The Pituitary (Pituitary (Melmed))

The Pituitary

Notice: The indications and dosages of all drugs in this book have been recommended in the medical literature and conform to the practices of the general community. The medications described do not necessarily have specific approval by the Food and Drug Administration for use in

the diseases and dosages for which they are recommended. The package insert for each drug should be consulted for use and dosage as approved by the FDA. Because standards for usage change, it is advisable to keep abreast of revised recommendations, particularly those concerning new drugs.

The Pituitary EDITED BY

SHLOMO MELMED, Senior Vice-President, Academic Affairs Cedars-Sinai Medical Center Professor and Director, Burns & Allen Research Institute UCLA School of Medicine Los Angeles, California

SECOND EDITION

Blackwell Science

MD

© 2002 by Blackwell Science, Inc. Editorial Offices: Commerce Place, 350 Main Street, Malden, Massachusetts 02148, USA Osney Mead, Oxford OX2 0EL, England 25 John Street, London WC1N 2BS, England 23 Ainslie Place, Edinburgh EH3 6AJ, Scotland 54 University Street, Carlton, Victoria 3053, Australia Other Editorial Offices: Blackwell Wissenschafts-Verlag GmbH, Kurfürstendamm 57, 10707 Berlin, Germany Blackwell Science KK, MG Kodenmacho Building, 7-10 Kodenmacho Nihombashi, Chuo-ku, Tokyo 104, Japan Iowa State University Press, A Blackwell Science Company, 2121 S. State Avenue, Ames, Iowa 50014-8300, USA

All rights reserved. No part of this book may be reproduced in any form or by any electronic or mechanical means, including information storage and retrieval systems, without permission in writing from the publisher, except by a reviewer who may quote brief passages in a review. Acquisitions: Beverly Copland Development: Angela Gagliano Production: Rosie Hayden Manufacturing: Lisa Flanagan Marketing Manager: Kathleen Mulcahy Cover design by Leslie Haimes Typeset by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound by Sheridan Books, Michigan, USA Printed in the United States of America 02 03 04 05 5 4 3 2 1 The Blackwell Science logo is a trade mark of Blackwell Science Ltd., registered at the United Kingdom Trade Marks Registry

Distributors: The Americas Blackwell Publishing c/o AIDC P.O. Box 20 50 Winter Sport Lane Williston, VT 05495-0020 (Telephone orders: 800-216-2522; fax orders: 802-864-7626) Australia Blackwell Science Pty, Ltd. 54 University Street Carlton, Victoria 3053 (Telephone orders: 03-9347-0300; fax orders: 03-9349-3016) Outside The Americas and Australia Blackwell Science, Ltd. c/o Marston Book Services, Ltd. P.O. Box 269 Abingdon Oxon OX14 4YN England (Telephone orders: 44-01235-465500; fax orders: 44-01235-465555) Library of Congress Cataloging-in-Publication Data The pituitary/edited by Shlomo Melmed.— 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN 0-632-04357-1 (hardcover) 1. Pituitary gland—Diseases. 2. Pituitary gland—Pathophysiology. 3. Pituitary gland— Physiology. [DNLM: 1. Pituitary Diseases— physiopathology. 2. Pituitary Diseases— diagnosis. 3. Pituitary Gland—physiology. 4. Pituitary Hormones, Anterior—physiology. WK 550 P6913 2002] I. Melmed, Shlomo. RC658 .P557 2002 616.4¢7—dc21 2001004591

Contents

Contributors vii Preface

Section 3. PITUITARY TUMORS

x

10.

Pituitary Surgery 405 Rudolf Fahlbusch, Michael Buchfelder and Panos Nomikos

11.

Acromegaly 419 Shlomo Melmed

12.

Prolactinoma 455 Mark E. Molitch

13.

Cushing’s Disease 496 Xavier Bertagna, Marie-Charles Raux-Demay, Brigitte Guilhaume, François Girard and Jean-Pierre Luton

14.

Thyrotropin-secreting Pituitary Tumors 561 Yona Greenman and Shlomo Melmed

15.

Gonadotroph Adenomas Peter J. Snyder

16.

Nonpituitary Tumors of the Sellar Region 592 Steffen Albrecht, Juan M. Bilbao and Kalman Kovacs

Section 1. HYPOTHALAMIC–PITUITARY FUNCTION 1.

2.

Functional Anatomy of the Hypothalamic Pituitary Axis 3 Sylvia L. Asa, Kalman Kovacs and Shlomo Melmed Adrenocorticotropin 45 Mark A. Herman and Joseph A. Majzoub

3.

Growth Hormone 79 Vivien S. Herman-Bonert and Shlomo Melmed

4.

Prolactin 119 Mark E. Molitch

5.

Thyroid-stimulating Hormone 172 Virginia D. Sarapura, Mary H. Samuels and E. Chester Ridgway

6.

Follicle-stimulating Hormone and Luteinizing Hormone 216 Shalender Bhasin, Charles E. Fisher and Ronald S. Swerdloff

7.

The Posterior Pituitary Daniel G. Bichet

279

Section 2. HYPOTHALAMIC–PITUITARY DYSFUNCTION 8.

The Hypothalamus 317 Glenn D. Braunstein

9.

Anterior Pituitary Failure Charles F. Abboud

349

575

Section 4. PITUITARY DISEASE IN SYSTEMIC DISORDERS 17.

Pituitary Function in Systemic Disorders 613 Harold E. Carlson

18.

The Pituitary Gland in Pregnancy and the Puerperium 628 Harold E. Carlson

19.

Drugs and Pituitary Function Harold E. Carlson

642 v

vi

Contents

Section 5.

DIAGNOSTIC PROCEDURES

20.

Pituitary Imaging 663 Barry D. Pressman

21.

Neuro-ophthalmologic Evaluation of Pituitary Disorders 687 Anthony C. Arnold

22. Index

Evaluation of Normal Pituitary Function 709 Gillian L. Booth, Afshan Zahedi and Shereen Ezzat 727

Contributors

Charles F. Abboud, MD David Eisenberg Professor of Medicine Consultant, Internal Medicine and Endocrinolgy Mayo Clinic, Mayo Medical School Rochester, MN Steffen Albrecht, MD FRCP(C) Department of Pathology Sir Mortimer B. Davis Jewish General Hospital Montreal, Canada Anthony C. Arnold, MD Associate Professor of Ophthalmology and Chief, Neuro-Ophthalmology Division Jules Stein Eye Institute, UCLA Department of Ophthalmology Los Angles, CA Sylvia L. Asa, MD, PhD Associate Professor of Pathology Mount Sinai Hospital University of Toronto Toronto, Canada Xavier Bertagna, MD Professor of Endocrinology Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Shalender Bhasin, MD Chief, Division of Endocrinology, Metabolism, and Molecular Medicine Professor of Medicine Charles R. Drew University School of Medicine and Science Los Angeles, CA

Daniel G. Bichet, MD, FRCP(C) Service de néphrologie et Centre de recherche Hôpital du Sarcé-Coeur de Montréal Department of Medicine Université de Montréal Montréal, Quebec Canada Juan M. Bilbao, MD, FRCP(C) Neuropathologist, Department of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada Gillian L. Booth, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada Glenn D. Braunstein, MD Chairman, Department of Medicine Cedars-Sinai Medical Center Professor of Medicine UCLA School of Medicine Los Angles, CA Michael Buchfelder, MD Associate Professor Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany vii

viii

Contributors

Harold E. Carlson, MD Chief, Endocrinology Section Northport Veterans Affairs Hospital, Northport Professor of Medicine State University of New York at Stony Brook Stony Brook, NY

Kalman Kovacs, MD, PhD, DSc, FRCP(C), FCAP, FRC Path. Professor of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada

Shereen Ezzat, MD, FRCP(C), FACP Assistant Professor of Medicine University of Toronto Consultant Endocrinologist Wellesley Hospital Toronto, Canada

Jean-Pierre Luton, MD Professor of Endocrinology and Director Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Dean, Cochin Medical School University René Descartes Paris, France

Rudolf Fahlbusch, MD Professor of Neurosurgery Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Charles Ellis Fisher, MD Division of Endocrinology and Metabolism King Drew Medical Center Los Angeles, CA François Girard, MD Professor of Physiology and Director Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France Yona Greenman, MD Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel Brigitte Guilhaume, MD Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Mark A. Herman, MD Fellow in Medicine Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, Texas Vivien S. Herman-Bonert, MD Division of Endocrinology Cedars-Sinai Medical Center Associate Professor of Medicine UCLA School of Medicine Los Angeles, CA

Joseph A. Majzoub, MD Chief, Division of Endocrinology Department of Pediatrics Children’s Hospital Professor of Pediatrics Harvard Medical School Boston, MA Shlomo Melmed, MD Senior Vice-President, Academic Affairs Director, Burns & Allen Research Institute Associate Dean, UCLA School of Medicine Cedars-Sinai Medical Center Los Angeles, CA Mark E. Molitch, MD Professor of Medicine Center for Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, IL Panos Nomikos, MD Neurosurgical Fellow Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Barry D. Pressman, MD, FACR Chief, Section of Neuroradiology and Chairman, Department of Imaging Cedars-Sinai Medical Center Los Angles, CA Marie-Charles Raux-Demay, MD Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France

Contributors

Chester E. Ridgway, MD Professor of Medicine and Head, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO Mary H. Samuels, MD Assistant Professor of Medicine Division of Endocrinology Oregon Health Science University Portland, OR Virginia D. Sarapura, MD Assistant Professor of Medicine Department of Medicine, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO

Peter J. Snyder, MD Professor of Medicine University of Pennsylvania School of Medicine Philadelphia, PA Ronald S. Swerdloff, MD Professor of Medicine and Chief, Division of Endocrinology Department of Medicine Harbor/UCLA Medical Center UCLA School of Medicine Torrance, CA Afshan Zahedi, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada

ix

Preface

The second edition of The Pituitary follows the successful initial 1995 debut of this comprehensive text devoted to understanding pathogenesis and treatment of pituitary disorders. The new edition is extensively revised to reflect the wealth of novel information derived from advances in molecular biology, biochemistry and therapeutics as they apply to the pituitary gland. Notably, a new chapter devoted to pituitary surgery has been added to complement a comprehensive overview of management options for patients harboring pituitary tumors. The wide spectrum of clinical disorders emanating from disordered function of the “master gland” are described in detail by experts in the field. Furthermore, fundamental mechanisms underlying disease pathogenesis are presented to provide the reader with an in-depth understanding of mechanisms underlying pituitary hormone secretion and action.

Thus the volume continues to reflect the cogent blend of basic science and clinical medicine which was the successful hallmark of the first edition. I am especially indebted to my erudite colleagues for their scholarly contributions and dedicated efforts in compiling this body of knowledge for physicians and scientists dedicated to caring for patients with pituitary disorders. Our desire is to provide medical students, clinical and basic endocrinology trainees, endocrinologists, internists pediatricians, gynecologists and neurosurgeons with a comprehensive single text devoted to the science and art of pituitary medicine. Shlomo Melmed, MD Los Angeles 2002

x

S e c t i o n 1

Hypothalamic–Pituitary Function

C h a p t e r

1 Functional Anatomy of the Hypothalamic Pituitary Axis Sylvia L. Asa Kalman Kovacs Shlomo Melmed

INTRODUCTION

Historical Review Descartes was the first to recognize that the brain was an organ integrating the functions of the mind and body [1]. The association was reinforced by Zander [2] who noted the connection between the adrenals and the brain, referring to observations on the absence of the adrenal cortex in anencephaly as recorded by Morgagni in 1733, Soemmering in 1792, and Meckel in 1802. It was only in 1849 that direct evidence of a role for the hypothalamus in endocrinology was provided by Claude Bernard [3] when he demonstrated that injury to the floor of the fourth ventricle, the “piqûre diabetique,’ caused polyuria and glucosuria; subsequently, numerous studies in the late 19th century and early 20th century confirmed that the hypothalamic–posterior pituitary system was the site of production of a major osmoregulatory substance. Galen (129–201 ad) had described the pituitary as the site of drainage of phlegm from the brain to the nasopharynx. Soon after the description of acromegaly by Pierre Marie in 1886 [4], the association of acromegaly with pituitary tumor was noted by Minkowski in 1887 [5], and the recognition of endocrine functions of the pituitary followed rapidly thereafter, with major contributions by Cushing [6] and Simmonds [7]. The early part of the 20th century saw the identification, isolation and characterization of the hormones of the anterior pituitary. Their regulation by the hypothalamus was the subject of a landmark monograph by Harris in 1948 [8]. The concept that neurons could release secretory materials into the bloodstream, proposed by Du Bois Reymond

in 1877 [9] and Schiefferdecker in 1905 [10], was revolutionary. The anatomic basis of a hypothalamic–pituitary vascular connection was already known to exist in the form of a system of capillaries, which were identified joining the medial basal hypothalamus to the anterior pituitary by Lieutaud in 1742 [11] and von Luschka in 1860 [12] and given the name “portal plexus” by Popa and Fielding in 1930 [13]. These vessels were thought at first to carry blood from the pituitary upwards to the hypothalamus; however, subsequent studies confirmed that they provided the main blood supply to the adenohypophysis. The concept of neurosecretion was suggested in the 1940s by Scharrer and Scharrer [14] and Bargmann and Scharrer [15], who demonstrated that peptide synthesized by neurons in the supraoptic and paraventricular nuclei of the hypothalamus passed through nerve fibers, was stored in nerve endings in the posterior pituitary and subsequently was released into the general circulation; the analysis and synthesis of oxytocin and vasopressin in 1954 [16] led to a Nobel Prize for du Vigneaud. In the 1950s and 1960s, evidence for the presence of hypothalamic releasing and inhibiting factors accumulated [17]. The isolation and characterization of the first of these yielded shared Nobel prizes for Guillemin and Schally in 1977 and by the mid 1980s, the isolation and characterization of many had been achieved [18–26].

Methodology Progress in the understanding of hypothalamic and pituitary endocrinology has utilized numerous techniques which have allowed the identification of hormones, their isolation and characterization, their localization, and recognition of their cell of origin. At the beginning of the 20th century, Benda [27], Erdheim [28], Cushing [6], and others [29,30] utilized histologic staining methods for the investigation of the human pituitary. Morphologic studies of the hypothalamus were 3

4

SECTION 1

Hypothalamic–Pituitary Function

dependent on conventional neurohistologic methods to demonstrate neurosecretory material and the silver stains that allowed visualization of neuronal cell processes [31]. The tinctorial properties of cell cytoplasm using hematoxylin–eosin, hematoxylin–phloxine, or hematoxylin– phloxine–saffron methods allowed the distinction of three cell types in the adenohypophysis. Clinical observations, correlated with morphologic changes, led to the classification of acidophils as cells thought to produce growth hormone (GH), basophils as those believed to contain adrenocorticotropin (ACTH) and chromophobes as functionally inactive cells. In the 1930s, Rasmussen [32] quantitated the three cell types in relation to sex, age, and body weight and determined their distributions within the gland. In 1940, Romeis [33] used a modification of the trichrome technique to establish that more than three cell types were identifiable in the adenohypo-physis. Application of the periodic acid–Schiff (PAS) method distinguished the PASpositive corticotrophs, thyrotrophs and gonadotrophs; similar results were obtained with lead hema-toxylin. Various trichrome stains, the aldehyde fuchsin and aldehyde thionin techniques were used to separate basophils [34] and the addition of Herlant’s erythrosin [35] and Brookes’ carmoisine [36] were useful to distinguish lactotrophs from the orange G-positive somatotrophs. Physiologic and biochemical studies provided evidence that six different hormones are produced by the anterior pituitary; however, histologic techniques failed to correlate cell type with clinical hormone production or secretory function. The search for correlations between structure and function led to the application of novel techniques including immunocytology [37,38] and electron microscopy [39,40] to the study of pituitary morphology. Ultrastructural immunocytochemistry [41], greatly improved by the use of the immunogold technique [40,42], and the application of the reverse hemolytic plaque assay [43], tissue culture methods [44] and molecular probes [45], have made pituitary and hypothalamic morphology significant for assessing pituitary function and pathology. HYPOTHALAMUS AND POSTERIOR PITUITARY

Topographic Anatomy The hypothalamus is a phylogenetically primitive structure. Understanding of its role in endocrine regulation is largely derived from animal studies; early data regarding functional localization were based on selective stimulation and ablation experiments. However, there are considerable anatomic and physiologic species variations which make extrapolation to the human difficult. Only the advent of sophisticated neurophysiologic methods and the recent applications of immunocytochemistry, as well as experiments of nature in which careful analyses of the location of pathologic lesions are performed, have allowed structure–function correlations in the human hypothalamus.

The hypothalamus is a poorly defined anatomic region which constitutes less than 1% of brain volume and weighs approximately 5 g [46,47]. The superior border of this region is the hypothalamic sulcus which courses from the interventricular foramen to the cerebral aqueduct, demarcating the hypothalamus from the thalamus (Fig. 1.1). The anterior delineation is roughly defined as a line through the anterior commissure, lamina terminalis, and optic chiasm. Posteriorly, it is bordered on the superior aspect by the midbrain tegmentum and inferiorly by the mamillary bodies. The lateral borders are defined conventionally as the substantia innominata, the internal capsule, the subthalamic nucleus, and the cerebral peduncle. The inferior aspect of the hypothalamus, known as the tuber cinereum, contains the median eminence and gives rise to the infundibulum, the neural stalk and the posterior lobe of the pituitary.

Vascular Supply A significant number of afferent and efferent signals of the hypothalamus are not neural, but rather represent bloodborne information, such as temperature, osmotic pressure, hormones, and glucose levels, thus endorsing the importance of the vascular supply of this area. The blood supply of the hypothalamus is derived from small arterial branches of the circle of Willis and the superior hypophysial artery. It is divided into three parts: (i) an anterior group which arises from the internal carotid artery, the anterior cerebral artery, and the anterior portion of the posterior communicating artery; (ii) an intermediate group from the posterior communicating artery; and (iii) a posterior group from the posterior communicating artery, posterior cerebral, and basilar arteries. Individual hypothalamic nuclei receive blood from more than one small artery. The microvascular pattern of the hypothalamus resembles that of the remainder of the brain with the exception of the magnocellular nuclei, the supraoptic and paraventricular nuclei. These are some of the most richly vascularized structures in the brain, in which the neurons are intimately associated with capillaries. The capillary endothelium is fenestrated and the blood–brain barrier is absent in many areas of the hypothalamus, including the subfornical organ, the organum vasculosum of the lamina terminalis (OVLT), the median eminence, and the neurohypophysis. The arterial supply of the median eminence and posterior pituitary has been extensively studied [48–51] (Fig. 1.2). It is derived from two (or three, in some species) paired arteries that arise from the intracranial portions of the internal carotid arteries. The superior hypophysial arteries branch into a plexus of small arteries that surrounds the upper half of the stalk (the external plexus), giving rise to a mesh of capillaries, and the gomitoli (the internal plexus). The latter are unique vascular structures composed of a central muscular artery surrounded by a spiral of capillaries, the arteriole communicating with the capillaries by way of small

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

5

FIGURE 1.1. Schematic representation of the human hypothalamus and pituitary, the disposition of hypothalamic nuclei and principal fiber tracts. From Scheithauer [47]

orifices surrounded by muscular sphincters. The gomitoli, which measure 1–2 mm in length and 0.1 mm in width, are found in large numbers in the infundibulum and proximal hypophysial stalk. Flow through these complex structures continues on through the portal vessels to the adenohypophysial capillaries. Although their function is not certain, the unique structure of gomitoli suggests that they may affect the rate of blood flow to the anterior pituitary, thereby influencing the entry of hypothalamic regulatory hormones into the portal circulation. In some individuals, a pair of middle hypophysial arteries also contributes to the trabecular or loral arteries which descend from the superior hypophysial arteries along the external surface of the pituitary stalk. These then divide to contribute to the subcapsular artery and the artery of the fibrous core (which provide a minor contribution to the blood supply of the adenohypophysis) and return upwards along the pituitary stalk as the long stalk arteries. The inferior hypophysial

arteries enter the sella just beneath its diaphragm and supply the pituitary capsule, the neural lobe, and the lower pituitary stalk. They enter the intralobar groove where they divide into ascending and descending branches that join their opposite partners to form an arterial circle about the neural lobe. They also give off a branch to the lower pituitary stalk, the communicating artery, which anastomoses with the trabecular arteries. Notably, the capillaries of the neurohypophysis are fenestrated and lie outside the blood–brain barrier. The earliest studies of the hypophysial portal vasculature assumed that blood flowed from the pituitary upwards to the hypothalamus [13]. Subsequently, it was found that the predominant blood flow is from the hypothalamus to the adenohypophysis, carrying hypothalamic regulatory factors. Recent studies suggest that the direction of blood flow is variable and there may be a component of reverse flow within the neurohypophysis, providing a route by which

6

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 1.2. Schematic representation of the blood supply of the hypothalamus and pituitary. From Scheithauer [47]

neuro- and adenohypophysial hormones gain access to the long portal system, the pars tuberalis of the adenohypophysis, the median eminence, and the cerebrospinal fluid of the third ventricle [50,51].

Nerve Supply A number of afferent neural pathways provide the hypothalamus with input from the forebrain, limbic system, visual cortex, thalamus, and brain stem [47,52]. The median forebrain bundle, a large unmyelinated structure lateral to the fornix, carries information to and from the brain stem and provides important input from the limbic system and olfactory forebrain structures. Olfactory connections are more extensively from the piriform cortex and

amygdaloid nuclear complex via the stria terminalis and ventral amygdalofugal pathway. The hippocampal–hypothalamic tract originates in the hippocampus, a limbic structure, and enters the anterolateral portion of the mamillary bodies. The amygdalo–hypothalamic tract provides entry of emotional data from the amygdaloid nucleus into the hypothalamus. The retino–hypothalamic tract originates in the ganglion cells of the retina and affects the pineal gland, playing a role in the regulation of circadian rhythms. In addition, a small number of cortico–hypothalamic fibers from the posterior orbital gyrus of the frontal lobe is a potential source of input from the neocortex into the hypothalamus.

Chapter 1

The hypothalamus sends efferent projections to the forebrain, brain stem, spinal cord and neurohypophysis. The median forebrain bundle carries fibers to the septal nuclei, integrating visceral and olfactory functions. The dorsal longitudinal fasciculus carries efferents to the midbrain tegmentum where it innervates visceral and sympathetic motor neurons. Fibers emanating from the mamillary body divide into the mamillo–thalamic tract, serving the anterior nucleus of the thalamus and memory function, and the mamillotegmental tract which projects to the midline tegmentum. Efferents from the ventral medial nucleus project to the amygdaloid nucleus via the stria terminalis. The hypothalamo–hypophysial tract consists primarily of nerve fibers from the supraoptic and paraventricular nuclei carrying vasopressin, oxytocin and their neurophysins to the posterior lobe of the pituitary, where the hormones are released into capillaries. The tubero–infundibular tract, originating from neurosecretory neurons which produce hypophysiotropic hormones, projects from several nuclei to the median eminence where the hormones are released into the hypophysial portal vascular system.

Nuclei and Nucleoinfundibular Pathways Within the hypothalamus, distinct clusters of neurons are called “nuclei.” The hypothalamic nuclei are generally divided into four anatomic regions, the preoptic, supraoptic and lateral, tuberal, and mamillary regions. In general, the nuclei are topographically discrete in many species and, although relatively well demarcated in the human fetus, they are poorly defined in the mature human hypothalamus [46]. In a few cases, the existence of some nuclei is inferred entirely from studies of experimental animals. A schematic illustration is provided in Fig. 1.1. Structure–function correlations are difficult because of cellular heterogeneity of many hypothalamic nuclei. Any given hypothalamic hormone is often produced in more than one nucleus and, in many cases, a single nucleus may express more than one hormone. The physiologic roles of many nuclei remain unknown. The most anterior nuclei are the paired medial and lateral nuclei that have been associated with autonomic function, particularly temperature control and olfaction. The suprachiasmatic nucleus is found in the preoptic area dorsal to the optic chiasm and anterior to the supraoptic nucleus. This sexually dimorphic cell group shows a striking decrease in volume and cell number with age [53]. It is located in an area that is essential for gonadotropin release and sexual behavior in lower animals and may play a role in maintaining circadian rhythms. It is thought that the nucleus also plays a role in the sexual differentiation of the brain which, in the absence of male gonadal hormones, remains female but, if exposed to these at a critical stage in development, becomes male [54]. It is also associated with fibers of the supraoptic commissure, and receives afferents from the retina and the lateral geniculate bodies.

Functional Anatomy of the Hypothalamic Pituitary Axis

7

The anterior hypothalamic nucleus is composed of small neurons which mediate parasympathetic effects; it has connections to the insular cortex, substantia innominata and thalamus. The lateral hypothalamic nuclei are poorly defined areas composed of larger neurons which receive fibers from and contribute efferents to the median forebrain bundle. The paraventricular nuclei lie adjacent to the third ventricle and ventromedial to the fornix. They are composed mainly of large “magnocellular” neurons along with the supraoptic nuclei and also contain a number of “parvicellular” neurons. Efferent fibers to the pituitary stalk terminate in the posterior lobe of the pituitary. These nuclei are a major site of oxytocin and vasopressin synthesis. In patients with traumatic or surgical stalk section and in those with long-standing hypopituitarism, there is a marked reduction in the number of magnocellular neurons and stalk nerve fibers [46]; the parvicellular component remains. The supraoptic nuclei are the other paired magnocellular nuclei of the hypothalamus; they have no significant parvicellular component. They overlie the anterior optic tract and posteriorly are divided by the optic tract into anterolateral and posteromedial components, united by a thin stream of cells. Patients with stalk section or long-standing hypopituitarism also have atrophy of these nuclei [55]. The dorsomedial and ventromedial nuclei are involved in autonomic function and emotional behavior. They are situated between the tuber cinereum and paraventricular nuclei. Stimulation of the dorsomedial and destruction of the ventromedial nuclei produces rage in experimental animals. The ventromedial nuclei are concerned with control of balance between hunger and satiety. Destruction of the ventromedial nucleus results in obesity [56,57]; conversely, destruction of the ventrolateral nucleus, known as the “feeding center,” is associated with anorexia and cachexia [56]. These nuclei show a large number of afferent connections from olfactory and retinal fibers, the reticular formation, and the nucleus of the solitary tract which receives input from the vagus. Afferents from the cortex enter by way of the thalamus. Ventral to the third ventricle and paraventricular nuclei lies the arcuate (infundibular) nucleus which is another important component of the hypophysiotropic region and plays a major role in the modulation of anterior pituitary function. The subventricular nucleus lies on the floor of the third ventricle posterior to the arcuate and anteromedial to the tuberal nuclei. This usually parvicellular nucleus shows marked hypertrophy of the neurons which become magnocellular in postmenopausal women [58,59], in young women suffering from postpartum hypopituitarism with complete gonadal atrophy [60], in hypogonadal men and women, in starvation [61], after hypophysectomy [62], and in late pregnancy. The neurons develop a distinctive nucleolar change considered a manifestation of feedback effect; a similar change is also observed in neurons of the arcuate

8

SECTION 1

Hypothalamic–Pituitary Function

nucleus [60]. In some of these conditions, the change is thought to reflect lack of estrogens [59,63,64]. The tuberal nuclei consist of irregularly grouped masses of large neurons inferior to the lateral nuclei that give rise to efferent fibers of the hypothalamus. After stalk section, a slight increase in coarsely granular basophilic cytoplasmic material may occur [46]. The posterior hypothalamic nucleus, situated between the third ventricle and the mamillo–thalamic tract, lies superior to the mamillary bodies. When stimulated, it produces sympathetic effects; it may play a role in temperature regulation and its large neurons may be the source of hypothalamic efferents that descend to the reticular formation of the brain stem. The paired mamillary nuclei and a variety of minor nuclei in the supramamillary area, including the nucleus intracalatus, form the most posterior portion of the hypothalamus. These nuclei integrate incoming information from the limbic system and the midbrain tegmentum and send out efferent fibers to the anterior nucleus of the thalamus and to the brain stem.

Embryology The forebrain of the human embryo can be identified at 4 weeks postconception; by 5 weeks it has divided into a central diencephalon and two lateral telencephalic structures. Three swellings appear in the diencephalon which represent the anlages of epithalamus, thalamus, and hypothalamus. The nuclei of the hypothalamus and the supraoptico-hypophysial tract are recognizable at 8 weeks of gestation [65]. The nerve cells originate from proliferating neuroblasts and form several closely associated nuclei in the hypothalamus. At 6 weeks, a thickening of neuroblasts projects caudally to become the median eminence and infundibulum and establishes direct contact with the upward invagination of the roof of the primitive oral cavity, Rathke’s pouch. By 3 months, the rudimentary neural lobe consists of a short wide sac with a thick wall and a lumen lined by columnar ependymal cells [66]. Proliferation of spongioblasts gives rise to the modified neuroglia of the neurohypophysis known as pituicytes while the ependymal cells become dispersed in the neuroglial substance. Downgrowth of unmyelinated axons of neurons of magnocellular nuclei of the hypothalamus reaches the median eminence by 3 months, passes through the stalk and terminates in the neural lobe by 6 months of gestation. Monoamine fluoresence can be detected at 10 weeks in hypothalamic nuclei and at 13 weeks in the median eminence [67]. The supraoptic nucleus matures earlier than the paraventricular nucleus; vasopressin is demonstrated earlier and in higher concentrations than oxytocin in the neural lobe [68]. Arginine vasopressin is identified by radioimmunoassay at 12 weeks of gestation [69]. Intracellular granules have been demonstrated in hypothalamic nuclei at 19 weeks and in the posterior lobe at 23 weeks of gestation. The

neurosecretory material is clearly visible in the supraoptic and paraventricular nuclei as well as the posterior lobe at 23–28 weeks of gestation [70–72]. Ultrastructurally, clusters of small clear vesicles and axon profiles have been identified in a fetus at 7.5 weeks of age. Granular vesicles become apparent in the axon profiles at 8.5 weeks of gestation. Fenestration of capillaries and expansion of perivascular spaces are noted with the appearance of intraaxonal vesicles [73]. Growth hormone-releasing hormone (GHRH) has been identified by immunohistochemistry in the fetal hypothalamus at 18 weeks of gestation [74]. Somatostatin (somatotropin release-inhibiting hormone; SRIH) is identified in fetal hypothalami in a large molecular form at 10 weeks of gestation and there is a gradual increase in both the number of somatostatin-containing neurons and the 14-amino acid form of this peptide with fetal age [75,76]. Dopamine is present in high concentrations in the fetal hypothalamus by 15 weeks of gestation [67]. Corticotropin-releasing hormone (CRH) has been detected in fetal median eminence at 16 weeks of gestation [77]. Thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH) are the first adenohypophysiotropic substances, being detectable in fetal hypothalami at 4.5 weeks and present in significant amounts by 10 weeks of gestation [78,79]. GnRH is localized by immunocytochemistry at 13 weeks and GnRH-containing nerve terminals are detected on portal vessels at 16 weeks [80]. Sexual dimorphism of GnRH concentration has been documented and correlates with sexual dimorphism of fetal gonadotropic hormones [81].

Functional Anatomy and Control of Anterior Pituitary Function Control of anterior pituitary hormone secretion is under a complex set of central and peripheral neural and chemical signals. The hypothalamus synthesizes and secretes unique releasing and inhibitory hormones that control the anterior pituitary hormones (Fig. 1.3). The polypeptide hormones are secreted into the hypothalamic pituitary portal vasculature, impinge upon specialized anterior pituitary cells and bind to specific surface receptors. The cell signal elicits regulation of pituitary trophic hormone gene transcription, translation, and/or secretion. The resultant secretion of anterior pituitary hormones into the systemic circulation depends upon a carefully controlled dynamic balance of hypothalamic hormone input, feedback regulation from peripheral target hormones, nonhormonal neurotransmitter agents, and paracrine or autocrine pituitary growth factors and peptides. These factors all contribute to the amount of each pituitary hormone secreted, as well as to their pattern of secretion. Complex feedback regulation loops interact to regulate the hypothalamic–pituitary axis. The target gland peripheral hormone usually exerts negative feedback regulation directly at the level of pituitary synthesis or secretion of the respective trophic hormone (Fig. 1.4). Pituitary

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

9

FIGURE 1.3. Amino acid sequences of human hypothalamic hormones. CRH, corticotropin-releasing hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; SRIH, somatostatin; TRH, thyrotropin-releasing hormone.

FIGURE 1.4. General control of anterior pituitary hormone release by central and hypothalamic inputs and feedback regulation by peripheral and trophic hormones. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; DA, dopamine; FSH, folliclestimulating hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; IGF-I, insulin-like growth factor-I; LH, luteinizing hormone; PRL, prolactin; SRIH, somatostatin; TRH, thyrotropin-releasing hormone; TSH, thyrotropin; VIP, vasoactive intestinal polypeptide.

trophic hormones may feed back in a very short loop to autoregulate their own secretion. They may also participate in short loop feedback regulation of the secretion of their respective hypothalamic releasing or inhibiting hormone. Finally, peripheral hormones may participate indirectly by feedback regulation of hypothalamic releasing and inhibiting hormones.

The central nervous system also influences the control of hypothalamic hormonal secretion by efferent signals emanating from exteroceptive organs including the ears, eyes, and skin, and from interoceptive senses of indices of metabolic and electrolyte homeostasis. The transduction of neuronal inputs to neurochemical signaling by the hypothalamus appears to occur in an oscil-

10

SECTION 1

Hypothalamic–Pituitary Function

latory fashion. The temporal nature of pituitary hormone secretion is a manifestation of this rhythmicity resulting in at least two patterns of circadian rhythm including sleeprelated hormone secretory cycles and 24-hour rhythm cycles entrained by a light–dark cycle. The peptidergic neurons of the hypothalamus are not classified into distinct cell types and there is no known correlation between cell structure and function. In fact, neurons of any anatomic area often contain more than one neuropeptide or candidate neurotransmitter. The functional anatomy of the hypothalamus is, therefore, best approached by discussion of each hormone or peptide. GHRH

The presence of the factor responsible for stimulating release of growth hormone (GH) was known for many years, but it was only in 1982 that GHRH was isolated and characterized [25,26]. It is now known to have two forms, a 40- and a 44-amino acid peptide; the active portion of the molecule is the 29-amino acid N-terminus. GHRH binds to the surface of the somatotrophs. The intracellular actions of GHRH are probably mediated by cyclic adenosine monophosphate (cAMP), resulting in stimulation of GH gene transcription, translation, and secretion. Somatostatin attenuates the secretion of GH by the somatotroph. GH stimulates predominantly the liver to produce insulin-like growth factor-I (IGF-I) which acts as a target hormone to induce tissue growth. GH itself also acts directly on the chondrocytes and possesses antiinsulin metabolic activity. Immunocytochemical studies have identified GHRH in neuronal cell bodies of the arcuate nucleus [82,83] (Fig. 1.5). Nerve fibers from this nucleus project mainly to the median eminence, are found in the external zone, and terminate on capillaries of the plexus of the hypothalamohypophysial portal system. In monkey hypothalamus, small groups of immunoreactive neurons are also found in ventral

portions of the ventromedial nucleus. In the rat, scattered cells are also seen in the lateral basal hypothalamus, the medial and lateral portion of the ventromedial nucleus, and the dorsal medial and paraventricular nuclei [84,85]. The primary anatomic localization of GHRH to the arcuate nucleus is in agreement with earlier physiologic studies which indicated that a region including the ventromedial and arcuate nuclei was facilitatory in GH regulation. Extrapituitary effects of GHRH have been reported in the rat. Administration of small doses of the peptide into the cerebral ventricles induces behavioral sedation as well as an increase in food intake [86]. No extra-hypothalamic sites of GHRH synthesis are known to exist in the human under normal circumstances, but endocrine tumors of lung, pancreas, gut, adrenal medulla, and C-cells of thyroid can give rise to GHRH excess and acromegaly [87] (see Chapter 11). Somatostatin (SRIH)

A hypothalamic factor with GH release-inhibiting activity was first described in 1968 [88]. In 1973, Brazeau and colleagues [23] isolated and characterized a 14-amino acid peptide which they called somatostatin, or somatotropin release-inhibiting hormone (SRIH). This peptide was also shown to inhibit thyrotropin (thyroid-stimulating hormone; TSH) secretion and to produce a variety of other effects not related to its action on the pituitary. The hypothalamic distribution of SRIH shows marked species variation [89–92]. In the human hypothalamus, SRIH-containing neurons are concentrated in the anterior portion of the arcuate nucleus [90]. No positivity has been found in the paraventricular nucleus, unlike the distribution observed in rodents where SRIH is found in the paraventricular nucleus in a narrow field extending 1–2 mm from the midline. Nerve fibers staining for SRIH are observed in the medial basal hypothalamus; nerve endings are concentrated in the external zone of the median eminence and the neurovascular zone of the pituitary stalk. Fibers containing SRIH are also localized in the ventromedial, arcuate, suprachiasmatic, and paraventricular nuclei. Somatostatincontaining neurons store the hormone in dense core cytoplasmic vesicles which measure 80–110 nm in diameter and are found in both the cell bodies and the nerve endings [92]. Somatostatin is also found in extrahypothalamic regions of the rat brain, including the preoptic region, central gray mesencephalon, amygdala, olfactory area, and spinal cord. In humans it is detectable in cortex, brain stem, pineal, retina, optic nerve, auditory nerve, and spinal cord. Somatostatin is also found in pancreatic islet cells, gut endocrine cells, thyroid C-cells, salivary gland, and placenta [93–95]. Prolactin-inhibiting Factor

FIGURE 1.5. Neurons containing immunoreactive growth hormone-releasing hormone are localized by immunocytochemistry in the arcuate nucleus. (Magnification ¥64)

Prolactin (PRL) secretion by the adenohypophysis is known to be under tonic inhibition by the hypothalamus [96]. The PRL-inhibiting activity of dopamine is well documented and, currently, it is widely accepted that dopaminergic

Chapter 1

control is the main PRL regulatory function of the hypothalamus. Dopamine is the only nonpeptidergic hypothalamic compound with a well-defined hypophysiotropic function. It exerts primarily inhibitory effects on the lactotroph and inhibits both PRL gene transcription and lactotroph mitotic activity. PRL acts to induce lactation in concert with other peripheral hormones. Although TRH also stimulates lactotroph function, its effect is probably not physiologic. Vasoactive intestinal peptide (VIP) has also been shown to stimulate the production of PRL although its physiologic role has not yet been clarified. Dopamine has been localized to the arcuate and periventricular nuclei of the mediobasal hypothalamus as well as being widespread throughout the brain [97,98]. The medium-sized dopaminergic neurons are scattered among other neurosecretory cells and are further subdivided into the tuberoinfundibular group, with terminals in the median eminence and pituitary stalk, and the tuberohypophysial group, with ends terminating in the neural and intermediate lobes of the pituitary. The dopamine projections to the intermediate and neural lobes of the pituitary originate primarily in the anterior and central portions of the arcuate nucleus, respectively. Dopamine-containing axon terminals are abundant in the zona externa of the median eminence and are characterized by dense core vesicles that measure 15–120 nm in diameter. They do not appear to form true synaptic connections. CRH

Despite being the first hypothalamic regulatory factor postulated, CRH was among the last identified. In 1981, a 41-amino acid peptide was extracted from the ovine hypothalamus, characterized and shown to stimulate specifically the release of ACTH and other pituitary peptides derived from proopiomelanocortin (POMC) [24]. CRH binds to the surface of the corticotroph and elicits transcription of the POMC gene, whose products include ACTH and bendorphin. ACTH stimulates the adrenal gland to synthesize corticosteroids which are critical for essential stress responses and cellular homeostasis. Antibodies against CRH have localized this peptide to neuronal cell bodies, mainly in the parvocellular portion of the paraventricular nucleus but also in a few magnocellular neurons [99–104]. In the rat hypothalamus, scattered cell bodies containing CRH are also detected in the lateral preoptic and hypothalamic areas and in some portions of the dorsal medial nucleus. CRH-immunoreactive fibers are observed in the area of the paraventricular nucleus, the neurovascular zone of the median eminence, the pituitary stalk, and the neurohypophysis. CRH is colocalized with arginine vasopressin in a subpopulation of magnocellular neurons of the paraventricular nucleus [103,104]; it is found together with enkephalin, peptide histidine isoleucine (PHI, a member of the secretin–glucagon family) and neurotensin in parvocellular neurons as well as in a discrete subset of oxytocinergic magnocelluar neurons [104]. Adrenalectomy

Functional Anatomy of the Hypothalamic Pituitary Axis

11

increases CRH immunoreactivity in parvocellular neurons of the paraventricular nucleus, consistent with established negative feedback effects of adrenal steroids on CRH production [104]. Vasopressin can also be found in the majority of CRH-containing parvocellular neurons after adrenalectomy [104]; it has been suggested that this plasticity allows for synergy of stimulation of ACTH secretion by the two peptides [104]. CRH is also widely distributed throughout the brain in the cerebral cortex, limbic system, brain stem, and spinal cord [98–101]. It is also found extracerebrally, particularly in placenta; some investigators have also localized CRH in endocrine cells of the pancreas, gut and lung; in liver, adrenal [105], and testicular Leydig cells [106]. TRH

The first adenohypophysiotropic hypothalamic hormone identified was the tripetide TRH [17,18]. At first demonstrated to release TSH, TRH was rapidly also shown to release PRL. TRH binds to specific receptors on the thyrotroph and stimulates the transcription and secretion of TSH. TSH stimulates the thyroid gland to secrete primarily thyroxine, and triiodothyronine. The major source of triiodothyronine, however, is hepatic deiodination of thyroxine. These hormones act to regulate thermogenesis and protein synthesis. Immunocytochemical studies have localized TRH in the rat brain, in neuronal cell bodies of the preoptic nucleus, the parvocellular portion of the paraventricular nucleus, the perifornical region, dorsomedial nucleus, and basolateral hypothalamus [107,108]. TRH-positive terminals that contain dense core vesicles are found in high concentrations in the median eminence as well as in several of the hypothalamic nuclei, which also contain TRH-positive neuronal cell bodies. The tripetide is widely distributed throughout the brain and spinal cord, suggesting that it may act as a neurotransmitter in other areas [107,108]. Several effects of TRH not mediated by the pituitary gland have been demonstrated, including hyperthermia, behavioral excitation and the ability to reverse or prevent anesthesia from barbiturates and other depressants [109]. TRH has been reported in fetal and neonatal pancreatic islet cells [110] and gut endocrine cells [111], where it is thought to inhibit gastric secretion, gastric motility, and exocrine pancreatic secretion; it has also been localized in several human tumors [112]. GnRH

The second hypothalamic adenohypophysiotropic peptide to be characterized was originally called luteinizing hormone (LH)-releasing hormone (LRH) but was subsequently shown to release both gonadotropins, follicle stimulating hormone (FSH), and LH, by the pituitary gland [20–22]. The gonads are thereby stimulated to produce sex steroids and secondary sex characteristics. GnRH is also thought to stimulate sexual activity in experimental animals of both

12

SECTION 1

Hypothalamic–Pituitary Function

sexes, an effect that appears to be independent of its hypophysiotropic action [113]. GnRH has been localized in the hypothalami of several species [113–115]. In primates, GnRH neurons are located in large areas extending caudally from the preoptic region to the premamillary nucleus [96]. The neurons are bipolar or multipolar cells with multiple dendrites. The highest concentration of these cells is found in the mediobasal hypothalamus (infundibular and premamillary nuclei) and in the preoptic area. These neurons give rise to an infundibular GnRH pathway ending on the capillaries of the pituitary portal plexus as well as to a preopticoterminal tract projecting to the capillaries of the OVLT. In rodents, the majority of neuronal cell bodies are located in the septal preoptic regions and the anterior hypothalamus, and are particularly in the dorsal and medial septal nuclei, the nucleus of the diagonal band of Broca and anterior hypothalamic nuclei, and the OVLT. These neurons are usually elongated with eccentric nuclei and are unipolar. They project to the external zone of the median eminence as well as to the OVLT. Immunoelectron microscopy has documented localization of GnRH in dense core vesicles which measure 75–95 nm in diameter and are found in cell bodies as well as nerve endings [115]. Extrahypothalamic localization of GnRH includes several areas of the limbic system (septum, hippocampus, and olfactory bulb), the breast, and placenta [116]. Vasopressin and Oxytocin

Vasopressin, oxytocin, and their binding proteins, neurophysins I and II, are detected by immunohistochemistry in the perikarya of magnocellular neurons in the supraoptic (Fig. 1.6) and paraventricular (Fig. 1.7) nuclei [117,118]. Fibers emanating from these neurons converge towards the median eminence, pass caudally through its internal zone and end in dilated nerve terminals of the pars nervosa of

the pituitary known as Herring bodies (Fig. 1.8). The nerve fibers, their axonal swellings and the exceedingly large and characteristic swellings which can exceed 20 mm in diameter contain secretory granules of variable electron density and electron-lucent vesicles (Fig. 1.9). The secretory granules originate in the perikarya of the neurons and mature, growing from 60 to 220 nm in diameter, as they travel to the nerve endings [119,120]. It has been clearly shown that vasopressin and oxytocin are produced by different magnocellular neurons in both nuclei. Immunostaining for vasopressin is markedly increased after adrenalectomy in the external zone but not the internal zone of the rat median eminence [121,122]. Vasopressin is colocalized in some neurons with CRH and is additive with that substance in the regulation of ACTH release. Interestingly, it is found in high concentrations in portal blood draining to the anterior pituitary, suggesting that it may play a physiologic role in ACTH release [104]. A subset of oxytocin-containing neurons also may contain CRH. Immunoreactive vasopressin has been demonstrated in various other areas of the central nervous system as well as testis, ovary, uterus, adrenal, and thymus [123]. VIP

This 28-amino acid peptide, first isolated from intestine, has been localized in mammalian brain [124]. Immunoreactive fibers are present in high concentrations in the cortex, hippocampus, dentate gyrus, amygdala, nucleus of the stria terminalis, suprachiasmatic nucleus, and periaqueductal gray matter. Fibers containing immunoreactive VIP and its related PHI are also detected in areas that do not contain cell bodies. In the hypothalamus, VIP has been localized in the ventromedial nucleus, where it may affect appetite [56], in the suprachiasmatic nucleus, where it is thought to play a role in circadian rhythmicity, and in parvocellular neurons of the paraventricular nucleus [125]. It is known to modulate secretion of GH, PRL, and LH at the level of their hypothalamic regulation [126–128] and also stimulates PRL release directly [126]; some investigators have suggested that VIP is the physiologic PRL-releasing factor. After adrenalectomy, the number and intensity of VIP-containing small neurons in the paraventricular nucleus increase, suggesting a possible role for this substance in PRL regulation during stress. Cholecystokinin (CCK)

FIGURE 1.6. Perikarya and axonal processes of magnocellular neurons in the supraoptic nucleus contain strong immunopositivity for vasopressin. (Magnification ¥64)

This hormone, which was first discovered in the gut, was detected in the brain by immunocytochemistry [129–131]. CCK-containing cell bodies are prominent in the cerebral cortex and are also widely distributed throughout the olfactory and limbic systems. In the hypothalamus, they are found in several nuclei, including the magnocellular paraventricular and supraoptic nuclei. The midbrain also contains a striking concentration of CCK-immunoreactive cell bodies in the ventral tegmentum and raphe. This peptide modulates dopaminergic, noradrenergic, and opioid systems. It also inhibits feeding, probably at the level

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

13

FIGURE 1.7. Adjacent to the third ventricle (V), magnocellular neurons in the paraventricular nucleus demonstrate strong immunoreactivity for vasopressin. (Magnification (a) ¥26; (b) ¥64)

FIGURE 1.8. The posterior lobe of the pituitary contains nerve fibers of the hypothalamohypophysial tract with axonal swellings and globular Herring bodies that stain with aldehyde thionin (a) and contain intense immunopositivity for vasopressin (b). (Magnification (a) ¥102; (b) ¥64)

of the ventromedial hypothalamus, and alters the pain threshold [132].

release, and to enhance GH release in response to GHRH in humans and rats [141], suggesting that hypothalamic galanin may modulate secretion of those hormones.

Galanin

Galanin is a 29-amino acid peptide that was originally isolated from intestine [133]. Immunoreactive galanin has been demonstrated in the central nervous system [134]; in hypothalamus, it colocalizes with dopamine and g-aminobutyric acid (GABA) [135]. It is also present in peripheral neural elements of the respiratory tract, gastrointestinal tract, pituitary, pancreas, urogentital tract, and adrenal medulla of humans and several mammalian species [136–139]. Galaninpositive neurons have been observed in several hypothalamic tumors [139]. Galanin is thought to act as a neurotransmitter although its functional roles have not been fully clarified. It has been shown to inhibit dopamine [140], thereby stimulating PRL

Gastrin

Gastrin is a gut peptide which is known to be produced in stomach and in numerous endocrine tumors of gut and pancreas. Specific antibodies have also localized gastrin in cerebral cortex, hypothalamus, and neurohypophysis [142,143]. Gastrin is known to modulate GH secretion at the hypothalamic level, either by reducing somatostatin release or by enhancing GHRH secretion [144]. Glucagon

Glucagon is expressed in the pancreatic islets and intestine, and has also been reported in other tissues, including thymus, thyroid, and adrenal medulla. A growing body of

14

SECTION 1

Hypothalamic–Pituitary Function

levels of NPY immunoreactivity are found in hypophysial portal blood. This peptide stimulates appetite when injected into the hypothalamus and appears to modulate the secretion of both LH and ACTH [150]. NPY-containing fibers also are known to contact TRH-synthesizing perikarya and neuronal processes in the paraventricular nucleus where it may mediate the neuroendocrine regulation of TRH [151]. Neurotensin

This 13-amino acid gut–brain peptide is found in the highest concentration in the ileum and hypothalamus [152]. In the rat, it is localized in the medial preoptic region, infundibular nucleus, paraventricular nucleus, and lateral hypothalamus with fibers most dense in the paraventricular and periventricular regions and in the median eminence. It has been shown to colocalize in neurons with catecholamines, possibly dopamine, in the periventricular and arcuate regions, and with CRH in the paraventricular nucleus. Some investigators have shown that it alters PRL release and blunts the response of that hormone to TRH [153]; it may also play a role in the regulation of GH and LH. Substance P

FIGURE 1.9. By electron microscopy, the normal human posterior lobe of pituitary contains numerous axonal processes filled with neurosecretory material (arrows). A large, dilated nerve terminal is the ultrastructural equivalent of the Herring body. (Magnification ¥7960)

evidence suggests that the brain is a site of glucagon biosynthesis. Neurons staining for glucagon and glucagon-like peptides have been detected in different regions of the brain including retina, hypothalamus, and medulla oblongata [145,146] and glucagon gene expression has been demonstrated in the brain stem and hypothalamus [147]. Intracerebral injections of glucagon produce dosedependent hyperglycemia, suggesting that this substance plays a role in the regulation of glucose metabolism centrally as well as peripherally [148]. Neuropeptide Y (NPY)

This 36-amino acid peptide is found by immunocytochemistry in a widely distributed pattern throughout the brain [149,150]. NPY-containing neurons are particularly numerous in the arcuate nucleus of the hypothalamus, striatum, hippocampus, and cortex. Nerve fibers are abundant in the paraventricular and suprachiasmastic nuclei and fibers are found in the internal zone of the median eminence. High

Substance P has been found in neurons in various regions of the brain. There are multiple substance P-containing afferents to the hypothalamus from the amygdala via the stria terminalis. Hypothalamic neurons containing this peptide are abundant in the arcuate, dorsomedial, ventromedial, and premamillary nuclei [152,154]. It is colocalized with vasopressin, is probably involved in pain perception within the limbic system, and may be secreted into the hypophysial portal vasculature where it is thought to affect blood flow. Substance P has been shown to alter GH regulation in vivo, but this is postulated to act via regulation of somatostatin neurons. It has variable effects on release of PRL, TSH, and LH. Bombesin/Gastrin-releasing Peptide (GRP)/Neuromedin

Bombesin was originally isolated from the skin of amphibians and stimulated great interest when it was shown to have potent biologic activity in mammals. Mammalian counterparts were soon discovered and given the names gastrin-releasing peptide and neuronedin. Bombesin-like immunoreactivity was found in neurons of the external zone of the median eminence near small blood vessels and colocalized with CRH [155]. Pharmacologic studies showed that bombesin can alter the release of adenohypophysial hormones including GH, PRL, and LH. The discovery of immunoreactivity for homologous peptides in hypothalamus suggests a possible role for these substances in the regulation of pituitary function. Calcitonin Gene-related Peptide (CGRP)

This 37-amino acid peptide is derived from the calcitonin gene by alternative messenger RNA (mRNA) processing.

Chapter 1

It is detected primarily in nervous tissue; CGRPimmunoreactive cell bodies are found in the supraoptic, paraventricular, and infundibular nuclei of the human hypothalamus [156]. CGRP inhibits GH release both in vitro and in vivo. It may also affect PRL release [157]. POMC-derived Peptides b-Endorphin

and other POMC-derived peptides are widespread in the central nervous system, their highest concentration being in the arcuate nucleus of the hypothalamus [158]. Nerve fibers containing b-endorphin and melanocyte-stimulating hormone-a (MSH-a) extend throughout many regions and innervate the median eminence, amygdala, preoptic area, ventromedial nucleus, and capillaries of the median eminence and portal system. There is another distinct and complex network of fibers containing the opiate methionine enkephalin (met-enkephalin). In addition to the multiple effects of these peptides on behavior, pain perception, immunomodulation, and many other functions too numerous to review here, they are also thought to modulate pituitary function [159], by inhibiting ACTH and LH release and stimulating PRL secretion. The effect on PRL is likely both direct and by reducing dopaminergic inhibition; the inhibition of LH appears to be only indirect via GnRH inhibition. The pituitary actions of endogenous opioids are complex, since they are themselves regulated by gonadal steroids and opioids may play a role in the negative-feedback effects of gonadal steroids on gonadotropins. Opioids also suppress the release of vasopressin and oxytocin, directly and possibly via noradrenergic regulation [160]. Cytokines

The close link between the neuroendocrine and immune systems has become widely recognized. Hormones are known to modulate the reactivity of immunocompetent cells and cytokines secreted by members of the immune system simulate secretion of hormones, most notably in the hypothalamus–pituitary–adrenal axis [161]. Interleukins are a family of peptides that mediate the immune response. Interleukin-1 (IL-1) is a monokine produced by macrophages in response to antigenic challenge. IL-1 stimulates ACTH and suppresses GH, LH and TSH secretion [162]. IL-1b immunoreactivity has been reported in neuronal fibers of the human brain, including the subfornical organ, stria terminalis, ventromedial nucleus of the hypothalamus, the posterior hypothalamus, and around the vessels of the median eminence. It is found in areas containing CRH-immunoreactive neurons and is known to increase CPH-mediated ACTH but is not found in the same synaptic vesicles as CRH. In fact, it is not released by depolarizing stimuli and it is not clear that IL-1b is even produced by neurons [161]. It also stimulates somatostatin biosynthesis in cultures of fetal rat brain [163], providing a

15

Functional Anatomy of the Hypothalamic Pituitary Axis

likely explanation for the in vivo reduction of GH and TSH secretion. Other interleukins are not known to be hypothalamic peptides but affect pituitary function. Interleukin-6 (IL-6) stimulates release of GH, PRL, and LH; tumor necrosis factor-a (TNF-a) and interferon-g also affect release of PRL and LH [164–166]. This information has significant neuroendocrinologic implications in the study of reproduction, autoimmunity, and other diseases. Growth Factors

A number of peptide growth factors, classified into several major families, are known to modulate cell proliferation. [167] Some are also known to affect hormone production (Table 1.1) and some are, in turn, modulated by hormones. Only a few have been identified in the hypothalamus and are thought to play a physiologic role in pituitary regulation; these include fibroblast growth factor (FGF) [168] and transforming growth factor-a (TGF-a) [169]. The former increases release of PRL and reduces basal GH; it also modifies the response of pituitary hormone release to TRH [170]. The latter has been localized by immunocytochemistry mainly in magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus. It is structurally homologous to epidermal growth factor (EGF) and may mediate some EGF effects on adenohypophysial production of GH, PRL, and TSH as well as cell proliferation [171].

Classification of Hypothalamic and Neurohypophysial Pathology Numerous pathologic processes involve the hypothalamus and neurohypophysis [46,47], giving rise to various symptoms, signs, and clinical syndromes. Among these, endocrine changes may be conspicuous. The local symptoms, such as visual defects, nerve palsy, and headache, may be secondary

Table 1.1. factors

Hormone regulation by pituitary-derived growth

Peptide

GH

PRL

EGF Activin FGF IGF-I IGF-II Insulin Endothelin Interleukin-1

Ø Ø

≠

Ø

FSH/LH

ACTH ≠

≠ ≠

Ø Ø Ø

TSH

≠

≠ ≠

≠ ≠

≠

GH, growth hormone; PRL, prolactin; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; ACTH, adrenocorticotropic hormone; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor.

16

SECTION 1

Hypothalamic–Pituitary Function

to increased intracranial pressure or may be due to compression, invasion, injury, or destruction of tissue components. Involvement of certain parts of the hypothalamus can give rise to nonendocrine pathology, including abnormalities of appetite and temperature regulation. The endocrine sequela of hypothalamic disorders can be classified into two categories: (i) those giving rise to hypothalamic hormone deficiency; and (ii) those giving rise to hypothalamic hormone excess. The major pathologic processes causing these disorders are reviewed briefly and their features are fully discussed in ensuing chapters. Hypothalamic Hormone Deficiency

Insufficient secretion of hypothalamic adenohypophysiotropic hormones may be caused by destructive lesions of the hypothalamus. Lesions such as granulomas, meningitis or other inflammatory processes, pituitary adenomas, craniopharyngiomas, lymphomas, histiocytosis-X, metastatic carcinomas, craniocerebral trauma or hypoxic tissue injury may impair the synthesis of numerous hormones, including vasopressin, oxytocin, and the various adenohypophysiotropic hormones. These lesions give rise to true hypothalamic hypopituitarism. Similarly, lesions affecting the pituitary stalk, such as surgical or traumatic sectioning of the hypophysial stalk or its interruption by various pathologic processes, block the transport of the hypothalamic hypophysiotropic hormones from their sites of production in the hypothalamus to the anterior pituitary. It is obvious that any disconnection of the direct link between the hypothalamus and pituitary results in various degrees of hypopituitarism. Hypocortisolism, hypogonadism, and/or hypothyroidism have been demonstrated conclusively in patients with destructive diseases of the hypothalamus. Deficiency of GHRH usually results in growth retardation or in dwarfism [172]. Destructive lesions may manifest as growth retardation of hypothalamic origin in which patients respond to administration of GHRH. Idiopathic GH deficiency may be associated with other pituitary insufficiency and hypothalamic hormone abnormalities. Of particular interest is an autosomal recessive form of hypothalamic dwarfism due to failure to produce GHRH [173,174]; this disorder represents approximately 10% of cases of inherited growth retardation. The limited morphologic studies of patients with GHRH deficiency have documented that pituitary somatotrophs have normal morphology and GH content and administration of GHRH elicits a normal GH response [175]. Destructive lesions of the hypothalamus may impair the synthesis and/or release of dopamine and therefore may give rise to hyperprolactinemia [96]. Elevated blood PRL levels are also found in patients with disruption of, or damage to, the pituitary stalk. As a result of decreased transport of dopamine, lactotrophs are relieved from their tonic hypothalamic inhibition, leading to hyperprolactinemia. Hypothalamic hypothyroidism is usually not an isolated deficiency; it is most often associated with multiple hypo-

thalamic hormone deficiencies caused by organic hypothalamic pathology. Hypothalamic hypogonadism may be attributed to lack of GnRH or to nonpulsatile release of that hormone [176]. It may be an isolated defect of GnRH neurons [177]; in patients with hemochromatosis it is attributable to a combination of iron-induced damage to GnRH neurons and pituitary abnormalities [178]. Kallmann’s syndrome is an uncommon variant of hypothalamic hypogonadism which occurs in both men and women. The hypogonadism is associated with anosmia [179]. These deficits are due to defective development of the GnRH-secreting part of the hypothalamus associated with hypoplasia or aplasia of the olfactory area of the brain that can be documented by magnetic resonance imaging (MRI) [180]. Kallman’s syndrome is now known to be attributed to deletion or mutations of the KAL gene, located at Xp22.3; this gene encodes a 680 amino acid protein, anosmin, that has homology to other molecules implicated in neural development [181]. The pathology of the hypothalamus in this disorder has been thoroughly described in a histologic study in which abnormalities were found in various hypothalamic nuclei [182]; the lateral tuberal nuclei were underdeveloped and the subventricular nucleus exhibited neuronal hypertrophy. The number of pituitary gonadotrophs was markedly decreased as documented by immunohistochemistry and electron microscopy [182]. Detailed pathologic examination of the hypothalamus is yet to be reported in most patients with no major organic hypothalamic disease and apparently idiopathic deficiency of hypothalamic adenohypophysiotropic hormones. Diabetes insipidus is an uncommon but distinct clinical syndrome characterized by polyuria and polydipsia and results from vasopressin deficiency [46,47]. The extent of disease varies with the location of tissue destruction. Because vasopressin is transported through the pituitary stalk to the posterior lobe where it is stored in nerve endings, destruction of the posterior pituitary may interfere with storage and release of the hormone, resulting in its deficiency. This situation is usually transient and selective lesions of the posterior lobe usually cause only mild and temporary polyuria and polydipsia. Destruction high in the infundibulum results in permanent diabetes insipidus due to retrograde axonal degeneration and atrophy of neurons in the supraoptic and paraventricular nuclei [183]. Destruction of the supraoptic and/or paraventricular nuclei gives rise to permanent disease; approximately 90% destruction is required to cause significant and permanent disease [184], explaining why section low in the stalk, which creates atrophy of a minority of neuronal axons, causes only a transient disorder. Rare forms of hereditary idiopathic diabetes insipidus are usually unassociated with gross destruction of the hypothalamus [185]; detailed histologic studies reveal severe atrophy of neurons in the supraoptic and paraventricular nuclei in most patients but the etiology of these disorders remains unknown. In a few instances, neurons of the

Chapter 1

two magnocellular nuclei have been microscopically normal but devoid of vasopressin [186]. Some studies have suggested that this disorder may in some cases represent autoimmune destruction of vasopressin-producing neurons [187]. Oxytoxin deficiency, resulting from a decrease in the production of oxytocin, causes no major clinical abnormalities. Hypothalamic Hormone Excess

Hypersecretion of hypothalamic hormones, although rare, can result in endocrine symptomatology. Certain endocrine diseases characterized by hyperfunction of the anterior pituitary may be due primarily to adenohypophysiotropic hormone excess as a result of excessive secretion of hypothalamic releasing hormones. As yet, there is no evidence that hypersecretion of hypothalamic inhibiting hormones causes pituitary hypofunction. Acromegaly, a syndrome resulting from sustained GH hypersecretion, is almost always the result of a pituitary adenoma. Only one case of proven ectopic GH production has been reported [188]. More numerous are cases of production of GHRH by tumors that result in somatotroph hyperplasia or adenoma in the pituitary and produce clinical features of GH excess [87]. Extrahypothalamic GHRH production has been documented in endocrine tumors of lung, pancreas, thymus, medullary thyroid carcinoma, and pheochromocytoma. Primary tumors of the hypothalamus, called variously hypothalamic neuronal hamartomas, hypothalamic gangliocytomas and adenohypophysial neuronal choristomas, are lesions consisting of neurons with hypothalamic differentiation. Some of these tumors have been shown to contain GHRH and have been associated with pituitary somatotroph adenoma and acromegaly [189]. Several have been shown to be plurihormonal, containing GnRH, glucagon, gastrin, and somatostatin. Gigantism with pituitary GH excess has been described occasionally in the McCune–Albright syndrome [190]; this disorder is characterized by polyostotic fibrous dysplasia, pigmentary abnormalities and a variety of endocrine abnormalities, most commonly precocious puberty in female children. The pituitary may show mammosomatotroph hyperplasia that is attributed to activation of the signal transduction pathway generating cyclic AMP in these cells. These various manifestations are now known to be due to activating mutations of the Gs alpha gene (gsp mutations) [191,192]. Mammosomatotroph hyperplasia has also been reported as a cause of gigantism in the absence of McCune–Albright syndrome [193]. No proof of GHRH excess, either of hypothalamic or ectopic origin, has been offered in these patients; however, the pathogenetic role of GHRH in mammosomatotroph proliferation has been proven in mice transgenic for GHRH who have mammosomatotroph hyperplasia resembling that seen in these human disorders [194]. Cerebral gigantism (Sotos’ syndrome) has also been postulated to be a primary hypothalamic endocrinopathy. However, in patients with this

Functional Anatomy of the Hypothalamic Pituitary Axis

17

disorder, hormone levels are normal and a morphologic study of the hypothalamus and pituitary showed no specific abnormality [47]. Cushing’s disease is known to be associated with pituitary corticotroph hyperplasia in some cases [195]; it has been suggested that the corticotroph hyperplasia may be secondary to excess production of CRH [196]. Ectopic production of CRH has been shown with increasing frequency and can cause Cushing’s disease; it must be considered in the differential diagnosis of that disorder [197,198]. In some cases, CRH has been elaborated in association with ACTH, suggesting autocrine regulation of some cases of ectopic ACTH syndrome. Rarely, a hypothalamic CRH-producing neuronal tumor has been reported in association with pituitary corticotroph hyperplasia or adenoma and Cushing’s disease [196,199–201]. Precocious puberty is an uncommon clinical syndrome which is in most instances idiopathic or attributable to androgen- or estrogen-producing neoplasms. Idiopathic precocious puberty is assumed to be hypothalamic in origin despite the absence of any anatomic lesion; less than 10% of cases are due to demonstrable hypothalamic pathology. Electroencephalographic abnormalities and behavioral disturbances have been observed in otherwise neurologically normal patients with this disorder [202]. Some patients respond to therapy directed against hypothalamic stimulation, but the disease may also be GnRH independent [203]. Structural lesions in and around the hypothalamus associated with precocious puberty fall into two groups. Some lesions actively participate in the production of precocious puberty by elaborating GnRH; these consist primarily of hypothalamic neuronal hamartomas which have been shown to contain GnRH in some cases [204,205]. The second group consists of hormonally inactive lesions which nonspecifically stimulate or inhibit hypothalamic centers involved in the regulation of sexual maturation. These are primarily lesions in the region of the posterior hypothalamus and include tumors of the pineal and hypothalamus, astrocytic gliomas, infections, hydrocephalus, head trauma, arachnoid cysts, and, occasionally, craniopharyngiomas. Precocious puberty is a prominent feature of the McCune–Albright syndrome [200]; in this disease, there is no demonstrable hypothalamic lesion and the patients may develop normal sexual function in adulthood. The hypothalamus has been implicated as the cause of some cases of hyperthyroidism but this has not been proven [206]. Vasopressin excess may cause water intoxication. The syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) may be due to idiopathic hypothalamic vasopressin excess or may be associated with various disease, including malignant tumors, most often bronchogenic carcinoma, and tuberculosis [207]. In the latter instances, this syndrome may be due to secretion of vasopressin or a similar peptide by tumoral or diseased pulmonary tissue [208,209]. In patients in whom no underlying cause is found, the

18

SECTION 1

Hypothalamic–Pituitary Function

idiopathic syndrome is likely due to posterior pituitary hyperfunction but no morphologic characteristics of this disorder have been described. The pathologic process in the brain may be in a number of areas; inhibitory osmoreceptors are found diffusely in the brain, as far away as the brain stem, and lesions can reduce the tonic inhibition of vasopressin, thereby increasing its release. Evidence of this widespread control is found in the increase in vasopressin release associated with congestive heart failure, cirrhosis, renal disease, brain trauma, positive pressure ventilation, and neurosurgical manipulation of the pituitary and hypothalamus. Age Related Changes in the Hypothalamus

Age related changes in the hypothalamus and neurohypophysis are few. The most striking is a morphologic curiosity, the accumulation of corpora amylacea, most often seen in the region of the supraoptic nuclei. The magnocellular nuclei are remarkably unaffected by age; immunohistochemistry shows no significant change in staining of vasopressin and oxytocin in older subjects [210]. The volume of the mamillary bodies is reduced but cell numbers appear normal [211]. Lipofuscin accumulation is commonly seen in the aging brain but does not appear to affect hypothalamic neuronal function. The suprachiasmatic nucleus decreases in volume and cell number with age [53]. The subventricular nucleus, in contrast, undergoes marked hypertrophy of its usually parvicellular neurons in postmenopausal women, as well as in starvation, posthypophysectomy, late pregnancy, and in patients suffering from prolonged postpartum hypopituitarism [58–64]. ADENOHYPOPHYSIS

Anatomy The adenohypophysis is composed of three parts, the pars distalis, the pars intermedia, and the pars tuberalis (Fig.

(a)

1.10). The pars distalis constitutes the largest portion of the gland; it is also known as the anterior lobe or the pars glandularis. The pars intermedia is rudimentary in the human pituitary; it is the vestigial posterior limb of Rathke’s pouch and is found in its underdeveloped form behind the medial cleft of the pituitary. It is also known as the intermediate lobe. The pars tuberalis is an upward extension of a few layers of adenohypophysial cells surrounding the external aspect of the lower hypophysial stalk; it is also known as the pars infundibularis. The adult human pituitary is bean shaped, bilaterally symmetrical, and measures approximately 13 mm transversely, 9 mm anteroposteriorly, and 6 mm vertically. It weighs approximately 0.6 g. The anterior lobe comprises about 80% of the pituitary. The pituitary of women is somewhat larger than that of men; moreover, the pituitary is heavier in multiparous than in nulliparous women. The increased size is attributable to the marked hyperplasia of the pituitary during pregnancy and lactation, which may increase the weight to 1 g or more; postlactational involution occurs but the gland does not return to its prepregnancy size. A slight to moderate weight reduction is seen with advancing age. The hypophysis is found within the sella turcica where it is lined by dura mater, a dense layer of connective tissue. The uppermost portion of the adenohypophysis is covered by the diaphragma sellae, a reflection of the dura that constitutes the roof of the sella turcica. The sellar diaphragm has a small central opening for the hypophysial stalk and protects the pituitary from the pressure of cerebrospinal fluid (CSF). Defective development or absence of this structure may lead to the so-called empty sella syndrome in which the pituitary is compressed by CSF pressure. The increased pressure results in enlargement of the sella turcica and flattening of the pituitary, which may be compressed to a thin layer of tissue at the bottom of the sella turcica. This lesion is usually unassociated with functional

(b)

FIGURE 1.10. (a) Sagittal section of an adult human pituitary obtained at autopsy illustrates the adenohypophysis (A) and neurohypophysis (N) attached to the pituitary stalk. (b) Horizontal cross-section of an adult human autopsy pituitary allows accurate identification of the neural lobe (N) and the anterior lobe (A) with subtle distinction between the two lateral lobes and the median wedge. The cystic remnants of Rathke’s cleft that represent the pars intermedia in humans are indistinct (arrow head).

Chapter 1

hypophysial abnormalities [206] but is important in differential diagnosis. Other anatomic variations in the shape of the hypophysis, the size and width of the sellar diaphragm, and relation to pituitary structures have been reported [207]. Minor changes have no endocrine significance.

Vascular Supply The blood circulation of the human hypophysis has major implications for the regulation of adenohypophysial hormone secretion [48–51]. The gland receives blood through the hypophysial portal circulation which carries the hypothalamic stimulatory and inhibitory hormones from the infundibulum to adenohypophysial cells. As described above (see Fig. 1.2), the branches of the superior hypophysial arteries penetrate the stalk, form a superficial network of vessels (external plexus) and give rise to a mesh of capillaries and gomitoli (internal plexus). The latter presumably regulate blood flow, thereby affecting transport of hypothalamic hormones to the adenohypophysis. The long portal vessels that arise from the infundibular plexuses and the short portal vessels that originate in the lower pituitary stalk and posterior lobe enter the pars distalis carrying adenohypophysiotropic hormones; the former carry 70–90% of the blood flow while only 10–30% originates in the short portal vessels. The trabecular or loral arteries transport blood to the adenohypophysis via the neural lobe, but, in addition, some arterial blood is directed to the adenohypophysis via two branches of the inferior hypophysial artery: (i) the capsular artery, which serves the connective tissue of the pituitary capsule and penetrates to the superficial cell rows of the adenohypophysis; and (ii) the artery of the fibrous core. In some individuals, the middle hypophysial artery may vascularize the adenohypophysis directly [212]. The volume of veins leading away from the adenohypophysis is considerably smaller than that of portal vessels entering the gland. The meager venous connections drain adenohypophysial and neurohypophysial blood to the cavernous sinus. It has been shown that the short portal vessels also serve as efferent channels, giving rise to reversal of blood flow in the neurohypophysial vascular bed. This implies that secretory products of the adenohypophysis may also enter the neurohypophysis and, by reverse flow to the median eminence, play a role in the regulation of hypothalamic factors via blood flow [50,51]. Pituitary capillaries are lined by fenestrated endothelium with a thin subendothelial space. Hormones released by adenohypophysial cells must therefore pass through the basement membrane of their cell of origin, capillary basement membrane, subendothelial space, and the endothelial cell layer to reach the bloodstream.

Nerve Supply The adenohypophysis has no direct nerve supply, apart from small sympathetic nerve fibers which are associated with and

Functional Anatomy of the Hypothalamic Pituitary Axis

19

presumably innervate capillaries [195]. In some species, the intermediate lobe has rich innervation [213]. Thus, although neural connections may affect blood flow to the adenohypophysis, it is unlikely that they are involved in the regulation of adenohypophysial hormone secretion, which is predominantly neurohumoral.

Embryology All three portions of the adenohypophysis are assumed to derive from Rathke’s pouch, an endodermal pouch of the primitive oral cavity. At the third week of gestation, endoderm from the roof of the stomodeum thickens and begins to invaginate; by 5 weeks, Rathke’s pouch is a long tube with a narrow lumen and a thick wall composed of stratified cuboidal epithelium. By 6 weeks, the connection with the oropharynx is totally obliterated and Rathke’s pouch establishes direct contact with the downward extension of the hypothalamus that gives rise to the infundibulum. The two tissues are enclosed by the cartilage anlage of the sphenoid bone, separating them from the stomodeum; the sella turcica is formed by 7 weeks [66]. It was suggested that Rathke’s pouch arises from the ventral neural ridge in the pharyngeal region, thus sharing with the hypothalamus and posterior pituitary a common neuroectodermal origin [214–217]. Some features of adenohypophysial cells suggest that they are members of the APUD (amine precursor uptake and decarboxylation) cell system. The use of avian allografts, biologic markers, and serial sections of early chick embryos has provided indirect evidence for this theory; however, further proof is required for its validation. As the cells of Rathke’s pouch proliferate, the anterior portion forms the pars distalis and pars tuberalis whereas the posterior wall lies in direct contact with the posterior lobe anlage and becomes the pars intermedia [218,219]. The growth of the anterior limb extends laterally and follows a triradiate pattern; the lateral borders become the lateral wings of the adult gland and the midline portion becomes the anteromedial “mucoid wedge.” By midgestation, the medial cleft becomes a residual lumen and growth of the pars nervosa reverses the convexity of the posterior wall of the cleft to a concave structure. The border between Rathke’s pouch and the pars nervosa becomes indistinct; it consists of remnants of the obliterating lumen, and a few cystic cavities lined by cuboidal or columnar epithelium. This represents the rudimentary pars intermedia of the human hypophysis. The pituitary grows rapidly in early fetal life: the mean weight at 10–14 weeks of gestation is 3 mg, at 25–29 weeks 50 mg, and at term approximately 100 mg [218]. The pituitary portal vascular system begins to form before 7 weeks of gestation, and by 12 weeks the anterior pituitary and median eminence are well vascularized. Portal vessels are recognized at 11.5–14 weeks, are well developed by 15–16 weeks, and are fully established by 18–20 weeks [71,72].

20

SECTION 1

Hypothalamic–Pituitary Function

The first cell type to develop in the human fetal pituitary is the corticotroph [219–221]; at 6 weeks of gestation, cells with ultrastructural features of differentiating corticotrophs are found, and by 7 weeks, ACTH immunoreactivity is detectable. At 8 weeks of gestation, somatotrophs are identified by ultrastructural criteria and contain intense GH immunoreactivity. a-Subunit of the glycoprotein hormones is found in cells which have features of the glycoprotein hormone cell line; differentiated thyrotrophs and gonadotrophs are found subsequently at 12 weeks when b-subunits of those hormones are immunolocalized. Gonadotrophs show sexual dimorphism in the fetus; the differences are most marked from 15–25 weeks when this cell type is more numerous in female glands. Throughout gestation, LH-containing cells predominate in male glands, unlike the more equal distribution of LH- and FSHcontaining cells in female tissues [220]. Lactotrophs are the last cells to differentiate in the human fetal pituitary. PRL is found only at 12 weeks of gestation and is localized in mammosomatotrophs, bihormonal cells which appear to be the sole source of PRL until 24 weeks of gestation [221]. Differentiated lactotrophs are found after that time and undergo a striking hyperplasia in the late third trimester, analogous to that seen in late gestation and lactation in the pituitary of the mother. Novel transcription factors that play a role in anterior primordial development are being identified at a rapid pace. Many of these are implicated in early pituitary organogenesis, including the bicoid-related pituitary homeobox factor Ptx1 [222], Pituitary homeobox factor 2 (Ptx2), structurally related to Ptx1 [223], two members of the Lhx gene family, a group of LIM homeobox genes, Lhx3 and Lhx4 [224] and P-LIM, another LIM homeobox protein transcription factor that is selectively expressed in the pituitary with highest levels at the early stages of Rathke’s pouch development [225]. Another early marker of pituitary differentiation is the Rathke’s pouch homeobox (Rpx) protein which is identified in the pituitary primordium prior to the onset of known pituitary hormone production [226]. The Prophet of Pit-1 (PROP-1) is a paired-like homeodomain protein that is expressed early in pituitary development. It induces Pit-1 expression and plays a role in downregulation of Rpx [227,228]. Inactivating mutations of PROP-1 have been identified as the cause of Pit-1 deficiency in Ames dwarf mice [227] and in humans with combined pituitary hormone deficiency [229,230]. Id, a member of the helix-loop-helix (HLH) family of transcription factors is also found early in development and in some pituitary tumor cell lines but is decreased or absent in differentiated cells [231]. Its role in pituitary cytodifferentiation remains unclear. The molecular factors that determine hormone production have now been identified as transcription factors that target specific hormone genes. These factors have clarified three main pathways of cell differentiation [232,233]. ACTHproducing corticotrophs are determined by a novel T box

factor in cooperation with Ptx1 and corticotropin upstream transcription-binding element (CUTE) proteins including neuroD1/beta2 [234,235]. Bihormonal gonadotrophs require expression of steroidogenic factor (SF)-1 [236,237]. Pit-1 [238] directs differentiation of a complex family of cells that can mature into somatotrophs, mammosomatotrophs, lactotrophs or thyrotrophs [239–241] with the additional expression of estrogen receptor (ER) a [242], which enhances PRL secretion, or thyrotroph embryonic factor (TEF) which stimulates TSH-beta production [243]. The recognition of these molecular determinants of adenohypophysial cytodifferentiation has clarified the patterns of plurihormonality which have been recognized in pituitary adenomas and provide a framework for classification of these tumors. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene; mutations in the pit-1 gene result in hypopituitarism [244–247] and hypoplasia of somatotrophs, lactotrophs and thyrotrophs [244]. An interesting observation is that Pit-1 mRNA and protein are highly expressed during human pituitary development at 17–19 weeks, when GH levels are extremely high, and near term when there is proliferation of lactotrophs [248]. These data suggest that Pit-1 plays an important role not only in the differentiation process, but also in the regulation of hormonal activity and possibly also of cell proliferation. Gonadotroph differentiation likewise requires SF-1; mice with disruption of SF-1 fail to develop pituitary gonadotrophs [236] as well as a ventromedial nucleus of the hypothalamus and steroidogenic glands [249].

Functional Anatomy In contrast with the hypothalamus, the cell types of the adenohypophysis are highly characterized with respect to structure and function. It is notable that with the exception of ACTH and a-subunit of the glycoprotein hormones, ectopic production of pituitary hormones is rare. Somatotrophs

Approximately 50% of the cells of the adenohypophysis produce GH. Most of these are located in the lateral wings of the anterior lobe; scattered somatotrophs are found in the median wedge [195]. These cells can usually be identified by light microscopy as medium-sized acidophilic cells that stain with eosin, phloxin, and orange G. They are spherical or oval in shape, with central, spherical nuclei. Immunohistochemistry reveals intense positivity for GH distributed throughout the cytoplasm [38] and for Pit-1 in the nucleus [221,250]. Occasionally, smaller cells contain GH positivity in a globular structure which represents the Golgi complex; these may be sparsely granulated, actively secreting cells. In situ hybridization localizes GH mRNA to both densely granulated acidophils and to occasional chromophobes (Fig. 1.11). By electron microscopy, somatotrophs are spher-

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

21

FIGURE 1.11. In situ hybridization localizes growth hormone messenger RNA in the normal pituitary in large cells with acidophilic cytoplasm and in occasional chromophobes. (Magnification ¥102)

ical or oval cells with centrally located spherical nuclei and cytoplasm of relatively low electron density [40]. The prominence of rough endoplasmic reticulum and Golgi regions varies with the secretory activity of the cell; active cells generally have well-developed, lamellar rough endoplasmic reticulum, and a large Golgi complex, whereas less active ones tend to have less conspicuous synthetic organelles. In the majority of somatotrophs, the cytoplasm is occupied by spherical, evenly electron-dense secretory granules which store hormone (Fig. 1.12). The limiting membrane of the granules is tightly opposed. The secretory granules range widely in size from 150 to 800 nm in diameter with an average of 350–500 nm. Secretory granules may be found accumulated at the cell periphery but granule extrusions are not detected. Ultrastructural immunocytology confirms the presence of GH in secretory granules and within the Golgi region of actively secreting cells. A subset of these cells has been recognized to contain a-subunit of glycoprotein hormones [40]. The incidence, distribution, morphology, and hormone content of somatotrophs are remarkably constant in the human pituitary. They do not appear to be affected by age, sex, various disease states, or drug therapies. Even in glands containing GH-producing adenomas the nontumorous somatotrophs show no evidence of suppression and cannot be distinguished from those in normal glands [40]. An exception to this morphologic uniformity is the case of long-standing hypothyroidism in which some degranulation of somatotrophs may occur; this change is much less marked than the almost complete degranulation of somatotrophs due to hypothyroidism which has been documented in rodents [250]. In pituitary adenomas, somatotrophs display a striking dimorphism [195]. Despite the morphologic differences between densely granulated and sparsely granulated soma-

FIGURE 1.12. A nontumorous somatotroph contains short profiles of rough endoplasmic reticulum (arrow head), a moderately developed Golgi complex (G) and numerous spherical, evenly electron-dense secretory granules with tightly fitted limiting membranes. (Magnification ¥5500)

totroph adenomas, both types have similar clinical features with elevated blood GH levels and acromegaly. Although the GH concentration in the tumor is proportional to the degree of granularity, neither of these correlate with blood GH levels. The morphine variant does, however, predict response to octreotide therapy [251]. Densely granulated somatotroph adenomas are composed of intensely acidophilic cells which contain strong immunoreactivity for GH throughout their cytoplasm. The tumor cells resemble resting nontumorous somatotrophs, with welldeveloped rough endoplasmic reticulum, prominent Golgi complexes and numerous spherical, evenly electron-dense secretory granules which measure 300–600 nm in diameter. These tumors may contain immunoreactivity for prolactin and/or a-subunit of glycoprotein hormones. The presence of these substances and GH in the same secretory granules has been documented by ultrastructural immunocytology using the double immunogold technique [252]. The use of keratin immunocytochemistry distinguishes these cells from

22

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 1.13. A sparsely granulated adenomatous somatotroph harbors a characteristic juxtanuclear fibrous body composed of intermediate filaments that trap smooth endoplasmic reticulum, Golgi saccules (arrowheads), centrioles (arrows) and pleomorphic forming secretory granules. (Magnification ¥11390)

other somatotroph adenomas. Densely granulated cells have perinuclear low molecular weight cytokeratin filaments. Sparsely granulated somatotroph adenomas are composed predominantly of chromophobic or only slightly acidophilic cells which may show considerable nuclear pleomorphism. The nuclei are generally concave and binucleate; multinucleate cells are easily found. By light microscopy, spherical light areas can be seen adjacent to the nuclei; these correspond to the fibrous bodies seen by electron microscopy and are known to contain immunoreactive keratin [254]. The application of cytokeratin staining allows recognition of fibrous bodies that characterize this tumor [253]. GH positivity is found generally in the juxtanuclear Golgi region, while secretory granules, which are few, can be seen usually only with oil immersion. By electron microscopy, this tumor has a highly characteristic ultrastructure which differs significantly from that of nontumorous somatotrophs (Fig. 1.13). The tumor cells are irregularly shaped with numerous indentations and have lobulated pleomorphic and eccentrically located nuclei. The globular cytoplasmic

fibrous body, found in up to 95% of cells in sparsely granulated somatotroph adenomas, consists of intermediate-sized filaments that accumulate in the Golgi region, close to the concave side of an indented eccentric nucleus. The nearly perfectly globular structure traps tubular smooth endoplasmic reticulum, Golgi saccules, centrioles, mitochondria, lysosomes, and secretory granules. The secretory granules in the cells are scattered throughout the cytoplasm and are smaller than in densely granulated cells, measuring 100 – 250 nm in diameter. No exocytoses are seen. GHRH has been shown to stimulate proliferation of GH-containing cells in vitro during long-term incubations [255]. The effects of chronic exposure to GHRH in humans can be deduced from the study of pituitaries of patients with tumors producing GHRH [40,87]. These patients have marked somatotroph hyperplasia; the somatotrophs are generally large with very well-developed rough endoplasmic reticulum profiles and large Golgi complexes. The hyperplastic cells have numerous large electron-dense secretory granules characteristic of nontumorous cells; sparsely granulated cells with fibrous bodies are generally not seen. Although short-term exposure to GHRH is known to deplete the GH pool [256] and to convert densely granulated cells to more sparsely granulated forms [257], cells of densely granulated somatotroph adenomas chronically exposed to GHRH in vitro show an increase in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes and a decrease in cytoplasmic volume density of secretory granules. However, they do not assume the characteristics of sparsely granulated cells and do not contain fibrous bodies [258]. Sparsely granulated adenomas undergo similar alterations during long-term exposure to GHRH in vitro and retain their characteristic fibrous bodies [258]. Although GHRH stimulates somatotroph proliferation in patients with GHRH-producing tumors, the hyperplastic adenohypophysial cell population in mice transgenic for this hormone is dominated by mammosomatotrophs as well as somatotrophs [194]. The presence of mammosomatotrophs may be attributed to exposure to GHRH excess in these animals during fetal life, at which time mammosomatotrophs are numerous, in contrast to the usual onset of tumoral GHRH excess in adulthood. Further studies are required to clarify the complex regulation of somatotroph and mammosomatotroph differentiation and proliferation by GHRH. The morphologic effects of somatostatin exposure on nontumorous somatotrophs has not been documented. The use of somatostatin analogs, such as SMS 201–995 (Sandostatin or Octreotide) in the therapy of acromegaly has allowed morphologic study of the effects of that substance on somatotroph adenoma cells. Both in vivo [259–261] and in vitro [262], somatostatin and its long acting analogs induce inconsistent morphologic alterations. In some tumors, there is no change, while in others, cell size is reportedly reduced. In some cases, there is an increase in the cytoplasmic volume density of lysosomes unassociated with significant alteration

Chapter 1

of other subcellular organelles. A few studies have indicated that these substances may induce fibrosis; others have found vascular changes which may cause cell necrosis and tumor shrinkage but direct cytotoxic effects have not been documented in vitro. No inhibition of hormone synthesis has been identified, as indicated by reduction in the cytoplasmic volume density of rough endoplasmic reticulum or Golgi complexes; this suggestion confirms molecular studies which have shown no suppression of mRNA in somatostatinexposed nontumorous adenohypophysial cells [263] or tumors [264]. The morphologic studies suggest that somatostatin suppresses hormone release and may stimulate lysosomal degradation of stored hormones. There is, however, differential response to octreotide in vivo by the two somatotroph adenoma types; densely granulated tumors exhibit greater reduction of growth hormone secretion than do sparsely granulated tumors [251]. Lactotrophs

Lactotrophs, or PRL cells, represent approximately 15% of the cells of the adenohypophysis. However, the number of these cells shows wide variation, related to age, sex, and parity in women. In adult men and nulliparous women they constitute approximately 9% of adenohypophysial cells, whereas in multiparous women they represent up to 31% of the cell population [266]. PRL cells are randomly distributed throughout the anterior lobe but are most numerous in the posteromedial portions, with large numbers extending to the posterolateral aspect [195]. Using conventional stains, PRL cells are acidophils that are indistinguishable from somatotrophs; however, Herlant’s erythrosin and Brookes’ carmoisine allow selective visualization of densely granulated lactotrophs, although these techniques are inconsistent and are not

FIGURE 1.14. Immunocytochemistry identifies prolactin diffusely in the cytoplasm of polyhedral and elongated densely granulated cells (arrow), and in the juxtanuclear globular Golgi region of sparsely granulated cells (arrow head). (Magnification ¥102)

Functional Anatomy of the Hypothalamic Pituitary Axis

23

sufficiently sensitive to detect sparsely granulated forms. Immunohistochemistry reveals larger numbers of PRLcontaining cells among which two populations can be distinguished [38] (Fig. 1.14). Densely granulated, polyhedral, or elongated cells are frequently found close to capillaries randomly distributed throughout the anterior lobe; they have abundant cytoplasm almost completely filled with dense granular positivity corresponding to secretory granules. Sparsely granulated cells, in contrast, are more numerous and are found predominantly in clusters at the posterolateral portion of the gland. They are elongated or angular cells with long cytoplasmic processes and strong immunoreactivity for PRL in a juxtanuclear globular Golgi complex. It has been postulated that the densely granulated cells store PRL, whereas the sparsely granulated cells are actively secreting forms. In addition to nuclear Pit-1 reactivity these cells may also exhibit staining for estrogen receptor a [265a,265b]. By electron microscopy, the two cell types are readily distinguishable [40] (Fig. 1.15). Densely granulated cells are

FIGURE 1.15. A nontumorous lactotroph has abundant cytoplasm with highly developed rough endoplasmic reticulum in parallel arrays (*) and few, small secretory granules. (Magnification ¥6080)

24

SECTION 1

Hypothalamic–Pituitary Function

rare in the adult pituitary but are more common in childhood and adolescence. They have ovoid or elongated cell bodies, well-developed rough endoplasmic reticulum at the cell periphery and a spherical or oval Golgi complex containing a few forming granules. The cell cytoplasm is almost completely filled with spherical, oval, or irregularly shaped large granules that have evenly electron-dense cores and measure up to 650 nm in diameter. Granule extrusions are seen occasionally, usually on the cell surfaces overlying basement membrane. The majority of lactotrophs in the adult gland are sparsely granulated, elongated or polygonal cells which may have multiple cell processes extending from the center of acini to the basement membrane and intimately surrounding gonadotrophs. They contain richly developed rough endoplasmic reticulum found in parallel arrays and occasionally forming concentric structures known as “Nebenkern” formations. The Golgi apparatus is prominent and contains pleomorphic immature secretory granules. Scattered secretory granules are fewer and smaller than in storing cells; the diameters range from 150 to 250 nm. Granule extrusions are common and are found not only at the basal cell surface but also on the lateral cell borders, distant from capillaries and basement membranes; the term “misplaced exocytosis” is used to designate this form of granule extrusion. Ultrastructural immunocytology localizes PRL in secretory granules of both cell types; cloudy positivity can also be found in the Golgi region of sparsely granulated forms. In the fetus [220] and newborn as well as during pregnancy and lactation [266,267] there is marked hyperplasia of PRL cells which has been attributed to stimulation by estrogens. During pregnancy and lactation the weight of the pituitary gland may increase to more than 1 g and almost 50% of the total pituitary cell population is composed of PRLcontaining cells. Exogenous estrogens are known to induce only mild hyperplasia of lactotrophs in the human pituitary; the increased cell population is composed predominantly of the small sparsely granulated cell type [268a]. With sustained stimulation of PRL synthesis due to decreased dopamine inhibition or increased estrogen levels, there is a progressive increase in cytoplasmic volume and accumulation of highly organized rough endoplasmic reticulum, enlarged Golgi, numerous forming granules, and few small stored granules [40]. In animals lacking the dopamine D2 receptor, there is extensive lactotroph hyperplasia that progresses to adenoma [268b]. Suppressed lactotrophs are found in some pituitary glands containing PRL-producing adenoma or in patients treated with dopamine agonists. These inactive cells have a reduced nuclear volume, irregular indented nucleus with coarse clumped heterochromatin, small cytoplasm with poorly developed organelles, and secretory granules which measure 50–300 nm; the granules have an increased relative cytoplasmic volume due to the markedly reduced total cytoplasmic volume. Occasional exocytoses may be found at the basal cell surface. These suppressed lactotrophs are identified by immunocytology at the electron microscopic level; they

cannot be characterized on the basis of their ultrastructural features alone, which are not unlike those of null cells. PRL cell adenomas also occur in densely and, more commonly, in sparsely granulated varieties [195]. There is no correlation between morphology and blood PRL levels, age, or sex of the patient. The cells resemble the densely and sparsely granulated cells of the nontumorous pituitary. Effects of dopamine agonists on the cells of lactotroph adenomas include significant reduction of cytoplasmic, nuclear, and nucleolar areas [269]. There is also a marked reduction in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes, whereas secretory granules increase in size and cytoplasmic volume density. The size reduction of bromocriptine-treated prolactinomas is therefore attributable to reduction of individual cell size rather than to a decrease in cell number. Protracted therapy may result in fibrosis which can adversely affect surgical outcome [270]. Mammosomatotrophs

The existence of bihormonal cells containing both GH and PRL in the nontumorous human pituitary has been recognized only in the last few years [40]. This cell type was initially identified in pituitary tumors [195]; it was subsequently found to be the cell of origin of PRL in the fetal pituitary during the first half of gestation [221]. Mammosomatotrophs are thought to be randomly distributed throughout the anterior lobe. They cannot be recognized using conventional histologic techniques; they are acidophils and are indistinguishable from somatotrophs. Immunohistochemistry reveals intense GH content within these densely granulated, polyhedral cells. PRL content is also identified but staining is usually less intense. Nuclear Pit-1 and ER staining is usually strong [239,240,265a,265b] and these cells often contain a-subunits [253]. By electron microscopy these cells resemble densely granulated somatotrophs (Fig. 1.16a). They are fairly large, polyhedral cells with ovoid or slightly irregular nuclei. They have abundant electron-lucent cytoplasm which contains well-developed rough endoplasmic reticulum and a prominent Golgi apparatus which often harbors numerous immature secretory granules. The mitochondria vary from ovoid to rod-shaped; they have a light matrix and lamellar cristae. The distinctive feature of these cells is their unique population of secretory granules. Some are small, spherical, or slightly ovoid, electron-dense granules which measure 150–400 nm; they have tightly fitting limiting membranes. The larger granules are irregular, often extremely elongated structures which can measure from 350 to 2000 nm; they contain secretory material of variable electron density and are bound by a loosely fitting membrane. These secretory granules frequently show misplaced exocytosis with fusion of the limiting membrane to the cell membrane. Unlike the granule extrusions of lactotrophs, the contents that are emptied into the intercellular space persist and retain electron density. Ultrastructural

Chapter 1

(a)

Functional Anatomy of the Hypothalamic Pituitary Axis

25

(b)

FIGURE 1.16. (a) By electron microscopy, mammosomatotrophs resemble densely granulated somatotrophs but have numerous conspicuous large secretory granules of irregular shape and variable electron density, as well as the hallmark of prolactin secretion, the misplaced exocytosis (arrow). (Magnification ¥7300) (b) For the definitive evidence of bihormonal differentiation of mammosomatotrophs, ultrastructural immunocytology localizes both growth hormone (15 nm gold particles) and prolactin (40 nm gold particles) to the same cell, often within the same secretory granule. (Magnification ¥20160) (b) From Felix et al. [271]

immunocytology using the double immunogold technique documents the presence of both GH and PRL in a single cell, frequently within the same secretory granule (Fig. 1.16b). Hyperplasia of mammosomatotroph cells has been identified as a cause of gigantism of early onset [193] and has been associated with acromegaly and hyperprolactinemia in the McCune–Albright syndrome [190]. Although the pathogenesis of this lesion in human patients remains unclear, an animal model for this disorder is provided by giant mice transgenic for GHRH in whom chronic exposure to excess GHRH causes mammosomatotroph hyperplasia [194]; continued long-term GHRH stimulation leads to the development of mammosomatotroph adenoma in old animals [272]. In human patients, tumors composed of mammosomatotrophs are more common than assumed in the past; they are slowly growing adenomas most often found in young patients with gigantism and mild hyperprolactinemia [195,252]. Corticotrophs

A single cell type in the human pituitary is responsible for the production of the POMC molecule and its various derivatives, including ACTH, MSH, lipotropic hormone (LPH), and endorphins [195]. Corticotrophs comprise approximately 15–20% of the adenohypophysial cell popu-

lation. The vast majority of these cells are found in clusters in the central mucoid wedge of the adenohypophysis; occasional scattered cells are also found in the lateral wings of the anterior lobe. Corticotrophs are also the predominant cell type in the poorly developed intermediate lobe of the human pituitary where they are found scattered in follicular structures. By light microscopy these medium-sized cells have varying degrees of cytoplasmic basophilia and stain strongly with PAS; the affinity is attributed to the carbohydrate moiety present in ACTH precursors. They also stain with lead hematoxylin. The most reliable method of identifying corticotrophs is the immunoperoxidase technique which reveals strong granular cytoplasmic positivity for ACTH. The presence of a large unstained perinuclear lysosomal vacuole known as the “enigmatic body” may be helpful in identifying these cells. Corticotrophs can also be identified by in situ hybridization which localizes the POMC mRNA [273]. Immunocytologic studies have indicated that almost all corticotrophs contain immunoreactive MSH, LPH, endorphins and other fragments of the POMC molecule which are derived by differing posttranslational processing [158]. By electron microscopy (Fig. 1.17), corticotroph cells are oval or slightly angular medium-sized cells with spherical or oval eccentric nuclei and a spherical nucleolus which is

26

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 1.18. In the pituitary of a patient treated with high doses of glucocorticoids, corticotrophs with Crooke’s hyaline change have glassy cytoplasm and their periodic acid–Schiff (PAS) positive secretory material is pushed to the rim of the cell; the large juxtanuclear globule (arrow head) is the enigmatic body. (PAS stain; magnification ¥256)

FIGURE 1.17. The nontumorous corticotroph has widely dispersed short rough endoplasmic reticulum membranes, a prominent Golgi complex (G) and numerous secretory granules of variable size, shape and electron density. The two highly characteristic features of this cell type are the perinuclear and cytoplasmic bundles of intermediate filaments (arrows) and the large lysosomal enigmatic body (EB). (Magnification ¥5150)

usually attached to the nuclear membrane [40]. The cytoplasm has varying electron density and is frequently moderately electron-opaque. The rough endoplasmic reticulum is moderately developed and takes the form of widely dispersed membranes. Numerous free ribosomes can be found. The Golgi apparatus is spherical or flattened and is often displaced by the “enigmatic body.” This large structure is membrane-bound and has an electron-dense periphery that exhibits acid phosphatase activity, confirming its lysosomal nature [274]. Mitochondria are spherical or oviod, with lamellar or tubular cristae and a moderately electron-dense matrix. Variable numbers of intermediate filaments of the cytokeratin type [275], previously described as type I microfilaments, are found in small bundles usually adjacent to the nucleus; they measure about 7 nm in width and show no periodicity. They vary considerably in amount and are not numerous under physiologic conditions. The secretory granules are usually numerous and extremely variable in size,

shape, and electron density. They may be spherical, flattened, dented, heart shaped, or tear-drop shaped. They vary in electron density and may be 150–700 nm in diameter, most measuring 150–400 nm. While secretory granules may be found lined up along the cell membrane, exocytosis is not described in this cell type. Immunoelectron microscopy identifies the various POMC-derived peptides in the secretory granules of corticotrophs; however, there is no evidence that the morphologic differences of granule populations reflect their content [38]. Corticotrophs are the first cell type to differentiate in the fetal pituitary [220,221]; however, they are found in significantly decreased numbers in anencephaly [276]; it has been suggested that after autonomous differentiation, they are dependent on hypothalamic factors for normal growth and development. Their numbers do not vary with age or changes in the hormonal environment; however, they do develop specific morphologic features that reflect changes in endocrine homeostasis. In the human pituitary exposed to glucocorticoid excess due to administration of exogenous corticosteroids or any cause of endogenous glucocorticoid hypersecretion (including ectopic secretion of ACTH), corticotrophs undergo a distinctive morphologic alteration known as Crooke’s hyaline change [195]. By light microscopy, the cells accumulate a glassy, homogeneous, slightly acidophilic substance in the cytoplasm; the PAS positivity and ACTH immunoreactivity, corresponding to secretory granules, are displaced to the perinuclear rim and the periphery of the cell [38] (Fig. 1.18). The hyaline material does not contain immunoreactive POMC derivatives. This material has been shown to be composed of keratin filaments and stains with several antibodies directed against low-molecular-weight keratin proteins [275]. By electron microscopy, Crooke’s hyaline

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

27

FIGURE 1.20. Corticotrophs invade the neural lobe of the pituitary (N) in a normal age-related phenomenon that is unassociated with endocrinopathy. This case, the same as that illustrated in Fig. 1.18, shows that the population of corticotrophs participating in “basophil invasion,” like those lining the pars intermedia cysts (arrow head), do not undergo Crooke’s hyaline change. (Periodic acid–Schiff stain; magnification ¥102)

FIGURE 1.19. Corticotroph with Crooke’s change in the pituitary of a patient with cortisol excess has a ring of intermediate filaments filling the cytoplasm, surrounding the nucleus and trapping numerous secretory granules in a juxtanuclear location. (Magnification ¥5090)

material is composed of an accumulation of intermediate filaments, resembling the smaller bundles seen in nontumorous corticotrophs (Fig. 1.19). The accumulation of microfilaments can be so extensive as to occupy almost the entire cytoplasm, leaving only a small juxtanuclear Golgi region and a thin rim of secretory granules adjacent to the cell membrane. Crooke’s hyaline change is reversible [195]. In the pituitaries of patients with inadequately treated adrenal insufficiency, the corticotrophs become hypertrophied, with large nuclei, prominent nucleoli, and a poorly granulated cytoplasm. In cases of long-standing Addison’s disease, focal nodular hyperplasia of corticotrophs and corticotroph adenomas may be present [277]. The ultrastructural appearance of these “adrenalectomy” cells in the human pituitary has not been well documented. However, in adrenalectomized rats, the markedly enlarged cells contain abundant rough endoplasmic reticulum and Golgi membranes and an increased number of secretory granules [278]. Chronic administration of CRH to rats increases the number of ACTH-containing cells, but no significant ultra-

structural changes have been documented [279]. Morphometric analysis indicated that there was no change in cell area; the nuclear area increased slightly and the secretory granule diameter was greater in corticotrophs of CRHexposed animals. These data suggest that the proliferation of corticotrophs in Addison’s disease may be mediated by CRH, but the morphologic characteristics of adrenalectomy cells cannot be attributed solely to CRH excess. The pars intermedia corticotrophs also are strongly (PASpositive and exhibit intense immunostaining for ACTH and other POMC derivatives [195]. The border between the pars intermedia and the pars distalis is often indistinct; occasionally a thin layer of connective tissue is noticeable between these two portions of the adenohypophysis. On the posterior aspect of the pars intermedia, basophil cells are frequently identified, often in clusters within the neuropil of the pars nervosa. These clusters vary in number and in some cases they may be found spreading deeply into the neural lobe (Fig. 1.20). This process, known as “basophil invasion,” is found in older individuals; it is more frequent and pronounced with advancing age and is said to be more prominent in men than in women. Its functional significance is not known but it is not thought to be associated with endocrine abnormalities. Some studies have suggested that the presence of a-MSH immunoreactivity is indicative of intermediate lobe differentiation [280,281]. By electron microscopy, corticotrophs of the pars intermedia are smaller, more dense, and contain fewer intermediate filaments [192]. They are also known to be less sensitive to the feedback effect of glucocorticoids, in most cases showing absent or only mild filament accumulation in patients with glucocorticoid excess and lacking the Crooke’s hyaline change that is found in pars distalis corticotrophs of those patients [195].

28

SECTION 1

Hypothalamic–Pituitary Function

The corticotrophs of the pars intermedia and those which form the basophil invasion of the pars nervosa may give rise to a specific group of corticotroph adenomas. These tumors may represent one form of the silent corticotroph adenomas which are unassociated with signs or symptoms of ACTH excess [252]; they are probably capable of secreting biologically and immunologically active substances that remain uncharacterized. As is the case with pars intermedia corticotrophs, these tumors do not show striking accumulations of intermediate filaments and, in some cases, may lack filaments totally. The derivation of corticotroph cell adenomas from this subpopulation of corticotrophs is difficult to prove. The morphology of corticotrophs in most adenomas associated with ACTH excess resembles that of nontumorous corticotrophs [195,252]. Occasional tumors are composed of cells with Crooke’s hyaline change [252], suggesting that the adenomatous cells are sensitive to feedback inhibition by glucocorticoids. Morphologic studies of functioning corticotroph adenomas in vitro [282] confirm the response of most such tumors to CRH stimulation associated with slight ultrastructural changes indicative of increased hormone synthesis. Incubation of tumor cells with glucocorticoids inhibits ACTH release and causes accumulation of intermediate filaments; however, it remains to be seen if the threshold for suppression is altered in tumor cells.

idase technique reveals granular positivity for TSH; this stain reveals the characteristic angular shape of these cells and their long cytoplasmic processes that establish contact with the basement membrane (Fig. 1.21). Ultrastructurally, these cells are characterized by their marked angularity and well-developed cytoplasmic processes [40] (Fig. 1.22). The nucleus is spherical and often eccentric while the cytoplasm contains numerous short, slightly dilated rough endoplasmic reticulum profiles, a globoid Golgi complex and small, spherical secretory granules that measure 100–200 nm in diameter. When scanty, the secretory granules characteristically are aligned under the plasmalemma and may contain rod-shaped or irregularly electron-dense contents; more densely granulated cells have granules scattered throughout the cytoplasm. Although the numbers of thyrotrophs do not appear to vary with age, these cells undergo morphologic changes in association with altered hormonal status. In patients with primary hyperthyroidism, thyrotrophs are few and small;

Thyrotrophs

The least common cell type in the adenohypophysis is the thyrotroph. These comprise approximately 5% of the total adenohypophysial cell population and are found primarily singly or in small clusters in the anteromedial portion of the gland. These medium-sized cells are basophilic when stained with conventional dyes, contain PAS positivity, stain with aldehyde fuchsin and aldehyde thionin. The immunoperox-

FIGURE 1.21. Immunohistochemical localization of thyrotropin identifies scattered angular thyrotrophs in the nontumorous human autopsy pituitary. (Magnification ¥102)

FIGURE 1.22. Nontumorous thyrotrophs have welldeveloped cytoplasm with short, slightly dilated profiles of rough endoplasmic reticulum (arrow heads), a large Golgi region (G), small secretory granules lined up along the plasma membrane and small numerous secondary lysosomes (L). (Magnification ¥7440)

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

29

subset of altered somatotrophs which are bihormonal [283]; the morphologic changes are reversible in the experimental animal. Gonadotrophs

FIGURE 1.23. In a patient with long-standing hypothyroidism, the cytoplasm of these “thyroidectomy cells” is dominated by the abundant widely dilated rough endoplasmic reticulum with flocculent contents; they also contain expanded Golgi complexes (G), few small secretory granules and prominent lysosomes (L). (Magnification ¥4380)

their ultrastructural features are not well documented. In cases of untreated primary hypothyroidism, thyrotrophs are released from the negative feedback effects of thyroid hormones and TSH secretion is increased. The number and size of thyrotrophs are increased; the enlarged cytoplasm is less strongly positive using the PAS, aldehyde fuchsin and aldehyde thionin stains, and TSH immunoreactivity is faint but diffuse. These cells exhibit strong nuclear positivity for Pit-1 [195,239]. By electron microscopy, these stimulated thyrotrophs, known as “thyroidectomy cells” or “thyroid deficiency cells,” have abundant dilated rough endoplasmic reticulum cisternae and an expanded Golgi complex, whereas the number of secretory granules is reduced [40] (Fig. 1.23). Large cytoplasmic spherical, PAS-positive lysosomal globules are prominent. With prolonged stimulation, nodular hyperplasia and thyrotroph adenomas may occur [195]; TRH may be implicated in the pathogenesis of this cell proliferation [252]. In the rat, there is evidence that at least some of the thyroidectomy cells may derive from a

The cells which produce the gonadotropins, FSH and LH are the gonadotrophs; these probably represent up to 10% of the human adenohypophysial cell population [195]. No reliable cell count is, however, available. In rats, the number of gonadotrophs containing each hormone varies with age, sex, and hormonal status [40]. Gonadotrophs are scattered throughout the pars distalis and comprise the major constituent of the pars tuberalis [284]. They stain with basic dyes, the PAS technique, aldehyde thionin, and aldehyde fuchsin. The immunoperoxidase technique reveals cytoplasmic positivity for FSH and LH, often both present in the same cell, indicating that one cell type is capable of producing both gonadotropins. There are, however, variations in the number of cells containing FSH and LH, indicating that some gonadotrophs contain only one of these two hormones and there are likely fluctuations with functional demand. Nuclear reactivity for SF-1 defines the gonadotroph population [237]. The ultrastructural features of gonadotrophs reveal a single cell population which is responsible for both FSH and LH production (Fig. 1.24). The large oval or elongated cells have spherical, eccentric nuclei which are oriented with distinct polarity at the base of the cell [40,252]. Their abundant, slightly dilated rough endoplasmic reticulum profiles contain a flocculent electron-lucent substance. The Golgi is prominent and globoid, composed of sacculi and vesicles associated with immature secretory granules. Mature secretory granules are unevenly distributed in the cytoplasm and fall into two groups which vary in size and density. The distribution of these two granule populations may show sexual dimorphism. In men, smaller secretory granules are usually numerous; they have an average diameter of 250 nm. In women, larger secretory granules predominate and measure 300–600 nm in diameter. Ultrastructural immunocytology localizes FSH and LH within the same cells and, in some cases, within the same secretory granule. Gonadotrophs are found in close proximity to the basement membrane and exhibit an intriguing intimate contact with lactotrophs. The latter frequently extend cell processes around gonadotrophs and there are intercellular junctions between the two cell types [285], suggesting paracrine interactions which are not well understood. Gonadotrophs are found in the pituitaries of fetuses and show sex-related dimorphism during gestation [220]. Between 15 and 25 weeks of gestation, pituitaries of female fetuses contain more numerous gonadotrophs than pituitaries of male fetuses. Throughout gestation, LH-containing cells predominate in male pituitaries, whereas the number of LHand FSH-containing cells are almost equal in females. This dimorphism correlates with differences in the levels of hypothalamic GnRH at the same stages of gestation. In the adult

30

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 1.24. A nontumorous gonadotroph is large and oval with short profiles of dilated rough endoplasmic reticulum containing electron-lucent contents (*), ring-like Golgi complex (G) and large, dense but irregular secretory granules. (Magnification ¥8170)

pituitary, no such dimorphism of gonadotroph numbers has been identified; however, the ultrastructural differences in males and females have been described above. Gonadotroph adenomas of the adult pituitary show an even more striking fine-structural sexual dimorphism [195,286]. Gonadotroph adenomas in women are usually well-differentiated tumors consisting of polyhedral cells with long cytoplasmic processes, dilated profiles of well-developed endoplasmic reticulum, and the characteristic “honeycomb” Golgi apparatus, a large saccular structure associated with developing secretory granules. The secretory granules in this tumor type are spherical and membrane-bound with varying electron density; they measure about 400–450 nm in diameter. The tumors in adult men are less differentiated, with poorly to moderately developed rough endoplasmic reticulum profiles, a moderately developed Golgi complex and fewer, generally smaller secretory granules. Strong nuclear SF-1 positivity is a reliable marker [237]. Gonadotrophs of the pars distalis show morphologic changes which reflect their hormonal environment; in con-

trast, the gonadotrophs of the pars tuberalis show signs of functional inactivity in most circumstances. In patients treated with pharmacologic doses of estrogen, the gonadotroph cells are small and dense [195]. During pregnancy, the number of cells immunoreactive for FSH and/or LH is also significantly reduced [264]. Gonadotrophs are morphologically abnormal in Kallmann’s syndrome, an uncommon variant of hypothalamic hypogonadism due to GnRH deficiency and associated with anosmia [182]; there is a marked decrease in the number of these cells and the remaining small cells are only weakly positive for FSH and LH. Castration leads to characteristic morphology in gonadotrophs [40,195,287]. Prolonged lack of the negative feedback effect of gonadal steroids results in stimulation of gonadotrophs to secrete FSH and LH in higher quantities. These stimulated cells are enlarged and are found in increased numbers. They have vacuolated cytoplasm which may take the form of several small vacuoles or one large vacuole; the latter displaces the nucleus to the cell periphery, giving the cell a “signet ring” appearance. By electron microscopy, these so-called “gonadectomy cells,” “gonadal deficiency cells,” or “castration cells” (Fig. 1.25) have markedly dilated rough endoplasmic reticulum, which is responsible for the cytoplasmic vacuoles seen by light microscopy. The Golgi complex is enlarged and hormone storage is decreased but not lost, despite the marked increase in secretory activity, since secretory granules are present in reduced numbers. It has been suggested that secretory granule formation is bypassed and that discharge of hormone takes place in an unconventional way not discernible by electron microscopy. An unusual prominence of large active gonadotrophs has been noted in the nontumorous adenohypophysis of some women with PRL-producing adenomas [40]. The factors underlying this change are not clear, but it has been suggested as the explanation for the LH hyperresponse to GnRH which has been found in these patients. Follicular Cells

Follicular cells are found throughout the adenohypophysis surrounding follicles, lumina lined mainly by agranular or poorly granulated cells that are joined at their apex by junctional complexes [195,288]. By electron microscopy, it was shown that granulated adenohypophysial cells can form follicles around damaged cells which have disrupted cell membranes. The surrounding cells form specialized intercellular attachments and the participating cells undergo degranulation and dedifferentiation. These follicular cells may derive from somatotrophs, lactotrophs, or corticotrophs. The follicular cells form specialized junctions, macula adhaerens, between each other and with adjacent granulated adenohypophysial cells; in contrast, granulated adenohypophysial cells form only the less prominent zonulae adhaerentes. Follicles are found in areas with increased cell destruction, particu-

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

31

FIGURE 1.26. Immunolocalization of S-100 protein identifies a pituitary stellate cell with long branched cytoplasmic processes surrounding granulated adenohypophysial cells. (Magnification ¥256)

FIGURE 1.25. After castration, “gonadectomy cells” have markedly dilated rough endoplasmic reticulum and large globoid Golgi regions (G) which harbor forming secretory granules. (Magnification ¥4620)

been implicated as the source of FGF and vascular endothelial growth factor [293], but are not found in the pituitaries of anencephalics [294]. They are numerous in the compressed adenohypophysis at the periphery of adenomas. Some investigators have found no S-100-reactive sustentacular cells within pituitary adenomas [290]; in contrast, others have identified S-100-containing cells within tumors of several types [295]. They are also found in large numbers at the periphery of other pituitary lesions, such as abscesses, amyloid deposits and in the residual hypophysis after surgery, but not adjacent to metastatic tumor deposits, infarcts, or Rathke’s cleft cysts [296]. Null Cells

larly surrounding tumors [40]. They are assumed to play a role in isolating and processing cell debris. Some pituitary adenomas contain follicles. Folliculostellate Cells

Immunocytochemical studies have localized S-100 protein to a specific subtype of cells in the normal human pituitary gland [289,290]. These cells are agranular and do not contain immunoreactive hormones. Some also contain immunoreactivity for glial fibrillary acidic protein (GFAP). They have a characteristic morphology, with long, branched cytoplasmic processes embracing granulated adenohypophysial cells (Fig. 1.26). Because of confusion with the follicular cells described above, some authors have suggested that these cells be called “stellate cells” [289]. They are believed to have a supportive role similar to that played by the S-100-positive sustentacular cells of the adrenal medulla and carotid body. In addition, they are thought to play a role in paracrine regulation [291] and have been shown to produce IL-6, a cytokine which may participate in local regulation of hormone secretion [292]. These cells have also

The term null cell was used to identify cells that show evidence of adenohypophysial differentiation, possessing organelles required for hormone synthesis and storage, but whose differentiation is incomplete and does not resemble any of the known adenohypophysial cell types [297]. The term was applied initially to a group of tumors which possessed no histologic, immunocytochemical, or ultrastructural markers revealing their cellular differentiation and which were unassociated with clinical evidence of hormone excess. Application of increasingly sensitive techniques of cell identification have shown that most tumors with morphologic features of null cells are in fact gonadotroph adenomas [298,237]; however, only a small subpopulation of tumor cells may be hormonally active at any one time, as indicated by the reverse hemolytic plaque assay [299]. Immunohistochemical analyses [221] and studies of hormone mRNA [300] have shown that the majority of these tumors contain cells capable of producing glycoprotein hormone subunits. Focal ultrastructural differentiation along several lines has also been documented [301]. It has been suggested that these tumors may originate from uncommitted or

32

SECTION 1

Hypothalamic–Pituitary Function

committed stem cells or from dedifferentiated adenohypophysial cells. Null cells may also represent unstimulated or resting cells that are not actively engaged in hormone production and cannot be recognized with currently available investigative techniques [302]. The nontumorous pituitary contains cells that cannot be identified conclusively by ultrastructural criteria. These may represent resting cells, uncommitted or committed stem cells. In the fetal pituitary, a population of cells with features of the glycoprotein hormone cell line can be identified prior to the recognition of differentiated thyrotrophs or gonadotrophs [221]; a-subunit immunoreactivity is present in fetal glands at the same stage of gestation and b-subunits are not yet detected [220]. It has been suggested that these primitive cells which resemble null cells may represent the source of a-subunit and may be precursors of the glycoprotein hormone cell line; they may be the cell of origin of some null cell adenomas. The null cells are chromophobic and by immunocytochemistry, usually contain no hormones [195,252]. A small number of these cells may contain various pituitary hormones, most commonly a-subunit of the glycoprotein hormones followed by b-subunits of FSH and/or LH. They usually exhibit SF-1 nuclear staining, indicating gonadotrophin differentiation [237]. By electron microscopy, the cells are polyhedral with irregular nuclei. The poorly developed cytoplasm contains short, scattered rough endoplasmic reticulum profiles, an inconspicuous or only moderately developed Golgi apparatus and small, dense, rodshaped mitochondria. Secretory granules are few and small, measuring less than 250 nm in diameter, frequently with central electron-dense cores, peripheral electron-lucent halos, and a prominent limiting membrane (Fig. 1.27). Oncocytes

Oncocytes are large cells which have abundant acidophilic granular cytoplasm due to the numerous mitochondria that take up acid dyes [195,252]. By electron microscopy, they are characterized by a striking increase in the number and size of mitochondria. A varying number of oncocytic cells may be found in the normal human hypophysis [303]. The number of these cells appears to increase with advancing age, a phenomenon also found in other organs including thyroid, parathyroid, and salivary glands. Cells in hormonally active adenomas only rarely show oncocytic transformation; the tumors usually retain their fine structural and immunocytochemical markers and can be recognized as showing various degrees of oncocytic change. In contrast, null cells, which have no other identifying markers, more frequently undergo oncocytic change. Gonadotroph adenomas regularly contain a varying number of cells with oncocytic change, and the number of oncocytes in any given tumor often increases with tumor recurrence [195]. Oncocytomas are found more commonly in elderly patients. In vitro studies using tissue culture techniques [298] and the reverse hemolytic plaque

FIGURE 1.27. A null cell in the nontumorous human pituitary has poorly developed organelles in a scant cytoplasm. However, there are short profiles of rough endoplasmic reticulum (arrow heads) and a few small secretory granules, indicating the potential for hormone synthesis. This cell has no features which allow recognition of its differentiation. (Magnification ¥5950)

assay [299] have indicated that there is no functional difference between null cell adenomas and oncocytomas of the human pituitary. The factors underlying oncocytic change are unknown, but they may lie in abnormalities of the mitochondrial DNA which is independent of the cellular DNA [304]. Oncocytes are large polyhedral cells with abundant granular cytoplasm that varies from strongly acidophilic to chromophobic. These cells may show ill-defined purple coloration with trichrome stains using aniline blue; this may lead to misinterpretation as basophilia. The phosphotungstic acid–hematoxylin method is helpful to demonstrate mitochrondrial abundance. Oncocytes showing ultrastructural features of differentiated adenohypophysial cells may contain the appropriate immunoreactive hormone. The majority of oncocytes, which are thought to be derived from null cells of gonadotrophs contain no immunoreactive hormones or may contain a-subunit, FSH LH. They often have nuclear SF-1 reactivity [237].

Chapter 1

FIGURE 1.28. In the nontumorous human pituitary, an isolated oncocyte has abundant cytoplasm which is almost totally occupied by numerous spherulated mitochondria. Rough endoplasmic reticulum (arrow heads), a small Golgi complex (G) and small secretory granules suggest that the cell is capable of hormone synthesis and storage. (Magnification ¥5590)

By electron microscopy, oncocytes are readily identified as large, polyhedral cells with irregular nuclei and a cytoplasm almost totally filled with numerous mitochondria. Histologically some oncocytes are dark or acidophilic; these cells contain tightly packed oblong mitochondria with granular matrix and numerous transverse cristae. Other oncocytes may be histologically light, i.e., only slightly acidophilic or chromophobic; their crowded mitochondria appear swollen and rarefied, with loss of cristae. Oncocytes contain small, short profiles of rough endoplasmic reticulum, inconspicuous Golgi regions and few scattered secretory granules (Fig. 1.28). Others

Acidophil Stem Cells are recognized primarily in pituitary adenomas producing both GH and PRL [195,305]. They are rare and are not known to occur in the nontumorous pituitary. The cell type is assumed to derive from a common precursor of somatotrophs and lactotrophs; while

Functional Anatomy of the Hypothalamic Pituitary Axis

33

mammosomatotrophs have been recognized as the source of GH and PRL early in gestation, no cells resembling tumorous acidophil stem cells have yet been identified in the human fetal pituitary [221]. Immunohistochemistry reveals the presence of PRL and GH in the cells. The positivity for PRL is usually stronger and patients with tumors composed of these cells have hyperprolactinemia; GH production may be reflected in the phenomenon known as “fugitive acromegaly.” By electron microscopy, these cells resemble sparsely granulated lactotrophs and have some features of somatotrophs. They are irregular in shape and have large nuclei containing fine chromatin and inconspicuous nucleoli. The cytoplasm contains scattered rough endoplasmic reticulum profiles and relatively small Golgi regions. The Golgi sacculi are flat and may contain a few forming granules. The mitochondria may be abundant, indicating oncocytic transformation, and often show focal cavitation as well as a unique mitochondrial gigantism not identified in other pituitary cell types. Giant mitochondria may reach the size of a nucleus and be visible by light microscopy as cytoplasmic vacuoles. The mitochondria retain their double limiting membranes but lose their cristae and are filled with electron-lucent granular matrix. Occasionally, they contain electron-dense tubular structures resembling tubules of centrioles and cilia. The secretory granules in this cell type are sparse and small, ranging from 50 to 300 nm. Characteristically, granule extrusions or “misplaced exocytoses” are identified, similar to those of sparsely granulated lactotrophs. In addition, these cells have a structural marker of sparsely granulated somatotrophs, the fibrous body, a juxtanuclear aggregate of smooth endoplasmic reticulum membranes, and keratin immunoreactive intermediate filaments. These features of lactotrophs and somatotrophs are frequently found in the same cell; ultrastructural immunocytochemistry confirms the localization of GH and prolactin in the same cell. A tumor type identified as silent subtype III adenoma is composed of chromophobic or slightly acidophilic cells which contain immunocytochemical evidence of multiple hormones. Staining for ACTH is generally weak, bendorphin immunoreactivity may be strong and the tumor cells may contain GH, PRL, and/or a-subunit immunoreactivity. In some cases, the tumors contain no recognizable hormones. By electron microscopy, large, elongated cells have an eccentric nucleus, a prominent nucleolus which may harbor spheridia, and a highly differentiated cytoplasm with abundant rough and smooth endoplasmic reticulum profiles and a well-developed Golgi apparatus. The secretory granules are small, measuring less than 200 nm in diameter, and have variable electron density; they are mainly spherical but occasionally irregular or dropshaped granules are found. These cells bear no similarity to any of the known nontumorous adenohypophysial cells and their cytodifferentiation is unknown. Other unclassified plurihormonal cells have been identified in the nontumorous pituitary and adenomas [40,195]. The

34

SECTION 1

Hypothalamic–Pituitary Function

most common types produce GH, a-subunit, TSH, and/or PRL in any combination. Some of these cells have ultrastructural features of somatotrophs or mammosomatotrophs, while others resemble thyrotrophs [40]. Studies of the rat pituitary have confirmed the existence of these cells and indicate a role for them in some pathologic states; for example, some somatotrophs transform into a population of thyroidectomy cells in hypothyroidism [283]. This common pattern of plurihormonality is easily explained by the expression of Pit-1, the transcription factor implicated in activation of the genes encoding GH, PRL and bTSH. However, occasionally other patterns of plurihormonality have been reported. Very rarely, cells producing POMC derivatives with ultrastructural features of corticotrophs may produce gonadotropins or other hormones both in human tumors [195,306] and in the rat nontumorous pituitary [307]. Conversely, cells with gonadotroph morphology may contain ACTH as well as gonadotropins [308]; it is intriguing, however, that ACTH, which is so commonly expressed in extrapituitary tumors, is rarely found in plurihormonal cells of the adenohypophysis.

Localization of Other Peptides in the Adenohypophysis A number of substances not initially thought to be of pituitary origin are detectable in adenohypophysial cells. Hypothalamic Adenohypophysiotropic Hormones

GHRH, somatostatin, TRH, and GnRH have been identified in pituitary adenohypophysial cells [308–311]. Large amounts of GHRH and somatostatin were measured in culture media of perifused normal pituitaries and GHsecreting adenomas and their release was stimulated by other peptides [309]. Preprosomatostatin mRNA has been detected in nontumorous adenohypophysis as well as several types of pituitary adenomas [310]. It is known that GnRH binds to gonadotrophs and becomes internalized [311]; however, it has also been shown that TRH and GnRH are released by pituitary cells maintained in culture for a prolonged period [308]. GnRH is colocalized with b-LH and some gonadotrophs also contain TRH. These data suggest production of hypothalamic peptides by de novo synthesis in a multipotential pituitary cell. Angiotensin II

Angiotensin II, as well as renin and angiotensin converting enzyme, have been localized in pituitary gonadotrophs [312]. These substances colocalize with b-LH. Angiotensin II has been shown to stimulate PRL release in vitro; this is a dosedependent phenomenon. It has also been found to stimulate ACTH and b-endorphin release. Lactotrophs and corticotrophs have high-affinity binding sites for angiotensin II. Bombesin/GRP

Bombesin-like immunoreactivity has been found in the pituitary gland [313]; however, cell localization has not been

demonstrated. Bombesin-related peptides stimulate pituitary hormone release, mediated by the hypothalamus. CGRP

CGRP is found in the pituitary in small amounts and the concentration is known to increase with age. It is unclear if this substance is present in adenohypophysial cells or in nerve terminals of the posterior lobe [314], since CGRP is found in the hypothalamus (see above). CCK

CCK is found in the adenohypophysis but in a form which differs from that present in other CCK-synthesizing tissues [315]. The pituitary is devoid of CCK-8 but contains substantial amounts of three large nonamidated pro-CCK fragments and small amounts of a-amidated CCK (CCK-58, CCK-33, component I). No immunocytochemical localization has been reported but CCK has been found in corticotroph adenomas in large amounts and in some tumors associated with acromegaly which also contained ACTH. It therefore seems that pituitary CCK derives from corticotrophs [316]. The form found in tumors is smaller and less sulfated than in the normal pituitary; tumors contain CCK-8-like forms similar to those found in brain and gut [315]. Galanin

In humans, galanin has been localized in corticotrophs of the nontumorous pituitary, including Crooke’s cells and cells of basophil invasion in the posterior lobe. Galanin is also present in some corticotroph adenomas [139,317]. In contrast, in rats, galanin mRNA is found in estrogen-induced pituitary tumors [318] and galanin has been localized in PRL- and GH-producing cells as well as some TSHcontaining cells [319]. The reason for this species-specific regulation is unknown. In the rat, galanin expression shows sexual dimorphism [319], is inducible by estrogen [318–320] and is altered by changes in adrenal, thyroid, or gonadal status [319]. It appears to be involved in the modulation of pituitary GH and PRL secretion, probably mediated by alterations in their hypothalamic regulation [140,141]. Gastrin

The neurohypophysis is known to contain gastrin [142], but there appears to be species-specific variation in the adenohypophysis. The human adenohypophysis contains only traces of this substance which is localized to corticotrophs [321] and corticotroph adenomas [322]. Analogy has been drawn to cosynthesis with POMC in other tissues; the significance of this colocalization is unknown and gastrin is not thought to act directly on the pituitary but rather in the hypothalamus. NPY

NPY immunoreactivity and mRNA are found in the pituitary and can be localized by immunocytochemistry

Chapter 1

in adenohypophysial cells consistent with a subset of thyrotrophs [323]. These cells increase in size and NPYimmunoreactivity after thyroidectomy. NPY may have autocrine or paracrine activity in the modulation of release of GH and gonadotropins. Neurotensin

Neurotensin has been localized to the anterior pituitary and is thought to be present in gonadotrophs. It is also known to be affected by thyroid status, being found in reduced amounts in both hypothyroidism and hyperthyroidism [151,324]. The role of neurotensin in the pituitary is not known. Substance P

Immunoreactive substance P has been found in the rodent anterior pituitary. In rats it is localized in gonadotrophs and lactotrophs [325], but in the guinea pig it is found in thyrotrophs [326]. Thyroidectomy increases and thyroid hormone treatment decreases the amount of substance P in the pituitary [152]; the amount also varies with levels of gonadal steriods. It is not clear if thyroid hormone and corticosteroids act directly to influence pituitary content. Substance P may play a local or paracrine role in the adenohypophysis. VIP

There is evidence that VIP is synthesized in the pituitary [327] in lactotrophs [127,328]; it is also inducible by estrogen and may be altered by other changes in the hormonal environment. Recently, it was found to be present in increased amounts in hypothyroidism [329] and VIP production by adenohypophysial cells in vitro was found to be stimulated by TRH as well as GHRH, but not by CRH or GnRH. Tissue culture studies have shown that VIP regulates tonic PRL secretion but does not affect TRH- or GHRHinduced PRL release [329]. Cytokines

Cytokines are known to modulate pituitary function and some have been shown to be produced in the hypothalamus (see above and references 161–166). IL-6 has been demonstrated in stellate cells within the pituitary [292] and is known to modulate GH and PRL responses to appropriate stimuli [164]. Growth Factors

Growth factors regulate cell replication and functional differentiation by directly altering the expression of specific genes. Several polypeptide growth factors regulate anterior pituitary hormone secretion. Growth factor action in the pituitary, however, must be understood in the context that the pituitary itself is a site of both synthesis and action of growth factors [330,331]. Several pituitary-derived growth factors are also regulated within the pituitary by peripheral hormones including triiodothyronine and hydrocortisone.

Functional Anatomy of the Hypothalamic Pituitary Axis

35

Pituitary-derived growth factors include EGF, FGF, TGF-a and TGF-b, IGF, endothelin, and IL-1 [331]. Several partially characterized growth factors have also been described, including thyroid hormone-inducible growth factor, vascular endothelial growth factor mammary cell growth factor, chondrocyte growth factor, mammary cell growth factor, chondrocyte growth factor, and adipocyte growth factor [331]. EGF, a potent stimulator of PRL and ACTH secretion in vitro, also attenuates GH secretion [332–334]. EGF may also play a role in the regulation of LH secretion [335]. EGF has been localized in the pituitary within cells that produce glycoprotein hormones. EGF-receptor is expressed by normal human anterior pituitary and is upregulated in some adenomas, usually recurrent somatotroph tumors [336]. It has been speculated that EGF may mediate proliferation of thyrotrophs in hypothyroidism and of lactotrophs in the lactotroph hyperplasia of pregnancy [337]. TGF-a is expressed by pituitary cells where it has a membrane-anchored localization [338,339]; by immunohistochemistry it colocalizes with PRL and possibly with GH in the bovine pituitary [340]. TGF-a interacts with the EGF receptor and inhibits replication of rat pituitary tumor cells [341]. The regulation of lactotroph growth and differentiation by this substance suggests a potential role in promotion or facilitation of pituitary tumorigenesis [339] and in transgenic mice, targeting of TGF-a overexpression to lactotrophs results in lactotroph adenoma formation [343]. Activin, a member of the TGF-b gene family, suppresses GH secretion and somatotroph replication, and stimulates FSH secretion [343–346]. The inhibin subunits which comprise activin and inhibin and their mRNAs have been localized in pituitary gonadotrophs [309]. Activin-binding protein, which shows homology to follistatin, has been isolated in the rat pituitary and appears to block the action of activin [347]. Follistatin is expressed by normal gonadotrophs and is reduced or absent in gonadotroph adenomas, suggesting a role for this growth factor in the tumorigenic process [348]. The pituitary contains high concentrations of basic FGF [349]. There are several indications that the FGF family plays an important role in the regulation of pituitary function. It may be an important paracrine regulator of PRL secretion [170]; nonmitogenic concentrations of FGF stimulate TRHinduced PRL and TSH secretion in pituitary cells. Pituitaryderived FGF stimulates the replication of human pituitary cells in vitro [350]. FGF has also been implicated in the new arteriolar vessel formation reported to accompany prolactinomas [351]. FGF expression is upregulated in aggressive pituitary adenomas and this growth factor has been shown to be released by the adenoma cells [352], indicating its potential role in the control of adenohypophysial cell proliferation. Moreover, altered FGF receptors have been implicated in the dysregulated growth of pituitary tumors [353].

36

SECTION 1

Hypothalamic–Pituitary Function

Endothelin, a powerful vasoconstrictor, is expressed in the pituitary and stimulates FSH and LH secretion [354]. Pituitary-derived endothelin-3 is stimulated by IGF-I [355]. IGF-I mediates most of the peripheral growth promoting actions of GH [356]. GH-dependent IGF-I is secreted predominantly by the liver, and IGF-I participates in feedback regulation of GH gene transcription and secretion [357]. Regulation of pituitary IGF-I receptors also determines the action of the growth factor on somatotroph function [356]. IGF-I also stimulates the in vitro secretion of hypothalamic SRIH, further delineating its role in feedback regulation of GH secretion [358]. Pituitary expression of GH, IGF-I and IGF-I receptors have been documented [359,360] and may therefore mutually interact by a paracrine or autocrine feedback mechanism [361]. Regulation of endogenous or circulating pituitary growth factors and their respective receptors may be important paracrine or autocrine determinants of pituitary cell function and trophic hormone secretion. The role of these growth factors in the development of pituitary tumors is being intensively studied [232].

Classification of Adenohypophysial Pathology Many diseases can affect tissues in and adjacent to the sella turcica, giving rise to a wide spectrum of clinical pathology. The clinical problems may be manifest as pituitary hormone deficiency or pituitary hormone excess. In addition, associated problems arise from compression, invasion, injury, or destruction of other tissue components; symptoms such as visual field defects, nerve palsies and headaches are characteristic of mass effects on the hypothalamus and neurohypophysis. Adenohypophysial Hormone Deficiency

The clinical problem of pituitary hormone deficiency may be due to decreased secretion or to increased degradation of hormone. Characteristic adenohypophysial changes are often associated with abnormalities of hormone secretion. Hypopituitarism involving one, two, or more adenohypophysial hormones is usually attributed to either a decreased number of hormone-secreting cells or to reduced endocrine activity. Organic lesions such as massive necrosis, inflammatory, or neoplastic processes can replace large portions of the pituitary and account for reduction of hormone-secreting parenchyma [195]. Reduced endocrine activity can result from target gland hormone excess or administration of exogenous hormones feedback negatively to suppress hypophysial function. Alternatively, it may be due to decreased hypothalamic stimulation or interference with receptors which govern secretion of hormones. Various developmental abnormalities can reduce the amount of adenohypophysial parenchyma. Some of these have been reviewed above (see embryology). It is now recognized that mutations of PROP-1 or Pit-1 result in various patterns of pituitary hormone deficiency and hypoplasia

[229,230,233,245,247]. In pituitary aplasia, there is total absence of the adenohypophysis due to a defect in the formation of Rathke’s pouch; pituitary hypoplasia is a milder variant of this disorder. In anencephaly, the brain, including the hypophysiotropic hypothalamus, is missing; due to lack of neurohumoral control, the adenohypophysis is smaller than normal and there is marked reduction in the number of corticotrophs associated with a less striking reduction of other adenohypophysial cell types [276a]. Cornelia de Lange syndrome, a rare congenital cyst of Rathke’s pouch, may compress the adenohypophysis and lead to various degrees of hypopituitarism. Pituitary dystopia or ectopia rarely affects function. Tissue destruction leads to a decrease or absence of pituitary hormone-producing parenchyma. This may be caused by tumors, inflammatory or infiltrative processes, or vascular lesions. Symptoms of hypopituitarism develop only with reduction of more than 60% of anterior lobe tissue and is clinically significant when there is more than a 90% reduction of hormone-producing cells. Hormonally inactive tumors of the adenohypophysis represent approximately 25% of surgically removed pituitary adenomas [195]. The null cell adenomas and oncocytomas fall into this category; these cell types have been discussed above. Craniopharyngiomas are less common than pituitary adenomas [195]. They represent 3–5% of intracranial neoplasms and occur usually in children or young adults. They are more frequently suprasellar than intrasellar and can cause varying degrees of hypopituitarism due to compression or destruction of the adenohypophysis, pituitary stalk, or hypophysiotropic hypothalamus. Other primary neoplasms in and around the sella turcica include fibromas, angiomas, gliomas, meningiomas, granular cell tumors, paragangliomas, chordomas, and many others (see Chapter 15) [195]. These tumors produce no hormones. The majority are benign and may be discovered incidentally at postmortem examination. Occasionally they cause hypopituitarism and local symptoms. Primary sarcomas have been reported in patients who have previously received irradiation for the treatment of pituitary adenomas [363]. Metastatic malignancies are found in patients with widely disseminated neoplasms; reported incidences vary from 1 to 14% [47,362]; however, the true incidence of adenohypophysial involvement is not known, since small deposits are easily overlooked. Pituitary involvement is rarely manifest before patients are overwhelmed by the malignancy. Inflammatory conditions in the sella turcica may occur as acute processes, with purulent hypophysitis and abscess formation, due to severe septicemia or direct extension from adjacent tissues. Granulomatous inflammation may be seen in tuberculosis, syphilis, sarcoidosis, and giant-cell granuloma. The latter is an uncommon disorder of unknown etiology which may involve the pituitary gland selectively; it may represent localized sarcoidosis [364]. Lymphocytic hypophysitis is an autoimmune disorder which may cause

Chapter 1

extensive destruction of the adenohypophysis and subsequent hypopituitarism [365,366]. Vascular lesions such as hemorrhages may occur as complications of traumatic head injuries or in tumors. Infarction is seen focally as an incidental autopsy finding but significant lesions may be associated with diabetes mellitus, generalized carcinomas, cranio-cerebral trauma, increased intracranial pressure, infection, and in patients maintained on mechanical ventilators. Postpartum pituitary necrosis, known as Sheehan’s syndrome [367], occurs in pregnancy, during which time the pituitary is predisposed to vasospasm and infarction. When damage is significant, hypopituitarism is proportional to the degree of tissue destruction. Infiltrative disorders such as amyloidosis, hemochromatosis, and mucopolysaccharidosis, may impair adenohypophysial cell function. Hemochromatosis can give rise to selective hypogonadism due to preferential deposition of iron in pituitary gonadotrophs [368]. Feedback inhibition may cause cytologic alterations in the adenohypophysis. These changes have been described above. Selective deficiencies of adenohypophysial hormones may result from inherited genetic abnormalities or selective damage to one cell type. Isolated GH deficiencies are a genetically heterogeneous group of disorders that cause dwarfism. In most cases, there is a normal pituitary with normal numbers of granulated somatotrophs; these are attributed to GHRH deficiency or abnormality [172–175]. Selective deficiency of a specific pituitary transcription factor, Pit-1, causes selective congenital GH and PRL deficiency associated with TSH deficiency [369–371]; several animal models of pituitary dwarfism, the Snell, Jackson and Ames dwarf mice, have also been shown to have specific abnormalities of the Pit-1 gene [372]. Congenital isolated ACTH deficiency has been attributed to T pit mutations. Lymphocytic hypophysitis has been reported to cause selective destruction of corticotrophs; antibodies to other pituitary cell types are also detectable in patients with endocrine autoimmune diseases and may give rise to isolated pituitary hormone deficiency [365,366]. Short stature with features of isolated GH deficiency but elevated GH blood levels was found to be due to a deficiency of GH-binding protein, the extracellular domain of the GH receptor [373,374]. Adenohypophysial Hormone Excess

Excess hormone production by the pituitary is usually due to a pituitary tumor [195]. The most common lesion is adenoma; carcinoma occurs exceedingly rarely and may be associated with hormone excess. Hyperplasia and hypertrophy of hormone-secreting cells may result from increased stimulation by hypothalamic releasing hormones, or from loss of inhibition by hypothalamic-inhibiting hormones, or excess target organ hormones. The cytologic features of each adenoma type have been described above. The features of hyperplastic adenohypophysial cells have also been provided. Tumor-like lesions may also be associated with hypersecretion of adenohypophysial hormones. Lymphocytic

Functional Anatomy of the Hypothalamic Pituitary Axis

37

hypophysitis may give rise to a mass lesion of the sella turcica associated with hyperprolactinemia [365,366]. The elevated PRL levels may be due to inflammation, injury of the stalk or hypothalamus or compression of those structures by the enlarged, inflamed gland. The disorder is frequently associated with pregnancy and it has been suggested that antibodies directed against hyperplastic lactotrophs may stimulate PRL release, interfere with dopamine receptors, or damage PRL cells causing release of stored PRL. Rarely, pituitary hormone excess has been associated with normal pituitary morphology [195]. The hyperfunction may be caused by intrinsic abnormalities of the cells which produce subtle changes that may not be detectable by conventional morphologic techniques.

REFERENCES 1 Descartes R. Des Passions de l’Ame. Amsterdam, 1649. 2 Zander R. Ueber functionelle und genetische Beziehungen der Nebennieren zu anderen Organen, speciell zum Grosshirn. Beitr Pathol Anat 1890;7: 439–534. 3 Bernard C. Physiologie; chiens rendus diabétiques. C R Soc Biol 1849;1:60. 4 Marie P. Sur deux cas d’acromégalie. Hypertrophie singuliére non congénitale des extremités supérieures, inférieures et céphaliques. Rev Méd 1886;6:297–333. 5 Minkowski O. Ueber einen Fall von Akromegalie. Berl Klin Wochenschr 1887;21:371–374. 6 Cushing H. The Pituitary Body and Its Disorders. Philadelphia: JB Lippincott, 1912. 7 Simmonds M. Zwergwuchs bei Atrophie der Hypohysen Vorderlappens. Munch Med Wochenscht 1919;45:487–488. 8 Harris GW. Neural control of pituitary gland. Physiol Rev 1948;28: 139–179. 9 Du Bois Reymond E. Gesammelte Abhandlungen zur allgemeinen Muskel und Nerven physic, vol. 2. Leipzig: Veit & Comp, 1877. 10 Schiefferdecker. Ueber die Neuronen und die innere Sekretion. Bonn: Sitzgsber Niederrhein Ges Natur Heilkde, 1905:46–54. 11 Lieutaud J. Essais Anatomiques, contenant l’histoire exact de toutes les parties qui composent le corps de l’homme, avec la manière de dissequer. Paris: Huart, 1742. 12 von Luschka H. Der Hirnanhang und die Steissdruese des Menschen. Berlin: G Reimer, 1860:1–97. 13 Popa GT, Fielding U. The vascular link between the pituitary and the hypothalamus. Lancet 1930;ii:238–240. 14 Scharrer E, Scharrer B. Secretory cells within the hypothalamus. Res Publ Assoc Nerv Ment Dis 1940;20:170–174. 15 Bargmann W, Scharrer E. The site of origin of the hormones of the posterior pituitary. Am Sci 1951;39:255–259. 16 du Vigneaud V, Gish DT, Katsoyannis PG. A synthetic preparation possessing biological properties associated with arginin-vasopressin. J Am Chem Soc 1954;76:4751–4752. 17 Guillemin R. Peptides in the brain: the new endocrinology of the neuron. Science 1978;202:390–402. 18 Bøler J, Enzman F, Folkers K et al. The identity of chemical and hormonal properties of the thyrotropin-releasing hormone and pyroglutamylhistidyl proline amide. Biochem Biophys Res Commun 1969;37:705–710. 19 Burgus R, Dunn TF, Desiderio D et al. Structure moléculaire du facteur hypothalamique TRH d’origine ovine: mise en évidence par spectrométrie de masse. C R Acad Sci 1969;269:1870–1873. 20 Baba Y, Matsuo H, Schally AV. Structure of the porcine LH- and FSHreleasing hormone. II. Confirmation of the proposed structure by conventional sequential analysis. Biochem Biophys Res Commun 1971;44:459–467. 21 Burgus R, Butcher M, Ling N et al. Structure moléculaire du facteur hypothalamique (LRF) d’origine ovine contrôlant la sécrétion de l’hormone gonadotrope hypophysaire de lutéinisation. C R Acad Sci 1971;173: 1611–1613. 22 Schally AV, Arimura A, Baba Y et al. Isolation and properties of the FSH and LH releasing hormone. Biochem Biophys Res Commun 1971;43:393–399.

38

SECTION 1

Hypothalamic–Pituitary Function

23 Brazeau P, Vale W, Burgus R et al. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 1973;179: 77–79. 24 Vale W, Spiess J, Rivier C et al. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and aendorphin. Science 1981;213:1394–1397. 25 Guillemin R, Brazeau P, Böhlen P et al. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982;218: 585–587. 26 Rivier J, Spiess J, Thorner M et al. Characterization of a growth hormone releasing factor from a human pancreatic islet tumour. Nature 1982;300: 276–277. 27 Benda C. Beitrage zur normalen und Pathologischen Histologie der menschlichen Hypophysis cereberi. Berl Klin Wochenschr 1900;37:1205–1210. 28 Erdheim J. Zur normalen und pathologischen Histologie der Glandula Thyreoidea, Parathyreoidea und Hypophysis. Beitr Pathol Anat Allg Pathol 1903;33:158–236. 29 Kraus EJ. Zur Pathologie der basophilen Zellen der Hypophyse. Zugleich ein Beitrag zur Pathologie des Morbus Basedowi und Addisoni. Virchows Archiv 1923;247:421–447. 30 Severinghaus AE. Cellular changes in the anterior hypophysis with special reference to its secretory activities. Physiol Rev 1937;17:556–588. 31 Le Gros Clark WE. Morphological aspects of the hypothalamus. In: Le Gros Clark WE, Beattie J, Riddoch G, Dott NM, eds. The Hypothalamus: Morphological, Functional, Clinical and Surgical Aspects. Edinburgh: Oliver & Boyd, 1938:1–68. 32 Rasmussen AT. The proportions of various subdivisions of the normal adult human hypophysis cerebri and the relative number of the different types of cells in pars distalis, with biometric evaluation of age and sex differences and special consideration of basophilic invasion into the infundibular process. Res Publ Assoc Res Nerv Ment Dis 1938;17:118–150. 33 Romeis B. Hypophyse. In: von Mollendorf W, ed. Handbuch der Mikroskopischen Anatomie des Menschen, vol. 6, part 3. Berlin: Springer-Verlag, 1940:1–625. 34 Phifer RF, Spicer SS, Hennigar GR. Histochemical reactivity and staining properties of functionally defined cell types in the human adenohypophysis. Am J Pathol 1973;73:569–588. 35 Herlant M. Étude critique de deux techniques nouvelles destinées à mettre en évidence les différentes catégories cellulaires présentes dans la glande pituitaire. Bull Micro Appl 1960;10:37–40. 36 Brookes LD. A stain for differentiating two types of acidophil cells in the rat pituitary. Stain Technol 1968;43:41–42. 37 Sternberger LA. Immunocytochemistry. Englewood Cliffs: Prentice-Hall, 1974. 38 Kovacs K, Horvath E, Ryan N. Immunocytology of the human pituitary. In: DeLellis RA, ed. Diagnostic Immunohistochemistry. New York: Masson Publishing Inc, 1981:17–35. 39 Rinehart JF, Farquhar MG. Electron microscopic studies of the anterior pituitary gland. J Histochem Cytochem 1953;1:93–108. 40 Horvath E, Kovacs K. Fine structural cytology of the adenohypophysis in rat and man. J Electr Microsc Tech 1988;8:401–432. 41 Moriarty GC. Adenohypophysis: ultrastructural cytochemistry. A review. J Histochem Cytochem 1973;21:855–892. 42 Bendayan M. Protein A—gold electron microscopic immunocytochemistry: methods, applications and limitations. J Electron Microsc Tech 1984;1:243–270. 43 Neill JD, Frawley LS. Detection of hormone release from individual cells in mixed populations using a reverse hemolytic plaque assay. Endocrinology 1983;112:1135–1137. 44 Asa SL. In vitro culture techniques. In: Kovacs K, Aas SL, eds. Functional Endocrine Pathology, 2nd ed. Boston: Blackwell Science, 1998:118–133. 45 Lloyd RV. Use of molecular probes in the study of endocrine diseases. Human Pathol 1987;18:1199–1211. 46 Sheehan HL, Kovacs K. Neurohypophysis and hypothalamus. In: Bloodworth JMB Jr, ed. Endocrine Pathology, General and Surgical, Baltimore: Williams & Wilkins, 1982:45–99. 47 Scheithauer BW. The hypothalamus and neurohypophysis. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology, 2nd ed. Boston: Blackwell Science, 1991:171–296. 48 Stanfield JP. The blood supply of the human pituitary gland. J Anat 1960;94:257–273. 49 Sheehan HL, Davis JC. Pituitary necrosis. Br Med Bull 1968;24:59–70. 50 Bergland RM, Page RB. Can the pituitary secrete directly to the brain? (Affirmative anatomical evidence). Endocrinology 1978;102:1325–1338. 51 Bergland RM, Page RB. Pituitary–brain vascular relations: a new paradigm. Science 1979;204:18–24.

52 Nieuwenhuys R. Chemoarchitecture of the brain. Berlin: Springer-Verlag, 1985. 53 Swaab DF, Fliers E. A sexually dimorphic nucleus in the human brain. Science 1985;228:1112–1115. 54 Gorski RA. Sexual differentiation of the brain. Hosp Pract 1978;13:55–62. 55 Whitehead R. The hypothalamus in post partum hypopituitarism. J Pathol Bacteriol 1963;86:55–67. 56 Cezayirli RC, Robertson JT. Pharmacologic modulation of hypothalamic control of appetite. In: Givens JR, ed. The Hypothalamus. Chicago: Year Book Medical Publishers Inc, 1984:115–128. 57 Liebelt RA, Perry JH. Action of gold thioglucose on the central nervous system. In: Handbook of Physiology, sect. 6, vol. 1. Alimentary canal control of food and water intake. Washington DC: American Physiological Society, 1967:271–285. 58 Sheehan HL. Le noyau subventriculaire de l’hypothalamus. Rev Fr Endocrinol Clin 1975;16:111–114. 59 Rance NE, McMullen NT, Smialek JE, Price DL, Young WS III. Postmenopausal hypertrophy of neurons expressing the estrogen receptor gene in the human hypothalamus. J Clin Endocrinol Metab 1990;71:79–85. 60 Ule G, Schwechheimer K, Tschahargane C. Morphological feedback effect on neurons of the nucl. arcuatus (sive infundibularis) and nucl. subventricularis hypothalami due to gonadal atrophy. Virchows Archiv (Pathol Anat) 1983;400:297–308. 61 Hart MN. Hypertrophy of human subventricular hypothalamic nucleus in starvation. Arch Pathol 1971;91:493–496. 62 Hommes OR. Effects of hypophysectomy and age on the infundibular nucleus in man. J Endocrinol 1974;62:479–488. 63 Sheehan HL, Kovacs K. The subventricular nucleus of the human hypothalamus. Brain 1966;89:589–614. 64 Sheehan HL. Variations in the subventricular nucleus. J Pathol Bacteriol 1967;94:409–416. 65 Weill J, Bernfeld J. Le Syndrome Hypothalamique. Paris: Masson & Cie, 1954. 66 Falin LI. The development of human hypophysis and differentiation of cells in its anterior lobe during embryonic life. Acta Anat 1961;44:188–205. 67 Hyyppä M. Hypothalamic monoamines in human fetuses. Neuroendocrinology 1972;9:257–266. 68 Goodyer CG, Guyda JH, Giroud CJP. Development of the hypothalamic– pituitary axis in the human fetus. In: Tolis G, Labrie F, Martin JB, Naftolin F, eds. Clinical Neuroendocrinology: a Pathophysiological Approach. New York: Raven Press, 1979:199–214. 69 Skowsky R, Fisher DA. Immunoreactive arginine vasopressin (AVP) and arginine vasotocin (AVT) in the fetal pituitary of man and sheep. J Clin Invest 1973;52:77a. 70 Benirschke K, McKay DG. The antidiuretic hormone in fetus and infant. Obstet Gynecol 1953;1:638–649. 71 Räiha N, Hjelt L. The correlation between the development of the hypophysial portal system and the onset of neurosecretory activity in the human fetus and infant. Acta Pediatr Scand 1957;72:610–616. 72 Rinne UK. Neurosecretory material passing into the hypophysial portal system in the human infundibulum, and its foetal development. Acta Neuroveg 1963;25:310–324. 73 Okado N, Yokota N. An electron microscopic study on the structural development of the neural lobe in the human fetus. Am J Anat 1980;159:261–273. 74 Bresson J-L, Clavequin M-C, Fellmann D et al. Ontogeny of the neuroglandular system revealed with HPGRF 44 antibodies in human hypothalamus. Neuroendocrinology 1984;39:68–73. 75 Bugnon C, Fellmann D, Bloch B. Immunocytochemical study of the ontogenesis of the hypothalamic somatostatin-containing neurons in the human fetus. Metabolism 1978;27(suppl. 1):1161–1165. 76 Ackland J, Ratter S, Bourne GL et al. Characterization of immunoreactive somatostatin in human fetal hypothalamic tissue. Regul Peptides 1983;5: 95–101. 77 Bugnon C, Fellmann D, Gouget A et al. Corticoliberin neurons: cytophysiology, phylogeny and ontogeny. J Steroid Biochem 1984;20:183–195. 78 Winters AJ, Eskay RL, Porter JC. Concentration and distribution of TRH and LRH in the human fetal brain. J Clin Endocrinol Metab 1974;39: 960–963. 79 Aubert ML, Grumbach MM, Kaplan SL. The ontogenesis of human fetal hormones. IV. Somatostatin, luteinizing hormone releasing factor, and thyrotropin releasing factor in hypothalamus and cerebral cortex of human fetuses 10–22 weeks of age. J Clin Endocrinol Metab 1977;44:1130–1141. 80 Bugnon C, Bloch B, Fellmann D. Cyto-immunological study of the ontogenesis of the gonadotropic hypothalamo–pituitary axis in the human fetus. J Steroid Biochem 1977;8:565–575.

Chapter 1 81 Siler-Khodr TM, Khodr GS. Studies in human fetal endocrinology. I. Luteinizing hormone-releasing factor content of the hypothalamus. Am J Obstet Gynecol 1978;130:795–800. 82 Bloch B, Brazeau P, Ling N et al. Immunohistochemical detection of growth hormone releasing factor in brain. Nature 1983;301:607–608. 83 Bloch B, Gaillard RC, Brazeau P et al. Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus. Regul Peptides 1984;8:21–31. 84 Bloch B, Brazeau P, Bloom F et al. Topographical study of the neurons containing hpGRF immunoreactivity in monkey hypothalamus. Neurosci Lett 1983;37:23–28. 85 Merchenthaler I, Vigh S, Schally AV et al. Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus. Endocrinology 1984;114:1082–1085. 86 Vaccarino FJ, Bloom FE, Rivier J et al. Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor. Nature 1985;314:167–168. 87 Sano T, Asa SL, Kovacs K. Growth hormone-releasing hormone-producing tumors: clinical, biochemical and morphological manifestations. Endocr Rev 1988;9:357–373. 88 Krulich L, Dhariwal APS, McCann S. Stimulatory and inhibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary in vitro. Endocrinology 1968;83:783–790. 89 Elde RP, Parsons JA. Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus. Am J Anat 1975;144:541–548. 90 Désy L, Pelletier G. Immunohistochemical localization of somatostatin in the human hypothalamus. Cell Tissue Res 1977;184:491–497. 91 Finley JCW, Maderchut JL, Roger LJ et al. The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system. Neuroscience 1981;6:2173–2192. 92 Pelletier G, Dubé D, Puviani R. Somatostatin. Electron microscopic immunohistochemical localization in secretory neurons of rat hypothalamus. Science 1977;196:1496–1470. 93 Hökfelt T, Efendic S, Hellerstrom C et al. Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the A1-cells of the pancreatic islets and to the hypothalamus. Acta Endocrinol 1975;80(suppl.):5–41. 94 Reichlin S. Somatostatin. Part I. N Engl J Med 1983;309:1495–1501. 95 Reichlin S. Somatostatin. Part II. N Engl J Med 1983;309:1556–1563. 96 Ben Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr Rev 1985;6:564–589. 97 Bjorklund A, Lindvall O, Nobin A. Evidence of an incertohypothalamic dopamine neuron system in the rat. Brain Res 1975;89:29–42. 98 Ajika K, Hökfelt T. Projections to the median eminence and the arcuate nucleus, with special reference to monoamine systems: effects of lesions. Cell Tissue Res 1975;158:15–35. 99 Olschowka JA, O’Donohue TL, Mueller GP et al. The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain. Peptides 1982;3:995–1015. 100 Merchenthaler I, Vigh S, Petrusz P et al. Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain. Am J Anat 1982;165: 385–396. 101 Swanson JW, Sawchenko PE, Rivier J et al. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 1983;36: 165–186. 102 Pelletier G, Désy L, Côté J et al. Immunocytochemical localization of corticotropin-releasing factor-like immunoreactivity in the human hypothalamus. Neurosci Lett 1983;41:259–263. 103 Roth KA, Weber E, Barchas JD. Immunoreactive corticotropin-releasing factor (CRF) and vasopressin are colocalized in a subpopulation of the immunoreactive vasopressin cells in the paraventricular nucleus of the hypothalamus. Life Sci 1982;31:1857–1860. 104 Sawchenko PE, Swanson LW. Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed Proc 1985;44:221–227. 105 Petrusz P, Merchenthaler I, Maderdrut JL et al. Central and peripheral distribution of corticotropin-releasing factor. Fed Proc 1985;44: 229–235. 106 Audhya T, Hollander CS, Schlesinger DH et al. Structural characterization and localization of corticotropin-releasing factor in testis. Biochim Biophys Acta 1989;995:10–16. 107 Brownstein MJ, Palkovits M, Saavedra JM et al. Thyrotropin-releasing hormone in specific nuclei of the brain. Science 1974;185:267–269.

Functional Anatomy of the Hypothalamic Pituitary Axis

39

108 Hökfelt T, Fuxe K, Johansson O et al. Thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry. Eur J Pharmacol 1975;34:389–392. 109 Horita A, Carino MA, Chestnut RM. Influence of TRH on drug-induced narcosis and hyperthermia in the rabbit. Psychopharmacology 1976;49:57–62. 110 Kawano H, Daikoku S, Saito S. Location of thyrotropin-releasing hormonelike immunoreactivity in rat pancreas. Endocrinology 1983;112:951–955. 111 Dolva LO, Hanssen KF, Aadland E et al. Thyrotropin-releasing hormone immunoreactivity in the gastrointestinal tract of man. J Clin Endocrinol Metab 1983;56:524–529. 112 Wilber JF, Spinella P. Identification of immunoreactive thryotropin-releasing hormone in human neoplasia. J Clin Endocrinol Metab 1984;59:432–435. 113 Baker BL, Dermody WC, Reell JR. Localization of luteinizing hormonreleasing hormone in the mammalian hypothalamus. Am J Anat 1974;139: 129–134. 114 Barry J, Dubois MP, Poulain P. LRF-producing cell of the mammalian hypothalamus. Z Zellforsch 1974;146:351–366. 115 Pelletier G, Labrie F, Puviani R et al. Immunohistochemical localization of luteinizing hormone-releasing hormone in the rat median eminence. Endocrinology 1974;1:157–166. 116 Siler-Khodr TM, Khodr GS. Luteinizing hormone releasing factor content of the human placenta. Am J Obstet Gynecol 1978;130:216–219. 117 Dierickx K, Vandesande F. Immunocytochemical demonstration of separate vasopressin–neurophysin and oxytocin–neurophysin neurons in the human hypothalamus. Cell Tissue Res 1979;196:203–212. 118 Antunes JL, Carmel PW, Zimmerman EA. Paraventricular nucleus: the origin of vasopressin secreting terminals in hypophysial portal vessels in the monkey. Brain Res 1977;137:1–10. 119 Lederis K. An electron microscopical study of the human neurohypophysis. Z Zellforsch 1965;65:847–868. 120 Silverman AJ. Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins. J Histochem Cytochem 1976;24: 816–827. 121 Stillman MA, Recht LD, Rosario SL et al. The effects of adrenalectomy glucocorticoid replacement of vasopressin and vasopressin–neurophysin in the zona externa of the median eminence of the rat. Endocrinology 1977;101: 42–49. 122 Dubé D, Leclerc R, Pelletier G. Electron microscopic immunohistochemical localization of vasopressin and neurophysin in the median eminence of normal and adrenalectomized rat. Am J Anat 1976;147:103–108. 123 Clements JA, Funder JW. Arginine vasopressin (AVP) and AVP-like immunoreactivity in peripheral tissues. Endocr Rev 1986;7:449–460. 124 Polak JM, Bloom SR. Distribution and tissue localization of VIP in the central nervous system and seven peripheral organs. In: Said S, ed. Vasoactive Intestinal Peptide. New York: Raven Press, 1982:107–120. 125 Hökfelt T, Fahrenkrug J, Ju G et al. Analysis of peptide histidineisoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus. Neuroscience 1987;23:827–857. 126 Watanabe H. The immunostaining for the hypothalamic vasoactive intestinal peptide but not for beta-endorphin, dynorphin-A or methionine-enkephalin, is affected by the glucocorticoid milieu in the rat: correlation with the prolactin secretion. Regulatory Peptides 1990;28:301–311. 127 Prysor-Jones RA, Jenkins JS. Vasoactive intestinal peptide and anterior pituitary function. Clin Endocrinol 1988;29:677–688. 128 Vijaryan E, Samson WK, Saide SD et al. Vasoactive intestinal peptide: evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone and prolactin in conscious ovariectomized rats. Endocrinology 1979;104:53–57. 129 Vanderhaeghen JJ, Signeau JC, Gepts W. New peptide in the vertebrate CNS reacting with gastrin antibodies. Nature 1978;257:604–605. 130 Crowley JN. Comparative distribution of cholecystokinin and other neuropeptides. Ann NY Acad Sci 1985;448:1–8. 131 Hökfelt T, Herrera-Marschitz M, Seroogy K et al. Immunohistochemical studies on cholecystokinin (CCK)-immunoreactive neurons in the rat using sequence specific antisera with special reference to the caudate nucleus and primary sensory neurons. J Chem Neuroanat 1988;1:11–51. 132 Albus M. Cholecystokinin. Prog Neuro-Psychopharmacol Biol Psych 1988; 12(suppl.):S5–21. 133 Tatemoto K, Rokaeus A, Jornvall H et al. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett 1983;164:124–128. 134 Skofitsch G, Jacobowitz DM. Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 1985;6:509–546.

40

SECTION 1

Hypothalamic–Pituitary Function

135 Melander T, Hökfelt T, Rokaeus A et al. Coexistence of galanin-like immunoreactivity with catecholamines, 5-OH-tryptamine, GABA and neuropeptides in the rat CNS. Neuroscience 1986;6:3640–3656. 136 Cheung A, Polak JM, Bauer FE et al. The distribution of galanin immunoreactivity in the respiratory tract in pig, guinea-pig, rat and dog. Thorax 1985;40:889–896. 137 Bauer FE, Christofides ND, Hacker GW et al. Distribution of galanin immunoreactivity in the genitourinary tract of man and rat. Peptides 1986;7:5–10. 138 Bauer FE, Hacker GW, Terenghi G et al. Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas. J Clin Endocrinol Metab 1986;63:1372–1378. 139 Sano T, Vrontakis ME, Kovacs K et al. Galanin immunoreactivity in neuroendocrine tumors. Arch Pathol Lab Med 1991;115:926–929. 140 Nordström O, Melander T, Hökfelt T et al. Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett 1987;73:21–26. 141 Murakami Y, Kato Y, Shimatsu A et al. Possible mechanisms involved in growth hormone secretion induced by galanin in the rat. Endocrinology 1989;124:1224–1229. 142 Rehfeld JF. Localization of gastrins to neuro- and adenohypophysis. Nature 1978;271:771–773. 143 Rehfeld JF, Hansen HF, Marley PD et al. Molecular forms of cholecystokinin in the brain and the relationship to neuronal gastrins. Ann NY Acad Sci 1985;448:11–23. 144 McCann SM. The role of brain peptides in the control of anterior pituitary hormone secretion. In: Müller EE, MacLeod RM, eds. Neuroendocrine Perspectives, vol. 1. New York: Elsevier, 1982:1–22. 145 Lorén I, Alumets J, Ha° kanson R, Sundler F, Thorell J. Gut-type glucagon immunoreactivity in nerves of the rat brain. Histochemistry 1979;61: 335–341. 146 Dorn A, Rinne A, Bernstein HG et al. Immunoreactive glucagon in neurons of various parts of the human brain. Demonstration by immunofluorescence technique. Acta Histochem 1981;69:243–247. 147 Drucker DJ, Asa SL. Glucagon gene expression in vertebrate brain. J Biol Chem 1988;263:13475–13478. 148 Inokuchi A, Oomura Y, Shimizu N et al. Central action of glucagon in rat hypothalamus. Am J Physiol 1986;250:R120–126. 149 Allen YS, Adrian TE, Allen JM et al. Neuropeptide Y distribution in the rat brain. Science 1983;221:8877–8879. 150 Danger JM, Tonon MC, Jenks BG et al. Neuropeptide Y: localization in the central nervous system and neuroendocrine functions. Fundam Clin Pharmacol 1990;4:307–340. 151 Toni R, Jackson IMD, Lechan RM. Neuropeptide Y-immunoreactive innervation of thyrotropin-releasing-hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus. Endocrinology 1990;126:2444–2453. 152 Aronin N, Coslovsky R, Leeman SE. Substance P and neurotensin: their roles in the regulation of anterior pituitary function. Annu Rev Physiol 1986;48:537–549. 153 Carraway R, Leeman S. Characterization of radioimmunoassayable neurotensin in the rat: its differential distribution in the central nervous system, small intestine and stomach. J Biol Chem 1976;251:7045–7052. 154 Panula P, Hsiu-Ying TY, Costa E. Comparative distribution of bombesin/GRP- and substance P-like immunoreactivities in rat hypothalamus. J Comp Neurol 1984;224:606–618. 155 Giraud AS, Smith AI, Rundle SE. Distribution and molecular forms of immunoreactive bombesin in the ovine median eminence. Mol Cell Endocrinol 1987;53:245–250. 156 Takahashi K, Mouri T, Sone M et al. Calcitonin gene-related peptide in the human hypothalamus. Endocrinol Jpn 1989;36:409–415. 157 Fahim A, Rettori V, McCann SM. The role of calcitonin gene-related peptide on the control of growth hormone and prolactin release. Neuroendocrinology 1990;51:688–693. 158 Smith AI, Funder JW. Proopiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues. Endocr Rev 1988;9:159–179. 159 Plosker SM, Marshall LA, Martin MC, Jaffe RB. Opioid, catecholamine, and steroid interaction in prolactin and gonadotropin regulation. Obstet Gynecol Surv 1990;45:441–453. 160 Zhao BG, Chapman C, Brown D, Bicknell RJ. Opioid––noradrenergic interactions in the neurohypophysis. II. Does noradrenaline mediate the actions of endogenous opioids on oxytocin and vasopressin release? Neuroendocrinology 1988;48:25–31. 161 Bateman A, Singh A, Kral T, Solomon S. The immune–hypothalamic– pituitary–adrenal axis. Endocr Rev 1989;10:92–112.

162 Bernton EW, Beach JE, Holaday JW et al. Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 1987;238:519–524. 163 Scarborough DE, Lee SL, Dinarello CA, Reichlin S. Interleukin-1b stimulates somatostatin biosynthesis in primary cultures of fetal rat brain. Endocrinology 1989;124:549–551. 164 Spangelo BL, Judd AM, Isakson PC, MacLeod RM. Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 1989;125:575–577. 165 Yamaguchi M, Sakata M, Matsuzaki N et al. Induction by tumor necrosis factor-alpha of rapid release of immunoreactive and bioactive luteinizing hormone from rat pituitary cells in vitro. Neuroendocrinology 1990;52: 468–472. 166 Walton PE, Cronin MJ. Tumor necrosis factor-a and interferon-gamma reduce prolactin release in vitro. Am J Physiol 1990;259:E672–676. 167 Rizzino A. Growth factors. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology. Boston: Blackwell Science, 1991:979–989. 168 Gospodarowicz D, Cheng J, Lui G-M et al. Isolation of brain fibroblast growth factor by heparin–sepharose affinity chromatography: identity with pituitary fibroblast growth factor. Proc Natl Acad Sci USA 1984;81: 6963–6967. 169 Kudlow JE, Leung AWC, Kobrin MS et al. Transforming growth factor-a in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA. J Biol Chem 1989;264:3880–3883. 170 Baird A, Mormede P, Ying SY et al. A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 1985;82:5545–5549. 171 Ikeda H, Mitsuhashi T, Kubota K et al. Epidermal growth factor stimulates growth hormone secretion from superfused rat adenohypophyseal fragments. Endocrinology 1984;115:556–558. 172 Schriock EA, Lustig RH, Rosenthal SM et al. Effect of growth hormone (GH)-releasing hormone (GHRH) on plasma GH in relation to magnitude and duration of GH deficiency in 26 children and adults with isolated GH deficiency or multiple pituitary hormone deficiencies: evidence for hypothalamic GRH deficiency. J Clin Endocrinol Metab 1984;58:1043–1049. 173 Rimoin DL. Hereditary forms of growth hormone deficiency and resistance. Birth Defects 1976;12:15–29. 174 Phillips JA III, Parks JS, Hjelle BL et al. Genetic analysis of familial isolated growth hormone deficiency type I. J Clin Invest 1982;70:489–495. 175 Rimoin DL, Schechter JE. Histologic and ultrastructural studies in isolated growth hormone deficiency. J Clin Endocrinol Metab 1973;37:725–735. 176 Santora N, Filicori M, Crowley WF Jr. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropin-releasing hormone. Endocr Rev 1986;7:11–23. 177 Toledo SP, Luthold W, Mattar E. Familial idiopathic gonadotropin deficiency: a hypothalamic form of hypogonadism. Am J Med Genet 1983;15:405–416. 178 Stocks AE, Martin FIR. Pituitary function in hemochromatosis. Am J Med 1968;45:839–845. 179 Soules MR, Hammond CB. Female Kallmann’s syndrome: evidence for a hypothalamic luteinizing hormone-releasing hormone deficiency. Fertil Steril 1980;33:82–85. 180 Klingmüller D, Dewes W, Krahe T et al. Magnetic resonance imaging of the brain in patients with anosmia and hypothalamic hypogonadism (Kallmann’s syndrome). J Clin Endocrinol Metab 1987;65:581–584. 181 Seminara SB, Hayes FJ, Crowley WF Jr. Gonadotropin-releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallman’s syndrome): pathophysiological and genetic considerations. Endocrine Rev 1998;19:521–539. 182 Kovacs K, Sheehan HL. Pituitary changes in Kallmann’s syndrome: a histologic, immunocytologic, ultrastructural, and immunoelectron microscopic study. Fertil Steril 1982;37:83–89. 183 Daniel PM, Prichard MML. Studies of the hypothalamus and the pituitary gland: with special reference to the effects of transection of the pituitary stalk. Acta Endocrinol 1975;201(suppl.):1–216. 184 Heinbecker P, White HL. Hypothalamico-hypophysial system and its relation to water balance in the dog. Am J Physiol 1941;133:582–593. 185 Green JR, Buchan GC, Alvord EC Jr, Swanson AG. Hereditary and idiopathic types of diabetes insipidus. Brain 1967;90:707–714. 186 Nagai I, Li CH, Hsieh SM et al. Two cases of hereditary diabetes insipidus, with an autopsy finding in one. Acta Endocrinol 1984;105:318–323. 187 Scherbaum WA, Bottazzo GF. Autoantibodies to vasopressin cells in idiopathic diabetes insipidus: evidence for an autoimmune variant. Lancet 1983;i: 897–901. 188 Melmed S, Ezrin C, Kovacs K et al. Acromegaly due to secretion of growth hormone by an ectopic pancreatic islet-cell tumor. N Engl J Med 1985;312: 9–17.

Chapter 1 189 Asa SL, Scheithauer BW, Bilbao JM et al. A case for hypothalamic acromegaly: a clinicopathologic study of six patients with hypothalamic gangliocytomas producing growth hormone-releasing factor. J Clin Endocrinol Metab 1984;58:796–803. 190 Kovacs K, Horvath E, Thorner MO, Rogol AD. Mammosomatotroph hyperplasia associated with acromegaly and hyperprolactinemia in a patient with the McCune–Albright syndrome: a histologic, immunocytologic and ultrastructural study of the surgically-removed adenophypophysis. Virchows Archiv (Pathol Anat) 1984;403:77–86. 191 Weinstein LS, Shenker A, Gejman PV et al. Activating mutations of the stimulatory G protein in the McCune–Albright syndrome. New Engl J Med 1991;325:1688–1695. 192 Schwindinger WF, Francomano CA, Levine MA. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune–Albright syndrome. Proc Natl Acad Sci USA 1992;89:5152–5156. 193 Moran A, Asa SL, Kovacs K et al. Pituitary mammosomatotroph hyperplasia associated with childhood gigantism. New Engl J Med 1990;323:322–327. 194 Stefaneanu S, Kovacs K, Horvath E et al. Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a histologic, immunocytochemical and electron microscopic investigation. Endocrinology 1989;125:2710–2718. 195 Asa SL. Tumors of the Pituitary Gland. Atlas of Tumor Pathology, Fascicle 22, Third Series. Washington DC: Armed Forces Institute of Pathology, 1998. 196 Krieger DT. Pathophysiology of Cushing’s Disease. Endocr Rev 1983;4:22–43. 197 Carey RM, Varma SK, Drake CR Jr et al. Ectopic secretion of corticotropinreleasing factor as a cause of Cushing’s syndrome: a clinical, morphologic, and biochemical study. N Engl J Med 1984;311:13–20. 198 Asa SL, Kovacs K, Vale W et al. Immunohistologic localization of corticotropin-releasing hormone in human tumors. Am J Clin Pathol 1987;87: 327–333. 199 Asa SL, Kovacs K, Tindall GT et al. Cushing’s disease associated with an intrasellar gangliocytoma producing corticotropin-releasing factor. Ann Intern Med 1984;101:789–793. 200 Puchner MJA, Lüdecke DK, Valdueza JM et al. Cushing’s disease in a child caused by a corticotropin-releasing hormone-secreting intrasellar gangliocytoma associated with an adrenocorticotropic hormone-secreting pituitary adenoma. Neurosurgery 1993;33:920–925. 201 Puchner MJA, Lüdecke DK, Saeger W et al. Gangliocytomas of the sellar region—a review. Exp Clin Endocrinol 1995;103:129–149. 202 Liu N, Grumbach MM, de Napoli RA, Morishima A. Prevalence of electroencephalographic abnormalities in idiopathic precocious puberty and premature pubarche: bearing on pathogenesis and neuroendocrine regulation of puberty. J Clin Endocrinol Metab 1965;25:1296–1308. 203 Rosenthal SM, Grumbach MM, Kaplan SL. Gonadotropin-independent familial sexual precocity with premature Leydig and germinal cell maturation (familial testotoxicosis): effects of a potent luteinizing hormone-releasing factor agonist and medroxyprogesterone acetate therapy in four cases. J Clin Endocrinol Metab 1983;57:571–579. 204 Judge DM, Kulin HE, Page R et al. Hypothalamic hamartoma: a source of luteinizing-hormone-releasing factor in precocious puberty. N Engl J Med 1977;296:7–10. 205 Hochman HI, Judge DM, Reichlin S. Precocious puberty and hypothalamic hamartoma. Pediatrics 1981;67:236–244. 206 Kamoi K, Mitsuma T, Sato H et al. Hyperthyroidism caused by a pituitary thyrotrophin-secreting tumor with excessive secretion of thyrotrophinreleasing hormone and subsequently followed by Grave’s disease in a middle aged woman. Acta Endocrinol 1985;110:373–382. 207 DeTroyer A, Demanet JC. Clinical, biological and pathogenic features of the syndrome of inappropriate secretion of antidiuretic hormone: a review of 26 cases with marked hyponatraemia. Q J Med 1976;45:521–531. 208 Vorherr H, Massry SG, Fallet R et al. Antidiuretic principle in tuberculous lung tissue of a patient with pulmonary tuberculosis and hyponatremia. Ann Intern Med 1970;72:383–387. 209 Pettengill OS, Faulkner CS, Wurster-Hill DH et al. Isolation and characterization of a hormone-producing cell line from human small cell anaplastic carcinoma of the lung. J Natl Cancer Inst 1977;58:511–518. 210 Bergeron C, Kovacs K, Bilbao JM. Primary empty sella. A histologic and immunocytologic study. Arch Intern Med 1979;139:248–249. 211 Bergland RM, Ray BS, Torack RM. Anatomical variations in the pituitary gland and adjacent structures in 225 human autopsy cases. J Neurosurg 1968;28:93–99. 212 Gorczyca W, Hardy J. Arterial supply of the human anterior pituitary gland. Neurosurgery 1987;20:369–378.

Functional Anatomy of the Hypothalamic Pituitary Axis

41

213 Cox BM, Baizman ER, Su T-P et al. Further studies on the nature and function of pituitary endorphins. Adv Biochem Psychopharmacol 1978;18: 183–189. 214 Ferrand R, Hraoui S. Origine exlusivement ectodermique de l’adénohypophyse chez la caille et le poulet; demonstration par la méthode des associations tissulaires interspécifiques. C R Soc Biol 1973;167:740–743. 215 Ferrand R, Pearse AGE, Polak JM, Le Douarin NM. Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions. Histochemistry 1974;38:133–141. 216 LeDouarin N. Particularités du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularités comme “marque biologique” dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogénèse. Bull Biol Fr Belg 1969;103: 435–452. 217 Takor Takor T, Pearse AGE. Neuroectodermal origin of avian hypothalamohypophyseal complex: the role of the ventral neural ridge. J Embryol Exp Morphol 1975;34:311–325. 218 Daikoku S. Studies on the human foetal pituitary. 1. Quantitative observations. Tokushima J Exp Med 1958;5:200–213. 219 Baker BL, Jaffe RB. The genesis of cell types in the adenohypophysis of the human fetus as observed by immunocytochemistry. Am J Anat 1975;143: 137–162. 220 Asa SL, Kovacs K, Laszlo FA et al. Human fetal adenohypophysis. Histologic and immunocytochemical analysis. Neuroendocrinology 1986;43:308–316. 221 Asa SL, Kovacs K, Horvath E et al. Human fetal adenohypophysis. Electron microscopic and ultrastructural immunocytochemical analysis. Neuroendocrinology 1988;48:423–431. 222 Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptxl (pituitary homeobox 1) acts in synergy with SF-1 and Pit-1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 1998;12:428–441. 223 Gage PJ, Camper SA. Pituitary Homeobox 2, a novel member of the bicoidrelated family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 1997;6:457–464. 224 Sheng HZ, Moriyama K, Yamashita T et al. Multistep control of pituitary organogenesis. Science 1997;278:1809–1812. 225 Bach I, Rhodes SJ, Pearse RV et al. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 1995;92:2720–2724. 226 Hermesz E, Machem S, Mahon KA. Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke’s pouch of the mouse embryo. Develop 1996;122:41–52. 227 Sornson MW, Wu W, Dasen JS et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327–333. 228 Gage PJ, Brinkmeier MLSLM, Knapp LT. The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation. Mol Endocrinol 1996;10: 1570–1581. 229 Wu W, Cogan JD, Pfäffle RW et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genet 1998;18:147–149. 230 Fofanova O, Takmura N, Kinoshita E. Compound heterozygous deletion of the prop-1 gene in children with combined pituitary hormone deficiency. J Clin Endocrinol Metab 1998;83:2601–2604. 231 Jackson SM, Barnhart KM, Mellon P et al. Helix-loop proteins are present and differentially expressed in different cell lines from the anterior pituitary. Mol Cell Endocrinol 1993;96:167–176. 232 Asa SL, Ezzat S. The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 1998;19:798–827. 233 Asa SL, Ezzat S. Molecular determinants of pituitary cytodifferentiation. Pituitary 1999;1:159–168. 234 Lamolet B, Pulichino AM, Lamonerie T et al. A pituitary cell-restricted T box factor, T pit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 2001;104:849–859. 235 Poulin G, Turgeon B, Drouin J. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 1997;17: 6673–6682. 236 Ingraham HA, Lala DS, Ikeda Y et al. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8:2302–2312. 237 Asa SL, Bamberger A-M, Cao B et al. The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 1996;81:2165–2170.

42

SECTION 1

Hypothalamic–Pituitary Function

238 Rosenfeld MG. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 1991;5:897–907. 239 Asa SL, Puy LA, Lew AM et al. Cell type-specific expression of the pituitary transcription activator Pit-1 in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1993;77:1275–1280. 240 Friend KE, Chiou Y-K, Laws ER Jr et al. Pit-1 messenger ribonucleic acid is differentially expressed in human pituitary adenomas. J Clin Endocrinol Metab 1993;77:1281–1286. 241 Pellegrini I, Barlier A, Gunz G et al. Pit-1 gene expression in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1994;79:189–196. 242 Day RN, Koike S, Sakai M et al. Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol 1990;4:1964–1971. 243 Drolet DW, Scully KM, Simmons DM et al. TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 1991;5:1739–1753. 244 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 245 Tatsumi K, Miyai K, Notomi T et al. Cretinism with combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Genet 1992;1:56–58. 246 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 247 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 248 Puy LA, Asa SL. The ontogeny of pit-1 expression in the human fetal pituitary gland. Neuroendocrinology 1996;63:349–355. 249 Ikeda Y, Luo X, Abbud R et al. The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 1995;9:478–486. 250 Yang H-J, Ozawa H, Kurosumi K. Ultrastructural changes in growth hormone cells in the rat anterior pituitary after thyroidectomy as studied by immunoelectron microscopy and enzyme nistochemistry. J Clin Electr Microsc 1989;22:269–283. 251 Ezzat S, Kontogeorgos G, Redelmeier DA et al. In vitro responsiveness of morphological variants of growth hormone-producing pituitary adenomas to octreotide. Eur J Endocrinol 1995;133:686–690. 252 Horvath E, Kovacs K. The adenohypophysis. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology 2nd ed. Boston: Blackwell Science, 1998; 247–281. 253 Sano T, Ohshima T, Yanad S. Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin on growth hormone-producing pituitary adenomas. Pathol Res Pract 1991;187:530–533. 254 Neumann PE, Goldman JE, Horoupian DS, Hess MA. Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med 1985;109:505–508. 255 Billestrup N, Swanson L, Vale W. Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 1986;83: 6854–6857. 256 Richardson SB, Twente S. Evidence that diminished pituitary responsitivity to GHRF is secondary to intracellular GH pool depletion. Am J Physiol 1988;254:E358–364. 257 Loras B, Yi JY, Durand A et al. GRF et adénomes somatotropes humains. Correlations in vivo et in vitro entre la libération de GH et les aspects morphologiques et immunocytochimiques. Ann Endocrinol 1985;46: 373–382. 258 Kawakita S, Asa SL, Kovacs K. Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation. J Endocrinol Invest 1989;12:443–448. 259 Beckers A, Stevenaert A, Kovacs K et al. The treatment of acromegaly with SMS 201–995. Advanc Biosci 1988;69:227–228. 260 Barkan AL, Lloyd RV, Chandler WF et al. Preoperative treatment of acromegaly with long-acting somatostatin analog SMS 201–995: shrinkage of invasive pituitary macroadenomas and improved surgical remission rate. J Clin Endocrinol Metab 1988;67:1040–1048. 261 Ezzat S, Snyder PJ, Young WF et al. Octreotide treatment of acromegaly. A randomized, multicenter study. Ann Intern Med 1992;117:711–718. 262 Asa SL, Felix I, Kovacs K, Ramyar L. Effects of somatostatin on somatotroph adenomas of the human pituitary: an in vitro functional and morphological study. Endocr Pathol 1990;1:228–235.

263 Baringa M, Bilezikjian LM, Vale WW, Rosenfeld MG, Evans RM. Independent effects of growth hormone releasing factor on growth hormone release and gene transcription. Nature 1985;314:279–281. 264 Herman V, Weiss M, Becker D, Melmed S. Hypothalamic hormonal regulation of human growth hormone gene expression in somatotroph adenoma cell cultures. Endocr Pathol 1990;1:236–244. 265a Zafar M, Ezzat S, Ramyar L et al. Cell specific expression of estrogen receptor in the human pituitary and its adenomas. J Clin Endocrinol Metab 1995;80:3621–3627. 265b Friend KE, Chiou TK, Lopes MBS et al. Estrogen receptor expression in human pituitary. J Clin Endocrinol Metab 1994;78:1497–1504. 266 Asa SL, Penz G, Kovacs K, Ezrin C. Prolactin cells in the human pituitary: a quantitative immunocytochemical analysis. Arch Pathol Lab Med 1982;106: 360–363. 267 Scheithauer BW, Sano T, Kovacs K et al. The pituitary gland in pregnancy: a clinicopathologic and immunohistochemical study of 69 cases. Mayo Clin Proc 1990;65:461–474. 268a Scheithauer BW, Kovacs K, Randall RV, Ryan N. Effects of estrogen on the human pituitary: a clinicopathologic study. Mayo Clin Proc 1989;64: 1077–1084. 268b Asa SL, Kelly MA, Grandy DK, Low MJ. Pituitary lactotroph adenomas develop after prolonged lactotroph hyperplasia in dopamine D2 receptordeficient mice. Endocrinology 1999;140:5348–5355. 269 Tindall GT, Kovacs K, Horvath E, Thorner MO. Human prolactin-producing adenomas and bromocriptine: a histological, immunocytochemical, ultrastructural, and morphometric study. J Clin Endocrinol Metab 1982;55: 1178–1183. 270 Esiri MM, Bevan JS, Burke CW, Adams CBI. Effect of bromocriptine treatment on the fibrous tissue component of prolactin-secreting and nonfunctioning macroadenomas of the pituitary gland. J Clin Endocrinol Metab 1986;63:383–388. 271 Felix IA, Horvath E, Kovacs K et al. Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. Acta Neuropathol 1986;71:76–82. 272 Asa SL, Kovacs K, Stefaneanu L et al. Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. Endocrinology 1992;131:2083–2089. 273 Lewis ME, Sherman TG, Burke S et al. Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe. Proc Natl Acad Sci USA 1986;83:5419–5423. 274 Horvath E, Ilse G, Kovacs K. Enigmatic bodies in human corticotroph cells. Acta Anat 1977;98:427–433. 275 Neumann PE, Horoupian DS, Goldman JE, Hess MA. Cytoplasmic filaments of Crooke’s hyaline change belong to the cytokeratin class. An immunocytochemical and ultrastructural study. Am J Pathol 1984;116:214–222. 276a Asa SL, Kovacs K. Functional morphology of the human fetal pituitary. Pathol Annu 1984;19(1):275–315. 276b Pilardzic D, Kovacs K, Asa SL. Pituitary morphology in human anencephalic fetuses. Neuroendocrinology 1997;65:164–172. 277 Scheithauer BW, Kovacs K, Randall RV. The pituitary gland in untreated Addison’s disease. A histologic and immunocytologic study of 18 adenohypophyses. Arch Pathol Lab Med 1983;107:484–487. 278 Siperstein ER, Allison VF. Fine structures of the cells responsible for secretion of adrenocorticotropin in the adrenalectomized rat. Endocrinology 1965;76: 70–79. 279 Asa SL, Kovacs K, Hammer GD et al. Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector. Endocrinology 1992;131:715–720. 280 Coates PJ, Doniach I, Hale AC, Rees LH. The distribution of immunoreactive a-melanocyte-stimulating hormone cells in the adult human pituitary gland. J Endocrinol 1986;111:335–342. 281 McNicol AM. A study of intermediate lobe differentiation in the human pituitary gland. J Pathol 1986;150:169–173. 282 Horvath SE, Asa SL, Kovacs K et al. Human pituitary corticotroph adenomas in vitro: morphologic and functional reponses to corticotropin-releasing hormone and cortisol. Neuroendocrinology 1990;51:241–248. 283 Horvath E, Lloyd RV, Kovacs K. Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells: a morphologic study of the rat pituitary including immunoelectron microscopy. Lab Invest 1990;63:511–520. 284 Asa SL, Kovacs K, Bilbao JM. The pars tuberalis of the human pituitary: a histologic immunohistochemical, ultrastructural and immunoelectron microscopic analysis. Virchows Archiv (Pathol Anat) 1983;399:49–59.

Chapter 1 285 Horvath E, Kovacs K. Gonadotroph adenomas of the human pituitary: sexrelated fine structural dichotomy. A histologic, immunocytochemical and electron microscopic study of 30 tumors. Am J Pathol 1984;117:429–440. 286 Denef C. Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 1986;15:1–32. 287 Kovacs K, Horvath E. Gonadotrophs following removal of the ovaries: a fine structural study of human pituitary glands. Endokrinologie 1975;66:1–8. 288 Horvath E, Kovacs K, Penz G, Ezrin C. Origin, possible function and fate of “follicular cells” in the anterior lobe of the human pituitary. Am J Pathol 1974;77:199–212. 289 Girod C, Trouillas J, Dubois MP. Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the anterior lobe of the normal human pituitary. Cell Tissue Res 1985;241:505–511. 290 Höfler H, Walter GF, Denk H. Immunohistochemistry of folliculo-stellate cells in normal human adenohypophyses and in pituitary adenomas. Acta Neuropathol 1984;65:35–40. 291 Baes M, Allaerts W, Denef C. Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary aggregates. Endocrinology 1987;120:685–691. 292 Vankelecom A, Carmeliet P, Van Damme J et al. Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 1989;49:102–106. 293 Ferrara N, Schweigerer L, Neufeld G et al. Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 1987;84: 5773–5777. 294 Coates PJ, Doniach I. Development of folliculo-stellate cells in the human pituitary. Acta Endocrinol 1988;119:16–20. 295 Lauriola L, Cocchia D, Sentinelli S et al. Immunohistochemical detection of folliculo-stellate cells in human pituitary adenomas. Virchows Archiv (Cell Pathol) 1984;47:189–197. 296 Nishioka H, Llena JF, Hirano A. Immunohistochemical study of folliculostellate cells in pituitary lesions. Endocr Pathol 1991;2:155–160. 297 Kovacs K, Horvath E, Ryan N, Ezrin C. Null cell adenomas of the human pituitary. Virchows Archiv (Pathol Anat) 1980;387:165–174. 298 Asa SL, Cheng Z, Ramyar L et al. Human pituitary null cell adenomas and oncocytomas in vitro: effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology. J Clin Endocrinol Metab 1992;74:1128–1134. 299 Yamada S, Asa SL, Kovacs K et al. Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab 1989;68:73–80. 300 Jameson JL, Klibanski A, Black PMcL et al. Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas. J Clin Invest 1987;80:1472–1478. 301 Kontogeorgos G, Horvath E, Kovacs K et al. Null cell adenoma of the pituitary with features of plurihormonality and plurimorphous differentiation. Arch Pathol Lab Med 1991;115:61–64. 302 Kovacs K, Asa SL, Horvath E et al. Null cell adenomas of the pituitary: attempts to resolve their cytogenesis. In: Lechago J, Kameya T, eds. Endocrine Pathology Update. Philadelphia: Field and Wood, 1990;17–31. 303 Kovacs K, Horvath E, Bilbao JM. Oncocytes in the anterior lobe of the human pituitary gland. A light and electron microscopic study. Acta Neuropathol 1974;27:43–54. 304 Carafoli E. Mitochondrial pathology: an overview. Ann NY Acad Sci 1986; 488:1–18. 305 Horvath E, Kovacs K, Singer W et al. Acidophil stem cell adenoma of the human pituitary: clinicopathological analysis of 15 cases. Cancer 1981;47: 761–771. 306 Sano T, Kovacs K, Asa SL, Smyth HS. Immunoreactive luteinizing hormone in functioning corticotroph adenomas of the pituitary. Immunohistochemical and tissue culture studies of two cases. Virchows Archiv (Pathol Anat) 1990;471: 361–367. 307 Moriarty GC, Garner LL. Immunocytochemical studies of cells in the rat adenohypophysis containing both ACTH and FSH. Nature 1977;265:356–358. 308 May V, Wilber JF, U’Prichard DC, Childs GV. Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures. Peptides 1987;8:543–558. 309 Joubert (Bression) D, Benlot C, Lagoguey A et al. Normal and growth hormone (GH)-secreting adenomatous pituitaries release somatostatin and GH-releasing hormone. J Clin Endocrinol Metab 1989;68:572–577. 310 Pagesy P, Li JY, Rentier-Delrue F et al. Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland. Mol Endocrinol 1989;3:1289–1294.

Functional Anatomy of the Hypothalamic Pituitary Axis

43

311 Duello TM, Nett TM, Farquhar MG. Fate of a gonadotropin releasing hormone agonist internalized by rat pituitary gonadotrophs. Endocrinology 1983;112:1–10. 312 Deschepper CF, Crumrine DA, Ganong WF. Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat. Endocrinology 1986;119:36–43. 313 Major J, Ghatei MA, Bloom SR. Bombesin-like immunoreactivity in the pituitary gland. Experientia 1983;39:1158–1159. 314 Zaidi M, Abeyasekera G, MacIntyre I. Calcitonin gene-related peptide: endocrine distribution and characterization of circulating forms. J Endocrinol Invest 1989;12:699–704. 315 Rehfeld JF. Preprocholecystokinin processing in the normal human anterior pituitary. Proc Natl Acad Sci USA 1987;84:3019–3023. 316 Rehfeld JF, Lindholm J, Andersen BN et al. Pituitary tumors containing cholecystokinin. N Engl J Med 1987;316:1244–1247. 317 Vrontakis ME, Sano T, Kovacs K, Friesen HG. Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary. J Clin Endocrinol Metab 1990;70:747–751. 318 Vrontakis ME, Peden LM, Duckorth MC, Friesen HG. Isolation and characterization of a complementary DNA (galanin) clone from estrogeninduced pituitary tumor messenger RNA. J Biol Chem 1987;262: 16755–16758. 319 O’Halloran DJ, Jones PM, Steel JH et al. Effect of endocrine manipulation on anterior pituitary galanin in the rat. Endocrinology 1990;127: 467–475. 320 Kaplan LM. Gabriel SM, Koenig JI et al. Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 1988;85:7408–7412. 321 Larson LI, Rehfeld JF. Pituitary gastrins occur in corticotrophs and melanotrophs. Science 1981;213:768–770. 322 Bardram L, Lindholm J, Rehfeld JF. Gastrin in pituitary tumors. Acta Endocrinol 1987;115:419–422. 323 Jones PM, Ghatei MA, Steel J et al. Evidence for neuropeptide-Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 1989;125:334–341. 324 Goedert M, Lightman SL, Nagy IT et al. Neurotensin in the rat anterior pituitary gland. Nature 1982;298:163–165. 325 Morel G, Chayvialle JA, Kerdelhue B, Dubois PM. Ultrastructural evidence for endogenous substance P-like immunoreactivity in the rat pituitary gland. Neuroendocrinology 1982;35:86–92. 326 de Palatis LR, Fiorindo RP, Ho RH. Substance P immunoreactivity in the anterior pituitary gland of the guinea pig. Endocrinology 1982;110: 282–284. 327 Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC. Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 1986;119: 2052–2057. 328 Steel JH, Gon G, O’Halloran DJ et al. Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 1989;93:183–189. 329 Lam SK, Reichlin S. Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 1989;50: 524–528. 330 Webster J, Ham J, Bevan JS, Scanlon MF. Growth factors and pituitary tumors. Trends Endocrinol Metab 1989;1:95–98. 331 Ezzat S, Melmed S. The role of growth factors in the pituitary. J Endocrinol Invest 1990;13:691–698. 332 White BA, Bancroft FC. Epidermal growth factor and thyrotropin-releasing hormone interact synergistically with calcium to regulate prolactin mRNA levels. J Biol Chem 1983;258:4618–4622. 333 Murdoch GH, Potter E, Nicolaisen AK et al. Epidermal growth factor rapidly stimulates prolactin gene transcription. Nature 1982;300:192–194. 334 Polk DH, Ervin MG, Padbury JF et al. Epidermal growth factor acts as a corticotropin-releasing factor in chronically catheterized fetal lambs. J Clin Invest 1987;79:984–988. 335 Miyake A, Tasaka K, Otsuka S et al. Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro. Acta Endocrinol 1985;108:175–178. 336 Leriche VK, Asa SL, Ezzat S. Epidermal growth factor and its receptor (EGFR) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab 1996;81:656–662. 337 Kasselberg AG, Orth DN, Gray ME, Stahlman MT. Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues. J Histochem Cytochem 1985;33:315–322.

44

SECTION 1

Hypothalamic–Pituitary Function

338 Mueller SG, Kobrin MS, Paterson AJ, Kudlow JE. Transforming growth factor-a expression in the anterior pituitary gland: regulation by epidermal growth factor and phorbol ester in dispersed cells. Mol Endocrinol 1989;3:976–983. 339 Ezzat S, Walpola IA, Ramyar L et al. Membrane-anchored expression of transforming growth factor-≤ in human pituitary adenoma cells. J Clin Endocrinol Metab 1995;80:534–539. 340 Kobrin MS, Asa SL, Samoondar J, Kudlow JE. a-Transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization. Endocrinology 1987;121:1412–1416. 341 Ramsdell JS. Transforming growth factor-a and -b are potent and effective inhibitors of GH4 pituitary tumor cell proliferation. Endocrinology 1991;128: 1981–1990. 342 McAndrew J, Paterson AJ, Asa SL et al. Targeting of transforming growth factor-a expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 1995;136: 4479–4488. 343 Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9:267–293. 344 Roberts V, Meunier H, Vaughan J et al. Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 1989;124:552–554. 345 Billestrup N, González-Manchón C, Potter E, Vale W. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 1990;4:356–362. 346 Corrigan AZ, Bilezikjian LM, Carroll RS et al. Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 1991;128:1682–1684. 347 Kogawa K, Nakamura T, Sugino K et al. Activin-binding protein is present in pituitary. Endocrinology 1991;128:1434–1440. 348 Penabad JL, Bashey HM, Asa SL et al. Decreased follistatin gene expression in gonadotroph adenomas. J Clin Endocrinol Metab 1996;81:3397–3403. 349 Gospodarowicz D, Ferrara N, Schweigener L, Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 1987;8:95–114. 350 Prysor-Jones RA, Silverlight JJ, Jenkins JS. Oestradiol, vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumour cells in vitro. J Endocrinol 1989;120:171–177. 351 Elias KA, Weiner RI. Direct arterial vascularization of estrogen-induced prolactin-secreting anterior pituitary tumors. Proc Natl Acad Sci USA 1984;81:4549–4553. 352 Ezzat S, Smyth HS, Ramyar L, Asa SL. Heterogeneous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 1995;80:878–884. 353 Ezzat S, Zheng L, Zhu XF, Wu GE, Asa SL. Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 2002;109:69–78. 354 Stojlkovic SS, Merelli F, Iida T et al. Endothelin stimulation of cystolic calcium and gonadotropin secretion in anterior pituitary cells. Science 1990;248:1663–1666. 355 Matsumoto H, Suzuki N, Shiota K et al. Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture. Biochem Biophys Res Commun 1990;172:661–668.

356 Prager D, Melmed S, Fagin J. Feedback regulation of growth hormone gene expression by insulin-like growth factor I. In: LeRoith D, Raizada MK, eds. Molecular and Cellular Biology of Insulin-like Growth Factors and Their Receptors. New York: Plenum Press, 1989:57–71. 357 Yamashita S, Melmed S. Insulin-like growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 1987;79:449–452. 358 Berelowitz M, Szabo M, Frohman LA et al. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1982;212:1279. 359 Han VKM, Lund PK, Lee DC, D’Ercole AJ. Expression of somatomedin/ insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol Metab 1998;66:422–429. 360 Yamashita S, Weiss M, Melmed S. Insulin-like growth factor I regulates GH secretion and mRNA levels in human pituitary tumor cells. J Clin Endocrinol Metab 1986;63:730–735. 361 Asa SL, Coschigano KT, Bellush L et al. Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic and GH receptor-deficient mice. Am J Pathol 2000;156:1009–1015. 362 Asa SL, Kovacs K. Histological classification of pituitary disease. J Clin Endocrinol Metab 1983;12:567–596. 363 Shi T, Farrel MA, Kaufmann JCL. Fibrosarcoma Complicating irradiated pituitary adenoma. Surg Neurol 1984;22:277–283. 364 Del Pozo JM, Roda JE, Montoya JG et al. Intrasellar granuloma. Case Report. J Neurosurg 1980;53:717–719. 365 Asa SL, Bilbao JM, Kovacs K et al. Lymphocytic hypophysitis of pregnancy resulting in hypopituitarism. A distinct clinicopathologic entity. Ann Intern Med 1981;95:166–171. 366 Thodou E, Asa SL, Kontogeorgos G et al. Lymphocytic hypophysitis: Clinicopathologic findings. J Clin Endocrinol Metab 1995;80:2302–2311. 367 Sheehan HL, Stanfield JR. The pathogenesis of post-partum necrosis of the anterior lobe of the pituitary gland. Acta Endocrinol 1961;37:479–510. 368 Bergeron C, Kovacs K. Pituitary siderosis. A histologic and immunocytologic study. Arch Intern Med 1979;139:248-249. 369 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 370 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 371 Tatsumi K, Miyai K, Notomi T et al. Cretinism with a combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Gen 1992;1:56–58. 372 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 373 Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA 1987;84:4636–4640. 374 Baumann G, Shaw MA, Winter RJ. Absence of the plasma growth hormonebinding protein in Laron-type dwarfism. J Clin Endocrinol Metab 1987;65: 814–816.

C h a p t e r

2 Adrenocorticotropin Mark A. Herman Joseph A. Majzoub

The anterior pituitary forms via invagination of the pharyngeal stomodeum in the region of contact with the diencephalon. By week 5 of human gestation, this invagination, termed Rathke’s diverticulum, has formed, but the downward migration of the diencephalic diverticulum, destined to be the neurophypophysis, has not yet commenced. It is at this time that adrenocorticotropin (ACTH) is first detectable by immunocytochemistry in the collection of cells within Rathke’s diverticulum which are furthest from contact with the diencephalon [1]. By 8 weeks of gestation, ACTH is detectable by radioimmunoassay of both fetal pituitary tissue and fetal blood [1]. The hypophyseal–portal vascular system forms between 8 and 14 weeks gestation, dating the earliest time after which hypothalamic corticotropin releasing factors may function to regulate fetal pituitary ACTH [1]. By 14 weeks gestation, release of ACTH from human fetal corticotrophs is highly responsive to exposure to corticotropin releasing hormone (CRH) in vitro [2]. The intensity of immunohistochemical ACTH staining in the anterior pituitary increases progressively from the mid-first through the end of the second trimester. In contrast, it is only after 21 weeks gestation that ACTH-positive cells begin to appear in the pars intermedia of the human fetal pituitary, defined as that region between Rathke’s cleft anteriorly and the neurohypophysis posteriorly. The ACTHcontaining cells in this region are epithelial-like, in contrast to the more angular, ovoid appearance of corticotrophs in the anterior pituitary [1].

those containing a-L-fucose and complex N-glyosylprotein, as well as terminal b-galactose. Corticotrophs constitute between 10 and 20% of the cell population of the anterior pituitary [3]. They are large cells with an ovoid or angular shape. They occur either singly or in clumps. They are most numerous in the midsagittal region of the pituitary (median wedge) but also occur in the lateral wings of the gland. Although the adult human pituitary does not contain an intermediate lobe, the junctional zone between the anterior and posterior lobes is known as the zona intermedia. This region, derived from the portion of Rathke’s diverticulum posterior to Rathke’s cleft, contains scattered cells immunopositive for ACTH. Some of these ACTHcontaining cells appear to extend into the posterior pituitary, a feature known as basophilic invasion [3]. These areas of apparent migration of corticotrophs from the zona intermedia into the posterior pituitary occur focally along the border between these two regions. In humans, in addition to the sellar pituitary, a pharyngeal pituitary exists which is located in either the sphenoid sinus or within the sphenoid bone. It consists of pituitarylike tissue approximately 2 to 5 mm by 0.2 mm in size. It is connected to the sellar gland by transsphenoidal vessels [4]. Only 1% to 2% of the cells in the pharyngeal pituitary contain immunoreactive ACTH, in contrast to approximately 14% of the cells in the sellar pituitary [4]. The pharyngeal pituitary is thought to arise as a rest of tissue resulting from the normal migration of cells from Rathke’s pouch to the sella turcica. There have been several reports of Cushing’s disease due to corticotroph adenomas arising in the pharyngeal pituitary [4,5].

Adult Anatomy

Molecular Embryology

Corticotrophs were initially identified by their basophilic staining. This has been subsequently found to be due to the presence of complex sugars in corticotrophs, principally

Tremendous progress has been made in recent years concerning the molecular mechanisms controlling pituitary organogenesis and pituitary cell-lineage specification in

CELLS OF ORIGIN

Fetal Anatomy

45

46

SECTION 1

Hypothalamic–Pituitary Function

animal models. A complex network of transcriptional events mediated by extrinsic and intrinsic signals has been implicated in the determination and a stereotypical spatiotemporal differentiation of the five trophic cell types of the mature anterior pituitary gland. Intrinsic Signals

RPX (for Rathke’s pouch homeobox), also known as HESX1 (for homeobox gene expression in embryonic stem cells) is the earliest known marker for the pituitary primordium [6]. HESX1/RPX is a member of the paired-like class of homeobox genes. Early HESX1/RPX expression occurs in the anterior midline endoderm and prechordal plate precursor, followed by expression in the cephalic neural plate [6]. Expression becomes progressively restricted anteriorly in the cephalic neural plate in a distribution consistent with tissue known by fate mapping studies to form the primordium of the anterior pituitary [7–10]. HESX1/RPX expression ultimately becomes restricted to Rathke’s pouch, and down regulation of HESX1/RPX in the pouch coincides with the differentiation of pituitary-specific cell types [6]. Embryonic mice lacking Rpx demonstrate variable abnormalities of hypothalamic and pituitary morphogenesis, reduced prosencephalon, anophthalmia or microophthalmia, and defective olfactory development. Neonatal mice have abnormalities in the corpus callosum, the anterior and hippocampal commisures, and the septum pellucidum [11]. These abnormalities are reminiscent of defects seen in a heterogeneous group of human disorders known as septooptic dysplasia (SOD). Deficits in SOD include optic nerve hypoplasia, abnormalities of the midline brain structures, and abnormalities of the hypothalamic-pituitary axis. Patients commonly present with endocrinopathies including hypoglycemia and adrenal crisis, which may signal growth hormone, ACTH, or thryoid-stimulating hormone deficiency [12]. Dattani and colleagues have identified two siblings with SOD who are homozygous for missense mutations within the HESX1 homeodomain which prevents it from binding target DNA [11]. The siblings identified with HESX1 mutations presented with hypoglycemia hours after birth and demonstrated hypopituitarism, substantiating a role for HESX1 in human pituitary development [11,13]. The corticotroph lineage appears to diverge relatively early from the other cell types of the anterior pituitary. LHX3, a LIM-type homeodomain protein, is essential for growth of Rathke’s pouch and determination of pituitary cell lineages [14]. In Lhx3-deficient mice, proopiomelanocortin (Pomc) was detected in a small cohort of cells at the ventral base of the pouch remnant which roughly corresponds to the position of the first presumptive corticotroph cells to differentiate in the wild-type pouch [14]. Although some pouch cells were able to differentiate and express Pomc, these cells failed to proliferate. An insufficient or non-functional corticotroph cell mass may have caused the hypoplastic adrenal cortices noted in these animals [14].

Ptx1 is a bicoid-related homeobox transcription factor identified based upon its ability to activate transcription of Pomc in the corticotroph derived AtT-20 cell line [15] and its ability to interact with the transactivation domain of Pit-1 [16]. It is expressed in the primordial Rathke’s pouch, oral epithelium, first branchial arch, the duodenum, and hindlimb [16]. Ptx1 is expressed in all mature pituitary cells, but is differentially expressed during pituitary development in different cell types [17]. In the mature pituitary, the highest levels of expression are found within corticotrophs [15]. In addition to activating transcription of Pomc, Ptx1 is required for sustained expression of Lhx3 [18], directly activates transcription of the common a-glycoprotein subunit (a-GSU) [16], synergizes with Pit-1 on the growth hormone and prolactin promoter [18], and synergizes with SF-1 on the promoter of the lutenizing hormone b (LH-b) gene [18]. Ptx2, also known as RIEG, is an additional bicoid-related homeobox gene 97% identical to Ptx1 [19]. Ptx2 is also differentially expressed in the pituitary and is excluded from corticotrophs [20], suggesting that some of the functions ascribed to Ptx1 may be performed by Ptx2. Expression of Pit-1, a POU domain transcription factor, is restricted to the anterior pituitary and required for the development of thyrotophs, lactotrophs, and somatotrophs [21]. Consistent with this, patients with mutations in PIT-1 have normal hypothalamic-pituitary-advanced (HPA) axis function [22]. Prophet of PIT-1 (PROP-1) is required for PIT-1 expression, and mutation of its gene is also associated with deficiencies in the development of thyrotrophs, lactotrophs, and somatotrophs in the Ames dwarf mouse and in humans. In a small number of patients with mutations in PROP1, modest impairment of ACTH secretion has been reported [23]. Developmentally essential, cell line-restricted factors have been identified for a number of different pituitary cell types. SF-1, an orphan nuclear receptor, is necessary for the development of gonadotrophs [24]. However, no essential corticotroph specific factor has yet been clearly identified. NeuroD, also known as Beta2, is a cell-restricted basic helixloop-helix (bHLH) transcription factor implicated in late neuronal differentiation [25] which has also been isolated as a cell-specific transcription factor of the insulin gene [26]. NeuroD is expressed in a number of tissues including pancreatic endocrine cells, the intestine, and brain including the mouse corticotroph AtT-20 cell line, but not the rat somatotroph pituitary cell line (GH3) [26]. Drouin and colleagues have recently demonstrated that NeuroD expression does appear to be restricted to corticotrophs in the mouse pituitary, and that NeuroD, in association with ubiquitous bHLH dimerization partners, specifically recognizes and activates transcription from the POMC promoter E box that confers transcriptional specificity of POMC to corticotrophs [27,28]. However, the necessity of this transcription factor for corticotroph specification or differentiation is still undetermined. Recently, a corticotroph-restricted transcription factor. TPIT, has been identified, which interacts with PTX1 and is required for POMC transcription, as discussed subse-

Chapter 2

quently. However, its role in corticotroph development is not known, and awaits further study. Because no instrinsic factor has yet been identified that is absolutely required for the development of corticotrophs in situ, it is possible that they arise elsewhere during fetal life and migrate into the anterior pituitary. One possible source could be neural crest, as this is the origin of POMC-producing melanocytes which migrate to the skin [29]. Extrinsic Signals

The oral ectoderm, from which Rathke’s pouch forms, and the neural ectoderm of the ventral diencephalon, from which the hypothalamus arises, are in direct contact at the time of the formation of Rathke’s pouch [30]. Classical explant experiments have indicated that inductive signals arising from mesenchyme and neural ectoderm adjacent to Rathke’s pouch are required for pituitary organogenesis and cell line specification [31]. The necessity for extrinsic signals for development of corticotrophs is supported by the demonstration that the expression of POMC in ectoderm explants requires coculture with mesoderm [32]. Direct evidence confirming the necessity of the ventral diencephalon for proper pituitary organogenesis has recently been obtained from examination of mice bearing null mutations in the Nkx2.1 homeobox gene, also known as T/EBP or TTF1 [33]. This gene is not expressed in the oral ectoderm or in the developing pituitary at any time during embryogenesis [34], but Nkx2.1-null mice demonstrate severe defects in the development of the diencephalon and also fail to develop a pituitary gland [33]. The nature of the extrinsic signals that promote pituitary organogenesis and cause cell-line specification is an area of active investigation. A number of secreted factors have been implicated in pituitary development and cell-line specification including bone morphogenic proteins (BMP4, BMP2), fibroblast growth factor 8 (FGF8), Sonic hedgehog (Shh) and Wnt5a [35–37]. A model of coordinate control of anterior pituitary progenitor cell identity, proliferation and differentiation imposed by FGF8 secreted from the dorsally located infundibulum and BMP2 from ventrally located mesenchyme has been proposed [37]. It has been suggested that corticotroph progenitors progress to a definitive corticotroph state only after escaping both FGF8 and BMP2 signaling [37]. While the precise signals and interactions required for pituitary organogenesis and corticotroph specification are incompletely understood, it is clear that signals from the mature hypothalamus like corticotropin releasing hormone (CRH) and vasopressin (AVP) are not required for corticotroph specification. The anterior pituitary appears to develop normally in a Crh-null mouse [38]. Furthermore, deletion of the class III POU transcription Brn-2 results in failed maturation of Avp-, Crh-, and oxytocin producing neurons of the hypothalamus and failed maturation of the posterior pituitary with no apparent defect in the maturation of any anterior pituitary cell type [39].

Adrenocorticotropin

47

Localization of Non-ACTH Peptides with Corticotrophs Many other neuropeptides have been found to be colocalized with ACTH within corticotrophs, although in most cases it is not clear whether this is due to synthesis or binding of the peptide within the cell. Neurophysin (but not vasopressin) colocalizes with ACTH in both normal and adenomatous corticotrophs [40]. Neurophysin immunocytochemical staining is especially prominent in corticotrophs in the zona intermedia which appear to project into the posterior pituitary. Chromogranin A has been described in the majority of corticotroph adenomas, although only a fraction of these patients have elevations of circulating plasma chromogranin A levels [41]. Galanin is present in all normal corticotrophs as well as in the majority of corticotroph adenomas which have been examined [42]. Galanin has also been found in those corticotrophs in the zona intermedia which appear to be migrating into the posterior pituitary. It has also been described in corticotrophs which have undergone Crooke’s hyalinization. Calpastatin, an inhibitor of the calcium-dependent cysteine proteases, calpain I, and calpain II, has been found in all ACTH-containing cells of the anterior pituitary, including those in the median wedge as well as those extending into the posterior pituitary [43]. Vasoactive intestinal peptide has been found in corticotroph adenomas, but only rarely in normal corticotrophs [44]. Normal corticotrophs contain small amounts of cytokeratin, whereas corticotrophs which have undergone Crooke’s hyalinization are markedly positive for this protein [45]. Corticotrophs appear to have few structural characteristics associated with neuronal cell types, for they are negative for neurofilament, vimentin, glial fibrillary acidic protein and desmon [45,46]. Likewise, corticotrophs in the zona intermedia, unlike those in the pars intermedia of rodents, appear not to be innervated by neurons, since they do not stain with any of these neuron-specific markers [47]. Similarly, synaptophysin, a 38 kD integral membrane glycoprotein found in presynaptic vesicles of neurons stains only weakly in corticotrophs [48]. These findings, together with a lack in humans of an anatomically discrete intermediate lobe with large numbers of a-MSH-containing cells, has led most investigators to conclude that there is no functional counterpart of the rodent pituitary intermediate lobe in humans.

Extra-pituitary Localization of ACTH and Related Peptides Although the vast majority of ACTH is synthesized in anterior pituitary corticotrophs, it is also expressed in several nonpituitary human tissues, both within and outside of the central nervous system. Within the central nervous system, ACTH and its related peptides are expressed to the greatest degree in cell bodies of the infundibular nucleus of the basal hypothalamus (analogous to the rodent arcuate nucleus) [49–51]. (See POMC Biosynthesis and Processing,

48

SECTION 1

Hypothalamic–Pituitary Function

pp. 52–53.) The cell bodies of these hypothalamic ACTH neurons are located at the base of the third ventricle, adjacent to the median eminence and pituitary stalk. These neurons project to limbic, diencephalic, mesencephalic, and amygdaloid sites [52]. Lesser amounts of ACTH and related peptides are found in substantia nigra, periventricular grey matter, and hippocampus [53]. Interestingly, POMC expression in the hypothalamus occurs in areas also known to express the orexigenic neuropeptides NPY and AGRP and project to many of the same hypothalamic targets [54,55]. The rodent arcuate nucleus prominently expresses the leptin receptor, implicating Pomc products in the regulation of appetite and energy homeostasis (see Melanocortin Receptor 4, p. 60) [56,57]. The brain areas containing ACTH also coincide with areas mediating stimulation analgesia, suggesting that expression of ACTH, or another product of the POMC gene (such as b-endorphin) in these sites may regulate pain perception [52]. Outside of the central nervous system, ACTH and other POMC gene products, including a-MSH and b-endorphin, are synthesized in a large number of human tissues, including in descending order of abundance, adrenal, testis, peripheral mononuclear cells, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum [58,59]. In most of these tissues, POMC peptides are translated from truncated messenger RNAs lacking signal peptide sequences, suggesting that they cannot be secreted extracellularly [60,61], raising the question of their functional significance. However, adrenal and testis in addition express full length POMC mRNA, suggesting that these tissues may also secrete POMC-related peptides [58]. Recently, several additional cell types have been shown to produce POMC peptides including monocytes [62], astrocytes [63], gastrin-producing cells of the gastrointestinal tract [64], keratinocytes [65], skin melanocytes [66], and atrial myocytes [67]. GENE STRUCTURE ACTH is derived from a 266 amino acid precursor, proopiomelanocortin (POMC), so named because it encodes opioid, melanotropic, and corticotropic activities [68]. The human POMC gene is a single copy gene located on chromosome 2p23 [69]. It and the highly homologous opioid peptides, preproenkephalin A and preproenkephalin B (dynorphin), are all located on different chromosomes [70]. The human POMC gene is 8 kilobases (kb) long (Fig. 2.1). It consists of a promoter of at least 400 basepairs (bp) at the 5¢ end of the gene, followed by three exons, 86 (exon 1), 152 (exon 2), and 833 (exon 3) bp long, and two introns, 3708 (intron 1) and 2886 (intron 2) bp in length [69]. Exon 1 is not translated. Perhaps because of this, exon 1 of the human and other mammalian POMC genes are less than 50% identical. The initiator methionine is located 20 bp into exon 2, and is followed by a 26 amino acid hydrophobic signal peptide. Except for the signal peptide and 18 amino acids of the amino-terminal glycopeptide, the majority of

the POMC precursor is encoded by exon 3 [71]. Exon 2 is close to 90% identical between the POMC genes of humans and other mammals. Within exon 3 of POMC are located all known peptide products of the gene, including N-terminal glycopeptide, gamma-melanotropin (gammaMSH), joining peptide, a-MSH, ACTH, corticotropin-like intermediate lobe peptide (CLIP), b-lipotropin (b-LPH), b-MSH, and b-endorphin. Within exon 3, the regions encoding the N-terminal glycopeptide, a-MSH, ACTH, and b-endorphin, are greater than 95% identical between the human and other mammals [71]. In contrast, joining peptide, the region between the N-terminal glycopeptide and ACTH, is very poorly conserved among mammals [72], which has suggested to some workers that it does not encode a biologically important function [73].

POMC Gene Promoter Structure The promoter of the human POMC gene contains typical TATA and CAAT boxes 28 and 62 bp, respectively, upstream from the transcription start site. Using in vitro transcription [74] and transfection of the human POMC gene into heterologous cells [75], the POMC gene promoter has been shown to contain DNA elements which mediate increased transcription by cyclic AMP and decreased transcription by glucocorticoids. Although these DNA elements have not been precisely localized in the human POMC gene promoter, they are present with in the 700 bp 5¢ to the transcription start site [75]. The rat Pomc gene promoter has been studied more extensively. Drouin and coworkers, using DNA-mediated gene transfer into transgenic mice and tissue culture cells, found that the DNA sequences needed for corticotroph-specific expression and negative transcriptional regulation by glucocorticoid are contained within 543 bp of the transcription start site. These workers have reported the presence of a DNA sequence which mediates negative regulation by glucocorticoids (nGRE), located in the region of the CAAT box, which also binds nuclear proteins of the chicken ovalbumin upstream promoter (COUP) family. Using DNAase footprint and gel retardation analysis of the rat Pomc gene promoter, multiple synergistic DNA elements have been reported to be necessary for correct pituitaryspecific expression of the gene [76]. Recently, these workers have identified NeuroD, Ptx1, and Tpit as transcription factors which cooperate to cause corticotroph-specific expression of POMC [77].

POMC mRNA Transcription, Splicing, and Polyadenylation In addition to the major transcriptional initiation site present in corticotrophs, at least six other start sites have been found in several non-pituitary tissues (Fig. 2.1), including adrenal, thymus, and testes of the human [60] and rat [78]. These sites are all between 41 and 162 nucleotides downstream from the 5¢ and of exon 3. The mRNAs transcribed from

Chapter 2

Adrenocorticotropin

49

FIGURE 2.1. Structure of pro-opiomelanocortin (POMC) gene, messenger RNA (mRNA), and peptide products. (a) schematic diagram of gene (top), mRNA (middle) and POMC peptide (bottom). The gene consists of the promoter, 3 exons (thick bar, 1–3) separated by two exons (thin broken lines). Translated regions are stippled. The mRNA consists of 3 exons and the polyadenylate (AAAA) tail. The POMC precursor can be divided into 3 domains: I (N-terminal glycopeptide, joining peptide), II (adrenocorticotropic hormone; ACTH); and III (b-lipotropin; b-LPH). (b) Structure of individual products of POMC. Individual peptides are enclosed in different boxes depending on proteolytic cleavage specificities: = similar processing in pituitary and brain; = pituitary pattern of processing; = brain pattern of processing. = dibasic amino acids at proteolytic cleave sites. = glycosylation sites. = N-terminal acetylation sites. CLIP, corticotropin-like intermediate lobe peptide; a-MSH, a-melanocyte stimulating hormone.

these sites thus would be intronless, and the only truncated molecules that might be translated would be devoid of a signal peptide, and therefore could not be secreted [78]. Tissues containing these shorter forms of POMC mRNA, including adrenal, testis, spleen, kidney, ovary, lung, thyroid, and gastrointestinal tract, express ACTH, N-terminal glycopeptide1–61, and b-endorphin [58]. These truncated mRNAs are capable of being translated both in cell-free translation systems and in heterologous cells transfected with the appropriate fragment of the human POMC gene, although the peptide products are not secreted [61]. The significance of the expression of these truncated forms of POMC mRNA and their translated peptide products in human nonpituitary tissue is not clear.

A canonical polyadenylation signal is present in human POMC mRNA 23 bases upstream from the poly (A) addition site [79]. The length of the polyadenylate tail, attached to the 3¢ end of POMC mRNA and believed to play a role in translational efficiency of mRNA stability, is much longer in hypothalamic compared with pituitary cells in the rat [78], although this has not been studied in the human. GENE REGULATION In human anterior pituitary corticotrophs, POMC mRNA levels are increased by CRH and inhibited by glucocorticoids [80]. Similar results are seen in normal and adenoma-

50

SECTION 1

Hypothalamic–Pituitary Function

tous corticotrophs, although the glucocorticoid inhibition of POMC mRNA expression is less in the latter cells [80]. The same results are seen in rat anterior pituitary corticotrophs, where the opposing effects of CRH and glucocorticoids are due to their opposite influence on the transcription rate of the POMC gene [81]. Of interest, glucocorticoids have no effect on the POMC mRNA content of rat intermediate pituitary lobe corticotrophs [81]. CRH, acting via the type 1 CRH receptor [82] increases cAMP content in anterior pituitary corticotrophs [83]. CRH, vasopressin, and glucocotricoids all inhibit expression of CRH receptor 1 mRNA [84], which may limit the effect of these agents during the stress response, as discussed subsequently. The CRH-induced rise in cAMP is responsible for both the increase in POMC transcription and peptide synthesis as well as for the rise in intracellular calcium which results in ACTH secretion [85]. CRH mediates its stimulation of POMC transcription via the POMC CRH responsive element (PCRH-RE), which binds PCRH-RE binding protein [86]. The negative effect of glucocorticoids upon POMC gene transcription is thought to be mediated by a glucocorticoid–glucocorticoid receptor complex binding to cis-acting DNA sequences within the POMC promoter, although definitive evidence for this is lacking. The possibility exists that the glucocorticoid receptor complex does not bind directly to the POMC gene, but instead to another protein such as a positive transcription factor, and in this way mediates its negative effect on POMC gene expression [87]. Glucocorticoid stimulates, rather than inhibits, POMC gene expression [88] in the arcuate nucleus of the hypothalamus, the site of alphaMSH production. The significance of this is not clear, but could be involved in the inhibition of appetite by glucocorticoids in the postabsorptive state, when glucocorticoid levels are high. Recently, new insights into the development and regulation of the HPA axis have come from the discovery that leukemia-inhibitory factor (LIF) has an important role in these events [89]. LIF is a cytokine expressed in corticotrophs and folliculostellate cells beginning as early as 14 weeks gestation [90]. LIF stimulates transcription of POMC and expression of ACTH [90]. In many tissues, including pituitary, LIF expression is unregulated by inflammatory stimuli. The LIF receptor is a member of the class I cytokine receptor superfamily which heterodimerizes with gp130. In common with other family members, the LIF receptor signals through the Jak-STAT pathway, particularly utilizing Jak1 and STAT-3 [91]. As with other receptors coupled via these mediators, SOCS-3 inhibits POMC expression following its simulation by LIF [92]. Mice which overexpress a LIF transgene develop Cushing’s syndrome [93]. Their pituitary glands have corticotroph hyperplasia and multiple Rathke-like cysts lined by ciliated cells. Mice with targeted deletion of LIF have secondary adrenal insufficiency [94]. All of these data point towards LIF playing an important role in the regulation of ACTH secretion, perhaps most

importantly by immune and inflammatory stimuli [95]. In addition, the data suggest that LIF might be involved in the pathogenesis of Rathke-cleft tumors. Vasopressin potentiates the action of CRH on ACTH secretion, both in vitro [96] and in vivo [97]. Vasopressin’s effect is mediated by the vasopressin V1b (or V3) receptor and protein kinase C [98]. Despite this positive effect on ACTH secretion, vasopressin has been reported to decrease both basal and CRH-stimulated POMC mRNA levels in anterior pituitary cells [99]. b-adrenergic catecholamines, like CRH, also increase POMC mRNA levels in corticotrophs via a cAMP mechanism [100]. Insulin-induced hypoglycemia also causes an increase in POMC mRNA content in rat anterior pituitary corticotrophs [101,102], but whether this is secondary to an increase in hypothalamic CRH [101], vasopressin [103], or catecholamines [104] is not known. The inhibitory neurotransmitter GABA causes a decrease in POMC mRNA levels in intermediate, but not anterior pituitary corticotrophs [105]. POMC BIOSYNTHESIS AND PROCESSING The human POMC precursor has the potential to encode several overlapping peptides of biological importance (Fig. 2.1). Within the precursor, these peptides are separated from one another by two or more basic amino acids which serve as recognition sites for prohormone cleavage enzymes. In addition, POMC-derived peptides contain potential signals for amidation, glycosylation, acetylation and phosphorylation. Because the nomenclature of the various proteolytic products of POMC has been derived from both peptidemapping studies as well as molecular biological studies in which putative peptides had been predicted from inspection of nucleotide sequences [72], the terminology can be confusing. To avoid confusion, amino acid (aa) positions in this chapter are numbered as superscripts with reference to the 240 aa-long human POMC precursor, formed after removal of the 26 aa-long signal peptide. The 240 aa POMC precursor can be considered to be composed of three domains (Fig. 2.1). Domain I (aa 1–111), the N-terminal domain, encodes the 76 aa long Nterminal glycopeptide1–76 within its first 78 aa, and the 30 aa long C-terminal joining, or hinge, peptide ( JP79–108) within its last 33 aa. The middle Domain II (aa 112–152), 41 aa long, encodes the 39 aa ACTH112–150 peptide, which may be further processed to a-melanocyte stimulating hormone (MSH)112–124. The C-terminal Domain III, termed b-LPH153–240, is 88 aa long. It contains within it the 31 aa long b-endorphin210–240. Besides these peptides, several other products have been identified, although evidence for their existence and/or biological importance in man is not clear [106]. These include gamma1-MSH51–61, gamma2-MSH51–62, and gamma3-MSH51–76 in Domain I, corticotropin-like intermediate lobe peptide (CLIP)130–150 in Domain II, and gamma-LPH153–206 and b-MSH191–206 in Domain III (Fig. 2.1).

Chapter 2

Glycosylation of POMC Within the endoplasmic reticulum (ER), POMC undergoes initial glycosylation. Human POMC is glycosylated solely at two sites in the N-terminal glycopeptide1–76 of Domain I. Carbohydrate is added via an O-linked glycosylation to Thr45 and via an N-linked glycosylation to Asn65.

C-terminal Amidation of POMC Three human POMC products undergo C-terminal amidation [107]. These include N-terminal glycopeptide1–61, JP79–108, and -MSH112–124. In addition, these three products are also present in their Gly-extended forms, which may be incompletely-processed intermediates. C-terminal amidation is common among neuropeptides, and is usually essential for bioactivity [108]. This reaction is mediated by a bifunctional enzyme consisting of peptidylglycinea-amidating monooxygenese (PAM) and peptidly-ahydroxyglycine a-amidating lyase (PAL) activities, which transfer the amino group of a C-terminal Gly to the carboxyl group of the adjacent amino acid. Human PAM/ PAL exists in both a membrane-bound and free cytoplasmic form [109].

N-terminal Acetylation of POMC Two human POMC products undergo N-terminal acetylation. These are (as described below) a-MSH and bendorphin. In humans a-MSH exists predominantly in the nonacetylated form [110]. N-terminal acetylation of ACTH1–13-amide to form a-MSH results in increased melanotrophic activity [111] and decreased corticotrophic activity.

Proteolytic Processing of POMC POMC gives rise to several smaller, biologically active peptide products. These are generated by posttranslational cleavage of POMC by trypsin-like prohormone convertase endopeptidase enzymes which cleave the precursor on the C-terminal side of regions of two or more basic amino acid residues [112]. These basic amino acids are subsequently removed by a carboxypeptidase activity. Some POMC proteolytic products subsequently undergo amidation at their C-terminus or acetylation at their N-terminus, as described above. The posttranslational processing of POMC exhibits a remarkable degree of tissue-specificity, which recently has been postulated to be due to the differential distribution of processing enzymes in the various tissues which synthesize POMC (see below). Proteolytic Processing Enzymes

All proteolytic processing of human POMC occurs at either lys–arg or arg–arg residues (Fig. 2.1). Every lys–arg and arg– arg site within the human precursor is capable of being cleaved in vivo [106], whereas in the mouse and rat, additional arg–lys and lys–lys sequences at the N-termini of

Adrenocorticotropin

51

gamma1-MSH and b-MSH, respectively, appear to be utilized [106]. It is likely that the proteolytic digestion of human POMC at all sites is mediated by either of two structurally related endopeptidases, prohormone convertase 1 (PC1) [113] or prohormone convertase 2 (PC2) [113]. These enzymes, best studied in rodents thus far, are part of a seven member family of subtilisin/kexin-like mammalian proteinases [114], and are distributed specifically within endocrine cells and neurons. Both enzymes are capable of cleaving neuropeptide precursors, including POMC, proinsulin, and proglucagon, at dibasic sites, and each appears to manifest distinct preferences for different sites within the same precursor prohormone, with PC2 able to cleave at a wider selection of available dibasic sites than PC1 [115]. The tissue distributions of PC1 and PC2 mRNAs are distinctly different. PC1 is abundant in approximately 20% of anterior pituitary cells (presumably including corticotrophs), in all intermediate lobe pituitary cells (of the rodent), and in the supraoptic nucleus of the hypothalamus [116]. In contrast, PC2 is absent from the anterior pituitary, but is highly expressed in rodent pituitary intermediate lobe, multiple sites within the central nervous system, including cerebral cortex, hippocampus, and thalamus, and in pancreatic islet cells [116]. The differential tissue-specific distribution of these enzymes matches nicely with the known tissue-specific differences in POMC proteolytic processing (see below), suggesting that PC1 is responsible for the POMC cleavage products found in anterior pituitary corticotrophs, whereas PC2 cleaves POMC in pituitary intermediate lobe (of lower mammals) and in the brain. This suggestion is supported by studies of the differential processing of POMC by PC1 and PC2 [115,116]. Recently, a patient with severe childhood obesity and hyperproinsulinemia with postprandial hypoglycemia has been identified with compound heterozygous mutations in the PC1 gene [117]. The patient presented with multiple endocrine abnormalities including impaired glucose tolerance and postprandial hypoglycemia which has been attributed to the secretion of large amounts of proinsulin given its partial insulin-like action and long biological half-life. She also suffered from hypogonadotropic hypogonadism with primary amenorrhea, but otherwise normal development of secondary sexual characteristics. Ovulation was induced with exogenous gonadotropins, and the patient delivered healthy quadruplets. The pregnancy was complicated by gestational diabetes requiring insulin treatment. She also suffered from mild adrenal cortical insufficiency with complaints of fatigue and excessive daytime somnolence reversed by glucocorticoid administration. The adrenal cortical insufficiency was attributed to defective POMC processing with elevated levels of serum ACTH precursors confirming a role for PC1 in human POMC processing [117]. Following the generation of peptide products by prohormone convertase enzymes, the C-terminal basic amino acids are removed by carboxypeptidase activities. Carboxypeptidase E (Cpe) is required for the excision of paired

52

SECTION 1

Hypothalamic–Pituitary Function

dibasic residues of various peptide prohormone intermediates, including those derived from proinsulin and POMC [118]. The mutation in a strain of hyperproinsulinemic, late-onset obese mouse, the fat/fat mouse, has recently been mapped to the carboxypeptidase E gene [118]. A missense mutation has been identified in the Cpefat allele and these mice demonstrated a 20-fold decrease in Cpe enzymatic activity in pituitaries and isolated islets. The sorting of POMC into secretory granules is probably mediated by a signal “patch” [119], located within the tertiary structure of the molecule, which directs it to the granules. The presence of all POMC processed products in equimolar amounts within secretory granules suggests that this sorting precedes proteolytic cleavage of POMC. This suggestion is supported by data which demonstrates that initiation of proteolytic processing of POMC begins in the trans-Golgi system and continues in secretory vesicles, and is also consistent with data demonstrating localization of PC1 and PC2 within the TGN and/or dense core secretory granules [120]. Since POMC as well as PC1 and PC2 all contain signal peptides and are presumably colocalized throughout the endoplasmic reticulum, prohormone convertase activity must be inhibited in the endoplasmic reticulum and cis-Golgi regions. Proconvertase activity may be regulated by the local intracellular environment of these compartments and/or by control of catalytic activation of prohormone convertase. 7B2 is a small, hydrophobic acidic protein originally isolated from porcine and human anterior pituitary glands [121] that is widely distributed in neuroendocrine tissues [122] and found to associate specifically with PC2 [123]. The 27-kDa 7B2 precursor protein is cleaved to a 21-kDa protein and a small carboxy-terminal peptide (CT peptide) [124]. Interaction of 7B2, particularly the 21-kDa fragment, with proPC2 appears necessary for the generation of mature and active PC2 in the trans-Golgi region [125]. The 7B2 CT peptide is a nanomolar inhibitor of PC2 in vitro, but its role in vivo has not been defined. The role of 7B2 in activating proPC2 has been confirmed in vivo in 7B2-null mice [126]. 7B2-null mice are devoid of PC2 activity and deficiently process islet hormones and Pomc. The mice are hypoglycemic and demonstrate hyperproinsulinemia and hypoglucagonemia. These mice also demonstrate profound intermediate lobe ACTH hypersecretion with minimal conversion of this peptide to a-MSH, resulting in a severe Cushing’s syndrome that causes death by 9 weeks of age [126]. Curiously, PC2 null mice demonstrate similar islet cell dysfunction resulting in hypoglycemia, but do not abnormally produce a Cushingnoid syndrome [127]. This discrepancy suggests additional functional roles for 7B2, which are further suggested by the localization of 7B2 in regions of the brain lacking PC2 [128]. Recently, the suggestion that 7B2 may represent one member of a family of related convertase inhibitor proteins has been proposed with the identification of the protein proSAAS [129]. ProSAAS is a 26 kDa granin-like neuroen-

docrine peptide precursor isolated from rodents and humans with structural similarity to 7B2, including a proline-rich sequence in the first half of the molecule and a C-terminal peptide (SAAS CT peptide) following a dibasic cleavage sequence [129]. Overexpression of proSAAS in AtT-20 cells reduces the rate of Pomc processing and the SAAS CT peptide is a nanomolar competitive inhibitor of PC1, but not PC2 [129]. Tissue Specificity of POMC Processing

In human corticotrophs, POMC is processed predominantly into N-terminal glycopeptide1–76, joining peptide ( JP)79–108, ACTH112–150, and b-LPH153–240 (Fig. 2.1) [130]. Much smaller amounts of a-MSH112–124, CLIP130–150, bendorphin210–240, and a truncated form of N-terminal glycopeptide1–61, also known as “big” gamma-MSH, are also present [107]. There is no evidence for cleavage after arg50, and therefore no evidence for the presence of gamma1MSH51–61 in the human pituitary. JP79–108 exists as both a monomer and homodimer, most likely joined via disulfide bonding between the single Cys87 of two molecules [73]. Although JP79–108 has been postulated to stimulate adrenal androgen steroidogenesis [131], some studies do not support this hypothesis [73]. A function for N-terminal glycopeptide has also not been assigned, although one intriguing study reported that it is capable of stimulating aldosterone release from adrenal cells [130]. Whereas the production of distinct POMC peptide derivatives is clearly segregated between the anterior and intermediate lobes of the rodent, in the human, small, acetylated POMC peptide derivatives colocalize with larger desacetylated POMC peptides in corticotrophs of the anterior pituitary, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in rodents and other species does not extend to human pituitaries [132]. Levels of desacetyl-a-MSH are elevated in pituitary corticotrophs [133] and plasma [134] of patients with Addison’s disease, Cushing’s disease, and Nelson’s syndrome. Desacetyla-MSH has approximately 75% of the melanotrophic activity as does a-MSH [111], whereas ACTH is only 5% as potent as a-MSH in this regard [111,135]. Because the serum levels of ACTH are 50- to 100-fold higher than levels of desacetyl-a-MSH in patients with Cushing’s disease, Addison’s disease, and Nelson’s syndrome, it is likely that the hyperpigmentation associated with these disorders is largely due to the melanotrophic effect of ACTH, and not MSH. Thus, in human anterior pituitary corticotrophs, the POMC precursor is predominantly cleaved at limited lys–arg sites into two peptides in Domain I (N-terminal glycopeptide1–76 and JP79–108), one peptide in Domain II (ACTH112–150) and one peptide in Domain III (b-LPH153–240) (see Fig. 2.1). As discussed above, human POMC is also expressed in several brain sites outside of the anterior pituitary, predominantly in the arcuate nucleus of the anterior hypothalamus. In these extra-pituitary locations, POMC is

Chapter 2

processed to a greater extent than in anterior pituitary. In brain, ACTH112–150 is cleaved to a-MSH112–124 and CLIP130–150 such that the amount of a-MSH relative to ACTH is 300-fold higher in hypothalamus, telencephalon, and mesencephalon than it is in anterior pituitary [136]. As in anterior pituitary, a-MSH is almost exclusively present in the desacetyl form [50]. Adding additional levels of complexity to the issue of tissue specificity of POMC processing are results from a dopamine D2 receptor (D2R) deficient mouse. Similar to dopaminergic tonic inhibitory control of prolactin expression in lactotrophs, POMC expression in the rodent intermediate lobe is under inhibitory dopaminergic control mediated via D2 receptors [137]. D2R-deficient mice demonstrate mild intermediate lobe hyperplasia accompanied by upregulation of both PC1 and PC2 [138]. These mice present with unexpectedly high levels of ACTH with corresponding adrenal hypertrophy and increases in corticosteroid secretion [138]. The altered prohormone convertase levels in these mice suggest the possibility of dynamic regulation of prohormone processing within specific tissues. HORMONE MEASUREMENT ACTH was one of the first substances to be measured by radioimmunoassay (RIA) [139]. In pioneering work using polyclonal antisera, Berson and Yalow described the measurement of ACTH and related peptides in normal persons and those with ectopic ACTH production by lung cancer [140]. These studies provided among the first data that ACTH was synthesized from larger precursors, which were termed “big” and “big–big” ACTH. The ACTH RIA can be performed on unextracted plasma, but suffers from a limit of sensitivity of approximately 25 pg/ml, and is therefore often unable to detect levels of plasma ACTH in the normal basal range. Radioimmunoassay remained the standard method for the measurement of plasma ACTH until the development of the immunoradiometric assay (IRMA). ACTH IRMAs employ two antibodies, one or both monoclonal, against ACTH. The solution-phase antibody is radiolabeled, and the solid-phase antibody is linked to a bead or other solid support. In general, the ACTH IRMA on unextracted plasma compares very favorably with RIA [141], being more sensitive, more reproducible, and more rapid [142]. Most IRMAs have lower limits of detection of 3–5 pg/ml and coefficients of variation of less than 10% up to 5000 pg/ml [141,142]. Depending on the antigenic specificity of the chosen antibodies, the ACTH IRMA may detect only intact ACTH [141], both ACTH and POMC precursor peptides [143,144], or only POMC precursors [145]. It is essential to know the sequence specificity of any IRMA in clinical use, for some, unlike most polyclonal RIAs, are incapable of detecting ACTH precursors which may be secreted by lung carcinomas [143]. Despite the wide availability of the ACTH IRMA, results from different laboratories are diffi-

Adrenocorticotropin

53

cult to compare because of lack of agreement on a suitable ACTH reference standard. An immunofluorometric assay (IFMA) for ACTH has also been developed [146]. Its sensitivity and accuracy are similar to those of the IRMA. Its principal advantage is its speed and the use of a nonradioactive label which is stable for over 1 year. Plasma assay of POMC products other than ACTH, including b-endorphin [147] and N-terminal glycopeptide, has been suggested as an adjunct in the evaluation of the hypothalamic-pituitary-adrenal (HPA) axis. In general, the levels of these hormones parallel that of ACTH in various HPA axis abnormalities [148]. However, except for use as a screening test for lung carcinoma associated with preferential secretion of N-terminal glycopeptide1–61 or proACTH1–150 [148], such tests are much less helpful than the ACTH IRMA because of the low concentration of other POMC peptide fragments compared with ACTH in most physiologic and pathologic settings. SECRETION OF ACTH: BIOCHEMISTRY AND PHARMACOLOGY Secretion of ACTH from the corticotrophs of the anterior pituitary is mediated by several factors (Fig. 2.2). CRH and vasopressin are the primary secretagogues for ACTH, although a number of other agents may also affect its release, and glucocorticoids are the major negative regulators of ACTH secretion. Once a ligand has bound to its receptor, release of ACTH from the corticortroph is mediated by second messengers through one of four signal transduction pathways, involving either protein kinase A, protein kinase C, glucocorticoids, or the Janus kinase/STAT system. (This

–

CRH

AVP

Catecholamines IL1β IL-6 TNFα

+

–

Cortisol

ACTH

Adrenal

+

+

LIF IL-6 IL1β TNFα

CRH IL1β IL-6 TNFα

FIGURE 2.2. Control of adrenocorticotropic hormone synthesis and secretion.

54

SECTION 1

Hypothalamic–Pituitary Function

last pathway, regulated by LIF, was described in a previous section, p. 50.) These pathways result in changes in the phosphorylation pattern of specific cellular proteins, and/ or in intracellular calcium levels, impacting on ACTH synthesis and release. Circulating ACTH then binds to receptors, primarily in the adrenal gland, leading to steroid biosynthesis.

Second Messenger Regulation of ACTH Secretion Protein Kinase A Pathway

CRH stimulates ACTH release via the cAMP-protein kinase A pathway. CRH stimulates adenylate cyclase activity, which then increases cAMP [83]. Forskolin, a direct stimulator of adenylate cyclase activity, and 8-bromo-cAMP, a cAMP analog, both markedly stimulate ACTH release, and increase CRH-stimulated ACTH release, but do not potentiate ACTH release from cells maximally stimulated by CRH [149]. The CRH-stimulated increase in cAMP activates cAMP-dependent protein kinase A [83]. Protein Kinase C Pathway

AVP V1b and V2 receptors are coupled to different second messenger systems. V1 receptors in the pituitary mobilize calcium through activation of phospholipase C, whereas V2 receptors activate adenylate cyclase, increasing cAMP [150]. AVP-stimulated ACTH release is mediated via the protein kinase C pathway [151]. Evidence that AVP acts through the protein kinase C pathway in the anterior pituitary includes the finding that AVP stimulates accumulation of inositol phosphates in rat anterior pituitary cells [152]. Phorbol esters, which activate protein kinase C directly by substituting for diacylglycerol and binding to protein kinase, induce ACTH release in rat pituitary cell cultures. Activation of protein kinase C appears more important in the sustained phase of AVP-stimulated ACTH release, not in the initial phase [153].

CRH CRH Stimulation of ACTH Secretion

Perfused human fetal pituitaries and cultured human fetal pituitary cells in culture secrete ACTH in response to CRH [2]. Rats have been used extensively in the study of ACTH secretion, as it is difficult to culture human corticotroph cells in vitro. In dispersed rat anterior pituitary cells, CRH stimulates a ninefold increase in ACTH release that is sustained for as long as the cells are exposed to CRH [83]. Even with maximal CRH stimulation, only 6% of cultured pituitary cells release ACTH [154]. CRH stimulates ACTH synthesis as well as release. In humans, the biphasic response may reflect secretion of a ready pool of ACTH, followed by later release of newly synthesized protein [155]. Exposing cells to CRH for a long period of time results in an increase in ACTH in the cell and in the medium. Thus, the sustained phase of ACTH

secretion may represent later release of newly synthesized ACTH peptide. Most of the CRH released into the hypophyseal blood is derived from the PVN [156]. Concentrations of oCRH that are similar to the concentrations of CRH found in rat portal plasma have been shown to increase secretion of ACTH in human fetal hemipituitaries in vitro [157]. The CRH content in the hypophyseal portal blood of anesthetized male rats is about 100 pM [158], which exceeds the in vitro threshold of 10–20 pM CRH to stimulate ACTH release [159]. In addition to stimulating ACTH expression and release, CRH can also directly stimulate secretion of glucocorticoids from the adrenal gland [160]. CRH Receptors

In human pituitaries, CRH has been shown to bind to sites in the anterior lobe [161]. The distribution of binding sites in human pituitary correlates with the distribution of corticotrophs [161]. CRH receptors in the anterior pituitary gland are low capacity, high affinity receptors, with a Kd for CRH binding of about 1 nM [162]. To date, two CRH receptor genes have been identified in humans and other mammals [163–165], with a third additional one being described in the catfish [166]. The type 1 receptor [163,165] is expressed predominantly in anterior pituitary corticotroph cells, whereas the type 2 receptor [164] is more widely distributed in the brain and periphery, particularly in cardiovascular tissue. The type 1 receptor binds and is activated by both CRH as well as the CRH-like peptide, urocortin [167]. This receptor mediates the actions of CRH at the corticoctroph. In addition, the type 1 receptor mediates fear and anxiety behaviors following stressors, even in CRHdeficient mice [168]. Mice with deletion of the CRH type 1 receptor gene show reduced fear and anxiety [169]. These data suggest that a CRH-related peptide, possibly urocortin or another unknown member of the CRH family, mediates fear responses via the CRH type 1 receptor. The CRH type 2 receptor binds urocortin with over 20-fold higher affinity compared to CRH. Its distribution, along with the hypotensive cardiovascular response to infused urocortin which is abolished in CRH type 2 receptor-deficient mice [170], suggests that the receptor may be involved in blood pressure control. This may underlie the hypotension observed during the CRH stimulation test. Recently, a nonpeptide, type 1-specific CRH antagonist has been developed which is orally active [171,172]. It should help to elucidate the role of CRH in ACTH regulation and other pathways.

AVP AVP Stimulation of ACTH Secretion

In rat anterior pituitary cells, AVP causes a twofold increase in ACTH release [173]. AVP elicits an initial rapid release of ACTH, observable within 5 seconds, reaching a maximum in less than 1 to 2 minutes, and lasting less than 3 to 6 minutes. This is followed by a second phase lasting

Chapter 2

for several hours. In humans, AVP infusion by itself has only a small effect on ACTH release. AVP is synthesized in the same parvocellular hypothalamic paraventricular nuclear neurons which express CRH, and appears to be coreleased with CRH at the median eminence into the portal hypophyseal system [174]. However, a substantial amount of AVP in the portal blood is released from projections from the supraoptic nuclei in the median eminence [156]. AVP from the posterior pituitary may also reach the anterior pituitary through portal vessels that connect the two [175]. This raises the possibility that increased vasopressin secretion from the posterior pituitary, in response to hyperosmolality, may also stimulate ACTH secretion. AVP Receptors

A single population of specific AVP receptors have been identified in rat anterior pituitaries, which are distinct from CRH receptors [176]. Most corticotrophs have AVP receptors, since 80% of the ACTH-secreting cells in the pituitary bind AVP [176]. The anterior pituitary AVP receptors are distinct from the V2 renal receptors and the V1 hepatic/pressor receptors [150]. This has led to the classification of hepatic/pressor receptors as V1a receptors and anterior pituitary receptors as V1b [150], or V3, receptors. V1a, V1b, and V2 receptors can be distinguished by their patterns of recognition of AVP analogues [177]. V1a binding sites in the rat anterior pituitary have a Kd of about 1 nM, and the minimal effective dose of AVP is 0.1 nM [173]. dDAVP (desmopressin), an AVP analog with V2 receptor affinity, has an insignificant effect on plasma ACTH levels, though it does increase, but is not additive to, CRHstimulated ACTH release [178]. During the past 6 years, the genes for all three vasopressin receptors (V1a, V2, and V1b) have been identified [179]. They are highly related members of the 7 transmembrane, G protein coupled, receptor family. As anticipated, V1b receptor mRNA is highly expressed in anterior pituitary corticotrophs, and is coupled to stimulation of POMC gene expression and ACTH secretion via the protein kinase C pathway [180,181].

Modulators of CRH and AVP Release CRH is the most important physiologic ACTH secretagogue [182]. Stressors, endogenous circadiam rhythms, and glucocorticoids influence CRH release [183]. In the rat, afferent inputs to the PVN may mediate the action of stressors by controlling the release of CRH [183]. Sources of neuronal afferents to the hypothalamus include the amygdala and hippocampus of the limbic system, and brainstem regions involved in autonomic functions [184]. In rats, acetylcholine, norepinephrine, angiotensin II, and possibly CRH itself, increase CRH concentrations in the hypophyseal portal plasma. On the other hand, AVP, b-endorphin, and GABA inhibit the ACTH response to stress [182,185–187].

Adrenocorticotropin

55

In rodents, central catecholamines stimulate ACTH release, and the effects of catecholamines on ACTH secretion appear to be mediated via secretion of CRH, and possibly AVP, into the hypophyseal portal circulation [188]. However, in humans, catecholamines have little direct effect on ACTH secretion from the pituitary. Peripheral catecholamines, increased by a variety of stresses, do not cross the blood–brain barrier to reach the hypothalamus, but do reach the pituitary, yet do not increase basal or CRHstimulated plasma ACTH levels. This suggests that the increased peripheral levels of epinephrine and norepinephrine generated during stress are probably not responsible for the increase in ACTH, and that catecholamines do not act directly on the pituitary to stimulate ACTH release [189]. In humans and other intact mammals, the impact of angiotensin II on basal or CRH-stimulated ACTH release is unclear. In humans, infusion of angiotensin II alone does not increase ACTH release [178]. However, in dispersed rat anterior pituitary cells, angiotensin II does stimulate release of ACTH [190]. Angiotensin II has a synergistic effect with CRH in stimulating ACTH release in humans in vivo [178]. Angiotensin II potentiates CRH-stimulate ACTH release from cultured anterior pituitary cells [191], although it is less effective than AVP, and potentiates the CRH-stimulated increase in cAMP. Angiotensin II and AVP do not potentiate each other’s effect on ACTH release, suggesting that they act via the same mechanism. Interleukin-1 beta stimulates ACTH release in conscious rats by acting on the hypothalamus [192] to induce secretion of CRH [193,194]. Interleukin-1 does not cross the blood–brain barrier [195], but activates noradrenergic neurons in the brainstem and hypothalamus, which may stimulate CRH secretion, especially in the median eminence. Prostaglandins may be involved in the response to interleukin-1, since ibuprofen, which blocks the formation of prostaglandins, blocks endotoxin-induced ACTH release in humans [196]. Tumor necrosis factor is a potent secretagogue for ACTH, and when administered to human subjects, leads to an increase in ACTH, cortisol, and AVP [197], but inhibits CRH-, AVP-, and angiotensin II-stimulated ACTH secretion [198]. Tumor necrosis factor may stimulate ACTH secretion by stimulating CRH release from the hypothalamus [199]. However, there is evidence that the site of action of tumor necrosis factor is peripheral to the pituitary and hypothalamus [199]. Bacterial endotoxin, when administered to human subjects, increases ACTH, cortisol, and AVP release [196]. Tumor necrosis factor may mediate the hormonal responses to endotoxin, since tumor necrosis factor levels increase after endotoxin administration [200]. Endotoxin does not increase ACTH release from cultured rat adenohypophyseal cells [201]. In rats and humans, interleukin-6 leads to ACTH secretion via CRH-dependent [202] and CRH-independent pathways, most likely via a prostaglandin-dependent pathway [203]. GABA inhibits the secretion ACTH by inhibiting

56

SECTION 1

Hypothalamic–Pituitary Function

the release of CRH and possibly AVP [184]. GABAnergic inputs into the PVN of the hypothalamus have been characterized in animals, including inputs from the hippocampus [184]. Opiates and opiate agonists decrease ACTH secretion, and may tonically inhibit ACTH secretion [204–211], although some studies demonstrate no effect of naloxone, an opiate antagonist, on the basal levels of cortisol [212,213]. Opiates inhibit CRH- and AVP-stimulated ACTH release, and different opiate agonists differentially effect CRH- versus AVP-stimulated release [214]. In humans, morphine blunts CRH-stimulated ACTH release without decreasing AVP or catecholamine levels [214]. Modulation of CRH-stimulated ACTH secretion by opiates most likely occurs at a level above the corticotroph [214]. Met-enkephalin analogs inhibit ACTH secretion, and controversy exists as to whether inhibition occurs at the hypothalamus or pituitary. In humans CRH-induced ACTH release is almost completely abolished with pretreatment with a met-enkephalin analog [215].

Synergism Between CRH and AVP Alone, AVP is a less important physiologic ACTH secretagogue than CRH [182]. AVP potentiates CRH-stimulated release of ACTH from cultivated rat anterior pituitary cells by twofold. When cells are first exposed to CRH, reaching the sustained phase of CRH-stimulated ACTH release, and AVP is then added, the usual initial spike of AVP-stimulated ACTH secretion is superimposed on top of the CRHinduced plateau of secretion. Despite continued exposure to AVP, ACTH secretion decreases down to the level of the plateau phase of CRH-stimulated secretion [216]. In cells exposed to AVP before CRH, CRH does not potentiate AVP-stimulated ACTH secretion [216]. This, together with the presence of AVP and CRH in the same parvocellular neurons of the PVN, suggests that the two neuropeptides cooperate to regulate ACTH release. However, this remains to be proven in humans, as there are no examples of persons with defects in AVP synthesis or release who have impaired ACTH secretion.

Oxytocin In humans, low-dose oxytocin perfusion decreases plasma ACTH and cortisol levels [217], and infusion of oxytocin completely inhibits CRH-stimulated ACTH release [218]. Oxytocin acts via a similar mechanism as AVP. Oxytocin binds competitively to AVP receptors in the anterior pituitary [219], but is much weaker than AVP at stimulating ACTH release [220]. Like AVP, oxytocin stimulates ACTH secretion through the protein kinase C pathway.

Glucocorticoids Glucocorticoids are the primary negative regulators of ACTH secretion. Glucocorticoids act on corticotrophs to

inhibit the secretion of ACTH induced by AVP and CRH, the synergism between CRH and AVP, and substances that provoke production of inositol phosphates and cAMP. Glucocorticoids’ negative impact on ACTH regulation is also due to their inhibition of the principal stimulators of ACTH, CRH and AVP. In the rat, glucocorticoid receptors are widely distributed in the brain, including the PVN. The PVN is a site for glucocorticoid negative feedback, since dexamethasone decreases the amount of basal CRH in the hypothalami explant and in the PVN, and the CRH response to secretagogues like serotonin and to stress. Glucocorticoids inhibit CRH release and decrease intracellular CRH in the rat PVN [182], and dexamethasone has a local effect on the hypothalamus, decreasing CRH mRNA expression [221], and preventing the rise in intracellular of CRH and AVP usually seen after adrenalectomy [183]. Glucocorticoids increase, and adrenalectomy decreases, the amount of GABA in the hypothalamus and the hippocampus [222]. Glucocorticoids appear to feedback to increase the GABA activity of the hippocampus and hypothalamus, and thus inhibiting CRH release [222]. In the anterior pituitary, glucocorticoid inhibition of ACTH secretion in vitro is mediated via glucocorticoid receptors, and lack of glucocorticoid effect in the intermediate lobe of the pituitary is most likely because functional receptors are not present in these cells. The negative effect of glucocorticoids on CRH-, AVP-, angiotensin II-, and norepinephrinestimulated ACTH release [223] is biphasic, which may reflect an initial inhibition of ACTH release, followed by an inhibition of POMC biosynthesis. Glucocorticoids inhibit POMC secretion, gene transcription and mRNA levels in the anterior pituitary. EFFECTS OF POMC-DERIVED PEPTIDES IN SKIN AND ADRENAL The melanocortin receptors are a family of seven transmembrane-spanning, G-protein coupled receptors that are activated by melanocortin derivatives of POMC including a-MSH and ACTH. Activation of all five receptors results in adenylate cyclase activity and cAMP production. Cloning of the melanocyte MSH receptor (melanocortin 1 receptor–MC1R) [224,225] and the adrenal ACTH receptor (melanocortin 2 receptor–MC2R) [225] were quickly followed by the cloning of three additional family members. The five known melanocortin receptors show distinct tissue distributions throughout the nervous system and periphery and distinct selectivities for the various melanocortin peptides. Prior to the cloning of this receptor family, the actions of melanocortins were primarily known through the effects of MSH on pigmentation and the effects of ACTH on glucocorticoid secretion from the adrenals. However, many additional roles including cognitive and behavioral effects, effects on the immune system, and effects on the cardiovascular system have also been attributed to the melanocortins.

Chapter 2

With the cloning of this family of receptors, the physiologic roles of ACTH, MSH and other melanocortin derivatives are beginning to be elucidated.

Ligand Specificity The pharmacology of melanocortin receptor activation with a large number of natural and synthetic melanocortin peptides is the subject of extensive investigation. All five melanocortin receptors are activated by ACTH. However, MC2R binds only ACTH and is not activated by other melanocortin peptides [226]. The synthetic agonist 4norleucine, 7-D-phenylalanine-a-MSH (NDP-MSH) is the most potent agonist of MC1R, MC3R, MC4R, and MC5R [227,228]. The endogenous non-ACTH melanocortin peptides generally bind the melanocortin receptors with an order of potency MC1R > MC3R > MC4R > MC5R when expressed in COS cells and measurements are obtained in competition with NDP-MSH [228–232]. g-MSH is relatively selective for MC3R over MC4R and MC5R [228,233]. Whether the differences in melanocortin receptor specificity for different melanocortin ligands are physiologically relevant are unknown. Interestingly, Org 2766 and BIM 22015, two ACTH4-10 analogs, have no activity at any of the cloned MCRs, but have potent effects on central and peripheral nervous systems suggesting the possibility of undiscovered melanocortin receptors [234,235].

Melanocortin 1 Receptor The MC1R was initially cloned from primary melanoma tumors [224,236]. MC1R gene expression has also been confirmed in primary human melanocytes by northern blotting [236]. Other cutaneous cells including keratinocytes [237] and dermal fibroblasts [238] have also been reported to express MC1R, although the presence of MC1R mRNA [239] and MC1R protein [240] in keratinocytes could not be confirmed in other studies. Recent evidence demonstrates that the MC1R is expressed in keratinocytes and that its expression is induced by calcium and UV light treatments [241]. Corresponding to its expression in the skin, its function is perhaps most fully understood with respect to its role in cutaneous pigmentation (for review see [242]). Activation of melanocyte MC1R, via activation of adenylate cyclase, stimulates tyrosinase activity, the rate-limiting enzyme in melanogenesis [239]. The activation of tyrosinase results in an increased proportion of eumelanin (brown–black pigment) formation over pheomelanin (red–yellow pigment) formation resulting in increased pigmentation [243]. Mutations and variant alleles in the MC1R gene have been linked to variation in mammalian pigmentation. The extension locus has long been known to regulate pigmentation in mammalian species. The extension locus has been shown to encode the mouse MC1R [244]. The recessive yellow allele (e) at this locus results from a frameshift that produces a prematurely terminated, nonfunctioning recep-

Adrenocorticotropin

57

tor [244]. The sombre (Eso and Eso–3j) alleles and tobacco darkening (Etob) alleles, which have dominant melanizing effects, result from point mutations that produce constitutively active or hyperactive receptors [244]. The human MC1R is highly polymorphic, and mutant alleles have been associated with fair skin and blond or red hair [245–247]. Analysis of five naturally occurring common variants of MC1R associated with fair skin and red hair have revealed decreased stimulation of cAMP synthesis with no changes in or only slightly reduced aMSH binding [248]. In addition, MC1R variants have recently been shown to determine sun-sensitivity in humans with dark hair [249]. Melanocytes, which express POMC and several of its processed peptides [250,251], are derived from the neural crest [29]. It is likely that a-MSH produced via POMC expression in melanocytes is responsible for the pigmenting effects of ultraviolet light, and together with variations in MC1R discussed above, for the different degrees of skin pigmentation observed among ethnic groups. MC1R mRNA [252] and protein [253] have been detected in Leydig cells of the testis, luteal cells of the corpus luteum, and in the placenta. In the central nervous system, detection of MC1R mRNA and protein has been confined to a few scattered neurons of the periaqueductal grey in rat and human brains [254]. With the cloning of the melanocortin receptors, the role of melanocortins as anti-inflammatory agents has gained renewed interest [255–257]. Systemic administration of a-MSH has been shown to be beneficial in animal models of arthritis [258], adult respiratory distress syndrome (ARDS) [259], and septic shock [260]. Consistent with this role, MC1R expression has been documented on macrophages and monocytes [261,262]. Treatment of activated macrophages with a-MSH resulted in decreased nitric oxide production by inhibiting the induction of nitric oxide synthase II [262]. Lipopolysaccharides (LPS) and interferon treatments of neutrophils induced increases in neutrophil MC1R mRNA and treatment with a-MSH inhibited neutrophil chemotaxis by a cAMP dependent process [263]. Constitutive expression of MC1R has also been found on dermal microvascular endothelial cells where its expression can be increased by stimulation with IL-1b or a-MSH itself [264]. A potential role for melanocortins to regulate local inflammation in the brain has been proposed based on the evidence that tumor necrosis factor (TNF)-alpha secretion by an anaplastic astrocytoma cell line (A-172) is decreased by aMSH and that these cells express MC1R [63].

Melanocortin 2 Receptor—the ACTH Receptor Eary in the study of receptor biology, Haynes demonstrated the action of ACTH in generating cyclic adenosine monophosphate (cAMP) in adrenal cells [265]. The ACTH receptor was the first receptor that was shown to bind its ligand with high affinity and specificity [266]. In human adrenal glands, the ACTH receptor has a Kd of 1.6 nmol/l

58

SECTION 1

Hypothalamic–Pituitary Function

and about 3500 sites/cell [267]. The ED50 of ACTH for cAMP production is 0.11 nmol/l, 20-fold less than the Kd for binding [267]. The ED50 of ACTH for cortisol production is 2 pmol/l, 720-fold less than the Kd for ACTH binding, and 35-fold less than the concentration of ACTH needed to obtain a half-maximal increase in cAMP production [267]. Only a small percentage of ACTH receptors need to be occupied to achieve a maximal effect on steroidogenesis, which occurs at an ACTH concentration of 0.01 nmol/l [268,269]. Because the adrenal gland may express more than one of the five melanocortin receptors (see below) it is not possible to assign the biochemical characterizations to a specific melanocortin receptor. The human ACTH receptor (MC2R) was cloned based on its homology to MC1R [225]. In situ hybridization with the adrenal gland of the rhesus monkey demonstrated the presence of messenger RNA in the zona glomerulosa and fasciculata cells, and a weaker signal in the zona reticularis [225]. Initial attempts at characterizing the ligand specificity and cAMP signal generation in response to ACTH and other melanocortins was confounded by either poor levels of expression or the presence of endogenous melanocortin receptors in transfected cells [225,270,271]. Recently, the human MC2R has been stably transfected into the Y6 cell line, a mutant derived from the mouse Y1 adrenocortical cell line, that fails to express any endogenous MC2R [272]. Y6 cells alone demonstrated no cAMP response to micromolar ACTH, and MC2R transfected cells displayed an EC50 of 6.8 nmol/l [272].

Adrenal Actions of Acth The critical role of ACTH in regulating the synthesis and secretion of steroids from the adrenal gland has long been recognized. In adrenocortical cells, ACTH regulates lipoprotein uptake by receptor-mediated endocytosis from the plasma to lipid droplets by stimulation of lipoprotein receptors [273]. Within the lipid droplets, ACTH regulates hydrolysis of cholesterol esters by activation of cholesterol esterases or suppression of cholesterol acyltransferase, through cAMP-dependent protein kinase [274]. ACTH stimulates the transport of cholesterol to the mitochondria [275–277], principally via stimulation of steroidogenic acute regulatory protein (StAR) [278]. The rate-limiting step in steroidogenesis is the side-chain cleavage of cholesterol to pregnenolone, and is catalyzed by cytochrome P450-side chain cleavage enzyme [279] in the inner membrane of mitochondria of the adrenal [280], probably on the matrix side [281]. ACTH stimulation results in long-term and short-term effects on steroid hormone biosynthesis in the mitochondria [282]. The long-term effect of ACTH leads to an increase in the amounts of steroid hormone enzymes by increasing transcription of these genes [282]. StAR is the key protein which regulates cholesterol transport into the mitochondion [278]. Mutations in this protein result in defects in adrenal and gonadal steroidogenesis, which had been previously attributed to defects in side chain cleavage

enzyme activity [278]. Beginning several hours after ACTH administration, ACTH increases the levels of steroidogenic enzyme mRNAs in primary cultures of human adrenals by several-fold, including cholesterol side chain cleavage enzyme, 17-a-hydroxylase/17,20-lyase, 11-b-hydroxylase/ 18-hydroxylase/18-methyl-oxidase, and 21-hydroxylase cytochrome P-450 enzyme [283]. ACTH has a positive regulatory effect on its own receptors, and on the cAMP response to binding of ACTH to the receptor [284]. With sustained stimulation, down-regulation does occur, but physiologically this effect is minor since ACTH causes proliferation as well as steroid secretion. In addition to its prominent role in regulating adrenal steroidogenesis, ACTH exerts profound trophic effects upon the adrenal. Hypophysectomy results in adrenal atrophy and ACTH replacement restores adrenal gland weight in a dosedependent manner [285–288]. While the role of ACTH in adrenal hypertrophy is well established, its role in adrenocortical mitogenesis and hyperplasia is incompletely understood. The absence of ACTH induces apoptotic cell death in the adrenal cortex [289]. Prolonged ACTH administration not only blocks apoptosis, but also increases the adrenal DNA content in the rat [290], ACTH increases mRNA levels for c-fos and b-actin, proteins involved in cellular proliferation [291]. However, ACTH paradoxically inhibits mitosis of adrenocortical cells in culture [292–294]. ACTH antiserum administered to intact rats caused a highly significant decrease in corticosterone levels, but had no effect on adrenal weight [295]. Furthermore, ACTH inhibits the rapid compensatory proliferation of the remaining adrenal that normally occurs after unilateral adrenalectomy [296]. Another anterior pituitary-derived candidate for the stimulation of adrenal proliferation is the 28 amino acid Nterminal pro-opiomelanocortin peptide (N-POMC). This peptide is mitogenic in vitro and in vivo for the adrenal cortex, and N-POMC antiserum significantly diminishes adrenal mitotic activity after enucleation [297,298]. The compensatory adrenal growth that occurs after unilateral adrenalectomy may be mediated by a neural reflex that includes afferent neurons originating from the disrupted adrenal gland, the ventromedial nuclei of the hypothalamus, and efferent neurons innervating the remaining gland [299,300]. In 1947, Albright coined the term adrenarche to denote the developmental increase in adrenal androgens that occurs several years before the onset of gonadal maturation [301]. Adrenal androgen secretion may be sufficient for the development of some secondary sexual changes, including the development of pubic and axillary hair [302] and the maturation of sebaceous glands [303]. A condition in which axillary and pubic hair develop prematurely as a result of early adrenal androgen secretion has been termed premature adrenarche [304–309]. The mechanisms controlling adrenarche have remained obscure. Many hypotheses have been advanced, and many factors including ACTH, estrogens, prolactin, gonadotropins, growth hormone, glucocorticoids, androgens, and other POMC-derived products have been

Chapter 2

suggested as modulators of adrenal androgen secretion (for review see [310]). ACTH is widely accepted as a modulator of adrenal androgen secretion although, after administration of corticotropin, increases in dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) tend to be small [311–313]. Furthermore, the increase in adrenal androgens that occurs in adrenarche is not accompanied by an increase in serum cortisol levels, leading to the suggestion that factors other than ACTH are responsible for adrenarche [307,309,314,315]. However, cortisol production rates do increase at the same time as does the increase in adrenal androgen secretion, suggesting a possible common link that could be due to ACTH stimulation of both steroid pathways [316]. Dissection of the physiology of adrenarche has proven difficult as the only animal showing an adrenarche similar to that of humans is the chimpanzee [317]. Recent data from patients with genetic defects in MC2R provide definitive evidence of the participation of this receptor in the process of adrenarche, as discussed subsequently. Familial Glucocorticoid Deficiency Syndrome

The familial glucocorticoid deficiency (FGD) syndrome is a rare autosomal recessive syndrome originally described by Shepard et al. in 1959 [318]. Patients typically present with symptoms resulting from their glucocorticoid insufficiency including hyperpigmentation, hypoglycemia, lethargy, and weakness [318–321]. The clinical course may be complicated by frequent infections [318,319]. Patients with FGD do not present with symptoms related to salt wasting, dehydration, or electrolyte disturbances, as the renin-aldosterone axis is preserved. The preservation of the renin-aldosterone axis clearly distinguishes this syndrome from childhood Addison’s disease. An unexplained feature of the syndrome is that many of the patients are reported to be unusually tall [318,319,321–325]. Patients with FGD typically have low or undetectable cortisol concentrations, but occasionally low, normal cortisol values, which respond subnormally to exogenous corticotropin [318,319,321–325]. Plasma ACTH levels are markedly elevated, often greater than 1000 pg/ml. The adrenal glands are atrophic, and only occasional cortical cells remained in the zona glomerulosa with no remnants of the zona fasciculata or reticularis, but the adrenal medulla appears normal [318,319,323]. This adrenal atrophy substantiates the physiologic relevance of ACTH’s trophic action on the adrenal gland. Recently, an examination of 11 patients with FGD revealed a discrepancy between partial glucocorticoid deficiency and significantly diminished DHEAS secretion confirming a significant contribution for ACTH (or at least for MC2R) in the onset of adrenarche [326]. A number of different homozygous or compound heterozygous missense and nonsense mutations in MC2R have been reported in patients with FGD, and in all cases, these mutations cosegregate with disease in the affected families [322,327–331]. Transient expression of the S741 mutant MC2R (ser74 Æ ile) in COS-7 cells resulted in a EC50 for cAMP production of 67 nmol/l, approximately 12-fold

Adrenocorticotropin

59

higher than the EC50 of the normal MC2R of 5.5 nmol/l. Expression of a Asp107 Æ Asn mutation in Cloudman S91 melanoma cells resulted in a six- to ninefold increase in the EC50 for cAMP generation. These results were confounded by endogenous melanocortin receptors making it difficult to determine whether the mutant receptor demonstrated defective ligand binding or defective coupling to adenylate cyclase [328]. Not all cases of the FGD syndrome can be accounted for by mutations in MC2R. Several families exhibiting the classic FGD signs and symptoms have no mutations in the coding regions of the MC2R gene [322,328,332,333]. These patients are clinically indistinguishable from patients with MC2R mutations except that they do not demonstrate increased height [334]. Recently, linkage of this form of FGD to a 12-cM region surrounding MC2R has been excluded in six families [332]. FGD, with ACTH resistance, also occurs in the Allgrove, also known as triple A (adrenal insufficiency, achalasia, and alacrima) syndrome [335]. Mutations in MC2R do not cause the resistance to ACTH. Recently, a novel gene, AAAS, has been identified on chromosome 12q13 in which several homozygous or compound heterozygous loss-of-function mutations are associated with this syndrome, in nine unrelated families [336,337]. This protein must in some way interfere with the expression or function of MC2R. ACTH-independent Activation of ACTH Receptor Pathways

Several rare causes of ACTH-independent Cushing’s syndrome are due to ACTH-independent pathological activation of ACTH receptor pathways. In the McCune-Albright syndrome, a mutation in the GTPase region of the stimulatory alpha subunit G protein, Gsa, can result in constitutive activation of protein kinase A in the adrenal cortex, leading to hypersecretion of cortisol and adrenal adenoma formation [338,339]. Ectopic expression of several other transmembrane G protein-coupled receptors, including those which bind gastric inhibitory polypeptide, luteinizing hormone, vasopressin, and catecholamines, in the adrenal cortex, may occur [340]. In this situation, Cushing’s syndrome results from the cognate hormone binding to the ectopically expressed receptor, leading to stimulation of adenylate cylase, activation of cyclic AMP, and cortisol hypersecretion [340]. Finally, patients with mutations in the regulatory subunit of protein kinase A, R1a, may develop Cushing’s syndrome secondary to micronodular adrenocortical hyperplasia [341,342]. This disorder, termed Carney complex, may be associated with myxomas of the cardiac atria and other tissues, and freckling of the skin [343]. EFFECTS OF POMC-DERIVED PEPTIDES OUTSIDE OF ADRENAL ACTH binds with high affinity to rat adipocytes and has potent lipolytic effects [344–347]. High levels of MC2R mRNA expression have been demonstrated in all murine

60

SECTION 1

Hypothalamic–Pituitary Function

adipose tissues examined, but MC5R mRNA expression was also found in a subset of these tissues [348]. Both MC2R and MC5R mRNA were identified in the 3T3-L1 cell line after these cells were induced to differentiate into adipocytes. The physiologic importance of the actions of melanocortins on adipose tissue is unclear. Primate adipose tissues have been reported to be insensitive to the lipolytic actions of ACTH [349]. MC2R mRNA expression was not detected in human adipose tissue [252]. In order to examine whether the MC2R might play roles in the hypothalamic–pituitary–adrenal axis in sites other than the adrenal gland, MC2R expression has been examined in the hypothalamus and the pituitary. MC2R mRNA could not be detected in the hypothalamus or the pituitary [350]. The expression of an ACTH receptor on human mononuclear leukocytes was suggested after a finding that high-affinity ACTH binding did not occur in a patient with FGD [351]. However, this finding is puzzling in retrospect as MC2R mRNA expression has not been demonstrated in leukocytes. Human skin cells express MC2R along with mRNA for three obligatory enzymes of steroid synthesis, the cytochromes P450scc (CYP11A1), P450c17 (CYP17), and P450c21 (CYP21A2) [352]. Slominski et al. have hypothesized that an equivalent of the HPA axis composed of locally produced CRH, CRH-receptor, POMC, and cortisol operate in mammalian skin as a local response to stress [353]. Recently, it was demonstrated that ACTH can induce DNA synthesis and cell proliferation in an oral keratinocyte cell line [354].

Melanocortin 3 Receptor Expression of the third cloned melanocortin receptor, MC3R, was originally identified in the brain, placenta, and gut [355]. Subsequently, MC3R expression was also documented in the heart [252]. Detailed mapping of the MC3R in the central nervous system by in situ hybridization localized the highest densities of MC3R mRNA to the hypothalamus and limbic system [356]. MC3R mRNA was found in the arcuate nucleus [356], the site of POMC expression within the hypothalamus. An autoradiographic approach using the non-selective [125I]NDP-MSH in competition with the relatively MC3R selective g-MSH or an MC4R selective synthetic agent HS014 has been used to visualize the MC3R and MC4R distributions within the central nervous system [357]. In the nucleus accumbens shell, the medial preoptic area, and the ventromedial nucleus of the hypothalamus, the MC3R dominates. In the lateral septum and the olfactory tubercle, both MC3R and MC4R seem to be present. The lack of overlap between the autoradiographic data and the data concerning MC3R mRNA expression may indicate the presence of MC3R on nerve terminals projecting from the arcuate nucleus. A physiologic role for MC3R has not yet been identified.

Recently, in a murine model of experimental gout, systemic treatment of mice with ACTH4–10 inhibited neutrophil accumulation [358]. This effect was blocked by he melanocortin receptor type 3/4 antagonist SHU9119. MC3R, but not MC4R, mRNA was detected in murine macrophages suggesting that MC3R may play a role in modulating inflammation. Recently, a mouse with targeted deletion of the MC3R gene has been created [359]. Although this mouse has a normal body weight, it has an increased fat to lean weight ratio, which seems to be due to an increased efficiency of converting ingested food into stored fuel. Thus, a function of melanocortins acting via MC3R may be to promote the conversion of energy in food into either lean body mass or forms of energy other than fat. This raises the possibility that in humans, mutations in MC3R may contribute to the “thrifty” genotype, as is found in Pima Indians [360].

Melanocortin 4 Receptor The MC4R is localized to the brain [361] and, in contrast to the MC3R, its expression has been documented throughout the central nervous system including the cortex, thalamus, hypothalamus, brainstem, and spinal cord [362]. In humans, the MC4R has not been detected in any peripheral tissue [252], although this distribution does not hold true for all animals as the MC4R is expressed in many peripheral tissues of the chicken [363]. The MC4R has garnered considerable attention as this melanocortin receptor subtype appears to play a central role in weight regulation. Mice homozygous for an Mc4r-null allele demonstrate autosomal dominant, maturity-onset obesity, hyperphagia, hyperinsulinemia, and hyperglycemia [364]. Heterozygotes have an intermediate phenotype. The Mc4r-null mouse also demonstrates increased linear growth [364], a feature unique to agouti yellow mice (discussed subsequently) and MC4Rnull mice among rodent obesity models. The MC4R has been implicated in leptin signaling as administration of the melanocortin receptor antagonist SHU9119 can block the reduction of food intake induced by central administration of leptin [365]. Leptin receptors are found on POMC expressing neurons in the arcuate nucleus and administration of exogenous leptin does not reverse obesity in Mc4rnull mice [366]. The role of MC4R in weight regulation in humans has been confirmed with the identification of individuals with dominantly inherited obesity segregating with mutations in the MC4R gene that result in frameshift errors [367–369]. Recent studies suggest that haploinsufficiency of MC4R may be a frequent, but incompletely penetrant, cause of human obesity [370,371]. The incomplete penetrance is highlighted by the absence of obesity in individuals with large deletions of chromosome 18q, a region that spans the MC4R gene [372]. A dominant-negative effect of mutant MC4R has been proposed, but cotransfection studies of mutant and wild-type MC4R in vitro showed that mutants

Chapter 2

affected neither signaling nor cell surface expression of wildtype MC4R [373].

Melanocortin 5 Receptor Expression of the fifth melanocortin receptor was originally recognized in the brain [374]. Subsequently, MC5R expression was documented widely at low levels in tissues including the adrenal glands, skin, adipocytes, skeletal muscle, kidneys, lung, stomach, liver, spleen, thymus, lymph nodes, mammary glands, ovary, pituitary, testis, and uterus [64,374–377]. In situ hybridization studies showed that within the adrenal, MC5R is predominantly expressed in the aldosterone-producing zona glomerulosa cells [376]. High levels of MC5R mRNA expression have been documented in the secretory epithelia of a number of exocrine and endocrine glands including Harderian, preputial, lacrimal, sebaceous, and prostate glands and pancreas [378,379]. Melanocortins have been reported to affect a number of exocrine glands. Removal of the neurointermediate lobe of the pituitary reduces sebaceous lipid production, and this reduction is restored by administration of a-MSH [380–382]. Exogenous ACTH and MSH increase secretion from the lacrimal gland [383,384]. Deletion of the murine Mc5r resulted in the loss of detectable binding of [125I]NDP-MSH to Harderian, lacrimal, and preputial glands, and skeletal muscle indicating that MC5R is the predominant melanocortin receptor in these tissues [378]. Development of the Mc5r-null mouse confirmed a physiologic role for the melanocortins in regulating exocrine gland function; the mice demonstrate severe deficits in water repulsion and thermoregulation as a result of decreased sebaceous lipid production [378]. MC5R also appears to be essential for hormonally regulated release of porphyrins from the Harderian gland [378]. The preputial gland is a specialized sebaceous gland implicated in pheromone production [385]. Exogenous a-MSH stimulates the release of a preputial odorant into the urine of male mice which stimulates aggressive attacks [386]. Chen and colleagues have hypothesized the existence of a hypothalamic– pituitary–exocrine axis which might provide a mechanism by which stress could alter behavior via the regulation of olfactory cues [378].

Diseases Caused by Melanocortin Receptor Ligands POMC Deficiency

Two individuals have been identified with genetic defects in the POMC gene [387] (Fig. 2.3). The first patient was a compound heterozygote for two mutations in exon 3 which interfere with appropriate synthesis of ACTH and a-MSH. The second patient was homozygous for a mutation in exon 2 which abolishes POMC mRNA translation. Both patients presented with early-onset obesity, adrenal insufficiency, and red hair pigmentation. The brother of patient one had died at the age of 7 months of hepatic failure following severe

Adrenocorticotropin

61

cholestasis and was found to have bilateral adrenal hypoplasia in the postmortem examination. All other anterior pituitary-derived hormones were normal. No symptoms related to b-endorphin deficiency have been noted. The heterozygous parents were normal in both families. Isolated ACTH deficiency of the pituitary due to other causes is also rare. It most commonly appears to be acquired in later life, and is more common in men [388–391]. Most cases have been reported in Japan, and are often associated with other autoimmune endocrinopathies, including Hashimoto’s thyroiditis and type 1 diabetes mellitus. It is likely that only pituitary ACTH, and not hypothalamic aMSH, is affected in these patients, as they are not obese and do not have red hair, unlike patients with POMC gene mutations [387]. Recently, the gene Tpit has been identified as a transcription factor specific to pituitary corticotrophs and melanotrophs, and absent in hypothalamic neurons which also express POMC [77]. It interacts with the POMC-specific transcription factor, Ptx1, to regulated POMC transcription. Two families with recessive mutations in TPIT have been described [77]. Consistent with its sites of expression, these patients have congenital, secondary adrenal insufficiency, but not obesity or red hair. Recently, a POMC-deficient mouse has been produced whose phenotype is similar to the human POMC-deficient syndrome and confirms the known functions of melanocortins [392]. The phenotype includes obesity, increased body length, yellow pigmentation, deficits in sebaceous gland function and thermoregulation, and adrenal hypoplasia and glucocorticoid deficiency. Adrenal glands could not be identified. In addition to undetectable corticosterone levels, aldosterone levels were also undetectable. The mutant mice lost 40% of their excess weight after 2 weeks of treatment with a stable a-MSH agonist. Although some of this weight loss was clearly attributable to decreased food intake, these same authors have clearly shown that a-MSH administration has additional lipolytic effects and results in increased energy expenditure [393]. Agouti and Agouti-related Protein

The agouti gene locus was identified 45 years ago as a genetic locus that controls the amount and distribution of eumelanin (brown–black) and pheomelanin (yellow–red) pigmentation in the mammalian coat [394]. However, analysis of mutations at the agouti gene locus have occupied investigators for nearly a century. The lethal yellow mutation at the agouti locus was the first murine embryonic lethal mutation and the first murine obesity syndrome to be characterized [395,396]. Agouti is a small 131 amino acid protein that is secreted by dermal papillae cells and acts to block melanocortin action on follicular melanocytes at Mc1r in the mouse [397,398]. The recombinant murine agouti protein is a potent nanomolar competitive antagonist for melanocortin receptors at Mc1r and Mc4r, relatively weaker at Mc3r, and only a micromolar inhibitor of Mc5r [397,399,400] and

62

Hypothalamic–Pituitary Function

SECTION 1

Girl's weight

Boy's weight

35

–P97 82

Patient

–P90

30

72

25

–P75 –P50 –P25 –P10 –P3

62

20

–P97 –P90 –P75 –P50 –P25 –P10 –P3

15

52

Patient

42 32

10 22 5

0

12

0

3 6 9 12 18 24 30 36 42 48 Age (months)

2

0

2

4

6

8 10 12 14 16 18

Age (years)

represents the first known endogenous antagonist for a Gprotein coupled-receptor. In wild-type rodents, agouti expression is restricted to the hair follicle [398]. The human agouti gene which is 85% identical to the mouse gene is expressed much more widely including in adipose tissue, testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin [401,402]. The human agouti protein, the agouti-signaling protein (ASIP), displays a similar pharmacologic profile for antagonism of melanocortin receptors as compared to the murine agouti protein except that ASIP may display both competitive and noncompetitive antagonism at MC4R and also appears to act as a noncompetitive antagonist of MC2R [403,404]. The heterozygous lethal yellow agouti mouse is characterized by a yellow coat-color, late onset obesity associated

FIGURE 2.3. Patients and weight curves of two unrelated patients with homozygous mutations in POMC. Note the obesity coupled with fair skin and red hair, as well as early onset obesity. From reference [387] with permission.

with hyperphagia, hyperinsulinemia and hyperglycemia, increased linear growth, and susceptibility to a wide variety of epithelial and mesenchymal tumors (reviewed in [405]). This phenotype is the result of a 170 kb deletion which removes all but the promoter and noncoding first exon of the Raly gene 5¢ to the exons of the agouti gene [406] and results in ubiquitous overexpression of agouti in all tissues examined to date [398,407]. The lethal yellow phenotype is presumably the result of agouti antagonism of the widely distributed melanocortin receptors and is consistent with the known actions of melanocortin receptors described above. An agouti-related protein (AGRP) gene was recently identified based on its homology to agouti and is expressed in the hypothalamus, adrenal medulla, and at low levels

Chapter 2

in the testis, lung, and kidney [408,409]. In contrast to the divergent expression patterns of murine and human agouti, the expression pattern of murine and human AGRP appears identical [408]. Within the hypothalamus, AGRP expression is confined to the arcuate nucleus and AGRP-immunoreactive terminals paralleled POMCimmunoreactive terminals projecting from the arcuate nucleus [55]. AGRP is a selective, nanomolar competitiveantagonist of MC3R and MC4R, clearly implicating it as the endogenous melanocortin antagonist involved in energy homeostasis [410–412]. Corticostatins are a family of related low molecular weight members of the defensin family of peptides that are competitive inhibitors of ACTH-induced steroidogenesis in the adrenal cortex, which act by blocking the ACTH receptor [413]. In rats, a dose of 8 mg/kg body weight blocks the corticosterone response to stress [414]. Corticostatins and the agouti peptides are the two known endogenous competitive inhibitors of melanocortin receptors, but do not appear to be structurally related [415].

Additional Potential Melanocortin Actions Additional physiologic roles for melanocortins have been proposed that do not clearly correlate with the known functions of specific melanocortin receptors as surmised from human or mouse mutants. A multitude of behavioral and psychological effects have been attributed to melanocortins (for review see [416]). Intracerebroventricular administration of ACTH or a-MSH elicits excessive grooming behavior [417], yawning, stretching, and penile erection [418]. The grooming, stretching, and yawning behavior, but not erectile function, may be mediated by MC4R as deduced through the use of MC4R selective antagonists [233,419,420]. Recently, in a double-blind, placebo controlled crossover study, a cyclic a-MSH analogue initiated erections in men with psychogenic erectile dysfunction [421]. Roles for the actions of melanocortins in neuromuscular development, promotion of the regeneration of crushed nerves, and CNS protection from injury have also been postulated [235,422–425]. A potential role for melanocortins in fetal growth and brain development has been demonstrated [426,427]. Data indicating a role for melanocortins as modulators of inflammation have been discussed previously. Centrally administered MSH is a potent antipyretic agent and, in an endogenous pyrogen-induced fever model in rabbits, is approximately 25,000-fold more potent than acetaminophen [428]. Physiologic roles for melanocortins in maintenance of cardiovascular homeostasis have also been proposed. Peripheral or central administration of g-MSH causes tachycardia and pressor effects [429,430]. Central administration of a-MSH results in bradycardia and depressor effects [429,430]. Melanocortins may further influence cardiovascular homeostasis through their effects on electrolyte regulation. a-MSH and g-MSH are potent natriuretics [431–434].

Adrenocorticotropin

63

SECRETION OF ACTH: PHYSIOLOGY Physiologically a number of factors interact to determine the final pattern of ACTH release, including circadian rhythms, stress, and negative feedback by glucocorticoids. These factors impact on each other in an integrated fashion to control ACTH release. The stage of development, from fetal life through puberty, and pregnancy and parturition, also impact on ACTH secretion. Finally, the immune system interacts with the HPA axis, adding another facet to the complexity of ACTH release.

Secretion Dynamics of ACTH in vivo ACTH secretion is characterized by pulsatile release of ACTH from the corticotroph in a burst-like pattern, with no interpulse secretion [435]. Fifteen-minute sampling reveals approximately 12 ACTH and cortisol pulses over a 24-hour period [436], whereas more frequent 10-minute sampling reveals 40 ACTH pulses in 24 hours [435]. Blood ACTH rises by an average of 24 pg/ml per pulse [435]. bendorphin secretion parallels the pulsatile release of ACTH [437]. Spontaneous ACTH and cortisol pulses correlate highly. There is a strong relationship between the magnitude of concomitant ACTH and cortisol pulses, particularly if a 15 minute phase delay in cortisol secretion is allowed for [435,438–440]. Not all spontaneous ACTH and cortisol pulses are concomitant: approximately 50% to 75% of spontaneous ACTH pulses are followed by a cortisol pulse, whereas approximately 60% to 90% of spontaneous cortisol pulses are preceded by an ACTH pulse [436,440]. The 24-hour pattern of ACTH pulses, but not the cortisol pattern, differs between males and females. Males have more pulses, greater mean peak ACTH amplitude, greater 24-hour ACTH secretion, and higher mean ACTH levels [438]. The sensitivity of the adrenal cortex, or the availability of ACTH to the adrenal cortex, may be greater in females. Alternatively, males and females may have different set points for cortisol feedback [438]. CRH may also be secreted in a pulsatile fashion, accounting for pulsatile ACTH secretion. When exogenous CRH is given continuously, there is a progressive desensitization of the ACTH response to CRH [441]. However, when CRH is given in a prolonged pulsatile manner, it does not desensitize the corticotroph CRH receptor, and the releasable ACTH pool is not depleted [436].

Negative Regulation of ACTH Secretion Negative feedback can be defined as long, short, or ultrashort depending on the location and nature of the hormone mediating the feedback. Long Feedback by Glucocorticoids

Long feedback refers to the effects of adrenal glucocorticoids on ACTH secretion at the pituitary and in the hypo-

64

SECTION 1

Hypothalamic–Pituitary Function

thalamus. Glucocorticoid-mediated negative feedback can be subdivided into fast and delayed, which can be further subdivided into intermediate and slow feedback. In animals, the initial fast negative effect of glucocorticoids occurs within seconds to minutes [183], involves inhibition of stimulated ACTH and CRH release, not synthesis, and occurs during the period when plasma glucocorticoid levels are increasing. Cortisol given to patients at the start of surgery attenuates the surgery-induced ACTH rise [211], and may be an example of fast feedback. Delayed feedback has two components, intermediate and slow [442]. Intermediate feedback is the component of delayed feedback that is due to inhibition of ACTH release, but not synthesis, and may be important after short durations of glucocorticoid exposure, or after noncontinuous, repeated exposures [442]. Intermediate delayed feedback develops after 45 to 120 minutes, and maximal inhibition occurs 2 to 4 hours after administration of one dose of glucocorticoids. Unlike ACTH, CRH synthesis as well as release may be affected by intermediate feedback [442]. The slow component of delayed feedback is most important after long exposures to a moderately high dose of glucocorticoid, and is a function of the total dose of glucocorticoids, the glucocorticoid level achieved, and the amount of time since the steroid was given [442]. Slow feedback occurs after more than 24 hours of exposure to glucocorticoids and can persist for days. POMC biosynthesis is inhibited, leading to inhibition of basal and stimulated ACTH secretion [442], and intracellular ACTH decreases implying decreased synthesis. In humans, negative feedback by glucocorticoids takes 30 to 60 minutes to manifest. When endogenous cortisol is suppressed with metyrapone, the effects of exogenous glucocorticoids on the morning ACTH rise, or on oCRHstimulated ACTH release, are not seen initially but appears 30 minutes after glucocorticoid administration [443]. In addition, glucocorticoids have little effect on the initial CRH-induced increase in ACTH release, but decrease CRH-induced ACTH release after 60 minutes. Cortisol given to patients at the start of surgery attenuates the surgery-induced ACTH rise [211]. Cortisol modulates the responsiveness of the pituitary. The corticotroph is dependent on CRH stimulation to maintain ACTH secretion, and glucocorticoids suppress CRH-induced ACTH secretion in vivo and in vitro. On the other hand, when endogenous cortisol levels are suppressed by metyrapone, basal ACTH and CRH-induced ACTH release are increased [444]. Glucocorticoid inhibition of ACTH secretion from the corticotroph may recover more quickly than CRH secretion from the hypothalamus [445]. Secondary adrenal insufficiency due to long-term glucocorticoid therapy may in part be due to continued suppression of hypothalamic CRH secretion 184. Adrenalectomized patients on exogenous glucocorticoid therapy have a blunted ACTH response to CRH that normalizes after several CRH boluses, suggesting that lack of stimula-

tion of the corticotroph by CRH suppresses the ACTH response [446]. On the other hand, corticotrophs of patients recovering from transsphenoidal surgery for Cushing’s disease are profoundly unresponsive to CRH, which can not be attributed solely to deficient CRH priming [447]. Glucocorticoids inhibit AVP secretion [448]. In most studies, patients with hypopituitarism or primary adrenal insufficiency are unable to maximally dilute their urine in response to a water load, and this is corrected by glucocorticoid administration [449]. However, it is unclear if the elevated AVP levels, or the lack glucocorticoids, is responsible for the inability to maximally dilute the urine in the hypocortisolemic state [448]. Glucocorticoids inhibit nitric oxide synthase, and nitric oxide is capable of stimulating the insertion of the water channel, aquaporin 2, in the luminal membrane of the renal collecting cell [450]. This may provide an explanation for why glucocorticoid deficiency is associated with decreased free water clearance. Short Feedback

Short feedback refers to the effect of pituitary ACTH to inhibit CRH release. In normal subjects, the administration of ACTH does not affect CRH levels, most likely because of the negative effects of cortisol present prior to ACTH administration [451]. However, in patients with elevated CRH levels due to Addison’s disease or hypopituitarism, ACTH decreases CRH and b-endorphin levels, suggesting that ACTH inhibits CRH secretion [451]. ACTH may act in the median eminence or in the hypothalamus to inhibit CRH release [451].

Circadian Regulation of ACTH Secretion There is an endogenous circadian rhythm to the pulsatile pattern of ACTH secretion which leads to a circadian rhythm of glucocorticoid release. The function of this circadian rhythm in cortisol secretion is not known, although one hypothesis suggests that the early morning rise in cortisol causes a delayed-phase rise in insulin resistance, which may play a role in altered glucose metabolism [452]. For instance, since the brain does not require insulin for glucose uptake, peripheral insulin resistance might cause a rise in glucose levels, leading to greater uptake in the brain. The circadian rhythm is generated in the suprachiasmatic nucleus (SCN), and the signals travel via efferent inputs to the PVN to modulate CRH release [183,453]. This circadian rhythm is due to variation in ACTH pulse amplitude, not frequency [435]. The amount of ACTH secreted per pulse varies by 3.8-fold over a 24-hour period [435]. Basal ACTH and cortisol levels parallel each other and are the highest upon awakening in the morning between 0600 hours and 0900 hours, decline through the day to intermediate levels at 1600 hours, and are lowest between 2300 hours and 0300 hours [435]. From 2300 hours to 0200 hours, there is a quiescent period of minimal secretory activity, corresponding to the nadir of ACTH and cortisol levels [454]. Secretion of

Chapter 2

ACTH and cortisol abruptly increases in the early morning [454]. The diurnal secretory pattern is similar for free and total cortisol, although the relative increase in free cortisol is about 1.5 times greater than the relative increase in total cortisol [454]. Alterations in feeding and sleep impact on cortisol secretion [455]. Cortisol briefly increases postprandially, especially after the midday meal [454]. Exercise or administration of ACTH at 1000 hours leads to a rise in cortisol and blunts the midday cortisol surge, and at 1400 hours leads to a rise in cortisol [454]. Overall, the major features of the diurnal cortisol pattern persist under conditions of complete fasting, continuous feeding, or total sleep deprivation [456]. However, the circadian rhythm of cortisol secretion fully adapts to permanent changes in environmental time and the sleep–wake pattern. This adaptation takes about 3 weeks, the limiting factor being the time it takes for the quiescent period of secretion to fully adapt [456]. The acrophase adapts much more quickly and is partially synchronized after one day and totally synchronized after 10 days [456]. There is circadian regulation of the sensitivity of the response of the adrenal cortex to ACTH [457,458]. Injection of ACTH at 0700 hours, just at the time of the endogenous cortisol peak, causes a significant increase in cortisol. The absolute increment in cortisol secretion in response to an ACTH stimulation test is greater when the test is performed at circadian nadir compared to the peak, although in one study this was true in males only [459]. The incremental increase in cortisol secretion in response to a CRH stimulation test is also greatest at night when basal levels are lowest, although the total amount of cortisol released is greatest in the morning when the basal cortisol levels are highest [460]. The basal cortisol level, not the time of day, seems to be the important factor determining the cortisol response to oCRH, and the higher the basal cortisol level, the lower the peak cortisol response to oCRH [436]. On the other hand, the maximum ACTH blood level in response to oCRH occurs at 0700 hours, the time of the minimum cortisol increment to oCRH [436]. The factors governing the circadian rhythm in ACTH release in humans are not clear. On the one hand, it may be regulated by a diurnal rhythm in CRH secretion [461]. The highest CRH levels occur at 0600 hours (7.0 pg/ml) and the lowest at 1800 hours and 2200 hours (about 1.8 pg/ml), which parallels the pattern of ACTH and cortisol secretion [462]. Serotoninergic and cholinergic pathways may play a role in the CRH circadian rhythm, and display circadian periodicity in their hypothalamic concentrations [463]. On the other hand, when the pituitary is exposed to constant levels of CRH 30-fold higher than those found in portal hypophyseal blood, such as in pregnancy [464] and during CRH infusion [441], the circadian rhythms in ACTH and cortisol persist. CRH clearly plays a role in the ACTH rhythm, as CRH-deficient mice have an absent or markedly attenuated diurnal rise in ACTH [465]. However, in these mice, constant infusion of CRH restores the ACTH

Adrenocorticotropin

65

rhythm. This indicates that changes in CRH amplitude are not necessary to drive the ACTH rhythm, but that a tonic level of CRH is required to maintain ACTH responsiveness [466] to circadian cues.

Physical Stress Regulation of ACTH Secretion Glucocorticoid release mediated by ACTH, plays a major role in the response to stress. An interaction exists between stress-mediated ACTH release, which leads to glucocorticoid secretion, and glucocorticoid-mediated negative feedback, which inhibits further ACTH and glucocorticoid release. A number of stressful stimuli lead to ACTH secretion. Most physical stressors activate the HPA axis. The magnitude of the rise in ACTH and cortisol is dependent upon many factors, including the nature of the stress, its magnitude including the rapidity of its appearance, and the time of day it is experienced. In general, stressors have a larger impact on ACTH release when they develop rapidly, are of high magnitude, and occur during the circadian nadir in ACTH release. Hypoglycemia

In humans, insulin induced hypoglycemia is associated with a increase in plasma ACTH levels [467], from a basal level of about 40 pg/ml to a peak of 250 pg/ml at 45 minutes [467]. Cortisol levels increase over twofold, from a basal level of about 11 ug/dl to a peak of about 25 ug/dl at 60 to 90 minutes [467]. Insulin-induced hypoglycemia causes a four- to fivefold greater increase in ACTH secretion than oCRH alone, and a 1.3-fold greater increase than AVP plus CRH [97,468]. CRH may play a permissive rather than a dynamic role in the ACTH response to hypoglycemia [182], whereas AVP may play a more direct role [467]. AVP levels have been shown to increase 2.5- to sevenfold at 30 to 45 minutes after the insulin. When AVP levels are raised endogenously by saline infusion or lowered to undetectable levels by waterloading, the hypoglycemia-induced AVP increase is greater after saline, even though saline blunts the hypoglycemic response to insulin [469]. Catecholamines increase in response to hypoglycemia, and may act at the hypothalamus to mediate ACTH release. Epinephrine appears to play more of a role than norepinephrine, increasing at least 13fold at 30 minutes after insulin, whereas norepinephrine increases 2.4-fold at 60 minutes. Exercise

Exercise increases ACTH and b-endorphin levels, and the response is dependent on the intensity of exercise and the level of training [470]. Exercising to exhaustion, or exercise of short duration and high intensity, increases ACTH, bendorphin, and cortisol levels. Hypercortisolism is seen in highly trained athletes, who need a higher level of oxygen consumption to stimulate ACTH release, and have elevated basal ACTH levels [471]. Physical exercise and stress both

66

SECTION 1

Hypothalamic–Pituitary Function

lead to analgesia in man [472]. Naloxone reverses exerciseinduced analgesia from certain types of pain, suggesting a role for endorphins. Dexamethasone reverses exerciseinduced analgesia from other types of pain, like dental pain, that are not reversed by naloxone, suggesting a role for ACTH, although dexamethasone also suppresses bendorphin release. Lower Body Negative Pressure and Acute Hemorrhage

Lower body negative pressure in humans simulates acute hemorrhage. Lower body negative pressure increases ACTH secretion to peak values of 60–250 pg/ml at 2 to 10 minutes after cessation of the stimulus, and the increase in ACTH is reversed by dexamethasone [473]. Hypovolemia may be the physiologically important stress. In animals, hemorrhage stimulates ACTH secretion primarily mediated by CRH and, to some degree, by AVP [182]. Hypovolemia increases portal blood CRH, AVP, epinephrine, and oxytocin, whereas hypotension, also a component of hemorrhage, induced by nitroprusside, increases portal blood CRH only. Surgical Stress

Surgery induces a large increase in plasma ACTH levels [284]. Patients undergoing surgery may have increased sensitivity of the adrenal cortex to ACTH [474,475]. Fentanyl, an opiate agonist, attenuates the ACTH response to surgery [211]. Evidence in adrenalectomized primates suggests that supraphysiological doses of glucocorticoids are not necessary for the animal to withstand surgical stress, but that a minimal level is necessary [474,476].

Psychological and Emotional Stress Psychological and emotional stress play a role in the hormonal stress response. ACTH levels are high in patients awaiting an insulin-tolerance test. During physical exercise, psychological and physical stress may act synergistically to increase b-endorphin and ACTH levels [477]. With increasing experience in performing a set level of exhaustive physical exercise, plasma ACTH and cortisol levels declined [478].

Fetal and Postnatal Regulation of ACTH Secretion In utero, maternal cortisol influences alterations of fetal heart rate, movement, and behavioral states [479]. By 35 weeks of gestation, there is a circadian rhythm in fetal behavioral states that is altered if the maternal diurnal variation in ACTH and cortisol secretion are abolished by maternal administration of triamicinolone [479]. Neonates, even when born prematurely, have an endogenous cortisol rhythm. Minimally and severely stressed neonates in the neonatal intensive care unit, born between 23 and 38 weeks of gestation, have a significant diurnal rhythm in cortisol and endorphin secretion, although ACTH levels do not vary sig-

nificantly [463]. Others have suggested that diurnal rhythmicity is a function of maturation, and is not present before 6 months of age [463,480,481].

Regulation of ACTH Secretion During Puberty Normal children between the ages of 1 year and 16 years do not differ from adults in ACTH, b-endorphin, and cortisol responses to CRH, and the responses of boys do not differ from girls [482]. Some of the other adrenal steroids and adrenal androgens demonstrate basal and stimulated variation with age. The CRH-stimulated androstenedi one to 17-hydroxyprogesterone ratio increases with sexual maturation, suggesting that the 17,20-desmolase activity increases with puberty [483]. The dehydroepiandrosterone response to CRH increases as children progress from stage 1 to stage 5 of puberty, and by stage 5 of puberty, dehydroepiandrosterone levels do not differ from adults [482].

HPA-Axis-Immune Interactions The immune system and the neuroendocrine system communicate with each other, and share a common set of structurally identical hormones and receptors [484–488]. Cells of the immune system synthesize biologically active neuroendocrine peptide hormones, immune cells contain receptors for neuroendocrine hormones, neuroendocrine hormones modulate immune function, and lymphokines modulate neuroendorine function. The neuroendocrine system and the immune system work together in the regulation of both the stress and the immune response, and the components include lymphoid cells, cholinergic and adrenergic neurons, cytokines and lymphokines, hormones and neuropeptides released by the endocrine glands and the CNS, receptors, and higher CNS activity which modulates these responses [489,490]. Effect of Immune System on HPA Axis

Immune cells, particularly monocytes, macrophages, and lymphocytes, produce cytokines involved in the immune response, and these cytokines activate the HPA axis. Bacterial-derived endotoxin and lipopolysacchride stimulate the release of interleukin-1, interleukin-6, and TNF-a, which are regulated by glucocorticoid feedback. The effects of cytokines on the HPA axis at the level of the brain, pituitary, and adrenal gland, are the best examples of immune modulation of a neuroendocrine system. Interleukin-1 is produced by stimulated macrophages and monocytes, and stimulates CRH release. Interleukin-1 also directly stimulates the adrenal cortex. Tumor necrosis factor is produced primarily by activated monocytes [491], and has many of the same biological activities and responds to many of the same immune challenges as interleukin-1. Tumor necrosis factor stimulates ACTH release, most likely at an extra-pituitary site. Interleukin-6 has actions similar to interleukin-1 and

Chapter 2

TNF, and stimulates the HPA axis [202,491]. Interleukin-2 is synthesized by T cells after an antigenic challenge, and increases ACTH and cortisol levels in humans, although this may be indirect and due to the stress response generated by fever and chills. Gamma-interferon causes an increase in steroid production by adrenal cells. As discussed above, leukemia-inhibitory factor (LIF) may be a major activator of POMC gene transcription and ACTH release following immune or inflammatory stimulation. Effect of HPA Axis on Immune System

Stress suppresses immune function. Following an infection or most immunization procedures, and the presence of bacterial endotoxin, a stress-like response of the pituitary occurs leading to the release of ACTH and cortisol which tends to suppress the immune system. However, in humans, there is no clear evidence that the rise in cortisol following an immune or inflammatory stimulus plays a significant role in modulating the subsequent immune response. In fact, prior to the treatment of patients with primary adrenal insufficiency, there were no substantive reports that these patients suffered from overactivity of their immune systems. Receptors for CRH, ACTH, and glucocorticoids most likely mediate the effect of these hormones on the immune system. ACTH receptors on human peripheral monocytes have been characterized, and glucocorticoid receptors are present on human lymphocytes. Glucocorticoids inhibit many aspects of immune function, establishing a negative feedback loop between the immune and neuroendocrine systems. Glucocortiocoids block lymphocyte activation, block the production and action of interleukin-2, interleukin-1, gamma-interferon, TNF, and prostaglandins, and interfer with the interaction of certain effector molecules with target cells. HPA Axis Within Immune System

Cells of the immune system produce CRH, ACTH and endorphins. Human peripheral mononuclear leukocytes synthesize three molecular forms of immunoreactiveACTH, and lymphocytes produce b-endorphins. Mouse spleen macrophages and virally infected mouse splenocytes contain b-endorphin and POMC mRNA [492–494]. CRH mRNA and peptide are found in monocytes in acute inflammatory reactions [495] and in T cell lymphocytes [496]. Releasing hormones and cytokines interact to stimulate ACTH production from immune cells. CRH and AVP stimulate a dramatic increase in biologically active ACTH and b-endorphin from human peripheral leukocytes in some studies. Unstimulated leukocytes produce little ACTH. In humans, only B lymphocytes secrete b-endorphin in response to CRH and AVP, and monocyte-secreted interleukin-1 mediates the effect. A feedback loop exists between the immune and neuroendocrine systems [490]. Cytokines released by immune cells stimulate secretion of ACTH and glucocorticoids which are active in the fight

Adrenocorticotropin

67

against infection. Subsequently, these hormones suppress the further synthesis of cytokines. SECRETION OF ACTH: PATHOPHYSIOLOGY Abnormal secretion of ACTH occurs in ACTH-secreting pituitary adenomas responsible for Cushing’s disease or Nelson’s syndrome, and in Cushing’s syndrome due to an ectopic pituitary adenoma or an ectopic ACTH-secreting tumor. Decreased or absent ACTH secretion due to genetic or autoimmune causes has been discussed in the above section on melanocortin actions.

Pituitary Adenomas ACTH immunoreactivity in an abnormal pituitary is usually due to a single functional adenoma, but can be associated with nodular hyperplasia or a silent cortiocotropic adenoma. Diffuse or nodular corticotroph hyperplasia, which could result from an ectopic or hypothalmic CRH-producing tumor, or Addison’s disease, is a rare cause of a pituitary adenoma or ACTH hyperfunction. Pituitary adenomas constitute about 15% of intracranial tumors [3]. Approximately 56% of pituitary adenomas are active, and about one third of active pituitary adenomas produce ACTH. Ten to 15% of pituitary adenomas are pleurihormonal, some of which secrete ACTH. When discovered, ACTH-producing adenomas are often functional, small, highly vascular, and prone to hemorrhage [497]. Corticotroph adenomas tend to be located in the central portion of the adenohypophysis, in the “mucoid wedge,” and form micronodular aggregates [497]. Invasive adenomas are more frequently found among undifferentiated, extermely laterally localized, or large adenomas, and recur more frequently than non-invasive tumors. There is no correlation between the size of an adenoma and the cortisol level or rate of recurrence [498]. The majority of adenomas responsible for Cushing’s disease or Nelson’s syndrome are microadenomas. ACTH-producing adenomas are usually monoclonal, but may be polyclonal [499]. Pleurihormonal adenomas are usually polyclonal [499]. The biochemical structure and ultrastructure of ACTHsecreting pituitary adenoma cells differ from nonadenomatous pituitary cells [500]. The cells may look normal, but be increased in number [497]. Cells are oval to polygonal in shape, with eccentric spherical nuclei and well-developed rough endoplasmic reticulim. Crooke’s hyaline is characteristic of ACTH-producing tumors [501], is associated with either endogenous or exogenous hypercortisolism, and is due to massive accumulation of intermediate cytoplasmic filaments that are normally present in small numbers [3]. Clinically, an increased number of Crooke’s cells is correlated with a longer postoperative replacement dose of cortisol requirement [498]. Pituitary adenomas may produce products in addition to ACTH. Some ACTH-producing corticotroph adenomas contain a form of gastrin that is smaller than gastrin found

68

SECTION 1

Hypothalamic–Pituitary Function

in the normal adenophypophysis. 7B2 is a secretory granule-associated protein, which may be involved in proconvertase activtion [502], which is sometimes secreted by ACTH-producing tumors, it is secreted in the highest levels from nonfunctional pituitary tumor. A variant of Cushing’s disease is due to ACTHproducing pituitary adenomas occurring ectopically [5], and have been described to arise in the mucosa of the sphenoid sinus [5,503], within a benign cystic ovarian teratoma [504], and intrahemispherically in a 6-year-old boy [505]. Rarely, both Cushing’s disease and Nelson’s syndrome have been preceded by generalized glucocorticoid resistance due to a mutation in the glucocorticoid receptor [506,507]. In these cases, the high rate of ACTH secretion, stimulated by the generalized glucocorticoid resistance, in some way led to adenoma formation, perhaps following a second oncogenic transformation. Pituitary carcinomas are malignant pituitary tumors associated with extracranial metastases, including liver and lung [508]. Pituitary carcinomas cannot be histologically differentiated from adenomas [3]. A large number of pituitary carcinomas produce Cushing’s disease [3]. In patients with Cushing’s disease due to a pituitary carcinoma, the primary tumor and metastases stain immunochemically for ACTH, b-LPH, b-endorphin, and a-MSH [508], and production of both CRH and ACTH from a pituitary carcinoma has been described [509].

Most nonpituitary POMC-secreting tumors do not produce CRH [515]. However, some patients with ectopic ACTH syndrome due to lung cancer have tumors that produce ACTH and CRH, and secrete ACTH in response to CRH [516]. Bronchial carcinoid tumors may contain CRH and ACTH, and be associated with high plasma ACTH and CRH levels and Cushing’s syndrome, or with normal plasma CRH levels. Cushing’s syndrome has been associated with ectopic ACTH secretion from a unilateral adrenal pheochromocytoma [517], and from bilateral pheochromocytomas in a case of multiple endocrine neoplasia type 2A [518]. A number of other ectopic tumors have been described that secrete ACTH, including an adenoid cystic carcinoma of the lung [519], a renal cell carcinoma [520], a neuroendocrine tumor of the nasal roof [521], and an ACTHproducing tumor metastatic to the liver [522]. In general, POMC mRNA from nonpituitary tumors responsible for the ectopic ACTH syndrome is identical to normal and to POMC mRNA from pituitary tumors [523]. However, some tumors contain a larger POMC mRNA species that is increased in amount from 0.3% of the overall POMC mRNA in normal pituitaries, to up to 35 to 40% in the tumor, and is transcribed from an alternative upstream promoter [523]. Pancreatic islet cell tumors responsible for Cushing’s syndrome have been demonstrated to contain ACTH and b-endorphin [524], and to express POMC mRNA [524].

Ectopic ACTH-secreting Tumors Several neuroendocrine neoplasms occur in the bronchopulmonary tract, including small-cell neuroendocrine carcinomas, carcinoids, well-differentiated neuroendocrine carcinomas, and intermediate-cell neuroendocrine carcinomas [510]. These neoplasms express neuroendocrine markers including chromogranins and synaptophysin [510]. Thirtyfour percent of small-cell carcinomas of the lung show immunoreactivity to one or more peptide hormones [511], and patients with peptide-positive small-cell lung carcinomas have a shorter mean survival than in patients with nonreactive tumors. ACTH is by far the most common hormone present, and is seen in 24% of small-cell carcinomas [511]. However, 56% of small-cell lung cancer cell lines secrete significant concentrations of ACTH precursors, with little, if any, processing to ACTH. Oat-cell lung carcinomas may also produce ACTH, leading to Cushing’s syndrome [512]. Carcinoid tumors often produce more than one hormone, and can be responsible for the ectopic ACTH syndrome [513]. Bronchial carcinoids occasionally contain ACTH and related opioid peptides, which does not alter the overall favorable prognosis of these tumors. Recently, Cushing’s syndrome caused by ACTH secretion by pulmonary tumorlets has been described [514]. Upon radiologic imaging, such tumorlets, which may be over 100 in number, present a very unusual appearance.

REFERENCES 1 Ikeda H, Suzuki J, Sasano N et al. The development and morphogenesis of the human pituitary gland. Anat Embryol (Berl) 1988;178:327–336. 2 Blumenfeld Z, Jaffe RB. Hypophysiotropic and neuromodulatory regulation of adrenocorticotropin in the human fetal pituitary gland. J Clin Invest 1986; 78:288–294. 3 Doniach I. Histopathology of the pituitary. Clin Endocrinol Metab 1985;14: 765–789. 4 Puy LA, Ciocca DR. Human pharyngeal and sellar pituitary glands: differences and similarities revealed by an immunocytochemical study. J Endocrinol 1986;108:231–238. 5 Lloyd RV, Chandler WF, Kovacs K et al. Ectopic pituitary adenomas with normal anterior pituitary glands. Am J Surg Pathol 1986;10:546–552. 6 Hermesz E, Mackem S, Mahon KA. Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke’s pouch of the mouse embryo. Development 1996; 122:41–52. 7 Osumi-Yamashita N, Ninomiya Y, Doi H et al. The contribution of both forebrain and midbrain crest cells to the mesenchyme in the frontonasal mass of mouse embryos. Dev Biol 1994;164:409–419. 8 Kawamura K, Kikuyama S. Evidence that hypophysis and hypothalamus constitute a single entity from the primary stage of histogenesis. Development 1992;115:1–9. 9 Couly GF, Le Douarin NM. Mapping of the early neural primordium in quail-chick chimeras. I. Developmental relationships between placodes, facial ectoderm, and prosencephalon. Dev Biol 1985;110:422–439. 10 Couly GF, Le Douarin NM. Mapping of the early neural primordium in quail-chick chimeras. II. The prosencephalic neural plate and neural folds: implications for the genesis of cephalic human congenital abnormalities. Dev Biol 1987;120:198–214. 11 Dattani MT, Martinez-Barbera JP, Thomas PQ et al. Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nat Genet 1998;19:125–133.

Chapter 2 12 Hellstrom A, Aronsson M, Axelson C et al. Children with septo-optic dysplasia—how to improve and sharpen the diagnosis. Horm Res 2000;53 Suppl S1:19–25. 13 Wales JK, Quarrell OW. Evidence for possible Mendelian inheritance of septooptic dysplasia. Acta Paediatr 1996;85:391–392. 14 Sheng HZ, Zhadanov AB, Mosinger B, Jr et al. Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 1996;272:1004–1007. 15 Lamonerie T, Tremblay JJ, Lanctot C et al. Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene. Genes Dev 1996;10:1284–1295. 16 Szeto DP, Ryan AK, O’Connell SM et al. P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc Natl Acad Sci USA 1996;93:7706–7710. 17 Lanctot C, Gauthier Y, Drouin J. Pituitary homeobox 1 (Ptx 1) is differentially expressed during pituitary development. Endocrinology 1999;140:1416–1422. 18 Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 1998;12:428–441. 19 Semina EV, Reiter R, Leysens NJ et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 1996;14:392–399. 20 Gage PJ, Camper SA. Pituitary homeobox 2, a novel member of the bicoidrelated family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 1997;6:457–464. 21 Li S, Crenshaw EB, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 22 Radovick S, Cohen LE, Wondisford FE. The molecular basis of hypopituitarism. [Review] [40 refs]. Horm Res 1998;49 Suppl 1:30–36. 23 Pernasetti F, Toledo SP, Vasilyev VV et al. Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301-302delAG) in the prophet of Pit-1 gene. J Clin Endocrinol Metab 2000;85:390–397. 24 Ingraham HA, Lala DS, Ikeda Y et al. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8: 2302–2312. 25 Lee JE, Hollenberg SM, Snider L et al. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 1995;268: 836–844. 26 Naya FJ, Stellrecht CM, Tsai MJ. Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev 1995;9: 1009–1019. 27 Therrien M, Drouin J. Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene. Mol Cell Biol 1993;13:2342–2353. 28 Poulin G, Turgeon B, Drouin J. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 1997;17: 6673–6682. 29 Dunn KJ, Williams BO, Li Y et al. Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development. Proc Natl Acad Sci USA 2000;97:10050–10055. 30 Schwind JL. The development of the hypophysis cerebri of the albino rat. Am J Anat 1928;41:295–315. 31 Daikoku S, Chikamori M, Adachi T et al. Effect of the basal diencephalon on the development of Rathke’s pouch in rats: a study in combined organ cultures. Dev Biol 1982;90:198–202. 32 Heideveld M, Ayoubi TA, van de Wiel MH et al. Proopiomelanocortin gene expression as a neural marker during the embryonic development of Xenopus laevis. Differentiation 1993;52:195–200. 33 Kimura S, Hara Y, Pineau T et al. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 1996;10:60–69. 34 Lazzaro D, Price M, de Felice M et al. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 1991;113:1093–1104. 35 Takuma N, Sheng HZ, Furuta Y et al. Formation of Rathke’s pouch requires dual induction from the diencephalon. Development 1998;125:4825–4840. 36 Treier M, Gleiberman AS, O’Connell SM et al. Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 1998;12: 1691–1704. 37 Ericson J, Norlin S, Jessell TM et al. Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 1998;125:1005–1015.

Adrenocorticotropin

69

38 Muglia L, Jacobson L, Dikkes P et al. Corticotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 1995;373:427–432. 39 Schonemann MD, Ryan AK, McEvilly RJ et al. Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2. Genes Dev 1995;9:3122–3135. 40 Kimura N, Andoh N, Sasano N et al. Presence of neurophysins in the human pituitary corticotrophs, Cushing’s adenomas, and growth hormone-producing adenomas detected by immunohistochemical study. Am J Pathol 1986;125: 269–275. 41 Deftos LJ, O’Connor DT, Wilson CB et al. Human pituitary tumors secrete chromogranin-A. J Clin Endocrinol Metab 1989;68:869–872. 42 Vrontakis ME, Sano T, Kovacs K et al. Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary. J Clin Endocrinol Metab 1990;70:747–751. 43 Kitahara A, Takano E, Ohtsuki H et al. Reversed distribution of calpains and calpastatin in human pituitary gland and selective localization of calpastatin in adrenocorticotropin-producing cells as demonstrated by immunohistochemistry. J Clin Endocrinol Metab 1986;63:343–348. 44 Hsu DW, Riskind PN, Hedley Whyte ET. Vasoactive intestinal peptide in the human pituitary gland and adenomas. An immunocytochemical study. Am J Pathol 1989;135:329–338. 45 Uei Y. Immunohistological study of Crooke’s cells. Pathol Res Pract 1988;183: 636–637. 46 Halliday WC, Asa SL, Kovacs K et al. Intermediate filaments in the human pituitary gland: an immunohistochemical study. Can J Neurol Sci 1990;17: 131–136. 47 Ogawa A, Sugihara S, Nakanishi Y et al. Intermediate filament expression in non-neoplastic pituitary cells. Virchows Arch [B] 1990;58:331–340. 48 Stefaneanu L, Ryan N, Kovacs K. Immunocytochemical localization of synaptophysin in human hypophyses and pituitary adenomas. Arch Pathol Lab Med 1988;112:801–804. 49 Desy L, Pelletier G. Immunohistochemical localization of alpha-melanocyte stimulating hormone (alpha-MSH) in the human hypothalamus. Brain Res 1978;154:377–381. 50 Parker CR, Jr, Barnea A, Tilders FJ et al. Characterization of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in human brain tissue. Brain Res Bull 1981;6:275–280. 51 Bugnon C, Bloch B, Lenys D et al. Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins, ACTH, MSH and beta-LPH. Cell Tissue Res 1979;199:177–196. 52 Pilcher WH, Joseph SA, McDonald JV. Immunocytochemical localization of proopiomelanocortin neurons in human brain areas subserving stimulation analgesia. J Neurosurg 1988;68:621–629. 53 Emson PC, Corder R, Ratter SJ et al. Regional distribution of proopiomelanocortin-derived peptides in the human brain. Neuroendocrinology 1984;38:45–50. 54 Haskell-Luevano C, Chen P, Li C et al. Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat. Endocrinology 1999;140:1408–1415. 55 Broberger C, Johansen J, Johansson C et al. The neuropeptide Y/agouti generelated protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci USA 1998;95:15043–15048. 56 Haskell-Luevano C, Rosenquist A, Souers A et al. Compounds that activate the mouse melanocortin-1 receptor identified by screening a small molecule library based upon the beta-turn. J Med Chem 1999;42:4380–4387. 57 Cheung CC, Clifton DK, Steiner RA. Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 1997;138:4489–4492. 58 DeBold CR, Menefee JK, Nicholson WE et al. Proopiomelanocortin gene is expressed in many normal human tissues and in tumors not associated with ectopic adrenocorticotropin syndrome. Mol Endocrinol 1988;2:862–870. 59 Buzzetti R, McLoughlin L, Lavender PM et al. Expression of proopiomelanocortin gene and quantification of adrenocorticotropic hormone-like immunoreactivity in human normal peripheral mononuclear cells and lymphoid and myeloid malignancies. J Clin Invest 1989;83:733–737. 60 Lacaze Masmonteil T, de Keyzer Y, Luton JP et al. Characterization of proopiomelanocortin transcripts in human nonpituitary tissues. Proc Natl Acad Sci USA 1987;84:7261–7265. 61 Clark AJ, Lavender PM, Coates P et al. In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA. Mol Endocrinol 1990;4:1737–1743. 62 Rajora N, Ceriani G, Catania A et al. Alpha-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line. J Leukoc Biol 1996;59:248–253.

70

SECTION 1

Hypothalamic–Pituitary Function

63 Wong KY, Rajora N, Boccoli G et al. A potential mechanism of local antiinflammatory action of alpha-melanocyte-stimulating hormone within the brain: modulation of tumor necrosis factor-alpha production by human astrocytic cells. Neuroimmunomodulation 1997;4:37–41. 64 Labbe O, Desarnaud F, Eggerickx D et al. Molecular cloning of a mouse melanocortin 5 receptor gene widely expressed in peripheral tissues. Biochemistry 1994;33:4543–4549. 65 Luger TA, Schauer E, Trautinger F et al. Production of immunosuppressing melanotropins by human keratinocytes. Ann NY Acad Sci 1993;680: 567–570. 66 Slominski A, Ermak G, Hwang J et al. Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin. FEBS Lett 1995;374:113–116. 67 Millington WR, Rosenthal DW, Unal CB et al. Localization of proopiomelanocortin mRNA transcripts and peptide immunoreactivity in rat heart [see comments]. Cardiovasc Res 1999;43:107–116. 68 Chretien M, Benjannet S, Gossard F et al. From beta-lipotropin to betaendorphin and “pro-opio-melanocortin”. Can J Biochem 1979;57: 1111–1121. 69 Whitfeld PL, Seeburg PH, Shine J. The human pro-opiomelanocortin gene: organization, sequence, and interspersion with repetitive DNA. DNA 1982;1:133–143. 70 Litt M, Buroker NE, Kondoleon S et al. Chromosomal localization of the human proenkephalin and prodynorphin genes. Am J Hum Genet 1988;42:327–334. 71 Eberwine JH, Roberts JL, Analysis of pro-opiomelancortin gene structure and function. DNA 1983;2:1–8. 72 Imura H. ACTH and related peptides: molecular biology, biochemistry and regulation of secretion. Clin Endocrinol Metab 1985;14:845–866. 73 Robinson P, Bateman A, Mulay S et al. Isolation and characterization of three forms of joining peptide from adult human pituitaries: lack of adrenal androgen-stimulating activity. Endocrinology 1991;129:859–867. 74 Notake M, Kurosaki T, Yamamoto T et al. Sequence requirement for transcription in vitro of the human corticotropin/beta-lipotropin precursor gene. Eur J Biochem 1983;133:599–605. 75 Israel A, Cohen SN. Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells. Mol Cell Biol 1985;5:2443–2453. 76 Therrien M, Drouin J. Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements. Mol Cell Biol 1991;11:3492–3503. 77 Lamolet B, Pulichino A, Lamonerie T et al. A pituitary cell-restricted t box factor, tpit, activates pomc transcription in cooperation with pitx homeoproteins. Cell 2001;104:849–859. 78 Jeannotte L, Burbach JP, Drouin J. Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly (A) tails in hypothalamus. Mol Endocrinol 1987;1:749–757. 79 Takahashi H, Hakamata Y, Watanabe Y et al. Complete nucleotide sequence of the human corticotropin-beta-lipotropin precursor gene. Nucleic Acids Res 1983;11:6847–6858. 80 Suda T, Tozawa F, Yamada M et al. Effects of corticotropin-releasing hormone and dexamethasone on proopiomelanocortin messenger RNA level in human corticotroph adenoma cells in vitro. J Clin Invest 1988;82:110–114. 81 Fremeau RT, Jr, Lundblad JR, Pritchett DB et al. Regulation of proopiomelanocortin gene transcription in individual cell nuclei. Science 1986;234:1265–1269. 82 Potter E, Sutton S, Donaldson C et al. Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc Natl Acad Sci USA 1994;91:8777–8781. 83 Aguilera G, Harwood JP, Wilson JX et al. Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells. J Biol Chem 1983;258:8039–8045. 84 Pozzoli G, Bilezikjian LM, Perrin MH et al. Corticotropin-releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger ribonucleic acid in rat anterior pituitary cell cultures. Endocrinology 1996;137:65–71. 85 Loeffler JP, Kley N, Pittius CW et al. Calcium ion and cyclic adenosine 3¢,5¢monophosphate regulate proopiomelanocorin messenger ribonucleic acid levels in rat intermediate and anterior pituitary lobes. Endocrinology 1986;119:2840–2847. 86 Jin WD, Boutillier AL, Glucksman MJ et al. Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein. Mol Endocrinol 1994;8:1377–1388.

87 Yang-Yen HF, Chambard JC, Sun YL et al. Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 1990;62:1205–1215. 88 Wardlaw SL, McCarthy KC, Conwell IM. Glucocorticoid regulation of hypothalamic proopiomelanocortin. Neuroendocrinology 1998;67:51–57. 89 Auernhammer CJ, Melmed S. Leukemia-inhibitory factor-neuroimmune modulator of endocrine function. Endocr Rev 2000;21:313–345. 90 Akita S, Webster J, Ren SG et al. Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion. J Clin Invest 1995;95:1288–1298. 91 Bousquet C, Zatelli MC, Melmed S. Direct regulation of pituitary proopiomelanocorin by STAT3 provides a novel mechanism for immunoneuroendocrine interfacing. J Clin Invest 2000;106:1417–1425. 92 Bousquet C, Susini C, Melmed S. Inhibitory roles for SHP-1 and SOCS-3 following pituitary proopiomelanocortin induction by leukemia inhibitory factor. J Clin Invest 1999;104:1277–1285. 93 Yano H, Readhead C, Nakashima M et al. Pituitary-directed leukemia inhibitory factor transgene causes Cushing’s syndrome: neuro–immune– endocrine modulation of pituitary development. Mol Endocrinol 1998; 12:1708–1720. 94 Akita S, Malkin J, Melmed S. Disrupted murine leukemia inhibitory factor (LIF) gene attenuates adrenocorticotropic hormone (ACTH) secretion. Endocrinology 1996;137:3140–3143. 95 Wang Z, Ren SG, Melmed S. Hypothalamic and pituitary leukemia inhibitory factor gene expression in vivo: a novel endotoxin-inducible neuro-endocrine interface. Endocrinology 1996;137:2947–2953. 96 Rivier CL, Plotsky PM. Mediation by corticotropin releasing factor (CRF) of adenohypophysial hormone secretion. Annu Rev Physiol 1986;48: 475–494. 97 DeBold CR, Sheldon WR, Decherney GS et al. Arginine vasopressin potentiates adrenocorticotropin release induced by ovine corticotropin-releasing factor. J Clin Invest 1984;73:533–538. 98 Oki Y, Nicholson WE, Orth DN. Role of protein kinase-C in the adrenocorticotropin secretory response to arginine vasopressin (AVP) and the synergistic response to AVP and corticotropin-releasing factor by perifused rat anterior pituitary cells. Endocrinology 1990;127:350–357. 99 Levin N, Blum M, Roberts JL. Modulation of basal and corticotropinreleasing factor-stimulated proopiomelanocortin gene expression by vasopressin in rat anterior pituitary. Endocrinology 1989;125:2957–2966. 100 Luini A, Lewis D, Guild S et al. Hormone secretagogues increase cytosolic calcium by increasing cAMP in corticotropin-secreting cells. Proc Natal Acad Sci USA 1985;82:8034–8038. 101 Tozawa F, Suda T, Yamada M et al. Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland. Endocrinology 1988;122:1231–1235. 102 Robinson BG, Mealy K, Wilmore DW et al. The effect of insulin-induced hypoglycemia on gene expression in the hypothalamic–pituitary–adrenal axis of the rat. Endocrinology 1992;130:920–925. 103 Plotsky PM, Bruhn TO, Vale W. Hypophysiotropic regulation of adrenocorticotropin secretion in response to insulin-induced hypoglycemia. Endocrinology 1985;117:323–329. 104 Mezey E, Reisine TD, Brownstein MJ et al. Beta-adrenergic mechanism of insulin-induced adrenocorticotropin release from the anterior pituitary. Science 1984;226:1085–1087. 105 Loeffler JP, Demeneix BA, Pittius CW et al. GABA differentially regulates the gene expression of proopiomelanocortin in rat intermediate and anterior pituitary. Peptides 1986;7:253–258. 106 Facchinetti F, Storchi AR, Furani S et al. Pituitary changes of desacetyl-alphamelanocyte-stimulating hormone throughout development. Biol Neonate 1988;54:86–92. 107 Fenger M, Johnsen AH. Alpha-amidated peptides derived from proopiomelanocortin in normal human pituitary [published erratum appears in Biochem J 1988 Jun 15;252(3):935]. Biochem J 1988;250:781–788. 108 Eipper BA, Mains RE. Structure and biosynthesis of pro-adrenocorticotropin/ endorphin and related peptides. Endocr Rev 1980;1:1–27. 109 Glauder J, Ragg H, Rauch J et al. Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells. Biochem Biophys Res Commun 1990;169:551–558. 110 Tilders FJ, Parker CR, Jr, Barnea A et al. The major immunoreactive alpha-melanocyte-stimulating hormone (alpha MSH)-like substance found in human fetal pituitary tissue is not alpha MSH but may be desacetyl alpha MSH (adrenocorticotropin1-13NH2). J Clin Endocrinol Metab 1981;52: 319–323.

Chapter 2 111 Spruijt BM, De Graan PN, Eberle AN et al. Comparison of structural requirements of alpha-MSH and ACTH for inducing excessive grooming and pigment dispersion. Peptides 1985;6:1185–1189. 112 Mains RE, Dickerson IM, May V et al. Cellular and molecular aspects of peptide hormone biosynthesis. Front Neuroendo 1991;11:52–89. 113 Seidah NG, Gaspar L, Mion P et al. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases [published erratum appears in DNA Cell Biol 1990 Dec;9(10):789]. DNA Cell Biol 1990;9:415–424. 114 Seidah NG, Chretien M. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr Opin Biotechnol 1997;8:602–607. 115 Benjannet S, Rondeau N, Day R et al. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991;88:3564–3568. 116 Korner J, Chun J, Harter D et al. Isolation and functional expression of a mammalian prohormone processing enzyme, murine prohormone convertase 1. Proc Natl Acad Sci USA 1991;88:6834–6838. 117 Jackson RS, Creemers JW, Ohagi S et al. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene [see comments]. Nat Genet 1997;16:303–306. 118 Naggert JK, Fricker LD, Varlamov O et al. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat Genet 1995;10:135–142. 119 Morris JF, Pow DV. Capturing and quantifying the exocytotic event. J Exp Biol 1988;139:81–103. 120 Malide D, Seidah NG, Chretien M et al. Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells. J Histochem Cytochem 1995;43:11–19. 121 Hsi KL, Seidah NG, De Serres G et al. Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide. Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60. FEBS Lett 1982;147:261–266. 122 Steel JH, Van Noorden S, Ballesta J et al. 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;122:270–282. 123 Braks JA, Martens GJ. 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 1994;78:263–273. 124 Paquet L, Bergeron F, Boudreault A et al. The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase. J Biol Chem 1994;269:19279–19285. 125 Muller L, Zhu X, Lindberg I. Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding. J Cell Biol 1997;139:625–638. 126 Westphal CH, Muller L, Zhou A et al. The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing’s disease. Cell 1999;96:689–700. 127 Furuta M, Yano H, Zhou A et al. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 1997;94:6646–6651. 128 Seidel B, Dong W, Savaria D et al. Neuroendocrine protein 7B2 is essential for proteolytic conversion and activation of proprotein convertase 2 in vivo. DNA Cell Biol 1998;17:1017–1029. 129 Fricker LD, McKinzie AA, Sun J et al. Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing. J Neurosci 2000;20:639–648. 130 Seidah NG, Chretien M. Complete amino acid sequence of a human pituitary glycopeptide: an important maturation product of pro-opiomelanocortin. Proc Natl Acad Sci USA 1981;78:4236–4240. 131 Parker LN. Control of adrenal androgen secretion. Endocrinol Metab Clin North Am 1991;20:401–421. 132 Evans VR, Manning AB, Bernard LH et al. Alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin immunoreactivites are localized in the human pituitary but are not restricted to the zona intermedia. Endocrinology 1994;134:97–106. 133 Coates PJ, Doniach I, Wells C et al. Peptides related to alpha-melanocytestimulating hormone are commonly produced by human pituitary corticotroph adenomas: no relationship with pars intermedia origin. J Endocrinol 1989;120:531–536. 134 Thody AJ, Fisher C, Kendal-Taylor P et al. The measurement and characterisation by high pressure liquid chromatography of immunoreactive

135 136 137

138

139 140 141 142

143

144

145

146

147

148 149 150 151

152

153

154 155

156

157

158

159

Adrenocorticotropin

71

alpha-melanocyte stimulating hormone in human plasma. Acta Endocrinol (Copenh) 1985;110:313–318. Thody AJ, Plummer NA. The melanocyte stimulating activity of several commercial corticotrophin preparations. Br J Dermatol 1971;84:361–365. Kleber G, Gramsch C, Hollt V et al. Extrahypothalamic corticotropin and alpha-melanotropin in human brain. Neuroendocrinology 1980;31:39–45. Hollt V, Haarmann I, Seizinger BR et al. Chronic haloperidol treatment increases the level of in vitro translatable messenger ribonucleic acid coding for the beta-endorphin/adrenocorticotropin precursor proopiomelanocortin in the pars intermedia of the rat pituitary. Endocrinology 1982;110:1885–1891. Saiardi A, Borrelli E. Absence of dopaminergic control on melanotrophs leads to Cushing’s-like syndrome in mice. Mol Endocrinol 1998;12: 1133–1139. Yalow RS. Radioimmunoassay: a probe for the fine structure of biologic systems. Science 1978;200:1236–1245. Yalow RS. Multiple forms of corticotropin (adrenocorticotropic hormone, ACTH) and their significance. Ciba Found Symp 1976;41:159–181. Gibson S, Pollock A, Littley M et al. Advantages of IRMA over IA in the measurement of ACTH. Ann Clin Biochem 1989;26:500–507. White A, Smith H, Hoadley M et al. Clinical evaluation of a two-site immunoradiometric assay for adrenocorticotrophin in unextracted human plasma using monoclonal antibodies. Clin Endocrinol (Oxf ) 1987;26:41–51. Fukata J, Naitoh Y, Usui T et al. Two-site immunoradiometric assay for adrenocorticotrophin: a cautionary study about the reactivity to its precursor molecules. Endocrinol Jpn 1989;36:155–161. Dobson SH, Gray C, Smith H et al. Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH. J Immunol Methods 1986;88:83–90. Crosby SR, Stewart MF, Ratcliffe JG et al. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay. J Clin Endocrinol Metab 1988;67:1272–1277. Dobson S, White A, Hoadley M et al. Measurement of corticotropin in unextracted plasma: comparison of a time-resolved immunofluorometric assay and an immunoradiometric assay, with use of the same monoclonal antibodies. Clin Chem 1987;33:1747–1751. Dean B, Kolavcic M, Tizian L et al. Should plasma beta-endorphin be measured in patients with disorders of the hypothalamic–pituitary–adrenal axis. Clin Chem 1986;32:895. Boscaro M, Giacromazzi P, Sonino N et al. Pro-gamma-MSH levels in various disorders of pituitary-adrenal axis. J Endocrinol Invest 1990;13:29–33. Sobel DO. Role of cyclic AMP in corticotropin releasing factor mediated ACTH release. Peptides 1985;6:591–595. Guillon G. [Vasopressin, oxytocin and angiotensin receptors in mammals]. Ann Endocrinol (Paris) 1989;50:425–433. Raymond V, Leung PC, Veilleux R et al. Vasopressin rapidly stimulates phosphatidic acid-phosphatidylinositol turnover in rat anterior pituitary cells. FEBS Lett 1985;182:196–200. Todd K, Lightman SL. Vasopressin activation of phosphatidylinositol metabolism in rat anterior pituitary in vitro and its modification by changes in the hypothalamo–pituitary–adrenal axis. Neuroendocrinology 1987;45: 212–218. Abou-Samra AB, Catt KJ, Aguilera G. Involvement of protein kinase C in the regulation of adrenocorticotropin release from rat anterior pituitary cells. Endocrinology 1986;118:212–217. Leong DA. A complex mechanism of facilitation in pituitary ACTH cells: recent single-cell studies. J Exp Biol 1988;139:151–168. DeBold CR, Decherney Gs, Jackson RV et al. Effect of synthetic ovine corticotropin-releasing factor: prolonged duration of action and biphasic response of plasma adrenocorticotropin and cortisol. J Clin Endocrinol Metab 1983;57:294–298. Antoni FA, Fink G, Sheward WJ. Corticotrophin-releasing peptides in rat hypophysial portal blood after paraventricular lesions: a marked reduction in the concentration of corticotrophin-releasing factor-41, but no change in vasopressin. J Endocrinol 1990;125:175–183. Gibbs DM, Stewart RD, Vale W et al. Synthetic corticotropin-releasing factor stimulates secretion of immunoreactive beta-endorphin/beta-lipotropin and ACTH by human fetal pituitaries in vitro. Life Sci 1983;32:547–550. Plotsky PM, Vale W. Hemorrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat hypophysial portal circulation and its inhibition by glucocorticoids. Endocrinology 1984;114:164–169. DeBold CR, Sheldon WR, Jr, Decherney GS et al. Effect of subcutaneous and intranasal administration of ovine corticotropin-releasing hormone in man: comparison with intravenous administration. J Clin Endocrinol Metab 1985;60: 836–840.

72

SECTION 1

Hypothalamic–Pituitary Function

160 Smith R, Mesiano S, Chan EC et al. Corticotropin-releasing hormone directly and preferentially stimulates dehydroepiandrosterone sulfate secretion by human fetal adrenal cortical cells. J Clin Endocrinol Metab 1998;83:2916–2920. 161 De Souza EB, Perrin MH, Whitehouse PJ et al. Corticotropin-releasing factor receptors in human pituitary gland: autoradiographic localization. Neuroendocrinology 1985;40:419–422. 162 Aguilera G, Wynn PC, Harwood JP et al. Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system. Neuroendocrinology 1986;43:79–88. 163 Chen R, Lewis K, Perrin M et al. Expression cloning of a human corticotropin-releasing factor receptor. Proc Natl Acad Sci USA 1993;90: 8967–8971. 164 Chang CP, Pearse RV, O’Connell S et al. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 1993;11:1187–1195. 165 Perrin M, Donaldson C, Chen R et al. Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA 1995;92:2969–2973. 166 Arai M, Assil IQ, Abou-Samra AB. Characterization of three corticotropinreleasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis. Endocrinology 2001;142:446–454. 167 Vaughan J, Donaldson C, Bittencourt J et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor [see comments]. Nature 1995;378:287–292. 168 Weninger SC, Dunn AJ, Muglia LJ et al. Stress-induced behavior require the CRH receptor, but not CRH. Proc Natl Acad Sci USA 1999;96:8283–8298. 169 Smith GW, Aubry JM, Dellu F et al. Corticotropin releasing factor receptor 1deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 1998;20:1093–1102. 170 Coste SC, Kesterson RA, Heldwein KA et al. Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat Genet 2000 Apr 2001;24:403–409. 171 Schulz DW, Mansbach RS, Sprouse J et al. CP-154,526: a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors. Proc Natal Acad Sci USA 1996;93:10477–10482. 172 Deak T, Nguyen KT, Ehrlich AL et al. The impact of the nonpeptide corticotropin-releasing hormone antagonist antalarmin on behavior and endocrine responses to stress. Endocrinology 1999;140:79–86. 173 Spinedi E, Negro-Vilar A. Arginine vasopressin and adrenocorticotropin release: correlation between binding characteristics and biological activity in anterior pituitary dispersed cells. Endocrinology 1984;114:2247–2251. 174 Gaillard RC, Schoeneberg P, Favrod-Coune CA et al. Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin-releasing factor. Proc Natl Acad Sci USA 1984;81:2907–2911. 175 Baertschi AJ. Portal vascular route from hypophysial stalk/neural lobe to adenohypophysis. Am J Physiol 1980;239:R463–R469. 176 Childs GV, Unabia G, Burke JA et al. Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP). Am J Physiol 1987;252:E347–E356. 177 Schmidt A, Audigier s, Barberis C et al. A radioiodinated linear vasopressin antagonist: a ligand with high affinity and specificity for V1a receptors. FEBS Lett 1991;282:77–81. 178 Gaillard RC, Riondel AM, Ling N et al. Corticotropin releasing factor activity of CRF 41 in normal man is potentiated by angiotensin II and vasopressin but not by desmopressin. Life Sci 1988;43:1935–1944. 179 Lolait SJ, O’Carroll AM, Brownstein MJ. Molecular biology of vasopressin receptors. Ann NY Acad Sci 1995;771:273–292. 180 Aguilera G, Rabadan-Diehl C. Regulation of vasopressin V1b receptors in the anterior pituitary gland of the rat. [Review] [47 refs]. Exp Physiol 2000 Mar 2001;85 Spec No:19S–26S. 181 Rabadan-Diehl C, Aguilera G. Glucocorticoids increase vasopressin V1b receptor coupling to phospholipase C. Endocrinology 1998;139:3220-3226. 182 Plotsky PM. Hypophysiotropic regulation of stress-induced ACTH secretion. Adv Exp Med Biol 1988;245:65–81. 183 Dallman MF, Akana SF, Cascio CS et al. Regulation of ACTH Secretion: Variations on a Theme of B. Recent Progress in Hormone Research 1987;43: 113–173. 184 Koenig JI. Pituitary gland: neuropeptides, neurotransmitters and growth factors. Toxicol Pathol 1989;17:256–265. 185 Hedge GA, Yates MB, Marcus R et al. Site of action of vasopressin in causing corticotropin release. Endocrinology 1966;79:328–340. 186 Jones MT, Gillham B, Altaher AR et al. Clinical and experimental studies on the role of GABA in the regulation of ACTH secretion: a review. Psychoneuroendocrinology 1984;9:107–123.

187 Jones MT, Hillhouse EW, Burden J. Effect on various putative neurotransmitters on the secretion of corticotrophin-releasing hormone from the rat hypothalamus in vitro – a model of the neurotransmitters involved. J Endocrinol 1976;69:1–10. 188 Rivier C, Vale W. Effects of corticotropin-releasing factor, neurohypophyseal peptides, and catecholamines on pituitary function. Fed Proc 1985;44: 189–195. 189 al-Damluji S. Adrenergic mechanisms in the control of corticotrophin secretion. J Endocrinol 1988;119:5–14. 190 Watanabe T, Oki Y, Orth DN. Kinetic actions and interactions of arginine vasopressin, angiotensin-II, and oxytocin on adrenocorticotropin secretion by rat anterior pituitary cells in the microperifusion system. Endocrinology 1989; 125:1921–1931. 191 Schoenenberg P, Gaillard RC, Kehrer P et al. cAMP-dependent ACTH secretagogues facilitate corticotropin releasing activity of angiotensin II on rat anterior pituitary cells in vitro. Acta Endocrinol (Copenh) 1987;114:118–123. 192 McCann SM, Rettori V, Milenkovic L et al. Role of monokines in control of anterior pituitary hormone release. Adv Exp Med Biol 1990;274:315–329. 193 Berkenbosch F, van Oers J, del Rey A et al. Corticotropin-releasing factorproducing neurons in the rat activated by interleukin-1. Science 1987;238: 524–526. 194 Sapolsky R, Rivier C, Yamamoto G et al. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987;238:522–524. 195 Matta S, Singh J, Newton R et al. The adrenocorticotropin response to interleukin-1 beta instilled into the rat median eminence depends on the local release of catecholamines. Endocrinology 1990;127:2175–2182. 196 Michie HR, Majzoub JA, ODwyer ST et al. Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans. Surgery 1990;108:254–259. 197 Michie HR, Spriggs DR, Manogue KR et al. Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery 1988;104:280–286. 198 Gaillard RC, Turnill D, Sappino P et al. Tumor necrosis factor alpha inhibits the hormonal response of the pituitary gland to hypothalamic releasing factors. Endocrinology 1990;127:101–106. 199 Sharp BM, Matta SG, Peterson PK et al. Tumor necrosis factor-alpha is a potent ACTH secretagogue: comparison to interleukin-1 beta. Endocrinology 1989;124:3131–3133. 200 Michie HR, Manogue KR, Spriggs DR et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988;318: 1481–1486. 201 Yasuda N, Greer MA. Evidence that the hypothalamus mediates endotoxin stimulation of adrenocorticotropic hormone secretion. Endocrinology 1978; 102:947–953. 202 Naitoh Y, Fukata J, Tominaga T et al. Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 1988;155:1459–1463. 203 Navarra P, Tsagarakis S, Faria MS et al. Interleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway. Endocrinology 1991;128:37–44. 204 Bochicchio D, Ambrosi B, Faglia G. Loperamide, an opiate analog, differently modifies the adrenocorticotropin responses to corticotropin-releasing hormone and lysine vasopressin in patients with Addison’s disease. Neuroendocrinology 1988;48:611–614. 205 Atkinson RL. Endocrine and metabolic effects of opiate antagonists. J Clin Psychiatry 1984;45:20–24. 206 Gaillard RC, Grossman A, Smith R et al. The effects of a met-enkephalin analogue on ACTH, beta-LPH, beta-endorphin and MET-enkephalin in patients with adrenocortical disease. Clin Endocrinol (Oxf ) 1981;14:471–478. 207 Blankstein J, Reyes FI, Winter JS et al. Effects of naloxone upon prolactin and costisol in normal women. Proc Soc Exp Biol Med 1980;164:363–365. 208 Grossman A, Gaillard RC, McCartney P et al. Opiate modulation of the pituitary–adrenal axis: effects of stress and circadian rhythm. Clin Endocrinol (Oxf) 1982;17:279–286. 209 Morley JE, Baranetsky NG, Wingert TD et al. Endocrine effects of naloxoneinduced opiate receptor blockade. J Clin Endocrinol Metab 1980;50:251–257. 210 Volavka J, Cho D, Mallya A et al. Naloxone increases ACTH and cortisol levels in man [letter]. N Engl J Med 1979;300:1056–1057. 211 Raff H, Flemma RJ, Findling JW. Fast cortisol-induced inhibition of the adrenocorticotropin response to surgery in humans. J Clin Endocrinol Metab 1988;67:1146–1148. 212 Spiler IJ, Molitch ME. Lack of modulation of pituitary hormone stress response by neural pathways involving opiate receptors. J Clin Endocrinol Metab 1980;50:516–520.

Chapter 2 213 Wakabayashi I, Demura R, Miki N et al. Failure of naloxone to influence plasma growth hormone, prolactin, and cortisol secretions induced by insulin hypoglycemia. J Clin Endocrinol Metab 1980;50:597–599. 214 Rittmaster RS, Cutler GB Jr, Sobel DO et al. Morphine inhibits the pituitaryadrenal response to ovine corticotropin-releasing hormone in normal subjects. J Clin Endocrinol Metab 1985;60:891–895. 215 Allolio B, Deuss U, Kaulen D et al. FK 33-824, a met-enkephalin analog, blocks corticotropin-releasing hormone-induced adrenocorticotropin secretion in normal subjects but not in patients with Cushing’s disease. J Clin Endocrinol Metab 1986;63:1427–1431. 216 Watanabe T, Orth DN. Detailed kinetic analysis of adrenocorticotropin secretion by dispersed rat anterior pituitary cells in a microperifusion system: effects of ovine corticotropin-releasing factor and arginine vasopressin. Endocrinology 1987;121:1133–1145. 217 Lowry PJ, Estivariz FE, Gillies GE et al. CRF: its regulation of ACTH and pro-opiomelanocortin peptide release and its extra hypothalamic occurrence. Acta Endocrinol Suppl (Copenh) 1986;276:56–62. 218 Page SR, Ang VT, Jackson R et al. The effect of oxytocin infusion on adenohypophyseal function in man. Clin Endocrinol (Oxf ) 1990;32:307–313. 219 Antoni FA, Holmes MC, Kiss JZ. Pituitary binding of vasopressin is altered by experimental manipulations of the hypothalamo–pituitary–adrenocortical axis in normal as well as homozygous (di/di) Brattleboro rats. Endocrinology 1985; 117:1293–1299. 220 Gibbs DM, Vale W, Rivier J et al. Oxytocin potentiates the ACTH-releasing activity of CRF(41) but not vasopressin. Life Sci 1984;34:2245–2249. 221 Kovacs KJ, Mezey E. Dexamethasone inhibits corticotropin-releasing factor gene expression in the rat paraventricular nucleus. Neuroendocrinology 1987;46:365–368. 222 Mishunina TM, Kononenko VIa. [Hormonal control of metabolism of gammaaminobutyric acid in the rat hypothalamus and hippocampus]. Ukr Biokhim Zh 1990;62:71–79. 223 Abou-Samra AB, Catt KJ, Aguilera G. Biphasic inhibition of adrenocorticotropin release by corticosterone in cultured anterior pituitary cells. Endocrinology 1986;119:972–977. 224 Chhajlani V, Wikberg JE. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett 1992;309: 417–420. 225 Mountjoy KG, Robbins LS, Mortrud MT et al. The cloning of a family of genes that encode the melanocortin receptors. Science 1992;257:1248–1251. 226 Schioth HB, Chhajlani V, Muceniece R et al. Major pharmacological distinction of the ACTH receptor from other melanocortin receptors. Life Sci 1996;59:797–801. 227 Adan RA, Oosterom J, Ludvigsdottir G et al. Identification of antagonists for melanocortin MC3, MC4 and MC5 receptors. Eur J Pharmacol 1994;269: 331–337. 228 Schioth HB, Muceniece R, Wikberg JE et al. Characterisation of melanocortin receptor subtypes by radioligand binding analysis. Eur J Pharmacol 1995;288: 311–317. 229 Schioth HB, Muceniece R, Wikberg JE. Characterisation of the melanocortin 4 receptor by radioligand binding. Pharmacol Toxicol 1996;79:161–165. 230 Schioth HB, Muceniece R, Larsson M et al. The melanocortin 1, 3, 4 or 5 receptors do not have a binding epitope for ACTH beyond the sequence of alpha-MSH. J Endocrinol 1997;155:73–78. 231 Schioth HB, Muceniece R, Mutulis F et al. Selectivity of cyclic [D-Nal7] and [D-Phe7] substituted MSH analogues for the melanocortin receptor subtypes. Peptides 1997;18:1009–1013. 232 Schioth HB, Muceniece R, Larsson M et al. Binding of cyclic and linear MSH core peptides to the melanocortin receptor subtypes. Eur J Pharmacol 1997;319:369–373. 233 Adan RA, Cone RD, Burbach JP et al. Differential effects of melanocortin peptides on neural melanocortin receptors. Mol Pharmacol 1994;46:1182–1190. 234 Strand FL, Lee SJ, Lee TS et al. Non-corticotropic ACTH peptides modulate nerve development and regeneration. Rev Neurosci 1993;4:321–363. 235 Strand FL, Saint-Come C, Lee TS et al. ACTH/MSH(4-10) analog BIM 22015 aids regeneration via neurotrophic and myotrophic attributes. Peptides 1993;14:287–296. 236 Cone RD, Mountjoy KG, Robbins LS et al. Cloning and functional characterization of a family of receptors for the melanotropic peptides. Ann N Y Acad Sci 1993;680:342–363. 237 Bhardwaj RS, Becher E, Mahnke K et al. Evidence of the expression of a functional melanocortin receptor 1 by human keratinocytes (abstr). J Invest Dermatol 1996;106:817. 238 Bohm M, Schulte U, Hartmeyer M et al. Human dermal fibroblasts express melanocortin-1 receptors and respond to alpha-melanoctye stimulating

239

240

241

242 243 244

245

246

247 248

249 250

251

252 253

254 255

256

257

258

259

260

261

262

263

Adrenocorticotropin

73

hormone with secretion of interleukin-6 and interleukin-8 (abstr). J Invest Dermatol 1997;108:593. Suzuki I, Cone RD, Im S et al. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 1996;137:1627–1633. Xia Y, Skoog V, Muceniece R et al. Polyclonal antibodies against human melanocortin MC1 receptor: preliminary immunohistochemical localisation of melanocortin MC1 receptor to malignant melanoma cells. Eur J Pharmacol 1995;288:277–283. Chakraborty AK, Funasaka Y, Pawelek JM et al. Enhanced expression of melanocortin-1 receptor (MC1-R) in normal human keratinocytes during differentiation: evidence for increased expression of POMC peptides near suprabasal layer of epidermis. J Invest Dermatol 1999;112:853–860. Thody AJ, Graham A. Does alpha-MSH have a role in regulating skin pigmentation in humans? Pigment Cell Res 1998;11:265–274. Hearing VJ, Tsukamoto K. Enzymatic control of pigmentation in mammals. Faseb J 1991;5:2902–2909. Robbins LS, Nadeau JH, Johnson KR et al. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 1993;72:827–834. Valverde P, Healy E, Jackson I et al. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat Genet 1995;11:328–330. Koppula SV, Robbins LS, Lu D et al. Identification of common polymorphisms in the coding sequence of the human MSH receptor (MCR) with possible biological effects. Hum Mutat 1997;9:30–36. Rana BK, Hewett-Emmett D, Jin L et al. High polymorphism at the human melanocortin 1 receptor locus. Genetics 1999;151:1547–1557. Schioth HB, Phillips SR, Rudzish R et al. Loss of function mutations of the human melanocortin 1 receptor are common and are associated with red hair. Biochem Biophys Res Commun 1999;260:488–491. Healy E, Flannagan N, Ray A et al. Melanocortin-1-receptor gene and sun sensitivity in individuals without red hair [letter]. Lancet 2000;355:1072–1073. Slominski A, Wortsman J, Luger T et al. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. [Review] [457 refs]. Physiol Rev 2000;80:979–1020. Slominski A, Szczesniewski A, Worstman J. Liquid chromatography-mass spectrometry detection of corticotropin-releasing hormone and proopiomelanocortin-derived peptides in human skin. J Clin Endocrinol Metab 2000;85:3582–3588. Chhajlani V. Distribution of cDNA for melanocortin receptor subtypes in human tissues. Biochem Mol Biol Int 1996;38:73–80. Thornwall M, Dimitriou A, Xu X et al. Immunohistochemical detection of the melanocortin 1 receptor in human testis, ovary and placenta using specific monoclonal antibody. Horm Res 1997;48:215–218. Xia Y, Wikberg JE, Chhajlani V. Expression of melanocortin 1 receptor in periaqueductal gray matter. Neuroreport 1995;6:2193–2196. Lipton JM, Ceriani G, Macaluso A et al. Antiinflammatory effects of the neuropeptide alpha-MSH in acute, chronic, and systemic inflammation. Ann NY Acad Sci 1994;741:137–148. Catania A, Lipton JM. The neuropeptide alpha-melanocyte-stimulating hormone: a key component of neuroimmunomodulation. Neuroimmunomodulation 1994;1:93–99. Luger TA, Scholzen T, Brzoska T et al. Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone. Ann NY Acad Sci 1998;840:381–394. Ceriani G, Diaz J, Murphree S et al. The neuropeptide alpha-melanocytestimulating hormone inhibits experimental arthritis in rats. Neuroimmunomodulation 1994;1:28–32. Lipton JM, Carnes K, Macaluso A et al. The peptide alpha-MSH inhibits white blood cell migration in experimental adult respiratory distress syndrome (ARDS). Anesthesiology 1993;79:A300. Lipton JM, McCoy D, Macaluso A et al. The neuropeptide alpha-MSH increases survival in septic shock in mice. Abstract. Anesthesia Research Society. Orlando: 1994. Bhardwaj R, Becher E, Mahnke K et al. Evidence for the differential expression of the functional alpha-melanocyte-stimulating hormone receptor MC-1 on human monocytes. J Immunol 1997;158:3378–3384. Star RA, Rajora N, Huang J et al. Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone. Proc Natl Acad Sci USA 1995;92:8016–8020. Catania A, Rajora N, Capsoni F et al. The neuropeptide alpha-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro. Peptides 1996;17:675–679.

74

SECTION 1

Hypothalamic–Pituitary Function

264 Hartmeyer M, Scholzen T, Becher E et al. Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone. J Immunol 1997;159:1930–1937. 265 Haynes RC. The activation of adrenal phosphorylase by the adrenocorticotropic hormone. J Biol Chem 1958;233:1220–1222. 266 Lefkowitz RJ, Roth J, Pricer W et al. ACTH receptors in the adrenal: specific binding of ACTH-125I and its relation to adenyl cyclase. Proc Natl Acad Sci USA 1970;65:745–752. 267 Catalano RD, Stuve L, Ramachandran J. Characterization of corticotropin receptors in human adrenocortical cells. J Clin Endocrinol Metab 1986;62: 300–304. 268 Buckley DI, Ramachandran J. Characterization of corticotropin receptors on adrenocortical cells. Proc Natl Acad Sci USA 1981;78:7431–7435. 269 Dallman MF, Akana SF, Jacobson L et al. Characterization of corticosterone feedback regulation of ACTH secretion. Ann NY Acad Sci 1987;512:402–14. 270 Weber A, Kapas S, Hinson J et al. Functional characterization of the cloned human ACTH receptor: impaired responsiveness of a mutant receptor in familial glucocorticoid deficiency. Biochem Biophys Res Commun 1993;197: 172–178. 271 Naville D, Penhoat A, Barjhoux L et al. Characterization of the human ACTH receptor gene and in vitro expression. Endocr Res 1996;22:337–348. 272 Elias LL, Huebner A, Pullinger GD et al. Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: poor correlation of phenotype and genotype. J Clin Endocrinol Metab 1999;84: 2766–2770. 273 Brown MS, Kovanen PT, Goldstein JL. Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Recent Prog Horm Res 1979;35:215–257. 274 Pedersen RC, Brownie AC. Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of procorticotropin/endorphin. Proc Natl Acad Sci USA 1980;77:2239–2243. 275 Jefcoate CR, Simpson ER, Boyd GS. Spectral properties of rat adrenalmitochondrial cytochrome P-450. Eur J Biochem 1974;42:539–551. 276 Privalle CT, Crivello JF, Jefcoate CR. Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland. Proc Natl Acad Sci USA 1983;80:702–706. 277 Papadopoulos V, Brown AS, Hall PF. Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. Mol Cell Endocrinol 1990;74:109–123. 278 Miller WL, Strauss JF. Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StAR. J Steroid Biochem Mol Biol 2001; 69:131–141. 279 Simpson ER. Cholesterol side-chain cleavage, cytochrone P450, and the control of steroidogenesis. Mol Cell Endocrinol 1979;13:213–227. 280 Yago N, Ichii S. Submitochondrial distribution of components of the steroid 11 beta-hydroxylase and cholesterol sidechain-cleaving enzyme systems in hog adrenal cortex. J Biochem (Tokyo) 1969;65:215–224. 281 Churchill PF, Kimura T. Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion. J Biol Chem 1979;254:10443–10448. 282 Orme-Johnson NR. Distinctive properties of adrenal cortex mitochondria. Biochim Biophys Acta 1990;1020:213–231. 283 Miller WL. Molecular biology of steroid hormone synthesis. [Review]. Endocrine Reviews 1988;9:295–318. 284 Saez JM, Begeot M, Durand P. [ACTH receptors]. Ann Endocrinol (Paris) 1989; 50:409–417. 285 Tepperman J, Engel FL, Long CNH. A review of adrenal cortical hypertrophy. Endocrinol 1943;32:373–402. 286 Smith PE. Hypophysectomy and a replacement therapy in the rat. Amer J Anta 1930;45:205–273. 287 Ney RL, Dexter RN, Davis WW et al. A study of mechanisms by which adrenocorticotropic hormone maintains adrenal steroidogenic responsiveness. J Clin Invest 1967;46:1916–1924. 288 Ingle DJ. The functional interrelationship of anterior pituitary and the adrenal cortex. Ann Intern Med 1951;35:652–672. 289 Wyllie AH, Kerr JF, Macaskill IA et al. Adrenocortical cell deletion: the role of ACTH. J Pathol 1973;111:85–94. 290 Farese RV, Reddy WJ. Observations on the interrelations between adrenal protein, RNA and DNA during prolonged ACTH administration. Biocem Biophys Acta 1963;76:145–148. 291 Imai T, Seo H, Murata Y et al. Adrenocorticotropin increases expression of c-fos and beta-actin genes in the rat adrenals. Endocrinology 1990;127: 1742–1747.

292 Gospodarowicz D, Ill CR, Hornsby PJ et al. Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor. Endocrinology 1977;100:1080–1089. 293 Masui H, Garren LD. Inhibition of replication in functional mouse adrenal tumor cells by adrenocorticotropic hormone mediated by adenosine 3¢:5¢cyclic monophosphate. Proc Natl Acad Sci USA 1971;68:3206–3210. 294 Ramachandran J, Suyama AT. Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin. Proc Natl Acad Sci USA 1975;72:113–117. 295 Rao AJ, Long JA, Ramachandran J. Effects of antiserum to adrenocorticotropin on adrenal growth and function. Endocrinology 1978;102:371–378. 296 Dallman MF, Engeland WC, Holzwarth MA et al. Adrenocorticotropin inhibits compensatory adrenal growth after unilateral adrenalectomy. Endocrinology 1980;107:1397–1404. 297 Estivariz FE, Iturriza F, McLean C et al. Stimulation of adrenal mitogenesis by N-terminal proopiocortin peptides. Nature 1982;297:419–422. 298 Estivariz FE, Morano MI, Carino M et al. Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary. J Endocrinol 1988;116:207–216. 299 Engeland WC, Shinsako J, Dallman MF. Corticosteroids and ACTH are not required for compensatory adrenal growth. Am J Physiol 1975;229: 1461–1464. 300 Dallman MF, Engelmand WC, Shinsako J. Compensatory adrenal growth: a neurally mediated reflex. Am J Physiol 1976;231:408–414. 301 Albright F. Osteoporosis. Annals of Internal Medicine 1947;27:861–882. 302 Ishihara F, Komatsu M, Yamada T et al. Role of dehydroepiandrosterone and dehydroepiandrosterone sulfate for the maintenance of axillary hair in women. Horm Metab Res 1993;25:34–36. 303 Stewart ME, Downing DT, Cook JS et al. Sebaceous gland activity and serum dehydroepiandrosterone sulfate levels in boys and girls. Archives of Dermatology 1992;128:1345–1348. 304 Rosenfield RL, Grossman BJ, Ozoa N. Plasma 17-ketosteroids and testosterone in prepubertal children before and after ACTH administration. J Clin Endocrinol Metab 1971;33:249–253. 305 Rosenfield RL. Plasma 17-ketosteroids and 17-beta hydroxysteroids in girls with premature development of sexual hair. J Pediatr 1971;79: 260–266. 306 August GP, Hung W, Mayes DM. Plasma androgens in premature pubarche: value of 17 alpha-hydroxyprogesterone in differentiation from congenital adrenal hyperplasia. J Pediatr 1975;87:246–249. 307 Korth-Schutz S, Levine LS, New MI. Serum androgens in normal prepubertal and pubertal children and in children with precocious adrenarche. J Clin Endocrinol Metab 1976;42:117–124. 308 Warne GL, Carter JN, Faiman C et al. Hormonal changes in girls with precocious adrenarche: a possible role for estradiol or prolactin. J Pediatr 1978;92:743–747. 309 Sizonenko PC, Paunier L. Hormonal changes in puberty III: Correlation of plasma dehydroepiandrosterone, testosterone, FSH, and LH with stages of puberty and bone age in normal boys and girls and in patients with Addison’s disease or hypogonadism or with premature or late adrenarche. J Clin Endocrinol Metab 1975;41:894–904. 310 Parker LN, Odell WD. Control of adrenal androgen secretion. Endocr Rev 1980;1:392–410. 311 Vaitukaitis JL, Dale SL, Melby JC. Role of ACTH in the secretion of free dehydroepiandrosterone and its sulfate ester in man. J Clin Endocrinol Metab 1969;29:1443–1447. 312 Rosenfeld RS, Hellman L, Roffwarg H et al. Dehydroisoandrosterone is secreted episodically and synchronously with cortisol by normal man. J Clin Endocrinol Metab 1971;33:87–92. 313 Irvine WJ, Toft AD, Wilson KS et al. The effect of synthetic corticotropin analogues on adrenocortical, anterior pituitary and testicular function. J Clin Endocrinol Metab 1974;39:522–529. 314 Migeon C, Keller A, Lawrence B et al. DHA and androsterone levels in human plasma. J Clin Endocrinol Metab 1957;17:1051–1062. 315 Parker LN, Sack J, Fisher DA et al. The adrenarche: prolactin, gonadotropins, adrenal androgens, and cortisol. J Clin Endocrinol Metab 1978;46:396–401. 316 Linder BL, Esteban NV, Yergey AL et al. Cortisol production rate in childhood and adolescence [see comments]. J Pediatr 1990;117:892–896. 317 Cutler Jr GB, Glenn M, Bush M et al. Adrenarche: a survey of rodents, domestic animals, and primates. Endocrinology 1978;103:2112–2118. 318 Shepard TH, Landing BH, Mason DG. Familial Addison’s disease. Case reports of two sisters with corticoid deficiency unassociated with hypoaldosteronism. Am J Dis Child 1959;97:154–162.

Chapter 2 319 Migeon CJ, Kenny EM, Kowarski A et al. The syndrome of congenital adrenocortical unresponsiveness to ACTH. Report of six cases. Pediatr Res 1968;2:501–513. 320 Spark RF, Etzkorn JR. Absent aldosterone response to ACTH in familial glucocorticoid deficiency. N Engl J Med 1977;297:917–920. 321 Stempfel RSJ, Engel FL. A congenital, familial syndrome of adrenocortical insufficiency without hypoaldosteronism. J Pediat 1960;57:443–451. 322 Weber A, Toppari J, Harvey RD et al. Adrenocorticotropin receptor gene mutations in familial glucocorticoid deficiency: relationships with clinical features in four families. J Clin Endocrinol Metab 1995;80:65–71. 323 Kelch RP, Kaplan SL, Biglieri EG et al. Hereditary adrenocortical unresponsiveness to adrenocorticotropic hormone. J Pediatr 1972;81:726–736. 324 Franks RC, Nance WE. Hereditary adrenocortical unresponsiveness to ACTH. Pediatrics 1970;45:43–48. 325 Thistlethwaite D, Darling JA, Fraser R et al. Familial glucocorticoid deficiency. Studies of diagnosis and pathogenesis. Arch Dis Child 1975;50:291–297. 326 Weber A, Clark AJ, Perry LA et al. Diminished adrenal androgen secretion in familial glucocorticoid deficiency implicates a significant role for ACTH in the induction of adrenarche. Clin Endocrinol (Oxf ) 1997;46:431–437. 327 Clark AJ, McLoughlin L, Grossman A. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet 1993;341:461–462. 328 Naville D, Barjhoux L, Jaillard C et al. Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency. J Clin Endocrinol Metab 1996;81:1442–1448. 329 Tsigos C, Arai K, Hung W et al. Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene [see comments]. J Clin Invest 1993;92:2458–2461. 330 Tsigos C, Arai K, Latronico AC et al. A novel mutation of the adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency, but no ACTH-R abnormalities in two families with the triple A syndrome. J Clin Endocrinol Metab 1995;80: 2186–2189. 331 Slavotinek AM, Hurst JA, Dunger D et al. ACTH receptor mutation in a girl with familial glucocorticoid deficiency. Clin Genet 1998;53:57–62. 332 Naville D, Weber A, Genin E et al. Exclusion of the adrenocorticotropin (ACTH) receptor (MC2R) locus in some families with ACTH resistance but no mutations of the MC2R coding sequence (familial glucocorticoid deficiency type 2). J Clin Endocrinol Metab 1998;83:3592–3596. 333 Weber A, Clark AJ. Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency. Hum Mol Genet 1994;3:585–588. 334 Clark AJ, Cammas FM, Watt A et al. Familial glucocorticoid deficiency: one syndrome, but more than one gene. J Mol Med 1997;75:394–399. 335 Allgrove J, Clayden GS, Grant DB et al. Familial glucocorticoid deficiency with achalaisa of the cardia and deficient tear production. Lancet 1978;1: 1284–1286. 336 Handschug K, Sperling S, Yoon SJ et al. Triple A syndrome is caused by mutations in AAAS, a new WD-repeat protein gene. Hum Mol Genet 2001; 10:283–290. 337 Tullio-Pelet A, Salomon R, Hadj-Rabia S et al. Mutant WD-repeat protein in triple-A syndrome. Nat Genet 2000;26:332–335. 338 Stratakis CA, Kirschner LS. Clinical and genetic analysis of primary bilateral adrenal diseases (micro- and macronodular disease) leading to Cushing syndrome. [Review] [84 refs]. Horm Metab Res 1998;30:456–463. 339 Boston BA, Mandel S, LaFranchi S et al. Activating mutation in the stimulatory guanine nucleotide-binding protein in an infant with Cushing’s syndrome and nodular adrenal hyperplasia. J Clin Endocrinol Metab 1994;79: 890–893. 340 Lacroix A, Ndiaye N, Tremblay J et al. Ectopic and abnormal hormone receptors in adrenal Cushing’s syndrome. Endocr Rev 2001;22:75–110. 341 Kirschner LS, Carney JA, Pack SD et al. Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 2000 Sep 2001;26:89–92. 342 Casey M, Vaughan CJ, He J et al. Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex. J Clin Invest 2000 Sep 2001;106:R31–R38. 343 Carney JA. The Carney complex (myxomas, spotty pigmentation, endocrine overactivity, and schwannomas). [Review] [15 refs]. Dermatol Clin 1995;13: 19–26. 344 Ramachandran J, Farmer SW, Liles S et al. Comparison of the steroidogenic and melanotropic activities of corticotropin, alpha-melanotropin and analogs with their lipolytic activities in rat and rabbit adipocytes. Biochim Biophys Acta 1976;428:347–354.

Adrenocorticotropin

75

345 Ramachandran J, Lee V. Divergent effects of adrenocorticotropin and melanotropin on isolated rat and rabbit adipocytes. Biochim Biophys Acta 1976; 428:339–346. 346 Oelofsen W, Ramachandran J. Studies of corticotropin receptors on rat adipocytes. Arch Biochem Biophys 1983;225:414–421. 347 White JE, Engel FL. Lipolytic action of corticotropin on rat adipose tissue in vitro. J Clin Invest 1958;37:1556–1563. 348 Boston BA, Cone RD. Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line. Endocrinology 1996;137:2043–2050. 349 Bousquet-Melou A, Galitzky J, Lafontan M et al. Control of lipolysis in intraabdominal fat cells of nonhuman primates: comparison with humans. J Lipid Res 1995;36:451–461. 350 Xia Y, Wikberg JES. Localization of ACTH receptor mRNA by in situ hybridization in mouse adrenal gland. Cell Tissue Res 1996;286:63–68. 351 Smith EM, Brosnan P, Meyer WJ et al. An ACTH receptor on human mononuclear leukocytes. Relation to adrenal ACTH-receptor activity. N Engl J Med 1987;317:1266–1269. 352 Slominski A, Ermak G, Mihm M. ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin. J Clin Endocrinol Metab 1996;81: 2746–2749. 353 Slominski A, Ermak G, Hwang J et al. The expression of proopiomelanocortin (POMC) and of corticotropin releasing hormone receptor (CRH-R) genes in mouse skin. Biochim Biophys Acta 1996;1289:247–251. 354 Kapas S, Cammas FM, Hinson JP et al. Agonist and receptor binding properties of adrenocorticotropin peptides using the cloned mouse adrenocorticotropin receptor expressed in a stably transfected HeLa cell line. Endocrinology 1996;137:3291–3294. 355 Gantz I, Konda Y, Tashiro T et al. Molecular cloning of a novel molanocortin receptor. J Biol Chem 1993;268:8246–8250. 356 Robbins LS, Nadeau JH, Johnson KR et al. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 1993;72:827–834. 357 Lindblom J, Schioth HB, Larsson A et al. Autoradiographic discrimination of melanocortin receptors indicates that the MC3 subtype dominates in the medial rat brain. Brain Res 1998;810:161–171. 358 Getting SJ, Flower RJ, Perretti M. Agonism at melanocortin receptor type 3 on macrophages inhibits neutrophil influx. Inflamm Res 1999;48:S140–S141. 359 Chen AS, Marsh DJ, Trumbauer ME et al. Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nat Genet 2000;26:97–102. 360 Cummings DE, Schwartz MW. Melanocortins and body weight: a tale of two receptors. Nat Genet 2000;26:8–9. 361 Gantz I, Miwa H, Konda Y et al. Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 1993;268: 15174–15179. 362 Mountjoy KG, Mortrud MT, Low MJ et al. Localization of the melanocortin4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 1994;8:1298–1308. 363 Takeuchi S, Takahashi S. Melanocortin receptor genes in the chicken-tissue distributions. Gen Comp Endocrinol 1998;112:220–231. 364 Huszar D, Lynch CA, Fairchild-Huntress V et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 1997;88:131–141. 365 Seeley RJ, Yagaloff KA, Fisher SL et al. Melanocortin receptors in leptin effects [letter]. Nature 1997;390:349. 366 Marsh DJ, Hollopeter G, Huszar D et al. Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nat Genet 1999;21:119–122. 367 Hinney A, Schmidt A, Nottebom K et al. Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly inherited obesity in humans. J Clin Endocrinol Metab 1999;84:1483–1486. 368 Yeo GS, Farooqi IS, Aminian S et al. A frameshift mutation in MC4R associated with dominantly inherited human obesity [letter]. Nat Genet 1998; 20:111–112. 369 Vaisse C, Clement K, Guy-Grand B et al. A frameshift mutation in human MC4R is associated with a dominant form of obesity [letter]. Nat Genet 1998;20:113–114. 370 Vaisse C, Clement K, Durand E et al. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity [see comments]. J Clin Invest 2000;106:253–262. 371 Farooqi IS, Yeo GS, Keogh JM et al. Dominant and recessive inheritance of morbid obesity associated with melanocortin 4 receptor deficiency [see comments]. J Clin Invest 2000;106:271–279.

76

SECTION 1

Hypothalamic–Pituitary Function

372 Cody JD, Reveles XT, Hale DE et al. Haplosufficiency of the melancortin-4 receptor gene in individuals with deletions of 18q. Hum Genet 1999;105: 424–427. 373 Ho G, MacKenzie RG. Functional characterization of mutations in melanocortin-4 receptor associated with human obesity. J Biol Chem 1999;274: 35816–35822. 374 Chhajlani V, Muceniece R, Wikberg JE, Molecular cloning of a novel human melanocortin receptor [published erratum appears in Biochem Biophys Res Commun 1996 Jan 17;218(2):638]. Biochem Biophys Res Commun 1993;195: 866–873. 375 Barrett P, MacDonald A, Helliwell R et al. Cloning and expression of a new member of the melanocyte-stimulating hormone receptor family. J Mol Endocrinol 1994;12:203–213. 376 Griffon N, Mignon V, Facchinetti P et al. Molecular cloning and characterization of the rat fifth melanocortin receptor. Biochem Biophys Res Commun 1994;200:1007–1014. 377 Gantz I, Shimoto Y, Konda Y et al. Molecular cloning, expression, and characterization of a fifth melanocortin receptor. Biochem Biophys Res Commun 1994;200:1214–1220. 378 Chen W, Kelly MA, Opitz-Araya X et al. Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides. Cell 1997;91:789–798. 379 van der Kraan M, Adan RA, Entwistle ML et al. Expression of melanocortin-5 receptor in secretory epithelia supports a function role in exocrine and endocrine glands. Endocrinology 1998;139:2348–2355. 380 Thody AJ, Cooper MF, Bowden PE et al. Effect of alpha-melanocytestimulating hormone and testosterone on cutaneous and modified sebaceous glands in the rat. J Endocrinol 1976;71:279–288. 381 Thody AJ, Shuster S. Possible role of MSH in the mammal. Nature 1973; 245:207–209. 382 Ebling FJ, Ebling E, Randall V et al. The synergistic action of alphamelanocyte-stimulating hormone and testosterone of the sebaceous, prostate, preputial, Harderian and lachrymal glands, seminal vesicles and brown adipose tissue in the hypophysectomized-castrated rat. J Endocrinol 1975;66: 407–412. 383 Leiba H, Garty NB, Schmidt-Sole J et al. The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter. Eur J Pharmacol 1990;181:71–82. 384 Jahn R, Padel U, Porsch PH et al. Adrenocorticotropic hormone and alphamelanocyte-stimulating hormone induce secretion and protein phosphorylation in the rat lacrimal gland by activation of a cAMP-dependent pathway. Eur J Biochem 1982;126:623–629. 385 Bronson FH, Caroom D. Preputial gland of the male mouse; attractant function. J Reprod Fertil 1971;25:279–282. 386 Nowell NW, Thody AJ, Woodley R. Alpha-Melanocyte stimulating hormone and aggressive behavior in the male mouse. Physiol Behav 1980;24:5–9. 387 Krude H, Biebermann H, Luck W et al. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat Genet 1998;19:155–157. 388 Kikuchi T, Yabe S, Kanda T et al. Antipituitary antibodies as pathogenetic factors in patients with pituitary disorders. Endocr J 2000 Aug 2001;47: 407–416. 389 Skodda S, Kraus M, Durwen H et al. Hashimoto’s encephalopathy associated with isolated ACTH deficiency and hyponatremia: a case report [letter]. J Neurol 1999;246:970–971. 390 Yamamoto T, Fukuyama J, Kabayama Y et al. Dual facets of hyponatraemia and arginine vasopressin in patients with ACTH deficiency. Clin Endocrinol (Oxf ) 1998;49:785–792. 391 Nishikawa M, Toyoda N, Miyaji M et al. Variant angina in isolated adrenocorticotropin deficiency, inappropriate vasopressin secretion and Hashimoto’s thyroiditis. Intern Med 1998;37;398–402. 392 Yaswen L, Diehl N, Brennan MB et al. Obesity in the mouse model of proopiomelanocortin deficiency responds to peripheral melanocortin [see comments]. Nat Med 1999;5:1066–1070. 393 Forbes S, Bui S, Robinson BR et al. Integrated control of appetite and fat metabolism by the leptin-proopiomelanocortin pathway. Proc Natl Acad Sci USA 2001;98:4233–4237. 394 Silvers WK, Russell ES. An experimental approach to action of henes at the agouti locus in the mouse. J Exp Zool 1955;130:199–200. 395 Cuneot L. Les races pures et leurs combinaisons chez les souris. Arch Xool Exp gen 1905;3:123–132. 396 Danforth CH. Hereditary adiposity in mice. J Hered 1927;18:153–162. 397 Lu D, Willard D, Patel IR et al. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature 1994;371:799–802.

398 Bultman SJ, Michaud EJ, Woychik RP. Molecular characterization of the mouse agouti locus. Cell 1992;71:1195–1204. 399 Kiefer LL, Veal JM, Mountjoy KG et al. Melanocortin receptor binding determinants in the agouti protein. Biochemistry 1998;37:991–997. 400 Willard DH, Bodnar W, Harris C et al. Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist. Biochemistry 1995;34:12341–12346. 401 Kwon HY, Bultman SJ, Loffler C et al. Molecular structure and chromosomal mapping of the human homolog of the agouti gene. Proc Natl Acad Sci USA 1994;91:9760–9764. 402 Wilson BD, Ollmann MM, Kang L et al. Structure and function of ASP, the human homolog of the mouse agouti gene. Hum Mol Genet 1995;4:223–230. 403 Yang YK, Ollmann MM, Wilson BD et al. Effects of recombinant agoutisignaling protein on melanocortin action. Mol Endocrinol 1997;11:274–280. 404 Kiefer LL, Ittoop OR, Bunce K et al. Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors. Biochemistry 1997;36:2084–2090. 405 Yen TT, Gill AM, Frigeri LG et al. Obesity, diabetes, and neoplasia in yellow A(vy)/-mice: ectopic expression of the agouti gene. Faseb J 1994;8:479–488. 406 Michaud EJ, Bultman SJ, Klebig ML et al. A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation. Proc Natl Acad Sci USA 1994;91:2562–2566. 407 Miller MW, Duhl DM, Vrieling H et al. Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation. Genes Dev 1993;7:454–467. 408 Shutter JR, Graham M, Kinsey AC et al. Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Genes Dev 1997;11:593–602. 409 Ollmann MM, Wilson BD, Yang YK et al. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein [published erratum appears in Science 1998 Sep 11;281(5383):1615]. Science 1997;278:135–138. 410 Rosenfeld RD, Zeni L, Welcher AA et al. Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein. Biochemistry 1998;37:16041–16052. 411 Yang YK, Thompson DA, Dickinson CJ et al. Characterization of Agoutirelated protein binding to melanocortin receptors. Mol Endocrinol 1999; 13:148–155. 412 Fong TM, Mao C, MacNeil T et al. ART (protein product of agouti-related transcript) as an antagonist of MC-3 and MC-4 receptors. Biochem Biophys Res Commun 1997;237:629–631. 413 Zhu Q, Bateman A, Singh A et al. Isolation and biological activity of corticostatic peptides (anti-ACTH). Endocr Res 1989;15:129–149. 414 Cervini LA, Gray WR, Kaiser R et al. Rat corticostatin R4: synthesis, disulfide bridge assignment, and in vivo activity. Peptides 1995;16:837–842. 415 Tatro JB. Receptor biology of the melanocortins, a family of neuroimmunomodulatory peptides. [Review] [224 refs]. Neuroimmunomodulation 1996;3:259–284. 416 de Wied D, Jolles J. Neuropeptides derived from pro-opiocortin: behavioral, physiological, and neurochemical effects. Physiol Rev 1982;62:976–1059. 417 Dunn AJ. Studies on the neurochemical mechanisms and significance of ACTH-induced grooming. Ann NY Acad Sci 1988;525:150–168. 418 Bertolini A, Gessa GA, Ferrari W. Penile erection and ejaculation: a central effect of ACTH-like peptides in mammals. In: Sandler M, Gessa GA. eds. Sexual Behavior, Pharmacology and Biochemistry. NY: Raven Press, 1975: 247–257. 419 Vergoni AV, Bertolini A, Mutulis F et al. Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats. Eur J Pharmacol 1998;362:95–101. 420 Argiolas A, Melis MR, Murgia S et al. 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: 425–431. 421 Wessells H, Fuciarelli K, Hansen J et al. Synthetic melanotropic peptide initiates erections in men with psychogenic erectile dysfunction: double-blind, placebo controlled crossover study. J Urol 1998;160:389–393. 422 Strand FL, Stoboy H, Cayer A. A possible direct action of ACTH on nerve and muscle. Neuroendocrinology 1973;13:1–20. 423 Strand FL, Kung TT. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1980;1:135–138. 424 Strand FL, Rose KJ, King JA et al. ACTH modulation of nerve development and regeneration. Prog Neurobiol 1989;33:45–85. 425 Gispen WH, Verhaagen J, Bar D. ACTH/MSH-derived peptides and peripheral nerve plasticity: neuropathies, neuroprotection and repair. Prog Brain Res 1994;100:223–229.

Chapter 2 426 Swaab DF, Visser M, Tilders FJ. Stimulation of intra-uterine growth in rat by alpha-melanocyte-stimulating hormone. J Endocrinol 1976;70:445–455. 427 Swaab DF, Martin JT. Functions of alpha-melanotropin and other opiomelanocortin peptides in labour, intrauterine growth and brain development. Ciba Found Symp 1981;81:196–217. 428 Murphy MT, Richards DB, Lipton JM. Antipyretic potency of centrally administered alpha-melanocyte stimulating hormone. Science 1983;221: 192–193. 429 Klein MC, Hutchins PM, Lymangrover JR et al. Pressor and cardioaccelerator effects of gamma MSH and related peptides. Life Sci 1985;36:769–775. 430 Li SJ, Varga K, Archer P et al. Melanocortin antagonists define two distinct pathways of cardiovascular control by alpha- and gamma-melanocytestimulating hormones. J Neurosci 1996;16:5182–5188. 431 Lymangrover JR, Buckalew VM, Harris J et al. Gamma-2MSH is natriuretic in the rat. Endocrinology 1985;116:1227–1229. 432 Orias R, McCann SM. Natriuretic effect of alpha melanocyte stimulating hormone (-MSH) in hypophysectomized or adrenalectomized rats. Proc Soc Exp Biol Med 1972;139:872–876. 433 Orias R, McCann SM. Natriuresis induced by alpha and beta melanocyte stimulating hormone (MSH) in rats. Endocrinology 1972;90:700–706. 434 Chen XW, Ying WZ, Valentin JP et al. Mechanism of the natriuretic action of gamma-melanocyte-stimulating hormone. Am J Physiol 1997;272: R1946–R1953. 435 Veldhuis JD, Iranmanesh A, Johnson ML et al. Amplitude, but not frequency, modulation of adrenocorticotropin secretory bursts gives rise to the nyctohemeral rhythm of the corticotropic axis in man. J Clin Endocrinol Metab 1990;71:452–463. 436 Desir D, Van Cauter E, Beyloos M et al. Prolonged pulsatile administration of ovine corticotropin-releasing hormone in normal man. J Clin Endocrinol Metab 1986;63:1292–1299. 437 Veldhuis JD, Iranmanesh A, Johnson ML et al. Twenty-four-hour rhythms in plasma concentrations of adenohypophyseal hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. J Clin Endocrinol Metab 1990;71:1616–1623. 438 Horrocks PM, Jones AF, Ratcliffe WA et al. Patterns of ACTH and cortisol pulsatility over twenty-four hours in normal males and females. Clin Endocrinol (Oxf ) 1990;32:127–134. 439 Iranmanesh A, Lizarralde G, Johnson ML et al. Circadian, ultradian, and episodic release of beta-endorphin in men, and its temporal coupling with cortisol. J Clin Endocrinol Metab 1989;68:1019–1026. 440 Krishnan KR, Ritchie JC, Saunders W et al. Nocturnal and early morning secretion of ACTH and cortisol in humans. Biol Psychiatry 1990;28: 47–57. 441 Schulte HM, Chrousos GP, Gold PW et al. Continuous administration of synthetic ovine corticotropin-releasing factor in man. Physiological and pathophysiological implications. J Clin Invest 1985;75:1781–1785. 442 Keller Wood ME, Dallman MF. Corticosteroid inhibition of ACTH secretion. Endocr Rev 1984;5:1–24. 443 DeBold CR, Jackson RV, Kamilaris TC et al. Effects of ovine corticotropinreleasing hormone on adrenocorticotropin secretion in the absence of glucocorticoid feedback inhibition in man. J Clin Endocrinol Metab 1989;68:431–437. 444 Totani Y, Niinomi M, Takatsuki K et al. Effect of metyrapone pretreatment on adrenocorticotropin secretion induced by corticotropin-releasing hormone in normal subjects and patients with Cushing’s disease. J Clin Endocrinol Metab 1990;70:798–803. 445 Watson AC, Rosenfield RL, Fang VS. Recovery from glucocorticoid inhibition of the responses to corticotrophin-releasing hormone. Clin Endocrinol (Oxf ) 1988;28:471–477. 446 Schopohl J, Hauer A, Kaliebe T et al. Repetitive and continuous administration of human corticotropin releasing factor to human subjects. Acta Endocrinol (Copenh) 1986;112:157–165. 447 Avgerinos PC, Nieman LK, Oldfield EH et al. The effect of pulsatile human corticotropin-releasing hormone administration on the adrenal insufficiency that follows cure of Cushing’s disease. J Clin Endocrinol Metab 1989;68: 912–916. 448 Raff H. Glucocorticoid inhibition of neurohypophysial vasopressin secretion. Am J Physiol 1987;252:R635–R644. 449 Oelkers W. Hyponatremia and inappropriate secretion of vasopressin (antidiuretic hormone) in patients with hypopituitarism [see comments]. N Engl J Med 1989;321:492–496. 450 Bouley R, Breton S, Sun T et al. Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells. J Clin Invest 2000;106:115–126.

Adrenocorticotropin

77

451 Suda T, Tomori N, Yajima F et al. A short negative feedback mechanism regulating corticotropin-releasing hormone release. J Clin Endocrinol Metab 1987;64:909–913. 452 Van Cauter E, Polonsky K, Scheen AJ. Role of circadian rhythm and sleep in human glucose regulation. Endocr Rev 1997;18:716–738. 453 Moore RY, Eichler VB. Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res 1972;42:201–206. 454 Follenius M, Brandenberger G. Plasma free cortisol during secretory episodes. J Clin Endocrinol Metab 1986;62:609–612. 455 Dallman MF, Strack AM, Akana SF et al. Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. [Review] [26 refs.] Front Neuroendocrinol 1993;14:303–347. 456 Van Cauter E, Refetoff S. Multifactorial control of the 24-hour secretory profiles of pituitary hormones. J Endocrinol Invest 1985;8:381–391. 457 Dallman MF, Engeland WC, Rose JC et al. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am J Physiol 1978;235:R210–R218. 458 Dijkstra I, Binnekade R, Tilders FJ. Diurnal variation in resting levels of corticosterone is not mediated by variation in adrenal responsiveness to adrenocorticotropin but involves splanchnic nerve integrity. Endocrinology 1996;137:540–547. 459 Iannotta F, Magnoli L, Visconti G et al. Differences in cortisol, aldosterone and testosterone responses to ACTH 1–17 administered at two different times of day. Chronobiologia 1987;14:38–46. 460 Sensi S, Capani F, De Remigis P et al. Circadian time structure of pituitary and adrenal response to CRF, TRH and LHRH. Prog Clin Biol Res 1990;341:535–542. 461 Suda T, Tomori N, Yajima F et al. Immunoreactive corticotropin-releasing factor in human plasma. J Clin Invest 1985;76:2026–2029. 462 Watabe T, Tanaka K, Kumagae M et al. Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects. Life Sci 1987;40:1651–1655. 463 Hindmarsh KW, Tan L, Sankaran K et al. Diurnal rhythms of cortisol, ACTH, and beta-endorphin levels in neonates and adults. West J Med 1989;151: 153–156. 464 Sasaki A, Shinkawa O, Yoshinaga K. Placental corticotrophin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans. J Clin Invest 1989;84:1997–2001. 465 Muglia LJ, Jacobson L, Weninger SC et al. Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone, in corticotropin-releasing hormone-deficient mice. Journal of Clinical Investigation 1997;99:2923–2929. 466 Muglia LJ, Jacobson L, Luedke C et al. Corticotropin-releasing hormone links pituitary adrenocorticotropin gene expression and release during adrenal insufficiency [see comments]. J Clin Invest 2000 May 2000;105:1269–1277. 467 Ellis MJ, Schmidli RS, Donald RA et al. Plasma corticotrophin-releasing factor and vasopressin responses to hypoglycaemia in normal man. Clin Endocrinol (Oxf ) 1990;32:93–100. 468 Decherney GS, DeBold CR, Jackson RV et al. Effect of ovine corticotropinreleasing hormone administered during insulin-induced hypoglycemia on plasma adrenocorticotropin and cortisol. J Clin Endocrinol Metab 1987;64: 1211–1218. 469 Adler GK, Majzoub JA. Influence of infused hypertonic saline on the response to insulin-induced hypoglycemia in man. J Clin Endocrinol Metab 1987;65: 116–121. 470 Luger A, Deuster PA, Gold PW et al. Hormonal responses to the stress of exercise. Adv Exp Med Biol 1988;245:273–280. 471 Deuster PA, Chrousos GP, Luger A et al. Hormonal and metabolic responses of untrained, moderately trained, and highly trained men to three exercise intensities. Metabolism 1989;38:141–148. 472 Janal MN, Colt EW, Clark WC et al. Pain sensitivity, mood and plasma endocrine levels in man following long-distance running: effects of naloxone. Pain 1984;19:13–25. 473 Pitts AF, Preston MA, II, Jaeckle RS et al. Simulated acute hemorrhage through lower body negative pressure as an activator of the hypothalamic–pituitary–adrenal axis. Horm Metab Res 1990;22:436–443. 474 Udelsman R, Chrousos GP. Hormonal responses to surgical stress. Adv Exp Med Biol 1988;245:265–272. 475 Udelsman R, Norton JA, Jelenich SE et al. Responses of the hypothalamic– pituitary–adrenal and renin-angiotensin axes and the sympathetic system during controlled surgical and anesthetic stress. J Clin Endocrinol Metab 1987; 64:986–994. 476 Udelsman R, Ramp J, Gallucci WT et al. Adaptation during surgical stress. A reevaluation of the role of glucocorticoids. J Clin Invest 1986;77: 1377–1381.

78

SECTION 1

Hypothalamic–Pituitary Function

477 Oltras CM, Mora F, Vives F. Beta-endorphin and ACTH in plasma: effects of physical and psychological stress. Life Sci 1987;40:1683–1686. 478 Voigt K, Ziegler M, Grunert Fuchs M et al. Hormonal responses to exhausting physical exercise: the role of predictability and controllability of the situation. Psychoneuroendocrinology 1990;15:173–184. 479 Arduini D, Rizzo G, Parlati E et al. Modifications of ultradian and circadian rhythms of fetal heart rate after fetal-maternal adrenal gland suppression: a double blind study. Prenat Diagn 1986;6:409–417. 480 Price DA, Close GC, Fielding BA. Age of appearance of circadian rhythm in salivary cortisol values in infancy. Arch Dis Child 1983;58:454–456. 481 Onishi S, Miyazawa G, Nishimura Y et al. Postnatal development of circadian rhythm in serum cortisol levels in children. Pediatrics 1983;72:399–404. 482 Attanasio A, Rosskamp R, Bernasconi S et al. Plasma adrenocorticotropin, cortisol, and dehydroepiandrosterone response to corticotropin-releasing factor in normal children during pubertal development. Pediatr Res 1987;22:41–44. 483 Ghizzoni L, Virdis R, Ziveri M et al. Adrenal steroid, cortisol, adrenocorticotropin, and beta-endorphin responses to human corticotropinreleasing hormone stimulation test in normal children and children with premature pubarche. J Clin Endocrinol Metab 1989;69:875–880. 484 McEwen BS. The neurobiology of stress: from serendipity to clinical relevance. [Review] [157 refs]. Brain Res 2000 Dec 15 2001;886:172–189. 485 Elenkov IJ, Wilder RL, Chrousos GP et al. The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system. [Review] [445 refs]. Pharmacol Rev 2000 Dec 2001;52:595–638. 486 Blalock JE. Proopiomelanocortin and the immune-neuroendocrine connection. [Review] [68 refs]. Ann NY Acad Sci 1999;885:161–172. 487 Ottaviani E, Franchini A, Genedani S. ACTH and its role in immuneneuroendocrine functions. A comparative study. Curr Pharm Des 1999;5: 673–681. 488 Elenkov IJ, Webster EL, Torpy DJ et al. Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: acute and chronic effects. [Review] [59 refs]. Ann NY Acad Sci 1999;876:1–11. 489 Cavagnaro J. Molecular basis for the bidirectional modulation of the neuroendocrine and the immune systems. Year Immunol 1986;2:303–322. 490 Reichlin S. Neuroendocrine-immune interactions. N Engl J Med 1993;329: 1246–1253. 491 Eskay RL, Grino M, Chen HT. Interleukins, signal transduction, and the immune system-mediated stress response. Adv Exp Med Biol 1990;274: 331–343. 492 Kavelaars A, Ballieux RE, Heijnen CJ. The role of IL-1 in the corticotropinreleasing factor and arginine- vasopressin-induced secretion of immunoreactive beta-endorphin by human peripheral blood mononuclear cells. J Immunol 1989;142:2338–2342. 493 Westly HJ, Kleiss AJ, Kelley KW et al. Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J Exp Med 1986;163: 1589–1594. 494 Lolait SJ, Clements JA, Markwick AJ et al. Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages. J Clin Invest 1986;77:1776–1779. 495 Karalis K, Sano H, Redwine J et al. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 1991;254:421–423. 496 Muglia LJ, Jenkins NA, Gilbert DJ et al. Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells. Journal of Clinical Investigation 1994;93:2066–2072. 497 Scheithauer BW. Surgical pathology of the pituitary: the adenomas. Part II. Pathol Annu 1984;19:269–329. 498 Saeger W, Geisler F, Ludecke DK. Pituitary pathology in Cushing’s disease. Pathol Res Pract 1988;183:592–595. 499 Herman V, Fagin J, Gonsky R et al. Clonal origin of pituitary adenomas. J Clin Endocrinol Metab 1990;71:1427–1433. 500 Hori T, Nishiyama F, Anno Y et al. Differences in glycoconjugates of adrenocorticotropic hormone-secretory granules between nonadenomatous pituitary cells and adenoma cells as detected by double labeling. Neurosurgery 1988;23:52–57. 501 Challa VR, Marshall RB, Hopkins MB et al. Pathobiologic study of pituitary tumors: report of 62 cases with a review of the recent literature. Hum Pathol 1985;16:873–884.

502 Apletalina V, Muller L, Lindberg I. Mutations in the catalytic domain of prohormone convertase 2 result in decreased binding to 7B2 and loss of inhibition with 7B2 C-terminal peptide. J Biol Chem 2000;275:14667–14677. 503 Schteingart DE, Chandler WF, Lloyd RV et al. Cushing’s syndrome caused by an ectopic pituitary adenoma. Neurosurgery 1987;21:223–227. 504 Axiotis CA, Lippes HA, Merino MJ et al. Corticotroph cell pituitary adenoma within an ovarian teratoma: A new cause of Cushing’s syndrome. Am J Surg Pathol 1987;11:218–224. 505 Neilson K, de Chadarevian JP. Ectopic anterior pituitary corticotropic tumour in a six-year-old boy. Histological, ultrastructural and immunocytochemical study. Virchows Arch [A] 1987;411:267–273. 506 Karl M, Lamberts SW, Koper JW et al. Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 1996;108:296–307. 507 Karl M, Von Wichert G, Kempter E et al. Nelson’s syndrome associated with a somatic frame shift mutation in the glucocorticoid receptor gene. J Clin Endocrinol Metab 1996;81:124–129. 508 Gabrilove JL, Anderson PJ, Halmi NS. Pituitary pro-opiomelanocortin-cell carcinoma occurring in conjunction with a glioblastoma in a patient with Cushing’s disease and subsequent Nelson’s syndrome. Clin Endocrinol (Oxf ) 1986;25:117–126. 509 Nawata H, Higuchi K, Ikuyama S et al. Corticotropin-releasing hormone- and adrenocorticotropin-producing pituitary carcinoma with metastases to the liver and lung in a patient with Cushing’s disease. J Clin Endocrinol Metab 1990;71:1068–1073. 510 Gould VE, Lee I, Warren WH. Immunohistochemical evaluation of neuroendocrine cells and neoplasms of the lung. Pathol Res Pract 1988;183:200–213. 511 Kasurinen J, Syrjanen KJ. Peptide hormone immunoreactivity and prognosis in small-cell carcinoma of the lung. Respiration 1986;49:61–67. 512 Sieber SC, Gelfman NA, Dandurand R et al. Ectopic ACTH and adrenal myelolipoma. Conn Med 1989;53:7–10. 513 Herbst WM, Kummer W, Hofmann W et al. Carcinoid tumors of the thymus. An immunohistochemical study. Cancer 1987;60:2465–2470. 514 Arioglu E, Doppman J, Gomes M et al. Cushing’s syndrome caused by corticotropin secretion by pulmonary tumorlets. N Engl J Med 1998;339:883–886. 515 Raux Demay MC, Proeschel MF, de Keyzer Y et al. Characterization of human corticotrophin-releasing hormone and pro-opiomelanocortin-related peptides in a thymic carcinoid tumour responsible for Cushing’s syndrome. Clin Endocrinol (Oxf ) 1988;29:649–657. 516 Suda T, Kondo M, Totani R et al. Ectopic adrenocorticotropin syndrome caused by lung cancer that responded to corticotropin-releasing hormone. J Clin Endocrinol Metab 1986;63:1047–1051. 517 Sakurai H, Yoshiike Y, Isahaya S et al. A case of ACTH-producing pheochromocytoma. Am J Med Sci 1987;294:258–261. 518 Mendonca BB, Arnhold IJ, Nicolau W et al. Cushing’s syndrome due to ectopic ACTH secretion by bilateral pheochromocytomas in multiple endocrine neoplasia type 2A [letter]. N Engl J Med 1988;319:1610–1611. 519 Southgate HJ, Archbold GP, el Sayed ME et al. Ectopic release of GHRH and ACTH from an adenoid cystic carcinoma resulting in acromegaly and complicated by pituitary infarction. Postgrad Med J 1988;64: 145–148. 520 Watanobe H, Yoshioka M, Takebe K. Ectopic ACTH syndrome due to Grawitz tumor. Horm Metab Res 1988;20:453–456. 521 Werner S, Jacobsson B, Bostrom L et al. Cushing’s syndrome due to an ACTH-producing neuroendocrine tumour in the nasal roof. Acta Med Scand 1985;217:235–240. 522 Long SI. ACTH-producing apudoma metastatic to the liver. J Natl Med Assoc 1987;79:122–123. 523 de Keyzer Y, Bertagna X, Luton JP et al. Variable modes of proopiomelanocortin gene transcription in human tumors. Mol Endocrinol 1989;3: 215–223. 524 Melmed S, Yamashita S, Kovacs K et al. Cushing’s syndrome due to ectopic proopiomelanocortin gene expression by islet cell carcinoma of the pancreas. Cancer 1987;59:772–778.

C h a p t e r

3 Growth Hormone Vivien S. Herman-Bonert Shlomo Melmed

GROWTH HORMONE (GH) GENE STRUCTURE

Somatotroph Development and Differentiation

The human GH genomic locus spans approximately 66 kb and contains a cluster of five highly conserved genes [1] located on the long arm of human chromosome 17 at bands q22–24 [2]. The 5¢ to 3¢ arrangement of these genes is hGHN, hCS-L, hCS-A, hGH-V, and hCS-B [1], all of which have the same basic structure consisting of five exons separated by four introns [1]. The hGH-N gene is transcribed only in somatotrophs of the anterior pituitary while the hCS-A and hCS-B genes are expressed in placental trophoblasts [3]. hGH-N codes for a 22-kDa protein consisting of 191 amino acids. Approximately 10% of pituitary GH is presented as a 20-kDa variant lacking amino acid residues 32–46 [4,5], and probably arising as a result of an alternate splicing mechanism [6]. hGH-V is expressed by the syncytiotrophoblast of the placenta during the second and third trimesters of gestation [7,8], hGH-V messenger RNA (mRNA) encodes 22kDa protein secreted form which can be detected in the maternal circulation from midpregnancy [9] and a minor form hGH-V2 which is predicted to be a 26-kDa protein product [10]. The role of hGH-V is unknown, however, the rise in hGH-V concentrations in maternal serum correlates with a fall in hGH-N concentrations, suggesting the possibility of a feedback loop on the maternal hypothalamic pituitary axis [9]. Postpartum, GH-V levels drop rapidly and are undetectable in the circulation after one hour [9]. hGH-V has a greater binding affinity than hGH-N for somatogen vs lactogen receptors [11]. hGH-V and hGH-N also differ in their ratio of somatogen to lactogen bioactivities, with hGH-V having the greater ratio [12]. In addition, hGH-V has been shown to influence carbohydrate and fat metabolism in rat adipose tissues in a manner similar to that of hGH-N [13]. The hCS-L gene is not known to yield a product [14].

GH is specifically expressed in the somatotroph cell of the anterior pituitary which develops in a time-and-space dependent manner. Expression of the a-subunit transcript in the hypophyseal placode within ectoderm of the pharynx prior to the formation of Rathke’s pouch defines the onset of pituitary organogenesis [15]. The mammalian anterior pituitary develops from Rathke’s pouch during the early stages of embryonic development [16]. Its ventral epithelium serves as the anterior pituitary anlagen while the dorsal epithelium generates the intermediate lobe of the pituitary. A process of cytodifferentiation gives rise to the different hormone-producing cells [17]. Acidophils are the progenitors for both GH-producing somatotrophs and prolactin producing lactotrophs (PRL), while basophils give rise to cells which secrete adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) [17]. Cells of the intermediate lobe produce pro-opiomelanocortin (POMC) gene-related peptides [17]. Experimental evidence obtained from transgenic mice studies suggests that most PRL-expressing cells arose from GH-producing cells [18]. Ablation of somatotrophs by expression of GH-diphtheria toxin and GH-thymidine kinase fusion genes inserted into the germ line of transgenic mice results in the elimination of the majority of lactotrophs; however a small percentage of lactotrophs escape destruction [18,19]. This suggests that the majority of PRLproducing cells arose from postmitotic somatotrophs. The Pit-I gene transcript and POUIFI protein are found in somatotrophs, lactotrophs and thyrotrophs [15]. This suggests an mRNA-specific translational control as part of the developmental process [15]. The actions of Pit-1 protein are complemented by other factors required to achieve the 79

80

SECTION 1

Hypothalamic–Pituitary Function

physiologic patterns of cell-specific gene activation [15]. The estrogen receptor, which is activated subsequent to the appearance of Pit-1, appears to mediate in part a progressive increase in PRL gene expression characteristic of mature lactotrophs. Inherited isolated GH deficiency and short stature may be attributed to mutations of transcription factors, the GH-1 gene, the GHRH receptor, the GH receptor or rarely IGF-related molecules.

Promoter Structure The hGH promoter region contains cis-elements which mediate both pituitary-specific and hormone-specific signaling [20,21]. DNase footprinting reveals several proteinbinding sites within 300 bp of the 5¢ flanking DNA. One one of these proteins is unique to pituitary cells, suggesting that it plays a major role in tissues-specific expression of the GH gene [20,22]. This factor, alternately named Pit-1 or GHF-1 [23,24], binds to two sites centered around sequences -80 and -120. Pit-1 is a 31- or 33-kDa protein important for GH promoter activity both in vivo and in vitro [23–25]. When transferred into nonexpressing cells, Pit-1 is capable of stimulating GH-directed gene expression in nonpituitary cells [20]. In addition, Pit-1 binds to the PRL promoter [26]. Pit-1 is a member of a family of transcription factors sharing two regions of homology [27]; a highly conserved POU-specific (POUs) domain and a more divergent homeodomain (POUHD). Initial recognition of this family resulted from the cloning of complementary DNAs (cDNAs) encoding several transactivators Pit-1 [23,24], Oct1 [28,29], Oct-2 [30,31], and unc-86 [32]. The POUs domain is unique to this family of proteins but the POUHD, which is divergent, shares sequence homology and the predicted a-helical structures with classic homeodomain-containing proteins [33]. Data from mutant Pit-1 proteins suggest that while POUHD is required and sufficient for low-affinity DNA binding, the POUs domain is necessary for highaffinity binding and accurate recognition of natural Pit-1 response elements. In addition, POU domain proteins, in contrast to homeodomain proteins, require that both the POUs and POUHD contact the DNA [27]. Transcriptional activity of Pit-1 is conferred by a serine- and threoninerich N-terminal region which is different to the transcriptional contacts of other types of activation domains [27]. A variant isoform of Pit-1, Pit-1B [34], contains a 26-amino acid insertion in the transactivation domain and, like the previously described Pit-I protein, is capable of inducing both GH and PRL promoters. PROP-1, a paired homeobox protein, is required for initial commitment of Pit-1 cell lineages [35]. PROP-1 represses Rpx expression, and missense and spliced mutations of PROP-1 leading to loss of DNA-binding or transactivation leads to pituitary failure with short stature and varying degrees of thyroid failure, hypogonadism, and ACTH deficiency [36].

POUIFI [37] mutations may also lead to pituitary failure. Patients with combined pituitary hormone deficiency have predominantly GH and PRL deficiency, with variable degrees of hypothyroidism [37]. Footprinting analysis reveals the presence of a second, ubiquitous factor binding to the distal Pit-1 site [38,39]. The protected template contains a consensus sequence for the binding of Sp1, a transcription factor that recognizes the glycine-cysteine box in several viral and eukaryotic gene promoters [40–42]. Pit-1 binds to its distal site with a lower affinity [23,24], and, in addition, Pit-1 and Sp1 bind in a mutually exclusive manner to the GH promoter [38,39]. In vivo, both binding sites contribute to rat GH promoter activation [39]. Mutation of the Sp1 binding site results in a 50% reduction of promoter activity, suggesting that activation of GH by Pit-1 and Sp1 may occur through a multistage mechanism. Sequences -266/-252 of the hGH promoter contain an 8 bp recognition site for the adenoviral major late transcription factor (MLTF), also termed upstream stimulating factor (USF) [43]. A USF-like protein also binds to the upstream hGH promoter [38,43,44]. This factor contributes to one of the protected sites in the third footprint of the hGH gene promoter. USF is a basal transcription factor initially identified in Hela cell extracts [45,46]. Known targets of USF include adenovirus-2 [45,46], fibrinogen-g and the metallothionein-1 genes [47,48]. The distal part of this protected region of the hGH promoter, -290/-272, binds an insulin-inducible nuclear factor [44] that may represent an NF1 family protein [49]. A locus control region (LCR) of the hGH gene located 5¢ to the transcription start site determines somatotroph and latotroph GH expression. This genetic control also involves appropriate regulation of a chromatin domain in these pituitary cell types [50].

Hormonal Regulation of the hGH Gene Synthesis and release of GH is under control of a variety of hormonal agents, including insulin growth factor-I (IGF-I), GH-releasing hormone (GHRH), Ghrelin, somatostatin, thyroid hormone, glucocorticoids, and insulin. IGF-I inhibits GH mRNA expression as well as GH secretion in dispersed human adenoma cell cultures [51]. This effect is blocked by the monoclonal IGF-I receptor antibody a-IR3 [51]. IGF-I also attenuates basal and stimulated GH gene expression when 500 bp of the hGH gene promoter was transfected into human choriocarcinoma cells ( JEG3) or HeLa cells [52]. GHRH stimulates the release of hGH, and cyclic adenosine monophosphate (cAMP) is thought to be the second messenger for this effect. Analysis of the hGH promoter for a cAMP-responsive element (CRE) fails to reveal a consensus sequence. Two groups have reported the presence of novel CREs within -83 and -150 bp of the 5¢ flanking DNA, respectively [21,53]. CREB binding protein (CBP) is also a co-factor for Pit-1 dependent human GH

Chapter 3

activation. CBP is phosphorylateral by protein kinase A, independently of CREB [54]. The role of thyroid hormone in hGH synthesis remains unclear. Release of hGH in response to provocative stimuli is usually reduced in hypothyroid patients; however, basal GH levels and responses may be normal [55,56]. In numerous hGH gene transfer studies, absence of a T3 response has been documented [57,58]. Glucocorticoid responsiveness of the hGH gene has been demonstrated with the strongest receptor binding site being located within the first intron of the hGH gene; however, a 5¢ GRE is also present and capable of weak induction [59–61]. Insulin suppresses hGH secretion by acromegaly tumor cells in culture [62], and also attenuates basal and stimulated transient expression of a transfected hGH gene [63]. Somatostatin is synthesized in the medial preoptic area of the hypothalamus and inhibits secretion of GH from human adenoma cell cultures [64]. Thus, numerous ubiquitous factors bind to the GH promoter. In the absence of Pit-1 the promoter region is inactive, and binding of Pit-1 to the transcriptional machinery facilitates the interaction between this factor and other ubiquitous activators already bound to the promoter or subsequently activated to enhance GH transcription. This model

Growth Hormone

81

of cooperative interaction provides an explanation for tissuespecific expression of the hGH gene by a single cell typespecific activator. CONTROL OF GH SECRETION The GH molecule, a single-chain polypeptide hormone consisting of 191 amino acids (Fig. 3.1), is synthesized, stored, and secreted by somatotroph cells. The crystal structure of human GH and GH-binding protein reveals four alpha helixes, and two identical GHBP molecules occurring within a single GH molecule [65]. Somatotrophs are located predominantly in the lateral wings of the anterior pituitary gland. Somatotrophs comprise 35% to 45% of pituitary cells and the gland contains a total of 5–15 mg of GH [66]. Circulating GH molecules are a heterogenous group of peptides [67] which comprises at least three monomeric forms and several oligomers. The monomeric moieties include a 22- and 20-kDa form, acetylated 22K, and two desamido GH’s. The 22-kDa peptide is the major physiologic GH component. The 20-kDa GH has a slower metabolic clearance than the 22-kDa form [68], which accounts for the plasma 20 : 22 ratio being higher than in the pituitary gland.

FIGURE 3.1. Amino acid structure of human growth hormone (GH). GH is a 191amino acid single-chain 21.5 kDa polypeptide with two intramolecular disulfide bonds. Fifteen percent of GH is deleted from amino acid (32–46) and is secreted as a 20 kDa protein.

82

SECTION 1

Hypothalamic–Pituitary Function

The 22-kDa peptide retains growth-promoting activity, but lacks diabetogenic effects, which are more pronounced with the 20-kDa form. The relative proportions of circulating GH molecular forms in children of various ages are quantitatively and qualitatively identical in adults. Monomeric GH forms found in the plasma of acromegalic patients are also qualitatively similar to those found in normal plasma [69].

Ontogeny of GH Levels Circulating GH is first detectable in fetal serum at the end of the first trimester, peaks at a concentration of 100 to 150 ng/ml at 20 weeks of gestation, and subsequently falls to 30 ng/ml at birth. GH levels continue to fall during infancy. During childhood, levels are similar to those in adulthood, until puberty, when circulating levels are elevated. GH levels decline after adolescent growth and remain stable until mid-adulthood, when they decline progressively through old age [70]. Decreased sensitivity of the pituitary to GHRH action with aging may contribute to these GH fluctuations with advancing age [71].

Physiologic Factors Involved in GH Secretion GH secretion is pulsatile, the anterior pituitary gland secreting bursts of GH, with almost undetectable basal levels occurring between these peaks [72]. The number of GH pulses detected depends on the frequency of blood sampling. When sampling frequency is reduced to three or fewer samples per hour, less than 0.4 GH pulses/hr are detected, and this rate increases as sampling is intensified [72]. Integrated GH levels are higher in women than in men [73], and are also enhanced in postmenopausal women following estrogen replacement [74]. In children and young adults, maximum GH secretion occurs within an hour of the onset of deep sleep (stage III or IV), with subsequent smaller peaks appearing during later sleep [75]. Effects of Sleep

A major GH secretory pulse occurs shortly after the onset of sleep, associated with the first episode of slow wave sleep [76]. Sleep stimulates GH secretion and 60% to 70% of daily GH secretion occurs during early sleep, in association with slow wave sleep [77] Rapid eye movement (REM) sleep is reduced by approximately 50% after age 50 years with significant sleep fragmentation [78]. The decline in slow wave sleep from early adulthood to midlife is paralleled by a major decline in GH secretion. This suggests that the age-related alterations in the somatotropic axis may partially reflect decreased sleep quality. Although increased PRL and ACTH concentrations occur later during sleep, their secretion is not as tightly linked to sleep patterns as is GH secretion. “Jet lag” transiently increases the height of GH peaks during the day and night, resulting in a transient increase of 24-hour

GH secretion. Jet lag also shifts the major GH secretory spike from early to late sleep [79]. Exercise

Exercise increases GH secretion probably mediated by a cholinergic mechanism [80]. Stress

GH release is stimulated by physical stress, including trauma with hypovolemic shock [81] and sepsis [82]. However, chronic debilitating diseases, including cancer, are not associated with increased GH levels [83]. Increased GHRH release, mediated by adrenergic pathways, is thought to mediate stress-induced GH secretion [84]. Emotional deprivation is associated with suppressed GH secretion [85], and subnormal GH responses to provocative stimuli have been described in endogenous depression [86]. Nutritional and Metabolic Effects

Nutritional and metabolic factors profoundly influence GH secretion. Chronic malnutrition [87] and voluntary 5-day fasting [88] are associated with elevated GH levels. Both pulse frequency and amplitude of GH secretory peaks increase with fasting [88] (Table 3.1). Obesity decreases basal and stimulated GH secretion. Insulin-induced hypoglycemia stimulates GH release 30 to 45 minutes after the glucose trough, whereas acute hyperglycemia inhibits GH secretion [89]. Diabetic patients with chronic hyperglycemia, however, do not have suppressed GH levels and in fact many poorly controlled diabetic patients have increased basal [90] and exercise-induced GH levels [91]. Central nervous system glucoreceptors appear to sense fluctuations, rather than absolute glucose levels. However, glucose homeostasis is not the major determinant of GH secretion, this being overridden by effects of sleep, exercise, stress, and by random GH bursts. A high-protein meal, and single amino acids (including arginine and leucine) administered intravenously stimulate GH secretion. Arginine may suppress endogenous somatostatin secretion and thereby stimulate GH secretion [92]. Decreased serum free fatty acid (FFA) levels cause acute GH release [93] and increased serum FFA blunt the effects of various stimuli, including arginine infusion, [94] sleep, Ldopa, and exercise [95–98] on GHRH-stimulated GH release [98].

Table 3.1. Deconvolution analysis of growth hormone (GH) secretion in adult males. From Thorner et al. [88a]

24 hr secretion Secretory bursts GH/burst

Normal

Fasting

Obesity

Middle-age

540 ± 44 12 ± 1 45 ± 4

2171 ± 333 32 ± 2 64 ± 9

77 ± 20 3 ± 0.5 24 ± 5

196 ± 65 10 ± 1 20 ± 6

Chapter 3

Growth Hormone

83

FIGURE 3.2. Schematic representation of the actions of leptin. Leptin acts either directly or by activating specific centers in the central nervous system to decrease food intake, increase energy expenditure, influence glucose and fat metabolism, and alter neuroendocrine function. From Mantzoros [100a].

FIGURE 3.3. Schematic representation of feedback loops involving leptin. Leptin, an adipocyte-derived hormone, circulates in the serum either in free form or bound to leptinbinding proteins, activates specific receptors in the hypothalamus, and alters expression of several neuropeptides; these in turn decrease appetite, increase energy expenditure by altering sympathetic and parasympathetic tone, and alter neuroendocrine function. Increasing leptin levels activate the thyroid, growth hormone, and gonadal axes and suppress the pituitary–adrenal axis. Leptin, acting directly or indirectly (by altering the levels of other hormones and neuropeptides), also influences hemopoiesis and immune function and improves glucose and fat metabolism. From Mantzoros [100a].

Leptin

Leptin, a 167 amino acid cytokine, is the product of the ob gene, expressed in white adipose tissue, stomach, placenta and possible mammary gland [99]. Leptin is thought to play a key role in the regulation of body fat mass [100]. Regulating food intake and energy expenditure (Fig. 3.2). GH has several metabolic effects, including lipolytic and anabolic effects. In turn, alterations in nutritional status, such as obesity and food deprivation, influence GH secretion. In obese subjects, GH secretion is markedly impaired. The mechanism by which metabolic status regulates GH secretion is poorly understood. As leptin receptors are present on

the hypothalamus, and leptin plays a role in hypothalamic–pituitary function in fasted animals [101], leptin may act as a metabolic signal to regulate GH secretion (Fig. 3.3). Leptin antiserum administered to normal fed rats, decreased plasma GH levels [102], implying that physiologic leptin levels may facilitate normal spontaneous GH secretion. Furthermore leptin administered to fasted rats reversed the inhibitory effect of food deprivation on in vivo GH secretion [102]. Increased SRIF release may be a factor in the suppressed GH secretion observed in fasted rats [103], possibly mediated by neuropeptide Y, as hypothalamic neuropeptide Y

84

SECTION 1

Hypothalamic–Pituitary Function

release is increased in fasted rats [104]. Leptin receptors are present in neuropeptide Y producing hypothalamic neurons [105], which synapse with somatostatin neurones [104]. Furthermore both antisera to neuropeptide Y and somatostatin reverse starvation-induced GH release [103,104]. Further studies are needed to elucidate the effects of leptin on neuropeptide Y, and somatostatin and its role as a metabolic signal in the regulation of GH secretion. In GH deficient hypopituitary adults, leptin concentrations are more elevated than would be expected from the degree of obesity [106].

Cholinergic and Serotonergic Neurotransmitters

Cholinergic and serotoninergic neurons have been implicated in the etiology of sleep-induced GH secretion [119]. Opiods

Endorphins and enkephalins stimulate GH secretion in animals [120], probably via interaction with other neurotransmitters in both the pituitary and hypothalamus. Opioids increase rat GHRH concentrations [121], and endogenous opiates probably increase GH release during severe physical stress and extreme exercise [122]. Gastrointestinal Neuropeptides

Pharmacologic Control of GH Secretion The central neurogenic control of GH is complex. Neuropeptides, neurotransmitters and opiates impinge on the hypothalamus and modulate GHRH and somatostatin (SRIF) release. The net effect of these complex influences determines the final secretory pattern of GH.

Several gastrointestinal neuropeptides stimulate GH secretion in animal models, including substance P, neurotensin, vasoactive intestinal polypeptide, peptide histidine isoleucine amide (PHI), motilin, galanin, cholecystokinin, and glucagon [123]. HYPOTHALAMIC HORMONES

Dopaminergic Neurotransmitters

Dopamine (DA) is a precursor of epinephrine and norepinephrine. Apomorphine, a central dopamine receptor agonist, stimulates GH secretion [107]. GH-deficient children have been shown to increase their growth velocity after 6 months of levodopa treatment [108]. Sixty to 90 minutes after oral l-dopa administration, adults increase their serum GH levels from 0 to 5–20 ng/ml [109]. Phentolamine, an a-adrenergic blocking agent, inhibits l-dopa-induced GH release, indicating that l-dopa may, in addition, require conversion to epinephrine or norepinephrine to exert its effect on GH secretion [110]. Catecholaminergic Neurotransmitters

Norepinephrine increases GH secretion via a-adrenergic pathways and inhibits GH release via b-adrenergic pathways [111]. Insulin-induced hypoglycemia increases GH secretion via an a2-adrenergic pathway [112], whereas clonidine acts on a1-adrenergic receptors to increase GH secretion [113]. Arginine administration, exercise, l-dopa, and antidiuretic hormone (ADH) facilitate GH secretion by a-adrenergic effects [114]. Animal studies using pharmacologic agents and passive immunization show that a-adrenergic blockade simultaneously inhibits GHRH release while stimulating SRIF release, resulting in suppressed GH secretion [115]. b2-adrenergic receptor stimulation decreases basal GH levels [116]. b-adrenergic blockade increases GHRH-induced GH release, possibly due to a b-adrenergic effect at the pituitary level or via decreased hypothalamic somatostatin release [117]. b-blockade also enhances GH release elicited by insulin-induced hypoglycemia, ADH, glucagon, and L-dopa [114]. Epinephrine may regulate GH release by decreasing somatostatin release [118], and metabolites of g-aminobutyric acid (GABA) may alter GH secretion by enhancing catecholaminergic release [110].

Thyrotropin-releasing hormone (TRH) does not stimulate GH secretion in normal subjects [124]; it does, however, induce GH secretion in about 70% of patients with acromegaly [125]. TRH also stimulates in vitro GH secretion in cultured pituitary tumor cells [126]. The discordant GH response to TRH has also been demonstrated in patients with liver disease [127], renal disease, ectopic GHRH-releasing carcinoid tumors [128], anorexia nervosa [129], and depression [130]. Administration of intravenous corticotropin-releasing hormone (CRH) increases GH secretion in a small percentage of patients with acromegaly [131] and in patients with chronic depression [132]. Gonadotropin-releasing hormone (GnRH) stimulates GH secretion in one-third of patients with acromegaly, but the mechanism is unclear [133].

Hormones Facilitating GH Secretion Physiologic glucocorticoid concentrations increase GH secretion in human pituitary cultures [134]. In acromegalic patients, dexamethasone suppresses GH secretion in vivo [135]. Supraphysiologic serum glucocorticoid concentrations retard growth in humans [136]. Cushing’s disease is also associated with growth retardation, decreased serum GH [137], and decreased pituitary GH content in the tissue surrounding the adenoma [138]. A single dose of dexamethasone administered to normal subjects suppresses GHRH-induced GH release within 12 hours. Glucocorticoids administered to normal subjects produce a dose dependent inhibition of GHRH-stimulated GH secretion, identical to that seen in Cushing’s syndrome [139,140]. Glucocorticoids have a greater inhibitory action on GHRHinduced GH secretion than clonidine or arginine [139]. In chronic hypocortisolism, GHRH-induced GH secretion is reduced [141]. To elucidate the mechanism of the blocking

Chapter 3

action of glucocorticoids on GH release, increased somatostatin secretion seems unlikely, as pyridostigmine (which blocks hypothalamic somatostatin secretion) does not increase suppressed GH levels in Cushing’s syndrome [142]; neither can IGF-I be incriminated, as IGF-I levels are normal in Cushing’s syndrome [143]. The above data exclude decreased endogenous GHRH release as the mechanism. When the glucocorticoid effect is measured acutely, i.e., at 3 hours, stimulated GH levels are measured, which remain elevated for 2 hours [144]. Acute glucocorticoidinduced GH secretion occurs with several different corticosteroid preparations including hydrocortisone, deflazacort, and dexamethasone. The short-term stimulatory effect of glucocorticoids on GH secretion is one of the most potent and also the most delayed stimulus to GH secretion, as it takes 3 hours to occur [144]. Thus glucocorticoids have short-term stimulatory effects on GH secretion and delayed inhibitory effects. Specific glucocorticoid responsive elements have been identified on the GH gene. Currently, the mechanism of action of the dual stimulation and inhibition of glucocorticoid on GH secretion has not been elucidated. Thyroid hormone increases basal and stimulated rat GH synthesis, acting directly at the level of gene expression [145]. This effect appears to be synergistic with glucocorticoids. T3 binds to nuclear receptors in rat pituitary tumor cells [146] and stimulates GH gene transcription [147]. In vitro data confirm that the hGH promoter is not thyroid hormone-responsive, while T3 strongly induces the rat GH promoter [21,148]. In hyperthyroid patients, GH secretion rate is decreased, but normalizes when patients are rendered euthyroid [149], suggesting that thyroid hormone suppresses GH secretion from the normal human somatotroph in vivo. GHRH

Hypothalamic GHRH was characterized initially by two different groups from ectopic pancreatic GHRH-secreting tumors causing acromegaly [128,150]. Analysis of one tumor revealed a 44-amino acid GHRH residue [151]; the other contained 37-, 40-, and 44-amino acid forms [152]. GHRH (1–40) and GHRH (1–44) are both found in extracts derived from the human hypothalamus. GHRH is secreted

Growth Hormone

85

from neurons in the hypothalamic arcuate nucleus and premammillary area, with axons that project to the median eminence [153]. The hGHRH gene encodes a 108-amino acid prepro-hormone for GHRH-44 [154,155], which has a free amino terminal and amidated carboxy terminal residue. The amino terminal tyrosine appears to bestow biologic activity on the GHRH molecule (Fig. 3.4). There is considerable structural homology between GHRH and several gut peptides. The highest is between GHRH and PHI, which have 12 amino acids in common in equivalent positions [156]. Varying degrees of homology exist between GHRH and VIP, glucagon, secretin, and GIP [157]. All of these peptides stimulate GH secretion in various physiologic systems, but with lower potency than GHRH. GHRH binds to specific sites on the somatotroph membrane, resulting in increased intracellular 3¢, 5¢cAMP [158]. The GHRH receptor gene has recently been cloned and sequenced, encoding a 47-kDa protein of 423 amino acids [159]. GHRH has a selective action on GH release; it does not release other anterior pituitary or gut hormones. GHRH increases GH synthesis as well as secretion, and stimulates transcription of GH mRNA [64,160]. GHRH stimulates GH release from both stored and newly synthesized intracellular GH pools, with a greater effect on stored pools [161]. There are a number of heterogeneous GH pools, varying according to time of GH synthesis [162–164], molecular size [163], and response to provocative stimuli [163]. Somatostatin suppresses both basal and GHRHstimulated GH release, but does not affect GH biosynthesis [165]. It is thus apparent that GHRH stimulates both release and synthesis of GH independently of any effects of somatostatin. GHRH administered to normal adults elicits a prompt increase in serum GH levels, with higher levels occurring in female subjects [166]. Most acromegalic patients retain an intact GH response to administered GHRH [167]. SRIF

SRIF, a cyclic tetradecapeptide, is recognized as a heterogeneous group of molecules including quantitatively predominant, but less bioactive SRIF-14, more bioactive SRIF-28,

FIGURE 3.4. Amino acid structure of growth hormone-releasing hormone (GHRH) (1–44). GHRH (1–44) and GHRH (1–40) have similar GH-releasing potency in vivo. The tyrosine at the amino terminus confers biologic activity. Fragments as short as GHRH (1–29) exhibit full biologic activity. The carboxy terminal fragment (28–44) has no biologic activity.

86

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 3.5. Amino acid structure of native somatostatin (SRIF). SRIF, a tetradecapeptide (14-amino acid peptide) designated SRIF-14, SRIF-28, which is extended at the amino terminal, and several larger forms range in size from 11.5 to 15.7 kDa.

sst

PHOSPHATASE

ADENYLCYCLASE K/Ca++ CHANNELS cAMP

Ca++

Calcineurin

Ser/Thr Phosphatase

– Exocytosis

INHIBITS SECRETION

FIGURE 3.6. SRIF receptor signaling pathway: somatostatin receptors mediate their responses via several cellular effectors such as adenylyl cyclase potassium and calcium channels and phosphatases. Adapted from Yogesh et al. [176a].

SRIF block the GH secretory response to insulin-induced hypoglycemia, exercise, arginine, morphine, levodopa, and sleep related GH release. Somatostatin exerts its biologic effects through specific membrane-bound high affinity receptors. At least five somatostatin receptor (SSTR) subtypes have been cloned [176], termed SSTRs1-5. Somatostatin receptors are coupled to guanine nucleotide protein (G), and have seven transmembrane domains. There is 42% to 60% amino acid homology among the five somatostatin receptor subtypes [177]. Somatostatin receptors mediate their responses via several cellular effectors such as adenylyl cyclase, protein phosphatases, Na+-H+ exchanger, cyclic GMP-dependent protein kinases, phospholipase C, potassium and calcium channels [176] (Fig. 3.6). The human pituitary gland expresses SSTR1, 2 and 5 [178,179], whereas human pituitary adenomas contain SSTR1, 2, 3 and [178–181]. Somatostatin analogs, octreotide and lanreotide, used to control GH hypersecretion in acromegaly, bind with high affinity to SSTR 2 and less efficiently to SSTR5 [182]. SSTR1-4 have highter affinity for SRIF-14, whereas SSTR5 bind more potently to SRIF-28 [176] (Table 3.2). GHRH and SRIF Interaction in Regulating GH Secretion

and still larger forms which range in molecular mass from 11.5 to 15.7-kDa [168] (Fig. 3.5). The SRIF precursor is a 116-amino acid prohormone consisting of a 24-amino acid signal peptide, a 64-amino acid connecting region, followed by SRIF-28 [169] which incorporates SRIF-14. Prosomatostatin is synthesized in neuronal perikarya of the anterior hypothalamic periventricular nuclei, and transported by axonal flow to the nerve terminals ending near the hypophyseal portal vessels. SRIF has also been isolated from pancreatic islets, gastrointestinal, neural and epithelial cells, and extrahypothalamic central nervous system neurons [170]. SRIF has a short plasma half-life of 2 to 3 minutes [168]. SRIF inhibits GH secretion, TSH release, TRH stimulation of TSH but not PRL [171], and pancreatic secretion of insulin and glucagon [172]. Dopamine [173], substance P, and neurotensin [174] stimulate SRIF release from the hypothalamus, while glucose inhibits SRIF secretion in cultured rat hypothalamus [174]. SRIF-28 binds to normal rat pituitary receptors with a threefold greater affinity than SRIF-14 [175]. Both SRIF14 and SRIF-28 block the effect of GHRH on GH release in vitro and in vivo in many different species. Infusions of

SRIF and GHRH secreted in independent waves from the hypothalamus interact to generate pulsatile GH release. SRIF inhibits GH secretion, while GHRH stimulates GH synthesis and secretion. GHRH stimulates pituitary GH synthesis and secretion, and also induces transcription of the GH gene [160]. SRIF suppresses GH secretion without altering GH mRNA levels [64]. GH secretion is further regulated by its target growth factor, IGF-I, which participates in a hypothalamic–pituitary–peripheral regulatory feedback system [183]. GH stimulates the liver and other peripheral tissues to produce IGF-I, which exerts a feedback effect on the hypothalamus and pituitary. IGF-I stimulates hypothalamic SRIF release [172] and inhibits pituitary GH gene transcription and GH secretion [51,184]. In vitro, IGF-I directly attenuates hGH gene expression [51]. Specific antibodies against GHRH and SRIF have been used to dissect the respective contributions of SRIF and GHRH in the generation of GH pulsatility in rats. Anti-SRIF antibody administration results in elevated baseline GH levels, with intact intervening GH pulses [185]. These studies imply that hypothalamic SRIF secretion generates GH troughs. AntiGHRH antibodies eliminate spontaneous GH surges. In humans, GH pulsatility persists when GHRH is tonically

Chapter 3 Table 3.2. receptors

Growth Hormone

87

Properties of the five cloned subtypes of human somatostatin

Properties

Somatostatin receptor Subtype 1

Subtype 2

Subtype 3

Subtype 4

Chromosomal location

14

17

22

20

16

G protein coupling

Yes

Yes

Yes

Yes

Yes

Effector system Adenylate cyclase activity Tryosine Phosphatase activity

Reduced Increased

Reduced Increased

Reduced ?

Reduced ?

Reduced ?

IC50 (nM) Somatostatin-14 Somatostatin-28 Octreotide Vapreotide (RC-160) Lanreotide (BIM-23014)

1.1 2.2 >1000 >1000 >1000

1.3 4.1 2.1 5.4 1.8

1.6 6.1 4.4–35 30.9 43

0.5 1.1 >1000 45 66

0.9 0.07 5.6 0.7 0.6

Brain, lungs, jejunum, kidneys, liver and pancreas

Brain and kidneys

Brain and pancreas

Brain and lungs

Brain, heart, adrenal glands placenta, pituitary, small intestine and skeletal muscle

Distribution in normal human tissue

Subtype 5

Adapted from Patel YC and Srikant CB [182].

elevated due to ectopic GHRH production by a tumor or during GHRH infusion [127,186], suggesting that hypothalamic SRIF is mainly responsible for GH pulsatility. The rat hypothalamus releases GHRH and SRIF 180° out of phase every 3 to 4 hours, resulting in pulsatile GH levels [185]. GHRH and SRIF also act synergistically, in that preexposure to SRIF has been shown to enhance somatotroph sensitivity to GHRH stimulation [187]. Hence, during a normal GH trough period, the high SRIF level probably primes the rat somatotroph to respond maximally to the subsequent GHRH pulse, thus optimizing GH release. In addition to exerting opposing and cooperative interactions on the pituitary somatotroph, SRIF exerts a central inhibitory effect on GHRH release via direct synaptic connections between SRIF-containing axons and GHRHcontaining perikarya in the rat hypothalamic arcuate nucleus [188]. GH Autoregulation

Chronic GHRH stimulation, either by continuous infusion or repeated bolus administration, eventually results in decreased GH release from cultured rat anterior pituitary cells [189] and in vivo [190] due to somatotroph desensitization. Loss of GH sensitivity to administered GHRH does not occur in acromegalic patients in vivo [191] or in somatotroph adenomas in vitro [192], possibly reflecting larger intracellular pools of GH or abnormal signaling. One possible mechanism of pituitary desensitization is depletion of a GHRH-sensitive pool of GH. GHRH pretreatment in

vitro also leads to a 50% decrease in somatotroph GHRH binding sites [193]. Feedback loops exist between GH and IGF-I and the release of SRIF and GHRH (Fig. 3.7). GH stimulates hypothalamic SRIF release in vitro [194], and in vivo, GH administration decreases GH responses to GHRH [195], most likely by increasing hypothalamic SRIF release [196]. GHRH and SRIF also autoregulate their own secretion. GHRH inhibits its own secretion but stimulates SRIF secretion in vitro [197], while SRIF inhibits its own secretion in vitro [198]. Growth Hormone Secretagogues (GHS) and Ghrelin

Small synthetic molecules termed growth hormone secretagogues (GHS) [199] stimulate and amplify pulsatile pituitary GH release [200], via a separate pathway distinct from GHRH/SRIF. A strategy of expression cloning was used to identify the GHS receptor, which is a heterotrimeric GTP-binding protein (G-protein)-coupled molecule [201]. This classic Gprotein coupled receptor has seven alpha helical membrane spanning domains and three intracellular and extracellular loops. The GHS receptor is most strongly expressed in the anterior pituitary and both the hypothalamic and nonhypothalamic regions of the brain. The ligand of the GHS receptor is a 28 amino acid peptide, Ghrelin isolated from stomach, with 3 serine residues and is n-octanoylated [202]. It releases GH both in vivo and in vitro and 0-n octanoylation appears to be essential for GH releasing activity. The

88

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 3.7. Growth hormone (GH), insulin-like growth factor-I (IGF-I) Growth Hormone secretagogues GHS (GHRH and Ghrelin), somatostatin (SRIF) feedback loop in the control of somatic growth. A stimulatory effect of GHRH and Ghrelin and inhibitory effect of SRIF interact to result in GH secretion from the anterior pituitary. GH stimulates IGF-I production from the liver. This, in turn, has a negative feedback effect on pituitary GH secretion and a positive feedback effect on hypothalamic SRIF release. Pituitary GH secretion has a negative autoregulatory feedback effect on hypothalmic GHRH secretion. GH exerts an effect on somatic growth by a direct as well as indirectly via IGF-I.

gene encoding human Ghrelin has been isolated, and found to be expressed in the stomach and hypothalamus [202]. The isolation of Ghrelin implicates a control system in addition to GHRH/SRIF in the regulation of GH secretion. Ghrelin is analogous to GHRH/SRIF in that it is also synthesized in peripheral tissues (stomach) as well as centrally (hypothalamus). It would be anticipated that hypothalamic Ghrelin would control GH secretion, while peripheral sources may have other biological effects requiring further elucidation. Several artificial GH secretagogues (GHS) release GH in vitro [203]. GHSs, administered alone or in combination with GHRH are potent and reproducible GH releasers and are useful tools for the diagnosis of GH deficiency. GHSs act directly on the pituitary somatrophs [204], stimulating GH secretion by activating the cAmp-dependent protein kinase pathway [205]. GHRH and GHS thus act through distinct receptors, via different intracellular signaling pathways on somatotrope subpopulations. GHS increases the number of somatotropes releasing GH, without altering the amount of hormone released from each individual cell

[206]. Somatostatin too decreases the number of somatotropes that secrete GH, and it is postulated that GHS and SRIF may respectively depolarize and hyperpolarize the same somatotroph cell. GHRH, on the other hand, stimulates the amount of GH secreted per cell, as well as the number of cells secreting GH [206]. Evidence that the main action of GHS occurs at the hypothalamic level, and that the direct pituitary activity is ancillary, is supported by the fact that GHSs require the presence of a functional hypothalamus to exert their GH secreting effect. In patients with intact pituitaries, who have hypothalamic dysfunction leading to stalk section, GHSinduced GH release is blocked and the synergistic effect of GHRH and GHS is absent [207]. Current evidence suggests that the dual control of GH secretion postulated for GHRH/SRIF should be expanded to incorporate Ghrelin. If indeed Ghrelin acts at the hypothalamic level, its mechanism(s) of action could include release of endogenous GHRH. However available data seems to indicate that Ghrelin does not act via GHRH release in that the GH response to GHS is greater than that of GHRH alone [208];

Chapter 3

FIGURE 3.8. A new physiologic model of the regulation of GH secretion involving Ghrelin, GHRH and somatostatin (SRIF). From Diguez and Casanueva [218a].

GHSs potentiate GH release in response to a maximal stimulating dose of exogenous GHRH [209]. GHSs are unable to release GHRH in rat hypothalamic explants [210]; GHRH infusions block GHRH mediated GH release without altering the action of GHS [211,212]; after administration of a saturating dose of GHRh, a second dose of GHRH is ineffective, while GHS is fully effective [213]. The above evidence does not support a role of GHRH in the mechanism of action of GHS. GHSs do not seem to inhibit somatostatin release [212,214–216]. However, GHS mediated GH release is relatively insensitive to the inhibitory action of SRIF [217]. The presence of GHS in hypothalamic structures and the evidence that GHS-elicited GH secretion is not mediated by changes in endogenous GHRH or SRIF, but requires a functional hypothalamus, suggests that exogenous GHS may induce the release of another hypothalamic factor with GH-releasing capability [218] (Fig. 3.8). Release of another unknown hypothalamic factor with GH releasing capability is supposed by observations that GHSs effectively release GH when administered intravenously, subcutaneously, orally or intranasally [219]. Hypoglycemia increases GHS stimulated GH release [214] as does decreased circulating FFAs [220]. GHSs are resistant to wellknown inhibitors of GH secretion including increased FFA levels, hyperglycemia, SRIF infusion, atropine or drugs that enhance hypothalmic SRIF secretion [221]. GHS-mediated GH release is not reduced by a previous rise in GH [213], glucocorticoid treatment [222], or thyroid hormone abnormalities [223]. GHS-mediated GH release is more potent and reproducible than that elicited by GHRH alone [224].

Growth Hormone

89

GHRH-mediated GH release is more erratic and less reproducible, possibly due to the influence of metabolic and hormonal factors. Functional GHs receptors are detected in the human pituitary by the fifth week of gestation [225]. GHS mediated GH-release is demonstratable at birth [221], continues through infancy, increases at puberty and then decreases thereafter. Estrogen and testosterone increase GHS mediated GH release in childhood. When administered with GHRH, GHS has a synergistic action on GH release. As GH secretion elicited by combined GHRH and GHS administration is minimally altered by age, sex, or adiposity and is devoid of potential side effects (unlike insulin-induced hypogylcemia) this test has the potential to become a widely used diagnostic tool in the diagnosis of adult GHD. No side effects have been reported with the GHSs. Although highly specific for GH release, slight increases in prolactin and ACTH/cortisol have been reported with some GHSs [226], leading to the development of new more selective GHSs with no ACTH or PRLreleasing effects e.g. ipamorelin [227]. GHSs are being evaluated as potential therapeutic agents for enhancing circulating GH levels in patients with intact hypothalamic–pituitary axs, including children with GHD as well as adults with catabolic states, AIDS and heart failure. Long-term studies are required with a large number of subjects to determine dosage efficacy.

GH REGULATION

GH Transgenic Animals Transgenesis allows for the transfer of inheritable functional genes between animals of different species. GH transgene models of endocrine hyperfunction have also been useful in investigating regulatory factors required for tissue-specific gene targeting. When a rat or hGH reporter gene driven by a potent metallothionein-I (MT) promoter is expressed in a transgenic progeny, rat or human GH is produced by a number of tissues including the pituitary [228,229]. Circulating GH levels in these animals are several hundred times higher than normal with an increase in transgenic mouse size to twice that of normal littermates [230]. Circulating IGF-I levels are elevated two- to threefold, consistent with the notion that IGF-I mediates the allometric growth of these transgenic animals [228,229]. Interestingly, the effects of excess GH are only apparent after 3 weeks of age and no further growth occurs after 3 months. Immunocytochemical and ultrastructural study of transgenic pituitary glands revealed a marked reduction in both size and number of GH-secreting cells [229,231]. Pituitary lactotroph cells were also suppressed in male transgenic animals, but not in females. In contrast, both sexes exhibited corticotroph cell hyperplasia. Male transgenics had high levels of LH and highly active gonadotrophs, resembling “gonadectomy” pituitary cells. When ovariectomized female

90

SECTION 1

Hypothalamic–Pituitary Function

hGH transgenics were studied, adult female mice developed hypoprolactinemia, but had high levels of LH due to PRL-like activity of GH [232]. Metallothionein rat GH (MTrGH), metallothionein bovine GH (MTbGH) or metallothionein human GH (MThGH) female transgenics are sterile; however, metallothionein ovine GH (MToGH) female transgenics are fertile [233,234]. In MTrGH transgenic animals [235] both the liver and spleen undergo a threefold increase in size. Furthermore, MToGH male transgenics contain hepatic intranuclear inclusions whose presence correlates with high circulating levels of oGH [233]. The heart, lungs, kidneys, and thyroid glands of GH transgenics grew significantly larger when compared to nontransgenic control littermates. Interestingly, no increase was observed in brain or adrenal size. In contrast, methallothionein-driven IGF-I gene (MTIGF-1) transgenics [230] have an increased growth ratio of one to three times that of control littermates, which is only apparent from 6 to 8 weeks after birth. Spleen, pancreas, and kidneys increased in size in direct proportion to the increase in body weight both in IGF-I and GH transgenic models. Compared to GH transgenics, IGF-I transgenic mice show an increase in brain size but no enlargement of the liver or heart [230]. GH transgenics develop renal glomerular sclerosis, similar to abnormalities seen in diabetics, whereas this lesion is not present in IGF-I transgenics. GH transgenics also have elevated serum insulin and lipid levels [236]. Interestingly, mesangial renal proliferation followed by progressive glomerulosclerosis was also seen in transgenic GHRH animals [237], suggesting that the hypersomatotrophism associated with excess GHRH secretion is predominantly GH-mediated. While many of the sequelae of chronic GH overexpression such as increased growth and pituitary somatotroph atrophy were predictable, the abnormalities of hepatic and renal function, and the reduced fertility of female transgenics, were unexpected. These novel findings may provide useful models of human disease. Transgenesis is a useful tool to investigate ontogeny of gene expression, as well as a method of exposing animals to growth at a time when they would not usually be accessible to manipulation of factors responsible for both allometric as well as organ-specific growth. GH Binding Protein (GHBP)

Circulating GH is attached to specific binding proteins [238]. Two circulating GHBPs have been identified, one of high affinity and one of low affinity [239]. Although the site of GHBP synthesis is as yet undetermined, the liver has been postulated as the primary source [240]. The 60-kDa high-affinity BP corresponds to the extracellular domain of the hepatic GH receptor [241]. Under basal conditions, half of the circulating 22-kDa GH is bound to the highaffinity BP when GH levels are up to 10–15 ng/ml [242]. The 20-kDa GH binds preferentially to the low-affinity BP [243]. Binding to plasma GHBP prolongs GH plasma half-

life by decreasing GH metabolic clearance rate, as the GHbinding protein complex is too large for renal glomerular filtration [243]. The high-affinity BP also inhibits GH binding to surface receptors by competing for the ligand [244]. Thus GHBP dampens acute oscillations in serum GH levels caused by pulsatile pituitary GH secretion. Highaffinity BP levels are low in the fetus and neonate, rise most rapidly in the first 1 to 2 years after birth [245,246], and stay constant throughout adult life, with similar levels found in males and females [245]. GH itself does not regulate either type of BP, as evidenced by normal BP levels found in patients with either hypopituitarism or acromegaly [245]. GH resistance, as demonstrated in short stature, Laron dwarfism, and African pygmies, is characterized by decreased plasma levels of high-affinity BP [247,248]. Although these conditions provide insight into the role of GHBP in GH action, Laron dwarfs are also GH receptor-deficient, therefore, no conclusions can be drawn about GHBP function per se from these findings. In African pygmies, GHBP levels, although low, vary widely with considerable overlap with normal subjects.

Peripheral GH Action GH Receptor

GH binds to its peripheral receptor and induces intracellular signaling by a phosphorylation cascade involving the JAK/STAT pathway. GH also acts indirectly by inducing synthesis of IGF-I, the potent growth and differentiation factor [249]. The GH receptor (GHR) is a 70 kd protein member of the class I cytokine/hematopoietin receptor superfamily [250]. GHR consists of an extracellular ligandbinding domain, a single membrane-spanning domain, and a cytoplasmic signaling component. The GH ligand complexes with two GHR components leading to receptor dimerization. This sequential ligand-receptor interaction and dimerization is critical for subsequent GH signaling. GHR dimerization is followed by rapid binding and activation of JAK2 tyrosine kinase, leading to phosphorylation of cytoplasmic signaling molecules, especially the signal transducing activators of transcription proteins (STAT) which comprise the critical signaling components for GH action. These cytoplasmic proteins are phosphorylated by JAK2 and directly translocated to the cell nucleus where they elicit GH-specific target gene expression by binding to nuclear DNA [250]. In addition, STAT1 and STAT5 may in fact interact directly with the GH receptor molecule [251]. Other target actions induced by GH include c-fos induction, IRS-1 phosphorylation, and insulin synthesis. As a differentiating and growth factor, IGF-1 is a critical protein induced by GH, and is likely responsible for most of the growth-promoting activities of GH [252]. IGF-I itself may also directly regulate GH [252] and GH receptor function [253]. The liver contains the highest abundance of GH receptors, and several peripheral tissues also express modest amounts of receptor, including muscle and fat [254].

Chapter 3

Mutations of the GH receptor may be associated with partial or complete GH insensitivity and growth failure. These syndromes are associated with normal or high circulating GH levels, decreased circulating GHBP levels and low levels of circulating IGF-I. Multiple homozygous or heterozygous exonic and intronic GHR mutations have been described, most of which occur in the extracellular ligandbinding domain of the receptor. Tissue responses to GH signaling may be determined by the pattern of GH secretion, rather than the absolute amount of circulating hormone. Thus, linear growth patterns, liver enzyme induction and STAT5b activity may be phenotypically distinct for male animals due to their higher rates of GH pulse frequency [255]. STAT5b is sensitive to repeated pulses of injected GH [256], unlike other GHinduced patterns which are desensitized by repeated GH pulsing. Mice harboring a disrupted STAT5b transgene exhibit impaired male pattern body growth [257] with IGFI and testosterone levels normally seen in female mice. Thus, the sexual dimorphic pattern of GH secretion and GH tissue targeting appears to be determined by STAT5b. The requirement for appropriate GH pulsatility to determine body growth also appears to be mediated by STAT5b [258]. In contrast, STAT5 does not appear to be critical for metabolic effects of GH on carbohydrate metabolism [259]. Unraveling STAT5 regulation in disease, especially those involving GH resistance, will provide novel mechanistic insights for dysregulated GH signaling in humans. Intracellular GH signaling is also abrogated by SOCS proteins, which disrupt the JAK/STAT pathway and thus exert a further level of control over the action of GH [260] (Fig. 3.9).

Growth Hormone

91

IGF-I and IGF-II Structure and Synthesis

IGF-I and IGF-II are single-chain polypeptide molecules with three intrachain sulfide bridges (Figs 3.10 and 3.11). IGF-I, composed of 70 amino acids, and IGF-II, consisting of 67 amino acids, have a sequence homology of 62% [261]. The IGFs consist of B and A peptide domains (structurally homologous with the insulin B and A chains), a C domain analogous to the connecting (C) peptide of proinsulin, and a D domain. IGF-I and IGF-II are single distinct gene loci, localized on chromosome 12 (12q22–q24.1) and chromosome 11 (11p15), respectively [262,263]. The IGF-I gene primary transcript can be alternately spliced to different products resulting in IGF-Ia (exons 1,2,3,5) or IGF-Ib (exons 1,2,3,4). Several IGF-I mRNA species have been isolated from adult and fetal tissues. The liver is an important source of circulating IGF-I levels [264]. The IGF-I gene is expressed in human fetal conective tissues and cells of mesenchymal origin [265]. This ubiquitous localization of IGFs favors a paracrine/autocrine function as well as an endocrine function of IGF-I. GH is the major regulator of IGF-I gene expression in adult liver, heart, lung, and pancreas [266], and acts at the level of IGF-I transcription [267]. Fetal IGF-I production is GHindependent [268], and platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) also stimulate IGF-I production from human fibroblasts in vitro [269]. ACTH, TSH, LH, and FSH stimulate the paracrine production of IGF-I in their respective target tissues. Nutritional status is also an important regulator of IGF-I production at all ages [270]. As discussed below, IGF-I and IGF-II are bound to carrier proteins in the serum. IGFs are found in lymph [271], breast milk [272], saliva [273], and amniotic fluid [274]. IGF-I levels are low before birth, rise during childhood to high levels during puberty, and decline with age [275]. IGF-binding Proteins (IGFBPs)

FIGURE 3.9. Role of STAT proteins in growth hormone signaling. From Herrington, Smith, Schwartz, Carter-Su [260a].

IGF-I and IGF-II are complexed to six specific binding proteins in biological fluids [276] (Fig. 3.12 and Table 3.3). The IGFBPs are cysteine-rich proteins, with very similar amino acid sequences. They have a unique ability to bind IGF’s with high affinity. Recently several groups of cysteine-rich proteins have been described [277] with structural and functional similarities to the IGFBP’s, called IGFBP-related proteins (IGFBP-rPs). The IGFBP-rPs bind IGFs with low affinity. The physiological role of these IGFBP-rPs in the IGF system is not known. However some of these proteins unequivocally bind IGFs [277]. An IGFBP superfamily has been proposed, composed of the IGFBPs and the IGFBPrelated proteins (IGFBP-rPs) (Fig. 3.13). The major form of binding protein present in the human circulation is IGFBPIII, a glycoprotein associated with an IGF molecule and an 80-kDa acid-labile subunit (ALS) to form a 150- to 200kDa complex [278] (Fig. 3.14).

92

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 3.10. Amino acid sequence of human insulin-like growth factor I (IGF-I). The black amino acids are identical to those in human insulin. The numbering corresponds to the numbering of residues in the proinsulin molecule. IGF-I consists of a 70-amino acid single peptide chain with A, B, C, and D, domains. A and B domains are structurally homologous to the A and B chains in the insulin molecule, and the C domain is equivalent to the connecting (C peptide in proinsulin). From Humbel [261a].

FIGURE 3.11. Primary structure of human insulin-like growth factor II (IGF-II). From Sara and Hall [261b].

Actions of the IGFBPs include modulation of IGF actions, storage of IGFs in extracellular matrices, and the carrier protein function of IGFBPs. In the serum most of the IGF circulates as a 150,000 dalton complex that consists of IGF-I or IGF-II plus IGFBP-III and a nonIGF binding component, the acid labile cubunit (ALS). Binding of IGF-I or II to IGFBP-III in the presence of ALS results in the formation of an IGF-IGFBP-ALS ternary complex, which is stabilized by IGF binding [279] 75% of circulating IGF-I and IGF-II is carried in this ternary 150-kDa complex. When associated with the 150-kDa complex, the IGFs do not readily leave the vascular compartment, and have prolonged half-lives of 12 to 15 hours [280] compared to the half-life of free IGF-I which is less than 10 minutes [281]. The exact function of the large store of IGF bound

to the 150-kDa ternary serum complex is unknown. It has been hypothesized to be a functional pool of material available for organisms to use during stress. The plasma concentrations of IGFBPs are hormonally regulated. Serum IGFBP-III levels correlate with IGF-I and II levels, double in patients with acromegaly and are reduced in hypopituitarism [282]. Malnutrition, insulin-dependent diabetes mellitus, and cirrhosis are associated with decreased IGF-I levels, as well as suppressed IGFBP-III levels [283]. IGFBP-I levels are high at birth and decline until puberty [284]. There is a diurnal variation with a nocturnal peak in serum IGFBP-I levels [285]. However, IGFBP-I levels are elevated in hypopituitarism [286] and decreased in acromegaly. IGFBP-I levels are regulated by insulin. The increased IGFBP-I levels associated with insulin-dependent

FIGURE 3.12. Simplified diagram of GH-IGF-I axis involving hypophysiotropic hormones controlling pituitary GH release, circulating GH-binding protein and its GH receptor source, IGF-I and its largely GHdependent binding proteins, and cellular responsiveness to GH and IGF-I interacting with their specific receptors. IGFR, IGF-I receptor; FFA, free fatty acids. From Rosenbloom [276a].

Table 3.3.

General characteristics of the human IGF binding protein No. of amino acids

Core molecular mass (kDa)

Chromosomal localization

IGF affinity

Modulation of IGF action

Source in biological fluids

IGFBP-I

234

25.3

7

I = II

Inhibition and/or potentiation

Amniotic fluid, serum, placenta, endometrium, milk, urine, synovial fluid, interstitial fluid and seminal fluid

IGFBP-II

289

31.4

2

II > I

Inhibition

CSF, serum, milk, urine, synovial fluid, interstitial fluid, lymph follicular fluid, seminal fluid, and amniotic fluid

IGFBP-III

264

28.7

7

I = II

Inhibition and/or potentiation

Serum, follicular fluid, milk, urine, CSF, amniotic fluid, synovial fluid, interstitial fluid, and seminal fluid

IGFBP-IV

237

25.9

17

I = II

Inhibition

Serum follicular fluid, seminal fluid, interstitial fluid and synovial fluid

IGFBP-V

252

28.5

5

II > I

Potentiation

Serum and CSF

IGFBP-VI

216

22.8

12

II > I

Inhibition

CSF, serum and amniotic fluid

Modified from Rajaram S et al. [278a].

94

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 3.13. The IGF system. The components of this system include the peptides IGF-I and -II, IGFBP-1 to -6, IGFBP-rPs, IGFBP proteases, type I and type II IGF receptors, and potential IGFBP(s) and IGFBP-rP(s) receptors. M6P, Mannose-6-phosphate. From Hwa, Oh, Rosenfeld [277a].

diabetes mellitus [287] are normalized by insulin, and insulinoma is associated with suppressed IGFBP-I levels. Octreotide (a long-acting somatostatin analog) has been shown to increase IGFBP-I levels in acromegalic patients [288]. When normal subjects ingest glucose, the fall in IGFBP-I levels correlates inversely with the rise in insulin levels [289]. Hypophysectomy is associated with elevated IGFBP-II levels in rats, which fall with GH administration [283]. Insulin infused into normal rats increases IGFBP-II levels [283]. PARACRINE GH ACTION GH and GH-releasing factors are also produced in tissues outside the hypothalamic pituitary axis. The extrapituitary actions of GH are likely autocrine/paracrine and complement the classic endocrine action between the GHreleasing factors, GH and target tissues. GH gene expression is not restricted to the pituitary gland. GH immunoreactivity and GH mRNA have been localized to several extrapi-

tuitary tissues [290] including placenta, mammary gland muscle, spleen lymphocyte, suggesting an extrapituitary paracrine/autocrine action for GH. Classic endocrine actions of GH include GH promoting effects on bone and several metabolic actions.

Effect on Growth Whether GH exerts its growth-promoting action on the skeleton via a direct GH effect on the growth plate, or indirectly by stimulating hepatic or local IGF-I production, remains unclear. The somatomedin hypothesis proposed an indirect effect of GH on growth mediated through plasmatransported IGF-I that is synthesized in the liver [291]. The dual effector theory postulates a direct effect of GH by stimulating epiphyseal growth plate precursor cells to differentiate and become responsive to IGF-I, and to induce local IGF-I synthesis that in turn stimulates the multiplication of differentiating chondrocytes [292]. This proposes a dual role for GH and IGF-I in the control of tissue mass. IGF-1 has

Chapter 3

Growth Hormone

95

which is rapidly restored by GH administration [299]. Hypophysectomized rats show a progressive decline in longitudinal bone growth rate [300], finally growing at only 0 to 10% of the normal rate. GH and IGF-I Effects on Bone in vitro

FIGURE 3.14. Relative distribution of various IGF pools in human serum. The distribution of IGFs between the 50-kDa, 150kDa, and the free pool as determined before and during continuous subcutaneous infusion of 30 mg/day of rhIGF-1 in healthy men. From Rajaram, Baylink, Mohan [278a].

a potent mitogenic action, especially on immature differentiating cells (adipocytes, chondrocytes), whereas GH is not a mitogen [293]. The dual effector theory proposes that although the overall effects of GH and IGF-I on growth at the tissue level appear to be the same, at the cellular level their effects are very different; GH has both a direct action and an indirect action mediated by IGF-I. The IGFBP regulatory hypothesis proposes that GH stimulates both IGF-I and IGFBP-III [294]. Circulating IGFBP-III regulates the bioavailability of IGF-I to the growth plate, and may inhibit IGF-I action. However, recently hepatic IGF-II production in the liver of mice was completely abolished using an IGF-I knockout mouse model, with complete inactivation of the IGF-I gene in the hepatocytes [295]. Serum IGF-I concentration was reduced by 75%. However the reduction in serum IGFI concentration had no discernible effect on postnatal body growth. Role of GH in Growth and Development

Congenital absence of the human pituitary gland does not result in abnormal birth or newborn weight [296], indicating that fetal and early postnatal development may occur independently of GH. Postpubertal mammalian longitudinal bone growth results from chondrocyte proliferation and differentiation in long bone epiphyseal growth plates. The proximal zone of the epiphyseal growth plate contains stem cells and is the layer of prechondrocyte origin [292]. These cells differentiate and enter the proliferative layer, where they undergo limited clonal expansion. After maturation, the cells become calcified and are incorporated into metaphyseal bone [297]. Hypophysectomy in the growing rat results in a marked reduction in tibial epiphyseal cartilage plate width [298],

Although earlier studies failed to demonstrate in vitro stimulatory effects of GH [301], subsequent studies have demonstrated significant direct effects of GH on chondrocyte and osteoblast cultures [302]. GH increases progenitor cell thymidine incorporation, chondrocyte differentiation, and osteoclast formation. The in vitro effect of IGF-I on chondrocytes has been more consistently demonstrated. IGF-I increases thymidine and sulfate incorporation into isolated chondrocytes [303], and this effect is additive with GH [302,303] occurring at different stages of maturation [304]. Cells from the proximal growth plate (prechondrocytes and young proliferating chondrocytes) form larger colonies in vitro, whereas chondrocytes from the more distal proliferative zone form smaller colonies [305]. Both GH and IGF-I induce chondrocyte colony formation [305] but exert differential effects on colonies of different sizes. GH preferentially potentiates formation of large size colonies, suggesting that GH stimulates prechondrocyte differentiation [304], while IGF-I induces smaller colonies, implicating IGF-I in epiphyseal chrondrocyte stimulation at a more mature stage of differentiation [304]. In vivo Effects of GH and IGF on Skeletal Growth

GH administered locally at the epiphyseal growth plate [306,307] or infused into a femoral artery [308] in hypophysectomized rats results in unilateral longitudinal bone growth and an increase in growth plate width. When IGFI antiserum is infused locally with GH this growth effect is eliminated [308], suggesting that GH exerts its growth promotion via local IGF-I. Local infusion of IGF-I alone into the epiphyseal growth plate results in longitudinal bone growth [307] and increased epiphyseal width. Local GH infusion at the growth plate additionally results in increased numbers of IGF-I-containing cells in the proliferative zone, as well as producing a simultaneous increase in plasma IGFI levels [309], suggesting that the in vivo GH effect may be mediated by locally produced IGF-I in cells that have been stimulated by GH. This may explain why GH-deficient children treated with GH grow significantly, even though their plasma IGF-I levels may not change appreciably [310]. The effects of systemic GH and IGF-I administration have been compared. IGF-I increases tibial epiphyseal width [311] and total body growth in hypophysectomized rats [312], but to a lesser degree than after replacement doses of GH. In mutant GH-deficient dwarf rats, hGH infusions had a more pronounced stimulatory effect on body weight and bone growth than hIGF-I administration, while hIGF-I caused a greater increase in kidney, adrenal, and spleen size than did hGH [313]. GH and IGF-I may also have

96

SECTION 1

Hypothalamic–Pituitary Function

differential tissue effects. Some of these observations suggest that GH acts directly on tibial plate cartilage via a locally produced paracrine or autocrine IGF-I action. The somatomedin hypothesis, however, suggests that plasma IGF-I levels are much higher than tissue levels, and that plasma IGF-I is indeed biologically active. The liver synthesizes more IGF-I in response to GH stimulation than does costal cartilage. Furthermore, IGF-I increases thymidine and sulfate uptake and incorporation in bone and cartilage cultures to a far greater degree than does GH. These findings favor the liver as the predominant site of IGF-I synthesis, with subsequent plasma transport of biologically active IGF-I to its target organ, bone, where it mediates growth. A model incorporating GH, IGF and IGFBP systems in the regulation of systemic growth integrates the dual effector theory and the somatomedin hypothesis (Fig. 3.15). GH stimulates hepatic/tissue IGF-I synthesis, and it is the endocrine action of this circulating IGF-I on the growth plate which regulates skeletal growth, by stimulating cell division. Furthermore, GH induces hepatic IGFBIII and ALS production, forming the circulating 150-kDa ternary

complex that binds IGF-I, thereby increasing its half-life. GH also directly induces prechondrocyte differentiation into chondrocytes at the growth plate, in addition to stimulating local IGF-I production, which acts in an autocrine/ paracrine fashion to further stimulate cell division. Thus the endocrine actions of circulating IGF-I largely contribute to cell division which results in intrauterine and post natal growth. This mechanism is augmented by the direct actions of GH stimulating prechondrocyte/chondrocyte differentiation at the growth plate as well as the local IGF-I stimulation by GH where it acts by an autocrine/ paracrine action to stimulate cell division.

GH and Metabolism In childhood, the multiple metabolic actions of GH contribute to linear growth. GH continues to be secreted in adulthood after growth cessation, implying that GH has important metabolic functions in adult life. The metabolic actions of GH are either acute insulin-like or chronic insulin antagonism. The metabolic actions of GH may be direct or indirectly mediated by IGF-I.

FIGURE 3.15. Integrated model of the GH-IGFBP-IGF axis in the growth process. Three mechanisms are proposed: (1) GH stimulates IGF-I production; circulating IGF-I (endocrine IGF-I) acts at the growth plate; (2) GH regulates hepatic production of IGFBP-III and ALS: IGF-I binds to IGFBP-III and thereafter with ALS, forming the 150 kDa ternary complex; proteases cleave IGFBP-III into fragments that release IGF-I in the intravascular space and at the growth plate; and (3) GH induces differentiation local IGF-I production, and IGF-I acts via an autocrine and paracrine mechanism to stimulate cell division.From Spagnol and Rosenfeld [313a].

Chapter 3 Table 3.4.

Acute metabolic effects of growth hormone

Enhanced lipolysis and lipid oxidation ÆMobilization of stored triglyceride Protein synthesis stimulation Antagonism of insulin action Water and sodium retention

Acute, transient, insulin-like effects of GH can be demonstrated on carbohydrate, protein, and lipid metabolism (Table 3.4). The insulin-like effects are more easily demonstrated in tissues that have not recently been exposed to GH (for example, those derived from hypophysectomized animals, or GH-deficient humans) than in normal tissues [314]; casting doubt on the physiologic relevance of the insulin-like effects of GH, because GH is secreted endogenously in frequent bursts, resulting in significant changes in ambient GH levels. The chronic antiinsulin effects of GH result in decreased glucose utilization, increased lipolysis, and tissue refractoriness to the insulin-like effects of GH. Antiinsulin effects occur at both hepatic and peripheral sites [315]. Effects on Carbohydrate Metabolism

GH-deficient children have decreased fasting glucose levels [316,317], decreased insulin secretion [316], contradictory impairment of glucose tolerance [318–321], and increased insulin sensitivity due to increased glucose utilization and blunted hepatic glucose release. GH replacement increases fasting glucose levels [317,318], insulin levels [318], and hepatic glucose production [317]. Endogenous physiologic GH secretion antagonizes insulin action [322]. Normally, GH secretion increases 3 to 5 hours after oral glucose ingestion, resulting in decreased disposal of a second oral glucose challenge, associated with hyperinsulinemia occurring 2 hours after GH levels peak. Both intravenous and oral glucose tolerance tests are impaired if performed during periods of increased GH secretion, such as sleep onset. GH-deficient adults have elevated fasting insulin levels and a positive correlation between fasting plasma insulin and both fat mass and waist girth [323], suggesting insulin resistance, which is confirmed by hyperinsulinemic euglycemic clamp [324]. GH replacement initially further increases insulin resistance, in the first 1 to 6 weeks of therapy, but subsequently, although hyperinsulinemia persists, carbohydrate metabolism returns to baseline after 3 months of GH treatment [325]. Acromegaly is the experiment of nature for effects of excess GH secretion on carbohydrate metabolism. Several stages of impaired glucose tolerance occur in acromegaly, possibly indicative of a stepwise deterioration in carbohydrate metabolism as the disease progresses [326]. Glucose tolerance after both intravenous [326] and oral glucose administration is impaired in association with hyperinsu-

Growth Hormone

97

linemia [327]. When acromegalic patients eventually manifest overt diabetes mellitus, postglucose insulin levels are depressed [327]. Effects on Protein Metabolism

Somatic growth is under the primary control of GH and, as such, GH is an anabolic hormone. GH causes urinary nitrogen retention and decreased plasma urea levels in GH deficient children [328], and in normal [329] and obese [330] adults. Furthermore, GH-deficient children increase their muscle mass following GH treatment [331]. Both insulin and IGF-I have been implicated in the anabolic effects of GH on protein metabolism, especially as insulin is a protein anabolic hormone and GH stimulates circulating insulin levels. However, GH-stimulated protein synthesis precedes the GH stimulated increase in insulin levels. Physiologically, GH may promote nitrogen retention during starvation, as GH levels rise during fasting.

Effects on Lipid Metabolism GH Effect on Adipose Tissue

GH plays an important role in fat metabolism. GHdeficient children are mildly obese [331,332], with a decreased total number of fat cells [333,334] that are larger in size, with a greater lipid content than normal. GH replacement therapy leads to a decrease in body fat and, eventually, a decrease in the size and lipid content of subcutaneous adipocytes. GH deficient adults have altered body composition, with increased fat mass and decreased lean body mass. Initial acute effects of GH on lipid metabolism are antilipolytic (insulin-like) and subsequently, GH exerts a lipolytic (antiinsulin) effect. Lipolysis

GH increases fat mobilization and decreases fat deposition, thereby decreasing body fat. GH activates hormonesensitive lipase [335], resulting in increased hydrolysis of triglycerides to free fatty acids and glycerol (lipolysis). GH also decreases fatty acid reesterification [336]. The lipolytic actions of GH can be counteracted by insulin and are additive with glucocorticoids [337], which decrease the antilipolytic effects of insulin [338]. In vivo Effects on Lipids and Lipoproteins

Physiologic doses of GH have multiple, inconsistently demonstrated effects on free fatty acids and ketones [339]. Lipolytic and ketogenic effects of GH are easily demonstrable in normal subjects with suppressed insulin levels [318] and in GH-deficient insulin-dependent diabetic patients infused with GH [340,341]. GH deficient adults have elevated total cholesterol, low density lipoprotein cholesterol (LDL) and apolipoprotein B (ApoB) [342], with low high density lipoprotein levels (HDL) and high triglyceride levels. This lipid profile is

98

SECTION 1

Hypothalamic–Pituitary Function

associated with premature atherosclerosis and cardiovascular disease. GH replacement treatment, decreases total cholesterol (342) LDL cholesterol and ApoB and increases HDL. Long term surveillance will be required to determine whether GH replacement therapy reverses premature atheroclerosis and reduces cardiovascular morbidity and mortality in GH-deficient adults.

the abdomen [323]. The reduction in abdominal fat mass is mainly due to reduced visceral fat mass [343,344]. GH therapy increases total body water [343] especially extracellular water within 3 to 5 days [345,351]. Total blood volume increases after 3 months of treatment [346]. A direct action of GH and/or IGF-I on renal tubular sodium reabsorption, as well as activation of the renin angiotensin system [345,352], contributes to the antinatiuretic GH effects.

GH Effects on Body Composition

The anabolic, lipolytic and antinatiuretic actions of GH impact body composition affecting fat mass, lean body mass and fluid volume in GH-deficient adults. Lean body mass (LBM) is reduced [323,343], and fat mass is increased in GH-deficient adults compared to predicted values for age, sex, and height matched normal controls in GH-deficient adults [323]. Excess fat accumulates mostly in the visceral component in a central distribution, mainly abdominal [343,344]. Total body water is reduced in adults with GH deficiency. Reduced plasma volume [345] and total blood volume [346], contribute to the reduced extracellular water [347,345]. GH replacement therapy reverses these effects on body composition by increasing LBM (Fig. 3.16) [343,344, 348–350]. There is a significant increase in skeletal muscle, the most important component of LBM [348]. GH replacement also reduces fat mass by 4–6 kg in GH deficient adults [343,344,348–350] with the most significant reduction in

GH Effects on Bone Metabolism and Bone Mineral Density (BMD)

GH-deficient adults have reduced bone mass, [353] measured by bone mineral density (BMD), resulting in a 2 to 3 times increased fracture rate [354]. The pathogenesis of the bone loss is unclear, with studies demonstrating unchanged [355], increased, [356] or reduced [357] bone formation as well as unchanged bone resorption [358] and delayed mineralization [359]. With GH replacement therapy there is predominant bone resorption in the first 3 to 6 months, resulting in initial reduced BMD after 6 to 12 months [360] with subsequent increased bone formation leading to a net gain in bone mass after 18 to 24 months of treatment [361]. GH Effects on Muscle Strength and Exercise Performance

GH deficiency is associated with reduced muscle strength, due to altered body composition [362]. Reduction in muscle cross-sectional area, as well as lack of conditioning and training, may contribute to the weakness. Prolonged GH replacement therapy for at least 12 months is required to significantly increase muscle strength [362] and for at least 3 years to normalize the muscle strength [363]. GH Effects on Cardiovascular Function

FIGURE 3.16. Effect of GH replacement on (a) lean body mass (LBM) and (b) fat mass in 24 adults with GHD. Reproduced with permission from Salomon et al. [347a].

Hypopituitary adults have increased vascular mortality [364] mostly attributable to cerebrovascular and cardiovascular disease [365]. GH deficiency has been implicated [366] based on the hypothesis that adult GH deficiency predisposes to the development of premature atherosclerosis, as evidenced by increased carotid artery intima-medial thickening and plaque formation [367], increased triglycerides, plasminogen activator inhibitor-1 activity and fibrinogen [368]. The role of GH in regulating cardiac structure and function has not been established. In GH-sufficient adults with dilated cardiomyopathy and congestive heart failure, GH has been shown to improve cardiac function and increase ventricular mass [369] by some workers and to have no beneficial effect by others. GH deficient adults have reduced left ventricular mass and impaired systolic function. Short term GH treatment for 6 months improves the deficits in cardiac function and structure, with a sustained effect on cardiac performance for up to 3 years of GH treatment [370]. Effects of GH excess in acromegaly manifest as left ventricular hypertrophy, hypertension, cardiac failure and increased mortality due to cardiovascular causes [371]. The long term effects of GH

Chapter 3

Growth Hormone

99

replacement on cardiac structure and function are unknown. GH replacement should aim to restore somatic deficiency, including cardiac size and function. In view of the deleterious cardiac effects of excessive GH, supraphysiological GH doses should be avoided.

surements to be consistently more reproducible and sensitive than repeated pharmacologic stimulation tests [377]. These conflicting findings may be attributable to differences in control groups in the discrepant studies.

TESTS OF GH SECRETION

An alternative approach to be assessment of spontaneous GH secretion is the measurement of GH concentrations in urine [378]. As urinary GH concentrations are very low, assays require high affinity antibodies [379]. Several different immunoassay methods for urinary GH measurement have been developed [380], none of which have the ability to reflect pharmacological GH testing or to adequately discriminate between normal and abnormal GH secretion [381–383]. Clinical utility of urinary GH measurements in the diagnosis of GH deficiency will require attainment of adequate-age-and-sex-matched controls and a decision as to whether GH excretion should be expressed relative to body weight or creatinine excretion [384]. 24-hour urinary collections have been assayed for GH in an attempt to diagnose acromegaly [385,386]. Overnight urinary GH measurements were, however, not useful in differentiating acromegalic patients from normal controls [385]. However, others have found 24-hour urine collections for GH measurement to be useful in diagnosis of acromegaly [385].

Urinary GH Measurement

Because of the pulsatile nature of pituitary GH secretion, a single random blood sample for GH measurement is not helpful in the diagnosis of GH hypersecretory or deficiency states, or GH neurosecretory disorders. Nonphysiologic provocative or suppression tests, or measurement of spontaneous GH secretion by 24-hour integrated serum GH concentration (IC-GH), are therefore employed to assess GH secretion.

Measurement of Spontaneous GH Secretion Twenty-four-hour Integrated Concentration of GH

Pituitary GH secretion occurs episodically during waking hours, as well as during sleep, necessitating continuous measurement of integrated secretion over 24 hours [372] rather than over shorter periods. Constant blood collection over a 24-hour period allows the determination of a true mean or IC-GH. Measurement of the 24-hour IC-GH concentration ideally requires a nonthrombogenic continuous withdrawal pump [373] or patent indwelling catheter from subjects whose food intake and physical activity are not limited. Sampling intervals of 20 minutes are most widely used [373], but 5-minute and 30-second sampling frequencies detect significantly more pulses per hour [374]. Samples from collection periods may be pooled, producing a combined aliquot in which the ICGH concentration is measured. The 24-hour IC-GH reflects the average GH concentration over a 24-hour period, eliminating peak or trough levels that might otherwise be obtained by single random sampling of GH when the latter is released in a pulsatile manner. The discriminating power of continuous GH measurement in the diagnosis of GH deficiency has been disputed [375,376]. Measuring spontaneous GH secretion in prepubertal short children was found to be an insensitive test, with no clear diagnostic advantage over GH stimulation tests and considerable overlap between values obtained in normal, short children and children with GHD [375]. As young normal controls have IC-GH levels which overlap with those of organic hypopituitary patients [377] measurement of spontaneous GH secretion has limitations in the diagnosis of organic GH deficiency in adults. As measurement of spontaneous GH secretion has limitations in the diagnosis of adult GH deficiency, 12-hour overnight spontaneous GH secretion has been measured. Night-to-night variation in results occurred when the test was repeated on consecutive nights, as compared with GH stimulation tests which proved to be more reproducible [376]. However, others have found 24-hour IC–GH mea-

Evaluation of GH Hypersecretion Increased serum IGF-I levels are a consistent finding in acromegaly [387]. Integrated 24-hour serum GH levels are elevated and show a log (dose) response correlation with serum IGF-I levels [388]. The currently accepted diagnostic test of GH hypersecretion is failure of GH levels to be suppressed to less than 1 ng/mL within 2 hours following a 75 g oral glucose load using an IRMA (two site immunoradiometric assay) or chemiluminescent assay [389]. In normal subjects receiving oral glucose loading, serum GH levels initially fall and then subsequently increase as plasma glucose declines. However, in acromegalic patients, oral glucose fails to suppress GH to the normal range [390]. GH levels may increase in response to an oral glucose load in acromegaly (in approximately 28%) [391], remain unchanged (in approximately 36%), or fall (in approximately 36% of patients). As GH secretion is pulsatile, random GH measurements may be misleading.

Evaluation of GH Deficiency Random GH and 1GF-I Measurements

Random GH measurements are not diagnostic of GH deficiency, as GH secretion is pulsatile and daytime levels are often low in normal subjects. Low IGF-I levels are suggestive of GH deficiency, but are also found in malnutrition [392], acute illness, celiac disease, poorly controlled diabetes mellitus, liver disease, and estrogen ingestion [393]. Fifteen

100

SECTION 1

Hypothalamic–Pituitary Function

percent of children diagnosed to be GH deficient by stimulation tests may have normal IGF-I levels [394]. IGF-I levels are normally very low before 3 years of age and highest in adolescence. Normal and GH-deficient children may have IGF-I levels which overlap with those observed in infancy [395]. Furthermore, both normal and low IGFI levels are present in children with growth delay and genetic short stature [396]. IGF-I levels do not always correlate with GH levels after provocative GH stimulation [397]. Low IGFBP-III levels are also encountered in patients with GH deficiency [398]. The diagnosis of adult GHD is more reliably established by provocative testing of GH secretion, as a single ageadjusted IGF-I level has limited diagnostic value [377]. Whereas, in acromegaly there is an excellent correlation between IC-GH and IGF-I [388], in adult GH deficiency, age-adjusted IGF-I should be regarded as a simple, low-cost screening test, because of the limitations outlined above. IGFBP-III is the major serum carrier protein for the IGF peptides, and is GH-dependent [282]. IGFBP-III assays have several advantages over IGF-I assays in that (i) they are technically simple, not requiring separation of IGF peptides from BPs; (ii) circulate in the plasma in high concentrations, so that assay sensitivity is not compromised; (iii) IGFBP-III plasma concentrations are less influenced by nutritional factors than IGF-I levels; (iv) plasma levels vary minimally with age, so that identification of abnormal concentrations in infancy or puberty is clear-cut [384,399]. IGFBP-III is a sensitive test for diagnosis of severe childhood GHD [518], with superior reproducibility to GH stimulation tests [400]. Provocative Tests

Dynamic testing of GH reserve involves stimulation of the somatotrophs to secrete GH in response to a pharmacologic stimulus. Several GH stimulatory agents have been utilized, including insulin, clonidine, arginine, L-dopa, GHRH, and propranolol. Insulin-induced Hypoglycemia (Insulin Tolerance Test, ITT)

This reliable stimulus for GH secretion is the gold standard provocative test [401]. The ITT is the best validated GH stimulation test and is recommended by the Growth Hormone Research Society as the test of choice [401]. Regular insulin 0.1 IU/kg is administered intravenously to decrease the basal glucose level by 50% to a value below 40 mg/dI. Maximal GH secretion peaks at 30 to 60 minutes. Patients may experience symptoms of hypoglycemia, including light headedness, anxiety, tremulousness, sweating, tachycardia seizures and rarely, unconsciousness. Insulin induced hypoglycemia is contraindicated in patients with a history of seizure disorder, coronary artery disease, or over the age of 55 years. The test should be performed under close supervision, and intravenous glucose (50%) should be readily at hand for rapid administration. The risk of inducing profound hypoglycemia is greater in GH-deficient patients because of their increased sensitivity to insulin.

Clonidine

This alpha-adrenergic agonist stimulates GH release via a central action [402]. Clonidine (0.15 mg/m2) is administered orally, with a maximum GH secretory peak occurring after 60 to 90 minutes. Patients may experience some drowsiness, with a decrease in systolic blood pressure in sodium depleted GH deficient adults at doses required to release GH (0.25–0.30 mg orally.) Clonidine, frequently used as a stimulus for GH release in children, is not reliable in adults [403]. L-dopa/propranolol

l-dopa, the immediate metabolic precursor of dopamine, stimulates GH release by stimulating hypothalamic dopaminergic receptors. Adrenergic blockade (propranolol) enhances the GH response to l-dopa [404]. l-dopa is administered orally according to the patient’s weight (125 mg if weighing <10 kg; 250 mg if 10–30 kg; 500 mg if >30 kg) together with propranolol 0.75 mg/kg (maximum dose 40 mg) after an overnight fast. Maximum GH secretion occurs after 60 to 90 minutes. l-dopa is very effective in stimulating GH release and rarely results in adverse effects [255]. Arginine/GHRH

Recently it has been shown that arginine potentiates maximal somatotroph responsiveness to GHRH [405]. After an overnight fast, GHRH 1 ug/kg is administered as an intravenous bolus at 0 minutes with Arginine 30 g in 100 ml infused from 0 to 30 minutes, with subsequent blood sampling for GH performed every 15 minutes for 90 minutes. Combined Arginine/GHRH responses are ageindependent and is a highly reproducible GH provocative test [405], at least as sensitive as insulin induced hypoglycemia [406]. GHRH/GHRP-6

GHRH 1 ug/kg plus GHRP-6 1 ug/kg is given intravenously at 0 minutes and blood is drawn for GH sampling at 0 and 120 minutes [407]. GH releasing peptide-6 (GHRP-6) is an artificial hexapeptide [203] that activates a hypothalmic and pituitary receptor whose natural ligand Ghrelin was recently characterized [202]. Combined administration of GHRP-6 and GHRH is the most potent stimulus to GH release, with excellent reproducibility and no side effects [203]. GHRH/GHRP-6 is a good alternative to the ITT in patients with organic pituitary disease, however there is some overlap between GH levels attained in the control group and severely GH-deficient patients. Since GHRH and GHRP act directly on the pituitary, it is possible that their administration restored GH secretion in patients who had a deficiency of these secretagogues because of hypothalamic disease [408]. Pitfalls of Provocative Testing

Provocative testing has several limitations. The diagnosis of childhood onset GH deficiency is determined by an inad-

Chapter 3

equate GH response to two distinct provocative tests. The definition of an adequate response is arbitrary. The availability of recombinant human GH in the mid-1980s resulted in a loosening of the diagnostic cut-off from 5–7 ng/ml to 10 ng/ml. Sequential tests on the same day have been attempted [409], but discordant individual results are obtained in approximately 30% of subjects. By altering the temporal relationship between the administration of provocative agents, the amount of GH released can be decreased or increased [410–412]. A combination of two provocative agents administered simultaneously has been tried, but this approach failed to identify some patients who demonstrated GH deficiency following separate testing with each agent individually [413]. The reproducibility of provocative tests has been questioned, as only a moderate correlation was found between pharmacologic tests carried out on two separate occasions, 6 weeks apart [414]. Many physical states alter stimulated GH levels. Increased GH response to various provocative stimuli during puberty [415] have been attributed to increased estrogens and androgens [416]. Sex steroid priming with either estrogens or testosterone 3 to 5 days prior to provocative testing in prepubertal children has therefore been recommended by some. Mildly impaired GH release in response to provocative stimuli in hypothyroid patients is a frequent finding [417]. Patients should thus be euthyroid at the time of provacative GH testing. Furthermore, 67% of patients with “idiopathic” GHD diagnosed in childhood normalize their GH secretion, when reevaluated after completion of treatment [418].

Variability of GH Assays The comparisons of results of various GH assays obtained in different laboratories is difficult because of differences between almost all aspects of the immunoassays. Plasma GH is usually measured by radioimmunoassay (polyclonal or monoclonal; RIA) or by immunoradiometric assay (dual monoclonal; IRMA). Comparative GH measurements are obtained using 11 commercial immunoassays varied by a factor of three [419]. As previously mentioned, there are several different circulating forms of GH, most of which are not measured in GH assays because reagents to detect all these forms do not exist. Because monomeric 22 k is the only GH form available as a standard in sufficient purity and quantity, and because monomeric 22 k is also the most abundant circulating form, it is used as the basis for GH measurement: however it accounts for only about 25% of circulating immunoreactivity [420]. The other GH forms are recognized to varying and largely unknown degrees. Thus an assay result is an averaged look at a variety of GH forms, and different antibodies or assay designs yield different answers. Polyclonal antibodies were used in the early RIAs, inducing higher estimates of GH, because they recognized several molecular forms of GH compared to newer immunometric

Growth Hormone

101

assays employing highly specific monoclonal antibodies [421,422]. GH standards also affect comparison of GH values in different laboratories. In 1994, the first international standard for somatotropin, IRP 88/624 [423], was prepared by the WHO, using recombinant technology in contrast to the previous standards prepared from pituitary extracts. Use of an international standard enables uniformity of calibration between different GH kits. GH-binding proteins (GHBPs) may influence GH estimates by interfering in some of the GH assays. In the circulation, approximately 50% of GH is complexed to GHBP, which corresponds to the extracellular domain of the GH receptor. Both high affinity and low-affinity BPs are present in the serum. As the low-affinity GHBP has a greater affinity for the 20-KD GH molecule, it presumably does not interfere in GH estimates in the new GH assays specific for the 22-KD GH molecule. The influence of GHBP in noncompetitive immunometric assays could lead to too low estimates of GH if the GHBP competes with one or both antibodies. In competitive assays like the RIAs, the error in the GH estimates will depend on whether the GHBPbound 125 I-GH is in the antibody-bound or in the free fraction. In RIAs using polyethylene glycol or double antibody for separation, the GHBP-complexed GH will end up in the free fraction if the antibodies are directed against epitopes in the GHBP-binding area and thus give spuriously high values [424]. Another factor contributing to the heterogeneity of results from GH immunoassays is the antibody used. As different antibodies employed in immunoassays bind to a different spectrum of GH isoforms, GH concentrations measured by immunoassay are likely to depend on the particular antibody used. Furthermore, as the distribution of the different GH isoforms varies between individuals, results from different GH immunoassays cannot be compared. The heterogeneity of GH immunoassay results poses a major problem in the definition of standards for the diagnosis of GHD. Consensus statements including cut-off recommendations refer to the polyclonal-antibody-based RIA. However RIA is now used infrequently, therefore clinicians should be aware of the GH assay used by their laboratory and how it compares to polyconal RIA. Recently, three new GH assays have been developed, two of which, ESTA and IFA, measure GH bioactivity rather than immunoactivity as measured by convential RIA. A third, GHEA, measures the different GH isoforms in the circulation [425]. ESTA or IFA bioassays could replace RIA if they become commercially available, as they more closely reflect biologically active GH than that measured by RIA. CLINICAL USE OF GH

Recombinant hGH (rhGH) The application of recombinant DNA technology has made available a sufficient quantity of hGH for treatment of GH

102

SECTION 1

Hypothalamic–Pituitary Function

deficiency [426]. Early trials investigating the effects of recombinant human GH compared to pituitary-derived hGH in adults showed comparable kinetics in the two preparations, as well as similar ability to stimulate IGF-I levels. Both compounds resulted in a similar decrease in serum blood-urea nitrogen (BUN) and cholesterol, and increased serum triglyceride levels [427] and clinical growth-promoting effects.

GH Therapy in Childhood Classic GH Deficiency

Childhood GH deficiency ranges from complete absence of GH associated with severe growth retardation to partial GH deficiency resulting in short stature. Diagnosis is based on decreased height (more than 2.5 standard deviations (SD) below the mean height for age-matched normal children), poor growth velocity (less than the twenty-fifth percentile), delayed bone age and a predicted adult height below mean parental height [428]. GH deficiency (GHD) is usually confirmed by inadequate pituitary GH responses to standard provocative stimuli (peak serum GH value of less then 10 mg/L) [429], with values less than 5 mg/L in severe GH deficiency. Combined clinical evaluation and provocative testing are used in the assessment of childhood GH deficiency. Concomitant endocrine deficiencies, especially hypothyroidism, should be corrected to maximize the growth-promoting benefits of hGH. GH replacement should be started as early as possible before height drops below the third percentile, as total height gain is inversely proportional to the pretreatment chronologic and bone age [430], as well as severity of GH deficiency. The most pronounced acceleration in linear growth rate occurs during the first two years of treatment [431]. Dose and frequency of administration of hGH both influence height velocity [432]. Currently, the recommended weekly dose is 0.18 mg/kg (0.54 iu/kg) divided into six or seven daily subcutaneous injections or as three injections/ weekly administered on alternate days. The maximal replacement weekly dosage is 0.3 mg/kg (0.9 iu/kg) divided into equal doses given on three alternate days. Idiopathic Short Stature

Idiopathic short stature describes otherwise normal children who are at or below the fifth percentile for height, with normal growth hormone responses to provocative stimuli. In the past, scarcity of human pituitary GH limited its therapeutic uses to children with severe GH deficiency diagnosed by a decreased growth rate and inadequate GH response to two provocative stimuli. As short stature may affect psychological development, trials of GH in “slow” growing children have been initiated to determine whether GH treatment can increase final adult height in normal children without complications of treatment. GH treatment in these children is controversial. Long-term studies in short

children treated for up to ten years have shown conflicting results, some showing no significant difference in the SD score for final height [433] while others report final heights exceeding the predicted height [434]. Approximately onethird of all children on GH treatment in the USA are nonGH deficient with idiopathic short stature [435]. In view of the contradictory data on the benefical effect of GH therapy on final height, non-GH-deficient children and their families should have realistic expectations. In children with no identifiable cause of growth failure GH therapy is indicated if growth is retarded more than 2.5 SD below the mean for age and very slow growth velocity (less than the twenty-fifth percentile).

GH Neurosecretory Dysfunction (GHND) A subgroup of GH-deficient children has been described who have normal responses to GH provocative tests, low serum IGF-I levels and abnormal 24-hour GH secretory patterns [436]. This syndrome, termed “growth hormone neurosecretory dysfunction,” is characterized by height below the first percentile, bone age delayed by 2 or more years, and a growth velocity of 4 cm/year or less, in combination with the serum abnormalities described above. Classical GH deficiency, GHND, and normal children can be distinguished by assessing the number of GH pulses during a 24-hour period, mean peak GH pulse amplitude, highest nocturnal peak, and mean 24-hour GH concentration. Patients with GHND may secrete less GH than patients with classical GH deficiency, and the nocturnal GH surge is maintained but with a lower magnitude [436]. GHND has also been associated with Turner’s and Noonan’s syndrome, empty sella syndrome, and precocious puberty [437]. hGH was administered intramuscularly three times weekly for at least 6 months to children with GHND [436]. This treatment resulted in a doubling of growth velocity, comparable to that seen in children with GH deficiency receiving GH. Turner’s Syndrome

Patients with Turner’s syndrome manifest dysmorphic body features, ovarian failure, and reduced growth rate, starting during intrauterine life and continuing through childhood and puberty, resulting in reduced final adult height [438]. Earlier studies evaluating the use of hGH in Turner’s syndrome were inconclusive, possibly due to inadequate dosage regimens and short-term follow-up [439]. Recently, GH therapy in girls with Turner’s syndrome has been shown to significantly increase predicted height, with a greater increase in height in girls treated with combined GH and oxandrolone (an anabolic steroid) [440]. Turner’s syndrome is an approved FDA indication for GH use. Low Birth Weight

Children with low birth weight generally have reduced adult height [441]. An early study investigating the use of

Chapter 3

hGH in the treatment of short children with low birth weight yielded inconclusive results, probably due to the inadequate dosage regimen. Subsequent studies using higher, more frequent hGH dosages [442,443] have shown an increased growth rate for these children, but no increase in final height [444]. Chronic Diseases

hGH has been used in an attempt to increase height in children with short stature associated with a variety of chronic nonendocrine disorders. Growth retardation may be due to specific effects of the disease of may be secondary to poor nutrition or infection. Increased height velocity has been demonstrated after hGH treatment in prepubertal children and infants with chronic renal failure and following renal transplantation [445,446]. Chronic renal insufficiency prior

Table 3.5. deficiency

Causes of adult growth hormone (GH)

Presenting in childhood Congenital Idiopathic Genetic GHRH receptor defect Transcription factor defect Embryologic defects GH resistance Laron dwarfism Pygmy Neurosecretory defects Radiation for juvenile brain tumors, leukemia Head trauma Perinatal birth injury Child abuse Accidental Inflammatory diseases Viral encephalitis Meningitis, bacterial, fungal, tuberculosis Acquired in adulthood Postpituitary surgery Pituitary mass-adenoma, craniophyringioma Hematologic disorders Hemochromatosis Sickle cell disease Thalassemia Autoimmunity Lymphocytic hypophysitis Irradiation: central nervous system radiation for tumors Histiocytosis Granulomas Sarcoidosis Idiopathic Infection Tuberculosis Syphilis Vascular

Growth Hormone

103

to transplantation is an FDA approved indication for GH therapy. Achondroplasia and Hypochondroplasia

Achondroplasia and hypochondroplasia result in short stature, with normal GH responses to provocative tests. GH therapy increases growth in both conditions, however there is worsening of body disproportion [447]. GH Treatment in Adults

As part of the normal aging process, adults experience loss of lean body mass, atrophy of skeletal muscle and organs, and increased adipose tissue [448]. Skeletal muscle atrophy is associated with a loss of muscle strength. These changes have been attributed to GH deficiency, as GH and IGF-I levels decline with age in healthy adults. With the increased availability of hGH, studies have been performed to evaluate the effects of rhGH on body composition in normal elderly individuals [449–451]. These studies have reported an increase in lean body mass, decreased adipose tissue content and increased retention of sodium, calcium, and nitrogen. None of these parameters translate into a measurable improvement in muscle size or strength. Adults may be truly GH-deficient due to isolated GH deficiency or to panhypopituitarism, from a number of causes (Table 3.5). GH-deficient adults have altered body composition with increased fat and decreased muscle volume and strength, lower psychosocial achievement, altered glucose and lipid metabolism, decreased bone mineral density and possibly increased mortality due to cardiovascular disease (Table 3.6). The same pharmacologic stimulating tests, used in children, have been used in adults. However, the definition of an adequate response differs. IGF-I, IGFBP-III, cholesterol and LDL levels can also be determined (Table 3.7). Mean 24-hour GH secretion sampled every 20 minutes, insulin-induced hypoglycemia and IGF-I levels have been compared to normals in the diagnosis of adult GHD [452]. IGF-I is a poor indicator of adult GHD as levels may overlap with normal. The diagnosis of adult GH deficiency is confirmed by provocative testing of GH secretion. Other hormonal deficits should be adequately replaced prior to GH provocative testing. The

Table 3.6. Physical findings in the adult growth hormone deficiency syndrome. Adapted from Cuneo et al. [451a] Truncal adiposity Waist/hip ratio increased Thin, dry, cool skin Exercise performance reduced Muscle strength reduced Reduced bone density Mood depressed Psychosocial impairment

104

SECTION 1

Hypothalamic–Pituitary Function

Table 3.7. Diagnosis of adult GH deficiency. Adapted from Cuneo et al. [451a] Stimulated GH <3 ng/mL Insulin Clonidine L-dopa Stimulated GH <9–16 ng/mL Arginine/GHRH IGF-I low or normal IGFBP-III low Hypercholesterolemia, with low HDL and often high LDL levels Reduced glomerular filtration rate Reduced lean body mass Reduced bone density

Table 3.8.

Table 3.9.

Complications of GH replacement therapy

Metabolic Glucose homeostasis Salt and fluid retention; peripheral edema Unmasking of thyroid dysfunction Joint Adults Children

Arthralgias and myalgias Carpal tunnel syndrome Slipped capital femoral epiphysis

Potential promotion of neoplastic growth Skin Pituitary adenoma Colonic polyps Other

GH therapy in adults Table 3.10.

Approved use GH Deficiency AIDS-associated muscle wasting Investigational use Frailty of aging Osteoporosis Catabolic states, cachexia Burns Postoperative recovery Wound healing Parenteral nutrition Ovulation induction Immune deficiency

insulin tolerance test is the current gold standard. Most normal subjects respond to insulin-induced hypoglycemia with a peak GH response of greater than 5 ng/mL. Severe GH deficiency is defined by a peak GH response to hypoglycemia of less than 3 ng/mL. Combined administration of arginine and GHRH is as sensitive as the insulin tolerance test. Childhood onset GH deficiency requires reconfirmation in adulthood by two GH provocative tests because growth hormone may no longer be deficient [453]. Adult patients with hypothalmic–pituitary disease and additional anterior pituitary hormone deficiencies require only one GH provocative test to confirm GH deficiency. Insulin induced hypoglycemia shows a clear separation of the peak GH response in GH-deficient adults compared to normals [454]. The scarcity of GH in the past has limited GH replacement therapy to GH-deficient children. The primary therapeutic goal has been to increase linear growth, and in the past treatment has been terminated after epiphyseal fusion occurs and a certain height has been achieved. However, GH secretion normally continues into adulthood, and GH influences many metabolic systems other than growth (Tables 3.8 and 3.9). The numerous beneficial effects of GH

Beneficial effects of GH replacement in adults

Body composition

Increased lean body mass Decreased fat mass Increased bone mass

Physical performance

Increased Max O2 uptake Increased max power

Metabolic effects

Increased IGF-I levels Increased BMR Decreased LDL with probable increased HDL Transient hyperglycemia Increased T3 levels Salt and water retention

Cardiovascular effects

Increased stroke volume Increased diastolic volume Increased LV wall mass Atherosclerosis impact

Psychological effects

Mood and energy uplift Enhanced vitality Improved physical mobility Social isolation improved

Adipose tissue effect

Decreased adipocyte size Increased lipolysis Decreased lipogenesis

replacement in adulthood have been previously described (Table 3.10). In addition, GH replacement in GH deficient adults is associated with increased energy levels, improved mood, vitality and emotional reactions and less feeling of social isolation [343]. GH replacement therapy is associated with significantly improved Nottingham Health Profile (NHP) energy scores (Fig. 3.17). GH doses in adulthood are adjusted to individual needs (Table 3.11). Elderly, male, or obese individuals require lower doses. Furthermore, the use of oral estrogen replacement effects the GH replacement dose. Premenopausal

Chapter 3

FIGURE 3.17. Effect of long term (10 years) treatment with GH on quality of life as measured by the Nottingham Health profile (Energy Level) in 21 GH deficient adults. (Note: higher scores indicate greater energy impairment). Reproduced with permission from Gibney et al. [343a].

Table 3.11. replacement

Factors determining side effects of GH

Patient age Enhanced IGF-I response Greater body weight and body mass index Adult onset vs. childhood onset of GH deficiency

women or postmenopausal women using transdermal estrogen replacement require lower doses of GH than postmenopausal women taking oral estrogen replacement [454]. Oral, but not transdermal estrogens antagonize the actions of GH and reduce IGF-I levels (Fig. 3.18). GH replacment therapy should be started at a low dose (0.15–0.30 mg/d) (GRS guidelines). GH is self-administered as a daily subcutaneous injection in the evening. Serum IGF-I levels and tolerability are used to guide dosing. IGF-I concentrations are monitored every 4 to 6 weeks, and GH doses are adjusted until levels reach mid-normal range for age and sex (Fig. 3.19). Side effects of GH replacement include edema and carpal tunnel syndrome due to fluid retention, which are usually transient, but may require dose reduction. Mild self-limiting arthralgias may also occur. As GH may reduce insulin sensitivity, glycemic control should be monitored. GH replacement therapy is contraindicated in patients with active malignancy, benign intracranial hypertension and proliferative diabetic retinopathy (Table 3.12). Changes in body composition, bone density and lipid profiles may only be observed after several months of therapy. These effects are best assessed by annual dual-energy x-ray absorptiometry
EXA and serum lipid level measurements. A baseline MRI is recommended prior to initial GH replacement. GH therapy does not impose a need for

Growth Hormone

105

FIGURE 3.18. Time course of GH dose and serum IGF-I concentration in a representative patient (38-year-old woman) who was switched from oral to transdermal estrogen therapy during the course of GH replacement. Reproduced with permission from Cook et al. [454].

more frequent pituitary MRI’s than would be done for the regular clinical follow-up of residual pituitary tumor. GH Treatment in Catabolic States

The well-recognized anabolic actions of GH have tempted the use of GH in catabolic states such as surgery, trauma burns and organ failure. The negative nitrogen balance in critically ill patients is partly attributable to GH resistance as well as decreased production and action of IGF-I [455]. Postsurgical recovery is associated with hypercatabolism. GH administered to postsurgical patients as well as to normal subjects receiving hypocaloric intravenous alimentation [456], results in positive nitrogen balance [457]. Beneficial effects of GH in patients with extensive burns has been demonstrated in children [458] and adults [459]. Chronic high-dose glucocorticoid treatment is associated with protein catabolism. These effects in normal subjects may be reversed by simultaneous GH administration [459]. A potential complication of combined GH and glucocorticoid administration is resultant hyperglycemia. GH treatment improved nitrogen balance in two small studies of patients with chronic obstructive pulmonary disease [460,461], with improved respiratory muscle function only reported in one group [460]. Short-term studies of GH treatment in cancer patients suggested improvement in nitrogen balance, most pronounced in the least malnourished patients [461]. Although an uncontrolled study in critically ill patients suggested that GH treatment decreased mortality [462], a large European trial suggested increased mortality in critically ill patients treated with large doses of GH, and was terminated prematurely [463]. GH Treatment in Osteoporosis

Bone mass declines after the second decade of life in healthy men and women, with accelerated bone loss in post-

106

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 3.19. Algorithm for the management of adult GH deficiency.

Table 3.12.

Contraindications to GH therapy in adults

Absolute Active neoplasm Intracranial hypertension Diabetic retinopathy Relative Uncontrolled diabetes Untreated thyroid dysfunction Cost benefit Need for injections

menopausal women. As declining GH secretion has been implicated in the pathogenesis of osteoporosis, GH has been administered to healthy men and women with idiopathic osteoporosis in an attempt to decrease bone loss [464]. Although GH therapy increased indices of bone formation and resorption [464] a small increase in only spine bone mineral density was observed in males, after 6 months [449], with an increase in spine mineral density in postmenopausal females when GH was administered with calcitonin [465]. A longer study in osteoporotic females showed increases in both spine and total hip bone mineral density [466], after 2 years of treatment with GH and calcitonin, although the response was less than with estrogen or biophosphonate therapy [467]. Limited efficacy, side effects and lack of comparative studies with beneficial therapies for osteoporosis, limit the use of GH in the treatment of osteoporosis.

[468], with an increase in extracellular water and lean body mass, a decrease in body fat and improved work output after 3 months [469]. Long term beneficial effects of GH on survival and quality of life have not been established yet. GH Therapy in Cardiac Failure

Beneficial effects of GH on cardiac function in GH deficiency led to its attempted use in congestive heart failure. GH administered for 3 months to a small group of patients with idiopathic dilated cardiomyopathy and moderate to severe congestive heart failure improved cardiac output [369] at rest and during exercise. A larger controlled study for a longer period of time did not confirm these findings [470]. Increased extracellular volume and edema, as well as the potential for cardiomyopathy as seen in acromegaly, must be considered in the use of GH in cardiac failure. GH and Wound Healing

GH treatment increases the strength and speed of collagen formation in incisional wounds in rats [471–473]. Fracture healing in rats is also facilitated by GH treatment [474]. No controlled data are yet available in human subjects. GH Treatment in Ovulation Induction

The growth hormone axis influences gonadal function [474] GH therapy reduces gonadotropin requirements in hypogonadotropic males and females [475,476]. Furthermore, improved implantation and pregnancy rates occur with the addition of GH to gonadotropin based protocols in women undergoing in vitro fertilization [477].

GH Treatment in AIDS-associated Wasting Syndrome

GH is FDA approved in adult patients with HIV associated cachexia. Short term GH administered to patients with HIV-associated cachexia results in positive nitrogen balance

GH use Competitive Sports

The ethical issues of GH abuse in sports have received much attention. GH has been used by many athletes to enhance

Chapter 3

muscle mass [478]. Whether this is accompanied by increased muscle strength is unknown. Continued pharmacologic use of GH by athletes would result in the adverse effects of acromegaly, which would decrease performance. COMPLICATIONS OF GH TREATMENT The complications of GH treatment have been described in GH-deficient patients treated with GH (see Table 3.9). Non-GH-deficient patients receiving GH can be likened to acromegalic patients, with increased GH levels. Adverse reactions, including arthralgias, myalgias, back ache, parasthesias, peripheral edema, carpel tunnel syndrome, headache, hypertension, and rhinitis are frequent, often transient or disappear with lowering of the GH dose and are more common in adult-onset than childhood-onset GH deficiency [479]. Although acromegalic patients have a potential increased risk of developing neoplasms of the gastrointestinal system, recent evidence is less compelling [480]. Colonic polyps occur more frequently, although their relationship to IGFI levels is not confirmed [481]. The true incidence of neoplasia in acromegalic patients is difficult to determine, although a potential link between GH and tumor development has been postulated in animal models. Elevated IGF-I levels in acromegaly may be responsible for cellular proliferation [482]. GH therapy in GH-deficient children has been implicated in the pathogenesis of leukemia. However, this was not confirmed in a worldwide survey [483]. Endogenous IGF-I concentrations have been epidemiologically linked to prostate cancer [484] and breast cancer risk [485]. ABNORMAL GH SECRETION GH hypersecretion, due to either excess GHRH (ectopic or eutopic) or to excess GH (ectopic or eutopic) secretion and GH deficiency (idiopathic or secondary) are documented fully in Chapter 10 (see also Table 3.13).

Table 3.13. acromegaly

Elevated GH levels not associated with

Associated decreased IGF levels Protein-calorie malnutrition Starvation Uncontrolled diabetes mellitus Anorexia nervosa GH insensitivity–Laron’s syndrome Decreased GH clearance Chronic renal failure Mucoviscidosis Physiologic (intermittent) Sleep Stress

Growth Hormone

107

Decreased IGF-I Levels Protein-calorie Malnutrition, Starvation, Anorexia Nervosa

Short-term fasting in normal healthy people results in moderately elevated basal GH levels [486]. However, proteincalorie malnutrition is associated with low IGF-I levels and markedly elevated GH levels [487]. This may reflect an uncoupling of the IGF-I feedback regulation of GH secretion. Low levels of IGF-I in normal fasted volunteers were normalized on refeeding, and caloric intake normalized IGF-I levels to a greater degree than did protein intake [488]. In patients with anorexia nervosa, basal GH levels are also elevated [489]. Furthermore, GH levels rise paradoxically after a glucose load [490] and after TRH injection in these patients [491]. The abnormalities in anorexia nervosa are due not only to the weight loss, but to the concomitant psychiatric disturbance, which may influence hypothalamic– pituitary function. Diabetes Mellitus

Poorly controlled diabetes mellitus (both insulin dependent and noninsulin-dependent) is associated with elevated basal GH levels [90] and increased GH response to exercise [492] and to TRH [493]. Elevated GH levels return to normal with improved diabetic control after insulin administration [494]. IGF-I levels are low in children with poorly controlled insulin-dependent diabetes, including those entering puberty, suggesting insulin resistance. Antiinsulin actions of the increased GH may worsen the resistance. Addition of IGF-I to insulin therapy in adolescents with poorly controlled diabetes was postulated to decrease GH secretion, increase insulin sensitivity and decrease insulin requirements. Daily subcutaneous IGF-I injections for a month normalize IGF-I levels with less fluctuations in blood glucose levels, and reduced insulin doses [495]. Insulin resistance in non-insulin-dependent diabetes is associated with increased insulin secretion in an attempt to overcome putative post-receptor defects in insulin action [496]. IGF-I therapy was proposed to stimulate peripheral glucose utilization and reduce insulin resistance and secretion, and was shown to decrease fasting blood glucose by 30% with a 50% decrease in insulin secretion [497]. A high incidence of side effects with dosages used initially discouraged adjunctive IGF-I therapy [498]. Diabetes and malnutrition are both conditions of nutrient deprivation. In malnutrition, there is inadequate intake of nutrients, whereas diabetic patients are unable to utilize available nutrients appropriately. Malnutrition and diabetes both manifest retarded growth, despite elevated GH levels and decreased IGF-I generation. Laron Syndrome

Laron syndrome, an autosomal recessive disorder, is a condition of peripheral unresponsiveness to GH. Serum GH levels are normal or elevated, circulating IGF-I is absent, and

108

SECTION 1

Hypothalamic–Pituitary Function

there is no IGF-I response to exogenously administered GH [499]. Inactivating mutations of the GH receptor [500] cause insensitivity to exogenous GH. IGF-I therapy increased height velocity, with improved body composition as evidenced by loss of fat mass [501].

Decreased GH Clearance Chronic Renal Failure

Basal GH levels are high in patients with chronic renal failure [502], and GH rises paradoxically in response to a glucose load in these patients [502]. GH is degraded by the kidney. However, elevated basal GH levels imply some change in GH regulation; if the feedback loop were intact, GH levels should fall to normal. Serum IGF-I levels are reported to be elevated or variable in chronic renal failure [503–505]. As IGFs circulate bound to large carrier proteins, serum samples should be pretreated with acid-ethanol to ensure reliable quantitation of total IGFs [506]. Uremic serum may contain excess carrier protein not completely removed by acid-ethanol extraction, resulting in underestimation of immunoreactive IGF-I levels [505]. A more specific assay employs a second antibody to separate bound from free radioligand [507]. Despite high GH levels, children with renal failure are short. Following renal transplantation, return to normal growth is variable. Growth retardation can be inversely correlated with posttransplant creatinine clearance [508]. REFERENCES 1 Miller WL, Eberhardt NL. Structure and evaluation of growth hormone gene family. Endocrinol Rev 1983;4:97–130. 2 George DL, Phillips JA III, Francke U, Seeburg PH. The genes for growth hormone and chorionic somatomammotropin are on the long arm of human chromosome 17 in the region of q21-qter. Human Genet 1981;57:138–141. 3 Chen EY, Yu-Cheng L, Smith DH et al. The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 1989;4:479–497. 4 Lewis UJ, Dunn JT, Bonewald LF et al. A naturally occurring structural variant of human growth hormone. J Biol Chem 1978;253:2679–2687. 5 Lewis UJ, Bonewald LF, Lewis VJ. The 20,000-dalton variant of human growth hormone: location of the amino acid deletions. Biochem Biophys Res Commun 1980;92:511–516. 6 DeNoto FM, Moore DD, Goodman HM. Human growth hormone DNA sequences and mRNA structure: possible alternative splicing. Nucleic Acids Res 1981;9:3719–3730. 7 Frankenne F, Rentier-Delrve F, Scippo ML et al. Expression of the growth hormone variant in human placenta. J Clin Endocrinol Metab 1987;64: 635–637. 8 Liebhaber SA, Urbanek M, Ray J et al. Characterization and histologic localization of human growth hormone—variant gene expression in the placenta. J Clin Invest 1989;83:1985–1991. 9 Frankeme F, Closset J, Gomez F et al. The physiology of growth hormone (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab 1988;66:1171–1180. 10 Cooke NE, Ray J, Watson MA et al. Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. J Clin Invest 1988;82:270–275. 11 Ray J, Okamura H, Kelly PA et al. Human growth hormone-variant demonstrates a receptor binding profile distinct from that of normal pituitary growth hormone. J Biol Chem 1990;265:7939–7944. 12 MacLeod JN, Worsley I, Ray J et al. Human growth hormone-variant is a biologically active somatogen and lactogen. Endocrinology 1991;91:1290–1302.

13 Goodman HM, Tai L-R, Ray J et al. Human growth hormone-variant produces insulin-like and lipolytic responses in rat adipose tissue. Endocrinology 1991;91:1779–1783. 14 Hirt H, Kimelman J, Birnbaum MJ et al. The human growth hormone gene locus: structure, evolution and allelic variations. DNA 1987;6:59–70. 15 Simmons DM, Voss JW, Ingraham HA et al. Pituitary cell phenotypes involve cell-specific Pit-I mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev 1990;4:695–711. 16 Gilbert MS. Some factors that influence the development of mammalian hypophysis. Anat Res 1935;62:337–359. 17 Chatelain A, Dupouy JF, Dubois MP. Ontogenesis of cells producing polypeptide hormones (ACTH, MSH, LPH, GH, prolactin) in the fetal hypophysis of the rat: influence of the hypothalamus. Cell Tissue Res 1979;196:409–427. 18 Behringer RR, Matthews LS, Palmiter RD, Brinster RL. Dwarf mice produced by genetic ablation of growth hormone-expressing cells. Genes Dev 1988;2:453–461. 19 Borrelli E, Heyman RA, Arias C et al. Transgenic mice with inducible dwarfism. Nature 1989;339:538–541. 20 Bodner M, Karin M. A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells. Cell 1987;50:267–275. 21 Brent GA, Harney JW, Moore DD, Larsen PR. Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3¢5¢-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids. Mol Endocrinol 1988;88:792–798. 22 Lefevre C, Imagawa M, Dana S et al. Tissue-specific expression of the human growth hormone gene is conferred in part by the binding of a specific transacting factor. EMBO J 1987;6:971–981. 23 Ingraham HA, Chen R, Mangalam HJ et al. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 1988;55:519–529. 24 Bodner M, Castrillo JL, Theill LE et al. The pituitary specific transcription factor GHF-1 is a homeobox-containing protein. Cell 1988;55:505–518. 25 Voss JW, Yao FP, Rosenfeld MG. Alternative translation initiation site usage results in two structurally distinct forms of Pit-1. J Biol Chem 1991;266: 12832–12835. 26 Mangalam HJ, Albert VR, Ingraham HA et al. A pituitary POU domain protein, Pit-1 activates both growth hormone and prolactin promoters transcriptionally. Genes Dev 1989;3:946–958. 27 Ingraham HA, Flynn SE, Voss JW et al. The POU-specific domain of Pit-1 is essential for sequence specific, high affinity DNA binding and DNAdependent Pit-1–Pit-1 interactions. Cell 1990;61:1021–1033. 28 Sturm RA, Herr W. The POU-domain is a bipartite DNA-binding structure. Nature 1988;336:601–604. 29 Sturm RA, Das G, Herr W. The ubiquitous octamer-binding protein Oct-1 contains a POU-domain with a homeobox subdomain. Genes Dev 1988;2: 1582–1599. 30 Ko HS, Fast P, McBride W, Staudt LM. A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain. Cell 1988;55:135–144. 31 Clerc RG, Corcoran LM, LeBowitz JH et al. The B-cell-specific Oct-2 protein contains POU box and homeo box-type domains. Genes Dev 1988;2:1570–1581. 32 Finney M, Ruykun G, Horvita HR. The elegans cell lineage and differentiation gene unc-86 encodes a protein containing a homeodomain and extended sequence similarity in mammalian transcription factors. Cell 1988; 55:757–769. 33 Laughan A, Scott M. Sequences of Drosophila segmentation gene protein homology with DNA binding proteins. Nature 1984;310:25–31. 34 Konzak KE, Moore DD. Functional isoforms of Pit-1 generated by alternative messenger RNA splicing. Mol Endocrinol 1992;6:241–247. 35 Sornson MW, Wu W, Dasen JS et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384(6607):327–333. 36 Wu W, Cogan JD, Pfaffle RW et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genetics 1998;18(2): 147–149. 37 Parks JS, Brown MR, Hurley DL et al. Heritable disorders of pituitary development. JCEM 1999;84:4362–4370. 38 Lemaigre FP, Courtois SJ, LaFontaine DA, Rousseau GG. Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human growth hormone gene. Eur J Biochem 1989;181: 555–561.

Chapter 3 39 Schaufele F, West BL, Reudethaber TL. Overlapping Pit-1 and Sp1 binding sites are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Sp1 binding. J Biol Chem 1990;265:17189–17196. 40 Dynan WS, Tijan R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. Nature 1985;316:774–777. 41 Kadonaga JT, Jones KA, Tijan R. Promoter-specific activation of RNA polymerase II transcription by Sp1. Trends Biochem 1986;11:20–23. 42 Kadonaga JT, Carner KR, Masiarz FR, Tijan R. Isolation of cDNA encoding, transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 1987;51:1079–1090. 43 Peritz LN, Fodor EJB, Silversides DW et al. The human growth hormone gene contains both positive and negative control elements. J Biol Chem 1988;263:5005–5007. 44 Prager D, Gebremedhin S, Melmed S. An insulin-induced DNA binding protein for the human growth hormone gene. J Clin Invest 1990;85: 1680–1685. 45 Sawadogo M, Van Dyke MW, Gregor PD, Roeder RG. Multiple forms of the human gene-specific transcription factor USF: I. Complete purification and identification of USF from HeLa cell nuclei. J Biol Chem 1988;283: 11985–11993. 46 Sawadogo M. Multiple forms of the human gene-specific transcription factor. II. DNA binding properties and transcriptional activity of the purified HeLa USF. J Biol Chem 1990;283:11944–12001. 47 Chodosh LA, Carthew RW, Morgan JG et al. The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter. Science 1987;238:684–688. 48 Carthew RW, Chodosh LA, Sharp PA. The major late transcription factor binds to and activates the mouse metallothionein I promoter. Genes Dev 1987;1:973–980. 49 Courtois SJ, Lafontaine DA, Lemaigre FP et al. Nuclear factor-1 and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and transactivate the human growth hormone gene. Nucleic Acids Res 1990;18:57–64. 50 Bennani-Baiti IM, Asa SL, Song D et al. DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression. Proc Natl Aca 1998;10655–10660. 51 Yamashita S, Weiss M, Melmed S. Insulin-growth factor I regulates growth secretion and messenger ribonucleic acid levels in human pituitary tumor cells. J Clin Endocrinol Metab 1986;63:730–735. 52 Yamashita S, Ong J, Melmed S. Regulation of human growth hormone gene expression by insulin-like growth factor I in transfected cells. J Biol Chem 1987;262:13254–13257. 53 Dana S, Karin M. Induction of human growth hormone promoter activity by the adenosine 3¢5¢-monophosphatase pathway involves a novel responsive element. Mol Endocrinol 1989;89:815–821. 54 Cohen L, Hashimoto Y, Zanger K et al. CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression. JCI 1999:1123–1130. 55 Katz HP, Youlton R, Kaplan SL, Grumbach MM. Growth and growth hormone. III. Growth hormone release in children with primary hypothyroidism and thyrotoxicosis. J Clin Endocrinol Metab 1969;29: 346–351. 56 Williams T, Maxon H, Thorner MO, Frohman LA. Blunted growth hormone (GH) response to GH-releasing hormone in hypothyroidism resolves in the euthyroid state. J Clin Endocrinol Metab 1985;61:454–456. 57 Cattini PA, Eberhardt NL. Regulated expression of chimaeric genes containing the 5¢-flanking regions or human growth hormone-related genes in transiently transfected rat anterior pituitary tumor cells. Nucleic Acids Res 1987;15:1297–1309. 58 Leidig F, Shephard AR, Zhang W et al. Thyroid hormone responsiveness in human growth hormone-related genes. Biol Chem 1992;267:913–921. 59 Robins DM, Paek I, Seeburg PH, Axel R. Regulated expression of human growth hormone genes in mouse cells. Cell 1982;29:623–631. 60 Slater EP, Rabenau O, Karin M et al. Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene. Mol Cell Biol 1985;5:2984–2990. 61 Moore DD, Marks AR, Buckley DI et al. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor. Proc Natl Acad Sci USA 1985;82:699–702. 62 Ceda GP, Hoffman AR, Silberberg GD et al. Regulation of growth hormone release from cultured human pituitary adenomas by somatomedins and insulin. J Clin Endocrinol Metab 1985;60:1204–1209.

Growth Hormone

109

63 Prager D, Melmed S. Insulin regulates expression of the human growth hormone gene in transfected cells. J Biol Chem 1988;263:16580–16585. 64 Herman V, Weiss M, Becker D, Melmed S. Hypothalamic hormonal regulation of human growth hormone gene expression in somatotroph adenoma cell cultures. Endocr Pathol 1990;1:236–244. 65 Cunningham BC, Ultsch M, De Vos AM et al. Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 1991;254(5033):821–825. 66 Cryer PE, Daughaday WH. Growth hormone. In: Martini L, Besser GM, eds. Clinical Neuroendocrinology. New York/San Francisco/London: Academic Press, 1977:243–277. 67 Frohman LA, Burek L, Stachura ME. Characterization of GH of different molecular weights in rat, dog and human pituitary. Endocrinology 1972;91:262–269. 68 Baumann G, Stolar MW, Buchanan TA. Slow metabolic clearance of the 20000-dalton variant of human growth hormone: implications for biological activity. Endocrinology 1985;117:1309–1313. 69 Baumann G, MacCart JC, Amburn K. The molecular nature of circulating growth hormone in normal and acromegalic man: evidence for a principal and minor monomeric forms. J Clin Endocrinol Metab 1983;56:946–952. 70 Rudman D, Kutner MH, Rogers M et al. Impaired growth hormone secretion in the adult population. J Clin Invest 1981;67:1361–1369. 71 Shibasaki T, Shizume K, Nakahara M et al. Age-related changes in plasma growth hormone response to growth hormone-releasing factor in man. J Clin Endocrinol Metab 1984;58:212–214. 72 Plotnick LP, Thompson RG, Kowarski A et al. Circadian variation of integrated concentration of growth hormone in children and adults. J Clin Endocrinol Metab 1975;40:240–247. 73 Ho KKY, Evans WS, Blizzard RM et al. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 1987;64: 51–58. 74 Dawson-Hughes B, Stern D, Goldman J, Reichlin S. Regulation of growth hormone and somatomedin-C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab 1986;63: 424–432. 75 Takahashi Y, Kipnis DM, Daughaday WH. Growth hormone secretion during sleep. J Clin Invest 1968;47:2079–2090. 76 Sassin JF, Parker DC, Mace JW et al. Human growth hormone release: relation to slow-wave sleep and sleep-walking cycles. Science 1969;165: 513–515. 77 Van Cauter E, Plat L, Copinsch G. Interrelations between sleep and the somatotropic axis. Sleep 1998;21:553–566. 78 Van Cauter E, Leproult R, Plat L. Age related changes in slow wave sleep and REM sleep and relationship with growth hormone and cortisol levels in healthy men. JAMA 2000;284:861–868. 79 Golstein J, Cauter EV, Desir D et al. Effects of “jet lag” on hormonal patterns. IV. Time shifts increase growth hormone release. J Clin Endocrinol Metab 1983;56:433–440. 80 Casanueva FF, Villanueva L, Cabranes JA et al. Cholinergic mediation of growth hormone secretion elicited by arginine, clonidine, and physical exercise in man. J Clin Endocrinol Metab 1984;59:526–530. 81 Carey LC, Cloutier CT, Lowery BD. Growth hormone and adrenal cortical response to shock and trauma in the human. Ann Surg 1971;174:451–460. 82 Sibbald WJ, Short A, Cohen MP, Wilson RF. Variations in adrenocortical responsiveness during severe bacterial infections. Ann Surg 1977;186:29–33. 83 Cornil A, Blinder D, Leclercq R, Copinschi G. Adrenocortical and somatotropic secretions in acute and chronic respiratory insufficiency. Am Rev Resp Dis 1975;112:77–81. 84 Vigas M, Malatinsky J, Nemeth S, Jurcovicova J. Alpha-adrenergic control of growth hormone release during surgical stress in man. Metabolism 1977;26: 399–402. 85 Powell GF, Brasel JA, Blizzard RM. Emotional deprivation and growth retardation simulating idiopathic hypopituitarism. II: Endocrinologic evaluation of the syndrome. N Engl J Med 1967;276:1279–1283. 86 Sachar EJ, Mushrush G, Perlow M et al. Growth hormone responses to L-dopa in depressed patients. Science 1972;178:1304–1305. 87 Soliman AT, Hassan AEHI, Aref MK et al. Serum insulin-like growth factors (IGF) I and II concentrations and growth hormone and insulin responses to arginine infusion in children with protein-energy malnutrition before and after nutritional rehabilitation. Pediatr Res 1986;20:1122–1130. 88 Ho KY, Veldhuis JD, Johnson ML et al. Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man. J Clin Invest 1988;81:968–975.

110

SECTION 1

Hypothalamic–Pituitary Function

88a Thorner MO, Vance ML, Horvath E, Kovacs K. The anterior pituitary. In: Wilson JD, Foster DW, eds. Williams Textbook of Endocrinology. Philadelphia: WB Saunders, 1992:221–310. 89 Roth J, Glick SM, Yalow RS, Berson S. Hypoglycemia: a potent stimulus to secretion of growth hormone. Science 1963;10:987–988. 90 Vigneri R, Squatrito S, Pezzino V et al. Growth hormone levels in diabetes. Correlation with the clinical control of the disease. Diabetes 1976;25: 167–172. 91 Hansen AP. Normalization of growth hormone hyperresponse to exercise in juvenile diabetics after “normalization” of blood sugar. J Clin Invest 1971;50:1806–1811. 92 Alba-Roth J, Muller OA, Schopohl J, VonWerder K. Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab 1988;67:1186–1188. 93 Pontirol AE, Lanzi R, Monti ID et al. Effect of acipimox, a lipid lowering drug on GH response to GH-releasing hormone in normal subjects. J Endocrinol Invest 1990;13:539–545. 94 Blackard WG, Hull EW, Lopez A. Effect of lipids on growth hormone secretion in humans. J Clin Invest 1971;50:1439–1441. 95 Fraser WH, Blackard WG. The effect of lipids on prolactin and growth hormone secretion. Horm Metab Res 1977;9:389–394. 96 Lucke C, Adelman N, Glick SM. The effect of elevated free fatty acids on the sleep-induced human growth hormone peak. J Clin Endocrinol Metab 1972;35:407–412. 97 Casanueva FF, Villanueva L, Penalva A et al. Free fatty acid inhibition of exercise-induced growth hormone secretion. Horm Metab Res 1981;13: 348–350. 98 Cassanueva FF, Vilanueva L, Dieguez C et al. Free fatty acids block growth hormone (GH) releasing hormone-stimulated GH secretion in man directly at the pituitary. J Clin Endocrinol Metab 1987;65:634–642. 99 Zhang Y, Proenca R, Maffei M et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432. 100 Pelleymounter MA, Cullen MJ, Baker MB et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269:540–543. 100a Mantzoros C. The role of leptin in human obesity and disease: a review of current evidence. Ann Intern Med 1999;130:671–680. 101 Ahima RS, Prabakaran D, Montzoros C et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250–255. 102 Carro E, Senaris R, Considine RV et al. Regulation of in vivo growth hormone secretion by leptin. Endocrinology 1997;138:2203–2206. 103 Tannenbaum GS, Rorstad O, Brazeau P. Effects of prolonged food deprivation on the ultradion growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat Endocrinology 1979;104:1733–1738. 104 Okada K, Sugihara H, Minami S, Wakabayashi I. Effect of parenteral administration of selected nutrients and central injection of antiserum to neuropeptide Y, on GH secretion in food-deprived rats. Neuroendocrinology 1993;57:678–686. 105 Mercer JG, Hoggard N, Williams LM et al. Coexpression of leptin receptor and preponeuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 1996;8:733–735. 106 Al-Shoumer KAS, Anyaoku V, Richmond W, Johnston DG. Elevated leptin concentrations in growth hormone-deficient hypopituitary adults. Clin Endocrinol 1997;47:153–159. 107 Lal S, Martin JB, De La Vega CE et al. Comparison of the effect of apomorphine and l-dopa on serum growth hormone levels in normal men. Clin Endocrinol 1975;4:277–295. 108 Huseman CA, Hassing JM. Evidence for dopaminergic stimulation of growth velocity in some hypopituitary children. J Clin Endocrinol Metab 1984;58: 419–425. 109 Boyd AE, Lebovitz HE, Pfeiffer JB. Stimulation of growth hormone secretion by l-dopa. N Engl J Med 1970;283:1425–1429. 110 Masala A, Delitalia G, Alagna S et al. Effects of pimozide on levodopainduced growth hormone release in man. Clin Endocrinol 1977;7:253–256. 111 Massara F, Camanni F. Effects of various adrenergic receptor stimulating and blocking agents on hGH secretion. J Endocrinol 1972;54:195–203. 112 Tater P, Vigas M. Role of alpha 1- and alpha 2-adrenergic receptors in the growth hormone and prolactin response to insulin-induced hypoglycemia in man. Neuroendocrinology 1984;39:275–280. 113 Mazzi C, Mainini E, Riva LP et al. Effect of clonidine in children with short stature: behavior of growth hormone, prolactin, and cortisol. In: Chuimello G, Sperling M, eds. Recent Progress in Pediatric Endocrinology. New York: Raven Press, 1983:305–312. 114 Martin JB. Neural regulation of growth hormone secretion. Medical progress report. N Engl J Med 1973;228:1384–1393.

115 Chikara K, Minamitani N, Kaji H et al. Noradrenergic modulation of human pancreatic growth hormone releasing factor (hpGHRF[I-44])-induced growth hormone release in conscious male rabbits: involvement of endogenous somatostatin. Endocrinology 1984;114:1402–1406. 116 McWilliams JR, Meldrum BS. Noradrenergic regulation of growth hormone secretion in the baboon. Endocrinology 1983;112:254–259. 117 Chihara K, Kodama H, Hidesuke K et al. Augmentation by propranolol of growth hormone-releasing hormone (1-44)-NH2induced growth hormone release in normal short and normal children. J Clin Endocrinol Metab 1985;61:229–233. 118 Terry LC, Crowley WR, Lynch C et al. Role of central epinephrine in regulation of anterior pituitary hormone secretion. Peptides 1982;3:311–318. 119 Martin JB. Hypothalamic regulation of growth hormone secretion. In: Black PM, Zervas NT, Ridgway EC, Martin JB, eds. Secretory Pituitary Tumors. New York: Raven Press, 1984:109–134. 120 Grossman A. Brain opiates and neuroendocrine function. Clin Endocrinol Metab 1983;12:725–745. 121 Miki N, Ono M, Shizume K. Evidence that opiatergic and alpha adrenergic mechanisms stimulate rat growth hormone release via growth hormonereleasing factor. Endocrinology 1984;114:1950–1952. 122 Moretti C, Fabri A, Gnessi L et al. Naloxone inhibits exercise-induced release of PRL and GH in athletes. Clin Endocrinol 1983;18:135–138. 123 Giustina A, Veldhuis JD. Pathophysiology of the Neuroregulation of GH secretion in experimental animals and the human. Endocr Rev 1998;19: 717–797. 124 Chihara K, Kato Y, Maeda K et al. Effects of thyrotropin-releasing hormone on sleep and sleep-related growth hormone release in normal subjects. J Clin Endocrinol Metab 1977;4:1096–2000. 125 Irie M, Tsushima T. Increase of serum growth hormone concentration following thyrotropin-releasing hormone injection in patients with acromegaly or gigantism. J Clin Endocrinol Metab 1972;35:97–100. 126 Enjalbert A, Epelbaum J, Aranciba S et al. Reciprocal interactions of somatostatin with thyrotropin-releasing hormone and vasoactive intestinal peptide on prolactin and growth hormone secretion in vitro. Endocrinology 1982;111:42–47. 127 Musseo M, Tiengso A, Fedele D, Crepaldi G. Altered control of growth hormone secretion in patients with cirrhosis of the liver. Arch Intern Med 1979;139:1157–1160. 128 Thorner MO, Perryman RL, Cronin MJ et al. Acromegaly with somatotroph hyperplasia: successful treatment by resection of a pancreatic tumor secreting a growth hormone (GH) releasing factor (GRF). J Clin Invest 1982;70: 965–977. 129 Maeda KY, Kato M, Yamaguchi K et al. Growth hormone release following thyrotropin releasing hormone injection into patients with anorexia nervosa. Acta Endocrinol 1976;81:1–8. 130 Maeda KY, Kato S, Ohgo K et al. Growth hormone and prolactin release after injection of thyrotropin-releasing hormone in patients with depression. J Clin Endocrinol Metab 1975;40:501–505. 131 Pieters GFFM, Hermus ARMM, Smals AGH, Kloppenberg PWC. Paradoxical responsiveness of growth hormone to corticotropin-releasing factor in acromegaly. J Clin Endocrinol Metab 1984;58:560–562. 132 Gold P, Kellner C, Rubinow D et al. Abnormal growth hormone responses to corticotropin-releasing factor in patients with primary affective disorders. Program of the 65th Annual Meeting of The Endocrine Society, San Antonio, Texas, 1983 (Abstract 443: 191). 133 Martin JB, Brazeau P, Tannenbaum GS et al. Neuroendocrine organization of growth hormone regulation. In: Reichlin S, Baldessarini R, Martin JB, eds. The Hypothalamus. New York: Raven Press, 1978:329–357. 134 Bridson WE, Kohler PO. Cortisol stimulation of growth hormone production by human pituitary tissue in culture. J Clin Endocrinol Metab 1970;30: 538–540. 135 Nakagawa K, Akikawa K, Matsuhara M et al. Effect of dexamethasone on growth hormone (GH) responses to growth hormone releasing hormone in acromegaly. J Clin Endocrinol Metab 1985;60:308–310. 136 Loeb JN. Corticosteroids and growth. N Engl J Med 1976;295:547–552. 137 Tyrrell JB, Wiener-Kronish J, Lorenzi M et al. Cushing’s disease: growth hormone response to hypoglycemia after correction of hypercortisolism. J Clin Endocrinol Metab 1977;44:217–221. 138 Suda T, Demura H, Demura R et al. Anterior pituitary hormones in plasma and pituitaries from patients with Cushing’s disease. J Clin Endocrinol Metab 1980;51:1048–1053. 139 Burguera B, Muruais C, Penalva A et al. Dual and selective actions of glucocorticoids upon basal and stimulated growth hormone release in man. Neuroendocrinology 1990;51:51–58.

Chapter 3 140 Casanueva FF, Burguera B, Tome MA et al. Depending on the time of administration, dexamethasone potentiates or blocks growth hormone releasing hormone-induced growth hormone release in man. Neuroendocrinology 1988;47:46–49. 141 Giustina A, Romanelli G, Candrina R et al. Growth hormone deficiency in patients with idiopathic adrenocorticotrophin deficiency resolves during glucocorticoid replacement. J Clin Endocrinol Metab 1989;68:120–124. 142 Leal-Cerro A, Pereira JL, Garcia-Luna PP et al. Effect of enhancement of endogenous cholinergic tone with pyriclostigmine on growth hormone (GH) responses to GH-releasing hormone in patients with Cushing’s syndrome. Clin Endocrinol 1990;33:291–295. 143 Gourmeler M, Girard F, Binoux M. Serum somatomedin/insulin like growth factor (IGF) and IGF carrier levels in patients with Cushing’s syndrome or receiving glucocorticoid therapy. J Clin Endocrinol Metab 1982;54:885–892. 144 Casanueva FF, Burguera B, Muruais C et al. Acute administration of corticoids: a new and peculiar stimulus of growth hormone secretion in man. J Clin Endocrinol Metab 1990;70:234–237. 145 Wegnez M, Schachter BS, Baxter JD, Martial JA. Hormonal regulation of growth hormone messenger RNA. DNA 1982;1:145–153. 146 Surks MI, Kumara-Siri MH. Increase in nuclear thyroid and glucocorticoid receptors and GH production during the deoxyribonucleic acid synthesis phase of the cell growth cycle. Endocrinology 1984;114:873–879. 147 Coulombe P, Schwartz HL, Oppenheimer JH. Relationship between the accumulation of pituitary growth hormone and nuclear occupancy by triiodothyronine in the rat. J Clin Invest 1978;62:1020–1028. 148 Cattini PA, Anderson TR, Baxter JD et al. The human growth hormone gene is negatively regulated by triiodothyronine when transfected into rat pituitary tumor cells. J Biol Chem 1986;261:13367–13372. 149 Sasaki N, Tsuyusaki T, Nakamura H et al. Sleep-related growth hormone release in thyrotoxic patients before and during propylthiouracil therapy. Endocrinol Jpn 1985;32:39–44. 150 Guillemin R, Brazeau P, Bohlen P et al. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982;218:585–587. 151 Esch FS, Bohlen P, Ling NC et al. Characterization of a 40 residue peptide from a human pancreatic tumor with growth hormone-releasing activity. Biochem Biophys Res Commun 1982;109:152–158. 152 Rivier J, Spiess J, Thorner M et al. Characterization of a growth hormonereleasing factor from a human pancreatic islet tumor. Nature 1982;300: 276–278. 153 Bloch B, Brazeau P, Ling N et al. Immunohistochemical detection of growth hormone-releasing factor in brain. Nature 1983;301:607–608. 154 Gubler U, Monahan JJ, Lomedico PT et al. Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin. Proc Natl Acad Sci USA 1983;80:4311–4314. 155 Mayo KE, Vale W, Rivier J et al. Expression, cloning, and sequencing of a cDNA encoding human growth hormone-releasing factor. Nature 1983;306:86–88. 156 Taternoto K, Mutt V. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon-secretin family. Proc Natl Acad Sci USA 1981;78:6603–6607. 157 Spiess J, Rivier J, Vale W. Characterization of rat hypothalamic growth hormone-releasing factor. Nature 1983;303:532–535. 158 Bilezikjian LM, Vale WW. Stimulation of adenosine 3¢,5¢monophosphate production by growth hormone-releasing factors and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 1983;113: 1726–1731. 159 Mayo KE. Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. Mol Endocrinol 1991;6: 1734–1744. 160 Barinaga M, Yamanoto G, Rivier G et al. Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor. Nature 1983; 306:84–86. 161 Fukata J, Diamond DJ, Martin JB. Effects of rat growth hormone (rGH)releasing factor and somatostatin on the release and synthesis of rGH in dispersed pituitary cells. Endocrinology 1985;117:457–467. 162 Stachura ME, Dhariwal APS, Frohman LA. Growth hormone synthesis and release in vitro: effects of partially purified ovine hypothalamic extract. Endocrinology 1972;91:1071–1078. 163 Stachura ME, Frohman LA. Growth hormone: independent release of big and small forms from rat pituitary in vitro. Science 1975;187:447–449. 164 Stachura ME. Basal and dibutyryl cyclic AMP-stimulated release of newly synthesized and stored growth hormone from perfused rat pituitaries. Endocrinology 1975;98:580–589.

Growth Hormone

111

165 Barinaga M, Bilezikjian LM, Vale WW et al. Independent effects of growth hormone-releasing factor on growth hormone release and gene transcription. Nature 1985;314:279–281. 166 Gelato MC, Pescovitz O, Cassorla F et al. Effects of a growth hormonereleasing factor in man. J Clin Endocrinol Metab 1983;57:674–676. 167 Chiodini PG, Liuzzi A, Dallabonzana D et al. Changes in growth hormone (GH) secretion induced by human pancreatic GH-releasing hormone-44 in acromegaly: a comparison with thyrotropin-releasing hormone and bromocriptine. J Clin Endocrinol Metab 1985;60:48–52. 168 Reichlin S. Somatostatin. N Engl J Med 1983;309:1495–1501. 169 Shen LP, Picket RL, Rutter WJ. Human somatostatin I: sequence of the cDNA. Proc Natl Acad Sci USA 1982;79:4575–4579. 170 Finley JWC, Maderchut JL, Roger LJ, Petrusz P. The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system. Neuroscience 1981;6:2173–2192. 171 Siler TM, Yen SSC, Vale W, Guillemin R. Inhibition by somatostatin of the release of TSH induced in man by thyrotropin-releasing factor. J Clin Endocrinol Metab 1974;38:742–745. 172 Koerker DJ, Ruch W, Chideckel E et al. Somatostatin; hypothalamic inhibitor of the endocrine pancreas. Science 1974;184:482–484. 173 Chihara K, Arimura A, Schally AV. Effect of intraventricular injection of dopamine, norepinephrine, acetylcholine, and 5-hydroxy-tryptamine on immunoreactive somatostatin release into rat hypophyseal portal blood. Endocrinology 1979;104:1656–1662. 174 Berelowitz M, Dudlak D, Frohman LA. Release of somatostatin-like immunoreactivity from incubated rat hypothalamus and cerebral cortex. Effects of glucose and glucoregulating hormone. J Clin Invest 1982;69: 1293–1301. 175 Shrikant CB, Patel YC. Receptor binding of somatostatin-28 is tissue specific. Nature 1981;294:259–260. 176 Reisine T, Bell GI. Molecular Biology of Somatostatin Receptors. Endocr Rev 1995;16:427–442. 176a Yogesh CP, Srikant CB, Sharma KP. C-terminal region of human somatostatin receptor 5 is required for induction of Rb and GI cell cycle arrest. Mol Endocrinol 1999;13:82–90. 177 Yamada Y, Kagimoto S, Kubota A et al. Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. Biochem Biophys Res Commun 1993;195:844–852. 178 Miller GM, Alexander JM, Bihhal NA et al. Somatostatin receptor subtype gene expression in pituitary adenoms. JCEM 1995;80:1386–1392. 179 Panetta R, Patel Y. Expression of MRNA for all five human somatostatin receptors (hSSTR1-5) in pituitary tumors. Life Sci 1995;56:333–342. 180 Greenman Y, Melmed S. Heterogeneous expression of two somatostatin receptor subtypes in pituitary tumors. JCEM 1994;78:398–403. 181 Greenman Y, Melmed S. Expression of Somatostatin receptor subtypes in pituitary adenomas: evidence for peferential SSTRs expression in the mammosomatotroph lineage. JCEM 1994;79:724–729. 182 Patel Y, Srikant CB. Subtype selectively of peptide analogs for all five cloned human somatotroph receptors (hSSTR1-5) Endocrinology 1994;135:2814– 2817. 183 Berelowitz M, Szabo M, Frohman LA et al. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1982;212:1279–1281. 184 Yamashita S, Melmed S. Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 1986;118:176–182. 185 Tannenbaum GS, Ling N. The interrelationship of growth hormone (GH)releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 1984;115:1952–1957. 186 Vance ML, Kaiser DL, Martha PM Jr et al. Lack of in vivo somatotroph desensitization or depletion after 14 days of continuous growth hormone (GH)-releasing hormone administration in normal men and a GH-deficient boy. J Clin Endocrinol Metab 1989;68:22–28. 187 Tannenbaum GS, Painson JC, Lengyel AMJ et al. Paradoxical enhancement of pituitary growth hormone (GH) responsiveness to GH-releasing factor in the face of high somatostatin tone. Endocrinology 1989;124:1380–1388. 188 Horvath S, Palkovits M, Gorcs T et al. Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormone-releasing hormone and somatostatincontaining neurons in the hypothalamus of the rat. Brain Res 1989;481:8–15. 189 Bilezikjian LM, Vale WW. Chronic exposure of cultured rat anterior pituitary cells to GRF causes partial loss of responsiveness to GRF. Endocrinology 1984;115:2032–2034.

112

SECTION 1

Hypothalamic–Pituitary Function

190 Davis JRE, Sheppard MC, Shakespear RA et al. Does growth hormonereleasing factor desensitize the somatotroph? Interpretation of responses of growth hormone during and after 10-hour infusion of GRF 1-29 amide in man. Clin Endocrinol 1986;24:135–140. 191 Losa M, Chiodini PG, Liuzzi A et al. Growth hormone-releasing hormone infusion in patients with active acromegaly. J Clin Endocrinol Metab 1986;63:88–93. 192 Spada A, Elahi FR, Arosio M et al. Lack of desensitization of adenomatous somatotrophs to growth hormone-releasing hormone in acromegaly. J Clin Endocrinol Metab 1987;64:585–591. 193 Bilezikjian LM, Seifert H, Vale W. Desensitization to growth hormonereleasing factor (GRF) is associated with down-regulation of GRF-binding sites. Endocrinology 1986;118:2045–2052. 194 Sheppard MC, Kronheim S, Pimstone BL. Stimulation by growth hormone of somatostatin release from the rat hypothalamus in vitro. Clin Endocrinol 1978;9:583–586. 195 Rosenthal SM, Hulse JA, Kaplan SL, Grumbach MM. Exogenous growth hormone inhibits growth hormone-releasing factor-induced growth hormone secretion in normal men. J Clin Invest 1986;77:176–180. 196 Ross RJM, Borges F, Grossman A et al. Growth hormone pretreatment in man blocks the response to growth hormone-releasing hormone. Clin Endocrinol 1987;26:117–123. 197 Aguila MC, McCann SM. Stimulation of somatostatin-release in vitro by synthetic growth hormone-releasing factor by a non-dopaminergic mechanism. Endocrinology 1985;117:762–765. 198 Peterfreund RA, Vale WW. Somatostatin analogs inhibit somatostatin secretion from cultured hypothalamus cells. Neuroendocrinology 1984;39: 397–402. 199 Smith RG, Van der Ploeg LH, Howard AD et al. Peptidomimetic regulation of growth hormone secretion. Endocr Rev 1997;18:621–645. 200 Akman MS, Girard M, O’Brien LF et al. Mechanisms of action of a second generation growth hormone-releasing peptide in rat anterior pituitary cells. Endocrinology 1993;132:1286–1291. 201 Howard AD, Feighner SD, Cully DF et al. A receptor in pituitary and hypothalamus release that functions in growth hormone release. Science 1996;273:974–977. 202 Kojima M, Hosoda H, Date Y et al. Ghrelin is a growth-hormone releasing acylated peptide from stomach. Nature 1999;402:656–660. 203 Casanueva FF, Diguez C. Growth hormone secretagogues: physiological studies and clinical utility. TEM 1999;10:30–38. 204 Bercu BB, Yang SW, Masuda R, Walker RF. Role of selected endogenous peptides in growth hormone releasing hexapeptide (GHRP) activity: analysis of GHRH, TRH and GnRH. Endocrinology 1992;130:2579–2586. 205 Chen C, Wu D, Clarke IJ. Signal transduction systems employed by synthetic GH-releasing peptides in somatotrophs. J Endocrinol 1996;148:381–386. 206 Goth MI, Lyons CE, Canny BJ, Thomer MO. Pituitary adenylate cyclase activating polypeptide growth (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors. Endocrinology 1992;130:939–944. 207 Popovic V, Damjanovic S, Micic D et al. Blocked growth hormone-releasing peptide (GHRP-6)-induced GH secretion and absence of the synergic action of GHRP-6 plus GH-releasing hormone in patients with hypothalamopituitary disconnection: evidence that GHR-6 main action is exerted at the hypothalmic level. J Clin Endocrinol Metab 1995;80: 942–947. 208 Torsello A, Grilli R, Luoni M et al. Mechanism of action of hexarelin I growth hormone releasing activity in the art. Eur J Endocrinol 1996;135: 481–488. 209 Penalva A, Pombo M, Carballo A et al. Influence of sex, age and adrenergic pathways on the growth hormone response to GHRP-6. Clin Endocrinol 1993;38:87–91. 210 Korbonits M, Grossman A. Growth hormone-releasing peptide and its analogues: novel stimuli to growth hormone release. Trends Endocrinol Metab 1995;6:43–49. 211 Robinson BM, Friberg RD, Bowers CY, Barkan AL. Acute growth hormone (GH) response to GH-releasing hexapeptide in humans is independent of endogenous GH-releasing hormone. J Clin Endocrinol Metab 1992;75: 1121–1124. 212 Jaffe CA, Ho PJ, Demott FR et al. Effect of prolonged growth hormone (GH)-releasing peptide infusion on pulsatile GH secretion in normal men. J Clin Endocrinol Metab 1993;77:1641–1647. 213 Micic D, Popovic V, Kendereski A et al. The sequential administration of GHRH followed 120 minutes later by hexarelin, as an effective test to assess the pituitary GH reserve in man. Clin Endocrinol 1996;45:543–551.

214 Penalva A, Carballo A, Pombo M et al. Effect of growth hormone (GH)releasing hormone (GHRH), atropine, pyridostigmine or hypoglycemic on GHRP-6-induced GH secretion in man. J Clin Endocrinol Metab 1993;76:168–171. 215 Guillaume V, Magnan E, Cataldi M et al. GH-releasing hormone secretion is stimulated by a new GH-releasing hexapeptide in sheep. Endocrinology 1994;135:1073–1076. 216 Yagi H, Kaji H, Sato M et al. Effects of intravenous or intracerebroventricular injection of His-D-trp-Ala-Trp-D-Phe-Lys-NH, on GH release in conscious, freely moving male rats. Neuroendocrinology 1996;63:198–206. 217 Arvat E, Gianotti L, Di Vito L et al. Modulation of growth hormonereleasing activity of hexarelin in man. Neuroendocrinology 1995;61:51–56. 218 Bowers CY, Sartor AA, Reynolds DG, Badger TAM. On the actions of the growth hormone-releasing hexapeptide, GHRP-6. Endocrinology 1991;128:2027–2035. 218a Diguez G, Casaneuva FF. Ghrelin: a step forward in the understanding of somatotroph cell function and growth regulation. Europ J Endocrinol 2000;142:413–417. 219 Ghigo E, Arvat E, Gianotti L et al. Growth hormone-releasing activity of hexarelin, a new synthetic hexapeptide, after intravenous, subcutaneous, and oral administration in man. J Clin Endocrinol Metab 1994;78:693–698. 220 Peino R, Cordido F, Penalva A et al. Acipimox-mediated plasma free fatty acid depression per se stimulated growth hormone (GH) secretion in normal subjects and potentiates the response to other GH-releasing stimuli. J Clin Endocrinol Metab 1996;81:909–913. 221 Ghigo E, Arvat E, Muccioli G, Camanni F. Growth hormone-releasing peptides. Eur J Endocrinol 1997;136:445–460. 222 Giustina A, Bussi AR, Deghenghi R et al. Comparision of the effects of growth hormone-releasing hormone and hexarelin, a novel growth hormonereleasing peptide-6 analog, on growth hormone secretion in humans with or without glucocorticoid excess. J Endocrinol 1995;146:227–232. 223 Ramos-Diaz JC, Pimentel-Filho F, Reis AF, Lengyel AMJ. Different growth hormone (GH) responses to GH-releasing peptide and GH-releasing hormone in hyperthroidism. J Clin Endocrinol Metab 1996;81:1343–1346. 224 Loche S, Colao A, Cappa M et al. The growth hormone response to hexarelin in children: reproducibility and effect of sex steroids. J Clin Endocrinol Metab 1997;82:861–864. 225 Shimon I, Yan X, Melmed S. Human fetal pituitary expresses functional growth hormone-releasing peptide receptors. J Clin Endocrinol Metab 1998;83:174–178. 226 Massoud AF, Hindmarsh PC, Brook DGD. Hexarelin-induced growth hormone, cortisol, and prolactin release: a dose-response study. J Clin Endocrinol Metab 1996;81:4338–4341. 227 Raun K, Hansen BS, Johansen NL et al. Ipamorelin, the first selective growth hormone secretagogue. Eur J Endocrinol 1998;139:552–561. 228 Palmiter RD, Brinster R, Hammer RE et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982;300:611–615. 229 Palmiter RD, Norstedt N, Gelinas RE et al. Metallothionein-human GH fusion genes stimulate growth of mice. Science 1983;222:809–814. 230 Matthews LS, Hammer RE, Brinster RL, Palmiter RD. Expression of insulin-like growth factor I in transgenic mice with elevated level of growth hormone is correlated with growth. Endocrinology 1988;123: 433–437. 231 Stefaneau L, Kovacs K, Horvath E et al. An immunocytochemical and ultrastructural study of adenohypophyses of mice transgenic for human growth hormone. Endocrinology 1990;126:608–615. 232 Chandrashelar V, Bartke A, Wagner TE. Neuroendocrine function in adult female transgenic mice expressing the human growth hormone gene. Endocrinology 1992;130:1802–1808. 233 Orian JM, Lee CS, Weiss LM, Brandon MR. The expression of a metallothionein ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathological lesions in the liver. Endocrinology 1989;124:455–463. 234 Bartke R, Steger LJ, Hodges SL et al. Infertility in transgenic female mice with human growth hormone expression: evidence for luteal failure. J Exp Zool 1988;218:121–124. 235 Shea BT, Hammer RE, Brinster RL. Growth allometry of the organs in giant transgenic mice. Endocrinology 1987;121:1924–1930. 236 Quaife CJ, Mathews LS, Pinkert CA et al. Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice. Endocrinology 1989;124:40–48. 237 Doi T, Striker LJ, Quaife C et al. Progressive glomerulosclerosis develops in transgenic mice chronically expressing growth hormone and growth hormone

Chapter 3

238

239

240

241

242

243

244

245

246

247

248

249 250 251

252 253

254 255

256

257

258

259

260 260a

releasing factor but not in those expressing insulin-like growth factor-I. Endocrinology 1988;131:398–403. Herington AC, Ymer S, Stevenson J. Identification and characterization of specific binding proteins for growth hormone in normal human sera. J Clin Invest 1986;77:1817–1823. Baumann G, Shaw MA. Plasma binding of the 20,000-dalton growth hormone variant is distinct from that the 22K-growth hormone. Endocrinology 1989;124(suppl.):436(Abstract). Smith WC, Kuniyoshi J, Talamantes F. Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 1989;3:984–990. Leung DW, Spencer SA, Cachianes G et al. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 1987;330:537–543. Barsano CP, Bauman G. Simple algebraic and graphic methods for the apportionment of hormone (and receptor) into bound and free fractions in binding equilibria; or how to calculate bound and free hormone? Endocrinology 1989;124:1101–1106. Bauman G, Shaw MA. Plasma transport of 20,000-Dalton variant of hGH. Evidence for a 20K-specific binding site. J Clin Endocrinol Metab 1990;71: 1335–1343. Baumann G, Stolar MW, Buchanan TA. The effect of circulating growth hormone-binding protein on metabolic clearance, distribution and degradation of human growth hormone. J Clin Endocrinol Metab 1987;64:657–660. Baumann G, Shaw MA, Amburn K. Regulation of plasma growth hormone-binding proteins in health and disease. Metabolism 1989; 38:683–689. Daughaday WH, Triveldi B, Andrews BA. The ontogeny of serum GHbinding protein in man: a possible indicator of hepatic GH receptor development. J Clin Endocrinol Metab 1987;65:1072–1074. Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA 1987;84:4636–4640. Baumann G, Shaw MA, Merimee TJ. Low levels of high-affinity growth hormone-binding protein in African pygmies. N Engl J Med 1989;320: 1705–1707. Carter-SU C, Schwartz J, Smit LS. Molecular mechanism of GH action. Annu Rev Physiol 1996;58:187–207. Herrington J, Smit L, Schwartz J, Carter-Su C. The Role of STAT protein in growth hormone signaling. Oncogene 2000;19(21):2585–2597. Xu BC, Wang X, Darns J et al. Growth hormone promotes the association of transcription factor: STAT5 with the GH receptor. J Biol Chem 1996;271: 19768–19773. Melmed S, Yamashita H, Yamasaki J et al. IGF-1 Receptor Signalling: Lessons from the somatotroph. Rec Prog Horm Res 1996;51:189–216. Leung KC, Waters MJ, Markus I et al. Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation. Cell Biology 1997;94:11381–11386. Barnard R, Waters MJ. The serum growth hormone binding protein: pregnant with possibilities. J of Endocrinology 1997;153:1–14. Udy GB, Towers RP, Snell RG et al. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc of Nat Acad of Sci USA 1997;94:7239–7244. Ram PA, Park SH, Choi HK, Waxman DJ. Growth hormone activation of Stat1, Stat3, and Stat5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation. Journal of Biological Chem 1996;271(10):5929–5940. Teglund S, McKay C, Schuetz E, van Deursen JM et al. Stat5a and Stat5b ptoteins have essential and nonessential, or redundant, roles in ccytokine responses. Cell 1998;93(5):841–850. Davey HW, Park SH, Grattan DR et al. STAT5b-deficient mice are growth hormone pulse-resistant. Role of STAT5b in sex specific liver P450 expression. J Biol Chem 1999;274:35331–35336. Fain JN, Ihle JH, Bahouth SW. Stimulation of lipolysis but not of leptin release by growth hormone is abolished in adipose tissue from Stat5a and b knockout mice. Biochem Biophys Res Comm 1999;263(1): 201–205. Starr R, Hilton DJ. SOCS: suppressors of cytokine signalling. International Journal of Biochem and Cell Biol 1998;30:1081–1085. Herrington J, Smith L, Schwartz J, Carter-Su C. Role of STAT proteins in growth hormone signalling. Oncogene 1996;119:2585–2597.

Growth Hormone

113

261 Rinderknecht E, Humbel RE. The amino acid sequence of human insulinlike growth factor I and its structural homology with pro-insulin. J Biol Chem 1978;253:2769–2776. 261a Humbel RB. Insulin-like growth factors I and II. Fur J Biochem 1990;190:445–462. 261b Sara VR, Hall K. Insulin-like growth factors and their blinding proteins. Physiol Rev 1990;70:591–613. 262 Bell GI, Gerhard DS, Fong NM et al. Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous. Proc Natl Acad Sci USA 1985;82:6450–6454. 263 Brissende JE, Ulllrich JE, Francke U. Human chromosomal mapping of genes for insulin-like growth factors I and II and epidermal growth factor. Nature 1984;310:781–784. 264 McConghey O, Sledge CB. Production of “sulfation factor” by the perfused rat liver. Nature 1970;225:1249–1250. 265 Han VKM, D’Ercole AJ, Lund PK. Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science 1987;236:193–197. 266 Mathews LS, Norstedt G, Palmiter RG. Regulation of insulin-like growth factor I gene expression by growth hormone. Proc Natl Acad Sci USA 1986;83:9343–9347. 267 Doglio A, Dani C, Frederikson G et al. Acute regulation of insulin-like growth factor I gene expression by growth hormone during adipose cell differentiation. EMBO J 1987;6:4011–4016. 268 Romanus JA, Rabinovitch A, Rechler MM. Neonatal rat islet cell cultures synthesize insulin-like growth factor I. Diabetes 1985;34:696–702. 269 Clemmons DR, Shaw DS. Variables controlling somatomedin production by cultured human fibroblasts. J Cell Physiol 1983;115:137–142. 270 Hall K, Sara VR. Growth and somatomedins. Vitam Horm 1983;40:175–233. 271 Binoux M, Hossenlopp L. Insulin-like growth factor and IGF binding proteins: comparison of human serum and lymph. J Clin Endocrinol Metab 1988;67:509–514. 272 Corps AN, Brown KD, Rees LH et al. The insulin-like growth factor I content in human milk increases between early and full lactation. J Clin Endocrinol Metab 1988;67:25–29. 273 Costigan DC, Guyda HJ, Posner BI. Free insulin-like growth factor I (IGF-I) and IGF-II in human saliva. J Clin Endocrinol Metab 1988;66: 1014–1018. 274 Chochinov RH, Mariz IE, Hajek AS, Daughaday WH. Characterization of a protein in mid-term amniotic fluid which reacts in the somatomedin-C radioreceptor assay. J Clin Endocrinol Metab 1977;44:902–908. 275 Hall K, Enberg G, Ritzen M et al. Somatomedin C levels in serum from healthy children and from children with growth hormone deficiency and delayed puberty. Acta Endocrinol 1980;94:155–165. 276 Moses AC, Nissley SP, Cohen KL, Rechler MM. Specific binding of a somatomedin-like polypeptide in rat serum depends on growth hormone. Nature 1976;263:137–140. 276a Rosenbloom A. Growth hormone insensitivity: physiologic and genetic basis, phenotype and treatment. J Pediatr 1999;135:280–289. 277 Kim HS, Nagalla SR, Oh Y et al. Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPS): characterization of connective tissue growth factor as a member of the IGFBP superfamily. Proc Natl Acad Sci USA 1997;94:12981–12986. 277a Hwa V, Oh Y, Rosenfeld RG. The IGFBP superfamily. Endocr Rev 1998;20: 761–787. 278 Baxter RC. Characterization of the acid-labile subunit of the growth hormone-dependent insulin-like growth factor binding protein complex. J Clin Endocrinol Metab 1988;67:265–272. 278a Rajaram S, Baylink DJ, Mohan S. IGFBPs in serum and other biological fluids: regulation and functions. Endocr Rev 1997;18:801–831. 279 Baxter RC, Martin JL. Structure of the Mr 140,000 growth hormone dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity labelling. PANS 1989;86:6898–6902. 280 Guler H-P, Zapf J, Schmid C, Froesch ER. Insulin-like growth factor I and II in healthy man. Estimations of half-lives and production rates. Act Endocrinol 1989;121:753–758. 281 Hogkinson SC, Davis SR, Moore LG et al. Metabolic clearance of insulinlike growth factor-II in sleep. J Endocrinol 1989;123:461–468. 282 Baxter RC, Martin JL. Radioimmunoassay of growth hormone dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 1986;78:1504–1512. 283 Zapf J, Hauri C, Waldvogel M et al. Recombinant human insulin-like growth factor I induces its own specific carrier protein in hypohysectomized and diabetic rats. Proc Natl Acad Sci USA 1989;86:3813–3817.

114

SECTION 1

Hypothalamic–Pituitary Function

284 Drop SLS, Kortleve DJ, Guyda HJ, Posner BI. Immunoassay of somatomedinbinding protein from human amniotic fluid: levels in fetal, neonatal, and adult sera. J Clin Endocrinol Metab 1984;59:908–915. 285 Degerblad M, Povoa G, Thoren M et al. Lack of diurnal rhythm of low molecular weight insulin-like growth factor binding protein in patients with Cushing’s disease. Acta Endocrinol 1989;120:195–200. 286 Busby WH, Snyder DK, Clemmons DR. Radioimmunoassay of a 26,000 dalton plasma insulin like growth factor binding protein: control by nutritional variables. J Clin Endocrinol Metab 1988;67:1225–1230. 287 Suikkari AM, Koivistinen VA, Rutanen EM et al. Insulin regulates serum levels of low molecular weight insulin-like growth factor binding protein. J Clin Endocrinol Metab 1988;66:266–272. 288 Ezzat S, Ren SG, Braunstein GD, Melmed S. Octreotide stimulates insulinlike growth factor binding protein (IGFBP-1) level in acromegaly. J Clin Endocrinol Metab 1991;73:441–443. 289 Holly JMP, Biddlecombe RA, Dunger DB et al. Circadian variation of GHindependent IGF-binding protein in diabetes mellitus and its relationship to insulin: a new role for insulin? Clin Endocrinol 1988;29:667–675. 290 Harvey S, Hull KL. Growth hormone: a paracrine growth factor. Endocrine 1997;7:267–279. 291 Daughaday WH, Hall K, Raben MS et al. Somatomedin: proposed designation for sulfation factor. Nature 1972;235:107. 292 Green H, Morikawa M, Nixon T. A dual effector theory of growth hormone action. Differentiation 1985;29:195–198. 293 Zapf J, Froesch ER, Humbrel RE. The insulin-like growth factors (IGF) of human serum: chemical and biological characterization and aspects of their possible physiological role. Curr Top Cell Regul 1981;19:257–309. 294 Blum WF, Albertsson-Wikland K, Rosberg S et al. Serum levels of IGF-1 and IGFBP-3 reflect spontaneous GH secretion. JCEM 1993;76:1610–1616. 295 Sjogren K, Liu JL, Skrtic S et al. Liver-derived insulin-like growth factor-1 is the principal source of IGF-1 in blood but is not required for postnatal body growth in mice. PANS 1999;96:7088–7092. 296 Reid JD. Congenital absence of the pituitary gland. J Pediatr 1960;56: 658–664. 297 Kember NF, Walker DVR. Control of bone growth in rats. Nature 1971;229: 428–429. 298 Evans HM. The function of the anterior hypophysis. Harvey Lectures 1924; 19:212–215. 299 Geschwind II, Li CH. The tibia test for growth hormone. In: Smith Jr, Gaebler OH, Long CNH, eds. The Hypophyseal Growth Hormone: Nature and Actions. New York: McGraw-Hill, 1955. 300 Thorngren K-G, Hansson LI, Menander-Sellman K, Stenstrom A. Effect of hypophysectomy on longitudinal bone growth in the rat. Calcif Tissue Int 1973;11:281–300. 301 Phillips LS, Weiss LJ. Circulating growth factor studies in growth plate versus resting cartilage in vitro: tissue responsiveness. Endocrinology 1982;111:1255–1262. 302 Madsen K, Friberg U, Roos P et al. Growth hormone stimulates the proliferation of cultured chondrocytes from rabbit ear and rat rib growth cartilage. Nature 1983;304:545–547. 303 Smith RL, Alathumpat V, Ku CW, Hintz RL. Growth hormone stimulates insulin-like growth factor I actions on adult articular chondrocytes. J Orthop Res 1989;7:198–207. 304 Lindahl A, Isgaard J, Carlsson L, Isaksson OGP. Differential effects of growth hormone and insulin like growth factor I on colony formation of epiphyseal chondrocytes in suspension culture in rats of different ages. Endocrinology 1987;121:1061–1069. 305 Lindahl A, Nilsson A, Isaksson OGP. Effects of growth hormone and insulin-like growth factor I on colony formation of rabbit epiphyseal chondrocytes at different stages of maturation. J Endocrinol 1987;115: 263–271. 306 Isaksson OGP, Jansson JO, Gause IAM. Growth hormone stimulates longitudinal bone growth directly. Science 1982;216:1237–1239. 307 Russell SM, Spencer EM. Local injections of human or rat growth hormone or of purified human somatomedin-C stimulate unilateral tibial epiphyseal growth in hypophysectomized rats. Endocrinology 1985;116: 2563–2567. 308 Schlechter NL, Russell SM, Spencer EM, Nicoll CS. Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin. Proc Natl Acad Sci USA 1986;83:7932–7934. 309 Nilsson A, Isgaard J, Lindahl A et al. Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate. Science 1986;233:571–574.

310 Rosenfeld RG, Kemp SF, Hintz RL. Constancy of somatomedin response to growth hormone treatment of hypopituitary dwarfism and lack of correlation with growth rate. J Clin Endocrinol Metab 1981;53:611–617. 311 Schoenle E, Zapf J, Humbel RE, Froesch ER. Insulin-like growth factor I stimulates growth in hypophysectomized rats. Nature 1982;296:252–253. 312 Guler P, Zapf J, Scheiwiller E, Froesch ER. Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats. Proc Natl Acad Sci USA 1988;85:4889–4893. 313 Skottner A, Clark RG, Fryklund L, Robinson ICAF. Growth responses in mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor I. Endocrinology 1989;124:2519–2526. 313a Spagnol A, Rosenfeld RG. The mechanism by which GH brings about growth. The relative contributions of GH and insulin-like growth factors. Endocrinol Metab Clin North Am 996;25:613–615, 680. 314 Grichting G, Levy LK, Goodman HM. Relationship between binding and biological effects of human growth hormone in rat adipocytes. Endocrinology 1983;113:1111–1120. 315 MacGorman LR, Rizza RA, Gerich JE. Physiological concentrations of growth hormone exert insulin-like and insulin-antagonistic effects on both hepatic and extrahepatic tissues in man. J Clin Endocrinol Metab 1981;53:556–563. 316 Lippe BM, Kaplan SA, Golden MP et al. Carbohydrate tolerance and insulin receptor binding in children with hypopituitarism: responses after acute and chronic human growth hormone administration. J Clin Endocrinol Metab 1981;53:507–513. 317 Bougueres PF, Artavia-Loria E, Ferre P et al. Effects of hypopituitarism and growth hormone replacement therapy on the production and utilization of glucose in childhood. J Clin Endocrinol Metab 1985;61:1152–1157. 318 Merimee TJ, Felig P, Marliss E et al. Glucose and lipid homeostasis in the absence of human growth hormone. J Clin Invest 1971;50:574–582. 319 Grunt JA, Crigler JF Jr, Slone D, Soeldner JS. Changes in serum insulin, blood sugar, and free fatty acid levels four hours after administration of human growth hormone to fasting children with short stature. Yale J Biol Med 1967;40:68–74. 320 Costin G, Kogut MD, Frasier SD. Effect of low-dose human growth hormone on carbohydrate metabolism in children with hypopituitarism. J Pediatr 1972;80:796–803. 321 Gold H, Spector S, Samaan NA, Pearson OH. Effect of growth hormone on carbohydrate metabolism in hypopituitary dwarfs. Metabolism 1968;17: 74–83. 322 Schnure JJ, Raskin P, Lipman RL. Growth hormone secretion during sleep: impairment in glucose tolerance and nonsuppressibility by hyperglycemia. J Clin Endocrinol Metab 1971;33:234–241. 323 Salomon F, Cuneo RD, Hesp R, Sonksen PH. The effects of treatment with recombinant human growth hormone on body composition and metabolism in GH deficient adults. N Engl J Med 1989;321:1797–1803. 324 Johansson JO, Fowelin J, Landin K, Lager I, Bengtsson BA. Growth hormone deficient adults are insulin resistant. Metabolism 1995;44:1126–1129. 325 O’Neal DN, Kalfas A, Dunning PL et al. The effect of 3 months of recombinant human growth hormone therapy on insulin and glucosemediated glucose disposal and insulin secretion in GH deficient adults: minimal model analysis. J Clin Endocrinol Metab 1994;79:975–983. 326 Sonksen PH, Greenwood FC, Ellis JP, Lowy C, Rutherford A, Nabarro JDN. Changes of carbohydrate tolerance in acromegaly with progress of the disease and in response to treatment. J Clin Endocrinol Metab 1967;27:1418–1430. 327 Trimble ER, Atkinson AB, Buchanan KD, Hadden DR. Plasma glucagon and insulin concentrations in acromegaly. J Clin Endocrinol Metab 1980;51: 626–631. 328 Bolinder J, Ostman J, Werner S, Arner P. Insulin action in human adipose tissue in acromegaly. J Clin Invest 1986;77:1201–1206. 329 Dahms WT, Owens RP, Kalhan SC, Kerr DS, Danish RK. Urea synthesis, nitrogen balance and glucose turnover in growth-hormone deficient children before and after GH administration. Metabolism 1989;38:197–203. 330 Manson J McK, Wilmore DW. Positive nitrogen balance with human growth hormone and hypocaloric intravenous feeding. Surgery 1986;100: 188–197. 331 Anon. Metabolic actions of growth hormone. Nutr Rev 1972;30:79–82. 332 Tanner JM, Hughes PCR, Whitehouse RN. Comparative rapidity of response of height, limb muscle and limb fat to treatment with human growth hormone in patients with and without growth hormone deficiency. Acta Endocrinol 1977;84:681–696. 333 Tanner JM, Whitehouse RH. The effect of human growth hormone on subcutaneous fat thickness in hyposomatotrophic and panhypopituitary dwarfs. J Endocrinol 1967;39:263–275.

Chapter 3 334 Bonnet F, Vanderschueren-Lodewyckx M, Eckels R, Malvaux P. Subcutaneous adipose tissue and lipids in blood in growth hormone deficiency before and after treatment with growth hormone. Pediatr Res 1974;8:800–805. 335 Birnbaum RS, Goodman HM. Studies on the mechanism of the antilipolytic effect of growth hormone. Endocrinology 1976;99:1336–1345. 336 Goodman HM. Growth hormone and the metabolism of carbohydrate and lipid in adipose tissue. Ann NY Acad Sci 1968;148:419–440. 337 Fain JN, Kovacev VP, Scow RO. Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J Biol Chem 1965;240:3522–3529. 338 Fineberg SE, Merimee TJ. Acute metabolic effects of human growth hormone. Diabetes 1974;23:499–513. 339 Sherwin RS, Schulman GA, Hendler R et al. Effect of growth hormone on oral glucose tolerance and circulating metabolic fuels in man. Diabetologia 1983;24:155–161. 340 Pearson OH, Dominquez JM, Greenberg E et al. Diabetogenic and hypoglycemic effects of human growth hormone. Trans Assoc Am Physicians 1960;73:217–226. 341 Gerich JE, Lorenzi M, Bier DM et al. Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. Studies involving administration of exogenous hormone during suppression of endogenous hormone secretion with somatostatin. J Clin Invest 1976;257:875–884. 342 Attanacio AF, Lamberts SWJ, Matranga AMC et al. Adult growth hormone deficient patients demonstrate hetrogeneity between childhood onset and adult onset before and during human GH treatment. J Clin Endocrinol Metab 1997;82:82–88. 343 Bengtsson BA, Eden S, Lonn L et al. Treatment of adults with growth hormone deficiency with recombinant human growth hormone. J Clin Endocrinol Metab 1993;76:309–317. 343a Gibney J, Wallace JD, Spinks et al. The effects of ten years of recombinant human GH in adult GH-deficient patients. J Clin Endocrinol Metab 1999;84:2596–2602. 344 Snel YE, Doerga ME, Brummer RM et al. Magnetic Resonance Imaging assessed adipose tissue and serum lipid and insulin concentrations in growth hormone deficient adults. Effects of growth hormone replacement. Arterioscler Thromb Vasc Biol 1995;15:1543–1548. 345 Moller J, Frandsen E, Fisker S et al. Decreased plasma and extracellular volume in growth hormone-deficient adults and the acute and prolonged effects of GH administration: a controlled experimental study. Clin Endocrinol 1996;44:533–539. 346 Christ ER, Cummings MH, Pearson TC et al. The importance of growth hormone in the regulation of crythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency. J Clin Endocrinol Metab 1997;82:2985–2990. 347 De Boer H, Blok GJ, Voerman B et al. The optimal growth hormone replacement dose in adults, derived from bioimpedance analysis. J Clin Endocrinol Metab 1995;80:2069–2076. 347a Salomon F, Cuneo RC, Hesp R, Sonksen PH. The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N Engl J Med 1989;321: 1797–1803. 348 Whitehead HM, Boreham C, McIlrath EM et al. Growth hormone treatment of adults with growth hormone deficiency: results of a 13month placebo controlled cross-over study. Clin Endocrinol (Oxf) 1992; 36:45–52. 349 Binnerts A, Swart GR, Wilson JHP et al. The effect of growth hormone administration in growth hormone deficient adults on bone, protein, carbohydrate and lipid homeostasis, as well as body composition. Clin Endocrinol (Oxf) 1992;37:79–87. 350 Baum HB, Biller BM, Finkelstein JS et al. Effects of physiologic growth hormone therapy on bone density and body composition in patients with adult-onset of growth hormone deficiency. A randomized, placebo-controlled trial. Ann Intern Med 1996;125:883–890. 351 De Boer H, Blok GJ, Voerman HJ et al. Body composition in adult growth deficient men assessed by anthropometry and bioimpedance analysis. Clin Endocrinol Metab 1992;75:833–837. 352 Hoffman DM, Crampton L, Sernia C et al. Short term growth hormone (GH) treatment of GH-deficient adults increases body sodium and extracellular water, but not blood pressure. J Clin Endocrinol Metab 1996;81:1123–1128. 353 Holmes SJ, Economou G, Whitehouse RW et al. Reduced bone mineral density in patients with adult onset growth hormone deficiency. J Clin Endocrinol Metab 1994;78:669–674.

Growth Hormone

115

354 Rosen T, Wilhelmsen L, Landin-Wilhelmsen K et al. Increased fracture frequency in adult patients with hypopituitarism and GH deficiency. Europ J of Endo 1997;137:240–245. 355 Johannsen JS, Pedersen SA, Jorgensen JOL et al. Effects of growth hormone on bone Gla protein in GH-deficient adults. J Clin Endocrinol Metab 1990;70:916–919. 356 Hyer SL, Rodin DA, Tobias JH et al. Growth hormone deficiency during puberty reduces adult bone mineral density. Arch Dis Child 1992;67: 1472–1474. 357 Nielsen HK, Jorgensen JOL, Brixen K, Christiansen JS. Serum osteocalcium and bone isoenzyme alkaline phosphatase in growth hormone-deficient patients: dose-response studies with biosynthetic human GH. Calcif Tissue Int 1991;46:82–87. 358 Sartario A, Conti A, Monzani M et al. Growth hormone treatment in adults with GH deficiency: effects on new biochemical markers of bone and collagen turnover. J Endocrinol Invest 1993;16:893–898. 359 Bravenboer N, Holzmann P, Blok GJ, Lips P. Histomorphometric analysis of bone mass and bone metabolism in growth hormone deficient adult men. Bone 1996;18:551–557. 360 Holmes SJ, Whitehouse RW, Swindell R et al. Effect of growth hormone replacement on bone mass in adults with adult-onset growth hormone deficiency. Clin Endocrinol (Oxf) 1995;42:627–633. 361 Johannsson G, Rosen T, Bosaeus I et al. Two years of growth hormone (GH) treatment increases bone mineral content and density in hypopituitary patients with adult-onset GH deficiency. J Clin Endocrinol Metab 1996;81:2865–2873. 362 Rutherford OM, Beshyah SA, Johnston DG. Quadriceps strength before and after growth hormone replacement in hypopituitary adults: relationships to changes in learn body mass and IGF-1. Endocrinol Metab 1994;1:41–47. 363 Jorgensen JOL, Thuesen L, Muller J et al. Three years of growth hormone treatment in growth hormone-deficient adults: near normalization on body composition and physical performance. Eur J Endocrinol 1994;130:224–228. 364 Bates AS, Van’t Hoff W, Jones PJ, Clayton RN. The effect of hypopituitarism on life expectancy. J Clin Endocrinol Metab 1996;81:1169–1172. 365 Rosen T, Bergtsson BA. Premature mortality due to cardiovascular ulcer disease in hypopituitarism. Lancet 1990;336:285–288. 366 Shahi M, Beshyah GA, Hackett D et al. Myocardial dysfunction in treated adult hypopituitarism: a possible explanation of increased cardiovascular mortality. Br Heart J 1992;67:92–96. 367 Markussis V, Beshyah SA, Fischer C et al. Detection of premature atherosclerosis by high-resolution ultrasonography in symptom-free hypopituitary adults. Lancet 1992;340:1188–1192. 368 Johansson JO, Landin K, Tengborn L et al. High fibrinogen and plasminogen activator inhibitor activity in growth hormone-deficient adults. Arterioscler Thromb 1994;14:434–437. 369 Fazio S, Sabatini D, Capaldo B et al. A preliminary study of growth hormone in the treatment of dilated cardiomyopathy. N Engl J Med 1996;334:809–814. 370 Johannsson G, Bengtsson BA, Andersson B et al. Long term cardiovascular effects of growth hormone treatment in GH-deficient adults. Preliminary data in a small group of patients. Clin Endocrinol (Oxf.) 1996;45:305–314. 371 Smallridge RC, Rajfer S, Davia J, Schaaf M. Acromegaly and the heart. Am J Med 1979;66:22–27. 372 Kowarski A, Thompson RG, Migeon CJ, Blizzard RM. Determination of integrated plasma concentrations and true secretion rates of human growth hormone. J Clin Endocrinol Metab 1971;32:356–360. 373 Thompson RG, Rodriguez A, Kowarski AA, Blizzard RM. Growth hormone: metabolic clearance rates, integrated concentrations and production rates in normal adults and the effect of prednisone. J Clin Invest 1972;51: 3193–3199. 374 Evans WS, Faria ACS, Christiansen E et al. Impact of intensive venous sampling on characterization of pulsatile GH release. Am J Physiol 1987;252:E549–E556. 375 Rose SR, Ross JL, Uriarte M et al. The advantage of measuring stimulated as compared with spontaneous growth hormone levels in the diagnosis of growth hormone deficiency. N Engl J Med 1988;319:201–207. 376 Donaldson DL, Hollowell JG, Pan F et al. Growth hormone secretory profiles: variation on consecutive nights. J Pediatr 1989;115:51–56. 377 Hoffman DM, O’Sullivan AJ, Baxter RC, Ho KY. Diagnosis of growth hormone deficiency in adults. Lancet 1994;343:1064–1068. 378 Hourd P, Edwards R. Current methods for the measurement of GH in urine. Clin Endocrinol 1996;40:155–170. 379 Hashida S, Ishikawa E, Nakagawa K et al. Demonstration of human growth hormone in normal urine by a highly specific and sensitive sandwich enzyme immunoassay. Anal Lett 1985;18:1623–1634.

116

SECTION 1

Hypothalamic–Pituitary Function

380 Hourd P, Edwards R. Current methods for the measurement of growth hormone in urine. Clin Endo 1994;40(2):155–170. 381 Albini CH, Quattrin T, Vandlen RL, McGillivray MH. Quantitation of urinary growth hormone in children with normal and subnormal growth. Pediatr Res 1988;23:89–92. 382 Hattori N, Shimatsu A, Yamanaka C et al. Nocturnal urinary growth hormone excretion in children with short stature. Acta Endocrinol (Copenh.) 1988;119:113–117. 383 Granada ML, Sanmarti A, Lucas A et al. Clinical usefulness of urinary growth hormone measurements in normal and short children according to different expressions of urinary growth hormone data. Pediatr Res 1992;32:73–76. 384 Rosenfeld RG, Albertsson-Wilkland K, Cassorl F et al. Diagnostic controversy: The diagnosis of childhood deficiency revisited. JCEM 1995;80:1532–1540. 385 Bercu BB, Shulman D, Root AW, Spiliotis BE. Growth hormone (GH) provocative testing frequently does not reflect endogenous GH secretion. J Clin Endocrinol Metab 1986;63:709–716. 386 Lunt H, Tucker AJ, Bullen H et al. Overnight urinary growth hormone measurement in the diagnosis of acromegaly. Clin Endocrinol 1990;33: 205–212. 387 Clemmons DR, Van Wyk JJ, Ridgway EC et al. Evaluation of acromegaly by radioimmunoassay of somatomedin-C. N Engl J Med 1979;301:1138–1142. 388 Barkan AL, Beitins IZ, Kelch RP. Plasma insulin-like growth factorI/Somatomedin-C in acromegaly: correlation with the degree of growth hormone hypersecretion. J Clin Endocrinol Metab 1988;67:69–73. 389 Melmed S, Jackson I, Kleinberg D et al. Current treatment guidelines for acromegaly. JCEM 1998;83:2646–2652. 390 Earll J, Sparks LL, Forsham PH. Glucose suppression of serum growth hormone in the diagnosis of acromegaly. JAMA 1967;201:628–630. 391 Beck P, Parker ML, Daughaday WH. Paradoxical hypersecretion of growth hormone in response to glucose. J Clin Endocrinol Metab 1966;26:463–469. 392 Phillips LS, Vassilopoulou-Sellin R. Somatomedins. Part I. N Engl J Med 1980;302:371–380. 393 Phillips LS, Vassilopoulou-Sellin R. Somatomedins. Part II. N Engl J Med 1980;302:438–446. 394 Dean HJ, Kellet JG, Bala RM et al. The effect of growth hormone treatment on somatomedin levels in growth hormone deficient children. J Clin Endocrinol Metab 1982;55:1167–1173. 395 Bala RM, Lopatka J, Leung A et al. Serum immunoreactive somatomedin-C levels in normal adults, pregnant women at term, children at various ages and children with constitutional delayed growth. J Clin Endocrinol Metab 1981;52:508–512. 396 Rubin KR, Lichtenfels JM, Ratzan SK et al. Relationship of somatomedin-C concentration to bone age in boys with constitutional delay of growth. Am J Dis Child 1986;19:975–980. 397 Reiter EO, Lovinger RD. The use of commercially available somatomedin-C radioimmunoassay in patients with disorders of growth. J Pediatr 1981;99: 720–724. 398 Blum WF, Ranke MB, Kietzmann K et al. A specific radioimmunoassay for growth hormone (GH)-dependent somatomedin-binding protein: its use for diagnosis of GH deficiency. J Clin Endocrinol Metab 1990;70:1292–1298. 399 Hasegawa Y, Hasegawa T, Aso T et al. Usefulness and limitation of measurement of insulin-like growth factor binding protein 3 for diagnosis of GH deficiency. Endocrinol Jpn 1992;39:585–591. 400 Hasegawa Y, Hasegawa T, Kotch S, Tsuchiya Y. Reproducibility of GH stimulation tests (arginine and insulin) IGF-1 and IGFBP-3 measurements. Clin Pediatr Endocrinol 2 1993;(Suppl):75–78. 401 Growth Hormone Research Society. Consensus guidelines for the diagnosis and treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society workshop on Adult Growth Hormone Deficiency. J Clin Endocrinol Metab 1998;83:379–381. 402 Gil-Ad I, Topper E, Laron Z. Oral clonidine as a growth hormone stimulation test. Lancet 1979;2:278–279. 403 Gil-Ad I, Gurewitz R, Marcovici O et al. Effects of aging on plasma GH response to aging. Mech Aging Dec 1984;27:97–100. 404 Fass B, Lippe BM, Kaplan SA. Relative usefulness of three growth hormone stimulation screening tests. Am J Dis Child 1979;133:931–933. 405 Ghigo E, Bellone J, Mazza E et al. Arginine potentiates the GHRH but not the pyridostigmine-induced GH secretion in normal short children. Further evidence for a somatostatin suppressing effect of arginine. Clin Endocrinol (Oxf.) 1990;32:763–767. 406 Aimaretti G, Corneli G, Razzore P et al. Comparison between insulininduced hypoglycemia and GHRH plus Arginine as provocative tests for the diagnosis of GH deficiency in adults. JCEM 1998;83:1615–1619.

407 Popovic V, Leal A, Dragan M et al. Lancet 2000;356:1137–1142. 408 Ho KKY. Diagnosis of adult GH deficiency. Lancet 2000;356:1125–1126. 409 Penny R, Blizzard RM, Davis WT. Sequential arginine and insulin tolerance tests on the same day. J Clin Endocrinol Metab 1969;29:1499–1501. 410 Reiter EO, Morris AH, Biggs DE. Modulation of GHRH-induced growth hormone release by an alpha-adrenergic agonist and hypoglycemia. J Pediatr Endocrinol 1988;3:21–25. 411 Shibasaki T, Hotta M, Masuda A et al. Plasma GH response to GHRH and insulin-induced hypoglycemia in man. J Clin Endocrinol Metab 1985;60: 1265–1267. 412 Woolf PD, Lantigua R, Lee LA. Dopamine inhibition of stimulated growth hormone secretion: evidence for dopaminergic modulation of insulin and L-dopa-induced growth hormone secretion in man. J Clin Endocrinol Metab 1979;49:326–330. 413 Weldon VV, Gupta SK, Klingensmith G et al. Evaluation of growth hormone release in children using arginine and L-dopa in combination. J Pediatr 1975;87:540–544. 414 Cacciari E, Tassoni P, Cigognani A et al. Value and limits of pharmacological and physiological test to diagnose GH deficiency and predict therapy response: first and second retesting during replacement therapy of patients defined as GH deficient. JCEM 79:1663–1669. 415 Sperling MA, Kenny FM, Drash AL. Arginine-induced growth hormone responses in children: effect of age and puberty. J Pediatr 1970;77: 462–465. 416 Illig R, Bucher H. Testosterone priming of growth hormone release. In: Laron Z, Butenandt O, eds. Evaluation of Growth Hormone Secretion. Basel: Karger, 1983:75–83. 417 MacGillivray MH, Aceto T Jr, Frohman LA. Plasma growth hormone responses and growth retardation of hypothyroidism. Am J Dis Child 1968;115:273–276. 418 Tauber M, Moulin P, Pierkowski C et al. GH retesting and auxiological data in 131 GH deficient patients after completion of treatment. JCEM 1997;82: 352–356. 419 Granada ML, Sanmarti A, Lucas A et al. Assay-dependent results of immunoassayable spontaneous 24 hr GH secretion in short children. Acta Ped Scand 1990;370(suppl):63–70. 420 Baumann G, Shaw MA, Amburn K et al. Heterogeneity of circulating growth hormone. Nucl Med Biol 1994;21:369–379. 421 Reiter EO, Morris AH, MacGillivray MH, Weber D. Variable estimates of serum GH concentrations by different radioassay systems. JCEM 1988;66: 68–71. 422 Orskov H, Weeke J, Frystyk J, Kaal A, Nielsen S et al. Growth hormone determination in serum from patients with growth disorders; in Juul A, Jorgenson JOL eds. Growth Hormone in Adults: Physiological and Clinical Aspects. Cambridge: Cambridge University Press, 1996:109–121. 423 Bristow AF, Gaines Das R, Jeffcoate SL, Schulster D. The first international standard for somatotropin: Report of an international collaborative study. Growth Regul 1995;5:133–141. 424 Fisker S, Orskov H. Factors modifying GH estimates in Immunoassays. Horm Res 1996;46:183–187. 425 Strasburger CJ, Dattani MT. New growth hormone assays: potential benefits. Acta Pediatric 1997;423(Suppl):5–11. 426 Goeddel DV, Heyneker HL, Hozumi T et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 1979;281:544–548. 427 Hintz RL, Rosenfeld RG, Wilson DM et al. Biosynthetic methionyl human growth hormone is biologically active in adult man. Lancet 1982;i:1276. 428 Vance ML, Mauras N. Drug therapy: growth hormone therapy in adults and children. N Engl J Med 1999;341:1206–1216. 429 Guidelines for the use of growth hormone in children with short stature; a report by the Drug and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society. L Pediatr 1995;127:857–867. 430 Joss E, Zuppinger K, Schwartz HP, Roten H. Final height of patients with pituitary growth failure and changes in growth variables after long-term hormonal therapy. Pediatr Res 1983;17:676–679. 431 Maes M, Lindberg A, Price DA et al. Long term growth response to growth hormone therapy in prepubertal children with idiopathic growth hormone deficiencies: analysis of Kabi International growth study. Kabi International Growth Study Biannual Report No 11. Mannheim, Germany: J and J Verlag, 1994:15–26. 432 Preece MA, Tanner JM, Whitehouse RH, Cameron N. Dose dependence of growth response to human growth hormone in growth hormone deficiency. J Clin Endocrinol Metab 1976;42:477–483.

Chapter 3 433 Loche S, Cambiaso P, Setzu S et al. Final height after growth hormone therapy in non-growth hormone deficient children with short stature. J Pediatr 1994;125:196–200. 434 Hintz RL, Attie KM, Baptista J, Roche A. Effect of growth hormone treatment on adult height of children with idiopathic short stature. N Engl J Med 1999;340:502–507. 435 Cuttler L, Silvers JB, Sing J et al. Short stature and growth hormone therapy: a national study of physician recommendation patterns. JAMA 1996; 276:531–537. 436 Spiliotis BE, August GP, Hung W et al. Growth hormone neurosecretory dysfunction: a treatable cause of short stature. JAMA 1984;251:2223–2230. 437 Bercu BB, Diamond F. Growth hormone neurosecretory dysfunction. J Clin Endocrinol Metab 1986;15:537–590. 438 Lyon AJ, Preece MA, Grant DB. Growth curve for girls with Turner syndrome. Arch Dis Child 1985;60:932–935. 439 Stahnke N. hGH treatment in short children without growth hormone deficiency. N Engl J Med 1984;310:925–926. 440 Rosenfeld RG, Attie KM, Frane J et al. Growth hormone therapy for Turner’s syndrome: beneficial effect on adult height. J Pediat 1998;132:319–324. 441 Davies PSW, Valley R, Preece MA. Adolescent growth and pubertal progression in the Silver-Russel syndrome. Arch Dis Child 1988;63: 130–135. 442 Lanes R, Plotnick LP, Lee PA. Sustained effect of human growth hormone therapy on children with intrauterine growth retardation. Pediatrics 1979;63:731–735. 443 Stanhope R, Ackland F, Hamill G et al. Physiological growth hormone secretion and response to growth hormone treatment in children with short stature and intrauterine growth retardation. Acta Paediatr Scand 1989;349(suppl.):47–52. 444 de Zegher F, Maes M, Gargosky SE et al. High dose growth hormone treatment of short children born small for gestational age. JCEM 1996;81:1887–1892. 445 Hokken-Koelega AC, Stijnen T, de Jong RC et al. A placebo-controlled, double-blind trial of growth hormone treatment in prepubertal children after renal transplant. Kidney Int Suppl 1996;53:S128–S134. 446 Fine RN. Recombinant human growth hormone in children with chronic renal insufficiency-clinical update: 1995. Kidney Int Suppl 1996;53: S115–S118. 447 Bridges NA, Brook CGD. Growth hormone in skeletal dysplasia. Horm Res 1994;42:231–234. 448 Rudman D. Growth hormone, body composition and aging. J Am Geriatr Soc 1985;33:800–807. 449 Rudman D, Feller AG, Nagraj HS et al. Effects of human growth hormone in men over 60 years old. N Engl J Med 1990;323:1–6. 450 Binnerts A, Wilson JHP, Lamberts SWJ. The effects of human growth hormone administration in elderly adults with recent weight loss. J Clin Endocrinol Metab 1988;67:1312–1316. 451 Marcus R, Butterfield G, Holloway L et al. Effects of short-term administration of recombinant human growth hormone to elderly people. J Clin Endocrinol Metab 1990;70:519–527. 451a Cuneo RC, Salomon F, Macgauley GA, Sonksen PH. The growth of hormone deficiency syndrome in adults. Clin Endocrinol 1992;37:387–397. 452 Hoffman DM, O’Sullivan AJ, Baxter RC, Ho KKY. Diagnosis of GH deficiency in adults. Lancet 1994;343:1064–1068. 453 Nicolson A, Toogood AA, Rahim A, Shalet SM. The prevalence of severe growth hormone deficiency in adults who received growth hormone replacement in childhood. Lancet 1996;44:311–316. 454 Cook DM, Ludlam WH, Cook MB. Route of estrogen administration helps to determine GH replacement dose in GH deficient adults. J Clin Endocrinol Metab 1999;84:3956–3960. 455 Jenkins RC, Ross RJM. Growth hormone therapy for protein catabolism. QJ Med 1996;89:813–819. 456 Manson JM, Wilmore DW. Positive nitrogen balance with human growth hormone and hypocaloric intravenous feeding. Surgery 1986;100:188–197. 457 Ponting GA, Halliday D, Teale JD, Sim AJW. Postoperative positive nitrogen balance with intravenous hyponutrition and growth hormone. Lancet 1988;i:438–440. 458 Herndon DN, Barrow RE, Kunkel KR et al. Effects of recombinant human growth hormone on donor-site healing in severely burned children. Ann Surg 1990;212:424–429. 459 Knox JB, Demling RH, Wilmore DW et al. Increased survival after major thermal injury: the effect of GH therapy in adults. J Trauma 1995;39:526–530.

Growth Hormone

117

460 Pape GS, Friedman M, Underwood LE et al. The effect of GH on weight gain and pulmonary function in patients with chronic obstructive lung disease. Chest 1991;99:1495–1500. 461 Tayek JA, Brasel JA. Failure of anabolism in malnourished cancer patients receiving growth hormone: a clinical research center study. J Clin Endocrinol Metab 1995;80:2082–2087. 462 Knox JB, Wilmore DW, Demling RH et al. Use of GH for postoperative respiratory failure. Ann J Surg 1996;171:576–580. 463 Takada J, Ruokonen E, Webster N et al. Increased mortality associated with growth hormone treatment in critically ill adults. N Engl J Med 1999;341:785–792. 464 Johansson AG, Lindh E, Blum WF et al. Effects of growth hormone and IGF in men with idiopathic osteoporosis. J Clin Endocrinol Metab 1996;81:44 –48. 465 Gonnelli S, Cepollaro C, Montomoli M et al. Treatment of postmenopausal osteoporosis with recombinant human growth hormone and salmon calcitonin: a placebo controlled study. Clin Endocrinol 1997;46:55–61. 466 Holloway L, Kohlmeier L, Kent K et al. Skeletal effects of cyclic recombinant human growth hormone and salmon calcitonin in osteoporotic postmenopausal women. J Clin Endocrinol Metab 1997;82:1111–1117. 467 Deal CL. Osteoporosis: prevention, diagnosis and management. Am J Med 1997;102:355–395. 468 Mulligan K, Grunfeld C, Hellerstein MK et al. Anabolic effects of recombinant human growth hormone in patients with wasting associated with HIV infection. J Clin Endocrinol Metab 1993;77:956–962. 469 Schambelan M, Mulligan K, Grunfield C et al. A controlled trial of recombinant human growth hormone in patients with HIV associated growth hormone wasting. Ann Inter Med 1996;125:873–882. 470 Osterziel KJ, Strohm O, Schuler J et al. Randomized, double-blinded, placebo-controlled trial of human recombinant growth hormone in patients with chronic heart failure due to dialated cardiomyopathy. Lancet 1998;351:1233–1237. 471 Hollander DM, Devereux DF, Marafino BJ, Hoppe H. Increased wound breaking strength in rats following treatment with synthetic human growth hormone. Surg Forum 1984;35:612–614. 472 Jorgensen PH, Andreassen TT, A dose-response study of the effects of biosynthetic human growth hormone on formation and strength of granulation tissue. Endocrinology 1987;121:1637–1641. 473 Belcher HJCR, Ellis H. Somatotropin and wound healing. J Clin Endocrinol Metab 1990;70:939–943. 474 Homburg R, Levy T, Ben-Rafael Z. Adjuvant growth hormone for induction of ovulation with GnRH agonist and gonadotropins in polycystic ovary syndrome: a randomized, doubleblind, placebo controlled trial. Hum reprod 1995;10:2550–2553. 475 Homburg R, Eshel A, Abdalla HI et al. Growth hormone facilitates ovulation induction by gonadotropins. Clin Endocrinol 1988;29:113–117. 476 Shoham Z, Conway GS, Ostergard H et al. Cotreatment with growth hormone for induction of spermatogenesis in patients with hypogonadotropic hypogonadism. Fertil Steril 1992;57:1044–1051. 477 Bergh C, Hillensjo T, Wikland M et al. Adjuvant growth hormone treatment during in vitro fertilization: a randomized, placebo controlled study. Fert Steril 1994;62:113–120. 478 Crist DM, Peake GT, Egan PA, Waters DL. Body composition response to exogenous GH during training in highly conditioned adults. J Appl Physiol 1988;65:579–584. 479 DeBoer H, Blok GJ, Van Der Veen EA. Clinical aspects of growth hormone deficiency in adults. Endo Rev 1995;16:63–86. 480 Melmed S. Acromegaly and cancer: not a problem? J Clin Endocrinol Metab 2000;86:2929–2934. 481 Shalet SM, Renehan AG, Painter JE et al. Circulating insulin-like growth factor II and colorectal adenomas. JCEM 2000;85(9):3402–3408. 482 Besser M, Jenkins PJ, Fairclough PD et al. Acromegalic, colonic polyps and carcinoma. Clinica Endocrinol 1997;47(1):17–22. 483 Daughaday WH. The possible autocrine/paracrine and endocrine roles of insulin-like growth factors of human tumors. Endocrinology 1990;127:1–4. 484 Stahnke N, Zeisel HJ. Growth hormone therapy and leukemia. Eur J Pediatr 1989;148:591–596. 485 Chan JM, Stampfer MJ, Giovannucci E et al. Circulating insulinlike growth factor I and prostate cancer risks a prospective study. Science 1998;279: 563–566. 486 Hankinson S, Pollak M, Michaud D et al. A prospective assessment of plasma insulin-like growth factor levels and breast cancer risk. Am J Epidemiol 1997;145:s72. 487 Cahill GR, Herrera MG, Morgan AP et al. Hormone-fuel interrelationships during fasting. J Clin Invest 1966;45:1751–1769.

118

SECTION 1

Hypothalamic–Pituitary Function

488 Grant DB, Hambley J, Becker D, Pimstone BL. Reduced sulfation factor in undernourished children. Arch Dis Child 1973;48:596–600. 489 Isley WL, Underwood LE, Clemmons DR. Dietary components that regulate serum somatomedin-C concentrations in humans. J Clin Invest 1983;71: 175-182. 490 Brown GM, Garfinkel PE, Jeuniewic N et al. Endocrine profiles in anorexia nervosa. In: Vigersky RA, ed. Anorexia Nervosa. New York: Raven Press, 1977:123–125. 491 Casper RC, Davis JM, Pandey GN. The effect of the nutritional status and weight changes on hypothalamic function tests in anorexia nervosa. In: Vigersky RA, ed. Anorexia Nervosa. New York: Raven Press, 1977:137–147. 492 Macaron C, Wilber JF, Green O, Freinkel N. Studies of growth hormone (GH), thyrotropin (TSH) and prolactin (PRL) secretion in anorexia nervosa. Psychoneuroendocrinology 1978;3:181–185. 493 Hansen AP. Abnormal serum growth hormone response to exercise in juvenile diabetics. J Clin Invest 1971;49;1467–1478. 494 Dasmahapatra A, Urdanivia E, Cohen MP. Growth hormone response to thyrotropin-releasing hormone in diabetes. J Clin Endocrinol Metab 1981;52:859–862. 495 Hansen AP, Johansen K. Diurnal patterns of blood glucose, serum free fatty acids insulin, glucagon and growth hormone in normal and juvenile diabetics. Diabetologia 1970;6:27–33. 496 Bach MA, Chin E, Bondy CA. The effects of subcutaneous insulin-like growth factor-I infusion in insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1994;79:1040–1045. 497 Moller DE, Flier JS. Insulin resistance-mechanisms, syndromes and implications. NEJM 1991;325:938–948. 498 Zenobi PD, Jaeggi-Groisman SE, Riesen WF et al. Insulin-like growth factor-I improves glucose and lipid metabolism in type 2 diabetes mellitus. J Clin Invest 1992;90:2234–2241. 499 Jabri N, Schalch DS, Schwartz SL et al. Adverse effects of recombinant human insulin-like growth factor I in obese insulin-resistant type II diabetic patients. Diabetes 1994;43:369–374.

500 Laron Z, Pertzelan A, Karp M et al. Administration of growth hormone to patients with familial dwarfism with high plasma immunoreactive growth hormone measurement of sulfation factor, metabolic and linear growth response. J Clin Endocrinol Metab 1971;33:332–342. 501 Godowski PJ, Leung DW, Meacham LR et al. Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci USA 1989;86:8083–8087. 502 Laron Z, Anin S, Klipper-Aurbach Y, Klinger B. Effects of insulin-like growth factor on linear growth, head circumference, and body fat in patients with Laron-type dwarfism. Lancet 1992;339:1258–1261. 503 Samaan NA, Freeman RM. Growth hormone levels in severe renal failure. Metabolism 1970;19:102–113. 504 Andress DL, Pandian MR, Endres DB, Kopp JB. Plasma insulin-like growth factors and bone formation in uremic hyperparathyroidism. Kidney Int 1989;36:471–477. 505 Goldberg A, Trivedi B, Delmez J et al. Uremia reduces insulin-like growth factor-I, increases insulin-like growth factor-II and modifies their serum protein binding. J Clin Endocrinol Metab 1982;55: 1040–1045. 506 Powell DR, Rosenfeld RG, Baker BK et al. Serum somatomedin levels in adults with chronic reneal failure: the importance of measuring insulin-like growth factor-I (IGF-I) and IGF-II in acid-chromatographed uremic serum. J Clin Endocrinol Metab 1986;63:1186–1192. 507 Daughaday WH, Mariz IK, Blethen SL. Inhibition of access of somatomedin to membrane receptor and immunobinding sites: comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J Clin Endocrinol Metab 1980;51:781–788. 508 Daughaday WH, Kapadia M, Mariz I. Serum somatomedin binding proteins: physiologic significance and interference of radioligand assay. J Lab Clin Med 1987;109:355–363. 509 Saenger P, Wiedermann E, Schwartz E et al. Somatomedin and growth after renal transplantation. Pediatr Res 1974;8:163–169.

C h a p t e r

4 Prolactin Mark E. Molitch

HISTORICAL OVERVIEW In the late 1920s and early 1930s, a number of groups found that pituitary extracts could induce milk secretion [1–3]. Riddle and coworkers found that this substance, which they named prolactin (PRL), could be differentiated from the known growth and gonad stimulating substances [3–5]. In these experiments, they showed that PRL stimulated milk production by guinea pig mammary glands and a milk-like substance from the crop sacs of pigeons and doves [3–6]. The pigeon crop sac assay for PRL [3–6] then became the standard assay procedure for PRL and was used in many experiments over the next 30 years. Over the ensuing years, PRL was characterized, sequenced and specific radioimmunoassays (RIAs) developed for PRL from a number of species [7–10]. Because of the high lactogenic activity of even very highly purified preparations of human growth hormone (GH) [11,12], however, it was impossible to separate human PRL from GH using the relatively crude pigeon crop assay and the evaluation of PRL physiology in humans proceeded slowly. Even the existence of a PRL separate from GH in humans was questioned [12]. Several experiments of nature provided strong evidence, however, that even in humans these two hormones were separate. For example, it was observed that most patients with pituitary tumors in whom galactorrhea and amenorrhea were the cardinal clinical features did not have acromegalic features [13] and patients who were known to have isolated, congenital GH deficiency were able to undergo postpartum lactation [14]. Finally, in 1970, Frantz and Kleinberg developed a sensitive in vitro bioassay which involved staining milk produced by cultured, lactating mouse mammary tissue in response to PRL that was capable of measuring ovine PRL levels as low as 5 ng/ml. In this

assay they added excess antibody to GH to neutralize any potential lactogenic effects it had and, for the first time, were able to demonstrate measurable PRL levels in women with puerperal and nonpuerperal galactorrhea but not in most normal men and women [15]. Shortly thereafter, still using the pigeon crop assay, Lewis et al. reported considerable progress in the purification of human PRL and its separation from GH via several sequential separation techniques, leading the way to the eventual development of heterologous [16] and homologous [17] RIAs for human PRL which could finally measure PRL levels in the sera of normal individuals. The RIA for human PRL finally permitted the eventual sequencing of human PRL [18] and determination of its cDNA sequence [19]. With the development of PRL RIAs, studies of PRL physiology and pathophysiology in animals and humans have progressed at an astonishing pace over the past 20 years.

CELL OF ORIGIN

Lactotroph Morphology PRL is made by the pituitary lactotrophs (also known as mammotrophs). In the normal human pituitary, the lactotrophs comprise about 15% to 25% of the total number of cells, are similar in number in both sexes and do not change significantly with age [20–22]. During pregnancy and subsequent lactation, however, considerable lactotroph hyperplasia may be found [23,24], presumably due to the stimulatory effect of the hormonal milieu of pregnancy (see below for a discussion of the effects of estrogen on PRL secretion). The hyperplastic process involutes within several months after delivery, although breast-feeding retards this process [24]. This stimulatory effect of pregnancy on the lactotrophs also holds true for prolactinomas and very 119

120

SECTION 1

Hypothalamic–Pituitary Function

significant pregnancy-induced tumor enlargement may occur (see Chapter 12, p. 483). The lactotrophs are of two types: (i) large, polyhedral elongated cells distributed throughout the gland but generally near capillaries; and (ii) small-to-medium-sized, angular or elongated cells appearing in clusters on the border between the lateral wings and the median wedge and in the posterolateral edges of the gland [25]. The larger cells correspond to the densely granulated (granules 250–800 nm in diameter) cells seen on electron microscopy that are thought to be in a resting, storing stage while the smaller cells correspond to the sparsely granulated (granules 200– 350 nm in diameter) cells seen on electron microscopy that are actively secreting hormone [22,25]. Within these cells, PRL synthesis on the ribosomes of the rough endoplasmic reticulum, packaging into secretory granules within the Golgi, and release into the perivascular space by exocytosis appears to proceed in a normal fashion similar to that of other secretory proteins [25,26], although exocytosis may also occur at the lateral cell membranes, a phenomenon referred to as “misplaced exocytosis” [25]. Although it is possible that the densely and sparsely granulated lactotrophs represent different cell types, it is more likely that they represent the same cells in different phases of storage and secretion [22]. Most PRL is secreted by lactotrophs in the normal individual; however, there is a small proportion that is secreted by mammosomatotrophs, that are also capable of secreting GH [20,22,27]. In these cells some secretory granules contain GH, some PRL and some both hormones [27]. The ability of individual adenohypophyseal cells to secrete PRL in culture can be assessed using the reverse hemolytic plaque assay (Fig. 4.1) [28]. In this assay, the PRL secreted by individual cells is ascertained by identifying zones of hemolysis (plaques) around individual lactotrophs when

incubated in a monolayer with staphylococcal protein-Acoated ovine erythrocytes in the presence of PRL antiserum and complement [28]. Initial experiments with this system revealed that stimulatory agents such as thyrotropin releasing hormone (TRH) could cause an increase in plaque number and size, and inhibitory agents such as dopamine (DA) could cause a decrease in plaque number and size [28]. Subsequent studies have shown that there is considerable heterogeneity in the responsiveness of lactotrophs to such stimuli. A subpopulation of lactotrophs, comprising about 65% of cells, forms large plaques and is readily suppressible by DA; the remaining cells form small plaques and are not suppressible by DA [29]. In other studies, only one-third of lactotrophs were shown to respond to TRH [30]. Further analysis revealed that cells taken from the peripheral rim of the pituitaries of lactating rats were greatly responsive to TRH but poorly responsive to DA, whereas cells from the central region of the pituitary were little influenced by TRH but highly suppressible by DA [31]. The reverse hemolytic plaque assay, applied in a sequential fashion to PRL and GH, reveals that in normal rats of the pituitary cells secreting both GH and PRL, about one-third secrete GH alone, one-third secrete PRL alone and onethird secrete both hormones (i.e. mammosomatotrophs) [32], although mammosomatotrophs were found in only 5% of cells in another study [33]. In neonatal rats, the proportion of these cells that secrete only PRL was only 1.7%, suggesting that PRL-secreting cells arise from GH-secreting cells, i.e. mammosomatotrophs [34]. Tissue obtained near the periphery contained a higher proportion of cells that produced both PRL and growth hormone (GH) than did tissue obtained from the central region of the pituitary [31]. Estradiol treatment causes some cells that had only secreted GH to also secrete PRL, i.e. they converted from somatotroph

FIGURE 4.1. Reverse hemolytic plaque assay. In this assay, the prolactin secreted by individual cells is ascertained by identifying these zones of hemolysis (plaques) when incubated in a monolayer with staphylococcal protein A-coated ovine erythrocytes in the presence of PRL antiserum and complement. From Neill and Frawley [28]

Chapter 4

to mammosomatotroph cells [35]. Added GH releasing hormone (GHRH) and gonadotropin releasing hormone (GnRH) caused an increase in the proportion of PRL secretors and a decrease in the proportion of GH secretors, but corticotropin releasing hormone (CRH) increased the proportion of PRL secretors and did not affect GH secretors [36]. As yet, few studies have been performed with the reverse hemolytic plaque assay with human pituitary tissue. In one study of human fetal pituitary tissue (gestational age 18 to 22 weeks), 8% of cells formed PRL plaques [37], a smaller number than would expected from studies of adult pituitaries (see above). Of cells secreting either GH or PRL, 70% secreted GH only, 9% secreted PRL only and 21% secreted both hormones [37]. If we assume that heterogeneity of basal secretion, the response to various stimulatory and suppressive substances, and the ability of the cell population to shift between GH, PRL and GH plus PRL secretion all also take place in the adult human, such findings have obvious importance with regard to the pathogenesis of prolactinomas. For example, such tumors may arise from cells that are minimally or not responsive to endogenous inhibition by DA and thus may explain the variability of suppression of PRL by tumors in response to dopaminergic agents such as bromocriptine.

Lactotroph Ontogeny Continuous hypothalamic–pituitary interaction takes place during embryologic development. During the formation of Rathke’s pouch, the primordium of the anterior pituitary, the ectodermal primordial cells of the anterior and intermediate lobes of the pituitary make contact with the neuroectoderm of the floor of the diencephalon and experimental studies have shown inductive interactions between these tissues that are necessary for their subsequent interdependent development [38]. Elegant studies using a series of targeted mutations have shown that there is a whole host of homeodomain transcription factors that are sequentially expressed in the developing hypothalamus and pituitary that lead to the final determination of the five mature pituitary cell types and the functional integration of the hypothalamic–pituitary system [38,39]. In the earliest phase, in which Rathke’s pouch first appears, the transcription factors Six-3 and Hesx1 (also known as Rpx) are necessary for the initial development of Rathke’s pouch and other midline brain structures [38,39]. In humans, mutations in the genes for these factors result in defects in the development of midline structures and failure of normal hypothalamic and pituitary development with clinical hypopituitarism [40,41]. The LIM homeobox gene Lhx3 begins to be expressed in Rathke’s pouch at the time of organ commitment, in part stimulated by bone morphogenic protein 4 (BMP4) that is expressed in the ventral diencephalon [39] and inactivation/deletion of both the BMP4 and Lhx3 genes in mice causes a failure of the pitu-

Prolactin

121

itary to develop, with the exception of the corticotroph lineage, despite the formation of Rathke’s pouch [39,42]. Lhx3 can synergize with Pit-1 in the transcriptional activation of the gene that encodes PRL and is required for the eventual proliferation and determination of the lactotroph, thyrotroph, gonadotroph, and somatotroph lineages [43]. Another homeobox-containing transcription factor gene, PTX1/P-OTX, is expressed in Rathke’s pouch early but later is restricted to corticotrophs and its role in the development of lactotrophs is still unclear [38]. A second BMP, BMP2, is transiently expressed in Rathke’s pouch and its temporal rise and fall both seem to be necessary for terminal pituitary cell differentiation including specification of the Pit-1 lineage [39]. Fibroblast growth factor 8 (FGF8) and Wnt4 have also been determined as important in the proliferation of the various pituitary cell types [39]. The POU homeodomain transcription factor Pit-1 gene (see below) becomes activated close to the same time as the Lhx3 gene and is also necessary for the activation of the PRL, GH, GHRH receptor, and TSHb genes as well as being necessary for the differentiation and proliferation of these cell lineages. Pit-1 also activates the c-fos promoter and Fos is known to be involved in cell cycle initiation [44]. A point mutation in the POU homeodomain of Pit-1 has been found to be the cause of the GH, PRL, and TSH deficiencies found in the Snell dwarf mouse, with absence of somatotroph, lactotroph, and thyrotroph cells [45]. Similar mutations in the POU-specific domain [46] and the POU homeodomain [47] of Pit-1 have now been found to cause a similar deficiency of GH, PRL and TSH in humans. A second paired-like homeodomain factor, known as Prophet of Pit-1 (Prop-1) has also been found to be necessary for the expression of Pit-1 and mutations of the Prop-1 gene causes the dwarfism in mice known as the Ames mouse (defects of somatotrophs, lactotrophs and thyrotrophs) [48] and similar mutations in humans cause variable deficiencies of GH, PRL, TSH, LH and FSH [49–51]. Although the glycoprotein a-subunit is the first hormone transcript to be expressed during the development of the pituitary, such cells are not the precursor of subsequent lactotrophs, as found by targeted ablation of a-subunit expressing cells [52]. Most lactotrophs arise from cells that at least at some point expressed the GH gene [53]. Subsequent lactotroph proliferation occurs once estrogen receptors appear [53]. Estrogen stimulates PRL gene transcription (see below) only if Pit-1 is bound to the PRL promoter [54]. Thus, the division of corticotroph from other cell premordia occurs very early. Stimulation by Prop-1 is necessary for the subsequent development of all noncorticotroph cells and Pit-1 is then necessary for the development and proliferation of thyrotrophs and somatomammotrophs, but the separation of these two cell lines occurs before the appearance of the ability to synthesize a-subunits. The final differentiation of somatomammotrophs occurs at least in part in relationship to estrogen stimulation.

122

SECTION 1

Hypothalamic–Pituitary Function

PROLACTIN GENE STRUCTURE

Chromosome Location The PRL gene in the human is located on chromosome 6, whereas those of the related hormones GH and human placental lactogen (HPL, also known as human chorionic somatomammotropin or HCS) are located on chromosome 17 [55,56]. In the rat, PRL similarly is dispersed to a separate chromosome from its relatives, PRL being located on chromosome 17 and GH on chromosome 10 [57]. These three genes (and possibly others, see below) are thought to have derived by gene reduplication from a common ancestral gene which diverged about 400 million years ago [19,58,59]. In both the human and the rat, the PRL genes and amino acid sequences show less homology with GH than placental lactogen does, implying an earlier divergence of PRL and GH and a more recent divergence of GH and PL [59].

PRL Gene Structure The human PRL (hPRL) and rat PRL (rPRL) genes are approximately 10 kb long from the 5¢ transcription initiation site to the poly A addition site at the 3¢ end and both consist of 5 exons separated by 4 large introns [60,61] (Fig. 4.2). Exon 1 consists of 56 bp that code for a portion of the 5¢ untranslated portion of the messenger RNA (mRNA) and the initial 28 aminoacids of the signal peptide. Exon 2 comprises 56 base pairs coding for the rest of the signal peptide and 120 base pairs coding for the first 40 amino acids of PRL itself. Exons 3 and 4 code for the amino acid sequences 41–76 and 77–136 respectively. Finally, Exon 5 codes for amino acids 137–199, the termination triplet TAA

FIGURE 4.2. details, see text.

and 118 base pairs before the AATAAA polyA signal [61]. The introns have sizes of 1.3, 1.5, 1.9 and 2.0 kb for A through D respectively [44]. The TATA sequence (Goldberg–Hogness box) is located 29 bp upstream of the start site of transcription [61]. The mature PRL mRNA that results after nuclear processing of the original heteronuclear RNA is only about 1 kb long and codes for a 227 amino acid sequence which includes an initial 28 amino acid signal peptide as well as the 199 amino acid structural peptide [19,61,62]. Almost 50% of the protein has an a-helix structure and the mature protein folds in a four-helix bundle similar to GH [63]. The 5¢ flanking region of the PRL genes contains an initial 500 base pairs region with considerable sequence homology between human, rat and bovine species, indicating that possible important regulatory elements are conserved there [61,64]. In the 5¢ flanking region, five highly conserved (79–90% homology between hPRL and rPRL) sequences for PRL are not found in hGH or rGH, indicating several specific sites that might be specific for PRL gene regulation in this regard [61].

5¢ Flanking Region and Tissue Specific Enhancers Detailed analyses of the 5¢ flanking region have been confined to the rat, using such models as the GH3, GH1, and GH4C1 tumor cell lines (Fig. 4.3). Preliminary analyses of the bovine PRL gene suggest common features in the 5¢ flanking regions across species [65], so that the findings to be discussed are likely also present in the human gene, with modifications.

Processing of the human prolactin gene. The exons are denoted by the numbers (1–5) and the introns by letters (A–D). For

Chapter 4

Prolactin

123

FIGURE 4.3. Detailed analysis of the 5¢ flanking region of the rat prolactin gene. ERE, estrogen response element; TRE, thyroid response element. 1P, 2P, 3P, and 4P refer to proximal binding sites for Pit-1; 1D, 2D, 3D and 4D refer to distal binding sites for Pit-1. For details see text.

There are two regions responsible for tissue-specific transcription activation, i.e. that allow expression of the PRL gene solely in the pituitary lactotrophs and not in other tissues; a distal enhancer element in the -1720 to -1580 bp region accounts for 98% to 99% of this activity while 1% to 2% of this activity is located at a more proximal promoter element at -422 to +33 [66,67]. Within these elements there are A,T—rich consensus sequences with a core of T/A T/A T/A ATANCAT that is critical and serves as a binding site for a tissue-specific transcription activator protein, termed Pit-1 [66,67]. Within the proximal region there are four binding sites for Pit-1, referred to as 1P (-38 to -62), 2P (-115 to -130), 3P (-142 to -162), and 4P (-180 to -200); similarly within the distal region the four binding sites for Pit-1 are referred to as 1D (-1550 to -1578), 2D (-1619 to -1636), 3D (-1645 to -1670), and 4D (-1694 to -1718) [67]. Although the distal and proximal regions are each capable of directing cell-specific expression of the PRL gene, they also appear to act synergistically [67]. The transcription enhancement of the PRL gene by the Pit-1 activator protein can be modified by other factors, such as TRH, epidermal growth factor (EGF), cAMP [68,69], and estrogens [54]. Others have also reported similar findings with respect to these distal and proximal sites [70]. Maurer et al. [71] have also found the tissue specific factor to bind to the sequence -1666 to -1659. Lemaigre et al. [72] demonstrated three pituitary specific protein binding sites in the proximal 217 bp of the PRL promoter in the 5¢ flanking region of the human PRL gene. Pit-1 consists of 291 amino acids with a molecular mass of 32.9 kDa [67]. Human Pit-1 cDNA has 85% DNA homology and greater than 95% amino acid sequence homology with the bovine, rat and mouse sequences

[46,73]. Pit-1 is part of the POU-domain gene family in which the DNA binding domain consists of a POUspecific domain and a POU-homeodomain [74]. Pit-1 is expressed in lactotrophs, somatotrophs and thyrotrophs and is the critical cell-specific transcription factor for activating expression of the PRL, GH, and TSH-b genes and for their respective cell development and proliferation [75,76] (see above).

Estrogen Also in the distal enhancer region lies the estrogen-response element, which has been localized to the position -1582 to -1569 [75,77,78]. This region binds the estrogen receptor and is required for the enhanced transcription of the PRL gene due to estrogen stimulation. The base sequence in this region of the rPRL gene represents an imperfect palindrome similar to the perfect conserved palindrome, GGTCANNNTGACC, which has been found to be necessary for the estrogen responsiveness of the vitellogenin gene in other tissues [75,78]. A second possible estrogen response element may also exist at -1722 to -1709 [77,79]. As noted above, estrogen regulates PRL gene transcription only if the PRL promoter is bound by Pit-1 [54].

Thyroid Hormone Two areas of the 5¢ flanking region have been found to be involved in the regulation of PRL gene transcription by thyroid hormones. The region -1565 to -1530 appears to be a stimulatory thyroid hormone response element that is distinct from the estrogen response element and can bind to the thyroid hormone receptor [80]. Day and Maurer [80] demonstrated by mutation analysis that the specific sequence

124

SECTION 1

Hypothalamic–Pituitary Function

of GGTCA at position -1555 to -1551 was shown to be critical and is the only portion of this region which displays sequence identity to a portion of the consensus sequence that has been derived for thyroid hormone response sequences [81]. A proximal region, -176 to -38, is involved in the ability of thyroid hormone to inhibit PRL gene transcription, but the thyroid hormone receptor does not bind to this region and it is likely that this inhibitory activity is indirect via some other trans-acting protein [80,82].

Intracellular Calcium Changes in cytosolic free calcium concentrations mediate the lactotroph responses to a number of extracellular stimuli (see below). A calcium response element has been demonstrated to reside within the initial 174 base pairs of the 5¢ flanking sequence [83]. The effects of intracellular calcium on enhancing PRL gene transcription may, in part, be mediated by the phosphorylation of a nuclear matrix protein kinase, topoisomerase II, by a calcium-calmodulin dependent protein kinase [84]. Four topoisomerase II recognition sequences were found in the 5¢ flanking region, at regions +1500 to +1600, -42 to -63, -1200 to -1300 and -2900 to -3000 base pairs with respect to the transcription start site [84]. Site 2, at -42 to -63 base pairs upstream from the transcription start site is within the region cited above as containing a calcium responsive element. Day and Maurer [85] have also found that both the proximal and distal enhancer regions are involved in mediating the transcriptional response to calcium. As discussed below, the activation of the phosphotidyl inositol pathway is probably a major route of stimulation of PRL gene transcription via an increase in intracellular calcium and the phosphorylation of protein kinase C. Direct stimulation of protein kinase C via phorbol ester causes PRL gene transcription via a regulatory region located at -74 to -30 of the 5¢ flanking region [68].

Glucocorticosteroids The region between -200 and +75 is necessary for glucocorticoid down-regulation of PRL gene transcription in the rat [77]. Sakai et al. [86] have provided evidence in the cow that the glucocorticoid response element (GRE) is a compound element with a constitutive enhancer-like component that activates transcription when glucocorticoids are not present and a sequence in which receptor binding reverses this enhancement. This negative GRE appears to lie between -310 and -152 of the 5¢ flanking region of the bovine PRL gene.

Cyclic AMP The sequence mediating the stimulatory effect of cAMP lies between -127 and +73 [70]. The sequence CCCCTCCC

lying between -78 and -71 in the rat gene has been shown to be a cAMP responsive element which binds to a transactivating factor termed Activator Protein 2 (AP-2) [87–89]. AP-2 has a molecular mass of 50–52 kDa [88]. In addition to this AP-2 binding region, the -97 to -84 region contains the sequence TGACGAAA which is similar to a cAMP response element (CRE) and may bind a CRE binding protein (CREB) [89]. There is also evidence that cAMP induced stimulation of PRL gene transcription may actually occur via the release of an inhibitory element downstream of -127 [70] or via intracellular calcium-dependent events [90]. Stimulation of PRL transcription by cAMP may also occur by augmenting the tissue specific enhancer element, Pit-1, discussed above [88,90].

PROLACTIN GENE REGULATION PRL secretion is influenced by a number of factors. Some of these factors, such as estrogen, have a direct action on the gene to alter transcription. Others, such as TRH and DA, act by binding to specific receptors on the lactotroph membrane, thereby activating intracellular second messenger signal transduction systems. This section will review these direct acting substances and these intracellular signal transduction systems (Fig. 4.4).

Estrogen The hormonal milieu of pregnancy has long been known to be stimulatory to lactotroph growth, causing hyperplasia of the normal pituitary lactotrophs [23,24] and enlargement of prolactinomas [91]. In early animal studies, it was shown that the PRL surge that occurred during proestrus was triggered by the prior estrogen peak [92] and that estrogen administration caused an increase in PRL synthesis [93–95]. Estrogen administration in vivo or when added to cell cultures results in an increase in lactotroph mRNA levels, paralleling the increase in PRL biosynthesis [96–101]. This stimulation of PRL gene transcription is biphasic. The first phase begins within 30 minutes of exposure and peaks within 1 hour, and involves the direct binding of estradiol to the nuclear estrogen receptor and subsequent binding of the estrogen-receptor complex to the specific estrogen response element discussed above. The second phase begins within 6 hours, continues for 24 to 48 hours and is indirect, in that it involves synthesis of a protein intermediate, as shown by this effect being blocked with protein synthesis inhibitors such as cycloheximide [100,102,103]. This second phase may be due to a modulation of the inhibitory effect of DA on PRL gene transcription, which itself is mediated by a decrease in intracellular cAMP [103,104]. Estrogen administration also stimulates mitotic activity, DNA synthesis and lactotroph proliferation [105–108].

Chapter 4

Prolactin

125

FIGURE 4.4. Signal transduction mechanisms involved in prolactin regulation. ER, estrogen receptor; TR, thyroid hormone receptor; GR, glucocorticoid receptor. In cytoplasm: ER, endoplasmic reticulum. For details, see text.

Thyroid Hormone

Adenyl Cyclase and Cyclic AMP

Thyroid hormones have long been known to suppress PRL synthesis and secretion from pituitary cell cultures [109]. Maurer [110] demonstrated a concomitant decrease in PRL mRNA levels, indicating an effect on PRL gene transcription. The roles of direct binding of the thyroid hormonereceptor complex to the distal thyroid hormone response element vs indirect effects via trans-acting factors at the more proximal site mentioned above in this inhibitory effect of thyroid hormone still need to be established.

Increases in intracellular cAMP result in an increase in PRL gene transcription and hormone synthesis and release in some cell systems but not others [103,114–116]. Agents which directly activate adenyl cyclase, such as forskolin, stimulate PRL gene transcription [116,117]. As discussed above, there may be both proximal and distal regions of the 5¢ flanking region that are responsible for the response to cAMP. It is likely that the effects of cAMP on gene transcription occur via the activation of a cAMP-dependent protein kinase and resultant phosphorylation of a specific DNA binding protein (AP-2, see above) associated with the cAMP response element (AP-2 binding site) of the PRL gene [118] or perhaps also via modulation of the effects of Pit-1 [70]. There are likely additional enzymes and proteins similarly phosphorylated that may be involved in eventual secretion of the mature PRL molecule. Furthermore,

Glucocorticoids Glucocorticoids exert an inhibitory action on PRL synthesis [111–113] and gene transcription. As discussed above, the inhibitory action may actually be a turning off of a constitutive enhancer-like component [86].

126

SECTION 1

Hypothalamic–Pituitary Function

protein phosphatases which interrupt signal transduction by decreasing phosphorylation are also important [119]. DA, the primary PRL inhibitory factor (PIF), acts through the D2 DA receptor, in part by inhibiting adenyl cyclase and decreasing intracellular cAMP [120,121]. Maurer found that adding monobutyryl cAMP to ergocryptine-pretreated cells resulted in a stimulation of PRL gene transcription [114] and Anderson et al. [122] were able to reverse the inhibitory effect of DA by adding forskolin, which increases intracellular cAMP levels. However, other experiments have shown that maintenance of intracellular cAMP levels does not abolish the inhibitory effects of DA on PRL release [123,124], so that other pathways may also mediate some of the inhibitory effects of DA. The intracellular phosphoinositide/calcium pathways may well be more important in mediating the inhibitory effect of DA on PRL release (see below). Vasoactive intestinal peptide (VIP) stimulates PRL release primarily through stimulation of adenyl cyclase and the production of intracellular cAMP [125–127]. However, it is unlikely that other secretogogues work through this pathway [128].

Phosphoinositide/Arachidonate Pathways A number of factors affecting PRL secretion act through the phosphoinositide pathways, including TRH, angiotensin II and DA [128]. As in other systems, after hormonereceptor interaction, coupled via a G protein, the initial step is the phospholipase C hydrolysis of phophatidylinositol 4,5biphosphate to yield inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Stimulation causes an increase in IP3 which then causes a rapid release of calcium from the endoplasmic reticulum and an activation of membrane calcium channels to increase transport of extracellular calcium, resulting in a net increase in intracellular calcium content. Changes in intracellular calcium result in a number of further changes (see below), including, with DAG, activation of protein kinase C, which then acts by phosphorylating other proteins. The role of the phosphoinositide-protein kinase C pathway in the regulation of PRL secretion has been investigated in great detail. Protein kinase C is located in the normal pituitary [129], various PRL-secreting rat pituitary lines [130,131], and human pituitary tumors [132]. Activation of protein kinase C by phorbol ester results in PRL gene transcription with an increase in PRL mRNA levels and eventual PRL release from pituitary cells in vitro [133–137]. The mechanism mediating this PRL gene transcription is probably the protein kinase C phosphorylation of intracellular proteins [138,139] that, in turn, act as transacting factors on DNA regulatory elements in the proximal promoter region of the 5¢-flanking region of the PRL gene [140]. The phosphoinositide-protein kinase C pathway is a primary mediator of the stimulatory effects of TRH on PRL

gene transcription and release. TRH results in a rapid, phospholipase C-mediated hydrolysis of phosphotidylinositol with generation of DAG and IP3 [141,142]. The released DAG stimulates protein kinase C, resulting in phosphorylation of intracellular [139] and specifically nuclear, chromatin-binding proteins [138]. DAG probably mediates the second phase of the PRL response to TRH that occurs between 5 and 60 minutes after stimulation [143]. The increase in IP3 stimulates release of intracellular calcium stores [136,144], another mechanism involved in PRL gene transcription and release (see below). In various in vitro preparations, angiotensin II, neurotensin and bombesin have been found to stimulate PRL release in a similar fashion [128]. Studies of the interaction of DA with the pituitary D2 DA receptor and the phosphoinositide-protein kinase C pathway have yielded conflicting results, in part due to differences in cell preparations and experimental designs (reviewed by Lamberts and MacLeod [128]). Because chronic D2 receptor activation results in an attenuation of the phosphorylation of phosphotidylinositides [145], Lamberts and MacLeod [128] have concluded that DA’s inhibitory activity on PRL secretion occurs via this effect on phosphorylation rather than through an effect on phospholipase C. A second pathway involving membrane phospholipids is the arachidonate pathway, although the specifics of this pathway have not been fully elucidated. Stimulation of membrane-bound phospholipase A2 by TRH, angiotensin II, neurotensin and other direct stimulators results in PRL and arachidonic acid release from pituitary cells in vitro [146,147]. This effect of arachidonic acid release is blocked by inhibitors of phospholipase A2 [146] and by DA [148]. Arachidonate itself can liberate PRL in such cultures, an effect blocked by inhibitors of the conversion of arachidonate to its metabolites [149]. As only inhibitors of the lipoxygenase and epoxygenase pathways of arachidonate metabolism have an inhibitory effect of PRL release, it is likely that one or more metabolites in these pathways are the proximal effectors of PRL release [150]. The fact that arachidonate metabolism inhibitors have a much greater effect on inhibiting PRL release in basal conditions compared to the stimulated state suggests that these eicosanoids may be more likely to mediate paracrine or autocrine modulations of secretory mechanisms, rather than to function as intracellular messengers [150]. The effect of arachidonic acid in increasing PRL release is primarily via an increase in calcium influx rather than by mobilizing intracellular calcium stores [151].

Intracellular Calcium As discussed above, activation of the phosphoinositide pathway results in a mobilization of intracellular calcium stores and an influx of extracellular calcium. The resultant increase in intracellular free calcium results in PRL gene

Chapter 4

transcription, PRL synthesis and release. The mobilization of intracellular calcium is rapid but transient, resulting in a high amplitude burst of PRL release [128,152]. The stimulation of calcium uptake via calcium channels is more prolonged and proceeds at a lower rate, resulting in sustained PRL release [128,152]. As mentioned above, calcium interacts directly with DAG to activate protein kinase C. Calcium also binds to topoisomerase II which then interacts with specific DNA binding sites and to calmodulin, which binds to a number of different intracellular enzymes [84]. Preston et al. [153] demonstrated that the increase in intracellular PRL mRNA is due not only to an increase in gene transcription but also to a posttranscriptional mechanism involving the posttranslational modification of a stable protein which then regulates PRL mRNA stability. Blockade of calmodulin activity with penfluridol or selective antagonists such as W7 results in a decrease in PRL release [154,155], whereas the calcium ionophore, A23187, stimulates PRL release [155]. Furthermore, A23187 stimulates intracellular cAMP accumulation and W7 blocks both the A23187 stimulation of intracellular cAMP accumulation and PRL release [155]. Thus mobilization of intracellular calcium may have effects on both protein kinase C and cAMP. Calcium channel antagonists, such as verapamil, diltiazem, nifedipine, nimodipine, pimozide, penfluridol and the negative enantiomer of Bay K8644 inhibit, and agonists, such as the positive enantiomer of BAY K8644, stimulate PRL gene transcription, synthesis and release in in vitro preparations [156–162]. The effects of manipulation of intracellular calcium levels on the stimulatory and/or inhibitory effects of a number of physiologic PRL regulators have been assessed. Agents which mobilize intracellular calcium stores, such as A23187 and ionomycin, do not add to the PRL releasing effects of TRH, whereas BAY K8644, which stimulates calcium channels, does show an additive effect [159,163], suggesting that the effect of TRH on the first phase of PRL release is primarily through release of calcium stores rather than by stimulating calcium channels [162–166]. However, when these experiments were examined with respect to the action of TRH on PRL gene transcription and PRL synthesis, none of these agents were additive with TRH. Thus,TRH appears to cause rapid release of PRL via activation of protein kinase C and calcium store mobilization but not calcium channel transport, and its effects on PRL gene transcription and PRL synthesis involve all three processes [159]. La Verriere et al. [159] postulate that the initial steps involve activation of protein kinase C and mobilization of intracellular calcium, which in turn control the activity of calcium channels. DA inhibits the PRL release induced by activation of calcium channels by BAY K8644 [157,160] and maitotoxin [123] and by mobilization of intracellular calcium stores by A23187 [167]. Conversely, the calcium channel blockers nimodipine, verapamil and diltiazem antagonize the inhibitory effect of DA in vitro [168]. DA administration blocks

Prolactin

127

the rise in intracellular calcium concentrations induced by TRH and angiotensin II [169], and DA withdrawal from the medium causes a rapid influx of calcium [170]. These findings are consistent with an inhibitory action of DA on calcium influx via calcium channels. Maitotoxin, which activates calcium channels, also blocks the DA induced inhibition of PRL secretion from lactotrophs in vitro [123]. Thus, at least part of the inhibitory action of DA on PRL secretion appears to be mediated by effects on intracellular calcium concentrations. In humans, administration of the calcium channel blocker verapamil causes an increase rather than a decrease in serum PRL levels [171,172] and does not inhibit the TRHinduced PRL rise [172]. This was shown to be due to a decrease in tuberoinfundibular generation of DA [173]. Because dihydropyridine and benzothiazepine calcium channel blockers had no effect on PRL levels in humans, it was hypothesized that the effect of verapamil is via action at N-type calcium channels present in neuronal tissues [173].

HORMONE BIOSYNTHESIS The primary translation product of the human PRL gene is 227 amino acids in length, consisting of a 28 amino acid signal peptide and a 199 amino acid hormone with a molecular mass of 23-kDa [19]. The structure predicted by the mRNA sequence delineated by Cook et al. [19] differs at eight positions from the early sequence of Shome and Parlow [18], which was determined by standard sequencing techniques. There are three disulfide bonds between cysteines at positions 4 and 11, 58 and 174 and 191 and 199, giving rise to small loops at the amino and carboxy terminal ends and a large loop in the middle (See Fig. 4.2). There is considerable heterogeneity in the final PRL product, depending upon variations in the degree of posttranslational modifications, which include cleavage, polymerization, glycosylation, phosphorylation and degradation (Table 4.1).

Cleavage Products Small amounts of 16 K and 8 K cleavage products are present in the normal pituitary and plasma of humans [174] as well as rats [175] and mice [176]. In the rat, the 16 K fragment has greater mitogenic activity than the intact hormone [177], but this has not yet been determined for hPRL. Several PRL-like proteins that are only a few amino acids shorter than the 23 K form and have a molecular weight of about 21 K are present in the rat pituitary. These smaller variants arise from carboxy (COOH)-terminus proteolytic clipping during storage and not from transcriptional modifications resulting from alternative splicing of the PRL pre-mRNA, as only a single 23 K band is found with in vitro translation systems [178–181]; these have not yet been described in the human.

128

SECTION 1

Table 4.1. of prolactin

Hypothalamic–Pituitary Function

Posttranslational processing and other variants

Cleavage 23K 21K (?) 16K 8K

Polymerization Monomer—80–90% Dimer—8–20% Polymer—1–5%

Glycosylation Circulating—50–100% Pituitary—13–25%

Phosphorylation

Decidual prolactin Lymphoblastoid prolactin

different types of glycosylation products have been described [197]. The glycosylated variants account for 13% to 25% of pituitary PRL and 50% to 100% of circulating PRL [196–199]. Glycosylated PRLs are less immunoreactive in the routine PRL RIA [196], bind less well to the PRL receptor [200] and are less bioactive, using a variety of bioassays [197,201,202]. On the other hand, Young et al. [203] found glycosylated porcine PRL to have greater lactogenic and binding affinity, despite decreased mitogenicity compared to nonglycosylated PRL. Interestingly, during pregnancy, subsequent lactation, and pathologic hyperprolactinemic states, when there is a high PRL secretion rate, the relative amount of nonglycosylated PRL increases compared to other times [199,201].

Placental variants

Phosphorylated Forms Larger Molecular Weight Forms About 80% to 90% of PRL extractable from pituitaries and in serum is monomeric, 8% to 20% is dimeric (molecular mass 45–50 kDa) and 1% to 5% is polymeric [182–186]. The dimeric and polymeric forms are linked by disulfide bonds [184] (see below) and can be largely dissociated under reducing conditions or with digestion with chymotrypsin [184,185]. Polymerization occurs primarily within storage granules and in stimulated lactotrophs PRL is synthesized and discharged rapidly, bypassing polymerization [187]. However, with stimulation the stored polymeric forms are released into plasma [188]. These larger molecular weight polymers have decreased binding to receptors and display decreased bioactivity in a variety of receptor assays [183,184] but have normal bioactivity in the Nb2 lymphoma cell bioassay (see below) [186]. Using highly purified, monomeric human PRL infused into normal male subjects, Molitch et al. [189] showed that some conversion of monomer to dimer also occurs in plasma. A number of patients have been described with elevated basal serum PRL levels but normal reproductive function who were found to have markedly elevated proportions of polymeric PRL, presumably thereby resulting in less PRL bioactivity [190–194]. However, in many such reported cases the proportion of monomeric PRL in the blood is still elevated. Preliminary screening of sera suspected of having a high proportion of polymers can be done by precipitating the large complexes with polyethylene glycol, but gel filtration chromatography remains the best procedure for quantitating the amount of polymers [195]. In clinical practice, screening for polymers is rarely carried out.

Glycosylated Products Glycosylation of human PRL occurs [196]. The carbohydrate unit is linked to the asparagine at position 31 and the complex has a molecular weight of about 25 K [196]. Two

PRL can be phosphorylated within secretory granules in rat and bovine lactotrophs [181,204,205]. These secretory granules appear to contain a PRL kinase, although the specifics of this remain to be determined. The role of this phosphorylation in secretion, whether phosphorylated forms circulate, and whether this process occurs in humans are unknown.

Placental Variants In mice and rats, there are a variety of PRL-like proteins produced by the placenta in addition to placental lactogen [206,207]. These PRL-like proteins are secreted at different times during gestation by the placenta and thus may have different functions, including alterations in blood vessel formation, hematopoiesis, and lymphocyte function [206,207]. No such proteins have been identified in the human and human placental lactogen is much closer in structure to GH than it is to PRL.

Decidual Prolactin PRL levels in maternal blood rise throughout gestation and are of pituitary origin [208,209]. However, PRL concentrations in amniotic fluid are 10–100 fold higher than either maternal or fetal blood levels [208]. Early biochemical studies found that amniotic fluid PRL was identical to serum PRL biologically, chemically and immunologically [210,211]. Friesen et al. [212] first showed that cultured human chorion released PRL and subsequently Riddick and Kusmik [213] found that cultured human decidual cells released it. Finally Golander et al. [214] documented that cultured human chorion-decidual explants synthesize and release a PRL that was shown to be identical to pituitary PRL. It was further documented that the decidua produced PRL [215], and that its bioactivity was equal to pituitary PRL [216]. Additional studies have shown that decidual PRL mRNA hybridizes with a pituitary PRL

Chapter 4

cDNA probe and Northern blot analysis shows that the 2 PRL mRNAs are indistinguishable, except for four silent nucleotide differences and the decidual PRL gene being about 150 nucleotides longer in the 5¢ untranslated region [217–219]. Similar to pituitary PRL, decidual PRL is also glycosylated [220] and some of the glycosylated PRL is bound to immunoglobulin by disulfide bonds in the amniotic fluid [221]. Similar rat decidual PRL-like substances have been reported that are structurally different from rat pituitary PRL but bind to PRL receptors and stimulate steroidogenesis by the corpus luteum [222–224]. The regulation of decidual PRL production differs from that of pituitary PRL (detailed below). DA agonists [225] and antagonists [226] given to the mother respectively decrease and increase maternal serum PRL levels but have no effect on amniotic fluid PRL levels. Similarly, DA, bromocriptine, and TRH have no effects on the decidual production of PRL in vitro [214]. Decidual PRL production is increased by progesterone and progesterone plus estrogen but not estrogen alone [218,227]. In 1988, Golander et al. [228] reported that a purified, 23.5-kd placental protein could stimulate the synthesis and release of PRL from decidual cultures. Insulin, through the insulin receptor, insulin-like growth factor I (IGF-I), through the IGF-I receptor and relaxin, a third peptide related to insulin and IGF-I have all been reported to stimulate synthesis and release of PRL [218,229,230]. The function of decidual PRL remains obscure, although there is some evidence that it may contribute to the osmoregulation of the amniotic fluid [231], fetal lung maturation [232] and uterine contractility [233].

Lymphoblastoid Cell Prolactin A human clonal lymphoblast cell line, IM-9-P3, has been established which produces a PRL that is identical to pituitary PRL in all aspects [234], including the sequence of their mRNA’s [235]. There are differences from pituitary but not decidual PRL, however, in the 5¢ untranslated region of the gene, suggesting that regulatory mechanisms may be different [235]. Indeed, preliminary studies have shown that PRL production by this cell line can be decreased by dexamethasone but other substances known to affect pituitary PRL secretion, such as estradiol, thyroxine, TRH and VIP, have no effect [236]. The similarities in the structure and regulation of the IM-9 and decidual PRL genes suggest that studies of the IM-9 cell line may prove fruitful in discerning the mechanisms regulating decidual PRL production, but it is doubtful that such studies will contribute much to our understanding of pituitary PRL regulation. HORMONE SECRETION—BIOCHEMISTRY Following synthesis on the rough endoplasmic reticulum, PRL is packaged into secretory granules in the Golgi and

Prolactin

129

PRL release occurs via exocytosis [237,238]. Nicoll and Swearingen in the early 1970s [239,240] demonstrated the existence of two pools of PRL within the rat lactotroph cell, one turning over rapidly and the other turning over slowly. Newly synthesized PRL is preferentially released compared to older, stored PRL in response to some stimuli and constitutes the rapidly turning over pool [238,241–243]. However, other stimuli, such as TRH, result in a preferential release of older, stored PRL [238]. Walker and Farquhar [238] found that these two types of secretion, rapid and slow, occurred not so much due to differences in the type of stimulation for a given cell but due to functional heterogeneity of the cells, so that some cells synthesize and secrete PRL rapidly while others secrete more slowly (see also the discussion above about the hemolytic plaque assay). Much of the storage pool of PRL in the pituitary appears to exist in a high molecular weight, disulfide-bonded, poorly immunoreactive polymeric form that is converted to a releasable, immunoreactive monomeric form within the secretory granule when processed for release [244]. This conversion involves a thio-disulfide interchange mechanism and can be stimulated by reduced glutathione and decreased by aminothiols such as cysteamine [244,245]. Treatment with cysteamine, in fact, reduces the amount of PRL detectable within cells using immunoprecipitation and RIA, but not when electrophoresis alone is used for detection [246,247]. These changes appear to explain a previous finding in rats that stimulation resulted in a “depletion” of the storage form of PRL in the pituitary with its “transformation” into the readily releasable, immunoreactive PRL [248]. Cysteamine has also been shown to reduce PRL release from pituitaries in humans and its therapeutic use in patients with hyperprolactinemia has been suggested [249], although its action on disulfide bonds in other peptides, such as somatostatin, would appear to preclude such use [250].

Measurement of PRL RIA

RIAs for PRL were initially developed for the rat [6], sheep [7] and cow [8]. After studies finally showed that in humans PRL and GH were separate molecules (see above), RIAs were developed for human PRL [16,17]. Most RIAs are double antibody assays, using as standard PRL that is either of commercial origin or a preparation available from the National Hormone and Pituitary Program in the U.S.A. or the National Institute for Biological Standards and Control in the UK. Because of the above noted differences in the kinds of PRL that exist in the pituitary and serum, it is not surprising that antibodies raised against one preparation of PRL may bind PRL differently from antibodies raised against a different preparation of PRL. Furthermore, PRL preparations for iodination and standards differ from one laboratory to another and therefore some means of standardizing one assay with another is

130

SECTION 1

Hypothalamic–Pituitary Function

important. Although antibodies still differ, at least the standards used can be standardized against a reference preparation, and this is usually done. First, second and third International Reference Preparations (IRPs) have been established by the National Institute for Biological Standards and Control in London [251,252] and similar reference preparations are made available by the National Hormone and Pituitary Program in Baltimore. Unfortunately, the supply of suitable standards does not preclude considerable variation from RIA to RIA in different laboratories in the measurement of PRL levels in both research studies and clinical evaluations of patients. In a survey of 10 different laboratories, the mean intraassay coefficient of variation was 9.6% but ranged from 5.6% to 25.3% and the mean interassay coefficient of variation was 7.9% [253]. Over the past decade, two-site immunoradiometric assays (IRMA) and chemiluminometric assays (ICMA) have come into wide use because of improved sensitivity and precision and short incubation periods. However, in patients with large prolactinomas with very high PRL levels, the very high level of PRL may saturate the antibodies, preventing the formation of the PRL-antibody sandwich, leading to loss of labeled antibody and a falsely low value [254,255]. St. Jean et al. [254] noted this high end “hook effect” in 5.6% of 69 patients thought to have clinically nonfunctioning adenomas. Therefore, in patients with large macroadenomas, PRL assessments should always be performed in both undiluted and 1 : 100 diluted serum to exclude the “hook effect” when two-site assays are used. Bioassays

The pigeon crop sac assay for PRL was the first assay used and was the standard until the late 1960s [1,3,4]. Although the procedure was refined and quantified substantially by Nicoll [4], it still proved cumbersome. Kleinberg and Frantz subsequently developed a highly sensitive bioassay which involved grading histologic changes in mouse mammary tissue that had been cultured with varying concentrations of PRL or serum samples [256]. It was with this assay that Kleinberg and Frantz first demonstrated that PRL is a separate hormone from GH in human blood [15]. This assay was further refined by Turkington [257], with quantitation of casein production by the mammary tissue being the endpoint measured. A radioreceptor assay using rabbit mammary gland membranes has been developed [258] which, as with other radioreceptor assays, essentially measures receptor binding but not true bioactivity. The most commonly used bioassay now is the Nb2 node rat lymphoma cell assay. In this assay cultured lymphoma cells replicate in response to added GH and/or PRL but not other hormones, with a sensitivity down to 0.1 ng/ml [259]. Using this assay, Tanaka et al. [260] and Rowe et al. [261] found bioassay/RIA ratios with a range of 0.53– 1.56 in sera from normal and hyperprolactinemic patients. As discussed above, in some patients with high amounts of polymeric PRL circulating in their blood, this ratio is

normal, whereas it is decreased in other bioassays. Thus this bioassay measures only one aspect of PRL function and when detailed assessments of biological function are necessary, more than one bioassay may be required. Frawley et al. [262] have revived the mammary gland casein production assay, achieving much better sensitivity. In this assay, they use a monolayer dispersed cell culture of rat mammary glands and then detect PRL induced casein production by a variant of their hemolytic plaque assay using casein antibodies, protein-A coated ovine RBC’s and complement, achieving a sensitivity of <1 pg/ml [262].

Physiology Metabolic Clearance and Production Rates of Prolactin

The metabolic clearance rate (MCR) and production rate (PR) of PRL have been determined by two methods: the infusion to equilibrium method using radioactively labeled PRL, and the infusion of exogenous hPRL into individuals in whom endogenous PRL production had been suppressed with a DA infusion. In two studies using the former method, the MCRs were found to be 46 ± 4 and 40 ± 6 mL/min/m2 [263,264] and with the latter method it was found to be 71 ± 19 mL/min/m2 [189]. The calculated PRs using the labeled PRL method were 200 ± 63 and 536 ± 218 mg/day/m2 and 802 ± 377 mg/day/m2 using the hPRL infusion method. The calculated disappearance half-lives varied from 26 to 47 minutes in these three studies. In another study the disappearance half-life was estimated to be 43.6 ± 11.2 minutes as determined from the rate of fall of PRL with individual secretory episodes [265]. The fate of PRL is unclear. Studies in patients with chronic renal failure have shown the MCR to be reduced by 33% [264] but Bratusch-Marrain et al. [266] found no hepatic uptake of PRL in man, in contrast to the liver’s being responsible for about 50% of total GH clearance in man and playing a substantial role in the uptake of PRL in rabbits [267]. Hormone Secretion Patterns

PRL is secreted episodically (Fig. 4.5). There is an innate pulsatility to pituitary PRL secretion with an interpulse interval of about 8 minutes, as determined by studies of plasma samples in rats in whom pituitaries were placed under the capsule of the kidney to escape hypothalamic regulation [268] and from media obtained from primate pituitaries cultured in vitro [269]. When plasma is sampled from normal individuals in whom hypothalamic function is superimposed upon this innate pulsatility, it becomes apparent that there are 4 to 14 secretory episodes per day. In two studies that used cluster analysis, 13 to 14 peaks per day in young subjects were found with a peak duration of 67 to 76 minutes, a mean peak amplitude of 3–4 ng/ml and an interpulse interval of 93 to 95 minutes [270,271]. Disinhibition caused by hypothalamic tumors causes an increase in

Chapter 4

FIGURE 4.5. Prolactin levels throughout the day in a single individual superimposed upon the range from five normal individuals. Note the episodic nature of secretion and the nocturnal rise.

basal PRL levels due to an increase in pulse amplitude and not pulse frequency [272]. There is an increase in the amplitude of the PRL secretory pulses that begins about 60 to 90 minutes after the onset of sleep; the secretory pulses increase with non-REM sleep and fall prior the next period of REM sleep [273]. Lowest PRL concentrations are found during REM sleep and highest concentrations are found during non-REM sleep [273]. When subjects are kept awake to reverse the sleep-waking cycle, PRL levels do not rise until sleep begins [274]. Thus, the diurnal variation of PRL secretion is not an inherent rhythm but depends on the occurrence of sleep. Interestingly, the diurnal variation of PRL with the sleep-induced rise persists despite other powerful physiologic influences such as breast-feeding [275]. Studies of PRL secretory patterns in twins show that there are both genetic and environmental influences on these patterns [276]. Weitzman et al. [277] noted small rises in PRL in the afternoon and evenings and they postulated that there may be a relationship to meals. Quigley and Ropert noted an increase in circulating PRL levels of 50% to 100% within 30 minutes of meals [278]. Subsequent work by Carlson related this increase to the amino acids generated from the protein component of the meals, phenylalanine, tyrosine and glutamic acid being the most potent in this regard [279–282]. Carlson et al. [282] have provided evidence that this stimulatory action of these amino acids is centrally mediated by showing that large neutral amino acids such as valine inhibit the transport of phenylalanine across the blood–brain barrier and blunt the stimulatory action of this amino acid. Changes in Prolactin with Age

PRL levels are elevated almost 10-fold in infants following delivery but then gradually decrease so that levels are normal by three months of age [283]. These high levels of PRL at birth are probably related to the stimulatory effect of high maternal estrogen levels. PRL levels are lowest between the ages of 3 months and 9 years and then rise modestly during

Prolactin

131

FIGURE 4.6. Serum prolactin (PRL) concentrations measured serially at weekly intervals as a function of gestation (n = 4). The dashed line represents the linear regression and the solid line represents the second order regression. NP, nonpregnant PRL values. From Rigg et al. [209]

puberty to adult levels [283]. In some studies there is a gradual fall of basal PRL levels with age, but in other studies no changes with age have been found [271,283–290]. One recent study found that PRL levels in women gradually fall by about 50% over the first 18 months following menopause, but this fall is considerably less in women treated with estrogen replacement therapy [287]. Other studies have shown no change in PRL levels with hormone replacement therapy [288]. In hyperprolactinemic women, estrogen replacement therapy causes no change in PRL levels [289]. A recent detailed study showed that mean serum PRL concentrations were decreased by 55% in older compared to younger men, and that this decrease was due to decreased basal secretion as well as the amount of PRL secreted with each secretory burst but that there was no change in pulse duration or frequency or interpulse interval [290]. Changes in Prolactin Levels during the Menstrual Cycle

A number of investigations of the changes in blood PRL levels during the menstrual cycle have revealed that some, but not all, subjects have higher levels at mid-cycle and lower levels in the follicular compared to the luteal phase [208,253,285,291–294]. In most of these studies, no correlations were found between PRL and estradiol, progesterone, LH and FSH levels. However, some studies have shown that PRL and LH secretion are often synchronous in the luteal phase and that very small doses of GnRH can cause the secretion of both PRL and LH at this time [295]. Changes in Prolactin Levels during Pregnancy

Basal PRL levels gradually increase throughout the course of pregnancy (Fig. 4.6) [208,209]. This has generally been attributed to the stimulatory effect of the hormonal milieu of pregnancy, primarily estrogenic, on the pituitary

132

SECTION 1

Hypothalamic–Pituitary Function

lactotrophs. There is a gradual increase in the number of pituitary lactotrophs during pregnancy [23,24] and by term, PRL levels may be increased 10-fold to levels over 200 ng/ml [208,209,283]. These elevated PRL levels found at term prepare the breast for lactation. Changes in Prolactin Levels with Postpartum Lactation

Within the first 4–6 weeks postpartum basal PRL levels remain elevated in lactating women and each suckling episode triggers a rapid release of pituitary PRL resulting in a 3–5 fold increase in serum PRL levels, peaking about 10 minutes after the end of suckling [208,296]. This increase in PRL secretion is accompanied by a suckling-induced increase in pituitary PRL mRNA levels which provide for increased PRL synthesis [297]. Following termination of suckling PRL levels gradually fall to reach prenursing levels by about 3 hours after the beginning of the suckling episode [296,298,299]. Over the next 4–12 weeks, basal PRL levels gradually fall to normal and the PRL increase which occurs with each suckling episode decreases [296,300]. Eventually there is little or no rise in PRL with suckling despite continued milk production [208]. The actual PRL levels achieved during suckling do not correlate with the amount of milk produced [298]. The decreases in basal and stimulated PRL levels between 3 and 6 months postpartum are largely the result of decreased breast-feeding as formula is introduced into the baby’s diet. If intense nursing behavior is maintained, basal PRL levels remain elevated and postpartum amenorrhea persists (Fig. 4.7) [301–303]. Eighty minutes of nursing per day with a minimum of six nursing episodes will usually result in persistent hyperprolactinemia and amenorrhea [303]. However, the delay in the onset of menses is more associated with high suckling duration and frequency than with a particular level of PRL [303]. High intensity lactation-induced failure to ovulate and menstruate has been used as a method of contraception in a number of developing countries for many years [304–306]. Breast stimulation may cause an increase in PRL levels in some non-breast-feeding normal women but not in men [296] except for one study showing PRL increases in men after breast stimulation by their wives [307]. Changes in Prolactin Secretion with Stress

PRL has long been known to be one of the pituitary hormones released by stress, along with adrenocorticotropic hormone (ACTH) and GH. Neill initially demonstrated the release of PRL from rats with ether stress [308] while Noel et al. initially showed a similar response to physical stress in humans [309]. The stress-induced rise in PRL generally consists of a doubling or tripling of PRL levels and lasts less than 1 hour. This rise in PRL may be limited by the concomitant release of cortisol which suppresses further PRL release, as evidenced by the fact that stress applied to adrenalectomized rats results in a PRL increase that is sustained for as long as the stressful stimulus is applied [310,311]. In

FIGURE 4.7. Mean 24-hour prolactin levels as a function of number of minutes of nursing in the same 24-hour period. Each circle represents one woman. The same amount of nursing results in higher levels of prolactin in the first year than in the second year postpartum.
= <12 months postpartum;  = >12 months postpartum. r = 0.54, P < 0.02. From Stern et al. [303]

humans, prolonged critical illness does not cause a sustained elevation of PRL; rather there is a reduction in the pulsatile secretion with an overall lowering of levels [312]. The teleologic significance for these stress-induced changes in PRL is not clear. The neuroendocrine mediation of the acute stress response is probably multifactorial but does not include a decrease in dopamine [313]. Corenblum and Taylor [314] attempted to dissect out the neurotransmitter regulation of the PRL stress response in humans by administering various blocking agents immediately prior to surgery. Blockade of histamine H1 receptors using chlorpheniramine, serotonin receptors using cyproheptadine, and dopamine receptors using pimozide had little effect on the peak PRL level reached during surgery. Blockade of opiate receptors with high dose naloxone resulted in a significant blunting, but not complete inhibition of the PRL response. These studies imply that the endogenous opiate-like peptidergic pathways may play a role in the PRL stress response. On the other hand, in humans naloxone has generally not been found to be able to block the PRL response to hypoglycemia [315]. Abe et al. found that VIP antisera inhibited the etherinduced PRL rise in rats [316] and Kaji et al. [317] found that antisera to VIP and PHI were additive in this regard. Other factors including serotonin, oxytocin and possibly unidentified posterior pituitary PRL releasing substances may also be involved, but their relative roles remain obscure [313].

Chapter 4

Prolactin

133

Hypoglycemia has been regarded as a form of stress, but whether it acts as a nonspecific stressor or has more specific effects is not clear. Among their tests of various types of stress, Noel et al. [309] showed that PRL did indeed rise with hypoglycemia. Woolf et al. [318] evaluated this effect systematically in normal men and women, finding that PRL levels increased by at least 10 ng/ml with a doubling of baseline levels in two-thirds of normal subjects, the maximal rise occurring between 40 and 90 minutes after the injection of intravenous insulin. 2-deoxy-d-glucose, which causes intracellular glucopenia, also causes a rise in PRL levels [319]. Studies using combinations of inhibitors of dopamine, histamine and serotonin suggest that at least some of the PRL response to hypoglycemia may be mediated by serotoninergic pathways [320]. Acute exercise has also been regarded as a form of stress and results in an acute, transient increase in PRL levels [309,321]. Although chronic, high-level exercise often results in menstrual disturbance, it is not associated with sustained hyperprolactinemia [322]. Effects of Thyroid Hormone Status on Prolactin Secretion

As noted above, thyroid hormones have a direct suppressing action on PRL gene transcription and hormone synthesis, so that in hypothyroidism this effect is decreased. As discussed below, in hypothyroidism TRH synthesis is increased, pituitary TRH binding is increased and pituitary VIP levels are increased. The result is that in hypothyroidism, PRL levels may be significantly increased. NEUROENDOCRINE REGULATION The hypothalamus exerts a predominantly inhibitory influence on PRL secretion through one or more PRL inhibitory factors (PIF) that reach the pituitary via the hypothalamic–pituitary portal vessels (Fig. 4.8). There are PRL-releasing factors (PRF) as well (Table 4.2). Disruption of the pituitary stalk leads to a moderate increase in PRL secretion as well as to decreased secretion of the other pituitary hormones.

PIFs Dopamine

In 1954, Everett [323] demonstrated that the luteotropic properties of the pituitary (due to PRL) were increased when pituitaries were transplanted to beneath the renal capsule, a site away from the regulation by the hypothalamus, thus demonstrating the predominance of the inhibitory component of hypothalamic regulation of PRL secretion. Pasteels [324] and Talwalker et al. [325] subsequently showed that hypothalamic extracts suppressed PRL secretion from pituitaries in vitro. At about the same time, Sawyer’s group [326,327] demonstrated an increase in PRL secretion in rats in vivo with use of catecholamine depleting agents such as reserpine or with DA receptor blockers, such as chlorpro-

FIGURE 4.8. Neuroendocrine regulation of prolactin secretion. GAP, gonadotropin-associated peptide; PHM, peptide histidine methionine; PIF, prolactin inhibitory factor; PRF, prolactin releasing factor; TRH, thyrotropin releasing hormone; VIP, vasoactive intestinal peptide.

mazine. In 1968 van Maanen and Smelik proposed that tuberoinfundibular DA (TIDA), released into the hypothalamic–pituitary portal vessels in the median eminence, was the physiologic PIF [328]. Shortly thereafter, the direct pituitary site of action for DA was confirmed by experiments in which it was demonstrated that DA could suppress PRL release from pituitaries in vitro [329–331] and Diefenbach et al. [332] demonstrated that the DA precursor L-dopa could suppress PRL secretion in intact and stalk-sectioned monkeys. A number of experiments have now firmly established that DA is the predominant, physiologic PIF. Gibbs and Neill [333] demonstrated that the concentration of DA found in the pituitary stalk plasma (about 6 ng/ml) was sufficient to decrease PRL levels in rats and was five to 10fold higher than levels found in peripheral plasma. Stimuli

134

SECTION 1

Hypothalamic–Pituitary Function

Table 4.2.

Substances affecting prolactin release

Stimulatory Thyrotropin-releasing hormone Vasoactive intestinal peptide Peptide histidine isoleucine Serotonin Opioid peptides Growth hormone-releasing hormone Gonadotropin-releasing hormone Oxytocin Vasopressin Histamine (H1) Bradykinin Angiotensin II Neurotensin Substance P

Inhibitory Cholecystokinin Bombesin Secretin Gastrin Galanin Calcitonin Calcitonin gene-related peptide Thymosin factor 5 Melatonin Other posterior pituitary factors (?) Platelet activating factor Epidermal growth factor a-melanocyte stimulating hormone Prolactin-releasing peptide

which result in an acute release of PRL usually also result in an acute decrease in portal vessel DA levels, including such stimuli as estrogen [334], cervical stimulation [335], and mammary nerve stimulation simulating suckling [336]. However, in many experiments it was found that the PRL increase obtained by simply reducing DA was considerably less than the elevation of PRL achieved by simultaneous stimulation by a PRF; similarly, the PRL level achieved with the simultaneous stimulation by a PRF with the reduction in DA is usually greater than that achieved by a PRF alone [337–339]. It is likely that in most physiologic circumstances that cause a PRL rise, such as lactation, there is a simultaneous fall in DA along with a rise in a PRF, such as VIP [316]. However, there may well be circumstances in which various PRF’s may stimulate PRL release with no concomitant lowering of DA levels, or DA may be lowered with no concomitant increase in a PRF. Newer work with mice in which the DA D2 receptor or DA transporter have been “knocked out” (KO) with inactivating mutations has confirmed these earlier studies that employed pharmacologic methods or lesioning. Thus, mice with the DA D2 receptor KO develop lactotroph hyperplasia without adenomatous transformation and sustained hyperprolactinemia [340,341]. DA action within the synapse terminates by DA reuptake by the DA-secreting neurons via the DA transporter. In contrast to the findings with the DA receptor KO, DA transporter KO mice have increased dopaminergic tone and lactotroph hypoplasia [342]. Although such mice have normal circulating levels of PRL, they cannot increase these levels with various stimuli and are unable to lactate [342]. Interestingly, the elevated DA levels in the DA transporter KO mice also depresses hypothalamic GH releasing hormone (GHRH) content resulting in decreased GH secretion and growth [342]. Although much of the direct work demonstrating DA in hypothalamic–pituitary portal vessels and the effects of DA

Dopamine Gonadotropin-associated peptide g-aminobutyric acid Somatostatin Acetylcholine

FIGURE 4.9. Time course of the serum prolactin response to the intravenous injection of 400 mg of synthetic thyrotropinreleasing hormone (TRH) in 36 normal men and 36 normal women. From Jacobs et al. [406]

on PRL release in vitro have been done in animals, it is clear that DA is the primary PIF in humans as well. Infusion of DA causes a rapid suppression of basal PRL levels [342–346] that can be reversed by metoclopramide, a DA receptor blocker [346]. Dopamine also blocks the PRL increments induced by such stimuli as hypoglycemia [347], arginine [348] and TRH [349]. Studies with low-dose DA infusions in humans have shown that DA blood concentrations similar to those found in rat and monkey hypothalamic–pituitary portal blood [333,350] are able to suppress PRL secretion [351–353]. Blockade of endogenous DA receptors by a variety of drugs, including phenothiazines, butyrophenones, metoclopramide and domperidone causes a rise in PRL

Chapter 4

[354,355]. Furthermore, blockade of the decarboxylation of l-dopa to DA by the dopa decarboxylase inhibitor carbidopa in the peripheral blood and median eminence causes an increase in PRL [356]. The axons responsible for the release of DA into the median eminence originate in perikarya in the dorsomedial portion of the arcuate nucleus and inferior portion of the ventromedial nucleus of the hypothalamus [357–359]. This pathway is known as the tuberoinfundibular DA (TIDA) pathway and DA axon terminals comprise about a third of all axon terminals in the zona externa of the median eminence [360]. Some TIDA neurons do not terminate in the median eminence but project to the posterior and intermediate lobes where their endings are found close to pituicytes, magnocellular axon terminals and precapillary spaces [361]. In some experiments, removal of the posterior lobe results in an increase in basal PRL levels that lasts for a few days [362]. However, the precise physiologic role of the posterior pituitary in inhibiting PRL secretion in the rat remains unknown [362]. There are no data in humans regarding such a role for the posterior pituitary in PRL regulation. The DA that traverses the TIDA pathway binds to the class of DA receptors referred to as D2 receptors [363–365] on the lactotroph cell membrane [366,367]. As discussed above, activation of this receptor results in: (i) an inhibition of adenyl cyclase with lowered intracellular cAMP levels; (ii) inhibition of phosphoinositide metabolism; and (iii) decreased intracellular calcium mobilization and inhibition of calcium transport through calcium channels. Some studies have also suggested that at least some of the inhibitory action of DA may be mediated by nitric oxide [368]. It has been proposed that these different actions of DA may actually be mediated by multiple similar D2 receptors that are produced by alternative RNA splicing [369]. The functional state of the D2 receptor is the high affinity form [364,367,370]. Although binding of DA to the D2 receptor appears to be sufficient to decrease PRL secretion mediated by the above pathways, DA can also be found intracellularly in association with PRL secretory granules [371–373]. The functional significance of this association of DA with the secretory granules is unknown. The inhibitory action of DA on PRL secretion is partially blocked by estrogen administration. This may be largely due to the direct action of estrogen on the estrogen response element of the PRL gene (see above). However, there may also be other mechanisms but with considerable interspecies differences. Estradiol is able to block the inhibitory action of dopamine on PRL release from rat lactotroph cells in vitro [374–376] but not from monkey lactotrophs [377]. In studies in humans, Judd et al. [344] found that the same dose of infused DA resulted in a greater suppression of PRL during the early follicular phase, when estrogen levels are low, compared to the late follicular or periovulatory phases, when estrogen levels are higher. PRL levels are also suppressed to a greater extent in women compared to men by the same dose of estrogen [343]. Estrogens

Prolactin

135

result in a decrease in DA receptor numbers in rats [378] but the DA receptor population showed no sex related differences in a limited number of human pituitaries [367]. Although portal vessel DA levels are higher in female rats or in male rats treated with estrogen [379], the direct effects of estrogen at the lactotroph level result in a blunting of the inhibitory effect of DA and increased PRL production [104,105]. Gonadotropin Associated Peptide (GAP)

Whether DA alone can account for all of the PIF activity of the hypothalamus has long been a question. In the mid-1970s a number of groups found considerable PIF activity in rat hypothalamic extracts in which DA had been removed [380–383]. In 1985 Nikolics et al. [384] reported the PRL inhibiting ability of a 56 amino acid polypeptide that is in the carboxyterminal region of the precursor to gonadotropin releasing hormone (GnRH) and which they termed GAP. In pituitary cultures, GAP was found to inhibit PRL secretion at concentrations of 10-11 M, whereas DA was found to inhibit PRL secretion only at much higher concentrations (10-7 M) [384]. Subsequent experiments showed a 100-fold lower potency of GAP for PRL suppression but at still lower molar levels than for DA [385,386]. Furthermore, passive immunization of rabbits against fragments of GAP resulted in marked elevations of endogenous PRL levels [384]. In these experiments, GAP was also shown to have weak gonadotropin stimulatory properties, giving rise to the concept of inverse stimulation/inhibition of these hormones in various physiologic circumstances. Antibodies raised to a synthetic peptide constituting residues 40–53 of GAP colocalized with GnRH in secretory granules of nerve terminals in the median eminence of the rat [387], and GnRH and GAP have been found to be cosecreted into the hypothalamic–pituitary portal system in sheep [388]. The gene and hypothalamic cDNA for the common precursor for GnRH and GAP were isolated from libraries derived from human and rat hypothalamic mRNA [389]. The GAP sequences in human and rat have 17 amino acid differences, of which eight are conservative in nature [389]. However, GAP had no PRL suppressing activity when tested against human prolactinomas cultured in vitro [390] and neither GAP nor fragments of GAP were able to inhibit PRL release from lactating or oophorectomized, hypothalamo–pituitary disconnected ewes [391]. At this point the physiologic significance of GAP as a PIF vis-à-vis DA is still not clear. Still less is known about its bioactivity in humans. Because of the inverse nature of its suppressing effect on PRL while having stimulating effects on gonadotropins, GAP remains an intriguing unknown. g -Aminobutyric Acid (GABA)

Schally et al. [392] initially demonstrated the inhibitory effect of GABA on PRL secretion in vivo and in vitro in rats, and Grandison and Guidotti demonstrated high-affinity

136

SECTION 1

Hypothalamic–Pituitary Function

GABA receptors on the pituitary [393]. A tuberoinfundibular GABAergic system has been described with perikarya located in the arcuate nucleus and nerve endings demonstrated in the median eminence [394]. GABA has also been demonstrated to be present in portal blood [395]. High concentrations (10-5 mol/L) of GABA and muscimol, a GABA receptor agonist, have been shown to inhibit PRL synthesis and release in vitro in rats but blockade of this inhibition with GABA receptor antagonists, such as picrotoxin and bicuculline, have given variable results [393,396,397]. Subsequent studies have shown GABA and muscimol to cause a decrease in lactotroph PRL mRNA levels with in vitro incubations [398]. Studies of the effects of GABA in vivo in rats have shown variable results, probably due to inactivation of GABA by GABA transaminase. When inhibitors of the catabolism of GABA are employed, GABA can be shown to decrease circulating PRL levels and lactotroph PRL mRNA levels [398]. Studies of the GABA system in humans have yielded conflicting results in studies of widely differing experimental designs. Bioactive metabolites of GABA, such as gammahydroxybutyric acid and gamma-amino-beta-hydroxy butyric acid, and muscimol cause modest increases in PRL levels when injected into humans [399–401]. However, GABA itself causes a modest decrease in PRL levels when given to humans for several days [402] and activation of the endogenous GABAergic system with sodium valproate causes a suppression in the PRL rise induced by mechanical breast stimulation in puerperal women [403]. The physiologic role of GABA, like that of GAP, remains to be fully elucidated in the human as well as various animal species.

PRFs TRH

Shortly after its initial isolation and characterization, TRH was demonstrated to cause a rapid release of PRL from rat pituitary cell cultures [404] and in humans after intravenous injection (Fig. 4.9) [405,406]. Release of PRL is biphasic, the initial peak being mediated by activation of intracellular phosphoinositide pathways with IP3 generation and mobilization of intracellular calcium causing release of stored hormone; the second, more sustained phase is mediated by influx of extracellular calcium through calcium channels, which causes sustained secretion and synthesis of new hormone [166,407]. However, Tashjian et al. [408] found the rise in intracellular calcium to precede the rise in intracellular IP3, so that other mediators of intracellular calcium mobilization may also be involved. TRH is also internalized within the lactotrophs [409] and can even be synthesized by lactotrophs [410] but the physiologic significance of this is not clear. A number of different experimental approaches have failed to clarify the physiologic role of TRH as a PRF. The smallest dose of TRH that releases TSH also releases PRL in humans [411]. Immunoneutralization of endogenous TRH

FIGURE 4.10. Blockade of the prolactin rise induced by ether stress in rats treated with anti-vasoactive intestinal polypeptide (VIP) antiserum or normal rabbit serum (NRS). From Abe et al. [316]

with TRH antisera causes a 50% suppression of basal PRL levels in rats in some studies [412] but not in others [413,414]. Neither did such immunization affect the PRL response to electrical stimulation of the paraventricular nucleus or suckling [414], and it delayed but did not affect the magnitude of the spontaneous surge of PRL on proestrus [415]. Active, chronic immunization of ewes with TRH conjugated to albumin resulted in only minimal decreases in PRL levels basally and after stimulation by stress, suckling and estrus although reductions in thyroid hormone levels were more marked [416]. In the mouse with targeted disruption of the TRH gene (TRH KO), mice became hypothyroid with elevated levels of TSH with reduced biological activity but had normal PRL levels [417], further casting doubt on the essential role of TRH in PRL regulation. Suckling causes an increase in hypothalamic and portal vessel TRH levels as well as a decrease in DA levels [336]. If TRH mediates the PRL response to suckling, even in part, it ought to be accompanied by an increase in TSH, unless there were a concomitant increase in somatostatin. Detailed evaluations of serum levels of PRL and TSH following suckling in rats showed elevations of TSH in some studies [418,419] but not in others [414,420]. Similar studies in humans failed to show any elevations of TSH [421,422]. Very small doses of TRH given systemically were effective in releasing PRL and TSH in lactating rats and women in those studies, however, so it is unlikely that failure to show a rise in TSH was due to an increase in somatostatin. In hypothyroidism, TRH synthesis is increased [423] and portal vessel TRH levels are increased but not statistically significantly [424]. In addition in hypothyroidism, pituitary TRH binding is increased due to an increased number of TRH receptors, but an effect on binding affinity is contro-

Chapter 4

versial [425–427]. In human hypothyroidism, basal TSH and PRL levels are increased as are their responses to injected TRH [428]. Correction of the hypothyroidism with thyroid hormone corrects both the elevated TSH and PRL levels and their responses to TRH, although the correction is more rapid for TSH [428–430]. Conversely, in hyperthyroidism in humans, PRL levels are not low basally but the PRL response to TRH is markedly blunted and returns to normal with correction of the hyperthyroidism [428–430]. The above conflicting data from passive immunization studies, TRH KO mice, observation of TSH levels during lactation and examination of PRL levels in various thyroid states support a role for TRH as a physiologic PRF, albeit not the primary one or even one of major importance. TRH is itself generated from a larger precursor with a molecular weight of 29.2-kDa [431] and other peptides generated from the precursor have been found in the brain, but their functions are unknown [432]. Although also found in other nuclei, the neuronal perikarya for TRH which project ventrally to terminate in the median eminence are located in the parvocellular division of the paraventricular nucleus [431–433]. A stable, bioactive metabolite of TRH, cyclo-His-Pro (also known as histidyl-proline-diketopiperazine), is found in rat hypothalamus and pituitary in concentrations similar to those of TRH [434]. Cyclo-His-Pro inhibits the PRL response to TRH from rat pituitaries in vitro but reports conflict as to whether it inhibits the PRL response to TRH or suckling in vivo [435–438]. In conscious monkeys it also suppresses basal and TRH stimulated PRL levels [439]. The mechanism by which cyclo-His-Pro suppresses PRL release is not clear, as it does not appear to act via the TRH receptor [436]. How cyclo-His-Pro integrates with other factors involved in the regulation of PRL in animals and humans is as yet unknown. VIP and Peptide Histidine Methionine (PHM)/PHI

VIP was initially found to stimulate PRL release in 1978 [440,441]. VIP neuronal perikarya are present in the parvocellular region of the paraventricular nucleus, with axons terminating in the external zone of the median eminence [442,443]. Its effects are selective for PRL and additive to TRH in causing PRL release [444,445] at concentrations (2–6 ¥ 10-10 M) found in hypothalamic–pituitary portal blood [446,447]. As discussed above, the effects of VIP appear to be mediated by stimulation of adenyl cyclase, although recent evidence suggests that transport of calcium through membrane calcium channels may also be important [448]. In addition to stimulating PRL release,VIP also stimulates pituitary PRL mRNA content and PRL synthesis [449]. In conditions of increased PRL synthesis, such as lactation, hypothalamic VIP mRNA levels are also increased [450]. In addition to these studies in rats, intravenously administered VIP has also been shown to increase PRL levels in monkeys [451] and humans [452–454] at serum levels similar to those demonstrated in rat portal blood.

Prolactin

137

FIGURE 4.11. Parallel fall in prolactin and growth hormone (GH) levels following resection of a growth hormone-releasing hormone producing pancreatic tumor that had caused acromegaly. From Thorner et al. [540]

A number of experiments have been performed using passive immunoneutralization techniques to determine the physiologic role of VIP as a PRF. Anti-VIP antisera administered to rats have been shown to partially inhibit the PRL responses to suckling, ether-induced stress (Fig. 4.10) [316,317], estrogen administration [455], intraventricular 5HT [456] and proestrus [457] and to suppress PRL pulsatile secretion in dopamine receptor blocked rats [458]. Similar results for suckling have been obtained with a VIP antagonist [459]. Part of the 20-kDa 170 amino acid VIP precursor is another similarly sized peptide known as PHM [460]. VIP has 28 and PHM has 27 amino acid residues. The terminal amino acid in the porcine equivalent of PHM is isoleucine, and therefore the peptide of porcine origin is known as PHI. PHI and VIP [461] and PHM and VIP [462] colocalize in the hypothalamus and median eminence. Interestingly, PHI is of similar potency to VIP in releasing PRL from rat pituitaries in vitro and in vivo [463,464]. Furthermore, passive immunoneutralization with anti-PHI plus anti-VIP antisera causes a greater suppression of the PRL responses to the serotonin precursor 5-hydroxytryptophan [465] and ether [317] than either anti-PHI or anti-VIP antisera alone. PHM given to humans has caused a PRL increment in some experiments [462,466] and not others [454]. Further complicating the role of VIP as a PRF is the finding that VIP is actually synthesized by anterior pituitary

138

SECTION 1

Hypothalamic–Pituitary Function

tissue [467]. Antisera to VIP inhibit basal PRL secretion from dispersed pituitary cells in vitro [468,469], suggesting a local “autocrine” role for VIP in PRL regulation within the pituitary. Interestingly, pituitary VIP concentration is increased in the pituitaries of hypothyroid rats along with a decreased PRL content [470] and TRH increased VIP and PRL secretion. However, immunoneutralization of the pituitary cultures with anti-VIP antiserum did not decrease the PRL response to TRH [471]. It is not yet clear whether the VIP secreting pituitary cells are lactotrophs [469] or nonlactotroph stellate cells [470]. The physiologic role of VIP as a PRF appears to be warranted by the experimental data. The precise roles of VIP vs. PHM and hypothalamic VIP vs. pituitary VIP still are not clear. How VIP/PHM interact with other PRF’s such as TRH are additional areas requiring clarification. Serotonin

A considerable number of experiments have demonstrated a role for serotonin as a neurotransmitter involved in the release of PRL. Most serotoninergic neuronal perikarya are in the dorsal and median raphe nuclei, and their axons project forward to the hypothalamus and other limbic and cortical areas [472]. Lesions of the dorsal but not the median raphe nuclei decrease forebrain serotonin levels and basal and stimulated serum PRL levels [472–475], while stimulation of the dorsal raphe nucleus results in PRL release [473]. Serotonin and its precursor, 5-hydroxytryptophan, cause a release of PRL in rats whether injected systemically [476,477] or into the third ventricle [478]. In a variety of experiments using blockers of serotonin synthesis, such as parachlorophenylalanine, blockers of serotonin receptors, such as methysergide, or blockers of serotonin reuptake by nerve terminals, such as fluoxetine, it has been shown that serotonin mediates, in part, the PRL elevations associated with suckling and proestrus [473,475,477,479–481]. Conversely, agents which are serotonin releasers, such as fenfluramine, or agonists, such as quipazine, result in a rapid increase in serum PRL levels [481–483]. Using specific serotonin receptor blockers, it has been found that it is the S2 receptor that mediates the serotonin action on PRL secretion [483]. Studies in humans also suggest a role for serotonin in PRL secretion. Infusion of the serotonin precursor, 5hydroxytryptophan, results in a prompt increase in PRL levels [484,485]. Nocturnal PRL secretion is inhibited by cyproheptadine [486]. Fenfluramine, a serotonin releasing agent caused a fourfold rise in PRL in humans that could be partially blocked by cyproheptadine [487]. Similarly, mchlorophenylpiperazine, a metabolite of the antidepressant drug, trazadone, has agonist activity at central serotonin receptors and causes a rise in PRL levels [488]. Fluoxetine, a serotonin reuptake inhibitor, also increases PRL levels modestly but still within the normal range [489]. On the other hand, pizotifen, a specific, non-ergot serotonin antag-

onist, had no effect on the suckling-induced PRL rise in postpartum women [490]. Whether serotonin’s effects are mediated solely through brain pathways or whether it has direct effects on the pituitary is controversial. One possibility is that serotonin causes a decrease in hypothalamic DA generation. Synaptic junctions between serotoninergic nerve terminals and dopaminergic perikarya in the arcuate nucleus have been demonstrated [491]. Furthermore, Pilotte and Porter [492] showed that intraventricular injections of serotonin decreased portal vessel DA concentrations and Johnston and NegroVillar [493] demonstrated that 5-hydroxytryptophan administration to rats results in a decrease in median eminence DA synthesis. However, when DA levels were maintained by infusion, serotonin was still able to cause a release of PRL [492]. It has also been proposed that serotonin acts by increasing VIP. Shimatsu et al. demonstrated that serotonin stimulates the release of VIP into portal blood [447] and passive immunization against VIP (see above) decreases the increase in PRL induced by 5-hydroxytryptophan [465]. Electrolytic lesions which destroy the paraventricular nucleus, the site of origin of VIPergic tuberinfundibular pathways, result in a marked blunting of the PRL responses to 5-hydroxytryptophan, ether stress and restraint stress [494], further buttressing the argument for a role for VIP in mediating the serotonin stimulatory effects on PRL secretion. Serotonin [495,496] and serotoninergic nerve fibers have been found within the anterior pituitary [497,498]. Serotoninergic nerve terminals have also been demonstrated within the median eminence [499]. Tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis is present in the pituitary [496], suggesting in situ synthesis. High affinity S2 serotonin receptors have been found in the anterior pituitary [500] as well uptake of labeled serotonin into cells of the pituitary [501]. In direct tests of the effects of serotonin on pituitary PRL release, Apfelbaum [502] found serotonin to increase basal and stimulated PRL secretion from pituitaries in vitro and Stobie and Shin [503] and Wehrenberg et al. [504] found that serotonin stimulated PRL from hypophysectomized pituitary grafted rats and stalk-section monkeys, respectively. On the other hand, Birge et al. [329], Lamberts and MacLeod [505] and Delitala et al. [506] found no effect of serotonin on PRL release from rat pituitary cells in vitro. Thus, although it is possible that serotonin is a direct secretagogue for PRL, via transport from the hypothalamus by the portal vessels or through an autocrine action within the pituitary, its role in this regard is still uncertain. It likely mediates the nocturnal surge of PRL, and may well participate in the suckling-induced rise in PRL via the ascending serotoninergic pathways from the dorsal raphe nucleus and mediated by activation of VIP release. Opioid Peptides

A detailed description of the various opioid peptides, their receptors and their neuronal pathways is beyond the scope

Chapter 4

Prolactin

139

of this discussion. Approaches to determining the roles of the opioid peptides and pathways in the regulation of PRL secretion have focused on using opioid agonists and antagonists in experimental animals and humans. In rats, morphine, met- and leu-enkephalin, bendorphin, dynorphin and leumorphin injected systemically or intracerebroventricularly have all been shown to cause release of PRL [507–518]. More recent studies employing specific agonists and antagonists operative on the m, d and k opioid receptors and antibodies directed against several opioid peptides have shown that it is the m receptor that is the predominant one involved in PRL release, the k receptor is involved to a lesser extent and the d receptor is not at all involved [518–521]. Most evidence suggests that the opioid peptides do not have a direct effect on the pituitary [508,521,522] and stimulate PRL release by inhibiting DA turnover and release by the TIDA pathway [523–526]. The recently identified opioid peptide, orphanin FQ (also called nociceptin), which binds to an opioid-like orphan receptor, also causes an increase in PRL levels in rats when administered intracerebroventricularly [527]. In humans, morphine and morphine analogs increase PRL release acutely [528–532] and chronically [522,523]. However, blockade of the m receptor with naloxone has minimal to no effect on PRL levels either basally or with stimulation by hypoglycemia, exercise, sleep, TRH or physical stress [315,533–537]. In contrast to these findings, two groups have reported an increase in PRL levels in response to naloxone given in the late follicular and midluteal phases of the menstrual cycle [538,539]. The interpretation of these changes is not clear. Overall, it appears that the endogenous opioid pathways play at most only a minor role in the regulation of PRL secretion, especially in humans. GHRH

A number of studies have found GHRH to have PRL releasing properties. The initial clue to this effect of GHRH was the finding that many of the patients with acromegaly due to GHRH secreting tumors were hyperprolactinemic and PRL levels fell in parallel with GH following excision of the GHRH-secreting tumor [540] (Fig. 4.11). Large doses of GHRH have been reported to release PRL in vivo in normal humans in most studies [541–546]. In our own studies, GHRH was found to cause small, consistent PRL responses in addition to larger GH responses in normal individuals (Fig. 4.12) [543]. Chronic therapy with GHRH in children with GH neurosecretory dysfunction results in a sustained elevation of PRL levels [547]. In rat pituitary cell cultures, GHRH causes PRL and GH release but an increase only in GH mRNA and not PRL mRNA, indicating no stimulation of PRL synthesis [548]. The similarity of GH and PRL responses to a variety of stimuli, such as exercise, stress, hypoglycemia, arginine infusion and sleep and the pathological conditions of renal failure and hepatic cirrhosis suggest, but do not prove, that GHRH may serve as a physiologic PRF under some circumstances.

FIGURE 4.12. Prolactin secretory responses to bolus injection of growth hormone-releasing hormone (GHRH1–40) 3.3 mg/kg. Modified from Goldman et al. [543]

Posterior Pituitary, Oxytocin and Vasopressin

A number of studies in animals have shown that oxytocin, in levels found in the hypothalamic–pituitary portal vessels [549], can stimulate PRL release when added to the medium of pituitary cell cultures or incubations or when given intravenously but it lowers PRL levels when directly injected into the third ventricle [550–554]. Studies in which endogenous oxytocin was eliminated by passive immunization with oxytocin antisera or by oxytocin antagonists show a reduction and a delay in the suckling induced PRL surges in some studies [551] but not others [552] and a reduction in the PRL rise associated with proestrus [552,555] but no effect on the PRL response to ether stress [552]. Antisera to oxytocin also block the PRL surge seen in response to intracerebroventricularly administered VIP [459]. In addition, the peak phase of the PRL surge accompanying proestrus can be blocked by either posterior pituitary

140

SECTION 1

Hypothalamic–Pituitary Function

lobectomy or antisera to VIP [457]. These last findings suggest that oxytocin and/or other posterior pituitary substances may be involved in at least one of what are turning out to be multiple routes by which VIP stimulates PRL release. Very limited studies in humans suggest that oxytocin administered intravenously has no effect on basal PRL levels and causes only a minimal increase in TRH stimulated PRL levels [556]. Vasopressin also has PRL releasing properties when injected intravenously into normal rats and sheep and rats with pituitaries transplanted to the renal capsule, but not sheep with hypothalamic–pituitary disconnection [553]. The neurophysin portions of the precursors to oxytocin and vasopressin also stimulate PRL secretion in rats [557]. Schally et al. [558] have shown that a peptide identical to the sequence of 109–147 of the vasopressin–neurophysin precursor has been isolated from porcine hypothalami and has PRL inhibiting properties. Nagy et al. [559] found that the suckling induced PRL rise is absent in Brattleboro rats (which have diabetes insipidus due to a single base deletion in the vasopressin gene) and is partially restored with exogenous vasopressin. There are no studies to date of the effects of vasopressin on PRL secretion in humans. Whether there are other PRF’s in the posterior pituitary in addition to oxytocin, vasopressin and their respective neurophysins has been a matter of controversy. Rats which have had the posterior pituitary removed and replaced with vasopressin and oxytocin have a PRL response to suckling that is greatly reduced [560]. Antagonists to TRH, angiotensin II, endorphin and inactivation of vasopressin and oxytocin by disrupting disulfide bonds do not affect this posterior pituitary PRF activity [560,561] and studies of its physical characteristics suggest that it has a molecular mass of 1–5-kDa [562]. Although Mori et al. [554] could attribute posterior pituitary PRF activity only to contained oxytocin, Samson and Mogg [563] agree with Ben-Jonathan and her coworkers that the posterior pituitary PRF activity is due to some additional factor, and recently Laudon et al. [564] have localized the PRF activity to the intermediate rather than the true posterior lobe. The posterior pituitary, therefore, may play a significant role in the regulation of PRL secretion in rats. Vasopressin, oxytocin, the neurophysins and possibly an additional factor may be involved, possibly including VIP in the concatenation of neural links. How these factors interact with dopamine, TRH and other release and inhibiting substances in PRL regulation and how relevant all of this is to human PRL regulation are aspects that remain to be determined. Gonadotropin Releasing Hormone

GnRH was initially found to release PRL from rat pituitary cells in vitro [565]. Subsequently, GnRH has been found to cause a release of PRL in anovulatory women [566–568] and in women with anorexia nervosa who were gaining weight [569]. In 24% to 78% of normal women there is a PRL response to GnRH, depending upon the phase of the

menstrual cycle, the highest number of responders being in the periovulatory phase [570]. Postmenopausal women also have a PRL response to GnRH that is augmented with estrogen supplementation [571]. There is no PRL release in response to GnRH in normal, eugonadal males but such a release does occur with high doses of estrogen pretreatment (given to transsexual men) [572]. Analysis of PRL and LH secretory pulses suggests a high degree of concordance in rats and women [295,573]. This cosecretion of LH and PRL suggests that the response to GnRH is physiologic and is evidence against the inhibitory cosecreted GAP having a physiologic role. A subset of human prolactinomas that also contain the glycoprotein a subunit have been shown to bind GnRH specifically and with high affinity and to release PRL in response to GnRH in vitro [574]. Renin–angiotensin System

Angiotensin converting enzyme (ACE) and angiotensin II receptors and activity have been identified in the rat pituitary and median eminence of the hypothalamus [575–577]. In the human pituitary, renin, ACE and angiotensinogen have been detected in normal lactotroph cells and in PRLsecreting adenomas [578]. Angiotensin II incubated with rat pituitary cells stimulates release of PRL, an effect blocked by AT1 but not AT2 antagonists [579,580a]. However, in humans, blockade of ACE with enalapril results in no change in basal PRL levels [581,582a], no change in the TRH and metoclopramide induced PRL rises [581] and only a minimal decrease in the PRL response to hypoglycemia [582a]. It is unlikely, therefore, that the endogenous reninangiotensin system of the hypothalamus and pituitary have significant physiologic effects on PRL regulation. PRL-releasing Peptide

Hinuma et al. [582b] recently discovered a new, 31 amino acid peptide capable of releasing PRL, termed PrRP31, by looking for endogenous ligands for an orphan receptor present in the human pituitary termed hGR3. In pituitary cell preparations, PrRP31 released PRL with a potency equal to that of TRH [582b]. However, although PrRP31 is found in neuronal perikarya in the paraventricular and supraoptic nuclei, PrRP31-immunoreactive nerve fibers are found only in the internal zone of the median eminence and not the external zone [582c], casting uncertainty on the physiologic significance of this new peptide. Other Neuroactive Peptides and Neurotransmitters

Neurotensin This has been found to increase PRL levels when given intravenously to urethane-anesthetized intact and steroid primed male rats [508,583]. In in vivo studies, diphenhydramine, an H1 histamine receptor blocker, was able to block this stimulatory effect of neurotensin [508] but other studies have found neurotensin to release PRL directly from rat hemipituitaries in vitro [584]. When neurotensin is injected intracerebroventricularly, however, it inhibits PRL release [583,585] and the intracerebroventricular injection of

Chapter 4

antisera to neurotensin results in a rise in PRL levels, suggesting that endogenous neurotensin may be playing a physiologic role in rats [586]. In contrast to these results in rats, the administration of neurotensin to humans has no effect on PRL levels [587]. Substance P This also stimulates PRL release when given intravenously to urethane-anesthetized rats [508,588] and when given intracerebroventricularly to monkeys [589]. No such inhibition occurred when Substance P was given intracerebroventricularly to rats [585]; indeed, inhibition of PRL release was reported when it is given in this fashion [590]. Substance P also stimulates PRL release directly in vitro with pituitaries from female [585] but not male [590] rats. Cholecystokinin (CCK) This has been shown to increase PRL release from in vitro rat preparations [445,591] but not from similar human pituitary cultures [445]. The intravenous route in rats has provided both positive [591] and negative results [592] with respect to PRL stimulation. Intracerebroventricular CCK results in a marked PRL release that can be antagonized with antisera to VIP [592], implying that its action may require the activation of VIPergic systems. The intracerebroventricular or intravenous administration of proglumide, a CCK antagonist, cause a marked fall in PRL levels in male but not female rats [593]. The reason for these varying results, the discrepancy in findings between the sexes and the relevance to humans are unknown. Somatostatin Receptors for somatostatin have been found on human PRL as well as GH-secreting adenomas [594]. Somatostatin has been found to inhibit adenyl cyclase activity of rat anterior pituitary homogenates and spontaneous [595,596], TRH-induced [596], and VIP-induced [597] PRL release. Furthermore, administration of somatostatin antiserum to rats causes a rise in PRL levels, implying a physiologic inhibitory action of somatostatin basally [594]. In humans, however, somatostatin administered exogenously has no effect on the TRH-induced PRL release [598]. Other Peptides Bombesin is present in the hypothalamus and stimulates PRL secretion in vivo from steroid-primed male rats [599] and in vitro from pituitary cell cultures [600,601]. However, when bombesin is administered intracerebroventricularly, it inhibits the PRL rise induced by restraint stress [602]. No studies of its effects in humans have been reported. Secretin has been found to have both stimulating [603] and inhibiting [604] effects on PRL release in rats. Gastrin causes an increase in PRL levels when injected intravenously in rats but causes a fall in PRL levels when injected intracerebroventricularly. It has no effect on PRL secretion in vitro [605]. Galanin is expressed in the PVN and the median eminence of the hypothalamus and in pituitary lactotrophs, where it is colocalized with PRL in the same secretory granules [606]. Galanin injected intravenously

Prolactin

141

causes an increase in PRL levels when given in high doses, but has no effect when injected intracerebroventricularly or in vitro [607]. Galanin KO mice have 30% to 40% lower PRL levels, are unable to lactate, and their lactotrophs fail to proliferate in response to estrogen [606]. Endothelin is produced by lactotrophs and also causes a release of PRL from lactotrophs, suggesting a possible autocrine regulation of PRL secretion [608]. Transforming growth factor-b1 (TGF-b1) is similarly produced by lactotrophs but inhibits PRL gene expression, synthesis and release in a paracrine rather than an autocrine manner [609,610]. Adenosine is produced by the folliculostellate cells of the pituitary and also stimulates PRL release in a paracrine fashion [611]. Several other substances have been found to increase PRL release from pituitary cell cultures, including bradykinin [612,613], calcitonin [614]; calcitonin generelated peptide (CGRP) [615]; relaxin [616], plateletactivating factor [617], and thymosin fraction 5 [618]. Melatonin has also been shown to increase PRL levels from humans when given orally [619]. On the other hand amelanocyte stimulating hormone (a-MSH), injected intracerebroventricularly causes a decrease in PRL levels and the administration of MSH antiserum causes a rise in PRL levels [620,621]. Basic fibroblast growth factor also inhibits PRL release in vitro [622]. A peptide identical to the 27–52 amino acid sequence of proopiomelanocortin has been isolated from the hypothalamus and is also inhibitory to PRL secretion [558]. Pituitary adenylate cyclase activating polypeptide (PACAP) also inhibits PRL secretion when injected intracerebroventricularly through a stimulation of DA release by TIDA neurons [623]. Histamine This neurotransmitter plays an uncertain role in PRL regulation [624]. Histamine neuronal perikarya are present in the posterior hypothalamic region and axons project to almost all of the nuclei of the hypothalamus [624]. Histamine administered systemically or intracerebroventricularly to rats causes a modest PRL rise [625–627]. H1 receptor antagonists suppress the suckling-induced PRL increment in rats [625] as well as the PRL rise caused by intravenous histamine [627], whereas H2 receptor antagonists, such as cimetidine and ranitidine, augment the intravenous histamine-induced PRL rise [628]. In contrast, intracerebroventricular histamine also increases PRL release, an effect blocked by H2 blockers but not H1 blockers [628]. These studies suggest that histamine exerts its PRLreleasing activity via H2 receptors when given intracerebroventricularly and via H1 receptors when given intravenously. However, histamine has no effect on PRL release from pituitaries in vitro or in stalk-sectioned rats [627]. Although intracerebroventricular histamine causes a 26% fall in stalk vessel dopamine levels, this is not sufficient to explain the large rise in plasma PRL levels [626]. In humans, intravenous H2 but not H1 blockers cause a rise in PRL levels [629–632] but prolonged oral administration of H2 blockers does not result in sustained PRL elevation

142

SECTION 1

Hypothalamic–Pituitary Function

[632]. Histamine alone can cause a rise in PRL secretion in man that is further augmented by H2 blockers and is blocked by H1 blockers [628]. The fact that the administration of high doses of H2 blockers causes an increase of PRL levels in humans, that histamine cannot cross the blood–brain barrier and that histamine has no effect on pituitaries in vitro suggests that histamine may play a physiologic facilitory role within the median eminence in PRL secretion. The roles of other bioamines in the regulation of PRL secretion are even less well established. Central adrenergic a2 agonists such as clonidine usually have no effect on PRL secretion although both increases and decreases have been reported, the differences possibly being due to the doses used [633–635]. The administration of a2 adrenergic antagonists such as yohimbine or piperoxan result in an increase in PRL levels that is blocked by clonidine [635,636], implying an overall inhibitory role of the a2 adrenergic system in the hypothalamus. a-methyl dopa, another central adrenergic agonist, causes a sustained elevation of PRL [637] but this may be due to inhibition of the synthesis of norepinephrine or dopamine centrally by inhibiting the enzyme l-aromatic acid decarboxylase, which is responsible for conversion of dopa to dopamine and by acting as a false neurotransmitter to decrease dopamine secretion or synthesis by a local feedback inhibitory action. However, monoamine oxidase inhibitors cause an increase in PRL levels [638]. Such an elevation is unexpected, since these drugs should increase the levels of norepinephrine and dopamine in the synapse. Acetylcholine This neurotransmitter inhibits adenyl cyclase and cAMP accumulation, lowers intracellular free calcium levels and decreases PRL release from pituitary cell cultures, acting through muscarinic and not nicotinic receptors [127,639]. In such cultures, coincubations of acetylcholine or the cholinergic agonist, carbachol, with VIP or TRH result in marked inhibition of the VIP- and TRHinduced PRL release [127,640]. Although atropine, a muscarinic receptor antagonist, blocks this acetylcholine inhibition of PRL in vitro [127,640], it had no effect on basal or TRH-induced PRL release in humans [641]. Moreover, pirenzepine, another muscarinic receptor blocker, actually caused a modest decrease of PRL levels in humans in vivo [642]. Another muscarinic receptor blocker, methscopolamine, had no effect on the sleep induced PRL rise while blocking the GH rise [643]. The widespread presence of acetylcholine as a neurotransmitter in the CNS and the possibility that pituitary tissue itself may synthesize acetylcholine [644] make interpretation of studies testing this system difficult, and the true role of acetylcholine in the regulation of PRL secretion is uncertain.

Prolactin Short Loop Feedback Considerable evidence in rats suggests that PRL is able to feed back negatively on its own secretion (short-loop feedback or autofeedback) [645–647]. Most evidence

suggests that such feedback occurs via augmentation of hypothalamic TIDA turnover [648–652], including direct measurements of dopamine in portal vessels [379]. Direct confirmation of the importance of short-loop feedback in rodents comes from recent studies using mice with targeted disruption of the PRL gene (PRL gene KO). Mice with the PRL-KO have no pituitary PRL and have markedly decreased DA in TIDA neurons along with hyperplasia of lactotrophs that do not make PRL [653–655]. Direct evidence for such PRL short-loop feedback in the human has not been demonstrated. In a number of reports, however, it has been suggested that altered regulation of gonadotropin and TSH secretion in hyperprolactinemic patients may constitute indirect evidence of PRL-induced augmented TIDA activity [656–663]. In hyperprolactinemic patients, decreases in gonadotropin pulse amplitude and frequency are usually found, being attributed to altered gonadotropin releasing hormone secretion (see below). Such an alteration of GnRH secretion has been postulated to be due, in part, to PRL-induced DA increase [656,664–667]. In a direct test of this hypothesis, however, no evidence was found of suppression of TSH, LH or FSH levels with short term administration of human PRL which resulted in a two- to threefold elevation of PRL levels [668]. However, these acute studies do not rule out the possibility that such feedback may occur with more prolonged states of hyperprolactinemia. Alternatively, such feedback might occur via other mechanisms, such as a decrease in a PRF such as VIP [669]. PRL ACTION PRL has a great diversity of actions in many species of animals from fish and birds to mammals, including osmoregulation, growth and developmental effects, metabolic effects, actions on ectodermal and integumentary structures and actions related to reproduction [670]. However, in humans it has as its primary physiologic action the preparation of the breast for lactation in the postpartum period [671]. A number of effects of increased levels of PRL may be seen on many tissues. Although the roles of physiologic levels of PRL in such tissues are quite speculative, considerable clarification of these roles has been elucidated from studies in PRL-KO and PRL receptor KO mice.

PRL Receptors The PRL and GH receptors are members of the class 1 cytokine receptor family. The human genes for the PRL and GH receptors have been localized to the short arm of chromosome 5, sublocalizing to region 5p13–p14 [672]. The human PRL receptor gene has 10 exons, with exons 3 to 10 encoding the full length of the long form of the receptor [673]. Two PRL receptor gene promoters, termed hPIII and hPN that direct alternative first exons of the HPRL receptor gene have been found in the 5¢ untranslated region [673]. The human PRL receptor is 598 amino acids long,

Chapter 4

has a theoretical, non-glycosylated molecular mass of 66.9kDa and an additional signal peptide of 24 amino acids [674]. The extracellular domain contains five cysteine residues and three potential N-linked glycosylation sites [674]. There is a hydrophobic region (amino acids 211–234) which corresponds to the single transmembrane-spanning region of the receptor [674]. The human PRL receptor has a much longer intracytoplasmic region than does the rat PRL receptor [63] but there is high sequence homology between the rat, rabbit and human PRL receptors and also between the human PRL and GH receptors [63]. Two isoforms of the PRL receptor result from alternative splicing and differ in the length and composition of the cytoplasmic tail, being referred to as the long and intermediate forms; the short form found in the mouse is not present in humans [63,633]. PRL binds to its receptor with high affinity, the dissociation constant (Kd) being 10-10 mol/L [63]. Half-saturation of the receptor occurs at a hormone concentration of 7 ng/ml [63]. Once PRL binds to the receptor there is dimerization of the receptor, a necessary step for activation of the receptor (Fig. 4.13) [63]. PRL receptors are widely distributed, being found in the breast, pituitary, liver, kidney tubules, adrenal cortex, prostate, ovary, testes, seminal vesicles, epididymis, intestine, skin, pancreatic islets, lymphocytes, lung, myocardium and brain [63]. PRL release caused by suckling increases PRL receptor levels in the breast and liver, resulting in a much greater PRL binding activity in lactating animals compared to those not lactating [63]. Hepatic PRL receptor synthesis and numbers increase with estrogen treatment in vivo, mediated by the effect of estrogen on increasing pituitary PRL secretion [675] (Table 4.3). Interaction of PRL binding site 1, encompassing several amino acids belonging to helices one and four, with one PRL receptor causes the formation of a one-hormone-onereceptor complex. This is a prerequisite for PRL binding site 2, involving helices 1 and 3, to interact with another receptor, ultimately causing the formation of the active trimeric complex [63]. Signal transduction of the activated receptor involves the JAK-Stat pathway. JAK2 is the particular Janus kinase involved and it is constitutively associated with the PRL receptor [63]. PRL-induced dimerization of the two receptor molecules brings the two associated JAK2 molecules close to each other so that they may be activated by transphosphorylation of tyrosines [63]. The two cytoplasmic domains of the receptor must be strictly identical, and the short receptor functions as a dominant negative isoform which inhibits activation of the receptor complex by heterodimerization [63]. The activated JAK2 phophorylates tyrosine residues on the receptor itself and on three members of the Stat (signal transducer and activator of transcription) family of proteins, Stat 1, Stat 3 and, particularly, Stat 5 [63]. Activation of the MAP kinase cascade has also been reported after receptor activation but whether this involves JAK2 is not known [63]. Subsequently, the hormone–receptor complex is internalized and localized in the golgi and vacuoles [676] but the physiologic

Prolactin

143

FIGURE 4.13. (a) Ribbon representation of the predicted 3D structure of hPRL, modeled on the basis of the crystallographic structure of porcine GH. HPRL is predicted to adopt the four-helix bundle folding described for GH. Location of binding sites 1 and 2 is indicated. Side chains of amino acids involved in binding site 1, as deduced from mutational studies are represented. (b) Human prolactin receptor extracellular domain. The extracellular domain folds in a b-sandwich formed by two antiparallel b-sheets and Cterminal ends are indicated by N and C respectively. Note that the structures depicted in panels A and B are not at the same scale. (c) Prolactin receptor activation induced by PRL-induced dimerization. Hormone binding to PRL receptor is sequential. First, the hormone (H) interacts with the receptor (R) through its binding site 1 (see A), forming an inactive H1 : R1 complex. Then, the hormone binds to a second receptor through its site 2, which leads to receptor homodimerization and formation of an active H1 : R1 complex. Hormone analogs whose binding site 2 is sterically blocked are unable to induce receptor homodimerization and are thus inactive; since they still bind to the receptor through site 1, they behave as antagonists of wild-type hormones. From Bole-Feysot et al. [63]

significance of this beyond degradation and scavenging is not known.

PRL Effects on Breast PRL, GH, cortisol, insulin, estrogen, progesterone and thyroxine all contribute to breast development. The high concentrations of estrogen and progesterone produced by the placenta, coupled with the estrogen-induced high concentration of circulating PRL and the high concentrations of hPL cause development of the lobular, alveolar tissue during

144

SECTION 1

Hypothalamic–Pituitary Function

Table 4.3.

Summary of phenotypes of PRL receptor +/- and receptor -/- mice*

+/-

-/-

Reproduction Female

Normal mating, pregnancy

Male

Normal

Sterility Ovulation decreased Oocyte maturation decreased No implantation Delayed fertility

Mammary gland development

Impaired development, efficient lactation after first pregnancy

No lactation Ductal branching greatly reduced Absence of alveoli

Behavior Maternal Eating, locomotor

Reduced Normal

Absent Normal

Immunity

Normal

Normal

* Modified from Bole-Feysot et al. [63]. +/-, heterozygous PRL receptor knockout, -/-, homozygous PRL receptor knockout.

pregnancy [671]. Once the breast is fully developed and hormonally primed, PRL stimulates the production of milk proteins and other components [671]. Prior to term, the high estrogen levels suppress the effects of the high PRL levels on milk production but the rapid fall off of estrogen levels following delivery allows milk production to proceed [677]. Suppression of this physiologic hyperprolactinemia in the puerperium by bromocriptine causes a rapid cessation of milk production [678]. The key role of PRL in milk formation has been demonstrated by the finding that mice with either the PRL gene KO [653] or the PRL receptor KO [679] are unable to lactate. One intriguing finding is the production by the lactating breast of the parathyroid hormonelike protein that has been implicated in the humoral hypercalcemia of some malignancies [680]. Suckling results in an increase in serum PRL and the appearance of the mRNA for this parathyroid hormone-like protein in breast tissue, both effects being blocked by bromocriptine treatment [680]. This protein is not present in the circulation in significant amounts but is present in breast milk in high concentrations [681], suggesting that it may act within the breast to facilitate calcium transport into the milk. Galactorrhea

Clinically, nonpuerperal galactorrhea has been regarded as being a sign of possible hyperprolactinemia. The presence of even minute amounts of milk expressible from one or both breasts justifies the diagnosis of galactorrhea. Its persistence for more than 1 year after normal delivery and cessation of breast-feeding or its occurrence in the absence of pregnancy generally is taken as a definition of inappropriate lactation. If the material expressible from the nipple looks like milk, it probably is milk; if there is any question

of diagnosis, examination of the breast secretion by staining of fat globules with Sudan IV is diagnostic. The incidence of galactorrhea has been variously reported in normal women as ranging from 1% to 45% of subjects tested [682–685]. This variability is probably the result of differences in the techniques used to express milk from the breast and the way in which nonmilky secretions are classified. The volume of milk expressed does not correlate with PRL levels. However, in individuals with hyperprolactinemia, lowering the blood PRL level to normal will almost always lead to a marked decrease or abolition of lactation. Inappropriate lactation may be an important clue to the presence of pituitary–hypothalamic disease, especially if accompanied by amenorrhea. In a series of 70 women with galactorrhea studied in the author’s clinic, 19 (27%) had normal menses [686]. Of those with normal menses, only one had elevated PRL levels. Combined data from 14 published series suggest that 27.9% of galactorrheic women with normal menses have elevated PRL levels [687]. More recent experience, however, suggests that galactorrhea may be present in about 5% to 10% of normally menstruating women and basal PRL levels are normal in more than 90% of these women.

PRL Effects on Gonadotropin Secretion The effects on gonadotropin secretion by PRL levels in the normal range are not known. However, PRL gene KO and PRL receptor KO female mice are sterile. Both types have disordered estrous cycles [653,679]. Females with the PRL receptor KO had fewer primary follicles, fewer eggs ovulated, fewer eggs fertilized, those that were fertilized had

Chapter 4

poor progression to the blastocyst stage and finally the uterus was refractory to implantation by the blastocyst [679]. Furthermore, they had decreased estradiol and progesterone levels [688]. Half of the males with PRL receptor mutations were fully fertile, but the remainder were either completely or partially infertile despite normal sexual behavior and normal spermatogenesis as determined by histologic evaluation of the testes [679]. Male mice with the PRL gene KO were fully fertile and had normal plasma testosterone levels and normal testosterone release from the testes, despite decreased plasma LH levels and LH and FSH secretion from the pituitary in vitro and decreased weights of the seminal vesicles and ventral prostate [689]. In normal women treated with short-term bromocriptine to lower PRL levels to about 5 ng/ml, there was no change in the pulsatile secretion of LH and FSH, but estradiol levels were higher the last 3 days of the follicular cycle and progesterone levels were lower during the luteal phase [690]. Hyperprolactinemia has a number of effects on various steps in the reproductive axis (Fig. 4.14). Hyperprolactinemia has been found in most studies to suppress LH pulsatile secretion by decreasing pulse amplitude and frequency [664,665,693–695]. With castration in rats or menopause in humans, hyperprolactinemia can prevent the expected rise in gonadotropins [696–699]; normalization of PRL levels with bromocriptine results in elevation of gonadotropin levels and hot flashes [699]. Hyperprolactinemia inhibits pulsatile gonadotropin secretion by a number of mechanisms. It had been postulated that the pulsatile gonadotropin secretion was directed by the hypothalamic GnRH pulse generator and that alteration of pulsatile secretion necessarily meant a direct hypothalamic action. However, direct measurement of portal vessel GnRH levels in rats showed a marked inhibitory effect of hyperprolactinemia in one study [700] but not in another [701]. The pituitary gonadotroph response to GnRH in hyperprolactinemia has generally been found to be decreased in rats [695,696,701] and normal, increased and decreased in humans [702–704]. In our own series, the gonadotropin response to GnRH was normal in 22 of 25 patients and decreased in the remaining three [686]. GnRH plays a major role in the regulation of the number of its own recep-

Prolactin

145

tors on the gonadotroph cell. The number of GnRH receptors on gonadotroph cells in hyperprolactinemic rats are reduced [705] even when endogenous GnRH is replaced with intraarterial pulses of GnRH [695,706,707]. In addition to these effects, hyperprolactinemia in women has been associated with loss of positive estrogen feedback on gonadotropin secretion [708].

PRL Effects on the Ovary PRL has long been known to be trophic to corpus luteum function in rats, giving rise to the name luteotrophic hormone [709]. As noted above, the ovaries of PRL receptor KO mice have a decreased number of primary follicles with a decrease in the number of eggs ovulated [679] and decreased estradiol and progesterone production [688]. The role of PRL in normal ovarian function in humans is less well established, however. McNatty et al. [710] demonstrated that low physiologic concentrations of PRL are necessary for progesterone synthesis by human granulosa cells but that high concentrations are inhibitory in vitro. Recent studies suggest that PRL can activate the expression of Type II 3bhydroxysteroid dehydrogenase, the final enzymatic step in progesterone biosynthesis [711]. Del Pozo et al. [712] found no effect on luteal function in women treated with bromocriptine to lower normal PRL levels. However, Schulz et al. [713], Mühlenstedt et al. [714], and Kauppila et al. [690] found that such a decrease in PRL resulted in lowered progesterone levels and short luteal phases. On the other hand, short luteal phases have also been reported in hyperprolactinemic women [715]. In humans, plasma PRL levels > 100 ng/ml have been found to cause an increase of antral fluid PRL levels and reductions in antral fluid FSH and estradiol levels along with a decrease in granulosa cells [716]. Perfusion studies of human ovaries in vitro reveal a direct suppressive effect of PRL on progesterone and estrogen secretion [717]. PRL can inhibit estrogen formation by antagonizing the stimulatory effects of FSH on aromatase activity [718,719] and direct inhibition of aromatase synthesis has recently been shown [720]. PRL also inhibits LH-stimulated androgen synthesis by ovarian interstitial cells in rats [721], thereby

FIGURE 4.14. How hyperprolactinemia produces hypogonadism. Schematic representation of sites where hyperprolactinemia interferes with the reproductive system and sex steroid action. CNS, central nervous system; FSH, folliclestimulating hormone; LH, luteinizing hormone. From Odell [691], as modified by Malarkey [692]

146

SECTION 1

Hypothalamic–Pituitary Function

depriving the ovary of the substrate for conversion to estrogen. On the other hand, no differences in the amount of exogenous gonadotropins are necessary to induce ovulation [722,723]. PRL levels have been found to be elevated in 19% to 50% of women with polycystic ovaries (PCO) [724–728]. Bromocriptine treatment of hyperprolactinemic patients with PCO usually results in a reduction of testosterone and LH levels and resumption of ovulatory cycles. Why many patients with PCO have hyperprolactinemia is not clear. Del Pozo and Falaschi [726] have hypothesized that the increased estrogen levels found in PCO stimulate increased PRL secretion; however, no correlation has been found between estrone levels and PRL levels in these patients. The increased androgen secretion associated with PCO may cause hirsutism and other excessive androgen action such as acne and seborrhea [729], although these features may also be caused by excessive androgen secretion by the adrenal (see below). Clinical Effects of Hyperprolactinemia on Menstrual Function

When amenorrhea or oligoamenorrhea is associated with galactorrhea, it is usually a manifestation of hyperprolactinemia. In our series of 51 women with galactorrhea– oligo/amenorrhea, 84% had hyperprolactinemia [686]. In combined series totaling 471 patients with galactorrhea– amenorrhea, 75.4% were found to have hyperprolactinemia III [687]. Although the amenorrhea caused by hyperprolactinemia usually is secondary, it also can be primary if the disorder begins before the usual age of puberty. Of 33 patients presenting with primary amenorrhea and low gonadotropin levels, 9 (27%) were found to have hyperprolactinemia in two series [730,731]. However, in a third series of 38 patients with primary amenorrhea, PRL levels were normal in all [732]. In patients with primary amenorrhea due to hyperprolactinemia, estrogen deficiency and failure to develop normal secondary sexual characteristics may be the presenting problem. Galactorrhea is variable in this setting because the breast may not have been exposed to appropriate priming with estrogen and progesterone. Patients with primary amenorrhea tend to have macroadenomas more commonly than those with secondary amenorrhea, for uncertain reasons (see Prolactinoma, Chapter 12, p. 470). Hyperprolactinemia has been found in many women with a short luteal phase, as noted above. It is likely that a short luteal phase is the first evidence of interference in the normal cycle by hyperprolactinemia. Three hyperprolactinemic patients with amenorrhea corrected by bromocriptine studied by Seppala et al. [733] displayed the following sequence of events when bromocriptine was discontinued: (i) as the serum PRL levels increased, the luteal phase became shorter; (ii) ovulation was missed; and (iii) amenorrhea ensued. In the initial period of shortened luteal phase, progesterone levels were subnormal, suggesting deficient corpus luteum function.

Infertility also may be a presenting symptom of patients with hyperprolactinemia and is invariable when gonadotropin levels are suppressed with anovulation. In three series of women (combined number of 367 cases) studied for infertility, one-third were found to have hyperprolactinemia [687]. Most of the women had amenorrhea and galactorrhea as well, but in one series of 113 cases of infertility, five of the 22 hyperprolactinemic women had neither amenorrhea nor galactorrhea [734]. That PRL excess may be important in this type of patient is suggested by the finding that treatment of similar patients with bromocriptine restored fertility [729]. In some of these women, transient hyperprolactinemia lasting for 1–2 days during the cycle can be documented; this subset usually responds to bromocriptine with increased progesterone during the luteal phase and improved fertility [735]. Reduced libido and orgasmic dysfunction are found in most hyperprolactinemic amenorrheic women when such complaints are specifically elicited [686,736]. Reduction of PRL levels to normal restores normal libido and sexual function in most of these women [737].

PRL Effects on the Testes The role of PRL in normal testicular function is unclear. In hypophysectomized mice, Bartke [738] found that PRL increased the testicular spermatogenic response to LH and the administration of LH and PRL to such mice increases testosterone levels more than treatment with LH alone [739]. Suppression of normal PRL levels causes a decrease in binding of LH to LH receptors in Leydig cells [740]. In animals with hereditary PRL deficiency, PRL administration increases the activities of 17-beta-hydroxysteroid dehydrogenase and 3-beta-hydroxysteroid dehydrogenase, enzymes necessary for testosterone synthesis [741,742]. However, as noted above, the PRL gene KO mice had normal fertility and normal testosterone production despite decreased LH and FSH secretion [689], and half of the males with PRL receptor mutations were fully fertile with the remainder subfertile despite normal sexual behavior and normal spermatogenesis [679]. Suppression of normal PRL levels by bromocriptine in normal men for 8 weeks results in suppression of basal and hCG-stimulated testosterone levels, implying a physiologic role for PRL in normal testosterone production in humans [743]. Although PRL is present in human semen in very high concentrations [744] and PRL has been shown to stimulate adenyl cyclase, fructose utilization and glycolysis and glucose oxidation in human spermatozoa [745], other functions have not been investigated in detail. Chronic hyperprolactinemia in males results in impotence and decreased libido in over 90% of cases [746–754]. Other findings of hypogonadism, such as decreased beard growth and strength are less common [749,754]. Galactorrhea in men has been reported in 10% to 20% of cases and is virtually pathognomonic of a prolactinoma [746,749,751,754,755].

Chapter 4

In hyperprolactinemic men there is a decrease in the pulsatile secretion of LH and FSH, as noted above, and testosterone levels are low or are in the lower part of the normal range [746–754]. The testosterone response to stimulation with hCG has been reported to be both decreased [750,756] and normal [746,757]; in those with decreased responses there is improvement in the response when PRL levels are lowered with bromocriptine [750]. If there is sufficient normal pituitary tissue, reduction of elevated PRL levels to normal usually results in a return of normal testosterone levels [752,753,758,759]. Although some studies have suggested that drug-induced elevated PRL levels cause a partial block in the enzyme 5-alpha reductase, resulting in a decrease in dihydrotestosterone levels [760] this has not been found in studies of men with prolactinomas [756]. Carter et al. [746] noted that testosterone therapy of hyperprolactinemic men does not always correct the impotence until PRL levels are brought down to normal. Whether this is due to a decrease in DHT levels has not been verified directly. Sperm counts and motility are decreased with an increase in abnormal forms [757] and histological studies reveal abnormal seminiferous tubule walls and altered Sertoli cell ultrastructure [761]. The semen analysis does not always return to normal despite a return to normal of testosterone levels with restoration of normoprolactinemia [758]. A number of surveys have attempted to assess the frequency of hyperprolactinemia among men with complaints of impotence or infertility. Between 2% and 25% of males with impotence have been found to be hyperprolactinemic in various series [729,750,752,762–764]. However, only 1% to 5% of men with infertility have been found to be hyperprolactinemic [765–768]. Thus, although these frequencies are relatively low, the cost of the test is low in terms of money and risk and the condition is generally easily treatable, so that measurement of PRL levels is worthwhile in the investigation of the man complaining of infertility or impotence, especially if there is associated loss of libido or documented low testosterone levels.

Prolactin Effects on the Adrenal Cortex Although PRL receptors are found on cells of the adrenal cortex, the physiologic role of PRL in adrenal steroidogenesis is unknown. Plasma dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) levels have been found to be mildly elevated in about 50% of women with hyperprolactinemia in most [769–771] but not all series [772–774]. In most of these studies, however, the investigators did not try to correlate the androgen levels with the presence of hirsutism or other indices of virilization. Glickman et al. [774] and Vermuelen et al. [775] found low plasma sex hormone-binding globulin and elevated free testosterone levels in 43% of hyperprolactinemic patients, but increased DHEAS levels in only 19%. When the patients studied by Glickman et al. [774] were divided into those who were hirsute and those who were not, the hirsute patients had

Prolactin

147

higher free testosterone levels but not higher DHEAS levels, although both groups had patients with elevated levels of both hormones. The abnormal androgen levels return to normal with correction of the hyperprolactinemia by bromocriptine [776].

Effect of Prolactin on Bones PRL may have a physiologic role in calcium and bone metabolism. PRL has been shown to increase intestinal calcium absorption even in vitamin D deficient rats [777] and can stimulate 1-a hydroxylation of 25 hydroxyvitamin D in the kidney, resulting in increased plasma levels of 1,25(OH)2D [778]. In humans, however, plasma 1,25(OH)2D levels and intestinal calcium absorption are normal in hyperprolactinemic subjects [779]. The PRL receptor KO mouse has a decrease in bone formation rate and bone mineral density in association with increased parathyroid hormone (PTH) levels, but also decreased estradiol and progesterone levels [688] so that it is difficult to say how much of the effect on bone is due to the lack of PRL. 1,25(OH)2 enhances PRL gene expression with dependence on extracellular calcium [780,781] and treatment of rats with 1,25(OH)2D causes an increase in serum PRL levels [782]. Recent studies suggest that this 1,25(OH)2D mediated increase in PRL is mediated by enhancement of calcium transport through membrane calcium channels [783]. The initial observation by Klibanski et al. that hyperprolactinemic women have a decreased bone mineral density [784] was confirmed by others [785,786] but whether this effect is mediated by estrogen deficiency [784] or is a direct effect of the hyperprolactinemia [785,787] has been controversial. Correction of the hyperprolactinemia results in an increase in bone mass [788,789]. Studies of hyperprolactinemic women who were not amenorrheic and hypoestrogenemic have shown that their bone mineral density is normal [790,791], confirming the initial hypothesis that it is the estrogen deficiency that mediates the bone mineral loss. A similar, androgen-dependent loss of bone mineral is found in hyperprolactinemic men that is reversible with reversal of the hypoandrogenic state [792,793].

Effect of Prolactin on Carbohydrate Metabolism PRL receptors have been found in islet cells [794] but the role of PRL in normal carbohydrate homeostasis is not known. Patients with hyperprolactinemia with or without evidence of prolactinoma have mildly increased (still within the normal range) blood glucose levels during glucose tolerance tests compared with normal controls [795,796]. Insulin levels are also increased in these patients, indicating mild insulin resistance [795,796]. The insulin resistance is due to a decrease in the number of receptors rather than a decrease in receptor affinity [797]. Treatment with bromocriptine causes a return to normal of both glucose and insulin levels [795]. However, in normoprolactinemic

148

SECTION 1

Hypothalamic–Pituitary Function

subjects with insulin-dependent diabetes, suppression of PRL levels with bromocriptine had no effect on glucose control [798].

Effects of Prolactin on the Kidney An extensive body of literature documents the importance of PRL in osmoregulation in fish, amphibians, reptiles, birds and lower mammals [799]. PRL effects include increased tubular reabsorption of sodium, chloride and water, increased glomerular filtration rate, increased thirst, and increased intestinal absorption of sodium, chloride, calcium, water, glucose and amino acids [799]. A number of experiments in humans, however, have failed to document a significant role for PRL in fluid and electrolyte balance or of osmotic stimuli for PRL release [800–803].

Effects of Prolactin on the Immune System Normal PRL levels appear to be essential for normal immune function in rats, although the precise role of PRL in humans in this regard is not clear. In hypophysectomized rats, antibody production against sheep red blood cells, the skin response to dinitrochlorobenzene and the rejection of skin grafts are all decreased [804]. These immune responses could be restored with treatment of the hypophysectomized rats with rPRL, rGH, bPRL, bGH, hPL and hGH [805]. Hypoprolactinemia induced with bromocriptine results in impaired lymphocyte proliferation and decreased production of macrophage activating factors by lymphocytes as well as decreased tumoricidal activation of macrophages after infections; these effects could be reversed with ovine PRL administration to the rats [806]. Neutralization of PRL with anti-PRL antibodies inhibits the in vitro lymphocyte proliferative response to T and B cell mitogens in both rat and human preparations, but neutralization with antibodies to other pituitary hormones had no effect [807]. However, when the lymphocytes were cultured in serum-free medium, the anti-PRL antibodies similarly inhibited lymphocyte proliferation, implying the presence of a necessary, lymphocyte-produced PRL-like protein [807]. In other studies, in response to stimulation with conconavalin A, a PRL-like substance has been found to be produced from murine splenic mononuclear cells that reacts with anti-PRL antibodies and is mitogenic in the Nb2 node lymphoma bioassay [808] and mRNA obtained from such lymphocytes hybridizes with PRL and GH cDNA probes [809]. Furthermore, the immunosuppressive drug cyclosporin A has been found to compete with PRL for a common binding site on rat lymphocytes and stimulation of PRL secretion reverses the immunosuppression induced by cyclosporin [809]. Presumably, this rat lymphocytic PRL-like substance is analogous to the lymphoblastoid PRL produced by IM9 lymphocytes discussed above [235]. In preliminary studies with the PRL gene KO and receptor KO mice, no effects were seen on B or T lymphocyte production but more

detailed studies on immune function have not yet been carried out [63,653]. Lymphoblastoid prolactin can also be produced by conconavalin A or phytohemag-glutinin stimulated human peripheral blood mononuclear cells and has been shown to cause lymphoproliferation, a phenomenon blocked by antihuman PRL antibodies [810]. Human T lymphocytes contain the genes for hPRL and the PRL receptor, but the lymphocyte hPRL gene has an extra 5¢ noncoding exon similar to that found for decidual PRL [811]. PRL mRNA has been localized to a variety of human immune tissues, including thymus, spleen, tonsils, and lymph nodes using in situ hybridization; within these tissues the mRNA was detected in lymphocytes, epithelial cells and vascular cells [812]. PRL receptors have been found in thymus, bone marrow, spleen, lymph nodes and peripheral macrophages and B lymphocytes [813]. A variety of cytokines affect pituitary PRL secretion and may be mediators, in part, of the PRL response to stress. In various experimental paradigms, interleukin-2 (IL-2) and IL-6 have been found to increase PRL secretion, and IL-1 and interferon-gamma inhibit PRL secretion [814–816]. However, in vivo these substances have little effect on PRL secretion in rats [816]. Conversely, PRL acts synergistically with IL-2 to induce maturation of nature killer cells [813]. Prolactin may have some clinically relevant effects on the immune system as well. Hyperprolactinemia caused by injections of ovine PRL into mice caused an increase in phagocytosis, intracellular killing of salmonella typhimurium, chemotaxis and overall survival [817]. Moreover, hyperprolactinemia caused by implanting pituitaries under the renal capsule caused increased autoimmune phenomena in the b/w mouse model [818]. In human patients with prolactinomas, polymorphonuclear cells show reduced chemotaxis [819,820] but monocytes display significantly increased bactericidal activity against Mycobacterium avium [821]. Yet other studies have shown that hyperprolactinemic patients display decreased natural killer T cell function and this effect is reversed with bromocriptine treatment [822]. Data on RPL levels in patients with various autoimmune disorders are conflicting. Nagy et al. [823] found a low PRL bioactivity to immunoreactivity ratio in patients with rheumatoid arthritis due to serum factors capable of inhibiting PRL bioactivity. In other studies hyperprolactinemia has been found in substantial portions of men and women with systemic lupus erythematosus [824–831]; in some of these studies PRL levels correlated with the extent of clinical disease and antinuclear antibody activity [826,828]. In many of these studies, no information is given regarding renal function, CNS involvement or medication use, so that whether the hyperprolactinemia is truly related to autoimmune disease or other factors known to elevate PRL levels is not clear. However, in several studies [827,828,831], lupus nephritis, hypothyroidism, medications and other known causes of hyperprolactinemia were specifically excluded. In

Chapter 4

one study of lupus in children, three of 33 had elevated PRL levels but all three had evidence of CNS lupus [829]. In an interesting report, Funauchi et al. [832] describe a patient with SLE and a prolactinoma whose serum PRL levels were significantly correlated to the serum anti-DNA titers and inversely correlated to the serum complement activity and whose flares and remission clinically correlated with stopping and resuming bromocriptine treatment. In studies of another inflammatory condition, Jara et al. [833] found PRL levels to be elevated in 36% of patients with Reiter’s syndrome. Furthermore, Bravo et al. found that four patients with Reiter’s syndrome had dramatic clinical improvement when treated with bromocriptine [834]. Giasuddin et al. [835] also reported elevated levels in patients with psoriasis. The clinical relevance of lymphoblastoid PRL and changes in immune function in normal individuals and those with hyperprolactinemia is still unclear. Although there is strong evidence in rodents that PRL is important in this regard, the evidence in humans is still very preliminary and what does exist is often contradictory and suggests that these effects of PRL are relatively minor compared to those of other immune regulators [836]. Nonetheless, the intriguing finding of increased PRL levels in many patients with autoimmune rheumatologic diseases merits continued study. PATHOLOGIC STATES OF PROLACTIN SECRETION Population studies of normal individuals reveal that PRL levels are not “normally” distributed and better fit a lognormal distribution contaminated by a small number of abnormally high measurements. Several studies have suggested that “elevated” PRL levels may be found in 1% to 10% of a random sample of individuals without evidence of endocrine abnormality [837–839]. When the frequency of abnormality in such a randomly sampled population is so high, it makes it difficult to determine what indeed is the upper limit of normal for that population and a number of mathematical formulations have been tried (for more complete discussions of this problem, see [837] and [840]). As discussed previously, PRL is secreted episodically, resulting in varying blood PRL levels throughout the day. Therefore, a PRL level in a single sample that is in the upper part of the normal range should be confirmed with one or two repeated tests before considering it to be abnormal.

Hypoprolactinemia As noted above, female mice with the PRL receptor KO are infertile with a number of defects occurring, including fewer primary follicles, fewer eggs ovulated, fewer eggs fertilized, impaired progression to the blastocyst stage, poor implantation and decreased estradiol and progesterone levels [679,688]. Male mice with the PRL receptor KO had only minimally impaired fertility [679] and those with the PRL gene KO had normal fertility [689].

Prolactin

149

Idiopathic PRL deficiency has been described in only a single human [841]. This was a 30-year-old woman who had had a minimally delayed puberty (thelarche, age 15 and menarche, age 17), regular menses and normal body hair and breast development who conceived but was unable to lactate postpartum. Her basal PRL levels were all <3 ng/ml, during pregnancy her highest PRL level was 7.8 ng/ml and during the first few days postpartum her highest PRL level was 11.7 ng/ml. After TRH stimulation her PRL levels increased from 3.8 to 13.2 ng/ml. Her pituitary function was otherwise normal. PRL levels in her two children and other family members were normal. The ability of this woman to develop primary and secondary sexual characteristics normally and then conceive and deliver a child speak to the minimal, if any, importance of PRL in normal reproductive function, save that of postpartum lactation. No cases have been described analogous to the PRL gene or receptor KO mice. PRL deficiency may occur in the setting of panhypopituitarism, generally as a result of pituitary infarction. When the cause of the hypopituitarism is hypothalamic or stalk dysfunction, PRL levels generally rise due to disinhibition of PRL secretion (see below). However, when the pituitary tissue is actually destroyed, as in Sheehan’s peripartum necrosis, PRL levels are usually low [842]. In this latter circumstance, the hypoprolactinemia is manifest as an inability to lactate postpartum along with other symptoms of hypopituitarism. A deficiency of PRL may also occur in association with deficiencies of GH and TSH due to mutations in the Pit-1 and Prop-1 transcription factors necessary for tissuespecific PRL gene activation [46,47,49–51] (see above). PRL levels are also low in the condition of hypoparathyroidism [843]. Presumably this in some way reflects the importance of calcium as an intracellular mediator of PRL secretion, but the specific details of this have not been elucidated.

Hyperprolactinemia The differential diagnosis of sustained hyperprolactinemia is broad (Table 4.4). This section discusses those causes of hyperprolactinemia other than prolactinomas, which are discussed in Chapter 12. Medications

Neuroleptics The neuroleptic agents (phenothiazines and butyrophenones) uniformly result in elevated RPL levels, generally to less than 100 ng/ml but some patients have been reported with levels as high as 365 ng/ml [844,845]. Levels begin to rise within a few hours of starting therapy, continue to rise for 6 to 9 days, and then fall to lower, though still elevated levels after long-term treatment [844,845]. Chlorpromazine, sulpiride, and metoclopramide, major tranquilizers and antiemetics with potent dopamine antagonist action, have in fact been used as diagnostic agents to evaluate PRL secretory reserve. Neuroleptic agents raise PRL levels by blocking dopamine receptors, both on the

150

SECTION 1

Table 4.4.

Hypothalamic–Pituitary Function

Etiologies of hyperprolactinemia

Pituitary disease

Medications

Prolactinomas Acromegaly “Empty sella syndrome” Lymphocytic hypophysitis Cushing’s disease

Phenothiazines Haloperidol Monoamine-oxidase inhibitors Tricyclic antidepressants Reserpine Methyldopa Metoclopramide Amoxepin Cocaine Verapamil Serotonin reuptake inhibitors

Hypothalamic disease Craniopharyngiomas Meningiomas Dysgerminomas Nonsecreting pituitary adenomas Other tumors Sarcoidosis Histiocytosis X Neuraxis irradiation Vascular Pituitary stalk section Neurogenic Chest wall lesions Spinal cord lesions Breast stimulation

Other Pregnancy Hypothyroidism Chronic renal failure Cirrhosis Adrenal insufficiency Pseudocyesis Idiopathic

pituitary lactotrophs and in the hypothalamus. PRL levels usually fall to normal within 48 to 96 hours of discontinuation of neuroleptic drug therapy [844]. Combined serotonin/dopamine receptor antagonists such as risperidol cause similar elevations of PRL [846]. Antidepressants Tricyclic antidepressants cause modest hyperprolactinemia in about 25% of patients [847]. Monoamine oxidase (MAO) inhibitors, when used chronically, may also cause a minimal elevation of PRL levels [638]. The mechanisms by which these drugs cause increased PRL levels are not certain; they probably facilitate several possible stimulatory pathways and their effects on portal vessel dopamine are uncertain. Serotonin reuptake inhibitors, by increasing synaptic serotonin levels, commonly cause a mild increase in PRL to levels still within the normal range; rarely does hyperprolactinemia occur [489,848,849]. Opiates and Cocaine Chronic opiate abuse is associated with mild hyperprolactinemia and menstrual dysfunction (see above). Cocaine abuse has also been associated with chronic, mild hyperprolactinemia [850]. Antihypertensive Drugs Alpha-methyldopa causes moderate hyperprolactinemia [639] by reducing the effect level of dopamine that reaches the pituitary by inhibiting the enzyme l-aromatic amino acid decarboxylase, which is responsible for converting l-dopa to dopamine and by acting as a false neurotransmitter to decrease dopamine synthesis. Reserpine, a little-used antihypertensive drug now,

causes hyperprolactinemia by interfering with the storage of hypothalamic catecholamines in secretory granules [851]. ACE inhibitors, such as enalapril, facilitate PRL release in some individuals [582a] but sustained hyperprolactinemia causing symptoms has not been reported with their use. Calcium Channel Blockers Verapamil acutely and chronically, in most but not all experiments, has been found to increase basal PRL secretion and the PRL response to TRH [172,852,853] and patients have been described with galactorrhea associated with sustained hyperprolactinemia due to verapamil [171] (Fig. 4.15). In a survey of patients taking verapamil in an outpatient clinic, PRL levels were found to be elevated in 8.5% of patients [854]. Verapamil does not block the PRL inhibition caused by graded lowdose dopamine infusion, thereby showing lack of effect at the lactotroph [173]. However, it does block the hypothalamic generation of dopamine, as judged by finding decreased inhibition of PRL secretion by l-dopa after pretreatment with the peripheral dopa-decarboxylase inhibitor carbidopa, a maneuver that allows the assessment of central dopamine generation [173]. Interestingly, other calcium channel blockers such as the dihydropyridines and benzothiazepines have no action on PRL secretion, implying that the action of the phenylakylamine, verapamil, likely is acting on the neuronal N-type calcium channel [173]. Protease Inhibitors A recent report has shown that some HIV positive patients treated with protease inhibitors have developed galactorrhea and hyperprolactinemia [854a]. The mechanism for this is unknown at present. Stress

As noted previously, physical stress such as physical discomfort, exercise and hypoglycemia causes an acute, transient rise in PRL levels (see above). Chronic hyperprolactinemia due to prolonged physical stress has not been reported. Psychologic stress may cause minimal elevations of PRL [855] but chronic hyperprolactinemia has not been reported with any chronic psychiatric state except that of pseudocyesis, in which PRL levels fall with psychotherapy [856]. Renal disease

Hyperprolactinemia occurs in 73% to 91% of women and 25% to 57% of men with end-stage renal disease [857–859] (Fig. 4.16). PRL suppression by dopamine infusion is decreased [857] but this may be, at least in part, the result of delayed degradation of PRL in renal failure, since longterm use of bromocriptine results in suppression of PRL levels [858]. Although metabolic breakdown of PRL is delayed in renal failure, there is also increased production [264]. Therefore, there is disordered regulation of PRL secretion and possibly short-loop feedback, although the exact nature of the defect has not been defined. About onequarter of individuals with renal insufficiency not requiring dialysis (serum creatinine 2.0–12.0 ng/ml) have PRL levels in the 25–100 ng/ml range [800] (Fig. 4.17). When such

Chapter 4

Prolactin

151

FIGURE 4.15. (a) Effect of verapamil on the prolactin (PRL) response to a submaximal dose (100 mg) of thyrotropinreleasing hormone given at time 0. The open boxes represent the values before verapamil and the filled boxes represent the values after 1 week of verapamil (240 mg daily, sustained release). Note the elevated basal values. (b) Effect of verapamil on the suppression of the PRL by L-dopa (500 mg). The open boxes represent the values before verapamil and the filled boxes represent the values after 1 week of verapamil (240 mg daily, sustained release). Despite the elevated basal values after verapamil, the precentage suppressions were similar. From Kamal and Molitch [172]

FIGURE 4.16. Prolactin levels in patients requiring dialysis. CAPD, continuous ambulatory peritoneal dialysis. From Hou et al. [859]

FIGURE 4.17. Prolactin levels in patients with renal insufficiency, including patients on medications (M, metoclopramide; all others designated receiving medications were taking methyldopa). From Hou et al. [859]

has occurred in some women treated with bromocriptine [858], but reports of restored ovulation are rare [857]. patients take medications known to alter hypothalamic regulation of PRL, such as methyldopa or metoclopramide, PRL levels may rise to over 2000 ng/ml [859]. Correction of the renal failure with transplantation causes a return of PRL levels to normal [857,859]. Hyperprolactinemia plays a role in the hypogonadism of chronic renal failure, but probably does not explain all of the abnormalities in every case. Return of normal menses

Cirrhosis

Basal PRL levels are increased in patients with alcoholic cirrhosis in frequencies varying from 16% to 100% [860–862] and in patients with nonalcoholic cirrhosis from 5% [863] to 13% [860]. In a study by Van Thiel et al. [861] the PRL response to TRH was normal in 75% of the hyperprolactinemic men with alcoholic cirrhosis and blunted in

152

SECTION 1

Hypothalamic–Pituitary Function

the remainder. In another study, PRL responses to TRH were greatly augmented in patients with a variety of types of cirrhosis [864]. In a third study, 50% of patients with hepatic encephalopathy were found to be hyperprolactinemic [865]. Although l-dopa caused a decrease in PRL levels in all, inhibition of peripheral decarboxylase with carbidopa resulted in an impaired PRL response to l-dopa, implying an underlying defect in hypothalamic dopamine generation as the cause of the hyperprolactinemia in these encephalopathic patients. In women without cirrhosis, acute alcohol withdrawal is associated with hyperprolactinemia in 50% [866]. Hypothyroidism

Primary hypothyroidism is associated with a modest increase in the level of PRL in 40% of patients, but levels greater than 25 ng/ml are reached in only 10% [867]. The mechanisms involved probably include increased TRH production, increased sensitivity of lactotrophs to TRH, and possibly increased pituitary VIP generation (see above). Because many patients with long-standing hypothyroidism may have evidence of pituitary enlargement on x-ray, the finding of hyperprolactinemia, galactorrhea and/or amenorrhea associated with an enlarged pituitary seen in hypothyroidism may be easily confused with a prolactinoma. Therapy with Lthyroxine will cause the PRL levels to return to normal and can even result in a regression of pituitary size [868]. Adrenal Insufficiency

As discussed above, glucocorticoids have a suppressive effect on PRL gene transcription and PRL release. Adrenalectomy has been reported to cause hyperprolactinemia in animals [869]. Rare cases in humans have been reported of hyperprolactinemia occurring in patients with adrenal insufficiency in whom the PRL levels return to normal with glucocorticoid replacement [870–874]. Neurogenic

As discussed above, sexual breast stimulation and suckling cause a reflex release of PRL that is mediated, in part, by afferent neural pathways going through the spinal cord. Chest wall and cervical cord lesions have been reported to result in elevated PRL levels and galactorrhea through stimulation of these afferent neural pathways [875,876]. Similar chronic elevations of PRL have been reported after mastectomy and thoracotomy [877] and with chronic spinal cord injuries [878]. Ectopic Prolactin Secretion

Ectopic production of PRL is exceedingly rare. In a careful evaluation of 215 patients with a variety of malignancies, PRL elevations were found in only 15 and in 12 of these the elevations could be explained by the use of phenothiazines, opiates, or prior irradiation to the chest wall or head [879]. In one of the remaining three patients subsequent samples all had normal PRL measurements and in the other two, the cancer (lung and breast) could also have caused the

modest hyperprolactinemia by stimulation of chest wall afferent nerves. However in another series of patients with uterine cervical carcinoma, 229 out of 743 patients had elevated serum PRL levels; surgery in 86 of these patients resulted in normalization of PRL levels [880]. In this same study, 22 out of 49 cervical carcinomas stained positively for PRL by immunohistochemistry and 5 out of 8 carcinomas produced PRL when cultured [880]. In none of these cases were all of these aspects put together, i.e. elevated serum levels that normalized after surgery and whose tumors both stained for PRL and produced PRL in vitro. However, symptomatic hyperprolactinemia due to well-documented PRL production from a renal cell carcinoma [881], a gonadoblastoma [882] and ectopic pituitary tissue in two ovarian teratomas [883,884] have been reported. Given the great frequency of prolactinomas, “idiopathic hyperprolactinemia” and other causes of hyperprolactinemia, a search for an ectopic source of PRL secretion is not warranted unless some other tumor shows up coincidentally. Hypothalamic/Pituitary Stalk Disease

Hyperprolactinemia caused by lesions of the hypothalamus and of the pituitary stalk is due to disturbance of the neuroendocrine mechanisms that control PRL secretion. From hypothalamic lesion work in animals, it has been assumed that this PRL elevation is due to disinhibition of the tonic PIF (dopamine) acting at the level of the pituitary lactotrophs. PRL secretory dynamics were studied systematically in a group of such patients with hypothalamic hyperprolactinemia (six with hypothalamic lesions such as craniopharyngiomas, eosinophilic granuloma), eight with large “nonfunctioning” pituitary adenomas with marked suprasellar extension, and seven with partially empty sellas [885]. Basal PRL levels were <100 ng/ml in all but 1 (Fig. 4.18). However, in one patient with a Rathke’s cleft cyst, preoperative PRL levels ranged between 93 and 148 ng/ml; she had normal TSH and ACTH function but following transsphenoidal resection she developed diabetes insipidus and panhypopituitarism, indicating high stalk section, and PRL levels fell to 25–28 ng/ml. Interestingly, of seven of these patients tested with insulin-induced hypoglycemia, three had normal PRL responses and seven out of 12 showed a significant inhibition in response to L-dopa even after peripheral conversion to dopamine was blocked by carbidopa pretreatment. Furthermore, most of these patients had normal TSH and ACTH function. These results imply that there still is significant transmission of hypothalamic releasing factors to the pituitary in most of these cases, despite increased PRL levels. The fall in PRL levels in the patient with the Rathke’s cleft cyst, along with the development of a true stalk section syndrome, implies that there is a difference in patients with total stalk section in whom the PRL rise is due solely to dopamine deficiency and those with partial stalk/hypothalamic dysfunction who have dopamine deficiency plus continued PRF activity, resulting in higher PRL levels. In addition to this case with a Rathke’s

Chapter 4

Prolactin

153

FIGURE 4.18. Basal prolactin levels in patients with hypothalamic disease. From Molitch and Reichlin [900]

cleft cyst with a basal PRL level of 122 ng/ml a number of cases have been reported in the literature by others with PRL levels between 104 and 219 ng/ml [886–891]. A case of a patient with a PRL of 662 ng/ml whose tumor did not stain for PRL has been well-documented [892], but cases with such high PRL levels must be extraordinarily rare. It is also possible that some cases of hyperprolactinemia due to hypothalamic disease could be due to increased PRF activity with normal PIF activity. Anterior hypothalamic lesions that are irritative could potentially stimulate PRF pathways, similar to findings in experimental animals [893,894]. The findings of a normal suppression of PRL with l-dopa alone and combined with carbidopa in two of our patients supports the concept of an intact TIDA system in these two patients [885]. Idiopathic Hyperprolactinemia

When no specific cause is found with the evaluation outlined below, the hyperprolactinemia is of uncertain etiology and has been designated to be idiopathic. It is recognized that in many such cases small prolactinomas that are too small to be detected by current radiologic techniques may be present. In other cases the hyperprolactinemia is due to presumed hypothalamic regulatory dysfunction, but no dysfunction specific to idiopathic hyperprolactinemia has been definitively elucidated. Long-term follow-up of such patients has found that in about one-third, PRL levels return to normal, in 10% to 15% there is a rise in PRL levels to >50% over baseline, and in the remaining patients prolactin levels remain stable [895,896]. Over a 2 to 6 year followup of 199 patients, only 23 developed evidence of microadenomas and none developed macroadenomas [895–899]. CLINICAL TESTING As mentioned above, PRL is secreted episodically and some PRL levels during the day may be above the upper limit of

normal established for a given laboratory. Thus the finding of minimally elevated levels in blood requires confirmation in several samples. As indicated in the previous section, there are a number of conditions that may cause moderate PRL elevations, although generally to levels <250 ng/ml. A careful history and physical examination, screening blood chemistries, thyroid function tests and a pregnancy test will exclude virtually all causes except for hypothalamic– pituitary disease. When there is no obvious cause of the hyperprolactinemia from the routine screening, a radiologic evaluation of the hypothalamic–pituitary area is mandatory to exclude a mass lesion. This includes patients with even mild PRL elevations. At present, such an evaluation is generally done using magnetic resonance imaging (MRI) with gadolinium enhancement or computed tomography (CT) with intravenous contrast enhancement (see Chapter 20). It should be emphasized here that it is very important to distinguish between a large nonsecreting tumor causing modest PRL elevations (usually <250 ng/ml, see above) from a PRL-secreting macroadenoma (PRL levels usually >>250 ng/ml), as the therapy may be quite different. PRLsecreting macroadenomas generally will respond quite readily to bromocriptine and other dopamine agonists with size reduction (see Chapter 11, p. 425) whereas only about 10% of nonsecreting pituitary tumors respond in this manner. Stimulation and suppression tests give nonspecific results. Although blunted PRL responses to TRH (Fig. 4.19) and hypoglycemia (Fig. 4.20) are characteristic of patients with prolactinomas, such blunted responses are also seen in patients with hypothalamic dysfunction and other causes of hyperprolactinemia and normal PRL responses may be seen in 10% to 15% of patients with prolactinomas [900–902]. Similar nonspecific results have been found with chlorpromazine, metoclopramide, nomifensine, and l-dopa and also carbidopa [900–902]. Thus, consensus has developed that such stimulation and suppression tests are worthless in the

154

SECTION 1

Hypothalamic–Pituitary Function

FIGURE 4.19. Prolactin responses to thyrotropin-releasing hormone in patients with hyperprolactinemia of different etiologies. Note the marked blunting of the responses of all categories of hyperprolactinemic patients in comparison to those of normal controls. From Molitch and Reichlin [900]

FIGURE 4.20. Prolactin responses to insulin-induced hypoglycemia in patients with hyperprolactinemia of different etiologies. Note the marked blunting of the responses of all categories of hyperprolactinemic patients in comparison to those of normal controls. From Molitch and Reichlin [900]

differential diagnosis of hyperprolactinemia and reveal no more information than just measurement of basal PRL levels [900–902]. REFERENCES 1 Stricker P, Grueter F. Action du lobe anterieure de l’hypophyse sur la montee laitesse. C R Soc Biol 1928;99:1978–1980. 2 Corner GW. The hormonal control of lactation. I. Non-effect of the corpus luteum. II. Positive action of extracts of the hypophysis. Am J Physiol 1930;95:43–55. 3 Riddle O, Braucher FP. Studies on the physiology of reproduction in birds. Am J Physiol 1931;97:614–625. 4 Riddle O, Bates WR, Dykshorn WS. A new hormone of the anterior pituitary. Proc Soc Exptl Biol Med 1932;29:1211–1212. 5 Riddle O, Bates WR, Dykshorn WS. The preparation, identification and assay of prolactin – a hormone of the anterior pituitary. Am J Physiol 1933;105: 191–216.

6 Nicoll SC. Bio-assay of prolactin: analysis of the pigeon crop-sac response to local prolactin injection by an objective and quantitative method. Endocrinology 1967;80:641–655. 7 Li CH, Dixon JS, Lo T-B, Pankov YA, Schmidt KD. Amino-acid sequence of ovine lactogenic hormone. Nature 1969;244:695–696. 8 Kwa HG, Verhofstad F. Radioimmunoassay of rat prolactin. Biol Biophys Acta 1967;133:186–188. 9 Arai Y, Lee TH. A double-antibody radioimmunoassay procedure for ovine pituitary prolactin. Endocrinology 1967;81:1041–1046. 10 Bryant DG, Greenwood FC. Radioimmunoassay for ovine, caprine and bovine prolactin in plasma and tissue extracts. Biochem J 1968;109:831– 840. 11 Chardwick A, Folley SJ, Gemzell CA. Lactogenic activity of human pituitary growth hormone. Lancet 1961;2:241–243. 12 Ferguson KA, Wallace ALC. Prolactin activity of human growth hormone. Nature 1961;190:632–633. 13 Forbes AP, Henneman PH, Griswold GC, Albright F. A syndrome characterized by galactorrhea, amenorrhea and low urinary FSH: comparison with acromegaly and normal lactation. J Clin Endocrinol Metab 1954;14:265– 272.

Chapter 4 14 Rimoin DL, Holzman GB, Merimee TJ, Rabinowitz D, Barnes AC, Tyson JEA, McKusick VA. Lactation in the absence of human growth hormone. J Clin Endocrinol Metab 1968;28:1183–1188. 15 Frantz AG, Kleinberg DL. Prolactin: Evidence that it is separate from growth hormone in human blood. Science 1970;170:745–747. 16 Hwang P, Guyda H, Friesen H. A radioimmunoassay for human prolactin. Proc Nat Acad Sci USA 1971;68:1902–1906. 17 Sinha YN, Selby FW, Lewis UJ, Vanderlaan WP. A homologous radioimmunoassay for human prolactin. J Clin Endocrinol Metab 1973;36:509–516. 18 Shome B, Parlow AF. Human pituitary prolactin (hPRL) the entire linear amino acid sequence. J Clin Endocrinol Metab 1977;45:1112–1115. 19 Cooke NE, Coit D, Shine J, Baxter JD, Martial JA. Human prolactin, cDNA structural analysis and evolutionary comparisons. J Biol Chem 1981;256: 4007–4016. 20 Zimmerman EA, Defendini R, Frantz AG. Prolactin and growth hormone in patients with pituitary adenomas. A correlative study of hormone in tumor and plasma by immunoperoxidase technique and radioimmunoassay. J Clin Endocrinol Metab 1974;38:577–585. 21 Halmi NS, Parson JA, Earlandsen SL, Duello T. Prolactin and growth hormone cells in the human hypophysis. A study with immunoenzyme histochemistry and differential staining. Cell Tiss Res 1975;158:497–507. 22 Kovacs K. The morphology of abnormal lactotrophs. In: Mena F, Valverde-R C, eds. Prolactin Secretion: a Multidisciplinary Approach. New York: Academic Press, Inc., 1984:353–369. 23 Goluboff LH, Ezrin C. Effect of pregnancy on the somatotroph and the prolactin cell of the human adenohypophysis. J Clin Endocrinol Metab 1969;29:1533–1541. 24 Scheithauer BW, Sano T, Kovacs KT, Young WF Jr, Ryan N, Randall RV. The pituitary gland in pregnancy. A clinicopathologic and immunohistochemical study of 69 Cases. Mayo Clin Proc 1990;65:461–474. 25 Horvath E, Kovacs K. Pathology of the pituitary gland. In: Kovacs K, Ezrin C, Weiss MH, Horvath E, Kaufman B eds. Pituitary Diseases. Boca Raton, Florida: CRC Press, 1980:1–83. 26 Pelletier G, Antakly T, Labrie F. The prolactin cell: structure and function. In: Mena F, Valverde-R C, eds. Prolactin Secretion: a Multidisciplinary Approach. New York: Academic Press, Inc., 1984:75–92. 27 Zurschmiede C, Landolt AM. Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis. Virchows Arch B 1987;53:308–315. 28 Neill JD, Frawley LS. Detection of hormone release from individual cells in mixed populations using a reverse hemolytic plaque assay. Endocrinology 1983;112:1135–1138. 29 Luque EH, De Toro MM, Smith PF, Neill JD. Subpopulations of lactotropes detected with the reverse hemolytic plaque assay show differential responsiveness to dopamine. Endocrinology 1986;118:2120–2184. 30 Boockfor FR, Hoeffler James P, Frawley SL. Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries: Evidence for functional heterogeneity within rat mammotrope and somatotrope populations. Neuroendocrinology 1986;42:64–70. 31 Boockfor FR, Frawley SL. Functional variations among prolactin cells from different pituitary regions. Endocrinology 1987;120:874–879. 32 Frawley SL, Boockfor FR, Hoeffler JP. Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 1985;116:734–737. 33 Leong DA, Lau SK, Sinha YN et al. Enumeration of lactotropes and somatotropes among male and female pituitary cells in culture. Evidence in favor of a mammosomatotrope subpopulation in the rat. Endocrinology 1985;116:1371–1378. 34 Hoeffler JP, Boockfor FR, Frawley LS. Ontogeny of prolactin secretors also release growth hormone. Endocrinology 1985;117:187–195. 35 Boockfor FR, Hoeffler JP, Frawley SL. Estradiol induces a shift in cultured cells that release prolactin or growth hormone. Am J Physiol 1986;250: 103–106. 36 Hoeffler JP, Frawley SL. Hypothalamic factors differentially affect the proportions of cells that secrete growth hormone or prolactin. Endocrinology 1987;120:791–795. 37 Mulchahey JJ, Jaffe RB. Detection of a potential progenitor cell in the human fetal pituitary that secretes both growth hormone and prolactin. J Clin Endocrinol Metab 1988;66:24–32. 38 Treier M, Rosenfeld MG. The hypothalamic-pituitary axis: co-development of two organs. Curr Opin Cell Biol 1996;8:833–843. 39 Treier M, Gleiberman AS, O’Connell SM et al. Multistep signaling requirements for pituitary organogenesis in vivo. Genes and Dev 1998;12: 1691–1704.

Prolactin

155

40 Dattani MT, Martinez-Barbera J-P, Thomas PQ et al. Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nature Genetics 1998;19:125–130. 41 Wallis DE, Roessler E, Hehr U et al. Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nature Genetics 199;22:196–198. 42 Sheng HZ, Zhadanov AB, Mosinger B Jr et al. Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 1996;272:1004–1007. 43 Bach I, Rhodes SJ, Pearse RV II et al. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergies with Pit-1. Proc Natl Acad Sci USA 1995;92:2720–2724. 44 Gaiddon C, de Tapia M, Loeffler J-P. The tissue-specific transcription factor Pit-1/GHF-1 binds to the c-fos serum response element and activates c-fos transcription. Mol Endocrinol 1999;13:742–751. 45 Li S, Crenshaw EBI, Rawson EJ et al. Dwarf locus mutants, which lack three pituitary cell types, result from mutations in the POU domain gene, Pit-1. Nature 1990;347:528–533. 46 Pf äffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 47 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 48 Sornson MW, Wu W, Dasen JS et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327–333. 49 Wu W, Cogan JD, Pfäffle RW et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genetics 1998;18:147–149. 50 Cogan JD, Wu W, Phillips JA III et al. The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency. J Clin Endocrinol Metab 1998;83:3346–3349. 51 Rosenbloom AL, Almonte A, Brown MR et al. Clinical and biochemical phenotype of familial anterior hypopituitarism from mutation of the PROP1 gene. J Clin Endocrinol Metab 1999;84:50–57. 52 Burrows HL, Birkmeier TS, Seasholtz AF, Camper S. Targeted ablation of cells in the pituitary primordial of transgenic mice. Mol Endocrinol 1996;10: 1467–1477. 53 Voss JW, Rosenfeld MG. Anterior pituitary development: short tales from dwarf mice. Cell 1992;70:527–530. 54 Schaufele F. Regulation of estrogen receptor activation of the prolactin enhancer/promoter by antagonistic activation of function-2-interacting proteins. Mol Endocrinol 1999;13:935–945. 55 Prager EM, Wilson AC, Lowenstein JM, Sarich VM. Genes for growth hormone, chorionic somatomammotropin, and growth hormone – like gene on chromosome 17 in humans. Science 1980;209:289–292. 56 Owerbach D, Rutter WJ, Cooke NE et al. The prolactin gene is located on chromosome 6 in humans. Science 1981;212:815–816. 57 Cooke NE, Szpirer C, Levan G. The related genes encoding growth hormone and prolactin have been dispersed to chromosomes 10 and 17 in the rat. Endocrinology 1986;119:2451–2454. 58 Niall HD, Hogan ML, Sauer R et al. Sequences of pituitary and placental lactogenic and growth hormones: Evolution from a primordial peptide by gene reduplication. Proc Nat Acad Sci USA 1971;68:866–870. 59 Walter LM, Norman LE. Structure and evolution of the growth hormone gene family. Endocrine Reviews 1983;4:97–130. 60 Chien YH, Thompson EB. Genomic organization of rat prolactin and growth hormone genes. Proc Natl Acad Sci USA, Biochemistry 1980;77: 4583–4587. 61 Truong AT, Duez C, Belayew A, Renard A. Isolation and characterization of the human prolactin gene. EMBO J 1984;3:429–437. 62 Cooke NE, Coit D, Weiner RI et al. Structure of cloned DNA complementary to rat prolactin messenger RNA. J Biol Chem 1980;255: 6502–6510. 63 Bole-Feysot C, Goffin V, Edery M et al. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocrine Revs 1998;19:225–268. 64 Rottman F, Camper S, Goodwin E et al. Structure and regulated expression of bovine prolactin and bovine growth hormone genes. Adv Exptl Med Biol 1985;205:281–299. 65 Wolf JB, David VA, Deutch AH. Identification of a distal regulatory element in the 5¢ flanking region of the bovine prolactin gene. Nucl Acids Res 1990; 18:4905–4911. 66 Nelson C, Albert VR, Elsholtz HP, Lu LIW, Rosenfeld MG. Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 1988;239:1400–1405.

156

SECTION 1

Hypothalamic–Pituitary Function

67 Ingraham HA, Albert VR, Chen R et al. A family of pou-domain and pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development. Annu Rev Physiol 1990;52:773–791. 68 Elsholtz HP, Mangalam HJ, Potter E et al. Two different cis-active elements transfer the transcriptional effects of both EGF and phorbol esters. Science 1986;234:1552–1557. 69 Day RN, Maurer RA. The distal enhancer region of the rat prolactin gene contains elements conferring response to multiple hormones. Mol Endocrinol 1989;3:3–9. 70 Keech CA, Gutierrez-Hartmann A. Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3¢,5¢-cyclic adenosine monophosphate-regulated gene expression in vivo. Mol Endocrinol 1989;3: 832–839. 71 Maurer RA, Kim KE, Day RN, Notides AC. Regulation of prolactin gene expression by estradiol. In: Sato GH, Stevens JL, eds. Molecular Endocrinology and Steroid Hormone Action. New York, Alan R. Liss, Inc., 1990:159–169. 72 Lemaigre FP, Peers B, Lafontaine DA et al. Pituitary-specific factor binding to the human prolactin, growth hormone, and placental lactogen genes. DNA 1989;8:1419–1423. 73 Lew AM, Elsholtz HP. Cloning of the human cDNA for transcription factor Pit-1. DNA 1989;1419–1423. 74 Rosenfeld MG. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 1990;5:879–907. 75 Crenshaw EB III, Kalla K, Simmons DM. Cell-specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between promoter and enhancer elements. Genes and Dev 1990;3:959–972. 76 Davis JRE. Tissue-specific regulation of prolactin gene expression. J Endocrinol 1990;125:171–173. 77 Somasekhar MB, Gorski J. Two elements of the rat prolactin 5¢ flanking region are required for its regulation by estrogen and glucocorticoids. Gene 1988;69:13–21. 78 Waterman ML, Adler S, Nelson C et al. A single domain of the estrogen receptor confers deoxyribonucleic acid binding and transcriptional activation of the rat prolactin gene. Mol Endocrinol 1988;2:14–21. 79 Lannigan DA, Notides AC. Estrogen regulation of transcription. In: Sato GH, Steven JL, eds. Molecular Endocrinology and Steroid Hormone Action. New York, Alan R. Liss, Inc., 1990:187–197. 80 Day RN, Maurer RA. Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation. Mol Endocrinol 1989;3:931–938. 81 Glass CK, Holloway JM, Devary OV, Rosenfeld MG. The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements. Cell 1988;54: 313–323. 82 Stanley F. Transcriptional regulation of prolactin gene expression by thyroid hormone–alternate suppression and stimulation in different GH cell lines. Mol Endocrinol 1989;3:1627–1633. 83 Jackson AE, Bancroft C. Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene. Mol Endocrinol 1988;2:1139–1147. 84 White BA, Preston GM. Evidence for a role of topoisomerase II in the Ca 2±-dependent basal level expression of the rat prolactin gene. Mol Endocrinol 1988;2:40–46. 85 Day RN, Maurer RA. Pituitary calcium channel modulation and regulation of prolactin gene expression. Mol Endocrinol 1990;4:736–742. 86 Sakai DD, Helms S, Carlstedt-Duke J et al. Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene. Genes and Dev 1988;2:1144–1154. 87 Cooke NE, Baxter JD. Structural analysis of the rat prolactin gene suggests a separate origin for its 5¢ end. Nature 1982;297:603–606. 88 Roesler WJ, Vandenbark GR, Hanson RW. Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 1988;263:9063–9066. 89 Iverson RA, Day KH, d’Emden M et al. Clustered point mutation analysis of the rat prolactin promoter. Mol Endocrinol 1990;4:1564–1571. 90 Waterman M, Murdoch GH, Evans RM, Rosenfeld MG. Cyclic AMP regulation of eukaryotic gene transcription by two discrete molecular mechanisms. Science 1985;229:267–269. 91 Molitch ME. Pregnancy and the hyperprolactinemic woman. N Engl J Med 1985;312:1364–1370. 92 Neill JD, Freeman ME, Tillson SA. Control of the proestrus surge of prolactin and luteinizing hormone secretion by estrogens in the rat. Endocrinology 1971;89:1448–1453. 93 Baker BL, Clark RH, Hunter RL. Starch gel electrophoresis of rat hypophysis in relation to prolactin activity. Proc Soc Exptl Biol Med 1963;114: 251–255.

94 MacLeod RM, Abad A, Eidson LL. In vivo effect of sex hormones on the in vitro synthesis of prolactin and growth hormone in normal and pituitary tumor-bearing rats. Endocrinology 1969;84:1475–1483. 95 Maurer RA, Gorski J. Effect of estradiol-17b and pimozide on prolactin synthesis in male and female rats. Endocrinology 1977;101:76–84. 96 Shupnik MA, Baxter LA, French LR, Gorski J. In vivo effects of estrogen on ovine pituitaries: prolactin and growth hormone biosynthesis and messenger ribonucleic acid translation. Endocrinology 1979;104:729–735. 97 Vician L, Shupnik MA, Gorski J. Effects of estrogen on primary ovine pituitary cell cultures: stimulation of prolactin secretion, synthesis, and preprolactin messenger ribonucleic acid activity. Endocrinology 1979;104: 736–743. 98 Maurer RA. Estradiol regulates the transcription of the prolactin gene. J Biol Chem 1982;257:2133–2136. 99 Maurer RA. Relationship between estradiol, ergocryptine and thyroid hormone: Effects on prolactin synthesis and prolactin messenger ribonucleic acid levels. Endocrinology 1982;110:1515–1520. 100 Shull JD, Gorski J. Estrogen stimulates prolactin gene transcription by a mechanism independent of pituitary protein sy