic ovarian syndrome:

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Relationship with insulin and luteinizing hormone secretion. J Clin Endocrinol Metab, 1995;80:3501–3506. Lundblad JR, Roberts JL. Regulation of proopiomelanocortin gene expression in pituitary. Endocr Rev, 1988;1:135–158. Margioris AN, Makrogiannakis E, Makrigiannakis A, Gravanis A. PC12 rat pheochromocytoma cells synthesize dynorphin. Its secretion is modulated by nicotine and nerve growth factor. Endocrinology, 1992;131:703–709. Marley PD, Livett BG. Effects of opioid compounds on desensitization of the nicotinic response of isolated bovine adrenal chromaffin cells. Biochem Pharmacol, 1987;36:2937–2944. Nikolarakis K, Pfeiffer A, Stalla GK, Herz A. The role of CRH in the release of ACTH secretion by opiate agonists and antagonists in rats. Brain Res, 1987;421:373–376. Nussdorfer GG. Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev, 1996;48:495–530. Oka K, Andoh T, Watanabe I, Kamiya Y, Ito H. Inhibition of the neuronal nicotinic receptor-mediated current by kappa opioid receptor agonists in PC12 cells. Pflug Arch, 1998;436:887–893. Racz K, Glaz E, Kiss R, Lada G, Varga I, Vida S, Di Gleria K, Medzihradszky K, Lichtwald K, Vecsei P. Adrenal cortex—a newly recognized peripheral site of action of enkephalins. Biochem Biophys Res Commun, 1980;97:1346–1353. Reid RL, Yen SS. Beta-endorphin stimulates the secretion of insulin and glucagon in humans. J Clin Endocrinol Metab, 1981;52:592–594. Rittmaster R, Cutler G, Sobel D, et al. Morphine inhibits the pituitary-adrenal response to ovine corticotropin-releasing

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SP mRNA in the rat and human anterior pituitary (AP) [8]. Radioimmunoassay (RIA) revealed the presence of SP and other TGRP in the AP of various species. In adrenocorticotropic hormone (ACTH)-secreting human pituitary tumors, notably higher levels of SP immunoreactivity (IR) were found than in tumors not secreting ACTH [27, 48]. Several groups reported the presence of NKA in the rat AP, whereas NKB could not be identified [8]. Synthesis of multiple TGRP in AtT20 cells infected with vaccinia virus recombinants was also demonstrated, and again NKB was not found [35]. Immunocytochemistry (ICC) showed that in the AP, SP-IR was contained in both nerve fibers and parenchymal cells. SP-positive nerve fibers are distributed mainly around the pituitary cells. In the monkey and dog, SPpositive fibers were found to be more closely related to AP cells than to blood vessels. In the rat, SP-positive fibers are nonmyelinated and varicose, and they form typical synaptic contacts with all types of AP cells [29, 33]. SP-IR is present in the AP cells, although the cell subtype is still a matter of discussion [9]. Some groups of investigators reported that SP-IR was present in rat lactotrophs and gonadotrophs, whereas others groups identified SP-positive cells as somatotrophs. By cell immunoblot assay, Arita et al. [4] demonstrated SP secretion by individual rat AP cells. SP secretion differed among AP cells by not less than 1000-fold and estimations indicated that SP-secreting cells accounted for approximately 0.5% of the total dispersed AP cells. This value was lower than the 12% of SP-IR cells present in the AP of male rats. In the guinea pig and humans, SP-IR was present mainly in thyrotrophs [9]. RT-PCR detected the expression of the NK1 receptor mRNA in the rat AP and AtT20 D16v cells [66]. Binding studies carried out in the rat and human AP revealed specific SP-binding sites. Based on titration curves, the receptor was classified as an NK1 receptor. Furthermore, receptor autoradiography combined with ICC

ABSTRACT The mammalian tachykinins are a family of peptides that act as neurotransmitters, neuromodulators, and probably as hormones. They are encoded by three genes, TAC1, TAC3, and TAC4. Tachykinin-gene-related peptides (TGRP), which include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), exert their biological actions through three G-protein-coupled receptors, named NK1, NK2, and NK3. Expression of preprotachykinin genes and TGRP receptors, TGRP localization, as well as their direct effects on the endocrine glands (pituitary, thyroid gland, parathyroid, pancreatic islets, adrenal gland, Leydig cells, and ovary) are surveyed in this chapter.

INTRODUCTION The mammalian tachykinins belong to an evolutionarily highly conserved family of small neuropeptides that are encoded by three genes, preprotachykinin (PPT) 1 (TAC1), TAC 3, and TAC4 (PPT genes: PPT-A, PPT-B, and PPT-C, respectively). Alternatively spliced mRNAs encode different combinations of peptides, and the entire group of biologically active mammalian tachykinins is described as tachykinin-gene-related peptides (TGRP) and includes substance P (SP), neurokinin A (NK A), and neurokinin B (NK B). The biological effects of TGRP are mediated through specific Gprotein-coupled receptors, named NK1, NK2, and NK3 (reviewed in Chapter 114 of the Brain Peptide section).

PITUITARY GLAND Reverse transcription (RT)-polymerase chain reaction (PCR) showed the expression of PPT-A gene and Handbook of Biologically Active Peptides

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834 / Chapter 114 revealed that both lactotrophs and gonadotrophs were provided with SP-binding sites [67]. Binding studies also suggested the presence of NK2 receptor at least in a subset of rat lactotrophs [54]. The direct effects of TGRP on AP secretion are controversial. Studies performed on cultured AP cells, pituitary halves, or AtT20 cells reported results that can be summarized as follows: (1) no effects on human, pig, and rat somatotrophs [22]; (2) no effects and stimulatory or inhibitory effects on human and rat lactotrophs [3, 26]; (3) no effects or stimulatory effects on human and rat thyrotrophs [20, 46]; (4) no effects or inhibitory effects on human, rat, and mouse corticotrophs [24, 41, 58]; and (5) no effect and stimulatory or inhibitory effects on human, pig, and rat gonadotrophs [17, 18]. Taken together, these findings suggest a direct stimulating effect of TGRP on prolactin secretion, but their effects on the secretion of other AP hormones is doubtful.

THYROID GLAND RT-PCR did not detect SP mRNA in extracts of bovine thyroid gland [13]. However, strong expression of TAC 4 gene was recently described in the human and rat thyroid gland [32]. In the human thyroid gland, δTAC 4 gene transcripts were expressed, suggesting that the gland may secrete hemokinins [52]. Earlier reports showed relatively low concentrations of SP-IR in the porcine and rat thyroid gland [14]. ICC indicated that localization of TGRP in the thyroid gland was intimately connected with the gland innervation. In several species Ahren et al. [1] demonstrated SP-positive nerve fibers around blood vessels and thyroid follicles, and a similar pattern of distribution was observed for NKA-positive fibers [23]. In the rat thyroid gland, SP-positive fibers originated from the jugular, cervical dorsal root, or trigeminal ganglia, and cervical vagotomy reduced the number of NKA-IR fibers by approximately 50% [1, 23]. Studies on the direct effects of TGRP on thyroid growth and function are few. SP caused a prompt but transient rise in cAMP levels and also increased basal, but not thyroid-stimulating hormone (TSH)-stimulated, release of thyroid hormones from canine thyroid slices [68]. On the contrary, SP did not influence either basal or TSH-stimulated cAMP levels in human thyroid cell cultures [7]. Taken together, these findings suggest that TGRP are primarily involved in the regulation of thyroid blood flow.

PARATHYROID GLAND RIA demonstrated the presence of SP-IR in extracts of sporadic human parathyroid adenomas, where the

SP-IR content correlated directly with that of parathyroid hormone [65]. In the bovine parathyroid gland, ICC localized SP-IR in the nerve fibers, which ended in contact with the tunica media of arteries and arterioles or were dispersed throughout the stroma of the gland. No intimate contact with chief cells was observed [45]. A similar pattern of nerve fibers distribution has been described in rat, guinea pig, cat, dog, and sheep parathyroid gland [34]. In parathyroid adenomas, SP-IR was confined to 5–10% of oxyphilic cells, and only a few SP-positive fibers were found. In porcine parathyroid cells, RT-PCR showed the expression of NK1 receptor mRNA [21]. Earlier reports indicated that SP had no effect on parathyroid hormone release from dispersed bovine parathyroid cells. However, recently Galvin et al. [21] found a potent, calcium-dependent, and NK1-receptormediated inhibitory effect of SP on parathyroid hormone release from these cells, thereby suggesting the involvement of this tachykinin in the regulation not only of blood flow but also of the secretory activity.

PANCREATIC ISLETS The endocrine cells of the adult rat pancreas did not appear to express PPT-A, whereas in the rat β-cellderived cell lines RIN5mF and RINr1046–38, normal transcription and posttranslational processing of the (PPT)-I gene and regulated release of SP and NKA have been demonstrated [39], suggesting that TGRP expression may be a feature of the transformed state of these cell lines. However, there is indication that TGRP may play a role in pancreatic islet embryogenesis. In fact, many endocrine cells of the fetal and neonatal rat pancreas were found to synthesize SP and NKA, and after postnatal day 20 the number of these cells declined and none was detected in the adult rat pancreas [40]. SP-IR was demonstrated by RIA in extracts of porcine and rat pancreas [16]. Moreover, capsaicin administration stimulated SP secretion by isolated perfused porcine pancreas; however, the source (endocrine and/or exocrine pancreas) of that peptide remains unknown [59]. ICC traced several SP-positive nerve fibers around pancreatic islets of rats, cats, pigs, and cows [2, 16, 59]. Reports on the effects of TGRP on insulin and glucagon secretion are controversial. The first functional studies carried out on isolated rat pancreatic islets either failed to demonstrate SP effects on insulin secretion or revealed an inhibitory effect on insulin release and a stimulatory one on glucagon output [44]. Subsequent investigations showed a stimulating and an inhibitory effect of SP on glucagon secretion from pancreatic tissue fragments obtained from normal and diabetic rats, respectively [56]. Inhibitory effects of SP on insulin

Role of Tachykinin-Gene-Related Peptides in the Local Regulation of Endocrine Glands / 835 and glucagon release were also observed in a model of perfused rat pancreas, whereas in similar canine and porcine preparations SP and NKA were shown to stimulate secretion of both insulin and glucagon, the effects being mediated by the NK1 receptor [59]. Collectively, these findings suggest that TGRP are involved in the local regulation of pancreatic islet secretion, although the effect depends on the species and the experimental model used.

ADRENAL GLAND High expression of TAC4 gene was found in human, rat, and mouse adrenals [52]. TAC4 encoded hemokinins and endokinins, which possess high selectivity and potency for NK1 receptors. In the rat, endokinins and SP displayed similar hemodynamic effects, and it has been suggested that endokinins are the endogenous peripheral SP-like endocrine/paracrine agonists where SP is not expressed. All four transcripts of TAC 4 (α, β, γ, and δ) were identified in the adrenal gland, and adrenal was the only tissue found to express all transcripts and hence able to produce all four endokinins. TAC1 expression was limited to the human adrenocortical carcinoma-derived H-295 cell line, and TAC 1 and TACR1 expression was not detected in the rat adrenal gland [52]. High-performance liquid chromatography (HPLC) and RIA have demonstrated SP-IR in the adrenal glands of several mammalian species, including humans, cows, sheep, cats, dogs, guinea pigs, rabbits, mice, and rats. A very high concentration of SP-IR was found in the human adrenal medulla [37, 48]. The presence of tachykinins other than SP has been frequently demonstrated in adrenal glands. RIA showed the presence of NKA and NPγ(1–9) in rat and porcine adrenals [64]. Moreover, HPLC demonstrated the presence of numerous tachykinins (among which are NKA and NKB) in the bovine adrenal medulla [6, 12]. In the rat, adrenal concentration of SP was markedly higher than that of NKA and NKB [51]. ICC detected tachykinin-IR in nerve fibers, as well as ganglion and chromaffin cells of adrenal medulla [37, 48]. In both humans and rats, SP-positive fibers penetrated the adrenal capsule and cortex to innervate the medulla; few collaterals were found in the cortex, whereas medullary cells were richly innervated. SP-IR has been also demonstrated in rat adrenal medulla where SP-positive chromaffin cells occurred alone or in small islets [63]. The origin of SP-positive fibers in the adrenal gland is not clear and probably differs among species. Systemic capsaicin treatment reduced by 80% SP-IR in the adrenal gland of the adult guinea pig and had no effect in the rat adrenals, whereas splanchnic

denervation caused a notable lowering in the SP content of the rat adrenal [11]. This finding suggests that a part of adrenal SP was localized in nerve fibers entering the gland via splanchnic nerves. Further studies on the origin of SP in rat adrenal cortex revealed that splanchnic nerve section followed by a 10-day recovery period caused an increase in the adrenal capsular content of SP compared with sham-operated controls, whereas the inner cortex/medullary content of this tachykinin was unaffected [25], suggesting that SP in the rat adrenal capsule/zona glomerulosa is regulated by splanchnic nerve activity. High concentrations of NK2 receptors and the absence of NK1 and NK3 subtypes have been shown in the rat adrenals [51]. Other authors, however, found the presence of NK1 receptors in medullary chromaffin cells [36]. Moreover, autoradiography demonstrated that [3H]SP binding sites were exclusively located on chromaffin cells of human [48]. Evidence has been provided that TGRP stimulates catecholamine release from adrenomedullary chromaffin cells, mainly acting via NK1 receptors [47, 48]. Results of studies on the direct TGRP effects on adrenal steroidogenesis depend on the experimental model applied. In freshly isolated rat adrenocortical cells, SP and other TGRP did not affect secretion of aldosterone and corticosterone. On the contrary, in adrenal slices (containing adrenal medulla) and perfused rat adrenals, SP and related peptides notably stimulated aldosterone and corticosterone secretion [48]. These findings suggest that the aldosterone secretagogue effect of SP requires the integrity of the adrenal tissue and especially the presence of medullary chromaffin cells. It has been well documented that catecholamines increase adrenal steroid secretion in mammals, zona glomerulosa (ZG) and aldosterone secretion being their main targets in rodents, bovine, and humans [47]. Hence, it is possible that SP may enhance aldosterone secretion by eliciting the release from chromaffin cells of catecholamines, which in turn stimulate ZG in a paracrine manner. Some pharmacological data strongly support this hypothesis [38, 47, 48].

LEYDIG CELLS RT-PCR demonstrated the presence of PPT-A mRNA in extracts of human, mouse, and bovine, but not in rat and boar, testes [13]. Furthermore, Northern blot analysis detected PPT-A gene expression in the Siberian hamster testis [18]. TAC 4 expression was recently found in human and rat testes, where βTAC 4 and αTAC4 splice variant 2 were present, a finding suggesting local endokinin synthesis [52]. The expression of TAC4 gene was found in the rat testis, whereas the expression of

836 / Chapter 114 TAC1 and TACR1 was virtually absent. Moreover, PCR analysis revealed also the presence of both SP and NKA receptor mRNAs in human testes [52]. Few results are available on the TGRP-IR concentration in the testes. In the rat, testicular SP and NKA concentrations displayed the tendency to decrease from ages day 21 to day 60 [62]. ICC detected the presence of SP-IR in both Leydig cells and nerve fibers [18]. SP-IR was found in Leydig cells of mice, gerbils, hamsters, guinea pigs, pigs, and marmosets [18, 31, 50] but not rats [18]. In the hamster and guinea pig testis during fetal and postnatal development, SP-IR was found in both fetal and adult generation of Leydig cells, suggesting a possible role of SP in the local paracrine control of gametogenesis [15, 42]. Numerous SP-positive fibers are found in both blood vessels and Leydig cell nests in the cat and pig testes [61]. Sporadic functional studies revealed that SP and other TGRP exert a potent in vitro inhibitory effect on testosterone secretion from mouse and hamster Leydig cells [18].

OVARY RT-PCR demonstrated SP and NK1-receptor mRNAs in the bovine corpus luteum at an early developmental stage [57]. Moreover, isolated cumulus cells of the mouse follicles expressed PPT-A, PPT-B, PPT-C, and low levels of NK1 and NK2 receptors [53]. In the rat ovary, the concentration of SP-IR varied in relation to the onset of puberty, with values increasing significantly between the late juvenile phase and the day of first proestrus [17]. The concentrations of SP and NKA in the ovaries of mice treated with pregnant mare serum (PMS) or PMS/hCG were significantly lower than in control ovaries, whereas the total ovarian content of NKA was not significantly different. These results show that gonadotrophin-stimulated ovaries have lower concentrations of tachykinins than agematched normal ones. In human follicular fluid obtained from hCG-stimulated infertile patients undergoing in vitro fertilization, SP concentration was approximately 10-fold higher than in the serum, and peptide concentrations did not vary with the size of follicles [28]. Moreover, in the human ovaries thecal and stromal concentrations of SP-IR were greater than those in the corpora lutea and tunica albuginea [5]. Perfusion studies demonstrated SP release from human and bovine ovaries, but the source of the ovarian TGRP remained unrecognized [43, 60]. Production of SP-IR from cultured rat granulosa was not detected under either basal or gonadotrophin-stimulated conditions [49, 55]. The bulk of ICC evidence indicates that ovarian TGRP were present primarily in nerve fibers. In ovaries

from juvenile and peripubertal rats, SP-positive fibers were found to be closely associated with the theca externa of antral follicles, as well as with the interstitial tissue and tunica adventitia of small blood vessels, mostly arterioles. SP-IR was not detected in the corpora lutea [19]. In the bovine ovary, sparse SP-positive fibers were present in all ovarian regions and especially in the medulla, where they innervated blood vessels. SPpositive fibers were sometimes observed near primordial and primary follicles and between granulosa lutein cells [30, 57]. SP-IR was also localized in a subpopulation of ovarian-vein endothelial cells both in situ and in culture [10, 60]. Functional studies on the role of TGRP in regulation of ovarian hormone synthesis are scarce. SP did not stimulate steroid secretion from either cultured rat granulosa cells or ovarian fragments in short-term incubation. SP also failed to stimulate prostaglandin E2 release from whole ovaries or to modify the secretory response of cultured granulosa cells to folliclestimulating hormone (FSH) and to β2-adrenoceptor agonists [49]. SP was found to exert a marked vasodilatory effect on preconstricted ring preparations of human ovarian vein, the effect being partially mediated by endothelium [30, 57]. This finding makes it likely that SP plays a role in the local control of vascular tone in the ovary.

CONCLUSION It is well established that TGRP may modulate endocrine gland functions by acting primarily at the hypothalamic level (reviewed in the Chapter 188 of the Brain Peptide section). But in addition to this mechanism, TGRP also have an effect that is exerted directly on endocrine glands, conceivably through autocrine/paracrine mechanisms. In this regard, TGRP released by nerve fibers may exert a major role in the neuroendocrine control of both blood flow and hormone release.

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Role of Tachykinin-Gene-Related Peptides in the Local Regulation of Endocrine Glands / 837 [4] Arita J, Kojima Y, Yamamoto I, Mazawa S, Kimura F. Somatotropes and thyrotropes in the rat anterior pituitary gland cosecrete substance P: analysis by the sandwich cell immunoblot assay. Neuroendocrinology 1994;60:567–74. [5] Barad DH, Ryan KJ, Elkind-Hirsch K, Makris A. Immunoreactive substance P in the human ovary. Am J Obstet Gynecol 1988; 159:242–6. [6] Basile S, Cheung NS, Livett BG. Chromatographic evidence for the presence of multiple tachykinins in the bovine adrenal medulla. J Neurochem 1992;58:1584–6. [7] Brandi ML, Toccafondi R. Neuropeptidergic control of cyclic AMP accumulation in human thyroid cell. Peptides 1985;6: 641–4. [8] Brown ER, Harlan RE, Krause JE. Gonadal steroid regulation of substance P (SP) and SP-encoding messenger ribonucleic acids in the rat anterior pituitary and hypothalamus. Endocrinology 1990;126:330–40. [9] Brown ER, Roth KA, Krause JE. Sexually dimorphic distribution of substance P in specific anterior pituitary cell populations. Proc Natl Acad Sci USA 1991;88:1222–6. [10] Brylla E, Aust G, Geyer M, Uckermann O, Loffler S, SpanelBorowski K. Coexpression of preprotachykinin A and B transcripts in the bovine corpus luteum and evidence for functional neurokinin receptor activity in luteal endothelial cells and ovarian macrophages. Regul Pept 2005;125:125–33. [11] Bucsics A, Saria A, Lembeck F. Substance P in the adrenal gland: origin and species distribution. Neuropeptides 1981;1: 329–41. [12] Cheung NS, Basile S, Livett BG. Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure. Neuropeptides 1993;24:91–7. [13] Chiwakata C, Brackmann B, Hunt N, Davidoff M, Schulze W, Ivell R. Tachykinin (substance-P) gene expression in Leydig cells of the human and mouse testis. Endocrinology 1991;128:2441–8. [14] Cremins JD, Michel J, Farah JM, Krause JE. Characterization of substance P-like immunoreactivity and tachykinin-encoding mRNAs in rat medullary thyroid carcinoma cell lines. J Neurochem 1992;58:817–25. [15] Davidoff MS, Schulze W, Middendorff R, Holstein AF. The Leydig cell of the human testis—a new member of the diffuse neuroendocrine system. Cell Tissue Res 1993;271:429– 39. [16] De Giorgio R, Sternini C, Anderson K, Brecha NC, Go VL. Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas. Peptides 1992;13:91–8. [17] Debeljuk L. Tachykinins in the normal and gonadotropinstimulated ovary of the mouse. Peptides 2003;24:1445–8. [18] Debeljuk L, Rao JN, Bartke A. Tachykinins and their possible modulatory role on testicular function: a review. Int J Androl 2003;26:202–10. [19] Dees WL, Kozlowski GP, Dey R, Ojeda SR. Evidence for the existence of substance P in the prepubertal rat ovary, II. Immunocytochemical localization. Biol Reprod 1985;33:471–6. [20] Duvilanski BH, Pisera D, Seilicovich A, del Carmen Diaz M, Lasaga M, Isovich E, et al. Interaction between substance P and TRH in the control of prolactin release. J Endocrinol 2000; 166:373–80. [21] Galvin RJS, Babbey LE, Hipskind PA, Lamar T, George CA, Baez M, et al. Substance P regulates PTH secretion through the neurokinin-1 receptor. Biochem Biophys Res Commun 2000;270:230–4. [22] Glavaski-Joksimovic A, Jeftinija K, Jeremic A, Anderson LL, Jeftinija S. Mechanism of action of the growth hormone secretagogue, L-692,585, on isolated porcine somatotropes. J Endocrinol 2002;175:625–36.

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838 / Chapter 114 [42] Middendorff R, Davidoff M, Holstein AF. Neuroendocrine marker substances in human Leydig cells—changes by disturbances of testicular function. Andrologia 1993;25:257–62. [43] Miyamoto A, Bruckmann A, von Lutzow H, Schams D. Multiple effects of neuropeptide Y, substance P and vasoactive intestinal polypeptide on progesterone and oxytocin release from bovine corpus luteum in vitro. J Endocrinol 1993;138:451–8. [44] Moltz JH, Dobbs RE, McCann SM, Fawcett CP. Effects of hypothalamic factors on insulin and glucagon release from the islets of Langerhans. Endocrinology 1977;101:196–202. [45] Mortimer S, Hanley D, Stell W. Immunohistochemical identification of calcitonin gene-related peptide and substance-P in nerves of the bovine parathyroid gland. Cell Tiss Res 1990; 261:339–45. [46] Moura EG, Santos CV, Santos RM, Pazos-Moura CC. Interaction between substance P and gastrin-releasing peptide on thyrotropin secretion by rat pituitary in vitro. Braz J Med Biol Res 1999; 32:1155–60. [47] Nussdorfer GG. Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev 1996;48:495–530. [48] Nussdorfer GG, Malendowicz LK. Role of tachykinins in the regulation of the hypothalamo-pituitary-adrenal axis. Peptides 1998;19:949–68. [49] Ojeda SR, Costa ME, Katz KH, Hersh LB. Evidence for the existence of substance P in the prepubertal rat ovary. I. Biochemical and physiologic studies. Biol Reprod 1985;33:286– 95. [50] Ortega HH, Lorente JA, Salvetti NR. Immunohistochemical study of intermediate filaments and neuroendocrine marker expression in Leydig cells of laboratory rodents. Anat Histol Embryol 2004;33:309–15. [51] Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;73:229–308. [52] Page NM. Hemokinins and endokinins. Cell Mol Life Sci 2004;61:1652–63. [53] Pintado CO, Pinto FM, Pennefather JN, Hidalgo A, Baamonde A, Sanchez T, et al. A role for tachykinins in female mouse and rat reproductive function. Biol Reprod 2003;69:940–46. [54] Pisera D, Candolfi M, De Laurentiis A, Seilicovich A. Characterization of tachykinin NK2 receptor in the anterior pituitary gland. Life Sci 2003;73:2421–32. [55] Pitzel L, Jarry H, Wuttke W. Effects of substance-P and neuropeptide-Y on in vitro steroid release by porcine granulosa and luteal cells. Endocrinology 1991;129:1059–65.

[56] Ponery AS, Adeghate E. Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues. Peptides 2000;21:1503–9. [57] Reibiger I, Aust G, Tscheudschilsuren G, Beyer R, Gebhardt C, Spanel-Borowski K. The expression of substance P and its neurokinin-1 receptor mRNA in the bovine corpus luteum of early developmental stage. Neurosci Lett 2001;299:49–52. [58] Richardson UI, Schonbrunn A. Inhibition of adrenocorticotropin secretion by somatostatin in pituitary cells in culture. Endocrinology 1981;108:281–90. [59] Schmidt PT, Tornoe K, Poulsen SS, Rasmussen TN, Holst J J. Tachykinins in the porcine pancreas: potent exocrine and endocrine effects via NK-1 receptors. Pancreas 2000;20:241–7. [60] Stones RW, Loesch A, Beard RW, Burnstock G. Substance P: endothelial localization and pharmacology in the human ovarian vein. Obstet Gynecol 1995;85:273–8. [61] Suburo AM, Chiocchio SR, Soler MV, Nieponice A, Tramezzani JH. Peptidergic innervation of blood vessels and interstitial cells in the testis of the cat. J Androl 2002;23:121–34. [62] Vazquez Moreno N, Debeljuk L, Diaz Rodriguez E, Fernandez Alvarez C, Diaz Lopez B. Seasonal changes of SP and NKA in frontal cortex, striatum and testes in the rat. Role of maternal pineal gland. Peptides 2004;25:997–1004. [63] Vinson JP, Hinson JP, Toth IE. The neuroendocrinology of the adrenal cortex. J Neuroendocrinol 1994;6:235–46. [64] Wang JM, de Ridder EFMM, de Potter WP, Weyns ALM. Localization of neurokinin A and chromogranin A immunoreactivity in the developing porcine adrenal medulla. Histochem J 1994; 26:431–6. [65] Weber CJ, O’Dorisio TM, Howe B, D’Agati V, Ward L, Russell J, et al. Vasoactive intestinal polypeptide-, neurotensin-, substance P-, gastrin-releasing peptide-, calcitonin-, calcitonin gene related peptide-, and somatostatin-like immunoreactivities in human parathyroid glands. Surgery 1991;110:1078–85. [66] Winkler A, Papsdorf G, Odarjuk J, Siems WE, Fickel J, Melzig MF. Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells. Eur J Pharmacol 1995;291:51–5. [67] Wormald PJ, Millar RP, Kerdelhue B. Substance P receptors in human pituitary: a potential inhibitor of luteinizing hormone secretion. J Clin Endocrinol Metab 1989;69:612–5. [68] Yamashita K, Koide Y, Aiyoshi Y. Effects of substance P on thyroidal cyclic AMP levels and thyroid hormone release from canine thyroid slices. Life Sci 1983;32:2163–6.

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115 Neuropeptide Y and the Regulation of Endocrine Function J. P. Hinson

actions of NPY on the tissues of the peripheral endocrine system.

ABSTRACT Neuropeptide Y (NPY) is widely distributed in the peripheral endocrine system and has been the subject of several studies in each tissue, with the adrenal being the most extensively studied. A remarkable pattern has emerged, with striking similarities among the tissues studied. In all tissues, NPY is found in nerves supplying the gland, with some instances of individual endocrine cells also expressing NPY. In all cases studied, NPY causes a decrease in the rate of blood flow through the endocrine gland. Although the effect on basal hormone secretion is highly variable and clearly dependent on the tissue preparation used, NPY has a common action to inhibit stimulated hormone secretion.

NPY AND ADRENAL FUNCTION The adrenal gland was the first endocrine organ reported to have a significant NPY content [4], and there is a considerable body of literature on the distribution and role of NPY in the mammalian adrenal gland, which has recently been reviewed [58, 64]. Neuropeptide Y gene expression is high in the rat adrenal medulla [19], and higher concentrations have been reported in the medulla of cat and cow compared with the cortex [4, 37], although in the rat adrenal the difference is smaller [23]. There appear to be species differences in the distribution of NPY in the adrenal, numerous medullary cells staining positive for NPY peptide in the cat and guinea pig adrenal but only nerve fibers staining in the pig adrenal [35]. In addition there is a network of nerve fibers supplying the adrenal cortex, which are mostly confined to the zona glomerulosa and are particularly associated with blood vessels (Fig. 1) [34]. Adrenal NPY content is actively regulated by a range of factors (for a comprehensive review see [58]). Splanchnic nerve stimulation causes both the release of NPY from the adrenal [5] and an increase in NPY gene expression [15]. Adrenal NPY content is also reported to increase with age [20] and in response to various forms of stress (reviewed in [58]) but to decrease in response to activation of the hypothalamo-pituitaryadrenal (HPA) axis (reviewed in [58]). It is presumed that the stress-induced increase in adrenal NPY is mediated through increased sympathetic activity rather than the HPA axis. As in other endocrine tissues, NPY causes a dosedependent decrease in blood flow through the gland

INTRODUCTION Neuropeptide Y (NPY) is a peptide with wellrecognized functions in the brain (see Chapter 95 by Dumont and Quirion in the Brain Peptides section of this book). Central administration of NPY brings about a wide range of effects, including effects on the endocrine system. However, NPY is also found in the peripheral sympathetic nervous system, with a wide distribution in the innervation of tissues in the endocrine system. Both NPY peptide and its receptors have been immunolocalized in tissues of the peripheral endocrine system, including the endocrine pancreas, ovary, testis, thyroid, and adrenal glands. It is clear that, in addition to its central actions, NPY exerts direct paracrine and autocrine effects on endocrine tissues. It should perhaps not be surprising to find that NPY exerts remarkably similar effects on different endocrine tissues. In research it is easy to focus solely on one tissue and thus fail to recognize common actions of a peptide on different tissues. This chapter focuses on the distribution and Handbook of Biologically Active Peptides

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840 / Chapter 115

FIGURE 1. Neuropeptide Y immunoreactive nerves in the rat adrenal cortex. The adrenal glands of male SpragueDawley rats were perfusion-fixed in situ with 4% paraformaldehyde. Adrenals were removed and post-fixed for 60 min in 4% paraformaldehyde and then cryopreserved in 15% w/v sucrose in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. Glands were frozen in embedding solution and 6 μm sections cut. Sections were thaw-mounted onto slides and incubated for 60 min in a blocking solution (10% donkey serum) at room temperature. Sections were incubated in anti-NPY serum (Biogenesis, Cambridge, UK) diluted in PBS containing 0.2% Triton X-100 at room temperature for 48 h. Sections were washed and then incubated in FITC-labeled donkey antirabbit secondary antiserum for 4 h. Sections were washed again and then mounted with PBS/glycerol (1 : 3) aqueous mountant containing the antifade agent DABCO and viewed using a fluorescence microscope. Images were imported into Photoshop and gray levels stretched to optimize contrast, but they were not manipulated in any other way. Scale: 1 cm = 100 μm. Figure kindly provided by Dr. D. Renshaw.

[22]. Which receptor mediates this effect has not been established, although both Y1 and Y4 receptors are present in the rat adrenal gland [58]. The actions of NPY on the adrenal cortex appear paradoxical. In intact adrenal tissue, both in vivo and in vitro, NPY causes an increase in basal aldosterone secretion, but does not consistently affect corticosterone (the major glucocorticoid of the rat) (for review see [58, 64]). However, when dispersed cells were used as the model system, NPY inhibited basal aldosterone secretion and caused attenuation of the response to maximal stimulation [21, 52], but enhanced the response to submaximal stimulation [21]. The differences between the tissue preparations may be accounted for by the observation that NPY causes a release of cate-

cholamines from medullary cells within the rat adrenal capsule, and these catecholamines exert a stimulatory effect on aldosterone, which presumably masks the direct effects of NPY itself [59]. Despite stimulating catecholamine release from adrenal capsular preparations, NPY has been reported to inhibit stimulated medullary catecholamine release [18, 42]. This is controversial, however, and depends at least in part on the tissue preparation used and the species under investigation (for review see [64]). It has proved difficult to establish the effects of NPY on adrenal growth because the in vivo infusion of this peptide is associated with changes in both the HPA axis and in the renin-angiotensin system and the experiments conducted have produced conflicting results [64]. Adrenal cells do not grow well in culture, and so this approach has not been possible. However, recent studies from our laboratory, using cultures of intact rat capsular tissue, have shown that NPY is a potent mitogen in the rat zona glomerulosa, the outermost layer of the adrenal cortex (Fig. 2) [68].

NPY IN THE PANCREAS Neuropeptide Y has been identified in nerves supplying the rodent pancreas [54, 65] and also in the endocrine cells of the islets of Langerhans [2]. Much of the innervation appears to be associated with the vasculature. A similar distribution has been reported in both normal and diabetic rats, although a wider distribution of NPY was found in the islets of diabetic rats with an increase in the total number of NPY-positive cells compared to controls [2]. In the bovine pancreas NPY immunoreactivity was confined to nerve fibers that were associated with the acini and the islets of Langerhans and around blood vessels [51]. The related peptide, PYY, has also been identified in nerves supplying the rat and porcine pancreas [36]. Comparative aspects of the distribution of NPY and PYY in the pancreas have been reviewed [12]. Little is known about the regulation of NPY release from the pancreas, but in vitro studies on rat islets have shown that glibenclamide, a sulfonylurea widely used to treat type 2 diabetes, stimulates NPY release [33]. Although NPY is classically a peptide of the sympathetic nervous system, evidence suggests that in the pancreas most of the nerve fibers belong to the parasympathetic innervation with relatively small amounts of NPY release by sympathetic stimulation [62]. The effects of NPY on pancreatic function are consistent in that NPY reliably inhibits glucose-stimulated insulin release. Using isolated rat islets NPY has been shown to inhibit glucose-stimulated insulin release without affecting basal secretion [7, 9, 49]. In an in situ

Neuropeptide Y and the Regulation of Endocrine Function / 841 A

B

ences in the physiological status of the animals tested may also have influenced the results obtained. In normal rat pancreatic tissue incubated in vitro, NPY caused a significant increase in basal insulin release, but this effect was no longer seen in tissue obtained from diabetic rats [2]. It appears likely that, as in other endocrine tissues, the Y1 receptor has a major role in mediating the effects of NPY. The Y1 receptor subtype has been identified in the mouse pancreatic islet [9] and in the rat pancreas, with some islet cells staining positive [41]. In mice with the NPY Y1 receptor knockout, fasted insulin levels were higher than controls, but there was a significantly impaired insulin response to feeding [8]. NPY may also have a role in maintenance of islet cell number as treatment of isolated islets with NPY in vitro causes proliferation of islet cells [9]. In terms of glucagon release, NPY has been shown to strongly stimulate basal release from normal rat pancreatic tissue, but to exert an inhibitory effect on tissue from diabetic rats [57]. In the mouse in vivo, however, NPY had no effect on basal secretion, but inhibited glucagon release stimulated by terbutaline, a betaadrenoceptor agonist [55].

NPY AND GONADAL FUNCTION

FIGURE 2. Section through a rat adrenal capsule incubated in vitro for 8 days (A) under control conditions and (B) in the presence of 1 μmol/l NPY. Following the culture period, media were removed and tissues incubated with bromodeoxyuridine for 6 h. Tissue was fixed, paraffin wax embedded, and sections cut at 6 μm. Dark staining shows BrDU staining, indicating actively dividing cells. Bar represents 50 μm. Figure kindly provided by Dr. E. J. Whitworth.

rat pancreatic perfusion system, however, NPY inhibited both basal and stimulated insulin release, although when administered intracerebroventricularly (ICV) NPY had a stimulatory effect [49]. NPY has also been shown to inhibit stimulated insulin secretion but to stimulate basal insulin secretion in the mouse in vivo [54]. Thus NPY has been reported to inhibit, stimulate, or have no effect on basal insulin release, with the effect of NPY appearing to vary depending, at least partly, on the tissue preparation tested. This is not unique to the pancreas (see adrenal section). It is possible that differ-

There is considerable evidence to suggest that the major role of NPY in reproductive function is centrally mediated. NPY acts on the hypothalamus to regulate gonadotrophin-releasing hormone (GnRH) release and also appears to act on the anterior pituitary to regulate luteinizing hormone (LH) and folliclestimulating hormone (FSH) secretion (for review see [43]). This peptide also appears to have a role in the initiation of puberty [47]. There is also, however, considerable evidence for a local effect of NPY-ergic innervation in the paracrine regulation of gonadal function. A significant gender difference in NPY innervation of the rat gonads has been noted, leading to the suggestion that this peptide is likely to be more important in ovarian than testicular function [6]. The distribution and possible roles of NPY in the female mammalian reproductive tract has been reviewed recently [40]. Although most studies have been carried out in mammals, there is also evidence for an NPY-ergic innervation to the testis of the toad (Bufo arenarum [1]), although the functional significance of this finding has not been established. In the bluehead wrasse (Thalassoma bifasciatum), a teleost fish that undergoes female-to-male sex transformation (i.e., is protogynous), intraperitoneal injection of NPY stimulated ovarian degeneration, a significant indicator of sex reversal [32].

842 / Chapter 115 Testis High concentrations of NPY have been found in nerves supplying the human male genital tract, although the levels in the testis were low compared with other tissues [3]. NPY-positive innervation has also been described in the rat [6] and pig testis [67]. The pattern of innervation changes during development. The fetal rat testis has no significant NPY-positive innervation, but innervation appeared from day 2, with more abundant nerves appearing from day 5, although this was fairly sparse [6]. In the pig testis, there is an increase in NPY innervation of the testis during early life, but this is followed by an almost total withdrawal of NPY-positive fibers with the onset of maturity [67, 69]. There is evidence that the gene encoding NPY may be expressed in rat Leydig and Sertoli cells in response to the appropriate hormonal stimulation [29]. The NPYinnervation is also affected by the hormonal milieu, with immunization against GnRH preventing the loss of NPY-positive testicular innervation normally seen in maturing animals [67]. Neuropeptide Y Y1 receptors have been identified in the blood vessels supplying the rat testis [30, 41] and there is some evidence that an intra-testicular injection of NPY causes a local decrease in blood flow [10]. NPY binding has also been described in both Leydig cell and Leydig-Sertoli cell tumors [31], although the significance of this finding is not clear. The effects of NPY on Leydig or Sertoli cell function do not appear to have been investigated.

although the magnitude of these effects varies depending on the stage of the estrous cycle [39]. NPY has also been shown to reduce ovarian blood flow in the rabbit [27]. NPY does not appear to have a role in regulating ovulation. In an in vitro rat ovarian preparation, NPY did not increase the number of ovulations and had no effect on the number of norepinephine-stimulated ovulations [26]. However, NPY may have a role in follicular and luteal function. In the pig, ovarian denervation, associated with a significant reduction in NPY-positive nerve fibers adjacent to follicles, resulted in a decrease in plasma progesterone in the late follicular and luteal phase of the cycle [25]. In bovine corpus luteum incubated in vitro, NPY increases both progesterone and oxytocin release [48]. However, in porcine luteal cell cultures NPY inhibited both basal and hCG-stimulated progesterone release [56]. Studies on ovarian tissue incubated in vitro from rats in diestrus revealed opposite effects of NPY depending on whether the tissue came from rats in D1 or D2 [16]. It is therefore possible that this observation may account for differences reported as the effects of NPY are clearly dependent on the estrous cycle. As in many other endocrine tissues, NPY Y1 receptors have been identified in the rat ovary, particularly associated with blood vessels [30] and are likely to mediate the effects of NPY on blood flow and, possibly, the other reported effects.

NPY AND THYROID FUNCTION Ovary Nerve fibers staining for NPY have been identified in the ovary of human [53], rat [6, 44], guinea pig, cow, and pig [28]. NPY-positive nerves are numerous in the ovary and are particularly associated with blood vessels [28, 39, 44]. Some NPY-positive fibers appear to directly enter the follicles in the rat ovary [44]. In the rat the concentration of NPY in the ovary appears to depend on the stage of the estrous cycle [26]. It is not clear whether this is true for other species. There is no NPY-positive innervation to the rat ovary during fetal life, and the innervation develops in the first two weeks of life [6]. The ovarian innervation does not appear to change with the onset of maturity [6]. Neuropeptide Y has several reported roles in the ovary. There is evidence that NPY modulates the sympathetic input to the ovary of the immature rat, causing a significant decrease in the amount of norepinephrine released in response to electrical stimulation [13]. NPY appears to regulate ovarian blood flow, acting to cause constriction of the porcine ovarian artery and to potentiate the vasoconstrictor effect of norepinephrine,

Neuropeptide Y or its mRNA have been identified in the thyroid gland of several different species including human [17, 50], rat and other rodent species [17, 60], and lower vertebrates [61]. In all species the NPY in the thyroid is located in nerve fibers [17], but there is some evidence that in rodent thyroids NPY is also found within parafollicular cells [60]. In the human thyroid these NPY-positive nerves are mostly associated with blood vessels, with relatively few extravascular fibers [50]. The clear association of NPY with thyroid arterioles has led to the proposal of a role for NPY in regulating thyroid blood flow. An infusion of NPY (0.625 μg over 2 minutes) into anesthetized rats did not cause any change in thyroid blood flow as measured by the radioactive microsphere technique [24]. However, this finding did not rule out the possibility of a tonic effect of endogenous NPY. A more recent study from the same group, in which researchers administered an NPY antiserum to rats and observed an increase in thyroid blood flow, suggests that endogenous NPY exerts a tonic vasoconstrictor effect in the rat thyroid gland [46]. It is

Neuropeptide Y and the Regulation of Endocrine Function / 843 likely that the effects of NPY on thyroid blood flow may be mediated by the NPY Y-1 receptor subtype, which is known to mediate vasoconstrictor effects of this peptide and which is abundant in the arterioles of the rodent thyroid gland [41]. The relationship between thyroid NPY and the sympathetic regulation of thyroid blood flow has been investigated and has produced some rather puzzling data. Sympathetic stimulation results in a decrease in thyroid blood flow but not in significant release of NPY into the thyroid vein [45], and further, this vasoconstriction was not blocked either by an NPY antagonist or by administration of an NPY antiserum [11]. Taken together, these findings suggest that thyroidal NPY is independent of the sympathetic nervous system, and it is not clear how its activity is regulated. It is perhaps worth noting at this point that all the studies on NPY and thyroid blood flow have been carried out by one research group, and so these findings still await confirmation. The widely reported effects on NPY on metabolism (e.g., [66]) may suggest a role for this peptide in regulating thyroid hormone secretion. Indeed, central administration of NPY has a significant inhibitory effect on the HPT axis [14]. There remains the question of whether the NPY present in the thyroid may have an additional effect on hormone secretion. However, in vivo studies suggest that NPY does not have a significant role in the regulation of thyroid hormone release in rats. Neither acute nor chronic infusion of NPY into rats had any effect on circulating thyroxine or T3 [24, 38]. Furthermore, administration of an NPY antiserum had no effect on plasma thyroxine or thyroid-stimulating hormone (TSH) [46]. However, a study in mice suggests that NPY may have a more subtle role in enhancing the thyroid hormone response to stimulation while having no effect on basal secretion rates [17]. Chronic intraperitoneal administration of NPY to rats (1 μg/rat/day for 4 days) had no effect on thyroid weight or serum T3/T4 or TSH levels [38]. However, an in vitro study, using thymidine incorporation as an indicator of growth, reported that low and high concentrations of NPY inhibited thymidine uptake into rat thyroid tissue incubated for 4 hours in vitro but enhanced the stimulatory effects of TSH [63]. These findings suggest a fine-tuning role for NPY in growth as well as hormone secretion in the thyroid gland.

CONCLUSION Neuropeptide Y is found in nerves supplying most endocrine tissues and also appears to be present in some endocrine cells. It appears to act to reduce blood flow through endocrine tissues and also has a role in

the regulation of hormone secretion. Although the effects of NPY on basal hormone secretion appear variable, and generally fairly minor, this peptide has a common action in several tissues in the inhibition of stimulated hormone secretion.

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[35] Lundberg JM, Terenius L, Hokfelt T, Goldstein M (1983) High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci Letts 42:167– 172. [36] Lundberg JM, Terenius L, Hokfelt T, Tatemoto K (1984) Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons. J Neurosci 4:2376–2386. [37] Majane EA, Alho H, Kataoka Y, Lee CH, Yang H-YT. (1985) Neuropeptide Y in bovine adrenal glands: Distribution and characterisation. Endocrinology 117:1162–1168. [38] Malendowicz LK, Miskowiak B (1990) Effects of prolonged administration of neurotensin, arginine vasopressin, NPY and bombesin on blood TSH, T3 and T4 levels in the rat. In Vivo 4:259–261. [39] Markiewicz W, Jaroszewski JJ, Barszczewska B, Sienkiewicz W (2003b) Localization of neuropeptide Y and norepinephrine in the porcine ovarian artery and their influence on the local blood pressure. Folia Histochem Cytobiol 41:73–81. [40] Markiewicz W, Jaroszewski JJ, Bossowska A, Majewski M (2003) NPY: its occurrence and relevance in the female reproductive system. Folia Histochem Cytobiol 41:183–192. [41] Matsuda H, Brumovsky PR, Kopp J, Pedrazzini T, Hokfelt T (2002) Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues. J Comp Neurol 449:390–404. [42] McCullough LA, Westfall TC (1995) Neuropeptide Y inhibits depolarisation-stimulated catecholamine synthesis in rat phaeochromocytoma cells. Eur J Pharmacol 287:271–277. [43] McDonald JK (1990) Role of NPY in reproductive function. Ann NY Acad Sci 611:258–272. [44] McDonald JK, Dees WL, Ahmed CE, Noe BD, Ojeda SR (1987) Biochemical and immunocytochemical characterization of neuropeptide Y in the immature rat ovary. Endocrinology 120: 1703–1710. [45] Michalkiewicz M, Dey M, Huffman LJ, Hedge GA (1998) The neuropeptides, VIP and NPY, that are present in the thyroid nerves are not released into the thyroid vein. Thyroid 8:1071– 1077. [46] Michalkiewicz M, Huffman LJ, Dey M, Hedge GA (1993) Endogenous neuropeptide Y regulates thyroid blood flow. Am J Physiol 264:E699–E705. [47] Minami S, Sarkar DK (1992) Central administration of neuropeptide Y induces precocious puberty in female rats. Neuroendocrinology 56:930–934. [48] Miyamoto A, Bruckman A, von Lutzow H, Schams D (1993) Multiple effects of neuropeptide Y, substance P and vasoactive intestinal polypeptide on progesterone and oxytocin release from bovine corpus luteum in vitro. J Endocrinol 138:451– 458. [49] Moltz JH, McDonald JK (1985) Neuropeptide Y: direct and indirect action on insulin secretion in the rat. Peptides 6:1155– 1159. [50] Munz H, Claas B (1997) Neuropeptide Y in the human thyroid gland. Ann Anat 179:553–558. [51] Myojin T, Kitamura N, Hondo E, Baltazar ET, Pearson GT, Yamada J (2000) Immunohistochemical localisation of neuropeptides in bovine pancreas. Anatomia, Histologia, Embryologia 29:167–172. [52] Neri G, Andreis PG, Nussdorfer GG. (1990) Effects of neuropeptide Y and substance P on the secretory activity of dispersed zona glomerulosa cells of rat adrenal gland. Neuropeptides 17:121–125. [53] Owman C, Stjernquist M, Helm G, Kannisto P, Sjoberg NO, Sundler F (1986) Comparative histochemical distribution of

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[62] Sheikh SP, Holst JJ, Skak-Nielsen T, Knigge U, Warberg J, Theodorsson-Norheim E, Hokfelt T, Lundberg JM, Schwarz TW (1988) Release of NPY in pig pancreas: dual parasympathetic and sympathetic regulation. Am J Physiol 255:G46–G54. [63] Skowronska-Jozwiak E, Lewinski A, Modrzejewska H, KunertRadek J, Greger J (1994) Changes of 3H-thymidine incorporation into DNA and thymidine kinase activity in rat thyroid lobes, following their exposure to neuropeptide Y. Cytobios 80:49– 54. [64] Spinazzi R, Andreis PG, Nussdorfer GG (2005) Neuropeptide-Y and Y-receptors in the autocrine-paracrine regulation of adrenal gland under physiological and pathophysiological conditions. Int J Mol Med 15:3–13. [65] Sundler F, Moghimzadeh E, Hakanson R, Ekelund M, Emson P (1983) Nerve fibers in the gut and pancreas of the rat displaying neuropeptide Y immunoreactivity. Intrinsic and extrinsic origins. Cell Tissue Res 230:487–493. [66] Szekely M, Petervari E, Pakai E, Hummel Z, Szelenyi Z (2005) Acute, subacute and chronic effects of central neuropeptide Y on energy balance in rats. Neuropeptides 39:103–115. [67] Wasowicz K, Kaleczye J, Sienkiewicz W, Czaja K, Zivcik A, Lakomy M (2001) Influence of active immunization against GnRH on VIP- and NPY-positive innervation of the porcine testis. Folia Histochem Cytobiol 39:269–274. [68] Whitworth EJ (2004) Adrenocortical proliferation and signal transduction in vitro. PhD thesis, University of London. [69] Wrobel KH, Brandl B (1998) The autonomous innervation of the porcine testis in the period from birth to adulthood. Ann Anat 180:145–156.

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116 Effects of PACAP in the Local Regulation of Endocrine Glands DAVID VAUDRY, AURÉLIA RAVNI, OLIVIER WURTZ, MAGALIE BÉNARD, BÉATRICE BOTIA, VALÉRIE JOLIVEL, ALAIN FOURNIER, BRUNO GONZALEZ, AND HUBERT VAUDRY

vasoactive intestinal polypeptide (VIP), identifying PACAP as a member of the VIP-glucagon-secretin superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordate to mammals, suggesting that the peptide is involved in the regulation of vital biological functions [3, 78]. Characterization of the PACAP precursor cDNA has revealed the existence of a PACAP-related peptide flanking PACAP on its N-terminal side, whose activity remains unknown. Two types of PACAP binding sites have been characterized: Type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1 receptor (PAC1-R), which is coupled to several transduction systems, and the two PACAP/VIP-mutual VPAC1 and VPAC2 receptors (VPAC1-R and VPAC2-R), which are primarily coupled to adenylyl cyclase. PACAP and its receptors are widely distributed in the brain and peripheral organs, notably in endocrine glands including the pituitary, thyroid, gonads, adrenal, and pancreas (Table 1).

ABSTRACT Pituitary adenylate cyclase–activating polypeptide (PACAP), a peptide of the vasoactive intestinal polypeptide (VIP)-glucagon superfamily, was initially characterized by virtue of its ability to stimulate cAMP formation in cultured rat anterior pituitary cells. Three PACAP receptors have been cloned so far: a PACAP-selective receptor, termed PAC1-R, and two PACAP/VIP common receptors, termed VPAC1-R and VPAC2-R. PACAP and its receptors are widely expressed in the brain and in peripheral organs, notably in the hypothalamus and in endocrine glands. Indeed, there is now clear evidence that PACAP exerts neuroendocrine, paracrine, and autocrine control of the activity of the pituitary, thyroid, testis, ovary, adrenal medulla, adrenal cortex, and endocrine pancreas. These observations suggest that selective PACAP agonists and antagonists could have therapeutic value for the treatment of various endocrine disorders.

INTRODUCTION Pituitary adenylate cyclase–activating polypeptide (PACAP) was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cAMP formation in cultured rat anterior pituitary cells [54]. Characterization of the peptide revealed that it is composed of 38 amino acids and is C-terminally α-amidated. PACAP38 exhibits an internal cleavage site (Gly28-Lys29Arg30) and can thus generate a 27-amino-acid αamidated peptide (PACAP27), which is present, in most tissues, at a much lower concentration than PACAP38 [55]. PACAP27 exhibits 68% sequence identity with Handbook of Biologically Active Peptides

EFFECTS ON THE PITUITARY GLAND Various hypothalamic nuclei, including the paraventricular and arcuate nucleus, contain PACAP-producing neurons that project toward the external zone of the median eminence [43, 53, 74], and high concentrations of PACAP have been measured in the portal blood [14], suggesting that PACAP may act as a hypophysiotrophic neuropeptide (see chapter on PACAP in the Brain Peptides section of this book). In addition the occurrence

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848 / Chapter 116 TABLE 1. Localization and Relative Abundance of PACAP and Its Binding Sites in Various Endocrine Tissues.a PACAP PACAP Binding Sites

Cell Bodies

Structures Anterior pituitary FS, GH, PRL, and ACTH cells TSH, LH, FSH cells Neurohypophysis Thyroid Parathyroid Testis Leydig cells epithelial cells from epididymal tubules Ovary Granulosa and cumulus cells Adrenal gland Cortex Medulla Chromaffin cells Subcapsular region Endocrine pancreas

Fibers

−/++ ++ +

PAC1-R

VPAC1/2-R

++/+++

++

++

+

+ +

− +

+ +

+

++ − −/+ −/+

+ −/+ −/+ + ++

− ++ ++

++ ++

−/+ −/+ ++

a The symbols provide a semi-quantitative evaluation of the level of expression: +++, high density; ++, moderate density; +, low density; −, no hybridization or immunohistochemical signal.

TABLE 2. Effects of PACAP on Pituitary Cells.a Cell Type

Second-Messenger Coupling

Gonadotroph cells Somatotroph cells Lactotroph cells Corticotroph cells

↑ ↑ ↑ ↑

cAMP, ↑ IP turnover, ↑ [Ca2+]i, ↑ cGMP cAMP, ↑ [Ca2+]i [Ca2+]i [Ca2+]i

Thyrotroph cells Folliculo-stellate cells Fibroblasts Melanotroph cells

↑ ↑ ↑ ↑

[Ca2+]i cAMP, ↑ [Ca2+]i cAMP cAMP

Hormone Release and/or mRNA Expression ↑/→ LH release, ↑/→ FSH release ↑ LH mRNA, → FSH mRNA ↑/→ GH release ↑/↓/→ PRL release, ↑/→ PRL mRNA expression ↑/→ ACTH release → TSH release ↑ IL-6 release ↑ α-MSH release

a ↑, stimulatory effect; ↓, inhibitory effect; →, no effect; ACTH, adrenocorticotrophic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; IL-6, interleukin-6; LH, luteinizing hormone; MSH, melanocyte-stimulating hormone; PRL, prolactin; TSH, thyroid-stimulating hormone.

of PACAP has also been detected in a subpopulation of gonadotroph cells [53], indicating that the peptide may also act within the adenohypophysis as a paracrine or autocrine regulator. Indeed, the ability of PACAP to stimulate cAMP formation in anterior pituitary cells provides clear evidence that the peptide controls the activity of the adenohypophysis [54]. The diversity of the actions of PACAP on the pituitary has been detailed in previous reviews [48, 61]. Among the different hypophysiotrophic neuropeptides identified so far, the situation of PACAP is rather unique in that functional PACAP receptors are present in all endocrine cell types as well as in folliculo-stellate cells of the adenohypophysis [80]. PACAP stimulates the release of growth

hormone, adrenocorticotrophic hormone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin [61, 78]. PACAP is even more potent than classical hypothalamic hormones such as gonadotrophinreleasing hormone at increasing nitric oxide synthase I (NOS I) levels in cultured rat anterior pituitary cells [21], indicating that PACAP exerts important biological functions in these cells. PACAP also enhances the secretion of α-melanocyte-stimulating hormone (αMSH) from cultured rat pars intermedia melanotroph cells [40]. The effects of PACAP on pituitary cells are mediated through activation of adenylyl cyclase, cGMP, phospholipase C (PLC), and mobilization of cytosolic calcium concentration (Table 2). A detailed description

Effects of PACAP in the Local Regulation of Endocrine Glands / 849 of the effects of PACAP on each cell type can be found elsewhere [78].

EFFECTS ON THE THYROID GLAND In human, VPAC1-R is the predominant type of PACAP receptor expressed in the thyroid, whereas in mouse thyroid follicular cells primarily express VPAC2R [31, 62]. The expression level of VPAC-Rs is increased in medullary thyroid carcinoma, a property that can be used for the visualization of thyroid tumors by VIP receptor scintigraphy [81]. In the human and porcine thyroid, PACAP stimulates cAMP production, which is probably involved in the activation of thyroxin secretion [9].

EFFECTS OF PACAP ON THE ENDOCRINE TESTIS AND OVARY The presence of PACAP and its receptors in the testis and ovary strongly suggests that the peptide may operate as a local regulator of gonadal activity [31, 41]. The rat testis contains the highest level of PACAP in any peripheral organs [2]. The concentration of PACAP in the testis is significantly reduced after hypophysectomy and is restored by FSH administration, indicating that the expression of the PACAP gene is under the control of pituitary gonadotrophins [69]. In vitro, PACAP induces a dose-dependent stimulation of testosterone secretion from isolated rat Leydig cells [63, 64] and regulates protein synthesis in spermatocytes and spermatids [82]. In Leydig cells, PACAP acting through PAC1-R stimulates both adenylyl cyclase and PLC [63]. The effect of PACAP on Leydig cells may also be mediated via a novel receptor subtype coupled to a sodium channel through a pertussis toxin-sensitive G-protein [64]. The effects of PACAP on protein synthesis in spermatocytes and spermatids are both mimicked by dbcAMP [82]. In cultured Sertoli cells, PACAP increases cAMP concentration and stimulates estradiol and inhibin secretion [33]. In the epididymal epithelium, PACAP stimulates chloride secretion, which is important for sperm activation and storage [91]. The occurrence of PACAP-immunoreactive material in epididymal tubules indicates that PACAP is locally synthesized and, thus, may act as a paracrine regulator of sperm maturation [91]. The epididymal epithelium-derived PACAP may also stimulate epididymal spermatozoa that have lost PACAP-synthesis ability [67] but still possess PACAP binding sites [68]. In the human cavernous tissue, PACAP dose-dependently relaxes norepinephrine- and electrically contracted isolated preparations of human corpus cavernosum, suggesting that the peptide may be involved in the induction

and maintenance of penile erection [32]. Consistent with this notion, a stearic acid VIP conjugate has been shown to increase the copulatory activity and penile reflex in testosterone-treated, castrated rats [23]. These results suggest that PACAP and/or VIP derivates could be developed for the treatment of impotence. In the rat ovary, most granulosa and cumulus cells from large preovulatory follicles contain both PACAP mRNA and PACAP peptide [26]. Human chorionic gonadotrophin (hCG) stimulates the expression of PACAP mRNA and progesterone receptor mRNA [39]. The peak of expression of progesterone receptor mRNA occurs 3 hours after hCG treatment and the peak of PACAP mRNA only after 6 hours, suggesting that progesterone receptor activation is required for PACAP gene expression [39]. Indeed, it has been shown that the progesterone receptor antagonist ZK98299 blocks the effect of hCG on PACAP gene expression [39]. The hCG-evoked stimulation of PACAP gene transcription is abolished by cycloheximide, indicating the requirement of protein synthesis for PACAP mRNA expression [39]. The exposure of cultured granulosa cells to PACAP causes a dose-dependent increase in progesterone production [1, 25]. Reciprocally, immunoneutralization of endogenous PACAP reduces progesterone formation and impairs subsequent luteinization, suggesting that PACAP plays an important role in LH-induced progesterone secretion during the periovulatory period [25], which could, at least in part, explain the reduced fertility observed in PACAPdeficient mice [66]. Incubation of immature rat preovulatory follicles with PACAP or VIP induces a dose-dependent inhibition of follicle apoptosis [19, 47] and reduces FSH-stimulated follicle growth [8]. In luteinized granulosa cells, PACAP appears to be more potent than LH in stimulating cAMP accumulation [34]. In the human female genital tract, PACAP is located in fibers innervating blood vessels and smooth muscle cells of the internal cervical os, suggesting that the peptide could regulate local blood flow and lubrication of the vagina [24, 70]. High concentrations of PACAP are also found throughout the human uteroplacental unit [74]. In vitro, PACAP induces relaxation of nonvascular smooth muscle strips from the fallopian tube and myometrium [72] as well as stem villous and intramyometrial arteries [74], indicating that PACAP may regulate the vascular tone in the human female reproductive tract. In placental cells, PACAP enhances cAMP formation, and hCG and interleukin-6 production [12].

EFFECTS OF PACAP ON THE ADRENAL PACAP and its receptors are actively expressed in the adrenal medulla [51, 72] and PACAP exerts a

850 / Chapter 116 stimulatory action on catecholamine secretion from chromaffin cells [37, 57, 78]. PACAP also stimulates the release of brain natriuretic peptide and enkephalins, two regulatory peptides that are co-sequestered with catecholamines in chromaffin granules [4, 28]. PACAP causes a robust increase in VIP mRNA expression in bovine chromaffin cells through a cAMP-dependent, PKA-independent pathway [29]. In vivo studies have shown that PACAP and VIP stimulate catecholamine release in anesthetized dogs through activation of dihydropyridine-sensitive L-type calcium channels [22, 45]. PACAP-induced catecholamine secretion is significantly enhanced by hypoglycemia, suggesting that PACAP may play a beneficial role in glucose counterregulatory mechanisms in the adrenal medulla during hypoglycemia [86]. The effect of PACAP on catecholamine secretion is mediated through PAC1-R and associated with an increase in adenylyl cyclase activity [37, 46, 52] and calcium influx [56]. Incubation of adrenomedullary cells in calcium-free medium or blockage of voltageoperated calcium channels suppresses the PACAPevoked stimulation of catecholamine secretion [35, 37, 59], indicating that the effect of PACAP on chromaffin cells is mediated through calcium influx. Concurrently, PACAP increases calcium mobilization from ryanodine/caffeine-sensitive calcium stores [35, 58, 73]. Treatment of chromaffin cells with PACAP activates the expression of tyrosine hydroxylase, dopamine βhydroxylase, and phenylethanolamine N-methyltransferase [78], and the stimulatory effect of PACAP on tyrosine hydroxylase activity is mediated through the activation of the adenylyl cyclase/PKA transduction pathway [49]. Divergent results have been reported regarding the possible effect of PACAP on the multiplication of adrenochromaffin cells: PACAP appears to stimulate proliferation of rat chromaffin cells in primary culture and to inhibit the mitogenic action of nerve growth factor on chromaffin cells [76]. High levels of PACAP and its receptors are expressed in most pheochromocytomas where the peptide could act in an autocrine manner to regulate the secretory activity or the differentiation of tumor cells [62]. The pheochromocytoma PC12 cell line has been widely used as a model to investigate the neuroendocrine and neurotrophic effects of PACAP [79]. In PC12 cells, PACAP acting through PAC1-R inhibits cell proliferation, stimulates neurite outgrowth, and prevents apoptosis [78]. The effect of PACAP on neuritogenesis is mediated through an extracellular-signal-regulated kinase (ERK)-dependent PKA-independent mechanism [5]. In PC12 cells, PACAP also stimulates tyrosine hydroxylase [11] and chromogranin A gene expression [75], and it activates the transcription of the transfected neuropetide Y and proenkephalin A genes [10]. The effect of PACAP on global gene expression has also

been investigated in PC12 cells using microarray approaches [27, 77]. Many of the known genes regulated by PACAP are associated with neuritogenesis (ornithine decarboxylase and annexin A2), cell growth (growth arrest specific 1 and cyclin B2), cell morphology remodeling (actin and tubulin), vesicle trafficking (synaptotagmin IV), or cell adhesion (Mcma and attractin). Functional analyses are now in progress to clarify the role of the hundred of genes that have been identified so far and to determine how these genes can interact with one another. Intravenous administration of PACAP causes elevation of plasma cortisol levels in dog and calf [16, 38]. PACAP stimulates corticosterone and aldosterone secretion from human, rat, and chicken adrenal slices, but does not affect the release of corticosteroids from dispersed fasciculata and glomerulosa cells [50, 57], suggesting that the response of adrenocortical cells to PACAP involves the contribution of another adrenal cell type. Exposure of human adrenal slices to the βadrenoreceptor blocker l-alprenolol totally suppresses the steroidogenic effect of PACAP [57]. Similarly, the action of PACAP on dehydroepiandrosterone and cortisol secretion by the fetal human adrenal gland is suppressed by the β-adrenoreceptor antagonist propranolol [7]. Altogether, these observations indicate that, in most mammalian species, the effect of PACAP on corticosteroid secretion can be ascribed to the stimulatory action of the peptide on catecholamine secretion. In contrast, PACAP stimulates corticosteroid release from dispersed bovine and frog adrenocortical cells [6, 90]. The facts that PACAP enhances cAMP and inositol phosphate formation in bovine glomerulosa cells [6] and calcium mobilization in individual frog adrenocortical cells [90] provide additional evidence for a direct stimulatory effect of the peptide on steroidogenesis in these two species.

EFFECTS OF PACAP ON PANCREATIC ISLETS In the pancreas, PACAP-immunoreactive fibers innervate both the exocrine acini and the islets of Langerhans, as well as the small arteries of the connective tissue (Table 1) [44]. Electrical stimulation of the vagus nerve causes the release of PACAP from the isolated perfused pig pancreas, suggesting that PACAP may control exocrine and endocrine pancreatic secretions. PACAP appears to be much more potent than VIP or other regulatory peptides in stimulating pancreatic hormone secretion. In vivo administration of PACAP stimulates insulin secretion in mice [17, 20], calf [15], dog [85], and human [18]. Mice with a targeted deletion of the PACAP gene have a more profound insulininduced hypoglycemia than wild-type animals [30]. The

Effects of PACAP in the Local Regulation of Endocrine Glands / 851 stimulatory effect of PACAP on insulin release has also been documented on perfused rat and pig pancreas [89] and on cultured islet cells [17, 83]. On these cells, PACAP and VIP activate VPAC2-R to increase the number of pancreatic β-cells [60, 65]. The role of PACAP in the control of β-cell proliferation has been confirmed in mice overexpressing PACAP in the pancreas under the control of a human insulin promoter [88]. It has also been reported that pancreatic β-cells express cell-surface ectopeptidases capable of degrading PACAP [36]. The amplitude and kinetics of the PACAP-evoked stimulation of insulin release depends on glucose concentration in the incubation medium [15, 89]. PACAP induces a biphasic effect on insulin secretion, that is, a rapid and transient stimulation (acute phase) followed by a rebound of the secretory response (plateau phase). The plateau phase could arise from the ability of PACAP to regulate insulin gene expression. The phosphatidylinositol 3-kinase inhibitor wortmannin inhibits the plateau phase but not the acute phase of the PACAP-evoked insulin release [71]. Exposure of pancreatic β-cells to PACAP causes calcium influx through L-type calcium channels [84], and the stimulatory effect of PACAP on insulin secretion is abolished by nitrendipine [42], indicating that activation of voltage-sensitive L-type calcium channels is involved in the insulinotropic effect of PACAP. Paradoxically, the combination of glucose, PACAP, and carbachol stimulates insulin release while being unable to elevate intracellular calcium [42]. Incubation of isolated rat islets with specific PACAP antisera inhibits the ability of glucose to stimulate insulin release [84], suggesting that endogenous PACAP acts as a physiological regulator of pancreatic β-cell activity. PACAP is also a potent stimulator of glucagon secretion. Intravenous injection of PACAP increases plasma glucagon concentration in the mouse [20], dog [87], and human [18]. Likewise, in the perfused rat pancreas, PACAP enhances glucagon secretion [89]. The stimulatory effect of PACAP on insulin and glucagon release is completely abolished by somatostatin [89]. In contrast, the endozepine octadecaneuropeptide (a potent inhibitor of insulin release) has no effect on PACAP-evoked insulin secretion [13].

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117 Endothelins in the Local Regulation of Endocrine Glands GIAN PAOLO ROSSI AND DOMENICO MONTEMURRO

ET receptors is discussed in the Chapter 64 of the Cardiovascular Peptides section.

ABSTRACT The endothelin (ET) peptides, their synthesizing enzymes, and their ETA and ETB receptors are expressed in several endocrine glands. Evidence exists that the ETs intervene in regulating hormones secretion and cell turnover. We herein briefly summarize the data supporting the existence and the functional role of the ET system in the anterior pituitary, thyroid, and parathyroid glands; pancreatic islets; adrenal gland; and the endocrine testis and ovary. The pathophysiological implications of the upregulation of this system are also discussed.

PITUITARY GLAND Immunoreactive (IR) ETs are present in the AP of rats and humans, but, at variance with most other tissues, ET-3-IR is more abundant than ET-1-IR in this gland [26, 42]. Whereas ET-3-IR was observed in 31% of human AP cells, which was attributed to the gonadotroph subpopulation no such ET-1-IR was found in such glands [20], [29]. Moreover, the fact that ET-3 was released by unidentified cultured rat AP cells on stimulation with insulinlike growth factor I suggests the possibility of a functional regulation of the ETs system in AP. Data on the expression and distribution of ETA and ETB receptor subtypes in the AP are limited. Autoradiography revealed that ETA receptors are abundant in the rat AP [45]. Specific binding of [125I]-ET-1 has also been demonstrated in primary cultures of AP cells and T3-1 gonadotroph cell line. In these cell types, ligand binding was time- and temperature-dependent and was followed by rapid internalization of the receptor–ligand complex. The rate and extent of internalization of the [125I]ET-1–receptor complex were also dependent on the incubation temperature [45]. The binding of [125I]ET-1 to AP cells was more effectively displaced by ET-1 than ET-3 and was abolished by the ETA-receptor antagonist BQ-123. Thus, rat AP cells exclusively express ETA receptors, in accord with the demonstration of functional ETA receptors in quiescent lactotrophs [46]. The functional role of ETs in the AP has been little investigated. Results suggest that ETs stimulate adrenocorticotrophin hormone (ACTH) release from pituitary explants and thyroid stimulating hormone (TSH),

INTRODUCTION The endothelin (ET) peptides, their synthesizing enzymes ECE-1 and chymase, and their ETA and ETB receptor subtypes have been detected not only in the vascular endothelium and smooth muscle cells but also in endocrine cells of several glands. A local synthesis of the ET peptides rather than their uptake from the bloodstream is suggested in these glands by the concomitant detection of mRNA of ETs and ECE1 and, in some glands, also of chymase. Accordingly, the ET peptides can play an important physiological role in regulating some endocrine functions, including hormones synthesis and secretion and cell turnover. They can also attain a pathophysiological role under disease conditions. The purpose of this chapter is to summarize the information on the presence and functional role of ETs and their receptor subtypes in the functional regulation of the anterior pituitary (AP), thyroid, and parathyroid glands; pancreatic islets; adrenal gland; and endocrine testis and ovary. The evidence that the ET system is implicated in endocrine disease is also discussed. The biology of ETs and Handbook of Biologically Active Peptides

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856 / Chapter 117 gonadotrophins, prolactin (PRL), and growth hormone (GH) release from perifused AP cells [45]. AP cells repeatedly exposed to ET or GnRH (100 nM) for 30 min, followed by 30 washing periods, were able to mount a full [Ca2+]i response to gonadotrophin-releasing hormone (GnRH) [44] but not to ET-1. Both peptides elicited rapid increases in luteinizing hormone (LH) release, with comparable peak amplitudes, but the response to ET decreased much more rapidly during sustained stimulation, and gonadotrophs became refractory to further ET stimulation, probably because of a desensitization of ET receptors. It is noteworthy in this context that several cell types, including endocrine cells, show marked desensitization of ET-induced responses, which goes along with a rapid internalization of the receptor–ligand complexes [45]. This desensitization can account for some differences of results across different studies investigating responses of endocrine cells to several agonists in vitro. As for gonadotrophin secretion, ETA receptors exclusively mediate the effects of ETs on PRL release [45]. However, in contrast to gonadotrophs and thyrotrophs, the transient stimulatory effect of ET-1 on PRL release is followed by a sustained inhibitory action [19, 41]. Although the stimulatory action of ET-1 on PRL release in AP cells does not require extracellular Ca2+ [45], its sustained inhibitory action depends on Ca2+ entry through L-type calcium channels [41]. It occurs via ETA receptors and was attributed to an uncoupling between Ca2+ mobilization and voltage-gated Ca2+ influx due to activation of inward rectifier K+ channels by pertussis-toxin-sensitive G-proteins [45]. As regards the pathophysiological implications, available information is consistent with a role of ETs in different pituitary tumors. The ET system has been found to be upregulated in nonfunctioning pituitary adenomas, as well as in GH-, ACTH-, and PRL-secreting tumors. ET-1-IR and ET-3-IR were found in 48 and 31%, respectively, of a relatively large collection of different human pituitary adenomas [21]. These findings were confirmed at the gene-expression level, thus suggesting production of the peptides by the tumor itself rather than passive uptake of the peptides from the circulation [22]. Therefore, an activation of ET-1 in pituitary adenomas may be involved in tumorigenesis. However, the precise role played by the different ET peptides as well as of their receptor subtypes remains to be ascertained.

THYROID GLAND ET-1-IR has been detected in thyroid follicular cells [5], thus indicating that they can produce ET-1. These cells also have specific ET receptors [17], and it has

been estimated that there are approximately 4000 ET receptors per cell [15], suggesting the possibility of an autocrine-paracrine regulation. It remains controversial whether TSH itself stimulates ET-1 synthesis by thyrocytes or whether it can only enhance the stimulatory action of other cytokines, as transforming growth factorβ, which enhance both synthesis and secretion of ET-1 [47]. The intravenous administration of ET-1 has no effect on circulating TSH levels in human [49], but ET1 induced a transient release of TSH in perifused rat AP cells [44]. [125I]ET-1 binding to cultured human thyrocytes revealed the presence of specific binding that was time- and temperature-dependent and was followed by rapid internalization of the receptor–ligand complex. Scatchard analysis of surface membrane binding at 4°C showed a single class of high-affinity ET-A receptors [17, 47]. ET-1 exerts a proliferogenic effect on thyrocytes through Ca2+ influx [8], but has an inhibitory effect on thyroglobulin release from human thyroid cells through a protein kinase C (PKC)-mediated mechanism [17]. No correlation was found between the plasma levels of ET-1-IR, thyroid hormones, or TSH in humans [20] with solid or cystic nodular pathology of the thyroid [12]. However, ET-1-IR was demonstrated in a wide collection of normal and pathologic thyroid specimens, including classic papillary carcinomas, 90% of which were positive and approximately 50% of which were intensely immunoreactive, suggesting that this peptide could be involved in the growth of these tumors [23]. Compared with euthyroid individuals, hyperthyroid patients had higher plasma levels of ET-1-IR, which normalized after treatment [4, 24]. Increased gene expression of ET-1, ECE-1, and ETA was described in thyroid carcinoma [7, 48]. Thus, collectively the available evidence suggests that in the thyroid, as in other endocrine glands, ETs operate in an autocrine-paracrine manner and might play a role in tumorigenesis.

PARATHYROID GLAND ET-1-IR and specific transcripts for ET-1 [14] and ppET-1 mRNA have been demonstrated in the chief cells of rat parathyroid gland [45]. Reverse transcription polymerase chain reaction (RT-PCR) analysis has shown that both ETA and ETB receptors are expressed in bovine parathyroid tissue and in the BPE-1 cell line; however, only ETA receptors were detected in the PT-r parathyroid cell line [14]. Autoradiographic studies of frozen human parathyroid glands with [125I]ET-1 and use of selective ET receptor antagonists confirmed the presence of both ETA and ETB receptors [3]. Northern blot analysis of poly(A)1 RNA from human parathyroid adenomas demonstrated expression of transcripts for

Endothelins in the Local Regulation of Endocrine Glands / 857 both ETA and ETB receptors [45]. It was also shown that parathyroid cells are endowed with receptors for natriuretic peptides and that the latter increased the synthesis and secretion of ET-1, an observation that might contribute to the understanding of the link between hyperparathyroidism and hypertension [11]. Intracellular calcium concentration ([Ca2+]) is held to be a primary regulator of parathyroid hormone (PTH) secretion; however, in contrast to other cell types, increases in [Ca2+]i rapidly inhibit PTH release. The observation that ET-1, which acts as a Ca2+mobilizing agonist, stimulates PTH release in a dosedependent manner, suggests that [Ca2+]i is regulated by ETs in a different manner in these cells. In accord with this hypothesis, parathyroid chief cells appear to respond to ET-1 in a unique way. In the majority of these cells, ET induces transient stimulation followed by a sustained decrease of [Ca2+]i that may serve to modulate the sensitivity of their Ca2+-dependent PTH release mechanism. Of interest, parathyroid cells, when exposed to a shift of the ion in either direction, transiently increase ppET-1 mRNA and release ET-1-IR in the extracellular space where the peptide is rapidly removed [9]. Thus, it is likely that this short-lived mechanism can contribute to rapid calcium control by ET-1.

PANCREATIC ISLETS Quite limited information on the ET system and its role on the endocrine pancreas exists. ET-1-IR was found in the pancreas and predominantly in islet cells, where it colocalized with glucagons and insulin but not with somatostatin or pancreatic polypeptide [18]. Although no study has investigated the presence of ETA and ETB receptors in the pancreatic islets, there is indirect evidence that at least ETA receptors are present on the β cells. In fact, ET-1 was shown to directly stimulate insulin secretion from isolated mouse pancreatic islets. Furthermore with use of the ETA-receptor-specific antagonist BQ-123 and of the ETB-receptor-specific agonist BQ-3020, Gregersen et al. [16] were able to show that only BQ-123 at concentrations ranging between 1 μM/L and 10 μM counteracted the stimulatory effect of ET-1 on insulin secretion. Thus, the ETA receptor most likely mediates the secretagogue effect of ET-1 on insulin in the mouse. On the other hand, insulin was shown to stimulate ET-1 gene expression in endothelial cells, mostly via the PKC-dependent pathway [31]. Insulin was also shown to double ETA receptor density and to increase thymidine incorporation, this latter effect being potently enhanced by concomitant insulin and ET-1 stimulation [13]. Thus, hyperinsulinemia potentiates ET-1 release and its receptor-mediated action thereby contributing to vascular disease in diabetes.

ADRENAL GLAND Prepro (pp) ET-1 is expressed both at the mRNA and peptide level in the adrenal cortex and medulla of different species, including rat and human [30, 39]. The ECE-1 is also expressed in the adrenal gland, both in the cortex and in the medulla [34], thus indicating that ET-1 can be synthesized locally in the adrenal gland, where it may act in an autocrine-paracrine fashion. Immunohistochemical studies have provided information on the localization of the peptides in the different zones of the cortex as well as in the medulla [30, 39]. Evidence for the presence of chymase, which cleaves big ET-1 to ET-1(1–31) has also been provided [36], thus suggesting a role also of this novel peptide within the adrenal gland. Both ETA and ETB receptor subtypes in the rat and human adrenal gland have been found at the mRNA and protein level with different techniques [30, 32]. In the cortex, both ETA and ETB receptors were found to be predominantly expressed in the zona glomerulosa cells; ETA receptors were also found in the arteriole tunica media, whereas ETB receptors were demonstrated in the endothelium of the sinusoids. Overall, the ratio of ETA-to-ETB sites in the bovine adrenal gland was 9:1 [45]. Expression of both ETA- and ETB-receptor genes was detected by different techniques in aldosteroneproducing adenomas [38] and adjacent normal cortex [30]. Specific ET binding sites were also found to heavily label the rat adrenal medulla, but these results were not confirmed by immunohistochemistry studies, suggesting that the ET receptor density in the medulla is not high enough to be visualized with this technique [30]. Whether both receptor subtypes are present in the medulla, their relative density, and their functional role also remain to be determined. ETs directly affects both the steroidogenesis and the growth of the adrenal cortex, but different ET peptides exert different effects. ET-1 was found to be as potent as angiotensin II in stimulating aldosterone release from rat and human adrenocortical cell preparations in vitro [2, 32, 35]. Stimulatory effects of ET on adrenal steroid hormone release were also observed in adrenocortical cells of other species [30, 39], albeit with some differences that might derive from the desensitization of ET receptors as a result of the rapid internalization of the receptor–ligand complexes [45]. Furthermore, ACTH stimulates the release of ET-1 in the perifused rat adrenal gland and in dispersed adrenocortical cells [30], suggesting that an autocrine/paracrine regulation of aldosterone secretion may occur in this system. The direct stimulation of aldosterone secretion by ET-1 involves exclusively the ETB receptors in the rat and both receptor subtypes in humans [30]. In human,

858 / Chapter 117 the relative importance of ETA and ETB receptor subtypes in mediating the secretagogue effect of ET-1 on aldosterone may vary depending on the prevailing degree of activation of the renin-angiotensin system [40]. By selectively activating either ETA and ETB receptor subtypes, we have identified some of the cellular events coupled to the stimulation of each subtype in Conn’s adenomas cells. In brief, whereas the stimulation of ETA involves activation of PKC, that of ETB receptors is coupled with both the PKC- and cyclooxygenase-dependent signaling pathways [41]. The biological action of ET-1 on the adrenal cortex is not confined to its steroidogenic effects because, by acting via ETA receptors coupled to the PKC and tyrosine kinase (TK) pathways, the peptide exerts a proliferogenic effect on rat adrenocortical cells [28], which is specific to zona glomerulosa (ZG) cells. Of further interest is that the novel ET peptide ET-1(1–31) can be synthesized in the human adrenal cortex from big ET-1 through the action of chymase, which has been located in both the arteriolar wall and cell islets of ZG [36]. At variance with the classic ET-1(1–21), ET-1(1–31) negligibly affected aldosterone secretion but was far more potent in stimulating cell growth, an effect that occurs via ETA receptors. It has been suggested that ET-1(1–31) can act as a selective ETA agonist enhancing proliferation of cultured ZG cells through the activation of TKdependent mitogen-activated protein kinase (MAPK) cascade [27]. The concept has been also put forward that alternative cleavage of big ET-1 either to ET-1 by ECE-1 or to ET-1(1–31) by chymase might play a pivotal role in determining whether the stem cells of the adrenal cambium layer will differentiate into a secretory phenotype synthesizing aldosterone or evolve into a regenerating/hyperplastic and possibly neoplastic phenotype [36]. ET-1 is involved also in the control of the secretion of adrenal medulla, inasmuch as the systemic administration of ET-1 increases the plasma levels of epinephrine (E) and norepinephrine (NE) in dogs [30]. ET-1 also caused increases in [Ca2+]i and released E and NE from cultured chromaffin cells, although to a smaller extent than high K+-induced depolarization or stimulation with nicotine, bradykinin, or prostaglandin E2 [30].

LEYDIG CELLS The rat and human testis contains ppET-1 mRNA, ET-1 peptide, and very low levels of ET-3-IR [30]. ET-1 is produced by cultured rat Sertoli cells under the control of FSH, but no ETs were found in rat Leydig cells [30]. Rat Leydig cells were found to express abundant ETA receptors [30].

A Leydig cell tumor line also expressed ET receptors; the data from competitive binding experiments yielded a single class of high-affinity binding sites [45]. In addition to exerting direct actions on GnRH neurons and gonadotrophs, ETs may also affect reproductive function by acting on gonadal cells in an autocrineparacrine fashion. In Sertoli cells, ET has been reported to attenuate FSH-stimulated cAMP and estradiol accumulation [43]. In contrast, ETs stimulate steroidogenesis in rodent Leydig cells. In transformed murine Leydig cells that lack 17α-hydroxylase, ET-1 stimulates progesterone production [10]. In rat Leydig cells, ET-1 increases basal and hCG-induced testosterone production and PGE2 release, being more potent than ET-3 in this regard. ET-1 is also more potent than ET-3 in stimulating [Ca2+]i responses in these cells [6]. This action of ETs is completely inhibited by BQ-123 and unaffected by the ETB receptor antagonist BQ-788, indicating that ETA receptors are expressed and functionally operative in rat Leydig cells [1].

OVARY ET-1-IR is present in porcine follicular fluid and in media conditioned by cultured porcine granulosa cells; it is also found in high concentrations in rat corpora lutea [30]. The immunostaining for ET-1 has also been located in the wall of follicles at different stages of maturation, especially in the cytoplasm of granulosa cells and, obviously, in endothelial cells [25]. Northern blot analysis has also demonstrated the expression of ppET-1 mRNA in granulosa cells, but not in the thecal compartment, of follicles at different stages of maturation [25]. The presence of ECE-1 in the menstrual corpora lutea has also been demonstrated with Western blot [50], and the ECE-1 mRNA was found in the theca interna cells of secondary, tertiary, and atretic follicles, as well as in the corpora lutea of human and monkey ovary [30]. Recent data obtained in rat with repeated electro-acupuncture showed that the content of ET-1-IR in the ovary can be modulated by sympathetic nerve activity [44]. Both ETA and ETB receptor transcripts were found in the rat ovary, the ETA receptor subtype being far more abundant than the ETB in the ovary of humans, pigs, and rats [30]. However, ETB mRNA is also abundant in the granulosa cells of developing follicules and low in atretic follicles [30]. ETs affect steroidogenesis in ovarian granulosa cells. Earlier studies reported that both ET-1 and ET-3 stimulated the production of progesterone, estradiol, and testosterone from pregnant rats. However, most subsequent investigations observed an inhibitory effect of ETs on ovarian steroidogenesis [30]. A marked concentration-dependent inhibitory effect of ET-1 on

Endothelins in the Local Regulation of Endocrine Glands / 859 FSH-stimulated progesterone and estradiol release by cultured granulocyte cells has been consistently described [30]. The progesterone antisecretagogue effect of ET-1 may result from the combined inhibition of the early step of steroid biosynthesis and stimulation of progesterone-inactivating enzymes (e.g., 20α-hydroxysteroid-dehydrogenase and 5α-reductase). There is also evidence that, in turn, ovarian steroids modulate ET-induced LH release, thus suggesting a feedback loop [45].

CONCLUSION Despite the fact that less than two decades have passed since the discovery of ETs, it has become quite evident that the peptides of the ET family exert multiple biological actions that go far beyond vasoconstriction. Among these nonvascular actions, those involving the endocrine glands appear to play an important physiological and pathophysiological role. Careful assessment of the endocrine effects of the ET peptides appears to be important at a stage when ET antagonists are being used in the treatment of pulmonary hypertension and their use is being exploited for other indications. The balance between beneficial effects and untoward endocrine actions should indeed be weighed, particularly when life-long treatment is planned. Finally, the data on the more recently identified ET peptides ET-1(1–31) and ET-1(1–21) in endocrine glands are quite limited, and therefore their physiological and pathophysiological roles remain to be thoroughly investigated.

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[7] Donckier JE, Michel L, Van Beneden R, Delos M, Havaux X. Increased expression of endothelin-1 and its mitogenic receptor ETA in human papillary thyroid carcinoma. Clin Endocrinol 2003;59:354–60. [8] Eguchi K, Kawakami A, Nakashima M, Ida H, Sakito S, Sakai M, Terada K, Kawabe Y, Fukuda T, Ishikawa N. Stimulation of mitogenesis in human thyroid epithelial cells by endothelin. Acta Endocrinol (Copenh) 1993;128:215–20. [9] Eguchi S, Hirata Y, Imai T, Kanno K, Akiba T, Sakamoto A, et al. Endothelin receptors in human parathyroid gland. Biochem Biophys Res Commun 1992;184:1448–55. [10] Ergul A, Glassberg MK, Majercik MH, Puett D. Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells. Endocrinology 1993; 132:598–603. [11] Feo MLD, Bartolini O, Orlando C, Maggi M, Serio M, Pines M, et al. Natriuretic peptide receptors regulate endothelin synthesis and release from parathyroid cells. Proc Natl Acad Sci USA 1991;88:6496–500. [12] Foppiani L, Porcella E, Cuttica CM, Fazzuoli L, Valenti S, Giordano G, et al. Immunoreactive endothelin in nodular pathology of the thyroid. Clin Endocrinol 1997;47:479–83. [13] Frank HJ, Levin ER, Hu RM, Pedram A. Insulin stimulates endothelin binding and action on cultured vascular smooth muscle cells. Endocrinology 1993;133:1092–7. [14] Fujii Y, Moreira JE, Orlando C, Maggi M, Aurbach GD, Brandi ML, et al. Endothelin as an autocrine factor in the regulation of parathyroid cells. Proc Natl Acad Sci USA 1991;88:4235–9. [15] Gandhi CR, Stephenson K, Olson MS. Endothelin, a potent peptide agonist in the liver. J Biol Chem 1990;265:17432–5. [16] Gregersen S, Thomsen JL, Hermansen K. Endothelin-1 (ET-1)potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype. Metabolism 2000;49:264–9. [17] Jackson S, Tseng YC, Lahiri S, Burman KD, Wartofsky L. Receptors for endothelin in cultured human thyroid cells and inhibition by endothelin of thyroglobulin secretion. J Clin Endocrinol Metab 1992;75:388–2. [18] Kakugawa Y, Giaid A, Yanagisawa M, Baynash AG, Melnyk P, Rosenberg L, et al. Expression of endothelin-1 in pancreatic tissue of patients with chronic pancreatitis. J Pathol 1996;178:78– 3. [19] Kanyicska B, Burris TP, Freeman ME. Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture. Biochem Biophys Res Commun 1991;174:338–43. [20] Lam HC, Lee JK, Lai KH, Chiang HT. Plasma endothelin levels are unaltered by thyroid hormone status in humans. J Cardiovasc Pharmacol 2000;36:S382–5. [21] Lange M, Pagotto U, Hopfner U, Ehrenreich H, Oeckler R, Sinowatz F, et al. Endothelin expression in normal human anterior pituitaries and pituitary adenomas. J Clin Endocrinol Metab 1994;79:1864–70. [22] Lange M, Pagotto U, Renner U, Arzberger T, Oeckler R, Stalla GK. The role of endothelins in the regulation of pituitary function. Exp Clin Endocrinol Diabetes 2002;110:103–12. [23] Lenziardi M, Viacava P, Fiorini I, Castagna M, Nardini V, Pollina L, et al. Presence of endothelin-1 in the normal and pathological human thyroid. J Endocrinol Invest 1995;18:336–40. [24] Letizia C, Centanni M, Cesareo R, De Ciocchis A, Cerci S, Scuro L, et al. Increased plasma levels of endothelin-1 in patients with hyperthyroidism. Metabolism 1995;44:1239–42. [25] Magini A, Granchi S, Orlando C, Vannelli GB, Pellegrini S, Milani S, et al. Expression of endothelin-1 gene and protein in human granulosa cells. J Clin Endocrinol Metab 1996;81:1428– 33. [26] Matsumoto H, Suzuki N, Shiota K, Inoue K, Tsuda M, Fujino M. Insulin-like growth factor-I stimulates endothelin-3 secretion

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118 Adrenomedullin and Related Peptides in the Local Regulation of Endocrine Glands ALFREDO MARTÍNEZ

Interestingly, a few years later AM recovered its status as a bona fide peptide hormone when a serum binding protein was found. Setting up a radioimmunoassay (RIA) protocol for AM is not trivial, and the sample needs to be passed through a C-18 Sep-Pak cartridge to obtain meaningful results. Trying to identify the sources of these difficulties, Elsasser et al. [7] found the existence of a binding protein of approximately 120–140 kDa in the serum of several species. This serum binding protein for AM was later identified as complement factor H, a central regulator of the complement cascade [39]. There are at least two AM binding sites per molecule of factor H, and the amount of circulating factor H is staggering, approximately 500 μg/ml [27]. It has been recently shown that factor H protects AM from protease degradation [25]. All these data suggest that the real amount of AM in the blood is much higher than the values obtained by regular quantification techniques and, although we do not have exact numbers yet, the circulating concentration of AM may well be in the proper range to activate its receptors in locations far away from its secretion sites. In any case, all authors agree on the great potential of AM and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), in local regulation. In this chapter, the physiological regulation of the main endocrine organs by these peptides is reviewed.

ABSTRACT Adrenomedullin (AM) is a 52-amino-acid regulatory peptide that is expressed in many structures of the body, including endocrine organs. Pituitary, adrenals, pancreas, and some other endocrine organs express the proadrenomedullin gene and have binding sites for AM and its gene-related peptide, proadrenomedullin Nterminal 20 peptide (PAMP), suggesting the existence of autocrine and paracrine loops within the organ. Both peptides influence secretion of several hormones such as ACTH, aldosterone, corticosterone, catecholamines, and insulin. The gland architecture is also relevant: where the peptide is expressed, what the direction of the blood flow is inside the gland, and where the receptors and binding proteins are located. These are all important factors in understanding the fine regulation of glandular physiology.

INTRODUCTION Since its discovery in 1993 [14], adrenomedullin (AM) has been considered a peptide hormone circulating in the bloodstream. Nevertheless, with the discovery of the AM receptor [31] (discussed in Chapter 106), the concept of AM as a circulating hormone became doubtful. The reason for these doubts was the low concentration of AM measured in the blood (between 2 and 20 pmol/liter in humans) and the relatively high concentration needed to bind to the receptor (it has a dissociation constant of approximately 2–3 nM). As a consequence, circulating AM could not elicit any meaningful response in cells containing AM receptors. There was always the possibility of finding much higher concentrations of the peptide in regions close to the secretion site, thus pointing to a potential paracrine or autocrine mechanism to explain AM-related physiology. Handbook of Biologically Active Peptides

PITUITARY AM is expressed in all components of the hypothalamo-pituitary-adrenal (HPA) axis [34], including the supraoptic and paraventricular nuclei of the hypothalamus, nerves of the hypothalamo-neurohypophysial tract, specific cells of the neural and anterior lobes of the pituitary, and adrenal cells both in the cortex and the medulla [18]. In the pituitary, AM immunoreactivity

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862 / Chapter 118 has been found in most cells of the anterior lobe, whereas PAMP was restricted to follicle-stimulating hormone (FSH)-containing cells [32]. This lack of colocalization between AM and PAMP, two peptides theoretically produced by the same prohormone, has been observed in several locations and may be explained by an alternative splicing mechanism acting on the mRNA [23]. Intermedin, also called AM-2, is also present in the pituitary. The presence of AM immunoreactivity in the hypothalamus and pituitary can be traced back to the phylogenetic origin of these organs in elasmobranch fishes (Fig. 1). AM has been also found in the pituitary of frogs, birds, and all kinds of mammals. Immunohistochemical studies were confirmed by RIA quantification and gene expression studies of tissue extracts, which showed higher levels of AM in the pituitary than in the brain [12]. AM and PAMP binding sites have been found in the pituitary, but they present rather low values [36]. Several studies have looked into the functional roles of AM in pituitary physiology. The addition of AM or PAMP to dispersed cells of the anterior pituitary resulted in a dose-dependent reduction of adrenocorticotrophin hormone (ACTH) both under basal conditions and following corticotrophin-releasing hormone (CRH)induced secretion [45]. There seems to be also a moderate increase in growth hormone (GH) secretion following exposure of rat and human somatotrophs to AM [33]. The HPA axis is involved in the regulation of the stress response, and AM seems to be part of it. Several situations of experimental and clinical stress, including students preparing for their final exams [2], resulted in

FIGURE 1. Adrenomedullin immunoreactivity in the hypothalamic region of the brown shark, Carcharhinus plumbeus. Hematoxylin was used to provide nuclear counterstaining (blue). Bar = 20 μm. (See color plate.)

an increase of AM expression that might have physiological impact in restoring homeostasis.

ADRENALS AM was initially isolated from tissue extracts of a pheochromocytoma, a tumor of the adrenal medulla [14]. The peptide and its mRNA have been found in both components of the adrenals, the cortex, and the medulla [13]. In the cortex, AM is highly expressed by the zona glomerulosa and, at lower levels, by the zonae fasciculata and reticularis. In the medulla, chromaffin cells synthesize high levels of AM (Fig. 2). AM and PAMP are co-stored with catecholamines in the secretory granules of chromaffin cells and are co-secreted with them on activation of these cells. Interestingly, the blood vessels supplying the adrenals do not express AM. It has been reported that immature adrenal glands have higher expression of AM than adult ones [40]. Binding sites for AM and PAMP have been found throughout the adrenals [3], suggesting the existence

FIGURE 2. Distribution of AM mRNA in a section of rat adrenal gland by in situ hybridization. AM expression is highest in the zona glomerulosa and in the medulla. Bar = 150 μm. Reproduced with permission from [14]. (See color plate.)

Adrenomedullin and Related Peptides in the Local Regulation of Endocrine Glands / 863 of autocrine-paracrine mechanisms in the regulation of AM/PAMP-mediated effects. These effects consist mainly in the regulation of the secretion of adrenal hormones. The main hormone secreted by the zona glomerulosa is aldosterone, and there are contradictory reports on whether AM induces or inhibits aldosterone secretion in the adrenal cortex. In support of an inhibitory function, AM has been shown to decrease the induction of aldosterone secretion elicited by angiotensin II, KCl, and ACTH (see [43], among others). PAMP has been also described as an inhibitor of aldosterone secretion, being more efficient than AM [44]. In contrast, there are reports of a stimulating role for both AM and PAMP (see [11], among others). The discrepancies among these reports may relate to different cell preparation methods, such as the use of dispersed cells versus the perfused organ [50]. In any case, this example clearly points out the complexity of the regulation pathways in endocrine organs. The relative situation of the source of the hormone, the blood flow pattern, and the localization of the receptors would greatly contribute to an understanding of the exact regulatory role played by AM and PAMP in the secretion of other hormones. There may be also alternative mechanisms that do not require the presence of a specific receptor in the target cell. This seems to be the case for the elevation of corticosterone secretion elicited by AM. This effect may be explained just by the increased perfusion of the adrenal cortex induced by the vasodilatory function of AM [30]. In the adrenal medulla, AM increases catecholamine release by stimulating Na+/Ca2+ exchange in the membrane of the chromaffin cells [5]. However, PAMP has the reverse effect, inhibiting catecholamine secretion by an independent mechanism [29]. Because both AM and PAMP are co-secreted with catecholamine from the chromaffin cells, PAMP would exert an autocrine feedback mechanism on chromaffin cell secretion, whereas AM would tend to multiply the initial secretory stimulus. In addition, AM has been shown to reduce tyrosine hydroxylase activity [49].

ENDOCRINE PANCREAS AM has been found in the endocrine pancreas of all vertebrates, from fish all the way to humans [17]. In lower vertebrates, AM colocalizes with all the pancreatic hormones in a complex pattern, but in birds and mammals the cells expressing higher levels of AM are the F cells, which also produce pancreatic polypeptide in the periphery of the islets of Langerhans [28] (Fig. 3). This phylogenetic variation in the distribution of AM immunoreactivity is almost identically recapitulated during ontogenesis. In early embryos, when the pancre-

FIGURE 3. Three-dimensional confocal microscopy reconstruction of a rat isolated pancreatic islet stained for AM (yellow) and insulin (red). Bar = 70 μm. (See color plate.)

atic anlage is being formed (at day E11.5 in the rat), AM expression is already present and is found throughout the rest of the organ’s development. In the first stages, AM colocalizes with insulin, glucagon, and somatostatin but, as the organ matures, AM gets restricted to specific cells and is finally confined to the F cells [21]. The receptors for AM and its binding protein, complement factor H, are expressed by the β-cells that synthesize insulin in the center of the islet [24, 26]. This architecture, and the fact that blood flow in the islet goes from the periphery to the center, suggests a clear model for local regulation of AM in pancreatic physiology and identifies the β-cells as the target for AM function. Indeed, AM has been shown to reduce insulin secretion when applied to isolated islets. Even more impressive is the observation that the application of a monoclonal antibody against AM to isolated islets increased insulin secretion fivefold, demonstrating that AM exerts a tonic inhibition of insulin secretion in normal conditions [28]. This physiological effect of AM has been also shown in vivo. Injection of AM in rats increased circulating glucose levels, whereas the monoclonal antibody reduced them [22]. As expected from the close relationship that exists between β-cell function and diabetes, important variations of AM levels have been reported in diabetic patients. Both type 1 and type 2 diabetic patients had elevated levels of AM when compared with normal controls [37]. Analysis of patients with a recent onset of the

864 / Chapter 118 disease revealed the existence of two subsets of patients, depending on their AM levels [22]. All diabetic patients had a higher concentration of AM than normal controls, but some of the diabetic patients exhibited an exceedingly high concentration of AM. In these patients, preexisting excessive AM may have triggered the onset of the disease [22]. Type 2 diabetes is characterized by a secretory defect at the islet of Langerhans and also by a desensitization of peripheral tissues (mainly liver, adipose tissue, and skeletal muscle) to insulin action. AM may play a regulatory role at both ends. At the islet, an excess of AM may reduce insulin secretion, thus producing hyperglycemia. This has been shown by the injection of rats with synthetic AM, which resulted in elevation of circulating glucose [22]. On the other hand, AM may also play a role in peripheral insulin resistance. Heterozygote mice for an AM knockout have circulating levels of AM of approximately one-half the amount found in wild-type controls. In these animals, there is an increased insulin resistance when compared with normal controls [46]. These observations suggest that AM may constitute an interesting target to design novel treatments for diabetes control. In fact, the application of a monoclonal antibody against AM to obese diabetic rats resulted in a decrease of circulating glucose and the elimination of postprandial hyperglycemia, a clear characteristic of type 2 diabetes [22].

DIFFUSE ENDOCRINE SYSTEM OF THE GUT The diffuse endocrine system is composed of a variety of individual cells with secretory endocrine morphology that appear scattered among other epithelial cells throughout the digestive tract (Fig. 4). These cells act as chemo- and mechanoreceptors of the conditions occurring in the gut and release their secretory peptides to neighboring cells to regulate digestive physiology [41]. Both AM and PAMP have been found in the secretory granules of a subset of these cells, colocalizing with serotonin and gastrin [15]. The number of PAMPpositive cells in the gut seems to be higher than the AM-positive ones, and they have been found in the stomach and the small and large intestines [47]. The study of the function of the diffuse endocrine system has always been complex given the dispersion of the cells that constitute it and the paracrine mechanism of action. Nevertheless, several functions have been ascribed to AM and PAMP that relate to digestive physiology, and in most cases they may be better understood by the local production of regulatory substances. For instance, AM and PAMP have an inhibitory effect on gastric acid secretion [42]. AM is able to promote proliferation of intestinal epithelial cells [38], and thus it

FIGURE 4. Paraffin section of the stomach of the lizard Anolis sagrei, stained for PAMP. Note the numerous PAMPpositive cells belonging to the diffuse endocrine system located in the deep portion of the gastric glands. Hematoxylin was used to provide nuclear counterstaining (blue). Bar = 25 μm. (See color plate.)

could help in the rapid cycle of epithelial turnover that occurs in the gut. In addition, AM promotes epithelial healing of the gastric mucosa [9]. AM may also influence peristalsis by inhibiting smooth muscle contraction [35]. Recently, AM and PAMP have been shown to influence the intestinal absorption of sugars through a mechanism involving the Na+-dependent glucose transporter, SGLT1 [8]. Additional information about the role of AM and the gastrointestinal system is presented in the Gastrointestinal Peptides section of this book, Chapter 137.

OTHER ENDOCRINE ORGANS AM has been found in the ovary, mainly in the granulosa and thecal layers of the follicles and in the corpora lutea, indicating some involvement in maturation of the

Adrenomedullin and Related Peptides in the Local Regulation of Endocrine Glands / 865 physiology in these locations. Unfortunately, attempts to generate knockout mice, which would answer important questions on the physiology of these peptides, resulted in fetal death [4]. We will have to wait for the generation of organ-specific targeted knockout models to finally unravel many of the questions remaining about these regulatory peptides.

Acknowledgments The author is supported by a grant from the Spanish Ministry for Science and Education, BFU2004-02838/ BFI.

References

FIGURE 5. Confocal microscopy image of a monkey thyroid gland stained for AM (green), calcitonin (red), and DAPI (blue). Colocalization of both immunoreactivities in the C-cells is manifested by the yellow hue. Bar = 30 μm. (See color plate.)

ova and maintenance of pregnancy [19]. Interestingly, in the monkey, AM immunoreactivity was found in the primary follicles but not in the more mature ones [51]. This is in agreement with the observation that gonadotrophins reduce AM expression in the ovary, implicating AM in the process of granulosa cell differentiation [1]. The level of AM in follicular fluid has been suggested to be a marker of decreased ovarian response [20]. AM levels are greatly elevated during pregnancy [6], probably to maintain a vasodilatory state during this period. The probable sources for this increased level of AM are the corpora lutea and the placenta. AM was also found in the thyroid gland, both in the follicular epithelium and in the C-cells, colocalizing with calcitonin (Fig. 5). No specific action in the thyroid has been reported for AM, but PAMP decreases thyroidstimulating hormone (TSH) levels and epithelium/ colloid ratio in rat thyroid glands [10]. AM and PAMP levels are increased in patients with hyperthyroidism [48], but the implications of the peptides in the pathophysiology of this disease are not yet clear. AM is not present in normal parathyroid glands [16].

CONCLUSION AM and PAMP are expressed by many endocrine organs and constitute important regulators of local

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[31] McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998;393:333–9. [32] Montuenga LM, Burrell MA, Garayoa M, Llópiz D, Vos M, Moody T, et al. Expression of proadrenomedullin derived peptides in the mammalian pituitary: co-localization of follicle stimulating hormone and proadrenomedullin N-20 terminal peptide-like peptide in the same secretory granules of the gonadotropes. J Neuroendocrinol 2000;12:607–17. [33] Nakamura Y, Shimatsu A, Murabe H, Mizuta H, Ihara C, Nakao K. Calcitonin gene-related peptide as a GH secretagogue in human and rat pituitary somatotrophs. Brain Res 1998;807:203– 7. [34] Nussdorfer GG. Proadrenomedullin-derived peptides in the paracrine control of the hypothalamo-pituitary-adrenal axis. Int Rev Cytol 2001;206:249–84. [35] Ochiai T, Chijiiwa Y, Motomura Y, Yasuda O, Harada N, Nawata H. Direct inhibitory effect of adrenomedullin, calcitonin generelated peptide, calcitonin, and amylin on cholecystokinininduced contraction of guinea-pig isolated caecal circular smooth muscle cells. Peptides 2001;22:909–14. [36] Owji AA, Smith DM, Coppock HA, Morgan DG, Bhogal R, Ghatei MA, et al. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 1995;136:2127–34. [37] Pío R, Martínez A, Cuttitta F. Cancer and diabetes: two pathological conditions in which adrenomedullin may be involved. Peptides 2001;22:1719–29. [38] Pío R, Martínez A, Elsasser TH, Cuttitta F. Presence of immunoreactive adrenomedullin in human and bovine milk. Peptides 2000;21:1859–63. [39] Pío R, Martínez A, Unsworth EJ, Kowalak JA, Bengoechea JA, Zipfel PF, et al. Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J Biol Chem 2001;276:12292– 300. [40] Rebuffat P, Forneris ML, Macchi C, Malendowicz LK, Trejter M, Nussdorfer GG. Adrenomedullin and its receptors are expressed in the zona glomerulosa of immature and adult rat adrenals: evidences of upregulation of ADM in immature glands. Int J Mol Med 2002;10:85–8. [41] Rindi G, Leiter AB, Kopin AS, Bordi C, Solcia E. The “normal” endocrine cell of the gut: changing concepts and new evidences. Ann NY Acad Sci 2004;1014:1–12. [42] Rossowski WJ, Jiang NY, Coy DH. Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats. Eur J Pharmacol 1997;336:51–63. [43] Salemi R, McDougall JG, Hardy KJ, Wintour EM. Effect of adrenomedullin infusion on basal and stimulated aldosterone secretion in conscious sheep with cervical adrenal autotransplants. J Endocrinol 2000;166:389–99. [44] Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999;61:363–89. [45] Samson WK, Murphy TC, Resch ZT. Proadrenomedullin Nterminal 20 peptide inhibits adrenocorticotropin secretion from cultured pituitary cells, possibly via activation of a potassium channel. Endocrine 1998;9:269–72. [46] Shimosawa T, Ogihara T, Matsui H, Asano T, Ando K, Fujita T. Deficiency of adrenomedullin induces insulin resistance by increasing oxidative stress. Hypertension 2003;41:1080–5. [47] Tajima A, Osamura RY, Takekoshi S, Itoh Y, Sanno N, Mine T, et al. Distribution of adrenomedullin (AM), proadrenomedullin N-terminal 20 peptide, and AM mRNA in the rat gastric

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119 Ghrelin in the Local Regulation of Endocrine Glands ORESTE GUALILLO, FRANCISCA LAGO, ROSALIA GALLEGO, TOMAS GARCIA-CABALLERO, JOSE RAMON GONZALEZ-JUANATEY, JUAN J. GOMEZ-REINO, FELIPE F. CASANUEVA, AND CARLOS DIEGUEZ

local regulation of some endocrine glands and other peripheral tissues.

ABSTRACT Ghrelin is the recently identified 28-amino-acid peptide that is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Ghrelin, in addition to its role in the regulation of growth hormone (GH) secretion and energy homeostasis, is now emerging as a pivotal pleiotropic factor involved in a host of biological actions, including endocrine and nonendocrine functions. This chapter highlights the evidence for the potential autocrine-paracrine functional role of ghrelin and its receptor in the regulation of endocrine glands and other peripheral tissues.

GHRELIN AND THE PITUITARY Several findings indicate that ghrelin is expressed in rodents [7] as well as in normal and abnormal human pituitary [26]. In rodents, by means of immunohistological techniques, which cell types express ghrelin was determined. By double immunofluorescence, ghrelin expression was found in specific anterior pituitary (AP) cell types, namely somatotrophs, lactotrophs, and thyrotrophs; it is absent in the remaining cell types. This specific pattern of ghrelin expression is highly interesting because the three cell types that express ghrelin are those whose differentiation is markedly dependent on Pit-1 gene expression. Therefore, there appears to be some noteworthy interrelationship between ghrelin and Pit-1. Such an interrelationship is based on ghrelin’s ability to increase Pit-1 gene transcription in neonatal rat AP cells through two cAMP response elements present in the Pit-1 promoter [18]. Furthermore, persistent expression of the ghrelin gene was found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-day-old rats onward; however, at 60 days old the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. These data provide direct morphological evidence

INTRODUCTION Ghrelin has emerged in the last years as a widespread hormone that brings us to a new understanding of the regulation of growth hormone (GH) secretion and energy balance [18, 44]. It is produced mainly in the stomach by the X/A-like cells within the oxyntic glands of the gastric fundus mucosa, although significant amounts are present elsewhere in the body [25]. The pituitary [7, 26], heart and cardiomyocytes [20], placenta [16], testis [41], adrenal cortex [1], pancreas [45], ovary [41], and cartilage [6] also express significant levels of ghrelin. The ubiquitous expression of ghrelin in a host of tissues is suggestive of local paracrine and autocrine actions. Some data about isolation, identification, structures, and main functions of ghrelin have been reviewed in several chapters in this handbook (see Chapters 101, 130, 146, and 164), so in the following paragraphs we focus on the role of ghrelin in the Handbook of Biologically Active Peptides

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870 / Chapter 119 that ghrelin might act in a paracrinelike fashion in the regulation of AP cell function. In humans, RNAs extracted from human pituitary tissues showed that both ghrelin and growth hormone secretagogue receptor (GHS-R) mRNA were expressed. Human pituitary corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry revealed positive staining in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate radioimmunoassay (RIA) reactions for the N-terminal and Cterminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with approximately 7% octanoylated (active) ghrelin present as a percentage of the total.

GHRELIN AND THE THYROID Although there is evidence that ghrelin gene expression is influenced by thyroid hormones, few data are available about the presence and the role of ghrelin in the thyroid gland. Polymerase chain reaction (PCR) amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines but not in a rat thyroid follicular cell line [23]. Moreover, it has been observed that ghrelin is expressed in the thyroid in the fetus but not in the adult [45] and is expressed in a high percentage of thyroid tumors of follicular origin. Moreover, autocrine interactions between ghrelin and putative receptors other than the GHS-R may be active in the thyroid because ghrelin binding sites were detected in both normal and neoplastic thyroid tissues in the absence of GHS-R 1 mRNA expression. The functionality of these possible alternative ghrelin receptors is also supported by the evidence of a specific, although modest, antiproliferative effect of ghrelin in two different follicular-derived thyroid carcinoma cell lines.

GHRELIN AND THE HEART AND CARDIOMYOCYTES The discovery of ghrelin as the endogenous GH secretagogue (GHS) immediately prompted research on its hemodynamic effects because GH is known to play a role in the maintenance of cardiovascular health [14]. The possibility that GHS has direct cardiovascular effects, independent of GH release, has been strongly supported by different experimental approaches. Ghrelin can be synthesized by the cardiomyocytes of both human and

murine origin, and it is secreted by HL-1 cells (a cultured line derived from murine atrial cardiomyocytes that maintains a heart-specific phenotype [9, 13]) and also by human cardiomyocytes in primary culture [20]. Moreover, it has been shown that HL-1 cardiomyocytes produce GHS-R that efficiently binds ghrelin at the cell surface, that human myocardium expresses GHS-R1a mRNA [20], and that GHS-R mRNA is present in rat left ventricle and left atrium [25], strongly suggesting that ghrelin has paracrine-autocrine activity in the cardiac muscle. Interestingly, cardiac GHS-R(s) display distinct binding properties compared with GHS-R1a, mediating GH secretion in the pituitary and hypothalamus. The existence of a novel GHS-R in heart, distinct from GHSR1a, is further supported by the finding that both ghrelin and des-acyl ghrelin inhibit the apoptosis of cardiomyocytes and endothelial cells through activation of an intracellular survival pathway [4]. That most of the hemodynamic and cardioprotective effects of ghrelin may be direct (i.e., not mediated by GH) is suggested not only by the evidence of a paracrine-autocrine mode of action but also, in some cases, by more direct evidence: Its vasodilatory effects are not affected by GH release inhibitors [31], the synthetic GHS-R ligand hexarelin prevents cardiac damage after ischemiareperfusion even in hypophysectomized rats, and other GHS, such as GHRP-6, attenuate left ventricular (LV) dysfunction and dilation in dilated cardiomyopathy hamsters at concentrations that had no effect on serum GH and insulinlike growth factor 1 (IGF-1) levels [21]. Direct action in vivo is also suggested by the observations that in vitro ghrelin stimulates H9c2 cardiomyocyte cell proliferation [33] and reduces doxorubicin-induced mortality of H9c2 cardiomyocytes and endothelial cells [4] and AraC-induced mortality of HL-1 cells [20]. Ghrelin mRNA levels, which were decreased by AraC, were increased by pretreatment with GH, which protects against AraC-induced apoptosis in these cells [20]. Exactly how GH and ghrelin interact in cardiomyocytes remains to be elucidated. That ghrelin has beneficial cardiovascular effects, together with the anti-apoptotic effects of ghrelin observed in several studies, suggests that part of this increased risk may be due to obesity-related reduction of plasma ghrelin levels [37, 43], which may reduce protection against the cardiomyocyte apoptosis that is known to contribute to progressive cardiomyocyte loss in heart failure [24]. Weight reduction, which is known to be essential for reducing cardiovascular risk in the obese [36], may therefore owe this effect in part to its restoring normal ghrelin levels. The administration of ghrelin has been found to reduce cardiac afterload and increase cardiac output without increasing heart rate in healthy volunteers [25] and to induce vasodilation [31, 47] and improve the

Ghrelin in the Local Regulation of Endocrine Glands / 871 hemodynamics of patients with chronic heart failure (CHF) [25]. CHF-associated cachexia is attenuated by ghrelin in rats and in humans is accompanied by abovenormal ghrelin levels, possibly as a compensatory mechanism in response to catabolic-anabolic imbalance [25]. Ghrelin also regulates cardiovascular function in rats suffering septic shock [8] and exerts a protective effect against ischemic injury in rat heart [11]. Similar beneficial cardiovascular effects have been observed in rabbits [29]. As already stated, in both humans and experimental animals ghrelin’s beneficial cardiovascular effects seem not to be mediated by GH. This suggests that one of the multiple mechanisms by which obesity favors cardiac pathology may consist in its being associated with low ghrelin levels, which may reduce cardioprotection.

GHRELIN—A NOVEL PEPTIDE FOR CARTILAGE AND BONE HOMEOSTASIS Until recently, the major regulation of the growth plate was thought to be due to systemic hormones, mainly those related to the GH–IGF-1 axis [18, 25]. However, it is likely that locally produced peptide factors play an important autocrine and paracrine role in ensuring that skeletal development and growth progress correctly. Thus, it is of great interest to clarify whether ghrelin, an important component of the regulatory network of the GH axis, can act in a paracrine fashion. Several studies indicated that growth hormone secretagogues evoked positive effects on bone, even though it is not clear whether the effects are direct or are mediated by growth hormone. In humans, GHS treatment affects biochemical markers of bone turnover and increases growth velocity in selected short children with or without GH deficiency. In rodents, GHS treatment increases bone mineral content, but it has not yet been shown that GHS treatment can affect bone mass in adult humans [25]. It is well known that gastrectomy is associated with generalized osteopenia in several species, including human, although the mechanism is far from being completely understood. The degree of gastric mucosa loss is probably related to a proportional decrease in serum ghrelin concentration (but also of other peptides such as pancreastatin), so it is reasonable that gastrectomyinduced osteopenia could be due to the loss of ghrelin. If the effects of ghrelin (and its analogs) on bone are few and very poorly understood, the biological actions of ghrelin on cartilage are completely unknown. Only recently [6] some data have become available clarifying the role of ghrelin in cartilage. Indeed, immunoreactive ghrelin was identified in rat cartilage, localized

mostly in the proliferative and maturative zone of the epiphyseal growth plate, and in mouse and human chondrocytic cell lines. Moreover, ghrelin mRNA was also detected in rat cartilage as well as in mouse and human chondrocyte cell lines. Ghrelin mRNA expression has been studied in rat during early life development and shows a stable profile of expression throughout. Although ghrelin expression in chondrocytes suggests the presence of an unexpected autocrineparacrine pathway, the authors have failed to identify the functional (GHS-R)1A by reverse transcription polymerase chain reaction (RT-PCR). On the other hand, binding analysis with 125I ghrelin suggests the presence of specific receptors different from the 1A isotype. Actually, Scatchard analysis revealed the presence of two receptors, one with high and one with low affinity. Finally, ghrelin, in vitro, significantly inhibited chondrocyte metabolic activity and strongly stimulated cAMP production both in human and murine chondrocytes. In addition, ghrelin actively decreased both spontaneous or insulin-induced long-chain fatty acid uptake in human and mouse chondrocytes. These studies provide evidence for the presence of this novel peptide in chondrocytes and suggest novel potential roles for this newly recognized component of the GH axis in cartilage metabolism.

GHRELIN AND THE PANCREAS Data about ghrelin physiological actions on the endocrine pancreas, as well ghrelin and GHS-Rs pancreatic cell localization, are quite controversial. For instance, some studies located ghrelin in the beta- or alpha-cells of the pancreas [25], whereas others described a new Langerhans islet cell, the epsilon-cell, which contains ghrelin but no other pancreatic hormones [46]. In human islets, ghrelin expression is highest prenatally and neonatally, and the onset of islet ghrelin expression preceded that of gastric ghrelin [46]. Animals lacking transcription factors important for pancreatic beta-cell development, Nkx2.2 and Pax4, have relatively normal-size islets, but the majority of their islet cells contain ghrelin [25], suggesting that insulin-secreting beta-cells and ghrelin-synthesizing epsilon-cells may have a common precursor. Messenger RNA for GHS-R has been detected in the pancreas by means of the RNA protection assay and RT-PCR analysis [12, 19]. A recent report [22] demonstrated that ghrelin and GHS-R-like immunoreactivity mainly colocalized with glucagon-like immunoreactivity in rat pancreatic islets and that GHS-R-like immunoreactivity was also localized in some of the pancreatic beta-cells. Ghrelin induces an increase in the cytosolic free Ca2+ concentration and insulin release from isolated rat beta-cells

872 / Chapter 119 under high glucose concentrations [25]. In addition, intravenously injected ghrelin also increased circulating insulin in rats [28]. However, ghrelin has been demonstrated to inhibit insulin secretion in rodent perfused pancreas [10, 35] and in humans [5, 40]. Recently, it has been obeserved that neuronal constitutive nitric oxide synthase in pancreatic islets might be involved in the ghrelin-inhibited insulin release as well as in the ghrelin-stimulated glucagon release [34]. Despite the host of controversial results about both ghrelin pancreatic localization and ghrelin physiological action, it is certain that ghrelin physiological relevance in the integrative regulation of endocrine pancreatic activity needs to be explored further.

GHRELIN AND THE ADRENAL CORTEX Human adrenal glands were found to possess abundant ghrelin-displaceable GHS-Rs [32] and to express both ghrelin and GHS-R1a mRNAs [12, 42]. Recent findings indicate that ghrelin and GHS-R are both expressed in the rat adrenal cortex [1]. In addition, ghrelin binding sites have been identified abundantly in the zona glomerulosa (ZG). Ghrelin administration does not affect the secretory activity of rat adrenocortical cells but significantly enhances the proliferation rate of cultured ZG cells, without affecting the apoptotic deletion rate. Finally, the proliferogenic action of ghrelin in the human cultured ZG cells involves tyrosine kinase–dependent activation of the p42/p44 mitogen-activated protein kinase (MAPK) cascade [30].

GHRELIN AND THE REPRODUCTIVE SYSTEM Ghrelin and the Ovary There is evidence for the expression of ghrelin in the cyclic ovary. In the rat, ovarian expression of the ghrelin gene was demonstrated throughout the estrous cycle, with the lowest levels in proestrus and peak expression at diestrus (i.e., during the luteal phase of the cycle). Moreover, ghrelin immunoreactivity was predominantly located in the luteal compartment of the ovary [41]. Likewise, strong ghrelin immunostaining was observed in young and mature corpora lutea of the human ovary, whereas the ghrelin signal was absent in ovarian follicles at any developmental stage [41]. In addition, ghrelin was also detected in interstitial hilus cells, an androgen-secreting cell type of the ovary that shows morphological characteristics identical to those of testicular Leydig cells, which are the source of ghrelin

expression within the testis. In addition to the ligand, expression of GHS-R1a protein has been recently demonstrated by immunohistochemistry in the human ovary, with a wide pattern of tissue distribution. The expression of GHS-R1a peptide in somatic cells from ovarian follicles roughly paralleled follicular development, suggesting a potential relationship between GHSR expression and follicle growth, which remains to be proven. Moreover, the presence of GHS-R1a immunoreactivity has been reported in the surface epithelium of the human ovary, where ghrelin expression was also detected [41].

Ghrelin and the Testis The expression of ghrelin in rat and human testis was demonstrated through molecular and immunological approaches [41]. In the rat testis, ghrelin expression was selectively detected in Leydig cells at advanced stages of maturation, regardless of their fetal or adult origin. Similarly, immunohistochemical analyses showed that ghrelin is strongly expressed in interstitial mature Leydig cells of the human testis. In addition, testicular expression of the putative ghrelin receptor, the GHSR1a, was also demonstrated in the rat and human [41]. Intriguingly, in the rat testis expression of GHS-R1a mRNA remained undetectable up to prepubertal stages in spite of the rather constant levels of net GHS-R gene expression throughout postnatal development, suggesting a change in the pattern of splicing of the GHS-R gene in rat testis throughout development that favors the expression of the biologically active type 1a form of the receptor. GHS-R1a mRNA was also detected in adult human testis, and location analyses in the rat and human revealed a scattered pattern of distribution of GHS-R1a, with specific expression in somatic Sertoli and Leydig cells as well as in germ cells, mainly in pachytene spermatocytes. Ghrelin and its cognate receptor expression in the male as well in female gonads strongly suggested potential actions of locally produced ghrelin in the regulation of gonadal function. Indeed, ghrelin is able to significantly inhibit, in a dose-dependent manner, human chorionic gonadotrophin (hCG)- and cAMP-stimulated testosterone secretion [41]. Ghrelin might also directly regulate seminiferous tubule function because the expression of GHS-R1a was demonstrated in the tubular compartment of the testis. Actually, ghrelin inhibits the expression of the gene encoding stem cell factor (SCF), which is a Sertoli cell product regarded as the major paracrine stimulator of germ cell development and a survival factor for spermatogonia, spermatocytes, and spermatids in the adult rat seminiferous epithelium [41]. In addition, testicular SCF has been involved in Leydig cell development and survival. Thus, the actions of ghrelin on tubular SCF

Ghrelin in the Local Regulation of Endocrine Glands / 873 mRNA expression may have implications not only for the control of spermatogenesis but also for Leydig cell proliferation. Indeed, with in vivo models, it has been recently demonstrated that ghrelin is able to inhibit the proliferative rate of immature Leydig cells during puberty.

Ghrelin and the Placenta Ghrelin has been found in human as well as in rat placenta [16], an organ that contains all the main regulatory elements of the somatotrophic axis (i.e., GH, GHRH, somatostatin, and IGF-1) [18]. Ghrelin mRNA and ghrelin peptide are present in the human as well as in rat placenta. In human placenta, ghrelin mRNA was detected both during the first trimester and after delivery. Although ghrelin peptide was not detected in human placenta at term, it was easily identified at the first trimester, mainly expressed in cytotrophoblast cells and less so in syncytiotrophoblast. Ghrelin was also identified in a human choriocarcinoma cell line, the BeWo cells. Ghrelin was found in the cytoplasm of labyrinth trophoblast of rat placenta, whereas other placental cell types seem to be negative for ghrelin immunostaining. Moreover, placental ghrelin mRNA, in pregnant rats, showed a characteristic profile of expression, being practically undetectable during early pregnancy, with a sharp peak of expression at day 16 and decreasing in the latest stages of gestation. In spite of the existence of a specific profile of expression of ghrelin mRNA throughout pregnancy, the physiological function of ghrelin in this organ is not fully known at present, although very recently it has been reported that ghrelin is involved in the decidualization of human endometrial stromal cells [39]. Another hypothesis about the role of placental ghrelin is that it contributes to the pool of circulating maternal ghrelin. Actually, increased ghrelin levels have been observed in foodrestricted pregnant rats [17], so ghrelin may have a role in mediating the physiological responses to undernutrition and could represent an adaptive response to prevent long-lasting alterations in energy balance and, in general, serve as a signal from the fetoplacental unit that contributes to the regulation of maternal-fetal food intake and energy homeostasis.

CONCLUSION Data gleaned over the last few years have conclusively shown that ghrelin plays an important role in the regulation of food intake and GH secretion. In addition, the expression of the ghrelin gene has been found in many different tissues, including most of the endocrine glands. Ghrelin has emerged as a paracrine hormone

that may influence different functions, including cell proliferation and differentiation as well as gene expression in these endocrine glands. However, many questions remain to be answered. Although it was generally accepted that the biological activity of ghrelin was completely dependent in its acylation at serine-3, the fact that nonacylated ghrelin may exhibit biological activity [15, 27], even activity opposite to acylated ghrelin in some cell systems, indicates that a full understanding of its physiological role may be more difficult to uncover than originally thought. In this regard data obtained with knockout mice for the ghrelin gene [25, 38] should be taken with caution because the inactivation of acylated ghrelin could compensate in biological terms by producing the same effect on acylated ghrelin. This could explain the paradoxical data in transgenic animals overexpressing exclusively nonacylated ghrelin [2, 3], which appears to behave as a functional antagonist in some tissues. Further studies identifying new receptors through which acylated and nonacylated ghrelin act together, with the characterization of the cell biology process involved in ghrelin acetylation, are needed in order to fully understand the ghrelin system.

Acknowledgments This work was supported in part by grants from Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Fondo de Investigación Sanitaria; Ministerio de Educación y Ciencia; Xunta de Galicia. Oreste Gualillo and Francisca Lago are the recipients of a research contract co-funded by SERGAS (Hospital Clinico Universitario de Santiago) and Instituto de Salud Carlos III (Exp 00/3051, 99/3040).

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874 / Chapter 119 [6] Caminos JE, Gualillo O, Lago F, Otero M, Blanco M, Gallego R, Garcia-Caballero T, Goldring MB, Casanueva FF, Gomez-Reino JJ, Dieguez C. The endogenous growth hormone secretagogue (ghrelin) is synthesized and secreted by chondrocytes. Endocrinology 2005;146(3):1285–92. [7] Caminos JE, Nogueiras R, Blanco M, Seoane LM, Bravo S, Alvarez CV, Garcia-Caballero T, Casanueva FF, Dieguez C. Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland. Endocrinology 2003; 144(11):5089–97. [8] Chang L, Du JB, Gao LR, et al. Effect of ghrelin on septic shock in rats. Acta Pharmacol Sin 2003;24:45–49. [9] Claycomb WC, Lanson NA Jr, Stallworth BS, et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA 1998;95:2979–84. [10] Egido EM, Rodriguez-Gallardo J, Silvestre RA, Marco J. Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion. Eur J Endocrinol 2002;146:241–4. [11] Frascarelli S, Ghelardoni S, Ronca-Testoni S, et al. Effect of ghrelin and synthetic growth hormone secretagogues in normal and ischemic rat heart. Basic Res Cardiol 2003;98:401–5. [12] Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, et al. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002;87:2988. [13] González-Juanatey JR, Iglesias MJ, Alcaide C, Piñeiro R, Lago F. Doxazosin induces apoptosis in cardiomyocytes cultured in vitro by a mechanism that is independent of alpha-adrenergic blockade. Circulation 2003;107:127–31. [14] González-Juanatey JR, Piñeiro R, Iglesias MJ, Gualillo O, Kelly PA, Diéguez C, Lago F. Growth hormone prevents apoptosis in cardiomyocytes cultured in vitro through a calcineurindependent mechanism. J Endocrinol 2004;180:325–35. [15] Gottero C, Broglio F, Prodam F, Destefanis S, Bellone S, Benso A, Gauna C, Arvat E, van der Lely AJ, Ghigo E. Ghrelin: a link between eating disorders, obesity and reproduction. Nutr Neurosci 2004;7(5–6):255–70. [16] Gualillo O, Caminos J, Blanco M, Garcia-Caballero T, Kojima M, Kangawa K, Dieguez C, Casanueva F. Ghrelin, a novel placental-derived hormone. Endocrinology 2001;142:788–94. [17] Gualillo O, Caminos JE, Nogueiras R, Seoane LM, Arvat E, Ghigo E, Casanueva FF, Dieguez C. Effect of food restriction on ghrelin in normal-cycling female rats and in pregnancy. Obes Res 2002;10(7):682–7. [18] Gualillo O, Lago F, Gomez-Reino J, Casanueva FF, Dieguez C. Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 2003;552(2–3):105–9. [19] Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, et al. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997;48:23–9. [20] Iglesias MJ, Pineiro R, Blanco M, Gallego R, Dieguez C, Gualillo O, Gonzalez-Juanatey JR, Lago F. Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes. Cardiovasc Res 2004;62:481–8. [21] Iwase M, Kanazawa H, Kato Y, et al. Growth hormone releasing peptide can improve left ventricular dysfunction and attenuate dilation in dilated cardiomyopathic hamsters. Cardiovasc Res 2004;61:30–8. [22] Kageyama H, Funahashi H, Hirayama M, Takenoya F, Kita T, Kato S, Sakurai J, Lee EY, Inoue S, Date Y, Nakazato M, Kangawa K, Shioda S. Morphological analysis of ghrelin and its receptor distribution in the rat pancreas. Regul Pept 2005;126(1–2):67– 71.

[23] Kanamoto N, Akamizu T, Hosoda H, Hataya Y, Ariyasu H, Takaya K, Hosoda K, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K. Substantial production of ghrelin by a human medullary thyroid carcinoma cell line. J Clin Endocrinol Metab 2001;86(10):4984–90. [24] Kang PM, Izumo S. Apoptosis and heart failure: a critical review of the literature. Circ Res 2000;86:1107–13. [25] Kojima M, Kangawa K. Ghrelin: Structure and function. Physiol Rev 2005;85:495–522. [26] Korbonits M, Bustin SA, Kojima M, Jordan S, Adams EF, Lowe DG, Kangawa K, Grossman AB. Presence of ghrelin in normal and adenomatous human pituitary. J Clin Endocrinol Metab 2001;86:881–7. [27] Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB. Ghrelin—a hormone with multiple functions. Front Neuroendocrinol 2004;25(1):27–68. [28] Lee HM, Wang G, Englander EW, Kojima M, Greeley GH Jr. Ghrelin, a new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations. Endocrinology 2002;143:185–90. [29] Matsumura K, Tsuchihashi T, Fujii K, Abe I, Iida M. Central ghrelin modulates sympathetic activity in conscious rabbits. Hypertension 2002;40:694–9. [30] Mazzocchi G, Neri G, Rucinski M, Rebuffat P, Spinazzi R, Malendowicz LK, Nussdorfer GG. Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects. Peptides 2004;25(8):1269–77. [31] Okumura H, Nagaya N, Enomoto M, Nakagawa E, Oya H, Kangawa K. Vasodilatory effect of ghrelin, an endogenous peptide from the stomach. J Cardiovasc Pharmacol 2002;39:779– 83. [32] Papotti M, Ghe C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G. Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab 2000; 85(10):3803–7. [33] Pettersson I, Muccioli G, Granata R, et al. Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation. J Endocrinol 2002;175:201–9. [34] Qader SS, Lundquist I, Ekelund M, Hakanson R, Salehi A. Ghrelin activates neuronal constitutive nitric oxide synthase in pancreatic islet cells while inhibiting insulin release and stimulating glucagon release. Regul Pept 2005;128(1):51–6. [35] Reimer MK, Pacini G, Ahren B. Dose-dependent inhibition by ghrelin of insulin secretion in the mouse. Endocrinology 2003; 144:916–21. [36] Schunkert H. Obesity and target organ damage: the heart. Int J Obes Relat Metab Disord 2002;26:S15–20. [37] Shiiya T, Nakazato M, Mizuta M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002;87:240–4. [38] Sun Y, Ahmed S, Smith RG. Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 2003;23(22):7973–81. [39] Tanaka K, Minoura H, Isobe T, Yonaha H, Kawato H, Wang DF, Yoshida T, Kojima M, Kangawa K, Toyoda N. Ghrelin is involved in the decidualization of human endometrial stromal cells. J Clin Endocrinol Metab 2003;88(5):2335–40. [40] Tassone F, Broglio F, Destefanis S, Rovere S, Benso A, Gottero C, Prodam F, Rossetto R, Gauna C, van der Lely AJ, Ghigo E, Maccario M. Neuroendocrine and metabolic effects of acute ghrelin administration in human obesity. J Clin Endocrinol Metab 2003;88(11):5478–83. [41] Tena Sempere M. Exploring the role of ghrelin as novel regulator of gonadal function. Growth Horm IGF Res 2005;15(2):83– 8.

Ghrelin in the Local Regulation of Endocrine Glands / 875 [42] Tortorella C, Macchi C, Spinazzi R, Malendowicz LK, Trejter M, Nussdorfer GG. Ghrelin, an endogenous ligand for the growth hormone-secretagogue receptor, is expressed in the human adrenal cortex. Int J Mol Med 2003;12(2): 213–7. [43] Tschöp M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50:707–9. [44] Van der Lely AJ, Tschöp M, Heiman ML, et al. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–57.

[45] Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, Papotti M. Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on tumor growth. Am J Pathol. 2003;162(2):645–54. [46] Wierup N, Yang S, McEvilly RJ, Mulder H, Sundler F. Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells. J Histochem Cytochem 2004;52(3):301–10. [47] Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin-1. Br J Pharmacol 2002;136:1146–52.

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120 Atrial Natriuretic Peptide in Local Regulation of Endocrine Glands JOLANTA GUTKOWSKA

ity was shown to depend on the hydromineral status of the body and was altered by adrenalectomy [19]. The relevance of this observation was confirmed by de Bold et al. [2], who performed landmark experiments and reported that, in anesthesized rats, the injection of rat atrial tissue extract causes rapid but short duration diuresis and natriuresis. The discovery of the cardiac hormone, atrial natriuretic peptide (ANP), was a major breakthrough in the search for natriuretic hormones.

ABSTRACT ANP was the first member of the natriuretic peptide (NP) family isolated from heart tissue, and other members—BNP, CNP, urodilatin, and DNP—were isolated from the brain, kidney cells, and snake venom. The physiological actions of NPs are mediated by two guanylyl cyclase (GC) receptors and a clearance (C) receptor, whose main role is to internalize and degrade NPs. Activation of GC receptors leads to the increase of cGMP and subsequent intracellular events. NPs were originally thought to be primarily involved in the maintenance of cardiovascular homeostasis, but their presence and that of their cognate receptors in a wide variety of tissues suggest a much broader range of functions. Indeed, NPs have antiproliferative, antifibrotic, and anti-inflammatory functions; are involved in the regulation of bone growth; and are essential for the growth and maturation of ovarian follicles.

STRUCTURE OF THE PRECURSOR mRNA GENE ANP was initially purified from the rat heart, and cDNA clones encoding the ANP precursor were isolated from atrial cDNA libraries. In the adult heart, ANP gene is expressed predominantly in the atria where ANP mRNA represents 1–3% of the total mRNAs. ANP is the first member of the natriuretic peptide (NP) family, which consists of four other peptides: BNP and CNP, both isolated from the porcine brain [34, 35]. The BNP and CNP genes appear to each synthesize only one peptide hormone, BNP-32 and CNP-22, within their respective prohormones (i.e., BNP1–108 and CNP1–103). The fourth, a 32-aa peptide known as urodilatin, a product of the alternative processing of proANP in kidney cells, was found in urine [29]. The recently discovered dendroaspis-type natriuretic peptide (DNP) is a 38-aa peptide isolated from venom of the Dendroaspis angusticeps snake, and it has structural and functional similarity to other NPs. Like ANP and BNP, DNP activates the guanylyl cylase-A (GC-A) receptor, demonstrating natriuretic and diuretic properties. DNP-like peptide has been reported to be present in human plasma and heart and is elevated in human congestive heart failure.

DISCOVERY The existence in the heart of a hormone regulating blood volume was predicted long before its discovery. Pioneering studies by Gauer and Henry [11] advanced the concept that volume regulation is an integral part of an overall cardiovascular system. The search for a natriuretic hormone was stimulated by the experiments of de Wardener et al. [4], who observed potent natriuresis in renal artery–clamped dogs that could not be explained by a change in aldosterone and the glomerular filtration rate that remained constant. Then, the advent of electron microscopy allowed the discovery and characterization of specific atrial granules, presumably of a secretory nature [16], and gave new direction to the search for a natriuretic hormone. Atrial granularHandbook of Biologically Active Peptides

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878 / Chapter 120 All NPs have structural homology in the 17-aa ring formed by a disulfide bond between cysteine residues at positions 105 and 121 in humans. Biological activity of the peptides depends on the integrity of the ring structure (Fig. 1). The ANP structure is highly conserved during evolution. There is only one amino acid difference in position 110 between human and rat ANP—methionine in human ANP replaces isoleucine in rats [25]. The gene encoding pro-ANP consists of three coding exon sequences separated by two intron sequences (Fig. 2). All NPs are synthesized by different genes and stored in the form of prohormones—ANP (126 aa), BNP (108 aa), CNP (126 aa)—but the DNP prohormone gene structure is unknown in humans. The ANP gene is present not only in the animal kingdom but also in plants [37]. The pro-ANP gene is located on the distal

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FIGURE 2. Structure of the ANP gene. ANP is synthesized as a prepropeptide from mRNA originating from three exons of ANP gene. Prepropeptide and its derivative ANP propeptide are stored in the cells. Proteolytic cleavage of the propeptide sequences results in the formation of mature (low-molecularmass) ANP99–126 and N-terminal N-proANP1–98.

short arm of chromosome 1 in band 1P36 in humans and on chromosome 4 in mice.

DISTRIBUTION OF ANP mRNA The availability of ANP-specific antibodies allowed the development of immunocytochemical and radioimmunoassay analyses for ANP. Molecular biology led to the mapping of ANP-expressing tissue. With these tools, ANP has also been detected in several extra-atrial tissues [14], where it exerts physiological paracrine or autocrine effects. Among the tissues, ANP is highly expressed in various regions of the brain, particularly in the rostral diencephalon, and the ability of NPs to interact with the secretion of hypothalamic-neuroendocrine systems regulating, releasing, and inhibiting hormones has been established [27]. Furthermore, ANP is synthesized and secreted by rat adrenal chromaffin cells and is detected in peripheral ganglia, in the superior cervical nodosa, and in cell bodies of superior celiac mesenteric ganglia [14]. ANP prohormone has been found in vena cavae (inferior and superior), in both extra- and intrapulmonary veins and in the lungs, from which ANP is released as a 28-aa peptide. The gastrointestinal tract contains ANP. Linkage between ANP and the immune system has been supported by the demonstration of ANP in thymus and lymphoid tissues [38]. Other tissues, such as the pancreas, thyroid, male and female reproductive organs, liver, and salivary glands, contain NPs, and in some of them, a physiological role of NPs has been postulated.

PROCESSING Since its discovery, enormous progress has been made in identifying the structural and functional properties of ANP. The human ANP precursor peptide gene encodes a 151-aa preprohormone, a molecule that contains a 25-aa signal peptide. The signal peptide is removed during transport of the peptide across the endoplasmic membrane reticulum, producing a prohormone ANP(1–126). The final proteolytic cleavage of ANP(1–126) to the major, biologically active peptide ANP(99–126) takes place during or just after release of the peptide by exocytosis. Several groups have purified biologically active ANP peptides of different lengths from rat atrial homogenates. These other peptides most likely are artifacts of proteolytic digestion during purification. Several enzymes have been shown to process pro-ANP(1–126) to ANP(99–126), such as carboxypeptidases, kallikrein, trypsin, thrombin, corin, and others, but which one is physiologically relevant has not yet been established [25].

Atrial Natriuretic Peptide in Local Regulation of Endocrine Glands / 879

RELEASE OF NPs ANP release is controlled by many neural and hormonal factors, but the principal stimulus controlling ANP synthesis and release is atrial and ventricular wall stretch. Angiotensin II, endothelin, glucocorticoids, norepinephrine, oxytocin, arginine-vasopressin (AVP), morphine, and other factors, acting via hemodynamic stress or by direct effects on cardiac cells, have been shown to stimulate ANP release [25].

RECEPTORS AND SIGNALING CASCADE All NPs exert most of their biological effects via binding to two membrane-bound guanylyl cyclase (GC) receptors, GC-A and GC-B. ANP and BNP bind with higher affinity to GC-A, whereas CNP binds to GC-B. All NPs bind with relatively similar affinity to a third, very abundant C (or clearance) receptor, whose role is to internalize and degrade NPs (Fig. 3). In addition, the C receptor may also mediate the biological actions of NPs by the inhibition of cAMP or stimulation of phosphoinositide hydrolysis [22]. The GC receptors belong to a family of seven isoforms of transmembrane enzymes that convert guanosine triphosphate into cyclic 3′,5′guanosine monophosphate (cGMP). The activation of intracellular cGMP subsequently modulates the activity of regulatory proteins such as cGMP-regulated phos-

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BIOLOGICAL ACTIVITIES OF NPs Originally, ANP was found to be a potent diuretic and natriuretic substance, but it and other NPs are now recognized as hormones with a wide spectrum of central and peripheral autocrine-paracrine functions, including reproductive functions, bone formation, and cell proliferation and differentiation in various tissues.

Heart

Extracellular Domain Membrane

phodiesterases, ion channels, and cGMP-dependent protein kinases types I and II. These proteins, which are differentially expressed in various cell types, modify the cellular functions of cGMP [33]. Several studies have established that ANP activating the GC-A–cGMP pathway regulates blood pressure (BP) and the cardio-renal axis through its ability to antagonize the renin-angiotensin-aldosterone and sympathetic nervous systems or through its direct vasodilatory and natriuretic responses, modulating endothelial permeability and fluid shift from vascular beds into the lymphatic system. Gene-disruption studies have revealed that mice lacking the GC-A receptor present marked cardiac hypertrophy and fibrosis, disproportionate to their increase in BP, and chronic antihypertensive treatment of these animals normalized BP without a marked effect on cardiac hypertrophy [23]. The targeted deletion of BNP gene has indicated that BNP may act as a local antifibrotic factor and may be involved in the mechanism that inhibits the development of cardiac hypertrophy and fibrosis [17].

protein kinases

NPs exert important autocrine-paracrine actions within the heart and coronary circulation. All NPs have effects on cardiac contractility, considered as being negatively inotropic, although conflicting results have been reported. They are involved in the regulation of coronary blood flow, cardiomyocyte growth, inhibition of cardiac fibroblast proliferation, and extracellular matrix secretion; have a cardioprotective anti-ischemic function; and present a vasorelaxant influence in the coronary circulation. Most of these diverse physiological actions within the heart are mediated by cGMP [6].

phosphodiesterases

FIGURE 3. Natriuretic peptide receptor subtypes and their signaling pathways. GC-A and GC-B are particulate GC receptor-producing cGMP. Receptor C internalizes bound NPs and subjects them to lysosomal degradation. Receptor C can also lead to G-protein-coupled inhibition of adenylyl cyclase, activation of phospholipase C, and G-protein-gated inwardly rectifying K+ channels.

Pancreas and Gastrointestinal Tract Both ANP and its receptors are expressed in the pancreas and gastrointestinal tract [26]. ANP is present in acinar and centroacinar cells and in nerve fibers in the pancreas of porcine and other species. Several findings support the participation of ANP in the

880 / Chapter 120 modulation of gastrointestinal physiology. ANP diminishes bile secretion in rats by a direct effect, independent of the autonomic nervous system. Similarly, ANP exerts a direct stimulatory action on the pancreatic secretion of fluid and protein, mediated by C receptors coupled to the phosphoinositide pathway [26], independent of nitric oxide or parasympathetic activity. ANP also increases amylase secretion from isolated pig pancreatic segments. Because of the close association between peptide localization in nerve fibers with pancreatic secretory cells, it has been suggested that nerve fibers release ANP, which subsequently stimulates protein and amylase secretion [1]. ANP has an inhibitory effect on glucagon secretion by a mechanism that involves cGMP inhibition of Ca2+ uptake, probably via C receptors. These observations indicate a neuromodulatory role of ANP in the control of pancreatic secretion.

Hypothalamo-Pituitary-Adrenocortical System NPs are specifically involved in the regulation of the hypothalamo-pituitary-adrenocortical (HPA) system. ANP is a hypothalamic neurohormone that regulates blood pressure and volume directly by affecting blood vessels and kidney and indirectly by inhibiting the secretion of arginine vasopressin (AVP), renin, and aldosterone. In various species, ANP inhibits AVP and oxytocin release in response to osmotic stimuli or after hemorrhage and reduces plasma AVP in euhydration and dehydration [13]. ANP is likely to play an important inhibitory part in the neural control of adrenocorticotrophic hormone (ACTH) release [8]. ANP is a potent inhibitor of basal ACTH secretion and of secretion induced by hemorrhage, central angiotensin II administration, or stress. The latter may be mediated, at least partly, by suppression of AVP release from the neural lobe. One important aspect of the hypothalamic and pituitary action of NPs is their ability to interrupt the mechanism controlling stress hormone secretion, the release of corticotrophin-releasing hormone (CRH). ANP, BNP, and CNP inhibit CRH-evoked ACTH secretion in mice hemi-pituitary preparations, with CNP being the most effective [12]. NPs also significantly reduce pituitary pro-opiomelanocortin (POMC) mRNA expression. ANP can act directly on the pituitary to stimulate luteinizing hormone (LH) and folliclestimulating hormone (FSH) release and to inhibit prolactin. ANP does not influence the thyrotrophinreleasing-hormone (TRH)-induced prolactin response, suggesting that it has no direct influence on lactotroph cells in inhibiting prolactin secretion but may activate the hypothalamic-dopaminergic system. On the other hand, endogenous ANP plays a pathophysiological role in augmenting prolactin release during stress, either by

inhibiting the release of prolactin-inhibiting factors or, alternatively, by enhancing the release of prolactinreleasing factors [10]. Intracerebroventricular injection of ANP increases plasma growth hormone (GH) levels in both conscious and urethane-anesthetized rats.

Adrenal Glands The human and rat adrenal medulla is a site of ANP and all three receptors. The adreno-medullary synthesis of ANP is increased in patients with primary aldosteronism, and ANP mRNA is upregulated in the adrenal medulla and cortex of deoxycorticosterone acetate (DOCA)-salt-treated rats [18]. ANP inhibits angiotensin II–induced aldosterone secretion [28]. The role of ANP in the regulation of aldosterone, which plays a central part in the control of BP, has been suggested in the development of hypertension in offspring from foodrestricted mothers. This plasma aldosterone elevation was associated with decreased plasma ANP and an increased density of adrenal C receptors, which could explain, at least in part, the observed high BP [30].

Thyroid and Parathyroid Glands The entire NP system has been demonstrated in the thyroid, and some results point to its functional role. Abnormalities in NPs have been found in hypothyroid patients who showed decreased plasma ANP, whereas hyperthyroid patients presented a twofold increase in circulating plasma ANP in comparison with normal subjects. T4 treatment of hypothyroid patients normalizes ANP circulating levels. ANP infusion in humans decreases T3 and T4 with a reciprocal increase in thyroidstimulating hormone (TSH), indicating that the ANP action occurs directly in the thyroid, not via the pituitary or hypothalamus; otherwise, TSH would decrease in the circulation if the inhibitory effects occurred at hypothalamic or pituitary sites [36]. ANP suppresses thyroglobulin production in cultured human thyroid cells [32], and this is correlated with a reduction of cAMP, indicating that the effect is mediated by C receptors. The role of GC receptors is supported by another study in which ANP and CNP altered the morphology of human thyroid cells, increasing cells of the retracted phenotype [31]. The upregulation of ANP gene expression by thyroid hormones together with the observation that T4 increases plasma ANP in hypothyroidism indicates the clinical relevance of NPs in the thyroid. The functional interaction between parathyroid hormone (PTH) and NPs has been observed in modulating cyclic nucleotides and cell proliferation in chondrocytes and osteoblasts. The direct action of ANP on the release of endothelin from cultured rat parathyroid

Atrial Natriuretic Peptide in Local Regulation of Endocrine Glands / 881 cells has been shown, indicating the presence of NP receptors in the cells. In addition, the volume decreased by bolus injection of ANP was associated with the decrease of PTH, suggesting an inhibitory role of ANP; however, the clinical relevance has yet to be shown [3].

Reproductive Organs Although all NPs are highly expressed in male and female reproductive organs, there is strong evidence that CNP acts as a local paracrine or autocrine regulator in female reproduction, pregnancy, and fetal development. CNP also plays a distinct role in male reproduction [39]. In rodent ovaries and uteri, both CNP and GC-B show time-dependent expression with maximal expression at proestrus [5]. The modulation of follicular atresia in follicles at preantral to antral transition has been postulated. In pregnancy, the highest expression of CNP mRNA is found in the placenta, indicating a role for this peptide in gestation, probably to antagonize overactive vasoconstrictive systems. Higher CNP levels have been found in the fetal than in the maternal circulation, suggesting the need for and ability of the fetus to synthesize de novo CNP. Pathological pregnancies, such as intrauterine growth, retardation, or preeclampsia, present differential regulation of CNP, a decrease in the placenta with an increase in the myometrium, compared with normal pregnancy, indicating an organspecific function of CNP. NPs stimulate testosterone production by Leydig cells [21]. In humans, acute alpha-hANP injection significantly increases testosterone concentrations in the spermatic vein but not in peripheral blood [9]. The role of NPs in the early phase of fetal testicular steroidogenesis has been postulated before the onset of pituitary LH secretion [7]. Furthermore, the paracrine action, particularly of CNP, includes the relaxation of seminiferous tubules necessary to regulate sperm transport and testicular blood supply. Support for ANP function in the maintenance of serum testosterone and testicular steroidogenesis was provided recently by the study of Leydig cells isolated from GC-A −/− and transgenic mice. ANP had no effect on testosterone production in GC-A −/− mice. However, Leydig cells isolated from animals having extra copies of GC-A gene showed increased steroidogenesis [24]. CNP and GC-B receptors are expressed in the testis [20] and in porcine seminal plasma. CNP has been shown to regulate testicular function, such as testosterone release, modulation of spermatozoa mobility, and testicular cell germ development. The possible involve-

ment of CNP in penile erection has also been hypothesized, because CNP activates GC-B receptors localized on the cavernosal membrane.

PATHOPHYSIOLOGICAL IMPLICATIONS In patients with congestive heart failure (CHF), plasma levels of ANP and BNP and their N-terminal fragments of prohormones are elevated because the cardiac hormonal system is activated by volume overload and pressure. NPs have a beneficial, compensatory action in CHF by inducing vasodilatation, natriuresis, and inhibition of the sympathetic nervous and reninangiotensin-aldosterone systems. Furthermore, the autocrine-paracrine effects, such as the regulation of coronary blood flow, proliferative responses contributing to vascular remodeling, and cardioprotection, could greatly benefit the patient with cardiovascular disease. These diverse actions may have a significant counterregulatory role to the various mechanisms activated in cardiovascular diseases, and the use of recombinant human BNP (nesiritide) and ANP (anaritide) has already been proven to be beneficial for acute decompensation in heart failure [15]. Ultimately, an understanding of autocrineparacrine actions of NPs may lead to their therapeutic application in various pathologies.

References [1] Adeghate E, Ember Z, Donath T, Pallot DJ, Singh J. Immunohistochemical identification and effects of atrial natriuretic peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas. Peptides 1996; 17(3):503–509. [2] De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1981; 28:89–94. [3] De Feo ML, Bartolini O, Orlando C, Maggi M, Serio M, Pines M, Hurwitz S, Fujii Y, Sakaguchi K, Aurbach GD, Brandi ML. Natriuretic peptide receptors regulate endothelin synthesis and release from parathyroid cells. Proc Natl Acad Sci USA 1991; 88(15):6496–6500. [4] De Wardener HE, Mills IH, Clapham WF, Hayter CJ. Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 1961; 21:249–258. [5] Dos Reis AM, Fujio N, Dam TV, Mukaddam-Daher S, Jankowski M, Tremblay J, Gutkowska J. Characterization and distribution of natriuretic peptide receptors in the rat uterus. Endocrinology 1995; 136(10):4247–4253. [6] D’Souza SP, Davis M, Baxter GF. Autocrine and paracrine actions of natriuretic peptides in the heart. Pharmacol Ther 2004; 101(2):113–129. [7] El Gehani F, Tena-Sempere M, Ruskoaho H, Huhtaniemi I. Natriuretic peptides stimulate steroidogenesis in the fetal rat testis. Biol Reprod 2001; 65(2):595–600. [8] Fink G, Dow RC, Casley D, Johnston CI, Lim AT, Copolov DL, Bennie J, Carroll S, Dick H. Atrial natriuretic peptide is a physiological inhibitor of ACTH release: evidence from immunoneutralization in vivo. J Endocrinol 1991; 131(3):R9–12.

882 / Chapter 120 [9] Foresta C, Mioni R, Miotto D, De Carlo E, Facchin F, Varotto A. Stimulatory effects of alpha-hANP on testosterone secretion in man. J Clin Endocrinol Metab 1991; 72(2):392–395. [10] Franci CR, Anselmo-Franci JA, McCann SM. The role of endogenous atrial natriuretic peptide in resting and stress-induced release of corticotropin, prolactin, growth hormone, and thyroid-stimulating hormone. Proc Natl Acad Sci USA 1992; 89:11391–11395. [11] Gauer OH, Henry JP. Circulatory basis of fluid volume control. Physiol Rev 1963; 43:423–481. [12] Guild SB, Cramb G. Characterisation of the effects of natriuretic peptides upon ACTH secretion from the mouse pituitary. Mol Cell Endocrinol 1999; 152(1–2):11–19. [13] Gutkowska J, Antunes-Rodrigues J, McCann SM. Atrial natriuretic peptide in the brain and pituitary gland. Physiol Rev 1997; 77(2):465–515. [14] Gutkowska J, Nemer M. Structure, expression, and function of atrial natriuretic factor in extraatrial tissues. Endocr Rev 1989; 10:519–536. [15] Keating GM, Goa KL. Nesiritide: a review of its use in acute decompensated heart failure. Drugs 2003; 63(1):47–70. [16] Kisch B. Electron microscopy of the atrium of the heart. Exp Med Surg 1956; 14:99–112. [17] Kuhn M. Molecular physiology of natriuretic peptide signaling. Basic Res Cardiol 2004; 99(2):76–82. [18] Lai FJ, Shin SJ, Lee YJ, Lin SR, Jou WY, Tsai JH. Up-regulation of adrenal cortical and medullary atrial natriuretic peptide and gene expression in rats with deoxycorticosterone acetate-salt treatment. Endocrinology 2000; 141(1):325–332. [19] Marie JP, Guillemot H, Hatt PY. Le degré de granulation des cardiocytes auriculaires—etude planimétrique au cours de différents apports d’eau et de sodium chez le rat. Pathol Biol 1976; 24:549–554. [20] Middendorff R, Davidoff MS, Behrends S, Mewe M, Miethens A, Muller D. Multiple roles of the messenger molecule cGMP in testicular function. Andrologia 2000; 32(1):55–59. [21] Mukhopadhyay AK, Bohnet HG, Leidenberger FA. Testosterone production by mouse Leydig cells is stimulated in vitro by atrial natriuretic factor. FEBS Lett 1986; 202:111–116. [22] Murthy KS, Teng BQ, Zhou H, Jin JG, Grider JR, Makhlouf GM. G(i-1)/G(i-2)-dependent signaling by single-transmembrane natriuretic peptide clearance receptor. Am J Physiol Gastrointest Liver Physiol 2000; 278(6):G974–G980. [23] Oliver PM, Fox JE, Kim R, Rockman HA, Kim HS, Reddick RL, Pandey KN, Milgram SL, Smithies O, Maeda N. Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Proc Natl Acad Sci USA 1997; 94(26):14730–14735. [24] Pandey KN, Oliver PM, Maeda N, Smithies O. Hypertension associated with decreased testosterone levels in natriuretic peptide receptor-A gene-knockout and gene-duplicated mutant mouse models. Endocrinology 1999; 140(11):5112– 5119.

[25] Ruskoaho H. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev 1992; 44(4):479–602. [26] Sabbatini ME, Villagra A, Davio CA, Vatta MS, Fernandez BE, Bianciotti LG. Atrial natriuretic factor stimulates exocrine pancreatic secretion in the rat through NPR-C receptors. Am J Physiol Gastrointest Liver Physiol 2003; 285(5):G929–G937. [27] Samson WK. Here the heart may give a useful lesson to the head [editorial]. Endocrinology 1996; 137(9):3629–3630. [28] Schiebinger RJ, Kem DC, Brown RD. Effect of atrial natriuretic peptide on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by rat adrenal glomerulosa cells. Life Sci 1988; 42:919–926. [29] Schulz-Knappe P, Forssmann K, Herbst F, Hock D, Pipkorn R, Forssmann WG. Isolation and structural analysis of “urodilatin,” a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine. Klin Wochenschr 1988; 66:752–759. [30] Sebaai N, Lesage J, Alaoui A, Dupouy JP, Deloof S. Effects of dehydration on endocrine regulation of the electrolyte and fluid balance and atrial natriuretic peptide-binding sites in perinatally malnourished adult male rats. Eur J Endocrinol 2002; 147(6):835–848. [31] Sellitti DF, Lagranha C, Perrella G, Curcio F, Doi SQ. Atrial natriuretic factor and C-type natriuretic peptide induce retraction of human thyrocytes in monolayer culture via guanylyl cyclase receptors. J Endocrinol 2002; 173(1):169–176. [32] Sellitti DF, Tseng YC, Wartofsky L. Receptors for atrial natriuretic peptide (ANP) and regulation of thyroglobulin secretion by ANP in human thyroid cells. Life Sci 1989; 45:793–801. [33] Soderling SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 2000; 12(2):174–179. [34] Sudoh T, Kangawa K, Minamino M, Matsuo H. A new natriuretic peptide in porcine brain. Nature 1988; 332:78–81. [35] Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 1990; 168(2):863–870. [36] Vesely DL, San Miguel GI, Hassan I, Gower WR, Jr., Schocken DD. Atrial natriuretic hormone, vessel dilator, long-acting natriuretic hormone, and kaliuretic hormone decrease the circulating concentrations of total and free T4 and free T3 with reciprocal increase in TSH. J Clin Endocrinol Metab 2001; 86(11):5438–5442. [37] Vesely MD, Gower WR, Jr., Perez-Lamboy G, Overton RM, Graddy L, Vesely DL. Evidence for an atrial natriuretic peptidelike gene in plants. Exp Biol Med (Maywood) 2001; 226(1):61– 65. [38] Vollmar AM, Lang RE, Hanze J, Schulz R. A possible linkage of atrial natriuretic peptide to the immune system. Am J Hypertens 1990; 3:408–411. [39] Walther T, Stepan H. C-type natriuretic peptide in reproduction, pregnancy and fetal development. J Endocrinol 2004; 180(1):17–22.

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121 Galanin, Neurotensin, and Neuromedins in the Local Regulation of Endocrine Glands GIUSEPPINA MAZZOCCHI, RAFFAELLA SPINAZZI, AND GASTONE G. NUSSDORFER

central nervous and digestive systems, where they act as neuromodulators. Here, we survey findings suggesting that these peptides play a role in the autocrine-paracrine regulation of endocrine glands in mammals. Mammalian NMs are divided into four groups: kassininlike tachykinins (reviewed in the Chapter 114 of this book) bombesin-like NMs, NT-like NMs, and NMU.

ABSTRACT Galanin (Gal) is expressed in lactotrophs, thyrotrophs, somatotrophs, gonadotrophs, and corticotrophs of the anterior pituitary (AP). Gal enhances prolactin (PRL) release from lactotrophs, and there is evidence of its involvement in the development of prolactinomas. Less clear is the role of Gal in the functional control of other AP cells. Gal-positive nerve fibers are distributed around pancreatic islets, and Gal was found to inhibit agonist-stimulated insulin release. Gal is expressed in the adrenal medulla, and it has been shown that Gal directly stimulates glucocorticoid secretion acting in a paracrine manner. Neurotensin (NT) expression has been detected in AP lactotrophs, thyrotrophs, and gonadotrophs. NT enhances PRL release from lactotrophs, although contrasting findings have been obtained on its effect on thyrotrophs and gonadotrophs. NT is expressed in the pancreas and adrenal medulla, and evidence indicates that NT modulates insulin release and inhibits mineralocorticoid secretion. Neuromedin (NM) B is mainly contained in AP thyrotrophs, where its expression is upregulated by thyroid hormones. NMB inhibits thyroid-stimulating hormone release from thyrotrophs, leading to the hypothesis that a central feedback mechanism is operative by which the thyroid gland dampens its own exceedingly high secretory activity. NMB and NMC are expressed in the adrenal medulla, but their effect on adrenal secretion is unclear. Conversely, NMU has been reported to raise adrenal steroid production through an indirect paracrine mechanism involving the adrenal medulla.

GALANIN Gal is a 29-amino-acid peptide (30 amino acids in humans), which exerts multiple actions acting via three subtypes of G-protein-coupled receptors (Gal-Rs). GalR1 and Gal-R3 are thought to inhibit adenylate cyclase (AC) and to activate inward K+ currents, whereas Gal-R2 appears to be positively coupled to phospholipase (PL)C (reviewed in Chapter 104 of this Book).

Gal and Gal-R Expression in Endocrine Glands Anterior Pituitary Compelling evidence shows that Gal is expressed in the mammalian anterior pituitary (AP) as mRNA and protein [14, 24, 57]. In situ hybridization (ISH) and immunocytochemistry (ICC) revealed that in female rats Gal-positive cells are predominantly lactotrophs [67], whereas in males they are mainly somatotrophs, thyrotrophs, and corticotrophs [13]. In adult male monkeys, Gal-positive cells are thyrotrophs and gonadotrophs [50], and in normal human AP and pituitary adenomas are almost exclusively corticotrophs [38]. Consistent findings indicate that estrogens, by inducing lactotroph hyperplasia, enhance Gal mRNA and protein expression in rats [10, 18, 57, 67]. This observation explains why Gal expression in AP and Gal release

INTRODUCTION Galanin (Gal), neurotensin (NT), and neuromedins (NMs) are regulatory peptides widely distributed in the Handbook of Biologically Active Peptides

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884 / Chapter 121 from cultured AP cells are much higher in female than male rats [30] and accords well with the rise in Gal expression during the development of estrogen-dependent prolactinomas [10, 56, 67]. Growth hormone (GH)-releasing hormone (RH) was found to stimulate Gal release from dispersed mouse AP cells, and transgenic mice harboring human GHRH gene develop a somatotroph adenoma associated with a marked increase in Gal mRNA expression [31, 48]. Thyroidstimulating hormone (TSH) has been reported to enhance Gal release from cultured rat AP cells [30], and surgical or chemical thyroidectomy has been shown to lower Gal content in the rat AP, the effect being reversed by T4 administration [27]. However, T3 was found to decrease Gal mRNA in cultured rat AP cells [18], and the development of [131I]-induced thyrotroph adenomas is associated with a lowering of Gal mRNA expression [31]. Pancreatic Islets Gal-positive nerve fibers have been demonstrated around islets of the rat and dog pancreas. They originate from celiac ganglion, and are thought to release Gal in an amount sufficient to evoke the effects described in the next section [62]. Gal-R3 mRNA expression has been detected in the human pancreas [35], and Gal-binding sites have been shown in rat βand δ-cell-derived cell lines [1, 36]. Adrenal Gland Gal mRNA expression has been detected in the fresh adrenal medulla of rats, cows, and pigs [17, 51], and evidence shows that in bovine adrenal chromaffin cells Gal gene transcription is enhanced by the activation of Ca2+ influx, protein kinase (PK) A, and PKC [3]. Gal immunoreactivity (IR) was detected by radioimmunoassay (RIA) in the adrenal medulla of rats, rabbits, cats, pigs, and humans, as well as in human pheochromocytomas [12, 26, 51, 66]. ICC showed the presence of Gal-IR in chromaffin cells and Gal-positive nerve fibers in the mammalian adrenal medulla [17, 26, 51].

Effect of Gal on Hormone Secretion Anterior Pituitary There is a general consensus that Gal, acting in an autocrine-paracrine manner, enhances prolactin (PRL) synthesis in and release from lactotrophs, estrogens playing a critical role in these Gal-mediated events [19]. This contention is based on the following pieces of evidence: Gal raises PRL secretion from cultured rat AP

cells, and Gal-positive lactotrophs secrete more PRL than Gal-negative ones [10]; Gal antiserum decreases PRL secretion from rat Gal-positive lactotrophs [10]; Gal-gene knock-out mice display lowered AP expression of PRL and decreased PRL secretion [67]; and, finally, intact human Gal-gene transgenic, but not ovariectomized, female mice exhibit lactotroph hyperplasia and enhanced AP expression of PRL mRNA, whereas transgenic males, if not treated with estrogens, do not differ from wild controls [11]. Findings also indicate that suckling evokes parallel increases in Gal and PRL expression in the rat AP, suggesting that Gal amplifies lactotroph stimulation during nursing via an autocrineparacrine mechanism [58]. The direct effect of Gal on gonadotrophs is well established, but different investigations have produced conflicting results. Gal has been reported to raise luteinizing hormone (LH) release from rat AP cells [37, 61]. In contrast, Todd et al. [64] observed that Gal and a Gal-R2 agonist inhibit LHRH-stimulated LH and follicle-stimulating hormone secretion from cultured rat AP cells. Because Gal immunoneutralization potentiates LH response to LHRH, these authors conclude that endogenous Gal exerts a paracrine inhibitory action on gonadotropin secretion in the rat. Gal was found to increase basal, but not LHRH-stimulated, LH release from cultured pig AP cells [23]. However, antisense oligonucleotides directed against the Gal gene do not alter LH response to LHRH, casting doubt on the physiological relevance of endogenous Gal in the fine-tuning of gonadotroph secretion in the pig. Sporadic findings suggest that Gal directly stimulates TSH and inhibits adrenocorticotrophin hormone (ACTH) secretion. Gal has been reported to raise thyrotrophin-releasing hormone (TRH)-stimulated, but not basal, TSH release from cultured rat AP cells [54] and to decrease ACTH release from the same preparations, the effect being quenched by Gal immunoneutralization [13]. The physiological relevance of these effects of Gal remains questionable [38]. Pancreatic Islets Convincing evidence indicates that Gal inhibits agonist-stimulated insulin release from islet β-cells [2, 25, 36, 39, 65]. The mechanism of this effect of Gal appears to involve inhibition of AC through functional Gal-Rs associated with pertussis toxin–sensitive GTPbinding protein mediating guanine nucleotide control of Gal binding [2, 36]. Additional mechanisms may include activation of K+ channels and an ensuing decrease in Ca2+ influx [22]. Gal has been also reported to blunt somatostatin release from perfused rat pancreas and a β-cell line, mainly via the inhibition of AC [1, 65].

Galanin, Neurotensin, and Neuromedins in the Local Regulation of Endocrine Glands / 885 Adrenal Gland Despite numerous studies indicating that Gal is expressed in the adrenal medulla, in vitro investigations on its possible effect on catecholamine secretion are lacking. In contrast, there is evidence that Gal secreted by adrenal chromaffin cells could affect cortical function in a paracrine manner [51]. In fact, Gal was found to increase corticosterone and cAMP production from dispersed rat inner adrenocortical cells, the effect being abrogated by the Gal-R antagonist galantide. The corticosterone secretagogue action of Gal was also blocked by the PKA inhibitor H-89, suggesting the involvement of Gal-Rs coupled to the AC/PKA signaling cascade [46].

NEUROTENSIN Neurotensin (NT) is a 13-amino-acid peptide that acts through two subtypes of G-protein-coupled receptors, NTS1 and NTS2. The former receptor is thought to activate AC and PLC, whereas the latter seems to stimulate PLC and arachidonate release (perhaps the activation of PLA2). A third NT receptor subtype, referred to as NTS3, is not coupled to a G-protein and has been identified with human sortilin (reviewed in the Chapter 102 in the Brain Peptide section).

NT and NT-R Expression in Endocrine Glands Anterior Pituitary NT is expressed, as mRNA and protein, in the rat AP [33, 40]. ICC showed that NT is colocalized with TSH and LH in the rat thyrotrophs and gonadotrophs [6]. NT expression has been reported to be downregulated in the AP of hypothyroid [32] and castrated rats [5]. [3H]NT binding sites have been shown in the rat AP [47]. Pancreatic Islets NT-IR has been demonstrated in the mouse pancreas. It seems to be negatively regulated by insulin, inasmuch as it reaches a maximum concentration in genetically diabetic animals at the time of β-cell failure [7]. Adrenal Gland RIA detected NT-IR in the adrenal medulla of cows, cats, rabbits, guinea pigs, and rats [51]. ICC showed that NT-IR is present in the medullary chromaffin cells and traced NT-positive nerve fibers in the rat adrenal medulla [17, 51]. Dexamethasone, nerve growth factor,

and AC activators were found to synergistically enhance NT content in cultured rat adrenomedullary and PC12 cells [34, 63]. ISH demonstrated that the interruption of splanchnic-nerve trasmission raises NT mRNA in rat adrenomedullary cells [17]. NT binding sites have been reported in the rat adrenals, especially at the corticomedullary junction [51].

Effects of NT on Hormone Secretion Anterior Pituitary Consistent findings indicate that NT modulates PRL release from rat lactotrophs via a twofold mechanism. It lowers PRL secretion indirectly by stimulating the hypothalamic release of dopamine and enhances PRL release by acting directly on lactotrophs [60]. The direct effect appears to involve stimulation of PLCdependent [41, 47] and perhaps arachidonate-dependent cascades [59]. Evidence has also been provided that NT decreases basal and TRH-stimulated TSH release [4] and increases basal, but not LHRH-stimulated, LH secretion from rat AP cells [37]. Pancreatic Islets Using dispersed or cultured rat islet cells, a dual effect of NT has been observed. At low glucose concentrations and over short-term incubation period (20 min), NT enhances insulin and somatostatin release, whereas at high glucose concentrations it inhibits hormone secretion [20]. Adrenal Gland NT was found to inhibit aldosterone response of dispersed rat zona glomerulosa cells to angiotensin-II and K+. Basal and ACTH-stimulated secretions are not affected, thereby suggesting that NT interferes with the transduction mechanism of agonists raising intracellular Ca2+ concentration [44]. As far as we are aware, the possible effects of NT on catecholamine secretion from adrenomedullary cells have not yet been investigated. However, there is evidence that NT may affect the function of the adrenal medulla. In fact, NT was found (1) to increase phosphatidylinositol turnover and to enhance enkephalin release from bovine adrenal chromaffin cells [9] and (2) to raise corticotrophinreleasing hormone (CRH) and ACTH release from rat adrenal medulla slices. The ACTH response is blocked by a CRH antagonist, suggesting that it is consequent to the stimulation of CRH release [45]. The physiological relevance of the NT-induced stimulation of the intramedullary CRH-ACTH system (for a review, see [51]) in the paracrine regulation of the adrenal cortex secretion remains to be addressed.

886 / Chapter 121 NEUROMEDINS The two mammalian bombesin (BN)-like neuromedins (NMs) are the 10-amino-acid peptides neuromedin B (NMB) and NMC, NMC being the C-terminal decapeptide of gastrin-releasing peptide (GRP). They act via three subtypes of receptors: the GRP-preferring receptor (GRP-R), the NMB-preferring receptor (NMBR), and the BN receptor 3, which shows low affinity for NMB and NMC [42]. NT-like NM (NMN) is a 6-aminoacid peptide whose C-terminal tetrapeptide is identical to that of NT. NMN shares with NT biological functions and receptors [42]. Two forms of NMU have been isolated, containing 25- and 8-amino-acid residues. NMU-8 is the C-terminal octapeptide of NMU-25. NMU acts via two G-protein-coupled receptors, NMU-R1 and NMUR2, that are thought to be positively coupled to PLC and PLA2 and perhaps negatively coupled to AC (reviewed in the Chapter 103 in the Brain Peptide section).

NM and NM Receptor Expression in Endocrine Glands Anterior Pituitary NMB has been detected in the rat AP as mRNA and protein [16, 29, 52, 53], and ICC showed that NMB-IR is mainly contained in thyrotrophs [55]. NMB-R mRNA has been demonstrated in the rat AP [29]. NMB expression decreases in hypothyroid rats and increases after thyroid hormone administration [53, 55]. Ovariectomy lowers and estradiol raises NMB mRNA and protein in the rat AP [49], and estradiol upregulates NMB and NMB-R expression in cultured rat AP cells [29]. AP NMB-IR content decreases in fasted rats, and increases in streptozotocin-induced diabetic animals [52]. NMU expression has been detected in rat and human AP and human pituitary adenomas [8], and prolonged TRH administration was reported to increase NMU-IR in the rat AP [21]. ICC showed NMU-IR in rat corticotrophs and to a lesser extent in thyrotrophs [15]. Adrenal Gland NMB-IR and NMC-IR have been found in the adrenal medulla of cows and rats, and elevated concentrations of NMN-IR have been found in the cat adrenal medulla [51]. No NMU expression has been detected in the adrenals, which do, however, contain NMU-R1 and NMU-R2 [8].

Effects of NMs on Hormone Secretion Anterior Pituitary Consistent findings show that NMB lowers basal and TRH-stimulated TSH release from rat thyrotrophs [49,

55]. This observation, along with the demonstration that thyroid hormones upregulate NMB synthesis in thyrotrophs, led to the hypothesis that a negative feedback mechanism is operative in the rat AP, by which NMB acting in a autocrine-paracrine manner dampens exceedingly high thyroid gland stimulation by the TRHTSH system [55]. Because hyper- and hypoestrogenism and diabetes alter pituitary NMB synthesis, this hypothesis may explain why dysregulation of thyroid gland secretion frequently occurs in these pathological events. NMC has been reported to enhance GH and PRL secretion from male rat AP cells, the effect being modulated by the endocrine status of animals. In fact, estrogens and high concentrations of dexamethasone potentiate the stimulating action of NMC on somatotrophs, whereas low concentrations of dexamethasone inhibit the effect of NMC on lactotrophs and block the potentiating effect of estrogens [28]. Adrenal Gland NMB does not alter corticosterone secretion from dispersed rat inner adrenocortical cells, thereby making it likely that the in vivo glucocorticoid secretagogue action of this peptide is indirectly mediated by the stimulation of the central branch of the hypothalamicpituitary-adrenal axis [42]. NMU-8 has no effect on steroid-hormone secretion from dispersed rat adrenocortical cells. However, it stimulates hormone secretion from adrenocortical slices but not from quarters of regenerated adrenocortical autotransplants deprived of chromaffin cells [43]. The hypothesis has been advanced that NMU-8 enhances steroid secretion indirectly by acting on medullary chromaffin cells, which in turn stimulate adrenocortical cells in a paracrine manner. Because the glucocorticoid secretagogue effect of NMU8 on adrenal slices is blocked by antagonists of both CRH and ACTH [43], it has been suggested that NMU-8, like NT, activates the intramedullary CRH-ACTH system. Studies have not been carried out on the possible effect of NMs on adrenomedullary catecholamine secretion.

CONCLUSION This survey has shown that Gal, NT, and NMs play a potentially important role in the local regulation of endocrine glands. However, much work still remains to be done. In fact, well-documented effects have been described only for AP, pancreatic islets, and adrenals; only sporadic studies have dealt with the expression and function of these peptides in the thyroid and parathyroid glands and endocrine gonads. Moreover, the expression and function of Gal, NT, and NM receptors in endocrine glands have been poorly investigated, in spite of the fact that these receptors have been cloned

Galanin, Neurotensin, and Neuromedins in the Local Regulation of Endocrine Glands / 887 and selective antagonists are available. We hope that these topics will be addressed by future investigations.

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122 Neuropeptide Y: A Conductor of the Appetite-Regulating Orchestra in the Hypothalamus SATYA P. KALRA AND PUSHPA S. KALRA

eating periods of varied duration. Based on the intermittency of the innate urge to consume food, animals are classified as either nocturnal (e.g., rodents) or diurnal (e.g., ungulates). In humans and in subhuman primates in the laboratory, feeding is voluntarily confined to two to three meals during the light phase and early dark phase of the photoperiod. Seasonal abundance of energy resources and period of droughts are additional environmental factors that modify periodic ingestive behavior in vertebrates [1, 16, 24]. The intricate interconnected neural network that operates in a timely manner to propagate the intermittent appetitive urges and sustain them at high levels during periods of food shortages or the high energy demands of physiological events such as reproduction and lactation has recently been mapped in the rodent brain [17, 27]. Since the first demonstration of its potent appetite-stimulating effects [5], evidence accumulated over the past 2 decades has established neuropeptide Y (NPY) as the most important physiological appetite transducer within the hypothalamic appetiteregulating network (ARN) in the brain of vertebrates [10, 17, 20, 25].

ABSTRACT Neuropeptide Y (NPY) is a long-sought after endogenous neurochemical signal that integrates the expression of the innate appetitive drive. Circadian and ultradian NPY neurosecretion in the discrete arcuate nucleus–magnocellular paraventricular nucleus (ARCmPVN) pathway in the hypothalamus are temporally linked with the timely onset of the periodic appetitive drive. NPY and cohorts propagate the urge to eat directly through activation of Y1, Y5, GABAA, and α1 adrenergic receptors and indirectly by the repression of anorexigenic melanocortin signaling within the ARCmPVN neuroaxis. The reciprocal circadian and ultradian secretions of two afferent hormonal signals, anorexigenic adipocyte-derived leptin and orexigenic gastric ghrelin, participate in the periodic increase in NPY discharge. Furthermore, both high and low abundance of NPY signal at target sites resulting from the breakdown at one or more loci in this tightly integrated appetite-regulating network confer hyperphagia, increased fat accretion, and obesity but never anorexia, inanition, or morbidity. This recognition that the hypothalamic NPY circuit is the conductor of the appetiteregulating network in the brain has paved the way for novel therapies designed to subjugate hypothalamic NPY signaling for tonically repressing appetite and, thereby, curb the current worldwide escalation in the incidence of obesity and the attending metabolic syndrome.

NPY AND APPETITE Stimulation of Feeding NPY, a 36-amino-acid peptide [34], is the only member of the pancreatic polypeptide family of peptides produced by neurons [8]. Produced by clusters of neurons distributed ubiquitously in the peripheral and central nervous systems, NPY regulates diverse physiological functions. Because NPY propagates the appetitive drive in a reliable fashion in vertebrates, it has topped the list of the most studied orexigenic peptide signals in the brain [14, 17, 20].

INTRODUCTION The appetitive drive is an organized periodic behavior. Periods of active eating during the arousal phase of the daily rest–activity cycle are interspersed among nonHandbook of Biologically Active Peptides

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890 / Chapter 122 Feeding begins within 10–15 minutes after central administration of NPY in sated animals; the magnitude and duration of the appetitive drive engendered is dose-dependent. When administered during ongoing spontaneous feeding, NPY amplifies the magnitude of feeding [5, 6]. NPY administered at intervals or infused continuously for long periods evokes relentless hyperphagia resulting in increased rate of weight gain and obesity without development of tolerance [14, 17]. Likewise, endogenous NPY hypersecretion is concomitant with persistent hyperphagia and excess weight gain in genetic models of obesity [14, 17, 27]. Stimulation of feeding in discrete bouts is another unique feature encoded by the NPY molecule. The amplitude and duration, but not the frequency, of feeding bouts are dependent on the strength and duration of the NPY signal [16]. Finally, NPY can override the restraint imposed by central and peripheral anorexigenic signals [14, 16, 17].

NPY Target Sites Evaluation of feeding in response to microinjection of the peptide in various hypothalamic sites and examination of neuronal c-fos activation following intracerebroventricular injection of NPY have identified the ARC, VMH, DMH, PVN, and medial preoptic area–anterior hypothalamic area as NPY target sites [17, 20, 23, 37]. However, the hard-core wiring that mediates NPY-induced appetite on a daily basis is coextensive in the magnocellular PVN (mPVN) and DMH because fluctuations in c-fos activation in these two sites parallel initiation and termination of feeding [17, 20, 37] (Fig. 1).

NPY Neurosecretion That the ARC-PVN neuroaxis is the final common pathway for eliciting the intermittent daily and fasting-

MECHANISM OF ACTION NPY Subpopulations Three subpopulations of NPY-expressing neurons in the hypothalamus and an extrahypothalamic subpopulation in the brain stem (BS) participate in varied ways to orchestrate the daily meal pattern and heightened appetite in response to abrupt increases in energy demands. The cluster of NPY-expressing neurons that span the entire arcuate nucleus (ARC) in the ventral medial hypothalamus and their innervations dorsally to the paraventricular nucleus (PVN) constitute the major pathway for the propagation and termination of the appetitive urge [8, 14, 17, 20]. A subgroup of NPYexpressing neurons in the dorsomedial hypothalamus (DMH) is also goaded into action to meet the enhanced energy demands of pregnancy and lactation and during the pre- and postnatal growth and development periods [4, 10, 26, 37]. NPY-producing neurons in the DMH and PVN elicit hyperphagia and obesity when signaling in the ARN is disrupted [17, 20]. Excessive weight gain and fat accrual in humans and rodents after either transient or permanent loss of regulatory communication from the ventromedial hypothalamus (VMH) to the ARC-PVN NPY-ergic signaling is well documented [7, 13, 17, 20]. The subpopulation of NPY-expressing neurons in the BS also participates in maintaining the daily energy homeostasis because disruption of the information relay from BS to the hypothalamus either pharmacologically or surgically promotes hyperphagia and increased weight gain [20, 29].

FIGURE 1. Representation of the interconnected hypothalamic appetite regulating circuitry modulated by the opposing afferent hormonal feedback actions of leptin from fat tissue and ghrelin from stomach.

Neuropeptide Y: A Conductor of the Appetite-Regulating Orchestra in the Hypothalamus / 891 induced appetite is supported by analyses of NPY synthesis, storage, and release [17, 20] (Fig. 1). In rodents, increased NPY synthesis in the ARC and enhanced storage of the peptide in PVN nerve terminals precede its release in the PVN to trigger and maintain episodic appetitive behavior during the dark phase [17, 18]. In ungulates, protracted peaks in NPY secretion in the hypothalamus are succeeded by spontaneous feeding episodes during the light phase [25]. In subhuman primates, NPY also plays a pivotal role in stimulation of appetite [10], and fluctuations in NPY signaling correlate with seasonal availability of energy resources [17, 25]. The important role of NPY in elicitation of appetitive behavior is also suggested by the observation that these anticipatory sequential events in synthesis, storage, and release can be desynchronized from the circadian clock and coupled to the timing of food availability. For example, a shift in timing or restriction of food availability to a few hours during the light phase rearranged the antecedent neurosecretory events in NPY signaling to precede the time of food availability [17, 18, 31]. These observations, together with the evidence that ultradian NPY secretion in the PVN is normally augmented in anticipation of meal time and during fasting, hypersecretion subsides as food is consumed [18], and blockade of NPY release or action suppressed food intake, are consistent with the notion that NPY is a physiological appetite transducer [14, 17, 20]. It is the intermittent waxing and waning of NPY discharge in the PVN that underlies propagation and termination of appetite [17, 18, 20].

The ARC and BS NPY subpopulations coexpress varied signaling molecules that cooperatively amplify NPY-induced appetite [17, 20]. The ARC NPY neurons express the orexigenic agouti-related peptide (AgrP) and γ-amino butyric acid (GABA) [17, 20] (Fig. 1). AgrP coreleased with NPY enhances feeding by antagonizing MC3/MC4 receptors located on NPY targets in the PVN and MC3 receptors on POMC-expressing neurons in the ARC [17, 27]. GABA coreleased with NPY exerts a similar dual synergistic action in the ARCmPVN axis mediated through GABAA receptors [30]. On the other hand, the BS subpopulation of neurons coexpress adrenergic neurotransmitters that, when coreleased with NPY in the ARC-mPVN axis and possibly in the DMH, amplify feeding through interplay between adrenergic and Y1/Y5 receptors [8, 17, 37]. In addition, the NPY network in the ARC-PVN axis communicates directly with four other orexigenic circuits in the hypothalamus—the opioid peptides and galanin-expressing neurons in the ARC and melaninconcentrating hormone (MCH)- and orexin-producing neurons in the lateral hypothalamus—to evoke the appetitive urge under varied physiological challenges [17, 20, 27] (Fig. 1). Thus, the convergence of evidence concurs with our proposal that the hypothalamic NPY circuit is an important conductor of the hypothalamic appetite-regulating orchestra composed of orexigenic and anorexigenic players of diverse chemical compositions [17, 20].

NPY Receptors

REGULATION OF NPY SIGNALING AND APPETITE

The cellular effects of NPY responsible for propagating the appetitive urge are mediated by disparate NPY receptor subtypes [9, 17] (Fig. 1). The appetitive drive is generated by direct activation of Y1/Y5 receptors located at target sites in the mPVN and DMH, simultaneous with restraining the release of the anorexigenic α-melanocyte-stimulating hormone (α-MSH) in the PVN through the activation of these very two receptor subtypes located on α-MSH, producing proopiomelanocortin (POMC) neurons in the ARC [9, 17, 20, 37]. Appetite termination, likewise, is conferred by a twoprong action of NPY within the ARC-mPVN axis [9, 17, 20]. First, reduction in the supply of releasable NPY in PVN nerve terminals is initiated by autoregulation via the inhibitory Y2 receptor subtype on the ARC NPYexpressing neurons. Second, diminution of NPY release locally in the ARC permits the reinstatement of the melanocortin restraint on NPY target sites in the mPVN (Fig. 1).

NPY and Cohorts

Distinct neural and hormonal feedback mechanisms impinge on the hypothalamic NPY system, not only to initiate and terminate episodic feeding but also to restrain feeding during prolonged intermeal intervals. Dark-phase feeding in rodents and light-phase feeding in ungulates is entrained to the circadian neural clock [17, 25, 31]. Apparently, the timely relay of signals from the circadian clock in the suprachiasmatic nucleus of the hypothalamus traverses the sub-PVN zone en route to the ARC NPY neurons to elicit antecedent increases in NPY synthesis and storage in the ARC-PVN axis and ultimately ultradian NPY release in the PVN to initiate feeding [17, 18]. Similarly, multiple feeding episodes during the light phase in ungulates result from the photoperiodic-clock-driven increase in NPY release in the hypothalamus [25]. As mentioned in the preceding section, shifts in the timing and scarcity of food supply can uncouple this tight temporal relationship of NPY

892 / Chapter 122 neurosecretory sequelae from the circadian-clockdriven appetitive behavior [17, 18, 31, 36]. The peripheral orexigenic hormonal signal, gastric ghrelin, probably coupled to the circadian clock or timing of meals, participates in initiating the sequential neurosecretory events in NPY-ergic signaling [17, 20, 21, 27] (Fig. 1). In addition, during food scarcity ghrelin hypersecretion independently augments NPY signaling to sustain appetite at high level [20, 21, 27]. On the other hand, termination of NPY release, due to the depletion of releasable NPY stores in the PVN NPY nerve terminals, is facilitated by peripheral anorexic hormonal signals, leptin secreted by adipocyte and PYY3-36 secreted by L cells of the distal intestine [18, 21, 27] (Fig. 1). Although the peripheral administration of each of these two peptides inhibits food intake in rodents by repressing NPY signaling, only leptin is currently considered as the primary physiological peripheral anorexigenic signal that imposes tonic restraint on NPY during the intermeal intervals [20, 21]. Experimental evidence suggests that PYY 3-36, together with leptin, may participate in termination of dark-phase feeding in rodents and humans by the activation of inhibitory Y2 receptors on NPY ARC neurons [20, 21, 27]. Despite the fact that central injection of pancreatic insulin inhibits food intake, coalescence of new evidence, and the observations that peripheral administration of insulin stimulates feeding and promotes adipogenesis and leptin hypersecretion during the postprandial interval, the role of insulin as a physiological peripheral anorexigenic signal appears unlikely [11, 20, 21]. Thus, evidence from various clinical and

animal paradigms is in keeping with the view that leptin is the primary peripheral signal involved, not only in terminating meals but also for exerting tonic restraint on feeding during the intermeal intervals via NPY regulation in the hypothalamus [20, 21]. Two circulating steroids have also been found to differentially impact feeding through NPY signaling in ARC-PVN axis [17]. Physiological levels of estrogens suppress feeding and weight by inhibiting NPY release in the PVN, a response mediated by estrogen receptors expressed on a subgroup of NPY neurons in the ARC [3, 17]. On the other hand, glucocorticoids stimulate feeding and promote weight gain by upregulating NPY signaling through the glucocorticoid receptor expressed on NPY neurons in the ARC [17, 22].

PATHOPHYSIOLOGY NPY, Eating Disorders, Obesity, and Metabolic Syndrome A detailed investigation of the synthesis, storage, and release of NPY in the ARC-PVN axis under a variety of paradigms that disturb the daily meal patterning unexpectedly revealed that both high abundance and low abundance of NPY in the hypothalamus engenders hyperphagia, increased weight gain, and fat accretion [12, 13, 17, 20, 21] (Fig. 2). Overactive NPY signaling, due either to increased NPY release in the PVN or to development of NPY receptor hypersensitivity to offset NPY deficit, promotes hyperphagia and weight gain.

NPY IN THE ARC-mPVN AXIS

VITY

FIGURE 2. Pathophysiological consequences of both high and low NPY abundance in the ARC-mPVN axis. With permission from [20].

Neuropeptide Y: A Conductor of the Appetite-Regulating Orchestra in the Hypothalamus / 893 The disruption of neurotransmission by either neurotoxins and lesions in the VMH and PVN or the neural transection to prevent bidirectional communication between the BS and hypothalamus produces a low abundance of NPY but upregulation of Y1 receptor response in the ARC-PVN axis, leading to hyperphagia and adiposity [7, 12, 17, 29] (Fig. 2). Similarly, high-fat diet-induced obesity is accompanied by diminished hypothalamic NPY levels, release, and increased sensitivity to NPY, the cluster of neural reorganizations also observed in various experimentally induced obesity models [20, 35]. Further, obesity produced either by gold thioglucose in mice or postnatal treatment of rats with monosodium glutamate is associated with substantially reduced NPY signaling in the ARC-PVN axis [17, 20]. On the other hand, complete loss of NPY in germline knockout mice, while maintaining daily food consumption, exhibit multiple defects [24, 28]. These include a decrease in dark-phase food intake and diminished hyperphagia induced by fasting and loss of insulin [2, 32, 33]. Obesity is also markedly attenuated after deletion of the NPY gene in leptin mutant mice [24, 28]. Thus, hypothalamic NPY is a pivotal component of neural derangements underlying hyperphagia. Other genetic approaches, including NPY receptor knockouts that partially or completely interrupt NPY signaling in discrete loci in the ARC-PVN axis, also support the notion that low abundance of NPY signaling is responsible for the progressive increase in adiposity in these mice [24, 28]. In aggregate, disruption in any one or more loci in the operation of NPY signaling in the ARCPVN axis leads gradually to adiposity and symptoms of metabolic syndrome, including hyperleptinemia, triglyceridemia, hyperglycemia, increased circulating free fatty acids, hyperinsulinemia, insulin resistance, and diabetes type 2 (Fig. 2). Seemingly, any excursion from the normal pattern of NPY synthesis, release, and receptor dynamics deranges the tightly regulated local homeostatic communication within the hypothalamus. A common outcome of these diverse reorganizations, advantageous for survival of the species, is relentless hyperphagia and increased energy storage in the form of fat but never anorexia, inanition, and morbidity [17, 20] (Fig. 2).

Subjugation of NPYergic Signaling to Control Appetite, Obesity, and Metabolic Syndrome A variety of approaches have been explored recently to repress NPY signaling in the hypothalamus in attempts to curb appetite on a long-term basis and, thereby, design therapies to suppress the age-related and high-calorie-induced increased rate of weight gain and obesity [18, 20]. Pharmacologic interventions focused on developing suitable Y1/Y5 receptor antago-

nists to inhibit endogenous NPY-induced feeding on a long-term basis have been unsuccessful because of poor bioavailability and brain penetration and nonselective action of these drugs [20]. The naturally occurring cytokines, interleukin-1 ciliary neurotrophic factor and leukemia inhibitory factor, suppress appetite for prolonged periods by decreasing both the availability of NPY at receptor sites and the abundance of Y1 receptors in the hypothalamus [15]. However, due to the diverse effects of cytokines and toxicity, the long-term efficacy of these naturally occurring anorectic signals is unlikely [15]. In contrast, the delivery of leptin, the naturally occurring anorectic signal and a member of the cytokine receptor family, to the hypothalamus with the aid of viral gene-transfer technology has been successful in suppressing appetite in both short-term and long-term experiments in rodents [19, 21]. A series of investigations has shown that enhanced leptin gene expression locally in the hypothalamus, transduced by a single central injection of nonimmunogenic and nonpathogenic recombinant adeno-associated virus vectorencoding leptin, concomitantly suppressed orexigenic NPY signaling and enhanced anorexigenic melanocortin signaling in the hypothalamus [19, 21]. The resultant tonic restraint on the ARN attenuated appetite, weight gain, and adiposity, and suppression of symptoms of metabolic syndrome, such as hyperinsulinemia, increased insulin sensitivity and hyperglycemia for the life of the rodents [19–21]. Success of the genetherapy paradigm has formulated a new understanding that long-term subjugation of NPY signaling to control appetite and weight with naturally occurring anorexigenic signals is feasible.

Acknowledgment This work was supported by grants from the National Institutes of Health (DK37273, NS32727, and HD08634).

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894 / Chapter 122 [5] Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1984;115:427–429. [6] Clark JT, Kalra PS, Kalra SP. Neuropeptide Y stimulates feeding but inhibits sexual behavior in rats. Endocrinology 1985;117:2435–2442. [7] Dube MG, Xu B, Kalra PS, Sninsky CA, Kalra SP. Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats. Brain Res 1999;816:38–46. [8] Everitt BJ, Hokfelt T. The coexistence of neuropeptide Y with other peptides and amines in the central nervous system. In: Everitt, BJ, Hokfelt, T, editors. Neuropeptide Y, Raven Press, New York 1989, 61–72. [9] Fetissov SO, Kopp J, Hokfelt T. Distribution of NPY receptors in the hypothalamus. Neuropeptides 2004;38:175–188. [10] Grove KL, Chen P, Koegler FH, Schiffmaker A, Susan Smith M, Cameron JL. Fasting activates neuropeptide Y neurons in the arcuate nucleus and the paraventricular nucleus in the rhesus macaque. Brain Res Mol Brain Res 2003;113:133–138. [11] Hidaka S, Yoshimatsu H, Kondou S, Oka K, Tsuruta Y, Sakino H, Itateyama E, Noguchi H, Himeno K, Okamoto K, Teshima Y, Okeda T, Sakata T. Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats. J Neurochem 2001;77:993–1000. [12] Kalra PS, Dube MG, Xu B, Farmerie WG, Kalra SP. Neuropeptide Y (NPY) Y1 receptor mRNA is upregulated in association with transient hyperphagia and body weight gain: evidence for a hypothalamic site for concurrent development of leptin resistance. J Neuroendocrinol 1998;10:43–49. [13] Kalra PS, Dube MG, Xu B, Kalra SP. Increased receptor sensitivity to neuropeptide Y in the hypothalamus may underlie transient hyperphagia and body weight gain. Regul Pept 1997;72:121–130. [14] Kalra S. Is neuropeptide Y a naturally occurring appetite transducer? Curr Opin Endocrinol Diabetes 1996;3:157–163. [15] Kalra SP. Circumventing leptin resistance for weight control. Proc Natl Acad Sci USA 2001;98:4279–4281. [16] Kalra SP, Dube MG, Kalra PS. Continuous intraventricular infusion of neuropeptide Y evokes episodic food intake in satiated female rats: effects of adrenalectomy and cholecystokinin. Peptides 1988;9:723–728. [17] Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 1999;20:68–100. [18] Kalra SP, Dube MG, Sahu A, Phelps CP, Kalra PS. Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc Natl Acad Sci USA 1991;88:10931–10935. [19] Kalra SP, Kalra PS. Keeping obesity and metabolic syndrome at bay with central leptin and cytokine gene therapy. In: Hamilton, I, editor. Current Medicinal Chemistry—Central Nervous System Agent, Bentham Science, San Francisco 2003;189–199. [20] Kalra SP, Kalra PS. NPY and cohorts in regulating appetite, obesity and metabolic syndrome: beneficial effects of gene therapy. Neuropeptides 2004;38:201–211. [21] Kalra SP, Kalra PS. Subjugation of hypothalamic NPY and cohorts with central leptin gene therapy alleviates dyslipidemia, insulin resistance, and obesity for life-time. In: Zukowska, Z, Feuerstein, G, editors. NPY Family of Peptides in Neurobiology, Cardiovascular and Metabolic Disorders: Genes, Functions and Therapeutics, Birhauser Verlag, Switerland 2006;157–169.

[22] Larsen PJ, Jessop DS, Chowdrey HS, Lightman SL, Mikkelsen JD. Chronic administration of glucocorticoids directly upregulates prepro-neuropeptide Y and Y1-receptor mRNA levels in the arcuate nucleus of the rat. J Neuroendocrinol 1994;6:153– 159. [23] Li BH, Xu B, Rowland NE, Kalra SP. c-fos expression in the rat brain following central administration of neuropeptide Y and effects of food consumption. Brain Res 1994;665:277–284. [24] Lin S, Boey D, Herzog H. NPY and Y receptors: lessons from transgenic and knockout models. Neuropeptides 2004;38:189– 200. [25] Mogi K, Yonezawa T, Chen DS, Li JY, Sawasaki T, Nishihara M. Correlation between spontaneous feeding behavior and neuropeptide Y profile in the third ventricular cerebrospinal fluid of goats. Domest Anim Endocrinol 2003;25:175–182. [26] Moran TH, Lee P, Ladenheim EE, Schwartz, GJ. Responsivity to NPY and melanocortins in obese OLETF rats lacking CCK-A receptors. Physiol Behav 2002;75:397–402. [27] Neary NM, Goldstone AP, Bloom SR. Appetite regulation: from the gut to the hypothalamus. Clin Endocrinol (Oxf) 2004;60:153– 160. [28] Pedrazzini T. Importance of NPY Y1 receptor-mediated pathways: assessment using NPY Y1 receptor knockouts. Neuropeptides 2004;38:267–275. [29] Pu S, Dube MG, Kalra SP, Kalra PS. Permanent interruption of information flow between hypothalamus and hindbrain produces obesity accompanied by a selective dark-phase hyperphagia and hyperinsulinemia. In Proceedings of the 33rd Annual Society for Neuroscience Meeting, New Orleans, LA, 2003, p. W-33, [Abstract 831.837]. [30] Pu S, Jain MR, Horvath TL, Diano S, Kalra PS, Kalra SP. Interactions between neuropeptide Y and gamma-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis. Endocrinology 1999;140:933–940. [31] Sahu A, White JD, Kalra PS, Kalra SP. Hypothalamic neuropeptide Y gene expression in rats on scheduled feeding regimen. Brain Res Mol Brain Res 1992;15:15–18. [32] Segal-Lieberman G, Trombly DJ, Juthani V, Wang X, MaratosFlier E. NPY ablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting. Am J Physiol Endocrinol Metab 2003;284:E1131–1139. [33] Sindelar DK, Palmiter RD, Woods SC, Schwartz MW. Attenuated feeding responses to circadian and palatability cues in mice lacking neuropeptide Y. Peptides 2005; May 26 [Epub ahead of print]. [34] Tatemoto K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA 1982;79:5485– 5489. [35] Wang H, Storlien LH, Huang XF. Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression. Am J Physiol Endocrinol Metab 2002;282: E1352–1359. [36] Xu B, Kalra PS, Farmerie WG, Kalra SP. Daily changes in hypothalamic gene expression of neuropeptide Y, galanin, proopiomelanocortin, and adipocyte leptin gene expression and secretion: effects of food restriction. Endocrinology 1999;140:2868–2875. [37] Yokosuka M, Kalra PS, Kalra SP. Inhibition of neuropeptide Y (NPY)-induced feeding and c-Fos response in magnocellular paraventricular nucleus by a NPY receptor antagonist: a site of NPY action. Endocrinology 1999;140:4494–4500.

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123 Hypothalamic Galanin and Ingestive Behavior: Relation to Dietary Fat, Alcohol, and Circulating Lipids SARAH F. LEIBOWITZ

STIMULATORY EFFECTS OF GAL INJECTION ON INGESTIVE BEHAVIOR

ABSTRACT Hypothalamic galanin (GAL), which stimulates feeding and alcohol intake, has distinct properties and functions. While being unresponsive to conditions of negative energy balance, GAL in the paraventricular nucleus (PVN) functions under conditions of positive energy balance related to dietary fat and alcohol. This peptide is stimulated by a high-fat diet, alcohol intake, and circulating lipids, and it is reduced by pharmacological blockade of fat oxidation. When injected into the PVN, GAL stimulates feeding and alcohol ingestion and causes stronger responses in rats consuming a fatrich diet. The opposite effect is produced by compounds that block GAL synthesis, release, or receptor function. This evidence supports the existence of nonhomeostatic, positive feedback circuits linking GAL to both fat and alcohol. These circuits produce excess consummatory behavior and through metabolic actions ultimately contribute to weight gain on fat-rich diets. Galanin (GAL) is a 29-amino-acid peptide that was originally isolated from the small intestine and is widely distributed throughout the hypothalamus and well conserved among species, including rats and humans [21]. It is expressed in a number of neuronal populations within the hypothalamus, including the paraventricular nucleus (PVN), arcuate nucleus (ARC), and other hypothalamic nuclei. A variety of evidence suggests that this peptide may have a role in feeding behavior and metabolism that influence long-term body weight regulation. Handbook of Biologically Active Peptides

Injection studies reveal a number of effects of GAL on behavioral responses, in addition to endocrine and metabolic systems [14, 21, 32, 33, 36, 37]. The first behavioral investigation with acute GAL injection revealed a stimulatory effect of this peptide on food intake in rats [30]. This effect is strongest when GAL is administered directly into the PVN, and the opposite effect, a suppression of food intake, is seen with injection of compounds that block GAL receptors or GAL synthesis. Robust and confirmed in multiple studies, this GAL response is found to be smaller and of shorter duration than that seen with injection of neuropeptide Y (NPY), another peptide found just 2 years earlier to stimulate feeding behavior [29, 37]. This provided the first evidence that these hypothalamic peptides with a stimulatory effect on food intake may function differently in their effects on energy balance. In addition to the magnitude of their feeding responses, GAL differs from NPY in the specific nature of its feeding response. Whereas GAL injection has little effect on an animal’s preference for the macronutrients, carbohydrate, fat, or protein, a variety of evidence links this peptide’s feeding-stimulatory response specifically to dietary fat [1, 5, 36, 40, 41, 45]. In contrast to NPY, the GAL-elicited feeding response is stronger and more prolonged in subjects maintained on a highfat (compared with a low-fat) diet or also in subgroups or strains of rats that naturally prefer fat, and it is greatly

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896 / Chapter 123 attenuated when fat is removed from the diet. It is suppressed by the pentapeptide enterostatin, which increases c-fos immunoreactivity in the PVN, and by the GAL antagonist M40, both of which reduce the consumption of fat. Further, repeated PVN injections of antisense oligonucleotides to GAL mRNA, which decrease GAL peptide levels, produce a marked reduction in fat ingestion. This is consistent with the idea that GAL-induced feeding has a specific relation to dietary fat and possibly to the large meal size and overeating generally associated with fat-rich foods. In addition to stimulating food intake, there is evidence that central GAL injection also increases the consumption of alcohol [39, 49]. This effect, which contrasts with the suppressive effect seen with NPY [56], occurs with GAL injection into the ventricles as well as directly into the PVN and both in the presence and absence of food and water. Also, the opposite effect, a suppression of alcohol intake, can be observed with injection of the GAL antagonist, M40. This stimulatory effect of GAL on alcohol intake may actually be linked to its feedingstimulatory action through mechanisms related to dietary fat and a fat-induced increase in circulating lipids. This is suggested by two additional findings showing, first, that alcohol consumption is closely associated with and affected by fat intake [9] and, second, that alcohol ingestion similar to dietary fat increases circulating levels of lipids [13].

EFFECTS PRODUCED BY MUTATIONS OF THE GAL GENE OR GAL RECEPTOR GENES To date, studies with GAL mutants indicate that mice with deletions of the GAL gene or GAL receptor genes or that overexpress GAL are able to defend their food intake and body weight when fed ad libitum on a low-fat diet or even fasted on this diet [15, 64]. Whereas this suggests that GAL may not have a major role under these conditions, this lack of response in GAL mutants, as with animals receiving chronic GAL injections, may be attributed to other factors. These may be the lack of sufficient dietary fat or the activation of compensatory mechanisms that help maintain nutrient homeostasis. Because GAL is believed to function specifically when dietary fat is in excess, GAL mutants may exhibit disturbances in feeding and body-fat accrual only when maintained on a high-fat diet or pure macronutrient diets that allow animals to express their natural preferences for fat as compared with carbohydrate or protein. Studies to date have examined only GAL mutants on standard lab chow diets that are low in fat. As for activation of compensatory mechanisms, the evidence obtained so far in GAL knockouts shows increased sensitivity to the inhibitory effects of chronic leptin

treatment on body weight and fat mass, suggesting that endogenous GAL plays a role in counteracting leptin’s actions [26].

EFFECTS OF CIRCULATING HORMONES AND DIETARY CONDITIONS ON ENDOGENOUS GAL Endogenous GAL is highly responsive to manipulations of hormones and diet, and its responses to these manipulations differ markedly from those seen with NPY, underscoring the functional differences between these peptides [6, 12, 29, 33, 36–38]. In contrast to NPY, which is highly responsive to conditions of negative energy balance, GAL is stimulated by conditions of positive energy balance related specifically to dietary fat. This is seen, for example, in their different responses to corticosterone, which rises when energy stores are low. This adrenal steroid has little impact on or transiently inhibits GAL gene expression in PVN neurons as well as the GAL-induced feeding response, whereas it potently stimulates endogenous NPY in the ARC as well as feeding stimulated by NPY injection. Conversely, the gonadal steroid estrogen, either alone or in combination with progesterone, is a potent stimulant of GAL but not of NPY. These two peptides also differ in their response to the adipose tissue hormone, leptin. This hormone has a strong inhibitory effect on NPY in the ARC but produces only a small suppression of PVN GAL mRNA and little or no change in GAL expression in the ARC, GAL release from hypothalamic explants, and GAL-induced corticotrophin-releasing factor release. This differential responsiveness of these peptides to leptin administration may be related to their different responses under physiological conditions involving a spontaneous rise or fall in leptin. That is, a deprivationinduced decrease in leptin has little effect on or suppresses GAL mRNA, whereas it markedly enhances NPY expression in the ARC. Also, an obesity-related rise in leptin suppresses NPY but stimulates PVN GAL mRNA. These clear differences between these orexigenic peptide systems suggest that they may function in different physiological states or conditions, as well as through distinct mechanisms. Marked differences between GAL and NPY are also seen in studies examining the effects on endogenous peptides of diet, as well as antimetabolites that affect nutrient metabolism. One main finding in a number of studies and animal models is that endogenous GAL gene expression and peptide production in the PVN, similar to the feeding response induced by exogenous GAL, are closely related to dietary fat [1, 32, 33, 37, 45]. They are positively correlated with the amount of fat consumed, stimulated by consumption of a mixed

Hypothalamic Galanin and Ingestive Behavior / 897 high-fat diet, and elevated to peak levels during the middle of the feeding cycle when preference for fat naturally rises. This is in contrast to NPY in the ARC, which is unaffected or reduced by fat consumption, is positively correlated with carbohydrate intake, and peaks 2 hours prior to onset of the nocturnal feeding cycle [37]. A major consequence of fat consumption is a rise in levels of circulating lipids, particularly triglycerides (TG) [3, 36, 52, 58]. This metabolite increases in direct proportion to the amount of fat consumed. This effect is evident under chronic feeding conditions, with hypertriglyceridemia a common trait in animals and humans that overeat a fat-rich diet. It is also seen in acute feeding paradigms, with TG levels rising higher and remaining elevated for longer periods after fat-rich meals. In recent studies, GAL in the PVN (but not NPY) is found to be strongly positively related to circulating levels of TG, in addition to their uptake and metabolism in muscle [36]. This relationship is robust, seen in different models of dietary obesity, even under conditions of acute exposure to a high-fat diet, for 1 day or even 2 hours, indicating its independence of changes in body fat and leptin. Injection of the fat emulsion, Intralipid, which raises TG levels, stimulates GAL expression and c-fos immunoreactivity in the PVN, but it has no effect on NPY expression or c-fos in the ARC [11]. Further, fat-preferring obesity-prone rats and mice compared with carbohydrate-preferring animals exhibit higher levels of circulating TG and elevated GAL in the PVN, whereas NPY is unaffected or reduced [2, 17, 18, 33, 36], and they are more responsive to the feedingstimulatory effects produced by GAL (but not NPY) injection [41]. A direct relationship between GAL and the metabolism of fat is suggested by evidence that pharmacological blockade of fat oxidation, while having no effect on NPY, reduces PVN GAL expression while suppressing fat intake and stimulates the expression of GALR1 in the PVN [20, 37]. Conversely, GAL shows little change after pharmacological blockade of glucose oxidation, which has a potent stimulatory effect on NPY. Thus, endogenous GAL appears to function specifically under dietary conditions rich in fat and in response to signals related to circulating TG and fat metabolism. This positive relation of PVN GAL to dietary fat, together with the finding that PVN GAL injection produces a stronger feeding response on fat-rich diets, suggests that this peptide may function within a nonhomeostatic, positive feedback loop involving a vicious cycle between GAL and fat. There is evidence that this bidirectional relationship between GAL and fat may be similarly seen for GAL in relation to alcohol. In addition to the stimulatory effect of GAL injection on the consumption of alcohol, recent studies demonstrate that GAL expression in the PVN (but not NPY in the ARC) is stimulated in rats injected

with 10% alcohol or induced to drink alcohol that raises blood levels to approximately 20 mg/dL [35]. Further, withdrawal from the opioid effects of alcohol ingestion, produced by naloxone injection, reverses this alcohol effect on GAL, significantly reducing peptide expression below baseline levels. These studies with GAL injection and measurements of endogenous GAL support the existence of a positive relationship between GAL and alcohol intake, which may contribute to the overconsumption of alcohol. This GAL-alcohol feedback loop, which may involve elevated TG levels induced by alcohol intake [13], may operate in a nonhomeostatic manner similar to that suggested for the GAL-fat feedback loop. In fact, these relationships linking GAL to circulating nutrients may actually interact, as suggested by evidence that fat intake stimulates the consumption of alcohol [9].

SITES OF ACTION AND NEURAL NETWORKS AFFECTED As already indicated, the feeding response induced by GAL injection is strongest when this peptide is administered directly into the PVN [33, 34, 37]. This feeding effect is most robust in the medial parvocellular division of this nucleus, and it sharply diminishes in magnitude as the injection site moves to the lateral PVN and then beyond this nucleus in all directions. The studies of endogenous GAL show that the stimulatory effect of dietary fat, Intralipid injection, or alcohol intake on GAL expression also occurs in the medial parvocellular division of the PVN, most particularly its anterior region [1, 11, 32, 36]. It is not detected in the caudolateral area PVN, where the magnocellular neurons are concentrated, nor is it seen in the ARC. These findings suggest that the PVN may be a key site involved in promoting ingestive behavior linked to the GAL-mediated, nonhomeostatic processes. The importance of this PVN GAL system in nonhomeostatic consummatory behavior is supported by evidence that GAL-induced feeding is attenuated by PVN lesions or GAL antagonists and that the consumption of a fat-rich diet or alcohol is reduced by local injections of compounds that block GAL synthesis, release, or receptor function [1, 32, 33, 39, 49]. This indicates that this nucleus contains the peptide neurons that respond to dietary fat or alcohol, as well as the peptide receptors that promote the overeating of palatable fat-rich foods or consumption of alcohol. Thus, GAL may function, in part, through local circuits involving cells and fibers of the peptide in the PVN that act as interneurons to regulate its own expression and release [31]. The PVN clearly differs from the ARC, which has a more prominent role in homeostatic control

898 / Chapter 123 mechanisms related to body fat stores and responds very differently to endocrine and dietary stimuli [29, 32, 37]. However, in the process of stimulating feeding, GAL in the PVN may act through neurocircuits involving the ARC. This is suggested by evidence that GAL stimulates the release of NPY in the ARC [6]. In addition, it has a direct inhibitory action on neurons in the ARC, perhaps acting through GALR1 receptors [47]. GAL may also function through opioid peptides in the hypothalamus, which, like GAL, stimulate feeding behavior most strongly on fat-rich diets.

RECEPTORS AND SIGNALING PATHWAYS MEDIATING GAL EFFECTS ON INGESTIVE BEHAVIOR There is evidence supporting the existence of several distinct GAL receptor subtypes, GALR1, GALR2, and GALR3, in the brain [21]. These receptors are found to activate different intracellular signal transduction pathways and, thus, are likely to mediate different physiological functions [7, 23, 59]. The promoter region of GALR1, but not GALR2 or GALR3, contains CRE-like binding sites, and GALR1 protein levels are upregulated in a cAMP/CREB-dependent manner that does not extend to GALR2 or GALR3. In contrast, GALR2, which is more widely distributed throughout the brain than GALR1, is coupled to G-proteins activating phospholipase C. There are several reports demonstrating that the feeding response induced by GAL is blocked by general GAL receptor antagonists, M35 or M40 [14, 33]. As for the specific receptor subtype involved in this effect, there is indirect evidence supporting a role for GALR1. This is suggested by a report indicating that a selective GALR2/GALR3 agonist fails to stimulate feeding behavior [59].

GAL IN RELATION TO OTHER PEPTIDERGIC AND AMINERGIC SYSTEMS CONTROLLING INGESTIVE BEHAVIOR Although evidence is limited, there are some studies suggesting the existence of additional neural substrates, relating GAL to opioid peptides, which are associated with dietary fat and possibly involved in fat-induced hyperphagia. Central injections of enkephalin (ENK) or dynorphin (DYN), similar to GAL, stimulate feeding behavior, and this effect is strongest in the PVN and also on diets rich in fat [34, 36, 66]. In addition, measurements of endogenous peptides in the PVN demonstrate that brief periods of fat consumption or injection of Intralipid, which elevates TG levels, stimulate ENK gene expression in the PVN [11]. The finding that

GAL-induced feeding is blocked by an antagonist of opioid receptors [5, 19] supports the idea that PVN GAL, in producing hyperphagia, acts through a local opioid circuit, even possibly within the same neurons that synthesize ENK. There is further evidence suggesting a role for mesolimbic dopamine (DA) in the overeating on a high-fat diet induced by PVN peptides. Whereas DA itself has only a weak to moderate effect in initiating feeding, this catecholamine is believed to have a specific function in producing arousal and enhancing an alreadyestablished feeding response [16, 63]. Its involvement specifically in fat-induced hyperphagia is supported by evidence that DA in the nucleus accumbens (NAc) is released by consumption of a high-fat diet [62]. Also, DA is stimulated by injection of GAL or the opioids [50, 55]. These hypothalamic peptides may act through projections coursing rostrally to DA terminal sites in the NAc or, more likely, caudally to DA neurons in the ventral tegmental area (VTA) [53]. This latter possibility is consistent with anatomical evidence linking the PVN and VTA [28]. These DA neurons in the VTA, which project to the NAc, control DA release in the shell of this nucleus, where it functions in relation to the novelty of a reward, and in the core where it relates to the directional/discriminative aspects of instrumental responding [16]. A role for this PVN-VTA-NAc neurocircuit in controlling food intake is supported by the findings that VTA injection of a general opioid antagonist blocks feeding induced by PVN injection of an opioid agonist [48] and that the GALR1 receptors possibly involved in the feeding response exist on DA neurons in the VTA [24]. Thus, DA signaling in the NAc, an important modulator of food-seeking behavior, is likely to have a role in mediating the stimulatory effects of PVN GAL and opioid peptides on the consumption of palatable, fat-rich diets. As suggested by evidence summarized here and the finding that alcohol stimulates NAc DA release [57], this catecholamine may function similarly in the alcohol consummatory response induced by GAL or opioid stimulation.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL CONSEQUENCES OF GAL’S ACTIONS There is extensive evidence in animals and humans showing that dietary fat produces hyperphagia, a 10– 15% increase in daily caloric intake [42, 58]. Alcohol ingestion also enhances caloric intake [25]. Fat-induced hyperphagia is evident in acute as well as chronic feeding paradigms, with meal size and total daily intake rising in direct proportion to the amount of fat in the diet. Dietary fat enhances the palatability of food,

Hypothalamic Galanin and Ingestive Behavior / 899 perhaps due to its caloric density and texture. It is also less satiating than protein and carbohydrate, as reflected by evidence that spontaneous fat-rich meals are followed by both shorter postmeal intervals and larger subsequent meals. In fact, preloads rich in fat compared with carbohydrate, even with equal palatability, are found to stimulate greater subsequent caloric intake regardless of whether the meals are presented in solid or liquid form, infused intragastrically, or given in a sham-feeding paradigm [43, 60, 61]. This may be due to the fact that fat ingestion is less tightly regulated than carbohydrate or protein intake, with lipids less readily oxidized and more easily stored in large fat depots [51]. The biological function of hyperphagia and nonhomeostatic, positive feedback signals on fat-rich diets may be required under conditions of scarce food supplies, when periods of gorging are essential to survival. The circulating TG levels that rise with fat and alcohol intake are known to interfere with normal behavioral and physiological processes, thereby promoting further consummatory behavior. They are associated with a variety of psychological disorders, such as stress-related overeating and certain eating disorders [10, 22, 46], and they contribute to complex metabolic disturbances along with cardiovascular disease and low-grade systemic inflammation [20, 27]. These lipids disturb the release, actions, and transport into the brain of hormones (e.g., leptin), which normally reduce food intake [4] and inhibit hypothalamic peptides that stimulate feeding. Direct support for the idea that elevated TG levels contribute to overeating on fat-rich diets is provided by evidence that food intake is significantly greater after injection of Intralipid compared with an equicaloric glucose solution [8]. Also, PVN GAL along with circulating TG is invariably increased in rats that show a preference for fat when given pure macronutrient diets or overeat calories on a mixed, fat-rich diet [32, 33, 36]. Thus, circulating lipids, through their effect on the brain and body, may be an important and active participant in the process of increasing caloric intake on diets rich in fat. A pathophysiological consequence of the overeating of fat is invariably an increase in weight gain and accrual of body fat [36, 58]. There is recent evidence demonstrating that chronic GAL stimulation in the PVN enhances weight gain along with daily food consumption, most predominantly under conditions when dietary fat is in excess. Chronic GAL injections in mice that are deficient in endogenous GAL are found to significantly increase daily caloric intake and body weight [26]. Also, whereas intracerebroventricular injections of GAL have little effect [54], a daily injection of GAL directly into the PVN of rats significantly stimulates food intake and body fat accrual and increases

leptin and TG levels in addition to lipoprotein lipase activity in adipose tissue [65]. Conversely, repeated PVN injections of antisense oligonucleotides to GAL mRNA markedly reduce fat intake and weight gain, in conjunction with a decline in PVN GAL levels [1, 33, 34]. The stimulatory effects of GAL on feeding and body weight occur specifically on a high-fat diet (45–60% fat) but not on a low-fat diet. The importance of fat in revealing GAL’s long-term effects is further evident in the finding that endogenous GAL is invariably stimulated in rats that become obese and have elevated TG on a high-fat diet [1, 17, 18, 36, 37, 58] but remains unaltered in rats that develop obesity with normal TG levels on a high-carbohydrate diet [17]. In light of evidence that GAL stimulates carbohydrate over fat metabolism and reduces energy expenditure [33, 65], it appears that the function of this peptide in states of positive energy balance related to fat is to compensate for the lack of dietary carbohydrate while counteracting the metabolic disturbances induced by excess fat consumption. Thus, PVN GAL may restore nutrient balance by stimulating food consumption to provide more dietary carbohydrate and also by stimulating muscle to increase their capacity to use the limited carbohydrate stores. In light of evidence that GAL and alcohol are also positively related, a further consequence of enhanced PVN GAL may be the stimulation of alcohol intake under conditions of excess dietary fat [9].

Acknowledgments The research described in this review was supported by U.S. Public Health Service grants MH 43422 (S.F.L.) and AA 12882 (S.F.L. and B.G. Hoebel of Princeton University). We thank Ms. Olga Karatayev and Valeriya Gaysinskaya for their invaluable help in the preparation of this manuscript.

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[56] Thiele TE, Sparta DR, Hayes DM, Fee JR. A role for neuropeptide Y in neurobiological responses to ethanol and drugs of abuse. Neuropeptides 2004;38(4):235–43. [57] Tuomainen P, Patsenka A, Hyytia P, Grinevich V, Kiianmaa K. Extracellular levels of dopamine in the nucleus accumbens in AA and ANA rats after reverse microdialysis of ethanol into the nucleus accumbens or ventral tegmental area. Alcohol 2003;29(2):117–24. [58] Wang J, Alexander JT, Zheng P, Yu HJ, Dourmashkin J, Leibowitz SF. Behavioral and endocrine traits of obesity-prone and obesity-resistant rats on macronutrient diets. Am J Physiol 1998;274:E1057–66. [59] Wang S, Ghibaudi L, Hashemi T, He C, Strader C, Bayne M et al. The GalR2 galanin receptor mediates galanin-induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation. FEBS Lett 1998;434(3):277–82. [60] Warwick ZS. Probing the causes of high-fat diet hyperphagia: a mechanistic and behavioral dissection. Neurosci Biobehav Rev 1996;20(1):155–61. [61] Warwick ZS, McGuire CM, Bowen KJ, Synowski SJ. Behavioral components of high-fat diet hyperphagia: meal size and postprandial satiety. Am J Physiol Regul Integr Comp Physiol 2000;278(1):R196–200. [62] Wilson C, Nomikos GG, Collu M, Fibiger HC. Dopaminergic correlates of motivated behavior: importance of drive. J Neuroscience 1995;15:5169–78. [63] Wise RA. Dopamine, learning and motivation. Nat Rev Neurosci 2004;5(6):483–94. [64] Wynick D, Bacon A. Targeted disruption of galanin: new insights from knock-out studies. Neuropeptides 2002;36(2–3):132–44. [65] Yun R, Dourmashkin JT, Hill JO, Gayles EC, Fried SK, Leibowitz SF. PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat. Peptides 2005; in press. [66] Zhang M, Gosnell BA, Kelley AE. Intake of high-fat food is selectively enhanced by mu opioid receptor stimulation within the nucleus accumbens. J Pharmacol Exp Ther 1998;285(2):908–14.

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124 Effects of Melanocortins on Ingestive Behavior PATRICIA RENE AND ROGER D. CONE

addition, the administration of SHU9119 alone caused an increase in nocturnal and fast-induced food intake, indicating that the endogenous melanocortin system exerts a tonic inhibitory effect on food intake via the centrally expressed MC3-R and MC4-R. The chronic administration of melanocortin agonists produces a sustained reduction in food intake, although tachyphylaxis is seen. Some evidence supports idea of melanocortins inducing satiety because melanocortin administration reduces meal size but not meal frequency, generally without inducing aversive effects [5, 108]. In contrast, the endogenous MC3-R/MC4-R antagonist AgRP has long-term effects on food intake [35, 65]. A low dose (0.01 nmol) of AgRP(83–132) can induce a potent hyperphagic response within 1 h of intracerebroventricular (ICV) administration and, remarkably, this effect persists an entire week. The acute effect of AgRP on food intake is mediated by antagonism/inverse agonism of the MC3/4-R and the long-term effect may be mediated by other mechanisms, potentially independent of the melanocortin pathway [35]. Moreover, the fact that a synthetic MC4-R antagonist lacks this long-term effect on food intake [55] supports a unique mechanism of AgRP action independent of α-MSH blockade. The observation of a small but measurable response of MC4-R(−/−) to centrally administred AgRP suggests that there are targets of AgRP action in addition to MC4-R [66]. The duration of the feeding response appears to be unique to AgRP among the other orexigenic substances. On a molar basis, AgRP(83–132) is less effective than NPY in its orexigenic effects during the first 24 h after administration. However, a single dose of NPY, even at very high doses, has little or no effect on food intake beyond 4 h. Similarly other orexigenic neuropeptides such as melanin-concentrating hormone, orexin, galanin, and opioids do not show such long-term effects on food intake. Such long-term effects are more likely

ABSTRACT The central melanocortin system is a novel peptidergic sytem involving one gene, proopiomelanocortin (POMC), encoding a family of peptide agonists of the melanocortin receptors and a second, agouti-related peptide (AgRP), that encodes a small protein antagonist. Together, POMC and AgRP are important regulators of ingestive behavior. Although mutations in the POMC gene are rare in humans, defects in melanocortin signaling due to heterozygous mutations in the MC4 account for up to 5% of severe early onset obesity. The melanocortin system appears to play a physiological role in both satiety signaling and adipostatic signaling under the control of leptin.

EFFECTS OF THE MELANOCORTINS ON FEEDING BEHAVIOR The central melanocortin system (Fig. 1), defined as hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neurons originating in the arcuate nucleus (ARC), brain-stem POMC neurons originating in the commissural nucleus of the solitary tract, and melanocortin-3 receptor (MC3-R) and melanocortin-4 receptor (MC4R) expressing target neurons, is a critical circuit in the regulation of body weight and composition [25, 47]. The central melanocortin peptides (described in the Brain Peptides Section of this book) were initially implicated in the regulation of energy homeostasis using a pharmacological approach. The central administration of melanocyte-stimulating hormone (MSH), or the MC3/4-R agonist melanotan II (MTII) reduced acute fasting-induced food intake in wild-type, Agouti, and leptin-deficient ob/ob mice, an effect that could be blocked by the MC3/4-R antagonist SHU9119 [25]. In Handbook of Biologically Active Peptides

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CNS LPB CEA

PVN

BST

AP NTS/POMC DMV

LH ARC POMC/AGRP (+) (–)

Adiposity Signals Leptin, Insulin

Vagal afferent Gut-released peptides CCK, Ghrelin, PYY 3-36

FIGURE 1. Central Melanocortin System.

to involve transcriptional changes and alterations in the synthesis of proteins by hypothalamic and extrahypothalamic neurons involved in the control of food intake, resulting in neural plasticity. AgRP might induce changes in protein synthesis leading to altered synaptic efficacy.

GENETIC STUDIES ON MELANOCORTIN ACTION The lethal yellow mouse heterozygous for the Ay allele is not only characterized by a yellow coat color but also by an obesity syndrome associated with hyperphagia, increased linear growth, and non-insulin-dependent diabetes [111]. Genetic analysis showed that Ay is in fact the result of a chromosomal rearrangement altering the normally restricted expression of agouti in the skin to ubiquitous ectopic expression of the protein, including the hypothalamus. Overexpression of agouti in multiple tissues is therefore the cause of the Ay phenotype. In vitro studies using recombinant mouse agouti protein proved that agouti is a potent melanocortin antagonist (in the nanomolar range) at MC1-R and MC4-R and a relatively weak antagonist at MC3-R and MC5-R [53, 63, 106]. This led to the hypothesis that the agouti obesity syndrome results from aberrant antagonism of the hypothalamic MC4-R [63]. It also led to the search for an agouti-like protein in the brain that might block signaling at central melanocortin receptors and stimulate food intake. Thus, the agouti-related protein (AgRP) gene was isolated based on its homology to agouti [82, 92] and has been characterized as equally potent in inhibiting signaling at MC3-R and MC4-R [30, 86, 109]. Along with the pharmacological studies already described, the disruption of MC4-R in mice causes an

obesity syndrome recapitulating the characteristic features of the agouti obesity syndrome, thus providing consistent evidence for a pivotal role of this receptor in the control of energy homeostasis [47]. This melanocortin obesity syndrome has been recapitulated in transgenic mice expressing agouti behind ubiquitous promoters [56], transgenic mice expressing AgRP behind the β-actin promoter [34, 82], and finally, in POMC knockout mice [110]. The relevance of those studies in rodents has been confirmed conclusively by the discovery of mutations in the POMC and MC4-R genes that are responsible for an inherited obesity syndrome in humans. The MC3-R knockout model has also provided clues to the respective role of each receptor in the control of energy homeostasis [13, 15]. The MC3-R (−/−) mouse is obese but without hyperphagia. This phenotype is attributed to a greater feeding efficiency. Also, mice lacking both MC3-R and MC4-R demonstrate greater obesity than caused by MC4-R deficiency alone. Thus, MC3-R serves a nonredundant role compared with MC4-R in the regulation of energy homeostasis. Evidence that the melanocortin obesity syndrome can occur in humans resulted from the astute recognition of an agouti-mouse-like syndrome in two families, resulting from null mutations in the proopiomelanocortin (POMC) gene [60]. These patients have a rare syndrome that includes adrenocorticotrophin hormone (ACTH) insufficiency, red hair, and obesity, resulting from the lack of ACTH peptide in the serum and a lack of α-MSH in skin and brain. These results demonstrated, for the first time, that the central melanocortin circuitry subserves energy homeostasis in humans as it does in the mouse. Shortly thereafter, heterozygous frameshift mutations in the human MC4R were reported, associated with nonsyndromic obesity in two separate families [101, 112]. Additional reports [28, 102] provide a clearer picture of the frequency and diversity of MC4R mutations and show that haploinsufficiency of the MC4R in humans is the most common monogenic cause of severe obesity at the present time, accounting for up to 5% of cases. Two recent reports provide a detailed clinical picture of the syndrome [10, 27]. Remarkably, the syndrome is virtually identical to that reported for the mouse [14, 26, 47], with increased adipose mass, increased linear growth and lean mass, hyperinsulinemia greater than that seen in matched obese controls, and severe hyperphagia. It is notable that most of the affected patients reported thus far have been heterologous for their respective mutations, suggesting that a partial reduction in MC4-R function resulting from haploinsufficiency can cause obesity, consistent with the rodent model results. Heterozygous mice with one MC4-R

Effects of Melanocortins on Ingestive Behavior / receptor allele deleted have an obese phenotype that is intermediate between that of the homozygous MC4-R knockout mouse and wild-type mouse [47]. Other reports have revealed that the intracellular retention of mutant receptors is a common characteristic. In general, intracellular retention of misfolded mutant receptors is one of the common reasons of loss-of-function phenotypes in G-protein-coupled receptors (GPCRs). Such observations have been found for the vasopressin receptor [72], the gonadotrophin-releasing hormone (GnRH) receptor [61], and several others. Naturally occurring mutations that severely impair NDP-MSH binding, AgRP binding, and cAMP generation generally occur within the region between the first transmembrane domain and the start of the fourth transmembrane domain. The functional consequences of this group of mutations are largely consistent with previous mutagenesis studies with both murine and human MC4-R, which have shown this region of the MC4-R to be critical for both α-MSH and AgRP binding [40]. The study of the functional properties of these mutant receptors has provided novel insights into the structure–function relationships of MC4-R, but has also revealed unexpected consequences of certain mutations. In particular a mutation in the C-terminal tail, I316S, appeared to selectively reduce receptor affinity for its natural agonist while the binding of its natural antagonist is preserved [113]. This selective loss of agonist affinity could be interpreted as the loss of constitutive activity displayed normally by the wild-type receptor, resulting in the maintenance of the mutant MC4-R in an inactive conformation that has a lower affinity for agonist. This suggests that the constitutive activity of MC4-R might be required to maintain normal body weight. Thus, the design and screening for agonists stabilizing the MC4-R at the cell surface may be important to treat obesity in general because many reports suggest that mutations associated with obesity show decreased MC4-R expression at the cell surface. Haploinsufficiency has been so far the preferential hypothesis for explaining the manifestation of an obese phenotype in heterozygous loss-of-function mutation carriers, but a D90N mutation with dominant-negative activity has also been reported [8]. Recently a novel concept supporting the notion that the constitutive activity of the MC4-R could play a role in the long-term energy balance in human has been proposed, based on obesity-associated mutations in the N-terminal domain of MC4-R that decrease its constitutive activity. Srinivasan et al. demonstrated that the MC4-R constitutive activity is provided by its N-terminal domain, which acts as a tethered intramolecular ligand for the receptor [94].

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FUNCTIONAL RESPONSE OF THE MELANOCORTINS TO DIFFERING METABOLIC AND FEEDING STATES Situated between the third ventricle and the median eminence, in which the portal vascular system functions to transport neuroendocrine-releasing factors to the anterior pituitary, the POMC and AgRP/NPY neurons of the arcuate nucleus and POMC neurons of the brain stem are uniquely positioned to sample factors in both blood and cerebrospinal fluid, as well as to receive vagal inputs in the case of the latter (Fig. 1). Two methods have been instrumental in characterizing the regulation of these neurons, the first being analysis of POMC/cocaine- and amphetamine-related transcript (CART) and AgRP/NPY gene expression, or c-fos expression as a marker of neuronal activation. POMC and NPY neurons were identified as two neurochemically defined sites of expression of the leptin receptor in the central nervous system (CNS) [36, 46], and sites of c-fos activation, in the case of the former, by both peripheral and central leptin administration [23]. Even before studies on the effect of the leptin gene product on NPY, results suggested that aberrant regulation of the orexigenic hypothalamic NPY was responsible for a component of the obesity syndrome in the leptin-deficient mouse [95]. This hypothesis was confirmed when it was demonstrated that crossing the Lepob/Lepob into the NPY −/− background reduced the obesity of the Lepob/Lepob by approximately 50% [24]. Fasting potently upregulates levels of AgRP and NPY mRNA (5–10 times) and modestly decreases POMC mRNA levels (20–50%), whereas leptin replacement to prefasting levels normalizes expression of these genes [70, 71, 89]. Like leptin, insulin also upregulates ARC NPY and AgRP expression, decreases ARC POMC gene expression, and activates ATP-sensitive K channels in what are likely to be ARC NPY neurons [93]. Although leptin and insulin activate different signal tranduction pathways, both hormones appear to activate PI3 kinase in the hypothalamus and to be dependent on PI3 kinase for their anorexigenic activity. A brain-specific insulinreceptor knockout mouse strain has been demonstrated to exhibit a mild obesity syndrome [12], arguing for a physiological role for central insulin action in energy homeostasis, and central insulin signaling also appears to be important for the regulation of glucose production by the liver [80]. Estrogen is another hormone that is likely to regulate POMC neurons in a manner relevant to energy homeostasis. The ARC and ventromedial hypothalamus (VMH) are major sites of estrogen receptor expression. POMC neurons are a presynaptic target of estrogens

906 / Chapter 124 [9] and provide synaptic input to GnRH neurons. Estrogens can also modulate the activity of G-protein– regulated inward-rectifying potassium (GIRK)-type K+ channels in POMC neurons [52]. Electrophysiological recordings in mouse hypothalamic slice preparations, in which the rare POMC/ CART and NPY/AgRP cell types could be identified with either POMC/green fluorescent protein (GFP) or NPY/GFP transgenes, have also provided a method for the direct analyses of the regulation of these cell types. Because both POMC and NPY/AgRP neurons comprise only a fraction of total arcuate neurons, a transgenic line, POMC–enhanced green fluorescent protein (EGFP), was created with the hypothalamic POMC promoter driving the expression of EGFP to identify POMC neurons [18]. Whole-cell patch clamp recordings and a loose-cell attached patch method with this system were then developed to characterize the responsiveness of these neurons to leptin and other agents [18, 19, 42, 48, 84]. All POMC neurons appear to exhibit spontaneous action potentials; leptin was found to inhibit the release of GABA from NPY terminals synapsing onto POMC neurons, and immunoelectronmicroscopy demonstrated that many POMC cell bodies were contacted by terminals containing both GABA and NPY [18]. In addition to characterization of leptin action, this preparation was also used to demonstrate that MC3-R is an inhibitory autoreceptor on the POMC/NPY circuit [18]. Remarkably, the levels of gene expression in the central melanocortin system reflect metabolic state and the electrical activity of the NPY/AgRP neurons do as well, and this property is maintained in hypothalamic slices used for electrophysiological studies [96]. The spontaneous firing rate of the NPY/AgRP neuron is generally quite low (0.5 Hz) and is activated threefold by fasting. The activation can be prevented by the administration of leptin to the fasted animal. The best evidence of a role for the hypothalamic melanocortin system in responding directly to acute satiety/hunger signals comes from results from ghrelin. Identified as an endogenous ligand for the growthhormone secretagog receptor (GHS-R) [57, 58], ghrelin is an acylated 28-amino-acid peptide predominantly secreted by the stomach and is regulated by the ingestion of nutrients [4, 20, 100], with potent effects on appetite [20, 98]. Ghrelin levels are markedly reduced with meal ingestion in both rodents and humans, but rebound to baseline before the next meal or increase after an overnight fast [20, 98, 100]. GHS-R has been demonstrated on arcuate NPY-containing neurons [107], and pharmacological doses of ghrelin injected peripherally or into the hypothalamus activate c-fos solely in arcuate NPY neurons in rats [44] and stimulate food intake and obesity, in part by stimulating NPY and AGRP expression [51, 77, 90], thus antagonizing leptin’s

anorexic effect [90]. On the cellular level, electrophysiological analyses suggest that ghrelin acts on the arcuate NPY/AgRP neurons to coordinately activate these orexigenic cells and inhibit the anorexigenic POMC cells by increasing GABA release onto them [19]. These properties strongly suggest that ghrelin is a candidate meal-initiating signal [20]. Stimulation of food intake by ghrelin administration is blocked by the administration of NPY/Y1 and Y5 antagonists [90] and reduced in the NPY −/− mouse. The administration of the melanocortin agonist MTII blocks further stimulation of weight gain by growth-hormone-releasing peptide-2, a synthetic GHS-R agonist, in the NPY −/− mouse [99]. Finally, peripheral administration of ghrelin activates c-fos expression only in arcuate NPY/ AgRP neurons, not in other hypothalamic or brain-stem sites [104], and ablation of the arcuate nucleus blocks the actions of ghrelin administration on feeding but not the elevation of growth hormone [97]. Despite activating only c-fos in arcuate NPY neurons, peripheral ghrelin may access the arcuate via vagal afferents because the growth hormone secretagog (GHS) receptor is expressed on vagal afferents, ghrelin suppresses firing of vagal nerves, and surgical or chemical vagotomy blocks the stimulation of feeding and c-fos activation in the arcuate by peripheral but not central ghrelin administration [22]. How can these findings be assembled into a single model? Peripheral ghrelin may largely suppress brain-stem satiety centers, thus explaining the lack of c-fos activation at these sites. The nucleus tractus solitarii (NTS) sends dense catecholaminergic projections to the arcuate, so although a convergence of ascending vagal afferent information arrives at the arcuate from both brain stem and intermediate hypothalamic sites, it is possible that NTS neurons inhibited by ghrelin synapse directly with NPY arcuate neurons, thus explaining the absence of other hypothalamic neurons activated by peripheral ghrelin. Recently, the idea has developed that the hypothalamus, in addition to responding to long-term adipostatic signals (leptin and insulin) and acute satiety/hunger hormones (ghrelin), may also depend on a direct nutrient sensor to regulate food intake and energy balance. This concept is essentially an extension of the glucostatic hypothesis, which postulated that meal initiation may be regulated, in part, by central sensing of blood glucose levels [67]. For example, recent results show that intracerebroventricular administration of oleic acid, a long-chain fatty acid, inhibits food intake and glucose production [79]. Furthermore, animals overfed a high-fat diet became resistant to the anorexigenic effects of oleic acid [73]. In addition, central inhibition of the rate-limiting enzyme of fatty acid oxidation, carnitine palmitoyltransferase-1, also has been found to decrease food intake and glucose production [81].

Effects of Melanocortins on Ingestive Behavior / These studies all involve ICV administration, which allows access to both forebrain and hindbrain sites, so additional work will be needed to determine the sites of action by which lipids or lipid oxidation may potentially act as an energy sensor. Another cellular energy sensor, the enzyme AMP kinase (AMPK), has also recently been demonstrated to play a role in energy homeostasis [2, 69]. Remarkably, the enzyme is not only a cellular energy sensor but, in certain hypothalamic nuclei, is inhibited by leptin, insulin, melanocortin agonists, high glucose, and refeeding. Furthermore, constitutively active AMPK blocks inhibition of feeding and weight loss induced by leptin. Although AMPK activity is inhibited in ARC and paraventricular nucleus (PVH) by refeeding [69], exercise in rats appears not to alter hypothalamic activity of the enzyme [3]. The expression and activity of AMPK in POMC-, AgRP-, or MC4-R-expressing neurons have not yet been reported, although the results suggested that AMPK levels in the PVH may be mediated by MC4R agonists. Thus far, one line of evidence shows directly that the melanocortin system has the potential of being a component of a hypothalamic nutrient sensor. POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits and, thus, are likely to have a functional ATP-sensitive K channel, a mechanism for linking neuronal activity to levels of cellular energy stores. Furthermore, the firing rate of the cells appears responsive to glucose concentrations in the bath [48]. Ultimately, formal proof of the physiological relevance of a nutrient sensor to hypothalamic control of energy homeostasis will require demonstrating that (1) hypothalamic systems such as the melanocortin system respond directly either to postprandial changes in blood nutrient levels or chronic changes resulting from obesity or starvation and (2) the degree of response is significant relative to responses to changes in leptin, ghrelin, insulin, glucocorticoids, cholecystokinin (CCK), and other hormones that have evolved to communicate aspects of metabolic state to the CNS.

NEUROANATOMY OF THE CENTRAL MELANOCORTIN SYSTEM The major site of POMC expression in the CNS originates in neurons of the arcuate nucleus (Fig. 1). In mice there are approximately 3000 such POMC-positive cells, most of which also express the anorectic peptide CART. The POMC/CART cell bodies are found throughout the rostrocaudal extent of the ARC and periarcuate area of the hypothalamus [49, 78, 105]. Proceeding from rostral to caudal, arcuate POMC cells send a dense bundle of fibers ventral to the anterior commissure to

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a number of nuclei in the septal region, including the bed nucleus of the stria terminalis and lateral septal nucleus, as well as to the nucleus accumbens in the caudate putamen. More caudally, fibers are seen projecting to the periventricular region of the thalamus and to the medial amygdala. Within the hypothalamus, the densest fibers project to the periventricular nucleus, the PVH, and the perifornical region, with some fibers seen in almost all hypothalamic regions. These hypothalamic POMC neurons also send two descending sets of projections to the brain stem, one via the periaqueductal gray and dorsomedial tegmentum that is thought to innervate the rostral NTS and lateral reticular nucleus (A5–C1 cell groups), and another via the ventral tegmental area believed to be the predominant descending bundle innervating the rostral NTS, ventrolateral medulla (A1 cell group) nucleus ambiguous, and spinal cord. Approximately one-half of the α-MSH immunoreactivity in the brain stem is thought to derive from hypothalamic POMC neurons; the other half derives from a smaller number (∼300) of POMC-expressing neurons in the brain stem [50, 83]. Although the POMC neurons of the arcuate are defined neurochemically by virtue of their expression of POMC and CART peptides, controversy continues to exist regarding neurotransmitters that may also be present in these cells. GABAergic currents have been identified in autaptic cultures of hypothalamic POMC neurons purified on the basis of fluorescence from transgenic mice expressing GFP under the control of the POMC promoter [43]. Furthermore, approximately one-third of ARC POMC neurons were demonstrated to express the mRNA for glutamic acid decarboxylase. However, the mRNA for the glutamate transporter is also expressed in some of these cells, implying some POMC neurons may be glutamatergic. However, POMC neurons have not yet been demonstrated to be functionally glutamatergic or GABAergic in a slice preparation. Because the arcuate NPY/AgRP neurons express the potent MC3-R/MC4-R antagonist, AgRP, they are also a critical component of the central melanocortin system. AgRP-immunoreactive fibers appear primarily in a subset of the same hypothalamic and septal brain regions that contain dense POMC innervation, with the densest fibers found innervating the PVH, dorsomedial hypothalamus (DMH), posterior hypothalamus, and septal regions around the anterior commissure [11, 39]. Within the rat hypothalamus, POMC fibers have a much wider distribution than AgRP, with moderate fiber density seen in just about every nucleus, with the possible exceptions of the VMH and supraoptic nucleus. AgRP-immunoreactive fibers are also notably absent in the rat from additional divisions of the neuraxis receiving POMC fibers, such as the rostral portion of the

908 / Chapter 124 brain stem, hippocampus, amygdala, corpus striatum, and olfactory cortex. Ultimately, understanding of the nature of the MC3-R- and MC4-R-expressing target cells is also essential to completing this anatomical picture of the central melanocortin system.

RECEPTORS AND SIGNALING PATHWAYS RESPONSIBLE FOR MELANOCORTIN ACTIONS ON INGESTIVE BEHAVIOR The main intracellular signal generated by stimulation of melanocortin receptors is cAMP production via coupling to adenylate cyclase, as has been shown in heterologous systems [32, 33, 74, 75, 85]. In accordance with these results, the membrane-permeable cAMP analog 8(4-chlorophenylthio)-cAMP increased the amplitude of the GABA current through a presynaptic action, mimicking the effect of α-MSH on a GABA interneuron and providing an indirect proof of the melanocortin-receptor coupling to the cAMP pathway in neuronal cells [17]. However, in contrast to the other melanocortin receptors, the MC3-R is also reported to be coupled to Gq, with modest activation of inositol 1,3,4-triphosphate turnover and induction of intracellular calcium in response to stimulation with α-MSH [59]. More recently, it has been reported that MC4-R can signal through mitogen-activated kinase (MAPK) in vitro and in rat hypothalamus [21]. These results show that in COS-1 cells transfected with MC3-R and MC4-R, MAPK is activated only in MC4-R COS-1-transfected cells after MTII exposure, despite the fact that cAMP was elevated in both cases. MTII is highly potent in activating MAPK in MC4-R-transfected cells, whereas even high saturating concentrations of MTII have no effect on this signaling pathway in cells transfected with MC3-R. An increase in the number of cells in the rat PVN that are immunoreactive for the phosphorylated MAPK (activated form) was also demonstrated when rats were treated with a dose of MTII that substantially decreases food intake. The activation of PVN neurons by MTII is consistent with reports of melanocortin agonist-induced Fos expression in this brain area [7, 68]. However, we cannot exclude that some of the activated MAPK observed in these experiments result from indirect activation. The demonstration that those PVN neurons that exhibit MTII-induced MAPK activation express MC4-R would support more convincingly the notion of a direct coupling between MC4-R and MAPK. Mountjoy et al. have also reported that α-MSH through MC4-R expressed in HEK293 cells can increase intracellular calcium concentration due to mobilization from intracellular stores by a mechanism that does not involve a transient increase in inositol triphosphate

(IP3), suggesting the involvement of an alternative second messenger [76]. A recent publication on the action of α-MSH on oxytocin release supports this. Sabatier et al. have measured a transient increase in intracellular calcium concentration in oxytocin neurons isolated from the supraoptic nucleus following α-MSH exposure. This response is still observed in low extracellular calcium concentrations, suggesting that this increase in intracellular calcium might reflect a mobilization from intracellular stores rather than entry via a voltage-gated channel [87]. Although α-MSH is regarded as the natural ligand for the MC4-R, other POMC-derived peptides are produced and secreted in the hypothalamus including desacetyl-α-MSH, β-MSH, ACTH, and β-lipotropin hormone (LPH). All of these peptides include the core His-Phe-Arg-Trp residues characteristic of melanocortin agonists and could therefore play physiological roles in the central melanocortin signaling pathway, given the high affinity of the MC4-R for all of these peptides. Although unique among the melanocortin receptors in its ability to respond to physiological levels of γ-MSH, the MC3-R does not show apparent selectivity in its response to stimulation by the various melanocortin peptides α-, β-, γ-MSH or ACTH [32, 85]. In contrast, γ-MSH is 100 times more potent at the MC3-R than MC4-R, therefore the most selective endogenous ligands of the MC3-R [32, 85]. AgRP and Agouti have been characterized as a competitive antagonists of α-MSH action at melanocortin receptors [63, 82], but further studies have defined AgRP as an inverse agonist as well [1]. AgRP has been shown to dose-dependently suppress the basal constitutive activity of MC4-R in tissue culture, a characteristic of an inverse agonist. This effect is blocked by SHU9119, a neutral receptor antagonist. These results provide experimental evidence that AgRP could act both independently or in concert with α-MSH. For instance, some neurons co-expressing the thyrotropin-releasing hormone (TRH) and MC4-R in the medial parvocellular part of the PVN are innervated by AgRP terminals but not by α-MSH nerve terminals. Hence, it is possible to imagine a regulation by AgRP in the absence of α-MSH. Another important level of regulation of this system might be the expression level of the MC4-R at the cell surface because it has been highlighted through the study of the MC4-R knockout mice and MC4-R mutations in humans that haploinsufficiency of the MC4-R can result in obesity [16, 28, 47]. Recently, a few reports have added some clues about which part in the Cterminus sequence of MC4-R is involved in its export to the plasma membrane [103] and in its desensitization and internalization [91]. However, the potential regulation of MC4-R expression at the plasma membrane

Effects of Melanocortins on Ingestive Behavior / suggests that specific regulators or partners might be involved, such as attractin-like protein [37].

INTERACTIONS OF THE MELANOCORTINS WITH OTHER PEPTIDERGIC/AMINERGIC SYSTEMS It is likely that the melanocortin system interacts at one or more levels with a wide variety of other circuits involved in energy homeostasis. Examples include circuits regulating motivated behaviors, circadian rhythm, reproductive function, and olfaction. Of course a fundamental aspect of this topic is the existence of the apparently asymmetrical innervation of POMC neurons by a dense array of NPY/AgRP fibers derived from adjacent ARC NPY/AgRP cells, as already discussed. As a consequence, the ARC POMC and NPY/AgRP cell bodies constitute a functional unit in which neural inputs to NPY/AgRP cells may rapidly affect both NPY/ AgRP and POMC neurons. Neural inputs to brain-stem POMC neurons remain largely uncharacterized, although these cells do receive direct inputs from vagal afferent nerves. In the hypothalamus, α-adrenergic and serotonergic inputs to the melanocortin system have been characterized. For example, POMC neurons express 5HT2C receptors and can be activated by dexfenfluramine, and the ability of this compound to inhibit food intake in the mouse is blocked by the melanocortin antagonist SHU9119 [42]. Serotonergic inputs to POMC neurons may also be important for the actions of cytokines on the melanocortin circuits during cachexia. Both ARC NPY/AgRP and POMC neurons also receive innervation from orexin neurons originating in the lateral hypothalamic area (LHA) [45], and tachykinin-immunoreactive fibers have been characterized synapsing onto ARC NPY/ Agrp cells as well. A number of functionally characterized cell groups have been demonstrated to be regulated by POMC and AgRP neurons, and a few of these are described here. α-MSH neurons innervate the TRH neurons in the PVH and centrally administered α-MSH prevents the drop in TRH expression induced by starvation [29] and can increase TSH levels in vivo and in hypothalamic slices [54]. Moreover, it has been demonstrated that AgRP inhibits the effect of leptin on TRH release from hypothalamic slices [82]. Taken together, these results suggest that engagement of the melanocortin pathway is required for leptin to regulate TRH expression. Furthermore, Harris et al. have shown by in situ hybridization that 48% of TRH neurons in the medial parvocellular division of the PVN co-express MC4-R [38]. However, only approximately 34% of medial parvocellular PVN neurons receive contacts by axon termi-

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nals containing α-MSH [29], and most neurons in the medial parvocellular subdivision of the PVN are innervated by axon terminals containing AgRP [62]. Evidence about a direct regulation of the TRH promoter by CREB after α-MSH exposure [38] and the increase of phospho-CREB (PCREB) immunoreactivity in the majority of TRH neurons after α-MSH ICV administration in fasted animals provided molecular insights into the regulation of TRH expression through MC4-R [88]. Similarly the percentage of corticotrophin-releasing hormone (CRH) neurons colocalizing with PCREB rose to 54% following α-MSH ICV infusion [88]. Moreover, 33% of CRH neurons in the ventromedial part of the parvocellular PVN contain MC4-R mRNA and central administration of the melanocortin agonist MTII stimulates CRH gene transcription [64]. Furthermore, blockade of CRH receptors with α-helical-CRH9-41 significantly attenuates MTII-induced anorexia. These results suggest that the central melanocortin system may modulate food intake in part through TRH and CRH neurons in the PVN. Interestingly, the ventral parvocellular subdivision of the PVN is involved in the regulation of the autonomic nervous system through descending projections to brain stem and the spinal cord targets. It is possible that the PCREB-responsive neurons in this subdivision of the PVN mediate the regulation of energy expenditure through the activation of the sympathetic outflow to brown fat, white fat, and muscle, regulating thermogenesis, lipolysis, and proteolysis, for example [6, 41].

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS The results described here clearly argue that the central melanocortin system plays a significant physiological role in ingestive behavior and energy homeostasis. The fact that up to 5% of individuals with severe early onset obesity have mutations in the coding region of the MC4-R, a small 1-kb exon, suggests that other mutations that might occur in the MC4-R promoter or in other proteins required for melanocortin signaling might ultimately account for a much larger percentage of cases of severe obesity. Although not discussed here, the central melanocortin system may also be involved in transmitting a component of the anorexic signal resulting from disease cachexia (for review, see [31]). The central melanocortin system thus has potential as a site for therapeutic intervention for disorders such as obesity, diabetes, and cachexia. However, to avoid potential side effects, it will also be important to learn more about the possible roles for these circuits in cardiovascular function, natriuresis, inflammation, sexual function, and other physiological actions attributed to the central melanocortin peptides.

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125 CART Peptide and Ingestive Behavior KELLY B. PHILPOT AND MICHAEL J. KUHAR

CART have been reported to increase feeding behavior in rats [1, 38]. Conversely, intranuclear injection of CART peptide into the nucleus accumbens shell (NA shell) has an inhibitory effect on feeding behavior [73]. Regardless of differing behavioral results, histochemical studies have reported that central administration of active CART peptide induces cFos expression in brain nuclei involved in feeding behavior and also increases circulating glucose, corticosterone, and oxytocin [67, 69].

ABSTRACT Cocaine- and amphetamine-regulated-transcript (CART) is an important neurohormone that inhibits feeding behaviors. The CART gene is conserved across many species, suggesting its relative importance in the regulation of basic ingestive behaviors. A variety of central and peripheral pathways may contribute to the satiety signal individually or in concert. Through rodent models of obesity, the generation of CART knockout (KO) mice and human genetic studies, CART peptide is now recognized as a novel drug target for potential regulation of ingestive behavior in humans.

STUDIES FROM GENETIC MANIPULATIONS OR MUTATIONS

DESCRIPTION OF CART PEPTIDE EFFECTS ON FEEDING BEHAVIOR

CART Deletion CART KO mice are prone to being phenotypically obese, especially later in life, and also are more susceptible to high-fat-diet-induced obesity compared to wild type [10, 49, 70]. Furthermore, the genetic deletion of CART suppresses glucose-stimulated insulin secretion, suggesting a role for CART in disorders of the pancreas [70].

Since the late 1990s, CART peptide has been recognized as an endogenous neuropeptide involved in ingestive behaviors and energy homeostasis [32, 33]. Central administration of CART peptide inhibits feeding in normal rats, and this inhibition is still apparent during starvation and neuropeptide Y (NPY)-induced feeding states [42, 45, 46]. Furthermore, the administration of a CART antibody increases feeding, suggesting that CART peptides exert an inhibitory tone on feeding behavior [42, 45, 46]. Chronic central infusion of CART peptide inhibits food intake and causes weight loss in lean and obese Zucker rats, as well as in lean and high-fat-fed obese rats [47, 55]. It should be noted, however, that disruption of locomotor activity was a concern in some studies after the central administration of CART [4, 5, 6, 47]. Although most reports conclude that intracerebroventricular (ICV) administration of CART results in anorectic effects, discrete hypothalamic injections of Handbook of Biologically Active Peptides

Targeted Gene Transfer In contrast, chronically increased CART peptide in the arcuate nucleus, resulting from targeted gene transfer, results in increased cumulative food intake and increased weight gain [38]. Likewise, fasting and food restriction of animals overexpressing CART cDNA is associated with increased weight loss compared with controls. These results contradict the generally accepted anorexigenic effects of CART peptide observed after ICV injection and suggest that the anorexigenic site in the brain has not yet been identified.

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914 / Chapter 125 FUNCTIONAL RESPONSE OF CART TO DIFFERING METABOLIC AND FEEDING STATES CART peptide exhibits a diurnal rhythm with levels being low in the morning and high in the evening in the nucleus accumbens, hypothalamus, amygdala, and serum in rats [65]. The diurnal rhythm is altered following 24-h fasting [65]. Fasting-induced decreases in hypothalamic CART mRNA were first identified in 1998 [42] and then again in 2005 with addition of the NA [8, 65, 73]. High-energy diets acutely, but not chronically, upregulate hypothalamic CART mRNA, theoretically to counteract the hypercaloric intake [8, 71]. Differing results have been reported following chronic exposure to high-

fat diets. CART mRNA and peptide expression is decreased in the arcuate nucleus in one account, whereas CART mRNA in the arcuate is increased in another [61, 62, 71]. The discrepancies may be due to differing diets and differing periods of exposure to each diet. This hypothesis is supported by the results from Davidowa et al. [21], stating that CART peptide’s effect on hypothalamic neuronal activity depends on the nutritional state of rats.

SITES OF ACTION AND NEURAL NETWORKS AFFECTED CART peptides affect body-weight regulation through a variety of possible mechanisms (Table 1). Some of

TABLE 1. Possible Mechanisms Involved in CART Peptide Body-Weight Regulation. Increasing Thermogenesis and Energy Expenditure Leptin activates POMC/CART neurons that innervate sympathetic preganglionic neurons in the thoracic spinal cord [29]. Leptin and CART levels are decreased in arcuate nucleus in animal models of anorexia [2, 3, 35, 53]. CART is increased in rats fed a high-fat diet and it is positively correlated with leptin levels [71]. CART-positive terminals are found in the intermediolateral cell column and other sympathetic autonomic nuclei [25, 26, 30, 39, 41] and induces cFos in oxytocin neurons [66, 67], which might synapse with preganglionic neurons of the intermediolateral cell column [7]. Repeated intra-arcuate CART peptide injections increase thermogenic response to BRL 35135 [38]. Mediation of Satiety Effects of Cholecystokinin (CCK) CART is expressed in vagal afferent neurons with CCKA receptors [15]. Meal intake induces cholecystokinin release in the gut, which stimulates vagal neurons expressing CCKA receptors. This suggests that CART might terminate food intake by acting as a mediator released from central vagal afferents after CCK stimulation. Regulation of Motor-pattern Generators That Control Ingestion Injections of CART into the fourth ventricle reduces liquid food and water intake, causing tremors and a conditioned taste aversion that might interfere with normal ingestion [5, 6]. Indeed, CART increases c-Fos-like immunoreactivity in the nucleus solitary tract and dorsolateral parabrachial nucleus [66], supporting the action of CART within the brainstem and mechanisms involving vagal satiety signals. Decreasing Rate of Gastric Emptying Injections of CART into the fourth ventricle decreases the rate of gastric emptying [9, 50]. CRH and cholecystokinin also reduce food intake and inhibit gastric emptying, and icv CART induces c-Fos expression in CRH-containing neurons in the PVN [67]. Other Issues The anatomical location of CART and the fact that CART is involved in the regulation of mesolimbic dopamine suggest a possible role for CART in the rewarding and reinforcing properties of foods [32, 33, 34, 52, 73]. Centrally administered CART increases circulating nonesterified fatty acid levels and decreases lipoprotein lipase activity in adipose tissue in rats on a high-fat diet [71], suggesting a mechanism of diminishing of lipid storage and promotion of lipid use. A missense mutation of the CART gene is associated with resting energy expenditure and obesity [22, 48]. CART variants in association with melanocortin-4 receptor (MC4R) mutations results in the lowest activity scores in a human population [48]. Peripheral CART may play a role in the regulation of food intake [70]. Identification of CART receptors will be important for therapeutic development.

CART Peptide and Ingestive Behavior / 915 these mechanisms may involve increasing thermogenesis and energy expenditure, mediation of satiety effects of cholecystokinin, regulation of motor-pattern generators that control ingestion, and decreasing the rate of gastric emptying [34].

RECEPTORS AND SIGNALING PATHWAYS RESPONSIBLE FOR INGESTIVE BEHAVIORS Since the discovery almost 10 years ago that CART peptides suppress ingestive behavior, academicians and pharmaceutical firms have avidly focused on identifying the CART receptor(s). Identifying a CART receptor(s) could potentially lead to designer drugs that could harness the ever-evolving obesity epidemic in developed countries, especially the United States. Until recently, little has been known about the CART receptor(s) and the signaling cascades responsible for ingestive behaviors. Although no receptor for CART has yet been identified, current research strongly suggests that the CART receptor(s) signals through G-protein-coupled receptors with negative and positive coupling to cAMP and mitogen-activated protein kinase (MAPK) signaling pathways, respectively [44, 56, 75]. Multiple CART receptors may exist. For example, differences of relative potencies for CART(55–102) and CART(62–102) have been shown in food consumption and other behaviors [14, 17, 60]. Furthermore, desensitization of the CART receptor(s) may occur in rats after chronic exposure to CART, because food consumption returns to baseline after 7 days [47]. This may be important in intracellular coupling of CART receptor(s) and functional consequences of an overactive system.

Glucocorticoids CART peptide is regulated by glucocorticoids at all levels of the hypothalamic-pituitary-adrenal (HPA) axis [12, 13, 58, 63, 64, 68]. CART mRNA and peptide in the paraventricular nucleus (PVN) and arcuate nucleus of the hypothalamus are reduced following adrenalectomy and fully restored after dexamethasone treatment or partially restored after corticosterone treatment [12, 13, 68]. Pituitary CART expression and release are regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids [58]. Furthermore, CART is partly localized to a subpopulation of corticotrophs containing adrenocorticotrophin hormone (ACTH) [58]. CART mRNA and peptide are located in the adrenal gland, and peripheral circulation of CART peptide is increased by corticosterone treatment [20, 40, 65]. Finally, serum CART peptide is decreased following adrenalectomy or administration of a glucocorticoid synthesis inhibitor, metyrapone [63, 64].

Endocannabinoids Although it seems counterintuitive, CART has recently been suggested as a downstream mediator of the orexigenic effect of endocannabinoids [43]. Endocannabinoids bind to CB1 receptors, which are colocalized with CART in the amygdala and co-expressed at the mRNA level in the PVN [19, 43]. Because the CB1 receptor is predominately presynaptic, it may inhibit the release of tonically active CART, decreasing the anorexigenic effect and increasing feeding behavior. The CB1 antagonist Rimonabant is currently being tested as an anti-obesity treatment due to its potent effect on weight reduction [37].

Other Systems INTERACTIONS WITH OTHER PEPTIDERGIC/AMINERGIC SYSTEMS Leptin CART is highly associated with modulating the actions of the anorectic peptide leptin [29, 42, 47]. The expression of CART mRNA is relatively low in mice with a disrupted leptin system, whereas the CART transcript is subsequently increased following leptin replacement [42]. Leptin activates hypothalamic CART neurons projecting to the spinal cord [29, 51], which may contribute to the thermogenesis and energy expenditure as well as the subsequent weight loss observed after leptin administration. Likewise, hypothalamic CART mRNA and serum leptin levels are dramatically decreased in a mouse model of anorexia (anx/anx) characterized by a much-reduced food intake [35].

Another potent orexigenic neuropeptide, orexin-A, is interconnected with the CART system. Orexininduced hyperphagia is mediated via orexin receptor -1 (OX1R) [54]. Orexin neurons innervate and receive innervations from CART/proopiomelanocortin (POMC) neurons in the arcuate nucleus [28]. Likewise, OX1R immunoreactivity is localized to these neurons, indicating cross-communication between these systems [11]. The transcript for an anorectic neuropeptide, thyroid-releasing hormone (TRH), is co-expressed with CART mRNA in cells of the PVN. Both of these neuropeptides inhibit depolarization-induced dopamine (DA) release in the hypothalamus, which may be a mechanism for blunting the rewarding effects of DA and, therefore, inhibiting DA-induced food intake [16].

916 / Chapter 125 PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS The human CART gene is a positional candidate for obesity because it maps to human chromosome 5q13– 14, which has been identified as a susceptibility locus for obesity [24, 27, 31]. Polymorphisms in the CART gene, as well as differing levels of CART peptide in the blood, have been implicated in diverse human populations with anorexia nervosa, hereditary obesity, variations in energy expenditure, hip-to-waist ratio, fasting plasma insulin levels, triglycerides, and even alcoholism, an overindulgent form of ingestive behavior [18, 22, 23, 36, 48, 57, 59, 72]. One of the most convincing human studies suggesting the CART gene plays a role in human obesity surfaced in the first couple years of this millennium [22]. A missense mutation in codon 34 of proCART was found to co-segregate with severe early-onset obesity over three generations of an obese Italian family and was not found in the control population [22]. The same mutation in a CART cDNA construct alters CART peptide levels in AtT20 cells, possibly through CART processing, sorting, and trafficking [23, 74]. These findings imply that changes in CART peptide levels occur in some tissues in members of an Italian family that carry the mutation and exhibit obesity. Further investigation of CART polymorphisms and circulating peptide levels in humans may lead to novel drug targets for the treatment of obesity, type 2 diabetes, and secondary complications related to these disorders, such as cardiovascular disease.

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[38] Kong, W.M., Stanley, S., Gardiner, J., Abbott, C., Murphy, K., Seth, A., Connoley, I., Ghatei, M., Stephens, D. and Bloom, S., A role for arcuate cocaine and amphetamine-regulated transcript in hyperphagia, thermogenesis, and cold adaptation. Faseb J 2003; 17:1688–90. [39] Koylu, E.O., Couceyro, P.R., Lambert, P.D. and Kuhar, M.J., Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol 1998; 391:115–32. [40] Koylu, E.O., Couceyro, P.R., Lambert, P.D., Ling, N.C., DeSouza, E.B. and Kuhar, M.J., Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J Neuroendocrinol 1997; 9:823–33. [41] Kozicz, T., Neurons colocalizing urocortin and cocaine and amphetamine-regulated transcript immunoreactivities are induced by acute lipopolysaccharide stress in the EdingerWestphal nucleus in the rat. Neuroscience 2003; 116:315–20. [42] Kristensen, P., Judge, M.E., Thim, L., Ribel, U., Christjansen, K.N., Wulff, B.S., Clausen, J.T., Jensen, P.B., Madsen, O.D., Vrang, N., Larsen, P.J. and Hastrup, S., Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 1998; 393:72–6. [43] Kunos, O.-H., Palkovits, Kuhar, Mackie Bannon, Endocannabinoids and appetite: possible interactions with other neurotransmitter systems. Symposium on the Cannabinoids, Burlington, VT, 2002. [44] Lakatos, A., Prinster, S., Vicentic, A., Hall, R.A. and Kuhar, M.J., Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors. Neurosci Lett 2005; 384:198–202. [45] Lambert, P.D., A role for novel CART peptide fragments in the central control of food intake. Neuropeptides 1997; 31:620–1. [46] Lambert, P.D., Couceyro, P.R., McGirr, K.M., Dall Vechia, S.E., Smith, Y. and Kuhar, M.J., CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 1998; 29:293–8. [47] Larsen, P.J., Vrang, N., Petersen, P.C. and Kristensen, P., Chronic intracerebroventricular administration of recombinant CART(42–89) peptide inhibits and causes weight loss in lean and obese Zucker (fa/fa) rats. Obes Res 2000; 8:590–6. [48] Loos, R.J., Rankinen, T., Tremblay, A., Perusse, L., Chagnon, Y. and Bouchard, C., Melanocortin-4 receptor gene and physical activity in the Quebec Family Study. Int J Obes Relat Metab Disord 2005; 29:420–8. [49] Moffett, H., Asnicar, Kuhar, CART knockout mice respond differently to cocaine and novelty than wild-type controls. CPDD, Orlando, FL, 2005. [50] Okumura, T., Yamada, H., Motomura, W. and Kohgo, Y., Cocaine-amphetamine-regulated transcript (CART) acts in the central nervous system to inhibit gastric acid secretion via brain corticotropin-releasing factor system. Endocrinology 2000; 141: 2854–60. [51] Oliveira, V.X., Jr., Fazio, M.A., Miranda, M.T., da Silva, J.M., Bittencourt, J.C., Elias, C.F. and Miranda, A., Leptin fragments induce Fos immunoreactivity in rat hypothalamus. Regul Pept 2005; 127:123–32. [52] Philpot, K.B., Dallvechia-Adams S, Smith Y, Kuhar M.J., A CARTcontaining projection from the lateral hypothalamus to the ventral tegmental area. Neuroscience 2005; to appear. [53] Robson, A.J., Rousseau, K., Loudon, A.S. and Ebling, F.J., Cocaine and amphetamine-regulated transcript mRNA regulation in the hypothalamus in lean and obese rodents. J Neuroendocrinol 2002; 14:697–709. [54] Rodgers, R.J., Halford, J.C., Nunes de Souza, R.L., Canto de Souza, A.L., Piper, D.C., Arch, J.R., Upton, N., Porter, R.A.,

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126 Orexins and Opioids in Feeding Behavior CATHERINE M. KOTZ, CHARLES J. BILLINGTON, AND ALLEN S. LEVINE

willing to work for sweet-pellet delivery and thus are motivated to eat after OxA [77]. However, 24-h fooddeprived rats complete more cycles of lever pressing than OxA treated rats, indicating that motivation to eat is greater after 24-h food deprivation than after stimulation with OxA [77]. OxA action has also been considered within the context of the well-described sequence of feeding behavior: eating, grooming, and resting. Both OX1R antagonist and OxA antibody inhibit normal feeding [88], and OxA increases face washing, grooming, searching, and burrowing [29], all behaviors associated with various aspects of normal feeding behavior. Mealpattern analyses show that OxA delays the onset of satiety, the transition period between eating and resting. The overall effect is to increase meal length rather than meal number, which, according to this analysis, indicates that OxA prolongs rather than initiates feeding [71]. However, when stimulated with OxA, rats clearly initiate meals because OxA treated rats have increased eating attempts (Fig. 1) and consume significantly more food than controls. One difference between our and others’ studies is that in our study OxA injections were given during the day, when animals are not normally eating; therefore, the effects on meal initiation may be more robust. Recent evidence strongly implicates elevated orexin neuron activity in meal initiation [30, 55]. Orexins are also associated with enhanced wakefulness and activity [88], leading to speculation that OxAinduced feeding may be a by-product of enhanced activity. Our operant studies refute this idea [77]. An important point to consider is that OxA-induced feeding occurs after injection into only some brain sites at certain times of day, whereas OxA-induced activity occurs after injection into all brain areas tested to date ([35, 39, 78] and unpublished data) and throughout the light-dark cycle, indicating that OxA-induced

ABSTRACT Orexins and opioids consistently stimulate food intake and interact to elicit feeding; blockade of one affects the response to the other. Most important, the strength of the feeding response for each depends on the brain site of administration. Together, these indicate a network model of feeding behavior in which both orexins and opioids participate. This review first presents evidence for orexins and opioids separately, establishing their role in feeding behavior. The final section discusses the interaction between opioids and orexins. Behavioral and neurobiological evidence demonstrates that orexins and opioids are an important part of the neural network that operates to maintain natural feeding behavior.

EFFECT OF OREXINS AND OPIOIDS ON FEEDING BEHAVIOR Orexins Orexins A and B (or hypocretins) acting at their two receptors (OX1R and OX2R) have well-documented effects on feeding behavior. Orexin A (OxA) increases feeding after injection into the ventricles and many specific brain sites [17, 39, 75, 76, 78, 97]. Orexin B has less consistent effects on feeding behavior, and thus most feeding studies are focused on OxA. The amount of food intake elicited by OxA is more modest than some neuromodulators of feeding behavior, such as neuropeptide Y (NPY), but is on par with that produced by central opioids. Daytime injections of OxA elicit more robust feeding responses than nighttime injections in nocturnally feeding rats [39, 79]. Operant studies using a progressive ratio of 5, a fairly difficult operant schedule, indicate that OxA treated rats are Handbook of Biologically Active Peptides

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FIGURE 1. OxA stimulates number of eating attempts and food intake, 1 h postinjection. Naloxone (nlx, 5 mg/kg i.p.) blocks the effect of orexin on food intake (B), but not on the number of eating attempts (A). *P < .001.

feeding, wakefulness, and activity are not inextricably linked but, rather, that OxA function depends on brain site. In a recent study of OxA effects in the nucleus accumbens shell (sNAcc), low doses of OxA elicited feeding but not activity, and at higher doses the time course of feeding and activity effects were incongruous, suggesting that OxA-induced feeding and activity occur via different circuitry [78]. OxA-induced waking may be a component of normal feeding behavior. Ventricular OxA infusion increases waking and feeding behavior and enhanced orexin neuron activity is associated with increased waking, although not all OxA-induced waking is associated with feeding behavior [18]. Orexin neurons are involved in waking associated with food-anticipatory behavior, which is thought to be mediated by histamine neurons in the tuberomammillary nucleus (TMN): OxA excites histaminergic TMN neurons and increases the firing rate of TMN neurons [88]; TMN histamine neuron activity is high during waking and low during sleep [84]; there is high cfos-ir in TMN neurons during meal anticipation [30]; an attenuation of food anticipatory activity is observed in orexin knockout mice [55]; and increased locomotor activity prior to meal anticipation coincides with activation of TMN neurons, the consummatory phase and activation of the lateral hypothalamic (LH) and perifornical (PFH) areas, where orexin neurons reside. Several orexigenic neuropeptides activate orexin neurons, suggesting that orexin neuron activation may be common to many forms of feeding behavior and OxA action may link wakefulness and feeding behavior.

Opioids Endogenous opioids and their receptors affect intake of a variety of foods and liquids [4, 44]. Opioid receptors are distributed in many brain sites, including those known to affect reward and food intake, and opioids appear to be particularly important in reward-related feeding [68]. For example, the opioid receptor antagonist naloxone decreases sucrose solution intake much more effectively than water intake [46]. Also, the hedonic property of sucrose solutions, as reflected by taste-reactivity tests, is decreased in rats treated with naltrexone, an opioid antagonist [66]. Such a decrease in the hedonic property of sucrose was also noted by humans given oral naltrexone [3]. Injection of opioid agonists increases the intake of sweet solutions. Although rats and humans “report” a decrease in hedonic properties of sweet solutions following blockade of opioid receptors, their ability to discriminate sweet taste is not affected [63]. Many investigators have suggested that opioids are involved in macronutrient intake, particularly in fat ingestion. Opioid ligands selectively increase or decrease intake of high-fat diets when presented concurrently with high-carbohydrate diets [53]. Injection of the muopioid ligand DAMGO into the sNAcc increases intake of a high-fat and a high-carbohydrate diet when they are presented individually [95]. However, when these diets were presented concurrently, DAMGO-injected rats increased intake of the high-fat diet but not the high-carbohydrate diet, and this effect was blunted by the injection of naloxone into the sNAcc. Gosnell et al.

Orexins and Opioids in Feeding Behavior / 921 found that a rat’s baseline preference for fat or carbohydrate altered the apparent selectively of high fat diets after morphine injection [26]. When rats were grouped into carbohydrate and fat preferrers based on their baseline intakes, morphine affected the intake of the preferred diets rather than intake of the high-fat versus the high-carbohydrate diet. We have also found that naloxone decreases the intake of preferred diets much more effectively than nonpreferred diets. For example, the injection of naloxone at only 0.01 mg/kg decreased intake of a preferred diet, whereas doses as high as 3 mg/kg did not alter the intake of a nonpreferred diet [24]. An area of some confusion relates to the effect of opioids on the development of preferences for sweet diets. We found that peripheral infusion of naltrexone via an osmotic minipump inhibited the development of a preference for a sweet diet in rats given a choice between a sweet (sucrose) and bland (starch) following a 10-day period with only the bland diet [45]. Those infused with vehicle clearly preferred the high-sucrose diet to the starch diet. Also Lynch noted that naloxone inhibited the development of a preference for a saccharin solution in rats [50]. However, Bodnar and Sclafani’s laboratories found that naltrexone failed to alter the acquisition or the expression of a conditioned flavor preference in sham-fed rats given a flavor paired with saccharin or sucrose solutions [1, 94]. Naltrexone did, however, decrease the intake of the solution containing the flavor paired with the sucrose solution. They also reported that naltrexone decreased the intake of, but not the acquisition or expression of, a preference associated with an intragastric infusion of a 16% sucrose solution. However, these conditioning studies were dependent on conditioning and learning, behaviors that are influenced by opioid receptors [9].

STUDIES FROM GENETIC MANIPULATIONS OR MUTATIONS Orexins Humans and dogs with narcolepsy were shown to be deficient in orexin neurons and OX2R, respectively [88]. Genetic knockout studies have repeated this finding because mice lacking prepro-orexin are also narcoleptic. However, narcolepsy is not the only phenotypic difference. Humans with narcolepsy have an increased body-mass index (BMI) and prevalence of obesity [15], and in mice, postnatal ablation of orexin neurons with ataxin toxin results in reduced locomotor activity and a late-onset obese phenotype, in spite of a 30% reduction in nighttime feeding (27). These results suggest that the reduced locomotor activity in these

mice has a proportionally more significant effect on body weight than the reduced food intake. Hypophagia and decreased food anticipatory activity are also observed in orexin knockout mice [11, 55, 88]. Orexin co-localizes with dynorphin and glutamate, and the consequences of orexin neuron ablation may be more profound than lack of prepro-orexin; some of the effects may be due to loss of other neurotransmitters. Obesity in orexin neuron-ablated mice is influenced by genetic background and environmental factors [27]. These studies have greatly aided progress in understanding orexin function, but these issues have limited the ability to interpret functional significance.

Opioids Few studies have been done with feeding in animals with genetic modifications of the opioid system. In one study, mu-receptor-deficient CXBK mice preferred saccharin less than intact mice [93]. Mice lacking betaendorphin and enkephalin work less for food on a progressive ratio schedule than wild-type mice [28]. Also, mu-opioid-receptor knockout mice have diminished food-anticipatory activity compared to wild-type mice, as measured by schedule-induced running wheel activity prior to food intake [31]. Thus, there is limited evidence from genetically modified mice indicating that opioids are involved in reward and feeding.

FUNCTIONAL RESPONSE OF OREXINS AND OPIOIDS TO METABOLIC AND FEEDING STATES Orexins Several studies demonstrate that orexin neurons respond to metabolic challenges. Orexin neurons are activated (as measured by cfos-immunoreactivity, cfosir) by 48-h food deprivation, food restriction, and dramatically lowered blood glucose concentrations. More specifically, orexin neurons are stimulated by low blood glucose and inhibited by high blood glucose [8, 41]. Insulin administration and glucopenia induced by 2deoxyglucose (2DG) increases cfos-ir in orexin neurons [6, 8]. Food deprivation also increases central orexin mRNA and peptide and OXR mRNA [6, 8, 49]. Paradoxically, orexin gene expression is elevated in states of hypertriglyceridemia [10, 65, 90], although one study showed no effect of high-fat diet feeding on orexin or OX1R gene expression [87]. That orexin signaling may be elevated during metabolic challenge is supported by studies testing the feeding response to OxA during food restriction and food deprivation. Sensitivity to OxA is increased in food-restricted rats [81] and with

922 / Chapter 126 increasing levels of food deprivation (e.g., 3, 6, 12, and 24 h) [80]. Further, the co-administration of subthreshold doses of 2DG and OxA enhanced feeding. Together, these suggest that OxA signaling and responsivity are elevated during nutritional duress, implicating OxA in feeding associated with maintaining homeostasis.

Opioids Opioid circuitry seems to be affected by the intake of palatable foods and by food deprivation. Consumption of sweet food causes the release of beta-endorphin in the hypothalamus of nondeprived rats [54]. Also, palatability-induced overeating results in an increase in the gene expression of dynorphin in the hypothalamic arcuate nucleus (ARC) as well as increased dynorphin levels in the hypothalamic paraventricular nucleus (PVN) [86]. When rats are exposed to a highly palatable diet and then switched to chow, both proopiomelanocortin (POMC) and dynorphin mRNA levels decrease in the ARC [43]. Restricted consumption of a palatable diet also decreases striatal enkephalin gene expression, resembling that seen during energy deprivation [33]. Restricting the intake of a palatable food also results in a decrease in gene expression of opioids, again mi-micking that seen with food restriction or deprivation [86]. Chronic exposure to glucose solution increases mu-opioid-receptor occupation in many brain regions, including the sNAcc [14]. Thus, manipulation of diet affects opioid circuits, as reflected by gene expression, opioid peptide levels, and ligand binding.

60, 75, 78, 97]. Ineffective sites include VMH, preoptic area, central nucleus of the amygdala (CeA), ventral tegmental area (VTA), and caudal nucleus of the solitary tract (cNTS) [17, 75, 78]. The magnitude of the feeding response varies by site. As discussed in other sections, OxA pathways interact with several other components of the neural network associated with feeding, including leptin, NPY, opioid, ghrelin, glucagon-like peptide, melanocortin, and urocortin-related pathways. OX1R and OX2R are G-protein-coupled receptors widely distributed throughout the brain [73, 82]. The highest hypothalamic concentrations of OX1R and OX2R are in VMH and PVN, respectively [82], although many other hypothalamic and extrahypothalamic regions also contain OXR mRNA [2, 82]. Binding studies indicate that OX1R is selective for OxA, whereas OX2R binds with similar affinity to both orexins [73]. Functionality of OXR subtypes is undefined, but OX2R is more strongly associated with wakefulness because it is the lack of this receptor that leads to narcolepsy in dogs [48]. Orexin B binds only OX2R and does not consistently stimulate feeding, whereas the stimulation of OX1R reliably induces feeding. Interestingly, blockade of OX1R is more effective in reducing food intake and body weight in an animal model of obesity compared with obesity-resistant strains [87]. However, blockade of OX1R also affects the locomotor effects of OxA [78]. Thus, receptor function is incompletely defined and probably depends on brain location, because each brain site has unique sets of neuronal populations, inputs, and efferent connections.

Opioids OxA AND OPIOID LIGANDS AND RECEPTORS: SITES OF ACTION AND NEURAL NETWORKS AFFECTED Orexins Orexins A and B are coded from the same prepromRNA, which is limited to the LH, PFH, dorsomedial (DMH), and posterior hypothalamic areas [16, 69, 73] (see also hypocretin chapter). Orexin neurons project throughout the hypothalamus, including PVN, ARC, LH, PFH, and ventromedial hypothalamus (VMH), as well as extrahypothalamic sites [69], including TMN, which is linked to wakefulness and activity associated with meal anticipation. Orexin peptides are abundant in orexin projection sites, in both hypothalamic and extrahypothalamic regions [61]. OxA injected into many of these projection sites affects feeding behavior [17, 75, 78]. Areas in which OxA administration increases food intake include the rostral LH area, DMH, ARC, PVN, PFH, sNAcc, and dorsal vagal complex [17,

Opioids are known to be active in a number of specific brain sites, and opioid site-specific stimulation studies, using microinjection, have been informative. Kelley and colleagues have shown that stimulation of DAMGO injection into the sNAcc increased the intake of a high-fat diet and also increased c-fos-ir in LH and DMH, midbrain, and gustatory-visceral relay areas [32]. Injection of muscimol, a GABAA agonist, into sNAcc projection sites, including DMH, LH, VTA, or intermediate NTS, blocked sNAcc DAMGO-induced feeding [32], suggesting that sNAcc DAMGO-stimulated efferent signals that are important to feeding behavior require functional activity at these sites. Many GABA neurons in the sNAcc and ventral striatum contain enkephalin and beta-endorphin and express mu-, delta-, and kappa-opioid receptors. Perhaps food consumption resulting from the activation of the sNAcc opioid system involves downstream regions that integrate motivational, autonomic, and metabolic aspects of ingestive behavior. MacDonald et al. found that feeding induced by unilateral injection of DAMGO into VTA was dose-

Orexins and Opioids in Feeding Behavior / 923 dependently decreased by bilateral injections of the dopamine receptor 1 antagonist SCH 23390 in sNAcc [51]. When DAMGO was injected into sNAcc, its orexigenic effects were diminished by SCH 23390 injected into VTA but not by administration of a dopamine receptor 2 antagonist. Injections of SCH 23390 into sNAcc inhibited feeding induced by intrasNAcc DAMGO but not by a delta-opioid agonist. Together, these results indicate the presence of an opioid-dopamine interaction important to feeding within sNAcc and VTA. Opioids in CeA, hypothalamus, and hindbrain also affect feeding. By injection of DAMGO into one site and naltrexone into a second site, the connections between these sites, including PVN, CeA, NTS, sNAcc, and VTA, have been investigated. In general, DAMGOinduced feeding in one site is blocked by naltrexone injection into a distant site. Pairs of sites tested include (1) CeA and NTS [22], (2) PVN and VTA [70], (3) sNAcc and CeA [34], (4) PVN and CeA [21], and (5) sNAcc and VTA [52]. The effect was bidirectional in most cases because DAMGO injection in all sites enhanced feeding and opioid-receptor blockade in the corresponding site pair reduced DAMGO-induced feeding. One exception occurred when DAMGO was injected into PVN and naltrexone in CeA, and vice versa [21]. In this case, intra-PVN naltrexone blocked intraCeA DAMGO, but naltrexone injected into CeA (and DAMGO injected into PVN) did not. Such studies help identify opioid feeding circuitry and indicate that action at multiple brain sites may be required for food intake. One behavioral study suggests that opioids do not uniformly affect food preference across all brain sites where opioids are active because intra-CeA naltrexone decreased preferred food more potently than the nonpreferred food [23]. However, intra-PVN naltrexone decreased most preferred and nonpreferred diets to the same degree. Together, these studies indicate that feeding is regulated in part by a widely distributed complex central opioid network.

INTERACTIONS WITH OTHER PEPTIDERGIC/AMINERGIC SYSTEMS Orexins OxA increases neuronal activation in several areas associated with feeding behavior [57], implying interactions with other parts of the feeding network. These results are supported by behavioral evidence. The most studied to date is the interaction between OxA and NPY: NPY antagonist partially blocks OxA-induced feeding [62, 92], cfos-ir is elevated in Arc NPY neurons after OxA injection [92], antibody to orexin inhibits NPYinduced feeding and cfos-ir in hypothalamic nuclei [62], orexin-containing axon terminals synapse with

Arc NPY and POMC neurons, and orexin injection into Arc increases feeding [60]. Simultaneous injections of subthreshold doses of cerebroventricular NPY and OxA [72] enhance food intake, suggesting that OxA and NPY interact to influence feeding behavior. Other interactions have also been studied. Urocortin, an anorectic neuropeptide, injected into the lateral septal area inhibits OxA-sensitive feeding pathways in the rostral LH area [85]. Ventricular injection of additional anorectic neuropeptides, including glucagon-like peptide 1, leptin, and alpha-melanocyte-stimulating hormone also partially block OxA-induced feeding [98]. Central injections of the orexigenic neuropeptides ghrelin [42, 64], agouti-related peptide (AgRP) [96], and neuropeptide W [47] activate orexin neurons. In the AgRP study, orexin neuron stimulation was coincident with the feeding response to AgRP (delayed by several hours after injection), suggesting that orexin neuron activation is required for AgRP-induced feeding behavior [96]. OxA also interacts with opioid feeding pathways. Whole-cell patch clamp recordings of orexin neurons indicate that several neuropeptides important to feeding influence orexin neuron activity, including corticotrophin-releasing factor [89], cholecystokinin [83], leptin, and ghrelin [91]. Orexin neurons are also sensitive to input from serotonin [59].

Opioids Opioid-receptor blockade decreases feeding induced by NPY, AgRP, and OxA. Injection of naltrexone into NTS or CeA decreases the intake stimulated by injection of NPY into PVN [38]. However, there is only a minor effect of naltrexone on NPY-induced feeding when both compounds are injected into PVN. Naltrexone administered chronically (subcutaneously) via minipump increased NPY mRNA levels in the ARC [37]. NPY stimulates beta-endorphin release from the mediobasal hypothalamus in vitro and decreases ARC POMC mRNA in vivo [20, 67]. Brugman et al. reported that combined blockade of mu- and kappa-opioid receptors prevents AgRP-induced feeding [7]. Such results demonstrate that opioids interact with a number of broadly distributed neuroregulators that alter feeding.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS Orexins As indicated earlier, orexins are important to normal sleep-wake patterns and feeding behavior. Thus, studies reporting on the loss of orexin neurons or overexpres-

924 / Chapter 126 sion studies will probably have effects on all of these behaviors. Normal physiology dictates that sleeping and feeding behaviors are linked, and a word often used for snacks, “refreshments,” indicates the association between food ingestion and feelings of wakefulness. Sleep deprivation reduces leptin, with restoration of normal circulating leptin upon return of normal sleeping patterns [58]. Narcoleptic patients, who lack OxA and have an elevated BMI, have low leptin levels [36], and an elevated BMI in narcolepsy may be biologically linked to lack of orexin [15]. NHANES I data indicate that length of sleep is inversely correlated with weight [19], and in children sleep loss was dose-dependently correlated to level of obesity [74]. Insomnia (<4 h sleep/night) and obesity increase mortality risk [40, 56]. Orexin neurons may integrate signals important to feeding and sleep-wake behavior and as such may be important in obesity. Orexins have been studied in animal models of obesity. The OX1R-specific antagonist SB-334867 is more effective in reducing food intake and body weight in obesity-prone versus obesity-resistant rats, indicating that elevated OX1R signaling may enhance the development of obesity [87]. Also, the effectiveness of OxA in increasing activity-induced energy expenditure was reduced or absent in obesity-prone rats (unpublished data). Mechanisms supporting enhanced feeding and reduced energy expenditure increase susceptibility to obesity, and together these results suggest that differences in orexin signaling may influence the propensity for obesity.

Opioids Opioids are synthesized in the central and peripheral nervous system and affect both physiology and behavior. They impact functions, including memory, nociception, immune status, gastric motility, cell death, renal excretion of sodium and water, hormone secretion, and food intake [25]. The wide distribution of opioids with a variety of receptor subtypes makes this a complex neural circuit. The effect of opioids on memory and learning could clearly affect food intake because learning is involved in consummatory behaviors [5]. Ingestion of sweet tastants affects nociception, perhaps through opioid receptors. Stress-induced changes in opioids can also affect feeding [4]. Thus, it is difficult to isolate the feeding effects of opioids without understanding the importance of these peptides on a large array of behaviors.

INTERACTION BETWEEN OxA AND OPIOIDS Orexin neurons may also mediate reward-based feeding via its interaction with opioids. OxA-induced

feeding is dependent on functional opioid pathways because central opioid antagonist injections block OxAinduced feeding [13, 76]. Peripheral naloxone also blocks OxA-induced feeding; however, this effect is due to action on central pathways because naloxone methiodide, a form of naloxone that does not cross the blood– brain barrier, does not block OxA-induced eating [76]. The data in Fig. 1 show that interfering with opioid signaling reduces the amount eaten but not the number of attempts to eat (measured by consumption of 0.1 g chow or more) after OxA, establishing opioids as interacting with OxA and as maintainers, but not initiators, of eating. What can also be said is that OxA initiates meals because the number of attempts to eat (∼80%) was significantly greater after animals received OxA (Fig. 1). More indirect evidence for orexin-opioid interactions also exists. Like opioids, OxA selectively enhances high-fat consumption when animals are given the choice of high- and low-fat diets [13]. However, also like opioids, when a low-fat diet is the only choice available, OxA enhances consumption. The finding that orexin neurons also contain dynorphin [12] indicates a neurobiological basis for possible coordinate effects of both OxA and dynorphin.

CONCLUSION In conclusion, strong evidence implicates both orexins and opioids in feeding behavior. Further, it is clear that these neuropeptides and their receptors interact in eliciting food intake. In the larger context of feeding behavior, orexins and opioids participate in a network of brain areas with distinct neuronal populations and connections, which provide unique backgrounds that define the effect of any given neuropeptide in each particular area. Further, none of the neuropeptides has the sole function of playing a role in feeding behavior because all have effects on other physiological processes. Advances in understanding the role of neuropeptide function in feeding behavior await more sophisticated models that simulate brain connections and can predict the physiological outcomes of the interactions.

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127 Melanin-Concentrating Hormone ELEFTHERIA MARATOS-FLIER

stress responses [36]. Indeed results from initial studies aimed at elucidating the function of this neuropeptide suggested that it played a role in processes as varied as sexual behavior [19] and auditory gating [32]. It is now known that MCH plays a critical role in energy homeostasis, regulating both food ingestion and energy expenditure.

ABSTRACT Melanin-concentrating hormone was initially discovered in teleost fish, in which it mediates color change by inducing the migration of pigment-containing granules into the perinuclear area. It is synthesized and secreted into the circulation by the pituitary. In mammalian brain, MCH is synthesized in magnocellular neurons of the lateral hypothalamus. These neurons make diffuse monosynaptic connections throughout the brain. The role of MCH in mammals is regulation of energy balance. Through studies of MCH action in rodents and of genetically altered mice, it is now known that MCH affects both feeding behavior and energy expenditure.

EFFECTS OF MCH ON FEEDING BEHAVIOR The potential role of MCH in mediating appetite and regulating energy expenditure was initially considered when a study reported increased expression of MCH mRNA (pMCH) in the hypothalamus of the genetically obese ob/ob mouse, as well as the orexigenic effect of MCH when administered to rats through an intracerebroventricular (ICV) route [38]. This feeding effect is rapid and robust. Thus animals injected with MCH show a substantial increase in food intake within an hour after treatment compared with animals injected with artificial cerebrospinal fluid (aCSF). In a typical experiment the net increment in food intake is two- to threefold in the first 6 hours [38, 55], an effect that persists for at least 6 hours. Afterward, the difference between MCH-treated and aCSF control animals diminishes, and 24 hours after treatment no net effect on total food intake is seen. This effect is dose-dependent and requires 1–1.5 μg injected into the lateral ventricle. A dose of 5 μg appears to produce a maximal effect, although higher doses may lead to more prolonged effects. In one study examining the results of twice a day repetitive injections in rats, increased feeding was observed for several days, but became attenuated over time and was not seen after 5 days; moreover, the rats did not gain weight [39]. In contrast, when MCH was administered by continuous ICV infusion, weight gain was seen in both Wistar and Sprague-Dawley rats [12].

INTRODUCTION Melanin-concentrating hormone (MCH) is a 19amino-acid neuropeptide that has emerged as a key regulator of energy homeostasis. In mammalian brain, MCH is expressed in a unique set of magnocellular neurons that are located in the lateral hypothalamus and zona incerta. Its peptide structure is highly conserved and is known to be identical in mice, rats, and humans as is the pattern of expression of the peptide and the projections of the MCH neurons [48]. The neurons are unusual in that they make monosynaptic projections throughout the neuraxis [4]. Fibers project rostrally to the cortex, hippocampus, olfactory bulb, and striatum, including the nucleus accumbens and caudate putamen; caudal projections are directed to the nucleus of the solitary tract, parabrachial nucleus, and various reticular structures including the reticular formation and the dorsal raphe. This pattern of projection suggested that MCH was involved in mediating complex motivated behaviors [35] such as feeding and Handbook of Biologically Active Peptides

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930 / Chapter 127 In addition to mediating food intake in rats, acute ICV injections of MCH increase food intake in sheep [58]. In rats, MCH has been shown to also increase water intake independent of food intake [11]. ICV injections also increase alcohol intake [13] as well as sucrose intake [42]. These effects of MCH may be related to its effects regulating energy balance or may reflect effects on reward. In mice, acute injections of ICV MCH appear to have no effect on feeding (E. Maratos-Flier, unpublished data). However, chronic infusion can lead to both increased food intake and increased adiposity. Interestingly in mice eating chow, chronic MCH infusions lead to mild obesity without an observable increase in food intake, whereas when MCH is infused into animals that are fed a high-fat diet providing 52% calories from fat an effect on food intake is seen [18].

STUDIES FROM GENETIC MANIPULATIONS OR MUTATIONS Substantial support for the physiological role of MCH in energy balance derives from genetic models. Thus, ablation of the MCH signal through deletion of the gene for MCH or its receptor leads to leanness, whereas overexpression of the hormone leads to mild obesity. Genetic ablation of the pMCH gene reported a lean phenotype that was hypophagic and had increased energy expenditure [44]. Consistent with leanness, mice had low leptin levels and low insulin levels. Expression of other neuropeptides in the hypothalamus revealed no difference in NPY, AgRP, or orexin expression. However expression of pre-proopiomelanocortin was decreased, an effect consistent with lowered leptin levels. Additional insight regarding the potential role of MCH derived from studies of mice lacking both MCH and leptin (ob/ob). Interestingly, mice lacking both genes were substantially leaner than mice lacking only leptin (ob/ob). The ob/ob mice are obese secondary to both hyperphagia and a marked decrease in energy expenditure. However, the attenuation of the obese state in ob/ob mice lacking MCH was entirely attributable to an increase in energy expenditure [43], which resulted from both increased activity and an increased metabolic rate. Thus, compared with ob/ob, MCH-KO-ob/ob mice had a threefold increase in locomotor activity and a 25% increase in resting metabolic rate. Furthermore, double null animals had higher core body temperatures and improved cold tolerance. Studies on mice lacking MCH in inbred strains further confirmed the importance of MCH in regulating energy expenditure. Both the C57BL/6 and 129

strains lacking MCH were lean compared with wild-type control animals [23]. In both strains, ablation of MCH was associated with substantial resistance to diet-induced obesity. However, hypophagia was not a feature. Indeed mice with the 129 background ate more than wild-type animals. Leanness was due to increased energy expenditure, an effect that was particularly noticeable when animals were placed on high-fat diets. The increase in energy expenditure reflected both increases in activity and in metabolic rate. Similar results were seen in mice lacking the receptor for MCH, MCHR-1. Thus these animals are also lean and resist diet-induced obesity. However, increased activity is more pronounced in animals [9, 30] without the MCH receptor than in animals without pMCH. The results on altered metabolic rate suggest that MCH may be important in regulating autonomic activity. Consistent with this hypothesis, increased heart rate has been reported in mice lacking MCHR-1 [2]. One study has reported the effects of increased expression of MCH in the lateral hypothalamus in a transgenic mouse model [29]. Transgenic mice expressed twofold higher levels of MCH mRNA in the hypothalamus and showed concomitant increases in MCH immunoreactivity. When bred onto an FVB background, homozygote mice were modestly obese when fed a high-fat diet. In addition, mice display significant hyperinsulinemia and islet cell hyperplasia that is out of proportion to their modest degree of obesity and suggests that MCH may play a direct or indirect role in the islet. On a C57BL/5 background, obesity was seen in heterozygous males fed chow. A summary of the biological effects of MCH and the results from genetic manipulation studies is shown in Table 1.

FUNCTIONAL RESPONSE OF THE PEPTIDE/ GENE TO DIFFERING METABOLIC AND FEEDING STATES As already noted, MCH as a potential regulator of energy expenditure comes from the finding that expression of the mRNA is increased in the ob/ob mouse compared with control animals [38, 53]. Increased expression has also been reported in the Zucker rat, which lacks leptin signaling secondary to a mutation in the leptin receptor [46]. MCH expression increases with fasting in both mice and rats [3]. Increased MCH expression, both in the fasted and leptin-deficient states, is inhibited by the administration of leptin [53], an effect that is similar to that of leptin on NPY expression. However, there are few leptin receptors in the lateral hypothalamus; thus it seems unlikely that the leptin acts directly on MCH neurons to regulate

Melanin-Concentrating Hormone / 931 TABLE 1. Summary of MCH Actions and Effects of Genetic Manipulations. MCH Actions Acute

Chronic

Increase feeding Increase drinking

MCH Gene Ablation

Increase adiposity Increase feeding Decrease energy expenditure Effects of Genetic Manipulations MCH Receptor Gene Ablation

Leanness Increased energy expenditure Resistance to diet-induced obesity Attenuation of ob/ob phenotype

Leanness Increased energy expenditure Resistance to diet-induced obesity Increased sympathetic activity

MCH Gene Overexpression Increased adiposity Increased sensitivity to diet-induced obesity Hyperinsulinemia

expression. Interestingly the MCH receptor also appears to be sensitive to leptin insufficiency; MCH receptor expression is markedly increased in ob/ob mice, and expression decreases when animals are treated with leptin [24], an effect that is seen throughout the brain but is particularly prominent in the arcuate nucleus. Although it is clear the MCH levels increase in the fasted state, the response to obesity is somewhat controversial. In mice lacking uncoupling protein, which are hyperphagic and hyperleptinemic, MCH expression is suppressed, as is expression of NPY and AgRP [51]. In rats, one study reported that obesity associated with lesions of the ventromedial hypothalamus (VMH) and paraventricular nucleus (PVN) was associated with obesity and decreased MCH immunoreactivity in the lateral hypothalamus [47]. This would be consistent with MCH rising in low leptin states and falling when there is hyperleptinemia. However, in one study in rats, MCH levels rose with diet-induced obesity and a positive correlation with leptin levels was seen [15]. Further studies will be required to address this discrepancy. Disagreement also exists on the potential effect of estrogens on MCH expression and the role of MCH in estrogen-induced anorexia. One group reported MCH may be a target of estrogen action; thus, animals treated with estrogen developed hypophagia, which was associated with decreased MCH expression [34]. In contrast, another study reported a rise in MCH expression in response to estrogen treatment and furthermore showed the expected anorectic response to estrogen in mice lacking MCH [54].

SITES OF ACTION AND NEURAL NETWORKS AFFECTED MCH neurons make monosynaptic projections throughout the central nervous system, and MCH receptors are expressed in corresponding areas. Although various sites that can mediate a feeding response by endogenous MCH have been defined, sites that may be critical for this stimulation remain unknown. These studies typically involve anatomic identification through studies examining behavioral-physiological effects of direct MCH injections into specific locations. Injections into the PVN induce feeding [39]. Injection into the arcuate nucleus leads to a robust fourfold increase in feeding, and injection into the dorsomedial nucleus induces a twofold increase in feeding [1]. MCH fibers project into striatal structures, and high levels of receptor expression have been described in these structures; thus, it is not surprising that injections of MCH into these areas induces biological effects. Thus, when injected into the nucleus accumbens, MCH induces increased feeding, an effect that can be partially blocked by MCH receptor antagonists [16]. It seems likely that the effects of MCH on energy expenditure are mediated through effects in anatomical locations distinct from those mediating feeding effects. Such a distinction is possible because both the lateral hypothalamus and zona incerta are heterogeneous in organization, with broad projections throughout the neuraxis [33]. The results from studies of mice lacking MCH or the MCH receptor suggested that MCH, in addition to regulating eating behavior, also

932 / Chapter 127 had direct effects on energy expenditure because animals had evidence of increased energy expenditure [3, 23, 43]. MCH neurons innervate brown adipose tissue, although both the stellate ganglion and the nucleus of the solitary tract, which receives direct projections of MCH neurons from the lateral hypothalamus [61]. Furthermore, increased brown adipose tissue (BAT) activity, as assessed by sympathetic nerve activity and increased tissue temperature, is seen when lateral hypothalamic area (LHA) neurons are disinhibited, although the peptidergic identity of the neurons involved is unknown [6]. Additional metabolic effects in the periphery may be mediated through effects on visceral organs that play a role in mediating energy balance, such as the liver and the stomach. Thus, the LHA appears to send signals to the liver through the vagus [56] and responds to signals from the gastric vagal afferent [60].

RECEPTORS AND SIGNALING PATHWAYS RESPONSIBLE FOR THE INGESTIVE EFFECTS In mammals, two high-affinity receptors for MCH have been identified and designated MCHR-1 and MCHR-2. MCHR-1 was initially described as an orphan G-protein-coupled receptor of the somatostatin family, designated SLC-1 [25] Several groups subsequently identified this receptor as the MCH receptor [8, 27, 41, 45]. This receptor is expressed widely throughout the central nervous system, including the cortex, hippocampus, substantia nigra, nucleus accumbens, amygdala, and olfactory tubercle, areas that also receive projections from the MCH neurons in the lateral hypothalamus. In the hypothalamus, high levels of MCHR-1 expression are seen in the dorsomedial and ventromedial nuclei and in the arcuate nucleus. MCH couples to G-proteins, and signal transduction is a complex process involving coupling to Gi, Go, and Gq [21, 37], as well as a synergistic interaction with other Gs-coupled receptors [37]. Thus, acting through Gi, MCH blocks the induction of cAMP by ligands that activate receptors through coupling to Gs. MCH stimulates Ca influx by coupling to Go and Gq and inositol phosphate production by coupling to Gi and Gq. MCH also induces extracellular-signal-regulated kinase (ERK) 1/2. These effects have been demonstrated in ex vivo brain-slice preparations, and it has been shown that MCH can act in the piriform cortex and olfactory tubercle to induce ERK 1/2 phosphorylation [37]. This effect is anatomically specific and is not seen in the cortex and hippocampus. However, the potential downstream effects of MCH injected into this area on appetite or other behaviors are not as yet defined.

MCHR-2 is expressed in humans, dogs, cats, and ferrets, but it is not expressed in rodents, rabbits, or guinea pigs, although a pseudogene is present in the latter two species [50]. Interestingly, both the fugu and zebrafish express orthologs of MCHR-2, suggesting that the initial gene duplication that resulted in two mammalian genes occurred early and that rodents have lost the second gene [28].

INTERACTIONS WITH OTHER PEPTIDERGIC/AMINERGIC SYSTEMS Interactions of MCH with other hypothalamic peptidergic systems were initially described in fish, where MCH acts to lighten skin color antagonistically with melanocyte-stimulating hormone (MSH), which acts to darken skin color [52]. Interestingly a similar, mutually antagonistic action is seen when these peptides are administered ICV to rats where MCH acts to increase feeding while MSH acts to decrease feeding [55]. The orexigenic actions of MCH can also be inhibited by glucagon-like peptide (GLP)-1 and neurotensin [55]. It seems unlikely that MCH neurons are directly downstream of leptin because there is little expression of leptin receptors in the lateral hypothalamus. MCH neurons are probably regulated indirectly via known projections from AgRP and proopiomelanocortin (POMC) neurons from the arcuate [14]. Fibers from neurons expressing galanin-like peptide also appear to contact MCH containing neurons [49]. MCH may interact with gonadotrophs and play a role in regulating reproductive behavior and gonadal function. MCH fibers come in close contact with 85–90% of GnRH cell bodies [59] and MCH acts to release LHRH from ex vivo brain slices [10]. Given MCH projections, it is likely that it has effects on other complex behavior and, given heavy innervation (enervated is what you feel after writing this chapter) of the nucleus accumbens, it may play a role in addictive behavior. The specific pathways and interactions that may be involved are now being explored but have not been fully defined.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS All of the aggregate animal results indicate that the MCH system is critical for maintaining normal body weight. However, at this point, confirmation of the likely role of MCH in humans has not occurred. Identification of a loss of function mutation in the peptide or the receptor is difficult for practical reasons. Loss of function is associated with leanness and resistance to

Melanin-Concentrating Hormone / 933 diet-induced obesity. Thus far, identification of human mutations in genes important in regulating energy balance have been limited to loss of function of genes that lead to morbid obesity. Notably, these include mutations in leptin, the leptin receptor, the melanocortin4 receptor, and the gene encoding for the precursor to MSH, POMC. All of these mutations lead to an obvious metabolic phenotype of early onset, morbid obesity, a pathological condition that brings individuals and families to medical attention. In a society with ready access to calories, leanness and resistance to dietinduced obesity, far from leading an individual to seek medical advice, is viewed as a fortunate state of being and is unlikely to be further remarked on. To date, no group has collected families with “genetic leanness” for genetic studies. A number of groups have searched for gain-offunction mutations in the MCH receptor. In general, mutations tend to be rare, and thus far studies have failed to identify mutations that may be relevant to the obese phenotype. Two missense mutations have been associated with obesity, but neither one was associated with a functional alteration of receptor function [17]. Another study found 12 single-nucleotide polymorphisms (SNPs) in MCHR-1 and 8 SNPs in MCHR-2, but none of these was associated with altered receptor function [20]. Another group found a potential association with two SNPs of MCHR-1 and obesity; however, additional population analysis did not confirm this association [57]. To date, there are no reports of genetic analysis of the gene for the ligand, in part because it is highly unlikely that a ligand mutation would lead to a gain-offunction that could be defined by an obesity phenotype. The current record levels of obesity in human populations have led to increased morbidity and mortality secondary to the diseases associated with increased body mass, including diabetes, hypertension, and cardiovascular disease. Furthermore, it is clear that successful treatment of obesity markedly diminishes these risks. At present, the only long-term therapy for the treatment of severe obesity remains gastric bypass surgery; pharmacological therapy remains disappointing. Thus far, direct evidence of the involvement of the MCH system in human energy balance is lacking. However, the results from animal studies are compelling. Therefore, a number of pharmaceutical companies are attempting to develop MCH receptor antagonists, and a diverse group of antagonists has been described [26]. These antagonists of MCHR-1 are effective in reducing feeding and body weight. In dietinduced obesity in rats, systemically administered MCH reduces obesity, resulting in decreased body weight in animals fed a palatable diet [5]. Similar results to antagonists have been reported in diet-induced obesity mice, whereas mice of the same strain of mice lacking MCH

receptors failed to respond [31]. Effects of antagonists beyond feeding and appetite have also been reported, including decreased anxiety [5, 7], an effect consistent with the decreased anxiety seen in mice lacking the MCH receptor [40]. However, these effects on anxiety are difficult to interpret because infusions of MCH itself are also reported to be anxiolytic [22].

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128 Corticotrophin-Releasing Hormone (CRH) and Ingestive Behavior MARY ANN PELLEYMOUNTER

responses in rodents and nonhuman primates [10, 21, 33]. CRH also produces a clear dose-dependent reduction in refeeding following an overnight fast in rodents [50] and in daily home-cage food intake in baboons [71]. Further, CRH has been reported to reduce feeding in sheep [65], pigs [57], rhesus monkeys [21], goldfish [19], and other species. The anorectic effects of CRH are most pronounced when it is administered centrally; it is not effective when administered subcutaneously [50] and only modestly effective when administered intraperitoneally [29, 83]. The effects of CRH on food intake do not diminish appreciably when it is administered by continuous central infusion [17, 28], but they do diminish when the peptide is administered as repeated bolus intraventricular injections [40] or as bolus intraperitoneal injections [29, 83]. Finally, the anorectic effects of CRH are apparent within 30 minutes to 2 hours after central [58] or peripheral [29, 50, 83] administration, but diminish rapidly after 2 hours. In addition to its anorectic effects, centrally infused CRH has also been reported to produce behavioral suppression [10] or activation [75], depending on the novelty of the test environment. Both behavioral suppressant and activation effects of CRH have been demonstrated in rodents [10, 75] as well as in nonhuman primates [21]. Finally, central CRH has been shown to produce conditioned taste aversion in rodents [5, 27], although the latter group also suggested that CRH could produce taste preference under certain conditions. Because the dose responses for these stresslike effects of CRH overlap with the dose range that is effective in producing anorexia, it has been suggested that the anorectic effects of CRH could be associated with its emotional effects [39, 58]. Urocortin I, which is a high-affinity ligand for both CRH receptors, has anorectic effects similar to CRH, in that it is effective when administered centrally or

ABSTRACT Corticotrophin-releasing hormone (CRH) and urocortin I are potent activators of central and peripheral responses to stress, one of which is anorexia. Recently, two other urocortin peptides (II and III) were characterized that do not activate the hypothalamic-pituitaryadrenal (HPA) aspect of the stress response but are equally potent anorectic agents. Whereas the HPA stress response is primarily mediated by the CRH1 receptor subtype, the anorectic activity of the CRH peptides may be mediated by the CRH2 receptor subtype (at least in rodents). CRH-induced anorexia has been associated with activation of the limbic system, hypothalamus, and brainstem and may play a downstream role in the pharmacological effects of many anorectic agents.

EFFECTS OF CRH AND UROCORTINS ON FEEDING BEHAVIOR CRH was first shown to be a centrally acting anorectic agent in the early 1980s [10, 23, 47]. Subsequently, several other members of the CRH family of peptides were also shown to reduce food intake, including the frog-derived sauvagine [50], the fish-derived urotensin I [50], and the mammalian paralogs, urocortin I [73], urocortin II [29, 62], and urocortin III (Ucn III) [29, 59]. Ucn II is the mouse homolog of human stresscopin-related peptide (SRP), and Ucn III is the rodent homolog of human stresscopin (SCP) [29, 42]. An indepth discussion of the discovery, genetics and biochemical processing of these peptides is provided in Lovejoy’s Chapter 92 in the Brain Peptides section of this book. The acute central administration of CRH produces a dramatic dose-dependent reduction in food intake regardless of the method used to assess feeding. CRH has been shown to reduce food-motivated Handbook of Biologically Active Peptides

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938 / Chapter 128 peripherally, although its potency is much greater when given centrally [29, 73, 83]. When administered intraventricularly, it has been reported to have a delayed onset of action (4–6 hours) in mice [7] and rats [33]. The effects of urocortin I on locomotor activity, anxiety, and freezing behaviors are somewhat unclear at this time; some investigators have found clear evidence of anxiogenic and behavioral suppressant effects in the same potency range as for food intake [46, 58], whereas others have found a dissociation or dose window between the anorectic and anxiogenic effects of urocortin [33, 73]. Like corticotrophin-releasing factor (CRF), urocortin has been shown to induce taste aversion [33], although it has been argued that there is a 10-fold dose window between the effects of urocortin on taste aversion and its effects on satiety using meal microstructure analysis [5, 33]. Urocortin II and urocortin III have been shown to dose-dependently reduce natural dark-cycle food intake and refeeding in food-deprived animals, whether administered peripherally or centrally [29, 33, 59, 62, 83]. In addition, the anorectic effects of Ucn II and Ucn III can be blocked by coadministration of the CRH2selective antagonist, ASV-30 [59]. Unlike CRH or urocortin, however, these peptides have not been shown to alter hypothalamic-pituitary-adrenal (HPA) activity, whether tested in pituitary culture systems [29], given peripherally [29], or when given centrally [59]. These peptides do not appear to have the behavioral suppressant effects of CRH or urocortin in rats or mice [62, 80, 81, 85]. Whether they have anxiogenic effects in the same dose range as their anorectic effects is controversial at this time; some investigators have shown anxiogenic effects using the plus maze task [59], audiogenic startle responses in mice [64], or the ability to induce learned helplessness behavior in rats [25], whereas other investigators have shown delayed anxiolytic effects in rats using the plus maze task [80, 81]. In general, Ucn II appears to be the more potent anorectic agent in that lower doses are required to reduce food intake than for Ucn III [59]. Another important aspect of the effects of CRH peptides on food intake is the impact of these peptides on gastric emptying and colonic motility, which is discussed in more detail in Wang’s Chapter 140 in the Gastrointestinal Peptides section of this book. Briefly, CRH and urocortin I are potent dose-dependent inducers of motility in the colon while acting to slow gastric emptying [83]. Ucn II, in contrast, does not seem to alter colonic motility, although it significantly slows gastric emptying [83]. Interestingly, the effect of CRH and Ucn I on the colon are reversed by CRH1 but not by CRH2 antagonists, whereas its effects on gastric emptying are reversed only by the CRH2 antagonist, ASV-30 [83]. These results have led to the hypothesis that CRH1

receptors mediate the effects of CRH on colonic motility, whereas CRH2 receptors mediate the effects of CRH on gastric emptying [83].

STUDIES FROM GENETIC MANIPULATIONS The genetic deletion of CRH does not produce a phenotype with altered feeding behavior. In addition, CRH knock-out (KO) mice do not show blunted responses to fenfluramine, stress-induced anorexia, or lipopolysaccharide (LPS)-induced anorexia [76], suggesting either that (1) CRH does not play a physiological role in feeding behavior or (2) biological compensation occurs during development in response to genetic deletion of CRH. Further, neither acute nor chronic intracerebroventricular (ICV) administration of CRH antisense oligonucleotides in adult animals produce significant changes in food intake or body weight [30].

METABOLIC REGULATION OF CRH AND CRH RECEPTORS The effects of food deprivation and restriction on CRH production may depend on the deprivation paradigm and measure of CRH induction. For example, 50% food restriction over a 12-day period has been shown to reduce CRH mRNA in the dorsal hypothalamus [43] or in the hypothalamic paraventricular nucleus (PVN) [8]. Acute food deprivation, however, increased the number of CRH-staining neurons with c-fos activation in the hypothalamic PVN, along with other areas of the hypothalamus, limbic system, and brainstem in obese and lean Zucker rats [78]. CRH mRNA also changes with normal pre- and postprandial status; it is reduced in the lateral hypothalamus, ventromedial hypothalamus (VMH), and amygdala after feeding; is increased in the VMH before feeding; and is increased in the amygdala during feeding [60]. Finally, anorexia induced by exposure to running wheels resulted in an elevation of CRH mRNA in the dorsomedial nucleus of the hypothalamus rather than the PVN [35]. Very little is known about the regulation of the urocortin peptides in response to metabolic status; one group, however, has shown that Ucn II mRNA was not altered in the PVN of food-restricted rats [77].

SITES OF ACTION AND NETWORKS MEDIATING CRH EFFECTS Central (ICV) injection of CRH activates c-fos in the hypothalamus (parvocellular PVN) [56]; this effect can be reversed by co-administration of the antagonist,

Corticotrophin-Releasing Hormone (CRH) and Ingestive Behavior / 939 alpha-helical CRH [38]. Exogenous CRH also induces Fos in limbic structures such as the lateral septal nucleus, the medial amygdala, and the bed nucleus of the stria terminalis (BNST), as well as in brainstem structures, including the nucleus tractus solitarius (NTS) [32]. Exogenous administration of Ucn II induces Fos in the BNST, PVN, parabrachial nucleus, and NTS [62]. Not all of these structures seem to mediate CRH-induced anorexia, however, because CRH has anorectic effects when infused into the PVN but not into the lateral hypothalamus or VMH [39]. Both CRH and Ucn modulate feeding when infused into the intermediate portion of the lateral septum [82]. Finally, fourthventricle injections of Ucn are as effective in reducing food intake and increasing plasma glucose in decerebrate rats as they are in neurologically intact rats [18]. Fos induction in the decerebrate rats was limited to the NTS in this study [18]. Taken together, these results could suggest a hypothetical neural circuit, which mediates the anorectic effects of CRH peptides consisting of a limbic (lateral septal nucleus), hypothalamic/endocrine (PVN), and brainstem component (NTS).

RECEPTORS AND PATHWAYS MEDIATING THE EFFECTS OF CRH AND THE UROCORTINS ON FOOD INTAKE Two known receptor subtypes are thought to mediate the biological effects of the CRH family of peptides, CRHR1 and CRHR2. Both receptor subtypes are expressed in brain and the periphery, but do have distinct distribution patterns. The CRH1 receptor is expressed most densely in anterior pituitary, cortex, cerebellum, amygdala, hippocampus, and olfactory bulb [12]. In rodents, CRH2R is most abundant in the VMH, lateral septum, amygdala, hippocampus, and retina. CRH2 receptors are also expressed in the periphery [2, 14, 70], as well as in brain arterioles and in choroid plexus [12]. In nonhuman primates, there is a less distinct distribution of the two receptor subtypes, even in the pituitary [66]. The hypothalamic localization for CRH2 has suggested that it may play a role in the anorectic effects of the CRH peptides. The genetic deletion of the CRH2 receptor, however, does not produce an obvious phenotype in terms of food intake or body weight [3, 16], although there are results demonstrating that, if challenged with a high-fat diet, CRH2 KO mice show increased food intake and reduced feed efficiency [4]. Neither CRH1 or CRH1/ CRH2 KO mice show a metabolic phenotype [61], although one investigator described altered circadian feeding [49]. The pharmacological inhibition of CRH1 receptor activity with selective antagonists has also failed to

produce anorexia. Neither NBI 30775 (30 mg/kg/day) nor CRA1000 altered food intake or body weight [52, 54] when administered on a chronic basis. Further, selective CRHR1 antagonists do not alter the acute effects of CRH and Ucn on food intake [58, 72, 83]. The anorectic effects of CRH and Ucn can be completely blocked, however, by nonselective CRH receptor antagonists, such as astressin [83] or by the selective CRH2 antagonist, antisauvagine-30 (ASV-30) [17, 58, 59, 83]. It is interesting to note that while ASV-30 can completely block the anorexia associated with chronic CRH infusion, it only partially inhibits the weight loss associated with chronic CRH. Consistent with this observation is pair-feeding data showing that CRH-induced weight loss can only partially be explained by anorexia [17]. ASV-30 itself produces only marginally significant elevations in food intake and body weight when administered on a chronic basis [17] and has no effect when administered on an acute basis at doses that reversed the effects of Ucn or CRH [83]. Finally, as stated earlier, the selective CRH2 agonists Ucn II and Ucn III produce anorectic effects that are comparable to CRH and Ucn [29, 33, 59, 62, 83]. Thus, the evidence to date suggests that the CRH2 receptor subtype mediates the anorectic effects of exogenous CRH and urocortin peptides. The failure of potent and selective CRH2 antagonists such as ASV-30 to produce biologically significant elevations in food intake or body weight and the lack of an obvious metabolic phenotype in CRH2 KO mice open the question of whether the CRH2 receptor mediates basal satiety. We could speculate that the CRH2 receptor mediates anorexia under conditions when it may be adaptive, such as environmental or psychological stress.

INTERACTIONS WITH NEUROPEPTIDE SYSTEMS The acute administration of leptin increases PVN CRH mRNA [68, 79] and stimulates leptin release in hypothalamic explants [15]. These effects of leptin on CRH mRNA are not sustained over chronic leptin administration, however [51]. Further, the acute anorectic effects of leptin can be attenuated by the CRH antagonist, D-Phe CRH12–41 [20]. Leptin has also been shown to facilitate the transport of urocortin across the blood–brain barrier [55], resulting in a synergistic effect on food intake when these peptides are systemically co-administered [34]. Central alpha-metanocyle-stimulating hormone (MSH) [67] or melanotan II (MTII) [44] also increases CRH mRNA in the PVN, as does central infusion of glucagonlike peptide (GLP)-1 [24] and neuromedin U receptor ligands [31]. The anorectic effects of MTII can be atten-

940 / Chapter 128 uated (50%) by the administration of the CRH antagonist, alpha-helical CRH9–41 [44], as are the anorectic effects of GLP-1 [24]. Further, neuromedin U is inactive in CRH KO mice [26]. CRH, then, may mediate the anorectic effects of neuromedin U and may act as a downstream component for the anorectic effects of alpha-MSH and GLP-1. Finally, CRH-induced anorexia can be inhibited by the oxytocin antagonist, d-CH2(5), Try (Me)2-Orn8-vasotocin [53], and potentiated by insulin [63]. Histamine has been shown to elevate CRH mRNA in the PVN [37], as has the acute peripheral administration of fenfluramine [41]. Subchronic administration of fenfluramine, however, reduced hypothalamic CRH levels while increasing plasma corticosterone after 4 days of administration [1]. Further, alpha-helical CRH9–41 reversed the weight loss induced by the selective serotonin reuptake inhibitor (SSRI), fluvoxamine [84]. Thus, many anorectic agents acutely increase CRH expression in the PVN, suggesting that these agents could at least initially enhance HPA activity. Indeed, MC4 and 5HT agonists have been shown to increase corticosterone, as previously noted. Interestingly, orexigenic agents, such as neuropeptide Y (NPY) [74] and galanin [6], have also been shown to increase hypothalamic CRH mRNA. CRH has been shown to attenuate NPY-stimulated food intake [48]. Further, a toxin targeted to a CRH monoclonal antibody enhanced NPY-stimulated food intake when infused into the PVN but not the VMH. This study also demonstrated that downregulation of CRH by dexamethasone increased NPY-stimulated food intake [45]. Taken together, these results suggest that NPY and CRH have a mutually inhibitory relationship rather than a serial downstream relationship, as suggested for the anorectic agents discussed earlier. Schwartz et al. [69] have suggested that the mutually inhibitory relationship between hypothalamic CRH and NPY may form the basis of a model for the hypothalamic response to starvation.

PHYSIOLOGY AND PATHOPHYSIOLOGICAL IMPLICATIONS There is no clear evidence of association or linkage of CRH, urocortin, or the two CRH receptors with human obesity. There is a “silent” polymorphism in the CRH1 receptor (C861T) that is associated with increased body-mass index (BMI) but is not independently associated with waist/hip ratio, plasma insulin, or blood pressure. It is not known whether this polymorphism has functional consequences for the CRH1 receptor [11]. There are results suggesting, however, that CRH is altered in anorexia nervosa. CSF levels of CRH are

elevated in underweight anorexia patients, as is plasma cortisol. These changes are normalized after weight recovery [36], although a significant correlation remained between CSF CRH and depression ratings in weight recovered subjects [36]. Further, anorexic patients showed a blunted adrenocorticotrophinc hormone (ACTH) response to CRH administration, which normalized with weight recovery, albeit over a period of 6 months. In contrast, bulimics, who do not show weight loss, displayed a normal ACTH response to CRH [22]. Much less is known, of course, about the human pathophysiology associated with the CRH2-selective urocortin peptides. There is growing evidence, however, that these peptides are potent vasodilators and are present in the human cardiovascular system, suggesting therapeutic potential in heart failure [13] and as cardioprotective agents in ischemic heart disease [9].

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129 Peptide YY (PYY) and Neuromedin U (NMU): Effects on Ingestive Behavior CAROLINE J. SMALL, PREETI H. JETHWA, AND STEPHEN R. BLOOM

appetite [35, 57]. Recently, some of the main neural circuits involved have been identified. The arcuate nucleus (Arc) of the hypothalamus plays an integrative role in appetite regulation, for example, by receiving signals from the periphery via the brainstem [11, 35, 57]. Two well-characterized neuronal populations integrate signals: appetite-inhibiting proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) co-expressing neurons and appetitestimulating neuropeptide Y (NPY) and agouti-related peptide (AgRP) co-expressing neurons [11, 13, 35, 57]. Both of these neuronal populations project to the paraventricular nucleus (PVN) and other important nuclei involved in the regulation of food intake. The PVN also receives important inputs from other hypothalamic nuclei, for example, melanin-concentrating hormone (MCH)-producing neurons from the lateral hypothalamic area. Neuromedin U (NMU) is one of the most abundant neuropeptides and has been found in significant concentrations in both the gastrointestinal tract and the CNS. Within the CNS, NMU cell bodies are found in the rostrocaudal part of the Arc of the hypothalamus with more widespread distribution of NMU fibers in the nucleus accumbens, medial thalamus, and brainstem and in the paraventricular nucleus, ventromedial nucleus, and dorsomedial nucleus, areas where the NMU2 receptor is expressed. It has been shown recently that NMU is a potent anorexigenic peptide and that alterations of NMU signaling can predispose to obesity. Early studies on the actions of peripherally administered PYY demonstrated numerous effects on the gastrointestinal tract. PYY administration significantly delayed gastric emptying, gastric and pancreatic secretion, and the cephalic phase of gallbladder emptying but increased ileal postprandial fluid and electrolyte absorption. However, recently PYY’s role in the regula-

ABSTRACT Many peptides are synthesized and released from the gastrointestinal (GI) tract and the central nervous system (CNS). It is now evident that these peptides influence eating behavior. Peptide YY (PYY) is released postprandially from the gastrointestinal L cells and neuromedin U (NMU) has been found in significant concentrations in both the GI tract and the CNS. Recently NMU has been shown to be a potent anorexigenic peptide and alterations of NMU signaling have been shown to predispose to obesity. Following peripheral administration of PYY3–36, the circulating form of PYY, to mouse, rat, or human, there is marked inhibition of food intake. Obese subjects have lower basal fasting PYY levels and have a smaller postprandial rise. However, obesity does not appear to be associated with resistance to PYY. The effects of NMU and PYY on ingestive behavior and their potential as anti-obesity agents are the focus of this review.

INTRODUCTION The World Health Organization estimates that over 1 billion adults are currently overweight worldwide, and the increasing prevalence in younger generations [2] suggests that this epidemic will continue to worsen. Obesity is causally associated with cardiovascular disease, noninsulin-dependent diabetes mellitus, obstructive pulmonary disease, and cancer [49]. Obesity costs the National Health Service over one-half billion pounds every year. Even modest weight loss can reduce the morbidity and mortality associated with diabetes and cardiovascular disease [2]. However, public health initiatives to improve diet and promote exercise have been ineffective. The hypothalamus and the dorsal vagal complex appear to be important CNS regions directly regulating Handbook of Biologically Active Peptides

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946 / Chapter 129 tion of food intake and body weight has been the focus of investigators. Here we examine the discovery, distribution, and receptors for PYY and NMU. In addition, we examine the role of these peptides in the control of food intake and body weight.

DISCOVERY Neuromedin U (NMU) was first isolated from porcine spinal cord in 1985 and named for its potent contractile activity on the uterus [44, 45]. Two molecular forms were purified: NMU-25 and NMU-8, identical to the C-terminal of NMU-25 [44, 45]. Both forms are biologically active, stimulating contraction of rat uterus in vitro and causing potent vasoconstriction in rats and dogs [22, 31, 61]. NMU has since been fully sequenced in several species, and most of the NMU peptides that have been isolated from different species are icosapentapeptides, with the exception of rat NMU, NMU-23. PYY was first isolated from porcine intestine in 1980 [63]. PYY belongs to the PP fold peptide family that also includes NPY and PP. These peptides have other common features: all have 36 amino acids containing several tyrosine residues and all undergo C-terminal amidation, which is necessary for biological activity. The PP fold configuration consists of a polyproline helix and α-helix connected by a β turn, resulting in a characteristic U-shaped peptide. There is also marked evolutionary conservation of amino acid sequence between the peptides, with 42% homology between rat PP and PYY.

DISTRIBUTION AND RELEASE NMU shows remarkable conservation throughout evolution, the C-terminal heptapeptide (Phe-Leu-PheArg-Pro-Arg-Asn-NH2) is conserved throughout all mammalian species [41]. This degree of conservation suggests the importance of the carboxyl-terminus sequence for biological activity [29, 56]. NMU is an abundant peptide, found at significant concentrations in both the GI tract [3] and CNS [4, 17, 23, 31, 33, 34, 58]. It is also found in other tissues such as the pituitary gland [17], the thyroid gland [15], and the urogenital tract [17]. However, NMU is not found in the circulation and is likely to act as a neurotransmitter rather than a hormone. In the rat brain, the highest levels of NMU-like immunoreactivity are found in the nucleus accumbens (Acb), hypothalamus, anterior pituitary, and thalamus [16, 17]. NMU-immunoreactive cell bodies are found in the rostrocaudal part of the Arc of the hypothalamus,

with more widespread distribution of immunoreactive fibers in the Acb, medial thalamus, PVN, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and supraoptic nucleus (SON) and also in the brainstem with dense terminal fields primarily in the nucleus of the solitary tract (NST) and parabrachial nucleus (PBN) [4, 31, 33, 58, 60]. In situ hybridization has shown NMU mRNA to be most abundant in the pituitary pars tuberalis and slightly less 50 in the Arc and median eminence, the caudal brainstem (NST; area postrema, AP; dorsal motor nucleus of the vagus, DMV), and the spinal cord [31, 33, 34, 58]. PYY is widely expressed in endocrine L cells throughout the GI tract, where it is co-localized with glucagonlike peptide (GLP-1) and Oxyntomodulin [1, 10]. PYY-immunoreactive cells are almost absent in the stomach, and there are relatively few in the duodenum and jejunum, but they dramatically increase in frequency in the ileum and colon and are at very high levels in the rectum [1]. PYY has been described in the myenteric plexus and endocrine pancreas of many species but not in humans. PYY-immunoreactivity has also been reported in human adrenal medulla. PYY is present in the CNS with PYY-immunoreactive nerve terminals in the hypothalamus, medulla, pons, and spinal cord [19]. PYY is released into the circulation in response to food intake, rising to a plateau after 1–2 hours. Release is partly proportional to calorie intake, but it is also influenced by meal composition. Higher plasma PYY concentrations are seen following isocaloric meals of fat compared with intake of protein or carbohydrate [1]. An intraduodenal meal increases plasma PYY even before nutrients have reached the PYY-containing cells of the ileum. This suggests release through a neural reflex, possibly via the vagus. In addition to nutrients, PYY release is also stimulated by gastric acid, cholecytokinin (CCK), and infusion of bile acids into the ileum or colon in animal studies. PYY is not released by gastric distension. Other factors also alter circulating PYY; plasma PYY concentrations are increased by insulin-like growth factor-1, bombesin, and calcitonin-gene-related peptide and decreased by GLP-1.

RECEPTORS In 1998 a G-protein-coupled receptor (GPCR) FM-3 was cloned from human and murine cDNA libraries. Cloning enabled a subsequent reverse pharmacological approach for the identification of NMU as an endogenous ligand for the orphan human GPCR, FM-3 [20, 30, 32, 33, 54, 58, 62]. NMU binds to and activates human FM-3 with subnanomolar affinity and potency. Simultaneously a second NMU receptor, a GPCR, FM-4 was

Peptide YY (PYY) and Neuromedin U (NMU): Effects on Ingestive Behavior / 947 cloned from the human and rat [32, 33, 54, 58], and more recently the murine receptor [21, 33] was cloned. FM-3 and FM-4 are cognate receptors and have been renamed NMU1R and NMU2R, respectively (nomenclature as recommended by International Union of Pharmacology, IUPHAR). Both receptors have significant abilities to distinguish between different forms of NMU and have species and tissue selectivity in the biological actions of NMU [20, 30, 32, 33, 54, 58, 62]. The mRNA for NMU1R is expressed in a wide variety of tissues, but levels are greatest in peripheral tissues, in particular the small intestine and stomach. There is relatively low expression of NMU1R in the human or rodent brain [20, 21, 33]. In humans, NMU2R mRNA is confined predominantly to specific regions within the brain, with the greatest expression observed in the substantia nigra, medulla oblongata, pontine reticular formation, spinal cord, and thalamus. More recently a detailed analysis of NMU2R expression was completed in the rat [23]. The NMU2R gene was found in the medial postal region of the Arc adjacent to the third ventricle, isolated neurons in other portions of the Arc [23], and the PVN. Five cloned receptors for the PP-fold peptide family have been described, Y1–Y5 (nomenclature as recommended by IUPHAR) [40]. They are all seventransmembrane-domain receptors coupled to Gi, resulting in the inhibition of adenylate cyclase. However, Y1 also increases intracellular calcium, and Y2 regulates both calcium and potassium channels. See Chapter 95 on Neuropeptide Y in the Brain Peptide section of this book. The receptors are classified according to their affinity for PYY, PP, and NPY fragments and analogs and have diverse distributions and functions. Whereas PYY binds with high affinity to all Y receptors, PYY (3–36), the active fragment, shows selectivity for Y2 and Y5 receptors.

THE ROLE OF NMU IN THE REGULATION OF ENERGY BALANCE Intracerebroventricular (ICV) administration of NMU reduces nocturnal food intake and decreases body weight [34, 38, 48, 50, 65]. In addition, ICV administration of NMU antiserum increases food intake [38], whereas fasting reduces levels of NMU mRNA in the ventromedial hypothalamic region [33]. Wren et al. [65] demonstrated a dose-dependent reduction of feeding 1 hour after administration of NMU into the PVN and Arc [33]. In addition to the immediate reduction in food intake observed following NMU administration into the PVN or Arc, NMU has a much delayed inhibitory (4–8 hours postinjection) effect following administration into the medial preoptic area (MPOA) of the hypo-

thalamus [65]. The MPOA is involved in reproductive function and c-fos expression and NPY levels increase after feeding, whereas α-melanocyte-stimulating hormone (MSH) reduces food intake following administration in the MPOA [37]. Thus, NMU may interact with other neuropeptides to mediate the delayed feeding effect [65]. The decrease in food intake following a single ICV injection of NMU is associated with a significant increase in gross locomotor activity, core body temperature, heat production, and oxygen consumption [26, 33, 34, 48, 65]. However, NMU does not cause shivering, suggesting that the effects on body temperature are mediated via changes in sympathetic outflow to brown adipose tissue and skeletal muscle. Ivanov et al. [34] proposed that the effects of NMU on feeding and energy expenditure are independent because they observed an increase in core body temperature in the absence of a decrease in food intake in satiated animals. ICV administration of NMU reduces gastric acid secretion [46]. This CNS effect of NMU appears to be independent of vagal innervation because NMU significantly suppressed pentagastrin secretion in vagotomized rats. Prostaglandin E2 reduces gastric acid secretion directly by inhibiting parietal cell secretion and indirectly by inhibiting gastric acid release. Peripheral administration of indomethacin, to block prostaglandin synthesis, prevents the inhibitory action of neuropeptides on gastric secretion. However, the administration of NMU suppressed acid secretion in indomethacin-treated rats, suggesting that NMU acts independently of prostaglandin E2 [46]. More recently NMU null (NMU−/−) mice [27] and the NMU-overexpressing (NMU Tg) mice [39] have been developed. NMU−/− mice show increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese NMU−/− mice develop hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia, and hyperlipidemia [27]. The NMU Tg mice, on the other hand, show decreased body weight and adiposity, hypophagia, and a modest increase in respiratory quotient during food deprivation and refeeding. Unlike the NMU−/− mice the NMU Tg mice also show improved insulin sensitivity when fed both a normal and high-fat diet [39]. In addition, many neuropeptides important to the regulation of feeding are altered in these transgenic animals. In the NMU−/− mice there is a decrease in POMC while NPY and AgRP remain unchanged [27]. However, in the NMU Tg mice, both POMC and NPY are increased while AgRP remains unchanged [39]. The phenotype of these animals suggests that alterations in the NMU system leads to altered energy metabolism. Corticotropin-releasing hormone (CRH) is another hypothalamic peptide located in the PVN that decreases

948 / Chapter 129 food intake, increases energy expenditure via stimulation of the sympathetic nervous system, induces stress responses, and increases locomotor and grooming behavior [35, 43]. ICV and intra-PVN administration of NMU increases plasma adrenocorticotropic hormone and corticosterone. In addition, the release of CRH and arginine vasopressin is increased from ex vivo hypothalamic explants incubated with NMU [53]. Consistent with these effects, NMU increases c-fos expression in CRH containing neurons in the parvocellular region of the PVN and SON [34] and augments CRH mRNA expression in the PVN [27]. The mechanism by which hypothalamic NMU alters feeding behavior and energy homeostasis is not clear. However, given that the ICV effects of CRH appear to be similar to those observed following NMU administration, Hanada et al. [28] hypothesized that the changes in feeding behavior and energy homeostasis following NMU may be via CRH. ICV administration of NMU did not alter dark-phase food intake or fasting-induced feeding in CRH null (CRH−/−) mice when compared to wild type [28]. In addition the increases in oxygen consumption, body temperature, and locomotor activity observed following NMU administration were suppressed in the CRH−/− mice. In further experiments, the effects of NMU were also abolished in wild-type mice pretreated with the CRH receptor antagonist alpha-helical-CRH(9–41) when compared with their controls [26]. Further supporting this, recently Hanada et al. [27] showed that CRH mRNA expression is reduced in the PVN of NMU− /− mice and showed a lower plasma corticosterone level. However, surprisingly, NMU Tg mice showed no changes in CRH mRNA expression [39]; this may be due to the differing method of detection. To further support the hypothesis, NMU-induced inhibition of gastric acid secretion is blocked by pretreatment with anti-CRH IgG, suggesting that NMUinduced acid inhibition is mediated by CRH. CRH inhibits gastric acid secretion by the activation of the sympathetic noradrenergic nervous system and not the vagal fibers [18]. This finding supports the possibility that CRH mediates NMU-induced acid inhibition through a vagus-independent pathway. ICV administration of NMU also inhibited gastric emptying in rats; however, peripheral NMU had no effect [46]. These findings suggest that NMU mediates its effects on food intake and energy expenditure via hypothalamic CRH, see Chapter 103 on Neuromed in U (NMU): Brain Peptide in the Brain Peptide of this book.

PYY Peripheral administration of PYY was first reported to decrease appetite in 1993 [51]. In addition, when

PYY3–36, the circulating form of PYY, is administered peripherally to mouse, rat, or human there is marked inhibition of food intake [5, 7]. The pattern of c-fos expression in the brain after the peripheral administration of PYY3–36 shows a marked induction of c-fos in the Arc. Injection of PYY3–36 directly into the Arc inhibits food intake and chronic administration of PYY3–36 leads to a decrease in food intake and body weight. Addition of PYY3–36 to ex vivo hypothalamic explants inhibits the release of NPY and stimulates the release of α-MSH. Peripheral administration of PYY3–36 in rats causes a decrease in expression of arcuate NPY mRNA. PYY3–36 has a high affinity for the Y2 receptor (Y2R). The inhibition of appetite is seen with a Y2R-specific agonist and is absent in the Y2R knockout mouse [5, 7]. It appears that circulating PYY3–36 inhibits appetite by acting directly on the Arc via the Y2R, a pre-synaptic inhibitory autoreceptor [5, 7]. These results were reproduced by Halatchev et al., who confirmed that intraperitoneal (IP) PYY3–36 dose-dependently inhibited food intake in rodents in both dark-phase food intake and following a fast. However, this effect was only seen in animals acclimatized to handling and IP injections [25]. Challis et al. [9] showed that IP injections of PYY3–36 reduced food intake at 6 and 24 hours postinjection following a 24hour fast in mice, but not in nonfasted, freely feeding animals, although the first measurement was taken 6 hours postinjection, when PYY3–36 may already have had its effect. Some investigators have not been able to reproduce the original observation that peripheral administration of PYY3–36 inhibited food intake in rodents [64]. However, many other investigators have confirmed the original findings [8, 9, 14, 25, 47, 55]. It is likely that the anorectic effects of PYY3–36 are influenced by stress [25]. In order to overcome this, extensive handling and habituation of the animals to the experimental procedures are necessary prior to PYY administration. PYY is hypothesized to mediate its anorectic actions by switching off the Arc NPY neuron, decreasing hypothalamic NPY and thus food intake. This is supported by the observation that PYY3–36 decreases NPY mRNA and NPY release from ex vivo hypothalamic explants [7]. This switching off of the NPY neuron leads to an activation of the arcuate POMC/CART neuron. This is supported by the observations that PYY3–36 decreases POMC mRNA and α-MSH release from hypothalamic explants and increases the electrical activity of the POMC neuron [7, 9]. However, recent evidence has shown that its mechanism of action may be more complex. POMC−/− mice have been found to retain a normal, acute anorectic response to peripherally administered PYY3–36 [25, 42], suggesting that melanocortin

Peptide YY (PYY) and Neuromedin U (NMU): Effects on Ingestive Behavior / 949 peptides may not be required for the actions of PYY3– 36. In addition, a recent study demonstrated that melanocortin-4 receptor knockout (MC4-R−/−) mice are responsive to the anorexigenic effects of PYY3–36 [25, 42]. Together, these results suggest that the melanocortin system is not essential for the anorectic actions of PYY3–36. The administration of PYY3–36 into the CNS has strikingly opposing actions to those seen peripherally. Injections of PYY3–36 into the third, lateral, or fourth cerebral ventricles [12, 52], the PVN [59], or the hippocampus [24] in rodents potently stimulate food intake. ICV injections of PYY3–36 also increase food intake but this orexigenic action is reduced in both Y1 mice and Y5 knockout mice [36], suggesting that these receptors may play a role in the CNS feeding effects of PYY3–36. Batterham et al. [7] proposed that the discrepancy between the CNS and peripheral effects of PYY3–36 can be explained by the activation of the Y2R in the hypothalamic Arc. This anatomical specificity was confirmed by showing that injections of Y2 agonist administered directly into the Arc inhibited food intake in a dose-dependent fashion. This effect was not reproducible with injections of Y2 agonist into the hypothalamic paraventricular nucleus. However, the exact mechanism of action of peripheral PYY3–36 remains to be established and is a focus of ongoing research. In humans, food intake in a free-choice meal is reduced by 30% following an intravenous infusion of PYY3–36, which results in plasma levels similar to those achieved physiologically after a meal [7]. Recently, whether obese subjects are resistant to the anorectic effect of PYY3–36 infusion [5] has been investigated. Caloric intake during a buffet lunch offered 2 hours after the infusion of PYY3–36 was decreased by 30% in obese subjects and 31% in lean subjects. Overall PYY significantly reduced 24-hour caloric intake in both the obese (16.5%) and lean groups (23.5%). This is in contrast to the marked resistance to the action of leptin in the obese, greatly limiting its therapeutic effectiveness. In this study, PYY3–36 infusion also caused a reduction in the fasting preprandial concentrations of the hunger hormone ghrelin, suggesting an interaction between these two gut hormones and a possible mechanism by which PYY reduces hunger in humans. In addition, they also show that endogenous fasting and postprandial PYY levels were significantly lower in obese subjects and that plasma PYY levels correlated negatively with body mass index. This suggests that PYY deficiency may contribute to the pathogenesis of this condition. Increasing plasma PYY levels, either by exogenous administration or by stimulating endogenous release, is therefore an attractive strategy for the treatment of obesity.

CONCLUSION Several new chemical entities are being developed by pharmaceutical companies to target receptor systems involved in appetite regulation. The development of the NMU null (NMU−/−) mice [27] and the NMU-overexpressing (NMU Tg) mice [39] supports the role of NMU as an endogenous anorexigenic peptide. The NMU−/− mice lack the gene encoding NMU and develop an obese phenotype, whereas NMU Tg mice have ubiquitous expression of the NMU transgene and develop a lean phenotype. These findings suggest NMU is an important regulator of energy balance as perturbation of NMU signaling can result in obesity. Thus, using NMU as a potential anti-obesity target may be successful. However, it is likely that NMU mediates its effects on body weight via alterations in hypothalamic CRH. Although NMU is an important and interesting hypothalamic circuit, stimulation of NMU would result in weight loss but also an overactive hypothalamopituitary-adrenal axis. Thus NMU may not prove to be a useful, long-term anti-obesity target. Obesity may be thought of as a state of chronic adaptation to the hormonal changes of increased fat mass. Results to date suggest that increased BMI is associated with increased plasma leptin and decreased plasma ghrelin and PYY. Obese people have a similar sensitivity to the appetite-inhibitory effects of exogenous PYY3–36 infusion as lean people [6] (i.e., no PYY resistance). Replacing PYY deficiency may be a more realistic therapeutic goal. The peripheral administration of natural gut hormones as therapeutic agents has the advantage of targeting only the relevant brain appetite systems. Gut hormones are released every day after meals without side effects and continue to exert their effects without escape. Thus, the administration of the naturally occurring gut hormone may offer a long-term therapeutic approach to weight control without deleterious side effects.

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obesity. Int J Obes Relat Metab Disord 1997 May;21(5):387– 392. Niimi, M.; Murao, K.; Taminato, T. Central administration of neuromedin U activates neurons in ventrobasal hypothalamus and brainstem. Endocrine 2001 Dec;16(3):201–206. Okada, S.; Ohshima, K.; Mori, M.; Tatemoto, K. Peripheral not central administered PYY decreases high fat diet intake. Endocrinology 1993;Suppl180. O’Shea, D.; Morgan, D. G.; Meeran, K.; Edwards, C. M.; Turton, M. D.; Choi, S. J.; Heath, M. M.; Gunn, I.; Taylor, G. M.; Howard, J. K.; Bloom, C. I.; Small, C. J.; Haddo, O.; Ma, J. J.; Callinan, W.; Smith, D. M.; Ghatei, M. A.; Bloom, S. R. Neuropeptide Y induced feeding in the rat is mediated by a novel receptor. Endocrinology 1997 Jan;138(1):196–202. Ozaki, Y.; Onaka, T.; Nakazato, M.; Saito, J.; Kanemoto, K.; Matsumoto, T.; Ueta, Y. Centrally administered neuromedin U activates neurosecretion and induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of rat. Endocrinology 2002 Nov;143(11):4320–4329. Raddatz, R.; Wilson, A. E.; Artymyshyn, R.; Bonini, J. A.; Borowsky, B.; Boteju, L. W.; Zhou, S.; Kouranova, E. V.; Nagorny, R.; Guevarra, M. S.; Dai, M.; Lerman, G. S.; Vaysse, P. J.; Branchek, T. A.; Gerald, C.; Forray, C.; Adham, N. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J Biol Chem 2000 Oct;275(42):32452–32459. Riediger, T.; Bothe, C.; Becskei, C.; Lutz, T. A. Peptide YY directly inhibits ghrelin-activated neurons of the arcuate nucleus and reverses fasting-induced c-Fos expression. Neuroendocrinology 2004;79(6):317–326. Sakura, N.; Ohta, S.; Uchida, Y.; Kurosawa, K.; Okimura, K.; Hashimoto, T. Structure-activity relationships of rat neuromedin U for smooth muscle contraction. Chem Pharm Bull (Tokyo) 1991 Aug;39(8):2016–2020. Schwartz, M. W.; Woods, S. C.; Porte, D., Jr.; Seeley, R. J.; Baskin, D. G. Central nervous system control of food intake. Nature 2000 Apr;404(6778):661–671. Shan, L.; Qiao, X.; Crona, J. H.; Behan, J.; Wang, S.; Laz, T.; Bayne, M.; Gustafson, E. L.; Monsma, F. J., Jr.; Hedrick, J. A. Identification of a novel neuromedin U receptor subtype expressed in the central nervous system. J Biol Chem 2000 Dec;275(50):39482–39486. Stanley, B. G.; Leibowitz, S. F. Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior. Proc Natl Acad Sci USA 1985 Jun;82(11):3940–3943. Steel, J. H.; Van, N. S.; Ballesta, J.; Gibson, S. J.; Ghatei, M. A.; Burrin, J.; Leonhardt, U.; Domin, J.; Bloom, S. R.; Polak, J. M. Localization of 7B2, neuromedin B, and neuromedin U in specific cell types of rat, mouse, and human pituitary, in rat hypothalamus, and in 30 human pituitary and extrapituitary tumors. Endocrinology 1988 Jan;122(1):270–282. Sumi, S.; Inoue, K.; Kogire, M.; Doi, R.; Takaori, K.; Suzuki, T.; Yajima, H.; Tobe, T. Effect of synthetic neuromedin U-8 and U-25, novel peptides identified in porcine spinal cord, on splanchnic circulation in dogs. Life Sci 1987 Sep;41(13):1585– 1590. Szekeres, P. G.; Muir, A. I.; Spinage, L. D.; Miller, J. E.; Butler, S. I.; Smith, A.; Rennie, G. I.; Murdock, P. R.; Fitzgerald, L. R.; Wu, H.; McMillan, L. J.; Guerrera, S.; Vawter, L.; Elshourbagy, N. A.; Mooney, J. L.; Bergsma, D. J.; Wilson, S.; Chambers, J. K. Neuromedin U is a potent agonist at the orphan G proteincoupled receptor FM3. J Biol Chem 2000 Jul;275(27):20247– 20250. Tatemoto, K.; Mutt, V. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 1980 Jun;285(5764):417–418.

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130 Ghrelin and Ingestive Behavior DIEGO PÉREZ-TILVE, RUBÉN NOGUEIRAS, AND MATTHIAS TSCHÖP

changes in longitudinal skeletal growth or an increase in lean (muscle) mass [61]. The contradictory action profile of ghrelin as a potent growth hormone releasing factor, as well as the strongest known circulating adipogenic hormone, on the other hand, has yet to be understood. One way to reconcile these two seemingly opposing actions of one hormone may be to hypothesize that teleologically ghrelin aims to ensure the provision of calories that GH requires for growth and repair. Changes in body weight induced by ghrelin administration become significant in rodents after no more than 48 h and are self-evident at the end of 2 weeks of continuous treatment [61, 62]. Increase of fat mass induced by administration of ghrelin or growth hormone secretagogues (GHS) has now also been confirmed using a number of different methods [37, 61, 62]. Although pharmacological effects of ghrelin as well as its origin from the stomach point to a physiological role in the regulation of food intake, targeted mouse mutagenesis indicates that ghrelin-deficient mice have normal food intake and differ in body weight and fat mass from wild-type littermates only if early and chronically exposed to a high-fat diet (M. Sleeman et al., personal communication). One reason why ghrelin nevertheless receives substantial recognition as a potentially important co-regulator of energy balance is that its gene expression, as well as its effect on food intake, is well preserved across numerous species. Ghrelin has been found [36] and observed to influence feeding related behavior in several species including nonmammals, such as goldfish and chicken [20, 65], and can encompass behaviors beyond actual caloric intake. Siberian hamsters represent a particularly interesting example because, although they do not develop an impressively increased food intake following ghrelin administration, they do exhibit significantly increased

ABSTRACT Ghrelin is the only known circulating hormone with strong orexigenic activity. No other peripheral endocrine agent has comparable effects on hunger and energy balance. In addition, both ghrelin has numerous other known, and probably a substantial number of yet unknown, functions. This overview specifically summarizes available scientific information on the putative role of ghrelin in the regulation of food intake, showing that its popular name “hunger hormone” is neither farfetched nor entirely correct.

GHRELIN ADMINISTRATION INDUCES FOOD INTAKE ACROSS SPECIES When the gastric hormone ghrelin was discovered by M. Kojima and colleagues (please also see Chapter 101 by Kojima and colleagues in the section on Brain Peptides) [34, 35], it was their original goal to identify the endogenous ligand for an orphan G-protein-coupled receptor called the growth hormone (GH) secretagogue receptor [28]. They successfully identified the so-far only known ligand for this receptor and called it ghrelin. In addition to its first known biological effect of potently triggering GH secretion, a major role for ghrelin in energy balance regulation was discovered soon after the peptide was first identified [61]. The administration of ghrelin induced a marked increase in orexigenic activity and body weight [29, 46, 73]. Furthermore, when ghrelin action was neutralized by the administration of specific anti-ghrelin IgG, food intake (fasting-induced refeeding as well as dark-phase feeding) was found to be reduced in rats when compared with vehicle injected controls [4, 46]. Interestingly, ghrelin-induced body-weight gain is exclusively based on the accretion of fat mass and occurs without Handbook of Biologically Active Peptides

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954 / Chapter 130 foraging and food-hoarding behavior [32]. Ghrelin is, moreover, involved in other functions of the digestive process such as gut motility and gastric acid secretion, which in turn may further influence food intake [19]. Finally, and possibly most important, ghrelin administration in clinical studies causes an acute increase in hunger and food intake in healthy human volunteers, as has now been shown in several studies [16, 71]. This finding is not only of substantial importance because there are a number of known peripherally circulating satiety hormones (cholecystokinin; peptide YY PYY; leptin; glucagonlike peptide 1, GLP-1; oxyntomodulin; etc.) but only one hunger-inducing hormone; it is also important because the arguably most important energybalance-regulating hormone, leptin, has no effects on food intake in healthy volunteers, whereas ghrelin clearly has a strong and reproducible effect on food intake in humans. Based on the currently available results and following analogies with leptin as an afferent signal that informs the central nervous system (CNS) about the chronic state of energy balance, ghrelin might reflect the acute state of energy balance and signal the CNS in times of food deprivation in order to indicate that increased energy intake and an energy-preserving metabolic state are desirable [27, 61].

THE GROWTH HORMONE SECRETAGOGUE RECEPTOR GHSR-1a MEDIATES GHRELIN-INDUCED FOOD INTAKE Results obtained from a series of studies with the endogenous GHSR-ligand ghrelin were consistent with earlier reports published throughout the previous decade, in which several synthetic ligands of the GHSR1a increased food intake in rodents. Today, all GHSs reported have been shown to increase food intake and create a positive energy balance. This list includes growth hormone releasing peptide-2 (GHRP-2), ipamorelin, GHRP-6, hexarelin, and several other GHSR-1a ligands [67]. R. Smith and colleagues treated GHSR-1a knockout (KO) mice with ghrelin and did not find an increase in food intake when they were compared with wild-type littermates, indicating that the GHSR-1a is the essential mediator of this pathway and for this phenomenon [58]. The GHSR-1a expression pattern as identified in rodents includes a number of brain areas involved in the control of food intake [24]. Statistical evidence for the important role of the GHSR-1a receptor in the control of food intake comes from rats that were engineered to overexpress antisense mRNA against the GHSR-1a under the control of the promoter for tyro-

sine hydroxylase (TH). These rats are consecutively lacking the GHSR-1a selectively in the hypothalamic arcuate nucleus and did not respond to GHS with increased food intake as observed in their wild-type littermates [56]. These transgenic rats overall showed less food intake and body weight as well as lower levels of fat in the first weeks after weaning, indicating that the GHSR-1a might have an endogenous role in energybalance regulation. Consistent with this finding and as already mentioned, mice lacking the GHSR-1a have been shown to be unreponsive to ghrelin administration in terms of changes in food intake [8].

GHRELIN TARGETS THE HYPOTHALAMUS TO INCREASE FOOD INTAKE AND BODY WEIGHT Ghrelin is expressed in several tissues, but the main source of endocrine-active plasma ghrelin is the digestive tract, specifically the oxyntic glands of the stomach. This finding has been confirmed in rodent [14, 34] as well as in human [1] studies. The first reports of hypothalamic ghrelin expression described ghrelinergic neurons in the ventrolateral part of the arcuate nucleus [34]. However, contradictory reports indicate that ghrelin might not be produced in the CNS—at least not in amounts that can clearly be considered as physiologically relevant [70]. A more detailed analysis of its neuroanatomical distribution in the brain [10] later showed bodies of inmunopositive-ghrelin cells in the interstitial areas among the dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), and the walls of the third ventricle. Ghrelin immunopositive fibers were identified in close proximity to neuropeptide Y (NPY)-immunoreactive, proopiomelanocortin (POMC), thyroid-releasing hormone (TRH), and corticotropin-releasing hormone (CRH)-immunoreactive cells, as well as in nuclei related with food-intake control, such as the lateral hypothalamus (LH) and the paraventricular hypothalamus (PVH). In this report, M. Cowley and colleagues conclude that hypothalamus-derived ghrelin stimulates the release of orexigenic peptides and neurotransmitters, thus representing an essential part of a novel regulatory circuit controlling energy homeostasis [10]. According to current knowledge, efferent projections of ghrelin-immunopositive neurons reach key circuits of central energy balance regulation. It seems that two major hypothalamic pathways are the predominant mediators of both central and peripheral ghrelin influence on energy balance. Ghrelin action is believed to balance the activity of orexigenic NPY-AgRP and anorectic POMC neurons to modulate their resulting efferent message. Ghrelin attenuates the melanocortinergic

Ghrelin and Ingestive Behavior / 955 anorexigenic brake by increasing the release of melanocortin receptor inverse agonist AgRP and indirectly decreasing the discharge of alpha-melanocytestimulating hormone (MSH) from POMC neurons onto melanocortinergic receptor neurons in the PVN. This message is believed to be in part mediated by TRH and CRH [67]. Simultaneously, ghrelin triggers the expression of the orexigenic neuropeptide NPY [31] at the same hypothalamic neurons in the arcuate nucleus that produce AgRP [25]. The important point is that, according to the current view, gastric ghrelin probably reaches at least a certain number of GHSR1a located in mediobasal parts of the hypothalamic arcuate nucleus to activate the same neuroendocrine circuitry that is targeted by leptin. Such action appears to be co-modulated by a population of mostly bipolar-shaped, specifically ghrelin-expressing neurons that co-influence the firing rates of the orexigenic NPY-AgRP neurons and the anorexigenic POMC neurons, both of which are targets for gastric ghrelin. This model is consistent with the effect of ghrelin on energy balance, neuropeptide expression, and neuronal activity, and points to a combined role for ghrelin as an afferent nutrient sensor and a central energy balance regulator. Both leptin and ghrelin have been clearly shown to modulate the electrophysiological activity of the NPY-AgRP neurons, and leptin reduces the ghrelin-triggered increase in calcium concentration in NPY cells [33]. Whereas ghrelin induces pacemaker activity in arcuate neurons containing NPY and AgRP, leptin suppresses such activity [66]. Similar opposing action was found in POMC neurons in which leptin raises the expression of POMC mRNA [54] and neuronal excitability [9], whereas ghrelin reduces the activity of the POMC neurons by increasing the inhibitory release of GABA onto these neurons [10]. These opposite neurophysiologic effects further support the notion that ghrelin and leptin may be endogenous opponents in the regulation of energy balance [74]. Binding of ghrelin on arcuate nucleus NPY-AgRP neurons induces an increase in the firing activity [10] and membrane excitability [66] as well as an increase in the expression of neuropeptides NPY and AgRP [55]. The potency of ghrelin to modulate POMC neurons is weaker than its effect on NPY-AgRP neurons. POMC expression is not changed after intracerebroventricular (ICV) infusion of ghrelin for 72 hours, but this treatment raises both NPY and AgRP mRNA levels [31]. However, treatment with the GHSR-1a agonist tabimorelin for 18 days decreased POMC mRNA levels in the arcuate nucleus [26]. Ghrelin receptors are not colocalized on POMC neurons but induce a decrease in POMC neuronal firing rate [10]. However, this effect disappeared and even an increase of the firing rate was achieved when a Y1 receptor antagonist and an inhibi-

tor of GABA release were added, suggesting that the effect on POMC neurons is indirect and mediated via the activation of GABA-ergic NPY-AgRP neurons. The integrity of the melanocortin signaling pathway seems to be essential for ghrelin to exert its effect on food intake and adiposity. Ghrelin does not increase food intake in mice deficient for the melanocortin receptors 3 and 4 [8]. Consistent with this finding, ghrelin loses its orexigenic effect in an obese mouse model with ectopic overexpression of the melanocortin receptor 4 inverse agonist AgRP [43]. Increased food intake following central administration of ghrelin can be completely prevented in mice lacking the both AgRP and NPY, as Chen and colleagues have recently shown [8]. Ghrelin-induced adiposity is not exclusively dependent on NPY because ghrelin increases the fat content of NPY deficient mice, which is identical to its effect in wild-type mice [62]. Because ghrelin immunoreactive fibers were found to be colocalized with corticotropin-releasing factor (CRF) inmunopositive body cells in the PVN of the rat [10], ghrelin has also been speculated to be involved in anxietylike behavior of rodents. In mice, both ICV and intraperitoneal administration of ghrelin potently induces anxiogenic activities via mechanisms involving the hypothalamic-pituitary-adrenal axis. In addition, peripherally administered ghrelin significantly increases CRH mRNA expression in the hypothalamus and the administration of a CRH receptor antagonist actually inhibits ghrelin-induced anxiogenic effects. These findings suggest that ghrelin may have a role in mediating neuroendocrine and behavioral responses to stressors. Ghrelin may directly stimulate hunger and appetite, and indirectly affect energy relevant behavior via effects on anxiety [2]. NPY might not only be involved in ghrelin action as a co-mediator of increased food intake but also participate in mechanisms mediating ghrelin-induced intestinal motor activity. Neutralization of NPY with a specific antiserum partially blocks the duodenal and antral motility induced by ghrelin [19], whereas ghrelin ability to decrease the colonic transit time was blunted when it was co-administered ICV with BIBP-3226, a selective Y1 antagonist [59].

OTHER HYPOTHALAMIC PATHWAYS INVOLVED IN GHRELIN-INDUCED HYPERPHAGIA Although the hypothalamic arcuate nucleus is believed to be the main target for the orexigenic activity of ghrelin, the paraventricular nucleus (PVN) seems to be an important site of ghrelin action as well. Using specific antibodies raised against octanoylated-ghrelin

956 / Chapter 130 in porcine hypothalamus, ghrelin-immunoreactive neurons have been detected in the PVN, indicating a source of ghrelin neuropeptide [53]. Injection of ghrelin directly in the PVN increases food intake [72] and ICV administration of ghrelin or GHRP-6 induces c-fos expression in this hypothalamic nucleus [38]. In rats, it has recently been reported that the orexigenic activity of ghrelin in the PVN may in part be mediated by endocannabinoids and that this effect can be abolished by systemic pretreatment with the CB1 cannabinoid receptor antagonist SR141716 [64]. The lateral hypothalamus (LHA) may be involved as well in ghrelininduced hyperphagia via activated orexin pathways. The expression of c-fos in orexin-expressing neurons in the LHA was observed to be increased following ghrelin treatment, and the co-administration of antiorexin A IgG partially blocked the increase of food intake [60]. Finally, orexigenic effects of ghrelin were found to be reduced in orexin A–deficient mice when compared with wild-type littermates [60], which again points to a role of circuits in the LHA as ghrelin targets.

EXTRA-HYPOTHALAMIC EFFECTS OF GHRELIN ON FOOD INTAKE Although the current view suggests that the main target site for the induction of ghrelin-induced food intake is the hypothalamus and in particular the arcuate nucleus, very elegant studies have recently reported orexigenic effects of ghrelin following the administration to extra-hypothalamic areas. The brain stem is an equally important CNS area where food intake is regulated and afferent signals such as cholecystokinin or leptin [23] are received. The brain stem has now been shown to be a target of ghrelin action and capable of inducing ghrelin-triggered increases in food intake. The administration of ghrelin in the fourth ventricle or directly on the dorsal vagal complex resulted in an hyperphagic response with a magnitude similar to the one obtained in the third ventricle, measured as total amounts of ingested food as well as by meal pattern analysis [18]. Consistent with this finding, the administration of ghrelin or GHRP-6 increases the number of c-fos cells not only in the hypothalamic nuclei but also in distal areas such as nuclei tractus solitarii (NTS) or area postrema (AP) [38]. This also fits with the expression pattern of the GHS-R1a, which is localized on neuronal cells of the NTS, the dorsal motor nucleus of the vagus (DMNV), the rostal ventrolateral medulla (RVLM), and the caudal ventrolateral medulla (CVLM) [42]. It has even been reported that the injection of ghrelin in areas that are not regarded as centers of direct food-

intake control, such as the hipoccampus or dorsal raphe nucleus [7], evoked an increase in food intake. The fact that the GHS-R1a receptor has been shown to be expressed [24] in these regions adds credibility to these reports. The important point is that, in addition to one set of receptors targeted by ghrelin in the hypothalamus, other subsets of GHS-R1a may mediate the orexigenic activity of ghrelin in a number of other CNS areas. Among those, the most impressive data point to the areas in the brain stem. Apart from effects directly caused by ghrelin binding in the brain, there is one published report indicating that ghrelin action might be mediated not only by efferent but also by afferent activity of the vagus nerve. Such reports include electrophysiological studies in which ghrelin-induced intravenously administered ghrelin has been shown to decrease the afferent activity of the gastric vagal nerve at low doses [3]. In addition, the expression of GHSR-1a was found in cells of the nodose ganglion with stomach-projected afferents [52]. Furthermore, it has been reported in humans that ghrelin has no effect on food intake in patients with surgical procedures involving vagotomy [39]. The effects described here are not consistent with those of gastrointestinal satiety peptides such as cholecystokinin [45] and may add additional pathways to the growing number of signaling routes with which ghrelin is connected [29]. More recently, it has been reported that blockade of the gastric vagal afferent abolished ghrelin-induced feeding, GH secretion, and activation of NPY-producing and GHRH-producing neurons in rats. This study, which has not yet been repeated, suggested that the gastric vagal afferent is the only pathway conveying ghrelin’s signals for both increased orexigenic drive and enhanced GH secretion to the CNS [15].

PERIPHERAL GHRELIN LEVELS ARE REGULATED BY FOOD INTAKE In mammals, circulating ghrelin levels are increased by food deprivation or caloric restriction and are decreased postprandially. This phenomenon, which has been confirmed in several recent studies, further supports the concept of ghrelin as an endogenous regulator of energy homeostasis [67]. The ontogeny of ghrelin in the stomach was studied in rats, and it was shown that it begins to develop after birth. Plasmatic levels and tissue ghrelin expression increases throughout the suckling period until reaching normal adult values around weaning of the rat pups [41]. This indicates that stomach-derived ghrelin is unlikely to be involved in the ingestive pattern of pups while their feeding is dependent on their mother.

Ghrelin and Ingestive Behavior / 957 Another extremely intriguing aspect about ghrelin as a potential regulator of food intake is the fact that ghrelin secretion is clearly regulated by fasting and feeding and shows a perfectly logical circadian rhythm with three premeal peaks and a nightly increase [12, 13]. These results, which were first reported by D. Cummings and colleagues, have been discussed as possible evidence to support a role for ghrelin in the initiation of the meals. The influence of the gastric volume as a determinant of ghrelin secretion was discarded because only the administration of glucose solution, but not water alone, could decrease the ghrelin levels [61]. The possibility of the access to the intestinal lumen of ghrelin-expressing cells was investigated [51], and it was shown that the number of open cells to the lumen was very low in the stomach but high in the downstream regions of the proximal gut. It seems tempting to speculate that ghrelin produced in the stomach may be the predominant origin of the circulating hormone fraction [14], whereas the existence of a physiological role of the small amount of ghrelin produced in the other parts of the gut still remains unclear. Several studies demonstrate that the signal that promotes the decrease of ghrelin levels after a meal does not directly originate from the lumen of the stomach. When glucose solution was delivered to the rat stomach, the suppression on ghrelin plasma levels was blunted when the pylorus was closed. When the pylorus was open, ghrelin levels decreased following the delivery of a glucose solution to the stomach. This indicates that nutrients need to be absorbed distal from the stomach to act on ghrelin-secreting cells via blood circulation. Ghrelin levels again did not decrease when water alone was delivered [68]. Consistent with these findings, it was shown that the decrease of plasma ghrelin was also observed after administration of nutrients distal of the stomach, including in the duodenum and the jejunum [48]. Even though carbohydrates, proteins, and lipids all suppress the ghrelin levels [22], lipids seem to be a less potent inhibitor of ghrelin secretion because a smaller decrease of ghrelin levels is observed when lipids are administered in isocaloric amounts and compared with the administration of glucose and amino acids [48]. The control of the increase of ghrelin levels in response to fasting seems to depend on the integrity of vagal efferences from the CNS, because that effect is suppressed in vagotomized rats. However, the intragastric administration of nutrients in vagotomized animals does lead to decreased ghrelin levels [69]. These results suggest that a regulatory feedback loop between the gut and the stomach, independent of the CNS, cannot be excluded as a potential mechanism for the regulation of ghrelin levels in response to a meal. Among the presumably numerous determinants of the control of

ghrelin secretion, the most important candidates appear to be an increase of postprandial levels of insulin and glucose [67]. Insulin is an independent determinant of circulating ghrelin concentration as has been shown by several study groups using hyperinsulinemic euglycemic clamps in humans [44, 50]. Other authors have reported that when increases in insulin levels are evoked without nutrient intake or glucose stimulation, the ghrelin levels remain unaltered [5, 6]. Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated that ghrelin levels can be suppressed after a meal in the absence of insulin [21], indicating that, for example, a calorie-dependent but insulin-independent mechanism may control ghrelin levels in response to ingestive behavior. The current opinion seems to favor a combined mechanism involving insulin as well as ingested macronutrients as signals influencing the ghrelin-secreting cell in concert. The fact that ghrelin is clearly regulated by feeding and fasting in many species adds further evidence for the notion that ghrelin is a hormone with a strong link to ingestive behavior. In humans, ghrelin has been shown to be elevated in Prader-Willi syndrome (PWS) [11], a genetic disease characterized by obesity and hyperphagic behavior. It was suggested that the high ghrelin levels could be involved in the pathophysiological underpinnings behind the strong hyperphagia displayed by PWS patients. When the adipogenic and orexigenic effects of ghrelin were first discovered, one of the most interesting matters was to define a possible role for ghrelin in a clinical syndrome with altered ingestive behavior and energy metabolism. When ghrelin levels were studied in obesity, these were found to be reduced, not increased, in obese individuals [63]. However, although ghrelin levels in obesity are reduced, ghrelin levels remain unaltered after a standard meal in obese patients [17] independent of the caloric intake, in contrast to those observed in lean subjects [40]. In contrary to what was observed in obesity, fasting ghrelin levels were found to be higher in anorexic patients, returning the levels to the normal range after a partial recovery of their body weight [47]. In an animal model of catabolic state, an STZ-induced diabetes rat model that shows clear hyperphagia, ghrelin levels were higher than in nondiabetic control rats [30]. When these hyperphagic STZ diabetic rats were pair-fed with nondiabetic controls, the ghrelin levels off higher than those of diabetic animals that were fed ad libitum. Such results suggest that ghrelin levels are regulated according to energy requirements, but do not respond with a predictable change in response to a specific amount of ingested calories [21]. The orexigenic activity of ghrelin is reduced in diet-induced obese (DIO) mice on a highfat diet (HFD) in comparison with mice fed chow. When obese DIO mice on an HFD are switched to low-

958 / Chapter 130 fat diet for as much time as necessary to evoke a loss in body weight, ghrelin increases its orexigenic activity compared with the group of DIO mice maintained on an HFD [49]. In humans, ghrelin stimulates food intake both in lean and obese subjects, showing that obese, but not lean, patients report an increase in the palatability of food under the influence of ghrelin [16]. In the first studies performed in mice with disruption of ghrelin gene expression, no significant change in food intake was observed [57, 70]. However, the absence of ghrelin reduces the respiratory quotient in animals on an HFD, reflecting a derease in fat oxidation. This finding suggests that endogenous ghrelin may play a role in nutrient portioning, causing a preference for glucose and protein use as metabolic fuel [70]. This could be one mechanism that could explain the resistance of ghrelindeficient transgenic mice against diet-induced obesity that was recently reported from ghrelin KO mice exposed to an HFD very early and for a very long period of time. These mice clearly gained less fat mass than their wild-type littermates under the same conditions (Wortley and Sleeman, personal communication). Although numerous questions surrounding a possible role for ghrelin in the regulation of food are still unanswered, ghrelin is the only known molecule circulating in mammalian blood with considerable orexigenic potency. Whether its physiological role in the control of ingestive behavior is that unique, or even physiologically relevant at all, remains to be shown.

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Ghrelin and Ingestive Behavior / 959 [23] Grill HJ, Schwartz MW, Kaplan JM, Foxhall JS, Breininger J, Baskin DG. Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake. Endocrinology 2002;143 (1):239–46. [24] Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van der Ploeg LH, Howard AD. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997;48 (1):23–9. [25] Hahn TM, Breininger JF, Baskin DG, Schwartz MW. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1998;1 (4):271–2. [26] Holm AM, Johansen PB, Ahnfelt-Ronne I, Romer J. Adipogenic and orexigenic effects of the ghrelin-receptor ligand tabimorelin are diminished in leptin-signalling-deficient ZDF rats. Eur J Endocrinol 2004;150 (6):893–904. [27] Horvath TL, Diano S, Sotonyi P, Heiman M, Tschop M. Minireview: ghrelin and the regulation of energy balance—a hypothalamic perspective. Endocrinology 2001;142 (10):4163–9. [28] Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273 (5277):974–7. [29] Inui A. Ghrelin: an orexigenic and somatotrophic signal from the stomach. Nat Rev Neurosci 2001;2 (8):551–60. [30] Ishii S, Kamegai J, Tamura H, Shimizu T, Sugihara H, Oikawa S. Role of ghrelin in streptozotocin-induced diabetic hyperphagia. Endocrinology 2002;143 (12):4934–7. [31] Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and agouti-related protein mRNA levels and body weight in rats. Diabetes 2001;50 (11): 2438–43. [32] Keen-Rhinehart E, Bartness TJ. Peripheral ghrelin injections stimulate food intake, foraging, and food hoarding in Siberian hamsters. Am J Physiol Regul Integr Comp Physiol 2005;288 (3): R716–22. [33] Kohno D, Gao HZ, Muroya S, Kikuyama S, Yada T. Ghrelin directly interacts with neuropeptide-Y-containing neurons in the rat arcuate nucleus: Ca2+ signaling via protein kinase A and Ntype channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 2003;52 (4):948–56. [34] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402 (6762):656–60. [35] Kojima M, Hosoda H, Matsuo H, Kangawa K. Ghrelin: Discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 2001;12 (3):118–22. [36] Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev 2005;85 (2):495–522. [37] Lall S, Tung LY, Ohlsson C, Jansson JO, Dickson SL. Growth hormone (GH)-independent stimulation of adiposity by GH secretagogues. Biochem Biophys Res Commun 2001; 280 (1):132–8. [38] Lawrence CB, Snape AC, Baudoin FM, Luckman SM. Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology 2002;143 (1): 155–62. [39] le Roux CW, Neary NM, Halsey TJ, Small CJ, Martinez-Isla AM, Ghatei MA, Theodorou NA, Bloom SR. Ghrelin does not stimu-

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[66] van den Top M, Lee K, Whyment AD, Blanks AM, Spanswick D. Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat Neurosci 2004;7 (5):493–4. [67] van der Lely AJ, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25 (3):426–57. [68] Williams DL, Cummings DE, Grill HJ, Kaplan JM. Meal-related ghrelin suppression requires postgastric feedback. Endocrinology 2003;144 (7):2765–7. [69] Williams DL, Grill HJ, Cummings DE, Kaplan JM. Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinology 2003;144 (12):5184–7. [70] Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M, Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci USA 2004;101 (21):8227–32. [71] Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 2001;86 (12):5992. [72] Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001;50 (11):2540–7. [73] Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 2000;141 (11):4325–8. [74] Zigman JM, Elmquist JK. Minireview: from anorexia to obesity— the yin and yang of body weight control. Endocrinology 2003;144 (9):3749–56.

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131 Cholecystokinin and Satiety TIMOTHY H. MORAN, JIE CHEN, AND SHENG BI

endogenously released CCK. The results of these investigations have demonstrated an important role for CCK as a within-meal negative-feedback satiety signal. This chapter details the current state of our understanding of the role of peripheral CCK in satiety and the mechanisms through which these actions are mediated. We also discuss a newly identified putative role for brain CCK in feeding control.

ABSTRACT Peripheral or central administration of the brain/ gut peptide cholecystokinin (CCK) inhibits food intake. The actions of peripheral CCK in feeding are consistent with a role in satiety. CCK is rapidly released during a meal, exogenous peptide terminates an individual meal without long-term effects on food intake, and CCK antagonists or genetic manipulations that alter CCK-A receptor signaling result in increases in meal size. A role for central CCK in energy balance is suggested from the ability of central CCK to inhibit food intake and the gene-expression changes underlying the hyperphagia and obesity of Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors. Alterations in CCK-A receptor signaling have recently been implicated in human obesity.

FEEDING ACTIONS OF PERIPHERAL CCK Exogenous peripheral administration of CCK results in a dose-related suppression of short-term food intake in a variety of species and a variety of experimental situations. CCK has been shown to effectively reduce food intake in birds, rodents, pigs, sheep, nonhuman primates, and humans. CCK affects both solid and liquid food intake and is effective in spontaneous food intake as well as feeding that is stimulated by access to a palatable food source or in response to deprivation. CCK inhibits both real and sham feeding, in which ingested food is swallowed but drains from the stomach through a gastric fistula, demonstrating that CCK in the absence of much postingestive feedback is a sufficient stimulus for reducing food intake [41]. Across a dose range that results in feeding suppressions of up to 50%, the actions of peripheral CCK result in an earlier termination of food intake rather than in a blockade of feeding [41]. Using a system that allowed repeated CCK administration in coordination with spontaneous meals, West and colleagues [45] demonstrated that repeated meal-contingent CCK administration that occurred at meal onset resulted in a reliable reduction in meal size without an overall change in food intake. That is, in response to the repeated CCKinduced reductions in meal size, rats compensated for the missed calories through increasing their meal

INTRODUCTION In their classic 1973 paper, Gibbs, Young, and Smith [19] demonstrated the ability of exogenous administration of the brain-gut peptide cholecystokinin (CCK) to inhibit food intake in rats. The pattern of behavioral changes produced by CCK was consistent with the production of satiety. CCK reduced meal size and meal duration, and it resulted in an earlier appearance of a behavioral sequence of satiety similar to that seen following ingestion of a normal-size meal [1]. From these results, Gibbs, Young, and Smith suggested that exogenously administered CCK was mimicking a physiological hormonal action of the endogenous peptide [19]. These initial reports have stimulated 30 years of work in a variety of laboratories aimed at demonstrating a physiological role for CCK in the control of food intake and at understanding the mechanisms underlying the satiety actions of both exogenously administered and Handbook of Biologically Active Peptides

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962 / Chapter 131 frequency. In response to CCK administration timed to meal termination, there was no effect on the duration of the intermeal interval and no overall changes in the amounts consumed [46]. Such meal-termination actions of CCK are consistent with the dynamics of CCK in circulation. Exogenously administered CCK is rapidly degraded with estimates of half-life in the circulation in the range of a few minutes. In response to a meal, plasma CCK levels rise rapidly, peaking within a few minutes of meal initiation and then falling to less elevated levels that are sustained over a longer time period [24]. This rapid release and short duration in circulation suggest a role in the termination of a single meal without actions on the reoccurrence of eating at the next meal. The behavioral specificity of CCK for producing satiety has received extensive experimental assessment. The satiating action is characterized by an inhibition of food intake without the elicitation of abnormal or competing behaviors. CCK elicits the normal behavioral sequence of satiety. Rats treated with CCK stop eating sooner but go through the normal sequence of postprandial behaviors that characterize response to a spontaneous meal. They go through a period of grooming and exploration followed by resting or sleep [1]. Doses of CCK that inhibit food intake in food-deprived rats do not affect water intake in water-deprived rats, demonstrating that the actions of CCK were not secondary to an abnormal alteration in oral motor patterns [19]. The possibility that the reductions in food intake produced by exogenous CCK administration were secondary to malaise was an issue right from the outset. Assessments of CCK’s ability to induce a conditioned taste aversion (CTA) produced differing results depending on the test design and the dosages of CCK employed. However, it

became clear that CCK could inhibit food intake at doses that did not produce a CTA [23] and, in fact, at the low end of the effective dose range for food intake, CCK could result in a conditioned preference [34]. Such data were interpreted to support an action of exogenous CCK that is specific to satiety [41]. Results from human experiments in which the subjective sensations of subjects receiving CCK were assessed are consistent with this interpretation. Using slow intravenous infusions of CCK, Kissileff and colleagues [35] have demonstrated significant suppression in food intake in the absence of significant reports of side effects. Together, these assessments of specificity lead to the hypothesis that the actions of exogenously administered CCK were mimicking a physiological action of the endogenous peptide [41]. The availability of CCK receptor antagonists provided the ability to critically assess the physiological significance of endogenous CCK in the control of meal size. Initial experiments with a nonspecific CCK antagonist produced mixed results. Shillabeer and Davison [40] reported that the administration of the CCK antagonist proglumide increased food intake in prefed rats, consistent with an action of endogenous CCK in limiting food intake. However, Schneider et al. [38] failed to replicate these findings, concluding that, because proglumide blocked the effect of exogenously administered CCK but failed to increased food intake after oral preloads, the role of endogenous CCK in feeding controls remained uncertain. The availability of more potent and receptorsubtype-specific CCK antagonists provided clear evidence for satiety actions of endogenous CCK. In a variety of testing paradigms across multiple species, CCK antagonists with specificity to the A receptor subtype can be shown to increase food intake [28, 37]. As demonstrated in Fig. 1, in nonhuman primates, the increases in food

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FIGURE 1. Dose-related increases in food intake in rhesus monkeys in response to intragastric administration of the CCK-A receptor antagonist devazepide. Monkeys had 4-h daily access to 1 g food pellets in response to level pulls. The maximal increase occurred at a dose of 100 μg/kg and represents a 40% increase in total food intake.

Cholecystokinin and Satiety / 963 intake are dose-related up to a maximum at a dose of 100 μg/kg, resulting in a 40% increase in total intake, and the majority of the effect is secondary to an increase in the size of the first meal consistent with an action of endogenous CCK in the control of meal size. Although results with CCK antagonists in human subjects have been mixed, Beglinger et al. [2] have demonstrated small but significant increases in caloric intake in response to the CCK-A antagonist loxiglumide. These increases corresponded to decreased reports of fullness and increased reports of hunger.

FEEDING ACTIONS OF CENTRAL CCK Assessments of the potential central actions of CCK in feeding control have provided mixed results. An original report by Della-Fera and Baile [8] demonstrated feeding inhibitory effects of low doses of CCK in sheep. Positive results have also been generated in baboons [14]. In both cases, feeding inhibitory actions were obtained at doses that were not effective when administered peripherally, providing support for a central site of action. In the rat, a number of investigators have demonstrated feeding inhibitory actions of intracerebroventricular CCK. However, for the most part, these were obtained with doses that also affected intake when administered peripherally, leading to the suggestion that the CCK was affecting intake through a peripheral rather than a central site of action [7]. However, specific brain-site-directed injections have produced positive results at significantly lower doses. Blevins et al. [5] have identified multiple brain sites at which CCK inhibits food intake. The magnitude of inhibition was greatest with CCK injections aimed at the dorsomedial hypothalamus (DMH). A role for central CCK in feeding has also been suggested from the results of studies employing central CCK antagonist injections. Intracerebroventricular injections of low doses of CCK antagonists increase feeding in rats at doses that are ineffective when given systemically [10]. Furthermore, peripheral injections of CCK antagonists that cross the blood–brain barrier increase feeding in vagotomized rats, whereas peptide antagonists that are limited to the periphery do not [36]. The specific site of action for central antagonists to affect food intake has not been determined.

STUDIES WITH OLETF RATS AND CCK-A RECEPTOR KNOCKOUT MICE In 1984, a spontaneous mutation arose in a colony of outbred Long Evans rats that resulted in obesity and diabetes. Selective mating resulted in an obese strain,

Otsuka Long Evans Tokushima Fatty (OLETF) rats, and a control strain, Long Evans Tokushima Otsuka (LETO) rats. In examining the overall pancreatic function in OLETF rats, researchers discovered that the rats lacked an amylase response to exogenous CCK, leading to the demonstration that OLETF rats have a 6-kb deletion in the gene for CCK-A receptors that spanned the promotor region and the first and second exons, resulting in the absence of CCK-A receptors in these rats [16]. Functional studies in OLETF rats have revealed that they do not reduce food intake in response to CCK administration, and examinations of their meal patterns have demonstrated that they have a satiety deficit [30]. OLETF rats consume meals that are about twice normal size. In response to this increase in meal size, meal number is decreased, but not sufficiently to normalize overall food intake, resulting in about a 40% hyperphagia (Fig. 2). The OLETF rats’ obesity is completely secondary to their hyperphagia. Pair-feeding OLETF rats to amounts consumed by LETO controls normalizes body weight, body fat, and plasma glucose, insulin, and leptin levels [3]. Analyses of patterns of hypothalamic gene expression have demonstrated changes in arcuate nucleus (Arc), neuropeptide Y (NPY), and proopiomelanocortin (POMC) expression that appear to be a response to the increased food intake and body weight. Arc NPY is reduced and Arc POMC is elevated. Both of these changes are normalized by pair-feeding. In contrast, DMH NPY is significantly elevated in pair-fed OLETF rats (Fig. 3), and this elevation is also found in ad lib– fed preobese OLETF rats, suggesting that it might play a role in driving the hyperphagia and obesity. Consistent with this view, we have demonstrated that CCK injected directly into the DMH reduces food intake and reduces DMH NPY gene expression, suggesting that CCK normally exerts an inhibitory action on DMH NPY [4]. We have proposed that the hyperphagia in OLETF rats is the consequence of two deficits: the first arising from the absence of peripheral CCK-A receptors, resulting in a deficit in the control of meal size, and the second arising from the absence of DMH CCK-A receptors, resulting in a deficit in the control of overall energy balance [2, 4]. CCK-A receptor knockout mice have also been produced. These mice do not reduce their food intake in response to exogenously administered CCK but, unlike OLETF rats, consume normal amounts of food intake over 24 h and do not become obese [22]. However, we have demonstrated that they do in fact have a satiety deficit. CCK-A receptor knockout mice consume larger meals than wild-type mice of the same background strain. The increase is relatively smaller than that seen in OLETF rat, and mice do compensate for the increase so that total food intake is not significantly elevated [4]. CCK-A receptor knockout mice do not overexpress

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DMH NPY and, in fact, we failed to localize CCK-A receptors in the DMH of wild-type mice [4]. Together, these data further support a role for elevated DMH NPY due to the absence of DMH CCK-A receptors in the OLETF rat.

FUNCTIONAL RESPONSES TO PERIPHERAL CCK IN DIFFERING METABOLIC STATES The potency and efficacy of peripheral CCK in inhibiting food intake depend on the metabolic status. CCK

is less effective under conditions of food deprivation than in ad lib feeding, and CCK is less effective in rats during the dark cycle, when the majority of feeding occurs. These state-induced changes in the feedinginhibitory efficacy of CCK have been proposed to be due to an alteration in leptin signaling. Leptin levels are low in response to food deprivation or prior to nighttime feeding. Leptin has been shown to affect food intake through a mechanism dependent on reduction in meal size, and some aspects of this action appear to depend on leptin-CCK interactions. Exogenously administered leptin at doses that alone do not affect food intake enhances the ability of CCK to both inhibit feeding and induce neural activation in CCK-activated pathways, as measured by the presence of the immediate early gene c-Fos [12]. In contrast, exogenously administered NPY, mimicking deprivation induced elevated NPY levels, significantly reduces the ability of CCK to inhibit food intake and induce c-Fos [26]. The feeding inhibitory actions of peripheral CCK are also modulated by estrogen [18]. In female rats and mice, the amounts and patterns of food intake vary across the estrous cycle. Food intake is lowest at time points corresponding to high estrogen levels, and these decreases are expressed as decreases in meal size. This effect appears to be mediated by altered sensitivity to CCK. In ovariectomized females, CCK is more effective in reducing food intake when females are estrogen primed, and sensitivity to CCK varies across the estrus cycle. Furthermore, CCK antagonists prevent the decreases in food intake that correspond to high estrogen levels.

Cholecystokinin and Satiety / 965

SITES OF ACTION FOR PERIPHERAL CCK IN SATIETY

RECEPTORS AND SIGNALING PATHWAYS MEDIATING CCK SATIETY

Peripherally administered CCK induces a variety of changes in gastrointestinal motility and secretion; some of these actions are direct local effects of the peptide and some depend on the activation of vagal afferent fibers [32]. A role for CCK-induced vagal activation in CCK satiety has been demonstrated in a variety of experimental settings. Total subdiaphragmatic vagotomy completely blocks the ability of CCK to inhibit food intake, and vagal deafferentation significantly attenuates the feeding inhibitory effects of CCK [42]. The nature of this attenuation is to eliminate the ability of low doses of CCK to affect food intake while reducing the efficacy of higher doses [29] (Fig. 4). Surgical removal of the pyloric sphincter also significantly attenuates CCK satiety. CCK contracts the pyloric sphincter, an action that contributes to CCK’s ability to inhibit gastric emptying. In contrast to the findings with vagal deafferentation, pylorectomy does not affect the ability of low doses of CCK to inhibit food intake but truncates the dose-response curve such that the additional reduction produced by higher doses are eliminated [33] (Fig. 4). The nature of these lesion-induced changes in the CCK dose-response curve have led to the proposal that CCK-induced activation of vagal and pyloric sites mediate different portions of the ability of various doses of CCK to inhibit food intake and together account for the complete extent of CCK-induced reductions in food intake.

Early work investigating the distribution of CCK binding sites demonstrated that different CCK fragments bind to brain and pancreatic CCK receptors with different patterns of affinity, leading to the suggestion that there might be multiple CCK receptor subtypes. Receptor autoradiography experiments using pharmacological methods confirmed the existence of two CCK receptor subtypes, demonstrated that both subtypes were found within the periphery and brain, and developed a nomenclature of CCK-A (for alimentary receptors) and CCK-B (for brain receptors) [31]. The two receptors have now been purified and cloned, and their relative distributions have been confirmed by in situ techniques. Binding to the CCK-A receptor requires sulfated tyrosine and a minimum length of CCK-7. Smaller CCK fragments and unsulfated CCK bind to CCK-B receptors with high affinity. Structure–activity studies examining the ability of various forms and fragments of CCK to inhibit food intake revealed that sulfated CCK-8 and CCK-33 were 100–1000 times more potent than unsulfated CCK, gastrin, or smaller CCK fragments [31]. This profile was consistent with CCK-A receptor mediation of the satiety actions of CCK. The availability of specific CCK agonists and antagonists confirmed these data. Agonists with high affinity and specificity for CCK-A receptors potently inhibited food intake, whereas those with affinity and specificity for CCK-B receptors did not. Specific CCK-A receptor antagonists blocked the ability of exogenous CCK to inhibit food intake. Antagonists that were specific for CCK-B receptors did not [37]. Studies of the actions of CCK in stimulating the release of pancreatic amylase demonstrated that CCK-A receptors in the rat appeared to exist in two functional affinity states. The activation of the high-affinity site resulted in the stimulation of amylase release. The activation of the low-affinity site suppressed such release. Thus, actions of CCK at the two affinity states resulted in an inverted-U-shaped dose-response curve. CCK analogs were developed that had specific actions, depending on the affinity state of the receptor. One such compound, CCK-JMV-180, is an agonist at the high-affinity CCK-A site but an antagonist at the lowaffinity site. This profile made CCK-JMV-180 a very useful tool for assessing whether the satiety actions of CCK were mediated through interactions with high- or low-affinity CCK-A receptors [44]. The logic of the experiments was that if the satiety actions of CCK were mediated by high-affinity CCK-A receptors, the administration of CCK-JMV-180 should reduce food intake similar to CCK. If the actions were mediated through

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FIGURE 4. Effects of specific afferent vagotomy and surgical pylorectomy on the ability of CCK to inhibit food intake. Afferent vagotomy eliminates the ability of low doses of CCK to affect food intake. Pylorectomy does not affect the inhibition of food intake by low doses of CCK but eliminates further increases by high doses.

966 / Chapter 131 low-affinity sites, CCK-JMV-180 should act as a CCK antagonist, blocking the ability of CCK to inhibit food intake. The results were clear. CCK-JMV18-0 alone had no effect on food intake. Given in combination with CCK, it prevented the CCK-induced reduction of food intake. This pattern of results led to the conclusion that exogenous peripheral CCK acts through low-affinity CCK-A receptors to inhibit food intake [44]. The affinity state of CCK-A receptors mediating the ability of central CCK to inhibit food intake has not been assessed. Assessments of the CCK receptor subtype mediating the actions of endogenous CCK in food intake have also pointed to a critical role for CCK-A receptors. Although an initial report demonstrated an action for CCK-B receptors in modifying food intake, that result has not been replicated [9]. CCK antagonists with specificity for the CCK-A receptor subtype have been demonstrated to increase food intake in a variety of species and across a range of testing paradigms [28, 37]. Consistent with the lesion data already discussed, vagal afferent fibers do contain CCK-A receptors and CCK activation of these fibers is mediated through lowaffinity sites [39]. Similarly, CCK-A receptors are found in the pyloric circular muscle and the activation of these receptors results in contraction of the sphincter [28]. Thus, the sites that have been demonstrated to mediate the satiety actions of CCK do contain the necessary receptor subtype. The pathways critical to CCK satiety have been investigated. Peripheral CCK administration induces c-fos activation in a variety of brain sites. Among these are the nucleus of the solitary tract (NTS), area postrema (AP), and hypothalamic paraventricular nucleus (PVN) [15]. Lesion studies have supported a role for the NTS as a critical site for mediating CCK satiety. Lesions of the NTS, but not the overlying AP, block the ability of peripheral CCK to inhibit food intake [11]. Activation of this site may also be sufficient for mediating CCK satiety. Decerebration, which disconnects forebrain from hindbrain sites, does not eliminate the ability of peripheral CCK to inhibit food intake [20]. These data have been interpreted to suggest that the hindbrain contains the sufficient neural network for mediating peripheral CCK effects on meal size. The neural systems mediating the ability of DMH CCK to inhibit food intake remain to be investigated.

ating the actions of other peptides with roles in feeding control. CCK-serotonin interactions have been well documented. The administration of the serotonergic antagonists significantly diminishes the feeding suppression produced by peripheral CCK. Similarly, the CCK-A antagonist devazepide blocks feeding inhibition in response to peripheral serotonin or the fenfluramine, suggesting an interdependence of 5-HT and CCK in feeding control [6]. CCK’s action also appears to involve melanocortin signaling. Peripheral CCK activates NTS POMC-containing neurons and CCK has recently been shown to be ineffective in melanocortin4 receptor knockout mice [13]. CCK also plays a role in mediating the feedinginhibitory actions of enterostatin. The CCK-A receptor antagonist lorglumide blocked the inhibition of feeding produced by enterostatin, and enterostatin was ineffective in OLETF rats [25].

PATHOPHYSIOLOGIAL IMPLICATIONS Consistent with the data from the OLETF rat, there have been a number of reports of obesity and diabetes associated with altered CCK-A receptor signaling in human subjects. Expression of a CCK-A receptor protein with a 262-bp deletion that resulted in a nonfunctional receptor has been associated with gallstones and obesity [27]. Two missense mutations resulting in amino acid substitutions in the CCK-A receptor gene have also been associated with individual cases of obesity [21]. Finally, a polymorphism in the promotor region for the CCK-A receptor has been associated with increased adiposity and serum leptin levels [17]. Although these findings demonstrate that alterations in CCK-A receptor signaling are associated with obesity and its consequences, the mechanisms underlying these associations have yet to be adequately investigated. CCK-A receptor agonists have been investigated as potential anti-obesity agents. A number of classes of compounds with high selectivity and oral activity have been generated. In animal studies, these have produced potent and long-lasting inhibitions of food intake and body weight across extensive testing intervals [43]. No CCK agonist compound has yet become available for human use.

References INTERACTIONS WITH OTHER SIGNALING SYSTEMS Peripheral CCK action in food intake has been demonstrated to depend on its interactions with a number of other signaling systems and CCK plays a role in medi-

[1] Antin J, Gibbs J, Jolt J, Young RC, Smith GP. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J Comp Physiol Psychol 1975;89:784–790. [2] Beglinger C, Degen L, Matzinger D, D’Amato M, Drewe J. Loxiglumide, a CCK-A receptor antagonist, stimulates calorie intake and hunger feelings in humans. Am J Physiol Regul Integr Comp Physiol 2001;280(4):R1149–1154.

Cholecystokinin and Satiety / 967 [3] Bi S, Ladenheim EE, Schwartz GJ, Moran TH. A role for NPY overexpression in the dorsomedial hypothalamus in hyperphagia and obesity of OLETF rats. Am J Physiol Regul Integr Comp Physiol 2001;281(1):R254–260. [4] Bi S, Scott KA, Kopin AS, Moran TH. Differential roles for cholecystokinin a receptors in energy balance in rats and mice. Endocrinology 2004;145(8):3873–3880. [5] Blevins JE, Stanley BG, Reidelberger RD. Brain regions where cholecystokinin suppresses feeding in rats. Brain Res 2000; 860(1–2):1–10. [6] Cooper SJ, Dourish CT, Clifton PG. CCK antagonists and CCKmonoamine interactions in the control of satiety. Am J Clin Nutr 1992;55(1 Suppl):291S–295S. [7] Crawley JN. Clarification of the behavioral functions of peripheral and central cholecystokinin: Two separate peptide pools. Peptides 1985;6(Suppl 2):129–136. [8] Della Fera MA, Baile CA. CCK-octapeptide injected in CSF causes satiety in sheep. Ann Rech Vet 1979;10(2–3): 234–236. [9] Dourish CT, Rycroft W, Iversen SD. Postponement of satiety by blockade of brain cholecystokinin (CCK-B) receptors. Science 1989;245(4925):1509–1511. [10] Ebenezer IS. Effects of intracerebroventricular administration of the CCK(1) receptor antagonist devazepide on food intake in rats. Eur J Pharmacol 2002;441(1–2):79–82. [11] Edwards GL, Ladenheim EE, Ritter RC. Dorsomedial hindbrain participation in cholecystokinin-induced satiety. Am J Physiol 1986;251(5 Pt 2):R971–977. [12] Emond M, Schwartz GJ, Ladenheim EE, Moran TH. Central leptin modulates behavioral and neural responsivity to CCK. Am J Physiol 1999;276(5 Pt 2):R1545–1549. [13] Fan W, Ellacott KL, Halatchev IG, Takahashi K, Yu P, Cone RD. Cholecystokinin-mediated suppression of feeding involves the brainstem melanocortin system. Nat Neurosci 2004;7(4): 335–336. [14] Figlewicz DP, Sipols AJ, Porte D, Jr., Woods SC, Liddle RA. Intraventricular CCK inhibits food intake and gastric emptying in baboons. Am J Physiol 1989;256(6 Pt 2):R1313–1317. [15] Fraser KA, Davison JS. Cholecystokinin-induced c-fos expression in the rat brain stem is influenced by vagal nerve integrity. Exp Physiol 1992;77(1):225–228. [16] Funakoshi A, Miyasaka K, Jimi A, Kawanai T, Takata Y, Kono A. Little or no expression of the cholecystokinin-A receptor gene in the pancreas of diabetic rats (Otsuka Long-Evans Tokushima Fatty = OLETF rats). Biochem Biophys Res Commun 1994; 199(2):482–488. [17] Funakoshi A, Miyasaka K, Matsumoto H, Yamamori S, Takiguchi S, Kataoka K, Takata Y, Matsusue K, Kono A, Shimokata H. Gene structure of human cholecystokinin type A receptor: Body fat content is related to CCK type A receptor gene promotor polymorphism. FEBS Lett 2000;466:264–266. [18] Geary N. Estradiol, CCK and satiation. Peptides 2001;22(8): 1251–1263. [19] Gibbs J, Young RC, Smith GP. Cholecystokinin decreases food intake in rats. J Comp Physiol Psychol 1973;84(3):488–495. [20] Grill HJ, Smith GP. Cholecystokinin decreases sucrose intake in chronic decerebrate rats. Am J Physiol 1988;254(6 Pt 2):R853– 856. [21] Inoue H, Iannotti CA, Welling CM, Veile R, Donis-Keller H, Permutt MA. Human cholecystokinin type A receptor gene: Cytogenetic localization, physical mapping and identification of two missense variants in patients with obesity and non-insulindependent-diabetes-mellitus. Genomics 1997;42:331–335. J Physiol 1988;255(6 Pt 2):R1059–1063. [22] Kopin AS, Mathes WF, McBride EW, Nguyen M, Al-Haider W, Schmitz F, Bonner-Weir S, Kanarak R, Beinborn M. The chole-

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968 / Chapter 131 [43] Szewczyk JR, Laudeman C. CCK1R agonists: A promising target for the pharmacological treatment of obesity. Curr Top Med Chem 2003;3(8):837–854. [44] Weatherford SC, Laughton WB, Salabarria J, Danho W, Tilley JW, Netterville LA, Schwartz GJ, Moran TH. CCK satiety is differentially mediated by high- and low-affinity CCK receptors in mice and rats. Am J Physiol 1993;264(2 Pt 2):R244–249.

[45] West DB, Fey D, Woods SC. Cholecystokinin persistently suppresses meal size but not food intake in free-feeding rats. Am J Physiol 1984;246(5 Pt 2):R776–787. [46] West DB, Greenwood MR, Sullivan AC, Prescod L, Marzullo LR, Triscari J. Infusion of cholecystokinin between meals into freefeeding rats fails to prolong the intermeal interval. Physiol Behav 1987;39:111–115.

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132 Enterostatin, a Peptide Regulator of Dietary Fat Ingestion DAVID A. YORK AND MIEJUNG PARK

been shown to be present in the gastroduodenal mucosa and in specific regions of the brain (amygdala, hypothalamus, and cortex) and enterostatinlike immunoreactivity has been identified in human cerebrospinal fluid [10, 15, 26, 31, 37].

ABSTRACT Enterostatin is a pentapeptide released from procolipase precursor in the exocrine pancreas, gastrointestinal (GI) tract, and specific brain regions. It acts peripherally and centrally to initiate an early satiety response with a selective reduction in dietary fat intake. Peripherally it acts in the gastroduodenal region to initiate an afferent vagal signaling pathway. Centrally, it acts in the amygdala to activate pathways that involve 5HT-1B receptors, cholecystokinin A (CCK-A) receptors, and alpha-melanocyte-stimulating hormone (MSH). The anorectic response requires a chronic signal related to dietary fat ingestion. The enterostatin receptor is the plasma-membrane-located beta-subunit of F1ATP synthase present in several tissues, including the brain and liver, where it initiates signaling through the p-extracellular-signal-regulated kinase (pERK) and mitogen-activated protein kinase (MAPK) pathways.

ENTEROSTATIN EFFECTS ON FEEDING BEHAVIOR The initial interest in enterostatin as a potential anorexic peptide came from observations that rabbits injected with procolipase to raise antibodies had a loss of appetite [7]. Subsequent injections of high doses of enterostatin into the brain ventricles of rats identified a dose-dependent inhibition of food intake in rats. Other studies have shown efficacy for enterostatin on food intake in a number of species, including sheep, mice, and baboons [6, 7], but, in studies using either intravenous or oral enterostatin, no effect has yet been identified in humans [13, 35, 36]. Behavioral studies suggest that enterostatin acts to initiate an early satiety response and that the major effect is on the length and size of the first meal when overnight-fasted rats are refed [21]. Enterostatin does not induce a conditioned taste aversion. Further, although enterostatin has effects on gastrointestinal motility and function, including reducing the rate of stomach emptying, the effects on food intake appear to be independent of these responses [23, 29]. Enterostatin has selective effects toward the regulation of dietary fat. In rats adapted to a three-choice macronutrient diet of fat, carbohydrate, and protein [30, 32] enterostatin reduces the intake of the fat macronutrient, but has no effects on either carbohydrate or protein intakes. On a two-choice high-fat (HF) and lowfat (LF) diet paradigm, enterostatin reduces the intake of the HF diet only, not of the LF diet [16]. Similarly,

INTRODUCTION Enterostatin is a natural peptide released by cleavage from its precursor protein procolipase [6, 7]. Its structure is conserved over a wide range of species, particularly in the presence of prolines in positions 2 and 4. In humans and rat, the sequence of enterostatin is alanineproline-glycine-proline-arginine [42, 43]. Procolipase, the parent 101-amino-acid precursor, was originally thought to be expressed only in the exocrine pancreas when tryptic digestion of the procolipase in the duodenal lumen after pancreatic secretion released the colipase to act as a cofactor in the digestion of ingested triacylglycerols by pancreatic lipase. Subsequently, by use of immunohistochemistry, Western and Northern blots, and reverse transcription polymerase chain reaction (RT-PCR), both procolipase and enterostatin have Handbook of Biologically Active Peptides

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970 / Chapter 132 enterostatin reduces the intake of single diets when the diet is high in fat but not when it is low in fat content [32]. The ability to selectively inhibit fat intake on a two- or three-choice feeding paradigm has been demonstrated after both intraperitoneal [17, 32], near celiac arterial [14], intracerebroventricular (ICV) [17, 18], and intra-amygdala [24] injection of enterostatin. Enterostatin is also effective in reducing the ingestion of HF diets after intragastric, intraduodenal, intravenous, and intracarotid [7, 14, 27] injection, but the ability to selectively inhibit fat intake in choice feeding studies has not been investigated for these routes of administration. Enterostatin preferentially suppresses the intake of dietary fat without causing a compensatory increase in the ingestion of other dietary components. The reduction in total caloric intake is explained virtually entirely by the reduction in intake of the fat macronutrient or the HF diet. Enterostatin is rapidly metabolized by both intestinal and brain membranes [4] to yield smaller fragments that are themselves biologically active [17, 34, 38] in suppressing dietary fat intake or insulin secretion from pancreatic islets. It is possible that one of these fragments (e.g., PGP or cGP) is the major biologically effective peptide in situ.

GENOMIC STUDIES Procolipase- (and enterostatin-)deficient mice (Clps −/−) have reduced postnatal survival and reduced weight gain in surviving animals [5]. Food intake is normal on high-carbohydrate diets but elevated on HF diets. Although this hyperphagia is consistent with the absence of enterostatin, the fat malabsorption and steatorrhea of the Clps −/− mice may also be a contributor to the hyperphagia.

FUNCTIONAL RESPONSES TO DIFFERING FEEDING AND METABOLIC CONDITIONS Dietary fat is required for the response to enterostatin. Despite the original observations in SpragueDawley rats fed a chow diet, subsequent studies using rats fed semipurified HF or high-carbohydrate diets indicated the lack of response in high-carbohydrate-fed rats. This suggested the need for a fat-related signal for the response to enterostatin. Studies in our laboratory confirmed this [22]. Rats adapted to an HF or an LF– high-carbohydrate diet for 2 weeks were tested for response to ICV enterostatin, either while maintained on their familial diet or after switching to the alternative diet. Rats maintained and tested on an HF diet showed the expected anorexic response to ICV enterostatin,

whereas those on the LF diet were unresponsive. However, when rats adapted to the LF diet were switched to an HF diet, they did not show a response to enterostatin for 14 days. In contrast, the rats adapted to the HF diet were still responsive to enterostatin when tested on a high-carbohydrate diet, but this response disappeared after 2 days on the reverse diet. The data suggest the necessity for a signal related to the chronic ingestion of dietary fat but having a relatively short half-life. The nature of this signal is unknown at the present time. It is not leptin because the Zucker fa/fa rats that have a defect in the leptin receptor are responsive to enterostatin [7]. The ability to be selective in macronutrient or dietary choice implies that an animal must be able to identify dietary fat. Orosensory perception of dietary fat may involve a specific slowly rectifying potassium channel [8]. However, inhibitors of this channel failed to block the response to enterostatin (Ling and York, unpublished observations).

The Relationship of Dietary Fat Intake to Enterostatin Secretion Lack of a good assay for plasma enterostatin has hindered investigations of peptide release. The level of colipase in the pancreas is inversely related to the level of dietary fat intake, either across rat strains differing in their preference for dietary fat and carbohydrate or with the natural variation in dietary fat intake across individual rats in an outbred strain [7, 30]. Because colipase and enterostatin are produced in a 1 : 1 molar ratio, these data imply that enterostatin release from the exocrine pancreas is inversely related to dietary fat ingestion. HF feeding also increases the activity of pancreatic and gastric colipase [45]. In another study, gastric procolipase expression was reduced by HF feeding [42], although the colipase activity in the gastric juice was unaltered. Enterostatin is released from gastric procolipase by the actions of acid and pepsin, suggesting that part of the intraduodenal enterostatin is of gastric origin. The secretion of enterostatin into the gastrointestinal lumen after feeding is rapid and is stimulated by cholecystokinin-8 (CCK-8) [31], but the rise in plasma enterostatin after a meal is not observed for 60–90 minutes; it is greater in rats fed dietary fat rather than carbohydrate [28]. This slow time response reflects the major route of absorption of enterostatin through the lymphatic system [40] and shows that circulating enterostatin is not responsible for the rapid feeding response to enterostatin. Likewise, clearance also appears to be slow, and enterostatin binds to several plasma proteins [43]. Further, the uptake of enterostatin across the blood–brain barrier appears to be very limited [12] or absent (Lin and York, unpublished observations), suggesting that the circulating

Enterostatin, a Peptide Regulator of Dietary Fat Ingestion / 971 enterostatin is not a signal for the central pathways that are responsive to enterostatin. The mechanism through which enterostatinprocolipase production is increased by HF diets is not known. A number of hormones have effects [45]. Gastric inhibitory polypeptide, released in the gastrointestinal tract during a fat-containing meal, stimulates procolipase synthesis. Insulin and corticosterone both inhibit procolipase mRNA production, whereas cAMP has a stimulatory effect. In contrast, the increase in procolipase expression, as well as procolipase synthesis, after adrenalectomy is associated with reduced intake of HF diet and body weight.

SITES OF ACTION AND NEURAL NETWORKS AFFECTED The Response to Peripheral Enterostatin Enterostatin is secreted into the duodenal lumen during digestion. Enterostatin intubated or injected into the stomach or proximal duodenum induces a rapid reduction in food intake [27, 41], as does intraperitoneal injection of enterostatin. This contrasts with the long delay in response to an intravenous bolus injection of enterostatin in which no reduction in food intake is seen for nearly 2 hours. Near-arterial injections have been used to show that the stomach–proximal duodenum region is a site at which enterostatin acts to inhibit dietary fat intake [14]. Near-celiac-arterial injections of enterostatin cause an immediate inhibition of feeding in overnight-fasted rats given an HF diet and an immediate selective inhibition of intake of the HF diet, but not the LF diet, in rats given a dietary choice. This peripheral response is dependent on afferent vagal firing because it is prevented by either selective hepatic vagotomy and by capsaicin [29, 39] and by local tetracaine nerve blockade [27]. Although the specific pathway has not been completely mapped, peripheral enterostatin induces c-fos expression in the brainstem, hypothalamic regions, and the amygdala, as well as decreasing the hypothalamic 5HT : 5HIAA ratio [11, 44], implying the involvement of hypothalamic serotonergic pathways in modulating a vagally mediated response to peripherally administered enterostatin.

after which food intake returns toward or to control levels [16, 45]. However, body weight and body fat are reduced, and this reflects the additional effect of central enterostatin to elevate the sympathetic stimulation of brown adipose tissue thermogenesis [2, 29]. Mapping experiments have identified the central nucleus of the amygdala as the most sensitive region for the foodintake response [20, 21]. Paraventricular nucleus (PVN) injections of enterostatin also reduce intake of dietary fat, but the response is delayed and less sensitive than that seen in the amygdala, whereas the stimulation of metabolic rate is seen after enterostatin injections onto the PVN but not onto the amygdala. This implies a separation of the pathways that regulate the feeding response from the metabolic response (Lin and York, unpublished observations). The neuronal pathways that are affected by enterostatin to cause the behavioral changes are not fully understood. However, a number of major observations underpin the hypothetical pathway shown in Fig. 1. These include: 1. The response to ICV enterostatin is blocked by kappa-opioidergic agonists in the nucleus tractus solitarius (NTS) [7, 45].

AMYGDALA

Enterostatin

DORSAL RAPHE N

PVN

5HT α MSH

5HT1BR

AFFERENT VAGUS

? NTS

The Central Response to Enterostatin Acute ICV injections of enterostatin inhibit the intake of HF diets or fat macronutrients in experimental choice paradigms. The response is rapid, within 5–10 minutes, and occurs in doses as low as 10 nmol with a maximal response at 1 μmol [17]. Chronic infusions of enterostatin reduce food intake for the initial few days,

κ-opioid

Enterostatin

ARCUATE

Fat Intake

FIGURE 1. Neuronal pathways activated by enterostatin in the amygdala to suppress dietary fat intake.

972 / Chapter 132 2. The reduction in fat intake in response to amygdala enterostatin is blocked by a 5HT-1B receptor antagonist GR55526 but not by ritanserin, a 5HT-2C receptor antagonist, given into the PVN. The anorexic response to enterostatin is also observed in 5HT-2C-receptor-null mice. This suggests a role for the 5HT-1B receptor in mediating the response to enterostatin [45; Lin and York, unpublished observations]. 3. A serotonergic inhibitory control of dietary fat intake has been shown in the PVN [9]. 4. The responses to peripheral and ICV enterostatin are both blocked when the CCK-A receptor antagonist lorglumide is given by the same route, suggesting that both peripheral and central CCK-A receptors are necessary for the response to enterostatin [18]. 5. Retrograde tracing and c-fos immunohistochemistry show that neurons that are activated in the amygdala in response to enterostatin innervate a number of brain regions, including the PVN, the NTS, and the arcuate nucleus. In the arcuate nucleus, the c-fos is localized into neuropeptidy Y–proopiomelanocortin (NPY-POMC) neurons. The anorexic response to enterostatin is also partially inhibited by the MC3MC4 receptor antagonist SHU9119 [24; Lin and York, unpublished observations]. Because procolipase and enterostatin have been localized in the central bed region of the amygdala, there appears to be an endogenous system within this nucleus that might regulate an animal’s preference for dietary fat. We propose (Fig. 1) that enterostatin activates a pathway from the amygdala to the PVN, where the neurons presynaptically stimulate serotonergic fibers from the raphe to release 5HT. Enterostatin also activates POMC neurons in the arcuate to release alpha-MSH in the PVN. These actions, through serotonergic activation of 5HT-1B receptors, effect a pathway to the NTS to inhibit neurons that are normally activated by kappa-opioid input, with a resultant selective inhibition of dietary fat intake. Peripheral vagal activity induced by enterostatin appears to effect the same serotonergic system in the PVN, suggesting that both the central and peripheral responses share a common distal pathway.

RECEPTORS AND SIGNALING PATHWAYS Ligand binding studies on neuronal membranes suggested that there were both high- and low-affinity binding sites for enterostatin [7]. Subsequent

conventional affinity chromatography was used to identify the mitochondrial F1ATP synthase beta-subunit (FATPSBS) as a protein that would bind enterostatin [1, 2]. Although this protein is recognized as a mitochondrial protein, a component of ATP synthase, the demonstration of its localization also on the plasma membrane [25] raised the possibility that it was the enterostatin receptor. Binding affinity for the lowaffinity site has been estimated at 1.5 × 10−7 M from BIACORE Surface Plasmon Resonance spectroscopy [34] or at 1.7 × 10−7 M using a two-phase partition assay with an iodinated enterostatin analog (YGGAPGPR) [1]. By comparing a range of peptide analogs of enterostatin that had variable efficacy to either reduce food intake or insulin secretion with the ability of these analogs to likewise effect the binding of an enterostatin antagonist, beta-casomorphin, to the recombinant protein in an in vitro assay, we provided substantive evidence to support the hypothesis that this protein is indeed the enterostatin receptor [34]. Further, we have shown that the receptor FATPSBS is present in plasma membranes prepared from amygdala and hypothalamus in addition to the liver [34]. Martinez et al. [25] showed the presence of FATPSBS protein and ATP hydrolase activity on HepG2 plasma membranes, but not on Chinese hamster ovary (CHO) cells, and showed its role as an apolipoprotein A-1 receptor. We have taken advantage of these observations to study the receptor protein by expressing a green fluorescent protein–tagged FATPSBS transcript in HepG2 cells and have shown that enterostatin promotes the trafficking of this receptor protein to the membrane. Further studies on HepG2 cells and human myocytes have shown that enterostatin activates AMP kinase (AMPK) and extracellular-signal-regulated kinase (ERK) phosphorylation, suggesting that these signaling pathways may play a role in the neuronal responses to enterostatin [Park, Hulver, and York, unpublished observations]. However, because the FATPSBS acts as an ATP hydrolase, regulating extracellular ATP levels, it is possible that enterostatin may initiate changes in these signaling pathways indirectly, through changes in extracellular concentrations of adenine nucleotides (ATP, ADP, and AMP), which act through the purinergic P2X or P2Y receptors to effect AMPK and mitogen-activated protein kinase (MAPK) pathways (Fig. 2). The ability to block the effects of enterostatin on AMPK in myocytes and HepG2 cells with the general P2 receptor antagonist suramin supports this suggested pathway. However, we have yet to show that this mechanism is also active in neuronal cells or is responsible for the feeding effects of enterostatin [Park and York, unpublished observations].

Enterostatin, a Peptide Regulator of Dietary Fat Ingestion / 973

When Enterostatin binds to F1-ATPase β-subunit.

ADP

Ca 2+

ATP

Ca 2+

P2X

EXTRACELLULAR

αβ

P2Y

Ca 2+ Ca 2+ Ca 2+

F 1-ATP Synthase

Ad Cyclase

Ca 2+

ATP INTRACELLULAR

Ca

2+

Flux

PI3K MAPK JNK

Insulin Secretion

cAMP

PKA

Dietary Fat Intake

FIGURE 2. Signaling pathways activated by enterostatin.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS Although the overconsumption of dietary fat has been related to increased risk for a number of chronic diseases, there is, as yet, no evidence that the preference for dietary fat can be related to lack of enterostatin activity or that enterostatin can reduce this appetite in humans. A reduction in urine enterostatin levels was reported in obese subjects compared to lean subjects after a meal [3]. However, neither oral nor intravenous (IV) enterostatin has been effective in reducing intake of HF meals in humans, although in one study a reduction in appetite was recorded prior to the provision of the meal [13, 35, 36]. However, in light of the animal data, it seems unlikely that circulating enterostatin is an important signal for feeding behavior and that only central nervous system (CNS) or intraduodenal changes may have an influence.

References [1] Berger K, Sivars U, Winzell MS, Johansson P, Hellman U, Rippe C, and Erlanson-Albertsson C. Mitochondrial ATP synthase—a possible target protein in the regulation of energy metabolism in vitro and in vivo. Nutr Neurosci 2002;5:201–10. [2] Berger K, Winzell M, Mei J, and Erlanson-Albertsson C. Enterostatin and its target mechanisms during regulation of fat intake. Physiol Behav 2004;83:623–30.

[3] Bowyer RC, Jenhanli AM, Pater G, and Herman-Taylor J. Development of enzyme-linked immunosorbent assay for free human procolipase activation peptide (APGPR). Clin Chim Acta 1991;200:137–52. [4] Bouras M, Huneau JF, Luengo C, Erlanson-Albertsson C, and Tome D. Metabolism of enterostatin in rat intestine, brain membranes and serum: differential involvement of proline specific peptidases. Peptides 1995;16:399–405. [5] D’Agostino D, Cordle RA, Kullman J, Erlanson-Albertsson C, Muglia LJ, and Lowe ME. Decreased postnatal survival and altered body weight regulation in procolipase-deficient mice. J Biol Chem 2002;277:7170–7. [6] Erlanson-Albertsson C. Pancreatic colipase. Structural and physiological aspects. Biochim Biophys Acta 1992;1125:1–7. [7] Erlanson-Albertsson C, and York DA. Enterostatin—a peptide regulating fat intake. Obes Res 1997;5:360–72. [8] Gilbertson TA, Liu L, York DA, and Bray GA. Dietary fat preferences are inversely correlated with peripheral gustatory fatty acid sensitivity. Ann N Y Acad Sci 1998;855:165–8. [9] Halford J, Smith BK, and Blundell J. Serotonin (5HT) and serotonin receptors in the regulation of macronutrient intake. In: Berthoud HR and Seeley RJ, editors. Neural and Metabolic Control of Macronutrient Intake. Boca Raton: CRC Press; 2000, p. 425–46. [10] Imamura M, Sumar N, Hermon-Taylor J, Robertson HJF, and Prasad C. Distribution and characterization of enterostatin-like immunoreactivity in human cerebrospinal fluid. Peptides 1998;19:1385–91. [11] Koizumi M, and Kimura S. Enterostatin increases extracellular serotonin and dopamine in the lateral hypothalamic area in rats measured by in vivo microdialysis. Neurosci Lett 2002;1; 320:96–8. [12] Koizumi M, Nakanishi Y, Sato H, Morinaga Y, Ido T, and Kimura S. Uptake across the blood-brain barrier and tissue distribution

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nervous system interactions. J Gastroenterol 2002;37(Suppl)14: 118–27. Okada S, York DA, and Bray GA. Procolipase mRNA: tissue localization and effects of diet and adrenalectomy. Biochem J 1993;292:787–9. Okada S, York DA, Bray GA, and Erlanson-Albertsson C. Enterostatin (Val-Pro-Asp-Pro-Arg) the activation peptide of procolipase selectively reduces fat intake. Physiol Behav 1991;49: 1185–9. Okada S, York DA, Bray GA, Mei J, and Erlanson-Albertsson C. Differential inhibition of fat intake in two strains of rat by the peptide enterostatin. Am J Physiol 1992;262:R1111–6. Ookuma K, Barton C, York DA, and Bray GA. Effect of enterostatin and kappaopioids on macronutrient selection and consumption. Peptides 1997;18:785–91. Park M, Lin L, Thomas S, Braymer HD, Smith PM, Harrison DH, and York DA. The F(1)-ATPase beta-subunit is the putative enterostatin receptor. Peptides 2004;25:2127–33. Rossner S, Barkeling B, Erlanson-Albertsson C, Larsson B, and Wahlin-Boll E. Intavenous enterostatin does not affect single meal food intake in man. Appetite 1995;24:37–42. Smeets M, Geiselman P, Bray GA, and York DA. The effect of oral enterostatin on hunger and food intake in human volunteers. FASEB J 1999;13:A871 (Abs). Sorhede M, Mulder H, Mei J, Sundler F, and Erlanson-Albertsson C. Procolipase is produced in rat stomach—a novel source of enterostatin. Biochim Biophys Acta 1996;1301:207–12. Tadayyon M, Liou S, Briscoe CP, Badman G, Eggleston DS, Arch JRS, and York DA. Structure-function studies on enterostatin inhibition of insulin release. Int J Diabetes & Metabolism 2002;10:14–21. Tian Q, Nagase H, York DA, and Bray GA. Vagal-central nervous system interactions modulate the feeding response to peripheral enterostatin. Obes Res 1994;2:527–34. Townsley MI, Erlanson-Albertsson C, Ohlsson A, Rippe C, and Reed RK. Enterostatin efflux in cat intestinal lymph: relation to lymph flow, hyaluronan and fat absorption. Am J Physiol 1996; 271:G714–21. White CL, Bray GA, and York DA. Intragastric β-casomorphin inhibits the suppression of fat intake by enterostatin. Peptides 2000;21:1377–81. Winzell MS, Lowe ME, and Erlanson-Albertsson C. Rat gastric procolipase: sequence, expression and secretion during high fat feeding. Gastroenterology 1998;115:1179–85. Wu YJ, Hughes D, Lin L, Braymer HD, and York DA. Comparative study of enterostatin sequence in five rat strains and enterostatin binding proteins in rat and chicken serum. Peptides 2002;23:537–44. York DA, Lin L, Smith BK, and Bray GA. Enterostatin and 5HT: modulation of fat intake. In: Pennington Symposium Series, Nutrition, Genetics, and Obesity. Baton Rouge: Louisiana State University Press; 1998,Vol. 9, p. 246–63. York DA, Lin L, Smith B, and Chen J. Enterostatin as a regulator of fat intake. In: Berthoud H and Seeley R, editors. Neural and Metabolic Control of Macronutrient Intake. Boca Raton: CRC Press; 2000, Chap. 20, p. 295–308.

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133 Regulation of Feeding Behavior by Glucagonlike Peptide 1 (GLP-1) PATRICIA M. VUGUIN AND MAUREEN J. CHARRON

litus (T2DM), GLP-1 has been shown to enhance satiety, reduce energy intake [22, 58], and cause a progressive and sustained reduction in body weight [66]. Contrary to human studies, in rodents the feeding response to the peripheral administration of GLP-1 has been ambiguous. GLP-1(7–36) amide did not modify food intake, although a dose-dependent loss of body weight was observed 24 h after acute administration [41]. In a different study, dose-dependent intravenous infusions of GLP-1 inhibited food intake and gastric emptying [10]. Others have shown short-term anorectic effects of subcutaneous GLP-1 administration [47]. The long-acting GLP-1 agonist NN2211 caused lasting and reversible anorexia accompanied by weight loss secondary to reduction in adiposity and secondary to a moderate drop in total energy expenditure [31]. Thus, in contrast to earlier beliefs, GLP-1 significantly decreased food intake in rodents via a peripherally accessible site. Higher doses of GLP-1 and the longer plasma half-life of the circulating protease (DPP-IV) [43] may explain the discrepancy between studies.

ABSTRACT Glucagonlike peptide 1 (GLP-1) is a posttranslational product of the proglucagon gene. GLP-1 is released in response to meals and acts as a satiety signal. High GLP1 concentrations as well as the widespread distribution of its receptor in the central nervous system have suggested a central role for GLP-1 in appetite suppression. The mechanism by which GLP-1 inhibits food intake is not clear. Does it mediate its effect directly by binding to peripheral or central receptors? Does GLP-1 act as a hormone or as a neurotransmitter? In this review, we present our current understanding of GLP-1 actions on postprandial satiety. Information about the discovery, structure of the precursor mRNA/gene, distribution of the mRNA, processing, receptors, and conformation is provided in the Chapter 145 by Holst in the GI Peptides section of this book.

EFFECTS OF GLP-1 ON FEEDING BEHAVIOR The worsening global epidemic of obesity has increased the urgency to understand the mechanisms of appetite regulation. One important aspect is the interaction between hormonal signals released from the gut in response to a meal and appetite centers in the brain. Specifically, glucagonlike peptide 1 (GLP-1) has been implicated in signaling satiety postprandially. GLP-1 concentrations within the physiological range reduced food intake by 21% in lean subjects [16, 37, 60]. In humans, GLP-1 infusion decreased postprandial feelings of hunger [33, 39], suggesting a decreased rate of entry of nutrients into the circulation by reducing the gastric emptying rate [17]. GLP-1 infusion rate was found to be the only independent predictor of the reduction in energy intake in both lean and obese subjects [60]. In healthy subjects and type 2 diabetes melHandbook of Biologically Active Peptides

STUDIES FROM GENETIC MANIPULATIONS (KNOCKOUTS, TRANSGENICS) AND SYNTHETIC ANALOGS Mice with a targeted disruption in the GLP-1R (GLP1R−/−) gene are viable and do not exhibit disturbances on body weight [53, 54]. GLP-1R−/− display delayed satiety, suggesting that GLP-1 signaling may be required for central control of postprandial satiety [55]. GLP-1 is inactivated rapidly in vivo and does not appear to be useful as a therapeutic option. Agents acting on GLP-1R have been found (such as exendin-4) or developed as GLP-1 derivatives (such as NN2211 or liraglutide, albugon, or GLP-1/CJC-1131). Exendin-4, a 39amino-acid peptide, is produced in the salivary gland of

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976 / Chapter 133 the Gila monster lizard. It has 53% homology to GLP-1 and causes a sustained reduction in food intake and weight gain [40]. In contrast, transgenic overexpression of exendin-4 is not associated with a sustained reduction in food intake or body weight [4]. Mode and time of delivery may explain the different responses to the peptide analog. Liraglutide NN2211, a long-acting injectable GLP-1 derivative, causes a profound inhibition of food intake and change of body weight. Adverse reactions after liraglutide administration are similar to those observed with GLP-1, and symptoms are usually transient [34]. Albugon, a human GLP-1–albumin recombinant protein, increases GLP-1R–dependent cAMP levels, causing a reduction in food intake after both intracerebroventricular (ICV) and intraperitoneal administration [5]. In contrast, the GLP-1 analog (CJC1131) does not significantly lower body weight [28], suggesting that this conjugate may not be transported across the blood–brain barrier, compromising its ability to reduce food intake in vivo.

FUNCTIONAL RESPONSE OF THE GLP-1 PEPTIDE/GENE TO DIFFERING METABOLIC AND FEEDING STATES Fasting levels of total GLP-1 are in the range 5– 10 pmol/liter to 25 pmol/liter after the ingestion of a mixed meal. GLP-1 is released in response to fat- [15] and carbohydrate-rich meals [12, 25, 40], especially glucose [25], from open-type L-endocrine cells distributed throughout the small and large intestine [37]. Other mechanisms such as neural transmission [25, 37, 40], paracrine activation of L cells by gastric inhibitory polypeptide (GIP) [46], and muscarinic receptors [31] may also be involved in mediating GLP-1 secretion. GLP-1 secretion is exaggerated in patients who undergo total gastrectomy, suggesting that intestinal exposure to nutrients is the major stimulus for L-cell activation [37]. After secretion, the GLP-1 is rapidly cleaved by the plasma enzyme dipeptide-peptidase IV (DPP4) into the metabolite GLP-1(9–36) amide, which possesses antagonistic properties to the GLP-1R [37]. The half-life for intact GLP-1(7–36) amide is 2 min, and 5 min for the metabolite (9–36) amide. The hepatoportal bed seems to be the site of major degradation of both amides, followed by renal extraction [37]. In obese subjects, GLP-1 secretion is thought to be reduced after a carbohydrate load, whereas fat ingestion evokes similar GLP-1 responses as with normal-weight subjects [45, 61]. A recent study has shown a similar GLP-1 response to glucose and fat between obese and lean subjects [15]. Some studies have found differences in gastric emptying between obese and lean subjects, suggesting that differences in the rate of absorption may influence

GLP-1 secretion [26, 64]. T2DM and impaired glucose tolerance have been associated with a slight reduction in the GLP-1 secretory response to oral glucose, especially at lower glucose concentrations [37, 57]. Differences in glucose concentrations could have a direct influence on the GLP-1 release. Although ATPdependent K+ channels have been described in GLP1–producing L cells [21], sulfonylurea pretreatment does not influence GLP-1 secretion. The administration of the lipase inhibitor orlistat has been shown to reduce GLP-1 secretion in patients with T2DM, implying that intraluminal fat digestion is required for normal release of GLP-1 [44]. First-degree relatives of patients with T2DM have normal GLP-1 secretion [37].

SITES OF ACTION AND NEURAL NETWORKS AFFECTED BY GLP-1 It is unclear whether the anorectic effects GLP-1 are mediated by direct action in the central nervous system or are related to the deceleration of gastric emptying. Anorexia induced by peripheral administration of GLP1 seems to involve vagal control of gastric motility [63], inhibition of gastrin secretion, and stimulation of somatostatin [14]. Decreased gastric motility may constitute a prandial satiety signal, mediated via vagal nerve fibers that terminate in the caudal medial and commissural part of the nucleus of the solitary tract (NTS) [29]. GLP-1 may cross the brain–blood barrier [31] directly by passive diffusion or by binding to the subfornical organ and area postrema, followed by transport into the brain by the choroid plexus, which has a high-density GLP-1R [2]. Centrally, GLP-1 acts as a potent dosedependent hypothalamic inhibitor of feeding behavior [11, 36]. GLP-1 acts modulating feeding in neurons sensitive to monosodium glutamate treatment (MSG) [31], not only in the paraventricular nuclei (PVN) [35] but also in other medial hypothalamic loci as well as in the lateral hypothalamus. The inhibitory role of GLP-1 can be demonstrated by specific GLP-1R blockade [50, 51]. Central administration GLP-1 leads to taste aversion [31], but, because this latter effect is unaffected by MSG treatment, it further stresses the specificity of central GLP-1–induced anorexia.

GLP-1 RECEPTORS AND SIGNALING PATHWAYS RESPONSIBLE FOR THE INGESTIVE EFFECTS GLP-1 meets the major criteria required for a neuropeptide [7]. It is synthesized from preproglucagon by noncatecholaminergic and leptin receptor positive neurons of NTS and the dorsal and ventral part of the

Regulation of Feeding Behavior by Glucagonlike Peptide 1 (GLP-1) / 977 reticular nucleus [31, 38]. GLP-1 is present in the synaptosome fraction, and potassium induces its release in a calcium-dependent manner [23, 30]. Chromatographic analysis of preproglucagon in the neurons of the NTS confirmed that processing of GLP-1 is likely to be mediated by prohormone convertase PC1/3 [31]. NTS neurons target numerous forebrain structures, including hypothalamic paraventricular (PVN) and dorsomedial nuclei involved in regulation of body weight and energy homeostasis. Interestingly, the subcortical diencephalic nucleus has all GLP-1 binding sites with a remarkable lack of GLP-1 binding in the neocortex and the hippocampus [31]. All of the nuclei containing GLP-1 binding sites also express high levels of mRNA encoding the GLP-1R. GLP-1R gene gives rise to a seven-transmembrane-domain (GLP-1R) protein with a regional distribution on β-cells of the pancreatic islets of Langerhans, as well as in various other tissues including the lung, heart, kidney, gastrointestinal tract, and certain areas of the brain [8, 32]. In rodents, specific binding of GLP-1(7–36) amide is detected in the nucleus of the hypothalamus and the NTS [7]. The expression of the GLP-1R gene in rodents gives rise to a protein [2] with effects on selective release of neurotransmitters that control appetite and water intake [41, 59]. In humans [62], GLP-1R is present in the cerebral cortex, caudate and putamen nucleus, hypothalamus including the ventromedial and arcuate nuclei (Arc), thalamus, and globus pallidum [1]. GLP-1R is functionally coupled to increase in intracellular cAMP and intracellular calcium signaling [24]. Glucagon is also a full agonist but is 200-fold less potent than GLP1(7–36) amide in stimulating the human GLP-1R [20]. This GLP-1–receptor interaction is characterized by a high affinity, specificity, and saturability. Corticotropin-releasing hormone (CRH) or hypothalamic neuronal histamine mediates the GLP-1induced suppression of feeding behavior [19], possibly by inhibiting the postsynaptic signal initiated by neuropeptide Y (NPY) [52, 68] in the PVN, via agouti-related protein (AgRP)-independent pathways [13] and not by suppression of NPY synthesis in the Arc [27]. Catecholaminergic neurons in the area postrema and the caudal portion of the NTS receive afferents from the gastrointestinal and cardiovascular system via the vagal and glossopharyngeal nerves. It is likely that interaction with vagal afferents and hepatoportal sensors may link peripheral GLP-1 to central autonomic control sites [56, 65]. GLP-1 activates c-fos expression in the rat PVN and central nucleus of the amygdala [31, 59], it increases the release of aspartate and glutamate on the ventromedial hypothalamus [9], and it decreases the levels of tryptophan and serotonin [42]. It has been suggested that the glucose transporter 2 (GLUT2), glucokinase (GK), and GLP-1R are expressed

in many of the same cells of the human hypothalamus and are located in areas involved in the regulation of energy homeostasis, feeding behavior, and glucose metabolism [1, 48]. Accordingly, increased glycemia after meals may be recognized by these hypothalamic cells. Because GLP-1R, GLUT2, and GK are proteins involved in the multistep process of glucose sensing in pancreatic β-cells, the colocalization of specific GLP-1R and glucose-sensing-related proteins in hypothalamic neurons supports a role for this peptide in the hypothalamic regulation of macronutrient intake.

INTERACTIONS OF GLP-1 WITH OTHER PEPTIDERGIC/AMINERGIC SYSTEMS Central administration of the anorectic neuropeptide GLP-1 activates the central CRH-containing neurons of the hypothalamo-pituitary-adrenocortical axis by increasing c-fos expression, as well as oxytocinergic neurons of the hypothalamo-neurohypophysial tract [31]. Thus, GLP-1R may represent an upstream component of the systems regulating the CRH–adrenocorticotropic hormone (ACTH)–adrenal axis. Consistent with this hypothesis, GLP-1 administration increases corticosterone secretion in rats [31] and cortisol in human subjects [49]. GLP-1 also has a potential role in the regulation of thyrotrophin release, which appears to involve direct actions of the peptide on the anterior pituitary gland [6]. Leptin stimulates GLP-1 secretion from rodent and human intestinal L cells [3] and increases hypothalamic GLP-1 in food-restricted mice [37]. Although leptin and GLP-1 actions overlap in the brain and the endocrine pancreas, disruption of GLP-1 signaling does not modify the response to leptin or the phenotype of leptin deficiency [55], suggesting that GLP-1 does not mediate leptin action in the hypothalamus.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS The administration of GLP-1 has been associated with a sustained weight loss by regulation of food intake in both normal subjects and subjects with T2DM [60]. The postprandial rise in GLP-1 levels may act as a meal-termination signal by increasing the feeling of fullness caused by delayed gastric emptying. It is likely that the postprandial rise in plasma GLP-1 levels in both normal and T2DM subjects plays an important role in the termination of meal ingestion. On the basis of these findings, there is no reason to suspect a defective GLP-1 action plays a major role in the pathogenesis of T2DM. If GLP1 causes postprandial satiety, we may speculate that GLP-

978 / Chapter 133 1 levels are decreased in obese subjects. In contrast, results regarding plasma GLP-1 concentrations in obesity have been conflicting [18, 37, 45]. Thus, we cannot conclude that alterations in GLP-1 secretion are the cause of the development of obesity. Although alterations in GLP1 or GLP-1R expression and/or action do not play a role in the pathogenesis of T2DM, maturity onset diabetes of the young [67], or obesity, the positive effects of GLP-1 on food intake, satiety, and body weight make it an attractive candidate for treating these diseases.

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[44] Pilichiewicz A, O’Donovan D, Feinle C, Lei Y, Wishart JM, Bryant L, Meyer JH, Horowitz M, Jones KL. Effect of lipase inhibition on gastric emptying of, and the glycemic and incretin responses to, an oil/aqueous drink in type 2 diabetes mellitus. J Clin Endocrinol Metab 2003;88(8):3829–3834. [45] Ranganath LR, Beety JM, Morgan LM, Wright JW, Howland R, Marks V. Attenuated GLP-1 secretion in obesity: cause or consequence? Gut 1996;38(6):916–919. [46] Roberge JN, Brubaker PL. Regulation of intestinal proglucagonderived peptide secretion by glucose-dependent insulinotropic peptide in a novel enteroendocrine loop. Endocrinology 1993;133(1):233–240. [47] Rodriquez de Fonseca F, Navarro M, Alvarez E, Roncero I, Chowen JA, Maestre O, Gomez R, Munoz RM, Eng J, Blazquez E. Peripheral versus central effects of glucagon-like peptide-1 receptor agonists on satiety and body weight loss in Zucker obese rats. Metabolism 2000;49(6):709–717. [48] Roncero I, Alvarez E, Chowen JA, Sanz C, Rabano A, Vazquez P, Blazquez E. Expression of glucose transporter isoform GLUT2 and glucokinase genes in human brain. J Neurochem 2004;88(5):1203–1210. [49] Ryan AS, Egan JM, Habener JF, Elahi D. Insulinotropic hormone glucagon like peptide-1 (7-37) appears not to augment insulin mediated glucose uptake in young men during euglycemia. J Clin Endocrinol Metab 1998;83(7):2399–2404. [50] Schick RR, Zimmermann JP, vorm Walde T, Schusdziarra V. Glucagon-like peptide 1 (7–36)-amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. Am J Physiol Regul Integr Comp Physiol 2003;284:627–632. [51] Schick RR, Zimmermann JP, vorm Walde T, Schusdziarra V. Peptides that regulate food intake: glucagon-like peptide 1-(7– 36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. Am J Physiol Regul Integr Comp Physiol 2003;284(6):R1427–1435. [52] Schusdziarra V, Zimmermann JP, Schick RR. Importance of orexigenic counter-regulation for multiple targeted feeding inhibition. Obes Res 2004;12(4):627–632. [53] Scrocchi LA, Brown TJ, MaClusky N, Brubaker PL, Auerbach AB, Joyner AL, Drucker DJ. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med 1996;2(11):1254–1258. [54] Scrocchi LA, Drucker DJ. Effects of aging and a high fat diet on body weight and glucose tolerance in glucagon-like peptide-1 receptor −/− mice. Endocrinology 1998;139(7):3127–3132. [55] Scrocchi LA, Hill ME, Saleh J, Perkins B, Drucker DJ. Elimination of glucagon-like peptide 1R signaling does not modify weight gain and islet adaptation in mice with combined disruption of leptin and GLP-1 action. Diabetes 2000;49(9):1552–1560. [56] Tang-Christensen M, Vrang N, Larsen PJ. Glucagon-like peptide containing pathways in the regulation of feeding behaviour. Int J Obes Relat Metab Disord 2001;25 Suppl (5):S42–47. [57] Toft-Nielsen MB, Damholt MB, Madsbad S, Hilsted LM, Hughes TE, Michelsen BK, Holst JJ. Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients. J Clin Endocrinol Metab 2001;86(8):3717–3723. [58] Toft-Nielsen MB, Madsbad S, Holst JJ. Continuous subcutaneous infusion of glucagon-like peptide 1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care 1999;22(7):1137–1143. [59] Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CM, Meeran K, Choi SJ, Taylor GM, Heath MM, Lambert PD, Wilding JP, Smith DM, Ghatei MA, Herbert J, Bloom SR. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379(6560):69–72. [60] Verdich C, Flint A, Gutzwiller JP, Naslund E, Beglinger C, Hellstrom PM, Long SJ, Morgan LM, Holst JJ, Astrup A. A

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[65] Yamamoto H, Kishi T, Lee CE, Choi BJ, Fang H, Hollenberg AN, Drucker DJ, Elmquist JK. Glucagon-like peptide-1responsive catecholamine neurons in the area postrema link peripheral glucagon-like peptide-1 with central autonomic control sites. J Neurosci 2003;23(7):2939–2946. [66] Zander M, Madsbad S, Madsen JL, Holst JJ. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallelgroup study. Lancet 2002;359(9309):824–830. [67] Zhang Y, Cook JT, Hattersley AT, Firth R, Saker PJ, Warren-Perry M, Stoffel M, Turner RC. Non-linkage of the glucagon-like peptide 1 receptor gene with maturity onset diabetes of the young. Diabetologia 1994;37(7):721–724. [68] Zhu Y, Yamanaka A, Kunii K, Tsujino N, Goto K, Sakurai T. Orexin-mediated feeding behavior involves both leptin-sensitive and -insensitive pathways. Physiol Behav 2002;77(2–3):251– 257.

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134 Role of Amylin and Calcitonin-Gene-Related Peptide (CGRP) in the Control of Food Intake THOMAS A. LUTZ

(CGRP), studies on the long-term effect of CGRP on food intake have not been performed so far. Amylin, or islet amyloid polypeptide (IAPP), was first isolated from pancreatic amyloid deposits, which typically occur in human type 2 diabetes mellitus, in feline diabetes mellitus, and in insulinomas in animals and humans. Amylin, however, is also a physiological product of pancreatic B cells from where it is co-secreted with insulin. Although not proven unequivocally, it is likely that the pancreatic B cells constitute the main source for circulating and postprandially released amylin. Pancreatectomized cats have virtually no meal-induced release of amylin and no postprandial increase in blood amylin levels (unpublished research). Unfortunately, no measures of food intake or meal pattern were determined in these studies. In rats, food intake results in a marked increase in the plasma amylin concentration. This meal-induced increase is correlated to the size of the pertinent meal and occurs in less than 5 min after meal onset. Although this classifies amylin as a potential satiating hormone, blood levels of CGRP show only a modest and late increase in response to food intake. Therefore, a role for circulating CGRP in satiation appears unlikely. The source for this increase in circulating CGRP is not completely clear, but CGRP may reach the blood by spillover from neuronal cells.

ABSTRACT This chapter focuses on the anorectic action of the pancreatic hormone amylin and its structurally related peptide calcitonin-gene-related peptide (CGRP). Both peptides produce potent anorectic effects and delay gastric emptying. The anorectic action of CGRP seems to parallel amylin’s effect on food intake in some aspects. In this chapter, more emphasis is put on the effects of amylin on food intake because numerous studies suggest that amylin’s effects may be of physiological relevance. This, however, has been elaborated less extensively and is clearly less certain for CGRP. Other effects of CGRP are mentioned in Chapter 138 in the Gastrointestinal Peptides Section of this book.

INTRODUCTION The anorectic action of amylin is one important factor in amylin’s overall role to control the influx of nutrients into the circulation. Apart from its anorectic effect, amylin’s action to reduce gastric acid secretion, to limit the rate of gastric emptying, and to diminish pancreatic glucagon and digestive enzyme secretion are other factors serving the same purpose [14, 22, 29]. Therefore, by regulating nutrient appearance and the postprandial glucose concentration, amylin seems to be a necessary and complementary factor to insulin in the control of nutrient flux. The best-investigated function of amylin in this context is its role as a hormone contributing to mealending satiation [14]. In addition to the immediate short-term effect of amylin to control meal size, several studies suggest that amylin may also be involved in the long-term control of food intake and body weight, similar to the established lipostatic factors leptin and insulin [13, 14, 27]. Whereas the former effects of amylin can in part be reproduced with calcitonin gene-related peptide Handbook of Biologically Active Peptides

CHARACTERIZATION OF THE EFFECTS OF AMYLIN AND CGRP ON FOOD INTAKE Amylin shares the typical characteristics of satiating hormones that are involved in the control of meal size. Amylin is released during food ingestion, and it dosedependently reduces meal size. Further, after intraportal or intraperitoneal injection, amylin has a rapid onset and brief duration of action.

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982 / Chapter 134 During chronic amylin infusion [1, 15], amylin not only reduced food intake by decreasing average meal size, but in some studies it also reduced meal number by increasing the duration of the intermeal interval. Similarly, amylin increases the latency to eat under certain experimental conditions. The latter effects may be taken as evidence that amylin not only acts as a satiating hormone but that it may also have an effect on postprandial satiety. However, neither chronically elevated amylin levels nor an acute increase in amylin prior to meal onset mimics the natural situation because physiologically amylin is acutely released during a meal. Therefore, the best characterized function of amylin is to reduce meal size. It remains to be investigated whether amylin also has an effect on postprandial satiety [14]. Qualitatively, CGRP reduces food intake by affecting the same meal pattern parameters as amylin; that is, CGRP has also been shown to reduce meal size after acute and chronic administration and to affect the intermeal interval during chronic infusion. Due to its release pattern, with an increase of plasma CGRP levels occurring only at least 1 h after meal onset, it appears to be rather unlikely that blood-borne CGRP constitutes a physiological satiating agent [10]. When performing meal pattern analysis after the injection of amylin or CGRP, another major difference was demonstrated in that higher doses of CGRP reduced the eating rate. This and other studies suggest that some aversive or illness-producing effect may at least partly underlie the anorectic action of CGRP, indicating that its action on feeding is not specific. This contrasts with the specific anorectic action of amylin because it has been shown repeatedly that amylin does not reduce feeding by producing a conditioned taste aversion or by an unspecific effect (e.g., via a reduction in drinking) [14, 20]. The lowest dose of exogenous amylin that produced a significant reduction in feeding yielded plasma amylin levels that were about two times higher than the concentrations measured postprandially [1]. Physiological amylin concentrations that occur at the end of spontaneous meals have not yet been shown to reduce feeding. This may be related to different kinetics of the blood amylin concentration after exogenous amylin delivery rather than endogenous secretion. Nevertheless, it is conceivable that amylin is a physiological regulator of meal size because several studies have clearly shown that peripherally or centrally delivered amylin antagonists produce an effect opposite to that of amylin—an increase in meal size. The role of amylin as a satiating hormone seems to depend on the meal-induced rise in plasma amylin levels and is mediated by neurons in the area postrema (AP). In addition to this immediate satiating effect, amylin may play a role in the long-term control of food

intake and/or body weight [13, 15, 27]. Similar to the lipostatic signals leptin and insulin, the basal levels of amylin, being co-secreted with insulin, depend on the prevailing body weight and body adiposity. In rats, chronic amylin infusion lowers food intake and body weight gain by reducing body adiposity, whereas chronic peripheral or central infusion of amylin antagonists increases body weight with a major effect on body fat mass while sparing lean body mass [15, 23, 27]. Therefore, the effect of a chronic modulation of amylin signaling is similar to that of leptin or insulin. The relative contribution of amylin versus leptin or insulin to the control of body weight is difficult to judge. However, even animals with defective leptin and insulin signaling (e.g., the Zucker fa/fa rat) keep their body weight relatively stable, even though this occurs at a higher level. When testing the effect of the amylin antagonist AC187 on food intake and body weight in the obese Zucker fa/fa rat as a model of basal hyperamylinemia, AC187 increased food intake in obese fa/fa rats but not in lean control animals, which have low baseline amylin levels. Amylin may therefore play some role as a lipostatic feedback signal similar to leptin and insulin, at least when the leptin and insulin feedback signaling systems are deficient [14].

FOOD INTAKE IN KNOCKOUT ANIMALS Amylin knockout animals have been developed to study the role of amylin in nutrient and bone metabolism [11, 21]. These animals have also been used to study the physiological role of amylin in the control of food intake and body weight. Although adult body weight did not differ between knockout and wild-type animals in some studies, amylin knockout mice show a higher rate of body weight gain compared with corresponding wild-type controls. The knockout mice grew significantly faster up to approximately 4 months of age. Food intake was slightly, although not significantly, higher than in control animals [14]. Interestingly, amylin knockout mice also differed from control animals in their interaction with other factors controlling food intake [18] because the satiating effect of cholecystokinin (CCK) was abolished in amylin knockout mice. This was restored by acute injection of subthreshold doses of amylin. Together, these studies suggest a role for endogenous amylin in the overall control of energy balance, most likely via an effect on food intake [18, 23, 27]. Food intake in amylin receptor knockout animals has not been determined so far. A recent study determined the effect of haplodeletion of amylin and its receptor protein (calcitonin receptor, CT-R) on bone metabolism. Heterozygous amylin+/− and heterozygous CT-R+/− mice were crossbred, but

Role of Amylin and Calcitonin-Gene-Related Peptide (CGRP) in the Control of Food Intake / 983 neither data on food intake nor on body weight were reported [21].

FOOD INTAKE IN ANIMALS OVEREXPRESSING AMYLIN To my knowledge, no detailed studies are available on the effect of amylin overexpression on food intake and/or body weight [21]. One recent study reported that rats that were transgenic for the overexpression of human amylin, had a slightly lower body weight than wild-type controls. This is, in principle, consistent with the effects of exogenous amylin on body weight. The rather small difference between transgenic and control animals may have been due to receptor desensitization or the largely redundant system controlling body weight. Because the major difference in body weight between amylin knockout and wild-type mice occurs during a certain period of life, the same may also hold true for animals overexpressing amylin.

FOOD INTAKE IN ANIMALS DEFICIENT OF CGRP OR OVEREXPRESSING CGRP To my knowledge, no detailed data are available related to the effect of genetic CGRP deficiency or CGRP overexpression on food intake or body weight [21].

REGULATION OF AMYLIN AND CGRP SYNTHESIS AND SECRETION The rapid four- to fivefold meal-related increase in plasma amylin levels seems to be mainly derived from pancreatic amylin secretion. Due to their co-synthesis in pancreatic B cells and co-localization in the same secretory vesicles, insulin and amylin are normally cosecreted on activation by the appropriate stimuli (i.e., nutrients, incretin hormones, or neural input). Under physiological conditions, the secretions of amylin and of insulin occur at a fixed ratio of approximately 1 : 10 to 1 : 100. Interestingly, however, divergent secretion of insulin and amylin is possible. It has, for example, been shown that human obesity, various rat models of obesity, diabetes mellitus, pancreatic cancer, and pharmacological intervention (e.g., dexamethasone) lead to relative oversecretion of amylin versus insulin. The exact mechanisms underlying this divergence are currently unknown. It seems, however, that the amylin : insulin ratio at the level of mRNA is similar to that of circulating peptides. Thus, the synthesis and secretion of the peptides do not diverge, and differential regulation of

amylin and insulin in insulin resistance and diabetes can also be observed at the level of mRNA. Clear evidence for a preferential transcription of amylin or increased stability of amylin mRNA under these conditions has not been found so far. It has, however, been hypothesized that differential regulation of amylin and insulin mRNA production may involve the cAMP and protein kinase A (PKA) pathways. Via multiple signaling pathways, different transcription factors for insulin and amylin may be involved. Obese individuals have been reported to have higher CGRP levels than lean controls [10], but a direct link between baseline CGRP levels and the degree of adiposity seems to be unclear because CGRP levels did not decrease in response to marked body weight loss. Plasma levels of CGRP increase in response to food intake mainly by spillover from sensory nerve endings rather than controlled release from endocrine cells. This was only seen after a high-fat meal but not after a high-carbohydrate meal, and the late increase argues against an effect of CGRP on individual meals. Whether CGRP contributes to the control of the duration of the intermeal interval, and hence meal initiation, is currently unknown.

CENTRAL PATHWAYS MEDIATING THE ANORECTIC EFFECTS OF AMYLIN AND CGRP Most experimental evidence supports the idea that the anorectic effect of peripheral amylin is mediated by direct humoral action on the AP in the hindbrain [15, 16, 17, 19]. While amylin binding sites have been described in various central nervous system (CNS) locations involved in the regulation of food intake, bloodborne amylin has easy access to AP neurons due to the lack of a functional blood–brain barrier in this area. After acute or chronic peripheral administration, amylin’s anorectic action is abolished in rats with lesions in the AP–nucleus tractus solitarius (NTS) region. The importance of the AP for mediating the anorectic effect of exogenous but also of endogenous amylin was demonstrated in a study showing that an infusion of amylin into the AP potently reduced feeding, whereas infusion of the amylin antagonist AC 187 increased food intake due to an increase in meal size. AC 187 infused into the AP also partly blocked the anorectic effect induced by a peripheral amylin injection. These experiments provide the best in vivo evidence for the importance of the AP in mediating peripheral amylin’s anorectic action [17]. Feeding studies were complemented by electrophysiological and immunohistochemical studies underlining the role of the AP [24–26]. A direct dose-dependent

984 / Chapter 134 stimulating effect of amylin on AP neurons underlines their sensitivity to amylin. Further, peripheral amylin produced a strong expression of the immediate early gene product Fos protein as a marker of neuronal activation in AP neurons. Both responses were effectively blocked by AC187. Similar to exogenously applied amylin, refeeding (2-h access to food following a 24-h food-deprivation period) also caused substantial neuronal activation in the AP. Refeeding-induced Fos activation in the AP is presumably caused by the release of endogenous amylin because AC187 significantly reduced the Fos expression in AP neurons of refed rats. In addition to the AP, amylin also elicits a strong Fos response in the NTS, the lateral parabrachial nucleus (lPBN), the central nucleus of the amygdala (CeNA), and the bed nucleus of the stria terminalis (BNST) [25, 26]. The AP, NTS, and the lPBN form a necessary part of the CNS pathway conveying amylin’s anorectic signal to higher brain structures, but their activation seems to be synaptically mediated and secondary to an action of amylin on AP neurons. Endogenous amylin as released by refeeding also caused substantial neuronal activation in the AP, NTS, lPBN, and CeA. At least in the AP, AC187 antagonized the feeding-induced activation. The site of action for CGRP’s anorectic action has been investigated in less detail than that of amylin. Similar to amylin, the anorectic action of CGRP administered peripherally (but not centrally into the lateral brain ventricle) is abolished in rats with an AP lesion [16]. Immunohistochemical and electrophysiological studies have not been performed to the same extent as for amylin, so it is difficult to integrate these findings into the whole brain network for the physiological control of food intake. An open question is the importance of forebrain receptors in mediating the anorectic action of amylin. Amylin infusion into the third brain ventricle produces a very potent and long-lasting reduction in feeding, and third ventricular infusion of AC187 increases food intake in rats [6, 27]. This was associated with a modest increase in body weight and a marked increase in the weight of the retroperitoneal fat pads by more than 30%. However, at present there is no substantial evidence to suggest that forebrain amylin receptors are involved in the regulation of food intake by peripheral amylin. Because CNS production of amylin has not been shown unequivocally, the endogenous ligand of forebrain amylin receptors, as well as their role in amylin’s effect on food intake, remains unknown. A recent study investigated the effect of an injection of CGRP into the paraventricular hypothalamic nucleus (PVN), which has been shown to contain CGRP binding sites [8]. CGRP produced a strong anorectic effect under these conditions, and it increased the concentration of various anorectic neuropeptides in the

hypothalamus, such as alpha-melanocyte-stimulating hormone, cocaine- and amphetamine-regulated transcript, and corticotrophin-releasing factor. However, very high doses of CGRP have been used in this study, exceeding effective concentrations (EC50) for amylin to elicit an anorectic effect after its administration into the third brain ventricle by a factor of more than 200. Further, the PVN content of CGRP was elevated in response to fasting, which is difficult to reconcile with the idea of CGRP acting as an anorectic hormone or neuropeptide. Therefore, the physiological relevance of these findings and the role of CGRP as a brainintrinsic neuropeptide in the regulation of feeding is not clear. Overall, the forebrain has binding sites for both amylin and CGRP, and the activation of these receptors produces a potent reduction in food intake and, if administered chronically, body weight. Nonetheless, the physiological ligand(s) of these binding sites and the physiological relevance of these effects remains to be determined in future studies.

HYPOTHALAMIC INVOLVEMENT IN THE ANORECTIC ACTION OF PERIPHERAL AMYLIN The hypothalamus is intimately interconnected with the AP-NTS-lPBN axis, which is activated by amylin [3, 25]. An involvement of the lateral hypothalamic area (LHA) in amylin’s anorectic effect seems likely because the fasting-induced Fos activation in LHA neurons was reversed both by refeeding rats and by injecting amylin in rats without access to food. The amylin-induced inhibition of LHA neurons may coincide with a downregulation of the expression of orexin and melaninconcentrating hormone (MCH) in the LHA. However, the true phenotype of LHA neurons, whose fastinginduced activation is downregulated by amylin, remains to be determined.

RECEPTORS AND SIGNALING PATHWAYS Amylin and CGRP binding sites belong to the family of G-protein-coupled receptors. The functional amylin receptor involves the calcitonin receptor (CT-R) as a core receptor, whose amylin specificity and affinity are due to the co-expression of one of several receptoractivity-modifying proteins (RAMPs). The CT-R without co-expression of RAMPs represents the classical CT receptor, whereas a typical amylin receptor arises from the interaction of RAMP1 or RAMP3 with the CT-R. A similar principle leads to functional receptors for CGRP [9]. These rely on the co-expression of the calcitonin receptorlike receptor (CL-R) with appropriate RAMPs.

Role of Amylin and Calcitonin-Gene-Related Peptide (CGRP) in the Control of Food Intake / 985 Amylin binding sites are widely distributed in the CNS [28] and also occur in high densities in brain areas involved in the regulation of food intake. Consistent with the studies discussed before, the AP contains all components of functional amylin receptors. RAMP1 and RAMP3 mRNA are present in the mouse and rat AP [4], and there is an almost complete overlap between amylin-sensitive AP neurons and neurons carrying the CT-R [5]. Preliminary immunohistochemical studies also indicate the presence of RAMP1 and RAMP3 protein in the rat AP. Detailed studies of the presence of CL-R protein as part of functional CGRP receptors in the brain, and especially in brain areas involved in the control of food intake, are not available. However, in situ hybridization studies have shown that CL-R mRNA is present in the rat brain. Other than in the AP, amylin and CGRP binding sites can be found throughout the CNS [28]. As discussed before, however, the physiological relevance of these binding sites for the anorectic action of amylin and CGRP remains unknown.

INTERACTION WITH OTHER PEPTIDES REGULATING INGESTIVE BEHAVIOR Subthreshold doses of peripheral insulin and amylin are capable of reducing food intake in rats when coadministered [14, 18]. Further, amylin and CCK or bombesin (BN), all being released in response to food intake, also seem to interact in their effect on food intake. Part of the satiating actions of CCK and BBS seems to be mediated by amylin because amylin antagonists attenuated their anorectic actions in rats. In addition, the anorectic effects of CCK and BN were almost abolished in amylin knockout mice. These results point to an important role of endogenous amylin in mediating CCK’s and BN’s anorectic effects. Amylin may have a neuromodulator function within the AP-NTS region regarding incoming satiating signals, such as those elicited by peripheral CCK and BN. This is consistent with the observation that amylin and CCK interact synergistically to reduce feeding in mice.

INTERACTION BETWEEN AMYLIN AND METABOLITES Higher doses of amylin are necessary to inhibit sham feeding than real feeding [2], which may be due to the lack of cooperative effects between amylin and metabolic cues under these conditions [24]. In line with this suggestion is the observation that physiological concentrations of amylin and glucose activate the same neurons

in the AP, the primary receptive site for peripheral amylin’s anorectic effect. The co-responsiveness of AP neurons to amylin and glucose is similar to that for glucose and CCK. AP neurons therefore seem to integrate metabolic (e.g., glucose) and hormonal (e.g., amylin or CCK) signals in the control of food intake and meal size.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS At present, only little is known about the possible role of amylin’s effect on feeding under pathophysiological conditions. It has been hypothesized that amylin contributes to cancer anorexia occurring during certain pancreatic neoplastic diseases that are associated with chronically supraphysiological plasma amylin levels. Further, an altered ratio between amylin and insulin may also play a role in eating disorders and hence contribute to abnormal feeding behavior in diabetes mellitus. The lack of amylin in type 1 diabetes and later stages of type 2 diabetes could result in higher food intake and/or body weight. Interestingly, recent clinical studies in humans showed that the amylin analog pramlintide causes a decrease in food intake and body weight in obese or diabetic patients [7, 12].

CONCLUSION In conclusion, both amylin and the structurally related neuropeptide CGRP reduce food intake. Whereas amylin may be a physiological regulator of meal size, this is less clear for CGRP. The effects of peripheral amylin and CGRP on food intake are centrally mediated, with the AP in the hindbrain as the most likely primary target area. Chronic exposure to amylin decreases food intake and body weight. Next to a satiating action, amylin may also constitute one of the body’s lipostatic feedback signals. The anorectic action of amylin is being pharmacologically exploited because amylin analogs are under clinical consideration for their effect in reducing food intake and body weight in humans.

References [1] Arnelo U, Reidelberger R, Adrian TE, Larsson J, Permert J. Sufficiency of postprandial plasma levels of islet amyloid polypeptide for suppression of feeding in rats. Am J Physiol 1998; 275: R1537–R1542. [2] Asarian L, Eckel LA, Geary N. Behaviorally specific inhibition of sham feeding by amylin. Peptides 1998; 19: 1711–1718. [3] Barth SW, Riediger T, Lutz TA, Rechkemmer G. Differential effects of amylin and salmon calcitonin on neuropeptide gene

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[16] Lutz TA, Senn M, Althaus J, Del Prete E, Ehrensperger F, Scharrer E. Lesion of the area postrema/nucleus of the solitary tract (AP/NTS) attenuates the anorectic effects of amylin and calcitonin gene-related peptide (CGRP) in rats. Peptides 1998; 19: 309–317. [17] Mollet A, Gilg S, Riediger T, Lutz TA. Infusion of the amylin antagonist AC 187 into the area postrema increases food intake in rats. Physiol Behav 2004; 81: 149–155. [18] Mollet A, Meier S, Grabler V, Gilg S, Scharrer E, Lutz TA. Endogenous amylin contributes to the anorectic action of cholecystokinin and bombesin. Peptides 2003; 24: 91–98. [19] Morley JE, Flood JF, Horowitz M, Morley PMK, Walter MJ. Modulation of food intake by peripherally administered amylin. Am J Physiol 1994; 267: R178–R184. [20] Morley JE, Suarez MD, Mattamal M, Flood JF. Amylin and food intake in mice: Effects on motivation to eat and mechanism of action. Pharmacol Biochem Behav 1997; 56: 123 –129. [21] Muff R, Born W, Lutz TA, Fischer JA. Biological importance of the peptides of the calcitonin family as revealed by disruption and transfer of corresponding genes. Peptides 2004; 25: 2027– 2038. [22] Reidelberger RD, Arnelo U, Granqvist L, Permert J. Comparative effects of amylin and cholecystokinin on food intake and gastric emptying in rats. Am J Physiol 2001; 280: R605–R611. [23] Reidelberger RD, Haver AC, Arnelo U, Smith DD, Schaffert CS, Permert J. Amylin receptor blockade stimulates food intake in rats. Am J Physiol 2004; 287: R568–R574. [24] Riediger T, Schmid HA, Lutz TA, Simon E. Amylin and glucose co-activate area postrema neurons of the rat. Neurosci Lett 2002; 328: 121–124. [25] Riediger T, Zünd D, Becskei C, Lutz TA. The anorectic hormone amylin contributes to feeding-related changes of neuronal activity in key structures of the gut-brain axis. Am J Physiol 2004; 286: R114–R122. [26] Rowland NE, Richmond RM. Area postrema and the anorectic actions of dexfenfluramine and amylin. Brain Res 1999; 820: 86–91. [27] Rushing PA, Hagan MM, Seeley RJ, Lutz TA, D’Alession DA, Air EL, Woods SC. Inhibition of central amylin signaling increases food intake and body adiposity in rats. Endocrinol 2001; 142: 5035 –5038. [28] Sexton PM, Paxinos G, Kenney MA, Wookey PJ, Beaumont K. In vitro autoradiographic localization of amylin binding sites in rat brain. Neurosci 1994; 62: 553–567. [29] Young AA, Gedulin B, Vine W, Percy A, Rink TJ. Gastric emptying is acelerated in diabetic BB rats and is slowed by subcutaneous injections of amylin. Diabetologia 1995; 38: 642–648.

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135 Leptin and the Regulation of Feeding MARTIN G. MYERS, JR.

ABSTRACT

THE ABSENCE OF LEPTIN ACTION

Leptin is an adipocyte-derived polypeptide hormone that acts in the central nervous system via the long form of its receptor (LRb) to inhibit feeding. Alterations in leptin levels during chronic changes in nutritional status regulate feeding and energy expenditure, increasing feeding and decreasing energy expenditure in low leptin states such as starvation. The major sites of leptin action are in the brain, and leptin regulates melanocortin and neuropeptide Y (NPY) tone in the hypothalamus. The activation of PI 3-kinase and STAT3 by LRb mediate specific aspects of leptin action. Alterations in central-nervous-system leptin action may contribute to the development of obesity.

Due to the decades-long existence of ob/ob mice, there is a long-standing literature regarding the phenotype of mice with loss-of-function mutations for leptin (or indeed of mice null for the leptin receptor, db/db mice). These ob/ob and db/db mice have indistinguishable phenotypes of hyperphagia, low metabolic rate, and consequent obesity. In addition, ob/ob and db/db mice display a stereotypical neuroendocrine starvation response (even in the face of nutrient excess and obesity) that is characterized by hypothyroidism, hypothalamic infertility, decreased growth, and decreased immune function [1]. In ob/ob, but not db/db, mice exogenous leptin therapy reverses all of these phenotypes. Similar phenotypes have been observed in rare human patients with null mutations for leptin or the leptin receptor [12]. Thus, leptin sufficiency not only suppresses feeding but increases metabolic rate and permits neuroendocrine energy expenditure; leptin deficiency results in increased drive to feed and initiates a series of energy-sparing events. Although leptin is produced primarily by adipocytes, it is also produced in smaller quantities by other tissues. Although no tissue-specific null mice have been generated to probe the physiologic relevance of leptin production by specific tissues, the phenotype of patients and rodent models with the almost complete lack of adipose tissue (lipodystrophy) is remarkably similar to the phenotype of leptin deficiency (with the exception of adipose mass) [25]. Lipodystrophy is characterized by low circulating leptin levels, increased feeding, decreased metabolic rate, and neuroendocrine dysfunction similar to that observed in starvation or genetic leptin deficiency. Indeed, most of these phenotypes are partially or completely remedied by treatment of lipodystrophy with exogenous leptin [25], suggesting that adipose tissue is indeed the main source of circulating leptin.

LEPTIN AND FEEDING BEHAVIOR Leptin was identified through the study of the spontaneous loss-of-function mutation (obese or ob gene) in mice that causes hyperphagia and obesity in the homozygous (ob/ob) state [13, 35]. Upon the positional cloning of the ob gene, it became clear that the gene product was a secreted polypeptide expressed primarily in adipocytes. This gene product was named leptin (Greek lepto “thin”), because the injection of leptin into various animals resulted in a profound anorexia (loss of appetite) and concomitant weight loss. Indeed, leptin is probably the most powerful anorectic hormone known and is a primary controller of many other central nervous system (CNS) peptides that regulate feeding and energy expenditure [11, 13]. Physiologically, leptin functions to communicate the status of body energy stores from adipocytes to the brain in order to control the intake and expenditure of calories [1, 28]. Leptin treatment results not only in decreased caloric intake but also in constant or increased energy expenditure. Handbook of Biologically Active Peptides

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988 / Chapter 135 FUNCTIONAL RESPONSES TO LEPTIN DURING ALTERED METABOLIC STATES In adipocytes, leptin production is regulated by nutritional status: generally speaking, more stored triglyceride results in greater production of leptin, whereas less stored fat results in decreased production of leptin [9]. The regulation of circulating leptin levels by bodyfat stores positions leptin well to function as an indicator of body energy stores. Thus, in times of inadequate energy stores (e.g., starvation) leptin levels fall; these low leptin levels enhance appetite and decrease energy use. Conversely, with adequate energy stores, high leptin levels decrease the drive to eat and permit increased use of energy. Leptin secretion is constitutive. Leptin is synthesized and extruded into the secretory pathway for release by mass action, as opposed to being packaged into secretory vesicles for release in response to an acute stimulus [9]. Thus, although there is modest diurnal variation in circulating leptin levels, leptin secretion and circulating leptin levels depend primarily on the rates of transcription and translation of the leptin mRNA, and circulating leptin levels vary little on a minute-to-minute or hour-to-hour basis. The alterations in leptin levels requiring prolonged (many hours to days) changes in nutritional status predict that leptin acts to set the chronic tone of the feeding and neuroendocrine systems, as opposed to acutely regulating the activity of these neural circuits (which is likely to be accomplished by more acutely regulated factors such as insulin and ghrelin).

appear to result from direct action on LRb-expressing T cells [20]. Within the Arc, LRb is found in at least two distinct populations of neurons: (1) neurons that co-express neuropeptide Y (NPY) and agouti-related peptide (AgRP) and (2) neurons that express proopiomelanocortin (POMC) [11, 28]. POMC is processed to α-melanocytestimulating hormone (α-MSH) in the LRb-POMC neuron. α-MSH mediates a powerful anorectic (appetitesuppressing) signal; LRb stimulates the expression of POMC and activates the LRb-POMC neuron [11, 28]. AgRP is an antagonist/inverse agonist of central melanocortin signaling, and NPY is itself an orexigenic (appetitestimulating) hormone that also acts to suppress the central LRb growth and reproductive axes [11, 28]. Leptin acts via LRb to inhibit the NPY-AgRP neurons and to suppress expression of these neuropeptides. Thus, LRb signaling stimulates the production of anorectic neuropeptides and suppresses levels of orexigenic peptides. Conversely, when leptin action is decreased or deficient (e.g., starvation or in ob/ob or db/db mice), appetite is stimulated via the suppression of anorectic neuropeptides (e.g., POMC-derived peptides) and by increased expression of orexigenic peptides (e.g., NPY and AgRP) [11, 28]. LRb-expressing Arc NPY-AgRP and/or POMC neurons also regulate energy expenditure and other elements of neuroendocrine function [11]. The neurochemical properties of LRb-expressing neurons in the DMH, VHM, and elsewhere (including the brainstem) are poorly defined.

LEPTIN RECEPTORS AND INTRACELLULAR SIGNALING PATHWAYS SITES OF LEPTIN ACTION Much of the action of leptin is attributable to effects in the central nervous system (CNS). CNS administration of leptin recapitulates the anorectic and neuroendocrine effects of peripherally applied leptin [13]. Furthermore, brain-specific transgenic expression of the long form of the leptin receptor (LRb) rescues the anorectic and neuroendocrine phenotypes of db/db mice [7], and brain-specific deletion of LRb increases feeding and adiposity [8]. Within the nuclei of the basomedial hypothalamus, including the arcuate (Arc), dorsomedial hypothalamic (DMH), and ventromedial hypothalamic (VMH) nuclei, leptin acts on neurons that regulate levels of circulating hormones (e.g., thyroid hormone, sex steroids, and growth hormone) [11]. Leptin action on these hypothalamic neurons also regulates the activity of the autonomic nervous system, although direct leptin action on brainstem LRb-expressing neurons may play a role in this and other leptin actions [11, 16]. Leptin actions on the immune system

Multiple LR isoforms exist; all are products of a single lepr gene and result from alternative mRNA splicing and/or proteolytic processing [6, 23]. LR isoforms fall into three classes: secreted, short, and long. The secreted forms are alternative splice products (e.g., the murine LRe) or proteolytic cleavage products of membrane-bound LR forms. These contain only extracellular leptin-binding domains and complex with circulating leptin, perhaps regulating free leptin concentrations [33]. Short-form (LRa, etc.) and long-form (LRb) receptors contain identical extracellular and transmembrane domains as well as the same first 29 intracellular amino acids and diverge in sequence secondary to alternative splicing of 3′-exons. LRb is highly conserved among species and possesses an intracellular domain of approximately 300 residues [6, 23]. Although the function of the short LR forms remains unclear, LRb is critical for leptin action. Indeed, the originally described db/db mice lack only LRb (as a consequence of a mutation that results in the missplic-

Leptin and the Regulation of Feeding / 989 There are three conserved tyrosine residues on the intracellular domain of LRb: Tyr985, Tyr1077, and Tyr1138 [2, 32]. In the human long-form LR, there exist two additional intracellular tyrosine residues, albeit within motifs that do not appear to be particularly hydrophilic and accessible to kinases [32]. Tyr985, Tyr1077, and Tyr1138 are phosphorylated during receptor signaling, and each contributes uniquely to leptin signaling and action [2, 19]. There are thus four primary intracellular signaling pathways that emanate from LRb: those originating directly from Jak2 tyrosine phosphorylation sites, those originating from Tyr985 of LRb, those from Tyr1077, and those originating from Tyr1138 of LRb. In cultured cells, Tyr985 interacts with SHP-2 to mediate the majority of extracellular-signal-regulated kinase (ERK) activation, with the remainder of ERK signaling mediated by Jak2 independently of tyrosine phosphorylation sites on LRb, presumably via tyrosine phosphorylation sites on Jak2 [2]. The role of Tyr1138 phosphorylation in recruiting STAT3 (Fig. 1) to the LRb-Jak2 complex was suggested by homology between LRb and other cytokine receptors of the IL-6 receptor family; indeed, mutation of this site abrogates STAT3 signaling by LRb [23]. In addition

ing of the LRb message), but exhibit a phenotype indistinguishable from that of leptin-deficient ob/ob animals and of db3J/db3J mice (which are deficient in all LR isoforms) [6, 14, 31]. The initially observed homology of LRb with other type I cytokine receptors (e.g., the gp130 subunit of the IL-6 receptor family) suggested that leptin binding to LRb activates Jak kinases to mediate cytokine-receptorlike signals. Of the four known members of the Jak kinase family, Jak2 represents the unique Jak kinase involved in signaling by the intracellular domain of LRb. All functional cytokine receptors contain a prolinerich Box 1 motif that is required for Jak kinase interaction and activation; additional, less conserved sequences (sometimes referred to as Box 2) COOH-terminal to Box 1 are also important for Jak kinase interactions and likely function in Jak kinase isoform selectivity [30]. In addition to Box 1 sequences, intracellular residues 31– 36 of LRb (i.e., immediately downstream of the alternative splice junction following amino acid 29) represent Box 2 and are required for Jak2 activation [23]. This motif is absent from all described short LR isoforms, explaining the inability of these molecules to mediate leptin action in db/db animals [23].

pY

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FIGURE 1. LRb signaling and the control of physiology. Leptin binding to LRb activates the associated Jak2 tyrosine kinase, resulting in the tyrosine phosphorylation (pY) and activation of Jak2 and, subsequently, the phosphorylation of LRb on Tyr985 and Tyr1138. Tyr1138 recruits STAT3 to mediate its transcriptional activation; one transcriptional target of STAT3 is the inhibitory SOCS3. STAT3 is also an important mediator of melanocortin action, feeding, and energy expenditure during leptin action. Tyr985 recruits SHP-2 and provides a binding site for SOCS3, participating in an important feedback loop to attenuate LRb signaling. Other LRb signals appear to be mediated via Jak2, including PI 3-kinase. Not only are these Jak2-dependent, LRb-tyrosine-phosphorylation-independent signals implicated in several neuroendocrine actions of leptin, but PI 3-kinase has also been shown to contribute to the anorectic action of leptin, suggesting that, although STAT3 signaling is important for this leptin effect, other signals participate, as well.

990 / Chapter 135 to mediating a variety of positive signals, the LRb-STAT3 pathway induces expression of the suppressor of cytokine signaling 3 (SOCS3, which is induced by prolonged LRb activation and mediates feedback inhibition of LRb signaling) [2, 5]. SOCS3 interacts via its SH2 domain with phosphorylated Tyr985 of LRb, and the sensitive blockade of LRb → STAT3 signaling is mediated by this interaction [5]. Other recent results suggest that another LRb → STAT3–dependent mechanism of feedback inhibition of LRb signaling may directly block Jak2 activation as well [10]. Jak2 tyrosine phosphorylation during LRb stimulation mediates some signals independently of tyrosine phosphorylation sites on LRb (e.g., a portion of ERK activation, insulin-receptor-substrate (IRS)-protein phosphorylation) [2]; unfortunately, most Jak2 tyrosine phosphorylation sites have not been defined, limiting our understanding of the mechanisms by which Jak2dependent signals are mediated. Identifying tyrosine phosphorylation sites on Jak2 and defining their roles in intracellular signaling will be critical to understanding the breadth and depth of LRb signaling. The IRS-protein → PI 3′-kinase pathway is one signal that clearly contributes to leptin action in vivo [24]. First described as insulin receptor substrates, the IRSproteins (IRS-1–4), are members of a class of intracellular signaling molecules termed docking proteins that are phosphorylated by a number of activated tyrosine kinases (e.g., insulin receptor and some cytokine receptors) and act to bind and activate phosphoinositide (PI) 3′-kinase. The blockade of PI 3-kinase activity abrogates leptin-mediated hyperpolarization and inhibition of (presumably) LRb-NPY-AgRP hypothalamic neurons [17, 23, 29]. Furthermore, leptin stimulates IRS-2associated PI 3′-kinase activity in the hypothalamus, and pharmacological blockade of PI 3′-kinase activity in the hypothalamus blocks the anorectic effect of leptin in vivo [24]. Of importance is that inhibition of PI 3′-kinase does not alter the anorectic effect of melanocortin agonists that operate downstream of LRb neurons, suggesting that this effect of PI 3′-kinase blockade is specific for LRb neurons. PI 3′-kinase activity is also required for leptin-regulated sympathetic nervous system function [26]. The functions of the LRb tyrosine phosphorylation sites and their binding partners have been investigated via brain-specific deletion of STAT3, SHP-2, and SOCS3 and by knock-in of LRb molecules mutant for specific phosphorylation sites [4, 15, 34]. Interestingly, the phenotype of the SHP-2 and SOCS3 brain knockouts have opposite phenotypes; SHP-2-deleted mice are obese with neuroendocrine failure, whereas the SOCS3deleted mice are lean and leptin sensitive [18, 22, 34]. Both of these results must be interpreted with caution, however, because SHP-2 and SOCS3 are also expressed

in a variety of non-LRb neurons and are important for signaling by myriad receptors in the CNS. Indeed, the complex phenotype of the SHP-2-deleted mice is difficult to attribute solely to altered LRb signaling. In contrast, the SOCS3 phenotype seems reasonably consistent with the known biology of LRb; mice mutant for LRb Tyr985 may thus turn out to be lean and leptin sensitive without major defects in leptin action. The role of STAT3 signaling in the brain in leptin action has been investigated by studying homologously targeted knock-in mice in which LRb is replaced by a mutant molecule (LRbS1138) that contains a substitution mutation of Tyr1138 (the STAT3 binding site) [4], as well as by studying mice with deletion of the STAT3 gene in the forebrain [4, 15]. Like db/db animals, mice homozygous for LRbS1138 (s/s) display hyperphagia and decreased energy expenditure, resulting in massive early-onset obesity that is associated with increased serum leptin levels. Thus, the LRb → STAT3 signal is central to the regulation of body weight by leptin and the dysfunction of this signal generates leptin resistance [4]. Similar hyperphagia and obesity have been observed in mice null for STAT3 in the brain [15]. Important differences exist between the phenotypes of s/s mice and db/db mice, however [4]. Whereas db/db animals are infertile and demonstrate decreased linear growth, s/s mice are fertile and demonstrate increased linear growth compared with wild-type animals. Furthermore, glycemic control is spared in s/s mice compared with db/db animals, suggesting that STAT3-independent LRb signals are major contributors to the regulation of glucose homeostasis by leptin [3]. Neurochemically, like db/db mice, s/s mice have decreased POMC and increased AgRP mRNA levels in the hypothalamus. In contrast, whereas db/db animals display dramatic induction of hypothalamic NPY mRNA, s/s animals have levels of NPY message that are near normal. These results suggest that LRb → STAT3 signaling is a critical regulator of hypothalamic melanocortin action and that dysregulated melanocortin signaling (as opposed to alterations in NPY) may account for the obesity of s/s animals. In addition, non-STAT3 LRb signals are critical regulators of NPY expression in the LRb -NPY neuron. The finding of infertility and reduced growth in mice with forebrain deletion of STAT3 [15] suggests that STAT3 is also required for proper neuroendocrine development or function independently of LRb.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS Over one-quarter of adult Americans are obese, and the incidence of obesity continues to rise in the United States as well as other industrialized nations. Obesity is

Leptin and the Regulation of Feeding / 991 a major risk factor for type 2 diabetes, cardiovascular disease, and some forms of cancer [27]. Because the administration of leptin to rodents decreases food intake and increases energy expenditure, resulting in loss of fat mass, leptin was initially hailed as a potential cure for obesity. With the exception of rare human patients with genetic leptin deficiency, however, circulating leptin levels correlate well with body mass index (BMI) and total body fat mass. Hence, obese individuals have elevated circulating leptin levels, but this abundant leptin fails to mediate weight loss, suggesting that most human obesity represents a form of leptin resistance. Indeed, although therapy with exogenous leptin does augment weight loss, the effects of leptin are modest at the doses that have been tested [21]. A number of potential mechanisms have been postulated to underlie leptin resistance, including defects in leptin access into the brain, in LRb signaling, or in pathways or neurons that mediate downstream leptin action. Dissecting the relevant mechanisms of leptin resistance in human obesity will be important for our understanding of this disease.

References [1] Ahima, R. S.; Prabakaran, D.; Mantzoros, C. S.; Qu, D.; Lowell, B. B.; Maratos-Flier, E.; Flier, J. S. Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252; 1996. [2] Banks, A. S.; Davis, S. M.; Bates, S. H.; Myers, M. G., Jr. Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572; 2000. [3] Bates, S. H.; Kulkarni, R. N.; Seifert, M.; Myers, M. G., Jr. Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis. Cell Metab 1:169–178; 2005. [4] Bates, S. H.; Stearns, W. H.; Schubert, M.; Tso, A. W. K.; Wang, Y.; Banks, A. S.; Dundon, T. A.; Lavery, H. J.; Haq, A. K.; MaratosFlier, E.; Neel, B. G.; Schwartz, M. W.; Myers, M. G., Jr. STAT3 signaling is required for leptin regulation of energy balance but not reproduction. Nature 421:856–859; 2003. [5] Bjorbaek, C.; Lavery, H. J.; Bates, S. H.; Olson, R. K.; Davis, S. M.; Flier, J. S.; Myers, M. G., Jr. SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem 275:40649–40657; 2000. [6] Chua, S. C., Jr.; Koutras, I. K.; Han, L.; Liu, S. M.; Kay, J.; Young, S. J.; Chung, W. K.; Leibel, R. L. Fine structure of the murine leptin receptor gene: Splice site suppression is required to form two alternatively spliced transcripts. Genomics 45:264–270; 1997. [7] Chua, S. C., Jr.; Liu, S. M.; Li, Q.; Sun, A.; DeNino, W. F.; Heymsfield, S. B.; Guo, X. E. Transgenic complementation of leptin receptor deficiency. II. Increased leptin receptor transgene dose effects on obesity/diabetes and fertility/lactation in lepr-db/db mice. Am J Physiol Endocrinol Metab 286:E384–E392; 2004. [8] Cohen, P.; Zhao, C.; Cai, X.; Montez, J. M.; Rohani, S. C.; Feinstein, P.; Mombaerts, P.; Friedman, J. M. Selective deletion of leptin receptor in neurons leads to obesity. J Clin Invest 108:1113–1121; 2001. [9] Coleman, R. A.; Herrmann, T. S. Nutritional regulation of leptin in humans. Diabetologia 42:639–646; 1999. [10] Dunn, S. L.; Bjornholm, M.; Bates, S. H.; Chen, Z.; Seifert, M.; Myers, M. G., Jr. Feedback inhibition of leptin receptor/Jak2

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992 / Chapter 135 [30] Taga, T.; Kishimoto, T. gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol 15:797–819; 1997. [31] Tartaglia, L. A. The leptin receptor. J Biol Chem 272:6093–6096; 1997. [32] Tartaglia, L. A.; Dembski, M.; Weng, X.; Deng, N.; Culpepper, J.; Devos, R.; Richards, G. J.; Campfield, L. A.; Clark, F. T.; Deeds, J.; Muir, C.; Sanker, S.; Moriarty, A.; Moore, K. J.; Smutko, J. S.; Mays, G. G.; Woolf, E. A.; Monroe, C. A.; Tepper, R. I. Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271; 1995.

[33] Yang, G.; Ge, H.; Boucher, A.; Yu, X.; Li, C. Modulation of direct leptin signaling by soluble leptin receptor. Mol Endocrinol 18:1354–1362; 2004. [34] Zhang, E. E.; Chapeau, E.; Hagihara, K.; Feng, G. S. Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci USA 101:16064–16069; 2004. [35] Zhang, Y.; Proenca, R.; Maffei, M.; Barone, M.; Leopold, L.; Friedman, J. M. Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432; 1994.

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also increased eating. The view that the brain per se does not respond directly to insulin was challenged when it was discovered that insulin enters the brain, where it reacts with specific insulin receptors and triggers diverse events including the net catabolic action of reducing food intake and body weight [26, 28].

ABSTRACT Plasma insulin is secreted by the pancreas and provides a signal that reflects circulating energy in the form of glucose as well as stored energy in the form of white adipose tissue, and particularly visceral adipose tissue. Insulin, like leptin, is therefore considered an adiposity signal, and it is transported from the plasma into the brain, where it interacts with insulin receptors on neurons in the mediobasal hypothalamus and elicits a net catabolic response. This includes reduced caloric intake (and body weight if prolonged) and decreased secretion of glucose into the blood by the liver. Insulin reduces food intake in the hypothalamus by enhancing the ability of meal-generated satiety signals to reduce meal size. The response is dose-dependent and more prominent in males than in females. Insulin therefore provides an important and indeed necessary input to the brain for the appropriate regulation of energy homeostasis.

EFFECTS OF INSULIN ON FEEDING BEHAVIOR Insulin secretion and levels in plasma are typically low after a fast (basal condition) and increase mainly during and immediately after meals (prandial condition) or glucose administration (stimulated condition). Basal, prandial, and stimulated levels are a direct function of the amount of white adipose tissue in the body, leaner individuals having lower levels and more obese individuals having higher levels [41]. Plasma insulin, along with plasma leptin (see Chapter 135 elsewhere in this Handbook) whose secretion is also directly proportional to white fat [11], therefore conveys an important signal to the brain indicating the degree of adiposity. The generally accepted model is that insulin provides a key negative feedback signal in the regulation of body fat (Fig. 1). When an individual has food withheld (or voluntarily diets) and loses weight, less insulin is secreted and reaches the brain, and the catabolic tone in the hypothalamus is reduced. Food intake increases until body weight (and the insulin signal) is restored. Analogously, if an individual overeats (or is force-overfed), body fat and the insulin signal increase, increasing the catabolic tone until the weight is lost. Insulin (like leptin) is therefore an important adiposity signal whose activity is integrated with diverse other information to determine food intake. If exogenous insulin is administered directly into the brain, near or directly within the mediobasal

INTRODUCTION Insulin, the peptide hormone from pancreatic B cells, is best known for its ability to enhance the cellular uptake of glucose by most tissues and consequently to decrease blood glucose. Diabetic patients take insulin to reduce their hyperglycemia, and a common adverse effect of taking too high a dose of insulin is hypoglycemia. Because hypoglycemia per se can induce eating, for more than 50 years after its discovery the major behavioral effect of insulin was thought to be increased food intake. The response was known to result from insufficient glucose reaching the brain because administering glucose along with insulin circumvented enhanced eating and because reducing glucose availability to the brain by means of nonmetabolizable glucose analogs Handbook of Biologically Active Peptides

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994 / Chapter 136 Catabolic: Reduce Food Intake Reduce Body Fat Anabolic: Increase Food Intake Increase Body Fat

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White Fat Insulin Transporter Insulin Pancreas Brain Capillary

FIGURE 1. Insulin is secreted into the blood from the pancreas in direct proportion to the amount of fat stored in white adipose tissue. As it circulates through brain capillaries, a small amount of insulin is transported into the brain, where it acts on insulin receptors on neurons with either net catabolic or anabolic activity, for example, in the arcuate nucleus of the hypothalamus. These neurons in turn influence energy homeostasis (food intake) and ultimately the amount of fat stored in the body.

hypothalamus, animals behave as if they have excess fat in the body; they eat less food and lose weight (see reviews in [29, 42]). The response is dose-dependent, has been documented in numerous species including nonhuman primates, and is not secondary to illness or incapacitation. Conversely, if insulin antibodies are administered in or near the mediobasal hypothalamus, animals overeat and gain weight [18]. When insulin levels in the brain are held constant for long intervals by means of slow, steady local infusions, body weight is maintained and defended at a level determined by the dose of insulin administered [7]. Although insulin has not been administered directly into the brain of humans, it has been administered intranasally to humans with a consequent increase of insulin in the cerebrospinal fluid [5]. Humans administered insulin intranasally eat less food and lose body fat [12]. Most if not all insulin in adult mammals is made in pancreatic B cells. Although scattered reports suggest that the brain synthesizes some insulin, the bulk of evidence implies the opposite, and it is generally acknowledged that most if not all insulin influencing the brain reaches it via the circulation [2, 43]. Insulin enters via insulin receptor–facilitated transport through capillary endothelial cells [2, 43]. The process is saturable, selective for insulin, and subject to regulation. The ease of transport of insulin into the brain is reduced

in fasted animals, in animals fed a high-fat diet [40], and in genetic and dietary-induced obesity [43]. Although insulin receptors are expressed in many specific areas of the brain, relatively high concentrations are found in the mediobasal hypothalamus, particularly in the arcuate nucleus [26], which also expresses the most active form of the leptin receptor [21]. This region is thought to mediate most of insulin’s catabolic action. Within the brain, insulin and leptin exert their anabolic action in part by increasing the efficacy of meal-generated satiety signals. These signals, such as cholecystokinin (CCK), are secreted during meals as partially digested food interacts with specialized endocrine cells lining the gastrointestinal tract (see reviews in [19, 36] and Chapter 131 in this Handbook). Their cumulated message is conveyed to the brain, where it interacts with other factors to terminate the meal and contribute to the sensation of satiety. When the insulin or leptin signal is increased locally within the brain, the potency of CCK and other satiety signals is greatly increased. Hence, adiposity signals, when they are elevated, help to regulate body weight by reducing the amount of food eaten during individual meals. When an individual is underweight, the reduced adiposity signal allows larger meals to be consumed until weight is restored [36].

Ingestive Peptides: Insulin / 995 To summarize, increased insulin in the mediobasal hypothalamus provides a signal that ample or excess energy is available in the body, and one consequence is to limit the amount of energy subsequently ingested. Simultaneously, the increased hypothalamic insulin signal also triggers a reflex to the liver via the vagus nerve to suppress the production of glucose [24]. Increased insulin (and probably leptin) is therefore analogous to other signals indicating a surfeit of energy, such as a local increase of long-chain fatty acids [23] or glucose [16] in the hypothalamus; and there is evidence that overlapping intracellular signaling pathways mediate the overall catabolic response to these diverse signals [21, 28].

GENETIC AND MOLECULAR BIOLOGICAL MANIPULATIONS Humans and animals with insulin-deficiency diabetes mellitus are hyperphagic. They cannot become obese because insulin is required for adipocytes to store fat. When insulin is administered locally into the brain of animals lacking insulin, the hyperphagia is attenuated with no change in the degree of diabetes [35]. Consistent with this, mice with a targeted deletion of insulin receptors selectively in neurons develop an obese phenotype [6], and reducing insulin signaling locally in the hypothalamus by use of antisense oligodeoxynucleotides directed against the insulin receptor precursor protein substantially increases food intake [22]. Thus, accurate sensing of signals indicative of energy level by hypothalamic receptors is required for effective control of energy intake, and this is also true of hepatic glucose output [15]. Genetically altered mice with no insulin receptors are hyperglycemic and have a short life span. Genetic rescue of insulin receptors in the pancreas and liver of these mice ameliorates but does not normalize the syndrome [25]. However, when the insulin receptor is also rescued in the brain, the mice become normoglycemic [25]. The important point is that insulin signaling in the hypothalamus is necessary for normal energy homeostasis. Like peripheral insulin receptors, hypothalamic insulin receptors rely on an intracellular signaling cascade using insulin receptor substrate-phosphatidylinositol 3-OH kinase (IRS-PI3K). Although most peripheral tissues and many brain cells use IRS-1, arcuate neurons and pancreatic B cells use IRS-2. Mice without IRS-2 have severe obesity and hyperglycemia, and this can be reversed by inserting the IRS-2 gene uniquely into the brain [17]. Administering drugs that block the IRSPI3K pathway near the arcuate nucleus prevents insulin from exerting its catabolic effect [20]. Likewise, administering an insulin mimetic that bypasses the extracel-

lular portion of the insulin receptor and stimulates the intracellular signaling cascade directly reduces food intake and body weight [1] as well as hepatic glucose production [24]. The implication from all of these experiments is that the insulin signaling system in the hypothalamus controls many aspects of energy homeostasis and that disruptions of the signaling cascade at any point can lead to overeating, obesity, and glucose dysregulation.

METABOLIC STATES As previously discussed, insulin secretion and level in the plasma are decreased during weight loss or fasting and the entry of insulin into the brain is disproportionately decreased even further, but is restored on refeeding. This is adaptive because, when available energy is low, circulating insulin has less access to key receptors in the hypothalamus that would limit food intake and, when energy is high, insulin more readily acts to reduce meal size. When rats are maintained on a high-fat diet, the ability of insulin to reduce food intake when administered directly into the brain is reduced [40], perhaps contributing to the obesity that often develops. When rats are allowed to select their own mix of macronutrients (protein, carbohydrate, and fat) and insulin is administered directly into the brain, food intake and body weight are decreased; however, the intake of carbohydrate and protein is protected, whereas there is a selective reduction of dietary fat [8], presumably because, when insulin is relatively high, carbohydrate is the preferred source of energy by most tissues but, when insulin is relatively low, fat is more readily oxidized. One important point from this literature is that the ability of insulin to exert its central catabolic action interacts with body fat and its metabolism. It also interacts with the location of fat in the body. Fat distribution differs between males and females. On average, men have more visceral and less subcutaneous fat, whereas the converse is true for women [37]. The distribution of fat in the body is in turn related to the risk for developing symptoms of the metabolic syndrome, with visceral obesity carrying a substantially higher risk than subcutaneous obesity. Hence, the metabolic comorbidities of obesity (including cardiovascular disease, type-2 diabetes, certain cancers, and hyperlipidemia) are more frequent in men [38]. The relative secretion of leptin and insulin also correlates with fat distribution. Insulin secretion is highly correlated with visceral fat and is therefore a risk factor for the metabolic syndrome whereas leptin secretion is better correlated with subcutaneous fat and does not carry the same metabolic

996 / Chapter 136 risk. Insulin is therefore a more relevant adiposity signal in males, and leptin is a more relevant adiposity signal in females. Consistent with this, the male brain is relatively more sensitive to the catabolic action of insulin than the female brain [9, 12], whereas the female brain is relatively more sensitive to leptin [9].

SITE OF ACTION AND NEURAL NETWORKS AFFECTED As already discussed, although insulin receptors are found in many areas of the brain, the mediobasal hypothalamus is thought to be the primary site for insulin’s catabolic action, with the arcuate nucleus (Arc) being especially prominent [31, 44]. Two types of arcuate neurons are particularly important. One synthesizes proopiomelanocortin (POMC), cleaving it to form αmelanocyte-stimulating hormone (α-MSH), an agonist at melanocortin-3 and melanocortin-4 receptors (MC3/4R) located on neurons in several hypothalamic and other brain areas. Stimulation of MC3/4R causes a net catabolic action [10]. Insulin, as well as leptin, stimulates POMC neurons, and the catabolic action of each adiposity signal requires α-MSH signaling through this circuit since administration of antagonists to MC3/4R blocks leptin and insulin action in the brain [3, 33]. Other arcuate neurons synthesize and secrete orexigenic neurotransmitters [32]. Agouti-related peptide (AgRP) is an antagonist at MC3/4R, and the administration of AgRP agonists causes a long-lasting increase of food intake by countering the catabolic activity of α-MSH. Arcuate AgRP neurons also synthesize neuropeptide Y (NPY), which acts at Y1-Y5 receptors to stimulate food intake. When either AgRP or NPY is administered chronically near the Arc, body weight increases. Insulin and leptin each suppresses the activity of NPY-AgRP neurons in the Arc [31, 44]. As previously discussed, there is also evidence that some Arc neurons are directly sensitive to local levels of energy-rich fuels, including glucose and lipid in the form of long-chain fatty acids. Because the Arc also receives information on the levels of satiety signals during meals [4], it is ideally situated to receive and integrate diverse information important in the overall regulation of energy homeostasis. Arc neurons project to many areas of the brain, including hindbrain areas that directly control eating behavior [4] and several nearby regions of the hypothalamus. The hypothalamic paraventricular nuclei (PVN) express MC3/4R and various Y receptors, making them targets for α-MSH, AgRP, and NPY. PVN neurons in turn synthesize and secrete several neuropeptides that have a net catabolic action, including corticotropinreleasing hormone (CRH) and oxytocin. The adminis-

tration of exogenous CRH or oxytocin directly into the brain near or within the hypothalamus causes reduced food intake [32]. Hence, a catabolic circuit exists in which excess body fat is signaled to the brain via increased insulin and leptin activity, this information acts on firstorder hypothalamic neurons to increase α-MSH and decrease NPY and AgRP activity, and this in turn is reflected as increased activity in second-order neurons that use CRH, oxytocin, and other catabolic signals. All of these lead, in turn, to reduced food intake and increased energy expenditure. Neurons in the lateral hypothalamic area (LHA) also receive direct inputs from the Arc, and they have a contrasting profile from PVN neurons. The LHA contains neurons that synthesize and secrete anabolic peptides, including melanin-concentrating hormone (MCH) and the orexins. The administration of MCH or orexin agonists in or near the hypothalamus increases food intake and body weight gain [32]. The presence of opposing hypothalamic circuits for controlling energy homeostasis enables rapid and fine-tuned control over energy homeostasis because the brain can simultaneously turn up one system (e.g., catabolic or anabolic) while turning down the other.

RECEPTORS AND SIGNALING PATHWAYS The brain insulin receptor is the same as the peripheral insulin receptor, and as already discussed, the activation of the brain insulin receptor initiates an intracellular signaling cascade involving IRS2-PI3K. Interference with brain insulin signaling at any point leads to a syndrome of hyperphagia and insulin resistance. The other important point is that the intracellular signaling cascades for both insulin and leptin overlap considerably [21].

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS It has been recognized for more than a half century that the brain exercises regulatory control over body-fat content and that this is manifest at least partly via the control of meal size. It has also long been known that the brain influences blood glucose levels as well, in part via direct control of the autonomic nervous system and the hypothalamic-pituitary axis. Only recently, however, has it become recognized that Arc neurons control both food intake and hepatic glucose production and that the same receptors and signals evidently control both responses. As we have seen, when insulin is administered near or directly into the Arc, animals become hypophagic

Ingestive Peptides: Insulin / 997 [31, 44], and when insulin is low or absent in the brain, animals become hyperphagic; and this can be reduced by the local administration of insulin into the brain [35]. Increased insulin in the Arc also reduces hepatic glucose production via the vagus nerve [24, 27]; likewise, Arc administration of oleic acid also reduces both food intake and hepatic glucose production [15, 23]. Thus, molecules that signify ample available energy (insulin or certain fatty acids) initiate a signal to the liver to stop producing so much glucose while simultaneously sending a message to eat less food. Evidence suggests that normal control of hepatic glucose output relies on both an adequate insulin signal and a fatty-acid signal locally within the hypothalamus because, when either signal is compromised, animals gain excess weight and are systemically insulin resistant, two key symptoms of the metabolic syndrome. Leptin elicits comparable actions as insulin in the hypothalamus [21], although it should be noted that the way that they innervate POMC and NPY-AgRP neurons differs [39]. As with insulin, leptin in the Arc reduces food intake and body weight, and reduced leptin signaling results in hyperphagia and obesity as well as systemic and central [14] insulin resistance. The administration of leptin into the brain reverses insulin-deficiency hyperphagia [34] and lessens systemic insulin resistance [13]. Hence, convergent signals from insulin and leptin act in the brain to regulate both energy and glucose homeostasis, and a defect in either leptin or insulin signaling in the brain can result in overeating, weight gain, insulin resistance, and other sequelae of the metabolic syndrome [28, 30].

[4] [5]

[6]

[7]

[8]

[9]

[10] [11]

[12]

[13]

[14]

[15]

[16]

CONCLUSION [17]

Plasma insulin is a signal that reflects both circulating energy in the form of glucose and stored energy in the form of visceral adipose tissue. Insulin is transported into the brain from the plasma and provides an important and, indeed, necessary input for the appropriate regulation of both stored energy and glucose secretion by the liver. When integrated with other signals, insulin in the brain has a potent catabolic influence reducing energy intake and storage.

References [1] Air EL, Strowski MZ, Benoit SC, Conarello SL, Salituro GM, Guan XM, Liu K, Woods SC, and Zhang BB. Small molecule insulin mimetics reduce food intake and body weight and prevent development of obesity. Nat Med 8: 179–183, 2002. [2] Banks WA. The source of cerebral insulin. Eur J Pharmacol 490: 5–12, 2004. [3] Benoit SC, Air EL, Coolen LM, Strauss R, Jackman A, Clegg DJ, Seeley RJ, and Woods SC. The catabolic action of insulin in the

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998 / Chapter 136 [24] Obici S, Zhang BB, Karkanias G, and Rossetti L. Hypothalamic insulin signaling is required for inhibition of glucose production. Nat Med 8: 1376 –1382, 2002. [25] Okamoto H, Nakae J, Kitamura T, Park BC, Dragatsis I, and Accili D. Transgenic rescue of insulin receptor-deficient mice. J Clin Invest 114: 214 –223, 2004. [26] Plum L, Schubert M, and Bruning JC. The role of insulin receptor signaling in the brain. Trends Endocrinol Metab 16: 59–65, 2005. [27] Pocai A, Lam TK, Gutierrez-Juarez R, Obici S, Schwartz GJ, Bryan J, Aguilar-Bryan L, and Rossetti L. Hypothalamic K(ATP) channels control hepatic glucose production. Nature (London), New Biol 434: 1026–1031, 2005. [28] Porte DJ, Baskin DG, and Schwartz MW. Insulin signaling in the central nervous system: a critical role in metabolic homeostasis and disease from C. elegans to humans. Diabetes 54: 1264–1276, 2005. [29] Schwartz MW, Figlewicz DP, Baskin DG, Woods SC, and Porte D, Jr. Insulin in the brain: a hormonal regulator of energy balance. Endocr Rev 13: 387–414, 1992. [30] Schwartz MW and Porte DJ. Diabetes, obesity, and the brain. Science 307: 375–379, 2005. [31] Schwartz MW, Woods SC, Porte DJ, Seeley RJ, and Baskin DG. Central nervous system control of food intake. Nature 404: 661– 671, 2000. [32] Seeley RJ and Woods SC. Monitoring of stored and available fuel by the CNS: implications for obesity. Nat Rev Neurosci 4: 901–909, 2003. [33] Seeley R, Yagaloff K, Fisher S, Burn P, Thiele T, van DG, Baskin D, and Schwartz M. Melanocortin receptors in leptin effects. Nature 390: 349, 1997.

[34] Sindelar DK, Havel PJ, Seeley RJ, Wilkinson CW, Woods SC, and Schwartz MW. Low plasma leptin levels contribute to diabetic hyperphagia in rats. Diabetes 48: 1275–1280, 1999. [35] Sipols AJ, Baskin DG, and Schwartz MW. Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression. Diabetes 44: 147–151, 1995. [36] Strader AD and Woods SC. Gastrointestinal hormones and food intake. Gastroenterology 128: 175–191, 2005. [37] Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 21: 697– 738, 2000. [38] Wajchenberg BL, Giannella-Neto D, da Silva ME, and Santos RF. Depot-specific hormonal characteristics of subcutaneous and visceral adipose tissue and their relation to the metabolic syndrome. Horm Metab Res 34: 616–621, 2002. [39] Wanting Xu A, Kaelin CB, Takeda K, Akira S, Schwartz MW, and Barsh GS. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest 115: 951–958, 2005. [40] Woods SC, D’Alessio DA, Tso P, Rushing PA, Clegg DJ, Benoit SC, Gotoh K, Liu M, and Seeley RJ. Consumption of a high-fat diet alters the homeostatic regulation of energy balance. Physiol Behav 83: 573–578, 2004. [41] Woods SC, Decke E, and Vasselli JR. Metabolic hormones and regulation of body weight. Psychol Rev 81: 26–43, 1974. [42] Woods SC and Seeley RJ. Insulin as an adiposity signal. Int J Obes Relat Metab Disord 25: S35–S38, 2001. [43] Woods SC, Seeley RJ, Baskin DG, and Schwartz MW. Insulin and the blood-brain barrier. Curr Pharm Design 9: 795–800, 2003. [44] Woods SC, Seeley RJ, Porte DJ, and Schwartz MW. Signals that regulate food intake and energy homeostasis. Science 280: 1378– 1383, 1998.

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137 Adrenomedullin in Gastrointestinal Function MITCHELL L. SCHUBERT

adrenal medulla [19]. The peptide circulates in blood and exerts sustained vasodilator and hypotensive effects. Adrenomedullin is expressed in a variety of extraadrenal sites, including the gastrointestinal tract where it may also influence function in a paracrine or autocrine manner. Adrenomedullin belongs to a family of peptides that include calcitonin, calcitonin-gene-related peptide (CGRP), and amylin. Although there is some crossreactivity among these peptides, there is now evidence for the existence of unique adrenomedullin receptors. Interest in adrenomedullin has grown rapidly, and it appears likely that this peptide is involved in a variety of physiological and pathophysiological gastrointestinal processes, especially the regulation of secretion, repair, motility, and vasodilation.

ABSTRACT Adrenomedullin, first isolated in 1993 from human pheochromocytoma, is a member of the calcitonin family of peptides that includes calcitonin, calcitoningene-related peptide (CGRP), and amylin. The adrenomedullin gene encodes a 185-amino-acid precursor, preproadrenomedullin, which is processed to form the biologically active peptides adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP). Adrenomedullin circulates in the plasma and is expressed in a variety of gastrointestinal tissues, including the liver, pancreas, stomach, duodenum, small intestine, and colon. The biologic activities of adrenomedullin are mediated via CGRP receptors and specific adrenomedullin receptors, termed AM1 and AM2, primarily through activation of adenylate cyclase. In the stomach, adrenomedullin (1) stimulates somatostatin secretion and thus inhibits histamine and acid secretion; (2) protects the stomach from injury by increasing mucosal blood flow, stimulating epithelial cell proliferation and restitution, and promoting angiogenesis; and (3) inhibits gastric emptying. Adrenomedullin inhibits sodium absorption in the colon and amylase secretion in the pancreas. It may also contribute to the hemodynamic alterations observed in cirrhotic portal hypertension. More potent and selective adrenomedullin receptor antagonists are required to define the physiological and pathophysiological roles of adrenomedullin in the regulation of gastrointestinal function.

STRUCTURE Human adrenomedullin is a 52-amino-acid peptide that belongs to the calcitonin family of peptides, which also includes calcitonin, CGRP, and amylin [2, 35]. The peptide family is characterized by the presence of a 6amino-acid (7 for calcitonin) ring structure linked by a disulfide bridge at the N-termini. The structure of adrenomedullin is conserved across species, the rat peptide only differing by six substitutions and two deletions from the human peptide. The human adrenomedullin gene is located on chromosome 11. The initial translation product of the gene is preproadrenomedullin, a 185-amino-acid peptide that is converted to proadrenomedullin. Proadrenomedullin undergoes proteolysis and amidation to yield adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) [44]. Recent evidence indicates that PAMP may also possess biological activity [6].

INTRODUCTION Adrenomedullin was first isolated in 1993 from human pheochromocytoma and was termed adrenomedullin based on the high concentration in the normal Handbook of Biologically Active Peptides

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1000 / Chapter 137 EXPRESSION AND DISTRIBUTION Adrenomedullin circulates in the plasma of normal subjects in picomolar concentrations (2–4 pmol/liter) [39]. Adrenomedullin and its mRNA are expressed not only in the adrenal gland but also in a variety of tissues and cells, including the lung, heart, kidney, brain, thyroid, bone, skin, nerve, vascular endothelial and smooth muscle cells, submandibular gland, liver, pancreas, stomach, duodenum, small intestine, and colon [15, 16, 51]. In human and rat pancreas, adrenomedullin is expressed predominantly in islet cells, although a few adrenomedullin immunoreactive cells are scattered among the exocrine parenchymatic cells [25]. In the hollow digestive system, adrenomedullin is concentrated in mucosal neuroendocrine cells [21]. In the stomach, adrenomedullin has been localized to histamine-containing enterochromaffin-like (ECL) cells, chief cells in oxyntic mucosa, and gastrin cells in pyloric mucosa [9, 20, 50]. In the colon, adrenomedullin immunoreactivity has been localized to serotonincontaining enterochromaffin (EC) cells [32].

RELEASE Plasma concentrations of adrenomedullin increase with fasting as well as a variety of disease states including acute myocardial infarction, ischemic heart disease, congestive heart failure, essential hypertension, chronic renal failure, hyperparathyroidism, sepsis, and cirrhosis [22, 42, 45]. One common denominator may be bacterial infection or inflammation because exposure of gastric epithelial cells to various strains of bacteria including Helicobacter pylori as well as various cytokines such as interleukin-1, interleukin-6, tumor necrosis factor, interferon, and lipopolysaccharide stimulate adrenomedullin production and secretion [1]. Circulating hormones such as corticosteroids, thyroid hormones, angiotensin II, and norepinephrine also stimulate adrenomedullin release [6]. In contrast, interferon-γ, vasoactive intestinal peptide (VIP), and thrombin suppress adrenomedullin production [39].

RECEPTORS AND SIGNAL TRANSDUCTION PATHWAYS Because of their structural similarities, there is some cross-reactivity among adrenomedullin, amylin, and CGRP. Adrenomedullin can bind to CGRP receptors with an affinity approximately 10-fold less than that of CGRP itself [12]. However, there is now evidence to support the existence of separate independent

receptors for each of the peptides. The calcitoninreceptorlike receptor (CRLR), a 7-transmembranedomain receptor, can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a family of single-transmembranedomain proteins, called receptor-activity-modifying proteins (RAMPs), are expressed [30]. RAMPs function as accessory proteins that are required for trafficking of CRLR to the plasma membrane and for pharmacological specificity [36]. When CRLR and RAMP1 were cotransfected into Xenopus oocytes, a CGRP pharmacological response was acquired, whereas transfection of CRLR and RAMP2 or RAMP3 resulted in an adrenomedullin receptor [27, 30, 31]. The subtype of adrenomedullin receptor formed by association with RAMP2 is termed AM1, whereas that formed by association with RAMP3 is termed AM2 (Fig. 1). Adrenomedullin22–52 (AM22–52) functions as a low-affinity selective adrenomedullin receptor antagonist in some but not all circumstances [3, 5, 52]. It seems to antagonize the effects of adrenomedullin but not CGRP and has significant selectivity for the AM1 receptor over the AM2 receptor [13]. The AM1 receptor recognizes adrenomedullin over CGRP with a selectivity of two to three orders of magnitude and is more effectively antagonized by AM22–52 than by CGRP8–37 [24]. The AM2 receptor is sensitive to both CGRP8–37 and AM22–52, although rat and mouse AM2 receptors show a preference for CGRP8–37 over AM22–52. The biological activities of adrenomedullin are mediated primarily through the activation of adenylate cy-

FIGURE 1. Model illustrating the adrenomedullin receptor and showing the interactions between the 7-transmembrane domain G-protein-coupled receptor known as the calcitonin receptorlike receptor (CRLR) and the single-domain receptor-associated membrane proteins (RAMPs). Their association forms a high-affinity receptor coupled to Gαs that activates adenylate cyclase (AC) to generate cAMP. The subtype of adrenomedullin receptor formed by the association of CRLR with RAMP2 is termed AM1, whereas that formed by association with RAMP3 is termed AM2. Adapted from [36].

Adrenomedullin in Gastrointestinal Function / 1001 clase, although in some cell types adrenomedullin may also release calcium from inositol 1,4,5-trisphosphate– sensitive intracellular stores (Fig. 1) [48].

BIOLOGICAL ACTIONS Gastric Acid Secretion Adrenomedullin circulates in the plasma [17], and intravenous infusion of the peptide inhibits basal, histamine-stimulated, pentagastrin-stimulated, and 2deoxy-d-glucose–stimulated acid secretion in conscious rats equipped with a gastric fistula [40, 41]. The effect is 50–60% blocked by a selective somatostatin receptor antagonist, suggesting that systemic adrenomedullin inhibits acid secretion via both somatostatin-dependent and -independent pathways. The adrenomedullin receptor antagonist, AM22–52, however, failed to significantly antagonize the effect of adrenomedullin. The findings are consistent with those reported in pylorusligated rats, in which the inhibitory effect on acid secretion of subcutaneous injection of adrenomedullin was more potently blocked by the CGRP receptor antagonist, CGRP8–37 than by AM22–52, suggesting that the antisecretory effect is mediated predominantly via CGRP receptors [43]. In the stomach, CGRP is capable of stimulating somatostatin and inhibiting gastrin and acid secretion [26, 38, 46]. It should be cautioned, however, that rodent AM2 receptors show a preference for CGRP8–37 [24]. Studies in the isolated mouse stomach and superfused rat fundic segments [14], preparations that retain intact intramural neural and paracrine pathways but eliminate hormonal influences such as gastrin, indicate that adrenomedullin acts locally (i.e., within the oxyntic mucosa) to stimulate somatostatin and inhibit histamine and acid secretion (Fig. 2). The effect is abolished by the axonal blocker, tetrodotoxin, indicating that adrenomedullin’s effect is mediated via activation of intramural neurons. The identity of the neurotransmitter responsible for adrenomedullin-induced somatostatin secretion is not yet known. In dog mesenteric arteries, adrenomedullin receptors have been identified on adrenergic nerve endings [7].

Gastric Protection and Repair In rats, intracisternal, but not intravenous, injection of adrenomedullin dose-dependently inhibits gastric lesions induced by 70% ethanol [18]. The effect was not blocked by intracisternal injection of the CGRP antagonist, CGRP8–37, but was significantly attenuated by subdiaphragmatic vagotomy as well as peripheral injection of atropine, indomethacin, and the nitric

(SRIF)

FIGURE 2. Model illustrating the regulation of acid secretion in gastric oxyntic mucosa by adrenomedullin. Adrenomedullin, acting via an as yet unidentified intramural neuron, stimulates somatostatin (SRI) and thus inhibits histamine secretion from enterochromaffin-like (ECL) cells and acid secretion from parietal cells. Reproduced by permission from [14].

oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME). The results suggest that adrenomedullin, acting via AM1 receptors, exerts a gastroprotective effect centrally that is mediated via the vagus nerve with activation of peripheral prostaglandin- and nitric oxide–dependent protective mechanisms. Central administration of adrenomedullin also protects against reserpine-induced gastric lesion in rats. However, in contrast to the previous study, the effect was blocked by CGRP8–37 [4]. Inhibition of acid secretion and increased mucosal blood flow play important roles in preventing gastric mucosal injury. In rats, subcutaneous injection of adrenomedullin prevents gastric erosions induced by reserpine or pylorus ligation [43]. The protection is accompanied by inhibition of acid secretion and vasodilation. Studies using CGRP8–37 to block the CGRP receptor and adrenomedullin22–52 to block the adrenomedullin receptor suggest that the acid effect is mediated predominantly via CGRP receptors, whereas the vasomotor effect is mediated predominantly via adrenomedullin receptors [43]. This notion is consistent with the finding that adrenomedullin and its receptor are expressed in endothelial and vascular smooth muscle cells [33]. Cell proliferation, migration, differentiation, and angiogenesis play important roles during repair of

1002 / Chapter 137 mucosal injury. Adrenomedullin is capable of stimulating epithelial cell proliferation and angiogenesis. The expression of adrenomedullin and its receptor is increased in rat gastric mucosa after direct instillation of ethanol [50] and during gastric ulcer healing [49], suggesting that adrenomedullin may contribute to gastric mucosal injury repair, perhaps in a paracrine or autocrine manner. In support of this notion, adrenomedullin promotes epithelial restitution, as measured by potential difference, in human oxyntic mucosal sheets mounted in an Ussing chamber following injury induced by 0.5 M saline [9].

Gastric Emptying Several peptides influence gastric motor activity on administration into the brain. Intracisternal injection of adrenomedullin dose-dependently inhibits gastric emptying in rats [28]. Central injection of PAMP, however, was ineffective in rats [28], but inhibited gastric emptying in mice [34]. Intravenous adrenomedullin has been reported to either inhibit [37] or have no significant effect [28] on gastric emptying in rats.

Colonic Transport Adrenomedullin is present in human and rat colonic mucosa. In rat distal colon mounted in an Ussing chamber, adrenomedullin inhibits sodium absorption and stimulates chloride secretion [8, 29]. The effects were inhibited by the axonal blocker, tetrodotoxin, implying that they were mediated via activation of intramural neurons.

Pancreatic Exocrine Secretion Adrenomedullin is present in pancreatic islet cells and capable of binding specifically to pancreatic acini [47]. In isolated rat pancreatic acini, adrenomedullin inhibits amylase secretion induced by cholecystokinin as well as the calcium ionophore A23187 [47].

Cirrhotic Portal Hypertension Peripheral arterial vasodilation leading to decreased systemic vascular resistance is the most outstanding hemodynamic alteration in liver cirrhosis. There is increasing evidence that arterial vasodilation contributes to portal hypertension and the formation of ascites through arterial hypotension, increased cardiac output, and activation of renal sodium retentive mechanisms. The precise cause of the vasodilation remains unknown, but overproduction or reduced degradation of endogenous vasodilators is thought to play a role. Adrenomedullin causes vasodilation by increasing cAMP in vascular smooth muscle cells and releasing

nitric oxide from vascular endothelial cells. Plasma levels of adrenomedullin are increased in patients with cirrhosis and are highest in patients with refractory ascites [10, 11, 23]. Studies in CCL4-induced cirrhotic rats with ascites suggest that overproduction of adrenomedullin may contribute to the observed vascular hyporeactivity to vasoconstrictors [22].

CONCLUSION Adrenomedullin is a relatively recently discovered peptide. Although we have made significant progress in understanding its structure, molecular biology, distribution, plasma levels in health and disease, receptors, second-messenger systems, and bioactivity in experimental circumstances, questions remain regarding its true physiological role. Although adrenomedullin circulates in plasma, it is likely that it acts also as an autocrine and paracrine agent. In the gastrointestinal tract, adrenomedullin inhibits gastric acid secretion, protects the stomach from injury, inhibits gastric emptying, inhibits sodium absorption in the colon, inhibits amylase secretion in the pancreas, and may contribute to the hemodynamic alterations observed in cirrhotic portal hypertension. More potent and selective adrenomedullin receptor antagonists are required to define the physiological roles of adrenomedullin in the regulation of gastrointestinal function.

Acknowledgment The author thanks Mary E. Beatty for the artwork.

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[47] Tsuchida T, Ohnishi H, Tanaka Y, Mine T, Fujita T: Inhibition of stimulated amylase secretion by adrenomedullin in rat pancreatic acini. Endocrinology 1999, 140:865–870. [48] Uezono Y, Shibuya I, Ueda Y, Tanaka K, Oishi Y, Yanagihara N, Ueno S, Toyohira Y, Nakamura T, Yamashita H, Izumi F: Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5trisphosphate in human oligodendroglial cell line KG-1C. Brain Res. 1998, 786:230–234. [49] Wang H, Tomikawa M, Jones K, Pai R, Sarfeh IJ, Tarnawski AS: Sequential expression of adrenomedullin and its receptor during gastric ulcer healing. Dig. Dis. Sci. 2000, 45:591–598. [50] Wang H, Tomikawa M, Jones MK, Sarfeh IJ, Tarnawski AS: Ethanol injury triggers activation of adrenomedullin and its receptor genes in gastric mucosa. Dig. Dis. Sci. 1999, 44:1390–1400. [51] Washimine H, Asada Y, Kitamura K, Ichiki Y, Hara S, Yamamota Y, Kangawa K, Sumiyoshi A, Eto T: Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue. Histochemistry 1995, 103:254. [52] Zimmermann U, Fischer JA, Fre K, Fischer AH, Reinscheid RK, Muff RA: Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastoma X glioma hybrid cells. Brain Res. 1996, 724:238–245.

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138 Calcitonin Gene-Related Peptide and Gastrointestinal Function VICENTE MARTINEZ AND YVETTE TACHÉ

stimulates sensory fibers, eliciting the release of CGRP, and at high doses, completely ablates CGRP innervation and sensory transmission; and (2) the CGRP fragment, CGRP8-37, which behaves as a CGRP1 receptor antagonist. Experimental findings suggest that CGRP plays important and multiple roles in the neural control of the GI function including mucosal homeostasis, blood flow, exocrine and endocrine secretory processes, motility, and visceral sensations (nociception). Recent observations suggest that CGRP might be implicated in the pathophysiology of several GI disorders involving alterations of the mucosal barrier, dysmotility states, inflammatory responses, and changes in visceral sensitivity. For detailed aspects of CGRP related to gene structure and regulation of its expression, synthesis, processing, structure and conformation of the mature peptide, and species-related differences, see the corresponding Chapter 106 by Sexton in the Brain Peptides section.

ABSTRACT Calcitonin gene-related peptide (CGRP) is a 37amino-acid peptide present in nerve terminals of extrinsic afferents and in intrinsic enteric neurons throughout the gastrointestinal tract. Following its release, the peptide acts locally on specific receptors (CGRP1) located in enteric neurons, smooth muscle, endocrine cells, and vascular structures. The stimulation of CGRP1 receptors elicits wide-ranging effects on gut function, including inhibition of gastric acid secretion and gut motility, increased gastric mucosal blood flow and mucosal resistance to injury, and modulation of visceral nociception. These observations suggest that CGRP plays an important role in the maintenance of gastrointestinal homeostasis and might be involved in pathophysiological alterations associated with secretory, motor, inflammatory, and sensory disturbances.

INTRODUCTION DISTRIBUTION OF CGRP IN THE GI TRACT Calcitonin gene-related peptide (CGRP) is a 37amino-acid neuropeptide that, together with calcitonin, adrenomedullin, and amylin, belongs to the calcitonin superfamily of regulatory peptides. CGRP and CGRP receptors have an ubiquitous distribution through the gastrointestinal (GI) tract, predominantly in neuronal structures of the peripheral nervous system. In particular, CGRP has been localized mainly in nerve terminals from both intrinsic and extrinsic sensory neurons, but it can also be detected in some endocrine and immune cells. In addition, functional CGRP receptors have also been localized in specific cell types of the GI tract. Most of the knowledge regarding the functionality of the CGRP system in the GI tract is based on the use of (1) the vanilloid compound, capsaicin, which at low doses Handbook of Biologically Active Peptides

The main sources of CGRP in the mammalian GI tract are extrinsic primary afferent nerve fibers and intrinsic neurons of the enteric nervous system [13]. These two systems contain different molecular forms of the peptide that occur by cell-specific alternative posttranslational processing. Rat extrinsic afferents contain α-CGRP (also known as CGRP-I), whereas enteric neurons only express β-CGRP (also known as CGRP-II), which differs by a single amino acid substitution (Fig. 1). Furthermore, only extrinsic primary afferent neurons are sensitive to the neurotoxic capsaicin, whereas autonomic and enteric neurons are not [8, 12]. Moreover, the content of CGRP-immunoreactive (IR) neurons, as well as the relative contribution of extrinsic

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DRG

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Aboral LM β-CGRP

MP CM

BV

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FIGURE 1. Schematic representation of the extrinsic and intrinsic CGRP-containing neurons in the rat intestine. Extrinsic spinal afferents have their cell bodies in the dorsal root ganglia and contain only α-CGRP. CGRP-containing extrinsic nerve terminals innervate the circular and longitudinal smooth muscle layers; the myenteric plexus; the submucosal plexus, where they densely innervate vascular structures; and the mucosa. CGRP-containing intrinsic enteric neurons express only β-CGRP. These neurons are localized in the submucosal and myenteric plexuses. Submucosal neurons innervate the mucosa, whereas myenteric neurons project orally and aborally, both within the myenteric plexus and to the circular muscle layer, and participate in the regulation of motility. BV, blood vessel; CM, circular smooth muscle; DRG, dorsal root ganglion; LM, longitudinal smooth muscle; MP, myenteric plexus; SMP, submucosal plexus.

and intrinsic innervation, varies greatly among different species and the different regions of the GI tract considered [8]. In rodents, CGRP present in the esophagus and stomach and pyloric sphincter is derived from extrinsic afferent neurons, mainly of splanchnic origin [8, 13]. For instance, 80–90% of the CGRP-containing nerve terminals in the rat stomach originate in the dorsal root ganglia. CGRP-IR vagal afferents arising from the nodose ganglia innervate the esophagus and the proximal part of the stomach, but make a relatively small contribution (less than 10%) to the gastric corpus, antrum, and intestine. Gastric CGRP-IR fibers are found in the myenteric plexus, circular smooth muscle, submucosal blood vessels, and mucosa. In the upper GI tract, there is no CGRP-IR in intrinsic neurons or endocrine cells. Contrasting with rodents, the density of CGRP fibers appears to be lower in healthy human gastric mucosa; however, in patients with gastric ulcers,

intense CGRP immunostaining is present in the antrum and body, particularly at the ulcer margins [32]. Whereas gastric CGRP is mainly of extrinsic origin, the small intestine and the colon contain a large proportion of CGRP-expressing neurons of intrinsic origin, namely from the myenteric and submucosal ganglia [33]. In guinea pigs, intrinsic CGRP neurons project mainly to the mucosa, whereas in rats they project orally and caudally within the myenteric plexus as well as to the muscle layers and the mucosa. These neurons are mainly involved in secretory and motor reflexes within the gut. The small intestine and colon also have abundant CGRP-positive nerve terminals from extrinsic afferent fibers originating from cell bodies in the dorsal root ganglia. These fibers reach the GI tract via sympathetic (splanchnic, colonic, and hypogastric) and sacral parasympathetic (pelvic) nerves while passing through prevertebral ganglia and forming collateral synapses with sympathetic ganglion cells. Within the gut wall, CGRP fibers primarily supply the arteries and arterioles, the lamina propria of the mucosa, the submucosal and myenteric nerve plexuses, and the circular and longitudinal muscle layers (Fig. 1). Many spinal afferents in the rat, guinea pig, and canine gut co-express CGRP and substance P, whereas in intrinsic enteric neurons of the same species substance P and CGRP do not co-exist [13]. The pancreas also presents a significant amount of CGRP-IR, localized mainly in peripheral terminals of afferent neurons that are abundantly associated with blood vessels [1]. Apart from neuronal structures, endocrine cells of the human GI mucosa, rat pancreas, and immune cells within the lamina propria of the rat stomach also contain CGRP-IR [33]. However, the functional significance of these sources of CGRP remains largely unknown.

CGRP RECEPTORS IN THE GI TRACT The biological effects of CGRP in the GI tract are mediated through the family B of specific G-proteincoupled receptors (secretin family). CGRP receptors are heterodimers formed by the association of a calcitonin-receptor-like receptor (CL) and a modifying single-transmembrane-domain protein belonging to a family called receptor-activity-modifying proteins (RAMPs) [18, 25]. Three RAMPs, RAMP1, -2, and -3, have been characterized so far [18, 25]. Only the heterodimer CL-RAMP1 forms selective CGRP1 receptors; however, studies suggest that the heterodimers formed by the calcitonin (CT) receptor and RAMP1 could be classified as CGRP2 receptors [18]. On the other hand, heterodimers of CL or CT receptors with RAMP2 or RAMP3 enable these same receptors to

Calcitonin Gene-Related Peptide and Gastrointestinal Function / 1007 behave as nonselective CGRP, adrenomedullin, or amylin receptors or as selective adrenomedullin or amylin receptors [18, 25]. In addition, the presence of an intracellular protein called “receptor component protein” (RCP) is required for efficient coupling of the CGRP receptor to the Gs–adenylate cyclase signaling pathway [25]. The presence of CGRP receptors in the GI tract has been inferred from pharmacological and receptor binding studies and from the localization of CL and RAMP1 gene expression. CGRP1, but not CGRP2, receptors are sensitive to the antagonist CGRP8-37. Because most of the biological effects of CGRP on the GI tract are blocked by CGRP8-37, CGRP1 receptors should be the predominant receptor subtype in the gut. The wideranging biological effects of CGRP on gut function are consistent with the presence of CGRP binding sites throughout the GI tract, including the vasculature, smooth muscle, neural structures, gut-associated lymph tissue, and lamina propria along with epithelialendocrine mucosal cells [6, 21]. So far, expression studies to establish the distribution of CL and RAMP1 mRNA (the two components of CGRP1 receptors) revealed expression in epithelial cells of the antropyloric region of the rat stomach, probably corresponding to somatostatin-producing cells (D cells), and in mucosal immune cells of the rat gut [11, 16].

RELEASE OF CGRP IN THE GI TRACT A number of studies established that CGRP is released from extrinsic afferents within the gut when vanilloid receptor type 1 (VR1 or TRPV1) is stimulated. The TRPV1 receptor is highly expressed on sensory neurons and epithelial cells all along the GI tract [7]. Numerous stimuli, including vanilloid compounds (such as capsaicin), moderate heat (42–53°C), low pH (pH 5–6), and mechanical distortion, stimulate TRPV1 receptors, eliciting through an axonal reflex, the local release of transmitters, including CGRP, from afferent nerve terminals [7]. The release of CGRP in the GI tract has been demonstrated in vitro in segments of rat stomach and guinea pig gallbladder after capsaicin stimulation [14, 27] and in rat colon in response to muscle stretch, serotonin, and stroking of the mucosa [9]. In addition, there is pharmacological evidence that CGRP is released following capsaicin, electrical field stimulation, and acidification of the gastric and duodenal mucosa [2, 14]. Experimental evidence suggests that in the stomach the activation of vagal efferents recruits CGRP-containing extrinsic afferents, eliciting the local release of CGRP, which might have physiological relevance in the central vagal control of gastric function [13, 17].

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS OF CGRP IN THE GI TRACT Motility The major motor effect of CGRP in the GI tract is the induction of smooth muscle relaxation through CGRP8-37-sensitive receptors [13]. The overall functional consequences are the inhibition of gastric emptying and the reduction of motility throughout the intestine [31]. CGRP inhibition of gut motor function is mediated by a direct action on smooth muscle cells [19] or indirectly through the stimulation of enteric neurons producing inhibitory mediators [31]. It is well recognized that CGRP released from sensory neurons by muscle stretch contributes, as a sensory neurotransmitter, to the peristaltic reflex in the small intestine [9]. This is supported by the fact that CGRP8-37 and capsaicin interfere with the normal peristaltic reflex. Growing evidence also suggests that CGRP released from extrinsic afferents might contribute to the disturbed motility observed in several pathophysiological conditions associated with reduced propulsive motor activity, such as postoperative or peritonitis-induced gastric and intestinal ileus [4, 23] and diabetic gastroparesis [20].

Gastric and Intestinal Secretions The effects of CGRP on secretion (electrolytes, mucus, enzymes, and fluid) vary with the region of the GI tract, the species, and the experimental conditions under consideration. Numerous observations support a role for CGRP as a neurotransmitter that, released from extrinsic afferents, regulates secretory processes in the gastroduodenal area [29–31]. First, CGRP, acting through CGRP8-37-sensitive receptors, is a potent suppressor of basal and secretagogues-stimulated gastric acid and pepsin secretion in humans, rats, dogs, and rabbits. The pathways mediating CGRP antisecretory effects are not completely elucidated and might reveal species-related differences. Multiple mechanisms seem to be implicated, involving the release of somatostatin and the inhibition of gastrin- and cholinergic-dependent stimulatory effects (Fig. 2). The physiological relevance of these observations is demonstrated by the fact that CGRP8-37 augments basal and stimulated acid secretion [15] and by the capacity of luminal acid to release CGRP from extrinsic afferents, eliciting a local inhibitory reflex [2]. In addition, CGRP stimulates the secretion of mucus and bicarbonate, increasing the protection of the gastroduodenal mucosa to the erosive effects of acid [2, 30, 31] (Fig. 2).

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H+ (low pH)

FIGURE 2. Schematic representation of the effects of CGRP in the stomach, modulating secretion and mucosal homeostasis in response to an increase in luminal acidity. When luminal pH is lowered excessively, luminal protons stimulate extrinsic CGRP-containing sensory afferent terminals in the mucosa, probably of both vagal and spinal origin. As a consequence, there is a local axonal reflex that results in the release of CGRP that stimulates CGRP1 receptors (CGRP8-37-sensitive) in effector cells of the mucosa and submucosa. CGRP activates inhibitory mechanisms (release of somatostatin, SST) and inhibits stimulatory mechanisms (gastrin, G; cholinergic activity, Ach) controlling acid secretion. As a result, the activity of the enterochromaffin-like cells (ECL) is diminished, which induces a reduced stimulation of parietal cells and secretion of protons to compensate the initial low luminal pH. Several mechanisms increasing gastric mucosal protection in response to the excess of acid are activated in parallel. CGRP increases the secretion of mucus and bicarbonate and releases nitric oxide (NO). Nitric oxide, in turn, produces local vasodilatation (VD), which increases mucosal blood flow to wash away the excess of acid, and activates repair mechanisms at a cellular level.

In the small intestine of the dog and the colon of the rat and guinea pig, CGRP stimulates electrolytes and fluid secretion [28]. In the guinea pig these effects are mediated through enteric neural reflexes, whereas in the rat colon the secretory responses are related to a direct stimulatory effect of the peptide on epithelial cells. The physiological relevance of these observations remains unclear. However, under pathophysiological conditions, CGRP might be involved in the fluid secretory responses associated with bacterial infections [24].

Vascular Effects A particularly well-characterized action of CGRP is its potent vasodilatory effects on gut vascular beds. The arteries and submucosal arterioles of the GI tract receive

a dense innervation from CGRP-containing extrinsic afferents. This, together with the localization of CGRP receptors on vascular endothelium and smooth muscle and the high potency of CGRP to induce vasodilatation in the gut vascular bed, supports an important vasoregulatory role for CGRP on the enteric circulation. In addition, there is evidence that the nonadrenergic noncholinergic dilation of the rat superior mesenteric artery is mediated through capsaicin-sensitive afferents releasing CGRP. Observations in α-CGRP knockout mice suggest that endogenous CGRP indirectly modulates vascular functions through the inhibitory modulation of the sympathetic tone. In the rat stomach, CGRP potently enhances mucosal blood flow through a CGRP8-37-sensitive mechanism operating in the submucosal arterioles and dependent upon the local release of nitric oxide (NO; Fig. 2) [13,

Calcitonin Gene-Related Peptide and Gastrointestinal Function / 1009 30]. CGRP seems to be an important component of the regulatory mechanisms of gastric mucosal blood flow during pathological situations associated with the disruption of the mucosal barrier. For instance, during ethanol or bile salt exposure, the mucous barrier is disrupted, the luminal acid enters in contact with the mucosa and diffuses into the extracellular spaces. This acid diffusion lowers the local pH and stimulates extrinsic afferents (probably through TRPV1 receptors) that release CGRP through an axonal reflex. As a result, among other effects of CGRP, there is an increase in mucosal blood flow that helps to neutralize and wash away the acid and plays a vital role in the protective mechanisms of the mucosa. Moreover, the antisecretory effects, the stimulation of bicarbonate and mucus secretion, and the activation of local repair mechanisms contribute to the protective effects of the peptide (Fig. 2) [13, 29–31]. Studies on the central regulation of gastric function suggest that gastric vagal efferent pathways interact with CGRP-containing afferents in the stomach. This interplay is instrumental for the stimulation of gastric mucosal blood flow observed during central vagal stimulation and might have physiological implications in the central control of gastric function [17]. The effects of CGRP on the microcirculation of the intestine are less clear. In the guinea pigs CGRP dilates submucosal arterioles, whereas in the rat the peptide does not affect mucosal blood flow in either the large or the small intestine.

Mucosal Homeostasis and Cytoprotection Gastrointestinal mucosal integrity and repair are largely under the modulation of extrinsic afferents releasing CGRP. This is supported by the ability of exogenous CGRP to protect the mucosa in different animal models of gastric injury and colonic inflammation and by the worsening of the responses under conditions of capsaicin pretreatment at neurotoxic doses depleting CGRP in gut afferent fibers. These observations might have an important physiological significance, because the gastroduodenal protective effects of capsaicin or chili powder can also be observed in humans. In the rat stomach, cytoprotective actions of CGRP are mediated through CGRP8-37-sensitive receptors and involve NO and KATP channels, whereas prostaglandins are not implicated [13, 30]. Accordingly, the blockade of CGRP receptors with CGRP8-37 or the immunoneutralization of CGRP prevents the protective effects of the peptide or the ability of intragastric capsaicin to reduce gastric damage and results in the exacerbation of experimentally induced injury. Cytoprotective effects of CGRP are largely vasodilatation- and hyperemia-

dependent. The hyperemia ensures the appropriate delivery of bicarbonate to the surface mucus layer and facilitates the repair of the mucosa. In addition, hyperemia-independent effects of the peptide (such as the activation of repair processes, inhibition of acid secretion, and enhancement of the secretion of bicarbonate and mucus) contribute to its protective effects [13, 29–31]. Interestingly, the activation of CGRP-containing afferents by vagal efferent mechanisms is important in centrally mediated cytoprotective responses in the gastric mucosa [13, 17]. CGRP-containing afferent nerves are not tonically active and they probably operate as an alarm system that, when recruited by mucosal stimuli (such as an excess of luminal protons) or vagal efferents, activates local defense or digestive mechanisms (postprandial gastric hyperemia). The fact that sensory neuropathies reduce the resistance to gastric injury supports the protective role of these mechanisms. Similar protective effects of CGRP might also apply to other parts of the GI tract. For instance, in the rat colon, CGRP released from sensory afferents dampens experimental colitis, whereas sensory denervation with capsaicin or pharmacological blockade of CGRP receptors worsens colitis [26].

Visceral Sensitivity and Nociception Mounting evidence points toward a predominant role for CGRP-containing capsaicin-sensitive afferent nerves in mediating a wide range of noxious sensations arising from visceral organs, including the GI tract. In rats, the administration of exogenous CGRP elicits a viscerosomatic response, contractions of the abdominal musculature, consistent with the presence of pain of visceral origin [5, 22]. In addition to mediating visceral pain responses in normal conditions, sensitization of CGRP-containing visceral afferents contributes to the visceral hyperalgesic responses observed during chemical [22] or mechanical colonic irritation [10]. Morphological and functional observations suggest an increased release of CGRP, both in the colon and the spinal cord, during colitis-associated hyperalgesic states in rats and noxious mechanical distention of the colon in mice [29, 34]. The direct implication of CGRP in visceral pain is further supported by the fact that either the blockade of CGRP1 receptors with CGRP8-37 or the in vivo immunoneutralization of the peptide prevented the hyperalgesic responses to colonic irritation [10, 22]. Convergent findings support that the site of CGRP action mediating colonic hyperalgesia is the spinal cord. First, morphological studies suggest an enhanced release of CGRP in the spinal cord during colonic irritation [34]. Second, the CGRP antagonist, CGRP8-37, is more effective in inhibiting colonic

1010 / Chapter 138 hyperalgesic responses after intrathecal than after intravenous administration [10, 22]. These observations might have a relevance to the understanding of the pathophysiological mechanisms underlying several unexplained colonic disorders that course with visceral hypersensitivity (including rectal hypersensitivity, fecal urgency, or irritable bowel syndrome). In fact, patients with rectal hypersensitivity and fecal urgency have an increased expression of TRPV1 receptors in sensory afferents as well as an increased number of CGRP-immunoreactive cell bodies in the submucosal ganglia [3], suggesting an increased stimulation and responsiveness of sensory mechanisms leading to the development of colonic hypersensitivity.

References [1] Adeghate E. Distribution of calcitonin-gene-related peptide, neuropeptide-Y, vasoactive intestinal polypeptide, cholecystokinin-8, substance P and islet peptides in the pancreas of normal and diabetic rats. Neuropeptides 1999; 33:227–35. [2] Akiba Y, Nakamura M, Nagata H, Kaunitz JD, Ishii H. Acidsensing pathways in rat gastrointestinal mucosa. J Gastroenterol 2002;37 (Suppl 14):133–8. [3] Chan CL, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, et al. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet 2003;361:385–91. [4] Freeman ME, Cheng G, Hocking MP. Role of alpha- and betacalcitonin gene-related peptide in postoperative small bowel ileus. J Gastrointest Surg 1999;3:39–43. [5] Friese N, Diop L, Chevalier E, Angel F, Riviere PJ, Dahl SG. Involvement of prostaglandins and CGRP-dependent sensory afferents in peritoneal irritation-induced visceral pain. Regul Pept 1997;70:1–7. [6] Gates TS, Zimmerman RP, Mantyh CR, Vigna SR, Mantyh PW. Calcitonin gene-related peptide-a receptor binding sites in the gastrointestinal tract. Neuroscience 1989;31:757–70. [7] Geppetti P, Trevisani M. Activation and sensitisation of the vanilloid receptor: role in gastrointestinal inflammation and function. Br J Pharmacol 2004;141:1313–20. [8] Green T, Dockray GJ. Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea pig. Neuroscience 1988;25:181–93. [9] Grider JR, Kuemmerle JF, Jin J-G. 5-HT released by mucosal stimulai initiates peristalsis by activation of 5-HT4/5-HT1p receptors on sensory CGRP neurons. Am J Physiol 1996;270: G778–G782. [10] Gschossmann JM, Coutinho SV, Miller JC, Huebel K, Naliboff B, Wong HC, et al. Involvement of spinal calcitonin gene-related peptide in the development of acute visceral hyperalgesia in the rat. Neurogastroenterol Motil 2001;13:229–36. [11] Hagner S, Knauer J, Haberberger R, Goke B, Voigt K, McGregor GP. Calcitonin receptor-like receptor is expressed on gastrointestinal immune cells. Digestion 2002;66: 197–203. [12] Holzer P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev 1991;43:143– 201.

[13] Holzer P. Control of gastric functions by extrinsic sensory neurons. In: Brookes S, Costa M, editors, Innervation of the Gastrointestinal Tract, London: Taylor and Francis; 2002, p. 103–70. [14] Holzer P, Peskar BM, Peskar BA, Amann R. Release of calcitonin-gene related peptide induced by capsaicin in the vascularly perfused rat stomach. Neurosci Lett 1990;108:195–200. [15] Kato K, Martinez V, St. Pierre S, Taché Y. CGRP antagonists enhance gastric acid secretion in 2-h pylorus-ligated rats. Peptides 1995;16:1257–62. [16] Kawashima K, Ishihara S, Karim Rumi MA, Moriyama N, Kazumori H, Suetsugu H, et al. Localization of calcitonin generelated peptide receptors in rat gastric mucosa. Peptides 2002;23:955–66. [17] Kiràly A, Sütö G, Livingston EH, Guth PH, St. Pierre S, Taché Y. Central vagal activation by TRH induces gastric hyperemia: role of CGRP in capsaicin-sensitive afferents in rats. Am J Physiol 1994;267:G1041–G1049. [18] Kuwasako K, Cao YN, Nagoshi Y, Tsuruda T, Kitamura K, Eto T. Characterization of the human calcitonin gene-related peptide receptor subtypes associated with receptor activity-modifying proteins. Mol Pharmacol 2004;65:207–13. [19] Maton PN, Sutliff VE, Zhou Z-C, Collins SM, Gardner JD, Jensen RT. Characterization of receptors for calcitonin-gene related peptide on gastric smooth muscle cells. Am J Physiol 1988;254: G789–G794. [20] Miyamoto Y, Yoneda M, Morikawa A, Itoh H, Makino I. Gastric neuropeptides and gastric motor abnormality in streptozotocininduced diabetic rats: observation for four weeks after streptozotocin. Dig Dis Sci 2001;46:1596–603. [21] Ozdemir FA, Berghofer P, Goke R, Goke B, McGregor GP. Specific calcitonin gene-related peptide binding sites present throughout rat intestine. Peptides 1999;20:1361–6. [22] Plourde V, St. Pierre S, Quirion R. Calcitonin gene-related peptide in viscerosensitive response to colorectal distension in rats. Am J Physiol 1997;273:G191–G196. [23] Plourde V, Wong HC, Walsh JH, Raybould HE, Taché Y. CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus. Peptides 1993;14:1225–9. [24] Pothoulakis C. Effects of Clostridium difficile toxins on epithelial cell barrier. Ann NY Acad Sci 2000;915:347–56. [25] Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, et al. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 2002;54: 233–46. [26] Reinshagen M, Flamig G, Ernst S, Geerling I, Wong H, Walsh JH, et al. Calcitonin gene-related peptide mediates the protective effect of sensory nerves in a model of colonic injury. J Pharmacol Exp Ther 1998;286:657–61. [27] Renzi D, Evangelista S, Mantellini P, Santicioli P, Maggi CA, Geppetti P, et al. Capsaicin-induced release of neurokinin A from muscle and mucosa of gastric corpus: correlation with capsaicin-evoked release of calcitonin gene-related peptide. Neuropeptides 1991;19:137–45. [28] Rolston RK, Ghatei MA, Mulderry PK, Bloom SR. Intravenous calcitonin gene-related peptide stimulates net water secretion in rat colon in vivo. Dig Dis Sci 1989;34:612–6. [29] Roza C, Reeh PW. Substance P, calcitonin gene related peptide and PGE2 co-released from the mouse colon: a new model to study nociceptive and inflammatory responses in viscera, in vitro. Pain 2001;93:213–9. [30] Taché Y. Inhibition of gastric acid secretion and ulcers by calcitonin gene-related peptide. Ann NY Acad Sci 1992;657: 240–7.

Calcitonin Gene-Related Peptide and Gastrointestinal Function / 1011 [31] Taché Y, Raybould H, Wei JY. Central and peripheral actions of calcitonin gene-related peptide on gastric secretory and motor function. Adv Exp Med Biol 1991;298:183–98. [32] Tani N, Miyazawa M, Miwa T, Shibata M, Yamaura T. Immunohistochemical localization of calcitonin generelated peptide in the human gastric mucosa. Digestion 1999; 60:338–43.

[33] Timmermans JP, Scheuermann DW, Barbiers M, Adriaensen D, Stach W, Van Hee R, et al. Calcitonin gene-related peptide-like immunoreactivity in the human small intestine. Acta Anat (Basel) 1992;143:48–53. [34] Traub RJ, Hutchcroft K, Gebhart GF. The peptide content of colonic afferents decreases following colonic inflammation. Peptides 1999;20:267–73.

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139 Peripheral Cholecystokinin JOSEPH R. REEVE, JR., DAVID A. KEIRE, AND GARY M. GREEN

their paper is the first description of two activities of cholecystokinin: stimulation of pancreatic secretion and gallbladder contraction. In the late 1920s, Ivy and Oldberg demonstrated that preparations with secretin activity could be separated from the stimulant of gallbladder contractions. Mellanby demonstrated that secretin stimulated water and inorganic electrolyte secretion by the pancreas that did not contain high concentrations of enzymes. Harper and Raper demonstrated that the mucosa of the upper small intestine contained a substance that was different from secretin because when this new activity was injected intravenously it stimulated pancreatic enzyme release. They named this new activity pancreozymin. Wang and Grossman studied pancreatic secretion after various substances were injected into the small intestine and observed that substances that released pancreozymin also released cholecystokinin (CCK). After Jorpes and Mutt purified secretin in 1961, they turned their attention to the purification and characterization of pancreozymin. They determined that pancreozymin was in a side fraction formed during the purification of secretin (pancreozymin was contained in the methanol-insoluble fraction of a concentrate of thermostable intestinal peptide, CTIP, portion of intestinal extracts). The pancreozymin was purified by several steps, including size exclusion chromatography and ion-exchange chromatography. The purified pancreozymin not only stimulated pancreatic secretion when injected intravenously, but it also stimulated gallbladder contraction. As the purification continued, the specific activity of pancreozymin and cholecystokinin increased in parallel, leading to the suggestion that the two activities were contained in the same peptide. The structure of CCK-33 was described in two publications by Mutt et al. in 1968 and 1971. The highly purified peptide retained both activities and is now known as cholecystokinin.

ABSTRACT As we enter the second century of cholecystokinin (CCK) research, it is apparent that there is still much to learn about the biochemistry, physiology, and pathophysiology of this gut peptide. For many hormones, peptide processing of a single gene product can result in multiple secreted molecular forms. For cholecystokinin, recent studies suggest that although there are many possible enzymatic processing products of preprocholecystokinin only one appears as a major circulating form (CCK-58). If these findings are confirmed and shorter forms of cholecystokinin are not only absent from mammalian tissues but are also aberrant in their actions compared to endogenous natural CCK, then past conclusions on the role of cholecystokinin in gut physiology will have to be reconsidered.

DISCOVERY Bayliss and Starling presented preliminary data to the Royal Society on January 23, 1902, that described a new substance that stimulated pancreatic secretion, and they named the substance secretin (see references in [29]). The secretin was extracted from the mucosa of the upper small intestine, and later that year they demonstrated that this preparation could also stimulate bile flow. They reasoned, “The question arises whether the substance exciting the liver is the same as that exciting the pancreas. It would be appropriate that the same body should perform both functions, but we must leave the question at the present undecided [1A].” In 1919 Braga and Campos found that a secretin preparation prepared in a similar manner to Bayliss and Starling’s preparation caused emptying of the gallbladder. If we assume the original secretin preparation of Bayliss and Starling contained both secretin and cholecystokinin, Handbook of Biologically Active Peptides

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1014 / Chapter 139 Many have isolated new molecular forms of cholecystokinin from intestinal extracts in amounts and in purities suitable for identification and assays of their biological activities. These new forms include CCK-83 [9], CCK-58 (from several species by multiple groups [14]), CCK-22 (from several species) [11], and CCK-8 [49] (references shown are for the first report on the molecular form). However, none of these forms would have been purified without the pioneering work that started with Bayliss and Starling and culminated with Jorpes and Mutt. Reading the descriptions of these early experiments shows how insightful and talented the experimenters were who laid the foundation for all of the cholecystokinin research taking place today.

duodenal and jejunal mucosa, where the peptide is localized to enteroendocrine cells [35]. Endocrine cells containing cholecystokinin, classified as I cells, are roughly trapezoidal, with their apical surfaces in contact with the intestinal lumen and with cholecystokinin-rich secretory granules clustered around the base [4]. This arrangement enables these I cells to be stimulated by luminal intestinal nutrients and to release their contents into the blood and adjoining tissue. In addition, cholecystokinin is observed in enteric nerves with the highest concentration of cholecystokinin-containing neurons found in the ileum and colon [35]. Neurons containing cholecystokinin are found in the myenteric plexus, submucosal plexus, and muscle layers of the small intestine and colon, and the celiac plexus and vagus nerve [36].

STRUCTURE OF THE PRECURSOR mRNA/GENE The approximately 7-kb rat cholecystokinin gene is made up of three exons separated by introns of approximately 1100 and 5300 bp [7]. The first exon contains the 5′ untranslated portion of the cholecystokinin mRNA, the second exon encodes the signal peptide and the prohormone, and the third exon is translated into the biologically important regions of the peptide. The mRNA has a size of 706 bases and is translated to create a 115-amino-acid peptide, preprocholecystokinin. For all species, there is only one gene encoding cholecystokinin, which is highly conserved.

CHOLECYSTOKININ PEPTIDE DISTRIBUTION Within the gastrointestinal tract, cholecystokinin is found in both secretory and neural tissues [36]. Cholecystokinin is observed in high concentrations in the

PROCESSING OF PREPROCHOLECYSTOKININ The nascent product of cholecystokinin mRNA translation is termed preprocholecystokinin. Multiple molecular forms of cholecystokinin are formed from preprocholecystokinin by a series of proteolytic and covalent modifications of preprocholecystokinin (Fig. 1). Six types of modifications of preprocholecystokinin produce the active peptides: (1) removal of the signal peptide by signal peptidase, (2) cleavage after double basic residues, (3) cleavage after single basic residues, (4) exopeptidase reactions, (5) carboxyl terminal amidation, and (6) covalent modification of amino acid side-chain groups.

Signal Peptide Cleavage The first step is the removal of the signal peptide that is on all peptides and proteins bound for export from

Preprocholecystokinin MKCGVCLCVVMAVLAAGALA

Prepeptide

QPVVPVEAVDPMEQRAEEAPRRQLRAVLRP

Cholecystokinin Forms

DSEPRARLGALLARYIQQVRKAPSGRMSVL KNLQGLDPSHRISDRDY*MGWMDF

(amide in fully processed peptide)

GRR

Amidation motif

SAEDY*EY*PS

Carboxyl terminal flanking peptide

FIGURE 1. Structure of preprocholecystokinin with single letter code for amino acids. The prepeptide or signal peptide is cleaved from preprocholecystokinin as the peptide crosses the endoplasmic reticulum. The resulting peptide, procholecystokinin, is processed by sulfation reactions at selected tyrosines (designated Y*), endopeptidase cleavages at one dibasic site (in amidation region) and five monobasic sites (forming the amino terminus of CCK-83, CCK-58, CCK-39, CCK-33, CCK-22, and CCK-8 designated by underlined Q, A, Y, K, N, and D with larger fonts, respectively). These molecular forms of cholecystokinin become biologically active when the glycine in the amidation region is cleaved to give F-amide as the carboxyl terminus. The carboxyl terminal flanking peptide may influence processing of procholecystokinin, but has no other known function.

Peripheral Cholecystokinin / 1015 their cell of synthesis. The signal peptide directs the insertion of preprocholecystokinin into the membrane of the endoplasmic reticulum, and the signal peptide is cleaved by signal peptidase that is attached to the luminal surface of the endoplasmic reticulum. The site of signal peptidase cleavage was determined when CCK83 was purified form human intestinal extracts [9]. Signal peptidase cleaves between residues 20 and 21 of preprocholecystokinin, yielding a signal peptide of 20 amino acids and the amino terminus of CCK-83 (see Fig. 1).

Processing at Double Basic Residues As the structures of prepropeptides were being discovered by determination of their mRNA, one of the first processing sites recognized was the cleavage after double basic residues. There are three pairs of basic residues in procholecystokinin (Fig. 1). Cleavage at the first pair of basic residues (R21R22 of procholecystokinin) leads to CCK-61 with an amino terminus blocked from sequencing by pyroglutamate. CCK-61 has not been detected in intestinal extracts, possibly due to a blocked amino terminus. In the new era of proteomics, a reexamination of the molecular forms of cholecystokinin by mass spectral analysis might lead to the discovery of CCK-61. The second pair of basic residues (R50K51 of procholecystokinin) is cleaved between the basic residues instead of after the basic pair to form CCK-33. The third pair (RR) in the amidation motif is cleaved after the pair of basic residues, as predicted, to form a carboxyl terminal Gly-Arg-Arg, which is an amidation motif.

Processing at Single Basic Residues Benoit et al. [2] have concluded that cleavages occur at single basic residues where there is an arginine (or occasionally a histidine) three, five, or seven residues before the residue cleaved if that residue is near a leucine or an alanine. In cholecystokinin, four single basic residues fulfill these criteria, and the K51 (in R50K51 pair of basic residues) of procholecystokinin is preceded by an arginine seven residues before it, suggesting procholecystokinin may be cleaved at five monobasic motifs. Peptides have been characterized that correspond to each of these potential sites. Peptides that could be formed from these five cleavage sites are CCK-58, CCK-39, CCK-33, CCK-22, and CCK-8. However, recent observations on molecular forms of cholecystokinin in rat intestinal extracts by our laboratory question the presence of peptides other than CCK58 (unpublished observations), and the single basic motif may be for enzymes not normally found in the cholecystokinin secretory pathway. However, the possi-

bility that only CCK-58 is present in intestinal extracts (unpublished observations) and blood suggests that the rules for processing at single basic residues do not accurately predict the processing of procholecystokinin. The predictions may likewise be unreliable for other propolypeptides.

Exopeptidase Reactions The only documented exopeptidase reaction occurring for cholecystokinin is the carboxypeptidase activity that removes the two basic residues in the amidation region (Fig. 1).

Carboxyl Terminal Amidation The nitrogen from the glycine that becomes the carboxyl-terminal residue after the exopeptidase reaction (Fig. 1) is the atom that forms the amide in carboxyl terminus of cholecystokinin. A peptidylglycine α-amine monooxygenase enzyme complex is responsible for the cleavage that produces the peptide amide and glyoxylate [10] from the carboxyl-terminal glycine.

Covalent Modifications of Amino Acid Side-Chain Groups There are four tyrosines in procholecystokinin, three of them are sulfated in the Golgi, presumably by either tyrosylprotein sulfotransferase (TSP)-1 or TSP-2 [1]. The three tyrosines have the consensus motif for sulfation. Interestingly, it has been believed for decades that the tyrosine in the carboxyl terminal biologically active region of cholecystokinin is fully sulfated, whereas the analogous tyrosine in gastrin is partially sulfated, suggesting different regulation of posttranslational sulfation of the two peptides. However, more recent reports demonstrated the presence of nonsulfated CCK-58 in porcine intestinal extracts. There has been discussion that this was an artifact of the extraction of pig CCK-58 on a large scale, when peptides are extracted with acidic buffers for days. However, more recently nonsulfated CCK-58 has been purified from canine intestinal extracts for which extractions were finished in hours rather than days. The results suggest that the homologous tyrosine in gastrin and cholecystokinin are not fully sulfated and that the physiology of nonsulfated cholecystokinin should be further studied. This suggestion is supported by the fact that in preliminary studies it has been shown that nonsulfated CCK-58 is as potent, but one-third as efficacious, as sulfated CCK-58 for stimulation of pancreatic secretion [54]. The methods recently used to show that CCK-58 is the only endocrine form of cholecystokinin [50] in the rat would not differentiate between the sulfated and nonsulfated peptides.

1016 / Chapter 139 MOLECULAR FORMS OF CHOLECYSTOKININ There has been some dispute about the molecular forms of cholecystokinin that are present in tissue and in the circulation. For tissue, we suggest that some of the discrepancy results from the proximity of the cells that synthesize cholecystokinin (the intestinal I cells) to the pancreatic duct, resulting in the exposure of extracted cholecystokinin to high concentrations of digestive enzymes. Other sources of degradation could be enzymes that are exposed to cholecystokinin products when cellular compartments are destroyed during the extraction process. Therefore, some of the species variability reported for the molecular forms of cholecystokinin may result in species differences in amount or activity of digestive or endosomal enzymes. We suggest that peptide characterization techniques have improved so much since the initial characterizations of the molecular forms of cholecystokinin that the time has come to reevaluate the molecular forms of cholecystokinin in the intestine and brain. This would not be important if all molecular forms of cholecystokinin had the same bioactivity, but various forms can have very different quantitative and qualitative expressions of biological activity. Cholecystokinin was first chemically characterized as a 33-amino-acid peptide [42, 43], and shortly thereafter a 39-amino-acid variant was described [41]. These reports by Mutt et al. were milestones not only for cholecystokinin but for all biologically active peptides. Using elegant biochemical analysis, it was shown that both cholecystokinin forms ended in phenylalanineamide and both contained a sulfated tyrosine seven amino acids from the carboxyl terminus. Amidation of the carboxyl terminus was described for other biologically active peptides before it was detected in cholecystokinin, but Tatemoto and Mutt were the first to recognize that carboxyl-terminal amidation might be used as a tool for discovering new biologically active peptides [66]. Using this tool, Mutt and Tatemoto discovered other bioactive peptides (e.g., neuropeptide Y, NPY [64]; and peptide YY PYY [63]). We are tempted to speculate that Mutt, in developing methods for detecting carboxyl terminal amides in secretin and cholecystokinin, may have contributed to the discovery of many other peptides. Most believed that CCK-33 or CCK-39 was the largest stored and circulating form of cholecystokinin until the early 1980s when a new postdoctoral fellow (Viktor Eysselein) showed an article to an investigator in Walsh’s laboratory who had just finished his postdoctoral fellowship ( Joe Reeve) that described a larger form of cholecystokinin in tissue and blood of pigs. Being a protein chemist, Reeve decided to enlighten Eysselein (a

medical doctor) about nonspecific binding of peptides to larger proteins. Experiments were designed to eliminate peptide-protein interactions for cholecystokinin extracted from canine intestine, but the cholecystokininlike immunoreactivity (CCK-LI) continued to elute before CCK-33 standards during gel-permeation chromatography. The new technique for the purification of peptides, reverse-phase high-performance liquid chromatography (HPLC), allowed the purification of this new molecular form of cholecystokinin from canine intestinal extracts. Microsequence analysis of this larger form showed that there were 19 additional amino acids that extended the amino terminus of CCK-39, demonstrating that canine intestines contained CCK-58 [14]. Working with very fresh intestinal extracts and rapid characterization of the CCK-LI by either gel-permeation chromatography or HPLC, our laboratory demonstrated that CCK-58 was the major stored form of cholecystokinin in canine intestine [49] even when the diminished cross-reactivity of CCK-58 was not taken into account. Many believe that other molecular forms predominate in the intestine of other species. However, CCK-58 has been purified and characterized from rat [67], rabbit (manuscript submitted), pig [3], human [13], cat (unpublished observations), and cow [12]. Although most of these studies did not study the relative amounts of CCK-58 to other molecular forms, it is important to note that most, if not all, amounts of CCK-58 are underestimated because this peptide is not as cross-reactive as smaller forms of cholecystokinin. The measurement of CCK-58 by bioassay does not yield better quantitative data because CCK-58 is less potent than CCK-8 [48] for release of amylase from pancreatic acini. Others have observed the same diminished radioiommunoreactivity and bioactivity for CCK-58 [24]. Therefore, appropriate standards are necessary for the quantification of cholecystokinin, and the lack of CCK-58 standards may lead to the underestimation of CCK-58 levels in tissue or blood samples. We suggest that the validation of standards requires the use of the proper species of cholecystokinin (see Table 1 for a comparison of crossreactivity of CCK-58 from various species), demonstration of the concentration of the standard by amino acid analysis or absorbance at 280 nm (not the use of powder weight as a valid estimate of peptide amount), and mass spectral analysis of the standard to ensure no modifications are present that might change its immunoreactivity.

CHOLECYSTOKININ RECEPTORS Two CCK receptors, now termed CCK-1 and CCK-2, were identified first by pharmacological properties [61]

Peripheral Cholecystokinin / 1017 TABLE 1. Immunoreactivity of CCK-8 compared to CCK-58 from various mammalian species. The underlined residues are those that differ in the various species of CCK-58 and must account for the differences in the epitope region (the last 10 amino acids of CCK-58). Since the epitope region is identical in all species differences in cross reactivity must result form various tertiary structures induced by underlined residues. CCK-8 Human CCK-58 Rat CCK-58 Dog CCK-58 Rabbit CCK-58

DYMGWMDF-amide VSQRTDGESRAHLGALLARYIQQARKAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF-amide AVLRPDSEPRARLGALLARYIQQVRKAPSGRMSVLKNLQGLDPSHRISDRDYMGWMDF-amide AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQNLDPSHRISDRDYMGWMDF-amide AAQRTDVESRGHLGALLARYIQQARRAPAGRMSIIKNLQSLDPSHRISDRDYMGWMDF-amide

Peptide CCK-8 Human CCK-58 Rat CCK-58 Dog CCK-58 Rabbit CCK-58

Antibody

ID50 CCK-8/ID50 CCK-58

5135 RO16 RO16 5135 5135

1.0 2.6 5.3 2.0 2.0 21.9

and finally by cloning [32]. Both natural and expressed CCK-1 receptors show a 100- to 1000-fold preference for sulfated cholecystokinin forms [57], whereas the CCK-2 receptor shows only a 10- to 20-fold preference for the sulfated forms [25]. In the rat gastrointestinal tract, CCK1 receptors are observed in pancreatic acinar cells, chief cells, and D cells of the gastric mucosa, smooth muscle cells of the gallbladder, pyloric sphincter, sphincter of Oddi, gastrointestinal smooth muscle cells, and enteric neuronal cells [27, 68]. By contrast, CCK-2 receptors are found broadly distributed in smooth muscle cells, parietal cells, enterochromaffinlike D cells and chief cells of the gastric mucosa, myenteric plexus neurons, pancreatic acinar cells, monocytes, and T-lymphocytes [23, 27, 39, 55, 56, 62, 68]. It is important that both the CCK-1 and CCK-2 receptors are present in two distinct populations of vagal afferent fibers projecting to different regions of the nucleus tractus solitarii (NTS) [6]. A common perception is that all sulfated forms of cholecystokinin bind equally to the CCK-1 and CCK-2 receptor subtypes. However, there is a significant difference in the binding of sulfated CCK-8 and sulfated CCK-58. More surprisingly, there is a large and significant difference in the binding of sulfated CCK-58 from various mammals to CCK-1 and CCK-2 receptors (unpublished results), even though their biologically active regions are identical in amino acid sequence. We hypothesize that the variation in receptor binding is due to differences in tertiary structure and that these differences are stable in the presence of receptors. This hypothesis is supported by the fact that a solution tertiary structure can also explain the differences in digestion of CCK-8 and CCK-58 by neural endopeptidase and the fact that several mammalian CCK-58 peptides differ

in cross reactivity (Table 1) to antibodies whose epitope is the carboxyl terminal region (a region that is identical in all mammals studied).

SOLUTION CONFORMATION The hypothesis that tertiary structure is important for receptor binding and activation is supported by the observation that CCK-8 has a different carboxyl terminal solution conformation from either canine or rat CCK-58 [30, 31]. Furthermore, the peptide that binds the mouse CCK-1 receptor most potently, CCK-58, is the weaker stimulant of amylase release from rat pancreatic acinar cells [53]. These studies need to be repeated using the same species for binding and bioactivity studies, but nevertheless the results demonstrate that the solution conformation is important for both receptor binding and receptor activation. These biological studies also need to be done with CCK-58 from various mammalian species. Such studies will help us understand the importance of tertiary structure in the expression of biological activity. There have been two groups that have attempted to predict the structure of CCK-1 receptor when it binds to cholecystokinin [8, 18]. In both sets of studies, short cholecystokinin agonists or antagonists have been used to study the interactions between the residues of the peptide with the residues of the receptor. For CCK-58, we suggest that the receptor’s activation of intracellular signaling pathways may be influenced by the amino terminus of CCK-58 because the 40 N-terminal residues modify the tertiary structure of the C-terminal residues involved in receptor binding.

1018 / Chapter 139

There are approximately 10 actions of endocrine peptides that have been firmly established as physiological, based on criteria suggested by Grossman and others in the 1970s [21]. For cholecystokinin, two of those actions are the regulation of pancreatic secretion and gallbladder contraction. In addition, cholecystokinin is increasingly recognized as an inhibitor of gastric motility. A more speculative physiological action for cholecystokinin is the inhibition of food intake, or satiety.

creas new studies will be required to determine the ratio of CCK-58ns and CCK-58s released after a meal, if various macronutrients can change this ratio, and if various ratios of the two peptides produce different patterns of pancreatic secretion. Most original studies of the physiological actions of cholecystokinin were done in rodents, for which there are two possible routes of actions of cholecystokinin: direct actions via the CCK-1 receptors on pancreatic acinar cells and indirect actions via CCK-1 receptors on vagal afferents [28]. Humans and many other species do not have CCK-1 receptors on their pancreas, so cholecystokinin must act indirectly via receptors on vagal afferents or via other yet undiscovered indirect pathways [28]. Because CCK-58ns stimulates pancreatic secretions in rat, it will be important to determine if it will cause similar stimulation in other species. One possible route of action of CCK-58ns is via CCK-2 receptors present on vagal afferents [6].

Regulation of Pancreatic Secretion

Gallbladder Contraction

One of the most interesting aspects of cholecystokinin is emerging evidence that various molecular forms of cholecystokinin have different quantitative and qualitative expressions of biological activity for pancreatic secretion. In conscious rats, CCK-8 causes a stimulation of pancreatic protein with little change in the rate of fluid secretion resulting in a marked increase in enzyme concentration in pancreatic juice [70]. A significantly different pattern is elicited by CCK-58. Both protein and fluid are stimulated by CCK-58, resulting in more total enzyme secretion with little change in enzyme concentration [70]. Similar results comparing CCK-8 and CCK-58 have been observed in the anesthetized rat [51]. Surprisingly, preliminary results show that nonsulfated CCK-58 (CCK-58ns) can cause the stimulation of pancreatic secretion even though it will not stimulate amylase release from purified pancreatic acinar cells [54]. These experiments suggest that CCK-58ns may act indirectly through CCK-2 receptors to stimulate pancreatic secretion. CCK-58ns stimulation is approximately one-third as efficacious as CCK-58s and stimulates fluid in a similar fashion to the sulfated peptide. There have been two reports [3, 52] that nonsulfated CCK-58 is present in intestinal extracts, but the relative amounts of CCK-58ns and CCK-58s were not directly determined in either study. Note that it is possible that other nonsulfated molecular forms of gastrin and cholecystokinin may not have the same actions as CCK-58ns because, unlike CCK-58, they are rapidly degraded before having a chance to activate the CCK-2 receptor. Clearly, to understand the physiological significance for the pan-

Again, the molecular form of cholecystokinin is critical for patterns of actions at the gallbladder. CCK-58 elicited a different pattern of gallbladder contraction from CCK-33 or CCK-8 [65].

PERIPHERAL BIOLOGICAL ACTIONS OF CHOLECYSTOKININ The peripheral actions of cholecystokinin can be divided into three categories: (1) regulation of a meal, (2) other actions not directly influencing a meal, and (3) pathophysiological actions.

Regulation of a Meal

Gastric Motility CCK-8 relaxes the smooth muscle of the forestomach and increases the contractions of the pylorus, resulting in a slowing of food passing from the stomach to the duodenum. Relaxing of the forestomach is mediated by capsaicin-sensitive vagal afferents [47]. CCK-58 evokes a qualitatively different pattern of vagal discharge and thus may express differences in actions in gastric motility [33]. Satiety Over 30 years ago, intraperitoneal administration of CCK-8 was shown to inhibit food intake in rats [17]. Inhibition of food intake in response to CCK-8 has also been demonstrated in mice, monkeys, and humans. However, in all these studies the results were short term and repeated CCK-8 did not produce significant decreases in food intake. In preliminary studies, Gibbs’s group has shown that CCK-8 causes reduction in meal size with a concomitant reduction in meal interval, resulting in no significant reduction in net food intake [46]. Like CCK-8, CCK-58 causes a reduction in meal size, but, unlike the shorter peptide, CCK-58 causes an extension of the interval between meals, resulting in a significant reduction in net food intake [46].

Peripheral Cholecystokinin / 1019

Peripheral Actions Not Directly Influencing Feeding and Digestion Regulation of Body Temperature by Cholecystokinin Previous studies showed that peripheral and intracerebroventricular injections of CCK-8 induced hypothermia in guinea pigs and rats. In the rat this response resembled the regulation of short-term food intake inhibition by CCK-8, particularly with respect to ablation of the response by the sensory neurotoxin capsaicin [59]. The reduction of rectal temperature by doses of CCK-8 administered intraperitoneally that were in the range known to suppress food intake and the effect of systemic capsaicin treatment on the responses were examined in rats by South [59]. Intraperitoneal injection of CCK-8 at 4 μg/kg significantly lowered rectal temperature, and the reduction in temperature coincided with the maximal suppression of food intake. This response and the suppression of food intake were significantly attenuated or abolished by intraperitoneal capsaicin pretreatment, even at doses of capsaicin (25 mg/kg) that did not alter corneal chemosensitivity, suggesting that the action of CCK-8 was limited to fine sensory fibers accessible to intraperitoneal capsaicin application. To further examine the mechanisms by which CCK-8 elicited hypothermia, Miyasaka and coworkers [45] investigated the role of CCK-1 receptor alterations in the regulation of body temperature in naturally occurring CCK-1 receptor knockout rats (Otsuka Long Evans Tokushima Fatty, OLETF, rats) and CCK-1 receptor knockout mice. OLETF rats had a normal circadian rhythm, but had a markedly altered response to changes in ambient temperature, marked by a large hysteresis. The authors concluded that the OLETF rat had a defect in detecting ambient temperature. The role of the CCK-1 receptor in this defect was further examined in knockout mice deficient in CCK-1 receptor and CCK-2 receptor and in mice with the combined CCK-1 and CCK-2 receptor knockout. The results were in agreement with results in OLETF rats in that mice deficient in the CCK-1 receptor, or the combined receptor deficit, had normal circadian rhythms but showed an abnormal response to changes in ambient temperature, exhibiting a large level of hysteresis. The investigators concluded that the CCK-1 receptor plays a role in the sensory pathway of transmitting ambient temperature information from the skin to the brain. Cardiovascular Actions of Cholecystokinin The peripheral administration of CCK-8 increases blood pressure in anesthetized and conscious rats and counters severe hemorrhagic shock in anesthetized rats

[26]. CCK-8 caused dose-dependent increases in blood pressure in conscious rats that were markedly inhibited by the CCK-1 receptor antagonist L364,718. The pressor effects of CCK-8 were mediated largely through alphaadrenoceptors [26]. The role of peripheral CCK-1 receptors on such actions of cholecystokinin was investigated in the pithed rat by Gaw et al. [16]. They reported that sulfated CCK-8 induced a dose-dependent bradycardia and increase in mean arterial blood pressure. These responses were inhibited by the blockade of the CCK-1 receptor but not by blockade of the CCK2 receptor. The authors concluded that CCK-8 acted via CCK-1 receptors to increase arterial blood pressure via the activation of alpha-adrenoceptors and through direct actions on the heart to produce bradycardia. The role of CCK-1 receptors in cardiovascular effects of CCK-8 was further studied using the natural CCK-1 receptor deficient OLETF rat by Kurosawa et al. [34]. In control Long-Evans Tokushima Otsuka (LETO) rats, the heart rate deceased after intravenous CCK-8 at 3 nmol/kg, and this was abolished by treatment with the CCK-1 receptor antagonist MK-329 but not after treatment with the CCK-2 receptor antagonist L-365,260. Predictably, the heart rate of OLETF rats lacking the CCK-1 receptor was unaffected by CCK-8. On the basis of CCK-1 receptor precursor mRNA location, the investigators suggested that CCK-8 decreases heart rate via CCK-1 receptors located in the atrium. Involvement of Cholecystokinin in Functions Associated with Reproduction The regulation of cholecystokinin blood levels and several actions of cholecystokinin are associated with the menstrual cycle, pregnancy, lactation, and copulation. In humans, fasting CCK levels were significantly increased during the luteal phase of the menstrual cycle and were also significantly higher during pregnancy than during the menstrual cycle [15]. The authors speculated that the higher levels of cholecystokinin might increase pancreatic function during pregnancy. Newborn human infants increase their plasma CCK concentrations shortly after initiating breast feeding, and this is closely associated with satiety behavior [40]. The authors concluded that peripheral CCK might be important as a satiety factor in the regulation of food intake in newborns. In lactating animals, suckling stimulated significant increases in plasma CCK levels, which were abolished by abdominal vagotomy [37]. Lesions that disrupted the oxytocin-mediated milk-ejection reflex also blocked the increase in plasma CCK in response to suckling in lactating animals. The authors concluded that peripheral CCK receptor mechanisms induce the release of CCK in the brain [37]. Sexual

1020 / Chapter 139 activity in male rats affects plasma CCK levels and CCK1 receptors may be involved in the satiety induced by ejaculation. The ingestion of food and ejaculation cause comparable increases in plasma CCK-8 levels and comparable inhibition in food intake [38]. Male fooddeprived rats ignored food pellets immediately after ejaculating with a sexually receptive female rat [38]. The effect of ejaculation on the latency to start eating was partially reversed by intraperitoneal injection of the CCK receptor antagonist proglumide.

Pathophysiology of Cholecystokinin Preliminary results show that, unlike CCK-8, CCK-58 is a poor inducer of pancreatitis [69]. Others have shown that part of the induction of pancreatitis is caused by activated pancreatic enzymes not being able to leave the cell [5]. Another stimulant of pancreatic secretion (e.g., bombesin) that produces a large stimulation of fluid secretion in conscious rats similar to CCK-58 [60] is similarly a poor inducer of pancreatitis [20]. Physiological and Pathophysiological Relevance of Effects of CCK-8 in Nondigestive Actions of CCK In most of the animal studies reviewed so far, CCK-8 was administered at dose levels considered to be pharmacological (microgram or nanomole amounts) and should therefore be interpreted cautiously. This caveat is even more important in light of recent studies that show that CCK-8 may not be a physiological endocrine form of cholecystokinin and that the actions of CCK-8 appear to be highly aberrant compared to CCK-58 [70], which is the only detectable endocrine form of cholecystokinin in the rat [50]. Because the amounts of CCK-8 used in studies of behavioral and other nondigestive functions of CCK are pharmacological, might this be nevertheless relevant? Are there conditions under which cholecystokinin is secreted at abnormally high levels, and are the effects of such high endogenous levels similar to effects produced by pharmacological amounts of CCK-8 used in behavioral and nondigestive studies? The most-studied pathological effect of high doses of cholecystokinin is induction of acute edematous pancreatitis by CCK-8 or cerulein [19]. The doses of CCK-8 or cerulein that induce acute pancreatitis are in the range frequently used in behavioral studies and in studies on nondigestive functions of cholecystokinin. Are the effects of abnormally high concentrations of endogenous cholecystokinin pathological? The strongest stimulant of endogenous CCK release in rats is the removal of pancreatic secretions and bile acids from the proximal intestine. Removal of each secretion alone is much less effective

on plasma CCK levels than the removal of both simultaneously [44]. Thus, it was predicted that prolonged exclusion of bile and pancreatic juice from the proximal small intestine would cause severe edematous pancreatitis in the rat. That this does not occur (even though pancreatic injury is observed) [58] was a mystery until it was shown that synthetic CCK-58, the endogenous form of cholecystokinin in the rat, at supramaximal doses was much less damaging to the pancreas than equivalent doses of CCK-8 or cerulein [69]. This suggests that levels of endogenous CCK that are equivalent to those produced by pharmacological doses of CCK-8 in behavioral and nondigestive function studies may not produce the same effects as CCK-8 or cerulein. Studies to test this possibility will be practical when large amounts of highly purified synthetic sulfated CCK-58 become available.

CONCLUSION In the past, a need for standardization of nomenclature for cholecystokinin became apparent because multiple names were used for a single peptide. The convention adapted was that cholecystokinin would be used at all times and that pancreozymin or cholecystokinin-pancreozymin (CCK-PZ) should be dropped from use [22]. Today, one name is used to describe many peptides (CCK-8, CCK-33, etc.), which might be appropriate if all forms of cholecystokinin had the same actions. However, because CCK-58 has significant qualitative differences in action from shorter forms of cholecystokinin, to avoid confusion we suggest authors designate which form is being described in titles and throughout the text of all articles on cholecystokinin. As we enter the second century of cholecystokinin research, a new chapter is unfolding in the story of cholecystokinin’s role in regulation of mammalian physiology. If the finding is confirmed that shorter forms of cholecystokinin are not only absent from mammalian tissues but are also atypical in their actions compared to endogenous natural CCK, which appears to be CCK-58, this will require our rethinking of regulation by cholecystokinin specifically and by bioactive peptides in general.

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140 Corticotrophin-Releasing Hormone (CRH) Family in the Gastrointestinal System LIXIN WANG, PU-QING YUAN, AND MULUGETA MILLION

pain and strengthened the concept of the brain-gut axis [34–36]. The discoveries of three new mammalian peptides in the CRH family, namely urocortin (Ucn) 1, 2, and 3, coupled with the development of selective antagonists of CRH receptors, have helped to unravel the CRH receptor types mediating CRH effects on different GI functions and also the existence of a peripheral CRH system in the GI organs. Ucn 2 and 3 are selective agonists of CRH receptor 2, whereas Ucn 1 has a higher binding affinity than CRH to both CRH receptors 1 and 2. The selective agonists and antagonists have helped with the characterization of CRH actions, identification of CRH receptor types, and recognition of the existence of a peripheral CRH system in the GI organs.

ABSTRACT Both central and peripheral corticotrophin-releasing hormone (CRH) signaling pathways are involved in the gut responses to stress. The CRH family of peptides and receptors exists in the gastrointestinal system. CRH1 and CRH2 receptors play different roles in gastrointestinal responses to stress and to nociceptive stimuli. CRH1 mediates increased colonic motor function and visceral sensitivity, whereas CRH2 activation leads to decreased gastric motility and an antinociceptive effect. Studies indicate that CRH, its related peptides, and CRH receptors are relevant to treating stress-related gastrointestinal disorders, such as irritable bowel syndrome (IBS).

EXPRESSION AND LOCATION OF CRH PEPTIDES AND RECEPTORS IN THE GI SYSTEM

INTRODUCTION Corticotrophin-releasing hormone (CRH) was originally discovered as a neuroendocrine factor released from neurons in the hypothalamus to stimulate adrenocorticotrophic hormone (ACTH) secretion from the pituitary (see Chapter 92 in the Brain Peptides section of this book). CRH is well recognized now as a multifunctional neuropeptide involved in neuroendocrine, autonomic, and behavioral stress responses. The first evidence of its effects on the gastrointestinal (GI) system was provided by Taché et al. in 1983 [33], showing that CRH injected into the cisterna magna inhibited pentagastrin- or thyrotrophin-releasing hormone (TRH)stimulated gastric acid secretion in fasted rats. Advances made in the last 3 decades show the involvement of both central and peripheral CRH signaling pathways in the GI response to stress, inflammation, and visceral Handbook of Biologically Active Peptides

CRH family peptides and receptors exist in the GI system. Recent studies demonstrate more specified tissue and cellular location in the GI system. However, there are some mismatches and a lack of a detailed mapping of mRNA and peptides. Identification of the mRNA variants and development of specific antibodies (especially those for the receptors) are needed.

CRH and Related Peptides A limited number of reports suggest that CRH is expressed in peripheral tissues and mainly produced by peripheral immunocytes. It is suggested that peripheral CRH mRNA has different forms from those in the brain

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1024 / Chapter 140 [39]. In human colonic tissues, CRH immunoreactivity and CRH mRNA are detected in the colonic mucosal cells near the base of the crypts. The mucosal cells that express CRH mRNA are also immunostained with 5hydroxytryptamine, suggesting that normal human colonic mucosal enterochromaffin cells produce CRH [11]. Ucn 1 mRNA is found in mucosal cells in the stomach and duodenum [1] and enteric neurons of rats [9]. Radioimmunoassay (RIA) demonstrates that Ucn 1 exists from the stomach to the colon in a higher concentration than that in the hypothalamus of rats [28]. The peptide has been shown to be present in parietal cells of the stomach, in nerve terminals in the myenteric and submucosal plexuses, and in some enteroendocrine cells in the rat small and large intestine [13]. In the human colon, immunoreactive Ucn 1 and Ucn 1 mRNA are predominantly detected in lamina propria macrophages and isolated lamina propria mononuclear cells [25]. The expression of Ucn 2 mRNA in the GI tract is restricted mainly to the stomach, with very low levels of expression in the duodenum, jejunum, ileum, and colon [4]. The cellular location of Ucn 2 in the GI tract is still unknown. In rats, the GI tract contains relatively high levels of Ucn 3 mRNA compared with other peripheral organs/ tissues. Significant levels of Ucn 3 mRNA are found in the colon, small intestine, and stomach but not in the cecum or esophagus [10, 18]. Immunohistochemical studies detect specific Ucn 3 signals in the muscularis mucosa of the mouse small intestine [10]. In the human large intestine, both immuno- and in situ hybridization histochemistry demonstrate Ucn 3 expression in myenteric and submucosal plexus cells, vascular endothelial cells, vascular smooth muscle cells, smooth muscle layers, and enterochromaffin cells [32]. Ucn 2 and Ucn 3, but not Ucn 1, are also found in the mouse pancreas [19]. The level of Ucn 3 mRNA appears to be higher than Ucn 2 mRNA. Ucn 3 mRNA and immunoreactivity are located in the pancreatic islets. Double immunofluorescent staining shows that the majority of Ucn 3-immunopositive cells are colocalized with insulin-positive cells but not with glucagon.

CRH Receptors The distribution and expression of two types of CRH receptors, CRH1 and CRH2, in the brain are well documented. However, information regarding their peripheral distributions, especially in the GI tract remains incomplete. The new findings indicate that CRH1 and CRH2 receptors, as well as some subtypes, have unique tissue distribution in the GI tract despite their considerable sequence similarity.

Although CRH1 receptor mRNA was first detected in the duodenum, few studies have revealed its existence in other parts of the GI tract. More recently, studies have demonstrated CRH1 immunoreactivity in the enteric plexus of the stomach and intestine of guinea pigs and rats [20, 30]. In the guinea pig, CRH1 receptor is co-localized with calbindin, choline acetyltransferase, and substance P in the myenteric and submucosal plexus and with neuropeptide Y in the submucosal neurons [20]. In rat, CRH1 immunoreactivity is displayed also in some endocrine cells and nerve bundles among smooth muscles of small intestine [30], in cells of the colonic crypts, and scattered in cells of the surface epithelium and the lamina propria of the proximal colonic mucosa [3]. CRH2α and CRH2β mRNA signals are low in the stomach and intestines compared with high levels of CRH2α in the brain and those of CRH2β in the heart, skin, and skeletal muscles in mice [5]. In humans, a study showed that CRH2α mRNA was detected in the isolated lamina propria mononuclear cells of the colonic mucosa, and CRH2β mRNA, a minor isoform in human tissues, was also detected at a low level [25].

ACTIONS OF CRH AND RELATED PEPTIDES IN GI FUNCTIONS Central or peripheral administration of CRH and related peptides mimic GI responses to stress, including increased gastric acid secretion, delayed gastric emptying, slow small intestine transit, and increased colonic motor activity. CRH receptor antagonists block the similar responses induced by stress as well as those by CRH peptides, suggesting the involvement of CRH signaling pathways in stress-related GI functional alterations.

Gastric and Duodenal Acid and Bicarbonate Secretion In the 1980s, immediately after the characterization of ovine CRH, its effects on gastric and duodenal secretion were reported. Central injection of CRH or sauvagine (the amphibian analog) in the rat and dog reduced gastric acid secretion stimulated by intravenous (IV) infusion of pentagastrin or by intracisternal (IC) injection of TRH [34]. On the other hand, gastric and duodenal bicarbonate secretion was increased after central and/or IV injection in rats, and the CRH receptor antagonist (α-helical CRH9–41) blocked the effect [15]. The CRH-inhibited gastric acid secretion was not associated with local gastric factors, such as gastrin and histamine, but related to autonomic nerve modulation [34]. The decreased gastric acid secretion and increased

Corticotrophin-Releasing Hormone (CRH) Family in the Gastrointestinal System / 1025 gastric and duodenal bicarbonate after CRH treatment indicated that CRH may have protective effects on the upper GI tract during stress.

GI Motor Alterations The gastric and colonic motility are altered differently depending on the type of stressors. However, most of them delay gastric emptying while stimulating colonic transit, and these alterations are reproduced with high fidelity by CRH and related peptides [21, 35, 37]. Delayed gastric transit occurs after either central or peripheral injection of CRH, sauvagine, Ucn 1, and Ucn 2 in rodents as revealed by the gastric-emptying rate with nutrient or nonnutrient food. Ucn 1 injected intracerebroventricular (ICV) or IV changes the fasted motor pattern of antral contraction to fedlike ones in conscious rats, as shown by manometric recording [21]. CRH administered ICV has higher potency than Ucn 1 and 2 in inhibiting gastric emptying, whereas by peripheral injection (intraperitoneal, IP, or IV), Ucn 1 and Ucn 2 have a stronger effect than CRH. Although Ucn 3 is a selective CRH2 agonist, it has a much smaller effect on gastric transit than Ucn 1 or Ucn 2 [21]. The potency disparity of CRH and Ucns on the stomach and colon motor functions might be associated with their binding affinity to CRH receptors as well as a difference in central and peripheral mechanisms engaged on activation of CRH receptors. The reason for the weak effect of Ucn 3 on the GI tract is unknown despite its high affinity to CRH2. However, it may be related to the low efficacy of Ucn 3 in activating signal transduction mechanisms and to its chemical and pharmacodynamic properties, which might be different from other CRH peptides. Evidence indicates that CRH2 receptors play a major role in mediating CRH and related peptide-induced inhibition of gastric motor functions. The nonselective CRH receptor antagonists (α-helical CRH9–41 and astressin) and the selective ones for CRH2 (antisauvagine-30 and astressin2-B), but not those for CRH1 (NBI27914, NBI-35965, CP-154,526, and antalarmin), administered peripherally block CRH and related peptide-induced, as well as stress-induced, inhibition of gastric motility [21, 35]. However, the central effect of CRH and Ucns on gastric emptying is not solely mediated by CRH2, as in the periphery, because CRH2 antagonists only show a partial blockade. Furthermore, CRH1 plays a role in postoperative gastric ileus. Nonselective astressin or the selective CRH1 receptor antagonist, CP154,526, blocks abdominal surgery-inhibited gastric emptying in rats and mice, and CRH1 deficient mice do not develop postoperative gastric ileus [35]. The CRH effects on small intestinal motility seem to be inconsistent, being reported as inhibitory or

excitatory. Central (ICV) CRH inhibits the occurrence of migrating motor complex (MMC) in the duodenum and jejunum of dogs [2] and delays small intestinal transit in rats [17]. The direct peripheral effects of CRH on the small intestine come mainly from in vitro (longitudinal and/or circular smooth muscle strips of guinea pig and rat) studies and demonstrate that CRH increases the contraction by activating the myenteric neurons [8]. However, a recent study using rat circular smooth muscle strips showed that CRH and Ucn 1 increased duodenal contraction and inhibited ileal contraction, and that the former action was blocked by a CRH1 antagonist and the latter by a CRH2 antagonist [30]. The inconsistency may have resulted from central versus peripheral, in vivo versus in vitro studies, as well as from different tissue preparations, intestinal segments, and species. More studies need to be conducted in whole animals to establish the effects of CRH on regional as well as whole small intestine. In the colon, the effects of CRH and related peptides are well documented to exert stimulatory effects [21, 35, 37]. CRH and Ucn 1 injected centrally or systemically in rats or mice mimic the colonic responses to stress—increased motility and defecation and decreased transit time—CRH being more potent than Ucn 1. In humans, IV CRH stimulates motility in the descending colon. The CRH2 receptor agonists, Ucn 2 and 3, administered peripherally in rodents and centrally in rats have no measurable effect on colonic motor functions by themselves. However, Ucn 2 stimulates colonic transit when injected ICV in mice, and it blunts CRH stimulatory effects when applied peripherally in rats. It implies that different mechanisms may be recruited in the central nervous system and in the periphery. Nevertheless, the CRH1 receptor mediates central and peripheral CRH and related peptides, as well as stress-stimulated colonic motor functions in experimental animals [35]. This is shown by the blockade of the colonic responses by CRH receptor nonselective (αhelical CRH9–41 and astressin) and CRH1 selective antagonists (CP-154,526, NBI-35965, NBI-27914, and CRA-1000) and the lack of blockade by CRH2 antagonists (antisauvagine-30 and astressin2-B).

Pancreatic Secretion CRH and Ucns have effects on both pancreatic exocrine and endocrine secretion. CRH and Ucn 1 stimulate the secretion of proteins from the pancreas, and the CRH receptor antagonist astressin, but not the CRH2 antagonist antisauvagine-30, blocked the effect, suggesting a CRH1 mediation [7, 16]. The CRH2 agonist, Ucn 3, which is expressed in pancreatic β cells, stimulates secretion of insulin and glucagon when it is injected

1026 / Chapter 140 IV in rats, whereas the CRH2 antagonist, astressin2-B, inhibits the effect [19]. These results suggest that CRH peptides stimulate pancreatic exocrine and endocrine secretions through the activation of CRH1 and CRH2 receptors, respectively.

NEURONAL PATHWAYS INVOLVED IN CRH ACTIONS IN GI FUNCTIONS The pathways for communication between the central CRH and GI system are mainly neuronal pathways. The brain areas primarily involved in the CRH regulatory effects on GI are mainly the paraventricular nucleus (PVN) of the hypothalamus, amygdala, lateral parabrachial nucleus (LPB), locus coeruleus (LC), Barrington’s nucleus, and dorsal vagal complex. The CRH-containing neurons in the PVN and Barrington’s nucleus project to the dorsal vagal complex and the preganglionic sympathetic and parasympathetic neurons in the spinal cord, where they terminate in densely CRH-containing nerve fibers. Microinjection of CRH into the PVN and LC–Barrington’s area in experimental animals inhibits gastric acid secretion and motility and increases colonic transit [23, 24]. The peripheral GI signals are sent to and integrated into the brain circuits linked to CRH neurons. For instance, distention in the stomach and colorectum activates neurons in the nucleus tractus solitarius and/ or those in the lumbosacral dorsal horn and intermediate zone, and higher centers including LC–Barrington’s complex, LPB, amygdala, and PVN. All those areas are in circuits that can be modulated by CRH signaling. CRH and Ucns injected in the periphery also activate neurons in most of those areas [21]. Moreover, chronic experimental colitis in rats induced by chemical stimulants alters the expression pattern of CRH transcripts with an increase in its heteronuclear (hn)RNA in the magnocellular PVN and supraoptic nucleus (SON) in the hypothalamus [14]. This shows that GI inflammation and visceral pain modify central CRH synthesis and that CRH may in turn regulate systemic functions. The central CRH effects on gastric acid secretion and motility and colonic functions are not affected by adrenalectomy, hypophysectomy or naloxone. They are fully blocked by vagotomy and celiac ganglionectomy and are partially attenuated by a noradrenergic blocker; the effect on the colon is only partially attenuated by vagotomy, but it is fully reversed by hexamethonium and atropine [17, 26, 29, 35]. In addition, CRH and sauvagine injected IC decrease the vagal efferent activity in rats [21]. In the last few years, interest in the peripheral CRH signaling pathways has gained momentum. CRH and

related peptides and receptors have been located in the GI system. One of the targets is myenteric neurons, and those in the intestine contain CRH1 receptors [20]. CRH applied to longitudinal muscle and myenteric plexus strips of guinea pig ileum and colon activate the myenteric neurons as shown by electrophysiological recording [8]. The systemic administration of CRH in rats also displays activation of myenteric neurons in the colon, as demonstrated by induction of an early gene product, c-Fos; the nonselective CRH1/CRH2 antagonist, astressin, and the CRH1 antagonist, CP-154,526, block this activation [35].

PATHOPHYSIOLOGICAL IMPLICATIONS CRH and its related peptides are key factors in stressor inflammation-induced GI dysfunction.

Intestinal Mucosal Pathophysiological Responses Stress influences the epithelia and mucosal glands in the intestine to affect barrier function and secretion, and CRH may be implicated in the mechanisms [31, 35, 38]. Although the roles could be complex depending on the subject and type of stressor, evidence indicates that central CRH plays an anti-inflammatory role in the GI tract, whereas peripheral CRH contributes to the inflammatory process. Lewis rats, which have lower CRH levels in the brain, develop a higher level of trinitrobenzene sulfonate (TNBS)-induced colonic inflammation than Fischer rats, which have a high brain CRH response to stress. Furthermore, CRH injected ICV exerts a protective effect. Central blockade of CRH receptors abolishes the CRH effect and also further enhances restraint stress-aggravated colitis induced by TNBS in rats [6, 22]. In the periphery, CRH injected IV or IP in rats enhances release of the colonic mucin, ions, and mast cell protease II and also enhances the uptake of macromolecules such as horseradish peroxidase across the epithelium. The CRH receptor antagonist, α-helical CRH9–41, injected peripherally in rats prevents stressstimulated colonic mucin secretion, prostaglandin E2 release, and mast cell degranulation [31]. In isolated ileum or colon, CRH receptor antagonists reduce toxin A–caused inflammatory reactions in the mucosa and also abolish the CRH-induced translocation of horseradish peroxidase [38]. It is of note also that ulcerative colitis patients and colitic rats show elevated luminal CRH [12, 40]. The receptor types involved in mucosal barrier functions and inflammation are yet to be determined.

Corticotrophin-Releasing Hormone (CRH) Family in the Gastrointestinal System / 1027

Visceral Pain A lowered threshold to visceral pain and discomfort are symptoms of irritable bowel syndrome (IBS) patients. Stress induces visceral hypersensitivity, and evidence suggests the CRH system is involved in the modulation of visceral sensitivity. In humans, IV administration of CRH increases the sensation of discomfort and pain to distention of the colon. Similarly, CRH injected ICV or IP in rats deceases the threshold of pain to intestinal distention. Studies involving treatment with CRH1 or CRH2 antagonists show that the CRH-CRH1 signaling pathway is implicated in the occurrence of visceral pain [27, 35], whereas CRH2 appears to play an antinociceptive role [21, 27]. The CRH2 agonist, Ucn 2, injected IV or intrathecally in rats reduces the pain response to colorectal or duodenal distention. CRH2 antagonists, antisauvagine-30 and astressin2-B, block, respectively, intrathecal CRH- and IV Ucn 2-induced visceral analgesic responses to duodenal or colorectal distention. More investigations need to be directed to the characterization of the peripheral forms or variants of CRHrelated peptides and receptors and to reveal their up- or downregulation under pathophysiological conditions. Taken together, CRH and its related peptides and their receptors could be potential pharmacological targets for the treatment of stress-related GI disorders, including IBS.

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Acknowledgment This work was supported by NIH grants R21 DK68155, R01 DK-57238, and Center Grant DK-41301. The authors are grateful to Teresa Olivas for her help with the preparation of the manuscript.

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of gastrointestinal function during stressful conditions. Peptides 2005;26:1196–1206. Taché Y, Goto Y, Gunion MW, Vale W, River J, Brown M. Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor. Science 1983;222:935–937. Taché Y, Gunion MW, Stephens RL. CRF: central nervous system action to influence gastrointestinal function and role in the gastrointestinal response to stress. In: De Souza, E. B.; Nemeroff, C. B., Eds. Corticotropin-Releasing Factor: Basic and Clinical Studies of a Neuropeptide. Boca Raton: CRC Press; 1990. pp. 299–307. Taché Y, Martinez V, Wang L, Million M. CRF1 receptor signaling pathways are involved in stress-related alterations of colonic function and viscerosensitivity: Implications for irritable bowel syndrome. Br J Pharmacol 2004;141:1321–1330. Taché Y, Million M. Stress and the gut: Peripheral influences. In: Spiller, R.; Grundy, D., Eds. Pathophysiology of the Enteric Nervous System: A Basis for Understanding Functional Diseases. Malden, UK: Blackwell Publishing; 2004. p. 99–101. Taché Y, Monnikes H, Bonaz B, Rivier J. Role of CRF in stressrelated alterations of gastric and colonic motor function. Ann NY Acad Sci 1993;697:233–243. Taché Y, Perdue MH. Role of peripheral CRF signalling pathways in stress-related alterations of gut motility and mucosal function. Neurogastroenterol Motil 2004;16 Suppl 1:137–142. Thompson RC, Seasholtz AF, Douglass JO, Herbert E. Cloning and distribtuion of expression of the rat corticotropin-releasing factor (CRF) gene. In: De Souza, E. B.; Nemeroff, C. B., Eds. Corticotropin-Releasing Factor: Basic and Clinical Studies of a Neuropeptide. Boca Raton: CRC Press; 1990. p. 1–12. van Tol EA, Petrusz P, Lund PK, Yamauchi M, Sartor RB. Local production of corticotropin releasing hormone is increased in experimental intestinal inflammation in rats. Gut 1996;39:385– 392.

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141 Paneth Cell α-Defensins ANDRE J. OUELLETTE

are 3–4 kDa and contain six cysteine residues with a characteristic spacing pattern that facilitates the formation of specific and invariant disulfide-bond pairings that define the peptides as α-defensins (Fig. 1). The first evidence for Paneth cell α-defensins was provided by genetic analyses of mouse small intestine. When 35S-Cys-labeled cell-free translation products of adult and fetal mouse small intestinal mRNAs were compared, a population of mRNAs that coded for 6– 8 kDa Cys-rich peptides were found to be abundant in the adult small bowel but absent from the fetal organ. Subsequently, the cDNA sequences of those abundant mRNAs showed that the deduced gene products were α-defensin precursors, as inferred from the spacing of the six cysteines in the encoded protein. Northern blot hybridization experiments showed that very high levels of the mRNAs existed in small intestine, and by in situ hybridization the putative pro-α-defensin sequences were localized to Paneth cells located at the base of the crypts of Lieberkühn (Fig. 2). Accordingly, the putative mouse α-defensin peptides were termed cryptdins (Crps) for “crypt defensin.” Human Paneth cell αdefensin mRNAs also were described over a decade ago by using a discovery strategy that was based on conserved sequences in α-defensin cDNAs and genes [15]. The predicted existence of Paneth cell α-defensin peptides was confirmed by the purification of several α-defensin peptides from mouse and human small intestine. Coupled with additional analyses of cDNAs from individual mouse crypts, mouse Paneth cells were shown to express numerous α-defensin variants that are coded by individual, closely linked genes. In contrast to the variety of α-defensins in mouse and rhesus macaque

ABSTRACT In the small bowel, Paneth cells at the base of the crypts of Lieberkühn secrete α-defensins and additional antimicrobial peptides at high levels in response to cholinergic stimulation and when exposed to bacterial antigens. Paneth cell α-defensins, having evolved to function in the extracellular environment, show broad-spectrum antimicrobial activities and constitute the majority of bactericidal peptide activity in Paneth cell secretions. The release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and to confer protection from enteric infection. The most compelling evidence for a Paneth cell role in enteric resistance to infection is evident from studies of mice transgenic for a human Paneth cell α-defensin, HD-5; these mice are completely immune to infection and systemic disease from orally administered Salmonella enterica serovar Typhimurium. α-Defensins in Paneth cell secretions also may interact with bacteria in the intestinal lumen above the crypt-villus boundary and influence the composition of the enteric microbial flora.

DISCOVERY OF ENTERIC PANETH CELL a-DEFENSINS The defensins make up one of the major antimicrobial peptide (AMP) families in mammals and were among the first AMPs to be described. They consist of three subfamilies of cationic, Cys-rich AMPs, termed α-, β-, and θ-defensins, all of which have broadspectrum antimicrobial activities [27]. The α-defensins Handbook of Biologically Active Peptides

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Human HD-5 Human HD-6 Cryptdin-1 Cryptdin-2 Cryptdin-3 Cryptdin-4 Cryptdin-5 Cryptdin-6 RED-1 RED-2 RED-3 RED-4 RED-5 RED-6

ATCYCRTGRCATRESLSGVCEISGRLYRLCCR AFTCHCRRS-CYSTEYSYGTCTVMGINHRFCCL LRDLVCYCRSRGCKGRERMNGTCRKGHLLYTLCCR LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR LRDLVCYCRKRGCKRRERMNGTCRKGHLMYTLCCR GLLCYCRKGHCKRGERVRGTC--G-IRFLYCCPRR LSKKLICYCRIRGCKRRERVFGTCRNLFLTFVFCCS LRDLVCYCRARGCKGRERMNGTCRKGHLLYMLCCR (R)TCRCRIRRCRGLESSFGNCILHGQFAKLCCR FTCHCRIGRCSWFETRFRSCTLLGLAANLCCR HTCYCRNKRCFTPEFHAGKCKVEGRTYKLCCR (R)TCYCRTGRCYTPEFHSGKCVFNGRTYKLCCR MICLCRIGRCSWREAHFGSCTKMGQFAKICCRRAS (R)NCHCRIGHCRRPAAPMGVCIIHGQFGKLCCR

FIGURE 1. Primary structures of representative Paneth cell α-defensins. The single-letter notation amino acid sequences of human Paneth cell α-defensins HD-5 and HD-6, mouse Crps 1–6, and rhesus macaque enteric α-defensins REDs 1–6, were positioned manually to align canonical Cys, Arg, Glu, and Gly residues (boxed). Dash characters denote spaces introduced to maintain the alignment of conserved Cys residues and the disulfide array. The tridisulfide array that defines the α-defensin peptide family is shown.

Paneth cells, human Paneth cells express only two αdefensin genes [21]. Antisera prepared against a synthetic congener of mouse cryptdin-1 showed that the Crp1 molecule immunolocalized exclusively to apically oriented, electron-dense secretory granules of Paneth cells in the small bowel [22].

FIGURE 2. The Paneth cell. The figure illustrates the prominent, apically oriented, electron-dense secretory granules of Paneth cells. Paneth cell secretion is mediated by increases in cytosolic Ca2+ ([Ca2+]i) by mobilization of intracellular stores, and mIKCa1 channels in the mouse Paneth cells open to provide the counterbalancing K+ efflux necessary to sustain Ca2+ entry from the external milieu. Electron micrograph was generously provided by Dr. Susan J. Hagen, Beth Israel Deaconess Medical Center, Boston, MA.

a-DEFENSIN GENE AND PRECURSOR STRUCTURES The predominant sites of α-defensin gene expression are cells of myeloid origin and Paneth cells. Paneth cell α-defensin genes are closely linked at chromosomal loci with myeloid α-defensin genes with the two human Paneth cell α-defensin genes mapping to 8p21-8pter and the mouse genes syntenic and located on the proximal region of chromosome 8 [27]. As shown in Fig. 3, α-defensin genes expressed in myeloid cells contain three exons, and those genes expressed in Paneth cells have a two-exon structure [9]. The 5′ untranslated region and the preprosegment of Paneth cell αdefensin genes are coded by exon 1, but all known myeloid α-defensin genes contain an additional intron that interrupts the 5′ untranslated region of the transcript [27]. In all cases, the most distal exon of α-defensin genes codes for the functional peptide [3]. A model for the evolution of human α-defensin genes has proposed that epithelial α-defensins predate their hematopoietic counterparts and that an early duplication of a

primordial α-defensin gene gave rise to the ancestral genes of present-day human α-defensins HD-5 and HD6 [4]. The model also predicts that a subsequent unequal meiotic crossover event produced a hybrid of the two parental genes, the proposed ancestor of the hematopoietic α-defensin genes. The exonic organization of hematopoietic and epithelial α-defensin genes is highly conserved among mammalian species, suggesting that the corresponding ancestral genes of each type existed prior to the evolutionary divergence of those species investigated [4]. The biosynthesis of α-defensins requires posttranslational activation by lineage-specific proteinases. Although the enzymes that mediate pro-α-defensin processing in myeloid and epithelial cells almost certainly differ, the overall processing schemes are the same. Both myeloid and Paneth cell α-defensins derive from ∼10-kDa pre-propeptides that contain canonical signal sequences, usually acidic proregions, and a ∼3.5-kDa

Paneth Cell α-Defensins mature α-defensin peptide in the C-terminal portion of the precursor. In human and rabbit neutrophils, αdefensins are almost fully processed by primary cleavage steps that leave major intermediates of 75 and 56 amino acids and the mature HNP-1 peptide [9]. The mechanisms of human and mouse Paneth cell α-defensin posttranslational activation are markedly different (Fig. 4). For example, human Paneth cells store unprocessed α-defensin precursors (e.g., proHD5(20– 94)), which are processed after secretion by anionic and meso isoforms of trypsin at R62↓A63 to produce the predominant form of HD-5 (Fig. 4) [11]. Additional, perhaps alternative, proHD5 processing sites include an HD5(37–94) variant that was isolated from secretions of human small intestinal crypts stimulated with carbamyl choline [7]. Trypsin also may process monkey Paneth cell pro-α-defensins after secretion [31], because all rhesus macaque Paneth cell α-defensin precursors

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deduced from cDNA sequences contain canonical trypsin cleavage sites at residue position 62, the junction of the proregion and the α-defensin N-terminus [31]. The rhesus Paneth cell α-defensin RED-4 has alternative N-termini that apparently result from activating cleavage steps at both Arg62↓Arg63 and at Arg63↓Thr64. In contrast to trypsin-mediating human Paneth cell α-defensin activation, pro-α-defensin processing in mouse Paneth cells is catalyzed by matrix metalloproteinase-7 (MMP-7) and takes place intracellularly and prior to secretion. In mouse small-intestinal epithelium, only Paneth cells express MMP-7 as components of dense secretory granules [35], and the bactericidal activity of mouse Paneth cell α-defensins depends completely on activation of 8.4-kDa proCrps by MMP-7catalyzed proteolysis [28, 36]. MMP-7 gene disruption ablates proCrp processing such that mature activated

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FIGURE 3. α-Defensin gene and precursor structure. Schematics of the myeloid and Paneth cell αdefensin genes and precursors are aligned at left. The signal peptide and proregions are crosshatched differentially, and the gray C-terminal region of the precursors denote the residues that constitute the mature α-defensin peptide. The 3D structure shown is that of rabbit α-defensin RK-1.

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DPIQNTDEETKTEEQPGEDDQAVS↓VSFGDPEGTS↓LQEES↓LRDLVCYCRSRGCKGRERMNGTCRKGHLLYLCCR

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ESLQERADEATTQKQSGEDNQDLAISFAGNGLSALR↓TSGSQAR↓ATCYCRTGRCATRESLSGVCEISGRLYRLCCR

FIGURE 4. Distinct mechanisms of pro-α-defensin activation in mouse and human Paneth cells. Human pro-HD5 is activated by anionic and meso-trypsins, which cleave the precursor at the sites depicted by arrows in pro-HD5. In contrast, MMP-7 cleaves all known mouse Paneth cell pro-α-defensins intracellularly at the sites depicted by arrows in pro-Crp1. In both sequences, proregions are underlined and the mature α-defensin peptides are italicized. (NB: Mouse proCrps are resistant to in vitro proteolysis by trypsins.)

1032 / Chapter 141 Crp peptides are absent from the small intestine and innate immunity to oral bacterial infection is impaired in MMP-7-null mice [36]. MMP-7 produces active 3.5-kDa α-defensins by cleaving precursors in vitro at conserved sites in the proregion and at the junction of the propeptide and the α-defensin moiety [28] (Fig. 4). ProCrps lack in vitro bactericidal activity, corresponding with a diminished ability to interact with model membranes and to permeabilize large unilamellar vesicles [25]. However, despite the fact that mouse and human Paneth cells use distinctly different mechanisms of α-defensin activation, the capability for delivery of functional microbicidal α-defensins to the lumen is conserved.

DISTRIBUTION OF α-DEFENSINS IN THE GASTROINTESTINAL TRACT In the small intestine, α-defensins occur only in Paneth cells, which occupy the base of the crypts of Lieberkühn of most mammals and contain high concentrations of α-defensins in their apically directed secretory granules (Fig. 2) [22]. Paneth cell α-defensin mRNAs accumulate during intestinal development coincident with differentiation of the lineage during intestinal crypt ontogeny [5]. Paneth cell differentiation is determined by continuous Wnt signaling via the frizzled-5 receptor, and transcription of the Paneth cell α-defensin and MMP-7 genes is mediated by β-catenin/ TCF-4 recognition sites in the 5′ upstream regions of the respective gene transcription start sites [32]. In humans, histologically normal Paneth cells develop prenatally as early as 13.5 weeks of gestation [19], and, as in mice, the expression of HD-5 and HD-6 genes in utero shows that Paneth cell α-defensin gene activation is independent of infectious stimuli. A subset of Crp genes also is differentially expressed in gobletlike cells that are dispersed in the immature epithelial cell monolayer of the fetal and newborn mouse small intestine [8, 32].

STRUCTURES OF PANETH CELL a-DEFENSINS The 3- to 4-kDa α-defensins contain six cysteine residues, which form specific and invariant disulfide bond pairings (Fig. 1) [9, 27]. The three-dimensional structures of several α-defensins have been determined by both nuclear magnetic resonance (NMR) and x-ray crystallographic techniques, and all reported structures contain a canonical triple-stranded antiparallel β-sheet motif [27, 34]. The crystal structure of the human neutrophil α-defensin HNP-3 is a noncovalent, amphipathic dimer in which Arg side chains lie equatorially

above a hydrophobic surface consisting of apolar monomer side chains [12]. In contrast, α-defensins from rabbit neutrophils, mouse Paneth cells, and rabbit kidney are monomeric in solution [27]. For example, mouse Crp4 is a monomer, and its three-dimensional solution structure consists of a remarkably amphipathic, triple-stranded antiparallel β-sheet with the β strands joined by a series of tight turns and a β-hairpin (Fig. 5) [14].

MECHANISMS OF ACTION α-Defensins from human and rabbit polymorphonuclear neutrophilis (PMN) achieve bacterial cell killing by differing membrane-disruptive mechanisms. Although there are reports of lectin-like activity for certain α-defensins [17], the bactericidal activities of most α-defensins appear not to be receptor-mediated. α-Defensins are microbicidal against gram-positive and gram-negative bacteria, certain fungi, spirochetes, protozoa, and enveloped viruses [9, 27]. Neutrophil α-defensins permeabilize the outer and inner membranes of Escherichia coli sequentially and induce ionchannel formation in lipid bilayers; the peptide-elicited effects are influenced by membrane energetics. The dimeric human neutrophil α-defensin HNP-2 forms stable, 20-Å multimeric pores after insertion into model membranes, but monomeric rabbit neutrophil NP-1 permeabilizes model phospholipid bilayers by creating short-lived defects [13, 34]. Mouse Crp4 exhibits strong interfacial binding to model membranes, and it induces fluorophore leakage from membrane vesicles via a graded mechanism [24]. Mouse Paneth cells secrete α-defensin-rich granules when exposed to bacteria, bacterial lipopolysaccharide, lipoteichoic acid, lipid A, and muramyl dipeptide or when stimulated pharmacologically with cholinergic agonists [1]. Crps account for approximately 70% of bactericidal peptide activity released by mouse Paneth cells, which occurs at initial concentrations of 25– 100 mg/ml [1]; these values are 1 × 104 times greater than their minimal bactericidal concentrations. Human α-defensin HD-5 is stored in the ileal mucosa at levels of 90–450 μg per cm2 of mucosal surface area [23], suggesting that luminal HD-5 concentrations may reach 50–250 μg/ml. Mouse Paneth cell secretion is modulated in part by the Ca2+-activated K+ channel, mIKCa1, which is expressed only by Paneth cells in mouse smallintestinal epithelium [2]. The secretion of total protein and lysozyme and the bactericidal peptide activities of elicited secretions are independent of acyl chain modifications of the glycolipid agonists [29]. In structure–activity studies of mouse Crp4, differential bactericidal activities of variant peptides were

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FIGURE 5. Solution structure and charge distribution of mouse Crp4. The basic regions of the Crp4 peptide are shaded dark gray, acidic regions are light gray, and neutral and hydrophobic regions shown in white, as produced by the program MOLMOL. A–E show five different perspectives, and F depicts the same view as E but in ribbon form. Reprinted from Jing, W., Hunter, H.N., Tanabe, H, Ouellette, A.J., Vogel, H.J.: Solution structure of cryptdin-4, a mouse Paneth cell α-defensin. Biochemistry, 43: 15759–15766, 2004, with permission.

directly related to peptide binding and disruption of phospholipid bilayers. The bactericidal activities of Nterminal Crp4 variants correlated directly with permeabilization of live E. coli, equilibrium binding to E. coli–membrane phospholipid bilayers and vesicles, and induced fluorophore leakage from phospholipid vesicles [24]. Also, Arg residues are determinants of mouse

Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. Charge-reversal or charge neutralization mutations at Arg residue positions induce loss-of-function in Crp4, attenuating or eliminating microbicidal activity against varied bacterial species, regardless of the Arg residue positions mutated [30].

1034 / Chapter 141 A functional analysis of Crp4 disulfide mutants has shown that the canonical α-defensin disulfide arrangement provides critical peptide protection during precursor activation, but it is not a determinant of α-defensin bactericidal activity [18]. Disulfide bond disruption does not induce loss of function; the in vitro bactericidal activities of certain α-defensin disulfide variants with disordered structures equal or exceed those of the native Crp4 peptide. However, although native Crp4 is completely resistant to proteolysis by the MMP-7 convertase, all disulfide variants of Crp4 were cleaved extensively, eliminating peptide bactericidal activity [18].

PATHOPHYSIOLOGICAL IMPLICATIONS The contribution of Paneth cell α-defensins to enteric mucosal immunity is evident from the phenotype of mice transgenic for human α-defensin HD-5 (tg-HD-5), which express the HD-5 minigene specifically in Paneth cells at a level similar to the endogenous Crp genes [23]. In contrast to control mice that are highly susceptible to oral challenge with wild-type S. enterica serovar Typhimurium, tg-HD-5 mice were immune to infection and few viable S. enterica serovar Typhimurium were recovered from the intestinal lumen and spleens of tg-HD-5 mice relative to wild-type controls [23]. Thus, the α-defensin composition of Paneth cell secretions may protect the crypt epithelium by the direct killing of invading pathogens or by influencing the composition of the resident microflora. In mice, crypt intermediate or granulomucous cells accumulate Paneth cell gene products in electron-dense granules under conditions of disrupted crypt cell biology. For example, in mice expressing attenuated diphtheria toxin A fragment or SV40 large T-antigen transgenes under control of the Crp2 gene promoter, a transient Paneth cell deficiency occurs in crypts of 3- to 4-week-old transgenic mice [10]. Intermediate cells and granule-containing goblet cells increase in number and accumulate electron-dense secretory granules that contain both Crps and sPLA2 during the Paneth cell–deficient period [10]. Normally, Paneth cells are found only in the small intestine, but in Barrett’s esophagus, Crohn’s disease, gastritis, and ulcerative colitis the cells appear ectopically along with lineage-specific markers [21]. Collectively, these disruptions of crypt cell biology apparently modify Wntregulated epithelial lineage differentiation programs, resulting in increased production of Paneth cell antimicrobial peptides for secretion. Defects in Paneth cell physiology and α-defensin biology may predispose individuals to infectious challenges and perhaps to inflammatory bowel diseases. For

example, MMP-7-null mice are deficient in functional α-defensins due to defective proCrp processing, and their host defense to oral enteric infection is compromised in vivo [36]. Cystic fibrosis mice, null for the cystic fibrosis transmembrane conductance regulator, accumulate undissolved Paneth cell secretory granules in mucus-occluded crypts, resulting in decreased resistance to bacterial colonization of the small intestine, bacterial overgrowth of small bowel and mucus colonization, and an overwhelming predominence of Enterobacteriaceae in the microflora [6, 20]. In human and mouse small intestinal epithelium, NOD2, an intracellular peptidoglycan pattern-recognition receptor and a susceptibility gene for Crohn’s ileitis [26], is expressed in the Paneth cell [16]. Although proinflammatory cytokine levels are unaffected by NOD2 status, ileal HD5 and HD-6 mRNA levels decline in Crohn’s ileum, especially in patients with NOD2 mutations [33]. Possibly, NOD2 mutations alter human Paneth cell biology and the composition of their secretions, leading to diminished innate mu-cosal immunity. Thus, defects in activation, secretion, dissolution, and function of Paneth cell α-defensins all may affect crypt innate immunity adversely. It follows that the impairment of innate mucosal immunity may arise from altered α-defensin levels and also from defects in Paneth cell or crypt cell biology associated with the delivery of functional αdefensins and additional AMPs to the microenvironment of the crypt lumen.

Acknowledgments Supported by NIH Grant DK044632, The Human Frontiers Science Program, and The United States– Israel Binational Science Foundation.

References [1] Ayabe, T., D. P. Satchell, C. L. Wilson, W. C. Parks, M. E. Selsted, and A. J. Ouellette. 2000. Secretion of microbicidal alphadefensins by intestinal Paneth cells in response to bacteria. Nat Immunol 1:113–8. [2] Ayabe, T., H. Wulff, D. Darmoul, M. D. Cahalan, K. G. Chandy, and A. J. Ouellette. 2002. Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel. J Biol Chem 277:3793– 800. [3] Bevins, C. L. 2004. The Paneth cell and the innate immune response. Curr Opin Gastroenterol 20:572–80. [4] Bevins, C. L., D. E. Jones, A. Dutra, J. Schaffzin, and M. Muenke. 1996. Human enteric defensin genes: chromosomal map position and a model for possible evolutionary relationships. Genomics 31:95–106. [5] Bry, L., P. Falk, K. Huttner, A. Ouellette, T. Midtvedt, and J. I. Gordon. 1994. Paneth cell differentiation in the developing intestine of normal and transgenic mice. Proc Natl Acad Sci USA 91:10335–9.

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142 Galanin in the Gastrointestinal Tract: Distribution and Function LAURA ANSELMI, ILARIA CAVALLI, AND CATIA STERNINI

receptor binding [10]. Details on galanin discovery, precursor structure, and peptide processing are covered in the Brain Peptide Section of this Handbook. Galanin has a widespread distribution in the nervous system and peripheral organs of both vertebrates and invertebrates and possesses a multitude of biological actions, including neurotransmitter and hormone release, feeding behavior, and digestive functions [5, 9, 27]. The diverse biological effects of galanin are mediated by its interaction with specific cell surface receptors that are members of the G-protein-coupled receptor superfamily, named galanin receptor 1 (GalR1), GalR2, and GalR3, which have been cloned from several species (human, rat, mouse) [9, 10]. This chapter focuses on the distribution, regulation, and function of galanin and its receptors in the gastrointestinal tract.

ABSTRACT In the gastrointestinal tract, galanin is localized to myenteric and submucosal neurons and it modulates many functions, including transmitter release, motility, and secretion. Galanin triggers cellular responses at three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2, and GalR3, which differ in their pharmacology, signaling, and distribution. Cellular responses to galanin depend on the receptor subtype activated. All three receptor mRNAs are expressed in the gut but at different levels. GalR1 and GalR2 mRNAs are the most abundant. GalR1 and GalR2 immunoreactivities are expressed by enteric neurons, endocrine cells (GalR1), and smooth muscle cells (GalR2). Galanin plays an important role in mediating the excess of fluid secretion with diarrhea in intestinal infection and inflammation that is likely to be due to an overexpression of GalR1, thus implicating the galanin system in the pathophysiology of inflammatory diseases.

GALANIN DISTRIBUTION Galanin is abundantly distributed in the gastrointestinal tract, where it is localized to myenteric and submucosal neurons and to fibers projecting to the muscle and mucosa, with an increasing density in the distal regions [12, 15, 22]. Galanin fibers are primarily intrinsic to the enteric nervous system, as demonstrated by the lack of effect of degeneration of extrinsic fibers to the galanin innervation pattern of the intestine [12]. Galanin fibers are quite abundant in the muscle layer, and they have long descending projections with a dual termination in the myenteric ganglia and in the smooth muscle. This projection pattern is consistent with a double site of action of galanin in the control of motor activity, including a direct myogenic action on gastrointestinal smooth muscle and a nerve-mediated effect involving the release of other transmitters.

INTRODUCTION Galanin is a brain-gut peptide that was isolated in the early 1980s from porcine intestine as a 29-amino-acid C-terminally amidated peptide, and it was named for its N-terminal glycine and C-terminal alanine residues [34]. Only the human species of galanin contains 30 amino acids and lacks C-terminal amidation. Galanin’s first 15 amino acids at the N-terminus are highly conserved across species (from mammals, including humans, to lower vertebrates and invertebrates) and the deletion of the first 16 amino acids causes the complete loss of its affinity for galanin receptors, suggesting that this region of galanin is critical for high-affinity Handbook of Biologically Active Peptides

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1038 / Chapter 142 BIOLOGICAL EFFECTS OF GALANIN IN THE GASTROINTESTINAL TRACT Galanin exerts multiple effects in the digestive system [27]. It suppresses the postprandial release of several substances, including somatostatin, pancreatic polypeptide, glucagon, and insulin [6]. Galanin inhibits gastric acid secretion, and it modulates gastric emptying and gastrointestinal motility. The effects of galanin on gastrointestinal motility are both stimulatory and inhibitory and are species-related and dependent on the gastrointestinal segment. For instance, galanin causes a contraction of rat fundus, ileum, and jejunum [11]; of mouse colon [13]; and of guinea pig ileum [8]. By contrast, galanin induces relaxation of the canine ileum [8, 14] and guinea pig stomach [16]. In humans, galanin inhibits gastrointestinal motility and causes a delay of gastrointestinal transit [6]. These effects are the result of a direct action on smooth muscle cells and indirect effect via the activation of intrinsic or extrinsic neuronal pathways. Indeed, galanin modulates gastrointestinal motility by acting prejunctionally through a nerve-mediated effect involving the release of other transmitters/modulators and postjunctionally by inhibiting or stimulating the muscle directly [1, 6, 11, 14, 23, 24, 32, 35]. Studies in vitro and in vivo have shown that galanin inhibits the excitatory neuroneuronal and neuromuscular transmission to the longitudinal and circular muscle layer acting on the neurogenic cholinergic contraction mediated by endogenous acetylcholine and substance P [1, 23, 32]. Galanin also might contribute to the noncholinergic, nonadrenergic transmission to the muscle [14, 24]. These effects are likely to be mediated by different receptor subtypes. Electrophysiological studies with intracellular microelectrode recording in myenteric neurons of the guinea pig small intestine have shown an inhibitory action of galanin consisting of membrane hyperpolarization, decrease in input resistance, and suppression of excitability [25, 33]. Galanin suppresses fast excitatory postsynaptic potentials in myenteric neurons [25, 33]. Furthermore, studies using whole cell recording approaches revealed that galanin suppresses voltageactivated Ca2+ conductance and opens inwardly rectifying K+ channels in myenteric neurons. Thus, the ionic mechanism beyond the inhibitory action of galanin appears to be an increase in K+ conductance that occurs in inwardly rectifying K+ channels [28]. These studies suggest that galanin effects on myenteric neurons might be mediated by the activation of two different receptor subtypes, one activating inwardly rectifying K+ current and one inhibiting calcium current. Whether these effects on the two membrane currents are due to the activation of a common recep-

tor or of different receptor subtypes remains to be elucidated. Galanin has also been shown to inhibit Ca2+ responses to electrical stimulation in cultured myenteric neurons, suggesting a presynaptic inhibition of cholinergic neurons [29]. We have shown that galanin (1–16), a galanin fragment with high affinity for GalR1 and GalR2, inhibits voltage-dependent Ca2+ influx in cultured myenteric neurons, which is likely to be mediated by GalR1, because GalR1 is coupled to Gi/o inhibitory proteins. Thus, it mediates galanin inhibitory effects [4]. GalR3 is also coupled to Gi/o proteins; however, because galanin (1–16) has low affinity for GalR3, the involvement of GalR3 is unlikely, although it cannot be ruled out completely. By contrast, GalR2 is predominantly coupled to Gq/11 excitatory proteins; thus its activation would more likely result in a direct stimulation of Ca2+ instead of an inhibition of evoked Ca2+. Indeed, activation of GalR2 by a fragment that is selective for this receptor induces Ca2+ influx in cultured myenteric neurons (Anselmi and Sternini, unpublished data). Galanin also mediates mucosal epithelial cell absorption and ion transport [7]. The effects of galanin on intestinal ion flux are variable, depending on the species and the gut region. Indeed, galanin increases shortcircuit current, a measure of net ion flux, in rat colon, which is likely to be due to decreased Na+ and Cl− absorption, but has no effect in rat jejunum or guinea pig colon; however, it decreases short-circuit current in rabbit ileum and pig jejunum. The galanin-induced inhibition of short-circuit current in rabbit ileum is due to induction of Na+ and Cl− absorption. Furthermore, in humans, galanin causes Cl− secretion by a Ca2+dependent mechanism through the activation of GalR1, the only galanin receptor subtype expressed by human colonocytes [7].

GALANIN RECEPTORS The actions of galanin are mediated through different receptor subtypes that have distinct pharmacological profiles [9, 10]. For instance, GalR2 and GalR3 are distinguished by higher affinity for galanin(2–29), which lacks Gly (approximately 44- and 8-fold higher), than for GalR1 [36]. On the other hand, galanin(1–16) has approximately a 10-fold-higher affinity for GalR1 and GAL-R2 compared with GalR3, whereas [D-Trp2]galanin(1–29) has a higher affinity for GalR2 and GalR3 (about 250- and 50-fold, respectively) compared with GalR1 [36]. However, none of these fragments is highly selective for any individual GalR subtype. More recently, a selective GalR2 agonist has been discovered that has a 500-fold-higher affinity for GalR2 compared with GalR1 [19]. In addition, pharmacological differences

Galanin in the Gastrointestinal Tract: Distribution and Function / 1039 between galanin receptors in the central nervous system and periphery have been indicated by studies using galanin fragments and chimerical galanin peptides. Indeed, the peptide chimera M15 (or galantide), M35, and M40 have been shown to act as antagonists in the central nervous system, whereas in the periphery they either fail to antagonize galanin effects or act as agonists [10]. The existence of different receptor subtypes has now been confirmed by molecular cloning, which has identified three distinct G-protein-coupled receptors: GalR1, GalR2, and GalR3 [9, 10]. All three receptors consist of seven putative transmembrane domains and belong to the G-protein-coupled receptor superfamily. GalRs have been cloned in several species, including rodents and humans, and appear to be conserved among species. For instance, rat GalR1 shares a 92% amino-acid identity to human GalR1, and the more recently discovered mouse GalR1 shares a 91% and 94% identity with human and rat GalR1, respectively. Rat and human GalR2 share an 85% identity, but display only a 38% degree of identity with rat and human GalR1. Rat and human GalR3 also share high homology (89% aminoacid identity), and display 35/36% and 52/54% homology (depending on the group that reported the discovery of the receptor) with GalR1 and GalR2, respectively [9, 10].

GalR Signaling Native galanin receptors activate multiple secondmessenger pathways to affect cell activity [9, 10]. The activation of GalR1 results in pertussis toxin–sensitive inhibition of adenylyl cyclase (AC) activity with subsequent reduction of intracellular cAMP and opening inwardly rectifying K+ channels through interaction with Gi/o proteins. GalR1 also mediates pertussis toxin– sensitive Gβγ signaling to activate mitogen-activated protein kinase (MAPK). The activation of GalR2 leads to the stimulation of multiple intracellular pathways.

GalR2 predominantly couples to phopholipase C through Gq/11 protein, causing hydrolysis of phosphatidyl inositol 4,5-bisphosphate to generate inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG). Elevation of intracellular IP3 levels results in the mobilization of Ca2+ from intracellular stores and the activation of Ca2+dependent Cl− channels. Both DAG and Ca2+ may activate protein kinase C. GalR2 can also mediate a weak pertussis toxin–resistant reduction of forskolinstimulated cAMP, presumably through Gαi proteins, and activate MAPK through Go. When activated, GalR3 couples to Gi/o proteins, resulting in the inhibition of cAMP and induction of hyperpolarization by opening inwardly rectifying K+ channels via a pertussis toxin– sensitive mechanism.

GalR Distribution GalR1, GalR2, and GalR3 mRNAs are expressed in the central and peripheral nervous system as well as in peripheral tissues, including the digestive system [37]. Using real-time reverse transcription polymerase chain reaction (RT-PCR), we found that mRNAs coding for all three GalRs are present in each segment of the gastrointestinal tract with different levels of expression [3, 4]. GalR1 and GalR2 mRNAs are the most abundant throughout the gut. In the intestine, they are both more abundant in the large intestine than in the small intestine. GalR2 mRNA is the most abundant in the stomach. The levels of the GalR3 mRNA are the lowest throughout the entire length of the gut, with the relative highest levels in the colon and the lowest in the stomach and ileum [3, 4]. GalR1 immunoreactivity is localized to the cell surface of enterochromaffinlike (ECL) cells and of the myenteric and submucous neurons (Fig. 1), as well as to fibers distributed to the plexuses, interconnecting strands, muscle layers, vasculature, and mucosa [2, 17, 26, 32]. GalR1 immunoreactive fibers form a dense network in the deep muscular plexus (Fig. 1) in very

FIGURE 1. GalR1 immunoreactivity in the myenteric plexus (mp), submucosal plexus (smp), and deep muscular plexus (dm) of the guinea pig ileum. Arrows point to myenteric and submucosal neurons. Note the dense neuropil in the myenteric plexus (mp) and deep muscular plexus (dm), where GalR1 immunoreactive fibers form dense networks. Calibration bar: 20 μm.

1040 / Chapter 142 close association with interstitial cells of Cajal. GalR1 enteric neurons comprise functionally distinct populations, which differ in different regions of the gastrointestinal tract and different species. Indeed, in the rat stomach, GalR1 immunoreactive myenteric neurons comprise both ascending cholinergic/tachikinergic neurons (∼50%) and descending neurons containing nitric oxide (NO) or vasoactive intestinal polypeptide (VIP) (∼40%) [17]. In the small intestine of rat and guinea pig, the vast majority of GalR1 myenteric neurons are cholinergic (>80%) [26, 32]. However, in the rat small intestine, GalR1 neurons do not appear to comprise descending inhibitory neurons, as indicated by the lack of colocalization of GalR1 with either nitric oxide synthase (NOS) [26], the enzyme synthesizing NO, or VIP, whereas in the guinea pig small intestine GalR1 myenteric neurons include a small proportion of VIP and NOS descending neurons (12–18%) [32]. In the submucosal plexus, GalR1 neurons comprise both tachykinergic and VIPergic neurons [26]. Finally, GalR1 enteric neurons do not comprise intrinsic primary afferent neurons [2]. The presence of GalR1 on neuronal cell surface and processes distributed to the muscle is consistent with pre- and postsynaptic actions of galanin via this receptor. The finding that the majority of GalR1 myenteric neurons are cholinergic/tachykinergic supports the concept that GalR1 myenteric neurons comprise ascending excitatory neurons. However, the presence of GalR1 on VIP and NOS immunoreactive neurons in the guinea pig suggests a modulatory role of this receptor also on descending pathways, at least in this species. All together, the localization of GalR1 immunoreactivity to functionally distinct enteric neurons and to ECL cells supports the hypothesis that GalR1 mediates galanin actions on gastrointestinal motility and secretion by modulating the release of other neurotransmitters and contributes to galanin-induced inhibition of gastric acid secretion via the suppression of endogenous histamine release. GalR2 immunoreactivity, visualized with a Cterminus antibody (sc-16219) of GalR2 (Santa Cruz) is localized to myenteric and submucosal neurons in all gastrointestinal segments with highest density in the stomach and colon and to smooth muscle cells (Fig. 2)

(Cavalli and Sternini, unpublished). Numerous GalR2 myenteric neurons are cholinergic, and some appear to contain GalR1. These findings support the hypothesis that GalR2 mediates galanin excitatory action on gut motility both prejunctionally on enteric neurons and postjunctionally on smooth muscle cells. There are no data at the moment on the cell sites of expression of GalR3 in the gastrointestinal tract.

GalR1 and GalR2 Activation The distribution of GalR1 and GalR2 on different types of cells, including endocrine cells (GalR1), enteric neurons (both GalR1 and GalR2), and smooth muscle cells (GalR2) is consistent with the concept that more than one GalR subtype on different targets mediates the galanin biological effects in the gut. For example, in the rat stomach, galanin exerts a short inhibition, followed by an excitatory effect on gastric motility in vivo, the latter being significantly reduced by a GalR1 antagonist (RWJ-57408 from Johnson Pharmaceutical Institute, Spring House, PA), indicating an involvement of GalR1. By contrast, in vitro galanin has only an excitatory, tetrodotoxin-independent effect on intragastric pressure, which is not affected by GalR1 antagonist. This is indicative of a nonneuronal direct effect on smooth muscle [17] that is not mediated by a GalR1 pathway. This effect is likely to be mediated by GalR2, the only GalR that signals through a stimulatory Gq/11 pathway; thus its activation by galanin could result in smooth muscle contraction [9, 10]. The participation of GalR2 in the effect of galanin on gastric motility would be consistent with our findings of high levels of GalR2 mRNA in the stomach [3, 4] and the localization of GalR2 on smooth muscle cells (Cavalli and Sternini, unpublished) and with functional studies indicating GalR2 as the main GalR implicated in the excitatory mechanism for smooth muscle contraction [35]. Furthermore, galanin has been shown to induce contraction of longitudinal smooth muscle cells of the jejunum and colon [35]. The GalR1s mediating the excitatory effect of galanin on intragastric pressure do not appear to be located on intrinsic neuronal pathways regulating gastric motility, because the galanin contractile effect in the stomach in

FIGURE 2. GalR2 immunoreactivity in the myenteric plexus (mp) of the rat stomach (cryostat section) and distal colon (whole mount) and in a smooth muscle cell in the circular muscle (cm) of the colon (whole mount). Calibration bar: 50 μm.

Galanin in the Gastrointestinal Tract: Distribution and Function / 1041 vitro is not suppressed by the GalR1 antagonist, but they are likely to be located on extrinsic vagal efferents innervating the stomach [17]. Because GalR1 couples to inhibitory proteins and its activation results in inhibition of transmitter release, it is likely that GalR1s mediating the excitatory effect of galanin on gastric motility in vivo are located on the vagal efferents innervating descending pathways and that their activation results in increased intragastric pressure due to the inhibition of release of inhibitory mediators regulating muscle relaxation. Finally, a different GalR on extrinsic vagal neuronal circuitry might be involved in the galanin short-lasting relaxing effect on gastric motility in vivo that is not blocked by the GalR1 antagonist and not observed in vitro. Activation of GalR1 on ECL cells of the gastric mucosa might be one of the mechanisms through which galanin inhibits gastric acid secretion through the inhibition of histamine release from ECL cells. Similarly, the activation of GalR1 on cholinergic neurons innervating the gastric mucosa could contribute to the galanin inhibition of gastric secretion. A GalR different from GalR1 and GalR2 could be responsible for the galanin inhibition of gastric G cells that has been suggested by Schepp et al. [30], because these GalR subtypes have not been visualized in G cells. GalR3 mRNA is present in the stomach at very low levels; however, we do not know its cellular localization. Functional studies are consistent with a role of galanin as an inhibitory neuromodulator on cholinergic and tachykinergic transmission through a pertussis toxin–sensitive mechanism [1, 11, 23] that involves, at least in part, GalR1 [32]. Indeed, exogenous galanin inhibits cholinergic transmission to the longitudinal muscle of the guinea pig ileum by acting prejunctionally, because submaximal contractions to exogenous acetylcholine in unstimulated neuromuscular preparations are not affected. Galanin appears to act at two distinct receptors, as indicated by a biphasic inhibitory curve of the electrically induced muscle twitch, one being the GalR1 because the low-potency phase is antagonized by GalR1 antagonist [32]. The highpotency phase might be mediated by GalR3, a receptor that binds inhibitory proteins and uses the same transduction mechanism as GalR1. GalR2, which predominantly couples to excitatory proteins, would induce direct muscle contraction and transmitter release; thus, it cannot be responsible for the inhibitory effect induced by galanin on cholinergic transmission. GalR1 activation is also responsible for the galanin inhibition of peristalsis efficiency and intestinal wall compliance decrease [32]. The reduction of peristalsis efficiency is probably due to galanin inhibition of cholinergic drive though the activation of GalR1. This is consistent with the abundant presence of GalR1 on ascending cholinergic neurons [26, 32]. By contrast, the decreased

compliance is likely to be due to the inhibition of descending neurons mediated by GalR1. This is in agreement with the presence of GalR1 on a subpopulation of descending neurons [32] or the activation of an excitatory muscle receptor that could be GalR2, which is supported by the presence of GalR2 on smooth muscle cells (Cavalli and Sternini, unpublished). Indeed, an excitatory receptor such as GalR2 located on smooth muscle cells could, in part, hinder the accommodation of the circular muscle during the preparatory phase of peristalsis. These functional and morphological findings are in agreement with the proposal that the diverse biological functions of galanin are mediated via distinct receptor subtypes [9, 10]. Thus, the different signaling profiles, pharmacological properties, and tissue distribution of GalRs are consistent with receptor selectivity of galanin modulation of various functions in different organs.

INFLAMMATION AND INJURY Galanin and its receptors have been implicated in injury and inflammation. For instance, peripheral nerve injury induces marked upregulation of galanin and a downregulation of both GalR1 and GalR2 in primary sensory neurons, whereas peripheral inflammation induces downregulation of galanin and GalR1 and a transient upregulation of GalR2 in primary sensory neurons [19, 31]. An increased expression of GalR1 by colonocytes has been described in the mouse and human colon following infection with different pathogens and inflammation [18, 20, 21]. This increased expression of GalR1 by the colonic epithelium lining has been proposed as a novel mechanism accounting for the increased colonic fluid secretion responsible for infectious diarrhea and for the increased colonic fluid secretion in colitis. These findings support a possible role for the galanin system in the pathophysiology of inflammatory bowel diseases [18, 20, 21].

Acknowledgment The original work presented here was supported by the National Institutes of Health grant DK 57307.

References [1] Akehira K, Nakane Y, Hioki K, Taniyama K. Site of action of galanin in the cholinergic transmission of guinea pig small intestine. Eur J Pharmacol 1995; 284: 149–55. [2] Anselmi L, Cervio E, Guerrini S, Vicini R, Agazzi A, Dellabianca A, et al. Identification of galanin receptor 1 on excitatory motor neurons in the guinea pig ileum. Neurogastroenterol Motil 2005; 17: 273–80.

1042 / Chapter 142 [3] Anselmi L, Lakhter A, Hirano AA, Tonini M, Sternini C. Expression of galanin receptor messenger RNAs in different regions of the rat gastrointestinal tract. Peptides 2005; 26: 815–9. [4] Anselmi L, Stella SJ, Lakhter A, Hirano A, Tonini M, Sternini C. Galanin receptors in the rat gastrointestinal tract. Neuropeptides 2005; 349: 349–52. [5] Bartfai T, Hokfelt T, Langel U. Galanin—a neuroendocrine peptide. Crit Rev Neurobiol 1993; 7: 229–74. [6] Bauer FE, Zintel A, Kenny MJ, Calder D, Ghatei MA, Bloom SR. Inhibitory effect of galanin on postprandial gastrointestinal motility and gut hormone release in humans. Gastroenterology 1989; 97: 260–4. [7] Benya RV, Marrero JA, Ostrovskiy DA, Koutsouris A, Hecht G. Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl-secretion. Am J Physiol 1999; 276: G64–72. [8] Botella A, Delvaux M, Fioramonti J, Frexinos J, Bueno L. Galanin contracts and relaxes guinea pig and canine intestinal smooth muscle cells through distinct receptors. Gastroenterology 1995; 108: 3–11. [9] Branchek TA, Smith KE, Gerald C, Walker MW. Galanin receptor subtypes. Trends Pharmacol Sci 2000; 21: 109–17. [10] Branchek T, Smith KE, Walker MW. Molecular biology and pharmacology of galanin receptors. Ann NY Acad Sci 1998; 863: 94–107. [11] Ekblad E, Hakanson R, Sundler F, Walhestedt C. Galanin: Neuromodulatory and direct contractile effects on smooth muscle preparations. Br J Pharmacol 1985; 86: 241–6. [12] Ekblad E, Rokaeus A, Hakanson R, Sundler F. Galanin nerve fibers in the rat gut: Distribution, origin and projections. Neuroscience 1985; 16: 355–63. [13] Fontaine J, Lebrun P. Ca2+-dependent contractile effects on the isolated mouse distal colon. Eur J Pharmacol 1989; 164: 583–6. [14] Fox JET, McDonald TJ, Kostolanska F, Tatemotot K. Galanin: An inhibitory neural peptide of the canine small intestine. Life Sci 1986; 39: 103–10. [15] Furness JB, Costa M, Rokaeus A, McDonald TJ, Brooks B. Galanin-immunoreactive neurons in the guinea-pig small intestine: Their projections and relationships to other enteric neurons. Cell Tissue Res 1987; 250: 607–15. [16] Gu ZF, Pradhan TK, Coy DH, Jensen RT. Smooth muscle cells from guinea pig stomach possess high-affinity galanin receptors that mediate relaxation. Am J Physiol 1994; 266: G839–45. [17] Guerrini S, Raybould HE, Anselmi L, Agazzi A, Cervio E, Reeve JR, Jr., et al. Role of galanin receptor 1 in gastric motility in rat. Neurogastroenterol Motil 2004; 16: 429–38. [18] Hecht G, Marrero JA, Danilkovich A, Matkowskyj KA, Savkovic SD, Koutsouris A, et al. Pathogenic Escherichia coli increase Cl− secretion from intestinal epithelia by upregulating galanin-1 receptor expression. J Clin Invest 1999; 104: 253–62. [19] Liu HX, Brumovsky P, Schmidt R, Brown W, Payza K , Hodzic L, et al. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: Selective actions via GalR1 and GalR2 receptors. Proc Natl Acad Sci USA 2001; 98: 9960–4. [20] Marrero JA, Matkowskyj KA, Yung K, Hecht G, Benya RV. Dextran sulfate sodium-induced murine colitis activates NFkappaB and increases galanin-1 receptor expression. Am J Physiol Gastrointest Liver Physiol 2000; 278: G797–804.

[21] Matkowskyj KA, Danilkovich A, Marrero J, Savkovic SD, Hecht G, Benya RV. Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens. Nat Med 2000; 6: 1048–51. [22] Melander T, Hokfelt T, Rokaeus A, Fahrenkrug J, Tatemoto K, Mutt V. Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species. Cell Tissue Res 1985; 239: 253–70. [23] Mulholland MW, Schoeneich S, Flowe K . Galanin inhibition of enteric cholinergic neurotransmission: Guanosine triphosphate-binding protein interactions with adenylate cyclase. Surgery 1992; 112: 195–201. [24] Muramatsu I, Yanaihara N. Contribution of galanin to noncholinergic, non-adrenergic transmission in rat ileum. Br J Pharmacol 1988; 94: 1241–9. [25] Palmer JM, Schemmann M, Tamura K, Wood JD. Galanin mimics slow synaptic inhibition in myenteric neurons. Eur J Pharmacol 1986; 124: 379–80. [26] Pham T, Guerrini S, Wong E, Reeve JR, Jr, Sternini C. Distribution of galanin receptor 1 immunoreactivity in the rat stomach and small intestine. J Comp Neurol 2002; 450: 292–302. [27] Rattan S. Role of galanin in the gut. Gastroenterology 1991; 100: 1762–8. [28] Ren J, Hu HZ, Starodub AM, Wood JD. Galanin suppresses calcium conductance and activates inwardly rectifying potassium channels in myenteric neurones from guinea-pig small intestine. Neurogastroenterol Motil 2001; 13: 247–54. [29] Sarnelli G, Vanden Berghe P, Raeymaekers P, Janssens J, Tack J. Inhibitory effects of galanin on evoked [Ca2+]i responses in cultured myenteric neurons. Am J Physiol Gastrointest Liver Physiol 2004; 286: G1009–14. [30] Schepp W, Prinz C, Tatge C, Hakanson R, Schusdziarra V, Classen M. Galanin inhibits gastrin release from isolated rat gastric G-cells. Am J Physiol 1990; 258: G596–602. [31] Sten Shi TJ, Zhang X, Holmberg K, Xu ZQ, Hokfelt T. Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: Effect of axotomy and inflammation. Neurosci Lett 1997; 237: 57–60. [32] Sternini C, Anselmi L, Guerrini S, Cervio E, Pham T, Balestra B, et al. Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum. Neuroscience 2004; 125: 103–12. [33] Tamura K, Palmer JM, Winkelmann CK, Wood JD. Mechanism of action of galanin on myenteric neurons. J Neurophysiol 1988; 60: 966–79. [34] Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett 1983; 164: 124–8. [35] Wang S, Ghibaudi L, Hashemi T, He C, Strader C, Bayne M, et al. The GalR2 galanin receptor mediates galanin-induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation. FEBS Lett 1998; 434: 277–82. [36] Wang S, Hashemi T, He C, Strader C, Bayne M. Molecular cloning and pharmacological characterization of a new galanin receptor subtype. Mol Pharmacol 1997; 52: 337–43. [37] Waters SM, Krause JE. Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral tissues. Neuroscience 2000; 95: 265–71.

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143 Gastrin G. J. DOCKRAY

C-terminally amidated gastrins (G-17) from porcine antral mucosa by Gregory and Tracy in the 1960s finally proved beyond doubt the existence the hormone [14]. The two peptides differ in whether or not a tyrosine residue is sulfated; subsequently, two 34-amino-acid gastrins corresponding to N-terminally extended forms of G-17 were also characterized [15].

ABSTRACT Gastrin was discovered by J. S. Edkins in 1905. The main human gastrins are C-terminally amidated peptides of 17 and 34 residues. They arise from a precursor of 101 amino acid residues via C-terminally glycineextended intermediates. Amidated gastrins act at CCK2 receptors to stimulate acid secretion by releasing histamine from enterochromaffinlike cells; they also stimulate gastric epithelial cell proliferation and parietal cell maturation. Recent work suggests that progastrin and Gly-extended gastrins also possess growth factor–like properties. Hypergastrinemia occurs in patients with gastrin-secreting tumors and conditions in which normal mechanisms of acid-inhibition of the G cell are absent, as in pernicious anemia.

STRUCTURE OF THE GENE AND PRECURSOR The gastrin gene consists of three exons, two in the coding region, located at chromosome 17q21 [21]. The human gene encodes a precursor of 101 amino acid residues (104 in the rat), with a characteristic Nterminal signal peptide. Following mRNA translation, the precursor is rapidly converted to progastrin, which passes through the Golgi complex to the trans-Golgi network where there is sulfation of Tyr at position 87 and phosphorylation of Ser at 96 [46]. Following sequestration in secretory vesicles the precursor is cleaved by subtilisinlike serine proteases (SPC1/3 and SPC2), acting at three pairs of basic residues, followed by carboxypeptidase E action to generate a peptide corresponding to G34 with a C-terminal Gly [47]. The latter peptide is then converted to G-34, the cleavage of which generates G-17. Expression of the gastrin gene is increased by food in the stomach and by epidermal growth factor (EGF); luminal acid inhibits gene expression [53]. The same factors regulate gastrin release. Prolonged increases in gastric pH, associated, for example, with the administration of proton pump inhibitors or the loss of parietal cells in pernicious anemia, are linked to sustained increases in gastrin gene expression, maintaining elevated plasma gastrin concentrations [4, 7].

DISCOVERY John Sidney Edkins first used the term gastrin in 1905 to describe a gastric acid secretagog extracted from the pyloric antral mucosa [10]. The idea that such a secretagog might exist had been recognized by Bayliss and Starling 3 years earlier when describing their discovery of the first hormone, secretin [1]. Subsequently, Popielski discovered that histamine was a strong stimulant of gastric acid secretion and the existence of gastrin was cast into doubt [37]. Well-conceived experiments by Komarov in the 1930s established that it was possible to prepare histamine-free extracts of pyloric antral mucosa that stimulated gastric acid secretion [26]. The proof-of-principle experiments performed by Grossman, Robertson, and Ivy in the 1940s ultimately established that stimulation of the pyloric antral mucosa, in vivo, was linked to release of an endocrine stimulant of acid secretion [16]. The isolation of two 17-amino-acid, Handbook of Biologically Active Peptides

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1044 / Chapter 143 DISTRIBUTION OF MRNA IN GUT Physiologically, the main site of gastrin gene expression is G cells in the pyloric antral mucosa. In human, there is also expression in duodenal G cells [48]. The gastrin gene is expressed in gastrinomas, and these tumors are generally able to convert the precursor to amidated gastrins. A number of other tumors, including colorectal carcinomas, express the gene. However, for the most part these are unable to execute the full range of posttranslational processing events and, as a consequence, tend to secrete progastrin or Gly-gastrins [32, 44].

RECEPTOR EXPRESSION AND REGULATION The amidated gastrins act at the cholecystokinin (CCK)-2 (also known as CCK-b or gastrin/CCK-B) receptor [22]. The gut-brain peptide CCK has similar affinity for this receptor, but it is present in the circulation in approximately 10-fold lower concentrations, so amidated gastrins are the main naturally occurring ligands in the periphery [9]. The receptor is, however, expressed by many central nervous system (CNS) neurons, and CCK is likely to be the natural ligand for this pool of receptors. In the stomach, the CCK-2 receptor is expressed by parietal and enterochromaffinlike (ECL) cells and by some smooth muscle cells. Increased CCK-2 receptor expression has been reported in response to damage of the gastric epithelium [41]. Clinically, increased expression has been reported in Barrett’s mucosa of the esophagus, in medullary carcinoma of the thyroid, and in some gastrointestinal tumors (e.g., pancreas and stomach) [17, 38, 39, 51]. The CCK-2 receptor belongs to the G-proteincoupled, seven-transmembrane-domain family of receptors. The CCK-2 receptor gene is localized to chromosome 11p15.4; the gene consists of five exons and gives rise to a protein of 447-amino-acid residues [27, 31]. It is related to the CCK-1 receptor (which has a high affinity for CCK but a low affinity for gastrin). Coupling is via Gαq/11, which activates phospholipase C, leading to increased inositol trisphosphate, release of intracellular calcium, and activation of protein kinase C. In addition, there is activation of the mitogen-activating protein kinase (MAPK) and phosphoinositide (PI)-3-kinase signaling pathways, which may occur secondary to transactivation of the EGF receptor via shedding of EGF receptor ligands such as HB-EGF, TGFα, and FGF [33]. A number of studies indicate that progastrin and Gly-gastrin act via receptors distinct from the CCK-2 receptor [5, 20, 42, 43], but the precise identity of receptors mediating the effects of progastrin and

Gly-gastrin remains to be determined. There is some evidence that fragments of progastrin that include the C-terminal 26-amino-acid residues are able to reproduce the effects of intact progastrin in stimulating colon proliferation [35].

BIOLOGICAL ACTIONS ON GI TRACT Acid Secretion The best-studied acute effect of gastrin is stimulation of acid secretion [48]. Basal plasma gastrin concentrations are typically <30 pmoles/liter and after a meal these increase about three-fold. The increase in gastric acid secretion in response to intragastric administration of amino acids is roughly comparable to that in response to the infusion of exogenous G-17 in a dose chosen to match the increase in plasma concentration of endogenous gastrin released by intragastric amino acids [12]. The administration of gastrin antibodies or CCK-2 receptor antagonists inhibits postprandial acid secretion [2, 28]. Although parietal cells express the CCK-2 receptor, the role of these receptors in acute stimulation of acid secretion by gastrin remains in doubt [3]. However, mice null for the gastrin or CCK-2 receptor genes are achlorhydric and are resistant to acute stimulation of acid secretion by gastrin, histamine, or cholinomimetic secretagogs [13, 25]. The administration of gastrin for 24 h or more reverses this insensitivity, suggesting that gastrin plays a role in parietal cell maturation, possibly by acting directly on immature parietal cells. It has been known for many years that the action of gastrin on acid secretion is inhibited by antagonists of the histamine (H)-2 receptor, consistent with the view that gastrin acts by releasing histamine from ECL cells [18, 40]. In the isolated perfused rat stomach, the release of histamine occurs with concentrations of gastrin that are in the physiological range. In addition, gastrin stimulates histamine synthesis by increasing the expression and activity of the enzyme histidine decarboxylase, and it increases the capacity for histamine storage in secretory vesicles by stimulating expression of vesicular monoamine transporter (VMAT)-2, which transports histamine from cytosol to secretory vesicles [7, 8, 50].

Growth Prolonged increases in plasma gastrin are associated with ECL cell hyperplasia [29, 30]. In the presence of either inflammation (which occurs, for example, in pernicious anemia) or mutations of the tumor-suppressor gene menin (leading to multiple endocrine neoplasia

Gastrin type 1), hypergastrinemia is linked to ECL cell carcinoid tumors [22, 36]. In pernicious anemia, the latter frequently resolve following surgical removal of the antrum to lower plasma gastrin concentrations [19]. There is also evidence that gastrin stimulates the proliferation of gastric epithelial cells after a meal [34], and that moderate hypergastrinemia is associated with gastric hyperproliferation [24]. These effects are thought to be mediated by CCK-2 receptors, possibly via release of EGF family members. In addition, however, it is clear that progastrin and the Gly-gastrins have growth factor properties; these are not mediated by CCK-2 receptors.

Other Targets In recent years functional genomics approaches have identified several previously unsuspected target genes for gastrin [11], including plasminogen activator inhibitor-2, matrix metalloproteinase-7 and -9, and trefoil factor-1 [23, 45, 52]. These are likely to play a role in tissue remodeling in hypergastrinemia and may be part of the response to tissue damage that includes increased expression of the CCK-2 receptor [11].

PATHOPHYSIOLOGICAL IMPLICATIONS As already noted, elevated concentrations of plasma gastrin occur in patients with gastrin-secreting tumors (gastrinomas) and in circumstances in which there is interruption of the normal inhibition of G-cell function by acid acting through the paracrine mediator somatostatin. Thus, hypergastrinemia also occurs in pernicious anemia (when parietal cells are lost), in subjects treated with proton pump inhibitors, and in rare surgical cases in which the antrum is excluded from acid [9]. In addition, there is modest hypergastrinemia in some subjects infected with Helicobacter pylori. In part, this may be due to locally raised pH in the antral mucosa secondary to ammonia production, but there is also evidence that the inflammation associated with H. pylori inhibits somatostatin-cell function and stimulates G-cell function [6]. In subjects with a normal corpus mucosa, hypergastrinemia is associated with the increased secretion of acid. However, inflammation in the corpus (as may occur with Helicobacter infection) inhibits acid secretion, and in these circumstances there is some evidence that gastrin might exacerbate the progression to gastric atrophy [49].

References [1] Bayliss WM, Starling EH. The mechanism of pancreatic secretion. J Physiol 1902;28:325–353.

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[2] Beltinger J, Hildebrand P, Drewe J, Christ A, Hlobil K, Ritz M, D’Amato M, Rovati L, Beglinger C. Effects of spiroglumide, a gastrin receptor antagonist, on acid secretion in humans. Eur J Clin Invest 1999;29:153–159. [3] Black JW, Shankley NP. How does gastrin act to stimulate oxyntic cell secretion. TIPS 1987;8:486–490. [4] Brand SJ, Stone D. Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria. J Clin Invest 1988;82:1059–1066. [5] Brown D, Yallampalli U, Owlia A, Singh P. pp60(c-Src) kinase mediates growth effects of the full-length precursor progastrin(1–80) peptide on rat intestinal epithelial cells, in vitro. Endocrinology 2003;144:201–211. [6] Calam J, Gibbons A, Healey ZV, Bliss P, Arebi N. How does Helicobacter pylori cause mucosal damage? its effect on acid and gastrin physiology. Gastroenterology 1997;113:S43– S49. [7] Dimaline R, Evans D, Varro A, Dockray GJ. Reversal by omeprazole of the depression of gastrin cell function by fasting in the rat. J Physiol 1991;433:483–493. [8] Dimaline R, Sandvik AK, Evans D, Forster ER, Dockray GJ. Food stimulation of histidine decarboxylase messenger RNA abundance in rat gastric fundus. J Physiol 1993;465:449–458. [9] Dockray GJ. Gastrin. Best Pract Res Clin Endocrinol Metab 2004;18:555–568. [10] Dockray GJ. The history of gastrin. In: Merchant, JL, Buchan, AM, Wang, TC, Eds., Gastrin in the New Millennium. Los Angeles: CURE Foundation; 2004:1–10. [11] Dockray G, Dimaline R, Varro A. Gastrin: old hormone, new functions. Pflugers Arch 2005;449:344–355. [12] Feldman M, Walsh JH, Wong HC, Richardson CT. Role of gastrin heptadecapeptide in the acid secretory response to amino acids in man. J Clin Invest 1978;61:308–313. [13] Friis-Hansen L, Sundler F, Li Y, Gillespie PJ, Saunders TL, Greenson JK, Owyang C, Rehfeld JF, Samuelson LC. Impaired gastric acid secretion in gastrin-deficient mice. Am J Physiol 1998;274:G561–G568. [14] Gregory RA, Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:103– 114. [15] Gregory RA, Tracy HJ. Isolation of two “big gastrins” from Zollinger-Ellison tumour tissue. Lancet 1972;2:797–799. [16] Grossman MI, Robertson CR, Ivy AC. The proof of a hormonal mechanism for gastric secretion—the humoral transmission of the distension stimulus. Am J Physiol 1948;153:1–9. [17] Haigh CR, Attwood SE, Thompson DG, Jankowski JA, Kirton CM, Pritchard DM, Varro A, Dimaline R. Gastrin induces proliferation in Barrett’s metaplasia through activation of the CCK2 receptor. Gastroenterology 2003;124:615–625. [18] Hakanson R, Ding XQ, Norlen P, Lindstrom E. CCK2 receptor antagonists: pharmacological tools to study the gastrin-ECL cellparietal cell axis. Regul Pept 1999;80:1–12. [19] Higham AD, Dimaline R, Varro A, Attwood S, Armstrong G, Dockray GJ, Thompson DG. Octreotide suppression test predicts beneficial outcome from antrectomy in a patient with gastric carcinoid tumor. Gastroenterology 1998;114:817– 822. [20] Hollande F, Choquet A, Blanc EM, Lee DJ, Bali JP, Baldwin GS. Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells. J Biol Chem 2001;276:40402–40410. [21] Ito R, Sato K, Helmer T, Jay G, Agarwal K . Structural analysis of the gene encoding human gastrin: the large intron contains an Alu sequence. Proc Natl Acad Sci USA 1984;81:4662– 4666.

1046 / Chapter 143 [22] Jensen RT. Involvement of cholecystokinin/gastrin-related peptides and their receptors in clinical gastrointestinal disorders. Pharmacol Toxicol 2002;91:333–350. [23] Khan ZE, Wang TC, Cui G, Chi AL, Dimaline R. Transcriptional regulation of the human trefoil factor, TFF1, by gastrin1. Gastroenterology 2003;125:510–521. [24] Koh TJ, Chen D. Gastrin as a growth factor in the gastrointestinal tract. Regul Pept 2000;93:37–44. [25] Koh TJ, Goldenring JR, Ito S, Mashimo H, Kopin AS, Varro A, Dockray GJ, Wang TC. Gastrin deficiency results in altered gastric differentiation and decreased colonic proliferation in mice. Gastroenterology 1997;113:1015–1025. [26] Komarov SA. Gastrin. Proc Soc Exp Biol Med 1938;38:514– 516. [27] Kopin AS, Lee YM, McBride EW, Miller LJ, Lu M, Lin HY, Kolakowski LF, Jr, Beinborn M. Expression cloning and characterization of the canine parietal cell gastrin receptor. Proc Natl Acad Sci USA 1992;89:3605–3609. [28] Kovacs TO, Walsh JH, Maxwell V, Wong HC, Azuma T, Katt E. Gastrin is a major mediator of the gastric phase of acid secretion in dogs: proof by monoclonal antibody neutralization. Gastroenterology 1989;97:1406–1413. [29] Lamberts R, Creutzfeldt W, Struber HG, Brunner G, Solcia E. Long-term omeprazole therapy in peptic ulcer disease: gastrin, endocrine cell growth, and gastritis. Gastroenterology 1993; 104:1356–1370. [30] Larsson H, Carlsson E, Mattsson H, Lundell L, Sundler F, Sundell G, Wallmark B, Watanabe T, Hakanson R. Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation. Studies with omeprazole and ranitidine in intact and antrectomized rats. Gastroenterology 1986;90:391–399. [31] Lee YM, Beinborn M, McBride EW, Lu M, Kolakowski LF, Jr, Kopin AS. The human brain cholecystokinin-B/gastrin receptor. Cloning and characterization. J Biol Chem 1993;268:8164– 8169. [32] Nemeth J, Taylor B, Pauwels S, Varro A, Dockray GJ. Identification of progastrin derived peptides in colorectal carcinoma extracts. Gut 1993;34:90–95. [33] Noble PJ, Wilde G, White MR, Pennington SR, Dockray GJ, Varro A. Stimulation of gastrin-CCKB receptor promotes migration of gastric AGS cells via multiple paracrine pathways. Am J Physiol 2003;284:G75–G84. [34] Ohning GV, Wong HC, Lloyd KC, Walsh JH. Gastrin mediates the gastric mucosal proliferative response to feeding. Am J Physiol 1996;271:G470–G476. [35] Ottewell PD, Varro A, Dockray GJ, Kirton CM, Watson AJ, Wang TC, Dimaline R, Pritchard DM. The C-terminal 26 amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo. Am J Physiol Gastrointest Liver Physiol 2005;288:G541–G549. [36] Peghini PL, Annibale B, Azzoni C, Milione M, Corleto VD, Gibril F, Venzon DJ, Delle FG, Bordi C, Jensen RT. Effect of chronic hypergastrinemia on human enterochromaffin-like cells: insights from patients with sporadic gastrinomas. Gastroenterology 2002;123:68–85. [37] Popielski L. Beta-imidazolylathylamin und die Organextrakte. I. Beta-imidazolylathylamin als machtiger erreger der Magendrusen. Pflugers Arch ges Physiol 1919;178:214–259.

[38] Reubi JC, Waser B. Unexpected high incidence of cholecystokinin-B/gastrin receptors in human medullary thyroid carcinomas. Int J Cancer 1996;67:644–647. [39] Reubi JC, Waser B, Schmassmann A, Laissue JA. Receptor autoradiographic evaluation of cholecystokinin, neurotensin, somatostatin and vasoactive intestinal peptide receptors in gastro-intestinal adenocarcinoma samples: where are they really located? Int J Cancer 1999;81:376–386. [40] Sandvik AK, Waldum HL. CCK-B (gastrin) receptor regulates gastric histamine release and acid secretion. Am J Physiol 1991;260:G925–G928. [41] Schmassmann A, Reubi JC. Cholecystokinin-B/gastrin receptors enhance wound healing in the rat gastric mucosa. J Clin Invest 2000;106:1021–1029. [42] Seva C, Dickinson CJ, Yamada T. Growth-promoting effects of glycine-extended progastrin. Science 1994;265:410–412. [43] Singh P, Lu X, Cobb S, Miller BT, Tarasova N, Varro A, Owlia A. Progastrin1-80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites. Am J Physiol Gastrointest Liver Physiol 2003;284:G328–G339. [44] van Solinge WW, Nielsen FC, Friis-Hansen L, Falkmer UG, Rehfeld JF. Expression but incomplete maturation of progastrin in colorectal carcinomas. Gastroenterology 1993;104:1099– 1107. [45] Varro A, Hemers E, Archer D, Pagliocca A, Haigh C, Ahmed S, Dimaline R, Dockray GJ. Identification of plasminogen activator inhibitor-2 as a gastrin-regulated gene: Role of Rho GTPase and menin. Gastroenterology 2002;123:271–280. [46] Varro A, Henry J, Vaillant C, Dockray GJ. Discrimination between temperature- and brefeldin A-sensitive steps in the sulfation, phosphorylation, and cleavage of progastrin and its derivatives. J Biol Chem 1994;269:20764–20770. [47] Varro A, Voronina S, Dockray GJ. Pathways of processing of the gastrin precursor in rat antral mucosa. J Clin Invest 1995;95:1642– 1649. [48] Walsh, JH. Gastrin. In: Walsh, JH, Dockray, GJ, Eds., Gut Peptides. New York: Raven Press; 1994:75–121. [49] Wang TC, Dangler CA, Chen D, Goldenring JR, Koh T, Raychowdhury R, Coffey RJ, Ito S, Varro A, Dockray GJ, Fox JG. Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer. Gastroenterology 2000;118:36–47. [50] Watson F, Kiernan RS, Deavall DG, Varro A, Dimaline R. Transcriptional activation of the rat vesicular monoamine transporter 2 promoter in gastric epithelial cells: regulation by gastrin. J Biol Chem 2001;276:7661–7671. [51] Weinberg DS, Ruggeri B, Barber MT, Biswas S, Miknyocki S, Waldman SA. Cholecystokinin A and B receptors are differentially expressed in normal pancreas and pancreatic adenocarcinoma. J Clin Invest 1997;100:597–603. [52] Wroblewski LE, Pritchard DM, Carter S, Varro A. Gastrinstimulated gastric epithelial cell invasion: the role and mechanism of increased matrix metalloproteinase 9 expression. Biochem J 2002;365:873–879. [53] Wu SV, Giraud A, Mogard M, Sumii K, Walsh JH. Effects of inhibition of gastric secretion on antral gastrin and somatostatin gene expression in rats. Am J Physiol 1990;258:G788– G793.

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144 Gastrin-Releasing Peptide F. S. LEHMANN and C. BEGLINGER

mammalian bombesinlike peptides are GRP and neuromedin-B. The bombesin subfamily has been studied much more extensively than the other two subfamilies.

ABSTRACT The neuropeptide bombesin and its mammalian homolog, gastrin-releasing peptide (GRP), exert different physiological functions. In the gut, they are involved in the regulation of exocrine secretions, motility, and hormone release. In addition, bombesin and its analogs enhance proliferation in normal tissue and in various tumors. The pathophysiological relevance of the bombesin/GRP receptor (BB2 receptor), which is expressed, for example, in 30% of human colon tumor cell lines and in 24–40% of native tumors, has not been clearly assessed at this time.

STRUCTURE OF THE PRECURSOR mRNA/GENE AND PEPTIDE VARIANTS Chemistry and Molecular Biology A single human GRP-encoding gene has been identified and found to be located on chromosome 18 [27]. The gene has been cloned from a human small-cell medullary carcinoma metastasis [38]; the predicted precursor corresponded to a peptide consisting of 148 amnio acids with a single copy of GRP [9]. Subsequently, three different mRNA species were identified. The consequence of these findings is that through alternate mRNA splicing three distinct human mRNAs are produced that encode precursors of slightly different chain lengths [3]. In contrast, only one GRP mRNA form has been identified in rats. The three GRP mRNAs all have the same signal peptide but differ in the region encoding the C-terminal extension peptide. In all tissues examined, they seem to be present in the same relative ratio. From PreproGRP, the signal peptidase cleaves away the signal peptide from the amino terminus. The resulting ProGRP is then cleaved by a series of enzymes to form GRP. In humans, cleavage occurs between arginine

INTRODUCTION The tetradecapeptide bombesin was originally isolated from the skin of two amphibians, Bombina bombina and Bombina variegata by Erspamer and coworkers [1]. Gastrin-releasing peptide (GRP) is the mammalian counterpart of bombesin and differs by only 1 of the 10 carboxyl-terminal residues (Table 1). Only a few years after its discovery, the occurrence of bombesinlike bioactivity and immunoreactivity was observed in the mammalian gastrointestinal tract [11,12]. Many bombesinlike peptides have since been isolated from different sources. Based on their carboxyl-terminal tripeptides, they have been classified into three subfamilies: the bombesins, the ranatensins, and the phyllolitorins. The only known Handbook of Biologically Active Peptides

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1048 / Chapter 144 TABLE 1. Amino acid sequences of bombesin and GRP. Bombesin pGlu-Gln-Arg-Leu-Gly-Asn-GlnTrp-Ala-Val-Gly-His-Leu-Met-NH2 Human GRP Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-LeuThr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His Trp-Ala-Val-Gly-His-Leu-Met-NH2

and glycine (positions 17 and 18). GRP1–27 can be further processed to form the decapeptide GRP18–27 [32]. A variety of bombesin analogs have been identified in amphibian species. They all share extensive homology over the carboxyl-terminal 8- to 10-aminoacid residues. GRP and neuromedin-B are the only bombesinlike peptides found in mammalian tissue [12, 32].

Isolation from Tissue In the mammalian gut, GRP-immunoreactivity is found in neurons but not in endocrine cells [18, 28]. Chromatography of mammalian gut extracts revealed two forms of bombesinlike immunoreactivity, one larger than amphibian bombesin (GRP1–27) and one smaller (GRP18–27). Using gastrin release as bioassay, GRP was originally isolated from porcine stomach by McDonald et al. [25]. They found a 27-amino-acid peptide homologous to the carboxyl terminus of bombesin and named it gastrin-releasing peptide. GRP has now been characterized and cloned from human, canine, rat, avian, and guinea pig species. The structure of human GRP has been established by amino acid sequencing and molecular cloning. GRP from human, canine, porcine, and chicken tissue contains 27 amino acids [32]. Human GRP shares an identical 15-aminoacid sequence at the carboxyl terminus with canine and porcine GRP, but has less sequence homology at the amino terminus. From canine tissue, Reeve at al. isolated a smaller form of GRP immunoreactivity and determined it was the carboxyl-terminal decapeptide of GRP [33]. GRP-10 or GRP18–27 seems to be present in all species that express GRP, and it has also been isolated from human stomach. GRP and GRP-10 reproduce all of bombesin’s biological effects and have therefore been considered the mammalian equivalents of amphibian bombesin.

DISTRIBUTION OF GRP-LIKE IMMUNOREACTIVITY, GRP mRNA, AND GRP RECEPTOR GRP-like immunoreactivity is widely distributed throughout the mammalian gastrointestinal (GI) tract and is found exclusively in neurons but not in endocrine cells. In the study by Price et al., the highest levels of GRP-like immunoreactivity were found in the fundus, antrum, pylorus, and pancreas, with lower concentrations in the duodenum, jejunum, terminal ileum, and colon [28]. In the antrum and colon, GRP-like immunoreactivity was observed in both the muscle and mucosal layers [18]. The study by Price was in line with data from Walsh et al., who found the highest GRP levels in rat fundus and antrum, with lower levels in the small bowel and colon [49]. Immunocytochemical studies in the small intestine and colon revealed that GRP is predominantly located in cell bodies within the submucosal and myenteric plexuses, as well as in nerve terminals in smooth muscle [18]. In gastric fundus and antrum, however, the highest concentration of GRP is found in the mucosa. In the pancreas, GRP-like immunoreactivity is found predominantly in intrapancreatic ganglia, mainly in the periacinar regions. In contrast, the endocrine islets contain only a few GRP-positive nerve fibers. Using Northern blots in human fetal and adult GI tissues, Sunday et al. found the highest levels of GRP mRNA in colon with moderate levels in stomach and lower levels in duodenum, jejunum, ileum, and pancreas [42]. The levels of GRP mRNA were lower in the adult than in the corresponding fetal tissues. Assessed by autoradiography studies, GRP receptors have been shown to be abundant in the circular muscle and myenteric plexus of the antrum, with lower levels in the circular muscle from the esophagus to the pylorus, in the longitudinal muscle in ileum and colon, and in the myenteric plexus in small intestine and colon [42]. Rettenbacher et al. examined the expression of GRP receptors in human colon tissue [35]. They found the highest concentrations of GRP receptors in the myenteric plexus, a high concentration in the longitudinal smooth muscle, a moderate density in the circular smooth muscle, and only low levels in the muscularis mucosae. GRP receptors in the circular smooth muscle were found predominantly in the mucosa-directed margin, where the interstitial cells of Cajal are located. The topical association of GRP receptors and the interstitial cells of Cajal could explain the effect of GRP on gut rythmic activity. Studies on GRP receptor expression, however, are complicated by considerable species differences [10]. The expression of GRP receptors in epithelial gastrointestinal cells is found exclusively in G cells of the

Gastrin-Releasing Peptide / 1049 gastric antrum. The presence of high-affinity GRP receptors on isolated canine G cells was first suggested by Vigna et al. [46]. It is currently believed that over 200,000 GRP receptors are present on a single G cell. In the study by Ferris et al., pinch biopsies were taken from esophagus, stomach, jejunum, ileum, and colon and GRP mRNA was extracted from the epithelial cells [15]. Results from that study confirmed that GRPexpression is limited to cells lining the gastric antrum. In contrast, the presence of high-affinity GRP binding sites has been found in the colonic epithelium of the mouse; in the ileal mucosa of rats, chickens, and guinea pigs; and in the jejunum and colon of pigs [34].

RECEPTOR SUBTYPES AND SIGNALING The effects of bombesin and GRP are mediated by at least four different receptor subtypes: the GRP receptor (GRP-R), or BB2 receptor; the neuromedin-B receptor (NMB-R), or BB1 receptor; and the BB3 and BB4 receptor subtypes. The GRP-R (BB2 receptor) has been cloned by Spindel [39] and Battey [3], the NMB-R (BB1) by Wada [47], the BB3 by Fahti [13] and Gorbulev [17], and the BB4 by Nagalla [26]. The BB3 receptor subtype is an orphan receptor that recognizes bombesin and other ligands only at nonphysiological concentrations. The BB4 has been cloned from amphibians and is not expressed in mammalian tissue. BB1, BB2, and BB3 have been shown to share approximately 50% homology in amino acid sequence (BB1 and BB2, 55% homology; BB2 and BB3, 51% homology) [34]. In humans, both BB2 and BB3 are located on the X chromosome, whereas BB1 maps to chromosome 6. Compared to the BB2 receptor, the distribution and function of the other bombesin/GRP receptor subtypes have only been poorly characterized. The BB2 receptor subtype is expressed widely both in the central nervous system and throughout the whole GI tract (pancreatic acinar cells, epithelial neuroendocrine cells, and smooth muscle cells and enteric neurons of stomach, intestine, and colon) [16]. The BB2 is also the active receptor on human antral G cells. The distribution of BB1 receptors within the GI tissue is different from BB2. BB1 receptors have been found in the esophageal smooth muscle tissue, as well as in smooth muscle cells from the circular muscle of human colon [18]. BB3 and BB4 are not expressed in GI tissue. All the BB receptors are coupled to G-protein via their intracellular domain and, thus, belong to the Gprotein-receptor superfamily. After ligand binding, multiple cellular signal transduction pathways are activated, including phospholipase C, generation of inositol triphosphate (IP3) and diacylglycerol, and elevation of intracellular calcium. On binding to a bombesin or

GRP receptor, the receptors are phosphorylated, internalized, and sometimes lost from the cell surface. Studies in the isolated perfused rat stomach, as well as in isolated canine G cells, have shown that the bombesin-induced gastrin release depends on the influx of extracellular calcium. Calcium mobilization seems to be one of the major species-independent mechanisms of bombesin-stimulated gastrin secretion [41]. Studies in isolated human antral G cells demonstrated that GRP receptor stimulation was associated with both the release of intracellular calcium and influx of extracellular calcium.

BIOLOGICAL ACTIONS WITHIN THE GI TRACT Bombesin and GRP have a wide spectrum of biological actions. In the central nervous system, they have been implicated in thermoregulation, satiety, and the regulation of circadian rhythm [22, 24, 40, 50]; in the GI tract, the effects include stimulation of gut motility, stimulation of pancreatic, gastric, and intestinal secretions, stimulation of gut hormone release, and stimulation of intestinal and pancreatic epithelial growth (Table 2). The serum concentrations of GRP are low and do not change after food intake. This observation is not surprising because GRP is a neurotransmitter and not a circulating hormone. After exogenous infusion, the half-life of GRP-10 in the circulation is only 1–2 min. In

TABLE 2. Biological effects of bombesin and GRP. Stimulation of exocrine secretions Gastric acid secretion Pepsinogen secretion Exocrine pancreatic secretion Stimulation of hormone release Gastrin Cholecystokinin Somatostatin Pancreatic glucagon Pancreatic polypeptide Insulin Enteroglucagon Neurotensin Gastric inhibitory peptide Stimulation of motility Gallbladder contraction Antral smooth muscles Intestinal smooth muscles Intestinal reflexes Inhibition of motility Gastric emptying Relaxation of gastric fundus and body Inhibition of food intake

1050 / Chapter 144 humans the biological activity of synthetic human GRP is equivalent to that of synthetic bombesin [21]: GRP and bombesin were equipotent with respect to gastric acid secretion, pancreatic enzyme output, gallblader contraction, and plasma hormone release (Fig. 1).

Effect on Gastrin and Acid Secretion The gastrin-releasing actions of GRP can be documented in most mammalian species [48]. GRP and bombesin are both potent stimulants of gastrin release and acid secretagogs: In rats, dogs, and humans, GRP and bombesin dose-dependently increase plasma gastrin concentrations and gastric acid secretion. Gastrin is

released from endocrine G cells of the antrum and proximal duodenum. In cultured human antral G cells, bombesin and GRP significantly stimulate gastrin release [4]. The intracellular mediators of gastrin secretion seemed to involve the adenylate cyclase and phosphatidyl inositide second-messenger systems. GRP-stimulated gastrin is released into the blood and stimulates acid production; during normal eating, gastrin contributes substantially to the overall acid output. Somatostatin, on the other hand, significantly inhibits bombesin-stimulated gastrin release, but has no effect on basal gastrin secretion. GRP can, however, stimulate somatostatin secretion from D cells in the

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Gastrin-Releasing Peptide / 1051 corpus and fundus; GRP/bombesin analogs can therefore act both as stimulants and inhibitors of acid secretion [14]. The stimulatory action of bombesin on gastric acid secretion in humans depends on gastrin release because the removal of gastrin-producing antral G cells by antrectomy completely abolished the effect [48]. Similarly, bombesin does not stimulate acid secretion in the dog if the antrum is not present. Bombesin may stimulate acid secretion directly from bullfrog fundus, but there is no evidence of a similar effect in humans or mammals. A maximal effect on gastric acid secretion is seen with low doses of GRP, whereas plasma gastrin is further

stimulated with increasing doses. Compared to stimulation with pentagastrin, the acid response to bombesin analogs is clearly submaximal [20]. The failure of bombesin analogs to increase both effects in parallel suggests that one or more inhibitory peptides (e.g., somatostatin) are released concomitantly at higher doses. As mentioned before, somatostatin has been found to be released by bombesin analogs [14]; bombesin and GRP neurons of the gastric fundus exert an inhibitory effect on acid secretion, which is mainly mediated by fundic somatostatin (Fig. 2). The classical concept that bombesin analogs stimulate acid secretion exclusively via endogenous gastrin

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1052 / Chapter 144 release has been challenged by two findings. First, bombesin nonapeptide stimulates the same acid output as bombesin tetradecapeptide but with only half the increase in serum gastrin. Second, GRP (BB2) receptor blockade inhibits acid secretion, but does not change plasma gastrin levels [20]. In human volunteers, the potent and specific peripheral BB2 receptor antagonist BIM26226 dose-dependently inhibited exogenous GRP-induced acid output and plasma gastrin release, suggesting that the compound is indeed a potent BB2 receptor antagonist in humans. When BIM26226 was used as a tool to block endogenous GRP, acid secretion after both an oral liquid meal and a sham feeding was significantly inhibited by GRP receptor blockade [20]. Surprisingly, plasma gastrin release remained unchanged under these conditions (Fig. 3). This striking observation points to an important role for GRP neurons in the regulation of acid secretion, most likely during the cephalic phase of acid secretion [14]. The cephalic phase of gastric acid secretion has been a topic of great interest since its first description by Pavlov more than 100 years ago. Pavlov’s experiments demonstrated that sham feeding elicited a potent gastric acid secretory response via the vagus nerve [14]. This hypothesis is extended with these findings: Endogenous GRP seems to participate in the physiological control of postprandial gastric acid secretion, but the effect is not

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exclusively mediated by gastrin. The identity of the stimulatory pathway remains to be determined.

Effect on Gastrointestinal Hormones and Pancreatic Secretion In addition to gastrin release, bombesin and GRP both stimulate the release of a variety of other GI peptides, including somatostatin, pancreatic glucagon, pancreatic polypeptide, pepsinogen, cholecystokinin, insulin, enteroglucagon, neurotensin, and gastric inhibitory peptide (Table 2). In addition, bombesin and GRP have been shown to have a potent stimulatory effect on exocrine pancreatic secretion and BB2 receptors have been identified on pancreatic acinar cells [5]. In healthy volunteers, bombesin and GRP dose-dependently stimulate biliary and pancreatic exocrine secretions (Fig. 1), including the output of amylase, lipase, and bilirubin [21]. The effects of GRP seem to be direct effects and not mediated by endogenous cholecystokinin (CCK).

Motility Bombesin and GRP have potent effects on gastrointestinal motility. Stimulatory effects include stimulation of human, feline, guinea pig, and canine gallbladder contraction. The effect can be inhibited by GRP receptor blockage, but indirect stimulation by CCK release has also been discussed. Gallbladder contraction is accompanied by the relaxation of the sphincter of Oddi. In healthy volunteers, bombesin and human GRP produce a dose-dependent contraction of the gallbladder [21]. Gallbladder contraction is maximal because gallbladder volumes were below 10% of the basal volume (Fig. 1) after exogenous GRP/bombesin administration. The effects of bombesin analogs on gastric emptying are complex. The administration of exogenous synthetic bombesin inhibits gastric emptying [8]. Using the specific BB2 receptor antagonist BIM26226 as a tool to block endogenous BB2 receptors during a physiological stimulation (meal intake), researchers showed that gastric emptying is dramatically decreased, gallbladder contraction is inhibited, and small bowel transit is accelerated [8] when endogenous GRP is blocked. These results point to an important physiological role of endogenous GRP in human GI motility. Several mechanisms whereby GRP could stimulate intestinal motility are possible: (1) GRP could directly activate its receptor on smooth muscle cells, (2) GRP could activate reflex pathways, or (3) GRP could stimulate a variety of other regulatory factors, which in turn could mediate smooth muscle contraction. In fact, GRP-containing nerves are believed to play an important role in the control of gastric and intestinal motor activity. At the cellular level,

Gastrin-Releasing Peptide / 1053 GRP is present in neuronal cell bodies. More specifically, GRP is present in neurons of the myenteric plexus and, to a lesser extent, the submucosal plexus [44]. Although the exact physiological function of GRPcontaining nerves is not known, the anatomy and functional results suggest a regulatory function in modulating motor reflexes [18]. Bombesin/GRP stimulate antral motility, relax gastric body and fundus, and stimulate contractions of intestinal smooth muscle, leading thereby to slower propulsion through the small intestine and colon. Finally, GRP has been shown to act as a neurotransmitter in the colon to regulate the peristaltic reflex [18].

Satiety Exogenous administration of bombesin and GRP have been shown to decrease food intake in rats, baboons, and humans [2, 22, 40]. In a previous study from our laboratory, increasing concentrations of GRP or saline as a control were administered by slow infusion to 12 nonobese men [19]. A decrease of food intake (by weight) was observed, resulting in a significant reduction in calorie consumption. In addition, GRP decreased self-reported hunger feelings in the premeal period and increased self-reported fullness in the same premeal period. Consistent with the possibility that endogenous GRP reduces food intake, mice deficient for the GRP receptor do not suppress their food intake when administered GRP; instead, they eat significantly larger meals and develop late-onset obesity [2, 24, 40]. Most GI satiety factors are thought to act by reducing the size of an ongoing meal. Hence, if they are administered between meals, they have no behavioral effects such as prolonging the time until the individual initiates a meal [2, 40]. Members of the bombesin family of peptides appear to be an interesting exception in that, when they are administered to animals between meals, they increase the amount of time until the subsequent meal begins [2, 22, 40]. At present, it is difficult to draw conclusions about the physiological nature of the feeding inhibitory effects of GRP, at least in humans. We have to accept the limitations of study designs that simulate physiology by using exogenous infusions. Experiments using potent and selective antagonists for GRP will be required to define the physiological role of GRP in the regulation of food intake and satiety in humans.

Trophic Effects Bombesinlike peptides have been found in the breast milk of some mammals. This observation is compatible with the hypothesis that bombesin influences the postnatal development of the digestive tract [45]. Studies in

neonatal and suckling rats have shown that oral and subcutaneous administration of bombesin stimulated cell growth in the stomach, pancreas, small intestine, and colon [45]. The effect in the colon was less pronounced than in the other tissues. It is possible that the bombesin-induced growth effects are mediated by other hormones (e.g., gastrin, CCK, enteroglucagon, or pancreatic polypeptide). In the study by Puccio, the increase in DNA synthesizing and dividing cells by oral bombesin disappeared after weaning [29]. Animal studies, mainly in adult rats, have also shown, that bombesin can stimulate the growth of normal gastrointestinal tract mucosa and pancreatic tissue [30, 31, 45]. In the upper GI tract, bombesin induces antral gastrin cell proliferation, increases the weight and DNA content of oxyntic and duodenal mucosa, and results in hyperplasia of the fundic mucosa. Somatostatin attenuates the proliferative effects of bombesin. Studies on the bombesin receptor antagonist RC-3095 suggest that the proliferation effects in the gastroduodenal mucosa are probably direct effects of the molecule [43]. Similar to their effects in the upper GI tract, bombesin and GRP stimulate jejunal, ileal, and colonic mucosal growth.

PATHOPHYSIOLOGICAL IMPLICATIONS Role of GRP in Tumor Growth GRP can activate the growth of cancer cell lines from the human stomach, pancreas, and colon [42, 45]. During the past years, numerous bombesin/GRP antagonists have been developed and evaluated for antitumoral effects. GRP receptor antagonists could be attractive targets for cancer treatment because they are more frequently expressed on cancer cells than in normal tissue. However, it is too early to decide whether this pathway is clinically relevant. GRP has also been proposed as contributing to gastric cancer growth. Clinical studies have shown that up to 56% of surgically resected gastric cancers have high-affinity binding sites for GRP. Carroll et al. demonstrated the expression of BB2 receptor mRNA in 40% of nonantral gastric cancers [4]. In that study, 80% of the receptors were mutated. GRP receptors have been described not only in gastric cancer specimens but also in the mucosa of patients with Menetrier’s disease, in human gastric cancer xenografts, and in multiple human gastric cancer cell lines [4, 20]. GRP has also been associated with pancreatic carcinogenesis [31]. An increased expression of GRP receptors has been demonstrated in peritumoral vessels surrounding exocrine pancreatic carcinomas. Furthermore, GRP mRNA and specific GRP binding sites have

1054 / Chapter 144 been observed in AR42j rat pancreatic cancer cells, azaserine-induced rat pancreatic carcinomas, and several human pancreatic cancer cell lines [9, 30]. Bombesin and GRP enhance not only the proliferation of and DNA synthesis in rat pancreatic acinar carcinoma cells but also the expression of c-fos, c-myc in the human pancreatic cell lines [9, 30, 34]. The three transcription factors are believed to be involved in cellular proliferation. From 25 to 40% of all colon cancers and approximately 30% of selected colon cancer cell lines have been shown to express high-affinity binding sites for GRP [6, 36, 37, 42]. Bombesinlike peptides increase proliferation and DNA synthesis in human colon cancer cells. Bombesin dose-dependently stimulates the invasive capacity and migration of colon carcinoma cells [6]. This effect can be fully reversed by specific bombesin/GRP receptor antagonists. Furthermore, the inhibition of colon cancer growth by specific bombesin antagonists has been shown in nude mice. Recent data suggest that bombesin expression could be a useful marker for aggressive colorectal cancers. Seretis and coworkers demonstrated an increased bombesin expression in colorectal cancers; this was associated with a poor histological grade and with liver metastases [37]. These data are in line with the observation that GRP receptor mRNA expression in colon cancer was related to tumor dedifferentiation and lymphatic vessel invasion [36]. In contrast, GRP receptor co-expression did not adversely affect the outcome of patients with colon cancer [6].

Protective Role of GRP In several animal models, bombesin has been shown to confer protective effects in the stomach and gut after experimental injury. This effect has been observed in ethanol-induced gastric lesions, in acetic acid–induced gastric ulcers, in methotrexate-induced enterocolitis, in trinitrobenzene sulfonic acid–induced colonic damage, and in burn-induced gut injury [23]. In these models, bombesin dose-dependently improved the healing of gastric or intestinal mucosa. The mechanisms by which bombesin induces mucosal protection are unclear, but they might involve the release of endogenous gastrin, enhancement of the immune system, or stimulation of endogenous prostaglandins. Under physiological conditions, eating stimulates the release of gut hormones, which help to maintain mucosal integrity. In critically ill patients without enteral nutritition, mucosal atrophy of the small intestine may develop. In these patients, the use of trophic peptides including GRP could be of clinical relevance. The administration of trophic gastrointestinal hormones

could also be useful in patients with short bowel syndrome. However, clinical data supporting a role for GRP in these pathological situations are lacking.

GRP Antagonists as Potential Acid Blockers The potential role of GRP receptor antagonists as gastric acid blockers has recently been discussed in detail [23]. In summary, only limited data are available investigating the effects of GRP antagonists on ulcer healing. It is currently not clear whether GRP antagonists can be developed into clinically useful drugs. The potential therapeutic application of these agents is limited not only by their short plasma half-life but also by the inhibition of other important gastrointestinal functions (e.g., gallbladder contraction, pancreatic secretion, and gastrointestinal motility).

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cancer and binding of bombesin to its receptors. J Cancer Res Clin Oncol 1994;120:519–28. Reeve JR Jr, Cuttitta F, Vigna SR, Shively JE, Walsh JH. Processing of mammalian preprogastrin-releasing peptide. Ann NY Acad Sci 1988;547:21–9. Reeve JR, Walsh JH, Chew P, Clark B, Hawke D, Shively JE. Amino acid sequences of three bombesin-like peptides from canine intestine extracts. J Biol Chem 1983;258:5582–8. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 2003;24(4):389–427. Rettenbacher M, Reubi JC. Localization and characterization of neuropeptide receptors in human colon. Naunyn Schmiedebergs Arch Pharmacol 2001;364:291–304. Saurin JC, Rouault JP, Abello J, Berger F, Remy L, Chayvialle JA. High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer. Eur J Cancer 1999;35:125 –32. Seretis E, Gavrill A, Agnantis N, Golematis V, VoloudakisBaltatzis IE. Comparative study of serotonin and bombesin in adenocarcinomas and neuroendocrine tumors of the colon. Ultrastruct Pathol 2001;25:445–54. Spindel ER, Chin WW, Price J, Rees LH, Besser GM, Habener JF. Cloning and characterization of cDNAs encoding human gastrin-releasing peptide. Proc Natl Acad Sci USA 1984;81:5699– 703. Spindel ER, Giladi E, Brehm P, Goodman RH, Segerson TP. Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin releasing peptide receptor. Mol Endocrinol 1990;4:1956 –63. Strader AD, Woods SC. Gastrointestinal hormones and food intake. Gastroenterology 2005 Jan;128(1):175–91. Sugano K, Park J, Soll AH, Yamada T. Stimulation of gastrin release by bombesin and canine gastrin-releasing peptides. Studies with isolated canine G cells in primary culture. J Clin Invest 1987 Mar;79(3):935–42. Sunday ME, Kaplan LM, Motoyama E, Chin WW, Spindel ER. Gastrin-releasing peptide (mamalian bombesin) gene expression in health and disease. Lab Invest 1988;59:5–24. Szepeshazi K, Schally AV, Cai RZ, Radulovic S, Milovanovic S, Szoke B. Inhibitory effect of bombesin/gastrin-releasing peptide antagonist RC-3095 and high dose of somatostatin analogue RC-160 on nitrosamine-induced pancreatic cancers in hamsters. Cancer Res 1991;51:5980–6. ter Beek WP, Muller ES, Van Hogezand RA, Biemond I, Lamers CB. Gastrin releasing peptide receptor expression is decreased in patients with Crohn’s disease but not in ulcerative colitis. J Clin Pathol 2004 Oct;57(10):1047–51. Thomas RP, Hellmich MR, Townsend CM Jr, Evers BM. Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocr Rev 2003;24(5):571–99. Vigna SR, Giraud AS, Soll AH, Walsh JH, Mantyh PW. Bombesin receptors on gastrin cells. Ann NY Acad Sci 1988;547:131–7. Wada E, Way J, Shapira H, Kusano K, Lebacq-Verheyden AM, Coy D, et al. cDNA cloning, characterization, and brain regionspecific expression of a neuromedin-B-preferring bombesin receptor. Neuron 1991;6:421–30. Walsh JH, Kovacs TO, Maxwell V, and Cuttitta F. Bombesin-like peptides as regulators of gastric function Ann NY Acad Sci 1988;547:217–224. Walsh JH, Wong HC, Dockray GJ. Bombesin-like peptides in mammals. Fed Proc 1979;38:2315–9. Zadina JE, Banks WA, Kastin AJ. Central nervous system effects of peptides, 1980–1985: A cross-listing of peptides and their central actions from the first six years of the journal Peptides. Peptides 1986;7(3):497–537.

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145 Glucagonlike Peptides 1 and 2, Enteroglucagon, Glicentin, and Oxyntomodulin JENS JUUL HOLST

ABSTRACT

glucagon plus eight additional amino acids at the Cterminus). Collectively, the two molecules were designated enteroglucagon. At that time, it was known that proglucagon, the biosynthetic precursor of glucagons was a rather large molecule, and it was hypothesized that glicentin constituted part of proglucagon [11]. In agreement with this, the N-terminal fragment corresponding to amino acid residues 1–30 of glicentin, designated glicentin-related pancreatic polypeptide (GRPP), was also isolated from the pancreas and demonstrated to be secreted in parallel with glucagon. Proglucagon cDNA was first cloned from anglerfish islets and was demonstrated to encode a glucagon-containing glicentinlike peptide plus an additional glucagonlike peptide that showed strong sequence homology with (anglerfish) glucagon. On cloning the mammalian and human proglucagon genes, it was clear that they encode yet another glucagonlike sequence, glucagonlike peptide 2 (GLP-2) [1]. (Actually, subsequent studies showed that the piscine proglucagon gene also contains the GLP-2 encoding sequence, but this is removed by differential splicing of the mRNA precursor, when the gene is expressed in the pancreas.) Further studies revealed that (1) only a single gene encodes proglucagon; (2) identical mRNAs are formed in mammalian species; (3) the gene is expressed in the gut, pancreas, and brain; and (4) the single identical precursor, proglucagon, is processed differentially in the different tissues so that different products are secreted from the different organs [14]. Thus, in the pancreas, proglucagon (PG), consisting of 160 amino acids, is cleaved to produce GRPP (PG1–30), glucagon (PG33–61), and major proglucagon fragment, corresponding to PG72– 158. The three molecules are secreted in parallel and in equimolar amounts. In the gut, the products are glicentin (PG1–69, part of which is cleaved further to

The glucagon gene is expressed not only in the pancreas but also in endocrine cells of the gut and in the brain stem. Here the precursor, proglucagon, gives rise to the peptides glicentin, oxyntomodulin, and glucagonlike peptides 1 and 2 (GLP-1 and -2). Whereas the physiological role of the former is unclear, the GLPs appear to be important hormones, GLP-1 as an incretin hormone stimulating insulin release and as an enterogastrone inhibiting appetite and gastrointestinal secretion and motility and GLP-2 as an intestinotrophic factor regulating gut mucosal growth. GLP-1 has shown great promise as a therapeutic for type 2 diabetes, and GLP-2 is currently being investigated for treatment of intestinal failure.

DISCOVERY AND STRUCTURE OF THE GENE It was already suspected in 1948, based on bioassay studies, that glucagon is produced in the gastrointestinal mucosa, and subsequent radioimmunoassay studies confirmed that a number of endocrine cells produced substances with glucagonlike immunoreactivity. Eventually, two of these substances were isolated and their structure elucidated [11]. The immunoreactivity was due to the presence in both peptides of the entire glucagon sequence of 29 amino acids. One was glicentin (from GLI for “glucagonlike immunoreactivity” and centin because the peptide was originally thought to contain 100 amino acids), which consists of 69 amino acids of which residues 33–61 constitute the glucagon sequence. The other, oxyntomodulin, is a fragment of glicentin, corresponding to amino acids 33–69 (i.e., Handbook of Biologically Active Peptides

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1058 / Chapter 145 produce GRPP (PG1–30) and oxyntomodulin (PG33– 69)) and each of the two glucagonlike peptides plus the so-called intervening peptide 2 (corresponding to PG111–123). By isolation and chemical analysis, human GLP-1 and GLP-2 were shown to correspond to PG78– 106 amide (residue 107 being lost in the amidation step) and PG126–158, respectively. Thus, the structure of GLP-1 predicted from the cDNA sequence and corresponding to PG72–107 differed from that of the fully processed peptide by being amidated and by the fact that the natural peptide is formed by cleavage at the monobasic site PG77 rather than the dibasic site at PG70–71. This cleavage pattern is catalyzed by the prohormone convertase PC1/3 (whereas the pancreatic pattern results from the activity of PC2). Small amounts of the N-terminally extended form, PG72–106 amide may be produced in the pancreas, but most investigators have found this molecule to be inactive. In contrast, the fully processed form, which today is designated

GLP-1(7–36) amide or simply GLP-1, was isolated on the basis of its potent insulinotropic activity [15]. The processing pattern in the brain stem resembles that in the gut. The processing is shown in Fig. 1.

EXPRESSION OF THE GENE The single proglucagon gene is expressed in the endocrine alpha-cells of the pancreatic islets; in a certain group of cells in the nucleus of the solitary tract of the brain stem projecting particularly to the hypothalamus (the arcuate and paraventricular nuclei); and in the L cells, open-type endocrine cells of the gastrointestinal mucosa. In some species (dogs, cats, and rats), there are A-like cells in the gastric mucosa that may produce fully processed glucagon (and this probably explains the finding of glucagonlike bioactivity in stomach extracts in 1948), but such cells are rare in

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GLICENTIN FIGURE 1. Proglucagon processing. Schematic representation of the differential processing of proglucagon as it occurs in the pancreatic islets and in the intestinal mucosa in humans. A. The vertical lines in proglucagon (PG) indicate the positions of the cleavage sites (basic amino acids). GRPP, glicentin-related pancreatic peptide; IP-1, intervening peptide 1; IP-2, intervening peptide-2. B. Note that in the pancreas, the main products are glucagon itself and GRPP (inactive) and major proglucagon fragment (inactive). Small amounts of N-terminally extended GLP-1 (inactive) may also be formed. C. In the gut the main products are glicentin, in which the glucagon sequence is buried (whereby glucagon-like bioactivity is lost); smaller amounts of oxyntomodulin and the same inactive N-terminal fragment as in the pancreas; GLP-1, the majority of which is amidated whereas the rest is extended by one glycine residue; and GLP-2, which functions as a growth factor for the gut. Thus, although the precursor is the same, the differential processing gives rise to three biologically active but completely different products in the two tissues, while terminal extensions inactivate the “other” active products.

Glucagonlike Peptides 1 and 2, Enteroglucagon, Glicentin, and Oxyntomodulin / humans (and pigs). In humans, the L cells are lacking from the duodenum, but appear with increasing frequency from the proximal jejunum toward the ileum. A large number of L cells are also found in the colon, particularly in the cecum and the rectum. Their function in the colon is unknown. The L cells have microvilli protruding into the intestinal lumen, and because the cells are stimulated to secrete their products—glicentin, oxyntomodulin (enteroglucagon), and GLP-1 and -2 (again, in parallel and in equimolar amounts)—by the luminal presence of nutrients, it is assumed that molecules in the villous membranes react to these and activate the secretory machinery [9]. Thus, in an L-cell-like cell line, glucose may activate GLP-1 secretion both by enhancing glucose metabolism in a KATP-channel-dependent manner and by transporting via the sodium-dependent glucose transporter, SGLT-1 [7]. However, lipids and protein also stimulate L-cell secretion. Because of the presence of L cells in the upper jejunum, secretion is increased rapidly (within 10–15 minutes on meal ingestion), and, in agreement with the increasing density of L cells distally, larger meals cause larger responses. Likewise, the responses correlate strongly with the gastric emptying rate. Hormonal mechanisms do not seem to participate, at least in pigs, where this has been studied in detail, and although secretion may be influenced by intramural enteric nervous activity, efferent vagal fibers do not stimulate secretion. The sympathetic innervation may inhibit secretion, and GLP-1 itself (but not the other proglucagon products) powerfully inhibits L-cell secretion, presumably by the activation of neighboring D cells, releasing somatostatin-28, which acts on the L cells via sstr five receptors.

ACTIVE CONFORMATION AND METABOLISM Each of the processed products from the pancreas and gut may be found in the circulation, where their concentrations reflect their rates of secretion and elimination. The organ mainly responsible for the elimination is the kidney, which effectively extracts the biologically active products (up to 70% for GLP-1). Thus, the half-lives of oxyntomodulin, glucagon, and GLP-2 are ∼6–7 min, whereas that of glicentin is ∼20 min. No details are known about metabolism of glicentin and oxyntomodulin in the circulation, but glucagon is subject to some degradation by neutral endopeptidase 24.11. GLP-1 and GLP-2 (but not glucagon) are both substrates for the ubiquitous enzyme, dipeptidyl peptidase IV (DPP-IV), which cleaves off the two N-terminal residues (His-Ala in both peptides) whereby the molecules are rendered inactive [3]. This results in a half-life

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for GLP-2 of ∼7 min, as mentioned, whereas that of the metabolite, GLP-2 3–33, is ∼27 min. GLP-1 is more extensively cleaved by DPP-IV, leaving the peptide with an apparent plasma half-life of 1.5–2 min and a clearance of 6–10 liters of plasma per minute, greatly exceeding cardiac output. This means that the peptide is degraded rapidly in the circulation and that a steady state is not reached. In fact, the peptide is degraded already as it leaves the endocrine organ (i.e., the gut). GLP-1 is stored in the L cells in the intact form, but approximately 75% is cleaved to GLP-1 9–36 amide as in enters the venous blood draining the gut. Because DPP-IV is localized to the endothelial surface of the capillaries, it is thought that the intact molecule, on exocytosis from the L cells, diffuses across the basal lamina and into the lamina propria, where it is taken up by a capillary and degraded [8]. This means that only approximately 25% reaches the portal circulation in the intact form. In the liver, half of the arriving GLP-1 is degraded further, so only 10–15% of what was secreted makes it to the systemic circulation and thereby the pancreatic islets. The metabolite, which has a half-life ∼4 min, is eliminated mainly in the kidneys. Also, GLP-1 may be degraded by neutral endopeptidase, although less extensively.

RECEPTOR EXPRESSION The glucagon receptor is dealt with elsewhere. In agreement with its structure, oxyntomodulin may activate the glucagon receptor, but its potency is only approximately 1/50 of that of glucagon [17]. GLP-1 may also interact with the glucagon receptor but with only about 1/100 of the potency of glucagon. A specific receptor for oxyntomodulin was hypothesized to exist, but it now appears that oxyntomodulin may also interact with the GLP-1 receptor but less potently than GLP-1. Glicentin has no known specific receptor and apparently does not interact with any of the other receptors of the family. The GLP-1 receptor was cloned by expression cloning [23] and is related to the glucagon receptor, to receptors for GLP-2, and to receptors for the other members of the glucagon-secretin family of peptides [17]. It is a class 2 G-protein-coupled receptor with seven transmembrane segments. Like the other receptors of the family, it appears to couple preferentially to adenylate cyclase. The receptor is expressed in several tissues, including the beta- and delta-cells of the pancreatic islets. It has been reported that 20% of the alpha-cells may also express the GLP-1 receptor. It is also expressed in the gastric mucosa, perhaps particularly by the parietal cells, and in the lungs where type II pneumocytes have a dense expression. Hepatocytes

1060 / Chapter 145 do not express GLP-1 receptors, but there is evidence that other cells, perhaps associated with the portal vessels, express the GLP-1 receptor. Recently, GLP-1-R mRNA has been demonstrated in cells of the nodose ganglion, suggesting that sensory vagal neurons may express the GLP-1 receptors. GLP-1 receptors have also been localized to the heart, where they may be responsible for some of the cardiovascular actions of GLP-1. Finally, numerous GLP-1 receptors are expressed in the brain, not only in the brain stem where the GLPproducing cells are found, but also in many other regions, notably the hypothalamus. These receptors are presumably the targets for GLP-1 produced in the neurons of the brain stem that project to the hypothalamus [16]. Receptors have also been localized in the subfornical organ and in the area postrema, circumventricular areas without a classical blood–brain barrier but with a tight ependymal barrier between the cerebrospinal fluid (CSF) and the rest of the brain. It has been demonstrated that GLP-1 injected into the peripheral circulation can access these restricted regions, suggesting that peripherally secreted GLP-1 may influence the central nervous system (CNS) via this pathway. The GLP-2 receptor was also cloned by expression cloning and was found to be highly homologous to the GLP-1 receptor [18]. It is expressed particularly in the small intestine, but there is some expression also in the colon and the stomach and in certain nuclei of the brain, particularly the dorsomedial nuclei of the hypothalamus. This receptor does not bind other members of the peptide family. Its exact localization in the gut is a matter of controversy. Initial reports described its expression by immunohistochemistry in endocrine mucosal cells, but subsequent careful studies have not confirmed this. Instead an expression in myenteric ganglia was found by use of immunohistochemistry as well as in situ hybridization, whereas reverse transcription polymerase chain reaction (RT-PCR) excluded expression in epithelial cells. A recent report described a clear expression by both immunohistochemistry and in situ hybridization in subepithelial myofibroblast of several species, including humans, and provided evidence that keratinocyte growth factor (KGF) produced by these cells could explain some of the growth effects of GLP-2.

BIOLOGICAL ACTIONS Glicentin has been reported to influence gastric acid secretion, and single reports on growth effects and other effects have been published, but most investigators have found the peptide to be inactive. Thus, in unpublished studies from the author’s laboratory, synthetic glicentin was without effects on gastric acid secre-

tion and insulin secretion in humans. Oxyntomodulin mimics most of the actions of glucagon, but is less potent and probably does not act as a glucagonlike agonist physiologically. Like glucagon, it inhibits gastric acid secretion but by an action that appears to be mediated by GLP-1 receptors (although their exact location is uncertain). However, it is doubtful whether the circulating concentrations of oxyntomodulin under normal circumstances reach sufficient levels to activate the receptors. Recently, intracerebrovascular administration of oxyntomodulin was shown to powerfully inhibit food intake in rats by actions that did not appear to be mediated via the GLP-1 receptor. It is possible that oxyntomodulin released from the fibers arising from the proglucagon-producing neurons in the nucleus of the solitary tract in parallel with GLP-1 (in agreement with the gutlike processing pattern of proglucagon in these cells) may contribute to the inhibitory effect on appetite and food intake apparently resulting from activation of these neurons, together with GLP-1 and GLP-2.

ACTIONS OF GLP-1 The most conspicuous effect of GLP-1 is the stimulation of glucose-induced insulin secretion [12]. Thus, GLP-1 is considered to be one of the incretin hormones, the gut hormones that increase insulin secretion on oral, as opposed to intravenous, administration of glucose. GLP-1 and GIP (gastric inhibitory polypeptide, also called glucose-dependent polypeptide) are thought to be responsible for the incretin effect, which may be responsible for as much as 70% of the insulin secreted after carbohydrate intake. Indeed, glucose tolerance and insulin secretion are impaired in mice with deletions of both the GLP-1 and the GIP receptor, and more so in animals with knockout of both receptors. However, GLP-1 has numerous other actions, and its physiologically most important role may be to act as an enterogastrone hormone, inhibiting gastrointestinal motility and secretion, reducing appetite and food intake, and enhancing peripheral deposition of already absorbed nutrients by stimulation of insulin secretion [10].

Effects on the Islets GLP-1 is one of the most potent insulin-releasing substances known. It is strongly insulinotrophic in mimicry experiments, and animal experiments involving an antagonist of the GLP-1 receptor have indicated that GLP-1 is responsible for a substantial part of the insulin response to oral glucose. Furthermore, experiments with the same antagonist in humans have sug-

Glucagonlike Peptides 1 and 2, Enteroglucagon, Glicentin, and Oxyntomodulin / gested that GLP-1 may be essential for normal glucose tolerance. As mentioned, mice with a targeted deletion of the GLP-1 receptor become glucose intolerant and may develop fasting hyperglycemia. GLP-1’s insulinotropic activity, which is strictly glucose dependent, is exerted via interaction with the GLP-1 receptor located on the cell membrane of the β-cells [17]. Binding of GLP-1 to the receptor causes activation—via a stimulatory G-protein—of adenylate cyclase, resulting in the formation of cAMP. The formation of cAMP leads to the activation of protein kinase A and the cAMP-regulated guanine nucleotide exchange factor II (cAMP-GEFII, also known as Epac2) and a plethora of events, including altered ion channel activity, intracellular calcium handling, and enhanced exocytosis of insulin containing granules [12]. GLP-1 stimulates all steps of insulin biosynthesis as well as insulin gene transcription, thereby providing continued and augmented supplies of insulin for secretion. The activation of pancreas duodenum homeobox 1 (PDX-1), a key regulator of islet growth and insulin gene transcription may be involved. In addition, GLP1 upregulates the genes for the cellular machinery involved in insulin secretion, such as the glucokinase and glucose transport protein 2 (GLUT-2) genes. GLP1 also has trophic effects on β-cells. Not only does it stimulate β-cell proliferation, it also enhances the differentiation of new β-cells from progenitor cells in the pancreatic duct epithelium. In addition, GLP-1 has been shown to be capable of inhibiting apoptosis of β-cells, including human β-cells. The complicated intracellular mechanisms whereby GLP-1 may exert these effects on the β-cells were reviewed recently [2]. GLP-1 also strongly inhibits glucagon secretion from the alpha-cells. The mechanism is still unclear, but may involve interaction with the alpha-cells directly (although in isolated alpha-cells secretion has been reported to be stimulated by GLP-1) or a paracrine action by neighboring D cells, whose secretion of somatostatin-14 is potently stimulated by GLP-1. Alternatively, it may be due to inhibitory effects of insulin or other β-cell products whose release is stimulated by GLP-1.

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of the reduced insulin secretion is the reduced gastric emptying of and reduced subsequent absorption of insulinotropic nutrients. Thus, the physiological role of GLP-1 may be to adjust the delivery of chyme to the digestive and absorptive capacity of the gut by retarding propulsion and digestion of the gastric contents [10]. Most recently, Schirra et al. [22] have probed the physiological actions of GLP-1 on gastric motility in humans using the GLP-1 receptor antagonist, exendin9– 39. The antagonist both increased fasting antral motility and meal-induced gastric and duodenal motility and decreased pyloric tone. This strongly suggests that endogenous GLP-1 is a physiological regulator of antroduodenal motility. The mechanism whereby GLP-1 exerts these actions appears to involve the nervous system. As already described, GLP-1 is degraded extensively in the gut before it reaches the systemic circulation. This has led to the hypothesis that GLP-1 must act locally in the lamina propria before it is degraded. Thus, on its way to the capillary, while still in the intact form, GLP-1 may interact with afferent sensory nerve fibers arising from the nodose ganglion, causing them to send impulses to the nucleus of the solitary tract and onward to the hypothalamus. It has been demonstrated that intraportal administration of GLP-1 causes increased impulse activity in the vagal trunks. These impulses may be reflexively transmitted to the pancreas. Studies employing ganglionic blockers have shown that the insulin response to intraportal administration of GLP-1 and glucose may be reduced to that elicited by glucose alone after ganglionic blockade. Thus, regarding the mechanism of GLP-1-stimulated insulin secretion under physiological circumstances, the neural pathway may be more important than the endocrine route. However, the concentration of intact GLP-1 does rise after meal intake and rises more the larger the meal is. Thus, it is possible that the endocrine route becomes more prominent after extensive L-cell stimulation. Also the inhibition of gastric motility and acid secretion and emptying seems to involve the regulation of efferent vagal activity via activation of vagal sensory afferents.

Effects on Appetite and Food Intake Effects on the Gastrointestinal Tract Further important effects of GLP-1 include the inhibition of gastrointestinal secretion and motility, notably gastric emptying [10]. Physiologically, it may be that these effects of GLP-1 are more important than the insulinotropic activity. Thus, when GLP-1 is infused intravenously during ingestion of a meal, the insulin responses are diminished dose-dependently rather than being enhanced. At the same time gastric emptying is being progressively retarded, so the explanation

Nutrients in the ileum are thought to have a satiating effect, curtailing food intake and also cause a release of GLP-1. Infusions of slightly supraphysiological amounts of GLP-1 significantly enhance satiety and reduce food intake in normal subjects [6]. The effect on food intake and satiety is preserved in obese subjects also as well as in obese subjects with type 2 diabetes. Furthermore, direct injections of GLP-1 into the cerebral ventricles also inhibit food intake. Here, the peptide presumably interacts with the cerebral GLP-1

1062 / Chapter 145 receptors previously described and likely to be targets for GLP-1 released from nerve fibers ascending from cell bodies in the nucleus of the solitary tract in the brain stem. The question arises whether these neurons are linked to meal-induced satiety. Rinaman et al. [21] analyzed the neurons of the nucleus of the brain stem that were activated by various procedures designed to model enteroceptive stress (lithium chloride administration, CCK injection, lipopolysaccharide) and observed c-fos expression in cell bodies that also stained for GLP-1, whereas neurons showing c-fos expression after meal ingestion were distinct from the GLP-1 neurons. In further experiments, they administered the GLP-1 receptor antagonist, exendin9–39, intracerebroventricularly to rats given lithium chloride as described previously and found that the antagonist could completely reverse the anorexigenic effect of systemic lithium chloride. Thus, it seems clear that GLP-1 from the brain stem functions as a mediator of the anorexic effects of enteroceptive stress, whereas its role in meal-induced satiety is less clear. However, the same group recently reported divergent results in mice, suggesting that differences between species may be of importance. It should be noted though that GLP-1 receptor knockout mice do not become obese, but this may reflect the redundancy of the appetite-regulating mechanisms rather than ineffectiveness of the signal.

Other Actions It has been known for some time that there are GLP1 receptors in the heart. A physiological function for these receptors was indicated in recent studies in mice lacking the GLP-1 receptor, which exhibit impaired left-ventricular contractility and diastolic functions as well as impaired responses to exogenous epinephrine. Recent studies in rats showed that GLP-1 protects the ischemic and reperfused myocardium in rats by mechanisms independent of insulin. These findings may have important clinical implications. Thus, Nikolaidis et al. studied patients treated with angioplasty after acute myocardial infarction but with postoperative leftventricular ejection fractions as low as 29%. In these patients, GLP-1 administration significantly improved the ejection fraction to 39% and improved both global and regional wall motion indices [19]. Recently, GLP-1 was reported to dramatically improve left-ventricular and systemic hemodynamics in dogs with induced dilated cardiomyopathy, and it was suggested that GLP1 may be a useful metabolic adjuvant in decompensated heart failure. Finally, GLP-1 was recently found to improve endothelial dysfunction in type 2 diabetic patients with coronary heart disease, again a finding with interesting therapeutic perspectives.

It has been reported that cerebral GLP-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons in rats, leading to downstream activation of cardiovascular responses. Furthermore, it has been suggested that catecholaminergic neurons in the area postrema expressing the GLP-1 receptor may link peripheral GLP-1 and central autonomic control sites that mediate the diverse neuroendocrine and autonomic actions of peripheral GLP-1. However, peripheral administration of GLP-1 in humans is not associated with changes in blood pressure or heart rate. Recent studies showed that intracerebroventricular GLP-1 administration was associated with improved learning in rats and also displayed neuroprotective effects, and GLP-1 has been proposed as a new therapeutic agent for neurodegenerative diseases, including Alzheimer’s disease [20]. The function of GLP-1 in the lungs is unclear, although it has been reported to increase the secretion of macromolecules from the neuroendocrine cells. The actions of GLP-2 remained elusive for some time until Drucker et al. [5] observed mucosal hypertrophy in mice carrying proglucagon-producing tumors with an intestinal processing pattern. In subsequent studies of the effects of injected proglucagon product, GLP-2 was found to be capable of producing a similar mucosal growth. GLP-2 presumably activates its receptor on either neuronal cells or subepithelial myofibroblast, which in turn produce growth factors that cause mucosal growth. GLP-2 enhances enterocyte proliferation as well as inhibiting enterocyte apoptosis and thereby produces a pronounced growth of the epithelium. In addition, it may increase the expression of nutrient transporters and thereby augment the absorptive capacity of the gut. GLP-2 is currently thought be an important regulatory factor for adaptive intestinal growth [4]. Recently, GLP-2 was also found to increase intestinal blood flow. The mechanism is unknown, but it may be related to an increased intestinal metabolic rate. GLP-2 may also act as an enterogastrone hormone. Thus, GLP-2 inhibits sham-feeding-induced acid secretion and may inhibit gastric emptying, but it is not as potent as GLP-1. Furthermore, as previously mentioned, it may act to inhibit appetite and food intake on release from brain-stem neurons projecting to the ventromedial nuclei of the hypothalamus. Finally, recent studies have suggested that GLP-2 may be one of possibly several intestinal signals responsible for meal-induced regulation of bone resorption. In studies in which bone resorption was monitored by measurements of cross-linked fragments of collagen-1, the secretion of GLP-1 and -2 was well correlated to the meal-induced inhibition of resorption, and on subcutaneous administration GLP-2 caused a similar reduction of bone resorption.

Glucagonlike Peptides 1 and 2, Enteroglucagon, Glicentin, and Oxyntomodulin /

PATHOPHYSIOLOGICAL IMPLICATIONS Disturbances in L-Cell Secretion The secretion of GLP-1 is clearly decreased in patients with type 2 diabetes, and it is likely that this decrease contributes to the severely impaired incretin effect seen in these patients and thereby also to their impaired insulin secretion [24]. Part of the reason why GLP-1 secretion is decreased may be the lower secretion often seen in obesity. In patients with morbid obesity, GLP-1 responses to meal ingestion may be almost eliminated. Because GLP-1 inhibits appetite, the impaired secretion may contribute to or aggravate the development of obesity. As already discussed, GLP-1 secretion is related to the gastric emptying rate, and in patients with accelerated gastric emptying GLP-1 secretion may be excessive. Several lines of evidence suggest that excessive GLP-1 secretion may be responsible for the occurrence of postprandial reactive hypoglycemia in such patients. Thus, accelerated gastric emptying causes high postprandial glucose concentrations coinciding with high GLP-1 concentrations, resulting in exaggerated insulin secretion, which in turn causes hypoglycemia. The inhibition of glucagon secretion may contribute. Tumors producing proglucagon products generally exhibit a pancreatic processing pattern, and the resulting glucagonoma syndrome is due to the excessive secretion of glucagon. However, in some cases, an intestinal processing pattern is observed and in these patients the most dramatic clinical symptoms (intestinal obstruction) are due to intestinal mucosal hyperplasia. This is likely to be a consequence of increased secretion of GLP-2.

Therapeutic Application of Proglugon-Derived Peptides The actions of GLP-1 have attracted great interest because almost all of them are considered to be beneficial in patients with type 2 diabetes [13]. Indeed, infusions of GLP-1 completely normalizes fasting blood glucose concentrations in virtually all patients with type 2 diabetes investigated. In clinical studies in which GLP-1 was administered by continuous subcutaneous infusion for 6 weeks, blood glucose levels were greatly reduced, blood lipids improved, body weight was reduced, and beta-cell secretory capacity and insulin sensitivity increased. Single subcutaneous injections, however, are ineffective because of the rapid degradation of the peptide by DPP-IV. In order to exploit the effects of GLP-1 receptor activation, several pharmaceutical companies have developed resistant analogs of GLP-1 or activators of the receptor. One of these is exendin-4, a peptide of 39 amino acids isolated from

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the salivary secretions of the lizard, Heloderma suspectum, which is 53% homologous with GLP-1 (with respect to the sequence of the first 30 amino acids) and is a full agonist of the GLP-1 receptor. This peptide is resistant to the actions of DPP-IV and is cleared by the kidneys by glomerular filtration, resulting in a plasma half-life of approximately 30 min. Single subcutaneous injections, therefore, result in a plasma exposure lasting 6–8 hours. Patients with type 2 diabetes have now been treated for more than 2 years with exendin-4 (designated exenatide in its synthetic form, and now registered as a drug for diabetes treatment under the name of Byetta by the Amylin Corporation and Eli Lilly in collaboration), and the most conspicuous effects are a sustained reduction of glycated hemoglobinA1c levels (a measure of the average blood glucose levels) to approximately 7%, a recommended target, and a continued average weight loss amounting to 8 kg (www. amylin.com). Other preparations currently under clinical development generally show similar effects, although not yet documented for a similar time span. An alternative approach is the inhibition of the enzyme DPP-IV. Unlike the GLP-1 agonists or analogs, DPP-IV inhibitors are small molecules that are orally available. DPP-IV inhibition increases the levels of intact GLP-1 and thereby amplifies the action of the endogenous hormone, presumably both with respect to insulin and glucagon secretion. DPP-IV inhibitors are currently in late-stage clinical development. One of them, Vildagliptin, developed by Novartis, has been given for 52 weeks to patients failing on their usual therapy with metformin, a biguanide. This resulted in reductions of hemoglobinA1c levels to approximately 7% (whereas levels increased in a placebo-treated control group), and meal-stimulated insulin secretion increased in the treated group but fell in the control group, suggesting a protective effects on beta-cell function of the inhibitor. The first inhibitors are expected to reach registration in 2006. Because of its intestinotrophic activities, GLP-2 is currently being investigated as a therapy for intestinal insufficiency. Because of its much slower metabolism compared with GLP-1, GLP-2 can be used in its natural form and has been demonstrated to enhance intestinal absorption and increase lean body weight in patients with short bowel syndrome in the course of a 5-week study with two daily subcutaneous injections. A DPP-IVresistant analog may be produced by the substitution of amino acid residue 2 (with Gly) and has been shown to have similar, but prolonged, effects. Studies in experimental animals have supported that GLP-2 may also be effective in the therapy of intestinal atrophy after parenteral nutrition and in intestinal failure after experimental enteritis and after radiation and chemotherapy. GLP-2 is also currently being investigated as a therapy

1064 / Chapter 145 for osteoporosis and was most recently demonstrated to strongly reduce the nightly increase in bone resorption in postmenopausal women.

References [1] Bell, G. I.; Sanchez-Pescador, R.; Laybourn, P. J.; Najarian, R. C. Exon duplication and divergence in the human preproglucagon gene. Nature 1983;304(5924):368–371. [2] Brubaker, P. L.; Drucker, D. J. Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology 2004 Jun; 145(6):2653–2659. [3] Deacon, C. F.; Johnsen, A. H.; Holst, J. J. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an Nterminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 1995;80(3):952–957. [4] Drucker, D. J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol 2003 Feb;17(2):161–171. [5] Drucker, D. J.; Erlich, P.; Asa, S. L.; Brubaker, P. L. Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA 1996 Jul;93(15):7911–7916. [6] Flint, A.; Raben, A.; Astrup, A.; Holst, J. J. Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998 Feb;101(3):515–520. [7] Gribble, F. M.; Williams, L.; Simpson, A. K.; Reimann, F. A novel glucose-sensing mechanism contributing to glucagon-like peptide-1 secretion from the GLUTag cell line. Diabetes 2003 May;52(5):1147–1154. [8] Hansen, L.; Deacon, C. F.; Orskov, C.; Holst, J. J. Glucagon-like peptide-1-(7–36)amide is transformed to glucagon-like peptide1-(9–36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine [In Process Citation]. Endocrinology 1999 Nov;140(11):5356–5363. [9] Holst, J. J. Enteroglucagon. Annu Rev Physiol 1997;59:257– 271. [10] Holst, J. J. Glucagon-like peptide 1(GLP-1): an intestinal hormone signalling nutritional abundance, with an unusual therapeutic potential. Trends Endocrinol Metab 1999;10(6): 229–234. [11] Holst, J. J. Gut glucagon, enteroglucagon, gut glucagonlike immunoreactivity, glicentin—current status. Gastroenterology 1983;84(6):1602–1613.

[12] Holst, J. J.; Gromada, J. Role of incretin hormones in the regulation of insulin secretion in diabetic and nondiabetic humans. Am J Physiol Endocrinol Metab 2004 Aug;287(2):E199–E206. [13] Holst, J. J.; Orskov, C. The incretin approach for diabetes treatment: modulation of islet hormone release by GLP-1 agonism. Diabetes 2004 Dec;53 Suppl 3:S197–S204. [14] Holst, J. J.; Örskov, C.; Hartmann, B.; Deacon, C. F. Posttranslational processing of proglucagon and postsecretory fate of proglucagon products. In: Fehmann, H. C.; Göke, B., Eds. The Insulinotropic Gut Hormone Glucagon-Like Peptide-1. Basel: Karger; 1997. 24–48. [15] Holst, J. J.; Orskov, C.; Nielsen, O. V.; Schwartz, T. W. Truncated glucagon-like peptide I, an insulin-releasing hormone from the distal gut. FEBS Lett 1987;211(2):169–174. [16] Larsen, P. J.; Tang-Christensen, M.; Holst, J. J.; Orskov, C. Distribution of glucagon-like peptide-1 and other preproglucagonderived peptides in the rat hypothalamus and brainstem. Neuroscience 1997 Mar;77(1):257–270. [17] Mayo, K. E.; Miller, L. J.; Bataille, D.; Dalle, S.; Goke, B.; Thorens, B.; Drucker, D. J. International Union of Pharmacology. XXXV. The glucagon receptor family. Pharmacol Rev 2003 Mar;55(1): 167–194. [18] Munroe, D. G.; Gupta, A. K.; Kooshesh, F.; Vyas, T. B.; Rizkalla, G.; Wang, H.; Demchyshyn, L.; Yang, Z. J.; Kamboj, R. K.; Chen, H.; McCallum, K.; Sumner-Smith, M.; Drucker, D. J.; Crivici, A. Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2. Proc Natl Acad Sci USA 1999 Feb;96(4):1569–1573. [19] Nikolaidis, L. A.; Mankad, S.; Sokos, G. G.; Miske, G.; Shah, A.; Elahi, D.; Shannon, R. P. Effects of glucagon-like peptide-1 in patients with acute myocardial infarction and left ventricular dysfunction after successful reperfusion. Circulation 2004 Mar;109(8):962–965. [20] Perry, T. A.; Greig, N. H. A new Alzheimer’s disease interventive strategy: GLP-1. Curr Drug Targets 2004 Aug;5(6):565–571. [21] Rinaman, L. Interoceptive stress activates glucagon-like peptide1 neurons that project to the hypothalamus. Am J Physiol 1999 Aug;277(2 Pt 2):R582–R590. [22] Schirra, J.; Goke, B. The physiological role of GLP-1 in human: incretin, ileal brake or more? Regul Pept 2005 Jun;128(2): 109–115. [23] Thorens, B. Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci USA 1992 Sep;89(18):8641–8645. [24] Vilsboll, T.; Holst, J. J. Incretins, insulin secretion and type 2 diabetes mellitus. Diabetologia 2004 Mar;47(3):357–366.

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other hand, because the question of the role of des-acyl ghrelin is receiving much attention recently, it may be noted that des-acyl ghrelin is in fact MTLRP. That motilin and ghrelin form a new family of peptides is further confirmed by the structural relationships between their receptors. There is even an interesting historic relation because the orphan receptor G-proteincoupled receptor (GPCR38), cloned based on structural similarities with the ghrelin receptor, was identified as the motilin receptor (see Chapter 148 in this section on motilin). If MTLRP (i.e., des-acyl ghrelin) also has biological activity, then it may be assumed to act via a third receptor related to the ghrelin and motilin receptors.

Ghrelin is the endogenous ligand for the growth hormone secretagog receptor (GHS-R), but the peptide is most abundant in the stomach, where it is produced in endocrine cells of the oxyntic mucosa. Ghrelin forms with motilin a new gut peptide family. Ghrelin seems to affect gastric acid secretion, has gastroprotective effects, and increases gastrointestinal motility—ghrelin induces the migrating motor complex and accelerates gastric emptying. These effects are due to the activation of central and peripheral ghrelin receptors. A better understanding of the physiology of ghrelin may lead to new therapeutic approaches in the treatment of hypomotility syndromes.

STRUCTURE OF THE PRECURSOR mRNA/GENE ALTERNATIVE DISCOVERIES OF GHRELIN The structure of the ghrelin precursor has already been described in the Brain Peptides Section. Note that the overall arrangement and length of the ghrelin precursor (signal peptide, bio-active peptide, C-terminal extension) corresponds to the arrangement and length of the motilin precursor and that the amino acid similarities relate to the complete precursor (Fig. 2). An unusual feature of the ghrelin gene is that the bioactive peptide is encoded by two exons. Interestingly, this is also the case for motilin. Moreover, in ghrelin an alternative splicing at the end of intron 2 may give rise to des-Gln14-ghrelin. Actually, this also corresponds to the boundary between the two exons in motilin, and in des-Gln14-ghrelin the number of homologous residues increases to eight (Fig. 1). As mentioned in the Brain Peptides Section, the ratio between ghrelin and des-Gln14-ghrelin may differ from species to species, and des-Gln14-ghrelin may not be produced in humans.

The discovery of ghrelin has been described in the Brain Peptides section of this book. However in relation to ghrelin’s effects on the gastrointestinal system, it is of interest to note that 7 months after the discovery of ghrelin, another group described a peptide with the same sequence but gave it the name motilin-related peptide (MTLRP) because the authors noted a similarity to the sequence of motilin [17]. This similarity (Fig. 1) had escaped the attention of Kojima and collaborators [8]. However, both groups overlooked the fact that their sequence had already been submitted under the name motilin homolog as part of a patent application in 1998 [14]. Still, because the sequences of the MTLRP and of the motilin homolog were derived from cDNA sequences, they did not include the octanoylation of serine 3, which is an important determinant of the biological activity of ghrelin. Therefore, the true discovery of ghrelin was indeed made by Kojima et al. [8]. On the Handbook of Biologically Active Peptides

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FIGURE 1. Homology between the amino acid sequence of ghrelin and motilin. A. Alignment of part of the ghrelin sequence with the complete motilin sequence showing six homologous residues (bold). B. In des-Gln14-ghrelin, which is produced from an alternative splice variant, the number increases to eight.

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FIGURE 2. Schematic representation of the ghrelin and motilin precursors. The sequence homologies between the corresponding parts of both proteins are indicated; identities and similarities are printed in plain and bold characters, respectively. From [17]. MAP, motilin associated peptide; MTLRPAP, motilin-related-peptide-associated peptide.

DISTRIBUTION IN THE GASTROINTESTINAL TRACT As detailed in the paper describing the discovery of ghrelin, the main source of ghrelin was the stomach. This was confirmed subsequently and applies to the peptide as well as to the mRNA. The drop in ghrelin plasma levels following gastrectomy and small bowel resection suggests that approximately two-thirds of the ghrelin circulating in the plasma is derived from the stomach and the remaining one-third from the small intestine [6, 9].

STRUCTURE–ACTIVITY RELATIONS OF GHRELIN AND OF THE GHRELIN RECEPTOR The amino acid sequence of ghrelin is well conserved, especially in the N-terminal part. For example, in seven mammalian species (human, monkey, cat, dog, pig, sheep, cow) the first 10 residues are identical. This

suggests that the biological activity is determined by the N-terminus. In motilin, the N-terminus is also well conserved, although to a lesser extent than in ghrelin; in the same seven species, only the first six residues are identical in motilin. However, there is a paradox here. As already mentioned, a unique and crucially important feature of ghrelin is the octanoylation of serine-3. If the octanoyl group is removed, the potency drops dramatically: more than 2300-fold. But if the side chains of the other well-conserved amino acids are removed, the effect on potency is quite small and the peptide can be shortened to an N-terminal fragment of only four residues without appreciable loss of biological activity [9, 19]. It has therefore been suggested that the evolutionary pressure may be due to the fact that the enzyme responsible for the octanoylation of serine-3 requires a large recognition site [19], but as yet this enzyme has not been identified. In the ghrelin receptor, the activation domain involves transmembrane domain 2 (TM2) and TM3, and this domain has been remarkably conserved during

Ghrelin more than 400 million years of evolution. Indeed, it is already present in related receptors in the pufferfish. In particular, glutamic acid residue 124 in TM2 of the GHS-R has a critical interaction with a positively charged nitrogen of ghrelin agonists [4]. In the motilin receptor, the corresponding residue may play a similar role [20], suggesting the possibility of cross-reactivity. A peculiar feature of the ghrelin receptor is its high level of constitutional activity. It shares this property with two other members of the ghrelin receptor family, the neurotensin receptor 2 and the orphan receptor GPR39. In contrast, the motilin receptor does not have this property, which seems to be related to an aromatic cluster in the inner face of the extracellular ends of TMs VI and VII [7].

BIOLOGICAL ACTION ON THE GASTROINTESTINAL TRACT Gastric Acid Secretion Because ghrelin is present in the oxyntic mucosa, a paracrine or endocrine effect on the cells involved in the regulation of gastric acid secretion—enterochromaffinlike (ECL) cells, parietal cells, and gastrin (G) cells—and present in the wall of the stomach may be considered. However, studies evaluating the effect of exogenous ghrelin on gastric secretion are equivocal; stimulation, inhibition, and lack of effect have been reported [12]. Still, the studies seem to agree in one respect: The previously mentioned hypothesis can be discarded. Thus, in isolated cell cultures ghrelin had no effect on G cells or ECL cells, and in the in vivo studies in which an effect was found it seemed to require a vagal or central pathway. Indeed, the stimulation of gastric acid secretion after intravenous ghrelin was abolished by vagotomy, and the inhibitory effect seen after intracerebroventricular (ICV) administration was abolished after cysteamine, suggesting the involvement of central somatotinergic pathways [12]. More studies are needed to determine the precise role of ghrelin in the regulation of gastric acid secretion.

Gastroprotection The first report of a role of ghrelin in gastroprotection was obtained in a model of ethanol-induced gastric ulcers in rats [15]. It was confirmed in the same model and was also found in models of stress-induced ulcers. The protective effect has been observed as well after intraperitoneal (IP), subcutaneous (SC), or ICV administration, suggesting that the mechanism can be mediated via peripheral and central ghrelin receptors [10, 12].

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Ghrelin released in the stomach wall may affect neighboring cells or reach them via the bloodstream. Thus, it has been shown that ghrelin acts directly on enteric neurons to produce nitric oxide (NO) and blockade of NO synthesis blocks the protective effect. The protective effect involves prostaglandin synthesis because it is reduced by indomethacin or the selective COX-2 inhibitor rofecoxib. In agreement with these observations, ghrelin does not offer protection in indomethacininduced ulcers. Immune cells may also be involved because ghrelin reduces the expression of the proinflammatory cytokine tumor necrosis factor (TNF)-α [10]. Exogenous ghrelin, or ghrelin produced in the stomach, may also reach the central nervous system via the bloodstream, see Chapter 203 on ingestive peptides and the blood-brain barrier in the Blood-Brain Barrier Peptides section of this Handbook, or the ghrelin signal may be conveyed to the brain via vagal afferents. However, although there seems to be agreement that sensory denervation blocks the effect of ghrelin, vagotomy blocked the effect in one study [1] but was without effect in another [15]. The effect of exogenous ghrelin is blocked by a ghrelin antagonist, but the role of endogenous ghrelin has not been investigated, although it has been noted that mucosal expression of ghrelin and ghrelin plasma levels is increased in stress- and ethanol-induced ulcers [12]. We may also hypothesize that ghrelin knockout (KO) mice will develop ulcers more easily, but these studies remain to be done. An important factor of ulcer formation in humans is Helicobacter pylori infection. There are studies indicating that H. pylori infection decreases the expression of ghrelin, the number of ghrelin-producing cells, and plasma ghrelin levels, factors that may contribute to the development of ulcers [12]. Following eradication, one study noted a rise of plasma ghrelin, but this was not confirmed in another study based on a larger group of patients. The discrepancy may be explained by the different topography (antrum or fundus, where ghrelin cells are located) and duration of the infection (reversible damage after a short infection, irreversible atrophy after long infection) in both studies [2]. The issue may be important because it has been suggested that eradication of H. pylori, via its effect on plasma ghrelin, may contribute to the development of obesity [11].

Gastrointestinal Motility Apparently ghrelin is related to motilin not only structurally but also functionally. Thus, the two bestcharacterized effects of motilin on the gastrointestinal tract, induction of the migrating motor complex and acceleration of gastric emptying, have now also been described for ghrelin. In fed rats, ghrelin, given intra-

1068 / Chapter 146 venously (IV) or ICV, shortens the migrating motor complex (MMC) cycle [5], and in healthy fasting volunteers IV infusion of ghrelin induces a premature phase III originating in the stomach. Ghrelin and the ghrelin agonist GHRP-6, accelerate gastric emptying in rats, mice, and dogs [12]. In humans, IV administration of ghrelin accelerates the gastric emptying of liquids in patients with gastroparesis [16]. The physiological importance of these findings may be questioned because in ghrelin KO mice the rate of gastric emptying is not affected by the absence of ghrelin [3]. Because using rather large doses is required, crossreactivity with the motilin receptor could be considered, in view of the role of the TM2 domain in both receptors. However, in the classical in vitro model for motilin-induced contractions, the rabbit duodenum, ghrelin is without effect. On the other hand, in rat smooth muscle preparations, the result is the opposite—motilin is without effect and ghrelin enhances cholinergic responses [12]. In fact, recent results show that the motilin and the motilin receptor genes are nonfunctional in rat and mouse, ruling out any interaction of ghrelin with the motilin receptor and suggesting that ghrelin has taken over the function of motilin in these species [13]. Finally, we have found, in collaboration with the laboratory of P. Robberecht (ULB, Brussels), that, in Chinese hamster ovary (CHO)-K1 cells expressing the human motilin receptor and in CHO-K1 cells expressing the ghrelin receptor, there is only very weak cross-reactivity of ghrelin, GHSs, motilin, and motilides (unpublished data). As for gastric acid secretion and gastroprotection, motor effects have been observed after central as well as after peripheral administration of ghrelin. Elegant experiments using various routes of administration of ghrelin and a ghrelin antagonist led to the conclusion that the motor effects depend on the activation of central receptors and efferent vagal pathways [5]. Yet these results should be interpreted with caution because the antagonist used in this study is rather weak and may cross-react with other receptors. The involvement of vagal afferents should also be considered because vagotomy blocks the effect of peripheral administration of ghrelin and there is evidence for GHS-R on vagal afferents [6, 9]. Alternatively, ghrelin may activate the enteric nervous system. Recent studies have localized the ghrelin receptor in the myenteric plexus of guinea pig ileum and in human and rat stomach and colon. In vitro studies provide evidence for the activation of cholinergic pathways in rat and mouse preparations [12]. It is possible that the enteric nervous system is activated when other pathways become nonfunctional. Thus, in control rats a ghrelin antagonist had no effect on fasted motor activ-

ity, but when ghrelin was given to vagotomized rats it completely blocked the MMC [5].

PATHOPHYSIOLOGICAL IMPLICATIONS AND THERAPEUTIC POTENTIAL Little is known about the mechanism controlling the release of ghrelin, but conditions have been described in which plasma ghrelin is increased or decreased [6, 9]. The most important factor seems to be the nutritional state, but how fasting increases ghrelin secretion and how a meal reduces it are unclear. Blood glucose may play a role and the contact of food components with the stomach wall may as well. Increased plasma levels have also been described in anorexia, with decreasing body mass index, in renal insufficiency, following leptin administration, and after vagotomy and hypophysectomy. Plasma ghrelin is decreased postprandially, after gastrectomy and small bowel resection. Because of the effect of ghrelin on appetite and adiposity (see Brain Peptides and Ingestive Peptides sections), ghrelin may play a role in the development of obesity. Some aspects of the physiology of ghrelin may be taken into account in the treatment of obesity. Thus, diet-induced weight loss causes a rise in plasma ghrelin, apparently an adaptive response that constrains weight loss. Gastric bypass surgery suppresses ghrelin levels, and this may contribute to the success of this procedure in controlling weight [9]. The ghrelin receptor may serve as a therapeutic target for obesity drugs. Considering the high level of constitutional activity of the ghrelin receptor, not just antagonists but also inverse agonists may be considered. Also, because of this phenomenon, not just ghrelin levels but also changes in the expression of the ghrelin receptor should be studied in pathological conditions. It has been proposed that the increasing efforts to eradicate H. pylori may contribute to the development of obesity, thereby increasing the risk for developing reflux disease, which increases the risk of developing Barrett’s esophagus, which in its turn increases the risk of esophageal adenocarcinoma [11]. Before considering the plausibility of this sweeping hypothesis, the extent of the effect of H. pylori infection and eradication on plasma ghrelin levels should be clarified. The effects of ghrelin on gastric emptying may find application in the treatment of hypomotility syndromes. Indeed, it has been observed in gastroparesis patients, and animal studies showed that ghrelin reverses the delay of gastric emptying in the postoperative ileus in the rat [18], dog, and septic mice [12]. A major problem for the development of successful agonists is the wide spectrum of activity ghrelin, and it remains to be seen

Ghrelin whether agonists for a specific effect, in this case motility, can be developed. Interaction with endogenous ghrelin may also need to be considered, because agonists may be positive or negative modulators of ghrelin [7].

Acknowledgments The author’s research on motilin and ghrelin is supported by grants from the Belgian Ministry of Science (GOA 03/11 and IAP P5/20), the Flemish Foundation for Scientific Research (FWO grant number G.0144.04), and the Ministry of the Flemish Community (International Scientific and Technological Cooperation with the P.R. China grant BIL 01/13). The author gratefully acknowledges stimulating discussions on ghrelin and motilin with Dr. Pierre Poitras, author of the chapter on motilin in this section of the book and, despite a long-standing competition in research, also a long-standing friend.

References [1] Brzozowski T, Konturek PC, Konturek SJ, Kwiecien S, Drozdowicz D, Bielanski W, Pajdo R, Ptak A, Nikiforuk A, Pawlik WW, Hahn EG. Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage. Regul Pept 2004;120:39–51. [2] Cummings DE. Helicobacter pylori and ghrelin: Interrelated players in body-weight regulation? Am J Med 2004;117: 436–9. [3] De Smet B, Depoortere I, Moreaux B, Tack J, Moechars D, Coulie B, Bosmans JP, Peeters T. Gastric emptying and food intake in ghrelin knockout mice. Gastroenterology 2004;126: A-90. [4] Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund R, Underwood D, Tata JR, Dean DC, Tan CP, McKee KK, Woods JW, Patchett AA, Smith RG, Van der Ploeg LH. Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues. Mol Endocrinol 1998;12:137–45. [5] Fujino K, Inui A, Asakawa A, Kihara N, Fujimura M, Fujimiya M. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats. J Physiol 2003;550:227–40.

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[6] Ghigo E, Broglio F, Arvat E, Maccario M, Papotti M, Muccioli G. Ghrelin: More than a natural GH secretagogue and/or an orexigenic factor. Clin Endocrinol (Oxf) 2005;62:1–17. [7] Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW. Common structural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 2004;279:53806–17. [8] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656–60. [9] Kojima M, Kangawa K. Ghrelin: Structure and function. Physiol Rev 2005;85:495–522. [10] Konturek PC, Brzozowski T, Pajdo R, Nikiforuk A, Kwiecien S, Harsch I, Drozdowicz D, Hahn EG, Konturek SJ. Ghrelin—a new gastroprotective factor in gastric mucosa. J Physiol Pharmacol 2004;55:325–36. [11] Nwokolo CU, Freshwater DA, O’Hare P, Randeva HS. Plasma ghrelin following cure of Helicobacter pylori. Gut 2003;52: 637–40. [12] Peeters TL. Ghrelin: a new player in the regulation of gastrointestinal function. Gut 2005;in press. [13] Peeters TL, Aerssens J, De Smet B, Mitselos A, Thielemans L, Coulie B, Depoortere I. The mouse is a natural knock-out for motilin and for the motilin receptor. Functionally they have been replaced by ghrelin. Neurogastroenterol Motility 2004;16:687. [14] Sheppard P. Motilin homologs. WO 1998;98-42840 01.10.1998. [15] Sibilia V, Rindi G, Pagani F, Rapetti D, Locatelli V, Torsello A, Campanini N, Deghenghi R, Netti C. Ghrelin protects against ethanol-induced gastric ulcers in rats: Studies on the mechanisms of action. Endocrinology 2003;144:353–9. [16] Tack J, Depoortere I, Bisschops R, Verbeke K, Janssens J, Peeters T. Influence of ghrelin on gastric emptying and meal-releated symptoms in gastroparesis. Alimentary Pharmacol Ther 2005;in press. [17] Tomasetto C, Karam SM, Ribieras S, Masson R, Lefebvre O, Staub A, Alexander G, Chenard MP, Rio MC. Identification and characterization of a novel gastric peptide hormone: The motilin-related peptide. Gastroenterology 2000;119:395–405. [18] Trudel L, Tomasetto C, Rio MC, Bouin M, Plourde V, Eberling P, Poitras P. Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat. Am J Physiol Gastrointest Liver Physiol 2002;282:G948–52. [19] Van Craenenbroeck M, Gregoire F, De Neef P, Robberecht P, Perret J. Ala-scan of ghrelin (1–14): interaction with the recombinant human ghrelin receptor. Peptides 2004;25:959–65. [20] Xu L, Depoortere I, Vertongen P, Waelbroeck M, Robberecht P, Peeters TL. Motilin and erythromycin-A share a common binding site in the third transmembrane segment of the motilin receptor. Biochem Pharmacol 2005;in press.

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food intake and energy expenditure, but also participates in the regulation of the hypothalamo-pituitary axis, angiogenesis, the immune system and inflammatory response, cell proliferation and apoptosis, and nutrient absorption. This multifunctional role of leptin is consistent with the production of this hormone by various tissues and organs, including the stomach [2, 11]. This review focuses on the stomach-derived leptin and its implication in the physiology of the gastrointestinal tract.

The initial view that leptin, a 16-kDa protein-encoded by the ob gene, is an adipocyte-derived signal, has been modified by the identification of other producing tissues, such as the stomach. The discovery of stomachderived leptin, mainly secreted in the lumen, has initiated several investigations leading to the suggested clues: (1) gut leptin may act locally within the gastrointestinal tract to regulate intestinal functions such as nutrient absorption, (2) leptin may integrate mealrelated signals from the gut and thus participate in the short-term regulation of energy balance, and (3) leptin secretion may have pathophysiological implications.

THE STOMACH PRODUCES LEPTIN The stomach serves as a reservoir in which ingested food accumulates and undergoes chemical and enzymatic primodigestion. The gastric mucosa is composed of several types of secretory epithelial cells. Among these cells are endocrine cells secreting hormones, the mucous cells secreting alkaline mucus that protects the epithelium, parietal cells secreting gastric acid, and chief cells secreting pepsinogen, a proteolytic enzyme. The leptin-secreting cells were initially identified as pepsinogen-secreting chief cells in rodents and humans. Subsequent studies have detected leptin in the secretory granules of endocrine P cells in addition to chief cells [12]. Furthermore, ultrastructural studies showed that leptin protein is present along the rough endoplasmic reticulum–Golgi-granules secretory pathways both in chief and endocrine cells [10]. During development, leptin production in the stomach starts at the onset of suckling in neonatal rats and it markedly increases at the transition from liquidto solid-food intake [31, 34]. This observation underlies the physiological importance of stomach-secreted

INTRODUCTION Leptin, secreted primarily from the white adipose tissue [44], has been recognized as an adipostatic signal. Thus, leptin administration in rodents with genetic or diet-induced obesity decreased body weight and adiposity and improved metabolic control by central nervous system (CNS)-mediated mechanisms. The leptin receptor Ob-R, encoded by the db gene [40], is a member of the gp130 family of cytokine receptors. It occurs in several isoforms resulting from the alternative splicing of the db gene [25]. Only the long isoform, Ob-Rb, can activate the signal transducers and activators of transcription (STAT) pathways, whereas both Ob-Rb and one of the short isoforms (Ob-Ra) can transduce signals through insulin receptor substrates (IRSs) and mitogen-activated protein kinase (MAPK) pathways [39]. Leptin is now considered to be involved in a wide range of biological processes. Leptin not only regulates Handbook of Biologically Active Peptides

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1072 / Chapter 147 leptin and the potential role that it could play in the control of gut-derived regulation of food intake in neonates. Leptin secretion in the stomach is regulated by the nutritional status, acetylcholine-released by the vagus nerve, and intestinal hormones (i.e., cholecystokinin and secretin) (reviewed in [19]). In addition, gastric leptin shows diurnal variations influenced by food intake rhythms, and the changes occurring just before the beginning of the feeding period are opposite to those of the appetite-stimulating peptide ghrelin [35]. Gut-derived leptin can be distinguished from fatderived leptin through its rapidly increased secretion by a meal and its main secretion into the gastric lumen, suggesting different functions for these two pools of leptin.

GASTRIC LEPTIN IS A NEUROENDOCRINE MOLECULE FOR SATIETY One of the first questions raised by gastric leptin was whether it can act locally in the stomach to generate signals contributing to meal-induced termination of food intake, the vagus nerve conveying those signals to the CNS. This implies, however, the expression of leptin receptors on the vagus nerve. Interestingly, mRNA and protein for leptin receptors have been described in the rat and human nodose ganglion [6, 8]. Moreover, it was reported that the vagal afferent neurons possess functionally active leptin receptor proteins and that leptin may activate intracellular STAT3 in these neurons [8]. Moreover, leptin receptors have also been described on the intestinal vagal mechanoreceptors [18]. These vagal receptors are responsive to leptin directly delivered in the stomach, as evidenced by the increase of the tonic activity of gastric-related neurons in the nucleus tractus solitarii (NTS) [41, 43]. These results correlated well with those obtained from electrophysiological studies, identifying two types, 1 and 2, of leptinresponsive gastric vagal afferent fibers [42]. Interestingly, pretreatment of cholecystokinin-8 (CCK-8) fibers was required to increase the sensitivity for leptin of the type 2 fibers [42]. This clearly provides a basis for the reported leptin-CCK potentiating effect in the firing frequency of gastric vagal terminals [42], in neuronal activity in the NTS, on food intake [4, 42], and finally on body weight [27]. Altogether, the vagus nerve appears to be the primary target of leptin produced by and secreted into the stomach. This leptin acts from the luminal side via a neurocrine mode of action through the vagal leptin receptors. The molecular basis of this neurocrine action of leptin remains to be elucidated.

GASTRIC LEPTIN ENTERS THE INTESTINE Some of the stomach-derived leptin is not fully degraded by proteolysis, reaching the intestine in an active form to initiate biological functions. Thus, high amounts of free leptin and leptin bound to highmolecular-weight proteins are present in duodenal lumen in rats [20]. Such leptin-binding proteins were reported as the soluble leptin receptor in the blood and also identified as clusterin, also termed apolipoprotein J [3], a protein whose function has not yet been defined. Interestingly, in the rat colonic lumen, leptin can only be detected as free leptin (Guilmeau et al., unpublished results). Although the significance and the precise nature of these high-molecular-weight leptin-binding proteins in the intestinal juices needs further investigation, they may have physiological implications in terms of the bio-availability of leptin in the gut lumen. Notably, the amount of leptin in the duodenum was greater than that expected from the stomach, suggesting contributions from other leptinproducing sites. In addition to gastric secretions, the duodenum receives hepatobiliary and exocrine pancreatic secretions, and it has been reported that the liver also produces leptin [23]. Thus, the liver can contribute to the levels of leptin trafficking in the intestinal lumen by direct hepatic synthesis of leptin or by increasing the release of free leptin from the hepatic stores of the complexes of leptin bound to OB-Re [26]. More precise quantification of leptin and additional molecular studies are needed to ascertain the respective contribution of the various parts of the digestive system to leptin in the intestinal lumen. In addition to the presence of leptin in the gut lumen, leptin receptors were found at the apical side of the enterocytes all along the small and large intestine, which argues for the viewpoint that leptin, acting from the luminal side, affects biological functions of the intestinal epithelium [19].

LEPTIN REGULATES INTESTINAL ENDOCRINE SECRETIONS Previous studies have shown that CCK-1 receptor antagonists can prevent both peripheral leptin-induced inhibition of food intake [4] and the stimulation of pancreatic exocrine secretions [21]. In addition, it was shown that intraduodenal leptin also stimulates pancreatic enzyme secretion in a CCK-1 receptor–dependent manner, probably through activation of duodenopancreatic reflexes [30]. These results suggest that endogenously released CCK may be involved in those leptin actions. In fact, it was demonstrated that circulating leptin (mainly reflecting adipocyte leptin) and, more

Leptin and the Gastrointestinal Tract / 1073 important, duodenal leptin increase plasma CCK at levels comparable to those induced by feeding [20]. This action of duodenal leptin is probably relayed via a direct activation on the CCK-producing endocrine I cells. Finally, the leptin-induced release of CCK determines a positive feedback loop because CCK was reported to stimulate the release of gastric leptin [2]. It is important to note that CCK is one of the mealgenerated molecules that generate satiety signals that are conveyed by the viscero-sensitive vagal afferent neurons to the nucleus of the NTS and then in the hypothalamus. Thus, the leptin stimulation of duodenal CCK secretion suggests that, under physiological conditions, both peptides may potentiate their own actions by cross-stimulating their secretions. This is in accordance with the reported dampening of CCK or leptin inhibitory action on food intake when either peptide is absent or their receptors are functionally inactive [4]. Leptin was also reported to stimulate the secretion of glucagonlike peptide 1 (GLP-1), another satiety signal, in vitro in enteroendocrine L cells and in vivo in rodents [1], providing evidence for the existence of an adipoenteroendocrine axis involved in the regulation of nutrient homeostasis.

GUT LEPTIN AND INTESTINAL ABSORPTION OF NUTRIENTS The small intestinal lining is composed of functionally and morphologically polarized enterocytes that play a central role in the absorption of nutrients after digestion. In the intestine, the chyme undergoes hydrolysis by proteolytic enzymes from pancreatic, bile, and intestinal juices, pursuing the primodigestion started in the stomach. Nutrients thus degraded into smaller molecules cross the intestinal brush-border by active transport, passive diffusion, or facilitated processes. The arrival of the meal in the intestine stimulates the release of gastrointestinal hormones that control the absorption of nutrients and are also signals for induction of postprandial satiety. Thus, it is conceivable to postulate that gut leptin rapidly secreted in the lumen after a meal (by contrast to adipocyte leptin) is a key molecule controlling the intestinal absorption of nutrients.

Jejunal Absorption of Proteins Under physiological conditions, dietary proteins are degraded in a series of steps by hydrolytic enzymes originating from the stomach, pancreas, and small intestine. This results in a mixture of free amino acids and small peptides that is efficiently absorbed by ente-

rocytes. These small peptides are cleared from the intestinal lumen by the brush-border transporter PepT-1, which co-transports di- and tripeptides peptides with protons [17]. Luminal, but not basolateral, leptin increases the absorption of dipeptides through PepT-1 in the enterocytelike Caco2 cells in vitro [7]. These results were confirmed in vivo in rats in which direct administration of leptin in the jejunum (mimicking gastric leptin) rapidly increases absorption of dipeptides [7]. The mechanism of this short-term action involves increased recruitment of membrane PepT-1 molecules from an intracellular preformed pool to the apical membrane. From a physiological point of view, the facilitation of protein absorption through PepT-l activation by gut leptin is consistent with reports of a satiety effect of dietary proteins and is in line with the aminostatic hypothesis.

Jejunal Absorption of Fats A major function of intestinal cells is absorbing large amount of dietary lipids. After a digestive phase, the free fatty-acid (FA) lipolytic products are absorbed by the enterocytes, in which sequential events result in their packaging as chylomicrons. The formation and secretion of these intestinal lipoproteins are key steps in the transport of dietary fats. The assembly of triglyceride (TG)-rich lipoproteins within the enterocytes involves multiple pathways including (1) the uptake of FAs by several specific carriers, such as fatty-acid transporter (FAT) and its human homolog CD36; (2) their translocation from the brush-border membrane to the endoplasmic reticulum by intestinal and liver fatty-acid binding proteins (I- and L-FABPs); and (3) their esterification in TG and subsequent assembly with apolipoprotein (apoB, apoA-IV) to form lipoprotein particles. Leptin appears to play a role in the regulation of the synthesis of apolipoproteins. In fact, leptin administrated to fat-loaded ob/ob mice induced STAT5 DNA binding and reduced apolipoprotein transcript levels in the mice jejunum [29]. Leptin was also involved in the regulation of circulating apo-AIV by suppressing apoAIV synthesis in the small intestine [13]. It remains unknown, however, if this function is assumed by leptin trafficking in the lumen of the intestine. In the enterocytelike Caco2 cells, leptin was reported to reduce the output of de novo–synthesized apolipoprotein ApoB-100 and ApoB-48, as well as that of newly formed chylomicrons and of low-density lipoproteins, supporting a role for leptin in the reduction of intestinal TG secretion into the circulation. Moreover, I-FABP expression was decreased by leptin in Caco2 cells [14]. These effects of leptin on FA uptake and assembly in the enterocyte are likely to be involved in the regulation of energy homeostasis.

1074 / Chapter 147 Jejunal Absorption of Sugars Dietary carbohydrates are digested in the intestine through the action of amylase and intestinal brushborder membrane disaccharidases into monosaccharides, d-glucose, d-galactose, and d-fructose. Sodium-dependent glucose transporter 1 (SGLT-1) is the specific transporter for d-glucose and d-galactose, whereas d-fructose is transported into the enterocytes by the pentose transporter glucose transport protein 5 (GLUT-5). After accumulation in the enterocytes, the monosaccharides exit the cell across the basolateral membrane by the Na+-independent transporter GLUT2. In the jejunum, it was shown that leptin can inhibit the active absorption of galactose mediated by the Na+glucose co-transporter SGLT-1 without affecting the passive component of the absorption. This leptin inhibition of galactose absorption in vitro is likely to involve leptin-receptor-coupled activation of protein kinase C and A [5]. On the other hand, systemic leptin administration to rats after massive small bowel resection (MSBR) leads to an increase in the amounts of GLUT-5 protein with no change in the levels of SGLT-1 [32]. Gut leptin also retains the ability to modulate the absorption of glucose-galactose in the rat jejunum. Indeed, luminal addition of leptin on rat jejunum, isolated in Ussing chambers, rapidly and dramatically decreased active glucose transport [15]. This rapid inhibition of glucose entry into the enterocyte by luminal leptin involves a reduced recruitment of SGLT-1 from an intracellular preformed pool to the apical membrane. On the other hand, the inhibition of active glucose transport by serosal leptin was slower and probably involved the release of CCK as a relay molecule. Because the small intestine is now recognized as an insulin-sensitive and gluconeogenic organ [28], a better understanding of the mechanisms by which leptin affects the intestinal absorption of monosaccharides may have physiological relevance in the management of diabetes, in particular non-insulin-dependent diabetes mellitus (NIDDM).

Colonic Absorption of Short-Chain Fatty Acids Butyrate, a short-chain fatty acid (SCFA), is one of the products of the microbial digestion of carbohydrates and dietary fibers in the large bowel, which represents a dominant energy source for the colonocytes. Gut leptin is involved in the regulation of butyrate uptake by the intestinal epithelial cells through the proton-linked monocarboxylate transporter type 1 (MCT-1) [22]. Indeed, in the human intestinal Caco2 cells, luminal leptin increases butyrate uptake by increasing MCT-1 mRNA levels and the amounts of MCT-1 protein on the apical membrane [9]. Such a control of MCT-1 activity by leptin, which affects the availability of

SCFA in the mucosa, probably modulates the intracellular events regulating normal differentiation and proliferation in the colonic mucosa. Along this line, it has been reported that in vitro leptin can protect cancer HT-29 cells from butyrate-induced apoptosis [33], suggesting potential implications for the diseased colon. More investigations are needed to establish the relevance of these current results in the pathophysiology of colon cancer.

LEPTIN AND GUT PATHOLOGIES Leptin is involved in maintaining gastric epithelial cell integrity and gastroprotection. In rats, systemic leptin was found to be effective in attenuating both ethanol- and aspirin-induced damage to the gastric mucosa. This was correlated with an increase of leptin production by the stomach during experimentally induced gastric damage in rats and during Helicobacter pylori infection in humans (reviewed in [24]). This gastric cytoprotective effect of leptin involved an increase in blood flow, local production of nitric oxide and prostaglandin E2 (PGE2), and vagus nerve– dependent mechanisms. One of the emerging functions of leptin is its role in the regulation of inflammatory processes. There is, indeed, a large body of evidence that leptin-mediated signal pathways play an active role in innate and adaptive immunity through the alteration of various target genes’ transcription [16]. Interestingly, leptin-deficient ob/ob mice are reported to be resistant to colonic inflammation induced by oral dextran sulfate sodium or trinitrobenzene sulfanate (TNBS), and the replacement of leptin converted their resistance to disease into susceptibility [36]. The mechanisms responsible for such an action largely involved the T-cell-activation capacity of leptin [37]. Recently, human inflamed colonic cells have been shown to exhibit a strong leptin-immunoreactivity that is concentrated at subapical part of the colonic cells, whereas normal colonic epithelial cells do not show this [38]. This supports the idea that fat- and gut-derived leptin may be a key component in the control of intestinal inflammation processes. Whether the aberrant expression of leptin is a cause or consequence of inflammation in colon mucosa and whether leptin acting from the luminal side contributes to the progression and maintenance of the chronicity of the inflammation remain to be further investigated.

CONCLUSION The gastrointestinal epithelial cells are in contact with submucosal immune cells that are tightly and finely controlled by the enteric nervous system. There is

Leptin and the Gastrointestinal Tract / 1075 increasing evidence pointing to the importance of leptin at the interface of the immune, endocrine, and enteric nervous systems and the epithelial barrier within the gut. These results make leptin, a hormone and a cytokine, a good candidate to link neuroendocrine and immune systems to metabolic status. A better understanding of the role of gut leptin in the physiology of gastrointestinal functions will provide a basis for the determination of its relevance in several diseases states such as obesity, diabetes, and irritable bowel syndrome (IBD).

Acknowledgments The authors’ work was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm), by Inserm grant APEX99 n°4X006E to A.B. S.G was supported by Fondation pour la Recherche Médicale (FRM).

References [1] Anini Y, Brubaker PL. 2003. Role of leptin in the regulation of glucagon-like peptide-1 secretion. Diabetes 52: 252–9. [2] Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, et al. 1998. The stomach is a source of leptin. Nature 394: 790–3. [3] Bajari TM, Strasser V, Nimpf J, Schneider WJ. 2003. A model for modulation of leptin activity by association with clusterin. FASEB J 17: 1505–7. [4] Barrachina MD, Martinez V, Wang L, Wei JY, Tache Y. 1997. Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc Natl Acad Sci USA 94: 10455–60. [5] Barrenetxe J, Sainz N, Barber A, Lostao MP. 2004. Involvement of PKC and PKA in the inhibitory effect of leptin on intestinal galactose absorption. Biochem Biophys Res Commun 317: 717–21. [6] Burdyga G, Spiller D, Morris R, Lal S, Thompson DG, et al. 2002. Expression of the leptin receptor in rat and human nodose ganglion neurones. Neuroscience 109: 339–47. [7] Buyse M, Berlioz F, Guilmeau S, Tsocas A, Voisin T, et al. 2001. PepT1-mediated epithelial transport of dipeptides and cephalexin is enhanced by luminal leptin in the small intestine. J Clin Invest 108: 1483–94. [8] Buyse M, Ovesjo ML, Goiot H, Guilmeau S, Peranzi G, et al. 2001. Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve. Eur J Neurosci 14: 64–72. [9] Buyse M, Sitaraman SV, Liu X, Bado A, Merlin D. 2002. Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE. J Biol Chem 277: 28182–90. [10] Cammisotto PG, Renaud C, Gingras D, Delvin E, Levy E, Bendayan M. 2005. Endocrine and exocrine secretion of leptin by the gastric mucosa. J Histochem Cytochem 53: 851–60. [11] Cinti S, Frederich RC, Zingaretti MC, De Matteis R, Flier JS, Lowell BB. 1997. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue. Endocrinology 138: 797–804. [12] Cinti S, Matteis RD, Pico C, Ceresi E, Obrador A, et al. 2000. Secretory granules of endocrine and chief cells of human stomach mucosa contain leptin. Int J Obes Relat Metab Disord 24: 789–93.

[13] Doi T, Liu M, Seeley RJ, Woods SC, Tso P. 2001. Effect of leptin on intestinal apolipoprotein AIV in response to lipid feeding. Am J Physiol Regul Integr Comp Physiol 281: R753–9. [14] Dube N, Delvin E, Yotov W, Garofalo C, Bendayan M, et al. 2001. Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines. J Cell Biochem 81: 613–20. [15] Ducroc R, Guilmeau S, Akasbi K, Devaud H, Buyse M, Bado A. 2005. Luminal leptin induces rapid inhibition of active intestinal absorption of glucose mediated by sodium-glucose cotransporter 1. Diabetes 54: 348–54. [16] Fantuzzi G, Faggioni R. 2000. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J Leukoc Biol 68: 437–46. [17] Fei Y J, Kanai Y, Nussberger S, Ganapathy V, Leibach FH, et al. 1994. Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368: 563–6. [18] Gaige S, Abou E, Abysique A, Bouvier M. 2004. Effects of interactions between interleukin-1beta and leptin on cat intestinal vagal mechanoreceptors. J Physiol 555: 297–310. [19] Guilmeau S, Buyse M, Bado A. 2004. Gastric leptin: A new manager of gastrointestinal function. Curr Opin Pharmacol 4: 561–6. [20] Guilmeau S, Buyse M, Tsocas A, Laigneau JP, Bado A. 2003. Duodenal leptin stimulates cholecystokinin secretion: Evidence of a positive leptin-cholecystokinin feedback loop. Diabetes 52: 1664–72. [21] Guilmeau S, Nagain-Domaine C, Buyse M, Tsocas A, Roze C, Bado A. 2002. Modulation of exocrine pancreatic secretion by leptin through CCK(1)-receptors and afferent vagal fibres in the rat. Eur J Pharmacol 447: 99–107. [22] Halestrap AP, Price NT. 1999. The proton-linked monocarboxylate transporter (MCT) family: Structure, function and regulation. Biochem J 343 Pt 2: 281–99. [23] Ikejima K, Takei Y, Honda H, Hirose M, Yoshikawa M, et al. 2002. Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat. Gastroenterology 122: 1399–410. [24] Konturek PC, Brzozowski T, Sulekova Z, Brzozowska I, Duda A, et al. 2001. Role of leptin in ulcer healing. Eur J Pharmacol 414: 87–97. [25] Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, et al. 1996. Abnormal splicing of the leptin receptor in diabetic mice. Nature 379: 632–5. [26] Liefers SC, te Pas MF, Veerkamp RF, Chilliard Y, Delavaud C, et al. 2003. Association of leptin gene polymorphisms with serum leptin concentration in dairy cows. Mamm Genome 14: 657–63. [27] Matson CA, Reid DF, Cannon TA, Ritter RC. 2000. Cholecystokinin and leptin act synergistically to reduce body weight. Am J Physiol Regul Integr Comp Physiol 278: R882–90. [28] Mithieux G. 2005. The new functions of the gut in the control of glucose homeostasis. Curr Opin Clin Nutr Metab Care 8: 445–9. [29] Morton NM, Emilsson V, Liu YL, Cawthorne MA. 1998. Leptin action in intestinal cells. J Biol Chem 273: 26194–201. [30] Nawrot-Porabka K, Jaworek J, Leja-Szpak A, Palonek M, Szklarczyk J, et al. 2004. Leptin is able to stimulate pancreatic enzyme secretion via activation of duodeno-pancreatic reflex and CCK release. J Physiol Pharmacol 55 Suppl 2: 47–57. [31] Oliver P, Pico C, De Matteis R, Cinti S, Palou A. 2002. Perinatal expression of leptin in rat stomach. Dev Dyn 223: 148–54. [32] Pearson PY, O’Connor DM, Schwartz MZ. 2001. Novel effect of leptin on small intestine adaptation. J Surg Res 97: 192–5. [33] Rouet-Benzineb P, Aparicio T, Guilmeau S, Pouzet C, Descatoire V, et al. 2004. Leptin counteracts sodium butyrate-induced apoptosis in human colon cancer HT-29 cells via NF-{kappa}B signaling. J Biol Chem 279: 16495–502.

1076 / Chapter 147 [34] Sanchez J, Oliver P, Miralles O, Ceresi E, Pico C, Palou A. 2005. Leptin orally supplied to neonate rats is directly uptaken by the immature stomach and may regulate short-term feeding. Endocrinology 146: 2575–82. [35] Sanchez J, Oliver P, Pico C, Palou A. 2004. Diurnal rhythms of leptin and ghrelin in the systemic circulation and in the gastric mucosa are related to food intake in rats. Pflugers Arch 448: 500–6. [36] Siegmund B, Lehr HA, Fantuzzi G. 2002. Leptin: A pivotal mediator of intestinal inflammation in mice. Gastroenterology 122: 2011–25. [37] Siegmund B, Sennello JA, Jones-Carson J, Gamboni-Robertson F, Lehr HA, et al. 2004. Leptin receptor expression on T lymphocytes modulates chronic intestinal inflammation in mice. Gut 53: 965–72. [38] Sitaraman S, Liu X, Charrier L, Gu LH, Ziegler TR, et al. 2004. Colonic leptin: Source of a novel proinflammatory cytokine involved in IBD. FASEB J 18: 696–8. [39] Tartaglia LA. 1997. The leptin receptor. J Biol Chem 272: 6093–6.

[40] Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, et al. 1995. Identification and expression cloning of a leptin receptor, OB-R. Cell 83: 1263–71. [41] Wang L, Barachina MD, Martinez V, Wei JY, Tache Y. 2000. Synergistic interaction between CCK and leptin to regulate food intake. Regul Pept 92: 79–85. [42] Wang YH, Tache Y, Sheibel AB, Go VL, Wei JY. 1997. Two types of leptin-responsive gastric vagal afferent terminals: An in vitro single-unit study in rats. Am J Physiol 273: R833– 7. [43] Yuan CS, Attele AS, Dey L, Xie JT. 2000. Gastric effects of cholecystokinin and its interaction with leptin on brainstem neuronal activity in neonatal rats [In Process Citation]. J Pharmacol Exp Ther 295: 177–82. [44] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. 1994. Positional cloning of the mouse obese gene and its human homologue [published erratum appears in Nature 1995 Mar 30;374(6521):479] [see comments]. Nature 372: 425– 32.

C

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148 Motilin PIERRE POITRAS

ABSTRACT

of the stomach” [2]. The structure of porcine motilin is shown in Table 1.

Motilin is released from the M cell of the duodenal mucosa into the blood circulation to help regulate the interdigestive motility of the stomach and small intestine. Motilin, as an interdigestive hormone, induces the phase III contraction of the migrating motor complex (MMC) during the fasting period. Motilin receptor stimulation is a potent pharmacological stimulus of gastric motor activity and can be used to accelerate the gastric emptying of a meal. Motilin is part of the newly discovered motilin-ghrelin peptides family.

STRUCTURE OF MOTILIN AND RELATED PEPTIDES Structural Heterogeneity and Species Species heterogeneity in the structure of motilin soon became a major issue in the physiological and pharmacological exploration of the peptide; indeed it had an impact on the detection of motilin by radioimmunoasay (RIA) or on the bioactivity of motilin on various experimental models. Biochemical characterization of motilin was, therefore, undertaken in numerous animal species. In the late 1970s, using successive chromatographies (gel; ion; and high-performance liquid chromatography, HPLC) of small intestinal extracts and detection of motilin-like-immunoreactivity by RIA with an antiserum against porcine motilin, we purified canine motilin and found differences in 5 of its 22 amino acids (see Table 1). From the mid-1980s, molecular biology allowed the isolation and characterization of cDNA clones encoding motilin precursors in numerous species. Human motilin was found to be similar to the pig peptide; but differences in the amino acid sequence were subsequently identified in rabbit, cat, chicken, sheep, monkey, horse, cow, and guinea pig (as shown on Table 1).

DISCOVERY OF MOTILIN The sequence of motilin was first published in 1973 by Brown et al. [2]. Inspired by observations made at the beginning of the twentieth century that gastric motility was inhibited by perfusing an acid solution into the duodenum, Brown discovered that instillation of pH 9 Tris buffer into the duodenum increased motor activity in a transplanted gastric pouch in dogs. This experiment, confirming the regulation of gastric motor activity by an endocrine factor released from the duodenum, led to the purification of this hormone. As starter material, he used a side fraction produced during the purification of porcine intestinal secretin by Victor Mutt at the Karolinska Institute. Tracing the purification steps with the in vivo model of canine transplanted gastric pouch, Brown, through successive chromatographies on CM cellulose, Sephadex G25, and DEAE cellulose, isolated a 22-amino-acid peptide with a molecular weight of 2700. He chose the name motilin “because of the original observation that it caused an increase in motor activity in pouches of gastric fundus gland area Handbook of Biologically Active Peptides

Mouse and Rat Motilin Confusion certainly arose from studies in rat and mouse; the two common laboratory animals could not be used for motilin research because motilin could not

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TABLE 1. Sequence of motilin in various species and year of discovery.a

PIG (1972) DOG (1979) MAN (1987) RABBIT (1991) CAT (1993) CHICKEN (1995) SHEEP (1997) MONKEY (1998) HORSE (1999) COW (1999) GUINEA PIG (2001) MOUSE (2004) RAT (2004) a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

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VAL

PRO

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TYR

GLY

GLU

LEU

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MET

GLN

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GLU

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x

—, identical AA; x, absent AA.

Motilin / 1079 be detected in these species and was without any biological activity in them. The efforts of many research teams to characterize motilin and motilin receptors in these rodents were in vain. An important step in the comprehension of this unusual situation in the field of peptide research was taken recently by T. L. Peeters using an “in silico” approach to identify motilin and the motilin receptor in the now-elucidated rat genome. Peptide conserved sequences were discovered, but the putative translation products contained stop codons that disable the formation of full prepro-motilin peptides. A partial 1–13 fragment that has amino acid substitutions in positions 2, 3, and 7 might eventually be formed. This fragment showed contractile activity similar to porcine motilin in the rabbit duodenum in vitro, confirming its motilin nature [11]. Reverse transcription polymerase chain reaction (RT-PCR) analysis with a probe made from mouse motilin of mouse duodenum, stomach, or brain failed to reveal motilin in these organs, suggesting that motilin is not expressed in mouse. Moreover, “in silico” analysis of the motilin receptor also suggested the presence of homologous regions but with many stop codons and a frame-shift, making the synthesis of the mature protein impossible. Mouse motilin, indeed, was inactive in the mouse duodenum in vitro and in the mouse stomach in vivo, suggesting motilin receptors were absent in mouse digestive tract. A similar observation has now been documented in the rat. Heterogeneity in the structure of motilin among species is unusual in the field of peptides.

receptors, suggests of course cross-reactivity between ghrelin and motilin analogs on their receptors. However, recent studies have failed to show a significant interaction between motilin ligands and the ghrelin receptor and between ghrelin ligands and the motilin receptor. The significance and implication of the motilin-ghrelin peptide family have still to be identified.

Gene Structure and Posttranslational Processing The human motilin gene was mapped to the P 21.2 → P 21.3 region of chromosome 6. It consists of five exons separated by four introns; the bioactive peptide structure is encoded in two distinct exons (exon 2 and exon 3). Nucleotide sequence analysis predicts that motilin is first synthesized as a precursor of 114–119 amino acids. A 25-amino-acid signal peptide precedes the 22-amino-acid sequence of the active peptide, followed by two lysine residues that precedes a 65-aminoacid sequence that has been called motilin-associated peptide (MAP), whose biological importance remains unknown. Northern blot analysis of human duodenal mRNA revealed a single band and suggested that the multiple forms of motilinlike-immunoreactive material found by gel permeation chromatographies were produced by posttranslational processing of a single motilin precursor. The importance of these promotilins, however, appears marginal; in tissues as well as in blood, the 22amino-acid peptide is by far the dominant molecular form encountered.

Motilin-Ghrelin Family Motilin was in a peculiar position because it was not identified as belonging to a family of peptides, as, for example, in peptide families such as gastrin– cholecystokinin (CCK) and secretin–vasointestinal polypeptide (VIP). However, ghrelin, a newly discovered peptide of the mammalian stomach, shares sufficient structural similarity with motilin to have been called motilin-related peptide [15]. Motilin and ghrelin (see other chapters on ghrelin in this book) have a number of biological actions in common. The motor effects of ghrelin on the gastrointestinal (GI) tract are indeed comparable to those observed for motilin; that is, induction of phase III of the migrating motor complex (MMC) in the fasting period and stimulation of gastric emptying during the postprandial period. In the central nervous system, motilin and ghrelin show comparable activities on the release of growth hormone (GH) or on appetite stimulation. Not only do motilin and ghrelin show structural similarity, but their receptors have sequence homology. Resemblance between motilin and ghrelin in the structure of the ligands, as well as in the structure of their

DISTRIBUTION IN THE GI TRACT The presence of motilin in the gut of numerous animal species has been verified by RIA measurement of motilinlike immunoreactivity (MLI) in tissue extracts, by immunostaining of motilin-containing cells or structures, or by Northern blots to detect motilin mRNA in various tissue regions of the GI tract. By far the most prevalent region for motilin expression is the proximal gut mucosa, mainly the duodenum and, to a minor extent, the jejunum. Motilin is produced in a specific endocrine cell of the intestinal mucosa. The M cell is well characterized in electron microscopy by its relatively small, solid secretory granules, often of irregular shape and density. The presence of motilin in a subpopulation of 5-hydroxytryptamine-enterochromaffin cells has been initially described, but remains controversial. The presence of motilin in extra-mucosal structures of the GI tract is also debated. MLI was detected in extracts from gut muscle, but this could be explained by mucosal contamination during tissue preparation.

1080 / Chapter 148 Some studies, most often unconfirmed, suggested that motilin was present in the vagus nerve as well as in neurons of the intestinal wall. Motilin appeared as one of the rare peptides not considered as a brain-gut peptide. Motilin concentrations measured in the brain were always much lower than those found in the gut and much lower than other brain peptides such as CCK or VIP. However, motilin mRNA has been detected in specific brain regions of various animal species, including humans. Motilin receptors also seem to be present in the brain, and we must now face the possibility that motilin could, indeed, like most other peptides, play a role as a neural transmitter.

Interestingly, the C-terminal structure, not required for in vitro bioactivity, was found to be important for in vivo bioactivity. N-terminal fragments of 15 amino acids, fully active in vitro, were indeed inactive in vivo, whereas longer fragments with 19, 20, or 21 amino acids could mimic the motor activity of the 22-amino-acid native peptide when injected in conscious dogs [13]. This is an interesting concept in the field of peptide pharmacology; the C-terminal structure seems to be important in protecting the whole molecule, probably against circulating degrading enzymes. Interestingly, a similar observation on the protective role of the C-terminal segment in vivo has been made for ghrelin [14], the other member of this peptide family.

STRUCTURE–ACTIVITY RECEPTOR Nuclear magnetic resonance analysis of the threedimensional structure of motilin revealed that the first seven amino acids of the N-terminal portion of the molecule are positioned in a curved fashion, whereas the C-terminal segment of the peptide displays an alphahelix appearance (see Fig. 1). The N-terminal portion of the molecule is responsible for receptor binding and in vitro bioactivity. Peptide fragments containing the first 12 amino acids of the molecule demonstrate full binding capacity and full contractile activity in vitro. Phe1, ILe4, and Tyr7 are key amino acids for the pharmacophore of motilin.

3 7

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FIGURE 1. Structure of the 22-amino-acid motilin peptide revealed by nuclear magnetic resonance. The N-terminal curved sequence binds to the receptor, whereas the α-helix C-terminal structure protects the molecule for in vivo bioactivity.

The motilin receptor was cloned in 1999. For years before that, all attempts to sequence the intestinal motilin receptor failed when using a goal-directed approach with a motilin probe for its recognition (of course, we now realize that strategies with mouse or rat as starting material were destined to fail, due to the now-documented absence of motilin receptors in these species). Researchers in Merck Laboratories [5] followed a different strategy by first cloning and sequencing a large variety of G-protein-coupled receptors from human genomic DNA libraries and then looking at the nature of the ligands for these receptors. Starting from an orphan receptor with a structure close to that of the receptor for the growth hormone secretagogues (GHS, ghrelin), mass screening of more than 500 peptide and nonpeptide molecules was performed to obtain positive results for motilin. Starting tissue for this work came from the thyroid, and the significance of this receptor localization remains an enigma. The motilin receptor gene was mapped to human chromosome 13 q14–q21. It is made of 412 amino acids with seven transmembrane domains and has 52% homology with the ghrelin receptor. Recent cloning of the motilin receptor of the rabbit confirmed the structural heterogeneity of motilin receptors among species, as was suspected in earlier pharmacological studies looking at the interaction of various fragments and analogs of motilin on various tissue membranes or organs in vitro and in vivo. In in situ hybridization studies, motilin receptor RNA was expressed in a subset of interstitial cells mostly apparent in human duodenum, jejunum, and colon. These cells also demonstrated immunoreactivity for neuron-specific enolase and, to some extent, for nitric oxide synthase (NOS) as well as choline acetyltransferase. In the duodenum, motilin receptor expression was also found in a separate population of cells having an

Motilin / 1081 elongated appearance consistent with smooth muscle cells. Functional experiments in humans as well as in animals suggested that motilin receptors indeed exist on nerves as well as on muscles of the gut. Receptors on neurons, sensitive to antimuscarinic blockade, are activated in humans by low doses of exogenous motilin to induce phase III contraction of the MMC during the fasting period, whereas muscle receptors, insensitive to atropine, seem to be activated by higher doses of ligands to accelerate gastric emptying of a meal [1, 3]. In receptor binding studies with solutions specifically enriched in muscle or in neural elements of the intestinal wall [9], specific and distinct responses to various motilin synthetic analogs and fragments suggested the existence of structurally heterogeneous motilin receptor subtypes specific to muscle or neural elements (which we have called M and N receptor subtypes). Motilin receptors in the brain remain an enigma. Biological responses (such as an increase in GH secretion, increase in food intake, or decrease in anxiety) to intracerebral injection of motilin were indeed observed, but, most of the time, in rats or mice, animals with a questionable contribution to motilin research. Motilin binding sites were, however, identified by autoradiographic studies of rabbit brain tissues, and receptor binding studies allowed the identification of high- and low-affinity binding sites for motilin on these tissues. The identification of motilin receptors in the rabbit brain as well as the documentation of motilin RNA in the brain of most species in which it has been investigated up to now suggest a role for motilin as a central neurotransmitter, a role that has still to be identified.

BIOLOGICAL ACTIONS Difficulties in Experimental Conditions Motilin was discovered more than 30 years ago, but many questions still appear unanswered. Exploration of its biological action was difficult for various reasons. 1. Experimental subjects: Rat and mouse are convenient laboratory animals for biological studies, but, as discussed previously, were of no help in motilin research. We therefore had to rely on more difficult experimental animals, including the dog, which was very important in the elucidation of motilin physiology. 2. Biological actions: The main biological action of motilin is the induction of phase III of the MMC. The recording of this gastrointestinal contractile activity is, therefore, essential for physiological studies on motilin, but it is a complicated task. It requires, in most cases, an invasive technology

(surgical implantation of recording electrodes, fluoroscopic positioning of nasally or orally introduced recording probes, etc.) that demands technical expertise from researchers as well as physical and even emotional tolerance from test subjects. Furthermore, the basal cyclical nature of the studied biological phenomenon requires prolonged and tedious experimental protocols. 3. Species heterogeneity: Species heterogeneity in the structure of motilin and of the motilin receptor, as well as in the regulatory mechanisms of the MMC, limited the possibility of extrapolating data from species to species and forced extreme caution in the transfer of information from one species to another.

Pharmacological Action As its name implies, motilin acts on digestive motility. When administered in pharmacological doses, motilin or motilin derivatives such as erythromycin can increase the contraction of the lower esophageal sphincter, of the fundic and antral stomach, of the duodenum, of the gallbladder, and, to a lesser extent, of the sigmoid colon (this poor action of motilin on the colon is surprising considering the high number of motilin receptors in this organ). Secretions from the pancreas as well as from the small intestine can also be stimulated by the exogenous administration of motilin. A recent study showed that motilin had no influence on visceral afferents involved in the sensation of rectal balloon distension. Stimulation of antroduodenal motility remains, however, the dominant action of the peptide. Initial in vitro studies with intestinal tissues from rabbits and humans revealed that motilin can stimulate smooth muscle contraction by a direct effect on muscle cells; the peptide action was seen despite the addition of all neural blockers, including tetrodotoxin. Experiments with isolated muscle cells have confirmed the presence of motilin receptors on muscle cell membranes. However, in vivo studies in dogs and humans clearly identified that the motor action of motilin is mediated by muscarinic transmitters.

Physiological Role The characteristic action of motilin is the induction of phase III contraction of the MMC. The MMC is the basic organization of motor activity of the gut during the fasting interdigestive period. It lasts 80–120 minutes and consists of three successive phases: phase I, no signification contractions are seen for 20–60 minutes; phase II, intermittent and irregular contractions start to occur 20–60 minutes before phase III; phase III,

1082 / Chapter 148 strong peristaltic contractions, lasting 3–10 minutes, start from the stomach and lower esophagus to migrate distally to the duodenum, jejunum, and ileum until the colon. This phase III peristaltic wave has been proposed as cleaning the digestive tract from bacteria or nutrients that could accumulate during the digestive period and could generate deleterious effects on the gut (e.g., bacterial overgrowth). Feeding interrupts the MMC and induces more constant motor activity of moderate amplitude to allow the optimal absorption of ingested nutrients because accelerated transit during phase III would indeed impair nutrient absorption by the small intestine. Regulatory peptides can exert their influence via different pathways: endocrine (hormonal), neurocrine, paracrine, or autocrine. At present, we believe that the main action of motilin is to regulate MMC activity through an endocrine mechanism, that is, through variation in its plasma concentrations. Motilin is in a unique position, as a GI regulatory peptide, of being active during the interdigestive period and therefore being not a digestive but an interdigestive hormone. Its role as an interdigestive hormone has been well identified in the dog, in which all of the Morton Grossman’s criteria [4] establishing the endocrine contribution of peptides have been fulfilled [12]. 1. Regulation of the cyclical pattern of the MMC is indeed under the control of circulating factors, as shown in various experiments in which MMC persisted in animals when their stomach had been completed denervated.

2. Plasma motilin levels are variable during the fasting period, and there is a perfect correlation between circulating peak levels of endogenous motilin in the blood and the initiation of phase III contractions from the stomach or proximal duodenum. 3. Administration of exogenous motilin induces phase III contraction of the MMC, and this can be obtained even when motilin is given in small doses reproducing physiological plasma variations. 4. Inhibition of circulating plasma motilin by the administration of specific motilin antisera blocked phase III contractions from the upper gut. Although the situation could not be explored under such perfect experimental conditions in humans, most evidence suggests that circulating motilin also plays a key role in the regulation of phase III initiated from the antrum in humans.

Release Mechanisms Most gastrointestinal hormones are released after a meal to allow or facilitate the digestion and absorption of nutrients. Motilin is a unique hormone; it is released periodically during the interdigestive fasting period, and its cyclical release is abolished after a meal (as shown schematically in Fig. 2). Therefore, a biological clock, still of unknown nature, somewhere in the organism periodically signals motilin cells to release the peptide into the circulation. In vitro preparations of intestinal mucosal cells enriched in motilin cells showed

meal

A GI MOTILITY

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PLASMA MOTILIN (pM)

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•

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• •• • •• •

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FIGURE 2. Schematic representation of canine plasma motilin variations. During the fasting interdigestive period, motilin is released cyclically every 80–120 min (B) to induce the phase III contraction of the migrating motor complex (MMC) from the stomach to the ileum (indicated by the dark boxes in A). After eating, motilin cyclical peak increases are abolished for 2–8 hours (depending on the content and nature of the meal) while the MMC is interrupted and the fed pattern motility profile is taking place.

Motilin / 1083 that muscarinic receptors are present on the motilin cell membrane and that protein kinase C activators are the most potent second messengers eliciting motilin release. In the ex vivo perfused canine intestine, bombesin has been identified as a direct stimulant of motilin release, whereas the stimulatory effect of opiates was mediated by acetylcholine. On the other hand, phenylephrine and somatostatin appear to act directly on M cell membrane receptors to block the release of the peptide. Vagus nerves seem to have some influence on the release of plasma motilin because acute vagal cooling inhibited the cyclical peak increases in plasma motilin; however, vagotomized dogs displayed a normal cyclical profile of motilin plasma release. As the stimulation of motilin cyclical peak release during the interdigestive period is incompletely understood; the inhibition of this cyclical release by a meal (see Fig. 2) is also incompletely understood. The situation is still more complicated by the fact that meal ingestion is followed in humans by a very early and brief increase in plasma motilin before the interdigestive release cycle is interrupted, as in the dog. This early release in humans can be mimicked experimentally by central stimulation with modified sham-feeding and by distension of the gastric fundus with an air-filled balloon. The contribution of this postprandial motilin release, not present in the dog, in the process of nutrient digestion in humans remains to be characterized.

PATHOPHYSIOLOGICAL IMPLICATION Disorder of Motilin Secretion Some GI hormones, such as gastrin and VIP, are important to clinicians because of the disease symptoms they can generate in response to tumoral hypersecretion by gastrinomas or VIPomas. One patient with a motilinoma has been reported [6]; suffering from liver metastases from a rectal neuroendocrine tumor, the patient had an isolated increase in plasma motilin, but was without any specific clinical manifestation. Up to now, there is no clinical phenotype attributed to motilin hypersecretion. High levels of circulating motilin have been documented, as co-secreted factors, in some patients with pancreatic tumors and Zollinger-Ellison syndrome as well as in patients with carcinoid tumors of the gut. Although it could be tempting to speculate on the role of motilin in the diarrhea found in these patients, its contribution to the biological alterations remains unknown. Because motilin hypersecretion could be expected to generate GI hypermotility and hypersecretion with probable diarrhea, it is logical to expect that hypomo-

tilinemia would induce GI hypomotility. Some investigators have indeed reported altered levels of plasma motilin in patients with idiopathic intestinal pseudoobstruction, idiopathic or postoperative gastroparesis, or irritable bowel syndrome (IBS). To this point, however, plasma motilin measurement has not been shown to be useful for inclusion in the workup diagnoses of any clinical situations.

Motilin Receptor Agonists and Antagonists Motilin was recently of major interest to medical clinicians because of the capacity of motilin receptor agonists to act as powerful stimulants of GI motor activity in patients with hypokinetic disorders. Zen Itoh was the first to observe that erythromycin, a macrolide antibiotic, could mimic the motor effect of motilin when injected in dogs [7]. It was soon established that erythromycin was in fact acting on motilin receptors, and Janssens et al. [8] made the capital observation that erythromycin was the most potent gastrokinetic ever tested to stimulate gastric emptying in diabetic patients with gastroparesis. Since then, erythromycin, whether administered intravenously (IV) or by mouth (p.o.), is used by many clinicians for the treatment of patients with gastroparesis or intestinal pseudo-obstruction. Motilides, or motilin receptor agonist substances derived from the erythromycin macrolide and with improved gastrokinetic activity but devoid of antibiotic properties, have been developed by many pharmaceutical companies [10]. At least three motilides have been tested in humans, but for various reasons (e.g., rapid tachyphylaxis, no significant clinical benefit, potential side effects), clinical trials with these newly derived molecules have failed to substantiate the impressive pharmacological potential seen with I V erythromycin. Whether the gastrokinetic capacity of motilin receptor agonists will be amenable to commercial development and clinical exploitation remains to be seen. Motilin receptor antagonists were developed recently. Based on our knowledge of the contractile capacity of motilin, we wonder whether these antagonists could have a therapeutic role in relaxing the small intestine or colon in patients with IBS or the gastric fundus in patients with functional dyspepsia.

Acknowledgments The author thanks Dr. Theo L. Peeters, author of the chapter on ghrelin in the GI section of this book, for his revision of this manuscript and for professional, as well as personal, friendship developed over 20 years of competitive research on motilin and ghrelin.

1084 / Chapter 148 References [1] Boivin M, Pinelo LR, St-Pierre S, Poitras P. Neural mediation of the motilin motor effect on the human antrum. Am J Physiol 1997; 272(1 Pt 1): G71–G76. [2] Brown JC, Cook MA, Dryburgh JH. Motilin, a gastric motor activity stimulating polypeptide: The complete amino acid sequence. Can J Biochem 1973; 51: 533–537. [3] Coulie B, Tack J, Peeters T, Janssens J. Involvement of two different pathways in the motor effects of erythromycin on the gastric antrum in humans. Gut 1998; 43(3): 395–400. [4] Drossman MI. Physiological effects of gastrointestinal hormones. Fed Proc 1977; 36: 1930–1931. [5] Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, MacNeil D, Cascieri MA, Nargund R, Bakshi R, Abramovitz M, Stocco R, Kargman S, O’Neill G, Van Der Ploeg LH, Evans J, Patchett AA, Smith RG, Howard AD. Receptor for motilin identified in the human gastrointestinal system. Science 1999; 284(5423): 2184–2188. [6] Fiasse R, Deprez P, Weynand B, De Clercq P, Wibin E, Pauwels S, Rahier J, Peeters T. An unusual metastatic motilin-secreting neuroendocrine tumour with a 20-year survival. Pathological, biochemical and motility features. Digestion 2001; 64(4): 255–260. [7] Itoh Z, Nakaya M, Suzuki T, Arai H, Wakabayashi K. Erythromycin mimics exogenous motilin in gastrointestinal contractile activity in the dog. Am J Physiol 1984 Dec; 247(6 Pt 1): G688–G694.

[8] Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Muls E, Bouillon R. Improvement of gastric emptying in diabetic gastroparesis by erythromycin. N Engl J Med 1990; 322(15): 1028–1031. [9] Miller P, Roy A, St-Pierre S, Dagenais M, Lapointe R, Poitras P. Motilin receptors in the human antrum. Am J Physiol Gastrointest Liver Physiol 2000; 278(1): G18–G23. [10] Peeters TL. Erythromycin and other macrolides as prokinetic agents. Gastroenterology 1993; 105: 1886–1899. [11] Peeters TL, Aerssens J, DeSmet B, Mitselos A, Thielemans L, Coulie B, Depoortere I. The mouse is a natural knock-out for motilin and for the motilin receptor. Neurogastro Mot 2004; 16: 687 (abstract). [12] Poitras P. Motilin is a digestive hormone in the dog. Gastroenterology 1984; 87: 909–913. [13] Raymond MC, Boivin M, St-Pierre S, Gagnon D, Poitras P. Studies on the structure-activity of motilin in vivo. Effect of motilin synthetic analogues in conscious dog. Regul Pept 1994; 50(2): 212–126. [14] Tolle V, Zizzari P, Tomasetto C, Rio MC, Epelbaum J, Bluet-Pajot MT. In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrinology 2001; 73(1): 54–61. [15] Tomasetto C, Karam SM, Ribieras S, Masson R, Lefebvre O, Staub A, Alexander G, Chenard MP, Rio MC. Identification and characterization of a novel gastric peptide hormone: The motilin-related peptide. Gastroenterology 2000; 119(2): 395– 405.

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149 Neurotensin in Regulation of Gastrointestinal Functions DEZHENG ZHAO AND CHARALABOS POTHOULAKIS

ABSTRACT

as a 13-amino-acid peptide was established soon thereafter [35]. Early on, it became evident that the gastrointestinal (GI) tract is a major source of NT and that the majority of the concentrations of NT in rats is localized in the intestine (∼90%), with a much smaller percentage (∼10%) present in the brain [35]. Among the different areas of the intestine, the jejuno-ileal section expresses the highest NT concentration, although other parts of the GI tract such as esophagus, stomach, duodenum, and large intestine also express this peptide [30, 35]. In mammals, NT-containing cells are scattered among epithelial cells of the jejunum and ileum, whereas in chicken NT cells are more evident in the antrum [56]. NT-containing cells are fairly evident in cat, dog, and human intestine and in most mammals are localized predominately on the villi, although in the dog intestine are more numerous in the crypts [56]. Ultrastructural analysis indicates that NT-producing neuroendocrine cells, also called N cells, contain cytoplasmic granules, predominantly in the basal portion of the cell that represent the storage site for this peptide [56]. This cellular localization facilitates the rapid release of NT following specific stimuli and enables this peptide via NT receptor binding to target intestinal cells to modulate a variety of intestinal functions. Three NT receptors have been identified, including two G-protein-coupled receptors (GPCRs), one high affinity (NTR1) and one low affinity (NTR2), and a third non-GPCR, NTR3 (now named gp95/sortilin) [63]. Among them, NTR1 mediates most of the intestinal NT responses, as shown by use specific nonpeptide NTR1 antagonists [10, 16, 17, 36, 47, 65]. In contrast to NTR2, high levels of NTR1 mRNA have been detected in the intestine [63]. Moreover, the amino acid sequence of the human NTR1, isolated from human colonic adenocarcinoma HT-29 cells, indicates its 84% identity to the rat NTR1 [63]. NTR1 is expressed in the colonic

Neurotensin (NT) is a brain-gut peptide expressed in many areas of the gastrointestinal (GI) tract, including stomach and small and large intestine. NT-producing cells, also called N cells, are distributed along the length of the intestine and release NT from cytoplasmic granules in response to several diverse stimuli, such as fat and bacterial products. Surface NT receptors are localized in nerves, epithelial cells, and immune and inflammatory cells of the GI and gastric mucosa. Of the three NT receptors identified so far, the high-affinity NTR1 appears to mediate most of the NT-induced GI responses. Accumulating evidence indicates that NTNT receptor interactions mediate several critical physiological and pathophysiological GI functions, such as motility, secretion of ions and fluid, inflammation, mucosal healing, and stress-related colonic responses. Thus, understanding the biology of NT receptors and identifying the signaling mechanisms following NT-NT receptor coupling in cells of the GI mucosa will help elucidate how this peptide modulates intestinal functions in physiological conditions and disease states. Such understanding might also provide important insights for therapeutic applications of NT antagonists in GI disorders, such as irritable bowel syndrome and inflammatory bowel disease. This review summarizes our accumulated knowledge of the biology of NT and signaling mechanisms of its receptors as they relate to its participation in the physiology and pathophysiology of the GI tract.

INTRODUCTION Neurotensin (NT) was first isolated by Carraway and Leeman from bovine hypothalami [14], and its identity Handbook of Biologically Active Peptides

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1086 / Chapter 149 mucosa of rats and humans in cells of the lamina propria, over myenteric and submucosal ganglia, and on colonic epithelial cells [6, 10, 16, 17, 65]. The binding of NT to NTR1 in a variety of GI cell types mediates several physiological and pathophysiological functions, including effects in motility, chloride secretion, intestinal cell growth, and intestinal inflammatory and stress responses. The NTR2 is primarily expressed on the brain and some nonintestinal organs, but its presence and participation in GI functions have not been yet clarified. Although the NTR3 is expressed in colonic adenocarcinoma HT29 cells [38], its functional importance in mediating NT-related GI responses remains unknown.

NT INHIBITS MOTILITY AND SECRETION IN THE STOMACH AND SMALL INTESTINE Several pieces of evidence indicate that NT inhibits gastrointestinal motility and secretion in animals and humans. NT administration reduces gastric acid secretion in response to pentagastrin, a test meal, or insulin hypoglycemia in dogs [2] and humans [8, 55]. Experiments in isolated gastric preparations also showed that NT inhibits spontaneous motor activity [3]. NT infusion in humans also inhibits gastric and duodenal motility [58, 59] and reverses the motility pattern of the duodenum and proximal jejunum from a fasting- to a fed-type [61], supporting a role for NT in postprandial regulation of motility in the upper intestine. Apart from peripheral NT, studies by Bueno et al. indicate that central NT might also exert inhibitory effects in gastric motility [11]. The administration of NT to normal volunteers reduces pressure of lower esophageal sphincter [50], suggesting that this peptide can affect esophageal function. Food intake, particularly fat, represents a major stimulus for NT release from the GI tract. For example, plasma levels of NT increase dramatically after a meal or administration of oleic acid [33, 34, 49]. The main source of NT following fat or meal ingestion appears to be the distal small intestine via signals generated at the proximal gut [64]. Infusion of several other lipids or fat emulsion preparations into the intestine of humans inhibits acid secretion and increases NT levels, indicating that NT might play a role in gastric secretion [44, 46]. Along these lines, NT infusion into the circulation increases the translocation of oleic acid from the intestinal lumen into the lymph of rats [4]. Intragastric fat intake also decreases lower esophageal sphincter pressure and increases NT concentrations [31]. Several mechanisms have been proposed that might mediate the effects of NT in gastric and small

intestinal secretion and motility, including release of somatostatin [28], norepinephrine [53], and direct effects on myocytes and cells of the myenteric plexus [1, 15]. Although a direct functional link for participation of NT in gastric acid output and gastric and upper small intestinal motility has not been firmly established, it does not appear to involve modulation of gastrin levels [40]. In support for an important role for NT in fat digestion, this peptide has also been implicated as a critical mediator of bile acid cycling. For example, NT administration to fasted rats enhances intestinal absorption of taurocholate, which represents an important conjugated bile acid [25]. Moreover, the modulation of intestinal bile acid concentration during feeding increases intestinal NT mRNA expression and circulating levels of NT [27], whereas the administration of the NTR1 antagonist SR-48692 to rats inhibits intestinal taurocholate (TC) absorption [27]. These results suggest a role for endogenous NT in enterohepatic bile acid cycling. Although the mechanism of this NT response is not entirely clear, recent pharmacological evidence indicates that NT-stimulated TC uptake in rat jejunum is mast cell-depended [26].

NT ENHANCES COLONIC MOTILITY AND STIMULATES SECRETION In contrast to its inhibitory effect in the upper GI tract, NT increases motility in the colon. Thus, peripheral NT administration significantly increases motility in dog [11] and cat [29, 51] colon. Studies in humans demonstrate that intravenous NT administration increases the duration of contractions in both the ascending colon and the rectosigmoid area [60] and stimulates defecation [12]. The mechanism(s) of these NT effects may be mediated by release of endogenous acetylcholine, although direct NT effects on smooth muscle cells have also been proposed [12, 43]. Two recent studies showed that NT can induce both excitatory and inhibitory responses in motor activity of the rat or human colon [42, 62], indicating the complexity of this response. NT might also play an important role in the mediation of secretory responses in the small intestine and colon. In an early study, Eklund et al. showed NT stimulates fluid secretion in the cat ileum [21]. Moreover, serosal administration of NT stimulates chloride secretion in guinea pig ileum [32], porcine jejunum [9], isolated rabbit colonocytes [52], and human colonic epithelial HT29 cell monolayers [41]. Recent results also indicate that NT might play a role in several colonic diarrheal disorders. Ussing chamber studies demon-

Neurotensin in Regulation of Gastrointestinal Functions / 1087 strate that NT induces chloride secretion in human colon and that this effect is mediated by nerve cells, adenosine, and prostaglandins [47]. Pharmacological and immunohistochemical studies also provided evidence that these NT chloride response is mediated by NTR1 expressed on nerve cells of the colonic mucosa [47].

ROLE OF NT AND NTR1 IN INTESTINAL INFLAMMATION The presence of NTR1 in the colon and the discovery of specific nonpeptide NTR1 antagonists provided the opportunity to examine the role of NT in acute intestinal inflammation. Experiments employing an in vivo colitis model using Clostridium difficile toxin A, an enterotoxin that mediates antibiotic-associated colitis in animals and humans, showed that the injection of toxin A into intestinal loops stimulated an early increase in NT and NTR1 expression in rat colonic mucosa [17]. Moreover, pretreatment of rats with the NTR1 antagonist, SR-48,692 significantly inhibited colonic fluid secretion, intestinal permeability, mucosal neutrophil infiltration, colonocyte damage, and mast cell activation following luminal toxin A exposure to colonic loops [17]. These results strongly indicate that NT is a proinflammatory peptide in acute colonic secretion and inflammation mediated by a bacterial enterotoxin. The mechanism of the proinflammatory responses to NT may involve mast cell activation [17] because mast cells contain NTR1 receptors [7] that mediate histamine release in response to NT [39]. Leukocytes might also represent another NT cell target implicated in intestinal inflammation, based on the reported direct phagocytic, chemotactic, and increased adherence activity of NT in neutrophils [23, 48], macrophages [19, 24], and lymphocytes [22]. Endothelial cells known to express NTR1 may also be a target for NT during intestinal inflammation [54]. NT appears to directly stimulate proinflammatory signaling in human colonocytes. NTR1 is localized on colonic epithelial cells in the rat, mouse, and human colon [10, 16, 17], as well as in several human colonic epithelial cancer cell lines [20, 37] and nontransformed human colonic epithelial NCM460 cells [65]. NT-NTR1 interactions in human nontransformed colonic epithelial cells stimulates mitogen-activated protein kinase (MAPK) activation and production of the proinflammmatory cytokine interleukin-8 (IL-8) by mechanisms involving the activation of NF-κB, mobilization of intracellular calcium, and activation of protein kinase CPKC and the Rho GTPases RhoA, Rac1, and Cdc42 [65, 66, 68].

NT PROMOTES CELL GROWTH, REGENERATION, AND HEALING OF THE GI MUCOSA NT is a trophic peptide, able to stimulate cell growth and proliferation in the gastric, small intestinal, and colonic mucosa [57]. NT also increases cellularity and reverses mucosal hypoplasia in the intestine of rats fed an elementary diet [57]. In rats, NT also restores gut mucosal integrity and reverses bacterial translocation after radiation-induced mucosal injury and small and large intestinal mucosal adaptation after colon resection [57]. Along the lines of a proliferative response, NT stimulates growth in several colonic cancer cell lines and enhances growth of human colon tumors in mice [36, 57]. It is important to note that the administration of a NTR1 antagonist reduces the development of experimental colonic tumors in nude mice [36], indicating that NT receptor antagonism may have a place in therapy of colonic cancer. Recent evidence also suggests that NT via NTR1 might participate in the pathophysiology of mucosal healing during intestinal inflammation. Brun et al. showed that NT promotes mucosal healing in experimental sodium sulfate (DSS) model of chronic colitis [10]. The administration of the NTR1 antagonist SR48642 worsened all aspects of colitis, whereas the continuous administration of NT reduced DSS-induced colitis after 5 days [10]. NTR1 and NT expression were elevated in the colon of patients with ulcerative colitis, as well as in the colon of mice with established colitis [10]. Interestingly, NTR1 antagonism during the repair phase of experimental colitis prevents mucosal healing via a mechanism involving NT-induced epithelial cell migration and prostaglandin release [10]. Exposure of human colonocytes to NT transactivates the epidermal growth factor receptor (EGFR) [67], a critical receptor in colon tissue repair. NT-induced EGFR phosphorylation is preceded by the release of matrix metalloproteinases and secretion of the EGFR ligand, transforming growth factor-α (TGFα), and mediates NT-induced MAPK activation and IL-8 secretion in colonocytes [67]. Thus, the NT-EGFR pathway may participate in mucosal healing and proinflammatory responses following NTNTR1 engagement in the human colonic mucosa, consistent with the ability of NT to stimulate growth of the intestinal mucosa in physiological and pathological conditions [57].

NT AND COLONIC STRESS RESPONSES Irritable bowel syndrome (IBS) is a common debilitating functional gastrointestinal disorder with a wide

1088 / Chapter 149 variety of symptoms known to be affected by psychological stress. Thirty minutes of immobilization stress in rats increases plasma NT levels and stimulates colonic mucin release and goblet cell depletion, prostaglandin E2 secretion, and mast cell activation [18, 45]. The administration of the NTR1 antagonist SR-48692 prior to restraining rats attenuates stress-mediated secretion of colonic mucin and prostaglandin E2 and activates colonic mucosa mast cells [18]. Although the mechanism of the NT-mediated colonic effects is not entirely clear, direct NT effects on colonic epithelial cells [5] and mast cells [13], leading to mucin and mast cell mediators release, respectively, might represent a likely mechanism. These results indicate that NT may mediate some of the colonic responses and symptoms in patients with IBS.

CONCLUSION The findings discussed here suggest that NT and its high-affinity receptor may play an important role in the physiology and pathophysiology of several GI functions and disease states, including colonic cancer, mucosal regeneration and healing, secretory diarrhea, inflammation induced by a bacterial enterotoxin, inflammatory bowel disease, and IBS. Other important intestinal functions associated with this peptide include modulation of motility, enterohepatic circulation of bile acids, and secretory diarrhea. Evidence indicates that NT can directly affect many intestinal cell types such as myocytes, enteric nerves, mast cells, and epithelial cells of the small intestine and colon via specific high-affinity receptors expressed in these cells. Thus, identification of the particular cells of the GI mucosa expressing NT receptors in humans and studies on the signaling cascades that are activated following exposure of the different intestinal cells to NT would help us understand the pathways activated by this peptide. Elucidation of these NT-related pathways is critical for designing effective therapeutic strategies for possible treatment of various GI diseases using NT receptor antagonists.

References [1] Al-Saffar, A., Analysis of the control of intestinal motility in fasted rats, with special reference to neurotensin, Scand J Gastroenterol, 19 (1984) 422–8. [2] Andersson, S., Chang, D., Folkers, K. and Rosell, S., Inhibition of gastric acid secretion in dogs by neurotensin, Life Sci, 19 (1976) 367–70. [3] Andersson, S., Rosell, S., Hjelmquist, U., Chang, D. and Folkers, K., Inhibition of gastric and intestinal motor activity in dogs by (Gln4) neurotensin, Acta Physiol Scand, 100 (1977) 231–5. [4] Armstrong, M.J., Parker, M.C., Ferris, C.F. and Leeman, S.E., Neurotensin stimulates [3H]oleic acid translocation across rat small intestine, Am J Physiol, 251 (1986) G823–9.

[5] Augeron, C., Voisin, T., Maoret, J.J., Berthon, B., Laburthe, M. and Laboisse, C.L., Neurotensin and neuromedin N stimulate mucin output from human goblet cells (Cl.16E) via neurotensin receptors, Am J Physiol, 262 (1992) G470–6. [6] Azriel, Y. and Burcher, E., Characterization and autoradiographic localization of neurotensin binding sites in human sigmoid colon, J Pharmacol Exp Ther, 297 (2001) 1074–81. [7] Barrocas, A.M., Cochrane, D.E., Carraway, R.E. and Feldberg, R.S., Neurotensin stimulation of mast cell secretion is receptormediated, pertussis-toxin sensitive and requires activation of phospholipase C, Immunopharmacology, 41 (1999) 131–7. [8] Blackburn, A.M., Fletcher, D.R., Bloom, S.R., Christofides, N.D., Long, R.G., Fitzpatrick, M.L. and Baron, J.H., Effect of neurotensin on gastric function in man, Lancet, 1 (1980) 987–9. [9] Brown, D.R. and Treder, B.G., Neurohormonal regulation of ion transport in the porcine distal jejunum. Actions of neurotensin and its natural homologs, J Pharmacol Exp Ther, 249 (1989) 348–57. [10] Brun, P., Mastrotto, C., Beggiao, E., Stefani, A., Barzon, L., Sturniolo, G.C., Palu, G. and Castagliuolo, I., Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation, Am J Physiol Gastrointest Liver Physiol, 288 (2005) G621–9. [11] Bueno, L., Fioramonti, J., Fargeas, M.J. and Primi, M.P., Neurotensin: A central neuromodulator of gastrointestinal motility in the dog, Am J Physiol, 248 (1985) G15–19. [12] Calam, J., Unwin, R. and Peart, W.S., Neurotensin stimulates defaecation, Lancet, 1 (1983) 737–8. [13] Carraway, R., Cochrane, D.E., Lansman, J.B., Leeman, S.E., Paterson, B.M. and Welch, H.J., Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels, J Physiol, 323 (1982) 403–14. [14] Carraway, R. and Leeman, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J Biol Chem, 248 (1973) 6854–61. [15] Carraway, R.E. and Mitra, S.P., Binding and biologic activity of neurotensin in guinea pig ileum, Peptides, 15 (1994) 1451– 9. [16] Castagliuolo, I., Leeman, S.E., Bartolak-Suki, E., Nikulasson, S., Qiu, B., Carraway, R.E. and Pothoulakis, C., A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats, Proc Natl Acad Sci USA, 93 (1996) 12611–15. [17] Castagliuolo, I., Wang, C.C., Valenick, L., Pasha, A., Nikulasson, S., Carraway, R.E. and Pothoulakis, C., Neurotensin is a proinflammatory neuropeptide in colonic inflammation, J Clin Invest, 103 (1999) 843–9. [18] Castagliuolo, I., Wershil, B.K., Karalis, K., Pasha, A., Nikulasson, S.T. and Pothoulakis, C., Colonic mucin release in response to immobilization stress is mast cell dependent, Am J Physiol, 274 (1998) G1094–100. [19] De la Fuente, M., Garrido, J.J., Arahuetes, R.M. and Hernanz, A., Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N, J Neuroimmunol, 42 (1993) 97–104. [20] Ehlers, R.A., Bonnor, R.M., Wang, X., Hellmich, M.R. and Evers, B.M., Signal transduction mechanisms in neurotensin-mediated cellular regulation, Surgery, 124 (1998) 239–46; discussion 246–7. [21] Eklund, S., Fahrenkrug, J., Jodal, M., Lundgren, O. and Sjoqvist, A., Mechanisms of neurotensin-induced fluid secretion in the cat ileum in vivo, Acta Physiol Scand, 129 (1987) 203–10. [22] Garrido, J.J., Arahuetes, R.M., Hernanz, A. and De la Fuente, M., Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes, Regul Pept, 41 (1992) 27–37.

Neurotensin in Regulation of Gastrointestinal Functions / 1089 [23] Goldman, R. and Bar-Shavit, Z., On the mechanism of the augmentation of the phagocytic capability of phagocytic cells by Tuftsin, substance P, neurotensin, and kentsin and the interrelationship between their receptors, Ann NY Acad Sci, 419 (1983) 143–55. [24] Goldman, R., Bar-Shavit, Z., Shezen, E., Terry, S. and Blumberg, S., Enhancement of phagocytosis by neurotensin, a newly found biological activity of the neuropeptide, Adv Exp Med Biol, 155 (1982) 133–41. [25] Gui, X. and Carraway, R.E., Enhancement of jejunal absorption of conjugated bile acid by neurotensin in rats, Gastroenterology, 120 (2001) 151–60. [26] Gui, X. and Carraway, R.E., Involvement of mast cells in basal and neurotensin-induced intestinal absorption of taurocholate in rats, Am J Physiol Gastrointest Liver Physiol, 287 (2004) G408–16. [27] Gui, X., Dobner, P.R. and Carraway, R.E., Endogenous neurotensin facilitates enterohepatic bile acid circulation by enhancing intestinal uptake in rats, Am J Physiol Gastrointest Liver Physiol, 281 (2001) G1413–22. [28] Hammer, R.A., Ochoa, A., Fernandez, C., Ertan, A. and Arimura, A., Somatostatin as a mediator of the effect of neurotensin on pentagastrin-stimulated acid secretion in rats, Peptides, 13 (1992) 1175–9. [29] Hellstrom, P.M. and Rosell, S., Effects of neurotensin, substance P and methionine-enkephalin on colonic motility, Acta Physiol Scand, 113 (1981) 147–54. [30] Holzer, P., Bucsics, A., Saria, A. and Lembeck, F., A study of the concentrations of substance P and neurotensin in the gastrointestinal tract of various mammals, Neuroscience, 7 (1982) 2919–24. [31] Horstmann, O., Nustede, R., Neufang, T., Lepsien, G. and Becker, H., Neurotensin-related regulation of the postprandial lower-oesophageal-sphincter pressure, Langenbecks Arch Surg, 384 (1999) 467–72. [32] Kachur, J.F., Miller, R.J., Field, M. and Rivier, J., Neurohumoral control of ileal electrolyte transport. II. Neurotensin and substance P, J Pharmacol Exp Ther, 220 (1982) 456–63. [33] Kihl, B., Rokaeus, A., Rosell, S. and Olbe, L., The effect of intraduodenal installation of oleic acid on plasma neurotensinlike immunoreactivity and on gastric acid secretion stimulated by betazole and sham feeding in man, Scand J Gastroenterol, 17 (1982) 633–9. [34] Kihl, B., Rokaeus, A., Rosell, S. and Olbe, L., Inhibition of pentagastrin stimulated gastric acid secretion and rise in the plasma concentration of neurotensin-like immunoreactivity (NTLI) by intraduodenal oleic acid in man, Acta Physiol Scand, 110 (1980) 329. [35] Leeman, S.E. and Carraway, R.E., Neurotensin: Discovery, isolation, characterization, synthesis and possible physiological roles, Ann NY Acad Sci, 400 (1982) 1–16. [36] Maoret, J.J., Anini, Y., Rouyer-Fessard, C., Gully, D. and Laburthe, M., Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice, Int J Cancer, 80 (1999) 448–54. [37] Maoret, J.J., Pospai, D., Rouyer-Fessard, C., Couvineau, A., Laboisse, C., Voisin, T. and Laburthe, M., Neurotensin receptor and its mRNA are expressed in many human colon cancer cell lines but not in normal colonic epithelium: Binding studies and RT-PCR experiments, Biochem Biophys Res Commun, 203 (1994) 465–71. [38] Martin, S., Navarro, V., Vincent, J.P. and Mazella, J., Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line, Gastroenterology, 123 (2002) 1135–43.

[39] Miller, L.A., Cochrane, D.E., Carraway, R.E. and Feldberg, R.S., Blockade of mast cell histamine secretion in response to neurotensin by SR 48692, a nonpeptide antagonist of the neurotensin brain receptor, Br J Pharmacol, 114 (1995) 1466–70. [40] Mogard, M.H., Maxwell, V., Sytnik, B. and Walsh, J.H., Regulation of gastric acid secretion by neurotensin in man. Evidence against a hormonal role, J Clin Invest, 80 (1987) 1064–7. [41] Morris, A.P., Cunningham, S.A., Benos, D.J. and Frizzell, R.A., Cellular differentiation is required for cAMP but not Ca(2+)dependent Cl− secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator, J Biol Chem, 267 (1992) 5575–83. [42] Mule, F. and Serio, R., Mode and mechanism of neurotensin action in rat proximal colon, Eur J Pharmacol, 319 (1997) 269–72. [43] Mule, F., Serio, R. and Postorino, A., Motility pattern of isolated rat proximal colon and excitatory action of neurotensin, Eur J Pharmacol, 275 (1995) 131–7. [44] Petersen, B., Christiansen, J., Rokaeus, A. and Rosell, S., Effect of intravenous and intrajejunal fat infusion on gastric acid secretion and plasma neurotensin-like immunoreactivity in man, Scand J Gastroenterol, 19 (1984) 48–51. [45] Pothoulakis, C., Castagliuolo, I. and Leeman, S.E., Neuroimmune mechanisms of intestinal responses to stress. In L.J. McCann SM, Sternberg EM, Chrousos GP, Gold PW, Smith CC (Ed.), Neuroimmunomodulation: Molecular, Integrative Systems, and Clinical Advances, Vol. 840, New York, 1998, pp. 635–648. [46] Read, N.W., McFarlane, A., Kinsman, R.I., Bates, T.E., Blackhall, N.W., Farrar, G.B., Hall, J.C., Moss, G., Morris, A.P., O’Neill, B. et al., Effect of infusion of nutrient solutions into the ileum on gastrointestinal transit and plasma levels of neurotensin and enteroglucagon, Gastroenterology, 86 (1984) 274–80. [47] Riegler, M., Castagliuolo, I., Wang, C., Wlk, M., Sogukoglu, T., Wenzl, E., Matthews, J.B. and Pothoulakis, C., Neurotensin stimulates Cl(−) secretion in human colonic mucosa In vitro: Role of adenosine, Gastroenterology, 119 (2000) 348–57. [48] Robbins, R.A., Nelson, K.J., Gossman, G.L. and Rubinstein, I., Neurotensin stimulates neutrophil adherence to bronchial epithelial cells in vitro, Life Sci, 56 (1995) 1353–9. [49] Rosell, S. and Rokaeus, A., The effect of ingestion of amino acids, glucose and fat on circulating neurotensin-like immunoreactivity (NTLI) in man, Acta Physiol Scand, 107 (1979) 263–7. [50] Rosell, S., Thor, K., Rokaeus, A., Nyquist, O., Lewenhaupt, A., Kager, L. and Folkers, K., Plasma concentration of neurotensinlike immunoreactivity (NTLI) and lower esophageal sphincter (LES) pressure in man following infusion of (Gln4)-neurotensin, Acta Physiol Scand, 109 (1980) 369–75. [51] Rothstein, R.D. and Ouyang, A., Mechanism of action of neurotensin at the ileocecal sphincter region, Life Sci, 45 (1989) 1475–82. [52] Sahi, J., Wiggins, M.P., Gibori, G.B., Layden, T.J. and Rao, M.C., Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes, J Cell Physiol, 168 (1996) 276–83. [53] Sakai, Y., Daniel, E.E., Jury, J. and Fox, J.E., Neurotensin inhibition of canine intestinal motility in vivo via alpha-adrenoceptors, Can J Physiol Pharmacol, 62 (1984) 403–11. [54] Schaeffer, P., Laplace, M.C., Savi, P., Pflieger, A.M., Gully, D. and Herbert, J.M., Human umbilical vein endothelial cells express high affinity neurotensin receptors coupled to intracellular calcium release, J Biol Chem, 270 (1995) 3409–13. [55] Skov Olsen, P., Holst Pedersen, J., Kirkegaard, P., Stadil, F., Fahrenkrug, J. and Christiansen, J., Neurotensin inhibits mealstimulated gastric acid secretion in man, Scand J Gastroenterol, 18 (1983) 1073–6.

1090 / Chapter 149 [56] Sundler, F., Hakanson, R., Leander, S. and Uddman, R., Light and electron microscopic localization of neurotensin in the gastrointestinal tract, Ann NY Acad Sci, 400 (1982) 94– 104. [57] Thomas, R.P., Hellmich, M.R., Townsend, C.M., Jr. and Evers, B.M., Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues, Endocr Rev, 24 (2003) 571–99. [58] Thor, K. and Rokaeus, A., Antroduodenal motor response induced by (Gln4)-neurotensin in man, Acta Physiol Scand, 118 (1983) 369–72. [59] Thor, K., Rokaeus, A., Kager, L. and Rosell, S., (Gln4)neurotensin and gastrointestinal motility in man, Acta Physiol Scand, 110 (1980) 327–8. [60] Thor, K. and Rosell, S., Neurotensin increases colonic motility, Gastroenterology, 90 (1986) 27–31. [61] Thor, K., Rosell, S., Rokaeus, A. and Kager, L., (Gln4)neurotensin changes the motility pattern of the duodenum and proximal jejunum from a fasting-type to a fed-type, Gastroenterology, 83 (1982) 569–74. [62] van der Veek, P.P., Schots, E.D. and Masclee, A.A., Effect of neurotensin on colorectal motor and sensory function in humans, Dis Colon Rectum, 47 (2004) 210–8.

[63] Vincent, J.P., Mazella, J. and Kitabgi, P., Neurotensin and neurotensin receptors, Trends Pharmacol Sci, 20 (1999) 302–9. [64] Walker, J.P., Fujimura, M., Sakamoto, T., Greeley, G.H., Jr., Townsend, C.M., Jr. and Thompson, J.C., Importance of the ileum in neurotensin released by fat, Surgery, 98 (1985) 224–9. [65] Zhao, D., Keates, A.C., Kuhnt-Moore, S., Moyer, M.P., Kelly, C. P. and Pothoulakis, C., Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes, J Biol Chem, 276 (2001) 44464–71. [66] Zhao, D., Kuhnt-Moore, S., Zeng, H., Wu, J.S., Moyer, M.P. and Pothoulakis, C., Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways, Am J Physiol Cell Physiol, 284 (2003) C1397–404. [67] Zhao, D., Zhan, Y., Koon, H.W., Zeng, H., Keates, S., Moyer, M.P. and Pothoulakis, C., Metalloproteinase-dependent TGFa release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells, J Biol Chem (2004). [68] Zhao, D., Zhan, Y., Zeng, H., Koon, H.W., Moyer, M.P. and Pothoulakis, C., Neurotensin stimulates interleukin-8 expression through modulation of IκBα phosphorylation and p65 transcriptional activity: Involvement of protein kinase Cα, Mol Pharmacol, 67 (2005) 2025–31.

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150 Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) PATRIZIA M. GERMANO AND JOSEPH R. PISEGNA

ABSTRACT

terminal amino acids are identical in these two peptides [1, 14, 21]. In other chapters of this book, descriptions regarding the localization and function of PACAP in diverse regions are included, such as in the pulmonary system, central nervous system, and endocrine system and in tumor biology [1, 35]. In the gastrointestinal tract, PACAP expression is present in the esophagus, stomach, duodenum, and intestine [4, 23]. On intestinal smooth muscle cells, PACAP is a potent nonadrenergic, noncholinergic (NANC) inhibitory neurotransmitter. Its actions in the intestine are thought to involve apaminsensitive potassium channels [30]; however, more recent evidence suggests that VIP and PACAP may be acting through the nitric oxide synthase pathway [24]. PACAP is also able to stimulate cellular growth and differentiation, as demonstrated in AR-42J cells, in which PACAP stimulates an increase in ornithine decarboxylase activity and cell proliferation [2], and in PC-12, a pheochromocytoma cell line in which PACAP stimulates neurite outgrowth [5]. PACAP has been specifically linked to some human motility disorders such as Hirschsprung’s disease, in which there is a reduction in PACAPimmunoreactive nerve fibers [32].

Pituitary adenylate cyclase activating polypeptide (PACAP) is expressed in the enteric nervous system of the gastrointestinal tract, where it functions to increase secretory activity and motility. Studies focusing on the role of PACAP in the gastrointestinal (GI) tract have identified it is as a key regulator in gastric acid secretion. Receptors for PACAP (PAC1) are expressed on the enterochromaffinlike (ECL) cells of the gastric corpus, where they regulate the release of histamine. PACAP is expressed in both gastric and colonic neurons, where it regulates gastrointestinal physiology. PACAP has also been demonstrated to regulate immune function in the GI tract and may be a key regulator of the inflammatory response associated with conditions such as inflammatory bowel disease. Receptors for PACAP have been discovered on tumors of the GI tract, and their stimulation is involved with growth of colonic tumors.

PACAP IS A NEWLY DISCOVERED NEUROENTERIC PEPTIDE Arimura and coworkers have demonstrated pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide identified in 1989, to be a potent activator of adenylyl cyclase in the endocrine system (see also Chapter 94 by Li, Nakamachi, and Arimura on VIP/ PACAP in the Brain Peptides section of this book). The peptide has been shown to belong to the larger class of peptides in the vasoactive intestinal polypeptide (VIP), secretin, and glucagon family of peptide hormones. Through the processing of the PACAP gene, two biologically active forms of the peptide are present in vertebrates, PACAP-38 and PACAP-27. The first 27 NHandbook of Biologically Active Peptides

CLONING AND CHARACTERIZATION OF PAC1 It had been previously hypothesized from radioligand studies in the pancreatic acinar carcinoma cell line, AR42J, the neuroblastoma NB-OK1 cell line, as well as in hypothalamic and brain membrane studies, that a specific receptor exists for PACAP [6, 27, 33]. Differential affinities for VIP and PACAP were observed, suggesting that there were at least two or three receptor subtypes in the VIP family. It is now known from the

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1092 / Chapter 150 differential affinities for the ligands VIP, helodermin, and PACAP-27/PACAP-38 that there are three separate receptor subtypes [14, 33]. The existence of a specific PACAP receptor was demonstrated in 1993 by several groups with the cloning of the rat PAC1 [27, 34]. The receptor cDNA encoded a putative protein containing 495 amino acids with a molecular weight of approximately 50 kDa [27]. Cloning the rat PAC1 gene determined that the receptor could exist at four major splice variants [34]. The homology of this cloned receptor was confirmed by the close examination of the deduced amino acid sequence, which placed PAC1 within the family of VIP, secretin, and glucagon receptors. Pharmacologically, transfected cell systems showed that the PAC1 has a high affinitiy for both PACAP-27 and PACAP-38, whereas there is little to no affinitiy for secretin and glucagon [27]. However, PACAP has similar affinity for either the VPAC1 and VPAC2 receptors and little affinity at the secretin receptor. An interesting feature of the PAC1 receptor, that was discovered initially by Spengler and coworkers, is that there are at least four splice variants that differ in the length of the third intracellular domain [26, 34]. These investigators showed that two exons (“hip” and “hop”) can be alternatively spliced and expressed as at least four major distinct splice variants [34]. A fourth splice variant, occurring in the transmembrane region of PAC1, has been shown not to couple to either adenylyl cyclate or phospholipate C, but couples to an L-type Ca2+ channel [39]. Of perhaps greater importance was the observation that each of the splice variants has differential tissue expression and differences in their ability to differentially activate adenylyl cyclate (AC) and phospholipase C [34]. A similar observation was made following the discovery of the human PAC1, wherein two exons can be differentially spliced and expressed. The differential activation of phospholipase C by one of the splice variants may have important consequences in the signal transduction cascades observed in the gastrointestinal tract and expressed on tumors [26]. Interestingly, the gene for the human PAC1 is localized to chromosome 7, but in the rat it is localized to chromosome 4. Following the cloning of PAC1, the VPAC2 receptor was identified, which appears to have high affinity for not only PACAP but also helodermin [13, 16].

SIGNAL TRANSDUCTION OF PAC1 From the earliest discovery of PACAP, this peptide was demonstrated by Deutsch and Sun, working with the pheochromocytoma cell line PC-12, to be dually coupled to both adenylyl cyclate and phospholipate C [5, 26, 34]. These observations were also confirmed using anterior pituitary–derived somatotrophs and

gonadotrophs, as well as cultures of adrenal chromaffin cells. In somatotrophs, PACAP stimulates cAMPdependant Ca2+ influx, whereas in gonadotrophs, PACAP stimulates phospholipase C-stimulated Ca2+ release [3, 20]. Within the carboxyl-terminal region, it has been demonstrated that two critical amino acids (Ser and Arg) are the regions of PAC1 responsible for signal transduction coupling [17]. In colonic smooth muscle, the PAC1 appears to activate apamin-sensitive K+ channels to induce relaxation [30]. It has been hypothesized that the ability of PAC1 to signal differentially via the Gαs and Gαq signal transduction pathways in a tissue-specific manner indicates that PACAP is able to influence the proliferation of gastrointestinal and nongastrointestinal tumor cells. This has been shown in the pheochromocytoma cell line, PC-12, as well as in cultured neuroblasts [5].

LOCALIZATION OF PAC1 As discussed in other sections of this book, the greatest density of receptors occurs in the hypothalamus, olfactory bulb, thalamus, and cerebellum, where the null splice variant is the predominant form [39]. In the anterior pituitary gland, the predominant splice variant of PAC1 is the “hop” type [29]. In peripheral tissues, the greatest density of PAC1 (predominantly the “hop” variant) occurs in the adrenal gland [34, 39]. It has also been previously demonstrated that the human prostate gland and testis express high levels of PAC1 [39]. The discovery of PAC1 in the gastrointestinal tract was perhaps the latest to be shown, due in part to the development of a specific anti-PAC1 polyclonal antibody, developed in collaboration with the late Dr. John Walsh and Helen Wong. Using these antibodies, PAC1 was discovered in the rat enterochromaffinlike (ECL) cells of the stomach and in lymphocytes [19, 40]. Confirmation of the expression of PAC1 has been shown using specific PAC1 primers and the highly sensitive techniques of reverse transcription polymerase chain reaction (RT-PCR) [40]. Recent data have demonstrated that PACAP is expressed in the enteric neurons of the stomach and colon [19]. In these studies, in whole-mount sections we studied, and demonstrated that PACAP and PAC1 were colocalized to the enteric neurons and ECL cells, respectively. In addition to PAC1, VPAC1 is also expressed in the gastrointestinal tract. The PAC1 receptor is expressed on the gastric ECL cells, whereas VPAC1 (classical VIP receptor) is expressed on the gastric D cells and chief cells [40]. The cell types described are principally involved in the secretory functions of the stomach and appear to play a key role in the regulation of gastric acid secretion. In addition to being expressed

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) / 1093 on secretory cells, both VPAC1 and PAC1 appear to be expressed on gastrointestinal smooth muscles [30]. More recent studies indicate that immunological lymphocytes also express PAC1, that may have a role in the regulation of responses. PAC1 has also been expressed on a number of tumor cell lines, including the human colon cancer cell lines such as the HCT-8 cell line [8, 15]. Interestingly, a differential coupling of the PAC1 SV1 splice variant on HCT-8 and HCT-116 human colonic tumors to stimulate cAMP, but not Ca2+, does not appear to activate tumor proliferation [8].

PHYSIOLOGY OF PAC1 IN THE GASTROINTESTINAL TRACT In the gastrointestinal tract, PACAP is expressed in the esophagus, stomach, duodenum, and small and large intestines [4, 23]. In the rat pancreas, PACAP has been previously demonstrated to be a potent stimulant of amylase release, and presumably PACAP is localized within nerve fibers innervating the pancreas. The existence of PACAP immunoreactivity in the pancreas may implicate a PACAP role in exocrine pancreatic function [28]. On gastrointestinal smooth muscles, PACAP is a potent NANC inhibitory neurotransmitter that is thought to act via apamin-sensitive potassium channels [30]. PACAP-containing nerve fibers are present in the small and large intestines, where they are involved in motility function [19]. In rats, PACAP stimulates apaminsensitive K+ channels [30]. In addition, both VIP and PACAP stimulate nitric oxide synthase (NOS) [9–11]. This has most recently been shown in the porcine jejunum [18]. In a recent study of the effects of PACAP on colon–inferior mesenteric ganglion (IMG) reflexes, it was observed that PACAP plays a key role in their regulation [7, 38]. The role of VIP and PACAP in relaxing the gastrointestinal smooth muscle is not clear. Some results indicate that the principal mechanism is via the release of NO [10, 24]. In dispersed rabbit gastric muscle cells, cross-competition by either VIP or PACAP suggests the potential for the interaction of both peptides and the same receptors and differential signal transduction pathways [36]. In studies of both gastric smooth muscle from rabbit and guinea pigs, it appears that VPAC2 is principally expressed [38]. More recently, using a guinea pig tenia coli model, it has been shown that there is specific expression of a subclass of VPAC receptor with specificity for VIP and this may represent a novel splice variant [22]. However, the role of PAC1 has been supported by a study using a mutant mouse model showing that the effects of PACAP in NANC relaxation of longitudinal muscles are mediated specifically by PAC1 [22]. At present, it is unclear whether the discrepancy

observed in these studies is species-dependent or whether there are muliple classes of VPAC and PAC receptors that are intimately involved in regulating gastrointestinal motor function. In humans, PACAP has been linked to motility disorders such as Hirschsprung’s disease, in which there is an observable decrease in the number of PACAP-immunoreactive nerve fibers [32]. In the stomach, PACAP-containing enteric nerve fibers have been described that are co-localized with PAC1 receptors [19]. The physiological significance of PACAP expression in the gastric epithelium appears to stimulate the gastric ECL cell to release histamine and thereby stimulate gastric acid secretion. However, PACAP activation of VPAC1 receptors expressed on the somatostatin-containing D cells has an opposite effect by inhibiting gastric acid secretion. The effects of PACAP stimulation of ECL cells, during the nocturnal hours, may account for the observed increase in gastric acid secretion that occurs in humans at approximately 2:00 to 4:00 a.m. and, therefore, may have clinical implication in the development of nocturnal gastroesophageal reflux symptoms [40] (Fig. 1). The development of pure cell populations of rat gastric ECL cells has permitted the understanding of the receptor expression and the physiological function of PACAP in the stomach. PACAP stimulation of pure populations of ECL cells results in intracellular Ca2+ release, cAMP stimulation, and histamine release. The use of microarray analysis confirmed the expression profile of genes predominantly involved in the ECL secretory pathways [25]. Similarly, using whole rabbit gastric glandular preparations, it has been demonstrated that PACAP activates intracellular Ca2+ release in the ECL cell and, through the release of histamine, the stimulation of parietal cell acid secretion [40]. PAC1 has been demonstrated to be expressed in the neuroendocrine tumor cell line, BON. This cell line is a model for the study of neuroendocrine tumors of the gastrointestinal cells. PACAP stimulation of the BON cell line results in a robust cAMP response, as well as the release of intracellular Ca2+. In response, there is a dose-dependent release of serotonin from intracellular stores. More recently, PAC1 has been identified by our group to be expressed on intestinal and circulating lymphocytes. To investigate the role of PAC1 expression, we used the well-defined dextran sulfate sodium (DSS) colitis model in eight wild-type and seven PAC1−/− mice. Following 4 days of DSS treatment, we examined the intact colon from both groups of mice. We observed a greater weight less in the PAC1–/– groups. Of greater significance was the histological evidence of more profound inflammation in the PAC1-deficient mice. The colonic length-to-weight ratio was significantly lower in PAC1–/– mice. The hematoxylin and eosin (H&E)-

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H+

G Cell

H2R OCH3 OCH3 O N

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Histamine N

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S N H

ECL Cell

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PACAP

FIGURE 1. Regulation of gastric acid secretion by PACAP.

stained slides of the distal and proximal colon indicated that in the PAC1−/− group there is a dense mucosal infiltrate, containing neutrophils, lymphocytes, and macrophages, that was significantly more abundant than in the PAC1+/+ group. These results indicate that the PAC1 receptor may be involved in colonic mucosal inflamation and that defeciency states may have a role in the evolution of inflammatory bowel disease (unpublished data).

CONCLUSION The discovery of the PACAP neuropeptides has ushered in a new era in the understanding of their complex role in the regulation of physiological processes. We have only recently begun to understand the role of these highly expressed neuropeptides in the gastrointestinal tract. PACAP and its receptor are expressed throughout the gastrointestinal tract. The neuronal expression of this peptide suggests that it is one of the key regulators of the neuroenteric nervous system. PACAP appears to stimulate secretion from the stomach through its actions on the PAC1 receptor,

whereas it clearly has a regulatory function in inhibiting gastric acid secretion through the VPAC1 receptor expressed on the gastric D cells. In the intestine, PACAP, acting at either the PAC1 or VPAC1, appears to be responsible for smooth muscle relaxation. In the pancreas, the role of PACAP is less clear, but it is presumably linked to the secretion of the exocrine pancreas.

Acknowledgments Supported by US VA Merit Review, AGA IRSA, VA Career Development Award, and a NIH supplement grant.

References [1] Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991; 129(5): 2787–9. [2] Buscail L, Cambillau C, Seva C, Scemama JL, De Neef P, Robberecht P, Christophe J, Susini C, Vaysse N. Stimulation of rat pancreatic tumoral AR4-2J cell proliferation by pituitary adenylate cyclase-activating peptide. Gastroenterology 1992; 103(3): 1002–8.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) / 1095 [3] Canny BJ, Rawlings SR, Leong DA. Pituitary adenylate cyclaseactivating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes. Endocrinology 1992; 130(1): 211–15. [4] Cox HM. Pituitary adenylate cyclase activating polypeptides, PACAP-27 and PACAP-38: stimulators of electrogenic ion secretion in the rat small intestine. Br J Pharmacol 1992; 106(2): 498–502. [5] Deutsch PJ, Sun Y. The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J Biol Chem 1992; 267(8): 5108–13. [6] Cauvin A, Buscail L, Gourlet P, DeNeef P, Gossen D, Arimura A, Miyata A, Coy DH, Robberecht P, Christophe J. The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the hum an neuroblastoma cell line NB-OK. Peptides 1990; 11(4): 773–7. [7] Ermilov LG, Schmalz PF, Miller SM, Szurszewski JH. PACAP modulation of the colon-inferior mesenteric ganglion reflex in the guinea pig. J Physiol 2004 Oct 1; 560(Pt 1): 231–47. Epub 2004 Jul 29. [8] Germano PM, Le SV, Oh DS, Fan R, Lieu S, Siu A, Pisegna JR. Differential coupling of the PAC1 SV1 splice variant on human colonic tumors to the activation of intracellular cAMP but not intracellular Ca2+ does not activate tumor proliferation. J Mol Neurosci 2004 Feb–Apr; 22(1–2): 83–92. [9] Grider JR. Interplay of VIP and nitric oxide in the regulation of the descending relaxation phase of peristalsis. Am J Physiol 1993; 264: G334–40. [10] Grider JR. Reciprocal activity of longitudinal and circular muscle during intestinal peristaltic reflex. Am J Physiol Gastrointest Liver Physiol 2003 May; 284(5): G768–75. [11] Grider JR, Katsoulis S, Schmidt WE, Jin JG. Regulation of the descending relaxation phase of intestinal peristalsis by PACAP. J Auton Nerv Syst 1994; 50: 151–9. [12] Grider JR, Makhlouf GM. Colonic peristaltic reflex: identification of VIP as a mediator of descending relaxation. Am J Physiol 1986; 251: G40–5. [13] Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 1998; 50(2): 265–70. [14] Lam HC, Takahashi K, Ghatei MA, Kanse SM, Polak JM, Bloom SR. Binding sites of a novel neuropeptide pituitary-adenylatecyclase-activating polypeptide in the rat brain and lung Eur J Biochem 1990; 193: 725–9. [15] Le SV, Yamaguchi DJ, Mc Ardle CA, Pisegna JR, Germano P. PAC1 and PACAP expression, signaling, and effect in the growth of HCT8, human colonic tumor cells. Regul Pept 2002 Nov 15; 109(1–3): 115–25. [16] Lutz EM, Sheward WJ, West KM, Morrow JA, Fink G, Harmar AJ. The VIP2 receptor: molecular characterization of a cDNA encoding a novel receptor for vasoactive intestinal peptide. FEBS Lett 1993; 334(1): 3–8. [17] Lyu RM, Germano PM, Choi JK, Le S, Pisegna, J. Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization. J Biol Chem 2000; 275(46): 36134–42. [18] Matsuda NM, Miller SM, Sha L, Farrugia G, Szurszewski JH. Mediators of non-adrenergic non-cholinergic inhibitory neurotransmission in porcine jejunum. Neurogastroenterol Motil 2004 Oct; 16(5): 605–12. [19] Miampamba M, Germano PM, Arli S, Wong HH, Scott D, Tache Y, Pisegna JR. Expression of pituitary adenylate cyclase-

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activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons. Regul Pept 2002 May 30; 105 (3): 145–54. Miyata A, Arimura A, Dahl RR, Minamino N, Uheara A, Jiang L, Culler MD, Coy DH. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989; 164(1): 567–74 Moody TW, Zia F, Makheja L. Pituitary adenylate cyclase activating polypeptide receptors are present on small cell lung cancer cells. Peptides 1993; 14(2): 241–6. Mukai K, Satoh Y, Fujita A, Takeuchi T, Shintani N, Hashimoto H, Baba A, Hata F. PAC1 receptor-mediated relaxation of longitudinal muscle of the mouse proximal colon. Jpn J Pharmacol 2002 Sep; 90(1): 97–100. Mungan Z, Arimura A, Ertan A, Rossowski WJ, Coy DH. Pituitary adenylate cyclase-activating polypeptide relaxes rat gastrointestinal smooth muscle. Scand J Gastroenterol 1992; 27(5): 375–80. Murthy KS, Jin JG, Grider JR, Makhlouf GM. Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells. Am J Physiol 1997 Jun; 272(6 Pt 1): G1391–9. Oh DS, Lieu SN, Yamaguchi DJ, Tachiki K, Lambrecht N, Ohning GV, Sachs G, Germano PM, Pisegna JR. PACAP regulation of secretion and proliferation of pure populations of gastric ECL cells. J Mol Neurosci 2005; 26(1): 85–98. Pisegna JR, Wank SA. Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C. J Biol Chem 1996; 271(29): 17267–74. Pisegna JR, Wank SA. Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor. Proc Natl Acad Sci USA 1993; 90(13): 6345– 9. Raufman JP, Malhotra R, Singh L. PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas. Regul Pept 1991 Oct 1; 36(1): 121–9. Rawlings SR, Hezareh M. Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 1996; 17(1): 4–29. Schworer H, Katsoulis S, Creutzfeldt W, Schmidt WE. Pituitary adenylate cyclase activating peptide, a novel VIP-like gut-brain peptide, relaxes the guinea-pig taenia caeci via apamin-sensitive potassium channels. Naunyn Schmiedebergs Arch Pharmacol 1992; 346(5): 511–14. Seki T, Shioda S, Izumi S, Arimura A, Koide R. Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina. Peptides 2000; 21(1): 109–13. Shen Z, Larsson LT, Malmfors G, Absood A, Hakanson R, Sundler F. A novel neuropeptide, pituitary adenylate cyclaseactivating polypeptide (PACAP), in human intestine: evidence for reduced content in Hirschsprung’s disease. Cell Tissue Res 1992; 269(2): 369–74. Shivers BD, Gorcs TJ, Gottschall PE, Arimura A. Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions. Endocrinology 1991; 128(6): 3055–65. Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L. Differential signal transduction by five splice variants of the PACAP receptor. Nature 1993; 365(6442): 170–5. Tatsuno I, Somogyvari-Vigh A, Arimura A. Developmental changes of pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor in the rat brain. Peptides 199415(1): 55–60.

1096 / Chapter 150 [36] Teng BQ, Grider JR, Murthy KS. Identification of a VIPspecific receptor in guinea pig tenia coli. Am J Physiol Gastrointest Liver Physiol 2001 Sep; 281(3): G718–25. [37] Teng B, Murthy KS, Kuemmerle JF, Grider JR, Makhlouf GM. Selective expression of vasoactive intestinal peptide (VIP)2/ pituitary adenylate cyclase-activating polypeptide (PACAP)3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Regul Pept 1998 Oct 16; 77(1–3): 127–34. [38] Vanneste G, Robberecht P, Lefebvre RA. Inhibitory pathways in the circular muscle of rat jejunum. Br J Pharmacol 2004 Sep; 143(1): 107–18. Epub 2004 Aug 9.

[39] Vaudry D, Gonzalez BJ. Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000; 52(2): 269–324. [40] Zeng N, Athmann C, Kang T, Lyu RM, Walsh JH, Ohning GV, Sachs G, Pisegna JR. PACAP type I receptor activation regulates ECL cells and gastric acid secretion. J Clin Invest 1999; 104(10): 1383–91. [41] Zizzo MG, Mule F, Serio R. Interplay between PACAP and NO in mouse ileum. Neuropharmacology 2004 Mar; 46(3): 449–55.

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151 Pancreatic Polypeptide XIAOYING DENG AND DAVID C. WHITCOMB

ABSTRACT

previously unknown peptide, which was named pancreatic polypeptide. Homologs of pancreatic polypeptide have been identified in many species [42], and a variety of actions have been reported related to inhibitory regulation of vagally mediated digestive function, regulation of carbohydrate metabolism, and effects on reproduction in rodents. Human PP has a molecular weight of 4184 daltons. Among the higher mammals, bovine, ovine, porcine, canine, and human PP differ from one another by 1–4 amino acid residues (but porcine and canine have identical structures). Avian PP differs significantly from mammalian PP in length (with 37 amino acids) and in sequence, with 17 out of 36 amino acids, compared to humans [36]. The biological activity of PP relies on the COOH-terminal hexapeptide fragment; removal of the amide radical on the COOH-terminal tyrosine residue results in loss of inhibiting pancreatic exocrine secretion by PP [52]. Pancreatic polypeptide is also similar to two other regulatory peptides, peptide YY (PYY) and neuropeptide Y (NPY), in amino acid number and sequence as well as three-dimensional structure.

Pancreatic polypeptide (PP) is a 36-amino-acid regulatory peptide with close structural similarities to peptide YY (PYY) and neuropeptide Y (NPY). PP is found in high concentrations in a subset of pancreatic islet cells and is released during a meal at rates proportionate to vagal activity. The action of PP is primarily inhibitory and directed toward organs innervated by the vagus, suggesting a major role in feedback regulation of foregut function. A primary site of action is the dorsal vagal complex, where PP crosses the blood–brain barrier and binds to Y4 receptors to directly modulate neurons involved in central regulation of visceral function. PP binding sites and Y4 receptors are also distributed in the stomach, intestine, liver, adrenal gland, sympathetic nerves, selected brain nuclei, and testis. The possibility of PP receptors other than Y4 has not been excluded. PP appears to modulate digestion, metabolism, weight, and reproduction, although mechanisms that are complex, incompletely understood, and probably different among the species studied.

Structure

DISCOVERY OF PANCREATIC POLYPEPTIDE AS IT RELATES TO THE GASTROINTESTINAL TRACT

The three-dimensional structure of various forms of PP have been determined by x-ray diffraction analysis and nuclear magnetic resonance [26, 41, 104]. The peptide takes on a U-shape (known as the PP-fold), which consists of two antiparallel helices, an NH2terminal polyproline type II helix (residues 1–6), and an amphiphilic alpha-helix (residue 15–30) that are connected by a type II beta-turn and held together by a core of interdigitating hydrophobic residues. Although the amino acid sequences of PP from various species differ, the three-dimensional structure appears to be

Pancreatic polypeptide (PP) is a 36-amino-acid peptide hormone primarily produced in Langerhans islets cells of the pancreas and released into the blood after the organism ingests a meal. PP was discovered by Kimmel et al. [36, 38] in chicken pancreas and by Lin and Chance [51] in bovine pancreas as a protein contaminant in pancreatic extracts during insulin purification. Sequence analysis by these groups revealed a Handbook of Biologically Active Peptides

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1098 / Chapter 151 conserved. The COOH-terminal hexapeptide extends out from the globular body of the molecule [26]. PP tends to form a dimer in concentrated solutions. Seven residues (Pro5, Pro8, Gly9 Ala12, Tyr27, Arg 33, and Arg35) are conserved among all the species studied to date.

In preproPP, the 36-amino-acid residue PP domain is preceded by a signal peptide sequence and followed by Gly-Lys-Arg plus a 16- to 30-residue (27 in human and 30 in rats) C-terminal extension peptide [93, 105]. The 36-residue product is generated by proteolytic cleavage and carboxyamidation.

Ontogeny STRUCTURE OF THE PRECURSOR mRNA/GENE AND PEPTIDE VARIANTS Precursor PP Multiple forms of PP are present in pancreatic extracts, tumors, and even the circulation [83, 99]. These forms probably reflect precursor proteins, splicing errors, or degradation products. Normally the pancreas releases only one form of PP [73]. The higher molecular forms probably represent the precursor and incompletely cleaved products, whereas the primary lower molecular form appears to be the COOHterminal 31–36 fragment [73]. Normally PP is derived from a 95-amino-acid precursor, prepro-pancreatic polypeptide (preproPP), with a molecular weight of 10,432. PreproPP is cleaved to generate three peptides: PP, an icosapeptide (20 amino acid residues), and a smaller peptide [47]. The gene coding PP is located on chromosome 17q21.1, which is 10 kb away from the gene for PYY [34]. A second PP-PYY cluster is seen in mammals and is located on chromosome 17q11 in humans [16]. However, the distance between the two genes is 20 kb, so these genes are not simple duplications. The PP, PYY, and NPY genes have four exons. In addition, the PP gene also includes a Goldberg-Hogness promoter consensus sequences separated by 24 or 25 bases from the cap site and a repetitive DNA element between 700 and 1500 bp downstream from the polyadenylation site [46, 47]. There is also a core enhancer sequence, GTGGAAAG in 5′-flank region. In human PP precursor, each domain in the protein is largely encoded by a separate exon [47]. Exon 1 encodes the 5′-untranslated region. Exon 2 encodes most of the functional domain terminating just before the Tyr36 residue. Exon 3 encodes the icosapeptide. Exon 4 encodes the carboxyl-terminal heptapeptide region of the precursor and the 3′-untranslated regions [47]. In addition, a mosaic form of preproPP has been observed [105]. The rat C-terminal peptide differs from the human precursor because it does not contain the icosapeptide sequence. After co-translational remove of a 29-amino-acid signal sequence, a 66-amino-acid prohormonal form is produced that contains the sequence of functional PP at the N-terminal [46]. This molecule is further cleaved and generates a 36-amino-acid PP.

A few PP-reactive cells can be detected in the pancreas as early as 10 weeks’ gestation [15, 27, 75]. However, the cell numbers remains relatively low during fetal development and infancy. Thereafter, the numbers increase progressively with age [88]. PP-immunoreactive cells can also be detected in the fetal gastrointestinal tract in regions that contain glucagon cells [45]. In rats, the mRNA for PP was detected as early as E21. The exclusive expression of PP in endocrine cells was established at P5 [70].

DISTRIBUTION OF THE mRNA AND PROTEIN PP is exclusively expressed in endocrine cells in the pancreas and gastrointestinal tract. PP was initially localized to endocrine cells in the outer rim of islet cells [43]. PP was specifically localized to F cells in the dog [24] and D cells in humans [33], and both cell types are now recognized to express PP [89]. It is unclear whether PP is expressed in nerves. PP-immunoreactive neuroendocrine cells have also been identified in the stomach of opossum, cat, and dog and are transiently expressed in human and rats during the postnatal period [20, 44, 97]. A few PP-reactive cells have been detected in intestine in most animal species studied [44]. The pancreas is the primary site for circulating PP. Approximately 93% of PP immunoreactivity, measured by bovine or avian PP antisera radioimmunoassay, was localized to within the pancreas, with the remaining PP being found in the mucosa of the gastrointestinal tract [2]. PP-immunoreactive cells are mainly distributed in pancreatic islets, but are also scatted in pancreatic parenchyma and ductal epithelium [43]. The relative distribution of these three regions depends on species. In most species, the PP cells and glucagon cells occupy a peripheral position in islets, with somatostatin cells interspaced among them and β-cells forming a central core [7, 43]. The density of PP cells is low in the pancreatic tail and higher in the head, whereas glucagon cells are more dense in the tail [7, 59]. This feature of the PP distribution might also reflect the ontogeny of PP cells in pancreas, with PP cells being predominant in the ventral bud and glucagon cells developed in the

Pancreatic Polypeptide / 1099 dorsal bud. In addition, the PP-immunoreactive cells in the human pancreas have two distinct morphology appearances: one has small electron-density granules (similar to rats) and the other has larger, more electronlucent granules (similar to cat and dog) [89]. No functional distinction is known.

RECEPTOR SUBTYPES, SIGNALING, AND DISTRIBUTION The distribution of PP receptors and the subsequent action continues to be an area of research and discovery. The challenges come from the close similarity of PP to PYY and NPY, with similar and overlapping functions; the complexity of the systems being investigated; and the co-expression of various receptors in the same tissue. Nevertheless, clear roles for PP are beginning to emerge. Initial clues came from binding studies, but they were greatly facilitated with the cloning of the PP receptor.

A

PP Binding Studies The first systematic study to determine PP binding sites was done by Kimmel et al. in chickens [37]. Radiolabeled avian PP (125I-aPP) was infused into groups of chickens with or without excess unlabled aPP. Displaceable binding was found in the spleen, duodenum, ileum, pancreas, and bone marrow, with lesser amounts in the proventriculus and liver [37]. In 1987, Schwartz et al. reported high-affinity binding of 125I-PP in the rat pheochromocytoma cell line, PC-12 [85]. Binding has been confirmed in vivo [87], with both the medulla and cortex expressing binding sites [103]. Although the density of PP binding sites in the rat adrenal gland is among the highest anywhere in the body, researchers were unable to demonstrate 125I-PP binding to human adrenal glands or membranes (Whitcomb, Vigna, and Taylor, unpublished data, 1990). PP binding sites were also noted in the suprarenal ganglion, a sympathetic ganglion associated with the adrenal gland (Whitcomb, Vigna, and Taylor, unpublished data, 1991; Fig. 1). The function of the PP receptors in the rat adrenal gland is not completely known, although NPY appears to modulate the release of catecholamines and aldosterone [90]. Thus, PP binding sites in the rat adrenal gland are consistent with the well-recognized association between the PP family of peptides and receptors on the sympathetic nervous system and in the regulation of metabolism. In 1990 Whitcomb et al. [102], using both an in vivo radioreceptor assay and receptor autoradiograph, identified PP receptors in the brain, including the dorsal vagal complex, areas of the hypothalamus, parabrachial

B

C FIGURE 1. Autoragiography showing 125I-PP binding sites in the rat suprarenal ganglia. A. Giemsa stain of retroperitoneal tissue from the rat, with structures as labeled. B. 125I-PP label alone. C. 125I-PP in the presence of unlabeled PP, demonstrating nonspecific binding. Note saturable binding of PP to the adjacent adrenal gland as well as to the nerve cell bodies within the sympathetic ganglia. The techniques are as described [103]. LN, lymph nodes; n, nerves.

nucleus, and intrapeduncular nucleus [96, 100, 102]. A more detailed study confirmed binding in the hypothalamus (arcuate and paraventricular nucleus), rostral forebrain (medial preoptic area, anterior olfactory nucleus, islands of Calleja, dorsal endopiriform nucleus,

1100 / Chapter 151 piriform cortex, and bed nucleus of the stria terminalis), medial amygdaloid nucleus, thalamus (anteromedial thalamic nucleus reuniens thalamic nucleus; and paraventricular thalamic nucleus), interpeduncular red nucleus, substantia nigra, parabrachial nucleus, locus coeruleus, mesencephalic trigeminal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract (NTS), and the area postrema [100]. The finding of specific PP receptors in the dorsal vagal complex was especially important because this complex regulates digestive function, satiety, and metabolism by integrating descending information from higher brain centers, sensory inputs, and ascending spinal cord signals in the NTS and relays an appropriate vagal drive through the dorsal motor nucleus of the vagus. The third component of this complex is the area postrema, one of the few central nervous system regions with an incomplete blood–brain barrier that allows peptide hormones to come into direct contact with central neurons [102]. The finding of PP binding sites in this area suggests that PP may regulate vagal function through a neuro-endocrine-neuro feedback loop [80, 101]. PP binding sites were also identified in regions of other circumventricular organs (e.g., hypothalamus), raising the possibility

A

B

that circulating PP could act at these sites. However, other central regions with PP binding are clearly excluded from the direct influence of circulating peptides. PP binding sites have also been identified in the liver. Initial studies failed to detect binding sites using isolated membranes [12] or in vivo radioreceptor assay [87], although binding was demonstrated in the ductus choledochus using this approach in rats [87] and binding was suggested with an in vivo approach in chickens [37]. Using more sensitive techniques, Nguyen et al. demonstrated high-affinity receptors on purified membranes [72]. We found faint binding in normal rat liver using receptor autoradiography, which was markedly upregulated in what appeared to be a subset of hepatocytes following bile duct ligation (Whitcomb, Vigna, and Taylor, unpublished observations, 1990; Fig. 2). PP binding to liver microsomes was also demonstrated to be upregulated in rats with chronic pancreatitis following oleic acid treatment [86]. These data confirm binding sites for PP in the liver, although the density is much less than the binding sites for insulin. The identity of the cells expressing PP binding sites is yet to be determined.

C

FIGURE 2. Autoradiography showing 125I-PP binding sites in the rat bile duct–ligated liver. A. Low-powered photomicrograph of a cross section of the rat liver 7–10 days after bile duct ligation. B. 125I-PP label alone. C. 125I-PP in the presence of unlabeled PP, demonstrating nonspecific binding. Note saturable binding of PP to a subset of hepatocytes. The techniques are as described [103].

Pancreatic Polypeptide / 1101 The intestine, including duodenum and ileum, appeared to have PP binding sites by in vivo receptor assay [87]. No binding to testis, epididymis, retroperitoneal fat, epididymal fat, muscle, or other tissues was identified using this in vivo binding technique.

PP (Y4) Receptor Clones In 1995 Lundell et al. [56] cloned the human PP receptor, which was quickly followed by the cloning of the PP receptor in other species [31, 57, 106]. A single PP receptor was identified, which is now called the Y4 receptor [64]. The Y4 receptors belong to the Y receptor family, which includes six subtypes, although others may also exist [49]. The human Y4 receptor shares ∼75% homology to rat Y4 receptors at the amino acid level [57, 106]. The Y4 receptor preferentially binds PP over PYY and NPY [64], with a rank order of human PP (affinity of 13.8 pM) = bovine PP ≥ human pro34 PYY > rat PP > human PYY (affinity of 1044 nM) = human NPY (affinity of 9.9 nM) [106]. The tissue distribution and ligand selectivity of the Y4 receptors differ among species [8, 106]. The Y4 receptor mRNA has been detected in the colon, small intestine, stomach, pancreas, prostate, and some brain subregions in human. In rats, Y4 receptor mRNA was detected in the colon at low levels and in the lung, testis, and brain [57] with expression in both epithelial and nonepithelial tissue of the small and large intestine [28]. Mouse mRNA was observed in heart and small intestine but not in the lung [31]. The Y receptors, including the Y4, are members of the seven-transmembrane-domain G-protein-coupled receptor family. The functional Y4 receptor may be a homodimer without the ability to form complexes with other Y receptors [9]. In stably transfected Chinese hamster ovary (CHO) cells, rat Y4 was shown, in the presence of agonists, to inhibit forskolin-stimulated cAMP synthesis and increase calcium influx [57]. Likewise, in cells cultured from the circular muscle layer of the rabbit stomach PP binding to the Y4 receptor induced concentration-dependent contraction through Gαq, which resulted in inositol 1,4,5-trisphosphate (IP3) formation and a subsequent increase in cytosolic free Ca2+ [66] (Fig. 3). Furthermore, the Y4 receptor was negatively coupled to adenylyl cyclase via Gαi2 [66].

BIOLOGICAL ACTIONS Gastrointestinal Tract A number of effects attributed to PP have been observed within the digestive system with infusion of PP or PP neutralizing antibodies, including on the stomach,

FIGURE 3. Illustration of the second-messenger signaling pathways for the Y2 and Y4 receptors in gastric smooth muscle. Note that activation of either Y2 or Y4 receptor results in similar downstream signaling. Modified from [66]. Gα, G protein subunits; AC, adenylate cyclase; PLC-B, phospholipage CB isoform; IP3, inasitol-1,4,5,-trisphosphate; CaM, calmodulin; MLCK, myosin light chain kinase; MLC20, myosin light chain phosphorylation site.

small and large intestine, pancreas, and gallbladder. The results were sometimes conflicting or inconsistent, a problem probably related to cross-reactivity between receptors and to species differences. For example, Schmidt et al. [82] recently noted clear effects of PP in humans related to gastric emptying that were not seen by others, which they attributed to their use of human PP rather than PP from other species. As already noted, PP is released from pancreas in direct relationship to feeding and efferent vagal activity. The pancreas does release a small amount of PP during fasting (basal PP secretion) and a much higher amount during all phases of digestion. Basal PP secretion fluctuates rhythmically, with peaks every 90 minutes coupled with the interdigestive myoelectric complex [35, 74]. Postprandial PP release is mainly mediated by efferent vagal stimulation and augmented by cholecystokinin (CCK) [48] and inhibited by anticholinergic substances or vagotomy [25, 84].

1102 / Chapter 151 Gastric Acid Secretion and Emptying Among the first reported physiological actions of bovine PP was the ability to stimulate gastric secretion in fasted dogs while inhibiting pentagastrin-stimulated gastric secretion at the pharmacological doses of 20– 100 μg/kg/h [52]. However, bovine PP failed to alter basal and pentagastrin-stimulated gastric secretion, pepsin output, and gastrin level in human volunteers at the dose of 218 pmol/kg/h, a dose designed to reproduce human postprandial plasma level of PP [29]. PP at higher dose (1–100 μg/kg) stimulated gastric emptying [50], but it failed to alter gastric emptying in human volunteers when infused at the physiological doses (1–2 pmol/kg/h) [4]. Recently, Schmidt et al. [82] demonstrated that human PP does delay gastric emptying of solids, but not liquids, in humans. The mechanism of PP’s action in vivo appears to involve integrated effects from multiple sites that differ depending on hormone levels. The Y4 receptors have been identified on the circular smooth muscle of rabbit stomach, which contracts with PP in a dose-dependent manner [66]. This would be expected to increase the emptying of fluids rather than solids, as seen by Lin et al. [50], at high doses. McTigue et al. [61, 63] demonstrated that the direct application of PP to the dorsal vagal complex strongly stimulated gastric acid secretion and motility. However, later studies demonstrated a mixture of response to PP injections in individual central neurons [62]. This is opposite of the effect on acid secretion seen in dogs [52], although the effect of regulatory peptides applied centrally are often the opposite of what is seen when there are given systemically.

Pancreatic Secretion Infusion of bovine PP in human volunteers and dogs and infusion of porcine PP in dogs significantly inhibit pancreatic exocrine secretion [1, 30, 52, 54, 95]. However, there are no PP receptors on pancreatic acinar cells [55]. Putnam et al. [79] investigated the mechainism of PP-dependent inhibition of pancreatic secretion by infusing rats with PP at the physiological dose of 50, 200, and 800 pmol/kg/h. PP significantly inhibited pancreatic responses to a variety of secretagogues. However, the inhibitory effect differed markedly, being most potent in inhibiting 2-deoxy-d-glucose (2-DG) > CCK >> efferent vagus electrical nerve stimulation or bethanechol infusion. The mechanism became clear with the identification of PP binding sites in the brain stem within the region that controls pancreatic secretion [102]. The site of inhibition is between the afferent and efferent vagus and this explains why PP [18] (or PYY [17]) is more potent at inhibiting CCKand 2-DG-stimulated secretion (proximal stimulation)

compared to electrical stimulation of the efferent vagus or bethanechol (distal stimulation). The reason that the area postrema within the dorsal vagal complex is important is because it acts as a port of entry from PP into this region of the brain, as was demonstrated with area postrema lesions. Infusion of PP (or PYY) with a physiological dose range, inhibits basal, 2-DG-, and CCK-stimulated pancreatic secretion only if the area postrema is intact [18, 19]. Of note, the effect of PP on the central inhibition of pancreatic secretion differs from PYY in that PYY appears to be more potent in inhibiting CCK-stimulated pancreatic secretion [17], whereas PP is more potent at inhibiting 2-DG-stimulated secretion [18]. PP also appears to have peripheral sites of action that project centrally because intraabdominal injection of PP causes c-fos activation in the NTS independent of the area postrema [19].

Intestinal Motility An initial study in the dog demonstrated that intravenous infusion of bovine PP at the dose of 50–100 μg/ kg enhanced intestinal motility [50]. Bolus injection of a higher dose of bovine PP increased gut motility in antrum, duodenum, and colon of the dog, whereas bolus injection of a low dose decreased intraluminal pressure in the canine antrum, pylorus, duodenum, ileocecal valve, and descending colon [50]. In humans, bovine PP reduced motilin levels by 80% [3]. In smooth muscle cells from the human jejunum, PP caused contraction of circular but not longitudinal muscles [65]. PP also produced a concentrationdependent transient inhibition of the spontaneous contractions of the rabbit ileum [21]. In the rat proximal colon, both Y2 and Y4 receptors were expressed at high levels and PP induced contractions, which were blocked by tetrodotoxin [22], suggesting that the effects on contraction may be neurally mediated. The cells expressing high levels of Y4 receptor in the colon need to be clarified.

Gallbladder PP has no effect on hepatic bile secretion, whereas bovine and porcine PP inhibits intraluminal gallbladder pressure in conscious dogs and pigs. The inhibition is indirect because PP does not alter the tension in strips of human gallbladder [53].

Metabolic Effects In experimental animals, intravenous infusion of human PP significantly decreased the insulin levels in both fasting and fed states without alteration of glucagon [68, 82]. Early studies in humans using intravenous

Pancreatic Polypeptide / 1103 infusion of bovine PP at the dose of 100 pmol/kg/min only slightly increased basal insulin levels and had no effect on glucose- and arginine-induced insulin and glucagon secretion [3]. When the dose of PP was decreased to 1–2 pmol/kg/min, there was no effect on insulin, glucagon, gastrin, secretin, enteroglucagon, and gastric inhibitory peptide [3]. However, when synthetic human PP was infused into humans, the postprandial rise in insulin was significantly delayed from that seen in animal models [82]. In addition to an effect on insulin release, PP appears to affect glucose metabolism. In dogs, the infusion of PP has no effect on glucose levels [52]. In humans, PP prolongs the postprandial rise in plasma glucose [82]. PP infusion in subjects with chronic pancreatitis and insulin resistance caused an improvement in oral glucose tolerance tests, suggesting that PP improves hepatic insulin sensitivity [13, 92]. In dogs with pancreatectomy or pancreatectomy with distal pancreas autotransplantation, the infusion of PP improved glucose disposal and hepatic glucose output, an effect that was lost over time [78]. PP also reduces insulin requirements in an artificial pancreas during the first 48 hours following surgery [39], raising the possibility that this effect may be most important during stress. The effects of PP were believed to be on the liver, a hypothesis that is supported by the demonstration of PP receptors in the liver (e.g., Fig. 2), which appear to act, in part, by increasing the density of the hepatic insulin receptors [86].

and hypothalamic orexin was decreased in PPoverexpressing mice [6]. The repeated administration of PP decreased body weight gain and ameliorated insulin resistance and hyperlipidemia in both ob/ob obese mice and ob/ob obese mice with a fatty liver [6]. Liver enzyme abnormalities in these mice were also ameliorated by PP [6]. Knockout mice have been produced that lack the PP Y4 receptor. These mice have elevated PP levels and develop the same phenotype as the transgenic mice with high PP levels (i.e., reduced food intake, body weight, and fat mass, as well as reduced glucose-induced insulin secretion and gastric emptying), with effects more evident in male compared to female transgenic mice [81]. This suggests that PP may have important actions through peripheral binding sites that are independent of the Y4 receptor. The relevance of these finding for humans may be seen in the excess body weight of male Pima Indians. Koska et al. [40] found that fasting and postprandial PP levels were negatively associated with body size and adiposity. When these subjects were followed prospectively for nearly 5 years, the original change in PP response to the meal was negatively associated with the change in body weight. In contrast, a high fasting PP level was positively associated with change in body weight [40]. These data suggest that PP does have important effects on metabolism and body fat, although the mechanism is complex.

Food Intake and Regulation of Weight

Reproduction

PP appears to have significant effects on food intake and use in rodents. In early studies, repeated injection of bovine PP reduced food intake in obese mice and returned blood glucose and body weight to normal [58, 67]. However, a careful dose-response study of the PP on feeding raised doubts about the physiological importance of acute levels of PP for satiety, food intake, and PP’s role in the ob/ob mouse [94]. Furthermore, the primary defect in the ob/ob mice was subsequently found to be related to leptin [76]. On the other hand, the central administration of PP stimulates food intake and accelerates gastric emptying [5, 10, 60, 63]. Recently, the effects of PP were demonstrated with repeated injections of PP in ob/ob mice and in transgenic mice. Transgenic mice that express PP at levels 20 times normal gain less weight with specifically reduced food intake and fat mass compared with controls, a result that was more evident in male than in female mice [98]. The decreased food intake and gastric emptying can be diminished by anti-PP serum in these mice. Furthermore, PP reduces leptin in white adipose tissue and corticotropin-releasing factor gene expression in obese mice [6]. The expression of gastric ghrelin

Cloning of the PP Y4 receptor resulted in the discovery of high levels of Y4 receptors in the testis [57]. Sainsbury et al. [81] crossed the Y4 knockout mice with ob/ob mice, which are normally infertile. This resulted in significantly increased plasma testosterone levels in males compared to ob/ob controls and significantly increased absolute testis and seminal vesicle weights to values not significantly different from those of OB/ob or wild-type control mice [81]. Furthermore, female mice had improved fertility, enhanced mammary gland development, and improved gonadotropin-releasing hormone production [81]. However, there was no change in the obesity in the ob/ob, Y4-/Y4- double knockout mice.

PHYSIOPATHOLOGICAL IMPLICATIONS Increased basal PP concentration (∼22%–77%) has been observed in a variety of pancreatic endocrine tumors [23, 69, 77, 83] . The frequency with which elevated PP concentration occurs varies with the origin and nature of the tumor [11]. Pancreatic tumors associ-

1104 / Chapter 151 ated with PP can be divided into three categories based on the abundance of PP in these tumors: tumors exclusively or predominantly consisting of PP cells, sometimes called PP-omas; tumors in which PP cells are part of a mixed tumor cell population; and hyperplasia of PP cells [83]. The typical tumors with elevated PP are vipoma and multiple endocrine neoplasia type 1 (MEN1) [69, 83]. In general, endocrine tumors that have PP as their predominant secretory product are usually silent. Other causes also elevate PP concentration, such as PP cell hyperplasia and chronic stimulation of PP cells secondary to sustained hypoglycemia in insulinoma [71]. However, the value of plasma PP as a tumor marker appears to be limited to MEN1 [69]. PP-omas account for 10% of pancreatic endocrine tumors [32]. As with other neuroendocrine tumors, PP-omas display a range of aggressiveness and malignant behavior. PP-omas are more frequently in the pancreatic head, but also could occur in any part of the pancreas [91], as would be expected by the distribution of PP cells in the pancreas. In addition, PP has been reported to be present in approximately 13% of carcinoid tumors [14].

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1106 / Chapter 151 [63] McTigue DM, Rogers RC. Pancreatic polypeptide stimulates gastric acid secretion through a vagal mechanism in rats. Am J Physiol 1995;269(5 Pt 2):R983–987. [64] Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 1998;50(1):143–150. [65] Misra S, Mahavadi S, Grider JR, Murthy KS. Differential expression of Y receptors and signaling pathways in intestinal circular and longitudinal smooth muscle. Regul Pept 2005;125(1–3): 163–172. [66] Misra S, Murthy KS, Zhou H, Grider JR. Coexpression of Y1, Y2, and Y4 receptors in smooth muscle coupled to distinct signaling pathways. J Pharmacol Exp Ther 2004;311(3): 1154–1162. [67] Mordes JP, Eastwood GL, Loo S, Rossini AA. Pancreatic polypeptide causes diarrhea and weight loss in obese mice but not in lean littermates. Peptides 1982;3(5):873–875. [68] Murphy WA, Fries JL, Meyers CA, Coy DH. Human pancreatic polypeptide inhibits insulin release in the rat. Biochem Biophys Res Commun 1981;101(1):189–193. [69] Mutch MG, Frisella MM, DeBenedetti MK, Doherty GM, Norton JA, Wells SA, Jr., Lairmore TC. Pancreatic polypeptide is a useful plasma marker for radiographically evident pancreatic islet cell tumors in patients with multiple endocrine neoplasia type 1. Surgery 1997;122(6):1012–1019; discussion 1019–1020. [70] Myrsen-Axcrona U, Ekblad E, Sundler F. Developmental expression of NPY, PYY and PP in the rat pancreas and their coexistence with islet hormones. Regul Pept 1997;68(3): 165–175. [71] Nelson RL, Service FJ, Ilstrup DM, Go VL. Are elevated pancreatic polypeptide levels in patients with insulinoma secondary to hypoglycaemia? Lancet 1980;2(8196):659–661. [72] Nguyen TD, Wolfe MS, Heintz GG, Whitcomb DC, Taylor IL. High affinity binding proteins for pancreatic polypeptide on rat liver membranes. J Biol Chem 1992;267(13):9416–9421. [73] O’Hare MM, Huda I, Sloan JM, Kennedy TL, Buchanan KD. Characterization of immunoreactive forms of pancreatic polypeptide in islet cell tumors using antisera with different regional specificities. Cancer 1985;55(9):1895–1898. [74] Owyang C, Achem-Karam SR, Vinik AI. Pancreatic polypeptide and intestinal migrating motor complex in humans. Effect of pancreaticobiliary secretion. Gastroenterology 1983;84(1): 10–17. [75] Paulin C, Dubois PM. Immunohistochemical identification and localization of pancreatic polypeptide cells in the pancreas and gastrointestinal tract of the human fetus and adult man. Cell Tissue Res 1978;188(2):251–257. [76] Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269(5223): 540–543. [77] Polak JM, Bloom SR, Adrian TE, Heitz P, Bryant MG, Pearse AG. Pancreatic polypeptide in insulinomas, gastrinomas, vipomas, and glucagonomas. Lancet 1976;1(7955):328–330. [78] Prillaman HM, Cox SB, Freedlender AE, Cornett GE, Jones HA, Flanagan TL, Chance RE, Hoffmann JA, Andersen DK, Elahi D, et al. The effect of pancreatic polypeptide on glucose disposal after surgical alterations of the pancreas. Ann Surg 1992;216(5):574–582. [79] Putnam WS, Liddle RA, Williams JA. Inhibitory regulation of rat exocrine pancreas by peptide YY and pancreatic polypeptide. Am J Physiol 1989;256(4 Pt 1):G698–703.

[80] Rogers RC, McTigue DM, Hermann GE. Vagovagal reflex control of digestion: afferent modulation by neural and “endoneurocrine” factors. Am J Physiol 1995;268(1 Pt 1):G1–10. [81] Sainsbury A, Schwarzer C, Couzens M, Jenkins A, Oakes SR, Ormandy CJ, Herzog H. Y4 receptor knockout rescues fertility in ob/ob mice. Genes Dev 2002;16(9):1077–1088. [82] Schmidt PT, Naslund E, Gryback P, Jacobsson H, Holst JJ, Hilsted L, Hellstrom PM. A role for pancreatic polypeptide in the regulation of gastric emptying and short-term metabolic control. J Clin Endocrinol Metab 2005;90(9):5241–5246. [83] Schwartz TW. Pancreatic-polypeptide (PP) and endocrine tumours of the pancreas. Scand J Gastroenterol Suppl 1979;53: 93–100. [84] Schwartz TW, Rehfeld JF, Stadil F, Larson LI, Chance RE, Moon N. Pancreatic-polypeptide response to food in duodenal-ulcer patients before and after vagotomy. Lancet 1976;1(7969): 1102–1105. [85] Schwartz TW, Sheikh SP, O’Hare MM. Receptors on phaeochromocytoma cells for two members of the PP-fold family— NPY and PP. FEBS Lett 1987;225(1–2):209–214. [86] Seymour NE, Spector SA, Andersen DK, Elm MS, Whitcomb DC. Overexpression of hepatic pancreatic polypeptide receptors in chronic pancreatitis. J Surg Res 1998;76(1):47–52. [87] Shetzline MA, Zipf WB, Nishikawara MT. Pancreatic polypeptide: identification of target tissues using an in vivo radioreceptor assay. Peptides 1998;19(2):279–289. [88] Shevell JL, Schweisthal MR. Regional distribution of pancreatic polypeptide cells in the 21-day fetal rat pancreas. Experientia 1982;38(9):1093–1095. [89] Solcia E, Fiocca R, Capella C, Usellini L, Sessa F, Rindi G, Schwartz TW, Yanaihara N. Glucagon- and PP-related peptides of intestinal L cells and pancreatic/gastric A or PP cells. Possible interrelationships of peptides and cells during evolution, fetal development and tumor growth. Peptides 1985;6(Suppl 3):223–229. [90] Spinazzi R, Andreis PG, Nussdorfer GG. Neuropeptide-Y and Y-receptors in the autocrine-paracrine regulation of adrenal gland under physiological and pathophysiological conditions (review). Int J Mol Med 2005;15(1):3–13. [91] Strodel WE, Vinik AI, Lloyd RV, Glaser B, Eckhauser FE, Fiddian-Green RG, Turcotte JG, Thompson NW. Pancreatic polypeptide-producing tumors. Silent lesions of the pancreas? Arch Surg 1984;119(5):508–514. [92] Sun YS, Brunicardi FC, Druck P, Walfisch S, Berlin SA, Chance RE, Gingerich RL, Elahi D, Andersen DK. Reversal of abnormal glucose metabolism in chronic pancreatitis by administration of pancreatic polypeptide. Am J Surg 1986;151(1): 130–140. [93] Takahashi H, Nakano K, Adachi Y, Aoki N, Hajiro K, Yamamoto T, Higashizawa T, Chikugo T, Suzuki T. Multiple nonfunctional pancreatic islet cell tumor in multiple endocrine neoplasia type I. A case report. Acta Pathol Jpn 1988;38(5):667–682. [94] Taylor IL, Garcia R. Effects of pancreatic polypeptide, caerulein, and bombesin on satiety in obese mice. Am J Physiol 1985;248(3 Pt 1):G277–280. [95] Taylor IL, Solomon TE, Walsh JH, Grossman MI. Pancreatic polypeptide. Metabolism and effect on pancreatic secretion in dogs. Gastroenterology 1979;76(3):524–528. [96] Trinh T, van Dumont Y, Quirion R. High levels of specific neuropeptide Y/pancreatic polypeptide receptors in the rat hypothalamus and brainstem. Eur J Pharmacol 1996;318(1): R1–3. [97] Tsutsumi Y. Immunohistochemical studies on glucagon, glicentin and pancreatic polypeptide in human stomach: normal and pathological conditions. Histochem J 1984;16(8):869–883.

Pancreatic Polypeptide / 1107 [98] Ueno N, Inui A, Iwamoto M, Kaga T, Asakawa A, Okita M, Fujimiya M, Nakajima Y, Ohmoto Y, Ohnaka M, Nakaya Y, Miyazaki JI, Kasuga M. Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice. Gastroenterology 1999;117(6):1427–1432. [99] Villanueva ML, Hedo JA, Marco J. Heterogeneity of pancreatic polypeptide immunoreactivity in human plasma. FEBS Lett 1977;80(1):99–102. [100] Whitcomb DC, Puccio AM, Vigna SR, Taylor IL, Hoffman GE. Distribution of pancreatic polypeptide receptors in the rat brain. Brain Res 1997;760(1–2):137–149. [101] Whitcomb DC, Taylor IL. A new twist in the brain-gut axis. Am J Med Sci 1992;304(5):334–338. [102] Whitcomb DC, Taylor IL, Vigna SR. Characterization of saturable binding sites for circulating pancreatic polypeptide in rat brain. Am J Physiol 1990;259:G687–G691.

[103] Whitcomb DC, Vigna S, McVey D, Taylor I. Localization and characterization of pancreatic polypeptide receptors in the rat adrenal gland. Am J Physiol 1992;262(25):G532– G536. [104] Wood SP, Pitts JE, Blundell TL, Tickle IJ, Jenkins JA. Purification, crystallisation and preliminary X-ray studies on avian pancreatic polypeptide. Eur J Biochem 1977;78(1): 119–126. [105] Yamamoto H, Nata K, Okamoto H. Mosaic evolution of prepropancreatic polypeptide. J Biol Chem 1986;261(14): 6156–6159. [106] Yan H, Yang J, Marasco J, Yamaguchi K, Brenner S, Collins F, Karbon W. Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors. Proc Natl Acad Sci USA 1996;93(10):4661– 4665.

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152 Peptide YY GUILLERMO GOMEZ, GUIYUN WANG, ELLA W. ENGLANDER, AND GEORGE H. GREELEY, JR.

ABSTRACT

STRUCTURE OF THE PYY GENE AND PYY BIOSYNTHESIS

Peptide YY (PYY) is a 36-amino-acid peptide hormone produced primarily in enteroendocrine L cells of the distal intestine. Dietary fat is a strong secretagogue for PYY secretion. The main function of PYY physiologically is to serve as an ileal brake and to facilitate digestion and absorption of ingested nutrients by its inhibition of gastric emptying and intestinal transit. PYY is also a strong enterogastrone and inhibitor of pancreatic secretion. More recently, PYY(3–36) has been shown to inhibit food intake in experimental animals and humans.

The PYY gene has been cloned from several species, including the rat and human [19, 22]. The conserved structural organization of the PYY, NPY, and PP genes indicates that these genes arose by duplication of a common ancestral gene. The PYY gene consists of four exons and three introns. Each exon encodes a functional domain of the mRNA:exon 1, the 5′ untranslated region; exon 2, the signal peptide and a majority of the mature form of the peptide; exon 3, the amidation region and much of the carboxy-terminal peptide; and exon 4, the carboxy-terminal heptapeptide and the 3′ untranslated region [19]. The prepro form of PYY is synthesized as a signal peptide followed by the PYY(1– 36), a Gly-Lys-Arg cleavage-sequence, and a carboxyterminal flanking peptide [22]. The PYY precursor form is processed at the carboxy-terminal by a prohormone convertase, the Lys-Arg sequence is removed by a carboxypeptidase, and the carboxy-terminal tyrosine is amidated.

INTRODUCTION Peptide YY (PYY) is a 36-amino-acid peptide hormone that is structurally similar to two other gut peptides, pancreatic polypeptide (PP), and neuropeptide Y(NPY ) [31]. PYY, PP, and NPY are each 36 amino acids in length and have an amidated carboxy-terminus. This family of peptides is called the PP-fold family of peptides because they have a hairpin-like three-dimensional structure called the PP-fold. PYY is produced primarily in enteroendocrine L cells in the intestinal epithelium of the ileum-colon and is a candidate enterogastrone because PYY is secreted into the systemic circulation in response to dietary fat and is a potent inhibitor of gastric acid secretion. PYY can also inhibit pancreatic exocrine secretion, gastric emptying, intestinal transit, and intestinal fluid secretion. Because of its potent antisecretory action, PYY may be an endogenous inhibitor of diarrhea. More recently, a variant of PYY, PYY(3–36), has been shown to inhibit food intake in experimental animals and in humans. Handbook of Biologically Active Peptides

GASTROINTESTINAL DISTRIBUTION AND ONTOGENY OF INTESTINAL PYY PYY is produced in the enteroendocrine L cells in the mucosal epithelium of the terminal ileum, colon, and rectum (Fig. 1) [7, 13]. Much lower amounts of PYY are produced in the stomach antrum, proximal small intestine, and pancreatic endocrine cells. In the distal intestine PYY is co-produced with proglucagon, and in the pancreas PYY may reside with glucagon or PP.

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FIGURE 1. Distribution of PYY immunoreactivity in the digestive tract of the (A) rat (full thickness) and (B) dog (mucosal and muscle layers). In the dog, the duodenum, jejunum, and ileum were divided into three segments of equal length. The colon was divided into two segments. A. * = p < 0.05 vs. antrum, fundus, duodenum, jejunum, and mid-ileum; † = p < 0.05 vs. distal ileum. B. * = p < 0.05 vs. antrum, fundus, duodenum, and proximal and distal jejunum; † = p < 0.05 vs. muscle layer of respective region; ‡ = p < 0.05 vs. distal jejunum and proximal and mid-ileal mucosal layers. Modified and reproduced from [13].

PYY is the first major gut peptide hormone to appear during development in the mouse colon [8, 32]. On embryonic day 15.5, colonic PYY expression is observed. In the rat, both PYY mRNA and peptide are detected initially at 17 days of gestation; their levels continue to increase during the postnatal period. After weaning, there are coordinated declines in levels of PYY mRNA and PYY peptide when pups switch from breast milk to an adult diet. The pattern of rat intestinal PYY gene expression agrees with the pattern of plasma PYY levels in human neonates. In newborn cord blood, plasma PYY levels are higher when compared with plasma PYY levels in normal fasting adults, and plasma PYY levels increase during the first 12 days postbirth in newborns maintained on either breast milk or formula. These observations suggest that intestinal PYY gene expression is controlled by dietary nutrients and that changes in

the intestinal PYY gene expression have a role in the adaptation of the bowel to the changes in dietary intake. PYY has been detected in the fetal mouse pancreas at embryonic day 9.5 and is detected in each of the four islet cell types. In the rat pancreas, PYY expression is first detected on day 15 of gestation and rises to its maximum on day 18. At this time, pancreatic PYY mRNA levels are sevenfold higher than in the fetal and adult colon, and 90-fold higher than those levels in the adult pancreas, in contrast to the intestine. Pancreatic PYY mRNA levels drop dramatically after birth. The functional role of PYY in the pancreas during development is not clear; however, PYY may participate in the regulation of pancreatic and gastrointestinal (GI) functions by endocrine and paracrine pathways. PYY is measurable by immunoassay in the adult canine pancreas, with the highest amounts in the right lobe and head of the dog pancreas [40]. The early developmental appearance of intestinal PYY gene expression implies a role for PYY in the development of the intestine and in the regulation of epithelial cell replication or differentiation. PYY may also have a developmental influence on the stomach and pancreas during this period. Although gastric acid and pancreatic secretion are low during the postnatal period and do not achieve adult levels until weaning, PYY may regulate their regulation because it is a potent inhibitor of gastric acid and pancreatic exocrine secretion in adult animals [11, 15].

REGULATION OF PYY SECRETION In humans, dietary fat is a potent stimulus for release of PYY from the intestine [1]. PYY is released from the intestine in proportion to the calories ingested. In dogs, the ability of different nutrients given either into the duodenum or colon to stimulate PYY secretion has been described [12]. Fatty acids (oleic acid) and glucose (2 g/kg) were shown to be the strongest PYY secretagogues when administered into the duodenum. Amino acids and intact protein do not cause PYY release when given intraduodenally. However, a mixture of the amino acids tryptophan and phenylalanine or intact protein administered into the colon of dogs will cause PYY secretion [39]. Short-chain fatty acids, which are normal constituents of feces, when given into the isolated perfused colon of the rabbit, will stimulate PYY secretion. Interestingly, plasma levels of PYY increase significantly within 15–30 min after the ingestion of nutrients or in response to luminal administration of a fatty acid in humans, dogs, and rats (Fig. 2) [14]. PYY secretion in response to the intraduodenal administration of fat causes PYY release even when the flow of chyme is

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Minutes FIGURE 2. Plasma PYY levels in response to an oral mixed meal in conscious dogs. Note that circulating PYY levels are elevated 15 min after feeding the meal. B1 and B2 are basal plasma PYY levels. Reproduced from Jeng Y-J, Hill FLC, Lluis F, Gomez G, Izukura M, Kern K, et al. Peptide YY release and actions. In: Thompson JC, Cooper CW, Greeley GH, Jr., Rayford PL, Singh P, Townsend CM, Jr., editors. Gastrointestinal Endocrinology: Receptors and PostReceptor Mechanisms, San Diego: Academic Press; 1990. p. 371–386.

prevented from reaching the distal intestine; the removal of the ileum-colon extinguishes the intestinal PYY response to fat. This rapid release of PYY resulted in the speculation that another gut hormone released from the upper gut triggered PYY release. It was shown that intravenous (IV) cholecystokinin (CCK) caused a dose-dependent release of PYY, and further experiments showed that a CCK-A-type-receptor antagonist prevented the PYY response to a fatty meal or to exogenous CCK, supporting the role of intestinal CCK in causing PYY secretion [14]. The influence of growth factors on regulation of intestinal PYY homeostasis has been studied [21, 35]. Both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) stimulate intestinal PYY expression and peptide levels in rodents. GH and IGF-1 also stimulate PPY secretion. In terms of a molecular mechanism, the elevation in intestinal PYY expression by IGF-1 involves Sp1 binding sites in the proximal promoter region of PYY [34]. Neural pathways may have a role in the regulation of intestinal PYY secretion. In dogs, either atropine, hexamethonium, or atropine plus hexamethonium administration decreases food-induced secretion of PYY [41]. Beta-adrenergic blockade with propranolol or depletion of nerve terminal stores of catecholamines with reserpine do not influence nutrient-induced PYY secretion. The IV administration of a β2-adrenergic agonist, terbutaline, prompts PYY secretion [18]. Truncal vagotomy elevates resting-fasted and food-

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stimulated release of PYY. The IV administration of bethanechol to dogs, a cholinergic agonist, stimulates the release of PYY, and direct electrical stimulation of the vagus stimulates PYY release. Together, these findings suggest that stimulated PYY levels are dependent on ganglionic transmission and an atropine-blockable postganglionic parasympathetic pathway. A vagal cholinergic mechanism may act in a tonic fashion to inhibit PYY secretion. Although adrenergic mechanisms do not participate in nutrient-stimulated PYY release, electrical stimulation of the splanchnic nerves will increase PYY secretion, implying that the sympathetic nervous system has a role in PYY secretion. Interruption of intestinal intramural neural pathways does not modify food-induced PYY secretion, implying that intramural neural mechanisms do not participate in food-induced release of PYY [14].

PYY RECEPTORS PYY, NPY, and PP bind to a family of G-protein-linked receptors (called Y receptors) belonging to the rhodopsin-like superfamily (class 1) of receptors [6, 9]. There are five different Y receptor subtypes cloned from mammals (Y1, Y2, Y4, Y5, and y6). All Y receptor subtypes are expressed in either the small or large intestine with specific distribution profiles [9]. Y1, Y2, and Y5 bind NPY and PYY preferentially, whereas Y4 binds PP preferentially. The responses to exogenous PYY, NPY, and PP may reflect the peptide’s activity on local neuronal receptors as well as direct activity on the cells of the targeted tissue. Y1 receptors are expressed only in nonepithelial colonic cells, and Y2 and Y4 receptors are expressed in epithelial and nonepithelial cells of both the small and large intestine. The Y5 receptor is found restricted to epithelial crypts of the small intestine and nonepithelial cells of the colon. Y1, Y2, Y4, Y5, and y6 receptors are coupled to inhibitory G-proteins (Gi) and cause the inhibition of cAMP synthesis [20, 30, 33]. In intestinal epithelial cells (IEC-6), the activation of the Y1 receptor is coupled to mitogen-activated protein kinase (MAPK). Y1, Y2, Y4, and Y5 receptors also couple to phospholipase C (PLC) and cause release of Ca2+ from intracellular stores [30]. The Y1 receptor is found in the GI tract and is thought to mediate a PYY-induced inhibition of fluid secretion in the intestine. The Y4 receptor is expressed in the pancreas and mediates an inhibition of pancreatic exocrine secretion. On rabbit gastric circular smooth muscle cells, Y1, Y2, and Y4 receptors are negatively coupled to adenylyl cyclase through activation of Gi2. Y2 and Y4 receptors have been shown to be coupled to inositol triphosphate (IP3)-dependent (Ca2+)i release and activation of PLC-β by activation of Gq [26].

1112 / Chapter 152 BIOLOGICAL ACTIONS ON THE GASTROINTESTINAL TRACT PYY has been called an ileal brake because the IV administration of PYY has been shown to inhibit gastric emptying and intestinal transit in rodents, dogs, and humans. IV PYY inhibits gastric acid secretion in the dog, rat, rabbit, cat, and human [3, 11]. In the dog, PYY inhibits pentagastrin-stimulated, but not histamine- and cholinergic-stimulated (i.e., bethanechol), gastric acid secretion [15]. In addition, evidence in the dog indicates that PYY may decrease gastric acid secretion by blocking the cephalic phase of gastric acid secretion [28]. Exogenous PYY is a strong inhibitor of the cephalic phase of gastric acid secretion in humans, indicating that the enterogastrone action of PYY is mediated partly via neural pathways. In humans, the inhibitory action of PYY on acid secretion has been shown to be independent of somatostatin (SRIF) and gastrin, whereas in the cat, PYY can inhibit pentagastrin-stimulated acid secretion by the stimulation of SRIF secretion. The full 36residue form of PYY is required for its inhibitory action on acid secretion because PYY(1–36) is more potent than PYY(3–36) and PYY(4–36) [37]. In isolated rat gastric enterochromaffin-like (ECL) cells, PYY can inhibit gastric acid secretion by blocking gastrin-induced histamine release and Ca2+ entry via the activation of the Y1 receptor [38]. PYY, given systemically, inhibits thyrotropin-releasing hormone (TRH) stimulation of vagal efferent cholinergic projections to the stomach and acid secretion. The administration of PYY directly into the cisterna magna and dorsal vagal complex (DVC) inhibits gastric transit and antral motility. In rats, PYY given into the dorsal motor nucleus (DMN) causes a dose-dependent and vagal-dependent activation of gastric acid secretion [36]. The medullary region contains vagovagal circuits that communicate between the GI tract and central nervous system (CNS). PYY receptors have been identified autoradiographically in the DVC of rats, and radiolabeled PYY given IV penetrates into the DVC [16]. These neural PYY receptors may be stimulated by PYY released from enteric sites or the CNS. The IV administration of PYY inhibits pancreatic exocrine secretion. PYY can lower the stimulatory actions of luminal nutrients as well as exogenous and endogenously released CCK, secretin, and neurotensin on the exocrine pancreas [25]. PYY inhibits the pancreatic secretion of juice, bicarbonate, and pancreatic enzymes. PYY given at physiological levels inhibits meal-induced pancreatic secretion, and the administration of anti-PYY serum elevates pancreatic secretion in the rat. PYY(1– 36), PYY(3–36), and PYY(4–36) inhibit pancreatic secretion with similar potencies in the dog [37]. The inhibitory effects of PYY on the pancreas may be mediated by

several pathways. PYY presumably exerts its inhibitory effects on pancreatic secretion by the activation of adrenergic pathways because phentolamine plus propranolol lowers the inhibitory effects of PYY. The delay in intestinal transit of nutrients by IV PYY is dependent on a β-adrenergic pathway that acts via serotonergic and opioid pathways [23]. There is also evidence to support the idea that PYY acts via intrapancreatic neural pathways as well as in the CNS to inhibit neurally mediated pancreatic secretion. In the rat, PYY has been shown to inhibit pancreatic secretion through the DVC of the CNS. The area postrema is the major site of PYYmediated inhibition of CCK and secretin-stimulated pancreatic secretion. Furthermore, PYY may regulate pancreatic exocrine secretion by the inhibition of intestinal CCK secretion because the IV administration of PYY blocks the release of CCK in response to intraduodenal infusion of a fatty acid in dogs [26] and in humans. IV PYY inhibits gastric emptying and intestinal transit. Unabsorbed nutrients, especially fats, in the lumen of the ileum inhibit upper-gut motility. This negativefeedback mechanism of the lower intestine is a major and fundamental mechanism for the regulation of upper-gut motility and most likely part of the ileal brake. PYY’s primary role in the GI tract is to be an ileal brake factor. PYY has all the qualifications for this role—PYY is produced in the distal intestine and inhibits gastric emptying and intestinal transit [29]. Additional evidence supports the ileal brake role of PYY [24]. In dogs, immunoneutralization of systemic PYY prolongs intestinal transit of ingested food. Furthermore, infusion of fat into the ileum in dogs increases the migrating motor (myoelectric) complex (MMC) cycle length, lowers the number of MMCs, and elevates plasma levels of PYY. The fasting motor patterns of the upper gut, the interdigestive MMC, are disrupted by ingested nutrients. The interdigestive MMC, cycle length, and blood levels of PYY are also coordinated. Because of its strong antisecretory action, PYY is considered an endogenous inhibitor of diarrhea. PYY has been shown to enhance intestinal absorption of fluid and electrolytes in the jejunum and ileum. In fact, the administration of PYY or PYY analogs into the intestinal lumen are proabsorptive. In support of the antisecretory action of PYY, PYY receptors can be localized in the intestinal crypt cells where chloride secretion occurs [34]. PYY inhibits intestinal chloride ion secretion by lowering basal and vasoactive intestinal polypeptide or prostaglandin-stimulated levels of cyclic AMP. A natural variant of PYY, PYY(3–36) [10], can inhibit appetite and food intake in normal and obese subjects as well as in experimental laboratory animals [5]. Exogenous PYY can lower plasma ghrelin levels (ghrelin stimulates food intake) and endogenous plasma PYY levels are reduced in obese subjects. This finding implies

Peptide YY that the PYY deficiency has a role in the etiology of obesity. PYY may stimulate the sensation of satiety by the inhibition of gut motility and by giving a feeling of fullness that is mediated by vagal afferents originating in the gut and projecting to the hindbrain, eventually terminating in the hypothalamus. In laboratory animal studies, PYY can inhibit hypothalamic NPY neurons and agouti-related protein (AgRP) neurons by the activation of inhibitory NPY-Y2 receptors that disinhibit adjacent proopiomelanocortin (POMC)-expressing neurons and decrease food intake. Systemically administered PYY has been shown to cross the blood–brain barrier, indicating that PYY released by the gut has the potential to influence central ingestive mechanisms.

GROWTH PROMOTING ACTIONS OF PYY ON THE GASTROINTESTINAL TRACT The early appearance of intestinal PYY during development suggests a role for PYY in the regulation of gut growth and development. In rodent studies, PYY has been shown to stimulate intestinal growth [8]. In adult female mice, the administration of PYY(1–36) increases intestinal growth. In nursing rat pups, the mitogenic action of PYY is observed only in the proximal small intestine. Evidence that the Y1 receptor is involved in intestinal cell growth is shown by a study in which the activation of the Y1 receptor in IEC-6 cells stably expressing this receptor caused cell growth and increased MAPK phosphorylation.

INHIBITORY ACTION OF PYY(3–36) ON APPETITE AND FOOD INTAKE Infusion of PYY(3–36), a natural variant of PYY [10], in subjects of normal weight and in rodents reduces appetite and food intake, and repeated administration of PYY reduces weight gain in rodents.

PATHOPHYSIOLOGY OF PYY The secretion of intestinal PYY is modified in some clinical conditions [2]. There are elevated plasma levels of PYY in patients having diarrhea associated with malabsorption of fat [2]. Gastric resection can cause rapid gastric emptying and result in the dumping syndrome; plasma PYY levels are also elevated in these patients. In patients with celiac and tropical sprue, and with massive small bowel resection, basal plasma and postprandial levels of PYY are increased. The increase in PYY secretion is an anticipated adaptation because there is an increased amount of unabsorbed nutrients in the lumen

/ 1113

of the distal intestine that provokes PYY secretion. In patients with morbid obesity, basal plasma and postprandial PYY levels are decreased despite an unabated food intake [4]. These findings agree with the concept that a decrease in PYY secretion underlies the pathogenesis of obesity, at least in part. In obese patients, the administration of PYY intravenously lowers appetite and food intake with magnitudes not different from those in normal subjects, indicating that obesity is not associated with an impaired response to PYY.

References [1] Adrian TE, Ferri GL, Bacarese-Hamilton AJ, Fuessl HS, Polak JM, Bloom SR. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 1985; 89: 1070–7. [2] Adrian TE, Savage AP, Bacarese-Hamilton AJ, Wolfe K, Besterman HS, Bloom SR. Peptide YY abnormalities in gastrointestinal diseases. Gastroenterology 1986; 90:379–84. [3] Adrian TE, Savage AP, Sagor GR, Allen JM, Bacarese-Hamilton AJ, Tatemoto K, et al. Effect of peptide YY on gastric, pancreatic, and biliary function in humans. Gastroenterology 1985; 89: 494–9. [4] Alvarez Bartolome M, Borque M, Martinez-Sarmiento J, Aparicio E, Hernandez C, Cabrerizo L, et al. Peptide YY secretion in morbidly obese patients before and after vertical banded gastroplasty. Obes Surg 2002; 12:324–7. [5] Batterham RL, Bloom SR. The gut hormone peptide YY regulates appetite. Ann NY Acad Sci 2003; 994:162–8. [6] Berglund MM, Hipskind PA, Gehlert DR. Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp Biol Med (Maywood) 2003; 228: 217–44. [7] Bottcher G, Sjolund K, Ekblad E, Hakanson R, Schwartz TW, Sundler F. Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut. Regul Pept 1984; 8:261–6. [8] Gomez G, Zhang T, Rajaraman S, Thakore KN, Yanaihara N, Townsend CM, Jr., et al. Intestinal peptide YY: ontogeny of gene expression in rat bowel and trophic actions on rat and mouse bowel. Am J Physiol 1995; 268:G71–81. [9] Goumain M, Voisin T, Lorinet AM, Laburthe M. Identification and distribution of mRNA encoding the Y1, Y2, Y4, and Y5 receptors for peptides of the PP-fold family in the rat intestine and colon. Biochem Biophys Res Commun 1998; 247:52–6. [10] Grandt D, Schimiczek M, Struk K, Shively J, Eysselein VE, Goebell H, et al. Characterization of two forms of peptide YY, PYY(1–36) and PYY(3–36), in the rabbit. Peptides 1994; 15:815–20. [11] Greeley GH, Jr., Guo YS, Gomez G, Lluis F, Singh P, Thompson JC. Inhibition of gastric acid secretion by peptide YY is independent of gastric somatostatin release in the rat. Proc Soc Exp Biol Med 1988; 189:325–8. [12] Greeley GH, Jr., Hashimoto T, Izukura M, Gomez G, Jeng J, Hill FL, et al. A comparison of intraduodenally and intracolonically administered nutrients on the release of peptide-YY in the dog. Endocrinology 1989; 125:1761–5. [13] Greeley GH, Jr., Hill FL, Spannagel A, Thompson JC. Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey. Regul Pept 1987; 19:365–72. [14] Greeley GH, Jr., Jeng YJ, Gomez G, Hashimoto T, Hill FL, Kern K, et al. Evidence for regulation of peptide-YY release by the proximal gut. Endocrinology 1989; 124:1438–43.

1114 / Chapter 152 [15] Guo YS, Fujimura M, Lluis F, Tsong Y, Greeley GH, Jr., Thompson JC. Inhibitory action of peptide YY on gastric acid secretion. Am J Physiol 1987; 253:G298–302. [16] Hernandez EJ, Whitcomb DC, Vigna SR, Taylor IL. Saturable binding of circulating peptide YY in the dorsal vagal complex of rats. Am J Physiol 1994; 266:G511–6. [17] Jeng Y-J, Hill FLC, Lluis F, Gomez G, Izukura M, Kern K, et al. Peptide YY release and actions. In: Thompson JC, Cooper CW, Greeley GH, Jr., Rayford PL, Singh P, Townsend CM, Jr., editors. Gastrointestinal Endocrinology: Receptors and Post-Receptor Mechanisms, San Diego: Academic Press; 1990. p. 371–386. [18] Kogire M, Izukura M, Gomez G, Uchida T, Greeley GH, Jr., Thompson JC. Terbutaline, a beta 2-adrenoreceptor agonist, inhibits gastric acid secretion and stimulates release of peptide YY and gastric inhibitory polypeptide in dogs. Dig Dis Sci 1990; 35:453–7. [19] Krasinski SD, Wheeler MB, Leiter AB. Isolation, characterization, and developmental expression of the rat peptide-YY gene. Mol Endocrinol 1991; 5:433–40. [20] Laburthe M, Chenut B, Rouyer-Fessard C, Tatemoto K, Couvineau A, Servin A, et al. Interaction of peptide YY with rat intestinal epithelial plasma membranes: binding of the radioiodinated peptide. Endocrinology 1986; 118:1910–7. [21] Lee HM, Udupi V, Englander EW, Rajaraman S, Coffey RJ, Jr., Greeley GH, Jr. Stimulatory actions of insulin-like growth factorI and transforming growth factor-alpha on intestinal neurotensin and peptide YY. Endocrinology 1999; 140:4065–9. [22] Leiter AB, Toder A, Wolfe HJ, Taylor IL, Cooperman S, Mandel G, et al. Peptide YY. Structure of the precursor and expression in exocrine pancreas. J Biol Chem 1987; 262:12984–8. [23] Lin HC, Neevel C, Chen JH. Slowing intestinal transit by PYY depends on serotonergic and opioid pathways. Am J Physiol Gastrointest Liver Physiol 2004; 286:G558–63. [24] Lin HC, Zhao XT, Wang L, Wong H. Fat-induced ileal brake in the dog depends on peptide YY. Gastroenterology 1996; 110: 1491–5. [25] Lluis F, Gomez G, Fujimura M, Greeley GH, Jr., Thompson JC. Peptide YY inhibits nutrient-, hormonal-, and vagally-stimulated pancreatic exocrine secretion. Pancreas 1987; 2:454–62. [26] Lluis F, Gomez G, Fujimura M, Greeley GH, Jr., Thompson JC. Peptide YY inhibits pancreatic secretion by inhibiting cholecystokinin release in the dog. Gastroenterology 1988; 94:137–44. [27] Misra S, Murthy KS, Zhou H, Grider JR. Co-Expression of Y1, Y2 and Y4 receptors in smooth muscle coupled to distinct signaling pathways. J Pharmacol Exp Ther 2004; 3:1154–62. [28] Pappas TN, Debas HT, Taylor IL. Enterogastrone-like effect of peptide YY is vagally mediated in the dog. J Clin Invest 1986; 77:49–53.

[29] Savage AP, Adrian TE, Carolan G, Chatterjee VK, Bloom SR. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut 1987; 28:166–70. [30] Servin AL, Rouyer-Fessard C, Balasubramaniam A, Saint Pierre S, Laburthe M. Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3′,5′-monophosphate production in rat small intestine: structural requirements of peptides for interacting with peptide-YY-preferring receptors. Endocrinology 1989; 124:692–700. [31] Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982; 296:659–60. [32] Upchurch BH, Fung BP, Rindi G, Ronco A, Leiter AB. Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice. Development 1996; 122:1157–63. [33] Voisin T, Couvineau A, Rouyer-Fessard C, Laburthe M. Solubilization and hydrodynamic properties of active peptide YY receptor from rat jejunal crypts. Characterization as a Mr 44,000 glycoprotein. J Biol Chem 1991; 266:10762–7. [34] Voisin T, Rouyer-Fessard C, Laburthe M. Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villuscrypt axis. Am J Physiol 1990; 258:G753–9. [35] Wang G, Leiter AB, Englander EW, Greeley GH, Jr. Insulinlike growth factor I increases rat peptide YY promoter activity through Sp1 binding sites. Endocrinology 2004; 145:659– 66. [36] Yang H, Tache Y. PYY in brain stem nuclei induces vagal stimulation of gastric acid secretion in rats. Am J Physiol 1995; 268: G943–8. [37] Yoshinaga K, Mochizuki T, Yanaihara N, Oshima K, Izukura M, Kogire M, et al. Structural requirements of peptide YY for biological activity at enteric sites. Am J Physiol 1992; 263: G695–701. [38] Zeng N, Walsh JH, Kang T, Wu SV, Sachs G. Peptide YY inhibition of rat gastric enterochromaffin-like cell function. Gastroenterology 1997; 112:127–35. [39] Zhang T, Brubaker PL, Thompson JC, Greeley GH, Jr. Characterization of peptide-YY release in response to intracolonic infusion of amino acids. Endocrinology 1993; 132: 553–7. [40] Zhang T, Sumi S, Thompson JC, Greeley GH, Jr. Release of peptide-YY from the dog pancreas. Endocrinology 1992; 130: 2025–30. [41] Zhang T, Uchida T, Gomez G, Lluis F, Thompson JC, Greeley GH, Jr. Neural regulation of peptide YY secretion. Regul Pept 1993; 48:321–8.

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153 Secretin WILLIAM Y. CHEY AND TA-MIN CHANG

ABSTRACT

twentieth century, research on secretin went through many major advances, including isolation, purification, structural determination, chemical synthesis, establishment of its hormonal status by radioimmunoassay and immunoneutralization, identification of a specific receptor, cloning of secretin and its receptor, and identification of a secretin-releasing peptide. The purification and structural determination of porcine secretin by Jorpes and Mutt [16, 25] is the key advancement leading to these accomplishments. It is now established that secretin is a hormone regulating the pancreatic exocrine secretion of fluid and bicarbonate, the secretion of gastric acid, and gastric motility. The release and actions of secretin are regulated by hormone-hormonal and hormone-neural interactions, particularly the vagal afferent pathway. Substantial amounts of information about the regulation of secretin gene and the property of secretin receptor have been accumulated. Although secretin is also present in tissues and organs outside the gastrointestinal tract, including the central nervous system, this chapter covers mainly its distribution and function in the digestive system.

Secretin is the first peptide hormone ever discovered. Secretin is a 27-amino-acid peptide of secretinglucagon-vasoactive intestinal polypeptide superfamily and is localized mainly in the upper small intestinal mucosa. The gene structures of secretin and its receptor have been determined. Compared with several bioactive prosecretins, secretin is the most active form. The physiological functions of secretin include the stimulation of pancreatic exocrine secretion of water and electrolytes, inhibition of gastric acid secretion, and motility. The physiological release and actions of secretin are subjected to hormone-hormonal and neural-hormonal regulations, among which the vagal afferent pathway plays a significant role. Secretin is used clinically to assess pancreatic function and test for pancreatic malignancy. Pathological states of both hyposecretinemia and hypersecretinemia have been well documented.

DISCOVERY OF SECRETIN In 1902, Bayliss and Starling [1] reported a historical observation that the infusion of 0.4% HCl into a denervated jejunal loop, but not intravenous infusion of the acid, resulted in continuous secretion from the pancreas for some minutes. They further demonstrated that the intravenous injection of an acid mucosal extract from the denervated jejunal loop stimulated pancreatic secretion. They named the active agent from the intestinal mucosa secretin and subsequently coined the term hormone to describe the active humoral substance such as secretin that is produced in one organ and carried through the circulation to another organ to exert its effect. The discovery of secretin thus started an epoch of hormone research in the following decades. In the Handbook of Biologically Active Peptides

STRUCTURE OF SECRETIN, ITS PRECURSOR TRANSCRIPT, AND GENE When first purified from porcine intestinal extract, secretin was shown to be a peptide of 27 amino acid residues, with an N-terminal histidine and C-terminal valine carboxamide. Subsequent purification and structural determination of secretin from other animal species including humans, pigs, dogs, rats, mouse, goats, rabbits, guinea pigs, and chickens have indicated that the structure of secretin, as shown in Fig. 1, is highly conserved among mammalian species that differ by one to three amino acid residues from bovine/ovine/

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Pig, cow, goat H S D G T F T S E L S R L R D S A R L Q R L L Q G L V * Dog H S D G T F T S E L S R L R E S A R L Q R L L Q G L V * Rat H S D G T F T S E L S R L Q D S A R L Q R L L Q G L V * Human H S D G T F T S E L S R L R E G A R L Q R L L Q G L V * Mouse H S D G M F T S E L S R L R D S A R L Q R L L Q G L V * Rabbit H S D G T L T S E L S R L R D R A R L Q R L L Q G L L * Guinea pig H S D G T F T S E K S R L R D S A R L Q R L L Q G L V * Chicken H S D G L F T S E Y S K M R G N A Q V Q K F I Q N L M* * Denotes -CONH2

porcine secretin (which are identical). The structure of secretin shares some degree of sequence homology with glucagon and several regulatory peptides isolated subsequently that form the glucagon–vasoactive intestinal peptide (VIP) superfamily. Several proforms of porcine secretin, including an N-terminal 9-amino-acid extended secretin (N-prosecretin), secretin-Gly, secretin-Gly-LysArg, and a C-terminal 44-amino-acid extended form that exhibit various levels of bioactivities, have been isolated. Their structures match that predicted from the cDNA sequence of porcine secretin mRNA first cloned by Kopin et al. [17], who also confirmed the C-terminal 44-amino-acid extended form as a product of alternative splicing of secretin transcript at exon 3 [18]. The deduced secretin cDNA sequences of rat and pig indicate that both precursors are composed of a signal peptide, a short N-terminal peptide, secretin, and a Cterminal extension of 72 amino acid residues (Fig. 2). The tripeptide sequence, Gly-Lys-Arg, which extends immediately after the C-terminal valine residue of secretin, appears to serve as the dipeptidyl cleavage site and amino donor for α-amidation during posttranslational processing of the secretin precursor. Subsequent determination of the structure of rat [14, 18] and porcine secretin genes [19] indicated that the coding sequences of both are composed of four exons separated by three introns. Similar secretin gene organizations were found in the mouse and human secretin genes that were cloned later.

DISTRIBUTION OF SECRETIN AND ITS mRNA Before radioimmunoassay and immunohistochemical methods became available, the distribution of secre-

FIGURE 1. Structure of secretin from various animal species. Single-letter codes for amino acid residues are used. The underlined residues are those that differ from porcine secretin.

tin was assessed by the determination of its bioactivity by applying acid along the intestinal mucosa in several segments or assaying the mucosal extracts of the segments. These early studies indicated that secretin bioactivity is most abundant in the proximal small intestinal mucosa and decreases with increasing distance from the pylorus. The results of subsequent radioimmunoassays of the intestinal extracts by several investigators were in general in good agreement with the bioassays. In most mammalian species, secretin is most concentrated in the mucosa of the duodenum and upper jejunum, with very low concentrations in the ileum and colon. However, a high concentration of secretin was also found in rat ileum. Immunohistochemical studies have also indicated that secretin-containing cells, denoted as S cells of the small intestinal mucosa, are most abundantly found in the mucosa of upper small intestine. The majority of S cells are found located in the villi, occurring at a frequency of six cells per 1000 mucosal epithelial cells. S cells in smaller numbers are also found in the mid- to upper crypt region, an observation that may not be surprising because S cells are differentiated from stem cells in the crypt and migrate upward during maturation. The concentration of S cells in the upper small intestinal mucosa thus fits well with its function as a pancreatic secretagogue released by gastric acid during the postprandial state. Although high levels of secretin mRNA are found in the duodenum and jejunum, as would be expected for production of secretin, the highest expression level was found in the distal ileum of the rat [17]. This observation appears to be related to the high concentration of secretin found in rat ileum. It was proposed that the unusually high level of secretin in rat ileum might be involved in a different release mechanism and function [17]. Apart from the small intestine, secretin mRNA is also found

Secretin / 1117 N-terminal peptide

Signal peptide Rat Porcine

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FIGURE 2. Comparison of amino acid sequences of rat and porcine secretin precursors predicted from their cDNA sequences. Identical amino acids are boxed. Dashes indicate gaps introduced to optimize the alignment. Predicted functional domains are indicated above the amino acid sequences. Arrows denote potential signal peptide cleavage sites. Adapted from [17] with permission.

in rat and mouse colon and many extraintestinal tissues. Low levels of immunoreactive and bioactive secretin are also found in the stomach. Like the presence of secretin in the colon, the presence of secretin in the stomach is supported by the presence of S cells, determined using immunohistochemistry, and mRNA, determined using reverse transcription polymerase chain reaction (RT-PCR). Similarly, many other extraintestinal tissues or organs, including the heart, lung, kidney, testis, spleen, thymus, and several areas of the brain, also contain secretin, and many of these are supported by the presence of secretin mRNA. It should be noted that in tissues of low secretin abundance controversial results might arise through different assay techniques. For example, a low-sensitivity immunoassay does not

detect secretin in the stomach or the brain, whereas a high-stringency Northern blot analysis does not detect secretin mRNA in tissues with low abundance. In addition, local autocrine or paracrine action by secretin in the extraintestinal tissues may not need a secretin concentration as high as that found in the duodenum and jejunum, where secretin functions as a hormone. During development, S cells are found as early as embryonic day 17 (E17) [19]. Secretin is present at relatively high levels at birth in the rat and guinea pig intestine, falling in the postnatal period. In the rat duodenum, both secretin and its mRNA levels are at the highest 2 days before birth and fall gradually after birth to reach the adult levels [18]. Secretin mRNA is also detected in the developmental pancreas, reaching the highest level at E19 and falling to an undetectable level

1118 / Chapter 153 by adulthood. The expression of secretin mRNA in the fetal pancreas is supported by immunohistochemical detection of secretin immunoreactivity in islet β cells [34].

SECRETIN RECEPTOR SUBTYPES, DISTRIBUTION, AND SIGNALING The presence of a high-affinity receptor for secretin was first demonstrated by Jensen et al. in guinea pig pancreatic acini [15]. Subsequent studies have indicated that secretin receptor is a glycoprotein receptor coupled to adenylate cyclase through an oligomeric G-protein and is widely distributed in many organs. Because of cross-reaction of secretin and several members of the secretin-glucagon-VIP superfamily to their corresponding receptors, it is not possible to use such ligand binding studies to determine the precise localization of secretin receptor or its subtype classification. The cloning of rat secretin receptor by Ishihara et al. [13] has permitted the fulfillment of these tasks. To date, human and rabbit secretin receptors have also been cloned. Except for a misspliced form of secretin receptor found in ductal pancreatic adenocarcinoma cell lines [8] and gastrinoma [7], no other secretin receptor subtypes have been found. Based on their observation in rats that secretin-Gly was as potent as secretin in binding the pancreatic secretin receptor and stimulating pancreatic exocrine secretion but substantially weaker than secretin in inhibiting gastric acid secretion and unable to inhibit somatostatin release from gastric somatostatin cells, Solomon et al. [31] proposed that pancreatic and gastric secretin receptors are of different subtypes. However, it is not clear whether these are two different proteins or the same protein with different functional properties due to different cellular environments. Secretin receptor is a seven-transmembrane Gprotein-coupled receptor with a long N-terminal extracellular tail, three extracellular and three intracellular loops, and a short hydrophilic cytoplasmic C-terminal chain. This receptor is classified as type II G-proteincoupled receptor. Extensive studies of the secretin receptor have been carried out by L. Miller’s and P. Robberecht’s groups. Their results have indicated that the N-terminal extracellular tail of the receptor is involved in binding secretin. The putative Nglycosylation site at position 72N is crucial for ligand binding, whereas extracellular loops are also essential, probably for maintaining the active conformation of the receptor’s extracellular binding domain. The cytoplasmic C-terminal tail is involved in the desensitization of the receptor through phosphorylation by G-proteincoupled receptor kinase. The secretin receptor is expressed in the pancreas, stomach, liver, kidney, colon,

heart, lung, spleen, thymus, testes, ovary, and brain of various species. In the pancreas, secretin receptor is present in both the ductal and acinar cells [33]. The binding of secretin to the receptor activates not only adenylate cyclase but also phospholipase C leading to the production of cAMP and elevation of intracellular calcium ion concentration through phosphatidylinositol hydrolysis [24]. However, it is not clear at present whether this dual signaling pathway occurs in all cells expressing secretin receptor. In pancreatic ducts, the activation of the secretin receptor increases the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) and chloride/bicarbonate exchanger to elicit electrolyte secretion and increase aquaporin 1 on the apical membrane to elicit water secretion. A similar mechanism is found for secretin-stimulated electrolyte and water secretion from liver chlorangiocytes.

BIOLOGICAL ACTIONS OF SECRETIN IN THE GASTROINTESTINAL TRACT Secretin is known to stimulate pancreatic exocrine secretion of water, bicarbonate, gastric mucus, and pepsinogen, as well as duodenal Brunner’s gland secretion, biliary secretion of water and bicarbonate, release of pancreatic islet hormones (insulin, glucagon, and somatostatin), and pancreatic growth. Secretin is also known to inhibit gastric acid secretion and emptying; motilities of the lower esophageal sphincter, small intestine, and colon; and growth of gastric mucosa. Among these actions, the stimulation of pancreatic exocrine secretion and inhibition of gastric acid secretion and emptying have been established as physiological and hormonal functions of secretin. The hormonal status of secretin was established by its release into the circulation following ingestion of a meal [6, 29] and by the abolishment of postprandial pancreatic exocrine secretion on immunoneutralization of circulating secretin with a specific antisecretin serum [4] (Fig. 3). Similarly, the intravenous administration of an antisecretin serum augmented postprandial gastrin release and acid output in dogs (Fig. 4) [5], suggesting that secretin is an enterogastrone that functions as a feedback inhibitor of gastrin release and gastric acid secretion. Also observed in dogs, gastric acid secretion in response to a liquid amino acid meal was inhibited dosedependently by secretin in physiological doses, whereas intravenous antisecretin serum augmented acid output, thereby confirming the physiological role of secretin in inhibiting gastric acid secretion. Moreover, secretin also inhibited dose-dependently the gastric emptying of the amino acid meal, whereas antisecretin serum accelerated gastric emptying, indicating that secretin is also a regulator of gastric motility.

Secretin / 1119 iv Rabbit Serum

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The release of secretin from the upper small intestinal mucosa is stimulated by gastric acid, digested products of fat and protein, bile acid, and herbal extracts [3]. Among these stimulants, gastric acid delivered to the duodenum appears to be the most important because ablation of gastric acid secretion with a histamine H2 blocker, cimetidine, resulted in the abolition of postprandial secretin release in both humans and dogs. It has become clear that acid-elicited secretin release is mediated by a secretin-releasing peptide (SRP) secreted in the intestinal lumen [22]. Canine pancreatic juice also contains an SRP that appears to be identical to pancreatic phospholipase A2 (PLA2) [2]. Although PLA2 is capable of stimulating secretin release from secretin-producing cells in vitro and is released into the intestinal lumen upon duodenal acidification in rats, intraduodenal infusion of PLA2 was unable to release secretin consistently, suggesting that a yet unidentified factor is required for its in vivo function. SRP also mediates the release of secretin and increase of pancreatic exocrine secretion elicited by bilepancreatic juice diversion, a feedback regulatory mechanism that is mediated by pancreatic protease, as first proposed by Green and Lyman [10]. The physiological actions of secretin are regulated by hormone-hormonal and neural-hormonal interactions. Pancreatic exocrine secretion after a meal is mainly stimulated by synergistic interaction between secretin and cholecystokinin (CCK), so that an adequate amount of pancreatic juice is produced for proper digestion. For example, pancreatic secretions of

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Time (min) FIGURE 3. Effect of rabbit antisecretin serum on postprandial pancreatic bicarbonate secretion in dogs. * denotes significant suppression of pancreatic secretin by the antisecretin serum. Adapted from [4] with permission.

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FIGURE 4. Effect of rabbit antisecretin serum on postprandial gastrin release and gastric acid secretion in dogs. * indicates significant decrease in plasma gastrin concentrations (A) or gastric acid output (B) in dogs treated with antisecretin serum compared with those treated with normal rabbit serum. Adapted from [5] with permission.

1120 / Chapter 153 bicarbonate in humans stimulated by a combination of secretin and CCK-8 in physiological doses are greater than the sum of bicarbonate output stimulated by each hormone alone [36]. Secretin also acts with neurotensin to potentiate pancreatic enzyme secretion. Insulin apparently plays a permissive role on the action of secretin and CCK. Thus, a profound inhibition of pancreatic exocrine secretion stimulated by secretin in combination with CCK (Fig. 5) or by a meal [20] was observed when circulating insulin was neutralized with a specific anti-insulin serum in rats. This effect of antiinsulin was also observed in secretin- and CCKstimulated secretion from isolated and vascularly perfused rat and dog pancreas. Except in the rat, the action of secretin on the exocrine pancreas in a physiological dose is highly sensitive to atropine, indicating an important mediation by cholinergic neurons. The physiological action of secretin is highly dependent on the vagal afferent pathway. Thus, chemical ablation of vagal afferent fibers by perivagal application of capsaicin in rats resulted in a profound inhibition of the pancreatic secretion stimulated by a physiological, but not by a pharmacological, dose of secretin [21]. The release and actions of SRP released by duodenal acidification are also neural-mediated and dependent on the vagal afferent pathway. Thus, the concentrate of duodenal acid perfusate (containing SRP activity) prepared from donor rats treated with tetrodotoxin (TTX), vagotomy, or perivagal capsaicin was unable to stimulate pancreatic exocrine secretion or the release of

secretin in recipient rats [22]. In addition, the concentrated acid perfusate prepared from untreated donor rats was also unable to stimulate secretin release from the recipient rats pretreated with TTX, vagotomy, or perivagal application of capsaicin. The electrical stimulation of the medial amygdala in rats augmented pancreatic bicarbonate and fluid secretion stimulated by duodenal acidification and a low dose of secretin. This effect of the medial amygdala stimulation was abolished by bilateral truncal vagotomy, suggesting that the stimulation of the medial amygdaloid elicited a stimulatory signal transmitted through the vagus nerve to potentiate the action of secretin. Some neuropeptides and neurotransmitters may modulate or mediate the release and action of secretin. Both metenkephalin (MEK) and somatostatin inhibit the release and action of secretin on the exocrine pancreas. MEK also inhibits the release of SRP and its action on secretin release through the release of somatostatin. In conscious rats and dogs, secretin-stimulated pancreatic exocrine secretion was inhibited by a nitric oxide (NO) synthase inhibitor, Nnitro-l-arginine, and the inhibition was reversed by the substrate of the enzyme, arginine, suggesting that NO mediates the action of secretin. In anesthetized rats, both the 5-HT2 antagonist ketanserin and the 5-HT3 antagonist ondansetron dose-dependently inhibited pancreatic volume and bicarbonate secretion and secretin release elicited by duodenal acidification, and both 5-HT antagonists inhibited pancreatic secretion stimulated by physiological doses of secretin. These observa-

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Iv secretin+CCK-8, CU or µg/kg/h FIGURE 5. Effect of rabbit anti-insulin serum on secretin and CCK-stimulated pancreatic exocrine secretin in rats. Adapted from [20] with permission.

Secretin / 1121 tions suggest that 5-HT mediates the release and action of secretin through the two 5-HT receptor subtypes. In isolated and perfused rat pancreas, electrical field stimulation enhanced the stimulation of pancreatic secretion by secretin. The enhanced secretion was reduced by atropine or a specific anti-gastrin-releasing peptide (GRP) serum and abolished by combination of atropine and the antiserum, suggesting that the enhancement of secretin-stimulated secretion was mediated by acetylcholine and GRP released from intrapancreatic neurons. The physiological stimulant of these intrapancreatic neurons is unknown at present. In isolated rat pancreatic ducts, secretin-stimulated fluid secretion was potentiated by acetylcholine [9], suggesting a possible interaction between the two stimulants. The physiological actions of secretin in the stomach are also mediated by other hormones and regulated by the vagus nerve. In both isolated and perfused rat stomach and the intact rats, the inhibition of gastric acid secretion by secretin is inhibited by treatment with an antisomatostatin serum and inhibitor of prostaglandin synthesis, suggesting mediation by these two mediators. Similarly to that observed in the pancreas, perivagal capsaicin treatment and vagotomy in conscious rats blocked the inhibition of pentagastrin-stimulated gastric acid secretion by secretin. Lu and Owyang [23] also demonstrated that the vagal afferent pathway mediates inhibition of gastric motility by a physiological dose of secretin, confirming a previous observation made by Raybould and Holzer [28]. In rats, vagotomy, vagal ligation, or perivagal colchicine (but not perivagal capsaicin treatment) decreased the number of high-affinity secretin binding sites in the rat forestomach and reduced secretin-elicited relaxation of the rat forestomach muscle strips. This observation suggested that the vagal efferent pathway also regulates secretin action through the modulation of secretin receptor in rat forestomach. The ability of peripheral secretin to interact with nerve terminals was well demonstrated by intravenous secretin-elicited expression of Fos in the central nucleus of the amygdala, area postrema, bed nucleus of the stria terminalis, supraoptic nucleus, dorsal motor nucleus of the vagus, and medial nucleus tractus solitarius [11]. These effects of peripheral secretin have been shown by Yang et al. [35] to be mediated through the vagal afferent pathway and the expression of secretin receptor in nodose ganglia that contain cell bodies of vagal afferent neurons.

PATHOPHYSIOLOGY AND CLINICAL APPLICATION OF SECRETIN Hypersecretinemia is found in patients with ZollingerEllison syndrome, duodenal ulcer with hypersecretion

of gastric acid secretion, renal failure [3], and a patient with a secretin-producing endocrine tumor in the pancreas that caused hypersecretion of pancreatic juice and watery diarrhea [30]. Secretin-producing cells were also found in the tumor of a patient with esophageal small cell carcinoma [26]. It is possible that secretinproducing endocrine tumors occur more frequently than is realized but are overlooked unless they cause hypersecretinemia and watery diarrhea. Hyposecretinemia is observed in two pathological states, namely in patients with achlorhydria and adult celiac sprue. In achlorhydric patients, the content of secretin cells in the intestinal mucosa is normal and hyposecretinemia can be corrected by providing acidic drinks such as orange juice. In adult celiac sprue, mucosal atrophy in the upper small intestine leads to loss of secretin cells and hyposecretinemia can be corrected only after mucosal regeneration with a gluten-free diet. In one report [27], the secretin and gastric inhibitory polypeptide contents in the duodenal bulb were found to be reduced and this correlated well with malnutrition in patients with familial amyloidotic polyneuropathy. Secretin has been widely used for pancreatic function tests for diseases involving the pancreas, particularly chronic pancreatitis. In recent years, secretin has been used to collect pancreatic juice for the analysis of molecular biological markers to diagnose pancreatic cancer. Secretin is also used to enhance noninvasive magnetic resonance cholangiopancreatography. A secretin-provocative test [12] is useful for detecting gastrinomas in the pancreas or extrapancreatic region, whereas a selective arterial secretin injection was found to be useful for detecting gastrinoma in the duodenal submucosa [32]. However, in some gastrin-producing tumors that express both secretin receptor and the misspliced receptor [7], a false negative result is produced due to the dominant-negative action of the misspliced receptor.

References [1] Bayliss HP, Starling EH. Mechanism of pancreatic secretion. J. Physiol. Lond. 1902; 28:325–53. [2] Chang TM, Lee KY, Chang CH, Li P, Song Y, Roth FL, et al. Purification of two secretin-releasing peptides structurally related to phospholipase A2 from canine pancreatic juice. Pancreas 1999; 19:401–5. [3] Chey WY, Chang TM. Secretin. In: Makhlouf G, editor. Handbook of Physiology; Section 6: The Gastrointestinal System. Bethesda, American Physiological Society, 1989, vol. 2, p. 359–402. [4] Chey WY, Kim MS, Lee KY, Chang TM. Effect of rabbit antisecretin serum on postprandial pancreatic secretion in dog. Gastroenterology 1979; 77:1268–75. [5] Chey WY, Kim MS, Lee KY, Chang TM. Secretin is an enterogastrone in the dog. Am. J. Physiol. 1981; 240:G239–44.

1122 / Chapter 153 [6] Chey WY, Lee YH, Hendrick JF, Rhodes RA, Tai HH. Plasma secretin concentration in fasting and postparadial state in man. Am. J. Dig. Dis. 1978; 23:981–8. [7] Ding W-Q, Bohmig KS, Wiedenmann B, Miller LJ. Dominant negative action of an abnormal secretin receptor arising from mRNA missplicing in a gastrinoma. Gastroenterology 2002; 122-500–11. [8] Ding W-Q, Cheng Z-J, McElhiney J, Kuntz SM, Miller LJ. Silencing of secretin receptor function by dimerization with a misspliced variant secretin receptor in ductal pancreatic adenocarcinoma. Cancer Res. 2003; 62:5223–9. [9] Evans RL, Ashton N, Elliott AC, Green R, Argent BE. Interaction between secretin and acetylcholine in the regulation of fluid secretion by isolated rat pancreatic ducts. J. Physiol. Lond. 1996; 496:265–73. [10] Green GM, Lyman RL. Feedback regulation of pancreatic enzyme as a mechanism for trypsin inhibitor-induced hypersecretion in rats. Proc. Soc. Exp. Biol. Med. 1972; 140:6–12. [11] Goulet M, Shromani PJ, Ware CM, Strong RA, Boismenu R, Rusche JR. A secretin IV infusion activated gene expression in the central amygdala of rats. Neuroscience 2003; 118:881–8. [12] Isenberg JI, Walsh JH, Passaro E Jr, Moore EW, Grossman MI. Unusual effect of secretin on serum gastrin, serum calcium, and gastric acid secretion in a patient with suspected ZollingerEllison syndrome. Gastroenterology 1972; 62:626–31. [13] Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Takahashi K, Nagata S. Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J. 1991; 10:1635–41. [14] Itoh N, Furuya T, Ozaki K, Ohta M, Kawasaki T. The secretin precursor gene. Structure of the coding region and expression in the brain. J. Biol. Chem. 1991; 266:12595–8. [15] Jensen RT, Charlton CG, Adachi H, Jones SW, O’Donohue TL, Gardner JD. Use of 125I-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini. Am. J. Physiol. 1983; 245:G186–95. [16] Jorpes JE, Mutt V. On the biological activity and amino acid composition of secretin. Acta Chem. Scand. 1961; 15: 1790–1. [17] Kopin AS, Wheeler MB, Leiter AB. Secretin: structure of the precursor and tissue distribution of the mRNA. Proc. Natl. Acad. Sci. USA 1990; 87:2299–303. [18] Kopin AS, Wheeler MB, Nishitani J, McBride EW, Chang T-M, Chey WY, Leiter AB. The secretin gene: evolutionary history, alternative splicing, and developmental regulation. Proc. Natl Acad. Sci. USA 1991; 88:5335–9. [19] Larsson LI, Sundler F, Alumets J, Hakanson R, Schaffalitzky de Muckadell OB, Fahrenkrug J. Distribution, ontogeny and ultrastructure of the mammalian secretin cell. Cell Tissue Res. 1977; 181:361–8. [20] Lee KY, Zhou L, Ren XS, Chang T-M, Chey WY. An important role of endogenous insulin on exocrine pancreatic secretion in rats. Am. J. Physiol. 1990; 258:G268–74. [21] Li P, Chang TM, Chey WY. Neuronal regulation of the release and action of secretin-releasing peptide and secretin. Am. J. Physiol. 1995; 269:G305–12.

[22] Li P, Lee KY, Chang TM, Chey WY. Mechanism of acid-induced release of secretin in rats: Presence of a secretin releasing factor. J. Clin. Invest. 1990; 262:8956–9. [23] Lu Y-X, Owyang C. Secretin at physiological doses inhibits gastric motility via a vagal afferent pathway. Am. J. Physiol. 1995; 268:G1012–46. [24] Mahapatra NR, Mahata M, O’Connor DT, Mahata SK. Secretin activation of chromogranin A gene transcription. Identification of the signaling pathways in cis and in trans. J. Biol. Chem. 2003; 278:19986–94. [25] Mutt V, Jorpes JE. Secretin: isolation and determination of structure. (Abstract) Proc. I. U. P. A. C. Fourth International Congress on the Chemistry of Natural Products, June 26–July 2, 1966, Stockholm, Sweden, Section 2C-3. [26] Nagashima R, Mabe K, Takahashi T. Esophageal small cell carcinoma with ectopic production of parathyroid hormone-related protein (PTHrp), secretin, and granulocyte colony-stimulating factor (G-CSF). Dig. Dis. Sci. 1999; 44:1312–6. [27] Nyhlin N, Anan I, El-Salhy M, Ando Y, Suhr OB. Endocrine cells in the upper gastrointestinal tract in relation to gastrointestinal dysfunction in patients with familial amyloidotic polyneuropathy. Amyloid 1999; 6:192–8. [28] Raybould HE, Holzer H. Secretin inhibit gastric emptying in rats via a capsaicin-sensitive vagal afferent pathway. Eur. J. Pharmacol. 1993; 250:165–7. [29] Schaffalitzky de Muckdadell OB, Fahrenkrug J. Secretion pattern of secretin in man: regulation of gastric acid secretion. Gut 1978; 19:812–28. [30] Schmitt MG Jr, Soergel KH, Hensley GT, Chey WY. Watery diarrhea associated with pancreatic islet cell carcinoma. Gastroenterology 1975; 69:206–16. [31] Solomon TE, Varga G, Zeng N, Wu SV, Walsh JH, Reeve JR Jr. Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280:G88–94. [32] Takasu A, Shimosegawa T, Fukudo S, Asakura T, Uchi M, Kimura K, et al. Duodenal gastrinoma-clinical features and usefulness of selective arterial secretin injection test. J. Gastroenterol. 1998; 33:728–33. [33] Ulrich II CD, Wood P, Hadac EM, Kopras E, Whitcomb D, Miller LJ. Cellular distribution of secretin receptor expression in rat pancreas. Am. J. Physiol. 1998; 275:G1437–44. [34] Wheeler MB, Nishitani J, Buchan AMJ, Kopin AS, Chey WY, Chang T-M, Leiter AB. Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene. Mol. Cell Biol. 1992; 12:2631–9. [35] Yang H, Wang L, Wu SV, Tay J, Goulet M, Boismenu R, et al. Peripheral secretin-induced Fos expression in the rat brain is largely vagal dependent. Neuroscience 2003; 128: 131–41. [36] You CH, Rominger JM, Chey WY. Potentiation effects of cholecystokinin octapeptide on pancreatic bicarbonate secretion stimulated by a physiologic dose of secretin in humans. Gastroenterology 1983; 85:40–5.

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154 Somatostatin MATHIAS GUGGER AND JEAN CLAUDE REUBI

ABSTRACT

SOMATOSTATIN-14, SOMATOSTATIN-28 AND CORTISTATIN IN THE GASTROINTESTINAL TRACT

Somatostatin is abundant in the mucosa and in the enteric nervous system of the gastrointestinal tract and in the pancreas. In these tissues, it exerts a broad range of mainly inhibitory physiological actions in multiple targets, including endocrine glands, exocrine glands, smooth muscles, blood vessels, and immune cells, mediated by up to six somatostatin receptor subtypes. Several diseases of the gastrointestinal tract are characterized by disturbances in the somatostatin production or by overexpression of somatostatin receptors. In particular, somatostatin receptors have been found to be overexpressed in neuroendocrine gastroenteropancreatic tumors. These tumors can be diagnostically and therapeutically targeted with somatostatin analogs. In addition, various nonneoplastic diseases, including bleeding in the upper gastrointestinal tract, fistulas, and diarrhea can also be treated with somatostatin analogs.

Somatostatin is a phylogenetically ancient peptide that is widely distributed throughout the human body. The central nervous system, gastrointestinal tract, and endocrine glands are the major sites of production and action [62, 63]. In the rat, the gastrointestinal tract accounts for ∼30% of the total body somatostatin, the pancreas for ∼8%, the brain for ∼61%, and the remaining organs for ∼1% [55]. Somatostatin-14, somatostatin28, and the recently discovered structurally related cortistatin [15] form the somatostatin peptide family.

Somatostatin Localization in the Gastrointestinal Tract More than 90% of immunoreactive somatostatin in extracts of human gastrointestinal tissue is measured in the mucosa, in particular in the stomach, the duodenum, and the jejunum; less than 10% occurs in the muscle layers [57]. Within the mucosa, somatostatin is localized in epithelial endocrine D cells, found preferentially in the upper gastrointestinal tract [59]. Conversely, somatostatin is present in neurons of the submucosal and myenteric plexus throughout the whole gastrointestinal tract [23]. In the pancreas, somatostatin is localized in D cells of the islets [59]. Whereas the shorter somatostatin-14 form is mainly synthesized in the stomach and the pancreas, the longer somatostatin-28 form is mainly found in the intestine, in particular in its proximal part [17]. The gastrointestinal tract is the main source for the picomolar concentration of somatostatin measured in the circulation [9]. Somatostatin, however, degrades rapidly in the circulation, with a half-life of 1–2 minutes [3].

INTRODUCTION Somatostatin was discovered in 1973 in sheep hypothalamic extracts and named hypothalamic growth hormone inhibiting factor [6]. Next to the numerous endocrine and central nervous actions, it was quite clear at an early stage that somatostatin had extensive gastrointestinal actions as well [5]. During the last 3 decades, basic knowledge on somatostatin has grown considerably, and this has led to clinical applications that make somatostatin and its analogs particularly interesting peptides for clinicians and the diseased gastrointestinal tract a particularly attractive target. This chapter is dedicated to somatostatin in the gastrointestinal tract, summarizing both basic research data and the resulting clinical applications. Handbook of Biologically Active Peptides

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1124 / Chapter 154 Cortistatin mRNA is expressed in the same gastrointestinal tissues as somatostatin mRNA but also in cells of the immune system [12], where it was proposed to function as the main endogenous somatostatin receptor ligand [14].

Regulation of Somatostatin Release in the Gastrointestinal Tract The somatostatin release in the gastroenteropancreatic system is regulated by intraluminal, neural, and endocrine factors in a complex network. Intraluminal nutritional factors in the stomach regulate somatostatin release in animals [79] and humans [18]. The exact signaling pathways by which these stimuli trigger somatostatin release from D cells are complex and not completely understood. They involve mucosal cells as well as afferent and efferent vagal nerves. The latter synapse intragastrically with cholinergic and peptidergic nerves, which have efferent fibers ending on most of the cells involved in the regulation of gastric acid production [78]. Endocrine factors, represented by many gut peptides, can also stimulate [77, 78] or inhibit [48] somatostatin release from D cells.

SOMATOSTATIN RECEPTORS IN THE GASTROINTESTINAL TRACT

cosa, in the smooth muscle layers, and in the submucosal and myenteric nerve plexus [38, 74]. Sst2 mRNA was specifically found in enterochromaffin-like cells (ECL) cells [1, 61] and parietal cells [1] of the rodent stomach. In humans, mRNAs for the various somatostatin receptor subtypes were found in the stomach [45], in the small intestine [52, 89], and in the epithelium and lamina propria of the colon [11, 44, 86, 87]. mRNAs for all subtypes were found in the endocrine and exocrine rat pancreas [38]; information is still incomplete for the human pancreas [72, 89]. At the protein level, somatostatin receptors have been detected primarily by immunohistochemistry and by in vitro receptor autoradiography in several regions of the gastrointestinal tract. It was confirmed that sst2 was present on ECL cells [76, 81] and parietal cells [76] in the rodent corpus and on gastrin cells in the human antrum [29]. In the exocrine pancreas, sst2 has been detected in acinar cells [20, 32, 47, 84] in the rodent but not in the human so far [60, 67]. In the endocrine pancreas, all somatostatin receptor subtypes have been detected in rodents [47] and in humans [40, 60, 67], in varying amounts; sst2 was found on human A cells and sst5 on B and D cells in particularly large amounts. Furthermore, in both rodents and humans sst2 have been shown on neurons of the myenteric and submucosal plexus and on interstitial cells of Cajal [29, 64, 81]. Finally, sst2 was detected in the mucosa-associated lymphoreticular tissue and in submucosal blood vessels of the human gastrointestinal tact [64, 66, 69, 71].

Receptor Subtypes and Postreceptor Signaling The six somatostatin receptor subtypes and the multiple postreceptor signaling systems have been described in most somatostatin target tissues, including the brain, gastrointestinal tract, and endocrine tissues. Refer to Chapter 91 by Epelbaum and Winsky-Sommerer for general information.

Somatostatin Receptor Localization in the Gastrointestinal Tract The exact localization of somatostatin receptors in the animal and human gastrointestinal tract is not yet completed. One reason is the complex physiology of somatostatin and cortistatin, linked to the fact that these peptides can induce their effects by interacting with up to six specific cell surface receptors in target tissues. It is also complicated by the fact that there are species differences in somatostatin receptor distribution and differences depending on whether the receptors are estimated at the mRNA or protein level. At the transcriptional level, mRNAs for all somatostatin receptor subtypes have been detected throughout the whole rat gastrointestinal tract in the epithelium and the lamina propria of the mucosa, in the submu-

BIOLOGICAL ACTION OF SOMATOSTATIN IN THE GASTROINTESTINAL TRACT Somatostatin mediates a broad range of physiological actions in the gastrointestinal tract through the various somatostatin receptor subtypes, preferably in a paracrine way but also as a hormone and as a neurotransmitter [63]. It inhibits the secretion of most gut hormones. It inhibits the exocrine secretion at the level of the stomach, the intestine, the pancreas, and gallbladder. It has complex modulating actions at the level of the enteric nervous system in the stomach, intestine, and gallbladder; it decreases splanchnic blood flow. Furthermore, it modulates immune responses [36]. The biological actions of cortistatin in the human gastrointestinal tract are not yet fully investigated. Cortistatin applied intravenously in humans inhibits pancreatic insulin secretion [26] and may be involved in immune responses [13].

Somatostatin Actions in the Stomach Gastric acid output, one of the main functions of the stomach, reflects a complex balance between stimula-

Somatostatin / 1125 tory factors (e.g., histamine from ECL cells, acetylcholine from neurons, and gastrin from G cells) and inhibitory factors (e.g., somatostatin from D cells). The inhibition of acid secretion depends mainly on somatostatin originating from antral and fundic D cells, acting through paracrine mechanisms; fundic D cells are closely related to parietal and ECL cells through cytoplasmic processes; antral D cells are similarly linked to G cells. It is not yet completely clear which somatostatin receptor subtypes are involved in the somatostatin effects; there is evidence that sst2 may mediate the fundic somatostatin action [1, 58, 76] in the mouse and the rat and the antral somatostatin action [29] in humans. Somatostatin has also a function during the interdigestive phase, namely a tonic paracrine restraint on acid secretion [77].

esophagus the perception threshold to balloon distension increases but not the pain threshold [33]; in the stomach the normal reflex response to volume increase is inhibited, emptying of a meal is slowed [22], and the perception of fullness is decreased [50]; in the small bowel the transit time of solid food is slowed down; and the tonic response of the colon is decreased [84]; and in the intestine the endogenous fluid secretion is decreased and the absorption of water and electrolytes is stimulated [43]. Collateral splanchnic blood flow is reduced up to 30% [88] without causing adverse systemic effects [3].

Somatostatin Actions in the Pancreas

Disturbances of the somatostatin system have been observed in pathologic human gastrointestinal tissues, both in neoplastic and in nonneoplastic tissues. They include disturbances of somatostatin production and of somatostatin receptor expression.

Somatostatin affects both endocrine and exocrine pancreatic secretion. It inhibits insulin secretion primarily via sst5 and glucagon secretion via sst2, as reported from studies using sst2 [83] and sst5 [82] knockout mice. Conversely, a recent in vitro study with human pancreatic islets suggested that sst2 mediated mainly the somatostatin inhibition of insulin release [7], unraveling a potential species difference. Both insulin and glucagon are on tonic interdigestive somatostatin control through a paracrine mechanism [35]. In the rat, somatostatin acts directly on acinar cells to inhibit amylase release [49], probably through sst2 [32, 47]. Although no somatostatin receptors have been identified in the human exocrine part so far [60, 71], it was shown that postprandial increments in serum somatostatin-28 can reduce the pancreatic exocrine secretion [31].

Somatostatin Actions in the Enteric Nervous System Somatostatin acts as an important neurotransmitter through somatostatin receptors in the submucosal and myenteric nervous plexus and ganglia of the enteric nervous system [19]. The physiological implications of these actions are complex because the enteric nervous system influences mechanisms as different as motility, exocrine and endocrine secretions, microcirculation, and the immune system of the gastrointestinal tract [28]. It was shown in the rodent [81] that sst2 in enteric ganglia may be involved in the regulation of peristalsis, that sst2 in the pacemaker cells of Cajal may be involved in the modulation of slow wave contractions [73], and that sst2 in submucosal ganglia may be involved in the regulation of mucosal function and blood flow. Comparable effects can also be detected in humans on application of somatostatin analogs such as octreotide. In the

SOMATOSTATIN AND SOMATOSTATIN RECEPTORS IN THE DISEASED GASTROINTESTINAL TRACT

Somatostatin Production in Neoplastic Disease Disorders of somatostatin production are found in neoplasms, in particular in some endocrine tumors of the gastrointestinal tract and, outside the gastrointestinal tract, pheochromocytomas and small cell lung carcinomas [80]. The classical example of somatostatinproducing tumors in the gastrointestinal tract is the somatostatinoma, originating mostly in the pancreas or the duodenum; in pancreatic tumors [34] this usually leads to somatostatinoma syndrome with the clinical triad of diabetes mellitus, cholelithiasis, and steatorrhea [37].

Somatostatin Production in Nonneoplastic Disease Helicobacter pylori infection of the gastric corpus can lead to a reduced number of somatostatin-producing D cells and hypergastrinemia [90]. Medical eradication of H. pylori infection is followed by structural and functional restoration of endocrine D cells [51]. Interestingly, in a mouse model, it was sufficient to apply the Thelper2-cytokine interleukin-4, which induced somatostatin release from D cells, to resolve H. felis–induced hypergastrinemia and to normalize D cell number [90]. Somatostatin may therefore play a role in this disease, not only by interacting with acid regulation but also by influencing the inflammatory status.

Somatostatin Receptor Expression in Neoplastic Disease Of much greater importance and clinical impact than the production of somatostatin is the expression

1126 / Chapter 154 of somatostatin receptors in neoplasms [65]. A very high incidence and often a high density of somatostatin receptors have been found in gastroenteropancreatic endocrine tumors with receptor autoradiography or in situ hybridization methods [65]. They include endocrine neoplasms of the pancreas and the intestinal tract that express predominantly sst2 with or without other subtypes [65, 71]. Among the nonendocrine tumors, gastric carcinomas show a high density of sst1, sst2, or sst5 [71]; in comparison, somatostatin receptors have been rarely detected in the carcinoma tissue of colorectal cancer, esophageal cancer, and exocrine pancreatic cancer [65]. It should be noted, however, that mRNA for all somatostatin receptor subtypes have been detected with nonmorphological methods in colorectal cancer [87] and in pancreatic adenocarcinoma [21]. A possible explanation for these divergent findings is that most of these cancers, while expressing minimal amounts of somatostatin receptors, express somatostatin receptors in the peritumoral vessels in high density [70]. This observation can be extended to gastric carcinomas, as well as to a large number of other human cancer types in which a high density of somatostatin receptors has been detected in peritumoral veins [16]. This may reflect an important hosttumor interaction [70].

Somatostatin Receptor Expression in Nonneoplastic Disease The overexpression of somatostatin receptors can also be found in specific compartments of nonneoplastic gastrointestinal diseases. For instance, blood vessels expressed somatostatin receptors in idiopathic inflammatory bowel disease. In florid Crohn’s disease or ulcerative colitis, intramural veins showed a high density of somatostatin receptors, limited to the diseased parts of the colon [68].

SOMATOSTATIN ANALOGS AND THEIR CLINICAL APPLICATIONS IN GASTROINTESTINAL DISEASE The natural somatostatins are very potent molecules, produced locally and degraded very rapidly. Thus, synthesized stable somatostatin agonist analogs, such as octreotide, lanreotide, and vapreotide, have been developed not only for diagnostic and therapeutic applications in neoplastic and nonneoplastic disease but also in order to elucidate the physiological role and mechanism of actions of the native hormones. These analogs can be used as radioactive or nonradioactive compounds. In addition, more recently, specific antagonists

as well as second generation agonists such as [111In][DOTA0, 1-Nal3,Thr8]-octreotide [25] have been synthesized.

Diagnostic Applications with Radioactively Labeled Somatostatin Somatostatin receptors were the first peptide receptors to exemplify the principle of in vivo targeting of human cancers expressing a high receptor density [39]. Radiolabeled somatostatin is injected intravenously into the patient and distributed in the whole body; a neoplasm with a high density of somatostatin receptors binds the radiopeptide that internalizes with the receptor into the cell. The accumulated radioactivity in the tumor can be detected with a γ-camera; the remaining radioactivity in the body will be cleared through the kidneys. Radioactively labeled synthetic somatostatin, such as 111In-DTPA-[D-Phe1]octreotide (Octreoscan), is now used diagnostically for in vivo scintigraphy of primary neoplasms overexpressing somatostatin receptors and their metastases, in particular gastroenteropancreatic cancers [41]. Octreoscan can detect resectable tumors otherwise missed by conventional imaging techniques; it may modify the therapeutic strategy, for instance by preventing surgery in metastatic disease not detected by conventional imaging, and it may influence the choice of medical therapy [24].

Therapeutic Applications The targeting of overexpressed somatostatin receptors by synthetic somatostatin analogs in diseased tissues can be used therapeutically in several ways: (1) as radioactive analogs for tumor radiotherapy, (2) as nonradioactive but cytotoxic analogs for tumor therapy, and (3) as nonradioactive noncytotoxic analogs for symptomatic tumor therapy or therapy of nonneoplastic diseases [65]. Radioactive Somatostatin Analogs The principle of radiotherapeutic targeting of tumor tissue overexpressing somatostatin receptors is the same as for diagnostic applications, only the radioactive isotopes are different. The most frequently used somatostatin analog has been 90Y-DOTA-Tyr3octreotide (90Y-DOTATOC) with 90Y as a β-emitter. Because sst2 appears to be the main receptor subtype in many human tumors, somatostatin analogs with a particularly high sst2 affinity such as [177Lu-DOTA]-TATE have been developed [42]. Critical limitations of receptormediated radiotherapy are the radiation-induced destruction of distant receptor-positive normal tissue, in particular radiosensitive tissues such as those of the

Somatostatin / 1127 immune system, but also the kidney, mainly because it eliminates large amounts of the radioligand. In animal tumor models, complete tumor destruction was achieved with 90Y-DOTATOC as a radiotracer, especially in smaller tumors. In humans, several pilot studies have shown encouraging results of radiotherapy with octreotide analogs in intestinal and pancreatic endocrine neoplasms [53]. The majority of the tumors remained stable or shrunk by less than 50% of the pretherapeutic volume; only a few progressed. Nonradioactive Cytotoxic Somatostatin Analogs An alternative approach to radiotherapy is the use of somatostatin analogs coupled to cytotoxic agents. One of the most efficient compounds is AN-238, the potent cytotoxic 2-pyrrolinodoxorubicin linked to the somatostatin octapeptide RC-121. In this compound, the ability of the peptide portion to bind the receptor specifically is preserved and the cytotoxicity of the attached radical is retained. This cytotoxic somatostatin analog, however, has not been tested in clinical trials yet [2]. Nonradioactive Noncytotoxic Somatostatin Analogs Stable somatostatin analogs, such as octreotide, lanreotide, or vapreotide, can be used to treat a variety of neoplastic and nonneoplastic diseases of the gastrointestinal tract.

ulcers, probably due to reduction in splanchnic blood flow and inhibition of acid secretion [4]. Fistulas, post–endoscopic retrograde cholangiopancreaticography pancreatitis. Octreotide can be applied during and after pancreatic resection to reduce the occurrence of postoperative complications [8, 56], such as, for example, pancreatic fistula. The beneficial action of octreotide for this includes the inhibition of exocrine pancreatic secretion. Octreotide is also effective in the prevention of post–endoscopic retrograde cholangiopancreaticography (ERCP) pancreatitis [27]. In addition, a beneficial effect of octreotide in enterocutaneous and biliary fistulas has been shown with respect to fistula secretory output and closure [54]. Diarrhea. Octreotide is a first-line therapy for dumping syndrome, especially when dietary manipulation fails; it inhibits the release of vasoactive peptides that trigger the typical vasomotor symptoms [10]. Octreotide improves secretory diarrhea, prolonging gastrointestinal transit time, decreasing endogenous fluid secretion, and stimulating the absorption of water and electrolytes [43]. Octreotide has been shown to improve diarrhea associated with irritable bowel syndrome by slowing intestinal transit time and increasing the rectal perception threshold for discomfort, probably mediated via the inhibition of exaggerated release of serotonin from enteroendocrine cells [46].

References Neoplasms of the gastrointestinal tract. Long-acting somatostatin analogs can be administered to treat symptoms caused by the oversecretion of hormones from gastroenteropancreatic endocrine tumors. Octreotide can, for example, alleviate hypokalemia, dehydration, and diarrhea in patients with vasoactive intestinal polypeptide-secreting tumors (VIPomas) and can reduce peptic ulcers and necrolytic skin lesions in patients with gastrin- or glucagonproducing tumors, respectively [43]. A true antiproliferative effect of somatostatin analogs in these tumors is, however, modest [30], in contrast to the effect of somatostatin analogs in xenografted human neoplasm and tumor cell lines [75]. Acute variceal and nonvariceal bleeding of the upper gastrointestinal tract. The combination of sclerotherapy and octreotide is more effective than sclerotherapy alone in controlling acute bleeding from esophageal varices in patients with liver cirrhosis [3]. This is probably due to the reduction in splanchnic blood flow by octreotide. The effect of octreotide is preferable to other vasoconstricting agents because it is not causing adverse systemic effects. Octreotide also reduces the risk of nonvariceal rebleeding, for instance, in peptic

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Somatostatin / 1129 [42] Kwekkeboom D, Krenning EP, and de Jong M. Peptide receptor imaging and therapy. J Nucl Med 2000;41:1704–13. [43] Kwekkeboom DJ, Bakker WH, Kooij PP, Konijnenberg MW, Srinivasan A, Erion JL, Schmidt MA, Bugaj JL, de Jong M, and Krenning EP. [177Lu-DOTAOTyr3]octreotate: Comparison with [111In-DTPAo]octreotide in patients. Eur J Nucl Med 2001;28:1319–25. [44] Lamberts SW, van der Lely AJ, de Herder WW, and Hofland LJ. Octreotide. N Engl J Med 1996;334:246–54. [45] Laws SA, Gough AC, Evans AA, Bains MA, and Primrose JN. Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae. Br J Cancer 1997;75:360–6. [46] Le Romancer M, Cherifi Y, Levasseur S, Laigneau JP, Peranzi G, Jais P, Lewin MJ, and Reyl-Desmars F. Messenger RNA expression of somatostatin receptor subtypes in human and rat gastric mucosae. Life Sci 1996;58:1091–8. [47] Lesbros-Pantoflickova D, Michetti P, Fried M, Beglinger C, and Blum AL. Metaanalysis: The treatment of irritable bowel syndrome. Aliment Pharmacol Ther 2004;20:1253–69. [48] Ludvigsen E, Olsson R, Stridsberg M, Janson ET, and Sandler S. Expression and distribution of somatostatin receptor subtypes in the pancreatic islets of mice and rats. J Histochem Cytochem 2004;52:391–400. [49] Martinez V, and Tache Y. Somatostatin. In: Encyclopedia of Gastroenterology 2004; 426–33. [50] Matsushita K, Okabayashi Y, Hasegawa H, Koide M, Kido Y, Okutani T, Sugimoto Y, and Kasuga M. In vitro inhibitory effect of somatostatin on secretin action in exocrine pancreas of rats. Gastroenterology 1993;104:1146–52. [51] Mearadji B, Straathof JW, Biemond I, Lamers CB, and Masclee AA. Effects of somatostatin on proximal gastric motor function and visceral perception. Aliment Pharmacol Ther 1998;12:1163–9. [52] Milutinovic AS, Todorovic V, Milosavljevic T, Micev M, Spuran M, and Drndarevic N. Somatostatin and D cells in patients with gastritis in the course of Helicobacter pylori eradication: A six-month, follow-up study. Eur J Gastroenterol Hepatol 2003;15:755–66. [53] O’Carroll AM, Raynor K, Lolait SJ, and Reisine T. Characterization of cloned human somatostatin receptor SSTR5. Mol Pharmacol 1994;46:291–8. [54] Otte A, Herrmann R, Heppeler A, Behe M, Jermann E, Powell P, Maecke HR, and Muller J. Yttrium-90 DOTATOC: First clinical results. Eur J Nucl Med 1999;26:1439–47. [55] Paran H, Neufeld D, Kaplan O, Klausner J, and Freund U. Octreotide for treatment of postoperative alimentary tract fistulas. World J Surg 1995;19:430–4. [56] Patel YC. Somatostatin and its receptor family. Front Neuroendocrinol 1999;20:157–98. [57] Patel YC, and Reichlin S. Somatostatin in hypothalamus, extrahypothalamic brain, and peripheral tissues of the rat. Endocrinology 1978;102:523–30. [58] Pederzoli P, Bassi C, Falconi M, and Camboni MG. Efficacy of octreotide in the prevention of complications of elective pancreatic surgery. Italian Study Group. Br J Surg 1994;81:265–9. [59] Penman E, Wass JA, Butler MG, Penny ES, Price J, Wu P, and Rees LH. Distribution and characterisation of immunoreactive somatostatin in human gastrointestinal tract. Regul Pept 1983;7:53–65. [60] Pfeiffer M, Koch T, Schroder H, Klutzny M, Kirscht S, Kreienkamp HJ, Hollt V, and Schulz S. Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A). J Biol Chem 2001;276:14027–36. [61] Piqueras L, Tache Y, and Martinez V. Somatostatin receptor type 2 mediates bombesin-induced inhibition of gastric acid secretion in mice. J Physiol 2003;549:889–901.

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155 Somatostatin Analogs in the Gastrointestinal Tract ALAN G. HARRIS, ADRIAN F. DALY, MARIA TICHOMIROWA, ALBERT BECKERS, AND STEVEN W. LAMBERTS

ABSTRACT

the most fully characterized analog. Other analogs such as lanreotide and vapreotide have also been used in the treatment of GI disorders. SSAs exert pharmacological effects on the GI tract over and above those of native somatostatin. SSAs decrease gastroenteropancreatic (GEP) hormone secretion, particularly of glucagon, vasoactive intestinal peptide (VIP), pancreatic polypeptide, and gastrin. For example, octreotide is about 10 times more potent than somatostatin in the inhibition of gastric acid secretion [24]. SSAs inhibit cholecystokinin/secretin- and meal-stimulated pancreatic and gallbladder function [14, 24, 46]. Gastric emptying can be delayed or accelerated by SSAs, and gut motility is stimulated at low doses and intestinal transit time is prolonged at moderate to high doses, resulting in greater fluid and electrolyte absorption [24]. The panoply of physiological roles of somatostatin and the pharmacological effects of octreotide and other analogs in GI disease mean that, unlike many peptide analogs, SSAs have found widespread use in the clinical setting in treating GI diseases (Table 1).

Somatostatin analogs (SSAs) are generally more potent and have a longer in vivo half-life than somatostatin-14, which allows them to be used in many clinical settings in gastrointestinal disease. The SSAs most widely used in the gastrointestinal tract are octreotide, the first clinically useful SSA-, lanreotide and vapreotide. Other radioactively labeled SSAs have been found to be useful as imaging or therapeutic tools for somatostatinreceptor-positive tumors. SSAs have been used clinically in the treatment of gut neuroendocrine tumors, esophageal variceal bleeding, pancreatic surgery, malabsorption syndromes, and secretory diarrheas. This chapter reviews the extensive clinical results available on the use of SSAs in the gastrointestinal tract.

INTRODUCTION Somatostatin has multiple physiological effects in the gastrointestinal (GI) tract, and it is expressed throughout glandular and luminal epithelial cells, visceral neurons, and neuroendocrine cells (please see Chapter 155 in this section of the Handbook). Somatostatin is heavily localized in D cells of the endocrine pancreas, gastric crypts, and the duodenum and somatostatin-containing neurons of the extrinsic and intrinsic visceral nervous system are present throughout the gut; intrinsic neuronal activity is present in both the submucosal and enteric plexuses. This widespread distribution explains the numerous endocrine, paracrine, meurocrine, and exocrine functions of somatostatin within the gut. The first clinically useful somatostatin analog (SSA) in the GI tract was octreotide, and as such it remains Handbook of Biologically Active Peptides

GASTROENTEROPANCREATIC NEUROENDOCRINE TUMORS GEP neuroendocrine tumors comprise a heterogeneous group of rare, slow-growing neoplasms that are derived from neuroendocrine cells, which typically present with symptoms and signs directly associated with the hypersecretion of one or more hormones [44, 66, 69]. Some GEP tumors remain clinically silent and may be diagnosed incidentally; the rate on autopsy is 0.5–1%, whereas 15–30% of all pancreatic neuroendocrine tumors are nonfunctional. Other tumor types,

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1132 / Chapter 155 TABLE 1. Effects of Somatostatin and Its Analogs in the Human Gastrointestinal Tract. Region of Gastrointestinal Tract Oral cavity Esophagus

Stomach

Small intestine

Pancreas Biliary system

Large intestine

Effect SSAs reduce salivary output Somatostatinergic neurons present among longitudinal muscles SSAs increase lower esophageal sphincter tone and inhibit postprandial and swallow-induced sphincter relaxation. SSAs increase esophageal contraction amplitude and velocity Somatostatin secreted from D cells; it is a potent inhibitor of gastrin-induced acid secretion while also inhibiting histamine secretion SSAs inhibit gastrin secretion SSAs delay gastric emptying of solids, increase fasting gastric volume, and decrease the sensation of fullness after a satiating meal SSAs can also accelarate gastric emptying SSAs reduce gastroduodenal mucosal blood flow and can increase intragastric pH Somatostatin and SSA decrease arterial blood flow to the stomach in patients with nonvariceal gastric bleeding Somatostatin stimulates migrating motor complexes in the small intestine SSAs provoke phase II–like complexes and rapidly propagated, long activity fronts in the small intestine SSAs increase net epithelial cell absorption of electrolytes in the normal jejunum and ileum by reducing active electrolyte secretion SSAs suppress duodenal trypsin and lipase increases in response to Lundh meal; CCK release is abolished Somatostatin secreted from D cells SSAs inhibit hormone and meal-stimulated exocrine pancreatic secretion SSAs inhibit secretion of glucagon and insulin from pancreatic islet cells SSAs inhibit postprandial gallbladder contractility mainly via abolition of the CCK response to duodenal fat SSAs modulate the sphincter of Oddi activity, impairing biliary and pancreatic flow SSAs promote increase in biliary cholesterol and deoxycholic acid content, promoting bile crystallization SSAs inhibit CCK-mediated contraction in colonic smooth muscle Somatostatin and SSA reduce colonic secretion and colonic mucosal and submucosal blood flow SSAs reduce sensation of rectal distension via inhibition of visceral afferent pathways

particularly carcinoids, often do not produce classical symptoms until they metastasize to the liver. The initial treatment of GEP neuroendocrine tumors is surgical removal of the primary tumor, but in many cases surgical cure is not feasible due to metastatic disease. SSAs are a major therapeutic option for GEP tumors [9, 44], which is based on the predominant expression of sst2 receptors on more than 80% of GEP tumors [12] and capacity of somatostatin to directly inhibit the release and effects of GI hormones. Indeed, the relatively high frequency of sst2 expression means that radioactively labeled versions of octreotide have found widespread use in the radiological diagnosis and treatment of GEP tumors [20].

Carcinoid Tumors Clinical studies using the intermittent subcutaneous injected form of SSAs, such as octreotide or lanreotide at doses of 50–500 μg subcutaneously three times a day, have demonstrated a decrease in the release of bioactive

secreted products (5-HT and 5-hydroxyindolacetic acid), with effective resolution of flushing and diarrhea in 70–80% of cases [35]. The introduction of long-acting depot SSAs has provided significant improvement in the tolerability of the drug with similar or better efficacy as compared with their intermittent subcutaneous counterparts [44]. The optimum therapeutic approach is to begin therapy with intermittent subcutaneous SSAs for 3–7 days before and for approximately 14 days after the administration of a long-acting formulation in order to provide adequate symptomatic benefit [41]. To reduce the risk of a symptomatic carcinoid crisis brought on by anesthesia and surgical interventions, preoperative administration of short-acting SSAs remains standard therapy. SSAs provide a degree of tumor stabilization in a proportion of patients with metastatic carcinoid syndrome; however, when tumor progression occurs, SSA therapy may be titrated up to maintain symptomatic control. The overall efficacy of SSAs in carcinoid syndrome comprises a biochemical response in up to 77%, symptomatic responses in 36–100%, and tumor responses

Somatostatin Analogs in the Gastrointestinal Tract / 1133 in 0–9%. An experimental octreotide pamoate preparation, when given at a high dosage of 160 mg intramuscularly every 2–4 weeks, showed stabilization of tumor growth and/or biochemical parameters and improvement of symptoms of carcinoid syndrome in 10 out of 12 patients treated for 12 months [67]. There are preclinical data suggest that the universal SSA (SOM230), which binds with high affinity to receptor types 1, 2, 3, and 5 versus the relative sst2 receptor selectivity of octreotide or lanreotide, may have more a beneficial profile in terms of efficacy during long-term treatment; SOM230 is presently in phase II clinical trials for midgut carcinoids [40]. As already noted, the high levels of sst2 receptor expression on GEP tumors has led to the development of radioactively labeled octapep-tide SSAs for molecular imaging and therapeutic purposes [59]. There are also some results on the clinical use of similar octapeptide analogs as radiotherapy in patients with GEP tumors, using compounds such as 111In-pentetreotide [90Y-DOTA0, Tyr3]-octreotide (OctreoTher), the 177 better tolerated [ Lu-DOTA0, Tyr3]-octreotate, and 111 [ In-DOTA0]-lanreotide [9, 41, 63].

Gastrinomas Symptoms attributable to gastrinoma are usually satisfactorily controlled with high-dose protonpump-inhibitor drugs, although these drugs do not have any anticancer activity. In addition to reducing gastrin secretion, a recent study of long-term octreotide treatment of progressive metastatic gastrinoma showed that 53% of tumors (8/15 patients) responded at 3 months, 47% (7/15 patients) demonstrated tumor stabilization, and 6% (1/15 patients) demonstrated a decrease in tumor size. Tumor responses did not correlate with clinical parameters. However, slow-growing tumors were significantly more likely to respond prior to treatment (86 vs. 0%) [51].

Insulinomas Patients with insulinoma are generally unsuitable for SSA therapy because it has been observed that octreotide treatment worsens hypoglycemia in insulinomas that do not express receptors sst2 and sst5. This can result in a failure to suppress insulin secretion by the tumor in parallel with the pharmacological inhibition by SSAs of counter-regulatory hormones, particularly glucagon [66]. However, a subset of insulinoma patients with proven sst2 or sst5 positive tumors (via scintigraphy) can exhibit therapeutic benefit following SSA therapy. Recently Vezzosi et al. showed that the suppression of insulin secretion following a single dose of subcutaneous octreotide could be used to screen insulinoma patients for SSA therapy [64].

VIPomas Patients with VIP-secreting tumors suffer from debilitating, cholera-like, watery diarrhea, which is accompanied by electrolyte disturbances. SSAs revolutionized the treatment of these patients, and in the clinical setting the inhibition of VIP secretion by an SSA can rapidly normalize patients’ fluid and electrolyte balance. SSA treatment is effective in preventing diarrhea in 80–90% of VIPoma patients [42, 56].

Glucagonomas SSAs improve the majority of symptoms in glucagonoma patients [66], with notable effects on necrolytic migratory erythema [52]. It has also been noted that in some patients symptoms can be improved during octreotide therapy without a decrease in plasma glucagon levels [49, 68].

SECRETORY DIARRHEAS The clinical utility of SSAs in the management of treatment-resistant diarrhea is based on the ability of somatostatin to directly and indirectly decrease GI motility, intestinal fluid, and electrolyte transport; in addition, somatostatin is present in some intestinal neurons that are considered to be pro-absorptive [16]. The best-studied diarrheal conditions are chemotherapy-related diarrhea and AIDS-related diarrhea.

Chemotherapy-Induced Diarrhea Diarrhea is a well-recognized side effect of a number of chemotherapeutic regimens, with overall estimated prevalence around 10% of patients with advanced cancer. Severe or intractable diarrhea that is poorly responsive to standard therapy (e.g., opiates) can slow treatment cycles, reduce quality of life, and lead to significant debilitation [66]. A series of open-label studies of the efficacy of octreotide in the treatment of refractory chemotherapy-induced diarrhea have been performed. A review of early studies with intermittent subcutaneous octreotide reported efficacy rates of up to 90% [66], and similar results were shown in recent studies, in which the complete resolution of diarrhea occurred with octreotide therapy in 94% of patients; the effect of octreotide was rapid, with most patients responding fully within 72 hours [70]. Results with depot SSAs have been similar to those achieved with intermittent daily injections [48]. In a related oncological setting, octreotide has also shown to be more effective than conventional therapy (diphenoxylate and atropine) in controlling acute radiation-induced diarrhea in patients with cancer [69].

1134 / Chapter 155 AIDS-Related Diarrhea During the 1980s and early 1990s, chronic diarrhea was a common problem in patients with acquired immune deficiency syndrome (AIDS) in the developed world, resulting in significant morbidity and mortality [36]. Studies from before the highly active antiretroviral treatment era showed that diarrhea occurred in 30–50% of North American and European patients and in up to 90% of HIV-positive patients in developing countries [55]. Since the introduction of highly active antiretroviral treatment in 1996, there has been a significant decrease in the number of HIV-positive patients with refractory diarrhea [31]. Octreotide was first shown to be useful in the management of AIDS-related diarrhea in 1988 [47]. In a systematic review, patients in uncontrolled prospective studies that received octreotide for 1–33 weeks had a median complete response rate of approximately 30%, whereas the median partial response rate was 35%. In randomized controlled (versus standard therapy) studies of 10 days to 3 weeks duration, SSAs were associated with a 43% partial or complete response rate, compared with 29% in control patients [61]. The effectiveness of octreotide in AIDS-related diarrhea could be explained by the in vitro finding that octreotide inhibited a short-circuit current due to active chloride secretion induced by fraction 5 of the gp-41 protein of HIV [17].

ESOPHAGEAL VARICEAL HEMORRHAGE In patients with chronic elevations in hepatic portal pressures due to liver cirrhosis, varices can form at the level of esophageal veins; the rupture of these varices is a life-threatening event. The current gold-standard treatment for bleeding esophageal varices is endoscopic banding or sclerotherapy; varices can also be treated using transjugular intrahepatic portosystemic shunts, a radiological approach. Because not all medical centers have immediate access to a gastroenterologist or interventional radiologist, other forms of therapy, including balloon tamponade and pharmacotherapy, are often necessary. SSAs are often used in the treatment for bleeding esophageal varices, because somatostatin reduces hepatic venous pressure and hepatic blood flow in cirrhotic patients [7]. Some studies have shown octreotide to lower portal pressure following acute dosing, although this effect was less pronounced during long-term treatment [45]. The precise mechanism by which somatostatin and its analogs control variceal bleeding remains controversial. Somatostatin and its analogs inhibit glucagon and other GI vasodilatory peptides via sstr2-mediated effects, and simultaneous infu-

sion of glucagon can abolish the hemodynamic effects of somatostatin. The immediate splanchnic vascular response to a bolus injection of somatostatin or octreotide appears to be attributable to different mechanisms, such as inhibition of systematic and splanchnic protein kinase C–dependent vasoconstrictors via sstr2. In vitro studies indicate that somatostatin blunts endothelin-1induced constriction of hepatic stellate cells through the activation sstr1, which may decrease hepatic resistance in cirrhosis [1, 45]. Octreotide has been compared in clinical trials with a variety of treatments for bleeding esophageal varices. Octreotide was equivalent to balloon tamponade in patients with endoscopically proven bleeding varices, with improved tolerability and survival in the octreotide group [34]. Open-label studies have also shown octreotide treatment for 48 hours was similar to immediate endoscopic sclerotherapy or banding [57, 58], with some benefits of octreotide in terms of lower rebleeding rates. A large (n = 199) randomized controlled study of sclerotherapy alone versus sclerotherapy and octreotide showed significantly greater survival and decreased blood transfusions in the group that received octreotide [5]. In comparison with other medical therapies, octreotide has been shown to have equivalent efficacy and better tolerability than vasopressin [27] and terlipressin/nitroglycerin [53]. A sizable number of systematic reviews and meta-analyses have compared the efficacies of the various available forms of therapy for bleeding esophageal varices. Overall, the efficacy of pharmacotherapy (all types pooled) is similar to that of sclerotherapy in terms of survival, rebleeding, and transfusion requirements [11]. Among the individual therapies, systematic review data suggest that octreotide and vasopressin/terlipressin have equivalent efficacies [28], whereas octreotide is at least equivalent to sclerotherapy [10]. The role of SSAs in the treatment of bleeding esophageal varices is as a holding therapy before definitive endoscopic approaches can be arranged and also as an adjunctive agent to improve endoscopic outcomes.

GI MOTILITY AND FUNCTIONAL GI DISORDERS Somatostatin stimulates migrating motor complexes in the small intestine, which are synchronized with secretory and motor activities of the gallbladder and pancreas [18]. A single 50-μg dose of octreotide inhibits antral motor function and provokes phase II–like complexes in the small bowel in patients with functional or organic motility disorders of the upper gut and in healthy controls. Octreotide induces rapidly propagated, long activity fronts, even in patients with neu-

Somatostatin Analogs in the Gastrointestinal Tract / 1135 ropathological lesions, and this may initially facilitate the intestinal propulsion of chyme. Propulsion may not occur, however, if octreotide induces simultaneous activity fronts or if the activity front is followed by prolonged quiescence [25]. This may contribute to the delay in small bowel transit. In a recent review, Kuiken et al. described several studies on viscerosensory effects of SSAs. In healthy volunteers, octreotide reduced the perception of physiological sensations (esophagus and stomach) and augmented the pain threshold and the maximum tolerated volumes during slow ramp volume distension of the rectum but not during isobaric distension (stomach and rectum). This suggests that SSAs reduces afferent signaling on activation of a subset of visceral mechanoreceptors [29]. Patients with functional GI disorders experience chronic abdominal pain or discomfort often associated with abnormal motility in the absence of any organic disease [62]. The most common of these disorders are the irritable bowel syndrome and functional dyspepsia.

Irritable Bowel Syndrome In early open-label studies octreotide therapy reduced abdominal symptoms in irritable bowel syndrome patients [6, 61]. In a controlled trial, octreotide effectively increased discomfort thresholds at baseline and during experimentally induced rectal hyperalgesia in seven irritable bowel syndrome patients but not in eight controls. This indicates that SSAs exerts an antihyperalgesic rather than an analgesic effect on visceral perception [50]. These results suggest the possible usefulness of SSAs in irritable bowel syndrome treatment, although large-scale randomized controlled studies are needed.

Functional Dyspepsia In an open-label prospective study, 17 patients with severe refractory functional epigastric pain were treated with subcutaneous intermittent octreotide. After 1 month, a progressive improvement of pain intensity was reported in 15/17 patients, and in 11 of these 15 improved patients, whose subsequent progress was followed for at least 6 months, symptomatic improvements and a median weight gain of 4 kg was noted [15].

POSTOPERATIVE COMPLICATIONS OF SURGERY Pancreatic Surgery Pancreatic surgery can be accompanied by postoperative complications such as pseudocysts and fistulas,

which complicate recovery and increase the risk of infection. These complications are caused by build-up of pancreatic exocrine secretions. SSAs are potent inhibitors of exocrine pancreatic secretion, and their efficacy in reducing postoperative complications in pancreatic surgery has been studied extensively in clinical trials. A series of multicenter, placebocontrolled studies have indicated that the perioperative administration of octreotide or lanreotide in patients undergoing surgery for chronic pancreatitis or periampullary tumors reduces the risk of postoperative pancreatitis, hemorrhage, leakage, or fistula/pseudocyst formation [3, 8, 19, 37, 54]. Evidence from these trials suggests that SSAs may function more effectively in higher-risk patients with pancreatic tumors, although this remains controversial. Based on the weight of data from more than 1000 patients treated in these trials, it has been suggested that SSAs can significantly reduce the postoperative complications of surgery in those with pancreatic cancer or chronic pancreatitis [4, 23].

Dumping Syndrome The dumping syndrome occurs typically after gastric surgery and involves meal-provoked symptoms consisting of abdominal discomfort or systemic vascular effects (early dumping) and reactive hypoglycemia (late dumping). The efficacy of SSAs (mainly octreotide) has been studied in individual, small clinical trials, which have been pooled as part of a systematic review [33]. It was shown that SSAs were effective in alleviating diarrhea, abdominal pain, dizziness, and palpitations and that they also minimized changes in systemic blood pressure by reducing orthostatic fluctuations. SSAs can also minimize negative influences on packed cell volume and plasma osmolarity and prevent late hypoglycemia by reducing peak insulin concentration and prolonging maximal plasma glucose concentration.

Short-Bowel Syndrome Following extensive resection of the small intestine, patients may have symptoms of malabsorption and fluid and electrolyte loss, termed short-bowel syndrome. In one controlled and two uncontrolled prospective studies, all patients showed significant improvements in response to intermittent or infused octreotide therapy [30, 38, 43]. A more recent study using the depot formulation of octreotide demonstrated significantly reduced intestinal output, decreased in-hospital care, and reduced intravenous fluid and electrolyte requirements; in 80% of patients, total parental nutrition was not required following octreotide therapy [21].

1136 / Chapter 155 GI Fistula GI fistulae are anomalous connections between any portion of the GI tract and an internal or external epithelial surface. In combination with nutritional replacement therapy, somatostatin has been reported to inhibit both basal and stimulated digestive secretion, with a reduction in fluid loss, electrolyte imbalance, and malnutrition, which can reduce fistula output and shorten the time to fistula closure compared with conventional supportive care [22, 26]. With respect to SSAs, octreotide has been shown to decrease fistula output in some studies [2, 39]. In a recent study of 51 patients with GI or pancreatic fistulae, resolution of the fistula occurred in 65% of patients in the octreotide group, compared with 27% in the control group [32].

BILIARY SYSTEM As noted in the previous sections, SSAs have potent inhibitory effects on GI hormones, exocrine secretion, and motility; they also have an important effect on blood flow. These effects are also seen in the biliary tract, with reduced gallbladder contractility via inhibition of cholecystokinin (CCK) and altered bile constituents promoting the development of cholelithiasis. SSAs promote the development of a cholesterol supersaturated bile, a high molar ratio of cholesterol to phospholipids in the bile, rapid nucleation of cholesterol microcrystals, and a doubling of the deoxycholic acid concentration [13]. Also the alteration by SSAs of intestinal motility and absorptive properties disrupts the normal patterns of biliary component metabolism and reabsorption [13]. The combination of these effects leads to the development of de novo gallstones or biliary sludge in 30% of patients receiving long-term SSA therapy; however, the management of SSA-induced gallstones does not differ from that of gallstones in general.

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156 Substance P and Related Tachykinins in the Gastrointestinal Tract PETER HOLZER

discovery of another tachykinin, neurokinin A (NKA), in the mammalian gut; the identification of three tachykinin receptors labeled as NK1, NK2, and NK3; and the development of highly selective nonpeptide antagonists for all three tachykinin receptors. As a result, the distribution, biological actions, and pathophysiological implications of tachykinins in the mammalian gut have been thoroughly investigated, and tachykinin receptor antagonists are currently explored for their utility in the treatment of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and other GI disorders [4–6, 9, 11].

ABSTRACT Within the gastrointestinal tract, the tachykinins substance P and neurokinin A occur in neurons of the enteric nervous system, in fibers of extrinsic afferent neurons, and in enterochromaffin and immune cells of the mucosa. Their biological actions are mediated by three different types of G-protein-coupled receptors, termed NK1, NK2, and NK3, and are involved in the regulation of gastrointestinal motility, secretion, vascular permeability, and pain sensitivity. Pathophysiologically, tachykinins appear to be involved in inflammation-induced disturbances of gut function such as dysmotility, secretory diarrhea, edema, and hyperalgesia. Expressed in a cell-specific manner, tachykinin receptors thus represent an attractive target for novel therapeutic strategies in gastroenterology.

DISTRIBUTION OF TACHYKININ mRNAS AND PEPTIDES IN THE GI TRACT The structures of the three known tachykinin genes and their protein products are discussed in Chapter 105 by N. M. Page. SP and NKA, the major tachykinins in the GI tract, are derived from the TAC1 gene (previously called PPT-A for preprotachykinin A). The primary RNA transcript of TAC1 is alternatively spliced to produce four different types of mRNA that encode either SP (α-TAC1, δ-TAC1) or SP plus NKA (β-TAC1, γ-TAC1) and some N-terminally extended forms of NKA such as neuropeptide K and neuropeptide γ. The TAC3 (PPT-B) gene gives rise to NKB, whereas the human TAC4 (PPT-C) gene is alternatively processed to yield hemokinin 1 and/or endokinin A, B, C, or D. Because TAC1 mRNA prevails in the gut, SP and NKA are the predominant tachykinins in this organ system, whereas NKB has proved difficult to detect in the bowel [4] and the precise localization of TAC4 gene products in the GI tract is yet to be determined. Although some SP is contained in enterochromaffin and immune cells of the GI mucosa, the major source of tachykinins in the gut is the enteric nervous system

DISCOVERY OF TACHYKININS IN THE GUT The biological activity of substance P (SP) was discovered in 1931 when Ulf S. von Euler and John H. Gaddum reported that both intestine and brain contained a substance that caused contraction of gastrointestinal (GI) smooth muscle but was different from any of the endogenous compounds known at that time. In the early 1950s, Bengt Pernow realized the wide distribution of SP in the GI tract, and in the 1960s Fred Lembeck and others found that SP causes copious salivary secretion in rats and dogs. This biological effect was the lead bioassay whereby Susan E. Leeman and her laboratory identified SP as an undekapeptide in 1970. SP turned out to be the mammalian counterpart of a family of amphibian and nonvertebrate peptides, named tachykinins by Vittorio Erspamer because they caused fast contraction of GI smooth muscle (see Chapter 26 by D. R. Nässel). This breakthrough was followed by the Handbook of Biologically Active Peptides

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1140 / Chapter 156 (ENS) [2–4]. In the guinea pig intestine, SP is typically present in intrinsic primary afferent neurons (IPANs) of the myenteric and submucosal plexus and, within the myenteric plexus, in ascending interneurons, as well as in excitatory motoneurons to the longitudinal and circular muscle (Fig. 1). Most enteric neurons containing SP are cholinergic because they coexpress choline acetyltransferase. The tachykininergic enteric neurons can be further subgrouped by their content of calbindin/calretinin, neurofilament protein triplet, and dynorphin/enkephalinlike immunoreactivity (Fig. 1). Primary afferent neurons that originate from dorsal root ganglia and reach the gut via sympathetic and sacral parasympathetic nerves also contribute to the tachykinin content of the gut. The peripheral fibers of these extrinsic sensory neurons project primarily to submucosal arterioles, but also supply the mucosa and enteric nerve plexuses (Fig. 1).

TACHYKININ RECEPTOR SUBTYPES, SIGNALING, AND DISTRIBUTION IN THE GI TRACT The biological actions of tachykinins in the GI tract are mediated by three tachykinin receptors [4, 7, 10], currently termed NK1, NK2, and NK3 receptors and encoded by three different genes, TACR1, TACR2, and TACR3. As outlined in Chapter 105 by N. M. Page, the membrane topology of these receptors is typical of metabotropic receptors with seven-transmembrane

domains. Being coupled to G-proteins, the tachykinin receptors use the phospholipase C–phosphoinositide system as a major transduction pathway [7, 10], although other signaling mechanims such as the adenylate cyclase pathway may also play a role. By the modification of the amino acid sequence of endogenous tachykinins, selective agonists for all three tachykinin receptors have been obtained, and this advance has been complemented by the design of potent and receptor-selective nonpeptide antagonists for NK1, NK2, and NK3 receptors [4, 7]. The distribution of tachykinin NK1, NK2, and NK3 receptors to GI neurons and effector cells (Fig. 2) enables tachykinins to modify GI motility, secretory activity, vascular diameter, vascular permeability, immune function, gut sensitivity, and nociception [3–8]. NK1 receptors are found on longitudinal and circular muscle cells, interstitial cells of Cajal (ICCs), IPANs, excitatory and inhibitory motoneurons, secretomotor neurons, epithelial cells, and granulocytes of the rodent gut. In the human GI tract, NK1 receptors have also been localized to the muscularis mucosae, the media of submucosal blood vessels (Fig. 2), and some immune cells. NK2 receptors are typically expressed by the longitudinal muscle, circular muscle, and muscularis mucosae and, in addition, are present on epithelial cells and enteric nerve endings (Fig. 2). NK3 receptors are largely confined to enteric neurons; in the rodent intestine, these NK3 receptor-bearing neurons include IPANs, ascending and descending interneurons, excitatory and inhibitory motoneurons, and secretomotor neurons (Fig. 2).

FIGURE 1. Chemical coding and projections of the most important classes of tachykinin (TK)-immunoreactive neurons in the mammalian gut. AIN, ascending interneuron; BV, blood vessel; CB, calbindin; CGRP, calcitonin gene-related peptide; ChAT, choline acetyltransferase; CM, circular muscle; CR, calretinin; DRG, dorsal root ganglion; ELMN, excitatory longitudinal muscle motoneuron; ENK, enkephalin; EPAN, extrinsic primary afferent neuron; IPANs, intrinsic primary afferent neurons; LAECMN, long ascending excitatory circular muscle motoneuron; LM, longitudinal muscle; MP, myenteric plexus; MU, mucosa; NF, neurofilament protein; SAECMN, short ascending excitatory circular muscle motoneuron; SMP, submucosal plexus.

Substance P and Related Tachykinins in the Gastrointestinal Tract / 1141 FIGURE 2. Cellular expression of tachykinin NK1, NK2, and NK3 receptors in the mammalian gut. Filled circles depict neuronal somata; filled triangles depict nerve endings. AECMN, ascending excitatory circular muscle motoneuron; AIN, ascending interneuron; BV, blood vessel; CM, circular muscle; DICMN, descending inhibitory circular muscle motoneuron; DIN, descending interneuron; ELMN, excitatory longitudinal muscle motoneuron; ICC, interstitial cell of Cajal; LM, longitudinal muscle; MIPANs, myenteric intrinsic primary afferent neurons; MM, muscularis mucosae; MP, myenteric plexus; MU, mucosa; SIPANs, submucosal intrinsic primary afferent neurons; SMN, secretomotor neuron; SMP, submucosal plexus.

BIOLOGICAL ACTIONS OF TACHYKININS IN THE GUT Transmitter Function SP and NKA are cotransmitters of intrinsic enteric and extrinsic afferent neurons, which is consistent with their vesicular localization and calcium-dependent release on nerve stimulation [4–8]. Within the ENS, tachykinins mediate slow postsynaptic excitation, a process that is relevant to the communication among IPANs (mediated primarily by NK3 receptors), between IPANs and ascending as well as descending interneurons (mediated by NK3 receptors), between ascending interneurons and excitatory motoneurons (mediated by NK3 receptors), and between IPANs and inhibitory motoneurons (mediated by NK1 receptors) [3, 4]. Furthermore, tachykinins participate in excitatory neuromuscular transmission (mediated by NK2 and to some extent NK1 receptors), although in this role they are subordinate to the principal transmitter acetylcholine [4, 8].

Motor Activity Tachykinins can both stimulate and inhibit GI motility, the net response depending on the type and site of tachykinin receptors that are activated [3, 4, 8]. The facilitation of GI motor activity is typically brought about by the activation of NK1 receptors on IPANs, ICCs, and muscle cells; NK2 receptors on muscle cells; and NK3 receptors on IPANs, cholinergic interneurons, and cholinergic motoneurons (Fig. 2). The stimulation

of NK1 receptors on ICCs enforces motility by prolonging the duration of the slow waves generated by these cells. Tachykinin-induced muscle contraction in the human gut in vitro is prominently mediated by muscular NK2 receptors, and in the circular muscle of the isolated human sigmoid it appears as if tachykinins acting via NK2 receptors are the main excitatory neurotransmitters released by nerve stimulation. NK2 receptors also make an important contribution to the effects of SP and NKA to stimulate motility in the human small intestine in vivo and to replace the regular pattern of interdigestive motor activity by a pattern of irregular activity. The contractile response to NK3 receptor stimulation in the guinea pig intestine is predominantly mediated by cholinergic neurons [4]. NK1 and NK3 receptors on inhibitory motor pathways within the ENS enable tachykinins to depress motor activity via release of nitric oxide and adenosine triphosphate [3, 4]. Despite the high pharmacological potency of tachykinins to modify GI motility, NK1, NK2, and NK3 tachykinin receptor antagonists have little effect on GI motor performance under physiological conditions both in vitro and in vivo. NK1 receptor antagonists cause a minor stimulation of peristaltic motility in the guinea pig isolated small intestine and accelerate propulsion in the rabbit isolated distal colon, which is consistent with their ability to cause mild diarrhea in humans [4]. NK2 receptor antagonists, to the contrary, do not alter small intestinal motility in humans, but lead to a minute inhibition of peristalsis in the guinea pig small bowel. Only when the overwhelming cholinergic component in the neural activation of smooth muscle has been

1142 / Chapter 156 compromised does the blockade of tachykinin receptors impair peristalsis in the guinea pig small intestine. SP and NKA thus function as a backup system in the cholinergic activation of GI muscle during peristalsis, a role that in the guinea pig small intestine is brought about by NK1 and NK2 receptors but in the human intestine is primarily mediated by NK2 receptors. When, however, all three tachykinin receptors are blocked simultaneously, peristalsis in the guinea pig distal colon is significantly depressed even without concomitant blockade of cholinergic transmission [4]. From these findings it appears that, in humans, tachykinins regulate motility primarily in the colon, a hypothesis that has not yet been systematically tested. Furthermore, multi- or pan-tachykinin receptor antagonists seem to be more efficacious than mono-receptor antagonists in modifying GI motor activity and, eventually, GI motor disorders [4].

Secretory Activity Tachykinins modify endocrine and exocrine secretory processes in the GI tract, including the stomach and pancreas [4, 6]. Electrolyte and fluid output in the rodent intestine can be stimulated through the activation of tachykinin NK1 and NK3 receptors on cholinergic and noncholinergic secretomotor neurons in the submucosal plexus as well as NK1 and NK2 receptors on epithelial cells. Mucosal ion transport in the isolated human colon is enhanced by both NK1 and NK2 receptor activation and subsequent stimulation of enteric neurons. The neurogenic actions of tachykinins to elicit electrolyte and fluid secretion in the intestine are consistent with a role of SP and NKA as transmitters of enteric secretory reflexes. The implication of tachykinins in these reflexes can be threefold [4]. First, tachykinins released from IPANs or interneurons can activate cholinergic and noncholinergic secretomotor neurons. Second, SP can be released from axon collaterals of IPANs close to the epithelial effector cells and elicit chloride secretion via a mechanism resembling an axon reflex. Third, tachykinins released from extrinsic sensory nerve endings in response to capsaicin, Clostridium difficile toxin A, or distension can stimulate enteric secretomotor neurons through the activation of NK1 and NK3 receptors.

Proinflammatory Function Tachykinins, particularly SP, are vasoactive peptides that can induce dilation or constriction of blood vessels in the digestive tract, the type of action depending on the vascular bed and species under study [4, 6]. The tachykinin-evoked vasodilation in the intestine of cat,

dog, and guinea pig is mediated by NK1 receptors. Both SP and NKA enhance blood flow in the proximal small intestine of humans, probably through the activation of NK1 receptors that have been localized to the media of submucosal blood vessels. Conversely, blood flow in the rat gastric mucosa is diminished by tachykinins via constriction of collecting venules, a mechanism that may depend on the release of proteases from mast cells. Another effect of substance P acting via endothelial NK1 receptors is to increase venular permeability in the intestine and thereby to facilitate the extravasation of plasma proteins and leukocytes. In addition, tachykinins can influence the activity of various immune cells in the gut. Thus, NK1 and NK2 receptors have been localized to monocytes/macrophages, granulocytes, lymphoid cells, and eosinophils, and stimulation of tachykinin receptors can lead to the recruitment and activation of granulocytes as well as mast cells in the GI tract [4, 6].

PATHOPHYSIOLOGICAL IMPLICATIONS OF TACHYKININS IN THE GI TRACT Pathological Changes in the GI Expression of Tachykinins and Tachykinin Receptors Many studies have shown that GI infection, inflammation, and mucosal injury are associated with timerelated changes in the expression and release of tachykinins in the gut. However, the alterations in SP and NKA expression are variable, given that the intestinal tachykinin levels in patients with IBD have been reported to be decreased, increased, or unchanged [4, 6]. It is important that whole-mount analysis of the myenteric plexus has revealed that the chemical coding of enteric neurons in ulcerative colitis is shifted inasmuch as the ratio of SP-positive versus SP-negative cholinergic neurons is significantly enhanced. Animal studies have demonstrated that experimental infection and inflammation also cause changes in the GI tachykinin levels that mirror those seen in IBD to a variable degree. IBD is, likewise, accompanied by an increased expression of NK1 and NK2 receptors in the inflamed and noninflamed regions of the human ileum and colon. The upregulation and ectopic occurrence of SP binding sites in pseudomembranous colitis due to Clostridium difficile infection has been reproduced by treatment of rats with C. difficile toxin A. Several other studies have shown that the expression of tachykinin receptors is either up- or downregulated under conditions of experimentally induced infection or inflammation [4].

Substance P and Related Tachykinins in the Gastrointestinal Tract / 1143

Implications of Tachykinins in GI Motor Disturbances

Implications of Tachykinins in GI Hypersecretion and Inflammation

The alterations in the expression of tachykinins and tachykinin receptors seen in GI inflammation raise the conjecture that an imbalance in the SP and NKA system contributes to GI disease such as pathological disturbances of motility. Experimental support for this hypothesis comes from the findings that the motor effects of tachykinins are changed in certain GI diseases and that tachykinin receptor antagonists are beneficial in experimental models of GI dysmotility [4]. For instance, the efficacy of NK2 receptor agonists to contract the colonic circular muscle in vitro is attenuated in IBD. Similarly, the responsiveness of the colonic musculature to tachykinin receptor agonists is depressed by trinitrobenzene sulfonic acid (TNBSA)-induced colitis in the rat and rabbit, whereas in ricin-evoked ileitis of the rabbit tachykinin-mediated neurogenic contractions are amplified. The ability of NK2 receptor agonists to stimulate colonic circular muscle activity is increased in some patients with chronic idiopathic constipation, whereas NK2 receptor–mediated transmission to the colonic circular muscle is deficient in children with slow-transit constipation. Tachykinin receptor antagonists are able to correct various types of experimentally disturbed motility in the gut [4]. Thus, NK1 receptor antagonists inhibit stressinduced defecation and correct the hypomotility and muscular hyporesponsiveness caused by anaphylaxis, inflammation, and pain. Postoperative and peritonitisinduced ileus is ameliorated by NK1 and NK2 receptor antagonists, whereas the giant colonic contractions associated with inflammatory diarrhea are effectively suppressed by NK2 receptor antagonists. Although the usefulness of NK3 receptor antagonists remains to be explored, it appears that NK1 receptor antagonists are particularly useful in alleviating GI motor inhibition, whereas NK2 receptor antagonists are beneficial in attenuating pathological hypermotility without causing constipation. Apart from acting on mast cells, enteric neurons, and muscle cells, tachykinin receptor antagonists may correct GI dysmotility by interfering with the function of extrinsic afferents that can contribute to GI motor dysregulation in two ways [4]. First, they participate in autonomic intestino-intestinal reflexes in which SP and NKA, released from the central endings of sensory neurons in the spinal cord, mediate transmission to the efferent reflex arc. Second, tachykinins released from sensory nerve endings in the gut can disturb GI motility, an instance that may be reflected by the ability of NK1 receptor antagonists to ameliorate dysmotility caused by esophageal acidification, anaphylaxis, and inflammation.

There is considerable evidence that the tachykinin system contributes to GI mucosal pathologies associated with infection, inflammation, and functional bowel disorders [4]. For example, the secretory response to SP is blunted in mucosal tissues isolated from patients with Crohn’s disease or ulcerative colitis, and tachykinin receptor antagonists display beneficial effects in various models of experimental GI infection, GI inflammation, GI injury, diarrhea, and pancreatitis. Tachykinins play a particular role in the inflammation (granulocyte, mast cell, and macrophage activation) and hypersecretion evoked by Clostridium difficile toxin A, which involves the activation of capsaicin-sensitive extrinsic afferent neurons, release of SP, and activation of NK1 receptors on enteric neurons. Many types of GI hypersecretion and inflammation depend on multiple tachykinin receptors, which is exemplified by the observations that the intestinal hypersecretion evoked by Cholera toxin, the diarrhea caused by castor oil, and the TNBSAinduced rectocolitis in the rat are inhibited by both NK1 and NK2 receptor antagonists [4]. Of further relevance is the question of whether tachykinins play a role in the initiation and maintenance of GI hypersecretion and inflammation. Experimental data indicate that SP and NKA participate primarily in the initial stage of TNBSA- and acetic acid–induced colitis. If so, tachykinin receptor antagonists may be more beneficial in the initiation or reactivation of inflammation than in the suppression of an ongoing inflammatory process. The effect of SP to increase vascular permeability in the GI tract is amplified in the inflamed tissue because inflammation leads to downregulation of neutral endopeptidase, which in the normal tissue maintains low levels of SP in the extracellular fluid and thus limits its proinflammatory effects [4]. Preclinical studies suggest that various immune cells in the gut are under the influence of SP and NKA; the tachykinin-responsive cells include mast cells, lymphocytes, and granulocytes [4]. Furthermore, immune cells are not only targets of tachykinin actions, but under pathological conditions they can themselves be induced to synthesize and release tachykinins. This is true for macrophages in the mucosa of the rat ileum exposed to C. difficile toxin A and for eosinophils from the mucosa of IBD patients.

Implications of Tachykinins in GI Hyperalgesia and Pain The majority of spinal afferents supplying the rodent GI tract contain SP and NKA, and tachykinin NK1, NK2, and NK3 receptors are expressed at many levels of the

1144 / Chapter 156 gut-brain axis [4]. A double blind pilot study has shown that the tachykinin NK1 receptor antagonist CJ-11,974 reduces IBS symptoms and attenuates the emotional response to rectosigmoid distension. This observation is in keeping with preclinical studies that attest to a role of tachykinin receptors in visceral pain and hyperalgesia. For instance, genetic deletion of NK1 receptors in mice prevents intracolonic acetic acid and capsaicin from eliciting pseudoaffective pain responses, inducing primary mechanical hyperalgesia in the colon and causing referred mechanical hyperalgesia in abdominal skin [4]. Experimental studies with selective tachykinin receptor antagonists indicate that all three tachykinin receptors play a role in visceral nociception and inflammation-induced hyperalgesia [4]. For instance, the visceromotor pain response to acute gastric and colorectal distension in the rat is inhibited by NK2 and NK3 receptor antagonists, but is left unaltered by NK1 receptor antagonists. In contrast, the infection-, inflammation-, or stress-induced hypersensitivity to distension in the rabbit, rat, and guinea pig jejunum or colon is reduced by either NK1 or NK2 receptor antagonists. Tachykinin receptor antagonists may target multiple relays in the nociceptive pathways from the periphery to the brain. One site of action is within the spinal cord where tachykininergic transmission from primary afferents is interrupted. This appears to be true for the antihyperalgesic effect of the NK1 receptor antagonist TAK-637 in the rabbit and guinea pig. Studies with other tachykinin receptor antagonists administered intrathecally indicate that the blockade of NK1 and NK3 receptors within the rat spinal cord is particularly effective in suppressing the visceromotor pain response to colorectal distension and the hypersensitivity caused by repeated distension or experimental inflammation. Further consistent with a central site of the antinociceptive action of NK1 receptor antagonists is the finding that experimental colitis or cystitis in the rat leads to an upregulation and de novo expression of NK1 receptors in the dorsal horn of the spinal cord [4]. Apart from blocking tachykininergic transmission in the spinal cord, NK1, NK2, and NK3 receptor antagonists may be antihyperalgesic by a peripheral site of action [4]. Although the expression of tachykinin receptors by primary afferent nerve fibers remains to be clarified, the NK2 receptor antagonist nepadutant has been observed to inhibit the enhanced firing that lumbosacral afferent neurons exhibit after distension of the experimentally inflamed rat colon. Because the activity in pelvic and somatic afferent neurons is not affected, nepadutant has been proposed to be antihyperalgesic by a peripheral action on hypersensitive afferents supplying the colon. A similar conclusion has been reached for the non-brain-penetrant NK3 receptor antagonist SB-235375, which is able to inhibit the visceromotor

reaction to painful colorectal distension in rats. Because colonic motility and compliance are little affected and mechanonociception in the skin is not altered, it has been inferred that NK3 receptor antagonists exhibit intestine-specific antinociceptive activity. Part of the antinociceptive action of NK1 receptor antagonists in the periphery may be indirect and due to their ability to modify GI motility and secretion and to promote inflammatory processes. Taking all experimental findings together, tachykinin receptor antagonists appear to have potential for the treatment of visceral pain and functional bowel disorders [4]. This may in particular apply to IBS and other disorders where the tachykinin system is deranged in several ways. By correcting hyper- or hypomotility, hypersecretion, and inflammation, tachykinin receptor antagonists may reduce the sensory gain of extrinsic afferents in the GI tract and, in addition, block tachykininergic transmission in the spinal cord. Furthermore, the effects of brainpenetrant NK1 receptor antagonists at the level of the gut and afferent system may favorably combine with their inhibitory actions on emesis, anxiety, depression, and stress reactions in the brain [4].

Nausea and Vomiting Preclinical studies have revealed that NK1 receptor antagonists inhibit vomiting caused by a variety of factors, including the anticancer agent cisplatin, irradiation, copper sulfate, morphine, apomorphine, and motion [1]. This broad-spectrum profile of activity against peripherally and centrally acting emetogenic stimuli and the requirement for brain penetration demonstrate that NK1 receptor antagonists interrupt the emetic reflex at a central site of action within the brainstem, close to the nucleus of the solitary tract and the Bötzinger complex. The experimental observation that NK1 receptor antagonists block both the acute and delayed phase of cisplatin-induced emesis has been reproduced in clinical trials. In particular, the NK1 receptor antagonist aprepitant enhances the antiemetic efficacy of 5-HT3 receptor antagonists and dexamethasone in humans and, in this combination, provides significant control over the delayed phase of chemotherapy-induced nausea and emesis [1]. As a consequence, aprepitant was licensed in 2003, which made this compound the first NK1 receptor antagonist in clinical use.

Acknowledgments The artistry of Evelin Painsipp in preparing the figures is greatly appreciated. Work in the author’s laboratory is supported by the Austrian Research Funds, the Jubilee Funds of the Austrian National Bank, the

Substance P and Related Tachykinins in the Gastrointestinal Tract / 1145 Austrian Federal Ministry of Education, Science and Culture, and the Zukunftsfonds Steiermark.

References [1] Andrews PLR, Rudd JA. The role of tachykinins and the tachykinin NK1 receptor in nausea and emesis. In: Holzer P, editor. Tachykinins. Handbook of Experimental Pharmacology, Volume 164. Berlin: Springer; 2004. p. 359–440. [2] Brookes SJH. Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec 2001;262:58–70. [3] Furness JB, Sanger GJ. Intrinsic nerve circuits of the gastrointestinal tract: identification of drug targets. Curr Opin Pharmacol 2002;2:612–622. [4] Holzer P. Role of tachykinins in the gastrointestinal tract. In: Holzer P, editor. Tachykinins. Handbook of Experimental Pharmacology, Volume 164. Berlin: Springer; 2004. p. 511–558.

[5] Holzer P, Holzer-Petsche U. Tachykinins in the gut. Part I. Expression, release and motor function. Pharmacol Ther 1997;73:173–217. [6] Holzer P, Holzer-Petsche U. Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation. Pharmacol Ther 1997;73:219–263. [7] Maggi CA. The mammalian tachykinin receptors. Gen Pharmacol 1995;26:911–944. [8] Maggi CA. Principles of tachykininergic co-transmission in the peripheral and enteric nervous system. Regul Pept 2000;93:53– 64. [9] Pernow B, Substance P. Pharmacol Rev 1983;35:85–141. [10] Regoli D, Boudon A, Fauchère JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994;46: 551–599. [11] Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V. The tachykinin peptide family. Pharmacol Rev 2002;54:285–322.

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157 TFF (Trefoil Factor Family) Peptides WERNER HOFFMANN

Historically, the discovery of the three mammalian (TFF1, TFF2, and TFF3) and various amphibian TFFs was the result of four groups working in different fields (for a review, see [19]). Mammalian TFFs consist of one (TFF1 and TFF3) or two TFF domains (TFF2), whereas amphibian TFFs contain up to four TFF domains. The amino acid sequence of human TFF1 (formerly: pS2/ BCEI/pNR-2/pNR-105/Md2) was deduced in 1984, and the primary structure of porcine TFF2 (formerly, [pancreatic] spasmolytic polypeptide, [P]SP) was reported in 1985. However, the high similarity between TFF1 and TFF2 was first recognized in 1988 by analysis of TFF domains from the frog integumentary mucin FIM-A.1 (formerly, spasmolysin). Human TFF2 was first described in 1990. TFF3 (formerly, intestinal trefoil factor, ITF, P1.B) was detected 1991 in rat and 1993 in human. The situation in amphibia is more complex because there are additional TFF peptides existing for which no mammalian ortholog is known (for review and references, see [19]). Typical of the gastrointestinal (GI) tract are xP1 (single TFF domain), xP4.1, and xP4.2 (four TFF domains). xP1 is the amphibian ortholog of TFF1, whereas xP4.1 seems to be the functional equivalent of TFF2.

ABSTRACT The TFF (trefoil factor family) domain is an ancient cysteine-rich shuffled module forming the basic unit for the family of TFF peptides (in short, TFFs). TFFs are secretory products typical of mucin-producing cells of the gastrointestinal (GI) tract. Three mammalian TFFs are known consisting of one (TFF1 and TFF3) or two TFF domains (TFF2); in amphibia the pattern is more complex—xP1 (single TFF domain), xP4.1 and xP4.2 (both consisting of four TFF domains) have been characterized in the GI tract thus far. TFFs play a key role in the maintenance of the GI surface integrity in health and disease by supporting a variety of different mucosal defense and repair mechanisms. Their multiple molecular functions include (1) formation of the mucous barrier, (2) enhancement of rapid mucosal repair (restitution), (3) modulation of mucosal differentiation processes, and (4) modulation of the mucosal immune response. However, TFFs are also connected with oncogenic pathways.

DISCOVERY Secretory TFF (trefoil factor family) peptides (for reviews and references, see [10, 18–20, 35, 43, 44, 54]) share a common structural motif; that is, they consist of one or more TFF domains (formerly, P-domain or trefoil-domain). This unique ancient shuffled module arose well before amphibian evolution and contains six conserved cysteine residues as its hallmark. TFF domains are also present in mosaic proteins associated with mucous surfaces (for a review, see [19]). The actual nomenclature of TFF peptides is based on an agreement reached at one of the Conférence Philippe Laudat [56] to avoid the single term trefoil because of its multiple meanings. Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The three human and the three mouse TFF genes, as well as part of the amphibian TFF genes, have been characterized (for review and references, see [19]). There are several structural features common to all TFF genes analyzed thus far, among them: (1) all TFF peptides are synthesized via precursors containing a cleavable N-terminal signal sequence typical of secretory peptides, and (2) TFF domains are typical shuffled modules always encoded by a single exon belonging to

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TFF1

E1

E2 E3

TFF2

TFF3 10 kb

E1 E2 E3 E4

E1 E2 E3 29 279 510

29 333 970

30

20

10

0

chromosome 21

FIGURE 1. Chromosomal localization of human TFF genes. Illustrated is the TFF cluster on chromosome 21q22.3. The nucleotide positions are according to [14]. The exons (E) encoding TFF domains are shown in black. Each of the first exons (E1) encodes the signal sequences typical of precursors of secretory proteins.

the class 1-1. Consequently, the TFF1 and the TFF3 genes consist of three exons, whereas the TFF2 gene contains four exons. All three human TFF genes are clustered on chromosome 21q22.3 in a head-to-tail arrangement within 54.5 kb in the order tel-TFF1-TFF2-TFF3-cen. Transcription of all three genes is directed towards the centromere (Fig. 1). A syntenic 40-kb region was characterized in the mouse genome on chromosome 17q. This locus also encodes an antisense TFF1-related transcript [15]. The situation in amphibia is rather complex because more than three TFF genes exist (there are additional TFF genes expressed, e.g., xP2 in the skin [19]). Whether these genes form a single cluster as in mammals is not yet known. The expression of the clustered mammalian TFF genes is coordinatedly regulated (for review and references, see [19]). This has been shown in particular for murine stomach by analysis of various transgenic TFFdeficient mice. There are also numerous studies structurally and functionally characterizing the promoter regions of the various mammalian and amphibian TFF genes (for detailed review, see [19]). Pathologically most important is probably the regulation of TFF1 by Ras/ERK signaling and TFF3 by STAT1/3 [51]. Furthermore, TFF1 expression is regulated by gastrin [23], and TFF3 expression is inducible by hypoxia.

EXPRESSION IN THE GI TRACT TFF peptides are characteristic exocrine products of mucous epithelia and, in particular, of the GI tract (for reviews and references, see [19, 20, 54]). Here, they are often synthesized together with secretory mucins (Tab. 1). However, this does not imply that expression of TFFs and mucins is regulated together; for example, the expression of TFF3 and MUC2 shows a marked diver-

gence along the rat intestine. Furthermore, TFFs are found in the human serum, and they are also typical neuropeptides [16] (for reviews, see [19, 20]). There are remarkable species differences concerning TFF expression in the GI tract. The major focus here is on the human GI tract and differences are discussed as they apply to other species. TFF expression on the protein level often does not parallel the corresponding mRNA levels [25, 32]. Similar results were reported for expression of mucins. The reason might be mRNA instability [25]. Degradation of TFFs seems rather unlikely because they are considered to be fairly protease-resistant. Consequently, the biosynthesis of TFFs is discussed here mainly on the basis of the protein (i.e., the functional) level.

Expression in the Human Alimentary Tract TFFs are secreted from all parts of the normal human alimentary tract but to variable amounts (Table 1). TFF3 seems to represent the standard TFF peptide found in all GI organs (as well as in other regions of the human body), whereas TFF1 and TFF2, as the predominant gastric TFF peptides, have probably more specialized functions. The synthesis of TFFs starts in the oral cavity, where mainly TFF3 is secreted by the submandibular glands. Thus, TFF3 represents an important constituent of the saliva (about 10−6M). TFF3 as well as TFF1 transcripts are also detectable in the sublingual gland and minor salivary glands. Recent studies showed that the normal esophagus is also a site of TFF peptide synthesis. Here, TFF3 is a typical secretory product of the submucosal glands. The most complex TFF expression pattern of the whole body is present in the stomach. Only here are all three TFF peptides synthesized. TFF1 and TFF2 predominate in the surface mucous cells (SMCs) and mucous neck cells (MNCs) or antral gland cells, respec-

TFF (Trefoil Factor Family) Peptides / 1149 TABLE 1. TFF Peptides Synthesized in the Normal Human GI Tract.a Organ Salivary glands Esophagus Stomach

Intestine Vater’s ampulla Pancreas Liver Gallbladder

Cell

TFF Peptide

Secretory Mucin

Submandibular glands Minor salivary glands Submucosal glands Cardiac SMCs Corpus SMCs Antral SMCs MNCs Antral glands Brunner’s glands Goblet cells Goblet cells (Duct epithelium), islets LBDs, SBDs, PBGs Epithelial cells

TFF3 TFF3 TFF3 TFF1, TFF3 TFF1 TFF1, TFF3 TFF2 TFF2 TFF2, TFF3 TFF3 TFF3 (TFF1), TFF3 TFF1, (TFF2), TFF3 (TFF1), (TFF2), TFF3

MUC7 or MUC5B MUC5B MUC5B MUC5AC MUC5AC MUC5AC MUC6 MUC6 MUC6 MUC2 MUC5AC/B, MUC6 (MUC6) MUC6 MUC5B, MUC6

a Scarce TFF peptide levels are indicated by parentheses. Also shown are the secretory mucins often co-expressed. SMC, surface mucous cell; MNC, mucous neck cell, LBD, large biliary duct; SBD, small biliary duct; PBG, peribiliary gland.

tively. Gastric TFF3 expression has been neglected for a long time and was localized at the cellular level only recently. There is significant synthesis at the gastric border zones (i.e., only at the cardia and the antrum) [25]. TFF1 secretion occurs primarily at superficial SMCs, whereas TFF3 secretion is also detectable at SMCs located deeper toward the isthmus. All three TFFs are also present in gastric juice with great individual variation. Here, a shortened form of TFF3 is found that is probably the result of peptic cleavage at the N-terminal region [25]. Remarkably, the concentration of TFF2 in gastric juice is subject to a dramatic diurnal variation [39]. There is an abrupt change of the TFF expression pattern at the antroduodenal transitional zone. TFF1 is absent on the protein level in the intestine, whereas TFF2 is secreted from duodenal Brunner’s glands. TFF3 is found in Brunner’s glands as well as in goblet cells of the small and large intestine. Furthermore, significant amounts of TFF3 can be detected in the meconium of human infants as early as 12 weeks gestation [27]. TFF2 expression in the pancreas is rather sparse, and there is little TFF1 synthesis in ductal cells. Only recently has abundant TFF3 immunoreactivity been reported in the pancreatic islets co-localized with insulin, indicating endocrine secretion [21]. Synthesis of TFFs also occurs in the biliary system. Here, mainly TFF1 and TFF3 are occasionally detectable in large and rarely in small bile ducts [38, 42], and TFFs are secreted from the gallbladder. Furthermore, TFF3 was identified in goblet cells of the hepatopancreatic ampulla, also known as the ampulla of Vater [32], where it is localized together with MUC5AC, MUC5B, MUC6.

Expression in the Alimentary Tract of Other Mammals Remarkable species differences have been observed in the TFF expression patterns (for detailed analysis of the mouse, see [15]). However, information from other mammals is rather limited and currently is restricted to the porcine, murine, and rat GI tracts. In contrast to human and porcine sublingual glands, TFF2 expression is reported to occur in mucous acini of the rat sublingual gland. Furthermore, considerable differences also exist for TFF2 expression in the pancreas. In contrast to the situation in human, the pancreas is the major source for porcine TFF2, where it is found in acinar cells. Relatively high TFF2 expression levels are also found in the mouse and rat pancreas. Furthermore, in the murine liver (and kidney) an antisense TFF1-related transcript has been identified that is not detectable in human [15].

Expression in the Amphibian Alimentary Tract The situation in amphibia is quite different from that in mammals (for a review and references, see [19]). There are differences in the histology of the GI organs as well as in the structure of the TFFs. Limited information is available only for the South African toad Xenopus laevis (GI TFFs: xP1, xP4.1, and xP4.2). xP1, the amphibian ortholog of TFF1, is detectable in gastric SMCs with a decreasing gradient from the corpus toward the antrum, whereas the pronounced amphibian gastric MNCs are completely devoid of xP1. xP4.1 is N-glycosylated and is composed of four TFF domains. It is localized together with mucins in the

1150 / Chapter 157 characteristic MNCs and a little in SMCs in all parts of the stomach. Based on its expression pattern, xP4.1 is considered a functional equivalent of TFF2. xP4.2 is very similar to xP4.1 (89.7% identity), but it is lacking the N-glycosylation site. This peptide is mainly expressed in a distinct population of esophageal goblet cells and in MNCs of the gastric fundus and corpus but not the antrum.

ACTIVE CONFORMATION, 3D STRUCTURE The major hallmarks of the unique TFF module are six conserved cysteine residues, various hydrophobic amino acid residues, and an arginine residue. The consensus structure is composed of approximately 42 amino acid residues (for a compilation, see [19]). The cysteine residues form intramolecular disulfide bridges in the order C1-C5, C2-C4, C3-C6 (for a review, see [44]), creating three characteristic loop structures (L1-L3); a planar projection of this structure resembles a trefoil. The complex pattern of disulfide bridges is probably one reason for the remarkable resistance of TFFs against proteolytic degradation. However, a shortened form of TFF3 is found in the human gastric juice [25]. The single-copy TFF peptides TFF1 and TFF3 form disulfide-linked homo- and heterodimers via an additional cysteine residue at their C-terminal regions (intermolecular disulfide bridge). The major intracellular form of TFF1 during the secretory pathway is an unusual heterodimer (Mr: 25 k) with the BRICHOS domain containing protein TFIZ1 [52]. Gastric TFF3 forms similar heterodimers. The topology of the two TFF domains in TFF2 (headto-tail linkage) is fundamentally different from that in TFF1 and TFF3 homodimers (tail-to-tail linkage). Furthermore, TFF2 is stabilized by an additional intramolecular disulfide bridge between cysteine residues outside the TFF domains at the N-terminal and Cterminal regions, respectively. In addition, TFF2 is posttranslationally modified by N-glycosylation. The three-dimensional structures of various mammalian TFFs were determined (for a review, see [19]). A hydrophobic cleft between loops L2 and L3 is a characteristic feature of all TFF domains and all the conserved amino acid residues are located here. The tryptophan residue just before C6 is particularly of great importance. However, the 3D structures of the three TFFs differ markedly and even the closely related TFF1 and TFF3 homodimers have different hydrodynamic properties, overall charge, and distribution of surface charge [29, 31]. Generally, the TFF3 homodimer has a more compact structure when compared with the TFF1 homodimer. These differences suggest highly specialized functions for TFF1, TFF2, and TFF3.

TFF RECEPTORS AND SIGNALING CASCADES Thus far, there are no molecular data published unambiguously describing TFF receptors in spite of circumstantial evidence for their existence on the basolateral side of mucous epithelia (for a review, see [20]). For example, injected TFF1 and TFF3 have been shown to bind predominantly to TFF2-positive MNCs of the stomach [34]. Furthermore, a β-subunit of the fibronectin receptor (i.e., a β-integrin) and a transmembrane protein with similarity to CRP-ductin/DMBT1 were identified from porcine stomach due to their TFF2binding capacity [46]. However, in the human only soluble forms of DMBT1 have been detected thus far, and they are implicated in mucosal defense and epithelial differentiation. There are only a few studies on signaling cascades triggered by TFFs (for review and references, see [10, 17, 20]) including Src, STAT3, ERK1/2, JNK, Akt, and NF-κB [13, 24]. Of major interest, TFF1 and TFF3 differ in their ability to induce activation of STAT3. Reports on TFF-triggered gene expression are also rare. Possible target genes include decay-accelerating factor, NOS2, COX2, and TFFs themselves. TFFs have occasionally been claimed to trigger phosphorylation of the epidermal growth factor (EGF) receptor in certain cell lines (for a review and references, see [20]); however, TFFs do not bind directly to the EGF receptor. Taken together, integrins are still interesting TFF receptor candidates because this agrees with binding studies [46] and they easily could account for the complex and differential signaling mechanisms observed, including the interaction with the EGF receptor system (for review, see [17]).

BIOLOGICAL ACTIONS ON THE GI TRACT Protective and Healing Effects in Vivo There are numerous in vivo studies clearly indicating the potent protective and healing effects of all three TFFs after various damage to the rodent GI tract [3, 12, 33, 41, 50, 57] (for a review and older references, see [19, 20]). TFFs were applied via different routes (luminally or systemically), or transgenic mice overexpressing TFFs were investigated. GI damage was induced chemically, by restraint, by hypoxia, or by radiation. Generally, only TFF2 and the dimeric forms of TFF1 and TFF3 were active but not the monomeric forms. The beneficial effect of TFFs was correlated with significant reductions in VCAM-1, IL-6, and TNF-α expression [41, 57]. Also a synergistic protective effect with EGF was shown [12].

TFF (Trefoil Factor Family) Peptides / 1151 There were conflicting results in the past with regard to the most effective route of administration. Most studies reported on protective effects of TFFs from both sides (i.e., after systemic or luminal application). Only recent detailed comparative studies clearly documented that luminal application is superior over systemic delivery [33, 41]. It was even shown that systemic TFFs (in particular the TFF3/monomer) aggravated colitis scores [33]. A further strong indication for the superior potential of the luminal route is the report on the effective prevention of induced colitis after intragastric administration of TFFs by genetically modified Lactococcus lactis [50]. In agreement with the observed protective role of TFFs, all transgenic TFF-deficient mice show abnormalities in the GI tract. TFF1-deficient mice fail to develop a functional gastric mucosa and develop obligatory antropyloric adenomas, 30% of which progressed to carcinomas [26]. TFF2-deficient animals have an increased number of parietal cells and show a modulation of crucial genes involved in innate and adaptive immunity [2, 11]. TFF3-deficient mice show a decreased resistance to colonic injury [28]. Significant progress has been made within the last years in understanding the molecular function of TFFs. There is a body of evidence now that TFFs support a

mucous barrier

variety of different mucosal defense and repair mechanisms enhancing synergistically the surface integrity of the GI mucosa (Fig. 2). Their multiple molecular functions include (1) formation of the mucous barrier, (2) enhancement of rapid mucosal repair (restitution), (3) modulation of mucosal differentiation processes, and (4) modulation of the mucosal immune response.

TFFs Are Integral Constituents of the Mucous Barrier TFF peptides are characteristic constituents of mucous gels, together with hydrated oligomeric mucins, and soluble forms of DMBT1 ([25]; for review and older references, see [19, 20]). Particularly TFF1 and TFF2, which are cosecreted with the mucins MUC5AC and MUC6, respectively, are thought to act as link peptides influencing the rheological properties of multilayered gastric mucus. This model is strengthened by various reports on the interaction of TFF1 and MUC5AC [37]. Furthermore, the TFF1-TFIZ1 heterodimer [52] might have a role for the complex oligomerization and/or packaging of MUC5A during the secretory pathway in gastric surface mucous cells. This view is supported by the observation that TFF1-deficient mice have an altered gastric mucous layer and show the

mucosal restitution TFF peptide

mucosal immunity

mucosal differentiation

FIGURE 2. TFF peptides support all four different lines of mucosal defense and repair. TFFs probably have a role for the complex oligomerization/packaging of mucins during the secretory pathway and then they become integral constituents of the mucous barrier. Furthermore, TFFs are present on the luminal side of the GI mucosa (e.g., gastric juice) where they can reach their basolateral receptors only when the epithelial integrity is lost, i.e., after local mucosal damage. This simple but highly effective mechanism enables that only those cells are activated by TFFs which are directly adjacent to the wounded area (mucosal restitution, mucosal immunity). Local synthesis of TFFs close to differentiating cells modulates their fate (mucosal differentiation).

1152 / Chapter 157 activation of the unfolded protein response [47]. TFF2 has been shown to interact with a gastric DMBT1 isoform (porcine CRP-ductin [46]) and to change specifically the viscoelastic properties of mucous gels [45]. This all clearly points to an important function of TFFs (particularly TFF1 and TFF2) in the first line of GI mucosal defense, the mucous barrier.

TFFs Enhance Rapid Mucosal Repair (Restitution) Migration of epithelial cells is particularly observed in the gastric and intestinal mucosa after superficial injury. This rapid repair is called restitution and starts within minutes after damage and well before extensive inflammatory processes and proliferation occur. TFFs have been shown in a variety of in vitro models that they are involved in different stages of this multistep process (for review and references, see [17]). Generally, TFFs are thought to act from the luminal side, reaching their basolateral receptors only when the epithelial integrity is lost, that is, after local mucosal damage (Fig. 2). This view is strongly supported by systematic protection studies in vivo [33]. The first step in mucosal restitution is the reduction of cell-cell contacts directly at the borders of the injured area, followed by acquisition of these cells into a migratory phenotype (epithelial-mesenchymal transition, EMT). There are multiple reports that TFFs modulate adherens junctions (e.g., by reducing E-cadherin and α- and β-catenin levels [30]), and there is emerging evidence that they influence tight junctions also (e.g., by regulating claudin expression). Furthermore, TFFs have been shown to speed up migration of epithelial cells and also immune cells in different in vitro model systems (motogenic effect; for a compilation and references, see [19]). This effect is critically dependent on activation of ERK1/2 [13]. Restitution is also supported by the anti-apoptotic effect of TFFs [4, 6, 40]. This effect is perfectly in line with the motogenic effect of TFFs because it is becoming increasingly clear that both cell migration and apoptosis are linked by common signaling mechanisms (e.g., Rac1/XIAP). Restitution in vivo is also dependent on uninterrupted mucosal blood flow. Consequently, the proangiogenic activity of TFFs [36] supports particularly the final period of restitution (i.e., remodeling), which is overlooked often and can take months.

TFFs Modulate Mucosal Differentiation Processes The continuous regeneration of the GI mucosa via proliferation and differentiation from stem cells followed

by directed migration is an essential mechanism for maintaining its surface integrity. Recently, a role of TFFs in the differentiation of gastric cells has been demonstrated. For example, TFF1-deficient mice show an increased number of SMCs, whereas the number of parietal cell is decreased [22]. This effect is probably due to altered differentiation from prepit cell to preparietal cells. Furthermore, SMCs in the gastric corpus and the antrum are generated via different differentiation programs [25]. There are indications that TFF3 plays a key role for the stepwise maturation of antral SMCs and that this process is also responsible for the higher turnover rate of the antral mucosa when compared with the body mucosa. However, the proliferation of cells is not enhanced by TFFs. There are rather reports on an antiproliferative effect of TFFs [4, 5, 48].

TFFs Modulate the Mucosal Immune Response There is an increasing body of evidence that TFFs are involved in immune responses and inflammatory processes. For example, TFFs are expressed in lymphoid tissues such as spleen, thymus, lymph nodes, and bone marrow, and they stimulate migration of monocytes [9]. Furthermore, TFF2-deficient mice show altered expression, particularly of genes involved in innate and adaptive immunity [2]. There are also results supporting an anti-inflammatory function of TFF2 as a negative regulator of interleukin 1 (IL-1) receptor signaling. However, tumor necrosis factor α–induced secretion of IL-6 and IL-8 is enhanced by TFFs in vitro, pointing to a possible function in the recruitment of leukocytes during mucosal inflammation [13].

PATHOPHYSIOLOGICAL IMPLICATIONS TFFs are of particular interest for diseases of the stomach. For example, TFF1 enhances the efficient colonization of the gastric mucosa with Helicobater pylori. This pathogen not only directly interacts with TFF1, supporting its adherence to the mucous layer, but it also upregulates TFF1 expression [8]. Furthermore, there is a vast literature on the ectopical expression of TFFs during various inflammatory diseases, particularly of the GI tract (such as gastroesophageal reflux disease, gastric ulcerations, ulcerative duodenitis and colitis, Crohn’s disease, inflammatory bowel disease, hepatolithiasis, cholecystitis, and pancreatitis; for reviews and references, see [19, 20, 35, 53, 54]). Here, a unique glandular structure known as the ulcer-associated cell lineage (UACL) is a prominent site of TFF synthesis. The UACL is thought to represent a natural repair kit that is activated after mucosal damage

TFF (Trefoil Factor Family) Peptides / 1153 [55]. Interestingly, a selective reduction in TFF3 expression has been reported in untreated celiac disease [7]. Inflammation is a critical component of tumor progression. Of interest is that the pathological expression of TFFs is also observed in premalignant conditions, such as Barrett’s metaplasia and metaplastic polyps, as well as in many epithelial tumors (e.g., of the esophagus, stomach, biliary tract, pancreas, and intestine), suggesting a role for TFFs in cancer progression (for reviews and references, see [10, 19, 20, 35]). For example, TFF1 and TFF3 are thought to represent key players in the different responses of the stomach and the intestine to inflammation. The differential regulation of TFF1 and TFF3 expression by SHP2/ERK and STAT3 signaling, respectively, helps to explain why cancer develops in the stomach and inflammatory bowel disease hits the intestine when the critical gp130regulated balance between SHP2/ERK and STAT3 signaling is disrupted [51]. Consequently, reduced TFF1 expression and increased TFF3 synthesis have been observed in many gastric cancers indicating a clear shift toward STAT3 signaling in gastric neoplasia. The upregulation of TFFs is also observed after incisional wounds of GI tissue, in various induced gastric ulcerations, and after irradiation. Particularly, the late and sustained TFF1 and TFF3 responses are indicative for a role in late-stage repair processes [1, 49, 53]. Taken together, TFFs play a key role for the maintenance of the GI surface integrity in health and disease by supporting all four different lines of mucosal defense and repair, and they are also connected with multiple oncogenic pathways. However, the precise molecular mechanisms still await their full elucidation.

Acknowledgments I thank the many colleagues for making available their data prior to publication and E. VoB for excellent assistance with image processing. I also regret that the number of references had to be kept to a minimum so that only selected recent literature could be cited. The work in the author’s laboratory was supported by grants from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF), the Land SachsenAnhalt, and the Fonds der Chemischen Industrie.

References [1] Balcer-Kubiczek EK, Harrison GH, Xu J-F, Gutierrez PL. Coordinate late expression of trefoil peptide genes (pS2/TFF1 and ITF/TFF3) in human breast, colon, and gastric tumor cells exposed to X-rays. Mol Cancer Ther 2002;1:405–415. [2] Baus-Loncar M, Kayademir T, Takaishi S, Wang T. Trefoil factor family 2 deficiency and immune response. Cell Mol Life Sci 2005;62:2947–2955.

[3] Beck PL, Wong JF, Li Y, Swaminathan S, Xavier RJ, Devaney KL, Podolsky DK. Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor. Gastroenterology 2004;126:796–808. [4] Bossenmeyer-Pourié C, Kannan R, Ribieras S, Wendling C, Stoll I, Thim L, Tomasetto C, Rio M-C. The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1-S phase transition and reducing apoptosis. J Cell Biol 2002;157:761– 770. [5] Calnan DP, Westley BR, May FEB, Floyd DN, Marchbank T, Playford RJ. The trefoil peptide TFF1 inhibits the growth of the human gastric adenocarcinoma cell line AGS. J Pathol 1999;188:312–317. [6] Chan MWY, Chan VYW, Leung WK, Chan KK, To KF, Sung JJY, Chan FKL. Anti-sense trefoil factor family-3 (intestinal trefoil factor) inhibits cell growth and induces chemosensitivity to adriamycin in human gastric cancer cells. Life Sci 2005;76:2581– 2592. [7] Ciacci C, Di Vizio D, Seth R, Insabato G, Mazzacca G, Podolsky DK, Mahida YR. Selective reduction of intestinal trefoil factor in untreated coeliac disease. Clin Exp Immunol 2002;130:526– 531. [8] Clyne M, Dillon P, Daly S, O’Kennedy R, May FEB, Westley BR, Drumm B. Helicobacter pylori interacts with the human singledomain trefoil protein TFF1. Proc Natl Acad Sci USA 2004;101:7409–7414. [9] Cook GA, Familari M, Thim L, Giraud AS. The trefoil peptides TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response. FEBS Lett 1999;456:155– 159. [10] Emami S, Rodrigues S, Rodrigue CM, Le Floch N, Rivat C, Attoub S, Bruyneel E, Gespach C. Trefoil factor family (TFF) peptides and cancer progression. Peptides 2004;25:885–898. [11] Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, Wang TC. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest 2002;109:193–204. [12] FitzGerald AJ, Pu M, Marchbank T, Westley BR, May FEB, Boyle J, Yadollahi-Farsani M, Ghosh S, Playford RJ. Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis. Peptides 2004;25:793– 801. [13] Graness A, Chwieralski CE, Reinhold D, Thim L, Hoffmann W. Protein kinase C and ERK activation are required for TFFpeptide-stimulated bronchial epithelial cell migration and tumor necrosis factor-α-induced interleukin-6 (IL-6) and IL-8 secretion. J Biol Chem 2002;277:18440–18446. [14] Hattori M, Fujiyama A, Taylor TD, Watanabe H, Yada T, Park H-S, et al. The DNA sequence of human chromosome 21. Nature 2000;405:311–319. [15] Hertel SC, Chwieralski CE, Hinz M, Rio M-C, Tomasetto W, Hoffmann W. Profiling trefoil factor family (TFF) expression in the mouse: Identification of an antisense TFF1-related transcript in the kidney and liver. Peptides 2004;25:755–762. [16] Hinz M, Schwegler H, Chwieralski CE, Laube G, Linke R, Pohle W, Hoffmann W. Trefoil factor family (TFF) expression in the mouse brain and pituitary: Changes in the developing cerebellum. Peptides 2004;25:827–832. [17] Hoffmann W. TFF (trefoil factor family) peptide-triggered signals promoting mucosal restitution. Cell Mol Life Sci 2005;62:2932–2938. [18] Hoffmann W. Trefoil factor family (TFF) peptides: regulators of mucosal regeneration and more. Peptides 2004;25:727–730. [19] Hoffmann W, Jagla W. Cell type specific expression of secretory TFF peptides: Colocalization with mucins and synthesis in the brain. Int Rev Cytol 2002;213:147–181.

1154 / Chapter 157 [20] Hoffmann W, Jagla W, Wiede A. Molecular medicine of TFFpeptides: From gut to brain. Histol Histopathol 2001;16:319– 334. [21] Jackerott M, Møllgård K, Lee YC, Kofod H, Thim L, Nielsen JH. Localization of trefoil factor-3 in endocrine cells in fetal and adult human pancreas. Diabetes 2005;54(Suppl. 1):A398. [22] Karam SM, Tomasetto C, Rio M-C. Trefoil factor 1 is required for the commitment programme of mouse oxyntic epithelial progenitors. Gut 2004;53:1408–1415. [23] Khan ZE, Wang TC, Cui G, Chi AL, Dimaline R. Transcriptional regulation of the human trefoil factor, TFF1, by gastrin. Gastroenterology 2003;125:510–521. [24] Kinoshita K, Taupin DR, Itoh H, Podolsky DK. Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor. Mol Cell Biol 2000;20:4680–4690. [25] Kouznetsova I, Peitz U, Vieth M, Meyer F, Vestergaard EM, Malfertheiner P, Roessner A, Lippert H, Hoffmann W. A gradient of TFF3 (trefoil factor family 3) peptide synthesis within the normal human gastric mucosa. Cell Tissue Res 2004;316:155–165. [26] Lefebvre O, Chenard M-P, Masson R, Linares J, Dierich A, LeMeur M, Wendling C, Tomasetto C, Chambon P, Rio M-C. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 1996;274:259–262. [27] Lin J, Nadroo AM, Chen W, Holzman IR, Fan Q-X, Babyatsky MW. Ontogeny and prenatal expression of trefoil factor 3/ITF in the human intestine. Early Hum Dev 2003;71:103–109. [28] Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 1996;274:262–265. [29] May FEB, Church ST, Major S, Westley BR. The closely related estrogen-regulated trefoil proteins TFF1 and TFF3 have markedly different hydrodynamic properties, overall charge, and distribution of surface charge. Biochemistry 2003;42:8250– 8259. [30] Meyer zum Büschenfelde D, Hoschützky H, Tauber R, Huber O. Molecular mechanisms involved in TFF3 peptide-mediated modulation of the E-cadherin/catenin cell adhesion complex. Peptides 2004;25:873–883. [31] Muskett FW, May FEB, Westley BR, Feeney J. Solution structure of the disulfide-linked dimer of human intestinal trefoil factor (TFF3): The intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins. Biochemistry 2003;42:15139–15147. [32] Paulsen F, Varoga D, Paulsen A, Tsokos M. TFF peptides of Vater’s ampulla. Cell Tissue Res 2005;321:67–74. [33] Poulsen SS, Kissow H, Hare K, Hartmann B, Thim L. Luminal and parenteral TFF2 and TFF3 dimer and monomer in two models of experimental colitis in the rat. Regul Pept 2005;126:163–171. [34] Poulsen SS, Thulesen J, Hartmann B, Kissow HL, Nexø E, Thim L. Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats. Regul Pept 2003;115:91–99. [35] Ribieras S, Tomasetto C, Rio M-C. The pS2/TFF1 trefoil factor, from basic research to clinical applications. Biochim Biophys Acta 1998;1378:F61–F77. [36] Rodrigues S, van Aken E, van Bocxlaer S, Attoub S, Nguyen QD, Bruyneel E, Westley BR, May FEB, Thim L, Mareel M, Gespach C, Emami S. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling. FASEB J 2003;17:7–16. [37] Ruchaud-Sparagano M-H, Westley BR, May FEB. The trefoil protein TFF1 is bound to MUC5AC in human gastric mucosa. Cell Mol Life Sci 2004;61:1946–1954. [38] Sasaki M, Tsuneyama K, Saito T, Kataoka H, Mollenhauer J, Poustka A, Nakanuma Y. Site-characteristic expression and

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induction of trefoil factor family 1, 2 and 3 and diseased intrahepatic bile ducts relates to biliary pathophysiology. Liver Int 2004;24:29–37. Semple JI, Newton JL, Westley BR, May FEB. Dramatic diurnal variation in the concentration of the human trefoil peptide TFF2 in gastric juice. Gut 2001;48:648–655. Siu L-S, Romanska H, Abel PD, Baus-Loncar M, Kayademir T, Stamp GWH, Lalani EN. TFF2 (trefoil family factor2) inhibits apoptosis in breast and colorectal cancer cell lines. Peptides 2004;25:855–863. Soriano-Izquierdo A, Gironella M, Massaguer A, May FE, Salas A, Sans M, Poulsom R, Thim L, Pique JM, Panes J. Trefoil peptide TFF2 treatment reduces VCAM-1 expression and leukocyte recruitment in experimental intestinal inflammation. J Leukoc Biol 2004;75:214–223. Srivatsa G, Giraud AS, Ulaganathan M, Yeomans ND, Dow C, Nicoll AJ. Biliary epithelial trefoil peptide expression is increased in biliary diseases. Histopathology 2002;40:261–268. Taupin D, Podolsky DK. Trefoil factors: Initiators of mucosal healing. Nat Rev Mol Cell Biol 2003;4:721–732. Thim L. Trefoil peptides: from structure to function. Cell Mol Life Sci 1997;53:888–903. Thim L, Madsen F, Poulsen SS. Effect of trefoil factors on the viscoelastic properties of mucus gels. Eur J Clin Invest 2002;32: 519–527. Thim L, Mørtz E. Isolation and characterization of putative trefoil peptide receptors. Regul Peptides 2000;90:61–68. Torres L-F, Karam SM, Wendling C, Chenard M-P, Kershenobich D, Tomasetto C, Rio M-C. Trefoil factor 1 (TFF1/pS2) deficiency activates the unfolded protein response. Mol Med 2002;8:273–282. Uchino H, Kataoka H, Itoh H, Hamasuna R, Koono M. Overexpression of intestinal trefoil factor in human colon carcinoma cells reduces cellular growth in vitro and in vivo. Gastroenterology 2000;118:60–69. Ulaganathan M, Familari M, Yeomans ND, Giraud AS, Cook GA. Spatio-temporal expression of trefoil peptide following severe gastric ulceration in the rat implicates it in late-stage repair processes. J Gastroen Hepatol 2001;16:506–512. Vandenbroucke K, Hans W, van Huysse J, Neirynck S, Demetter P, Remaut E, Rottiers P, Steidler L. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 2004;127:502– 513. Wang TC, Goldenring JR. Inflammation intersection: gp130 balances gut irritation and stomach cancer. Nature Med 2002;8:1080–1082. Westley BR, Griffin SM, May FEB. Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a Brichos domain-containing protein with homology to SP-C. Biochemistry 2005;44:7967–7975. Wong WM, Playford RJ, Wright NA. Peptide gene expression in gastrointestinal mucosal ulceration: Ordered sequence or redundancy? Gut 2000;46:286–292. Wong WM, Poulsom R, Wright NA. Trefoil peptides. Gut 1999;44:890–895. Wright NA. Aspects of the biology of regeneration and repair in the human gastrointestinal tract. Phil Trans R Soc Lond B 1998;353:925–933. Wright NA, Hoffmann W, Otto WR, Rio M-C, Thim L. Rolling in the clover: Trefoil factor family (TFF)-domain peptides, cell migration and cancer. FEBS Lett 1997;408:121–123. Zhang B-H, Yu H-G, Sheng Z-X, Luo H-S, Yu J-P. The therapeutic effect of recombinant human trefoil factor 3 on hypoxiainduced necrotizing enterocolitis in immature rat. Regul Peptides 2003;116:53–60.

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158 Signaling by Vasoactive Intestinal Peptide in Gastrointestinal Smooth Muscle 1

KARNAM S. MURTHY AND SATISH C. RATTAN

acterized by their transmitter content and divided into two broad categories. One population (∼60%) contains the major excitatory transmitter, acetylcholine (ACh), often found together with tachykinins, substance P (SP), and neurokinin A (NKA) [4, 15, 16, 54]. The second main population (∼25%) contains the major inhibitory transmitters vasoactive intestinal peptide (VIP) and/or its homolog, pituitary adenylyl cyclaseactivating polypeptide (PACAP), often found together with neuronal nitric oxide synthase, an enzyme responsible for the generation of nitric oxide (NO) [4, 15, 16] (see chapters by the discoverers of these peptides elsewhere in this book). VIP neurons also contain a homolog peptide derived from the same precursor designated peptide histidine isoleucine (PHI) in animals and peptide histidine methionine (PHM) in humans [15, 16]. Gastrointestinal motility is mediated by the contractile activity of the smooth muscle cells that line the gastrointestinal wall. Smooth muscle of the gut possesses distinct regional and functional properties. Smooth muscle of the proximal stomach and gallbladder exhibits sustained tone with some rhythmic contractions, whereas smooth muscle of the distal stomach, small intestine, and colon exhibits variable tone on which are superimposed rhythmic contractions [32, 57]. The smooth muscle of the sphincters on the other hand is purely tonic in nature [47]. The rhythmic contractions are driven by cycles of membrane depolarization and repolarization (slow waves) that originate in pacemaker cells known as interstitial cells of Cajal [53, 57, 64]. Contraction is mainly regulated by acetylcholine and substance P and is characterized by acceleration of depolarization, whereas relaxation is mainly regulated by VIP/PACAP and NO and is characterized by the suppression of basal tone or rhythmic electrical and mechanical activities [4, 52]. The term inhibitory reflects the ability of the transmitters to inhibit

ABSTRACT Relaxation of smooth muscle of the gastrointestinal tract reflects the interplay of nitric oxide (NO) and the peptide transmitters, mainly vasoactive intestinal peptide and its homolog, pituitary adenylyl cyclase– activating polypeptide (PACAP). In smooth muscle cells, VIP (and PACAP) interact with cognate seventransmembrane VPAC2 receptors to generate cAMP and with the single-transmembrane NPR-C to generate NO and cGMP. Concurrent generation of cAMP and cGMP and activation of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, are the physiological norms. Both PKA and PKG act on various downstream targets to inhibit Ca2+ mobilization and MLC20 phosphorylation and, thus, induce muscle relaxation.

INTRODUCTION The main function of gastrointestinal motility is to propel ingested food and to mix the food with digestive enzymes for optimal digestion and absorption and excretion of undigested waste. The propulsion of intestinal contents depends on the peristaltic contractions and relaxations and the coordinated opening of tonically contracted sphincters. The process of propulsion is initiated by food, which triggers a propagated peristaltic reflex consisting of contraction orad (ascending contraction) and relaxation caudad (descending relaxation) to the site of stimulation caused by the distension of the gut wall by the bolus [4]. The peristaltic reflex is regulated by the enteric nervous system that is contained within the wall of the gut and that receives input from the autonomic and central nervous systems [15, 16]. Enteric neurons of the myenteric plexus, the main source of innervation of smooth muscle, are best charHandbook of Biologically Active Peptides

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1156 / Chapter 158 the muscle tone as well as rhythmic contractions. VIP, PACAP, and NO each induces the relaxation of smooth muscle from all regions of the gastrointestinal tract [2, 3, 6, 7, 17, 20, 45, 48, 65]. These neurotransmitters fulfill the criteria for inhibitory transmitters: They are present in the neurons innervating the muscle of stomach, intestine, and various sphincters and are released by pharmacological and electrical field stimulation of myenteric neurons with the release coupled to peristaltic reflex and muscle relaxation. In addition, exogenous addition of VIP, PACAP, or NO induces muscle relaxation mimicking the response induced by nerve stimulation. VIP-containing neurons also innervate epithelial cells, exocrine glands, and other neurons of the gastrointestinal tract. In addition to smooth muscle relaxation, the biological effects of VIP on the gastrointestinal system include stimulation of water and electrolyte secretion, enzyme secretion, and mucous secretion, as well as the inhibition of absorption and of acid secretion [29, 50]. VIP and PACAP belong to a family of structurally related peptides that also includes secretin, glucagons glucagon-like peptide, gastric inhibitory peptide, PHI/ PHM, growth hormone–releasing factor, and helodermin-like peptides [50]. VIP is a 28-amino-acid peptide originally isolated from the porcine duodenum and is synthesized as a 170-amino-acid precursor that also contains PHI/PHM [50, 53]. Radioligand binding and functional studies have demonstrated that the entire molecule of VIP is required for its maximal action on smooth muscle [5]. The molecular mechanisms underlying VIP-induced relaxation in gastrointestinal smooth muscle are discussed in greater detail in this chapter. PACAP is closely related to VIP and possesses identical properties to VIP in smooth muscle [10, 36].

INTERPLAY OF INHIBITORY NEUROTRANSMITTERS The neuropeptides VIP and PACAP, and neuronal nitric oxide synthase (nNOS), the enzyme responsible for formation of NO by enteric nerves, are co-localized in a subset of myenteric neurons that innervate the muscle, and their release is functionally linked [4, 15, 16, 47, 56]. VIP and PACAP are stored in vesicles in the nerve endings, whereas NO is synthesized on demand by the nNOS. NO formed in the nerve terminals by nNOS regulates VIP and PACAP release, and, in turn, VIP and PACAP stimulate smooth muscle eNOS to generate NO within the smooth muscle [21, 22, 37, 38, 40]. In isolated myenteric ganglia devoid of smooth muscle, the release of VIP and PACAP and the production of NO are inhibited by the blockade of nNOS activity implying NO-dependent VIP and PACAP release

[21]. Consistent with this notion, exogenous NO increases VIP and PACAP release, whereas exogenous VIP or PACAP had no effect on NO production [21]. In contrast, in isolated smooth muscle cells devoid of neural elements, VIP and PACAP induce NO production [22, 37, 38]. The ability of NO to stimulate VIP and PACAP from nerve terminals and the ability of VIP and PACAP to stimulate NO production in smooth muscle were evident in studies with innervated muscle preparations. In these preparations, nerve stimulation elicited the stimulation of VIP (and PACAP) release, NO formation, and relaxation [18, 25]. NOS inhibitors abolished NO formation and partly inhibited VIP release and relaxation, whereas VIP antagonists partly inhibited NO formation and relaxation. The ability of NOS inhibitors to block relaxation reflects the inhibition of NOS in both myenteric neurons and smooth muscle cells and the inhibition of NO-dependent VIP and PACAP release from the myenteric neurons. The residual relaxation in the absence of NO production reflects the direct effect of VIP and PACAP, which is independent of NOS activity in the smooth muscle. The ability of a VIP antagonist to block relaxation reflects the suppression of VIPinduced stimulation of NOS and adenylyl cyclase activities in smooth muscle. The residual relaxation, in the absence of NO production and cAMP generation in smooth muscle, reflects the direct effect of NO derived from myenteric neurons on smooth muscle cells. The interplay between VIP and NO during relaxation can be summarized by the following results: (1) NO stimulates VIP from nerve terminals and VIP, in turn, stimulates NO formation in muscle; (2) relaxation is mediated by NO and VIP release from nerve terminals and by NO formation in smooth muscle cells; and (3) the effect of NOS inhibitors and VIP antagonists reflects their effects on both VIP release and NO formation. Rattan and co-workers [8, 9] have demonstrated that, in the internal anal sphincter of the opossum, VIP acts on both myenteric neurons and smooth muscle cells to generate NO. The major source of NO in response to VIP during relaxation, however, is from myenteric neurons with some contribution from the smooth muscle cells. Studies by Goyal and co-workers [26, 33, 34] using knockout animals have demonstrated that VIP-induced hyperpolarization in the lower esophageal sphincter is abolished in nNOS-deficient mice but is not affected in eNOS-deficient mice, suggesting that nNOS is the major source of NO in the hyperpolarizing actions of VIP.

VIP RECEPTORS AND THEIR SIGNALING IN SMOOTH MUSCLE The biological actions of VIP and PACAP are mediated by a family of G-protein-coupled receptors, which

Signaling by Vasoactive Intestinal Peptide in Gastrointestinal Smooth Muscle / 1157 are designated VPAC1, VPAC2, and PAC1 receptors [23, 29, 62]. These receptors belong to family B of the Gprotein-coupled receptors (also known as the secretin receptor family), which include receptors for secretin, gastric inhibitory peptide, glucagon, glucagon-like peptide 1, calcitonin, parathyroid hormone, growth hormone–releasing factor, and corticotrophinreleasing hormone. The PAC1 receptors exhibit high affinity for PACAP and low affinity for VIP, whereas VPAC1 and VPAC2 receptors exhibit equal affinity for VIP and PACAP. Although VPAC1, VPAC2, and PAC1 are expressed throughout the gastrointestinal tract, the various VIP/PACAP receptor subtypes exhibit differential expression patterns. VPAC1 receptors are expressed in the mucosa and myenteric neurons; VPAC2 receptors are expressed in neuroendocrine cells, blood vessels, and smooth muscle; and PAC1 receptors are expressed in myenteric neurons [23, 29, 59, 62]. Neural VIP/ PACAP receptors are present in two locations: on nerve terminals, where they act as autoinhibitory receptors to suppress further neurotransmitter release, and on cholinergic/tachykinin neurons that innervate the longitudinal muscle layer. The activation of these receptors by VIP/PACAP released from myenteric neurons stimulates the release of ACh and tachykinins and induces the contraction of longitudinal muscle [19].

In gastrointestinal smooth muscle VIP and PACAP interact with two distinct classes of receptors: cognate VPAC2 receptors coupled via Gs to the stimulation of adenylyl cyclase V/VI, resulting in cAMP formation and activation of cAMP-dependent protein kinase (PKA), and a single-transmembrane natriuretic peptide clearance receptor (NPR-C) coupled via Gi1/Gi2 to the stimulation of Ca2+ influx and sequential activation of Ca2+/calmodulin-dependent eNOS and NO-dependent soluble guanylyl cyclase [37, 38, 40] (Fig. 1). In eNOSdeficient tenia coli muscle cells, NPR-C inhibited adenylyl cyclase via the α subunits of Gi1 and Gi2 and activated phospholipase C-β3 isoform via βγ subunits. In the smooth muscle of the gut, NPR-C is co-expressed with NPR-B but not NPR-A. Unlike NPR-A and NPR-B, NPRC is devoid of intracellular cyclase-activating domain and consists of a short 37-amino-acid sequence capable of activating pertussis toxin–sensitive Gi1 and Gi2 [1, 40]. With receptor-derived synthetic peptides, a 17-aminoacid G-protein-activating sequence (469RRNHQEESNIGKHREL485R) characterized by two NH2-terminal basic residues and a COOH-terminal motif BBXXB, where B and X represent basic and nonbasic residues, respectively, has been identified in the intracellular domain of NPR-C [39, 40] (Fig. 1). In tenia coli smooth muscle cells, this 17-amino-acid sequence selectively activated

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FIGURE 1. Signaling pathways initiated by VIP in gastrointestinal smooth muscle. In gastrointestinal smooth muscle VIP activate cognate seven-transmembrane VPAC2 receptors coupled via Gs to adenylyl cyclase V/VI, cAMP generation and activation of cAMP-dependent protein kinase (PKA). They also activate a singletransmembrane natriuretic peptide receptor C (NPR-C) coupled via Gαi1 and Gαi2 to Ca2+ influx and Ca2+/ calmodulin activation of NOS III expressed in smooth muscle cells, leading to NO-dependent activation of soluble guanylyl cyclase (sGC), cGMP generation, and activation of cGMP-dependent protein kinase (PKG). Activation of Gαi1/2 is mediated by a 17-amino-acid sequence in the middle region of the intracellular domain of NPR-C. cAMP activates PKA and cGMP activates PKG, but, in the presence of cGMP, cAMP crossactivates PKG also. Both PKA and PKG act separately upstream to induce desensitization of VPAC2 and NPR-C receptors, inhibition of adenylyl cyclase and soluble guanylyl cyclase, and activation of cAMP-specific PDE3A and PDE4 and cGMP-specific PDE5. Stimulation (+); inhibition (−).

1158 / Chapter 158 Gi1 and Gi2, inhibited adenylyl cyclase via the α subunit, and activated PLC-β3 via the βγ subunits and induced contraction in a similar fashion to the NPR-C ligand [41]. The substitution of either NH2-terminal or Cterminal basic residues with nonbasic residues by site-directed mutagenesis or the deletion of the entire 17-amino-acid sequence blocked the ability of receptor to activate G-proteins, suggesting that this region in the intracellular domain is both necessary and sufficient for activation of G-proteins [68]. It is worth noting that similar sequences have been identified in other G-protein-coupled seven-transmembrane receptors, such as muscarinic and adrenergic receptors [46]. In gastrointestinal smooth muscle, VPAC2 receptors are desensitized and internalized by a mechanism distinct from those used by VPAC1 or secretin receptors. VPAC2 receptors are phosphorylated by G-proteincoupled receptor kinase 2 (GRK2), resulting in desensitization and internalization of receptors. GRK2 is phosphorylated by PKA at Ser685, and this phosphorylation greatly augments the ability of GRK2 to phosphorylate the receptor and induce receptor internalization [70]. NPR-C receptors are also desensitized by a PKGdependent mechanism. A unique threonine residue (Thr466) outside the G-protein-activating sequence in the intracellular domain of NPR-C is selectively phosphorylated by PKG leading to desensitization of responses mediated by NPR-C [70]. In gastric and intestinal smooth muscle cells VAPC2 receptors exhibit equal affinity for both VIP and PACAP. In tenia coli smooth muscle cells, however, VIP and PACAP interact with distinct receptors [24]. These cells express a VIP-specific receptor that does not recognize PACAP, and a PACAP-specific receptor that does not recognize VIP. The VIP-specific receptor, but not the PACAP-specific receptor, is coupled to the activation of adenylyl cyclase. The PACAP-specific receptor, but not the VIP-specific receptor, is coupled to activation of apamin-sensitive K+ channels. The cDNA of the receptor cloned from tenia coli encodes a 437-amino-acid protein with a calculated molecular weight of 49,560 kDa. All amino acid residues required for VIP binding including aspartate, tryptophan, and glycine residues (corresponding to D68, W73, and G109 of human VPAC1 receptor), and all Nterminal cysteine residues are conserved [58, 67]. The amino acid sequence differed from that of the cloned guinea pig gastric receptor by only two amino acid residues (Phe40/Phe41 in lieu of Leu40/Leu41) in the ligandbinding N-terminal and showed 87% homology to the sequence of rat and mouse VPAC2 receptors. The mutation of these two residues to Leu40/Leu41 so as to mimic gastric VIP receptor resulted in an equal high affinity for VIP and PACAP.

Mechanism of Smooth Muscle Relaxation Induced by VIP VIP- and PACAP-induced activation of adenylyl cyclase by Gs-coupled VPAC2 receptors and soluble guanylyl cyclase by Gi1/2 coupled to NPR-C via NO generation leads to concurrent formation of cAMP and cGMP and activation of PKA- and cGMP-dependent protein kinase (PKG). The intracellular levels of cAMP and cGMP, and the strength and duration of their physiological effects depend on the rates of their synthesis by cyclases and degradation of phosphodiesterases (PDEs) [13, 14]. The activity of these enzymes, in turn, is regulated by PKA and PKG, the downstream kinase of cAMP and cGMP. Cyclic AMP levels are regulated by feedback activation of cAMP-specific PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI via PKA [44]. cGMP levels are regulated by feedback activation cGMPspecific PDE5 and inhibition of soluble guanylyl cyclase via PKG [35, 49, 61]. When there is concurrent generation of cAMP and cGMP, cGMP increases cAMP levels by inhibiting its degradation via PDE3A, whereas cAMP decreases cGMP levels by augmenting its degradation via PKA-mediated phosphorylation of PDE5 [35, 44]. Thus cAMP and cGMP levels are regulated by coordinated interplay among cyclases, phosphodiesterases, and protein kinases. cGMP selectively activates PKG, whereas cAMP at lower concentrations activates PKA and at higher concentrations cross-activates PKG [39]. Autophosphorylation of PKG in the presence of cGMP greatly augments the affinity of cAMP for PKG [30]. The ability of cAMP to cross-activate PKG is potentiated by the concurrent generation of cGMP. Thus, the concurrent generation of cAMP and cGMP and activation of PKA and PKG are the physiological norm in smooth muscle. PKA and PKG act on several downstream targets in the cascades that mediate initial and sustained contraction, resulting in MLC20 (20-kDa regulatory myosin light chain, MLC) dephosphorylation and muscle relaxation [11, 12, 27, 31, 42, 43] (Fig. 2). The relaxation of the initial Ca2+-dependent phase by PKA and PKG correlates with the decrease in [Ca2+]i, which is achieved by decreasing inositol 1,4,5-trisphosphate (IP3) generation and/or decreasing IP3-dependent Ca2+ release. Both PKA and PKG inhibit Gαq-dependent phospholipase C (PLC)-β1 activity. However, neither Gαq nor PLC-β1 is directly phosphorylated by PKA or PKG. The mechanism of inhibition involves the acceleration of Gαq inactivation by phosphorylation of two regulatory proteins, RGS4 (regulator of G-protein signaling 4) and GRK2 (G-protein-coupled receptor kinase 2). Phosphorylation of RGS4 and GRK2 increases their binding to Gαq and enhances the inactivation of Gαq leading to inhibition of PLC-β1 activity and IP3 generation [69].

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FIGURE 2. Downstream targets of PKA and PKG during VIP-induced relaxation of smooth muscle. PKG and PKA induce relaxation by targeting various components of the contractile signaling pathways. Both PKA and PKG phosphorylate RGS4 and stimulate inactivation of Gαq, leading to inhibition of PLC-β1 activity and IP3 formation. In addition, only PKG can phosphorylate IP3R-I and the sarcoplasmic Ca2+/ATPase pump, leading to inhibition of inhibition Ca2+ release and stimulation Ca2+ reuptake. These mechanisms act to reduce intracellular Ca2+ and thus inhibit MLCK activity, MLC20 phosphorylation, and initial contraction. Both PKA and PKG inactivate RhoA by phosphorylating RhoA at Ser188, leading to blockade of inhibition of MLC phosphatase via PKC/CPI17 and Rho kinase/MYPT1. A distinct mechanism involves phosphorylation of telokin, an endogenous activator of MLC phosphatase, by both PKA and PKG, leading to dephosphorylation of MLC20.

PKG, but not PKA, in addition, induces phosphorylation of type IP3 receptors expressed in smooth muscle, resulting in inhibition of Ca2+ release and relaxation of initial Ca2+-dependent contraction [28, 42] (Fig. 2). The relaxation of sustained Ca2+-independent phase of muscle contraction is mediated by the disinhibition of MLC phosphatase via PKA- and PKG-mediated phosphorylation of membrane-bound RhoA at Ser188 [43, 55] (Fig. 2). Phosphorylation of RhoA stimulates its translocation back to the cytosol and inhibits the activity of downstream membrane-bound targets, Rho kinase, and phospholipase D (PLD). Inhibition of Rho kinase blocks the phosphorylation of the regulatory subunit of MLC phosphatase (MYPT1) at Thr696, whereas the inhibition of PLD results in the decrease in diacylglycerol formation, protein kinase C (PKC) activity, and PKCmediated CPI-17 (17-kDa endogenous inhibitor of MLC phosphatase) phosphorylation at Thr38. Thus, the blockade of two pathways, Rho kinase/MYPT1 and PLD/PKC/CPI-17, that converge to inhibit MLC phosphatase leads to the activation of MLC phosphatase and dephosphorylation of MLC20, leading to the relaxation of sustained Ca2+-independent, RhoA-dependent contraction. Recent studies have shown that PKA and PKG can directly phosphorylate telokin, an endogenous 17kDa activator of MLC phosphatase, which results in dephosphorylation of MLC20 and muscle relaxation [63, 66] (Fig. 2).

Acknowledgments Supported by grants DK15564 and DK28300 (to K.S.M.), and DK 35385 (to S.R.) from the National Institute of Diabetes, and Digestive and Kidney Diseases.

References [1] Anand-Srivastava MB, Sehl PD, and Lowe DG. 1996. Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase: involvement of pertussis toxin-sensitive G protein. J. Biol. Chem. 271: 19324–19329. [2] Biancani P, Walsh JH, and Behar J. 1985. Vasoactive intestinal peptide. A neurotransmitter for lower esophageal sphincter. J. Clin. Invest. 73: 963–967. [3] Bitar KN, and Makhlouf GM. 1982. Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide. Science 216: 531–533. [4] Bornstein JC, Costa M, and Grider JR. 2004. Enteric motor and interneuronal circuits controlling motility. Neurogastroenterol. Motil. 16: 34–38. [5] Chakder S, and Rattan S. 1993. The entire vasoactive intestinal peptide molecule is for the activation of VIP receptor functional and binding studies on opossum internal sphinter smooth muscle. J. Pharmacol. Exp. Ther. 266: 392–399. [6] Chakder S, and Rattan S. 1993. Involvement of cAMP and cGMP in relaxation of internal anal sphincter by neural stimulation, VIP and NO. Am. J. Physiol. 264: G702–G707. [7] Chakder S, and Rattan S. 1993. Release of nitric oxide by activation of non-adrenergic and non-cholinergic neurons of internal anal sphincter. Am. J. Physiol. 264: G7–G12.

1160 / Chapter 158 [8] Chakder S, and Rattan S. 1995. Distribution of VIP binding sited in opossum in anal sphincter circular smooth muscle sites of actions. J. Pharmacol. Exp. Ther. 272: 385–391. [9] Chakder S, and Rattan S. 1996. Evidence for VIP-induced increase in NO production in myenteric neurons of opossum internal anal sphincter. Am. J. Physiol. 270: G492–G497. [10] Chakder S, and Rattan S. 1997. Excitatory and inhibitory actions of PACAP in the internal anal sphincter smooth muscle: sites of actions. J. Pharmacol. Exp. Ther. 283: 722–728. [11] Cornwell TL, Pryzwansky KB, Wyatt TA, and Lincoln TM. 1991. Regulation of sarcoplasmic reticulum protein phosphorylation by localized cGMP-dependent protein kinase in vascular smooth muscle cells. Mol. Pharmacol. 40: 923–931. [12] Etter EF, Eto M, Wardle RL, Brautigan DL, and Murphy RA. 2001. Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation. J. Bio. Chem. 276: 34681–34685. [13] Francis SH, and Corbin JD. 1999. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit. Rev. Clin. Lab. Sci. 36: 275–328. [14] Francis SH, Turko IV, and Corbin JD. 2001. Cyclic nucleotide phosphodiesterases: relating structure and function. Prog. Nucleic. Acid. Res. Mol. Biol. 65: 1–52. [15] Furness JB, Young HM, Pompolo S, Bornstein JC, Kunze WAA, and McConalogue K. Plurichemical transmission and chemical coding of neurons in the digestive tract. Gastroenterology 1995; 108: 554–563. [16] Goyal RK, and Hirano L. 1996. The enteric nervous system. N. Engl. J. Med. 334: 1106–1115. [17] Goyal RK, Rattan S, and Said SI. 1980. VIP as a possible neurotransmitter of non-cholinergic and non-adrenergic inhibitory neurons. Nature 288: 378–380. [18] Grider JR. 1994. Interplay of the inhibitory neurons in the regulation of VIP release and NO production during peristalsis. Am. J. Physiol. 267: G696–G701. [19] Grider JR. 1994. Reciprocal activity of longitudinal and circular muscle during intestinal peristaltic reflex. Am. J. Physiol. 284: G768–G775. [20] Grider JR, Cable MB, Bitar KN, Said SI, and Makhlouf GM. 1985. Vasoactive intestinal peptide: relaxant neurotransmitter in tenia coli of the guinea pig. Gastroenterology 89: 36–42. [21] Grider JR, and Jin JG. 1993. VIP release and L-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of VIP release by NO. Neuroscience 54: 521–526. [22] Grider JR, Murthy KS, Jin J-G, and Makhlouf GM. 1992. Postjunctional stimulation of nitric oxide in muscle cells by the relaxant neurotransmitter VIP. Am. J. Physiol. 262: G774– G778. [23] Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberrecht P, Said SI, Sreedharan SP, Wank SA, and Waschek JA. 1998. International union of pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol. Rev. 50: 265–270. [24] Jin J-G, Katsoulis S, Schmidt WE, and Grider JR. 1994. Inhibitory transmission in tenia coli mediated by distinct vasoactive intestinal peptide and apamin-sensitive pituitary adenylyl cyclase activating peptide. J. Pharmacol. Exp. Ther. 270: 433–439. [25] Jin J-G, Murthy KS, Grider JR, and Makhlouf GM. 1996. Stoichiometry of VIP release, nitric oxide formation and relaxation induced by nerve stimulation of rabbit and rat gastric muscle. Am. J. Physiol. 271: G357–G369. [26] Kim, CH, Goyal RK, and Mashimo H. 1999. Neuronal NOS provides ntriengic inhibitory neurotransmitter in mouse lower esophageal sphincter. Am. J. Physiol. 277: G280–G284.

[27] Koh SD, Sanders KM, and Carl A. 1996. Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A. Pflugers Arch. 432: 401–412. [28] Komalavilas P, and Lincoln TM. 1996. Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta. J. Biol. Chem. 271: 21933– 21938. [29] Laburthe M, Couvineau A, and Voisin T. 1999. Receptors for peptides of the VIP/PACAP and PYY/NPY/PP. Gastronintest. Endocrinol. 9: 125–157. [30] Landgraf W, Hulin R, Gobel C, and Hofmann F. 1986. Phosphorylation of cGMP-dependent protein kinase increases the affinity for cAMP. Eur. J. Biochem. 154: 113–117. [31] Lincoln TM, Dey N, and Sellak H. 2001. cGMP-dependent protein kinase signaling mechanism in smooth muscle: from the regulation of tone to gene expression. J. Appl. Physiol. 91: 1421–1430. [32] Makhlouf, GM. 2003. Smooth muscle of the gut. In: Textbook of Gastroenterology, 4th Edition, Chapter 6 (Ed. T. Yamada). Lippincott Williams and Wilkins, Philadelphia, pp 92–116. [33] Mashimo H, He XD, Huang PL, Fishman MC, and Goyal RK. 1996. Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission. J. Clin. Invest. 98: 8–13. [34] Mashimo H, He XD, Huang PL, Fishman MC, and Goyal RK. 1999. Lessons from genetically engineered animal models IV. Nitric oxide synthase gene knockout mice. Am. J. Physiol. 277: G745–G750. [35] Murthy KS. 2001. Activation of PDE5 and inhibition of guanylyl cyclase by cGMP-dependent protein kinase in smooth muscle. Biochem. J. 360: 199–208. [36] Murthy, KS, Jin J-G, Grider JR, and Makhlouf GM. 1997. Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells. Am. J. Physiol. 272: G1391– G1399. [37] Murthy KS, Jin J-G, Teng B-Q, and Makhlouf GM. 1998. G protein-dependent activation of smooth muscle eNOS mediated by the natriuretic peptide-C receptor. Am. J. Physiol. 275: C1409–C1416. [38] Murthy KS, and Makhlouf GM. 1994. VIP/PACAP-mediated activation of membrane-bound NO synthase in smooth muscle is mediated by pertussis toxin-sensitive Gi1–2. J. Biol. Chem. 269: 15977–15980. [39] Murthy KS, and Makhlouf GM. 1995. Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cell. Am. J. Physiol. 268: C171–C180. [40] Murthy KS, and Makhlouf GM. 1999. Identification of the G protein-activating domain of the natriuretic peptide clearance receptor (NPR-C). J. Biol. Chem. 274: 17587–17592. [41] Murthy KS, Teng BQ, Zhouo H, Jin J-G, Grider JR, and Makhlouf GM. 2000. Gi1/Gi2-dependent signaling by singletransmembrane natriuretic peptide clearance receptor. Am. J. Physiol. 278: G974–G980. [42] Murthy KS, and Zhou H. 2003. Selective phosphorylation of IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle. Am. J. Physiol. Gastrointest. Liver Physiol. 284: G221– G230. [43] Murthy KS, Zhou H, Grider JR, and Makhlouf GM. 2003. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially meditated by phosphorylation of RhoA. Am. J. Physiol. Gastrointest. Liver Physiol. 284: G1006–G1016. [44] Murthy KS, Zhou H, and Makhlouf GM. 2002. Regulation of phosphodiesterase 3 (PDE3) and adenylyl cyclase by cAMP-dependent protein kinase. Am. J. Physiol. 282: C508– C517.

Signaling by Vasoactive Intestinal Peptide in Gastrointestinal Smooth Muscle / 1161 [45] Nurko S, and Rattan S. 1988. Role of vasoactive intestinal peptide in the anal sphincter relaxation of the opossum. J. Clin. Invest. 81: 1146–1153. [46] Okamoto T, and Nishimoto I. 1992. Detection of G proteinactivator regions in m4 subtype muscarinic, cholinergic, and α2 adrenergic receptors based upon characteristics in primary structure. J. Biol. Chem. 267: 8342–8346. [47] Rattan S. 2005. The internal anal sphincter: regulation of smooth muscle tone and relaxation. Neurogastroenterol Motil. 17 (Suppl 1): 50–59. [48] Rattan S, and Chakder S. 1992. Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am. J. Physiol. 262: G107–G112. [49] Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, and Beavo J. 2002. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J. Biol. Chem. 277: 3310–3317. [50] Said SI. 1991. Vasoactive intestinal peptide. Biologic role in health and disease. Trends Endocrinol. Metab. 2: 107–112. [51] Said MI, and Mutt V. 1970. Polypeptide with broad biological activity: isolation from small intestine. Science 169: 1217–1218. [52] Said SI, and Rattan S. 2004. The multiple mediators of neurogenic smooth muscle relxation. Trends Endocrinol. Metab. 15: 189–191. [53] Sanders KM, Koh SD, Ordog T, and Ward SM. 2004. Ionic conductances involved in generation and propagation of electrical slow waves in phasic gastrointestinal muscles. Neurogastroenterol. Motil. Suppl 1 16: 100–105. [54] Sarna SK. 1999. Tachykinins and in vivo gut motility. Dig. Dis. Sci. 44: 114S–118S. [55] Sauzeau V, Ve Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, and Loriand G. 2000. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J. Biol. Chem. 275: 21722–21729. [56] Shah V, Lyford G, Gores G, and Farrugia G. 2004. Nitric oxide in gastrointestinal health and disease. Gastroenterology 126: 903– 913. [57] Szurszewski JH. Electrical basis of gastrointestinal motility. In: Physiology of the Gastrointestinal Tract, 2nd Edition (Ed. L.R. Johnson). Raven Press, New York, 1987, p 383. [58] Teng B-Q, Grider JR, and Murthy KS. 2001. Identification of VIP-specific receptor in guinea pig tenia coli. Am. J. Physiol. 281: G718–725.

[59] Teng B-Q, Murthy KS, Kuemmerle JF, Grider JR, and Makhlouf GM. 1998. Selective expression of VIP2/PACAP3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Regul. Pept. 77: 124–134. [60] Teng B-Q, Murthy KS, Kuemmerle JF, Grider JR, Michel T, and Makhlouf GM. 1998. Constitutive endothelial nitric oxide synthase: Expression in human and rabbit gastrointestinal smooth muscle cells. Am. J. Physiol. 275: G342–351. [61] Turko IV, Francis SH, and Corbin JD. 1998. Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation. Biochem. J. 329: 505–510. [62] Ulrich CD, Holtmann M, and Miller LJ. 1998. Secretin and vasoactive intestinal peptide receptors: members of a unique family of G protein-coupled receptors. Gastroenterology 114: 382–397. [63] Walker LA, MacDonald JA, Liu X, Nakamoto RK, Haystead TAJ, Somlyo AV, and Somlyo AP. 2001. Site-specific phosphorylation and point mutations of telokin modulates its Ca2+desensitizing effect in smooth muscle. J. Biol. Chem. 276: 24519– 24524. [64] Ward SM, Sanders KM, and Hirst GD. 2004. Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles. Neurogastroenterol. Motil. Suppl 1 16: 112–117. [65] Wiley JW, O’Dorisio TM, and Owyang C. 1988. Vasoactive intestinal peptide mediated CCK-induced relaxation of sphincter of Oddi. J. Clin. Invest. 81: 1920–1924. [66] Wu X, Haystead TAJ, Nakamoto RK, Somlyo AV, and Somlyo AP. 1998. Acceleration of myosin light chain dephosphorylation and relaxation in smooth muscle by telokin. J. Biol. Chem. 273: 11362–11369. [67] Zhou H, Huang J, Grider JR, and Murthy KS. 2005. Molecular cloning of a VIP-specific receptor. Gastroenterology 128: A193. [68] Zhou H, and Murthy KS. 2003. Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C). Am. J. Physiol. 284: C1255– C1261. [69] Zhou H, and Murthy KS. 2003. Relative contribution of RGS4 and RGS domain of GRK2 to inhibition of PLC-β activity by PKA: direct evidence from site-directed mutagenesis of RGS4 and GRK2. Gastroenterology 124: A23. [70] Zhou H, and Murthy KS. 2004. PKA-dependent phosphorylation of GRK2 augments VPAC2 receptor phosphorylation, internalization, and homologous desensitization in smooth muscle cells. Gastroenterology 126: A555.

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159 Adrenomedullin and Its Related Peptides KAZUO KITAMURA AND JOHJI KATO

amylin, with which AM shares some structural homology. As shown in Fig. 1, the sequence homology of AM with human CGRP and amylin is not high, although they share the C-terminal amide and a six-residue ring structure formed by the intramolecular disulfide linkage. Nevertheless, given the slight sequence homology and pharmacological activities that are similar to those of CGRP, it is likely that AM belongs to the CGRP superfamily. In addition to the human peptide, the amino acid sequences of AM from murine, canine, porcine, and bovine species have now been determined. Porcine AM is nearly identical to the human peptide, with a single substitution (Gly for Asn) at position 40. Rat AM has 50 amino acids, with two deletions and six substitutions, as compared with the human peptide. Notably, among all these species, the ring structure and C-terminal amide, both of which are essential for biological activity, are well conserved. Very recently a new member of AM family, adrenomedullin 2 (AM2)/intermedin was identified by two groups [18, 23]. Although the sequence identity between AM2/intermedin and AM is relatively low (approximately 30%), as shown in Fig. 1, the pharmacological activities are similar. One of the discoverers, Takei, discusses this peptide in the Renal Peptides Section of this Handbook.

ABSTRACT Adrenomedullin (AM) is a potent vasodilator peptide that exerts major effects on cardiovascular function. AM, initially isolated from human pheochromocytoma tissue, is biosynthesized in a wide variety of organs and cells. In addition to AM, proadrenomedullin Nterminal 20 peptide (PAMP) is found to be processed from the AM precursor. Both AM and PAMP show hypotensive effects in anesthetized rats but exhibit different hypotensive mechanisms. Further, AM possesses multiple biological effects closely related to cardiovascular homeostasis. Plasma AM concentration is increased in patients with several cardiovascular diseases such as hypertension, congestive heart failure, renal failure, and septic shock. It has been recognized that AM is one of the important vasoactive peptides involved in the physiology and pathophysiology of circulation and body fluid control.

DISCOVERY AND STRUCTURE OF AM We have been searching for peptides that may be relevant to circulation control, using an assay system that monitors the elevating activity of rat platelet cAMP. By isolating and sequencing all of the bioactive peaks in high-performance liquid chromatography (HPLC) analysis of a human pheochromocytoma tissue extract, we were able to discover the novel biologically active peptide. Because this peptide is also abundant in normal adrenal medulla, it was designated adrenomedullin (AM) [1, 7]. Human AM consists of 52 amino acids and has one intramolecular disulfide bond [1, 7]. In addition, the C-terminal Tyr is amidated, which has been observed in a number of other biologically active peptides, including calcitonin gene–related peptide (CGRP) and Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE The precursor for human AM (human preproAM) consists of 185 amino acid residues, including the AM sequence [9]. The predicted sequence of proAM contains a Gly-Lys-Arg segment immediately adjacent to the C-terminal tyrosine residue of AM. GIy-X-Y, where X and Y are basic residues, can serve as signals for Cterminal amidation, a process in which the glycine

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H2N-YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNV–APRSKISPQGY–CONH2

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H2N–ACDTATCVTHRLAGLLSRSGGVVKNNF–VPTNVGSKA–F–CONH2 * ** ** * * * * H2N–KCNTATCATQRLANFLVHSSNNFGALL–SSTNVGSNT–Y–CONH2 * ** * * * * * H2N–TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY–CONH2 * ** * ** *** * * * * ** *

FIGURE 1. Comparison of amino acid sequence of human adrenomedullin with human CGRP, amylin, and adrenomedullin 2/intermedin.

residue donates an amide moiety to the free carboxylic acid group in a reaction catalyzed by the enzyme peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3). In addition to AM, proadrenomedullin (proAM) contains a unique 20-amino-acid sequence followed by Gly-Lys-Arg, a typical amidation signal, in the Nterminal region. It is possible that a novel 20-residue peptide, termed proadrenomedullin N-terminal 20 peptide (PAMP), whose carboxy terminus is ArgCONH2, is processed from the AM precursor. We have clarified that PAMP exists in vivo and elicits a potent hypotensive activity in anesthetized rats. The genes for human and mouse AM were isolated and its structure was determined [3, 16]. The genomic DNA of human AM consists of four exons and three introns, as shown in Fig. 2. The mature AM peptide is coded in the fourth exon, whereas PAMP is interposed by the second intron. In addition, the AM gene is found to be situated in a single locus of chromosome 11. The 5′ flanking region of the gene contains TATA, CAAT, and GC boxes, and there are multiple binding sites for activator protein 2, a cAMP-regulated enhancer element, nuclear factor-kappa (κ) B, and hypoxia response elements. These indicate that the human AM gene contains components for its functional expression and that the expression may be subject to the activity of protein kinase C and feedback from cAMP levels.

DISTRIBUTION OF THE mRNA AND PEPTIDE IN THE CARDIOVASCULAR SYSTEM Although AM was discovered from pheochromocytoma tissue arising from adrenal medulla, AM has been shown to be widely distributed in tissue, including cardiovascular organs. Figure 3 summarizes the distribution of AM mRNA and immunoreactivity in rat tissue. A high level of AM mRNA was found in cardiovascular tissues such as atrium, aorta, kidney, and lung as well as in adrenal gland. A high concentration of immunoreactive AM was observed in lung and atrium as well as

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FIGURE 2. The schematic presentation of adrenomedullin (AM) gene and precursor, structures and biosynthesis of AM and proadrenomedullin N-terminal 20 peptide (PAMP).

adrenal gland. Immunoreactive AM was found ubiquitously in all tissue examined. The concentration of immunoreactive AM in aorta, ventricle, and kidney was less than 5% of that in adrenal gland, yet high levels of AM mRNA were found in these tissues [5]. This discrepancy may be explained by the possibility that AM biosynthesized in these tissues may be rapidly and constitutively secreted into the blood or function as an autocrine or paracrine regulator. In contrast, AM synthesized in adrenal medulla is thought to be stored in the granules and secreted in a regulatory pathway. Therefore, the biosynthetic and excretion systems of AM may be different from tissue to tissue. Many different cultured cell lines produce AM. The reverse transcription polymerase chain reaction (RTPCR) revealed the presence of AM in a variety of cells and tissues such as human pulmonary cells, pancreatic islet cells, cardiac myocytes, and vascular endothelial and smooth muscle cells [5]. Endothelial cells (ECs) actively synthesize and secrete AM [22]. In the cul-

Adrenomedullin and Its Related Peptides / 1165

FIGURE 3. Distribution of AM mRNA and immunoreactive AM in rat tissue.

ture medium of rat ECs, the secretion rate of AM was almost comparable to that of endothelin-1. In addition to ECs, vascular smooth muscle cells (VSMCs) were found to produce AM. The presence of specific AM receptors on VSMCs and ECs is consistent with the notion that AM secreted from ECs and VSMCs functions as an autocrine or paracrine regulator in vascular cell communication.

RECEPTORS AND THEIR DISTRIBUTION IN THE CARDIOVASCULAR SYSTEM AM has been shown to elevate intracellular cAMP levels in many but not all cells and tissues, including blood vessels, where it exerts biological actions, although identification of the AM receptor subtype has been controversial. McLatchie et al. identified three subtypes of receptor-activity-modifying protein (RAMP1–3), an accessory protein required for the transport of calcitoninreceptorlike receptor (CRLR) to the cell membrane [13]. CRLR was originally discovered as an orphan receptor that shows a 55% identity with calcitonin receptor. CRLR can function as either an AM receptor or a CGRP receptor, depending on the subtype of RAMP expressed [10]. RAMP2 enables CRLR to form an adrenomedullin (AM)-specific receptor that is sensitive to AM(22– 52) (AM1 receptor). RAMP3 enables CRLR to form an AM receptor sensitive to both CGRP(8–37) and AM(22–

52) (AM2 receptor), although rat and mouse AM2 receptors show a clear preference for CGRP(8–37) over AM(22–52). RAMP1 enables CRLR to form the CGRP(8–37)-sensitive CGRP1 receptor, which can also be activated by higher concentrations of AM. CRLR mRNA is extremely abundant in the rat lung and is expressed in blood vessels by in situ hybridization studies. CRLR protein was also shown to be expressed in vascular endothelial cells by immunocytochemistry. As to the distributions of RAMPs mRNA expression in the human, rat, and mouse, RAMP1 is abundantly expressed in the brain, fat, thymus, and spleen and RAMP2 in the lung, spleen, fat, and aorta; RAMP3 is most abundant in the kidney and lung and is expressed ubiquitously [13]. Recently Kuwasako et al. clearly demonstrated that CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway [11].

BIOLOGICAL ACTIONS IN THE CARDIOVASCULAR SYSTEM AM was discovered in human pheochromocytoma extract by monitoring activity that elevated rat platelet cAMP [7]. To date, however, it is known that AM is really multifunctional peptide. As summarized in Table 1, several biological effects of AM have been described

1166 / Chapter 159 in vivo and in vitro. We describe here the biological actions of AM in cardiovascular systems. The basic characteristic effect of AM is a potent, long-lasting hypotension that is dose-dependent in several species, including humans. AM dilates resistance vessels in the kidney, brain, lung, hind limbs, and mesentery in animals. In conscious sheep, Parkes has reported details on the cardiovascular and hemodynamic changes induced by human AM [17]. AM produced a dose-dependent decrease in blood pressure accompanied by an increase in heart rate and cardiac output. Decreased peripheral resistance and blood pressure (BP) induce reflex tachycardia; however, the heart rate increases less than after other vasodilators inducing comparable hypotension. Several papers show that the vasodilating effect by AM is abrogated by blockade of nitric oxide (NO) synthase activity with l-NAME, suggesting that the decrease in total peripheral resistance subsequent to AM infusion is in part due to NO generation [19]. AM activates endothelial nitric oxide synthase (eNOS) by at least two mechanisms. First, AM elevates the intracellular calcium level, which increases eNOS activity. Second, ADM activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt, which phosphorylate eNOS and increase its activity even at low calcium concentration. However, the diminution of vasodilation by l-NAME seems to vary greatly from study to study. Pulmonary vasodilator responses are significantly reduced by l-NAME in rats, but l-NAME had no significant effect in the pulmonary vascular bed of the cat [15]. Hence, it appears that

TABLE 1. Cardiovascular Actions of Adrenomedullin. Vasculature

Heart

Lung Adrenal gland Kidney Pituitary Brain

Hypotension, antiproliferation, survival factor inhibition of Ca increase by endothelin, decreased endothelin production Positive chronotropism and inotropism, increased coronary blood flow, increased ANP gene transcription, antimitogenesis, increased hypertrophy Vasodilation, bronchodilation, antiinflammatory Inhibition of aldosterone secretion Increased renal flow, diuresis, natriuresis, inhibition of mesangial cell proliferation Inhibition of ACTH secretion, inhibition of AVP secretion Increased collateralization and cerebral blood flow, inhibition of thirst and salt appetite, stimulation of sympathetic outflow (hypertension)

nitric oxide may at least be an important mediator for AM despite its regional and interspecies variation. In cultured cardiac myocytes and fibroblasts, AM may inhibit the protein synthesis and hypertrophy of myocytes, proliferation of fibroblasts, and production of extracellular matrix. Because AM is synthesized and secreted by cultured cardiac myocytes and fibroblasts, this peptide may regulate myocardial hypertrophy and remodeling in arterial hypertension or heart failure in a paracrine/autocrine manner. AM may bidirectionally regulate VSMC proliferation. It stimulates the proliferation of quiescent VSMC in the absence of other stimulating factors, but inhibits proliferation induced by PDGF or fetal bovine serum. AM inhibits endothelial cell apoptosis induced by serum deprivation. This effect is mediated by nitric oxide, but is cGMP-independent. In addition, AM stimulates the proliferation of endothelial cells that may be involved in angiogenesis and reendothelialization of injured vessels. Considering the results on vascular tissue expression of AM mRNA, AM seems to regulate vascular proliferation and remodeling as well as vascular tone.

PROADRENOMEDULLIN N-TERMINAL 20 PEPTIDE PAMP consists of 1–20 amino acids of proadrenomedullin (pro-AM) whose C-terminus is Arg-CONH2. The distribution of PAMP in mammalian tissues is similar to AM, consistent with their origin from a common precursor. The PAMP/AM ratio in tissue extracts and cell culture homogenates varies depending on the cell studied, from 1–2% in the lung to ∼50% in the heart atria. PAMP elicits a potent hypotensive effect in anesthetized rats [6]. An intravenous bolus injection of human PAMP causes a rapid and strong hypotensive effect in a dose-dependent manner, although this peptide is less potent than AM. PAMP is found to inhibit carbachol-induced catecholamine secretion in cultured bovine adrenal medullary cells, but AM showed no effect on catecholamine secretion. Fujita et al. demonstrated that AM infused into pithed rat showed hypotensive action dose-dependently but PAMP did not [20]. After BP was increased to a level of 80–100 mmHg by electrical stimulation of the pithed rat, PAMP exhibited hypotensive effects. Furthermore, during augmentation of peripheral sympathetic nerve activity with periarterial electrical stimulation, norepinephrine released in the perfusate was measured as an indication of neural transmission. PAMP decreased norepinephrine overflow dosedependently, whereas AM did not. These results suggest that the hypotensive effect of PAMP may be due to the inhibition of neural transmission at nerve endings

Adrenomedullin and Its Related Peptides / 1167 rather than via a direct vasodilating effect. Recently, it has been reported that PAMP may antagonize the stimulatory effect of AM on endothelial NO production, suggesting a more complex role of this peptide in the regulation of vascular tone [12]. Very recently, it has been reported PAMP exhibits an extremely potent angiogenic potential. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. Consequently, PAMP as well as AM is biosynthesized from the AM precursor and may participate in circulation control in different mechanisms (Fig. 2).

INFORMATION ON MOLECULAR FORM OF AM The mature form of AM (mAM, AM[1–52] NH2) is produced by C-terminal amidation of glycine-extended AM, AM-glycine (AM-Gly). AM-Gly, an intermediate form of AM (iAM), is processed from proAM. Biological activity is exerted only by mAM. We have found that most of the circulating AM in human plasma is not mAM but iAM [8]. The plasma concentration of mAM was much lower than that of AM-Gly or of total AM (t AM), which is mAM and iAM. All forms of AM increased significantly as the severity of congestive heart failure increased [2]. The ratio of mAM/tAM did not differ significantly in any of the study groups, suggesting that the amidation process of AM is unaffected in the patients with congestive heart failure. On the other hand, the major immunoreactive AM in tissue or cell is determined to be mAM.

CLINICAL IMPLICATION OF AM IN CARDIOVASCULAR DISEASES AM circulates in human blood at a considerable concentration, and the plasma AM levels were increased in patients with a variety of diseases, including congestive heart failure, renal diseases, hypertensive diseases, diabetes mellitus, and septic shock [1]. The plasma AM concentration in patients with essential hypertension or primary aldosteronism was significantly higher than that in normotensive controls. The plasma AM levels increase with the severity of hypertension [4]. Furthermore, in the patients with malignant hypertension and renovascular hypertension, the increase in plasma AM is marked in comparison with the increases in primary aldosteronism and essential hypertension. AM, therefore, seems to act as a humoral regulator in blood pressure control and to counteract

pressor factors that may cause and promote hypertension. In patients with hypertension complicated with chronic renal failure, the increment of AM is obvious and the plasma AM concentration was elevated in relation to the degree of renal failure. In patients with congestive heart failure, the plasma AM was significantly correlated with pulmonary artery pressure, pulmonary capillary wedge pressure, left atrial dimension, plasma renin activity, and plasma concentrations of atrial and brain natriuretic peptides. Intravenous infusion of human AM into patients with congestive heart failure predominantly improved cardiac function [14]. The AM elicited dilatation of the resistance arteries and increases in cardiac stroke index and urinary sodium excretion. An improvement of the cardiac pre- and after-loads and cardiac contractility elicited by AM is the mechanism for recovery of function of the failing heart. Intravenous injection of lipopolysaccharide (endotoxin) produces a marked increase in plasma immunoreactive AM in a dose-dependent fashion in the anesthetized rat, suggesting AM may be involved in sepsis. Actually, plasma AM concentration was markedly increased in patients with septic shock. As suggested by an experimental model of septic shock in transgenic mice [21], the large amount of plasma AM observed in patients with septic shock may play a role in protecting against peripheral circulatory failure, organ damage, and mortality characteristic of endotoxic shock. In summary, regarding the clinical features of a variety of diseases, the plasma and tissue concentrations of AM are increased in response to pathological conditions such as hypervolemia, hypertension, tissue ischemia or hypoxia, and inflammatory damage by cytokines. The increased plasma and tissue concentrations of AM seems to function as a counterregulator for pathologically altered circulation, tissue ischemia, or tissue injury by toxic factors through its humoral or paracrineautocrine actions.

CONCLUSION AM is a potent vasodilatory peptide discovered in 1993 from the extract of human pheochromocytoma. AM consists of 52 amino acids and belongs to the calcitonin, CGRP, and amylin family. In addition to AM, the AM precursor generates another bioactive peptide, proadrenomedullin N-terminal 20 peptide (PAMP). Though AM and PAMP show hypotensive effect, their mechanisms are different. During the 12 years since its discovery, over 1600 papers concerning AM and PAMP have been published. Considering the multifunctional characteristics of AM and significant increase in plasma

1168 / Chapter 159 immunoreactive AM levels in patients with cardiovascular diseases, AM should be recognized as an important factor regulating circulation and involved in cardiovascular diseases.

References [1] Eto T. A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides. Peptides 2001;22(11):1693–1711. [2] Hirayama N, Kitamura K, Imamura T, et al. Molecular forms of circulating adrenomedullin in patients with congestive heart failure. J Endocrinol 1999;160(2):297–303. [3] Ishimitsu T, Kojima M, Kangawa K, et al. Genomic structure of human adrenomedullin gene. Biochem Biophys Res Commun 1994;203(1):631–639. [4] Ishimitsu T, Nishikimi T, Saito Y, et al. Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 1994;94(5): 2158–2161. [5] Kitamura K, Kangawa K, Eto T. Adrenomedullin and PAMP: Discovery, structures, and cardiovascular functions. Microsc Res Tech 2002;57(1):3–13. [6] Kitamura K, Kangawa K, Ishiyama Y, et al. Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP). FEBS Lett 1994;351(1):35–37. [7] Kitamura K, Kangawa K, Kawamoto M, et al. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192(2): 553–560. [8] Kitamura K, Kato J, Kawamoto M, et al. The intermediate form of glycine-extended adrenomedullin is the major circulating molecular form in human plasma. Biochem Biophys Res Commun 1998;244(2):551–555. [9] Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H, Eto T. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem Biophys Res Commun 1993;194(2):720–725. [10] Kuwasako K, Cao YN, Nagoshi Y, Kitamura K, Eto T. Adrenomedullin receptors: Pharmacological features and possible pathophysiological roles. Peptides 2004;25(11):2003–2012. [11] Kuwasako K, Shimekake Y, Masuda M, et al. Visualization of the calcitonin receptor-like receptor and its receptor activity-

[12]

[13]

[14]

[15]

[16]

[17] [18]

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[21]

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modifying proteins during internalization and recycling. J Biol Chem 2000;275(38):29602–29609. Li J, Ren Y, Dong X, Zhong G, Wu S, Tang C. Roles of different peptide fragments derived from proadrenomedullin in the regulation of vascular tone in isolated rat aorta. Peptides 2003;24(4):563–568. McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998;393(6683):333–339. Nagaya N, Satoh T, Nishikimi T, et al. Hemodynamic, renal, and hormonal effects of adrenomedullin infusion in patients with congestive heart failure. Circulation 2000;101(5):498– 503. Nishimatsu H, Suzuki E, Nagata D, et al. Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta. Circ Res 2001;89(1):63–70. Okazaki T, Ogawa Y, Tamura N, et al. Genomic organization, expression, and chromosomal mapping of the mouse adrenomedullin gene. Genomics 1996;37(3):395–399. Parkes DG. Cardiovascular actions of adrenomedullin in conscious sheep. Am J Physiol 1995;268(6 Pt 2):H2574–2578. Roh J, Chang CL, Bhalla A, Klein C, Hsu SY. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activitymodifying protein receptor complexes. J Biol Chem. Feb 20 2004;279(8):7264–7274. Shimekake Y, Nagata K, Ohta S, et al. Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem 1995;270(9):4412–4417. Shimosawa T, Ito Y, Ando K, Kitamura K, Kangawa K, Fujita T. Proadrenomedullin NH(2)-terminal 20 peptide, a new product of the adrenomedullin gene, inhibits norepinephrine overflow from nerve endings. J Clin Invest 1995;96(3):1672–1676. Shindo T, Kurihara H, Maemura K, et al. Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 2000;101(19):2309–2316. Sugo S, Minamino N, Kangawa K, et al. Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 1994;201(3):1160–1166. Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, Miyano S. Identification of novel adrenomedullin in mammals: A potent cardiovascular and renal regulator. FEBS Lett 2004;556 (1–3):53–58.

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160 Angiotensin II and Its Related Peptides YASUKATSU IZUMI AND HIROSHI IWAO

In the early 1970s, polypeptides either inhibiting the formation of angiotensin II or blocking angiotensin II receptors were discovered, but these were not orally active. Cushman and Ondetti succeeded in the development of captopril, the orally active ACE inhibitor in 1977. After that, many experimental and clinical studies with ACE inhibitors uncovered additional roles for the RA system in the pathophysiology of hypertension, heart failure, and vascular diseases. Furthermore, losartan (Dup 753), an orally active, highly selective, and potent nonpeptide angiotensin II receptor blocker (ARB), was developed in 1988, and the cloning of angiotensin II receptors AT1 and AT2 was accomplished in the early 1990s.

ABSTRACT Much evidence supports the notion that angiotensin II, the central product of the renin-angiotensin system, may play a central role not only in the etiology of hypertension but also in the pathophysiology of cardiovascular diseases in humans. Angiotensin II, via the AT1 receptor, directly causes cellular phenotypic changes and cell growth, regulates the gene expression of various bioactive substances, and activates multiple intracellular signaling cascades in cardiac myocytes and fibroblasts, as well as vascular endothelial and smooth muscle cells. These actions are supposed to participate in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, and atherosclerosis.

DISCOVERY

STRUCTURE OF THE PEPTIDE AND COMPONENT OF RA SYSTEM

Research on the renin-angiotensin (RA) system began with the discovery of renin from the kidney by Tiegerstedt and Bergman in 1898. In 1940, a peptide that had vasoconstrictive effects in the RA system was discovered, and it was named hypertensin by BraunMenendez in Argentina, and angionin by Page and Helmer in the United States. These two terms persisted for about 20 years, until it was agreed to rename the pressor substance angiotensin. In the 1950s, two forms of angiotensin were recognized, the first a decapeptide (angiotensin I) and the second an octapeptide (angiotensin II). Skeggs et al. in the United States reported that angiotensin II was formed by enzymatic cleavage of angiotensin I by another enzyme, termed angiotensinconverting enzyme (ACE). Schwyzer and Bumpus succeeded in the synthesis of angiotensin II in 1957. Gross suggested that the renin-angiotensin system was involved in the regulation of aldosterone secretion in 1958, and then Davis, Genet, Laragh et al. proved his hypothesis.

The RA system plays an important role in the regulation of arterial blood pressure. Renin is an enzyme that acts on angiotensinogen to catalyze the formation of angiotensin I. Then, angiotensin I is cleaved by ACE to yield angiotensin II. A representation of the biochemical pathways of the RA system is shown in Fig. 1. The major element of the rate of angiotensin II production is the amount of renin released by the kidney. Renin is synthesized, stored, and secreted into the renal arterial circulation by the granular juxtaglomerular cells. The secretion of renin is controlled predominantly by three pathways. The first mechanism controlling renin release is the intrarenal macula densa pathway, and the second is the intrarenal baroreceptor pathway. The third mechanism is the β-adrenergic receptor pathway, which is mediated by the release of norepinephrine from postganglionic sympathetic nerve terminals. An increase in renin secretion enhances the formation of angiotensin II, and angiotensin II stimu-

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lates the AT1 receptors on juxtaglomerular cells to inhibit renin release. The substrate for renin is angiotensinogen, an abundant α2-globulin that circulates in the plasma. The primary structure of angiotensinogen has been deduced by molecular cloning. Angiotensinogen is synthesized primarily in the liver, although mRNA that encodes the protein is abundant in fat, certain regions of the central nervous system, and kidney. The rate of angiotensin II synthesis can be influenced by changes in angiotensinogen levels. ACE was discovered in plasma as the factor responsible for conversion of angiotensin I to angiotensin II. The ACE gene contains, in intron 16, an insertion/ deletion polymorphism that explains 47% of the phenotypic variance in serum ACE levels [20]. Individuals homozygous for the deletion allele have higher levels of serum ACE. Angiotensin I is rapidly converted to angiotensin II, when given intravenously. Angiotensin III can be formed by the action of aminopeptidase on angiotensin II. Angiotensin III and angiotensin II cause qualitatively similar effects. Angiotensin III is approximately as potent as angiotensin II in stimulating the secretion of aldosterone. However, angiotensin III is only 25% as potent as angiotensin II in elevating blood pressure [17]. There are bypass pathways in the RA system to produce angiotensin II in addition to the main enzymes such as renin and ACE. Arakawa et al. showed that trypsin and kallikrein can produce angiotensin II [2]. One of the most famous enzymes in such bypass pathways is chymase. However, it is still not well known what the pathophysiological significance is via angiotensin II production of these bypass pathways.

sin I in the plasma; circulating angiotensin I is converted by plasma ACE and pulmonary endothelial ACE to angiotensin II. Then, angiotensin II is delivered to its target organs via blood flow. This traditional pathway is called the circulating RA system. On the other hand, heart, blood vessels, and several other tissues contain and/or synthesize components of the RA system, called the tissue (local) RA system. Many tissues, including the heart, blood vessels, brain, kidney, and adrenal gland, express mRNAs for renin, angiotensinogen, and/or ACE, and various cultured cell types from these tissues produce renin, angiotensinogen, ACE, and/or angiotensin I, II, and III [3, 7, 19]. Angiotensin I and II have been localized in atria and ventricles. Therefore, it appears the tissue RA system exists independently of the renal-hepatic-based system. Several serine proteases, such as tonin and cathepsin G, have been shown to hydrolyze angiotensin II precursors. An aspartyl protease with cathepsin D–like properties was shown to convert angiotensinogen to angiotensin I. The conversion of angiotensin I to angiotensin II in human and dog cardiac ventricles may occur by heart chymase. Unlike ACE, human heart chymase shows high specificity for angiotensin I and does not degrade bradykinin or vasoactive intestinal peptide. It is likely that the relative contribution of ACE and chymase to cardiac angiotensin II formation varies with the cardiac chamber in the human heart because ACE levels are highest in the atria and chymase levels are highest in ventricles. Participation of heart chymase in cardiac angiotensin II formation remains to be fully assessed using in vivo animal models in molecular and pharmacological methods.

DISTRIBUTION OF RA SYSTEM, PROCESSING, AND ENDOGENOUS FORM IN THE CARDIOVASCULAR SYSTEM

RECEPTORS AND THEIR DISTRIBUTION IN THE CARDIOVASCULAR SYSTEM

Circulating renin of renal origin acts on circulating angiotensinogen of hepatic origin to produce angioten-

The effects of angiotensins are exerted through specific cell surface receptors. There are two subtypes of angiotensin II receptors, angiotensin II types 1 and 2

Angiotensin II and Its Related Peptides / 1171 (AT1 and AT2) [6]. The successful cloning of the AT1 receptor in 1991 and the AT2 receptor in 1993 allowed the development of further research on the structure and function of this receptor. The AT1 receptor consists of two subtypes, AT1a and AT1b, which have 94% homology with regard to amino acid sequence and have similar pharmacological properties and tissue distribution patterns. AT1 receptor is a member of the seven-transmembrane-spanning Gprotein-coupled receptor family, binds to heterotrimeric G-proteins, and lacks intrinsic tyrosine kinase activity. The human AT1 receptor gene is mapped to chromosome 3, and the AT1a and AT1b receptor genes in rats are mapped to chromosomes 17 and 2, respectively. The AT1 receptor is ubiquitously and abundantly distributed in adult tissues, including the blood vessel, heart, kidney, adrenal gland, liver, brain, and lung. The AT1 receptor mediates all the classic well-known effects of angiotensin II, such as the elevation of blood pressure, vasoconstriction, increase in cardiac contractility, aldosterone release from the adrenal gland, facilitation of catecholamine release from nerve endings, and renal sodium and water absorption. Therefore, the AT1 receptor has a role in atherosclerosis, congestive cardiac failure, and several acute and chronic inflammatory diseases, conditions in which inflammation is known to play a significant role [5]. Numerous selective and potent nonpeptide AT1 receptor antagonists have been developed, such as losartan, candesartan, valsartan, irbesartan, eprosartan, telmisartan, tasosartan, and others. In contrast, the AT2 receptor has a high affinity for PD123177, PD123319, CGP42112, L-162,686, L-162,638, and CGP42112A. The cDNA and genomic DNAs of human, rat, and mouse AT2 receptors have been cloned. The AT2 receptor is mainly expressed in developing fetal tissues and is believed to have an essential role in physiological vascular development. AT2 receptor expression rapidly decreases after birth [9, 14], and in the adult the expression of this receptor is limited mainly to the uterus, ovary, certain brain nuclei, heart, and adrenal medulla. Unlike the AT1 receptor, which has been shown to have subtypes in rats and mice, there is no evidence for subtypes of the AT2 receptor. Although a comparison of amino acid sequences of AT1a and AT2 receptors in rats, deduced from nucleotide sequences, shows a low homology between these receptors (32%), AT2 receptor is also a seven-transmembrane-domain receptor. In various cell lines, AT2 receptor-activated protein tyrosine phosphatase was shown to inhibit cell growth or induce programmed cell death (apoptosis) [13, 26]. The AT2 receptor inhibited AT1 receptor– mediated cell growth, demonstrating an antagonistic action. It is thought that the expression balance of AT1 and the AT2 is important for cardiovascular diseases [6,

23]. There have also been conflicting findings regarding these receptors. For example, it is controversial whether cardiac hypertrophy is promoted by the AT2 receptor [21, 22]. However, the AT2 receptor plays a cardioprotective role on left ventricular function after myocardial infarction [1, 16, 27]. In contrast to extensive data on the molecular and cellular functions and the pathophysiological significance of the AT1 receptor, the role of the AT2 receptor in cardiovascular diseases remains to be defined. At present, an AT2 receptor ligand has not been developed for clinical use.

BIOLOGICAL ACTIONS AND PATHOPHYSIOLOGICAL IMPLICATION IN THE CARDIOVASCULAR SYSTEM Molecular Characteristics of Pathological Cardiac Hypertrophy Generally, pathological left-ventricular hypertrophy is characterized not only by an increase in myocyte size (quantitative change) but also by myocyte gene reprogramming (qualitative change), as shown by enhanced expression of fetal phenotypes of genes such as β-myosin heavy chain (β-MHC), skeletal α-actin, and atrial natriuretic peptide (ANP). In the cardiac ventricle of most mammalian species, MHC consists of two isoforms, αand β-MHCs. In the rat, α-MHC is the predominant isoform in adult hearts, whereas β-MHC is the predominant isoform in fetal hearts. Therefore, changes in the ratio of β-MHC to α-MHC in the cardiac ventricle significantly alter the contractile properties of the heart. Cardiac sarcomeric actin is also composed of two isoforms: cardiac α-actin and skeletal α-actin. Cardiac αactin is predominantly expressed in adult rat hearts, whereas skeletal α-actin is normally expressed in fetal and neonatal rat hearts. The ratio of skeletal α-actin to cardiac α-actin in the ventricle plays a significant role in cardiac function because skeletal α-actin has greater contractility than cardiac α-actin. Furthermore, in addition to showing enhanced expression of β-MHC and skeletal α-actin, hypertrophic ventricular myocytes are also characterized by significant upregulation of ANP, which is scarcely expressed in normal adult ventricular myocytes. Another important property of pathological cardiac hypertrophy is the increased accumulation of extracellular matrix (ECM) proteins such as collagen (particularly collagen types I and III) and fibronectin in the interstitium and around blood vessels within the heart. These changes play a central role in ventricular fibrosis or remodeling [18, 24]. Increased interstitial collagen deposition in the heart enhances cardiac stiffness and results in diastolic dysfunction. Fibronectin is localized

1172 / Chapter 160 on the surface of cardiac myocytes, connects cardiac myocytes to perimyocytic collagen, and is thought to affect cardiac systolic and diastolic functions. Thus, increased ECM accumulation, as well as the previously mentioned ventricular myocyte gene reprogramming, plays a critical role in the impairment of cardiac performance and pathophysiology of cardiac failure. ECM proteins within the heart are predominantly produced by fibroblasts. Unlike cardiac myocytes, cardiac fibroblasts proliferate and increase the production of ECM proteins when the heart is exposed to hypertrophic stimuli such as hemodynamic overload. Thus, cardiac fibroblasts and cardiac myocytes play key roles in the development of pathological cardiac hypertrophy and dysfunction. Accumulating in vitro and in vivo evidence supports the concept that angiotensin II is involved in all of these important processes of pathological cardiac hypertrophy, including myocyte hypertrophy, myocyte gene reprogramming, fibroblast proliferation, and ECM protein accumulation.

Effects of in Vivo Angiotensin II Infusion on the Heart Figure 2 illustrates the cardiac molecular and cellular effects of angiotensin II in vivo. Angiotensin II infusion in rats can induce cardiac hypertrophy via the AT1 receptor, independent of its blood pressure–elevating

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FIGURE 2. Proposed mechanism of AT1 receptor-mediated pathological cardiac hypertrophy. Angiotensin II induces cellular hypertrophy, gene reprogramming, or necrosis in cardiac myocytes and cellular proliferation and upregulation of fibrosis-associated genes in cardiac fibroblasts, which are all mediated via the AT1 receptor.

effect. Furthermore, continuous infusion of angiotensin II in adult rats, while causing a moderate increase in blood pressure, produced both myocyte necrosis and myocytolysis, as shown by labeling of cardiac myocytes with exogenously administered monoclonal antimyosin antibody; this subsequently caused cardiac fibroblast proliferation and resulted in significant scar formation, indicating the cardiotoxic effects of angiotensin II in vivo. Angiotensin II infusion in rats caused a small and gradual increase in blood pressure; elevated leftventricular mRNAs for skeletal α-actin, β-MHC, ANP, and fibronectin, preceding an increase in left-ventricular mass; and elevated TGF-β1 and types I and III collagen mRNA levels. These increases were completely inhibited by ARB but not by hydralazine. Thus, angiotensin II directly induces cardiac myocyte hypertrophy and gene reprogramming, and probably fibroblast proliferation and subsequent fibrosis as well, independent of the elevation of blood pressure, indicating the key role of angiotensin II in the development of pathological cardiac hypertrophy. Investigations of the in vivo effects of angiotensin II on cardiac intracellular signaling cascades are essential to elucidating the molecular mechanism underlying angiotensin II–induced pathological cardiac hypertrophy. Accumulating in vitro evidence on cultured cardiac myocytes or fibroblasts suggests that mitogen-activated protein kinases (MAPKs), including extracellular-signalregulated kinase (ERK), c-jun amino terminal kinase (JNK), and p38MAP kinase (p38), may be responsible for myocyte hypertrophy and gene reprogramming or fibroblast proliferation. Yano et al. showed that angiotensin II–induced cardiac activation of JNK occurs in a more sensitive manner than that of ERK and that JNK activation by angiotensin II without ERK activation is followed by activation of activator protein-1 (AP-1) [28]. It is important that AP-1 regulates the expression of various genes by binding the AP-1 consensus sequence present in their promoter regions. Interestingly, fetal phenotypes of cardiac genes, such as skeletal α-actin and ANP, and cardiac fibrosis-associated genes, such as TGF-β1 and collagen type I, have AP-1-responsive sequences in their promoter regions. Indeed, AP-1 activation has been demonstrated to lead to increased promoter activity of skeletal α-actin and TGF-β1. Therefore, it is intriguing to postulate that JNK activation, in part through activation of AP-1, may be implicated in angiotensin II-induced cardiac hypertrophic response in vivo. Wenzel at al. reported that angiotensin II–induced TGF-β1 expression is regulated by a p38-dependent pathway [25]. An in vivo study showed that apoptosis signal-regulating kinase 1 (ASK1), one of the MAPK kinase kinases, plays an important role in angiotensin II–induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hyper-

Angiotensin II and Its Related Peptides / 1173 trophy–related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation [10]. Angiotensin II is well known as a powerful inducer of oxidative stress to cardiovascular tissues, and the reactive oxygen species (ROS) generated participate in angiotensin II–induced intracellular signaling pathways [8]. It has been suggested that angiotensin II–induced hypertrophic response is mediated by superoxide anion [10, 15]. Angiotensin II, via AT1 receptor, is known to facilitate the release of norepinephrine from cardiac sympathetic nerve terminals. In angiotensin II–infused rats, surgical cardiac sympathectomy or treatment with β1-adrenergic receptor blocker significantly prevented cardiac myocyte necrosis, showing that angiotensin II–induced cardiac damage is, at least in part, mediated by catecholamine release from cardiac sympathetic neurons. Thus, the activation of cardiac sympathetic neurons by angiotensin II also contributes to pathological cardiac hypertrophy.

Effects of in Vivo Angiotensin II Infusion on Vascular Tissues Numerous in vivo experiments have shown that angiotensin II can induce vascular smooth muscle cell (SMC) proliferation in vivo [11]. Angiotensin II infusion increased mesenteric vascular media width, media cross-sectional area, and media/lumen ratio. Despite detailed investigations into the molecular mechanism of angiotensin II–mediated vascular SMC growth in vitro, this process is poorly understood. Angiotensin II infusion, at least in part independent of its blood pressure–elevating effect, increased aortic mRNA and protein expression of fibronectin, which is an ECM protein that induces phenotypic change of vascular SMCs from a contractile to a synthetic phenotype. Basic fibroblast growth factor (bFGF) may play a key role in angiotensin II–mediated vascular SMC replication in vivo, as shown by the observation that the injection of anti-bFGF antibody significantly inhibited the mitogenic effect of angiotensin II infusion on rat carotid arteries. Angiotensin II infusion in rats doubled superoxide production in rat aorta by activation of NAD(P)H oxidase. On the other hand, norepinephrine infusion did not increase vascular superoxide production, despite a hypertensive effect comparable to that of angiotensin II, suggesting that SMC growth due to angiotensin II may be specifically mediated by increased superoxide generation. Angiotensin II infusion in rats increased heme oxygenase 1 (HO-1) mRNA and protein in the endothelium [12]. Because HO-1 is an oxidantsensitive gene, it is possible that increased oxidative stress is a trigger for HO-1 mRNA upregulation in the

angiotensin II–infused rat aorta and that HO-1 may serve to abrogate this increased stress caused by angiotensin II. Angiotensin II infusion stimulated aortic thrombin receptor mRNA expression in rats, which was blocked by either ARB or the heparin-binding chimera of human Cu/Zn superoxide dismutase but not by normalization of blood pressure with hydralazine treatment, suggesting that angiotensin II increases vascular thrombin receptor by AT1 receptor–mediated superoxide production and may be implicated in the pathophysiology of atherosclerosis by thrombin cascade activation [4]. The injection of angiotensin II resulted in increased oxidized low-density lipoprotein uptake by peritoneal macrophages and increased macrophage proteoglycan content, suggesting that angiotensin II may accelerate atherosclerosis by promoting foam cell formation and cholesterol accumulation in the vascular wall.

References [1] Adachi Y, Saito Y, Kishimoto I, et al. Angiotensin II type 2 receptor deficiency exacerbates heart failure and reduces survival after acute myocardial infarction in mice. Circulation 2003; 107: 2406–8. [2] Arakawa K, Maruta H. Ability of kallikrein to generate angiotensin II-like pressor substance and a proposed “kinin-tensin enzyme system.” Nature 1980; 288: 705–6. [3] Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system. Annu Rev Physiol 1992; 54: 227–41. [4] Capers QT, Laursen JB, Fukui T, Rajagopalan S, Mori I, Lou P, et al. Vascular thrombin receptor regulation in hypertensive rats. Circ Res 1997; 80: 838–44. [5] Das UN. Is angiotensin-II an endogenous pro-inflammatory molecule? Med Sci Monit 2005; 11: RA155–62. [6] de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 2000; 52: 415–72. [7] Dzau VJ. Vascular renin-angiotensin system and vascular protection. J Cardiovasc Pharmacol 1993; 22 Suppl 5: S1–9. [8] Griendling KK, Ushio-Fukai M. Reactive oxygen species as mediators of angiotensin II signaling. Regul Pept 2000; 91: 21–7. [9] Henrion D, Kubis N, Levy BI. Physiological and pathophysiological functions of the AT(2) subtype receptor of angiotensin II: From large arteries to the microcirculation. Hypertension 2001; 38: 1150–7. [10] Izumiya Y, Kim S, Izumi Y, Yoshida K, Yoshiyama M, Matsuzawa A, et al. Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling. Circ Res 2003; 93: 874–83. [11] Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev 2000; 52: 11–34. [12] Li Volti G, Seta F, Schwartzman ML, Nasjletti A, Abraham NG. Heme oxygenase attenuates angiotensin II-mediated increase in cyclooxygenase-2 activity in human femoral endothelial cells. Hypertension 2003; 41: 715–9. [13] Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res 1998; 83: 1182–91.

1174 / Chapter 160 [14] Miura S, Karnik SS. Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis. Embo J 2000; 19: 4026– 35. [15] Nakagami H, Takemoto M, Liao JK. NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy. J Mol Cell Cardiol 2003; 35: 851–9. [16] Oishi Y, Ozono R, Yano Y, Teranishi Y, Akishita M, Horiuchi M, et al. Cardioprotective role of AT2 receptor in postinfarction left ventricular remodeling. Hypertension 2003; 41: 814–8. [17] Peach MJ. Renin-angiotensin system: Biochemistry and mechanisms of action. Physiol Rev 1977; 57: 313–70. [18] Pelouch V, Dixon IM, Golfman L, Beamish RE, Dhalla NS. Role of extracellular matrix proteins in heart function. Mol Cell Biochem 1993; 129: 101–20. [19] Phillips MI, Speakman EA, Kimura B. Levels of angiotensin and molecular biology of the tissue renin angiotensin systems. Regul Pept 1993; 43: 1–20. [20] Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86: 1343–6. [21] Senbonmatsu T, Ichihara S, Price E, Jr., Gaffney FA, Inagami T. Evidence for angiotensin II type 2 receptor-mediated cardiac myocyte enlargement during in vivo pressure overload. J Clin Invest 2000; 106: R25–9.

[22] Sugino H, Ozono R, Kurisu S, Matsuura H, Ishida M, Oshima T, et al. Apoptosis is not increased in myocardium overexpressing type 2 angiotensin II receptor in transgenic mice. Hypertension 2001; 37: 1394–8. [23] Suzuki J, Iwai M, Nakagami H, Wu L, Chen R, Sugaya T, et al. Role of angiotensin II-regulated apoptosis through distinct AT1 and AT2 receptors in neointimal formation. Circulation 2002; 106: 847–53. [24] Weber KT. Extracellular matrix remodeling in heart failure: A role for de novo angiotensin II generation. Circulation 1997; 96: 4065–82. [25] Wenzel S, Taimor G, Piper HM, Schluter KD. Redox-sensitive intermediates mediate angiotensin II-induced p38 MAP kinase activation, AP-1 binding activity, and TGF-beta expression in adult ventricular cardiomyocytes. Faseb J 2001; 15: 2291–3. [26] Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 1996; 93: 156–60. [27] Yang Z, Bove CM, French BA, Epstein FH, Berr SS, DiMaria JM, et al. Angiotensin II type 2 receptor overexpression preserves left ventricular function after myocardial infarction. Circulation 2002; 106: 106–11. [28] Yano M, Kim S, Izumi Y, Yamanaka S, Iwao H. Differential activation of cardiac c-jun amino-terminal kinase and extracellular signal-regulated kinase in angiotensin II-mediated hypertension. Circ Res 1998; 83: 752–60.

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161 Bradykinin and Its Related Peptides DUNCAN JOHN CAMPBELL

likrein were activated by trypsin. The biological activity of submandibular kallikrein was found to be due to its enzymatic release of a low-molecular-weight thermostable compound from a serum protein. This low-molecular-weight compound, thought to be a polypeptide, was initially called DK and later called kallidin [26]. In the laboratory of Roche e Silva, bradykinin was initially identified as a hypotensive and smooth muscle– stimulating factor released from plasma globulin by snake venoms and by trypsin [24]. It was given the name bradykinin because it produced a slow movement of the gut [24]. Trypsin digestion of an ammonium sulfate fraction from ox serum produced the nonapeptide bradykinin; the sequence for this was reported by Elliott et al. in 1960 [10]. However, digestion of human plasma with human urinary kallikrein produced two peaks of bioactivity, one (named kallidin I) corresponding to bradykinin and a second (named kallidin II) corresponding to lysine-bradykinin [22]. Because human urinary kallikrein can hydrolyze synthetic arginine esters but not lysine esters, Pierce and Webster concluded that human urinary kallikrein produced lysinebradykinin (now known as kallidin) [22].

ABSTRACT The kallikrein kinin system (KKS) generates a family of bioactive peptides with varying biological activities. These include hydroxylated and nonhydroxylated bradykinin and kallidin (Lys0-bradykinin) peptides and their respective carboxypeptidase metabolites, des-Arg9bradykinin and des-Arg10-kallidin. Whereas bradykinin and kallidin act mainly via the bradykinin type 2 (B2) receptor, des-Arg9-bradykinin and des-Arg10-kallidin act mainly via the bradykinin type 1 (B1) receptor. Kinins are potent vasodilators, promote natriuresis and diuresis, and have beneficial cardiovascular effects; however, kinins may also promote inflammation. Peptidases are important determinants of kinin levels, and increased kinin levels may contribute to both the beneficial and adverse effects of peptidase inhibitor therapy.

DISCOVERY Bradykinin and its related peptides are generated by the action of kallikrein enzymes on kininogen substrates. Initial discoveries of the kallikrein kinin system (KKS) took place in the laboratories of Frey and Kraut in Germany (reviewed by Werle [26]) and Roche e Silva in Brazil [24]. In the laboratory of Frey and Kraut, kallikreinlike activity was initially revealed by the hypotensive effects of intravenous injection of urine [26]. It was also found that plasma or serum could inactivate this activity. Subsequent isolation of this hypotensive material in large amounts from the pancreas led to it being named kallikrein, although it was evident that pancreas was not the only source of kallikrein. Kallikrein could be extracted from the pancreas in both active and inactive forms. Kallikrein could also be isolated from plasma in both active and inactive forms, and this was named plasma kallikrein. Moreover, the inactive forms of kalHandbook of Biologically Active Peptides

STRUCTURE OF KININOGEN mRNA AND GENE Kininogens are glycoproteins that contain the bradykinin sequence in their midportion. There are two forms of kininogen that differ in structure, size, and susceptibility to cleavage by plasma and tissue kallikreins [2]. Human high-molecular-weight kininogen (HMWK) has 626 amino acids, with a mass of 88–120 kDa, whereas human low-molecular-weight kininogen (LMWK) has 409 amino acids, with a mass of 50–68 kDa [25]. The human kininogen gene encompasses 11 exons spanning

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1176 / Chapter 161 TABLE 1. Blood Kinin Peptide Levels in Humans.a

Kininogen gene 10BK

Exons 1-9

10HMW

LMWK mRNA

11

Peptide

HMWK mRNA

Exons 1-9 10BK 11

Exons 1-9

10BK

10HMW

poly A

poly A

LMWK Heavy chain BK Light chain 362 aa 9 aa 38 aa

HMWK Heavy chain 362 aa

BK 9 aa

Light chain 255 aa

FIGURE 1. Diagrammatic representation of differential splicing of the kininogen gene that produces high-molecularweight kininogen (HMWK) and low-molecular-weight kininogen (LMWK) mRNA. HMWK represents exons 1–9 spliced to the whole of exon 10, whereas LMWK represents exons 1–9 and the first 78 bp of exon 10 spliced to exon 11 [15, 25]. Both HMWK and LMWK contain the bradykinin (BK) sequence in their midportion, between their respective heavy and light chains. aa, amino acid. Not drawn to scale.

approximately 27 kb and codes for both HMWK and LMWK by differential splicing of the initial mRNA transcript (Fig. 1) [15, 25]. In the rat, T-kininogen (major acute-phase protein) is a potential precursor for Ile,Serbradykinin and Met,Ile,Ser-bradykinin [11]. However, there is no evidence that T-kininogen is a precursor of kinin peptides in vivo [6, 12].

Kallikreins Tissue kallikrein and plasma kallikrein are both serine proteases. Plasma kallikrein is initially secreted as the inactive prekallikrein, and tissue kallikrein is initially secreted as inactive prokallikrein. Both plasma prekallikrein and tissue prokallikrein are activated by serine protease activity. Whereas a single gene codes for plasma prekallikrein, there is a large family of tissue prokallikrein genes, although KLK1 is the only tissue kallikrein known to generate kinin peptides [27].

DISTRIBUTION OF KININOGEN AND KALLIKREIN mRNA AND KININ PEPTIDES Liver is the main site of kininogen production and, in humans, HMWK mRNA is also found in the kidney, pancreas, placenta, heart, and colon, whereas LMWK mRNA is found in the kidney, brain, placenta, testis, pancreas, thymus, heart, spleen, lung, colon, and small intestine [19]. Plasma prekallikrein mRNA is present in all tissues examined and is most abundant in the liver and pan-

BK(1–7) BK(1–8) BK(1–9) Hyp3-BK(1–7) Hyp3-BK(1–8) Hyp3-BK(1–9) KBK(1–7) KBK(1–8) KBK(1–9) Hyp3-KBK(1–7) Hyp3-KBK(1–8) Hyp3-KBK(1–9)

fmol/ml 1.4 (0.5–4.3) 0.08 (0.02–0.41) 0.18 (0.02–1.90) <0.14 0.53 (0.12–2.37) 0.18 (0.04–0.91) 0.46 (0.07–3.33) 0.10 (0.01–1.35) 0.22 (0.02–2.70) 0.04 (0.01–0.49) <0.03 0.04 (0.01–0.26)

Data shown as geometric mean (95% confidence interval), n = 19 for bradykinin (BK) peptides; n = 18 for kallidin (KBK) peptides. Hyp, hydroxyproline. Data from [7]. a

creas [19]. The KLK1 gene is highly expressed in the kidney, pancreas, salivary gland, endometrium, ovary, skin, and pituitary [8]. Other sites of KLK1 gene expression include the colon, heart, blood vessels, and adrenal [17, 20, 21]. Bradykinin peptides are present in all tissues studied (blood, heart, aorta, brown adipose tissue, adrenal, lung, and brain) [6]. In humans, circulating levels of kinin peptides are low, usually less than 3 fmol/ml (Table 1). Tissue levels of bradykinin are higher than blood levels in rats [6], and bradykinin and kallidin peptide levels are higher in venous than in arterial blood [3], consistent with tissue being the main site of formation of these peptides. Bradykinin peptides are more abundant than kallidin peptides in the blood and atrial tissue of humans [3, 7]. In urine, the levels of kinin peptides are several orders of magnitude higher than in blood, and kallidin peptides are more abundant than bradykinin peptides [3].

FORMATION OF KININ PEPTIDES In humans, plasma kallikrein forms bradykinin from HMWK, whereas tissue kallikrein forms kallidin from HMWK and LMWK (Figs. 2 and 3). By contrast, both plasma and tissue kallikrein generate bradykinin in rodents [2, 3]. Bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin. Kinins act via two types of bradykinin receptor, type 1 (B1) and type 2 (B2) receptors. Bradykinin and kallidin are more potent on the B2 receptor, whereas their carboxypeptidase (kininase I) metabolites des-Arg9-bradykinin and des-Arg10-kallidin, respectively, are also bioactive and more potent on the B1 receptor (Fig. 2) [23]. In humans, a proportion of kininogens is hydroxylated on Pro3 of

Bradykinin and Its Related Peptides / 1177 Kininogen gene

LMWK

Tissue kallikrein

HMWK

Nonhydroxylated hydroxylated

Tissue kallikrein Plasma kallikrein Aminopeptidase Kallidin KBK-(1-9) Hyp3-KBK-(1-9)

Carboxypeptidase

Bradykinin BK-(1-9) Hyp3-BK-(1-9)

Carboxypeptidase

Plasma kallikrein HMW & LMW kininogen -Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Leu-Met-Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ser0 1 2 3 4 5 6 7 8 9 B2 receptor agonists Bradykinin [BK-(1-9)] Kallidin [KBK-(1-9)] Hyp3-BK-(1-9) Hyp3-KBK-(1-9) B1 receptor agonists Bradykinin [BK-(1-8)] Kallidin [KBK-(1-8)]

B2 receptor

Hyp3-BK-(1-8)

KBK-(1-8) Hyp3-KBK-(1-8)

Aminopeptidase

Hyp3-KBK-(1-8)

BK-(1-8) Hyp3-BK-(1-8)

B1 receptor

FIGURE 2. Overview of the kallikrein kinin system in humans. LMWK, low-molecular-weight kininogen; HMWK, high-molecular-weight kininogen; Hyp, hydroxyproline. A proportion of kininogen is hydroxylated on the third amino acid (Hyp3) of the bradykinin sequence of the precursor. Thus, bradykinin peptides may be nonhydroxylated [BK(1–9)] or hydroxylated [Hyp3-BK(1–9)]. Kallidin is Lys0-bradykinin and may also be either nonhydroxylated [KBK(1–9)] or hydroxylated [Hyp3-KBK(1–9)]. Similarly, the carboxypeptidase metabolites des-Arg9-BK(1–9) and des-Arg9-KBK(1–9) may also be either nonhydroxylated [BK(1–8), KBK(1–8)] or hydroxylated [Hyp3-BK(1–8), Hyp3-KBK(1–8)].

the bradykinin sequence, leading to the formation of hydroxylated kinin peptides (Hyp3-bradykinin and Hyp4-kallidin). Hydroxylated kinins have similar biological activity to nonhydroxylated kinins [23]. Thus, there are eight bioactive kinin peptides in humans, hydroxylated and nonhydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites (Fig. 3). The proteins HMWK, plasma prekallikrein, and Factor XII are grouped together as the contact system because they require contact with artificial, negatively charged surfaces for zymogen activation in vitro. Recent studies indicate that tissue prokallikrein may also participate in the contact system by binding to HMWK [4]. In vivo, the contact system is assembled on endothelial and neutrophil cell membranes, where it may participate in basal kinin peptide production. In addition to kinin peptide formation, the contact system activates the intrinsic coagulation, complement, and fibrinolytic systems. There is little appreciation of how this system is activated in vivo.

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe

FIGURE 3. Amino acid sequences of the agonists of the type 1 (B1) and type 2 (B2) bradykinin receptors. Hyp, hydroxyproline. Bradykinin [BK(1–9)] and kallidin [Lys0bradykinin, KBK(1–9)] are excised from the interior of the kininogen molecules. Tissue kallikrein cleaves both HMWK and LMWK between Met379 and Lys380 (the same numbering for human HMWK and LMWK precursors) to produce the N-terminus and between Arg389 and Ser390 to produce the carboxy-terminus of kallidin. Plasma kallikrein cleaves HMWK between the Lys380 and Arg381 to produce the N-terminus and between Arg389 and Ser390 to produce the carboxy-terminus of bradykinin.

Kinin production in vivo is controlled in part by endogenous inhibitors of the kallikrein enzymes. The main inhibitors of plasma kallikrein are C1 inhibitor, α2-macroglobulin, and antithrombin III [2]. An important inhibitor of tissue kallikrein is kallistatin, although there is continuing uncertainty about the function of kallistatin in vivo [3]. Alternative pathways of kinin formation involving enzymes other than kallikreins may operate in disease states [3]. Although LMWK is a poor substrate for plasma kallikrein, it forms bradykinin in the presence of neutrophil elastase that, by cleaving a fragment from LMWK, renders it much more susceptible to cleavage by plasma kallikrein. Moreover, the combination of mast cell tryptase and neutrophil elastase releases bradykinin from oxidized kininogens, which are resistant to cleavage by kallikreins.

METABOLISM OF KININ PEPTIDES The metabolism of kinin peptides is described by Stewart and Gera in Chapter 63 of this volume. The efficiency of the metabolism of kinin peptides is an important determinant of their levels in blood and tissue. Angiotensin converting enzyme (ACE) plays an important role in

1178 / Chapter 161 bradykinin metabolism, producing bradykinin (1–5) [(BK(1–5)], the most abundant bradykinin metabolite [18], by sequential cleavage of dipeptides from the carboxy-terminus of bradykinin. Other peptidases have a more important role during ACE inhibition.

KININ RECEPTORS AND THEIR DISTRIBUTION IN THE CARDIOVASCULAR SYSTEM The B1 and B2 receptors are both G-protein-coupled, seven-transmembrane-spanning receptors that act through many different second messenger systems, in particular nitric oxide and prostaglandins [16]. Kinin receptors are described by Stewart and Gera in Chapter 63 of this volume.

Conformation In aqueous solution bradykinin is in rapid equilibrium between multiple conformations and does not show any persistent structural features [9].

Biological Actions of Kinin Peptides in the Cardiovascular System The role of kinins in amphibia is described by Conlon in Chapter 44, and in cancer is described by Stewart and Gera in Chapter 63, in this volume. Kinins are potent vasodilators and also promote diuresis and natriuresis. Kinins protect against ischemia-reperfusion injury by decreasing endothelial adherence of leukocytes, leading to the attenuation of postischemic leukocyte adherence, attenuation of disruption of the microvascular barrier, and reduced tissue injury [2]. Endogenous kinins may also have a role in the regulation of coronary vascular tone [3]. However, kinins also participate in the cardinal features of inflammation, producing vasodilatation, vascular permeability, neutrophil chemotaxis, and pain [2]. BK(1–5) has antithrombin activity [13]. However, it is unlikely that endogenous BK(1–5) levels inhibit thrombin in vivo because plasma BK(1–5) levels (30– 40 pmol/liter) [18] are at least six orders of magnitude below the micromolar concentrations required to inhibit thrombin [13].

ROLE OF KININ PEPTIDES IN HEALTH AND DISEASE STATES Some information about the role of kinin peptides in health and disease states is provided by genetic deficiencies of KKS components. Kininogen deficiency in

humans is relatively asymptomatic [3]. However, studies in kininogen-deficient rats and B1 and B2 receptor gene knockout mice provide evidence for an important role for kinin peptides in the regulation of blood pressure and sodium homeostasis, in renal response to vasopressin, in insulin sensitivity, and in contributing to inflammatory processes [3]. Given the vascular and renal actions of kinin peptides, the KKS has long been a candidate mechanism for essential hypertension. Recent genetic studies show an association between polymorphisms of the regulatory region of the KLK1 gene and hypertension among Chinese individuals [14]. However, heterozygous individuals with a loss-of-function polymorphism (R53H) of the KLK1 gene have normal blood pressure, although they do have an alteration of the geometry of the brachial artery [1]. However, as yet there is no evidence that these polymorphisms of the KLK1 gene influence kinin peptide levels. C1 inhibitor deficiency can result in unpredictable activation of the contact system, leading to angioedema and increased circulating bradykinin levels [3]. The activation of the contact system and kinin peptide formation also occurs during cardiopulmonary bypass [3] and may similarly occur in situations of systemic inflammation such as sepsis. Both ACE inhibitors and angiotensin type 1 receptor blockers increase kinin peptide levels [3, 7] that may contribute to the therapeutic effects of these agents. For example, B2 receptor antagonism attenuates the hypotensive effects of ACE inhibition in hypertensive humans [3]. However, increased kinin peptide levels may also contribute to the increased incidence of angioedema associated with these therapies [3, 7]. Kinin levels are increased further when neutral endopeptidase 24.11 (NEP) inhibition is combined with ACE inhibition. Consequently, the incidence of angioedema is higher with combined ACE and NEP inhibition than with ACE inhibition alone [5]. Patients with heart failure have reduced circulating levels of bradykinin and kallidin peptides [3]; this may account for the lower incidence of angioedema in heart failure subjects treated with ACE inhibitors or vasopeptidase inhibitors [5].

References [1] Azizi M, Boutouyrie P, Bissery A, Agharazii M, Verbeke F, Stern N, et al. Arterial and renal consequences of partial genetic deficiency in tissue kallikrein activity in humans. J Clin Invest. Mar 2005;115:780–7. [2] Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: Kallikreins, kininogens, and kininases. Pharmacol Rev. 1992; 44:1–80. [3] Campbell DJ. The kallikrein-kinin system in humans. Clin Exp Pharmacol Physiol. 2001;28:1060–5.

Bradykinin and Its Related Peptides / 1179 [4] Campbell DJ. The renin-angiotensin and the kallikrein-kinin systems. Int J Biochem Cell Biol. Jun 2003;35:784–91. [5] Campbell DJ. Vasopeptidase inhibition: A double-edged sword? Hypertension. Mar 2003;41:383–9. [6] Campbell DJ, Kladis A, Duncan A-M. Bradykinin peptides in kidney, blood, and other tissues of the rat. Hypertension. 1993;21:155–65. [7] Campbell DJ, Krum H, Esler MD. Losartan increases bradykinin levels in hypertensive humans. Circulation. 2005;111:315–20. [8] Clements JA, Willemsen NM, Myers SA, Dong Y. The tissue kallikrein family of serine proteases: Functional roles in human disease and potential as clinical biomarkers. Crit Rev Clin Lab Sci. 2004;41:265–312. [9] Denys L, Bothner-By AA, Fisher GH, Ryan JW. Conformational diversity of bradykinin in aqueous solution. Biochemistry. Dec 7 1982;21:6531–6. [10] Elliot DF, Lewis GP, Horton EW. The structure of bradykinin— a plasma kinin from ox blood. Biochem Biophys Res Commun. 1960;3:87–91. [11] Furuto-Kato S, Matsumoto A, Kitamura N, Nakanishi S. Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and a1-cysteine proteinase inhibitor. J Biol Chem. 1985;260:12054–9. [12] Gardes J, Baussant T, Corvol P, Ménard J, Alhenc-Gelas F. Effect of bradykinin and kininogens in isolated rat kidney vasoconstricted by angiotensin II. Am J Physiol. 1990;258:F1273– F81. [13] Hasan AAK, Amenta S, Schmaier AH. Bradykinin and its metabolite, Arg-Pro-Pro-Gly. Phe, are selective inhibitors of athrombin-induced platelet activation. Circulation. 1996;94:517– 28. [14] Hua H, Zhou S, Liu Y, Wang Z, Wan C, Li H, et al. Relationship between the regulatory region polymorphism of human tissue kallikrein gene and essential hypertension. J Hum Hypertens. Apr 28 2005;19:715–21. [15] Kitamura N, Kitagawa H, Fukushima D, Takagaki Y, Miyata T, Nakanishi S. Structural organization of the human kininogen gene and a model for its evolution. J Biol Chem. Jul 15 1985;260:8610–17. [16] Leeb-Lundberg LM, Marceau F, Muller-Esterl W, Pettibone DJ, Zuraw BL. International union of pharmacology. XLV. Classifi-

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cation of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol Rev. Mar 2005;57:27–77. Meneton P, Bloch-Faure M, Hagege AA, Ruetten H, Huang W, Bergaya S, et al. Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice. Proc Natl Acad Sci USA. 2001;98:2634–9. Murphey LJ, Eccles WK, Williams GH, Brown NJ. Loss of sodium modulation of plasma kinins in human hypertension. J Pharmacol Exp Ther. Mar 2004;308:1046–52. Neth P, Arnhold M, Nitschko H, Fink E. The mRNAs of prekallikrein, factors XI and XII, and kininogen, components of the contact phase cascade are differentially expressed in multiple non-hepatic human tissues. Thromb Haemost. Jun 2001;85:1043– 7. Nolly H, Carbini LA, Scicli G, Carretero OA, Scicli AG. A local kallikrein-kinin system is present in rat hearts. Hypertension. 1994;23:919–23. Nolly H, Saed G, Carretero OA, Scicli G, Scicli AG. Adrenal kallikrein. Hypertension. 1993;21:911–15. Pierce JV, Webster ME. Human plasma kallidin: Isolation and chemical studies. Biochem Biophys Res Commun. 1961;5:353– 7. Regoli D, Rhaleb N-E, Drapeau G, Dion S, Tousignant C, D’Orléans-Juste P, et al. Basic pharmacology of kinins: Pharmacologic receptors and other mechanisms. Adv Exp Med Biol. 1989;247A:399–407. Roche e Silva M, Beraldo WT, Rosenfeld G. Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am J Physiol. 1949;156:261–73. Takagaki Y, Kitamura N, Nakanishi S. Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens. J Biol Chem. Jul 15 1985;260:8601–9. Werle E. Discovery of the most important kallikreins and kallikrein inhibitors. In: Erdos EG, ed. Handbook of Experimental Pharmacology, Volume 25: Bradykinin, Kallidin and Kallikrein. Berlin: Springer-Verlag; 1970:1–6. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: Structure, function, and association to disease. Endocr Rev. 2001;22:184–204.

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162 Calcitonin Gene-Related Peptides CHRISTINA W. L. TAM AND SUSAN D. BRAIN

chromosome 11, CALC I forming calcitonin and αCGRP, and CALC II forming βCGRP [2]. The two isoforms share greater than 90% homology and differ in only 1 (rat) and 3 (human) amino acids [52]. αCGRP and βCGRP are believed to have similar biological activities with a substantially greater predominance of αCGRP. The αCGRP isoform is more abundantly distributed in the central and peripheral nervous systems compared with βCGRP. However, βCGRP is found in the enteric nerves [46]. Consequently, most research has been carried out with αCGRP, and thus, in the course of this discussion, αCGRP is referred to as CGRP. CGRP belongs to a family that includes adrenomedullin, most usually localized to nonneuronal tissues, such as vascular tissues [41] and amylin that is produced in the pancreas [14]. They share 25–50% structural homology and some overlapping biological activities [5]. CGRP has a wide distribution throughout the central and peripheral nervous systems and is usually colocalized with other peptides, including substance P, in C-fiber neurons [39]. Basal plasma concentrations for CGRP have been found to be in the low picomolar range (mean = 44 pM) in healthy volunteers. However, in the circulation CGRP has a half-life of less than 10 minutes [35]. Although CGRP is abundant in the nervous system, it is commonly found in the cardiovascular system, with greater occurrence around the arteries than the veins. CGRP has also been detected in the heart, with higher levels found in the atria than the ventricles [4]. The study of CGRP receptors has revealed a unique family of receptors. Pharmacological studies led originally to the classification of the receptors as CGRP1 and CGRP2. These receptors were distinguished by their sensitivity to the CGRP receptor antagonist, CGRP8–37 [10]. The CGRP1 receptor is antagonized by CGRP8–37 in the guinea pig atria, whereas CGRP8–37 is considerably less effective at antagonizing the effects of CGRP

ABSTRACT Calcitonin gene-related peptide (CGRP) is a 37amino-acid peptide that belongs to a family of structurally related peptides that include adrenomedullin and amylin. CGRP is predominantly found in sensory nerves. These nerves innervate blood vessels, particularly those found at the arteriolar level. CGRP is a potent vasodilator, especially in the microvasculature. CGRP acts via its own family of receptors consisting of a G-protein-linked binding site (CL) and one of three single-transmembrane-spanning proteins (RAMPs). CGRP circulates at low levels in normal individuals, but has been detected at increased levels in disease states (e.g., migraine). Thus CGRP, as a consequence of its unique receptor structure and potent vasodilator ability, is a key peptide within the cardiovascular system.

CALCITONIN GENE-RELATED PEPTIDE In 1982, an alternative peptide from the calcitonin gene was discovered in the thyroid of aging rats [3] and 2 years later in medullary thyroid carcinoma tissue in humans [45]. The alternative peptide, named calcitonin gene-related peptide (CGRP), was produced by alternative splicing of the calcitonin mRNA transcript [3]. Thus, the calcitonin mRNA is formed of exons 1, 2, 3, and 4 of the calcitonin gene, and the CGRP mRNA is formed from exons 1, 2, 3, 5, and 6, as shown in Fig. 1 [57]. CGRP immunoreactivity was found localized to sensory C and Aδ fibers in both the central and peripheral nervous systems in a range of species. It soon became established as a sensory neuropeptide [46]. CGRP is known to exist in two forms, named αCGRP (or CGRPI) and βCGRP (or CGRPII). Although these two forms have similar biological activities, they are formed from two distinct genes at different sites on Handbook of Biologically Active Peptides

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Exon 3

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in the rat vas deferens, where the CGRP2 receptor is considered to be found [15]. Interestingly, the CGRP linear analog [acetamidomethyl-Cys2,7]hCGRP ([Cys(ACM)2,7]hCGRP), an agonist, was far more effective at the rat vas deferens than the guinea pig atria [16]. This gave rise to the possibility of different CGRP receptor subtypes in different tissues [48]. Another linear analog, [Cys(Et)2,7]hCGRP, was identified [18], which possesses greater potency (ED50 = 3.4 ± 1.2 nM) than the original linear analog (ED50 = 82 ± 7.5 nM) for the CGRP2 receptor [18]. However, the [Cys(ACM)2,7]hCGRP agonist is still used in many studies [44], often in comparison with the ethylamide analog. Despite the use of proposed selective ligands to classify the CGRP2 receptor, the knowledge of the relevance of this receptor remains poor [49], and it has not been defined at the molecular level. The CGRP1 receptor is now considered primarily as the important cardiovascular receptor. The CGRP family of receptors consists of a seventransmembrane G-protein-coupled calcitonin receptorlike receptor (CL), important for ligand binding, complexed with one of three single-membrane-spanning receptor-activity-modifying proteins (RAMP) [42], essential for the phenotype of the receptor and its location at the cell surface. This heterodimer complex is, in addition, linked to a CGRP receptor component (RCP), which is suggested to enhance receptor coupling and activation [21]. There are three RAMPs (RAMP1, RAMP2, and RAMP3). CL, when present with RAMP1, is a CGRP receptor that is selectively blocked by BIBN4096BS and/ or CGRP8–37. CL with RAMP2 produces an adrenomedullin (AM) receptor that is blocked by AM22–52. CL with RAMP3 leads to a mixed CGRP-AM receptor, about which least is known. Vascular relaxation occurs through the activation of the CGRP receptor (CL/RAMP1). This can occur via a range of reported mechanisms that include nitric oxide (NO)-dependent endothelium-dependent mechanisms or cAMP endothelium-independent pathways. The latter mechanism appears common in the microcirculation. The rise in [cAMP]i stimulates protein kinase A (PKA), phosphorylation, and the opening of potassium channels. David Poyner and colleagues have systemati-

3’

FIGURE 1. Arrangement of exons that form either calcitonin or CGRP mRNA. Adapted from [57].

cally analyzed the pharmacology of these receptors in cell systems [29]. Expression of the CL has been found in many human tissues, including the endothelium of many types of blood vessels, heart muscle, and alveolar capillaries [27]. In addition, CL was also detected in human hairy skin, arteriolar smooth muscle, and venular endothelium [26]. Since their discovery, the RAMPs have been found to be expressed in a vast number of tissue types. Many studies have examined the gene, mRNA and protein expression of RAMPs. The results suggest that the RAMPs are an abundant protein, although the distribution of the separate RAMPs varies in different tissues. High gene expression of RAMP1 was found in the uterus, bladder, brain, pancreas, and gastrointestinal tract, whereas the gene expression of RAMP2 and -3 was more strongly expressed in the lung, breast, immune system, and fetal tissues [42]. A key method in learning more about receptors is through the use of antagonists, of which there are highly specific agents for the CGRP receptor. The only potent small-molecule CGRP antagonist described to date has been BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2oxoethyl]- 4 -[1,4 - dihydro -2 - oxo - 3(2H)-quinazolinyl]-, [R-(R*,S*)]. It is a potent competitive antagonist of the human and marmoset CGRP1 receptor [17] and has a 200-fold greater affinity in human than in rodent tissues. RAMP1 is important for determining species selectivity for this antagonist through a single amino acid residue (tryptophan at position 74) [40]. A report about a structurally similar substance, compound 1, (4-(2-Oxo-2,3dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenylpiperazin-1-yl)-ethyl]-amide) has also been published. This is a weak antagonist of vascular responses mediated via human and guinea pig CGRP receptors [19]. A further nonpeptide CGRP1 receptor antagonist has been described. SB-273779 [N-methyl-N-(2methylphenyl)-3-nitro-4-(2-thiazolylsulfinyl) nitrobenzanilide] is much weaker than BIBN4096BS in human tissues, but acts in a non-species-dependent manner to displace CGRP [1].

Calcitonin Gene-Related Peptides / 1183 In addition to its specificity for human CGRP receptors, BIBN4096BS has also been shown to exhibit receptor subtype specificity. In the rat left atrium, BIBN4096BS was shown to be 10-fold more potent at inhibiting CGRP-induced responses than in the rat vas deferens [59]. This suggests that the two tissues display different receptor populations and the fact that BIBN4096BS is more effective at one and not the other further supports the theory of the existence of CGRP1 and CGRP2 receptor subtypes [18, 56]. The CGRP1 and CGRP2 receptor terminology remains a widely used term, although the existence of the subtypes remains under heavy debate and molecular identification is awaited [49]. CGRP has a number of biological activities, but it is best known as a vasodilator that is active at all levels of the cardiovascular system but with potent and longlasting effects in the microcirculation. This is clearly observed in skin, where the intradermal injection of femtomolar amounts leads to an increased blood flow and of picomolar doses causes increased skin blood flow that lasts several hours [7]. It was realized that CGRP, probably as a consequence of its vasodilator activity, potentiated the ability of substance P to increase microvascular permeability and thus edema formation [6]. CGRP, when given intravenously, causes facial flushing at nonhypotensive doses and at greater doses induces hypotension [22]. Therefore, it is generally considered that the major activity of CGRP is local to the site of release. CGRP, perhaps surprisingly, does not appear to play an important role in the physiological regulation of blood pressure, although it is suggested that it is involved in the pathophysiological vascular aspects of a number of diseases, including Raynaud’s disease and migraine [5]. Plasma levels of CGRP are in the low picomolar level in normal volunteers [23], but have shown to be increased in diseases, of which migraine is notable [25]. Interestingly, CGRP has also been shown to have protective effects in a number of heart conditions, including ischemia [38]. The protective effect that CGRP seems to possess is thought to be due to its vasodilator ability, which allows increased blood flow to the affected area. Interestingly, CGRP is related to the vascular peptide, adrenomedullin (AM), as a consequence of structural homology and receptor components (as shown in Fig. 2). AM consists of 52 amino acids and has approximately 24% structural homology with CGRP [33]. CGRP and AM are both potent microvascular vasodilators and so influence many important processes such as inflammation; there is also evidence for a role of CGRP in wound healing [34]. AM, like CGRP, appears to exhibit its most potent activities within the microvasculature. The administration of AM also causes facial flushing at doses where blood pressure remains unchanged,

1

2 RCP

3 G protein

FIGURE 2. Diagrammatic representation of CLR (shown in blue) and RAMPs (shown in yellow). The dashed blue lines indicate the possible dimerization of either of the RAMPs with the CLR. The diamonds indicate intracellular signaling downstream of the receptor, where RCP indicates receptor component protein and the G-protein is most commonly Gαs.

showing that the microvasculature is more sensitive than the vasculature to AM [43]. The main difference between the microvascular activity of AM and CGRP is their comparative potency difference to one another. The potency of AM is significantly less than CGRP, by 3- to 10-fold in the rat skin [13], by 20-fold in the hamster cheek pouch [28], and by approximately 300fold in the mouse mesentery [54]. In consideration of their overlapping biological activities, it is therefore not surprising that, in some tissues, AM is able to act via either CGRP or AM receptors [28, 30] and that AM-mediated effects can be inhibited by CGRP antagonists. For example, in the rat cerebral arteries [36], the AM response can be inhibited by the CGRP antagonist CGRP8–37, suggesting that AM acts via the CGRP receptor. However, in the anesthetized rat the AM response was insensitive to CGRP receptor antagonism [31]. In other tissues, such as the human coronary artery [55], the AM response is antagonized by the AM receptor antagonist AM22–52 [20], implying that in this case AM is acting via the AM receptor. Another peptide in the same family is amylin, a 37amino-acid peptide that shares approximately 50% structural homology with CGRP [58] and is generally believed to have vasodilator actions [11]. Amylin is secreted from the pancreas and is largely involved in the metabolism of carbohydrates and in inhibition of glycogen synthesis; thus, it may be of some relevance in diabetes mellitus [50]. Amylin has also been linked to the calcitonin receptor because amylin can bind to the calcitonin receptor in vitro; thus, it was hypothesized that amylin may be another endogenous ligand for the calcitonin receptor [12]. Raynaud’s phenomenon refers to an extreme pain syndrome that occurs in the digits, most commonly in

1184 / Chapter 162 the fingers, which can lead to prolonged ischemia and subsequent amputation if left untreated. It was shown that infusion of CGRP led to increases in blood flow in the face and hands in studies performed in patients suffering from Raynaud’s phenomenon, whereas normal subjects only showed increases in blood flow in the face. This suggested that either a super-sensitivity to CGRP may exist in Raynaud’s sufferers or that endogenous levels of CGRP may be unusually low [51]. Deficient levels of CGRP were shown in the digits of Raynaud’s sufferers compared with normal subjects as well as raised levels of circulating endothelin 1 (ET-1) [8, 9]. Interestingly, at room temperature the response to infusion of CGRP was similar in normal subjects and Raynaud’s patients; however, at 5°C the response to CGRP was significantly less in Raynaud’s patients than in normal subjects, as shown by planimetry [8] and laser Doppler flowmetry. These studies suggest that it may be a combination of increased sensitivity to ET-1 and decreased sensitivity to CGRP that lead to the onset of Raynaud’s phenomenon. Therefore, alongside ET-1 antagonists [32], CGRP agonists (or AM1 agonists) could be employed or CGRP genes administered via gene therapy to alleviate the symptoms of Raynaud’s phenomenon. The most exciting disease-related studies to date for CGRP have been associated with migraine. Migraine is common, experienced by up to 15% of the population at some point, and is an extremely debilitating primary headache characterized by a unilateral throbbing pain with a range of associated symptoms. CGRP is commonly found in the trigeminovascular system where it has potent effects. It is important that BIBN4096BS was shown to inhibit neurogenic vasodilatation in animal models of relevance to migraine [17]. The administration of CGRP is associated with a migraine-type headache [37]. Indeed, it has been known for some time that CGRP levels are increased in blood sampled from the draining jugular vein of patients with migraines (92 pM compared with 40 pM control values), ipsilateral to the attack [25]. This evidence was used in the 1990s to strengthen the hypothesis that migraine possesses an important sterile neurogenic inflammatory component, in keeping with the finding that treatment with the 5-HT1B/1D agonist sumatriptan reduced the release of CGRP in animal models of migraine as well as in migraine patients [24]. It is suggested that intracranial extracerebral blood vessels (e.g., middle meningeal artery and its dural arterioles) that supply the dura mater relax and that this is pivotal in mediating the pain following stimulation of perivascular sensory nociceptive nerve fibers. However, it is becoming increasingly less likely that CGRP is acting purely to facilitate mechanisms via its vasodilator activity. There is evidence for a neuronal site of action for CGRP [53]. It is exciting

that the small-molecule CGRP antagonist BIBN4096BS, given by the intravenous route, has been shown to be successful in phase II clinical trials [47]. It is perhaps surprising, considering the large number of nonpeptide substance P NK1 antagonists that are available that more nonpeptide CGRP antagonists are not available. However, it appears, from the experience of a number of pharmaceutical companies, that it is difficult to develop an orally available high-affinity antagonist for this receptor. With the advent of new receptor antagonists, the full scale of the capabilities of CGRP may soon become clear. Further research into its novel receptor structure may lead to additional sites where CGRP is important. In summary, CGRP has wide-ranging activity and, due to its potency, is a critically important peptide within the cardiovascular system.

Acknowledgments We thank the British Heart Foundation for financial support.

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[28] Hall JM, Siney L, Lippton H, Hyman A, Kang-Chang J, Brain SD. Interaction of human adrenomedullin 13-52 with calcitonin gene-related peptide receptors in the microvasculature of the rat and hamster. Br J Pharmacol 1995; 114(3): 592–7. [29] Hay DL, Conner AC, Howitt SG, Takhshid MA, Simms J, Mahmoud K et al. The pharmacology of CGRP-responsive receptors in cultured and transfected cells. Peptides 2004; 25(11): 2019–26. [30] Hay DL, Smith DM. Knockouts and transgenics confirm the importance of adrenomedullin in the vasculature. Trends Pharmacol Sci 2001; 22(2): 57–9. [31] Haynes JM, Cooper ME. Adrenomedullin and calcitonin generelated peptide in the rat isolated kidney and in the anaesthetised rat: in vitro and in vivo effects. Eur J Pharmacol 1995; 280(1): 91–4. [32] Herrick AL. Treatment of Raynaud’s phenomenon: new insights and developments. Curr Rheumatol Rep 2003; 5(2): 168–74. [33] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H et al. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993; 192(2): 553–60. [34] Kjartansson J, Dalsgaard CJ. Calcitonin gene-related peptide increases survival of a musculocutaneous critical flap in the rat. Eur J Pharmacol 1987; 142(3): 355–8. [35] Kraenzlin ME, Ch’ng JL, Mulderry PK, Ghatei MA, Bloom SR. Infusion of a novel peptide, calcitonin gene-related peptide (CGRP) in man. Pharmacokinetics and effects on gastric acid secretion and on gastrointestinal hormones. Regul Pept 1985; 10(2–3): 189–97. [36] Lang MG, Paterno R, Faraci FM, Heistad DD. Mechanisms of adrenomedullin-induced dilatation of cerebral arterioles. Stroke 1997; 28(1): 181–5. [37] Lassen LH, Haderslev PA, Jacobsen VB, Iversen HK, Sperling B, Olesen J. CGRP may play a causative role in migraine 818. Cephalalgia 2002; 22(1): 54–61. [38] Lechleitner P, Genser N, Mair J, Dienstl A, Haring C, Wiedermann CJ et al. Calcitonin gene-related peptide in patients with and without early reperfusion after acute myocardial infarction. Am Heart J 1992; 124(6): 1433–9. [39] Lundberg JM, Franco-Cereceda A, Hua X, Hokfelt T, Fischer JA. Co-existence of substance P and calcitonin gene-related peptidelike immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin. Eur J Pharmacol 1985; 108(3): 315–19. [40] Mallee JJ, Salvatore CA, LeBourdelles B, Oliver KR, Longmore J, Koblan KS et al. Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists. J Biol Chem 2002; 277(16): 14294–8. [41] Marutsuka K, Hatakeyama K, Sato Y, Yamashita A, Sumiyoshi A, Asada Y. Immunohistological localization and possible functions of adrenomedullin. Hypertens Res 2003; 26 Suppl: S33–S40. [42] McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; 393(6683): 333–9. [43] Meeran K, O’Shea D, Upton PD, Small CJ, Ghatei MA, Byfield PH et al. Circulating adrenomedullin does not regulate systemic blood pressure but increases plasma prolactin after intravenous infusion in humans: A pharmacokinetic study. J Clin Endocrinol Metab 1997; 82(1): 95–100. [44] Moreno MJ, Terron JA, Stanimirovic DB, Doods H, Hamel E. Characterization of calcitonin gene-related peptide (CGRP) receptors and their receptor-activity-modifying proteins (RAMPs) in human brain microvascular and astroglial cells in culture. Neuropharmacology 2002; 42(2): 270–80.

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C

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163 Endothelins KATSUTOSHI GOTO

stimulated by thrombin and suggested that this EDCF is a peptide with molecular mass of ∼3000 kDa [7]. We performed similar experiments and confirmed that the supernatant from confluent monolayer cultures of porcine aortic endothelial cell contained a slowly developing and long-lasting vascular constricting factor(s), peptide in nature, because the vascular constricting activity was abolished by pretreatment of the conditioned medium with trypsin. The activity was also detected in serum-free conditioned medium, and no appreciable change in activity was found even after long-term (2–3 weeks) maintenance of the endothelial cell culture in serum-free condition. The successful attempt at serum-free maintenance and detection of vascular constricting activity prompted us to isolate and purify the active peptide in the supernatant because of the absence of interference with proteins and/or peptides in the serum itself. Because it was originally discovered from the culture supernatant of endothelial cells, the peptide was termed endothelin [12].

ABSTRACT A potent vasoconstrictor peptide, endothelin (endothelin-1), was discovered from culture supernatant of porcine endothelial cells in 1988. From human genomic DNA analysis, two other family peptides, endothelin-2 and endothelin-3, were found. They showed different effects and distribution, suggesting that each peptide may play separate roles in different organs. Endothelins act via the activation of two receptor subtypes, ETA and ETB, both of which are coupled to various guanosine triphosphate (GTP)-binding proteins depending on cell types. Although endothelin-1 plays no appreciable role in maintaining blood pressure in normal conditions, it is significantly implicated in causing or worsening cardiovascular diseases when production is increased.

DISCOVERY Vasoconstriction dependent on or enhanced by intact endothelium has been observed in response to various chemical and physical stimuli, such as norepinephrine, thrombin, hypoxia, increased transmural pressure, and mechanical stretch. These observations lead to the speculation that endothelial cells might release certain vascular constricting substance(s), endothelium-derived contracting factors (EDCF). In 1985, Hickey et al. attempted to test the biological activity of the culture medium of bovine aortic endothelial cells on isolated porcine coronary arteries and found that the culture supernatant contained peptidelike factor(s) that triggered a slowly developing and longlasting contraction of the coronary arteries [4]. Gillespie et al. also detected vascular constricting activity in the culture supernatant of porcine aortic endothelial cells. They subsequently reported that the activity was increased when the cultured endothelial cells were Handbook of Biologically Active Peptides

STRUCTURE OF THE PRECURSOR mRNA/GENE AND PROCESSING Preproendothelin and Intracellular Processing Based on the amino acid residues 7–20 of endothelin, several kinds of oligonucleotide were synthesized according to the mammalian codon use statistics. Using one of the synthesized oligonucleotides as a single optimal DNA probe, ∼2 × 106 clones were screened from a λ gt10 cDNA library constructed for porcine endothelial cell mRNA and a complete nucleotide sequence of porcine preproendothelin cDNA and the deduced amino acid sequence were determined. The 203-residue porcine preproendothelin contains 19 residues of deduced N-terminal amino acid sequence,

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FIGURE 1. The biosynthesis intracellular processing of endothelin 1 (ET-1) from prepro ET-1. Endothelin-converting enzyme (ECE) specifically catalyzes the conversion of big ET-1 to ET-1. CysCys represents intramolecular disulfide bond.

characteristic of a secretory signal sequence, that is, a hydrophobic core followed by residues with small polar side chains. As anticipated, paired basic amino acid residues Lys51-Arg52, which are recognized by the usual processing endopeptidase, furin, directly precede the

endothelin sequence, but no dibasic pair is found thereafter until Ara92-Arg93. This indicates that the intermediate peptide of 39 amino acids, called big endothelin (big ET) (Cys53-Arg92), may be generated first and that subsequently mature endothelin may be generated via

Endothelins unusual proteolytic processing between Try73 and Val74 in big endothelin by an endopeptidase exhibiting chymotrypsinlike specificity, termed endothelin-converting enzyme (ECE). This presumptive pathway of endothelin biosynthesis in endothelial cells was confirmed later. The human preproendothelin cDNA was soon cloned, and its sequence of 212 amino acid residues containing big endothelin with 38 amino acid sequences was determined (Fig. 1). The cDNA of ECE was also cloned, and it was revealed that ECE is a 785-amino-acid metalloprotease containing a single-membrane-spanning sequence with only a 56-residue N-terminal cytoplasmic tail and an extracellular C-terminal of 681 amino acid residues that contains the catalytic domain [11]. Amino acid residues 593–601 match the highly conserved consensus sequence of a zinc-binding motif, HEXXH, which is shared by many known metalloproteases. The ECE protein has 10 predicted sites for N-glycosylation in the extracellular domain, suggesting that ECE behaves as a highly glycosylated protein.

Endothelin Family Peptides and mRNA Distribution Southern blot analysis of human genomic DNA under low hybridization stringency with a 42-mer synthetic oligonucleotide probe corresponding to amino acid residues 7–20 of endothelin, demonstrated that

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three different restriction fragments were always detected, regardless of the restriction endonucleases used. The nucleotide sequences encoding amino acid residues of the three endothelins are highly conserved among the three genes, with 77–82% of the nucleotide residues being identical. In contrast, the nucleotide sequences upstream from the mature peptides are very poorly conserved. The three peptides were designated endothelin 1, 2, and 3 [5]. Endothelin 1 is the original peptide corresponding to that detected in the culture medium of porcine aortic endothelial cells. Although vascular endothelial cells are the major source of endothelin 1, Northern blot analysis revealed that the genes encoding the three endothelin isopeptides are expressed with different patterns in a wide variety of cell types, including cardiac myocytes, vascular smooth muscle cells, pituitary cells, macrophages, and mast cells, suggesting that the peptides may participate independently in complex regulatory mechanisms in various organs (Fig. 2).

PLASMA CONCENTRATION AND INACTIVATION OF ENDOTHELIN 1 Because endothelin 1 was discovered first and showed a wide variety of actions not only on the cardiovascular system but also on various other tissues, more information has accumulated on endothelin 1 compared with

FIGURE 2. Northern blot analysis of mRNA of endothelin 1, 2, and 3 and β-actin. Poly(A)RNA was extracted from various human tissues, and 8 μg of each RNA was subjected to electrophoresis and subsequent Northern blot analysis using each cDNA as the probe. (In the case of endothelial cells, total RNA was employed.)

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the two other peptides. The method for measurement of endothelin 1 levels in plasma or various tissues by means of radioimmunoassay (RIA) or enzyme immunoassay (EIA) [10] was established soon after the discovery of endothelin 1. At an early stage, the reported plasma levels of endothelin 1 in humans were fairly divergent, ranging from 0.2 to 18.5 pg/ml in healthy subjects. In order to avoid unnecessary confusion, the reason for the difference in the values of plasma endothelin 1 level was examined thoroughly and, as a result, it was generally agreed that the divergence originated mostly from differences in the specificity of the antibodies used and in the recovery rate during extraction of endothelin 1 from plasma or tissues. It is accepted now that the plasma concentration of endothelin 1 in healthy subjects is 1.0 to ∼2.0 pg/ml, which is at least one order of magnitude lower than that of circulating human natriuretic peptides and several times less than that of angiotensin II. The plasma levels of endothelin 1 increase drastically in the case of various cardiovascular diseases. Endothelin 1 may be released in both luminal and abluminal directions from endothelial cells in vivo. Luminal-released endothelin 1 may be diluted by the bloodstream, and its circulating concentration (1.0 to ∼2.0 pg/ml) is below the threshold concentration producing vasoconstriction. Although the exact concentration in the abluminary space (vascular smooth muscle surface) is not known, endothelin 1 is more likely to be a locally acting than a circulating hormone. When endothelin 1 production and secretion are increased drastically in certain places at pathophysiological conditions, severe local vasoconstriction might be anticipated. When endothelin 1 is intravenously administered as a bolus, it disappears quite rapidly from the bloodstream with a half-life of a few minutes. This rapid removal of endothelin 1 from the circulation is due to its uptake into various tissues, including the lung, kidney, spleen, and liver. The lung appears to be one of the most important tissues for uptake because approximately 60% of endothelin 1 is removed after a single passage through the pulmonary circulation. In lung tissues, ETB receptor is highly expressed and endothelin 1–ETB receptor complex may be taken up into cells through an internalization process and degraded by lysozomal enzymes (e.g., aspartic proteases). Neutral endopeptidase and lysozomal cathepsin G are also concerned with enzymatic degradation of endothelin 1. In spite of such a rapid disappearance from the circulation, the blood pressure–elevating effect of endothelin 1 continues for an extremely long period; the stabilization of the endothelin 1–ETA receptor complex may contribute, but the exact reason for this phenomenon is unknown (Fig. 3).

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FIGURE 3. Disappearance curve of 125I-endothelin 1 from circulation (upper curve) and blood pressure response to intravenous injection of endothelin 1. Under urethane anesthesia, a small amount of 125I-endothelin 1 with cold endothelin 1 (1.5 nmol/kg) was injected from femoral vein, a 0.3-ml blood sample was collected at appropriate intervals after the injection through a catheter previously inserted into the right atrium, and the radioactivity was measured. The blood pressure was measured in a separate rat at the same condition.

RECEPTORS AND THEIR DISTRIBUTION Endothelin-induced responses can be divided into two major groups. In the first group of responses, including vascular constriction, broncoconstriction, uterine smooth muscle constriction, and stimulation of aldosterone secretion, endothelin 1 and endothelin 2 act as far more potent agonists than endothelin 3. In the second group, which includes endotheliumdependent vascular relaxation, astrocyte proliferation, and inhibition of ex vivo platelet aggregation, the three isopeptides possess almost equal potency. These findings suggest that there are at least two distinct endothelin receptor subtypes. The existence of multiple receptors was also supported by biochemical studies, for example, cross-linking experiments and a radioligand binding affinity study. Subsequently, two cDNAs encoding for endothelin receptors were cloned and their amino acid sequences were deduced [1, 9]. The order of affinity of the three endothelin isopeptides for one receptor type, designated ETA, is endothelin 1 ⭌ endothelin 2 >> endothelin 3. The other type of receptor,

Endothelins designated ETB, shows equipotent affinity for all three endothelins. These results are consistent with the results of previous pharmacological and biochemical studies and, thus, definite molecular evidence for the existence of two distinct subtypes of endothelin receptors was provided. The existence of more subtypes of endothelin receptor has been a matter of controversy. Much pharmacological evidence has accumulated to suggest that there may exist an ETC receptor specific for endothelin 3 or further subsubtypes of ETA and ETB receptors. However, there has been no report thus far on isolation of the cDNA clone encoding for such a subtype or subsubtype of endothelin receptor from mammalian tissues. One possible explanation is posttranslational modification of ETA or ETB receptor proteins, but in order to solve such discrepancies further pharmacological, biochemical, or molecular biological analysis may be necessary. In addition to causing constriction of vascular tissue, endothelin isopeptides exert pleiotropic effects on nonvascular tissues, for example, the respiratory and gastrointestinal tissues, kidney, endocrine glands, and peripheral and central nervous systems. Each isopeptide seems to play specific physiological and/or pathophysiological roles independently in various tissues. The pharmacological effects of endothelin 1 on various

tissues and distribution of ETA and ETB receptors is collectively shown in Table 1.

BIOLOGICAL ACTIONS IN THE CARDIOVASCULAR SYSTEM Endothelin 1 causes extremely potent and longlasting vasoconstriction in most mammalian species, including humans, both in vivo and in vitro. The in vivo hemodynamic responses to intravenously injected endothelin 1 are complex and, depending on the vascular bed, include both direct vasoconstriction and indirect endothelium-mediated vasodilatation and reflex-mediated responses. In most arterial and some venous smooth muscle cells, endothelin 1 causes constrictor responses via stimulation of ETA receptors on the cell membrane. In contrast, endothelin 1 stimulates ETB receptors on endothelial cells and the release endothelium-derived relaxing factor (EDRF; nitric oxide), thereby producing vasodilatation. In cardiac muscle cells, endothelin 1 produces positive inotropic and chronotropic responses via the stimulation of mostly ETA receptors. Both ETA and ETB receptors are coupled to various GTP-binding proteins (Gq, Gs, Gi, Go, etc.), suggesting that the downstream signal

TABLE 1. Effects of Endothelin 1 on Various Tissues and Distribution of ETA and ETB Receptors.a Tissue (Effect) Vascular tissues Arterial smooth muscle (contraction) Venous smooth muscle (contraction) Endothelial cell (release of EDRF) Cardiac muscle (Positive inotropism) (Positive chronotropism) Coronary vessel (contraction) Nonvascular smooth muscle (contraction) Airway, gastrointestinal tract, bladder, uterus, etc. Lung (clearance receptor) Other than airway and vascular smooth muscle Endocrine glands Adrenal cortex (release of aldosterone) Anterior pituitary (release of ACTH and prolactin) Ovary (stimulation of female hormone synthesis) Kidney Afferent and efferent arterioles (contraction) Renal tuble (Na+ excretion and diuresis) Mesangial cell (contraction and proliferation) Central nervous system Mesencephalon (control of blood pressure) Astrocyte (proliferation)

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1192 / Chapter 163 transduction may differ in various cells depending on the type of GTP-binding proteins coupled. In addition to vasoconstrictor actions (acute effects), endothelin 1 exerts potent mitogenic actions on vascular smooth muscle cells and cardiac myocytes (chronic effects), hence causing vascular and cardiac hypertrophy. These effects are mediated via stimulation of either ETA or ETB receptors and might be provoked through the sequential activation of intracellular kinase cascade, including raf-1, mitogen-activated protein kinase (MAPK), MAPK kinase, and S6 kinase [3]. The development of ETA and ETB receptor antagonists as well as gene-manipulated animals greatly accelerated the elucidation of physiological and pathophysiological roles of endogenous endothelin 1. In normal states, the release of endothelin 1 appears to be sparse, and it plays no appreciable physiological role in the maintenance of blood pressure. When the production and release of endothelin 1 are increased due to certain reasons, endothelin 1 stimulates various cardiovascular diseases. Although the involvement of endothelin 1 in mild to moderate hypertension remains controversial, it is undoubtedly implicated in certain types of hypertension, particularly in malignant stages of the disease. Pulmonary hypertension (PH) is a progressively deteriorating condition characterized by an increase in pulmonary vascular tone and enhanced proliferation of pulmonary vascular smooth muscle cells. There has been no effective therapeutic method for the treatment of this disease. PH is associated with the increase in plasma endothelin 1 levels, which is strongly correlated with the severity of the disease [6]. The endothelin receptor antagonists are remarkably efficacious in all animal models thus far investigated. Bosentan (ETA and ETB receptor antagonist) is now available for clinical treatment of PH. In atherosclerosis as well as various kinds of vascular remodeling, endothelin 1 plays a role in causing or progressing the disease. Endothelin 1 is also deeply concerned in cardiac diseases, including cardiac hypertrophy and acute and chronic heart failure [8]. Endothelin receptor antagonists have been shown to exhibit desirable preventive or curative effects in various animal

models of these cardiovascular diseases [2], suggesting that they are of potential therapeutic value, and some of them are already under clinical investigation.

References [1] Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding and endothelin receptor. Nature 1990; 348: 730–732. [2] Goto K. Basic and therapeutic relevance of endothelinmediated regulations. Biol Pharm Bull 2001; 24: 1219–1230. [3] Goto K, Hama H, Kasuya Y. Molecular pharmacology and pathophysiological significance of endothelin. Jpn J Pharmacol 1996; 72: 261–290. [4] Hickey KA, Rubanyi GM, Paul RJ, Highsmith RF. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol 1985; 252: C550–C556. [5] Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Nat Acad Sci USA 1989; 86: 2863– 2867. [6] Miyauchi T, Yorikane R, Sakai S, Sakurai T, Okada M, Nishikibe M, Yano M, Yamaguchi I, Sugisita Y, Goto K. Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rat with monocrotaline-induced pulmonary hypertension. Circ Res 1993; 73: 887–897. [7] O’Brien RF, Robbins RJ, McMurthy IF. Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 1987; 132: 263–270. [8] Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature 1996; 384: 353–355. [9] Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide selective subtype of the endothelin receptor. Nature 1990; 348: 732–735. [10] Suzuki N, Matsumoto H, Kitada C, Masaki T, Fujino M. A sensitive sandwich-enzyme immunoassay to detect immunoreactive big endothelin in plasma. J Immunol Method 1989; 118: 245– 250. [11] Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, deWit D, Yanagisawa M. ECE-1: a membrane-bound metalloprotease that catalyze the proteolytic activation of big endothein-1. Cell 1994; 78: 473–385. [12] Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411–415.

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164 Ghrelin: Its Therapeutic Potential in Heart Failure NORITOSHI NAGAYA AND KENJI KANGAWA

GH and its mediator, insulinlike growth factor 1 (IGF-1), are anabolic hormones that are essential for skeletal and myocardial growth and metabolic homeostasis [1, 15]. Considering the hemodynamic and anabolic effects of GH/IGF-1, ghrelin may have beneficial effects on left-ventricular (LV) function and energy metabolism in congestive heart failure (CHF) through GH-dependent mechanisms. On the other hand, ghrelin may have direct cardiovascular and metabolic effects through GH-independent mechanisms: GHS-R mRNA is detected not only in the hypothalamus and pituitary but also in the heart and vessels [27]; stimulation of GHS-R has been shown to prevent cardiac damage after ischemia-reperfusion in hypophysectomized rats [23]; ghrelin inhibits apoptosis of cardiomyocytes and endothelial cells in vitro [4]; intravenous injection of ghrelin decreases arterial pressure and increases cardiac output in healthy humans [27]; and ghrelin inhibits sympathetic nerve activities [25]. These findings raise the possibility that the administration of ghrelin may have beneficial hemodynamic effects in patients with CHF. In patients with end-stage CHF, both LV dysfunction and cardiac cachexia are observed [2]. Cardiac cachexia, which is a catabolic state characterized by weight loss and muscle wasting, is associated with hormonal changes and cytokine activation in patients with CHF. It is important that the presence of cardiac cachexia is a strong independent risk factor for mortality in patients with CHF [3]. Thus, cardiac cachexia and LV dysfunction are therapeutic targets in the treatment of CHF. Interestingly, peripheral and intracerebroventricular administration of ghrelin stimulated food intake and increased body weight through GH-independent mechanisms [33]. In addition, ghrelin decreases fat utilization and increases carbohydrate utilization through a GHindependent mechanism [40]. Taking these results together with the GH-dependent anabolic effects of

ABSTRACT Ghrelin is a novel growth hormone-releasing peptide that also causes a positive energy balance by stimulating food intake and inducing adiposity through growth hormone (GH)-independent mechanisms. Ghrelin has some cardiovascular effects, including vasodilation, as indicated by the presence of its receptor in blood vessels and the cardiac ventricles. Ghrelin also inhibits cell apoptosis and sympathetic nerve activities. Short-term infusion of ghrelin decreases systemic vascular resistance and increases cardiac output in patients with heart failure. Repeated administration of ghrelin improves cardiac structure and function and attenuates the development of cardiac cachexia. These results suggest that ghrelin has cardiovascular effects and regulates energy metabolism through GH-dependent and -independent mechanisms.

INTRODUCTION Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary [10, 24, 37]. They act through the GHS receptor (GHS-R), a G-proteincoupled receptor with seven-transmembrane domains [17]. Using a reverse pharmacology paradigm with a stable cell line expressing GHS-R, ghrelin was purified as an endogenous ligand for GHS-R from rat stomach [21]. Ghrelin is a peptide hormone in which the third amino acid, usually a serine but in some species a threonine, is modified by a fatty acid; this modification is essential for ghrelin’s activity. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GHreleasing hormone but also by ghrelin derived from the stomach. Handbook of Biologically Active Peptides

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1194 / Chapter 164 ghrelin, ghrelin causes a positive energy balance through GH-dependent and independent mechanisms. These findings raise the possibility that the administration of ghrelin may be beneficial in cachectic patients with CHF. This chapter summarizes the biological actions and therapeutic potential of ghrelin for the treatment of LV dysfunction and the resultant cachexia in CHF.

DISCOVERY, STRUCTURE, AND SYNTHESIS Ghrelin was purified as an endogenous ligand for GHS-R from rat stomach in 1999 [21]. Human ghrelin is a 28-amino-acid peptide containing an n-octanoyl modification at serine 3; it is homologous to rat ghrelin apart from two amino acids (Fig. 1). The noctanoylation at ser3 is considered to be essential for the activity of ghrelin. Ghrelin is produced mainly in X/A-like cells of the stomach [8]. Ghrelin is not secreted into the gastrointestinal tract, but is secreted into blood vessels. Thus, the plasma ghrelin level is relatively high

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(100–120 fmol/ml) [16]. These results suggest that ghrelin serves as a circulating factor as well as an autocrine/paracrine factor. Ghrelin level in the blood and mRNA in the stomach are increased by fasting and decreased by feeding [6, 36, 38]. Oral or intravenous administration of glucose decreases plasma ghrelin level [32, 40]. Plasma ghrelin level is low in obese people and high in lean people [7, 41]. The plasma ghrelin level was significantly elevated in cachectic patients with CHF, although it did not significantly correlate with LV ejection fraction [31]. The plasma ghrelin level was also elevated in cachectic patients with chronic obstructive pulmonary disease (COPD) [19]. Considering the anabolic effects of ghrelin, increased plasma ghrelin may represent a compensatory mechanism under conditions of anabolic/catabolic imbalance in cachectic patients with CHF or COPD.

RECEPTORS AND THEIR DISTRIBUTION Before the discovery of the endogenous ligand, a specific receptor for ghrelin, GHS-R, was discovered in 1996 using an expression cloning strategy [17]. It is a G-protein-coupled receptor with seven-transmembrane domains. GHS-R is present in a variety of tissues, including the pituitary and hypothalamus, and is distinct from the growth hormone receptor–like hormone (GHRH) receptor. Interestingly, GHS-R is detected in the cardiac ventricles and blood vessels [27], suggesting that ghrelin may cause cardiovascular effects through GHindependent mechanisms.

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G H r el ea se FIGURE 1. A. Structure of human ghrelin. Human ghrelin is a 28-amino-acid peptide containing an n-octanoyl modification. B. Stimulation of GH release by GHRH and ghrelin. GHRH acts on the GHRH receptor through a cAMPdependent mechanism, whereas ghrelin and GHSs bind to the GHS receptor, followed by the release of calcium from intracellular stores.

GH-Releasing Activity Ghrelin stimulates GH release both in vitro and in vivo. Ghrelin increased GH release from cultured pituitary cells in a dose-dependent manner [21]. Intravenous injection of ghrelin markedly increased circulating GH in rats and humans, with greater potency than GHRH [39]. The peak level of GH occurred at 15– 20 min after a bolus of ghrelin, and the elevation of GH level lasted longer than 60 min after bolus injection [27]. These results suggest that ghrelin elicits potent, long-lasting GH release. A recent study demonstrated that blockade of the gastric vagal afferents abolished ghrelin-induced GH secretion and feeding [9]. In addition, ghrelin receptors are synthesized in vagal afferent neurons and transported to the afferent terminals. These findings suggest that gastric vagal afferents are the major pathway conveying ghrelin’s signals for GH secretion and starvation to the brain.

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Hemodynamic Effect GHS-R mRNA is detectable in the heart and blood vessels in rats and humans [20, 27]. To clarify whether ghrelin has direct vasodilatory effects in humans, the response of forearm blood flow to intra-arterial infusion of ghrelin was examined using a plethysmograph. Ghrelin increased forearm blood flow in a dosedependent manner [34]. A single injection of ghrelin significantly decreased mean arterial pressure both in sham-operated rats and CHF rats [30]. This hypotensive effect was also observed in GH-deficient rats. Interestingly, Wiley et al. have demonstrated that ghrelin is an endothelium-independent vasodilator of the longlasting constrictor endothelin 1 in isolated human arteries [42]. These results suggest that ghrelin has direct vasodilatory effects. In patients with CHF, intravenous infusion of human ghrelin significantly decreased mean arterial pressure without a significant change in heart rate [28]. Ghrelin significantly increased cardiac index and stroke volume index. In vitro, fractional cell shortening was not significantly altered by each dose of ghrelin, suggesting that ghrelin has no direct inotropic effects. Therefore, the increase in cardiac output may be mediated by a reduction of cardiac afterload and a GH/IGF-1-dependent inotropic effect.

Orexigenic Effect The hypothalamic arcuate nucleus is the main active site of ghrelin. Hypothalamic neuropeptide Y (NPY) mRNA expression was increased in rats that received intracerebroventricular injection of ghrelin [22, 33]. Ghrelin’s orexigenic effect was abolished dosedependently by co-injection of NPY Y1 receptor antagonist. Thus, ghrelin elicits potent stimulation of food intake via activation of NPY neurons in the hypothalamic arcuate nucleus.

Inhibition of Cell Apoptosis Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GHS-R. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHS-R. Recently, Baldanzi et al. showed that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through the activation of extracellularsignal-regulated kinase 1 and 2 and Akt serine kinases [4]. In addition, ghrelin and des-acyl ghrelin recognize common high-affinity binding sites on H9c2 cardiomyocytes, which do not express GHS-R. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell

death, and activate mitogen-activated protein kinase (MAPK) and Akt. These findings provide the evidence that, independent of its acylation, the ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor that is distinct from GHS-R.

Attenuation of Sympathetic Nerve Activity Microinjection of ghrelin into the nucleus of the solitary tract significantly decreased the mean arterial pressure and heart rate [22, 25]. The microinjection of ghrelin into the nucleus of the solitary tract also suppressed the renal sympathetic nerve activity. Pretreatment with intravenous injection of pentolinium, a ganglion-blocking agent, eliminated these cardiovascular responses induced by the microinjection of ghrelin into the nucleus of the solitary tract; however, pretreatment with intravenous injection of atropine sulfate, an antagonist of muscarinic acetylcholine receptors, failed to prevent them. Immunohistochemical study revealed that GHS-R, the receptor for ghrelin, was expressed in the neuronal cells of the nucleus of the solitary tract and the dorsal motor nucleus of the vagus but not in the cells of the area postrema. These results suggest that ghrelin acts at the nucleus of the solitary tract to suppress sympathetic activity and to decrease arterial pressure in rats. Similar to beta-blocker therapy, inhibition of sympathetic nerve activity by ghrelin may have a beneficial effect on cardiac performance in chronic heart failure.

CLINICAL APPLICATION OF GHRELIN Heart Failure and Cardiac Cachexia LV dysfunction and remodeling and cardiac cachexia are often observed in patients with end-stage CHF. GH and its mediator, IGF-1, are essential for skeletal and myocardial growth and metabolic homeostasis [1, 15]. Earlier studies have shown that GH supplementation may have beneficial effects on myocardial structure and function in some patients with CHF [5, 12, 43]. Thus, we investigated the effects of ghrelin on LV function, exercise capacity, and muscle wasting in patients with CHF [29]. Human synthetic ghrelin (2 μg/kg twice a day) was intravenously administered to patients with CHF for 3 weeks. Ghrelin increased LV ejection fraction in association with an increase in LV mass and a decrease in LV end-systolic volume. Treatment with ghrelin increased peak workload and peak oxygen consumption during exercise. Ghrelin improved muscle wasting, as indicated by increases in muscle strength and lean body mass. These preliminary results suggest that the repeated administration of ghrelin improves LV

1196 / Chapter 164 function, exercise capacity, and muscle wasting in patients with CHF. Ghrelin increased posterior wall thickness, inhibited progressive LV enlargement, and thereby reduced LV wall stress in rats with CHF [30]. GH and its mediator, IGF-1, have been shown to enhance physiological compensatory hypertrophy in rats with CHF, resulting in a decrease in LV wall stress, leading to improvement in cardiac function. Thus, ghrelin may also improve cardiac function partly through GHdependent mechanisms. On the other hand, ghrelin inhibits apoptosis of cardiomyocytes and endothelial cells through activation of extracellular-signal-regulated kinase 1 and 2 and Akt serine kinases [4], implying that improvement in cardiac function may be related to the direct effects of ghrelin on the myocardium. It is important that ghrelin significantly decreased plasma norepinephrine in patients with CHF. A recent study has demonstrated that ghrelin acts directly on the central nervous system to decrease sympathetic nerve activity [22, 25]. Thus, the inhibitory effects of ghrelin on sympathetic nerve activity may contribute to a decrease in plasma norepinephrine, which may have beneficial effects on cardiac performance in patients with CHF. Cardiac cachexia, which is a catabolic state characterized by weight loss and muscle wasting, occurs frequently in patients with end-stage CHF and is a strong independent risk factor for mortality in patients with CHF [3]. Thus, cardiac cachexia may be a therapeutic target in the treatment of heart failure. The 3-week administration of ghrelin tended to increase body weight and significantly increased lean body mass and muscle strength. These results suggest that treatment with ghrelin improves muscle wasting in patients with CHF. These effects may be mediated, at least in part, by GH/ IGF-1, which is considered to be essential for skeletal muscle. Earlier studies have shown that ghrelin induces orexigenic effects via the activation of NPY neurons in the hypothalamic arcuate nucleus [33]. The intravenous administration of ghrelin increased food intake in patients with CHF, which may contribute to the anabolic effects of ghrelin. Although many animal studies have documented beneficial effects of GH [5, 12, 43], controlled studies in humans have been predominantly negative [18, 35]. Nevertheless, ghrelin may have additional therapeutic potential because it has GH-independent effects such as attenuation of sympathetic nerve activities, vasodilatory actions, inhibition of cell apoptosis, and orexigenic effects. Thus, the administration of ghrelin may be a new therapeutic approach to the treatment of cardiac cachexia (Fig. 2).

Other Catabolic States Patients with COPD often show a certain degree of cachexia. Cachexia is an independent risk factor for

Severe heart failure

L V d y sf u n c t io n Vasodilation

Cachexia

Muscle growth

Muscle growth

GH release

Anti-apoptotic effect Inhibition of sympathetic nerve activity

Ghrelin

Feeding Adiposity Inhibition of sympathetic nerve activity

FIGURE 2. Characteristics of end-stage CHF and the therapeutic potential of ghrelin. Ghrelin may improve cardiac function and cardiac cachexia not only through GH-dependent mechanisms but also through GH-independent mechanisms: vasodilation, inhibition of sympathetic nerve activity, antiapoptotic effects, feeding, and adiposity.

mortality in COPD. The repeated administration of ghrelin improved body composition, muscle wasting, functional capacity, and sympathetic augumentation in cachectic patients with COPD [26]. Thus, the administration of ghrelin may be a new therapeutic approach to the treatment of pulmonary cachexia. Other potential clinical applications of ghrelin are in osteoporosis, aging, and catabolic states, including those seen in postoperative patients and in AIDS- and cancer-associated wasting syndromes [11, 13, 14].

CONCLUSION Ghrelin is a novel GH-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for GHS-R. This peptide also has a variety of GH-independent effects such as orexigenic effects, attenuation of sympathetic nerve activities, vasodilatory actions, and inhibition of cell apoptosis. The repeated administration of ghrelin improves body composition, muscle wasting, functional capacity, and sympathetic augumentation in cachectic patients with CHF. These beneficial effects were mediated by both GHdependent and -independent mechanisms. Thus, supplementation with ghrelin may be a new therapeutic approach to the treatment of heart failure and cardiac cachexia.

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[21] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402: 656–660. [22] Lin Y, Matsumura K, Fukuhara M, Kagiyama S, Fujii K, Iida M. Ghrelin acts at the nucleus of the solitary tract to decrease arterial pressure in rats. Hypertension 2004; 43: 977–982. [23] Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna V, Bernareggi M, et al. Growth hormone-independent cardioprotective effects of hexarelin in the rat. Endocrinology 1999; 140: 4024–4031. [24] Locatelli V, Torsello A. Growth hormone secretagogues: focus on the growth hormone releasing peptides. Pharmacol Res 1997; 36: 415–423. [25] Matsumura K, Tsuchihashi T, Fujii K, Abe I, Iida M. Central ghrelin modulates sympathetic activity in conscious rabbits. Hypertension 2002; 40: 694–699. [26] Nagaya N, Itoh T, Murakami S, et al. Treatment of cachexia with ghrelin in patients with chronic obstructive pulmonary disease. Chest 2005; 128: 1187–1193. [27] Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, et al. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol 2001; 280: R1483– 1487. [28] Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda H, et al. Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol Metab 2001; 86: 5854–5859. [29] Nagaya N, Moriya J, Yasumura Y, Uematsu M, Ono F, Shimizu W, et al. Effects of ghrelin administration on left ventricular function, exercise capacity, and muscle wasting in patients with chronic heart failure. Circulation 2004; 110: 3674– 3679. [30] Nagaya N, Uematsu M, Kojima M, Ikeda Y, Yoshihara F, Shimizu W, et al. Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 2001; 104: 1430– 1435. [31] Nagaya N, Uematsu M, Kojima M, Kojima M, Date Y, Nakazato M, et al. Elevated circulating level of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors. Circulation 2001; 104: 2034– 2038. [32] Nakagawa E, Nagaya N, Okumura H, Enomoto M, Oya H, Ono F, et al. Hyperglycaemia suppresses the secretion of ghrelin, a novel growth-hormone-releasing peptide: responses to the intravenous and oral administration of glucose. Clin Sci (Lond) 2002; 103: 325–328. [33] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, et al. A role for ghrelin in the central regulation of feeding. Nature 2001; 409: 194–198. [34] Okumura H, Nagaya N, Enomoto M, Nakagawa E, Oya H, Kangawa K. Vasodilatory effect of ghrelin, an endogenous peptide from the stomach. J Cardiovasc Pharmacol 2002; 39: 779–783. [35] Osterziel KJ, Strohm O, Schuler J, Friedrich M, Hanlein D, Willenbrock R, et al. Randomised, double-blind, placebocontrolled trial of human recombinant growth hormone in patients with chronic heart failure due to dilated cardiomyopathy. Lancet 1998; 351: 1233–1237. [36] Ravussin E, Tschop M, Morales S, Bouchard C, Heiman ML. Plasma ghrelin concentration and energy balance: overfeeding and negative energy balance studies in twins. J Clin Endocrinol Metab 2001; 86: 4547–4551. [37] Smith RG, Cheng K, Schoen WR, Pong SS, Hickey G, Jacks T. A nonpeptidyl growth hormone secretagogue. Science 1993; 260: 1640–1643.

1198 / Chapter 164 [38] Sugino T, Hasegawa Y, Kikkawa Y, Yamamura J, Yamagishi M, Kurose Y, et al. A transient ghrelin surge occurs just before feeding in a scheduled meal-fed sheep. Biochem Biophys Res Commun 2002; 295: 255–260. [39] Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M, et al. Ghrelin strongly stimulates growth hormone (GH) release in humans. J Clin Endocrinol Metab 2000; 85: 4908–4911. [40] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: 908–913.

[41] Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001; 50: 707–709. [42] Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin-1. Br J Pharmacol 2002; 136: 1146– 1152. [43] Yang R, Bunting S, Gillett N, Clark R, Jin H. Growth hormone improves cardiac performance in experimental heart failure. Circulation 1995; 92: 262–267.

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165 Natriuretic Peptides in the Cardiovascular System NAOTO MINAMINO, TAKESHI HORIO, AND TOSHIO NISHIKIMI

atriopeptin, cardionatrin, and auriculin, but it is now generally called atrial natriuretic peptide (ANP) [16]. The second member of the NP family was isolated from pig brain extracts by monitoring the relaxant activity of chick rectum in 1988, and it was named brain natriuretic peptide (BNP) [35]. After a few years, this peptide was demonstrated to be a cardiac peptide mainly expressed in the ventricle, and BNP concentration in the brain was undetectable or extremely low in rodents and humans [20, 28]. BNP is now recognized as B-type NP of the NP family. The third NP was also identified in the pig brain extracts by the same procedures and named C-type natriuretic peptide (CNP) in 1990 [36]. CNP is present in the brain, not at a high concentration but at the highest concentration of the tissues examined. CNP is considered to be a true brain NP, but recent studies showed that this peptide is produced in the vasculature, heart, bone, macrophages, and elsewhere [33]. CNP has been verified as an ancestral molecule of the NP family, as described in Chapter 110 in this book [7]. Figure 1 shows amino acid sequences of active molecular forms of human ANP, BNP, and CNP. All NPs have two cysteine residues in the molecule, and a 17-residue ring structure formed by a disulfide linkage is essential for exerting biological activity. In this ring structure, the amino acid sequence is well conserved among the three NPs. In the case of ANP and BNP, the C-terminal extension from the ring structure is essential for their biological activity, whereas CNP does not have it. In all NPs, the N-terminal extension does not alter the primary potency of biological activity. In mammalian ANPs, αANP of 28 amino acid residues is an endogenous and active form, and only one amino acid replacement (Met → Ile) is observed. There are many amino acid replacements in the case of BNPs, and the length of a major endogenous form is rather different in mammals. CNP is present as CNP-22 and CNP-53, and CNP-53 is gener-

ABSTRACT The natriuretic peptide family in mammals consists of three homologous peptides: atrial (A-type) natriuretic peptide (ANP), brain (B-type) natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP and BNP bind to natriuretic receptor-A, whereas CNP acts on natriuretic receptor B. Natriuretic receptor A and B are receptor-guanylyl cyclases, and these peptides dilate blood vessels and induce diuresis/natriuresis by increasing an intracellular cGMP concentration. ANP and BNP are most abundantly synthesized in cardiac myocytes, whereas CNP is expressed in endothelial cells and cardiac fibroblasts. Natriuretic peptide receptors are widely expressed in the peripheral tissue, and these peptides not only regulate blood pressure and body fluid volume but also inhibit remodeling of heart and blood vessels. The plasma concentrations of ANP and BNP are increased in the cardiovascular disease. Thus, natriuretic peptides are crucial regulatory factors in maintaining the homeostatic balance of the cardiovascular system.

DISCOVERY OF NATRIURETIC PEPTIDES Mammalian atrial cardiac myocytes exhibit the morphology of endocrine cells and contain specific atrial granules. By the beginning of the 1980s, atrial extracts were shown to induce potent diuresis/natriuresis and hypotension when injected into rats. Soon after confirmation of the correlation between diuretic/natriuretic activity and smooth muscle relaxant activity (in chick rectum and rabbit aorta) of the atrial extracts, the diuretic/natriuretic and hypotensive factor was identified as a single component in 1983 and 1984 [11]. As the result of the competition of many research groups, this peptide was first given a variety of names, such as Handbook of Biologically Active Peptides

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1200 / Chapter 165 ally a major endogenous form. The amino acid sequence of CNP-22 is identical in all mammals, and two replacements are observed between human and the other mammalian CNP-53s [27, 30, 32].

STRUCTURE OF THE PRECURSORS, mRNAs, AND GENES OF NPs The exon-intron organization, structure of mRNA, and precursor protein of three mammalian NPs are schematically represented in Fig. 2. Three mammalian NP genes are each coded in three exons. Only the third exon of CNP is not well characterized because it only codes the 3′ untranslated region (UTR) [27]. Human ANP and BNP genes are neighboring genes located at 11,840,035–11,842,106 bp and 11,851,790–

11,853,254 bp in chromosome 1, and no transcript from the 9.7-kb intervening sequence has been recorded in the database. The human CNP gene, except for putative exon 3, is located at 232,315,640–232,616,464 bp in chromosome 2. In the 5′ flanking region (FR) of human ANP gene, GATA4, Nkx2.5/Csx, Tbx5, and SRF are present as cisacting elements, and the transcriptional factors (TFs) binding to these elements are reported to be important for ANP gene transcription. In the 5′ FR of the human BNP gene, GATA, TEF1, and NFAT binding sites are observed, and binding of TFs to these elements increases BNP gene transcription [17]. All these TFs are also known to be required for embryogenesis and morphogenesis of heart tissue. Neuron-restrictive silencer factor has been recently reported to suppress the gene transcription of ANP and BNP, whose binding sites are

Atrial (A-type) natriuretic peptide (ANP) human/pig/cow rat/mouse

SLRRSSCFGGRMDRIGAQSGLGCNSFRY SLRRSSCFGGRIDRIGAQSGLGCNSFRY

Brain (B-type) natriuretic peptide (BNP) Human pig cow rat mouse

SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH SPKTMRDSGCFGRRLDRIGSLSGLGCNVLRRY GPKMMRDSGCFGRRLDRIGSLSGLGCNVLRRY SQDSAFRIQERLRNSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF SQGSTLRVQQRPQNSKVTHISSCFGHKIDRIGSVSRLGCNALKLL

C-type natriuretic peptide (CNP) human DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC rat/mouse/pig/cow DLRVDTKSRAAWARLLHEHPNARKYKGGNKKGLSKGCFGLKLDRIGSMSGLGC

FIGURE 1. Amino acid sequences of mammalian ANP, BNP, and CNP. Underline indicates amino acid replacement compared with human NP; double underline indicates CNP-22 sequence. Bracket over top NP sequence of each group is a disulfide linkage that forms the ring structure.

gene

mRNA

pr ec ur s or pro-form ? ?

active form a-ANP

b-ANP

BNP-32

CNP-53

CNP-22

FIGURE 2. Schematic representation of genes, mRNAs, precursors, and active forms of human ANP, BNP, and CNP. Boxes in the gene row indicate exons, and black and white regions represent coding and noncoding regions, respectively. In the precursor, pro-form, and active form rows, hatched bar, white bar, and ring structure indicate signal peptide, N-terminal intervening peptide, and active unit of each NP, respectively. In ANP, two arginines are inserted after α-ANP as shown by a white bar.

Natriuretic Peptides in the Cardiovascular System / 1201 located in the 3′ UTR of ANP gene and the 5′ FR of BNP gene. In the 5′ FR of the human CNP gene, an inverted CCAAT box, two GC boxes, and a cAMP response element–like sequence are present. Shear stress and TGF-β have been shown to regulate its gene transcription through their respective responsive elements. The apparent length of human NP mRNAs is approximately 850–900 bp for ANP and BNP and 1300 bp for CNP, and the difference in the mRNA length is mainly due to the 3′ UTR. In the 3′ UTR of the BNP gene, AUUUA repeats are present, which are known to accelerate the degradation of mRNA. Because these repeats are often observed in the 3′ UTR of lymphokine genes and oncogenes, the BNP gene is considered to be designed for dynamic physiological alterations. The precursor proteins of human ANP, BNP, and CNP are 153, 134, and 126 amino acids in length, and the active structure is located at the C-terminus in each precursor. Only in the case of the ANP precursor, two arginine residues are inserted after α-ANP [16, 27, 30].

DISTRIBUTION OF THE mRNA AND PEPTIDE IN THE CARDIOVASCULAR SYSTEM ANP mRNA is found in a variety of tissues, but is most abundant in the heart [16, 27, 30]. Within the heart, the atrium is a major site of ANP synthesis. The primary stimulus for ANP secretion is an increase in atrial-wall tension, which is attributable to elevation of venous blood pressure induced by an increased venous return. Tachycardias including supraventricular tachyarrhythmia also promote atrial ANP secretion. Several hormones and neurotransmitters, such as endothelin (ET), vasopressin, and catecholamines (especially αadrenergic agonists), directly stimulate ANP secretion from atrial and ventricular myocytes. Peptide concentrations of ANP in human, rat, and pig cardiac ventricles are 250 to 1000-fold less than those in atria. ANP mRNA levels are also much lower in the ventricles than in the atria. Although little ANP is produced by ventricular tissue in normal adults, a high level of ventricular ANP is present in the fetus [30]. Gene expression and synthesis of ANP are enhanced in both atrium and ventricle during early embryogenesis, but its expression in the ventricle is decreased after the prenatal period. ANP expression in the ventricle is reinducible postnatally in hypertrophied and failing hearts. BNP is found in the circulation and its highest concentration is observed in heart tissue, although this peptide was first identified in pig brain extracts. In rats,

the atrium expresses the BNP gene at a high level, and a comparable level of BNP mRNA is present in the ventricle [27, 28]. When tissue weight is taken into account, the total BNP mRNA content is three-fold higher in the ventricle than in the atrium, whereas ANP mRNA content in the ventricle is only 7% of that in the atrium. The peptide concentration of BNP in the ventricle is less than 1% of that in the atrium. In an experiment with isolated perfused rat hearts, however, the majority of BNP secretion (approximately 60%) was maintained even after atrial removal, in contrast to the observation that ANP secretion was reduced to less than 5% by atrial removal. Similar findings are observed in human hearts [20]. BNP and its mRNA level in the ventricle are less than those in the atrium, but the total content of BNP and its mRNA in the ventricle correspond to 30 and 70% of those in the whole heart, respectively. Predominant BNP secretion from the ventricle has been confirmed by a step-up of plasma BNP level in the anterior interventricular vein compared with that in the aorta. Thus, BNP is a cardiac hormone that is predominantly synthesized in and secreted from the ventricle. In patients with congestive heart failure, myocardial BNP mRNA and circulating BNP levels dramatically increased compared with ANP. Therefore, BNP appears to act as an emergency hormone in disease states. CNP was initially thought to act as a neuropeptide because CNP and its CNP mRNA are found predominantly in the brain. However, subsequent studies have shown that CNP is synthesized in peripheral tissues, including the kidney, bone, blood cells, vasculature, and heart. Significant gene expression and peptide secretion of CNP are observed in cultured vascular endothelial cells (ECs), and transforming growth factor markedly increases its expression and secretion [37]. CNP secretion from the ECs is also stimulated by other cytokines, such as tumor necrosis factor, interleukin-1, basic fibroblast growth factor, and lipopolysaccharide, suggesting that CNP plays an important role within the vascular wall under various pathological states. As for the production of CNP in the heart, discrepant findings have been reported [9]. Initial studies failed to detect CNP mRNA in human or rat heart. In addition, low levels of CNP detected in pig and human hearts have been considered to reflect cross-reactivity with ANP or products of coronary arterial ECs. However, subsequent studies confirmed the presence of CNP and its mRNA in both atrium and ventricle by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR). A recent in vitro study has verified significant gene expression and secretion of CNP from cultured rat cardiac fibroblasts but not from myocytes, indicating a distinct profile of cardiac CNP [6].

1202 / Chapter 165 PROCESSING AND ENDOGENOUS FORMS OF NPs Human ANP is stored in the cardiac myocytes as a 126-amino-acid protein called γ-ANP, which lacks the C-terminal two arginines from pro-ANP. In the normal heart tissue, ANP is almost exclusively present as γ-ANP. When secreted, it is cleaved after a Pro-Arg sequence by corin, a specific processing enzyme for ANP, to generate α-ANP [40]. In the atrium of patients with heart failure, α-ANP is present in the myocytes along with a unique antiparallel dimer of β-ANP. In the plasma, αANP is a major circulating and active form. A 98-residue N-terminal intervening peptide of pro-ANP (N-ANP) is also present in the plasma at a concentration higher than that of α-ANP. In mammals other than human, γ-ANP and α-ANP are major forms in the heart and plasma, respectively. In the case of human BNP, pro-BNP (or γ-BNP) is processed probably by furin to BNP-32. In heart tissue, BNP is both pro-BNP and BNP-32, and BNP-32 is a major form in some cases. In the plasma, BNP-32 is a major form. In the pig, BNP is present as pro-BNP in the heart and as BNP-32 and BNP-26 in the plasma. In the rat, BNP is present as pro-BNP in the heart and as BNP-45 in the plasma. As expected from the sequence heterogeneity of mammalian BNP, its endogenous form is different in each animal. An N-terminal 76-residue peptide of pro-BNP (N-BNP) is also present in the human plasma at a level higher than BNP-32 [16, 27, 32].

S S

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R R G G MD S F R S C I C D G G S A L F G S Q R

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Y

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CNP is present in all mammals in the uniform molecular forms as deduced from its high sequence identity [27]. In the cardiovascular system, ECs, cardiac fibroblasts, and the monocyte-macrophage system are known to produce CNP [2]. In the case of ECs, CNP is mainly present as CNP-53 along with CNP-22 in a manner similar to that in the brain. Both CNP-22 and CNP-53 elicit comparable potency and no critical difference has been reported between them. Furin is also considered to process pro-CNP into CNP-53 or CNP-22.

RECEPTORS OF NPs AND THEIR DISTRIBUTION IN THE CARDIOVASCULAR SYSTEM Natriuretic receptor A (NPR-A) and natriuretic receptor B (NPR-B) are guanylyl cyclase-linked receptors, and they use cGMP as the intracellular messenger (Fig. 3). Both ANP and BNP bind preferentially to NPRA, whereas CNP specifically binds to NPR-B. NPR-B has an extremely low affinity for ANP and BNP, whereas CNP does not bind to NPR-A even at a high concentration. NPs stimulated cGMP accumulation via NPR-A with a rank order potency of ANP ≥ BNP >> CNP, whereas that via NPR-B was CNP > ANP ≥ BNP. All three NPs bind to a third receptor NPR-C, known as a clearance receptor. NPR-C is not linked to guanylyl cyclase, and it mainly acts to clear the NPs from the circulation. Thus, the NP system consists of three ligands and three receptors [12, 30]. One NP molecule is now known to bind to each dimer-formed NPR [26].

K L G L D R I G S L G S M

CNP

cGMP

NPR-B (GC-B)

NPR-C (Clearance receptor)

FIGURE 3. Schematic representation of NPRs and ligand-receptor relationship of the NP family. Black arrows indicate interaction with active receptors, NPR-A and NPR-B, and gray arrows indicate interaction with clearance receptor, NPR-C. One NP molecule binds to each dimerformed NPR. NPR-A and NPR-B are also called GC-A and GC-B, respectively.

Natriuretic Peptides in the Cardiovascular System / 1203 An earlier study of whole-body autoradiography using [125I]-ANP demonstrated that high levels of radioactivity were detected in renal cortex, especially in the glomeruli, as well as in the endocardium, zona glomerulosa, and adrenal medulla. In the kidney, high-affinity 125IANP binding sites are concentrated over glomeruli and to a lesser extent over the arterial vasculature, and loweraffinity binding sites are observed over proximal tubules and inner medullary collecting ducts. As for the heart, there was a long-standing controversy regarding whether or not cardiac myocytes themselves are the targets for NPs. By using RT-PCR analysis after single cell collection, Lin et al. demonstrated that both cardiac myocytes and fibroblasts express NPR mRNA [14]. Interestingly, myocytes predominantly express NPR-A, whereas fibroblasts express NPRA and NPR-B. Indeed, ANP and BNP increase an intracellular cGMP level in myocytes, whereas ANP, BNP, and CNP increase it in fibroblasts. These results indicate that the NPs also act as autocrine and/or paracrine factors in the heart tissue. RT-PCR analysis showed that NPR-A and NPR-B transcripts were widely detected in all peripheral tissues. NPR-B is actually expressed in the vasculature, heart, adrenal gland, kidney, lung, ovary, and bone, in addition to the brain. In blood vessels, vascular smooth muscle cells (VSMCs) express NPR-B at a high level, which is close to that of ECs producing CNP. All three NPs binds to NPR-C with high affinity. The order of affinity to NPR-C is ANP > CNP > BNP, but their differences in the affinity are much smaller than those to NPR-A and NPR-B. Although NPR-C is recognized to primarily function as a clearance receptor in the circulation and in various tissues, recent studies raise a possibility that NPR-C may also be responsible for some actions of the NPs through second messengers other than cGMP.

INFORMATION ON ACTIVE AND/OR SOLUTION CONFORMATION OF NPs According to the nuclear magnetic resonance (NMR) spectroscopic study, NPs are very flexible and do not have specific conformations in the water. In the presence of organic solvents or lipid micelles, higher degrees of structure (β-sheet and β-turn arrangement) were observed, but the proposed structure was not consistent in each report [31]. The conformational study of NPs by circular dichroism spectroscopy also presented a random-coil conformation in water and a more ordered secondary structure in the presence of organic solvents [18]. Human and rat α-ANP and active forms of BNPs, except rat BNP, have a tendency to show similar conformational features, whereas CNP-22 is deduced to

have a separate preferential conformation. Methionine oxidation of α-ANP reduces its biological activity, and the solution conformation of oxidized α-ANP is different from that of ANP. Thus, conformational analysis of the NPs is expected to provide basic information of the flexibility and stability necessary for evaluating their potency and specificity. Recent crystallographic studies show that the extracellular domains of NPR-A and NPR-C form 2 : 1 complexes with α-ANP and that the structure of α-ANP in the complex of NPR-A is similar to that of NPR-C (Fig. 3) [26]. Because the dimers of the extracellular domains of NPR-A and NPR-C have a twofold rotational symmetry and α-ANP is located along the center of the receptor dimer axis, the interaction between α-ANP and one of the two extracellular domains is neither symmetrical nor equivalent. Detailed crystallographic analysis of the interaction between NPs and receptors, as well as of the conformational changes induced by the ligand binding, will afford us a solid base for designing the specific agonists and antagonists of the NPRs.

BIOLOGICAL ACTIONS OF NPs IN THE CARDIOVASCULAR SYSTEM Because NPR-A is widely distributed in the various tissues, ANP and BNP have a wide range of physiological actions. In the kidney, intrarenal arterial infusion of ANP in anesthetized dogs increased renal blood flow, glomerular filtration rate, and urine flow without changing blood pressure. However, a lower dose of ANP produced only diuresis and natriuresis, whereas renal hemodynamics remained unchanged. Thus, ANP inhibits tubular reabsorption even at a low dose and induces renal vasodilation at a higher dose [42]. Renal functions of the NPs are discussed in Chapter 171 in this book. ANP acts on multiple sites to reduce blood pressure. ANP at first exerts a relaxing effect on the vasculature. The acute effects of ANP also include a shift of fluid to the extravascular compartment. The reduction of blood volume and an increase in venous capacitance are considered to be responsible for the reduced cardiac output. This reduction of cardiac output may cause a reflex activation of the sympathetic nervous system and an increase in peripheral resistance, thereby overriding the vasodilator effects of ANP. Thus, ANP appears to have a depressor effect rather than a direct vasodilator effect. A lowering of peripheral resistance in response to ANP is not observed in normotensives, but is readily seen in hypertensives associated with an increased vascular tone. This most likely explains the discrepancy in the hemodynamic responses to ANP in normotensives and hypertensives [13]. In the endocrine/neuroendocrine system, ANP diminishes the secretion of renin, aldosterone,

1204 / Chapter 165 adrenocorticotropic hormone, and vasopressin along with sympathetic tone. All these actions lead to a lowering of the blood pressure. Intracerebroventricular (ICV) administration of ANP attenuates water intake and pressor response induced by ICV injection of angiotensin II. Centrally injected ANP produced a greater reduction of water intake in spontaneously hypertensive rats, and ICV infusion of ANP for 1 week also reduced the salt appetite of spontaneously hypertensive rats. Thus, the ANP-NPR-A system antagonizes the renin-angiotensin system in both central and peripheral systems. The effects of the NPs in the endocrine and central nervous systems are discussed in detail in Chapters 110 and 120 in this book. Recent studies showed that NPs regulate the growth of multiple cell types. ANP inhibits the proliferation of mesangial cells induced by angiotensin II or ET-1. The ANP-NPR-A system negatively regulates mitogen-activating protein kinase (MAPK)-ERK2 activity and proliferation of mesangial cells through a cGMP-dependent protein kinase pathway. In studies with cultured neonatal myocytes and fibroblasts, NPs elicited marked antihypertrophic and antifibrotic actions [3]. A study using an NP antagonist, HS-142-1, also showed that endogenous NPs have antihypertrophic and antifibrotic effects [5]. Indeed, mice lacking NPR-A develop ventricular hypertrophy and fibrosis independent of their blood pressure [7]. These results suggest that ANP and BNP are autocrine and/or paracrine antiremodeling factors. In cultured VSMCs, NPs inhibit proliferation and migration induced by growth factors or angiotensin II, suggesting that NPs act as antiatherosclerotic factors. In fact, doubly deficient NPR-A(−/−) and ApoE(−/−) mice showed greater atherosclerotic lesion size and more advanced plaque morphology compared with NPR-A(+/+) and ApoE(−/−) mice. Aortic medial thickness was also increased in NPR-A(−/−) and ApoE(−/−) mice [1]. CNP is a vasorelaxant with particularly venodilator effects, contrasting with the arterial bias of ANP and BNP [2]. In vitro studies with canine vascular preparations show that CNP causes the relaxation of saphenous, femoral, and renal venous tissue, but does not consistently cause arterial relaxation. The systemic administration of low-dose CNP in humans induces no hemodynamic response, but higher-dose CNP lowers blood pressure. Although the natriuretic effect of CNP is still controversial, it does not appear to have any marked influence on renal sodium handling. Thus, the systemic hemodynamic effects of CNP are evidently less than those of ANP and BNP. Because CNP is produced in ECs, endotheliumderived CNP can elicit direct vasodilation through NPRB abundantly expressed in VSMCs. CNP is well known to suppress various growth factor–stimulated VSMC migration and proliferation, which is more potent than

ANP and BNP. Furthermore, CNP directly inhibits ET-1 secretion from ECs. Because ET-1 is not only a potent vasoconstrictor but also a stimulator of VSMC proliferation, CNP could also inhibit VSMC proliferation via an ET-1-mediated pathway. These findings suggest that CNP regulates vascular tone and growth as an autocrine/paracrine factor in the vascular wall [2, 9]. Some in vitro studies have shown the direct effects of CNP on cardiac myocytes and fibroblasts [33, 34]. CNP inhibits fibroblast proliferation and extracellular matrix production more potently than ANP and BNP. ET-1 secretion from cardiac fibroblasts and ET-1induced hypertrophy of cardiac myoctes are also inhibited by CNP. Taken together with the CNP production in cardiac fibroblasts, CNP may also act as a local autocrine/paracrine regulator in the heart. Collectively, these results indicate that the NP-cGMP system plays a pivotal role in the growth regulation of various cells under certain physiologic and pathologic conditions.

PATHOPHYSIOLOGICAL IMPLICATION OF NPs IN THE CARDIOVASCULAR SYSTEM It is well known that plasma concentrations of ANP and BNP are increased in various pathological conditions such as heart failure, acute myocardial infarction (AMI), hypertension, left-ventricular hypertrophy, coronary artery disease, pulmonary hypertension, and renal failure. In heart failure, initial studies demonstrated that plasma ANP levels are elevated in patients with symptomatic congestive heart failure in proportion to the severity of the disease. Later, several studies showed that plasma ANP and N-ANP levels are also elevated in asymptomatic patients with left ventricular dysfunction. In addition, plasma BNP levels have been used to evaluate heart failure severity because plasma BNP levels in patients with heart failure correlate with the New York Heart Association (NYHA) functional class and hemodynamics [41]. Like ANP and N-ANP levels, plasma BNP and N-BNP levels are also useful for identifying patients with asymptomatic left ventricular dysfunction. A number of studies have examined the diagnostic value of ANP, BNP, N-ANP, and N-BNP in relation to left ventricular systolic dysfunction and shown that the levels of these peptides are useful biomarkers for the detection of left ventricular systolic dysfunction both in the general population and in patients with cardiovascular disease. Furthermore, the measurement of BNP can rule out congestive heart failure in newly symptomatic subjects. A plasma BNP level of 22 pmol/liter or higher indicates a sensitivity of 97%, a specificity of 84%, positive predictive value of 70%, and negative predictive value of 98% for heart failure. In the urgent-

Natriuretic Peptides in the Cardiovascular System / 1205 care setting, BNP is useful to discriminate acute dyspnea due to congestive heart failure from other causes. In patients whose plasma BNP levels are normal, other causes of dyspnea should be considered. Thus, the measurement of plasma NP levels is useful not only for evaluating the severity of heart failure, but also for a rule-out and screening test for left ventricular dysfunction and heart failure. Increased expression and secretion of NPs in heart failure are considered to be a compensatory mechanism. Indeed, an NPR antagonist, HS-142-1, decreased urinary sodium excretion in animal models of heart failure without altering hemodynamics, suggesting that the NP system exclusively compensates for heart failure via its natriuretic actions [24]. Furthermore, a recent study showed that the induction of heart failure in NPR-A(−/−) mice exhibited a more severe outcome than that in NPR-A(+/+) mice, providing the genetic evidence of a protective role of NP/NPR-A signaling against heart failure [23]. In AMI, plasma ANP levels were already increased at the time of admission and decreased thereafter. In contrast, plasma BNP levels were increased on admission and reached a peak at 12–24 hours after admission. Thereafter, the BNP level decreased and then made a second peak on day 5–7, possibly reflecting the left-ventricular remodeling. The BNP level gradually decreased, but was still higher than that of controls in the fourth week [19]. The height of the second peak of BNP may be a good index for evaluating left-ventricular remodeling in patients with AMI. In a rat AMI model, tissue concentrations of BNP increased in the noninfarcted region as well as in the infarcted region. The surviving myocytes in and around the necrotic tissues of the infarcted region were intensely stained with the anti-BNP antiserum, indicating the augmented production of BNP in the remaining myocytes [4]. Because ANP and BNP have antihypertrophic and antifibrotic effects, augmented production of NPs in the ventricle may be aimed to inhibit the remodeling. A number of studies showed that plasma ANP and BNP are increased in patients with hypertension compared with normotensive subjects. In hypertension, plasma ANP and BNP level are higher in patients with left-ventricular hypertrophy than in those without it. In addition, plasma BNP levels in essential hypertension are increased in patients with left-ventricular concentric hypertrophy compared with those with eccentric hypertrophy, concentric remodeling, or normal left ventricular structure [25]. Elevated plasma levels of NPs are considered to contribute to the pathophysiology of hypertension. In fact, ANP knockout mice exhibit saltdependent hypertension, suggesting that ANP regulates blood pressure via its natriuretic actions [8]. Interestingly, NPR-A knockout mice show salt-independent hypertension [15, 29], whereas BNP knockout mice

show normal blood pressure with slight focal fibrosis in the ventricle [38]. The differences in phenotype between ANP knockout mice and BNP knockout mice may be due to different sites of action and/or different gene expression regulatory systems of ANP and BNP. The increased production of NPs in the hypertrophied ventricle is thought to inhibit cardiac hypertrophy and fibrosis via their direct effects on the myocytes and fibroblasts, because NPR-A knockout mice exhibit cardiac hypertrophy and fibrosis [29]. Thus, ANP and BNP play important roles in the pathogenesis of hypertension and subsequently occurring left-ventricular hypertrophy. In patients with right-ventricular pressure overload due to pulmonary hypertension, plasma ANP and BNP levels are higher than those in patients with right-ventricular volume overload due to atrial septal defect. The plasma ANP and BNP levels correlated with mean pulmonary artery pressure, right atrial pressure, rightventricular end-diastolic pressure, and total pulmonary resistance in these patients [21]. After long-term vasodilator treatment, their plasma levels decreased according to a reduction of total pulmonary resistance [22]. Plasma BNP levels are also high in patients with acute pulmonary embolism, in whom BNP levels are associated with right-ventricular overload as well as increased mortality. Thus, both right atrial and ventricular pressure overloads stimulate ANP and BNP secretion independent of etiology, and plasma ANP and BNP levels are good indices especially for evaluation of the severity and judgment of the treatment efficacy in patients with right-ventricular overload. In a recent study with isolated perfused lungs of NPR-A(+/+) and (−/−) mice, ANP and BNP counteracted the hypoxia-induced pulmonary vasoconstriction and reduced pulmonary artery pressure in NPR-A(+/+) mice, whereas only this effect was attenuated in NPR-A(−/−) mice. Chronic hypoxia produces a greater increase in right ventricular systolic pressure and more muscularization of distal pulmonary arterioles in NPR-A(−/−) mice compared with NPRA(+/+) mice. Thus, the NPs-NPR-A system regulates the pulmonary vascular tone, remodeling of pulmonary vasculature, and right-ventricular hypertrophy in pulmonary hypertension. Elevated plasma ANP and BNP levels are prognostic indicators of heart failure, AMI, and pulmonary hypertension. Medical therapy can reduce the ANP and BNP levels in parallel with hemodynamic and clinical improvement in these patients. These findings strongly indicate that the measurement of NPs can be used to optimize therapy. Troughton et al. reported BNP-guided heart failure therapy. When targets (N-BNP level <200 pmol/ liter) were not met, drug therapy was increased in a stepwise fashion [39]. The results showed that N-BNP-guided treatment of heart failure reduced total cardiovascular

1206 / Chapter 165 events compared with intensive clinically guided treatment. Thus, ANP/BNP/NPR-A signaling is deeply involved in the pathophysiology of cardiovascular disease, and the measurement of ANP and BNP is a highly useful test for screening, detection of the progression, and optimizing treatment in cardiovascular disease. CNP potently inhibits VSMC proliferation and migration, suggesting that this peptide may play a protective role against abnormal growth of VSMCs in the disorders such as atherosclerosis and postangioplasty restenosis. In fact, CNP can limit neointimal formation of arteries subjected to balloon injury, due to the inhibition of VSMC proliferation and migration as well as rapid reendothelialization of the injured vessel. Elevated circulating levels of CNP are found in hypoxemia, sepsis, and chronic renal failure [2]. Endothelial damage caused by hypoxia and endotoxin may provoke CNP secretion from ECs. Marked elevation in plasma CNP observed in renal failure is probably attributable to impaired clearance. On the other hand, plasma CNP concentrations are not elevated in essential hypertension. Several studies have reported no significant increase in plasma CNP level of patients with chronic heart failure, in contrast to remarkable elevations of circulating ANP and BNP. However, tissue levels of CNP are increased along with ANP and BNP in the ventricular myocardium of patients with heart failure. Furthermore, a recent study demonstrated the significant cardiac production of CNP in chronic heart failure by detecting the step-up in plasma CNP concentration from the aorta to the coronary sinus [10]. In addition, coronary sinus CNP levels correlated with mean pulmonary capillary wedge pressure. On the other hand, both cardiac myocytes and fibroblasts express NRP-B, and the NPR-B mRNA level is markedly increased in the hypertrophied left ventricle. Although further investigations are required to clarify the pathophysiological role of CNP in the heart, cardiac CNP may act locally but not systemically in some pathological states.

References [1] Alexander MR, Knowles JW, Nishikimi T, Maeda N. Increased atherosclerosis and smooth muscle cell hypertrophy in natriuretic peptide receptor A−/− apolipoprotein E−/− mice. Arterioscler Thromb Vasc Biol 2003; 23: 1077–82. [2] Barr CS, Rhodes P, Struthers AD. C-type natriuretic peptide. Peptides 1996; 17: 1243–51. [3] Calderone A, Thaik CM, Takahashi N, Chang DLF, Colucci WS. Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J Clin Invest 1998; 101: 812–8. [4] Hama N, Itoh H, Shirakami G, Nakagawa O, Suga S, Ogawa Y, et al. Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction. Circulation 1995; 92: 1558–64.

[5] Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K. Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes. Hypertension 2000; 35: 19–24. [6] Horio T, Tokudome T, Maki T, Yoshihara F, Suga S, Nishikimi T, et al. Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts. Endocrinology 2003; 144: 2279–84. [7] Inoue K, Naruse K, Yamagami S, Mitani H, Suzuki N, Takei Y. Four functionally distinct C-type natriuretic peptides found in fish reveal new evolutionary history of the natriuretic system. Proc Natl Acad Sci USA 2003; 100: 10079–84. [8] John SW, Krege JH, Oliver PM, Hagaman JR, Hodgin JB, Pang SC, et al. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 1995; 267: 679–81. [9] Kalra PR, Anker SD, Struthers AD, Coats AJS. The role of C-type natriuretic peptide in cardiovascular medicine. Eur Heart J 2001; 22: 997–1007. [10] Kalra PR, Clague JR, Bolger AP, Anker SD, Poole-Wilson PA, Struthers AD, et al. Myocardial production of C-type natriuretic peptide in chronic heart failure. Circulation 2003; 107: 571–3. [11] Kangawa K, Matsuo H. Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide. Biochem Biophys Res Commun 1984; 118: 131–9. [12] Koller KJ, Goeddel DV. Molecular biology of the natriuretic peptides and their receptors. Circulation 1992; 86: 1081–8. [13] Lang RE, Unger T, Ganten D. Atrial natriuretic peptide: a new factor in blood pressure control. J Hypertens 1987; 5: 255–71. [14] Lin X, Hanze J, Heese F, Sodmann R, Lang RE. Gene expression of natriuretic peptide receptors in myocardial cells. Circ Res 1995; 77: 750–8. [15] Lopez MJ, Wong SK, Kishimoto I, Dubois S, Mach V, Friesen J, et al. Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide. Nature 1995; 378: 65–8. [16] Matsuo H, Nakazato H. Molecular biology of atrial natriuretic peptides. Endocrinol Metab Clin North Am 1987; 16: 43–61. [17] McBride K, Nemer M. Regulation of the ANF and BNP promoters by GATA factors: lessons learned for cardiac transcription. Can J Physiol Pharmacol 2001; 79: 673–81. [18] Mimeault M, De Lean A, Lafleur M, Bonenfant D, Fournier A. Evaluation of conformational and binding characteristics of various natriuretic peptides and related analogs. Biochemistry 1995; 34: 955–64. [19] Morita E, Yasue H, Yoshimura M, Ogawa H, Jougasaki M, Matsumura T, et al. Increased plasma levels of brain natriuretic peptide in patients with acute myocardial infarction. Circulation 1993; 88: 82–91. [20] Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, et al. Brain natriuretic peptide as a novel cardiac hormone in humans. J Clin Invest 1991; 87: 1402–12. [21] Nagaya N, Nishikimi T, Okano Y, Uematsu M, Satoh T, Kyotani S, et al. Plasma brain natriuretic peptide levels increase in proportion to the extent of right ventricular dysfunction in pulmonary hypertension. J Am Coll Cardiol 1998; 31: 202–8. [22] Nagaya N, Nishikimi T, Uematsu M, Satoh T, Kyotani S, Sakamaki F, et al. Plasma brain natriuretic peptide as a prognostic indicator in patients with primary pulmonary hypertension. Circulation 2000; 102: 865–70. [23] Nishikimi T, Hagaman JR, Takahashi N, Kim HS, Matsuoka H, Smithies O, et al. Increased susceptibility to heart failure in response to volume overload in mice lacking natriuretic peptide receptor-A gene. Cardiovasc Res 2005; 66: 94–103. [24] Nishikimi T, Miura K, Minamino N, Takeuchi K, Takeda T. Role of endogenous atrial natriuretic peptide on systemic and renal

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[35] Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature 1988; 332: 78–81. [36] Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type natriuretic peptide: a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 1990; 168: 863–70. [37] Suga S, Nakao K, Itoh H, Komatsu Y, Ogawa Y, Hama N, et al. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-β. J Clin Invest 1992; 90: 1145–9. [38] Tamura N, Ogawa Y, Chusho H, Nakamura K, Nakao K, Suda M, et al. Cardiac fibrosis in mice lacking brain natriuretic peptide. Proc Natl Acad Sci USA 2000; 97: 4239–44. [39] Troughton RW, Frampton CM, Yandle TG, Espiner EA, Nicholls MG, Richards AM. Treatment of heart failure guided by plasma aminoterminal brain natriuretic peptide (N-BNP) concentrations. Lancet 2000; 355: 1126–30. [40] Yan W, Wu F, Morser J, Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptideconverting enzyme. Proc Natl Acad Sci USA 2000; 97: 8525–9. [41] Yasue H, Yoshimura M, Sumida H, Kikuta K, Kugiyama K, Jougasaki M, et al. Localization and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Circulation 1994; 90: 195–203. [42] Yukimura T, Ito K, Takenaga T, Yamamoto K, Kangawa K, Matsuo H. Renal effects of a synthetic alpha-human atrial natriuretic polypeptide in anesthetized dogs. Eur J Pharmacol 1984; 103: 363–6. [43] Zhao L, Long L, Morrell NW, Wilkins MR. NPR-A-Deficient mice show increased susceptibility to hypoxia-induced pulmonary hypertension. Circulation 1999; 99: 605–7.

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166 Urotensin and Its Related Peptides KAZUHIRO TAKAHASHI AND KAZUHITO TOTSUNE

ABSTRACT

UROCORTIN (UROTENSIN I)

Urotensins are peptide hormones originally discovered in the fish neuroendocrine organ urophysis, which is located in the caudal spinal cord. Human homologs of urotensins have recently been identified. Urocortins (urocortin 1, 2, and 3) are human homologs of urotensin I and belong to the corticotropin-releasing hormone (CRH) family peptides. Urocortins have cardiovascular effects, such as vasodilatation and positive inotropic action, which are mediated by CRH type 2 receptors. Human urotensin II is a cyclic peptide consisting of 11 amino acids. Human urotensin II acts on the G-protein-coupled receptor GPR14 and exerts potent vasoconstrictor effects. In certain blood vessels, it also causes vasodilatation, possibly via the release of vasodilator substances, such as NO, from the vascular endothelium. Plasma concentrations of urotensin II are elevated in patients with chronic renal failure, heart failure, or diabetes mellitus. The expression of urotensin II is enhanced in cardiac tissue obtained from patients with heart failure or in blood vessels with atherosclerotic lesions. Thus, human homologs of urotensins (urocortins and urotensin II) are novel cardiovascular peptides that may be related to the pathophysiology of cardiovascular disease.

Discovery of Urocortins Urocortin 1 (Ucn1), a 40-amino-acid peptide, was discovered from the rat brain as a mammalian homolog of the fish peptide, UI [29]. Human Ucn1 has 63% identity with fish UI and 43% identity with rat/human CRH at the amino acid level. Ucn1 binds to both CRH type 1 and type 2 receptors (CRH-R1 and CRHR2) (Fig. 1). Urocortin 2 (Ucn2)/stresscopin-related peptide (SRP) and urocortin 3 (Ucn3)/stresscopin (SCP) were discovered by searching the public genome databases as specific agonists for CRH-R2 (Fig. 1) [9, 12, 19]. Ucn1, 2, and 3 form the CRH family together with CRH, fish UI, and frog sauvagine.

Distribution of the Ucn mRNAs and Peptides in the Cardiovascular System Ucn1 and 3 are expressed in various human tissues, including the brain, pituitary, and heart [11, 15, 25, 26]. Both Ucn1 and 3 are expressed in the heart, particularly cardiomyocytes [11, 25, 26]. On the other hand, the presence of Ucn2 peptide in human tissues has not been established because the predicted amino acid sequence of human Ucn2 precursor lacks a consensus proteolytic cleavage site that would allow for C-terminal processing for the peptide [9, 19].

INTRODUCTION Urotensins were originally discovered from urophysis, a fish neuroendocrine organ located in the caudal spinal cord. Urotensin I (UI) was considered to be a fish CRH-like peptide, whereas urotensin II (UII) was a somatostatin-like fish peptide. Recent studies have identified human forms of urotensins and revealed their (patho)physiological roles in various human diseases, particularly cardiovascular disease [1, 7, 9, 12, 19, 29]. Handbook of Biologically Active Peptides

Processing and Endogenous Form in the Cardiovascular System, Including Plasma Sephadex G50 column chromatography showed that Ucn1-like immunoreactivity in the heart was mostly eluted in a larger-molecular-weight region (corresponding to an approximately 45- to 50-amino acid peptide) [11]. Only a small amount of Ucn1-like immunoreactivity in the heart was eluted in the same position

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1210 / Chapter 166 P e pt i d e s ( Li g a n ds )

Rece pt ors

Representative biological actions

CRH CR H t y p e 1 Ucn 1

receptor

ACTH release anxiety-like effect

Ucn 2/SRP C R H ty p e 2 Ucn 3/SCP

re cep to r

A nx i ol y s i s Appetite-inhibiting effect Vasodilatation Positive inotropic action on myocardium D e a r o u s al

as authentic Ucn1. These findings suggested that most of Ucn1-like immunoreactivity in the heart was a larger-molecular-weight form, possibly a precursor fragment. Ucn3 is a 38-amino-acid peptide that corresponds to the sequence 3–40 of human SCP, a 40-amino-acid peptide. Reversed-phase high-performance liquid chromatography (HPLC) of Ucn3-like immunoreactivity in the heart, brain, and kidney extracts showed a large broad peak eluting in the position of Ucn3, with one smaller peak in the position of SCP and two smaller peaks eluting earlier [25]. This finding suggests that Ucn3-like immunoreactivity in these tissues consists of multiple molecular forms including Ucn3 and SCP. Reversed-phase HPLC of the plasma extract showed that a peak eluting earlier was larger than two peaks eluting in the positions of Ucn3 and SCP. Ucn3-like immunoreactivity in plasma may be modified to the hydrophilic form.

Receptors and Their Distribution in the Cardiovascular System The actions of the CRH family peptides are mediated by at least two types of G-protein-coupled receptors: CRH-R1 [6] and CRH-R2 [13, 14]. CRH-R1 mediates adrenocorticotropic hormone (ACTH) responses to stress, whereas CRH-R2 mediates stress-coping responses including anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium, and dearousal (Fig. 1). CRH-R2 is composed of at least three different isoforms: CRH-R2α, CRH-R2β, and CRH-R2γ. In the rat, CRH-R2α is expressed predominantly in the brain, whereas CRH-R2β is expressed in both the brain and peripheral tissues, especially in heart and skeletal muscle tissues [14]. In contrast to rodents, CRH-R2α appears to be the major CRH-2R in the brain, heart, and skeletal muscle tissues, whereas CRH-R2β is considered to be the minor isoform in humans [11, 13]. Actually, CRH-R2α was

FIGURE 1. Relationship between corticotropin-releasing hormone (CRH) family peptides and CRH receptors, and representative biological actions. SCP, stresscopin; SRP, stresscopin-related peptide; Ucn, urocortin. Reproduced from [26] Takahashi et al. Peptides 2004; 25: 1723–1731 with kind permission from the Elsevier Inc. Copyright 2004 Elsevier Inc.

expressed both in atria and ventricles obtained at autopsy in all cases examined [11].

Biological Actions in the Cardiovascular System Ucns have been demonstrated to have potent coronary vasodilatory and cardiac inotropic effects, and these effects have been shown to be more potent than CRH [2, 17]. Ucns have protective effects on cardiac myocytes from ischemic or reperfusion injury [5]. These protective effects of Ucns were mediated by the upregulation of p42/p44 mitogen-activating protein kinase (MAPK) signaling pathway, activation of protein kinase B/Akt, and induction of K(ATP) channel gene expression. Ucn2 and Ucn3 were more potent in anti-apoptotic effects on cardiomyocytes than Ucn1. Ucn1 has a stimulatory effect on proliferation of cardiac nonmyocytes and myocytes. Moreover, Ucn1 stimulates ANP and BNP secretions from neonatal rat cardiomyocytes [10]. The vasodilator effects of Ucn1 may be caused not only by its direct effect on vascular smooth muscle cells but also by its effect on mast cell degranulation [23]. Ucn1 and CRH induced rat skin mast cell degranulation and increased vascular permeability. This effect appeared to be mediated not by the CRH-R1, CRH-R2α, or CRH-R2β but perhaps by the CRH-R2γ. Furthermore, Ucn1 and CRH are synthesized and secreted by human mast cells.

Pathophysiological Implications Including Alterations of Plasma Concentrations in Physiological and Pathological Conditions Plasma concentrations of Ucn1 are elevated in patients with heart failure [16]. The expression of Ucn1 was increased in the heart with diseases, such as dilated cardiomyopathy [15]. In animals with experimental heart failure, the intravenous infusion of Ucn1 or Ucn2 showed beneficial hemodynamic, endocrine, and renal

Urotensin and Its Related Peptides / 1211 effects, such as increased cardiac output; decreased peripheral resistance; decreased plasma concentrations of renin, aldosterone, and endothelin-1; and increased urine volume and sodium excretion [2, 18]. Ucn1-deficient mice displayed an impaired acoustic startle response, suggesting that Ucn1 modulates the acoustic startle response through the Ucn1-expressing neuron projections from the region of the EdingerWestphal nucleus [30]. On the other hand, there has been no report showing abnormal cardiovascular function in Ucn1-deficient mice. Ucn2 and/or Ucn3 may compensate for the cardiovascular effects of Ucn1 in Ucn1-deficient mice. Another possibility is that Ucn1 (as well as Ucn2 and Ucn3) may have important regulatory actions on the cardiovascular system only in certain aspects of stresses, such as myocardial ischemia.

UROTENSIN II Discovery The cloning of the human UII cDNA has revealed that human UII is composed of only 11 amino acid residues [7], whereas fish and frog UII possess 12 and 13 amino acid residues, respectively. Urotensin-II-related peptide (URP) was identified as the UII-immunoreactive molecule in the rat brain [24] and binds to urotensin II receptor (UT receptor). URP is an 8-amino-acid peptide (ACFWKYCV). cDNA cloning showed that the amino acid sequence of human and mouse URP is the same as that of rat URP.

Distribution of the UII mRNA and Peptide in the Cardiovascular System UII mRNA and peptide are distributed widely in the cardiovascular system, including the heart and vascular vessels [8, 28]. The expression of UII was enhanced in the cardiac tissue obtained from patients with heart failure [8]. Furthermore, strong UII immunostaining was observed in the blood vessels with atherosclerotic lesions, including endothelial, smooth muscle, and inflammatory cells of the atherosclerotic plaques [4].

Processing and Endogenous Form in the Cardiovascular System Including Plasma Reversed-phase HPLC of the plasma extract showed three peaks: one eluting earlier, one in the position of authentic UII, and one eluting later [27]. The nature of materials eluting earlier or later has not been clarified. The tissue concentrations of UII are very low in the human tissues, including the brain and heart, and

UII-like immunoreactivity in these tissues has not been chromatographically characterized.

Receptors and Their Distribution in the Cardiovascular System The reverse pharmacological approach has shown that human UII is an agonist for GPR14, an orphan receptor with seven-transmembrane domains [1] that is now named UT receptor. The UT receptor is widely distributed in various tissues including heart.

Biological Actions in the Cardiovascular System UII is a potent vasoconstrictor peptide and its vasoconstrictive potency is one order of magnitude greater than that of endothelin 1 [1]. On the other hand, UII elicited vasodilatation in some arteries, possibly through the release of endothelium-derived hyperpolarizing factor and nitric oxide [3]. In addition to actions on vascular vessels, this peptide has a positive inotropic action [21], stimulates the proliferation of vascular smooth muscle cells [31], and induces hypertrophic responses in cultured cardiomyocytes. Furthermore, it inhibited insulin release from the perfused rat pancreas [22].

Pathophysiological Implications Including Alterations of Plasma Concentrations in Physiological and Pathological Conditions Plasma levels of UII-like immunoreactivity were elevated in patients with chronic renal failure [28]. Plasma concentrations of UII-like immunoreactivity were twofold higher in patients not on dialysis and threefold higher in those on hemodialysis than in healthy individuals (Fig. 2). Decreased elimination of UII from blood by kidney may explain increased plasma levels of UII-like immunoreactivity in patients with chronic renal failure. Increased production and/or secretion of UII, however, could not be excluded as one of the reasons for the elevated plasma levels of UII-like immunoreactivity in this condition. For example, hypertension accompanied by renal failure may stimulate the production and/or secretion of UII from the vascular tissues. Furthermore, increased expression of UII in the atherosclerotic lesion has been reported [4]. UII-like immunoreactivity was found in human urine [27], so the UII secreted by renal tubular cells into urine may account for UII-like immunoreactivity in urine. UII produced by the kidneys may also have roles in the renal circulation and the water-electrolyte transport in the kidney. Plasma levels of UII-like immunoreactivity were also increased in patients with heart failure [20], liver cirrhosis and portal hypertension, and diabetes mellitus

1212 / Chapter 166 p < 0.0001 p < 0.0001 p < 0.0063

Urotensin II concentration (fmol/mL)

20

effects or UII antagonists are expected to be effective in the treatment of cardiovascular diseases, such as hypertension and heart failure.

References 15

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FIGURE 2. Plasma concentrations of urotensin II in patients with chronic renal failure. Open circles and bars show mean and SEM, respectively. Reproduced from [28] Totsune et al. Lancet 2001; 358: 810–811 with kind permission from the Elsevier Inc. Copyright 2001 Elsevier Inc.

[27]. UII was strongly expressed in the cardiomyocytes in patients with end-stage congestive heart failure, suggesting that cardiomyocytes are one of the major sources of elevated plasma UII in these patients [8]. Plasma levels of UII-like immunoreactivity were elevated in patients with diabetes mellitus without renal failure (with normal serum creatinine) [27]. Because UII was expressed in cultured vascular endothelial cells, vascular endothelial damage accompanied by diabetes mellitus may account for the elevation of plasma UII-like immunoreactivity. Interestingly, the association of certain polymorphisms in the UII gene with type 2 diabetes mellitus has been reported [32]. UII may be related not only to the diabetic vascular complications but also to the control of carbohydrate metabolism.

CONCLUSION Human homologs of urotensins (Ucns and UII) are cardiovascular peptides that are expressed in cardiovascular organs including the heart. Ucns appear to protect the heart against various stresses via the CRH-R2, whereas UII may worsen heart diseases and promote the atherosclerotic process via its potent vasoconstrictor and cell proliferative effects. Drugs with urocortinlike

[1] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 401: 282–6. [2] Bale TL, Hoshijima M, Gu Y, Dalton N, Anderson KR, Lee KF, et al. The cardiovascular physiologic actions of urocortin II: acute effects in murine heart failure. Proc Natl Acad Sci USA 2004; 101: 3697–702. [3] Bottrill FE, Douglas SA, Hiley CR, White R. Human urotensin-II is an endothelium-dependent vasodilator in rat small arteries. Brit J Pharmacol 2000; 130: 1865–70. [4] Bousette N, Patel L, Douglas SA, Ohlstein EH, Giaid A. Increased expression of urotensin II and its cognate receptor GPR14 in atherosclerotic lesions of the human aorta. Atherosclerosis 2004; 176: 117–23. [5] Brar BK, Jonassen AK, Stephanou A, Santilli G, Railson J, Knight RA, et al. Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway. J Biol Chem 2000; 275: 8508–14. [6] Chen R, Lewis KA, Perrin MH, Vale WW. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 1993; 90: 8967–71. [7] Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint H, Beauvillain JC, et al. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci USA 1998; 95: 15803–8. [8] Douglas SA, Tayara L, Ohlstein EH, Halawa N, Giaid A. Congestive heart failure and expression of myocardial urotensin II. Lancet 2002; 359: 1990–7. [9] Hsu SY, Hsueh AJ. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat Med 2001; 7: 605–11. [10] Ikeda K, Tojo K, Sato S, Ebisawa T, Tokudome G, Hosoya T, et al. Urocortin, a newly identified corticotropin-releasing factorrelated mammalian peptide, stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes. Biochem Biophys Res Commun 1998; 250: 298–304. [11] Kimura Y, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, Darnel AD, et al. Expression of urocortin and corticotropinreleasing factor receptor subtypes in the human heart. J Clin Endocrinol Metab 2002; 87: 340–6. [12] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, et al. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001; 98: 7570–5. [13] Liaw CW, Lovenberg TW, Barry G, Oltersdorf T, Grigoriadis DE, de Souza EB. Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid. Endocrinology 1996; 137: 72–7. [14] Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, et al. Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 1995; 92: 836–40. [15] Nishikimi T, Miyata A, Horio T, Yoshihara F, Nagaya N, Takishita S, et al. Urocortin, a member of the corticotropin-releasing

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factor family, in normal and diseased heart. Am J Physiol Heart Circ Physiol 2000; 279: H3031–9. Ng LL, Loke IW, O’Brien RJ, Squire IB, Davies JE. Plasma urocortin in human systolic heart failure. Clin Sci (Lond) 2004; 106: 383–8. Parkes DG, Weisinger RS, May CN. Cardiovascular actions of CRH and urocortin: an update (review). Peptides 2001; 22: 821–7. Rademaker MT, Charles CJ, Espiner EA, Fisher S, Frampton CM, Kirkpatrick CM, et al. Beneficial hemodynamic, endocrine, and renal effects of urocortin in experimental heart failure: comparison with normal sheep. J Am Coll Cardiol 2002; 40: 1495–505. Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, et al. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001; 98: 2843– 8. Richards AM, Nicholls MG, Lainchbury JG, Fisher S, Yandle TG. Plasma urotensin II in heart failure. Lancet 2002; 360: 545–6. Russell FD, Molenaar P, O’Brien DM. Cardiostimulant effects of urotensin-II in human heart in vitro. Brit J Pharmacol 2001; 132: 5–9. Silvestre RA, Rodriguez-Gallardo J, Egido EM, Marco J. Inhibition of insulin release by urotensin II—a study on the perfused rat pancreas. Horm Metab Res 2001; 33: 379–81. Singh LK, Boucher W, Pang X, Letourneau R, Seretakis D, Green M, et al. Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin-releasing hormone receptors. J Pharmacol Exp Ther 1999; 288: 1349–56. Sugo T, Murakami Y, Shimomura Y, Harada M, Abe M, Ishibashi Y, et al. Identification of urotensin II-related peptide as the

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[32]

urotensin II-immunoreactive molecule in the rat brain. Biochem Biophys Res Commun 2003; 310: 860–8. Takahashi K, Totsune K, Murakami O, Saruta M, Nakabayashi M, Suzuki T, et al. Expression of urocortin III/stresscopin in human heart and kidney. J Clin Endocrinol Metab 2004; 89: 1897–903. Takahashi K, Totsune K, Murakami O, Shibahara S. Urocortins as cardiovascular peptides. Peptides 2004; 25: 1723–31. Totsune K, Takahashi K, Arihara Z, Sone M, Murakami O, Ito S, et al. Elevated plasma levels of immunoreactive urotensin II and its increased urinary excretion in patients with Type 2 diabetes mellitus: association with progress of diabetic nephropathy. Peptides 2004; 25: 1809–14. Totsune K, Takahashi K, Arihara Z, Sone M, Satoh F, Ito S, et al. Role of urotensin II in patients on dialysis. Lancet 2001; 358: 810–1. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995; 378: 287–92. Vetter DE, Li C, Zhao L, Contarino A, Liberman MC, Smith GW, et al. Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior. Nat Genet 2002; 31: 363–9. Watanabe T, Pakala R, Katagiri T, Benedict CR. Synergistic effect of urotensin II with mildly oxidized LDL on DNA synthesis in vascular smooth muscle cells. Circulation 2001; 104: 16–8. Wenyi Z, Suzuki S, Hirai M, Hinokio Y, Tanizawa Y, Matsutani A, et al. Role of urotensin II gene in genetic susceptibility to type 2 diabetes mellitus in Japanese subjects. Diabetologia 2003; 46: 972–6.

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167 Vasoactive Intestinal Peptide DARRELL R. SAWMILLER AND ROBERT J. HENNING

During cardiomyopathy and heart failure due to coronary artery disease, the myocardial concentration of VIP, VIP receptor density, and VIP-elicited myocardial contractility can decrease as much as 85%. In addition, VIP receptor density and/or coupling to adenylyl cyclase can decrease with hypertension, obesity, diabetes, and hypothyroidism. A decrease in myocardial VIP concentration and VIP receptor density can enhance coronary vascular resistance, reduce myocardial contractility and heart rate, and lead to myocardial fibrosis. Therapies that enhance the coronary and myocardial VIP concentration and/or VIP receptor expression may be effective in increasing coronary artery blood flow in patients with angina pectoris and may also improve symptoms in patients with cardiomyopathy and limit the progression of heart failure.

ABSTRACT Vasoactive intestinal peptide (VIP) is a 28-amino-acid neuropeptide discovered by Said and Mutt in 1970 that has diverse cardiovascular effects, including vasodilation of the cerebral and coronary arteries and augmentation of myocardial contractility and heart rate. VIP immunoreactive nerve fibers originate from the central, peripheral, and the intrinsic nervous systems of the gastrointestinal tract, tracheobronchial tree, genitourinary tract, pancreas, and heart. These fibers innervate blood vessels throughout the body as well as the conduction system of the heart and the myocardium. VIP directly causes coronary vasodilation with a median effective concentration (EC50) of 4 –5 × 10−11 M, which appears to be mediated by activation of VPAC1 and VPAC2 receptors, adenylyl cyclase, and potassium channels. At concentrations greater than that required for coronary vasodilation, VIP can increase atrial and ventricular contractility and heart rate, decrease the atrioventricular conduction time, and decrease atrial and ventricular refractory periods. In anesthetized dogs, endogenous VIP release elicited by vagal nerve stimulation increases left coronary artery blood flow by as much as 62%, increases right atrial and right ventricular contractile performance by as much as 28%, and increases heart rate by as much as 50%. These VIPelicited effects can be blocked by [4Cl-d-Phe6,Leu17]VIP, a sensitive and selective antagonist of VIP receptors. The concentration of VIP in the heart reflects a balance between local neurogenic VIP synthesis and release and removal by the circulatory and lymphatic systems. Circulating VIP is metabolized in the liver and lung by neutral endopeptidase. VIP is released from neurons in the coronary vasculature and myocardium after acute coronary occlusion and acts as a coronary vasodilator and free-radical scavenger to limit myocardial ischemia. Handbook of Biologically Active Peptides

INTRODUCTION Vasoactive intestinal peptide (VIP) is a 28-amino-acid neuropeptide discovered by Said and Mutt in 1970 that has diverse cardiovascular effects, including vasodilation of the cerebral and coronary artery circulations and an increase in myocardial contractile performance [7, 23, 24, 25, 50]. VIP appears to exert its effects by activating signaling processes involving VPAC1 and VPAC2 receptors, adenylyl cyclase, guanylyl cyclase, and potassium channels [7, 22, 25, 49]. VIP immunoreactive nerve fibers originate from the central, peripheral, and intrinsic nervous systems and innervate blood vessels throughout the body as well as the myocardium [21, 58]. VIP is metabolized in the circulation by neutral endopeptidase [16, 53]. The structure of the VIP gene and precursor mRNA, the processing of the VIP precursor, and VIP solution conformation are covered within Chapter 94 on PACAP/VIP in the Brain Peptides Section.

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Copyright © 2006 Elsevier

1216 / Chapter 167 DISCOVERY After the seminal discovery in 1902 by Bayliss and Starling of an intestinal extract that had vasodepressor properties, Said and Mutt isolated and sequenced an acid-extractable peptide from the duodenum that produced a long-lasting decrease in the systemic arterial pressure of anesthetized dogs [48]. When exogenous VIP was administered, femoral and mesenteric arterial blood flow, the stroke volume of the heart, and heart rate increased. Because the action of the endogenous peptide was initially thought not to extend beyond the intestine due to metabolism by the liver, the peptide was named vasoactive intestinal peptide. Subsequent studies found this peptide in high concentrations in the plasma and in tumor tissues of patients with pheochromocytoma and ganglioneuroblastoma, in neuroblastoma and astrocytoma cell lines, and widely distributed in the central and peripheral nervous systems [47, 49]. In addition to VIP immunoreactive nerve fibers, numerous VIP immunoreactive cell bodies have been discovered in the gastrointestinal tract, tracheobronchial tree, genitourinary tract, pancreas, and heart, indicating that there is also a peripheral intrinsic VIP-ergic system [34, 58]. The observation that VIP immunoreactive fibers innervate blood vessels in the small intestine, brain, and atrial and ventricular myocardium, as well as the conduction system of the heart, led to additional studies to determine the role of VIP in the regulation of blood flow and cardiac performance.

DISTRIBUTION VIP immunoreactive nerve fibers innervate blood vessels throughout the central and peripheral nervous systems as well as the heart. In the heart of guinea pigs, rats, and dogs, VIP-ergic fibers primarily innervate the sinoatrial node, cardiac glomeruli of the proximal coronary arteries, ascending aorta and pulmonary trunk, and intramural and terminal arteries of the atria [58]. A moderate number of VIP-ergic fibers innervate the atrioventricular node, the perivascular nerve plexuses of the major coronary arteries, and the atrial myocytes. Some VIP-ergic fibers innervate the intracardiac ganglia and the intramural and terminal vasculature of the ventricles. The intracardiac ganglia also contain VIPimmunoreactive cell bodies [58]. Although these fibers may be primarily of intrinsic origin, some extrinsic parasympathetic preganglionic efferent, parasympathetic and sympathetic postganglionic efferent, and parasympathetic and sympathetic afferent VIP-ergic fibers also exist [21]. An extrinsic VIP-ergic innervation exists particularly in the canine right ventricle [3]. The concentration of VIP in the left-ventricular epicardial

coronary arteries varies between 0.7 and 2.2 pmol g−1 tissue, with the highest concentrations in the proximal coronary regions [6]. A VIP-ergic innervation of the epicardial coronary arteries may play a role in maintaining coronary vasorelaxation. In the cerebral circulation, interneurons containing VIP innervate arterioles and receive input from acetylcholine and serotonin containing neural projections that contact cell soma and proximal dendrites [7]. Electrical stimulation of these interneurons produces vasodilation of adjacent arterioles, supporting a role for VIP-ergic neurons in the control of cerebral blood flow.

PROCESSING VIP is metabolized by neutral endopeptidase 24.11 (NEP), a transmembrane metallopeptidase that is abundant in the central nervous system, kidneys, liver, lung, and intestinal epithelium [46]. This enzyme cleaves a variety of peptides in addition to VIP, including enkephalins, angiotensin I, angiotensin II, substance P, neurotensin, and atrial natriuretic peptides. The topical application of two NEP inhibitors, phosphoramidon and thiorphan, enhances the vasodilatory effect of VIP in resistance vessels of the hamster cheek pouch, demonstrating a role for vascular NEP in limiting the vasoreactivity of VIP [53]. The plasma concentration of VIP increases 3.6-fold, from 30 fmol ⋅ ml−1, after a single dose of the NEP blocker UK77-568, but then returns to baseline after four consecutive daily doses of the NEP blocker due to feedback inhibition of VIP release [16]. The major sites of VIP metabolism in the circulation are the lungs, liver, and kidneys, which contribute to a VIP half-life of 2–5 min [28]. Other tissues, such as the intestine, brain, and heart, play little role in VIP metabolism. In the heart, the concentration of VIP reflects a balance between local neurogenic synthesis and release in the myocardium and removal by the circulatory and lymphatic systems. During chronic NEP inhibition, the myocardial VIP concentration can increase twofold [16]. The rate of VIP metabolism is decreased by increased dietary sodium intake and by angiotensin II [12, 13]. Angiotensin-converting enzyme (ACE) inhibition can increase the myocardial VIP concentration by as much as 4.5-fold, probably resulting from increased local VIP synthesis and release in the myocardium [17]. This increased concentration of VIP in the myocardium may contribute to the improvement of cardiac function in patients with cardiomyopathy who are treated with ACE inhibitors. However, chronic ACE inhibition does not change the concentration of VIP in the plasma, kidneys, and lung because it increases the synthesis and release, as well as the synthesis and release metabolism of VIP [17].

Vasoactive Intestinal Peptide / 1217

RECEPTORS Two subtypes of VIP receptors, VPAC1 and VPAC2, have been identified in the cardiovascular system. These G-protein-coupled receptors are members of the secretin receptor family and bind VIP and pituitary adenylate cyclase–activating peptide (PACAP) with equal affinity [25, 33]. In general, the density of VIP receptors in

arteries is greater than that in veins, accounting for VIP’s greater vasodilatory effect on arteries than veins [37]. VPAC1 and VPAC2 receptors have been identified in the endothelium and smooth muscle of the major arteries and arterioles of the right and left ventricle [50] (Fig. 1). VPAC1 and VPAC2 receptors are also present in the endothelium and smooth muscle of cerebral arteries and arterioles [7]. These observations

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FIGURE 1. Presence of (A,C) VPAC1 and (B,D) VPAC2 receptors in the (A,B) left-ventricular coronary artery and (C,D) coronary arterioles of the left ventricle of the rat as determined by immunohistochemistry. Negative controls are also shown (E,F) [50]. The primary stain is Dako DAB and the counterstain is hematoxylin. Scale bar represents 50 μm for A and B and 25 μm for C–F. Large arrow denotes location of artery or arteriole. Dashed arrow denotes regions expressing high levels of VPAC1 or VPAC2.

1218 / Chapter 167 suggest that both receptors may be involved in the control of the coronary and cerebral arterial blood flow. The involvement of VPAC1 and VPAC2 receptors in vascular regulation has been recently demonstrated by blocking pressure-induced cutaneous vasodilation with either VPAC1 or VPAC2 receptor antagonists, PG97-269 or PACAP6-38, respectively [20]. This implies a series relationship in which both receptors are necessary for the full expression of this neurally mediated cutaneous vasodilatory reflex. However, the specific involvement of VPAC1 and VPAC2 receptors and their interaction in the control of coronary or cerebral arterial blood flow has not yet been determined. Both VPAC1 and VPAC2 receptors are also present in atrial and ventricular myocardium [39, 56, 57]. The expression of both receptors in the coronary arteries, arterioles, and myocardium contrasts with that in intrinsic cardiac neurons, where primarily VPAC2 receptors are expressed [14].

BIOLOGICAL ACTIONS VIP produces vasodilation in the coronary, cerebral, mesenteric, pulmonary, and cutaneous circulations. In isolated coronary arterial strips and whole arteries, VIP produces vasorelaxation independent of the endothelium [5, 21, 25]. VIP also produces coronary vasodilation and increases coronary blood flow in intact animals and in isolated heart preparations, which is mediated by a direct action on the coronary vasculature. In isolated perfused rat hearts, for example, VIP produces coronary vasodilation with an EC50 of 4 –5 × 10−11 M without increasing heart rate, myocardial contractility, or myocardial oxygen consumption [50]. As a coronary vasodilator, VIP is 200-fold more potent than adenosine and 8000-fold more potent than sodium nitroprusside [50]. In anesthetized dogs maintained with a fixed cardiac output, systemic arterial pressure, and heart rate, the injection of VIP into the left main coronary artery increases coronary blood flow by as much as 84% but increases myocardial oxygen consumption by only 32% [2]. In humans, the infusion of VIP at rates of 9– 30 pmole min−1 into the left coronary artery decreases arterial-coronary sinus O2 content difference and myocardial O2 extraction by as much as 41% without changing the left-ventricular preload, afterload, contractility, or heart rate [41]. In addition to a direct coronary arterial vasodilatory effect, intravenous VIP may also increase coronary blood flow in humans due to increases in heart rate and stroke volume, probably resulting from a reduction in afterload [51]. Additional studies indicate that endogenous VIP is released in the coronary vasculature after parasympathetic nerve stimulation and produces significant coronary vasodilation. Vagal nerve stimulation in anesthetized dogs, after blockade of muscarinic

and β-adrenergic receptors, can increase left coronary artery blood flow by as much as 62% without changing left-ventricular contractility [18, 19]. This increase in blood flow is blocked by [4Cl-d-Phe6,Leu17]VIP, a sensitive and selective antagonist of VIP receptors. The coronary vasodilatory response to VIP involves the activation of calcium-activated potassium channels and voltage-sensitive potassium channels, sequestration of calcium in thapsigargin-sensitive stores, and a decrease in the calcium sensitivity of the contractile apparatus [32]. Cyclic AMP is involved during maximal VIP-elicited coronary vasodilation; however, the vasodilatory response to VIP vasodilation. The vasodilatory response, however, does not appear to involve cyclic GMP [50] (Fig. 2). VIP-elicited vasodilation in the cerebral, mesenteric, ovarian, and pulmonary circulations may also involve the activation of adenylyl cyclase [25]. However, the degree of VIP-elicited activation of adenylyl cyclase varies with the type of vessel studied as well as the species (rat, guinea pig, rabbit, cat, cow, or pig). Moreover, the vasodilatory effect of VIP may not be precisely correlated with an increase in cyclic AMP. VIP-elicited vasodilation in the cerebral circulation also involves nitric oxide and cyclic GMP through a direct action on vascular smooth muscle [22] and, in the mesenteric circulation, involves the inhibition of intracellular calcium mobilization independent of cyclic AMP [30]. In the pulmonary circulation, VIP-elicited vasodilation is dependent on the endothelium and involves both cyclic AMP and cyclic GMP, and is probably increased by prostacyclin and nitric oxide, respectively [29]. At concentrations greater than that required for coronary vasodilation, VIP can increase myocardial contractility and heart rate. In canine trabeculae, isolated from the right and left atria and ventricles, VIP at concentrations greater than 1 × 10−9 M can increase contractile force by as much as 100% [43]. Similar concentrations of VIP increase contractility of human atrial and ventricular trabeculae by 15–25% [39]. This increase in contractility appears to be mediated by the activation of adenylyl cyclase [9, 25]. In anesthetized dogs, VIP release elicited by vagal nerve stimulation, after blockade of muscarinic and β-adrenergic receptors, increases right atrial and right ventricular contractility by as much as 28%, but does not increase left-ventricular contractility [23, 24]. This positive right-, but not left-, ventricular inotropic response is best explained by the fact that abundant VIP-immunoreactive fibers innervate the right but not the left ventricle in the dog [3, 23, 24]. The increase in myocardial contractility elicited by exogenous VIP, but not the increase in coronary blood flow, can decrease after repetitive intracoronary infusions of VIP due to VIP receptor desensitization [1]. This suggests that the myocardial and coronary vascular effects of VIP may be mediated by different receptors or signal-

Vasoactive Intestinal Peptide / 1219 6

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Cyclic GMP Release (pmol/min/g)

Cyclic AMP Release (pmol/min/g)

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Coronary Vascular Resistance (Percent Change)

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FIGURE 2. Effect of VIP at 1 × 10 , 1 × 10−10, and 1 × 10−9 M on cyclic AMP release and the effect of VIP at 1 × 10−8 M and sodium nitroprusside (SNP) at 1 × 10−5 M on cyclic GMP release from the heart [50]. Also shown are the effects of these agents on coronary vascular resistance. Values are means ± SE of three hearts for cyclic AMP and three hearts for cyclic GMP. Asterisk indicates P < 0.05. −11

ing mechanisms. Additional studies indicate that VIP increases adenylyl cyclase activity, L-type calcium channel current, and contractility in rat ventricular myocytes [4, 54]. However, the increase in cyclic AMP is small compared with that elicited by isoproterenol, and not all cells respond to VIP. These effects of VIP on adenylyl cyclase activity and contractility in rat ventricular myocytes appear to be mediated by the activation of nonselective receptors that recognize both VIP and secretin [4, 9]. The discovery that VIP-immunoreactive nerve fibers occur in high density in and around the sinus node and

the atrioventricular node of the heart strongly suggests that VIP affects heart rate. In the anesthetized dog, the injection of VIP into the sinoatrial artery after bilateral vagotomy, stellectomy, and blockade of muscarinic receptors increases the heart rate in a dose-dependent manner by as much as 96% [42]. This increase in heart rate is similar to that produced by norepinephrine, but VIP is longer acting and twice as potent as norepinephrine. VIP also decreases the atrioventricular conduction time by as much as 30% and decreases atrial and ventricular refractory periods by 4–15% [44]. In addition, VIP can induce a shift of the pacemaker site to

1220 / Chapter 167 Bachman’s bundle and can potentiate sinus arrhythmia [38, 40]. After the blockade of muscarinic and β-adrenergic receptors, vagal nerve stimulation increases heart rate in a frequency-dependent manner by as much as 50% [23, 24, 27]. This increase in heart rate is accompanied by an increase in VIP release from the right atrium and is attenuated or blocked by several VIP/ antagonists, including [4Cl-d-Phe6,Leu17]VIP. This heart rate response to VIP may play a role in “excess tachycardia,” the increase in heart rate observed after muscarinic receptor blockade minus that observed after vagotomy. VIP may also play a role in “postvagal tachycardia,” the increase in heart rate following intense vagal nerve stimulation after the initial bradycardia has subsided [23].

PATHOPHYSIOLOGY Early studies showed that hypertension, obesity, diabetes, and hypothyroidism reduce the ability of VIP to increase myocardial adenylyl cyclase activity [8, 9, 45]. This attenuation appears to be due to a specific decrease in the density of VIP receptors or to uncoupling of the receptors from adenylyl cyclase. In hearts isolated from 10-weeks-old, spontaneously hypertensive rats of the Wistar-Kyoto strain (SHR), VIP-elicited adenylyl cyclase activity is reduced by as much as 69% compared with normotensive controls (WKY) [8]. In contrast, the adenylyl cyclase activity elicited by the β-adrenergic agonist, isoproterenol, is reduced by only 17%. These reductions are somewhat more pronounced in 14- and 30week-old rats. Furthermore, the reduction of VIP-elicited adenylyl cyclase activity precedes the reduction of isoproterenol-elicited adenylyl cyclase activity. This selective impairment of VIP-elicited adenylyl cyclase activity is specific for the heart and occurs concomitantly with the development of hypertension and myocardial hypertrophy [8, 45]. An impairment of VIP-elicited adenylyl cyclase activity may reduce VIP-elicited vasodilation and VIP effects on myocardial contractility and heart rate and contribute to the development of heart failure. The release of VIP from the heart and the VIP concentration in the coronary sinus blood are increased during coronary artery occlusion and subsequent coronary reperfusion. In the isolated perfused rat heart, the interruption of coronary perfusion for 30 min increases the VIP concentration in the coronary effluent by as much as 250% during the ensuing 60-min reperfusion period [31]. Moreover, coronary perfusion with VIP immediately prior to the induction of ischemia significantly limits the effects of ischemia on the myocardium, as evidenced by a decrease in the release of creatine kinase, reduced formation of hydroxyl radicals, limita-

tion of calcium overload in ventricular myocytes, and enhancement of myocardial contractile performance. These results indicate that VIP is released from neurons in the coronary vasculature and myocardium after acute coronary occlusion and acts as a vasodilator and freeradical scavenger to reduce myocardial ischemia and reperfusion injury. In patients with acute myocardial infarction, the VIP plasma concentration increases by 33% within 6 hours after the onset of symptoms, from normal values of 12.2 fmol ml−1, but then abruptly decreases below normal values to 8.9 fmol ml−1 after 24 hours [36]. This abrupt decrease may be due to the depletion of VIP from nerve endings and the impairment of neurogenic synthesis of VIP in the heart. In patients who die from acute myocardial infarction, the VIP plasma concentration is significantly lower than in those who survive after myocardial infarction. Normal plasma concentrations of VIP vary with a circadian rhythm, which peaks at 2000 h in adults ages 25–50 years and at 1800 h in older adults ages 65–75 years [10]. Moreover, the VIP plasma concentration in older adults of 3.3–4.4 fmol ml−1 is significantly less than the plasma concentration in younger adults of 12.7– 17.0 fmol ml−1. Patients with heart transplants for treatment of primary dilated cardiomyopathy also have reduced VIP plasma concentrations and lack a circadian rhythm [11]. In animal models of heart failure and in patients with cardiomyopathy secondary to coronary artery disease, the left-ventricular concentration of VIP can decrease as much as 85% [55]. Furthermore, in patients with severe heart failure, the VIP receptor density in the left ventricle and the contractile response to VIP in rightventricular trabeculae can decrease by as much as 62% [26]. These decreases in myocardial VIP concentration, receptor density, and function may impair myocardial contractility and enhance coronary vascular resistance, thereby perpetuating heart failure. In addition, a decrease in myocardial VIP concentration may lead to myocardial fibrosis. In animal models of myocardial fibrosis, the myocardial concentration of VIP is inversely correlated with the degree of fibrosis present in the heart [60]. Normotensive (WKY) and hypertensive (SHR) rats fed a high-salt diet have lower myocardial VIP concentrations and greater myocardial interstitial and perivascular fibrosis than rats fed a normal or lowsalt diet. In WKY rats fed a high-salt diet and treated with the nitric oxide synthase inhibitor l-nitro-ωmethylarginine (l-NAME) to induce hypertension, the vasopeptidase inhibitor omapatrilat increases the myocardial VIP concentration and reduces the degree of interstitial and perivascular fibrosis [61]. However, the myocardial VIP concentration and the degree of fibrosis are not altered by the ACE inhibitor enalapril in this rat model. These findings suggest that enhancing the

Vasoactive Intestinal Peptide / 1221 myocardial VIP concentration may reduce the progression of myocardial fibrosis. One mechanism by which VIP may reduce myocardial fibrosis is by reducing the synthesis of one or more profibrotic agents, such as angiotensin II, tumor necrosis factor α (TNFα), and tumor transforming growth factor β (TGFβ) [15, 52, 59]. These studies suggest that myocardial dysfunction and heart failure may be due, at least in part, to myocardial VIP depletion. Our understanding of the cardiovascular effects of exogenously administered and endogenously released VIP has evolved since the initial discovery of VIP in 1970 and suggests a role for VIP in the regulation of blood flow and cardiac performance. In addition, myocardial VIP depletion appears to be important in the development of cardiomyopathy and myocardial fibrosis. Additional studies are needed to determine the importance of endogenous VIP in the physiological regulation of blood flow, heart rate, and myocardial contractility. Studies are also needed to determine the roles of VPAC1, VPAC2, cyclic AMP, and other receptors and signaling mechanisms in the cardiovascular effects of VIP. These studies will be instrumental in the further development of endopeptidase inhibitors and other novel VIP-related therapies for the treatment of patients with angina pectoris, cardiomyopathy, and heart failure.

Acknowledgments This work was supported, in part, by grants from the Bugher Foundation, the Florida Affiliate of the American Heart Association, and the Office of Research and Development, Department of Veterans’ Affairs.

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[43] Rigel DF, Grupp IL, Balasubramaniam A, Grupp G. Contractile effects of cardiac neuropeptides in isolated canine atrial and ventricular muscles. Am J Physiol 1989;257:H1082–7. [44] Rigel DF, Lathrop DA. Vasoactive intestinal polypeptide facilitates atrioventricular nodal conduction and shortens atrial and ventricular refractory periods in conscious and anesthetized dogs. Circ Res 1990;67:1323–33. [45] Robberecht P, Gillet L, Chatelain P, De Neef P, Camus JC, Vincent M, Sassard J, Christophe J. Specific decrease of secretin/VIP-stimulated adenylate cyclase in the heart from the Lyon strain of hypertensive rats. Peptides 1984;5:355–8. [46] Roques BP, Noble F, Dauge V, Fournie-Zaluski MC, Beaumont A. Neutral endopeptidase 24.11: Structure, inhibition, and experimental and clinical pharmacology. Pharmacol Rev 1993; 45:87–146. [47] Said SI, Faloona GR. Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the watery-diarrhea syndrome due to pancreatic, bronchogenic and other tumors. NEJM 1975; 293:155–60. [48] Said SI, Mutt V. Polypeptide with broad biological activity: Isolation from small intestine. Science 1970;169:1217–8. [49] Said SI, Rosenberg RN. Vasoactive intestinal polypeptide: Abundant immunoreactivity in neural cells lines and normal nervous tissue. Science 1976;192:907–8. [50] Sawmiller DR, Henning RJ, Cuevas J, DeHaven WI, Vesely DL. Coronary vascular effects of vasoactive intestinal peptide in the isolated perfused rat heart. Neuropeptides 2004;38:289–97. [51] Smitherman TC, Popma JJ, Said SI, Krejs GJ, Dehmer GJ. Coronary hemodynamic effects of intravenous vasoactive intestinal peptide in humans. Am J Physiol 1989;257:H1254–62. [52] Sun W, Tadmori I, Yang L, Delgado M, Ganea D. Vasoactive intestinal peptide (VIP) inhibits TGF-β1 production in murine macrophages. J Neuroimmunol 2000;107:88–99. [53] Suzuki H, Gao XP, Olopade CO, Rubinstein I. Neutral endopeptidase modulates VIP-induced vasodilation in hamster cheek pouch vessels in situ. Am J Physiol 1996;271:R393–7. [54] Tiaho F, Nerbonne JM. VIP and secretin augment cardiac L-type calcium channel currents in isolated adult rat ventricular myocytes. Eur J Physiol 1996;432:821–30. [55] Unverferth DV, O’Dorisio TM, Miller MM, Uretsky BF, Magorien RD, Leier CV, et al. Human and canine ventricular vasoactive intestinal polypeptide: Decrease with heart failure. J Lab Clin Med 1986;108:11–6. [56] Usdin TB, Bonner TI, Mezey E. Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distributions. Endocrinol 1994;135:2662–80. [57] Wei Y, Mosjov S. Tissue specific expression of different human receptor types for pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide: implications for their role in human physiology. J Neuroendocrinol 1996;8:811–17. [58] Weihe E, Reinecke M, Forssmann WG. Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart. Interrelation with neurotensin- and substance P-like immunoreactive nerves. Cell Tissue Res 1984;236:527–40. [59] Ye VZC, Duggan KA. Vasoactive intestinal peptide downregulates the intrahepatic renin-angiotensin system in the anesthetized rat. Clin Sci 2000;99:201–6. [60] Ye VZC, Hodge G, Yong JLC, Duggan KA. Early myocardial fibrosis is associated with depletion of vasoactive intestinal peptide in rat heart. Exp Physiol 2002;87:539–46. [61] Ye VZC, Hodge G, Yong JLC, Duggan KA. Vasopeptidase inhibition reverses myocardial vasoactive intestinal peptide depletion and decreases fibrosis in salt sensitive hypertension. Euro J Pharmacol 2004;485:235–42.

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168 Cardiovascular Peptides: Vasopressin NATALIE N. RIZK, NOREEN F. ROSSI, AND JOSEPH C. DUNBAR

demonstrated to have their origin in the posterior pituitary. The antidiuretic hormone (ADH) effects of the neurohypophyseal extract was established later [13] in classic experiments by Verney [18]. Research on the cardiovascular actions (pressor effects) of vasopressin and the antidiuretic actions (water balance) continued until chemical analysis revealed in the 1950s that vasopressin and ADH were the same hormone. Since that time, the vasopressin and oxytocin peptides have been isolated from seven major vertebrate phyla as well as several invertebrate phyla. Virtually all vertebrate species have been demonstrated to have vasopressin or vasopressinlike peptides [13].

ABSTRACT Vasopressin has widespread actions and is essential in maintaining cardiovascular balance by acting at many sites, including the blood vessels, heart, kidney, and central nervous system. The multiplicity of these actions is based on the distribution of the classic vasopressin receptors: V1, vascular receptor; V2, renal receptor; V3, pituitary receptor; and oxytocin receptor. One of vasopressin’s most prominent actions is its potent vasoconstriction of most vascular beds via stimulating the V1 receptors. This vasoconstrictor response to vasopressin is consistent with the distribution of the V1 receptors. In addition to vasoconstrictor activity, vasopressin acts directly to decrease both heart rate and cardiac output. Vasopressin also increases the sensitivity of the baroreflex, thereby leading to an enhanced suppression of heart rate. Vasopressin modulates other major systems that play a role in cardiovascular function, such as potentiating the action of catecholamines and decreasing renin release by the kidney. The other major cardiovascular function of vasopressin is to protect blood volume and osmolality via stimulation of V2 receptors. From a chronic perspective, this leads to increased water retention and enhanced blood volume, thus adding protection against blood volume depletion or volume loss due to dehydration. Vasopressin agonists and antagonists are rapidly emerging as therapeutic agents for cardiovascular disorders such as vasodilatory shock states, hypertension, and heart failure.

STRUCTURE OF THE PRECURSOR mRNA/GENE The peptide hormone vasopressin is synthesized predominately in the magnocellular neurons of the supraoptic nucleus and the paraventricular nucleus of the hypothalamus and is released by the posterior pituitary or neurohypophysis. The ancestral gene for vasopressin appears to have risen early in the evolutionary scheme. Vasopressin is a neuropeptide with a disulfide bridge between the two cysteine amino acids. A further description can be found in the Brain Peptides section of this book.

DISTRIBUTION OF THE mRNA See section on Brain Peptides.

DISCOVERY Vasopressin was one of the first hormones to be identified. It was first identified by Oliver and Schäfer in 1895 [11] based on the ability of pituitary extracts to produce pressor effects. These pressor substances were Handbook of Biologically Active Peptides

PROCESSING See section on Brain Peptides.

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Copyright © 2006 Elsevier

1224 / Chapter 168 RECEPTORS

BIOLOGICAL ACTIVITY

The cellular effects of vasopressin are mediated by the stimulation of tissue-specific G-protein receptors. Vasopressin can interact with V1, vascular receptors; V2, renal receptors; V3, pituitary receptors; and oxytocin receptors. The V1 receptors were subclassified as V1a and V1b, which is currently V3. The G-protein-coupled receptors comprise seven-transmembrane α-helices. The receptors consist of intracellular and extracellular loops, an extracellular amino terminal domain, and a cytoplasmic carboxyl-terminal domain [5]. The stimulation of the vasopressin receptor leads to a receptor subtype-specific interaction with G-protein-coupled receptor kinase or protein kinase C. The G-proteins are signal transducers that mediate intracellular pathways. A single vasopressin receptor can activate multiple second-messenger pathways by interacting with different G-proteins. The vasopressin signal is mediated through Gs as well as Gq subtypes. The activation of the Gs pathway is characterized by the stimulation of adenyl cyclase and increase in cAMP; cAMP, in turn, modulates multiple cellular activities. The activation of the Gq leads to the mobilization of calcium [5]. The sequence is that the receptor agonist vasopressin stimulates phospholipase-C to produce the intracellular messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 releases calcium and DAG activates protein kinase C (PKC). The V1 receptor is the most widespread subtype of vasopressin receptors. It is found in the vascular smooth muscle cells, myometrium, bladder, adipocytes, hepatocytes, platelets, lymphocytes, monocytes, adrenal cortex, testis, myocardium, and many central nervous system (CNS) structures. The gene is located on chromosome 12 and maps to region 128, 14–15. The binding of vasopressin to the V1 receptors located on vascular smooth muscle cells results in arteriolar vasoconstriction and consequently an increase in systemic vascular resistance. V2-receptor-mediated actions are related to water and sodium regulation and are found primarily in the principal cells of the renal-collecting duct. The binding of vasopressin to V2 receptors results in the insertion of aquaporin 2, which is one of several water-channel renal proteins that increase the permeability of vascular membranes to water [1, 3], into the apical membrane of the renal-collecting duct cells. The V3 receptors regulate the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary [3].

Vasopressin regulates body fluid osmolality by adjusting both the rate of water intake and the rate of solute free water excretion by the kidney. The primary and most sensitive physiological stimulus for vasopressin secretion is an increase in plasma osmolality. The release of vasopressin is stimulated by osmoreceptors located in the organum vasculosum of the lamina terminalis juxtaposed to the anterior hypothalamus [1, 3]. A change in osmolality of 1–2% of the extracellular fluid compartment stimulates the release of vasopressin. The osmolality threshold for secretion is approximately 280 mOsm/kg water. Vasopressin induces an antidiuretic effect by increasing the permeability of the collecting ducts to water. This activation occurs via signaling through the Gs-protein-coupled receptor and activating the enzyme adenylyl cyclase, which increases intracellular levels of cyclic AMP. Consequently, the activation of cyclic AMP-dependent protein kinase (PKA) mediates the hydroosmotic effects of vasopressin [3]. Through protein phosphorylation, vasopressin allows the rapid shuttling of vesicles containing aquaporin 2 proteins into the luminal membrane of the principal cells and ultimately allows for water reabsorption to occur. The synthesis of aquaporin 2 is also stimulated [2, 3, 8]. V2 activation also increases urea permeability in the terminal portions of the medullary collecting duct through vasopressin-regulated urea transporters. As a result of vasopressin’s stimulation of an increase in water reabsorption, the plasma osmolality is decreased. This regulation of water reabsorption, blood osmolality, and blood volume plays a significant role in the overall regulation of cardiovascular function [5]. V2 receptors have also been implicated in platelet function and hemostasis via release of von Willebrand factor [6] (also see Chapter 169 on renal effects of neurohypophyseal peptides—Renal Peptides Section of this book). A decrease in blood volume also stimulates vasopressin release. However, the neuronal pathways that mediate the osmoregulation of vasopressin release are different from those involved in hemodynamic regulation [7]. Baroreceptors located in the left atrium, left ventricle, and pulmonary veins sense blood volume (filling pressures), and baroreceptors in the carotid sinus and aorta sense arterial blood pressure. Nerve impulses reach brainstem nuclei predominantly through the vagus and glossopharyngeal nerves, and these signals are relayed to the nucleus of the solitary tract, then to the A1-noradrenergic cell group in the caudal ventrolateral medulla, and finally to the supraoptic nucleus and paraventricular nucleus where vasopressin is released [1]. Although osmoregulation of vasopressin release requires a small difference (1–2%) in plasma osmolality, the hemodynamic regulation of

ACTIVE OR SOLUTION CONFORMATION No known changes.

Cardiovascular Peptides: Vasopressin / 1225 vasopressin release requires a large decrease (6–30%) in blood volume and/or pressure to elicit vasopressin release. Thus, the acute effects of vasopressin on the heart become essential only during episodes of severe hypovolemia and/or hypotension (e.g., hemorrhage, heart failure, hypotensive drugs, and diuretics) [7]. V1 receptors located in the vascular smooth muscles mediate the vasoconstrictor effect of vasopressin. When vasopressin binds to V1 receptors, a G-protein-coupled activation of phospholipase-C occurs and through the hydrolysis of phosphatidylinositol-4,5-bisphosphate to DAG and IP3. IP3 binds to its receptor on the endoplasmic reticulum and allows calcium to pass through into the cytoplasm. DAG promotes the translocation of protein kinase C from the cytosol to the plasma membrane, where it phosphorylates key proteins that elicit a biological response [3]. The biological effects mediated through the V1 receptor include vasoconstriction, myocardial hypertrophy, platelet aggregation, and growth of vascular smooth muscle cells [3, 11, 15]. The vascular smooth muscles located in the skin, skeletal muscle, fat, pancreas, and thyroid gland are the most sensitive to vasopressin actions [9]. The effects of vasopressin on the heart have been demonstrated to decrease cardiac output and heart rate. These effects, for the most part, are both direct and indirect and are the result of coronary vasoconstriction, decreased coronary blood flow, and alterations in vagal and sympathetic tone [5, 7]. The extracellular fluid volume as well as the composition of the extracellular fluid are important components in maintaining the integrity of the cardiovascular system. A reduction in blood volume increases the release of vasopressin. This decrease in blood volume leads to an inhibition of water diuresis. Studies have clearly demonstrated that stepwise decreases in blood volume can lead to stepwise increases in circulatory vasopressin. The threshold for this response is approximately 6%. On the other hand, an expansion in blood volume leads to a reduction in plasma vasopressin. Clearly evident in these studies is that the sensitivity of vasopressin to volume changes was less than the sensitivity to osmolality changes. The major receptor systems for vasopressin release in response to changes in blood volume and arterial pressure are stretch receptors located in the atria of the heart and arterial baroreceptors [3]. The atrial baroreceptors are stimulated when the blood pressure is reduced. The atrial receptors are sensitive to changes in atrial volume or blood volume. A number of studies using carotid occlusion, carotid sinus stimulation, vagotomy, or sino-aortic denervation confirm these relationships [14]. Although the oxytocin receptor is characterized as a nonselective vasopressin receptor, the oxytocin receptor has equal affinity for oxytocin and vasopressin. The

activation of this receptor stimulates Gq and increases phospholipase-C with an increase in IP3 and DAG. This pathway leads to stimulation of nitric oxide (NO) synthase and increased NO production. Activation of cardiac oxytocin receptors stimulates atrial naturetic peptide release, which also regulates diuresis and blood pressure [5] (see Chapter 165 on Natriuretic Peptides by Minamino et al. elsewhere in this section of the book). Vasopressin also exerts an important influence on ACTH secretion via the V3 receptor by potentiating the response to corticotropin-releasing hormone (CRH). This action of vasopressin is negligible without CRH [3]. The increase in ACTH can stimulate cardiovascular dynamics by increasing the production of adrenal hormones.

PATHOPHYSIOLOGICAL IMPLICATIONS Vasopressin plays a role in cardiovascular diseases, including hypertension, congestive heart failure (CHF), and shock. It has been reported that in some forms of hypertension plasma vasopressin levels are elevated and that this elevation correlates with the severity of hypertension [12, 16]. Studies have demonstrated that vasopressin levels in patients with advanced CHF were higher (9.5 pg/ml vs. 4.7 pg/ml) than in healthy agematched controls [16]. The increase in vasopressin observed in CHF appears to be independent of osmotic stimuli. The adverse effects of increased levels of vasopressin include increased free-water reabsorption, cardiac contractility, and vascular tone. This increase in vasopressin potentiates other pathways, including the renin-angiotensin-aldosterone system to promote retention of sodium and water and also the release of norepinephrine from the adrenal gland to increase inotropy [13]. The central renin-angiotensin system plays an important role in the regulation of arterial pressure and in the development of certain forms of clinical and experimental hypertension. This has also demonstrated that there is increased aquaporin 2 protein excretion in urine of patients with chronic heart failure [10]. It has been observed that in rat models with severe CHF the mRNA expression of aquaporin 2 was significantly increased [8, 10]. Also, rats with an elevated left-ventricular end-diastolic pressure and reduced plasma sodium concentrations had a significant increase in aquaporin 2 protein compared with rats with mild CHF without elevated left-ventricular end-diastolic pressure or reduced plasma sodium concentrations [19]. As a result of V2 receptor-mediated water retention, preload is increased, leading to increases in pulmonary capillary pressure and leftventricular filling pressure. V1 activation increases

1226 / Chapter 168 afterload and the subsequent arterial vasoconstriction can also contribute to hemodynamic changes associated with vasopressin release. These mechanisms suggest that the adverse hemodynamic changes that occur in CHF are not limited to one signaling pathway but that it is vasopressin’s activity at both the V1 and V2 receptors that compounds the adverse hemodynamic changes. The selective blockade of V1 receptors has been strongly suggested as a potential therapy for hypertension, CHF, and peripheral vascular disease [8]. Therapeutic agents that block V1 or V2 receptors, or both, are being developed, tested, and used to treat the fluid retention and vascular dynamics of heart failure [4]. On the other hand, shock is characterized by an initial increase in vasopressin secretion that plays a significant role in maintaining vascular tone and blood pressure. The progressive loss of sensitivity or downregulation of the vasopressin receptors may be an important factor in progressive loss of pressor effects in some shock situations. Vasopressin agonists could provide an additive pressor to maintain cardiovascular dynamics in these situations [17]. Tahara et al. [15] showed a time- and concentrationdependent vasopressin-stimulated increase in the secretion of vascular endothelial growth factor (VEGF) in vascular smooth muscle cells [15]. This V1 receptor– mediated action of vasopressin may contribute to cardiac remodeling that occurs following cardiac injury. Diabetes insipidus is a disease of impaired conservation of water by the kidney due to an inadequate secretion of vasopressin. The treatment, vasopressin agonist, is well known [2, 3]. Disorders that can induce syndrome of inappropriate antidiuretic hormone secretion (SIADH) include malignancies, pulmonary diseases, CNS diseases or injuries, and drugs. The hypersecretion of vasopressin in these patients can exacerbate cardiovascular disorders. The effect of vasopressin on the heart is controversial. Vasopressin has been demonstrated to both dilate and constrict coronary arteries. Vasopressin has been demonstrated to enhance cardiac resuscitation. Vasopressin has a negative direct inotropic effect on the heart, but it promotes cardiac hypertrophy. All of these actions lead to potential sites of clinical manipulation. Many of the cardiovascular actions of vasopressin are complex because hormonal action can potentiate the responses to many vasoactive agents such as norepinephrine and angiotensin II. Vasopressin also paradoxically causes vasodilation in selected vascular beds. Thus, because vasopressin can cause vascular constriction, vascular dilation, altered nervous system sensitivity, and responses to cardioactive agents, it is a very fertile site for future clinical approaches to cardiovascular regulation.

References [1] Cunningham E, Sawchenko P. Reflex control of magnocellular vasopressin and oxytocin secretion. Trends Neurosci. 1991; 14:406–411. [2] Deen P, Verdijk M, Knoers N, Wieringa B, Monnens L, Van Os CH, Oost B. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 1994; 264:92–95. [3] Ganong WF. Central regulation of visceral function. In Review of Medical Physiology. Connecticut; Appleton and Lange 1997: 225–233. [4] Gheorghiade M, Gattis WA, O’Connor CM, Adams Jr KF, Elkayam U, Barbagelata A, Ghali JK, Benza RL, McGrew FA, Klapholz M, Ouyang J, Orlandi C. Effects of talvaptan, a vasopressin antagonist, in patients hospitalized with worsening heart failure. JAMA 2004; 291:1963–1971. [5] Holmes CL, Landry DW, Granton JT. Science review: Vasopressin and the cardiovascular system Part 1. Receptor Physiol. 2003; 7:427–434. [6] Kaufmann JE, Oksche A, Wollheim CB, Günther G, Rosenthal W, Vischer UM. Vasopressin-induced von Willebrand factor secretion from endothelial cells involves V2 receptors and cAMP. J. Clin. Invest. 2000; 106:107–116. [7] László FA, László Jr F, De Wied D, Pharmacology and clinical prospectives of vasopressin antagonists. Pharmacol. Rev. 1991; 43:73–108. [8] Lee CR, Watkins ML, Patterson JH, Gattis W, O’Connor CM, Gheorghiade M, Adams Jr KF. Vasopressin: A new target for the treatment of heart failure. Am. Heart J. 2003; 146:9–18. [9] Liard JF, Deriaz O, Schelling P, Thibonnier M. Cardiac output distribution during vasopressin infusion or dehydration in conscious dogs. Am. J. Physiol. 1982; 243:H663–H669. [10] Martin PY, Abraham WT, Lieming X, Olson BR, Oren RM, Ohara M, Schrier RW. Selective V2-receptor vasopressin antagonism decreases urinary aquaporin-2 excretion in patients with chronic heart failure. J. Am. Soc. Nephrol. 1999; 10:2165– 2170. [11] Oliver G, Schafer EA. On the physiological secretion of extracts of the pituitary body and certain other glandular secretions. J. Physiol. (London) 1895; 18:277–279. [12] Packer M, Lee WH, Kessler PD. Role of neurohormonal mechanisms in determining survival in patients with severe chronic heart failure. Circulation 1987; 75(suppl 4):80–92. [13] Russell SD, Dewald T. Vasopressin receptor antagonists, therapeutic potential in the management of acute and chronic heart failure. Am. J. Cardiovasc. Drugs 2003; 3(1):13–20. [14] Share L. Vasopressin in cardiovascular regulation. Physiol. Rev. 1988; 68:1248–1284. [15] Tahara A, Saito M, Tsukada J, Ishii N, Tomura Y, Wada K, Kusayama T, Yatsu T, Uchida W, Tanaka A. Vasopressin increases vascular endothelial growth factor secretion from human vascular smooth muscle cells. Eur. J. Pharmacol. 1999; 368:89– 94. [16] Thibonnier M. Vasopressin receptor antagonists in heart failure. Curr. Opin. Pharmacol. 2003; 3:683–687. [17] Thrasher TN. Baroreceptors, baroreceptors unloading, and the long-term control of blood pressure. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 288:R819–R827. [18] Verney EB. The antidiuretic hormone and the factors which determine its release. Proc. R. Soc. Lond. 1947; 135:25– 106. [19] Xu DL, Martin PY, Ohara M, St John J, Pattison T, Meng X, Morris K, Kim JK, Schrier RW. Upregulation of aquaporin-2 water channel expression in chronic heart failure rat. J. Clin. Invest. 1997; 99:1500–1505.

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169 Renal Effects of Neurohypophyseal Peptides JOSEPH G. VERBALIS

studies, suggesting that this represents a speciesspecific effect of OT.

ABSTRACT The neurohypophyseal hormones vasopressin (VP) and oxytocin (OT) have existed throughout much of evolution, and in all species their most prominent physiological function is participation in the maintenance of water homeostasis. VP and OT receptors are expressed in various organs, including the kidney. The most abundant VP receptor in the kidney is V2R, which is responsible for VP-induced antidiuresis via changes in collecting duct water permeability. This process is initiated by V2R ligand binding in the collecting duct principal cells, leading to increased intracellular cAMP levels and fusion of aquaporin-2 (AQP2)-containing intracytoplasmic vesicles with the apical plasma membranes of the principal cells, a process that increases apical water permeability by markedly increasing the number of water-conducting pores in the apical plasma membrane. These effects are complemented by V2Rmediated increases in sodium and urea reabsorption into the renal interstitium, thus facilitating the generation of the corticopapillary osmotic gradient that is necessary for maximal urinary concentration. VP also acts at V1aR in the vasa recta and/or the intramedullary pericytes, which complements the effects of VP acting at V2R in the nephron to produce maximally concentrated urine by limiting blood flow into the inner medulla. Abundant data in animals and humans have implicated renal effects of OT in pathological antidiuresis, primarily via cross-reactivity at V2R at high concentrations of plasma OT. In contrast, the natriuretic effects of OT appear to occur at physiological plasma concentrations of OT and can be blocked by OT-specific antagonists, probably as a result of OTRligand binding in the macula densa. However, natriuretic effects of OT have not been observed in human Handbook of Biologically Active Peptides

BRIEF HISTORY AND OVERVIEW OF THE NEUROHYPOPHYSEAL PEPTIDES As described in the Brain Peptides Section of this Handbook, the neurohypophyseal hormones VP and OT are nonapeptides consisting of a 6-amino-acid ring with a cysteine to cysteine bridge, and a 3-amino-acid tail. Some form of these nonapeptides has existed throughout the evolutionary species from amphibia and reptiles to birds and mammals, and in all species their most prominent physiological function is participation in the maintenance of water homeostasis (although the target organ may vary from skin to bladder to kidney [2]). It is assumed that OT and VP derive from a single ancestral gene that is expressed in the lamprey and hag fish as only a single gene and hormone, vasotocin, which consists of a ring identical to oxytocin and a tail identical to vasopressin [2]. All mammals synthesize and secrete both OT and arginine vasopressin (AVP) with the exception of the pig; in the pig a lysine is substituted for arginine in position 8 of vasopressin, producing lysine vasopressin. Similar to other peptide hormones, VP is synthesized as part of a prohormone, including a midmolecule neurophysin and a carboxy-terminal glycopeptide. For OT the peptide products are the nonapeptide and a neurophysin but no glycopeptide [17]. At the physiologic pH of plasma, there is no binding of either OT or AVP to their respective neurophysins, so each peptide circulates independently in the bloodstream. Although each neurophysin and the VP glycopeptide circulate in

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1228 / Chapter 169 plasma, no biological functions have ever been demonstrated for these peptides [39]. Therefore, this chapter focuses exclusively on the actions of VP and OT in the kidney; effects at other organs, and specifically the reproductive functions of OT and the vascular and metabolic actions of VP, are covered elsewhere in this handbook.

BODY FLUID HOMEOSTASIS Because the most important homeostatic function of VP is the maintenance of normal body water stores, the renal effects of VP that serve to accomplish this function can best be appreciated and understood within the context of the overall mechanisms that regulate body fluid homeostasis. The maintenance of normal water balance in humans is achieved principally through an integration of thirst, VP secretion, and renal responsiveness to VP. Plasma osmolality is maintained within narrow limits by osmotically regulated VP secretion and thirst. Despite large variations in water intake, the osmolality of body fluids in healthy individuals is maintained within a relatively narrow range (275–295 mOsm/kg H2O). To maintain plasma osmolality at such a constant level, VP secretion must vary in response to relatively small changes in plasma osmolality, which is achieved through the activation of the osmoreceptors located in the anterior hypothalamus. The primary stimulus for VP release is an increase in plasma osmolality. There are individual variations in the osmotic threshold, or set point, for VP release, probably reflecting variations in the sensitivity of individual osmoreceptor cells. At all plasma osmolalities below this set point, plasma VP is suppressed to low or undetectable levels. Above this set point, the secretion of the hormone increases in direct proportion to the increasing plasma osmolality. Therefore, with plasma hypoosomolality VP is suppressed and urine osmolality is maximally dilute (diuresis), whereas with plasma hyperosmolality VP levels rise and the urine is progressively more concentrated (antidiuresis). The sensitivity of this response can be appreciated by noting that an increase in plasma osmolality of only 1% leads to an increase in plasma VP of 0.4 –0.8 pg/ml, a concentration sufficient to significantly increase urinary concentration and decrease urine flow, whereas maximum antidiuresis occurs at a plasma VP concentration of only 5 pg/ml [36]. In general, the osmotic threshold for thirst is usually set approximately 5–10 mOsm/kg H2O above that for VP release. This has the effect of allowing the kidney to regulate body water homeostasis in response to very small changes in plasma osmolality, whereas thirst is activated only by larger and more threatening

osmotic perturbations to maintain the total fluid balance by the ingestion of fluid. In addition to changes in plasma osmolality, VP secretion is also stimulated by hypotension and volume depletion, mediated largely by baroreceptors located in the cardiac atria and large arteries. In contrast to the extremely sensitive osmotic regulation of VP secretion, a reduction in effective circulating volume of approximately 10% is required before a significant VP response occurs. VP is also released in response to other nonosmotic stimuli such as nausea, pain, hypoxia, hypercapnia, hypoglycemia, physical exercise, and various medications. Although nonosmotic secretion of VP is not involved with normal body fluid homeostasis, such secretion is of importance because the renal effects of VP are the same regardless of the stimulus that caused the secretion of the hormone, leading to potential disturbances in water homeostasis from inappropriate degrees of antidiuresis [5].

VASOPRESSIN AND OXYTOCIN RECEPTORS Three known receptor subtypes mediate the peripheral actions of VP. They are classified according to the second-messenger system to which they are coupled. The vasopressin V1a (V1aR) and V1b (V1bR) receptors are linked to the phosphoinositol signaling pathway, with intracellular calcium acting as the second messenger. In contrast, the vasopressin V2 receptors (V2R) are linked to the adenylate cyclase signaling pathway with intracellular cAMP acting as the second messenger [45]. The V1aR subtype is ubiquitous and is present on vascular smooth muscle cells, hepatocytes, and platelets, where it mediates vascular constriction, glycogenolysis, and platelet aggregation, respectively. V1aR also appears to be the major AVP receptor subtype found within the brain, where it presumably mediates central effects of this peptide. The V1bR subtype is found predominantly in the anterior pituitary, where it mediates adrenocorticotropin release in concert with corticotropin-releasing hormone. The V2R subtype is present predominately in the kidney, where it mediates free water reabsorption. In addition, there is now evidence indicating the presence of extrarenal V2R in the endothelium that are involved in the secretion of von Willebrand factor and factor VIIIc [22]. In contrast to vasopressin, only one oxytocin receptor (OTR) has been identified. Similar to the V1aR and V1bR, the OTR is coupled to the phosphoinositol signaling pathway, with intracellular calcium acting as the second messenger [18]. Although some studies have suggested the presence of a second OTR in the brain, to date a second OT receptor has not been identified or sequenced [47]. The OTR subtype is present

Renal Effects of Neurohypophyseal Peptides / 1229 predominantly in breast and uterine tissue, with lesser expression in the brain, kidney, and heart [18].

LOCALIZATION OF VASOPRESSIN AND OXYTOCIN RECEPTORS IN THE KIDNEY V2R Expression V2R is the predominant neurohypophyseal receptor expressed in the kidney, apropos its crucial role in the urinary concentrating process. Prominent expression has been found by autoradiography [25, 34], immunohistochemistry [16], and receptor mRNA expression [28] throughout all portions of the collecting duct (cortical, and outer and inner medullary), the connecting segment, the distal convoluted tubule, and the thick ascending limb of the loop of Henle.

V1aR Expression High levels of V1aR expression have also been found in the renal medulla by autoradiography [34, 42], Western analysis [32], and receptor mRNA in situ hybridization histochemistry [28]. However, in contrast to the V2R, the V1aR is localized predominantly among the medullary interstitial cells and vascular elements of the vasa recta rather than the collecting ducts; sparser labeling is found in the renal cortex but not in the glomeruli.

OTR Expression In the adult rat, OTR expression by receptor autoradiography occurs predominantly in the macula densa cells of the juxtaglomerular apparatus, with a lesser degree of expression in the medulla on the loops of Henle of juxtamedullary nephrons [41]. The cortical OT binding sites express higher selectivity for OT than AVP compared with the medullary sites; this initially led to speculation about two different receptor subtypes [3], but it could also represent the binding of OT to medullary V2R. Estrogen treatment of ovariectomized rats induced OTR mRNA expression in the outer stripe of the outer medulla and increased expression in macula densa cells [29]. The induction of outer stripe OTR mRNA expression by estradiol was blocked by the antiestrogen, tamoxifen, but was not significantly affected by high levels of circulating oxytocin [29].

RENAL EFFECTS OF VASOPRESSIN The primary renal response to increased circulating VP is an increase in water permeability of the kidney collecting tubules, leading to antidiuresis. Although an

increase in solute reabsorption (primarily urea, but also sodium) occurs as well, the total solute reabsorption is proportionally much less than water. Consequently, a decrease in urine flow and an increase in urine osmolality occur as secondary responses to the increased net water reabsorption. With refinement of radioimmunoassays for VP, the unique sensitivity of this hormone to small changes in osmolality, as well as the corresponding sensitivity of the kidney to small changes in plasma VP levels, has become apparent [36]. Although some debate still exists with regard to the exact pattern of osmotically stimulated AVP secretion, most studies to date have supported the concept of a discrete osmotic threshold for VP secretion above which a linear relationship between plasma osmolality and VP levels occurs. The slope of the regression line relating VP to plasma osmolality can vary significantly across individual human subjects, in part because of genetic factors [37]. In general, each 1 mOsm/kg H2O increase in plasma osmolality causes an increase in plasma VP from 0.4 to 0.8 pg/ml. The renal response to circulating VP is similarly linear, with urinary concentration that is directly proportional to VP levels from 0.5 to 4–5 pg/ml, after which urinary osmolality is maximal and cannot increase further despite additional increases in VP levels. Thus, changes of 1% or less in plasma osmolality are sufficient to cause significant increases in plasma VP levels with proportional increases in urine concentration, and maximal antidiuresis is achieved after increases in plasma osmolality of only 5 to 10 mOsm/kg H2O (2–4%) above the threshold for VP secretion. However, even this analysis underestimates the sensitivity of this system to regulate free water excretion for the following reason. Urinary osmolality is directly proportional to plasma VP levels as a consequence of the fall in urine flow induced by the VP, but urine volume is inversely related to urine osmolality. Thus, an increase in plasma VP concentration from 0.5 to 2 pg/ml has a much greater relative effect on decreasing urine flow than does a subsequent increase in VP concentration from 2 to 5 pg/ml, thereby further magnifying the physiological effects of small initial changes in plasma VP levels [38]. The net result of these relations is a finely tuned regulatory system that adjusts the rate of free water excretion accurately to the ambient plasma osmolality via small changes in pituitary VP secretion. Furthermore, the rapid response of pituitary VP secretion to changes in plasma osmolality, coupled with the short half-life (10–20 minutes) of VP in human plasma, enables this regulatory system to adjust renal water excretion to changes in plasma osmolality on a minuteto-minute basis. Over the last decade the cellular mechanisms responsible for this exquisitely sensitive mechanism for renal

1230 / Chapter 169 water conservation have been elucidated following the identification of the aquaporin family of water channels by Agre et al. [8]. Renal concentrating ability is determined primarily by proximal water reabsorption, the corticopapillary osmotic gradient, and VP-mediated changes in collecting duct water permeability, all of which involve molecular water channels of the aquaporin family. Four major aquaporin water channels are expressed in the renal tubules [23]. Aquaporin-1 (AQP1) is primarily responsible for the high constitutive water permeability of the proximal tubules and thin descending limbs of the loop of Henle. Aquaporin-3 (AQP3) and aquaporin-4 (AQP4) are expressed in the basolateral membrane of collecting duct principal cells, and confer this epithelial membrane with high water permeability. The most unique water channel is aquaporin-2 (AQP2), which is expressed exclusively in renal collecting duct principal cells and whose activity is largely regulated by VP. VP induces changes in collecting duct water permeability via both short-term and long-term effects that regulate renal aquaporin synthesis and function [23]. Short-term regulation of renal aquaporins is associated with increases in water permeability within a few minutes of VP exposure, an effect that is rapidly reversible. VP triggers this response by binding to the V2R in the collecting duct principal cells and increasing intracellular cAMP levels by activating adenylate cyclase. Recent studies have demonstrated that the increase in collecting duct water permeability is a consequence of fusion of AQP2-containing intracytoplasmic vesicles with the apical plasma membranes of the principal cells, a process that increases apical water permeability by markedly increasing the number of waterconducting pores in the apical plasma membrane [27]. The dissociation of VP from the V2R allows intracellular cAMP levels to decrease, and the water channels are re-internalized into the intracytoplasmic vesicles, thereby terminating the increased water permeability. By virtue of the subapical membrane localization of the AQP2-containing vesicles, they can be quickly shuttled into and out of the membrane in response to changes in intracellular cAMP levels. This mechanism therefore allows minute-to-minute regulation of renal water excretion through changes in ambient plasma VP levels. Long-term regulation of collecting duct water permeability represents a sustained increase in collecting duct water permeability in response to prolonged high levels of circulating VP. This response requires at least 24 hours to elicit and is not as rapidly reversible. Recent studies have demonstrated that this conditioning effect is due largely to the ability of vasopressin to induce large increases in the abundance of AQP2 and AQP3 water channels in the collecting duct principal cells [12,

14]. Greater total expression of the number of AQP2 and AQP3 water channels, when combined with the short-term effect of vasopressin of shifting AQP2 into the apical plasma membrane, allows the collecting ducts to achieve higher water permeabilities during conditions of prolonged dehydration, thereby further enhancing the urine-concentrating capacity in response to equivalent levels of VP. In addition to these effects on water permeability of the collecting duct cells to regulate the degree of antidiuresis in response to plasma VP levels, VP also acts to enhance the other major factor regulating renal concentrating ability, namely the establishment and maintenance of the corticopapillary osmotic gradient that is the determinant of maximal urine concentration. In this regard, studies have shown VP-dependent reabsorption of sodium into the renal interstitium in the thick ascending limb of the loop of Henle loop that is regulated by the V2R in this region of the nephron via the upregulation of the Na-K-2Cl cotransporter expression in the outer medulla [21] and also the V2R-mediated increased expression of the epithelial sodium channel (ENaC) in the distal convoluted tubule [4]. Similarly, VP causes increased expression of the UT-A1 urea transporter in rats, either directly or indirectly via effects at V2R in the inner medulla of the kidney [40]. Thus, each of these factors in addition to AQP2 regulation can contribute to the achievement of maximal urinary concentration in response to chronic VP stimulation via long-term upregulation of the countercurrent multiplication system that is responsible for generation of the corticopapillary osmotic gradient. In contrast to the well-understood effects of VP acting at V2R receptors in the kidney, much less is understood about the effects of the V1aR that are also clearly expressed in the kidney. Early studies suggested that V1aR acted at the collecting duct principal cells to antagonize the V2R effects of VP by virtue of increasing intracellular Ca2+ by phosphoinositide hydrolysis [6] or stimulating the synthesis and release of prostaglandins such as PGE2 and nitric oxide. However, subsequent results have implicated V2R in many of these effects [13, 20, 24], and although some in vitro studies have suggested the presence of functional V1aR activity in isolated collecting tubules, no studies to date have conclusively demonstrated the expression of V1aR in collecting duct principal cells. Alternatively, in view of the localization of V1aR in the renal cortical and medullary microcirculation combined with results indicating that VP reduces renal medullary blood flow [10], it is reasonable to postulate an intrarenal hemodynamic effect for VP actions at kidney V1aR. Recent studies using reverse transcription polymerase chain reaction and Western blot analyses have confirmed that V1aR mRNA

Renal Effects of Neurohypophyseal Peptides / 1231 and protein, but not V2R mRNA and protein, were present in isolated cortical and medullary vasculature [32]. Thus, VP effects at V1aR are probably responsible for the vasoconstrictor actions on renal medullary blood flow [10]. The physiological ramifications of this effect are multiple, but recent results have suggested that regulation of the descending vasa recta microcirculation is a determinant of Na+ and water excretion and possibly also the development of hypertension [10, 31]. The data support the idea that regulation of descending vasa recta blood flow occurs via the actions of multiple vasoactive substances on the contractile pericytes surrounding these vessels [30, 46]. Substances known to reduce descending vasa recta blood flow via vasoconstriction include angiotensin II, vasopressin, endothelins, norepinephrine, acetylcholine, and adenosine (via adenosine A1R); substances known to increase descending vasa recta blood flow through vasodilatation include nitric oxide, prostaglandin E2, bradykinin, acetylcholine, and adenosine (via adenosine A2R) [30]. The complexity of this regulation is illustrated by the fact that some stimuli activate opposing systems. For example, both angiotensin II and VP have been shown to stimulate nitric oxide production in the kidney [26, 33], which may serve to counterbalance the otherwise unopposed vasoconstrictive effects of these hormones [11, 44]. Decreases in vasa recta blood flow have the effect of maximizing the corticopapillary concentration gradient, whereas increases in vasa recta flow enhance a washout of the solutes that establish this gradient. Thus, VP acting at V1aR in the vasa recta and/or the intramedullary pericytes complements the effects of VP acting at V2R in the nephron to produce maximally concentrated urine by limiting the blood flow into the inner medulla.

RENAL EFFECTS OF OXYTOCIN Because OT has been associated primarily with reproductive functions, studies of the renal effects of OT have been much more limited. However, abundant data in animals and humans have implicated renal effects of OT in pathological antidiuresis and in rats in physiological natriuresis. Antidiuretic effects of OT were first suggested by cases of hyponatremia in females infused with OT for the induction of labor or induced abortion [9]. Studies of OT infusion in normal subjects confirmed the antidiuretic effects of OT infusions [1], and other studies suggested an antidiuretic potency of OT approximately 1/200 that of VP [49]. Subsequent studies in Brattleboro rats indicated that the limited urinary-

concentrating abilities in this animal with genetic absence of VP synthesis were due to OT effects in the kidney [15]. Early studies did not differentiate whether these effects on urinary concentration were due to the effects of OT at OTR or due to cross-reactivity of OT at V2R. Subsequent in vitro studies using isolated collecting ducts from rats have indicated that the antidiuretic effects of OT could be blocked by the administration of V2R-specific antagonists, consistent with OT cross-reactivity at V2R to account for the antidiuretic effects of high concentrations of plasma OT [7]. In contrast to the antidiuretic effects of OT, the natriuretic effects of this peptide appear to occur at physiological concentrations of plasma OT [48] and are blocked by OT-specific antagonists [50]. Initial studies suggested that OT-induced natriuresis may be due to the stimulation of atrial natriuretic peptide (ANP) secretion from the cardiac atria [19]. However, subsequent studies have indicated that the natriuresis produced by low doses of OT involves a nitric oxide mechanism, unlike ANP-induced natriuresis, and therefore likely represents a direct effect of OT at the OTR on cells of the macula densa (43), presumably by altering the set point of glomerulotubular feedback mechanism. However, similar natriuretic effects have not been observed in human studies, suggesting that this represents a species-specific effect of OT [1, 35].

References [1] Abdul-Karim, R.; Assali, N. S. Renal function in human pregnancy. V. Effects of oxytocin on renal hemodynamics and water and electrolyte excretion. J.Lab.Clin.Med. 57:522–532; 1961. [2] Acher, R. Evolution of neurohypophysial control of water homeostasis: Integrative biology of molecular, cellular and organismal aspects. In: Saito, T.; Kurakawa, K.; Yoshida, S., Eds. Neurohypophysis: Recent progress of vasopressin and oxytocin research. Amsterdam: Elsevier; 1995:7–16. [3] Arpin-Bott, M. P.; Waltisperger, E.; Freund-Mercier, M. J.; Stoeckel, M. E. Two oxytocin-binding site subtypes in rat kidney: pharmacological characterization, ontogeny and localization by in vitro and in vivo autoradiography. J.Endocrinol. 153:49–59; 1997. [4] Bankir, L. Antidiuretic action of vasopressin: quantitative aspects and interaction between V1a and V2 receptor-mediated effects. Cardiovasc.Res. 51:372–390; 2001. [5] Bartter, F. C.; Schwartz, W. B. The syndrome of inappropriate secretion of antidiuretic hormone. Am.J.Med. 42:790–806; 1967. [6] Burnatowska-Hledin, M. A.; Spielman, W. S. Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule. Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate. J. Clin.Invest 83:84–89; 1989. [7] Chou, C. L.; Di Giovanni, S. R.; Luther, A.; Lolait, S. J.; Knepper, M. A. Oxytocin as an antidiuretic hormone. ii. Role of v2 vasopressin receptor. Am.J.Physiol. 269:F78–F85; 1995.

1232 / Chapter 169 [8] Chrispeels, M. J.; Agre, P. Aquaporins: Water channel proteins of plant and animal cells. Trends Biochem.Sci. 19:421–425; 1994. [9] Cluitmans, F. H.; Meinders, A. E. Acute volume and electrolyte disorders in obstetrics and gynaecology. Neth.J.Med. 36:265– 266; 1990. [10] Cowley, A. W., Jr. Control of the renal medullary circulation by vasopressin V1 and V2 receptors in the rat. Exp.Physiol. 85 Spec No:223S–231S; 2000. [11] Dickhout, J. G.; Mori, T.; Cowley, A. W., Jr. Tubulovascular nitric oxide crosstalk: Buffering of angiotensin II-induced medullary vasoconstriction. Circ.Res. 91:487–493; 2002. [12] DiGiovanni, S. R.; Nielsen, S.; Christensen, E. I.; Knepper, M. A. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc.Natl.Acad.Sci.U.S.A. 91:8984– 8988; 1994. [13] Ecelbarger, C. A.; Chou, C. L.; Lolait, S. J.; Knepper, M. A.; Di Giovanni, S. R. Evidence for dual signaling pathways for v2 vasopressin receptor in rat inner medullary collecting duct. Am.J.Physiol. 270:F623–F633; 1996. [14] Ecelbarger, C. A.; Terris, J.; Frindt, G.; Echevarria, M.; Marples, D.; Nielsen, S.; Knepper, M. A. Aquaporin-3 water channel localization and regulation in rat kidney. Am.J.Physiol. 269:F663– F672; 1995. [15] Edwards, B. R.; LaRochelle, F. T., Jr. Antidiuretic effect of endogenous oxytocin in dehydrated Brattleboro homozygous rats. Am.J.Physiol. 247:F453–F465; 1984. [16] Fahrenholz, F.; Jurzak, M.; Gerstberger, R.; Haase, W. Renal and central vasopressin receptors: Immunocytochemical localization. Ann.N.Y.Acad.Sci. 689:194–206; 1993. [17] Gainer H.; Wray, S. Cellular and molecular biology of oxytocin and vasopressin. In: Knobil E.; Neill J. D., Eds. The Physiology of Reproduction, 2nd edition. New York: Raven Press; 1994:1099–1129. [18] Gimpl, G.; Fahrenholz, F. The oxytocin receptor system: Structure, function, and regulation. Physiol.Rev. 81:629–683; 2001. [19] Haanwinckel, M. A.; Elias, L. K.; Favaretto, A. L.; Gutkowska, J.; McCann, S. M.; Antunes-Rodrigues, J. Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat. Proc.Natl.Acad.Sci.U.S.A. 92:7902–7906; 1995. [20] Kaufmann, J. E.; Iezzi, M.; Vischer, U. M. Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptor- and cAMP-mediated signaling. J.Thromb.Haemost. 1:821–828; 2003. [21] Kim, G. H.; Ecelbarger, C. A.; Mitchell, C.; Packer, R. K.; Wade, J. B.; Knepper, M. A. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop. Am.J.Physiol. 276:F96–F103; 1999. [22] Kinter, L. B.; McConnell, I.; Goodwin, B. T.; Campbell, S.; Huffman, W. F.; Arthus, M. F.; Lonergan, M.; Bichet, D. G. Vasopressin antagonist inhibition of clotting factor release in the rhesus monkey (Macaca mulatta). J.Pharmacol.Exp.Ther. 261:462–469; 1992. [23] Knepper, M. A. Molecular physiology of urinary concentrating mechanism: Regulation of aquaporin water channels by vasopressin. Am.J.Physiol. 272:F3–F12; 1997. [24] Martin, P. Y.; Bianchi, M.; Roger, F.; Niksic, L.; Feraille, E. Arginine vasopressin modulates expression of neuronal NOS in rat renal medulla. Am.J.Physiol.Renal Physiol. 283:F559–F568; 2002. [25] Mimura, Y.; Ogura, T.; Hayakawa, N.; Otsuka, F.; Hashimoto, M.; Yamauchi, T.; Makino, H.; Ogawa, N. In vitro macro- and microautoradiographic localization of v1 and v2 receptors in the rat kidney using opc-21268 and opc-31260. Nephron 76:331–336; 1997.

[26] Navar, L. G.; Ichihara, A.; Chin, S. Y.; Imig, J. D. Nitric oxideangiotensin II interactions in angiotensin II-dependent hypertension. Acta Physiol.Scand. 168:139–147; 2000. [27] Nielsen, S.; Chou, C. L.; Marples, D.; Christensen, E. I.; Kishore, B. K.; Knepper, M. A. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-cd water channels to plasma membrane. Proc.Natl.Acad.Sci. U.S.A. 92:1013–1017; 1995. [28] Ostrowski, N. L.; Lolait, S. J.; Bradley, D. J.; O’Carroll, A. M.; Brownstein, M. J.; Young, W. S. 3. Distribution of v1a and v2 vasopressin receptor messenger ribonucleic acids in rat liver, kidney, pituitary and brain. Endocrinology 131:533–535; 1992. [29] Ostrowski, N. L.; Young, W. S. 3.; Lolait, S. J. Estrogen increases renal oxytocin receptor gene expression. Endocrinology 136:1801–1804; 1995. [30] Pallone, T. L.; Silldorff, E. P. Pericyte regulation of renal medullary blood flow. Exp.Nephrol. 9:165–170; 2001. [31] Pallone, T. L.; Zhang, Z.; Rhinehart, K. Physiology of the renal medullary microcirculation. Am.J.Physiol.Renal Physiol. 284: F253–F266; 2003. [32] Park, F.; Mattson, D. L.; Skelton, M. M.; Cowley, A. W., Jr. Localization of the vasopressin v1a and v2 receptors within the renal cortical and medullary circulation. Am.J.Physiol. 273:R243– R251; 1997. [33] Park, F.; Zou, A. P.; Cowley, A. W., Jr. Arginine vasopressin-mediated stimulation of nitric oxide within the rat renal medulla. Hypertension 32:896–901; 1998. [34] Phillips, P. A.; Abrahams, J. M.; Kelly, J. M.; Mooser, V.; Trinder, D.; Johnston, C. I. Localization of vasopressin binding sites in rat tissues using specific V1 and V2 selective ligands. Endocrinology 126:1478–1484; 1990. [35] Rasmussen, M. S.; Simonsen, J. A.; Sandgaard, N. C.; HoilundCarlsen, P. F.; Bie, P. Effects of oxytocin in normal man during low and high sodium diets. Acta Physiol.Scand. 181:247–257; 2004. [36] Robertson, G. L. The regulation of vasopressin function in health and disease. Rec.Prog.Horm.Res. 33:333–385; 1976. [37] Robertson, G. L. Posterior pituitary. In: Felig, P.; Baxter, J.; Frohman, L., Eds. Endocrinology and Metabolism. New York: McGraw-Hill; 1995:385–432. [38] Robinson, A. G. Disorders of antidiuretic hormone secretion. Clin.Endocrinol.Metab. 14:55–88; 1985. [39] Robinson, A. G.; Verbalis, J. G. The posterior pituitary. In: Larsen, P. R.; Kronenberg, H. M.; Melmed, S.; Polonsky, K. S., Eds. Williams Textbook of Endocrinology, 10th edition. Philadelphia: W.B. Saunders; 2003:281–329. [40] Sands, J. M. Renal urea transporters. Curr.Opin.Nephrol. Hypertens. 13:525–532; 2004. [41] Schmidt, A.; Jard, S.; Dreifuss, J. J.; Tribollet, E. Oxytocin receptors in rat kidney during development. Am.J.Physiol. 259:F872– F881; 1990. [42] Serradeil-Le Gal, C.; Raufaste, D.; Marty, E.; Garcia, C.; Maffrand, J. P.; Le Fur, G. Autoradiographic localization of vasopressin V1a receptors in the rat kidney using [3H]-SR 49059. Kidney Int. 50:499–505; 1996. [43] Soares, T. J.; Coimbra, T. M.; Martins, A. R.; Pereira, A. G.; Carnio, E. C.; Branco, L. G.; Albuquerque-Araujo, W. I.; de Nucci, G.; Favaretto, A. L.; Gutkowska, J.; McCann, S. M.; Antunes-Rodrigues, J. Atrial natriuretic peptide and oxytocin induce natriuresis by release of cGMP. Proc.Natl.Acad.Sci. U.S.A. 96:278–283; 1999. [44] Szentivanyi, M., Jr.; Park, F.; Maeda, C. Y.; Cowley, A. W., Jr. Nitric oxide in the renal medulla protects from vasopressininduced hypertension. Hypertension 35:740–745; 2000.

Renal Effects of Neurohypophyseal Peptides / 1233 [45] Thibonnier, M.; Conarty, D. M.; Preston, J. A.; Wilkins, P. L.; Berti-Mattera, L. N.; Mattera, R. Molecular pharmacology of human vasopressin receptors. Adv.Exp.Med.Biol. 449:251–276; 1998. [46] Turner, M. R.; Pallone, T. L. Vasopressin constricts outer medullary descending vasa recta isolated from rat kidneys. Am.J.Physiol. 272:F147–F151; 1997. [47] Verbalis, J. G. The brain oxytocin receptor(s)? Front Neuroendocrinol. 20:146–156; 1999.

[48] Verbalis, J. G.; Mangione, M. P.; Stricker, E. M. Oxytocin produces natriuresis in rats at physiological plasma concentrations. Endocrinology 128:1317–1322; 1991. [49] Vorherr, H.; Vorherr, U. F.; Solomon, S. Contamination of prolactin preparations by antidiuretic hormone and oxytocin. Am.J.Physiol. 234:F318–F324; 1978. [50] Windle, R. J.; Judah, J. M.; Forsling, M. L. Effect of oxytocin receptor antagonists on the renal actions of oxytocin and vasopressin in the rat. J.Endocrinol. 152:257–264; 1997.

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170 Renal Renin-Angiotensin System L. GABRIEL NAVAR, MINOLFA C. PRIETO-CARRASQUERO, AND HIROYUKI KOBORI

ulation. When inappropriately stimulated, an enhanced intrarenal renin-angiotensin system (RAS) can markedly alter renal function by acting on vascular and tubular components and thus contribute to the development and maintenance of hypertension and associated cardiovascular diseases. The systemic RAS has already been discussed in two preceding chapters by Tallant et al. (Angiotensin Peptides and Cancer, Chapter 66) in the Cancer/Anticancer Section and by Izumi and Iwao (Angiotensin II and Its Related Peptides, Chapter 160) in the Cardiovascular Section. These presentations cover the basic cascade of the RAS, the formation of several angiotensin peptides by the various enzymes, and the characterization of angiotensin receptors, including AT1a, AT1b, and AT2, for Ang II and III and the Mas receptor for Ang 1–7. In addition, the discovery aspects of the RAS and the basic molecular and biochemical aspects of the precursor genes are covered by Izumi and Iwao in Chapter 160. To avoid redundancy, this review focuses on the novel renal aspects of the RAS.

ABSTRACT All the components of the renin-angiotensin system (RAS) are present within the kidney, and intrarenal formation of angiotensin (Ang) II occurs independently from the systemic RAS. Intrarenal Ang II is compartmentalized such that concentrations much higher than those existing in the circulation are maintained in the renal interstitial fluid and the proximal tubular compartment. The mRNA for angiotensinogen (AGT) is present in proximal tubular cells, and the protein is secreted into the tubules to increase intratubular formation of Ang I and II. Ang II is internalized via AT1 receptor–mediated mechanisms into renal cellular endosomes. Ang II also stimulates AGT mRNA, thus leading to a positive amplification mechanism and further enhancement of intrarenal Ang II. Ang II has pleiotropic actions in the kidney regulating both cortical and medullary blood flow, vascular tone of the afferent and efferent arterioles, mesangial cells, and the sensitivity of the tubuloglomerular feedback mechanism via activation of AT1 receptors. At the level of the tubules, Ang II regulates the transport rate of several transport systems in proximal and distal nephron segments via the activation of luminal and basolateral AT1 receptors. Collectively, the actions of Ang II regulate many aspects of renal function, including renal blood flow, glomerular filtration rate, and sodium excretion.

DISTRIBUTION OF RENIN, ANGIOTENSINCONVERTING ENZYME, AND ANGIOTENSINOGEN IN THE KIDNEY The kidneys are unique in having every component of the RAS as well as compartmentalization specialized to the tubular and interstitial networks. Because kidney Ang II tissue concentrations are much greater than can be explained by the concentrations delivered in the arterial blood flow, it is generally acknowledged that most of the Ang II in the intrarenal components is generated locally from angiotensinogen (AGT) delivered to the kidney and from AGT locally produced by proximal tubule cells [28, 30, 51, 52, 75]. The presence of abundant sources of renin, as well as AGT and angiotensin-converting enzyme

INTRODUCTION Although Ang II and related peptides affect many tissue and organ systems throughout the body, its role in the regulation of renal function is unique because renal functional alterations caused by angiotensin (Ang) II influence overall sodium balance and cardiovascular status thus contributing to blood pressure regHandbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

1236 / Chapter 170 (ACE), in the kidney provides an environment conducive to high levels of Ang II synthesis.

Renin Renin, synthesized by the juxtaglomerular apparatus ( JGA) cells, is the primary source of both circulating and intrarenal renin levels. Renin concentrations are particularly high in the renal interstitium because the JGA cells secrete renin into the renal interstitium, thus providing a stimulus for the local generation of Ang I [81]. The secreted active form of renin contains 339– 343 amino acid residues after proteolytic removal of the 43 amino acid residues at the N-terminus of prorenin. Circulating active renin and prorenin are derived mainly from the kidney; circulating prorenin is taken up by some tissues where it may contribute to the local synthesis of Ang peptides [61]. Although the only wellestablished role of renin is to act on AGT, recent studies have indicated that renin or prorenin, its precursor, may directly elicit cellular effects via a renin receptor that activates extracellular-signal-regulated (ERK) 1/ and 2 as well as contribute to the local formation of Ang peptides [54]. This recently described renin receptor appears to bind renin and prorenin, leading to an increase in the catalytic efficiency of Ang I formation from AGT [54]. There is growing awareness of the possible roles of renin in other intrarenal structures where renin has been localized. ACE inhibition induces a recruitment of cells in afferent arterioles beyond the JGA, even though they were not expressing the renin gene in the basal state [21]. Positive renin immunoreaction has also been observed in cells of glomeruli and in proximal and distal tubular segments [62, 70, 71]. More recently, increased attention has been focused on findings of renin mRNA and protein expression in distal nephron segments, specifically the principal cells of the connecting tubules and collecting ducts, suggesting local formation and secretion into the distal tubular fluid [39, 62, 64]. Renin expression in the principal cells of collecting ducts is further increased by chronic infusions of Ang II [62].

Angiotensin-Converting Enzyme Intrarenal ACE is located not only on endothelial cells throughout the vasculature as in other organs but also on membranes of both proximal and distal nephron segments with the greatest abundance found on the brush border of proximal tubules, particularly in the S3 segment [18, 76]. Marked upregulation of ACE in brush borders occurs in kidneys from Goldblatt hypertensive rats and Ang II–infused hypertensive rats [76]. Humans predominantly express ACE in the brush border of

proximal tubular segments with very little ACE expression on vascular endothelial cells or in the vasculature of the glomerular tuft [43]. The lower renal vascular endothelial expression in humans helps explain the much lower Ang I to Ang II conversion rates that have been reported for human kidneys as compared with other species [15, 52]. The recently described ACE2, which acts on Ang II to form Ang 1–7 or on Ang I to form Ang 1–9, is a membrane-associated and secreted enzyme expressed predominantly on endothelium but is highly restricted in humans to heart, kidneys, and testis [14]. Ang 1–7 may serve as an endogenous antagonist of the Ang II– induced actions mediated via AT1 receptors, and changes in ACE2 activity could affect the balance of Ang peptides found in the kidney [69]. This helps explain the elevated Ang II levels in the ACE2 knockout mice [66]. Collectrin, a novel homolog of ACE2 has been identified in mouse, rat, and human [72, 83]. Both ACE2 and collectrin have tissue-restricted expression in the kidney. Collectrin is localized on the luminal surface and in the cytoplasm of collecting duct cells, and its mRNA is expressed in renal collecting ducts cells [83], whereas ACE2 is present throughout the endothelium and in proximal tubular epithelial cells [72].

Angiotensinogen Although most of the circulating AGT is produced and secreted by the liver, renal AGT mRNA and protein have been localized to proximal tubule cells, indicating that much of the intratubular Ang II could be derived from locally formed and secreted AGT [16, 29, 35, 36, 39, 51]. Furthermore, AGT is regulated by an amplification mechanism such that AGT mRNA and protein are stimulated by Ang II, which maintains or further increases the production of Ang II in Ang II–dependent hypertension [29, 35, 36]. AGT produced in proximal tubule cells appears to be secreted directly into the tubular lumen, in addition to producing its metabolites intracellularly and secreting them into the tubule lumen [38, 39, 51, 64]. Proximal tubule AGT concentrations in anesthetized rats are in the range of 300 nM, which greatly exceed the free Ang I and Ang II tubular fluid concentrations [52]. Because of its molecular size, it seems unlikely that much of the plasma AGT filters across the glomerular membrane, further supporting the concept that proximal tubule cells secrete AGT directly into the tubule [16, 17, 33, 64]. The formation of Ang I and II in the tubular lumen subsequent to AGT secretion may be possible because some renin is filtered and/or secreted from JGA cells. The identification of renin in distal nephron segments also indicates a possible pathway for Ang I generation from proximally delivered AGT [62,

Renal Renin-Angiotensin System / 1237 64]. Intact AGT in urine reflects its presence throughout the nephron and, to the extent that renin and ACE are available along the nephron, substrate availability supports continued Ang I generation and Ang II conversion in distal segments [17, 39, 64]. Once Ang I is formed, conversion readily occurs because there are abundant amounts of ACE associated with the proximal tubule brush border. ACE activity is present in tubular fluid throughout the nephron except in the late distal tubule, being higher at the initial portion of the proximal tubule but then decreasing to the distal nephron and increasing again in the collecting ducts and urine [8]. Therefore, intratubular Ang II formation may occur not only in the proximal tubule but also beyond the distal convoluted tubule. Renal tissue ACE activity is critical to maintaining the steadystate Ang II levels in the kidney [3]. Tissue-ACE knockout mice exhibit 80% lower intrarenal Ang II levels than do wild-type mice [47].

Intrarenal Angiotensin II Receptors Ang II receptors are widely distributed in various regions and cell types of the kidney. Two major categories of Ang II receptors, AT1 and AT2, have been described, pharmacologically characterized, and cloned [32, 48, 49]. As indicated in Fig. 1, most of the Ang II–mediated hypertensinogenic actions are generally attributed to the AT1 receptor [10, 31]. In rodents, there are two AT1 receptor subtypes, with the AT1a being the predominant subtype in all nephron segments, and

the AT1b being more abundant than AT1a in the glomerulus [4, 65]. In mature kidneys, AT1a receptors have been localized to the renal microvasculature in both the cortex and medulla, smooth muscle cells of afferent and efferent arterioles, thick ascending limb of the loop of Henle, proximal tubular apical and basolateral membranes, mesangial cells, distal tubules, collecting ducts, and macula densa cells [25, 46, 59, 84]. The afferent arteriole has both AT1a and AT1b receptors, whereas the efferent arteriole primarily expresses AT1a receptors [24]. The essential role of the AT1a receptor in mice is apparent from studies showing that 2-kidney, 1-clip (2K1C) Goldblatt hypertension does not develop in AT1a knockout mice [9]. Vascular and tubular receptors respond differently during high Ang II states [50]. High Ang II levels associated with a low-salt diet decrease glomerular AT1 receptor expression but increase proximal tubular AT1 receptor levels [13]. In 2K1C Goldblatt hypertensive rats, glomerular AT1 receptors and AT1a receptor protein were reduced in both clipped and contralateral kidneys of 2K1C Goldblatt and 2-kidney, 1-wrap hypertensive models and in the kidneys of Ang II–infused rats [1, 80]. In the TGR(mRen2) harboring the mouse renin2 gene, AT1 receptor binding was increased in the vascular smooth muscle of afferent and efferent arterioles, JGA, glomerular mesangial cells, proximal tubular cells, and renomedullary interstitial cells, suggesting that upregulation of AT1 receptors may contribute to the pathogenesis of hypertension in these rats [87]. In Ang II–infused rats studied with in vitro autoradiography,

Ang II, III

Subtype 1A Receptor

Subtype 1B Receptor

Arterial Pressure Aldosterone Release Na Reabsorption Afferent and Efferent Vasoconstriction Mesangial Cell Contraction Sensitivity of TGF Mechanism Na+/H+ Exchanger Activity Proximal and Distal Reabsorption Renin Secretion ET, TxA2, Reactive Oxygen Species Activation of Cytokines and Growth Factors (ICAM1, MCP1, IL-6, TGFβ, PAI-1, NFκB, VEGF)

Ang 1-7

AT2 Type Receptor Vasodilator Effect Inhibit Cell Proliferation Stimulate Bradykinin Tubular Reabsorption (?) Stimulate Nitric Oxide Synthase (endothelial) Pro-inflammatory & pro-fibrogenic in some cells, stimulates RANTES, NF-κB, VEGF, angiopoetin 1 and 2

FIGURE 1. Major Ang II receptor subtypes and their renal actions.

Ang 1-7 Receptor Vasodilation Neural Actions Natriuresis Tubular Reabsorption

1238 / Chapter 170 there were differential responses with significant decreases in glomeruli and inner stripe but not in proximal tubules [26]. The AT2 receptor is highly expressed in human and rodent kidney mesenchyme during fetal life and decreases dramatically after birth [57]. AT2 receptors have been localized to the glomerular epithelial cells, proximal tubules, collecting ducts, and parts of the renal vasculature of the adult rat [46, 80]. AT2 receptor activation is thought to counteract AT1 receptor effects by stimulating the formation of bradykinin and nitric oxide, leading to increases in interstitial fluid concentration of cyclic guanosine monophosphate (cGMP) [67]. AT2 receptor activation appears to influence proximal tubule sodium reabsorption either by a cell membrane-receptor-mediated mechanism or via an interstitial nitric oxide–cGMP pathway [34].

fluid, and intracellular compartments. The interstitial and the intratubular compartments contribute to the disproportionately high intrarenal Ang II levels. Ang II concentrations in interstitial fluid are much higher than the plasma concentrations, with recent results indicating values in the range of 3–5 pmol/ml [55, 67, 68]. Increases in renal interstitial fluid Ang II levels have been reported in the wrapped kidney of rats with Grollman hypertension and in hypertensive rats infused with Ang II for 2 weeks [56, 67]. The high renal interstitial values indicate local regulation of Ang II formation in the renal interstitial compartment and an enhancement of interstitial Ang II production in Ang II dependent hypertension.

Tubular Ang II As shown in Fig. 2, the proximal tubule fluid concentrations of Ang I and Ang II are also much greater than the plasma concentrations and have been found to be in the range of 5–10 pmol/ml [5, 11, 44, 51, 78]. Results from perfused tubules indicate that the proximal tubule secretes Ang II or a precursor into the proximal tubule fluid [5]. In addition to AGT, proximal tubule cells also have renin that can act on AGT to generate Ang I [70]. Distal nephron renin mRNA and protein [39, 62, 64] also provide a pathway for Ang I generation from proximally delivered AGT, but the concentrations of Ang II in distal tubular fluid have not been measured.

INTRARENAL LEVELS OF ANGIOTENSIN II Interstitial Angiotensin II Intrarenal Ang II is not distributed in a homogeneous fashion but is compartmentalized in both a regional and segmental manner [50, 52]. Medullary Ang II levels are higher than the cortical levels in normal rats and increase further in Ang II–infused hypertensive rats [52]. Within the cortex, there is a distribution of Ang II in the interstitial fluid, tubular

EA

PT

ANG I ~0.4 pmol/ml

DT and CD ANG II ≅ 6-10 pmol/ml

ANG I ≅ 4-8 pmol/ml

AGT ≅ 300 pmol/ml

AGT~100 pmol/ml ANG II ~0.2 pmol/ml

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Interstitium ANG I ≅ 1 pmol/ml ANG II ≅ 3 pmol/ml AGT (from circulation) ACE

FIGURE 2. Intratubular processing of the renin-angiotensin system. All components of the renin-angiotensin system are present in the kidney, providing multiple pathways for enhanced intrarenal Ang II formation. AA, afferent arteriole; ACE, angiotensin converting enzyme; AGT, angiotensinogen; CD, collecting duct; DT, distal tubule; EA, efferent arteriole; JGA, juxtaglomerular apparatus; PT, proximal tubule.

Renal Renin-Angiotensin System / 1239

Intracellular Angiotensin II Ang II is internalized via AT1 receptor mediated endocytosis [28, 30, 74, 86]. Endosomal accumulation of Ang II in intermicrovillar clefts and endosomes is increased further in Ang II–infused hypertensive rats [85]. AT1 receptor blockade prevents the endosomal accumulation even though plasma Ang II increases. The presence of Ang II in renal endosomes indicates that some of the internalized Ang II remains intact and contributes to the total Ang II content measured in tissue homogenates [12, 27, 28, 74, 85]. Endocytosis of the Ang II–AT1 receptor complex seems to be required for the full expression of functional responses coupled to the activation of signal transduction pathways, but the full extent of the functions exerted by intracellular Ang II has not been determined [2]. In Ang II–dependent hypertension, a higher fraction of the total kidney Ang II is internalized into intracellular endosomes consisting of both light endosomes and intramicrovillar clefts via an AT1 receptor mediated process [85]. The functions of internalized Ang II remain unresolved. Ang II may be recycled and secreted in order to exert further actions by binding to Ang II receptors on the cell membranes. Ang II may act on cytosolic receptors to stimulate inositol triphosphate (IP3), as has been described for vascular smooth muscle cells [22]. Ang II may also migrate to the nucleus to exert genomic effects [12]. Nuclear binding sites of the AT1 subtype in renal cells have been reported [41]. Because Ang II exerts a positive stimulation on AGT mRNA and protein production, it is possible that intracellular Ang II may have genomic actions to regulate AGT or renin mRNA expression in proximal tubule cells.

BIOLOGICAL ACTIONS OF INTRARENAL ANG II In view of the near-ubiquitous distribution of Ang II receptors in the various structures in the kidney, it is not surprising that Ang II exerts pleiotropic effects at multiple levels within the kidney. The actions of Ang II span both cortical and medullary, vascular and tubular, and can be stimulatory or inhibitory [11, 45, 50, 51]. At the level of the vasculature, Ang II exerts important direct and indirect effects to regulate vascular tone of the afferent and efferent arterioles and of the mesangial cells [7, 20, 42]. Ang II also regulates afferent arteriolar tone through its influence on the tubuloglomerular feedback mechanism, with elevated Ang II levels increasing the sensitivity of the feedback mechanism, thus regulating the amount of filtrate that escapes the proximal nephron and enters the distal nephron segments [6, 45, 53]. In the postglomerular circulation,

Ang II regulates both the cortical and medullary capillary blood flow and helps to regulate the relative blood flowing to the medullary tissues through its control of the tone of the pericytes [58]. Interestingly, although Ang II constricts both afferent and efferent arterioles, the mechanisms are different. The afferent arteriolar constrictor responses to Ang II involve activation of L-type Ca2+ channels to provide sustained Ca2+ entry and vasoconstriction [53]. In contrast, efferent arterioles of normal rats do not vasodilate or vasodilate much less in response to L-type Ca2+ channel blockers, and the vasoconstrictor effects of Ang II are not blocked by L-type Ca2+ channel blockers. In contrast, T-type channels have greater functional significance in regulating the efferent arteriolar constrictor responses to Ang II [20, 23]. T-type Ca2+ channel blockers markedly attenuated the efferent vasoconstrictor responses to Ang II. It has also been suggested that stores-operated Ca2+ channels in efferent arterioles and afferent arterioles are activated by Ang II [19, 42]. Thus, although both afferent and efferent arterioles respond to Ang II, the mechanisms for Ca2+ entry and activation pathways are different. The extent to which these differences are attributable to the differential abundance of AT1A and AT1B receptors remains undetermined. Virtually all aspects of the filtration and tubular reabsorptive processes are regulated by Ang II. Ang II directly regulates the glomerular filtration coefficient with high Ang II concentrations, reducing and low levels or blockade of AT1 receptors increasing the filtration coefficient [45, 50]. At the level of the proximal tubule, Ang II binds to receptors at both the apical and basolateral membranes, stimulating the activity of the sodium-hydrogen exchanger and the sodium bicarbonate cotransporter and thereby exerting an important influence on net proximal reabsorption rate [50, 51, 63]. Transport mechanisms in the proximal tubule, loop of Henle, and distal nephron segments are also influenced by Ang II [37, 40]. Under conditions in which the activity of the RAS is stimulated and greater amounts of AGT are secreted from the proximal tubule, the locus of influence shifts to include the distal nephron segments where increased intratubular formation of Ang II stimulates the sodiumhydrogen exchanger and the sodium chloride cotransporter in distal tubules and the amiloride-sensitive sodium channel in principal cells of connecting tubules and collecting duct segments [37, 60, 79]. This enhanced stimulatory effect allows the kidney to have maximum capability to conserve sodium by decreasing fractional sodium reabsorption to the last 0.1%. At the whole kidney level, this is manifested as a marked suppression of the pressure-natriuresis relationship [45, 77]. Ang II also regulates acid-base balance through its

1240 / Chapter 170 effects on the Na+-H+ exchanger as well as by influencing the activity of the H+ ATPase in intercalated cells [73]. Collectively, the actions of Ang II allow the kidney to maximize sodium conservation without compromising its ability to regulate its many other regulatory functions.

CONCLUSION Recent findings have stimulated interest in the molecular mechanisms regulating the various components of the intrarenal RAS, particularly the interstitial and intratubular concentrations of Ang II and related peptides. It is becoming apparent that intratubular Ang II and interstitial Ang II are regulated independently from the circulating Ang II. The powerful actions of intrarenal Ang II acting via stimulation of AT1 receptors on the vascular, glomerular, and tubular structures provide a synchronous cascade of effects contributing to the ability of the kidney to retain over 99% of the filtered sodium. From a functional perspective, the effects of Ang II not only on proximal nephron reabsorption but also on distal nephron transport function, coupled with the associated actions of elevated aldosterone levels, markedly increase the sodium-retaining capability of the kidney. When activated in a physiologically appropriate setting under conditions of volume contraction or salt-deficient states, these actions can be life saving. When inappropriately maintained or augmented, however, these effects contribute to the development and maintenance of hypertension. Furthermore, the sustained increases in intrarenal Ang II in a setting of hypertension can lead to progressive renal injury, proliferation, and fibrosis associated with the activation of several major cytokines and growth factors [82].

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171 Renal Natriuretic Peptide System and Actions of Urodilatin MARKUS MEYER, JOCHEN R. HIRSCH, AND WOLF-GEORG FORSSMANN

and coworkers demonstrated by immunohistochemistry that urodilatin is present in the human distal tubule [26] and is released by a variety of intracellular stimuli, as well as osmotic effects and increased extracellular Na+ and Cl− [30]. However, the demonstration of a specific mRNA is still required. Thus, the working hypothesis is that urodilatin is derived from ANP gene product with a processing pattern different to that in the heart. Urodilatin, after being secreted into the lumen, interacts with luminally located natriuretic peptide receptors in the kidney, exerting its natriuretic and diuretic effects (Fig. 2) [26, 35, 41]. To characterize the relative physiological importance of urodilatin and ANP, these two peptides were measured in different experimental settings. Several observations suggest that urodilatin plays a key role in the physiological regulation of renal function, especially in the control of renal Na+ and water excretion [11, 23, 24, 27]. Our group [34] measured the effect of long-term Na+ load in healthy volunteers, where a close correlation between natriuresis and urodilatin excretion was found. A stepwise increase in Na+ intake induced a concomitant increase in urodilatin excretion parallel to Na+ excretion. Furthermore, an increased urodilatin and Na+ excretion was observed after acute volume load by saline infusion [8]. Drummer and coworkers investigated the role of urodilatin in six healthy volunteers over a period of 9 days and nights. In this period, an acute isotonic saline infusion was performed. The closest relation with Na+ excretion was observed for urodilatin excretion into the urine, which was considerably increased during the long-term period of up to 22 h postinfusion but not for ANP plasma levels. These results suggest that urodilatin might participate in renal response to an acute saline infusion

ABSTRACT Urodilatin, a natriuretic peptide with four additional amino acids compared to atrial natriuretic peptide (ANP), is generated in distal tubule cells of the renal nephron. Whereas the fundamental role of ANP in the kidney, namely the regulation of natriuresis and diuresis, is questionable, urodilatin may be responsible for this task when secreted into the lumen of the distal tubule. More stable than ANP, it can act as a paracrine regulator and bind to its specific receptors located in the luminal membrane of cortical collecting duct (CCD) and inner medullary collecting duct (IMCD), thus, displaying its effects on cellular pH, Na+ transport, and H2O homeostasis. Urodilatin might also play a future role in the treatment of acute decompensated heart failure, acute renal failure, and bronchial asthma.

DISCOVERY AND FIRST DESCRIPTIONS OF URODILATIN In 1988, a new member of the natriuretic peptide family was discovered by Schulz-Knappe and coworkers [20, 44] from human urine. A purification procedure from 1000 liters of human urine was performed and resulted in a peptide that was fully homologous to the C-terminal 32 amino acids found in the prohormone atrial natriuretic peptide (ANP) and exhibited an Nterminal extension by four amino acids compared to the circulating ANP (Fig. 1). This peptide was called urodilatin. The N-terminal extension suggests that the urinary peptide discovered (urodilatin) does not originate from the circulation but is synthesized in the kidney and excreted into the urine. In 1998, Herten Handbook of Biologically Active Peptides

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1244 / Chapter 171 A Arg

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FIGURE 1. Amino acid sequence of circulating (A) ANP(99–126) and (B) urodilatin(99–126). Urodilatin differs from ANP by an N-terminal extension of four amino acids (indicated by black bar).

[8]. Furthermore, Goetz and coworkers [24] investigated the endogenous production of urodilatin and ANP in conscious dogs following balloon dilation of the left atrium after cardiac denervation compared to normal dogs. Only urodilatin excretion in urine paralleled the natriuresis induced by balloon dilation, whereas the plasma levels of ANP showed no positive correlation. Emmeluth and coworkers [11] showed an increase in urodilatin excretion after a selective increase in cephalic Na+ concentration, suggesting that cephalic Na+ concentration receptors regulate the rate of Na+ excretion via urodilatin. Norsk and coworkers [37] studied the effects of 12-h head-out water immersion in seated normal human subjects and found that the excretion of urodilatin followed a temporal pattern similar to the increases in urine flow and Na+ excretion produced by immersion. Drummer and coworkers [7] investigated the role of urodilatin in the regulation of Na+ homeostasis and demonstrated that the circadian rhythm of

urinary Na+ excretion paralleled urodilatin excretion. Acute saline infusion [8] induced natriuresis that correlated more closely to urodilatin excretion than to ANP plasma levels. Similar results were obtained by Hansell and coworkers [25], who showed that natriuresis occurring after the intracerebroventricular application of hypertonic saline is not associated with an increase in ANP plasma levels. Taken together, these observations indicate that urodilatin may be the natriuretic factor primarily responsible for Na+ and water regulation. Urodilatin closely mimics the receptor-binding and agonist properties of ANP and binds to natriuretic peptide receptors at several nephron sites, including especially the glomerulus and the inner medullary collecting duct (IMCD) [41, 48]. Valentin [48] showed similar cGMP generation characteristics of urodilatin and ANP in glomeruli and IMCD cells, whereas Saxenhofer [41] reported a lower potency of urodilatin for cGMP stimulation compared to ANP in papillary

Renal Natriuretic Peptide System and Actions of Urodilatin / 1245

Endogenous ANP Exogenous Urodilatin Vas afferens (relaxation) Vas efferens (constriction)

Enhanced GFR

Synthesis and processing of Urodilatin

Interaction with NPR-A

Degradation of ANP Urodilatin is not affected

Inhibition of Na+- and H2Oreabsorption

Urodilatin

FIGURE 2. Mechanism of physiological and pharmacological effects of the natriuretic peptides ANP and urodilatin. In the proximal tubule, enzymatic degradation of filtered ANP(99–126) can occur to prevent an interaction of ANP further downstream with luminal receptors located in the distal tubule and collecting duct. In contrast, urodilatin is secreted by distal tubule cells into the lumen and can exert its renal effects in the distal parts of the nephron.

collecting-duct homogenates. However, these results demonstrate that urodilatin interacts with the same receptors and signaling pathways that mediate the actions of ANP. The stimuli for urodilatin synthesis and secretion in the kidney are, however, still far from being clarified. Whether the urodilatin secretion is a humoral- or neural-mediated response is unclear. To further clarify

this issue, Emmeluth and coworkers [11] performed experiments on dogs with surgically denervated kidneys. Again, Na+ excretion increased, but urodilatin excretion was not significantly different to that induced by isotonic saline infusion in control animals, which tended to be higher. Thus, the natriuretic response modulated by urodilatin appears to be independent of renal nerves favoring a humoral factor. However, the authors could

1246 / Chapter 171 demonstrate a strong correlation between natriuresis and endothelin excretion [12], suggesting that the role of urodilatin is still unclear. Comparable to the efforts that had been undertaken to characterize the renal function of ANP, the exact mechanisms by which urodilatin acts in the kidney were investigated by administering urodilatin intravenously. The initiation of profound diuresis and natriuresis as well as a slight reduction in blood pressure are the most prominent effects [1, 27, 42]. The strength and duration of these effects differed considerably from those of ANP. In bolus injections in healthy volunteers, the resulting natriuresis and diuresis were double the effect of ANP [1, 3, 27, 42]. In volume-expanded dogs, it was also demonstrated that urodilatin induced a stronger diuresis and natriuresis than that seen by ANP [3]. The precise way in which urodilatin interacts in the kidney and leads to diuresis and natriuresis was investigated in pharmacological studies. Although the available literature about urodilatin is not extensive compared to that of ANP, the mode of action of urodilatin in the kidney appears to be well established. Based on the similar affinity of urodilatin and ANP to intrarenal natriuretic peptide receptors [41, 47], most of the renal actions of urodilatin can be explained in analogy to those induced by ANP. Figure 3 shows the distribution of the NPR-A/GC-A receptor in different

FIGURE 3. RT-PCR analysis of NPR-A/GC-A distribution throughout the kidney. From cortex to papilla the receptor for ANP and urodilatin is present in every layer of the kidney. Modified from [21].

layers of the kidney that is bound by ANP and urodilatin. Using hydronephrotic rat kidneys, Endlich and coworkers [13] demonstrated that urodilatin leads to a dilation of preglomerular vessels, constriction of efferent arterioles, and subsequently to an increased glomerular filtration rate (GFR). These results were confirmed in humans [42] and animals [1], whereas Hildebrandt and coworkers [27] could not find any changes in GFR after injection into the renal artery of dogs. The same group found no changes in fractional lithium excretion as a marker for proximal tubular reabsorption. Therefore, the induced diuresis and natriuresis is similar to ANP, probably due to an enhancement of GFR and an inhibition of Na+ reabsorption in the IMCD.

METABOLISM OF URODILATIN IN THE KIDNEY AND ITS PHYSIOLOGICAL AND PHARMACOLOGICAL SIGNIFICANCE The removal of natriuretic peptides from circulation occurs via different mechanisms. In addition to the binding of circulating natriuretic peptides to clearance receptors, enzymatic degradation in the lung, liver, and kidney also takes place [6, 14]. The main enzyme responsible for degradation is the metalloendoprotease EC 24.11. One of the main location sites is the brush border of the proximal tubule. This enzyme cleaves the loop structure of ANP between positions Cys-105 and Phe-106, reducing receptor affinity and thus biological activity [22, 28, 38, 45]. Thus, under physiological conditions at least, the filtered ANP reaching the proximal tubule may be degraded and is unlikely to be detected as an intact molecule further downstream in the urine. Urodilatin, on the other hand, is synthesized in the distal tubule and secreted into the lumen (see Fig. 4) and almost completely removed from the renal tubular system [3, 19, 26, 30, 47]. From the pharmacological point of view, the following finding was very important: In contrast to ANP, studies demonstrated a high resistance of urodilatin to enzymatic degradation when both peptides were incubated with kidney cortex membranes from the dog, which are known to be rich in endoproteases [22]. This relative resistance of urodilatin to the endoproteases may be due to its N-terminal extension of four additional amino acids [15, 22, 28]. This structural difference may induce configurational changes, preventing the enzyme from attacking the cleavage site. Thus, administered intravenously, urodilatin could reach nephron sites that are located distal from the proximal tubule whereas ANP is inactivated there. This could also explain the stronger natriuretic and diuretic effects of urodilatin compared to ANP described by several groups

Renal Natriuretic Peptide System and Actions of Urodilatin / 1247

Acute Renal Failure

FIGURE 4. Immunostaining of urodilatin in distal tubules of porcine kidney. Arrows indicate strongest labeling for urodilatin in different segments of the distal tubule. Modified from [21].

[1, 27, 42, 49]. Bub and coworkers [32] infused radioactively labeled urodilatin and ANP in situ into the renal artery prior to freeze-sectioning and autoradiography. They found binding sites for both peptides predominantly located in the cortex related to glomeruli and arterial blood vessels. Furthermore, urodilatin was also labeled in the medullary structures, indicating a binding at the IMCD receptors. This finding supports the data of the relative resistance of urodilatin to the endoproteases and in addition appears to be of great clinical importance when considering the potential renal effects of intravenously administered ANP and urodilatin.

URODILATIN—CLINICAL IMPLICATIONS Urodilatin induces diuresis and natriuresis and has antagonistic effects on the renin-angiotensin-aldosterone system [4]. Thus, among other things, urodilatin regulates salt and water homeostasis and blood pressure. These physiological effects point to urodilatin’s pharmacological and therapeutic potential in various clinical indications. In animal models, urodilatin’s role as a potential therapeutic effector was established a decade ago. When urodilatin was given intravenously in acute renal failure models, it induced strong diuretic and natriuretic effects [43] whereas in congestive heart failure (CHF) beneficial hemodynamic effects were demonstrated [39, 40]. It was also shown that urodilatin relaxes the bronchi in animal models with drug-induced bronchoconstriction [18]. These renal, pulmonary, and cardiovascular effects indicate that urodilatin is potentially a drug for the treatment of related diseases.

Urodilatin has been used in the treatment and prophylaxis of acute renal failure following major surgery and heart transplantation. Summarizing, there are clinical studies using urodilatin with different results, significantly reducing the incidence of hemodialysis/ hemofiltration (HD/HF) in some [5, 50] and exhibiting no beneficial effects in another [33]. Similar results with opposite effects generated in clinical studies have also been demonstrated for ANP [2, 31]. These data generally reflect the problem in demonstrating the beneficial effects of drugs in prospective trials of acute renal failure. However, thorough analyses of data from acute renal failure studies with urodilatin and ANP have led to a better understanding of various factors, such as (1) multimorbidity of the patient collective, (2) start of including patients into the study, (3) definition of acute renal failure, (4) dosage-balancing treatment and hypotensive effects, and (5) duration of study drug. These issues were then considered and addressed in a recently published randomized placebo-controlled study in which ANP was infused in patients suffering from ischemic acute renal failure [46]. Here, ANP was demonstrated to enhance renal excretory function, to decrease probability of dialysis, and to improve dialysisfree survival in early ischemic acute renal failure. In contrast to more negative results in the past, reasons that may have contributed to the positive outcome of this study were the maintenance of hemodynamic stability, intervention in the early phase of acute renal failure, and the extended duration of drug application [9].

Bronchial Asthma Urodilatin exerts bronchodilator activity by the stimulation of intracellular cGMP as an alternative pathway to β2-agonists, such as albuterol, one of the first-line bronchodilators, increasing the intracellular concentrations of cAMP and thereby inducing bronchodilation. In patients with mild to moderate bronchial asthma, urodilatin given intravenously exerts a bronchodilatory effect on the central and peripheral airways, as shown by increasing lung function parameters such as forced expiratory volume in 1 second (FEV1), maximal expiratory flow (MEF), and peak expiratory flow (PEF) [16]. In a double-blind, randomized, placebo-controlled study with crossover design, urodilatin, given as monotherapy as well as in combination with albuterol, exerted bronchodilatory effects in clinically stable asthmatic patients with severe disease [17]. In conclusion, urodilatin may improve pulmonary function in patients with bronchial asthma and other obstructive pulmonary diseases. Particularly in patients with cardiovascular risk

1248 / Chapter 171 and in patients in whom the cAMP-mediated mechanism of bronchodilation is disturbed or exhausted due, for example, to receptor downregulation and desensitization, the cGMP-mediated bronchodilatory mechanism induced by urodilatin may be a potential treatment alternative.

Heart Failure In its first pharmacological studies, urodilatin was administered to cardiomyopathic dogs. The study revealed a beneficial effect in contrast to ANP [39, 40]. Urodilatin induced strong diuresis and natriuresis, whereas ANP caused no significant changes in renal excretory function at equimolar doses. In a study with patients suffering from CHF, the hemodynamic effects induced by urodilatin and ANP were compared [29]. Urodilatin stimulated diuresis and natriuresis more efficiently than ANP. In addition, the reduction of systemic vascular resistance (SVR) and pulmonary capillary wedge pressure (PCWP) and the increase of stroke volume index were more prominent after urodilatin administration. Based on these initial pharmacological studies, a double-blind, placebo-controlled study was performed with urodilatin in patients suffering from CHF [10]. Urodilatin was infused in a dose of 15 ng/kg body weight/min for 10 hours in 12 patients with CHF. Urine flow and urinary Na+ excretion were significantly increased. Furthermore, urodilatin significantly reduced systolic blood pressure and right atrial pressure, whereas diastolic blood pressure remained unchanged. A recent phase IIa ascending dose safety trial (SIRIUS I) in patients suffering from acute decompensated CHF (ADHF) demonstrates beneficial hemodynamic effects of urodilatin as a balanced vasodilator that relaxes both arterial and venous blood vessels and thus reduces pre- and afterload [36]. In 24 patients with ADHF, urodilatin infused at doses of 7.5, 15, and 30 ng/kg/min for 24 hours was well tolerated and the two higher doses resulted in significant reductions of PCWP and tended to improve dyspnea. These effects were associated with decreases in plasma Nt-proBNP levels, suggesting that urodilatin has potential as a new treatment for acute decompensated heart failure (ADHF). With the isolation of urodilatin, a new structural analog of ANP and the latest member of the natriuretic peptide family in mammals was discovered. Due to its stability toward endopeptidases and its place of origin, the distal tubule, urodilatin seems to be responsible for the regulatory effects of the late distal tubule and cortical and inner medullary collecting duct. Furthermore, urodilatin has the potential for being an important pharmacological and therapeutic effector in diseases

such as acute renal failure and ADHF and as a bronchodilator in bronchial asthma.

References [1] Abassi ZA, Powell JR, Golomb E and Keiser HR. Renal and systemic effects of urodilatin in rats with high-output heart failure. Am J Physiol 262: F615–F621, 1992. [2] Allgren RL, Marbury TC, Rahman SN, Weisberg LS, Fenyes AZ, Lafayette RA, Sweet RM, Genter FC, Kurnik BR, Conger JD and Sayegh MH. Anaritide in acute tubular necrosis. Auriculin Anaritide Acute Renal Failure Study Group. N Engl J Med 336: 828–834, 1997. [3] Bestle MH, Olsen NV, Christensen P, Jensen BV and Bie P. Cardiovascular, endocrine, and renal effects of urodilatin in normal humans. Am J Physiol 276: R684–R695, 1999. [4] Carstens J, Jensen KT and Pedersen EB. Metabolism and action of urodilatin infusion in healthy volunteers. Clin Pharmacol Ther 64: 73–86, 1998. [5] Cedidi C, Meyer M, Kuse ER, Schulz-Knappe P, Ringe B, Frei U, Pichlmayr R and Forssmann W-G. Urodilatin: a new approach for the treatment of therapy-resistant acute renal failure after liver transplantation. Eur J Clin Invest 24: 632–639, 1994. [6] Crozier IG, Nicholls MG, Ikram H, Espiner EA, Yandle TG and Jans S. Atrial natriuretic peptide in humans. Production and clearance by various tisues. Hypertension 8: II11–II15, 1986. [7] Drummer C, Fiedler F, König A and Gerzer R. Urodilatin, a kidney-derived natriuretic factor, is excreted with a circadian rhythm and is stimulated by saline infusion in man. J Am Soc Nephrol 1: 1109–1113, 1991. [8] Drummer C, Gerzer R, Heer M, Molz B, Bie P, Schlossberger M, Stadaeger C, Rocker L, Strollo F, Heyduck B, Bauer K, Warberg J, Baisch F, Christensen NJ, König A and Norsk P. Effects of an acute saline infusion on fluid and electrolyte metabolism in humans. Am J Physiol 262: F744–F754, 1992. [9] du Cheyron D. Atrial natriuretic peptide to prevent acute renal failure: old concept with new promise. Crit Care Med 32: 1421– 1422, 2004. [10] Elsner D, Muders F, Muntze A, Kromer EP, Forssmann W-G and Riegger GA. Efficacy of prolonged infusion of urodilatin [ANP(95–126)] in patients with congestive heart failure. Am Heart J 129: 766–773, 1995. [11] Emmeluth C, Drummer C, Gerzer R and Bie P. Roles of cephalic Na+ concentration and urodilatin in control of renal Na+ excretion. Am J Physiol 262: F513–F516, 1992. [12] Emmeluth C, Goetz KL, Drummer C, Gerzer R, Forssmann W-G and Bie P. Natriuresis caused by increased carotid Na+ concentration after renal denervation. Am J Physiol 270: F510–F517, 1996. [13] Endlich K, Forssmann W-G and Steinhausen M. Effects of urodilatin in the rat kidney: comparison with ANF and interaction with vasoactive substances. Kidney Int 47: 1558–1568, 1995. [14] Erdös EG and Skidgel RA. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J 3: 145–151, 1989. [15] Feller SM, Bub A, Gagelmann M and Forssmann W-G. Natriuretic peptides from the heart, brain, and kidney: localization, processing, vasoactivity, and proteolytic degradation. In: Heart Failure Mechanisms and Management, edited by Lewis BS and Kimchi A. Berlin-Heidelberg: Springer-Verlag, 1991, p. 398– 407. [16] Flüge T, Fabel H, Wagner TO, Schneider B and Forssmann WG. Urodilatin (ularitide, INN): a potent bronchodilator in asthmatic subjects. Eur J Clin Invest 25: 728–736, 1995.

Renal Natriuretic Peptide System and Actions of Urodilatin / 1249 [17] Flüge T, Forssmann W-G, Kunkel G, Schneider B, Mentz P, Forssmann K, Barnes PJ and Meyer M. Bronchodilation using combined urodilatin-albuterol administration in asthma: a randomized double-blind, placebo-controlled trial. Eur J Med Res 4: 411–415, 1999. [18] Flüge T, Hoymann HG, Hohlfeld J, Heinrich U, Fabel H, Wagner TO and Forssmann W-G. Type A natriuretic peptides exhibit different bronchoprotective effects in rats. Eur J Pharmacol 271: 395–402, 1994. [19] Forssmann W-G, Meyer M and Forssmann K. The renal urodilatin system: clinical implications. Cardiovasc Res 51: 450–462, 2001. [20] Forssmann W-G, Meyer M and Schulz-Knappe P. Urodilatin: from cardiac hormones to clinical trial. Exp Nephrol 2: 318– 323, 1994. [21] Forssmann W-G, Richter R and Meyer M. The endocrine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system. Histochem Cell Biol 110: 335–357, 1998. [22] Gagelmann M, Hock D and Forssmann W-G. Urodilatin (CDD/ ANP-95–126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (alpha-hANP/CDD-99–126). FEBS Lett 233: 249–254, 1988. [23] Goetz KL. Evidence that atriopeptin is not a physiological regulator of sodium excretion. Hypertension 15: 9–19, 1990. [24] Goetz K, Drummer C, Zhu JL, Leadley R, Fiedler F and Gerzer R. Evidence that urodilatin, rather than ANP, regulates renal sodium excretion. J Am Soc Nephrol 1: 867–874, 1990. [25] Hansell P, Goransson A, Leppaluoto J, Arjamaa O, Vakkuri O and Ulfendahl HR. CNS-induced natriuresis is not mediated by the atrial natriuretic factor. Acta Physiol Scand 129: 221–227, 1987. [26] Herten M, Lenz W, Gerzer R and Drummer C. The renal natriuretic peptide urodilatin is present in human kidney. Nephrol Dial Transplant 13: 2529–2535, 1998. [27] Hildebrandt DA, Mizelle HF, Brands MW and Hall JE. Comparison of renal actions of urodilatin and atrial natriuretic peptide. Am J Physiol 262: R395–R399, 1992. [28] Kenny AJ, Bourne A and Ingram J. Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptides and some C-receptor ligands by endopeptidase-24.11. Biochem J 291: 83–88, 1993. [29] Kentsch M, Ludwig D, Drummer C, Gerzer R and Müller-Esch G. Haemodynamic and renal effects of urodilatin bolus injections in patients with congestive heart failure. Eur J Clin Invest 22: 662–669, 1992. [30] Lenz W, Herten M, Gerzer R and Drummer C. Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line. Kidney Int 55: 91–99, 1999. [31] Lewis J, Salem MM, Chertow GM, Weisberg LS, McGrew F, Marbury TC and Allgren RL. Atrial natriuretic factor in oliguric acute renal failure. Anaritide Acute Renal Failure Study Group. Am J Kidney Dis 36: 767–774, 2000. [32] Meyer M and Forssmann W-G. Renal actions of atrial natriuretic peptide. In: Contemporary endocrinology: Natriuretic peptides in health and disease, edited by Samson WK and Levin ER. Totowa, NJ: Humana Press Inc., 1997, p. 147–170. [33] Meyer M, Pfarr E, Schirmer G, Uberbacher HJ, Schope K, Bohm E, Flüge T, Mentz P, Scigalla P and Forssmann W-G. Therapeutic use of the natriuretic peptide ularitide in acute renal failure. Ren Fail 21: 85–100, 1999.

[34] Meyer M, Richter R, Brunkhorst R, Wrenger E, Schulz-Knappe P, Kist A, Mentz P, Brabant EG, Koch KM, Rechkemmer G and Forssmann W-G. Urodilatin is involved in sodium homeostasis and exerts sodium-state-dependent natriuretic and diuretic effects. Am J Physiol 271: F489–F497, 1996. [35] Meyer M, Richter R and Forssmann W-G. Urodilatin: a natriuretic peptide with clinical implications. Eur J Med Res 3: 103– 110, 1998. [36] Mitrovic V, Lüss H, Nitsche K, Forssmann K, Maronde E, Fricke K, Forssmann W-G and Meyer M. Effects of the renal natriuretic peptide urodilatin (ularitide) in patients with decompensated chronic heart failure: A double-blind, placebo-controlled, ascending-dose trial. Am Heart J 150: 1239, 2005. [37] Norsk P, Drummer C, Johansen LB and Gerzer R. Effect of water immersion on renal natriuretic peptide (urodilatin) excretion in humans. J Appl Physiol 74: 2881–2885, 1993. [38] Olins GM, Spear KL, Siegel NR and Zurcher-Neely HA. Inactivation of atrial natriuretic factor by the renal brush border. Biochim Biophys Acta 901: 97–100, 1987. [39] Riegger GA, Elsner D, Forssmann W-G and Kromer EP. Effects of ANP-(95–126) in dogs before and after induction of heart failure. Am J Physiol 259: H1643–H1648, 1990. [40] Riegger GA, Elsner D, Kromer EP, Daffner C, Forssmann W-G, Muders F, Pascher EW and Kochsiek K. Atrial natriuretic peptide in congestive heart failure in the dog: plasma levels, cyclic guanosine monophosphate, ultrastructure of atrial myoendocrine cells, and hemodynamic, hormonal, and renal effects. Circulation 77: 398–406, 1988. [41] Saxenhofer H, Fitzgibbon WR and Paul RV. Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells. Am J Physiol 264: F267–F273, 1993. [42] Saxenhofer H, Raselli A, Weidmann P, Forssmann W-G, Bub A, Ferrari P and Shaw SG. Urodilatin, a natriuretic factor from kidneys, can modify renal and cardiovascular function in men. Am J Physiol 259: F832–F838, 1990. [43] Schramm L, Heidbreder E, Schaar J, Lopau K, Zimmermann J, Götz H, Ling H and Heidland A. Toxic acute renal failure in the rat: effects of diltiazem and urodilatin on renal function. Nephron 68: 454–461, 1994. [44] Schulz-Knappe P, Forssmann K, Herbst F, Hock D, Pipkorn R and Forssmann W-G. Isolation and structural analysis of “urodilatin,” a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine. Klin Wochenschr 66: 752–759, 1988. [45] Stephenson SL and Kenny AJ. The hydrolysis of alpha-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11. Biochem J 243: 183–187, 1987. [46] Sward K, Valsson F, Odencrants P, Samuelsson O and Ricksten SE. Recombinant human atrial natriuretic peptide in ischemic acute renal failure: a randomized placebo-controlled trial. Crit Care Med 32: 1310–1315, 2004. [47] Valentin JP and Humphreys MH. Urodilatin: a paracrine renal natriuretic peptide. Semin Nephrol 13: 61–70, 1993. [48] Valentin JP, Sechi LA, Qui C, Schambelan M and Humphreys MH. Urodilatin binds to and activates renal receptors for atrial natriuretic peptide. Hypertension 21: 432–438, 1993. [49] Villarreal D, Freeman RH and Johnson RA. Renal effects of ANF (95–126), a new atrial peptide analogue, in dogs with experimental heart failure. Am J Hypertens 4: 508–515, 1991. [50] Wiebe K, Meyer M, Wahlers T, Zenker D, Schulze F, Michels P, Dalichau H, Mohr FW, Borst H and Forssmann W-G. Acute renal failure following cardiac surgery is reverted by administration of urodilatin (INN: ularitide). Eur J Med Res 1: 259–265, 1996.

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172 ANP and Its Role in the Regulation of Renal Tubular Transport Processes JOCHEN R. HIRSCH, MARKUS MEYER, AND WOLF-GEORG FORSSMANN

the first to isolate the hormone from the human heart. The peptide was named α-human atrial natriuretic peptide (α hANP). Since then, remarkable advances have been made regarding the characterization of its gene and protein nature, synthesis, intracellular processing, release, metabolism, second-messenger systems, and receptors [14, 38]. An explanation for ANP-induced natriuresis and diuresis has not been found yet, but most research groups favor a combination of hemodynamic and tubular effects. Because the hemodynamic effects are well established, the following paragraphs focus on tubular effects of ANP.

ABSTRACT The main task of atrial natriuretic peptide (ANP) in the kidney is the induction of natriuresis and diuresis, but the mechanism behind these effects is far from being clarified. The involvement of ANP in the regulation of the cardiovascular system is well established, but its regulatory role in transport processes in the nephron is still unresolved. Here, we take a closer look at the regulation of a variety of transport proteins in the renal nephron by ANP from the proximal tubule to the cortical and inner medullary collecting duct.

DISCOVERY OF ATRIAL NATRIURETIC PEPTIDE ACTIONS IN THE KIDNEY

EFFECTS OF ANP ON TUBULAR TRANSPORT SYSTEMS

For nearly half a century an involvement of the heart in renal functions has been postulated [22]. In 1969, this hypothesis was supported by Lockett, who mentioned for the first time a hormonal influence of the heart on the kidney [37]. In 1974, Marie and coworkers [40] observed that the granular index of myoendocrine cells is affected during experiments, altering the body fluid and electrolyte homeostasis. By applying atrial extracts, Sonnenberg and colleagues [11] reported for the first time a previously unknown strong diuresis and natriuresis. A further biological effect of atrial extracts, relaxation of vascular smooth muscle, was discovered next [8, 15, 18]. The extract has a strong vasorelaxant effect on a cortical arteriole. Based on these two main biological effects, the first isolation of atrial natriuretic peptides (ANPs) was performed. Depending on the bioassay used for isolation, the peptides were designated cardiodilatin (from the pig) [15] or cardionatrin (from the rat) [13]. Kangawa and coworkers [34] were Handbook of Biologically Active Peptides

Effects of ANP on Proximal Tubules Early investigations on proximal tubules regarding the possible actions and effects of ANP resulted in controversial results. Many in vivo and in vitro microperfusion studies of proximal convoluted and straight tubules in the rat or rabbit have failed to find any effects of ANP on Na+, bicarbonate, and chloride transport, and only a few managed to demonstrate ANP-induced transport changes. It was shown that the solute delivery out of the proximal tubule increased by approximately 30–35% [7, 30]. Furthermore, Harris and coworkers [20] reported that the stimulatory effect of angiotensin II on fluid reabsorption was inhibited by peritubular ANP at physiological concentrations and abolished by higher concentrations of ANP. In contrast to the results of Cogan and coworkers [7, 30], the authors concluded that, at physiological concentrations, ANP acts within the kidney to decrease proximal reabsorption by the inhibition of

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1252 / Chapter 172 angiotensin II–stimulated Na+ and water transport; this was also supported by Garvin and coworkers [16]. With the introduction of modern techniques such as patch clamping, molecular biology, and protein chemistry, more reports showed direct effects of ANP and related natriuretic peptides such as urodilatin, B natriuretic peptide (BNP), and C natriuretic peptide (CNP) on various transport systems, ion channels, and pumps in the nephron and its various tubular structures. Thus, it was demonstrated that ANP inhibited several Na+-dependent transport systems. In proximal tubular brush border membrane vesicles, the Na+/H+antiporter, which is responsible for approximately 50% of the Na+ intake in this segment, is inhibited, as well as the Na+-coupled PO43− transport [58]. Na+-coupled PO43− transport has been extensively researched by Murer and his group, and in 2001 they reported that the inhibitory effect of ANP was due to the membrane retrieval containing the Na-Pi type Ila cotransporter [3]. Recently, it was shown that the low-affinity high-capacity Na+-glucose cotransporter (SGLT2) activity was drastically reduced when ANP was administered together with endothelin 3 [39]. This transport system is the most important retrieval pathway for sugar in the kidney and accounts for 90% of the Na+-coupled sugar intake. The Na+/K+ATPase, essential for all secondary active transport processes, was also reported to be deactivated in the proximal tubule by phosphorylation/dephosphorylation due to ANP-induced cGMP and dopamine-induced cAMP increase [2, 5]. However, not just Na+-dependent processes are involved in the regulatory pattern of ANP. The organic cation transporter of the basolateral membrane of isolated human proximal tubules showed a 20–30% increase in transport after being stimulated by ANP or 8-Br-cGMP [49]. In the meantime, Cl− as well as K+ channels have also been found to be the target of regulation by natriuretic peptides. In cultured rat proximal convoluted tubule (PCT) cells, a DIDS-inhibitable Cl− conductance was activated by ANP, urodilatin or their second messenger cGMP [10], whereas in the human proximal tubule cell line RPTEC, an inwardly rectifying K+ channel was found to be phosphorylated by a cGMP-dependent protein kinase (PKG) due to the application of ANP [43]. Several inwardly rectifying K+ channels have been identified in the proximal tubule [28, 41, 43]. Earlier, the authors described a K+ conductance that was inhibited by various natriuretic peptides such as ANP, urodilatin, BNP, and CNP in a cGMPdependent and -independent fashion in IHKE-1 cells, another human proximal tubule cell line [24]. This K+ conductance turned out to be the first directly cGMPdependent K+ channel described in humans [28]. Interestingly, in IHKE-1 cells this K+ channel was regulated by cGMP, not only intracellularly but also from the outside, demonstrating that ANP and cGMP can also display regulatory effects from the luminal side of the

tubule system [27]. Therefore, ANP, urodilatin, and other cGMP-generating factors have three possible ways to regulate transport in the tubular system: (1) binding to glomerular cells and subsequently releasing cGMP into the proximal lumen, (2) binding to its specific receptor (NPR-A/GC-A) and increasing intracellular cGMP, and (3) binding to its specific receptor, increasing intracellular cGMP with the consecutive extrusion of cGMP via a specific transport system [57] into the lumen, and acting from the outside on the target transmembrane protein. An overview of transport proteins involved in the regulatory pathway of ANP in the proximal tubule cell is given in Fig. 1.

Effects of ANP on Henle’s Loop The functional responses of ANP in Henle’s loop are unknown. The administration of pharmacological

lumen

blood

cGMP Na+ X Y cGMP

Na+ GTP

GC-A

cGMP

ATP

ATP

K+

PKG

K+ K+ Cl-

OC+

FIGURE 1. Cell model of the proximal tubule. Shown are the transport systems that are regulated via ANP and its second messenger cGMP. In the luminal membrane, the guanylyl cyclase receptor (GC-A) for ANP, urodilatin, and BNP (Y) is located and, after being activated, generates cGMP. This second messenger can activate a cGMP-dependent kinase (PKG), which phosphorylates several Na+coupled transporters, such as the basolateral Na+/K+-ATPase and the Na+/H+ exchanger, Na+-glucose and Na+-PO43− cotransporter (X) of the luminal membrane but also the Na+independent organic cation transporter (OC) of the basolateral membrane. Furthermore, PKG activates a Cl−-channel in the luminal membrane and an inwardly rectifying K+ channel (location is not verified and the basolateral membrane was chosen accidentally). cGMP can also act inhibitively on a K+ channel in the luminal membrane by itself. When transported to the lumen by a specific pump, it can also inhibit this channel from the outside. This mechanism also works when cGMP is released from glomerular cells due to stimulation by ANP or related natriuretic peptides.

ANP and Its Role in the Regulation of Renal Tubular Transport Processes / 1253 amounts of ANP resulted in an increase in urinary magnesium excretion [19]. Magnesium is principally reabsorbed in the cortical thick ascending limb and urinary magnesium excretion has been used as a marker for loop function [9]. Accordingly, it is inferred that ANP may alter salt transport in this segment. However, other studies have shown little effect of ANP in the loop. Peterson and coworkers [48] investigated the effects of these hormones on thick ascending limb Na+Cl− reabsorption in vivo. Using the microstop-flow conductivity technique, despite the presence of a dramatic natriuretic response, ANP did not significantly increase the minimum Na+Cl− concentration reached by the thick ascending limb. In contrast, vasopressin increased the rate at which luminal Na+ concentration declined by 23%. Up to now, the only direct effects of ANP on Na+Cl− transport are a report on a decrease in Cl− reabsorption [4] and an inhibitory effect of ANP on Na+/K+ATPase [55]. An explanation might be the low response of the cells of Henle’s loop in cGMP formation following ANP incubation [6, 56]. Comparable to other segments of the nephron, where ANP is able to modulate intracellular Ca2+ levels [45], a putative physiological importance of ANP could also be demonstrated in this nephron portion [9]. Using primary cell cultures, this group reported an ANP-induced receptor-mediated Ca2+ transient in cortical thick ascending limb cells resulting from internal release and influx across the plasma membrane. The authors discussed whether this receptor is most likely to be the clearance receptor that initiates Ca2+ signals. They concluded that these ANP-induced Ca2+ transients mediated by the putative clearance receptor may have significant physiological actions within Henle’s loop. The rather moderate effects of ANP, urodilatin, or cGMP in Henle’s loop do not necessarily mean that natriuretic peptides are not involved in the regulatory processes of this tubular segment. Recently, it was shown that a splice variant of the CNP-activated NPR-B/GC-B receptor, NPR-Bi, is highly expressed in human thick ascending limb and cortical collecting duct. This receptor lacks guanylyl cyclase activity and most likely acts as a tyrosine kinase [26].

Effects of ANP on the Collecting Duct System There is evidence that ANP affects natriuresis and diuresis in part by inhibiting net Na+ and water reabsorption in the collecting duct. In the following, the actions of ANP investigated in in vivo experiments are presented. In 1979, Jamison et al. [31] reviewed the day-to-day regulation of fluid and electrolyte balance, in which the collecting tubule system “appears to occupy a paramount position among segments of the renal tubule.” At the same time, the authors referred to the dis-

crepancies in reported results due to different methods, and they concluded that “controversy has arisen concerning the quantitative contribution by the collecting tubule system to the regulation of individual solute excretion, which in part may be due to differences among the investigative techniques (microcatheterization technique and the micropuncture method) employed.” In a fundamental work, Sonnenberg and coworkers [53] described the effects of ANP on the tubular system. Micropuncture and microcatheterization were performed in anesthetized rats to collect tubular fluid from the proximal and distal tubules and from the outer medullary collecting duct. Samples were taken from the same sites before and after intravenous (IV) administration of atrial tissue extract. Na+ excretion increased 17-fold after atrial extract was injected, whereas the clearances of insulin and single nephron filtration rates did not change significantly. The infusions of ANP did not alter the delivery of Na+ and water to Henle’s loop but led to increases in Na+ and water delivery out of the outer medullary duct. Because, between the outer medullary collecting duct and the final urine, there was a large increment of fractional Na+ excretion, it appeared likely that the atrial extracts interfered with the capacity of solute load reabsorption in the inner medullary collecting duct. The same working group [54] reported that the infusion of ANP was associated with increased Na+ delivery and reduced fractional reabsorption in the duct, whereas K+Cl− infusion had no effect on fractional reabsorption, suggesting that ANP has a highly specific inhibitory effect on net Na+ transport in this part of the nephron. Even if the in vivo studies strongly indicated the actions of ANP by inhibition of net Na+ and water reabsorption in the collecting duct, it cannot be excluded that these actions could also be caused by alterations of transepithelial driving forces. ANP has been shown to preferentially increase blood flow to juxtamedullary nephrons and to augment vasa recta blood flow. It has been demonstrated that ANP-induced alterations in transepithelial driving forces in the renal medulla cause unfavorable hydraulic gradients for net Na+ reabsorption in the inner medullary collecting duct (IMCD) [42]. These findings suggest that ANP can increase the hydraulic gradient from the vasa recta to papillary collecting duct, favoring reduced net volume and Na+ reabsorption in the IMCD.

Effects of ANP on the Late Distal Tubule HEK-293 cells are known to display a variety of features of the late distal tubule. Furthermore, they have the ability to release urodilatin, the structural analog to ANP. A recent study demonstrated that these human distal tubule cells contain specific mRNA for the ANP/

1254 / Chapter 172 urodilatin receptor NPR-A/GC-A, whereas the receptors for further members of the natriuretic family, GCB and GC-C, were not detectable [25]. ANP inhibited a K+ conductance via PKG that was rather inactive, but the effect became apparent when genistein activated a different K+ conductance in these cells and thus potentiated the ANP-induced inhibition [25].

Effects of ANP on the Cortical Collecting Duct Water reabsorption takes place in the cortical collecting duct (CCD) cells, induced by the peptide hormone vasopressin. An increase in 3′5′-cyclic adenosine monophosphate (cAMP) levels in these cells leads to an increasing permeability of the CCD apical membrane to water. The ANP-induced diuresis is probably due to the direct inhibition of renal tubular epithelial water transport, first investigated by Dillingham and coworkers [12]. The results that ANP leads to an inhibition of the hydraulic conductivity response to the hormone vasopressin but not to either cAMP or forskolin suggested that ANP acts proximally to cAMP formation. A similar result was found for aquaporin (AQP-1) [47], which is also localized in the kidney but predominantly in the proximal tubule, descending limb of Henle’s loop, and vasa recta [44]. Patil and colleagues showed that AQP-1 is upregulated when expressed in Xenopus laevis oocytes and treated with AVP. Water permeability increased and could be inhibited by ANP or incubation with 8-Br-cGMP [47]. A competing phosphorylation mechanism between protein kinase A (PKA) and PKG has been shown previously for dopamine and ANP in regulating the Na+/K+-ATPase of the proximal tubule [2, 5] and might be a likely mechanism in the CCD as well. However, in several cell types it has been shown that natriuretic peptides decrease cAMP concentration via the activation of the NPR-C receptor to which all natriuretic peptides can bind. This effect can either be mediated due to the activation of the receptor’s coupling to G-protein [1] or by regular cGMP increase and subsequent stimulation of cAMP-sensitive phosphodiesterases [52]. What is the effect of ANP on ion transport processes at this nephron site? A few groups were able to convincingly show that ANP neither has an effect on Na+ transport in the CCD [21, 50] nor regulates electrogenic electrolyte transport in principal cells of rat CCD [51]. With the localization of the NPR-A/GC-A receptor in the luminal membrane of intercalated cells, the authors demonstrated that ANP, urodilatin, and its membranepermeable second messenger 8-Br-cGMP inhibited Na+/H+ exchange activity [23], thus regulating the acidbase equilibrium of these cells (see Fig. 2).

FIGURE 2. Immunohistochemistry of NPR-A/GC-A in the cortical collecting duct of rat. The white arrows show typical intercalated cells protruding into the lumen. Note that the staining is prominent in the luminal membrane.

Effects of ANP on the Inner Medullary Collecting Duct There is strong evidence that most of the natriuretic effects of ANP contribute to the inhibition of Na+ reabsorption in the IMCD. The working group of Zeidel has made great efforts to clarify the mechanisms by which ANP acts and induces the natriuretic response in the IMCD. They demonstrated that ANP inhibits transportdependent oxygen consumption (QO2) in the IMCD, yet has no effect on the outer medullary collecting duct [61]. Further studies investigating the interaction of ANP with amiloride, ouabain, and amphotericin using QO2 measurements suggest that ANP inhibits Na+ entry into these cells [61]. ANP reduced isotopic Na+ uptake in suspensions of IMCD cells by two-thirds at concentrations identical to those found effective in oxygen consumption studies [60]. To characterize precisely the ANP-induced effect on Na+ reabsorption in IMCD, the patch-clamp technique was used [36]. This study revealed that ANP and cGMP inhibited an apically located cation channel in cell-attached patches. Thus, it seems likely that the natriuretic action of ANP is related in part to the cGMP-mediated inhibition of electrogenic Na+ absorption by the IMCD. This finding was also supported by Nonoguchi and coworkers [46]. In some points, the contradictory results of several groups and some open questions may reflect the uncertainties and difficulties in precisely characterizing and defining the role of ANP in Na+ homeostasis. Some authors even question the functional role of ANP in the kidney and favor a regulatory role primarily in the cardiovascular system [17]. However, this view is highly unlikely, because the application of a NPR-A/GC-A antagonist counterregulates natriuresis induced by acute volume expansion [59]. Mice lacking the ANP gene demonstrate salt-sensitive arterial hypertension [33] and elevated extracellular fluid volume [32]. Fur-

ANP and Its Role in the Regulation of Renal Tubular Transport Processes / 1255 thermore, ANP knock-out mice are unable to inhibit Na+ reabsorption in the collecting duct when kept on a high-salt diet and intravenous salt loading [29], and the natriuretic effect is virtually absent in NPR-A knock-out mice [35]. In addition to the established hemodynamic effects of ANP, there is a growing number of reports demonstrating direct inhibitory regulatory effects on transport proteins throughout the nephron. Among those are several Na+-dependent transport systems such as the Na+/H+ exchanger, which accounts for 50% of the Na+ intake in this segment; the Na+-glucose and Na+phosphate transporters from the proximal tubule; and the Na+/K+-ATPase from the proximal tubule and the thick ascending limb of Henle’s loop. In the collecting duct system, ANP is also responsible for inhibiting a nonselective cation channel and the Na+/H+exchanger responsible for cellular pH regulation. Taken together, these direct effects on tubular transport proteins add to the natriuretic effect of ANP because all mentioned Na+-coupled processes are inhibited by ANP and a diuretic effect is coupled with an increased Na+ loss. Therefore, a combination of hemodynamic and direct tubular effects is the most likely explanation for the natriuretic and diuretic effects of ANP. Whether ANP is solely responsible for these effects is questionable because there is a renal natriuretic peptide known as urodilatin that might be the key regulator in the kidney (see also Chapter 171 on urodilatin).

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19] [20]

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C

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173 Adrenomedullin as a Renal Peptide MICHIHISA JOUGASAKI

distal tubules might represent the AM peptide bound to the AM receptors or internalized by the distal tubular cells [23]. In cell culture studies, evidence of gene expression and secretion of AM was reported in the renal tubular cells [44, 56], mesangial cells [6, 33, 40, 44], and glomerular podocytes [17]. AM is detected both in plasma and urine, and urinary AM concentration is much higher than plasma AM concentration [55], indicating that the kidney is a major organ for AM production. There are two types of AM in plasma and urine, an amidated mature form and a nonamidated immature form. The percentages of the amidated mature form of AM are significantly higher in urine than in plasma, indicating that urine contains a higher percentage of active AM than plasma [2, 47].

ABSTRACT Although blood vessels are considered the major source of plasma adrenomedullin (AM), the kidney produces and secretes AM and the kidney is also a target organ for AM. AM has renal vasorelaxing actions and increases regional blood flow to the renal medulla and cortex. AM promotes natriuresis and diuresis, which are mediated through an increase in glomerular filtration rate and a decrease in distal tubular sodium reabsorption. Renal AM is modulated in various renal diseases, such as chronic renal failure, glomerulonephritis, IgA nephropathy, and urinary tract infection. Gene delivery of AM and chronic administration of AM have renoprotective actions in cardiorenal disorders. AM is a renal peptide that plays an important autocrine and/or paracrine role in the regulation of renal function.

AM RECEPTOR ADRENOMEDULLIN PEPTIDE

The AM receptor system is present in the kidney. AM receptor mRNA was colocalized with the AM peptide in the renal vessels, glomeruli, distal tubules, and inner medullary collecting duct cells [23]. McLatchie et al. has elucidated that the calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) 2 and 3 generate an AM receptor [39]. CRLRlike immunoreactivity was detected in the juxtaglomerular arteries, glomerular capillaries, and chief cells of the collecting duct in the kidney [14]. RAMP3 was abundantly expressed in the kidney [43]. The expression and distribution of AM peptide and AM receptor system in other organs are described in detail in this book, for example, in the Endocrine Peptide Section (Chapter 118 by Dr. Martinez) and the Gastrointestinal Peptide Section (Chapter 137 by Dr. Schubert).

Adrenomedullin (AM) immunoreactivity and gene expression are widely distributed in mammalian organs, including the kidney [20, 53]. Immunohistochemical study has revealed the localization of AM in the glomeruli, cortical distal tubules, and medullary collecting duct cells in the kidney [1, 28]. By use of the methods of in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR), renal localization of AM mRNA was also observed in the glomeruli, cortical distal tubules, and collecting duct cells of the medulla and cortex [3, 52]. Other investigators reported that AM mRNA was detected in the renal vessels, glomeruli, inner medullary collecting duct cells, and proximal tubules, suggesting that the immunoreactivity in the Handbook of Biologically Active Peptides

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1258 / Chapter 173 RENAL BIOLOGICAL ACTIONS OF AM Vasorelaxation AM has renal vasorelaxing actions [16]. Organ chamber studies showed that AM induced dosedependent relaxation in the canine renal artery with and without endothelium and that the relaxing effects were greater in the endothelium-intact artery than in the denuded artery [45]. The administration of AM caused dose-dependent increases in renal blood flow [13, 19]. The vasodilator response to AM in the isolated perfused rat kidney was mediated by endotheliumderived hyperpolarizing factor (EDHF) and the nitric oxide (NO)-cGMP pathway to a similar extent [66]. Indeed, other studies confirmed the involvement of NO-cGMP pathway in the mechanism of AM-induced vasorelaxation [15].

subdepressor dose of AM improved renal function in association with improved renal morphological findings in malignant hypertensive rats [42, 46], AM itself has renoprotective effects.

IgA Nephropathy Acute AM administration increased urine volume and urinary sodium excretion; it also tended to decrease proteinuria in patients with IgA nephropathy [38].

Renal Ischemia Renoprotective properties of AM were also demonstrated against ischemic or reperfusion injury by using transgenic and heterozygote knockout mouse models [50].

Natriuresis and Diuresis

Diabetes Mellitus

One of the major effects of AM on the kidney is its natriuretic and diuretic action [11, 12, 28, 34, 35, 64]. Intrarenal infusion of AM resulted in a marked diuretic and natriuretic response, and these significant natriuretic and diuretic actions in the AM-infused kidney were associated with an increase in glomerular filtration rate and a decrease in distal tubular sodium reabsorption [28]. The natriuretic action of AM was blocked by NO synthase inhibitor, indicating the involvement of NO system in AM-induced natriuresis [35, 41]. AMinduced natriuresis is also mediated through the renal prostaglandin system and renal nerves [25]. In congestive heart failure, the natriuretic actions of AM were attenuated [26].

AM gene transfer using adenoviral vectors significantly attenuated renal glycogen accumulation and tubular damage in streptozotocin-induced diabetic rats [8].

Other Actions AM reduces mesangial cell mitogenesis and proliferation [7, 29]. AM also stimulates renin secretion from juxtaglomerular cells [24].

RENOPROTECTIVE ACTIONS OF AM Hypertension Gene delivery or chronic administration of AM alleviates renal injuries in hypertensive rats [9, 65, 68, 71]. Human AM gene delivery enhanced renal function, as indicated by increases in renal blood flow and glomerular filtration rate [9]. Histological examination of the kidney revealed significant reductions in glomerular sclerosis, tubular injury, luminal protein cast accumulation, and interstitial fibrosis, as well as urinary protein excretion [9]. Because the chronic administration of a

Aortocaval Shunt AM administration resulted in beneficial effects on renal function, such as increases in urine volume, glomerular filtration rate, and renal blood flow in an aortocaval shunt model in rats [69].

CLINICAL IMPLICATIONS OF AM IN ASSOCIATION WITH THE KIDNEY Chronic Renal Failure The plasma concentration of AM is increased in patients with chronic renal failure [5, 22, 36, 55, 59– 61]. An amidated mature form of AM is also increased in chronic renal failure [21, 62]. Urinary excretions of both amidated mature form of AM and nonamidated immature form of AM were decreased in patients with chronic renal failure compared with normal subjects [47]. There are several reports concerning the effects of hemodialysis on plasma AM concentration. Some reports showed that plasma AM concentration decreased with hemodialysis [36, 59, 61], but other reports described no changes in AM concentration across hemodialysis [55, 67]. In the studies that showed reduced AM concentration through hemodialysis, the plasma AM concentration was not changed immediately by hemodialysis but decreased 14–20 hours after

Adrenomedullin as a Renal Peptide / 1259 hemodialysis [60], suggesting reduced AM synthesis rather than increased clearance of AM through dialysis membranes. On the other hand, other investigators showed an increase in plasma AM concentration across hemodialysis [37]. Many factors might influence the plasma concentration of AM, including different dialysis membranes, leukocyte activation by dialysis membranes, and the different status of blood volume in patients with chronic renal failure.

Glomerulonephritis Plasma AM concentration was increased, whereas the urinary AM concentration was decreased in patients with chronic glomerulonephritis compared with normal healthy subjects [31]. In rat models of glomerulonephritis, plasma and renal AM concentrations and renal AM mRNA expression were increased compared with control rats [57, 58].

IgA Nephropathy Plasma AM concentration was increased in patients with IgA nephropathy compared with healthy subjects, and there was a positive correlation between plasma AM concentration and serum creatinine levels [30]. However, other investigators reported that plasma AM concentration was not changed, whereas urinary AM concentraion was significantly lower in patients with IgA nephropathy compared with normal subjects [32].

Diabetic Nephropathy In animal models of streptozotocin-induced diabetes mellitus, AM mRNA and RAMP2 mRNA in the kidney were increased [18]. Plasma AM concentrations were decreased in these diabetic rats compared with the normal rats, whereas urinary AM excretion was much higher in the diabetic rats than in the normal rats, suggesting that AM was actively transcribed and translated in the kidney and immediately secreted into urine in the diabetic rats. Reduced circulating AM could be due to glomerular hyperfiltration in the diabetic rats.

Urinary Tract Infection Urinary AM concentration was significantly higher in children with urinary tract infection than in controls and was positively correlated with white cell count in urine [10]. Urinary AM concentration in patients with cystitis was significantly elevated compared with healthy subjects and correlated positively with the number of urine leukocytes [51].

Cardiovascular Disorders Renal AM is also modulated in various cardiovascular disorders, such as hypertension and congestive heart failure. Plasma AM concentration, urinary AM excretion, and renal AM mRNA were markedly increased in severely hypertensive rats compared with normal rats [49]. CRLR, RAMP2, and RAMP3 in the kidney were also increased in hypertensive rats compared with normal rats [48]. Renal AM was also activated in experimental congestive heart failure [27] and aortocaval shunt model [70].

Others Plasma AM concentration was increased during chronic salt loading in the rats, and chronic salt loading also upregulated gene expressions of AM, CRLR, and RAMP3 in the kidney [4]. Other investigators reported that the expressions of CRLR, RAMP1, and RAMP2 in the kidney were activated in the rats with obstructive nephropathy [43]. CRLR mRNA and RAMP3 mRNA levels significantly decreased in the remnant kidney after 5/6 nephrectomy compared with the shamoperated rats [63]. The application of shockwave lithotripsy caused an increase in plasma AM concentration, which might reflect an organized response of the kidney to this type of trauma [54].

References [1] Asada Y, Hara S, Marutsuka K, Kitamura K, Tsuji T, Sakata J, Sato Y, Kisanuki A, Eto T, Sumiyoshi A. Novel distribution of adrenomedullin-immunoreactive cells in human tissues. Histochem Cell Biol 112: 185–191, 1999. [2] Asakawa H, Nishikimi T, Suzuki T, Hara S, Tsubokou Y, Yagi H, Yabe A, Tsuchiya N, Horinaka S, Kangawa K, Matsuoka H. Elevation of two molecular forms of adrenomedullin in plasma and urine in patients with acute myocardial infarction treated with early coronary angioplasty. Clin Sci 100: 117–126, 2001. [3] Cameron VA, Fleming AM. Novel sites of adrenomedullin gene expression in mouse and rat tissues. Endocrinology 139: 2253– 2264, 1998. [4] Cao YN, Kitamura K, Kato J, Kuwasako K, Ito K, Onitsuka H, Nagoshi Y, Uemura T, Kita T, Eto T. Chronic salt loading upregulates expression of adrenomedullin and its receptors in adrenal glands and kidneys of the rat. Hypertension 42: 369–372, 2003. [5] Cheung B, Leung R. Elevated plasma levels of human adrenomedullin in cardiovascular, respiratory, hepatic and renal disorders. Clin Sci 92: 59–62, 1997. [6] Chini EN, Chini CCS, Bolliger C, Jougasaki M, Grande JP, Burnett JC, Jr., Dousa T. Cytoprotective effects of adrenomedullin in glomerular cell injury: Central role of cAMP signaling pathway. Kidney Int 52: 917–925, 1997. [7] Chini EN, Choi E, Grande JP, Burnett JC, Jr., Dousa TP. Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway. Biochem Biophys Res Commun 215: 868–873, 1995.

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[57] Shi Y, Yoshihara F, Nakahama H, Goto R, Sada M, Kawano Y, Moriyama T, Yazawa K, Ichimaru N, Takahara S, Kangawa K. Mycophenolate mofetil prevents autoimmune glomerulonephritis and alterations of intrarenal adrenomedullin in rats. Eur J Pharmacol 489: 127–133, 2004. [58] Shi Y, Yoshihara F, Nakahama H, Ichimaru N, Yazawa K, Sada M, Goto R, Kawano Y, Moriyama T, Takahara S, Okuyama A, Kangawa K. A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis. Regul Pept 127: 233– 238, 2005. [59] Suda T, Osajima A, Iwamoto M, Anai H, Tamura M, Kabashima N, Ota T, Watanabe Y, Kanegae K, Okazaki M, Nakashima Y. The mature form of adrenomedullin correlates with brain natriuretic peptide in plasma of chronic hemodialysis patients. Clin Nephrol 57: 444–451, 2002. [60] Toepfer M, Schlosshauer M, Sitter T, Burchardi C, Behr T, Schiffl H. Effects of hemodialysis on circulating adrenomedullin concentrations in patients with end-stage renal disease. Blood Purif 16: 269–274, 1998. [61] Tokura T, Kinoshita H, Fujimoto S, Hisanaga S, Kitamura K, Eto T. Plasma levels of mature form of adrenomedullin in patients with haemodialysis. Nephrol Dial Transplant 16: 783–786, 2001. [62] Tokura T, Kinoshita H, Fujimoto S, Kitamura K, Eto T. Plasma levels of proadrenomedullin N-terminal 20 peptide and adrenomedullin in patients undergoing hemodialysis. Nephron Clin Pract 95: c67–c72, 2003. [63] Totsune K, Takahashi K, Mackenzie HS, Arihara Z, Satoh F, Sone M, Murakami O, Ito S, Brenner BM, Mouri T. Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3. Peptides 22: 1933–1937, 2001. [64] Vari RC, Adkins SD, Samson WK. Renal effects of adrenomedullin in the rat. Proc Soc Exp Biol Med 211: 178–183, 1996. [65] Wang C, Dobrzynski E, Chao J, Chao L. Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats. Am J Physiol 280: F964–F971, 2001. [66] Wangensteen R, Quesada A, Sainz J, Duarte J, Vargas F, Osuna A. Role of endothelium-derived relaxing factors in adrenomedullin-induced vasodilation in the rat kidney. Eur J Pharmacol 444: 97–102, 2002. [67] Washimine H, Yamamoto Y, Kitamura K, Tanaka M, Ichiki Y, Kangawa K, Matsuo H, Eto T. Plasma concentration of human adrenomedullin in patients on hemodialysis. Clin Nephrol 44: 389–393, 1995. [68] Wei X, Zhao C, Jiang J, Li J, Xiao X, Wang DW. Adrenomedullin gene delivery alleviates hypertension and its secondary injuries of cardiovascular system. Hum Gene Ther 16: 372–380, 2005. [69] Willenbrock R, Pagel I, Krause EG, Scheuermann M, Dietz R. Acute hemodynamic and renal effects of adrenomedullin in rats with aortocaval shunt. Eur J Pharmacol 369: 195–203, 1999. [70] Yoshihara F, Nishikimi T, Okano I, Horio T, Yutani C, Matsuo H, Takishita S, Ohe T, Kangawa K. Alterations of intrarenal adrenomedullin and its receptor system in heart failure rats. Hypertension 37: 216–222, 2001. [71] Zhang JJ, Yoshida H, Chao L, Chao J. Human adrenomedullin gene delivery protects against cardiac hypertrophy, fibrosis, and renal damage in hypertensive Dahl salt-sensitive rats. Hum Gene Ther 11: 1817–1827, 2000.

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174 Adrenomedullin 2/Intermedin YOSHIO TAKEI

(AM) is one of the important vasodepressor and diuretic/natriuretic hormones. To evaluate the role of homologous AM in fish osmoregulation, we sought an AM ortholog in the genome database of tiger pufferfish, Takifugu rubripes, using the human AM sequence as query. The BLAST search resulted in the identification of five AM-like peptides in the pufferfish [11]. Molecular phylogenetic analyses revealed that five AM-like peptides form a subfamily in the calcitonin gene-related peptide (CGRP) family. Thus, we named them AM1–5 (Fig. 1). Five AMs also have been identified in other teleost species such as the zebrafish, Danio rerio [16]. Fish AM1 is an ortholog of mammalian AM, as determined by the synteny of neighboring genes and the presence of the proadrenomedullin N-terminal 20 peptide (PAMP)-like sequence in the N-terminus of the prohormone. The five fish AMs can roughly be divided into two groups, AM1, 4, and 5 and AM2 and 3, in terms of gene structure (exon-intron organization), precursor sequence deduced from the mRNA, and tissue distribution pattern of the gene transcripts. Thus, we hypothesized that the peptide in the AM2/3 group should exist also in mammals. In order to identify mammalian AM2/3, we developed a new search program and applied it to the genome and EST database of mammals, which resulted in the identification of an AM2/3-like sequence in the human, rat, and mouse [17]. Judging from the similarity of exon-intron organization, deduced precursor structure, and neighboring genes on the same chromosome, we determined that the novel peptide is a mammalian ortholog of fish AM2. Roh et al. (2004) also found this peptide in fishes and mammals and named it intermedin (IMD) because of its dense localization in the intermediate lobe of the

ABSTRACT Adrenomedullin 2 (AM2), or intermedin (IMD), is a novel peptide discovered from the comparative genomic approach. In mammals, AM, calcitonin generelated peptide (CGRP), and amylin are members of the CGRP family. In teleost fish, however, five AMs, named AM1–5, form an independent subfamily. Based on the fish sequence, AM2/IMD was identified in the human, rat, and mouse. AM2/IMD is expressed abundantly in the kidney, digestive tracts, heart, and brain, and it exerts a spectrum of renal, cardiovascular, gastrointestinal, and appetite-related actions either directly or via the central nervous system. AM2/IMD increases intracellular cAMP by binding to calcitonin receptor or calcitonin receptor-like receptor associated with receptor activity-modifying proteins, but another AM2specific receptor may also exist.

DISCOVERY Vertebrates have expanded their habitats from inland fresh water to the seas and onto the land during evolution. The transition from aquatic to terrestrial life accompanied physiological evolution, particularly in the cardiovascular and osmoregulatory systems. Terrestrial animals develop the cardiovascular system to pump the blood against gravity and the osmoregulatory system to cope with desiccation on the dry land. In this context, vasopressor and antidiuretic hormones predominate in terrestrial species, whereas vasodepressor and ion-regulating hormones are dominant and diversified in fishes, particularly Na-extruding hormones in marine species, as exemplified by the natriuretic peptide family [5, 15]. Adrenomedullin Handbook of Biologically Active Peptides

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1264 / Chapter 174 Adrenomedullin 1 Takifugu

SKNLVNQSRKNGCSLGTCTVHDLAFRLHQLGFQYKIDIAPVDKISPQGY-NH2

Tetraodon

SKNSGNQTRRQGCSLGTCIVHDLAHRLHQLGNKYKFGNAPEDKMSPQGY-NH2

Zebrafish

SKNSINQSRRSGCSLGTCTVHVLAHRLHDLNNKLKIGNAPVDKINPYGY-NH2

Carp

SKNSISQSRRSGCSLGTCTVHVLAHRLHDLNNKLKIGNAPADKINPFGY-NH2

Catfish

SKNSVNQSRRSGCSLGTCTVHDLAHRLHILNNKNKVSSAPADKISPLGY-NH2

Rainbow trout

SKISVNQAWRPGCSLGTCTVHDLAHRIHQLNNKLKIGSAPIDKISPQGY-NH2

Adrenomedullin 2 Takifugu

HANNGGGRSHGQLMRVACVLGTCQVQNLSHRLYQLIGQSGKEDSSPMNPHSPHSY-NH2

Zebrafish

HAFRGS-RGHPQLMRVGCVLGTCQVQNLSHRLYQLNSQSRRQE-SPINPRSPHSY-NH2

Rainbow trout HANGSGGRGQGQLMRVGCVLGTCQVQNLSHRLYQLIGQSGRQDSSPINPRSPHSY-NH2

Adrenomedullin 3 Takifugu

HIHSRGHHYPHPNQLIRAGCALGTCQVQNLSHRLYQLIGQSGRDDSSPINPKSPHSY-NH2

Tetraodon

YVHSRGSRGHHQNQLMRVGCVLGTCQVQNLSHRLYQLIGQSGREDSSPMNPQSPHSY-NH2

Zebrafish

HVHSRGHHSHHHPQLMRVGCVLGTCQVQNLSHRLYQLVGQSGREDS-PINPRSPHSY-NH2

Adrenomedullin 4 Takifugu

AA-GCALFMCAYHDLLQRLNHIYNKQKEVTAPKNKILSTGY-NH2

Tetraodon

AASGCFLFLCVHHNLLSRMEHFNNNQKDKLAPKNKIDGRGY-NH2

Zebrafish

A--GCNLATCSVHELAHLLNIMHAKTN--NAPTDKIGSNGY-NH2

Rainbow trout

PGCSLGTCTLQNLVHRLHILNNRLKVNRAPEDKISSQGY-NH2

Adrenomedullin 5 Takifugu

APQRGCQVGTCQVHNLANKLYQIG-RQGKDESTKVNDPQGY-NH2

Tetraodon

APQRGCHVGTCQVHNLANTLFQMGQRRGKDGSPEVNDPQGY-NH2

Zebrafish

AAQRGCQLGTCQVHNLVNKLYRMGQSNGKDESKKANDPTGY-NH2

Rainbow trout

APQRGCQLGTCQLHNLANTLYQMGKTNGKDESKKAHDAHGY-NH2

FIGURE 1. Amino acid sequences of adrenomedullin family in fishes. Amino acid sequences conserved in more than half of the species are shaded. Brackets show disulfide bridges.

pituitary. However, the name intermedin has been used for melanocyte-stimuating hormones, which is processed from the proopiomelanocortin in the intermediate lobe [6, 9, 18]. Because several papers already have been published with both names, the name AM2/IMD is used in this chapter to avoid confusion.

STRUCTURE OF THE PRECURSOR mRNA AND GENE The AM2/IMD gene is allocated on chromosome 22 in the human, chromosome 15 in the mouse, and chromosome 7 in the rat, but not on the chromosomes in fishes. The AM2/IMD gene consists of three exons, which differs from the AM/AM1 gene, which consists

of four exons. The AM2/IMD precursors deduced from the mRNA sequences have 147–151 amino acid residues, including the signal sequence of 24–25 amino acids in mammals and 169 amino acids with a 20 amino-acid signal peptide in the pufferfish (Fig. 2). The mature AM2/IMD is at the C-terminus of the prohormone, and its C-terminal glycine residue is followed by a stop codon. This precursor sequence is characteristic only for AM2 and -3 of fish, because AM1 and -4 precursors are followed by a C-terminal peptide connected by 4–5 consecutive arginine residues and an AM5 precursor with an additional arginine residue [11]. Molecular phylogenetic analyses with mammalian and fish peptides showed that AM2/IMD is in the same cluster with the AM subfamily and distinct from the clusters of CGRP and amylin [12, 17] (Fig. 3).

Adrenomedullin 2/Intermedin / 1265 Human Rat Mouse Pufferfish

1:MARIP--TAALGCISLLCLQLPGSLSRSLGGDPRPVKPREPPARSPSSSLQPRHPAPRPV 1:MAQLLMVTVTFGCISLLYL-LPGTLSGSLGKGLR---PREPPAKIPSSGPQPGHPSLRPV 1:MAQLLMVTVTLGCISLLYL-LPGTLSGSLGKGLRHSRPREPPAKIPSSNLQPGHPSLQPV 1:MRVLLPVWLLLSLLPLEVQARALSQQN-LGLPHRFSLLRTL--KIPKSSFIVIGPAASDP

58 56 59 57

Human Rat Mouse Pufferfish

59:VWKLH-RALQAQRG--------------------AGLAPVMGQPLRDGGRQHSGPRRHSG 57:VWKPP-HALQPQGR--------------------GNPALATVHLPQGGGSRHPGPQRHVG 60:VWKSRRHAPQPQGR--------------------GNRALAMVHLPQGGGSRHPGPQRPTG 58:PEVTYHHVAQGDGRVTWMAWLRGKPLLGSSDPPLGQTDRVLRDTPAWRGRSRGRRHANNG

97 95 99 117

Human 98:PRRTQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSYG* Rat 96:SRRPHAQLLRVGCVLGTCQVQNLSHRLWQLVRPSGRRDSAPVDPSSPHSYG* Mouse 100:SRRPHAQLLRVGCVLGTCQVQNLSHRLWQLVRPAGRRDSAPVDPSSPHSYG* Pufferfish 118:GGRSHGQLMRVACVLGTCQVQNLSHRLYQLIGQSGKEDSSPMNPHSPHSYG*

149 147 151 169

FIGURE 2. Amino acid sequences of adrenomedullin 2/intermedin precursors from mammals and pufferfish, Takifugu rubripes. Amino acid sequences conserved in more than half of the species are shaded. Brackets show disulfide bridges. A line and an arrow above the sequences are the signal sequence and processing site, respectively, in mammals; those below the sequences are for pufferfish.

AM or AM1

Adrenomedullin

Human

Pufferfish-1

Pufferfish-5

Mouse

953

441

327

Pufferfish-4

297

Pufferfish-2 Pufferfish-3

977

911

514

AM2 or 3 Human 2

989

Mouse 2

CGRP

771

Human a Human b 356

875

409 498

Pufferfish 2 909 Mouse a Pufferfish 1 Mouse b

Pufferfish 961

Mouse

Amylin

0.1

Human

FIGURE 3. Molecular phylogenetic analyses of adrenomedullins (AMs), calcitonin gene-related peptides (CGRPs), and amylin from mammals and pufferfish, Takifugu rubripes. Precursor sequences are analyzed by the Neighbor-Joining method. Numbers on the tree are bootstrap values.

1266 / Chapter 174 DISTRIBUTION OF mRNA Tissue distribution of mRNA showed that pufferfish AM2 gene is expressed almost exclusively in the brain [11]. In mice, however, AM2/IMD transcripts were detected in various tissues; high expression was observed in the kidney, submaxillary gland, stomach, and mesentery, which was followed by the pituitary, lung, pancreas, intestines, spleen, and thymus [17]. Radioimmunoassay for AM2/IMD also measured high concentrations in the kidney and stomach [19]. Thus, the kidney is the major organ that synthesizes and stores a large amount of AM/IMD. Immunoreactive AM2/IMD is localized in the glomerulus and vasa recta of the mouse kidney using AM2/IMD-specific antiserum [16]. The heart also contains a moderate amount of AM2/IMD, where only endothelial cells of the coronary vessels exhibit positive staining. The brisk expression in the pituitary suggests its paracrine action on the pituitary hormones [20]. Immunoreactive AM2/IMD was localized in the intermediate lobe of the pituitary in a few species [12]. However, AM2/IMD concentration in the pituitary was lower than in the hypothalamus, where the peptide was concentrated compared with other brain regions [19]. Thus, low expression in the whole brain of mice may be due to its expression only in the restricted region of the brain. There is also a possibility that AM2/IMD synthesized in the hypothalamic neurons is transported to the axon terminals in the intermediate lobe of the pituitary. In addition, AM2/IMD gene is expressed in the ovary but not in the testis, indicating some specific role for the female reproductive system [17].

PROCESSING OF THE PRECURSOR The inferred mature sequence of AM2/IMD is composed of 47 amino acid residues, assuming that proAM2/IMD is cleaved after two consecutive arginine residues by prohormone convertases and that the Cterminal glycine residue is used for amidation. AM2/ IMD-47 was more potent than N-terminally truncated AM2/IMD-42 for biological actions, including vasodepressor and chronotropic effects as well as suppressive effects on food intake and gastric emptying [12]. However, the shorter AM2/IMD-42 had stronger effects on cAMP accumulation in cells expressing calcitonin receptor-like receptor (CLR) and receptor activitymodifying protein (RAMP), indicating the presence of specific receptors other than CLR. There are additional dibasic amino acids in the prosegment of rat and mouse AM2/IMD, and the cleavage at this site produces AM2/ IMD-53. This longer peptide seems to have comparable cardioprotective activity [21]. Mature fish AM2 may be cleaved by a processing enzyme, furin, because its pro-

cessing signal (Arg-X-X-Arg) exists in the prosegment in all fish AMs. The truncation at this site produces mature AM2 of 55 amino acid residues in the tiger pufferfish. The processed, mature AM2/IMD possesses an intramolecular ring structure of six amino acid residues flanked by a single disulfide bond in the N-terminal region and an amidated C-terminus (Fig. 1). The length of the N-terminal sequence extended from the intramolecular ring varies among species and has little effect on its biological activity. However, the amidated Cterminus may be essential for activity, as is the case with AM. The circulating form of AM2/IMD has not yet been determined. With respect to AM, the C-terminally nonamidated (inactive) molecule circulates in the blood in amounts much larger than that of the amidated mature AM [8]. As mentioned earlier, the AM2/ IMD precursor terminates in a glycine residue, but the AM precursor is followed by four arginine residues and a C-terminal peptide. Thus, a complex processing sequence is required before C-terminal amidation of AM with endopeptidase and carboxypeptidase. These differences may influence the efficiency of amidation.

RECEPTORS AND SIGNALING CASCADE The CGRP family members bind the calcitonin receptor (CTR) or CLR associated with one of the RAMPs and use the cAMP signaling cascade for biological action [1, 4]. cAMP accumulation is increased by CGRP when given to cells co-expressing CLR and RAMP1, by AM in cells co-expressing CLR and RAMP2– 3, and by amylin in cells co-expressing CTR and RAMP1–3. AM2/IMD is effective in cAMP production in cells co-expressing CLR/CTR with one of the RAMPs, but in no case was it more potent than other CGRP family members [12, 16]. These results indicate the presence of AM2/IMD-specific receptors other than CTR and CLR as judged by its potent renal and cardiovascular actions. Only a weak inhibition of AM2/IMD effects by AM22-53 and CGRP8-37, competitive antagonists for CLR, supports the presence of other receptors [3, 19]. In fish, genes coding for two types of CLRs (CLR1– 2), one CTR, and five types of RAMPs (RAMP1–5) are identified in the pufferfish [10]. Molecular phylogenetic analyses and linkage analyses indicate that RAMP1 and 4, RAMP2 and 5, and RAMP3 are akin to RAMP1, RAMP2, and RAMP3 of mammals, respectively. The coexpression of each combination of CLR and RAMP showed that pufferfish AM2 increased cAMP production in cells expressing CLR1 and RAMP3, whereas pufferfish AM1, an ortholog of mammalian AM, was more widely effective in cells expressing CLR1 and

Adrenomedullin 2/Intermedin / 1267 RAMP2, -3, or -5, and CLR2 and RAMP2 [10]. Thus, the AM2-specific combination of CLR and RAMP was not found even in fishes with such diversified CLRs and RAMPs. These results further indicate the presence of an AM2-specific receptor other than CLR.

Biological Actions AM2/IMD exerts a spectrum of peripheral actions similar to AM in mammals. The intravenous injection of hypotensive dose of AM2/IMD was antidiuretic and antinatriuretic in mice [17], but its infusion into the renal artery of rats at nondepressor doses was diuretic and natriuretic [2]. Because the glomerular filtration rate did not change during diuretic/natriuretic dose of AM2/IMD infusion in rats, a direct tubular action is likely. Diuresis and natriuresis were never induced in mice at lower nondepressor doses, probably because of the developed water-conserving mechanisms in this species, which originated in an arid habitat [14]. For instance, atrial natriuretic peptide, which is a potent diuretic and natriuretic hormone in mammals, is antidiuretic in seawater-adapted eels, which have a welldeveloped mechanism for water retention (see [15]). Intravascular administration of AM2/IMD decreased arterial pressure and increased heart rate in rats [2, 12] and mice [17]. Unlike AM, the hypotension is not mediated by local production of nitric oxide [19]. The cardiovascular and renal effects of AM2/IMD appear to be somewhat less potent and shorter-lasting than AM [2, 19]. AM2/IMD also inhibits food intake and stomach motility in rats when given by intraperitoneal injection [12]. The effect on stomach may be mediated by locally produced AM2/IMD because the stomach abundantly synthesizes and stores this peptide. AM2/IMD has a protective effect on the heart against ischemia/reperfusion injury in the rat [21]. AM2/IMD also exhibits potent central actions. When administered into the cerebral ventricle, AM2/IMD causes immediate hypertension and delayed tachycardia in the rat through the activation of the sympathetic nerve system [19]. The central cardiovascular effect of AM2/IMD is more potent than that of AM, which is in contrast to the peripheral effect. Further, CGRP8-37, a CLR antagonist, only partially inhibited the cardiovascular effect of central AM2/IMD. It seems that some unknown receptor for AM2/IMD, different from CLR, exists in the central nervous system. In addition, intracerebroventricular (ICV) injection of AM2/IMD inhibited water and food intake in both satiated rats and those deprived of water and food overnight [19]. The ICV injection of AM2/IMD stimulated prolactin and adrenocorticotropic hormone (ACTH) secretion and inhibited growth hormone secretion from the ante-

rior pituitary of the rat [20]. The effect on ACTH secretion appears to be mediated by CRH secreted from the hypothalamus. Consistently, ICV injection of AM2/IMD increased the plasma concentration of the glucocorticoid corticosterone in the rat. The ICV AM2/IMD also increased plasma vasopressin and oxytocin concentrations [20]. On the other hand, similar injection of AM2/IMD augmented c-fos gene expression of the neurons in the supraoptic nucleus, paraventricular nucleus, arcuate nucleus, and nucleus tractus solitarius as shown by in situ hybridization in the rat [3]. Immunohistochemical staining of the same section with antiserum specific to vasopressin or oxytocin revealed that most c-fos-positive cells are oxytocin-producing cells and only a few are vasopressin-producing cells. Consistent with this observation, plasma oxytocin concentrations were profoundly increased after the ICV administration of AM2/IMD, but plasma vasopressin concentration increased only slightly [3]. The augmented c-fos expression in the nucleus tractus solitarius is consistent with its effect on the sympathetic activation. The AM2/IMD effects were blocked only slightly by pretreatment with CGRP8-37 and AM22-52, again indicating a new AM2-specific receptor different from CLRRAMP combination.

PATHOPHYSIOLOGICAL IMPLICATIONS The organ that synthesizes and stores AM2/IMD most abundantly is the kidney [17, 19], and immunoreactive AM2/IMD is localized in the glomerulus and vasa recta of mice. Because exogenously administered AM2/IMD displays potent renal actions at nondepressor doses [2], it is likely that locally produced AM2/ IMD acts in a paracrine/autocrine fashion to regulate renal function. Together with the potent antidipsogenic effect of AM2/IMD, this peptide may be involved in the body fluid regulation, as suggested for AM in mammals [13]. Osmoregulatory actions of AM2, including renal action, are under investigation in fishes. AM2/IMD mRNA was initially registered in the expressed sequence tag (EST) database (accession number: AK024788) as an mRNA from the primary vascular smooth muscle cells of human coronary artery, even though a different protein was predicted from the mRNA. In the mouse heart, immunoreactive AM2/IMD was detected in the endothelial cells of coronary vessels [16]. Further, AM2/IMD protects the heart from ischemic injury [21]. These results suggest that AM2/IMD may be secreted from the coronary artery to protect the heart as a local paracrine system at the onset of ischemic heart failure as suggested with AM [7]. The vasorelaxant effect in the general circulation is also beneficial for the congestive heart failure by decreasing the afterload.

1268 / Chapter 174 Thus the time-course changes in plasma AM2/IMD concentrations should be measured after the onset of heart failure. The efforts to produce AM2/IMD knockout mice are now in progress, which will delineate more clearly the physiological action of AM2/IMD in the near future.

References [1] Fischer JA, Muff R, Born W. Functional relevance of Gprotein-coupled-receptor-associated-proteins, exemplified by receptor-activity-modifying proteins (RAMPs). Biochem Soc Trans 2002; 30:455–60. [2] Fujisawa Y, Nagai Y, Miyatake A, Takei Y, Miura K, Shoukouji T, Nishiyama A, Kimura S, Abe Y. Renal effects of a new member of adrenomedullin family, adrenomedullin 2, in rats. Eur J Pharmacol 2004; 497:75–80. [3] Hashimoto F, Hyodo S, Kawasaki M, Mera T, Chen L, Soya A, Saito T, Fujihara H, Higuchi T, Takei Y, Ueta Y. Centrally administered adrenomedullin 2 activates hypothalamic oxytocinsecreting neurons causing elevated plasma oxytocin level in rats. Am J Physiol 2005; 289:E753–61. [4] Hay DL, Christopoulos G, Christopoulos A, Poyner DR, Sexton PM. Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol Pharmacol 2005; 67:1655–65. [5] Inoue K, Naruse K, Yamagami S, Mitani H, Suzuki N, Takei Y. Four functionally distinct C-type natriuretic peptides found in fish reveal new evolutionary history of the natriuretic system. Proc Natl Acad Sci USA 2003; 100:10079–84. [6] Johnsson S, Hogberg B. Observations on the connexion between intermedin and adrenocorticotropic hormone. Nature 1952; 169:286. [7] Jougasaki M, Grantham JA, Redfield MM, Burnett Jr JC. Regulation of cardiac adrenomedullin in heart failure. Peptides 2001; 22:1841–50. [8] Kitamura K, Kato J, Kawamoto M, Tanaka M, Chino N, Kangawa K, Eto T. The intermediate form of glycine-extended adrenomedullin is the major circulating molecular form in human plasma. Biochem Biophys Res Commun 1998; 244:551–5.

[9] Krayer O, Astwood EB, Waud DR, Alper MH. Rate-increasing action of corticotrophin and of alpha-intermedin in the isolated mammalian heart. Proc Natl Acad Sci USA 1961; 47:1227–36. [10] Nag K, Kato A, Nakada T, Hoshijima K, Mistry Ac, Takei Y, Hirose S. Molecular And Functional Characterization Of Adrenomedullin Receptors In Pufferfish. Am J Physiol 2006; 290:R467–78. [11] Ogoshi M, Inoue K, Takei Y. Identification of a novel adrenomedullin gene family in teleost fish. Biochem Biophys Res Commun 2003; 311:1072–7. [12] Roh J, Chang CL, Bhalla A, Klein C, Hsu SY. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activitymodifying protein receptor complexes. J Biol Chem 2004; 279:7264–74. [13] Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999; 61:363–89. [14] Schwarz E, Schwars HK. The wild and commensal stocks of the house mouse, Mus musculus Linnaeus. J Mammal 1943; 24:59– 72. [15] Takei Y, Hirose S. The natriuretic peptide system in eels: a key endocrine system for euryhalinity? Am J Physiol 2002; 282: R940–51. [16] Takei Y, Hyodo S, Katafuchi T, Minamino N. Novel fish-derived adrenomedullin in mammals: structure and possible function. Peptides 2004; 25:1643–56. [17] Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, Miyano S. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator. FEBS Lett 2004; 556:53–8. [18] Taylor JD. The effects of intermedin on the ultrastructure of amphibian iridophores. Gen Comp Endocrinol 1968;12:405– 16. [19] Taylor MM, Bagley SL, Samson WK. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake. Am J Physiol 2005; 288:919– 27. [20] Taylor MM, Samson WK. Stress hormone secretion is altered by central administration of intermedin/adrenomedullin-2. Brain Res 2005; 1045:199–205. [21] Yang J-H, Qi Y-F, Jia Y-X, Pan C-S, Zhao J, Yang J, Chang J-K, Tang C-S. Protective effects of intermedin/adrenomedullin 2 on ischemia/reperfusion injury in isolated rat hearts. Peptides 2005; 26:501–7.

C

H

A

P

T

E

R

175 Renal Endothelin JAN MICHAEL WILLIAMS AND DAVID M. POLLOCK

including the kidney. Although they appear to be derived from the same gene, these ETB receptors can be distinguished pharmacologically and thus have been referred to as ETB1 and ETB2, located on endothelium and vascular smooth muscle, respectively [10].

ABSTRACT Considerable progress has been made in our understanding of different aspects of endothelin (ET) physiology and pathophysiology in the kidney. We know that the kidney is one of the major sites of ET synthesis and that ET is involved in the regulation of tubular transport and fluid-electrolyte balance. Investigation of the ET system in the kidney has been challenging due to the contrasting nature of receptor function, but perhaps even more so because ET is an autocrine and/or paracrine factor. The development of specific receptor antagonists and genetically manipulated rat and mouse models has provided considerable new information in terms of the physiological role of the ET system in the kidney.

LOCALIZATION OF THE ET-1 SYSTEM IN THE KIDNEY Although there is little known about the physiological regulation of ET-1 synthesis, a variety of factors such as systemic hormones, cytokines, shear stress, hypoxia, and sodium intake are among the reported stimulators [25, 49]. A variety of cell types in the renal cortex can produce ET-1, such as the vascular smooth muscle, glomerular endothelium, mesangial cells, and tubular epithelial cells [25, 49]. Within the renal medulla, ET-1 synthesis appears to occur in the vasa recta and collecting duct cells and perhaps the thick ascending limb [25, 49]. Similar to the immunoreactivity studies, gene expression experiments confirmed the idea that sites of ET-1 mRNA expression include the glomeruli, proximal tubules, thick ascending limbs, vasa recta, and outer and inner medullary collecting ducts and that relatively higher amounts of ET-1 are synthesized within the renal medulla [25, 49]. In rat studies, ECE-1 mRNA and protein have been identified in the glomeruli, proximal straight tubule, cortical and medullary thick ascending limb, and inner medullary collecting duct [49]. In humans, ECE-1 immunoreactivity was observed in the vascular and glomerular endothelial cells and vasa recta capillaries [49]. The tubular epithelium of the outer and inner medullary collecting ducts also contains ECE-1. Both ET receptor subtypes are present in the kidney, and these receptors are localized at sites where ET synthesis occurs, consistent with the peptide’s role as an

BRIEF HISTORY OF ENDOTHELIN Chapter 163 in this text by Goto provides an excellent overview of the history of the endothelin system, so the history is not reviewed here in detail [24]. In brief, three distinct endothelin (ET) peptides, ET-1, ET-2, and ET-3, are synthesized by their respective precursors; for example, big ET-1 is synthesized by the endothelinconverting enzyme (ECE). A variety of different cell types in the kidney synthesize all three ET peptides; however, little is known about the specific ET-2 or ET-3 isopeptide function. ET-1 and ET-2 bind with high affinity to the ETA receptors, whereas all three ET isopeptides bind with equal affinity to the ETB receptor [24]. Within the vascular wall, ETA receptors are primarily located on vascular smooth muscle to cause constriction and ETB receptors are primarily located on endothelial cells to cause vasodilation but can also account for non-ETAmediated vasoconstriction in certain vascular beds, Handbook of Biologically Active Peptides

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Copyright © 2006 Elsevier

1270 / Chapter 175 autocrine and/or paracrine factor. Radioligand binding studies revealed the presence of receptors in the renal vascular smooth muscle, mesangial cells, and tubular cells [49]. A high number of binding sites have been identified in the inner medulla and glomeruli, as well as at low levels in the outer cortex [49]. Specific binding sites are also localized to endothelial cells of glomeruli and peritubular capillaries of the cortex [49].

RENAL HEMODYNAMIC EFFECTS OF ET-1 The intravenous infusion of ET-1 in dogs and rats decreases the renal blood flow and glomerular infiltration rate (GFR) [7, 42], but direct infusion into the renal artery of dogs stimulates vasodilation followed by a prolonged vasoconstriction [3]. Overt vasodilation is not evident in the rat kidney, although blockade of ETB receptors increases the vasoconstrictor response to ET1 [66]. These findings first suggested that ETB receptors produce vasodilation in the renal circulation, but the use of selective ETA antagonists revealed that non-ETA receptors contribute to some of the ET-1-mediated renal vasoconstriction [52]. This group went on to show that low doses of ET-1 decrease renal blood flow and GFR via ETA receptors, whereas ETB-receptor-mediated changes are evident only at higher doses [53]. In terms of the medullary circulation, Gurbanov et al. used single optical fibers to observe that an intravenous bolus of ET-1 actually increases medullary blood flow while at the same time decreasing cortical blood flow [26]. Studies attempting to localize ET-1-receptor-dependent action in the pre- and postglomerular vessels have resulted in inconsistent results. In isolated rabbit renal arterioles, ET-1 has been shown to be equipotent at constricting afferent and efferent arterioles [12]. In contrast, another study provided evidence that ET-1 was more potent in efferent arterioles than in afferent arterioles [39]. The ET-1-mediated vasoconstriction at what might be considered physiological concentrations appears to be primarily via the ETA receptor because ET-3 produced vasoconstriction, but with significantly less potency, and because ETA receptor blockade completely inhibits the decrease in renal blood flow [53]. In the hydronephrotic kidney, Endlich and colleagues reported that ET-1-induced preglomerular constriction was largely due to ETA receptor activation, whereas ETBreceptor-dependent vasoconstriction was more evident in postglomerular vessels [14]. ET-1 has been shown to increase cytosolic Ca2+ in vascular smooth muscle by activating T- and L-type channels [23]. However, there is some uncertainty as to the role of intracellular Ca2+ in mediating the renal vasoconstrictor actions of ET-1. Several studies have shown that ET-1 increases intracellular Ca2+ concentra-

tions in isolated afferent arterioles or isolated preglomerular vascular smooth muscle cells [12, 60]. In the hydronephrotic kidney, Loutzenhiser et al. demonstrated that Ca2+ channel antagonism prevented ET-1induced renal vasoconstriction [40]. In contrast, blockade of L-type Ca2+ channels in vivo inhibits ET-1induced increases in arterial pressure, yet has no effect on the associated renal vasoconstriction [5]. Similarly, Pollock et al. reported only a minor role for L-type Ca2+ channel activation in the renal vasoconstrictor response to ET-1 and sarafotoxin 6c, an ETB-receptor-selective agonist [51]. Although it is apparent that ET-1 increases intracellular Ca2+ levels in vascular smooth muscle, it appears as though L-type Ca2+ channel activation does not play a major role in the renal vasoconstriction produced by ET-1 via either the ETA or ETB receptor. New information about the actions of endogenous ET-1 has been discerned from the use of receptorselective antagonists. An infusion of bosentan, the nonselective ETA/ETB receptor antagonist, had no effect on arterial pressure or renal blood flow, but produced a small decrease in GFR [57]. These investigators also observed that the blockade of both ETA and ETB receptors stimulated a fall in glomerular blood flow caused by a significant increase in preglomerular arteriole resistance [56]. However, the administration of an ETA receptor blocker produced the same effect on arterial pressure and renal blood flow as bosentan, but did not change vascular resistance [41, 57]. These studies suggest that ET-1 in the renal vasculature exerts a tonic vasodilator influence on GFR, perhaps via the ETB receptor, by release of vasodilator agents such as NO or prostacyclin [28, 56, 68].

ET-1 EFFECTS ON EXCRETORY FUNCTION Initial studies investigating the influence of ET-1 on the renal excretion of salt and water suggested that ET1-induced increases in renal perfusion pressure account for the natriuretic and diuretic effects of ET-1 [34, 65]. However, at relatively low doses that do not produce decreases in GFR, the systemic administration of ET-1 has been reported to have potent diuretic and natriuretic effects [27, 59]. The diuretic and natriuretic response first appeared to be mediated via the ETB receptor because an ETA antagonist blocked big ET-1induced increases in blood pressure but not the diuresis and natriuresis [53]. Increases in medullary blood flow produced by ET-1 through the ETB receptor may account for increased excretion of salt and water [26, 66]. However, there has been considerable in vitro, and more recently in vivo, data generated to demonstrate direct actions of ET-1 in a variety of nephron segments.

Renal Endothelin / 1271

Proximal Tubule In proximal tubule preparations, ET-1 has been observed to have a biphasic effect on ion transport by stimulating influx at low concentrations and inhibiting at high concentrations in which both effects are protein kinase C (PKC)-dependent [17]. There is evidence supporting both effects. The stimulatory effect of ET-1 on fluid transport at low concentration can be attributed to the stimulation of the Na+/H+ exchanger [67]. This appears to be due to the activation of ETB receptors because Na+/H+ exchanger activity is increased in opossum kidney clone (OKP) cells transfected with ETB receptors, not ETA receptors [8]. In these same cells, ET-1 increased intracellular Ca2+ levels, decreased adenylate cyclase activity, and increased tyrosine phosphorylation of several proteins, suggesting that the fluid transport effect is dependent on of these distinct pathways [8]. The ability of ET-1 to inhibit sodium reabsorption in the proximal tubule is supported by the observation that ET-1 inhibited Na+/K+-ATPase activity along with increasing cGMP concentration in cultured proximal tubule cells [31, 46]. Again, these actions are probably due to ETB receptor activation because ET-1 and ET-3 were equally potent in reducing Na+/ K+-ATPase activity [46]. Other studies have shown that ET-1 inhibits proximal tubule fluid reabsorption by augmenting the production of arachidonic acid metabolites [47]. At high concentrations, ET-1 not only stimulates PKC but also enhances phospholipase A2 metabolite generation. There is also evidence that 20-hydroxyeicosa5,8,11,14-tetraenoic acid (20-HETE), a cytochrome P450 metabolite, contributes to ET-1 mediated natriuretic responses [15]. The blockade of the enzyme responsible for 20-HETE synthesis (CYP4A) prevented the increase in urinary sodium excretion produced by ET-1 [45]. Similarly, the inhibition of 20-HETE formation in isolated proximal tubules prevented the blockade of ion transport produced by ET-1, indicating that 20-HETE acts as a second messenger of ET-1 in this nephron segment [45]. More recently, Williams et al. showed that chronic ETB receptor blockade reduces CYP4A expression in the kidney, providing an additional link between the ET and cytochrome P450 systems [70].

Thick Ascending Limb of Henle’s Loop Isolated segments of thick ascending limb express ET-1 mRNA and secrete ET-1 [6, 35]. ETB receptors appear to decrease chloride reabsorption in isolated mouse medullary and cortical thick ascending limbs via a cAMP-independent and Ca2+-insensitive diacylglycerol-responsive PKC pathway [9]. Plato et al. observed

that the activation of ETB receptors in thick ascending limbs inhibits chloride influx via a nitric oxide (NO)dependent mechanism [48]. Exogenous ET-1 decreased chloride influx, and NO inhibition with l-NAME completely blocked the ET-1-induced decrease in chloride reabsorption [48]. The blockade of ETB receptors with BQ-788 inhibited these effects of exogenous ET-1.

Cortical Collecting Duct The renal collecting duct is a major site of ET-1 synthesis, receptor binding, and action [49]. ET-1 receptor expression and production increases along the length of the collecting duct from the cortex to the inner medulla. The activation of ETB receptors inhibits vasopressin (AVP)-stimulated water transport by decreasing cAMP levels through the activation of PKC [63]. Mineralocorticoid and AVP-stimulated sodium and chloride transport is diminished by ET-1 via the inhibition of apical sodium and chloride entry [16, 63]. In vitro studies have shown that ET-1 inhibits sodium transport by decreasing amiloride-sensitive sodium channel activity and increasing channel mean closed time [16]. Similar to the proximal tubule, ET-1 appears to have a biphasic response in the cortical collecting duct. A concentration of ET-1 in the picomolar range decreases sodium channel activity through ETB activation, whereas a concentration in the nanomolar range increases sodium channel activity through ETA activation; however, this mechanism is not well understood.

Inner Medullary Collecting Duct The inner medullary collecting duct produces up to ten times as much ET-1 than any other cell type in the nephron [6, 36, 55, 64]. This confirms the fact that the inner medullary collecting duct contains the highest concentration of ET-1 and its receptors than any other organ in the body [49, 72]. The effects of ET-1 in the inner medullary collecting duct appear to be very similar to those seen in the cortical collecting duct. Cells from this region of the kidney are able to secrete and bind ET-1 on the basolateral side of the membrane, providing evidence that ET-1 functions as a strong autocrine modulator of inner medulla function [38]. Resembling its effects in the cortical collecting duct, ET-1 blocks water transport by inhibiting AVP-stimulated cAMP accumulation and osmotic water permeability in the inner medullary collecting duct [11, 37, 62]. The ability of ET-1 to inhibit water reabsorption involves the inhibition of adenylate cyclase activity because administration of a cAMP mimetic to increase water reabsorption is unaffected by ET-1 [44]. Unfortunately, it has been very difficult to measure actual sodium transport in this nephron segment due to technical limitations.

1272 / Chapter 175 In freshly isolated inner medullary collecting duct cells, ET-1 reduces sodium transport by inhibiting the Na+/K+-ATPase on the basolateral side of the membrane [73]. This effect seems to involve the stimulation of prostaglandin E2 production and is attributed to ETB receptor activation [37, 73]. ET-1 is also thought to decrease sodium transport by negatively regulating epithelial sodium channel (ENaC) activity. Gilmore and colleagues observed that ET-1 decreases ENaC activity through the activation of Src kinase and is attributed to the activation of ETB receptors [22]. ETA receptors are also present in the collecting duct at fairly low levels [11]. Their function remains unknown, although it has been suggested that they regulate tubular transport by increasing endothelial nitric oxide synthase expression [71]. Ge and colleagues observed that collecting-duct-specific ETA receptor knockout (CD ETA KO) mice have similar arterial pressure, sodium excretion, and water excretion to their control counterparts when challenged with normal and high-salt diets [21]. However, CD ETA KO mice are unable to excrete an acute water load appropriately and have an increased sensitivity to AVP-induced cAMP accumulation [20]. These results suggest that ETA receptors in the collecting duct are not involved in the physiological regulation of arterial pressure or sodium excretion but more specifically help to regulate water reabsorption. Very recent results have provided more compelling evidence that ET-1 produced by the collecting duct plays a key role in fluid-volume balance and blood pressure regulation. Collecting duct-specific ET-1 knockout (CD ET-1 KO) mice have altered renal sodium and water handling suggesting a physiological role of collecting-duct-derived ET-1 in regulating tubular fluid [1, 20].

RENAL ET-1 IN BLOOD PRESSURE REGULATION Early evidence that renal ET-1 contributes to blood pressure regulation through the control of sodium and water balance came from numerous reports that administration of an ETA-receptor-antagonist-reduced hypertension in salt-dependent models [2, 4, 33, 58]. In contrast, blocking ETB receptors exacerbates the elevated arterial pressure in the DOCA-salt hypertensive rat [50]. In normotensive rats, chronic ETB receptor blockade elevates blood pressure in a salt-dependent manner [54, 69]. ETB receptors in the renal medulla are upregulated in response to salt loading, which is consistent with the hypothesis that ETB receptors in the kidney regulate sodium balance [49]. Furthermore, urinary ET, thought

to be derived from intrarenal sources, is elevated in animals placed on a high-salt diet [54]. Increased ETB receptor function in rats on a high-salt diet is also evidenced by the observation that increases in medullary blood flow produced by big ET-1 are more pronounced in rats on a high-salt diet [66]. Gene-targeting strategies have provided more specific evidence that ET-1 regulates salt and water excretion. The spotting lethal rat carries a naturally occurring deletion of the ETB receptor gene that results in deadly megacolon [19]. Gariepy et al. rescued these rats with a dopamine-β-hydroxylase promoter that directed tissue-specific ETB transgene expression to support normal enteric nervous system development yet remain without ETB receptors on endothelial or renal tubular epithelial cells [18]. Rats homozygous for the ETB receptor deletion have a higher basal arterial pressure than their control counterparts [13, 43, 61]. Similar to pharmacological ETB receptor blockade, ETB deficient rats become hypertensive when challenged with a highsalt diet [13, 18, 43, 61]. CD ET-1 KO mice have provided key evidence in determining the role of ET-1 in the collecting duct on sodium and water handling [1, 20]. These mice express Cre recombinase under control of the aquaporin-2 promoter and are homozygous for loxP-flanked exon 2 of the ET-1 gene. Because aquaporin-2 is expressed specifically in the collecting duct, these mice do not have the ability to produce ET-1 in the collecting duct. CD ET-1 KO mice have an impaired ability to excrete a sodium load, which causes them to have higher basal pressures as well as hypertension when challenged with a salt load [1]. The administration of the diuretics, amiloride, or furosemide decreases blood pressure in these mice [1, 20]. CD ET-1 KO mice also have an impaired diuresis in response to chronic water loading and have higher vasopressin-mediated cAMP accumulation in the inner medullary collecting ducts, suggesting that ET-1 decreases vasopressin sensitivity by preventing the increase in cAMP accumulation [20]. Under highsalt conditions, these mice also have a significantly lower ET-1 excretion than their control counterparts, confirming a predominant renal origin of urinary ET-1 [1]. Observations in humans with salt-sensitive hypertension reveal a significantly lower ET-1 excretion compared to normotensive controls, suggesting a defect in the ET-1 system could account for an inability to handle the salt load without an elevated arterial pressure [29, 30]. CD ETA KO mice, which were generated in the same manner as CD ET-1 KO mice, have no differences in systemic blood pressure and sodium excretion in response to a normal or high-salt diet compared to controls [20]. However, CD ETA KO mice have increased plasma AVP levels with no change in water excretion

Renal Endothelin / 1273 during chronic water loading [21]. Therefore, ETA receptors in the collecting duct may function to decrease renal sensitivity to AVP, but not participate in long-term arterial pressure regulation and sodium transport.

CONCLUSION Evidence that the renal ET system plays a role in the control of fluid-electrolyte balance is very strong. The localized action of the renal ET system explains how a potent vasoconstrictor can also produce vasodilation and facilitate the excretion of salt and water. Although human kidneys have similar ET-1 production and distribution of ET receptors, there have been no studies to determine the intrarenal actions of ET-1. One study examined the influence of ET-1 infusion on renal function; however, the dose used produced an extremely large degree of renal vasoconstriction and reductions in GFR that masked any potential influence of ETB receptors to promote natriuresis [32]. Clearly, the factors that regulate the production of ET, receptor expression, and activity in the human kidney remain to be elucidated.

References [1] Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE: Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 2004, 114:504–511. [2] Allcock GH, Venema RC, Pollock DM: ETA receptor blockade attenuates the hypertension but not renal dysfunction in DOCAsalt rats. Am J Physiol 1998, 275:R245–252. [3] Banks RO: Effects of endothelin on renal function in dogs and rats. Am J Physiol 1990, 258:F775–780. [4] Barton M, d’Uscio LV, Shaw S, Meyer P, Moreau P, Luscher TF: ETA receptor blockade prevents increased tissue endothelin-1, vascular hypertrophy, and endothelial dysfunction in salt-sensitive hypertension. Hypertension 1998, 31:499–504. [5] Cao LQ, Banks RO: Cardiovascular and renal actions of endothelin: effects of calcium-channel blockers. Am J Physiol 1990, 258:F254–258. [6] Chen M, Todd-Turla K, Wang WH, Cao X, Smart A, Brosius FC, Killen PD, Keiser JA, Briggs JP, Schnermann J: Endothelin-1 mRNA in glomerular and epithelial cells of kidney. Am J Physiol 1993, 265:F542–550. [7] Chou SY, Porush JG, Faubert PF: Renal medullary circulation: hormonal control. Kidney Int 1990, 37:1–13. [8] Chu TS, Peng Y, Cano A, Yanagisawa M, Alpern RJ: EndothelinB receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells. J Clin Invest 1996, 97:1454–1462. [9] de Jesus Ferreira MC, Bailly C: Luminal and basolateral endothelin inhibit chloride reabsorption in the mouse thick ascending limb via a Ca2+-independent pathway. J Physiol 1997, 505 (Pt 3):749–758. [10] Douglas SA, Beck GR, Jr., Elliott JD, Ohlstein EH: Pharmacological evidence for the presence of three distinct functional endothelin receptor subtypes in the rabbit lateral saphenous vein. Br J Pharmacol 1995, 114:1529–1540.

[11] Edwards RM, Stack EJ, Pullen M, Nambi P: Endothelin inhibits vasopressin action in rat inner medullary collecting duct via the ETB receptor. J Pharmacol Exp Ther 1993, 267:1028–1033. [12] Edwards RM, Trizna W, Ohlstein EH: Renal microvascular effects of endothelin. Am J Physiol 1990, 259:F217–221. [13] Elmarakby AA, Dabbs Loomis E, Pollock JS, Pollock DM: ETA receptor blockade attenuates hypertension and decreases reactive oxygen species in ETB receptor-deficient rats. J Cardiovasc Pharmacol 2004, 44:S7–S10. [14] Endlich K, Hoffend J, Steinhausen M: Localization of endothelin ETA and ETB receptor-mediated constriction in the renal microcirculation of rats. J Physiol 1996, 497 (Pt 1):211–218. [15] Escalante BA, McGiff JC, Oyekan AO: Role of cytochrome P-450 arachidonate metabolites in endothelin signaling in rat proximal tubule. Am J Physiol Renal Physiol 2002, 282:F144–150. [16] Gallego MS, Ling BN: Regulation of amiloride-sensitive Na+ channels by endothelin-1 in distal nephron cells. Am J Physiol 1996, 271:F451–460. [17] Garcia NH, Garvin JL: Endothelin’s biphasic effect on fluid absorption in the proximal straight tubule and its inhibitory cascade. J Clin Invest 1994, 93:2572–2577. [18] Gariepy CE, Ohuchi T, Williams SC, Richardson JA, Yanagisawa M: Salt-sensitive hypertension in endothelin-B receptor-deficient rats. J Clin Invest 2000, 105:925–933. [19] Gariepy CE, Williams SC, Richardson JA, Hammer RE, Yanagisawa M: Transgenic expression of the endothelin-B receptor prevents congenital intestinal aganglionosis in a rat model of Hirschsprung disease. J Clin Invest 1998, 102:1092–1101. [20] Ge Y, Ahn D, Stricklett PK, Hughes AK, Yanagisawa M, Verbalis JG, Kohan DE: Collecting duct-specific knockout of endothelin1 alters vasopressin regulation of urine osmolality. Am J Physiol Renal Physiol 2005, 288:F912–920. [21] Ge Y, Stricklett PK, Hughes AK, Yanagisawa M, Kohan DE: Collecting duct-specific knockout of the endothelin A receptor alters renal vasopressin responsiveness, but not sodium excretion or blood pressure. Am J Physiol Renal Physiol 2005, 289:F692–F698. [22] Gilmore ES, Stutts MJ, Milgram SL: SRC family kinases mediate epithelial Na+ channel inhibition by endothelin. J Biol Chem 2001, 276:42610–42617. [23] Gordienko DV, Clausen C, Goligorsky MS: Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. Am J Physiol 1994, 266:F325–341. [24] Goto K: Endothelins. In Handbook of Biologically Active Peptides. Edited by Kastin AJ: Elsevier; 2006. [25] Granger JP: Endothelin. Am J Physiol Regul Integr Comp Physiol 2003, 285:R298–301. [26] Gurbanov K, Rubinstein I, Hoffman A, Abassi Z, Better OS, Winaver J: Differential regulation of renal regional blood flow by endothelin-1. Am J Physiol 1996, 271:F1166–1172. [27] Harris PJ, Zhuo J, Mendelsohn FA, Skinner SL: Haemodynamic and renal tubular effects of low doses of endothelin in anaesthetized rats. J Physiol 1991, 433:25–39. [28] Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, Marumo F: Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest 1993, 91:1367–1373. [29] Hoffman A, Grossman E, Goldstein DS, Gill JR, Jr., Keiser HR: Urinary excretion rate of endothelin-1 in patients with essential hypertension and salt sensitivity. Kidney Int 1994, 45:556–560. [30] Hwang YS, Hsieh TJ, Lee YJ, Tsai JH: Circadian rhythm of urinary endothelin-1 excretion in mild hypertensive patients. Am J Hypertens 1998, 11:1344–1351. [31] Ishii K, Warner TD, Sheng H, Murad F: Endothelin increases cyclic GMP levels in LLC-PK1 porcine kidney epithelial cells via formation of an endothelium-derived relaxing factor-like substance. J Pharmacol Exp Ther 1991, 259:1102–1108.

1274 / Chapter 175 [32] Kaasjager KA, Koomans HA, Rabelink TJ: Endothelin-1-induced vasopressor responses in essential hypertension. Hypertension 1997, 30:15–21. [33] Kassab S, Miller MT, Novak J, Reckelhoff J, Clower B, Granger JP: Endothelin-A receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats. Hypertension 1998, 31:397–402. [34] King AJ, Brenner BM, Anderson S: Endothelin: a potent renal and systemic vasoconstrictor peptide. Am J Physiol 1989, 256: F1051–1058. [35] Kohan DE: Endothelin synthesis by rabbit renal tubule cells. Am J Physiol 1991, 261:F221–226. [36] Kohan DE, Fiedorek FT, Jr.: Endothelin synthesis by rat inner medullary collecting duct cells. J Am Soc Nephrol 1991, 2:150– 155. [37] Kohan DE, Hughes AK: Autocrine role of endothelin in rat IMCD: inhibition of AVP-induced cAMP accumulation. Am J Physiol 1993, 265:F126–129. [38] Kohan DE, Padilla E: Endothelin-1 is an autocrine factor in rat inner medullary collecting ducts. Am J Physiol 1992, 263:F607– 612. [39] Lanese DM, Yuan BH, McMurtry IF, Conger JD: Comparative sensitivities of isolated rat renal arterioles to endothelin. Am J Physiol 1992, 263:F894–899. [40] Loutzenhiser R, Epstein M, Hayashi K, Horton C: Direct visualization of effects of endothelin on the renal microvasculature. Am J Physiol 1990, 258:F61–68. [41] Matsuura T, Miura K, Ebara T, Yukimura T, Yamanaka S, Kim S, Iwao H: Renal vascular effects of the selective endothelin receptor antagonists in anaesthetized rats. Br J Pharmacol 1997, 122:81–86. [42] Miller WL, Redfield MM, Burnett JC, Jr.: Integrated cardiac, renal, and endocrine actions of endothelin. J Clin Invest 1989, 83:317–320. [43] Ohkita M, Wang Y, Nguyen ND, Tsai YH, Williams SC, Wiseman RC, Killen PD, Li S, Yanagisawa M, Gariepy CE: Extrarenal ETB plays a significant role in controlling cardiovascular responses to high dietary sodium in rats. Hypertension 2005, 45:940–946. [44] Oishi R, Nonoguchi H, Tomita K, Marumo F: Endothelin-1 inhibits AVP-stimulated osmotic water permeability in rat inner medullary collecting duct. Am J Physiol 1991, 261:F951–956. [45] Oyekan AO, McGiff JC: Cytochrome P-450-derived eicosanoids participate in the renal functional effects of ET-1 in the anesthetized rat. Am J Physiol 1998, 274:R52–61. [46] Ozaki S, Ihara M, Saeki T, Fukami T, Ishikawa K, Yano M: Endothelin ETB receptors couple to two distinct signaling pathways in porcine kidney epithelial LLC-PK1 cells. J Pharmacol Exp Ther 1994, 270:1035–1040. [47] Perico N, Cornejo RP, Benigni A, Malanchini B, Ladny JR, Remuzzi G: Endothelin induces diuresis and natriuresis in the rat by acting on proximal tubular cells through a mechanism mediated by lipoxygenase products. J Am Soc Nephrol 1991, 2:57– 69. [48] Plato CF, Pollock DM, Garvin JL: Endothelin inhibits thick ascending limb chloride flux via ETB receptor-mediated NO release. Am J Physiol Renal Physiol 2000, 279:F326–333. [49] Pollock DM: Renal endothelin in hypertension. Curr Opin Nephrol Hypertens 2000, 9:157–164. [50] Pollock DM, Allcock GH, Krishnan A, Dayton BD, Pollock JS: Upregulation of endothelin B receptors in kidneys of DOCAsalt hypertensive rats. Am J Physiol Renal Physiol 2000, 278:F279– 286. [51] Pollock DM, Jenkins JM, Cook AK, Imig JD, Inscho EW: L-type calcium channels in the renal microcirculatory response to endothelin. Am J Physiol Renal Physiol 2005, 288:F771–777.

[52] Pollock DM, Opgenorth TJ: Evidence for endothelin-induced renal vasoconstriction independent of ETA receptor activation. Am J Physiol 1993, 264:R222–226. [53] Pollock DM, Opgenorth TJ: ETA receptor-mediated responses to endothelin-1 and big endothelin-1 in the rat kidney. Br J Pharmacol 1994, 111:729–732. [54] Pollock DM, Pollock JS: Evidence for endothelin involvement in the response to high salt. Am J Physiol Renal Physiol 2001, 281: F144–150. [55] Pupilli C, Brunori M, Misciglia N, Selli C, Ianni L, Yanagisawa M, Mannelli M, Serio M: Presence and distribution of endothelin-1 gene expression in human kidney. Am J Physiol 1994, 267: F679–687. [56] Qiu C, Baylis C: Endothelin and angiotensin mediate most glomerular responses to nitric oxide inhibition. Kidney Int 1999, 55:2390–2396. [57] Qiu C, Samsell L, Baylis C: Actions of endogenous endothelin on glomerular hemodynamics in the rat. Am J Physiol 1995, 269: R469–473. [58] Schiffrin EL, Turgeon A, Deng LY: Effect of chronic ETAselective endothelin receptor antagonism on blood pressure in experimental and genetic hypertension in rats. Br J Pharmacol 1997, 121:935–940. [59] Schnermann J, Lorenz JN, Briggs JP, Keiser JA: Induction of water diuresis by endothelin in rats. Am J Physiol 1992, 263: F516–526. [60] Schroeder AC, Imig JD, LeBlanc EA, Pham BT, Pollock DM, Inscho EW: Endothelin-mediated calcium signaling in preglomerular smooth muscle cells. Hypertension 2000, 35:280– 286. [61] Taylor TA, Gariepy CE, Pollock DM, Pollock JS: Gender differences in ET and NOS systems in ETB receptordeficient rats: effect of a high salt diet. Hypertension 2003, 41:657–662. [62] Tomita K, Nonoguchi H, Marumo F: Effects of endothelin on peptide-dependent cyclic adenosine monophosphate accumulation along the nephron segments of the rat. J Clin Invest 1990, 85:2014–2018. [63] Tomita K, Nonoguchi H, Terada Y, Marumo F: Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat. Am J Physiol 1993, 264:F690–696. [64] Ujiie K, Terada Y, Nonoguchi H, Shinohara M, Tomita K, Marumo F: Messenger RNA expression and synthesis of endothelin-1 along rat nephron segments. J Clin Invest 1992, 90:1043– 1048. [65] Uzuner K, Banks RO: Endothelin-induced natriuresis and diuresis are pressure-dependent events in the rat. Am J Physiol 1993, 265:R90–96. [66] Vassileva I, Mountain C, Pollock DM: Functional role of ETB receptors in the renal medulla. Hypertension 2003, 41:1359– 1363. [67] Walter R, Helmle-Kolb C, Forgo J, Binswanger U, Murer H: Stimulation of Na+/H+ exchange activity by endothelin in opossum kidney cells. Pflugers Arch 1995, 430:137–144. [68] Warner TD, Mitchell JA, de Nucci G, Vane JR: Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J Cardiovasc Pharmacol 1989, 13 Suppl 5:S85–88; discussion S102. [69] Williams JM, Pollock JS, Pollock DM: Arterial pressure response to the antioxidant tempol and ETB receptor blockade in rats on a high-salt diet. Hypertension 2004, 44:770–775. [70] Williams JM, Zhao X, Wang MH, Imig JD, Pollock DM: Peroxisome proliferator-activated receptor-alpha activation reduces salt-dependent hypertension during chronic endothelin B receptor blockade. Hypertension 2005, 46:366–371.

Renal Endothelin / 1275 [71] Ye Q, Chen S, Gardner DG: Endothelin inhibits NPR-A and stimulates eNOS gene expression in rat IMCD cells. Hypertension 2003, 41:675–681. [72] Yukimura T, Notoya M, Mizojiri K, Mizuhira V, Matsuura T, Ebara T, Miura K, Kim S, Iwao H, Song K: High resolution

localization of endothelin receptors in rat renal medulla. Kidney Int 1996, 50:135–147. [73] Zeidel ML, Brady HR, Kone BC, Gullans SR, Brenner BM: Endothelin, a peptide inhibitor of Na+-K+-ATPase in intact renal tubular epithelial cells. Am J Physiol 1989, 257:C1101–1107.

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176 Prolactin and Kidney Function WILLIS K. SAMSON

groundbreaking work in unique animal models [35, 44, 46]. Numerous excellent reviews of the roles played by PRL in fishes, birds, and other species have been published [11, 29, 31, 37–39, 41], and those reviews are not recapitulated here. Instead, this review highlights both the evidence for and the evidence against actions of PRL in the mammalian kidney.

ABSTRACT Prolactin, a large peptide produced in and secreted from lactotrophs of the anterior pituitary gland, traditionally has been considered a reproductive hormone in humans. In submammalian species, however, it plays an important role in osmoregulation. Recently, prolactin receptors have been described in mammalian kidney, and the peptide appears to be produced there as well. Thus, prolactin may exert renotropic actions in humans similar to those observed in lower species. Indeed, the effects of prolactin on renal sodium and water handling have been reported, and the controversy over the physiological relevance of those observations is detailed in this review.

CLASSICAL PRODUCTION SITE AND ACTIONS OF PROLACTIN As reviewed recently by several recognized experts in the field [5, 10, 21], PRL is produced primarily in the anterior pituitary gland where it is both stored and released as the mature 199-amino-acid polypeptide after the removal from the prohormone of a 28-aminoacid signal peptide. It circulates as both a 23-kDa protein and, after enzymatic cleavage and reduction of one of its disulfide bonds in a variety of tissues sites, a 16-kDa biologically active fragment [4, 16]. In fact, several of the nontraditional actions of PRL have been ascribed to this 16-kDa fragment [3, 5, 6, 15, 17]. In the pituitary gland, PRL is produced in a unique population of cells, termed lactotrophs because the primary action of the protein in the mammary gland is to stimulate milk production [37]. This is the hallmark action of PRL and, together with its effects on corpus luteal function in the ovary [37, 39], formed the basis of the predominant view that PRL was a reproductive hormone, associated primarily with female reproductive function. Why then do males make, store, and release PRL from the anterior pituitary gland? Why is the PRL gene transcribed and protein produced in extrapituitary sites? Why then are receptors for the protein found in numerous tissue sites other than the ovary and mammary gland?

INTRODUCTION Because prolactin (PRL) was considered by most endocrinologists to be primarily a hormone associated with reproductive function, for years the prevailing dogma drew attention away from consistent findings that receptors for the peptide were localized to nonreproductive tissues, including kidney. And yet in submammalian species a clear role was indicated for the peptide in fluid and electrolyte homeostasis. What has emerged from years of sporadic interest in the possible renotropic actions of PRL is a body of evidence that suggests little significance in humans of this pituitaryderived peptide; however, much promise still exists for renal actions of endogenous PRL, in particular PRL produced within the kidney acting in a local paracrine fashion to control tubular function. These effects have not been observed in the human studies published to date, but were in fact predicted by not only the comparative studies in lower species but also by some Handbook of Biologically Active Peptides

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1278 / Chapter 176 PROLACTIN RECEPTORS Prolactin receptors belong to the class 1 cytokine receptor superfamily, which includes receptors for a wide variety of peptides (e.g., interleukins, erythropoietin, leukemia inhibiting factor, and the hormone leptin). As reviewed by Kelly and colleagues [10], the PRL receptor itself exists in at least three major isoforms, all splice variants of the same gene product. They share homology in the extracellular N-terminal domain and in the single-chain transmembrane portion, but differ in the length of their cytoplasmic C-terminal tails. The extracellular domain establishes the homology to the cytokine receptors and is approximately 200 amino acids in length. The intracellular domains of the receptor contain not only the sequences necessary for appropriate signal transduction but also those responsible for receptor internalization. Ligand binding to the PRL receptor results in the stimulation of the tyrosine kinase activity of Janus kinase 2 (JAK2) and, except in the case of the short-form isoform, receptor phosphorylation and the subsequent activation of signal transducer and activator of transcription (Stat) proteins, in particular Stat5. The interactions of the activated Stat proteins with DNA binding sites underlies the specificity of the genomic effects of PRL. Signaling via the mitogen-activated protein kinase (MAPK) pathway has also been identified and an interaction with the epidermal growth factor (EGF) family of receptors has been suggested. As previously stated, the primary actions of PRL were for a long time considered to be in mammary gland and ovary, tissues containing abundant receptors for the protein. However, a much broader distribution of PRL receptors has been cataloged in what were largely unexpected sites, including the brain, adrenal gland, bone, adipocytes, liver, pancreas, gastrointestinal tract, a variety of lymphoid tissues, and the kidney [10]. Not surprisingly, the pharmacological effects of PRL have now been described in those tissues as well, opening the possibility that PRL is not just a hormone of reproduction but instead one with diverse biological potential. Best characterized to date are the actions of PRL in the immune cells, bone, brain, and pancreas [5, 10, 21]. Some of those actions may be a reflection of circulating PRL, whereas others may require local production of the peptide, as in the case of the potential renal actions [46].

PROLACTIN AND OSMOREGULATION PRL plays a significant role in osmoregulation in species exposed to harsh environments [10, 29, 37, 39]. Indeed, the protein is an important protective factor in

fish in both the fresh and seawater environments and in the transition between those two osmotic conditions. The importance of PRL in osmoregulation in fish has been recently reviewed by Manzon [31] and its role in birds and amphibians described [11, 38, 41, 44]. Evidence for a role in osmoregulation in mammals has also accumulated and indeed the presence of PRL receptors in the mammalian kidney detailed. However, most clinically based studies have failed to present a clear picture of PRL effects in humans, and only some of the animal trials have been able to at least partially explain the possible similarity of PRL actions in mammals with those observed in submammalian species. What has been missing perhaps has been an appreciation of the importance of locally produced PRL as opposed to the circulating pituitary-derived peptide.

PROLACTIN RECEPTORS IN THE MAMMALIAN KIDNEY In the 1980s [36] it was already known that highaffinity PRL binding could be demonstrated in rat kidney membrane preparations (medullary and cortical fragments) and that maximal binding was present in epithelial cells of the glomerulus and proximal tubule as identified by autoradiography. Faint but detectable labeling was observed in tubule cells of the cortical and medullary nephron as well. That work extended the original observations of Marshall, Gelato, and Meites [32], which demonstrated by Scatchard analysis the presence of PRL binding activity in rat kidney. The proximal tubule site of autoradiographic PRL labeling suggested an action of the peptide on sodium handling in the kidney; however, the original paper [32] failed to demonstrate a significant effect of uninephrectomy or salt loading on PRL binding in kidney. Similarly, serum PRL levels failed to be significantly altered by the manipulation of osmolality in several studies [1, 8, 33]. With the cloning of the mammalian PRL receptors, much more was learned about the potential roles played by circulating or locally produced PRL in renal function. In situ hybridization histochemistry revealed the presence of both the short and long forms of the PRL receptor in the rat kidney. Unlike in the other tissues (e.g., adrenal, spleen, thymus, skin, and heart) in which the long receptor isoform predominated, in the kidney the short form was more abundantly detected, with highest signal found in the glomeruli and in tubules of the outer medulla [40]. This was later confirmed by Freemark and colleagues [23] by reverse transcription polymerase chain reaction, which also demonstrated the relative increased levels of the short versus long isoform of the PRL receptor in fetal rat kidney. The

Prolactin and Kidney Function / 1279 same group of investigators [45] extended those initial studies with a combination of in situ hybridization histochemistry, immunohistochemistry, and radioligand binding studies that demonstrated the presence of PRL receptors in fetal rat kidney by day 19–20 of gestation. At day 20, PRL binding was best observed in cortical and subcortical renal tubules. In human fetal kidney, PRL receptor immunoreactivity was detected at embryonic day 52 in tubules and at gestational day 96 in tubules and collecting ducts [22]. Staining was particularly intense on the luminal surfaces of the tubules and ducts. No immunoreactivity was observed in glomeruli. The luminal localization of PRL receptors was confirmed in murine kidney [46], in particular in the proximal tubule and the parietal epithelium of Bowman’s capsule, where mRNA encoding PRL itself also was detected, raising the possibility of local actions of endogenously produced PRL within the kidney. This is further supported by the failure of fetal growth restriction to alter renal PRL receptor messages in the face of suppressed synthesis and secretion of PRL in the pituitary gland [42].

RENAL PRODUCTION OF PROLACTIN How does PRL access its apparent binding sites on the luminal epithelium of the tubule? Certainly circulating PRL of pituitary origin may be filtered through the glomerulus. An arteriovenous gradient of PRL across the human kidney [18] has been described, suggesting either removal by filtration or metabolism by the kidney [19]; however, micropuncture studies describing the concentrations of PRL in tubular urine are lacking. It is clear that at least in the mouse [46] PRL mRNA and protein can be detected in the luminal epithelial cells of the proximal tubule. In addition, those cells also express the message for Pit-1, a transcription factor known to be important in the regulation of PRL gene expression [10], further suggesting significant local production of the peptide.

RENAL ACTIONS OF PROLACTIN Although there is clear consensus that PRL receptors exist in kidney, there is little agreement in the literature concerning the potential renal actions of the peptide. This may be a result of differing experimental protocols or the use of heterologous peptide preparations. The lack of agreement may also be a result of the commonly held belief that all PRL effects in kidney are a result of the peptide delivered by the general circulation. This may not be the case, and a unique isoform of PRL may in fact be the effective agent.

Horrobin and colleagues [25] injected semipurified sheep PRL intramuscularly into five male volunteers. In addition to a long-lasting malaise, they observed a decrease in urine volume and urinary sodium and potassium excretion that was matched by increased plasma sodium and osmolality. The volunteers also expressed a sensation of thirst. The absolute purity of the peptide preparation could not at the time be assured, and thus contamination with other anterior lobe hormones, as well as the neurohypophyseal peptides oxytocin and vasopressin could not be ruled out. That human study was designed to mirror work by Lockett and coworkers [28] in which the antinatriuretic, antidiuretic, and antikaliuretic effects of purified PRL preparations were observed in conscious rats, in the absence of demonstrable actions on glomerular filtration rate. Thus, tubular effects to increase sodium reabsorption were the first suggested actions of PRL. The decreased urine volume observed in human volunteers led many to speculate that PRL was a unique antidiuretic agent. This was further suggested by the observation in rats that ovine PRL infusions reduced the diuresis and natriuresis of an intravenous saline load [30]. However, that same year a report appeared that suggested that the antidiuretic effect of PRL was due to the occupancy of the vasopressin (AVP) receptors in kidney [20]. In fact the antidiuretic effect of PRL may be secondary to a direct tubular effect on sodiumpotassium ATPase activity, which by increasing medullary tonicity facilitates the action of AVP [43]. In that later study, the possible contamination of the PRL preparation was ruled out because of preabsorption by antiAVP antiserum. Later, it was demonstrated that pituitary transplants, which drastically and chronically elevated plasma PRL levels, resulted in increased urine flow and decreased osmolality [2]. Because those animals had been adrenalectomized, the authors concluded that the increased water excretion was a direct effect of PRL and not due to increased glucocorticoids or thirst, as had been suggested by others [27]. Thus the controversy over the physiological relevance of the pharmacological effects of purified PRL continued. One study did attempt to compare the effects of circulating versus postentially locally produced PRL, although at the time it had not yet been demonstrated that PRL was produced in the mammalian kidney. Stier and colleagues [47] examined the effects of ovine PRL (cross-species) on renal function in anesthetized, volume-expanded male rats pretreated with bromocryptine to lower the circulating levels of pituitary-derived endogenous PRL. Bolus, followed by constant infusion of, ovine PRL succeeded in elevating circulating hormone levels into the high physiological range for females, but certainly higher than normally observed

1280 / Chapter 176 for rat PRL in males. Just the same significant, transient effects were observed. Urinary sodium, potassium, and water excretion were reduced compared with controls, and although there was detectable AVP contamination in the preparation of the ovine PRL, the authors did not conclude that the contamination significantly contributed to the antidiuretic effect of the peptide preparation. This, of course, does not agree with the conclusions of earlier studies. It is important that Stier and colleagues further reported that at least with ovine PRL no changes in glomerular filtration rate, renal plasma flow, filtration fraction, renal blood flow renal vascular resistance, or arterial pressure were observed, suggesting that any effect on sodium excretion or urine volume was due to direct tubular actions. In micropuncture studies, no significant differences in single nephron glomerular filtration rate or reabsorption were observed, suggesting that any significant tubular effect of PRL must be exerted beyond the proximal tubule, deeper in the renal medullary interstitium. It should be remembered, however, that these studies were necessarily conducted in an anesthetized animal and then using a cross-species form of the peptide under acute conditions. Furthermore, plasma AVP levels were already elevated in these animals due to the anesthesia. It was not until more recently [35] that the story was clarified to some extent. In the absence of AVP (Brattleboro rats or Sprague Dawley rats, fluid-loaded to decrease endogenous AVP secretion), recombinant mouse PRL exerts antinatriuetic and antidiuretic effects. However, when AVP is present, the action is diuretic. These excellent studies demonstrate several important shortcomings of earlier work: the use of recombinant material obviating the potential for contamination with other renotropic factors, the use of species-specific polypeptide, and the separation of the potentiative effects of PRL on AVP actions. The study does not, however, isolate the problem of identifying the actions of circulating (pituitary origin) versus locally produced peptide.

PROLACTIN AND RENAL FUNCTION IN HUMANS There are three potential approaches to the study of the role of PRL in human renal function. One such approach is the administration of exogenous peptide, which was attempted earlier with ovine PRL, with significant side effects [25]. Now that multiple tissue sites of action of PRL have been described, administration into the general circulation of human volunteers may be unwise and certainly the results will be difficult to interpret. A second approach is to examine hormone secretion in response to changes in fluid and electrolyte

homeostasis. This has been attempted with varying results. Buckman and colleagues [12] reported that oral water loading suppressed serum PRL levels in normal subjects but not patients with pituitary tumors (disregulated PRL production and secretion). The same group later reported that hypotonic saline infusions lowered and hypertonic saline infusion elevated serum PRL levels in a small cadre of subjects [13]. Adler and colleagues [1] in a somewhat larger population of volunteers detected, on the other hand, a small rise in plasma PRL levels following oral water loading. No effects of intravenous hypotonic or hypertonic saline infusions were observed. Berl and colleagues [8] similarly failed to detect changes in serum PRL levels following experimentally induced increases or decreases in serum osmolality. Furthermore, experimentally elevated PRL levels (thyrotropin-releasing hormone, TRH, infusion) in healthy water-loaded volunteers did not result in any significant changes in renal hemodynamics or urinary sodium and water excretion. Thus, PRL or pituitary origin has not been convincingly demonstrated to significantly alter renal function in humans.

THE JURY IS STILL OUT ON THE RENAL ACTIONS OF PROLACTIN Despite abundant suggestive evidence for renotropic actions of PRL, these still have not been conclusively demonstrated to exist. Perhaps the approach taken has led to the confusion. Almost all the studies described in the literature focused on PRL of pituitary origin as the potential renotropic agent. Now that it is known that the gene for PRL is expressed in kidney [46] and that PRL receptors are expressed on the luminal epithelium of the renal tubule [21], attention should be focused on the potential for PRL of renal origin to act in the tubule to alter sodium and water handling. Micropuncture studies using homologous, recombinant peptide should be conducted to include the infusion of PRL into the tubular fluid. The potential actions of PRL variants in the kidney need to be further examined. For example, the effect of glycosylated PRL or the smaller 16-kDa isoform should be examined. In particular, the 16-kDa form may hold promise; this is the posttranslationally modified form of PRL that is created by enzymatic cleavage followed by reduction of the disulfide bridge between amino acids 58 and 174. The resultant 16-kDa isoform is poorly recognized by the classical PRL receptors [16], but does express unique biologic activities, including antiangiogenesis and antitumorigenesis [6, 15, 17, 23]. We have preliminary results indicating the detection by autoradiography of iodinated 16-kDa PRL binding in rat kidney slices and potent diuretic

Prolactin and Kidney Function / 1281 actions in anesthetized animals (R. C. Vari and W. K. Samson, unpublished observations). Clearly this modified form of PRL may be produced in kidney, where the necessary cathepsin-D-like enzymes and reductases are [4], and it also may play a role in the local regulation of sodium and water transport.

[11]

[12]

[13]

HAVE TRANSGENE METHODS CLARIFIED THE ISSUE? Two excellent models for the study of the potential renotropic actions of PRL have been developed: the PRL [25] and the PRL receptor [10] knockout mice. Recognizing that embryonic gene deletion may not uncover the physiological relevance of a protein that plays a role, along with other unique proteins that may compensate for its deletion, in renal function, these models if properly studied may provide insight into the tubular actions of 23-kDa or 16-kDa PRL. Likewise the recent availability of a modified PRL homolog with antagonistic activity [14] may well explain the confusion of the natiuretic/antinatriuretic or diuretic/ antinatriuretic effects of PRL and may clarify the potential role played by endogenous PRL in mammals that seems predicted by the important osmoregulatory actions of the peptide in submammalian species.

[14]

[15]

[16]

[17]

[18]

[19]

[20]

References [21] [1] Adler, R.A., G.L. Noel, L. Wartofsky, A.G. Frantz. Failure of oral water loading and intravenous hypotonic saline to suppress plasma prolactin in man. J Clin Endocrinol Metab 41: 383–389, 1975. [2] Adler, R.A., V.L. Herzberg, T. Brinck-Johnsen, H.W. Sokol. Increased water excretion in hyperprolactinemic rats. Endocrinology 118: 1519–1524, 1986. [3] Aranda, J., J.C. Rivera, M.C. Jeziorski, J. Riesgo-Escovar, G. Nava, F. López-Barrera, H. Quiróz-Mercado, P. Berger, G. Martínez de la Escalera, C. Clapp. Prolactins are natural inhibitors of angiogenesis in the retina. Invest Ophthalmol Vis Sci 46: 2947– 2953, 2005. [4] Baldocci, R.A., L. Tan, C.S. Nicoll. Processing of rat prolactin by rat tissue explants and serum in vitro. Endocrinology 130: 1653–1659, 1992. [5] Ben-Jonathan, N., J.L. Mershon, D.L. Allen, R.M. Steinmetz. Extrapituitary prolactin: distribution, regulation, functions and clinical aspects. Endocr Rev 17: 639–669, 1996. [6] Bentzien, F., I. Struman, J.F. Martini, J. Matial, R.I. Weiner. Expression of the antiangiogenic 16K hPRL in human HCT116 colon cancer cells inhibits tumor growth in Rag1(-/-) mice. Cancer Res 61: 7356–7362, 2001. [8] Berl, T., N. Brautbar, M. Ben-David, W. Czaczkas, C. Kleeman. Osmotic control of prolactin release and its effect on renal water excretion in man. Kidney Int 10: 158–163, 1976. [9] Bliss, D.J., C.J. Lote. Prolactin release in response to infusion of isotonic or hypertonic saline. J Endocrinol 92: 273–278, 1982. [10] Bole-Feysot, C., V. Goffin, M. Edery, N. Binart, P.A. Kelly. Prolactin (PRL) and its receptor: actions, signal transduction path-

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ways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19: 225–268, 1998. Brown, S.C, E.A. Horgan, L.M. Savage, P.S. Brown. Changes in body water and plasma constituents during bullfrog development: effects of temperature and hormones. J Exp Zool 237: 25–33, 1986. Buckman, M.T., N. Kaminsky, M. Conway, G.T. Peake. Utility of L-dopa and water loading in evaluation of hyperprolactinemia. J Clin Endocrinol Metab 36: 911–919, 1973. Buckman, M.T., G.T. Peake. Osmolar control of prolactin secretion in man. Science 181: 755–777, 1973. Chen, T.J., C.B. Kuo, K.F. Tsai, J.W. Liu, D.Y. Chen, A.M. Walker. Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone. Endocrinology 139: 609–615, 1998. Clapp, C., J. Martial, R.C. Guzman, F. Rentier-Delrue, R.I. Weiner. The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology 133: 1292–1299, 1993. Clapp, C., P.S. Sears, C.S. Nicoll. Binding studies with intact rat prolactin and a 16K fragment of the hormone. Endocrinology 125: 1054–1059, 1989. Corbacho, A., G. Martinez de la Escalera, C. Clapp. Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis. J Endocrinol 173: 219–238, 2002. Cowden, E.A., W.A. Ratcliffe, J.G. Ratcliffe, J.W. Dobbie, A.C. Kennedy. Hyperprolactinemia in renal disease. Clin Endocrinol 9: 241–248, 1978. Emmanouel, D., V.S. Fang, A.I. Katz. Prolactin metabolism in the rat: role of the kidney in degradation of the hormone. Am J Physiol 240: F437–F445, 1981. Evan, A.P., G.C. Palmer, M.S. Lucci, S. Solomon. Prolactininduced stimulation of rat renal adenylate cyclase and autoradiographic localization to the distal nephron. Nephron 18: 266–276, 1977. Freeman, M.E., B. Kanyicska, A. Lerant, G. Nagy. Prolactin: structure, function, and regulation of secretion. Physiol Rev 80: 1523–1631, 2000. Freemark, M., P. Driscoll, R. Maaskant, A. Petryk, P.A. Kelly. Ontogenesis of prolactin receptors in the human fetus in early gestation. J Clin Invest 99: 1107–1117, 1997. Freemark, M., M. Nagano, E. Edery, P.A. Kelly. Prolactin receptor gene expression in the fetal rat. J Endocrinol 144: 285– 292, 1995. Gutierrez de la Berrera, M., B. Trejo, P. Luna-Perez, L. LopezBerrera, G. Martinez de la Escalera, C. Clapp. Opposite association of serum prolactin and survival in patients with colon and rectal carcinomas: influence of preoperative radiotherapy. Dig Dis Sci 51: 54–62, 2006. Horrobin, D.F., P.G. Bursyn, I.J. Lloyd, N. Durkin, A. Lipton, K.L. Muiruri. Actions of prolactin on human renal function. Lancet 2: 352–354, 1971. Horseman, N.D., W. Zhao, E. Montecino-Rodriguez, M. Tanaka, K. Nakashima, S.J. Engle, F. Smith, E. Markoff, K. Dorskind. Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16: 6926–6935, 1997. Kaufman, S., B.J. MacKay, J.Z. Scott. Daily water and electrolyte balance in chronically hyperprolactinemic rats. J Physiol 321: 11–19, 1981. Lockett, M.F., B. Nail. A comparative study of the renal actions of growth and lactogenic hormones in rats. J Physiol 180: 147– 156, 1965. Loretz, C.A., H.A. Bern. Prolactin and osmoregulation in vertebrates. Neuroendocrinology 35: 632–637, 1987.

1282 / Chapter 176 [30] Lucci, M.S., H.H. Bengele, S. Solomon. Suppressive action of prolactin on renal response to volume expansion. Am J Physiol 229: 81–85, 1975. [31] Manzon, L.A. The role of prolactin in fish osmoregulation: a review. Gen Comp Endocrinol 125: 291–310, 2002. [32] Marshall, S., M. Gelato, J. Meites. Serum prolactin levels and prolactin binding activity in adrenals and kidneys of male rats after dehydration, salt loading, and unilateral nephrectomy. Proc Soc Exp Biol Med 149: 185–188, 1975. [33] Mattheij, J.A.M. Evidence against a role for prolactin in osmoregulation in the rat: water balance studies. Endo Res Commun 4: 1–9, 1977. [34] Matthews, B.F. Effects of hormones, placental extracts and hypophysectomy on inulin and para-aminohippurate clearance in the anaesthetized rat. J Physiol 165: 1–9, 1963. [35] Morrissey, S.E., T. Newth, R. Rees, A. Barr, F. Shora, J.F. Laycock. Renal effects of recombinant prolactin in anesthetized rats. Eur J Endocrinol 145: 65–71, 2001. [36] Mountjoy K., E.A. Cowden, J.W. Dobbie, J.G. Ratcliffe. Prolactin receptors in the rat kidney. J Endocr 87: 47–54, 1980. [37] Nicoll, C.S. Physiological actions of prolactin. In: Handbook of Physiology, vol 7, edited by E. Knobil and W.H. Sawyer. Washington DC, American Physiological Society, 1974, pp. 253–292. [38] Nicoll, C.S. Role of prolactin in water and electrolyte balance in vertebrates. In: Current Endocrinology—Prolactin, edited by R.B. Jaffe, Amsterdam, Elsevier, 1981, pp. 127–166. [39] Nicoll, C.S., H. Bern. On the actions of PRL among the vertebrates: Is there a common denominator? In: Lactogenic Hor-

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mones, edited by G.E.W. Wolstenholme and J. Knight, London, Churchill-Livingstone, 1972, pp. 299–337. Ouhtit, A., G. Morel, P.A. Kelly. Visualizaion of gene expression of short and long forms of prolactin receptor in the rat. Endocrinology 133: 135–144, 1993. Peaker, M.J., J.G. Phillips, A. Wright. The effect of prolactin on the secretory activity of the nasal salt-gland of the domestic duck (Ansa platythynchos). J Endocrinol 47: 123–127, 1970. Phillips, I.D., R.V. Anthony, G. Simonetta, J.A. Owens, J. Robinson, I.C. McMillan. Restriction of fetal growth has a differential impact on fetal prolactin and prolactin receptor mRNA expression. J Neuroendocrinol 13: 175–181, 2001. Pippard, C., P.H. Baylis. Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat. J Endocrinol 108: 95–99, 1986. Roberts, J.R., W.H. Danzler. Micropuncture study of avian kidney: effect of prolactin. Am J Physiol 262: 933–937, 1992. Royster, M., P. Driscoll, P.A. Kelly, M. Freemark. The prolactin receptor in the fetal rat: cellular localization of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation. Endocrinology 136: 3892–3900, 1995. Sakai, Y., Y. Hiraoka, M. Ogawa, Y. Yakeuchi, S. Aiso. The prolactin gene is expressed in mouse kidney. Kid Int 55: 833–840, 1999. Stier, C.T., E.A. Cowden, H.G. Friesen, M.E.M. Allison. Prolactin and the rat kidney: a clearance and micropuncture study. Endocrinology 115: 362–367, 1984.

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177 Therapeutic Potential of Adrenomedullin for Pulmonary Hypertension NORITOSHI NAGAYA

pulmonary vascular bed causes pulmonary vasoconstriction, smooth muscle cell proliferation, and in situ thrombosis. Despite therapeutic medical advances including prostacyclin therapy [14], many patients ultimately require heart-lung or lung transplantation. Therefore, a novel therapeutic strategy is desirable for the treatment of pulmonary hypertension, including IPAH. This chapter summarizes the pathophysiological significance and therapeutic potential of AM in pulmonary arterial hypertension.

ABSTRACT Adrenomedullin (AM) is a potent, long-acting vasodilator peptide that was originally isolated from human pheochromocytoma. Immunoreactive AM is detected in plasma and a variety of tissues, including the blood vessels and lungs. There are abundant binding sites for AM in the lungs. AM inhibits the migration and proliferation of vascular smooth muscle cells. These findings suggest that AM plays an important role in the regulation of pulmonary vascular tone and vascular remodeling. In fact, the administration of AM by intravenous or intratracheal delivery has beneficial effects on pulmonary hemodynamics in pulmonary hypertension. Thus, AM supplementation may be a new therapeutic strategy for the treatment of pulmonary hypertension.

DISCOVERY, STRUCTURE, AND SYNTHESIS AM was originally isolated from human pheochromocytoma [10] (see chapter by the discoverer in the Cardiovascular Peptides Section of this Handbook). The peptide consists of 52 amino acids with an intramolecular disulfide bond, sharing slight homology with calcitonin gene–related peptide and amylin (Fig. 1). Immunoreactive AM has subsequently been detected in plasma and a variety of tissues, including the heart and lungs [4]. Plasma AM level is increased in patients with pulmonary hypertension [6], and its level increases in proportion to the severity of this disorder. Circulating levels of AM in the pulmonary artery are significantly higher than in the pulmonary vein [25], suggesting that AM is partially metabolized in the lungs of patients with pulmonary hypertension.

INTRODUCTION Adrenomedullin (AM) is a potent, long-acting vasodilator peptide that was originally isolated from human pheochromocytoma [10]. Immunoreactive AM has subsequently been detected in plasma and in a variety of tissues including the blood vessels and lungs [4]. Abundant binding sites for AM have been reported in the lungs [21]. AM has been shown to inhibit the migration and proliferation of vascular smooth muscle cells [3, 7]. These findings suggest that AM may play an important role in the regulation of pulmonary vascular tone and vascular remodeling [2, 11, 16, 19]. Idiopathic pulmonary arterial hypertension (IPAH) is a rare but lifethreatening disease characterized by progressive pulmonary hypertension, ultimately producing rightventricular failure and death. Endothelial injury in the Handbook of Biologically Active Peptides

RECEPTORS AND THEIR DISTRIBUTION A seven-transmembrane G-protein-coupled receptor, calcitonin receptorlike receptor (CRLR), and

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1284 / Chapter 177 Arg

Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly Cys Cys Asp-Thr-Phe-Gln-Tyr-Ile-Gln-His-Ala-Leu-Lys-Gln-Val-Thr

Phe Gly Thr

Lys Asp Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH2

TABLE 1. Biological Effects of Adrenomedullin for the Treatment of Pulmonary Hypertension. Vasodilation Positive inotropic effect Diuresis and natriuresis Inhibition of endothelial cell apoptosis Inhibition of smooth muscle cell proliferation and migration Inhibition of myocyte hypertrophy and fibroblast proliferation Induction of angiogenesis Anti-inflammation

receptor activity-modifying proteins (RAMPs) have been recognized as integral components of the AM signaling system [13]. CRLR has demonstrated the expression of the transcript predominantly in microvascular endothelial cells and smooth muscle cells. This finding supports the view that CRLR is potentially a major mediator of the effects of AM on the vasculature. The effect of AM on CRLR is modified by RAMP2 and RAMP3. Thus, the pulmonary vasodilator effect of AM is mediated by CRLR/RAMP2 and CRLR/RAMP3 receptors. It should be noted that there are many binding sites for AM in the lung [21] (Fig. 2) and that circulating AM is partially metabolized in the lungs of patients with pulmonary hypertension [25]. These findings raise the possibility that AM plays a role in the regulation of pulmonary vascular tone.

BIOLOGICAL ACTIONS AM has a variety of biological actions, including vasodilation, an inotropic effect, diuresis and natriuresis, and the inhibition of smooth muscle cell proliferation (Table 1). These actions may have beneficial effects in pulmonary hypertension.

Vasodilation AM directly binds to vascular smooth muscle cells to induce vasodilation [5]. The increase in cAMP in smooth muscle cells by AM activates protein kinase A, resulting in the decrease in calcium content in smooth

FIGURE 1. Structure of adrenomedullin (AM). The peptide consists of 52 amino acids with an intramolecular disulfide bond, sharing slight homology with calcitonin gene–related peptide and amylin.

muscle cells. Thus, AM relaxes vascular smooth muscle through a cAMP-protein kinase A–dependent mechanism. On the other hand, Nossaman et al. have shown that AM regulates pulmonary vascular tone in rats through an endothelium-derived nitric oxide– dependent mechanism [19] (Fig. 3). Therefore, the vasodilatory effects of AM appear to be mediated by both cAMP-dependent and nitric oxide–dependent mechanisms. Intralobar arterial infusion of AM causes dose-related decreases in pulmonary vascular resistance under conditions of high pulmonary vascular tone [2]. In humans, intravenous infusion of AM significantly decreased pulmonary vascular resistance [16, 18]. The infusion of AM caused a greater and more prolonged vasodilation than that of an equimolar dose of atrial natriuretic peptide [22]. Thus, AM is one of the most potent endogenous vasodilators in the pulmonary vascular bed.

Inotropic Effect Earlier studies demonstrated that the infusion of AM markedly increased cardiac index and stroke volume index in animals and humans [18, 23]. Considering the strong vasodilator effect of AM, a significant decrease in mean arterial pressure may be responsible for the increased cardiac index during infusion. On the other hand, AM increases cardiac cAMP, which is known to mediate the positive inotropic action of β-adrenergic stimulants. Alternatively, Szokodi et al. have shown that AM produces a positive inotropic action through cAMPindependent mechanisms [24]. These findings suggest that the increases in cardiac index and stroke volume index by AM may be attributable not only to the fall in cardiac afterload but also to a direct, positive, inotropic action of AM.

Diuretic and Natriuretic Effects Systemically administered AM slightly, but significantly, increased urine volume and urinary sodium excretion in dogs with heart failure [12]. Although AM does not significantly increase creatinine clearance, it increases intracellular cAMP levels in the cortical thick ascending limb and distal convoluted tubule dissected

Therapeutic Potential of Adrenomedullin for Pulmonary Hypertension / 1285 Kidney

Lung

Liver

Adrenal gland

Bone marrow

Vasculature

Pituitary Thyroid gland Heart FIGURE 2. Binding sites of AM immediately after intravenous administration of

from rat kidney [1]. These findings suggest that AM can directly inhibit tubular sodium resorption. However, renal responses to AM were smaller than those to atrial natriuretic peptide (ANP), which increases renal blood flow and glomerular filtration rate in a dose-dependent manner. Thus, the renal effects of systemically administered AM may be relatively weak.

125

I-AM in rats.

Thus, AM has protective effects on the vasculature and myocardium, which may be beneficial in patients with pulmonary hypertension.

CLINICAL APPLICATION OF AM FOR PULMONARY HYPERTENSION Intravenous Administration

Other Vasoprotective Effects In vitro studies have shown that AM inhibits the migration and proliferation of vascular smooth muscle cells [3, 7]. AM has anti-apoptotic effects on vascular endothelial cells and cardiomyocytes via the phospatidylinositol 3-kinase (PI3-K)-Akt pathway [8, 20] and it induces angiogenesis through the activation of this pathway [9]. AM is a possible endogenous suppressor of myocyte hypertrophy and fibroblast proliferation.

AM is one of the most potent endogenous vasodilators in the pulmonary vascular bed. However, little information is available regarding the hemodynamic effects of intravenously administered AM in patients with pulmonary hypertension. Accordingly, we examined the hemodynamic and hormonal responses to intravenous infusion of AM (0.05 μg/kg/min) in patients with pulmonary arterial hypertension, including IPAH [16]. The infusion of AM significantly

1286 / Chapter 177 AM

AM

CRLR RAMP 2,3 cAMP

PLC

Endothelial cell

IP3 Ca 2+ eNOS

PI

ER ?

NO

Smooth muscle cell cAMP cGMP Vasodilation

decreased pulmonary vascular resistance by 32% without inducing a marked systemic hypotension, suggesting that AM has potent and selective pulmonary vasodilator activities in patients with pulmonary hypertension. We have shown that administered AM increases plasma cAMP, but not cGMP, in patients with pulmonary hypertension. It is therefore possible that AM relaxes vascular smooth muscle mainly through a cAMP-protein kinase A–dependent mechanism. Intravenous infusion of AM markedly increased cardiac index in patients with pulmonary hypertension, consistent with our previous results in patients with left-sided heart failure [18]. Considering the strong vasodilator activity of AM in the systemic and pulmonary vasculature, the significant decrease in cardiac afterload may be responsible for the increased cardiac index. On the other hand, AM produces a positive inotropic action through cAMP-independent mechanisms. These findings suggest that the increase in cardiac index may be attributable not only to a fall in cardiac afterload but also to a direct positive inotropic action of AM.

Inhalation Therapy The goals of vasodilator therapy for patients with IPAH are to reduce pulmonary vascular resistance without producing systemic hypotension and to improve the quality of life and survival. In clinical settings, inhalation therapy would be simpler, noninvasive, and more convenient than continuous intravenous infusion

FIGURE 3. Signaling pathway of AM in vascular endothelial cells and smooth muscle cells. AM induces vasodilation via cAMP-dependent and nitric oxide (NO)-dependent mechanisms.

therapy. In addition, inhalant application of vasodilators does not impair gas exchange because the ventilation-matched deposition of drug in the alveoli causes pulmonary vasodilation matched to ventilated areas. Thus, we examined the effect of inhaled AM on pulmonary hypertension. Inhalation of AM significantly decreased pulmonary vascular resistance in patients with pulmonary hypertension, whereas it did not alter systemic arterial pressure or systemic vascular resistance [15]. The ratio of pulmonary vascular resistance to systemic vascular resistance was significantly reduced by AM inhalation. These results suggest that inhaled AM improves hemodynamics with pulmonary selectivity. We examined the long-term effects of inhaled AM in monocrotaline (MCT)-induced pulmonary hypertension in rats [17]. AM or saline was inhaled as an aerosol using an ultrasonic nebulizer, for 30 min, four times a day. Repeated inhalation of AM for 3 weeks markedly decreased pulmonary vascular resistance in MCT rats. The inhalation of AM also inhibited an increase in the medial wall thickness of peripheral pulmonary arteries of MCT rats. Given the known potent vasoprotective effects of AM such as vasodilation and inhibition of smooth muscle cell migration and proliferation, it is interesting to speculate that AM trapped in the bronchial epithelium or alveoli leaks to the pulmonary arteries to maintain pulmonary vascular integrity in MCT rats. Thus, treatment with aerosolized AM may be an alternative approach for severe pulmonary hypertension that is refractory to conventional therapy.

Therapeutic Potential of Adrenomedullin for Pulmonary Hypertension / 1287

CONCLUSION This chapter describes the pathophysiological significance of AM and its therapeutic potential in pulmonary hypertension. Baseline plasma AM is significantly higher in these patients. Nevertheless, exogenously administered AM at a pharmacological level induces hemodynamic improvement. The beneficial effects of AM are mediated by vasodilation, positive inotrophic effects, diuresis, and inhibition of smooth muscle cell proliferation. Thus, AM supplementation may be a new therapeutic strategy for the treatment of pulmonary hypertension. Further studies are necessary to examine whether long-term administration of AM improves survival in such patients.

References [1] Edwards RM, Trizna W, Stack E, Aiyar N. Effect of adrenomedullin on cAMP levels along the rat nephron: comparison with CGRP. Am J Physiol 1996; 271: F895–F899. [2] Heaton J, Lin B, Chang JK, Steinberg S, Hyman A, Lippton H. Pulmonary vasodilation to adrenomedullin: a novel peptide in humans. Am J Physiol 1995; 268: H2211–2215. [3] Horio T, Kohno M, Kano H, Ikeda M, Yasunari K, Yokokawa K, et al. Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells. Circ Res 1995; 77: 660–664. [4] Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 1994; 338: 6–10. [5] Ishizaka Y, Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, et al. Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun 1994; 200: 642–646. [6] Kakishita M, Nishikimi T, Okano Y, Satoh T, Kyotani S, Nagaya N, et al. Increased plasma levels of adrenomedullin in patients with pulmonary hypertension. Clin Sci (Lond) 1999; 96: 33– 39. [7] Kano H, Kohno M, Yasunari K, Yokokawa K, Horio T, Ikeda M, et al. Adrenomedullin as a novel antiproliferative factor of vascular smooth muscle cells. J Hypertens 14: 209–213. [8] Kim W, Moon SO, Sung MJ, Kim SH, Lee S, Kim HJ, et al. Protective effect of adrenomedullin in mannitol-induced apoptosis. Apoptosis 1996; 7: 527–535. [9] Kim W, Moon SO, Sung MJ, Kim SH, Lee S, So JN, et al. Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells. FASEB J 2003; 17: 1937–1939. [10] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993; 192: 553–560.

[11] Lippton H, Chang J-K, Hao Q, Summer W, Hyman AL. Adrenomedullin dilates the pulmonary vascular bed in vivo. J Appl Physiol 1994; 76: 2154–2156. [12] Majid DSA, Kadowitz PJ, Coy DH, Navar LG. Renal responses to intra-arterial administration of adrenomedullin in dogs. Am J Physiol 1996; 270: F200–205. [13] McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; 393: 333– 339. [14] McLaughlin VV, Genthner DE, Panella MM, Rich S. Reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension. N Engl J Med 1998; 338: 273–277. [15] Nagaya N, Kyotani S, Uematsu M, Ueno K, Oya H, Nakanishi N, et al. Effects of adrenomedullin inhalation on hemodynamics and exercise capacity in patients with idiopathic pulmonary arterial hypertension. Circulation 2004; 109: 351–356. [16] Nagaya N, Nishikimi T, Uematsu M, Satoh T, Oya H, Kyotani S, et al. Haemodynamic and hormonal effects of adrenomedullin in patients with pulmonary hypertension. Heart 2000; 84: 653– 658. [17] Nagaya N, Okumura H, Uematsu M, Shimizu W, Ono F, Shirai M, et al. Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats. Am J Physiol Heart Circ Physiol 2003; 285: H2125–2131. [18] Nagaya N, Satoh T, Nishikimi T, Uematsu M, Furuichi S, Sakamaki F, et al. Hemodynamic, renal, and hormonal effects of adrenomedullin infusion in patients with congestive heart failure. Circulation 2000; 101: 498–503. [19] Nossaman BD, Feng CJ, Kaye AD, DeWitt B, Coy DH, Murphy WA, et al. Pulmonary vasodilator responses to adrenomedullin are reduced by NOS inhibitors in rats but not in cats. Am J Physiol 1996; 270: L782–789. [20] Okumura H, Nagaya N, Itoh T, Okano I, Hino J, Mori K, et al. Adrenomedullin infusion attenuates myocardial ischemia/ reperfusion injury through the phosphatidylinositol 3-kinase/ Akt-dependent pathway. Circulation 2004; 109: 242–248. [21] Owji AA, Smith DM, Coppock HA, Morgan DG, Bhogal R, Ghatei MA, et al. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 1995; 136: 2127–2134. [22] Oya H, Nagaya N, Furuichi S, Nishikimi T, Ueno K, Nakanishi N, et al. Comparison of intravenous adrenomedullin with atrial natriuretic peptide in patients with congestive heart failure. Am J Cardiol 2000; 86: 94–98. [23] Parkes DG, May CN. Direct cardiac and vascular actions of adrenomedullin in conscious sheep. Br J Pharmacol 1997; 120: 1179–1185. [24] Szokodi I, Kinnunen P, Tavi P, Weckstrom M, Toth M, Ruskoaho H. Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide. Circulation 1998; 97: 1062–1070. [25] Yoshibayashi M, Kamiya T, Kitamura K, Saito Y, Kangawa K, Nishikimi T, et al. Plasma levels of adrenomedullin in primary and secondary pulmonary hypertension in patients <20 years of age. Am J Cardiol 1997; 79: 1556–1558.

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178 Endothelin in the Airways BENGT W. GRANSTRÖM AND LARS EDVINSSON

the range of 1–30 nM, depending on species. Among the highest tissue levels of endothelin-like immunoreactivity measured in any tissue have been observed in the lung of the pig [26]. ET-1 has the ability to mimic the signal features of asthma, chronic obstructive pulmonary disease (COPD), and other respiratory diseases. Most experimental studies have been performed on the trachea or central airways because these are more readily available and the muscle preparations can be isolated more easily. However, the contraction of smooth muscle cells in peripheral airways may be more important both in normal conditions and in diseases. Under pathological conditions, upregulation of endothelin synthesis and release can be shown, suggesting a mediator role [32]. The contractile effect on bronchial airway smooth muscle is mediated via two subtypes of receptors, ETA and ETB. The relative proportion of ETA and ETB receptors varies considerably among species. In rat and mice the proportion seems to be approximately equal, whereas in humans, bronchial smooth muscle cells contain primarily ETB receptors [16]. Another important factor is that there are significant regional differences in subreceptor proportions within the lung [14]. The first studies of ET-1 in vessels observed that prior to contraction an endothelin-mediated relaxation was seen, indicating an important role in the regulation of vascular tonus. The same type of reaction can be seen in airways with intact epithelium. Relaxation within the airways seems to be mediated by ETB receptors located on the epithelial cells and mediated via nitric oxide (NO) release, because the response can be abolished by l-NMMA, an NO synthase inhibitor. Considering the vascular system in the lung, ET-1 has a pathogenic role in pulmonary arterial hypertension (PAH), and recent clinical studies have shown that the blockade of the ET receptors by bosentan (a combined

ABSTRACT After the discovery of endothelins, it was soon obvious that these neuropeptides have an important role both in the vascular system and in the airways. Endothelin production and receptors are found within the lung where endothelin 1 (ET-1) acts locally. Furthermore, ET-1 is found in bronchial alveolar lavage (BAL) and 80% of circulating plasma ET-1 is cleared by its first pass in the lung. Hypoxia and sheer stress stimulate to increased production and release, leading to increased pulmonary vascular resistance. Endothelin antagonists have been used successfully during cardiopulmonary operations. Inflammatory states such as bronchial asthma are associated with elevated ET-1 levels in BAL. During prolonged inflammatory conditions, the mitogenic effect of ET-1 can result in remodeling of the airways and pulmonary arteries, which has important pathophysiological consequences.

INTRODUCTION Endothelin 1 (ET-1) is known as an extremely potent smooth muscle spasmogen that has effects on vasomotor and bronchomotor tone as well as having mitogenic and inflammatory properties, not only in the vascular system but also in the airways. Purification, cloning, and initial pharmacological characterization was made by Yanagisawa and colleagues [36]. Early questions concerned the role of ET-1 as a mediator of a number of vascular diseases such as systemic and pulmonary hypertension, coronary vasospasm, Raynaud’s disease, preeclampsia, and other cardiac diseases. In most of these conditions the venous plasma level of ET-1 is raised. When compared with other known airway spasmogens such as histamine or leukotrienes, ET-1 is extremely potent, with a median effective concentration (EC50) in Handbook of Biologically Active Peptides

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1290 / Chapter 178 ETA and ETB receptor antagonist) improves pulmonary hemodynamics, exercise capacity, and survival [23]. ET-1 has a potent mitogenic action that may be relevant to features of lung disease such as airway smooth muscle cell and fibroblast hyperplasia. It can act as a mitogen alone, but in the human epithelium it is more important as a co-mitogen together with factors such as epidermal growth factor (EGF) [25]. The importance of ET-1 as a growth factor and in tissue differentiation has been clarified with gene knockout mice [6]. Furthermore, ETB receptors in the lung are considered to be the main site for the elimination of endothelin from the general circulation.

SYNTHESIS AND ELIMINATION OF ET-1 The major sources of ET-1 formation in the lung are the epithelium and vascular endothelium; inflammatory cells contribute less. ET-1 is not stored in these cells but production starts within minutes in response to sheer stress, hypoxia, or ischemia [20]. The synthesis of ET-1 begins with endopeptidase-induced cleavage of the 212-amino-acid precursor prepro ET-1 to a 38amino-acid residue peptide known as big ET-1, which is finally cleaved via endothelin-converting enzyme (ECE) to the 21-amino-acid peptide ET-1. Release seems preferably to occur abluminally, and the effect is local as a paracrine factor although significant amounts can be measured in bronchial alveolar lavage (BAL) as well as in plasma. Approximately 80% of a bolus injection of ET-1 in isolated perfused rat lung is retained by ETB receptors in the lung after one passage [12]. The plasma half-life of ET-1 is about 4–7 min [21].

ENDOTHELIN RECEPTOR SUBTYPES AND LOCALIZATION Three isoforms of endothelin receptors have been demonstrated (ETA, ETB, and ETC), where ETA and ETB are the most important once in humans. The genes map to different chromosomes, but they are thought to originate from the same ancestral gene and cloning has shown 60% homology. Subreceptor density and nerve fiber innervations differ between peripheral and proximal airways. The innervations of peripheral airways are sparser in small than in large airways. To make a proper appraisal of study results, it is important to take into consideration the species that is involved. Thus, human and pig peripheral lung tissue contain a mixture of these ET receptor subtypes, with approximately 40% being ETA receptors [15]. Thus, the field of endothelin research in the respiratory system may be hampered by the wide

range of tissue-specific expression. In general, it seems that ETB receptors predominate in human bronchi, whereas ETA receptors play a more important role in smaller laboratory animals such as mouse and rat [17]. The two subreceptors ETA and ETB are present in the airways on cholinergic nerves, but their influence on cholinergic function appears to be speciesdependent [9]. Also ET-1 can induce the potentiation of electrical-field-stimulation-evoked cholinergic contractions [10]. In the pulmonary arteries, the constriction is normally mediated through ETA receptors situated on the smooth muscle cells. In the lung, there is a mixture of ETA and ETB receptors on the smooth muscle cells mediating constriction, whereas ETB receptors on endothelial or epithelial cells mediate dilatation via the release of NO and prostacyclin (PGI2). The ETB receptors that mediate relaxation are termed ETB1. The ETB receptors mediating constriction, both in vessels and in bronchi, are termed ETB2, suggesting a role both in normal physiology and in pathophysiological processes. ETA and ETB receptors have been identified in lung tissue of humans and animals [1, 3]. In rat airways, there are equal proportions of contractile ETA and ETB receptors [19].

PATHOPHYSIOLOGICAL ROLE Changes in ET-1 or in endothelin receptor levels during pathological situations have been observed in many studies. Changes of transcription and translation mechanisms or clearance have been observed. One of the most common and important respiratory-tract pathogens in infants and young children is respiratory syncytial virus (RSV). The airway epithelial cells are the prime target for this infection, which can cause severe obstruction. Studies of ET-1 release by a RSV-infected bronchial epithelial cell line have shown enhanced production of ET-1 because ET-1 mRNA was increased significantly [29]. Bronchial asthma is considered to be an inflammatory disease with a combination of bronchospasm, edema, and hypersecretion during an acute attack. In asthmatics without bronchoconstriction, ET-1 in sputum was found unaltered, but it was apparent that ET-1 was produced or released locally within the respiratory tract because the concentration was more than four times higher in sputum than in plasma [5]. Levels of ET-1 were found to be elevated in broncial alveolar lavage fluid (BALF) [22] and in bronchial biopsies [27, 35] of asthmatic patients without steroid

Endothelin in the Airways / 1291 treatment, and, interestingly, treatment with oral glucocorticoids reduced ET-1 by 75% [22]. Pro-inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) have been found to promote an increase in the production of ET-1 in cultured guinea pig tracheal airway epithelial cells [37] and in murine tracheal segments. IL-1β upregulates the mRNA expression of ET-1 in mouse airways with intact epithelium, but at the same time causes the attenuation of ETB receptor expression [38]. After treatment of human temporal arteries with IL-1β, a pro-inflammatory cytokine, both maximal contraction and potency mediated by ETB receptors were increased [34]. Pretreatment with bosentan, a nonselective endothelin receptor antagonist, inhibited the eosinophilic inflammatory response to Sephadex administration in BAL fluid and in lung tissue [11]. Plasma endothelin levels in humans increased significantly after smoking [2]. A very important outcome of a long-standing inflammatory process in the airways, such as in bronchial asthma, is airway remodeling [28]. This consists of increased airway wall thickness due to the hypertrophy of the smooth muscle cell layer, fibroblast hyperplasia, elevation of the number of mucous glands, and augmented cholinergic neurotransmission. Soon after the discovery of endothelin, it was postulated that endothelins act in a paracrine/autocrine manner in growth regulation. In addition, they may cause increased secretion from both mucous and serous cells [31]. ET-1 has been shown to be a mitogen in ovine airway smooth muscle cells [13], but to a lesser extent than epidermal growth factor (EGF), for cultured guinea pig airway smooth muscle [33]. A study of human airway smooth muscle cell proliferation showed that ET-1 mainly potentiates mitogenesis induced by EGF, apparently via an ETA receptor–mediated mechanism, suggesting a potentiating role for other mitogens, and that it contributes to the airway smooth muscle hyperplasia associated with chronic severe asthma [25]. ET-1induced mitogenesis of cultured ovine airway smooth muscle cells suggests that both ETA and ETB receptor subtypes are involved in this process [4]. Smoking is one of the most important health issues in the world [8]. Cigarette smoke causes rapid cell proliferation in small airways and in associated pulmonary arteries [30]. Cigarette smoke–induced mitogenesis in rat airways can be blocked by BQ-610, a selective ETA receptor antagonist [7]. The importance of ET-1 as a growth factor and in tissue differentiation has been clarified in gene knockout mice [6]. Considering the vascular system in the lung, ET-1 has a pathogenic role in PAH, and recent clinical studies have shown that blockade of the ET receptors by bosentan improves pulmonary hemodynamics, exercise capacity, and survival [23].

We have shown that bronchial biopsies from patients with asthma and chronic airway obstruction demonstrate significantly higher levels of endothelin ETB receptor mRNA than endothelin ETA receptor mRNA [24]. Upregulation of bronchial constrictor endothelin receptors in airway smooth muscle cells may contribute to hyperreactivity during airway inflammation. Recently, we tested this hypothesis by quantitative endothelin receptor mRNA analysis and functional responses in ring segments of rat trachea and bronchi after 24 h of Sephadex-induced pulmonary inflammation [18]. We observed a significant increase in endothelin ETB receptor mRNA expression in bronchial smooth muscle cells, and functional myograph studies revealed an increase of the maximum contractile effects mediated by ETB receptors in bronchial, but not tracheal, ring segments. These results imply a role for ETB receptors in airway hyperreactivity during airway inflammation [18]. Inflammation induced by the pro-inflammatory cytokine IL-1β in the human temporal artery caused increased maximal contraction and potency to sarafotoxin 6c (S6c, a specific ETB agonist) but no change in the ETA/ETB receptor mRNA ratio [34]. The suggested explanation was that IL-1β might further stimulate translation of the mRNA to active receptors. Because asthmatics exhibit hyperreactivity to ET-1 and have elevated levels of ET-1 in BALF, altered levels of endothelin receptors and changes in phenotype distribution may contribute to the disease progression. Interestingly, the elevated levels of ET-1 in the lung are reduced during therapeutically induced remission [22]. If, as we hypothesize, receptor dynamics also play an important role, then the proper regulation of their expression may be another way to alleviate the patient’s symptoms.

References [1] Adner, M., Cardell, L. O., Sjoberg, T., Ottosson, A., Edvinsson, L.: Contractile endothelin-B (ETB) receptors in human small bronchi. Eur Respir J 1996, 9(2):351–355. [2] Borissova, A. M., Tankova, T., Kirilov, G., Dakovska, L., Krivoshiev, S.: The effect of smoking on peripheral insulin sensitivity and plasma endothelin level. Diabetes Metab 2004, 30(2):147–152. [3] Cardell, L. O., Uddman, R., Edvinsson, L.: Analysis of endothelin-1-induced contractions of guinea-pig trachea, pulmonary veins and different types of pulmonary arteries. Acta Physiol Scand 1990, 139(1):103–111. [4] Carratu, P., Scuri, M., Styblo, J. L., Wanner, A., Glassberg, M. K.: ET-1 induces mitogenesis in ovine airway smooth muscle cells via ETA and ETB receptors. Am J Physiol 1997, 272(5 Pt 1): L1021–1024. [5] Chalmers, G. W., Thomson, L., Macleod, K. J., Dagg, K. D., McGinn, B. J., McSharry, C., Patel, K. R., Thomson, N. C.: Endothelin-1 levels in induced sputum samples from asthmatic and normal subjects. Thorax 1997, 52(7):625–627.

1292 / Chapter 178 [6] Clouthier, D. E., Hosoda, K., Richardson, J. A., Williams, S. C., Yanagisawa, H., Kuwaki, T., Kumada, M., Hammer, R. E., Yanagisawa, M.: Cranial and cardiac neural crest defects in endothelinA receptor-deficient mice. Development 1998, 125(5):813–824. [7] Dadmanesh, F., Wright, J. L.: Endothelin-A receptor antagonist BQ-610 blocks cigarette smoke-induced mitogenesis in rat airways and vessels. Am J Physiol 1997, 272(4 Pt 1):L614–618. [8] Ezzati, M., Lopez, A. D.: Estimates of global mortality attributable to smoking in 2000. Lancet 2003, 362(9387):847–852. [9] Fernandes, L. B., D’Aprile, A. C., Henry, P. J., Spalding, L. J., Pudney, C. J., Goldie, R. G.: Detection of endothelin receptors in rat and guinea-pig airway nerves by immunohistochemistry. Pulm Pharmacol Ther 1999, 12(5):313–323. [10] Fernandes, L. B., Henry, P. J., Goldie, R. G.: Endothelin-1 potentiates cholinergic nerve-mediated contraction in human isolated bronchus. Eur Respir J 1999, 14(2):439–442. [11] Finsnes, F., Skjonsberg, O. H., Tonnessen, T., Naess, O., Lyberg, T., Christensen, G.: Endothelin production and effects of endothelin antagonism during experimental airway inflammation. Am J Respir Crit Care Med 1997, 155(4):1404–1412. [12] Fukuroda, T., Fujikawa, T., Ozaki, S., Ishikawa, K., Yano, M., Nishikibe, M.: Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Commun 1994, 199(3):1461– 1465. [13] Glassberg, M. K., Ergul, A., Wanner, A., Puett, D.: Endothelin-1 promotes mitogenesis in airway smooth muscle cells. Am J Respir Cell Mol Biol 1994, 10(3):316–321. [14] Goldie, R. G., D’Aprile, A. C., Cvetkovski, R., Rigby, P. J., Henry, P. J.: Influence of regional differences in ETA and ETB receptor subtype proportions on endothelin-1-induced contractions in porcine isolated trachea and bronchus. Br J Pharmacol 1996, 117(4):736–742. [15] Goldie, R. G., D’Aprile, A. C., Self, G. J., Rigby, P. J., Henry, P. J.: The distribution and density of receptor subtypes for endothelin-1 in peripheral lung of the rat, guinea-pig and pig. Br J Pharmacol 1996, 117(4):729–735. [16] Goldie, R. G., Henry, P. J., Knott, P. G., Self, G. J., Luttmann, M. A., Hay, D. W.: Endothelin-1 receptor density, distribution, and function in human isolated asthmatic airways. Am J Respir Crit Care Med 1995, 152(5): 1653–1658. [17] Goldie, R. G., Knott, P. G., Carr, M. J., Hay, D. W., Henry, P. J.: The endothelins in the pulmonary system. Pulm Pharmacol 1996, 9(2):69–93. [18] Granstrom, B. W., Xu, C. B., Nilsson, E., Bengtsson, U. H., Edvinsson, L.: Up-regulation of endothelin receptor function and mRNA expression in airway smooth muscle cells following Sephadex-induced airway inflammation. Basic Clin Pharmacol Toxicol 2004, 95(1):43–48. [19] Henry, P. J.: Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems. Br J Pharmacol 1993, 110(1):435–441. [20] Kuchan, M. J., Frangos, J. A.: Shear stress regulates endothelin1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol 1993, 264(1 Pt 2):H150–156. [21] Levin, E. R.: Endothelins. N Engl J Med 1995, 333(6):356–363. [22] Mattoli, S., Soloperto, M., Marini, M., Fasoli, A.: Levels of endothelin in the bronchoalveolar lavage fluid of patients with symptomatic asthma and reversible airflow obstruction. J Allergy Clin Immunol 1991, 88(3 Pt 1):376–384. [23] McLaughlin, V. V., Sitbon, O., Badesch, D. B., Barst, R. J., Black, C., Galie, N., Rainisio, M., Simonneau, G., Rubin, L. J.: Survival

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with first-line bosentan in patients with primary pulmonary hypertension. Eur Respir J 2005, 25(2):244–249. Möller, S., Uddman, R., Granstrom, B., Edvinsson, L.: Altered ratio of endothelin ET(A)- and ET(B) receptor mRNA in bronchial biopsies from patients with asthma and chronic airway obstruction. Eur J Pharmacol 1999, 365(1):R1–3. Panettieri, R. A., Jr., Goldie, R. G., Rigby, P. J., Eszterhas, A. J., Hay, D. W.: Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon. Br J Pharmacol 1996, 118(1):191–197. Pernow, J., Hemsen, A., Lundberg, J. M.: Tissue specific distribution, clearance and vascular effects of endothelin in the pig. Biochem Biophys Res Commun 1989, 161(2):647–653. Redington, A. E., Springall, D. R., Ghatei, M. A., Lau, L. C., Bloom, S. R., Holgate, S. T., Polak, J. M., Howarth, P. H.: Endothelin in bronchoalveolar lavage fluid and its relation to airflow obstruction in asthma. Am J Respir Crit Care Med 1995, 151(4):1034–1039. Rennard, S. I.: Repair mechanisms in asthma. J Allergy Clin Immunol 1996, 98(6 Pt 2):S278–286. Samransamruajkit, R., Gollapudi, S., Kim, C. H., Gupta, S., Nussbaum, E.: Modulation of endothelin-1 expression in pulmonary epithelial cell line (A549) after exposure to RSV. Int J Mol Med 2000, 6(1):101–105. Sekhon, H. S., Wright, J. L., Churg, A.: Cigarette smoke causes rapid cell proliferation in small airways and associated pulmonary arteries. Am J Physiol 1994, 267(5 Pt 1):L557–563. Shimura, S., Ishihara, H., Satoh, M., Masuda, T., Nagaki, N., Sasaki, H., Takishima, T.: Endothelin regulation of mucus glycoprotein secretion from feline tracheal submucosal glands. Am J Physiol 1992, 262(2 Pt 1):L208–213. Springall, D. R., Howarth, P. H., Counihan, H., Djukanovic, R., Holgate, S. T., Polak, J. M.: Endothelin immunoreactivity of airway epithelium in asthmatic patients. Lancet 1991, 337(8743):697–701. Stewart, A. G., Grigoriadis, G., Harris, T.: Mitogenic actions of endothelin-1 and epidermal growth factor in cultured airway smooth muscle. Clin Exp Pharmacol Physiol 1994, 21(4):277– 285. White, L. R., Leseth, K. H., Moller, S., Juul, R., Adner, M., Cappelen, J., Bovim, G., Aasly, J., Edvinsson, L.: Interleukin1beta potentiates endothelin ET(B) receptor-mediated contraction in cultured segments of human temporal artery. Regul Pept 1999, 81(1–3):89–95. Vittori, E., Marini, M., Fasoli, A., De Franchis, R., Mattoli, S.: Increased expression of endothelin in bronchial epithelial cells of asthmatic patients and effect of corticosteroids. Am Rev Respir Dis 1992, 146(5 Pt 1):1320–1325. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T. X., Sakakibara, S., Goto, K. et al: Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci USA 1988, 85(18):6964–6967. Yang, Q., Laporte, J., Battistini, B., Sirois, P.: Effects of dexamethasone on the basal and cytokine-stimulated release of endothelin-1 from guinea-pig cultured tracheal epithelial cells. Can J Physiol Pharmacol 1997, 75(6):576–581. Zhang, Y., Adner, M., Cardell, L. O.: Interleukin-1beta attenuates endothelin B receptor-mediated airway contractions in a murine in vitro model of asthma: roles of endothelin converting enzyme and mitogen-activated protein kinase pathways. Clin Exp Allergy 2004, 34(9):1480–1487.

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brain stem is of major interest. Many neurons within the solitary nucleus, the dorsal motor vagal nucleus, the nucleus ambiguus, the ventrolateral medulla, and the caudal raphe nuclei express PACAP-like immunoreactivity. PACAP-rich nerve fibers are present in the area postrema, the solitary nucleus, and the dorsal vagal and raphe pallidus nuclei [13]. Autonomic ganglia important for the parasympathetic innervation of peripheral organs, such as the sphenopalatine and otic ganglia, do express PACAP [28]. Within the lung, PACAPcontaining nerve fibers have been detected in the respiratory tract of various mammals, such as rats, guinea pigs, pigs, sheep, monkeys, and humans [12, 27, 28]. In the human upper-respiratory tract, PACAP immunoreactivity is found in nerve fibers in close association with blood vessels and glandular structures [12]. In bronchi of guinea pigs and rats, PACAP-containing nerve fibers are found beneath the epithelium, among bundles of smooth muscle cells of the bronchial wall, around blood vessels, and in seromucous glands [27, 28]. Nerve fibers with PACAP-like immunoreactivity are also observed close to small bronchioles [28]. PACAP is present in cholinergic nerve fibers that do co-express the closely related peptide VIP (vasoactive intestinal peptide) and the majority of VIP-containing nerve fibers within the lung expresses PACAP. Taken together, the expression data for PACAP within lungs derived from different mammals suggest a role for this peptide in secretory activities and in the regulation of smooth muscle tone of bronchi and pulmonary blood vessels.

ABSTRACT In this chapter, we review the expression of pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the respiratory system. Here, we focus on experiments addressing the role of PACAP in relaxation of airway and pulmonary vascular smooth muscle (and pulmonary arteries), as well as in mucus production and modulation of airway inflammatory processes. Animal models lacking PACAP or its specific type I receptor have been developed and impressively demonstrate the vital importance of PACAP-induced signaling for pulmonary function in vivo. Most likely, these findings will stimulate the development of potent PACAP receptor agonists or PACAP analogs for the treatment of asthma and pulmonary hypertension.

GENE STRUCTURE AND DISTRIBUTION OF THE PRECURSOR mRNA The tissue distribution of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA and protein has been extensively studied in various species (for a review see [2, 29]). [For more general information regarding PACAP and its receptors, please refer to Chapter 94 within the Brain Peptides Section of this book.] It turned out that the patterns of PACAP mRNA expression are pretty well conserved across different mammals. In general, PACAP mRNA expression is found to be higher in the central nervous system than in the peripheral organs. Within the central nervous system, the highest levels of PACAP mRNA are present in the neurons of hypothalamic nuclei, a finding that is in line with the proposed hypophysiotrophic role of PACAP. Apart from influencing pituitary function, PACAP seems to be involved in the central regulation of visceral organs, such as the lung, heart, and intestine. With regard to this aspect, PACAP mRNA expression in the Handbook of Biologically Active Peptides

RECEPTORS Two subsets of PACAP receptors have been identified: PACAP type II receptors (VPAC1-R and VPAC2-R), which show equal high binding affinity for PACAP and its close relative VIP, and PACAP-specific PACAP type I receptors (PAC1-R), which bind PACAP with a

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1294 / Chapter 179 thousandfold-higher affinity than VIP (for a review see [2, 29]). Extremely high levels of VPAC1-R mRNA can be detected in the lung, whereas moderate levels are found in the liver and gastrointestinal tract and only weak expression is seen in the thymus and brain of rats. VPAC2-R mRNA is expressed in insulin-producing cells of the pancreas, and in the lung, brain, stomach, and colon, but it is absent in the liver, kidney, and lung. Extremely strong expression of the PAC1-R is seen within the central nervous system. Here, the receptor is detected in neurons of the olfactory bulb, the dentate gyrus of the hippocampus, and the hypothalamus and several brain stem areas that are important for the regulation of visceral organs, such as the area postrema, the dorsal motor nucleus of the vagal nerve, the raphe nucleus, and pontine nuclei. Notably, these brain stem regions also express PACAP mRNA [13]. In the peripheral organs PAC1-R mRNA is detected in the adrenal glands, pituitary, ovary, heart, lung, testis, thymus, spleen, and uterus. PAC1-R mRNA levels in these organs reach approximately 5–15% of the PAC1-R mRNA levels present in brain. With reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, all three PACAP receptors, VPAC1-R, VPAC2-R, and PAC1-R, were shown to be expressed in human lung [3]. However, on the immunohistochemical level it was not possible to detect a signal for PAC1-R [4]. Conversely, VPAC1 and VPAC2 proteins were present in lymphocytes during the extravasation process from the blood vessels and macrophages. Some, but not all, smooth muscle cells in vascular walls expressed VPAC1 and VPAC2 proteins [4]. Using nonradioactive in situ hybridization, VPAC2 mRNA expression was studied in human lung [11]. Epithelial cells in extra- and intrapulmonary bronchi as well as in small bronchioles expressed VPAC2 mRNA. In addition, VPAC2 mRNA was detected in submucosal gland cells, alveolar macrophages, and peribronchial clusters of immune cells. VPAC2 mRNA was absent in airway or vascular smooth muscle cells and in endothelial cells of pulmonary vessels. This result is in contrast to published immunohistochemical data [4] and to recent findings demonstrating VPAC1 and VPAC2 mRNA and protein expression in cultured human primary pulmonary artery smooth muscle cells [20]. In summary, all three PACAP receptors, VPAC1-R, VPAC2-R, and PAC1-R, are present in the lung; the exact cellular localization of PAC1-R remains to be determined.

BIOLOGICAL ACTIONS WITHIN THE LUNG In line with the expression patterns of PACAP and its receptors in the lung, the biological effects of PACAP

within this organ fall into four different categories: (1) relaxant effects on airway smooth muscle cells, (2) relaxant activities on pulmonary arteries, (3) effects on airway mucous secretion, and (4) modulation of inflammatory cell activity. (Please also refer to Chapter 181 on VIP in this Section of the book.)

Relaxant Effects on Airway Smooth Muscle Cells During the last 13 years, the effects of PACAP on airway smooth muscle cells have been extensively studied in various species (for a review see [14, 23, 29]). In vitro studies using precontracted tracheal strips from guinea pigs demonstrated that PACAP38 was one-third as potent and 70% as efficacious as VIP in producing relaxation of tracheal smooth muscle cells. Interestingly, the effect of PACAP38 in relaxing smooth muscle lasted much longer than that of PACAP27 and VIP. This sustained effect of PACAP38 might be due to its greater resistance to enzymatic degradation by neutral endopeptidases. In line with this argument is the finding that the addition of protease inhibitors led to an enhancement of VIPinduced, but not PACAP38-induced, relaxation of precontracted tracheal rings. With regard to the mechanism underlying PACAP’s relaxant effect on smooth muscle cells, several hypotheses exist, depending on the experimental condition. On the one hand, it has been suggested that PACAP38 and PACAP27 lead to cAMPmediated activation of calcium-dependent potassium channels, because charybdotoxin, a potent calciumdependent potassium-channel blocker, partly inhibited the relaxation of tracheal smooth muscle cells induced by both PACAPs. On the other hand, with epithelialintact and -denuded tracheal strips, it could be demonstrated that major parts of the PACAP effect depend on the presence of the airway epithelium but do not involve nitric oxide synthesis. With smooth muscle preparations from the trachea and main bronchi of guinea pigs, the effect of PACAP38 on muscle contractions evoked by electric field stimulation was examined to a greater extent [27]. From these experiments, it has been suggested that within the trachea, PACAP38 suppressed smooth muscle contraction prejunctionally, presumably by reducing the release of acetylcholine from vagal nerve termini innervating the smooth muscle bundles. In the main bronchi, electric field stimulation in the presence of atropine and guanethidine led to smooth muscle contractions that were induced by the excitatory nonadrenergic noncholinergic (eNANC) component of the vagal nerve. At concentrations in the nanomolar range, PACAP38 was able to block these eNANC-induced contractions prejunctionally in the main bronchi. Assuming that this prejunctional mechanism of PACAP’s action on smooth muscle cells is true, we must conclude that the PACAP receptors mediating this effect are most

PACAP’s Role in Pulmonary Function / 1295 likely expressed on nerve termini but not on smooth muscle cells in the bronchial walls. Taking into account all the studies, PACAP receptors within the nerve termini innervating smooth muscle bundles as well as PACAP receptors within the airway epithelium may contribute to smooth muscle relaxation. Interestingly, PACAP38 also leads to a concentration-dependent relaxation of precontracted bronchial smooth muscle segments from humans. Whereas PACAP38 was effective in all bronchial segments tested, VIP failed in two-thirds of the preparations. Most likely this finding again reflects the greater susceptibility of VIP to degrading neutral endopeptidases. In vivo, the longer-lasting bronchodilator effect of PACAP-38, compared with PACAP-27 and VIP, was also observed in anesthetized, ventilated guinea pigs [25]. The animals were injected with histamine, leading to an increase in total pulmonary resistance, which decreased due to bronchodilation after the inhalation of VIP and PACAP. Interestingly, the bronchodilatory effect of PACAP38 lasted for more than 50 minutes, whereas after PACAP27 and VIP administration the same effect diminished within 10 minutes [25].

Relaxant Activities on Pulmonary Arteries Studies performed in vitro using precontracted pulmonary artery segments from humans and guinea pigs demonstrated that PACAP and VIP were even more potent dilators of pulmonary arteries than acetylcholine (for a review see [14, 23, 29]). Preincubation of the pulmonary rings with the nitric oxide (NO) synthase inhibitor NG-monomethyl-l-arginine inhibited PACAPinduced relaxation, indicating that PACAP-induced vasorelaxation depends on a mechanism involving endothelial NO synthase. This finding was further substantiated by elegant experiments using pulmonary artery segments exhibiting or lacking an intact endothelium. In these experiments it was clearly demonstrated that, in sharp contrast to VIP, PACAP-induced vasorelaxation involves an endothelium-dependent mechanism [6]. This result may imply that the dilatory effects of VIP and PACAP on pulmonary artery segments are mediated via different receptors and that the cellular localization of these two receptors is probably not the same. It is tempting to speculate that the receptor mediating VIP’s dilating effects is directly localized on smooth muscle cells, whereas the receptor mediating PACAP’s effects is present on pulmonary epithelial cells. The endothelium-dependent dilation of pulmonary artery segments by PACAP is unique for this vessel type because in the aorta and in coronary arteries PACAP activates an endothelium-independent mechanism (for a review see [14, 23, 29]). In vivo, it was also possible to demonstrate the vasodilatory effects of

PACAP on the pulmonary arteries using intact anesthetized cats [7].

Effects on Airway Mucous Secretion Mucus protects the surface of the airways by exerting a primary defense role against inhaled agents. Airway mucous secretion is regulated by a complex network of humoral and neuronal mediators (for a review see [24, 30]). Mucus-secreting cells (i.e., epithelial goblet cells and submucosal glands) synthesize respiratory tract mucins that appear to be responsible for the viscoelastic properties of airways mucus. These cells are innervated and possess surface receptors that interact with secretory stimuli, so that neural stimulation increases secretion. Three different neuronal pathway components innervate the airways: the sympathetic (adrenergic), parasympathetic (cholinergic), and the nonadrenergic noncholinergic (NANC) neuronal pathway component. By definition, the NANC system comprises all excitatory or inhibitory responses that remain after adrenoreceptor and acetylcholine receptor blockade. Small bioactive peptides and gases such as NO belong to the NANC neurotransmitters within the airways. The involvement of the tachykinins substance P and neurokinin A, as well as of VIP and PACAP, in airway mucus and electrolyte secretion has been clearly demonstrated. The exogenous administration of VIP or PACAP inhibits cholinergic and tachykininergic mucous output in ferret trachea acting through VPAC1 receptors. PACAP27 is the most potent stimulator of airways mucous secretion from isolated rat trachea, compared with the related peptides VIP, helospectin I and II, peptide histidine isoleucine (PHI), exendin-4, helodermin, and PACAP38 [9]. The effect of PACAP27 is not enhanced by the addition of thiorphan, a protease inhibitor. Whereas PACAP38 does not induce chloride currents, both PACAP27 and VIP enhance cystic fibrosis transmembrane conductance regulator (CFTR)dependent chloride secretion in human bronchial epithelial Calu-3 cells, acting through VPAC1 receptors [9]. CFTR is a plasma membrane protein that works as a chloride channel, and Calu-3 cells have the characteristics of serous gland cells, the major site of chloride secretion in the airways. Both mucous and electrolyte secretion contribute to the mucociliary transport system that clears the airways of pathogens. Thus, available data suggest the importance of PACAP-mediated signaling in airway secretion, but new and potent antagonists of PACAP receptors are needed to firmly establish the physiological role of PACAP at this level.

Modulation of Inflammatory Cell Activity Inflammation is an important process that mainly involves macrophages and lymphocytes and comprises

1296 / Chapter 179 antigen-specific and nonantigen-specific mechanisms. Available data on PACAP and, to a greater extent, on VIP have demonstrated that both peptides behave as pleiotropic neuroimmunomodulators of inflammatory responses (for a review see [10]). VIP and PACAP act on activated macrophages as potent endogenous antiinflammatory neuropeptides. They inhibit NO and pro-inflammatory cytokine production, but they induce anti-inflammatory cytokines. Mechanistically, VPAC1-Rmediated regulation of c-Jun, cAMP-response-elementbinding protein (CREB), CREB-binding protein (CBP), nuclear factor κB (NF-κB), and other molecules seems to be involved [22]. Moreover, VIP and PACAP inhibit the expression of various chemokines with macrophage inflammatory activity, so both peptides modulate the nature of the inflammatory infiltrate. PACAP has been shown to inhibit the release of thromboxane-B2 induced by leukotriene-D4 in chopped guinea pig lung, suggesting a possible anti-inflammatory action at this level. PACAP and VIP reduce apoptotic death of rat alveolar L2 cells treated with cigarette-smoke extract [18]. The attenuation of cytotoxicity is achieved via VPAC2 receptors and results, at least in part, in the inhibition of the activities of metalloproteinases and caspases. This is of interest because cigarette smoke is widely accepted as a major causative factor in the development of inflammatory lung diseases. In addition, both VIP and PACAP protect against glutamate toxicity in the lung. This effect may be mediated by antioxidant and antiapoptotic actions and by the suppression of glutamateinduced upregulation of its own receptor [26]. Thus, PACAP has been clearly identified as a potent antiinflammatory factor involved in airway inflammation.

PATHOPHYSIOLOGICAL IMPLICATIONS Considering the diverse biological roles of PACAP within the lung, it is obvious that impaired or dysregulated PACAP signaling may contribute to several pathological situations directly or indirectly affecting this organ. Here, we address the involvement of PACAPmediated signaling in the pathogenesis of pulmonary hypertension, bronchitis, asthma, and lung cancer.

Pulmonary Hypertension Mice deficient in VIP, PACAP, VPAC2-R, or PAC1-R have been generated. Whereas VIP- and VPAC2-Rdeficient mice do not show any signs of postnatal lethality, mice lacking PACAP [8] or the PAC1-R [19] display a very similar complex phenotype that results in death of the majority of mutant animals within the second postnatal week. Detailed analysis of the PACAP-deficient mice revealed that the mutant animals suffered

from reduced ventilation with blunted responses to hypoxia and hypercapnia. The authors suggested that PACAP may play a pivotal role in the central regulation of respiration. The expression of PACAP and the PAC1R in brain stem regions important for chemorespiratory control [2, 13, 29] and the finding that the injection of PACAP in anesthetized dogs stimulated breathing are in line with the observed phenotype of PACAP-deficient mice. It was further concluded that these mice could serve as a model for sudden infant death syndrome (SIDS), because subjecting the mutants to stressors such as hypothermia provoked their death, but the phenotype was rescued by elevation of the temperature in the animal house. In addition, some of the mutant mice shortly before death displayed steatosis of the liver and elevated fatty acids, symptoms that are characteristically seen in approximately 23% of human SIDS cases. PAC1-R-deficient mice displayed a very similar phenotype [19], although the percentage of mutants not surviving the second postnatal week was found to be dependent on the genetic background analyzed. Given a mixed genetic background, only 20% of mutant animals were lost until weaning, the surviving mutants displaying altered emotional behavior and a deficit in hippocampus-dependent associative learning. Conversely, given a C57BL/6 genetic background, almost all mutant animals died within the second postnatal week, exhibiting the same symptoms as those mutants that were lost given a mixed genetic background. At birth, mutants were indistinguishable from their wildtype littermates, but around postnatal day 6 they started to develop weight loss and progressive weakness. Finally, mutant animals died from rapidly developing right heart failure. Right heart failure was caused by pulmonary hypertension that was evidenced by the loss of lung capillaries, increased muscularization of the small pulmonary vessels, and elevated right-ventricular endsystolic pressure. Shortly before death, the mutant animals displayed signs of metabolic decompensation such as liver steatosis and elevated fatty acids, reflecting enhanced catabolism due to cardiac cachexia but not a specific involvement of PAC1-R-mediated signaling in lipid and carbohydrate metabolism. Because the animals were very small at the time of analysis, technical limitations did not allow an answer to the question of whether the animals suffered from primary pulmonary hypertension (due to primary vasoconstriction of pulmonary vessels) or whether pulmonary hypertension was secondary to primary alveolar hypoxia (due to bronchoconstriction/hypoventilation followed by vasoconstriction). The phenotype of the PACAP-deficient mice argues for secondary pulmonary hypertension as a consequence of central hypoventilation. However, hypoxia occurred very late in PAC1-R-deficient mice and thus may lead to the conclusion that these mice suffered

PACAP’s Role in Pulmonary Function / 1297 from primary pulmonary hypertension. Further studies using different strains of mice harboring tissue-specific inactivation of PAC1-R either in the neurons of the central nervous system, endothelial cells, or smooth muscle cells are required to definitely answer this important question. Considering the ex vivo experiments, the analysis of the relaxation of airway smooth muscle and pulmonary arteries leads to the suggestion that the relaxation of airway smooth muscle is mediated via PACAP type II receptors, whereas pulmonary artery relaxation shows different mechanisms for VIP and PACAP and thus requires the PAC1-R in endothelial cells apart from PACAP type II receptors within smooth muscle cells. It is therefore tempting to speculate that most likely either the endothelium-specific inactivation of the PAC1-R or the inactivation of this receptor within the central nervous system, but not the inactivation of PAC1-R within smooth muscle cells, leads to pulmonary hypertension. Taken together, the phenotypes of VIP-, PACAP-, PAC1-R-, and VPAC2-R-deficient mice demonstrate clearly that in vivo PACAP and its specific receptor PAC1-R are of pivotal importance for normal pulmonary function. The VPAC1-R and VPAC2-R that are still expressed in PAC1-R-deficient mice are not able to compensate for the lack of the PAC1-R. In light of the enormous conservation of PACAP during evolution, it might be worthwhile to examine whether subsets of patients suffering from pulmonary hypertension show mutations or epigenetic modifications within PACAP and/or the PAC1-R gene or in other genes that are required for the correct expression and processing of these two genes.

Asthma Bronchial asthma is one of the major airway pathologies. Taking into account the physiological and pharmacological properties of PACAP within the lung, it is conceivable that a decreased activity (i.e., low expression of PACAP and/or its receptors or a high extent of peptide degradation) may contribute to the pathogenesis of bronchial asthma. In fact, PACAP is a cotransmitter of the NANC relaxant system, the main relaxant system in human airways. Lack of NANC-mediated relaxation due to decreased PACAP activity could result in the airway hyperreactivity typically seen in bronchial asthma. As already mentioned, PACAP inhibits airways inflammation, an important feature of this chronic inflammatory disorder of the airways in which many cells play a role. Moreover, PACAP may be considered a modulator of airway smooth muscle cell proliferation, another important feature of bronchial asthma that contributes to increased airway resistance in this pathology. Interestingly, the inhibitory effects induced by

PACAP38 on histamine-induced respiratory resistance in guinea pigs are more prolonged than those exerted by PACAP27 or VIP, and they can be enhanced by the addition of the endopeptidase inhibitor phosphoramidon [25]. Exogenous PACAP decreases ozone hyperstimulation of airway responsiveness to histamine in guinea pigs, but it does not exert any effect on plasma extravasation, which suggests the potential effectiveness of PACAP in reversing this situation [1].

Cancer Lung carcinoma is the leading cause of cancerrelated deaths in the Western world. Lung cancer comprises small-cell lung cancer (SCLC, 25% of total cases) and non-small-cell lung cancer (NSCLC). There is growing evidence of the importance of neuropeptides in the pathogenesis and progression of SCLC and a fraction of NSCLC tumors. PACAP has been reported to stimulate both SCLC and NSCLC cell growth (for a review see [17]). PACAP stimulates clonal cell growth of the SCLC cell line NCI-H345 through the activation of VPAC receptors. The same response has been seen in NSCLC cells, and the mechanism appears to involve intracellular increases of Ca2+ and cAMP. These data, together with observations that describe PACAP27induced increases in phosphorylation of mitogenactivated protein kinase and expression of nuclear oncogenes (c-fos, c-jun, and c-myc mRNAs) in human lung cancer cells, suggest that PAC1 and VPAC1 receptors are involved in the stimulatory effect of PACAP on lung cancer cell growth. PACAP27 increases vascular endothelial growth factor (VEGF) mRNA in human lung cancer lines, suggesting the involvement of PACAP in the angiogenesis of lung cancers. Interestingly, the function of PACAP and VIP receptors has been demonstrated in human lung cancer biopsy specimens [5], which gives value to the potential regulatory role of these peptides on growth and differentiation of lung tumor cells. The opposite effects have been reported for VIP on SCLC and NSCLC growth and proliferation; please refer to Chapter 181 on VIP.

THERAPEUTIC POTENTIAL The involvement of PACAP-mediated signaling in diseases such as asthma, lung cancer, and pulmonary hypertension suggests that synthetic PACAP analogs could be of major therapeutic value. Apart from the already-existing synthetic PACAP analogs, more potent and more selective agonists/antagonists of the diverse PACAP receptors (i.e., small molecules or more stabilized peptide preparations) are required to fully

1298 / Chapter 179 exploit therapeutic approaches based on PACAP and its receptors.

Asthma Much of the knowledge of PACAP’s therapeutic potential in asthma has been stimulated by previous studies using VIP. The relief of airway constriction of bronchial asthma is achieved by the relaxation of airway smooth muscles, and it is a desirable feature of the therapeutic agent. The therapeutic effectiveness of PACAP27 and -38 is enhanced by adding an endopeptidase inhibitor such as phosphoramidon, indicating the important degradative activity of peptidases and, possibly, other enzymes [25]. New PACAP analogs (i.e., [Arg15,20,21-Leu17]-PACAP-Gly-Lys-Arg-NH2) have been developed to be less susceptible to cleavage by peptidases than the original peptide PACAP27, thus giving sustained, long-lasting bronchial smooth muscle relaxation in human bronchi [31]. The administration of the selective VPAC2 receptor agonist Ro 25-1553 by inhalation in patients with stable asthma produces important bronchodilatory effects that promise therapeutic potential to this synthetic peptide in this disease [15]. Extended clinical trials in patients suffering from asthma using PACAP analogs given by intravenous (IV) infusion or by inhalation as aerosol will define the therapeutic potential of this peptide in asthma and airway inflammation.

Cancer Potential antitumor strategies to reduce the proliferation of tumor cells in lung cancer may benefit from the availability of new PACAP and VIP receptor antagonists because both peptides stimulate the growth of lung cancer cells (for a review see [5]). Using VIPhyb, a VIP hybrid synthetic peptide that blocks PAC1, VPAC1, and VPAC2 receptors, the proliferation of a high number (51 of 56) of human lung cancer cell lines was inhibited. This peptide derivative inhibits basal cell proliferation, behaves as a cytostatic, and slows tumor proliferation, but it does not cause tumor regression. Interestingly, VIPhyb and VIP itself inhibit xenograft proliferation in nude mice [16] and cause smaller and a reduced number of blood vessels in tumors. Other potentially useful peptides for lung cancer therapy are some antagonistic analogs of growth hormone– releasing hormone (GHRH), named JV-1-51, 52, and 53, that are also endowed with VPAC receptor antagonistic activity [21]. These are discussed by Schally and colleagues in Chapter 60 in the Cancer/Anticancer Peptides Section of this book.

Pulmonary Hypertension Recently a study has been published describing reduced VIP serum levels, a reduction in VIP-positive nerve fibers within the media of pulmonary arteries, and strongly elevated PACAP type II receptors in patients suffering from primary pulmonary hypertension. Treatment with daily inhalations of VIP decreased the pulmonary vascular resistance by 50% and significantly increased the mixed venous oxygen saturation as well as the 6-minute-walk distance in these patients [20]. In preliminary studies, it has been shown that the inhalative application of VIP was as efficient as intravenous application and that there were almost no side effects in patients treated by inhalation. Interestingly, PACAP levels and expression of PAC1-R have not been analyzed yet in patients suffering from pulmonary hypertension [20]. Taking into account the phenotypes of the animal models mentioned here, we suggest that even in humans PACAP and PAC1-R might be the crucial players in the pathogenesis of pulmonary hypertension. This has to be carefully analyzed in the future. Once an involvement of PACAP and its specific receptor in subsets of human pulmonary hypertension has been demonstrated, the inhalation of PACAP and synthetic analogs could be of the utmost benefit for those patients. In addition, the development of small-molecule agonists for the PAC1-R could be very helpful.

References [1] Aizawa H, Shigyo M, Matsumoto K, Inoue H, Koto H, Hara N. PACAP reverses hyperresponsivenesss induced by ozone exposure in guinea pigs. Respiration 1999;66:538–542. [2] Arimura A. Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 1998;48:301–331. [3] Busto R, Carrero I, Guijardo LG, Solano RM, Zapatero J, Noguerales F et al. Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung. Am J Physiol 1999;277:L42–L48. [4] Busto R, Prieto JC, Bodega G, Zapatero J, Carrero I. Immunohistochemical localization and distribution of VIP/PACAP receptors in human lung. Peptides 2000;21:265–269. [5] Busto R, Prieto JC, Bodega G, Zapatero J, Fogué L, Carrero I. VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens. Peptides 2003;24:429– 436. [6] Cardell LO, Hjert O, Uddman R. The induction of nitric oxidemediated relaxation of human isolated pulmonary arteries by PACAP. Br J Pharmacol 1997;120:1096–1100. [7] Cheng DY, McMahon TJ, Dewitt BJ, Carroll GC, Lee SS, Murphy WA et al. Comparison of responses to pituitary adenylate cyclase activating peptides 38 and 27 in the pulmonary vascular bed of the cat. Eur J Pharmacol 1993;243:79–82. [8] Cummings KJ, Pendlebury JD, Jirik FR, Sherwood NM, Wilson RJA. Sudden neonatal death in PACAP-deficient mice is associated with reduced respiratory chemoresponse and susceptibility to apnoea. J Physiol 2004;555:15–26.

PACAP’s Role in Pulmonary Function / 1299 [9] Dérand R, Montoni A, Bulteau-Pignoux L, Janet T, Moreau B, Muller JM et al. Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTRdependent chloride secretion. Br J Pharmacol 2004;141:698– 708. [10] Ganea D, Delgado M. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) as modulators of both innate and adaptive immunity. Crit Rev Oral Biol Med 2002;13:229–237. [11] Groneberg DA, Hartmann P, Dinh QT, Fischer A. Expression and distribution of vasoactive intestinal polypeptide receptor VPAC2 mRNA in human airways. Lab Invest 2001;81:749–755. [12] Hauser-Kronberger C, Hacker GW, Albegger K, Muss WH, Sundler F, Arimura A et al. Distribution of two VIP-related peptides, helospectin and pituitary adenylate cyclase activating peptide (PACAP), in the human upper respiratory system. Regul Pept 1996;65:203–209. [13] Legradi G, Shioda S, Arimura A. Pituitary adenylate cyclaseactivating polypeptide-like immunoreactivity in autonomic regulatory areas of the rat medulla oblongata. Neurosci Lett 1994;176:193–196. [14] Linden A, Cardell LO, Yoshihara S, Nadel JA. Bronchodilation by pituitary adenylate cyclase-activating peptide and related peptides. Eur Respir J 1999;14:443–451. [15] Linden A, Hansson L, Andersson A, Palmqvist M, Arvidsson P, Lofdahl CG et al. Bronchodilation by an inhaled VPAC(2) receptor agonist in patients with stable asthma. Thorax 2003;58:217–221. [16] Maruno K, Absood A, Said SI. Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proc Natl Acad Sci USA 1998;95:14373–14378. [17] Moody TW, Hill JM, Jensen RT. VIP as a trophic factor in CNS and cancer cells. Peptides 2003;24:163–177. [18] Onoue S, Ohmori Y, Endo K, Yamada S, Kimura R, Yajima T. Vasoactive intestinal peptide and pituitary adenylate cyclaseactivating polypeptide attenuate the cigarette smoke extractinduced apoptotic death of rat alveolar L2 cells. Eur J Biochem 2004;271:1757–1767. [19] Otto C, Lutz H, Brede M, Jahns R, Engelhardt S, Gröne HJ et al. Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptordeficient mice. Circulation 2004;110:3245–3251. [20] Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K et al. Vasoactive intestinal peptide as a new drug

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for treatment of primary pulmonary hypertension. J Clin Invest 2003;111:1339–1346. Plonowski A, Varga JL, Schally AV, Krupa M, Groot K, Halmos G. Inhibition of PC-3 human prostate cancers by analogs of growth hormone-releasing hormone (GH-RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity. Int J Cancer 2002;98:624–629. Pozo D. VIP- and PACAP-mediated immunomodulation as prospective therapeutic tools. Trends Mol Med 2003;9:211–217. Prieto JC. Function of PACAP in the respiratory system. In: Pituitary Adenylate Cyclase-Activating Polypeptide (Vaudry H, Arimura A, eds); Kluwer Academic Publishers, Norwell, MA, USA, 2003; pp 289–303. Rogers DF. Pharmacological regulation of the neuronal control of airway mucus secretion. Curr Opin Pharmacol 2002;2:249– 255. Saguchi Y, Ando T, Watanabe T, Yamaki K, Suzuki R, Takagi K. Inhibitory effects of pituitary adenylate cyclase activating polypeptide on histamine-induced respiratory resistance in anesthetized guinea pigs. Regul Pept 1997;70:9–13. Said SI, Dickman K, Dey RD, Bandyopadhyay A, De Stefanis P, Raza S et al. Glutamate toxicity in the lung and neuronal cells: Prevention or attenuation by VIP and PACAP. Ann NY Acad Sci 1998;865:226–237. Shigyo M, Aizawa H, Inoue H, Matsumoto K, Takata S, Hara N. Pituitary adenylate cyclase activating peptide regulates neurally mediated airway responses. Eur Respir J 1998;12:64–70. Uddman R, Luts A, Arimura A, Sundler F. Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive (VIP)-like peptide in the respiratory tract. Cell Tissue Res 1991;265:197– 201. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52:269– 324. Wagner U, Bredenbröker D, Storm B, Tackenberg B, Fehman HC, Von Wichert P. Effects of VIP and related peptides on airway mucus secretion from isolated rat trachea. Peptides 1998;19:241–245. Yoshihara S, Yamada Y, Abe T, Kashimoto K, Lindén A, Arisaka O. Long-lasting smooth-muscle relaxation by a novel PACAP analogue in human bronchi. Regul Pept 2004;123:161–165.

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180 Tachykinins and Their Receptors in the Lung STEFANIA MEINI AND ALESSANDRO LECCI

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NH2), its elongated forms (namely, endokinin A and B), and other tachykinin-gene related peptides (namely, endokinin C and D). In agreement with its sequence homology with SP, HK-1 displays selectivity for the NK1 as compared with NK2 and NK3 receptors [27]. The three tachykinin receptor types, cloned from several different mammalian species, belong to class 1 of the G-protein-coupled receptors [13] phylogenetically related to rhodopsin, bearing seven α-helical transmembrane domains [1]. The activation of tachykinin receptors by agonists leads to multiple signaling events that are mediated by coupling with the trimeric Gq or Gs proteins, which, in turn, activate intracellular downstream pathways. The Gq-protein-mediated activation of phospholipase C leads to inositol phosphate hydrolysis, producing inositol-1,4,5-triphosphate, diacylglycerol, and intracellular calcium increases. On the other hand, Gs protein is responsible for the activation of adenylylcyclase and cAMP production. As anticipated, SP and NKA are the preferred ligands for the tachykinin NK1 and NK2 receptors, respectively, but in the last decade several studies have addressed ligand-receptor pharmacology in terms of agonist-directed receptor trafficking [19]. Two high-affinity binding sites have been associated with the NK1 receptor in both recombinant and native cell systems. The most abundant is coupled to the Gs protein, and the second is responsible for the Gq-protein-activated second messenger. Thus, experimental evidence using recombinant cell systems, Gprotein fusion, and mutagenesis has demonstrated that SP and some full SP-sequence analogs can activate the tachykinin receptor Gs-protein coupling, whereas NKA and truncated forms of SP can activate only the tachykinin NK1 receptor conformer linked to the Gq-proteinlinked signaling [3]. A similar pharmacological paradigm has been shown even for the tachykinin NK2 receptor, which when activated by NKA can elicit both calcium ions and cAMP

The tachykinins substance P (SP) and neurokinin A (NKA) have been identified in lung cells of different origin and are involved in neurogenic inflammation. In the airways SP and NKA can produce several effects, including smooth muscle contraction, epithelial and submucosal gland secretion, vasodilation, increase in vascular and epithelial permeability, stimulation of cholinergic nerves, and recruitment of inflammatory cells. Although the bulk of released tachykinins is expressed in capsaicin-sensitive afferent neurons, there is evidence for their expression in other kinds of neurons and in epithelial, smooth muscle, and immune cells. All these sources of tachykinins can contribute to airway pathologies such as asthma and chronic obstructive pulmonary disease.

TACHYKININ RECEPTOR PHARMACOLOGY Mammalian tachykinins are short peptides that share the common C-terminal sequence Phe-Xaa-Gly-LeuMet-NH2. They include substance P (SP; H-Arg-Pro-LysPro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), neurokinin A (NKA; H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2), and neurokinin B (NKB; H-Asp-Met-His-Asp-Phe-PheVal-Gly-Leu-Met-NH2) and exhibit a preferential activity to the tachykinin NK1, NK2, and NK3 receptors, respectively. Much of this first section has been reviewed in Chapter 105 by Page in the Brain Peptides chapter and elsewhere in this book, but some of it is repeated here to facilitate understanding of the role of tachykinins in the lung. These peptides are encoded by three genes: TAC1, which encodes SP and NKA; TAC3, which encodes NKB; and the more recently identified TAC4, which encodes a new tachykinin termed hemokinin 1 (HK-1; H-Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-MetHandbook of Biologically Active Peptides

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1302 / Chapter 180 increase, whereas its C-terminal truncated form (NKA(4–10)) is able to produce the only calcium response [3]. Interestingly, no pharmacological differences were observed in the agonist-induced NK1 receptor-β-arrestin complex [22], involved in the stimulation of the mitogen-activated protein kinase (MAPK), which is known to have a role in the proliferative effect induced by SP [11, 14]. It remains to be determined how these molecular mechanisms are involved in the pathophysiological roles that tachykinins exert in the airways.

TACHYKININ LOCALIZATION IN THE LUNG SP and NKA are normally expressed in and released from a subset of primary afferent neurons (C-type, capsaicin-sensitive) of several mammalian species, but under the influence of inflammatory stimuli (involving nerve growth factor), their synthesis may also occur in other kinds of airway sensory fibers (Aδ-type, capsaicinresistant) [25]. Moreover, large species-related quantitative differences in airway expression of tachykinins (particularly in capsaicin-sensitive primary afferent nerves) have been described. In particular, the content of tachykinins in guinea pig airways is relatively abundant, whereas in other species, including humans, tachykinin levels are much lower. A peculiarity of capsaicin-sensitive primary afferent nerves (in contrast to Aδ-fibers) is their ability to release tachykinins at both peripheral and central nervous system levels on excitation by adequate stimuli. Further sources of tachykinins are represented by epithelial and immune cells. In these cell systems, tachykinin expression appears to be upregulated by inflammatory stimuli. However, from a functional point of view, the evidence for the participation of nonneuronal tachykinins in biological processes is still limited [17].

TACHYKININ RECEPTOR DISTRIBUTION IN THE LUNG Tachykinin receptors in the airways have been mapped by use of mRNA detection, immunohistochemical, autoradiographic, and radioligand techniques. These studies have highlighted a differential distribution for the NK1 and NK2 receptors, the former being predominantly, but not exclusively, present at the level of bronchial vessels, epithelial cells, and submucosal glands and the latter on the airway smooth muscle. No conclusive morphological or molecular evidence for the expression of NK3 receptors has been presented, but its expression in intrinsic neurons and inflammatory cells has been deduced from functional studies. In

humans, tachykinin receptor expression can vary depending on disease state (asthma) or life habits (cigarette smoke) [17].

TACHYKININ-MEDIATED BIOLOGICAL EFFECTS IN THE LUNG A variety of stimuli, such as cigarette smoke, citric acid, low pH, increase in temperature, H2S, and inflammatory mediators such as bradykinin, prostaglandins, and leukotrienes, can activate capsaicin-sensitive neurons and the peripheral release of SP and NKA, which in turn produce neurogenic inflammation. In the airways, neurogenic inflammatory responses induced by endogenously released SP/NKA include plasma protein extravasation, bronchoconstriction, mucous secretion, and recruitment/activation of inflammatory and immune cells, thus contributing to the airway pathophysiology of such diseases as chronic bronchitis and asthma [2, 17]. The availability of potent and selective antagonists for blocking the NK1, NK2, or NK3 receptors has facilitated the determination of the functional role of these receptors in different animal models of asthma/bronchial hyperreactivity [21]. Thus, in acute models of pulmonary injury the NK1 receptor mediates the increase in postcapillary venular permeability, participates in recruiting of inflammatory cells, plays a role in bronchoconstriction, and stimulates mucous secretion from epithelial goblet cells and submucosal glands. The endothelium-dependent vasodilating effect involves the release of nitric oxide and hyperpolarizing factors. Furthermore, the stimulation of NK1 receptors is associated with neovascularization, which seems to occur following the activation of a calcium-dependent nitric oxide synthase in endothelial cells. Tachykinin-containing nerves, as well as NK1 receptors, have been localized in different lymphoid organs and in human blood lymphocytes; their likely role is to stimulate their proliferation in human diseases, and the involved mechanism has been related to the activation of MAPK pathways. The possibility that NK1 receptors could be involved in tissue remodeling is suggested by studies showing that SP induces airway smooth muscle and fibroblast proliferation. NK1 receptor--dependent production of superoxide ions has been reported in guinea pig and human alveolar macrophages. This effect, beyond having a protective role against airway infections, could contribute to the sensitization of sensory nerves associated with bronchial asthma. The most evident functional role of NK2 receptors is linked to bronchomotor responses. Bronchoconstriction induced by capsaicin, citric acid, allergen, H2S,

Tachykinins and Their Receptors in the Lung / 1303 ozone, cigarette and wood smoke, and physical exercise is prominently reduced by tachykinin NK2 receptor antagonists, whereas NK1 receptors only play a minor role [21]. There is evidence of the involvement of NK2 receptors in animal models of airway hyperreactivity, and, interestingly, the inhibition of hyperreactivity is associated with a reduction of inflammatory cells in the bronchoalveolar lavage. Finally, NK2 receptor-mediated increase in epithelial permeability has been demonstrated through the use of selective antagonists following ozone or cigarette smoke challenge [29]. Results from the literature do not support the presence of NKB in the lung. However, functional evidence for the expression of NK3 receptor has been obtained through the use of selective antagonists that reduce citric acid--induced cough, inflammatory cell recruitment, airway hyperreactivity, and microvascular leakage [9, 10, 15, 26]. These results imply that endogenously released NKA and SP activate NK3 receptors. A further NK3 receptor--mediated effect has been described in parasympathetic airway neurons following sensory nerve stimulation in guinea pig and human airways [24]. Molecular evidence for NK3 receptor expression has been recently provided in human lungs [28] as well as in membranes prepared from lungs of antigensensitized guinea pigs, a model in which a selective NK3 antagonist blocked airway hyperreactivity to methacholine [23].

THERAPEUTIC POTENTIAL OF TACHYKININ NK1 RECEPTOR BLOCKADE Two clinical studies have described the effect of NK1 receptor blockade in asthmatic patients by means of the selective antagonists FK888 and CP99994 [12, 16]. In both studies, neither an improvement of the basal lung function nor a reduction of the bronchoconstriction peak induced by physical exercise or hypertonic aerosolized solution was obtained but, rather, a faster recovery was observed. These results may confirm, in agreement with animal pathophysiological models, that NK1 receptors only play a minor role in airway motor responses. The fact that SP inhalation increases the concentration of several proteins in the sputum of asthmatics may indicate that SP can indeed increase plasma protein extravasation [30]. In this context, the possibility that NK1 receptor antagonists might reduce inflammatory components in asthma or chronic obstructive pulmonary disease cannot be ruled out. However, this hypothesis can only be assessed following the long-term administration (months) of tachykinin NK1 receptor antagonists.

THERAPEUTIC POTENTIAL OF TACHYKININ NK2 RECEPTOR BLOCKADE As occurs in animal models, the bronchoconstrictor response induced by NKA in asthmatics is antagonized by selective NK2 receptor antagonists [17, 31], and this could suggest a direct participation of NK2 receptors in asthmatic bronchoconstriction. However, Saredutant (administered for 9 days at a dose inhibiting the bronchoconstrictor effect induced by NKA) neither induced a bronchodilator effect nor prevented the hyperreactivity to the bronchoconstrictor effect induced by adenosine in subjects with allergic asthma [20]. Although these results exclude that NK2 receptor antagonists can possess direct bronchodilator effects, the possibility that these drugs may exert an anti-inflammatory effect cannot be ruled out. The fact that anti-inflammatory drugs currently used for asthma such as the corticosteroid fluticasone or the leukotriene D4 antagonist montelukast also reduced the NKA-induced bronchoconstriction suggests that NK2 receptors may be involved in airway inflammation in asthmatics [7, 32]. The discovery of a synergy between tachykinins released by sensory neurons and those released by inflammatory cells in the development of airway inflammation in a murine model [5] further supports the concept that the main target of tachykinin antagonists in diseased lungs is inflammation rather than bronchoconstriction. More recently, it has been shown that a challenge with adenosine induces bronchial release of both SP and NKA associated with bronchoconstriction in subjects with allergic asthma [8]. The fact that Saredutant was found to be inactive against adenosine-induced airway hyperreactivity [20] indicates that the blockade of NK2 receptors is not sufficient to reduce the stimulation (direct or mediated by mast cells) of afferent nerves by AMP. On the other hand, the possibility that part of the bronchoconstriction induced by adenosine in asthmatics is mediated by a direct effect of tachykinins on airway smooth muscle cannot be ruled out.

THERAPEUTIC POTENTIAL OF DUAL TACHYKININ NK1-NK2 RECEPTOR BLOCKADE On the basis of preclinical findings, it can be speculated that a dual NK1-NK2 receptor antagonist offers a broader therapeutic potential of selective antagonists because of a simultaneous reduction of both airway hypermotility and inflammation. An interesting interaction between NK1 and NK2 receptors has been recently described in guinea pig airways, where the blockade of

1304 / Chapter 180 both NK1 and NK2 receptors antagonizes the response of selective receptor (NK1 or NK2) agonists more prominently than the respective selective antagonists [6]. Indeed, the dual NK1-NK2 receptor antagonist DNK333 (Novartis) antagonized NKA-induced bronchoconstriction in asthmatics, and its effect was apparently larger than that of selective NK2 receptor antagonists [18], suggesting that the existence of a cooperation between NK1 and NK2 receptors in mediating bronchoconstriction could also occur in humans. However, there are no results to indicate whether a dual NK1-NK2 antagonist could have a therapeutic advantage over selective antagonists.

THERAPEUTIC POTENTIAL OF BLOCKADE OF THE TACHYKININ NK3 RECEPTOR A recent study identified the expression of NK3 on human lungs [19], and as already outlined, some of these receptors are expressed on parasympathetic postganglionic neurons in human airways [24]. Animal studies indicate that a selective NK3 receptor antagonist could share the anti-inflammatory and antibronchoconstrictor effects of NK1 and NK2 antagonists; however, no clinical results with NK3 antagonists are available to support the utility of NK3 antagonists in airway diseases.

CONCLUSION Animal studies have documented an important role for tachykinins in mediating inflammation, bronchoconstriction, hypersecretion, and inflammatory cell recruitment. Although this evidence has been mainly gathered in acute models of airway inflammation, the upregulation of the tachykinin system (peptides and receptors) induced by more persistent inflammation further supports the importance of tachykinin peptides in chronic inflammatory airway diseases. Up to now, clinical expectations have been elusive because no improvement of symptoms has been reported following the administration of selective NK1, NK2, or mixed NK1-NK2 receptor antagonists in asthmatics. On the other hand, the lack of bronchodilator effects does not exclude the hypothesis that tachykinin receptor antagonists might belong to the class of controllers that display beneficial effects only following long-term treatment. However, it should also be considered that this hypothesis might turn out to be wrong. In this context, the recent finding that sputum collected during exacerbations in chronic obstructive pulmonary disease patients contains less SP and NKA than in the stable

phase of the disease and that no differences in basal levels of these peptides were found in healthy nonsmokers, healthy smokers, or patients with stable chronic obstructive pulmonary disease brings into question the pro-inflammatory role of tachykinins in human subjects [4].

References [1] Almeida TA, Rojo J, Pinto FM, Hernandez M, Martín JD, Candenas ML. Tachykinins and tachykinin receptors: structure and activity relationships. Curr Med Chem 2004;11:2045--81. [2] Barnes PJ. Inflammatory mediators of asthma: an update. Pharmacol Rev 1998;50:515--96. [3] Beaujouan J-C, Torrens Y, Saffroy M, Kemel ML, Glowinskin J. A 25 year adventure in the field of tachykinins. Peptides 2004;25:339--57. [4] Boschetto P, Miotto D, Bonomi I, Faggian D, Plebani M, Papi A, Creminon C, De Rosa E, Fabbri LM, Mapp CE. Sputum substance P and neurokinin A are reduced during exacerbations of chronic obstructive pulmonary disease. Pulm Pharmacol Ther 2005;18:199--205. [5] Chavolla-Calderon M, Bayer MK, Fontan JJ. Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation. J Clin Invest 2003;111:973--80. [6] Corboz MR, Fernandez X, Rizzo CA, Tozzi S, Monahan ME, Hey JA. Increased blocking activity of combined NK1 and NK2 receptor antagonists on tachykinergic bronchomotor responses in guinea-pig. Auton Autacoid Pharmacol 2003;23:79--93. [7] Crimi N, Pagano C, Palermo F, Mastruzzo C, Prosperini G, Pistorio MP, Vancheri C. Inhibitory effect of a leukotriene receptor antagonist (montelukast) on neurokinin A-induced bronchoconstriction. J Allergy Clin Immunol 2003;111:833-39. [8] Crummy F, Livingston M, Ardill JES, Adamson C, Ennis M, Heaney LG. Mast cell mediator release in nonasthmatic subjects after endobronchial adenosine challenge. J Allergy Clin Immunol 2004;114:34--9. [9] Daoui S, Cognon C, Naline E, Emonds-Alt X, Advenier C. Involvement of tachykinin NK3 receptors in citric acid-induced cough and bronchial responses in guinea pigs. Am J Respir Crit Care Med 1998;158:42--8. [10] Daoui S, Cui YY, Lagente V, Emonds-Alt X, Advenier C. A tachykinin NK3 receptor antagonist, SR 142801 (osanetant), prevents substance P-induced bronchial hyperreactivity in guinea-pigs. Pulm Pharmacol Ther 1997;10:261--70. [11] DeFea KA, Vaughn ZD, O’Brian EM, Nishijima D, Déry O, Bunnett NW. The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex. Proc Natl Acad Sci USA 2000;97:11086-91. [12] Fahy JV, Wong HH, Geppetti P, Reis JM, Harris SC, Maclean DB, Nadel JA, Boushey HA. Effect of an NK1 receptor antagonist (CP-99,994) on hypertonic saline-induced bronchoconstriction and cough in male asthmatic subjects. Am J Respir Crit Care Med 1995;152:879--84. [13] Foord SM, Bonneer TI, Neubig RR, Rosser EM, Pinn J-P, Davenport AP, Spedding M, Harmar AJ. 2005. International union of pharmacology. XLVI. G protein-coupled receptor list. Pharmacol Rev 57:279--88. [14] Harrison NK, Dawes KE, Kwon OJ, Barnes PJ, Laurent GJ, Chung KF. Effects of neuropeptides on human lung fibroblasts proliferation and chemotaxis. Am J Physiol 1995;268:L278--83.

Tachykinins and Their Receptors in the Lung / 1305 [15] Hay DW, Giardina GA, Griswold DE, et al. Nonpeptide tachykinin receptor antagonists. III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyperreactivity in guinea pigs. J Pharmacol Exp Ther 2002;300:314-23. [16] Ichinose M, Miura M, Yamauchi H, Kageyama N, Tomaki M, Oyake T, Ohuchi Y, Hida W, Miki H, Tamura G, Shirato K. A neurokinin 1-receptor antagonist improves exercise-induced airway narrowing in asthmatic patients. Am J Respir Crit Care Med 1996;153:936--41. [17] Joos GF, De Swert KO, Pauwels RA. Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists. Eur J Pharmacol 2001;429:239--50. [18] Joos GF, Vinken W, Louis R, Schelfout VJ, Wang JH, Shaw MJ, Cioppa GD, Pauwels RA. Dual tachykinin NK1/NK2 antagonist DNK333 inhibits neurokinin A-induced bronchoconstriction in asthma patients. Eur Resp J 2004;23:76--81. [19] Kenakin T. Agonist-receptor efficacy: agonist trafficking of receptor signals. Trends Pharmacol Sci 1995;16:232--8. [20] Kraan J, Vink-Klooster H, Postma DS. The NK-2 receptor antagonist SR 48968C does not improve adenosine hyperresponsiveness and airway obstruction in allergic asthma. Clin Exp Allergy 2001;31:274--8. [21] Lecci A, Maggi CA. Peripheral tachykinin receptors as potential therapeutic targets in visceral diseases. Expert Opin Ther Targets 2003;7:343--62. [22] Martini L, Hastrup H, Holst B, Fraile-Ramos A, Marsh M, Schwartz TW. NK1 receptor fused to β-arrestin displays a singlecomponent, high affinity molecular phenotype. Mol Pharmacol 2002;62:30--7. [23] Mukaiyama O, Morimoto K, Nosaka E, Takahashi S, Yamashita M. Involvement of enhanced neurokinin NK3 receptor expres-

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sion in the severe asthma guinea pig model. Eur J Pharmacol 2004;498:287--94. Myers AC, Goldie RG, Hay DWP. A novel role for tachykinin neurokinin-3 receptors in regulation of human bronchial ganglia neurons. Am J Resp Crit Care Med 2005;171:212--16. Myers AC, Kajekar R, Undem BJ. Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways. Am J Physiol Lung Cell Mol Physiol 2002;282: L775--81. Nenan S, Germain N, Lagente V, Emonds-Alt X, Advenier C, Boichot E. Inhibition of inflammatory cell recruitment by the tachykinin NK(3)-receptor antagonist, SR 142801, in a murine model of asthma. Eur J Pharmacol 2001;421:201--5. Page NM. Hemokinins and endokinins. Cell Mol Life Sci 2004;61:1652--63. Pinto FM, Almeida TA, Hernandez M, Devillier P, Advenier C, Candenas ML. mRNA expression of tachykinins and tachykinin receptors in different human tissues. Eur J Pharmacol 2004;494:233--9. Tagawa A, Kaneko T, Nishiyama H, Shinohara T, Sato T, Geppetti P, Ishigatsubo Y. Cigarette smoke increases mucosal permeability in guinea pig trachea via tachykinin NK2 receptor activation. Eur J Pharmacol 2005;507:223--8. Van Rensen EL, Hiemstra PS, Rabe KF, Sterk PJ. Assessment of microvascular leakage via sputum induction: the role of substance P and neurokinin A in patients with asthma. Am J Respir Crit Care Med 2002;165:1275--9. Van Schoor J, Joos GF, Chasson BL, Brouard RJ, Pauwels RA. The effect of the NK2 tachykinin receptor antagonist SR 48968 (saredutant) on neurokinin A-induced bronchoconstriction in asthmatics. Eur Respir J 1998;12:17--23. Van Schoor J, Joos GF, Pauwels RA. Effect of inhaled fluticasone on bronchial responsiveness to neurokinin A in asthma. Eur Respir J 2002;19:997--1002.

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181 Vasoactive Intestinal Peptide SAMI I. SAID

also include peptide with N-terminal histidine and Cterminal isoleucine amide (PHI) and its counterpart in human tissues, peptide with N-terminal histidine and C-terminal methionine amide (PHM), secretin, glucagon, gastric inhibitory peptide or glucose-dependent insulinotropic peptide (GIP), corticotropin-releasing hormone (CRH), growth hormone–releasing hormone (GHRH), sauvagine, urotensin I, helodermin, and pituitary adenylate cyclase–activating peptide (PACAP).

ABSTRACT Vasoactive intestinal peptide (VIP) is a neuropeptide that is widely distributed in the central and peripheral nervous systems. VIP and its receptors are expressed in practically all pulmonary structures, including airway epithelial cells, airway and pulmonary vascular smooth muscle, secretory glands, and immune and inflammatory cells. VIP actions in the lung include the relaxation of airway and vascular smooth muscle and inhibition of their proliferation, modulation of inflammatory and immune responses, and protection against acute injury. Strong evidence, recently supported by findings in VIP knockout mice, suggests that VIP has important physiological roles in the transmission of airway and pulmonary vascular smooth muscle relaxation, the modulation of immune and inflammatory responses, and the defense of the lung against bronchial asthma and acute injury. VIP therefore is of potential therapeutic benefit in bronchial asthma, pulmonary hypertension, acute lung injury, the acute respiratory distress syndrome, and the multiorgan failure syndrome.

DISTRIBUTION AND LOCALIZATION The expression of VIP in different cells and organs has been defined by immunofluorescence, radioimmunoassay, and, more recently, by measurement of VIP mRNA. In the brain the peptide is found in its highest concentrations in the cerebral cortex, suprachiasmatic nucleus, hippocampus, amygdala, and striatum, but it is also present in the midbrain periaqueductal gray and the sacral spinal cord. In the peripheral nervous system, VIP is localized in the sympathetic ganglia; the vagus nerve; predominantly motor nerves such as the sciatic nerve; and nerves supplying exocrine glands, blood vessels, and nonvascular smooth muscle. In the lungs, VIP-containing nerve fibers and nerve terminals are principally localized in the smooth muscle layer of the airways from the trachea through the small bronchioles, around submucosal mucous and serous glands, and in the walls of both pulmonary and bronchial arteries. Immunoreactive VIP is also present in neuronal cell bodies forming microganglia that provide a source of intrinsic innervation of pulmonary structures. In addition to its neuronal localization, VIP is richly present in the immune system, including inflammatory cells such as mast cells, eosinophils, and B and T lymphocytes.

INTRODUCTION: VIP IN THE LUNG Although vasoactive intestinal polypeptide (VIP) was first isolated from intestinal extracts, its vasodilator activity, which guided its purification, had earlier been discovered in lung tissue. VIP was later rediscovered as a neuropeptide and a neurotransmitter and is now recognized as a widely distributed peptide with a wide variety of biological actions and functions.

VIP AND ITS FAMILY OF PEPTIDES VIP is a 28-amino-acid-residue peptide (Fig. 1) that belongs to a family of structurally related peptides that Handbook of Biologically Active Peptides

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1308 / Chapter 181 Colocalization with Other Peptides and Neurotransmitters VIP coexists with acetylcholine in many postganglionic cholinergic neurons supplying exocrine glands and blood vessels. VIP may also be colocalized with other peptides. It coexists with PHI or PHM, its human counterpart, both peptides being synthesized from the same precursor molecule, and may be colocalized with substance P. Other neurotransmitters that coexist with VIP in some of the neurons of bronchial ganglia include nitric oxide (nitric oxide synthase) and glutamate.

BIOLOGICAL ACTIONS PERTINENT TO THE LUNG These actions are summarized in Fig. 2.

Airways Airway smooth muscle tone: VIP relaxes airway smooth muscle, both in vitro and in vivo, in guinea pigs, rats,

His

HOOC

Ser

Asp Ala

Val

Phe

Thr

Asp Asn

Pulmonary Circulation

Tyr Ala

Met Gln

Lys

Arg

Lys

Tyr

Asn Ser

Leu

Arg

Thr

lle

Leu

Asn

Val Lys Leu

FIGURE 1. Amino acid sequence of VIP.

mice, rabbits, dogs, and humans. VIP also prevents or attenuates airway smooth muscle contraction by a variety of bronchoconstrictor agents, including histamine, prostaglandin F2α, kallikrein, leukotriene D4, neurokinin A, serotonin, and endothelin. VIPinduced airway relaxation is long-lasting and is unaffected by the blockade of adrenergic or cholinergic receptors or of cyclooxygenase activity. In limited trials in human asthmatics, VIP was generally less effective than expected in relieving bronchoconstriction, probably because of its degradation by proteases in airway secretions. Airway smooth muscle proliferation: VIP inhibits human airway smooth muscle proliferation, an action that is potentially beneficial in reducing the airway remodeling of bronchial asthma. Tracheobronchial secretion: VIP stimulates water and ion transport in canine tracheal epithelium and may also promote macromolecular secretion. The coexistence of VIP and acetylcholine in cholinergic neurons innervating bronchial and other exocrine glands brings together the blood flow–promoting and secretagogue effects of VIP and acetylcholine, respectively.

NH2

VIP dilates the vessels supplying the nose, upper airways, trachea, and bronchi, as well as pulmonary vessels. As a pulmonary vasodilator, VIP is 50 times as potent as prostacyclin, and its action is independent of endothelium. Given intravenously in sufficient concentrations, VIP reduces both pulmonary and systemic vas-

VIP Mediation of neurogenic airway smooth muscle relaxation

Modulation of airway & pulmonary vascular smooth muscle proliferation

Modulation of immune responses

Mediation of pulmonary vascular relaxation

Modulation of inflammation

Protection against apoptotic cell death, promotion of cell survival

FIGURE 2. Physiological roles of VIP in the lung.

Vasoactive Intestinal Peptide / 1309 cular resistance. Delivered as an aerosol, however, it is a selective pulmonary vasodilator. As with airway smooth muscle, VIP suppresses human pulmonary vascular smooth muscle proliferation, a major feature of pulmonary hypertensive disease.

Immune and Inflammatory Responses VIP modulates several aspects of the immune and inflammatory responses. First, it has a moderate inhibitory effect on antigen-induced release of histamine from guinea pig lung. Because the peptide is normally present in mast cells, the peptide may act as a natural modulator of mast-cell degranulation, an important mechanism underlying acute asthmatic and anaphylactic reactions. Second, VIP also modulates T-cell activation and inhibits the expression of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, IL-18] and chemokines (e.g., RANTES), inducible nitric oxide synthase (iNOS), and proteases (MMP-2 gelatinase), while upregulating the expression of anti-inflammatory cytokine IL-10.

Anti-Injury and Anti-Apoptotic Actions VIP prevents or attenuates experimental acute lung injury by a number of actions, including its antiapoptotic, pro-survival properties. The anti-apoptotic activity is attributable to three complementary mechanisms: inhibition of the activation of caspases, key effectors of apoptosis; upregulation of the anti-apoptotic protein bcl2; and suppression of cytoplasmic translocation of cytochrome c, a critical step in mitochondrialmediated apoptosis.

RECEPTORS Specific, high-affinity receptors for VIP have been identified in membrane preparations of normal lungs and human lung tumor cells. These receptors, localized immunocytochemically and by the increased cAMP levels resulting from activation by VIP, exist in a variety of pulmonary cells. Molecular and pharmacologic studies have led to the identification, characterization, and cloning of at least three receptors for VIP and the related peptide PACAP. These receptors, known as VPAC receptors, belong to a distinct family of seventransmembrane-domain receptors coupled to Gproteins. The three VPAC receptors are VPAC1 and VPAC2, both of which respond to VIP and PACAP with comparable affinity, and PAC1, which has greater affinity for PACAP than for VIP.

SIGNAL TRANSDUCTION PATHWAYS The actions of VIP are mediated principally by adenylyl cyclase stimulation and cAMP production. VIP biosynthesis itself is promoted by higher cAMP levels, and thus the peptide is potentially capable of stimulating its own generation. In some VIP actions, additional second messengers and signal transduction pathways are involved, for example, phospholipase C activation in the case of the PACAP-preferring PAC 1 receptor. Some actions of VIP, such as the relaxation of airway and vascular smooth muscle, are mediated in part by NO released from neurons or smooth muscle cells. Through its activation of a constitutive NOS in neural elements and smooth muscle, leading to activation of cytosolic guanylyl cyclase, VIP can stimulate intracellular cGMP formation. The contribution of neuronal NOS to VIP-induced relaxation of the trachea was recently examined in neuronal NOS knockout mice and was estimated to account for a significant proportion of the relaxation. It is now believed that both NO and carbon monoxide (CO) mediate VIP-induced relaxation of airway, gastrointestinal, and other smooth muscle. The combined activation of cAMP and cGMP pathways ensures greater smooth muscle relaxation, inhibition of mitogenesis, and smooth muscle proliferation.

PHYSIOLOGICAL ROLES A wide variety of experimental studies, including recent observations in VIP knockout mice, suggest that VIP has a number of important physiological roles in the lung (see Fig. 2), including: 1. Mediation of the dominant component of neurogenic (noncholinergic, nonadrenergic) airway smooth muscle relaxation. 2. Mediation of the same component of pulmonary vascular relaxation. 3. Modulation of airway and pulmonary smooth muscle proliferation. 4. Modulation of inflammation. 5. Modulation of the pulmonary immune response. 6. Protection against apoptotic cell death and promotion of cell survival.

VIP IN PULMONARY DISEASE Bronchial Asthma A possible causative link between VIP deficiency and the pathogenesis of asthma is suggested by two sets of

1310 / Chapter 181

VIP VIP

Pulmonary Circulation

Airways

•Relaxation of smooth muscle

•Relaxation of smooth muscle

•Inhibition of smooth muscle proliferation

•Inhibition of smooth muscle proliferation

•Suppression of inflammation & hyperreactivity

Potentially beneficial in bronchial asthma (VIP-immunoreactive nerves may be lacking in asthmatic airways)

Potentially beneficial in Primary Pulmonary Hypertension

Parenchymal Lung Cells

•Inhibition of apoptotic cell death; promotion of cell survival

Small Cell Lung Cancer Cells

•Suppression of proliferation in vitro & in vivo

•Modulation of inflammatory/immune damage

Protection against acute lung injury

Potentially beneficial in Small Cell Lung Cancer

(VIP-containing nerves are lacking in pulmonary arteries in PPH)

FIGURE 3. Potential therapeutic uses of VIP in pulmonary diseases, based on its known actions on various components of the respiratory system.

findings: (1) VIP-immunoreactive nerves were absent in airways of several severely asthmatic subjects who died accidentally, and (2) mice with targeted deletion of the VIP gene exhibited airway hyperresponsiveness and histopathologic features of airway inflammation.

TABLE 1. Possible Therapeutic Applications of VIP. Disorder Bronchial asthma

Cystic Fibrosis

Pulmonary hypertension

VIP-containing nerves that normally supply sweat glands were absent in a group of children with cystic fibrosis. But it is still not clear how this deficiency may relate to the pathophysiology of cystic fibrosis.

Acute lung injury/ARDS Small-cell lung cancer

Potential Benefit from VIP Suppresses bronchoconstriction, airway hyperreactivity, airway inflammation, airway remodeling Reduces pulmonary hypertension, inhibits pulmonary vascular smooth muscle proliferation Attenuates or prevents experimental acute lung injury Inhibits growth and proliferation of small-cell lung cancer cells

Primary Pulmonary Hypertension As is the case of asthma, a deficiency of VIP may be a factor in the pathogenesis of primary pulmonary hypertension (PPH). Serum VIP levels were low and VIP-containing nerves were absent in pulmonary arterial walls from PPH patients. At the same time, VIP receptors were upregulated in pulmonary arterial smooth muscle cells, and VIP inhibited the proliferation of these smooth muscle cells in vitro. Also, VIP knockout mice have pulmonary arterial hypertension and thickened arterial walls.

THERAPEUTIC POTENTIAL Preclinical data and early clinical trials support a therapeutic role for VIP or, in some instances, more suitable agonists in several lung disorders (Table 1).

Bronchial Asthma The airway-relaxant, anti-inflammatory, and smoothmuscle-proliferation-inhibitory effects of VIP are all

Vasoactive Intestinal Peptide / 1311 desirable anti-asthma effects. As already mentioned, early clinical trials of VIP as an aerosol in asthmatics showed modest benefit, probably because of its rapid inactivation by airway proteases. Longer-acting VIP-like peptides, which are more resistant to degradation, such as helodermin, PACAP, or synthetic analogs, may prove more effective.

Acute Lung Injury/Acute Respiratory Distress Syndrome VIP prevents or delays lung injury in 10 experimental models of acute lung injury (ALI), both in isolated lungs and in vivo. This protection is attributable to a combination of anti-inflammatory, anti-oxidant, and anti-apoptotic properties. In addition, male VIP knockout mice died earlier than wild-type mice as a result of endotoxemia. Clinical trials of VIP in acute respiratory distress syndrome (ARDS) are in progress, with the peptide delivered by intravenous (IV) infusion or as an aerosol.

Pulmonary Hypertension Given as an aerosol over 12–24 weeks, VIP markedly improved hemodynamics and exercise tolerance in patients with PPH.

Lung Cancer VIP suppressed the growth of small-cell lung cancer cells in culture and in athymic nude mice in vivo and may offer an effective and less toxic alternative to conventional chemotherapy in the management of this highly malignant form of cancer. But no clinical trials have been conducted with VIP in this disease.

References [1] Delgado M, Abad C, Martinez C, Leceta J, Gomariz RP. Vasoactive intestinal peptide prevents experimental arthritis by downregulating both autoimmune and inflammatory components of the disease. Nature Medicine, 2001. 7: 563–568. [2] Fahrenkrug J. Transmitter role of vasoactive intestinal peptide. Pharmacology and Toxicology, 1989. 72: 354–363. [3] Goetzl EJ, Voice JK, Shen S, Dorsam G, Kong Y, West KM, Morrison CF, Harmar AJ. Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide. Proceedings of the National Academy of Sciences USA, 2001. 98: 13854–13859. [4] Laburthe M, Couvineau A, Marie JC. VPAC receptors for VIP and PACAP. Receptors & Channels, 2002. 8: 137–153. [5] Maruno K, Absood A, Said SI. Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proceedings of the National Academy of Sciences USA, 1998. 95: 14373–14378. [6] Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K, Funk GC, Hamilton G, Novotny C, Burian B, Block LH. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. Journal of Clinical Investigation, 2003. 111: 1339–1346. [7] Said SI. Anti-inflammatory actions of VIP in the lungs and airways. In: Said SI, Editor Pro-Inflammatory and Anti-Inflammatory Peptides (vol. 112 in Lung Biology in Health and Disease, C. Lenfant, Exec. Editor), New York: Marcel Dekker, Inc., 1998, pp. 345– 361. [8] Said SI. Molecules that protect: the defense of neurons and other cells (editorial). Journal of Clinical Investigation, 1996. 97: 2163–2164. [9] Said SI. Vasoactive intestinal polypeptide and asthma (editorial). New England Journal of Medicine, 1989. 320: 1271–1273. [10] Said SI, Rattan S. The multiple mediators of neurogenic smooth muscle relaxation. Trends in Endocrinology and Metabolism, 2004. 15: 189–191.

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that was blocked by the opioid antagonist naloxone. Almost immediately, Simantov and Snyder [28] isolated and identified the same two pentapeptides using radiolabeled ligands in the brain. [Met5]-enkephalin has the amino acid structure of Tyr-Gly-Gly-Phe-Met (i.e., YGGFM), whereas [Leu5]-enkephalin differs in the fifth amino acid position and has a leucine molecule instead of methionine (i.e., YGGFL). In addition to [Met5]-enkephalin and [Leu5]enkephalin, there are a number of larger peptide fragments that include all or some of the enkephalin sequence. Some of these peptides have functions similar to that of the pentapeptides (e.g., octapeptide), whereas other peptide combinations do not resemble the functions of the five amino acid compounds.

ABSTRACT Proenkephalin-derived opioid peptides include [Met5]enkephalin and [Leu5]-enkephalin, as well as other derivatives. These peptides are processed from the preproenkephalin A gene and are distributed widely throughout the brain, but are also located in non-CNS structures, such as the gastrointestinal system, cardiovascular system, and placenta. These opioid peptides bind to classical μ and δ opioid receptors, and function as neuromodulators. However, [Met5]-enkephalin also serves as a potent inhibitor of cell proliferation and utilizes a novel nuclear-associated receptor, the opioid growth factor (OGF) receptor, for these growth-related actions.

DISCOVERY OF THE ENKEPHALINS

STRUCTURE OF THE PRECURSOR GENE

The discovery of opioid peptides in 1975 followed the identification of native opioid receptors in the brain and gastrointestinal tract in 1973 somewhat simultaneously in three laboratories [25, 29, 31]. Once the receptors were discovered, over the next 2 years researchers were in active pursuit of the endogenous ligands that bound to these receptors. Two different approaches were used for identifying native opioid compounds. In one approach, brain extracts were analyzed for morphinelike effects on smooth muscle, with special emphasis on actions that exhibited naloxone-reversible properties [10]. In another approach [24], radiolabeled binding assays were used to distinguish compounds that effectively competed for 3Hopioids such as dihydromorphine in opioid receptor binding assays. By 1975, brain extracts containing opioidlike activity were isolated and characterized as endogenous opioids [10, 11]. Hughes et al. [10] structurally identified two enkephalins, [Met5]-enkephalin and [Leu5]-enkephalin, that mimicked morphine in a manner Handbook of Biologically Active Peptides

The gene for the enkephalin pentapeptides is preproenkephalin A (PPE) [21, 22] from which both [Met5]enkephalin and [Leu5]-enkephalin are derived. The opioid products from all three genes associated with endogenous opioids (proopiomelanocortin, prodynorphin, and proenkephalin) share an N-terminal core sequence, but have different C-terminal amino acids and bind to different opioid receptors [4]. A comparison of the genes for proenkephalin, proopiomelanocortin (POMC), and prodynorphin indicates a highly conserved open reading frame (ORF) with nearly 90% of the ORF in each gene being coded on a single terminal exon [4].

DISTRIBUTION OF ENKEPHALIN mRNA AND PEPTIDE The determination of the location of the peptides was integral to the process of isolating them. Their

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1314 / Chapter 182 coexistence with opioid receptors was a key factor in their identification. The use of autoradiographic studies to determine the location of receptors provided a good estimate of where the enkephalins were distributed [29]. Once the peptides were isolated and purified, antibodies were produced that facilitated the visualization of enkephalins [27]. Early immunohistochemical studies detected enkephalins in axon terminals and cell bodies strongly supporting the role of the enkephalins as neurotransmitters [7, 8], as did the presence of enkephalins in small interneurons of the spinal cord [9]. Not until the advent of highly specific antisera for each peptide could a distinct location for [Met5]-enkephalin be discerned from that for [Leu5]enkephalin. Although each pentapeptide has distinct locations such as the [Met5]-enkephalin-enriched hippocampus and thalamus, neuronal populations frequently share both peptides. [Met5]-enkephalin is located in the central nervous system (CNS) structures, including the globus pallidus, hypothalamus, periaquaductal gray area, amygdala, and spinal cord. Because the two pentapeptides differ by only one amino acid that is coded by only one different base pair, it can be conjectured how the two peptides might be co-localized. Tissue distribution of the PPE gene has been identified predominately in the posterior pituitary (unlike proopiomelanocortin, which is found in the anterior pituitary), as well as in the spinal cord and sympathetic nervous system. PPE gene expression has also been detected by Northerns and in situ hybridization studies in other non-CNS structures such as the adrenal medulla, ocular tissue, gastrointestinal system including pancreas and intestine, cardiovascular system, and the placenta (e.g., [13, 19, 49, 54]). Gene expression of PPE may, in fact, coincide with the presence of the peptide in a variety of tissues, including cancers.

PROCESSING The PPE gene undergoes transcription, RNA processing, and translation to the preprohormone and prohormone of the same name—preproenkephalin A and proenkephalin, respectively. This prohormone undergoes further proteolysis and signal peptide cleavage to give rise to a variety of active proteins including [Leu5]-enkephalin, [Met5]-enkephalin, a heptapeptide termed Met-enkephalin-Arg-Phe, and an octapeptide termed proenkephalin. PPE codes for six copies of [Met5]-enkephalin and one copy of [Leu5]-enkephalin. The primary site for the processing of these peptides is within the secretory granules of the Golgi apparatus. Typically the cleavage occurs between pairs of basic amino acids such as Arg-Arg, Arg-Lys, and Lys-Lys.

Despite different phosphorylation and posttranslational processing, the enkephalins share the same four initial amino acids—tyrosine, glycine, glycine, and phenyalanine (YGGF)—with the N-terminus signifying distinctions in the resulting peptides. The processing of the prohormones is an ongoing event in order to sustain levels of peptides because both of the pentapeptides have very short half-lives, ranging from 2 to 5 minutes, in circulating blood [42]. A discussion on the processing of opioid genes would be remiss if gene duplication were not mentioned [4]. The striking similarity in the genes for proopiomelanocortin, prodynorphin, and proenkephalin suggests that gene duplication led to the formation of gene families in which there was some divergence in function as well as duplication of functional end products. Evidence supports that peptides from both prodynorphin and proenkephalin genes serve as analgesic molecules.

RECEPTORS Classical Opioid Receptors Enkephalins bind to opioid receptors, a seventransmembrane G-coupled-signaling molecule [30]. Enkephalins bind predominantly to δ (delta)- or μ (mu)-opioid receptors, of which there are several subsets with various terminology, including DOR1, DOR2 or δ1, δ2 and MOR1, MOR2 or μ1, μ2. The interaction between the agonists and their receptors activates transduction pathways that result in intracellular events such as calcium concentration changes, increased calcium influx into cells, increases in cGMP levels, and inhibition of phosphotidylinositol production [30]. Along with the natural endogenous ligands, there are several well-known exogenous ligands, including DPDPE ([D-Pen2, D-Pen2]enkephalin), DADLE (D-ala2, D-Leu5]enkephalin), DSLET ([D-Ser2, Leu5] enkephalin), and DTLET ([D-threonine2, Leu5] enkephalin), deltorphin I (Tyr-D-Ala-Phe-Asp-Val-ValGly-NH2) and deltorphin II (Tyr-D-Ala-Phe-Glu-Val-ValGly-NH2). Antagonists for δ- and μ-receptors, and thus for blocking the action of enkephalins, include nonspecific antagonists such as naloxone and naltrexone and more specific antagonists such as naltrindole, ICI174,864, and TIPP-ψ [30]. Classic opioid receptors for enkephalins are primarily located in the olfactory system, neocortex, dorsal horn of the CNS, primary afferent terminals, and limbic nuclei. Delta receptors, and thus presumably binding sites for enkephalins, have also been identified in cardiovascular tissues, cancerous cells, gastrointestinal

Proenkephalin-Derived Opioid Peptides / 1315 tract, and a variety of other nonneural related cells and tissues [30].

Nonclassical Opioid Receptor—Opioid Growth Factor Receptor In addition to the classic δ and μ receptors serving as binding sites for enkephalins, the opioid growth factor receptor (OGFr), also identified as zeta (ζ)opioid receptor, has been identified, isolated, characterized, cloned, and sequenced in mouse, rat, and human (see [53] for review). This receptor was initially discovered by pharmacological studies with radiolabeled OGF in developing rat brain [34, 35]; specific and saturable binding, with a one-site model of kinetics, was identified in the nuclear-enriched fraction of rat brain or cerebellar homogenates. Subcellular fractionation studies show that OGFr is an integral membrane protein associated with the nucleus [17, 36]. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse [51–53]. Classic opioid receptors, predominately δ, and OGFr differ from one another in a variety of structural and molecular characteristics [51, 52] (see [53] for review). The commonality between them is their hallmark opioid pharmacology. That is, their ability to bind the same endogenous opioid peptide in a specific and saturable manner and to do so in a naloxone-reversible way. Binding to OGFr was dependent on protein concentration, time, temperature, and pHs was sensitive to 100 nM Na+, Ca2+, and Mg2+; and was not markedly reduced by addition of guanyl nucleotides such as GppNHp. Optimal binding required protease inhibitors, and the pretreatment of the cell homogenates with trypsin markedly reduced [3H]-[Met5]-enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met5]-enkephalin was the most potent displacer of [3H]-[Met5]-enkephalin, although [Met5, Arg6, Gly7, Leu8]-enkephalin and [D-Ala5, D-Leu6]enkephalin also had Kis within approximately threefold to [Met5]-enkephalin in whole cell homogenate preparations. Drug displacement studies revealed a requirement of an N-terminus tyrosine group. Moreover, the binding of radiolabeled [Met5]-enkephalin displayed stereospecificity, with (−)-naloxone being 18-fold more potent than (+)-naloxone. The molecular and protein structures of OGFr have no resemblance to that of any classical opioid receptor and have no significant homologies to known domains or functional motifs, with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it

interfaces with OGF [48]. It is currently postulated that the peptide-receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in concert with the inner nuclear matrix or at the periphery of nuclear heterochromatin. Using immunoelectron microscopy and post-embedding technique, double-labeling experiments have revealed that complexes of OGF-OGFr co-localized on the outer nuclear envelope, in the paranuclear cytoplasm, perpendicular to the nuclear envelope in a putative nuclear pore complex, and in the nucleus adjacent to heterochromatin [48]. These data support the existence of an additional receptor for the endogenous peptide [Met5]enkephalin (i.e., OGF) and also indicate that signal transduction for cell proliferation appears to involve an OGF-OGFr complex that interfaces with chromatin in the nucleus.

BIOLOGICAL ACTIVITY OF ENKEPHALINS Neurotransmission The role of enkephalins has been widely studied and continues to develop as more specific antagonists and agonists are discovered. In general, enkephalins play a role in neurotransmission and pain modulation [1]. Their role as neurotransmitters was initially determined from localization studies in which immunohistochemical mapping revealed that enkephalins were in close proximity to autoradiographic mapping of opiate receptors [27]. The cellular localization of enkephalin suggested that its role was to act on opiate receptors positioned on terminals of sensory pain fibers and inhibit the release of neurotransmitters such as substance P [5, 12], vasopressin [14], and dopamine [23]. Along with the inhibitory role of enkephalins in preventing the release of transmitters is the modulatory role of enkephalins in altering calcium influx [20]. The direct hyperpolarization of neurons by enkephalins also supports their inclusion in the long list of neurotransmitters. Nonetheless, enkephalins have been implicated in a variety of functions in addition to classic neurotransmission. One such function is the regulation of cell proliferation as an inhibitory growth factor.

Enkephalins as Growth Factors During the early 1980s, the hypothesis was put forth that endogenous opioid peptides, particularly [Met5]enkephalin, are involved in the growth regulation of normal and abnormal cells and tissues. [Met5]-enkephalin was renamed OGF in order to distinguish the function of the peptide as a neuromodulator [1] and as an inhibitory growth factor [37–41, 44, 46, 50, 55]. In

1316 / Chapter 182 support of this hypothesis, a large number of laboratories have put forth evidence to support the role of OGF in governing the growth of developing, neoplastic, renewing, and healing tissues, with documentation of growth regulation in both prokaryotes and eukaryotes [2, 3, 6, 15, 16, 18, 26, 32, 33, 41, 42, 44, 46, 50, 54, 55]. OGF is a potent, reversible, species-unspecific, tissuenonspecific negative growth regulator with action that is opioid-receptor-mediated [42]. The peptide is autocrine and paracrine produced, secreted, and effective at concentrations consistent with physiological behavior. OGF is rapid in biological action and obedient to intrinsic rhythms of the cell (e.g., circadian rhythm). Our knowledge and understanding of OGF as a growth factor emanates from a decade or more of studies on normal neural tissue, namely the developing rat cerebellum and brain, and on abnormal neural tissues, namely neuroblastoma cells grown in vitro or transplanted into nude mice to produce tumors (e.g., [37–42, 44]). Nearly all of the characteristics of OGF as an inhibitory growth factor have been reproduced in all three models (normal brain, cancer cells in vitro, and tumors). To briefly summarize, OGF inhibits DNA synthesis in a receptor-mediated (i.e., OGFr) manner. The blockade of the OGF-OGFr interaction results in accelerated proliferation. OGF has been detected immunologically in tissues where the growth effects have been documented, and the specificity of OGF has been studied extensively. Using murine neuroblastoma cells (both in vitro and tumor transplantation) [37, 38] and in subsequent studies with the developing rat cerebellum [40], [Met5]-enkephalin (OGF) was discovered to be the most potent opioid peptide associated with growth. In the case of tumor cells, peptide concentrations as low as 10−10 M inhibited the growth of log-phase S20Y neuroblastoma cells exposed to drug for 48 h. Other PPE peptide derivatives such as the heptapeptide ([Met5, Arg6, Phe7]-enkephalin, octapeptide/proenkephalin ([Met5, Arg6, Gly7, Leu8]-enkephalin, and [Leu5]-enkephalin also exhibited inhibitory properties to some extent. The chemical structure of [Met5]enkephalin was also selective and specific. For example, no changes in cell growth occurred when the amino acids Tyr1 or Met5 were deleted, when smaller fragments of the [Met5]-enkephalin molecule such as TyrGly, Tyr-Gly-Gly, or even Met alone were used. Alterations in the basic structure of [Met5]-enkephalin (e.g., [Met5]enkephalinamide and [Met5]-enkephalin sulfoxide) did not yield compounds that were effective in inhibiting cell proliferation. Other opioid peptides and analogs related to the prodynorphin and proopiomelanocortin genes (e.g., β-endorphin, α-neo-endorphin, dynorphin B/rimorphin) and compounds selective for μ, δ, or κ opioid receptors (e.g., β-FNA, DADLE, EKC) had no significant influence on cultured cells even at concen-

trations at 10−6 M. In a wide variety of cells and tissues examined (e.g., colon cancer, heart, squamous cell carcinoma of the head and neck, pancreatic cancer, and developing nervous system) in humans and animals, both in vitro and in vivo, [Met5]-enkephalin has been identified as the primary opioid peptide involved with growth. The mechanism of OGF activity is currently a target of investigation. OGF is not cytotoxic and does not induce apoptosis as studied in a variety of neoplasias [45]. OGF does not play a major role in differentiation, as determined from a variety of assays using neuroblastoma cell lines and squamous cell carcinomas. Neither process outgrowth nor the distributions of βIII-tubulin, involucrin, or actin were altered by OGF treatment [43]. One target for the mechanism of OGF activity appears to be cell replication, with the G0/S phases of the cell cycle being likely candidates for pathways mediating the inhibitory effects of OGF [47]. Studies on the regulation of DNA synthesis and endogenous opioid peptides showed that [Met5]-enkephalin had a marked inhibitory effect on the labeling index of external germinal (granule) cells in the cerebellum of 6-day-old rats [39, 40], as well as in developing heart [18]. The inhibition of cell proliferation was eliminated with concomitant treatment of [Met5]-enkephalin and the short-acting, low-potency opioid antagonist naloxone, suggesting that the inhibitory effects on DNA synthesis were opioid-receptor-mediated. Moreover, there is a distinct developmental profile of binding to radiolabeled [Met5]enkephalin in human and rat brain, with maximal binding occurring at the time of cell proliferation and differentiation [34]. These results supported the hypothesis that endogenous opioid peptides, particularly [Met5]-enkephalin functioned as growth modulators.

PATHOPHYSIOLOGICAL IMPLICATIONS The basic science and clinical implications of the enkephalins are wide-ranging and have enormous potential. The native pentapeptides are not only produced by multiple genes, but have divergent functions and different sites of activity (i.e., receptors). Known to be conserved phylogenetically, enkephalins play a role in two very necessary processes of animal survival: growth and neurotransmission. As such, enkephalins have very broad-reaching implications in maintenance of mammalian homeostasis, as well as in various disease states. OGFr mRNA has been detected to a certain level in all organs of adult rodents. It may be that the homeostatic functions in these organs are minimal but that the genetic machinery is retained for cell restoration in case of injury. The OGF-OGFr axis has been shown

Proenkephalin-Derived Opioid Peptides / 1317 to be involved in the regulation of neoplasia, wound healing, normal homeostatic functions, embryonic development, and angiogenesis; other areas of activity have simply not yet been discovered. Mediation of enkephalin activity and harnessing the inhibitory action of enkephalins for therapeutic treatment and/or diagnostics have great clinical potential.

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[18] McLaughlin PJ, Zagon IS, Skitzki J. Human neuroblastoma cell growth in tissue culture is regulated by opioid growth factor. Int J Oncol 1999;14:373–380. [19] Messell TM, Iversen LL. Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 1977;268:549–551. [20] Mudge AW, Leeman SE, Fischbach GD. Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration. Proc Natl Acad Sci USA 1979;77:526–530. [21] Noda M, Furutani H, Takahashi M, Toyosato M, Notake S, Hakanishi S, Numa S. Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature 1982;295:202– 206. [22] Noda M, Furutani H, Takahashi M, Toyosato M, Notake S, Hakanishi S, Numa S. Isolation and structural organization of the human pre-proenkephalin gene. Nature 1982;297:431– 434. [23] North RA, Tonini M. The mechanism of action of narcotic analgesics in the guinea-pig ileum. Brit J Pharmacol 1977;61: 541–549. [24] Pasternak GW, Goodman R, Snyder SH. Characterization of an endogenous morphine-like factor (enkephalin) in mammalian brain. Mol Pharmacol 1975;12:504–513. [25] Pert CB, Snyder SH. Opiate receptor: Demonstration in nervous tissue. Science 1973;179:1011–1014. [26] Shahabi NA, Sharp BM. Antiproliferative effects of δ opioids on highly purified CD4+ and CD8+ murine T cells. J Pharmacol Exp Ther 1995;273:1105–1113. [27] Simantov R. Opioid peptide enkephalin: Immunohistochemical mapping in rat central nervous system. Proc Natl Acad Sci USA 1977;74:2167–2171. [28] Simantov R, Snyder SH. Morphine-like peptides in mammalian brain: Isolation, structure elucidation, and interactions with the opiate receptor. Proc Natl Acad Sci USA 1976;73:2515– 2519. [29] Simon EJ, Hiller JM, Edelman I. Stereospecific binding of the potent narcotic analgesic [3H]-etorphine to rat-brain homogenate. Proc Natl Acad Sci USA 1973;70:1947–1949. [30] Snyder SH. 2004. Opiate receptors and beyond: 30 years of neural signaling research. Neuropharmacology 47:274–285. [31] Terenius L. Characteristics of the ‘receptor’ for narcotic analgesics in synaptic plasma membrane from rat brain. Acta Pharmacol Toxicol 1973;33:377–384. [32] Vertes A, Kornyei JL, Kovacs S, Vertes M. Opioids regulate cell proliferation in the developing uterus: Effects during the period of sexual proliferation. J Steroid Biochem Mol Biol 1996;59:173– 178. [33] Villiger PJ, Lotz M. Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation. EMBO J 1992;11:135–143. [34] Zagon IS, Gibo DM, McLaughlin PJ. Zeta (ζ), a growth-related opioid receptor in developing rat cerebellum: Identification and characterization. Brain Res 1991;551:28–35. [35] Zagon IS, Gibo DM, McLaughlin PJ. Ontogeny of zeta (ζ), the opioid growth factor receptor, in the rat brain. Brain Res 1992;596:149–156. [36] Zagon IS, Goodman SR, McLaughlin PJ. Zeta (ζ), the opioid growth factor receptor: Identification and characterization of binding subunits. Brain Res 1993;605:50–56. [37] Zagon IS, McLaughlin PJ. Endogenous opioids and the growth regulation of a neural tumor. Life Sci 1988;43:1313–1318. [38] Zagon IS, McLaughlin PJ. Endogenous opioids regulate growth of neural tumor cells in culture. Brain Res 1989;490:14–25. [39] Zagon IS, McLaughlin PJ. Endogenous opioid systems regulate cell proliferation in the developing rat brain. Brain Res 1987;412:68–72.

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was approximately 700 times more potent than Leuenkephalin. For this reason, it was named dynorphin (1–13), from the greek prefix dyn “strength or power.” Later, the same laboratory reported the complete sequence of the endogenous heptadecapeptide named Dyn A(1–17). Soon thereafter, other laboratories described the sequence and distribution of the other dynorphins [8].

ABSTRACT The dynorphin family is composed of multiple peptides generated by posttranslational processing of prodynorphin. They are widely distributed in the central nervous system and occur in peripheral tissues, such as the heart, the gastrointestinal tract, and the adrenal gland. Dynorphin A(1–17) has been widely investigated because it can represent an endogenous ligand for the κ-opioid receptor. It displays several biological functions, including the regulation of pain transmission, feeding, and pituitary hormone release, and it may influence myocardial function. C-terminal fragments of dynorphin A(1–17), lacking the N-terminal tyrosine, do not bind to opioid receptors and are excitotoxic by interacting with glutamate receptors. Increased prodynorphin expression, observed in several pathophysiological conditions such as neuropathic pain and heart ischemia, may tilt the balance toward neurotoxic dynorphin derivatives that may contribute to tissue damage.

STRUCTURE OF THE PRECURSOR mRNA/GENE The cloning of the prodynorphin cDNA from a porcine hypothalamic library [14] confirmed that all dynorphins are contained and produced within the same protein-inactive precursor and are generated by cleavage at basic residues. The sequence of the prodynorphin gene has been reported for several species, including human. The coding gene contains four exons separated by three introns (Fig. 1). As reported in other opioid peptide genes, exons 1 and 4, which contain nontranslated sequences, are very large (1.4 and 2.2 kb, respectively). Exons 3 and 4 contain the translated regions; finally, prodynorphin mRNA possesses a very long nontranslated 3′-terminal end of approximately 1550 nucleotides. A variety of mechanisms have been shown to lead to species-, tissue-, and treatment-specific differences in the structure of prodynorphin mRNA transcripts, and some of these different structural forms affect function. For example, in rat testis, alternative splicing of the preprodynorphin mRNA yields a mature mRNA that lacks exon 2. This exon codes for part of the 5′ untranslated region; the lack of this exon has no apparent effect on translational efficiency [18]. At the peptide level, Dyn A(1–17) is completely conserved across all mammalian species in which its sequence has been determined; however, at the level of the mRNA

DISCOVERY Dynorphins include multiple bioactive peptides generated by posttranslational processing of the large precursor prodynorphin [8]; they include α-neoendorphin (α-NE), big dynorphin (Dyn A 1–32), leumorphin (Dyn B 1–29), dynorphin A (Dyn A 1–17), dynorphin B (Dyn B 1–13), leucine-enkephalin-arginine (Leuenkephalin-Arg), and potentially leucine-enkephalin (Leu-enkephalin) (Fig. 1). In 1979, Avram Goldstein and his colleagues reported the characterization and partial sequence of a highly potent opioid peptide obtained from pituitary extracts. This tridecapeptide contained the amino acid sequence for Leu-enkephalin at its N-terminus [9] and in the guinea pig ileum longitudinal muscle preparation it Handbook of Biologically Active Peptides

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1320 / Chapter 183 EXON 1 (148 bp)

EXON 2 (60 bp)

Intron

EXON 3 (147 bp)

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AAAAAA mRNA (2.4 Kb ca)

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Dyn A(1-32) Big Dynorphin

Dyn B(1-29)

β-NE Dyn A(1-17)

Dyn B(1-13)

Dyn A(1-8) Leu-Enk-Arg

Dyn A(2-17) C-Terminal Fragment

Leu-Enk

FIGURE 1. Prodynorphin (ProDyn) gene, messenger RNA, protein precursor structure, and processing pathways. Basic amino acid cleavage sites in the precursor are denoted by their single letter codes. K, lysine; R, arginine. Solid box corresponds to the sequence of Leu-enkephalin.

primary structure an assortment of mechanisms, including alternative splicing, differential transcription initiation, and site-specific cleavage, lead to the generation of species-, tissue-, and treatment-specific mRNAs for this peptide precursor [18]. The study of mRNA transcripts from the prodynorphin gene continues to provide examples of general and specific mechanisms of regulation of mRNA structure and also, in some instances, mRNA function. The functional significance of the longer form of the preprodynorphin mRNA is as yet unknown, but may possibly alter translational efficiency or mRNA stability. Several transcription factor binding sites were identified in the promoter region of the prodynorphin gene and found to be responsible for the enhanced prodynorphin mRNA expression in response to AMP or protein kinase A in the rat [6].

PROCESSING The most common processing signal sequences of the prodynorphin precursor, which is highly basic, are the lysine-arginine (KR), the lysine-lysine (KK), and the arginine-argine (RR) residues; these are cleaved on the carboxyl side of the basic amino acids and followed by removal of the C-terminal basic residue. An endopeptidase-designated dynorphin-converting enzyme (DCE) cleaves neuropeptides such as dynorphins at monobasic sites. Following endopeptidase activity, carboxypeptidases remove the basic amino acids from the C-terminus of peptides. Aminopeptidases capable of removing N-terminal Arg or Lys residues from the processed peptides have also been reported [5]. The relative levels of endopeptidase can

Prodynorphin-Derived Opioid Peptides / 1321 have a large effect on the bioactivity of neuropeptides because differential proteolysis of a single precursor can result in the generation of several neuropeptides that activate a diverse set of receptors. Thus, processing enzymes play a key role in the modulation of neuropeptide gene expression. Additional bioactive derivatives of prodynorphin probably exist in addition to those previously indicated. Many of the C-terminal fragments of dynorphin A [most likely Dyn A(2–17)], which are intrinsically neurotoxic, have been isolated from neural tissue in vivo or in vitro [11]. Moreover, neuropeptide processing may occur following secretion. Initial studies carried out to isolate dynorphins focused on the characterization of small fragments corresponding to the end products of prodynorphin precursor processing. However, using antibodies directed against Dyn A or Dyn B, radioimmunoassay analysis of gel-fractioned tissue extracts revealed that in certain tissues large-molecular-weight intermediates that were subsequently characterized [4]. The anterior pituitary gland contains several prodynorphin-processing intermediates: a 4-kDa peptide containing Dyn A and Dyn B [Dyn A (1–32)]; a 6-kDa fragment comprising Dyn A(1–32) and a C-terminal fragments an 8-kDa peptide having α-neoendorphin at the N-terminal position of the 6-kDa fragment; and, finally, a 16-kDa fragment comprising the N-terminal portion of the precursor plus α-neoendorphin and a 10-kDa fragment containing α-neoendorphin and Dyn A(1–32). Other intermediates, occurring in a small amount (approximately 10–20%) have not been well characterized. In other brain areas containing dynorphins, these intermediates are present only in trace amounts and the major components are the small dynorphins such as Dyn A(1–17), Dyn B, and α-neoendorphin [4]. Thus, this suggests that a tissue-specific processing requires the expression of particular processing enzymes.

RECEPTORS Radioreceptor binding assays in brain membrane fragments and bioassays in isolated intact tissues have ascertained that Dyn A(1–17) and the other dynorphins bind to κ-opioid receptors (KOR) with somewhat higher affinity than μ- or δ-opioid receptors (MOR and DOR, respectively), and Dyn A(1–17) is considered to be an endogenous ligand for the KOR [8]. Dyn A(1–8) and α-neoendorphin have lower affinities for the KOR, with significant affinity for the MOR and DOR sites. In agreement with the preferential binding to KOR, Dyn A(1–17)–elicited effects on isolated tissue preparations can be blocked by selective KOR antagonists such as nor-binaltorphine [3].

CONFORMATIONAL ANALYSIS OF DYN A(1–17) Structural studies of dynorphin in the solid-state and the membrane environment show that the N-terminus forms an α-helical structure and is inserted into the membrane with the helical axis almost perpendicular to the membrane surface. It has been suggested that the helical region of the extracellular loop II of the KOR may interact with the helical region of dynorphin with a high affinity in the membrane environment [19].

DYNORPHIN EXPRESSION IN THE CENTRAL NERVOUS SYSTEM AND POSSIBLE BIOLOGICAL ACTIONS Initial immunocytochemical studies of dynorphins were carried out in the rat hypothalamus, where Dyn (1–13) was found colocalized with vasopressin in magnocellular neurons of the supraoptic and paraventricular nuclei [15]. The immunocytochemical distribution of peptides derived from the prodynorphin precursor in the brain of the rhesus monkey (Macaca mulatta) indicates a widespread neuronal localization of immunoreactivity from the cerebral cortex to the caudal medulla. Immunoreactive perikarya are located in numerous brain loci, including the cingulate cortex, caudate nucleus, amygdala, hypothalamus, thalamus, substantia grisea centralis, parabrachial nucleus, nucleus tractus solitarius, and spinal cord dorsal gray laminae. In addition, fiber and terminal immunoreactivity are seen in varying densities in the striatum and pallidum, substantia innominata, hypothalamus, substantia nigra pars reticulata, parabrachial nucleus, spinal trigeminal nucleus, and other areas. The distribution of prodynorphin peptides in the brain of the monkey is similar to that described for the rat brain; however, significant differences also exist. In situ hybridization histochemistry has been adopted as a technique to investigate the localization of prodynorphin mRNA in the central nervous system; this approach has confirmed that prodynorphin-containing cells are relatively widespread throughout brain regions that contain dynorphin peptides [15]. Dynorphins play a role in a wide variety of physiological parameters, including motor activity, cardiovascular regulation, respiration, temperature regulation, feeding behavior, and hormone release [25]. Although they do not elevate pain threshold when injected in the brain, they antagonize opioid analgesia in naive animals and potentiate it in tolerant animals. Dynorphins have beneficial effects on stroke that are like those of

1322 / Chapter 183 opioid antagonists rather than like those of agonists [25]. Prodynorphin-positive neurons are widely distributed in brain and spinal cord areas involved in the transmission of nociceptive stimuli [15]. Dynorphins may be involved in a local circuit within the spinal cord, as well as in supraspinal functions. Interestingly, dynorphins were also detectable in cutaneous nerves with a distribution similar to that of calcitonin gene–related peptide, a specific marker for sensory neurons. A moderate density of KOR binding sites has been seen in the central and peripheral neuronal nociceptive system and in various immune cells [12]. A lack of an antinociceptive action of dynorphin after its administration into the lateral brain ventricle [21] and some slight antinociceptive activity after intrathecal injection [21, 25] have been reported. Other KOR agonists also possess some antinociceptive activity after their intrathecal administration. However, the effect is much weaker on a molar basis than that evoked by MOR or DOR agonists. On the other hand, in electrophysiological experiments in spinalized rats, KOR agonists reduced reflexes stimulated by thermal and mechanical nociceptive stimuli to the same extent and in a dose-dependent manner [17, 21]. Dynorphin-mediated analgesia has been ascribed to its inhibitory action on neurons at KOR. Electrophysiological evidence supports a KOR-mediated inhibitory effect of dynorphins on synaptic transmission of nociceptive neurons in the spinal dorsal horn [21]. Dynorphins, as well as other neurotransmitters, can be released and can induce the hyperpolarization of neurons, potentially through a KOR-coupled enhancement of potassium conductance. In addition, dynorphin-mediated activation of KOR suppresses calcium currents and calcium-dependent secretion [2]. Furthermore, dynorphin has been shown to inhibit substance P release in the spinal cord in a KOR-mediated manner [17]. The endogenous dynorphin-KOR system has been suggested to elicit antinociception during inflammation, pregnancy [21], and acupuncture [10] and to mediate cannabinoid-induced antinociception [24]. These studies support an antinociceptive function of dynorphin by negatively modulating transmission of nociceptive information.

PATHOPHYSIOLOGICAL IMPLICATIONS Observation of the antinociceptive actions of dynorphin in the spinal cord is hindered by its neurotoxic effects. These can result from a nonopioidergic mechanism of action of the peptide [11, 17]. The involvement of spinal dynorphins in animal models of inflammatory pain have been studied in great detail. Profound alterations in the spinal prodynorphin system has been observed when there is peripheral inflammation or

chronic arthritis [23, 29]. The levels of immunoreactive dynorphin were dramatically increased in the lumbar part of the spinal cord of rats with local inflammation of a hind limb. Furthermore, an increase in prodynorphin mRNA levels induced by acute and chronic inflammation has also been observed in the spinal cord. Thus, many forms of peripheral inflammation induce a dramatic upregulation of prodynorphin biosynthesis in nociceptive neurons of the spinal dorsal horn, which parallels the behavioral hyperalgesia associated with the inflammation. There is much evidence that nerve injury is associated with elevated levels of spinal dynorphins. Spinal injury, which may cause neuropathic pain, also increases the dynorphin level in the spinal cord. Both prodynorphin mRNA and dynorphin levels were significantly increased, whereas those of proenkephalin mRNA and Met-enkephalin remained unchanged 3 days after injury. The increase in the spinal levels of prodynorphin mRNA was highest in the areas close to the side of transaction [17]. Thus, endogenous dynorphin levels increase under various conditions associated with neuropathic pain following damage to the spinal cord and injury of peripheral nerves. The site and time of increase of the dynorphin content in the spinal cord after peripheral nerve injury appear to correlate with the appearance of neuropathic pain. Furthermore, a single intrathecal injection of Dyn A(1–17) to mice induced mechanical allodynia and cold allodynia (acetone applied to the dorsal hind paw) lasting several days [21]. Interestingly, Dyn A(2–17), a nonopioid peptide, induced cold and tactile allodynia analogous to that induced by Dyn A(1–17), indicating the involvement of nonopioid receptors. Furthermore, Dyn A(1–17) has nonopioid activity and may damage the spinal cord when given in high doses and may produce hind-limb paralysis when administered intrathecally to rats. This effect is mediated at least partly via nonopioid receptors because the des–Tyr fragment of dynorphin can induce a similar effect [21]. Recently, Dyn A(1–32), intrathecally administered in mice, was shown to be a more potent nociceptive peptide than Dyn A(1–17), and its effect was not mediated by opioid receptors; rather, it involved NMDA receptors [11]. Considerable evidence reveals that dynorphin is actually pronociceptive in chronic pain states. The idea that dynorphins may contribute to the maintenance of neuropathic pain states has been tested in prodynorphin knockout mice [22]. They develop neuropathic pain after nerve injury, but fail to sustain it for extended periods of time and, by 10 days after injury, they are no more sensitive than they were prior to injury. In contrast, wild-type animals continue to display neuropathic pain. Thus, spinal dynorphins are critical for the maintenance of neuropathic pain, and their upregulation may represent a mechanism for the maintenance of the neuropathic pain state. Several investigators have dem-

Prodynorphin-Derived Opioid Peptides / 1323 onstrated that neurological damage observed following intrathecal dynorphin administration can be prevented by antagonists and modulators of the NMDA receptor, which is known to be an important mediator of neuropathic pain states. Moreover, the long-lasting hyperresponsiveness to both innocuous and noxious stimuli that is elicited by intrathecal dynorphin is not blocked by the opioid receptor antagonist naloxone, but it is prevented by pretreatment with the NMDA receptor antagonist MK-801 [17]. This finding suggests that the NMDA receptor is a likely mediator of the nonopioid actions of dynorphin. The interaction of endogenous dynorphins and NMDA receptors in promoting neuropathic pain may be a consequence of binding of dynorphins to NMDA or they may act as a positive allosteric modulator. Interestingly, Dyn A(2–17) binds to NMDA receptor R1/R2A subunits showing high-affinity binding. Substantial evidence suggests that dynorphin facilitates excitatory amino acid release, and because the binding of dynorphin to NMDA receptors is attenuated by NMDA agonists, dynorphin may not interact appreciably with NMDA receptors under neuronal hyperexcitable conditions. It has been proposed that physiological concentrations of dynorphins are antinociceptive and neuroprotective through opiate receptor activation, whereas extremely elevated levels are pronociceptive and even excitotoxic in an NMDA-receptor-dependent fashion [11]. Recent findings indicate that dynorphins can exert protective or proapoptotic effects depending on whether they activate opioid receptors or nonopioid mechanisms (i.e., glutamate receptors or proteinprotein interactions) and that they seem to contribute to nervous system pathology through complex interactions involving multiple receptors and pathways [11].

DRUG ABUSE AND DYNORPHINS Rewarding effects and compulsive drug-seeking behavior, characterizing drug addiction, are induced by various drugs of abuse and may require an increase of dopamine neurotransmission in the nucleus accumbens and in the striatum and a glutamate-dependent neuroplasticity. Dynorphins, locally released, may modulate dopamine neurotransmission and interfere with psychostimulants and other drugs that increase it [16, 24]. Furthermore, these drugs of abuse may induce prodynorphin gene expression in both the nucleus accumbens and striatum through the activation of D1 dopamine receptors [24]. Interestingly, in rats exposed to the anabolic androgenic steroid nandrolone, elevated dynorphin levels were observed in brain regions regulating emotions, dependence, and defensive reactions [13]. The authors have proposed that brain dynorphins may participate in behavioral modifications

induced by anabolic steroids. Therefore, it has been suggested that the increase of endogenous dynorphins, caused by drugs of abuse, may counteract the effects elicited by these compounds on dopamine neurotransmission [11]. However, it cannot be excluded that considerable higher levels of dynorphins may also interfere with glutamate receptors and may promote, instead of counteract, neuroplastic changes that promote drugseeking behavior. Exposure to high doses of psychostimulants may cause a neutotoxic loss of neurons and apoptotic cell death [11]. Therefore, as previously described for the spinal effects of dynorphins, it remains to be established whether these peptides may have a dichotomy of actions: At lower levels they counteract drug addiction, whereas at supraphysiological levels they may contribute to drug abuse toxicity. In this regard, it should be mentioned that dynorphin dysregulation may be associated with various neurological disorders such as Parkinson’s and Huntington’s disease; moreover, dynorphin alternations have been associated with the pathogenesis of refractory seizures [11].

DYNORPHIN EXPRESSION IN PERIPHERAL TISSUES In the periphery, numerous tissues express prodynorphin-derived peptides. Initally, Spampinato and Goldstein [27] reported the presence of immunoreactive dynorphin in the following rat tissues: stomach, intestine, adrenal gland, ovary, testis, skeletal muscle, and heart. Gel-permeation chromatography revealed a larger apparent molecule than that of Dyn A(1–17). Moreover, dynorphins have been detected in rat and human plasma [27, 28]. However, the processing and the physiological role played by dynorphins in peripheral tissues are still largely unknown. The heart has drawn particular attention for its capacity to biosynthesize dynorphins in the rat atrial and ventricular tissue; dynorphins also occur in the human heart [26]. In single cardiac myocites, Dyn A(1–17) elicited an increase of cytosolic calcium via KOR. These peptides could play an early but crucial role in ischemic tolerance; in the rabbit model, this was explained by the unique role kappa-opioids provide in adrenergic regulation. Only KOR are found on presynaptic nerve terminals, and nerve terminal secretory vesicles contain dynorphin peptides. Both dynorphins and norepinephrine are released from nerve terminals during ischemia, but dynorphin activation of KOR then results in negative feedback on norepinephrine release [20]. As a governor of catecholamine stimulation during ischemia, dynorphins may reduce postischemic injury caused by deleterious, adrenergically-enhanced cellular metabolism and contractile function. Nonselective blockade of opioid receptors increases the canine cardiac response

1324 / Chapter 183 to adrenergic stimulation, supporting the regulatory role of endogenous opioids. The influence of KOR agonists may thus be confined to early events in ischemic injury to temper sympathetic stimulation. However, in the isolated heart Dyn A(1–17) may exacerbate infarct size and increase hydroxyl radical production [1]. Thus, this peptide may exert opposing effects on the ischemic heart depending on concentration, exposure time, and experimental model. Finally, circulating dynorphins may contribute to pressor effects observed in hypertensive patients during episodes of stress-induced blood pressure increase [7].

PERSPECTIVE Dynorphins are multifunctional peptides acting through different receptors in either a protective or toxic way. These interactions depend on the manner in which dynorphins are processed and degraded. Dynorphins display multiple effects in the central nervous system as well as in peripheral tissues. In recent years, novel approaches based on the use of prodynorphin knock-out mice and animal models of diseases have provided new vistas on the role played by these peptides as modulators of injury and disease outcome in the central nervous system and in the heart.

References [1] Aitchison KA, Baxter GF, Awan MM, Smith RM, Yellon DM, Opie LH. Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning. Basic Res Cardiol 2000; 95: 1– 10. [2] Chavkin C. Dynorphins are endogenous opioid peptides released from granule cells to act neurohumorly and inhibit excitatory neurotransmission in the hippocampus. Prog Brain Res 2000; 125: 363–67. [3] Corbett AD, Paterson SJ, Kosterlitz HW. Selectivity of ligands for opioid receptors. In: Herz A, editor. Opioids I, Berlin: SpringerVerlag; 1993; p. 645–79. [4] Day R, Trujillo KA, Akil H. Prodynorphin biosynthesis and posttranslational processing. In: Herz A, editor. Opioids I, Berlin: Springer-Verlag; 1993; p. 449–70. [5] Devi LA. Dynorphin processing endoprotease. In: Woessner F, Rawling N, Barrett AJ, editors. Handbook of Proteolytic Enzymes, San Diego: Academic Press; 1998, p. 1449–50. [6] Douglass J, McKinzie AA, Pollock KM. Identifiction of multiple DNA elements regulating basal and protein kinase A-induced transcriptional expression of the rat prodynorphin gene. Mol Endocrinol 1994; 8: 333–44. [7] Fontana F, Bernardi P, Spampinato S, Boschi S, De Iasio R, Grossi G. Pressor effects of endogenous opioid system during acute episodes of blood pressure increases in hypertensive patients. Hypertension 1997; 29: 105–10. [8] Goldstein A. The peptides: analysis, synthesis, biology. In: Udenfriend S, Meienhofer J, editors. New York: Academic Press; 1984, p. 95–123.

[9] Goldstein A, Tachibana S, Lowney LI, Hunkapillar M, Hood L. Dynorphin (1–13), an extraordinary potent opioid peptide. Proc Natl Acad Sci USA 1979; 76: 6666–70. [10] Han JS. Acupuncture and endorphins. Neurosci Lett 2004; 361: 258–61. [11] Hauser KF, Aldrich JF, Anderson KJ, Bakalkin G, Christie MJ, Hall ED, et al. Pathobiology of dynorphins in trauma and disease. Frontiers Biosci 2005; 10: 216–35. [12] Herz A. Peripheral opioid analgesia—facts and mechanisms. Prog Brain Res 1996; 110: 95–104. [13] Johansson P, Hallberg M, Kindlundh A, Nyberg F. The effect on opioid peptides in the rat brain, after chronic treatment with the anabolic androgenic steroid, nandrolone decanoate. Brain Res Bull 2000; 51: 413–18. [14] Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, Hirose T, et al. Cloning and sequence analysis of cDNA for porcine beta-neoendorphin/dynorphin precursor. Nature 1982; 298: 245–49. [15] Khachaturian H, Schaefer MKH, Lewis ME. Anatomy and function of the endogenous opioid systems. In: Herz A, editor. Opioids I, Berlin: Springer-Verlag; 1993; p. 471–97. [16] Kreek MJ. Cocaine, dopamine and the endogenous opioid system. J Addict Dis 1996; 15: 73–96. [17] Lai J, Ossipov MH, Vanderah TW, Malan TP Jr, Porreca F. Neuropathic pain: the paradox of dynorphin. Mol Interventions 2001; 1: 160–67. [18] Mayer P, Hollt V. Allelic and somatic variations in the endogenous opioid system of humans. Pharmacol Ther 2001; 91: 67–177. [19] Naito A, Nishimura K. Conformational analysis of opioid peptides in the solid states and the membrane environments by NMR spectroscopy. Curr Top Med Chem 2004; 4: 135–45. [20] Pepe S, van den Brink OW, Lakatta EG, Xiao RP. Cross-talk of opioid peptide receptor and beta-adrenergic receptor signalling in the heart. Cardiovasc Res 2004; 63: 414–22. [21] Przewlocki R, Przewlocka B. Opioids in chronic pain. Eur J Pharmacol 2001; 429: 79–91. [22] Sharifi N, Diehl N, Yaswen L, Brennan MB, Hochgeschwender U. Generation of dynorphin knockout mice. Mol Brain Res 2001; 86: 70–75. [23] Sharma HS, Olsson Y, Nyberg F. Influence of dynorphin A antibodies on the formation of edema and cell changes in spinal cord trauma. Prog Brain Res 1995; 104: 401–16. [24] Shippenberg TS, Chefer V. Opioid modulation of psychomotor stimulant effects. In: Maldonado R, editor. Molecular Biology of Drug Addiction, Totowa, NJ: Humana Press; 2002, p. 107– 33. [25] Smith AP, Lee NM. Pharmacology of dynorphin. Ann Rev Pharmacol Toxicol 1988; 28: 123–40. [26] Spampinato S, Canossa M, Ventura C, Bachetti T, Venturini R, Bastagli L, Bernardi P, Ferri S. Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guineapig heart. Life Sci 1991; 48: 551–59. [27] Spampinato S, Goldstein A. Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 1983; 3: 193–212. [28] Spampinato S, Paradisi R, Canossa M, Campana G, Frank G, Flamigni F, Ferri S. Immunoreactive dynorphin A-like material in extracted human hypothalamic-hypophysial plasma. Life Sci 1992; 52: 223–30. [29] Weihe E, Millan MJ, Hollt V, Nohr D, Herz A. Induction of the gene encoding pro-dynorphin by experimentally induced arthritis enhances staining for dynorphin in the spinal cord of rats. Neuroscience 1989; 31: 77–95.

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184 POMC Opioid Peptides MARGARET E. SMITH

differs from that of other mammalian species in that a tyrosine rather than a histidine residue is present at position 27. Figure 1 compares human and bovine βendorphin (1–31).

ABSTRACT The major opioid peptide derived from proopiomelanocortin (POMC) is β-endorphin(1–31). It is expressed in the pituitary and in various regions of the brain, notably the arcuate nucleus in the hypothalamus. It contains the pentapeptide opioid sequence at its Nterminal, which is responsible for its analgesic potency and its roles in euphoria and drug dependence. The C-terminal dipeptide probably also confers biological activity, including trophic actions and reduction of muscle fatigue during exercise. β-Endorphin can be processed in some tissues by C-terminal proteolysis to the biologically active derivatives β-endorphin(1–27) and glycylglutamine. It can also be α,N-acetylated, which reduces its analgesic potency.

DISCOVERY Opioid receptors were discovered in 1973, before their endogenous ligands were identified, in pharmacological studies of morphine analogs. The discovery of these receptors led to a search for their natural ligands, and in 1975 Hughes et al. [16] isolated two pentapeptides, Met- and Leu-enkephalin, that could compete with morphine for opiate receptors in the brain and exhibited pharmacological actions similar to those of morphine. The Met-enkephalin amino acid sequence was then identified in β-lipotropin (β-LPH), and, at around the same time, α-, β-, and γ-endorphins, which consisted of amino acid sequences present in β-LPH, were isolated. These peptides, in particular β-endorphin, also exhibited morphinelike actions.

INTRODUCTION The opioid peptides derived from proopiomelanocortin (POMC) are β-endorphin(1–31), α-endorphin (β-endorphin 1–16), and γ-endorphin (β-endorphin 1–17) (see Fig. 1). These peptides contain the Nterminal pentapeptide opioid sequence Tyr-Gly-GlyPhe-Met, which is also the sequence of Met-enkephalin, an opioid peptide derived from proenkephalin. Other opioid peptides, Leu-enkephalin, and dynorphins (derived from prodynorphin) differ from the endorphins and Met-enkephalin in a leucine, not a methionine residue at position 5. β-Endorphin is an opiate by virtue of its N-terminal pentapeptide sequence and a pleiotropic hormone by virtue of various regions of its molecular sequence, including the C-terminal di- or tetrapeptide region that is involved in biological activity but not analgesia. In most mammalian species, the Cterminal amino acid is glycyl-glutamine, but in the human it is glutamic acid. Human β-endorphin also Handbook of Biologically Active Peptides

DISTRIBUTION The POMC gene is described in Chapter 96 of the Brain Peptides Section of this book. The most potent opioid peptide derived from POMC is β-endorphin(1–31). Its immediate precursor is β-lipotropin (β-LPH), which is processed to γ-LPH (β-LPH 1–60) and β-endorphin (β-LPH 61–91). In the pituitary β-LPH and β-endorphin are expressed in the corticotrophs in the anterior lobe and the melanotrophs of the intermediate lobe. In adult humans, a discrete intermediate lobe is lacking, and β-endorphin is synthesized in the anterior pituitary corticotrophs as well as in scattered POMC-expressing cells in the zona

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Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-LysLys-Gly-Glu

B

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-His-LysLys-Gly-Gln

FIGURE 1. Amino acid sequence of (A) human (B) bovine, camel, and sheep β-endorphin(1–31).

intermedia (the vestigial intermediate lobe). βEndorphin(1–31) is the principal endorphin peptide in corticotrophs, but attenuated and acetylated derivatives of β-endorphin(1–31) are expressed in melanotrophs. In the brain, POMC-derived peptides have a more restricted distribution than enkephalins or dynorphins. The highest concentrations of β-endorphin are in the arcuate nucleus of the mediobasal hypothalamus, where it is strongly expressed in a group of large cell bodies. The arcuate nucleus neurons project to the nucleus accumbens, the periaqueductal gray matter (PAG), the reticular formation, and the medial amygdala. Many of the areas that express β-endorphin have been implicated in the pain response, including the hypothalamic, limbic, raphe, and pontine nuclei. β-Endorphin is also present in the median eminence, the ventromedial border of the third ventricle, the thalamus, midbrain, hippocampus, dorsal colliculae, the nucleus tractus solitarius (NTS) of the caudal medulla, and the spinal cord. In the spinal cord some POMC-peptide expression is in neuronal processes of supraspinal origin, but some is in neurons intrinsic to the spinal cord. β-Endorphin(1–31) is the principal form expressed in the hypothalamus, midbrain, and amygdala, but smaller amounts of β-endorphin(1–26) are also present in the midbrain and amygdala. A different pattern is seen in the hippocampus, colliculae, and some other brain-stem areas, where mainly N-acetylated forms of β-endorphin(1–27) and β-endorphin(1– 26) are expressed [39]. In addition β-endorphin is present in many peripheral tissues, including the gastrointestinal tract, pancreas, adrenal medulla, skeletal muscle, and skin.

PROCESSING The processing of the POMC prohormone yields a number of biologically active neuropeptides. An outline of POMC processing is given in Chapter 96 on melanocortins. It is accomplished in a tissue-specific manner

that enables functional diversity. In the corticotrophs of the anterior pituitary and the melanotrophs of the intermediate pituitary, POMC is cleaved to adreno corticotrophic hormone (ACTH) 1–39 and β-lipotropin (β-LPH) in a reaction catalyzed by prohormone convertase 1/3 (PC1/3), which hydrolyzes peptides at pairs of basic residues. Further processing of β-LPH to γ-LPH (β-LPH 1–60) and β-endorphin(1–31) (β-LPH 61–91) is accomplished by prohormone convertase 2 (PC2). The concentration of this enzyme is lower in the anterior lobe than the intermediate lobe, and consequently β-LPH is present in higher concentrations than βendorphin(1–31) in the corticotrophs. Nevertheless, only small amounts of β-endorphin(1–31) (and β-LPH) are present in melanotrophs, where secondary processing of β-endorphin gives rise to relatively high concentrations of β-endorphin(1–27) and β-endorphin(1–26), together with their acetylated forms. Thus, β-endorphin can be further processed in some tissues by cleavage at basic (lysine) residues to βendorphin(1–26) and β-endorphin(1–27). The conversion of β-endorphin(1–31) to β-endorphin(1–27) is a two-step process involving a specific endopeptidase and a carboxypeptidase B (Fig. 2). Two lysine molecules and the C-terminal dipeptide glycyl-glutamine (or glycylglutamate in humans) are also produced. Glylglutamine immunoreactiviy has been demonstrated in the intermediate pituitary [31]. Both β-endorphin(1–27) and the dipeptide are biologically active, although they exhibit negligible analgesic activity. Glycylglutamine was first shown to be biologically active by Parish et al. [30], who showed that it inhibited the firing of cells in the brain-stem reticular formation. β-Endorphin and its attenuated peptides all undergo N-acetylation in melanotrophs in most mammalian species, and a β-endorphin N-acetyltransferase has been detected in secretory granules of the intermediate lobe. C-terminal proteolysis and α, N-acetylation alter the biological activity of β-endorphin, causing a marked reduction in analgesic potency. Indeed, these reactions may be physiological mechanisms for modifying the activity of β-endorphin.

POMC Opioid Peptides / 1327 1

27

31

N-acetyltransferase a, N-acetyl b-EP 1-31 His-Lys-Lys-Gly-Gln endopeptidase His-Lys-Lys

+ Gly-Gln

carboxypeptidase B His

+

Lys

+

Lys

N-acetyltransferase a, N-acetyl b-EP 1-27

FIGURE 2.

Processing of β-endorphin.

The pattern of processing of β-LPH in the hypothalamus, midbrain, and amygdala resembles that in the anterior pituitary and hypothalamus, where βendorphin(1–31) is the principal β-endorphin-like product, although some β-endorphin(1–26) is also present in the midbrain and amygdala. The pattern of expression in the spinal cord is similar, although some proteolysis and acetylation of β-endorphin occurs, and this processing increases in the caudal direction. However N-acetyl β-endorphin(1–27) and N-acetyl βendorphin(1–26) are the principal forms in the hippocampus, colliculae, and some brain-stem regions. It is interesting however, that, although the scheme for β-endorphin processing represented in Fig. 2 has been generally accepted, the biologically active C-terminal tetrapeptide Lys-Lys-Gly-Glu/Gln may be present in some brain tissues. However, its distribution in the central nervous system (CNS) is unknown.

RECEPTORS β-Endorphin is an agonist for μ-, ∂-, and to a lesser extent κ-opioid receptors, as well as for the putative ε-receptor. It also binds to the nonopioid σ-receptor and possibly also to a β-endorphin C-terminal peptide receptor.

Opioid Receptors The existence of three main opioid receptors, μ, κ, and ∂, has been confirmed by cloning. They are all blocked by the opioid antagonist naloxone. In addition, there is pharmacological evidence for subtypes of each of these receptors, although so far their existence has not been confirmed by cloning. Indeed they may actually be alternatively spliced variants or oligomerized

receptors, which are known to exhibit unique properties [17]. β-Endorphin has a higher affinity for μreceptors than for ∂-receptors. μ-Opioid receptors subserve most of the central analgesic effects of βendorphin (and endomorphin and morphine) as well as its effects on euphoria, dependence, and respiratory depression. The activation of ∂-receptors also causes analgesia (mainly at the spinal level), but they are probably more important for the peripheral actions of β-endorphin. There are considerable variations in the distribution of opioid receptors in the CNS. It is highest in the striatum and lowest in the cerebellum. There are high densities in the substantia gelatinosa of the spinal cord, the locus coerelius, and clusters of high density in the caudate putamen, amygdala, and PAG.

Cellular Effects The μ-, κ-, and ∂-opioid receptors are G-protein coupled, and their activation results in the inhibition of adenylcyclase activity, reduction of cAMP concentration, and increase in the activity of phospholipase C and the protein kinases Erk-1 and Erk-2 [8]. Opioid receptor activation also inhibits voltage-gated Ca2+ N-type and L-type channels. The inhibition of Ca2+ channels in presynaptic nerve terminals causes a reduction in the release of many neurotransmitters, including acetylcholine, norepinephrine, dopamine, glutamate, somatostatin, serotonin, and substance P. The activation of opioid receptors also opens inwardly rectifying K+ channels in postsynaptic neurons, causing hyperpolarization and inhibition of cell firing.

The Putative e-Receptor A receptor with a high affinity for β-endorphin(1– 31), designated the ε-receptor, was first characterized in rat vas deferens. Evidence that β-endorphin(1–31) is the endogenous ligand for the ε-receptor in the brain is based on pharmacological and distributional studies [26]. Thus, although antinociception produced by intrathecally administered β-endorphin(1–31) in mice is mediated by μ- and κ-receptors, that produced by the intracerebroventricular (ICV) administration could not be blocked by μ, ∂, or κ antagonists but could be blocked by the ICV administration of β-endorphin(1–27). Furthermore the distribution of β-endorphin-containing fibers is different from that of μ- or ∂-receptors in the brain. Notably, the concentrations of β-endorphin in the hypothalamus and the PAG are high, but there are very few μ- or ∂-receptors in these regions. Nevertheless, the existence of the ε-opioid receptor remains controversial because selective antagonists have not yet been developed and it has not yet been cloned. Furthermore,

1328 / Chapter 184 Contet et al. [2] found no evidence for a Gprotein-coupled ε-receptor in brain of triple knockout mice lacking μ, ∂-, and κ-receptors.

has also been shown to activate opioid receptors (μ and ∂) on peripheral afferent nerve terminals in inflamed tissue to induce antinociception. Inflammation increases the potency of β-endorphin.

BIOLOGICAL ACTIONS

Tolerance, Dependence, and Euphoria

Analgesia Opioids reduce both the sensation and the affective (emotional) component of pain. The nociceptive sensory pathway is extremely complex. An oversimplified description is that it involves the activation of nociceptive neurons by noxious stimuli and the transmission of impulses in these neurons to interneurons, which are mainly located in the dorsal horn of the spinal cord. The interneurons, in turn, synapse with neurons with projection fibers in the spinothalamic tracts. These synapse mainly in the ventral and medial thalamus, with neurons whose axons project to the somatosensory cortex. Pain can be controlled at the level of the dorsal horn by descending inhibitory pathways from the cortex, via the PAG and the nucleus raphe magnus (NRM) of the medulla, to the dorsal horn. The pathways are activated by impulses transmitted from neurons originating in the thalamus, which stimulate the PAG, and indirectly by impulses in neurons originating in the spinothalamic tract, which stimulate the nucleus reticularis paragigantocellularis (NRPG), which in turn sends impulses to the NRM. Opioids cause analgesia by activating the descending pathways, thereby inhibiting the transmission in the dorsal horn, and by inhibiting the excitation of sensory nerve terminals in the periphery. β-Endorphin, in particular, has potent actions in the control of pain. It is present in the PAG, hypothalamus, NRM, and dorsal horn and therefore could stimulate the descending inhibitory pathways at several sites. In humans, β-endorphin produces long-lasting analgesia when administered ICV, intrathecally, or epidurally (but not when administered intravenously) and these effects are blocked by naloxone. Neurons of the arcuate nucleus in the hypothalamus play a central role in βendorphin-mediated analgesia; lesions of this area weaken poststress analgesia and reduce the antinociceptive effect produced by the electrical stimulation of the PAG. Part of the antinociceptive effect of β-endorphin may be due to an action to release enkephalins in the dorsal horn because it has been shown that ICV β-endorphin stimulates neurons in midline nuclei of the medulla to cause release of Met-enkephalin from nerve terminals in the dorsal horn [26]. Met-enkephalin then acts on ∂-opioid-receptors on dorsal horn neurons. This mechanism may be partly responsible for the antinociception produced by some types of stress. Finally, β-endorphin

Long-term administration of β-endorphin causes tolerance, or loss of responsiveness, as well as physical dependence. Tolerance is partly due to a reduction in the number of opioid receptors and blockade of receptor-effector coupling [3]. Physical dependence is indicated by the onset of withdrawal symptoms when treatment is discontinued. An important part of the analgesic effects of β-endorphin and other opioids is their ability to cause feelings of contentment, which reduces the anxiety associated with pain. Euphoria is mediated via μ-opioid receptors. β-Endorphin is particularly important in these reward systems, as evidenced by the fact that it is self-administered by laboratory animals [38]. A number of brain pathways play a role in drug dependence, including the mesolimbic reward pathway and pathways involved in the stress response, obsessive-compulsive behavior, and habitforming behavior. Endogenous peptides may also have a role in drug reinforcement by modulating the dependence that can develop for opioid and nonopioid drugs of abuse [9]. The pathways involved are unknown, but neurons in the ventral tegmental area may have an important role. The nonopioid drugs include alcohol, which appears to potentiate opioid neurotransmission. Endogenous βendorphin in particular has been implicated in the development and maintenance of alcoholism [27].

Neuroendocrine Effects The administration of opioids, including βendorphin, enhances the release of certain anterior pituitary hormones and suppresses the release of others. These actions are exerted at the level of the hypothalamus. Thus, β-endorphin administration in rats increases the release of prolactin and growth hormone via opioid receptors [32]. It inhibits the secretion of gonadotrophins [20] by inhibiting luteinizing hormone–releasing hormone (LHRH) release and thyrotrophin secretion by inhibiting thyrotrophin-releasing hormone (TRH) release [11]. β-Endorphin also has peripheral effects on hormone release. Thus, it stimulates insulin and glucagon release from the pancreas and inhibits somatostatin release. β-Endorphin also plays an important role in pregnancy and parturition. Plasma β-endorphin concentration rises during labor and remains elevated during the early postpartum period. Furthermore, in late preg-

POMC Opioid Peptides / 1329 nancy and parturition, there is an increase in the density of β-endorphin-immunoreactive cells in the arcuate nucleus and in β-endorphin-immunoreactive fibers in the supraoptic nucleus, to which they project [4]. This may be related to the inhibition of oxytocin neurons by β-endorphin, which occurs during pregnancy. Moreover, in late pregnancy and parturition β-endorphin stimulates the release of prolactin from the anterior pituitary via the regulation of dopaminergic neurons in the hypothalamus. Prolactin then stimulates progesterone secretion from the ovary.

probably by an action on μ-receptors, indicating an important role for β-endorphin in cardiovascular homeostasis [36]. Glycyl-glutamine, on ICV administration in the brain, inhibited the respiratory depression produced by morphine or β-endorphin but not the antinociception induced by morphine [29, 37]. A synthetic cyclic, nonpolar derivative cyclo(Gly-Gln) produced similar effects. Glycyl-glutamine also attenuated hemorrhagic hypotension and increased heart rate in conscious rats. Furthermore it has been proposed that this dipeptide is a functional antagonist for some actions of β-endorphin in the CNS.

Exercise and Stress β-Endorphin is released from the pituitary into the blood during stress [33] and exercise [1]. It has been reported to mediate the euphoria experienced in prolonged exercise and to inhibit postexercise pain. Plasma β-endorphin has a number of actions on the periphery during exercise. β-Endorphin(1–31), β-endorphin(1– 27), glycylglutamine, and the stabilized synthetic melanotrophin-potentiating factor (MPF) analog, Nacetyl-Lys-D-Lys-Sar-Glu, all increased the contractile response in isolated muscles of the rat [5]. β-Endorphin, glycylglutamine, and the MPF analog also reduced fatigue in isolated muscles stimulated at high frequency via the motor nerve. Furthermore, β-endorphin can be released by electrical stimulation of motoneurons [19], and POMC mRNA is upregulated in vivo in chronically stimulated motoneurons [12]. In addition β-endorphin, glycylglutamine, and the MPF analog stimulated glucose uptake in isolated skeletal muscles. β-Endorphin itself was more potent in increasing glucose uptake in contracting muscles than noncontracting muscles [5]. Its effect on glucose uptake was mediated partly via a ∂opioid receptor [7]. Together these observations indicate an important role for β-endorphin in the insulin-independent uptake of glucose during exercise. β-Endorphin is released from the pituitary into the blood under physical and emotional stress. Various nonopiate actions of human β-endorphin on the immune system have been reported. It binds to components of human complement and thymoma cells and stimulates the proliferation of lymphocytes. These actions may represent a modulation by β-endorphin of the immune system in stress situations. They were ascribed to the C-terminal tetrapeptide region of βendorphin [35].

Cardiorespiratory Effects and Thermogenesis β-Endorphin causes severe hypotension, bradycardia, and respiratory depression when injected into the cerebrospinal fluid (CSF) or directly into the NTS or the vasopressor region of the ventrolateral medulla,

Feeding Behavior β-Endorphin stimulates feeding behavior [24], and β-endorphin concentrations are higher in the plasma of obese individuals than of nonobese individuals [34]. POMC neurons in the arcuate nucleus of the hypothalamus, which project to other hypothalamic and brain-stem nuclei that control feeding, are involved in this feeding effect [18]. The arcuate nucleus neurons are targets for leptin, the adipostatic hormone that inhibits feeding via the release of melanocortins. The actions of β-endorphin in inducing feeding are very complex, but they oppose those of melanocortins. βEndorphin may also be involved in overeating caused by administration of neuropeptide Y because this effect is blocked by naloxone [23].

Development and Regeneration of the Nervous System Morley and Ensor showed that injection of βendorphin or MPF restored the ability to regenerate amputated limbs in hypophysectomized newts (in which the ability is lost). A stable synthetic MPF analog of this peptide was even more potent [25]. The same group [28] showed that MPF stimulates the proliferation of astrocytes and neurite outgrowth in cultures of neuroneocortical cholinergic and mesencephalic dopaminergic neurons. β-Endorphin is widely expressed in neurons and nerve fibers in the developing spinal cord of rodents during the first few weeks of life, but thereafter it markedly declines. Furthermore, the expression of POMC peptides is increased in injured regenerating motoneurons [13]. β-Endorphin and its C-terminal peptides have been shown to exert neurotrophic actions in the development and maintenance of synapses: βEndorphin inhibits acetylcholinesterase (AChE) expression at the neuromuscular junction during development [10], and glyclglutamine acts to maintain both AChE [22] and cholineacetyltransferase [21] in the denervated superior cervical ganglion of the cat.

1330 / Chapter 184 PATHOPHYSIOLOGICAL IMPLICATIONS Neuropathic Pain In humans, most opioids (including β-endorphin) are less effective in ameliorating neuropathic pain than acute pain. This may be partly due to the reduction in the number of opioid receptors that results from nerve injury [40]. Interestingly endomorphins are effective in alleviating neuropathic pain.

[9]

[10]

[11]

[12]

Neuromuscular Disease The expression of POMC peptides, including βendorphin, is increased in diseases of the neuromuscular system in rodents, such as muscular dystrophy [13]; in diabetes mellitus [15]; and following motoneuron injury [13] or intoxication [14]. Moreover, an increase in the density of ∂-opioid receptors has been demonstrated in muscular dystrophy [6] and diabetes [15]. Furthermore, it has been shown in dystrophic mice that β-endorphin can be released by the stimulation of motoneurons [19]. The roles of β-endorphin in these diseases is not clear, but the increased expression of the peptide and its receptor may be part of a regenerative response because both β-endorphin and an analog of the C-terminal tetrapeptide (MPF) increased the contractile response of isolated muscles of dystrophic mice [19]. Furthermore these peptides also increased glucose uptake in both obese (ob/ob) diabetic mice [7] and dystrophic mice [19].

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

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POMC Opioid Peptides / 1331 [28] Owen DB, Morley JS, Ensor DM, Allen YS, Miles JB. Trophic effects of melanotropin-potentiating factor (MPF) on cultures of cells of the central nervous system. Peptides 1997;18:1015– 21. [29] Owen MD, Unal CB, Callaghan MF, Trivedi K, York C, Millington WR. Glycyl-glutamine inhibits the respiratory depression, but not the antinociception produced by morphine. Am J Physiol Regul Integ Comp Physiol 2000;279:R1944–8. [30] Parish DC, Smyth DG, Normanton JR, Wolstencroft JH. Glycylglutamine, an inhibitory neuropeptide derived from βendorphin. Nature 1983;306:267–70. [31] Plishka RJ, Cangro CB, Neale JH. Immunohistochemical localization of the pro-opiomelanocortin-gene product, glycylglutamine, in the intermediate pituitary. Brain Res 1985;360:403–6. [32] Rivier C, Vale W, Ling N, Brown M, Guillemin R. Stimulation in vivo of the secretion of prolactin and growth hormone by β-endorphin. Endocrinology 1977;100:238–41. [33] Rossier J, French ED, Rivier C, Ling N, Guillemin R, Bloom FE. Foot shock-induced stress increases β-endorphin levels in blood but not in brain. Nature 1977;270:618–20. [34] Scavo D, Barletta C, Vagiri D, Burla F, Fontana M, Lazzari R. Hyperendorphinemia in obesity is not related to the affective state. Physiol Behav 1990;48:681–3.

[35] Schweigerer L, Teschemacher H, Bhakdi S, Lederle M. Interaction of human β-endorphin with nonopiate binding sites on the terminal SC5b-9 complex of human complement. J Biol Chem 1983;258:12287–92. [36] Sitsen JMA, van Ree JM, de Jong W. Cardiovascular and respiratory effects of β-endorphin in anaesthetized and conscious rats. J Cardiovasc Pharmacol 1982;4:883–8. [37] Unal CB, Owen MD, Millington WR. β-Endorphin-induced cardiorespiratory depression is inhibited by glycylglutamine, a dipeptide derived from β-endorphin. J Pharm Exp Ther 1994;271:952–8. [38] van Ree JM, Smyth DG, Colpaert FC. Dependence-creating properties of lipotropin C-fragment (β-endorphin): evidence for its internal control of behaviour. Life Sci 1979;24:495–502. [39] Zakarian S, Smyth DG. Distribution of β-endorphin-related peptides in rat pituitary and brain. Biochem J 1982;202:561–71. [40] Zhang X, Bao L, Shi TJ, Ju G, Elde R, Hokfelt T. Downregulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy. Neuroscience 1998;82:223–40.

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185 Endomorphins as Endogenous Peptides for μ-Opioid Receptor: Their Differences in the Pharmacological and Physiological Characters SHINOBU SAKURADA AND TSUKASA SAKURADA

selectivity and efficacy at the MOP-Rs. Before 1997, no mammalian opioid peptide was known that exhibited both high affinity and selectivity for MOP-Rs. The structure of casomorphin containing Tyr-Pro-aromatic isolated from milk is similar to that of Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), which had been isolated from bovine and human brain. This Tyr-Pro-Trp-Gly-NH2, a peptide with opioid-related activity in the central nervous system, is highly selective for MOP-Rs, but its affinity is relatively low [43]. Selective μ-opioid peptides with the highest affinity and selectivity were sought from peptides in which each of the possible natural amino acids substituted in the fourth position, according to the formula Tyr-Pro-Trp-X. Of the 20 peptides tested (Tyr-Pro-TrpX-NH2, X: Phe, Leu, Ile, Met, Val, Pro, Gln, Trp, Cys, Thr, Tyr, Asn, Ser, Ala, Gly, Arg, Lys, His, Asp, Glu), Tyr-Pro-Trp-Phe-NH2 showed a high affinity and selectivity for MOP-R. This peptide also showed very potent analgesia after intracerebroventricular and intrathecal administration [42]. Specific MOP-R antagonists reversed both the in vitro and in vivo effects of the new endogenous peptide. Zadina et al. generated a specific antibody against the tetrapeptide sequence that showed no cross-reactivity for over 40 opioids and used it in combination with high-performance liquid chromatography (HPLC) to screen extracts of bovine brain for immunoreactivity. Multiple purification steps yielded two peptide sequences, Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2. Because these were the first endogenous mammalian peptides that had a high affinity and a clear specificity for MOP-R, they were named endomorphin-1 (EM-1) and endomorphin-2 (EM-2) [42].

ABSTRACT Endomorphins (EMs) are endogenous μ-opioid peptides discovered by Zadina and his colleagues in the mammalian brain in 1997. Although both peptides are broadly localized in the central nervous system, there are some differences in their distributions. EMs have several pharmacological and physiological characters that are identical with other endogenous opioid peptides and μ-opioid receptor (MOP-R) agonists. Interestingly, EMs, which have only one difference in amino acid residue at position 3, show some characteristic differences from one another in their pharmacological and physiological effects. We describe the pharmacological and physiological characteristics of EMs, with emphasis on their differences.

DISCOVERY OF ENDOMORPHINS Since the discovery of opioid receptors, neuroscientists have searched for their endogenous ligands. The search led to the discovery of enkephalins [11], endorphins [4], and dynorphins [9] in the 1970s. Three families of opioid peptides were discovered to contain a common N-terminus amino acid residence of TyrGly-Gly-Phe. The enkepkalins ([Met5]enkephalin and [Leu5]enkephalin) are the endogenous ligands for δopioid receptors (DOP-Rs), and dynorphin A(1–17) is the endogenous ligand for κ-opioid receptors (KOPRs). β-Endorphin binds μ-opioid receptors (MOP-Rs) and DOP-Rs. These compounds display relatively low Handbook of Biologically Active Peptides

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1334 / Chapter 185 DISTRIBUTION OF THE ENDOMORPHIN-2LIKE IMMUNOREACTIVE NEURONS IN THE CENTRAL NERVOUS SYSTEM Soon after the discovery of EMs, the distribution of the EM-2-like immunoreactive (EM-2-LI) neurons was shown in the central nervous system. Fibers of EM-2-LI varicosities extend from the medulla oblongata to the sacral spinal cord. EM-2-LI was present in the superficial layers of dorsal horn of the spinal cord and medulla [16]. The dorsal horn of the spinal cord is an important site for nociceptive transmission. It constitutes the first relay station for incoming somatosensory information and contains many transmitters, such as substance P and glutamate. These regions, where primary afferents from the dorsal root ganglia and trigeminal nerve terminate, contain the highest densities of both MOP-Rs and EM-2-LI. Mechanical disruption of the primary afferents by the dorsal rhizotomy dramatically diminished the EM-2 immunostaining. Similarly, chemical disruption of primary sensory afferents by the neurotoxin, capsaicin, eliminated staining in the dorsal horn [15, 25]. Colchicine pretreatment failed to lead to the detection of perikarya within the lower brain stem and spinal cord, but EM-2-LI neuronal peirkarya were found in the dorsal root ganglia in all segments of spinal cord [14]. Colocalization studies showed that EM-2-LI was present in fibers containing immunoreactivity for MOPRs, substance P, and calcitonin gene-related peptide [15]. Taken together, these results provide evidence that the EM-2-LI fibers originate from the dorsal root ganglion neurons. Perikarya expressing EM-2-LI were present in the hypothalamus especially in the dorsomedial hypothalamus and arcuate hypothalamic nucleus, whereas those expressing EM-1-LI were present in both the posterior hypothalamus and the nucleus of solitary tract [14]. The MOP-Rs are expressed by primary afferent nociceptors that terminate in lamina I and II of the dorsal horn [3]. Based on this distribution and on functional studies of opioid-induced activity, two major mechanisms for producing opioid-induced analgesia have been proposed: presynaptic inhibition of neurotransmitter release from primary afferent nociceptors and postsynaptic hyperpolarization of excitatory neurons [1, 12]. Opioids are known to regulate the release of glutamate, substance P, and calcitonin gene–related peptide from primary afferents. EM-2 modulates release of these excitatory transmitters presynaptically [37, 38]. EM-2-LI is present in dense-cored vesicles in cord dorsal horn axon terminals [33] and is released by stimulation of the dorsal roots. The morphological demonstration and synaptic relationship provide evidence that EM-2LI functions as a transmitter and/or modulator regulating pain processes.

DISTRIBUTION OF THE EM-1-LI NEURONS IN THE CENTRAL NERVOUS SYSTEM Although both EM-1- and EM-2-LI are present in most areas, several differences in the distributions and staining intensity were observed. EM-2-LI is more prevalent in the spinal cord and lower brain stem, whereas EM-1-LI is more widely and densely distributed throughout the brain than was EM-2-LI. MartinSchild et al. [14] found a very wide distribution of EM-1-LI fibers in the central nervous system but a very limited region containing EM-1-LI cell bodies. Both EM-1-LI and EM-2-LI neuronal perikarya were found in the dorsomedial hypothalamic nucleus, arcuate hypothalamic nucleus, and ventromedial hypothalamic nucleus. But in the nucleus of solitary tract, EM-1-LI neuronal perikarya were reported. The EM-1-LI fibers were found widely distributed throughout the brain and spinal cord. In the telencephalon, a moderately dense plexus of EM-1- and EM-2-LI nerve fibers were seen in the nucleus accumbens, lateral septum, and stria terminalis [14, 33]. EM-1-LI was evident in the dorsal lateral septal nucleus, nucleus of the vertical limb of the diagonal band of Broca, and the bed nucleus of the stria terminalis. Thus, the EM-2-LI distribution closely paralleled that of EM-1-LI, but its contribution to the telencephalic structures was much smaller. In general, EM-1-LI was extensively present in hypothalamic structures. EM-1-LI fibers were detected in the organum vasculosum lamina terminalis and in the median preoptic and posterior hypothalamic nuclei. Other regions containing relatively large numbers of EM-1-LI fibers include the medial preoptic area, the lateral preoptic area, anterior hypothalamic area, lateral hypothalamic area, ventromedial hypothalamic nucleus, dorsal hypothalamic area, dorsomedial hypothalamic nucleus, arcuate nucleus, and ventral premammillary and supramammillary nuclei. Unlike EM-1-LI fibers, EM-2-LI fibers were infrequently detected in hypothalamic regions outside the regions containing EM-2-LI cell bodies. In the midbrain, EM-1-LI fibers were also reported in the superior and inferior colliculii, periaqueductal gray, dorsal raphe nucleus, laterodorsal tegmental nucleus, ventral tegmental area, and the substantia nigra pars reticularis. In the lower brain stem, most of the EM-1-LI fibers were found to spread over different parts of the locus coeruleus and the parabrachial nucleus. The principal sensory nucleus of the trigeminal, the nucleus of the spinal trigeminal tract, and the nucleus of the solitary tract also contained some EM-1LI fibers but at a lower density than those immunostained with EM-2-LI.

Endomorphins as Endogenous Peptides for μ-Opioid Receptor

RECEPTOR SELECTIVITY AND ITS MODULATION The receptor selectivities of EMs have been well established in several studies. As Zadina et al. described when they first reported the existence of EMs in mammalian brain, EMs show very high affinity and selectivity for the MOP-Rs [42]. The affinity of EM-1 to MOP-Rs mimics that of selective MOP-Rs agonist DAMGO, and its selectivity for MOP-Rs is more than 4000 times and 15,000 times higher than those for DOP-Rs and KOPRs, respectively. On the other hand, the affinity of EM2 to MOP-Rs is approximately two times lower than that of DAMGO, but its selectivity for MOP-R is still more than 13,000 times and 7000 times higher than those for DOP-Rs and KOP-Rs, respectively. The extremely high affinity and selectivity of EMs for MOP-Rs have been confirmed in the membrane preparations obtained from several brain regions and the spinal cord in mice and rats [10]. With high affinity and selectivity for the MOP-Rs, in addition to some biological action mimicking traditional MOP-Rs agonists, EMs were predicted to be the long-sought endogenous μ-opioid peptides [42]. Interestingly, EMs are the only endogenous opioid peptides to show high receptor selectivity; other endogenous opioid peptides, including β-endorphin [Met5]enkephalin, [Leu5]enkephalin, and dynorphin A(1–17), show selectivity to multiple opioid receptors [26]. Because opioid receptors are coupled with Gi/Goproteins, the intrinsic activity of opioid receptor agonists has been preferentially evaluated in the [35S]GTPγS binding assay. Like other MOP-R agonists, both EM-1 and EM-2 produce a marked increase in the [35S]GTPγS binding in membrane preparations obtained from several brain regions and the spinal cord in mice and rats [10]. The EMs-induced increase in [35S]GTPγS binding is completely eliminated by the co-incubation with selective MOP-R antagonists but not DOP-R and KOP-R antagonists [21]. Consistent with their selectivity to opioid receptors, G-protein activation induced by EMs is selectively mediated by MOP-Rs. In fact, the EMsinduced increase in [35S]GTPγS binding is completely abolished in the membrane preparation obtained from MOP-R knockout mice [18]. The maximal increase in [35S]GTPγS binding by EMs (the intrinsic activities of EMs) is approximately half that of DAMGO, a MOP-R full agonist [21]. Moreover, the maximal increase in [35S]GTPγS binding by the MOP-R full agonists, DAMGO and β-endorphin, is partially attenuated by coincubation with high concentrations of EMs [2, 19]. The evidence clearly suggests that EMs, as well as morphine and fentanyl, are partial agonists for MOP-Rs. It is of interest to note that EMs are the only endogenous opioid peptides to have a partial agonistic property,

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whereas other endogenous opioid peptides are full agonists for their respective opioid receptors [2, 44]. The physiological role of EMs may be somewhat different from other endogenous opioid peptides.

BIOLOGICAL ACTIONS OF ENDOMORPHINS Like other endogenous opioid peptides or selective MOP-R agonists, EMs have a variety of biological actions. As expected, a major biological action of EMs is its remarkable antinociceptive effect. After intrathecal and intracerebroventricular injections, EMs show as potent antinociception as the other endogenous opioid peptides [Met5]enkephalin, [Leu5]enkephalin, and dynorphin A(1–17) [32]. The antinociception induced by both EM-1 and EM-2 is mediated by MOP-Rs. However, their antinociceptive effects are characteristically somehow different. The antinociception induced by EM-2, but not EM-1, is attenuated by the μ1-opioid receptor antagonist naloxonazine [28, 32], whereas the antinociception induced by EM-1, but not EM-2, is attenuated by the recently identified μ2-opioid receptor antagonist d-Pro2-Tyr-W-MIF-1 [40]. These results clearly suggest that the antinociceptions induced by EM-1 and EM-2 are characteristically distinct from one another and selectively mediated through the μ2- and μ1-opioid receptors, respectively. This theory is also supported by the evidence that the antinociception induced by EM-1 and EM-2 is selectively attenuated by the μ2opioid receptor antagonist d-Pro2-endomorphin-1 and the μ1-opioid receptor antagonist d-Pro2-endomorphin2, respectively, but not vice versa [31]. Moreover, EM-2-induced antinociception is attenuated by 3methoxynaltrexone, which is a heroin-sensitive and morphine-insensitive antagonist, suggesting that EM-2 has selectivity to multiple MOP-Rs, including the μ1opioid receptor and other MOP-Rs as a third party of the MOP-R, but not to the μ2-opioid receptor [28, 29]. More interestingly, the antinociceptive effect of EM-2, but not EM-1, is mediated by DOP-R and KOP-R in addition to MOP-R [30], even though EM-2-induced antinociception is completely abolished in the MOP-R knockout mice [18]. This controversial result is shown by the ability of EM-2 to lead to the release of the other endogenous opioid peptides, [Met5]enkephalin and dynorphin A(1–17), by its stimulation of MOP-Rs [30]. β-Endorphin is an another endogenous opioid peptide known to release [Met5]enkephalin [39]. However, in the present situation, EM-2 is the only endogenous opioid peptide that releases endogenous κ-opioid peptides. Like other endogenous opioid peptides, the duration of EM-induced antinociception is short-lived, and it completely disappears within a half hour [18, 32]. Unfortunately, enzymatic degradation of EMs cannot

1336 / Chapter 185 explain this phenomenon. The process of enzymatic degradation of EMs by enzymes identified in the cerebrospinal fluid and central nervous systems has already been described [24, 27, 35]. The enzymatic degradation of EMs in cerebrospinal fluid, like that described earlier for the related peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), proceeds more slowly than that of other endogenous opioid peptides; it requires more than 2 h to degrade EMs [27, 35]. The identical character of EMs on acute tolerance may help explain their short-lived effects. As is well known, a single injection of opioid receptor agonists leads to a shortlived desensitization to the same agonists, called the acute tolerance effect, and the time course of acute tolerance is identical to the receptor that the agonist binds [20]. Unlike the traditional MOP-R agonist, DAMGO, whose acute antinociceptive tolerance shows up 3 h after the treatment and lasts for 6 h, the acute antinociceptive tolerance to EMs rapidly develops within a half hour and disappears by 3 h [41]. The rapid development of the acute antinociceptive tolerance to EMs may be involved in its short-lived antinociceptive effect. The other traditional biological activities of MOPR agonists, including rewarding effect, locomotor enhancement, inhibition of gastrointestinal transit, and cardiovascular and respiratory depression have also been described for EMs. Like other MOP-R agonists, both EM-1 and EM-2 produce potent inhibition of gastrointestinal transit [8] and cardiovascular depression [10] with similar efficacy. Both EM-1 and EM-2 also produce respiratory depression; however, approximately a 12-times-higher dose of EM-1 is required to produce respiratory depression in comparison with EM-2 [7]. The difference in the sensitivity for respiratory depression may be caused by the different distribution of EM-1- and EM-2-containing neurons. Although EM-1-LI and EM-2-LI both densely distribute in areas associated with respiratory function, such as the nucleus tractus solitarius and the parabrachial nucleus, EM-2-LI is more prominent than EM-1-LI in the ventrolateral nucleus tractus solitarius and external medial parabrachial nucleus, which are the most crucial regions for respiratory regulation [14]. Regarding the rewarding effect and locomotor enhancement, EM-1 and EM-2 show more significant differences from one another. EM-1, as well as other traditional MOP-R agonists, produces a remarkable rewarding and locomotor-enhancing effect in a dose-dependent manner [6, 22]. In contrast, EM-2 shows a bell-shaped dose-response curve for the rewarding and locomotor-enhancing effects, and prominently produces an aversive rather than rewarding effect [5, 22]. As is well known, the rewarding and locomotor-enhancing effects of MOP-R ago-

nists are mediated by the disinhibition of mesolimbic and nigrostriatal dopaminergic neurons via the activation of MOP-Rs located on the GABAergic neurons in the ventral tegmental area and substantia nigra, respectively [22]. In the terminal of mesolimbic and nigrostriatal dopaminergic neurons on the nucleus accumbens and striatum, respectively, dynorphinergic neurons are localized to inhibit the release of dopamine. Unlike EM-1 and other traditional MOP-R agonists, EM-2 has a characteristic pharmacological ability to release dynorphin A [30]. The release of dynorphin A in the nucleus accumbens and striatum may be involved in the lack of the remarkable rewarding effect and locomotor enhancing effect of EM-2. In fact, EM-2 shows a remarkable rewarding effect when dynorphin A is blocked by the use of its antiserum [22].

PATHOPHYSIOLOGICAL IMPLICATIONS OF ENDOMORPHINS The pathophysiological role of EMs still has not been well characterized. However, the progressing investigation of the functional changes of EMs in several pathological conditions may be helpful. In a neuropathic pain model in which the sciatic nerve is ligated, EM-2-LI is dramatically decreased in the superficial layer of the dorsal horn [34]. This evidence strongly suggests that EM-2 may regulate the transmission of nociceptive stimulati in the spinal cord, and the reduction of EM2-containing neurons may be associated with the nerve ligation-induced neuropathic pain. In contrast, EM-2-LI in the periaqueductal gray matter is greatly increased by spinal nerve ligation [36]. Taken together with the evidence that EM-2 is particularly more effective than morphine in nerve ligation-induced neuropathic pain, EM-2 in the periaqueductal gray matter may be involved in the regulation of the pain sensation induced by nerve injury via activation of the descending pain-control system. In peripheral inflammation, especially knee joint inflammation by adjuvant, EM-1-LI is greatly increased in the synovium of the knee joint [17]. The increase of EM-1-LI is also observed in the synovium of adjuvantinflamed paws [13]. At present, increased EM-1-LI is limited to the peripheral tissues. Unlike EM-2, which seems to control pain sensation in the central nervous system, EM-1 may control painful inflammation in the peripheral tissues. The EMs are the only endogenous opioid peptides identified as partial agonists [19]. The other endogenous opioid peptides are all full agonists for their respective receptors. Theoretically, a high dose of partial agonists can partially attenuate the effect of full

Endomorphins as Endogenous Peptides for μ-Opioid Receptor agonists working on the same receptors. As expected, EMs partially attenuate the effects of β-endorphin, another endogenous μ-opioid peptide [19]. In the specific physiological condition in which the μ-opioid system is overstimulated, EMs may prevent excessive stimulation of MOP-R by β-endorphin by a negative feedback mechanism in tissues in which EMs and βendorphin are co-localized.

CONCLUSION A growing body of evidence indicates some differences between EM-1 and EM-2. Overall, the pharmacological characteristics of EM-1 are relatively close to those of the traditional MOP-R agonists, whereas EM-2 has several differences in its pharmacological characteristics from other μ-opioid receptor agonists. It has been recently demonstrated that there are more than 15 subclasses of MOP-Rs based on the splice variants of MOPR mRNA [23]. The differences in their pharmacological and physiological character may be revealed by the different selectivity of EMs for MOP-R subclasses and by the different co-localization of EMs and their preferred MOP-R subclasses.

Acknowledgments Research in the author’s laboratory was supported by The Science Research Promotion Fund from The Promotion and Mutual Aid Corporation for Private Schools of Japan; a Grant-in-Aid for Scientific Research (C) (KAKENHI 16590058 and 17590065) from the Japan Society for the Promotion of Science; and a Grant-inAid for High Technology Research Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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186 Casomorphins and Hemorphins—Opioid Active Peptides Released by Partial Hydrolysis of Structural Proteins FRED NYBERG

acting on these receptors were identified and found to be present in the central nervous system (CNS) and also in peripheral tissues and in a variety of body fluids. The CNS distribution of β-endorphin, enkephalins, dynorphins, and endomorphins are described in other chapters (Chapters 182–186) in this section of this book. Interestingly, most opioid peptides present in the CNS have also been identified in the cerebrospinal fluid (CSF) [27]. The presence of opioid active material in bovine milk was originally reported by Brantl and co-workers [7, 8]. They isolated a peptide fragment from an enzymatic digest of the milk protein β-casein. This fragment exhibited binding affinity for opioid receptors and was shown to produce opioid effects in the guinea pig ileum (GPI) assay. Due to this and its origin, the isolated peptide was named β-casomorphin. Sequence analysis of β-casomorphin revealed that the structure of this compound diverges from that of the classic endogenous opioids; for example, the milk-derived peptide contains a Pro residue next to the N-terminal Tyr (Table 1). In subsequent studies, β-casomorphin was found to exist in bovine plasma as a naturally occurring compound both in truncated and extended forms of the first isolated entity [44] and also to exist in human body fluids. For instance, β-casomorphin-8-like immunoreactivity was measured in human plasma [20, 29], in human CSF [29, 30], and in human milk [38]. The β-casomorphins are fragments of the bovine β-casein sequence 60–68 and in sheep, water buffalo, and human whey they are found in analogous positions. In human β-casein, the casomorphin unit also resides in the sequence 60–68. Moreover, the human β-casomorphin differs from its bovine analog in having a Val-Glu instead of Pro-Gly in position 4–5 (Table 1). An analog, β-casomorphin-4 amide (named morphiceptin; see Table 1) isolated from a commercial hydro-

ABSTRACT In addition to the classic opioid peptides (βendorphin, dynorphins, and enkephalins), with the Nterminal Tyr-Gly-Gly-Phe tetrapeptide sequence, opioid active peptides containing the Tyr-Pro dipeptide residues in their N-terminal sequence have been identified and characterized. Other than the endomorphins, these so-called atypical opioid peptides are derived by partial hydrolysis of endogenous existing functional proteins and are mainly produced in peripheral tissues and body fluids. Among the atypical endogenously produced opioid active peptides are the β-casomorphins and the hemorphins. These peptides are formed through the hydrolysis of the milk protein β-casein and hemoglobin, respectively, and many of their biological effects have been characterized. They are less potent than the classic peptides but share most of their typical opioid effects. Moreover, the detected concentrations of β-casomorphins and hemorphins in body fluids highly exceed those of the β-endorphin and enkephalins. Originally, the β-casomorphins and the hemorphins were isolated from enzymatically treated bovine milk and blood, respectively. Later they were identified as naturally occurring compounds in human plasma and cerebrospinal fluid. This chapter gives a brief review of past and current research on the βcasomorphins and hemorphins regarding the mechanisms for their release, their distribution, their biological actions, and their pathophysiological roles.

DISCOVERY OF THE b-CASOMORPHINS AND THE HEMORPHINS Soon after the discovery of opioid receptors in mammalian brain, the presence of endogenous compounds Handbook of Biologically Active Peptides

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1340 / Chapter 186 TABLE 1. Amino Acid Sequence of β-Casomorphins, Hemorphins, and Related Peptides. Peptide β-casomorphin-5 (bovine) β-casomorphin-7 (bovine) β-casomorphin-8 (bovine) β-casomorphin-5 (human) β-casomorphin-7 (human) β-casomorphin-8 (human) Morphiceptin Hemorphin-4 (human) Hemorphin-7 (human) LVV-Hemorphin-4 LVV-Hemorphin-6 LVV-Hemorphin-7 Valorphin Spinorphin Endomorphin 1 Endomorphin 2

Amino Acid Sequence H-Tyr-Pro-Phe-Pro-Gly-OH H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-OH H-Tyr-Pro-Phe-Val-Glu-OH H-Tyr-Pro-Phe-Val-Glu-Pro-Ile-OH H-Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro-OH H-Tyr-Pro-Phe-Pro-NH2 H-Tyr-Pro-Trp-Thr-OH H-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH H-Leu-Val-Val-Tyr-Pro-Trp-Thr-OH H-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-OH H-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH H-Val-Val-Tyr-Pro-Trp-Thr-Gln-OH H-Leu-Val-Val-Tyr-Pro-Trp-Thr-OH H-Tyr-Pro-Trp-Phe-NH2 H-Tyr-Pro-Phe-Phe-NH2

lysate of bovine casein, appears as a highly potent and μ-opioid receptor (MOP-R) selective peptide [9]. Furthermore, opioid active peptides in milk lacking the Tyr-Pro sequence in their N-terminal have also been found. Among these are the Arg-Tyr-Leu-Gly-TyrLeu-Glu sequence from bovine α-casein, the whey protein–derived α-lactorphin (Tyr-Gly-Leu-Phe), and β-lactorphin (Tyr-Leu-Leu-Phe). These last peptides behave as MOP-R agonists, but they exhibit much weaker potency than that of the β-casomorphins [24]. The first report on the hemorphins also came from Brantl and co-workers [6]. This time they isolated and determined the sequence of an opioid active tetrapeptide (Tyr-Pro-Trp-Thr) from bovine blood treated with gastrointestinal enzymes. From a computer search, this peptide was identified as fragment 34–37 in the β-chain of bovine hemoglobin. In analogy with βcasomorphin, this tetrapeptide was named hemorphin-4, and it was also found to reside in position 35–38 of the β-, κ-, ∂-, and ε-chains of human hemoglobin. During the past decades, a number of opioid active peptides containing the Tyr-Pro-Trp-Thr core have been identified and characterized (Table 1). The existence of naturally occurring peptides containing the hemorphin-4 sequence in humans was reported in 1991 [16]. In subsequent studies similar structures in human CSF [18] and plasma [17] were found. All these hemorphin structures (LVV-hemorphin-6, LVVhemorphin-7, and hemorphin-7) that were shown to retain biological activity were found to exhibit affinity for opioid receptors. Among other fragments containing the hemorphin-4 sequence that were shown to be

released from the β-chain of hemoglobin are peptides given the names valorphin [14] and spinorphin [45] (Table 1).

THE FORMATION OF b-CASOMORPHINS AND HEMORPHINS In contrast to the classic opioid peptides, the pathways for the formation of the atypical opioids βcasomorphins and the hemorphins are not yet fully clarified. For instance, whereas the enkephalins or dynorphins are formed within and released from neurons in which their precursors are processed, the atypical opioids are not necessarily formed at the site of their precursor production. Pre-stages or intermediately processed entities of both β-casomorphins and hemorphins have been identified in the circulation [44] and CSF [29]. Extended forms of β-casomorphin-8 were found both in the CSF [29, 30] and in the blood [20, 29]. Therefore, the release of β-casomorphins from β-casein was suggested to occur stepwise. In the CSF, a radioimmunoassay (RIA)-active component chromatographically characterized as β-casomorphin-8 was identified in addition to a larger RIA-active congener [30]. In plasma and milk, the β-casomorphin-8-like RIA activity was mainly due to structures of larger size than the octapeptide [29]. However, in a later study the presence of βcasomorphin-8 in milk from a patient with postpartum psychosis was confirmed [38]. The presence of large fragments of the β-chain of hemoglobin containing the hemorphin sequence in

Casomorphins and Hemorphins / 1341 human pituitaries [16] and plasma [31] was believed to indicate that the blood protein subunit is processed or degraded in sequential steps. It was thus suggested that a chymotrypticlike cleavage at the Phe41-Phe42 bond in the β-chain was followed by a similar cleavage to generate LVV-hemorphin-7 [32], which can be further hydrolyzed by exopeptidases to yield smaller hemorphin peptides [21, 33]. An endopeptidase present in the erythrocyte membrane was shown to release the decapeptide LVVhemorphin-7 [2]. Also, the role of macrophage proteolytic complexes in the hemorphin generation has been investigated. When hemoglobin was exposed to rat liver lysosomes, a rapid release of hemorphin-related peptides including hemorphin-7, LVV-hemorphin-7, and VVhemorphin-7 was observed [15].

RECEPTOR SELECTIVITY AND ITS MODULATION Like most of the atypical opioid peptides containing the Tyr-Pro dipeptide in their N-terminal sequence, the β-casomorphins and the hemorphins show binding preference for the MOP-R [6–8, 16]. On this opioid receptor subunit, these peptides seems to act agonistically, as shown by means of the GTPγS assay. Their opioid activity was also confirmed in the GPI bioassay [6, 8, 16]. Although these peptides are endogenously found in quite high amounts, their agonistic actions on opioid receptors are comparatively weak. For instance, compared with β-endorphin, the affinity of the hemorphins for the MOP-R is approximately 500-fold lower. Interestingly, two more recently discovered opioid peptides, endomorphin 1 and endomorphin 2 [46], with structures similar (Table 1) to hemorphin and βcasomorphin, respectively, exhibit a very high potency to stimulate the MOP-R. Studies have also indicated that the β-casomorphins may act on nonopioid sites. For example, the bovine pentapeptide was shown to induce behavioral effects in the rat [19, 28] that were not completely reversed by appropriate doses of naloxone [19]. In recent years, studies have revealed that LVVhemorphin-7 may act as an endogenous ligand for the angiotensin AT4 receptor in the brain [25]. The AT4 receptor was originally defined as the specific highaffinity binding site for the angiotensin (Ang) II fragment Ang IV. This peptide is known to be released from both Ang I and Ang II. However, in addition to Ang IV, the hemoglobin fragment LVV-hemorphin-7 was found to exhibit high binding affinity for and strong agonistic effect on the AT4 receptor [25]. Moreover, the central administration of LVV-hemorphin-7 was shown to enhance learning and memory capabilities in normal rodents and reverse memory deficits observed in ani-

mal models of amnesia. The presence of high-affinity binding sites for this peptide was observed in the brain, including areas such as the hippocampus that are involved in memory processing. However, later these high-affinity Ang IV binding sites represented by the putative AT4 receptor were characterized as a transmembrane enzyme, known as insulin-regulated membrane aminopeptidase (IRAP). Both LVV-hemorphin-7 and Ang IV are competitive inhibitors of this enzyme activity and do not serve as substrates of the IRAP [1].

BIOLOGICAL ACTIONS AND FUNCTIONAL RELEVANCE OF THE b-CASOMORPHINS AND HEMORPHINS Like other endogenous opioid peptides, the βcasomorphins and hemorphins are shown to retain several effects related to opiates. These include analgesic effects, respiratory effects, constipation, sedative effects, and effects on various behaviors. The analgesic effects of the β-casomorphins were demonstrated in animals subjected to intracerebral injections of the peptides [9] or in the hot-plate test using mice [3]. Likewise, in an animal model for phasic nociception (tail-flick assay) both hemorphin-4 and hemorphin-5 produced a dose-related and naloxone-reversible antinociceptive effect in the mice [13].

Specific Actions and Functions of the b-Casomorphins The β-casomorphins have been shown to produce effects in the gastrointestinal tract. For instance, βcasomorphin-7 was found to markedly induce mucus release in the rat jeujenum [10, 43], an effect leading to the protection of the mucosa. The effect, which was dose-dependent, was reversible by naloxone. It was suggested that peptides from dairy products provide dietary prospects for improving gastrointestinal protection [43]. Given orally, the β-casomorphins were shown to influence postprandial metabolism by stimulating the secretion of insulin and somatostatin [40] and to modulate intestinal transport of amino acids [5]. Also, the β-casomorphins and their derivatives have been shown to prolong gastrointestinal transit time and to be useful in the treatment of diarrhea [12]. The β-casomorphins have also been shown to affect hypoxia. Studies on the effects of β-casomorphins on immediate and delayed changes in electrocardiography (ECG) in female rats subjected to acute hypobaric hypoxia during pregnancy were carried out [23]. Hypoxia was shown to induce alterations in the structure of ECG that reflected the development of arrhythmias and conduction disturbances. Intranasal administration of

1342 / Chapter 186 β-casomorphin-7 to pregnant female rats promoted their recovery from acute hypoxia and normalized ECG in the posthypoxic period. Studies examining the ability of β-casomorphins to induce various behaviors have been reported [19, 28, 34]. The effect on rotational behavior in the rat of βcasomorphins of bovine and human origin was compared with that produced by three reference compounds: morphine, D-ala2D-leu5-enkephalin, and U50,488H. These are ligands for MOP, δ-opoid (DOP), and κopoid (KOP) receptors, respectively. In this assay, the bovine β-casomorphin-5 appeared to be more potent than a human analog (β-casomorphin-4). The effects of both morphine and bovine β-casomorphin-5 in producing rotational behavior were partly antagonized by naloxone. With respect to their functional relevance, it has been speculated that the β-casomorphins may be involved in the interplay between mother and child during lactation. In fact, studies dealing with these milkderived peptides have been directed to both mothers and infants. At-term-pregnancy β-casomorphin-8 was found to be elevated both in the plasma and CSF, and this elevation was seen to further increase 6 months later in the puerperal period [29]. Interestingly, a positive correlation between plasma concentration and CSF levels of the peptide was observed. This indicated that β-casomorphin crosses the breast parenchyma– blood barrier into plasma and subsequently penetrates the blood–brain barrier to reach the CNS. It was suggested that the peptide may have a function in maternal behavior [29]. Studies also suggest that the milk-derived opioid may also reach the brain of the infant. Using a peptide extraction procedure combined with highperformance liquid chromatography and RIA, Pasi and co-workers [35] detected rostrocaudally increasing levels of RI-active β-casomorphin-8 in functional relevant brain areas including the mesencephalon, pons cerebri, and medulla oblongata. Indeed, it has been discussed that the β-casomorphins also may affect the child during lactation, and it has been assumed that the milk opioids may induce sedation.

Specific Actions and Function of the Hemorphins Several actions have been studied with regard to the hemorphins. They are shown to affect the blood pressure [26], to produce anti-inflammatory effects [39], to affect the immune system [2], and to inhibit cell proliferation [4]. The vasoconstrictive effect may result from the ability of the peptides to inhibit angiotensin-converting enzyme (ACE). A potent inhibitory effect on ACE has been demonstrated for hemorphin-7 and its N-terminal extensions LVV-hemorphin-7 and also for LVVhemorphin-4 and -6 [21, 45, 47]. The effect of hemorphin-7 on inflammatory processes is likely to be

mediated through its action on MOP-R. Using a blister model of inflammation in the rat hind footpad, the local administration of the heptapeptide was shown to inhibit the acute but not chronic peripheral inflammatory response [39]. The effects were reversible by naloxone. The hemorphins have been shown to be released in exercising horses [11] and in marathon runners [17]. The hemorphins are also shown to induce an in vitro release of β-endorphins in the pituitary or hypothalamus [32], and in humans a concomitant enhancement of hemorphin-7 and β-endorphin was observed in runners after a completed marathon run [17] and in patients subjected to open-heart surgery [32]. In the case of marathon runners, a significant positive correlation between plasma levels of hemorphin-7 and βendorphin was observed. This relationship suggests a link between the heptapeptide and β-endorphin, as was later found in studies of patients undergoing openheart surgery using a heart–lung machine [32]. During this treatment, which continues for several hours, the red blood cells are lysed as a result of their mechanical contact with the instrument and hemoglobin is released into the arterial blood flow and degraded by enzymes to generate hemorphins. Indeed, RIA-detectable hemorphin-7 was increased in the arterial blood flow with time but not in the jugular venous bulb. In contrast, the level of RIA-active β-endorphin in the arterial blood did not change during surgery but was significantly increased over time in blood collected from the venous bulb [32]. In fact, it is known that patients subjected to cardiopulmonary bypass generally have a sense of well-being during the first postoperative day, and this could be explained by the released peptides, which may reach brain nuclei associated with reward and produce euphoria through their actions on opioid receptors.

IMPLICATIONS OF b-CASOMORPHINS AND HEMORPHINS IN PATHOPHYSIOLGY There are still limited studies on the β-casomorphin and hemorphins in relation to pathophysiology. The β-casomorphins have been suggested to be involved in the mechanism underlying childhood autism [34] and apnea in relation to sudden infant death syndrome (SIDS). A recent review highlighted the possible link between β-casomorphins and SIDS in the context of the passage of these peptides through the gastrointestinal tract and the blood–brain barrier [42]. Thus, it was suggested that, in infants predisposed to respiratory apnea because of abnormal autonomic nervous system development and respiratory control mechanisms, milk-derived opioid peptides such as the βcasomorphins might be one of the etiological factors for SIDS [37, 41]. Furthermore, studies have implicated β-casomorphin-like peptides in postpartum psychosis

Casomorphins and Hemorphins / 1343 [22]; in fact, the presence of high levels of β-casomorphin-8 in milk from a patient with postpartum psychosis was reported [38]. As already mentioned, the hemorphins have been found at high levels within the central and peripheral nervous systems. In addition to opioid receptor binding, hemorphins have been shown to have a number of effects on the renin-angiotensin system, including the inhibition of ACE [21] and AT4 receptor binding [25]. However, only a few studies have examined the role of hemorphins in pathophysiology. Recently, a study [36] on hemorphins in Alzheimer’s disease (AD) brains was reported. By quantitative MALDI-TOF, mass spectrometry levels of both LVV- and VV-hemorphin-6 and -7 were assessed in brain tissues from control and AD patients. The authors found that LVV-hemorphin-6 and total hemorphin levels were elevated in the AD temporal neocortex but not in the hippocampus, occipital lobe, or frontal lobe. They suggested that the observed elevation of hemorphins could reflect a vascular abnormality resulting from cerebral amyloid angiopathy associated with both neurodegenerative disease and aging [36]. Increased levels of hemorphins in the circulatory system, also previously mentioned, have been seen under certain clinical condition, such as hemodialysis [31] and during open-heart surgery [32].

CONCLUSION Although the past decades have seen great progress in opioid peptide research, there are comparatively few studies directed to the atypical opioid peptides such as β-casomorphins and hemorphins and their role in physiology or pathophysiology. Although they are produced in relatively high amounts, their weak activity on opioid receptors may have resulted in their receiving less attention. However, currently the β-casomorphins seem to be receiving new interest regarding their role in nutrition [24] and in apnea related to SIDS [42]. The hemorphins have become very attractive compounds since their link to the renin-angiotensin system was discovered. Therefore, it is likely that both these peptide systems will receive renewed interest in years to come.

Acknowledgments This work was supported by the Swedish Medical Research Council (Grant 9459).

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[35] Pasi A, Mahler H, Lansel N, Bernasconi C, Messiha FS. Betacasomorphin-immunoreactivity in the brain stem of the human infant. Res Commun Chem Pathol Pharmacol 1993;80:305– 322. [36] Poljak A, McLean CA, Sachdev P, Brodaty H, Smythe GA. Quantification of hemorphins in Alzheimer’s disease brains. J Neurosci Res 2004;75:704–714. [37] Ramabadran K, Bansinath M. Opioid peptides from milk as a possible cause of sudden infant death syndrome. Med Hypotheses 1988;27:181–187. [38] Renlund S, Erlandsson I, Hellman U, Silberring J, Wernstedt C, Lindstrom L, Nyberg F. Micropurification and amino acid sequence of beta-casomorphin-8 in milk from a woman with postpartum psychosis. Peptides 1993;14:1125–1132. [39] Sanderson K, Nyberg F, Khalil Z. Modulation of peripheral inflammation by locally administered hemorphin-7. Inflamm Res 1998;47:49–55 [40] Schusdziarra V, Schick R, de la Fuente A, Specht J, Klier M, Brantl V, Pfeiffer EF. Effect of b-casomorphins and analogs on insulin release in dogs. Endocrinology 1983;112:885–889. [41] Storm H, Reichelt CL, Rognum TO. Beta-endorphin, human caseomorphin and bovine caseomorphin immunoreactivity in CSF in sudden infant death syndrome and controls. Prog Clin Biol Res 1990;328:327–330. [42] Sun Z, Zhang Z, Wang X, Cade R, Elmir Z, Fregly M. Relation of beta-casomorphin to apnea in sudden infant death syndrome. Peptides 2003;24:937–943. [43] Trompette A, Claustre J, Caillon F, Jourdan G, Chayvialle JA, Plaisancie P. Milk bioactive peptides and β-casomorphins induce mucus release in rat jejunum. J Nutr 2003;133:3499–3503. [44] Umbach M, Teschemacher H, Praetorius K, Hirschhauser R, Bostedt H. Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake. Regul Pept 1985;12:223–230. [45] Yamamoto Y, Ono H, Ueda A, Shimamura M, Nishimura K, Hazato T. Spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: roles in pain and inflammation. Curr Protein Pept Sci 2002;3:587–599. [46] Zadina JE, Hackler L, Ge LJ, Kastin AJ. A potent and selective endogenous agonist for the μ-opiate receptor. Nature 1997; 386:499–502. [47] Zhao Q, Sannier F, Garreau I, Guillochon D, Piot JM. Inhibition and inhibition kinetics of angiotensin converting enzyme activity by hemorphins, isolated from a peptic bovine hemoglobin hydrolysate. Biochem Biophys Res Commun 1994;204:216– 223.

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187 Anti-Opioid Peptides JOHN Q. WANG, EUGENE E. FIBUCH, SHINOBU SAKURADA, AND JI-SHENG HAN

which provides an anatomical basis for direct interactions between CCK and opioid systems at the receptor and postreceptor levels. Among all anti-opioid peptides, CCK has been most thoroughly studied and best characterized in terms of its pharmacological effects, gene expression, and peptide release in response to repeated opioid administration and its intracellular interactions with opioid receptorassociated signaling pathways. This peptide, therefore, is mainly reviewed in this chapter regarding its antiopioid action, followed by a concise summation of three other anti-opioid peptides (neuropeptide FF, NPFF; nociceptin; and the Tyr-MIF-1 family of peptides).

ABSTRACT The first demonstration in 1979 of an anti-opioid peptide and prediction of the existence of other endogenous anti-opioid peptides in the central nervous system (CNS) has prompted extensive research endeavors exploring and identifying these peptides during the past two and a half decades. Several peptides that fall into this category include cholecyctokinin (CCK), neuropeptide FF (NPFF), angiotensin II, Tyr-MIF-1 (TyrPro-Leu-Gly-NH2)-related peptides, and more recently nociceptin (orphanin FQ). Hypothetically, they are collectively viewed as a negative feedback system to antagonize the excessive activation of the opioid receptors induced by the repeated administration of opiates or excessive release of opioid peptides. From a practical point of view, the blockade of the anti-opioid peptides may have clinical implications for the treatment of intractable pain.

REGULATION OF OPIOID ANALGESIA BY CCK CCK, a member of the gastrin family of peptides (see Chapter 131 by Moran et al. and Chapter 139 by Reeve et al. in earlier sections of this book), is present in the central nervous system (CNS) mainly in the form of a carboxy-terminal octapeptide (CCK-8). Faris et al. [10] in 1983 first reported that CCK, when administered systemically or perispinally, antagonized opiate analgesia. The anti-opioid action of CCK was afterward confirmed and characterized in a large number of pharmacological and behavioral studies [15, 16, 18]. For instance, the ICV administration of CCK-8 suppressed morphine analgesia in rats [16, 18]. IT CCK-8 injection achieved the same effect [16]. Apparently, CCK-8 possesses the ability to antagonize morphine analgesia at both the supraspinal and spinal levels. Acupuncture and electroacupuncture (EA) have been documented to release endogenous opioid peptides in the CNS and thus produce strong analgesia [17]. Like morphine analgesia, EA-induced analgesia was substantially attenuated by ICV or IT injec-

INTRODUCTION Numerous pharmacological and behavioral studies have demonstrated a potent antagonizing effect of the anti-opioid peptides on various forms of opioid analgesia following systemic, intracerebroventricular (ICV), or intrathecal (IT) injection. Most of these anti-opioid peptides also contribute to the development of tolerance to opioid analgesia because (1) their expression and release are upregulated in the brain regions important for pain modulation and opioid analgesia and (2) the antagonism of anti-opioid peptide systems by the selective receptor antagonists, antiserum, or antisense oligonucleotides is able to reverse opioid tolerance. At the cellular level, chole cysto kinin (CCK) receptors colocalize with opioid receptors in the same neurons, Handbook of Biologically Active Peptides

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1346 / Chapter 187 tion of CCK-8 [16]. Thus, CCK-8 exerts its influence over analgesia induced by both exogenous and endogenous opioids in an inhibitory fashion. Furthermore, CCK-8 was observed to reverse morphine- or EA-induced inhibition of the pain-excitatory neurons and the excitation of the pain-inhibitory neurons in the rat nucleus parafascicularis [2] in addition to its ability to inhibit C-fiber-evoked firing [24]. The antagonizing effect of exogenous CCK suggests that endogenous CCK may act physiologically as an opioid antagonist to dose-dependently suppress opioid analgesia. Indeed, the CCK receptor antagonist proglumide or MK329 (L364,718) when given by the ICV, IT, or systemic route potentiated various forms of analgesia produced by morphine and endogenous opioids and seemed to reverse tolerance to morphine analgesia [33, 43]. The antiserum against CCK-8 produced similar effects [11]. In addition, the antiserum could reverse tolerance to EA analgesia [6]. Even clinical placeboinduced analgesia, which may be opioid in nature, was potentiated by proglumide [1]. Moreover, rats receiving CCK antisense RNA [34] and mice lacking CCK2 receptors [37] show increased opioid analgesia. These results suggest that in response to upregulated opioidergic activities the endogenous CCK system can act as a negative feedback controller to return pain sensitivity to its basal level. Because neither proglumide nor CCK8 antiserum produced measurable analgesia in the absence of morphine [6, 43], the endogenous CCK system seems not to be tonically active in affecting basal pain perception. Two subtypes of CCK receptors have been identified: CCKA and CCK1 receptors, predominantly located in the periphery, and CCKB or CCK2 receptors, in the CNS. In the attempts to verify the receptor mechanism, a large number of pharmacological studies have been conducted, and results from most of these studies support the concept that the central type CCKB rather than the peripheral type CCKA receptor is primarily responsible for the anti-opioid effect of CCK. The CCKB subtype-selective antagonist L365,260, L740,093, or CI988 readily mimicked the effect of the non-subtypeselective CCK receptor antagonists and CCK antiserum in augmenting morphine- and EA-induced analgesia and preventing tolerance to morphine analgesia [8, 47, 54]. In mice lacking CCKB receptors, morphine induced a significantly stronger antinociceptive effect as compared with wild-type animals [37]. Noticeably, the role of the CCKB receptor in modulating opioid analgesia varies with different opioid receptor subtypes. There is evidence showing that the CCKB receptor plays a significant role in antagonizing the analgesia mediated by μ- and κ-opioid receptors, with a minimal influence over δ-opioid-receptor-mediated analgesia [40].

CCK GENE EXPRESSION IN RESPONSE TO OPIOID ANALGESIA CCK mRNAs and CCK-like immunoreactive materials are broadly distributed in the CNS. Generally, substantial overlap in the anatomical distribution of CCK, endogenous opioid peptides, and their receptors exists in the brain and spinal cord, providing an anatomical basis for the interaction between the two systems. Alterations in CCK peptide gene expression in the CNS were investigated in rodents exposed to opioids or that had developed tolerance to opioid or EA analgesia. Basal levels of CCK mRNAs were elevated in the whole brain of rats rendered tolerant to repeated injections of morphine [55]. The increased levels of CCK mRNAs indicate an accelerated synthesis of CCK peptides in the brain at the transcription level, probably leading to enhanced CCK activity. As a compensatory response, enhanced CCK activity could then engage in counteracting the analgesic effect of opioids. The results from the pharmacological studies previously described support this notion. The region-specific increase in CCK mRNAs was also investigated by in situ hybridization. Pu et al. [30] found that CCK mRNA levels were elevated in the amygdala in morphine-tolerant rats. Moreover, the increase in CCK mRNA levels in the amygdala kinetically corresponded well with the development of tolerance to morphine [30]. Thus, CCK in the amygdala may play a functional role in regulating morphine tolerance based on the fact that its expression can be upregulated in this nucleus in response to morphine administration. This was substantiated by the finding that the CCKB receptor antagonist locally injected into the amygdala augmented the analgesic effect of systemic morphine [30]. The periaqueductal gray (PAG) of the midbrain represents another strategic site in the CNS where CCK-8 can exert its anti-opioid action [36]. Normally, CCK peptides are expressed in neurons in this region at variable levels, which may account for the high or low analgesic responsiveness to morphine and EA in rats with a low or high level of CCK in the PAG, respectively. The reduction of cellular levels of CCK-8 through an antisense approach converted low responder rats into high responders to morphine and EA analgesia and delayed the development of analgesic tolerance to prolonged EA stimulation or repeated morphine administrations [33]. Similarly, the CCK receptor antagonist injected into the PAG reversed tolerance to morphine analgesia [36]. These results demonstrate a strong antagonizing effect of endogenous CCK-8 in the PAG on opioid analgesia, although individual variation may exist in this action due to variable levels of CCK-8 expressed in this region.

Anti-Opioid Peptides / 1347 Chronic treatment with morphine increased CCK mRNA levels as well as CCK immunoreactive peptides in the hypothalamus, the spinal cord, and the brain stem [5]. Increased CCK synthesis may then provide the substrate for enhanced CCK activity in those areas to suppress the analgesic effect of morphine. Pharmacological studies have demonstrated an inhibitory role of CCK in those areas in regulating opioid analgesia [44]. The analgesic effect of morphine varies in different pain states. Patients or experimental animals with neuropathic pain following injury to the nervous system usually respond poorly to opioids. This poor response may be related to the anti-opioid action of CCK. It was found that axotomy caused an upregulation of CCK and CCKB receptor mRNAs in rat dorsal root ganglion neurons [38, 46]. The CCKB receptor antagonist CI988 was able to reverse the reduction of morphine analgesia in axotomized rats [45]. CI988 could also reverse the lack of effect of IT morphine in alleviating neuropathic painlike symptoms in rats after complete or partial peripheral nerve injury [46]. Thus, insensitivity to opioid analgesia in rats with nerve injury may result from enhanced CCK and CCK receptor expression and, as a result, their functions.

CCK RELEASE IN RESPONSE TO OPIOID ANALGESIA The extracellular level of CCK under different conditions determines its function of modulating opioid action. Through in vivo microdialysis technique in freely moving and conscious animals combined with radioimmunoassay, Gustafsson et al. [14] found that the basal CCK level was usually below or close to the detection limit of the radioimmunoassay in the normal rat spinal cord. The normal extracellular CCK levels could be markedly elevated by depolarization caused by perfusion of the microdialysis probe with Krebs-Ringer solution containing a high concentration of K+ [14]. In addition to depolarization, morphine is a potent stimulator for facilitating CCK release. When injected intravenously or applied topically on the spinal cord, morphine significantly increased CCK in the dialysate [25]. Because the release of CCK by morphine was sensitive to naloxone blockade, opioid receptors are believed to mediate the morphine-induced CCK release. CCK-8 was also detectable in the cerebrospinal fluid (CSF) of rats with a normal range of 14.4–15.1 fmol/ml [54]. EA stimulation resulted in a naloxone-sensitive increase in CCK-8 levels in the CSF [54]. This increase lasted for at least 6 h. Such a long-lasting increase in CCK-8 release may require a parallel increase in biosynthesis of the peptide as already noted.

The mechanism(s) underlying the opioid facilitation of CCK release are unclear. There is evidence that opioid receptors may facilitate the formation of inositol 1,4,5-phosphate (IP3) through their coupling to phospholipase C [3]. Because IP3 is a strong stimulator of intracellular Ca2+ release, the activation of opioid receptors may lead to a Ca2+-dependent CCK release, although it needs to be proven experimentally. It can be argued that the CCK release may not be mediated by a direct action of opioids because generally opioids exert an inhibitory modulation for a given cellular activity. The ability of opioids to facilitate CCK release can therefore be explained by a disinhibitory mechanism. It is possible that both direct and indirect mechanisms mediate CCK release by opioids. Using an electrophysiological approach, Heinricher et al. [19] showed that, although morphine activated the OFF cell and inhibited the ON cell simultaneously in the rostral ventromedial medulla (RVM), microinjection of CCK within the RVM prevented opioid activation of OFF cells; however, it did not prevent the suppression of spontaneous ON-cell firing by morphine.

CELLULAR AND MOLECULAR MECHANISMS FOR CCK-OPIOID INTERACTIONS The cellular and molecular mechanisms underlying the CCK anti-opioid action have been explored to a certain extent [15a]. At the receptor level, Wang et al. [40] have observed that CCK-8 reduced the ligand binding affinity to μ-opioid receptors. Considerable colocalization of CCK receptors in the dorsal horn neurons of spinal cord with μ-opioid receptors seems to provide a morphological basis for this receptorreceptor interaction in the same neuron [53]. At the postreceptor level, cross-talk between CCK and opioid receptor–associated signaling pathways may exist, and the activation of CCK receptor–derived signaling cascades has been found to suppress intracellular events downstream to opioid receptor activation [39].

REGULATION OF OPIOID ANALGESIA BY NPFF NPFF (FLFQPQRFamide; F8Famide; Phe-Leu-PheGln-Pro-Gln-Arg-Phe-NH2) is another important endogenous octapeptide that possesses CCK-like anti-opioid action. NPFF and NPFF receptors are densely distributed in the CNS, especially in the spinal dorsal horn and the PAG, areas important in opioid-mediated pain perception. ICV injection of NPFF reduced the

1348 / Chapter 187 analgesic effect of systemic morphine [23]. Similar results were replicated by other investigators. NPFF antisense oligonucleotides (injected ICV) decreased tolerance to morphine analgesia [13]. Electrophysiological studies also showed an antagonizing effect of NPFF on μ-opioid receptor–mediated inhibition of the dorsal horn neuronal activity evoked by C-fiber stimulation [26]. These results indicate a significant role played by NPFF in antagonizing opioid analgesia. At the supraspinal level, several brain areas have been identified as important sites for the NPFF-opioid interaction. Local infusion of NPFF into the hippocampus reduced the electrophysiological response of hippocampal neurons to opioids [27]. The NPFF analogs when injected into the nucleus raphe dorsalis also suppressed the antinociceptive effect of morphine [7]. Other central sites where application of NPFF or NPFF analogs produced anti-opioid action include the ventral tegmental area of the midbrain and the parafascicular nucleus. Remarkable similarities in several key anti-opioid actions are noted between CCK and NPFF. First, the two peptides when applied alone cause no significant pronociceptive effect, and they mainly exert a dosedependent antagonism of opioid-induced analgesia. Second, like CCK and CCK receptors, NPFF and NPFF receptors strategically overlap with the opioid receptor distribution in many brain regions related to pain perception and modulation. This provides a morphological and cellular basis for the interaction of NPFF and opioids. Finally, opioids can increase NPFF gene expression and NPFF release [4]. Thus, the NPFF system, like the CCK system, can be upregulated and mobilized to exert its anti-opioid action in response to enhanced opioidergic transmission.

REGULATION OF OPIOID ANALGESIA BY NOCICEPTIN Nociceptin (orphanin FQ) and its regulatory role in pain modulation is discussed by Meunier, one of its discoverers, in Chapter 188 in this section of the book. The available results show that nociceptin has a complex pharmacology in rodents, eliciting either an antiopioid/hyperalgesic action or analgesia depending on the dose, testing paradigm, and brain regions surveyed. As to its anti-opioid action, ICV injection of nociceptin blocked the supraspinal antinociception produced by ICV injection of all three opioid receptor subtype (μ, δ, and κ)-selective agonists [28]. Recent studies showed that nociceptin also antagonizes analgesia induced by nonopioid substances such as paracetamol [32] and 5-HT [41]. Therefore, it is not only an anti-opioid peptide, but may also be an anti-analgesic peptide. In

contrast to its supraspinal effect, IT nociceptin was completely ineffective against antinociception induced by IT or systemic injection of morphine [35]. In fact, nociceptin produced an antinociceptive effect in the spinal cord [35]. Thus, the anti-opioid action of nociceptin seems to be restricted to supraspinal sites. Nociceptinergic terminals are located in the PAG, and intra-PAG injection of nociceptin attenuated morphine analgesia [29], indicating the PAG as a site for nociceptin to antagonize opioid actions. Further studies show that continuous use of high doses of morphine accelerated the release and biosynthesis of nociceptin in rat brain, which antagonizes the analgesic effect of opioids and promotes the development of morphine tolerance [49]. The mechanisms underlying the anti-opioid effect of nociceptin are not clear. Rady and Fujimoto [31] pointed out that the anti-analgesic action of nociceptin is mediated by spinal PGE2 and attenuated by IT PGD2 or indomethacin. At the cellular level, μ-opioid receptor–mediated inhibition of calcium currents can be antagonized by nociceptin applied at the same neuron [52]. At the circuitry level, nociceptin, which profoundly inhibits all three classes of RVM neurons (ON, OFF, and neutral), blocked the opioid activation of OFF cells and interfered with opioid antinociception [19].

REGULATION OF μ-OPIOID SYSTEMS BY MIF-1, TYR-MIF-1, AND TYR-W-MIF-1 Another family of endogenous peptides that shows anti-opioid properties was identified by Kastin and his colleagues. In 1979, they identified an endogenous peptide, Pro-Leu-Gly-NH2 (MIF-1) from bovine brain [22] and later identified two more endogenous peptides, Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1) in 1989 [20], and Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) in 1992 [9] from human brain. These peptides show inhibitory actions against several pharmacological effects of morphine, including antinociception [22], hypothermia [48], and inhibition of guinea pig ileum contraction [51], and therefore these peptides have been considered to be endogenous anti-opioid peptides. Their antiopioid properties seem to be mediated by μ-opioid receptors because these peptides show low, but significant, affinity and very high selectivity for μ-opioid receptors [50]. Correlating with their affinity to μ-opioid receptors, their inhibitory effects against morphine are relatively weaker than those of traditional μ-opioid receptor antagonists, such as naloxone or CTOP [51]. Interestingly, these peptides show an inverted U-shape dose-response curve for the inhibition of morphineinduced pharmacological effects [21, 48]. As expected, these peptides later were reported to have agonistic

Anti-Opioid Peptides / 1349 properties for μ-opioid receptors in several experimental paradigms, including analgesia [42] and electrically stimulated guinea pig ileum contraction [51]. Their agonistic effects are completely attenuated by μ-opioid, but not δ- and κ-opioid, receptor antagonists. Moreover, the potencies of their agonistic effects are relatively lower than those of morphine. The evidence clearly suggests that these peptides are partial agonists for μ-opioid receptors with relatively low intrinsic activity and affinity, although effects at the Tyr-MIF-1 receptor itself may be involved. Physiologically, these peptides may biphasically, both negatively and positively, control the μ-opioid systems as a partial agonist.

Acknowledgment Supported by a grant (2003CB515407) from the Ministry of Science and Technology, PRC to JSH.

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188 Nociceptin JEAN-CLAUDE MEUNIER AND BRICE BES

an orphan receptor. The peptide is hereafter in this chapter referred to as NOP (nociceptin orphanin FQ peptide), in compliance with the latest recommendations of the International Union of Pharmacology (IUPHAR). The peptide’s precursor and receptor are referred to as prepro-NOP (ppNOP) and NOP receptor, respectively. The authors apologize to all colleagues whose important contribution is not directly cited because of space limitations.

ABSTRACT The heptadecapeptide nociceptin (NOP; FGGFTGARKSARKLANQ) was discovered as the natural ligand of the NOP receptor, the fourth cloned member of the opioid receptor family. NOP is derived from a larger precursor polypeptide (prepro-NOP) which is widely distributed in the nervous system. NOP is primarily an inhibitory neuropeptide that acts on neurons to depress synaptic transmission. NOP is reported to modulate pain, stress and anxiety, feeding, learning and memory, reward and addiction, and the functioning of the heart and vessels, airways, kidney, urinary bladder, intestine, and immune cells. The broad spectrum of pharmacological effects of NOP suggests potential therapeutic applications for NOP receptor agonists and/or antagonists.

STRUCTURE OF PRECURSOR MRNA/GENE NOP is derived from a larger precursor polypeptide, ppNOP, whose gene is composed of at least four exons separated by three introns. Exon I contains exclusively a 5' noncoding sequence, exons II and III share the open reading frame of the gene, and exon IV contains most of the 3' noncoding region of the message (Fig. 1). The nucleotide sequence of the murine and human ppNOP genes displays organizational and structural features that are very similar to those of the genes encoding the precursors to the endogenous opioid peptides, prepro-enkephalin, -dynorphin, and -opiomelanocortin, suggesting that the nociceptin and opioid peptide genes have evolved in parallel from a common ancestor gene. The ppNOP gene localizes to the short arm of human chromosome 8 (8p21). The cloning and sequencing of a 1.7-kb promoter and upstream regulatory region of the human ppNOP gene has revealed the presence of a number of potential binding sites for transcription factors such as, in particular, TF-IID, cAMP response element binding proteins, and glucocorticoid and estrogen receptors [32]. The ppNOP gene encodes a protein, ppNOP, whose amino acid sequence is highly conserved across murine, bovine, and human species (Fig. 2), especially the Cterminal third for NOP itself. The ppNOP polypeptide

DISCOVERY The discovery of nociceptin [19], or orphanin FQ [31], is one of the earliest examples of the successful application of reverse pharmacology or functional genomics. The neuropeptide was isolated from brain extracts as a component that inhibited forskolininduced production of cAMP in chinese hamster ovary cells made to stably express the opioid receptorlike 1 (ORL1) receptor, an orphan receptor whose cDNA had been previously cloned from a human brain-stem cDNA library. Nociceptin is a heptadecapeptide (FGGFTGARKSARKLANQ) bearing a close resemblance to opioid peptides, the natural ligands of the opioid receptors. The peptide was named nociceptin because of its ability to elicit apparent hyperalgesia when administered intracerebroventricularly in mice, and it was named orphanin FQ to designate a peptide framed with Phe (F) and Gln (Q) amino acid residues that activated Handbook of Biologically Active Peptides

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:signal peptide; :Met-enkephalin; :Leu-enkephalin FIGURE 1. A. General organization of the ppNOP gene. The ppNOP gene consists of four exons (numbered I to IV) interspersed by three introns (A, B, and C). The filled boxes correspond to the coding region. ATG, STOP, and poly(A) are the transcription start, stop, and polyadenylation sites, respectively. B. Comparison between the translated regions of the human preproNOP (hppNOP), prepro-opiomelanocortin (hppOMC), prepro-enkephalin (hppENK), and prepro-dynorphin (hppDYN) genes. Boxes indicate the coding frame and the interrupted lines materialize the introns. ME, Met-enkephalin; LE, Leu-enkephalin.

signal peptide

mouse rat bovine human

MKILFCDVLLLSLLSSVFSSCPRDCLTCQEKLHPAPDSFNLKTCILQCEEKVFPRPLWTVCTKVMASGSGQLSPADPELV MKILFCDVLLLSLLSSVFSSCPEDCLTCQERLHPAPGSFNLKLCILQCEEKVFPRPLWTLCTKAMASDSEQLSPADPELT MKILFCDLLLLSLFSSVSSSCQKDCLVCREKLRPTLDSFSLEMCILECEEKAFTSPLWTPCTKVMARGSWQLSPADPDHV MKVLLCDLLLLSLFSSVFSSCQRDCLTCQEKLHPALDSFDLEVCLIECEEKVFPSPLWTPCTKVMARSSWQLSPAAPEHV ** * ** ***** *** *** *** * * * * ** * * **** * **** *** ** * ***** * nocistatin NOP

080 080 080 080

mouse rat bovine human

SAALYQPKASEMQHLKRMPRVRSLVQVLDAEPGADAEPGADAEPGADDAEEVEQKQLQK RFGGFTGARKSARKLANQKR SAALYQSKASEMQHLKRMPRVRSVVQARDAEPEA------DAEPVADEADEVEQKQLQK RFGGFTGARKSARKLANQKR AAALDQPRASEMQHLKRMPRVRSLFQRQ-----------KRTEPGLEEVGEIEQKQLQKRFGGFTGARKSARKLANQKR AAALYQPRASEMQHLRRMPRVRSLFQEQE-EPEP----------GMEEAGEMEQKQLQKRFGGFTGARKSARKLANQKR *** * ******* ******* * * ***************************

159 153 148 148

hppNOP-[149-176] hppNOP-[149-165] hppNOP-[169-176] mouse rat bovine human

FSEFMRQYLVLSMQSSQRRRTLHQNGNV IQVIPRTACVHSKTCRPGVRIPPSPRH FSEFMRQYLVLSMQSSQRRRTLHQNGNV IQVSPRTACVHTKTCRPGVRIPPSLRH FSEFMRQYLVMSMQSSQRRRTLHQNGNA FSEFMRQYLVLSMQSSQRRRTLHQNGNV ********** ********** *** ******* ************ **

187 212 181 206 176 176

FIGURE 2. Alignment of the sequences of mouse, rat, bovine, and human NOP precursors. Asterisks denote amino acid identities. The putative proteolytic cleavage motifs are underlined. The sequences of the longer variant precursor from mouse and rat (see [33]) is also shown. There is evidence that a similar variant occurs in humans (not shown).

Nociceptin

which replaces exons I and II found in neuronal transcripts [2].

contains a putative N-terminal signal peptide of approximately 20 amino acids and one single copy of NOP framed by canonical Lys-Arg proprotein convertase excision motifs. However, in addition to those framing NOP, the ppNOP sequence contains other polybasic cleavage sites, suggesting that ppNOP may be the precursor not only to NOP but also to other biologically relevant neuropeptides. These include peptide 17 (bovine), 30 (human), 35 (rat), and 41 (mouse) amino acid residues in length located immediately upstream to NOP and known as nocistatin and the downstream peptides ppNOP(149–176), eventually -(149–165) and -(169–176) (human precursor numbering). In addition to nocistatin, which blocks NOP action in pain transmission, ppNOP(149–165) appears to be pharmacologically active, suggesting that it is physiologically relevant [20]. Alternative splicing of the ppNOP gene between exons III and IV generates a shorter transcript that encodes a precursor (ppNOP-L) which is 25 amino acid residues longer than ppNOP. The ppNOP and ppNOP-L transcripts have been found to be upregulated in mouse NS20Y neuroblastoma cells undergoing cAMP-induced neuronlike differentiation and, when transfected into undifferentiated NS20Y cells, to promote neurite outgrowth [33]. In peripheral human blood lymphocytes, ppNOP transcripts contain a unique exon, ImEx2b,

DISTRIBUTION OF MRNA AND PEPTIDE The ppNOP mRNA and NOP are broadly yet discretely distributed in the central nervous system of the rat (Fig. 3). mRNA and peptide are particularly abundant in the cortex and limbic structures (hippocampus, dentate gyrus, septal areas, and amygdala), a number of hypothalamic and brain-stem nuclei, the dorsal and ventral horns of the spinal cord, and cell bodies of the dorsal root ganglion. In their thorough study, Neal and colleagues [24] found only very few regions where mismatch occurred between peptide immunostaining and mRNA expression, indicating that NOP is produced essentially in interneurons. In several pain-modulatory areas, including the superficial dorsal horn of the spinal cord, sensory trigeminal complex, and periaqueductal gray matter, antisera to NOP and opioid peptides label different fiber systems, consistent with the differential actions of NOP and the opioids on nociception. In the cord, NOP-like immunoreactivity is resistant to unilateral dorsal rhizotomy, indicating that the peptide resides in central, rather than primary, afferent neurons [32].

Cx Hi

CC OB CPut

S

Th PAG

BST Hpt PVN DMH

Acb DBB

SON VMH Amy

high

high to moderate

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moderate

DR SN

Arc NL ME IL AP

C PBN LC NST Amb LRN

low

FIGURE 3. Distribution of ppNOP mRNA in the adult male rat. Adapted from [24]. Acb, nucleus accumbens; Amb, nucleus ambiguous; AP, anterior lobe of pituitary gland; Arc, arcuate nucleus of hypothalamus; BST, bed nucleus of stria terminalis; C, cerebellum; CC, corpus callosum; CPut, caudate putamen; Cx, cortex; DBB, diagonal band of Broca; DR, dorsal raphe nucleus; Hi, hippocampus; Hpt, hypothalamus; IL, intermediate lobe of pituitary gland; LC, locus coreuleus; LRN, lateral reticular nucleus; ME, median eminence; NL, neural lobe of pituitary gland; NST, nucleus of solitary tract; OB, olfactory bulb; PAG, periaqueductal gray matter; PBN, parabrachial nucleus; PVN, paraventricular nucleus of hypothalamus; S, septum; SON, supraoptic nucleus; SN, substantia nigra; Th, thalamus; VMH, ventromedial hypothalamus.

1354 / Chapter 188 The distribution of NOP in the central nervous system is consistent with the peptide being potentially involved in the regulation of a number of brain functions, including the stress response, learning and memory, locomotion, reinforcement and reward, sexual behavior, pain perception, and autonomic functions such as control of cardiovascular function and control of water and electrolyte balance.

BIOSYNTHESIS AND DEGRADATION The processing of ppNOP has so far been only marginally addressed. In the brain of prohormone convertase 2 (PC2) deficient mice, NOP production is significantly lowered and there is an accumulation of ppNOP and biosynthetic intermediates. This suggests that PC2 is directly involved in the biogenesis of mature NOP [1]. However, another study has reported that, in the NG108-15 hybridoma, prohormone convertase 1 (PC1) but not PC2 enhances processing at the Lys-Arg motif located between the C-terminus of nocistatin and the N-terminus of NOP [29]. In mouse brain cortical slices, NOP is degraded by the metallopeptidases aminopeptidase N and endopeptidase 24.15 but not by endopeptidase 24.11, which is involved in enkephalin catabolism. Indeed, simultaneous inhibition in vivo of aminopeptidase N and of endopeptidase 24.15 potentiates NOP-induced reduction in locomotor activity in mice [27]. However, endopeptidase 24.11 has been reported to play a major role in nociceptin catabolism in the mouse spinal cord [34].

RECEPTOR The NOP receptor belongs to the superfamily of Gprotein-coupled receptors, characterized by seven transmembrane helices interconnected by alternate intraand extracellular loops. It displays high sequence similarity with respect to opioid receptors, yet has little or no affinity for opioids. The primary structure of ORL1 is highly conserved across mammalian species, the mouse and human sequences being >95% identical. The NOP receptor gene is located in the distal region of mouse chromosome 2 and in the q13.2-13.3 region of human chromosome 20. Its coding sequence is organized into introns and exons in a similar manner to the opioid μ-opioid (MOP), δ-opioid (DOP), and κ-opioid (KOP) receptor genes, suggesting that the four genes have all evolved from a common ancestor, that is, they belong to the same family. Like other members of the opioid receptor family, the NOP receptor gene undergoes alternative splicing. The biological significance of these splice variants is unknown because pharmacological studies have not clearly established the existence

of functionally distinct NOP receptor subtypes. The 5'untranslated region of the mouse NOP receptor gene contains putative binding sites for glucocorticoid receptors and for Ying Yang 1 and myb transcription factors. Human lymphocytes express a NOP transcript whose 5'-untranslated region is clearly distinct from that of the brain transcript, which suggests a distinct regulation of the NOP receptor gene [18]. The NOP receptor mRNA is particularly abundant in the central nervous system, notably in the cortical and corticolimbic areas, hypothalamus, brain stem, dorsal and ventral horns of the spinal cord, and cell bodies of the dorsal root ganglion [25]. Extensive immunohistochemical mapping of the NOP receptor protein shows similar distribution patterns to those of mRNA, indicating that the receptor is expressed predominantly in local-circuit neurons. Moreover, antisera to the NOP and MOP receptors label different fiber systems in a number of areas involved in pain processing [21], consistent with the different pharmacological profiles of NOP and the opioids.

BIOLOGICAL ACTIONS At the cellular level, NOP activates the NOP receptor and a pertussis-sensitive G-protein to inhibit adenylate cyclase and calcium currents and to open potassium channels. These actions of NOP, especially those on ion channels, reduce synaptic efficacy, either presynaptically by reducing transmitter release and/or postsynaptically by reducing neuron excitability. Indeed, many in vitro and in vivo studies have shown that NOP inhibits the basal and/or stimulated release of many neurotransmitters, including acetylcholine, dopamine, GABA, glutamate, and substance P, in central and peripheral nerve tissue [9, 36]. Likewise, when tested electrophysiologically on individual neurons such as in particular spinal dorsal horn neurons, NOP is often, if not always, found to inhibit basal and/or stimulated electrical activity [22]. The cellular inhibitory action of NOP, as well as its inherent circuit-induced disinhibitory actions, suffice to explain most if not all of the peptide’s acute pharmacological effects [10].

NOP Actions on Pain and Inflammation The abundant literature addressing the function of NOP in pain and inflammation is highly contentious, with conflicting observations on almost every question examined [10]. One reason for this, among many others, is that NOP actions on pain transmission are dependent on dose and site of administration. Thus, NOP has been variously reported to cause hyperalgesia, allodynia, analgesia, and even nocifensive behavior.

Nociceptin Supraspinal Effects NOP was initially reported to cause thermal hyperalgesia when injected intracerebroventricularly (ICV) in mice. However, this effect was soon shown to actually reflect the reversal of a stress-induced, largely opioidmediated analgesia rather than a decrease of nociceptive threshold. Indeed, ICV-administered NOP attenuates opioid-induced analgesia, thus confirming the notion that NOP has the functional properties of a supraspinal anti-opioid peptide. One mechanism whereby NOP attenuates opioid-induced analgesia is by directly inhibiting a descending antinociceptive pathway, which is itself indirectly stimulated (disinhibited) by opioids [10, 20]. There is growing evidence that NOP receptor antagonists restore near-normal sensitivity to morphine analgesia in animals rendered tolerant to the opiate [20, 40]. Spinal Effects When administered intrathecally, very low doses of NOP cause essentially pro-nociceptive effects, whereas higher doses produce antinociceptive effects. Intrathecal (IT) injections of low (nanogram) doses of NOP in mice cause allodynia, a pain response to innocuous (tactile) stimulation, whereas IT injections of even lower (picogram) doses elicit hyperalgesia. These pronociceptine effects have been attributed to the disinhibition by NOP of spinal glycinergic transmission, but this remains contentious [41]. Likewise, femtomolar IT doses of NOP elicit typical nocifensive behavior (licking, scratching, and biting) in mice, and this appears to involve disinhibition by NOP of spinal histaminergic transmission [35]. In marked contrast, many studies have shown that IT administration of higher doses (microgram) of NOP produces antinociceptive effects (analgesia) in a wide variety of animal models of phasic (mechanical or thermal) and tonic (inflammatory or neuropathic) pain [39, 41]. The spinal analgesic actions of NOP are very much consistent with the well-documented ability of the peptide to block both normal and sensitized excitatory (glutamate) transmission in the dorsal horn of the spinal cord. Interestingly, NOP appears to be more efficacious in alleviating chronic neuropathic or inflammatory than acute pain states [39, 41]. Peripheral Effects Intradermal administration of picogram doses of NOP stimulates the flexor reflex in mice, an indication that the peptide may be endowed with peripheral pronociceptive activity. This effect involves the induction by NOP of the release of substance P from peripheral nerve endings [11]. In rats, intradermal application of NOP has also been reported to increase vascular perme-

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ability, an effect that is likely to involve stimulation by NOP of the release of histamine from mast cells [14]. This effect suggests that NOP may act as a proinflammatory cytokine, and this is consistent with the peptide’s reported ability to stimulate neutrophil chemotaxis and recruitment [37]. It is also consitent with the recent observation that experimental (dextran sodium sulfate–induced) colitis is substantially less severe in NOP receptor–deficient than in wild-type mice [13].

NOP Actions on Reward and Addiction The rewarding properties of NOP have been examined using the conditioned place preference test that pairs the administration of the drug with a particular set of environmental cues. In rats, ICV-administered NOP does not induce place preference or aversion, an indication that the peptide lacks intrinsic motivational properties. Most significantly, however, NOP was later shown to block morphine-induced place preference in rats, an effect that turned out to generalize to other abused drugs, all known to acutely stimulate the dopamine mesocorticolimbic pathway, such as alcohol, amphetamine, and cocaine [7, 23]. One mechanism whereby NOP attenuates reward elicited by drugs of abuse is by directly inhibiting the dopamine mesocorticolimbic neurons, which express the NOP receptor [16]. Of particular relevance to addiction are the recent observations that NOP blocks the reinstatement of alcohol-seeking behavior in alcohol-preferring rats [7] and that the nonpeptidic NOP receptor agonist Ro 646198 blocks the reinstatement of morphine-elicited place preference in mice [38].

NOP Actions on Stress and Related Behaviors Perhaps the most fundamental action of NOP is its action on stress and stress-related behaviors. An early study showed that ICV-administered NOP is anxiolytic in several benzodiazepine-sensitive behavioral tests [12]. Yet a more recent study has reported just the opposite [8]. The antistress/anxiolytic actions of NOP appear to be brought about by central inhibition of the hypothalamic-pituitary-adrenal axis, with the reduction of circulating corticosterone. Indeed, in comparison with wild-type animals, NOP-deficient mice have a greater tendency toward anxietylike behavior when exposed to a novel and threatening environment and have increased basal and stress-induced levels of plasma corticosterone [30]. Likewise, ICV-administered NOP causes hyperphagia, even in satiated rats, an effect that is mediated by corticosterone and central glucocorticoid receptors [26]. One mechanism whereby NOP induces feeding in these animals is by functionally antagonizing anorexia elicited by corticotrophin-releasing hormone (CRH),

1356 / Chapter 188 the primary mediator of stress, especially in the bed nucleus of stria terminalis [6]. Yet NOP may also increase food intake by decreasing the release of the anorectic peptide CART (cocaine- and amphetamineregulated transcript) and increasing the release of the orexigenic peptide AgRP (agouti-related protein), especially in the arcuate nucleus of hypothalamus [4].

Nociceptin Actions on Learning and Memory It was initially shown that microinjection of NOP into the CA3 region of the dorsal hippocampus caused profound impairment of spatial learning in male rats trained in the Morris water task. Later, NOP was reported to inhibit long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse in rat hippocampal slices. These observations receive strong support from gene invalidation because mice lacking the NOP receptor gene not only possess greater learning ability and have better memory than control mice but also show larger LTP in the hippocampal CA1 region [28]. Improvement of cognitive abilities in mice lacking the NOP receptor gene has been linked to hyperfunctioning of the NMDA receptor [17].

NOP Actions on Peripheral Organs By acting on both the central and peripheral nervous systems, NOP modulates the functioning of the heart and vessels, airways, kidney, urinary bladder, intestine, and perhaps also immune cells in rodents. Thus, NOP has been shown to possess vasorelaxant/hypotensive properties, to inhibit the cough reflex, to be a potent diuretic and antinatriuretic, to block micturition, and to stimulate neutrophil chemotaxis. Most of these actions have been reviewed in a special issue of the journal Peptides (volume 21, number 7, July 2000). These actions of NOP may be as important as its action on the central nervous system, especially in terms of potential clinical exploitation.

PATHOPHYSIOLOGICAL IMPLICATIONS To date, not even one pathological condition has been linked to a dysfunctioning of the NOP–NOP receptor system. Attempts to correlate pathological states, in particular chronic pain states, with modified levels of circulating peptide in biological fluids of human origin have not yielded consistent results [3]. Nevertheless, the broad pharmacological spectrum of NOP actions, even though it has been established so far only in rodents, and most often using a route of administration (ICV) that is impractical in routine medical practice, points to a number of potential therapeutic applications for NOP receptor agonists or antagonists.

Thus, based on available pharmacological results, NOP receptor agonists are claimed to be potentially useful for treating stress and anxiety, drug addiction, anorexia, cough and asthma, edema, urinary incontinence, and hypertension. Indeed, one advantage of NOP receptor agonists over currently used drugs such as the benzodiazepine anxiolytics or the antitussives would be lack of abuse potential. However, given the broad pharmacological spectrum of NOP actions, NOP receptor agonists may also be anticipated to have many unwanted effects. In one clinical trial, intravesical instillation of NOP was reported to inhibit the voiding reflex in patients presenting with overactive bladder due to spinal cord injury [15]. On the other hand, NOP receptor antagonists could be useful for enhancing cognitive function, as adjuncts for preventing morphine analgesic tolerance, and perhaps also as novel anti-inflammatory agents. Antagonists are expected to have fewer side effects than agonists, as suggested by the fact that NOP receptor-deficient mice appear to be outwardly normal. In a recent patent application, NOP receptor antagonists have been claimed to have potent antipruritic activity. Finally, although the actions of NOP on nociception have been most extensively studied, it is still highly contentious whether NOP receptor agonists or antagonists have a future as analgesics. The reported opposite pro- and antinociceptive actions of NOP, depending on dose and site of administration, begs the question as to what would be the effect (pain or analgesia?) of a ligand able to target all NOP receptor sites simultaneously. Drug companies have been quite successful in developing such ligands, agonists as well as antagonists [5], but these need to determine NOP-receptor versus opioidreceptor selectivity, and/or oral bioavailability. Only highly NOP receptor–selective and orally available ligands will allow for the unbiased validation of the NOP receptor as a therapeutic target.

References [1] Allen RG, Peng B, Pellegrino MJ, et al. Altered processing of pro-orphanin FQ /nociceptin and pro-opiomelanocortinderived peptides in the brains of mice expressing defective prohormone convertase 2. J Neurosci 2001; 21:5864–70. [2] Arjomand J, Cole S, Evans CJ. Novel orphanin FQ /nociceptin transcripts are expressed in human immune cells. J Neuroimmunol 2002; 130:100–8. [3] Barnes TA, Lambert DG. Editorial III: Nociceptin/orphanin FQ peptide-receptor system: Are we any nearer the clinic? Br J Anaesth 2004; 93:626–8. [4] Bewick GA, Dhillo WS, Darch SJ, et al. Hypothalamic cocaine and amphetamine regulated transcript (CART) and agouti related protein (AgRP) neurons co-express the NOP1 receptor and nociceptin alters CART and AgRP release. Endocrinology 2005; in press.

Nociceptin [5] Bignan GC, Connolly PJ, Middleton SA. Recent advances towards the discovery of ORL-1 receptor agonists and antagonists. Exp Opin Ther Patents 2005; 357–88. [6] Ciccocioppo R, Cippitelli A, Economidou D, et al. Nociceptin/ orphanin FQ acts as a functional antagonist of corticotropinreleasing factor to inhibit its anorectic effect. Physiol Behav 2004; 82:63–8. [7] Ciccocioppo R, Economidou D, Fedeli A, et al. Attenuation of ethanol self-administration and of conditioned reinstatement of alcohol-seeking behaviour by the antiopioid peptide nociceptin/orphanin FQ in alcohol-preferring rats. Psychopharmacology (Berl) 2004; 172:170–8. [8] Fernandez F, Misilmeri MA, Felger JC, Devine DP. Nociceptin/ orphanin FQ increases anxiety-related behavior and circulating levels of corticosterone during neophobic tests of anxiety. Neuropsychopharmacology 2004; 29:59–71. [9] Giuliani S, Lecci A, Maggi CA. Nociceptin and neurotransmitter release in the periphery. Peptides 2000; 21:977–84. [10] Heinricher MM. Orphanin FQ/nociceptin: from neural circuitry to behavior. Life Sci 2003; 73:813–22. [11] Inoue M, Kobayashi M, Kozaki S, et al. Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice. Proc Natl Acad Sci USA 1998; 95:10949–53. [12] Jenck F, Wichmann J, Dautzenberg FM, et al. A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: Anxiolytic profile in the rat. Proc Natl Acad Sci USA 2000; 97:4938–43. [13] Kato S, Tsuzuki Y, Hokari R, et al. Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis. J Neuroimmunol 2005; 161:21–8. [14] Kimura T, Kitaichi K, Hiramatsu K, et al. Intradermal application of nociceptin increases vascular permeability in rats: The possible involvement of histamine release from mast cells. Eur J Pharmacol 2000; 407:327–32. [15] Lazzeri M, Calo G, Spinelli M, et al. Urodynamic effects of intravesical nociceptin/orphanin FQ in neurogenic detrusor overactivity: A randomized, placebo-controlled, double-blind study. Urology 2003; 61:946–50. [16] Maidment NT, Chen Y, Tan AM, et al. Rat ventral midbrain dopamine neurons express the orphanin FQ/nociceptin receptor ORL-1. Neuroreport 2002; 13:1137–40. [17] Mamiya T, Yamada K, Miyamoto Y, et al. Neuronal mechanism of nociceptin-induced modulation of learning and memory: Involvement of N-methyl-D-aspartate receptors. Mol Psychiatry 2003; 8:752–65. [18] Meunier J, Mouledous L, Topham CM. The nociceptin (ORL1) receptor: Molecular cloning and functional architecture. Peptides 2000; 21:893–900. [19] Meunier JC, Mollereau C, Toll L, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 1995; 377:532–5. [20] Mogil JS, Pasternak GW. The molecular and behavioral pharmacology of the orphanin FQ /nociceptin peptide and receptor family. Pharmacol Rev 2001; 53:381–415. [21] Mollereau C, Moulédous L. Tissue distribution of the opioid receptor-like (ORL1) receptor. Peptides 2000; 907–17. [22] Moran TD, Abdulla FA, Smith PA. Cellular neurophysiological actions of nociceptin/orphanin FQ. Peptides 2000; 21:969–76. [23] Murphy NP. Nociceptin/orphanin FQ, hedonic state and the response to abused drugs. Nihon Shinkei Seishin Yakurigaku Zasshi 2004; 24:295–8. [24] Neal CR Jr, Mansour A, Reinscheid R, et al. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the

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central nervous system of the rat. J Comp Neurol 1999; 406:503– 47. Neal CR Jr, Mansour A, Reinscheid R, et al. Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I[(14)Tyr]-orphanin FQ binding. J Comp Neurol 1999; 412:563– 605. Nicholson JR, Akil H, Watson SJ. Orphanin FQ-induced hyperphagia is mediated by corticosterone and central glucocorticoid receptors. Neuroscience 2002; 115:637–43. Noble F, Roques BP. Association of aminopeptidase N and endopeptidase 24.15 inhibitors potentiate behavioral effects mediated by nociceptin/orphanin FQ in mice. FEBS Lett 1997; 401:227–9. Noda Y, Mamiya T, Manabe T, et al. Role of nociceptin systems in learning and memory. Peptides 2000; 21:1063–9. Otsuji T, Okuda-Ashitaka E, Kojima S, et al. Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase. Anal Biochem 2004; 329:230–7. Reinscheid RK, Civelli O. The orphanin FQ /nociceptin knockout mouse: A behavioral model for stress responses. Neuropeptides 2002; 36:72–6. Reinscheid RK, Nothacker HP, Bourson A, et al. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 1995; 270:792–4. Reinscheid RK, Nothacker HP, Civelli O. The orphanin FQ / nociceptin gene: Structure, tissue distribution of expression and functional implications obtained from knockout mice. Peptides 2000; 901–6. Saito Y, Maruyama K, Kawano H, et al. Molecular cloning and characterization of a novel form of neuropeptide gene as a developmentally regulated molecule. J Biol Chem 1996; 271:15615–22. Sakurada C, Sakurada S, Orito T, et al. Degradation of nociceptin (orphanin FQ) by mouse spinal cord synaptic membranes is triggered by endopeptidase-24.11: An in vitro and in vivo study. Biochem Pharmacol 2002; 64:1293–303. Sakurada S, Watanabe H, Mizoguchi H, et al. Involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord. Pain 2004; 112:171–82. Schlicker E, Morari M. Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system. Peptides 2000; 21:1023–9. Serhan CN, Fierro IM, Chiang N, Pouliot M. Cutting edge: Nociceptin stimulates neutrophil chemotaxis and recruitment: Inhibition by aspirin-triggered-15-epi-lipoxin A4. J Immunol 2001; 166:3650–4. Shoblock JR, Wichmann J, Maidment NT. The effect of a systemically active ORL-1 agonist, Ro 64-6198, on the acquisition, expression, extinction, and reinstatement of morphine conditioned place preference. Neuropharmacology 2005; in press. Xu X, Grass S, Hao J, Xu IS, et al. Nociceptin/orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions. Peptides 2000; 21:1031–6. Zaratin PF, Petrone G, Sbacchi M, et al. Modification of nociception and morphine tolerance by the selective opiate receptorlike orphan receptor antagonist SB-612111. J Pharmacol Exp Ther 2004; 308:454–61. Zeilhofer HU, Calo G. Nociceptin/orphanin FQ and its receptor—potential targets for pain therapy? J Pharmacol Exp Ther 2003; 306:423–9.

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189 Role of Tachykinins in Spinal Nociceptive Mechanisms and Their Interactions with Opioids XIAO-JUN XU AND ZSUZSANNA WIESENFELD-HALLIN

neurotransmitters, such as glutamate [14, 35, 37]. Neurokinin B [NKB], on the other hand, appears to be present mainly in dorsal horn interneurons but not in the DRG [30, 43]. Three subtypes of tachykinin receptors have been identified and cloned. The NK-1 receptor is concentrated in the superficial laminas of the dorsal horn, particularly in lamina I (LI) where internalization of this receptor has been shown to occur after noxious stimulation [25]. NK-2 receptor-LI has also been observed in the outer part of LI and in the area around the central canal. These receptors are, however, exclusively astrocytic [53]. NK-3 receptor-LI has been localized in lamina I, II, and X [8, 42, 54]. Neurons containing NK-3 receptor-LI in the superficial laminas have been shown to be of two distinct populations, coexisting with the μ-opioid receptor or nitric oxide synthase, respectively [8].

ABSTRACT The mammalian tachykinins and their receptors are present in sensory neurons and the dorsal horn of the spinal cord, areas that are important in the transmission and modulation of nociceptive information. Multiple lines of evidence support an excitatory role for tachykinins in spinal pain mechanisms in animals. Tachykinins also interact with opioids in nociceptive modulation. Tachykinin NK1 receptor antagonists have, however, failed to show analgesic efficacy in clinical pain states.

DISTRIBUTION OF TACHYKININS IN DORSAL ROOT GANGLIA AND THE SPINAL CORD It was Lembeck who first suggested that substance P (SP) may have a role in sensory transmission, based on the finding that there is a higher concentration of SP in dorsal roots and the dorsal horn than in ventral roots and the ventral horn [18]. Following the structural characterization of SP as an undecapeptide, immunohistochemical and, more recently, in situ hybridization studies have localized SP in a subpopulation of dorsal root ganglion (DRG) cells, primarily with small somata [12]. In addition, SP-like immunoreactivity (lamina I, LI) has been localized in the fiber network of the dorsal horn, mainly in the outer laminae, but also in bulbospinal tract fibers and in dorsal horn interneurons [35, 37]. After dorsal rhizotomy, SP is markedly depleted from the dorsal horn, indicating that afferents are the major source of SP [35, 37]. As in other structures of the nervous system, neurokinin A (NKA) is co-localized with SP in DRG neurons and the dorsal horn [14]. SP and NKA are also colocalized with other peptides, such as calcitonin gene– related peptide and galanin, and with classic Handbook of Biologically Active Peptides

PLASTICITY OF THE EXPRESSION OF TACHYKININS AND THEIR RECEPTORS FOLLOWING INFLAMMATION AND NERVE INJURY The expression of peptides and peptide receptors in the DRG and dorsal horn exhibit complex plasticity following peripheral nerve injury or inflammation, conditions known to be associated with chronic pain [13, 14]. SP is the first peptide that was shown to be downregulated in the DRG after axotomy [16]. Such downregulation occurs primarily in small-size DRG neurons, and the loss of target-derived nerve growth factors is believed to be responsible for this effect [45]. Paradoxically, it has been reported that nerve injury triggers a de novo synthesis of SP in large DRG neurons [33]. NK-1 receptors are upregulated in the dorsal horn after nerve injury [1]. Peripheral nerve injury also down-

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1360 / Chapter 189 regulates NKA in the DRG. In contrast to nerve injury, inflammation appears to be associated with a moderate increase in the synthesis of SP and NKA in the DRG [14]. The synthesis of NK-1 and NK-3 receptors is also increased in the dorsal horn after peripheral inflammation [1, 20, 28].

RELEASE OF TACHYKININS IN THE SPINAL CORD In 1976, Otsuka and Konishi demonstrated calciumdependent release of SP from isolated rat spinal cord on electrical or chemical stimulation [34]. Following these pioneering studies, the in vitro and in vivo release of SP in the spinal cord on noxious peripheral stimulation were repeatedly shown by a number of methods [35, 37]. Release occurs primarily at the superficial laminas of the dorsal horn, as seen with the antibody microprobe method [29] and with the internalization of NK-1 receptors in this area following noxious stimulation [25]. The release of SP is increased following inflammation [46]. NKA is also released in the spinal cord following noxious stimulation and, unlike SP whose release is confined in the superficial laminas, released NKA appears to spread into deeper laminas and persists much longer than SP [9]. This has led to the suggestion that NKA may be responsible for a more prolonged increase in spinal excitability than SP.

INVOLVEMENT OF TACHYKININS IN SPINAL NOCICEPTION It has long been known that SP, and to some extent NKA and NKB, depolarize and thus excite dorsal horn neurons [40, 56]. The excitatory effect of SP is typically delayed in onset, but has a prolonged duration, which differs from classic excitatory transmitters such as glutamate, which have rapid onset of effect with short duration [40, 56]. Behaviorally, intrathecal (IT) tachykinins evoke a characteristic response in rodents, consisting of caudally directed biting, scratching, and licking, and this has been suggested to be indicative of a pain-like reaction [15]. IT SP has also been consistently shown to produce hyperalgesia in many nociceptive tests. Finally, tachykinins increase the excitatory effect of glutamate and other neuropeptides such as calcitonin gene–related peptide [41, 50, 56]. The development of selective nonpeptide antagonists of tachykinin receptors has greatly facilitated the identification of the role of tachykinins in nociception. It soon became clear that NK-1 receptor antagonists are not antinociceptive in classic pain tests, such as the hot

plate or tail flick tests [3, 11]. These compounds also have minimal effect on baseline responses of dorsal horn neurons to noxious stimulation. Thus, NK-1 antagonists behave differently than strong analgesics such as opioids [3, 11]. On the other hand, the activation of tachykinin receptors, particularly that of the NK-1 type, have been shown to be a critical component in the activity-dependent increase (central sensitization) in spinal cord excitability after intense nociceptive stimulation [41, 50]. Furthermore, NK-1 receptor antagonists are also able to reduce hyperalgesia in rodent models of inflammatory pain [41, 50]. Because central sensitization is believed to play an important role in the generation of chronic pain states under pathological conditions, this indicates a potential analgesic effect of NK-1 antagonists in the clinic. This activity profile of NK-1 receptor antagonists has been confirmed in studies with knockout mice lacking genes for the NK-1 receptor or prepro-tachykinin [4, 7]. Earlier studies with selective NK-2 receptor antagonists have indicated that this receptor may also play a role in long-term central sensitization after the activation of muscle afferents or inflammation [21, 31, 49]. However, NK-2 receptors are not expressed in neurons in the spinal cord [53], and although one cannot rule out an involvement of NK-2 receptors on glial cells, recent work suggests that the pronociceptive effect of NKA in the spinal cord may be mediated largely by the activation of NK-1 receptors [39, 44]. It seems that NKA can bind to a segment of the NK-1 receptors that purported NK-2 selective antagonists may also bind, producing a selective antagonism of NKA-mediated effect on NK-1 receptors [44]. The activation of NK-3 receptors by selective agonists such as senktide has been shown to be associated with a transient hypoalgesia in behavioral studies, an effect that is naloxone reversible, indicating the involvement of the endogenous opioid system [5]. This may be due to the coexistence of NK-3 and μ-opioid receptors on dorsal horn neurons [8]. Electrophysiological studies, on the other hand, showed that NKB and other NK-3 agonists produced a mainly excitatory effect on dorsal horn neurons [2, 5, 52]. Studies with NK-3 selective antagonists also support a role for this receptor in mediating central sensitization, probably in concert with an increased release of SP and activation of the NK-1 receptor [2, 5, 52]. There is also some evidence for an increased role of NK-3 receptors after inflammation [19].

NK-1 RECEPTOR AND NEUROPATHIC PAIN In recent years it has been suggested that SP, by activating the NK-1 receptor, may also be involved in

Role of Tachykinins in Spinal Nociceptive Mechanisms / 1361 neuropathic pain after peripheral nerve injury. This is primarily based on the finding that nerve injury caused increased synthesis and release of SP in and from largediameter sensory neurons [24, 33]. Using a partial nerve injury model, it has also been possible to demonstrate increased SP synthesis in uninjured neighboring neurons in the DRG [22]. It is, however, important to note that nerve injury is associated with downregulation of SP in small-diameter C-afferents [16], and the role of SP in mediating central sensitization was diminished after nerve injury [48]. Functional studies examining the effect of NK-1 antagonists in rodent models of neuropathic pain have produced mixed results [6, 51], and NK-1 antagonists are ineffective in human neuropathic pain conditions [3]. Moreover, depleting the NK-1 receptor did not influence the development of neuropathic painlike behaviors after nerve injury [27]. Overall, it is unlikely that NK-1 receptor antagonists could be useful as analgesics in neuropathic pain.

SP-SAPORIN AND NK-1 RECEPTOR EXPRESSING LAMINAE I NEURONS IN NOCICEPTION The binding of SP to NK-1 receptors on neurons in the superficial dorsal horn causes profound internalization of the NK-1 receptors [25]. Using this observation, Mantyh et al. developed an elegant method using SP coupled with the toxin saporin to selectively destroy neurons that receive input from nociceptive afferents [26]. This has led to strong evidence for the involvement of this group of neurons, but not necessarily NK-1 receptors, in a variety of chronic pain conditions [32].

INTERACTION BETWEEN TACHYKININS AND OPIOIDS Opioids administered exogenously in the form of, for example, morphine or released endogenously produce antinociception in the spinal cord. The spinal release of SP is inhibited by morphine in vitro and in vivo, demonstrating a presynaptic inhibitory effect of opioids that may partly explain opioid-mediated antinociception [34]. However, later studies have shown that systemic morphine at analgesic doses did not inhibit SP release in the spinal cord [17, 29]. This observation, together with the fact that tachykinin receptor antagonists are not effective in traditional assays of nociception, indicate that the antinociceptive effect of opioids are not likely to be mediated by the inhibition of tachykinin release in the spinal cord. Repeated morphine injection leads to a reduced analgesic effect and tolerance, and it induces increased

SP synthesis in the DRG [23] and increased release of SP in the dorsal horn during naloxone-precipitated withdrawal [10]. It has been shown recently that NK-1 antagonists prevent the development of tolerance to morphine [38]. Chronic morphine administration also alters the activity of SP endopeptidase (SPE), and there is a significant correlation between SPE activity and some signs of withdrawal from morphine [55]. Finally, NK-1 receptor knockout mice also display decreased morphine withdrawal symptoms [36]. These results suggest that tachykinins may play a role in opioid tolerance and withdrawal. Tachykinins and opioids may also interact in a more complex and paradoxical manner in nociception. Thus, it is has shown that low-dose morphine releases SP in the spinal cord [47], and in some studies SP has been shown to enhance the antinociceptive effect of morphine, which has led to the development of chimeric peptides with opioid activity and SP activity as analgesics (see Chapter 191 by Lipkowski et al. in this section of the book).

THE FAILURE OF NK-1 ANTAGONISTS AS ANALGESICS IN HUMANS Several clinical trials have been conducted to examine the analgesic efficacy of NK-1 receptor antagonists in humans in various pain conditions, such as postoperative dental pain, osteoarthritis, diabetic neuropathy, postherpetic neuralgia, and migraine, and the results have been largely disappointing [3, 11]. Thus, in only one trial did the NK-1 antagonist CP-99994 show analgesic efficacy in dental pain comparable to ibuprofen [3]. The lack of clinical efficacy of these compounds is not due to insufficient dosing or lack of brain penetration, and these compounds are selective toward the human variant of NK-1 receptors. There may be several reasons for this lack of analgesic efficacy for NK-1 receptor antagonists. First, these compounds have not been tested in the most appropriate clinical pain models because in rodent models NK-1 antagonists are most effective in inflammatory pain whereas most clinical trials were done on acute or neuropathic pain. Second, this may be due to species differences between humans and rodents in the distribution of NK-1 receptors, particularly at supraspinal sites and in the physiology of tachykinins. Third, the negative results may indicate that monotherapy with NK-1 antagonists may not be sufficient to suppress pain involving a large number of transmitters and modulators and the complex interaction among them. Thus, a combination of tachykinin antagonists with, for example, NMDA antagonists, may still be a useful clinical approach in improving analgesic efficacy.

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Role of Tachykinins in Spinal Nociceptive Mechanisms / 1363 [34] Otsuka M, Konishi S. Release of substance P-like immunoreactivity from isolated spinal dorsal horn in rats. Brain Res 1987;403:350–354. [35] Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;229–308. [36] Passarelli F, Merante A, Pontieri FE, Margotta V, Venturini G, Palladini G. Rewarding effects of opiates are absent in mice lacking the receptor for substance P. Nature 2000;405:180– 183. [37] Pernow B. Substance P. Pharmacol Rev 1983;35:84–141. [38] Powell KJ, Quirion R, Jhamandas K. Inhibition of neurokinin-1substance P receptor and prostanoid activity prevents and reverses the development of morphine tolerance in vivo and the morphine-induced increase in CGRP expression in cultured dorsal root ganglion neurons. Eur J Neurosci 2003;18:1572– 1583. [39] Radhakrishnan V, Henry JL. Electrophysiological evidence that neurokinin A acts via NK-1 receptors in the cat dorsal horn. Eur J Neurosci 1997;9:1977–1985. [40] Randic M, Miletic V. Effect of substance P in cat dorsal horn neurons activated by noxious stimuli. Brain Res 1977;128:161– 169. [41] Saria A. The tachykinin NK1 receptor in the brain: pharmacology and putative functions. Eur J Pharmacol 1999;375:51–60. [42] Seybold VS, Grkovic I, Porbury AL, Ding YQ, Shigemoto R, Mizuno N, Furness JB, Southwell BR. Relationship of NK3 receptor-immunoreactivity to subpopulation of neurons in rat spinal cord. J Comp Neurol 1997;381:439–448. [43] Too HP, Maggio JE. Immmunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord. Peptides 1991;12:431–443. [44] Trafton JA, Abbadie C, Basbaum AI. Differential contribution of substance P and neurokinin A to spinal cord neurokinin-1 receptor signaling in the rat. J Neurosci 2001;15:3656–3664. [45] Verge VMK, Richardson PM, Wiesenfeld-Hallin Z, Hökfelt T. Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide expression in adult sensory neurons. J Neurosci 1996;15:2081–2096. [46] Warsame AA, Gustafsson H, Olgart L, Brodin E, Stiller CO, Taylor BK. Capsaicin-evoked substance P release in rat dorsal

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190 Exorphin-Opioid Active Peptides of Exogenous Origin MASAAKI YOSHIKAWA

Pro (gluten exorphin-A4), Tyr-Gly-Gly-Trp-Leu (gluten exorphin-B5), and Tyr-Gly-Gly-Trp (gluten exorphinB4), from the pepsin-thermolysin digest of wheat gluten [14]. All of these were rather selective for the δreceptor. Their median inhibitory concentration (IC50) values in the MVD assay were 60, 70, 0.017, and 3.4 μM, respectively. Gluten exorphins-A5, -B5, and -B4 were also released by the action of pepsin and pancreatic elastase [11]. Large amounts of des-Gly1-gluten exorphin-A5 (Tyr-Tyr-Pro-Thr) showing marginal δ-opioid activity in MVD assay (IC50 = 800 μM) was also found in the same digest. Gluten exorphin-A5 sequence was found 15 times in the primary structure of high-molecular-weight gluten. However, gluten exorphin-A5 is released from only one of these sites (-SGYYPTS-) by the action of pepsin and pancreatic elastase. Among two Tyr residues in gluten exorphins-A5, the Tyr3 was absolutely essential for the activity, whereas Tyr2 could be replaced with Phe. Therefore, gluten exorphinA could be classified as an opioid peptide containing Tyr-Pro. Gluen exorphin-B5, which corresponds to [Trp4][Leu]-enkephalin, showed the most potent δ-opioid activity in the MVD and δ-receptor binding assays among gluten exorphins. However, in most in vivo assays, gluten exorphin-A was weaker than gluten exorphin-B, probably because of susceptibility to peptidases. In the single-trial passive-avoidance experiment with stepthrough cages, gluten exorphin-A5 stimulated the acquisition of memory after the oral administration at doses of 300 mg/kg [31]. However, gluten exorphin-B5 was ineffective at the same dose. Gluten exorphins-A5 and -B5 stimulated postprandial insulin secretion after oral administration at a dose of 30 and 300 mg/kg, respectively, in rats [12]. On the other hand, gluten exorphin-B5 is reported to stimulate prolactin secretion after intravenous administration at a dose of 3 mg/kg in rats [10].

ABSTRACT Exorphin is a term for opioid peptides derived from protein of exogenous origin, such as milk or plants. Many peptides having opioid activities have been shown to be released from proteins other than the three precursor proteins for endogenous opioid peptides (proopiomelanocortin, proenkephalin, and prodynorphin). Most of these exogenous opioid peptides have Tyrresidue at their amino-termini. However, they differ from the typical endogenous ones by an absence of the enkephalin sequence (Tyr-Gly-Gly-Phe-Met/Leu). Such atypical opioid peptides are released from various proteins of animal and plant origin. Exorphins in the true sense might be those from plant proteins because those from animal proteins might work as endogenous peptides under certain conditions. In this chapter, however, atypical opioid peptides of both plant and animal origin are described. Exogenous opioid peptides are classified into two groups depending on whether they contain the Tyr-Pro sequence, and they are subdivided into four groups. Among them are β-casomorphin and hemorphin, a class of atypical opioid peptides containing a Tyr-Pro-aromatic sequence, as well as endomorphins. Opioid antagonist and anti-opioid peptides are also released from exogenous proteins and are also described.

OPIOID PEPTIDES DERIVED FROM PLANT PROTEINS Gluten Exorphins By using the mouse vas deferens (MVD) assay, Zioudou et al. found opioid activity in enzymatic digest of wheat gluten [50]. We isolated four opioid peptides, Gly-Tyr-Tyr-Pro-Thr (gluten exorphin-A5), Gly-Tyr-TyrHandbook of Biologically Active Peptides

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1366 / Chapter 190 Another opioid peptide Tyr-Pro-Ile-Ser-Leu was isolated from the pepsin-chymotrypsin-trypsin digest of wheat gluten. This peptide was named gluten exorphinC [13]. Gluten exorphin-C was slightly selective for δreceptor. Its IC50 value in the MVD assay was 13.5 μM. It should be noted that the Tyr-Pro sequence in gluten exorphin-C was not followed by an aromatic amino acid, which has been thought to be essential for the opioid activity, but instead by an aliphatic amino acid. We found the Tyr-Pro-Leu-Gly-Gln sequence in the primary structure of wheat gliadin as an analog of TyrPro-Leu-Gly-NH2 (Tyr-MIF-I) and synthesized it. The peptide was rather selective for the μ-receptor because it showed opioid activity in the guinea pig ileum (GPI) assay (IC50 = 7 μM) as did Tyr-MIF-I, an anti-opioid peptide [15, 49]. We named it gluten exorphin-D. Based on homology to β–casomorphin-7, the TyrPro-Gln-Pro-Gln-Pro-Phe sequence found in α-gliadin was synthesized and named gliadorphin [16]. Although the peptide did not show opioid activity in the MVD assay, its binding to human peripheral lymphocytes was blocked by naloxone and an enkephalin derivative.

Rubiscolins Ribulose bisphosphate carboxylase/oxygenase (rubisco) is a key enzyme in photosynthesis, catalyzing carbon dioxide fixation. Rubisco is ubiquitus for photosynthetic organisms and regarded as the most abundant protein on earth. From a nutritional point of view, the large subunit of Rubisco has an exceptionally ideal composition of essential amino acids among plant proteins. Therefore, plant Rubisco is expected to be a large source of food protein in the future. We found the TyrPro-Leu sequence, which satisfies the requirements for opioid activity as already described, in the conserved region in the large subunit of plant Rubisco. Then, we synthesized the corresponding sequences, Tyr-Pro-LeuAsp-Leu-Phe and Tyr-Pro-Leu-Asp-Leu. Both peptides were active in the MVD assay; IC50 values were 24.4 and 50 μM, respectively [38]. We named them rubiscolins-6 and -5, respectively. Their opioid activities were very weak in the GPI and μ-receptor bindng assays. Results from the receptor binding assay also suggested that rubiscolins are δ-selective opioid peptides. We examined the conditions for the enzymatic release of rubiscolins from spinach Rubisco by using a model peptide and partially purified protein. Rubiscolins-6 and -5 were released by the action of pepsin, pancreatic elastase, and leucine amino peptidase. The yield of rubiscolin-5 was higher than that of rubiscolin6. In certain algae species, the residue corresponding to Leu3 of rubiscolin-6 is replaced by Ile. Tyr-Pro-Ile-

Asp-Leu-Phe was about four times more active than Tyr-Pro-Leu-Asp-Leu-Phe in the MVD assay [40]. The Asp4 residue in rubiscolin was essential for δ-opioid activity; it could not be replaced with Glu. The replacement of Leu3 or Ile3 with Phe or Trp also resulted in a reduction of δ-opioid activity. The rubiscolin sequence has been in Rubisco far longer than the appeareance of δ-receptors in animals. The sequence might be essential for enzymatic activity. It might be attractive to speculate that the δ-receptor in animals has evolved to fit the rubiscolin sequence in Rubisco; however, this possibility might be remote. We think that the rubiscolin sequence might have affinity for δ-receptor in animals just by chance. This might be the case for most plantderived peptide sequences acting on animal receptors. In this sense, these bioactive peptides could be regarded as those obtained from a sort of random library. Rubiscolin-6 showed antinociceptive activity in mice after oral administration at a dose of 300 mg/kg. Rubiscolin-6 stimulated the acquisition of memory after oral administration at doses of 100 mg/kg in a passive avoidance experiment [39]. In the elevated-plus maze test in mice, rubisolin-6 showed anxiolytic activity after oral administration at a dose of 30 mg/kg. Thus, rubiscolin-6 is about three times more potent than gluten exorphin-A5 in vivo [42]. Both μ- and δ-opioids have been reported to inhibit memory consolidation, whereas κ-opioid stimulated it. It is an interesting observation that food-derived δopioid such as gluten exorphin and rubiscolin stimulate memory consolidation after oral administration.

OPIOID PEPTIDES DERIVED FROM ANIMAL PROTEINS b-Casomorphin, Cytochrophin, and Hemorphin β-Casomorphin-7 Tyr-Pro-Phe-Pro-Gly-Pro-Ile, which is derived from amino acid residues 60–66 of bovine β-casein, was isolated by Brantl et al. from commercial casein peptone as the first example of exogenous opioid peptide [5]. β-Casomorphin-5 Tyr-Pro-Phe-Pro-Gly, which was obtained by carboxypeptidase Y treatment of the heptapeptide, showed higher activity than βcasomorphin-7 [18]. β-Casomorphin-4 amide Tyr-ProPhe-Pro-NH2, which was synthsisized at first and then isolated from in vivo digest, was the most potent among the derivatives [6, 7]. It was the first peptide ligand selective for μ receptor and named morphiceptin. Elongated forms such as β-casomorphins-9, -11, -13, and -21 have been isolated from in vitro or in vivo digests of β-casein [20, 28]. Opioid activities of these elongated forms were all weaker than β-casomorphin-7. It should

Exorphin-Opioid Active Peptides of Exogenous Origin / 1367 be noted that β-casomorphin-7 is released from β-casein A1, B, and C, minor genetic variants in which Pro67 is replaced with His, by the action of pepsin, pancreatic elastase, and leucine aminopeptidase [20]. In human β-casein, β-casomorphin with modified sequence TyrPro-Phe-Val-Glu-Pro-Ile and weaker activity is conserved [2, 25, 47]. However, similar opioid peptides are not released from murine β-casein because residues essential for opioid activity are not conserved in them. From enzymatic digest of bovine blood two opioid peptides, Tyr-Pro-Phe-Thr and Tyr-Pro-Trp-Thr, were isolated by Brantl et al. [3, 4]. They were derived from cytochrome b and hemoglobin β and named cytochrophin and hemorphin, respectively. These peptides might work as endogenous opioid peptides under certain conditions. Hemorphin-5, Tyr-Pro-Trp-Thr-Gln, is released by the action of pancreatic elastase from hemoglobin [21]. Thus, β-casomorphin, cytochrophin, and hemorphin have the Tyr-Pro-aromatic sequence in common, which is also found in endomorphins 1 and 2 (Tyr-Pro-TrpPhe-NH2 and Tyr-Pro-Phe-Phe-NH2 [48]).

Chapter 187 on anti-opioid peptides in this section of the book and Chapter 63 on bradykinin and cancer in the Cancer/Anticancer Section).

a-Casein Exorphin

Opioid Peptides Derived from Other Animal Proteins

Loukas et al. isolated two opioid peptides, RYLGYLE and RYLGYL, from the pepsin digest of α-casein [27]. These are derived from the amino acid residues 90–96 and 90–95 of αs1-casein. These peptides are selective for the δ-receptor, and their IC50 values in the MVD assay are 30 and 70 μM, respectively. Miclo et al. isolated a decapeptide YLGYLEQLLR, which corresponds to αs1-casein(91–100) and contains a part of α-casein exorphin sequence, from a trypsin digest of αs1-casein [29]. This peptide shows anxiolytic and antidepressive activities after oral administration in mice at a dose of 0.4 mg/kg. This peptide was named α-casozepine because of its weak affinity for GABAA receptor, to which anxiolytic benzodiazepines also bind.

as1-Casomorphin Kampa et al. found that synthetic human αs1casein(158–162), Tyr-Val-Pro-Phe-Pro, had affinity for κ1 and κ2 receptors and named it αs1-casomorphin [22]. The αs1-casomorphin sequence is found in the structure of casoxin D, Tyr-Val-Pro-Phe-Pro-Pro-Phe, which has been isolated from the pepsin-trypsinchymotrypsin digest of human casein as an anti-opioid and ileum-contracting peptide. Casoxin D has affinity for bradykinin B1 receptor [41, 45]. αs1-Casomorphin inhibited growth of T47D breast cancer cells [17] (see

Neocasomorphin and Other Casein-Derived Peptides We isolated an opioid peptide, Tyr-Pro-Val-Glu-ProPhe, which corresponds to β-casein(194–119), from a pepsin-pancreatin digest of bovine β-casein and named it neocasomorphin [20]. The peptide showed weak opioid activity in the GPI assay (IC50 = 59 μM). This peptide is classified as an opioid containing the Tyr-Proaliphatic residue. We synthesized Tyr-Pro-Ser-Phe-NH2 and Tyr-GlyPhe-Leu-Pro, which are found in the primary structure of human β-casein, residues 59–63 and 41–44, respectively. Their IC50 values in the GPI assay were 30 and 270 μM, respectively. They were almost inactive in the MVD assay. We named the latter one β-casorphin [46].

In order to look for possible opioid peptide sequences in whey proteins, we synthesized Tyr-Gly-Leu-Phe-NH2 and Tye-Leu-Leu-Phe-NH2, which correspond to bovine α-lactalbumin(50–53) and β-lactoglobulin(102–105), respectively, and tested them in the GPI assay system. Both peptides inhibited electrically induced twitches of the ileum preparation with IC50 values 50 μM and 160 μM, respectively [20]. We named them αlactorphin and β-lactorphin, respectively. Whereas the inhibition of the twiches by α-lactorphin was blocked by naloxone, those by β-lactorphin was unaffected. Nonamidated α-lactorphin was found in the pepsin digest of whey protein [1]. Interestingly, it has hypotensive activity [30]. We isolated an opioid peptide Tyr-Gly-Phe-Gln-AsnAla from a pepsin digest of bovine serum albumin and named it serorphin [36]. This peptide is derived from amino acid residues 399–404 of the protein. Serorphin is selective for δ-receptor because its IC50 values in the MVD and GPI assays were 8.5 and 230 μM, respectively. As for similar opioid peptide containing Tyr-Gly-Phe sequences, Tyr-Gly-Phe-Gly-Gly and Tyr-Gly-Phe-Ile-Leu have been synthesized by Khachenko et al. [23]. These are found in the primary structures of histone H4 and carboxypeptidases A and B and are named historphin and valentorphin, respectively. Historphin showed opioid activity in the MVD assay (IC50 = 2.5 μM). They

1368 / Chapter 190 also synthesized a similar peptide Tyr-Ser-Phe-Gly-Gly found in the primary structure of immunoglobulin κ–chain and named it kapporphin [24].

INTRAPROTEIN OPIOID SEQUENCE Most of endogenous and opioid peptides have Tyr residue at the amino terminus, and a few of them, such as gluten exorphin A, have Tyr residue in the second position from the terminus. This is because a positive charge near the phenol group of Tyr is essential for the opioid activity. Lipkowski et al. found that Tyr residue in the midst of peptides could bind to an μ-receptor if it is followed by basic residues [26]. They found that this is the case in GAG protein of the HIV virus and speculated that HIV might infect immuno- and neurocells via the μ-receptor expressed on them.

CLASSIFICATION OF EXORPHINS ACCORDING TO THEIR STRUCTURE In Table 1, atypical opioid peptides derived from proteins are classified into two groups: those containing the Tyr-Pro sequence and others. Then, the Tyr-Pro group is further classified into two groups, those containing Tyr-Pro-aromatic amino acid residues and those containing Tyr-Pro-nonaromatic amino acid residues. The non-Tyr-Pro group is also subdivided into two groups according to the distance between Tyr and the aromatic amino acid residues. In in vitro assay systems, the potency of exorphins is usually 1/100 to 1/1000 that of endogenous opioid peptides. However, some exogenous opioid peptides are active after oral administration, in which none of the endogenous opioid peptides are active. One of the reasons for this might be that the Tyr-Pro-X sequence is more resistant to peptidases than enkaphalin sequence.

TABLE 1. Opioid Peptides Derived from Natural Proteins.a Structure

Peptides

Sources

Tyr-Pro type Tyr-Pro-aromatic type H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH H-Tyr-Pro-Phe-Val-Glu-Pro-Ile-OH H-Tyr-Pro-Phe-Thr-OH H-Tyr-Pro-Trp-Thr-OH Tyr-Pro-nonaromatic type H-Gly-Tyr-Tyr-Pro-Thr-OH H-Tyr-Pro-Ile-Ser-Leu-OH H-Tyr-Pro-Leu-Gly-Gln-OH H-Tyr-Pro-Leu-Asp-Leu-Phe-OH H-Tyr-Pro-Val-Glu-Pro-Phe-OH H-Tyr-Pro-Leu-Gly-NH2

β-Casomorphin hβ-Casomorphin Cytochrophin Hemorphin

[5] [2, 25, 47] [3] [4]

Gluten exorphin-A5 Gluten exorphin-C Gluten exorphin-D Rubiscolin-6 Neocasomorphin Tyr-MIF-I

[14] [13] [15] [38] [20] [49]

non-Tyr-Pro type Tyr-X1-X2-aromatic type H-Arg-Tyr-Leu-Gly-Tyr-Leu-Asp-OH H-Tyr-Gly-Gly-Trp-Leu-OH H-Tyr-Gly-Leu-Phe-NH2 H-Tyr-Leu-Leu-Phe-NH2 H-Tyr-Pro-Ser-Phe-NH2 H-Tyr-Val-Pro-Phe-Pro-OH Tyr-Gly/Ser-aromatic type H-Tyr-Gly-Phe-Gln-Asn-Ala-OH H-Tyr-Gly-Phe-Leu-Pro-OH H-Tyr-Gly-Phe-Gly-Gly-OH H-Tyr-Gly-Phe-Ile-Leu-OH H-Tyr-Ser-Phe-Gly-Gly-OH Intraprotein opioid sequence Ac-Asp-Ile-Tyr-Arg-Arg-Trp-Ile-Ile-Leu-NH2 Prefix h indicates human origin.

a

α-Casein exorphin Gluten exorphin-B5 α-Lactorphin β-Lactorphin hCasorphin hαsl-Casomorphin

[27] [14] [46] [46] [46] [22]

Serorphin hβ-Casein(41–44) Historphin Valentorphin Kapporphin

[36] [46] [23] [23] [24]

HIV GAG protein 24 fragment

[26]

Exorphin-Opioid Active Peptides of Exogenous Origin / 1369 TABLE 2. Opioid Antagonist and Anti-Opioid Peptides Derived from Natural Proteins.a Structure Opioid antagonists H-Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-OH H-Pro-Ty-Pro-Tyr-Tyr-OH H-Ser-Ty-Pro-Tyr-Tyr-OH (Peptide methylester) H-Ser-Arg-Tyr-Pro-Ser-Tyr-OCH3 H-Arg-Tyr-Pro-Ser-Tyr-OCH3 H-Tyr-Pro-Ser-Tyr-OCH3 H-Tyr-Leu-Gly-Ser-Gly-Tyr-OCH3 H-Arg-Tyr-Tyr-Gly-Tyr-OCH3 H-Lys-Tyr-Leu-Gly-Pro-Gln-Tyr-OCH3 Anti-opioid peptides H-Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg-OH H-Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg-OH H-Tyr-Val-Pro-Phe-Pro-Pro-Phe-OH

Peptides

Sources

Casoxin A Casoxin B hCasoxin B

[8] [8] [8]

Casoxin-6 Casoxin-5 Casoxin-4 hLactoferroxin A hLactoferroxin B hLactoferroxin C

[43] [43] [43] [35] [35] [35]

Oryzatensin Casoxin C hCasoxin D

[32, 34] [8, 33] [41, 45]

Prefix h indicates human origin.

a

OPIOID ANTAGONIST AND ANTI-OPIOID PEPTIDE DERIVED FROM PROTEINS During the screening of opioid peptides in enzymatic digests of proteins, we found that some peptides showed anti-opioid activity in the GPI and antinociceptive assays. These are classified into antagonists acting on opioid receptor and anti-opioid peptides acting at the postreceptor level (Table 2).

Opioid Antagonist Peptides The first example of an opioid antagonist is Ser-ArgTyr-Pro-Ser-Tyr-OCH3, a peptide carrying Tyr-methyl ester at the carboxyl-terminus. This peptide was obtained as an artificial product from a pepsin digest of bovine κ-casein and named casoxin-6 [9, 43]. The esterification of Tyr occurred during the extraction of the lyophilized digest with anhydrous chloroform-methanol. The peptide showed weak affinity for μ- and κ-receptors (IC50 = 5.5 and 10.1 μM, respectively), and antagonized opioid activities of DAMGO and dynorphin A (1–13) at 20 μM in the GPI assay. Interestingly, a truncated peptide Tyr-Pro-Ser-Tyr-OCH3 (casoxin-4) had no affinity for κ-receptor. From the fact that basic residues at the amino-terminus region direct the antagonist for κreceptor, we speculated that casoxin-6 binds κ-receptor reversewise to κ agonist peptides such as dynorphin, in which basic residues in the carboxyl-terminus region are essential for κ selectivity [44]. Affinities of nonesterified peptides for opioid receptors were less than 1/10 those of esterified from. As for natural peptides showing opioid antagonist activitiy, we obtained Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-Tyr

from pepsin-trypsin digest of bovine κ-casein [8]. This peptide was named casoxin A, and antagonized opioid activity of DAMGO at 50 μM in the GPI assay. From a synthetic approach, we found that Pro-Tyr-Pro-Tyr-Tyr and Ser-Tyr-Pro-Tyr-Tyr, derived from bovine and human κ-casein, respectively, showed opioid antagonist activity under the same conditions. We named them bovine and human casoxin B, respectively. As for opioid antagonists having Tyr-methyl ester, we obtained three peptides, Tyr-Leu-Gly-Ser-Gly-Tyr-OCH3, Arg-Tyr-Tyr-Gly-Tyr-OCH3, and Lys-Tyr-Leu-Gly-ProGln-Tyr-OCH3, from pepsin digest of human lactoferrin and named them lactoferroxins A, B, and C [35]. Because of basic amino acid residues situated at their N-termini, lactoforroxins B and C showed affinity for the κ receptor in addition to the μ receptor. The physiological significance of these peptides might be small because opioid antagonist activities of nonesterified forms are very weak.

Anti-Opioid Peptides Using GPI assay, we isolated an anti-opioid peptide Tyr-Ile-Pro-Gln-Tyr-Val-Leu-Ser-Arg from a trypsin digest of bovine κ-casein [8]. This peptide was derived from amino acid residue 25–34 of the protein and named casoxin C. Casoxin C showed weak affinity for the μ receptor (Ki = 200 μM). Its anti-opioid activity in the GPI was more potent than expected from receptor affinity. Furthermore, casoxin C was different from a typical opioid antagonist, such as naloxone and casoxinA, in inducing contraction of the ileum preparation in the absence of electrical stimuli. Casoxin C proved to have affinity for the receptor for complement C3a [33].

1370 / Chapter 190 Complement C3a is made of 75 amino acid residues and is released from complement C3 on activation of complement system to stimulate immune systems. It induces ileum contraction and inflammation and is called anaphylatoxin. The peptapeptide sequence at the carboxyl terminus of C3a, Leu-Gly-Leu-Ala-Arg, is the minimally essential structure required for C3a activity. This structural requirement could be generalized to the hydrophobic residue-X1-Leu-X2-Arg. Casoxin C has affinity for the complement C3a receptor (IC50 = 40 μM) because it meets this requirement. In the GPI, casoxin C and complement C3a antagonized opioid activities of DAMGO and dynorphin A at 10 μM [33]. However, they did not antagonize DPDEP in the MVD. In GPI, casoxin C and C3a induce biphasic contraction, rapid and slow ones. The rapid contaction is mediated by histamine release, whereas the slow contraction is mediated by prostaglandin E2 and acetylcholine. The antiopioid activities of casoxin C and C3a might be mediated by prostaglandin E2 and/or acetylcholine because they are related to the slow contraction. After intracerebrovascular (ICV) administration, casoxin C antagonized the antinociceptive activity of morphin and LI-50. 488H A but not DTLET. Furthermore, it reversed amnesia induced by scopolamine or cerebral ischemia in mice. Based on this observation, we also found for the first time that complement C3a itself also had anti-analgesic and anti-amnesic activities [19]. This suggests that C3a might work as an anti-opioid peptide in vivo. We obtained a peptide Gly-Tyr-Pro-Met-Tyr-Pro-LeuPro-Arg, which showed similar anti-opioid activity in the GPI, from a trypsin digest of rice albumin and designated it oryzatensin [32, 34]. In addition to affinity for the μ-opioid receptor (IC50 = 39 μM), oryzatensin has affinity for C3a receptors (IC50 = 44 μM) because it satisfies the structural requirements as already described. Another peptide Tyr-Val-Pro-Phe-Pro-Pro-Phe, which showed anti-opioid activity in GPI and MVD assays, was obtained from chymotrypsin digest of human casein [41, 45]. This peptide is derived from human αs1-casein and named casoxin D. Median effective concentration (EC50) values for anti-opioid activity in the GPI and MVD assays were 10−6 M and 10−5 M, respectively. However, affinities of casoxin D for μ and δ receptors were very weak (IC50 = 250 and 150 μM, respectively). Casoxin D has weak ileum-contracting activity (EC50 = 80 μM). Casoxin D also showed relaxing activity in canine mesenteric artery (EC50 = 3 μM), which was blocked by des-Arg9-[Leu8]-bradykinin, an antagonist for bradykinin B1 receptor, and indomethacin. In fact, casoxin D showed affinity for B1 receptor. However, its anti-opioid and ileum-contracting activities were not blocked by the B1 antagonist. Thus, receptors mediating anti-opioid and ileum-contracting activities of casoxin D are still unknown.

CONCLUSION Thus, agonists and antagonists for opioid receptors, as well as anti-opioid peptides, are released from various proteins. Stimulated by the concept of exorphin, many kinds of bioactive peptides acting through various types of receptors other than opioid have been also isolated from the enzymatic digest of animal and plant proteins. Those derived from animal proteins might have teleological function. However, those of plant origin could be regarded as those derived from a sort of random library. Studies on the physiological and pathological significance of those exogenous bioactive peptides will contibute to the development of foods and drugs to prevent diseases and promote our health.

Acknowledgments This work was supported in part by Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science and the PROBRAIN grant.

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191 Opioid-Substance P Chimeric Peptides ANDRZEJ W. LIPKOWSKI, DANIEL B. CARR, IWONA BONNEY, AND PIOTR KOSSON

distinct tachykinin receptors, NK1, NK2, and NK3, have been cloned in different species, including the human [24]. The three tachykinin receptors are recognized with moderate selectivity by endogenous tachykinins. Substance P, neurokinin A, and neurokinin B act as full agonists on the three tachykinin receptors, although they exhibit preferential binding to the NK1, NK2, and NK3 receptors, respectively. Within neuronal tissues, tachykinins act as excitatory neurotransmitters [Chapter 105 by Page, this volume]. However, substance P and other tachykinins are also widely distributed in nonneural tissues, where they mediate multiple homeostatic functions [Chapter 114 by Malendowicz, this volume]. The role of tachykinins in host defense systems has been extensively studied. Immune cells (T and B cells, endothelial cells, and macrophages) not only express NK1 receptors but also (in macrophages, eosinophils, and endothelial cells) synthesize substance P. The concentration of substance P increases in inflamed tissues [23], suggesting its importance in the mammalian host defense system. Indeed, available data suggest a multifunctional role for substance P, in which it activates immunological responses, generates a pain signal alarm, and also may itself have direct antibacterial and antifungal properties [12]. Numerous endogenous pathways are involved in neurologic and other disease pathology. Modern therapies therefore commonly rely on multidrug therapies that supplant selective drug use. However, using combinations of drugs has disadvantages, including differences in pharmacodynamic and pharmacokinetic profiles of each component drug. Therefore, the combination of multiple active pharmacophores in one molecule is a promising approach. A good example of such a molecule, based on the sequence of substance P, is the anticancer peptide Arg-D-Trp-Phe(N-Me)-DTrp-Leu-MetNH2, which simultaneously blocks receptors for substance P, bombesin/gastrin-releasing

ABSTRACT Chimerization of tachykinin and opioid pharmacophores offers a new avenue for analgesic development. The complexities of such design are illustrated by the analgesic efficacy (via different mechanisms) of chimeras that combine pharmacophores with opioid activity and substance P activity, as well as those that combine opioid agonist and substance P antagonist moieties. Although the interaction between substance P and opioid neural systems is more complex than a simple one-way inhibition, the relative balance of activities between tachykinin and opioid pharmacophores will generally determine the net effect of the chimeric molecule as pro-nociceptive, antinociceptive, or neutral. Intriguingly, endomorphins—μ-opioid receptor agonists with high intrinsic activity—may owe some of this high activity to weak but significant antagonist properties at tachykinin NK2 receptors, implying that these native peptides are endogenous chimeric opioid agonist–tachykinin antagonist compounds.

INTRODUCTION The discoveries of tachykinins and endogenous opioids were major milestones in the clarification of the role of neuropeptides in nociceptive signal formation, transmission, and perception. The undecapeptide substance P was the first recognized mammalian tachykinin; its amino acid sequence was successfully elucidated [4] in 1971. Subsequently, other members of the tachykinin family and superfamily, and their receptors and variants, have been well characterized. Tachykinin peptides are widely distributed in neuronal central nervous system (CNS) and peripheral tissues. Tachykinins interact with specific membrane receptors belonging to the family of G-protein-coupled receptors. Currently, three Handbook of Biologically Active Peptides

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1374 / Chapter 191 peptide, vasopressin, cholecystokinin, and neurotensin [9]. Multitarget therapies have became popular in pain treatment as well (multimodal analgesia) [30]. However, available analgesics in clinical practice are naturally, or have been crafted to be, maximally selective for one type of receptor. This chapter describes a new avenue for the development of new analgesics—the chimerization of tachykinin and opioid pharmacophores.

TABLE 1. Primary Sequences of Opioid Agonist– Substance P Antagonist Chimeric Compounds.a

AWL60

TACHYKININ ANTAGONIST–OPIOID AGONIST CHIMERIC COMPOUNDS The majority of neurophysiological and pharmacological studies have indicated that substance P and opioid systems are functionally antagonistic in the mediation of nociception and antinociception in vivo [20]. Thus, the simultaneous blockade of substance P receptors and activation of opioid receptors seems quite a reasonable approach to the construction of chimeric peptides. Indeed, co-injection of a weak peptide substance P antagonist together with an opioid peptide strongly enhances opioid analgesia [22]. The conclusion from this study indicated that incomplete blockade of substance P receptors enhances opioid analgesia. Studies of the effects of mixtures of peptides prompted the design of compounds that in one molecule combine substance P antagonist and opioid agonist pharmacophores. The first of this series was AWL60, a peptide in which an endomorphinlike moiety is hybridized (head-to-tail) with a substance P antagonist

Tyr-Pro-D-Phe-Phe-D-Phe-D-Trp-Met-NH2 [-endorphinlike---] [-------substance P7–11 antagonist --] Tyr-D-Ala-Gly-Phe-NH-NH←Trp←Cbz [----biphalin active fragment-----] [-substance P antagonist]

AA501

CROSS-INTERACTION OF TACHYKININ AND OPIOID SYSTEMS

Cbz, benzyloxycarbonyl-.

a

Saline

100

AWL60

80 % MPE

A large body of experimental evidence indicates that regulatory neuropeptides play modulatory roles in the afferent transfer and postsynaptic processing of nociceptive information within the dorsal horn of the spinal cord [3, 20] (see also Chapter 189 by Xu and Wiesenfeld-Hallin this volume). The superficial dorsal horn of the spinal cord is an area that receives primary synaptic input from sensory fibers originating from dorsal root ganglion (DRG) neurons. This area is a potentially important site for functional regulation (by modulatory opioid peptides) of nociceptive input mediated by substance P and excitatory amino acids released from primary afferent terminals. Anatomical studies indicate a similar distribution of substance P– and opioid-containing neural elements within this area [1, 25, 27]. Ligands of the μ receptor are highly effective in blocking or reducing nociceptive transmission at the spinal level [31], and this antinociception is thought to be caused at least partially by inhibition of substance P release [8].

Amino Acid Sequence and Indication of Respective Active Peptide Fragments

Compound

AWL60+NLX

60 40 20

0 –10

5

15

30 Time (min)

60

120

FIGURE 1. Antinociception in rats after intrathecal application of AWL60 (2.5 nmol) [15].

(Table 1) [15]. The resultant compound expressed properties of both hybridized components in an in vitro guinea pig ileum preparation. Although AWL 60 expressed moderate affinity to μ-opioid receptors (median inhibitory concentration, IC50, 1 μM), the in vivo intrathecal application of AWL produced intense, prolonged antinociception (Fig. 1). Preinjection of naloxone strongly reduced this antinociceptive effect, but did not abolish it completely. Further structure–activity relationship studies of chimeric morphiceptin-endomorphin molecules led to the conclusion that extension of the tetrapeptide with hydrophobic amino acids greatly diminished its affinity for opioid μ receptors. Therefore, a second, different molecule of this type of chimera was constructed. Biphalin, which hybridizes two opioid tetrapeptides head-tohead, is one of the most potent opioid analgesics. Structure–activity studies of biphalin concluded that one opioid pharmacophore could be replaced by one of the hydrophobic amino acids or their derivatives or mimetics [17]. An independent search for substance P antagonists resulted in the development of small aromatic tryptophan derivatives [16]. The consolidation of information from these several series of structure–activity studies allowed the development of a new

Opioid-Substance P Chimeric Peptides / 1375 compound, AA501, in which an opioid agonist—a substance P antagonist chimera was synthesized by head-to-head hybridization of a tetrapeptide with Nbenzyloxycarbonyl-tryptophan [21]. In this chimeric compound, the opioid agonist and tachykinin antagonist pharmacophores partially overlap (Table 1). AA501 has high affinity for opioid receptors and moderate affinity for tachykinin receptors. In vivo, after intrathecal injection AA501 provided intense antinociception in acute as well as in inflammatory and neuropathic pain models. Prolonged application of AA501 resulted in a slower rate of tolerance development than produced by administration of pure opioid. The more recently discovered endomorphins possess unique analgesic potency and selectivity for one opioid receptor type—μ. Further studies revealed that these peptides also express weak but significant antagonist properties at tachykinin NK2 receptors [11], implying that these peptides should be considered as endogenous chimeric opioid agonist–tachykinin antagonist compounds. The importance of the tachykinin antagonist component in their unique biological profile should be addressed in further studies.

antinociceptive effects of substance P may also be explained by its activation of a number self-regulatory mechanisms [18], including an influence on peptidase activities [2], modulation of the binding of opioids to their receptors [13], and influences on serotonin [10] or NMDA [29] pathways. Selective pharmacological activation of the opioid system may perturb the complex interactions between tachykinin and opioid systems. Simultaneous activation of the opioid system along with partial compensatory supplementation of the tachykinin system is the rationale for the development of chimeras that combine an opioid agonist and a tachykinin agonist. Because the principal effects of substance P and opioids are opposite, the relative balance of activities between tachykinin and opioid pharmacophores will determine the net effect of the chimeric molecule as pro-nociceptive, antinociceptive, or neutral. In the first compound of this series, SPF [14, 19] (Table 2), substance P activity predominated and intrathecal injection of the compound induced nociceptive symptoms characteristic of substance P (Fig. 2). In that compound, the presence of an opioid component provided only partial antinociception, and co-injection of the opioid antagonist naltrexone further augmented the nociceptive properties of the compound. In the second compound synthesized (ESP7), in which the endomorphin 2 and substance P C-terminal fragments were hybridized, opioid activity predominated [6]. Although, ESP7 expressed moderate affinity for opioid (Ki(μ) 218 nM) and NK1 (Ki 289) receptors, after intrathecal application in rats antinociception occurred at a very low dose (0.05 μg). Interestingly, because the molecule comprised two agonists at functionally opposite receptors, in rats a ceiling effect was observed at 40% of the maximal possible effect (MPE). The application of ESP7 for several days does not produce any evident tolerance. In fact, the application of ESP7 produced analgesia in morphine-tolerant animals.

TACHYKININ AGONIST–OPIOID AGONIST CHIMERIC COMPOUNDS The interaction between substance P and opioid neural systems is more complex than a simple one-way inhibition. In the spinal cord, these two systems are engaged in reciprocal regulation. Although the application of substance P in high doses is algesic, this molecule and its metabolites possess pain-alleviating effects when given in lower doses [26, 28]. To some extent, the analgesic effects described for low doses of substance P seem to reflect an opioidlike action because naloxone neutralizes substance P analgesia [7]. The

100

B Number of Behaviors

A

SPF SPF 2·10–8M 2·10–7M Naloxone Naloxone 10–6M 5·10–7M

SP

80 60

SPF+Naloxone SPF

40 20 0 0.1

1

10

100

1000

Dose of Peptide (pmol, i.t./mouse)

FIGURE 2. Examples of biological activities of SPF. A. In vitro electrically stimulated mouse vas deference: Spontaneous contraction characteristic to substance P and inhibition of electrical stimulation characteristic to opioids, reversed with naloxone [19]. B. Nociceptive behaviors in mice after intrathecal application. Weak nociception of SPF was potentiated by naloxone [14].

1376 / Chapter 191 TABLE 2. Primary Sequences of Opioid Agonist–Substance P Agonist Chimeric Compounds. Compound SPF ESP7 ESP6 AWL3106

Amino Acid Sequence and Indication of Respective Active Peptide Fragments Tyr-D-Ala-Gly-Phe-Phe-Gly-Leu-Met-NH2 [-enkephalin like-----] [-------substance P7–11-------] Tyr-Pro-Phe-Phe-Gly-Leu-Met-NH2 [-endomorphin-] [---substance P7–11-------] Tyr-Pro-Phe-Phe-Pro-Leu-Met-NH2 [-endomorphin-] [-[Proq]substance P7–11-] Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-D-Ala-Phe-Phe-Gly-Leu-Met-NH2 [-----dermorphin------------][--spacer--][-------substance P7–11--------]

B

100

80

80

60

60

% MPE

% MPE

A

100

40 20 0

Morphine

40 20

5

15

30 60 120 Time (min) 2 micromol/kg 6 micromol/kg 10 micromol/kg

0

5

AWL3106

15

30 Time (min)

1 micromol/kg

60

2 micromol/kg

120 5 micromol/kg

FIGURE 3. Comparison of antinociceptive effects of (A) morphine and (B) AWL3106 after intravenous application in rats.

The C-terminal fragment of substance P is shared by all three endogenous tachykinins. Previous structure– activity studies of tachykinins resulted in the conclusion that substitution of glycine [9] with proline increases selectivity for NK1 receptors. Therefore, in subsequent analogs the glycine of ESP7 was replaced with proline. The resultant peptide, ESP6, expressed higher affinity for opioid (Ki(μ) 92 nM) and similar affinity for NK1 (Ki 305 nM) receptors compared with ESP7 [5]. Intrathecal co-administration of morphine and ESP6 clearly slows the development of tolerance to morphine. Although, ESP6 itself has low antinociceptive potency, when it was administered during concurrent NK-1 receptor blockade, a decay in analgesic potency resulted, similar to that seen with tolerance to morphine alone. It was concluded that ESP6 represents a novel tolerance-eliminating adjuvant for the maintenance of opioid analgesia over time.

Both ESP6 and ESP7 compounds were designed as chimeric compounds with minimal size. Because substance P and endomorphin pharmacophores overlap in these molecules, the size of chimeras was reduced to a hexapeptide. Nevertheless, the extension of the original tetrapeptide sequence of endomorphin with additional hydrophobic peptides resulted in significant decreases of affinity to opioid receptors. Therefore, in the next generation of analogs two pharmacophores were hybridized in such a way that each pharmacophore was structurally independent and yet also connected by the spacer (Table 2). The resultant peptide, AWL3106, expressed high affinity for opioid (Ki(μ) 2.5 nM) and NK1 (Ki 7.5 nM) receptors and in addition was very potent when given intrathecally, with an analgesic ceiling at nearly 100% of MPE (Fig. 3). Chronic application of these peptides resulted in the very slow development of tolerance compared to morphine.

Opioid-Substance P Chimeric Peptides / 1377 Interestingly, this peptide was also very potent after intravenous application. It is very likely that the hydrophobic tachykinin domain of the peptide plays an additional role as a vector that helps it to cross the blood–brain barrier. This latter observation allows the creation of still more templates for the further design of chimeric drugs.

CONCLUSION The construction of opioid-tachykinin chimeric peptides seems to be a very promising avenue for the design of new analgesics. Hybridization of an opioid agonist with a substance P antagonist results in compounds with a synergistic antinociceptive interaction of both hybridized components. The modification of such compounds to provide a lesser degree of activation of the opioid system gives rise to the desirable property that the development of opioid tolerance during repeated administration is significantly slowed. The hybridization of opioid agonists with substance P agonists may also result in effective analgesics without apparent opioid tolerance during chronic application. The possible application of such compounds for the treatment of opioid tolerance and/or dependence is obvious, but further studies to evaluate their utility are needed.

References [1] Aicher S.A., Punnoose A., Goldberg A., “Mu-opioid receptors often colocalize with the substance P receptor (NK1) in the trigeminal dorsal horn.” J Neurosci 20 (2000) 4345–4354. [2] Barcklay R.K., Phillipps M.A., “Inhibition of enkephalin-degrading aminopeptidase activity by certain peptides.” Biochem Biophys Res Commun 96 (1980) 1732–1738. [3] Carr D.B., Cousins M.J., “Spinal route of analgesia: Opioids and future options.” In: Neural blocade in clinical anesthesia and management of pain (M.J. Cousins, P.O. Bridenbaugh, eds.), 3rd ed. Lippincott Raven, Philadelphia 1998, pp. 915–983. [4] Chang M.M., Leeman S.E., Niall H.D., “Amino-acid sequence of substance P.” Nature New Biol 232 (1971) 86–87. [5] Foran S.E., Carr D.B., Lipkowski A.W., Maszczynska I., Marchand J.E., Misicka A., Beinborn M., Kopin A.S., Kream R.M., “Inhibition of morphine tolerance development by a substance P-opioid peptide chimera.” J Pharm Exp Ther 295 (2000) 1142–1148. [6] Foran S.E., Carr D.B., Lipkowski A.W., Maszczynska I., Marchand J.E., Misicka A., Beinborn M., Kopin A.S., Kream R. M., “A substance P-opioid chimeric peptide as a unique nontolerance-forming analgesic.” Proc Natl Acad Sci USA 97 (2000) 7621–7626. [7] Hall M., Stewart J.M., “Substance P and antinociception.” Peptides 4 (1983) 31–35. [8] Jessel T.M., Iversen L.L., “Opiate analgesics inhibit substance P release from rat trigeminal nucleus.” Nature 268 (1977) 549– 551. [9] Jones D.A., Cummings J., Langdon S.P., Smyth J.F., “Preclinical studies on the broad-spectrum neuropeptide growth factor antagonist G.” Gen Pharmacol 28 (1997) 183–189.

[10] Klusa V.E., Abissova N.A., Muceniece R.K., Sirskis S.V., Bienert M., Lipkowski A., “Comparative study of substance P and its fragments with special reference to the analgesic properties, influence on the behavior and monoaminergic processes.” Biull Ekspe Biol Med 92 (1981) 692–694. [11] Kosson P., Bonney I., Carr D.B., Lipkowski A.W., “Endomorphins interact with tachykinin receptors.” Peptides 26 (2005), 1667–1669. [12] Kowalska K., Carr D.B., Lipkowski A.W., “Direct antimicrobial properties of substance P.” Life Sci 71 (2002) 747–750. [13] Krumnis S.A., Kim D.C., Igwe O.J., Larson A.A., “DAMGO binding to mouse brain membranes: Influence of salts, guanine nucleotides, substance P, and substance P fragments.” Peptides 14 (1993) 309–314. [14] Lei S.Z., Lipkowski A.W., Wilcox G.L., “Opioid and neurokinin activities of substance P fragments and their analogs.” Eur J Pharmacol 193 (1991) 209–215. [15] Lipkowski A.W., Carr D.B., Misicka A., Misterek K., Biological activities of a peptide containing both casomorphin-like and substance P antagonist structural characteristics.” In: β-Casomorphins and related peptides: Recent developments (V. Brantl, H. Teschemacher, eds.), VCH, Weinheim 1994, pp. 113–118. [16] Lipkowski A.W., Misicka A., Carr D.B., Ronsisvalle G., Kosson D., Maszczynska Bonney I., “Neuropeptide mimetics for pain management.” Pure Appl Chem 76 (2005) 941–950. [17] Lipkowski A.W., Misicka A., Davis P., Stropova D., Janders J., Lachwa M., Porreca F., Yamamura H.I., Hruby V.J., “Biological activity of fragment and analogues of the potent dimeric opioid peptide, biphalin” Bioorg Med Chem Lett 9 (1999) 2763–2766. [18] Lipkowski A.W., Osipiak B., Czlonkowski A., Gumulka W.S., “An approach to the elucidation of self-regulatory mechanism of substance P action. I. Synthesis and biological properties of pentapeptides related both to the Substance P C-terminal fragment and enkephalins.” Polish J Pharmacol Pharm 34 (1982) 63–68. [19] Lipkowski A.W., Osipiak B., Gumulka W.S., “An approach to the self regulatory mechanism of substance P actions: II. Biological activity of new synthetic peptide analogs related both to enkephalin and substance P.” Life Sci 33 sup. I (1983) 141–144. [20] Maszczynska I., Lipkowski A.W., Carr D.B., Kream R.M., “Dual functional interaction of substance P and opioids in nociceptive transmission: Review and reconciliation.” Analgesia 3 (1998) 259–268. [21] Maszczynska Bonney I., Foran S.E., Marchand J.E., Lipkowski A.W., Carr D.B., “Spinal antinociceptive effects of AA501, a novel chimeric peptide with opioid receptor agonist and tachykinin receptor antagonist moieties.” Eur J Pharmacol 488 (2004) 91–99. [22] Misterek K., Maszczynska I., Dorociak A., Gumulka S.W., Carr D.B., Szyfelbein S.K., Lipkowski A.W., “Spinal co-administration of peptide substance P antagonist increases antinociceptive effect of the opioid peptide biphalin.” Life Sci 54 (1994) 939– 944. [23] O’Connor T.M., O’Connell J., O’Brien D.I., Goode T., Bredin C.P., Shanahan F., “The role of substance P in inflammatory disease.” J Cellul Physiol 201 (2004) 167–180. [24] Pennefather J.N., Lecci A., Candenas M.L., Patak E., Pinto F.M., Maggi C.A., “Tachykinins and tachykinin receptors: A growing family.” Life Sci 74 (2004) 1445–1463. [25] Regoli D., Boudon A., Fauchere J.L., “Receptors and antagonist for substance P and related peptides.” Pharmacol Rev 46 (1994) 551–599. [26] Sakurada C., Watanabe C., Sakurada T., “Occurrence of substance P(1–4) in the metabolism of substance P and its antinociceptive activity at the mouse spinal cord level.” Met Find Exp Clin Pharmacol 26 (2004) 171–176.

1378 / Chapter 191 [27] Sanderson Nydahl K., Skinner K., Julius D., Basbaum A.I., “Colocalization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: A light and electron microscopic study in the rat.” Eur J Neurosci 19 (2004) 1789– 1799. [28] Stewart J.M., Getto C.J., Neldner K., Reeve E.B., Krivoy W.A., Zimmermann E., “Substance P and analgesia.” Nature 262 (1976) 784–785.

[29] Urban L., Nagy I., “Is there a nociceptive carousel?” Trends Pharmacol Sci 18 (1997) 223–224. [30] Walker S.M., Goudas L.C., Cousins M.J., Carr D.B., “Combination of spinal analgesic chemotherapy: A systemic review.” Anesth Analg 95 (2002) 674–715. [31] Yaksh T.L., Rudy T.A., “Analgesia mediated by a direct spinal action of narcotics.” Science 192 (1976) 1357–1358.

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192 VIP- and PACAP-Related Neuroprotection ILLANA GOZES

STRUCTURE OF THE PRECURSOR mRNA, DISTRIBUTION, AND PROCESSING

ABSTRACT Pituitary adenylate cyclase–activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuropeptides implicated in neuroprotection, and they are both widely distributed throughout the body. This review is a summary of selected original and recent developments in PACAP and VIP research that pertain to neuroprotection. It concludes with a discussion of NAP as a neuroprotective drug candidate, a clinical application stemming from VIP research.

Original studies identified the precursor mRNA structure for VIP [42] and established the existence of a separate exon for VIP and an additional exon for peptide histidine methionine amide (PHM) a VIPrelated peptide encoded by the same gene [10]. VIP distribution and processing have been previously reviewed [27, 29]. VIP and PACAP are discussed in Chapters 94 and 181 in this book by their discoverers [2, 55].

DISCOVERY VIP NEUROPROTECTION

The 28-amino-acid-long vasoactive intestinal peptide (VIP) was originally discovered as a gastrointestinal tract peptide [55] and later identified as a major brain peptide [27]. VIP patterns of expression and neuroprotective activity prompted research into the development of specific VIP analogs and the identification of downstream-acting neuroprotective proteins [11, 12, 34, 35]. Pituitary adenylate cyclase–activating polypeptide (PACAP, including a 38-amino-acid form, PACAP38, and a 27-amino-acid form, PACAP27) is highly homologous to VIP [2] and, like VIP, was shown to provide potent neuroprotection [2, 11, 65]. VIP neuroprotection has been associated with glial cell interaction to activate the synthesis and secretion of neuroprotective proteins [5, 12, 13, 24]. This review emphasizes a selected synopsis of VIP and PACAP neuroprotection, concluding with the discovery and development of NAP, an eight-amino-acid drug candidate for neuroprotection in Alzheimer’s disease [5, 6, 19, 28, 30, 36, 37, 43, 44, 51, 58, 59, 63–65]. Handbook of Biologically Active Peptides

The primary working hypothesis is that VIP (at low, probably physiological, concentrations) stimulates glial cells to release multiple neuroprotective substances, including a complex array of cytokines and growth factors [12, 13, 24]. Recent studies claim VIP brain bioavailability following specific formulations and systemic exposure [20] or nasal administration [21]. Improved bioavailability/stability and neuroprotective potency was claimed for nasally applied lipophilic VIP analogs [26, 32, 34]. The design of these analogs included (1) the addition of a stearyl moiety to the Nterminal side; and (2) the exchange of the labile methionine in position 17 with norleucine (an unnatural amino acid), yielding stearyl-Nle-VIP (SNV) [26, 32, 34] and the shortening of VIP to a backbone of four amino acids, yielding stearyl-KKYL [34] (Fig. 1). Interestingly, KKYL is a conserved epitope, identical to VIP and PACAP.

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ADNP, ADNF NAP

expression in adulthood is associated with reduced spatial learning. This was corroborated by studies with VIP antagonism [25]. Furthermore, VIP antagonist administration during development inhibited behavioral circadian rhythmicity [32]. The VPAC2 receptor was implicated in circadian behavior, and recent studies established that VIP coordinates daily rhythms in the suprachiasmatic nucleus by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons [4].

Toward a Better Understanding of Mechanisms FIGURE 1. Protection as a consequence of neuroglial interaction protein and peptide derivatives.

RECENT RESEARCH ON VIP: CONCENTRATING ON RECEPTORS AND MECHANISMS Receptors There are three receptor subtypes that recognize VIP and PACAP: VPAC1, VPAC2, and PAC1. PAC1 binds PACAP with a much higher affinity than it binds VIP, and as a result of alternative RNA splicing, the protein appears in several different splice forms (e.g., [52] for human PAC1). VPAC1 has been associated with cell proliferation [38]. VPAC2 has been associated with neurodevelopment and protection during development [53]. For example, the regulation of the synthesis of neuroprotective proteins (e.g., activity-dependent neuroprotective protein, ADNP) in glial cells was shown to be mediated in part by the activation of VPAC2 [65]. The hop2 splice variant of PAC1 may be implicated in neuroprotection [3], and differential receptor expression may contribute to cellular stability in response to environmental cues. Receptor expression patterns allow for differential effects of VIP and PACAP toward selective neuronal differentiation of mouse embryonic stem cells [14]. VPAC2 and PAC1 receptors were found to be expressed on embryonic stem cells that can aggregate into embryoid bodies, whereas embryoid bodies-derived cells expressed only a functional PAC1 receptor. The results suggest a developmental switch in VIP/PACAP receptor phenotype that may allow VIP and PACAP to differentially affect neuronal precursor proliferation and neuronal differentiation.

Lessons from Genetically Manipulated or Antagonist-Treated Rodents Original studies [31] showed that manipulation of VIP expression in transgenic mice that results in reduced

The neuroprotective actions of VIP can be divided into effects on cell division, impact on development, and protection against environmental stress. As already suggested, the control of cell division may depend on the variety of receptors and signaling mechanisms that are available to the cell or neuron. VIP stimulates brain growth during neurogenesis and embryogenesis, probably through ADNP [51] and activity-dependent neurotrophic factor (ADNF)-like molecules [5, 12, 16, 49]. The neurotrophic effects of VIP have been associated with synaptogenesis [8, 35], and VIP may be a neurotrophic factor, influencing neurite outgrowth through interaction with the expression of the cytoskeletal network [41]. As for neurodevelopment, original studies showed that VIP neuroprotection to developing neurons in culture may be independent of cAMP formation [32, 33]. Several reports showed that VIP protects against white-matter lesions in newborn and postnatal day-5 mice that form on the intracerebral administration of ibotenate, a glutamate agonist [39, 40, 53]. This neuroprotection was mediated by protein kinase C (PKC) through VPAC2 receptors and was shown to be independent from cAMP formation [52]. Interestingly, VPAC2 receptor activation was previously associated with mouse neocortical astrocytogenesis [62] that can be activated by neuroprotective lipophilic VIP analogs such as SNV [39]. The link to astrocytes is further emphasized by the ability of VIP to stimulate the synthesis and secretion of novel growth factors such as ADNF [12] and ADNP [24], potentially through activation of VPAC2 [65] and perhaps PAC1 splice variants [3]. In turn, ADNF-derived peptides [12, 13, 30, 40] and ADNP-derived peptides provide potent neurotrophic and neuroprotective effects [6, 30, 43, 44, 58]. Some of the ADNF/ADNP-related activity is inhibited by VIP blockade [8, 59]. Although neuroprotection by VIP was associated with VPAC2 binding followed by PKC activation, other studies have shown that VIP enhances synaptic transmission to CA1 hippocampal pyramidal cell dendrites through VPAC1-mediated actions that are dependent on PKC activity and that VPAC2-mediated actions are

VIP- and PACAP-Related Neuroprotection / 1381 responsible for the PKA-dependent actions [18]. Thus, the signaling mechanisms may depend on temporal and regional influences. VIP synthesis may increase as a consequence of neuronal injury, such as axotomy [9]. This increase may represent a compensatory mechanism. A recent example suggests that culturing porcine myenteric neurons results in increased expression of VIP and galanin and addition of VIP increases the survival of cultured porcine myenteric neurons, whereas the addition of galanin decreased their survival. The presence of VIP increased the number of galanin-producing neurons, whereas the majority of galanin-immunoreactive neurons did not express VIP [1]. The regulation of galanin is of interest because galanin is tightly associated with epilepsy and has been suggested as an antiepileptic compound [47]. Taken together, VIP provides a neuroprotective milieu and interacts with other potential growth factors. The precise mechanism of neuroprotection depends on the cell population and the receptors that are expressed.

RECENT RESEARCH ON PACAP Original work established that PACAP, like VIP, is neurotrophic [2]. It was further established that PACAP, specifically its 38-amino-acid, biologically active form PACAP38, acts to protect hippocampal neurons in the case of ischemia [60, 61]. Ischemia is a major focus of recent studies with PACAP. Original work was performed in rats that were given bolus injections of PACAP38 intracerebroventricularly and then underwent permanent middle cerebral artery occlusion. Results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia [54]. In further studies, Farkas et al. have established the dose-response curves for PACAP administration in traumatic axonal injury, demonstrating that a single postinjury intracerebroventricular injection of 100 μg PACAP significantly reduced the density of damaged, beta-amyloid precursor proteinimmunoreactive axons in the corticospinal tract [22]. The protective effects of PACAP during cerebral ischemia could be associated with its direct antiapoptotic effects or indirect effects through modulations of blood flow. For example, PACAP treatment reduced the amount of apoptosis caused by moderate injury to the spinal cord of adult rats. Although PACAP did not reduce the amount of apoptosis caused by severe injury, extended nerve fibers and motor neurons in the rostral and caudal regions of the spinal cord were observed [15]. Furthermore, the effects of PACAP on mean arterial pressure (MBP), regional cerebral blood flow (rCBF), and cerebral oxygen content (pO2) were

measured in mice to show that PACAP acts as a vasodilator to prevent neuronal cell death during cerebral ischemia. PACAP (5 × 10−8 mol/kg) produced a longlasting decline of MBP, rCBF, and cerebral pO2, whereas smaller doses produced only a transient decline in those values [50].

Lessons from Genetically Manipulated or Antagonist-Treated Rodents PACAP(−/−) mice exhibit increased postnatal mortality, increased novelty-seeking behavior, and abnormal explosive jumping in a novel environment [56]. It was suggested that neonatal death in PACAP-deficient mice is a result, in part, of PACAP localization in brainstem regions associated with respiratory chemosensitivity. Reduced ventilation causes prolonged apneas that precede the atrioventricular block, which leads to death [17]. PACAP(−/−) mice have also been used to demonstrate that PACAP promotes the coupling of neuronal nitric oxide synthase (NOS) to N-methyl-d-aspartate (NMDA) receptors for inflammatory and neuropathic pain to occur [45]. Mice lacking PACAP and PAC1R receptors, PACAP(+/−) and PAC1R(−/−) mice, were used to show that PACAP and PAC1R contribute to the establishment of long-term potentials essential to learning and memory [46].

Mechanism of PACAP Action PAC1, the PACAP receptor, is expressed in the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus, the neurogenic regions of the adult mouse brain. In the absence of other growth factors, PACAP promotes the proliferation of neural stem cells as neurospheres through the PAC1 receptor. Selective inhibition of PKC (Gö6976) confirmed that PACAP, through the PAC1 receptor, mediates neural stem cell proliferation through the phospholipase C (PLCγ)–PKC pathway [48]. One study confirmed the role of the cAMP–protein kinase A (PKA) pathway and the phosphatidylinositol 3-kinase (PI3-K) pathway as the routes of PACAP neuroprotection through selective inhibition. The inhibition of PKA with H-89 or Pp-cAMPS or of the PAC1 receptor with PACAP6–38 reversed the neuroprotection reduced by PACAP, confirming the cAMPPKA pathway. MEK inhibition with PD98059 and PLC inhibition with U73122 did not affect the neuroprotection of PACAP, suggesting that mitogen-activating protein kinase (MAPK) and PLC may not be involved as the major pathways of PACAP neuroprotection under certain circumstances. Inhibition of the Trk receptor with K-252a did not reverse the effects of PACAP. Gprotein subunits that activate PI 3-kinase attenuated the

1382 / Chapter 192 effects of PACAP, indicating that the PI3-K pathway is involved in PACAP neuroprotection [7].

Neuroprotective Peptide Analogs Ac-PACAP, a PACAP derivative, and IK, a VIP derivative, could cross the blood–brain barrier to prevent neural cell damage in the hippocampus after brain ischemia. Brain ischemia was simulated by the 10-min two vessel occlusion (2VO) model and the 30-min cerebral artery occlusion (MCAO) model. On the 2VO model, IK was effective in the 1–100 pmol/kg range and Ac-PACAP was effective in the 10 fmol/kg–1 pmol/ kg range, both in a dose-dependent manner. Using the MCAO model, IK was effective in the same range and Ac-PACAP was effective in the 1 fmol/kg–1 pmol/kg range, again in a dose-dependent manner [60]. These results emphasize the potential increased protective effects and bioavailability of lipophilic derivatives of the VIP/PACAP family of peptides, as was originally shown for SNV [26, 34] and stearyl-KKYL [34]. Both peptide analogs were shown to provide neuroprotection in an animal model of cholinotoxicity by nasal administration at a dose in the range of microgram(s) peptide/rat. Other in vivo models of neuroprotection for the neuroprotective lipophilic analogs (SNV and KKYL) included apolipoprotein E–deficient mice [30, 34] and a model of cerebral palsy [39]. In an in vitro model of ischemia, SNV was shown to increase ADNP expression, suggesting modulation through ADNP activity [57].

NAP: TREATMENT THROUGH A VIP-RELATED MECHANISM ANDP, a protein regulated by VIP, is essential for brain formation. ADNP (−/−) knockout mice die E8.5-9 because of cranial neural tube closure failure [51]. ADNP expression has shown plasticity during the estrus cycle [23] and has been shown to be regulated as a consequence of brain injury (e.g., [37, 63]). Peptide activity scanning identified NAP (NAPVSIPQ) as a small active fragment of ADNP that provides neuroprotection at very low concentrations. In cell culture, protection has been demonstrated against toxicity associated with Aβ (the β-amyloid peptide), NMDA, electrical blockade (tetrodotoxin), gp120 (the envelope protein of the AIDS virus), H2O2, nutrient starvation, tumor necrosis alpha toxicity, and zinc intoxication (e.g., [5, 6, 19, 43, 59]). In animal models of apolipoprotein E deficiency, cholinergic toxicity, closed head injury, and stroke, NAP provided neuroprotection (e.g., [5, 6, 30, 44]). The structure of NAP allows cell penetration, the inhibi-

tion of toxic protein β-sheet formation, and the stimulation of proper protein assembly. NAP binds to tubulin and facilitates microtubule polymerization, leading to enhanced cellular (astrocyte) survival that is associated with fundamental brain cell elements, the cytoskeleton [19]. A mass spectrometry assay determined that NAP reaches the brain on nasal administration and reaches rat and dog plasma. In a battery of toxicological tests including 1-month repeated-dose toxicity in rat and dog (and 90-day repeated-dose toxicity in rats), cardiopulmonary tests in dog, and functional behavioral assays in rats, no adverse side effects were observed with NAP concentrations that were similar or approximately 500-fold higher than the biological active dose. No genotoxicity was associated with NAP. A mass spectrometry assay has been validated for tests in human plasma (MPI Reasearch). The first- in-human exposure (phase I clinical trials sponsored by Allon Therapeutics, Inc.) indicated no drug-related adverse side effects in healthy young volunteers.

CONCLUSION VIP, PACAP, and the respective G-protein-coupled receptors modulate effects on neuroprotection. Some of these effects may be associated with changes in the cytoskeletal network that offer cellular protection or activity in the brain. ADNP was discovered as a VIP/ PACAP-regulated protein that is essential for brain formation. A downstream derivative of VIP/PACAP action, NAP is a very short 8-amino-acid peptide derived from ADNP that provides neuroprotection. NAP has been selected based on potent neuroprotective properties and brain bioavailability for further clinical development by Allon Therapeutics, Inc.—from the laboratory bench to clinical trials (Fig. 1).

Acknowledgments NAP is patented and licensed to Allon Therapeutics, Inc., where IG serves as CSO. Other support include the Llily and Avraham Gildor Chair for the Investigations of Growth Factors, The Dr. Diana and Zelman Elton (Elbaum) Laboratory for Molecular Neuroendocrinology, NICHD, NIA, BSF, and ISF. I thank Deborah Moody, David Dangoor, and Miriam Holtser-Cochav for their invaluable help.

References [1] Arciszewski MB, Ekblad E. Effects of vasoactive intestinal peptide and galanin on survival of cultured porcine myenteric neurons. Regul Pept 2005;125:185–92.

VIP- and PACAP-Related Neuroprotection / 1383 [2] Arimura A, Somogyvari-Vigh A, Weill C, Fiore RC, Tatsuno I, Bay V, Brenneman DE. PACAP functions as a neurotrophic factor. Ann NY Acad Sci 1994;739:228–43. [3] Ashur-Fabian O, Giladi E, Brenneman DE, Gozes I. Identification of VIP/PACAP receptors on rat astrocytes using antisense oligodeoxynucleotides. J Mol Neurosci 1997;9:211–22. [4] Aton SJ, Colwell CS, Harmar AJ, Waschek J, Herzog ED. Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons. Nat Neurosci 2005;8:476–83. [5] Bassan M, Zamostiano R, Davidson A, Pinhasov A, Giladi E, Perl O, et al. Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide. J Neurochem 1999;72(3):1283–93. [6] Beni-Adani L, Gozes I, Cohen Y, Assaf Y, Steingart RA, Brenneman DE, Eizenberg O, Trembolver V, Shohami E. A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice. J Pharmacol Exp Ther 2001;296(1):57– 63. [7] Bhave SV, Hoffman PL. Phosphatidylinositol 3′-OH kinase and protein kinase A pathways mediate the anti-apoptotic effect of pituitary adenylyl cyclase-activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol. J Neurochem 2004;88:359–69. [8] Blondel O, Collin C, McCarran WJ, Zhu S, Zamostiano R, Gozes I, Brenneman DE, McKay RD. A glia-derived signal regulating neuronal differentiation. J Neurosci 2000;20:8012–20. [9] Boeshore KL, Schreiber RC, Vaccariello SA, Sachs HH, Salazar R, Lee J, Ratan RR, Leahy P, Zigmond RE. Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis. J Neurobiol 2004;59:216–35. [10] Bodner M, Fridkin M, Gozes I, Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome. Proc Natl Acad Sci USA 1985;35:3548–51. [11] Brenneman DE, Eiden LE. Vasoactive intestinal peptide and electrical activity influence neuronal survival. Proc Natl Acad Sci USA 1986;83:1159–62. [12] Brenneman DE, Gozes I. A femtamolar-acting neuroprotective peptide. J Clin Invest 1996;97:2299–307. [13] Brenneman DE, Phillips TM, Hauser J, Hill JM, Spong CY, Gozes I. Complex array of cytokines released by vasoactive intestinal peptide. Neuropeptides 2003;37:111–19. [14] Cazillis M, Gonzalez BJ, Billardon C, Lombet A, Fraichard A, Samarut J, Gressens P, Vaudry H, Rostene W. VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells. Eur J Neurosci 2004;19:798–808. [15] Chen WH, Tzeng SF. Pituitary adenylate cyclase-activating polypeptide prevents cell death in the spinal cord with traumatic injury. Neurosci Lett 2005;384:117–21. [16] Chiba T, Hashimoto Y, Tajima H, Yamada M, Kato R, Niikura T, Terashita K, Schulman H, Aiso S, Kita Y, Matsuoka M, Nishimoto I. Neuroprotective effect of activity-dependent neurotrophic factor against toxicity from familial amyotrophic lateral sclerosis-linked mutant SOD1 in vitro and in vivo. J Neurosci Res 2004;15:542–52. [17] Cummings KJ, Pendlebury JD, Jirik FR, Sherwood NM, Wilson RJ. A SIDS-like phenotype is associated with reduced respiratory chemoresponses in PACAP deficient neonatal mice. Adv Exp Med Biol 2004;551:77–83. [18] Cunha-Reis D, Ribeiro JA, Sebastiao AM. VIP enhances synaptic transmission to hippocampal CA1 pyramidal cells through activation of both VPAC(1) and VPAC(2) receptors. Brain Res 2005;1049:52–60.

[19] Divinski I, Mittleman L, Gozes I. A femtomolar-acting octapeptide interacts with tubulin and protects astrocytes against zinc intoxication. J Biol Chem 2004;279:28531–8. [20] Dufes C, Gaillard F, Uchegbu IF, Schatzlein AG, Olivier JC, Muller JM. Glucose-targeted niosomes deliver vasoactive intestinal peptide (VIP) to the brain. Int J Pharm 2004;285:77–85. [21] Dufes C, Olivier JC, Gaillard F, Gaillard A, Couet W, Muller JM. Brain delivery of vasoactive intestinal peptide (VIP) following nasal administration to rats. Int J Pharm 2003;255:87–97. [22] Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept 2004;15:69–75. [23] Furman S, Hill JM, Vulih I, Zaltzman R, Hauser JM, Brenneman DE, Gozes I. Sexual dimorphism of activity-dependent neuroprotective protein in the mouse arcuate nucleus. Neurosci Lett 2005;373:73–8. [24] Furman S, Steingart RA, Mandel S, Hauser JM, Brenneman DE, Gozes I. Subcellular localization and secretion of activitydependent neuroprotective protein in astrocytes. Neuron Glia Biol 2004;1:193–9. [25] Glowa JR, Panlilio LV, Brenneman DE, Gozes I, Fridkin M, Hill JM. Learning impairment following intracerebral administration of the HIV envelope protein gp120 or a VIP antagonist. Brain Res 1992;570:49–53. [26] Gozes I, Bardea A, Reshef A, Zamostiano R, Zhukovsky S, Rubinraut S, Fridkin M, Brenneman DE. Neuroprotective strategy for Alzheimer disease: intranasal administration of a fatty neuropeptide. Proc Natl Acad Sci USA 1996;9:427–32. [27] Gozes I, Brenneman DE. VIP: molecular biology and neurobiological function. Mol Neurobiol 1989;125:201–36. [28] Gozes I, Divinsky I, Pilzer I, Fridkin M, Brenneman DE, Spier AD. From vasoactive intestinal peptide (VIP) through activitydependent neuroprotective protein (ADNP) to NAP: a view of neuroprotection and cell division. J Mol Neurosci 2003;20:315– 22. [29] Gozes I, Fridkin M, Hill JM, Brenneman DE. Pharmaceutical VIP: prospects and problems. Curr Med Chem 1999;6:1019– 34. [30] Gozes I, Giladi E, Pinhasov A, Bardea A, Brenneman DE. Activity-dependent neurotrophic factor: intranasal administration of femtomolar-acting peptides improve performance in a water maze. J Pharmacol Exp Ther 2000;293:1091–8. [31] Gozes I, Glowa J, Brenneman DE, McCune SK, Lee E, Westphal H. Learning and sexual deficiencies in transgenic mice carrying a chimeric vasoactive intestinal peptide gene. J Mol Neurosci 1993;4:185–93. [32] Gozes I, Lilling G, Glazer R, Ticher A, Ashkenazi IE, Davidson A, Rubinraut S, Fridkin M, Brenneman DE. Superactive lipophilic peptides discriminate multiple vasoactive intestinal peptide receptors. J Pharmacol Exp Ther 1995;273:161–7. [33] Gozes I, McCune SK, Jacobson L, Warren D, Moody TW, Fridkin M, Brenneman DE. An antagonist to vasoactive intestinal peptide affects cellular functions in the central nervous system. J Pharmacol Exp Ther 1991;257:959–66. [34] Gozes I, Perl O, Giladi E, Davidson A, Ashur-Fabian O, Rubinraut S, Fridkin M. Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: neuroprotective drug design. Proc Natl Acad Sci USA 1999;96:4143–8. [35] Gozes I, Shani Y, Rostene WH. Developmental expression of the VIP-gene in brain and intestine. Mol Brain Res 1987;2:137–48. [36] Gozes I, Steingart RA, Spier AD. NAP mechanisms of neuroprotection. J Mol Neurosci 2004;24:67–72. [37] Gozes I, Zaltzman R, Hauser J, Brenneman DE, Shohami E, Hill JM. The expression of activity-dependent neuroprotective

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193 Insulin-Like Growth Factor 1 ITAY BENTOV AND HAIM WERNER

sulfation factor [66]. The list of effects of the sulfation factor on cartilage development expanded in the following decade to include the stimulation of thymidine incorporation into DNA, proline into collagen, and uridine into RNA [14, 15, 68]. Furthermore, the global nature of the sulfation factor was established by studies showing that its growth-promoting activities were not limited to the cartilage. Subsequently, the term somatomedin was proposed to replace the operational sulfation factor [13]. The new term reflected the fact that the serum factor mediated the effects of somatotrophin (GH) at somatic target tissues. During the 1970s, the list of actions of somatomedin further expanded to incorporate a number of activities that were not restricted to growth, including an insulinlike activity in the diaphragm and the stimulation of glucose uptake in adipose tissue [30, 67]. Furthermore, two closely related peptides with growth-promoting activities were discovered in the serum fraction [62]. Although these peptides displayed a striking homology between their N-terminal domains and the insulin B chain, their insulin-like activities were not suppressed by insulin antibodies [61]. Thus, the factors that were originally termed sulfation factor, somatomedins, and NSILA (nonsuppressible insulin-like activity) were finally named insulin-like growth factor 1 and 2 (IGF-1 and IGF-2). Although bearing a strong structural and functional resemblance, both peptides represent distinct entities that are the products of two different genes, the IGF-1 gene located on chromosome 12 and the IGF-2 gene on chromosome 11 [8]. This chapter focuses on IGF-1.

ABSTRACT Insulin-like growth factor 1 (IGF-1) is a pleiotropic growth factor with multiple roles in various aspects of normal and pathological growth and differentiation. The biological actions of IGF-1 are mediated by the IGF-1 receptor (IGF1R), a cell-surface heterotetramer structurally related to the insulin receptor. In addition, the effects of IGF-1 are modulated by a family of binding proteins (IGFBPs) that carry the ligand in the circulation and extracellular fluids. IGF-1 is widely expressed in the central nervous system (CNS), where it promotes proliferation, survival, and differentiation of neuronal and nonneuronal cells. Furthermore, IGF-1 is a potent neurotrophic factor, rescuing neurons from apoptosis and enhancing neuronal growth and myelination. Understanding the role and regulation of the IGF-1 system will be of significant basic and clinical relevance.

DISCOVERY The discovery of insulinlike growth factor 1 (IGF-1) dates to the late 1950s, when the anabolic effects of growth hormone (GH) in hypophysectomized rats were studied. The model measured the incorporation of radioactive sulfate (in the form of chondroitin sulfate and keratin sulfate) into the hyaluronan macromolecule, the protein core of cartilage. Serum from these animals (with low GH levels) failed to stimulate the incorporation of sulfate into cartilage. Moreover, the addition of GH to the incubation medium had no effect on sulfate incorporation. In contrast, serum of GHtreated hypophysectomized rats significantly enhanced sulfate uptake. Therefore, it was concluded that the incorporation of sulfate was not a direct effect of GH but rather that GH stimulated the action or production of a secondary serum-borne factor, which was termed Handbook of Biologically Active Peptides

STRUCTURE OF PRECURSOR mRNA/GENE The IGF-1 gene, located on chromosome band 12q22–q24, is approximately 100 kb long and contains six exons. Due to alternative transcription initiation,

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FIGURE 1. The rat IGF-1 gene and transcripts. The gene is shown schematically at the top, and below the various transcripts are shown that result from alternative initiation, splicing, and polyadenylation. Arrows above exon 1 and exon 2 indicate transcription start sites. ORF, the putative open reading frame in the exon 1 5′-UTR. Black areas are the preproIGF-1 coding sequences. Met-22 indicates the exon 3 translation start site. Eb and Ea indicate the stop codons for the Eb and Ea forms of preproIGF-1. The arrows below exon 6 indicate the polyadenylation sites, and the small rectangles below show the resulting lengths of 3′-UTR and their terminating poly A tails. From [71].

alternative splicing, and different polyadenylation sequences, numerous IGF-1 mRNAs are produced. These transcripts differ slightly in their coding sequences but mostly diverge in their 5′ and 3′ untranslated regions (UTRs) [77]. Exons 1 and 2 are alternative first exons, each one encoding a different 5′-UTR and an alternative N-terminal sequence of the IGF-1 precursor. Extrahepatic production of IGF-1 mRNA is almost exclusively from exon 1, whereas hepatic production, although predominantly from exon 1, can occur also from exon 2 [2]. The differences between transcripts stem not only from the use of either exon 1 or 2. Both exons contain a cluster of start sites (four start sites in exon 1 and two sites in exon 2), which indicates the existence of a complex control mechanism. Furthermore, the use of different transcription start sites in the IGF-1 gene is modulated by the nutritional status of the animal as well as by GH levels [55]. Exon 3 encodes part of the signal peptide, whereas exon 4 encodes a common N-terminal domain that is similar in humans, rats, and mice. Exons 5 and 6 each encode a different C-terminal product, each of them containing a stop codon. Alternative splicing results in two isoforms of the E peptide moiety of the prohormone, Ea and Eb. In humans, exon 4 is

spliced directly either to exon 5 to encode the Eb isoform or to exon 6 to produce the Ea isoform [65]. In rodents, IGF-1 mRNA contains either exons 4, 5, and 6, or exon 4 spliced directly to exon 6 [44]. Exon 6 contains multiple polyadenylation sites, resulting in molecules containing alternative 3′-UTR domains of several hundred nucleotides in length. IGF-1 mRNA in humans and rats exists in three sizes: 0.8–1.2, 2.2, and 7.5 kb [63]. The large isoform contains a long (∼6.5-kb) AU-rich sequence, which results from the use of a downstream polyadenylation site and which renders the 7.5kb IGF-1 mRNA molecule remarkably unstable [32].

DISTRIBUTION OF THE mRNA In rodents, the prenatal period shows very low IGF-1 levels and high IGF-2 levels, whereas postnatal stages are characterized by an increase in circulating IGF-1 values and disappearance of IGF-2. These early observations might have led to an erroneously broad interpretation of the roles of IGF-2 and IGF-1 as fetal and pubertal growth factors, respectively. In humans, however, this expression pattern does not exist and

Insulin-Like Growth Factor 1 both ligands are produced from prenatal to postnatal periods. In fact, circulating IGF-2 levels in adults are higher than IGF-1 levels. Circulating (endocrine) IGF-1 levels are mostly dependent on liver production, which is tightly controlled by GH [16]. In addition to its hepatic production, many extra-hepatic tissues also produce IGF-1, which usually exhibits paracrine and autocrine modes of action in these organs. Numerous factors were shown to control circulating IGF-1 levels. These factors include both direct effectors (e.g., GH, protein calorie intake, thyroxine, catabolic stressors) and indirect factors (e.g., age, body fat, exercise, various hormones). An important contribution to the understanding of the equilibrium and relative importance of liver-produced (endocrine) IGF-1 versus locally produced (paracrine/ autocrine) IGF-1 came from a mouse model in which the liver IGF-1 gene was knocked out using the cre-lox recombinase system [75]. In this animal model, circulating IGF-1 levels were reduced by ∼75% and GH levels were elevated compared to normal littermates. However, there was almost no phenotypic difference, nor was there a disparity in tissue IGF-1 mRNA production that might have acted as a local compensatory mechanism. The significance of IGF-1 mRNA distribution and paracrine effect is presently under extensive investigation.

PROCESSING The IGFs are ∼7-kDa peptides that share a remarkable structural homology with proinsulin [76]. The amino acid sequence of IGF-1 has been elucidated for more than 10 mammalian species and for approximately the same number of nonmammalian vertebrate species. In most species, including humans, IGF-1 is a 70-amino-acid, linear, basic peptide. It consists of B and A domains that are homologous to the B and A domains of insulin. The N-terminal B domain contains 29 residues, whereas the A domain contains 21 residues. In addition, between the A and B domains lies a 12-residue C domain. Unlike insulin, the C domain is not cleaved out, and this may explain the immunological difference between IGFs and insulin that led to the historical finding of the nonsuppressible insulin-like activity of IGF-1. The IGF’s 8-residue D domain, located in the C-terminal end, is not found in insulin. The E domain, located in the C-terminus, is cleaved during secretion in the Golgi apparatus. The sizes of the E domain differ; thus, the IGF-1A protein contains a 35-residue E domain, whereas IGF-1B contains a 77-residue E domain. Because part of the E domain is encoded by exon 4 in both proteins, the initial 16 residues are identical. Although the mammalian IGF-1 is highly conserved, a more significant variability is observed among IGF-1

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molecules derived from nonmammalian vertebrates. Conserved amino acids include six cysteins that form three disulfide bonds (two between the A and B domains and one inside the A domain), suggesting that the overall spatial 3D topography of the molecule is conserved. In addition, three tyrosine residues are conserved (analogous to positions B24, C31 and A60 in the human IGF-1 protein), and it was suggested that these residues are critical in high-affinity binding to the receptor [7].

RECEPTORS IGF-1 and IGF-2 activate a common, ubiquitously expressed receptor, the IGF-1 receptor (IGF1R) or type 1 IGF receptor, which signals mitogenic, antiapoptotic, and transforming activities [43]. The IGF-2/mannose 6-phosphate receptor (IGF-2/M6PR) does not participate in IGF signaling, and its main role is to target IGF-2 for lysosomal degradation [39]. The IGF1R gene is located on the long arm of chromosome 15 (15q25– q26) and it encodes a 1367-amino-acid precursor protein that is proteolytically processed to yield mature α and β subunits. The functional cell-surface receptor is composed of two extracellular α-subunits, involved in ligand binding, and two transmembrane β-subunits containing a cytoplasmic tyrosine kinase domain. The IGF1R is coupled to several intracellular second messenger pathways, including the ras-raf mitogenactivating protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) signaling cascades [43]. IGF1R action is vital for cell survival, as illustrated by the lethal phenotype of mice in which the IGF1R gene was disrupted by homologous recombination [3]. During normal ontogenesis, the IGF1R is expressed at every developmental period, including the oocyte stage. Late embryonic and adult stages, in which the percentage of dividing cells declines, are associated with an overall reduction in IGF1R mRNA levels [73]. The seminal role of the IGF1R in normal and pathological growth has been demonstrated in animal models as well as in patients with chromosomal abnormalities that disrupt the IGF1R locus and in patients with specific IGF1R gene mutations. In mice, the disruption of the IGF1R gene by homologous recombination resulted in animals weighing 45% of control littermates at the time of birth [3]. These animals exhibited generalized developmental abnormalities, including hypoplasia, abnormal skin formation, delayed bone development, and abnormal central nervous system (CNS) morphology, and they died in the immediate postnatal period from respiratory failure. Chromosomal aberrations involving the 15q26 locus (e.g., ring chromosome 15) were shown to be

1388 / Chapter 193 associated with hemizygosity for the IGF1R locus and severe growth failure [56]. On the other hand, a patient with three copies of the gene due to partial duplication of the long arm of chromosome 15, presented with height and weight above the ninety-seventh percentile and showed accelerated cellular growth [54]. Combined, these studies underscore the link between IGF1R number and cellular proliferation. Recently, two children with mutations in the IGF1R gene were described in two pediatric cohorts; the first cohort consisted of children with unexplained intrauterine growth retardation and subsequent short stature, and the second group included children with short stature and high circulating IGF-1 levels [1]. A girl from the first group was identified as a compound heterozygote for point mutations in exon 2 of the IGF1R gene. Fibroblasts cultured from the patient had decreased IGF1R binding and phosphorylation. In the second cohort a boy was identified with a nonsense mutation, leading to reduced IGF1R expression. Noteworthy, although both cases showed prenatal and postnatal growth retardation, they were phenotypically different, suggesting that distinct domains of the IGF1R molecule are responsible for different aspects of growth and differentiation. Finally, the examination of various tumors (including breast, ovary, prostate, colon, hematopoietic, rhabdomyosarcoma, and kidney), showed an abundant expression of IGF1R mRNA and protein, suggesting that upregulation of the IGF1R gene constitutes a common paradigm in different types of cancer [72].

ACTIVE AND/OR SOLUTION CONFORMATION The vast majority of the IGF-1 peptide in the circulation is not found in a free form but, rather, in a ternary complex that includes, in addition to the IGF-1 molecule, a liver-derived glycoprotein (the acid-labile subunit, ALS) and a high-affinity binding protein, the IGF binding protein 3 (IGFBP3) [34]. Six IGFBPs (IGFBP1–6) and a number of IGFBP-related proteins have been characterized to date [37, 58]. IGFBP3, the predominant IGFBP in serum, is the largest and, therefore, the only one that cannot traverse the capillary membrane. The ternary complex between IGF-1, IGFBP3, and ALS modulates IGF-1 action by protecting the growth factor from proteolysis and prolonging its half-life in the circulation [6]. In addition, some IGFBPs exert their biological effects in an IGF-independent manner [27]. In general, the IGFBPs inhibit the metabolic and proliferative actions of IGF, although some IGFBPs may display IGF-potentiating effects as well [12]. IGFBP3 in particular is recognized as an inhibitor of proliferation, driving the cell to apoptosis. Several

potential mechanisms of action were postulated to explain IGFBP3 inhibition of IGF action, including the sequestration of IGF-1 from the receptor and binding competition with the IGF1R [51].

BIOLOGICAL ACTIONS The structural and functional similarities between insulin and IGF-1 suggest that both molecules are derived from a common ancestral precursor that probably participated in regulation of food intake and cellular growth [60]. A divergence of functions most likely occurred before the appearance of the first vertebrates, with insulin mostly active in the regulation of metabolism and IGF-1 in growth processes. In view of their common evolutionary origins and semi-conserved architecture, however, there is a certain degree of cross-talk among insulin, IGF-1, and their receptors. Accordingly, insulin exhibits a number of IGF-1-like activities, including growth stimulation, and IGF-1 exerts certain metabolic effects. In addition, the fact that IGF-1, but not insulin, is carried in the circulation and extracellular fluid by IGFBPs may further contribute to the divergent actions of both ligands [36]. At the cellular level, IGF-1 stimulates a mitogenic response and inhibits cell death in a wide variety of cell types, including primary cultures and cancer cell lines [47]. Quiescent cells in G0 can be induced to enter G1 by competence factors (e.g., platelet-derived growth factor, PDGF; basic fibroblast growth factor, bFGF). Once the cell enters into G1, subphysiological doses of IGF-1 will allow it to evade arrest in G1 and to progress through the cell cycle [45]. Thus, IGF-1 functions as a progression factor [52]. IGF-1 can also induce differentiation, and this activity can be blocked by specific antisense oligonucleotides [22]. IGF-1 exhibits a variety of cellular functions, including the regulation of hormone synthesis and secretion, chemoattractant migration, immune cell recognition, and neuromodulation [9, 25, 69]. The metabolic effects of IGF-1 include the elevation of glucose uptake and hypoglycemia, without lowering free-fatty-acid levels [29, 36]. In addition, it has been suggested that IGF-1 improves renal function by increasing renal blood flow and glomerular filtration rate [28]. The essential role played by the IGF system in growth and development was demonstrated by the severe growth deficits observed in mice in which various components of the IGF system were disrupted by homologous recombination [3]. Thus, heterozygous mice for a disrupted IGF-1 allele had a body weight at the time of birth that was approximately 10–20% less than wild-type animals, whereas null homozygotes weighed approximately 40% less than wild-type [57]. Furthermore, null

Insulin-Like Growth Factor 1 homozygotes had very high perinatal mortality rates and a number of phenotypic aberrations. Interestingly, screening of IGF levels in species with distinct phenotypic features revealed that in different breeds of dogs circulating IGF-1 levels are matched with breed size [20]. As a general rule, smaller breeds have lower IGF1 levels than larger breeds. Furthermore, a number of epidemiological observations in human populations highlight the pivotal role of the IGF axis in normal growth. For example, circulating IGF-1 levels in African pygmies are markedly lower than in the general population [48]. Moreover, GH and IGF-2 levels are normal in pygmies, whereas IGF-1 levels in adolescent males and females are significantly lower. As a result of this reduction, the growth spurt associated with puberty is absent [49].

PATHOPHYSIOLOGICAL IMPLICATIONS Growth Disorders The importance of IGF-1 in normal growth is classically illustrated by the Laron syndrome (LS) [64]. LS patients are characterized by mutations in the GH receptor, leading to resistance or insensitivity to GH action and diminished IGF-1 production (despite normal or even high GH levels) [26]. LS patients’ height is 4–10 standard deviations below median normal height, and acceleration of linear growth was demonstrated by continuous long-term treatment of affected patients with recombinant IGF-1 [41]. More recently, a homozygous partial deletion of the IGF-1 gene has been reported in a 15-year-old boy with severe prenatal and postnatal growth deficiency, sensorineural deafness, and mental retardation [74]. An additional component of the IGF system whose mutation was shown to have an impact on growth processes is the ALS of the ternary complex. Mutation of the ALS gene in a 17-year-old boy resulted in undetectable levels of circulating IGF-1 (due to instability of the ternary complex), delayed onset of puberty, and slow pubertal progress [18]. This mutation, however, had only a minimal effect on linear growth. The effect of mutations in the IGF1R gene have already been described.

Cancer The proliferative effect of IGF-1 led to several observational studies that were intended to elucidate the possible connection between circulating IGF-1 levels and an increased risk of neoplasia. Large epidemiological studies have suggested that high circulating IGF-1 concentrations are associated with an increased risk for breast, prostate, lung, and colorectal cancer.

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Specifically, in a prospective nested control study (the Nurse’s Health Study) the relative risk (RR) of breast cancer in premenopausal women was 4.6 in the upper tertile of IGF-1 values, in comparison to women in the lower tertile [31]. The RR increased to 7.3 when the concentrations of IGFBP3 were included in the analysis. Likewise, the RR of prostate cancer in men (evaluated in the Physician’s Health Study) in the upper quartile of IGF-1 values was 2.4 (4.3 when normalized for IGFBP3) [10]. In addition to hormone-dependent prostate and breast carcinomas, the importance of IGF-1 as a risk factor was evaluated in various nonhormonedependent types of cancers. Analyses of colon cancer risk in the Nurse’s Health Study and the Physician’s Health Study showed an increased risk in individuals with the highest IGF-1 values (although a lower RR was observed) [24, 46]. Several mechanisms have been suggested to explain the possible role of the IGF system in the initiation and/or progression of neoplasia [33]. Although IGF-1 was shown to increase chromosomal fragility under experimental conditions [11], it is usually considered to be nongenotoxic. The proliferative actions of IGF-1, however, have been well characterized. Once a malignant transformation has occurred, cell survival in transformed cells depends on IGF-1 action [5]. The disruption of internal checks and control mechanisms associated with the neoplastic phenotype is further emphasized by the finding that IGF-1 action can override the cellular signals of apoptosis [4]. An encouraging observation for a possible role of the IGF system not only in screening for cancer but as an interventional target is that the blockade of the IGF-1 receptor by monoclonal antibodies and specific IGF1R tyrosine kinase inhibitors reduced the proliferation of several carcinoma cell lines including colon, breast, and others [40, 42, 50]. Furthermore, when nude mice were implanted with colon cancer cells, the expression of a dominant-negative truncated IGF1R resulted in reduced tumor progression [59].

Neuroprotection IGF-1 is widely expressed in the CNS where it promotes proliferation, survival, and differentiation of neuronal and nonneuronal cells [23]. IGF-1 is a potent trophic factor for motor and sensory neurons as well as for glial cells. Furthermore, circulating IGF-1 is an important determinant of brain function, and its effects range from classic neurotrophic actions, such as housekeeping and anti-apoptotic/pro-survival effects, to modulation of blood–brain barrier permeability, neuronal excitability, myelination, and new neuron formation [19, 21]. Upregulation of IGF-1 in the CNS was observed

1390 / Chapter 193 1–7 days after various insults such as hypoxic-ischemic brain injury, brain contusion, and penetrating brain trauma, suggesting a role for IGF-1 in repair processes [38, 53, 70]. In a model of cerebral infarct, IGF-1 treatment reduced ischemia and improved neurological function. Interestingly, the administration of IGF-1 in combination with erythropoietin (a neuroprotective agent in itself) resulted in a synergistic neuroprotective effect, whereas the blockade of IGF-1 entry into the brain prevented the exercise-induced increase in neurogenesis [17]. The effect of IGF-1 is not limited to brain injury. Thus, central administration of IGF-1 has been shown to result in an antidepressant effect [35]. In conclusion, the potential therapeutic applications of IGF-1 in the treatment of common debilitating neurological conditions is showing great promise and is currently under extensive investigation.

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[51] Mohseni-Zadeh S, Binoux M. Insulin-like growth factor (IGF) binding protein-3 interacts with the type 1 IGF receptor, reducing the affinity of the receptor for its ligand: an alternative mechanism in the regulation of IGF action. Endocrinology 1997; 138:5645–8. [52] Moschos SJ, Mantzoros CS. The role of the IGF system in cancer: from basic to clinical studies and clinical applications. Oncology 2002; 63:317–32. [53] Nordqvist AC, Holmin S, Nilsson M, Mathiesen T, Schalling M. MK-801 inhibits the cortical increase in IGF-1, IGFBP-2 and IGFBP-4 expression following trauma. Neuroreport 1997; 8:455–60. [54] Okubo Y, Siddle K, Firth H, O’Rahilly S, Wilson LC, Willatt L, et al. Cell proliferation activities on skin fibroblasts from a short child with absence of one copy of the type 1 insulin-like growth factor receptor (IGF1R) gene and a tall child with three copies of the IGF1R gene. J Clin Endocrinol Metab 2003; 88:5981–8. [55] Pell JM, Saunders JC, Gilmour RS. Differential regulation of transcription initiation from insulin-like growth factor-I (IGF-I) leader exons and of tissue IGF-I expression in response to changed growth hormone and nutritional status in sheep. Endocrinology 1993; 132:1797–807. [56] Peoples R, Milatovich A, Francke U. Hemizygosity at the insulinlike growth factor I receptor (IGF1R) locus and growth failure in the ring chromosome 15 syndrome. Cytogenet Cell Genet 1995; 70:228–34. [57] Powell-Braxton-L, Hollingshead P, Warburton C, Dowd M, PittsMeek S, Dalton D, et al. IGF-I is required for normal embryonic growth in mice. Genes Dev 1993; 7:2609–17. [58] Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factorbinding proteins in serum and other biological fluids: regulation and functions. Endocrine Rev 1997; 18:801–31. [59] Reinmuth N, Liu W, Fan F, Jung YD, Ahmad SA, Stoeltzing O, et al. Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer. Clin Cancer Res 2002; 8:3259–69. [60] Rinderknecht E, Humbel RE. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 1978; 253:2769–76. [61] Rinderknecht E, Humbel RE. Amino-terminal sequences of two polypeptides from human serum with nonsuppressible insulinlike and cell-growth-promoting activities: evidence for structural homology with insulin B chain. Proc Natl Acad Sci USA 1976; 73:4379–81. [62] Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible insulin-like and cell-growth promoting activities in human serum: isolation, chemical characterization, and some biological properties of forms I and II. Proc Natl Acad Sci USA 1976; 73:2365–9. [63] Roberts CT Jr, Lasky SR, Lowe WL Jr, Seaman WT, LeRoith D. Molecular cloning of rat insulin-like growth factor I cDNAs: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues. Mol Endocrinol 1987; 1:243–8. [64] Rosenfeld RG. Insulin-like growth factors and the basis of growth. N Engl J Med 2003; 349:2184–6. [65] Rotwein P. Two insulin-like growth factor I messenger RNAs are expressed in human liver. Proc Natl Acad Sci USA 1986; 83:77– 81. [66] Salmon WD Jr, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 1957; 49:825–36. [67] Salmon WD Jr, DuVall MR. In vitro stimulation of leucine incorporation into muscle and cartilage protein by a serum fraction

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194 Erythropoietin—A Hematopoietic Hormone with Emerging Diverse Activities SARA PRUTCHI-SAGIV, MOSHE MITTELMAN, AND DRORIT NEUMANN

it is currently among the most valuable biotechnology products on the market.

ABSTRACT Erythropoietin (Epo), the major hormone that regulates erythropoiesis, is produced mainly in the adult kidney in response to hypoxia. Recombinant human Epo (rHuEpo) is thus considered a major treatment for various types of anemias. The past decade has revealed extra-hematopoietic sites of Epo production along with an abundance of Epo receptors in various tissues and cell lines, suggesting that this hormone may have pleiotropic activities. The unexpected discovery that Epo and its receptor are expressed also in cells of the central nervous system has opened new avenues for research regarding the biological effects of Epo in the brain. This chapter reviews the classic well-known features of Epo, as well as the more novel findings on its various effects and the possible underlying mechanisms.

STRUCTURE OF THE PRECURSOR Epo mRNA/GENE The gene encoding human Epo was cloned in 1985 by two groups independently [24, 34]. Subsequently, the Epo gene was cloned from several mammalian species, including nonhuman primates, rodents, ruminants, felines, and fish [7, 63]. The results revealed a high degree of conservation of gene structure among species. The human Epo gene, which spans 2.9 kb, is a single-copy gene located on the long arm of chromosome 7. The gene consists of five exons that encode a 193-amino-acid polypeptide, of which the 27-aminoacid leader peptide is cleaved prior to secretion [34]. The protein sequence of Epo from various mammals displays 80–90% identity to the human Epo [63]. Despite the fact that the Epo protein in remote species, such as fish, displays only 32% identity to human Epo, the overall structure of the gene, as well as the hydrophilicity plots of the molecule, displays striking similarities to those of human Epo [7]. Multiple transcription-start sites have been identified in human Epo that are differentially utilized in the kidney, liver, and brain in response to anemia. The initiation of transcription occurs within 250–650 bp upstream of the coding axon, and all contain the same first axon [14, 54].

DISCOVERY The first milestones in the discovery of erythropoietin (Epo) were the pioneering experiments by Carnot and Deflandre in 1906. They demonstrated that plasma from a donor rabbit subjected to bleeding stimulus elicited reticulocytosis in a recipient rabbit. The phenomenon was attributed to a hemopoietin factor. Its production in the kidney was revealed from the observation that bilaterally nephroctomized rabbits or rats did not respond to hemorrhage with the expected increase in erythropoietic activity. The paucity of Epo necessitated several thousands of liters of human urine to obtain a pure Epo preparation (carried out in the late 1970s) [17]. Partial amino acid sequences were then deduced. These experiments led to the cloning of the human Epo gene [24, 34]. Soon, recombinant human Epo (rHuEpo) became a therapeutic agent, and Handbook of Biologically Active Peptides

DISTRIBUTION OF Epo mRNA Initially, it was maintained that Epo functions chiefly in the stimulation of the erythroid compartment and that its biosynthesis in the embryo is in the fetal liver

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1394 / Chapter 194 and in the adult it occurs predominantly in the kidney. In the fetal liver, Epo mRNA is detected primarily in hepatocytes and in nonparenchymal cells. In the adult kidney, the primary sites of Epo production are the proximal tubules of the cortex, the capsular epithelium [47, 58], and the interstitial cells surrounding the tubules [27]. Multiple Epo gene regulatory elements control cell-type-specific expression and inducibility by hypoxia, which is the fundamental physiological stimulus [14, 55]. Unexpectedly, Epo mRNA was detected in nonerythroid compartments, suggesting that this hormone is by far more pleiotropic than initially anticipated. Notably, low levels of Epo mRNA have been demonstrated in the liver, central nervous system (CNS), bone marrow, spleen, heart, lung, ovary, and testis, where it is mainly induced by hypoxic conditions [25]. In most of these organs the Epo receptor (EpoR) is expressed in cells that are in close proximity to the cells expressing the Epo protein, thus pointing to a local paracrine feedback system [39]. A striking observation is that fish (fugu) Epo mRNA is primarily located in the heart and also found in the liver and brain [7]. The brain transcript of fugu includes an alternatively spliced first axon, yet a mammalian counterpart alternative axon has not been identified so far. The shift of Epo synthesis from the heart in fugu [7] to the kidney in mammals [15] is of interest from the standpoint of evolution. It may reflect the facts that the kidney is the primary site of erythropoiesis in primitive vertebrates (e.g., fish) [64] and the bone marrow acts as the major site of erythropoiesis in mammals.

PROCESSING OF Epo Mature Epo is a glycoprotein of molecular mass 30.4 kDa. It consists of a 165-amino-acid (aa) polypeptide core, which is generated by the cleavage of the 27-amino-acid signal peptide from the 193-aa precursor [25]. Further analysis based on nuclear magnetic resonance (NMR) spectroscopy revealed that the Epo molecule consists of four α-helical bundles connected by two long cross-over loops and one short loop [5]. The polypeptide moiety of Epo is sufficient for receptor binding. The carbohydrate portion, which consists of three N-linked and one O-linked acidic oligosaccharide side chains, comprises approximately 40% of the Epo molecule. The latter is required for secretion and in vivo stability of the mature hormone [25], and it can protect the molecule from damage caused by oxygen radicals. Minor quantitative differences in the composition of the N and O glycans exist between urinary and serum Epo. The functional significance of these differences is not clear. Two different rHuEpo products have been developed, alpha and beta. These recombinant products are identical to the natural human Epo with

respect to their primary and secondary structures; however, they differ slightly in their carbohydrate structure, which facilitates the detection of rHuEpo doping in athletes [25]. The contribution of carbohydrate side chains to Epo stability promoted the development of a new hyperglycosylated rHuEpo analog (darbepoietin, aranesp), into which two new N-glycosylation sites were introduced. Although the affinity of darbepoietin-α for the Epo receptor is lower than that of the natural ligand, it has an advantage over other commercially available rHuEpo preparations, due to its threefold higher plasma half-life and its increased biological potency in vivo [25].

THE Epo RECEPTOR The murine EpoR was isolated by expression cloning [11]. It encodes a 66-kDa single-membrane-spanning molecule that is activated on stabilization of preformed homodimers by ligand binding [52]. EpoR belongs to the cytokine receptor superfamily, as based on small regions of homology in the extracellular domain. These regions include four conserved cysteines that form two disulfide bonds and the WSXWS motif near the membrane-spanning segment. Because EpoR lacks intrinsic enzymatic activity, it necessitates complex formation with other signaling partners in order to function. The Epo-R consists of an ∼230-aa extracellular ligand binding domain, a single transmembrane segment, and an ∼230-aa cytosolic domain. The proximal region of the cytosolic domain contains three conserved segments: box 1, box 2, and the region between box 1 and 2. These regions are implicated in the association of the EpoR with Janus kinase 2 ( Jak2) Tyr kinase. Jak2 is activated by transphosphorylation following receptor dimerization, and it can then phosphorylate the Tyr residues on the receptor’s cytoplasmic domain. This process creates docking sites for SH-2-containing adaptor molecules, which subsequently recruit the transcription factor signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), phospholipase-Cγ, phosphatidylinositol 3-kinase (PI3-K), and the protein tyrosine phosphatases SHP1 and SHP2 [52]. Numerous other adaptors and intermediates are involved in Epo receptor signaling, generating multiple interactions that produce a complex interplay of signaling cascades [52] (Fig. 1).

Epo BIOLOGICAL ACTIONS Erythropoiesis The main biological action of Epo in erythropoiesis has been well known for the past 40 years. However, the

Erythropoietin—A Hematopoietic Hormone with Emerging Diverse Activities / 1395

Epo

Ca2+

EpoR

Epo

PLC-γ1 PIP2 Jak2

Jak 2

Jak 2

PIP3

βCR

Jak 2

Jak 2 Ca2+

EpoR

MAPK

PI3-K

STAT 5

Mitochondria

GSK-3Β

AKT

FOXO3a Bad NFκΒ

Caspase activation Nucleous Gene transcription

DNA fragmentation APOPTOSIS

PtdSer exposed on membrane

Bcl -XL

FIGURE 1. Epo-mediated intracellular signal transduction pathways. It is proposed that signaling pathways in the neural system are activated via Epo binding to its cognate EpoR or to an EpoR-βCR complex.

underlying mechanisms have been elucidated only in the last decade. Elaborate reviews have been published regarding the role of Epo in erythropoiesis, the signal transduction pathways controlling the process, and the interaction with additional cytokines that lead to normal red cell production [6, 23, 28, 52, 59]. Oxygen tension causes the activation of hypoxiainduced factor 1 (HIF-1), thus leading to Epo production in the kidney and to its release into the plasma [62]. Epo then circulates to the bone marrow, where it binds to EpoRs on the surface of erythroid progenitor cells [3, 59]. Ligand binding to the EpoR initiates a series of second-messenger complex cascades that ultimately transmit signals to the nucleus. These signals orchestrate the complex regulation of erythroidspecific gene expression to prevent erythroid apoptosis and to support terminal proliferation and differentia-

tion [6]. Molecules that play a key role in these processes include Jak2, STAT, MAPK, phospholipase-Cγ, PI3-K, AKT1, Ras, and Ca2+ channels. The observation that in vitro and in vivo erythroid cells in the absence of Epo are very susceptible to apoptosis [9, 20, 31, 37] may indicate that Epo acts as a main trophic factor that prevents apoptosis rather than merely as a growth-promoting mediator. Two main signaling pathways activated by EpoR are responsible for preventing apoptosis: (1) the Jak2-STAT5 signal transduction pathway, which activates numerous target genes, including the apoptosis inhibitor Bcl-XL, a member of the Bcl-2 proteins and required for the survival of erythroid cells at different differentiation and maturation stages; and (2) the PI3-K pathway, which activates the protein kinase B (PKB)/AKT1 and subsequently a member of the Forkhead family (FH) of

1396 / Chapter 194 transcription factors FKHRL1, which apparently regulates the generation of Epo-dependent anti-apoptotic signals [52].

neurotransmitters such as dopamine, acetylcholine, and choline acetyltransferase [10]. The mechanisms by which Epo acts as a neuroprotectant can be summarized as follows.

Beyond Erythropoiesis

1. Decrease in glutamate toxicity. Glutamate toxicity is regarded as the main cause of ischemia-induced neuron death. This effect is accomplished by inducing a rapid, transient increase in intracellular Ca2+ concentration in neuronal cells by raising the Ca2+ influx from outside of the cells. Morever, Epo can reduce the excitotoxic effect of cortical neuron cultures treated with glutamate or glutamate receptor agonists [40]. 2. Effects on apopotosis. Epo acts by upregulation of anti-apoptotic genes or by suppression of caspases. This is similar to the case in erythroid precursor cells where, as previously mentioned, Epo activates the expression of anti-apoptotic genes such as Bcl2 and Bcl-XL [40]. In addition, Epo-increased gene expression of XIAP and c-IAP2, members of apoptosis-inhibitor genes, was demonstrated in cerebrocortical cells following pre-incubation with Epo [13]. 3. Effects on inflammation. Epo acts by reducing inflammatory processes that play a key role in many forms of brain injury. Studies on mice showed that Epo causes the reduction of astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion [39], in cases of traumatic injury, and also in the experimental model of autoimmune encephalomyelitis [2, 10]. Noteworthy, ischemia-induced production of the pro-inflammatory cytokines TNFα, IL-6, and monocyte chemoattractant protein 1 concentration was reduced after rHuEpo administration [39, 40]. Because rHuEpo did not directly affect EpoRexpressing inflammatory cells, the premise is that it might attenuate the ischemia-induced inflammation by reducing the levels of neuronal cell death. In addition, Epo prevents phagocytosis of cells that are tagged by phosphatidylserine membrane exposure induced by anoxia and NO, and it can thus precipitate a latent cellular inflammation. These processes are involved in various neurodegerative diseases such as Alzheimer’s disease and other dementias [38]. 4. Decrease in NO-mediated injury. Overactivation of glutamate receptors on neurons leads to overproduction of NO, which induces neuronal cell death after reaction with superoxide and the formation of peroxynitrite [48]. Epo may thus decrease the NO-mediated injury in the absence of direct effects on its levels [33].

The finding of additional Epo-producing organs such as the liver, uterus, heart, immune system cells, and brain has led to the discovery of additional biological functions of Epo beyond erythropoiesis, as summarized in Table 1.

Epo and EpoR in the CNS The unexpected discovery that Epo and its receptor are expressed in the brain has elicited great interest within the scientific and medical communities, due to its potential activity as a neuroprotectant agent and its possible clinical relevance in various brain pathologies. In the brain, as in other tissues, hypoxia is a major stimulus of Epo transcription mediated by the HIF-1 pathway. Other factors such as insulin [38], reactive oxygen species (ROS) [17], and certain cytokines, for example, insulin-like growth factor-1 (IGF-1), tumor necrosis factor α (TNFα), interleukin (IL)-1β, and IL-6, may also regulate Epo and EpoR expression in the brain [38]. Neurons, microglia, and astrocytes are the main sites of Epo production in the CNS. Epo and EpoR mRNA have been identified in the temporal cortex, hippocampus, internal capsule, cerebellum, amygdala, and midbrain cerebral endothelial cells, and their expression is increased after exposure to hypoxia. Moreover, EpoR is expressed in brain-derived endothelial cells and on the myelin sheath of radicular nerves in the peripheral nervous system [39, 40]. Brain-derived Epo and EpoR molecules are smaller than their peripheral counterparts due to differences in sialic acid content [2]. Notably, in vitro, brain-derived Epo is more active than serum-Epo [41]. The observation that Epo is produced by astrocytes and subsequently binds to the EpoR on neighboring neurons [30] suggests that Epo mediates paracrine and autocrine effects in neurons. Neurotrophic and neuroprotective effects of Epo were shown in vivo in response to neuronal injury caused by hypoxia [33], ischemia [2], or brain hemorrhage, as well as by a variety of stressors such as glutamate, nitric oxide (NO) [33], and glucose deprivation [53]. These were shown when Epo was administered directly into sections of the brain or systemically by crossing the blood–brain barrier [2]. In vitro studies in neural cell lines and primary cultures have demonstrated that Epo stimulates neuronal function and viability by activating calcium channels and the release of

Erythropoietin—A Hematopoietic Hormone with Emerging Diverse Activities / 1397 TABLE 1. Summary of the Biological Actions of Epo. Organ/Cell Type Erythroid progenitors

CNS Neurons

Glial cells

CNS vasculature Spinal cord

Endothelial cells

Megakaryocytes Immune system

Fetal liver Heart Vascular smooth muscle Cardiomyocytes Uterus

Epo Action Inhibition of apoptosis Support of final stages of erythropoiesis in fetal liver, bone marrow, and spleen

Sources [52] [2, 19, 33, 38–40]

In vitro: Neurotrophic effects Increase in choline acetyltransferase activity in primary cultured neurons Regeneration of cholinergic neurons Enhanced survival and dopaminergic differentiation of neural precursors Neurogenesis after hypoxia Regulation of calcium flux in neural cell lines Elevation of intracellular concentration of monoamines Stimulation of neurotransmitters release Survival promotion by the activation of anti-apoptotic genes (Bcl-2, Bcl-XL, XIAP, c-IAP2) Decrease of DNA fragmentation and membrane PtdSer exposure Decrease of glutamate toxicity Decrease of NO-mediated injury In vivo: Neuronal survival and recovery Improvement in neuronal functional activity Neuroprotection Maturation and differentiation of oligodendrocytes and proliferation of astrocytes Survival Reduction of astrocyte activation Reduction of the release of pro-inflammatory and neurotoxic factors Decrease in BBB permeability and integrity maintenance Angiogenesis Decreased motor neuron apoptosis and inflammation Improvement in functional recovery Improvement in blood flow to ischemic cells Mitotic and chemotactic effect Survival Angiogenesis Increased endothelin 1 production Promotion of megakaryocytes colony formation Maturation Normalization of CD4:CD8 T cell ratio Increased antibody production in response to antigen Decreased pro-inflammatory cytokines (IL-6, TNFα, MP1) Polymorphonuclear cells (PMN) survival Decreased SO production from PMN Anti-inflammatory effects in CNS models Epo produced by liver is essential for fetal erythropoiesis Mitogenesis and protection of cardiomyocytes Improved myocardial energy preservation and function Rise of cytosolic Ca2+ concentration Increased response to norepinephrine, angiotensin II, endothelin-1 Mitogenic effects Enhanced proliferation (neonatal) Stimulation of Na+, K+ activity Promotion of uterine angiogenesis during the estrous cycle

[2, 19, 29, 33, 38–40, 43, 51, 60] [8] [29, 43, 51, 60]

[46] [56]

[66]

1398 / Chapter 194 5. Effects on angiogenesis. Epo leads to increased angiogenesis, thus enabling additional transport of erythrocytes and thereby increasing the amount of oxygen delivered to the hypoxic brain [40]. The signal transduction pathways by which Epo protects neuronal cells from apoptosis have not yet been fully elucidated; however, it has been shown that the signal transduction pathways leading to CNS cell protection involve Jak2, MAPK, and PI3-K. Phosphorylation of AKT1 through the PI3-K pathways is central to the ability of Epo to prevent several pathways of cell death. It includes those pathways that involve glycogen synthase kinase 3β, caspases 9, and the Bcl-2 antagonist of cell death (Bad). Forkhead transcription factor FOXO3a thus retains neuronal nuclear DNA integrity through the downstream modulation of mitochrondrial membrane potential; cytochrome c release; and caspase 1-, 3-, and 8-like activities. Epo can significantly enhance the activity of AKT1 during oxidative stress and prevent inflammatory activation of microglia. This pathway also leads to phosphorylation of the inhibitor of NF-κB (IκB), subsequent nuclear translocation of the transcription factor NF-κB, and NF-κB-dependent transcription of neuroprotective genes. As in erythroid cells, Epo upregulates Bcl-XL mRNA and protein in cultured cortical neurons [2, 19, 39]. Recently, an alternative EpoR signaling pathway has been postulated [2]. This was based on the generation of Epo derivatives (e.g., carbamylated Epo; CEpo) that bind to the EpoR and display a tissueprotective effect but not an erythropoietic one [2]. The premise is that CEpo-mediated tissue protection in the CNS and in myocardial tissue is carried out through a heteroreceptor complex comprising an EpoR monomer and a dimer of the common β receptor (βcR) subunit (CD131), which is the signal-transducing element common to the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 receptors. βcR provides increased ligand-binding affinity to the receptor complex [2]. Figure 1 illustrates these possible functions of Epo. Epo also plays a role in neural progenitor cell survival and neuronal cell generation in the developing brain [26, 67]. The lack of EpoR signaling affects brain development as early as E10.5, resulting in a reduction of neural progenitor cells and increased apoptosis and in a marked reduction in the survival of neuronal cell cultures exposed to hypoxia [67].

PATHOPHYSIOLOGICAL IMPLICATIONS Abnormal Serum Levels of Epo Low serum levels of Epo are typical in patients with end-stage renal failure. Indeed, such patients suffer

from anemia, and they respond to injections of rHuEpo [16]. This treatment results in increased hemoglobin, hematocrit, and red blood cells, as well as the reduction of blood-transfusion requirements, and on the whole an improved quality of life. Other clinical situations of interest include conditions such as the anemia associated with chronic disease, found in various chronic infections and especially in malignancies. In these cases, the level of serum Epo— albeit higher than normal—is still inadequate, or rather low relative to the value predicted due to the anemia [42]. These patients, suffering from symptomatic anemia with relatively low serum Epo levels (in contrast to the true Epo deficiency found in end-stage renal disease) also respond to the treatment with rHuEpo. Epo treatment leads to a significant improvement of the anemia and the quality of life in about 60% of these patients [4, 35, 44, 57]. High serum levels of Epo are commonly detected in patients with chronic hypoxia [50], often associated with chronic lung diseases. High serum levels of Epo have also been documented in rare cases such as renal cell cancer [21], hepatoblastoma [61], and polycystic kidney disease [21]. The high serum Epo levels are associated with polycythemia (erythrocytosis), resulting in the related clinical implications. Recently, the exogenous use of rHuEpo injections (doping) has become popular among athletes, especially bike riders [25], due to the higher hemoglobin and hematocrit levels that improve their physical performance. Unfortunately, young athletes are not always aware of the iatrogenic risks of such treatment.

Epo and EpoR in Experimental and Clinical States Once the Epo and EpoR genes and proteins have been characterized and cloned, attempts were made to identify related abnormalities that could explain certain clinical disorders. One possibility to be considered is an abnormal protein structure of Epo. However, this has not been reported. A more complex picture emerges regarding the involvement of EpoR in disease states. An EpoR in which Arg at position 129 of the extracellular domain is replaced with a Cys residue resulted in a constitutively active EpoR that could elicit erythroleukemia in a mouse model [36]. Also, an EpoR that had a C-terminus truncation was more sensitive to low concentrations of Epo [65]. It is noteworthy that a familial type of polycythemia was found to be related to a 3′ deletion of a negative control region in the EpoR gene [12]. Interestingly, some members of the affected family were actually ski champions [12].

Erythropoietin—A Hematopoietic Hormone with Emerging Diverse Activities / 1399

Clinical Uses of rHuEpo—Perspectives Cloning of the Epo gene has led to the introduction of rHuEpo into clinical practice in various anemias, including cases that are associated with kidney disease and cancer [35, 44, 49]. Treatment with rHuEpo has improved the quality of life in anemic, cancer, and hemodialysis patients. Apart from alleviating anemia, Epo might have additional effects such as antineoplastic [43, 45], neuroprotective, and immunomodulatory [18]. Finally, it should be noted that some concerns have been raised recently about possible adverse effects of Epo in clinical practice. Hence, studies on the effects of Epo in cases of head and neck [22] and breast cancer [32] have suggested deleterious effects of the treatment. This was in contrast with other reports, suggesting not only that Epo is safe but also that it might have a positive effect on the prognosis and survival [1, 35, 44]. The detection of EpoRs on tumor cell lines and cancers [30] points to the need for further critical studies in that respect. Given these concerns, on one hand, and the beneficial effects of Epo, on the other, emphasis should be directed to devising methods that will enable identifying the cases in which Epo might have adverse effects and distinguishing them from those in which rHuEpo treatment will be beneficial.

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[51]

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are also produced from the NRG-2 gene [51, 65]. The most important portion of the NRG molecule is the epidermal growth factor (EGF)-like domain shared by all the isoforms because it alone is sufficient for receptor binding and the stimulation of intracellular signaling pathways; hence, the EGF domain is both necessary and sufficient for biological activity. This domain contains approximately 50 amino acids and is characterized by three pairs of cysteines that are important for its tertiary structure and biological function. The alternate splice variant at this domain gives rise to α and β isoforms [3, 6]. Three types of NRG-1 (type I, II, and III) are also divided based on differences of the extracellular domains; types I and II have an immunoglobulin-like (IgG-like) domain followed by either a glycosylation-rich region (type I) or a GGF-specific (kringle) domain (type II), whereas type III has a cysteine-rich domain (CRD). In addition, all three types of NRG-1 have both transmembrane forms and secreted forms depending on whether the isoform is initially synthesized as a transmembrane or nonmembrane protein [3, 17].

ABSTRACT Neuregulins are pleiotrophic growth factors that influence cell survival, proliferation, differentiation and organogenesis throughout the body. Their effects are mediated via interactions with the ErbB family of transmembrane receptor protein tyrosine kinases and the subsequent activation of downstream intracellular signaling events. This chapter focuses on the expression and emerging neurotrophic, neuroprotective, and neuromodulatory roles of neuregulins in the central nervous system.

DISCOVERY AND STRUCTURE Neuregulins (NRGs) are a family of structurally related signaling proteins that bind to receptor tyrosine kinases of the ErbB family and mediate a myriad of cellular functions, including survival, proliferation, and differentiation in both neuronal and nonneuronal systems. Discovered independently over a decade ago by several different groups, these peptide growth factors were originally described as neu differentiation factor (NDF), heregulins, glial growth factors (GGFs), acetylcholine receptor–inducing activity (ARIA), and sensory and motor neuron-derived factor (SMDF) (for review, see [17]). It is now known that all these NRG proteins are encoded by the same gene, named NRG-1. Four genes (termed NRG-1, NRG-2, NRG-3, and NRG-4) have been identified that encode NRGs in vertebrates, and the best characterized of these is the NRG-1 gene [1, 3, 18]. Recently, the entire human NRG-1 gene was sequenced [56]. At least 15 isoforms are generated by multiple promotor use and alternative splicing of the NRG-1 gene, including the NDFs [48, 63], heregulins [31], GGFs [25, 42], ARIA [18], and SMDF [27]. Several isoforms Handbook of Biologically Active Peptides

DISTRIBUTION AND PROCESSING Neuregulin-1 transcripts are broadly but discretely expressed throughout the nervous system during development and in adulthood [8, 10, 35, 37, 44], with highest levels of expression in brainstem motor and sensory nuclei, spinal cord motor neurons, and sensory ganglia. Neuregulin-1 mRNAs are also found in the cerebral cortex, diencephalon, hippocampus, basal forebrain, substantia nigra and cerebellum, and the proliferative forebrain subventricular zone. Neuregulin-2 and -3 mRNAs are also expressed in the brain, but exhibit distinct expression patterns from NRG-1, with NRG-2 primarily restricted to the olfactory bulb, cerebellum,

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1402 / Chapter 195 and hippocampal dentate gyrus in the adult, whereas NRG-3 expression appears widespread [41]. Both NRG1 and NRG-2 mRNAs, as opposed to NRG-3 mRNA, are developmentally regulated, with the highest levels of expression found neonatally. Neuregulin-4 transcripts have not been detected in the central nervous system [26]. For the most part, immunohistochemical demonstrations of NRG-1 and NRG-2 proteins correspond fairly well with their mRNA localization in both the rodent and human brain [35, 37, 41]. The proteolytic processing of NRG proproteins has recently been reviewed [17]. Briefly, most (but not all) NRGs in the nervous system are initially synthesized as single transmembrane proproteins with an extracellular N-terminal ectodomain (containing the biologically active EGF domain), a juxtamembrane stalk region, the transmembrane domain, and the intracellular cytoplasmic tail. A bioactive ectodomain fragment is generated and released via the metalloprotease-mediated cleavage of the proprotein in the stalk region. Other NRGs are produced directly as soluble secreted proteins (e.g., GGF2). The type III NRG proproteins have two transmembrane domains, including an N-terminal region that loops back through the membrane resulting in an intracellular N-terminus, in addition to the intracellular C-terminus. Proteolytic cleavage at the stalk region results in an anchored N-terminal fragment (with the bioactive EGF-like region); further juxtamembrane proteolytic processing may release the fragment [17]. Another mode of NRG processing recently described is back-signaling, in which, on NRG type III/ErbB binding, the intracellular C-terminal domain is proteolytically released into the cytoplasm where it subsequently travels to the nucleus and modulates gene expression [2]. The ultimate result of these various avenues of NRG isoform processing is that NRGs garner the capacity to signal via autocrine, paracrine, juxtacrine, and possibly intracrine mechanisms.

RECEPTORS Neuregulins interact directly or indirectly with members of a subfamily of receptor tyrosine kinase proteins called ErbB receptors. The four members of the ErbB family include the EGF receptor (also known as ErbB1; the human homolog is human EGF receptor 1, HER1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/ HER4. All family members have in common an extracellular ligand-binding N-terminal domain, a single transmembrane portion, a cytoplasmic tyrosine kinase domain, and a cytoplasmic C-terminal domain containing autophosphorylation sites. Upon ligand binding, the receptors form homo- or heterodimers, which consequently stimulate the intrinsic tyrosine kinase activity

of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain (for recent reviews see [5, 9, 66]). These phosphorylated residues serve as docking sites for signaling molecules involved in the regulation of intracellular signaling cascades, mainly through the stimulation of the Ras-Raf-ERK mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3 kinase (PI3-K) pathway, the PLCγ-PKC pathway, and the JAKSTAT pathway [9]. An alternative signaling mechanism for ErbB4 upon ligand binding includes presenilindependent intramembrane proteolysis by γ-secretase to release an intracellular ErbB4 C-terminal fragment that translocates to the nucleus to regulate gene expression [38, 45]. The ErbB receptors form a complex hierarchical network based on their relative affinity for the ligands, intrinsic tyrosine kinase activities, ability to recruit specific signaling molecules, and cellular distribution [61]. This network is critical to the ErbB signaling pathway because ErbB2, which exhibits the highest degree of kinase activity, has no known direct ligands and ErbB3 is kinase-defective; therefore, neither of them can function by homodimerization. However, by heterodimerizing with other ErbB receptors, they can form potent signaling assemblies (i.e., ErbB2/ErbB3, ErbB3/ErbB4, etc.) and ErbB2 has been demonstrated to be the most potent partner to any ErbB family member. ErbB4 itself contains moderate kinase activity; therefore, its presence alone is sufficient for biological activity, and it can function via both homo- and heterodimerization. Neuregulins can directly bind to only the ErbB3 and ErbB4 receptors, which can then heterodimerize with ErbB2 (an orphan receptor) and ErbB1 to activate and/or potentiate subsequent signaling activity. All four NRG gene products can bind ErbB4, whereas only NRG-1 and NRG-2 proteins can bind ErbB3 [5, 9, 66]. ErbB4 is also noteworthy for the fact that multiple isoforms of the receptor may exist based on sequence alterations in the extracellular stalk region and in the cytoplasmic C-terminal domain; the latter variation can influence PI3-K function [33]. Interestingly, some members of the EGF ligand family, including betacellulin, epiregulin, and heparin-binding EGF-like growth factor (HB-EGF), can bind and activate the ErbB4 receptor [5, 30]. Thus, not only are there complex interactions among the ErbB receptor family members after NRG binding, but the possibility of receptor crossactivation with select EGF family ligands must be taken into consideration. ErbB receptor mRNAs are differentially expressed throughout the nervous system during development, both spatially and temporally, and are continuously expressed throughout adulthood. ErbB2, ErbB3, and ErbB4 transcripts and protein are broadly expressed

Neuregulins / 1403 throughout all areas of the adult rat brain and spinal cord, including the cortex, amygdala, hippocampus, medial habenula, reticular thalamic nucleus, substantia nigra pars compacta, ventral tegmental area, cerebellum, pons, and medulla [8, 10, 19, 23, 36, 44, 49, 57]. These three receptors are differentially expressed in neurons and glia. In general, ErbB4 is more prevalent in neurons and ErbB3 in glia in white matter tracts, whereas ErbB2 is present in both neural cell types at lower density. Notably, ErbB4 expression defines a large subset of GABAergic interneurons in forebrain regions [19, 55] and dopaminergic neurons in the ventral midbrain [57, 67, 69], suggesting some specific neurotransmitter-related functions for NRG-ErbB4 signaling in the brain. Moreover, the widespread expression of ErbB receptors in the adult brain points to numerous roles for NRG in the mature nervous system.

NRG-2 versus NRG-1 The overall domain structure of NRG-2 gene products resembles NRG-1 proteins (such as GGF2) and includes EGF-like, Ig, transmembrane, and cytoplasmic sequences. As in NRG-1, there appear to be two versions (α and β) of the EGF domain, which have distinct activities. Recent evidence, however, suggests important distinctions between NRG-1 and NRG-2, leading to the possibility of differing functions on central nervous system (CNS) neurons (see [59] for review). Studies have shown that coupling to downstream signaling molecules, including differential phosphorylation of select tyrosine residues on ErbB4, differs among various NRG1, -2, -3, and -4 ligands [28, 29, 58]. Differential signaling and bioactivities of NRG-1 versus NRG-2 ligands have been observed in various cell lines [12, 50]. Two consistent findings from these studies were that NRG-2β was more potent than other NRG-2 and NRG-1 isoforms and that some specific NRG receptor (ErbB1-4) combinations appeared to be more responsive than others to isoforms of NRG-2. For example, cells expressing ErbB4 or the ErbB4-ErbB1 combination showed a particularly robust response to NRG-2 proteins. These results could shed light on potential cellular targets for NRG-2 throughout the CNS. Within the rodent CNS, intriguing differences between NRG-1 and NRG-2 have recently been identified [41]. For example, with respect to mRNA expression, the pattern of NRG-2 is essentially distinct from and sometimes complementary to sites mapped for NRG-1 expression. Thus, the products of these two different genes could have distinct functions in different brain circuits. The temporal patterns of expression in the brain also differ between the two genes. NRG-1 mRNA expression is relatively high perinatally, followed by downregulation during maturation into adulthood.

In contrast, NRG-2 mRNA expression in the brain is the opposite, low prenatally and high in adulthood [41]. It thus has been hypothesized that NRG-1 might function preferentially in early prenatal and perinatal developmental events, whereas NRG-2 may be the preferred NRG ligand in the mature brain [59]. Finally, the subcellular distribution of the two NRGs differ in that NRG-2 is targeted to dendrites, whereas NRG-1 is mainly localized to axonal and somatic compartments [41], suggesting different functional roles for the ligands in the establishment, maintenance, and plasticity of synaptic connections. Taken together, the specific and distinct molecular and biochemical interactions of the two NRG ligands with their receptors, as well as their differential spatial, temporal, and subcellular expression patterns in the brain, point to some unique aspects of NRG-2 versus NRG-1 neural signaling.

BIOLOGICAL ACTIONS First recognized as glial cell mitogens, the NRGs are now believed to play essential roles in cell survival, migration, and differentiation in both neural and nonneural cells. Outside the nervous system, NRGs are essential, for example, for proper development of the heart, mammary gland, and skin. In terms of dysfunction, NRG-ErbB signaling is associated with many different cancers, including those of the breast, lung, bladder, ovary, and brain [5, 9, 17, 30]. The remainder of this review focuses on the emerging neuroactive functions of NRGs and their receptors in the nervous system, and particularly in the CNS. The most well-characterized functions of NRGs associated with the nervous system include roles in glial cell proliferation, maturation, and survival; neuromuscular acetylcholine receptor induction; and early neuronal migration and survival (see [1, 3, 17, 43] for reviews). Moreover, NRGs may have important neurotrophic functions in the brain in addition to their well-known actions in the peripheral nervous system [1, 3]. The analysis of mutant (knockout) mice deficient in either NRG-1, ErbB2, ErbB3, or ErbB4 reveals a severe reduction in several neural crest-derived cell populations, including Schwann cells, neural crest-derived cranial sensory neurons, and sympathetic neurons, and demonstrate critical roles for the receptors and ligand in aspects of hindbrain development (e.g., [15, 24, 53]). Mice deficient in heregulin (heterozygous mutants) exhibited hyperactivity in multiple behavioral tasks, suggesting abnormalities in motor performance [21]. The findings in cerebellar slices and cultures that NRG increases the expression of an NMDA receptor subunit (NR2C) and the GABAA receptor β2 subunit demonstrate that NRGs can regulate neurotransmitter

1404 / Chapter 195 receptors in the brain, as well as at the neuromuscular junction [46, 47, 52]. A trophic function is also suggested by the demonstration that NRGs enhance neurite outgrowth from cultured cerebellar granule cells [52], hippocampal neurons [22], and PC12 cells [62]. The potential role of NRGs and their receptors in activitydependent neuronal mechanisms is underscored by the demonstration that several forms of activity (seizures; forced locomotion; long-term potentiation, LTP) induce an upregulation of NRG and ErbB4 expression in hippocampal, neocortical, and amygdaloid neurons [14]. In hippocampal slices, NRG inhibited the induction of LTP [32]. In hippocampal cultures, NRGs increased the α7 subunit of nicotinic acetylcholine receptors and enhanced excitory synaptic transmission in GABAergic interneurons [39]. Finally, in the adult rat in vivo, NRGs have recently been found to differentially modulate synaptic transmission in two hippocampal circuits [54]. These results, combined with results showing that ErbB4 is enriched in brain postsynaptic densities and interacts functionally with PSD-95 in hippocampal synapses [20, 31], suggest a possible modulatory role for neuregulins in adult brain synaptic plasticity. The proliferation of neuronal progenitor cells in the brain is also regulated by NRGs [40]. Taken together, these findings provide a strong basis to postulate neurotrophic and neuromodulatory functions for NRGs and their receptors in brain neuronal systems.

Neuregulins Are Neurotrophic for Nigrostriatal Dopaminergic Neurons ErbB4 mRNA is expressed within a substantial population of dopaminergic neurons within the substantia nigra and ventral tegmental area of both rodent and monkey [23, 57, 67] (K. B. Seroogy, unpublished). These results suggest that ventral mesencephalic neurons could be responsive to the trophic effects of NRGs. Indeed, as determined by in vivo microdialysis, a single supranigral injection of the NRGs heregulin-β1 or GGF2 induced a substantial increase in dopamine overflow in the ipsilateral striatum [68]. This was among the first in vivo demonstrations of NRG actions in the brain and provided initial evidence that NRGs can augment dopaminergic nigrostriatal function. Consistant with these findings, recent in vitro results indicate substantial neurotrophic and neuroprotective roles for GGF2 in primary cell cultures derived from fetal rat mesencephalon [69]. The effects of GGF2 were tested on developing dopaminergic neurons in the midbrain cultures under both serum-free and 6OHDA-challenged conditions. It was found that GGF2 significantly promoted dopaminergic neuron survival, increased dopamine uptake, and enhanced dopaminergic fiber outgrowth under both conditions compared

with controls. Glial cells are absent or extremely rare in these serum-free midbrain cultures, suggesting that the neurotrophic effects of GGF2 on the dopaminergic cells are direct, and not mediated via glia. Notably, in separate preliminary experiments in midbrain cultures, the NRG-2 protein NRG-2β also enhanced dopamine neuron survival and process outgrowth, and protected the dopamine cells against 6-OHDA-induced degeneration (D. M. Yurek and K. B. Seroogy, unpublished).

Neuregulins and Neurological Disorders Increasing evidence of roles for NRG-ErbB signaling in the etiology and treatment of several neurological disorders is rapidly accumulating. In a chronic relapsing experimental model of multiple sclerosis, administration of GGF2 resulted in significant beneficial effects, including a reduction in inflammation and demyelination and enhanced remyelination [4]. Supported by the well-known trophic effects of NRGs on oligodendrocytes, and the suppression of activated microglial responses by GGF2 [13], these findings raise the possibility of NRGs as a treatment for demyelinating disorders. In animal models of stroke, both NRG and ErbB receptors are upregulated in the peri-infarct region in both neurons and macrophages/microglia. Treatment with NRG-1 in these paradigms reduced infarct volume, blocked secondary neuronal degeneration and proinflammatory responses, and improved functional outcome, indicating a neuroprotective role for NRGs in ischemic brain injury [64]. A potential role for NRGs in brain trauma is suggested by the upregulation of NRG ligands and receptors in neurons, astrocytes, and microglia in animal models of experimental brain injury [16, 60]. Similarly, the expression of NRG-1 within neuritic plaques and of both NRG-1 and ErbB4 within reactive astrocytes and microglia surrounding the neuritic plaques in human Alzheimer’s disease brain raises the possibility of NRG-ErbB mechanisms in this debilitating neurodegenerative disorder [7]. Much excitement has been generated within the realm of psychiatric disease with the identification of the NRG-1 gene as a probable susceptibility gene for schizophrenia [56]. The intriguing possibility that defects in NRG/ErbB signaling contribute to the molecular or cellular basis of schizophrenia has been recently reviewed [11]. Finally, the recent demonstration that NRG-1 can cross the blood– brain barrier in a receptor-mediated manner [34] holds promise in terms of the potential therapeutic delivery of NRGs for the treatment of CNS disorders.

Acknowledgments Supported by the National Institutes of Health (NS39128) and the Department of Defense (DAMD17-

Neuregulins / 1405 02-1-0174). We thank Kerstin Lundgren for invaluable assistance.

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synaptic transmission in GABAergic interneurons of the hippocampus. J Neurosci 2001;21:5660–9. Liu Y, Ford B, Mann MA, Fischbach GD. Neuregulin-1 increases the proliferation of neuronal progenitors from embryonic neural stem cells. Dev Biol 2005;283:437–45. Longart M, Liu Y, Karavanova I, Buonanno A. Neuregulin-2 is developmentally regulated and targeted to dendrites of central neurons. J Comp Neurol 2004;472:156–72. Marchionni MA, Goodearl AD, Chen MS, BerminghamMcDonogh O, Kirk C, Hendricks M et al. Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature 1993;362:312–8. Marchionni MA, Grinspan JB, Canoll PD, Mahanthappa NK, Salzer JL, Scherer SS. Neuregulins as potential neuroprotective agents. Ann NY Acad Sci 1997;825:348–65. Meyer D, Birchmeier C. Distinct isoforms of neuregulin are expressed in mesenchymal and neuronal cells during mouse development. Proc Natl Acad Sci USA 1994;91:1064–68. Ni CY, Murphy MP, Golde TE, Carpenter G. γ-Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science 2001;294:2179–81. Ozaki M, Sasner M, Yano R, Lu HS, Buonanno A. Neuregulin-β induces expression of an NMDA-receptor subunit. Nature 1997; 390:691–4. Ozaki M, Tohyama K, Kishida H, Buonanno A, Yano R, Hashikawa T. Roles of neuregulin in synaptogenesis between mossy fibers and cerebellar granule cells. J Neurosc Res 2000; 59:612–23. Peles E, Bacus SS, Koski RA, Lu HS, Wen D, Ogden SG et al. Isolation of the neu/HER-2 stimulatory ligand: A 44 kD glycoprotein that induces differentiation of mammary tumor cells. Cell 1992;69:205–16. Pinkas-Kramarski R, Eilam R, Alroy I, Levkowitz G, Lonai P, Yarden Y. Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation. Oncogene 1997:15:2803–15. Pinkas-Kramarski R, Shelly M, Guarino BC, Wang LM, Lyass L, Alroy I et al. ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network. Mol Cell Biol 1998;18:6090–101. Ponomareva ON, Ma H, Dakour R, Raabe TD, Lai C, Rimer M. Stimulation of acetylcholine receptor transcription by neuregulin-2 requires an N-box response element and is regulated by alternative splicing. Neuroscience 2005;134:495–503. Rieff HI, Raetzman LT, Sapp DW, Yeh HH, Siegel RE, Corfas G. Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells. J Neurosci 1999; 19:10757–66. Riethmacher D, Sonnenberg-Riethmacher E, Brinkmann V, Yamaai T, Lewin GR, Birchmieir C. Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 1997; 389:725–30. Roysommuti S, Carroll SL, Wyss JM. Neuregulin-1β modulates in vivo entorhinal-hippocampal synaptic transmission in adult rats. Neuroscience 2003;121:779–85.

[55] Seroogy KB, Herman JP, Lundgren KH. ErbB4 neuregulin receptor and GAD mRNAs are extensively colocalized in forebrain, but not ventral midbrain, GABAergic neurons. Soc Neurosci Abst 2004; #38.5. [56] Stefansson H, Sigurdsson E, Steinthorsdottir V, Bjornsdottir S, Sigmundsson T, Ghosh S et al. Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet 2002;71:877–92. [57] Steiner H, Blum M, Kitai ST, Fedi P. Differential expression of ErbB3 and ErbB4 neuregulin receptors in dopamine neurons and forebrain areas of the adult rat. Exp Neurol 1999;159: 494–503. [58] Sweeney C, Lai C, Riese DJ II, Diamonti J, Cantley LC, Carraway KL III. Ligand discrimination in signaling through an ErbB4 receptor homodimer. J Biol Chem 2000;275: 19803–7. [59] Talmage DA, Role LW. Multiple personalities of neuregulin gene family members. J Comp Neurol 2004;472:134–9. [60] Tokita Y, Keino H, Matsui F, Aono S, Ishiguro H, Higashiyama S et al. Regulation of neuregulin expression in the injured rat brain and cultured astrocytes. J Neurosci 2001;21:1257– 64. [61] Tzahar E, Waterman H, Chen X, Levkowitz G, Karunagaran D, Lavi S et al. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 1996;16:5276–87. [62] Vaskovsky A, Lupowitz Z, Erlich S, Pinkas-Kramarski R. ErbB-4 activation promotes neurite outgrowth in PC12 cells. J Neurochem 2000;74:979–87. [63] Wen D, Peles E, Cupples R, Suggs SV, Bacus SS, Luo Y et al. Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 1992;69:559–72. [64] Xu Z, Croslan DR, Harris AE, Ford GD, Ford BD. Extended therapeutic window and functional recovery after intraarterial administration of neuregulin-1 after focal ischemic stroke. J Cereb Blood Flow Metab 2006;26:527–35. [65] Yamada K, Ichino N, Nishii K, Sawada H, Higashiyama S, Ishiguro H et al. Characterization of the human NTAK gene structure and distribution of the isoforms for rat NTAK mRNA. Gene 2000;255:15–24. [66] Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–37. [67] Yurek DM, Seroogy KB. Neurotrophic factor protection of dopaminergic neurons. In: Mochetti I, editor. Neurobiology of the neurotrophins, New York: Sulburger & Graham Publishing; 2001, p 355–97. [68] Yurek DM, Zhang L, Fletcher-Turner A, Seroogy KB. Supranigral injection of neuregulin1-β induces striatal dopamine overflow. Brain Res 2004;1028:116–9. [69] Zhang L, Fletcher-Turner A, Lundgren KH, Marchionni M, Yurek DM, Seroogy KB. Neurotrophic and neuroprotective effects of the neuregulin glial growth factor-2 on dopaminergic neurons in rat primary midbrain cultures. J Neurochem 2004;91:1358–68.

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are in competition for limited supplies of trophic factors made by the target organ; those that make appropriate connections and obtain growth factors live, and the ones that do not die. Following the discovery of NGF and its physiological roles in the central and peripheral nervous systems, other neuron survival-promoting neurotrophic factors were cloned and identified, including brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5).

ABSTRACT The neurotrophins are homodimeric polypeptide growth factors that regulate the development and functioning of neurons and other cell types. Nerve growth factor (NGF) is the prototype. It is synthesized as a precursor pro-NGF that is further processed to a mature polypeptide, and each form has distinct activities. On binding to its receptors—the common p75 receptor (tumor necrosis factor, TNF, superfamily member) and a selective Trk tyrosine kinase receptor—the neurotrophins activate signaling that regulates cell growth, survival, and differentiation and regulates synaptic strength and plasticity in the adult nervous system. Neurotrophins and their receptors are implicated in human neurodegenerative diseases, pain, inflammation, and certain types of cancer, making these proteins targets for treatment.

STRUCTURE OF THE mRNA/GENE The human NGF gene is located on the short arm of chromosome 1 (1p22) with the human β NGF gene being highly homologous to mouse NGF gene. The NGF gene is more than 43 kbp and contains four exons: exons IA and B, exon II, exons IIIA and B, and exon IV. Part of exons IA, II, IIIB, and IV code for NGF precursor. Exon IV codes for all mature NGF (118 amino acids) and an additional 125 amino acids in the amino terminus [13]. Exon IIIA is noncoding (Fig. 1). There are four different NGF transcripts, with transcripts A and B being the most abundant and differing at the alternative splicing at exon II. There is a characteristic expression pattern for these transcripts, with B being more abundant than A in all the tissues except for the submaxillary gland and placenta [14]. Therefore, the NGF gene is regulated by transcription initiation and RNA processing. There are several sets of basic residues in the NGF precursor, and they correspond to cleavage sites that, once processed, result in the mature NGF protein.

DISCOVERY OF NERVE GROWTH FACTOR Nerve growth factor (NGF) was discovered in the 1950s by Rita Levy Montalcini and Viktor Hamburger while studying the developing nervous system and the peripheral structures it innervates. A nerve growthpromoting factor essential for the survival and maintenance of sensory and sympathetic neurons was identified on the basis of a functional assay that used a mouse sarcoma implanted on the body wall of chick embryos in proximity of the spinal cord. The sarcoma secreted a soluble factor that enabled fiber outgrowth and hypertrophy of sensory neurons from dorsal root ganglia but not motor neurons [7]. In vitro experiments by Cohen et al. led to the identification of a low-molecular-weight nerve-growth-promoting protein that was later cloned and identified as NGF [3]. From this work the neurotrophic factor hypothesis was developed, which states that developing neurons Handbook of Biologically Active Peptides

DISTRIBUTION OF THE mRNA Distribution of NGF mRNA was determined by in situ hybridization and Northern blot analysis in

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NGF 142 bp

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FIGURE 1. Structure of the NGF gene. Introns are shown as a line, and exons as a box. The size of exons and introns in base pairs (bp) is shown. There are three ATG start codons in different exons: exon IA, II, and IV. Black box represents exon IV coding for mature NGF. The structure of pre-proNGF precursor is shown underneath. Data summarized from [14].

different tissues. NGF mRNA is expressed by different cells of the peripheral and central nervous systems; it is expressed in the peripheral targets of sensory and sympathetic ganglion neurons. The highest levels of NGF mRNA are present in tissues densely innervated by sympathetic neurons such as heart and spleen [14]. NGF mRNA is also present in the epithelial cells, smooth muscle cells, fibroblasts, and male germ cells. In the brain, the highest levels of NGF mRNA are expressed at postnatal week 3. NGF mRNA–expressing cells were found in all areas of the hippocampus, almost exclusively in neurons.

PRECURSOR PROTEIN: PROCESSING AND ACTIVITY NGF is synthesized as precursor, 31–35 kDa preproNGF, which is processed until it is secreted as the mature homodimeric NGF of 28 kDa. The pre-mRNA sequence codes for a signal sequence that directs the synthesis of the protein at endoplasmic reticulum (ER) ribosomes. Subsequently, the protein is sequestered in the ER, with cleavage of the signal peptide resulting in pro-NGF, which then forms spontaneous non-covalently-linked pro-NGF homodimers. Further processing of NGF occurs in the ER with N-linked glycosylation in the prodomain that affords protein stability to NGF. Pro-NGF homodimers than transit to the Golgi and accumulate in the membrane stacks of the trans-Golgi network [16]. There is sulfation of N-linked oligosaccharides and cleaving of the N-terminal domain of pro-NGF to give rise to mature NGF. Pro-NGF is cleaved by furin and

proconvertases, PACE4 and PC5/6B, to yield Cterminal mature neurotrophin dimers. Furin is ubiquitously expressed and produced early in embryonic development. The intracellular cleavage of pro-NGF to produce mature NGF is at the Arg-Ser-Lys/Arg-Arg sequence [13]. From the Golgi, NGF can undergo secretion through the constitutive or secretory pathways, depending on the cell type. Both constitutive and regulated cells have the ability to process and secrete biologically active NGF, but pro-NGF can also be secreted by cells. Curiously, whereas mature NGF binds 2 receptors (TrkA and p75 = 2) and can afford neuronal survival, pro-NGF binds with a more restricted manner (p75 only) or to a different subset of receptors and signals toward death in neuronal cells, smooth muscle cells, and oligodendrocytes. Recently, it has been shown that p75 apoptosis signals are carried through the p75 neurotrophin receptor and sortilin. The pro-domain of NGF interacts with the cysteine-rich region of the sortilin extracellular domain, whereas the mature NGF domain interacts with p75 [9].

CONFORMATION NGF was sequenced 30 years ago, and its primary structure was elucidated. Mature NGF is cleaved from the prepro-NGF precursor (240–260 amino acids). It is a noncovalent homodimer with each monomer having a mass of 13 kDa, or 118–129 amino acids. NGF is a member of the cysteine knot family because of a double loop formed by two disulfide bonds, which is then penetrated by a third disulfide bond. The crystal structure of NGF was determined revealing the monomer struc-

The Neurotrophins / 1409 ture consisting of mostly β-structure domains, with seven β-strands A, A′, A″, A⵮, B, C, and D, which contribute to three antiparallel pairs of β-strands. The core of the NGF monomer is formed by a pair of twostranded, twisted β-sheets with a cysteine knot motif and a reverse turn on one hand and three β-hairpin loops at the other side. The large twist between each pair of β strands and the interface between the A′–A″ and B–C double-stranded sheets form the hydrophobic core. The cysteine knot motif and several side-chain to sidechain hydrogen bonds also contribute to the hydrophobic core and give rise to the same overall three-dimensional structure of NGF and other members of the neurotrophin family. Two NGF homodimers interact in a parallel manner through hydrophobic interactions, and this explains the high association constant for the two subunits. When comparing neurotrophin family proteins, most of the variable residues are located in the N-terminus, the three β-hairpin loops (residues 29–35, 43–48, and 92–98), and the reverse turn from residues 59–66 [8]. The other characteristic feature of the NGF dimer is that it is a basic protein with isoelectric point above 9 and clustered positive charged surface close to β-hairpin Asp-30 to Lys 34 that might be involved in binding to the acidic p75 receptor that has an isoelectric point of 4.4. Reportedly, variable residues are involved in the selective receptor binding of NGF to its selective TrkA receptor, where conserved residues between all the members of the neurotrophin family might enable the binding to common neurotrophin receptor p75. The crystal structure of the human TrkA IgC2 domain in complex with human NGF has been elucidated. The ligand-receptor interface consists of two patches: one involving the central β-sheet at the core of dimeric NGF and the loops at the TrkA-IgC2 domain C-terminal end and the other patch having contacts between the Nterminal region of NGF and the ABED sheet of TrkAIgC2 [15]. The crystal structure of the extracellular domain of p75 in complex with NGF was also determined. p75 binds along the homodimeric interface of NGF, which disables the second p75 binding site through an allosteric conformational change. Therefore, p75 receptors bind NGF in 1 : 2 stoichiometry [4]. Surprisingly, the crystal structure has shown no evidence of p75-Trk or p75-NGF-Trk heterocomplexes.

RECEPTORS—BINDING SITES Even though NGF was discovered 70 years ago, it was not until much later that its cellular receptors were identified. NGF elicits its function by binding to two families of transmembrane receptors—the selective and specific TrkA tyrosine kinase receptor and the common

(shared) p75 receptor. The first NGF binding protein that was cloned was p75; later it was discovered that it binds all members of the neurotrophin family (BDNF, NT-3, and NT-4). TrkA was initially discovered as an oncogenic fusion protein and was an orphan receptor until NGF was found to induce its fast tyrosine phosphorylation. Cells expressing TrkA alone display a small number of high-affinity NGF binding sites (10−12 M), but the majority of sites have intermediate binding affinity (10−10–10−11 M) with very slow rates of association. The p75 receptor binds all mature neurotrophins with low affinity (10−9 M) but with different kinetics. Co-expression of p75 with TrkA receptors leads to the formation of a larger number of NGF high-affinity binding sites (Kd = 10−12) and a faster association rate. The formation of high-affinity NGF binding sites depends on the ratio of the numbers of p75 and TrkA receptors on the cell surface and on the juxtamembrane and transmembrane domains of TrkA and p75. Other members of the Trk receptor family include TrkB, which binds preferentially BDNF, and TrkC, which binds preferentially NT-3. The picture is even more complex because high-affinity NT-3 binding to p75 has been reported, and p75 in complex with sortilin also creates high-affinity binding sites for pro-NGF. Indeed, pro-NGF can activate p75 signaling more efficiently than does mature NGF. The biological consequences of pro-NGF activation of p75 can be either death or survival.

TRKA AND p75 RECEPTORS—STRUCTURE All Trk receptors have an extracellular ligand-binding domain, a single-transmembrane domain and a cytoplasmic part with tyrosine kinase activity. The extracellular domain is a ligand-binding domain and it is glycosylated with 40 kDa of carbohydrate moiety. The extracellular domain has a characteristic subdomain organization. On the N-terminus, there is one cysteinerich domain (also known as D1) that is separated from the second cysteine-rich domain (also known as D3) by three consecutive leucine-rich motifs (also known as D2). The C-terminal region of the extracellular domain has two immunoglobulin (IgG)-like domains, IgC1 and IgC2 (also termed D4 and D5, respectively) (Fig. 2). Mutant-protein and domain-swapping studies identified the IgC2 domain as the ligand-binding domain; and this was confirmed by crystallography. The IgC1 domain has a role in regulating receptor dimerization and constitutive receptor activation, probably as a hinge region. The transmembrane domain linking the extracellular and intracellular domains plays an important role in the ligand activation of the receptor. There is 75% sequence identity in the tyrosine kinase catalytic

1410 / Chapter 196 NGF Cys-1 LRM Cys-2 IgC1 IgC2

Shc Shc Grb2 Sos

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Gab-1

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Protein kinase C

Ca2+ Akt

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domains of Trk family members and around a 50% sequence homology in the extracellular domains. The p75 receptor is a member of tumor necrosis factor (TNF) superfamily. Structurally, it has a ligandbinding extracellular domain, a single transmembrane domain, and an intracellular domain. The extracellular domain has four cysteine repeats (CR1–CR4) that create binding sites for all neurotrophins. The cytoplasmic domain has no catalytic activity and has a strong sequence homology to the death domain of the proteins of the TNF superfamily. Thus, p75 can signal through several adaptor proteins.

RECEPTOR SIGNALING The first step in Trk tyrosine kinase receptor activation is a ligand-induced receptor dimerization followed by the transphosphorylation of tyrosine residues in the activation loop that leads to the autophosphorylation of tyrosine residues that serve as recruitment sites for specific signaling proteins and adaptors. The binding of adaptor proteins containing phosphotyrosine binding (PTB) or Src-homology (SH-2) motifs to phosphorylated tyrosines on Trk couples the receptor to intracellular signaling pathways, including the Ras– mitogen-activating protein kinase (MAPK) pathway, phosphatidylinositol-3 kinase (PI-3 kinase)–Akt kinase pathway, and phospholipase C-γ (PLC-γ) pathway. On TrkA activation, the phosphorylation of tyrosine 490 leads to the recruitment and phosphorylation of Shc, which recruits the Grb-SOS complex via the SH-2 domain, leading to the activation of MAPK kinase family

FIGURE 2. Trk receptor structure and signaling.

extracellular signal-related kinases 1 and 2 (Erk1–2) via Ras-Raf-MEK-1 activation. On activation, Erk1–2 translocate to the nucleus where they activate transcription of genes regulated by NGF leading to cell survival. Direct activation of PI-3 kinase by Ras is a major survival-promoting pathway in most neurons. Phosphatidylinositides generated by phosphatidylinositol 3kinase (PI3-K) activate the protein kinase B (Akt) via phosphoinositide-dependent kinase 1 (PDK-1). Phosphorylation of Akt by PDK-1 on threonine 308 followed by autophosphorylation on Ser 473 activates Akt and allows it to facilitate survival by phosphorylating different substrates, including transcription factors that modulate cell survival [5]. The phosphorylation of TrkA at Y785 recruits and activates PLC-γ directly. Activated PLC-γ induces the hydrolization of phosphatidylinositide-4,5-bisphosphate to generate inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces Ca2+ release, resulting in the activation of various protein kinases and phosphatases activated by cytoplasmic Ca2+. DAG activates DAGregulated protein kinase C isoforms (PKC). PKCs are a family of intracellular serine-threonine kinases that can activate Raf and MAPK cascade, regulating cell survival or neuronal differentiation (Fig. 2).

NEUROTROPHINS—RETROGRADE TRANSPORT During development, retrograde-transported NGF promotes the survival of appropriately connected neurons. NGF bound to TrkA at the axon terminals is

The Neurotrophins / 1411 internalized into signaling endosomes and retrogradely transported to the cell bodies. Activated phospho-TrkA in signaling endosomes is linked to downstream signaling pathways that promote neuronal survival and differentiation, whereas surface NGF-bound TrkA receptors regulate axonal growth [6]. After endocytosis, signaling receptors move into early endosomes. From early endosomes, Trk receptors can either return to the plasma membrane for reutilization or go along the degradation pathway in a microtubule-dependent fashion, reaching late endosomes and the physical destruction of Trk receptor takes place in lysosomes. These studies show the relevance of receptor location in stimulation and afford a possible explanation of how different biological outcomes may be achieved by activation of Trks.

p75 RECEPTOR SIGNALING p75 receptors can modulate Trk receptor signaling, but they also signal independently of Trks. Depending on the cell type, developmental stage, whether they are unbound or ligand bound, and the type of ligand, p75 can activate apoptosis or survival and regulate Schwann cell migration, myelination, axonal growth, and regeneration. During development, p75 signals toward apoptosis to eliminate unnecessary neurons. The signaling pathways of p75-induced apoptosis involve the activation of Jun N-terminal kinase ( JNK) via the recruitment of p75 interacting proteins: neurotrophin receptor– interacting factor (NRIF), neurotrophin-associated cell death executor (NADE), neurotrophin receptor–interacting MAGE homolog (NRAGE), and TNF-associated

BDNF

factors (TRAFs). Neurotrophin binding to p75 can activate the MAPK pathway leading to cell survival or apoptosis, depending on the cell type or ligand type (Fig. 3). Recent findings also suggest that different outcomes of p75 signaling can be caused by differential subcellular localization and transport of p75 complexes. It has been shown that mature NGF induces a very slow internalization of p75, whereas pro-NGF in the presence of sortilin enables much faster kinetics of internalization of p75-NGF complex. Another signaling paradigm where p75 participates includes a receptor complex with Nogo receptor and Lingo-1 that mediates growth inhibition in response to myelin-based growth-inhibitory proteins (Nogo), myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (Omgp). It has also been shown that p75 plays a role in RhoA activation, which is required for growth inhibition. Recently, the role of regulated intramembrane proteolysis (RIP) of p75 by α and γ secretases has been shown to be important in the p75-mediated activation in response to myelin-based growth-inhibitory proteins [2]. The field of p75 signaling is evolving, with new ligands and intracellular interactors being identified, but we are still far from completely delineating the molecular pathways that enable p75 to regulate neuronal life, death, motility, and growth inhibition.

NEUROTROPHINS—PHYSIOLOGICAL ROLE AND ROLES IN PATHOLOGY Neurotrophins induce cell survival and neurite outgrowth on different neuronal populations. NGF

NT-3 NT4/5

NGF

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ARMS NRAGE

MAPK Akt Survival

RhoA

Axonal outgrowth

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FIGURE 3. p75 receptor structure and signaling.

1412 / Chapter 196 supports DRG neurons during development, supports the maintenance of mature cholinergic neurons, and regulates arborization and neurotransmitter release. One of the major roles of NGF is to mediate the selective survival of a proportion of developing sympathetic and sensory neurons as they innervate their particular target tissues. NGF also influences synaptic function and plasticity, and phenotypic (and probably also functional) maintenance in the mature cholinergic system. Neurotrophic factors also play key roles in pathophysiological mechanisms of neuropathies [11]. NGF is trophic to small-diameter sensory fibers and regulates nociception. Being a pro-inflammatory protein, NGF also plays a role in inflammatory pain. Thus, NGF antagonists could be useful in the treatment of certain forms of pain. The TrkA ectodomain fused to the Fc portion of an immunoglobulin has been shown to reduce inflammatory pain, presumably by mapping NGF. Anti-NGF antibodies have been used with similar results [10, 12]. Neurotrophins and their receptors have been also shown to play a role in the progression and differentiation of certain types of cancer—neuroblastoma (TrkA, NGF), prostate cancer (TrkA, NGF), and medulloblastoma (TrkC), just to name a few [11]. There is evidence for neurotrophic factors being effective at delaying neurodegenerative diseases in animal models and humans [11]. NGF has been shown to have an effect in models of diabetic peripheral neuropathy and Alzheimer’s disease. NGF induced positive results in phase II trials in diabetic neuropathy, but in a larger scale phase III trial there was no beneficial effect according to the predetermined clinical outcomes. Problems associated with the use of neurotrophins as drugs are numerous: Proteins are not orally bioavailable, they do not cross the blood–brain barrier in large amounts (see Chapter 200 by Pan in the next section of this book), they have a poor pharmacokinetic profile, and they can elicit an immune reaction. Because neurotrophins have been shown to be of potential use in the treatment of neurodegenerative diseases, several approaches have been used to circumvent some of these problems [11, 12], including gene therapy, cell therapy, and microencapsulated slow-release implants. Gene therapy has entered a nonrandomized, nonplacebo clinical trial with somewhat promising results. However, all these approaches require surgery and are still in the early stages. A more traditional pharmaceutical approach has been the development of small molecule nonpeptidic neurotrophin mimetics [12]. Such mimetic compounds, if defined on the basis of their activity, can have different mechanisms of action: activating receptors directly as ligands, activating them indirectly by upregulating the production of endogenous neurotrophins, affect-

ing receptor signaling pathways, or affecting coreceptors that regulate neurotrophin receptor activity [10, 12]. The first rationally derived TrkA agonist is a peptidomimetic compound termed D3. D3 is a proteolytically stable monovalent small molecule, a selective TrkA partial agonist that binds the Ig-C2 (D5) domain of TrkA. In vivo D3 binds to TrkA receptors and affords a significant and long-term rescue of cholinergic neurons in the cortex and in the nucleus basalis that correlates well with an enhanced cholinergic phenotype and a significant improvement of memory and learning in cognitively impaired aged rats [1]. Because a quantitative decline in TrkA expression in the cortex and nucleus basalis correlates well with cognitive decline and the stage of the disease in Alzheimer’s disease, age-associated cognitive impairment, and Down’s syndrome, it is conceivable that neurotrophic factors may be useful as therapeutics or diagnostics.

CLINICAL PROSPECTS Neurotrophins are a family of polypeptide growth factors that control the apoptotic death or survival, growth, and differentiation of neurons. NTFs also regulate several other cell populations such as lymphoid, epithelial, oligoglia, and mast cells [10, 11]. Altered activity (up- or downregulated) of neurotrophins or their receptors plays a well-defined role in certain human pathologies. Consequently, the neurotrophins and their receptors are important therapeutic targets, and pharmacological modulation may have applications ranging from the treatment of chronic or acute neurodegeneration and some forms of cancer and chronic pain with agonists, to the treatment of some forms of cancer or acute pain with antagonists. Several clinical trials have been attempted with neurotrophins, some of them seemingly premature and burdened with serious pharmacokinetic and pharmacodynamic issues, low stability, low efficacy, pleiotrophic effects due to the binding of multiple receptors, and high cost. Probably, as we learn more about these important ligands and receptors, and as pharmacological tools evolve and are refined, we may still see the successful targeting of these proteins in human disease.

References [1] Bruno MA, Clarke PB, Seltzer A, Quirion R, Burgess K, Cuello AC, and Saragovi HU. Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA. J Neurosci 2004; 24:8009–18. [2] Ceni C and Barker PA. Getting RIP’d stunts your growth. Neuron 2005; 46:839–40.

The Neurotrophins / 1413 [3] Cohen S. Purification of a nerve growth promoting protein from the mouse submaxillary gland and its neurocytotoxic antiserum. Proc Natl Acad Sci USA 1960; 46:302–11. [4] He XL and Garcia KC. Structure of nerve growth factor complexed with the shared neurotrophin receptor p75. Science 2004; 304(5672):833–4. [5] Huang EJ and Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem 2003; 72:609–42. [6] Kuruvilla R, Zweifel LS, Glebova NO, Lonze BE, Valdez G, Ye H, and Ginty DD. A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling. Cell 2004; 118:243–55. [7] Levi Montalcini R and Hamburger V. Selecting growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J Exp Zool 1951; 116: 321–62. [8] McDonald NQ, Lapatto R, Murray Rust J, Gunning J, Wlodawer A, and Blundell TL. New protein fold revealed by a 2.3—a resolution crystal structure of nerve growth factor. Nature 1991; 354:411–4. [9] Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, et al. Sortilin is essential for pro-ngf induced neuronal cell death. Nature 2004; 427:843–8.

[10] Pollack SJ and Harper SJ. Small molecule Trk receptor agonists and other neurotrophic factor mimetics. Curr Drug Targets CNS Neurol Disord 2002; 1:59–80. [11] Saragovi HU and Gehring K. Development of pharmacological agents for targeting neurotrophins and their receptors. Trends Pharm Sci 2000; 21:93–8. [12] Saragovi HU and Zaccaro MC. Small molecule peptidomimetic ligands of neurotrophin receptors, identifying binding sites, activation sites and regulatory sites. Curr Pharm Design 2002; 8:99–110. [13] Seidah NG, Benjannet S, Pareek S, Savaria D, Hamelin J, Goulet B, Laliberte J, Lazure C, Chretien M, and Murphy RA. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem J 1996; 314: 951–60. [14] Selby MJ, Edwards R, Sharp F, and Rutter WJ. Mouse NGF gene: structure and expression. Mol Cell Biol 1987; 7(9): 3057–64. [15] Wiesmann C, Ultsch MH, Bass SH, and de Vos AM. Crystal structure of nerve growth factor in complex with the ligand binding domain of the TrkA receptor. Nature 1999; 401: 184–8. [16] Zhou A, Webb G, Zhu X, and Steiner DF. Proteolytic processing in the secretory pathway. J Biol Chem 1999; 274:20745–8.

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197 Amino Acid Transport Across the Blood–Brain Barrier QUENTIN R. SMITH, HARITHA MANDULA, AND JAGAN M. R. PAREPALLY

disease and by the neuronal degeneration and death that occurs with excessive excitotoxic amino acid release in hypoxia, hypoglycemia, ischemia, and seizures. Brain amino acid pools are insulated from changes in plasma due in part to the presence of the blood– brain barrier (BBB). The BBB is a system of tissue sites including brain vascular endothelial cells, choroid plexus epithelial cells and arachnoid membrane, which together restrict and regulate the exchange of polar solutes between plasma and brain extracellular fluid [28]. The physical barrier at each site is formed by a single layer of cells joined together by multiple bands of tight junctions [17]. These junctions essentially seal adjacent cells together and block the aqueous paracellular diffusion pathway. In the absence of paracellular diffusion, polar solutes, such as amino acids, are forced to cross the BBB either by passive diffusion through lipoid BBB membranes or by carrier-mediated transport across the membranes via one of more than 12 BBB amino acid transport proteins. Only approximately half of the 20 amino acids that are required for brain development and function can be synthesized within the central nervous system (CNS). This includes both the small neutral and anionic amino acids that serve as neurotransmitters or neuromodulators. The remaining large neutral and cationic amino acids are nutritionally essential and must be supplied ultimately from the diet via gastrointestinal absorption and transport across the BBB. In the past 12 years, marked advances have been made in the identification and characterization of the specific transport proteins that mediate amino acid flux across the BBB and within the CNS. This chapter summarizes the current knowledge of the BBB amino acid transporters and how they function to regulate brain extracellular fluid amino acid concentrations. The primary focus is on the transporters of the brain

ABSTRACT Amino acids serve multiple roles in brain as neurotransmitters, neurotransmitter precursors, and building blocks of peptides and proteins. Their levels in brain are closely regulated in part by controlled transport across the blood–brain barrier (BBB). The BBB is located primarily at the brain capillary endothelium, which restricts and regulates the flux rates of 20 or more amino acids between the plasma and brain interstitial fluid. This regulation is achieved in good part through the selective action of 12 or more amino acid transporter proteins, which are highly expressed at the BBB and function predominantly to shuttle amino acids into or out of brain. This review summarizes the current knowledge of these BBB amino acid transport systems and their impact on brain metabolism and function.

BRAIN AMINO ACID REGULATION The brain depends on a diverse array of amino acids for normal development and function. Over 20 are required to sustain cerebral protein and peptide synthesis. Four (glutamate, aspartate, glycine, and GABA) serve as neurotransmitters, three (tryptophan, histidine, and tyrosine) as neurotransmitter precursors, and two thyronine derivatives (T3 and T4) as hormones. Further, an additional 20 perform critical roles as neuromodulators, intermediary metabolites, and essential precursors for other pathways (e.g., creatine, βalanine, taurine, quinolinic acid, and kynurenic acid). Most must be tightly regulated within brain neuronal, astrocytic, extracellular, and synaptic compartments. Imbalances profoundly influence brain function, as shown by the irreversible mental retardation that occurs in phenylketonuria and maple syrup urine Handbook of Biologically Active Peptides

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1416 / Chapter 197 capillary endothelium, which is thought to be the principal site of exchange for most solutes between brain interstitial fluid and the circulation.

Most BBB amino acid transport carriers have been shown to follow Michaelis-Menten transport kinetics [27, 41–42]. Unidirectional influx rates for amino acid uptake from plasma can be estimated with the Michaelis-Menten equation adapted for multiple substrate competition as

BLOOD–BRAIN BARRIER AMINO ACID TRANSPORT

Influx = (V maxC ) [ K m (1 + Σ (Ci K mi )) + C ]

Figure 1 illustrates the amino acid transporters of the BBB. Separate mechanisms have been identified for large neutral amino acids (System L and LNAA), small neutral amino acids (Systems A and ASC), cationic amino acids (System y+), anionic amino acids (System X−), β-amino acids (System β), creatine, and thyroid hormones. Transport is either active (against the electrochemical gradient and energized by linkage to ion movement) or passive (flowing down the electrochemical gradient without a requirement for additional energy). Some carriers are located at both the capillary luminal and abluminal membranes and mediate rapid bidirectional exchange across the BBB (e.g., Systems L and y+), whereas others (e.g., System A and LNAA) are located only at the capillary abluminal membrane and appear to mediate primarily active amino acid efflux from the CNS. The properties of the individual carriers are summarized in Table 1.

Phe, Trp Leu, Met Ile, Tyr His, Thr Val, Gln BCH

LAT1

LAT1

Arg Lys Orn

CAT1

CAT1

Ala Ser Pro Gln Asn His MeAIB

where C = plasma concentration of the amino acid of interest, Vmax (maximal transport velocity) and Km (half saturation concentration) are the transport constants of the amino acid, and Ci and Kmi are the plasma concentration and corresponding half saturation constant of each competing amino acid.

TRANSPORT SYSTEMS System y+/Cationic Amino Acid Transporter System y+/cationic amino acid transporter (CAT)1 was the first amino acid transport protein identified molecularly at the BBB. It mediates the brain uptake of three cationic amino acids: l-arginine, l-lysine, and lornithine. The cDNA for CAT1 was cloned serendipi-

Brain Side Ala Ser Cys Met Val

ATA2

ASCT 2

Na+

Na+

Na+

Na+

N

EAAT

Glu

Glu Asp

Gln Asn His

Glu Asp

N

Na+

Leu, Ile Val, Trp Taurine Tyr, Phe GABA Hypotaurine Met, Ala His, BCH Betaine β-Alanine

EAAT LNAA 1,2,3

GAT2

Na+

Na+

Na+

(1)

Na+

Cl–

CRT

Creatine

Cl–

TAUT

Na+

T4 rT3

Weak T4 T3

Oatp 14

Oatp 2

Cl–

Na+

ASCT 1

Ala, Ser Cys,Thr during development

T3 ?

Oatp 14

Oatp 2

T3

Blood Side FIGURE 1. Diagram of amino acid transport systems at the brain capillary endothelium and their localization to the capillary luminal (blood-facing) or abluminal (brain-facing) membranes. Shaded systems are Na+-dependent (two arrows) or Na+- and Cl−-dependent (three arrows). Unshaded systems are sodium independent. Amino acid assignments are from [27, 30–34, 41–42].

Amino Acid Transport Across the Blood–Brain Barrier / 1417 TABLE 1. Brain Capillary Amino Acid Transport Systems. Transport System

Gene Protein

Ion Dependence

Localization

Representative Amino Acid Substrates

System L

LAT1/SLC7A5

Neutral amino acid Both

—

System A

ATA2/SLC38A2

Abluminal

Na

System ASC System N System LNAA

ASCT2/SLC1A5

Abluminal Abluminal Abluminal

Na Na Na

System y+

CAT1/SLC7A1

Basic amino acid Both

—

Arg, Lys, Orn

System X− System ASC

EAAT 1-3/SLC1A1-3 ASCT2/SLC1A5

Acidic amino acid Luminal Abluminal

Na Na

Glu, Asp L-Asp

β-Amino acid GABA Creatine Oatp2

TAUT/SLC6A6 GAT2/SLC6A13 CRT/SLC6A8 Old—Oatp2/SLC21A5 New—Oatp1a4/SLCO1A4 Old—Oatp14/SLC21A4 New—Oatp1a4/SLCO1A4

Miscellaneous systems Abluminal Abluminal Luminal Both

Na, Cl Na, Cl Na, Cl —

Taurine, β-alanine GABA, Betaine Creatine Weak—T3, T4

—

T4, rT3 (Weak T3)

Oatp14

Present, but not localized to membrane

tously by Albritton et al. [3] in 1989 and subsequently shown independently by Kim et al. [22] and Wang et al. [54] to mediate high-affinity cationic amino acid transport. Stoll et al. [42] in 1993 demonstrated high levels of CAT1 mRNA in brain capillaries. The CAT1 protein was later shown to be present at both the brain capillary luminal and abluminal membranes [46]. CAT1 (SLC7A1) is a member of a family of cationic amino acid transporters (CAT 1-4) [16, 53]. Human CAT1 is a 629-amino-acid glycoprotein with 12–14 transmembrane-spanning regions [2]. The kinetic properties and substrate specificity of in vivo BBB cationic amino acid uptake match that of the cloned CAT1 transporter protein with Km values for l-arginine, l-lysine, and l-ornithine of between 50 and 120 μM. BBB CAT1 transport is Na+ independent and facilitated, mediating bidirectional exchange across both the capillary luminal and abluminal membranes. Table 2 summarizes plasma concentrations and BBB Michaelis-Menten kinetic constants for amino acids that are transported into brain. Because of the presence of System y+/CAT1 at the BBB, essential cationic amino acids, including l-lysine and -arginine, are taken up and equilibrate in brain with a half-life of <15 min. However, due to the high affinity of CAT1, it is essentially saturated with cationic amino acids as a group at normal

Phe, Trp, Leu, Met, Ile Tyr, His, Thr, Val, Gln BCH Ala, Ser, Pro, Gln, Asn His, MeAIB Ala, Cys, Ser, Met, Val Gln, Asn, His Leu, Ile, Val, Trp, Tyr Phe, Met, Ala, His, BCH

plasma concentrations. The saturation percentage can be calculated by the formula: Saturation (%) = 100 × Σ (C K m ) [1 + Σ (C K m )] With this, BBB CAT1 is predicted to be >85% saturated with cationic amino acid substrates at normal plasma concentrations. As a consequence, total cationic amino acid influx is predicted to remain fairly stable (within ∼twofold), with fluctuations in plasma concentration. However, the transport rates for individual cationic amino acids can change if plasma concentrations vary so as to significantly modify the fractional occupation of transporter binding sites by the amino acid. Such has been shown to occur following administration of a single cationic amino acid [6]. With transport saturation, the selective elevation in the plasma concentration of one cationic amino acid reduces the BBB influx of other amino acids sharing the same carrier by competitive inhibition. The presence of CAT1 at the BBB allows for the rapid bidirectional exchange of nutritionally essential cationic amino acids between plasma and brain interstitial fluid to support brain protein and peptide synthesis as well as nitric oxide generation. A CAT1 knockout mouse model has been developed that

1418 / Chapter 197 TABLE 2. Blood–Brain Barrier Transport Constants for Brain Uptake of Neutral Amino Acids, Basic Amino Acids, Acidic Amino Acids, and Thyroid Hormones. Amino Acid PHE TRP LEU MET ILE TYR HIS VAL THR GLN ARG LYS ORN

Plasma Concentration (μM) 81 82 175 64 87 63 95 181 237 485 117 245 98

GLU ASP

Km (μM)

Vmax (nmol/min/g)

Km(app) (μM)

Neutral amino acids (System L1) 11 41 170 15 35 330 29 59 500 40 25 860 56 60 1210 64 96 1420 100 61 2220 210 49 4690 220 17 4860 880 43 19900 Basic amino acids (System y+) 56 24 70 22 109 26

302 279 718

Influx (nmol/min/g) 13.2 8.2b 14.5 1.7 4.0 4.1 2.5 1.8 0.8 1.0 6.7 10.3 3.1

Acidic amino acids (System X −) 24 0.21 101 0.13 Thyroid hormones 0.26 0.16

T3

As measured by the in situ rat brain perfusion technique. Values are taken from [27, 41, 42]. b Estimated assuming ∼70% of albumin-bound TRP contributes to brain uptake. Vmax is the maximal saturable transport capacity, Km is the half-saturation concentration in the absence of competitors, Km(app) is the apparent Km under normal physiological conditions (i.e., in the presence of normal concentrations of plasma amino acids: Km(app) = Km (1 + Σ(Ci/Kmi)), and influx is the unidirectional amino acid flux rate from plasma to brain. Apparent Km values in vivo are much greater than true Km values because of transport saturation and competition [27, 41]. a

produces homozygous pups that die at birth [36]. The results suggest that CAT1 is critical for cellular cationic amino acid uptake and regulation.

System L/Large Neutral Amino Acid Transporter 1 System L/large neutral amino acid transporter (LAT)1, like CAT1, mediates the bidirectional exchange of essential amino acids across the BBB. System L was first described by Oxender and Christensen [35] and was subsequently shown to facilitate BBB uptake of more than eight large neutral amino acids, including l-phenylalanine, l-tryptotophan, l-leucine, l-methionine, l-tyrosine, l-isoleucine, l-histidine (neutral), lvaline, and threonine [27, 30–31, 38–39, 41]. The transporter is Na+- and H+-independent, and has traditionally been defined by sensitivity to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH). However, sub-

sequent work has demonstrated that BCH can interact with other transport proteins (e.g., B0+ and LNAA). The gene for the System L/LAT1 carrier was cloned in 1995 and shown by Kanai et al. [20] to encode for a ∼41-kDa light-chain protein (LAT1) that mediates Naindependent, BCH-sensitive, large neutral amino acid transport. The substrate affinity and selectivity of the cloned transporter [20] match well those reported for System L transport at the BBB [41]. LAT1 mRNA is highly expressed at the BBB, and the LAT1 protein has been shown to be present at both the luminal and abluminal membranes of brain capillaries [11, 18]. LAT1 was the first member (SLC7A5) discovered of a large family of heteromeric amino acid transporters consisting of different light and heavy chains [5, 9–10]. LAT1 has 12 predicted transmembrane-spanning regions and is found in cells linked via a disulfide bond to a heavy chain subunit (4F2/CD98). Other members of the LAT family (LAT2-4) are present in brain, but

Amino Acid Transport Across the Blood–Brain Barrier / 1419 distinct roles for these transporters have not been established at the BBB. Like CAT1, BBB LAT1 is heavily (>95%) saturated with amino acid substrates as a group at normal plasma concentrations and exhibits potent amino acid competition effects. Selective elevation of the plasma concentration of a single large neutral amino acid dramatically reduces brain influx rates of competing amino acids. Such has been shown to occur in phenylketonuria. Dietary supplementation with large neutral amino acids can partially overcome this inhibition and reduce brain phenylalanine to levels closer to normal [26].

System N l-Glutamine and asparagine were initially proposed to be taken up into brain by System L based on crossinhibition with l-phenylalanine in saline [31]. Consistent with this, the Km values for l-glutamine and l-asparagine (1.6 and 2.1 mM) with cloned LAT1 expressed in Xenopus oocytes [55] match those reported for the BBB L System (0.9 and 3.8 mM) in the absence of competitors [41]. However, in plasma, System L is more than 95% saturated with neutral amino acids as a group, and thus the flux rate for a given amino acid is only a small fraction of that from competitor-free saline. By the use of the reported BBB System L transport Vmax and Km values of Smith et al. [41], a unidirectional brain influx rate for l-glutamine of 15.3 nmol/min/g is predicted at 485 μM in competitor-free saline. In contrast, with rat serum, the predicted influx rate is only 1.0 nmol/min/g (6.5%). Using artificial blood perfusate with balanced amino acids, Ennis et al. [12] obtained a unidirectional brain influx rate for l-glutamine of 4.9 nmol/min/g, of which, from the calculation, the BBB L System is expected to contribute less than 20%. They found that the majority of BBB l-glutamine uptake was mediated by a Na+-dependent mechanism with properties similar to System N of the liver. Lee et al. [25] and O’Kane et al. [33] confirmed presence of System N activity at the BBB using isolated bovine brain endothelial cell membranes. However, in their preparation, they found Na+-dependent glutamine transport only at the capillary abluminal membrane and of this, 80% was mediated by System N and the remainder by System A. It was suggested that active BBB System N transport serves the brain by removing excess glutamine from brain and thereby contributing to the brain nitrogen metabolism and homeostasis [25].

System A/ATA2 Small neutral amino acids, including l-alanine, glycine, and proline, show very limited uptake into the brain and instead are effluxed from brain via the active

transport by one or more Na+-dependent transport systems. The first such mechanism to be demonstrated at the BBB was System A (ATA), which is localized solely to the BBB capillary abluminal membrane [8, 34, 38– 39]. System A mediates Na+-dependent transport of lalanine, l-serine, l-proline, l-asparagine, and l-glutamine [8, 34]. N-methylaminoisobutyric acid (MeAIB) is a selective System A substrate. Of the family of System A transporters (ATA1–3), the ATA2 isoform (SLC38A2) is predominantly expressed at the BBB [45]. System A is proposed to aid in the homeostasis of brain amino acids by clearing small neutral amino acids from brain extracellular fluid. Together with other Na-dependent transporters, it helps maintain brain extracellular fluid amino acid concentrations at one-tenth the levels in plasma [34, 45].

System ASC/ASCT1 and -2 System ASC is an additional Na-dependent, small neutral amino acid carrier that has been demonstrated to be present at brain capillary endothelial cell membranes. It expresses affinity for l-alanine, l-serine, lcysteine, l-methionine, l-glycine, l-threonine, and, to a lesser extent, l-valine, l-isoleucine, and l-leucine [34]. It can be distinguished from System A because it shows no activity towards MeAIB. Tayarani et al. [49] first noted this component using isolated rat cerebral microvessels and attributed it to System ASC, due to its preference for the small neutral amino acids alanine, serine, and cysteine. In adult animals, System ASC is localized almost exclusively to the capillary abluminal membrane and is mediated by the ASCT2 isoform (SLC1A5), which has a critical role in effluxing small neutral amino acids from brain [50]. In developing animals, ASCT1 (SLC1A4) is also transiently expressed at the BBB at both the capillary luminal and abluminal membranes and may have a role in delivering small neutral amino acids to the growing brain [37].

System B0+ or LNAA In addition to the Na+-dependent small neutral amino acid transporters localized at the brain capillary abluminal membrane, Hawkins’ group has proposed that a Na+-dependent large neutral amino acid transporter is also present at the capillary abluminal membrane that contributes to large neutral amino acid efflux across the BBB [32, 34, 39]. This transporter was initially identified as System B0+, but eventually this designation was changed because of the lack of crossinhibition between basic and neutral amino acids. A similar reasoning excluded System y+L. The Na+-dependent component of large neutral amino acid transport at the capillary abluminal membrane was found to be

1420 / Chapter 197 BCH-sensitive, high affinity (Km = 21 μM for l-leucine), and active for most all the substrates that are transported by the Na+-independent BBB System L.

System X-/EAAT 1–3 Anionic amino acids (i.e., glutamate and aspartate) are taken up into the brain and brain microvessels at low rates by a sodium-dependent, high-affinity, lowcapacity system tentatively identified as System X− (Km = 1.9 μM for l-glutamate) [4, 31]. Because these amino acids are neuroexcitatory and toxic at high concentrations, influx from blood must be tightly limited. Thus, the transport capacity is a small fraction (<1/100) of that for large neutral and basic amino acids. Brain regulation of acidic amino acids is also obtained by a highlevel expression of sodium-dependent excitatory amino acid transporters—EAAT1, -2, and -3 (SLC1A1–3)—at the BBB abluminal membrane, which actively efflux anionic amino acids across the BBB to the circulation [33]. The l-glutamate Km for abluminal membrane uptake is 14 μM, with a maximal transport capacity similar to that reported for System L [33, 39]. In addition, Tetsuka et al. [50] also demonstrated that ASCT2 contributes to l-aspartic acid efflux from brain. Together, these systems form a potent barrier to excitatory amino acid neurotoxicity from the circulation.

System TAUT Taurine, hypotaurine, β-alanine, and other β-amino acids are slowly taken up into brain at the BBB by a high-affinity, low-capacity, Na+- and Cl−-dependent transporter [7, 23, 47, 48]. This transporter mediates active uptake into the brain [47] of β-amino acids with Km values of 10–50 μM. Taurine transport at the BBB has been shown to be mediated by the TauT transporter (SLC6A6), which is a 620-amino-acid protein with a molecular weight of 70 kDa and 12 predicted transmembrane-spanning regions [21, 52]. This transporter appears to be expressed on both the capillary luminal and abluminal membranes for active uptake and efflux from the CNS [24].

System CRT Creatine is actively taken up into brain at the BBB by the Na- and Cl-dependent CRT transporter (SLC6A8) and can be inhibited by β-guanidinopropionate and guanidoacetate [29].

System Betaine-GABA/GAT2 GABA is actively effluxed from brain at the BBB by the Na+- and Cl−-dependent GABA-Betaine transporter

(GAT2, SLC6A13) [19, 44]. The Km is 680 μM with dense expression at the capillary abluminal membrane. The GAT2 transporter differs from that in neurons and glia (GAT1 and -3) and has been shown to have a significant role in removal of GABA from the brain extracellular fluid. Betaine, β-alanine, taurine, and quinidine all inhibited GABA uptake at the BBB by this system [44].

Thyroid Hormones The biologically active thyroid hormones triiodothyronine (T3) and thyroxine (T4) are lipophilic l-amino acids that taken up into brain across the BBB by saturable transport. Over 10 different gene products have been discovered that mediate saturable thyroid hormone transport [15]. The first was LAT1, which has been shown to mediate L-T3, and to a lesser extent L-T4, transport across biological membranes. LAT1 thyroid hormone transport is sensitive to competitive inhibition by the l-system selective substrate BCH but not by triiodothyroacetic acid [13]. This, however, does not match in vivo BBB L-T3 uptake, which is blocked by 10 μM triiodothyroacetic acid but not by 1 mM BCH, suggesting that in vivo the bulk of BBB T3 transport is mediated by a separate carrier. Consistent with this, LAT1 is predicted to contribute less than 15% of measured BBB T3 flux based on the expressed LAT1 T3/phenylalanine transport ratio of Friesema et al. [13] and the in vivo BBB phenylalanine transport rate [27, 41]. Alternate BBB transport carriers that have been shown to mediate thyroid-hormone transport and to be expressed at some site of the BBB include organic anion transporter protein (Oatp)2 and Oatp14 at the brain capillaries and MCT8 at the choroid plexus [1, 14, 43, 51]. Oatp14 transports T4 preferentially over T3 [43]. Monocarboxylic acid transporter (MCT)8 is highly expressed at the choroid plexus and the circumventricular organs [14]. Final demonstration of the mechanism that mediates brain T3 uptake awaits additional experiments.

CONCLUSION Over the past 15 years, significant progress has been made in the identification and characterization of molecular transport systems that mediate amino acid transport across the BBB. The genes and proteins of at least 12 BBB amino acid transporters have been identified. Work is under way to identify additional transporters and to resolve the contributions of transporters that have already been identified. The current results have documented the marked polarization of BBB amino acid transport processes, with passive facilitated carriers (LAT1 and CAT1) localized on the capillary luminal

Amino Acid Transport Across the Blood–Brain Barrier / 1421 membrane and most Na-dependent active transporters (ATA2, ASCT2, System N, System LNAA, EAAT1–3, TAUT, and GAT2) localized on the capillary abluminal membrane for active efflux from brain. Future work will address more closely how these systems work as a unit to regulate brain amino acid concentrations in health and disease. These BBB amino acid transporters also offer a valuable doorway for targeted drug delivery to brain. For example, high-affinity LAT1 drug analogs have been developed that showed significantly improved LAT1 affinity and brain uptake over existing LAT1 drug substrates (e.g., l-dopa, melphalan, and gabapentin) [40].

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1422 / Chapter 197 [35] Oxender DL, Christensen HN. Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J Bio Chem 1963; 238: 3686–99. [36] Perkins CP, Mar V, Shutter JR, del Castillo J, Danilenko DM, Medlock ES, et al. Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1. Genes Dev 1997; 11: 914–25. [37] Sakai K, Shimizu H, Koike T, Furuya S, Watanabe M. Neutral amino acid transporter ASCT1 is preferentially expressed in L-Ser-synthetic/storing glial cells in the mouse brain with transient expression in developing capillaries. J Neurosci 2003; 23: 550–60. [38] Sánchez del Piño MM, Hawkins RA, Peterson DR. Neutral amino acid transport by the blood-brain barrier. J Biol Chem 1992; 267: 25951–7. [39] Sánchez del Piño MM, Hawkins RA, Peterson DR. Neutral amino acid transport characterization of isolated luminal and abluminal membranes of the blood-brain barrier. J Biol Chem 1995; 270: 14913–8. [40] Smith QR. Carrier-mediated transport to enhance drug delivery to brain. Int Congr Ser 2005; 1277: 63–74. [41] Smith QR, Momma S, Aoyagi M, Rapoport SI. Kinetics of neutral amino acid transport across the blood-brain barrier. J Neurochem 1987; 49: 1651–8. [42] Stoll J, Wadhwani KC, Smith QR. Identification of the cationic amino acid transporter (System y+) of the rat blood-brain barrier. J Neurochem 1993; 60: 1956–9. [43] Sugiyama D, Kushuhara H, Taniguchi H, Ishikawa S, Nozaki Y, Aburtani H, et al. Functional characterization of the rat brainspecific organic anion transporter (Oatp14) at the blood-brain barrier. J Biol Chem 2003; 278: 43489–95. [44] Takanaga H, Otsuki S, Hosoya K, Terasaki T. GAT2/BGT-1 as a system responsible for the transport of gamma-aminobutyric acid at the mouse blood-brain barrier. J Cereb Blood Flow Metab 2001; 21: 1232–9. [45] Takanaga H, Tokuda N, Ohtsuki S, Hosoya K, Terasaki T. ATA2 is predominantly expressed as System A at the blood-brain

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198 Oligopeptide Transport at the Blood–Brain and Blood–CSF Barriers RICHARD F. KEEP AND DAVID E. SMITH

ABSTRACT

BLOOD–BRAIN BARRIERS: GENERAL PROPERTIES

Knowledge of oligopeptide transport at the blood– brain and blood–cerebrospinal fluid (CSF) barriers is important in understanding both endogenous neuropeptide activity and the delivery of peptide or peptidomimetic drugs to the brain. Although some oligopeptides use specific transport systems to gain entry into brain, the low lipophilicity of many small peptides and the presence of peptidases at the blood–brain barriers limit the entry of oligopeptides into the central nervous system. In addition, the presence of a number of transporters that clear peptides from the brain or CSF to the blood, such as PEPT2, P-glycoprotein, and PTS-1, have a major impact on brain peptide concentrations.

Passive Permeability The movement of peptides between blood and brain is regulated by two barrier systems, the blood– brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB), although both barriers are usually referred to by the single term BBB. The BBB is formed by the cerebral endothelial cells and the tight junctions that link them. The BCSFB is formed by choroid plexus (CP) epithelial cells, situated in the lateral third and fourth cerebral ventricles, and the cells of the arachnoid membrane that overlies the surface of the brain. These cells are also linked by tight junctions that limit paracellular permeability of polar compounds (such as many peptides). In contrast, even some large compounds, such as horseradish peroxidase, can permeate between the brain and cerebrospinal fluid (CSF) across the ependyma. Thus, homeostasis of the brain microenvironment relies on the coordinated action of both the BBB and the BCSFB. Although tight junctions limit paracellular diffusion at the BBB and the BCSFB, there are major quantitative differences between these two barrier systems. Thus, the transendothelial electrical resistance of the BBB is approximately 3000– 8000 Ω ⋅ cm2, whereas the transepithelial resistance of the CP is approximately 150–175 Ω ⋅ cm2. This is reflected in the passive diffusion of polar compounds across the barriers. The permeability of mannitol (molecular weight, MW, 182) and inulin (MW 5000) is about 10- and 100-fold greater at the BCSFB than at the BBB [18].

INTRODUCTION The ability of some peptides to affect cell function means that their concentrations in the extracellular space are critical. This may be particularly true in brain where many peptides are neurotransmitters or neuromodulators [8]. Perturbations in peptide concentration may come from brain parenchymal cell release, protein/peptide degradation within the brain, and blood-borne peptides. The aim of this chapter is to summarize our knowledge of the mechanisms that may influence the movement of oligopeptides (focusing on peptides with fewer than 10 amino acids) between the blood and brain and, thus, regulate peptide activity within the brain. Knowledge of such mechanisms is important in understanding the effects of endogenous neuropeptides, but it is also important for designing ways of delivering peptide or peptidomimetic therapeutic agents to the brain. Handbook of Biologically Active Peptides

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1424 / Chapter 198 CSF Sink Action In addition to the low permeability of the BBB and BCSFB to polar compounds, the continual replenishment of CSF also helps limit the penetration of polar compounds from the blood to brain or CSF. The CSF is primarily produced by the CPs and drains back into blood at the arachnoid villi and other drainage sites. This turnover of CSF continually clears compounds that have entered the brain from the blood, termed the CSF sink action. Thus, even during chronic administration, inulin concentrations in CSF only reach 1–3% of levels found in plasma, whereas sucrose (MW 342) may only reach 4–9% of plasma levels [18].

Transport Properties Although the blood–brain barriers were first described in terms of their ability to exclude dyes from the central nervous system (CNS), brain homeostasis requires more than a simple physical barrier. Thus, for example, the brain requires the acquisition of nutrients from and removal of waste products to the blood. Both of these functions are potentially hindered by the occlusion of the BBB and BCSFB paracellular pathways by tight junction proteins. There are, therefore, a series of transporter proteins at the BBB and BCSFB to fulfill these functions. Thus, Glut1 is present at the BBB to facilitate the entry of glucose from the blood to the brain, and there is an array of organic anion transporters at the BBB and BCSFB to remove metabolic waste products [6]. Whereas some barrier transporters are facilitative and enhance the rate of transport without imparting directionality (e.g., Glut1), others are coupled directly (e.g., P-glycoprotein) or indirectly (e.g., the peptide transporter, PEPT2) to ATP utilization. Many transporters are polarized in distribution (i.e., present on the blood-facing or brain-facing membrane of the barrier tissue) and involved specifically in blood to brain/CSF or brain/CSF to blood transport [6]. For larger molecules, such as polypeptides and proteins, blood to brain uptake can involve either fluid-phase, adsorptive, or receptor-mediated endocytosis [4]. It should be noted, however, that the level of endocytosis in cerebral endothelial cells is low compared with that in systemic capillaries and that endocytosis does not necessarily indicate transcytosis across the barrier tissue into the brain parenchyma or CSF. Thus, endocytosed compounds may accumulate in the barrier tissue, undergo degradation, or be recycled back to the blood.

Enzymatic Barriers Apart from the low paracellular permeability of the BBB and BCSFB and the presence of efflux transporters

at the barrier tissues that clear compounds from brain, the entry of organic compounds into the brain from the blood can also be prevented by enzymatic degradation at the level of the BBB or BCSFB. For example, the BBB contains a variety of neurotransmittermetabolizing enzymes such as monoamine oxidase, cholinesterases, GABA transaminase, aminopeptidases, and endopeptidases that help prevent the entry of neurotransmitters from the blood to the brain [7].

OLIGOPEPTIDE TRANSPORT AT THE BBB Blood to Brain Transport at the BBB Because of potential pharmacological and physiological importance, many studies have examined whether a variety of peptides can cross the BBB [4, 12, 23]. The BBB permeabilities of the dipeptides Gly-Phe, Gly-Leu, and carnosine are all very low [23]. It appears that these compounds enter the brain by passive diffusion because their permeabilities are not different from mannitol, a compound of similar molecular weight. Cyclo-(His-Pro) has a greater BBB permeability, although it is still low [1]. Its uptake into the brain is nonsaturable and the greater brain uptake is due to its greater lipophilicity and its resistance to enzymatic degradation [1]. Although it has a low permeability, cyclo-(His-Pro) has CNS effects when administered systemically, demonstrating the important point that peptides may have physiological effects within the brain at very low concentrations. The tripeptide glutathione has a much greater brain uptake than the dipeptides studied. This reflects the presence of a specific sodium-dependent glutathione transporter at the BBB [23]. Several larger oligopeptides also have a higher BBB than are predicted from their molecular weight and lipophilicity [23]. Thus, for example, Leuenkephalin and delta-sleep-inducing peptide have much higher permeabilities than expected. This, as with glutathione, reflects the presence of carrier-mediated transport at the luminal (blood-facing) membrane of the BBB [23]. It should be noted, however, that for Leu-enkephalin peptidase activity may prevent the appearance of intact peptide in the brain parenchyma [23]. It is now evident that the BBB can selectively transport a variety of oligo- and polypeptides [4, 12, 23]. In terms of small oligopeptides, the BBB expresses a number of different solute carriers that transport organic compounds [6]. Often these transporters have a wide range of substrates and overlapping substrate specificities. The affinity of these different transporters for different types of peptides has not been examined in detail. However, the organic anion trans-

Oligopeptide Transport at the Blood–Brain and Blood–CSF Barriers / 1425 porting polypeptide-2 (Oatp2) has affinity for the opioid peptide [D-pen2,5]enkephalin (DPDPE). Oatp2 is thought to act as a bidirectional transporter on both the luminal and abluminal (brain-facing) membranes of the BBB and to participate in DPDPE uptake into brain [6]. Human OATP transports DPDPE and deltorphin II [6]. For larger peptides and polypeptides, which are outside the scope of this chapter, transcytosis is an important mechanism for the blood to brain transport [4, 12, 23].

Brain to Blood Transport at the BBB In the past decade, considerable attention has focused on efflux transporters at the BBB, particularly in relation to P-glycoprotein (Pgp) and its role in preventing the access of a wide range of drugs into the brain. The brain : blood concentration ratio of the opioid peptide DPDPE after systemic administration is three- to fourfold greater in mice deficient in Pgp [5]. This effect of Pgp on peptide distribution is, however, likely to be limited to those peptides that are relatively lipophilic. Other efflux transporters at the BBB can also affect the distribution of peptides. Thus, peptide transport system (PTS)-1 clears Tyr-MIF-1, Met-enkephalin, and oxytocin from the brain [4]. Somatostatin and its analogs also have a saturable efflux system that is separate from PTS-1 [4]. Such transporters may play an important role in clearing endogenous neuropeptides from the brain, but they may also play an important role in limiting the entry of neuroactive peptides (endogenous or exogenous) from the blood to the brain.

PEPTIDASES AT THE BBB The cerebrovasculature expresses a wide range of proteins that degrade peptides, including carboxy-, amino-, and endopeptidases [21]. Some of these enzymes may be enriched both with respect to the brain and with respect to systemic capillaries. The metabolic substrates for such peptidases include a wide range of compounds including enkephalins, neurotensin, bradykinin, angiotensin I and II, substance P, endothelin, dynorphin, luteinizing hormone–releasing hormone, growth hormone–releasing factor, and kallidin [21]. Along with the low passive diffusion across the BBB, the presence of these peptidases on the cerebrovasculature may aid in preventing the entry of potentially neuroactive peptides into the brain from the blood. It should be noted, however, that some peptides are vasoactive as well as neuroactive (e.g., angiotensin) and some peptidases may be present to limit vascular effects.

OLIGOPEPTIDE TRANSPORTERS AT THE CHOROID PLEXUS Molecular Basis for Oligopeptide Transporter Heterogeneity In mammals, the proton-coupled oligopeptide transporter (POT) family consists of four members (i.e., PEPT1, PEPT2, PHT1, and PHT2) and is responsible for the transport of small peptides/mimetics across biological membranes via an inwardly directed proton gradient and negative membrane potential [2, 3, 13]. The POT proteins vary in size from 572 to 729 amino acids and are predicted to contain 12 transmembrane domains, with the N- and C-termini facing the cytosol. The encoded proteins have a number of potential protein kinase recognition domains (i.e., 0–3 for protein kinase A, PKA; 1–11 for PKC) and glycosylation sites (i.e., 2–7); however, their relevance has not been proven experimentally. Although PEPT1 and PEPT2 have high homology among species (about 80% in rat, rabbit, human, and mouse), the homology between these transporters for a given species is low (approximately 50%). Rat PHT1 and PHT2 have an amino acid identity of approximately 50%, but they show little homology to either PEPT1 or PEPT2 (less than 20%). PEPT1 is found primarily in the intestine and, to a lesser extent, in the kidney [13]. It has been shown to be a high-capacity, low-affinity transporter (i.e., Km values in the millimolar range). PEPT2 is believed to be the predominant POT in the kidney and, in contrast to PEPT1, was found to be a low-capacity, high-affinity transporter (i.e., Km values in the micromolar range) [3]. Together, PEPT1 and PEPT2 work in concert at the apical surface of epithelial cells to efficiently absorb and conserve protein digestive products arising in the intestine and kidney. However, PEPT2 has a wider tissue distribution than PEPT1 and, as demonstrated by immunolocalization in rats, is found extensively throughout the brain [15]. Although PEPT2 has its strongest expression in cerebral cortex, strong expression is also observed in the olfactory bulb, basal ganglia, cerebellum, and hindbrain. PEPT2 is also expressed abundantly in epithelial cells of the CP as well as in ependymal cells. This transporter is expressed exclusively on the apical membrane (i.e., CSF-facing) of CP epithelia in both adult and neonatal animals. PEPT2 is differentially expressed in cerebral cortex as a function of age, with much greater levels in fetal and neonatal tissue than in adult tissue. Moreover, PEPT2 is expressed in neurons (adult and neonate) and in astrocytes (neonate but not adult). The age-related decline of PEPT2 in cerebral cortex may reflect a loss of astrocytic PEPT2. There is no evidence for PEPT2 in endothelial cells of the BBB. Likewise, PEPT1 appears to be absent

1426 / Chapter 198 from the brain. The apical expression of PEPT2 in CP suggests (along with functional studies) that it is involved in the export of neuropeptides, peptide fragments, and peptidelike drugs from CSF. Its expression in neurons and astrocytes further suggests that PEPT2 may play a role in regulating the concentration of neuropeptides in extracellular fluid, especially around birth.

Basolateral Transporter The cloning, membrane localization, and functional activity of PEPT2 in CP epithelium have provided the fundamental basis for how peptides/mimetics are transported from CSF into the cell. However, the precise nature of the basolateral peptide transporter in CP has yet to be resolved (as is also the case in the kidney and intestine). Still, a low-affinity transporter was observed for glycylsarcosine (GlySar) uptake at the basolateral membrane of rat CP epithelial cells [17]. It has been suggested [19] that a single facilitative peptide transporter is expressed at the basolateral membrane of Caco-2 cells and that PEPT1 and the basolateral peptide transporter cooperate in the efficient transepithelial transport of small peptides and peptidelike drugs. Thus, PEPT1 (at the apical membrane of intestine) mediates the active uptake of these substrates from the lumen against a concentration gradient, followed by the facilitated cellular efflux into the blood down a concentration gradient. We speculate that the same mechanism could apply for the transepithelial transport of peptides/mimetics across the CP. Notwithstanding this uncertainty, the CP basolateral transporter does not appear to be PEPT1 [15, 17].

Intracellular Transporters Information on the tissue distribution, cellular localization, and transport properties of the peptide/histidine transporters, PHT1 and PHT2, is limited. PHT1 transcripts are abundantly expressed in rat eye and brain, including the CP [22]. Moreover, PHT1 expressed in Xenopus laevis oocytes transports l-histidine with high affinity (Km 17 μM) and the dipeptide carnosine. In contrast, PHT2 transcripts are abundant in the rat lung, spleen, and thymus but detected faintly in the brain [14]. Although the uptake of l-histidine and histidylleucine was observed when PHT2 protein was reconstituted into liposomes, the transport kinetics was not determined. PHT1 and PHT2 are unlikely to be involved in peptide/mimetic transport at the BCSFB (apical or basolateral membrane) because functional studies in rat CP primary cell cultures have failed to show the inhibition of GlySar or carnosine uptake by excess lhistidine [17, 20]. Despite the uncertain function of

PHT1 and PHT2 in the brain, they may have a role in the intracellular trafficking of small peptides, as suggested by lysosomal expression studies in rat PHT2transfected BHK and HEK-293T cells [14].

Larger Peptide Transporters Three members of the organic anion-transporting polypeptide (Oatp) gene family of membrane transporters have been shown to transport the δ-opioid peptides DPDPE (five amino acids) and deltorphin II (seven amino acids) [6]. Although rat Oatp1 and Oatp2 are localized at the apical and basolateral domains, respectively, of CP epithelium, Oatp2 is also expressed strongly at the BBB. Moreover, the human OATP (called OATPA) exhibits especially strong expression in brain microvessels and capillary endothelial cells. These findings suggest that certain members of the Oatp/ OATP gene family may play an important role in carriermediated translocation of opioid peptides across the mammalian BBB and BCSFB.

PEPTIDASES AT THE CHOROID PLEXUS A wide range of peptidases have been identified at the CP [18]. Some are located on the blood vessels of the CP (e.g., angiotensin-converting enzyme) and may be involved in regulating vasoactive hormones. Others, however, are present on the epithelium plasma membrane and have a polarized distribution. Thus, both endopeptidase 24.11 and aminopeptidase N are found on the CP apical membrane; these peptidases cleave enkephalins (as well as other substrates). It is probable that such peptidases serve to keep the concentrations of such neuroactive compounds low in CSF. They may act in concert with PEPT2 present on the apical membrane, with PEPT2 clearing peptide fragments produced by peptidase action. As well as cleaving peptides present in CSF, CP peptidases may be involved in preventing the entry of peptides from the blood to CSF. Peptidases on the CP vasculature or the epithelial basolateral membrane could prevent access of neuroactive peptides to the CSF. In addition, intracellular CP peptidases may degrade peptides taken up by endocystosis and other processes. Thus, cathepsin D and tripeptidyl peptidase I are both present in the CP lysosomal compartment [18].

PEPTIDE AND PEPTIDOMIMETIC DRUG TRANSPORT Although our understanding of blood–CNS barriers has increased greatly over the past few years, a signifi-

Oligopeptide Transport at the Blood–Brain and Blood–CSF Barriers / 1427 cant gap still remains in our appreciation of drug efflux transporters and their role in affecting the CNS exposure and pharmacological response to nutrients, drugs, and potential neuropharmaceuticals. Because of its unique localization within the brain, PEPT2 may represent an important determinant of CNS exposure and response to peptide (or mimetic) drugs, as well as endogenous peptides and their oligo fragments (some of which may be pharmacologically active or toxic). Studies in CP whole tissue from rats and transgenic mice suggest that PEPT2, located at the apical (CSFfacing) as opposed to basolateral (blood-facing) membrane, is the primary member of the POT family responsible for the removal of neuropeptides, peptide fragments, and peptidelike drugs from the CSF [18]. In particular, PEPT2-deficient mice have marked reductions (≥90%) in their ability to transport GlySar [11, 16], carnosine [20], and 5-aminolevulinic acid [11] in CP whole tissue. Moreover, the CP uptake of cefadroxil (a β-lactam antibiotic), as determined in wild-type and PEPT2-null mice [10], is mediated primarily by PEPT2 (80–85%) and to a minor extent by organic anion transporters (10–15%) and nonspecific mechanisms (5%). This finding suggests that aminocephalosporins, which are recognized by PEPT2, may have reasonable access to the CSF but are rapidly removed by drug efflux transporters making them ineffective for the treatment of bacterial infections in the CNS. Recently, the in vivo pharmacokinetics, tissue distribution, and systemic exposure of GlySar, a hydrolysis/peptidase-resistant dipeptide, was studied in wild-type and PEPT2-null mice [9]. Null mice had a twofold greater concentration of GlySar in CSF, despite having much lower blood concentrations (as a result of a greater clearance by the kidney). Thus, the CSF/ blood concentration ratio of GlySar was fourfold greater in PEPT2-null animals than in wild-type controls (p < 0.001; Fig. 1). In the absence of PEPT2, the CSF/blood ratio was approximately 0.9, indicating that GlySar can achieve a concentration in the CSF similar to that in blood. However, when PEPT2 is present, GlySar’s persistence in the CSF is substantially reduced (i.e., CSF/blood concentration ratio of approximately 0.2). Although a modest 40% increase was observed in the cerebral cortex/blood concentration ratio of GlySar in PEPT2-deficient mice (0.28 in wild-type vs. 0.39 in PEPT2-null animals, p < 0.01; Fig. 1), there was no difference between genotypes in the CP/blood ratio (0.62 in wild-type vs. 0.68 in PEPT2-null animals, p > 0.50; Fig. 1). However, a substantial difference was observed in the CP/CSF concentration ratio of GlySar, where wild-type animals had

FIGURE 1. Tissue (or CSF)/blood concentration ratios of GlySar in the choroid plexus (c. plexus), cerebral cortex (c. cortex), and CSF of wild-type and PEPT2 null mice, 60 min after an intravenous bolus dose of dipeptide (0.05 μmol/g body weight). Data are expressed as mean ± SE (n = 6–7). ** p < 0.01, and *** p < 0.001 compared with wild-type mice.

a fivefold greater tissue accumulation relative to medium at the transporter interface (4.2 in wild-type vs. 0.8 in PEPT2-null animals, p < 0.01). These results clearly demonstrate that under physiologic in vivo conditions PEPT2 operates as an efflux transporter in CP. Moreover, the findings suggest that PEPT2 may have profound effects on the sensitivity and/or toxicity of peptides and peptidelike drugs in the brain. A proposed model for the transepithelial transport of diand tripeptides (and peptidomimetic drugs) in CP is shown in Fig. 2.

CONCLUSION Although some peptides use specific transport systems at the BBBs to gain entry into the brain, the low lipophilicity of many small peptides and the presence of peptidases at the BBB and BCSFB limit the entry of oligopeptides into the CNS. The presence of efflux transporters may further limit such entry. In particular, the activity of transporters such as Pgp and PTS-1 at the BBB, and PEPT2 at the BCSFB serves to clear neuropeptides, peptide fragments, and potential peptidomimetic drugs from the brain. As a result, a better understanding of efflux transport will be critical to the successful design, delivery, and targeting of neuropharmaceuticals. Moreover, such knowledge will facilitate insight into the regulation of endogenous neuropeptide activity.

1428 / Chapter 198 CSF pH 7.3 2 mV

Epithelial Cell pH 7.0 - 60 mV

Blood pH 7.4 0 mV

2K+ Na+ 3Na+ H+

Amino Acids

Na

+

? H+

PT

Peptides

PEPT2 Dipeptides Tripeptides Mimetics

Acknowledgments This work was supported in part by grants R01 GM035498 (D.E.S.) and R01 NS034709 and P01 HL018575 (R.F.K.) from the National Institutes of Health.

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[14]

References [1] Banks WA, Kastin AJ, Akerstrom V, Jaspan JB. Radioactively iodinated cyclo (His-Pro) crosses the blood-brain barrier and reverses ethanol-induced narcosis. Am J Physiol 1993;264: E723–9. [2] Daniel H, Kottra G. The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflugers Arch 2004;447:610–8. [3] Daniel H, Rubio-Aliaga I. An update on renal peptide transporters. Am J Physiol 2003;284:F885–92. [4] Eggleton RD, Davis TP. Bioavailability and transport of peptides and peptide drugs into brain. Peptides 1997;18:1431–9. [5] Golden PL, Pollack GM. Blood-brain barrier efflux transport. J Pharm Sci 2003;92:1739–53. [6] Graff CL, Pollack GM. Drug transport at the blood-brain barrier and the choroid plexus. Curr Drug Metab 2004;5:95–108. [7] Hardebo JE, Owman C. Barrier mechanisms for neurotransmitter monoamines and their precursors at the blood-brain barrier. Ann Neurol 1979;8:1–31. [8] Mains RE, Eipper BA. Peptides. In: Siegel GJ, Agranoff BW, Albers RW, Fisher SK, Uhler MD, eds. Basic Neurochemistry (6th ed.) Philadelphia: Lippincott-Raven; 1999. p. 363–82. [9] Ocheltree SM, Shen H, Hu Y, Keep RF, Smith DE. Role and relevance of PEPT2 in the kidney and choroid plexus: In vivo studies with glycylsarcosine in wild-type and PEPT2 knockout mice. J Pharmacol Exp Ther 2005;315:240–47. [10] Ocheltree SM, Shen H, Hu Y, Xiang J, Keep RF, Smith DE. Mechanisms of cefadroxil uptake in the choroid plexus: Studies in wild type and PEPT2 knockout mice. J Pharmacol Exp Ther 2004;308:462–7. [11] Ocheltree SM, Shen H, Hu Y, Xiang J, Keep RF, Smith DE. Role of PEPT2 in the choroid plexus uptake of glycylsarcosine and

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[22]

[23]

FIGURE 2. Proposed model for the transepithelial transport of di- and tripeptides (and peptidomimetic drugs) in choroid plexus. It is uncertain whether an acid microclimate (an area of low pH adjacent to the apical membrane) exists at choroidal brush border membranes, such as that observed in the intestine and kidney. However, in the absence of a pH microclimate, accumulation could be driven by the negative membrane potential that exists in choroid plexus epithelium. Note that, in epithelial cells of the intestine and kidney, the polarity of Na+/K+ ATPase and the Na+/H+ exchanger are reversed; still, PEPT2 is localized to the apical membrane [2]. PT is the basolateral peptide transporter and PEPT2 is the apical proton-coupled oligopeptide transporter.

5-aminolevulinic acid: Studies in wild-type and null mice. Pharm Res 2004;21:1680–5. Pan W, Kastin AJ. Why study transport of peptides and proteins at the neurovascular interface. Brain Res Rev 2004;46:32–43. Rubio-Aliaga I, Daniel H. Mammalian peptide transporters as targets for drug delivery. Trends Pharmacol Sci 2002;23: 434–40. Sakata K, Yamashita T, Maeda M, Moriyama Y, Shimada S. Cloning of a lymphatic peptide/histidine transporter. Biochem J 2001;356:53–60. Shen H, Smith DE, Keep RF, Brosius FC. Immunolocalization of the proton-coupled oligopeptide transporter PEPT2 in developing rat brain. Mol Pharm 2004;1:248–56. Shen H, Smith DE, Keep RF, Xiang J, Brosius FC. Targeted disruption of the PEPT2 gene markedly reduces dipeptide uptake in choroid plexus. J Biol Chem 2003;278:4786–91. Shu C, Shen H, Teuscher N, Lorenzi P, Keep RF, Smith DE. Role of PEPT2 in peptide/mimetic trafficking at the blood-CSF barrier: Studies in rat choroid plexus epithelial cells in primary culture. J Pharmacol Exp Ther 2002;301:820–9. Smith DE, Johanson CE, Keep RF. Peptide and peptide analog transport systems at the blood-CSF barrier. Adv Drug Deliv Rev 2004;56:1765–91. Terada T, Sawada K, Saito H, Hashimoto Y, Inui K-I. Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2. Am J Physiol 1999;276: G1435–41. Teuscher NS, Shen H, Shu C, Xiang J, Keep RF, Smith DE. Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice. J Neurochem 2004;89:375–82. Witt KA, Gillespie TJ, Huber JD, Egleton RD, Davis TP. Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability. Peptides 2001;22:2329–43. Yamashita T, Shimada S, Guo W, Sato K, Kohmura E, Hayakawa T, Takagi T, Tohyama M. Cloning and functional expression of a brain peptide/histidine transporter. J Biol Chem 1997;272: 10205–11. Zlokovic BV. Cerebrovascular permeability to peptides: Manipulations of transport systems at the blood-brain barrier. Pharm Res 1995;12:1395–406.

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199 Opiate Peptides and the Blood–Brain Barrier RICHARD D. EGLETON, KEN A. WITT, AND THOMAS P. DAVIS

ABSTRACT

STABILIZATION

Opioid peptides have substantial potential for the treatment of pain. Despite this promise, few opioid peptides have shown true clinical viability for the alleviation of centrally mediated pain. The leading factor for the observed inactivity of these peptides is the blood–brain barrier (BBB). The BBB acts as a metabolic and transport barrier, preventing the delivery of substances to the central nervous system (CNS). In this chapter we discuss some of the mechanisms at the BBB that modulate opioid peptide delivery to the brain, as well as some promising methods that have been used to circumvent these impediments.

The metabolic stability of opioid peptides is a critical factor in the determination of brain bioavailability. Regardless of any other modification, if a peptide is not metabolically stable, it will not cross the BBB intact; thus, increasing the plasma half-life of a peptide increases the potential amount delivered to the CNS. There are a number of peptidases, both in the serum and within the membranes of blood vessels that metabolize peptides. The BBB itself has an elevated concentration of these peptide-degrading enzymes. Peptidases such as aminopeptidase A are present in serum and vary in concentration depending on clinical condition [30]. Aminopeptidase M (N) and enkephalinase (neutral endopeptidase), two zinc metallopeptidases involved in the inactivation of the opioid peptide enkephalins, show an elevated concentration within the brain vasculature [10]. In vitro and in vivo studies have shown that the inhibition of these enzyme systems can promote the delivery of peptides across the BBB [10, 14]. In the presence of enzyme inhibitors, a significant increase in Met-enkephalin permeability was observed without any effect on the basal permeability of the microvascular endothelial cell monolayers [10]. Structural design to reduce enzymatic degradation is also a common method used to enhance opioid peptide bioavailability. Because opioid peptide activity is dependent on the N-terminal tyrosine (Tyr), modification of this region of the peptide (to decrease Tyr hydrolysis) can be highly beneficial. Enzyme-masking strategies to prevent the cleavage of the Tyr include the modification of the N-terminus with N-acylation or pyroglutamyl residues, which reduce aminopeptidase M activity [9, 45]. Another modification for opioid peptides is to substitute the Glycine2 (Gly2) residue with a d-amino acid. The substitution with a d-amino acid leads to a stearic inhibition of the enzyme, thus reducing its ability to

INTRODUCTION The blood–brain barrier BBB maintains brain homeostasis by selectively regulating the entry of nutrients to the brain, as well as removing or degrading potential toxicants. The presence of the BBB also limits the distribution of peptides to the central nervous system (CNS), corresponding to a reduced efficacy. The innate physical makeup of the BBB (tight junctions and limited pinocytotic activity) directly limits the transport of peptides, which are generally hydrophilic in nature, into the brain. In addition, the BBB forms a highly active metabolic barrier that rapidly degrades peptides. Finally, the BBB expresses a number of efflux systems that actively remove peptides from the CNS. Through the alteration of the biochemical and structural composition of opioid peptides or by the inhibition or masking of mechanisms that reduce brain bioavailability we may be able to significantly enhance the efficacy of opioid peptides for the treatment of pain. Table 1 identifies several drug modifications currently applied to opioid peptides to enhance CNS bioavailability. Handbook of Biologically Active Peptides

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1430 / Chapter 199 TABLE 1. Opioid Peptide Modifications to Enhance CNS Bioavailability. Opioid Peptide

Modification

Mode of Enhancement

Sources

Biphalin Biphalin Biphalin Biphalin β-endorphin DPDPE DPDPE DPDPE DPDPE DPDPE DPDPE DPDPE DPDPE DALDA DALDA DPLPE Dalargin Deltorphin Dermorphin Dermorphin Leu-enkephalin Met-enkephalin

Hydrazide bridge Halogenation Pegylation Liposome Cationization Disulfide bridge Acetylation Pegylation Liposome Methylation Amino acid addition Halogenation Stereoselective design Adamantane Retrometabolic design Halogenation Nanoparticles Glycosylation Glycosylation Antibody attachment Glycosylation Glycosylation

Stability, transporter affinity Lipophilicity Reduced clearance, stability Lipophilicity, reduced clearance Membrane directed Enzymatic stabilization Lipophilicity, stability Reduced clearance, stability Lipophilicity, reduced clearance Lipophilicity Lipophilicity Lipophilicity Lipophilicity, stability Lipophilicity Lipophilicity, stability Lipophilicity Absorption, reduced clearance, stability Amphipathic, stability, reduced clearance Amphipathic, stability, reduced clearance Permeability, site directed Amphipathic, stability, reduced clearance Amphipathic, stability, reduced clearance

[1, 31] [2] [26] [50] [29, 36] [10, 47] [46] [51] [50] [24, 52] [23] [46, 48] [52] [27] [38] [22] [28] [32, 44] [32] [5] [8] [19, 37]

cleave the N-terminal Tyr. For Met-enkephalin, the substitution of D-Ala for L-Gly at the N-terminus and amidation of its C-terminus produce greater enzymatic stability [39]. Enhancing peptide stability can also be achieved by altering its biochemical structure through constrainment or bridging. The met-enkephalin analog [dPen2,5]-enkephalin (DPDPE) has a serum half-life greater than 500 min and a brain homogenate half-life of a similar magnitude. This extended stability is derived via its conformational constraint by the use of a disulfide bridge [10, 47]. Another variation on conformational bridging is shown with the opioid peptide biphalin, which contains two enkephalin sequences linked by a hydrazide bridge. Biphalin is protected from aminopeptidase activity by D-Ala residues in the 2 and 2′ positions, and is protected from carboxy peptidase activity by the hydrazide bridge [25, 31].

TARGETING Several approaches have been used to target opioid peptides to the brain. These can be divided into three basic strategies: (1) synthetic strategies that improve general membrane permeability via enhanced lipophilicity, (2) specific targeting of transport systems at the BBB via vectorization/pro-drug modifications, and (3) masking peptides from or inhibiting efflux pumps.

Strategies to Increase Lipophilicity One of the most common synthetic strategies is to increase the lipid solubility. Lipid solubility is a key factor in determining the rate at which a drug can passively cross the BBB endothelium. Peptide drugs generally contain polar functional groups that impart a degree of dipolarity and hydrogen bonding. The overall balance of polar to nonpolar groups within an opioid peptide can be reduced either by the removal of a polar group or the addition of a nonpolar group. A commonly used nonpolar group is the addition of methyl groups, which reduces the overall hydrogen-bond potential. Dimethylation of DPDPE on the Tyr has been shown to significantly enhance analgesia due to an increased bioavailability [24]. In another study, a DPDPE analog with three methyl groups attached to the phenylalanine (Phe) residue resulted in four purified diastereoisomeric conformations. These four methylated conformations showed an enhanced lipophilicity, yet only the (2S,3S)-TMP configuration exhibited a significant increase in uptake to the brain [52]. Halogenation of opioid peptides has also been used to enhance lipophilicity and BBB permeability. Halogenation of peptides such as DPDPE [46, 48], DPLPE [22], and biphalin [2] has shown to significantly enhance BBB permeability in vitro, depending on which halogen is conjugated (Cl, Br, F, or I). When chlorine was added to the phenylalanine residue(s) of either DPDPE [46, 48] or biphalin [2], BBB permeabil-

Opiate Peptides and the Blood–Brain Barrier / 1431 ity increased significantly. The addition of two chlorine atoms to DPDPE further increased BBB permeability beyond that of the single chlorine [46, 48]. However, the addition of fluorine to DPDPE did not increase permeability, whereas the addition of fluorine to biphalin greatly diminished BBB permeability [2]. The addition of lipophilic amino acids such as Phe can also be used to improve lipophilicity. The addition of a Phe to the amino terminal of DPDPE significantly increased permeability across bovine brain endothelial cell monolayers [23], whereas the addition of Phe to the C-terminus had no significant effect on permeability, compared to the parent peptide. Phe-DPDPE (Nterminus) also demonstrated a shortened half-life in both serum and brain homogenates [23]. Again, this study demonstrates that the location of the addition is as important as what is being added to the peptide. In addition, because highly lipid soluble drugs may be extensively plasma-protein bound, there is the potential for a reduction in the amount of free or exchangeable drug in the plasma, thereby compromising brain uptake [3].

Strategies to Target Transporter Mechanisms The BBB expresses a number of nutrient transporters that can be targeted to improve delivery to the brain. Transport systems that have been targeted for opioid peptide delivery include the large neutral amino acid transporter (LNAA) [1], receptor-mediated endocytosis/transcytosis [5], and adsorptive endocytosis/ transcytosis [35, 41]. For any drug to take advantage of the nutrient transports at the BBB, it must exhibit the appropriate molecular and biochemical nature, mimicking the endogenous nutrient. The difficulty in designing such peptide drugs to target specific transporters is that a great deal of knowledge of both the drug and transporter are required. The potent opioid peptide biphalin has been shown to use the large neutral amino acid carrier system to gain entry into the brain [1], although this was a case of serendipity rather than a plan. For CNS uptake, the hexose and large neutral amino acid carriers have the highest capacity and presently are the best candidates for the delivery of peptides to the brain, although lower-capacity carriers may also be used for highly potent peptide drugs. Receptors may also be targeted via antibody-peptide conjugation. The chimeric peptide (i.e., the conjugation of a peptide to a drug transport vector) approach is one such method. An example of a successful receptor-mediated transport vector is the monoclonal antibody OX26, which recognizes an external epitope of the transferrin receptor. The plasma protein transferrin is able to bind and undergo endothelial endocytosis in

the brain capillaries. An analog of the opioid peptide dermorphin ([Lys7]dermorphin) was conjugated to the OX26 vector, which demonstrated analgesia and was reversed by naloxone [5, 6]. Several methods have been used to target adsorptive endocytotic modes of transport, including cationization, glycosylation, and vectorization. Cationization of peptides results in the peptide interacting with the anionic sites on BBB endothelium (i.e., glycocalyx) [11]. The dynorphinlike analgesic peptide E-2078 is a polycationic peptide at physiologic pH shown to internalize into brain capillaries via adsorptive endocytosis [43]. Peptides containing different numbers of basic and neutral amino acids, with various C-terminal structures, were shown to significantly enhance affinity and capacity of adsorptive endocytosis at brain capillaries [41]. The basicity of the molecules and the C-terminal structure of the peptides were the most significant determinants for uptake by adsorptive endocytosis [41]. Cationized albumin has also been used as a vector to enhance peptide uptake via adsorptive-mediated endocytosis. When cationized albumin was conjugated to βendorphin, it yielded increased uptake into isolated brain endothelial cells, as compared to β-endorphin alone [29, 36]. Cationized albumin also displayed longer serum half-life and a general selectivity to the brain [7]. A number of studies have shown that the glycosylation of peptides not only increases the stability of peptides, but also the transport across the BBB [19, 49], probably via an adsorptive endocytotic mechanism [17, 18]. Several families of opioid peptides have been glycosylated to improve delivery, including dermorphins [32, 33], Met-enkephalins [19, 21, 37], and βendorphins [17]. The studies by Polt and colleagues highlight the importance of the location of the glycosylation on biological activity [37]. Opioid peptides require the alignment of the phenol rings of both the Tyr1 and Phe4 for maximal binding; placement of a Ser βD-glucose between these groups reduced the binding to the μ– opioid receptor from 30 nM to approximately 50,000 nM. The placement of the Ser βD-glucose outside of the active region at position 6 led to a μ–opioid receptor of 53 nM and only a minor change in binding [37], indicating that the glycosylation did not affect the alignment of the Tyr and Phe groups. Since the initial studies on glycosylated enkephalin opioids, a number of opioid analogs have been tested. The most promising peptides are based on linear Leu-enkephalin analogs. The monoglycosylated peptide Tyr-D-Thr-Gly-Phe-Leu-Ser(β-Dglucose) produces good analgesia on both central and peripheral administration, with an A50 value comparable to that of IV morphine administration [8]. Subsequent studies revealed that the glycopeptide had both

1432 / Chapter 199 higher metabolic stability and double the brain penetration of the parent peptide [20]. Recently, larger peptides based on the structure of β-endorphin have also been glycosylated [17]. These peptides have been shown to have an amphipathic structure and strong interactions with membranes, crucial for penetration of the BBB via endocytosis.

[2]

[3]

Strategies for Efflux Transporter Inhibition and Peptide Masking

[4]

The BBB expresses a number of efflux transporters responsible for limiting the delivery of a number of peptides. The efflux transporter P-glycoprotein (P-gp) is the most commonly examined efflux transporter at the BBB. P-gp has been shown to limit the delivery of a number of opioid peptides, including DPDPE [12, 16, 52], DADLE [42], and DAMGO [34, 40]. Several of the previously mentioned methods work in part by masking these peptides from P-glycoprotein or by inhibiting Pgp activity. Chen and Pollack [12] showed that DPDPE efficacy is impaired in Mdr1a–/– (P-gp knockout) mice, and that these animals are hypersensitive to DPDPE-induced analgesia. An inhibitor of P-gp, GF120918, coadministered with DPDPE was shown to increase analgesic effect [13], and GF120918 enhanced the permeability of DALDE across the BBB [15]. PEG conjugation has also been shown to reduce the P-pg affinity of DPDPE, potentially via a masking effect [51]. Another example of masking is through the use of liposome encapsulation of the peptide. One such liposome formulation is the pluronic copolymer P85. The P85 formulation shows an enhanced analgesic profile for biphalin and DPDPE [50], with the mechanism of action theorized to be the inhibition of P-gp [4].

[5]

CONCLUSION Present technologies are advancing our ability to alter peptides in manners previously unforeseen. These technologies inhibit mechanisms that diminish peptide efficacy and may provide potent pharmaceutics for the alleviation of CNS-mediated pain. This review has highlighted several modalities presently used to enhance the brain bioavailability of opioid peptides. The future of opioid peptide delivery to the CNS will likely be a composite of many of the approaches covered in this review.

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[34] Oude Elferink, R.P. and Zadina, J., MDR1 P-glycoprotein transports endogenous opioid peptides. Peptides, 22 (2001) 2015– 20. [35] Palian, M.M., Boguslavsky, V.I., O’Brien, D.F. and Polt, R., Glycopeptide-membrane interactions: Glycosyl enkephalin analogues adopt turn conformations by NMR and CD in amphipathic media. J Am Chem Soc 125 (2003) 5823–31. [36] Pardridge, W.M., Triguero, D. and Buciak, J.L., Beta-endorphin chimeric peptides: Transport through the blood-brain barrier in vivo and cleavage of disulfide linkage by brain. Endocrinology 126 (1990) 977–84. [37] Polt, R., Porreca, F., Szabo, L.Z., Bilsky, E.J., Davis, P., Abbruscato, T.J., Davis, T.P., Harvath, R., Yamamura, H.I. and Hruby, V.J., Glycopeptide enkephalin analogues produce analgesia in mice: Evidence for penetration of the blood-brain barrier. Proc Natl Acad Sci USA 91 (1994) 7114–8. [38] Prokai-Tatrai, K., Prokai, L. and Bodor, N., Brain-targeted delivery of a leucine-enkephalin analogue by retrometabolic design. J Med Chem 39 (1996) 4775–82. [39] Roemer, D. and Pless, J., Structure activity relationship of orally active enkephalin analogues as analgesics. Life Sci 24 (1979) 621–4. [40] Sarkadi, B., Muller, M., Homolya, L., Hollo, Z., Seprodi, J., Germann, U.A., Gottesman, M.M., Price, E.M. and Boucher, R.C., Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J 8 (1994) 766–70. [41] Tamai, I., Sai, Y., Kobayashi, H., Kamata, M., Wakamiya, T. and Tsuji, A., Structure-internalization relationship for adsorptivemediated endocytosis of basic peptides at the blood-brain barrier. J Pharmacol Exp Ther 280 (1997) 410–5. [42] Tang, F. and Borchardt, R.T., Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of the coumarinic acid-based cyclic prodrug of the opioid peptide DADLE. Pharm Res 19 (2002) 787–93. [43] Terasaki, T., Hirai, K., Sato, H., Kang, Y.S. and Tsuji, A., Absorptive-mediated endocytosis of a dynorphin-like analgesic peptide, E-2078 into the blood-brain barrier. J Pharmacol Exp Ther 251 (1989) 351–7. [44] Tomatis, R., Marastoni, M., Balboni, G., Guerrini, R., Capasso, A., Sorrentino, L., Santagada, V., Caliendo, G., Lazarus, L.H. and Salvadori, S., Synthesis and pharmacological activity of deltorphin and dermorphin-related glycopeptides. J Med Chem 40 (1997) 2948–52. [45] Veber, K. and Friedlinger, R.M., The design of metabolicallystable peptide analogs. Trends Neurosci (1985) 392–6. [46] Weber, S.J., Abbruscato, T.J., Brownson, E.A., Lipkowski, A.W., Polt, R., Misicka, A., Haaseth, R.C., Bartosz, H., Hruby, V.J. and Davis, T.P., Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs. J Pharmacol Exp Ther 266 (1993) 1649–55. [47] Weber, S.J., Greene, D.L., Hruby, V.J., Yamamura, H.I., Porreca, F. and Davis, T.P., Whole body and brain distribution of [3H]cyclic [D-Pen2,D-Pen5] enkephalin after intraperitoneal, intravenous, oral and subcutaneous administration. J Pharmacol Exp Ther 263 (1992) 1308–16. [48] Weber, S.J., Greene, D.L., Sharma, S.D., Yamamura, H.I., Kramer, T.H., Burks, T.F., Hruby, V.J., Hersh, L.B. and Davis, T.P., Distribution and analgesia of [3H][D-Pen2,D-Pen5] enkephalin and two halogenated analogs after intravenous administration. J Pharmacol Exp Ther 259 (1991) 1109–17. [49] Williams, S.A., Abbruscato, T.J., Szabo, L., Polt, R., Hruby, V.J. and Davis, T.P., The effect of glycosylation on the uptake of an enkephalin analog into the central nervous system. In P.O. Couraud and D. Scherman (Eds.), Advances in Behavioral Biology: Biology and Physiology of the Blood-Brain Barrier. Plenum, New York, 1996, Vol. 46, pp. 69–78.

1434 / Chapter 199 [50] Witt, K.A., Huber, J.D., Egleton, R.D. and Davis, T.P., Pluronic p85 block copolymer enhances opioid peptide analgesia. J Pharmacol Exp Ther 303 (2002) 760–7. [51] Witt, K.A., Huber, J.D., Egleton, R.D., Roberts, M.J., Bentley, M. D., Guo, L., Wei, H., Yamamura, H.I. and Davis, T.P., Pharmacodynamic and pharmacokinetic characterization of poly(ethylene

glycol) conjugation to met-enkephalin analog [D-Pen2, D-Pen5]enkephalin (DPDPE). J Pharmacol Exp Ther 298 (2001) 848–56. [52] Witt, K.A., Slate, C.A., Egleton, R.D., Huber, J.D., Yamamura, H.I., Hruby, V.J. and Davis, T.P., Assessment of stereoselectivity of trimethylphenylalanine analogues of delta-opioid [D-Pen(2), D-Pen(5)]-enkephalin. J Neurochem 75 (2000) 424–35.

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200 Permeability of the Blood–Brain Barrier to Neurotrophic Peptides WEIHONG PAN

within the central nervous system (CNS) and elsewhere, yet their induced expression after an insult to the CNS may not be sufficient to rescue neurodegeneration by themselves; (2) like typical cytokines, they are multipotent and may not always be beneficial or neuroprotective; and (3) endogenous neurotrophic peptides can be autocrine or paracrine signals and serve as neuromodulators, whereas exogenous neurotrophic peptides introduced by targeted delivery across the blood–brain barrier (BBB) or by implanted cells and tissue could have a greater impact on CNS functions. Here, I discuss the interactions of these peptides with the BBB. This complicated biological barrier, considered a neurovascular interface (as illustrated by other chapters in this section of the Handbook), provides a bidirectional route of communication. An efflux transport system explains part of the peripheral effects of CNS peptides, determines whether a peptide is retained in the CNS after influx, and is also important for the clearance of some peptides that are potentially cytotoxic, such as β-amyloid. The deposition and specific interactions of peptides with the cerebral and spinal vasculature precede the influx transfer of selective peptides. Because of their importance in vascular pathology and therapeutic potential in CNS diseases, the bloodto-brain (and spinal cord) influx of neurotrophic peptides is the focus of this chapter.

ABSTRACT Partially related to insufficient neurotrophic support, irreversible degenerative changes in the central nervous system (CNS) occur after various insults such as trauma, ischemia, and inflammation. Neurotrophic peptides not only serve important physiological functions in the normal CNS, but also rescue cell death. Their delivery from the peripheral circulation to the CNS involves the permeation of the blood–brain barrier (BBB). This review summarizes current understanding of the specific transport systems for neurotrophic peptides in physiological conditions. It then discusses the partial disruption of the BBB and increased paracellular permeability in pathological states and focuses on the regulation of specific transport systems for selective neurotrophic peptides and cytokines. Naturally occurring neurotrophic peptides for which interactions with the BBB have been studied include epidermal growth factor (EGF), transforming growth factor (TGF) α and β, insulinlike growth factor (IGF) I and II, acidic and basic fibroblast growth factor (FGF), colony-stimulating factors, and pituitary adenylate cyclase–activating polypeptide (PACAP). Overall, the ability of a neurotrophic peptide to cross the BBB depends on its stability to enzymatic degradation, its interactions with the cell surface that determine endocytosis, and the intracellular trafficking pathways that lead to exocytosis. The diversity of mechanisms indicates the complexity of the interactions of peptides with the BBB.

METHODS TO STUDY PERMEATION OF PEPTIDES ACROSS THE BBB: IN VIVO AND IN VITRO MODELS

INTRODUCTION Although the endothelial cells forming the BBB make a thin monolayer (less than 20 μm), they are joined by tight junctions and lined by a continuous basement membrane. This is further reinforced by

The functions of neurotrophic peptides are discussed in Chapters 192–196 in this Handbook. Three points should be stressed: (1) They can be produced both Handbook of Biologically Active Peptides

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1436 / Chapter 200 pericytes, astroglial endfeet, and extracellular matrix. In addition to the anatomical structure, intracellular degradation enzymes and extracellular matrix proteases also reinforce a biochemical barrier. If the overall structure is considered a semipermeable membrane, the permeation of peptides and proteins is slow because they are large and relatively lipid insoluble. Permeation is often incomplete, which means that proper mathematical modeling and correction for partial degradation are necessary. With the exception of several vasoactive peptides, the permeation of peptides and proteins across the BBB is not dependent on blood flow. Modified from Blasberg, Fenstermacher, and Patlak [14, 52], Banks and Kastin established the method of multiple-time regression analysis for in vivo studies [2–3, 5–8, 10]. The peptide to be studied is radioactively labeled to ensure sensitive and specific detection. This peptide is delivered to the mouse (or rat or other species) by an intravenous bolus injection. The study group consists of at least seven mice, each representing one time point of study. During a period when the majority of the peptide is still intact in the blood circulation, a brain/serum ratio is obtained for each time point and the linear portion of the kinetics of transfer indicates the permeability of the BBB to the particular peptide. To correct for disappearance of the peptide in the blood circulation over time and provide a steady-state value, the theoretical exposure time is calculated [28]. Two parameters of permeability can thus be derived from multiple-time regression analysis: the unidirectional influx rate (how fast) and the volume of distribution (how much). The analysis can be extended to different regions of brain and spinal cord, and it is applicable to the peripheral organs because their permeability to large peptides and proteins is also relatively low. With this method, permeability of different peptides can be compared and the effects of potential modulators tested. There are certain questions beyond the resolution of the two-compartmental model of the BBB that are answered by other techniques. The capillary depletion studies assist in addressing the events in the endothelial cell compartment in addition to cerebral vascular space and brain parenchyma [60]. The analysis of isolated cerebral microvessel endothelial cells in vitro further provides powerful tools to identify the mechanisms of permeation. The internalization of a peptide can be determined after the removal of specific surface binding by mild acid wash, trypsin digestion, or other similar procedures. Cultured primary cerebral microvessel endothelial cells can form tight junctions. This enables the measurement of apical-to-basolateral flux (simulating blood-to-brain transfer) and basolateral-to-apical flux (mimicking brain-to-blood permeation) when the cells

are grown on a supportive monolayer, such as the side-by-side diffusion chamber or the transwell insert. Because a relatively large quantity of viable cells is needed, many researchers choose bovine or porcine primary cells; however, studies are also performed on cells of rat or mouse origin. A tight monolayer is a prerequisite for in vitro permeability studies, so the nonspecific, paracellular diffusion is low. Nonetheless, endocytosis studies can also be performed on freshly isolated microvessels or immortalized endothelial cells. These cell lines include RBE4 and TR-BBB cells from rat, and TM-BBB4, b.End3, and b.End5 cells from mouse. There are also several emerging human cerebral microvessel endothelial cell lines. In addition to the kinetic assays of BBB permeability, intracellular trafficking studies elucidate the interactions of the peptide with cell membranes, cytoplasm, intracellular organelles, and molecular motors. Fluorescent-based as well as radioactive probes are valuable tools, and the specimen can be examined by the use of confocal microscopy, real-time imaging, and electron microscopy.

BINDING PROTEINS THAT AFFECT THE STABILITY AND PERMEATION KINETICS: INTRAVENOUS DELIVERY VERSUS IN SITU BRAIN PERFUSION A complex of the peptide with its binding proteins usually makes the peptide more stable and less susceptible to degradation enzymes in the blood circulation and provides a reservoir for its permeation across the BBB. A specific example is epidermal growth factor (EGF) [42]. Binding to proteins and cell surface heparin sulfate proteoglycans (HSPG) can also accelerate the peripheral degradation and reduce BBB permeation, such as occurs with receptor-associated protein (RAP) [48]. Further, the aggregation and polymerization of a peptide may deter or even abolish the permeation across the BBB, such as the chemokine MIP-1 [9] and the feeding-related peptide AgRP(83– 132) [25]. To accurately assess the permeability of the BBB, the first two issues can be resolved by in situ brain perfusion in blood-free physiological buffer. The radioactive-labeled tracers are delivered at a constant rate to the brain at a time when the BBB is still viable and functional (usually within 10 min), and multiple-time regression analysis can be performed. This method also detects rapid influx within the first few minutes that might have been missed by in vivo studies [38]. The last issue can be addressed by chemical methods such as gel chromatography or capillary electrophoresis [26].

Permeability of the Blood–Brain Barrier to Neurotrophic Peptides / 1437

PRESENCE OF RECEPTORS AT THE APICAL OR BASOLATERAL SURFACE OF THE BBB: USEFUL OR NOT FOR PEPTIDE TRANSPORT? Cell surface biotinylation and ligand binding methods can determine the relative distribution of receptors on the apical and basolateral surfaces. There are also experimental approaches that differ from whole-cell biology, involving the preparation of apical or basolateral membranes of the endothelial cells [18] or plasma membrane–derived inside-out vesicles [56]. Thus far, EGF and basic fibroblast growth factor (bFGF) are the two neurotrophic peptides for which the distribution of receptors in endothelial cell subdomains is well known. The EGF receptor (EGFR) is a peptide with characteristic six half cystines in the primary structure, which is mainly located at the basolateral surface of the epithelial cells [15, 23]. It is known that EGFR mediates the endocytosis of EGF from the basolateral surface [23]. However, the majority of trafficking studies has been carried out on nonpolarized cells, in which the sorting motifs in the EGFR (located at the apical surface) determine its fate, including lysosomal degradation [33], and sorting nexin 1 also is involved in the trafficking [34]. Intact EGF can be exocytosed from the basolateral to apical side in intact form in MDCK cells, with exocytosis being the rate-limiting step of transport [57, 59]. In animal studies of the BBB, the blood-tobrain transfer of EGF is a saturable process, indicating the presence of a specific transport system [42]. Nonetheless, this influx does not seem to be a receptormediated process because transforming growth factor (TGF)α, which binds to the same receptor, does not cross the BBB efficiently. Rather, it is retained in the vascular space of the brain [49]. Thus, in this case, the cell surface receptor for EGF and TGFα does not mediate blood-to-brain transport. Acidic fibroblast growth factor (aFGF, or FGF-1) has limited stability in the blood circulation and therefore cannot be studied by our multiple-time regression analysis. However, bFGF (also called FGF-2) has been shown to be endocytosed from the apical side of cerebral microvessel endothelial cells’ BBB by interactions with perlecan, a heparin sulfate proteoglycan [17]. Thus, the receptor does not play a significant role in the transport of bFGF across the BBB. Although a receptor is not always the transporter for a peptide across the BBB, there are many examples of receptor-mediated transport, such as for insulin, insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF)α, and transferrin [4, 11, 44–45]. The cross-inhibition of insulin and IGF-1 permeability indicates that the transport systems are at least partially shared.

ADSORPTIVE ENDOCYTOSIS AND CHEMICAL MODIFICATIONS THAT ENHANCE DELIVERY ACROSS THE BBB Several neurotrophic peptides are basic and therefore bear positive charges in solution. For instance, the isoelectric point (pI) of mouse bFGF is 9.59, making it possible to enter cells by adsorptive endocytosis. Binding of bFGF to heparin facilitates HSPG-mediated interaction with endothelial cells. For peptides crossing the BBB by simple diffusion, physiochemical properties such as size, lipophilicity, hydrogen bonding, conformation, and polymerization are important determinant factors of permeability. Thus, chemical modifications to enhance stability, lipophilicity, and selective interactions with cerebral endothelial cells will help. Among the posttranslational modifications, glycosylation increases the permeability of some proteins [53]. Pegylation enhances the stability of a peptide in the blood but might reduce its permeation across the BBB [54, 55]. Some of the modifications, especially immunoliposomes, have been used successfully to increase the bioavailability of a peptide drug to the CNS.

THE INTRACELLULAR TRAFFICKING OF PEPTIDES THAT LEADS TO EXOCYTOSIS The intracellular trafficking of neurotrophic peptides in the endothelial cells composing the BBB has not been extensively studied. Our unpublished results with urocortin indicate that substantial amounts of fluorescently labeled urocortin is co-localized with endosomes within 2 min and translocates to the Golgi complex and proteasomes at a later time. Urocortin is a 4-kDa peptide that binds to corticotrophin-releasing hormone (CRH) receptors and is primarily involved in feeding behavior and energy expenditure, as well as being neurotrophic in certain circumstances. More than 10% of radioactively labeled urocortin can be exocytosed by cultured cerebral microvessel endothelial cells over a time course of 5 min–1 h in intact form (Tu et al., manuscript in preparation). It is yet to be confirmed whether endocytosis is the rate-limiting step for transport, and much needs to be learned about the trafficking routes and any cytoskeletal motors, ions, or energy processes driving the exocytosis. Exocytosis of endocytosed peptides and proteins has not received much attention, but valuable information can be learned from studies of those newly synthesized. Although not a complete analogy, the trafficking patterns of a newly synthesized peptide or protein illustrate the diversity of potential pathways encoded by its structure and its interacting partners. The classical secretory

1438 / Chapter 200 route involves the Golgi complex. Sorting may be related to the signal peptide that is cleaved from the mature peptide, associated with sorting nexins, and dependent on chaperone proteins. There are also alternative exocytosis pathways, such as multivesicular bodies and exosomelike vesicles, exovesicles related to membrane blebbing, transporters residing at plasma membrane, and lysosomal secretion [56].

SPECIFIC EXAMPLES OF THE PHARMACOKINETICS OF BBB PERMEATION It seems that cerebral endothelial cells render to neurotrophic peptides a different fate than what has been shown in other cells such as fibroblasts, lymphocytes, and hepatocytes. Earlier work with radioactively labeled EGF in fibroblasts as well as other cell types clearly showed its intracellular degradation [16]. Later, the polarized sorting was shown to be different when EGF was applied apically versus basolaterally. Kozu et al. provided strong evidence that EGF can be exocytosed in its intact form and that exocytosis is the ratelimiting step of transport [32]. In vivo studies in mice also showed that EGF is relatively stable in blood and crosses the BBB by a specific transport system. The amount of EGF in brain parenchyma at 10 min accounts for over 50% of total radioactivity, as contrasted with only 20% in blood vessels [42]. TGFα, although sharing the same receptor as EGF, is retained in the cerebral vasculature without any meaningful penetration of the BBB after IV injection [49]. TGFβ1 is stable in circulating blood, but does not show influx from blood to the brain [27]. IGF1 delivered into blood is mainly protein-bound; however, it has a saturable transport system at the BBB. The passage of IGF1 across the BBB might partially explain the effect of peripheral growth hormone to the CNS because growth hormone itself has limited the permeation of the BBB by simple diffusion [50]. The rate of entry of 125I-IGF1 from the blood to the CNS shows apparent regional differences, and excess unlabeled IGF1 paradoxically increases the influx of the radioactively labeled tracer in each region tested, probably explained by the displacement of 125I-IGF1 from its binding proteins in the periphery [44]. Our unpublished in vitro and in situ brain perfusion study also showed that the transport system for IGF1 is partially shared with that of insulin. The receptor-mediated endocytosis of insulin in cerebral microvessel endothelial cells has been studied both in vivo and in vitro by several groups. Regional differences of insulin transport and its regulation in diabetes and various models of obesity have been reviewed in detail elsewhere [1]. Based on its capability

to transport insulin, the insulin receptor has been a target for the design of vehicles (antibody-based cargos) to deliver other neurotrophic peptides and proteins from the blood to the brain. Granulocyte monocyte colony–stimulating factor (GM-CSF) enters the brain and spinal cord by a saturable transport system in both mice and rats [36]. Its presence in the blood and relative stability in both the blood and CNS indicate that GM-CSF plays an important role in modulating the physiological functions of neurons [22] and astrocytes [21]. Pituitary adenylate cyclase–activating polypeptide (PACAP) is a peptide related to secretin, glucagon, glucagonlike peptide (GLP)1, and growth hormone–releasing factor (GRF) and it binds to PACAP/vasoactive intestinal peptide (VIP) receptors (as discussed elsewhere in this book). PACAP illustrates how the size of the primary structure does not determine the behavior of its permeation of the BBB. PACAP1–27 has a slower influx that is not saturable, whereas the larger PACAP1–38 has a faster and saturable influx [12]. The efflux of these two PACAP peptides from brain to blood is impaired in a mouse model of Alzheimer’s disease (SAMP8) [13]. The structurally related VIP is more vasoactive; it is able to enter the brain from the blood by simple diffusion and is retained in the CNS [20]. The interactions of many other peptides with the BBB and blood–CSF barrier are covered in greater detail in other chapters of this section of the Handbook.

THE SITUATION OF THE PARTIALLY DISRUPTED BBB AND SELECTIVE REGULATION OF THE TRANSPORT SYSTEM IN PATHOPHYSIOLOGY Neurotrauma, ischemia, and inflammation are associated with increased permeability of the BBB, altered axonal transport, and disrupted neuronal and glial functions. When the BBB is partially disrupted, certain transport systems for cytokines are not abolished but upregulated. This has been specifically shown by TNFα transport after spinal cord injury [39, 43, 46, 51], head trauma [47], experimental allergic encephalomyelitis (EAE) [40], and lipopolysaccharide (LPS) administration [37]. The enhanced transport is related to increased mRNA expression of the transporting receptors [41, 45]. Similar regulation of TNFα transport is also present after focal cerebral ischemia (a mouse model of stroke, unpublished observations). BBB permeability of PACAP1–38 is decreased at selected times (24 h) in rats after 2 h of middle cerebral artery occlusion followed by reperfusion, and the change appears to be bilateral rather than unilateral

Permeability of the Blood–Brain Barrier to Neurotrophic Peptides / 1439 [58]. One common feature found for both TNFα and PACAP1–38 is that regulation is not just confined to the damaged area but also extends to remote regions, such as the hemisphere contralateral to ischemia. This suggests a general and possibly hormonal factor in the regulation of transport. However, for neurotrophic proteins that enter brain and spinal cord by adsorptive endocytosis or other nonreceptor-mediated mechanisms, regulation does not always lead to increased uptake. Ebiratide, a synthetic analog of an adrenocorticotropic hormone (ACTH) peptide and potent neurotrophic factor, has a high baseline penetration across the BBB, and spinal cord injury does not further facilitate its permeation from the blood to the brain and spinal cord [39]. EGF uptake by the injured spinal cord also fails to show a significant increase over time in different CNS regions (unpublished observations).

ENHANCED DELIVERY OF NEUROTROPHIC PEPTIDES: HOW IT AFFECTS NEUROREGENERATION Unlike classical hormones and neurotrophins, the neurotrophic peptides have a broader distribution in both synthesis and targets of action. Because insults to the CNS often affect multiple populations of cells and extracellular matrix, the provision of trophic support by these peptides is extremely useful. To deliver neurotrophic peptides as CNS therapeutics, the peripheral route is more advantageous than invasive application directly to the CNS. However, there is only a small proportion of neurotrophic peptides crossing the BBB by specific transport systems; the rest depend on the timeand space-related partial disruption of the BBB. Even for those normally entering the CNS by saturable transport, there does not seem to be substantial enhancement of transport in the diseased state. With the additional concern of peripheral degradation, peptides are often linked to delivery vehicles for adequate transfer. These experimental approaches include immunoliposomes, phage display, viral vectors, stem cells, fusion proteins, and stable, more lipophilic synthetic analogs [61]. Apart from delivery of blood-borne neurotrophic peptides, such as by intravenous, subcutaneous, and intraperitoneal injections and by IV infusions, an alternative pathway is nasal delivery. Although the detailed mechanism of nasal mucosa– and olfactory nerve– mediated transfer is not clear, nasal-delivered IGF1 can reduce the infarct volume and neurological deficit in rats after focal stroke [35]. Transmission within the CSF has enabled neuropeptides to rescue neuronal cell death after neurodegeneration and cerebral ischemia [24].

Despite progress in the attempts to use neurotrophic peptides as CNS therapeutics, there have not been abundant successful long-term effects. Part of the overall picture is that their effects are time- and situationdependent and that other factors, especially inhibitory molecules for neuroregeneration, are also present in the local environment [31]. Nonetheless, the effects of neurotrophic peptides probably extend beyond their roles as cell survival factors to contribute to neuroplasticity both structurally and physiologically [19].

CONCLUSION Neurotrophic peptides not only affect the functions of endothelial cells and other components of the BBB, but also may cross the BBB themselves. The permeation of neurotrophic peptides across the BBB is determined by physiochemical properties of the peptides (for those permeating by simple diffusion) or the presence of a carrier system (for those possessing receptor-mediated transport, adsorptive endocytosis, or other specific interactions with the cell surface). There are multiple approaches to enhancing the permeability of these peptides across the BBB, to conjugating them to targeted delivery systems, or to bypassing the BBB. The long-term effect of exogenous neurotrophic peptides on CNS regeneration and neurobehavioral changes deserves more extensive study, almost 30 years after the first notion that peripheral peptides have direct CNS effects [29, 30]. It is now established that BBB permeation of neurotrophic peptides can be involved in neuroendocrine changes and neuroplasticity.

Acknowledgment Current grant support is from NIH (NS45751 and NS46528 from NINDS, and DK54880 from NIDDK). The author wishes to thank the present BBB Lab members in PBRC for their excellent work, and collaborators and past lab members for valuable contributions.

References [1] Banks, W. A. The source of cerebral insulin. Eur J Pharm 2004 490:5–12. [2] Banks, W. A.; Audus, K. L.; Davis, T. P. Permeability of the bloodbrain barrier to peptides: An approach to the development of therapeutically useful analogs. Peptides 1992;13:1289–1294. [3] Banks, W. A.; Fasold, M. B.; Kastin, A. J. Measurement of efflux rates from brain to blood. In: Irvine, G. B.; Williams, C. H., Eds. Methods in Molecular Biology, Neuropeptide Protocols. Totowa, NJ: Humana Press Inc.; 1997. 353–360. [4] Banks, W. A.; Jaspan, J. B.; Kastin, A. J. Effect of diabetes mellitus on the permeability of the blood-brain barrier to insulin. Peptides 1997;18:1577–1584.

1440 / Chapter 200 [5] Banks, W. A.; Kastin, A. J. Interactions between the blood-brain barrier and endogenous peptides: emerging clinical implications. Am J Med Sci 1988;295:459–465. [6] Banks, W. A.; Kastin, A. J. Measurement of transport of cytokines across the blood-brain barrier. Method Neurosci 1993;16:67– 77. [7] Banks, W. A.; Kastin, A. J. Passage of peptides across the bloodbrain barrier: pathophysiological perspectives. Life Sci 1996; 59:1923–1943. [8] Banks, W. A.; Kastin, A. J. Permeability of the blood-brain barrier to neuropeptides: the case for penetration. Psychoneuroendocrinol 1985;10:385–399. [9] Banks, W. A.; Kastin, A. J. Reversible association of the cytokines MIP-1α and MIP-1β with the endothelia of the blood-brain barrier. Neurosci Lett 1996;205:202–206. [10] Banks, W. A.; Kastin, A. J.; Broadwell, R. D. Passage of cytokines across the blood-brain barrier. Neuroimmunomodulation 1995; 2:241–248. [11] Banks, W. A.; Kastin, A. J.; Fasold, M. B.; Barrera, C. M.; Augereau, G. Studies of the slow bidirectional transport of iron and transferrin across the blood-brain barrier. Brain Res Bull 1988;21:881–885. [12] Banks, W. A.; Kastin, A. J.; Komaki, G.; Arimura, A. Passage of pituitary adenylate cyclase activating polypeptide1–27 and pituitary adenylate cyclase activating polypeptide1–38 across the blood-brain barrier. J Pharmacol Exp Ther 1993;267:690–696. [13] Banks, W. A.; Robinson, S. M.; Verma, S.; Morley, J. E. Efflux of human and mouse amyloid β proteins 1–40 and 1–42 from brain: Impairment in a mouse model of Alzheimer’s disease. Neuroscience 2003;121:487–492. [14] Blasberg, R. G.; Fenstermacher, J. D.; Patlak, C. S. Transport of α-aminoisobutyric acid across brain capillary and cellular membranes. J Cereb Blood Flow Metab 1983;3:8–32. [15] Carpenter, G.; Cohen, S. Epidermal growth factor. J Biol Chem 1990;265:7709–7712. [16] Carpenter, G.; Cohen, S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol 1976;71:159–171. [17] Deguchi, Y.; Okutsu, H.; Okura, T.; Yamada, S.; Kimura, R.; Yuge, T.; Furukawa, A.; Morimoto, K.; Tachikawa, M.; Ohtsuki, S.; Hosoya, K.; Terasaki, T. Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. J Neurochem 2002;83:381–389. [18] Delpino, M. M. S.; Hawkins, R. A.; Peterson, D. R. Biochemical discrimination between luminal and abluminal enzyme and transport activities of the blood-brain barrier. J Biol Chem 1995;270:14907–14912. [19] Ding, Y.; Kastin, A. J.; Pan, W. Neural plasticity after spinal cord injury. Curr Pharm Des 2005;11:1441–1450. [20] Dogrukol-Ak, D.; Banks, W. A.; Tuncel, N.; Tuncel, M. Passage of vasoactive intestinal peptide across the blood-brain barrier. Peptides 2003;24:437–444. [21] Guillemin, G.; Boussin, F. D.; Le Grand, R.; Croitoru, J.; Coffigny, H.; Dormont, D. Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brain. Glia 1996;16:71–80. [22] Ha, Y.; Kim, Y. S.; Cho, J. M.; Yoon, S. H.; Park, S. R.; Yoon, D. H.; Kim, E. Y.; Park, H. C. Role of granulocyte-macrophage colony-stimulating factor in preventing apoptosis and improving functional outcome in experimental spinal cord contusion injury. J Neurosurg—Spine 2005;2:55–61. [23] He, C.; Hobert, M.; Friend, L.; Carlin, C. The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants, including a dominant signal with a polyproline core. J Biol Chem 2002;277: 38284–38293.

[24] Johanson, C. E.; Duncan, J. A.; Stopa, E. G.; Baird, A. Enhanced prospects for drug delivery and brain targeting by the choroid plexus-CSF route. Pharmaceutical Res 2005;22(7):1011–1037. [25] Kastin, A. J.; Akerstrom, V.; Hackler, L. Agouti-related protein(83–132) aggregates and crosses the blood-brain barrier slowly. Metabolism 2000;49:1444–1448. [26] Kastin, A. J.; Akerstrom, V.; Hackler, L.; Pan, W. Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier. J Neurochem 2003;87:7–12. [27] Kastin, A. J.; Akerstrom, V.; Pan, W. Circulating TGF-β1 does not cross the intact blood-brain barrier. J Mol Neurosci 2003; 21:43–48. [28] Kastin, A. J.; Akerstrom, V.; Pan, W. Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: Meaningful controls. Peptides 2001;22:2127–2136. [29] Kastin, A. J.; Coy, D. H.; Schally, A. V.; Miller, L. H. Peripheral administration of hypothalamic peptides results in CNS changes. Pharmacol Res Commun 1978;10:293–312. [30] Kastin, A. J.; Olson, R. D.; Schally, A. V.; Coy, D. H. CNS effects of peripherally administered brain peptides. Life Sci 1979;25: 401–414. [31] Kastin, A. J.; Pan, W. Targeting neurite growth inhibitors to induce CNS regeneration. Curr Pharm Des 2005;11:1247–1253. [32] Kozu, A.; Kato, Y.; Shitara, Y.; Sugiyama, Y. Kinetic analysis of transcytosis of epidermal growth factor in madin-Darby canine kidney epithelial cells. Pharm Res 1997;14:1228–1235. [33] Kurten, R. C. Sorting motifs in receptor trafficking. Advanced Drug Delivery Rev 2003;55:1405–1419. [34] Kurten, R. C.; Cadena, D. L.; Gill, G. N. Enhanced degradation of EGF receptors by a sorting nexin, SNX1. Science 1996; 272:1008–1010. [35] Liu, X. F.; Fawcett, J. R.; Thorne, R. G.; Frey, W. H. Non-invasive intranasal insulin-like growth factor-I reduces infarct volume and improves neurologic function in rats following middle cerebral artery occlusion. Neurosci Lett 2001;308:91–94. [36] McLay, R. N.; Kimura, M.; Banks, W. A.; Kastin, A. J. Granulocytemacrophage colony-stimulating factor crosses the blood-brain and blood-spinal cord barriers. Brain 1997;120:2083–2091. [37] Osburg, B.; Peiser, C.; Dömling, D.; Schomburg, L.; Ko, Y. T.; Viogt, K.; Bickel, U. Effect of endotoxin on expression of TNF receptors and transport of TNF-α at the blood-brain barrier of the rat. Am J Physiol 2002;283:E899–E908. [38] Pan, W.; Banks, W. A.; Fasold, M. B.; Bluth, J.; Kastin, A. J. Transport of brain-derived neurotrophic factor across the blood-brain barrier. Neuropharmacology 1998;37:1553–1561. [39] Pan, W.; Banks, W. A.; Kastin, A. J. BBB permeability to ebiratide and TNF in acute spinal cord injury. Exp Neurol 1997;146: 367–373. [40] Pan, W.; Banks, W. A.; Kennedy, M. K.; Gutierrez, E. G.; Kastin, A. J. Differential permeability of the BBB in acute EAE: enhanced transport of TNF-α. Am J Physiol 1996;271:E636–E642. [41] Pan, W.; Csernus, B.; Kastin, A. J. Upregulation of p55 and p75 receptors mediating TNFα transport across injured blood-spinal cord barrier. J Mol Neurosci 2002;21:173–184. [42] Pan, W.; Kastin, A. J. Entry of EGF into brain is rapid and saturable. Peptides 1999;20:1091–1098. [43] Pan, W.; Kastin, A. J. Increase in TNFα transport after SCI is specific for time, region, and type of lesion. Exp Neurol 2001; 170:357–363. [44] Pan, W.; Kastin, A. J. Interactions of IGF-1 with the blood-brain barrier in vivo and in situ. Neuroendocrinology 2000;72:171– 178. [45] Pan, W.; Kastin, A. J. TNFα transport across the blood-brain barrier is abolished in receptor knockout mice. Exp Neurol 2002;174:193–200.

Permeability of the Blood–Brain Barrier to Neurotrophic Peptides / 1441 [46] Pan, W.; Kastin, A. J.; Bell, R. L.; Olson, R. D. Upregulation of tumor necrosis factor α transport across the blood-brain barrier after acute compressive spinal cord injury. J Neurosci 1999;19: 3649–3655. [47] Pan, W.; Kastin, A. J.; McLay, R. N.; Rigai, T.; Pick, C. G. Increased hippocampal uptake of TNFα and behavioral changes in mice. Exp Brain Res 2003;149:195–199. [48] Pan, W.; Kastin, A. J.; Zankel, T.; van Kerkhof, P.; Terasaki, T.; Bu, G. Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier. J Cell Sci 2004;117:5071–5078. [49] Pan, W.; Vallance, K.; Kastin, A. J. TGFα and the blood-brain barrier: accumulation in cerebral vasculature. Exp Neurol 1999; 160:454–459. [50] Pan, W.; Yu, Y.; Cain, C. M.; Nyberg, F.; Couraud, P.-O.; Kastin, A. J. Permeation of growth hormone across the blood-brain barrier. Endocrinology 2005;146:4898–4904. [51] Pan, W.; Zhang, L.; Liao, J.; Csernus, B.; Kastin, A. J. Selective increase in TNFα permeation across the blood-spinal cord barrier after SCI. J Neuroimmunol 2003;134:111–117. [52] Patlak, C. S.; Blasberg, R. G.; Fenstermacher, J. D. Graphical evaluation of blood-to-brain transfer constants from multipletime uptake data. J Cereb Blood Flow Metab 1983;3:1–7. [53] Poduslo, J. F.; Curran, G. L. Glycation increases the permeability of proteins across the blood-nerve and blood-brain barriers. Mol Brain Res 1993;23:157–162. [54] Reddy, K. R. Controlled-release, pegylation, liposomal formulations: New mechanisms in the delivery of injectable drugs. Ann Pharmacother 2000;34:915–923.

[55] Sakane, T.; Wu, D.; Pardridge, W. M. Neuropeptide pegylation: reduction in peripheral peptide metabolism causes parallel reduction in apparent brain neuropeptide uptake. FASEB J 1997;11:3628. [56] Schafer, T.; Zentgraf, H.; Zehe, C.; Brugger, B.; Bernhagen, J.; Nickel, W. Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells. J Biol Chem 2004;279:6244– 6251. [57] Shitara, Y.; Kato, Y.; Sugiyama, Y. Effect of brefeldin A and lysosomotropic reagents on intracellular trafficking of epidermal growth factor and transferrin in Madin-Darby canine kidney epithelial cells. J Controll Release 1998;55:35–43. [58] Somogyvari-Vigh, A.; Pan, W.; Reglodi, D.; Kastin, A. J.; Arimura, A. Effect of middle cerebral artery occlusion on the passage of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier in the rat. Regul Pept 2000;28: 89–95. [59] Sugiyama, Y.; Kato, Y. Pharmacokinetic aspects of peptide delivery and targeting—importance of receptor-mediated endocytosis. Drug Dev Industr Pharm 1994;20:591–614. [60] Triguero, D.; Buciak, J.; Pardridge, W. M. Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins. J Neurochem 1990; 54:1882–1888. [61] Xiang, S.; Pan, W.; Kastin, A. J. Strategies to create a regenerating environment for the injured spinal cord. Curr Pharm Des 2005;11:1267–1277.

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201 Transport of Basic Peptides at the Blood–Brain Barrier YOSHIHARU DEGUCHI AND TETSUYA TERASAKI

initially indicated by the finding that cationization of native albumin effectively enhanced internalization in the brain capillaries and transcytosis through the BBB [6, 11]. Subsequently, it was found that many basic peptides, including small oligopeptides [13, 14, 17, 18], also undergo transcytosis through this system at the BBB (Table 1). This basic peptide transport system at the BBB may be a useful pathway for delivering peptides and macromolecules to the brain, and so an understanding of its mechanism should be helpful for rational design of effective neuroactive peptides to treat CNS disorders. In this chapter, our recent research regarding transport of basic peptides at the BBB is presented. In addition, the mechanism of the BBB basic peptide transport system is discussed and a strategy for delivering CNS-acting peptides is proposed.

ABSTRACT Although the blood–brain barrier (BBB) is impermeable to almost all peptides, a possible strategy to deliver peptides as neuropharmaceuticals is to use the basic peptide transport system at the BBB. In this chapter, we describe several successful examples, including [d-Arg2]dermorphin analogs and basic fibroblast growth factor. We also discuss the mechanism of this transport process and a potential strategy to modify peptides to BBB-permeable neuropharmaceuticals.

INTRODUCTION Prospects are bright in the twenty-first century for the treatment of central nervous system (CNS) disorders, such as depression, Alzheimer’s dementia, and stroke, with novel CNS-acting drugs, including peptides, recombinant proteins, monoclonal antibodies, and gene medicines. However, systemically administered hydrophilic peptides and large macromolecules are generally not efficiently distributed into the brain due to the existence of the blood–brain barrier (BBB), which consists of brain capillary endothelial cells linked with tight junctions. One possible strategy to overcome this problem is to use endogenous peptide transport systems at the BBB. Many influx and efflux transport systems are expressed at the BBB, including transporter-mediated translocation systems and receptor-mediated transcytosis systems (Fig. 1). These transporter/receptor systems have comparatively strict substrate specificity. On the other hand, there is a unique transport system (known as the basic peptide transport system) that mediates the size-independent BBB transport of a wide variety of basic peptides and proteins (Table 1). Its existence at the BBB was Handbook of Biologically Active Peptides

Basic Oligopeptides and Proteins Not only synthetic cationized proteins such as cationized albumin but also basic natural peptides and proteins, including protamine [8], histone [10], the extracellular domain of the lymphocyte CD4 receptor [9], and basic fibroblast growth factor (bFGF) [4], are transported by the basic peptide transport system at the BBB. These basic peptides have a preponderance of arginine and lysine residues, relative to glutamate and aspartate residues. The basic peptide transport system at the BBB also mediates the transport of relatively small basic oligopeptides such as E-2078 (a dynorphin analog) [17, 18] and ebiratide (an adrenocorticotropic hormone, ACTH, analog) [13, 14, 19] (Table 1). Recently, we reported that dermorphin analogs with arginine in their structure (i.e., [d-Arg2]dermorphin analogs) are also transported by the basic peptide transport system (Table 1) [2, 3].

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1444 / Chapter 201 Astrocyte Foot Process Brain Capillary Endothelium Blood

?

1

2

3

4

Transport System 1 Carrier-mediated influx system 2 Carrier-mediated efflux system 3 Receptor-mediated influx system 4 Receptor-mediated efflux system 5 Basic peptide transport system 6 TAT-related protein transport system

5

6

Brain Parenchyma

Transporter/Receptor Suggested Oatp/OATP, PTSs P-glycoprotein (MDR1), PTSs Insulin receptor, Transferrin receptor Leptin receptor, AT1 receptor Apo-transferrin receptor, IgG2a receptor

FIGURE 1. Peptide transport systems at the blood–brain barrier. Oat/OATP, organic anion transporting-polypeptides; PTSs, peptide transport systems.

[d-Arg2]dermorphin Analogs TAPA (a μ1-specific opioid peptide, H-Tyr-d-Arg-Phe-βAla-OH) has been designed based on structure–activity relationship studies of dermorphin (H-Tyr-d-Ala-Phe-GlyTyr-Pro-Ser-NH2) and [d-Arg2]-kyotorphin (Tyr-d-Arg) [12]. TAPA produces potent antinociceptive activity with low physical and psychological dependence when administered subcutaneously. The isoelectric point of TAPA is calculated to be 9.6, and the molecule is cationic. Following an intravenous injection to mice, 125I-TAPA (7.3 pmol/g BW) was taken up into the brain with the BBB permeation influx rate (PSBBB,inf) of 0.265 ± 0.025 μl/(min⋅g of brain). The PSBBB,inf of 125I-TAPA was significantly inhibited by 70% following the coadministration of unlabeled TAPA (33 nmol/ g BW), suggesting that TAPA is transported across the BBB via a specific transport mechanism in vivo. In addition, the internalization of 125I-TAPA into the conditionally immortalized mouse brain capillary endothelial cells (TM-BBB4) showed temperature- and concentration-dependency with a half-saturation constant (Kd) of 10.0 ± 1.7 μM. The internalization of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor), and poly-l-lysine and protamine (polycations). These results are consistent with transport across the BBB by adsorptive-mediated endocytosis. Furthermore, the relationship between transport activity and the chemical structure of [d-Arg2]dermorphin analogs was examined using

ADAB, ADAMB, and TMPA (Table 1). The internalization of 125I-TAPA into TM-BBB4 cells was concentration-dependently inhibited by ADAM and ADAMB (pI 11.3), both of which have arginine residue in their structures. TMPA (pI 5.7; H-Tyr-D-MetO(RS)-Phe-Me-βAla-OH), which does not contain arginine, did not inhibit the internalization of 125ITAPA in TM-BBB4 cells. These results suggest that the arginine residue plays an important role in the BBB permeation of [d-Arg2]dermorphin analogs.

bFGF bFGF is an 18-kDa polypeptide composed of 154 amino acid residues with an isoelectric point of 10.1, and it is a candidate agent for treatment of neurogenerative disorders, such as brain ischemia. We demonstrated that bFGF is transcytosed through the BBB after the intravenous administration of the labeled peptide [4]. This transcytosis was significantly inhibited by simultaneously perfusing the brain with poly-l-lysine (300 μM), suggesting that bFGF is transported by the basic peptide transport system at the BBB. In addition, the internalization of 125I-bFGF in TM-BBB4 cells was time-, temperature-, osmolarity-, and concentrationdependent and was significantly inhibited by poly-llysine and compounds that contain sulfate moieties, such as heparin and chondroitin sulfate-B [5]. The internalization was also significantly reduced by the

TABLE 1. Kinetic Parameters for Basic Oligopeptides and Proteins in in Vitro Blood–Brain Barrier Models.a Sequence

M.W.

TAPA ADAMB ADAM E-2078 Ebiratide

H-Tyr-D-Arg-Phe-β-Ala-OH Nα-amidino-Tyr-D-Arg-Phe-methyl-βAla-OH Nα-amidino-Tyr-D-Arg-Phe-βAla-OH CH3-Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5 H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2

663.7 719.7 705.7

C-AVP4-9 001-C8 bFGF

pGlu-Asn-Cys(Cys-Pro-His-Arg)-Pro-Arg-Gly-NH2 H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2

Cationized albumin Histone

1165.0 760.0 17 kDa 210 kDa

Kd (μM)

Bmax (pmol/mg P)

10.0 5.68 3.76 4.62 62.1 15.9 16.4 1.33 0.076 0.0365 0.8 15.2

4.98 22.5 12.8 147 144 7.96 14.7 15.3 183 206 79 7700

pI 9.6 11.3 11.3 10.0 10.0 9.8 12.5 10.1 8.5–9

In Vitro Model

Source

TM-BBB4 TM-BBB4 TM-BBB4 B-CAP B-CAP BCEC MBEC4 BCEC TM-BBB4 B-CAP BCEC B-CAP

[2] [3] [3] [17] [13, 14] [19] [16] [15] [5] [4] [6] [7]

a Bmax, maximal binding capacity; B-CAP, isolated bovine brain capillaries; BCEC, primary cultured bovine brain capillary endothelial cells; Kd, half-saturation constant; MBEC4, immortalized mouse brain endothelial cells; M.W., molecular weight; pI, calculated or experimentally determined isoelectric point; TM-BBB4, conditionally immortalized mouse brain capillary endothelial cells.

Transport of Basic Peptides at the Blood–Brain Barrier / 1445

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1446 / Chapter 201 removal of heparan sulfate proteoglycan (HSPG) from the extracellular matrix of the cells, suggesting HSPGmediated adsorptive-mediated endocytosis of bFGF. However, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analyses suggested that the expression of perlecan, an HSPG core protein, was greater in the abluminal membrane than in the luminal membrane of the mouse brain capillary cells. Therefore, the in vitro studies are, at least in part, consistent with endocytosis from the abluminal side of the brain capillary cells.

TRANSPORT MECHANISM The mechanism underlying basic peptide transport at the BBB has been suggested to be adsorptivemediated endocytosis which is triggered after the binding of peptides to cell-surface anionic sites. Ultracytochemical examination has suggested that the anionic sites located on the luminal membrane of the brain capillary endothelium are mainly sialic acid residues of acidic glycoproteins [20]. A transendothelial pathway of adsorptive-mediated transcytosis has been proposed, based on a study using horseradish peroxidase (HRP) conjugated with the lectin wheat germ agglutinin (WGA) [1]. WGA-HRP is internalized in the endocytotic vesicles, which may, in part, directly undergo exocytosis. Other endocytotic vesicles are conveyed to the Golgi complex, and subsequently transport vesicles from the Golgi saccule may engage in exocytosis at both the abluminal and luminal surfaces. Arginine-rich basic peptides, such as TAT protein transduction domain (TAT-PTD) may be internalized by lipid raft-dependent macropinocytosis, which is independent of caveolar- and clathrin-mediated endocytosis [21] (Fig. 1). The adsorptive-mediated endocytosis at the BBB has been functionally characterized as time-, temperature-, and energy-dependent. Internalization is inhibited by endocytosis inhibitors, such as phenylarsine oxide and dansylcadaverine. The values of half-saturation constant (Kd) and maximum binding capacity (Bmax) of basic peptides and proteins, measured in in vitro BBB models, are listed in Table 1. The Kd values obtained are much greater than those for receptor-mediated endocytosis (e.g., insulin 1.2 nM, transferrin 5.6 nM, and leptin 5.1 nM) [7]. Tamai et al. have investigated the relationship between peptide structure and internalization activity by adsorptive-mediated endocytosis at the BBB, using the novel synthetic peptide 001-C8 (Table 1) and its derivatives with various numbers of basic and neutral amino acids and with various C-terminal structures [15]. They suggested that not the number of constitu-

ent amino acids of the peptide but rather the Cterminal structure and the basicity of the peptide are the key determinants of internalization activity for 001-C8 analogs.

PEPTIDE DELIVERY TO THE BRAIN VIA ADSORPTIVE-MEDIATED TRANSPORT Most neuropeptides do not cross the BBB when they are systemically administered. A possible approach to solve this problem is a prodrug approach by adding basic peptide fragment into the peptide structure in order to enhance the rate of adsorptive-mediated transcytosis at the BBB. This strategy was successfully applied to a cationic arginine-vasopressin fragment 4–9 (C-AVP4–9) analog, a prodrug of AVP4–9, which possesses a potent memory-facilitative effect in vivo [16]. CAVP4–9 (pI 9.8) was synthesized by introducing Arg-HisPro into AVP4–9. Because C-AVP4–9 is converted to the active peptide by the postproline cleaving enzyme (PPCE) [22], C-AVP4–9 is more effective than AVP4–9 if C-AVP4–9 is transported through the BBB more efficiently than AVP4–9 (Fig. 2). 125I-labeled C-AVP4–9 was taken up by the brain with threefold greater BBB permeability than 35S-labeled AVP4–9. An in vitro study with mouse-brain capillary endothelial cells immortalized by SV40 infection (MBEC4) demonstrated that C-AVP4–9 was internalized into the cells by adsorptive-mediated endocytosis.

CONCLUSION The chemical modification of neuropeptides to achieve high basicity can significantly enhance BBB transport via the basic peptide transport system. An increase in the basicity of a peptide may result in increased uptake by peripheral tissues in parallel with increased BBB permeability because adsorptivemediated endocytosis operates in the peripheral tissues. This problem might be overcome by further clarifying the mechanism underlying adsorptive-mediated transcytosis at the BBB and by identifying brain capillaryspecific surface binding sites for basic peptides, which make adsorptive-mediated transcytosis work efficiently at the BBB. Research on the basic peptide transport system at the BBB is still at an early stage.

Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research, and a 21st Century Center of Excellence (COE) Program from Japan Society for the Promotion of Science.

Transport of Basic Peptides at the Blood–Brain Barrier / 1447

Blood-Brain Barrier Brain Parenchymal Side

Blood Side

CNS effect Arginine-vasopressin (AVP)

AVP4-9 AVP4-9, active fragment of AVP

AME Arg-His-Pro

Cys pGlu-Asn-Cys-Pro-Arg-Gly-NH2

PPCE AME C-AVP4-9, a prodrug of AVP4-9 Arg-His-Pro-Cys

C-AVP4-9

pGlu-Asn-Cys-Pro-Arg-Gly-NH2

FIGURE 2. A prodrug approach for delivering peptide to the brain was successfully applied to the study of a cationic arginine-vasopressin fragment 4–9 (C-AVP4–9) analog, designed as a prodrug of AVP4–9, which possesses a potent memory-facilitative effect in vivo.

References [1] Broadwell RD, Balin BJ, Salcman M. Transcytotic pathway for blood-borne protein through the blood-brain barrier. Proc Natl Acad Sci USA 1988; 85: 632–636. [2] Deguchi Y, Miyakawa Y, Sakurada S, Naito Y, Morimoto K, Ohtsuki S, Hosoya K, Terasaki T. Blood-brain barrier transport of a novel μ1-specific opioid peptide, H-Tyr-d-Arg-Phe-β-Ala-OH (TAPA). J Neurochem 2003; 84: 1154–1161. [3] Deguchi Y, Naito Y, Ohtsuki S, Miyakawa Y, Morimoto K, Hosoya K, Sakurada S, Terasaki T. Blood-brain barrier permeability of novel [D-Arg2]dermorphin (1–4) analogs: Transport property is related to the slow onset of antinociceptive activity in the central nervous system. J Pharmacol Exp Ther 2004; 310: 177–184. [4] Deguchi Y, Naito T, Yuge T, Furukawa A, Yamada S, Pardridge WM, Kimura R. Blood-brain barrier transport of 125I-labeled basic fibroblast growth factor. Pharm Res 2000; 17: 63–69. [5] Deguchi Y, Okutsu H, Okura T, Yamada S, Kimura R, Yuge T, Furukawa A, Morimoto K, Tachikawa M, Ohtsuki S, Hosoya K, Terasaki T. Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. J Neurochem 2002; 83: 381–389. [6] Kumagai AK, Eisenberg JB, Pardridge WM. Absorptivemediated endocytosis of cationized albumin and a β-endorphincationized albumin chimeric peptide by isolated brain capillaries. J Biol Chem 1987; 262: 15214–15219. [7] Pardridge WM. Lipid-mediated transport and carrier-mediated transport of small molecules. In: Pardridge WM, editor. Brain Drug Targeting. Cambridge: Cambridge University Press; 2001, pp. 82–125. [8] Pardridge WM, Buciak JL, Kang YS, Boado RJ. Protaminemediated transport of albumin into brain and other organs in the rat. Binding and endocytosis of protamine-albumin complex by microvascular endothelium. J Clin Invest 1993; 92: 2224–2229.

[9] Pardridge WM, Buciak JL, Yoshikawa T. Transport of recombinant CD4 through the rat blood-brain barrier in vivo. J Pharmacol Exp Ther 1992; 261: 1175–1180. [10] Pardridge WM, Triguero D, Buciak J. Transport of histone through the blood-brain barrier. J Pharmacol Exp Ther 1989; 251: 821–826. [11] Pardridge WM, Triguero D, Buciak J, Yang J. Evaluation of cationized rat albumin as a potential blood-brain barrier drug transport vector. J Pharmacol Exp Ther 1990; 255: 893– 899. [12] Sakurada S, Takeda S, Sato T, Hayashi T, Yuki M, Kutsuwa M, Tan-No K, Sakurada C, Kisara K, Sakurada T. Selective antagonism by naloxonazine of antinociception by Tyr-d-Arg-Phe-βAla, a novel dermorphin analogue with high affinity at mu-opioid receptors. Eur J Pharmacol 2000; 395: 107–112. [13] Shimura T, Tabata S, Ohnishi T, Terasaki T, Tsuji A. Transport mechanism of a new behaviorally highly potent adrenocorticotropic hormone (ACTH) analog, ebiratide, through the blood-brain barrier. J Pharmacol Exp Ther 1991; 258: 459– 465. [14] Shimura T, Tabata S, Terasaki T, Deguchi Y, Tsuji A. In vivo blood-brain barrier transport of a novel adrenocorticotropic hormone analogue, ebiratide, demonstrated by brain microdialysis and capillary depletion methods. J Pharm Pharmacol 1992; 44: 583–588. [15] Tamai I, Sai Y, Kobayashi H, Kamata M, Wakamiya T, Tsuji A. Structure-internalization relationship for adsorptive-mediated endocytosis of basic peptides at the blood-brain barrier. J Pharmacol Exp Ther 1997; 280: 410–415. [16] Tanabe S, Shimohigashi Y, Nakayama Y, Makino Y, Fujita T, Nose T, Tsujimoto G, Yokokura T, Naito M, Tsuruo T, Terasaki T. In vivo and in vitro evidence of blood-brain barrier transport of a novel cationic arginine-vasopressin fragment 4–9 analog. J Pharmacol Exp Ther 1999; 290: 561–568.

1448 / Chapter 201 [17] Terasaki T, Deguchi Y, Sato H, Hirai K, Tsuji A. In vivo transport of a dynorphin-like analgesic peptide, E-2078, through the blood-brain barrier: An application of brain microdialysis. Pharm Res 1991; 8: 815–820. [18] Terasaki T, Hirai K, Sato H, Kang YS, Tsuji A. Absorptivemediated endocytosis of a dynorphin-like analgesic peptide, E-2078, into the blood-brain barrier. J Pharmacol Exp Ther 1989; 251: 351–357. [19] Terasaki T, Takakuwa S, Saheki A, Moritani S, Shimura T, Tabata S, Tsuji A. Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary

cultured bovine capillary endothelial cells. Pharm Res 1992; 9: 529–534. [20] Vorbrodt AW. Ultracytochemical characterization of anionic sites in the wall of brain capillaries. J Neurocytol 1989; 18: 359–368. [21] Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 2004; 10: 310–315. [22] Yoshimoto T, Ogita K, Walter R, Koida M, Tsuru D. Post-proline cleaving enzyme: Synthesis of new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluid of man. Biochim Biophys Acta 1979; 569: 184–192.

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202 Fibroblast Growth Factor and the Blood–Brain Barrier CONRAD E. JOHANSON, JOHN E. DONAHUE, EDWARD G. STOPA, AND ANDREW BAIRD

effects are exerted by FGF1 and FGF2 administered in the brain, resulting in significant protection against ischemia and enhanced recovery from trauma. On the other hand, there is growing evidence that these FGFs can regulate water balance at various loci in the CNS [17]. Because FGF1 and -2 have been the most extensively analyzed in normal brain, they are the two peptides that we discuss here. Both FGF1 and FGF2 and their high-affinity receptors are widely synthesized by several types of cells in the brain. Peptide and receptor variance is based on the region of the brain examined (e.g., cortex vs. subventricular nuclei), the course of development (e.g., fetal vs. adult life), and the physiological state [33] evaluated (normal vs. Alzheimer’s disease). In one example, a systematic mapping of FGF2 in the rat brain by Gonzalez et al. [10] found that FGF2 and one of its high-affinity receptors (FGFR1) localized to magnocellular neurons in the third ventricle, the ependyma of the choroid plexus, and glia in the cortex. In another, Stopa et al. [34] showed that FGF2 appears sequestered by amyloid deposits in the brains of Alzheimer’s subjects. Moreover, exogenous FGF2 crosses the blood– brain barrier (BBB) and exerts actions on the cerebrovascular wall, astrocytes, and neurons. FGF2 is also manufactured by the choroid epithelial cells and in certain situations can be detected in CSF. Although this chapter focuses on FGF2 and the BBB, it is important to note that FGF2 also interacts with the blood– cerebrospinal fluid barrier (BCSFB) and the ependyma found at the CSF–brain borders. This may be an important feature of FGF2 function in the CNS because many stem cells are localized in the periventricular region. Here, FGF2 appears to have significant biological effects on neuronal and glial progenitor cell proliferation. The diversity of FGF actions in the CNS is intimately linked to transport at several interfaces between the blood, CSF, and brain.

ABSTRACT Fibroblast growth factor (FGF) peptides in the brain regulate a plethora of metabolic, behavioral, and compensatory phenomena. FGF1 (acidic) and FGF2 (basic) peptides can permeate the three major transport interfaces in the central nervous system (CNS): the blood– brain barrier (BBB), choroid plexus, and the ependymal wall. BBB penetration of exogenous FGF can be expedited to enhance neuroprotection in the face of cerebral ischemia and trauma and to stimulate neurogenesis. FGF synthesized and secreted by the astroglia and choroidal epithelium modulate cerebral endothelial functions (angiogenesis and BBB permeability) and CSF formation (fluid balance), respectively. A better understanding of peptide delivery to the hypothalamus and other periventricular regions should enable more effective management of neuroendocrine diseases and the conversion of stem cells to new neurons.

INTRODUCTION There are now nearly two dozen genes known to encode proteins of the fibroblast growth factor (FGF) family [37]. Yet, even though it has been over two decades since acidic FGF (FGF1) and basic FGF (FGF2) were characterized, their physiological function in tissues such as the brain has remained enigmatic. As multifunctional peptides [14] that are present with spatiotemporal variance in neurons, glia, and microvasculature of the central nervous system (CNS), the FGFs are thought to play a role in the brain’s development, the maintenance of its structural integrity, and the progression of some neurodegenerative diseases. FGF peptides regulate angiogenesis, cell growth, differentiation, and cellular homeostasis. On one hand, neurotrophic Handbook of Biologically Active Peptides

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1450 / Chapter 202 FGF-INDUCED PROTECTION OF CEREBRAL ENDOTHELIUM AND NEURONS FGF2 has been thoroughly evaluated for its ability to attenuate neuronal injury and cell death in the face of various stressors [19]. When available to its target cells in the CNS, FGF2 is a powerful anti-apoptotic factor that significantly protects cells. It is therefore interesting to postulate that FGFs in the brain microvasculature could protect the BBB from noxious stimuli while at the same time providing trophic relief to neurons. FGF2 is certainly upregulated after CNS injury. Moreover, the BBB is the foremost physical line of defense used by the CNS against circulating toxins and pathogens. When damaged by cytotoxic proteins (e.g., human immunovirus, HIV, gp120), the barrier becomes less capable of excluding foreign elements from the brain. Studies with FGF target cells, including brain capillary endothelium, reveal that pretreating cells with FGF2 [23] greatly protects the endothelial cells from physical and chemical stressors (e.g., gp120 angiotoxicity). Such findings intimate that FGF2 plays a significant role in maintaining the integrity of the BBB challenged with noxious stimulants. Similarly, FGFs are powerful neuroprotective agents in vitro and in vivo, leading numerous investigators to propose that they have homeostatic roles in the CNS. FGF-mediated angioprotection to maintain endothelial fitness is another pharmacological strategy for maximizing the protection of neurons against systemic disorders.

FGF1 AND FGF2 TRANSPORT ACROSS THE BLOOD–BRAIN BARRIER Many neurotrophic factors such as the FGFs provide protection for neurons in culture. Few are like FGF2, that is, capable of modulating the function of cells derived from mesenchyme, endoderm, and neuroectoderm. As such, the presence of FGFs at specific loci can have profound effects on cellular homeostasis. Thus, FGF delivery can be critical to activity. In vivo, the FGFs and neurotrophic factors are most effective when the BBB is circumvented by administration directly into the brain parenchyma or via the CSF [18]. Their large size and hydrophilicity generally preclude the rapid transport of growth factors across cerebral capillaries to gain access to potential neuronal targets. Moreover, because of their highly specific spatiotemporal distribution, growth factors are normally produced in local tissues in order to exert confined actions. Nevertheless, certain growth factors, including FGF2, can slowly penetrate the BBB. Using a 125I-FGF2 marker, Deguchi et al. demonstrated the transport of FGF2 across the BBB [5]. Because this transcytotic

mechanism is inefficient, pharmacological manipulations, including intranasal deliveries, can be used to enhance FGF2 transfer into the brain. With a chimeric peptide approach, vectors can expedite the normally sluggish passage of FGF2 and other neurotrophic peptides, such as brain-derived neurotrophic factor (BDNF), across cerebral microvessels [38]. Most important, when FGF2 was conjugated to a BBB delivery vector and injected intravenously, it enhanced protection against regional ischemia [32]. In this regard, biotinylated FGF2 conjugated to a monoclonal antibody against the transferrin receptor was able to efficiently penetrate the BBB through binding and uptake at the transferrin receptor in the luminal membrane of the endothelial cell. Again, the bioactivity of the penetrating FGF2 conjugate was observed by its ability to reduce infarct volume in the rat model of permanent focal ischemia [32]. Information about peptide transport and distribution pathways in the CNS is essential to the appreciation of peptidergic regulatory systems and compensatory responses. Intravenously injected peptides induce a variety of behaviors, metabolic outcomes, and reparative processes. Infusion of intravenous (IV) FGF2 into rats with unilateral entorhinal lesions resulted in an increase in hippocampal sprouting in the septodentate pathway [27]. Similarly, the IV injection of FGF1 into gerbils following 5 min of transient brain ischemia provided neuroprotection in the hippocampus [3]. Moreover, the penetration of blood-borne FGF into CA1 neurons was demonstrated autoradiographically [3]. Thus, at least in animals, pharmacological doses of IV FGF given postinjury cross the BBB and protect neurons from delayed cell death. This prompts the consideration of stimulating peripheral systems to upregulate endogenous FGF2 to counter CNS pathology. That FGF peptides penetrate the BBB has also been deduced from studies of regulatory systems for pain, sleep, and neurogenesis. Three different administration routes leading to vascular, then brain, uptake of FGF peptides are represented here. First, the intraperitoneal administration of FGF1 to rats induced analgesia as measured by tail-flick latency [4]. Interestingly, neither hybrid (CYLT-FGF1) nor heat-inactivated FGF1, although bioactive, crossed the BBB or protected against the pain stimulus. Second, an intravenous bolus of FGF1 to rabbits promoted an increase in sleep duration [9]. This somnogenic effect, which was dependent on nitric oxide synthase activity, lasted for approximately 1 hour after the apparent penetration of the BBB by FGF1. Third, a subcutaneous injection of FGF2 stimulated neurogenesis in both the immature and mature rat CNS [36]. Such peripheral FGF2 presentation led to the incorporation of labeled thymidine by cells of the hippocampus, dentate gyrus, and subventricular zone. The ability of systemic FGF2 to penetrate brain capillaries

Fibroblast Growth Factor and the Blood–Brain Barrier / 1451 and stimulate neuron production indicates that neurogenesis is controlled by peripheral as well as central factors. Therefore, peptide transport mechanisms in the cerebral endothelium are an integral part of feedback loops and other endocrinelike means to integrate the function of distant organs with that of the brain.

FGF SECRETION BY MICROVASCULAR ENDOTHELIAL CELLS The concept that in vivo BBB endothelium is a potential target for vectors to expedite FGF delivery for angiogenesis and neuronal health stems from earlier work using FGF2-transfected fibroblasts for effective FGF2 gene delivery to promote recovery of dopaminergic neurons in the substantia nigra of lesioned animals. These studies led to the possibility that microvascular cell lines transfected with genes encoding FGFs could be transplanted into brain. Johnston et al. used a chimeric FGF gene in rat microvascular endothelial cells to enhance the secretion of bioactive FGF1 [21]. The surgical implantation of these FGF1-secreting endothelial cells remained viable in rat caudate-putamen for over 3 weeks. Although such findings demonstrate that endothelial cells can be modified to enhance FGF peptide delivery to neurons, it also introduces the possibility of manipulating the endogenous brain capillary endothelium to augment the delivery of growth factors to the CNS. If appropriately engineered and regulated, the genes for these trophic factors could be used to control drug delivery by regulating the barrier itself.

FGF MODULATION OF CEREBRAL ENDOTHELIAL CELLS The BBB not only transports FGF peptides but is also modulated by them in order to maintain structural and functional integrity of the endothelium. Indeed the permeability and transport capabilities of the BBB are regulated by a variety of cytokines and growth factors. There are debilitating effects of diseases on various components of the BBB. Consequently, it is pertinent to gain insight on the diverse actions of FGF in view of the possible therapeutic reconstitution of disruptions in brain microvessels. In vivo and culture studies of the BBB have provided evidence for significant plasticity of the cerebral endothelial cells in health and disease because FGF2, in particular, exerts a wide spectrum of effects on the cerebromicrovasculature. FGF2 has potent angiogenic effects. The proliferation and differentiation of the endothelium are integral to angiogenesis. Mouse brain capillary endothelial cells of the H-2Kb-tsA58 line uniquely responded (compared

to other growth factors) to exogenous FGF1 or FGF2 by proliferating and differentiating. This angiogenic response was mediated by the endothelial FGF receptor 1 [22]. Endogenous FGF2 was found in the endothelial cytoplasm of the immature invading capillaries but not in the adult-counterpart vessels [30]. Thus, FGF2 has a primary role in regulating brain endothelial cell division and specialization. FGF2 modulates the expression of endothelial proteins involved in transport and permeability phenomena. The BBB GLUT1 glucose transporter has a key role in facilitating the movement of glucose from blood to brain. FGF2 moderately induced GLUT1 gene expression in cultured bovine brain capillary endothelial cells [1]. FGF2 has also been implicated in preventing permeability increases in the hypoxia-stressed in vitro BBB. Conditioned media containing FGF2 and vascular endothelial growth factor (VEGF) thwarted permeability increases in hypoxia, presumably through the upregulation of the tight junction proteins actin and claudin-1 [2]. Because astrocytes help to regulate BBB permeability, it is equally important to recognize the role played by astrocyte-derived FGF2 in the maintenance of BBB intregrity. Cerebral endothelial cells receive astrocytesecreted FGF2 at the abluminal membrane, which expresses perlecan. There is evidence that perlecan, a core protein of heparan sulfate proteoglycan, helps to internalize FGF2 and thereby stabilize BBB permeability [6]. Transgenic mice deficient in FGF2 and FGF5 displayed a defect both in the perivascular astroglial endfeet and in BBB permeability [29]. This phenotype is rescued by exogenous FGF2, thus pointing to a possible future role of supplemental FGF2 for repairing a leaky BBB in diseases.

FGF2 TRANSPORT AND REGULATORY PHENOMENA AT THE CHOROID PLEXUS Another route for FGF2 delivery into the CNS is through the choroid plexus and into CSF. Barriers within the choroid plexus (CP) have structural and functional characteristics that are anatomically and functionally different from those of the cerebral endothelium [16]. Within the choroidal capillaries the BBB is absent, but a barrier is created via the choroidal epithelium. This BCSFB of the CP is considered generically to be part of the BBB system, albeit more specialized in that it regulates the trafficking of water-soluble molecules (including peptides) into and out of the CNS. FGF2 may be transported across CP, from blood to CSF, in addition to being synthesized and secreted from the BCSFB into the ventricles [31]. Normally the CSF titer of FGF2 is barely detectable. However, the increasing

1452 / Chapter 202 concentration of FGF2 in CSF in certain pathological states points to a role of this growth factor in repairing injured brain. Forebrain ischemia destabilizes both the hippocampal regions and the lateral ventricle CPs [8, 19]. The rapid repair of the ischemically injured epithelial cells in the plexuses is a key factor in minimizing damage to the CA regions of the hippocampi [19]. The upregulation of growth factors in the CP-CSF system postischemia/reperfusion is evidently a major event in stabilizing the BCSFB, including the reconstitution of the choroid epithelial cells harmed by the stroke insult [19]. This has been demonstrated in a model of transient forebrain ischemia (6–10 min) in adult rats. Thus, exogenous FGF2 infused into a lateral ventricle attenuated the damage to both the choroidal epithelium and CA1 [12]. Accordingly, FGF2 and a host of other growth factors secreted by the CP confer protection not only to neuronal networks but also to other cellular elements defending the functional integrity of the brain interior. The impressive ability of the CP in adult animals to recover from acute ischemic episodes [19] is probably rooted in the same growth factor systems (e.g., FGF2 and its receptors) that appear early in embryonic development and guide the formation of the CSF-brain. There are multiple receptors for FGF isoforms in the fetal choroidal tissues [28]. Early modulation of the BCSFB by FGF2 (and other growth factors) leads to epithelial differentiation, CSF and ventricle formation, and hydrostatic pressure development. These physiological events are critical to the orderly progression of ontogenetic phenomena that underlie CNS growth and well-being. Subsequently, in healthy young adults there seems to be a waning of FGF2 expression at the CNS transport interfaces and in the parenchyma to relatively quiescent levels; but such downregulation of FGF2 may later be boosted (upregulated) in response to ischemia, trauma, and fluid-imbalance disorders [12, 13, 17, 19]. There is emerging evidence that FGF2 regulation at the CP includes a role for effecting water balance in the CNS. FGF2 expression in the choroidal epithelium is markedly augmented by dehydration [17]. Because arginine vasopressin (AVP) in the plexus is also upregulated during hyperosmolality [39], it is tempting to link FGF2 with AVP in a peptide-coupled mechanism that regulates CSF formation rate. In support of this idea are the observations that FGF2 [11, 20], like AVP [7], reduces fluid production at the BCSFB. Moreover, the FGF2–FGF receptor system in the CP colocalizes with AVP in a subpopulation of choroid epithelial cells [35]. The action of FGF2 as a fluid-regulating peptide is a newly appreciated concept in neuroendocrinology. In this regard, FGF2 may work by controlling the release

of AVP, which then downregulates CSF formation [35]. Growth factor interaction with neuropeptides such as AVP thus adds a further layer of complexity to the model of peptidergic modulation of fluid transfer at CNS transport boundaries.

THE CSF-EPENDYMAL-BRAIN INTERFACE FGF2 secreted by CP can exert autocrine and paracrine effects on the BCSFB [35], but this growth factor can also be carried by bulk flow (volume transmission) to target sites in periventricular regions [15]. Choroidally derived and secreted FGF2 is likely conveyed to the subventricular zone, where it exerts neuroendocrinelike trophic and mitogenic effects on stem cells [24]. When this CSF-to-brain transport pathway is blocked, as in some hydrocephalus disorders, faulty neurogenesis may ensue due to the compromised delivery of growth factors to progenitor cells [24]. In the case of forebrain ischemia and damage to the hippocampus, the CP secretion of growth factors and other proteins evidently helps to attenuate injury to the CA1 regions [19]. This notion is reinforced by experimental findings that intracerebroventricularly administered exogenous FGF2 reduces damage to CA1 neurons [12]. The significant role of the CP in stabilizing the hippocampus in stroke has been emphasized by Johanson et al. [19] and by Ferrand-Drake [8]. Peptides have a major role in mediating these homeostatic processes at the BCSFB [31]. The CP-CSF-ependyma nexus is thus a critical delivery pathway in supplying growth factors such as FGF2 to the interior of the brain to buffer damage in the face of hypoperfusion and other oxidative insults. Moreover, there are regional differences in the ependyma [13] with regard to the transport and distribution of FGF2 into subventricular zones and white matter. Clearly the brain is dependent on receiving FGF2 and other factors from the CSF as well as the cerebral capillaries in order to maintain health and repair stressed neuronal networks [18].

MODELING OF PEPTIDE REGULATORY SYSTEMS: INTEGRATING MULTIPLE LIGANDS AND TRANSPORT INTERFACES Peptide-regulatory systems for behaviors such as feeding, satiety, sleep, and reproduction are extremely complex and interactive. Elsewhere, Pan and Kastin [26] have reviewed how the BBB can mediate interactions of cytokines and growth factors between the brain and peripheral systems. It is equally likely that the BCSFB mediates comparably important functions.

Fibroblast Growth Factor and the Blood–Brain Barrier / 1453 Systemic peptide signals are transferred across barrier interfaces from the blood to the CSF [31] and brain interstitial fluid. From there, they diffuse or are convected to their cognate receptors [15]. Some of these receptors reside in the hypothalamus and other subventricular zones and are therefore accessible to ligands from both the CSF and blood. Feeding behavior, for example, is affected by several trophic peptides that include the circulating hormone leptin and even FGF2. Leptin is transported from blood into the CNS via saturable mechanisms at both the BCSFB (choroidal epithelium) and the BBB (arcuate endothelium) (see Chapter 203 by Banks et al. in this section of the Handbook). Experimentally, blood-borne FGF2 can permeate both the CPs and cerebral capillaries. Moreover, endogenous FGF2 may derive directly from the parenchymal cells of the respective barriers. The ependyma at the CSF-brain interface also participate in the feeding-satiety response. In reaction to an ingested meal, ependymal cells release FGF1 into ventricular CSF [25] and, although the location of high-affinity receptors for this ependymal-delivered FGF1 is not known, the findings suggest that FGF within the ependymal lining contributes to neuroendocrine regulation.

CONCLUSION With the FGFs and other peptides delivered to the brain, a complete model for CNS delivery should account for peptide fluxes at three major transport interfaces: the BBB the BCSFB, and the CSF-brain interface. The last 30 years has clearly seen tremendous progress in understanding the role played by the BBB in affecting drug delivery to the CNS. To complement the BBB, however, it is becoming clearer that peptide transfer at other sites may prove to be salient in brain homeostasis. Specifically, studies with the FGFs point to integrative neuroendocrine roles played by the CP, the tanycytes (specialized epithelium in the ependymal wall), and the ependyma surrounding the circumventricular organs. A better understanding of the peptide transport performed by these CSF structures and the BBB will enable a more effective pharmacological manipulation of endogenous peptidergic secretory systems, a better management of CNS diseases, and the development of novel strategies for drug delivery to the brain [16].

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[2] Brown RC, Mark KS, Egleton RD, Huber JD, Burroughs AR, Davis TP. Protection against hypoxia-induced increase in bloodbrain barrier permeability: role of tight junction proteins and NFkappaB. J Cell Sci 2003;116(Pt 4):693–700. [3] Cuevas P, Carceller F, Munoz-Willery I, Gimenez-Gallego G. Intravenous fibroblast growth factor penetrates the blood-brain barrier and protects hippocampal neurons against ischemiareperfusion injury. Surg Neurol 1998;49(1):77–83. [4] Cuevas P, Prieto R, Saenz de Tejada I, Gimenez-Gallego G. Analgesic effects of fibroblast growth factor in the rat. Neurosci Lett 1996;207(3):175–8. [5] Deguchi Y, Naito T, Yuge T, Furukawa A, Yamada S, Pardridge WM, Kimura R. Blood-brain barrier transport of 125I-labeled basic fibroblast growth factor. Pharm Res 2000;17(1):63–9. [6] Deguchi Y, Okutsu H, Okura T, Yamada S, Kimura R, Yuge T, Furukawa A, Morimoto K, Tachikawa M, Ohtsuki S, Hosoya K, Terasaki T. Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. J Neurochem 2002;83(2):381–9. [7] Faraci FM, Mayhan WG, Heistad DD. Effect of vasopressin on production of cerebrospinal fluid: possible role of vasopressin (V1)-receptors. Am J Physiol 1990;258:R94–8. [8] Ferrand-Drake M. Cell death in the choroid plexus following transient forebrain global ischemia in the rat. Microsc Res Tech 2001;52(1):130–6. [9] Galan JM, Cuevas B, Dujovny N, Gimenez-Gallego G, Cuevas P. Sleep promoting effects of intravenously administered acidic fibroblast growth factor. Neurol Res 1996;18(6):567–9. [10] Gonzalez AM, Logan A, Ying W, Lappi DA, Berry M, Baird A. Fibroblast growth factor in the hypothalamic-pituitary axis: differential expression of fibroblast growth factor-2 and a high affinity receptor. Endocrinology 1994;134(5):2289–97. [11] Hakvoort A, Johanson CE. Growth factor modulation of CSF formation by isolated choroid plexus: FGF-2 vs. TGF-beta1. Eur J Pediatr Surg 2000;10 Suppl 1:44–6. [12] Hayamizu TF, Chan PT, Doberstein CE, Sunwoo LW, Guglielmo MA, Johanson CE. The role of FGF-2 in the response to cerebral ischemia: FGF-2 expression in the hippocampus and choroid plexus, and the neuroprotective effects of intraventricular FGF2 infusion in a rat model of transient forebrain ischemia. Soc Neuroscience Abs 1999;25:1851. [13] Hayamizu TF, Chan PT, Johanson CE. FGF-2 immunoreactivity in adult rat ependyma and choroid plexus: responses to global forebrain ischemia and intraventricular FGF-2. Neurol Res 2001;23(4):353–8. [14] Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet 2004;20(11):563–9. [15] Johanson C. The choroid plexus-CSF nexus: gateway to the brain. In Neuroscience in Medicine, P. Michael Conn, ed., Humana Press, Totowa, NJ, 2003;165–195. [16] Johanson CE, Duncan JA, Stopa EG, Baird A. Enhanced prospects for drug delivery and brain targeting by the choroid plexus-CSF route. Pharm Res 2005;22(7):1011–37. [17] Johanson CE, Gonzalez AM, Stopa EG. Water-imbalance-induced expression of FGF-2 in fluid-regulatory centers: choroid plexus and neurohypophysis. Eur J Pediatr Surg 2001;11 Suppl 1:S37–8. [18] Johanson CE, McMillan PN, Palm DE, Stopa EG, Doberstein CE, Duncan JA. Volume transmission-mediated protective impact of choroid plexus-CSF growth factors on forebrain ischemic injury. In Blood-Spinal Cord and Brain Barriers in Health and Disease, H.S. Sharma and J. Westman, eds., Academic Press, San Diego, 2003; 361–84. [19] Johanson CE, Palm DE, Primiano MJ, McMillan PN, Chan P, Knuckey NW, Stopa EG. Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms. Cell Mol Neurobiol 2000;20(2):197–216.

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brain barrier permeability: evidence from mouse mutants. J Neurosci 2003;23(16):6404–12. Schechter J, Pattison A, Pattison T. Basic fibroblast growth factor within endothelial cells during vascularization of the anterior pituitary. Anat Rec 1996;245(1):46–52. Smith DE, Johanson CE, Keep RF. Peptide and peptide analog transport systems at the blood-CSF barrier. Adv Drug Deliv Rev 2004;56(12):1765–91. Song BW, Vinters HV, Wu D, Pardridge WM. Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector. J Pharmacol Exp Ther 2002;301(2):605–10. Stopa EG, Berzin TM, Kim S, Song P, Kuo-LeBlanc V, RodriguezWolf M, Baird A, Johanson CE. Human choroid plexus growth factors: what are the implications for CSF dynamics in Alzheimer’s disease? Exp Neurol 2001;167(1):40–7. Stopa EG, Gonzalez AM, Chorsky R, Corona RJ, Alvarez J, Bird ED, Baird A. Basic fibroblast growth factor in Alzheimer’s disease. Biochem Biophys Res Commun 1990;171(2):690–6. Szmydynger-Chodobska J, Chun ZG, Johanson CE, Chodobski A. Distribution of fibroblast growth factor receptors and their co-localization with vasopressin in the choroid plexus epithelium. Neuroreport 2002;13(2):257–9. Wagner JP, Black IB, DiCicco-Bloom E. Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor. J Neurosci 1999;19(14):6006–16. Wiedlocha A, Sorensen V. Signaling, internalization, and intracellular activity of fibroblast growth factor. Curr Top Microbiol Immunol 2004;286:45–79. Wu D. Neuroprotection in experimental stroke with targeted neurotrophins. NeuroRx 2005;2(1):120–8. Zemo DA, McCabe JT. Salt-loading increases vasopressin and vasopressin 1b receptor mRNA in the hypothalamus and choroid plexus. Neuropeptides 2001;35:181–8.

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203 Ingestive Peptides and the Blood–Brain Barrier WILLIAM A. BANKS, RYOTA NAKAOKE, AKIHIKO URAYAMA, AND THANH Q. VO

mined by the presence or absence of saturable transporters, the physicochemical properties of the substance, which, in turn, determine the rate of nonsaturable transmembrane diffusion, the pharmacokinetic parameters (clearance from blood and volume of distribution), capillary sequestration, and binding in plasma. Efflux rates and CNS retention are determined by many factors that include the presence or absence of saturable efflux transporters, cerebrospinal fluid (CSF) reabsorption, degradation with the CNS, and uptake and sequestration by CNS tissues. The saturable transport systems are themselves regulated by physiological parameters and can be affected in disease states. Taken together, this means that the BBB is a pivotal point in the humorally based communications between the CNS and the GI tract. Understanding this role of the BBB is necessary to understanding how the GI tract and CNS interact under physiological conditions and is needed to explain some aspects of disease. In this chapter, we discuss selected examples of peptides and regulatory proteins and some of the major factors regulating their interactions with the BBB.

ABSTRACT The passage of substances such as leptin, ghrelin, and insulin across the blood–brain barrier (BBB) has emerged in the last decade as a major mechanism by which the central nervous system (CNS) and gastrointestinal (GI) tract communicate. Saturable transport systems in the brain or blood directions exist for many of these substances, and others cross by nonsaturable transmembrane diffusion. The transporters are themselves regulated and dysregulation results in disease. For example, the immune regulation of insulin transport could explain the insulin resistance of sepsis and the impaired transport of leptin as an early cause of the leptin resistance seen in obesity.

INTRODUCTION The discovery in the late 1970s that the central nervous system (CNS) and gastrointestinal (GI) tract use many of the same peptides and regulatory proteins renewed speculations about how these regions communicate [6]. The blood–brain barrier (BBB) plays a major role in communication between the GI tract and the CNS [8, 9]. Clear examples of this communication are provided by peptides and regulatory proteins that arise from the GI tract and reach their targets in the CNS by being transported by saturable systems across the BBB. It was assumed for many years that even small peptides were too large to cross the BBB. However, when experiments were carefully conducted, it was discovered that many of the principles governing the passage of small molecules also applied to peptides and to even the larger regulatory proteins [2, 4]. Specifically, the accumulation of substances by the brain from the blood is a balance between influx and efflux rates. Influx rates and CNS accumulation are primarily deterHandbook of Biologically Active Peptides

INSULIN The question of whether insulin crosses the BBB was posed as early as 1954. Insulin has powerful effects on feeding and energy regulation mediated through its receptors in peripheral tissues [11]. However, insulin also has receptors in the brain. Within the CNS, the largest concentration of insulin, insulin receptors, and insulin-degrading enzymes is in the olfactory bulb. Insulin through its CNS receptors has effects on body weight, feeding, glucose, and energy regulation, which are often opposite to those mediated through its peripheral receptors [13]. Insulin within the CNS thus acts as

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1456 / Chapter 203 its own counterregulatory hormone. CNS insulin has other effects on brain function, including effects on cognition. Margolis and Altszuler in 1967 proposed that insulin crossed the BBB because levels of insulin in the CSF increased after peripheral administration of insulin. Because the relation between CSF and blood levels of insulin was not linear, they proposed that insulin crossed the BBB by a saturable transport system. However, several other laboratories could not find insulin in the CSF or found insulin in the CNS of streptozotocintreated rodents. These studies appeared well into the 1980s and were interpreted as showing that insulin did not cross the BBB and that it was produced within the CNS. In a series of classic studies beginning in 1977, Woods, Porte, and collaborators clearly showed the presence of insulin in the CSF and its nonlinear relation with serum. As it became increasingly clear that little or no insulin is produced in the CSF, it also became clear that these studies showed that there was a saturable transporter for insulin. Studies from the laboratory of Pardridge reinforced this idea of insulin transport. His laboratory demonstrated binding sites on the endothelial cells that comprise the BBB. At least some of these binding sites represent insulin receptors coupled to intracellular machinery, thus providing a mechanism explaining why insulin has effects on brain endothelial cell function. But some of these binding sites could also represent a transporter mechanism. Further work by several laboratories, including our own, characterized many aspects of insulin transport across the BBB [3]. The use of radioactive insulin and of species-specific insulin radioimmunoassays not only definitely showed that insulin crosses the BBB, but also measured its influx rate and demonstrated the saturable nature of its passage. The insulin transporter is not static but it is regulated in various conditions. For example, the correlation between CSF and serum insulin is lost during hibernation, suggesting that the transporter is turned off. Because the hibernating animal is using but not acquiring energy, this finding suggests that CNS insulin is more important in the regulation of aspects of energy intake than in states of energy use. Insulin transport is also impaired in obesity, as is leptin transport. Unlike leptin, this obesity-related inhibition is not mediated by triglycerides. Insulin transport is also increased in streptozotocin-induced diabetes. This is not just an apparent increase caused simply by less endogenous insulin being available to compete for transport with the radioactive insulin used to measure influx rate. The increased transport of insulin is seen in diabetic mice even when assessed by the brain perfusion method in buffer rather than blood, which obviates the effects of serum insulin.

Recent in vitro work has shown that glucose itself is a regulator of insulin transport at the BBB. Hyperglycemia induces a faster rate of transport across brain endothelial cells cocultured with pericytes and astrocytes. This induction, however, does not appear to be mediated directly at the level of the endothelial cell but is dependent on crosstalk among the endothelial cells, astrocytes, and pericytes. Insulin transport is also modulated in mice treated with lipopolysaccharide (LPS), a derivative of the cell wall from gram-negative bacteria [14]. LPS induces an immune reaction mediated by cytokine release. LPS has multiple effects on BBB function, including the disruption of the BBB in extreme cases and effects through various mechanisms on some saturable transporters. LPS induces an increase in the saturable transport of insulin that is mediated through nitric oxide. The inhibition of nitric oxide synthase potentiates the effects of LPS on insulin transport, although the inhibition of nitric oxide synthase in non-LPS treated mice has no effect on insulin transport. This suggests that LPS induces two pathways affecting insulin transport across the BBB: One enhances insulin transport and the other, mediated through nitric oxide, inhibits it. Because CNS insulin counters the effects of peripheral insulin, the enhanced transport of insulin into the CNS by LPS could be a mechanism by which sepsis induces insulin resistance. A possible role for the BBB in mediating the insulin resistance of sepsis is discussed in Chapter 206 “Diseases Mediated by the BBB.”

LEPTIN Leptin is a 16-kDa protein secreted from fat cells, which acts at the arcuate nucleus in the hypothalamus to inhibit feeding and to promote thermogenesis [7]. An alternate route proposed for leptin uptake that does not exist in the adult is leakage out of the adjacent median eminence. The median eminence contains capillaries that do not form a BBB, and so leptin can leak into this tissue. However, the median eminence is isolated from the ventricular CSF and adjacent hypothalamic tissue by epithelial and tanycytic arms of the BBB [10]. These barriers are so robust that even a small molecule such as glutamate cannot penetrate them. The tanycytic barrier develops in rodents after birth. It is for this reason that monosodium glutamate can destroy the hypothalamic feeding centers when given to neonates but not when given to adults. Resistance to the anorectic and thermogenic effects of leptin is a hallmark of human obesity and of dietinduced obesity in many strains of rodents [7]. This resistance can occur at three levels: impaired transport at the BBB, receptor/postreceptor impairments, and

Ingestive Peptides and the Blood–Brain Barrier / 1457 impairments in the anorectic downstream neuronal circuitries stimulated by leptin. Studies with diet-induced obesity show that resistance first occurs at the level of the BBB [1]. At this stage, rodents can still respond to leptin given directly into the brain but no longer respond to leptin given peripherally. The analysis of the CSF and serum levels obtained from humans with moderate obesity also showed that the major level of resistance is at the BBB. As discussed in Chapter 206 “Diseases Mediated by the BBB,” this resistance at the BBB may be a major cause of leptin-related obesity. The leptin transporter is also not static but modulated by various substances and by various conditions. Obesity, starvation, and loss of ovarian function decrease the transport of leptin across the BBB. Glucose, insulin, and alpha1-adrenergics increase leptin transport, whereas triglycerides inhibit it. There is also a diurnal rhythm to leptin’s transport rate.

GHRELIN Ghrelin is a 28-amino-acid-residue peptide hormone characterized by a novel posttranslational acylation that is required for its bioactivity [12]. Ghrelin is secreted by the fundus of the stomach into the circulation before the onset of meals. Current thinking suggests that ghrelin is a major orexigenic peptide stimulating the sensation of hunger from sites within the CNS. As such, it is logical to assume that ghrelin crosses the BBB. Human octanoyl ghrelin is transported across the mouse BBB bidirectionally; that is, there is a saturable component to both its influx and its efflux [5]. Paradoxically, however, mouse ghrelin is treated differently than human ghrelin. Mouse octanoyl ghrelin, the biologically active form of ghrelin, and mouse des-octanoyl ghrelin are not transported by the influx system. Because human and mouse ghrelin differ by only two amino acids, this shows that these two amino acids are crucial for recognition by the influx transporter. Mouse octanoyl ghrelin, but not the des-octanoyl form, was transported by the efflux system. These results show that, as expected, ghrelin crosses the BBB. However, the paradoxical interactions of mouse octanoyl ghrelin with the BBB show that there is an aspect to the pathophysiology yet to be clarified.

OTHER INGESTIVE PEPTIDES AND PROTEINS: SPECIAL INTERACTIONS WITH THE BBB A number of other ingestive peptides and proteins have been examined for their ability to cross the BBB. Several of these have saturable transport systems, and

others, although able to penetrate the BBB, could not be shown to be inhibited by unlabeled peptide. These are categorized in Table 1 as having crossed the BBB by the nonsaturable mechanism of transmembrane diffusion. However, it is always possible that saturation may have been shown if a higher dose of unlabeled material had been used. Many of these peptides and proteins have special aspects affecting their brain uptake and BBB penetration. For example, whereas orexin A crosses the BBB by a nonsaturable mechanism, orexin B is so rapidly degraded in blood that it is difficult to study. Whereas the nonsaturable transport of orexin A provides a mechanism explaining how it can stimulate appetite after peripheral administration, the rapid degradation of orexin B makes it unlikely that it reaches the brain. Similarly glucagon-like peptide (GLP) is very unstable in blood, but it can be shown to cross the BBB when studied by brain perfusion. A more stable analog, [Ser8]GLP, also crosses by a nonsaturable system. Amylin is co-secreted with insulin from the pancreas and, like insulin, crosses the BBB. Insulin and amylin have distinct distribution patterns in the brain and, whereas insulin clearly is transported across the BBB by a saturable transporter, amylin transport has not been shown to be self-inhibitable. However, amylin transport is inhibited in mice pretreated with aluminum, a maneuver known to inhibit some saturable transporters. It may be that an amylin transporter exists but with a very high capacity. Binding in serum, either by serum proteins or by some factor in serum promoting aggregation, influences the BBB permeation of some ingestive peptides and proteins. Insulin-like growth factor (IGF)-1, galaninlike peptide, and adrenomedullin cross the BBB, but a saturable component to that passage is not demonstrable after their intravenous injection. However, the saturable transporter can be demonstrated when studied by the brain perfusion method. This method precludes the influence of circulating substances and allows the study of interactions directly between the BBB and the substance of interest. In the cases of IGF-1, galaninlike peptide, and adrenomedullin, it is likely that binding to serum proteins competes with the BBB transporters in such a way as to make it difficult to show the saturable nature of blood-to-brain uptake. Such a mechanism has been shown to be operable for free fatty acids. Binding in serum also interferes with uptake of Phe13,Tyr19-melanin-concentrating hormone (MCH). No uptake can be observed when studied after intravenous (IV) injection, but a low nonsaturable entry can be seen when studied by brain perfusion. Urocortin does not cross the BBB under normal conditions. However, a saturable transporter for it can be induced by treatment with either leptin or glucose.

1458 / Chapter 203 TABLE 1. Transport Systems for Ingestive Peptides and Proteins.a Substance Leptin Insulin Ghrelinb Des ghrelinb Orexin A Orexin B GLP Exendin PP Ser8-GLP ARP83-132 Amylin IGF-1 Adrenomedullin Mahogany1377-1428 PYY3-36 MSH MCH Galanin-like peptide CART NPY Urocortin Secretin

Influx

Efflux

Action with CNS

Sat Sat Nonsat Nonsat Nonsat None None Sat Sat Nonsat Nonsat Nonsat? Sat Sat Nonsat Nonsat Nonsat None Sat Nonsat Nonsat None/inducible Sat Sat (CSF) Nonsat (Vascular)

Nonsat Ret Sat Nonsat Nonsat NS NS Nonsat Nonsat NS Nonsat NS NS Nonsat

Anorectic Anorectic Orexigenic Inactive Orexigenic Orexigenic

Nonsat NS Nonsat Nonsat Nonsat Nonsat Nonsat NS

Anorectic Anorectic Orexigenic Anorectic Anorectic Anorectic Anorectic Orexigenic Anorectic Anorectic Orexigenic Anorectic

a ARP, agouti-related peptide; CART, cocaine- and amphetamine-related transcript; GLP, glucagon-like peptide; IGF-1, insulin-like growth factor; MCH, Phe13,Tyr19-melaninconcentrating hormone; MSH, alpha-melanocyte-stimulating hormone; None, no blood-tobrain uptake after intravenous injection; Nonsat, passage by nonsaturable mechansims (transmembrane diffusion for influx and bulk flow for efflux); NPY, neuropeptide Y; NS, not studied; PP, pancreatic polypeptide; Ret, retained by brain (little or no efflux) after intracerebroventricular injection; Sat, saturable transport. b Murine ghrelin as studied in the mouse.

Galaninlike peptide transport is increased with glucose and inhibited by food deprivation. Secretin was the first hormone to be discovered; Bayliss and Starling first described its actions on pancreatic exocrine secretions in 1902. Approximately 80 years later, secretin was discovered in the brain. Very low doses of secretin administered into the brain also induce pancreatic secretions. Secretin crosses the vascular BBB at a low rate by a nonsaturable mechanism, but crosses the blood–CSF barrier at a much higher rate and by a saturable system. Many other peptides and regulatory proteins that are not considered ingestive substances nevertheless have profound effects on appetite and metabolism. These include cytokines, opiate peptides, and corticotropinreleasing hormone (CRH). These are not considered here except to contrast their BBB interactions with the substances already discussed. For example, most cytokines found to have significant entry into the brain are transported across the BBB by a saturable system. CRH

and endogenous opiate peptides are substrates for various efflux systems, most notably P-glycoprotein and peptide transport system 1. No saturable influx system has yet been described for an opiate peptide, and only one saturable efflux system for a cytokine (IL-2) has been described.

CONCLUSION In conclusion, the BBB is a major regulatory interface coordinating a humoral-based communication between the GI tract and the CNS. Leptin, insulin, and ghrelin are important examples of substances involved in this communication. Their interactions with the BBB illustrate several principles, including the importance of saturable and nonsaturable mechanisms of passage, the regulation of BBB transporters, and the occurrence of disease with dysregulation of passage across the BBB.

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References [1] Banks WA. Is obesity a disease of the blood-brain barrier? Physiological, pathological, and evolutionary considerations. Curr Pharmaceut Des 2003;9:801–809. [2] Banks WA. Physiology and pathophysiology of the blood-brain barrier: Implications for microbial pathogenesis, drug delivery and neurodegenerative disorders. J Neurovirol 1999;5:538– 555. [3] Banks WA. The source of cerebral insulin. Eur J Pharmacol 2004;490:5–12. [4] Banks WA, Kastin AJ. Editorial review: Peptide transport systems for opiates across the blood-brain barrier. Am J Physiol 1990;259: E1–E10. [5] Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002;302:822–827. [6] Bloom SR. Gut and brain-endocrine connections. The Goulstonian Lecture 1979. J R Coll Physicians London 1980;14:51–57.

[7] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–770. [8] Kastin AJ, Pan W. Feeding peptides interact in several ways with the blood-brain barrier. Curr Pharmaceut Des 2003:9:789–794. [9] Morley JE. Minireview: The ascent of cholecystokinin (CCK)— from gut to brain. Life Sci 1982;30:479–493. [10] Peruzzo B, Pastor FE, Blazquez JL, Schobitz K, Pelaez B, Amat P, Rodriguez EM. A second look at the barriers of the medial basal hypothalamus. Exp Brain Res 2000;132:10–26. [11] Schwartz MW, Figlewicz DP, Baskin DG, Woods SC, Porte D Jr. Insulin in the brain: A hormonal regulator of energy balance. Endocr Rev 1992;13:387–414. [12] van der Lely AJ, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–457. [13] Woods SC, Seeley RJ, Baskin DG, Schwartz MW. Insulin and the blood-brain barrier. Curr Pharmaceut Des 2003;9:795–800. [14] Xaio H, Banks WA, Niehoff ML, Morley JE. Effect of LPS on the permeability of the blood-brain barrier to insulin. Brain Res 2001;896:36–42.

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204 Functional Aspects of Vasoactive Peptides at the Blood–Brain Barrier ARVIND K. CHAPPA, KELLY E. DESINO, SUSAN M. LUNTE, AND KENNETH L. AUDUS

Although Ang I is biologically inactive, Ang II has a wide range of physiological functions. It is generally believed that brain-derived Ang II functions in several ways, including as a hormone to control blood pressure, water intake, and salt appetite; as a regulator of reproductive hormones; and as a neuromodulator interacting with various neurotransmitters such as catecholamines and serotonin. Ang III exhibits vasopressor- and aldosterone-stimulation effects similar to its precursor Ang II. Ang IV binds to a different receptor than Ang I, II, and III and is suspected to play a role in the regulation of blood flow, motor activities, and even learning and memory [64]. The fragment Ang(1–7) produces effects that oppose those of Ang II. Ang(1–7) is found in brain tissue, including the hypothalamus and the medulla oblongata [59]. The two major receptors mediating the effects of the angiotensins are the AT1 and AT2 receptors. The AT1 receptor is believed to mediate the majority of the actions of Ang II [58]. Rose and Audus demonstrated that the uptake and transport of Ang II by bovine brain microvessel endothelial cells is mediated by the AT1 receptor [47]. The role of AT2 receptor is not as well understood. It may play an important role in embryonic development and tissue repair because it is widely expressed in fetal tissue and is increased under certain pathological conditions, respectively [59]. AT1 receptors are found in a variety of tissues including the kidney, brain, and heart. High densities of AT1 receptors occurring behind the blood–brain barrier (BBB) provide supporting evidence of neurally derived angiotensin [2]. It has been shown that angiotensin fragments do not cross the BBB when systemically administered [17]. Systemic angiotensin can, however, affect brain function through the AT receptors but only in circumventricular organs that lack the BBB [59]. Two additional receptors, AT3 [8] and AT4 [57], have also been reported. It is believed that AT3 may mediate

ABSTRACT The blood–brain barrier (BBB) maintains the stability of the brain microenvironment by controlling the selective passage of peptides and other molecules between the blood and the brain. Many peptides generated in the periphery are known to permeate the BBB and influence its properties. Vasoactive peptides such as angiotensin, bradykinin, substance P, and adrenomedullin elicit a variety of effects in the central nervous system (CNS) by binding to specific receptors on the BBB. Some of the actions produced by these peptides include vasoconstriction, vasorelaxation, regulation of the inflammatory and immunological functions, and alteration in BBB permeability. In this chapter, the central actions of the vasoactive peptides and their interactions with the BBB are described. A list of vasoactive peptides and their effects in the brain appears in Table 1.

ANGIOTENSIN Angiotensin (Ang) I and Ang II along with the precursor protein angiotensinogen and the enzymes renin and angiotensin-converting enzyme (ACE) compose the renin-angiotensin system (RAS). A separate RAS exists in the brain that is distinct from the peripheral RAS [69]. Angiotensinogen is cleaved by renin to form the decapeptide Ang I, which is further metabolized to Ang II via ACE in both the brain and periphery. The conversion of Ang I to Ang II is very rapid, and Ang II is considered the most significant metabolite. In addition to ACE, a variety of enzymes are capable of processing Ang I and Ang II to create other bioactive fragments. For example, in the brain Ang II is converted to Ang III by amino peptidase A and Ang III is converted to Ang IV by amino peptidase B [70]. Handbook of Biologically Active Peptides

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1462 / Chapter 204 TABLE 1. Major Vasoactive Peptides and Their Important Effects in the Brain. Peptide

Receptor Subtype

Ang II

AT1

Ang II Ang III Ang IV Ang (1–7) Bradykinin

AT2 AT1, AT3 AT4 AT3 B1, B2

Substance P

NK-1

Atrial natriuretic peptide Endothelin Adrenomedullin

A-type and B-type ETa and ETb CRLR with RAMP2 or RAMP-3

Effect(s) in Brain Vasoconstrictor, water intake, salt appetite, hormone regulator, neurotransmitter Embryonic development, injury Vasoconstrictor, aldosterone stimulation Vasodilator, memory, behavior modification Vasodilator Vasodilator, NO production, smooth muscle contraction, inflammatory response Neurotransmitter, neuromodulator, vasodilator, antigen presentation, inflammatory response Vasodilator, neuromodulator Vasoconstrictor Vasodilator

the function of Ang(1–7) as a vasodilator [53]. Ang IV binds to AT4 with a much greater affinity than the other angiotensin fragments [64]. The existence of AT4 receptors in cerebral blood vessels suggests that Ang IV may be involved in the regulation of blood flow, whereas their existence in the motor and sensory nuclei suggests involvement in motor activities and their localization within the hippocampus and amygdala suggests involvement in learning and memory [64]. Like Ang(1–7), there is evidence that Ang IV also contradicts the vasoactive effects of Ang II. Ang IV, via AT4 receptors, is shown to increase cerebral blood flow; however, Ang II via AT1 receptors demonstrates vasoconstrictive behavior [27]. For the most part, central and peripheral receptors mediate the same effects; however, it should be mentioned that central Ang II appears to induce natriuresis (salt excretion) but peripheral Ang II leads to sodium retention [44]. With the wide range of function of angiotensin fragments and receptors within the brain, it appears that angiotensin is an important neuropeptide and that its significance may go well beyond its classic role as a regulator of blood pressure. (Additional information about the role of angiotensin in the renal and cardiovascular systems can be found in Chapters 170 and 160, respectively, within this book.)

BRADYKININ The kallikrein-kinin system (KKS) is composed of kallikrein, kinins, kininase I and II, and enkephalinase. An intracerebral KKS has been described. Bradykinin acting from either the tissue or plasma can affect cerebral circulation. Bradykinin is a potent dilator of the cerebral arteries and, as a result, greatly increases the permeability of the BBB. This characteristic has made

bradykinin and bradykinin analogs of pharmacologic interest. The use of bradykinin analogs as transient disruptors of the BBB for delivery of therapeutic agents has been studied [5]. One bradykinin analog, lobradimil, also known as Cereport and RMP-7, has been tested clinically as a CNS-delivery-enhancing agent [6]. The transient disruption of the BBB was observed with restoration occurring 2–5 min after cessation of the infusion. Furthermore, tachyphylaxis resulted from continuous infusion of the analog. It was found that this increase in permeability was more pronounced in the brain-tumor vasculature than nontumor brain tissue. The actions of bradykinin are mediated by the receptors B1 and B2 and include vasodilation, as well as stimulation of endothelial production of nitric oxide, prostaglandin I2, and endothelium-derived hyperpolarizing factor. Bradykinin has a wide range of physiological activities that also include contraction of smooth muscle and mediation of the inflammatory response. Within the brain, bradykinin initiates conventional second-messenger signals via the B2 receptor, which stimulate G-proteins, calcium fluxes, and phosphoinositide. These substances then induce changes in the cellular tight junctions [5]. Bradykinin’s effect on cerebral microcirculation has also been studied. Endogenous neuronal bradykinin produces the dilation of microvessels; however, the intracarotid infusion of bradykinin results in no change in vessel diameter, suggesting bradykinin does not cross the BBB, and that the kinin receptors must be reached from the extravascular side [65]. Bradykinin does, however, disrupt the BBB, allowing the permeation of small molecules. This increase in brain microvascular permeability is believed to be mediated by the B2 receptor and can occur after either superfusion or intracarotid infusion [60]. (Additional information on bradykinin and its role in the cardiovascular system as well as its involvement in cancer can be found in Chapters 63 and 161 within this book.)

Functional Aspects of Vasoactive Peptides at the Blood–Brain Barrier / 1463

INTERPLAY BETWEEN ANGIOTENSIN AND BRADYKININ Angiotensin and bradykinin have several documented interactions and may offset one another physiologically. These counterbalancing effects can be seen in vascular functions, including regulation of blood pressure, inflammation, thrombosis, fibrinolysis, and angiogenesis. Angiotensin-converting enzyme (ACE), known as kininase II in the KKS, has the dual function of converting Ang I to the bioactive Ang II and degrading bradykinin. Ang(1–7), which has been shown to be biologically active, binds the bradykinin B2 receptor and decreases blood pressure, opposing the actions of Ang II [1]. Studies show that the biological effects of Ang(1–7) are unaffected by AT1 and AT2 antagonists. Furthermore, the vasodilation and nitric oxide (NO) formation caused by Ang(1–7) potentiate the effects of bradykinin as a substrate of the B2 receptor. Much of the communication between these two systems seems to be facilitated by the AT2 receptor. For example, RAS stimulates renal bradykinin production though the AT2 receptor, and the inhibition of RAS decreases bradykinin levels during salt depletion [52].

SUBSTANCE P Substance P (SP) was discovered in 1931 by von Euler and Gaddum when they found a substance in the equine brain and intestine that produced a distinct hypotensive effect and potent stimulant action on rabbit jejunum [50, 54]. The chemical structure of SP was later determined to be the 11-amino-acid sequence (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met NH2) in 1970 by Chang and Leeman [54]. For more than half a century since its discovery, SP has enjoyed the status of being the most extensively studied neuropeptide. For a detailed discussion on SP gene expression, mRNA distribution, and its proteolytic formation from precursor peptide see Chapters 114 and 180 (see also [50, 54]). SP binds with high affinity to the neurokinin-1 receptor (NK-1), a G-protein-coupled receptor. It also exhibits lower affinity for other neurokinin receptors, NK-2 and NK-3. (Further details on tachykinin receptors and their signal transduction mechanisms can be found in Chapter 180 by Meini et al.). The three NK receptors have a sequence homology of 50–66% and a significant overlap in function [54]. Wide variation in the tissue distribution and affinity of these receptors in different species has also been reported [54]. Interesting observations have been made regarding the existence of multiple binding sites on the NK-1 receptor for SP. Studies by Sagan et al. have demonstrated that SP binds with

high affinity (picomolar to subnanomolar) to the scarce NK-1m binding site and with low affinity (nanomolar) to the more abundant NK-1M binding site. These multiple binding sites have been postulated to correspond to different conformations of the NK-1 receptor that exhibit different functional properties. The binding site corresponding to the NK-1M conformer was shown to be temperature-dependent and resistant to the effect of endocytic inhibitors, whereas the internalization of NK1-m conformer is temperature-independent [23, 48]. These observations were confirmed in our studies involving the transport of SP across bovine brain microvessel endothelial cell (BBMEC) monolayers. Consistent with the diversity of the actions produced by SP, NK-1 receptors are distributed widely in both the CNS (olfactory bulb, parts of hypothalamus, cerebellum, spinal cord, and sympathetic ganglia) and the peripheral tissues (respiratory tract, gastrointestinal tract, salivary glands, lymphoid tissue, skin, and vascular smooth muscle) [54]. In addition to affecting these organ systems, SP also acts as a neurotransmitter and neuromodulator in the peripheral nervous system, producing long-lasting neuronal depolarization and modulating the actions of acetylcholine, glutamate, and N-methyl-d-aspartate [39]. SP is also one of the wellknown pain mediators and general inflammatory agents. SP is widely distributed in the nociceptive pathways in the periphery and the spinal cord for the transmission of pain information via the NK-1 receptors [33]. As an inflammatory agent, it acts as a neurogenic mediator of immune hyperactivity, also via the NK-1 receptors, inducing the proliferation and activation of immune cells [35, 54]. In the periphery, SP acts as a potent vasodilator by eliciting vascular permeability and plasma extravasation [54]. It also causes the amplification of inflammatory response by the chemotaxis of lymphocytes, monocytes, neutrophils, and fibroblasts and by stimulating the release of inflammatory mediators that further enhance leukocyte migration [29]. Further details on the mechanisms and role of SP in pain and inflammation is discussed in Chapter 189 by Xu et al. Observations similar to these were noted with SP in the cerebral endothelium. Inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)α were found to upregulate the gene expression of SP [3, 10]. Recent studies have demonstrated that SP mediates the increase in BBB permeability caused by IL-1β, TNFα, and human immunovirus (HIV)-1 envelope-protein gp120 [3]. SP has also been found to have a possible role in enhancing the class II MHC antigen presentation and lymphocyte adhesion to the brain endothelium [3]. Results from the studies by Stumm et al. on the expression of NK-1 receptor in cerebral endothelium suggest that the induction of NK-1 receptor contributes

1464 / Chapter 204 to the impairment of BBB permeability in the ischemic brain, leading to edema and leukocyte diapedesis, two important components of stroke [55]. In summary, all these observations highlight the central role played by SP in the breakdown of the BBB. The wide array of pharmacological actions mediated by SP makes it an attractive target for regulation in various disease conditions. A variety of antagonists for tachykinin receptors have been evaluated in the past decade for the clinical management of a number of disease states, including chronic pain, Parkinson’s disease, depression, arthritis, irritable bowel syndrome, asthma, and idiopathic lower back pain. Research in this area has a promising potential for the treatment and management of disease conditions for which the therapy still remains elusive.

ATRIAL NATRIURETIC PEPTIDE In 1981, De Bold et al. demonstrated that rats infused with atrial homogenates exhibited massive diuresis and natriuresis. The substance responsible for this effect was later determined to be atrionatriuretic peptide (ANP), also known as atrial natriuretic factor, atripeptin, and atrial natriuretic hormone [54]. The structure of ANP was identified by Kangawa and Matsuo in 1984 [54]. Further details on the expression, regulation, and distribution of ANP can be found in Chapter 165 by Minamino et al. ANP is one of the potent diuretic agents known, with a potency 10,000 times more than any available diuretic. In addition to its effects on sodium and water homeostasis, ANP produces vasodilatation and increases vascular permeability, thus affecting the regulation of blood pressure. Also, ANP has the ability to inhibit aldosterone and renin secretion [54]. ANP exerts a majority of its physiological actions in the periphery by binding to the natriuretic receptor A (NPR-A) (high affinity) and to the natriuretic receptor B (NPR-B) (low affinity). The natriuretic receptor C (NPR-C) acts as a clearance receptor by binding ANP and other natriuretic peptide fragments with high affinity and removing them from circulation. In addition to these receptors, specific binding sites for natriuretic peptides have been reported in various tissues, including vascular smooth muscle cells, endothelial cells, and organs such as the lung, liver, kidney, adrenal gland, intestine, and the central nervous system (CNS) [31]. The physiological function of ANP in the brain is that of a neuromodulator of cardiovascular function. ANP-containing cell bodies are found mainly in the antroventrical third ventricle that controls cardiovascular function, fluid, and electrolyte balance. ANP has been reported to inhibit the firing of periventricular

nucleus (PVN) neurons, leading to a lowering of blood pressure [40]. In addition, ANP produces vagoexcitatory and sympathoinhibitory actions by indirect mechanisms that affect heart rate, intra-atrial conduction time, and refractory period [40]. It has also been shown that ANP can exhibit a protective effect on the brain in cerebral ischemia by inhibiting the sodium transport and coupled water influx [40, 42]. Saturable binding of [125I] ANP at the BBB was demonstrated both in vitro and in vivo using isolated brain microvessels and carotid artery infusion, respectively [14, 67]. Studies by Levin et al. suggested that the uptake of plasma-borne ANP into the brain is minimal and that local synthesis of this peptide mainly accounts for the levels in brain [30]. ANP in general was shown not to affect the tightness of the BBB [38]; however, in traumatic brain injury it has been demonstrated that ANP exacerbates brain edema by increasing BBB permeability [16]. Similarly, under normal conditions, although ANP does not produce arteriolar or venular dilation, hyperosmolar disruption of BBB was shown to result in significant ANP-induced arteriolar (but not venular) dilation [21]. Recent evidence suggests that the actions of ANP in the brain are well integrated with those of other neuropeptides such as bradykinin, angiotensin, and vasopressin, which are involved in the control of water and salt metabolism [4].

ENDOTHELINS Endothelins (ETs) are a group of polypeptides that are found in vascular endothelial cells and other tissues in the body and that are strong vasoconstrictors. Three distinct genes encode for each of the three endothelins: ET-1, ET-2, and ET-3 [20]. Of the three endothelin peptides, ET-1 represents the most potent, widely distributed, and well-studied endothelin. It is reported to be 100 times more potent than norepinephrine in its vasoconstrictor effect and exerts its actions mainly via two distinct endothelin receptors, ETA and ETB [28]. In addition to the blood vessels, the major sites for the synthesis of the endothelins include the kidney, heart, brain, lung, pancreas, and spleen. The synthesis and secretion of endothelin are influenced by factors such as stress, hypoxia, transforming growth factor (TGF)-β, IL-1, Ang II, and thrombin [28]. The strong vasoconstrictor effects of ETs emphasize their important role in cerebral ischemia and subarachnoid hemorrhage as well as other cardiovascular disorders such as systemic and pulmonary hypertension and congestive heart failure. Cerebral endothelial cells produce ET and express ET-converting enzymes [71]. ET receptors (ETA and ETB) are also expressed on the capillary endothelium, indicating that ET-1 exerts its

Functional Aspects of Vasoactive Peptides at the Blood–Brain Barrier / 1465 vasoconstrictor effects at the same place where it is produced [62]. Endothelin has been reported to affect BBB permeability and contribute to cerebral edema leading to ischemic brain injury. An increase in BBB permeability and the inhibition of p-glycoprotein-mediated efflux have been observed with ET-1 in brain capillaries [18, 41]. Studies by Matsuo et al. have shown that the administration of an ETA antagonist minimizes the brain edema formation and cerebral injury in rats after cerebral artery occlusion [34]. These results support the hypothesis that ETs may contribute to cerebral ischemia and reperfusion injury by increasing the BBB permeability [63]. ET antagonists thus show potential as promising drugs in the treatment of cerebral ischemic disorders. Clinical studies with mixed and selective endothelin receptor antagonists are currently in progress [46].

ADRENOMEDULLIN Adrenomedullin (AM), a 52-amino-acid peptide with hypotensive activity, was originally identified in the pheochromocytoma cells in 1993 [26]. Later studies showed that AM is produced by a wide variety of cell types, including the vascular endothelial and smooth muscle cells [56]. Studies with primary cultures of rat cerebral endothelial cells demonstrate a higher expression of AM at both the peptide and the mRNA levels [25]. AM production in the cerebral vasculature is induced by astrocyte-derived factors, whereas cytokines and bacterial lipopolysaccharide promote its expression in the periphery [22]. Receptor systems for AM include the combination of calcitonin receptor-like receptor (CRLR) with either the receptor activity–modifying protein (RAMP)-2 or RAMP-3 [36]. The expression of all these receptors has been verified in the cerebral vasculature of rats as well as humans [7]. The high AM concentration in cerebral circulation and the expression of AM receptors in cerebral vasculature suggest a role for AM in the regulation of cerebral hemodynamics. AM exhibits a potent vasodilator effect on the cerebral vasculature, and it has been implicated in various cerebrovascular disorders. Exogenously administered AM was found to reduce the endothelial permeability by promoting the tightening of intercellular junctions. Also, AM was shown to decrease fluid phase endocytosis and activate p-glycoprotein efflux activity in the rat cerebral endothelial cells [24]. Further studies with rat cerebral endothelium have shown that AM exerts a protective effect against oxidative injury both in vitro and in vivo [9, 66]. AM thus plays an important role in regulating the BBB properties, maintenance of cerebral blood flow, and protection against ischemic brain injury.

MISCELLANEOUS PEPTIDES WITH VASOACTIVE PROPERTIES IN THE CNS Calcitonin Gene–Related Peptide Calcitonin gene–related peptide (CGRP) is a 37amino-acid neuropeptide isolated from the thyroid tissue of patients with medullary thyroid carcinoma. CGRP exhibits potent vasodilatory effects with a potency approximately 10 times greater than prostaglandins and 100–1000 times greater than other classic vasodilators such as acetylcholine, adenosine, 5-hydroxytryptamine, and substance P [7]. CGRP exhibits a long duration of action of approximately 5–6 h. CGRP and its receptors are widely distributed in the CNS and are often co-stored with substance P. Specific binding sites for CGRP are expressed in vascular smooth muscle and endothelial cells [19]. CGRP was shown to decrease cerebral venular permeability in the rat, and the administration of synthetic CGRP(1–37) has been reported to minimize ischemic damage to the brain after subarachnoid hemorrhage [68].

Cytokines Cytokines are polypeptides that serve as signaling molecules between the leukocytes and other cells. Cerebral endothelial cells express receptors for cytokines [61]. The binding of cytokines to these receptors has been reported to bring about a variety of effects, such as altering the permeability of BBB [37], upregulation of class I and II myosin heavy-chain (MHC) antigens [32], and stimulation of the secretion of vasoactive and inflammatory mediators such as the prostaglandins and endothelins [11].

Vasoactive Intestinal Peptide Vasoactive intestinal peptide (VIP) is a 28-amino-acid neuropeptide that was first isolated from porcine intestinal extracts. VIP is widely distributed in the central and peripheral nervous system and acts primarily as a potent cerebrovascular vasodilator with a protective role in inflammatory reactions, oxidative stress, and apoptosis [49]. Studies by Dogrukol-Ak et al. have demonstrated that VIP traverses the BBB mainly by transmembrane diffusion [13].

Vasopressin Vasopressin (VP) is a nonapeptide hormone synthesized in the hypothalamus. Increased plasma osmolality and hypovolemia serve as the most potent stimuli for the release of VP [12]. Three types of VP receptors, V1a, V1b, and V2, have been identified so far, with most CNS

1466 / Chapter 204 VP receptors being the V1a type [54]. Saturable transport of VP across the BBB, followed by rapid metabolism in the brain parenchyma, was demonstrated in the studies by Zlokovic et al. [72]. In a separate study, Reichel et al. reported that vasopressin alters the kinetics of the transport of l-tyrosine and l-valine across the BBB [45]. These results suggest that vasopressin-receptor interactions at the BBB induce changes in the properties of the transporter resulting in the suppression of the blood–brain transfer of large neutral amino acids.

Integration of the Effects of Vasoactive Peptides The central effects of some of the important vasoactive peptides discussed in the previous pages appear to be well integrated and balanced with one another. The distribution of ANP and ANP receptors in proximity to other neuropeptides such as VP and Ang II suggests that ANP influences water and electrolyte balance. The diuretic actions produced by ANP balance the antidiuretic effects of ET and VP. ET has strong vasoconstrictor activity and is reported to modulate the circulating levels of ANP and VP [15, 51]. The release of ET in water deprivation opposes the actions of ANP, whereas the central administration of ANP inhibits spontaneous drinking, basal secretion of VP, and dehydrating effects of Ang II. A close neuronal network involving the three neuropeptides ANP, VP, and Ang II in the subfornical organ-preoptic-hypothalamic axis has been reported to be involved in the regulation of body fluid volume [43, 54].

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205 Hypothalamic Neuropeptides and the Blood–Brain Barrier DAVID J. BEGLEY

are released at synaptic endings terminating outside of the blood–brain barrier (BBB) within anatomical sites where tight junctions between endothelial cells do not form and minimal BBB function exists. In the case of the hypothalamic neuropeptides, these release structures are the median eminence and the neurohypophysis (posterior pituitary). These release areas, including the pineal gland, transfer neurohormones to the blood and are often referred to as neurohemal organs; these form part of the larger complex of circumventricular organs (CVOs), which are specialized sites in the brain where no BBB exists [32].

ABSTRACT Until the mid-1980s, it was generally believed that peptides did not cross the blood–brain barrier because it was held that their relatively polar nature and high molecular weight would simply lead to their physical exclusion from the central nervous system. In the intervening years, this notion has been quite clearly demonstrated to be mistaken, with many peptides exerting central actions by virtue of either passive or carriermediated movement across the blood–brain barrier. This realization, that peptides are part of the complex regulation of physiology via feedback phenomena between blood and brain, and vice versa, is in full accordance with the modern concept of the blood–brain barrier as a dynamic, reactive, and adaptive interface between the brain and the peripheral tissues. In this chapter, the interaction of a number of peptides with the blood–brain barrier, better known for their hypophysiotropic regulatory function, are reviewed.

THE CIRCUMVENTRICULAR ORGANS The overall function of the CVOs is to act as windows in the BBB and thus, by virtue of their lack of BBB, permit the localization and equilibration of a small volume of parenchymal interstitial fluid to occur with plasma in terms of the lower-molecular-weight solutes. Within the CVOs, either sensitive dendrites or nerve endings can respond to changes in extracellular solute content or neurosecretory neuronal endings can specifically secrete their product into systemic blood (see Table 1). The interstitial fluid within the CVOs is effectively isolated from the bulk of brain extracellular fluid by a layer of ependymal cells with tight junctions between them [32]. Without CVOs, the brain would be sealed off by the BBB from direct influence by bloodborne solutes and in addition could not secrete neurohormones into the systemic circulation. The combination of BBB and the CVOs allows the brain to maintain its own specialized independent internal environment but still communicate freely and specifically with the soma via blood-borne signals. A number of the active neuropeptides found in the hypothalamus are known to interact with the BBB at

INTRODUCTION A large spectrum of biologically active peptides are synthesized by and released by the hypothalamus. These peptides are neuropeptides synthesized by neurons with neurotransmitter or neurohormonal roles that either regulate the function of the anterior pituitary or, in the case of vasopressin and oxytocin, are released by the posterior pituitary into the systemic circulation. In addition, within both the central nervous system (CNS) generally and within the hypothalamus a number of these peptides have more traditional neurotransmitter roles acting at synapses between neurons [37]. All the neuropeptides synthesized by the neurons of the hypothalamus as regulatory hypophysiotropic hormones of the anterior pituitary or as systemic hormones Handbook of Biologically Active Peptides

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1470 / Chapter 205 TABLE 1. Functions of Circumventricular Organs. Function Median eminence Neurohypophysis Pineal gland Choroid plexus Area postrema Subcommissural organ Subfornical organ Organum vasculosum lamina terminalis

large and are able to either penetrate it directly by passive diffusion or are transported across the endothelial cells by specific saturable mechanisms. These BBB transport processes also may form part of a mechanism whereby feedback mechanisms for systemically secreted CNS peptides can operate and may provide a means by which peripherally released peptides from other endocrine sites can alter and influence CNS function. The ability of some hypothalamic peptides to cross the BBB also opens a door for the clinical use of these peptides or their analogs as therapeutic agents [11]. A large variety of peptides are recognized as endogenous to the hypothalamus although this brain structure is not the exclusive site of their synthesis and release. The remainder of this chapter contains brief reviews of the interactions of selected hypothalamic peptides with the BBB. Some other peptides, which could be considered hypothalamic neuropeptides and whose interactions with the BBB are covered elsewhere in this section, are not included here. The concept of hypophysiotropic hormones has also been somewhat modified in recent years with the realization that a single regulatory peptide may influence the release of more than one anterior pituitary hormone. For example, growth hormone can be released by growth hormone–releasing hormone (GHRH), ghrelin, thyrotropin-releasing hormone (TRH), and pituitary adenylcyclase–activating polypeptide (PACAP), and its release is inhibited by somatostatin; in mammals TRH also releases prolactin in addition to thyrotropin [35] and gonadotropin-releasing hormone (GnRH) releases follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Clearly the influences are complex and much depends on the micro-anatomically specific release of neuropeptide delivered to a specific target either within the CNS or to a specific group of secondary endocrine response cells. In addition, neuropeptide influence on the CNS via the systemic route differs depending on whether it is via direct influence through non-BBB areas or via transport across the BBB.

Neurohemal Neurohemal Neurohemal Neurohemal/sensory/secretory (CSF) Sensory Sensory Sensory Sensory

TRH TRH is a tripeptide synthesized in the paraventricular nucleus that releases predominantly thyrotropin (TSH) from the anterior pituitary. Intact TRH appears to cross the BBB passively with a permeability three times that of mannitol [41]. TRH is also subject to hydrolysis, apparently on contact with the BBB because in the above experimental system there is no plasma protein present containing circulating peptidases. Its extraction, when radiolabeled at the proline residue, can be reduced by some 50% if the peptidase inhibitor bacitracin (2 mM) is added to the experimental perfusate. The BBB permeability to TRH cannot be reduced further by the addition of 1 mM TRH in addition to the enzyme inhibitor, indicating a lack of a saturable component to the transport [41]. A further series of experiments with the perfused sheep choroid plexus also shows similar passive movement of TRH with peptide hydrolysis on contact with the tissue [42, 43]. TRH has central actions after intravenous (IV) administration and has antidepressive properties and improves cognition. However, its short plasma halflife greatly limits its potency. The addition of methyl groups to the molecule to give a 3-methylprolinamide (RX74355) or a 3,3-dimethylprolinamide (RX77368) extends the plasma half-life, increases lipid solubility, and as a direct result enhances the CNS penetration and activity [9].

GnRH GnRH is a decapepetide synthesized by neurons with cell bodies located in the ventral hypothalamus, and in the mammal it releases both FSH and LH from the anterior pituitary [36, 37]. The intravenous administration of GnRH potentiates sexual behavior in both male and female rodents and enhances the central actions of dopamine and serotonin. These effects are evident in castrated and hypophysectomized animals and are highly suggestive of transport across the BBB [8].

Hypothalamic Neuropeptides and the Blood–Brain Barrier / 1471 GnRH has been shown to accumulate in many regions of the rat brain after intravenous injection [18]. Further studies demonstrate that GnRH has saturable bidirectional transport from blood to brain. Inclusion of excess (10 nM/ml) GnRH into the vascular perfusate significantly inhibited brain uptake of the peptide by the BBB. Arginine vasopressin (AVP) and Tyr-MIF 1 were without influence on blood-to-brain GnRH transport. Transport out of the cerebrospinal fluid (CSF) occurs to an extent greater than is expected from CSF turnover after intraventricular injection of the peptide. The introduction of excess GnRH, d-Tryp6GnRH, and GnRH(7–10) inhibited efflux from CSF. GnRH1–3 and tyrosine did not inhibit efflux from CSF. It is not clear if transport in both directions is the result of a single transporter or if two separate transporters are involved [8].

CORTICOTROPIN-RELEASING HORMONE Corticotropin-releasing hormone (CRH; 41aa) is a classic hypophysiotropic hormone releasing adrenocorticotropic hormone (ACTH) from the pituitary. It is synthesized and released by the parvicellular neurons of the paraventricular nucleus [12]. CRH is a member of a peptide family that includes urocortin (40aa), urocortin II (38aa), and urocortin III (38aa). The peptides have a high degree of homology. CRH and urocortin I bind to the same receptor, CRH-R1, whereas urocortins I, II, and III bind to CRH-R2 [25]. All members of the family are found in brain and modulate endocrine, autonomic, and behavioral responses to stress [25]. The peptides all inhibit feeding behavior but to different extents [22]. Their interactions with the BBB are also very different. Urocortin I enters the brain only after a specific saturable carrier for the peptide is activated by circulating leptin. Urocortin II enters the brain passively as a result of its inherent lipophilicity by a nonsaturable mechanism, whereas urocortin III is degraded by BBB enzymes and does not appear to enter the brain intact. CRH itself enters the CNS by a separate saturable transport system that is not sensitive to urocortins I, II, and III or leptin [22]. CRH is the only member of this peptide family to have a saturable efflux system outside the brain with a half-life of 15 minutes, compared with 50 minutes for albumin [26]. Transport is inhibited by verapamil, ouabain, colchicine, tumor necrosis factor (TNF)α, and β-endorphin, which suggests the involvement of an ATP-binding cassette (ABC) transporter. It is also interestingly enhanced by corticosterone, but appears to be cyclosporin insensitive [26].

VASOPRESSIN (ANTIDIURETIC HORMONE) Vasopressin is a nonapeptide synthesized in the magnocellular cells of the supraoptic and parventricular nuclei of the hypothamus. Following posttranslational processing of the pro-protein, the hormone is transported to the posterior pituitary in the axons forming the hypothalamic-hypophysial tract, where it is released into systemic circulation following appropriate osmotic and baroreceptor stimulation of the CNS. Its major target is usually considered to be the posterior convoluted tubule and the collecting duct of the kidney via V2 receptors, where it stimulates aquaporin insertion into the tubular cell membranes and thus enhances water resorption. The name vasopressin, however, arises from the peptide’s effect of promoting arteriolar smooth muscle contraction in response to significant hemorrhage by acting on the smooth muscle V1 receptors. In addition, vasopressin is recognized as having significant CNS activity associated with an elevated plasma level, and V1 receptors are found in the brain. Vasopressin has long been associated with the reinforcement of memory and attention and avoidance behavior [14, 27]; a rise in circulating vasopressin has been shown to reduce the blood-to-brain transport of certain large neutral amino acids [13, 33, 34]. This effect appears to result from a direct change in the activity of the large neutral amino acid transporter mediated by vasopressin at the BBB. After intracarotid injection, vasopressin accumulates in all brain regions with a BBB to an extent greater than the reference marker inulin. This accumulation is more marked in the pineal gland, the choroid plexus, the neural lobe, and the anterior pituitary, all lacking an endothelial BBB; the extraction ratio of vasopressin to inulin remains similar, suggesting no preferential extraction of vasopressin [17, 23]. The CNS effects of circulating vasopressin centering around the consolidation and reinforcement of memory processes, however, suggests a central mode of action and a number of studies have demonstrated a carrier-mediated transport of vasopressin from blood to brain and CSF [4, 28, 39, 40]. The blood-tobrain transport of vasopressin is significantly greater than mannitol, is saturable, and is inhibited with a V1 receptor antagonist [40]. Capillary depletion shows that vasopressin is transported intact across the BBB [39]. Vasopressin injected into the brain ventricles of the rat and guinea pig shows a rate of clearance from CSF with a terminal half-life of 26 min [21, 28]. This is in excess of the rate of turnover of CSF in these species produced by the secretion and suggests specific clearance mechanisms for this peptide.

1472 / Chapter 205 OXYTOCIN Oxytocin is a nonapeptide structurally closely related to vasopressin. It is also synthesized in the same hypothalamic nuclei as vasopressin but in separate neurons, and it is secreted into the systemic circulation by the posterior pituitary. Oxytocin acts on smooth muscle bearing appropriate oxytocin receptors and promotes contraction. In this context, its action on the uterus during parturition and the smooth muscle of the lactating mammary gland are well described. It is, however, stored in the posterior pituitary in significant quantities in the nonpregnant and nonlactating state and in both sexes. It is released into the circulation of both sexes during orgasm. Oxytocin has similar reinforcing effects on memory processes as vasopressin [14, 27], reduces food intake, and also reduces opioid self-administration in tolerant rats [16]. It also has effects on BBB permeability to orotic acid [24] and possibly other solutes. It accumulates in the brain after intracarotid injection with a similar regional distribution to vasopressin [18] and also appears to penetrate the BBB and appears in CSF after intravenous injection [28]; however, a specific influx transporter has yet to be identified [27]. Oxytocin is also cleared from CSF [21] with a half-life of 19 min [16, 28] by a carrier-mediated mechanism [16]. The clearance mechanisms for vasopressin and oxytocin appear to be distinct [4], PTS1 appears to transport vasopressin and PTS2 appears to transport oxytocin [2].

SOMATOSTATIN Somatostatin is a 14aa peptide cyclized by virtue of a disulfide bridge between residues 3 and 14. As its name implies, it inhibits anterior pituitary release of growth hormone. It appears to penetrate the BBB to a moderate extent, apparently by a nonsaturable passive process [1]. The volume of distribution is greater than that of radioiodinated serum albumin. The BBB transport of a number of octapeptide analogs of somatostatin has also been investigated in view of their ability to bind to somatostatin receptors expressed on brain tumors. Of three analogs (RC161, -160, and -121), RC161 appears to enter the brain most readily [7]. Interestingly, the brain entry of RC160 increased more than 200% when administered in a serum-free solution, suggesting that protein binding may be a significant factor limiting the brain uptake of some of these analogs. The BBB uptake of octreotide (SMS201–995) was also studied after IV injection and found to have a similar brain uptake to RC160 and RC121 [10]. For these three analogs of somatostatin, entry to the brain is passive. An in vitro study of octreotide movement across a monolayer of bovine cerebral

endothelial cells also suggests passive movement with a significant contribution from the paracellular pathway, which is presumably not available in the intact BBB [20]. Interestingly, when injected intracerebrally (ICV), the clearance of RC160 and RC121 was significantly faster than could be accounted for by turnover of CSF. The efflux of RC160 was saturable and thus carriermediated [6]. A confocal study with isolated porcine cerebral microvessels suggested that octreotide efflux is verapamil-, cyclosporin A-, and PSC833-sensitive and is probably mediated by the ABC transporters P-glycoprotein and multidrug-resistance-associated protein 2 [19].

PACAP PACAP is a member of a family of peptides that includes vasoactive intestinal peptide (VIP) (28aa), secretin (22aa), glucagon (29aa), glucagonlike peptides 1 (36aa) and 2 (33aa) (GLP1–2), gastric-inhibiting peptide (GIP) (42aa), and GHRH (45aa). These various peptides, described in detail in other chapters of this book, show different transport characteristics at the BBB. PACAP has two principal forms: PACAP27 and PACAP38. PACAP27 shows passive diffusive entry into the CNS, whereas the larger PACAP38 enters 30% faster and has a self-saturable component to entry [5, 15]. PACAP entry to the brain was not inhibited by an analog peptide-T, which antagonizes binding to vascular receptors [5]. Once within brain PACAP38 appears to be more resistant to degradation than PACAP27, but in spite of its passive entry a greater percentage of the injected dose enters brain of PACAP27, because the area under the curve (AUC) and pharmokinetics favor its entry [5]. PACAP38 is transported into the CNS by peptide transport system 6 [3, 29]. PACAP38 is a potent neurotropic and neuroprotective hormone, and its rate of BBB entry is significantly faster in the hypothalamus and hippocampus (four- to eightfold) than in other brain regions, the pons medulla showing no measurable PACAP38 penetration [30]. This suggests that it may have clinical applications in neurodegenerative diseases, such as Alzheimer’s, involving the hippocampus [31] and memory loss, and this may underpin its protective effect on CA1 neurons after experimental ischemia [38]. VIP, secretin, and GLP1 all appear to enter the CNS by passive diffusion [15]. Both glucagon and GHRH appear to cross the BBB, but the mechanism has not been examined [1].

References [1] Banks, W.A., Kastin, A.J. Peptides and the blood-brain barrier: Lipophilicity as a predictor of permeability. Brain Res Bull 15: 287–292; 1985.

Hypothalamic Neuropeptides and the Blood–Brain Barrier / 1473 [2] Banks, W.A., Kastin, A.J. Peptide transport system 1. In: New Concepts of a Blood-brain Barrier. Eds. Greenwood J., Segal M.B. and Begley D.J. pp 111–117, Plenum Press, NY. 1995. [3] Banks, W.A., Kastin, A.J., Arimura, A. Effects of spinal cord injury on the permeability of the blood brain and blood-spinal cord barriers to the neurotrophin PACAP. Exp Neurol 151: 116–123; 1998. [4] Banks, W.A., Kastin, A.J., Horvath, A., Michals, E.A. Carriermediated transport of vasopressin across the blood-brain barrier of the mouse. J Neurosci Res 18: 326–332; 1987. [5] Banks, W.A., Kastin, A.J., Komaki, G., Arimura, A. Passage of pituitary adenylate cyclase activating peptide 1-27 and pituitary adenylate cyclase activating polypeptide 1-38 across the bloodbrain barrier. J Pharm Exp Ther 267: 690–696; 1993. [6] Banks, W.A., Kastin, A.J., Sam, H.M., Cao, V.T., King, B., Maness, L.M., Schally, A.V. Saturable efflux of the peptides RC-160 and Tyr-MIF-1 by different parts of the blood-brain barrier. Brain Res Bull 35: 179–182; 1994. [7] Banks, W.A., Schally, A.V., Barrera, C.M., Fasold, M.B., Durham, D.A., Csernus, V.J., Groot, K, Kastin, A.J. Permeability of the murine blood-brain barrier to some octapeptide analogs of somatosatin. Proc Natl Acad Sci USA 87: 6762–6766; 1990. [8] Barrera, C.M., Kastin, A.J., Fasold, M.B., Banks, W.A. Bidirectional saturable transport of LHRH across the blood-brain barrier. Am J Physiol 261: E312–E318; 1991. [9] Begley, D.J. The blood-brain barrier: Principles for targeting peptides and drugs to the central nervous system. J Pharm Pharmacol 48: 136–146; 1996. [10] Begley, D.J. Peptides and the blood-brain barrier. In: Physiology and Pharmacology of the Blood-brain Barrier. Handbook of Experimental Pharmacology 103. Ed. M.W.B. Bradbury. pp 151–203. Springer Verlag, Berlin. 1992. [11] Begley, D.J. Peptides and the blood-brain barrier: The status of our understanding. In: Models of Neuropeptide Action. Eds. Strand, F.L., Beckwith, W., Chronwall B., Sandman C.A. Ann NY Acad Sci 739: 89–100; 1994. [12] Bruhn, T.O., Plotsky, P.M., Vale, W.W. Effect of paraventricular lesions on corticotrophin-releasing factor (CRF-like immunoreactivity) in the stalk-median eminence: Studies on the adrenocorticotropin response to ether stress and exogenous CRF. Endocrinology 114: 57–62; 1984. [13] Brust, P., Diemer, N.H. Blood-brain transfer of L-phenylalanine declines after peripheral but not central administration of vasopressin. J Neurochem 55: 2098–2104; 1990. [14] De Weid, D., Bohus, B., Urban, I., van Wimersma Greidanus, Tj. B., Gispen, W.H. Pituitary peptides and memory. In: Peptides: Chemistry, Structure and Biology. Proceedings of the Fourth American Peptide Symposium. Eds: Walter, R., Meinhofer, J. pp 635–643. University of Michigan Press, Ann Arbor Science Publ. Inc. 1975. [15] Doggrukol-Ak, D., Tore F., Tuncel, N. Passage of VIP/PACP/ secretin family across the blood-brain barrier: Therapeutic effects. Curr Pharm Des 10: 1325–1340; 2004. [16] Durham, D.A., Banks, W.A., Kastin, A.J. Carrier-mediated transport of labelled oxytocin from blood-to brain. Neuroendocrinology 53: 447–452; 1990. [17] Ermisch, A., Barth, T., Rühle, H-J., Sˇ kopková, J., Hrbas, P., Landgraf, R. On the blood-brain barrier to peptides: Accumulation of labelled vasopressin, DesGlyNH2-vasopressin and oxytocin by brain regions. Endocrinol Exp 19: 29–37; 1985. [18] Ermisch, A., Ruhle, H.J., Klasschenz, E., Kretzschmat, R. On the blood-brain barrier to peptide: [3H]-Gonadotropin releasing hormone accumulation by eighteen regions of the rat brain and by anterior pituitary Exp Clin Endocrinol 84: 112–16; 1984.

[19] Fricker, G., Nobmann, S., Miller, D.S. Permeability of the porcine blood-brain barrier to somatostatin analogues. Brit J Pharmacol 135: 1308–1314; 2002. [20] Jaehde, U., Masereeuw, R., De Boer, A.G., Fricker, G., Nagelkerde, J.F., Vonderscher, J., Breimer, D.D. Quantification and visualization of the transport of octreotide, a somatostatin analogue, across monolayers of cerebrovascular endothelial cells. Pharm Res 11: 442–448; 1994. [21] Jones, P.M., Robinson, I.C.A.F. Differential clearance of neurophysin and neurohypophysial peptides from the cerebrospinal fluid in conscious guinea pigs. Neuroendocrinology 34: 54–458; 1983. [22] Kastin, A.J., Akerstrom, V. Differential interactions of urocortin/corticotrophin-releasing hormone peptides with the bloodbrain barrier. Neuroendocrinology 75: 367–374; 2002. [23] Landgraff, R., Ermisch, A., Hess, R. Indications for a brain uptake of labelled vasopressin and oxytocin and the problem of the blood brain barrier. Endokrinologie 73: 77–81; 1979. [24] Landgraff, R., Hess, J., Ermisch, A. The influence of vasopressin on the regional uptake of [3H] orotic acid by the rat brain. Acta Biol Med Germ 37: 655–658; 1978. [25] Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E., Vale, W.W. Identification of urocortin III, an additional member of the corticotrophin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 98: 7570–7575; 2001. [26] Martins, M., Banks, W.A., Kastin, A.J. Acute modulation of active carrier-mediated brain-to-blood transport of corticotrophin releasing hormone. Am J Physiol 272: E312–E319; 1997. [27] McEwen, B.B. Brain-fluid barriers: Relevance for theoretical controversies regarding vasopressin and oxytocin memory research. Adv Pharmacol 50: 531–592; 2004. [28] Mens, W.B.J., Witter, A., van Wimersma Greidanus, T.B. Penetration of neurohypophysial hormones from cerebrospinal fluid (CSF): Half-times of disappearance of these neuropeptides from CSF. Brain Res 262: 143–149; 1983. [29] Mizushima, H., Banks, W.A., Dohi, K., Shioda, S., Matsumoto, H., Matsumoto, K. The effect of cardiac arrest on the permeability of the mouse blood-brain barrier and blood-spinal cord barrier to pituitary adenylate cyclase activating polypeptide (PACAP). Peptides 20: 1337–1340; 1999. [30] Nonaka, N., Banks, W.A., Mizushima, H., Shioda, S., Morley, J.E. Regional differences in PACAP transport across the bloodbrain barrier in mice: A possible influence of strain, amyloid β protein, and age. Peptides 23: 2197–2202; 2002. [31] Nonaka, N., Shioda, S., Banks, W.A. Effects of lipopolysaccharide on the transport of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier. Exp Neurol 191: 137–144; 2005. [32] Prescott, L., Brightman, M.W. Circumventricular organs of the brain. In: Introduction to the Blood-brain Barrier. Ed. Pardridge, W.M. pp 270–276. Cambridge University Press, Cambridge, UK 1998. [33] Reichel, A., Begley, D.J., Ermisch, A. Arginine vasopressin reduces the blood-brain transfer of L-tyrosine and L-valine: Further evidence for the effect of the peptide on the L-system transporter at the blood-brain barrier. Brain Res 713: 232–239; 1996. [34] Reichel, A., Begley, D.J., Ermisch, A. Changes in amino acid levels in rat plasma, cisternal cerebrospinal fluid, and brain tissue induced by intravenously infused arginine vasopressin. Peptides 16: 965–971; 1995. [35] Scanes, C.G., Jeftina, S., Glavaski-Joksimovic, A., Proudman, J., Arámburo, C., Anderson, L.L. The anterior pituitary gland: Lessons from livestock. Dom Animal Endocrinol 29: 23–33; 2005.

1474 / Chapter 205 [36] Schally, A.V., Arimura, A., Kastin, A.J. Hypothalamic regulatory hormones. Science 179: 341–350; 1973. [37] Strand, F. Neuropeptides: Regulators of Physiological Processes. MIT Press, Cambridge, MA. 1999. [38] Uchida, D., Arimura, A., Somogyvari-Vigh, A., Shioda, S., Banks, W.A. Prevention of ischemia-induced death of hippocampal neurons by pituitary adenylate cyclase activating polypeptide. Brain Res 736: 280–286; 1996. [39] Zlokovi´c, B.V., Banks, W.A., El Kadi, H., Erchegyi, J., Mackic, J.B., McComb, J.G., Kastin, A.J. Transport, uptake, metabolism of blood-borne vasopressin by the blood-brain barrier. Brain Res 590: 213–218; 1992. [40] Zlokovi´c, B.V., Hyman, S., McComb, J.G., Lipovac, M.N., Tang, G., Davson, H. Kinetics of arginine-vasopressin uptake at the

blood brain barrier. Biochim Biophys Acta 1025: 191–198; 1990. [41] Zlokovi´c, B.V., Lipovac, M.N., Begley, D.J., Davson, H., Raki´c, Lj. Slow penetration of thyrotropin releasing hormone across the blood-brain barrier of an in situ perfused guinea pig brain. J Neurochem 51: 252–257; 1988. [42] Zlokovi´c, B.V., Segal, M.B., Begley, D.J. Permeability of the isolated choroid plexus of the sheep to thyrotropin-releasing hormone. In: Carrier Mediated Transport of Solutes from Blood to Tissue. Eds. Yudilevich, D.L., Mann, G.E. pp 307–312. Longman, London 1985. [43] Zlokovi´c, B.V., Segal, M.B., Begley, D.J., Davson, H., Raki´c, Lj. Permeability of the blood-cerebrospinal fluid and blood-brain barriers to thyrotropin releasing hormone. Brain Res 358: 191–199; 1985.

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206 Diseases Mediated by the BBB: From Alzheimer’s to Obesity WILLIAM A. BANKS

this chapter emphasizes a different category of disease— those in which the BBB plays a central, even a causal, role in disease.

ABSTRACT The blood–brain barrier (BBB) acts as a regulatory interface between the central nervous system (CNS) and circulation by controlling the exchange of informational molecules, such as peptides and regulatory proteins. When this control is faulty, diseases can result. Examples of BBB dysfunctions and associated diseases are impaired transport of amyloid beta protein out of the brain and Alzheimer’s disease, impaired transport of leptin into brain and obesity, recovery of methionine enkephalin efflux in animals dependent on alcohol and alcohol withdrawal seizures, impaired transport of glucose into the brain and developmental retardation, and enhanced insulin transport into brain and the insulin resistance of sepsis.

LEPTIN AND OBESITY Leptin is a 16-kDa protein secreted from fat cells that acts at the arcuate nucleus in the hypothalamus to inhibit feeding and to promote thermogenesis [11]. Leptin at one-fourth the size of albumin would be too large to cross the BBB without the aid of a saturable transporter. Such a transporter has been demonstrated in vivo and in vitro in brain endothelial cells and in vivo at the choroid plexus. Leptin is transported across the BBB at all major brain regions, but transport is especially high at the arcuate nucleus. Resistance to the CNS actions of leptin arises in obese humans and in rodents with diet-induced obesity [11]. This resistance first occurs at the level of the BBB [2]. At this stage, rodents can still respond to leptin given directly into the brain but no longer respond to leptin given peripherally. Analyses of the cerebrospinal fluid (CSF) and serum levels of leptin show that in obese humans the major level of resistance is also at the BBB [6]. Several laboratories have demonstrated the decrease in BBB transport seen in obese animals. Exogenous leptin is blocked from entering the CNS in part because of competitive inhibition by the high levels of leptin in the serum of obese animals. However, this selfinhibition explains only a minor part of the BBB defect seen in obesity. Other components, including those not immediately ascribable to circulating factors, are major contributors to an impaired transport of leptin across the BBB. These defects in BBB transport are acquired in tandem with increasing obesity. They are also reversible with weight loss.

INTRODUCTION The blood–brain barrier (BBB) prevents the unrestricted exchange of substances between the blood and the fluids of the central nervous system (CNS; the brain’s interstitial fluid and the cerebrospinal fluid). The BBB also transports into the brain the glucose, amino acids, vitamins, minerals, and other nutrients required for brain function and transports out of the brain toxins, electrolytes, and xenobiotics. Some peptides and regulatory proteins are able to cross the BBB, either in the blood-tobrain direction, the brain-to-blood direction, or both directions. Immune cells are increasingly recognized as being able to cross the BBB under physiological, not just pathological, conditions. Taken together, this means that the BBB has protective, nutritive, homeostatic, and communication roles to play toward the brain. When the BBB fails to perform properly, diseases can arise. It has long been appreciated that the BBB can be affected as diseases attack other tissues of the body. But Handbook of Biologically Active Peptides

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1476 / Chapter 206 These findings of inducible and reversible defects in leptin transport demonstrate that the transporter is not static but is responsive to metabolic needs. This, in turn, implies that the leptin transporter is itself regulated. To date, two major factors have been noted to modulate leptin transport; both are themselves major players in energy utilization in general and involved in other aspects of leptin metabolism. These two factors are α1adrenergics and triglycerides [1, 4]. Leptin transport is enhanced by administration of α1-adrenergics [1]. Transport can be more than tripled with the intravenous or intraperitoneal administration of epinephrine but not after central administration. This could be one mechanism by which peripheral administration of epinephrine induces weight loss. Leptin enhances sympathetic tone to fat and other tissues and stimulates the release of epinephrine into blood by binding to leptin receptors located on the adrenal gland’s epinephrine-secreting cells. Hence, mice that lack leptin have decreased blood epinephrine levels. This increased sympathetic tone, in turn, suppresses leptin secretion from fat cells by binding to β3 adrenergic receptors. This forms a negative feedback loop between leptin and epinephrine and explains the inverse relation between blood levels of epinephrine and leptin. Epinephrine-enhanced transport of leptin reinforces this inverse relation between serum epinephrine and leptin levels in two ways. First, by elevating brain levels of leptin, increased serum levels of epinephrine increase the anorectic signal to the brain. This eventually leads to a decrease in body fat, resulting in lower leptin levels. Second, leptin and epinephrine have opposite effects in the CNS than in the periphery. Specifically, leptin inhibits epinephrine release from neurons and CNS epinephrine stimulates feeding. Thus, peripheral epinephrine reinforces its anorectic effect by indirectly, through leptin, inhibiting its antianorectic CNS levels. Leptin transport is inhibited by the peripheral administration of triglycerides [4]. Triglycerides are increased in obesity, and so this is probably a major mechanism by which obesity mediates a decrease in leptin transport. In fact, the rate of leptin transport is inversely related to serum triglyceride levels in dietinduced obesity as well as in fasted animals. Unlike most other metabolic effects induced by triglycerides, the effects on leptin transport are mediated by the triglycerides themselves and not by the free fatty acids (FFA) that can be hydrolyzed from them. The effects of triglycerides on leptin transport can also be demonstrated by lowering the triglycerides pharmacologically and by the direct administration of triglycerides in vivo and in vitro. As with epinephrine, the interaction between the BBB transporter for leptin and triglycerides is impor-

tant in a negative feed back loop between leptin action and triglycerides. Leptin decreases triglyceride levels by promoting triglyceride hydrolysis and FFA oxidation and inhibiting FFA synthesis. Patients with severe lipodystrophy or lipoatrophy (that is, little or no fat and, hence, little or no leptin) also have very severe hypertriglyceridemia, which is reversed by treatment with leptin. The ability of triglycerides to induce leptin resistance at the level of the BBB blocks the leptin-induced shift toward the use of triglycerides as an energy source and so helps to conserve fat stores and keep triglyceride levels elevated. Triglyceride inhibition of leptin transport may also explain the fundamental paradox of leptin resistance in obesity: Why should leptin resistance develop in obesity when otherwise it is so effective in maintaining body weight? Triglycerides are also elevated in starvation. During most of evolution, animals have been confronted with the problem of obtaining enough calories in the diet rather than having to deal with too many. Thus, healthy baboons living in the wild in nonfamine conditions have body fat mass and serum leptin levels seen only with anorectic conditions in Western humans [14]. Only when food sources become abnormally abundant do leptin levels approach those seen in Westerners and, with it, evidence of obesity-related diseases. Through evolutionary pressures, hypertriglyceridemia probably has come to represent to the brain a signal of starvation rather than of obesity. The ability of triglycerides to inhibit the transport of an anorectic signal to the brain provides a great survival value to the starving animal.

AMYLOID b PROTEIN EFFLUX AND ALZHEIMER’S DISEASE Amyloid β protein (Abeta) has emerged as playing a major role in the cause of Alzheimer’s disease. Abeta is neurotoxic and both transgenics and spontaneous mutations (the SAMP8 mouse) overexpressing Abeta develop cognitive impairments [12]. Reversals of histological changes and cognitive impairments have been shown in transgenics after active or passive vaccination against Abeta. In the SAMP8, antisense or antibodies directed against Abeta reverse cognitive impairments and reduce Abeta levels in brain. The level of Abeta in brain is, like most substances, an equilibrium between synthesis and degradation. For Abeta, two additional factors are also important: transport into the brain from blood and efflux from the brain into blood [18]. Both of these events are mediated by saturable transporters located at the BBB. The receptor for advanced glycation end products (RAGE) is the likely influx transporter and both

Diseases Mediated by the BBB: From Alzheimer’s to Obesity / 1477 low-density lipoprotein (LDL) receptor–related protein 1 and p-glycoprotein have been suggested as efflux transporters. Evidence from both humans and animal models suggests that efflux of Abeta is impaired in Alzheimer’s disease. Levels of LDL receptor–related protein are reduced in the brain vasculature of patients with Alzheimer’s disease [15]. Efflux is impaired in both transgenics and SAMP8 mice. Efflux of Abeta1–42, the more toxic form of Abeta, is impaired more than is the efflux of Abeta1–40 in the aged SAMP8 [7]. However, LDL receptor–related protein 1 has a preference for Abeta1–40. Whether separate transporters exist for the 1–40 and 1–42 forms is not currently established. The rate of reabsorption of cerebrospinal fluid into blood, known as bulk flow, is also impaired in the SAMP8 model. Thus, both the saturable and the nonsaturable clearances of Abeta from brain are impaired. Whether decreased efflux is caused from alternate splicing of Abeta versus alterations in transporter synthesis and function remains unknown. Either is possible, as mutations of Abeta, such as at codon 22, can result in proteins not transported by the BBB and mutations in LDL receptor–related protein results in the accumulation of Abeta by the brain. Work with SAMP8 mice suggests another mechanism, that of impairment induced by Abeta itself. Treatment of SAMP8 with antisense directed to Abeta restores both the saturable efflux of Abeta and bulk flow. Lack of clearance from the brain could be a major contributor to the accumulation of Abeta levels in brain. This lack of clearance of Abeta from brain, in turn, could explain how potential therapies work. Passive and active immunization against Abeta results in antibodies in the circulation that bind Abeta and so prevent it from crossing from the blood to the brain [13]. This prevents the influx mechanism from contributing to brain levels of Abeta. It could also prevent any Abeta that is effluxed from the brain into the blood from returning to the brain. CSF shunting has also been proposed as a mechanism by which unidentified toxins contributing to Alzheimer’s disease are rid from the brain. Shunting could restore a type of clearance of Abeta from brain. In summary, Abeta is transported by saturable mechanisms both into and out of the CNS. Impairment of the BBB’s mechanisms for efflux of Abeta from brain is postulated to be a major mechanism by which this protein accumulates in the brain of Alzheimer’s disease patients. Antisense directed toward Abeta can restore the BBB’s efflux mechanisms. Furthermore, treatment with antibodies or with CSF shunting may act by restoring a sort of balance between the influx and efflux rates of Abeta.

METHIONINE ENKEPHALIN AND ALCOHOL WITHDRAWAL An inverse relation exists between the levels of methionine enkephalin in brain and the amount of alcohol an animal will voluntarily drink [8]. This relation has been shown to occur across strains that are alcohol-naive as well as in animals whose brain levels of methionine enkephalin has been manipulated. Physical dependence to alcohol also lowers brain levels of methionine enkephalin. A major determinant of the brain levels of methionine enkephalin is peptide transport system 1 (PTS-1). This is an efflux (brain-to-blood) saturable transport system located at the BBB that has methionine enkephalin as one of its major ligands [5]. Studies across strains and among individuals within a strain suggest that PTS1 accounts for one-third to one-half of the variation in levels of methionine enkephalin in the brain. PTS-1 activity is greatly reduced in rodents made physically dependent on alcohol. Because these animals have reduced levels of methionine enkephalin, the decrease in PTS-1 activity can be seen as adaptive because this conserves brain levels of methionine enkephalin. In physically dependent animals, mRNA and protein levels of preproenkephalin no longer correlate with brain levels of methionine enkephalin. Thus, posttranslational mechanisms become the sole determinants of brain levels of methionine enkephalin. Mice made physically dependent on alcohol have a reduction in their brain levels of methionine enkephalin; paradoxically, these mice have a further reduction in brain methionine enkephalin levels when they stop drinking. This further reduction is thought to underlie the withdrawal seizures these animals undergo. PTS-1 is a likely cause for this paradoxical decrease in brain methionine enkephalin levels. Although brain levels of methionine enkephalin remain under posttranslational control, PTS-1 recovers its activity within hours of cessation of alcohol drinking. Thus, while the mechanisms for increasing the synthesis of methionine enkephalin (increases in transcription and translation) have not recovered, the mechanism for reducing brain levels (saturable efflux) has. In theory, blockade of this early recovery of PTS-1 could prevent alcohol withdrawal seizures.

GLUT-1 DEFICIENCY SYNDROME De Vivo and colleagues have described a familial syndrome in a number of patients caused by mutations in the Glut-1 transporter [10]. These patients are characterized by infantile seizures, developmental delays, acquired microencephalopathy, spastic ataxia, and low

1478 / Chapter 206 levels of glucose in the cerebrospinal fluid. Typically, the glucose levels in cerebrospinal fluid are one-half to one-third of normal levels. A number of missense, nonsense, insertion, and deletion mutations have been described that can lead to this syndrome. Because the red blood cell also has the GLUT-1, a test measuring the uptake of methyl-d-glucose by red blood cells is useful in the diagnosis of this syndrome. The Vmax for the red cell GLUT-1 is approximately 50% of normal with no change in Km.

INSULIN RESISTANCE IN SEPSIS Insulin and insulin receptors are found throughout the CNS. Originally, the source of CNS insulin was debated, but subsequent work has suggested that little or no mRNA for insulin is found in the CNS [3]. Thus, under normal circumstances, the majority, if not all, of CNS insulin originates from the pancreas. Seminal work by Margolis and Altszuler in the 1960s and by Woods, Porte, colleagues, and others showed correlations between blood and CSF levels of insulin and established that insulin in the CNS has important effects on feeding and metabolism [16]. Work from several laboratories showed that many of the effects of CNS insulin are opposite to those of peripheral insulin. For example, CNS insulin decreases the levels of insulin in the blood, increases glucose levels in blood, and inhibits feeding. Thus, insulin can act as it own counterregulatory hormone. In the short term, CNS insulin is then thought to produce a kind of insulin resistance. Paradoxically, the long-term effect of CNS insulin seems to be to prevent insulin resistance [9]. This latter conclusion is based on work in which transgenic animals with a selective knockout of CNS insulin receptors are prone to diet-induced obesity, increased serum insulin levels, resistance to the hypoglycemic effects of insulin, and increased body fat. The paradox that both short- and long-term insulin have opposite effects on insulin resistance is easily reconciled if the most important effect of short-term insulin in the CNS is suppression of appetite. By acting to prevent obesity, CNS insulin safeguards against this major cause of insulin resistance. CNS insulin probably plays important roles in several pathophysiological conditions, including insulin resistance and body weight control in inflammatory conditions. These conditions are associated with both insulin resistance and body weight loss. The treatment of mice with lipopolysaccharide (LPS) enhances insulin transport across the BBB [17]. LPS is derived from the cell walls of gram-negative bacteria and induces the release of pro-inflammatory cytokines. These cytokines probably are responsible for the effects of LPS on the BBB transport of insulin. The enhanced transport of

insulin across the BBB counters peripheral insulin’s effects and so provides a mechanism for insulin resistance in sepsis.

CONCLUSION The BBB is pivotal in an endocrine-like communication between the CNS and peripheral tissues. The BBB does this by regulating the exchange of information molecules such as peptides and proteins between the CNS and peripheral tissues. When the BBB does not perform this function correctly, diseases can occur. Here, five examples of BBB dysfunction and associated diseases are discussed. In most of these cases, the exact magnitude of the contribution of BBB dysfunction to the overall disease process is not established. However, these examples serve to illustrate the concept that the BBB is intimately involved in onset and promotion of disease processes.

References [1] Banks WA. Enhanced leptin transport across the blood-brain barrier by α1-adrenergic agents. Brain Res 2001;899:209–17. [2] Banks WA. Is obesity a disease of the blood-brain barrier? Physiological, pathological, and evolutionary considerations. Curr Pharm Des 2003;9:801–9. [3] Banks WA. The source of cerebral insulin. Eur J Pharmacol 2004;490:5–12. [4] Banks WA, Coon AB, Robinson SM, Moinuddin A, Shultz JM, Nakaoke R, Morley JE. Triglycerides induce leptin resistance at the blood-brain barrier. Diabetes 2004;53:1253–60. [5] Banks WA, Kastin AJ. The role of the blood-brain barrier transporter PTS-1 in regulating concentrations of methionine enkephalin in blood and brain. Alcohol 1997;14:237–45. [6] Banks WA, LeBel C. Strategies for the delivery of leptin to the CNS. J Drug Targeting 2002;10:297–308. [7] Banks WA, Robinson SM, Verma S, Morley JE. Efflux of human and mouse amyloid β proteins 1-40 and 1-42 from brain: Impairment in a mouse model of Alzheimer’s disease. Neuroscience 2003;121:487–92. [8] Blum K, Elston SFA, DeLallo L, Briggs AH, Wallace JE. Ethanol acceptance as a function of genotype amounts of brain [met]enkephalin. Proc Natl Acad Sci USA 1983;80:6510–12. [9] Bruning JC, Gautam D, Burks DJ, Gillette J, Schubert M, Orban PC, Klein R, Krone W, Muller-Wieland D, Kahn CR. Role of brain insulin receptor in control of body weight and reproduction. Science 2000;289:2122–25. [10] De Vivo DC, Trifiletti RR, Jacobson RI, Ronen GM, Behmand RA, Harik SI. Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. N Engl J Med 1991;325:703–9. [11] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–70. [12] Morley JE, Farr SA, Kumar VB, Banks WA. Alzheimer’s disease through the eye of a mouse: Acceptance lecture for the 2001 Gayle A. Olson and Richard D. Olson prize. Peptides 2002;23: 589–99. [13] Pan W, Solomon B, Maness LM, Kastin AJ. Antibodies to βamyloid decrease the blood-to-brain transfer of β-amyloid peptide. Exp Biol Med 2002;227:609–15.

Diseases Mediated by the BBB: From Alzheimer’s to Obesity / 1479 [14] Rutenberg GW, Coelho J, Lewis DS, Carey KD, McGill J. Body composition in baboons: Evaluating a morphometric method. Am J Primat 1987;12:275–85. [15] Shibata M, Yamada S, Kumar SR, Calero M, Bading J, Frangione B, Holtzman DM, Miller CA, Strickland DK, Ghiso J, Zlokovic BV. Clearance of Alzheimer’s amyloid-β1–40 peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest 2000;106:1489–99.

[16] Woods SC, Seeley RJ, Baskin DG, Schwartz MW. Insulin and the blood-brain barrier. Curr Pharm Des 2003;9:795–800. [17] Xaio H, Banks WA, Niehoff ML, Morley JE. Effect of LPS on the permeability of the blood-brain barrier to insulin. Brain Res 2001;896:36–42. [18] Zlokovic BV, Yamada S, Holtzman D, Ghiso J, Frangione B. Clearance of amyloid β-peptide from brain: Transport or metabolism? Nat Med 2000;6:718–19.

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207 Prebiotic Peptides BERND M. RODE AND KRISTOF PLANKENSTEINER

but since then specific and interdisciplinary research in physics, chemistry, and earth sciences has widely improved our knowledge. With this knowledge, it has become possible to achieve quite trustworthy reconstructions of the processes forming amino acids and subsequently linking them to peptides, almost ubiquitously and within a relatively wide variation of environmental conditions. Because we cannot get any direct access to remnants of that time, our only chance to obtain conclusive results is to attempt to experimentally repeat the evolutionary processes under laboratory conditions mimicking the primordial scenario. Any of these experiments can be scrutinized, therefore, by challenging the availability of necessary components, environmental conditions, and stability of products in the most likely scenario predicted by geochemical sciences for the primitive Earth.

ABSTRACT Based on modern geochemistry’s view of the atmospheric and geological conditions on the primordial Earth ∼3.8–4 billion years ago, the most realistic scenario for the formation of amino acids and peptides in chemical evolution is discussed, including possible reasons for sequence preferences in early proteins and for biohomochirality. Arguments for a peptide/protein world as primary origin of life, preceding RNA/DNAbased evolution, are presented.

INTRODUCTION Looking at the astonishing variety and complexity of peptides and proteins in today’s living organisms, the question of their origin and subsequent evolution is one of the most fundamental ones for chemists and biologists. Providing the pathways to these compounds long before life in its modern form developed, chemical evolution has become a most challenging research topic after Oparin’s hypothesis [42] of a primordial soup, containing numerous organic compounds synthesized by nature under the conditions of the primitive Earth some 3.8 billion years ago, when the Earth had cooled down sufficiently to allow the existence of liquid water. As the key role of peptides in the origin of life [46, 51] is increasingly better understood, the question of how amino acids and their first polymers came into being became crucial. Was the formation of the first prebiotic peptides a matter of chance with arbitrary results, or did chemistry and the given scenario point the way to specific, almost compulsory syntheses? The main difficulty of prebiotic research is our limited knowledge about this scenario, the conditions prevailing on the primitive Earth. Fifty years ago, these conditions were, in the literal sense, still mostly terra incognita, Handbook of Biologically Active Peptides

THE PRIMITIVE EARTH SCENARIO According to present geochemical knowledge, the Earth should have cooled down below the boiling point of water approximately 4 billion years ago, forming the first stable hydrosphere in its history. At that time, due to the weak gravitational field, the Earth had already lost its primary atmosphere of hydrogen and helium, and a secondary atmosphere was formed by gases of volcanic origin [18, 19, 22, 25, 40, 41, 59] that were stable enough to withstand the constant ultraviolet (UV) radiation of the sun and, thus, mainly consisted of CO2, N2, and water vapor, with smaller amounts of SO2 and possibly also some CO, H2S, and traces of noble gases [8, 23, 63]. Even certain amounts of oxygen formed by the decomposition of CO2 and water should have been present [5, 15, 24, 30, 48, 61]. Due to the high temperature, weather phenomena such as

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1482 / Chapter 207 thunderstorms and rain must have been much more violent than today. For the same reason, evaporation and recondensation in the coastal regions of the primitive ocean and lakes must have been common and frequent processes. Many minerals—partly still identifiable in precambrian rocks—could have served as reactants and catalysts for chemical reactions, together with inorganic salts dissolved in the sea and lakes, where they could reach considerable concentrations.

THE FORMATION OF AMINO ACIDS In 1953, Stanley Miller initiated the quickly expanding field of experimental simulations of prebiotic chemistry with his famous experiment investigating the possible formation of organic molecules in an assumed primitive atmosphere consisting of methane, hydrogen, ammonia, and water under the influence of electric discharges [35]. Soon some amino acids were identified among the products. However, after it had been realized that this reducing atmosphere could not have existed on the primordial Earth, the experiment had to be repeated, varying atmospheric composition and sources of energy [12, 16, 36, 43]. It was shown only very recently [46, 48] that amino acids can readily be formed in a neutral or even mildly oxidizing atmosphere of the type previously outlined as the most likely geochemical scenario of the primitive Earth. Up to now, numerous amino acids, such as Gly, Val, Ala, Ser, Pro, His, and Lys have been identified as products ([48] and unpublished results), and the fragments found by means of proton transfer reaction mass spectroscopy in the plasma of the artificial lightning in these experiments (unpublished results) point to a variety of synthetic pathways to these and other types of precursor molecules of today’s biochemistry.

THE FORMATION OF PEPTIDES Because the synthesis of a variety of amino acids on the primitive Earth thus appears to have been an easy and common process, the subsequent major evolution problem is the synthesis of peptides from these amino acids in aqueous solution. To form a peptide bond, water has to be removed, which makes the process thermodynamically very unfavorable. A second problem for peptide synthesis in water is the kinetic barrier because amino acids are rather unreactive concerning a nucleophilic substitution by an amino nitrogen at the carbonyl carbon. Therefore, the reaction partners would have to be activated somehow to lower the activation energy of the reaction [55]. When prebiotic chemistry research aimed at the formation of peptides, the main concern, therefore, was

to find ways to overcome both the thermodynamic and kinetic obstacles. The evaporation of water, condensation reagents, and catalysts were the main tracks along which solutions were researched. Fox and Harada proposed melts of dried amino acids to form peptides [13, 15, 46, 51]. At the melting temperatures of amino acid mixtures, all formed water evaporates and were thus removed from the reaction. However, a large excess of acidic and/or basic amino acids is necessary to allow polymerization and to avoid decomposition. Because these amino acids are rarely synthesized in atmospheric processes and the temperature range between melting and decomposition is very narrow, the prebiotic feasability of this reaction appears highly questionable. Further, a more thorough analysis of the resulting polymers (initially termed proteinoids) showed that they are polymers that only occasionally contain peptide bonds [1]. Another approach to prebiotic peptide formation was the use of condensation agents to facilitate the reactions between amino acids [46, 51]. The substances used in these experiments included cyanamides, cyanates, trimetaphosphate, ATP, GTP, CTP, UTP, linear and cyclic inorganic polyphosphates, and imidazoles, all of which must have been rather scarce or unstable in a primordial Earth setting, due to their rapid hydrolytic decomposition and precipitation as insoluble salts. There is, therefore, only a very minor probability that these types of peptide synthesis contributed to chemical evolution. Under assumed hydrothermal vent conditions [62], amino acids can also condense to peptides, but this scenario would be restricted to specific locations in the deep sea and especially high partial pressures of carbon monoxide, and thus its relevance for large-scale prebiotic peptide formation is rather questionable. A further possibility is peptide formation on the surface of minerals such as clays, silica, and alumina [46, 51]. In principle, it is possible to form peptides from adsorbed amino acids, but the yields are very low and the applicability is apparently restricted to very few amino acids such as Gly and Ala. However, these adsorption processes are of great importance for prebiotic peptide formation because peptides formed in other processes can be protected from decomposition [2] on the surface of such minerals and, furthermore, short peptides can be elongated to longer chains by this mechanism [46, 51]. For example, the salt-induced peptide formation (SIPF) reaction [51, 55], which efficiently forms peptides, perfectly harmonizes with clay-catalyzed peptide elongation in a one-pot reaction [46, 51]. This SIPF reaction is a special case among the many proposals for peptide formation under primordial Earth conditions [51]. It allows for peptide formation

Prebiotic Peptides / 1483 in aqueous solution directly from amino acids or from amino acids and short peptides, as long as sodium chloride, Cu(II) ions, and thermal energy are available. Sodium chloride has always been ubiquitously present [18, 19] in the oceans, in salt lakes, and in lagoons and puddles along the sea shore. In concentrations above 3 M, which can be reached either in large-volume salt lakes or lagoons (simulated by the constant volume type [46, 51] of SIPF reaction with constantly high sodium chloride concentrations) or in evaporating small puddles and small lagoons (simulated by evaporation cycle experiments [46, 51] in which the active concentration is reached by evaporation and subsequent redilution), sodium ions do not have a saturated first hydration shell and can, therefore, act as a dehydrating agent to overcome the thermodynamical barrier of peptide formation in aqueous solution [55]. Computational investigations of sodium chloride solutions [46, 51, 55], showing this effect in detail, were the basis for the discovery of the SIPF reaction. Subsequent simulations and experiments showed that the complexation of amino acids to metal ions, in particular to Cu(II) [51, 55], can activate them and thus lower the activation energy for the peptide formation process. Because precambrian rock formations contain large amounts of Cu(II)-containing minerals [17, 38, 48], the availability of these ions on the primitive Earth appears secured and the proper oxidation state could have been reached easily under the assumed atmospheric conditions [39, 48]. Since the late 1980s, the SIPF reaction has been and still is being investigated in many areas [46, 51], continuously revealing new favorable aspects and proving its very general applicability for prebiotic peptide formation under the conditions of the primordial Earth scenario just outlined [46]. The ideal temperature for the SIPF reaction is between 60 and 90°C [46, 51], a very realistic condition for recently condensed oceans on the cooling surface of the Earth. The reaction prefers the biologically relevant α-amino acids to amino acids containing a more distant amino group [46, 51]. Amino acids such as glycine and histidine, but also small linear or cyclic peptides such as diglycine and diketopiperazine, show a yield-increasing catalytic effect in the reaction [44, 45, 46, 49, 51], which can produce peptides even with otherwise almost nonreactive amino acids, thus resulting in peptide formation with all amino acids investigated so far. The catalytic mechanism [49, 51] apparently involves the intermediary formation of a peptide with the catalyst, followed by its condensation with another educt amino acid and the release of the catalyst by hydrolysis to the product peptide and the catalyst. Quite striking evidence for the prebiotic relevance of the SIPF reaction resulted from a comparison of the specific dipeptide yields of the reaction with the fre-

quency of the respective peptide linkages in membrane proteins of Archaea and Procaryonta, some of the oldest still existing organisms on the Earth; a correlation of the preferred sequences was observed that could hardly have occurred only by chance [46, 51, 52], indicating an evolutionary pathway from the SIPF process to the proteins of the first living organisms. It is interesting that the (in)famous proteinlike prions show an even higher degree of similarity with the most preferred SIPF sequences (Gly-Gly > Gly-Leu > Ala-Ala > Ala-Gly > AlaVal > Pro-Pro > Pro-Gly > Val-Val > Gly-Ala > Val-Gly > Leu-Gly > His-Gly > Leu-Leu > Val-Ala > Gly-His > ValLys > Lys-Val), in particular the characteristic dominance of the Gly-Gly linkage, thus indicating an origin of the prions at a very early stage of evolution [53]. As mentioned previously, the oligopeptides formed in the SIPF reaction could have been stabilized and elongated to longer chains with the help of clay minerals such as montmorillonite or hectorite in the same environment [46, 51]. Thus, the role of clay minerals in peptide evolution could have been quite a significant one, not so much for the primary formation of peptides from amino acids but for the further development of larger and more complex peptides and for their survival against hydrolytic attack. Another quite important feature of the SIPF reaction refers to chirality-related aspects; during the reaction, the optical purity of the amino acids is almost fully conserved [46, 50], especially at low concentrations, as we would expect in a very dilute prebiotic soup. This preservation of chirality, albeit quite important, is not the only characteristic of the reaction with respect to optical activity. The active complex in the SIPF reaction [32, 46, 51, 60] has been found to be a copper(II) complex with one amino acid coordinated as a chelate, another amino acid or oligopeptide bound end-on via its carboxylic group, a chloride ion, and two water molecules at elongated distance (see Fig. 1). Quantum chemical calculations have predicted a pseudotetrahedral arrangement of the amino acid and chloride ligands [46], thus leading to a central chirality at the Cu(II) ion that seems to amplify the small inherent chirality of copper due to the parity violating effect, which because of its Z 5-dependence (Z being the atomic number) is considerably larger than in organic compounds. This chirality effect can lead in some cases, especially with simple aliphatic amino acids such as Ala and Val, to a preference of l-amino acids over d-amino acids in the SIPF reaction [44, 45, 46, 47, 50] and might be, together with yet unknown amplification mechanisms, a basis for the processes having led to the phenomenon of biohomochirality. Yield differences according to the chirality of the educt amino acids, differences in the complex formation constants of l- and d-alanine, and circular dichroism (CD) spectroscopy

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FIGURE 1. Ab initio geometry optimized structure of the tetrahedrally distorted SIPF complex [CuCl(alaH2)(H2O)2]+ [46, 47].

have supplied independent indications toward this particular stereoselectivity in the SIPF reaction in favor of l-amino acids [52].

A PEPTIDE WORLD AS THE ORIGIN OF LIFE? Any discussion on the origin of life naturally involves a definition of a living system. It has become a convention to characterize life by the essential properties metabolism, replication, propagation of information, and the possibility of mutation, allowing evolution to adapted and more complex forms. Although a kind of metabolism can be assigned to a number of chemical systems, the other characteristics can be achieved according to our contemporary chemical knowledge by two types of biomolecules only, namely peptides/proteins and nucleic acids, which are the key players in all of today’s living organisms. Nucleic acids are the modern information-carrying molecules and replicators, but peptides and proteins could have preceded them in that task because peptides can also work as carriers of information and can replicate, as shown recently [20, 29], albeit not as efficiently as nucleic acids. Under the harsh conditions of the primordial Earth, however, evolution of lifelike processes based on peptides would have had many favorable aspects. In addition to various problems with the formation of nucleic acids under primordial Earth conditions [11, 14, 56], the first argument to support this statement is the chemical and physical (in)stability of nucleic acids; in a hot salty ocean irradiated by the UV light of the sun, nucleic acids and their building blocks decompose very rapidly [6, 21, 27, 31, 33, 57, 58] and thus would not have had

time to pass on their information and to chemically evolve. Peptides, on the other hand, could be formed much more efficiently [46, 51] and, once formed, would be much more stable [3, 4, 26, 64] and well protected from UV damage by dissolved ions and other organic molecules in the prebiotic soup that would have absorbed a great part of the harmful UV radiation in the wavelength region below 220 nm [6]. Furthermore, peptides are far less prone to errors caused by mutations, because their informational content and their functionality are less harmed by an exchange of amino acids in the sequence. For example, a hydrophobic amino acid would mainly be replaced by another hydrophobic amino acid, which in most cases would not have a strong influence on the characteristics of the peptide, whereas the replacement of one or several nucleobases by another of the four possibilities could have dramatic effects, leading even to a total loss of function. Without the help of sophisticated enzymatic repair mechanisms (which demand the existence of sophisticated proteins!) nucleic acid evolution would quickly have reached a dead end. Peptides can deal with many more errors by themselves. Based on information theory, Freeman Dyson’s model of chemical evolution [9] has predicted that, given the low autocatalytic efficiency of the first prebiomolecules, four basic building blocks, as in the case of nucleic acids, would be too few to reach a state in which the development of autocatalytic biochemical cycles would be possible; at least 8–10 would be required. This figure is realistic for early peptides, even considering a still limited number of amino acids formed during prebiotic chemical evolution. Finally, follow-up investigations on Eigen’s RNA hypercyles [10] quickly showed through computational simulations [37] that such cycles would quickly be terminated by various catastrophic conditions and thus not provide the possible development of living systems. Therefore, an initial peptide/protein world [46], as opposed to an RNA world scenario, appears to be a much more likely starting point for life on Earth. Amino acids and peptides would have been formed first and then have chemically evolved to assume the function of primary information carriers and replicators. Nucleic acids could probably only have been synthesized and have chemically evolved after some kind of protection against environmental influences had become available, most probably in the form of peptide/protein membranes or shells [54]. Inside this protected environment, further evolution of the nucleic acids to nucleosides and nucleotides and the necessary enzymatic repair mechanisms could take place, passing over the functions of the first peptides and proteins to RNA and DNA and ultimately leading to the genetic apparatus of today’s organisms. This later involvement of RNA/DNA in evolution was actually anticipated by

Prebiotic Peptides / 1485 numerous scientists in the past 3 decades [7, 28, 34, 46], but only the recent developments in peptide research have shown a plausible and more convincing alternative for the evolution of life on our planet.

References [1] Andini S, Benedetti E, Ferrara L, Paolillo L, Temussi PA. NMR studies of prebiotic polypeptides. Origins Life 1975;6: 147–53. [2] Bernal JD. The physical basis of life. London: Routledge and Kegan Paul; 1951. [3] Blank JG, Miller GH, Ahrens MJ, Winans RE. Experimental shock chemistry of aqueous amino acid solutions and the cometary delivery of prebiotic compounds. Origins Life Evol Biosphere 2001;31:15–51. [4] Brack A. Selective emergence and survival of polypeptides in water. Origins Life Evol Biosphere 1987;17:367–79. [5] Carver JH. Prebiotic atmospheric oxygen levels. Nature 1981; 292:136–8. [6] Cleaves HJ, Miller SL. Oceanic protection of prebiotic organic compounds from UV radiation. Proc Natl Acad Sci USA 1998; 95:7260–3. [7] Crick FHC. The origin of the genetic code. J Mol Biol 1968; 38:367–79. [8] Delano JW. Redox history of the earth’s interior since ∼3900 Ma: Implications for prebiotic molecules. Origins Life Evol Biosphere 2001;31:311–41. [9] Dyson F. Origins of life (rev. ed.). Cambridge, UK: Cambridge University Press; 1999. [10] Eigen M, Schuster P. The hypercyle. A principle of natural selforganization. Part A: Emergence of the hypercycle. Naturwissenschaften 1977;64:541–65. [11] Eschenmoser A. Chemical etiology of nucleic acid structure. Science 1999;284:2118–24. [12] Fox SW, Dose K. Molecular evolution and the origin of life. San Francisco: Freeman; 1972. [13] Fox SW, Harada K. Thermal copolymerization of amino acids to a product resembling protein. Science 1958;128:1214. [14] Gibson LJ. Did life begin in an “RNA world?” Origins 1993;20: 45–52. [15] Grandstaff DE. Origin of uraniferous conglomerates at Elliot Lake, Canada and Witwatersrand, South Africa: Implications for oxygen in the Precambrian atmosphere. Precambrian Res 1980;13:1–26. [16] Harada K, Fox SW. Thermal synthesis of natural amino acids from a postulated primitive terrestrial atmosphere. Nature 1964;201:335–6. [17] Hofmann HJ, Grey K, Hickman AH, Thorpe RI. Origin of 3.45 Ga coniform stromatolites in Warrawoona Group, Western Australia. Geol Soc Am Bull 1999;111:1256–62. [18] Holland HD. The chemical evolution of the atmosphere and oceans. Princeton, NJ: Princeton University Press; 1984. [19] Holland HD. The chemistry of the atmosphere and oceans. New York: Wiley; 1978. [20] Isaac R, Chmielewski J. Approaching exponential growth with a self-replicating peptide. J Am Chem Soc 2002;124:6808–9. [21] Isbell HS, Frush HL, Wade CWR, Hunter CE. Transformations of sugars in alkaline solutions. Carbohydr Res 1969;9:163–75. [22] Kasting JF. Archaean atmosphere and climate. Nature 2004;432: doi:10.1038/nature03166. [23] Kasting JF. Earth’s earliest atmosphere. Science 1993;259: 920–6. [24] Kasting JF. The evolution of the prebiotic atmosphere. Origins Life Evol Biosphere 1984;14:75–82.

[25] Kasting JF, Ackerman TP. Climatic consequences of very high carbon dioxide levels in the earth’s early atmosphere. Science 1986;234:1383–5. [26] Lai MC, Topp EM. Solid-state chemical stability of proteins and peptides. J Pharm Sci 1999;88:489–500. [27] Larralde R, Robertson MP, Miller SL. Rates of decomposition of ribose and other sugars: Implications for chemical evolution. Proc Natl Acad Sci USA 1995;92:8158–60. [28] Lederberg J, Lederberg EM. Replication plating and indirect selection of bacterial mutants. J Bacteriol 1952;63:399–406. [29] Lee DE, Granja JR, Martinez JA, Severin K, Ghadiri MR. A selfreplicating peptide. Nature 1996;382:525–8. [30] Levine JS, Augustsson TR, Natarajan M. The prebiological paleoatmosphere: stability and composition. Origins Life 1982; 12:245–59. [31] Levy M, Miller SL. The stability of the RNA bases: implications for the origin of life. Proc Natl Acad Sci USA 1998;95:7933– 98. [32] Liedl KR, Rode BM. Ab initio calculations concerning the reaction mechanism of the copper(II) catalyzed glycine condensation in aqueous sodium chloride solution. Chem Phys Lett 1992;197:181–6. [33] Lindahl T. Instability and decay of the primary structure of DNA. Nature 1993;362:709–15. [34] Margulis L. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof. Symp Soc Exp Biol 1975;29:21–38. [35] Miller SL. A production of amino acids under possible primitive earth conditions. Science 1953;117:528–9. [36] Miyakawa S, Yamanashi H, Kobayashi K, Cleaves HJ, Miller SL. Prebiotic synthesis from CO atmospheres: implications for the origins of life. Proc Natl Acad Sci USA 2002;99: 14628–31. [37] Niesert U, Harnasch D, Bresch C. Origin of life between Scylla and Charybdis. J Mol Evol 1981;17:348–53. [38] Nutman AP, McGregor VR, Friend CRL, Bennett VC, Kinny PD. The Itsaq gneiss complex of southern West Greenland; the world’s most extensive record of early crustal evolution (3900– 3600 Ma). Precambrian Res 1996;78:1–39. [39] Ochiai EI. The evolution of the environment and its influence on the evolution of life. Origins Life 1978;9:81–91. [40] Ohmoto H, Watanabe Y. Archaean paleosols and archaean air. Nature 2004;432:doi:10.1038/nature03168. [41] Ohmoto H, Watanabe Y, Kumazawa K. Evidence from massive siderite beds for a CO2-rich atmosphere before ∼1.8 billion years ago. Nature 2004;429:395–9. [42] Oparin AI. The origin of life on earth. Berlin: Dt. Verl. d. Wiss.; 1957. [Originally pub. 1936.] [43] Oró J. Synthesis of organic molecules by physical agencies. J Br Interplanet Soc 1968;21:12–25. [44] Plankensteiner K, Reiner H, Rode BM. Catalytic effects of glycine on prebiotic divaline and diproline formation. Peptides 2005;27:1109–12. [45] Plankensteiner K, Reiner H, Rode BM. Catalytically increased prebiotic peptide formation: ditryptophan, dilysine, and diserine. Origins Life Evol Biosphere 2005;35:411–9. [46] Plankensteiner K, Reiner H, Rode BM. From earth’s primitive atmosphere to chiral peptides—the origin of precursors for life. Chem Biodiv 2004;1:1308–15. [47] Plankensteiner K, Reiner H, Rode BM. Stereoselective differentiation in the salt-induced peptide formation reaction and its relevance for the origin of life. Peptides 2005;26: 535–41. [48] Plankensteiner K, Reiner H, Schranz B, Rode BM. Prebiotic formation of amino acids in a neutral atmosphere by electric discharge. Agnew Chem Int Ed 2004;43:1886–8, Agnew Chem 2004;116:1922–4.

1486 / Chapter 207 [49] Plankensteiner K, Righi A, Rode BM. Glycine and diglycine as possible catalytic factors in the prebiotic evolution of peptides. Origins Life Evol Biosphere 2002;32:225–36. [50] Plankensteiner K, Righi A, Rode BM, Gargallo R, Jaumot J, Tauler R. Indications towards a stereoselectivity of the saltinduced peptide formation reaction. Inorg Chim Acta 2004; 357:649–56. [51] Rode BM. Peptides and the origin of life. Peptides 1999;20: 773–86. [52] Rode BM, Eder AH, Yongyai Y. Amino acid sequence preferences of the salt-induced peptide formation reaction in comparison to archaic cell protein composition. Inorg Chim Acta 1997;254:309–14. [53] Rode BM, Flader W, Sotriffer C, Righi A. Are prions a relic of an early stage of peptide evolution? Peptides 1999;20:1513–6. [54] Santoso S, Hwang W, Hartman H, Zhang S. Self-assembly of surfactant-like peptides with variable glycine tails to form nanotubes and nanovesicles. Nano Lett 2002;2:687–91. [55] Schwendinger MG, Rode BM. Possible role of copper and sodium chloride in prebiotic evolution of peptides. Anal Sci 1989;5:411–4.

[56] Shapiro R. The improbability of nucleic acid synthesis. Origins Life Evol Biosphere 1984;14:565–70. [57] Shapiro R. Prebiotic cytosine synthesis: a critical analysis and implications for the origin of life. Proc Natl Acad Sci USA 1999;96:4396–401. [58] Shapiro R. The prebiotic role of adenine: a critical analysis. Origins Life Evol Biosphere 1995;25:83–98. [59] Sleep NH. Palaeoclimatology: archaean palaeosols and archaean air. Nature 2004;432:doi:10.1038/nature03167. [60] Tauler R, Rode BM. Reactions of Cu(II) with glycine and glycylglycine in aqueous solution at high concentrations of sodium chloride. Inorg Chim Acta 1990;173:93–8. [61] Towe KM. Precambrian atmospheric oxygen and banded iron formations: a delayed ocean model. Precambrian Res 1983; 20:161–70. [62] Wächtershäuser G. Life as we don’t know it. Science 2000;289: 1307–8. [63] Walker JCG. Carbon dioxide on the early Earth. Origins Life Evol Biosphere 1985;16:117–27. [64] Weber AL, Miller SL. Reasons for the occurrence of the twenty coded protein amino acids. J Mol Evol 1981;17:273–84.

C

H

A

P

T

E

R

208 Mixture-Based Combinatorial Libraries R. A. HOUGHTEN, C. T. DOOLEY, AND J. R. APPEL

are a rapid cost-effective means for the identification of extremely active, highly specific individual compounds [17]. Table 1 lists the highly specific opioid ligands that have been identified from mixture-based combinatorial libraries. One of two methods is used to generate the approximate equimolarity required for mixture-based libraries. In the first, equimolarity is achieved by mixing precoupled resins (a process termed divide-couple-recombine (DCR) [18], split synthesis [20], or portioning-mixing [13]). In the second, mixtures of incoming reagents are used during coupling [29]. The second method can be more readily used to incorporate multiple functionalities at diverse positions within a combinatorial library, which is critical for the design of positional scanning libraries. Also, the ability to exhaustively transform entire libraries of compounds to libraries of entirely different pharmacophores has been developed in the author’s laboratory [22–25, 27, 28]. Postsynthetic chemical modifications of resin-bound peptide libraries enables the generation of peptidomimetics and low-molecularweight heterocyclic combinatorial libraries [26].

ABSTRACT Mixture-based combinatorial libraries have many uses. They provide a fast inexpensive way to discover specific biologically active peptides. This chapter shows the widespread utility of combinatorial libraries by covering their use in opioid binding assays for the identification of a wide variety of new ligands for the opioid receptors. The ability to screen mixtures free in solution confers decided advantages in that not only can a broad spectrum of assays be used without alteration but the concentration of the library mixtures being screened can be varied to adjust to the needs of the particular assay.

INTRODUCTION Thousands of analogs of the natural opioid peptides enkephalin, dynorphin, endorphin, dermenkephalin, dermorphin, and the endomorphins have been synthesized over the past 20 years and used to determine the workings of the opioid receptor family. More recently, highly selective compounds (in most cases structurally related to the classic opiates, e.g., morphine) have been identified and used as research tools. To date, only a single gene has been identified for each of the known μ- [1, 12], δ- [11, 19], and κ- [35] opioid receptors. New ligands specific for, rather than selective for, the receptor subtypes would greatly facilitate binding studies. Such ligands may also prove more useful as tools for in vivo study. Structurally diverse ligands will aid in the study of analgesic efficacy, addiction, and tolerance and will delineate central and peripheral antinociception, thus leading to more effective therapeutic painmodulating agents. Studies carried out by our own and other laboratories, however, have shown that mixture-based libraries Handbook of Biologically Active Peptides

GENERAL METHODS The two most widely used deconvolution strategies for soluble combinatorial libraries are iterative [14, 18] and positional scanning [7, 30]. The iterative deconvolution approach uses a step-by-step selection and enhancement process to identify individual compounds. The most active sequence(s) in this process is thus identified by the systematic reduction in the number of compounds in the most active mixture. The positional scanning deconvolution approach is a more rapid means to gather information about all possible variable positions in a soluble mixture library and was first presented by the author’s laboratory in 1992 [30].

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Copyright © 2006 Elsevier

Total Compounds

Deconvolution

Mu

6 × 106

Iterative

YmFA-NH2

IC50 = 2

Agonist

[4]

Mu

6 × 106

PosScan

Y(DNve)G(L-Nal)-NH2

Ki = 0.4

Agonist

[9]

Mu Delta Kappa Mu

6 × 106 6 × 106 6 × 106 52 × 106

PosScan PosScan PosScan Iterative

YrAW-NH2 Wy(aAba)R-NH2 ff(DNle)r-NH2 YGGFMA-NH2

Ki = 1.8 Ki = 7 Ki = 1 IC50 = 28

Agonist Agonist Agonist Agonist

[9] [9] [9] [15]

Mu Mu Delta Mu

52 52 52 52

× × × ×

Iterative Iterative Iterative Iterative

YPFGFR-NH2 WWPR-NH2 YGGFLV-NH2 Ac-RFMWMK-NH2

IC50 IC50 IC50 IC50

13 10 10 5

Agonist Agonist Agonist Antagonist

[8] [8]

Mu Mu Mu

52 × 106 52 × 106 34 × 106

Iterative Iterative PosScan

Ac-RWIGWR-NH2 Ac-FRWWYM-NH2 YGGFMY-NH2

IC50 = 5 IC50 = 33 IC50 = 17

Antagonist Agonist Agonist

[8] [8] [7]

Delta Delta Delta

34 × 106 34 × 106 52 × 106

PosScan PosScan PosScan

YHGWLV-NH2 YGMHLV-NH2 YGFDLV-NH2

IC50 = 61 IC50 = 45 IC50 = 15

Not reported Not reported Agonist

[31] [31] [31]

Kappa

3.2 × 1012

PosScan

Ac-YRTRYRRYRR-NH2

IC50 = 28

Not reported

Orphanin

52 × 106

PosScan

Ac-RYYRWK-NH2

IC50 = 23

Agonist

[10]

Mu

52 × 106

Iterative

Ac-rfwink-NH2

IC50 = 18

Agonist

[3]

Mu

52 × 106

Iterative

iftwyr-NH2

IC50 = 5

Not reported

Kappa

7.3 × 106

PosScan

Rh-aOrn-R-7Aha-r-NH2

Ki = 5.7

Not reported

Library

Receptor

Tetrapeptide OXXX-NH2 Tetrapeptide OXXX-NH2

Hexapeptide OOXXXX-NH2

Hexapeptide Ac-OOXXXX-NH2 Hexapeptide OXXXXX-NH2 Hexapeptide OOXXXX-NH2 Decapeptide Ac-OXXXXXXXXX-NH2 Hexapeptide Ac-OOXXXX-NH2 Hexapeptide D-amino acids Ac-ooxxxx-NH2 Hexapeptide D-amino acids ooxxxx-NH2 Rhodamine-OXXX-NH2

106 106 106 106

Individual Compound

Activity (nM)

= = = =

Agonist/ Antagonist

Source

[2]

[16]

1488 / Chapter 208

TABLE 1. Opioid Ligands Identified from Combinatorial Libraries.

Mixture-Based Combinatorial Libraries / 1489 As an illustration of the method for a simple tetrapeptide combinatorial library, envision four different amino acids (D, F, I, and N) incorporated at each of four different variable positions, resulting in 256 (= 44) possible peptides. When this diversity is arranged as a positional scanning synthetic combinatorial library (PSSCL), 16 peptide mixtures (four separate mixtures for each of the four positions) are synthesized. The four positional sublibraries, namely O1XXX-NH2, XO2XXNH2, XXO3X-NH2, and XXXO4-NH2, contain all 256 peptides, but differ in the arrangement of peptides within the mixtures and hence the location of the position defined. Thus, the four mixtures in O1XXX-NH2 are DXXX-NH2, FXXX-NH2, IXXX-NH2 and NXXXNH2. Mixture DXXX-NH2 contains the 64 peptides containing D at position one. In sublibrary XO2XX-NH2, mixture XDXX-NH2 contains the 64 peptides with D at position 2. The only peptides in common between mixtures DXXX-NH2 and XDXX-NH2 are the 16 that have D in both positions (DDXX-NH2). In this example, assume that the sequence FIND-NH2 is the sole tetrapeptide having activity, with the other 255 being completely inactive. Because each positional sublibrary contains all 256 peptides, the individual tetrapeptide is present in only one mixture in each of the four positional sublibraries. Thus, the mixtures having activity will be FXXX-NH2, XIXX-NH2, XXNX-NH2, and XXXDNH2 due to activity of FIND-NH2. These four amino acids in their respective positions yield the sequence FIND in the initial screening.

m-RECEPTOR LIGANDS The use of libraries with membrane-bound receptors has so far been limited to those that can be screened with compounds free in solution. Such a library was used in a case study carried out in the authors’ laboratory employing a μ-opioid receptor binding assay and a hexapeptide synthetic combinatorial library composed of 400 different mixtures, each of which contained an equimolar mixture of 130,321 hexapeptides for a library total of 52,128,400 l-amino acid hexapeptides. The final deconvolution yielded sequences in which the first five residues corresponded to the naturally occurring opioid peptide sequences of methionine and leucine enkephalin (YGGFMA-NH2, median inhibitory concentration, IC50 = 28 nM, and YGGFLG-NH2, IC50 = 59 nM) [15]. Positional scanning deconvolution was also demonstrated by the μ-opioid receptor binding assay as a case study [7]. A hexapeptide PS-SCL consisting of six separate sublibraries, each having a single position defined with the remaining five positions as mixtures (O1XXXXX-NH2, XO2XXXX-NH2, XXO3XXX-NH2,

XXXO4XX-NH2, XXXXO5X-NH2, and XXXXXO6-NH2; 108 separate peptide mixtures), was screened. Peptides making up all possible combinations of the amino acids active at each position were synthesized. The IC50 values obtained for these peptides ranged from 17 nM to 3276 nM. Nine peptides had activities below 200 nM, five of which possessed the sequences of methionine- or leucine-enkephalin within the first five residues. The screening of an N-acetylated library, AcOOXXXX-NH2, in a μ-binding assay yielded new opioid antagonists with no homology to known opioid peptides [2, 4]. The individual peptides, namely AcRFMWMR-NH2, Ac-RFMWMK-NH2, and Ac-RFMWMTNH2, were termed Acetalins. These peptides were found to be potent μ-receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a new class of opioid receptor ligands. This study exemplifies the power of mixture-based combinatorial libraries for the identification of new ligands. A mixture-based combinatorial library containing all d-amino acid hexapeptides (Ac-ooxxxx-NH2) was used to identify a novel ligand for the μ-opioid receptor, Acrfwink-NH2 (IC50 = 18 nM) [3]. Although the first two amino acid side chains (Arg and Phe) and the presence of the N-terminal acetyl is similar to that found in the Acetalins, the d form of the amino acids was found to be essential. Ac-rfwink-NH2 had a Ki value of greater than 1500 nM for δ-receptors and had very low affinity in assays for the κ1 (Ki > 2000 nM) and κ2 (Ki > 5000 nM) receptor subtypes and only modest affinity in assays for κ3 receptors (Ki = 288 ± 71 nM). Ac-rfwink-NH2 was shown to be a full agonist in the guinea pig ileum assay (median effective concentration, EC50 = 433 ± 43 nM). This activity was antagonized by naloxone at a low concentration (Ke = 3.80 nM), indicating that it was an opioid effect mediated through interaction with μopioid receptors. Ac-rfwink-NH2 is a potent agonist at the μ-receptor and induces long-lasting analgesia in mice. The in vivo potency of Ac-rfwink-NH2 (median effective dose, ED50 = 0.6 nmol, intracerebrovascularly, ICV) was found to be approximately twice that obtained for morphine (ED50 = 1.29 nmol, ICV). Ac-rfwink-NH2 produced antinociception equal to that of morphine following intraperitoneal (IP) administration. Because analgesia produced by IP administered Ac-rfwink-NH2 is blocked by ICV administration of naloxone, this peptide is most likely able to cross the blood–brain barrier. A nonacetylated library made up entirely of d-amino acid hexapeptides (ooxxxx-NH2) was also found to have activity at the μ-receptor. On completion of the iterative deconvolution process, the two peptides found to bind with the highest affinity to the μ-receptor were iftwyrNH2 (IC50 = 5 nM) and imswwg-NH2 (IC50 = 10 nM).

1490 / Chapter 208 Two new series of nonacetylated hexapeptides not related to the enkephalins were identified, YPFGFONH2 and WWPKHO-NH2 (where O = 1 of 20 l-amino acids). Both possessed high affinity (IC50 values of the most active peptides were 10–15 nM) and selectivity for the μ-receptor, individuals of which were found to be μ-agonists. Two additional series were identified from the acetylated library, Ac-FRWWYO-NH2 and AcRWIGWO-NH2 (IC50 values of the most active peptides were 5–10 nM). The peptide Ac-FRWWYM-NH2 was determined to be an agonist for the μ-receptor, whereas the peptide Ac-RWIGWR-NH2 was found to be an antagonist at this receptor. In total, six different families of sequences were identified from the two libraries [8].

d-RECEPTOR LIGANDS A positional scanning format of the hexapeptide library (OOXXXX-NH2, XXOOXX-NH2, and XXXXOONH2) was used to identify a new ligand for the δreceptor, YGFDLV-NH2 (IC50 = 15 nM). This peptide is similar to the sequences found when a single position defined positional scanning library was used in an earlier study, YHGWLV-NH2 and YGMHLV-NH2 [31]. YGFDLVNH2 was found to be an agonist at the δ-receptor.

k-RECEPTOR LIGANDS An N-acetylated positional scanning decapeptide library was screened in binding assays specific for the κ-receptor. The library was made up of approximately 3 trillion decapeptides, with each of the 200 mixtures of these two separate libraries composed of approximately 200 billion decapeptides per mixture. From this extremely large pool of compounds, two peptide sequences were identified from the N-acetylated library having high affinities for the κ-receptor: AcYRTRYRYRRR-NH2 (IC50 = 28 nM) and Ac-RGWFHYKPKR-NH2 (IC50 = 30 nM). In a recent study, κ-selective peptides having intrinsic fluorescent properties were identified from a rhodamine-labeled tetrapeptide positional scanning synthetic combinatorial library (PS-SCL) [16]. From a library of approximately 7.3 million rhodamine-labeled tetrapeptides, more than 250 individual peptides were synthesized from the most active mixtures. Eight individual rhodamine-labeled peptides were identified that were specific for the κ-opioid receptor, having binding affinities ranging from 5 to 20 nM. The majority of the most active peptides identified contained at least two positively charged residues. Such strongly positively charged compounds have the potential for nonspecific binding to the receptor through ionic interactions. This was

found not to be the case because the same peptides lacking a rhodamine label did not inhibit binding of the radioligand to the κ-receptor (Ki > 10 μM). Because rhodamine and rhodamine sulfonyl amide had no inhibitory activity, the peptide sequence together with the rhodamine moiety were necessary components of a single molecule responsible for receptor binding. We are currently pursuing a range of other fluorescent groups in a similar manner with both peptide and nonpeptide libraries for the identification of opioid receptor–specific ligands.

m-, d-, AND k-LIGANDS A tetrapeptide positional scanning library composed of mixtures of 50 different l-, d-, and unnatural amino acids was screened against all three opioid receptors. Following an extensive study [4, 9], individual compounds were identified for μ-, δ-, and κ-opioid receptors. The most active peptide for the μ-receptor was Y(d-Nve-)G(l-Nal)-NH2 (Ki = 0.4 nM), and the most μselective peptide found was YrAW-NH2 with μ : δ and μ : κ ratios of greater than 1000. The most selective peptide identified for the δ-receptor was Wy(aAba)RNH2 (Ki = 7 nM). The most active peptide identified for the κ-receptor was ff(d-Nle)r-NH2 (Ki = 1 nM). The tetrapeptides identified for the κ-receptor were highly selective for this receptor, with κ : μ and κ : δ ratios of greater than 10,000 [9]. The κ-selective peptide Kaffiralin (ffir-NH2) is a full agonist in the GTPγS binding assay (EC50 = 10 nM hKOR-CHO cells). Kaffiralin generated a full dose-response curve in the mouse 55°C warm-water tail-flick assay and in the acetic acid writhing test after ICV administration. It was approximately 10-fold more potent in the writhing test than in the tail-flick test. After IP administration, Kaffiralin did not produce a full dose-response curve in the tail-flick assay, but did in the writhing test. It was antagonized by an IP injection of nor-BNI but not an ICV injection of nor-BNI. When given by IP administration, ffir-NH2 may produce antinociception at spinal or peripheral receptors instead of supraspinal receptors. There was no evidence of the peptide crossing the blood–brain barrier, and it is now in phase I clinical trials.

ORPHAN RECEPTOR LIGANDS In October and November 1995, two separate reports were published [21, 32] on the identification of the endogenous ligand for the orphan receptor ORL1, a potential member of the opioid family of receptors. After establishing a binding assay in rat brain with tritiated Orphanin FQ [7], a hexapeptide positional scan-

Mixture-Based Combinatorial Libraries / 1491 ning library containing two defined positions (1200 mixtures) was used to identify hexapeptide ligands for this receptor. The most active peptide found was AcRYYRWK-NH2. The five most active compounds were found to be agonists by use of three separate biochemical bioassays: inhibition of cAMP accumulation, stimulation of [35S]GTPγS binding, and inhibition of electrically induced contractions in the mouse vas deferens [10].

TESTING MIXTURES IN VIVO A series of preliminary experiments have been conducted as a proof of concept study to validate the use of mixture-based libraries reported to exhibit opioid activity in an in vivo model. In this study, we compared the activity of mixtures of tetrapeptides each containing 125,000 tetrapeptides. These peptide mixtures were based on the known active dermorphin analog 2,6-dimethyltyrosine-DALDA ([Dmt]-DALDA, H-Dmtd-Arg-Phe-Lys-NH2). Dmt-DALDA was identified through an outstanding classical synthetic medicinal chemistry effort by Schiller’s group through many years of effort [33]. These compounds have high μopioid receptor affinity [33, 37] with proven antinociceptive properties in mice [34]. The Dmt-tyrosine group of [Dmt]-DALDA has been shown to be an essential component, as demonstrated by both in vitro and in vivo empirical data. Therefore, a positively biased mixture of compounds was synthesized in which Dmt-tyrosine was fixed at the first position (Dmt-XXX). This mixture was examined for its activity and compared with the antinociceptive effect of the known active individual Dmt-DALDA and two negatively biased tetrapeptide mixtures (F-XXX and k-XXX— 800 and 3580 times less active in vitro, respectively). To assess the antinociceptive effects of these mixtures, the mouse tail-flick assay was used because it is well established, yields clear and reproducible end points, and can differentiate between μ and κ activity. DmtXXX, at a dose of 50 mg/kg, was active and had a duration of action comparable to the individual compound [Dmt]-DALDA at 10 mg/kg. This effect appears to be specific because F-XXXNH2 was inactive at comparable doses. It was also observed that Dmt-XXX was longer acting than morphine (i.e., 5 h vs. 1 h), a property already reported for [Dmt]-DALDA [34] and found to be maintained for the mixtures containing Dmt on the N-terminal. As anticipated, the mixtures required greater absolute doses than the individual compounds. Of clear significance in this study was that k-XXX, although having very poor activity in vitro, was clearly active in the tail-flick test. This may be due to a number of factors, including the

interaction at an unknown nonopiate receptor, the inhibition or stimulation of an enzyme, or translational factor or signaling event. Following these results, we carried out a single iteration by synthesizing the anticipated next Dmt-r-XX mixture, the second position was defined with darginine, which is the amino acid found in DALDA. This iteration decreases the complexity of the mixtures from 125,000 compounds (OXXX) to 2500 compounds (OOXX). The antinociceptive effects of these mixtures were tested in the tail-flick assay as before. As the tetrapeptide mixture becomes more defined, we expect an increase in activity when tested at the same concentration. It is of note that when decreasing the mixture size from 125,000 for Dmt-XXX to 2500 for Dmt-r-XX the increase in activity was modest (four-fold in vitro), whereas the decrease in mixture size was 50-fold. This discrepancy can be explained by several differing mechanisms. Clearly more than one substitution at the second position can be accepted and activity retained (lysine may be an acceptable replacement for arginine). In addition, it is important to point out that we fixed darginine at the second position simply as a test study to expand this proof of concept for the use of mixturebased combinatorial libraries directly in vivo. There is, in fact, no reason to assume that d-arginine is necessarily the most functionally useful amino acid at the second position, especially in vivo. At 5 hours postinjection at a dose of 25 mg/kg, this peptide mixture of 2500 tetrapeptides was clearly more active than morphine. Six others were tested in vivo (Y-XXX, F-XXX, p-XXX, KXXX, k-XXX, and D-XXX), and the activities of these mixtures in binding and cAMP assays are shown in Table 2. This expected correlation between the in vitro and in vivo activities was found for all except k-XXX, which had significant tail-flick activity from 30 minutes all the way through the 5 hour cut-off point. This result illustrates perhaps the central most exciting aspect of the proposed studies, namely the direct in vivo identification of mixtures that may be active as pain-modulating agents at sites other than the expected three opiate receptors. These preliminary results are encouraging, and the study must be moved forward to determine the final sequences.

CONCLUSION A wide range of combinatorial libraries, from peptides to low-molecular-weight heterocyclic compounds, have been successfully screened in assays specific for the opioid receptors and have enabled the identification of new ligands for these receptors. New opioid peptides found from combinatorial libraries range in length from tetramers to decamers. The compounds described

1492 / Chapter 208 TABLE 2.

μ-Binding Ki and cAMP IC50 Data for Selected Individual and Tetrapeptide Mixtures. μ-Binding Ki (nM)

cAMP IC50 (μM)a

Number of Compounds

L-(Dmt)-Tyr L-Tyr L-Phe D-Pro L-Lys D-Lys D-Asp

X X X X X X X

X X X X X X X

X X X X X X X

16.2 592.2 13,000 41,238 55,920 58,030 84,380

6.8 651.2 1027 N.D. N.D. N.D. N.D.

125,000 125,000 125,000 125,000 125,000 125,000 125,000

L-(Dmt)-Tyr L-Tyr

D-Arg D-Arg

X X

X X

4.5 136.0

0.4 91.3

2500 2500

Dmt-DALDA: L-(Dmt)-Tyr L-Tyr DALDA:

D-Arg D-Arg

L-Phe L-Phe

L-Lys L-Lys

0.2 5.5

0.002 0.8

1 1

N.D., no data.

a

in this chapter are the most active in a series and represent only a fraction of the number of individual peptides synthesized and tested. Some of the peptides resemble the classical opioids—they possess a tyrosine at position 1 and a tertiary amino group on the Nterminus. The peptide YPWFPO-NH2 is a hexapeptide analog of the recently described tetrapeptide endomorphin-1 (YPWF-NH2), thought to be the endogenous ligand for the μ-receptor [36] (see Chapter 185 in the Opioid Peptides section of this book). The δ selective hexapeptides identified possess both Tyr1 and Leu5, but differ from one another and the enkephalins at the remaining positions. YHGWLV-NH2 has a conservative replacement of Phe4 by Trp4; thus, it differs from the enkephalin mainly at the His2. Although not yet tested, it is assumed Val6 is redundant. The two remaining δ-peptides have replacements at the fourth and fifth positions (YGMHLV-NH2 and YGFDLV-NH2). Novel peptides identified for the opioid receptors were found to contain basic residues (arginine and lysine), and many also contain tryptophan. Basic residues are found in position 1 in the Acetalins and in the all damino acid peptides, Ac-rfwink-NH2. They are also found on the C-terminal residue in peptides selective for μ, δ, or κ (WWPR-NH2, Wy(Nve)R-NH2, and f(Dnal)(D-nle)r-NH2, respectively). The agonist activity of the Ac-rfwink-NH2 peptide was unexpected, in that it was incorrectly assumed that compounds identified from the libraries would in most instances be identified as antagonists. Furthermore, the Acetalins (AcRFMWMK-NH2), which have sequence similarities to Ac-rfwink-NH2 (although composed entirely of l-amino acids), were found to be antagonists. Certainly the identification of the opioid peptide Ac-rfwink-NH2, which does not require a phenolic hydroxyl nor a tertiary amino group to be an agonist, challenges the precon-

ception that these pharmacophores are required for agonist activity. Since then, we have found that more than 80% of the new sequences identified are agonists at the opioid receptors. Whether the high percentage of agonists found is due to a bias resulting from use of an agonist in the initial library screen has yet to be determined.

FUTURE STUDIES The high numbers of different compounds identified raise the question of the plasticity of the opioid receptors. The plasticity of the μ-receptor may account for the high numbers of new agonists identified. The data obtained from in vivo studies on these new opioid ligands should generate interesting insights into this question. Finally, it is now imperative to explore the enormous potential inherent in the direct in vivo testing of mixture-based chemical diverse libraries. The objective of these future studies is to demonstrate that in vivo screening and deconvolution of mixture-based combinatorial libraries can yield a more direct approach for the identification of fundamentally more advanced therapeutic candidates. If successful, this will result in savings of both time and resources relative to the typical target-based drug-discovery process.

References [1] Chen, Y., Mestek, A., Liu, J., Hurley, J.A., and Yu, L. Molecular cloning and functional expression of a μ-opioid receptor from rat brain. Mol. Pharmacol. 1993, 44, 8–12. [2] Dooley, C.T., Chung, N.N., Schiller, P.W., and Houghten, R.A. Acetalins: Opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries. Proc. Natl. Acad. Sci. USA 1993, 90, 10811–10815.

Mixture-Based Combinatorial Libraries / 1493 [3] Dooley, C.T., Chung, N.N., Wilkes, B.C., Schiller, P.W., Bidlack, J.M., Pasternak, G.W., and Houghten, R.A. An all D-amino acid opioid peptide with central analgesic activity from a combinatorial library. Science 1994, 266, 2019–2022. [4] Dooley, C.T., Hope, S.K., and Houghten, R.A. Identification of tetrameric opioid peptides from a combinatorial library composed of L-, D- and non-proteinogenic amino acids. In Peptides 94: Proceedings of the 23rd European Peptide Symposium; Maia, H.L.S., Ed.; ESCOM: Leiden, 1995; pp. 805– 806. [5] Dooley, C.T., Hope, S., and Houghten, R.A. Rapid identification of novel opioid peptides from an N-acetylated synthetic combinatorial library. Regul. Pept. 1994, 54, 87–88. [6] Dooley, C.T. and Houghten, R.A. Orphanin FQ: Receptor binding and analog structure activity relationships in rat brain. Life Sci. 1996, 59, PL23–PL29. [7] Dooley, C.T. and Houghten, R.A. The use of positional scanning synthetic peptide combinatorial libraries for the rapid determination of opioid receptor ligands. Life Sci. 1993, 52, 1509– 1517. [8] Dooley, C.T., Kaplan, R.A., Chung, N.N., Schiller, P.W., Bidlack, J.M., and Houghten, R.A. Six highly active mu-selective opioid peptides identified from two synthetic combinatorial libraries. Pept. Res. 1995, 8, 124–137. [9] Dooley, C.T., Ny, P., Bidlack, J.M., and Houghten, R.A. Selective ligands for the μ, δ, and κ opioid receptors identified from a single tetrapeptide positional scanning combinatorial library. J. Biol. Chem. 1998, 273, 18848–18856. [10] Dooley, C.T., Spaeth, C.G., Craymer, K., Adapa, I.D., Brandt, S.R., Houghten, R., and Toll, L. Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1. J. Pharmacol. Exp. Ther. 1997, 283, 735– 741. [11] Evans, C.J., Keith, D.E., Jr., Morrison, H., Magendzo, K., and Edwards, R.H. Cloning of delta opioid receptor by functional expression. Science 1992, 258, 1952–1955. [12] Fukuda, K., Kato, S., Mori, K., Nishi, M., and Takeshima, H. Primary structures and expression from cDNAs of rat opioid receptor δ- and μ-subtypes. FEBS Lett. 1993, 327, 311– 314. [13] Furka, A., Sebestyen, F., Asgedom, M., and Dibo, G. General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Pept. Protein Res. 1991, 37, 487–493. [14] Houghten, R.A., Appel, J.R., Blondelle, S.E., Cuervo, J.H., Dooley, C.T., and Pinilla, C. The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides. BioTechniques 1992, 13, 412–421. [15] Houghten, R.A. and Dooley, C.T. The use of synthetic peptide combinatorial libraries for the determination of peptide ligands in radio-receptor assays: Opioid peptides. BioMed. Chem. Lett. 1993, 3, 405–412. [16] Houghten, R.A., Dooley, C.T., and Appel, J.R. De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library. Bioorg. Med. Chem. Lett. 2004, 14, 1947–1951. [17] Houghten, R.A., Pinilla, C., Appel, J.R., Blondelle, S.E., Dooley, C.T., Eichler, J, Nefzi, A., and Ostresh, J.M. Mixture-based synthetic combinatorial libraries. J. Med. Chem. 1999, 42, 3743– 3778. [18] Houghten, R.A., Pinilla, C., Blondelle, S.E., Appel, J.R., Dooley, C.T., and Cuervo, J.H. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 1991, 354, 84–86. [19] Kieffer, B.L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C.G. The δ-opioid receptor: Isolation of a cDNA by expression cloning

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[32]

[33]

[34]

and pharmacological characterization. Proc. Natl. Acad. Sci. USA 1992, 89, 12048–12052. Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M., and Knapp, R.J. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991, 354, 82– 84. Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C., Molsand, C., Alvinerle, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazargull, H., Vassart, G., Parmentier, M., and Costentin, J. Isolation and structure of the endogenous agonist of the opioid receptor-like ORL1 receptor. Nature 1995, 377, 532–535. Nefzi, A., Dooley, C.T., Ostresh, J.M., and Houghten, R.A. Combinatorial chemistry: From peptides and peptidomimetics to small organic and heterocyclic compounds. BioMed. Chem. Lett. 1998, 8, 2273–2278. Nefzi, A., Giulianotti, M., and Houghten, R.A. Solid phase synthesis of 2,4,5-trisubstituted thiomorpholin-3-ones. Tetrahedron Lett. 1998, 39, 3671–3674. Nefzi, A., Ostresh, J.M., and Houghten, R.A. Solid phase synthesis of 1,3,4,7-tetrasubstituted perhydro-1,4-diazepine-2,5-diones. Tetrahedron Lett. 1997, 38, 4943–4946. Nefzi, A., Ostresh, J.M., Meyer, J.-P., and Houghten, R.A. Solid phase synthesis of heterocyclic compounds from linear peptides: Cyclic ureas and thioureas. Tetrahedron Lett. 1997, 38, 931–934. Nefzi, A., Ostresh, J.M., Yu, Y., and Houghten, R.A. Combinatorial chemistry: Libraries from libraries, the art of the diversityoriented transformation of resin-bound peptides and chiral polyamides to low molecular weight acyclic and heterocyclic compounds. J. Org. Chem. 2004, 69, 3603–3609. Ostresh, J.M., Husar, G.M., Blondelle, S.E., Dörner, B., Weber, P.A., and Houghten, R.A. “Libraries from libraries”: Chemical transformation of combinatorial libraries to extend the range and repertoire of chemical diversity. Proc. Natl. Acad. Sci. USA 1994, 91, 11138–11142. Ostresh, J.M., Schoner, C.C., Hamashin, V.T., Nefzi, A., Meyer, J.-P., and Houghten, R.A. Solid phase synthesis of trisubstituted bicyclic guanidines via cyclicization of reduced N-acetylated dipeptides. J. Org. Chem. 1998, 63, 8622–8623. Ostresh, J.M., Winkle, J.H., Hamashin, V.T., and Houghten, R. A. Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings. Biopolymers 1994, 34, 1681–1689. Pinilla, C., Appel, J.R., Blanc, P., and Houghten, R.A. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. Biotechniques 1992, 13, 901–905. Pinilla, C., Appel, J.R., Blondelle, S.E., Dooley, C.T., Eichler, J., Ostresh, J.M., and Houghten, R.A. Versatility of positional scanning synthetic combinatorial libraries for the identification of individual compounds. Drug Dev. Res. 1994, 33, 133– 145. Reinscheid, R.K., Nothacker, H.P., Bourson, A., Ardati, A., Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H., Monsma, F.J. Jr., and Civelli, O. Orphanin FQ: A neuropeptide that activates opioidlike G protein-coupled receptor. Science 1995, 270, 792–794. Schiller, P.W., Nguyen, T.M., Berezowska, I., Dupuis, S., Weltrowska, G., Chung, N.N., and Lemieux, C. Synthesis and in vitro opioid activity profiles of DALDA analogues. Eur. J. Med. Chem. 2000, 35, 895–901. Shimoyama, M., Shimoyama, N., Zhao, G.M., Schiller, P.W., and Szeto, H.H. Antinociceptive and respiratory effects

1494 / Chapter 208 of intrathecal H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA) and [Dmt1] DALDA. J. Pharmacol. Exp. Ther. 2001, 297, 364– 371. [35] Yasuda, K., Raynor, K., Kong, H., Breder, C.D., Takeda, J., Reisine, T., and Bell, G.I. Cloning and functional comparison of kappa and δ opioid receptors from mouse brain. Proc. Natl. Acad. Sci. USA 1993, 90, 6736–6740.

[36] Zadina, J.E., Hackler, L., Ge, L.-J., and Kastin, A.J. A potent and selective endogenous agonist for the μ-opiate receptor. Nature 1997, 386, 499–502. [37] Zhao, G.M., Qian, X., Schiller, P.W., and Szeto, H.H. Comparison of [Dmt1]DALDA and DAMGO in binding and G protein activation at mu, delta, and kappa opioid receptors. J. Pharmacol. Exp. Ther. 2003, 307, 947–954.

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209 Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV JÖRG VOTTELER, KARSTEN BRUNS, PETER HENKLEIN, VICTOR WRAY, AND ULRICH SCHUBERT

ties sufficient for structural and biochemical studies. In this synopsis of viral peptides, we do not go into any further detail regarding the methods of synthesis, purification, and quality control of such peptides already published in the literature cited here; instead, we dwell on the use of such peptides in structural and functional investigations. Space and convenience dictate that we concentrate on those systems of direct concern to our ongoing research of the human immunodeficiency virus type 1 (HIV-1) such as the regulatory proteins Vpu, Vpr, and Tat and the virus structural protein p6 Gag. In addition, we briefly cover the synthesis and characterization of the HIV-1 protease (PR), the nucleocapsid (NC), and the matrix (MA) proteins. Finally, we discuss the advantage of peptide synthesis in the molecular characterization of small virus proteins.

ABSTRACT Efficient solid-state synthesis now affords ready access, particularly in situations in which recombinant protocols usually fail, to high-quality synthetic peptides that are equivalent to many full-length biologically active systems or domains thereof, including posttranslational modifications such as phosphorylation and acetylation. Here we survey recent applications of such peptides to the investigations of human immunodeficiency viruses (HIV), with the focus on the regulatory peptides Vpu, Vpr, and Tat, as well as the protease, the Gag peptides nucleocapsid, matrix, and p6 from HIV-1. The importance of the full-length synthetic viral peptides for molecular characterization that involves both detailed biochemical and structural studies at the atomic level and their potential for therapeutic applications are emphasized.

HIV-1-DERIVED SYNTHETIC PEPTIDES All retroviruses contain at least three major genes that encode the main virion structural components, which are each synthesized as three polyproteins that produce either the virion interior (Gag, group-specific antigen), the viral enzymes (Pol, polymerase), or the glycoproteins of the virion envelope (Env). Viruses of the lentivirus genus including HIV-1, HIV-2, and simian immunodeficiency virus (SIV) replicate preferentially in lymphocytes and in differentiated macrophages and cause long-lasting and mostly incurable chronic diseases (for review see [8]). In addition to the canonical retroviral Gag, Pol, and Env proteins common to other replication-competent retroviruses, HIV-1 and other lentiviruses encode further small proteins with either essential regulatory (Tat and Rev) or accessory (Vpu, Vif, Nef, and Vpr) functions that serve to accelerate viral replication. These latter proteins are also called auxiliary because they are not essential for HIV-1 repli-

INTRODUCTION This chapter illustrates the use of synthetic full-length biologically active virus peptides, and the relevant domains thereof, that exhibit the amino acid sequence of the native counterpart. Of necessity, we do not consider synthetic peptide fragments that do not correspond to known domains of virus proteins, do not exhibit biological activity, or have been merely used as biochemical tools, such as for epitope mapping or for antibody generation. Inasmuch as the limit in solidphase peptide synthesis is around 100 amino acids, this chapter is limited to relatively small virus peptides with mostly regulatory functions in the virus replication cycle. Currently, two protocols exist for peptide synthesis, namely the step-by-step and the chemical ligation of peptide fragment procedures, that can be optimized to afford convenient, highly efficient products in quantiHandbook of Biologically Active Peptides

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1496 / Chapter 209 cation in certain cell lines, yet a growing number of in vivo studies report important roles of these HIV-1 gene products in the pathology and spread of HIV-1 (for review see [68]). Synthetic peptides have played a significant role in unraveling the molecular mechanisms of these six HIV-1 proteins in the virus replication cycle, and some of these have been chemically synthesized in their full-length form, including various mutants and fragments thereof.

THE HIV-1-SPECIFIC VIRUS PROTEIN U The virus protein U (Vpu) is unique to HIV-1. No structural homologs have been detected in primate lentiviruses, even in closely related species such as HIV-2 or SIV, except for the HIV-1 related isolate SIVCPZ. Little was known of the molecular structure of Vpu at the time of its first biochemical and functional characterization, and it was soon realized that such studies were only possible through the use of synthetic peptides because recombinant DNA techniques afforded only low yields of mostly toxic and insoluble amounts of these hydrophobic peptides when expressed in bacteria. Considerable biochemical data now exist that characterize Vpu as a type I oriented integral oligomeric membrane phosphoprotein consisting of an amphipathic sequence of 80–82 amino acids making up a hydrophobic Nterminal membrane anchor (VpuTM) proximal to a polar C-terminal cytoplasmic tail (VpuCYTO); each domain exhibits distinct regulatory functions in the viral life cycle (for review see [5, 22, 35]). Vpu has two main functions: the augmentation of virus particle release from a post–endoplasmatic reticulum (ER) compartment by a cation selective ion channel activity mediated by VpuTM [58, 59], and the regulation of the cell surface expression of several glycoproteins involved in host immune response mediated by VpuCYTO. The bestcharacterized mechanism of the latter function is the Vpu-induced degradation of the primary virus receptor CD4 in the ER that requires the casein kinase2 (CK-2)dependent phosphorylation [60, 61]. Identification of the phospho-acceptor sites in positions 52 and 56 within a highly conserved dodecapeptide extending from Glu47 to Gly-58 was achieved by employing a number of synthetic peptides [61]. With these peptides of VpuCYTO, it was shown that CK-2 preferentially phosphorylates Ser56, which through a positive cooperative mechanism stimulates the phosphorylation of Ser52 [60]. Also for the deciphering of Vpu’s second molecular activity (e.g., its ion channel function), the use of synthetic peptides were indispensable. Initially, the reconstitution of the synthetic VpuTM in planar lipid bilayers identified a cation-selective ion-channel activity [59] that was also demonstrated for full-length Vpu in bilay-

ers [19] and in amphibian oocytes [59]. The idea that Vpu functions as an ion channel was further supported by the observation that Vpu enhances membrane permeability when expressed in pro- and eukaryotic cells [28] or when a recombinant mutant version of Vpu expressed in Escherichia coli was reconstituted in bilayers [45]. Although the synthesis of full-length Vpu was initially accompanied by several problems, mostly due to its amphipathic nature, ion channel activity was finally shown also for full-length synthetic Vpu following reconstitution in lipid bilayers [40]. Over the last decade, structural studies with synthetic Vpu have involved the use of a combination of highresolution 1H nuclear magnetic resonance (NMR) spectroscopy in solution and solid-state 15N NMR spectroscopy in oriented bilayers. The failure of Vpu peptides to crystallize precludes the use of x-ray crystallographic techniques, although structural and orientational studies on Vpu peptides in lipid monolayers were conducted by x-ray scattering recently [78]. In initial structural studies conducted in buffered aqueous solution, only transitory stable structures were identified [34, 73]. Subsequently, secondary structures of Vpu peptides could be stabilized in a membranelike milieu afforded by organic solvents and micelles. The nature of the solution structure of the synthetic VpuCYTO under these conditions was established by two-dimensional 1H NMR spectroscopy and simulated annealing calculations: It is predominantly monomeric and adopts welldefined helix-interconnection-helix-turn conformations in which the four regions are bounded by residues 37– 51, 52–56, 57–72, and 73–78 [20]. Almost identical regions of secondary structure were determined from 2 H-, 13C-, or 15N-labeled recombinant Vpu analyzed in lipid micelles by multidimensional heteronuclear NMR spectroscopy [45]. Both helices are amphipathic in character, but they show different charge distributions. The flexibility of the interconnecting hinge region permits orientational freedom of the helices and exposes the highly conserved CK-2 phosphorylation sites. Subsequently, attention was on the N-terminal hydrophobic domain VpuTM, which is primarily associated with an ion channel activity. Although a compact well-defined U-shaped tertiary structure involving a number of helices was determined for the N-terminal synthetic peptide Vpu1–39 in 50% trifluorethanol (TFE) solution [74], subsequent proton-decoupled 15N crosspolarization solid-state NMR spectroscopy was employed to investigate synthetic, specifically 15N-labeled, VpuTM oriented in phospholipid bilayers [33, 74]. The 15N data are consistent with a transmembrane alignment of a helical polypeptide, implying that the nascent helices in the folded solution structure reassemble to form a linear tilted α-helix involving residues 6–29 that lies parallel to the bilayer normal, and the placement

Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV / 1497

C

N

Oligomeric Forms

Ion channel activity associated with transmembrane helix

Host Proteins

Interaction of phosphorylated cytoplasmic domain with CD4

FIGURE 1. Dynamic model of the membrane-bound structures of full-length Vpu. The combined experimental data indicate the three helices are bounded by residues 6–29 in VpuTM, and 31–51, as well as 57–72 in VpuCYTO. CK-2 phospho-acceptor sites are at serines 52 and 56.

of Trp-22 near the Glu-28-Tyr-Arg-motif is predicted to be important for helix termination and pore selectivity at the membrane-cytoplasm interface. Similar results were afforded by a novel 15N solid-state NMR technique using universally 15N-labeled recombinant material [53] and site-specific Fourier transform infrared dichroism data for the 13C-labeled peptide Vpu1–31 [43]. For further detailed analysis of the orientation of the various secondary structure elements of Vpu with respect to the membrane, full-length chemically synthesized Vpu and site-specific 15N-labeled analogs produced by native chemical ligation methodologies were studied by solid-state NMR spectroscopy in aligned and hydrated lipid bilayers [40]. These and previous orientational studies [33, 45] have provided a starting point for molecular dynamic simulations that have afforded a model of the ion channel activity of Vpu and other viral ion channels (for review see [23]). Thus, the model of Vpu shown in Fig. 1, based on the biochemical and structural studies of a large number of synthetic peptides and recombinant material in combination with theoretical calculations, suggests that Vpu exists in vivo either as a phosphoprotein in multiprotein complexes involving CD4 and several other host proteins [46] or in homooligomeric nonphosphorylated forms acting as an ion channel embedded in the cell membrane.

STRUCTURE AND FUNCTION OF THE LENTIVIRAL PROTEIN R Another HIV-1 accessory gene product, the highly conserved 96-amino-acid lentiviral peptide R (Vpr), which was produced synthetically as a full-length bio-

logically active molecule, has received considerable attention and a number of functions have been attributed to its presence in various cellular and extracellular compartments. The most intensively investigated biological functions of Vpr are those inducing the arrest of the infected cell in the G2 phase of the cell cycle, and mechanisms affecting the translocation of the viral preintegration complex (PIC) of the incoming virus from the cytoplasm to the nucleus, an essential step for the integration of the pro-viral genome into the chromosome (for a recent comprehensive review, see [1, 63]). The nuclear targeting function of Vpr, which is essential for transport of the PIC into the nucleus of cells that do not undergo mitosis and thus do not exhibit disruption of the nuclear membrane, has been associated with HIV-1 infection of terminally differentiated macrophages. Therefore, a major role has been attributed to Vpr for the productive infection of cells of the monocyte/macrophage lineage. Among others, these cells represent the virus reservoir in which HIV persists even in the course of effective antiretroviral therapy. Regarding the second function of Vpr, which leads to G2 cell cycle arrest in HIV-1-infected T cells, it was suggested that this activity provides an intracellular milieu conducive for enhanced viral replication by increasing the HIV long terminal repeat (LTR)-driven gene expression [27]. This response has recently been linked with the capacity of Vpr to alter the structure of the nuclear lamina, leading to transient DNA-containing herniations of the nuclear envelope that intermittently rupture [11]. Other studies suggest that the prolonged G2 arrest induced by Vpr ultimately leads to apoptosis of the infected cell. Conversely, early anti-apoptotic effects of Vpr have also been described, which are superseded later by its pro-apoptotic effects. These pro-apoptotic effects of Vpr may result from either effects on the integrity of the nuclear envelope or direct mitochondrial membrane permeabilization, perhaps involving Vprmediated formation of ion channels in cellular membranes (for review see [21]). Functions of Vpr and their cellular and viral binding partners are summarized in Fig. 2B. To date, structural studies of Vpr have been hampered by the fact that this protein does not crystallize, and the use of NMR techniques is complicated by the strong tendency for Vpr to undergo self-association. In addition, Vpr’s structure depends critically on the solution conditions [32, 69]. However, a model of secondary structure elements in Vpr is slowly emerging from studies of Vpr fragments [18, 44, 62] and, more recently, of full-length synthetic forms of Vpr [32, 69]. In order to detect the most structured forms of Vpr, these studies were performed in aqueous solution that required the presence of either detergents or micelles. The

1498 / Chapter 209 17

1

A

*

*

33

α1

*

40

*

55

48

α2

83

96

α3 B

transactivation of promoters:

HIV-1 components:

cyclinT, Sp1, TFIIB glucocorticoid receptor p300/CREB-binding protein

Gag (p6, NC), MA, Vpr

HIV-1 reverse transcription:

Lys-tRNA-synthetase

apoptosis:

HHR23A ANT

Vprmultifactor interacting protein

host cellular chaperones:

CypA, Hsp70 cell cycle arrest G2/M:

hVIP/mov34 HHR23A 14-3-3 proteins

mutation rate of HIV-1 genome:

uracil DNA glycosylase nuclear translocation:

RIP, Importin α nucleoporin cellular DNA

secondary structures in Vpr emerging from these analyses (summarized in Fig. 2A) suggest the presence of an α-helix-turn-α-helix motif between residues 17 and 48 and an amphipathic α-helix located between amino acids 53–55 and 78–83 [6, 69, 70]. With synthetic Vpr, a physical interaction of Vpr with the prolyl cis/trans isomerase (PPIase) cyclophilin A (CypA) was shown. Although it was known for a decade that both CypA and Vpr are present in HIV-1 virions, a functional relation of Vpr and this major host cell chaperone has not been defined until recently ([6, 77], and references therein). This interaction, which involves the N-terminal region of Vpr including an essential role for proline in position 35, regulates functional expression of Vpr and can be blocked by CypA inhibitors. Compatible with this, NMR data on N-terminal Vpr peptides also demonstrated that all of the conserved proline residues in positions 5,10,14, and 35 undergo a cis/trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism indicates that the folding and function of Vpr should depend on a cis/trans-proline isomerase activity [6]. These studies provided the first evidence that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr [77].

FIGURE 2. A. Structural model of HIV-1 Vpr. The extent and position of α-helical secondary structure are depicted according to NMR investigations on synthetic Vpr [69]. The four highly conserved Nterminal proline residues at positions 5, 10, 14, and 35 involved in the interaction of Vpr with CypA are indicated by asterisks [6, 77]. B. Summary of functional interaction of Vpr with cellular as well as viral proteins during the HIV-1 replication cycle.

THE TRANS-ACTIVATOR OF TRANSCRIPTION (Tat) OF HIV-1 It is now generally accepted that the HIV transactivator (Tat) plays an important role in the pathogenesis of AIDS. Although originally described as an activator of the HIV-1 LTR-promoter, it was later shown to regulate reverse transcription, to affect the expression of various cellular and viral genes, and to be released from infected cells. This extracellular Tat, which is acting as a cell membrane–transducing peptide in the sense of a so-called trojan molecule, can affect neighboring cells, both uninfected and infected target cells. Indeed, there is accumulating evidence that Tat in its extracellular form plays a major role in AIDS-associated diseases such as Kaposi’s sarcoma and HIV-associated dementia (for a recent review see [36]). Tat can be expressed in two forms, as the 72-aminoacid one-exon Tat and as the 86- to 101-amino-acid (depending on the HIV-1 isolate) two-exon Tat expressed primarily early during infection. The 14- to 15-kDa Tat binds to an RNA stem-loop structure forming the Tat-responsive element (TAR) at the 5′ LTR region. Tat represents the first positive regulator of viral gene expression that controls the elongation by RNA polymerase II. It activates transcriptional elongation by stimulating the protein kinase TAK (Tat-associated kinase) resulting in hyperphosphorylation of the RNA

Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV / 1499 polymerase II. Although the initiation of transcription from the LTR promoter is efficient, the polymerase separates from the DNA template prematurely because it cannot elongate efficiently in the absence of Tat. Thus, Tat stimulates the production of full-length HIV transcripts and is, therefore, essential for HIV replication (for review see [25]). Until now, no crystal structure of Tat has been obtained; the current model of its 3D structure is based on NMR experiments [2]. Synthetic Tat peptides have been used in various aspects of molecular analyses, most importantly for vaccine development. Considering the fact that Tat is released into the circulation and that extracellular Tat contributes to AIDS pathogenesis, makes it a bona fide candidate for an anti-HIV vaccine (for review see [7]). Several other points also support the development of a Tat vaccine. For instance, Tat is expressed early in infection, it is essential for virus replication, it is conserved among different HIV-1 isolates (compared with other highly divergent HIV proteins), and it can transduce different tissues and cells throughout the organism, including the central nervous system. However, the application of native Tat is arguable inasmuch as the molecule is immunosuppressive and impairs cell viability. For instance, an 86-amino-acid synthetic Tat was shown to induce cytotoxic effects in lymphocytes at concentrations as low as 900 nM [3]. Nevertheless, in another study synthetic Tat administered transcutaneously was shown to breach the skin barrier and induce effective cellular and humoral immune response [54]. In order to increase the safety of Tat vaccine, a chemically modified, biologically inactive but highly immunogenic Tat molecule (Tat toxoid) was developed, which is currently under investigation in clinical phase I and II trials. Although no signs of toxicity were observed in humans, Tat toxoid induced T-cell-mediated immunity and anti-Tat neutralizing antibodies. However, a disadvantage to this strategy was reported in studies in rhesus macaques in which immunization with Tat or Tat toxoid did not protect against infection [64]. Synthetic Tat was also used for various functional studies, the first report can be traced back to 1990 [37]. Recently, synthetic one-exon Tat acetylated at position Lys-50 was used to characterize the histone acetyltransferase p300/CBP-associated factor (PCAF) as a coactivator of the HIV LTR promoter [16]. In another approach, the proliferative activity of extracellular Tat on epithelial cells was studied with a two-exon synthetic (86-amino-acid) Tat peptide [4]. Further, six different Tat proteins in the range of 86–101 amino acids derived from different field isolates of HIV-1 were synthesized and tested for biological activity. Most interestingly, synthetic Tat derived from highly virulent African strains was significantly more active in LTR promoter activation when compared with peptides that originate from

less pathogenic HIV-1 isolates [55]. In general, the relatively effective protocols for solid-phase peptide synthesis of Tat that have been developed by different laboratories over the years afford the efficient production of various full-length Tat peptides and mutants thereof. Thus, the use of synthetic Tat in functional studies, molecular analyses, and even therapeutical applications can be foreseen in the future in HIV research.

THE HIV-1 PROTEASE HIV PR is initially synthesized as part of the p180 Gag-Pol polyprotein, the translation of which results from a rare (approximately 5% compared with Pr55 Gag expression) frame-shift event in the 3′-end of the gag gene. In concert with virus budding and release, the enzyme is autocatalytically released from p180, and subsequently processes Gag and Gag polyproteins in an ordered fashion, yielding the mature structural Gag (MA, NC, CA, and p6) and enzymatic active Pol (PR; RT; RNase H; and integrase, IN) proteins (for review see [8]). The HIV-1 PR was identified as a homodimer, with each dimer related to aspartic peptidases. Because many natural variants have been studied, the HIV-1 PR is one of the most widely studied enzymes in the history of protein crystallography. The original structural studies on the HIV-1 PR, both in its nonliganded (empty) and liganded forms in complex with substrate inhibitors, were conducted on the crystallized enzyme obtained by total chemical synthesis (for review see [38, 72]). Soon after the discovery of HIV, it was rationalized that PR provides an important target for drug intervention. Thus, the enzyme was the first viral protein of HIV-1 to be synthesized in its full length and biologically active form. Following the discovery of HIV-1 and HIV-2 as the causative agent for the AIDS pandemic, and subsequently the deciphering of the viral genomes, a competitive search was initiated to define an unlimited source of highly purified and correctly folded HIV PR enzymes. Clearly, the availability of biologically active PR in itself enabled the design of PR-inhibitors developed as the first chemotherapeutic drugs that proved to be effective for the treatment of HIV infections. Schneider and Kent first reported in 1988 the chemical synthesis of the HIV-1 PR. The 99-amino-acid peptide sequence was deduced from an open-readingframe search of the HIV-1 genome [57]. The peptide exhibited substrate specificity for the HIV-1 Gag polyprotein, and further evidence was provided that the enzyme belongs to the family of aspartic proteases. In the same year, Darke et al. also reported the total synthesis of a 99-amino-acid HIV-1 PR and compared the

1500 / Chapter 209 activity of synthetic PR with that of recombinant PR expressed in bacteria [10]. They demonstrated that both the synthetic and recombinant PR enzymes specifically processed Gag and Gag-Pol polyproteins but not Env of HIV or other related retroviral polyproteins. At the same time Nutt et al. reported the synthesis of a 99-amino-acid HIV-1 PR that after folding under reducing conditions revealed a dimerization of the enzyme [49]. The authors also demonstrated that the folded enzyme cleaved the HIV-1 Gag precursor Pr55 into the correct processing products p17 MA and p24 CA. Studies on small peptide substrates and the use of specific inhibitors further supported the notion that viral PR functions as an aspartic protease. Also in 1988, Copeland et al. reported the chemical synthesis of both HIV-1 and HIV-2 PR enzymes, which were able to process the respective Gag polyproteins in a correct manner [9]. These early steps in the development of antiretroviral PR-inhibitors, which are one of the major components in antiretroviral therapy, proved to be very effective in granting rapid access to the viral proteases in a very short time from the prediction of the open reading frame to the first setup of the high-throughput assays necessary to search for specific PR-inhibitors. For comparison, in 1989 Kräusslich et al. reported the application of recombinant PR (90% purity at best!) for functional studies, confirming that the E. coli expressed enzyme shares the same substrate specificity and aspartic characteristics with that reported for the chemically synthesized PR peptides [42]. Another important milestone in the design of inhibitors specific for the HIV-1 PR was the deciphering of the high-resolution structure of the enzyme in its active form. In 1989, the structure of synthetic HIV-1 PR crystallized in complex with a peptide inhibitor at a 2.3 Å resolution was solved, demonstrating for first time the interacting domains of the catalytic core of the homodimeric enzyme. The result of this concerted and intensive investigation finally resulted in the unraveling of the 3D structure of the HIV-1 PR, which basically laid down the groundwork for the structure-based drug design leading to the class of PR-inhibitors currently used at the forefront of antiretroviral therapy (for review see [38, 39]). Total synthesis is still the method of choice for use in the molecular understanding of HIV PR. For instance, site-specific 13 C-labeling enabled the characterization of the catalytic active aspartic side chain and, thus, the chemical mechanism of HIV-1 PR [65].

THE HIV-1 p6 Gag PROTEIN The first HIV-1 structural protein that has been chemically synthesized is the p6 Gag protein that forms the very C-terminal part of the Gag precursor protein

Pr55. The Gag polyprotein Pr55 of HIV-1, which forms the virion interior, is required and sufficient for virusparticle assembly and budding, although genomic RNA and envelope proteins are obligatory for the production of infectious progeny virions. The processing of Pr55 by the viral PR generates the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The function of p6 during virus entry and its location in mature HIV-1 virus particles are not known, although it appears not to be associated with the virus core [71]. However, several functions have been ascribed to p6. It facilitates virus release [29] and is required for the incorporation of the viral accessory protein Vpr into the virus particle. It has also been implicated in the incorporation of the viral Pol and Env proteins [50, 76] and in the control of particle size [26]. Recently, p6 was reported to be the major phosphoprotein of HIV-1 particles [48] and there is evidence that the host cell mitogenactivated protein kinase (MAPK) ERK-2 regulates viral assembly and release by phosphorylation of Thr-23 in p6 [31]. Furthermore, p6 becomes monoubiquitinylated at conserved Lys residues in positions 27 and 33 and sumoylated at position 27. The biological function of these posttranslational modifications is still enigmatic, inasmuch as the mutation of Lys residues does not affect the replication of HIV-1 in T-cell culture [30, 51, 52]. Although p6 is one of the most variable Gag domains among primate lentiviruses, two highly conserved motifs can be discerned: the C-terminal LXXLF sequence which governs virus incorporation of Vpr [41] and the tetrapeptide primary L-domain PT/SAP motif located near the N-terminus of p6, which is crucial for the detachment of budding virions from the cell membrane and from one another [15, 29]. This motif has been identified as the primary sequence determinant for binding Pr55 to the tumor susceptibility gene product (Tsg101), an E2-type ubiquitin ligaselike protein. Another region of p6 comprising a cryptic YPXL-type L-domain mediates the binding to AIP1/ALIX, a class E vacuolar protein sorting factor that also interacts with Tsg101. AIP1/ALIX also binds to late-acting components of the endosomal sorting complex required for transport (ESCRT) and is necessary for the formation of multivesicular bodies (MVB) at endosomal membranes [66]. Among known lentiviruses, the 52-amino-acid HIV-1 p6 peptide is by far the smallest. Although p6 fulfills a major function in the formation of infectious viruses and represents a docking site for several cellular and viral binding factors, the molecular structure of this virus peptide, which can be synthesized relatively easily by standard solid-phase peptide synthesis, has not been properly defined. From previous studies of synthetic full-length p6 by circular dichroism (CD) and 1H NMR

Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV / 1501 spectroscopy, it was concluded that the molecule, although soluble in water, adopts only a random conformation without any preference for secondary structure [67]. Recently, the solution structure of the ubiquitin E2 variant (UEV) domain of Tsg101 was resolved in complex with the peptide binding site of HIV-1 p6, embodied as the nine-residue L-domain containing peptide PEPTAPPEE. The L-domain, which itself is believed to reside within an unstructured region of p6, connects to a cavernous pocket of the UEV domain of Tsg101 that bears a resemblance to the binding sites of SH3 and WW domains [56]. In our recent work we have reexplored the highresolution structure and folding of synthetic p6 under various solution conditions using a combination of CD and NMR spectroscopy [24]. In its most structured state, p6 adopts a helix-flexible-helix structure: a short helix-1 (residues 14–18) is connected to a pronounced helix-2 (residues 33–44) by a flexible hinge region. Synthetic peptides were then used to show that helix-2 of p6, which comprises the LXXLF binding motif for Vpr, specifically interacts with Vpr. An overall model of the molecular structure of p6 in the context of known functional modules is summarized in Fig. 3.

genome, MA is part of the PIC and thus regulates the transport of virus genome into the nucleus at an early stage of infection (for review, see [63]). In the final stages of the replication cycle during virus assembly, the N-terminally myristoylated MA targets the Gag polyproteins to the cell membrane. It was postulated that the two distinct targeting functions of MA are regulated by its proteolytic release from the Gag polyproteins. This myristoyl switch hypothesis was recently challenged by using a 131-amino-acid MA totally synthesized and modified by N-myristoylation. Results indicated that structural changes are not involved but rather the oligomerization state of MA is affected by this modification (see [75], and references therein). The first total synthesis of a 72-amino-acid HIV-1 NC peptide was reported by Roques and co-workers in 1991. Since then, several studies on synthetic NC were reported from this laboratory. Similar to the biological function of its viral counterpart, the synthetic NC, which contains two zinc-finger domains, was able to stimulate HIV-1 RNA dimerization and annealing of the tRNA replication primer for reverse transcription [12, 13]. Further, synthetic NC was used for NMR experiments [47] and binding studies with other viral components such as HIV-1 RT [17] and Vpr [14].

THE HIV-1 Gag PROTEINS NC AND MA CONCLUSION Two other Gag proteins have been chemically synthesized, the nucleocapsid (p7NC) and the matrix (p17MA) proteins. Whereas MA mediates the plasma membrane targeting of the Gag polyprotein and lines the inner shell of the mature virus particle, NC regulates the packaging and condensation of the viral genome. In addition, together with IN, Vpr, and the pro virus

The skilled use of modern instrumentation and highly efficient synthesis protocols now affords access to most small linear peptides up to approximately 100 amino acids in length. These methods also have the advantage of reliably producing high-quality material that has the same biological properties as those of the natural product

FIGURE 3. A. The amino acid sequence of the p6 Gag protein derived from the HIV-1, isolate NL4-3, is shown. Phosphorylation sites, positions of positively and negatively charged side chains, predicted phosphorylation sites, sites for covalent attachment of ubiquitin (Ub) and SUMO-1, as well as known binding domains for cellular (class E vacuolar protein sorting factors Tsg101, AIP1/ALIX, and protein kinase ERK-2) and viral (Vpr) proteins are indicated. B. Schematic depiction of experimental secondary structure elements detected by NMR experiments in synthetic full-length sp6 are shown.

1502 / Chapter 209 in essentially unlimited amounts and that is usually stable for relatively long periods of time, particularly during NMR experiments. As is apparent from the discussion, synthetic peptides have now been used for almost every aspect of functional and structural analyses of HIV proteins. Most important, they have played a significant role in the development of PR-inhibitors. Both RT- and PRinhibitors are still the most effective anti-HIV drugs used in standard therapy. In summary, synthetic HIV peptides have been used for various applications, such as for structural studies; for AIDS-vaccine development; for functional studies such as those involving ion channels (Vpu) or HIV LTR promoter activation (Tat); for studies on cellular uptake, such as the characterization of Vpr and Tat as trojan molecules; for characterization of posttranslational modifications such as phosphorylation (Vpu) and acetylation (Tat); for proteomic approaches as binding partners for pull-down experiments; and, last but not least, for the generation and characterization of HIV-specific antibodies. The production of synthetic peptides has a number of general advantages. They are much faster to produce, starting from the predicted amino acid sequence up to purified and functionally folded peptides. Indeed, several viral proteins, particularly those that either integrate into (Vpu) or temporarily associate with (Vpr) a membrane, are amphipathic and hydrophobic in character and are usually difficult to produce by use of recombinant protocols. In many cases, it was observed that high-yield expression of recombinant proteins in E. coli results in the formation of insoluble inclusion bodies that have to be de- and renatured, a harsh treatment that is particularly complicated if enzymatically active proteins such as HIV PR are the proteins of interest. Refolding of insoluble and aggregated recombinant proteins often leads to heterogeneous products that complicate structural studies, particularly those requiring NMR analyses. An example of such a problematic protein is the HIV-1 Vpr, for which all structural and most of the function analyses conducted in vitro have used synthetic material. Further, peptide synthesis allows the rapid production of specific mutants as well as the generation of randomized sequences (applied in the case of the VpuTM region); posttranslational modification such as phosphorylation, myristoylation and acetylation by simple changes in the synthesis procedure; the selective labeling with fluorescent markers; and the selective 15N- and/or 13 C-labeling. Synthetic peptides are easy to produce under standardized good manufacturing practice (GMP) conditions, contain no bacterial contaminants, and hence are suitable for therapeutic application (for instance, the development of the Tat vaccine). It must be remembered, however, that there are disadvantages to peptide synthesis that have to be considered. Clearly, peptide synthesis (in contrast to alternative

biosynthetic, recombinant material) is limited to a chain length of approximately 100 residues. Although new ligation techniques of presynthesized fragments might decrease this barrier, any extension above the 100-amino-acid limit will be accompanied by a drop in yield and efficiency that is paralleled by an exponential increase in costs. Indeed, for NMR structural investigations requiring uniform or selective 13C- or 15Nlabeling, it might be easier and less costly to produce the desired protein by recombinant techniques. Nevertheless, further improvement of the chemical ligation procedures of presynthesized peptide fragments will decrease the size barrier and will eventually lead to the involvement of total chemical synthesis for all viral proteins in functional, structural, and therapeutic studies.

Acknowledgments We are indebted to Karin Metzner for a critical reading of the manuscript. Work that led to the experimental data discussed in this review was supported by NIH/NIDDK RO1 grant DK59537-01, by grant Schu11/2-1, by grants SFB 466 and SFB 643, by a Heisenberg grant from the Deutsche Forschungsgemeinschaft, and by grant IE-S08T06 from the German Human Genome Research Project to U.S. We thank Nicole Studtrucker, Steffi Mayer, Prisca Kunert, Barbara Brecht, and Christel Kakoschke for their excellent technical assistance during our experimental work cited here.

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1504 / Chapter 209 [47] Morellet, N, Jullian, N, De Rocquigny, H, Maigret, B, Darlix, JL, Roques, BP. Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR. Embo J 1992;11(8):3059–65. [48] Muller, B, Patschinsky, T, Krausslich, HG. The late-domaincontaining protein p6 is the predominant phosphoprotein of human immunodeficiency virus type 1 particles. J Virol 2002; 76(3):1015–24. [49] Nutt, RF, Brady, SF, Darke, PL, Ciccarone, TM, Colton, CD, Nutt, EM, et al. Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease. Proc Natl Acad Sci USA 1988;85(19):7129–33. [50] Ott, DE, Chertova, EN, Busch, LK, Coren, LV, Gagliardi, TD, Johnson, DG. Mutational analysis of the hydrophobic tail of the human immunodeficiency virus type 1 p6(Gag) protein produces a mutant that fails to package its envelope protein. J Virol 1999;73(1):19–28. [51] Ott, DE, Coren, LV, Chertova, EN, Gagliardi, TD, Schubert, U. Ubiquitination of HIV-1 and MuLV Gag. Virology 2000;278(1): 111–21. [52] Ott, DE, Coren, LV, Copeland, TD, Kane, BP, Johnson, DG, Sowder, RC, II, et al. Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus. J Virol 1998;72(4):2962–8. [53] Park, SH, Mrse, AA, Nevzorov, AA, Mesleh, MF, Oblatt-Montal, M, Montal, M, et al. Three-dimensional structure of the channelforming trans-membrane domain of virus protein “u” (Vpu) from HIV-1. J Mol Biol 2003;333(2):409–24. [54] Partidos, CD, Moreau, E, Chaloin, O, Tunis, M, Briand, JP, Desgranges, C, et al. A synthetic HIV-1 Tat protein breaches the skin barrier and elicits Tat-neutralizing antibodies and cellular immunity. Eur J Immunol 2004;34(12):3723–31. [55] Peloponese, JM, Jr., Collette, Y, Gregoire, C, Bailly, C, Campese, D, Meurs, EF, et al. Full peptide synthesis, purification, and characterization of six Tat variants. Differences observed between HIV-1 isolates from Africa and other continents. J Biol Chem 1999;274(17):11473–8. [56] Pornillos, O, Alam, SL, Davis, DR, Sundquist, WI. Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein. Nat Struct Biol 2002;9(11):812–17. [57] Schneider, J, Kent, SB. Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell 1988;54(3):363–8. [58] Schubert, U, Bour, S, Ferrer-Montiel, AV, Montal, M, Maldarell, F, Strebel, K. The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains. J Virol 1996;70(2):809–19. [59] Schubert, U, Ferrer-Montiel, AV, Oblatt-Montal, M, Henklein, P, Strebel, K, Montal, M. Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV-1-infected cells. FEBS Lett 1996;398(1):12–18. [60] Schubert, U, Henklein, P, Boldyreff, B, Wingender, E, Strebel, K, Porstmann, T. The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK2) at positions Ser52 and Ser56 within a predicted alpha-helixturn-alpha-helix-motif. J Mol Biol 1994;236(1):16–25. [61] Schubert, U, Schneider, T, Henklein, P, Hoffmann, K, Berthold, E, Hauser, H, et al. Human-immunodeficiency-virus-type-1encoded Vpu protein is phosphorylated by casein kinase II. Eur J Biochem 1992;204(2):875–83.

[62] Schuler, W, Wecker, K, de Rocquigny, H, Baudat, Y, Sire, J, Roques, BP. NMR structure of the (52–96) C-terminal domain of the HIV-1 regulatory protein Vpr: Molecular insights into its biological functions. J Mol Biol 1999;285(5):2105–17. [63] Sherman, MP, Greene, WC. Slipping through the door: HIV entry into the nucleus. Microbes Infect 2002;4(1):67–73. [64] Silvera, P, Richardson, MW, Greenhouse, J, Yalley-Ogunro, J, Shaw, N, Mirchandani, J, et al. Outcome of simian-human immunodeficiency virus strain 89.6p challenge following vaccination of rhesus macaques with human immunodeficiency virus Tat protein. J Virol 2002;76(8):3800–9. [65] Smith, R, Brereton, IM, Chai, RY, Kent, SB. Ionization states of the catalytic residues in HIV-1 protease. Nat Struct Biol 1996; 3(11):946–50. [66] Strack, B, Calistri, A, Craig, S, Popova, E, Gottlinger, HG. AIP1/ ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 2003;114(6):689–99. [67] Stys, D, Blaha, I, Strop, P. Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame. Biochim Biophys Acta 1993;1182(2):157–61. [68] Subbramanian, RA, Cohen, EA. Molecular biology of the human immunodeficiency virus accessory proteins. J Virol 1994;68(11): 6831–5. [69] Wecker, K, Morellet, N, Bouaziz, S, Roques, BP. NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol. Comparison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains. Eur J Biochem 2002;269(15):3779–88. [70] Wecker, K, Roques, BP. NMR structure of the (1–51) N-terminal domain of the HIV-1 regulatory protein Vpr. Eur J Biochem 1999;266(2):359–69. [71] Welker, R, Hohenberg, H, Tessmer, U, Huckhagel, C, Krausslich, HG. Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1. J Virol 2000; 74(3):1168–77. [72] Wlodawer, A, Miller, M, Jaskolski, M, Sathyanarayana, BK, Baldwin, E, Weber, IT, et al. Conserved folding in retroviral proteases: Crystal structure of a synthetic HIV-1 protease. Science 1989;245(4918):616–21. [73] Wray, V, Federau, T, Henklein, P, Klabunde, S, Kunert, O, Schomburg, D, et al. Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and 1H NMR spectroscopy. Int J Pept Protein Res 1995;45(1):35–43. [74] Wray, V, Kinder, R, Federau, T, Henklein, P, Bechinger, B, Schubert, U. Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy. Biochemistry 1999;38(16):5272–82. [75] Wu, Z, Alexandratos, J, Ericksen, B, Lubkowski, J, Gallo, RC, Lu, W. Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation. Proc Natl Acad Sci USA 2004;101(32): 11587–92. [76] Yu, XF, Dawson, L, Tian, CJ, Flexner, C, Dettenhofer, M. Mutations of the human immunodeficiency virus type 1 p6Gag domain result in reduced retention of Pol proteins during virus assembly. J Virol 1998;72(4):3412–17. [77] Zander, K, Sherman, MP, Tessmer, U, Bruns, K, Wray, V, Prechtel, AT, et al. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J Biol Chem 2003; 278(44):43202–13. [78] Zheng, S, Strzalka, J, Jones, DH, Opella, SJ, Blasie, JK. Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering. Biophys J 2003;84(4):2393–415.

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210 Pheromone Peptides MIRIAM ALTSTEIN

concentration, and order of presentation of the signal molecules, as well as the physiological state of the receiver. It is believed that, during the course of evolution, the pheromones of different organisms have become very distinctive in order to avoid ambiguity and to ensure that the channels of communication by which they exert their activity do not overlap, causing an even higher order of complexity. The chemical nature of pheromones ranges from small-molecule volatile compounds to highmolecular-weight, water-soluble, nonvolatile peptides and proteins. Usually, small molecules are used for communication, which requires rapid dispersal of the signal, whereas the larger molecules of less volatile compounds tend to function in attraction and stimulation, for which prolonged exposure is necessary. Whereas air-borne signal molecules have been studied extensively, identified, and applied commercially to disrupt mating and to monitor insect pests, much less is known about the chemical nature of the equivalent water-borne cues. Pheromone peptides are widely distributed throughout the prokaryotic and eukaryotic organisms, and are used to mediate a variety of functions in grampositive and gram-negative bacteria, fungi, and many different animals that belong to the arthropod, annelid, mollusk, and vertebrate (amphibians and mammals) phyla. The great abundance of this route of signaling, in such a large number of widely diverse organisms, hints at the evolutionary importance of this mode of communication. Pheromone peptides have been studied for many years and in several systems the nature of the peptides, their origins, and their modes of action are quite well understood. In other systems, however, our understanding is still very nascent, and the chemical, molecular, physiological, and biological aspects are still to be explored. Recently, several review articles on pheromone peptides in organisms belonging to the bacterial,

ABSTRACT Pheromones are chemical messages known to elicit changes in behavioral reactions that are followed by physiological alterations in conspecific receiver organisms. Pheromones comprise a wide range of compounds, varied in their chemical nature, ranging from smallmolecule volatile compounds to high-molecular-weight, water-soluble, nonvolatile peptides and proteins. Although volatile pheromone molecules have been studied extensively, much less is known about the chemical nature of the equivalent nonvolatile water-soluble cues. In this chapter, we summarize the state of our knowledge of pheromone peptides and a few proteins in a variety of species belonging to different phyla and classes.

INTRODUCTION Pheromones are chemical messages involved in communication that induce a behavioral reaction or a developmental process among individuals of the same species. Pheromones can elicit in the receiver organism an immediate behavioral reaction such as alarm, defense, aggregation, attraction, kin and colony recognition, marking of territories and egg deposition sites, mating behavior, recruitment, and thermoregulation; or they can induce complex behavioral and physiological alterations (such as the development of a particular caste or sexual maturation) via long-term endocrine changes. Chemical communication among organisms is a highly sophisticated mechanism that involves the regulation of many genes in the donor and the initiation of complex processes in the recipient, in which the interpretation of the individual chemical constituents or signals depends on the particular combination, ratio, Handbook of Biologically Active Peptides

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1506 / Chapter 210 fungal, and animal kingdoms have been published; they cover the chemical nature of these peptides, their mechanisms of production and reception, and their modes of action, as well as the behavioral changes elicited in the recipient organisms. This chapter presents a short summary of the state of our knowledge on pheromone peptides and a few proteins in a variety of species. The limited scope of the chapter does not allow for a detailed description of each system and the reader is referred for further information to the references (mostly recent review articles) in each section. A summary of the peptides mentioned in this chapter is presented in Table 1.

BACTERIAL PHEROMONE PEPTIDES One of the most complex and diverse systems of pheromone peptides is found in bacteria. Both unicellular organisms and individual cells of metazoans have evolved complex signaling mechanisms by which they respond to the environment and communicate with one another. These mechanisms involve the production and release of small molecules that are sensed by organisms with cognate sensors, with each signal molecule setting in motion a more or less complicated response pathway. There are two basic types of bacterial communication systems: those in which the signal is detected solely by organisms other than the producer, and those in which the signal is sensed by the producing organism as well. The first is typified by the mating pheromones of the enterococcal bacteria (e.g., Enterococcus faecalis), in which a potential recipient releases a small peptide that is taken up by a potential donor in which it initiates a plasmid-coded response that leads to cellular aggregation and transfer of the plasmid from donor to recipient. The enterococcal pheromone is processed from a chromosomally encoded lipoprotein precursor. Most strains produce multiple pheromones with differing spectra of activities, but each plasmid responds specifically to its cognate pheromone with extremely high specificity and sensitivity. Details on the synthesis of the enterococcal mating pheromone molecules, the components of the regulatory machinery that interact specifically with the peptides, and the molecular basis for their exquisite sensitivity and specificity have recently been reviewed [14, 19]. The second group of bacterial pheromone peptides comprises the auto-induction or quorum-sensing (QS) systems, which are widespread among bacteria, in which they elicit population-wide responses (cell-cell or cellenvironment responses) to low-molecular-weight signaling molecules, depending on cellular density. The structures of the signaling molecules differ between gram-negative and gram-positive bacteria. In gram-

positive bacteria, the mating pheromones and most QS signals are peptides that are sensed by transmembrane receptor components that activate an intracellular response pathway. Examples of QS-induced processes in gram-positive bacteria include virulence in Staphylococcus aureus [38, 42] and E. faecalis [38, 44], genetic competence in Streptococcus pneumoniae [18, 38] and Bacillus subtilis [38, 52], and the production of antimicrobial peptides (lantibiotics) in many lactic acid bacteria [25, 33–35, 38]. The production of antimicrobial peptides can be regulated in a cell-density-dependent manner by the antimicrobial peptides themselves (as in the case of nisin and subtilin production in Lactococcus lactis and B. subtilis, respectively) or by signaling molecules that are distinct from the antimicrobial peptides and that include the plantaricin A (PlnA) [36] or BlpC peptides (also termed SpiP peptides) that regulate bacteriocin production in Lactobacillus plantarum and S. pneumoniae, respectively [38]. Another example of QS occurs in the regulation of cytolysin production in E. faecalis, in which a subunit of the bacteriocin, CylLs regulates the transcription of the toxin [48]. The mechanisms regulating each of the QS systems are very complex and differ from one another with respect to the secretion and the sensing of the autoinducer and in the regulatory cascade that results from the perception of the autoinducer [50]. A detailed review on these QS peptides in gram-positive bacteria and of a few other QS peptides has recently been published [25, 27, 29, 33, 34, 38]. In gram-negative bacteria, most QS signals are N-acyl homoserine lactones, which are internalized by diffusion and bind to an intracellular receptor molecule to activate the response [55]. However, recent studies have revealed the existence of signaling peptides in gram-negative bacteria too. Studies based on an in silico strategy to screen the gram-negative bacterial genome for the presence of leader double-glycine sequences (a conserved leader motif sequence of many gram-positive pheromone peptides [41, 43]) have revealed the presence of genes coding for putative peptides that contain such motifs and that also show structural similarity to bacteriocins and pheromone peptides of gram-positive bacteria [24]. The role of such peptides in signal transduction and the possible mechanisms by which they can exert their activity in gram-negative bacteria is unclear at present.

FUNGAL PHEROMONE PEPTIDES The pheromone peptides of yeasts are among the best-documented pheromones in fungi. Two pheromone peptides that initiate mating in the yeast Saccharomyces cerevisiae—the α-factor and the a-factor

TABLE 1. Major Pheromone Peptides or Protein Families in Bacteria, Fungi, and the Animal Kingdom. Pheromone Peptide or Protein Family

Organism or Group Studied (Order/Genus/Species)

Bacterial pheromone peptides Mating pheromones cCF10, cAD1, cPD1, cAM373, cOB1

Enterococcus faecalis

Cellular aggregation and plasmid transfer

[14, 19, 38]

Staphylococcus aureus Enterococcus faecalis Enterococcus faecalis Streptococcus pneumoniae Bacillus subtilis Lactococcus lactis Bacillus subtilis Lactobacillus plantarum Streptococcus pneumoniae

Virulence peptides Virulence peptides Virulence peptides Genetic competence Genetic competence Production of bacteriocins Production of bacteriocins Production of bacteriocins Production of bacteriocins

[38] [38] [48] [38] [38] [25, 27, 29, 33–35] [33–35] [36, 38] [38]

Various bacterial spp. (see list in [24], Table 4)

Signaling peptides

[24]

Fungal pheromone peptides α-Factor a-Factor

Saccharomyces cerevisiae Saccharomyces cerevisiae

Mating initiation Mating initiation

[40] [16]

Arthropod pheromone peptides Insect peptides Sex peptide (accessory gland protein—Acp70A)

Drosophila melanogaster

[15, 17] [1, 45]

Quorum sensing peptides Gram-positive peptides Autoinducing peptide (AIP) Autoinducing peptide (AIP) Cytolysin subunit (CylLs″) Competence stimulating peptides (CSP-1, CSP-2) Competence stimulating factor (CSF)-ComX, Nisin Subtilin Plantaricin A (PlnA) peptides BlpC (also termed SpiP) peptides Gram-negative peptides Double Gly (G-G) containing peptides

Function

Sources

Various moth spp. (see list in [1], Table 1) Barnacles (e.g., Balanus amphitrite) Various decapod crustaceans (brachyuran crabs)

Colony aggregation and settlement Synchronization of larval release

[46] [46]

Annelid pheromone peptides Nereithione

Nereis succinea

Control of reproductive behavior, mate recognition, and gamete release

[28]

Mollusk pheromone peptides Attractins, enticin, temptin, seductin

Aplysia spp., Bursatella leachii

[20–22, 51]

ILME (Ile-Leu-Met-Glu)

Loligo pealeii, Sepia officinalis

Egg laying, mating aggregation, and initiation of respiratory pumping Transport of oocytes in the genital tract

[51]

Pheromone Peptides / 1507

Pyrokinin/Pheromone biosynthesis activating neuropeptide (PK/PBAN) family Crustacean peptides Settlement pheromones (e.g., arthropodin) Larval release pheromones

Variety of reproductive and postmating functions Sex pheromone biosynthesis

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TABLE 1.

(Continued)

Pheromone Peptide or Protein Family

Organism or Group Studied (Order/Genus/Species)

Sperm attractin peptide (SepSAP)

Sepia officinalis

Sperm attraction and facilitation of external fertilization

[51]

Cynops pyrrhogaster; Triturus carnifex Cynops ensicauda Litoria splendida Plethodon jordani Leptodactylus fallax

Female attraction Female attraction Female attraction Shortening of courtship process Stimulation of male aggression

[12, 30, 31, 53] [53] [54] [47] [32]

Rodents Rodents

Induction of copulatory behavior Communication of individual identity and ownership via scent marks Assistance in processing of pheromonal cues associated with social discriminations

[9, 10] [5, 6, 13]

Vertebrate pheromone peptides Amphibian peptides Sodefrin Silefrin Splendipherin Courtship pheromone Leptodactylus aggression-stimulating peptide (LASP) Mammalian peptides Aphrodisin Major urinary proteins (MUPs) Vasopressin and oxytocin

Rodents and sheep

Function

Sources

[7]

Pheromone Peptides / 1509 (containing 13 and 12 amino acids, respectively)—are currently known. The peptides are secreted by donor cells and act on the recipient via G-protein-coupled receptors (GPCR). The mating pheromones are absolutely required to trigger the mating cycle and cells that cannot produce these molecules or that lack their cognate receptors are sterile. The α-factor is a tridecapeptide pheromone (WHWLQLKPGQPMY) synthesized constitutively by S. cerevisiae mating type α cells, and it acts on S. cerevisiae mating type α cells. The peptide was isolated and characterized by Stotzler and Duntze [49] and later found to be encoded by two genes MFα1 and MFα2 [37]. The a-factor of S. cerevisiae is a dodecapeptide pheromone (YIIKGVFWDPAC [farnesyl]-OCH3) [2] in which posttranslational modification with a farnesyl isoprenoid and carboxymethyl group is required for its full biological activity [39]. The biosynthesis of these mating pheromones has been studied extensively and used as a model for posttranslational processing modification, and secretion of mammalian peptide hormones and proteins [8, 11, 16, 23]. The structure–activity relationship (SAR) studies of the α-factor peptide and the extensive biological, biochemical, molecular, and biophysical analyses of this peptide provided a model for the structure of the αfactor and for its interaction with its receptor (for an overview see [40]). The downstream intracellular signal transduction pathway by which the yeast responds to the presence of peptide mating pheromone in its vicinity and the peptide’s mode of action at the cellular level have been examined thoroughly and are summarized in reviews by Bardwell [4] and Flatauer et al. [26].

ARTHROPOD PHEROMONE PEPTIDES Insects One of the largest and best-documented groups of pheromones comprises those of insects. Chemical cues are major sources of the information used by most insects to interpret environmental stimuli; they lead to the initiation of gregarious behavior, formation of aggregations at food sites, dispersal behavior during predator attack, synchronized sexual maturity, mating attraction, and so forth. However, none of the pheromones that elicit these functions is of a peptidic nature. There are only two examples of peptides (and proteins) being related to pheromonal activity in insects. One involves Drosophila melanogaster, in which peptides play a role as primer pheromones that cause physiological changes associated with reproductive activity in the fly. The other involves peptides that originate in the central nervous system of Lepidopteran species and stimulate sex pheromone biosynthesis in female moths.

The first system involves proteins and peptides that are transferred together with sperm and seminal fluid; they act within and outside the reproductive tract of their female targets and elicit a wide variety of responses such as increased oogenesis and ovulation, decreased female receptivity, enhanced sperm storage, and formation of the mating plug [15]. The very first seminal fluid protein to be identified was the sex peptide or accessory gland peptide (Acp70A) [17]. During the 1960s to the 1980s, the nature of Acp70A was gradually revealed and the Acp70A gene that encodes the 36-amino-acid Acp70A peptide was identified (for an overview see [15] and references therein). The ongoing study of seminal-fluid molecules reveals that they have an unexpected variety of functions and in addition, some of the genes that encode these molecules show evidence of extremely rapid evolutionary change. These findings suggest that seminal-fluid molecules may be strong targets for natural or sexual selection. At present, new technologies and approaches (e.g., homologous recombination and RNA interference, along with the use of microarrays and yeast two-hybrid systems) are being used to gain a better insight into the functions of these molecules. Information on the currently known functions of the seminal-fluid proteins and peptides of D. melanogaster and the approaches used to study their role in the fruit fly’s reproductive and postmating functions have recently been published [15]. The second system involves a subesophageal ganglion neuropeptide, termed pheromone biosynthesisactivating neuropeptide (PBAN), that acts via a GPCR mechanism to initiate sex pheromone biosynthesis in the pheromone glands of female moths. Studies of the regulation of sex pheromone biosynthesis in moths have revealed that this function can be elicited by additional neuropeptides, all of which share the common C-terminal pentapeptide FXPRL-amide (X = S, T, G, or V) and belong to a large family of peptides termed the pyrokinin (PK)/PBAN family. In the past 2 decades, extensive studies were carried out on the chemical, cellular, and molecular aspects of PBAN and the other peptides of the PK/PBAN family, aiming to understand the mode of their action on sex pheromone biosynthesis (for an overview see [1, 45] and references therein). A detailed review of some of these topics is presented in Chapter 32 on insect pyrokinin/PBAN by Predel and Nachman in the Invertebrate Peptide Section of this book.

Crustaceans Another group of pheromone peptides in arthropods comprises those of the crustaceans. Crustacean pheromone peptides have been implicated in two different behavioral patterns: gregarious settlement of

1510 / Chapter 210 barnacles (involving settlement pheromones, e.g., arthropodin) and synchronization of larval release (larval release pheromones). It is currently well accepted that all known crustacean pheromone peptides are based on serine protease degradation products that have basic C-termini that are essential for bioactivity. The settlement pheromones are peptides released from intact living barnacles and the larval release pheromones are released from the brood. Most of the studies of the signal molecules used crude extracts or synthetic peptides because the natural pheromone peptides themselves have not yet been identified. Knowledge of the biological activity and SAR of synthetic settlement pheromone and of the larval release pheromone is presently quite elusive; the current state of knowledge has recently been summarized in [46].

ANNELID PHEROMONE PEPTIDES Females of the ragworm Nereis succinea employ a tetrapeptide, cysteinyl-glutathione (CSSG), as a materecognition and gamete-release pheromone during reproduction. The tetrapeptide pheromone (termed nereithione) controls reproductive behavior and the release of gametes. The pheromone is released into the water by swimming ripe females and induces males to increase their swimming speed and to release sperm. The role of peptide-based pheromones in the worms and the time course of their appearance as related to the worm’s sexual maturation have recently been reviewed in [28].

MOLLUSK PHEROMONE PEPTIDES Pheromone peptides and proteins have also been implicated in the control of a number of behaviors in mollusks—in both gastropods and cephalopods—but only a few have been characterized. A family of waterborne pheromonal protein attractants (termed attractins [20, 51]), which were found in several Aplysia spp. and recently also in Bursatella leachii [20], are released during egg-laying and act in concert with other waterborne protein pheromones (enticin [21], temptin [21], and the recently discovered protein seductin [22]) to stimulate other Aplysia to increase egg-laying, to form mating aggregations, and to initiate respiratory pumping. Other pheromone peptides are a tetrapeptide (IleLeu-Met-Glu, termed ILME) in the cephalopod Sepia (eluted from egg masses), which is thought to stimulate transport of oocytes in the genital tract, and a Sepia sperm-attractin peptide, termed SepSAP, that is released from oocytes during egg-laying and facilitates external fertilization by attracting spermatozoa. Evidence for the

pheromonal role of these peptides and proteins in various Aplysia and Sepia species, their origin, SAR, and bioactivity has recently been reviewed in [20–22, 51].

VERTEBRATE PHEROMONE PEPTIDES Amphibians In recent years it has become apparent that, although communication in amphibians depends primarily on auditory signals, pheromonal signals are used by both males and females for mating attraction. The first pheromone identified from amphibians was the female attractant sex pheromone of the newt Cynops pyrrhogaster, sodefrin. In 2000, a second sex pheromone was isolated from another Cynops species, C. ensicauda, and termed silefrin (for review of both peptides, see [31, 53]). Recently, a cDNA comparable to the cDNA encoding Cynops sodefrin was isolated from another newt, Triturus carnifex [12]. Cynops sodefrin and silefrin, are decapeptides with amino acid sequences of SIPSKDALLK and SILSKDAQLK, respectively; they are secreted from the abdominal glands through the cloacae of the animals, their synthesis is regulated by prolactin and androgens, and each pheromone attracts only conspecific females (for review see [3, 30, 31, 53] and Chapter 48 by Kikuyama in the Amphibian Peptides Section of this book). Another sex pheromone isolated from Litoria splendida is a 25-residue peptide termed splendipherin [54], which is produced in the highest levels during the mating season. Splendipherin attracts conspecific females and has no effect on males or on other species. Recently, a 22-kDa proteinaceous courtship pheromone has been discovered in a terrestrial salamander, Plethodon jordani [47]. This protein hormone is deposited directly onto the skin of the female by the male from his mental glands, located under the chin. This pheromone is thought to shorten the courtship process. In 2005, another novel amphibian pheromonal peptide was characterized [32]; it was termed leptodactylus aggression-stimulating peptide (LASP) and was isolated from male norepinephrine-stimulated skin secretions. LASP exhibited a chemoattractive effect on males, in which it stimulated aggressive behaviors that resulted in the emergence of dominant animals that subsequently attracted females to nesting sites.

Mammals Needless to say, that the most complex, interesting, and as yet unidentified signaling molecules are those of mammals. All mammals emit chemical cues into the environment via urine, saliva, and diverse secreted fluids. So far, only a few mammalian pheromones, espe-

Pheromone Peptides / 1511 cially of rodents, have been identified, and the examination of their chemical nature has revealed a wide diversity of compounds that range from small organic molecules to large proteins. Three proteinaceous pheromonotropic families are described in this chapter: aphrodisin (for a review, see [9, 10]), major urinary proteins (MUPs) [6], and the neuropeptides vasopressin (AVP) and oxytocin (OT) [7]. Aphrodisin is a protein that belongs to the lipocalin family; it is found in hamster vaginal secretions and when it is detected by the male accessory olfactory system-copulatory behavior is induced. At present, it is not clear whether aphrodisin itself performs the pheromonal function or whether it is simply a carrier for hydrophobic, small pheromone molecules that have not been identified yet. Recent studies have focused on the aphrodisin structure, its biological properties, and the associated signal transduction processes in the vomeronasal organ [9, 10]. Other members of the lipocalin family are the MUPs of mice, rats, and some other rodents. These proteins are thought to be responsible for the binding and release of low-molecular-weight pheromones, thereby providing a slow-release mechanism for volatile components of scent marks. However, the proteins may function as chemosignaling molecules in their own right, filling one or more roles in the communication of individual identity and ownership. A few recent reviews [5, 6, 13] summarize the current understanding of the structure and function of these urinary proteins and speculate about their role as supporters or key participants in the elaboration of the complex chemosensory properties of a rodent scent mark. Last but not least are the neurohypophysial peptides, AVP and OT, which do not serve directly as pheromonal cues in mammalian species but are, rather, critical to the mammalian ability to process such cues in an appropriate manner in the olfactory circuit and throughout the brain. In a recent review by Bielsky and Young [7] the roles of OT and AVP in social recognition in rodents, in offspring recognition in sheep, and in mate preference among pair-bonding voles are discussed.

CONCLUSION There is no doubt that the wide variety of pheromone peptides described here emphasizes their crucial role in a large variety of behavioral patterns that are associated mostly, but not exclusively, with courtship and mating (or transfer of genetic material). Despite the vast amount of information that is already available to us, the study of pheromone peptides is still nascent and requires further exploitation, and it seems that we have only just begun to understand some of the lan-

guages of a few inhabitants of this fascinating world. Understanding the interplay between behavioral and biochemical factors in the deposition and reception of scents, the identification of key signal molecules and elucidation of their chemical nature, and the exploitation of their modes of action and regulation in donor and recipient organisms presents a challenge that is still to be faced. However, even with the limited information in hand, it is obvious that pheromones of a peptidic nature play an important role in speciesspecific communication, either directly or via neural processing of the olfactory signal. Their high solubility, specificity, and variability (which is achieved by the existence of multiple genes encoding for peptide variants) make them highly efficient pheromonal candidates. There is no doubt that the novel methods and approaches that have been developed by chemists, biochemists, and biologists will enable us to determine further the chemical nature of pheromone peptides, to dissect their behavioral sequence of events, and to study their mechanisms of action at the cellular and molecular levels. It is anticipated that such studies will widen our understanding of the extraordinary diversity that surrounds us and will stimulate further studies that may lead to the unraveling of the chemical signaling mediated by peptides and hence lead to practical applications, such as the development of new therapies against bacterial infections or of behavior-modifying compounds for agrochemical, aquacultural, and medical applications.

References [1] Altstein M. Role of neuropeptides in sex pheromone production in moths. Peptides 2004;25(9):1491–1501. [2] Anderegg RJ. Betz R. Carr SA. Crabb JW. Duntze W. Structure of Saccharomyces cerevisiae mating hormone A-factor—identification of S-farnesyl cysteine as a structural component. Journal of Biological Chemistry 1988;263(34):18236–18240. [3] Apponyi MA. Pukala TL. Brinkworth CS. Maselli VA. Bowie JH. Tyler MJ. Booker GW. Wallace JC. John A. Separovic F. Doyle J. Llewellyn LE. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 2004;25(6):1035–1054. [4] Bardwell L. A walk-through of the yeast mating pheromone response pathway. Peptides 2005;26(2):339–350. [5] Beynon RJ. Hurst JL. Multiple roles of major urinary proteins in the house mouse, Mus domesticus. Biochemical Society Transactions 2003;31:142–146. [6] Beynon RJ. Hurst JL. Urinary proteins and the modulation of chemical scents in mice and rats. Peptides 2004;25(9):1553– 1563. [7] Bielsky IF. Young LJ. Oxytocin, vasopressin, and social recognition in mammals. Peptides 2004;25(9):1565–1574. [8] Bourbonnais Y. Germain D. Latchiniansadek L. Boileau G. Thomas DY. Prohormone processing by yeast proteases. Enzyme 1991;45(5–6):244–256. [9] Briand L. Blon F. Trotier D. Pernollet JC. Natural ligands of hamster aphrodisin. Chemical Senses 2004;29(5):425–430.

1512 / Chapter 210 [10] Briand L. Trotier D. Pernollet JC. Aphrodisin, an aphrodisiac lipocalin secreted in hamster vaginal secretions. Peptides 2004;25(9):1545–1552. [11] Caldwell GA. Naider F. Becker JM. Fungal lipopeptide mating pheromones—a model system for the study of protein prenylation. Microbiological Reviews 1995;59(3):406–422. [12] Cardinali M. Iwata T. Toyoda F. Kikuyama S. Polzonetti-Magni AM. Cloning of sodefrin-like cDNA of the newt, Triturus carnifex Laur. 20th Conf Europ Comp Endocrinol (Faro, Portugal) 2000;96. [13] Cavaggioni A. Mucignat-Caretta C. Major urinary proteins, alpha(2u)-globulins and aphrodisin. Biochimica et Biophysica Acta 2000;1482(1–2):218–228. [14] Chandler JR. Dunny GM. Enterococcal peptide sex pheromones: synthesis and control of biological activity. Peptides 2004;25(9):1377–1388. [15] Chapman T. Davies SJ. Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies. Peptides 2004;25(9):1477–1490. [16] Chen P. Sapperstein SK. Choi JD. Michaelis S. Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor. Journal of Cell Biology 1997;136(2):251–269. [17] Chen PS. Stummzollinger E. Aigaki T. Balmer J. Bienz M. Bohlen P. A male accessory-gland peptide that regulates reproductive-behavior of female Drosophila-melanogaster. Cell 1988;54(3):291–298. [18] Cheng Q. Campbell EA. Naughton AM. Johnson S. Masure HR. The com locus controls genetic transformation in Streptococcus pneumoniae. Molecular Microbiology 1997;23(4):683–692. [19] Clewell DB. Francia MV. Flannagan SE. An FY. Enterococcal plasmid transfer: sex pheromones, transfer origins, relaxases, and the Staphylococcus aureus issue. Plasmid 2002;48(3):193–201. [20] Cummins SE. Schein CH. Xu Y. Braun W. Nagle GT. Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness. Peptides 2005;26(1):121–129. [21] Cummins SF. Nichols AE. Amare A. Hummon AB. Sweedler JV. Nagle GT. Characterization of aplysia enticin and temptin, two novel water-borne protein pheromones that act in concert with attractin to stimulate mate attraction. Journal of Biological Chemistry 2004;279(24):25614–25622. [22] Cummins SF. Nichols AE. Warso CJ. Nagle GT. Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction. Peptides 2005;26(3): 351–359. [23] Davey J. Davis K. Hughes M. Ladds G. Powner D. The processing of yeast pheromones. Seminars in Cell & Developmental Biology 1998;9(1):19–30. [24] Dirix G. Monsieurs P. Dombrecht B. Daniels R. Marchal K. Vanderleyden J. Michiels J. Peptide signal molecules and bacteriocins in gram-negative bacteria: A genome-wide in silico screening for peptides containing a double-glycine leader sequence and their cognate transporters. Peptides 2004;25(9): 1425–1440. [25] Eijsink VGH. Axelsson L. Diep DB. Havarstein LS. Holo H. Nes IF. Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek 2002;81(1–4):639–654. [26] Flatauer LJ. Zadeh SF. Bardwell L. Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae. Molecular and Cellular Biology 2005;25(5):1793–1803. [27] Guder A. Wiedemann I. Sahl HG. Posttranslationally modified bacteriocins—the lantibiotics. Biopolymers 2000;55(1):62–73. [28] Hardege JD. Bartels-Hardege H. Muller CT. Beckmann M. Peptide pheromones in female Nereis succinea. Peptides 2004; 25(9):1517–1522.

[29] Hechard Y. Sahl HG. Mode of action of modified and unmodified bacteriocins from gram-positive bacteria. Biochimie 2002;84(5–6):545–557. [30] Iwata T. Conlon JM. Nakada T. Toyoda F. Yamamoto K. Kikuyama S. Processing of multiple forms of preprosodefrin in the abdominal gland of the red-bellied newt Cynops pyrrhogaster: regional and individual differences in preprosodefrin gene expression. Peptides 2004;25(9):1537–1543. [31] Kikuyama S. Nakada T. Toyoda F. Iwata T. Yamamoto K. Conlon JM. Amphibian pheromones and endocrine control of their secretion. Ann NY Acad Sci 2005 Apr;1040: 123–130. [32] King JD. Rollins-Smith LA. Nielsen PF. John A. Conlon JM. Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs. Peptides 2005;26(4):597–601. [33] Kleerebezem M. Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis. Peptides 2004;25(9):1405–1414. [34] Kleerebezem M. Bongers R. Rutten G. de Vos WM. Kuipers OP. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters. Peptides 2004;25(9):1415–1424. [35] Kleerebezem M. Quadri LEN. Kuipers OP. de Vos WM. Quorum sensing by peptide pheromones and two-component signaltransduction systems in gram-positive bacteria. Molecular Microbiology 1997;24(5):895–904. [36] Kristiansen P. Fimland G. Mantzilas D. Nissen-Meyer J. Structure and mode of action of the membrane-permeabilizing antimicrobial peptide pheromone plantaricin A. Journal of Biological Chemistry 2005;280:22945–22950. [37] Kurjan J. Herskowitz I. Structure of a yeast pheromone gene (MF alpha): A putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell 1982 Oct;30(3): 933–943. [38] Lyon G. J. Novick RP. Peptide signaling in Staphylococcus aureus and other gram-positive bacteria. Peptides 2004;25(9):1389– 1403. [39] Marcus S. Caldwell GA. Miller D. Xue CB. Naider F. Becker JM. Significance of C-terminal cysteine modifications to the biological-activity of the Saccharomyces-cerevisiae A-factor mating pheromone. Molecular and Cellular Biology 1991;11(7):3603– 3612. [40] Naider F. Becker JM. The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors. Peptides 2004;25(9):1441–1463. [41] Nes IF. Diep DB. Havarstein LS. Brurberg MB. Eijsink V. Holo H. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuwenhoek 1996;70(2–4):113–128. [42] Novick RP. Muir TW. Virulence gene regulation by peptides in staphylococci and other gram-positive bacteria. Current Opinion in Microbiology 1999;2(1):40–45. [43] Pestova EV. Havarstein LS. Morrison DA. Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Molecular Microbiology 1996;21(4):853– 862. [44] Qin X. Singh KV. Weinstock GM. Murray BE. Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF. Journal of Bacteriology 2001;183(11):3372–3382. [45] Rafaeli A. Jurenka RA. PBAN regulation of pheromone biosynthesis in female moths. In: Insect pheromone biochemistry and molecular biology. New York: Academic Press; 2003:107– 136.

Pheromone Peptides / 1513 [46] Rittschof D. Cohen JH. Crustacean peptide and peptide-like pheromones and kairomones. Peptides 2004;25(9):1503–1516. [47] Rollmann SM. Houck LD. Feldhoff RC. Proteinaceous pheromone affecting female receptivity in a terrestrial salamander. Science 1999;285(5435):1907–1909. [48] Shankar N. Coburn P. Pillar C. Haas W. Gilmore M. Enterococcal cytolysin: Activities and association with other virulence traits in a pathogenicity island. International Journal of Medical Microbiology 2004;293(7–8):609–618. [49] Stotzler D. Duntze W. Isolation and characterization of four related peptides exhibiting alpha factor activity from Saccharomyces cerevisiae. Eur J Biochem 1976 May;65(1):257–262. [50] Sturme MHJ. Kleerebezem M. Nakayama J. Akkermans ADL. Vaughan EE. de Vos WM. Cell to cell communication by autoinducing peptides in gram-positive bacteria. Antonie Van Leeuwenhoek 2002;81(1–4):233–243.

[51] Susswein AJ. Nagle GT. Peptide and protein pheromones in molluscs. Peptides 2004;25(9):1523–1530. [52] Tortosa P. Dubnau D. Competence for transformation: A matter of taste. Current Opinion in Microbiology 1999;2(6):588–592. [53] Toyoda F. Yamamoto K. Iwata T. Hasunuma I. Cardinali M. Mosconi G. Polzonetti-Magni AM. Kikuyama S. Peptide pheromones in newts. Peptides 2004;25(9):1531–1536. [54] Wabnitz PA. Bowie JH. Tyler MJ. Wallace JC. Smith BP. Differences in the skin peptides of the male and female Australian tree frog Litoria splendida—the discovery of the aquatic male sex pheromone splendipherin, together with Phe8 caerulein and a new antibiotic peptide caerin 1.10. European Journal of Biochemistry 2000;267(1):269–275. [55] Whitehead NA. Barnard AML. Slater H. Simpson NJL. Salmond GPC. Quorum-sensing in gram-negative bacteria. FEMS Microbiology Reviews 2001;25(4):365–404.

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Handbook, see Chapters 98, 109, 127, 128, 174, and 188. This chapter focuses on the stories of these peptides from fish to human. Stanniocalcins are glycoproteins [40] and the molecular masses are larger than other peptides. However, stanniocalcins are important glycoprotein hormones in fish, and probably in humans, and are therefore described briefly in this chapter.

ABSTRACT Melanin-concentrating hormones (MCH), urotensins, and stanniocalcin were originally discovered in fish (from the pituitary, urophysis, and corpuscles of Stannius, respectively). Now these peptides are known to be present in mammals, including humans, and to play very important physiological roles. For example, MCH is a hypothalamic neuropeptide that has a potent stimulatory action on appetite. Urotensin II is one of the most potent vasoconstrictor peptides, whereas urocortins (mammalian homologs of urotensin I) are vasodilator peptides. MCH and urotensins may therefore be involved in the pathophysiology of human diseases, such as obesity and hypertension. Furthermore, adrenomedullin 2/intermedin, a novel member of the calcitonin/ CGRP family, has been discovered in the process of searching the public human genome databases for the homology of the fish peptide. The stories of fish peptides may be of great help in the understanding of these peptides in human physiology and diseases.

MELANIN-CONCENTRATING HORMONE A dual hormonal control of color change by two antagonistic pituitary melanophorotropic hormones was first postulated in amphibia by Hogben and Slome in 1931 [7]. Although α-melanocyte-stimulating hormone (αMSH) was shown to have the skin-darkening effect, a hormone with an antagonistic action against αMSH had been unknown for a long time. Kawauchi et al. discovered MCH from salmon pituitaries in 1983 [12]. MCH, a cyclic peptide consisting of 17 amino acids, induces the aggregation of melanophores and paling of teleost fish skin. MCH is expressed in the neurons of teleost hypothalamus, and MCH nerve fibers project abundantly to the neurohypophysis [11]. Plasma levels of MCH increase in response to a white background or stress in fish. Rat, mouse, and human MCH is the identical peptide consisting of 19 amino acids [21, 25, 38] and differs from salmon MCH by an N-terminal extension of two amino acids and four additional substitutions. In mammals, MCH neurons are expressed in the lateral hypothalamus and zona incerta, and MCH nerve fibers project to almost all areas of brain [11, 20, 43]. In contrast to the teleost MCH neurons, MCH nerve fibers do not extend abundantly to the neurohypophysis in mammals. Studies using reverse transcriptase differential display and intracerebroventricular administration of MCH

INTRODUCTION Melanin-concentrating hormone (MCH), urotensins, and stanniocalcin were originally discovered in fish [12, 14, 24, 40]. There is accumulating evidence that shows the importance of these peptides in human physiology and diseases, such as cardiovascular disease and metabolic syndrome [1, 33]. Furthermore, adrenomedullin 2/intermedin (AM2/IMD) has recently been discovered as an ortholog of the fish peptide, adrenomedullin 2 [28, 34]. MCH, urotensins, and adrenomedullins belong to the families of either brain peptides, cardiovascular peptides, or both. MCH, urotensins (urocortins and urotensin II), and AM2/IMD are described in detail in The Brain and Ingestive Peptides sections of this Handbook of Biologically Active Peptides

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1516 / Chapter 211 [26] and studies using MCH gene knockout mice [30] all showed the appetite-stimulating role of MCH in mammals. MCH is now considered to be one of the most potent appetite-stimulating peptides in mammals. The effects of MCH on the mammalian brain and ingestion are discussed in the Brain and Ingestive Peptides sections of this Handbook, see Chapters 98 and 127. Thus, MCH is a circulating hormone that regulates skin color in the fish, whereas it acts primarily as a neurotransmitter or a neuromodulator in the mammal. Recent studies have suggested that MCH act as a stimulator of appetite also in fish [31]. In fasted fish, MCH gene expression in the brain was greater than in controls. In white-reared fish, MCH gene expression in the brain was greater than in black-reared fish. Finally, white-reared fish grew faster than black-reared fish [31]. Two types of MCH receptors, MCH type 1 and type 2 receptors, were identified at first in mammals [11]. Both MCH receptors belong to the G-protein-coupled receptors with seven transmembrane domains. MCH type 2 receptor was identified in humans and rhesus monkeys but not in mice or rats. The genes encoding fish orthologs of MCH receptors have recently been discovered [16]. The search in the whole genome shotgun datasets showed that Fugu contains two MCH receptor genes (MCHR1 and MCHR2) and zebrafish has three (MCHR1a, MCHR1b, and MCHR2) [16]. It is noteworthy that αMSH is expressed in the arcuate nucleus (infundibular nucleus) of the hypothalamus and suppresses the appetite in mammals [13]. Thus, MCH and αMSH act in opposite ways in the regulation of both skin color and appetite, although each has its own receptors (MCH type 1 and 2 receptors, and melanocortin 1–5 receptors, respectively). The skin color change may be an important strategy for survival against stress and enemies in fish and amphibia. MCH and αMSH may therefore constitute the essential protective mechanism against enemies through the regulation of the skin color and the appetite, at least in the species from fish to human.

UROTENSINS Urotensins are peptide hormones that were originally discovered from the fish neuroendocrine organ, urophysis. Urotensin I (UI) belongs to the corticotropin-releasing hormone (CRH) family [14], whereas urotensin II (UII) is a somatostatinlike cyclic peptide [14]. Human homologs of urotensins have recently been identified and their pathophysiological roles have been demonstrated, particularly in cardiovascular disease. There are three urocortins, urocortin (Ucn)1, Ucn2 (stresscopin-related peptide), and Ucn3 (stresscopin), which are mammalian homologs of UI [8, 15, 27,

37]. Urocortins have a potent vasodilator action, which is mediated by the CRH type 2 receptor. UII has been shown to be one of the most potent vasoconstrictor peptides in mammals [1], whereas it acts as a vasodilator in some arteries [2]. It is worth recalling the biological actions of urotensins in fish before reviewing these peptides in human. UI was found to be a hypotensive and corticotropinreleasing neuropeptide in fish [14]. Both UI and UII have effects on the intestinal absorption of water and NaCl in tilapia and are considered to have physiological roles in the adaptation to freshwater or seawater [18]. UI caused significant decreases in water and NaCl absorption in freshwater fish but not in seawater-adapted fish, whereas UII increased water and NaCl absorption in seawater-adapted fish but not in freshwater fish [18]. On the other hand, active chloride transport is stimulated by UI and inhibited by UII in the skin of a marine teleost [19]. Furthermore, UII stimulates sodium transport across the teleost urinary bladder [17]. Thus, urotensins are considered to play a role in osmoregulation by acting on the skin, intestinal tract, and urinary bladder of the teleost fish. Fish UII has a vasoconstrictor action in rat [9]. UII inhibits prolactin release from the organ-cultured rostral pars distalis of the tilapia [6]. UII induces hypoglycemia and hyperinsulinemia, enhances glucose mobilization, and stimulates hyperlipidemia in juvenile freshwater coho salmon [29]. These biological actions of urotensins in the fish remind us of the high expression of urotensins in human cardiovascular and renal tissues and their altered plasma levels in patients with renal or heart diseases [33, 36], discussed in the Cardiovascular Peptides Section of this Handbook, see Chapter 166. They also recall the relation of UII with human diabetes mellitus [35, 41].

ADRENOMEDULLIN 2/INTERMEDIN Adrenomedullin (AM) is a multifunctional peptide originally isolated from pheochromocytoma tissue [32]. AM and its receptor are expressed in almost all types of cells and organs. AM has various biological actions, including a potent vasodilator action, modulatory actions on hormone secretion and cell proliferation, anti-apoptotic actions, and neurotransmitter/neuromodulator actions. The AM receptor consists of the complex of calcitonin receptorlike receptor (CRLR) and receptor activity–modifying protein (RAMP)2 or -3. The complex of CRLR and RAMP1 generates receptors for CGRP. Takei et al. identified five AM cDNAs from the pufferfish, Takifugu rubripes, and named them AM1, AM2, AM3, AM4, and AM5 [22]. AM1 is the ortholog of mammalian AM. They then identified AM2 in mouse, rat,

Fish Peptides / 1517 and human [34]. Almost simultaneously Roh et al. identified the same peptide as a novel member of the calcitonin/CGRP peptide family and named it intermedin (IMD) because of the high expression of this peptide in the intermediate lobe of pituitary [28]. AM2/IMD can bind to the complex of CRLR and one of three RAMPs nonselectively. In addition to the intermediate lobe of the pituitary, AM2/IMD is highly expressed in the lung, gastrointestinal tract, kidney, and submaxillary gland. AM2/IMD stimulates cAMP production and has a potent vasodilator action like AM and CGRP. AM2/IMD has effects on natriuresis in the kidneys. Furthermore, AM2/IMD suppressed gastric-emptying activity and food intake in mice when administered intraperitoneally [28].

STANNIOCALCINS Discovery of Stanniocalcins Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland associated with the kidneys of bony fish [40]. The first mammalian homolog of STC (STC1) was isolated independently in two laboratories: Chang et al. [3] discovered STC1 by mRNA differential display during studies aimed at identifying genes involved in the control of cellular proliferation in SV40-transfected human fibroblasts, and Olsen et al. [23] found it during a random sequencing screen of human lung cDNAs. A human gene encoding a second STC-like protein (STC2) was identified in a search for related sequences in expressed sequence tag (EST) databases [5].

Structure of the Precursor mRNA/Gene of STCs The human STC1 cDNA encodes a protein of 247 amino acids, which shows approximately 50% homology with fish STC [4]. The human STC2 cDNA encodes a protein of 302 amino acids, which has a lower identity (∼35%) with STC1 and the fish counterpart [4]. The human STC1 gene is on the short arm of chromosome 8 (8p11.2–p21) and contains four exons spanning 13 kb. The STC2 gene has been localized to chromosome 5q33 or 5q35 and also contains four exons. On the other hand, the fish STC gene, which was isolated from sockeye salmon, contains five exons.

Distribution of the mRNA and Proteins of STCs STC1 and STC2 are expressed in a wide variety of mammalian tissues, including the endocrine glands, reproductive organs, and kidney [4]. High expression of STC1 was found in the human kidney, ovary, pros-

tate, and thyroid. In the kidney, STC1 was localized to principal and α-intercalated cells in the distal half of the nephron. STC1 mRNA expression was found in secondary interstitial and theca interna cells of the ovary. STC1 expression in the ovary increases during pregnancy and lactation. Furthermore, STC1 expression was observed in osteoblasts and chondrocytes of the bone, fully differentiated neurons, breast ductal epithelium, myocardiocytes, megakaryocytes, adipocytes, and so forth. Human STC2 is also widely expressed in various tissues, including the kidney, heart, pancreas, and spleen. The expression of STC2 is inducible by estrogen and repressed by anti-estrogen.

Receptors for STCs Saturable, displaceable, high-affinity (0.25–0.8 nM) STC1 binding activity was detected in the plasma membrane and inner mitochondrial matrix of liver and kidney. The STC receptor cDNA or gene has not been cloned, however.

Biological Actions of STCs STC elicits a typical antihypercalcemic and antihypophosphatemic response on undefined target cells in the gill, intestine, and kidney in the fish. STC inhibits whole-body Ca2+ influx in the gill and intestine, and stimulates the resorption of inorganic phosphorus by proximal tubule epithelium cells from fish kidney [4, 42]. In mammals, STC1 protected neurons against ischemic damage. Transgenic mice overexpressing STC1 showed growth retardation. On the other hand, STC1 stimulates osteoblast differentiation in rat calvaria cell cultures. STC1 is a selective modulator of hepatocyte growth factor–induced endothelial migration and morphogenesis. STC1 attenuated chemokinesis and diminished the chemotactic response to monocyte chemotactic protein 1 (MCP-1) and stromal cell–derived factor 1α in murine macrophagelike (RAW264.7) and human monoblastlike (U937) cells [10], suggesting that it modulates the immune/inflammatory response. Thus, STC1 appears to have multiple functions in mammals [42].

Pathophysiological Implications There is increasing evidence suggesting that STCs have a role in human cancer [4]. The expression of STC1 is induced in breast ductal epithelium by BRCA1, a tumor suppressor gene that has an important role in breast and ovarian cancers. Furthermore, in primary early-stage breast cancer patients, the detection of STC1 mRNA in the bone marrow and blood significantly

1518 / Chapter 211 correlated with multiple histopathological prognostic factors, including primary tumor size, number of positive lymph nodes, and clinical stages, whereas STC1 mRNA was not detected in the blood or bone marrow of volunteers without cancer [39]. Thus, STC1 was proposed as a novel, specific, and clinically useful molecular marker for detecting occult breast cancer cells in the bone marrow and blood.

CONCLUSION Fish peptides appear to have essential roles in the survival of the fish. The color change regulated by MCH and αMSH must be critical to protect against enemies. Osmoregulation controlled by urotensins is also essential for the fish living in both freshwater and seawater. The protection against calcium concentration change in water by STCs must be essential for the fish, which lives in water with a broad range of calcium concentrations. It may not be surprising that these fish peptides also have important roles in human physiology for circulation, appetite control, and cell differentiation and in the pathophysiology of human diseases, such as the metabolic syndrome, cardiovascular disease, and cancers. Could this be because all lives, including human, began in the water?

References [1] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 401: 282–6. [2] Bottrill FE, Douglas SA, Hiley CR, White R. Human urotensin-II is an endothelium-dependent vasodilator in rat small arteries. Brit J Pharmacol 2000; 130: 1865–70. [3] Chang AC, Janosi J, Hulsbeek M, de Jong D, Jeffrey KJ, Noble JR, Reddel RR. A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol Cell Endocrinol 1995; 112: 241–7. [4] Chang AC, Jellinek DA, Reddel RR. Mammalian stanniocalcins and cancer. Endocr Relat Cancer 2003; 10: 359–73. Review. [5] Chang AC, Reddel RR. Identification of a second stanniocalcin cDNA in mouse and human: Stanniocalcin 2. Mol Cell Endocrinol 1998; 141: 95–9. [6] Grau EG, Nishioka RS, Bern HA. Effects of somatostatin and urotensin II on tilapia pituitary prolactin release and interactions between somatostatin, osmotic pressure Ca++, and adenosine 3′,5′-monophosphate in prolactin release in vitro. Endocrinology 1982; 110: 910–5. [7] Hogben LI, Slome S. The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian colour change. Proc R Soc London Ser B 1931; 108: 10–53. [8] Hsu SY, Hsueh AJ. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropinreleasing hormone receptor. Nat Med 2001; 7: 605–11. [9] Itoh H, Itoh Y, Rivier J, Lederis K. Contraction of major artery segments of rat by fish neuropeptide urotensin II. Am J Physiol 1987; 252: R361–6.

[10] Kanellis J, Bick R, Garcia G, Truong L, Tsao CC, Etemadmoghadam D, et al. Stanniocalcin-1, an inhibitor of macrophage chemotaxis and chemokinesis. Am J Physiol Renal Physiol 2004; 286: F356–62. [11] Kawauchi H, Baker BI. Melanin-concentrating hormone signaling systems in fish. Peptides 2004; 25: 1577–84. [12] Kawauchi H, Kawazoe I, Tsubokawa M, Kishida M, Baker BI. Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 1983; 305: 321–3. [13] Krude H, Biebermann H, Luck W, Horn R, Brabant G, Gruters A. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat Genet 1998; 19: 155–7. [14] Lederis K, Letter A, McMaster D, Moore G, Schlesinger D. Complete amino acid sequence of urotensin I, a hypotensive and corticotropin-releasing neuropeptide from Catostomus. Science 1982; 218: 162–5. [15] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, et al. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001; 98: 7570–5. [16] Logan DW, Bryson-Richardson RJ, Pagan KE, Taylor MS, Currie PD, Jackson IJ. The structure and evolution of the melanocortin and MCH receptors in fish and mammals. Genomics 2003; 81: 184–91. [17] Loretz CA, Bern HA. Stimulation of sodium transport across the teleost urinary bladder by urotensin II. Gen Comp Endocrinol 1981; 43: 325–30. [18] Mainoya JR, Bern HA. Effects of teleost urotensins on intestinal absorption of water and NaCl in tilapia, Sarotherodon mossambicus, adapted to fresh water or seawater. Gen Comp Endocrinol 1982; 47: 54–8. [19] Marshall WS, Bern HA. Active chloride transport by the skin of a marine teleost is stimulated by urotensin I and inhibited by urotensin II. Gen Comp Endocrinol 1981; 43: 484–91. [20] Mouri T, Takahashi K, Kawauchi H, Sone M, Totsune K, Murakami O, et al. Melanin-concentrating hormone in the human brain. Peptides 1993; 14: 643–6. [21] Nahon JL, Presse F, Bittencourt JC, Sawchenko PE, Vale W. The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 1989; 125: 2056–65. [22] Ogoshi M, Inoue K, Takei Y. Identification of a novel adrenomedullin gene family in teleost fish. Biochem Biophys Res Commun 2003; 311: 1072–7. [23] Olsen HS, Cepeda MA, Zhang QQ, Rosen CA, Vozzolo BL. Human stanniocalcin: A possible hormonal regulator of mineral metabolism. Proc Natl Acad Sci USA 1996; 93: 1792–6. [24] Pearson D, Shively JE, Clark BR, Geschwind II, Barkley M, Nishioka RS, Bern HA. Urotensin II: A somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc Natl Acad Sci USA 1980; 77: 5021–4. [25] Presse F, Nahon JL, Fischer WH, Vale W. Structure of the human melanin concentrating hormone mRNA. Mol Endocrinol 1990; 4: 632–7. [26] Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, et al. A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 1996; 380: 243–7. [27] Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, et al. Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001; 98: 2843–8.

Fish Peptides / 1519 [28] Roh J, Chang CL, Bhalla A, Klein C, Hsu SY. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes. J Biol Chem 2004; 279: 7264–74. [29] Sheridan MA, Plisetskaya EM, Bern HA, Gorbman A. Effects of somatostatin-25 and urotensin II on lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen Comp Endocrinol 1987; 66: 405–14. [30] Shimada M, Tritos NA, Lowell BB, Flier JS, Maratos-Flier E. Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 1998; 396: 670–4. [31] Takahashi A, Tsuchiya K, Yamanome T, Amano M, Yasuda A, Yamamori K, Kawauchi H. Possible involvement of melaninconcentrating hormone in food intake in a teleost fish, barfin flounder. Peptides 2004; 25: 1613–22. [32] Takahashi K. Adrenomedullin from a pheochromocytoma to the eye: Implications of the adrenomedullin research for endocrinology in the 21st century. Tohoku J Exp Med 2001; 193: 79–114. Review. [33] Takahashi K. Translational medicine in fish-derived peptides: From fish endocrinology to human physiology and diseases. Endocr J 2004; 51: 1–17. Review. [34] Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, Miyano S. Identification of novel adrenomedullin in mammals: A potent cardiovascular and renal regulator. FEBS Lett 2004; 556: 53–8. [35] Totsune K, Takahashi K, Arihara Z, Sone M, Ito S, Murakami O. Increased plasma urotensin II levels in patients with diabetes mellitus. Clin Sci (Lond) 2003; 104: 1–5.

[36] Totsune K, Takahashi K, Arihara Z, Sone M, Satoh F, Ito S, et al. Role of urotensin II in patients on dialysis. Lancet 2001; 358: 810–1. [37] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995; 378: 287–92. [38] Vaughan JM, Fischer WH, Hoeger C, Rivier J, Vale W. Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 1989; 125: 1660–5. [39] Wascher RA, Huynh KT, Giuliano AE, Hansen NM, Singer FR, Elashoff D, Hoon DS. Stanniocalcin-1: A novel molecular blood and bone marrow marker for human breast cancer. Clin Cancer Res 2003; 9: 1427–35. [40] Wendelaar Bonga SE, Pang PK. Control of calcium regulating hormones in the vertebrates: Parathyroid hormone, calcitonin, prolactin, and stanniocalcin. Int Rev Cytol 1991; 128: 139–213. [41] Wenyi Z, Suzuki S, Hirai M, Hinokio Y, Tanizawa Y, Matsutani A, et al. Role of urotensin II gene in genetic susceptibility to type 2 diabetes mellitus in Japanese subjects. Diabetologia 2003; 46: 972–6. [42] Yoshiko Y, Aubin JE. Stanniocalcin 1 as a pleiotropic factor in mammals. Peptides 2004; 25: 1663–9. Review. [43] Zamir N, Skofitsch G, Bannon MJ, Jacobowitz DM. Melaninconcentrating hormone: Unique peptide neuronal system in the rat brain and pituitary gland. Proc Natl Acad Sci USA 1986; 83: 1528–31.

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212 Peptides and Sleep A. STEIGER

HPA systems, GH-releasing hormone (GHRH) and corticotropin-releasing hormone (CRH), appears to play a major role in sleep regulation.

ABSTRACT Between the electrophysiological activity of sleep as recorded by sleep electroencephalogram (EEG) and nocturnal hormone secretion there exists a bidirectional interaction. Various neuropeptides participate in sleep regulation. A reciprocal interaction of GHRH and CRH plays a key role in sleep regulation. Growth hormone releasing hormone (GHRH) promotes sleep and growth hormone and inhibits cortisol, whereas corticotropin-releasing hormone (CRH) exerts opposite effects. Age and gender appear to modulate these effects. Also ghrelin and galanin promote sleep, whereas somatostatin impairs sleep. Neuropeptide Y regulates sleep onset. VIP is involved in the temporal organization of sleep.

HYPOTHALAMIC-PITUITARYSOMATOTROPIC SYSTEM Basic Activity In humans, the major GH peak is found near sleep onset. This GH surge is associated with the first SWS period [82]. The GH surge is widely sleep-dependent and is suppressed during sleep deprivation [72]; however, a weak circadian component in the regulation of GH release was found [85]. As early as during the third decade of the life span, parallel decreases of SWS, SWA, and GH secretion start [10]. Hypothalamic GHRH mRNA depends on a circadian rhythm. In rats, its highest concentration occurs at the onset of the light period when sleep propensity reaches its maximum [17]. Hypothalamic GHRH contents display sleep-related variations with low levels in the morning, increases in the afternoon, and decreases at night [34]. In patients with isolated GH deficiency, SWS is lower than in normal controls [7]. Excessive GH levels are found in patients with acromegaly. One year after adenectomy, REMS and SWS time increased in these patients [8].

INTRODUCTION Human sleep is characterized by the cyclic occurrence of periods of non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) and by distinct patterns of hormone secretion. During the first NREMS period, the major portion of slow wave sleep (SWS) and in electroencephalogram (EEG) spectral analysis the major portion of slow wave activity (SWA) occur. During the first half of the night, the growth hormone (GH) surge preponderates, whereas corticotropin (ACTH) and cortisol levels are low. During the second half of the night, ACTH and cortisol concentrations are high, whereas GH release is low [90]. This pattern points to (1) a reciprocal interaction of the hypothalamic-pituitary-somatotrophic (HPS) and the hypothalamic-pituitary-adrenocortical (HPA) systems and (2) the existence of common regulators of sleep EEG and nocturnal hormone secretion. A reciprocal interaction of the key hormones of the HPS and Handbook of Biologically Active Peptides

Growth Hormone-Releasing Hormone GHRH is an important endogenous sleep-promoting substance. In the mouse, the GHRH gene is found in the region linked to SWA [32]. Intracerebroventricular (ICV) GHRH increases SWS in rats and rabbits [25, 56]. The same effect occurs after injection of GHRH into the medial preoptic area in rats [94] or intravenous (IV) administration in rats [61].

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1522 / Chapter 212 Similarly, after repetitive hourly IV injections of GHRH around sleep onset SWS and GH increase, whereas cortisol is blunted in young men [77]. Mimicking the pulsatile endogenous release appears to be crucial because sleep remained unchanged after GHRH infusion [50]. Sleep promotion in young males after GHRH was confirmed after IV [46, 50] and intranasal [69] administration. The effects of GHRH on human sleep were examined in three states with a change of the GHRH/CRH ratio in favor of CRH: (1) After repetitive IV GHRH between 4:00 and 7:00 a.m., sleep remains unchanged [73]; (2) only a weak sleep-promoting effect of GHRH was found in the elderly [38]; and (3) the effect of IV GHRH was tested in drug-free depressed patients of both sexes (19–76 years old) and in matched controls. A sexual dimorphism in the response to GHRH was found. In male patients and controls, GHRH decreased ACTH and cortisol, whereas these hormones were enhanced in normal and depressed females. Similarly, NREMS increased and wakefulness decreased in males, whereas sleep-impairing effects occurred in women [4, 5]. These results point to a reciprocal antagonism of GHRH and CRH in men, whereas a synergism of GHRH and CRH is suggested in women. In rats, NREMS decreases and sleep latency increases after GHRH receptor antagonists [65]. NREMS is reduced after antibodies to GHRH [66]. Sleep deprivation is a powerful stimulus for sleep. GHRH appears to mediate this effect. It is antagonized by GHRH antibodies in rats [66] and by microinjections of a GHRH antagonist into the preoptic area of rats [94]. After sleep deprivation, a depletion of hypothalamic GHRH and low hypothalamic GHRH contents are observed [34], whereas hypothalamic GHRH mRNA increases in rats [83, 94]. This high rate of release is thought to stimulate transcription. The rise in hypothalamic GHRH mRNA concentration is associated with decreases in hypothalamic somatostatin [93]. GHRH receptor mRNA and GHRH binding decline distinctly in the rat hypothalamus after sleep deprivation, whereas pituitary GHRH receptors remain unchanged. In mice, viral infections enhance NREMS. After influenza A infection, NREMS increases in wild-type mice. However, in the GHRH-receptor-deficient lit/lit mice NREMS and SWA decrease after influenza inoculation [1]. NREMS decreases after negative feedback inhibition of GHRH by GH in humans [51], cats [80], and rats [23, 64] or after higher dosages of ICV insulinlike growth factor (IGF)-1 [63]. On the other hand GH antagonism impairs sleep [59].

Somatostatin Selective increases of REMS were reported in rats after ICV administration of somatostatin [20]. Systemic

and ICV administration of the somatostatin analog octreotide in rats decreases NREMS and GH [9]. Similarly, SWS declines and wakefulness increases in young men after subcutaneous octreotide [95]. Octreotide is long acting and more potent than somatostatin. This explains why repetitive IV somatostatin impaired sleep in normal elderly subjects [33] but not in young men [77]. A reciprocal interaction of GHRH and somatostatin in sleep regulation, similar to their opposite effects on GH release, is likely.

Ghrelin Similar to GHRH, repetitive IV administration of ghrelin enhances SWS and GH in young men [89]. In contrast to GHRH, which blunted cortisol in young men [77], ACTH and cortisol increase after the administration of ghrelin [90]. Also, in mice ghrelin promotes NREMS [57]. An intact GHRH receptor is the prerequisite for this effect because ghrelin does not modulate sleep in mice with nonfunctional GHRH receptors. Bodosi et al. [11] investigated the relationships among plasma ghrelin and hypothalamic ghrelin contents and sleep and feeding in rats. They found a major influence of feeding on the diurnal rhythm of ghrelin. Variations in the hypothalamic ghrelin contents suggest an association with the sleep-wake activity. Dzaja et al. [24] examined interactions of sleep and ghrelin levels in young men who were semirecumbent during 24 hours. When they were allowed to sleep, ghrelin increased by sleep onset. This was followed by a decline throughout the night. The nocturnal rise was blunted during sleep deprivation. Ghrelin levels were determined between 8:00 p.m. and 7:00 a.m. in normal female and male control subjects who were active during daytime. In males, ghrelin increases continuously between 8:00 and 11:00 p.m. In females, however, ghrelin levels at 8:00 p.m. were already in the same range as during the sleeping period [74]. In a sample of more than 1000 subjects, short sleep time was associated with higher ghrelin levels [81]. Similarly, the comparison between extended (12 hours) and restricted (4 hours) sleep time during 2 days showed increases in ghrelin after sleep restriction during the daytime [76].

Animal Models of HPS System Changes In giant transgenic mice, GH is permanently elevated. During the light period, NREMS is higher and REMS is almost doubled in these mice. After sleep deprivation, these mice sleep more than normal mice [39]. In dwarf rats with deficits in the central GHRHergic transmission and reduced hypothalamic, GHRH NREMS is reduced compared with the wild type [60]. In dwarf homozygous (lit/lit) mice with nonfunc-

Peptides and Sleep tional GHRH receptor, NREMS and REMS are lower than in control mice. Chronic infusion of GH leads to the normalization of REMS but not of NREMS. Ghrelin, GHRH, and octreotide exert no effect on sleep EEG in dwarf mice. These results suggest that (1) GHRH deficiency is linked with decreases in NREMS, (2) decreases in GH lead to decreases in REMS, and (3) the actions of GHRH, ghrelin, and octreotide on sleep EEG require intact GHRH receptor signaling [64].

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decreases in the striatum and in the pituitary [28]. When rats are sleep deprived during intervals up to 92 h, ACTH and corticosterone plasma levels increase from 24 h of sleep deprivation and decrease during the recovery period [2]. The expectation of waking up at a certain time induces a marked increase in ACTH before the end of sleep [13]. The anticipatory increase in ACTH may facilitate spontaneous waking.

Corticotropin-Releasing Hormone HYPOTHALAMIC-PITUITARYADRENOCORTICAL SYSTEM Basic Activity In rats CRH gene transcription levels increase during the dark period, when the animals are active, and decrease throughout the light period [88]. In humans during sleep, both the nadir and the major portion of the secretion of ACTH and cortisol occur. Between 2:00 and 3:00 a.m., the first pulse of cortisol occurs. It is followed by further pulses until awakening [90]. Although ACTH is the prime regulator of nocturnal cortisol secretion in humans, the secretion of ACTH and cortisol may dissociate [30].

Sleep in Disorders with Pathological Changes of HPA Activity Characteristic sleep-EEG findings in depressed patients are disturbed sleep continuity and a decrease of NREMS and REMS disinhibition. Well-documented endocrine changes include signs of HPA overactivity (reviewed in [43]) and HPS dysfunction (reviewed in [79]). Most sleep-endocrine studies in depressed patients report elevated cortisol and ACTH [5, 48, 79] compared to normal controls. GH was blunted in most, [45, 79, 87] but not in all [48], studies. These findings suggest a causal relationship between shallow sleep, low GH, and HPA hyperactivity in depression. Furthermore, there are similarities in the sleep-endocrine changes during depression and during normal aging. Linkowski et al. [48] compared HPA hormones longitudinally between acute depression and recovery. These authors reported a decrease of ACTH and cortisol during the 24 h after recovery. Similar to the findings in depression, 24-h ACTH and cortisol are higher in young insomniacs than in controls [86].

Effects of Changes in Sleep-Wake Behavior on HPA Hormones In rats after 72 h of sleep deprivation, CRH levels increase in the striatum, limbic areas, and pituitary, whereas hypothalamic CRH decreases. CRH binding

After the ICV administration of CRH, SWS decreases in rats [25] and rabbits [68]. Even after 72 h of sleep deprivation, CRH reduces SWS in rats. Furthermore, sleep latency and REMS increase [49]. Similarly after repetitive IV administration of CRH around sleep onset in young men, SWS and REMS decrease. Furthermore, GH is blunted and cortisol increases [44]. A dose of CRH that does not affect sleep in young men impairs sleep in middle-aged men [86]. Obviously, the vulnerability of sleep to CRH increases during aging. After two different CRH antagonists, α-helical CRH and astressin, wakefulness declines [18]. In contrast, another study reported an effect of α-helical CRH only in stressed animals. In these rats, REMS increases and decreases to values of the nonstressed condition after the substance. In sleep-deprived rats, α-helical CRH diminishes the REMS rebound during recovery sleep. Stress acting via CRH is thought to induce the REMS rebound after sleep deprivation [36]. In the rat, amygdala kindling at light onset decreases SWS and REMS and enhances corticosterone. The ICV administration of astressin or α-helical CRH antagonizes the decrease of SWS after kindling [91]. After a 4-week trial with α-CRH-1 receptor antagonism, the characteristic sleepEEG changes in patients with depression are counteracted. The number of awakenings and REMS density decrease and SWS increases [40]. These results suggest that CRH is involved in the pathophysiology of sleepEEG changes during depression and that CRH-1 receptor antagonism helps to treat symptoms of depression including impaired sleep.

Vasopressin Vasopressin administered ICV increases wakefulness in rats [6]. After acute infusion of the peptide to humans, stage 2 sleep increases and REMS decreases (reviewed in [70]). Chronic intranasal vasopressin for 3 months improves sleep in elderly subjects [70].

ACTH Infusions of ACTH suppress REMS in normal subjects [31, 35], whereas cortisol and GH increase [14]. The

1524 / Chapter 212 synthetic ACTH(4–9) analog ebiratide shares several behavioral effects of ACTH, but it has no influence on peripheral hormone secretion. Accordingly, after repetitive IV administration of ebiratide, GH and cortisol remain unchanged in young men. A set of sleep-EEG changes is found after ebiratide corresponding to a general central nervous system (CNS) activation [78].

Animal Models of HPA System Changes In the Lewis rat, the synthesis and the release of CRH is reduced compared with other rat strains. Lewis rats spend more time in SWS and less time awake than the intact strains. After the ICV administration of CRH, waking increases similarly in Lewis and Sprague-Dawley rats. A role of CRH in the maintenance of wakefulness and the sleep-disturbing effects of CRH are confirmed by this study [67]. Similarly, the spontaneous wakefulness of rats decreases after CRH antisense [19].

HYPOTHALAMIC-PITUITARYTHYROID SYSTEM The release of thyroid-stimulating hormone (TSH) is related to circadian rhythm [16]. The lowest TSH levels are found during the daytime. TSH rises during the night and reaches its maximum by midnight. Pulsatile IV thyrotropin-releasing hormone (TRH) prompts decreases of sleep efficiency and the earlier occurrence of the cortisol morning rise in young male subjects [42].

OTHER PEPTIDES Cortistatin. After ICV administration, NREMS distinctly increases in rats [21]. d-sleep-inducing peptide (DSIP). DSIP was first detected in the cerebral venous blood of rabbits that had been subjected to sleep by electric stimulation of the intralaminal thalamic area [52]. The promotion of SWS after DSIP was reported in several species, although not all preclinical studies reproduced these effects (for review, see [12, 37]). Only one study investigated the effects of DSIP on sleep EEG in normal men and found only minor effects. Studies on the efficiency of DSIP in the treatment of insomnia produced conflicting results. DSIP-like immunoreactivity was compared in young men in baseline sleep, sleep deprivation, and recovery sleep. Plasma DSIP-like immunoreactivity decreased at the transition from wakefulness to sleep in both evening sleep and morning recovery sleep. These results suggest that DSIP is induced by mechanisms that are involved in the initiation of sleep [75].

Galanin. Sleep in the rat remains unchanged after ICV galanin, whereas REMS deprivation induces galanin gene expression [84]. After repetitive IV galanin administration in young men, SWS and REMS periods increase [53]. A cluster of GABAergic and galaninergic neurons was identified in the ventrolateral preoptic area, which may stimulate NREMS [71]. IV galanin or placebo was given to patients with depression during a steady state of antidepressive therapy with trimipramine. After galanin, REMS latency increased and the severity of depression decreased [55]. Neuropeptide Y (NPY). After ICV administration of NPY, EEG spectral activity changes in rats, similar to the effects of benzodiazepines [26]. The prolongation of sleep latency by CRH is antagonized dosedependently by NPY in rats [27]. In young men, repetitive IV NPY decreases sleep latency, the first REMS period, and cortisol and ACTH levels, and it increases stage 2 sleep and sleep-period time [3]. In depressed patients of both sexes with a wide age range and age-matched controls, the sleep latency decreases and prolactin increases after NPY, whereas cortisol and ACTH levels and the first REMS period remain unchanged [41]. These results suggest that NPY participates in sleep regulation, particularly in the timing of sleep onset as an antagonist of CRH acting via the GABAA receptor. Pituitary adenylate cyclase activating polypeptide (PACAP). The ICV administration of PACAP enhances REMS in rats [29]. Substance P. The microinjection of substance P into the ventrolateral preoptic area of rats enhances SWS. This effect is blocked by a phospholipase C inhibitor and by 3-mercaptopropionic acid, an inhibitor of GABA synthesis and release [92]. In young men, the repetitive IV administration of substance P increases wakefulness and REMS latency [47]. Vasoactive intestinal polypeptide (VIP). The ICV administration of VIP enhances REMS [22]. VIP given to rats during the dark period enhances NREMS and REMS [58]. Also, VIP microinjections into the pontine reticular tegmentum enhance REMS in rats [15]. The immunoneutralization of prolactin inhibits REMS promotion by systemic VIP in rats. The stimulation of prolactin appears to be involved in the promotion of REMS after VIP [62]. In young men, two doses of IV VIP prompt different effects [54]. After the lower-dose, prolactin decreases, but sleep EEG remains unchanged. In contrast, after the higher dose of VIP, prolactin increases. Furthermore, the NREMS-REMS cycles are decelerated. Each of the NREMS and REMS periods is prolonged, the cortisol nadir appears advanced, and the GH surge is blunted [54]. It is thought that VIP exerts a specific effect on the temporal organization of the sleep

Peptides and Sleep cycles and the sleep-related hormone secretion. VIP appears to affect the circadian clock, resulting in longer sleep cycles and the early occurrence of the cortisol nadir. The blunted GH surge may be due to the advanced elevated HPA activity.

in several species including humans. In Fig. 1 a model of peptidergic sleep regulation in humans is given. It is well documented that a reciprocal interaction of the neuropeptides GHRH and CRH plays a key role in sleep regulation. GHRH promotes sleep, at least in males, and stimulates GH, whereas CRH enhances vigilance, enhances the secretion of ACTH and cortisol, and impairs sleep. Changes in the CRH/GHRH ratio in favor of CRH contribute to shallow sleep, elevated cortisol, and blunted GH during depression and aging. GHRH participates in sleep promotion after sleep

CONCLUSION The data reviewed in this chapter show that various neuropeptides exert specific effects on the sleep EEG

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FIGURE 1. Model of peptidergic regulation. Characteristic hypnograms and patterns of cortisol and GH secretion are shown in (A) a young and (C) an elderly control subject and in (B) a depressed patient. It is thought that GHRH is released during the first half of the night, whereas CRH preponderates during the second half of the night. GHRH contributes to the high amounts of GH and SWS after sleep onset, whereas CRH is linked with cortisol release and REM sleep in the morning hours. NPY is a signal for sleep onset. In addition to GHRH, galanin and ghrelin are sleeppromoting factors, whereas somatostatin is a sleepimpairing factor. During depression (CRH overactivity) and during normal aging, similar changes of sleep-endocrine activity occur. It is thought that changes in the GHRH/CRH ratio in favor of CRH play a key role in these alterations. CRH, corticotropin-releasing hormone; GHRH, growth hormone-releasing hormone; NPY, neuropeptide Y; SRIF, somatostatin.

1526 / Chapter 212 deprivation. Recent data suggest CRH-like effects of GHRH in women. Several findings, particularly the effects of CRH-1 receptor antagonism in patients with depression, suggest that CRH promotes REMS; however, other studies do not support this view. NPY is another endogenous antagonist of CRH. Its major role appears to be the timing of sleep onset. In addition to CRH, somatostatin is a sleep-impairing peptide. At least in males, GHRH and somatostatin exert opposite actions on GH and on sleep. In addition to GHRH, galanin and ghrelin promote SWS. Intact GHRH receptors are the prerequisite for sleep promotion by ghrelin. In contrast to GHRH, ghrelin stimulates HPA hormones in males and may act as an interface between the HPA and HPS systems. Galanin is colocalized with GABA in the ventrolateral preoptic nucleus. Many hypothalamic GHRH responsive neurons are GABAergic. Galanin, ghrelin, and GHRH may either act in a synergistic fashion, or these peptides may be part of a cascade resulting in the promotion of NREMS. Probably, GABAergic neurons mediate the effects of these peptides. In young normal men after VIP administration, the NREMS/REMS cycle is decelerated, probably by action on the suprachiasmatic nucleus.

Acknowledgment Studies from the author’s laboratory were supported by grants from the Deutsche Forschungsgemeinschaft (Ste 486/1-2, 5-1, 5-2 and 5-3).

References [1] Alt JA, Obál F Jr, Traynor TR, Gardi J, Majde JA, Krueger JM. Alterations in EEG activity and sleep after influenza viral infection in GHRH receptor-deficient mice. Journal of Applied Physiology 2003; 95:460–8. [2] Andersen ML, Martins PJF, D’Almeida V, Bignotto M, Tufik S. Endocrinological and catecholaminergic alterations during sleep deprivation and recovery in male rats. J Sleep Res 2005; 14:83–90. [3] Antonijevic IA, Murck H, Bohlhalter S, Frieboes RM, Holsboer F, Steiger A. NPY promotes sleep and inhibits ACTH and cortisol release in young men. Neuropharmacology 2000; 39:1474–81. [4] Antonijevic IA, Murck H, Frieboes RM, Barthelmes J, Steiger A. Sexually dimorphic effects of GHRH on sleep-endocrine activity in patients with depression and normal controls—part I: The sleep EEG. Sleep Res Online 2000; 3:5–13. [5] Antonijevic IA, Murck H, Frieboes RM, Steiger A. Sexually dimorphic effects of GHRH on sleep-endocrine activity in patients with depression and normal controls—part II: Hormone secretion. Sleep Res Online 2000; 3:15–21. [6] Arnauld E, Bibene V, Meynard J, Rodriguez F, Vincent JD. Effects of chronic icv infusion of vasopressin on sleep-waking cycle of rats. Am J Physiol 1989; 256:R674–84. [7] Åström C, Lindholm J. Growth hormone-deficient young adults have decreased deep sleep. Neuroendocrinology 1990; 51:82–4.

[8] Åström C, Trojaborg W. Effect of growth hormone on human sleep energy. Clin Endocrinol 1992; 36:241–5. [9] Beranek L, Hajdu I, Gardi J, Taishi P, Obál F Jr, Krueger JM. Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses. Am J Physiol 1999; 277:R1297–304. [10] Bliwise DL. Sleep in normal aging and dementia. Sleep 1993; 16:40–81. [11] Bodosi B, Gardi J, Hajdu I, Szentirmai E, Obál F Jr, Krueger JM. Rhythms of ghrelin, leptin, and sleep in rats: Effects of the normal diurnal cycle, restricted feeding, and sleep deprivation. Am J Physiol Regul Integrative Comp Physiol 2004; 287: R1071–9. [12] Borbély AA, Tobler I. Endogenous sleep-promoting substances and sleep regulation. Physiol Rev 1989; 69:605–70. [13] Born J, Hansen K, Marshall L, Mölle M, Fehm HL. Timing the end of nocturnal sleep. Nature 1999; 397:29–30. [14] Born J, Späth-Schwalbe E, Schwakenhofer H, Kern W, Fehm HL. Influences of corticotropin-releasing hormone, adrenocorticotropin, and cortisol on sleep in normal man. J Clin Endocrinol Metab 1989; 68:904–11. [15] Bourgin P, Lebrand C, Escourrou P, et al. Vasoactive intestinal polypeptide microinjections into the oral pontine tegmentum enhance rapid eye movement sleep in the rat. Neuroscience 1997; 77:351–60. [16] Brabant G, Brabant A, Ranft U, et al. Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J Clin Endocrinol Metab 1987; 65:83–8. [17] Bredow S, Taishi P, Obál F Jr, Guha-Thakurta N, Krueger JM. Hypothalamic growth hormone-releasing hormone mRNA varies across the day in rat. NeuroReport 1996; 7:2501–5. [18] Chang FC, Opp MR. Blockade of corticotropin-releasing hormone receptors reduces spontaneous waking in the rat. Am J Physiol 1998; 275:R793–802. [19] Chang FC, Opp MR. A corticotropin-releasing hormone antisense oligodeoxynucleotide reduces spontaneous waking in the rat. Regul Pept 2004; 117:43–52. [20] Danguir J. Intracerebroventricular infusion of somatostatin selectively increases paradoxical sleep in rats. Brain Res 1986; 367:26–30. [21] de Lecea L, Criado JR, Prospero-Garcia O, et al. A cortical neuropeptide with neuronal depressant and sleep-modulating properties. Nature 1996; 381:242–5. [22] Drucker-Colin R, Bernal-Pedraza J, Fernandez-Cancino F, Oksenberg A. Is vasoactive intestinal polypeptide (VIP) a sleep factor? Peptides 1984; 5:837–40. [23] Drucker-Colin RR, Spanis CW, Hunyadi J, Sassin JF, McGaugh JL. Growth hormone effects on sleep and wakefulness in the rat. Neuroendocrinology 1975; 18:1–8. [24] Dzaja A, Dalal MA, Himmerich H, Uhr M, Pollmächer T, Schuld A. Sleep enhances nocturnal plasma ghrelin levels in healthy subjects. Am J Physiol Endocr M 2004; 286:E963–7. [25] Ehlers CL, Reed TK, Henriksen SJ. Effects of corticotropinreleasing factor and growth hormone-releasing factor on sleep and activity in rats. Neuroendocrinology 1986; 42:467–74. [26] Ehlers CL, Somes C, Lopez A, Kirby D, Rivier JE. Electrophysiological actions of neuropeptide Y and its analogs: New measures for anxiolytic therapy? Neuropsychopharmacology 1997; 17:34–43. [27] Ehlers CL, Somes C, Seifritz E, Rivier JE. CRF/NPY interactions: A potential role in sleep dysregulation in depression and anxiety. Depression Anxiety 1997; 6:1–9. [28] Fadda P, Fratta W. Stress-induced sleep deprivation modifies corticotropin releasing factor (CRF) levels and CRF binding in rat brain and pituitary. Pharmacol Res 1997; 35:443–6.

Peptides and Sleep [29] Fang J, Payne L, Krueger JM. Pituitary adenylate cyclase activating polypeptide enhances rapid eye movement sleep in rats. Brain Res 1995; 686:23–8. [30] Fehm HL, Klein E, Holl R, Voigt KH. Evidence for extrapituitary mechanisms mediating the morning peak of cortisol secretion in man. J Clin Endocrinol Metab 1984; 58:410–4. [31] Fehm HL, Späth-Schwalbe E, Pietrowsky R, Kern W, Born J. Entrainment of nocturnal pituitary-adrenocortical activity to sleep processes in man—a hypothesis. Exp Clin Endocrinol 1993; 101:267–76. [32] Franken P, Chollet D, Tafti M. The homeostatic regulation of sleep need is under genetic control. J Neurosci 2001; 21:2610–21. [33] Frieboes RM, Murck H, Schier T, Holsboer F, Steiger A. Somatostatin impairs sleep in elderly human subjects. Neuropsychopharmacology 1997; 16:339–45. [34] Gardi J, Obál F Jr, Fang J, Zhang J, Krueger JM. Diurnal variations and sleep deprivation-induced changes in rat hypothalamic GHRH and somatostatin contents. Am J Physiol 1999; 277: R1339–44. [35] Gillin JC, Jacobs LS, Snyder F, Henkin RI. Effects of ACTH on the sleep of normal subjects and patients with Addison’s disease. Neuroendocrinology 1974; 15:21–31. [36] Gonzalez MMC, Valatx JL. Involvement of stress in the sleep rebound mechanism induced by sleep deprivation in the rat: Use of alpha-helical CRH (9–41). Behav Pharmacol 1998; 9:655–62. [37] Graf MV, Kastin AJ. Delta-sleep-inducing peptide (DSIP): A review. Neurosci Biobehav Rev 1984; 8:83–93. [38] Guldner J, Schier T, Friess E, Colla M, Holsboer F, Steiger A. Reduced efficacy of growth hormone-releasing hormone in modulating sleep endocrine activity in the elderly. Neurobiol Aging 1997; 18:491–5. [39] Hajdu I, Obál F Jr, Fang J, Krueger JM, Rollo CD. Sleep of transgenic mice producing excess rat growth hormone. Am J Physiol Regul Integrative Comp Physiol 2002; 282:R70–6. [40] Held K, Künzel H, Ising M, et al. Treatment with the CRH1receptor antagonist R121919 improves sleep EEG in patients with depression. J Psychiatr Res 2004; 38:129–36. [41] Held K, Murck H, Antonijevic IA, Künzel H, Steiger A. Neuropeptide Y (NPY) shortens sleep latency and enhances prolactin but does not suppress ACTH and cortisol in depressed patients and controls. Psychoneuroendocrinology 2006; 31:100–7. [42] Hemmeter U, Rothe B, Guldner J, Holsboer F, Steiger A. Effects of thyrotropin-releasing hormone on the sleep EEG and nocturnal hormone secretion in male volunteers. Neuropsychobiology 1998; 38:25–31. [43] Holsboer F. The rationale for corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety. J Psychiatr Res 1999; 33:181–214. [44] Holsboer F, von Bardeleben U, Steiger A. Effects of intravenous corticotropin-releasing hormone upon sleep-related growth hormone surge and sleep EEG in man. Neuroendocrinology 1988; 48:32–8. [45] Jarrett DB, Miewald JM, Kupfer DJ. Recurrent depression is associated with a persistent reduction in sleep-related growth hormone secretion. Arch Gen Psychiatry 1990; 47:113–8. [46] Kerkhofs M, Van Cauter E, Van Onderbergen A, Caufriez A, Thorner MO, Copinschi G. Sleep-promoting effects of growth hormone-releasing hormone in normal men. Am J Physiol 1993; 264:E594–8. [47] Lieb K, Ahlvers K, Dancker K, et al. Effects of the neuropeptide substance P on sleep, mood, and neuroendocrine measures in healthy young men. Neuropsychopharmacology 2002; 27:1041–9. [48] Linkowski P, Mendlewicz J, Kerkhofs M, et al. 24-hour profiles of adrenocorticotropin, cortisol, and growth hormone in major depressive illness: Effect of antidepressant treatment. J Clin Endocrinol Metab 1987; 65:141–52.

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[49] Marrosu F, Gessa GL, Giagheddu M, Fratta W. Corticotropinreleasing factor (CRF) increases paradoxical sleep (PS) rebound in PS-deprived rats. Brain Res 1990; 515:315–8. [50] Marshall L, Derad L, Strasburger CJ, Fehm HL, Born J. A determinant factor in the efficacy of GHRH administration in the efficacy of GHRH administration in promoting sleep: high peak concentration versus recurrent increasing slopes. Psychoneuroendocrinology 1999; 24:363–70. [51] Mendelson WB, Slater S, Gold P, Gillin JC. The effect of growth hormone administration on human sleep: A dose-response study. Biol Psychiatry 1980; 15:613–8. [52] Monnier M, Hoesli L. Humoral transmission of sleep and wakefulness. II. Hemodialysis of sleep-inducing humor during stimulation of the thalamic hypnogenic area. Pflugers Arch 1965; 282:60–75. [53] Murck H, Antonijevic IA, Frieboes RM, Maier P, Schier T, Steiger A. Galanin has REM-sleep deprivation-like effects on the sleep EEG in healthy young men. J Psychiatr Res 1997; 33:225–32. [54] Murck H, Guldner J, Colla-Müller M, et al. VIP decelerates nonREM-REM cycles and modulates hormone secretion during sleep in men. Am J Physiol 1996; 271:R905–11. [55] Murck H, Held K, Ziegenbein M, Künzel H, Holsboer F, Steiger A. Intravenous administration of the neuropeptide galanin has fast antidepressant efficacy and affects the sleep EEG. Psychoneuroendocrinology 2004; 29:1205–11. [56] Obál F Jr, Alföldi P, Cady AB, Johannsen L, Sary G, Krueger JM. Growth hormone-releasing factor enhances sleep in rats and rabbits. Am J Physiol 1988; 255:R310–6. [57] Obál F Jr, Alt J, Taishi P, Gardi J, Krueger JM. Sleep in mice with non-functional growth hormone-releasing hormone receptors. Am J Physiol Regul Integrative Comp Physiol 2003; 284:R131–9. [58] Obál F Jr, Beranek L, Brandenberger G. Sleep-associated variations in plasma renin activity and blood pressure in the rat. Neurosci Lett 1994; 179:83–6. [59] Obál F Jr, Bodosi B, Szilagyi A, Kacsóh B, Krueger JM. Antiserum to growth hormone decreases sleep in the rat. Neuroendocrinology 1997; 66:9–16. [60] Obál F Jr, Fang J, Taishi P, Kacsóh B, Gardi J, Krueger JM. Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat. J Neurosci 2001; 21:2912–8. [61] Obál F Jr, Floyd R, Kapás L, Bodosi B, Krueger JM. Effects of systemic GHRH on sleep in intact and in hypophysectomized rats. Am J Physiol 1996; 270:E230–7. [62] Obál F Jr, Kacsóh B, Alföldi P, et al. Antiserum to prolactin decreases rapid eye movement sleep (REM sleep) in the male rat. Physiol Behav 1992; 52:1063–8. [63] Obál F Jr, Kapás L, Gardi J, Taishi P, Bodosi B, Krueger JM. Insulin-like growth factor-1 (IGF-1)-induced inhibition of growth hormone secretion is associated with sleep suppression. Brain Res 1999; 818:267–74. [64] Obál F Jr, Krueger JM. GHRH and sleep. Sleep Med Rev 2004; 8:367–77. [65] Obál F Jr, Payne L, Kapás L, Opp M, Krueger JM. Inhibition of growth hormone-releasing factor suppresses both sleep and growth hormone secretion in the rat. Brain Res 1991; 557:149–53. [66] Obál F Jr, Payne L, Opp M, Alföldi P, Kapás L, Krueger JM. Growth hormone-releasing hormone antibodies suppress sleep and prevent enhancement of sleep after sleep deprivation. Am J Physiol 1992; 263:R1078–85. [67] Opp MR. Rat strain differences suggest a role for corticotropinreleasing hormone in modulating sleep. Physiol Behav 1997; 63:67–74. [68] Opp M, Obál F Jr, Krueger JM. Corticotropin-releasing factor attenuates interleukin 1-induced sleep and fever in rabbits. Am J Physiol 1989; 257:R528–35.

1528 / Chapter 212 [69] Perras B, Marshall L, Köhler G, Born J, Fehm HL. Sleep and endocrine changes after intranasal administration of growth hormone-releasing hormone in young and aged humans. Psychoneuroendocrinology 1999; 24:743–57. [70] Perras B, Pannenborg H, Marshall L, Pietrowsky R, Born J, Fehm HL. Beneficial treatment of age-related sleep disturbances with prolonged intranasal vasopressin. J Clin Psychopharmacol 1999; 19:28–36. [71] Saper CB, Chou TC, Scammell TE. The sleep switch: hypothalamic control of sleep and wakefulness. Trends Neurosci 2001; 24:726–31. [72] Sassin JF, Parker DC, Mace JW, Gotlin RW, Johnson LC, Rossman LG. Human growth hormone release: Relation to slow-wave sleep and sleep-waking cycles. Science 1969; 165:513–5. [73] Schier T, Guldner J, Colla M, Holsboer F, Steiger A. Changes in sleep-endocrine activity after growth hormone-releasing hormone depend on time of administration. J Neuroendocrinol 1997; 9:201–5. [74] Schüessler P, Uhr M, Ising M, Schmid D, Weikel J, Steiger A. Nocturnal ghrelin levels—relationship to sleep EEG, the levels of growth hormone, ACTH, and cortisol—and gender differences. J Sleep Res 2005; 14:329–36. [75] Seifritz E, Müller MJ, Schönenberger GA, et al. Human plasma DSIP decreases at the initiation of sleep at different circadian times. Peptides 1995; 16:1475–81. [76] Spiegel K, Tasali E, Penev P, Van Cauter E. Brief communication: Sleep curtailment in healthy young men is associated with decreased leptin levels, elevated ghrelin levels, and increased hunger and appetite. Ann Intern Med 2004; 141: 846–50. [77] Steiger A, Guldner J, Hemmeter U, Rothe B, Wiedemann K, Holsboer F. Effects of growth hormone-releasing hormone and somatostatin on sleep EEG and nocturnal hormone secretion in male controls. Neuroendocrinology 1992; 56:566–73. [78] Steiger A, Guldner J, Knisatschek H, Rothe B, Lauer C, Holsboer F. Effects of an ACTH/MSH(4–9) analog (HOE 427) on the sleep EEG and nocturnal hormonal secretion in humans. Peptides 1991; 12:1007–10. [79] Steiger A, von Bardeleben U, Herth T, Holsboer F. Sleep EEG and nocturnal secretion of cortisol and growth hormone in male patients with endogenous depression before treatment and after recovery. J Affect Disord 1989; 16:189–95. [80] Stern WC, Jalowiec JE, Shabshelowitz H, Morgane PJ. Effects of growth hormone on sleep-waking patterns in cats. Horm Behav 1975; 6:189–96. [81] Taheri S, Lin L, Austin D, Young T, Mignot E. Short sleep duration is associated with reduced leptin, elevated ghrelin, and increased body mass index. PLoS Med 2004; 1:e62.

[82] Takahashi Y, Kipnis DM, Daughaday WH. Growth hormone secretion during sleep. J Clin Invest 1968; 47:2079–90. [83] Toppila J, Alanko L, Asikainen M, Tobler I, Stenberg D, PorkkaHeiskanen T. Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus. J Sleep Res 1997; 6:171–8. [84] Toppila J, Stenberg D, Alanko L, et al. REM sleep deprivation induces galanin gene expression in the rat brain. Neurosci Lett 1995; 183:171–4. [85] Van Cauter E, Kerkhofs M, Caufriez A, Van Onderbergen A, Thorner MO, Copinschi G. A quantitative estimation of growth hormone secretion in normal man: Reproducibility and relation to sleep and time of day. J Clin Endocrinol Metab 1992; 74:1441–50. [86] Vgontzas AN, Bixler EO, Wittman AM, et al. Middle-aged men show higher sensitivity of sleep to the arousing effects of corticotropin-releasing hormone than young men: Clinical implications. J Clin Endocrinol Metab 2001; 86:1489–95. [87] Voderholzer U, Laakmann G, Wittmann R, et al. Profiles of spontaneous 24-hour and stimulated growth hormone secretion in male patients with endogenous depression. Psychiatry Res 1993; 47:215–27. [88] Watts AG, Tanimura S, Sanchez-Watts G. Corticotropinreleasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: Daily rhythms and their interactions with corticosterone. Endocrinology 2004; 145:529–40. [89] Weikel JC, Wichniak A, Ising M, et al. Ghrelin promotes slow-wave sleep in humans. Am J Physiol Endocr M 2003; 284:407–15. [90] Weitzman ED. Circadian rhythms and episodic hormone secretion in man. Annu Rev Med 1976; 27:225–43. [91] Yi PL, Tsai CH, Lin JG, Lee CC, Chang FC. Kindling stimuli delivered at different times in the sleep-wake cycle. Sleep 2004; 27:203–12. [92] Zhang G, Wang L, Liu H, Zhang J. Substance P promotes sleep in the ventrolateral preoptic area of rats. Brain Res 2004; 1028: 225–32. [93] Zhang J, Chen Z, Taishi P, Obál F Jr, Fang J, Krueger JM. Sleep deprivation increases rat hypothalamic growth hormonereleasing hormone mRNA. Am J Physiol 1998; 275:R1755–61. [94] Zhang J, Obál F Jr, Zheng T, Fang J, Taishi P, Krueger JM. Intrapreoptic microinjection of GHRH or its antagonist alters sleep in rats. J Neurosci 1999; 19:2187–94. [95] Ziegenbein M, Held K, Künzel H, Murck H, Antonijevic IA, Steiger A. The somatostatin analogue octreotide impairs sleep and decreases EEG sigma power in young male subjects. Neuropsychopharmacology 2004; 29:146–51.

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213 Peptide Chronomics FRANZ HALBERG, GERMAINE CORNÉLISSEN, EUGENE KANABROCKI, ROBERT B. SOTHERN, ERHARD HAUS, SAMUEL ZINKER, RITA JOZSA, WEIHONG PAN, ROBERTO TARQUINI, FEDERICO PERFETTO, CRISTINA MAGGIONI, AND EARL E. BAKKEN

tropic hormone, ACTH) network (Figs. 1 and 2). Another source of confusion was due to an intergroup difference in the period of rhythms in blinded mice compared with controls that led to the concept of free-running rhythms (Figs. 3 and 4). In each case, information on time structure was indispensable as the control. The means for avoiding such blunders (as shown in Figs. 1C and 3B) while getting a usually better average (Fig. 5) and, in addition to the mean, some dynamic parameters (Fig. 6), the period τ and at each τ an amplitude A, an acrophase φ, and a waveform—the (A,φ) pairs of harmonics of the fundamental τ—and, by quantifying these parameters, to find potentially useful new facts (Figs. 7–9) prompted our acceptance of an invitation to write this review, the limited and fragmentary nature of available time-series for peptides notwithstanding.

ABSTRACT Chronomics, the mapping of broad time structures (chronomes), provides the indispensable control whether studies aim at examining the effect of a given intervention such as dietary restriction, at optimizing the timing of administration of treatment, or at assessing an elevated risk of developing a disease such as cancer. An increase in circadian amplitude exceeding any effect on the mean is likely to yield controversial results from single spot checks. An analog of the adrenocorticotropic hormone (ACTH 1–17), administered in the evening may help patients with rheumatoid arthritis, but may be ineffective when given around awakening. A daytime single sample of prolactin may not be discriminatory in assessing breast cancer risk when large differences are found during the rest span. The merits of assessing peptide chronomics are perhaps best illustrated by the facts that a peptide drug may have a major effect at one circadian stage but not at another, that a major effect on a polypeptide may be exerted again at one circadian stage but not at another, and that, by the design and the software of chronomics, relatively small numbers of patients are needed to validate such effects by inferential statistics.

INTRODUCTION The interpretation and statistical analysis of the data on peptides as time-varying biological functions are nearly always performed without regard for any broad chronome, or time structure consisting primarily of (1) multifrequency rhythms; (2) trends, when the data are long enough; and (3) probabilistic or other chaos, when the data are dense enough [36] (Fig. 10). Purely static interpretations of measurements, often based on a single “snapshot” in the clinic and interpreted without any temporal reference, may not suffice for diagnostic aims and certainly do not suffice for research aims in biomedicine or bioscience more generally. Even today, many physicians rely on blood prolactin or other measurements at one or very few arbitrary time points, although it has long been known that the values vary greatly along several time scales in health and disease,

PREAMBLE In seemingly rigorous tests comparing two concomitantly sampled groups at some to us then-convenient times, we obtained, in our own replications, some very contradictory results. Such puzzles were solved in the early 1950s [36]. One problem was due to an initially unrecognized intergroup difference in phase in a study of the effect of a restriction in dietary calories on breast cancer incidence, possibly involving the hypothalamic-pituitary-adrenocortical (adrenocorticoHandbook of Biologically Active Peptides

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FIGURE 1. Importance of rhythms in assessing intervention effects, notably in relation to stress or allergy. A. Eosinophil counts were found to be lowered by fasting (and/or stress), when a 50% reduction in dietary carbohydrates and fats (with proteins, vitamins, and minerals similar to control group) was fed in the morning to C3H mice, a model in which the naturally high incidence of breast cancer is lowered by a diet reduced in calories (not shown). The result could have been interpreted as an adrenocortical activation and then assessed by eosinophil depression, with applications for treating breast cancer and for prolonging life. Steroids that depress eosinophil cell counts and perhaps mitoses could be a mechanism through which caloric restriction and ovariectomy act in greatly reducing cancer incidence. B. In view of the importance of this finding to the etiology of cancer, results had to be replicated on a larger group of animals. One week later a follow-up study with more animals started at an earlier clock hour. Confusing results were obtained, showing no difference between the two groups of mice. C. After another week, another study starting at an even earlier clock hour yielded opposite results, which could have been interpreted as a response to allergy when considered alone. D. Studies at intervals of a few hours hinted at the reason for the confusion: By sampling at different clock hours, two groups of mice were found to be characterized by a circadian rhythm with different phases. Opposite effects thus became predictable. E. Abstract illustration of two circadian rhythms in antiphase. Differences in opposite direction or no effect are anticipated when sampling at different clock hours. From [36].

Peptide Chronomics / 1531

FIGURE 2. Effect of food restriction on circulating eosinophil counts in mice. Follow-up study on Fig. 1, with a phase difference greatly reduced by offering the restricted diet in the evening. A. Even after log10-transformation of the data expressed as percentage of mean, great interindividual variability is apparent in the raw data. B. Plots of timepoint mean for mice in each group reveal different circadian patterns. C–E. Parameter tests quantify differences, indicating that calorie restriction is associated with a lower MESOR (C–D), a larger circadian amplitude (D–E), and only a slight difference in acrophase (D). The difference in acrophase in this study, where calorie-restricted mice were fed in the evening, is much smaller than the almost antiphase observed in prior studies (Fig. 1A), where calorie-restricted mice were fed in the morning. From [50].

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with age, geography, and lifestyle. Diagnosing gross clinical-chemical pathology based on spot checks at an unspecified time is time-honored but ignores timing. Chronomics can do better [11, 12, 14]. This concluding chapter of the Handbook tries to motivate investigators in the routine applications of methods of regression and time-series analysis to biological peptide series as these are practiced in many other sciences such as time-microscopic chronobiology, physics, engineering, and economics. By the same token, the relationships of chronomic (time-structural) components among several biological variables need not be regarded from a static point of view, as more or less immutable features for a given person and set of conditions. Hence, when statistical methods are involved in data analysis, any time-dependence should usually be

FIGURE 3. Importance of assessing the rhythm’s period. Whereas circadian rhythms are usually synchronized by environmental schedules, they may free-run, for example, in the absence of the eyes, thereby assuming a period that deviates slightly but statistically significantly from precisely 24 h. When free-running occurs, results from two-timepoint studies may yield confusing results. A. This is illustrated for circulating eosinophil counts of mice sampled twice a day, during daytime and nighttime, after sham operation or bilateral optic enucleation. Results from four different (nonconsecutive) days postoperation are shown. Whereas sham-operated mice display a consistent pattern of higher daytime and lower nighttime counts, blinded mice exhibit erratic patterns, the nighttime counts being sometimes higher than, sometimes lower than, and sometimes almost equal to daytime counts. B. Abstract illustration of two components with slightly different periods accounts for the changing relation between the two curves depending on whether they are in or out of phase with one another. From [49].

sought. A store of methods proposed and published over several decades [5, 12, 22, 23, 25] is illustrated further here, in particular for work on short and sparse time-series of peptides, which are commonly used in biomedicine.

AVOIDING BLUNDERS When a curtain of ignorance is drawn over a so-called normal range (Table 1) by ignoring any rules of variation within this range, two circadian, or any other, rhythms with the same frequency may happen to be out of phase or may differ in frequency, and these facts will not be recognized (Figs. 1 and 3). Blunders of interpretation are then unavoidable (Fig. 1), but are readily

Peptide Chronomics / 1533

FIGURE 4. Blinding studies (I) led to the concept of free-running, as shown in chronograms of rectal temperature of sham-operated and blinded mice (I.A), seen to peak earlier and earlier each day in the blinded mice. Accordingly, the circadian acrophases of the blinded mice gradually drift as a function of time whereas those of the control mice remain about the same (I.B). Histograms of best-fitting periods estimated from records of groups of mice show that on the average, the circadian period of blinded mice is slightly shorter than 24 h (I.C). Internal phase relations among several variables are also shown (I.D). The components of the chronome are internally coordinated through feed-sideward, in a network of spontaneous, reactive, and modulatory rhythms (II). Apart from the spontaneous rhythms characterizing functions such as serum corticosterone or melatonin (II.A–B), reactive rhythms are found in response to a given stimulus applied under standardized controlled conditions of the laboratory: the adrenal response to ACTH is a case in point (broken line in II.A). Such response rhythms have been named β-rhythms, the spontaneous rhythms being called α-rhythms, whether they are 24-h or otherwise synchronized. Much controversy can be resolved by studying the effect of the interaction by more than two variables at different rhythm stages. A third entity may modulate, in a predictable insofar as rhythmic fashion, the effect of one entity on the other. Predictable sequences of attenuation, no-effect, and amplification can then be found. A case in point is corticosterone production by bisected adrenals stimulated by ACTH1–17 in the presence vs. absence of pineal homogenate (II. C). Such chronomodulation is also observed for the effect of ACTH1–17 on the metaphyseal bone DNA labeling in the rat (II.D). Some of these multiple entity interactions involve more than one frequency; this is the case for the effect of the immunostimulator cefodizime (HR221) on corticosterone production by the adrenals stimulated by ACTH1–17 (II.E). Chronomodulations involving one or several frequencies are known as γ- or δ-rhythms, respectively. They are part of feed-sidewards, rhythmic sequences of attenuation, amplification, and no-effect by a modulator on the interaction of an actor and a reactor (III).

1534 / Chapter 213 A Datum

Arithmetic Mean

MESOR (More Accurate) Equality of Areas

Rhythmic Function

B SE

SE Mean

MESOR (More Precise)

38.5% Reduction in Standard Error

00:00 Dec 22

Time (Calendar Date), ti 00:00 Jan Feb MarAprMayJun Jul AugSepOctNovDec Dec 22

00:00 Sunday

Sun Mon Tue Wed Thu

Fri

Sat

FIGURE 5. The MESOR usually provides a more accurate estimate of location than the arithmetic mean (A). When sampling is denser near the maximum (or minimum) of a periodic function than at other times, the arithmetic mean is biased toward that extremum, whereas the MESOR tends to be closer to the true average value of the given function (obtained when sampling is equidistant over an integer number of cycles). The MESOR usually also provides a more precise estimate of location than the arithmetic mean, notably when the data are characterized by a large-amplitude rhythm (B). This stems from the fact that the error around the MESOR is estimated after the variability accounted for by the rhythmic behavior of the data has been portioned out.

00:00 Sunday

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240 Period (τ): e.g., 24 hours, 7 days, 1 year Acrophase (ø) “Overfit’’

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FIGURE 6. Illustration of the single-cosinor method. One or several cosine curves with known (or approximately known) periods are fitted by least squares to the data, yielding estimates for the following parameters: the MESOR, a rhythm-adjusted (or rather chronome-adjusted) mean value; the amplitude, a measure of half the extent of predictable change within a cycle; and the acrophase, a measure of the timing of overall high values recurring in each cycle. When a multiple-component model is fitted, estimates of the amplitude and acrophase are obtained for each component. When the different components are harmonically related, the amplitudes and acrophases of the harmonic terms qualify the waveform of the fundamental component, which can in turn be characterized by a magnitude and orthophase, measures of the predictable extent and timing of change of the complex waveform. If the period is not known a priori, nonlinear least squares are needed to estimate M and (A, φ) of each component together with its respective period, the period being defined as the duration of one cycle. The acrophase is usually expressed in (negative) degrees, with 360° equated to the period length and 0° being set to the reference time (such as midnight preceding the start of data collection or the time of light onset in the case of circadian rhythms assessed in laboratory experiments, notably when staggered lighting regimens are used to facilitate the work during office hours without the need for around-the-clock sampling).

Peptide Chronomics / 1535 A

B

FIGURE 7. Circadian stage-dependence of effect of ACTH1–17 on urinary cortisol from patients with rheumatoid arthritis documented in an N-of-5 study, with only one subject assigned to receive the same dose of the same treatment at one of five different circadian stages, 4.8 h apart. Five men, 45–47 years of age, with rheumatoid arthritis, each provided five urine samples in unequal daily fractions (19:30–06:00, 06:00–08:30, 08:30–11:30, 11:30–13:15, 13:15–17:30, and 17:30– 19:30) for 24 h before and after medication. MESOR determined from fit of 24-h cosine curve to each man’s data before and after medication. PR = percent rhythm; P = p value from zero amplitude test. A. ACTH1–17 administered in the evening was effective, but during the night or on awakening the effect was minimal, if any. B. A cosinor plot indicates that the maximal effect occurred on the average around 18:00 and that the circadian stage-dependence of the effect was statistically significant, by the nonoverlap of the pole (center of the plot) by the error ellipse located around the tip of the (A, φ) vector (the pole representing an amplitude of zero or no rhythm). Measures of uncertainties for the amplitude and acrophase of the circadian response can be obtained by drawing concentric circles and radii tangent to the error ellipse, respectively [6]. From [20].

corrected by abandoning the concept of homeostasis, that constitutes this curtain, and by estimating parameters of the underlying rhythms (Figs. 2 and 4). The solution of these puzzles in Minnesota led to chronobiology as a sine qua non to avoid a mistaken interpretation, as in Fig. 1A–C, in which three different conclusions are drawn from a comparison of the same two concomitantly sampled groups only because they were investigated at different, then-convenient times. Unrecognized differences in frequency (Figs. 3 and 4) can also play havoc, yielding faulty interpretations until they are resolved. By contrast, when the rhythms are recognized, a new rule (nomos) of behavior in time (chronos) may emerge as chronomics [11, 31, 36, 37].

CHRONOMICS SPAWNED BY CHRONOBIOLOGY MAPS DYNAMICS Chronobiology is the study of mechanisms underlying chronomes, structures in time (Fig. 10) found in organisms, in populations, and in the environment [16, 36, 38, 39, 42]. The development of chronomics from chronobiology can be compared with that of genomics from genetics. Genetic focus on factors underlying interorganismic diversity in space needs the comple-

mentary chronobiological realization of intraindividual and intrapopulation diversity in time. Genetics led to genomics, the mapping of genomes; chronobiology led to chronomics, the mapping of chronomes in us, which were found to be near-matches of environmental chronomes near and far [16, 36, 38, 39].

END POINTS Computer-based chronomics provides, by cosine fitting, the probability of the occurrence of a cycle with a given anticipated τ length and of its uncertainties, and inferential statistical estimations of the parameters (including 95% confidence intervals for each), the τ (if there are replications of a cycle in the data analyzed); the A, a measure of extent of change; the acrophase, φ, a measure of timing; and the waveform given by the (A, φ) pairs of the harmonics of a given τ. Chronomics also assesses trends, for example, by the coefficients of polynomials fitted to the data. When time-series are dense, with observations numerous enough, nonlinearity and end points such as an approximate entropy or a correlation dimension, preferably analyzed further by curvefitting [8, 74], can also be determined. Trends are usually superposed on cycles, and if the data are long

C

1536 / Chapter 213

A

B

D

FIGURE 8. Circadian stage-dependence of effect of bromocriptine mesylate on circulating prolactin (A–B) and TSH (C–D) of patients with prostatic hypertrophy. Individual responses of patients at one of four different circadian stages are shown on the left. A summary by one-way analysis of variance shown on the right indicates that the treatment was effective in lowering prolactin when given at 1, 13, and 19 h after awakening but not at 7 h after awakening. By the same token, the treatment was more effective in lowering TSH at 13 and 19 h than at 7 h after awakening, and it was not effective at 1 h after awakening. From [87].

A

C

FIGURE 9. Rhythm characteristics can be useful for risk assessment, as illustrated for the case of circannual changes in prolactin and TSH in relation to breast cancer risk. Clinically healthy women at different familial and personal risk of developing breast cancer later in life were sampled around the clock in each of four seasons for the determination of a battery of hormones, including prolactin and TSH. A. A comparison of group means in each season indicates that circulating prolactin is discriminatory in winter but not in spring or summer. There is a large difference in the circannual amplitude of this variable between the two groups, being larger in Japanese women who are at a lower breast cancer risk than in North American women. B. When the circannual amplitude is estimated separately for each woman, it is found to correlate negatively with a breast cancer risk score. A similar analysis reveals that the circannual amplitude of circulating TSH correlates positively with a breast cancer risk score. C. In a separate study of women with or without fibrocystic mastopathy, the circannual rhythm of serum prolactin is again found to be discriminatory, whereas the circadian rhythm in this variable is not.

Peptide Chronomics / 1537

B

1538 / Chapter 213 FIGURE 10. Homeostasis recognizes that physiological processes remain largely within relatively narrow (but hardly negligible) ranges in health and that departure from such normal ranges may be associated with overt disease; and it still serves that purpose. It can be improved on by replacing time-unqualified ranges by time-varying reference limits as prediction and tolerance intervals (chronodesms). It is important to recognize that variability within the normal range is not dealt with as if it were random. The body strives for both seemingly unstructured and structured variation rather than for constancy. Learning about the rules of trends and about rhythmic and chaotic variations that take place within the usual value ranges is not needed for the postulation of a biological clock that enables the body to keep track of time. The fact that single cells and bacteria are genetically coded for a spectrum of rhythmic variation indicates, however, that the concept of clock needs to be extended beyond the year as a calendar and beyond the day as a clock. The concept of a broad chronome (time structure) takes the view that changes occurring within the usual value range resolvable as chronomes, with a predictable multifrequency rhythmic element, measure the essence of the dynamics of everyday life and are essential to obtain warnings before the fait accompli of disease, so that prophylactic measures can be instituted in a timely way. To paraphrase R. L. Stevenson, the world was made before homeostasis and according to slightly different time structures. Inferential statistical methods map chronomes as molecular biology maps genomes; biological chronomes await resolution of their interactions around us, for example, with magnetic storms in the interplanetary field (IMF). * Indicates the master switch; ** indicates several switches, including helio-magnetics.

TABLE 1. Chronobiological Concepts, Tools, and Long-Term Goals.*

Homeostatic Response Physiology [3, 4, 9, 18, 19, 21, 29, 32]

Chronome Physiology [2, 6, 7,10, 13–15, 17, 19, 22, 24, 26, 28–30, 32–35, 41, 44–46, 51, 52, 59, 60, 65, 73, 74, 77–79, 81, 84, 90, 92] Positive: parametric and nonparametric assessment Individualized: P values for statistical significance and for scientific (e.g., clinical) signification Chronomes: consisting of • rhythms • trends • deterministic and other chaos • any residuals and interactions among these

4. Variability 5. Biosystems’ behavior if perturbed (19)

Confounder (foe) Settling down to a steady state (constancy) or limited random hunting, as (mistakenly anticipated) when a single blood pressure is taken after some (≤30) minutes of rest [29, 32]

6. Analogy 7. Physiological or normal ranges of variation

Thermostats with hunting noise Broad, random, indivisible; equated to noise; current standard for diagnosis and treatment

Of interest in its own right (friend) Dynamic chronomes that characterize health within chronobiological limits set by the intermodulation of the chronomes’ α-, β-, γ-, and δ(spontaneous, reactive, and modulating) rhythms (e.g., a large circadian change in blood pressure during ≥24-h bed rest) [14, 29, 32] Pendulums in resolvable chronomes Structured, predictablec; resolved into reference ranges (chronodesms) for chronomes

8. Action?

Confounder elimination; incompatible with detection of circadian blood pressure disorder [35, 81]

3. Interpretation of reality

Monitoring and as-one-goes analyses and, on this basis, action

Time structure: the control in whatever we do Recognizing risk of abnormality before the fait accompli of catastrophe Chronorisk syndromes: • circadian overswinging of blood pressure • chronome alteration with heart rate jitter deficit • circadian rhythm alteration • altered about-yearly rhythms in circulating prolactin and TSH signaling breast and prostatic cancer risk elevation maintaining normal dynamics [10, 24, 35, 41, 74] As a tool and source of informationb Positive individualized quantification of health [14, 24]

Prediction Circadian blood pressure amplitude or circadian standard deviation for detecting effect of in utero exposure to betamimetics [84] Detects treatable overswinging of blood pressure amplitude, which carries a 720% increase in risk of ischemic stroke; improves cancer treatment [26, 29, 46]

Peptide Chronomics / 1539

Negative: absence of abnormality (e.g., of disease)a Population-based: percent abnormality, e.g., morbidity and mortality Putative (imaginary) set points

1. Definition of normalcy (e.g., health) 2. Quantification of normalcy (e.g., health)

Utility of Chronome Physiology

TABLE 1.

9. End points

10. Sources of variation

Original values: casual measurementsd at times of convenience, not necessarily of pertinence (e.g., of the blood pressure with >40% uncertainty in diagnosis in cases of borderline hypertension); time-unspecified: mean and standard error [35]

12. Hierarchy

Exogenous responses to stimuli from proximity mostly from the habitat niche Feedbacks along axes: unstructured modulation such as the deus ex machina in a physiological tragedy because outcomes may be unpredictable Up/down

13. Teleonomy 14. Simplified analogy 15. Biological evolution

Righting and regulation Thermostat Darwinian, externally adaptive

16. Health and environmental care

Medical treatment often limited and late, given mostly after the diagnosis of overt diseasef

17. Animal husbandry, apiculture, aquaculture, and economic entomology 18. Value 19. Seeking inanimate and animate origins

Convenience

11. Mechanism

Often wasteful Stratigraphy for identifying, in geologically analyzed space, sequences in time; radiocarbon dating

Chronome Physiology [2, 6, 7,10, 13–15, 17, 19, 22, 24, 26, 28–30, 32–34, 41, 44–46, 51, 52, 59, 60, 65, 73, 74, 77–79, 81, 84, 85, 90] Time-specified chrones in chronomes, time-coded: • original values • standard deviations (e.g., 6-h, 24-h) • MESOR(s) • amplitude(s) • acrophase(s) • chaotic dimensions • residuals • periods • waveform(s) • trends Endogenous and exogenous: responses to stimuli from near and far, including cosmos [7, 14, 17, 28, 34, 52, 78] Feed-sideward in networks with alternating outcomes: predictable (insofar as rhythmic) as a chronomodulation [14, 24, 28, 44, 45, 79] Collateral: alternating primacy among intermodulating multifrequency rhythms in chronomes Anticipatory, preparatory coordination Pendulum More and more internal and integrative while externally adaptive to both nature and nurture Optimization according to marker chronomes (of interventions by drugs and/or devices, e.g., pacemakers, with diagnosis and treatment refined by narrowed reference range and assessment within that range of chronorisk leading to preventive treatment timed by marker rhythms (that also serve to validate effect) [10, 14, 51, 73, 92] Chronome-basedg Cost-effective Additional tracing of chronomo-ontogeny and chronomo-phylogenyh in the context of glimpses of cycles in corresponding spans of a figurative cosmo-ontogeny [7, 17, 28, 52, 78]

Utility of Chronome Physiology Chronobiological software: • provides information (e.g., on interpretation of reality and on variability) • guides timed treatment that has greatly prolonged the survival of cancer patients [26, 30, 46, 59]

Resolution of impact of storms in space on myocardial infarctions on Earth: space weather report?e Predictable because rhythmic neuroendocrino-vascular intermodulations can account for outcomes that may be as different as stimulation vs. inhibition of immunity [14] Focusing on selected tasks at different times Greater flexibility Instrumented self-help For example, catastrophic and iatrogenic disease prevention [14, 84]

Optimization: greater efficacy; fewer undesirable effects Waste reduced Adds to knowledge of the past to better optimize the future

1540 / Chapter 213

Homeostatic Response Physiology [3, 4, 9, 18, 19, 21, 29, 32]

(Continued)

20. Life in the scheme of physical and cultural things

Survival of the fittest with humans dominating food chains viewed in the perspective of bioenergetics in a mostly terrestrial ecology

Physically and socially chronomodulating and thus informatively and integratively evolving biota molded by human culture; homo not only faber but cosmoinformans and chronomodulans in a budding broad chronocosmoecology [7, 14, 17, 28,34, 52, 78]

21. Investigator satisfaction

Frustrating work when (without specification of chronobiological timing, even at the same clock-hours) we get confusing, obscuring, or even opposite results from the same intervention

Sheer fun: long-standing controversy is resolved by accounting for both the genetic and broadly environmental bases of the feed-sideward among inanimate and animate cycles that constitute life; disease risk recognition promises to lead to the prevention or timed treatment of catastrophic events, such as stroke, cancer, and sudden death

Humans safeguard the integrity of the biosphere as it extends into the cosmos and as we speculatively yet by joining the approaches by ablations, superimposed epochs, and resonance tests concomitantly explore the temporal aspects of our origins, possibly represented by our chronomes that in turn may reflect a long-past environment [7, 17, 28, 34, 78] Increased productivity

Peptide Chronomics / 1541

*Just as contempary physics, by fission and fusion, gathers more and more energy by splitting the atom, biomedicine gathers more and more information by splitting the normal value range into time structures, thereby resolving, e.g., rhythms (fission) and looking at their feed-sideward interrelations (fusion) for a better understanding of our interdigitated, indivisible, Janus-faced inseparable soma and psyche. a Health promotion is a step in the right direction, by its recommendations of attention to diet, exercise, and relaxation, as long as it is then followed by a chronobiological assessment of the effect of recommended procedures rather than merely by the old reliance of ruling out the occurrence of values outside the normal range. b An international womb-to-tomb chronome initiative with aims primarily at stroke and other catastrophic vascular disease prevention by focusing on chronocardiology in general and blood pressure and heart rate dynamics in particular (see the chronobiology home page on the Web at http://msi.umn.edu/~halberg). c Information from the physiological range for prevention, diagnosis, or treatment is much refined when this range is individualized and interpreted in the light of a personalized background as well as in the context of gender, age, ethnicity, and chronome stage specification. d Location and dispersion indices include the determination from histogram of values, of means (arithmetic, geometric, harmonic), median, mode, minimum, maximum, 100% and 90% ranges, interquartile range, standard deviation, standard error; these end points are computed from time-unspecified single values in the context of the homeostatic approach, whereas in the chronobiological framework the location and dispersion indices are used as such on time-specified samples and on time series–derived parameters, that is, on each of the end points (chrones: M, A, φ, [An, φn], etc.) of the chronomes. e The need for forecasting storms in space should be explored further on the basis of systematic studies aligning physiological lifetime monitoring and clinical and archival statistical studies with ongoing physical data collection near and far, both for ascertaining effects and in studying countermeasures [7, 17, 28, 34, 52, 78]. Blood pressure, heart rate, and other physiological and psychological monitoring also provide basic information on any cross-spectral and other associations (feedsideward) within and among biological and environmental chronomes while further providing reference values of medical interest. f Even if some preventive measures have also been long implemented (e.g., by vaccination) and even if recently more and more hygienic measures (such as exercise and caloric, fat, and sodium restriction) are also popular, all can be greatly improved by timing designed with chronobiologic individualization. The alternative, current action based on group results, its unquestionable overall merits notwithstanding, fails to recognize, for instance, that the blood pressure response to salt may differ as a function of circadian stage [60] and that there are indeed individuals in whom the addition of salt lowers rather than raises blood pressure [6]. g Even after the death of a cockroach, when bacteria take over, periodicities (e.g., in oxygen consumption) may not be eliminated but instead continue with increased amplitude. Critical information may be lost by filtering variation deemed to be undesirable because it lies beyond our conventional scope. h Development from the egg of rhythms (some may be much older than shards) and of other constituents of chronomes to trace their homeo- or heterochronically roughly recapitulatory development across species, with both ontogeny and phylogeny, perhaps tracing in their turn the concomitant development of the geocosmic environment [7, 17, 28, 34, 52, 78]. This distant basic goal can be pursued with the immediate reward of obtaining indispensable reference values for the diagnosis of two chronobiological risk syndromes, circadian hyperamplitude-tension (CHAT), and chronome alterations of heart rate variability (CAHRVs), just as an extreme deficit in heart rate jitter [2, 74] associated with an increase in the risk of ischemic stroke or of myocardial infarction of 720 and 550%, respectively.

1542 / Chapter 213 enough, whether in a population or in the individual, they may turn out to be just parts of rhythms with a period τ longer than the data span [42]. Circadecadal cycles characterize individuals (whose vital functions are monitored for up to 3 or more decades). In populations, rhythms, again including circadecadals, are found in end points that are unique for the individual, such as birth and death. Thus, approximately 24-h natality or mortality rhythms can be aligned with decadal cycles in the same end points for a given city or state and still longer waxing and waning cyclicities [37].

SUSCEPTIBILITY RHYTHMS For a few variables, chronomics has already mapped a very broad spectrum of rhythms with τ covering cycles (e.g., in a second, in fractions of a minute, a day, a year, and a decade or still longer spans in populations). In each of these rhythms, which are often if not usually sampled over one or at most a few cycles, such as one or a few days, weeks, or decades, there is a cycle stage-dependent set of responses (Figs. 7 and 8). Not all serial data to be collected can be sampled over many multifrequency cycles; usually the data cannot be collected densely enough to document all aspects of the stage dependence of the effects of stimuli on the cycles involved. Single experiments are usually done along a system time of 24 h or of 1 year or along other time scales; but such sampling suffices to document that timing accounts for the difference between a large, a small, or no response. The importance of these findings for new drug development seems obvious, notably today when peptide drugs are in the forefront [72]. Many a promising molecule may be discarded because it is tested at the wrong time. Testing for timing should precede the regular three phases of drug testing and has, hence, been called a phase-zero design [27].

ATLAS There is a need for an atlas of maps for international use to gain a proper time horizon so that the whole mapped spectrum can be consulted instead of repeating the work of mapping in each study; results can then consider the context of a much broader time horizon that includes, in addition to the rhythm investigated, other cycles already mapped for the same variable. Other cycles that may be present but are not known may lead, because of insufficient sampling, to bioaliasing (Fig. 11). Furthermore, in dense and long time-series, chaos and trends that are organized by rhythms may also be resolved [8, 74].

BEGINNINGS OF OPPORTUNISTIC MAPPING ALONG THE SCALE OF YEARS Chronomics may find associations of certain circannual rhythmic patterns of the polypeptide prolactin with an elevated breast cancer risk (Fig. 9) [41, 86], or it may find that a fixed dose of another peptide such as ACTH acts in one stage of a circadian system but not in another (Fig. 7) [20]. Circadian peptide rhythms, such as those of ACTH [67] and prolactin [41], are well known. An 8-h (Fig. 12) and a 3.5-day cycle of circulating endothelin 1 (ET-1) in human blood have both been reported [58, 85, 88], with 8-h components in other circulating peptides such as substance P and neuropeptide Y (Fig. 13) [68]. In the case of ET-1, extracircadian rhythms are also found in the population densities of endotheliocytes, the cells producing ET-1 [64]. These findings in blood and pinnal ear tissue contrast with earlier findings of circadians in circulating ET-1 [1, 89]. Indeed, some circadian rhythms may undergo changes in frequency with the solar cycle stage to the point that the same variable a few years apart can be primarily circadian [1, 89] or primarily extracircadian [37, 58, 64, 85, 88]. In view of the ubiquity of circadians, which is now widely accepted, a line of thought that allows for their disappearance seems unreasonable, just as a half century ago the opposite, namely the possibility of a circadian rhythmic RNA and DNA synthesis, seemed unreasonable because nucleic acids were then regarded as the most constant material in the organism (for a history, see [36]). Ascertaining the stable versus transient features of the maps here presented is also a task for the future. As yet, there are no densely longitudinally sampled and replicated rhythms of approximately 10, 20, and 50 years mapped for peptides, but a circadecadal was demonstrated for a steroidal metabolic variable coordinated by the peptide ACTH [42]. Table 2 summarizes a candidate circadecadal prolactin and other rhythms [42] (cf. [69, 70, 82]). These rhythms are tentative not only because of the sparsity of sampling but also because each inferentially statistically validated cycle, however ample its underlying sampling, is first mapped as a candidate rhythm and needs a separate confirmation. Approximately 30-day cycles, primarily in women, are complemented by data on circatrigintans in men [14]. Then there are half-yearly cycles, some changing with geographic (geomagnetic) latitude dependence. A circannual rhythm characterizes the (peptide) vasopressincontaining nuclei in the hypothalamus [76]. There is no single peptide variable in which we can quantify, with uncertainties of parameters, ultradians with a τ in the second, minute, or few-hour domain and ∼10-year rhythms, as can be done for heart rate and in

alias (artifact)

real rhythm

Peptide Chronomics / 1543

FIGURE 11. Bioaliasing is defined as the ambiguity or misrepresentation of a periodic component related to inappropriate (too sparse) sampling. Two abstract examples are shown. A. A free-running circadian component with a period of 27.43 hours sampled once a day at the same clock hour each day (to control for the circadian variation) is spuriously seen (aliased) as an 8-day component. B. Another free-running circadian component with a period of 24.8 hours sampled once a day at the same clock hour each day is spuriously seen (aliased) as a 31-day component.

1544 / Chapter 213 A

B

FIGURE 12. Usually the circadian component is prominent in most variables assessed under usual conditions of daytime activity and nocturnal rest. There are only a few, perhaps transient and hence noteworthy, exceptions. In clinically healthy students, (A) endothelin 1 is characterized by an about 8-h component, whereas (B) cortisol in the same blood samples is circadian periodic. Left side, chronograms (time-point means; 95% confidence limits); right side, polar representations (three harmonics with periods of 24, 12, and 8 h). Subjects are two women and seven men, 22–27 years of age. Note that the 8-h component (C) is most significant for ET-1 (p < 0.001) but that it is not detected for cortisol (p > 0.4). From [58].

Peptide Chronomics / 1545 A

100 + 8.24 cos(360t/24 – 29) + 6.25 cos(360t/8 – 157) + 6.56 cos(360t/6 – 278)

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100 + 3.26 cos(360t/12 – 151) + 4.04 cos(360t/8 – 2)

B

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FIGURE 13. Components of approximately 8 h have been documented in clinical health in (A) substance P and (B) neuropeptide Y, together with other components with periods of 24, 12 and 6 h. From [68].

Variable

Units

WN

Monthly

1 2 3 4 5 6 7 8 9

Body temp Pulse Systolic BP Diastolic BP Pulse pressure Mean art press Double product Ocu press—rt Ocu press—lft

°F bts/min mmHg mmHg mmHg mmHg SBPxHR mmHg mmHg

1 2 3 4 5 6 7

Cortisol Insulin LH Melatonin Prolactin T3 uptake T4

μg/dl μIU/ml μIU/ml ng/L ng/ml % μg/dl

Degrees from Solar Ø

N of Data

%R

p

MESOR ± SE

Amp ± SE

(%amp)

Ø

6901–9805

353

77.4

<0.001

72.8 ± 1.4

65.3 ± 1.97

(90)

−36°

69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98 69,79,88,93,98

423 446 439 439 439 439 422 434 434

1.5 6.6 8.8 12.3 5.9 11.6 9.6 1.4 3.9

0.040 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.045 <0.001

Vital signs 97.66 ± 0.07 70.9 ± 0.7 125.4 ± 1.0 83.2 ± 0.7 42.2 ± 0.7 97.3 ± 0.7 8948 ± 137 16.12 ± 0.3 16.02 ± 0.3

0.21 6.3 66.7 7.7 4.8 7.1 1166 0.90 1.12

± ± ± ± ± ± ± ± ±

0.08 0.91 1.52 0.84 0.82 0.96 185 0.37 0.38

(0) (9) (53) (9) (11) (7) (13) (6) (7)

−86° −199° −158° −197° −76° −185° −184° −3° −333°

(−43°, −158°) (−182°, −212°) (−131°, −179°) (−184°, −207°) (−58°, −103°) (−170°, −198°) (−164°,−199°) (−297°, −40°) (−292°, −3°)

−50° −163° −123° −161° −41° −150° −149° −33° −298°

9.58 10.75 9.58 9.83 10.17 9.83 10.58 9.58 9.58

88,93,98 79,88,93,98 79,93,98 79,88,93,98 79,88,93,98 79,88,93 79,93,98

256 369 283 336 368 274 265

2.4 2.3 6.8 11.0 13.8 3.8 5.6

0.046 0.015 <0.001 <0.001 <0.001 0.006 <0.001

Hormones 9.40 ± 0.37 34.02 ± 4.00 10.06 ± 1.24 23.12 ± 1.88 6.68 ± 0.37 29.69 ± 0.22 8.48 ± 0.12

1.15 18.85 8.57 15.99 5.79 0.89 0.92

± ± ± ± ± ± ±

1.62 4.14 1.47 2.51 0.41 0.34 0.12

(12) (55) (85) (69) (87) (3) (11)

−85° −62° −70° −259° −38° −263° −54°

(−36°, −207°) (−38°, −119°) (−53°, −99°) (−241°, −290°) (−30°, −45°) (−237°, −342°) (−39°, −76°)

−49° −27° −35° −224° −2° −228° −19°

spurious? 8.58 10.67 13.66 10.41 5.17 6.66

Study Years

95% Limits

Best Fit (Y)

(−32°, −39°)

a Data from Kanabrocki et al., Hines VA Hospital, Chicago, IL. Single cosinor summary at solar cycle period of 10.42 yrs (91,312.5 h). Blood and vital signs in men, ages 23–77 years, sampled every 3 h for 24 h in May 1979 (n = 13), 1988 (n = 11), 1993 (n = 11) and/or 1998 (n = 11) (some men studied more than once). Vital signs also measured every 3 h in May 14–15, 1969 (n = 13). Sleep and/or rest: 2230–0630 h. LANP = long-acting natriuretic peptide (a.a. 1–30); VSDL = vessel dilator (a.a. 31–67); ANF = atrial natriuretic factor (a.a. 99–126 of the 126 a.a. prohormone). Single cosinor applied to all individual data. Acrophase (Ø) in degrees from reference: Jan. 1, 1969. 95% limits for Ø given if p < 0.05 from cosinor analysis.

1546 / Chapter 213

TABLE 2. Statistical Evaluation of Circadecadal Characteristics for Circulating Hormones and Vital Signs in Men and in Wolf’s Relative Sunspot Numbers (WN)a.

Peptide Chronomics / 1547 the infradian and circadian range for 17-ketosteroid excretion. Mapping for circulating prolactin is more limited in extent. The need for mapping is introduced today to show that we should seek proper reference values for tomorrow. Our goal herein is to illustrate how chronomics—the systematic mapping of chronomes (time structures), based on long and dense serial peptide data—can complement genomics and proteomics.

[43] and second by averaging both the original data and those expressed as percentages of the series mean, the latter referred to as relative values. After these time-macroscopic steps, an approach by cosinor follows, which yields parameters with their uncertainties. Maps such as the acrophase map in Fig. 19 are desirable for many peptides not analyzed in this figure and for many frequencies beyond the circadian.

MAPS OF PEPTIDES IN MEN

PEPTIDE CHRONOMICS IN WOMEN

One set of maps of peptide chronomics stems from clinically healthy or symptom-free men studied by one of us (EK) with help by another (RBS) around the clock for 24 h on six occasions (always in May) at 5- to 10-year intervals over a 34-year span (1969–2003) as part of an ongoing study of biological rhythms of more than 140 variables, checking vital signs, blood, plasma, serum, saliva, and urine. The number of subjects and age range per study were:

A Kyushu-Minnesota study involved sampling three age groups of women in Minnesota, each group consisting of two subgroups, one at high and the other at low familial risk of developing breast cancer, and a seventh group of women at low risk in Japan [41]. Two dozen, mostly endocrine, variables were sampled around the clock, at four stages of the menstrual cycle in the two menstruating groups in four seasons. From this international study, Fig. 20 shows, for circulating prolactin, 3-hour means of determinations at 20-minute intervals; by simply computing these averages, probable geographic, ethnic, and other differences emerge and should be kept in mind by those concerned with reference values. Note in Fig. 20A that there were peaks by night in the Japanese women above 50 ng/ml and hardly more than half that value in the Minnesotans. But Fig. 20A also shows a very marked within-day variation, which in this case overwhelms all other sources of variability. With harmonic interpolation, a second peak in the late afternoon can also be found for the Japanese subjects and has been discussed in the context of the literature [41]. We emphasize that the smooth curves describe only results from a given season. But first, let us point out the errors that can occur if such rhythms are ignored, as shown in Fig. 20C. This dense (20-min) sampling suggests both a larger circadian amplitude and a larger rhythm-adjusted, or rather chronome-adjusted, mean or MESOR of the circadian rhythm in blood plasma prolactin in Japanese as compared with North American women (p < 0.002 from Hotelling T 2 test). Judged on the basis only of daytime samples, erroneous conclusions may be drawn, as noted in this figure. Measures of the timing of circadian prolactin rhythms in the form of vectors, whose length corresponds to amplitude and whose direction indicates acrophases (timing) are shown in Fig. 20D. Circadian acrophases of plasma prolactin of adolescent, young adult, and postmenopausal women from Japan and Minnesota in each of the four seasons show a relatively high degree of synchronization. The vectorial cosinor summary achieves at a glance, objectively and quantitatively, what a display of all original data cannot readily convey.

Study Study Study Study Study Study

1: 2: 3: 4: 5: 6:

n n n n n n

= = = = = =

13, 14, 11, 11, 10, 10,

ages ages ages ages ages ages

22–27 23–51 41–60 46–72 52–77 57–81

years years years years years years.

Four of the original subjects participated in each of the five studies. During the study, participants were free to move about during the day without napping, with bed rest between 11:00 p.m. and 7:00 a.m., being briefly awakened for the 1:00 and 4:00 a.m. samplings. Meals were served at 4:30 p.m., 7:30 a.m., and 1:30 p.m. No food or liquids, except for water, were allowed between meals. Measurements (8–9 per subject) were obtained at 3-hour intervals beginning at 7:00 p.m., with subsequent sampling beginning at 10:00 p.m., 1:00 a.m., 4:00 a.m. 7:00 a.m., 10:00 a.m., 1:00 p.m., and 4:00 p.m. (and at 7:00 p.m. in Study 2). At each test time, subjects voided their bladder for a urine sample, followed by the measurement of their vital signs before drawing of blood samples. Because variables were added or eliminated from the study over the years, not every variable was measured in each study or in each subject. Therefore, as few as 10 and up to 46 time-series for some of the variables are available for analyses, yielding, as noted, the candidate rhythms in Table 2. The same study, however, also yielded replications, as summarized in Figs. 14–19 [61–63, 83, 91]. Changes with age notwithstanding, we are making a transition from candidate to replicated rhythms, first by the simple but very useful data transformation as percentages of the series mean

220

A: Circadian Profile by Study Year: µg/dl

150

B: Circadian Profile by Study Year: percent of mean

GASTRIN KEY 1 3 4

185

Study Year 1 1979 3 1993 4 1998

140 130

150 14

1

4

1 4

115

1

4 1

80

1

4

1

3 4

1

3

100

4

1 13

4 3

1

90

3

45

3

3

22

01

3

3

3

3

3

3 80

1 34

4

14 3

4

0

0 19

04 07 10 Time (Local clock Hour)

KEY 1 3 4

#Subj 1 3 4

13 11 11

%R

p

MESOR ± SE

50.7 0.015 53.6 0.003 50.7 0.079

13

16

19

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR

150

4

1

4

1

120 110

4

1

34

107.1 36.3 105.5

Amplitude ±SE

32.3 2.3 51.6

13.7 7.1 31.6

KEY 1 3 4

21:30h 19:12h 19:00h

C: Overall Circadian Profile: µg/dl

01

04 07 10 Time (Local clock Hour)

135

130

125

110

115

90

105

70

95

50

85

1 3 4

#Subj 13 11 11

p %R 50.7 <0.001 53.6 <0.001 50.7 <0.001

13

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR

Acrophase

9.0 1.4 17.5

22

MESOR ± SE 99.1 0.14 100.2 0.04 100.3 0.06

Amplitude ±SE 8.4 1.7 18.9 2.8 24.4 3.7

Acrophase 20:32h 19:04h 19:40h

D: Overall Circadian Profile: percent of mean

Timepoint Mean±SE

Best-fitting 24h Cosine

0

0 19

22

01

04 07 10 Time (Local clock Hour)

#Subj 35

%R

p

51.6 0.028

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR KEY

13

MESOR ± SE

Amplitude ±SE

84.4

16.5

20.3

6.0

Acrophase 19:45h

19

22

01

04 07 10 Time (Local clock Hour)

#Subj 35

p %R 51.6 <0.001

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR KEY

13

MESOR ± SE 99.8 0.11

Amplitude ±SE 1.9 16.6

FIGURE 14. Gastrin was measured around the clock for 24 h in 11–13 men, followed-up longitudinally in three studies in 1979, 1988, and 1993. A. There is great variability in timepoint means from one study year to another. B. This variability can be reduced by expressing the data as a percentage of each series’ mean value. C. Averaging across studies at each circadian stage is associated with large error bars obscuring the circadian variation. D. The rhythm comes readily to the fore when averaging the relative values at each circadian stage. From studies by E. Kanabrocki.

Acrophase 19:37h

Peptide Chronomics / 1549 32

A: 1979

36

64

AF23 RG33 J034 LT34 DL36 ML36 RM36 SB37 TM38 IS44 JP51 LS58

28

24

20

32

28

24

20

16

16

12

12

8

8

4

4

0 32

19

22

01

04

07

10

13

16

C: 1993

19 RS46 RG47 JO48 GK50 DL50 RM50 BZ50 IS58 RB65 EK71 LS72

28

24

20

0 32

subj,age

19

22

01

04

07

10

13

24

20

16

12

12

8

8

4

4

16 RG52 JO53 DL55 ML55 RM55 BZ55 IS63 RB70 JG72 EK76 LS77

D: 1998

28

16

0

RS41 RG42 JO43 DL45 ML45 RM45 BZ45 IS53 SB46 TM47 RB60

B: 1988

0 19

22

01

04

07

10

13

16

19

22

01

04

07

10

13

16

Sampling Time (Local Clock Hour)

FIGURE 15. Large interindividual variability in the circadian pattern of serum prolactin in men followed-up longitudinally in four studies conducted in 1979 (A), 1988 (B), 1993 (C), and 1998 (D). Key shows subject ID and age; dark horizontal bar indicates sleep or rest. From studies by E. Kanabrocki.

1550 / Chapter 213 A: Circadian Profile by Study Year: ng/ml 25

B: Circadian Profile by Study Year: percent of mean 200

PROLACTIN KEY

21

1 2 3 4

1 1

1 2 3 4

Study Year 1979 1988 1993 1998

175

17

150

13

125

1

1

3 2 4

3 2 9

5

23

1 2 3

1 3 2

4

4

4

4

1

4 1

3

100

1 1

24 234

2 34

1 2 34

75

2 3 4

2

3 2 4 1

1

24 4 1 2 3

4 12

13

3

0

0 19

22

01

04 07 10 Time (Local clock Hour)

KEY #Subj 1 1 13 2 2 11 3 3 11 4 4 11

%R 58.4 45.5 52.7 57.2

p 0.006 0.005 0.003 0.064

13

16

19

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR

15

3

2 34

MESOR ± SE 11.5 1.1 6.8 0.8 7.2 1.0 5.6 0.4

Amplitude ± SE 6.1 1.5 0.4 2.0 0.6 3.1 1.4 0.5

22

01

04 07 10 Time (Local clock Hour)

#Subj KEY 1 1 13 2 2 11 3 3 11 4 4 11

C: Overall Circadian Profile: ng/ml

%R

p

58.4 <0.001 45.5 <0.001 52.7 0.001 57.2 0.038

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR

Acrophase 03:20h 02:50h 03:38h 03:44h

13

MESOR ± SE 102.7 0.8 99.8 0.04 99.0 0.9 2.5 103.8

Amplitude ± SE 51.0 28.5 43.4 26.8

Acrophase

03:12h 02:39h 03:31h 02:59h

8.7 4.0 7.3 8.7

D: Overall Circadian Profile: percent of mean 170

13

150

11

130

9

110

7

90

5

70

0

0 19

22

01

04 07 10 Time (Local clock Hour)

POPULATION-MEAN COSINOR p #Subj %R KEY MESOR ± SE 46 53.7 <0.001 8.0 0.6

13

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours Amplitude ± SE 3.3

0.5

Acrophase 03:22h

Timepoint Mean±SE Best-fitting 24h Cosine 19

22

01

04 07 10 Time (Local clock Hour)

#Sub 46

%R

p

53.7 <0.001

16

REF. TIME: 00:00h FITTED PERIOD = 24.0 hours

POPULATION-MEAN COSINOR KEY

13

MESOR ± SE 101.4

0.7

Amplitude ± SE 37.9

3.9

Acrophase 03:09h

FIGURE 16. A. Average circadian profiles of serum prolactin in men studied in 1979, 1988, 1993, and 1998 show large variability. B. This interstudy variability is greatly reduced by expressing the data as a percentage of each series’ mean value. C. The marked overall circadian variation apparent from the averaged data. D. It is even more prominent in the relative data. From studies by E. Kanabrocki.

A

B

24h Acrophases

(Age,ID,Yr)

Polar Plot

00

166

23AF79 33RG79 34JO79 34LT79 36DL79 36RM79 36ML79 37SB79 38TM79 41RS88 42RG88 43JO88 44IS79 45DL88 45RM88 45ML88 45BZ88 46SB88 46RS93 47TM88 47RG93 48JO93 50DL93 50RM93 50BZ93 50GK93 51RB79 51JP79 52RG98 53IS88 53JO98 55DL98 55RM98 55ML98 55BZ98 58LS79 58IS93 60RB88 63IS98 65RB93 70RB98 71EK93 72LS93 72JG98 76EK98 77LS98

00

C

Circadian Waveform

03

21 148 52

% of Mean

130

Double Amplitude (%)

26 06

18

112

Best-fitting 24h cosine

94

15

76

09

Timepoint Mean±SE 12

0 03

06

09 12 15 TIME (Clock Hour)

18

21

00

19

22

01 04 07 10 Time (Local Clock Hour)

13

16

Peptide Chronomics / 1551

FIGURE 17. Circadian rhythm in circulating prolactin in men. A. Individual and group 24-h acrophases. B. Chronogram with best-fitting 24-h cosine. C. Polar plot. The large-amplitude circadian variation in serum prolactin of men has a relatively stable acrophase when assessed individually, clustering during the night. The polar display summarizing the results by population-mean cosinor shows the statistical significance of the circadian rhythm and the relatively small uncertainties for the average amplitude-acrophase pair by the small error ellipse away from the pole (right). Blood samples every 3 h for 24 h in May 1979, 1988, 1993, and 1998. Individual data converted to percentage of mean for the group analysis. Black dots indicate individual φ from single cosinor; vertical shaded area indicates 95% confidence limits of group φ from population-mean cosinor in (A); joint amplitude and 95% confidence region shown in (C); dark horizontal bars indicate sleep or rest. From studies by E. Kanabrocki.

1552 / Chapter 213 (Age,ID,Yr)

A: 24h Acrophases

41RS88 43JO88 45BZ88 45DL88 45ML88 45RM88 46RS93 46SB88 47RG93 47TM88 48JO93 50BZ93 50DL93 50GK93 50RM93 52RG98 53IS88 53JO98 55BZ98 55DL98 55ML98 55RM98 58IS93 60RB88 63IS98 65RB93 70RB98 71EK93 72JG98 72LS93 76EK98 77LS98

00

119

107

95

83

0 03

06

09

12

15

18

21

00

19

22

01

04

07

10

13

16

13

16

13

16

142

Serum ANF31-67

131

120

Best-fitting 24h cosine

109

98

Timepoint Mean±SE

87

0 03

06

41RS88 43JO88 45BZ88 45DL88 45ML88 45RM88 46RS93 46SB88 47RG93 47TM88 48JO93 50BZ93 50DL93 50GK93 50RM93 52RG98 53IS88 53JO98 55BZ98 55DL98 55ML98 55RM98 58IS93 60RB88 63IS98 65RB93 70RB98 71EK93 72JG98 72LS93 76EK98 77LS98

00

B: Circadian Waveform (% of mean)

131

41RS88 43JO88 45BZ88 45DL88 45ML88 45RM88 46RS93 46SB88 47RG93 47TM88 48JO93 50BZ93 50DL93 50GK93 50RM93 52RG98 53IS88 53JO98 55BZ98 55DL98 55ML98 55RM98 58IS93 60RB88 63IS98 65RB93 70RB98 71EK93 72JG98 72LS93 76EK98 77LS98

00

143

Serum ANF1-30

09

12

15

18

21

00

19

22

01

04

07

10

19

22

01 04 07 10 Time (Local clock Hour)

144

Serum ANF99-126

132

120

108

96

84

0 03

06

09 12 15 TIME (Clock Hour)

18

21

00

FIGURE 18. Circadian variations in atrial natriuretic peptides in men. A. 24-h acrophases. B. Circadian waveforms. The prominent circadian variation in atrial natriuretic peptides (ANF1–30, top; ANF31–67, middle; and ANF99–126, bottom) has consistent individual acrophases clustering during the night. Blood samples every 3 h for 24 h in May 1988, 1993, and 1998. Individual data converted to percentage of mean for the group analysis. Black dots indicate individual from single cosinor; vertical shaded area indicates 95% confidence limits of group from populationmean cosinor; dark horizontal bars indicate sleep or rest. From [91].

Peptide Chronomics / 1553 Variable (N series) ANF1-30 (32) ANF31-67 (32) ANF99-126 (32) Cortisol (33) C-Peptide (11) Gastrin (35) Insulin (46) Melatonin (42) Prolactin (46) Renin (11) T3 Uptake (33) Testosterone (22) TSH (46) 00

03

06 09 12 15 18 Sampling Times (Clock Hour)

Fig. 21A compares plasma prolactin in clinically healthy Japanese and Minnesotan women in four seasons. Samples taken at 20-min intervals over a 24-h span in Fukuoka, Japan, and Minneapolis, Minnesota, are shown. The difference in plasma prolactin between clinically healthy Japanese and Minnesotan women is seen again in a replication of part of the study in March 1978 (15 Whites of mixed ethnic background, 18–24 years old, in Minnesota and 20 Japanese, ∼20 years old, in Kyushu, Japan.) Note how time-macroscopy by a chronogram (Fig. 21B) is complemented by time-microscopy in a cosinor (Fig. 21C). The 95% error ellipses do not overlap with the circadian amplitude in nanograms per milliliter; the values are 5.4 (4.2–9.6) for the Minnesotans and 19.3 (12.1; 26.8) for the Japanese.

BREAST CANCER RISK ASSESSMENT Circannual chronopathology is introduced in Fig. 9. An obliteration of the circannual, but not of the circadian, rhythm in prolactin is seen by the overlap of the center of the graph (the pole) by the 95% error ellipse for data on fibrocystic mastopathy (FM) as compared with clinical health (H) for women sampled in Florence, Italy. Although the circadian amplitude (expressed in nanograms per milliliters or as percentage of MESOR) is not affected in the presence of benign breast disease, the circannual amplitude is drastically reduced. The alteration of a circannual prolactin

21

00

FIGURE 19. Circadian acrophase chart of different hormones determined in serum of men studied longitudinally in 1979, 1988, 1993, and 1998. Each dot indicates the timing of overall high values recurring each day, along with a measure of uncertainty (horizontal bars). It can be seen that TSH, melatonin, prolactin, ANP, and T3 uptake peak during the rest span. They are followed by cortisol and testosterone peaking shortly after awakening. C-petide, insulin, and gastrin peak later during the day, as does renin, which has a much wider 95% confidence interval of its circadian acrophase indicating a greater uncertainty. From studies by E. Kanabrocki.

rhythm may be a harbinger of increased breast cancer risk in Europe as in the United States. A negative correlation between the total relative breast cancer risk evaluated from epidemiologic criteria and the circannual prolactin amplitude seems to corroborate the finding that an elevation of breast cancer risk is associated with a decrease in circannual amplitude (based on the least-squares fit of a 365.25-day cosine curve to circadian MESORs assessed in each of the four seasons) (see Fig. 9). Note further the positive correlation between epidemiologically-assessed breast cancer risk and the circannual amplitude of thyroidstimulating hormone (TSH). Clinical hypothyroidism has empirically been associated with breast cancer risk. The circannual stage-dependence of the correlation seems pertinent and may prove to be useful for treating an elevated cancer risk, rather than the disease, to the extent that the interaction of rhythms with different frequencies can be untangled.

PROSTATE CANCER RISK ASSESSMENT In prostate cancer (a condition characterized by geographic differences in morbidity and mortality similar to those of breast cancer), the extent of circannual variation also changes as a function of risk and/or cancer. In blood sampled with serial independence as to subjects in the morning at different times of the year, a prominent circannual rhythm in TSH of healthy

B

C

D

FIGURE 20. Circadian variation of circulating prolactin in clinically healthy women at different risk of developing breast cancer (in Minnesota and Japan) sampled in winter. A. Three-hour averages. B. Smoothed curves obtained by harmonic interpolation. C. Superposition of the fitted 24-hour cosine curves indicates a lack of difference between the two groups of women during the daytime when a spot check is most likely to be obtained. A difference is only observed during the night when spot checks are unlikely to be obtained. D. The consistency of the circadian acrophase of circulating prolactin among different women sampled in different seasons is apparent from the polar displays. From [41].

1554 / Chapter 213

A

Peptide Chronomics / 1555

A

B

C

FIGURE 21. A. Circadian profiles of circulating prolactin in 42 clinically healthy women at different risks of developing breast cancer in Minnesota and Japan are compared in each season (1976–1977). Results from this longitudinal study are replicated in winter in another transverse study (March 1978; 15 North American women, 18–24 years of age, and 20 Japanese women, ∼20 years of age). B. Chronograms. C. Polar display. From [41].

1556 / Chapter 213 subjects at low risk of prostate cancer is lost in prostate cancer, and even in men at high risk of prostate cancer (Table 3). For prolactin, a circannual rhythm becomes demonstrable in the case of prostate cancer, although it is not demonstrable with serially-independent sampling in healthy men of low or high prostate cancer risk (Table 4) [41, 80]. Thus, TSH and prolactin show opposite behavior along the 1-year scale in cancers of both the breast and prostate (rather than responding in the same fashion, as is the case along the scale of minutes to hours, following the application of stimuli such as thyrotropinreleasing hormone, TRH). It is also noteworthy that the circannual relations of plasma TSH and prolactin to the risk of breast cancer are opposite with respect to prostate cancer (and as noted, opposite to each other in each cancer).

DISCUSSION The correlations shown in Fig. 9 are just part of a large correlation matrix. Moreover, by the use of Monte

Carlo methods, Ramon Hermida has greatly advanced chronome assessment more broadly [53–57]. The circumstance is also noted that correlations emerged as statistically significant for the very hormones that clinicians have long considered had some relation to breast cancer yet thus far could not rigorously establish such a relation as biologically significant, perhaps because of too-limited sampling. Although the conclusions herein rest on large samples, they describe only a small number of subjects. Moreover, all the conditions required to apply a linear regression between two variables are not satisfied. A test for lack of fit indicates that the model is not adequate for TSH; the error term is not normally distributed. In addition, the assumption of homogeneity of variance is not verified for prolactin or for TSH. In the case of prolactin, there seems to be an age effect on both the circannual amplitude (it decreases with age) and the breast cancer risk (it increases with age). This may contribute to the negative correlation illustrated in Fig. 9; hence, the correlations in the figure are of ordering rather than documenting value. They are intended to emphasize that circannual rhythmicity

TABLE 3. Chronomics: Obliteration (Desynchronization) of Circannual Thyroid-Stimulating Hormone Rhythmicity with Risk of Prostatic Cancer or the Actual Disease.a

Controls

Kind

Criterion

N of Subjectsb

P Valuec

Low

Family history negative Absence of known BPHd Family history positive Presence of known BPHd

244 257 11 41 149

0.003 0.001 0.670 0.268 0.475

High Cases

Adapted from [80] Schuman LM, Mandel JS, Radke A, Seal US, Halberg F., In: Magnus K, ed. Trends in Cancer Incidence: Causes and Practical Implications. Washington, D.C.: Hemisphere; 1982. p. 345–353. b Serially-independent sampling. c From tests of zero-amplitude assumption. d BPH: Benign Prostatic Hypertrophy. a

TABLE 4. Chronomics: Induction (Synchronization) of Circannual Prolactin Rhythmometry with Risk of Prostatic Cancer or the Actual Disease.a

Controls

Kind

Criterion

N of Subjectsb

P Valuec

Low

Family history negative Absence of known BPHd Family history positive Presence of known BPHd

200 217 9 29 124

0.665 0.784 0.559 0.495 0.040

High Cases

Adapted from [80] Schuman LM, Mandel JS, Radke A, Seal US, Halberg F., In: Magnus K, ed. Trends in Cancer Incidence: Causes and Practical Implications. Washington, D.C.: Hemisphere; 1982. p. 345–353. b Serially-independent sampling. c From tests of zero-amplitude assumption. d BPH: Benign Prostatic Hypertrophy. a

Peptide Chronomics / 1557 deserves further study in relation to carcinogenesis. If such correlations can be confirmed and if the circannual rhythms involved prove to be determinants of carcinogenesis in the human breast, these same correlations will point to the possibility of a chemoprevention of breast cancer. But in this same context, the new finding of spectral components longer than a year deserves emphasis [39]. Such components are the transyears; some are the near-transyears, exceeding the 1-year length by only a few weeks; and others are far-transyears, exceeding the year length by a few months. Both transyear and far-transyear patterns are found in 17-ketosteroid excretion, a variable influenced by peptides, among other variables, but not yet mapped for peptides. Should they be found in prolactin, they will qualify a stepwise theoretical reconstruction of a partial spectral structure of human plasma prolactin (Fig. 22A). For a group of Japanese women, it is based on parameter estimates obtained from two separate least-squares fittings of cosine curves with periods of 24 h (circadian), 28 days (circatrigintan), and 365 days (circannual) to data on plasma prolactin obtained every 20 min for 24 h, four times a year on a few women. This purely didactic example models the interaction of human plasma prolactin rhythms with different frequencies, including a modulation of the circadian MESOR and amplitude by circatrigintan and circannual rhythms. Fig. 22B is similarly qualified. It is also a theoretical reconstruction of a partial spectral structure of human plasma prolactin in three age groups, based on parameter estimates obtained from separate least-squares fittings of cosine curves with periods of 24 h (circadian), 28 days (circatrigintan), and 365 days (circannual) to data on plasma prolactin obtained every 20 min for 24 h, four times a year on a few women. This purely didactic example is also modeled on data collected in the Japan-Minnesota investigation [41]; it suggests age-related differences in the interaction of human plasma prolactin rhythms with different frequencies, including the circadian MESOR and the amplitude both modulated by circatrigintan rhythms (except for postmenopausal group) and circannual and probably many other rhythms, some of them probably transannual ones. Theoretical reconstructions, as yet, ignore the occasionally nonsinusoidal waveform, requiring multiple cosine fits that may lead to the objective quantification of a circadian rhythm’s waveform; sometimes more than a single cosine may be required for a given waveform, as in the case of cortisol in human circulating blood (Fig. 12) or in the case of a variable from the crayfish, Cherax quadricarinatus (Fig. 23) [71] or of a rodent, Mus musculus (Fig. 24) [75]. Harmonic terms may also contribute to the difference in the circadian pattern

between health and disease, as suggested in Fig. 25 for the case TNF-α compared between female patients with multiple sclerosis not treated with interferon (IFN) and healthy men, a circumstance that still requires inquiry into a possible gender difference. Apart from the waveform and the trends with age, shown for circulating prolactin of both men and women, there is also probabilistic chaos. A chaotic regime has been proposed in accounting for a modulation by neuropeptides of information processing at the level of a single neuron [66]; apart from modeling, the use of algorithms for the analysis of probabilistic chaos in time-series collected with appropriate density and length will constitute another challenge for students of peptide dynamics.

CONCLUSION Alchemy seemed to be less complex than chemistry; nonetheless, we are now in an age of clinical and other chemistry and automation runs smoothly. We successfully diagnose gross deviations from a normal range of peptide values with time-unspecified samples! These spot checks are the routine of our day for sampling blood for peptides in the clinic (cortisol is a nonpeptide exception) and in research. The task of determining budding chronodesms, time-specified tolerance and prediction intervals [47, 48], not only for interpreting single values but also for time-structural characteristics, still lies ahead for peptides. But biological variability has to be known, not only to reduce the width of the reference interval for single samples beyond that achieved by reducing the sources of technical variation, and not only because what may be acceptable at one time can be unacceptable at another (Fig. 26) but also to avoid blunders and, equally important, to make new discoveries. A great opportunity for applications of peptide chronomics lies in the pharmaceutical industry. Figure 7 shows that a peptide drug, at a fixed dose, acts at one time but not at another. Figure 8 shows that another drug’s action on a polypeptide, again prolactin, also depends on the timing of its administration [87]. How many useful potential drugs are lost, and how many effects on peptides go unrecognized, because timing is ignored? There are reportedly 720 candidates in the global peptide market in various stages of development; 44% have not yet reached the stage of advanced clinical trials [72]. For all of them, timing will be important, and the earlier the stage of development of the drug, the greater is the promise of testing its timing. It is not commonly realized that distributing six test animals over six circadian test times (or five patients over five equidistant test times,

B

FIGURE 22. A. Apart from the prominent circadian variation in human circulating prolactin, the study revealed other components, notably about-monthly and about-yearly variations in MESOR and circadian amplitude, as well as age trends. B. Simulations based on these results. From [41].

1558 / Chapter 213

A

Peptide Chronomics / 1559 A 35

RPCH (cDNA in pixels/mg tissue)

30

25

12.1 + 3.45.cos(2πt/24-4.28) + 3.00.cos(2πt/8-4.36) PR = 37%, P = 0.003

20

15

10

5

0 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00

Time (clock hours; LD regimen)

B 35

RPCH (cDNA in pixels/mg tissue)

30

25

15.2 + 4.54.cos(2πt/24-4.28) + 3.98.cos(2πt/8-4.14) PR = 75%, P = 0.002

20

15

10

5

0 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00

Time (clock hours; DD regimen)

FIGURE 23. Circadian pattern of red-pigment-concentrating hormone (RPCH) mRNA as cDNA in crayfish. A. In 12 h of light alternating with 12 h of darkness. B. Continuous darkness. In addition to the circadian variation, an approximately 8-h component is detected with statistical significance, contributing to the circadian waveform under both conditions. Data from [71].

1560 / Chapter 213 24 14.14 + 2.41 cos(2πt/24 - 0.91) + 1.64 cos(2πt/12 - 2.53) [PR=44%; P<0.001]

TNF-α (μL/g)

20

16

12

8 00

04

08

12

16

20

24

Time (clock hours) FIGURE 24. Statistically significant circadian rhythm of TNFα uptake by the total spinal cord of adult male CD1 mice is approximated by a two-component model. From [75].

MS patients Healthy men

7

Fitted Models

6

TNF-α (pg/ml)

5

4

3

2

Mean ± SE

1

0 18:00

22:00

02:00

06:00

10:00

14:00

18:00

Time (clock hours)

FIGURE 25. Reduced circulating TNF-α concentrations in female patients with multiple sclerosis not treated with IFN compared to those in healthy men. The apparent difference in timing of circadian variation of TNFα between the two groups needs to be qualified by the failure to reject the zero-amplitude (no rhythm) assumption with the limited sample available for analysis and by the confounding by gender. Data from E. Kanabrocki et al.

Peptide Chronomics / 1561

FIGURE 26. Abstract illustration of chronodesm (time-specified reference limits). When a variable is circadian rhythmic, spot checks are best interpreted in the light of time-varying rather than fixed limits. Single values within the homeostatic fixed limits can either be too low or too high (false negatives) when interpreted by means of the chronodesmic limits that account for the rhythmic variation in the data. By the same token, values outside the homeostatic fixed limits can be acceptable (false positives) once the rhythmic variation is accounted for.

as in Fig. 7) [20] allows the fit of a cosine curve and the detection of optimal timing. A potentially valuable molecule may be lost because it is tested at the wrong time! Peptides in organisms are not constants; they are variables. We can ignore their variability and assume with a putative homeostasis that, like the deus ex machina, the organism is presumed to be able to right itself, irrespective of time. So far so good, in the realm of philosophy. The trouble arises when the investigator of a putative homeostasis also assumes that the mechanism of righting assures relative constancy in a timeinvariant way and hence undertakes a study at some convenient but unspecified and not necessarily pertinent time. Fig. 1, dealing with a partly ACTHmediated effect, shows the problem. Some solutions have been outlined [36]. Indeed, the alternative to a putative homeostasis (Table 1) is to study the dynamics in the normal range and thus eventually to treat risk elevation rather than actual disease, whether we are dealing with severe vascular disease [35], cancer [41], or the basic science of our relation to our cosmos [38, 39].

Acknowledgments Support was provided by the U.S. Public Health Service (GM-13981) (FH).

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1564 / Chapter 213 [74] Otsuka K, Cornélissen G, Halberg F. Circadian rhythmic fractal scaling of heart rate variability in health and coronary artery disease. Clin Cardiol 1997; 20: 631–638. [75] Pan WH, Cornélissen G, Halberg F, Kastin AJ. Functional Genomics of Sleep and Circadian Rhythm. Selected Contribution: Circadian rhythm of tumor necrosis factor-α uptake into mouse spinal cord. J Appl Physiol 2002; 92: 1357–1362. [76] Portela A, Cornélissen G, Halberg Franz, Halberg Francine, Halberg J, Hofman MA, Swaab DF, Ikonomov OC, Stoynev AG. Metachronanalysis of circannual and circasemiannual characteristics of human suprachiasmatic vasopressin-containing neurons. In Vivo 1995; 9: 347–358. [77] Reinberg A, Smolensky MH. Biological Rhythms and Medicine: Cellular, Metabolic, Physiopathologic, and Pharmacologic Aspects. New York: Springer; 1983. [78] Roederer JG. Are magnetic storms hazardous to your health? Eos Trans Am Geophys Union 1995; 76: 441, 444–445. [79] Sánchez de la Peña S. The feedsideward of cephalo-adrenal immune interactions. Chronobiologia 1993; 20: 1–52. [80] Schuman LM, Mandel JS, Radke A, Seal US, Halberg F. Some selected features of the epidemiology of prostatic cancer: Minneapolis-St. Paul, Minnesota, case control study, 1976–1979. In: Magnus K, ed. Trends in Cancer Incidence: Causes and Practical Implications. Washington, D.C.: Hemisphere; 1982. p. 345–353. [81] Sheps SG, Canzanello VJ. Current role of automated ambulatory blood pressure and self-measured blood pressure determinations in clinical practice. Mayo Clin Proc 1994; 69: 1000–1005. [82] Soroko SI, Lushnov MS. Effects of long-term variations in cosmic rhythms on human biochemical parameters. Hum Physiol 2004; 30: 72–84. [83] Sothern RB, Vesely DL, Kanabrocki EL, Hermida RC, Bremner FW, Third JLHC, McCormick JB, Dawson S, Ryan M, Greco J, Bean JT, Nemchausky BM, Shirazi P, Scheving LE. Circadian relationships between circulating atrial natriuretic peptides and serum sodium and chloride in healthy humans. Am J Nephrol 1996; 16: 462–470.

[84] Syutkina EV, Cornélissen G, Halberg F, Grigoriev AE, Abramian AS, Yatsyk GV, Morozova NA, Ivanov AP, Shevchenko PV, Polyakov YA, Bunin AT, Safin SR, Maggioni C, Alvarez M, Fernandez O, Tarquini B, Mainardi G, Bingham C, Kopher R, Vernier R, Rigatuso J, Johnson D. Effects lasting into adolescence of exposure to betamimetics in utero. Clin Drug Inves 1995; 9: 354–362. [85] Tarquini B, Cornélissen G, Perfetto F, Tarquini R, Halberg F. About-half-weekly (circasemiseptan) component of the endothelin-1 (ET-1) chronome and vascular disease risk. Peptides 1997; 18: 1237–1241. [86] Tarquini B, Gheri R, Romano S, Costa A, Cagnoni M, Lee JK, Halberg F. Circadian mesor-hyperprolactinemia in fibrocystic mastopathy. Am J Med 1979; 66: 229–237. [87] Tarquini B, Halberg F, Seal US, Benvenuti M, Cagnoni M. Circadian aspects of serum prolactin and TSH lowering by bromocriptine in patients with prostatic hypertrophy. Prostate 1981; 2: 269–279. [88] Tarquini B, Perfetto F, Tarquini R, Cornélissen G, Halberg F. Endothelin-1’s chronome indicates diabetic and vascular disease chronorisk. Peptides 1997; 18: 119–132. [89] Tarquini B, Pini ME, Lapucci C, Navari N, Tarquini R, Curradi C, Perfetto F. Endothelin-1 circadian acrophase: A potential co-determinant in cardiovascular chronorisk. Abstract, 21st Conference, International Society for Chronobiology, Quebec, 11–15 July 1993: XV–8. [90] Touitou Y, Haus E, eds. Biological Rhythms in Clinical and Laboratory Medicine, Springer-Verlag: Berlin; 1992. [91] Vesely DL, Sothern RB, Scheving LE, Bremner WF, Third JLHC, McCormick JB, Dawson S, Kahn SJ, Augustine G, Ryan M, Greco J, Nemchausky BA, Shirazi P, Kanabrocki EL. Circadian relationships between circulating atrial natriuretic peptides and serum calcium and phosphate in healthy men. Metabolism 1996; 45 (8): 1021–1028. [92] Zaslavskaya RM. Chronodiagnosis and Chronotherapy of Cardiovascular Diseases. 2nd ed. Translation into English from Russian. Moscow: Medicina; 1993.

Index

A AA501 compound, 1375 ABC (ATP-binding cassette) transporters, 1471–1472 Absorptive-mediated endocytosis at the blood brain barrier, 1446 AC187 amylin antagonist, 982–984 ACE (Angiotensin-converting enzyme), 174, 361, 443, 1169–1170, 1177–1178, 1216, 1342–1343, 1461–1463 ACE (angiotensin-converting enzyme) inhibitors, 410, 460–461, 1178 ACE (angiotensin-converting enzyme) receptor polymorphisms, 460 Acid-sensing ion channels (ASIC), 376 ACTH (adrenocorticotrophic hormone/ corticotrophin), 689–694, 862–863, 884–885, 1267, 1523–1524, 1542 Actinoporins, 365 Activator of transcription (STATs) proteins, 1278 Activators of transcription (STATs), 1071–1072 Activity-dependent neuroprotective protein (ADNP), 1380–1382 Acute lung injury/acute respiratory distress syndrome, 1311 Acute myocardial infarction (AMI), 1204–1205 Acute renal failure, 1247 A. cylindracea ubiquitin-like peptide, 142 Acyl-modification of ghrelin, 733–734 AD. See Alzheimer’s disease (AD) Adaptive thermogenesis, 680 α-defensins. See Paneth cell α-defensins ADH (Antidiuretic hormone), 1223 ADNP (Activity-dependent neuroprotective protein), 1380–1382 adrenocorticotrophic hormone/ corticotrophin (ACTH), 689–694, 862–863, 884–885, 1267, 1523–1524, 1542 adrenomedullin (AM), 453–456, 1183 angiogenesis, 455 antiapoptosis, 454–455 biological actions of, 1284–1285 diuretic and natriuretic effects, 1284–1285 inotropic effect, 1284 other vasoprotective effects, 1285 overview, 1284 vasodilation, 1284

clinical application for pulmonary hypertension, 1285–1287 inhalation therapy, 1286 intravenous administration, 1285–1286 overview, 1285 clinical implication in cardiovascular diseases, 1167 clinical implications in kidney, 1258–1261 cardiovascular disorders, 1259 chronic renal failure, 1258–1259 diabetic nephropathy, 1259 glomerulonephritis, 1259 IgA nephropathy, 1259 others, 1259 overview, 1258 urinary tract infection, 1259 discovery of, 1163, 1283 expression, 453–454 in gastrointestinal function, 999–1002 biological actions of, 1001–1004 distribution of, 1000 expression of, 1000 overview, 999 receptors and signaling pathways, 1000–1001 release of, 1000 structure of, 999 growth regulation, 454 immune regulation, 455–456 migration/invasion, 455 molecular form of, 1167 mRNA and peptide distribution in cardiovascular system, 1164–1165 overview, 453, 1163, 1257, 1283 peptides, 1257 precursor mRNA/gene structure, 1163–1164 proadrenomedullin N-terminal 20 peptide, 1166–1167 receptors, 1165, 1257, 1283–1284 in regulation of endocrine glands, 861–865 adrenals, 862–863 diffuse system of gut, 864 other endocrine organs, 864–865 overview, 861 pancreas, 863–864 pituitary, 861–862

1565

renal biological actions of, 1258–1261 renoprotective actions of, 1258 structure of, 1163, 1283 synthesis of, 1283 therapeutic strategies, 456 adrenomedullin 2 (AM2), 1516–1517 discovery of, 1263–1264 mRNA distribution, 1266 overview, 1263 pathophysiological implications, 1267–1268 precursor mRNA and gene structure, 1264–1265 precursor processing, 1266 receptors and signaling cascade, 1266–1267 Adrenomedullin binding protein-1 (AMBP-1), 455 AEP (Asparagin endo-proteinase), 614 Agatoxins, 375 AGH (Androgenic gland hormone), 227 agonist peptides of formylpeptide receptors (FPR), 548–549 microbial peptides, 549 from peptide libraries, 549 Agouti-related peptide (AgRP), 692, 903–909, 923, 954–955, 996–997 Agrocybin, 127–128 AgRP (Agouti-related peptide), 692, 903–909, 923, 954–955, 996–997 Aib (Alpha-aminoisobutyric acid residues), 83–84 AIDS-related diarrhea, 1134 airways, endothelin in, 1289–1291 elimination of, 1290 overview, 1289 pathophysiological role, 1290–1291 receptor subtypes and localization, 1290 synthesis of, 1290 AKH/RPCH family. See invertebrate AKH/RPCH α-latrotoxin,(α Lx), 377 Alkanization assay, 33 allatostatins (ASTs) in crustacean bioactive Mollusca peptides, 225–226 biological actions of, 225–226 discovery of, 225 insect, 201–205 biological actions of, 205 discovery of, 201–202

1566 / Index allatostatins (ASTs) (continued) mRNA and peptides distribution, 202–203 overview, 201 precursors structure, 201–202 processing of, 203 receptors, 203–204 structure-activity and active conformations, 204 alleles, 43 Alpha-aminoisobutyric acid residues (Aib), 83–84 Altered peptide ligands (APL), 591–592 Alternaria alternata f. sp. mali, 152–154 Alternaria brassicae, 152 Alzheimer’s disease (AD), 653, 758–759, 1343, 1476–1477 peptide vaccine for, 535–539 EFRH phage elicits antibodies against β-amyloid peptide, 536–537 future of, 539 immunization of hAPP transgenic mice with EFRH-phage, 538–539 overview, 535 prevention and/or reduction of amyloid plaques in AD transgenic mice, 537–538 in vitro modulation of β-amyloid formation, 535–536 AM. See adrenomedullin (AM) amatoxins, 133–134 AMBP-1 (Adrenomedullin binding protein-1), 455 α-melanocyte stimulating hormone (αMSH), 689–694, 891, 947–948, 996, 1515–1516 American Society of Histocompatibility and Immunogenics (ASHI), 586 AMI (Acute myocardial infarction), 1204–1205 Amino acid residue substitutions, 416, 418 Aminopeptidase N, 1354 amphibian neurohypophysial peptides, 327–330 arginine vasotocin (AVT) and mesotocin (MT) distribution, 328 biological actions of AVT and MT, 328–329 discovery of, 327 distribution of, 328 overview, 327 precursor mRNA/gene structure, 327 receptor structure, 328 structure and function of hydrins, 329–330 neuropeptides, 283–287 caerulein peptides, 283–285 caerulein related peptides, 285–286 disulfide neuropeptides from genus Crinia, 287 overview, 283 tryptophyllin peptides, 286–287 opioid peptides biological actions of, 271–272 history of, 269–270 overview, 269

structure and conformation of, 270–271 amphibian bombesin-like peptides. See bombesin-like peptides, amphibian amphibian opioid peptides. See opioid peptides, amphibian amphibian tachykinins (TKs). See tachykinins (TKs), amphibian AMP kinase (AMPK), 907, 972 AMPs (Antimicrobial peptides), 1029 α-MSH (α-melanocyte stimulating hormone), 689–694, 891, 947–948, 996, 1515–1516 AM-toxins, 152–153 amylin, in control of food intake, 981–985 anorectic action of peripheral amylin, hypothalamic involvement in, 984 food intake effects, 981–982 food intake in animals deficient of CGRP or overexpressing CGRP, 983 food intake in animals overexpressing amylin, 983 food intake in knockout animals, 982–983 interaction between amylin and metabolites, 985 interaction with other peptides, 985 mediating anorectic effects of, 983–984 overview, 981 physiological and pathophysiological implications, 985 receptors and signaling pathways, 984–985 secretion regulation, 983 synthesis regulation, 983 analgesia, 412, 1328 anaphylatoxins, 553–557 biological actions of, 556–557 anaphylatoxic effect, 556–557 chemotactic effect, 557 phagocyte-activating effect, 557 expression, 554 generation of C3a and C5a, 554–555 historical perspective and scope, 553 overview, 553 pathophysiological implications of, 557 precursor mRNA/gene structure, 553–554 receptors for, 556 regulation of, 554 structure of, 555 Androgenic gland hormone (AGH), 227 Androgen vasotocin (AVT), 326 Androgen vasotocin receptor subtypes VT1R and VT2R, 328 Anemia, 1398 α-neurotoxins, 355–356 biological activity of, 356 conformation of, 356 discovery of, 355 precursor mRNA/gene structure, 355–356 receptors, 356 angiogenesis, 449, 455 angioneogenesis, 438

angiotensin, 1461–1462 angiotensin-converting enzyme in renal distribution of RAS, 1236 and bradykinin interactions, 1463 Angiotensin 1-7 (ANG-1-7) human cancer cell growth inhibition by, 462–463 and inhibition of angiogenesis, 463 Angiotensin-converting enzyme (ACE), 174, 361, 443, 1169–1170, 1177–1178, 1216, 1342–1343, 1461–1463 angiotensin-converting enzyme (ACE) inhibitors, 410, 460–461, 1178 angiotensin-converting enzyme (ACE) receptor polymorphisms, 460 Angiotensin I (Ang I), 459, 1170 angiotensin II (Ang II), 1169–1173, 1231 discovery of, 1169 interstitial, 1238 intracellular, 1239 intrarenal, 1239–1240 intrarenal levels of, 1238–1239 intrarenal receptors, 1237–1238 overview, 1169 pathophysiological implication in cardiovascular system, 1171–1173 receptor actions, 461–462 receptors and distribution in cardiovascular system, 1170–1171 structure of peptide and component of renin-angiotensin (RA) system, 1169–1170 angiotensin II receptor blockers (ARBs), 460–461 Angiotensinogen, 1170, 1236–1237 angiotensin peptides, and cancer, 459–463 ACE inhibitors, effect on cancer cell growth and tumorigenesis, 460–461 ACE receptor polymorphisms, 460 ANG-(1-7) human cancer cell growth inhibition by, 462–463 and inhibition of angiogenesis, 463 ANG II receptor actions, 461–462 cell signaling, 461 stimulation of angiogenesis by, 461–462 stimulation of apoptosis by, 462 ARBs, effect on cancer cell growth and tumorigenesis, 460–461 AT1 receptor polymorphisms, 460 overview, 459–460 Angiotensin receptor blockers (ARBs), 459–462 annelid (Glycera) neurotoxin, 399 Annexin I, 550 Anopheles gambaie, 165 anoplin, 393 Anorexia, 937–939 ANP. See atrial natriuretic peptide (ANP) Anterior pituitary (AP), 855, 883–886 Anteroventral periventricular nucleus (AVPN), 826 Anthopleurin-A (AP-A), 364

Index / 1567 ANTHO-RFamide-LIKE, 224 antiapoptosis, 454–455 antiarrhythmic agents, 410 Antibacterial peptide factors, 252 Antibiotic resistance, 508 anticholinesterases, 356–357 biological activity of, 357 conformation of, 357 discovery of, 356 precursor mRNA/gene structure, 356 receptors, 357 anticoagulants, 403–404 Antidiuretic hormone (ADH), 1223 antifungal peptides, 125–128 biological actions of, 128 discovery of, 125–127 overview, 125 structure of, 127–128 Antigen presenting cells (APC), 611–618 antihypertensive agents, 410 antimicrobial peptides, 252–253 discovery and structure of, 252 expression and function of, 252–253 as therapeutic property of venom peptides, 410–411 Antimicrobial peptides (AMPs), 1029 antimicrobial peptide toxins, 378–379 Anti-opioid effect of nociceptin, 1348 anti-opioid peptides, 1345–1349 cellular and molecular mechanisms for CCK-opioid interactions, 1347 cholecyctokinin (CCK) gene expression in response toopioid analgesia, 1346–1347 cholecyctokinin (CCK) release in response to opioid analgesia, 1347 overview, 1345 regulation of μ-opioid systems by MIF1, TYR-MIF-1, and TYR-W-MIF-1, 1348–1349 regulation of opioid analgesia by cholecyctokinin (CCK), 1345–1346 by neuropeptide FF (NPFF), 1347–1348 by nociceptin, 1348 Antipsychotic drugs (APDs), 739–740 Antisense-RNA-overlapping-melaninconcentrating hormone-gene (AROM), 707 antitumor peptides, 411–412 ants, insect venom peptides from, 393–394 ectatomin, 394 Myr p peptides and pilosulin, 393 poneratoxins, 393–394 ponericins, 394 AP (Anterior pituitary), 855, 883–886 AP-A (Anthopleurin-A), 364 apamin, 391 APC (Antigen presenting cells), 611–618 APDs (Antipsychotic drugs), 739–740 apelin, 787–792 biological actions within brain and pituitary gland, 789–792 food intake regulation, 789–790

overview, 789 water balance regulation, 790–792 discovery of, 787–788 distribution and receptor in rat brain, 789 overview, 787 peripheral cardiovascular actions, 792 precursor mRNA/gene structure, 788 precursor processing, 788 receptors and signaling cascades, 788–789 APGWamide, 243 Apitheraphy, 411 APJ receptor, 787–788 APL (Altered peptide ligands), 591–592 apoptosis, 449 Aquaporin (AQP-1), 1254 Aquaporin water channels, 1230 AR-42J cells, 1091 ARBs (Angiotensin receptor blockers), 459–462 Arcuate nucleus (ARC), 690, 895–897, 903–905 Arginine vasopressin (AVP), 787–792, 880, 1227–1229 Arginine-vasopressin fragment 4-9 (AVP4-9), 1446 AROM (Antisense-RNA-overlappingmelanin-concentrating hormonegene), 707 ART active tripeptide, 595 ASHI (American Society of Histocompatibility and Immunogenics), 586 ASIC (Acid-sensing ion channels), 376 Asparagin endo-proteinase (AEP), 614 asthma, 1247–1248, 1309–1311 ASTs. See allatostatins (ASTs) AT1 receptor, 1171 AT1 receptor polymorphisms, 460 AT2 receptor, 1171 ATP-binding cassette (ABC) transporters, 1471–1472 AtPep1 peptides, 5–8 biological actions of, 7–8 discovery of, 5 oveview, 5 ProAtPep1 gene encoding precursor protein, 6 mRNA distribution, 6–7 processing of, 7 receptors, 7 solution conformation, 7 structure of, 5–6 Atrial baroreceptors, 1224–1225 atrial natriuretic peptide (ANP), 1171–1172, 1199–1206, 1231, 1243–1248, 1251–1255 discovery of actions in kidney, 1251 effects on tubular transport systems, 1251–1256 collecting duct system, 1253 cortical collecting duct, 1254 Henle’s loop, 1252–1253 inner medullary collecting duct, 1254–1255

late distal tubule, 1253–1254 overview, 1251 proximal tubules, 1251–1252 overview, 1251 in regulation of endocrine glands, 877–881 biological activities of, 879–881 discovery of, 877 mRNA distribution, 878 overview, 877 pathophysiological implications, 881 precursor mRNA gene, 877–878 processing of, 878 receptors and signaling cascades, 879 release of, 879 as vasoactive peptide at blood–brain barrier (BBB), 1464 A-type natriuretic peptide receptor (NPRA), 809–810 Auxin, 26 AVIT peptides, 361 biological activity, 361 conformation, 361 discovery, 361 receptors, 361 structure of precursor mRNA/gene, 361 AVP (Arginine vasopressin), 787–792, 880, 1227–1229 AVP4-9 (Arginine-vasopressin fragment 4-9), 1446 AVPN (Anteroventral periventricular nucleus), 826 AVT (Androgen vasotocin), 326 B bacterial/antibiotic peptides. See colicins Bacteriocin leader sequences, 109 BALF (Bronchial alveolar leakage fluid), 1290–1291 Basal pancreatic polypeptide (PP) secretion, 1101 basidiomycete fungi toxins. See mushroom toxins BBB. See blood–brain barrier (BBB) β-casein, 1339 β-casomorphins and hemorphins, 1339– 1343 biological actions of, 1341–1342 overview, 1341 specific actions and function of hemorphins, 1342 specific actions and functions of βcasomorphins, 1341–1342 discovery of, 1339–1340 formation of, 1340–1341 implications in pathophysiolgy, 1342–1343 overview, 1339 β-cells of the pancreas, 850–851 BCSFB (blood-cerebrospinal fluid barrier), 1423–1424 BDNF (Brain-derived neurotrophic factor), 606 β-endorphin, 1325–1330 Benzodiazepine receptor (CBR), 815–817

1568 / Index Benzodiazepines (BZD), 813 BIBN4096BS CGRP antagonist, 1182–1184 Big endothelin (big ET), 1188 BIM26226 gastrin-releasing peptide receptor antagonist, 1052 bioactive Mollusca peptides, 366 crustacean, 221–227 allatostatin (AST), 225–226 Antho-RFamide-like, 224 crustacean cardioactive peptide (CCAP), 221–224 FLRFamide, 224 kinins, 226 neuropeptide F, 224 opioid–enkephalin, 224–225 orcokinin and orcomyotropin, 225 overview, 221–223 proctolin, 221 Pyrokinin/PBAN, 226 RFamide peptides, 224 sifamide, 226–227 sulfakinins, 224 tachykinin-related peptides (TRPs), 226 molluscan, 235–239 FMRFamide, FMRFamide-related peptides (FaRPs), 235–236 overview, 235 peptides and feeding behavior, 237–238 peptides and metabolism, 236–237 peptides and renal function, 238 tachykinins (TKs), 238–239 Biphalin, 1430 BK. See bradykinin (BK) BL (Brassinolides), 52 β-lipotropin, 1325–1327 blood–brain barrier (BBB) and fibroblast growth factor (FGF), 1449–1453 CSF-ependymal-brain interface, 1452 FGF1/FGF2 transport across BBB, 1450–1451 FGF2 transport and regulatory phenomena at choroid plexus, 1451–1452 FGF-induced protection of cerebral endothelium and neurons, 1450 modulation of cerebral endothelial cells, 1451 overview, 1449 peptide regulatory system modeling, 1452–1453 secretion by microvascular endothelial cells, 1451 and hypothalamic neuropeptides, 1469–1472 circumventricular organs, 1469–1470 corticotropin-releasing hormone, 1471 GnRH, 1470–1471 overview, 1469 oxytocin, 1472

PACAP, 1472 somatostatin, 1472 TRH, 1470 vasopressin (antidiuretic hormone), 1471 and ingestive peptides, 1455–1458 ghrelin, 1457 insulin, 1455–1456 leptin, 1456–1457 overview, 1455 oligopeptide transport at, 1424–1425 blood to brain transport, 1424–1425 brain to blood transport, 1425 overview, 1424 peptidases, 1425 and opiate peptides, 1429–1432 overview, 1429 stabilization of, 1429–1430 targeting, 1430–1432 efflux transporter inhibition and peptide masking, 1432 lipophilicity increase, 1430–1431 transporter mechanisms, 1431–1432 vasoactive peptides at, 1461–1466 adrenomedullin, 1465 angiotensin, 1461–1462 angiotensin and bradykinin interplay, 1463 atrial natriuretic peptide, 1464 bradykinin, 1462 CNS peptides with vasoactive properties, 1465–1466 endothelins, 1464–1465 overview, 1461 substance P, 1463–1464 blood–brain barrier (BBB) amino acid, 1415–1421 overview, 1415 regulation, 1415–1416 transport, 1416 transport systems, 1416–1421 overview, 1416 System A/ATA2, 1419 System ASC/ASCT1 and -2, 1419 System B0+ or LNAA, 1419–1420 System Betaine-GABA/GAT2, 1420 System CRT, 1420 System L/large neutral amino acid transporter 1, 1418–1419 System N, 1419 System TAUT, 1420 System X-/EAAT 1–3, 1420 System y+/cationic amino acid transporter, 1416–1418 thyroid hormones, 1420–1421 blood–brain barrier (BBB) basic peptide transport, 1443–1447 [d-Arg2]dermorphin analogs, 1444 basic oligopeptides and proteins, 1443–1444 bFGF, 1444–1446 overview, 1443–1446 peptide delivery via adsorptivemediated transport, 1446–1447 transport mechanism, 1446

blood–brain barrier (BBB) mediated diseases, 1475–1478 alcohol withdrawal and methionine enkephalin, 1477 Alzheimer’s disease and amyloid β protein efflux, 1476–1477 GLUT-1 deficiency syndrome, 1477–1478 insulin resistance in sepsis, 1478 obesity and leptin, 1475–1476 overview, 1475 blood–brain barrier (BBB) permeability, 1435–1439 apical or basolateral surface receptor usefulness, 1437 delivery enhancing adsorptive endocytosis and chemical modifications, 1437 neurotrophic peptide enhanced delivery affecting neuroregeneration, 1439 overview, 1435 partially disrupted, and selective regulation of transport system in pathophysiology, 1438–1439 peptide intracellular trafficking leading to exocytosis, 1437–1438 pharmacokinetic examples of, 1438 blood–brain barrier (BBB) properties, and oligopeptide transport, 1423–1424 cerebrospinal fluid (CSF) sink action, 1424 overview, 1423 passive permeability, 1423 transport properties, 1424 blood-cerebrospinal fluid barrier (BCSFB), 1423–1424 Blood pressure (BP) regulation, 879 β-MHC (β-myosin heavy chain), 1171–1172 β-myosin heavy chain (β-MHC), 1171–1172 BN. See Bombesin (BN) B natriuretic peptide (BNP), 1252 BNP. See B-type natriuretic peptide (BNP) Body fluid homeostasis in the rat, 810 Bombesin (BN), 285, 985, 1049–1054 bombesin (BN)-related peptides (GRP, NMB), 429–431 BN-receptor activation on cancer effects, 430 expression of, and receptors in cancer, 429 induced growth effects on cancer cells, 430 overview, 429 in treatment of human tumors, 430–431 bombesin-like peptides, amphibian, 277–281 bombesins, 277–279 gastrin-releasing peptide (GRP), 280 overview, 277–278 phyllolitorins, 279 phylogenetic considerations for additional mammalian bombesinlike peptides, 280–281 ranatensin-like peptides, 279–280

Index / 1569 Bombinakinin M, 293 Bombina variegata (Bv8), 272–274 bombinins, 333–337 biological activity of, 335–336 discovery of, 333–334 mode of action studies, 336–337 overview, 333 precursor mRNA/gene structure, 335 solution conformation, 335 Bombinins H, 334–337 bombolitins, 391 Bombyx mori, 164–165 Bone natriuretic peptide (BNP), 1199–1206 Bosentan receptor antagonist, 450 BP (Blood pressure) regulation, 879 bradykinin (BK), 1175–1178 and angiotensin interactions as vasoactive peptide at BBB, 1463 and cancer, 443–446 antagonists, 444–445 kinin biology, 444 kinin chemistry, 443 overview, 443 discovery, 1175 kallikrein mRNA distribution, 1176 kininogen mRNA and gene structure, 1175–1176 kallikreins, 1176 overview, 1175–1176 kininogen mRNA distribution, 1176 kinin peptide distribution, 1176 kinin peptide formation, 1176–1177 kinin peptide metabolosm, 1177–1178 kinin receptors and distribution in cardiovascular system, 1178 overview, 1175 role of kinin peptides in health and disease states, 1178 as vasoactive peptide at BBB, 1462 bradykinin (BK)-related peptides, 291–294 biological activities of, 293–294 biosynthesis of, 291–292 discovery of, 291 distribution of, 291 molecular variants of, 292–293 overview, 291 in solitary wasp venom peptides, 393 bradykinin-potentiating peptides biological activity, 361 conformation, 361 discovery, 360–361 receptors, 361 snake venom peptides, 360–361 structure of precursor mRNA/gene, 361 Brain-derived neurotrophic factor (BDNF), 606 Brassinolides (BL), 52 Brattleboro rats, 626–627 breast cancer, 424 Bronchial alveolar leakage fluid (BALF), 1290–1291 bronchial asthma, 1247–1248, 1309–1311

Bronchoconstriction, 1302–1303 B-type natriuretic peptide (BNP), 805–811 biological actions in brain, 810 discovery of, 805–807 mRNA distribution, 807–809 overview, 805 pathophysiological implications, 810–811 precursor mRNA and gene structure, 807 precursor processing, 809 receptors and signaling cascade, 809–810 B-type natriuretic peptide receptor (NPRB), 809–810 Bufokinin-LI, 263 Bungarotoxins, 356–357 bursicon, 215–219 biological actions of, 219 discovery of, 215–217 distribution of mRNA, 217–218 precursor mRNA/gene structure, 217 processing of, 218 receptors, 218–219 Bv8 (Bombina variegata), 272–274 BZD (Benzodiazepines), 813 BZR-cotoxins, 154–155 C C3a anphylatoxin receptor (C3aR), 556 C5a anphylatoxin receptor (C5aR), 556 Ca2+ channel blockers, 358–359 biological activity of, 357, 359 conformation of, 357, 359 discovery of, 357, 358 precursor mRNA/gene structure, 357, 358 receptors, 357, 358–359 Caco2 enterocyte like cells, 1073 caerulein peptides, 283–285 caerulein related peptides, 285–286 Calcaelin, 148 Calcicludine, 358–359 Calciseptin, 357 Calcitonin (CT), 771–772 calcitonin-gene-related peptide (CGRP), 263, 581, 771–777, 999–1001, 1163, 1181–1184, 1263–1266, 1465 biological actions within brain, 776–777 AM, 777 CGRP, 776 in control of food intake, 981–985 anorectic action of peripheral amylin, hypothalamic involvement in, 984 food intake effects, 981–982 food intake in animals deficient of CGRP or overexpressing CGRP, 983 food intake in animals overexpressing amylin, 983 food intake in knockout animals, 982–983 interaction with other peptides, 985 overview, 981

physiological and pathophysiological implications, 985 receptors and signaling pathways, 984–985 secretion regulation, 983 synthesis regulation, 983 discovery of, 771 and gastrointestinal function, 1005–1010 distribution of, 1005–1006 overview, 1005 physiological and pathophysiological implications of, 1007–1011 mRNA/peptide distribution, 773 adrenomedullin, 774 calcitonin gene-related peptide (CGRP), 773–774 overview, 771, 1181 pathological implications, 777 peptide conformation, 776 precursor mRNA/gene structure, 771–772 precursor processing, 774–775 receptors and signaling cascades, 775–776 AM, 775 CGRP, 775 molecular basis of CGRP and AM receptor phenotype, 775–776 signaling, 776 Calcitonin gene-related peptide antagonists (CGRP antagonists), 1182 Calcitonin receptor (CTR), 775, 984–985, 1266 Calcitonin receptor-like receptor (CRLR), 775–776, 985, 1182, 1257–1259, 1266–1267, 1283–1284 Calcium-dependent antibiotic synthetase (CDA), 90–92 Callus formation in plants, 29 Calnexin, 613 cAMP (cyclic AMP), 776, 849–850, 1224, 1284–1286 cancer, 1297, 1389 and angiotensin peptides, 459–463 ACE AND AT1 receptor polymorphisms, 460 ACE inhibitors and ARBs, effect on cancer cell growth and tumorigenesis, 460–461 ANG-(1-7), human cancer cell growth inhibition by, 462–463 ANG II receptor actions, 461–462 overview, 459–460 and bradykinin (BK), 443–446 antagonists, 444–445 kinin biology, 444 kinin chemistry, 443 overview, 443 and gastrin, 467–470 expression of in normal tissue, 467–468 expression of in tumors, 468–469 overview, 467 receptor(s), 468 as tumor growth factor, 469

1570 / Index cancer (continued) and growth hormone–releasing hormone (GHRH) antagonists, 483–488 antagonistic analogs of, 484–485 effects of, on human experimental cancers in vivo, 486–487 and GHRH receptors in human cancers, 485–486 mechanisms of tumor inhibition by, 486 overview, 483 therapeutic indications of, 484 and oxytocin (OT), 479–481 as modulator of cell growth in OTRexpressing tumors, 480–481 OT/OTR system, 481 overview, 479 physiology to neoplastic pathology, 479–480 and somatostatin, 435–437 antiangiogenic effects of, 436 direct/indirect antiproliferative effects of in tumors, 436 inhibition of hormone release from tumors by, 435 molecular signaling of receptors, 436–437 somatostatin receptors in, 437–438 in human tumors, 437 targeted tumor imaging, 437 targeted tumor therapy, 437–438 and VIP/PACAP, 473–476 overview, 473 proliferation, 475 second messengers, 474–475 tumor imaging, 475 VIP/PACAP peptides, 473–474 VIP receptors, 474 cancer-testis antigens, 500 Candesartan, 460 canine narcolepsy, 726 carboxyl terminal amidation, 1015 Carboxy terminal peptide of neuropeptide Y (CPON), 684–685 carcinoid tumors, 1132–1133 cardiac cachexia, 1195–1196 cardiotoxins, 357–358 biological activity of, 357–358 conformation of, 357 discovery of, 357 precursor mRNA/gene structure, 357 receptors, 357 Carpet mechanism of plasma membrane disruption, 301–302 CART. See cocaine- and amphetamineregulated transcript (CART) Casein kinase-2 (CK-2), 1496 Casoxin D, 1370 cathelicidins, 67–72 active and/or solution conformation, 70–71 biological functions of, 71–72 discovery of, 67 distribution of, 68–69 oveview, 67

precursor mRNA/structural gene structure, 67–68 processing of, 68–69 receptors, 69–70 cationic antimicrobial peptides, 55–65 active conformation, 59–65 biological actions of, 59 discovery of, 55 mRNA distribution, 58 mRNA expression, 56–57 oveview, 55 precursor mRNA/gene structure, 55–56 processing of, 58 receptors, 58–59 Cat S gene, 614 caudodorsal cell hormone genes (CDCH), 241–242 caudodorsal cell system (CDC), 241 CCAP. See crustacean cardioactive peptide (CCAP) C. carbonum race 1, 154 CCD (cortical collecting duct), 1254–1255 CCK. See cholecystokinin (CCK) CD4+ T cells, 585–591, 598 CD8+ T cells, 585–591, 598 CDA (Calcium-dependent antibiotic synthetase), 90–92 CDC (caudodorsal cell system), 241 CDCH (caudodorsal cell hormone genes), 241–242 CDR (Complementary determining region) of T cell receptor, 585 central nervous system (CNS), 1415, 1435–1439, 1443, 1449–1453, 1455–1456, 1469–1472 cerebral oxygen content (pO2), 1381 cerebral spinal fluid (CSF), 726–727 cerebratulus A toxins, 399 cerebratulus B toxins, 398–399 cerebrospinal fluid (CSF), 1339–1340, 1423–1427, 1455–1457 cerebrospinal fluid barrier (BCSFB), 1449–1453 Cerulein, 1020 Cetrorelix, 422–423 CgA (Chromogranin A), 311 cGMP (cyclic 3’5’-guanosine monophosphate), 879–880 CGRP. See calcitonin-gene-related peptide (CGRP) CGRP8-37 calcitonin-gene-related peptide receptor antagonist, 1001 Cg/Sg. See chromogranins/secretogranins (Cg/Sg) chemokines, 559–564 biological actions of, 563–564 brain development, 563 neuromodulation, 563–564 conformation, 563 discovery of, 560 overview, 559 pathophysiological implications, 564 precursor mRNA/genestructure, 560 precursor processing, 562 receptor distribution, 562 receptor expression, 562

receptors and signaling cascades, 562 SDF-1 mRNA and protein distribution, 560–561 chemotactic peptide ligands, 547–551 formylpeptide receptors (FPR), 547–548 mammalian peptides, 549–550 associated with amyloidogenic diseases, 549 associated with inflammatory and antibacterial responses, 549–550 frog skin peptides, 550 overview, 547 perspectives, 551 chemotherapy-induced diarrhea, 1133 CHF (Congestive heart failure), 881, 1193–1196, 1225–1226, 1247–1248 CHH (crustacean hyperglycemic hormone) peptide family, 229–233 cholecystokinin (CCK), 994, 1052, 1101–1102, 1111, 1119–1120 and satiety, 961–966 feeding actions of central CCK, 963 feeding actions of peripheral CCK, 961–963 functional responses to peripheral CCK, 964 interactions with other signaling systems, 966 overview, 961 pathophysiologial implications, 966 receptors and signaling pathways, 965–966 sites of action, 965 studies with OLETF rats and CCK-A receptor knockout mice, 963–964 cholecystokinin (CCK)/gastrin, 715–719 active and/or solution conformation, 718 biological actions within brain and pituitary, 718–719 discovery of, 715 distribution of, 715–716 overview, 715 pathophysiological implications, 719 precursor mRNA/gene structure, 715 precursor processing, 716–717 receptors and signaling cascades, 717–718 cholecystokinin (CCK) receptor antagonists, 1346–1347 cholecystokinin 8 (CCK-8), 284–285, 1073 cholecystokinin A (CCK-A) receptors, 965–966 cholecystokinin B (CCK-B) receptors, 965 cholecystokinin receptors, 285 choleocystokinin-like immunoreactivity (CCK-LI), 1016 choroid plexus, oligopeptide transport at, 1425–1426 basolateral transporter, 1426 intracellular transporters, 1426 larger peptide transporters, 1426 molecular basis for oligopeptide transporter heterogeneity, 1425–1426

Index / 1571 overview, 1425 peptidases, 1426 chromatophorotrophic hormones, crustacean, 229–230 biological actions of, 230 precursor mRNA/gene structure, 229–230 Chromogranin A (CgA), 311 chromogranins/secretogranins (Cg/Sg), 311–318 biological actions of, 316–318 extracellular functions, 317–318 intracellular functions, 316 discovery of, 311 mRNA distribution, 314–315 overview, 311 precursor mRNA/gene structure, 311–314 chronic obstructive pulmonary disease (COPD), 1194–1196 chronomics. See peptide chronomics Circadian rhythms, 1547–1561 cirrhotic portal hypertension, 1002 C-jun amino terminal kinase (JNK), 1172 CK-2 (Casein kinase-2), 1496 class II–associated invariant chain peptide. See CLIP Clavariopsin A and B, 125 CLAVATA3, 9–14 biological actions of, 13–14 CLV3 function model, 13–14 CLV3 regulation, 13 CLV signaling, 13 downstream target(s), 13 discovery of, 9–10 CLAVATA3 locus, 9 shoot apical meristem organization, 9–10 overview, 9 precursor mRNA/gene structure, 10 CLV3/ESR-related gene family, 12 CLV3 expression and function, 12 CLV3 gene and protein, 10–11 receptors, 12–13 Clay minerals in peptide formation, 1482–1483 CLE domain protein family, 12 CLIP (class II–associated invariant chain peptide), 611–619 as antagonist of TH1 polarization, 617–618 chaperone facilitating release, 615–616 flanking residues, 615 generation of, 614 membrane microdomains, 616–617 CLIP in pseudodimer model, 617 CLIP in tetraspan microdomains, 617 tetraspan microdomains, 616–617 MHC class II molecules, 611–614 impact of CLIP on MHC II peptide binding groove, 613 parent protein of CLIP: invariant chain, 613–614 structure of MHC class II peptide complexes, 612 overview, 611

proteolysis of Ii, 614 self-release of, 615 Clonorin, 400 CLV3 and ESR-related gene family, 12 expression and function, 12 function model, 13–14 gene and protein, 10–11 regulation, 13 C natriuretic peptide (CNP), 1252 CNP. See C-type natriuretic peptide (CNP) CNS (central nervous system), 1415, 1435–1439, 1443, 1449–1453, 1455–1456, 1469–1472 cocaine- and amphetamine-regulated transcript (CART), 697–701 cocaine- and amphetamine-regulatedtranscript (CART), 913–916 cocaine- and amphetamine-regulated transcript (CART) biological actions of CARP peptides within brain and pituitary, 699–701 feeding and energy metabolism, 699–700 hypothalamic hypophysiotropic neuron neuroendocrine regulation, 700 pain, 700 reward, 699 vegetative functions, 700–701 CART mRNA and peptide distribution, 698–699 CART peptides processing, 697–698 discovery of, 697 cocaine- and amphetamine-regulatedtranscript (CART) and ingestive behavior functional response to differing metabolic and feeding states, 914 interactions with other peptidergic/ aminergic systems, 915 overview, 913 physiological and pathophysiological implications, 916 receptors and signaling pathways, 915 sites of action and neural networks affected, 914–915 studies from genetic manipulations or mutations, 913 overview, 697 physiopathological implications, 701 precursor mRNA/gene structure, 697 receptors and signaling cascades, 699 Cockroach hindgut myotropic bioassay, 185–186 colicins, 115–122 classification of, 115 discovery of, 115 domain concept, 116–117 import mechanisms across outer membrane of, 122 overview, 115 precursor mRNA/gene structure, 115–116

research areas, 122 sequence comparisons of, 117–120 x-ray structures of, 120–122 Colon cancer, 1054 colonic transport in adrenomedullin gastrointestinal function, 1002 Complement activation cascade, 554 Complementary determining region (CDR) of T cell receptor, 585 Congestive heart failure (CHF), 881, 1193–1196, 1225–1226, 1247–1248 conus snail, venom peptides, 381–387 biodiversity of venomous molluscs, 381 biological mechanisms, 386–387 conotoxin families definined, 384–385 discovery of conotoxins, 381–382 overview, 381 precursor structure, expression, and processing, 382–384 receptor targets definined, 386 structures definined, 386 therapeutic applications, 387 COPD (chronic obstructive pulmonary disease), 1194–1196 copolymer 1 (Cop-1), 603–608 clinical trials with, 605 effects of on graft rejection, 607–608 experimental autoimmune encephalitis (EAE) suppression by glatiramer acetate (GA), 604 glatiramer acetate (GA) action mechanism, 605–606 immunomodulation and neurogenration in CNS, 606–607 other peptides related to MS, 604–605 overview, 603–604 corazonin, 163–164 corazonin-like immunoreactivity (CRZIR), 166 cortical collecting duct (CCD), 1254–1255 corticotrophin-releasing hormone (CRH), 581–582, 748–750, 784, 829, 885, 947–948, 954–955, 996, 1471 in gastrointestinal system, 1023–1027 biological actions of, 1024–1026 neuronal pathways, 1026 overview, 1023 pathophysiological implications, 1026–1028 hypothalamic-pituitary-adrenocortical system, 1523 and ingestive behavior, 937–940 feeding behavior effects, 937–938 interactions with neuropeptide systems, 939–940 metabolic regulation of, 938 overview, 937 physiology and pathophysiological implications, 940 receptors and signaling pathways, 939 sites of action and neural networks affected, 938–939 studies from genetic manipulations, 938

1572 / Index corticotrophin-releasing hormone (CRH) family, 655–661 biological actions in central nervous system, 660 discovery of, 655–656 mRNA distribution, 658 mRNA expression, 658 overview, 655 pathophysiological implications, 661 precursor mRNA/gene structure, 656–657 preprohormone processing, 658–660 receptors and binding proteins, 660 corticotrophin-releasing hormone (CRH) neurons, 700 corticotrophin-releasing hormone receptor 1 (CRH1R), 660, 939 corticotrophin-releasing hormone receptor 2 (CRH2R), 660, 939 cortistatin (CST), 645, 651 CPON (Carboxy terminal peptide of neuropeptide Y), 684–685 crabrolin, 392 CREB (cyclic-AMP response element binding protein), 699 CRH. See corticotrophin-releasing hormone (CRH) CRISP family toxins, 359 CRLR (Calcitonin receptor-like receptor), 775–776, 985, 1182, 1257–1259, 1266–1267, 1283–1284 crotamine-like myotoxins, 359 Crp 4 bactericidal activity, 1033–1034 crustacean cardioactive peptide (CCAP), 221–224 biological actions of, 224 discovery of, 221–224 crustacean hyperglycemic hormone (CHH) peptide family, 229–233 crustaceans bioactive Mollusca peptides, 221–227 allatostatin (AST), 225–226 Antho-RFamide-like, 224 crustacean cardioactive peptide (CCAP), 221–224 FLRFamide, 224 kinins, 226 neuropeptide F, 224 opioid–enkephalin, 224–225 orcokinin and orcomyotropin, 225 overview, 221–223 proctolin, 221 Pyrokinin/PBAN, 226 RFamide peptides, 224 sifamide, 226–227 sulfakinins, 224 tachykinin-related peptides (TRPs), 226 chromatophorotrophic hormones, 229–230 biological actions of, 230 precursor mRNA/gene structure, 229–230 hyperglycemic hormones, 230–233 biological actions of, 233 discovery of, 230

gene expression, 232 gene organization, 230–232 processing of, 233 receptors, 232 CRZ-IR (corazonin-like immunoreactivity), 166 CsA (Cyclosporin A), 607–608 CSαβ-motif (Cysteine Stabilized αβ motif), 341 CSF (cerebral spinal fluid), 726–727 CSF (cerebrospinal fluid), 1339–1340, 1423–1427, 1455–1457 CST (cortistatin), 645, 651 CT (Calcitonin), 771–772 C-terminal isoleucine amide (PHI), 1307–1308 C-terminal methionine amide (PHM), 1307–1308 C-terminal octapeptide of octadecaneuropeptide (OP), 816–817 CTFR (cystic fibrosis transmembrane conductance regulator), 1295 CTLs (cytotoxic T cells), 492–493 CTR (Calcitonin receptor), 775, 984–985, 1266 C-type lectins and lectin-like proteins, 360 C-type natriuretic peptide (CNP), 805–811, 1199–1206 biological actions in brain, 810 discovery of, 805–807 mRNA distribution, 807–809 overview, 805 pathophysiological implications, 810–811 precursor mRNA and gene structure, 807 precursor processing, 809 receptors and signaling cascade, 809–810 C-type natriuretic peptide receptor (NPRC), 809–810 Cupiennins, 378 C. victoriae, 154 CXCL12 (stromal derived factor 1-α and 1-β), 569–570 CXCR4 chemokine receptor, 562–564 cyclic 3′5′-guanosine monophosphate (cGMP), 879–880 cyclic AMP (cAMP), 776, 849–850, 1224, 1284–1286 cyclic-AMP response element binding protein (CREB), 699 cyclic gmp-dependent protein kinase (PKG), 1158 Cyclophilin A (CypA), 1498 Cyclosporin A (CsA), 607–608 CypA (Cyclophilin A), 1498 Cys patterns of conotoxins, 384–385 Cysteine Stabilized αβ motif (CSαβmotif), 341 cystic fibrosis, 1310 cystic fibrosis transmembrane conductance regulator (CTFR), 1295 cytokines, 1465

cytolysins, 26, 365–366 cytolytic peptide toxins, 378–379 cytotoxic GRP analogs, 430 cytotoxic luteinizing hormone-releasing hormone (LHRH) analog, 423–425 breast cancer, 424 endometrial cancer, 425 epithelial ovarian cancer, 424 melanoma, 425 non-Hodgkin’s lymphoma (NHL), 425 other cancers, 425 renal cell carcinoma (RCC), 425 side effects of, 425 cytotoxic T cells (CTLs), 492–493 D D3 compound, 1412 DA (Dopamine), 898, 915 δ-ACTXs (δ-atracotoxins), 373 DAG (diacylglycerol), 640–641 DAMGO μ-opioid ligand, 920–923 DAMGO μ-opioid receptor agonist, 1336 δ-atracotoxins (δ-ACTXs), 373 DCE (dynorphin-converting enzyme), 1320 DDH (disulfide-directed β-hairpin), 370 deconvolution strategies for soluble combinatorial libraries, 1487–1489 defensins. See cationic antimicrobial peptides dehydroamino acids, 97 deltorphins, 272 dendroaspis natriuretic peptide (DNP), 360 dendrotoxins, 358 depsilairdin, 155 depsipeptide and peptide phytotoxins defined, 152–155 dioxopiperazines from Leptosphaeria maculans, 155 host-selective toxins (HSTs) from Bipolaris zeicola race 3, 154–155 host-selective toxins (HSTs) from genus Alternaria, 152–154 Alternaria alternata f. sp. mali, 152–154 Alternaria brassicae, 152 host-selective toxins (HSTs) from genus Cochliobolus, 154 host-selective toxins (HSTs) from Leptosphaeria maculans, 155 dermaseptins, 295–303 microbicidal activity structural features and mechanisms, 301–303 overview, 295 preprodermaseptin-derived peptide activities, 300–301 preprodermaseptins, 297–300 superfamily, 295–297 Dermaseptin S1, 301 Dermorphin, 271 Des-Gln14-ghrelin peptides, 732 Destruxin B, 152 developmental peptides, 163–168 active and/or solution conformation, 167

Index / 1573 biological actions of, 167–168 corazonin, 168 ecdysis triggering hormones (ETHs), 167 prothoracicotropic hormone (PTTH), 168 discovery of, 163–164 corazonin, 163–164 ecdysis triggering hormones (ETHs), 163 prothoracicotropic hormone (PTTH), 164 mRNA and peptides distribution, 166 overview, 163 precursor mRNA/gene structure and peptide processing, 164–166 corazonin, 165 ecdysis triggering hormones (ETHs), 164–165 prothoracicotropic hormone (PTTH), 165–166 receptors, 166–167 corazonin receptors (CRZR), 167 ecdysis triggering hormone (ETH) receptors, 166–167 prothoracicotropic hormone (PTTH) receptor, 167 diabetes mellitus, 412, 1258 diabetic nephropathy, 1259 diacylglycerol (DAG), 640–641 diarrhea AIDS-related diarrhea, 1134 chemotherapy-induced diarrhea, 1133 overview, 1133 di-epitope multiple antigen peptides, 543 diet-induced obese (DIO) mice, 957–958 DIO (diet-induced obese) mice, 957–958 dipeptidylaminopeptidase III (DPP-III), 178–179 dipeptidyl aminopeptidase IV/CD26, 567–571 effector peptides, 568–570 in immune system, 567–568 overview, 567–568 peptide inhibitors, 571 secondary immunorelevant DP IV peptide substrates, 570–571 dipeptidyl peptidase IV (DPP-IV), 1059, 1063 discrete arcuate nucleus-magnocellular paraventricular nucleus (ARCmPVN), 889–891 disintegrin-like peptides, mamba, 357 disintegrins, 360, 403–404 disulfide-directed β-hairpin (DDH), 370 disulfide neuropeptides, 287 diuretic and antidiuretic hormones, insect, 157–161 biological actions of, 160–161 discovery of, 157–158 mRNA distribution, 159 overview, 157 peptide distribution, 159 peptide processing, 159 precursor mRNA/gene structure, 158–159

receptors, 159–160 structure-activity and active conformation, 160 δ-ligands, 1490 DMH (dorsomedial hypothalamus), 890–891, 963 DNP (dendroaspis natriuretic peptide), 360 Dopamine (DA), 898, 915 δ-opioid receptors (DOR), 1321–1322, 1335 DOR (δ-opioid receptors), 1321–1322, 1335 dorsal root ganglion (DRG), 1359–1361 dorsal vagal complex (DVC), 1112 dorsomedial hypothalamus (DMH), 890–891, 963 downstream target(s), 13 Doxorubicin (DOX), 423–426 δ-PaluITs (δ-palutoxins), 374 δ-palutoxins (δ-PaluITs), 374 DPDPE met-enkephalin analog, 1430–1432 DPP-III (dipeptidylaminopeptidase III), 178–179 DPP-IV (dipeptidyl peptidase IV), 1059, 1063 DRG (dorsal root ganglion), 1359–1361 Drome-CAP2b, 161 Drome-DH31, 160 Drome-DH44, 160 Drosophila melanogaster, 165 dumping syndrome, 1135 DVC (dorsal vagal complex), 1112 DVL1 mRNA distribution, 18–19 DVL peptides, 17–21 biological actions of, 20–21 discovery of, 17–18 DVL1 mRNA distribution, 18–19 oveview, 17 precursor mRNA/gene structure, 18 processing of, 19–20 receptors, 20 dynorphin-converting enzyme (DCE), 1320 dynorphins, 1319–1324 discovery of, 1319 drug abuse and, 1323 expression in central nervous system, 1321–1322 expression in peripheral tissues, 1323–1324 overview, 1319 pathophysiological implications, 1322–1323 possible biological actions, 1321–1322 precursor mRNA/gene structure, 1319–1320 processing of, 1320–1321 receptors, 1321 E EA (electroacupuncture) induced analgesia, 1345–1347 EAE (experimental autoimmune encephalitis), 603–608

ecdysis triggering hormones (ETHs), 163 ECE (endothelin-converting enzyme), 1189, 1269 ECL (enterochromaffinlike) cells, 1039–1041, 1044–1045, 1066, 1092, 1124–1125 ECM (extracellular matrix), 1171–1172 ECs (endothelial cells), 1164–1165, 1201, 1206 ectatomin, 394 EEG (electrocardiography), 1341–1342 EEG (electroencephalogram), 1521–1524 effector peptides, 568–570 EFRH (Glu-Phe-Arg-His) amyloid antigen peptide, 537–539 EGF (epidermal growth factor), 1150, 1290–1291, 1401–1402, 1437–1439 EGFR (epidermal growth factor receptor), 1087, 1437–1438 egg-laying hormones (ELHs), 241 EKE motifs, 5–7 electroacupuncture (EA) induced analgesia, 1345–1347 electrocardiography (EEG), 1341–1342 electroencephalogram (EEG), 1521–1524 electro-olfactogram (EOG) response, 323 ELHs (egg-laying hormones), 241 EMP-AF (eumenine mastoparan-AF), 393 EMs. See endomorphins (EMs) endocannabinoids, 915 endocrine gland regulation adrenomedullin in, 861–865 adrenals, 862–863 diffuse system of gut, 864 other endocrine organs, 864–865 overview, 861 pancreas, 863–864 pituitary, 861–862 atrial natriuretic peptide (ANP) in, 877–881 biological activities of, 879–881 discovery of, 877 mRNA distribution, 878 overview, 877 pathophysiological implications, 881 precursor mRNA gene, 877–878 processing of, 878 receptors and signaling cascade, 879 release of, 879 endothelins in, 855–859 adrenal, 857–858 Leydig cells, 858 ovary, 858–859 overview, 855 pancreatic islets, 857 parathyroid, 856–857 pituitary, 855–856 thyroid, 856 galanin (Gal) in, 883–885 expression of, 883–884 hormone secretion, 884–885 overview, 883 ghrelin in, 869–873 adrenal cortex, 872 cartilage and bone homeostasis, 871 heart and cardiomyocytes, 870–871

1574 / Index endocrine gland regulation (continued) overview, 869 pancreas, 871–872 pituitary, 869–870 reproductive system, 872–875 thyroid, 870 neuromedins (NMs) in, 886 neuropeptide Y (NPY) in, 839–843 adrenal, 839–840 gonadal function, 841–842 overview, 839 pancreas, 840–841 thyroid, 842–843 neurotensin (NT) in, 885 opioid peptides in, 829–831 adrenal gland, 830–831 overview, 829 pancreatic islets, 830 pituitary gland, 829–830 pituitary adenylate cyclase–activating polypeptide (PACAP) in, 847–851 adrenal, 849–850 ovary, 849 overview, 847 pancreatic islets, 850–851 pituitary, 847–849 testis, 849 thyroid, 849 tachykinin-gene-related peptides in, 833–836 adrenal, 835 Leydig cells, 835–836 ovary, 836 overview, 833 pancreatic islets, 834–835 parathyroid, 834 pituitary, 833–834 thyroid, 834 endometrial cancer, 425 endomorphins (EMs), 1333–1337 biological actions of, 1335–1336 discovery of, 1333 distribution in central nervous system, 1334 overview, 1333 pathophysiological implications, 1336–1337 receptor selectivity and modulation, 1335 Endonuclease colicins, 122 Endopeptidase 24.15, 1354 endothelial cells (ECs), 1164–1165, 1201, 1206 endothelial nitric oxide synthase (eNOS), 1166 endothelin-converting enzyme (ECE), 1189, 1269 endothelins, 447–450, 1187–1192 in airways, 1289–1291 elimination of, 1290 overview, 1289 pathophysiological role, 1290–1291 receptor subtypes and localization, 1290 synthesis of, 1290 and angiogenesis, 449 antagonism in vivo, 450

and apoptosis, 449 biological actions in cardiovascular system, 1191–1192 clinical trials, 450 discovery of, 1187 endothelin-1 (ET-1), 856–859, 1184, 1542 associated signal transduction pathways, 447–448 and tumor progression/metastases, 450 expression in cancer, 447–448 as mitogen, 449 overview, 447, 1187 plasma concentration and inactivation of endothelin 1, 1189–1190 precursor mRNA/gene processing, 1187–1189 precursor mRNA/gene structure, 1189 receptor distribution, 1190–1191 receptor expression in cancer, 448–449 in regulation of endocrine glands, 855–859 adrenal, 857–858 Leydig cells, 858 ovary, 858–859 overview, 855 pancreatic islets, 857 parathyroid, 856–857 pituitary, 855–856 thyroid, 856 renal, 1269–1273 as vasoactive peptide at BBB, 1464–1465 endozepines, 813–818 biological actions within brain, 817 control of biosynthesis and release of, 816 DBI mRNA and immunoreactivity distribution, 813–815 diazepam-binding inhibitor (DBI) processing, 815 discovery of, 813 overview, 813 pathophysiological implications, 817–818 precursor mRNA/gene structure, 813 receptors and signaling cascades, 815–816 solution conformation information, 816–817 enkephalins, 898, 1313–1317, 1429–1430 biological activity of, 1315–1316 growth factors, 1315–1316 neurotransmissions, 1315 overview, 1315 discovery of, 1313 mRNA and peptide distribution, 1313–1314 overview, 1313 pathophysiological implications, 1316–1317 precursor gene structure, 1313 processing of, 1314–1315 receptors, 1314–1315 classical opioid receptors, 1314–1315 nonclassical opioid receptor, 1315 overview, 1314

eNOS (endothelial nitric oxide synthase), 1166 enteric nervous system (ENS), 1139–1141 enterochromaffinlike (ECL) cells, 1039–1041, 1044–1045, 1066, 1092, 1124–1125 enteroglucagon, 1057–1064 active conformation and metabolism, 1059 biological actions of, 1060 discovery of, 1057–1058 expression of gene, 1058–1059 gene structure, 1057–1058 overview, 1057 receptor expression, 1059–1060 enterostatin, 969–973 feeding behavior effects, 969–970 functional responses to differing feeding and metabolic conditions, 970–971 genomic studies, 970 overview, 969 physiological and pathophysiological implications, 973 receptors and signaling pathways, 972–973 sites of action and neural networks affected, 971–972 central enterostatin response, 971–972 overview, 971 peripheral enterostatin response, 971 enzymatic active Pol (PR), 1499–1500 enzyme inhibitors, 358 EOG (electro-olfactogram) response, 323 Eotaxin (CCL11), 570 epidermal growth factor (EGF), 1150, 1290–1291, 1401–1402, 1437–1439 epidermal growth factor receptor (EGFR), 1087, 1437–1438 Epinephrine, 1476 epithelial ovarian cancer, 424 epitopes, 493 Epo. See erythropoietin (Epo) Equinatoxin II (EqII), 365–366 ERK (extracellular-signal-related kinase), 824, 931, 989, 1172 erythropoietin (Epo), 1393–1399 biological actions of, 1394–1398 beyond erythropoiesis, 1396 Epo and EpoR in CNS, 1396–1398 erythropoiesis, 1394–1396 overview, 1394 discovery of, 1393 mRNA distribution, 1393–1394 overview, 1393 pathophysiological implications, 1398–1399 abnormal serum levels, 1398 clinical uses of rHuEp, 1399 Epo and EpoR in experimental and clinical states, 1398 overview, 1398 precursor mRNA/gene structure, 1393 processing of, 1394 receptors, 1394 esophageal variceal hemorrhage, 1134

Index / 1575 ESP6 compound, 1376 ESP7 compound, 1375–1376 ET-1 endothelin precursor, 1290 ETA endothelin receptor, 447–449, 1190–1192, 1289–1291 ETA receptor knockout (KO) mice (CD ET-1 KO mice), 1272 ETB endothelin receptor, 447–449, 1190–1192, 1289–1291 ETHs (ecdysis triggering hormones), 163 eumenine mastoparan-AF (EMP-AF), 393 exercise and stress, 1329 exopeptidase reactions, in processing of preprocholecystokinin, 1015 exorphin-opioid active peptides of exogenous origin, 1365–1366 classification of exorphins according to their structure, 1368–1369 intraprotein opioid sequence, 1368 opioid antagonist and anti-opioid peptide derived from proteins, 1369–1371 anti-opioid peptides, 1369–1371 opioid antagonist peptides, 1369 overview, 1369 opioid peptides derived from animal proteins, 1366–1368 α-casein exorphin, 1367 αs1-casomorphin, 1367 β-casomorphin, cytochrophin, and hemorphin, 1366–1367 neocasomorphin and other caseinderived peptides, 1367 opioid peptides derived from other animal proteins, 1367–1368 overview, 1366 opioid peptides derived from plant proteins, 1365–1366 gluten exorphins, 1365–1366 overview, 1365 Rubiscolins, 1366 overview, 1365 experimental autoimmune encephalitis (EAE), 603–608 extracellular matrix (ECM), 1171–1172 extracellular-signal-related kinase (ERK), 824, 931, 989, 1172 F FaRPs. See FMRFamide-related peptides (FaRPs) Fasciculins, 356–357 FATPSBS protein, 972 FCAPs (feeding-circuit activating peptides), 237–238 feeding-circuit activating peptides (FCAPs), 237–238 feeding regulation amylin and calcitonin-gene-related peptide (CGRP) in, 981–985 anorectic action of peripheral amylin, hypothalamic involvement in, 984 central pathways, 983–984 food intake effects, 981–982 food intake in animals deficient of CGRP or overexpressing CGRP, 983

food intake in animals overexpressing amylin, 983 food intake in knockout animals, 982–983 interaction between amylin and metabolites, 985 overview, 981 physiological and pathophysiological implications, 985 receptors and signaling pathways, 984–985 synthesis regulation, 983 by glucagonlike peptide 1 (GLP-1), 975–978 feeding behavior effects, 975 functional response of to differing metabolic and feeding states, 976 interactions with other peptidergic/ aminergic systems, 977 overview, 975 physiological and pathophysiological implications, 977–978 receptors and signaling pathways, 976–977 sites of action and neural networks affected by, 976 studies from genetic manipulations and synthetic analogs, 975–976 and leptin, 987–991 absence of, 987 feeding behavior, 987 functional responses to during altered metabolic states, 988 overview, 987 physiological and pathophysiological implications, 990–991 receptors and signaling pathways, 988–990 sites of action, 988 FFA (free fatty acids), 1476 FGLamide family of ASTs, 205 fibroblast growth factor (FGF), and blood–brain barrier (BBB), 1449–1453 CSF-ependymal-brain interface, 1452 FGF1/FGF2 transport across BBB, 1450–1451 FGF2 transport and regulatory phenomena at choroid plexus, 1451–1452 FGF-induced protection of cerebral endothelium and neurons, 1450 modulation of cerebral endothelial cells, 1451 overview, 1449 peptide regulatory system modeling, 1452–1453 secretion of by microvascular endothelial cells, 1451 Fibronectin, 576 fish peptides, 1515–1519 adrenomedullin 2/intermedin, 1516–1517 melanin-concentrating hormone, 1515–1516 overview, 1515

stanniocalcins (STC), 1517–1519 biological actions of, 1517 discovery of, 1517 mRNA and protein distribution, 1517 overview, 1517 pathophysiological implications, 1517–1518 precursor mRNA/gene structure, 1517 receptors, 1517 urotensins, 1516 FL/IRFamides, extended biological actions of, 198 discovery of, 193–194 mRNA distribution, 195 precursor mRNA/gene structure, 194–195 processing of, 196 receptors, 197 structure-activity and active conformation, 198 FLPs. See FMRFamide-like peptides (FLPs) FLRFamide, 224 biological actions of, 224 discovery of, 224 FM-3 G-protein coupled receptor, 746–747 FM-4 G-protein coupled receptor, 747 FMRFamide-like peptides (FLPs) in free-living nematode peptides, 247–250 discovery and structure of, 247–249 expression of, 249 receptors and functions of, 249–250 in parasitic nematode peptides, 255–259 behavior and body length effects: Ascaris suum, 258 cAMP effects, 259 discovery of, 255–256 effect on muscle, 257–258 nerve effects, 258–259 peptide/mRNA distribution, 256–257 precursor mRNA/gene structure, 256 receptors, 257 FMRFamide-related peptides (FaRPs), 235–236 biological actions of, 236 discovery of, 235 mRNA distribution, 235–236 precursor mRNA/gene structure, 235 receptors, 236 FMRFamides, 235–236 biological actions of, 236 discovery of, 235 extened biological actions of, 198 discovery of, 193–194 mRNA distribution, 195 precursor mRNA/gene structure, 194–195 processing of, 196 receptors, 197 structure-activity and active conformation, 198 mRNA distribution, 235–236

1576 / Index FMRFamides (continued) precursor mRNA/gene structure, 235 receptors, 236 follicle-stimulating hormone (FSH), 635, 824–825, 1470–1471 formylpeptide receptors (FPR), 547–548 4-kDa peptide, 1–4 active and/or solution conformation, 2 biological actions of, 2–4 discovery of, 1 mRNA distribution, 1–2 oveview, 1 precursor mRNA/gene structure, 1 processing of, 2 receptors, 2 43-kDa basic glycoprotein (43 k-P), 1–2 FPR (formylpeptide receptors), 547–548 free fatty acids (FFA), 1476 free-living nematode peptides. See nematode peptides, free-living FRUITFUL MADS-box gene (FUL/ AGL8), 21 FS2, 357 FSH (follicle-stimulating hormone), 635, 824–825, 1470–1471 FUL/AGL8 (FRUITFUL MADS-box gene), 21 functional dyspepsia, 1135 fungal ribosome inactivating proteins, 145–148 biological actions of, 146–148 discovery of, 145 overview, 145 structure of, 146 Furin enzyme, 809 FXPRLamide (Pyrokinin/PBAN), 207–211 biological actions of, 211 discovery of, 207–208 mRNA and peptides distribution, 208–209 overview, 207 precursor mRNA/gene structure, 207–208 processing of, 209–210 receptors, 210 structure–activity relationships and active conformation, 210–211 G GABA (γ-amino butyric acid), 891, 906–907 GABAergic interneurons, 647 galanin (Gal) in gastrointestinal tract, 1037–1041 biological effects of, 1038 distribution of, 1037 inflammation and injury, 1041 overview, 1037 receptors, 1038–1041 hypothalamic, 895–899 in regulation of endocrine glands, 883–885 expression of, 883–884 hormone secretion, 884–885 overview, 883

galanin/GALP systems, 753–760 central actions of galanin/GALP in normal and pathophysiology, 756–760 Alzheimer’s disease, 758–759 anxiety, 759 feeding and metabolism, 757 galanin—normal physiology, 756–757 galanin—pathophysiology, 758 GALP—normal physiology, 759–760 GALP—pathophysiology, 760 learning and memory, 757 neural injury and repair, 759 nociception, 757–758 osmotic regulation, 757 seizures and epilepsy, 758 discovery of, 753 gene structure and regulation, 753–754 mRNA and peptide-immunoreactivity distribution, 754–755 overview, 753 precursor nature and processing, 754–755 receptors, 755–756 galanin knockout (KO) mice, 757–759 galanin-overexpressing (OE) mice, 757–759 Galanin receptor 1 (GalR1), 755–757, 1040–1041 Galanin receptor 2 (GalR2), 755–757, 1040–1041 Galanin receptor 3 (GalR3), 755–756 gallbladder, 1018, 1102 GalR, 1039–1040 γ-amino butyric acid (GABA), 891, 906–907 gastric acid secretion, 1001, 1007–1008, 1024–1025, 1067, 1102, 1118 gastric cancer, 1053 gastric inhibitory peptide (GIP), 1060 gastric protection and repair, 1001–1002 gastrin, 1043–1045 biological actions on GI tract, 1044–1045 acid secretion, 1044 growth, 1044–1045 overview, 1044 and cancer, 467–470 expression of, 467–469 overview, 467 receptor(s), 468 as tumor growth factor, 469 discovery of, 1043 gene and precursor structure, 1043 mRNA distribution in gut, 1044 overview, 1043 pathophysiological implications, 1045 receptor expression and regulation, 1044 gastrin/cholecystokinin (CCK). See cholecystokinin (CCK)/gastrin gastrinomas, 468, 1133 gastrin-releasing peptide, 1047–1054 biological actions within GI tract, 1049–1053 acid secretion effects, 1050–1052 gastrin release effects, 1050–1052

gastrointestinal hormone effects, 1052 motility, 1052–1053 overview, 1049–1050 pancreatic secretion effects, 1052 satiety, 1053 trophic effects, 1053 distribution of GRP-like immunoreactivity, 1048–1049 mRNA, 1048–1049 receptor, 1048–1049 overview, 1047 pathophysiological implications, 1053–1055 GRP antagonists as potential acid blockers, 1054 overview, 1053 protective role of, 1054 tumor growth role of, 1053–1054 precursor mRNA/gene and peptide variant structure, 1047–1048 chemistry and molecular biology, 1047–1048 isolation from tissue, 1048 overview, 1047 receptor subtypes and signaling, 1049 gastrin-releasing peptide (GRP), 277, 280, 1121 gastrin-releasing peptideimmunoreactivity, 1048 gastroenteropancreatic (GEP) hormone, 1131–1132 gastroenteropancreatic neuroendocrine tumors, and somatostatin analogs, 1131–1133 carcinoid tumors, 1132–1133 gastrinomas, 1133 glucagonomas, 1133 insulinomas, 1133 overview, 1131–1132 VIPomas, 1133 gastrointestinal fistula, 1136 gastrointestinal function adrenomedullin in, 999–1002 biological actions of, 1001–1004 distribution of, 1000 expression of, 1000 overview, 999 receptors and signaling pathways, 1000–1001 release of, 1000 structure of, 999 and calcitonin gene-related peptide (CGRP), 1005–1010 distribution of, 1005–1006 overview, 1005 physiological and pathophysiological implications of, 1007–1011 receptors, 1006–1007 release of, 1007 regulation, neurotensin (NT) in, 1085–1090 colonic motility enhancement, 1086 colonic stress responses, 1087–1088 GI mucosa healing, 1087

Index / 1577 intestinal inflammation, 1087 motility and secretion inhibition in stomach and small intestine, 1086 overview, 1085 promotion of cell growth and regeneration, 1087 secretion stimulation, 1086–1087 gastrointestinal hormone effects, 1052 gastrointestinal motility, 1067–1068, 1134–1135 gastrointestinal system, corticotrophinreleasing hormone (CRH) family in, 1023–1027 biological actions of, 1024–1026 gastric and duodenal acid/ bicarbonate secretion, 1024–1025 GI motor alterations, 1025 overview, 1024 pancreatic secretion, 1025–1026 neuronal pathways, 1026 overview, 1023 pathophysiological implications, 1026–1028 intestinal mucosal pathophysiological responses, 1026 overview, 1026 visceral pain, 1027 receptor expression and location, 1024 related peptide expression and location, 1023–1024 gastrointestinal tract, 1101 galanin in, 1037–1041 biological effects of, 1038 distribution of, 1037 inflammation and injury, 1041 overview, 1037 receptors, 1038–1041 and leptin, 1071–1076 intestinal endocrine secretion regulatation, 1072–1073 intestine induction of, 1072 leptin and gut pathologies, 1074 neuroendocrine molecule for satiety, 1072 nutrient absorption, 1073–1074 overview, 1071 stomach production of, 1071–1072 somatostatin analogs in, 1131–1136 biliary system, 1136 esophageal variceal hemorrhage, 1134 gastroenteropancreatic neuroendocrine tumors, 1131–1133 GI motility and functional GI disorders, 1134–1135 overview, 1131 postoperative complications of surgery, 1135–1136 secretory diarrheas, 1133–1134 substance P in, 1139–1144 biological actions of, 1141–1142 discovery of, 1139 mRNAS and peptides distribution, 1139–1140 overview, 1139 pathophysiological implications of, 1142–1145

gastroprotection, 1067 G cells, 1044–1045 GEP (gastroenteropancreatic) hormone, 1131–1132 GFR (glomerular fi ltration rate), 1246 GGF2, 1404 GH (growth hormone), 731–734, 869–873, 953, 1193–1196, 1385–1387, 1521–1524 GH-releasing activity, 1194 ghrelin, 731–735, 1065–1069 alternative discoveries of, 1065 biological action on gastrointestinal tract, 1067–1069 gastric acid secretion, 1067 gastrointestinal motility, 1067–1068 gastroprotection, 1067 overview, 1067 biological actions within brain, 734–735 discovery of, 731–732 distributionin gastrointestinal tract, 1066 and ingestive behavior, 953–958 extra-hypothalamic effects on food intake, 956 inducing food intake with administration of, 953–954 mediating ghrelin-induced food intake with growth hormone secretagogue receptor GHSR-1, 954 other hypothalamic pathways involved in ghrelin-induced hyperphagia, 955–956 overview, 953 regulating levels of by food intake, 956–958 targeting hypothalamus, 954–955 mRNA distribution, 732 overview, 731, 1065 pathophysiological implications, 735, 1068 precursor and mRNA/gene strucure, 731–733, 1065–1066 precursor processing, 732–734 receptor, 734 in regulation of endocrine glands, 869–873 adrenal cortex, 872 cartilage and bone homeostasis, 871 heart and cardiomyocytes, 870–871 overview, 869 pancreas, 871–872 pituitary, 869–870 reproductive system, 872–875 thyroid, 870 structure–activity relations of ghrelin and of ghrelin receptor, 1066–1067 therapeutic potential in heart failure, 1193–1196 biological actoins of, 1194–1195 clinical application of, 1195–1196 discovery of, 1194 overview, 1193 receptors and their distribution, 1194 structure of, 1194 synthesis of, 1194 therapeutic potential of, 1068–1069

ghrelin knockout (KO) mice, 1067–1068 ghrelin receptor (GHS-R), 734 GHRH. See growth hormone-releasing hormone (GHRH) GHS-R. See growth hormone secretagogue receptor (GHS-R) GHSs (growth hormone secretagogues), 953–956, 1193 Gigantin, 148 GIH (Gonad-inhibiting hormone), 230–233 GIP (gastric inhibitory peptide), 1060 glicentin, 1057–1064 active conformation and metabolism, 1059 biological actions of, 1060 discovery of, 1057–1058 expression of gene, 1058–1059 gene structure, 1057–1058 overview, 1057 receptor expression, 1059–1060 glomerular filtration rate (GFR), 1246 glomerular infiltration rate, 1270–1273 glomerulonephritis, 1259 glucagon-like peptide-1, 567 glucagonlike peptide 1 (GLP-1), regulation of feeding behavior by, 975–978 feeding behavior effects, 975 functional response to differing metabolic and feeding states, 976 interactions with other peptidergic/ aminergic systems, 977 overview, 975 physiological and pathophysiological implications, 977–978 receptors and signaling pathways, 976–977 sites of action and neural networks affected by, 976 studies from genetic manipulations and synthetic analogs, 975–976 glucagonlike peptide 1 receptor (GLP1R), 975–978 glucagonlike peptides 1 and 2 (GLP-1 and GLP-2), 1057–1064 active conformation and metabolism, 1059 biological actions of, 1060 discovery of, 1057–1058 expression of gene, 1058–1059 gene structure, 1057–1058 GLP-1 actions, 1060–1062 effects on appetite and food intake, 1061–1062 effects on gastrointestinal tract, 1061 effects on islets, 1060–1061 other actions, 1062 overview, 1060 overview, 1057 pathophysiological implications, 1063–1064 L-cell secretion disturbances, 1063 overview, 1063 therapeutic application of proglugonderived peptides, 1063–1064 receptor expression, 1059–1060

1578 / Index glucagon like protein-1 receptor (GLP-1 receptor), 1059–1060 glucagon like protein-2 receptor (GLP-2 receptor), 1059–1060 glucagonomas, 1133 glucocorticoids, 915 Glucokinase (GK), 977 glucose transporter 2 (GLUT-2), 977 GLUT-1 deficiency syndrome, 1477–1478 GLUT-2 (glucose transporter 2), 977 glutamate receptors/transporters, 376 Glutathione, 1424 Gluten exorphin-B5, 1365 Glycogen synthase kinase-3βGSK3β, 633 GM-CSF (granulocyte monocyte colonystimulating factor), 1438 GnRH. See gonadotrophin-releasing hormone (GnRH) Gonad-inhibiting hormone (GIH), 230–233 gonadotrophin-releasing hormone (GnRH), 635–644, 1470–1471 background, 635 biological actions of, 642–643 discovery of, 635 distribution of, 637–638 in molluscan peptides and reproduction, 242–243 biological actions of, 242–243 mRNA distribution, 242 precursor mRNA/gene structure, 242 overview, 635 pathophysiology, 643–644 physical structure of, 642 precursor processing, 638–639 precursor structure, 635–637 primary structures, 635–637 three-dimensional structures, 637 receptors agonist binding to, 640 desensitization, 642 genes, 642 insights into tertiary structure of, 640 overview, 638–640 signalling cascades gonadotrophin gene expression regulation, 641–642 intracellular signalling pathways, 640–641 overview, 638–640 gonadotropin releasing hormone (GnRH), 421, 824–825 GPCRs (G protein-coupled receptors), 174–175, 196, 203–204, 639–640, 775, 787 GPI (Guinea pig ileum) assay, 1367–1370 GPR54 (G-protein-coupled receptor 54), 821–827 G-protein-coupled receptor 54 (GPR54), 821–827 G protein-coupled receptors (GPCRs), 174–175, 196, 203–204, 639–640, 775, 787 gram-positive bacteria, nonlantibiotic heat-stable bacteriocins in, 107–112 biological actions of, 110 discovery of, 107–109

future trends, 112 overview, 107 receptors, 110–111 structure, 111–112 synthesis of, 107–110 granulocyte monocyte colony-stimulating factor (GM-CSF), 1438 Group A colicins, 117 Group B colicins, 117 growth disorders, 1389 growth factors, 1315–1316 growth hormone (GH), 731–734, 869–873, 953, 1193–1196, 1385–1387, 1521–1524 growth hormone-releasing hormone (GHRH), 663–669, 1521–1522 analog conformation and synthesis, 668 biological activity of, 668–669 discovery of, 663 expression in brain, 664–665 overview, 663 pathophysiological implications, 669 precursor mRNA/gene structure, 663–664 precursor processing, 665 receptor, 665–667 signaling, 667–668 growth hormone–releasing hormone (GHRH) antagonists in cancer, 483–488 antagonistic analogs of, 484–485 effects on human experimental cancers in vivo, 486–487 and GHRH receptors in human cancers, 485–486 mechanisms of tumor inhibition by, 486 overview, 483 therapeutic indications of, 484 growth hormone secretagogue receptor (GHS-R), 731, 870, 906, 1193–1196 growth hormone secretagogues (GHSs), 953–956, 1193 GRP (gastrin-releasing peptide), 277, 280, 1121 GsMtx-4, 410 Guinea pig ileum (GPI) assay, 1367–1370 H HABPs (high activity binding peptides), 515–523 hainantoxins (HNTX), 372–373 half-cystine pairs, 417–418 HbsAg (Hepatitis B virus antigen), 597 HC-toxin, 154 heart failure, as clinical implication of urodilatin, 1248 Heme oxygenase-1 (HO-1), 1173 Hemodyalisis, 1258–1259 Hemorphin-7, 570 hemorphins. See β-casomorphins and hemorphins Henle’s loop, 1252–1253 heparin sulfate proteoglycan (HSPG), 1446 Hepatitis B virus antigen (HbsAg), 597 HER-2/neu, 492, 502–503

Herceptin, 492 Heterocyclization, 89 HF (high-fat)LF ( vs. low-fat diets), 969–971 HGAC 39.G3 monoclonal antibody, 597 HIF-1 (hypoxia inducible factor-1), 454, 1395–1396 high activity binding peptides (HABPs), 515–523 high-fat (HF) vs. low-fat diets (LF), 969–971 highly immunogenic Tat molecule (Tat toxoid), 1499 high molecular weight kininogen (HMWK), 443, 1175–1177 high-voltage-activated Cav channels (HVA), 375 histidine-rich proteins (HRP), 518 Historphin, 1367 HIV-1-derived synthetic peptides, 1495–1496 HIV-1 Gag proteins NC and MA, 1501 HIV-1 p6 Gag protein, 1500–1501 HIV-1 protease, 1499–1500 HIV-1-specific virus protein U, 1496–1497 HLA (human leukocyte antigen), 585–588 HLA II (human leukocyte antigen class II), 575–576 HMWK (high molecular weight kininogen), 443, 1175–1177 HNTX (hainantoxins), 372–373 HO-1 (Heme oxygenase-1), 1173 Homodestruxin B, 152 host defense peptides. See cathelicidins; neuropeptides, amphibian host-selective toxins (HSTs), 151 from Bipolaris zeicola race 3, 154–155 from genus Alternaria, 152–154 alternaria alternata f. sp. mali, 152–154 alternaria brassicae, 152 from genus Cochliobolus, 154 C. carbonum race 1, 154 C. victoriae, 154 from Leptosphaeria maculans, 155 HPA (hypothalamo-pituitary-adrenal) axis, 712, 839–840, 861 H. pylori, 1068 HRP (histidine-rich proteins), 518 HSPG (heparin sulfate proteoglycan), 1446 HSTs. See host-selective toxins (HSTs) human leukocyte antigen (HLA), 585–588 human leukocyte antigen class II (HLA II), 575–576 human low-molecular-weight kininogen (LMWK), 1175–1177 human narcolepsy, 726–727 HVA (high-voltage-activated Cav channels), 375 Hydroxydestruxin B, 152 Hydroxyproline-rich glycopeptides (HypSys peptides), 49 hyperalgesia and pain, 1143–1144

Index / 1579 hyperglycemic hormones, crustacean, 230–233 biological actions of, 233 discovery of, 230 gene expression, 232 gene organization, 230–232 processing of, 233 receptors, 232 Hyperphagia, 899 hypersecretion and inflammation, 1143 hypertension, 1205, 1258 Hypocretin receptors, 723–724 hypocretins, 721–727 active and/or solution conformation, 724 biological actions within brain, 724–726 discovery of, 721 mRNA distribution, 722 overview, 721 pathophysiological implications, 726–727 canine narcolepsy, 726 human narcolepsy, 726–727 mouse knockout mutants, 726 precursor mRNA/gene structure, 721–722 precursor processing, 723 receptors and signaling cascades, 723–724 hypothalamic dorsomedial (DMN), 699 hypothalamic galanin (GAL), and ingestive behavior, 895–899 circulating hormones and dietary conditions on, 896–897 GAL gene or GAL receptor gene mutation effects, 896 injection on ingestive behavior, 895– 896 overview, 895 physiological and pathophysiological consequences, 898–899 receptors and signaling pathways, 898 in relation to other peptidergic and aminergic systems, 898 sites of action and neural networks affected, 897–898 hypothalamic neuropeptides, and blood– brain barrier (BBB), 1469–1472 circumventricular organs, 1469–1470 corticotropin-releasing hormone, 1471 GnRH, 1470–1471 overview, 1469 oxytocin, 1472 PACAP, 1472 somatostatin, 1472 TRH, 1470 vasopressin, 1471 hypothalamic-pituitary-adrenocortical system, 1523–1524 basic activity, 1523 corticotropin-releasing hormone, 1523 effects of changes in sleep-wake behavior on HPA hormones, 1523 overview, 1523 sleep in disorders with pathological changes of HPA activity, 1523 vasopressin, 1523

hypothalamic-pituitary- somatotropic system, 1521–1523 animal models of HPS system changes, 1522–1523 basic activity, 1521 ghrelin, 1522 growth hormone-releasing hormone, 1521–1522 overview, 1521 somatostatin, 1522 hypothalamo-pituitary-adrenal (HPA) axis, 712, 839–840, 861 hypoxia inducible factor-1 (HIF-1), 454, 1395–1396 I IBD (irritable bowel disease), 1142–1143 IBS (irritable bowel syndrome), 1087–1088, 1135, 1144 ICAM-1 (intercelllular adhesion molecule-1), 575 ICK (inhibitory cysteine knot motif), 370 ICV administration. See intracerebroventricular (ICV) administration Idiopathic pulmonary arterial hypertension (IPAH), 1283–1286 IgA nephropathy, 1258, 1259 IGF. See Insulin-like growth factor IGFBP3 (insulin-like growth factor binding protein-3), 1388 Ii polypeptide, 613–614 Il-1 (Interleukin-1), 694 IL-6 (Interleukin-6), 1396 IMCD (inner medullary collecting duct), 1244–1247, 1253, 1254–1255 IMD (intermedin). See adrenomedullin 2 (AM2) immune and inflammatory responses, 1309 immune regulation, 455–456 immune response regulating neuropeptides, 579–582 immunomodulatory peptides, 411 immunoreactive endothelin peptides (ETIR), 855–856 immunotoxins, 418 inflammation, in gastrointestinal tract galanin, 1041 ingestive behavior and cocaine- and amphetamineregulated-transcript (CART), 913–916 functional response to differing metabolic and feeding states, 914 interactions with other peptidergic/ aminergic systems, 915 overview, 913 physiological and pathophysiological implications, 916 receptors and signaling pathways, 915 sites of action and neural networks affected, 914–915 studies from genetic manipulations or mutations, 913 and corticotrophin-releasing hormone (CRH), 937–940

feeding behavior effects, 937–938 interactions with neuropeptide systems, 939–940 metabolic regulation of, 938 overview, 937 physiology and pathophysiological implications, 940 receptors and signaling pathways, 939 sites of action and neural networks affected, 938–939 studies from genetic manipulations, 938 and ghrelin, 953–958 extra-hypothalamic effects on food intake, 956 inducing food intake with administration of, 953–954 mediating ghrelin-induced food intake with growth hormone secretagogue receptor GHSR-1, 954 other hypothalamic pathways involved in ghrelin-induced hyperphagia, 955–956 overview, 953 regulating levels by food intake, 956–958 targeting hypothalamus, 954–955 and hypothalamic galanin (GAL), 895–899 circulating hormones and dietary conditions on, 896–897 GAL gene or GAL receptor gene mutation effects, 896 injection on ingestive behavior, 895–896 overview, 895 physiological and pathophysiological consequences, 898–899 receptors and signaling pathways, 898 in relation to other peptidergic and aminergic systems, 898 sites of action and neural networks affected, 897–898 and melanin-concentrating hormone (MCH), 929–933 feeding behavior effects, 929–930 functional response to differing metabolic and feeding states, 930–931 interactions with other peptidergic/ aminergic systems, 932 overview, 929 physiological and pathophysiological implications, 932–933 receptors and signaling pathways, 932 sites of action and neural networks affected, 931–932 studies from genetic manipulations or mutations, 930 and melanocortins, 903–909 feeding behavior effects, 903–904 functional response to differing metabolic and feeding states, 905–907

1580 / Index ingestive behavior (continued) genetic studies on, 904–905 interactions with other peptidergic/ aminergic systems, 909 overview, 903 physiological and pathophysiological implications, 909 receptors and signaling pathways, 908–909 system neuroanatomy, 907–908 and opioids feeding behavior effects, 920–921 functional response of, 922 interaction between OxA and opioids, 924 interactions with other peptidergic/ aminergic systems, 923 physiological and pathophysiological implications, 924 sites of action and neural networks affected, 922–923 studies from genetic manipulations or mutations, 921 and orexins feeding behavior effects, 919–920 functional response of, 921–922 interaction between OxA and opioids, 924 interactions with other peptidergic/ aminergic systems, 923 physiological and pathophysiological implications, 923–924 sites of action and neural networks affected, 922 studies from genetic manipulations or mutations, 921 and peptide Y Y (PY Y) and neuromedin U (NMU), 945–949 discovery, 946 distribution of, 946 introduction, 945–946 NMU role in energy balance regulation, 947–948 overview, 945 PY Y, 948–949 receptors, 946–947 release of, 946 ingestive peptides, and blood–brain barrier (BBB), 1455–1458 ghrelin, 1457 ingestive peptides and proteins, 1457–1458 insulin, 1455–1456 leptin, 1456–1457 overview, 1455 inhibitory cysteine knot motif (ICK), 370 inner medullary collecting duct (IMCD), 1244–1247, 1253, 1254–1255 Inositol-1-4-5-trisphosphate (IP3), 640 insects antidiuretic and diuretic hormones, 157–161 biological actions of, 160–161 discovery of, 157–158 mRNA distribution, 159 overview, 157

peptide distribution, 159 peptide processing, 159 precursor mRNA/gene structure, 158–159 receptors, 159–160 structure-activity and active conformation, 160 myosuppressins biological actions of, 198 discovery of, 193–194 mRNA distribution, 195 precursor mRNA/gene structure, 194 processing of, 196 receptors, 196–197 structure-activity and active conformation, 197–198 pigment dispersing factor (PDF), 213–215 biological actions of, 214–215 discovery of, 213–214 mRNA distribution, 213–215 precursor mRNA/gene structure, 213–214 processing of, 213 receptors, 214 insulin, 250–252, 983, 993–997 discovery and structure of, 250–252 expression, 252 feeding behavior effects, 993–995 genetic and molecular biological manipulations, 995 metabolic states, 995–996 overview, 993 physiological and pathophysiological implications, 996–997 receptors and functions of, 252 receptors and signaling pathways, 996 resistance, 996–997, 1478 secretion, 851 site of action and neural networks affected, 996 Insulinlike growth factor-1 (IGF-1), 1193–1196 insulin-like growth factor-1 (IGF-1), 1385–1390, 1457 active and/or solution conformation, 1388 biological actions of, 1388–1389 discovery of, 1385 mRNA distribution, 1386–1387 overview, 1385 pathophysiological implications, 1389–1390 cancer, 1389 growth disorders, 1389 neuroprotection, 1389–1390 overview, 1389 precursor mRNA/gene structure, 1385–1386 processing of, 1387 receptors, 1387–1388 insulin-like growth factor-2 (IGF-2), 1385–1387 insulin-like growth factor binding protein-3 (IGFBP3), 1388 insulinomas, 1133

insulin receptor (IR), 2–3 insulin-receptor-substrate (IRS), 990 integrins, 573 intercelllular adhesion molecule-1 (ICAM-1), 575 Interleukin-1 (Il-1), 694 Interleukin-6 (IL-6), 1396 intermedin (IMD). See adrenomedullin 2 (AM2) interstitial angiotensin II, 1238 intestinal motility, 1102 intestinal mucosal pathophysiological responses, 1026 intracellular angiotensin II, 1239 intracellular Ca2+ levels ([Ca2+]i), 323–325 intracerebroventricular (ICV) administration, 693–694, 724 of enterostatin, 971 of melanin-concentrating hormone, 929–930 of neuromedin U, 748–750, 947–948 of urotensin II, 800 intrarenal Ang II, 1239–1240 intrarenal angiotensin II receptors, 1237–1238 intrinsic primary afferent neurons (IPANs), 1140–1141 invertebrate AKH/RPCH, 189–192 biological actions of, 191–192 discovery of, 189–190 distribution of, 190 overview, 189 peptide and precursor structure and processing of, 189–190 receptors, 191 solution conformation of, 190–191 invertebrate tachykinins (invTKs), 171–176 biological actions of, 175 discovery of, 171–172 mRNA and peptides distribution, 173–174 overview, 171 precursor mRNA/gene structure, 172–173 processing of, 174 receptors, 174–175 structure-activity and active conformation, 175 invTKs. See invertebrate tachykinins (invTKs) ion channel blockers and modulators, 363–365 potassium channel toxins, 364–365 sodium channel toxins, 363–364 IP3 (Inositol-1-4-5-trisphosphate), 640 IPAH (Idiopathic pulmonary arterial hypertension), 1283–1286 IPANs (intrinsic primary afferent neurons), 1140–1141 IR (insulin receptor), 2–3 irritable bowel disease (IBD), 1142–1143 irritable bowel syndrome (IBS), 1087–1088, 1135, 1144 IRS (insulin-receptor-substrate), 990 Isarfelin, 125

Index / 1581 J Jak kinase family, 989–990 JGA (juxtaglomerular apparatus) cells, 1236 juvenile hormone (JH), 201 juxtaglomerular apparatus (JGA) cells, 1236 JV-1-63 growth hormone-releasing hormone antagonist, 484 K K+ channel specific toxins (KTxs), 339–342 K2K (Lys2Lys) peptide dendrimer cores, 542 Kallikrein-kinin system (KKS), 1175, 1462–1463 KB7 strain of Pseudomonas aeruginosa, 510–513 K-coil peptide, 513 kidney function, and prolactin, 1277–1281 human renal function, 1280 osmoregulation, 1278 overview, 1277 production site and actions of, 1277 receptors, 1278–1279 renal actions of, 1279–1281 renal production of, 1279 transgene method clarification, 1281 Kinin B1 receptors, 444 Kinin B2 receptors, 444 kinin-related peptides, 392 kinins, 226 KiSS-1/metastin, 821–827 biological actions within brain and pituitary, 824–826 discovery of, 821 gene structure, 822 GPR54 as receptor in structure and signaling cascades, 824 mRNA/protein distribution within brain, 822–823 overview, 821 pathophysiological implications, 826–827 precursor processing, 822 Kisspeptin-14, 822 KKS (Kallikrein-kinin system), 1175, 1462–1463 κ-ligands, 1490 κ-opioid receptors (KOR), 1321–1322, 1335–1336 KOR (κ-opioid receptors), 1321–1322, 1335–1336 KTxs (K+ channel specific toxins), 339–342 L LAB (lactic acid bacteria), 107 lactic acid bacteria (LAB), 107 LanP proteases, 98 lantibiotics, 97–103 active and/or solution conformation, 98–102 biological actions of, 100–103 discovery of, 97

mRNA distribution, 97–98 oveview, 97 precursor mRNA/gene structure, 97 processing of, 98 receptors, 98 large dense-cored vesicles (LDCV), 622 Laron syndrome (LS), 1389 latent inhibition (LI) model of schizophrenia, 739 lateral hypothalamic area (LHA), 931, 956, 984 lateral parabrachial nucleus (IPBN), 984 lateral parabrachial nucleus (LPB), 1026 lateral ventricular recess (LVR), 708 LB1 group peptides, 529–532 LDCV (large dense-cored vesicles), 622 LDL (low-density lipoprotein) receptor, 1477 left ventricular (LV) function, 1193–1196 LeHypSys subfamily, 50–51 lentiviral protein R, 1497–1498 leptin, 740, 893, 915, 930, 993–997, 1103 and gastrointestinal tract, 1071–1076 intestinal endocrine secretion regulatation, 1072–1073 intestine induction of, 1072 leptin and gut pathologies, 1074 neuroendocrine molecule for satiety, 1072 nutrient absorption, 1073–1074 overview, 1071 stomach production of, 1071–1072 and regulation of feeding, 987–991 absence of, 987 feeding behavior, 987 functional responses during altered metabolic states, 988 overview, 987 physiological and pathophysiological implications, 990–991 receptors and signaling pathways, 988–990 sites of action, 988 LeSys receptor, 52 LeSys subfamily, 50 leucine-rich repeat kinases (LRR), 12–13 leucine-rich repeat receptor-like kinases (LRR-RLK), 31 leukocyte function associated antigen-1 (LFA-1), 575 leuteinizing hormone (LH), 635, 740, 760, 824–825, 829–830 leuteinizing hormone releasing hormone (LHRH), 884 Leydig cells, 872–873 LFA-1 (leukocyte function associated antigen-1), 575 LH (leuteinizing hormone), 635, 740, 760, 824–825, 829–830 LH (luteinizing hormone), 1470–1471 LHA (lateral hypothalamic area), 931, 956, 984 LH self-stimulation (LHSS), 725 LHSS (LH self-stimulation), 725 LI (latent inhibition) model of schizophrenia, 739

Lipid rafts, 481 Lipodystrophy, 987 Lipopolysaccharide (LPS), 1456 LMWK (low molecular weight kininogen), 443 long form of the leptin receptor (LRb), 988–991 long-term potentiation (LTP), 679, 1356 low-density lipoprotein (LDL) receptor, 1477 low molecular weight kininogen (LMWK), 443 low-voltage-activated Cav channels (LVA), 375 Loxoscelism, 378 LPB (lateral parabrachial nucleus), 1026 LPS (Lipopolysaccharide), 1456 LRb (long form of the leptin receptor), 988–991 LRR (leucine-rich repeat kinases), 12–13 LRR-RLK (leucine-rich repeat receptorlike kinases), 31 LS (Laron syndrome), 1389 LTP (long-term potentiation), 679, 1356 lung, tachykinins in, 1301–1304 localization of, 1302 overview, 1301 receptor distribution, 1302 receptor pharmacology, 1301–1302 tachykinin-mediated biological effects, 1302–1303 therapeutic potential of dual tachykinin NK 1-NK 2 receptor blockade, 1303–1304 therapeutic potential of tachykinin NK 1 receptor blockade, 1303 therapeutic potential of tachykinin NK 2 receptor blockade, 1303 therapeutic potential of tachykinin NK 3 receptor blockade, 1304 lung cancer, 1311 luteinizing hormone (LH), 1470–1471 luteinizing hormone-releasing hormone (LHRH) analogs, 421–426 antagonists of, 422 cytotoxic, 423–425 breast cancer, 424 endometrial cancer, 425 epithelial ovarian cancer, 424 melanoma, 425 non-Hodgkin’s lymphoma (NHL), 425 other cancers, 425 renal cell carcinoma (RCC), 425 side effects of, 425 effects on tumors, 423 overview, 421–422 receptors for, on tumors, 422 LVA (low-voltage-activated Cav channels), 375 LVR (lateral ventricular recess), 708 M Mabs (monoclonal antibodies), 596–597 MAC-1 macrophage receptor of integrins, 575

1582 / Index Macrocyclization, 89 MAGE-1 (melanoma antigen-1), 500 magnocellular neurons (MCN), 621–627 major histocompatibility complex (MHC) class II, 520–521 major urinary proteins (MUPs), 1511 malaria, peptide vaccines for, 515–524 malarial antigens in target cell binding, 517–518 malarial vaccines, 516–517 overview, 515–516 vaccine design based on malarial antigens’ structural modification, 522–524 based on MHC-peptide-TCR complex conformation, 520–521 based on structural-function relationship, 518–520 based on structurally modified antigen binding to HLA-DRβ1* molecules, 521–522 mamba disintegrin-like peptides, 357 mamba intestinal toxin (MIT1), 361 Mambin, 357 mammalian peptides, 549–550 associated with amyloidogenic diseases, 549 associated with inflammatory and antibacterial responses, 549–550 frog skin peptides, 550 Manduca sexta, 164–165 MAP Ks (mitogen-activated protein kinases), 70, 447, 640–642, 908, 1039, 1172, 1410–1411 mast cell degranulating peptide (MCD), 391 mast cells (MC), 455–456 mastoparans, 392 matrix metalloproteinase-2 (MMP-2), 455 matrix metalloproteinase-7 (MMP-7), 1031–1032 matrix-metalloproteinases (MMPs), 449–450 Maurotoxin, 417 MBP (mean arterial pressure), 1381 MBP (myelin basic protein), 590–592, 598–599, 603–607 MC (mast cells), 455–456 MC3-R (melanocortin-3 receptor), 903–904, 908 MC4-R (melanocortin-4 receptor), 903–905, 908–909 MCD (mast cell degranulating peptide), 391 MCH. See melanin-concentrating hormone (MCH) MCN (magnocellular neurons), 621–627 MC-Rs (melanocortin receptors), 689–695 MCT (monocrotaline)-induced pulmonary hypertension in rats, 1286 MCT-1 (monocarboxylate transporter type one), 1074 mean arterial pressure (MBP), 1381 mechanosensitive ion channels (MSCs), 376

medial preoptic area (MPOA), 947 median eminence (ME), 634 MeJA (methyl jasmonate), 6 melanin-concentrating hormone (MCH), 560–564, 705–713, 1515–1516 biological actions of, 711–712 feeding behavior and energy homeostasis, 711–712 other functions, 712 pigmentation, 711 stress response, 712 discovery of, 705–706 and ingestive behavior, 929–933 feeding behavior effects, 929–930 functional response to differing metabolic and feeding states, 930–931 interactions with other peptidergic/ aminergic systems, 932 overview, 929 physiological and pathophysiological implications, 932–933 receptors and signaling pathways, 932 sites of action and neural networks affected, 931–932 studies from genetic manipulations or mutations, 930 mRNA/peptide distribution, 708–709 overview, 705 pathophysiological implications, 712–713 peptide structure and receptor conformation, 711 precursor mRNA/gene structure, 706–708 precursor processing, 709 receptors and signaling cascades, 709–710 melanin-concentrating hormone receptor1 (MCH-R1), 706, 930–932 melanin-concentrating hormone receptor2 (MCHR-2), 931–932 melanocortin-3 receptor (MC3-R), 903–904, 908 melanocortin-4 receptor (MC4-R), 903–905, 908–909 melanocortin receptors (MC-Rs), 689–695 melanocortins, 689–695 biological actions within brain, 693–695 antipyretic and anti-inflammatory activities, 694 cardiovascular effects, 694–695 cognitive functions, 694 energy homeostasis, 693 nerve regeneration, 694 opiate interactions, 695 sexual and social behavior, 693–694 stretching-yawning syndrome and grooming, 693 biosynthesis control and release of brain melanocortins, 692 discovery of, 689 and ingestive behavior, 903–909 feeding behavior effects, 903–904

functional response to differing metabolic and feeding states, 905–907 genetic studies on, 904–905 interactions with other peptidergic/ aminergic systems, 909 overview, 903 physiological and pathophysiological implications, 909 receptors and signaling pathways, 908–909 system neuroanatomy, 907–908 overview, 689 physiological and pathophysiological implications, 695 proopiomelanocortin (POMC) mRNA distribution and melanocortins in brain, 690–691 mRNA/gene structure, 689–690 processing, 691–692 receptors, 692–693 structure–activity relationships, 692 melanocyte-stimulating hormone (MSH), 903–909, 931 melanoma, 425 melanoma antigen-1 (MAGE-1), 500 melanotrophin-potentiating factor (MPF), 1329–1330 melittin, 390–391 MEN1 (multiple endocrine neoplasia type 1), 1104 merozoite surface protein-1 (MSP-1), 522–523 mesotocin receptor (MTR), 328 metastin. See KiSS-1/metastin metastin-like immunoreactivity (irMT), 823 methyl jasmonate (MeJA), 6 MHC (major histocompatibility complex) class II, 520–521 MHC class II molecules, 611–614 impact of CLIP on MHC II peptide binding groove, 613 parent protein of CLIP: invariant chain, 613–614 structure of MHC class II peptide complexes, 612 Michaelis-Menton equation, 1416–1417 microbial macrocyclic peptides, nonribosomally synthesized. See nonribosomally synthesized microbial macrocyclic peptides Microcin B17 synthetase enzyme complex, 77–78 microcins, 75–79 active and/or solution conformation, 78 biological actions of, 79 discovery of, 75 mRNA distribution, 76 oveview, 75 precursor mRNA/structural gene structure, 75–76 processing of, 76–78 receptors, 78 migrating motor complex (MMC), 1068, 1077–1082, 1112

Index / 1583 MIPs (molluscan insulin-related peptides), 236–237 MIT1 (mamba intestinal toxin), 361 mitogen, 449 mitogen-activated protein kinases (MAP Ks), 70, 447, 640–642, 908, 1039, 1172, 1410–1411 mixture-based combinatorial libraries, 1487–1492 μ-, δ-, and κ-ligands, 1490 μ-receptor ligands, 1489–1490 δ-receptor ligands, 1490 future studies, 1492 general methods, 1487–1489 κ-receptor ligands, 1490 orphan receptor ligands, 1490–1491 overview, 1487 testing mixtures in vivo, 1491–1492 MK-869 tachykinin agonist, 769 MLI (motilin-like immunoreactivity), 1080 MMC (migrating motor complex), 1068, 1077–1082, 1112 MMP-2 (matrix metalloproteinase-2), 455 MMP-7 (matrix metalloproteinase-7), 1031–1032 MMPs (matrix-metalloproteinases), 449–450 MNCs (mucus neck cells), 1148–1150 MoAbG6 monoclonal antibody, 454 MOG (myelin oligodendrocyte glycoprotein), 603–607 molluscan bioactive Mollusca peptides, 235–239 FMRFamide, FMRFamide-related peptides (FaRPs), 235–236 overview, 235 peptides and feeding behavior, 237–238 peptides and metabolism, 236–237 peptides and renal function, 238 tachykinins (TKs), 238–239 molluscan insulin-related peptides (MIPs), 236–237 molluscan reproduction, 241–245 biological actions of, 242 discovery, 243 biological actions of, 243 mRNA distribution, 243 precursor mRNA/gene structure, 243 egg-laying hormones (ELHs): discovery, 241 gonadotrophin-releasing hormone (GnRH), 242–243 biological actions of, 242–243 mRNA distribution, 242 precursor mRNA/gene structure, 242 mRNA distribution, 242 overview, 241 pheromones, 244–245 biological actions of, 244–245 precursor mRNA/gene structure, 244 precursor mRNA/gene structure, 241 vasopressin (VP)/oxytocin (OT), 243–244 biological actions of, 244 mRNA distribution, 244

precursor mRNA/gene structure, 243–244 receptors, 244 molt-inhibiting hormone (MIH), Error! No page number monocarboxylate transporter type one (MCT-1), 1074 monoclonal antibodies (Mabs), 596–597 monocrotaline (MCT)-induced pulmonary hypertension in rats, 1286 mono-epitope multiple antigen peptides, 543 monophosphoryl lipid A (MPL), 532 monosodium glutamate (MSG) treatment, 976 μ-opioid receptor binding assay, 1489 μ-opioid receptors (MOR), 1321–1322, 1340–1341 MOR (μ-opioid receptors), 1321–1322, 1340–1341 morphine, 1348 motilin, 1065–1066, 1077–1083 biological actions of, 1081–1083 experimental condition difficulties, 1081 overview, 1081 pharmacological action, 1081 physiological role, 1081–1082 release mechanisms, 1082–1083 discovery, 1077 distribution in GI tract, 1079–1080 overview, 1077 pathophysiological implication, 1083–1084 overview, 1083 receptor agonists, 1083 receptor antagonists, 1083 secretion disorder, 1083 receptors, 1080–1081 structure–activity, 1080 structure of, 1077–1079 gene structure and posttranslational processing, 1079 motilin-ghrelin family, 1079 mouse and rat motilin, 1077–1079 overview, 1077 structural heterogeneity and species, 1077 motilin-like immunoreactivity (MLI), 1080 motility, 1007, 1052–1053 mouse knockout mutants, 726 mouse vas deferens (MVD), 1365–1370 MPF (melanotrophin-potentiating factor), 1329–1330 MPL (monophosphoryl lipid A), 532 MPOA (medial preoptic area), 947 MS (multiple sclerosis), 590–592, 603–608 MSCs (mechanosensitive ion channels), 376 MSG (monosodium glutamate) treatment, 976 MSH (melanocyte-stimulating hormone), 903–909, 931 MSP-1 (merozoite surface protein-1), 522–523 MTR (mesotocin receptor), 328

mucins, 1148 mucosal homeostasis and cytoprotection, 1009 mucus neck cells (MNCs), 1148–1150 multiple endocrine neoplasia type 1 (MEN1), 1104 multiple sclerosis (MS), 590–592, 603–608 MUPs (major urinary proteins), 1511 muscarinic toxins, 356 mushroom toxins, 131–135 discovery of, 131–133 overview, 131 structures of, 133 toxic actions of, 133–135 amatoxins, 133–134 phallotoxins, 134–135 virotoxins, 135 MVD (mouse vas deferens), 1365–1370 myelin basic protein (MBP), 590–592, 598–599, 603–607 myelin oligodendrocyte glycoprotein (MOG), 603–607 myosuppressins, insect biological actions of, 198 discovery of, 193–194 mRNA distribution, 195 precursor mRNA/gene structure, 194 processing of, 196 receptors, 196–197 structure-activity and active conformation, 197–198 Myr p peptides and pilosulin, 393 N Na+ channel specific toxins (NaScTxs), 339–342 Na+Cl- reabsorption in vivo, 1253 N-acetylcysteamine (SNAC), 93–94 NANC (Nonadrenergic noncholinergic) neuronal pathway, 1294–1297 natriuresis and diuresis, 1258 natriuretic peptide clearance receptor, 1157 natriuretic peptides, 359–360, 410 biological activity, 360 in cardiovascular system, 1199–1206 biological actions of, 1203–1204 discovery of, 1199–1200 gene structure, 1200–1201 information on active and/or solution conformation, 1203 mRNA and peptide distribution, 1201 mRNA structure, 1200 overview, 1199 pathophysiological implications, 1204–1206 precursor structure, 1200 processing, 1202 receptors, 1202–1203 conformation, 360 discovery, 359–360 receptors, 360 structure of precursor mRNA/gene, 360

1584 / Index natriuretic receptor A (NPR-A), 1202 natriuretic receptor B (NPR-B), 1202 nausea and vomiting, 1144 NE (norepinephrine), 858 NEI (neuropeptide-glutamic acidisoleucine), 706, 709 nematode peptides free-living, 247–253 antimicrobial peptides, 252–253 FMRFamide-like peptides (FLPs), 247–250 insulins (INSs), 250–252 neuropeptide-like peptides (NLPs), 250 overview, 247 parasitic, 255–259 FMRFamide-like peptides (FLPs), 255–259 overview, 255 TKQELE, 259 nemertine peptide neurotoxins, 398–399 NEP (neural endopeptidase 24.11), 1216 nerve growth factor (NGF), 1407–1412 nervous system development, 1329 nervous system regeneration, 1329 neural endopeptidase 24.11 (NEP), 1216 neuregulins, 1401–1404 biological actions of, 1403–1404 neurological disorders, 1404 neurotrophic for nigrostriatal dopaminergic neurons, 1404 overview, 1403–1404 discovery of, 1401 distribution of, 1401–1402 overview, 1401 processing of, 1401–1402 receptors, 1402–1403 NRG-2 versus NRG-1, 1403 overview, 1402–1403 structure of, 1401 neurohypophyseal peptides, 1227–1233 body fluid homeostasis, 1228 history and overview of, 1227–1228 overview, 1227 oxytocin receptor localization in kidney OTR expression, 1229 receptors, 1228–1229 renal effects of, 1231–1233 vasopressin receptor localization in kidney V1aR expression, 1229 receptor localization in kidney V2R expression, 1229 receptors, 1228–1229 renal effects of, 1229–1231 neurohypophysial peptides, amphibian, 327–330 arginine vasotocin (AVT) and mesotocin (MT) distribution, 328 biological actions of AVT and MT, 328–329 discovery of, 327 distribution of, 328 overview, 327

precursor mRNA/gene structure, 327 receptor structure, 328 structure and function of hydrins, 329–330 neurokinin-1 receptor (NK-1 receptor), 1463 neurokinin A (NKA), 833–836, 1301–1302, 1359–1360 neurokinin B (NKB), 1359–1360 neuromedin B (NMB), 277 neuromedin N (NMN), 738 neuromedins (NMs), 886 neuromedin U (NMU), 745–750 biological actions in brain, 748–750 energy balance, 748 gastric acid secretion and emptying, 748 HPA axis and stress response regulation, 748–750 discovery of, 745 distribution of, 745–747 neuromedin U (NMU) receptors, 746–747 NMU1R, 747 NMU2R, 747–748 overview, 745 pathophysiological implications, 750 precursor mRNA/gene structure, 745–746 processing of, 745–747 neuromuscular disease, 1330 neuronal nitric oxide synthase (nNOS), 1156 neuropathic pain, 1330 neuropeptide AF (NPAF), 779–783 neuropeptide F, 224 neuropeptide FF (NPFF), 779–784 neuropeptide F-like peptides biological actions of, 198–199 discovery of, 193–194 mRNA distribution, 195–196 precursor mRNA/gene structure, 195 processing of, 196 receptors, 197 structure-activity and active conformation, 198 neuropeptide-glutamic acid-isoleucine (NEI), 706, 709 neuropeptide-glycine-glutamic acid (NGE), 706 neuropeptide-like peptides (NLPs), 250 neuropeptides amphibian, 283–287 caerulein peptides, 283–285 caerulein related peptides, 285–286 disulfide neuropeptides from genus Crinia, 287 overview, 283 tryptophyllin peptides, 286–287 that regulate immune responses, 579–582 neuropeptide Y (NPY), 438–440, 570, 683–687, 817, 841–842, 889–893, 895–897, 940, 963, 996–997, 1097–1101, 1195–1196 angioneogenesis effects, 438

and appetite, 889–890 overview, 889 signaling regulation, 891–892 stimulation of feeding, 889–890 biological actions of, 686–687 discovery of, 683 mechanism of action, 890–891 coexpression, 891 neurosecretion, 890–891 overview, 890 receptors, 891 subpopulations, 890 target sites, 890 mRNA and protein distribution, 685–686 normal and tumor cell proliferation, 438 overview, 683, 889 pathophysiological implications, 687 pathophysiology, 892–894 eating disorders, obesity, and metabolic syndrome, 892–893 NPYergic signaling subjugation, 893 overview, 892 precursor mRNA/gene structure, 683–684 precursor processing, 685 receptors expression in human tumors, 439–440 molecular signaling, 438 and signaling cascades, 686 subtypes, 438 and tumor targeting, 440 in regulation of endocrine glands, 839–843 adrenal, 839–840 gonadal function, 841–842 overview, 839 pancreas, 840–841 thyroid, 842–843 structure of, 686 neuroprotection, 1389–1390 VIP- and PACAP-related, 1379–1382 discovery of, 1379 distribution of, 1379 NAP, 1382 overview, 1379 PACAP research, 1381–1382 precursor mRNA structure, 1379 processing of, 1379 VIP neuroprotection, 1379–1380 VIP receptor and mechanism research, 1380–1381 neurotensin (NT), 431–432, 737–741 biological actions within brain, 739–741 amphetamine sensitization, 740 appetite control, 740 endogenous antipsychotic, 739–740 pain, 740 reproductive functions, 740–741 stress responses, 740 discovery of, 737–738 expression of in cancer cells, 431

Index / 1585 in gastrointestinal function regulation, 1085–1090 colonic motility enhancement, 1086 colonic stress responses, 1087–1088 GI mucosa healing, 1087 intestinal inflammation, 1087 motility and secretion inhibition in stomach and small intestine, 1086 overview, 1085 promotion of cell growth and regeneration, 1087 secretion stimulation, 1086–1087 induced growth effects on tumor cells, 431 information on active or solution conformation, 739 mRNA expression in brain, 738 overview, 431, 737 pathophysiological implications, 741 precursor mRNA/gene structure, 737–738 precursor processing, 738 proliferative effect of NT in cancer, 431 receptors and signaling cascades, 739 in regulation of endocrine glands, 885 expression of, 885 hormone secretion, 885 overview, 885 in treatment of human cancer, 431–432 neurotensin receptor-1 (NTR-1), 739–740 neurotensin receptor-2 (NTR-2), 739–740 neurotensin receptor-3 (NTR-3), 739 neurotransmissions, 1315 neurotrophins, 1407–1412 clinical prospects, 1412 conformation, 1408–1409 mRNA distribution, 1407–1408 mRNA/gene structure, 1407 nerve growth factor discovery, 1407 neurotrophins, 1410–1411 overview, 1407 physiological role and roles in pathology, 1411–1412 precursor protein activity, 1408 precursor protein processing, 1408 receptor signaling, 1410, 1411 NGE (neuropeptide-glycine-glutamic acid), 706 NGF (nerve growth factor), 1407–1412 NHL (non-Hodgkin’s lymphoma), 425 nicotinic acetylcholine receptors, 356 Nisin, 103 nitric oxide (NO), 1008–1009, 1067, 1155–1156, 1166, 1258, 1289–1290, 1295–1296, 1309 nitric oxide synthase (NOS), 1040, 1309 NK1-3 (tachykinin receptors), 767 NK-1 receptor (neurokinin-1 receptor), 1463 NKA (neurokinin A), 833–836, 1301–1302, 1359–1360 NKB (neurokinin B), 1359–1360 NLPs (neuropeptide-like peptides), 250 NLT (nucleus lateralis tuberis), 708 NMB (neuromedin B), 277 NMN (neuromedin N), 738 NMs (neuromedins), 886

NMU. See neuromedin U (NMU) NMU1R, 747 NMU2R, 747–748 nNOS (neuronal nitric oxide synthase), 1156 NO (nitric oxide), 1008–1009, 1067, 1155–1156, 1166, 1258, 1289–1290, 1295–1296, 1309 nociceptin (NOP), 1351–1356 biological actions of, 1354–1356 learning and memory, 1356 overview, 1354 pain and inflammation, 1354–1355 peripheral organs, 1356 reward and addiction, 1355 stress and related behaviors, 1355–1356 biosynthesis of, 1354 degradation of, 1354 discovery of, 1351 mRNA and peptide distribution, 1353–1354 overview, 1351 pathophysiological implications, 1356 precursor MRNA/gene structure, 1351–1353 receptors, 1354 nocorticotrophic hormone (ACTH), 880 NOD2 receptor, 1034 Nonadrenergic noncholinergic (NANC) neuronal pathway, 1294–1297 non-Hodgkin’s lymphoma (NHL), 425 nonlantibiotic heat-stable bacteriocins, 107–112 biological actions of, 110 discovery of, 107–109 future trends, 112 overview, 107 receptors, 110–111 structure, 111–112 synthesis of, 107–110 nonobese diabetic mice (NOD), 599 non-rapid-eye-movement sleep (NREMS), 1521–1526 nonribosomally synthesized microbial macrocyclic peptides, 89–95 chemoenzymatic approaches toward novel cyclopeptides, 94–95 excised thioesterase domains biochemical characterization, 93–94 macrocyclization, 92 modular structure of, 90–92 oveview, 89 structural diversity of, 89–90 surfactin thioesterase domain structure, 92–93 TE domains combinatorial potential to synthesize cyclic peptide libraries, 95 nonribosomal peptide synthesis complexes (NRPS), 85 nonselective toxins (NSTs), 151 non-small-cell lung cancer (NSCLC), 1297 nontypeable Haemophilus influenzae (NTHI), 527–532 NOP. See nociceptin (NOP)

norepinephrine (NE), 858 NOS (nitric oxide synthase), 1040, 1309 NPAF (neuropeptide AF), 779–783 NPFF (neuropeptide FF), 779–784 NPR-A (A-type natriuretic peptide receptor), 809–810 NPR-A (natriuretic receptor A), 1202 NPR-B (B-type natriuretic peptide receptor), 809–810 NPR-B (natriuretic receptor B), 1202 NPY (neuropeptide Y), 438–440 NREMS (non-rapid-eye-movement sleep), 1521–1526 NRM (nucleus raphe magnus), 1328 NRPS (nonribosomal peptide synthesis complexes), 85 NSCLC (non-small-cell lung cancer), 1297 NSTs (nonselective toxins), 151 NT. See neurotensin (NT) NTHI (nontypeable Haemophilus influenzae), 527–532 NTR-1 (neurotensin receptor-1), 739–740 NTR-2 (neurotensin receptor-2), 739–740 NTR-3 (neurotensin receptor-3), 739 NTS (nucleus of the solitary tract), 690, 906, 1100 nucleus accumbens shell (snAcc), 920–923 nucleus lateralis tuberis (NLT), 708 nucleus of the solitary tract (NTS), 690, 906, 1100 nucleus raphe magnus (NRM), 1328 O Oatp2 (organic anion transporting polypeptide-2), 1425–1426 obesity, 949, 1475–1476 octadecaneuropeptide (ODN), 815–816 OctreoScan, 437 Octreoscan, 1126 Octreotide, 1127, 1133 ODN (octadecaneuropeptide), 815–816 OLETF (Otsuka Long Evans Tokushima Fatty) rats, 1019 oligopeptide transport, 1423–1427 at blood–brain barrier (BBB), 1424–1425 blood to brain transport, 1424–1425 brain to blood transport, 1425 overview, 1424 blood–brain barrier (BBB) properties, 1423–1424 cerebrospinal fluid (CSF) sink action, 1424 overview, 1423 passive permeability, 1423 transport properties, 1424 at choroid plexus, 1425–1426 basolateral transporter, 1426 intracellular transporters, 1426 larger peptide transporters, 1426 molecular basis for oligopeptide transporter heterogeneity, 1425–1426 overview, 1425 overview, 1423 peptide and peptidomimetic drug transport, 1426–1427

1586 / Index OMP (outer membrane protein), 527–528 ONPG chromogenic substrate, 337 opiate peptides, and blood–brain barrier, 1429–1432 overview, 1429 stabilization of, 1429–1430 targeting, 1430–1432 opioid–enkephalin, 224–225 biological actions of, 225 discovery of, 224–225 receptors, 225 opioid peptides amphibian, 269–274 biological actions of, 271–272 history of, 269–270 overview, 269 structure and conformation of, 270–271 in regulation of endocrine glands, 829–831 adrenal gland, 830–831 overview, 829 pancreatic islets, 830 pituitary gland, 829–830 opioids, 579–581 and ingestive behavior feeding behavior effects, 920–921 functional response of, 922 interaction between OxA and opioids, 924 interactions with other peptidergic/ aminergic systems, 923 physiological and pathophysiological implications, 924 sites of action and neural networks affected, 922–923 studies from genetic manipulations or mutations, 921 orcokinin, 225 orcomyotropin, 225 Orexin A (OxA), 919–924 Orexin B (OxB), 919–922 orexins, and ingestive behavior feeding behavior effects, 919–920 functional response of, 921–922 interaction between OxA and opioids, 924 interactions with other peptidergic/ aminergic systems, 923 physiological and pathophysiological implications, 923–924 sites of action and neural networks affected, 922 studies from genetic manipulations or mutations, 921 organic anion transporting polypeptide-2 (Oatp2), 1425–1426 orphan receptor ligands, 1490–1491 OT. See oxytocin (OT) OTA (oxytocin agonist), 480 otitis media, peptide vaccine for, 527–532 available therapies, 527–528 contributions to area, 531–532 disease target, 527 future of, 532 overview, 527

OTR (oxytocin receptor), 479–481, 625–626 Otsuka Long Evans Tokushima Fatty (OLETF) rats, 1019 outer membrane protein (OMP), 527–528 outward-rectifying K+ channels (Kv), 374–375 OxA (Orexin A), 919–924 OxB (Orexin B), 919–922 oxyntomodulin, 1057–1064 active conformation and metabolism, 1059 biological actions of, 1060 discovery of, 1057–1058 expression of gene, 1058–1059 gene structure, 1057–1058 overview, 1057 receptor expression, 1059–1060 Oxypinins, 378 oxytocin (OT), 243–244, 996, 1227–1231, 1472, 1511 biological actions of, 244 and cancer, 479–481 as modulator of cell growth in OTRexpressing tumors, 480–481 OT/OTR system, 481 overview, 479 physiology to neoplastic pathology, 479–480 mRNA distribution, 244 precursor mRNA/gene structure, 243–244 receptors, 244 and vasopressin (VP), 621–627 active and/or solution conformation, 626 biological actions within brain and pituitary, 626 discovery of, 621–622 mRNA distribution, 623–624 overview, 621 pathophysiological implications of, 626–627 precursor mRNA/gene structure, 622–623 precursor processing, 624–625 receptors, 625 oxytocin agonist (OTA), 480 oxytocin receptor (OTR), 479–481, 625–626 P PACAP. See pituitary adenylate cyclase activating polypeptide (PACAP) PAG (Periaqueductal gray matter), 1326–1328 PAG (Periaqueductal gray) of the midbrain, 1346–1347 PAK strain of Pseudomonas aeruginosa, 510–513 PAMP (proadrenomedullin N-terminal 20 peptide), 455, 772–775, 861–865, 999–1002 PAMPs (Pathogen-associated molecular patterns), 58

Panbo-RPCH, 189, 191 Pancreastatin (PST), 311 Pancreatic B cells, 993–994 pancreatic polypeptide, 1097–1104 biological actions of, 1101–1107 food intake and regulation of weight, 1103 gallbladder, 1102 gastric acid secretion and emptying, 1102 gastrointestinal tract, 1101 intestinal motility, 1102 metabolic effects, 1102–1103 overview, 1101 pancreatic secretion, 1102 reproduction, 1103 discovery in gastrointestinal tract, 1097–1098 overview, 1097 structure, 1097–1098 mRNA and protein distribution, 1098–1099 overview, 1097 physiopathological implications, 1103–1104 precursor mRNA/gene and peptide variant structure, 1098 receptors subtypes, signaling, and distribution, 1099–1101 overview, 1099 PP (Y4) receptor clones, 1101 PP binding studies, 1099–1101 Pancreatic polypeptides (PPs), 683 pancreatic secretion as biological action of CRH in gastrointestinal system, 1025–1026 as biological action of pancreatic polypeptide, 1102 regulation, 1018 pancreatic surgery, 1135 Paneth cell α-defensins, 1029–1034 action mechanisms, 1032–1034 discovery of enteric, 1029–1030 distribution in gastrointestinal tract, 1030–1032 gene and precursor structures, 1030–1032 overview, 1029 pathophysiological implications, 1034 structures of, 1032 PAO strain of Pseudomonas aeruginosa, 510–513 parasitic nematode peptides. See nematode peptides, parasitic Parathyroid hormone (PTH), 857, 880–881 Paraventricular nuclei (PVN), 621–626, 895–898, 915, 938–940, 996, 1026 Parvocellular neurons (PCN), 621–623 Pathogen-associated molecular patterns (PAMPs), 58 PBAN (Pheremone biosynthesis-activating neuropeptide), 1509 PBR (Peripheral-type benzodiazepine receptor), 815–818

Index / 1587 PC12 cells, 830 PC2 (prohormone convertase-2), 1354 PCN (Parvocellular neurons), 621–623 PCOS (Polycystic ovary syndrome), 830 PCs (prohormone-convertases), 709 PCT (procalcitonin), 570 PcTx1 (Psalmotoxin 1), 376 PCWC (pulmonary capillary wedge pressure), 1248 PDH (Pigment dispersing hormone), 213, 229–230 Pea-pyrokinin-5, 210 Pediocin-like bacteriocins, 110–112 Peptaibol antibiotics, 127–128 peptaibols, 83–87 biological origins of, 85 biosynthesis of, 85 characteristic features of, 83–85 functions and activities of, 86–87 oveview, 83 structures of, 85–86 peptide-based cancer vaccines, 499–504 antigenic peptides recognized by T cells, 501 clinical trials, 502–503 overview, 499 strategies for enhancing efficacy of peptide-based vaccines, 503–504 tumor antigens recognized by T cells, 500–502 cancer-testis antigens, 500 overexpressed antigens, 501 tissue-specifi c differentiation antigens, 500–501 tumor-specific antigens resulting from genetic alterations, 501–502 viral proteins, 501 tumor-specific T cell features, 502 using immune system to treat cancer, 499 peptide chronomics, 1529–1561 atlas, 1542 avoiding blunders, 1532–1535 beginnings of opportunistic mapping along scale of years, 1542–1547 breast cancer risk assessment, 1553 developed by chronobiology maps dynamics, 1535 discussion, 1556–1557 end points, 1535–1542 overview, 1529–1532 peptide chronomics in women, 1547–1553 peptide maps in men, 1547 prostate cancer risk assessment, 1553–1556 susceptibility rhythms, 1542 peptide cytolysins, 400 peptide dendrimers, 541–546 background and discovery, 541–542 cell-mediated responses induced by lipidated MAPs, 543–544 design of as immunogens, 542–543 future of, 546 immunological property of MAPs, 543 overview, 541–542

reasons for increased immunogenicity, 544 synthesis, 544–545 Peptide histidine isoleucine (PHI), 674 Peptide histidine methionine (PHM), 674 Peptide/histidine transporters (PHTs), 1426 peptide immunogenicity, 495 peptide inhibitors, 571 Peptide transport system 1 (PTS1), 1477 peptide vaccines for Alzheimer’s disease (AD), 535–539 EFRH phage elicits antibodies against β-amyloid peptide, 536–537 future of, 539 immunization of hAPP transgenic mice with EFRH-phage, 538–539 overview, 535 prevention and/or reduction of amyloid plaques in AD transgenic mice, 537–538 in vitro modulation of β-amyloid formation, 535–536 for malaria, 515–524 malarial antigens in target cell binding, 517–518 malarial vaccines, 516–517 overview, 515–516 vaccine design, 518–522 for otitis media, 527–532 available therapies, 527–528 contributions to area, 531–532 disease target, 527 future of, 532 overview, 527 Peptide Y Y (PY Y), 570 peptide Y Y (PY Y), 1109–1113 biological actions on gastrointestinal tract, 1112–1113 biosynthesis of, 1109 gastrointestinal distribution and ontogeny of intestinal PY Y, 1109–1110 gene structure, 1109 growth promoting actions on gastrointestinal tract, 1113 and neuromedin U (NMU), 945–949 overview, 1109 pathophysiology of, 1113 receptors, 1111 secretion regulation, 1111 Peptidoglycan precursor lipid II, 103 Periaqueductal gray (PAG) of the midbrain, 1346–1347 Periaqueductal gray matter (PAG), 1326–1328 peripheral cholecystokinin (CCK), 1013–1020 discovery of, 1013–1014 distribution of, 1014 indirect peripheral actions influencing feeding and digestion, 1019–1020 body temperature regulation, 1019 cardiovascular actions, 1019 involvement in reproduction functions, 1019–1020

overview, 1019 physiological/pathophysiological relevance of CCK-8 effects in nondigestive CCK actions, 1020 molecular forms of, 1016 overview, 1013 peripheral biological actions of, 1018–1022 overview, 1018 regulation of meal, 1018 precursor mRNA/gene structure, 1014 processing of preprocholecystokinin, 1014–1015 carboxyl terminal amidation, 1015 covalent modifications of amino acid side-chain groups, 1015 exopeptidase reactions, 1015 overview, 1014 processing at double basic residues, 1015 processing at single basic residues, 1015 signal peptide cleavage, 1014–1015 receptors, 1016–1017 solution conformation, 1017 Peripheral-type benzodiazepine receptor (PBR), 815–818 PG (Proglucagon), 1057–1058 PG (prothoracic gland), 215 P-glycoprotein (Pgp), 1425 PH (pulmonary hypertension), 1192, 1296–1297, 1311 Phalloidin, 134 phallotoxins, 134–135 Pheremone biosynthesis-activating neuropeptide (PBAN), 1509 pheromone peptides, 1505–1511 annelid, 1510 arthropod, 1509–1510 crustaceans, 1509–1510 insects, 1509 overview, 1509 bacterial, 1506 fungal, 1506–1509 mollusk, 1510 overview, 1505–1506 vertebrate, 1510–1511 amphibians, 1510 mammals, 1510 overview, 1510 pheromones, 244–245 biological actions of, 244–245 precursor mRNA/gene structure, 244 PHI (Peptide histidine isoleucine), 674 PHM (Peptide histidine methionine), 674 Phomalide, 155 Phospadidylinositol-4-5 bisphosphate (PIP2), 640 Phospholipase A2 (PLA2), 640–642, 1119 Phospholipase C (PLC), 625, 676–677, 799, 848–849, 1111 Phospholipase Cβ (PLCβ), 640–641 Phospholipase D (PLD), 640–642, 1159 PHTs (Peptide/histidine transporters), 1426 Phyllokinin, 293

1588 / Index phyllolitorins, 279 Phylloseptins, 300 Phytosulfokine (PSK), 29–32 biological actions, 29–30 discovery of, 29 oveview, 29 precursor mRNA/gene structure, 30–31 receptors, 31 tyrosine sulfation, 31 phytotoxins, 151–152 pigment dispersing factor (PDF), insect, 213–215 biological actions of, 214–215 discovery of, 213–214 mRNA distribution, 213–215 precursor mRNA/gene structure, 213–214 processing of, 213 receptors, 214 Pigment dispersing hormone (PDH), 213, 229–230 PIII phage protein, 538 PIP2 (Phospadidylinositol-4-5 bisphosphate), 640 PISCF family of ASTs, 205 pituitary adenylate cyclase activating polypeptide (PACAP), 570, 663, 816, 1091–1096, 1155–1158, 1438, 1472 characterization of, 1092 cloning of, 1091–1092 discovery of, 1091 localization of, 1092–1093 overview, 1091 physiology in gastrointestinal tract, 1093–1094 in pulmonary function, 1293–1298 biological actions of, 1294–1296 gene structure, 1293 overview, 1293 pathophysiological implications, 1296–1297 precursor mRNA distribution, 1293 receptors, 1293–1294 therapeutic potential of, 1297–1298 in regulation of endocrine glands, 847–851 adrenal, 849–850 ovary, 849 overview, 847 pancreatic islets, 850–851 pituitary, 847–849 testis, 849 thyroid, 849 signal transduction of, 1092 Pituitary adenylate cyclase-activating polypeptide (PACAP)/VIP, 673–680 biological actions in brain and pituitary, 677–679 and cancer, 473–476 overview, 473 proliferation, 475 second messengers, 474–475 tumor imaging, 475 VIP/PACAP peptides, 473–474 VIP receptors, 474

discovery of, 673 mRNA distribution, 674–675 overview, 673 pathophysiology of, 679–680 precursor mRNA/gene structure, 673–674 precursor processing, 674–676 receptors and signaling cascades, 676–677 solution structure comparison, 677 PK (pyrokinin), 1509 PKA (protein kinase A), 667, 799, 1308–1381 PKC (protein kinase C), 640–642, 724, 799, 1159, 1271, 1380–1381 PLA2 (Phospholipase A2), 640–642, 1119 Plant shoot meristem (SM), 9 plasmodium falciparum acidic-basic repeat antigen (ABRA), 518 PLC (Phospholipase C), 625, 676–677, 799, 848–849, 1111 PLCβ (Phospholipase Cβ), 640–641 PLD (Phospholipase D), 640–642, 1159 PLP (proteolipid protein), 603–605 PMTXs (pompilidotoxins), 393 Polanrazines A-E, 155 POLARIS peptides, 23–27 biological actions of, 26–27 discovery of, 23–24 mRNA distribution, 25–26 oveview, 23 precursor mRNA/gene structure, 25 processing of, 26 Polycystic ovary syndrome (PCOS), 830 POMC. See proopiomelanocortin (POMC) pompilidotoxins (PMTXs), 393 poneratoxins, 393–394 ponericins, 394 positional scanning synthetic combinatorial peptide libraries (PS-SCL), 595–600 definition of, 595–596 to determine antibody ligands, 596–598 to identify MHC binding motifs, 599–600 overview, 595 for T cell epitope mapping, 598–599 postoperative complications of surgery, and somatostatin analogs, 1135–1136 dumping syndrome, 1135 GI fistula, 1136 overview, 1135 pancreatic surgery, 1135 short-bowel syndrome, 1135 POT (proton-coulpled oligopeptide transporter) family, 1425–1427 potassium channel toxins, 364–365 PPE (preproenkephalin A), 1313–1314 PPI (prepulse inhibition) model of schizophrenia, 739 ppNOP (prepro-NOP), 1351–1353 PPs (Pancreatic polypeptides), 683 P. pulmonarius RNase, 139, 142 Prader-Willi syndrome (PWS), 735, 957

prebiotic peptides, 1481–1485 amino acid formation, 1482 as origin of life, 1484–1485 overview, 1481 peptide formation, 1482–1484 primitive Earth scenario, 1481–1482 preprocholecystokinin processing, 1014–1015 carboxyl terminal amidation, 1015 covalent modifications of amino acid side-chain groups, 1015 exopeptidase reactions, 1015 overview, 1014 processing at double basic residues, 1015 processing at single basic residues, 1015 signal peptide cleavage, 1014–1015 preprodermaseptins, 297–300 Preproendothelin, 1187 preproenkephalin A (PPE), 1313–1314 Preprogalanin, 754–755 PreproGastrin-releasing peptide, 1047 Preprohypocretin, 723 prepro-nerve growth factor precursor (prepro-NGF), 1408 prepro-NGF (prepro-nerve growth factor precursor), 1408 prepro-NOP (ppNOP), 1351–1353 Pre-propeptides, 68 Preprotachykinins, 763–764 Preprotemporins, 306 Prepro-urotensin II, 795 Prepro-urotensin II-related peptide, 795 prepulse inhibition (PPI) model of schizophrenia, 739 presynaptic toxins, 377 primary pulmonary hypertension, 1310 proadrenomedullin N-terminal 20 peptide (PAMP), 455, 772–775, 861–865, 999–1002 Proapelin, 788 ProAtPep1 gene encoding precursor protein, 6 mRNA distribution, 6–7 processing of, 7 procalcitonin (PCT), 570 proctolin, 177–181 biological actions of, 180–181 discovery of, 177 mRNA distribution, 177–178 overview, 177 precursor mRNA/gene structure, 177 processing of, 178–179 receptors, 179 structure—activity and active conformation, 179–180 prodynorphin-derived opioid peptides. See dynorphins proenkephalin-derived opioid peptides. See enkephalins Progastrin, 469 Proglucagon (PG), 1057–1058 ProHD5 α-defensin precursor, 1031 prohormone convertase-2 (PC2), 1354 prohormone-convertases (PCs), 709 proinflammatory function, 1142

Index / 1589 Prokineticin 1 and 2, 273 prolactin (PRL), 740–741, 856, 883–885, 1329, 1547–1553 and kidney function, 1277–1281 human renal function, 1280 osmoregulation, 1278 overview, 1277 production site and actions of, 1277 receptors, 1278–1279 renal actions of, 1279–1281 renal production of, 1279 transgene method clarification, 1281 Promoter trapping strategy, 23 proopiomelanocortin (POMC), 314, 817, 891, 903–909, 954–955, 1313–1314 proopiomelanocortin (POMC) opioid peptides, 1325–1330 biological actions of, 1328–1329 analgesia, 1328 cardiorespiratory effects and thermogenesis, 1329 dependence, 1328 euphoria, 1328 exercise and stress, 1329 feeding behavior, 1329 nervous system development, 1329 nervous system regeneration, 1329 neuroendocrine effects, 1328–1329 overview, 1328 tolerance, 1328 discovery of, 1325 distribution of, 1325–1326 overview, 1325 pathophysiological implications, 1330 processing of, 1326–1327 receptors, 1327–1328 cellular effects, 1327 opioid, 1327 overview, 1327 putative ε-receptor, 1327–1328 protease inhibitor homologs, 358–359 Ca2+ channel blockers, 358–359 biological activity of, 359 conformation of, 359 discovery of, 358 precursor mRNA/gene structure, 358 receptors, 358–359 dendrotoxins, 358 enzyme inhibitors, 358 protein kinase A (PKA), 667, 799, 1308–1381 protein kinase C (PKC), 640–642, 724, 799, 1159, 1271, 1380–1381 protein tyrosine-phosphatases (PTPs), 650 proteolipid protein (PLP), 603–605 prothoracic gland (PG), 215 prothoracicotropic hormone (PTTH), 164 proton-coulpled oligopeptide transporter (POT) family, 1425–1427 proximal tubules, 1251–1252 P. sajor-caju RNase, 142 Psalmotoxin 1 (PcTx1), 376 Pseudecin toxin, 359

pseudogenes, 43 pseudomonas aeruginosa (Pa) synthetic peptide vaccine, 507–513 available therapies, 507–508 conventional treatment, 508 immunotherapy, 508 microbiology, 507–508 contributions, 510–513 disease target, 507 future of, 513 overview, 507 type IV pilus, 508–510 PSK. See Phytosulfokine (PSK) PS-SCL. See positional scanning synthetic combinatorial peptide libraries (PS-SCL) PST (Pancreastatin), 311 Psuedeche toxin, 359 PTH (Parathyroid hormone), 857, 880–881 PTPs (protein tyrosine-phosphatases), 650 PTS1 (Peptide transport system 1), 1477 PTTH (prothoracicotropic hormone), 164 P. tuber-regium Rnase, 142 pulmonary artery relaxant activities, 1295 pulmonary capillary wedge pressure (PCWC), 1248 pulmonary function, pituitary adenylate cyclase activating polypeptide (PACAP) in, 1293–1298 biological actions of, 1294–1296 airway smooth muscle cell relaxant effects, 1294–1295 effects on airway mucous secretion, 1295 inflammatory cell activity modulation, 1295–1296 overview, 1294 pulmonary artery relaxant activities, 1295 gene structure, 1293 overview, 1293 pathophysiological implications, 1296–1297 asthma, 1297 cancer, 1297 overview, 1296 pulmonary hypertension, 1296–1297 precursor mRNA distribution, 1293 receptors, 1293–1294 therapeutic potential of, 1297–1298 asthma, 1298 cancer, 1298 overview, 1297–1298 pulmonary hypertension, 1298 pulmonary hypertension (PH), 1192, 1296–1297, 1311 putative peptide ligand. See CLAVATA3 PVN (Paraventricular nuclei), 621–626, 895–898, 915, 938–940, 996, 1026 PWS (Prader-Willi syndrome), 735, 957 pyrokinin (PK), 1509 Pyrokinin/PBAN, 226 PY Y. See Peptide Y Y (PY Y)

Q QS (Quorum sensing) systems in bacteria, 1506 Quorum sensing (QS) systems in bacteria, 1506 R Radioimmunoassay (RIA) of sodefrin, 322 RALF (Rapid Alkalinization Factor) peptides, 33–35 biological actions of, 35 discovery of, 33 mRNA distribution, 34 oveview, 33 processing of, 34 receptors, 34–35 solution conformation, 35 structure of, 33–34 RAMPs (receptor-activity-modifying proteins), 775–776, 1000, 1006–1007, 1165, 1182, 1257–1259, 1266–1267, 1284, 1516–1517 Ranatensin, 280 ranatensin-like peptides, 279–280 Rapid Alkalinization Factor peptides. See RALF (Rapid Alkalinization Factor) peptides rapid eye movement (REM) sleep, 726, 1521–1526 RAS. See renin-angiotensin system (RAS) rCBF (regional cerebral blood flow), 1381 RCC (renal cell carcinoma), 425 RCP (receptor component protein), 775–776 receptor-activity-modifying proteins (RAMPs), 775–776, 1000, 1006–1007, 1165, 1182, 1257–1259, 1266–1267, 1284, 1516–1517 receptor component protein (RCP), 775–776 recombinant human erythropoietin (rHuEpo), 1393–1394, 1399 red pigment concentrating hormone (RPCH), 229–230 regional cerebral blood flow (rCBF), 1381 regulatory T cells (T[reg]), 504 relative risk (RR) of cancer, 1389 REM (rapid eye movement) sleep, 726, 1521–1526 renal cell carcinoma (RCC), 425 renal endothelin, 1269–1273 ET-1 blood pressure regulation, 1272–1273 excretory function effects, 1270–1272 renal hemodynamic effects, 1270 system localization, 1269–1270 history of, 1269 overview, 1269 renal function, and molluscan bioactive Mollusca peptides, 238 biological actions of, 238 discovery of, 238 mRNA distribution, 238 precursor mRNA/gene structure, 238 receptors, 238

1590 / Index renal ischemia, 1258 renin, 1236 renin-angiotensin system (RAS), 1235–1240 angiotensin II intrarenal levels, 1238–1239 interstitial angiotensin II, 1238 intracellular angiotensin II, 1239 overview, 1238 tubular Ang II, 1238 biological actions of, 1239–1240 distribution in kidney, 1235–1238 angiotensin-converting enzyme, 1236 angiotensinogen, 1236–1237 intrarenal angiotensin II receptors, 1237–1238 overview, 1235–1236 renin, 1236 overview, 1235 reproduction, 1103 RESA (ring-infected erythrocyte surface antigen), 518 respiratory syncytial virus (RSV), 1290 RFamide peptides, 224 RFamide-related peptides (RFRPs), 779–784 biological actions within brain and pituitary, 783–784 discovery of, 779 distribution in brain, 781 overview, 779 precursor processing, 781 precursor structure, 779–781 receptors, 783 solution conformation information, 783 RGD-peptides, 573–577 antiadhesive RGD-peptides discovery, 573–574 biologically active conformation of, 575 in immunological phenomena, 575–576 in other pathological phenomena, 576–577 overview, 573 and pathogen invasions, 576 in platelet aggregation, 574 retrosequences in proteins, 577 rHuEpo (recombinant human erythropoietin), 1393–1394, 1399 RIA (Radioimmunoassay) of sodefrin, 322 ribonucleolytic peptides, 137–142 biological actions of, 139–142 discovery of, 137–138 overview, 137 structure of, 137–141 ribosome inactivating proteins, fungal. See fungal ribosome inactivating proteins rickets gene in Drosophila, 218–219 ring-infected erythrocyte surface antigen (RESA), 518 rostral ventomedial medulla (RVM), 718 ROT FOUR LIKE genes (RTFL), 37 ROTUNDIFOLIA4, 37–40 biological actions of, 39–40 discovery of, 37

mRNA distribution, 38 oveview, 37 precursor mRNA/gene structure, 37–38 processing of, 38–39 receptors, 39 RPCH (red pigment concentrating hormone), 229–230 RSV (respiratory syncytial virus), 1290 RTFL (ROT FOUR LIKE genes), 37 Rubisco, 1366 RVM (rostral ventomedial medulla), 718 S salt-induced peptide formation (SIPF) reaction, 1482–1483 sarafotoxins, 359 satiety, 1018, 1053. See also cholecystokinin (CCK), and satiety SCF (stem cell factor), 872 SCLC (small-cell lung cancer), 445, 1297 scorpion, venom peptides, 339–343 discovery of, 339 molecular basis of activity, 341–342 overview, 339 receptor sites, 341 scorpion toxin precursors, 341 three-dimensional folding, 340–341 SCPs (small cardioactive peptides), / 237–238 SCR/SP11 peptide, 41–46 biological actions of, 46 discovery of, 41–43 criteria for identification of alleles, 43 pseudogenes in self-fertile Brassicaceae, 43 sequence polymorphism, 42–43 mRNA distribution, 43–44 oveview, 41 precursor mRNA/gene structure, 43 processing of, 44 receptors, 44 structure of, 44–46 SDF-1 chemokine, 559–564 sea anemone, venom peptides, 363–366 bioactive Mollusca peptides, 366 cytolysins, 365–366 ion channel blockers and modulators, 363–365 overview, 363 Secapin, 391 Secondary metabolites, 151 secretin, 1115–1121 biological actions in gastrointestinal tract, 1118–1121 clinical application of, 1121 discovery of, 1115 gene structure, 1116 mRNA distribution, 1116–1118 overview, 1115 pathophysiology of, 1121 precursor transcript, 1116 receptor subtypes, distribution, and signaling, 1118 structure of, 1115–1116 secretin-releasing peptide (SRP), 1119–1120

secretin-vasointestinal polypeptide (VIP), 1079–1083 Secretoneurin (SN), 312, 318 secretory diarrheas, 1133–1134 AIDS-related diarrhea, 1134 chemotherapy-induced diarrhea, 1133 overview, 1133 SEEPLY, 235–236 SEG (subesophageal ganglion), 208–210 self-incompatibility in Brassicaceae (SI), 41 self-major histocompatibility complex (self-MHC), 585–590 self-sustaining status epilepticus (SSSE), 758 SERA (serine repeat antigen protein), 517–518 SEREX (serological recombinant expression cloning), 500 serine repeat antigen protein (SERA), 517–518 serological recombinant expression cloning (SEREX), 500 Serorphin, 1367 SFO (subfornical organ), 274 SGLT-1 glucose co-transporter, 1074 ShK toxin, 365 short-bowel syndrome, 1135 SI (self-incompatibility in Brassicaceae), 41 SIADH (syndrome of inappropriate ADH secretion), 626–627 SIAN (stress-induced antinociception), 740 SIDS (sudden infant death syndrome), 1296, 1342–1343 sifamide, 226–227 biological actions of, 227 discovery of, 226–227 precursor mRNA/gene structure and distribution, 227 signal peptide cleavage, 1014–1015 Silefrin, 322–323 SIPF (salt-induced peptide formation) reaction, 1482–1483 SKPYMRFamide, 235 sleep, and peptides, 1521–1526 ACTH, 1523–1524 animal models of HPA system changes, 1524 overview, 1523 hypothalamic-pituitary-adrenocortical system, 1523–1524 basic activity, 1523 corticotropin-releasing hormone, 1523 effects of changes in sleep-wake behavior on HPA hormones, 1523 overview, 1523 sleep in disorders with pathological changes of HPA activity, 1523 vasopressin, 1523 hypothalamic-pituitary- somatotropic system, 1521–1523 animal models of HPS system changes, 1522–1523 basic activity, 1521

Index / 1591 ghrelin, 1522 growth hormone-releasing hormone, 1521–1522 overview, 1521 somatostatin, 1522 hypothalamic-pituitary-thyroid system, 1524 other peptides, 1524–1525 cortistatin, 1524 δ-sleep-inducing peptide (DSIP), 1524 galanin, 1524 neuropeptide Y (NPY), 1524 overview, 1524 pituitary adenylate cyclase activating polypeptide (PACAP), 1524 substance P, 1524 vasoactive intestinal polypeptide (VIP), 1524–1525 overview, 1521 SLG (S-locus glycoprotein), 44 s-LNv (small ventrolateral neurons), 213 S-locus glycoprotein (SLG), 44 S-locus receptor kinase (SRK), 41, 44 slow wave sleep (SWS), 1521–1524 small cardioactive peptides (SCPs), 237–238 small-cell lung cancer (SCLC), 445, 1297 small ventrolateral neurons (s-LNv), 213 SMases (sphingomyelinases) D toxins, 377–378 SMCs (smooth muscle cells), 1173 SMCs (surface mucus cells), 1148–1152 smooth muscle cells (SMCs), 1173 smooth muscle nitric oxide synthase (eNOS), 1156–1157 SN (Secretoneurin), 312, 318 SNAC (N-acetylcysteamine), 93–94 social hymenoptera, venom peptides from, 390–391 apamin, 391 bumblebee venoms, 391 honeybee venom, 390–391 social wasps, venom peptides from, 391–392 chemotactic peptides, 392 crabrolin, 392 kinin-related peptides (wasp kinins), 392 mastoparans, 392 sylverin, 392 sodefrin, 321–326 biological actions of, 325–326 discovery of, 321–322 mRNA distribution, 323 overview, 321 physiological implication of, 326 precursor mRNA/gene structure, 322–323 precursor processing, 323 receptors, 323–325 structure–activity relationships, 325 Sodefrin variants, 325 sodium channel toxins, 363–364 sodium sulfate (DSS) model of chronic colitis, 1087

solitary wasps, venom peptides from, 392–393 anoplin, 393 bradykinin (BK)-related peptides, 393 eumenine mastoparan-AF (EMP-AF), 393 pompilidotoxins (PMTXs), 393 SOM230 somatostatin analog, 1133 somatostatin (SRIF), 1112, 1123–1127, 1522 analogs and clinical applications in gastrointestinal disease, 1126–1130 diagnostic applications with radioactively labeled somatostatin, 1126 overview, 1126 therapeutic applications, 1126–1130 biological action in gastrointestinal tract, 1124–1125 overview, 1124 somatostatin actions in enteric nervous system, 1125 somatostatin actions in pancreas, 1125 somatostatin actions in stomach, 1124–1125 and cancer, 435–437 antiangiogenic effects of, 436 direct/indirect antiproliferative effects of in tumors, 436 inhibition of hormone release from tumors by, 435 molecular signaling of receptors, 436–437 receptors in cancer, 437–438 as hypothalamic neuropeptides, 1472 overview, 1123 production in neoplastic/nonneoplastic disease, 1125 receptor expression in neoplastic disease, 1125–1126 receptor expression in nonneoplastic disease, 1126 receptors in gastrointestinal tract, 1124 somatostatin-14, somatostatin-28 and cortistatin in gastrointestinal tract, 1123–1124 localization, 1123–1124 overview, 1123 release regulation, 1124 somatostatin analogs in gastrointestinal tract, 1131–1136 biliary system, 1136 esophageal variceal hemorrhage, 1134 gastroenteropancreatic neuroendocrine tumors, 1131–1133 GI motility and functional GI disorders, 1134–1135 overview, 1131 postoperative complications of surgery, 1135–1136 secretory diarrheas, 1133–1134 somatostatin receptor subtype 2 (sst2), 436

somatostatin-related peptides, 645–653 biological actions in brain and pituitary, 651–652 discovery of, 645–646 information on active and/or solution conformation, 650 mRNA and peptides distribution, 646–647 overview, 645 pathophysiological implications, 652–653 precursor mRNA/gene structure, 645–646 precursor processing and degradation of peptide, 648 receptors and signaling cascades, 648–651 SP. See substance P (SP) SPf66, 515 sphingomyelin (SM), 365–366 sphingomyelinases (SMases) D toxins, 377–378 spiders, venom peptides from, 355–361 acting on acid-sensing ion channels (ASIC), 376 acting on glutamate receptors/ transporters, 376 acting on mechanosensitive ion channels (MSCs), 376 acting on neurotransmitter release (presynaptic toxins), 377 antimicrobial and cytolytic peptide toxins from, 378–379 approaches in isolation of, 369–370 AVIT peptides, 361 bradykinin-potentiating peptides, 360–361 CRISP family toxins, 359 crotamine-like myotoxins, 359 C-type lectins and lectin-like proteins, 360 disintegrins, 360 miscellaneous peptides, 361 modulating Nav channel function, 370–374 modulating on Cav channels, 375–376 modulating voltage-gated potassium (K+) channels, 374–375 natriuretic peptides, 359–360 with nonselective actions on voltagegated ion channels, 376 overview, 355, 369 precursor structure and posttranslational processing of, 370 protease inhibitor homologs, 358–359 sarafotoxins, 359 sphingomyelinases (SMases) D toxins from, 377–378 structural organization of, 370 three-finger toxins, 355–358 waglerins, 360 SRIF. See somatostatin (SRIF) SRK (S-locus receptor kinase), 41, 44 SRP (secretin-releasing peptide), 1119–1120

1592 / Index SRP (stresscopin-related peptide), 1209–1210 Ss2A receptor, 648–650 SSSE (self-sustaining status epilepticus), 758 sst2 (somatostatin receptor subtype 2), 436 stanniocalcins (STC), 1517–1519 biological actions of, 1517 discovery of, 1517 mRNA and protein distribution, 1517 overview, 1517 pathophysiological implications, 1517–1518 precursor mRNA/gene structure, 1517 receptors, 1517 STAT3, 989–990 STATs (Activators of transcription), 1071–1072 STC. See stanniocalcins (STC) stem cell factor (SCF), 872 streptozotocin (STZ)-induced diabetic rats, 957 stresscopin-related peptide (SRP), 1209–1210 stress-induced antinociception (SIAN), 740 stretching-yawning syndrome (SYS), 693 stromal derived factor 1-α and 1-β (CXCL12), 569–570 subesophageal ganglion (SEG), 208–210 subfornical organ (SFO), 274 substance P (SP), 570, 763–769, 833–836, 1301–1302, 1359–1361 chimeric peptides, 1373–1377 overview, 1373 tachykinin agonist-opioid agonist chimeric compounds, 1375–1377 tachykinin and opioid systems crossinteraction, 1374 tachykinin antagonist-opioid agonist chimeric compounds, 1374–1375 in gastrointestinal tract, 1139–1144, 1463–1464 biological actions of, 1141–1142 discovery of, 1139 mRNAS and peptides distribution, 1139–1140 overview, 1139 pathophysiological implications of, 1142–1145 receptor subtypes, signaling, and distribution, 1140–1141 sudden infant death syndrome (SIDS), 1296, 1342–1343 sulfakinins, 183–186, 224 biological actions of, 186 discovery of, 183 overview, 183 precursor mRNA/gene structure, 183–184 processing of, 184 receptors, 185 structure/conformation-activity relationships, 185–186

tissue distribution, 184–185 mRNA level, 184 protein level, 184–185 surface mucus cells (SMCs), 1148–1152 SWS (slow wave sleep), 1521–1524 SYF-PEITHI prediction tool of MHC peptides, 588 sylverin, 392 sympathetic nerve activity attenuation, 1195 syndrome of inappropriate ADH secretion (SIADH), 626–627 synergistic toxins, 358 biological activity of, 358 conformation of, 358 discovery of, 358 precursor mRNA/gene structure, 358 receptors, 358 synthetic peptides, 491–497 active immunotherapeutic strategies using peptides, 493 B cell epitopes, 493 T cell epitopes, 493 for analyses of HIV-like viruses, 1495–1504 HIV-1-derived synthetic peptides, 1495–1496 HIV-1 Gag proteins NC and MA, 1501 HIV-1 p6 Gag protein, 1500–1501 HIV-1 protease, 1499–1500 HIV-1-specific virus protein U, 1496–1497 lentiviral protein R, 1497–1498 overview, 1495 trans-activator of transcription (Tat) of HIV-1, 1498–1499 antitumor immunity with peptide vaccines, 496 cancer immunotherapy, 491–492 current vaccination strategies, 492–493 passive immunotherapy with monoclonal antibodies, 492–493 passive versus active immunotherapy, 492 overview, 491 rational design of peptide vaccines, 495–496 molecular mimicry to cognate antigen, 495–496 peptide immunogenicity, 495 T and B cell epitopes identification, 494–495 B cell mimotopes, 494–495 epitope mapping, 494 SYS (stretching-yawning syndrome), 693 System A transport, 1418 systemins, 49–53 biological actions of, 52–53 discovery of, 49–50 LeSys receptor, 52 oveview, 49 precursor proteins processing, 51–52 solution conformations of subfamily members, 52 structures of, 50–51 subfamily mRNAs distribution, 51 System L transport, 1418

T T3 (Triiodothyronine), 1420 T4 (Thyroxine), 1420 TA (Temporin A), 550 TAAs (tumor-asssociated antigens), 491– 492 Tachykinin 1 (TAC1), 764–767 Tachykinin 2 (TAC2), 764–765 Tachykinin 3 (TAC3), 764–766 Tachykinin 4 (TAC4), 764–766 tachykinin agonist-opioid agonist chimeric compounds, 1375–1377 tachykinin antagonist-opioid agonist chimeric compounds, 1374–1375 tachykinin-gene-related peptides (TGRP), 833–836 tachykinin receptors (NK1-3), 767 tachykinin-related peptides (TKRPs), 171–176 biological actions of, 175, 226 discovery of, 171–172, 226 mRNA and peptides distribution, 173–174 overview, 171 precursor mRNA/gene structure, 172–173 precursor mRNA/gene structure and distribution, 226 processing of, 174 receptors, 174–175 structure-activity and active conformation, 175 tachykinins (TKs), 238–239, 285, 1373 amphibian, 261–267 actions in species of origin, 265 actions on mammalian animal species, 265–267 active and/or solution conformation, 264 discovery of, 261–262 distribution of, 263–264 overview, 261 pathological implications, 267 precursor mRNA/gene structure, 262–263 receptors, 264 brain, 763–769 active and solution conformation, 767 biological actions within brain, 768–769 discovery of, 763–764 mRNA distribution, 764–766 overview, 763 pathophysiological implications, 769 precursor mRNA/gene structure, 763–765 precursor processing, 766–767 receptors and signaling cascades, 767 in lung, 1301–1304 localization of, 1302 overview, 1301 receptor distribution, 1302 receptor pharmacology, 1301–1302 tachykinin-mediated biological effects, 1302–1303

Index / 1593 therapeutic potential of dual tachykinin NK 1-NK 2 receptor blockade, 1303–1304 therapeutic potential of tachykinin NK 1 receptor blockade, 1303 therapeutic potential of tachykinin NK 2 receptor blockade, 1303 therapeutic potential of tachykinin NK 3 receptor blockade, 1304 in spinal nociceptive mechanisms, 1359–1361 distribution in dorsal root ganglia and spinal cord, 1359 involvement in spinal nociception, 1360 NK-1 antagonists failure as analgesics in humans, 1361 NK-1 receptor and neuropathic pain, 1360–1361 opioid interactions and, 1361 overview, 1359 receptor expression following inflammation and nerve injury, 1359–1360 release of, 1360 SP-saporin and NK-1 receptor expressing laminae I neurons in nociception, 1361 tachykinin-gene-related peptides in regulation of endocrine glands, 833–836 adrenal, 835 Leydig cells, 835–836 ovary, 836 overview, 833 pancreatic islets, 834–835 parathyroid, 834 pituitary, 833–834 thyroid, 834 Tacrolimus (FK-506), 607–608 TAPA μ1-specific opioid peptide, 1444 Tapetum cell layer, 43 Targeted chemotherapy, 423 Tat (trans-activator of transcription) of HIV-1, 1498–1499 taurocholate (TC), 1086 T-cell clones (TCC), 598–599 T cell receptor (TCR), 585–591, 617 temporal lobe epilepse (TLE), 652 Temporin A (TA), 550 Temporin L, 307 temporins, 305–308 biological activities of, 307–308 biosynthesis of, 306–307 discovery of, 305–306 molecular heterogeneity of, 307 overview, 305 potential clinical and commercial applications of, 308 Tertiapin, 391 Tetraspanins, 616–617 TFF. See trefoil factor family (TFF) TG (triglycerides), 897–899, 1476 TGFα (transforming growth factor α), 1437–1439 TGRP (tachykinin-gene-related peptides), 833–836

3′5′-cyclic adenosine monophosphate (cAMP), 1254 three-finger toxins, 355–358 α-neurotoxins, 355–356 biological activity of, 356 conformation of, 356 discovery of, 355 precursor mRNA/gene structure, 355–356 receptors, 356 anticholinesterases, 356–357 biological activity of, 357 conformation of, 357 discovery of, 356 precursor mRNA/gene structure, 356 receptors, 357 Ca2+ channel blockers, 357 cardiotoxins, 357–358 biological activity of, 357–358 conformation of, 357 discovery of, 357 precursor mRNA/gene structure, 357 receptors, 357 disintegrin-like peptides, mamba, 357 homologs of unknown activity, 358 muscarinic toxins, 356 synergistic toxins, 358 threonine-bradykinin (Thr6-BK), 393 thrombolytic agents, 404–410 Thymopentin (RKDV Y), 575–576 thyroid-releasing hormone (TRH), 909 thyroid-stimulating hormone (TSH), 834, 843, 855–856, 880, 884–885, 1553–1556 thyroid–stimulating-hormone receptor (TSHR), 597 thyrotrophin-releasing hormone (TRH), 629–633, 1023–1024, 1470 biological actions within brain and pituitary, 631–633 discovery of, 629 overview, 629 pathophysiological implications, 633 precursor mRNA/gene structure, 629–630 precursor processing, 630 pre-proTRH mRNA and pre-pro-TRHderived peptide distribution, 630 receptor and degrading enzyme distribution, 630–631 TRH receptor signaling cascades, 631 thyrotropin (TSH), 631–632 thyrotropin-releasing hormone receptor-1 (TRH-R1), 631 thyrotropin-releasing hormone receptor-2 (TRH-R2), 631 Thyroxine (T4), 1420 TIDA (tuberinfundibular dopamine) neurons, 741 tissue-specifi c differentiation antigens, 500–501 TKQELE, 259 TKRPs. See tachykinin-related peptides (TKRPs) TKs. See tachykinins (TKs) TLE (temporal lobe epilepse), 652 TM-BBB4 cells, 1444

TMN (tuberomammillary nucleus), 920–921 TNBS (Trinitrobenzene sulfonic acid), 607 TNF (tumor necrosis factor) superfamily, 1410 TNFα (tumor necrosis factor α), 1396 TonB system, 78 Toxin cabals, 3 Toxoglossate snails, 381 trans-activator of transcription (Tat) of HIV-1, 1498–1499 transforming growth factor α (TGFα), 1437–1439 Transplant rejection, 590 trefoil factor family (TFF), 1147–1153 active conformation, 3D structure, 1150 biological actions on GI tract, 1150– 1154 enhancing rapid mucosal repair (restitution), 1152 modulating mucosal differentiation processes, 1152 modulating mucosal immune response, 1152 mucous barrier constituent, 1151–1152 overview, 1150 protective and healing effects in vivo, 1150–1151 discovery of, 1147 expression, 1148–1150 in alimentary tract of other mammals, 1149 in amphibian alimentary tract, 1149–1150 in human alimentary tract, 1148–1149 overview, 1148 overview, 1147 pathophysiological implications, 1152–1153 precursor mRNA/gene structure, 1147–1148 receptors and signaling cascades, 1150 TRH. See thyroid-releasing hormone (TRH) TRH neurons, 700 Triakontatetraneuropeptide (TTN), 815–816 Tricholin, 146, 148 triglycerides (TG), 897–899, 1476 Triiodothyronine (T3), 1420 Trinitrobenzene sulfonic acid (TNBS), 607 TRPV1 (vanilloid receptor type 1), 1007 tryptophyllin peptides, 286–287 TSHR (thyroid–stimulating-hormone receptor), 597 TTN (Triakontatetraneuropeptide), 815–816 tuberinfundibular dopamine (TIDA) neurons, 741 tuberomammillary nucleus (TMN), 920–921 tubular Ang II, 1238 tumor-asssociated antigens (TAAs), 491–492

1594 / Index tumor necrosis factor (TNF) superfamily, 1410 tumor necrosis factor α (TNFα), 1396 tumor-specific antigens resulting from genetic alterations, 501–502 20-hydroxyeicosa-5,8,11,14-tetraenoic acid (20-HETE), 1271 Type 1 bradykinin receptors (B1), 1176 Type 2 bradykinin receptors (B2), 1176 Type 2 diabetes mellitus (T2DM), 977–978 Type A lantibiotics, 98 Type B lantibiotics, 98 tyrosine sulfation, 31 Tyrosylprotein sulfotransferase, 31, 1015 U UACL (ulcer-associated cell lineage), 1152 Ubiquitin, 577 Ubiquitin E2 variant (UEV), 1501 Ucn (Urocortin), 655, 658, 1023–1025 Ucn1 (Urocortin 1), 1209–1211 Ucn2 (Urocortin 2), 656–660, 1209–1211 Ucn3 (Urocortin 3), 656–660, 1209–1211 UcnI (Urocortin I), 937–938 UcnII (Urocortin II), 937–938 UcnIII (Urocortin III), 937–938 UEV (Ubiquitin E2 variant), 1501 ulcer-associated cell lineage (UACL), 1152 ultraviolet (UV) radiation, 1484 uPA (urokinase-type plasminogen activator), 549–550 urinary tract infection, 1259 Urocortin (Ucn), 655, 658, 1023–1025 Urocortin 1 (Ucn1), 1209–1211 Urocortin 2 (Ucn2), 656–660, 1209–1211 Urocortin 3 (Ucn3), 656–660, 1209–1211 Urocortin I (UcnI), 937–938 Urocortin II (UcnII), 937–938 Urocortin III (UcnIII), 937–938 urodilatin, 1243–1248 clinical implications of, 1247–1249 acute renal failure, 1247 bronchial asthma, 1247–1248 heart failure, 1248 overview, 1247 discovery of, 1243–1246 overview, 1243 physiological and pharmacological significance of, 1246–1247 renal metabolism of, 1246–1247 urokinase-type plasminogen activator (uPA), 549–550 urotensin, 1209–1212, 1516 overview, 1209 urocortin (urotensin I) in cardiovascular system, 1209–1211 biological actions of, 1210 discovery of, 1209 overview, 1209 pathophysiological implications, 1210–1211

processing and endogenous form, including plasma, 1209–1210 receptor distribution, 1210 Ucn mRNA and peptide distribution, 1209 urotensin II in cardiovascular system, 1211–1212 biological actions of, 1211 discovery of, 1211 overview, 1211 pathophysiological implications, 1211–1212 processing and endogenous form, including plasma, 1211 receptor distribution, 1211 UII mRNA and peptide distribution, 1211 urotensin II (UII)/urotensin II–related peptide (URP), 795–800 biological actions within brain, 799–800 behavioral responses, 800 cardiovascular function regulation, 799–800 motor functions, 800 neuroendocrine actions, 800 overview, 799 discovery of, 795 localization of, 796–799 overview, 795 pathophysiological significance, 800 physiological significance, 800 receptors, 799 signaling cascades, 799 structure of, 795–796 genes, 795 overview, 795 peptides, 796 precursor processing, 795–796 urotensin receptor (UT receptor), 799 UT receptor (urotensin receptor), 799 UV (ultraviolet) radiation, 1484 V V2R (V2 receptors) expression, 1229 V2 receptors (V2R) expression, 1229 vaccines. See peptide vaccines vanilloid receptor type 1 (TRPV1), 1007 vascular endothelial growth factor (VEGF), 449, 455–456, 474, 492–493 vascular smooth muscle cells (VSMCs), 449, 1203–1206 vasoactive intestinal peptide (VIP), 570, 847–850, 1091, 1155–1159, 1215–1221, 1293–1298, 1307–1311, 1465. See also Pituitary adenylate cyclase-activating polypeptide (PACAP)/VIP biological actions of, 1218–1220, 1308–1309 discovery of, 1216 distribution of, 1216, 1307 interplay of inhibitory neurotransmitters, 1156 localization of, 1307–1308 overview, 1155, 1215, 1307 pathophysiology of, 1220–1221

physiological roles, 1309 processing of, 1216 in pulmonary disease, 1309–1310 pulmonary disease bronchial asthma, 1309–1310 cystic fibrosis, 1310 overview, 1309 primary pulmonary hypertension, 1310 receptors, 1156–1161, 1217–1218, 1309 signal transduction pathways, 1309 therapeutic potential of, 1310–1311 vasoactive peptides, at blood–brain barrier (BBB), 1461–1466 adrenomedullin, 1465 angiotensin, 1461–1462 angiotensin and bradykinin interplay, 1463 atrial natriuretic peptide, 1464 bradykinin, 1462 CNS peptides with vasoactive properties, 1465–1466 endothelins, 1464–1465 overview, 1461 substance P, 1463–1464 vasoconstriction, 1187 vasodilation, 1218, 1284 vasopressin (VP), 243–244, 581, 1223–1231, 1271, 1279–1280, 1452, 1511, 1523 active or solution conformation, 1224 biological actions of, 244 biological activity of, 1224–1225 discovery of, 1223 as hypothalamic neuropeptides, 1471 mRNA distribution, 244, 1223 overview, 1223 and oxytocin (OT), 621–627 active and/or solution conformation, 626 biological actions in brain and pituitary, 626 discovery of, 621–622 mRNA distribution, 623–624 overview, 621 pathophysiological implications of, 626–627 precursor mRNA/gene structure, 622–623 precursor processing, 624–625 receptors, 625 pathophysiological implications, 1225–1226 as peptide with vasoactive properties in central nervous system (CNS), 1465–1466 precursor mRNA/gene structure, 243–244, 1223 receptors, 244, 1224 vasopressin pituitary receptors (V3 receptors), 1224 vasopressin receptor (VR), 625 vasopressin renal receptors (V2 receptors), 1224–1226 vasopressin vascular receptors (V1 receptors), 1224–1226

Index / 1595 vasorelaxation, 1258 vasostatins (VS), 317 VEGF (vascular endothelial growth factor), 449, 455–456, 474, 492–493 venom peptides ants, 393–394 ectatomin, 394 Myr p peptides and pilosulin, 393 poneratoxins, 393–394 ponericins, 394 conus snail, 381–387 biodiversity of venomous molluscs, 381 biological mechanisms, 386–387 conotoxin families definined, 384–385 discovery of conotoxins, 381–382 overview, 381 precursor structure, expression, and processing, 382–384 receptor targets definined, 386 structures definined, 386 therapeutic applications, 387 scorpion, 339–343 discovery of, 339 molecular basis of activity, 341–342 overview, 339 receptor sites, 341 scorpion toxin precursors, 341 three-dimensional folding, 340–341 sea anemone, 363–366 bioactive Mollusca peptides, 366 cytolysins, 365–366 ion channel blockers and modulators, 363–365 overview, 363 social hymenoptera, 390–391 apamin, 391 bumblebee venoms, 391 honeybee venom, 390–391 social wasps, 391–392 chemotactic peptides, 392 crabrolin, 392 kinin-related peptides (wasp kinins), 392 mastoparans, 392 sylverin, 392 solitary wasps, 392–393 anoplin, 393 bradykinin (BK)-related peptides, 393 eumenine mastoparan-AF (EMP-AF), 393 pompilidotoxins (PMTXs), 393 spider, 355–361 acting on acid-sensing ion channels (ASIC), 376

acting on glutamate receptors/ transporters, 376 acting on mechanosensitive ion channels (MSCs), 376 acting on neurotransmitter release (presynaptic toxins), 377 antimicrobial and cytolytic peptide toxins from, 378–379 approaches in isolation of, 369–370 AVIT peptides, 361 bradykinin-potentiating peptides, 360–361 CRISP family toxins, 359 crotamine-like myotoxins, 359 C-type lectins and lectin-like proteins, 360 disintegrins, 360 miscellaneous peptides, 361 modulating Nav channel function, 370–374 modulating on Cav channels, 375–376 modulating voltage-gated potassium (K+) channels, 374–375 natriuretic peptides, 359–360 with nonselective actions on voltagegated ion channels, 376 overview, 355, 369 precursor structure and posttranslational processing of, 370 protease inhibitor homologs, 358–359 sarafotoxins, 359 sphingomyelinases (SMases) D toxins from, 377–378 structural organization of, 370 three-finger toxins, 355–358 waglerins, 360 therapeutic properties of, 403–412 analgesia, 412 antiarrhythmic agents, 410 anticoagulants, 403–404 antihypertensive agents, 410 antimicrobial peptides, 410–411 antitumor peptides, 411–412 congestive heart failure, 410 diabetes mellitus, 412 immunomodulatory peptides, 411 overview, 403–404 thrombolytic agents, 404–410 worm, 397–400 annelid (Glycera) neurotoxin, 399 nemertine peptide cytolysins, 399 nemertine peptide neurotoxins, 398–399 overview, 397–398 ventral tegmental area (VTA), 699, 898

ventricular natriuretic peptide (VNP), 806–807 VGSC (voltage-gated sodium channels), 364 Victorin C, 154 VIP. See vasoactive intestinal peptide (VIP) VIPomas, 1133 viral preintegration complex (PIC), 1497 viral proteins, 501 Viroicin, 135 virotoxins, 135 visceral pain, 1027 visceral sensitivity and nociception, 1009–1010 VNP (ventricular natriuretic peptide), 806–807 Voltage gated ion-channels, 86 voltage-gated K+ channels (Kv), 365 voltage-gated sodium channels (VGSC), 364 vomiting, 1144 VP. See vasopressin (VP) VPAC1 receptor of vasoactive intestinal peptide, 1215–1218 VPAC2 receptor of vasoactive intestinal peptide, 1215–1218 VR (vasopressin receptor), 625 VS (vasostatins), 317 VSMCs (vascular smooth muscle cells), 449, 1203–1206 VTA (ventral tegmental area), 699, 898 W waglerins, 360 wasps social, 391–392 solitary, 392–393 Wistar-Kyoto hypertensive rats (SHR), 1220 WKYMVm hexapeptide, 549 worm venom peptides, 397–400 annelid (Glycera) neurotoxin, 399 nemertine peptide cytolysins, 399 nemertine peptide neurotoxins, 398–399 overview, 397–398 WUSCHEL homeodomain-containing transcription factor (WUS), 13–14 W(X)6Wamide precursor of AST, 202 Y Y2 receptor (Y2R), 948 Y receptors, 1101–1102 Z Zona glomerulosa (ZG) cells, 858, 872

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