The Cardiovascular System, Volume 12A (Fish Physiology)

FISH PHYSIOLOGY VOLUME XII, Part A The Cardiovascular System

CONTRIBUTORS P. G. BUSHNELL WILLIAM R. DRIEDZIC ANTHONY P. FARRELL LEIF HOVE-MADSEN DAVID R. JONES

J. P. LOMHOLT CHRISTOPHER D. MOYES GEOFFREY H. SATCHELL J. F. STEFFENSEN GLEN F. TIBBITS

FISH PHYSIOLOGY Edited by W. S. H O A R DEPARTMENT OF ZOOLOGY T H E UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, BRITISH COLUMBIA, CANADA

D. J. RANDALL DEPARTMENT OF ZOOLOGY T H E UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, BRITISH COLUMBIA. CANADA

A. P. FARRELL DEPARTMENT OF BIOLOGICAL SCIENCES SIMON FRASER UNIVERSITY BURNABY. BRITISH COLUMBIA, CANADA

VOLUME X I I , Part A The Cardiovascular System

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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This book is printed on acid-free paper. @

Copyright 0 1992 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means. electronic or mechanical. including photocopy. reoording, or any information storage and retrieval system, without permission in writing fromthe publisher.

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United Kingdom Edition published by

Academic Press Limited 24-28 Oval Road, London NW17DX Library of Congress Cataloging-in-PublicationData (revised for vol. 12)

Hoar,William Stewart, date Fish physiology.

Vols.8-12 edited by W.S.Hoar [et al.] Includes bibliographies and index. Contents: v. 1. Excretion,ionic regulation, and metabolism -- [etc.] -- v. 11. The physiology of developing fish. pt. B. Viviparity and posthatching juveniles -- v. 12, pt. A-B. The cardiovascular system. 1. Fishes--Physiology--Collectedworks. I. Randall, David J,. date. II. Conte. Frank P.. date. 111. Title. QL639.1.H6 597l.01 76-84233 ISBN 0-12-350435-X (v. 12A) CIP

PRINTED IN THE UNITED STATES OF AMERICA MV 9 8 7 6 5 4 3 2 1

9 2 9 3 9 4 9 5 9 6 9 1

CONTENTS CONTENTS OF PARTB

vii

CONTRIBUTORS

ix

PREFACE CONTENTS OF OTHERVOLUMES

xi

xv

1. The Heart Anthony P . Farrell and David R. Jones 1 2 24 73

I. Introduction 11. Cardiac Anatomy and Morphology 111. Cardiac Physiology

References

2.

The Arterial System P . G . Bushotell, David R. Jones, and Anthony P. Fawell I. Introduction

11. Physical Factors Affecting Blood Flow 111. Roles of the Conus and Bulbus Arteriosus

IV. Pressure and Flow Relationships in the Ventral and Dorsal Aortas V. Blood-Flow Distribution and Vascular Resistance VI. Heat-Exchange Retid Systems References

89 91 96 105 110 120

3. The Venous System Geoffrey H. Satchel1 141 143 150

I. Introduction 11. The Capillaries 111. The Structure of Fish Veins V

CONTENTS

vi IV. V. VI. VII. VIII. IX. X.

Blood Pressure in Fish Veins The Capacitative Function of the Venous System Valves in Fish Veins The Veins of the Somatic System The Veins of the Hepatic Portal System The Secondary Veins of the Skin Discussion and Conclusion References

151 153 156 157 162 165 177 179

The Secondary Vascular System J . F . Steffensen and J . P. Lomholt

4.

I. Introduction 11. Morphology of the Secondary Vascular System 111. In Vtuo Microscopic Observations

IV. Physiological Experiments V. Functional Aspects of the Secondary Vascular System VI. Evolution of Lymphatic Vessels References

185 187 196 202 209 211 213

5. Cardiac Energy Metabolism William RDriedzic I. Introduction 11. Energy Demand and Supply under Normoxic Conditions 111. Fuels of Aerobic Metabolism

IV. Metabolism in Epicardial and Endocardia1 Layers V. Metabolism under Dysoxic Conditions VI. Impact of Low Temperature on Metabolism References

6.

219 220 223 236 238 250 258

Excitation-Contraction Coupling in the Teleost Heart

Glen F. Tibbits, Christopher D . Moyes, and Leif Hove-Madsen I. Introduction 11. Myocardial Contraction 111. Regulation ofCa2+ Delivery to Myofibrils

IV. Myocardial Relaxation V. Conclusions References

267 270 278 290 296 297

AUTHORINDEX

305

SYSTEMATICINDEX

319

SUBJECT INDEX

329

CONTENTS OF PART B Fish Blood Cells Ragnar Funge Chemical Properties of the Blood D . G. McDonald and C. L. Milligan Blood and Extracellular Fluid Volume Regulation: Role of the Renin-Angiotensin System, Kallikrein-Kinin System, and Atrial Natriuretic Peptides Kenneth R. Olson Catecholamines D . J . Randall and S . F . Perry Cardiovascular Control by Purines, 5-Hydroxytryptamine,and Neuropeptides Stefan Nilsson and Susanne Holmgren Nervous Control of the Heart and Cardiorespiratory Interactions E . W. Taylor Afferent Inputs Associated with Cardioventilatory Control in Fish Mark L. Burleson, Neal J. Smatresk, and William K . Milsom Author Index-Systematic

Index-Subject Index

vii

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CONTRIBUTORS Numbers in parentheses indicute the pages on which the authors’ contributions begin.

P. G . Bushnell (89), Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A9 William R. Driedzic (219), Department of Biology, Mount Allison Uniuersity, Sackville, New Brunswick, Canada EOA 3G0 Anthony P. Farrell(1, 89), Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1 S6 Leif Hove-Madsen (267),Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus, Denmark David R. Jones (1,89), Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A9

J .P. Lomholt (185),Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus, Denmark Christopher D. Moyes (267),Department of Kinesiology, Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1 S6 Geoffrey H. Satchel1 (141), Department of Physiology, University of Otago, Dunedin, New Zealand J . F. Steffensen (185), Marine Biological Laboratory, University of Copenhagen, DK 3000 Helsingor, Denmark Glen F. Tibbits (267), Department of Kinesiology, Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1 S6

ix

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PREFACE A considerable amount of new information has accumulated in recent years concerning the cardiovascular system of fishes. As a result we now have a better understanding of the cardiovascular diversity among fishes, and a number of unifying concepts have emerged regarding both design and function. Our present understanding of the cardiovascular system is presented in Volumes XIIA and XIIB. Fish are the most successful vertebrate group both in terms of biomass and number of species. They also occupy a wide range of environments. As a result, the basic cardiovascular design shows a multiplicity of modifications. As in all vertebrates, it appears that the underlying tenet is that the design of the cardiovascular system primarily reflects the need for oxygen transfer. In fact, the influence of activity pattern on cardiovascular design is such that correlations often transcend phylogeny. Thus, we find that fish with a higher oxygen consumption also have hearts that are bigger, have a more complex anatomy, beat faster, and generate higher blood pressures (Chapters 1A and 2A). Cardiac metabolism and excitation-contraction coupling are correspondingly fine-tuned to these overall demands (Chapters 5A and 6A). Earlier studies were essentially descriptive and drew from our knowledge of mammalian cardiovascular systems. The inherent danger of this approach is that similarities between systems tend to be emphasized and what is special is often ignored. Fish live in a different environment from most mammals, one in which the effects of gravity are relatively minor because the fish has a density similar to the medium. Instead fish must meet the challenge of moving through this viscous medium. The circulatory system of fish is divided into primary and secondary circulations (Chapters 2A, 3A, and 4A). Fish do not have a lymphatic system. The primary circulation consists of branching arterial, capillary, and venous networks. The secondary circulation arises from narrow vessels that connect with primary arteries. This secondary circuxi

xii

PREFACE

lation is a low-pressure and low-hematocrit system serving a primarily nutritive rather than respiratory function to surface structures that exchange gases directly with the water (Chapter 3A). Consequently, the secondary circulation is particularly prone to the hydrodynamic forces acting on the body surface. As a fish moves forward, pressure waves pass backward down the body squeezing blood beneath the skin toward the tail. This is a major problem for the design of the venous system, analogous to gravitational effects on the circulation of terrestrial vertebrates. Thus, the venous system, into which the secondary circulation empties, incorporates a number of accessory hearts to aid in the return of blood to the branchial heart via the central core of the body, which is less influenced by surface pressure waves (Chapter 4A). The cellular components of fish blood are well established but the mechanisms involved in blood cell production, differentiation, and release are still being defined (Chapter 1B). With respect to plasma, its ionic composition is relatively well documented (Chapter 2B). However, fish must cope with periodic changes in their environment, especially light and temperature, and in some species, salinity. Many of the mechanisms for responding to these changes involve the endocrine system. Thus there are circadian and seasonal variations in blood hormone levels, as well as many other components. We are in the process of describing these variations (Chapter 2B), but the nature of the control systems governing these circadian and seasonal rhythms is, in most cases, vague. The control of blood volume and its effect on venous return to the heart are intriguing questions. Some fish are tight skinned, such as tuna and flat fish. Others, such as the sea raven, are baggy skinned, probably so they can gorge meals that are about 50% of their body weight, presumably without raising intraperitoneal pressure which would affect venous return to the heart. Whether or not the tight skin of, for example, tuna has a functional parallel with the encapsulation of the mammalian kidney is not clear. That is, the volume of a fish may be limited by the lack of distensibility of the skin, requiring only systems that keep the body inflated. It appears, however, that fish do have mechanisms for monitoring venous pressure (Chapter 7B), but it is not known if such mechanisms are linked to the coptrol of blood volume. Fish also possess a renin-angiotensin system and atrial natriuretic peptides, but again exactly what role they play in volume regulation is not known (Chapter 3B). The whole question of fluid exchange across capillary walls and the regulation of blood volume in fish remains largely unanswered.

PREFACE

xiii

Cardiovascular regulation centers around control of the heart’s activity, modulation of central blood pressure, and alterations to vascular resistance to effect regional control of blood flow (Chapters 1A and 2A). Very little is known about the control of blood flow through capillaries with the exception of gill lamellae. Gill blood flow was reviewed recently in Volume X and, therefore, is not discussed in the present volume. Because of the paucity of information on other capillary beds in fish, we have not reviewed the subject in this volume. The emerging and complex area of vasoactive peptides and their associated nerves, however, has been reviewed (Chapter 5B). Respiratory and cardiac control are intimately coupled in vertebrates, and perhaps most obviously in fish. There is a sequential grouping of neurons in the central nervous system driving ventilation, with the most posterior neurons involved in cardiac control. This linear arrangement of neurons may allow coupled rhythm generation to be more easily studied than in other vertebrate groups. An understanding of peripheral receptors involved in the control of respiration and circulation is gradually evolving. Both peripheral and central nervous, as well as humoral, control are reviewed in this volume (Chapters 4B, SB,6B, and 7B). We hope that this volume of Fish Physiology sheds some light on these problems. W. S. HOAR D. J. RANDALL A. P. FARRELL

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CONTENTS OF OTHER VOLUMES

Volume I The Body Compartments and the Distribution of Electrolytes W. N . Holmes and Edward M . Donaldson The Kidney Cleveland P . Hickman, Jr., and Benjamin F . Trump Salt Secretion Frank P . Conte The Effects of Salinity on the Eggs and Larvae of Teleosts F . G . T . Holliday Formation of Excretory Products Roy P . Forster and Leon Goldstein Intermediary Metabolism in Fishes P . W. Hochachka Nutrition, Digestion, and Energy Utilization Arthur M . Phillips, Jr.

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume I1 The Pituitary Gland: Anatomy and Histophysiology J. N . Ball and Bridget 1. Baker The Neurohypophysis A . M . Perks Prolactin (Fish Prolactin or Paralactin) and Growth Hormone J . N . Ball xv

xvi

CONTENTS OF OTHER VOLUMES

Thyroid Function and Its Control in Fishes Aubrey Gorbman The Endocrine Pancreas August Epple The Adrenocortical Steroids, Adrenocorticotropin and the Corpuscles of Stannius I. ChesterJones,D. K. 0.Chan, I . W . Henderson, and J . N . Ball The Ultimobranchial Glands and Calcium Regulation D. Harold Copp Urophysis and Caudal Neurosecretory System Howard A. Bern AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume I11

Reproduction William S . Hoar Hormones and Reproductive Behavior in Fishes N . R. Liley Sex Differentiation Toki-o Yamamoto Development: Eggs and Larvae J . H . S . Blaxter Fish Cell and Tissue Culture Ken Wolfand M . C . Quimby Chromatophores and Pigments Ryozo Fujii Bioluminescence J . A. C . Nicol Poisons and Venoms Findlay E . Russell

AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECTINDEX Volume J Y

Anatomy and Physiology of the Central Nervous System JeraldJ . Berstein

CONTENTS OF OTHER VOLUMES

The Pineal Organ James Clarke Fenwick Autonomic Nervous System Graeme Campbell The Circulatory System D . J . Randall Acid-Base Balance C . Albers Properties of Fish Hemoglobins Austen Riggs Gas Exchange in Fish D . J . Randall The Regulation of Breathing G . Shelton Air Breathing in Fishes Gel1 Johansen The Swim Bladder as a Hydrostatic Organ ]ohan B . Steen Hydrostatic Pressure Malcolm S . Gordon Immunology of Fish John E. Cushing AUTHORINDEX-SYSTEMATICINDEX-SUBJECTINDEX

Volume V Vision: Visual Pigments F . W . Munz Vision: Electrophysiology of the Retina T . Tomita Vision: The Experimental Analysis of Visual Behavior David Ingle Chemoreception Toshiaki]. Hara Temperature Receptors R. W. Murray

xvii

xviii

CONTENTS OF OTHER VOLUMES

Sound Production and Detection William N . Tavolga The Labyrinth 0.Lowenstein The Lateral Line Organ Mechanoreceptors Ake Flock The Mauthner Cell J . Diamond Electric Organs M . V . L. Bennett Electroreception M . V . L. Bennett AUTHORINDEX-SYSTEMATIC INDEX-S UBJECT INDEX

Volume VI The Effect of Environmental Factors on the Physiology of Fish F . E . J . Fry Biochemical Adaptation to the Environment P. W . Hochachka and G . N . Somero Freezing Resistance in Fishes Arthur L. DeVries Learning and Memory Henry Gleitman and Paul Rozin The Ethological Analysis of Fish Behavior Gerard P. Baerends Biological Rhythms Horst 0. Schwassmann Orientation and Fish Migration Arthur D.Hasler Special Techniques D. J . Randall and W. S . Hoar AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

CONTENTS OF OTHER VOLUMES

Volume VII

Form, Function, and Locomotory Habits in Fish C. C. Lindsey Swimming Capacity F . W . H . Beamish Hydrodynamics: Nonscombroid Fish Paul W. Webb Locomotion by Scombrid Fishes: Hydromechanics, Morphology, and Behavior John]. Magnuson Body Temperature Relations of Tunas, Especially Skipjack E . Don Stevens and William H . Neil1 Locomotor Muscle Quentin Bone The Respiratory and Circulatory Systems during Exercise David R . Jones and David J . Randall Metabolism in Fish during Exercise William R. Driedzic and P . W. Hochachka

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume VIII Nutrition C . B . Cowey andJ.R. Sargent Feeding Strategy Kim D. Hyatt The Brain and Feeding Behavior Richard E . Peter Digestion Ragner Fange and David Grove Metabolism and Energy Conversion during Early Development Charles Terner Physiological Energetics 1.R . Brett and T . D . D . Groves

xix

xx

CONTENTS OF OTHER VOLUMES

Cytogenetics j . R. Gold Population Genetics Fred W. Allendorf and Fred M . Utter Hormonal Enhancement of Growth Edward M . Donaldson, Ulf H . M . Fagerlund, David A. Higgs, and]. R . McBride Environmental Factors and Growth J, R . Brett Growth Rates and Models W. E . Ricker AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume IXA Reproduction in Cyclostome Fishes and Its Regulation Aubrey Gorbman Reproduction in Cartilaginous Fishes (Chondrichthyes) J. M . Dodd The Brain and Neurohormones in Teleost Reproduction Richard E . Peter The Cellular Origin of Pituitary Gonadotropins in Teleosts P. G . W.j . van Oordt and J. Peute Teleost Gonadotropins: Isolation, Biochemistry, and Function David R. ldler and T . Bun N g The Functional Morphology of Teleost Gonads Yoshitaka Nagahama The Gonadal Steroids A. Fostier, B.]alabert, R . Bfllard,B . Breton, and Y.Zohar

Yolk Formation and Differentiation in Teleost Fishes T . Bun N g and David R . Idler An Introduction to Gonadotropin Receptor Studies in Fish Glen Van Der Kraak AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

CONTENTS OF OTHER VOLUMES

Volume IXB Hormones, Pheromones, and Reproductive Behavior in Fish N . R. Liley and N . E . Stacey Environmental Influences on Gonadal Activity in Fish T.J . Lam Hormonal Control of Oocyte Final Maturation and Ovulation in Fishes Fredrick W. Goetz Sex Control and Sex Reversal in Fish under Natural Conditions S. T . H . Chan and W. S . B . Yeung Hormonal Sex Control and Its Application to Fish Culture George A. Hunter and Edward M . Donaldson Fish Gamete Preservation and Spermatozoan Physiology Joachim Stoss Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish Edward M . Donaldson and George A. Hunter Chromosome Set Manipulation and Sex Control in Fish Gary H . Thorgaard

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume XA General Anatomy of the Gills George Hughes Gill Internal Morphology Pierre Laurent Innervation and Pharmacology of the Gills Stefan Nilsson Model Analysis of Gas Transfer in Fish Gills Johannes Piiper and Peter Scheid Oxygen and Carbon Dioxide Transfer across Fish Gills David Randall and Charles Daxboeck Acid-Base Regulation in Fishes Norbert Heisler

xxi

xxii

CONTENTS OF OTHER VOLUMES

Physicochemical Parameters for Use in Fish Respiratory Physiology Robert G . Boutilier, Thomas A. Heming, and George K . Iwama AUTHORINDEX-SYSTEMATIC

INDEX-SUBJECT

INDEX

Volume XB Water and Nonelectrolyte Permeation Jacques Zsaia Branchial Ion Movements in Teleosts: The Role of Respiratory and Chloride Cells P . Payan, J , P . Girard, and N . Mayer-Gostan Ion Transport and Gill ATPases Guy de Renzis and Michel Bornancin Transepithelial Potentials in Fish Gills W . T. W. Potts The Chloride Cell: The Active Transport of Chloride and the Paracellular Pathways J . A. Zadunaisky Hormonal Control of Water Movement across the Gills J . C . Rankin and Liana B o h Metabolism of the Fish Gill Thomas P . Mommsen The Roles of Gill Permeability and Transport Mechanisms in Euryhalinity David H . Evans The Pseudobranch: Morphology and Function Pierre Laurent and Suzanne Dunel-Erb Perfusion Methods for the Study of Gill Physiology S . F . Perry, P . S . Davie, C . Daxboeck, A . G . Ellis, and D . G. Smith AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT

INDEX

Volume XIA Pattern and Variety in Development J . H . S . Blaxter Respiratory Gas Exchange, Aerobic Metabolism, and Effects of Hypoxia during Early Life Peter J. Rombough

CONTENTS OF OTHER VOLUMES

xxiii

Osmotic and Ionic Regulation in Teleost Eggs and Larvae D . F . Alderdice Sublethal Effects of Pollutants on Fish Eggs and Larvae H . von Westernhagen Vitellogenesis and Oocyte Assembly Thomas P . Mommsen and Patrick J . Walsh

Yolk Absorption in Embryonic and Larval Fishes Thomas A. Heming and Randal K . Buddington Mechanisms of Hatching in Fish Kenjiro Yamagami

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume XIB The Maternal-Embryonic Relationship in Viviparous Fishes John P. Worums, Bryon D. Grove, andjulian Lombardi First Metamorphosis John H . Youson Factors Controlling Meristic Variation C . C . Lindsey The Physiology of Smolting Salmonids W . S . Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes David L. G . Noakes andJean-GuyJ . Godin

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

This Page Intentionally Left Blank

THE HEART ANTHONY P . FARRELL Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada

DAVZD R . JONES Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

I. Introduction 11. Cardiac Anatomy and Morphology A. Sinus Venosus B. Atrium C. Ventricle D. Innervation Patterns E. Myocytes F. Conus and Bulbus Arteriosus G. Coronary Circulation 111. Cardiac Physiology A. Cardiac Cycle B. Control of Stroke Volume C. Control of Heart Rate D. Cardiac Output E. Myocardial Oxygen Consumption F. Control of Coronary Blood Flow References

I. INTRODUCTION Since the descriptions of the cardiovascular system and the cardiovascular changes associated with exercise that appeared in earlier volumes of this series (Randall, 1970; Jones and Randall, 1978),several FISH PHYSIOLOGY, VOL. XIIA

1

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

ANTHONY P. FARRELL AND DAVID R. JONES

informative reviews, monograms, and perspectives have appeared, dealing with either general or specific aspects of cardiovascular physiology and anatomy. These works include those of Johansen and Burggren (1980),Tota (1983),Nilsson (1983),Laurent et al. (1983), Farrell (1984), Santer (1985), Farrell (1985), Wood and Perry (1985), Butler (1986), Johansen and Gesser (1986), Butler and Metcalfe (1988), Tota (1989), Forster et al. (1991), Davie and Farrell (1991a), Brill and Bushnell (1991), Farrell (1991a), and Satchell (1991). This chapter focuses on the heart, whereas the primary arterial circulation is dealt with in the following chapter. Cardiac anatomy and morphology are described in Section 11. The cardiac cycle and the control of cardiac output (0) are described in Section 111. Since the heart also generates sufficient pressure to overcome vascular resistance, pressure development and myocardial power output are also considered in Section 111. Accessory hearts, which assist venous return, are described in Chapter 3. 11. CARDIAC ANATOMY AND MORPHOLOGY

The fish heart is a four-chambered organ contained within a pericardial sac. Together, the sinus venosus, atrium, ventricle, and either an elastic bulbus arteriosus (in teleosts), or a contractile conus arteriosus (in elasmobranchs) raise the potential and kinetic energy of the blood. Detailed descriptions of fish hearts in the recent literature include Santer and Greer Walker (1980),Yamauchi (1980), Tota (1983), Santer (1985), Greer Walker et al. (1985), Emery et al. (1985), SanchezQuintana and Hurle (1987),and Tota (1989).What follows is a brief functional description of the anatomy, fine structure, and morphometrics of the heart.

A. Sinus Venosus The sinus venosus has been described as a thin-walled chamber usually 60-90 pm thick (Santer, 1985). Its volume is similar to that of the atrium in teleosts, but less than that of the atrium in elasmobranchs (Satchell, 1971). The sinus venoms receives venous blood via paired Cuverian ducts, hepatic veins, and anterior jugular veins. The sinoatrial ostium is guarded by a large valve, and the openings of the hepatic veins into the sinus venosus are guarded by muscular sphincters. However, the Cuverian ducts are not valved; they freely

1. THE HEART

3

communicate with the major systemic veins, which are particularly capacious in elasmobranchs (see Chapter 3). The principal tissue component of the sinus wall is connective tissue with bounding inner endothelial and outer epicardial linings. The amount of cardiac muscle present in the sinus venosus varies considerably among species. The European eel (Anguilla anguilla) has an almost complete layer of cardiac muscle (Yamauchi, 1980). In contrast, cardiac muscle is limited to a sparse arrangement of small bundles of 4 to 5 cells in plaice (Pleuronectes platessa) and bull rout (Myoxocephalusscorpius). The sinus venosus is virtually amuscular in loach (Misgurnus anguillicaudatus), brown trout (Salrno trutta), and zebra fish (Zebra danio).Goldfish (Carassius auratus) and carp (Cyprinus carpio) have only smooth muscle elements (Yamauchi, 1980). The major functional role of the sinus venosus is related to the initiation and control of the heart beat. The sinus venosus is the site of specialized cardiac pacemaker tissue in many fish. Histological and neurophysiological evidence for the location of pacemaker tissue has been reviewed previously (Laurent, 1962; Yamauchi, 1980; Laurent et al., 1983; Santer, 1985; Satchell, 1991). What emerges from these reviews is that a ring of specialized myocardial cells (nodal tissue) is usually located at the base of the sinoatrial ostium and connects with atrial myocardium. Nodal tissue can have other locations. In the eel (A. vulgaris) and lungfish (Protopterus ethiopicus), the pacemaker is located at the junction of the sinus venosus and Cuverian ducts. Specialized pacemaker cells have also been reported in the atrium of plaice, brown bullhead (IctuZurusnebulosus), and Pacific hagfish ( E p tatretus stouti), as well as in the atrioventricular region. Even in the absence of specialized pacemaker tissue, myogenic activity of the atrium, ventricle, or atrioventricular region can also initiate contraction, but the contraction rate is likely to be slower and more irregular than the pacemaker rate.

B. Atrium Water-breathing fish have a single atrium. The atrium is an irregularly shaped chamber with thin trabecular walls (Santer, 1985).Atrial volume is similar to or larger than ventricular volume (see Section III,B,2). Atrial mass generally constitutes 8-25% of ventricular mass and 0.01-0.03% of body mass (Table I). However, exceptionally large atria are reported for the New Zealand hagfish (Eptatretus cirrhatus), tunas, and two species of red-blooded Antarctic fishes (Table I). In E.

4

ANTHONY P. FARRELL AND DAVID R. JONES Table I Cardiac Morphometrics in Selected Fishes Relative ventricular Compacta Relative atrial (%) mass (a) Source” mass (%)

Teleosts with compacta Katsuwonus pelamis (skipjack tuna) Thunnus albacares (yellowfin tuna) Thunnus thynnus (northern bluefin tuna) Thunnus maccoyii (southern bluefin tuna) Thunnus obesus (bigeye tuna) Makaira nigricans (Pacific blue marlin) Scomber scombrus (mackerel) Engraulis encrasicolus (anchovy) Clupea harengus (herring) Seriola grandis (kingfish or yellowtail) Salrno gairdneri (rainbow trout) Cyrinus carpio (carp) Anguilla anguilla (European eel) Anguilla dieffenbachii (longfinned eel) Conger conger (conger eel) Elasmobranchs Carcharodon carcharias (great white shark) lsurus oxyrinchus (shortfin mako shark) Galeocerdo cuoieri (tiger shark) Prionance glauca (blue shark) Squalus acanthias (dogfish) Squatina squatina (monkfish) Raja claoata (thornback ray) Raja hyperborea (Arctic skate) Chimaera monstrosa Teleosts with only spongiosa Eptatretus cirrhatus (New Zealand hagfish) Chaenocephalus aceratus (hemoglobin-free icefish) Chionodraco humatus (hemoglobin-free icefish) Pagothenia borchgreoinki (red-blooded Antarctic fish)

0.38

65.6

0.061

0.29

55.4

0.056

0.31

39.1

-

0.29

-

48.5 73.6

0.053

0.087 0.18

48.0 43.0 61.7 24.8

0.013

42.3 30-39 37.0 34.0

0.030 0.017

0.03-0.10

-

40.9 16.0

0.007-0.013

-

36.0

-

0.14

0.028

-

41.5 19.8 16.7 22.1 34.5 35.8 17.3 5.0

0.096

none

0.039

0.30

none

-

0.39

none

-

0.16

none

0.040

-

-

0.11 0.08-0.13

-

-

0.086

-

-

-

-

-

0.017

-

-

-

(continued )

1.

5

THE HEART

Table I Continued Pagothenia bernacchi (red-blooded Antarctic fish) Hernitripterus americanus (sea raven) Chelidonichthys kumu (gurnard) Pleuronectes platessa (flounder) Coryphaenoides rupestris (deep-sea fish)

0.11

none

0.050

(4

0.07 0.05 0.035

none none none

-

(0)

0.017

-

(P)

0.06”

none

-

(9)

(4

(a) Farrell et al. (1992); (b) Poupa et al. (1981);(c )Santer and Greer Walker (1980); (d) P. S. Davie and co-workers (unpublished data); (e) Davie (1987); ( f ) Davie and Hutchinson (unpublished data); ( 9 ) Farrell et al. (1988a); (h) Bass et al. (1973); (i) Davie et al. (1992);(j) Emery et al. (1985); (k) Davie and Farrell (unpublished data); (1) Holeton (1970); (m) Tota et al. (1991); (n) Axelsson et al. (1992); (0)Farrell et al. (1985); (p) Santer et al. (1983); (9)Greer Walker et al. (1985) Total heart weight.

cirrhatus, Pagothenia bernacchii, and P . borchgrevinki, atrial mass is an unusually large proportion (33-50%) of ventricular mass. Skipjack tuna (Katsuwonus pelamis) have an exceptionally large ventricle, and the atrial to ventricular ratio is similar to that in other fish. As a result, their unusually large (0.06%) atrial mass relative to body mass is greater than the relative ventricle mass of some benthic fish (Table I). The variable form of the atrium in part reflects the variable distance that exists between the atrioventricular and ventricular-bulbar orifices and the extent of the S-shaped folding of the heart. Inside the atrium, there are two arcuate fans of muscular trabeculae in many teleosts (Santer, 1985). The trabeculae (19-35 pm in diameter) arise at the atrioventricular ostium and form a meshlike network. When they contract, they help pull the roof and sides of the atrium toward the atrioventricular ostium (Satchell, 1971). The atrioventricular valve is supported by a ring of cardiac tissue. In benthopelagic teleosts, the more spacious arrangement of trabeculae gives the atrium a frail appearance (Greer Walker et al., 1985). C. Ventricle

The ventricle shows considerable species variability with respect to its relative mass, gross morphology, histology, and vascularity. Santer (1985) emphasizes the fact that there is no “typical” shape to the ventricle in fishes. It is equally appropriate to state that there is no typical ventricular mass, histology, or vascular network. Instead, cer-

6

ANTHONY P. FARRELL AND DAVID R. JONES

tain patterns are useful to categorize fish hearts. These categories reflect functional rather than phylogenetic correlates, and some of the categories have hazy boundaries. Two features used to categorize fish hearts are (1) ventricular form, and (2) the relative development of an outer layer of compacta tissue and its associated coronary circulation.

1. VENTRICLE FORM Santer (1985)proposed three major categories of ventricular form in fishes: 1. A saclike ventricle, which is rounded in shape with an indistinct apex. This form is the most common shape in elasmobranchs and many marine teleosts, and the only shape found in 29 species of benthopelagic teleosts (Santer et al., 1983; Greer Walker et al., 1985). 2. A tubular ventricle with a cylindrical cross-section. This form is found only in fish with an elongate body shape, but all fish with an elongate body shape do not necessarily have a tubular heart. 3. A pyramidal ventricle with a triangular base forming the caudal aspect. The pyramidal ventricle is restricted to species with an active life style, e.g., the salmonid and scombrid families. A pyramidal ventricle always has an outer compacta layer, but not all fish with compacta have a pyramidal ventricle, e.g., elasmobranchs. The functional significance of these shapes, beyond the tubular ventricle reflecting body form, is not entirely clear. While the saclike ventricle is clearly the most common form, the possession of a pyramidal ventricle may be advantageous in terms of pressure development, because the apices have small radii of curvature as well as an unusual arrangement of fiber bundles.

2. SPONGIOSA AND COMPACTA Tota (1989)proposed that fish hearts can be divided into four major categories based on the possession and arrangement of spongiosa and compacta. The ventricles have two basic forms: whereas one form has an arrangement of muscle trabeculae that span the entire ventricle to form a spongelike network, the spongiosa, the other form has an outer compacta layer enclosing an inner spongiosa. The ventricle is bounded externally by epicardium and internally by endocardium. The compacta is always associated with a coronary circulation. The principal features distinguishing the four categories of ventricles are the extent of spongiosa versus compacta and the pattern of vasculariza-

1. THE HEART

7

Fig. 1. A schematic illustration of the main features that distinguish the four ventricle types in fishes. The basic anatomical design of the fish heart has been divided into quadrants with each quadrant illustrating particular features of a given ventricle type. Type I is characterized by having a single myocardial type (spongiosa) and no capillaries in the ventricular muscle. Most type I hearts have no coronary vessels whatsoever. Type I1 is characterized by two muscle layers in the ventricle (inner spongiosa and outer compacta), a coronary circulation, and capillaries only in the outer compacta. Type 111 is similar to type 11, but capillaries are found in both the spongiosa and compacta, and to a limited extent in the atrium. Type IV has a larger percentage ofthe ventricle as compacta (>30%) and a more extensive capillarization of the atrium. The coronary circulation, regardless of type, is derived from a cranial supply, as depicted in the lower left quadrant, and an additional pectoral supply in some fishes, as depicted in the lower right quadrant. Adapted with permission from Davie and Farrell (1991a).

tion (Fig. 1).The four categories reflect increasingly more complex arrangements. The majority of fish species have a type I venticle. The type I ventricle has only spongiosa myocardium (also given terms such as the inner, spongy, trabecular, or trabeculated layer, the subendocardium,

8

ANTHONY P. FARRELL AND DAVID R. JONES

and the endocardium in the literature). Variations of the type I ventricle exist and reflect differences in vascular development, although there are no capillaries per se in the myocardium of any type I hearts. Most fish have a subtype Ia ventricle in which venous blood contained in the lumen and intertrabecular spaces of the ventricle (luminal blood) provides the only blood supply (hence the terms venous, lacunary, or avascular hearts). Subtypes Ib and Ic possess a superficial coronary artery, but arterial vessels either are confined to the epicardium (subtype Ib), e.g., plaice, or connect directly to the intertrabecular spaces (subtype Ic), e.g., hemoglobin-free Antarctic fish (Tota, 1989). The three other categories of ventricles (types 11,111, and IV) have compacta and spongiosa myocardial tissue as well as capillaries in the myocardium (Tota, 1989; Davie and Farrell, 1991a).Type I1 ventricles are characterized by the capillaries of the coronary circulation being found only in the compacta. In ventricle types I11 and IV, the coronary circulation reaches the spongiosa as well as the cornpacta. The distinguishing feature between types I11 and IV is that type I11 ventricles have less than 30% compacta tissue. Those ventricles with more than 30%compacta and coronaries in the spongiosa are arbitrarily described as type IV. Most teleosts possessing compacta have type I1 ventricles. Most elasmobranchs have type I11 ventricles. Endothermic sharks and active teleosts have type IV ventricles. Ontogenetically, the cornpacta (also given terms outer, compact, or cortical layer, the epicardium, and the subepicardium in the literature) appears early in development (Kitoh and Oguri, 1985) and increases disproportionately with body size in juvenile fish (Poupa et al., 1974; Farrell et al., 1988a). The proportion of cornpacta in the ventricle (and its thickness) is quite variable between species. The relative proportion of compacta is loosely correlated with activity. Endothermic sharks have approximately twice the amount of cornpacta compared with ectothermic sharks (36-40% versus 15-24%; Emery et al., 1985). Among the active teleosts that have compacta, the most athletic ones generally have higher proportions of compacta (Table I). The bigeye tuna (Thunnus obesus) is reported to have 73.6% cornpacta tissue, the largest proportion recorded for any fish. Beyond a genetic constraint, the factors setting the upper limit for the proportion of compacta in the ventricle of mature fish are not clear. However, the proportion of compacta in the ventricle does vary somewhat with smoltification in Atlantic salmon (Salmo salar) and with changing seasons in rainbow trout (Oncorhynchus mykiss), but not with exercise training (Ostadl and Schiebler, 1972; Poupa et al., 1974; Farrell et al., 1988a; Farrell et al., 1990a).

1. THE HEART

9

The greater the proportion of compacta, the more layers of different fiber bundles it has, each layer having a different architecture (Santer, 1985; Sanchez-Quintina and Hurle, 1987). Dogfish (Gazeorhinus galeus) has 15%compacta, and blue shark (Prionace glauca) has 17% compacta; both species have a single layer of looped fiber bundles in the compacta. The more athletic Atlantic shortfin mako shark (Zsurus oxyrinchus) has 28%compacta arranged in two layers. The outer layer is looped (Fig. 2A), whereas the inner layer forms a sac of circular fibers. Similarly, there are two layers in swordfish (Xiphias gladius) and albacore (Thunnus alalunga), which have 29 and 26% compacta, respectively. Again the outer layer is arranged in loops (Fig. 2B), but these teleosts have a pyramidal rather than a saclike ventricle, and the inner layer forms rings encircling the vertices of the ventricular pyramid. Three layers of fiber bundles are present in Atlantic bluefin tuna (Thunnus thynnus),which has 39% compacta. The inner (Fig. 2C) and outer layers have a similar fiber architecture as swordfish and albacore, whereas the additional layer between inner and outer layers has the shape of a stirrup that surrounds the cranial apex, the lateral borders, and the caudal face of the ventricle. These observations suggest that up to three layers can be found, with looped fibers being the most fundamental fiber arrangement in the compacta. In teleosts, the dominant longitudinal arrangement of looped fibers on the ventral faces and a dominant transverse arrangement on the caudal face would act to reduce the longitudinal and transverse diameters of the ventricle during systole (Sanchez-Quintina and Hurle, 1987). The addition of inner fibers either as a circular arrangement around the saclike elasmobranch ventricle, or as coils around the vertices of the pyramidal teleost ventricle is clearly a characteristic of fish that are more active, and perhaps provides a mechanical advantage for developing higher blood pressures. Certain features distinguish the elasmobranch ventricle from that in teleosts. First, all elasmobranchs have compacta and a coronary circulation, whereas only a small proportion of active teleosts do. Second, the fibers of the compacta and spongiosa are continuous in elasmobranchs, but are separated b y a layer of connective tissue in teleosts (Sanchez-Quintina and Hurle, 1987). Third, the elasmobranch spongiosa has a precise arrangement of arcuate trabeculae running from the atrioventricular to the conoventricular ostia. In contrast, teleosts have an anarchial array of trabeculae except near the bulboventricular region, where a longitudinal arrangement exists. The trabecular arrangement of spongiosa accounts for the greater proportion of ventricular mass in almost all fishes (Table I). The idea that this trabecular arrangement allows the individual lacuna of the

1. THE HEART

11

spongiosa to function as “many small hearts” within the larger ventricle was put forward by Johansen (1965).Since the radius ofeach lacuna is small, LaPlacian relationships dictate that pressure will be generated at a considerable mechanical advantage compared with that of hearts consisting solely of compact myocardium. However, this idea has yet to be experimentally verified. Davie and FarreIl (1991a) suggested that the diameter of the trabeculae is a compromise between minimizing the distance for 0 2 diffusion and maximizing the crosssectional area for tension development. The basis for this suggestion is that atrial and ventricular trabeculae of most fish derive 0 2 from luminal blood, and a trabecular muscle structure increases the surface area and reduces the diffusion distance for 0 2 transfer compared with a compact muscle structure. In view of this suggestion, it would be interesting to learn whether the trabeculae of the atrium and the spongiosa in elasmobranchs and certain active teleosts (types I11 and IV) have larger diameters, since these trabeculae have a coronary circulation. 3. RELATIVEVENTRICLE MASS

Cardiac mass in fish scales almost in direct proportion to body mass (Poupa and Lindstrom, 1983), a situation which is similar to that in other vertebrates. However, this allometric relationship tends to obscure the fact that relative ventricular mass (ventricle mass divided by body mass) varies considerably among fishes and even within a species. Relative ventricular mass varies over a 10-fold range, being 0.035%of body mass in flounder to 0.38%of body mass in skipjack tuna (Table I). The following generalizations can be made regarding relative ventricle mass.

1. Active species have larger ventricles. Active teleosts (e.g., Clupidae, 0.249%) have a larger relative ventricular mass Fig. 2. The myocardial fiber architecture in the compada offish hearts. (A) A lateral view of the ventricle and conus arteriosus of the mako shark. Note the transverse arrangement of superficial fibers that loop around the sacular heart. An additional inner layer of fiber bundles in the compacta (not shown here) has a more circular arrangement. (B) A dorsal view of the swordfish ventricle and bulbus arteriosus showing the superficial fiber layers of the pyramidal ventricle. An inner layer consists of rings encircling the vertices, similar to but not so pronounced as those shown in C. (C)A cranial view of the Atlantic blue fin tuna ventricle showing the deep fiber layer of the compacta. The rings of fibers encircling the vertices of the ventricle are very pronounced. The atrioventricular and bulboventricular valves are evident. With permission from SanchezQuintina and Hurle (1987).

12

ANTHONY P. FARRELL AND DAVID R. JONES

than do active elasmobranchs (e.g., sharks, 0.196%) (Poupa and Lindstrom 1983). Even at the species level, larger hearts are observed in anadromous compared with lake varieties of 0. mykiss (Graham and Farrell, 1992). However, benthic elasmobranchs (e.g., Selachii, 0.098%) have a larger relative ventricular mass than do benthic teleosts (e,g., Pleuronectids, 0.061%) (Poupa and Lindstrom, 1983). 2. Endothermic sharks have larger ventricles than ectothermic sharks (Emery et al., 1985). 3. Exercise training has variable effects on cardiac growth. Some training regimens produce small increases in relative ventricle mass, whereas other regimens produce only isometric cardiac growth (Davison, 1989; Farrell et al., 1990a). 4. Cold-acclimation increases relative ventricular mass in rainbow trout, carp, and goldfish (Tsukuda et al., 1985; Farrell, 1987; Goolish, 1987; Graham and Farrell, 1989). In rainbow trout, ventricle mass increases by 70% when fish are acclimated to temperatures 10°C apart. Seasonal variations in relative ventricle mass of rainbow trout also correlate well with changes in water temperatures (Farrell et al., 1988a). Antarctic fishes have relatively large ventricles (Holeton, 1970;Johnston et al., 1983; Tota et al., 1991). These observations implicate a large relative ventricular mass as being important in (1) developing higher blood pressures in active fishes, (2) compensating for the negative inotropic effect of low temperature, and (3)accommodating large cardiac stroke volumes. These physiological considerations are covered in subsequent sections.

D. Innervation Patterns The literature on the innervation patterns of fish hearts and pacemaker tissue has been comprehensively reviewed by Laurent et al. (1983), Nilsson (1983), and Santer (1985).The following summarizes the cardiac distribution of cholinergic and adrenergic fibers. Other aspects of cardiac innervation are described in Part B of this volume, Chapters 5 and 6. Vagal cardiac innervation is found in all fishes except the hagfishes. In all cases the vagus carries small, myelinated, cholinergic efferent fibers. These efferents stimulate inhibitory muscarinic cholinoceptors in all species except lampetroids, which have excitatory nicotinic cholinoceptors. Only the sinus venosus is innervated in lampreys. In

1. THE HEART

13

elasmobranchs and teleosts, the sinoatrial region is richly innervated, and it is possible that every muscle cell has at least one nerve profile. The muscle fibers of the atrium and the atrioventricular region are innervated to a lesser extent whereas those of the ventricle are only sparsely and partially innervated. Large myelinated fibers are found in the vagal trunks and throughout the atrium and ventricle. These fibers are thought to be sensory, cholinergic afferent fibers, but their role has not been extensively studied (see Laurent, 1962). Adrenergic cardiac innervation is found in the majority of teleosts studied, but not in cyclostomes and elasmobranchs. Unmyelinated adrenergic efferents have various sizes, and their distribution pattern varies between species. Typically, the sinus venosus and atrium, especially the sinoatrial and atrioventricular regions, are well innervated. The compacta of the ventricle, especially near the atrioventricular region, the coronary vessels, and the bulbus arteriosus also receive adrenergic fibers. Benthic teleosts, such as plaice, lack adrenergic innervation. From these distribution patterns, we can expect significant cholinergic and adrenergic neural influences on pacemaker rates, atrioventricular conduction, and atrial contractility. In addition, adrenergic influences may affect ventricular contractility to a greater degree than cholinergic influences, and adrenergic coronary vasoactivity is to be expected.

E. Myocytes

A number of features distinguish fish cardiac muscle cells (myocytes) from those in mammals. These include: (1) small size (1-12.5 pm) compared with 10-25 pm in mammals, (2) limited anatomical development of the sarcoplasmic reticulum (SR), (3) peripheral arrangement of myofibrils, and (4) variable amount of intracellular myoglobin. The fine structure of myocardial cells is described in considerable detail by Yamauchi (1980), Helle (1983), and Santer (1985). The morphometric characteristics of myocytes from various fish atria and ventricles were compiled by Santer (1985). Myocyte diameters are most often between 2.5 and 6.0 pm and range from 1.0 to 12.5 pm. The differences that exist in the diameters of atrial and ventricular myocytes are small relative to the overall range in myocyte diameters. Tota (1989) notes that myocyte diameters are less in elasmobranchs than in teleosts. The largest myocyte diameters occur in A. vulgaris, Cyprinus carpio, Misgurnus anguillicaudatus, and two species of osteoglossids; all are species that tolerate aquatic hypoxia.

14

ANTHONY P. FARRELL AND DAVID R. JONES

Myocytes in the compacta have larger diameters than in the spongiosa in Arapairna gigas (Hochachka et al., 1978) and albacore (Breisch et al., 1983).Among the functional advantages of a narrow myocyte are a shorter diffusion distance from the outside to the center of the cell, and a higher ratio of sarcolemma area to intracellular volume. However, smaller diameters probably mean higher electrical resistance and slower conduction velocity. Poupa and Lindstrom (1983)proposed that myocyte diameter in homoeotherms is proportional to stroke volume, inversely proportional to heart rate, and inversely proportional to the maximum activity state of the animal. Thus, the “high speed” cardiac pump of small mammals is characterized by small myocytes. The small myocytes of the “low speed” fish heart clearly contrast with this dogma (Farrell, 1991a). The effect of cardiac growth on myocyte size has been assessed for perch (Perca perca; Karttunen and Tirri, 1986)and rainbow trout (Farre11 et al., 1988a). In perch, mean myocyte diameter (4.2 pm) and length (133 pm) were independent of fish size (50-250 g), implying that cardiac growth is entirely hyperplastic over this range of body mass for perch. In rainbow trout, myocyte length increased from 44 km to 103 pm, and myocyte diameter increased from 6.6 to 9.0 p m for a larger range of body mass (12-1750 g). From these dimensions it was estimated that myocyte volume increased 6-fold for a 100-fold increase in ventricle mass (Fig. 3A). Hence, hyperplastic cardiac growth occurs to a much greater extent than hypertrophic growth in rainbow trout. Hyperplastic growth of fish myocytes contrasts with the situation in mammals in which myocyte growth is hypertrophic, except in the embryonic period and sometimes in the first few weeks of neonatal life. Hyperplastic growth means that relatively small myocyte diameters are maintained during ontogeny. The SR is sparse in fish hearts and the sarcolemma (SL) lacks the T-tubule system typical of all skeletal muscle and mammalian cardiac muscle. Instead, there are small (150 nm diameter), flask-shaped infoldings of the SL termed calveolae (Santer, 1985). Tota (1989) notes that elasmobranch myocytes have less SR compared with teleosts. Four types of SR are recognized (Fig. 3B). Subsarcolemmal cisternae are in contact with tubules that encircle the myofibrils. Longitudinal tubules connecting the circular tubules are particularly sparse in fish. Patches of reticular lattices are also limited, but are more prominent in active species of fishes (Santer, 1985). The impact of small myocyte size, absence of T-tubules, and limited anatomical development of the SR with respect to excitation-contraction coupling is discussed more fully in Chapter 6.

15

1. THE HEART 0

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Fig. 3. (A) The relationship between ventricular mass and estimated myocyte volume for rainbow trout weighing between 10 and 1800 g. Since myocyte volume increases at only about one sixth the rate of ventricular mass, considerable hyperplastic myocyte growth is likely. From Farrell et al. (1988).(B) The sarcoplasmic reticulum (SR) is much reduced in fish myocardium. This schematic illustrates the four types of SR that have been identified in fishes and their anatomical relationship to two myofibrils: subsarcolemmal cisterna (sc); longitudinal tubules (It);circular tubules (ct); reticular SR (rSR). Adapted from Santer (1985).

Ventricular myocytes contain more myofibrils than atrial myocytes and can occupy 55% of myocyte volume in active species, approaching proportions found in mammalian ventricles (Yamauchi and Burnstock, 1968; Midtun, 1980; Breisch et al., 1983).Myofibrillar volume is particularly low in Antarctic fishes (Johnston et al., 1983). Myofibrils of some fish species are located in the peripheral part of the cell, close to the

16

ANTHONY P. FARRELL AND DAVID R. JONES

SL, with mitochondria being more centrally located. An advantage of peripherally located myofibrils is that, in the absence of a T-tubule system, calcium diffusion from the extracellular space to troponin-C is facilitated, which may be more critical for cardiac muscle performance than is the diffusion of oxygen to mitochondria (see Farrell, 1991b). Myoglobin is particularly variable in fish hearts. Some myocytes appear white as a result of being essentially devoid of myoglobin, e.g., ocean pout (Macrozoarces americanus), which has 5.2 nmol per g ventricular mass. However, most fish species possess myoglobin in the range of 50-150 nmol per g ventricular mass. The highest concentrations of myoglobin are found in active fishes, such as striped bass (400 nmol per g ventricular mass; Driedzic, 1983) and Atlantic bluefin tuna (up to 1300 nmol per g ventricular mass; Giovane et al., 1980),and hypoxia-tolerant species, such as the North American eel (A. rostrata, 239 nmoI per g ventricular mass; Bailey et at., 1990).Ventricular mass increases with body size in Atlantic bluefin tuna, and myoglobin concentrations do likewise (Poupa et al, 1974). Clearly, high myoglobin concentrations in large ventricles (activity related, hypoxia related, and growth related) reflect a balance between fine architecture of the cell and adequate 0 2 diffusion. Chapter 5 by Driedzic discusses the functional importance of myoglobin in facilitating 0 2 delivery to the myocardium of hypoxic, but not normoxic, fish hearts. Other cardiac cell inclusions, such as atrial naturetic factors, glycogen, and fat, are discussed in Chapter 5 and Part B, Chapter 3. F. Conus and Bulbus Arteriosus A cyclindrical-, pear-, or onion-shaped chamber lying within the pericardium is interposed between the ventricle and ventral aorta in chondrichthyes and osteichthyes. In selachians this chamber is rhythmically contractile and referred to as the conus arteriosus, whereas in teleosts it is elastic and called the bulbus arteriosus. Structurally, the bulbus resembles the ventral aorta, and this, combined with the fact that more ancient fishes have both conal and bulbar regions, has given rise to the notion that phylogenetically the primitive conus has been telescoped into the ventricle by backward growth of the ventral aorta. However, this view is not subscribed to by all, and Priede (1976), for instance, suggests that the teleost bulbus is of cardiac origin. Certainly, Senior’s (1909) study of heart development in the shad (Alosa sapidissima) implies that the conus develops within the pericardium. Nevertheless, the nomenclature of the bulb in cyclostomes as a “conus”, when its structure is aortic and it resides outside the pericardium, reflects the view of the conus as phylogenetically more primitive.

1.

THE HEART

17

Embryologically, the conus has four endocardial ridges, which are capable of occluding its lumen during contraction, although the ventral one is usually vestigial (Goodrich, 1930). Pocket valves, with the concavity to the front, develop from the dorsal and lateroventral ridges in transverse rows or tiers. Up to six tiers can be present, although in “advanced” selachians, there are only two tiers of well-developed valves. In contrast, multiple tiers of valves occur in the conus of primitive teleosts (Acipenser and Amia). In Polypterus and Lepidoseus, the conus is elongated, and there are multiple rows and tiers of valves (Goodrich, 1930). In most teleosts, a narrow muscular ring with two valves at the ventriculobulbar junction represents the only vestige of the conus. However, in the sticklebacks, Gasterosteus and Pungitius, there may be an exceptional arrangement in that valves have been described at both the ventriculobulbar and bulboaortic junctions (Benjamin et al., 1983). In most teleosts, the luminal wall of the bulbus is smooth or ridged although in trout and tuna (fish with high arterial blood pressures) there is a series of chordae-like structures (longitudinal elements), which run along the internal wall. Over most of their length, the obliquely running radial fibers connect the longitudinal elements to the bulbar wall (Priede, 1976). Deep sea fishes (Macrouridae) also have a unique bulbar anatomy. At the ventriculobulbar junction, there is a layer of gelatinous and collagenous tissue that forms an “inner tube” within the bulbus running toward the ventral aorta (Fig. 4A; Greer Walker et al., 1985).This “inner tube” even contains hyaline cartilage in Chalinura profundicola. The “inner tube” becomes continuous with spaces between anastornosing trabeculae in the main cavity of the bulbus. In cyclostomes the “conus” has no cardiac muscle in its walls and is not rhythmically contractile. The hagfish conus is much thinner than that in the lamprey, which is probably a reflection of much lower blood pressures in hagfishes than in lampreys (Wright, 1984; Davie et al.,

1987). The selachian conus contains cardiac muscle, and teleost bulbus contains smooth muscle in the walls. Hence, the nomenclature of wall structure for the conus is cardiac (i.e., epi-, myo-, and endocardium), whereas the corresponding three layers of the bulbus are described using blood vessel terminology (i.e., adventitia, media, and intima). Priede (1976) has argued that blood vessel terminology is inappropriate since it implies arterial homology, which exists only in the sense that bulbus and heart are both embryologically derived from the subintestinal blood vessel. The luminal surface (intimal or endocardial) consists of squamous, cuboid, or elongate (tall) cells, which do not appear to be aligned along

1. THE

HEART

19

the major axis of blood flow (Benjamin et al., 1983; Langille et al., 1983; Leknes, 1981, 1985).In the dogfish Scyliorhinus stellaris, there is a subendocardial layer of chromaffin cells associated with myelinated and unmyelinated nerve endings. It has been suggested that the chromaffin cells are part of a neurosecretory system that influences cardiac performance (Saetersdal et al., 1975; Zummo and Farina, 1989). In teleosts, smooth muscle and collagen fibers may be associated with the subintimal layer (Santer, 1985). The outermost layer of both bulbus and conus is usually described as visceral pericardium. Collagen is concentrated in this Iayer along with blood vessels and ganglion cells. Sympathetic fibers appear to innervate smooth muscle of the medial layer of the bulbus (Gannon and Burnstock, 1969; Watson and Cobb, 1979). As the nerves in the bulbar wall are generally associated with blood vessels, species lacking a well-developed coronary blood supply have few or no signs of neural innervation (i.e., plaice, Pleuronectes platessa). The myocardial layer of the conus is made up of circularly arranged cardiac myocytes, some collagen fibers, and many capillaries. The medial layer looks very much like the compact myocardium of the heart (Santer, 1985; Zummo and Farina, 1989). In contrast, the bulbar medial wall consists of smooth muscle cells and a fibrillar elastic reticular network (elastica). Smooth muscle cells miy be arranged either in a spiral of flat sheets (Watson and Cobb, 1979) or circumferentially around an inner layer of interdigitizing smooth muscle fasciles (Licht and Harris, 1973).The latter arrangement is reminiscent of the division of the myocardium into compact and trabeculate muscular regions. In rainbow trout and salmon, three muscle layers are recognized: outer and inner, which run longitudinally to the main axis of the bulbus, sandwiching a layer of circumferentially orientated cells (Serafini-Fracassini et al., 1978). Elastic fibrils (20-50 nm in diameter) are found in close association with smooth muscle cells (Fig. 4B). These fibrils form an isotropic, three-dimensional network and are not usually in sheets or lamellae (Fig. 4B). Occasionally fibrils anastomose to form a fibrillar core, although amorphous elastin of the type seen in higher vertebrates is Fig. 4. (A) Reconstruction ofa longitudinal section through the bulbus arteriosus of Ventrifossa occidentalis, based on a study of serial transverse sections. Ventricle (v), bulbus arteriosus (ba), inner tube (it), bulboventricular valves (bv). From Greer Walker et al. (1985), with permission. (B) Electronmicrograph (~20,000) ofthe bulbar wall in a yellowfin tuna. C, collagen; FE, fibrillar elastin; EL, elastic lamina; SM, smooth muscle. Jones, Wheaton, and Brill, 1991.

20

ANTHONY P. FARRELL A N D DAVID R. JONES

conspicuously lacking in fish. Some form of microfibrillar (4-15 nm in diameter) network may be associated with all elastic elements (Isokawa et al., 1988, 1990) as the skeleton on which they are laid. However, cyclostomes are a unique case because the “conus” contains microfibrils (Wright, 1984),that are not even made of elastin (Sage and Gray, 1979, 1980). G. Coronary Circulation The anatomical origin of the coronary circulation is well described in the older literature (e.g., Parker, 1886; Parker and Davis, 1899; Grant and Regnier, 1926; O’Donoghue and Abbot, 1928; Foxon, 1950). Developmental and evolutionary perspectives of the coronary circulation are presented by Halpern and May (1958). The distribution patterns of the coronary arteries are described for lamnid shark ventricles by De Andres et al. (1990) and for tuna by Tota (1978). Terminal distribution patterns within the ventricular wall and the functional significance of the luminal versus coronary circulations are covered in Tota et al. (1983), Tota (1989), and Davie and Farrell (1991a). The coronary circulation has two sides of origin: a cranial (cephalad) circulation (e.g., rainbow trout), and in some fish an additional pectoral (caudal) circulation (e.g., eel and swordfish). One to three pairs of arteries branch from the efferent branchial arch arteries and meet medially to form the hypobranchial artery from which the cranial coronary circulation is derived as one or more arteries. Cranial coronary arteries reach the ventricle across the surface of the bulbus arteriosus or conus arteriosus. The pectoral coronary circulation arises from the first branch of dorsal aorta, the coracoid artery, and vessels penetrate the pericardium, reaching the apical and sometimes lateral aspects of the ventricle via pericardial ligaments. The cranial and pectoral circulations are functionally interconnected at the arterial level (Davie and Farrell, 1991b). The anatomical origins of both the cranial and pectoral coronary circuits are such that oxygenated blood is delivered directly from the gills to the heart at the highest possible postbranchial blood pressure. In elasmobranchs, multiple coronary arteries are found on the conus arteriosus, often with a dominant ventral or dorsal vessel. On the ventricle, the coronary arteries form a visible, subepicardial (extramural) network and short perforating branches supply the compacta and spongiosa (Tota, 1989; De Andres et al., 1990). The compacta is well vascularized with capillaries connecting to veins that drain into the atrial chamber close to the atrioventricular region. The trabeculae of

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the spongiosa in type I11 and IV ventricles have centrally located arteries, terminal arterioles, a few capillaries, and no venous system (Voboril and Schiebler, 1970; Ostadal and Schiebler, 1971). Terminal arterioles and capillaries open directly to the lumen, thereby forming arteriolacunary connections analogous to the Thebesian system of the mammalian heart (Tota, 1989). In the type IV ventricle of the lamnid shark, the arterial branches that go to the inner spongiosa are formed in the region of the conoventricular and atrioventricular grooves rather than from the subepicardial network (De Andres et al., 1990), and the atrium receives small branches from arteries located in the atrioventricular region. Numerous very small, perpendicular branches circle and supply the conal cardiac muscle. Coronary veins also run parallel to the main coronary arteries on the conus arteriosus of elasmobranchs and presumably drain the conal cardiac muscle (A. P. Farrell, 1990). In teleosts, the cranial coronary artery is often a single vessel running along the dorsal (e.g., skipjack tuna) or ventral (e.g., rainbow trout) surface of the ventral aorta. The extramural network, so prominent in elasmobranchs, is largely lacking in teleost ventricles. The arterial distribution pattern has not been studied with respect to the various layers of fiber bundles in the compacta. Capillaries are limited to the compacta in type I1 ventricles, and venous drainage is to the atrium in the atrioventricular region. A Thebesian system, similar to that in elasmobranchs, is found in type IV teleost ventricles (Tota et al., 1983). In view of this anatomical diversity, it is most likely that the coronary circulation evolved more than once (Davie and Farrell, 1991a) with the cranial origin being the most primitive (Tota, 1989).All of the elasmobranchs and about one quarter of the teleosts have coronaries, and no single factor adequately accounts for the presence of a coronary circulation. Typically, teleosts that either tolerate environmental hypoxia or are active swimmers have both a coronary circulation and high myoglobin levels. Santer and Greer Walker (1980)postulated that the cost of swimming was higher in elasmobranchs because of the absence of a swimbladder, which may explain why all elasmobranchs have a coronary circulation: a continuous mode of swimming may lower the luminalO2 supply to a critical level for long periods (see Section 11,E). The histological structure of the fish coronary artery is characterized by an external parenchyma surrounding a medial layer of vascular smooth muscle; an internal elastic lamina separates the media from the intima, which normally has a single layer of endothelial cells (Fig. 5A). Robertson et al. (1961) observed arteriosclerotic lesions in and confined to the coronary vessels of mature Pacific salmon. These lesions have since been characterized as intimal proliferations of vascular

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smooth muscle with a disrupted elastic lamina (Fig. 5B).They resemble the early forms of mammalian arteriosclerotic lesions, and although the proliferation of intimal smooth muscle is just as extensive as in mature mammalian lesions, calcium and lipid inclusions typically found in mature mammalian lesions are absent (Moore et al., 1976; McKenzie et al., 1978; House and Benditt, 1981). Lesions are particularly prevalent and severe in mature migratory salmonids (Pacific salmonids, genus Oncorhynchus), Atlantic salmon, and steelhead trout (0.mykiss), but are less severe or absent in other fish species (Vastesaeger et al., 1965; Santer, 1985). Coronary lesions are typically found in more than 95% and often 100% of a sample population of migratory salmonids (Robertson et al., 1961; Maneche et al., 1973; Farrell et at., 1986a; Farrell et al., 1990b). In addition, severe lesions are located on 66-80% of the length of the main coronary artery, often obstructing the arterial lumen by 10 to 30%. Since the lesions are in a main arterial conduit, the potential exists for a substantial increase in vascular resistance as a result of lesion formation. Whether or not this leads to an ischemic state is unclear (Farrell et al., 1990b). The etiology of coronary lesions in salmonids is unclear. Sexual maturation has been implicated based on the observations that lesions are found in mature fish (Robertson et at., 1961; Schmidt and House, 1979) and that injections of sex hormones promote lesion formation (House et al., 1979). However, lesions are well developed at least 1 year before maturation in Atlantic salmon (Farrell et al., 1986a). Factors related to growth are also implicated in lesion development. The prevalence and severity of coronary lesions accelerates in parallel with rapid bodily growth in the ocean (Saunders et al., 1992; Kubasch and Rourke, 1990). The correlation between growth rate and lesion formation may explain the observation that lesions are fewer and less severe in the slower-growing, land-locked species of salmonids compared with the migratory varieties. Diet also influences lesion formation

Fig. 5. Histological cross-sections of the main coronary artery of salmonids. (A) A normal coronary artery from an adult Atlantic salmon. The internal elastic membrane (EM) is differentially stained to demarcate the inner limit ofthe medial vascular smooth muscle layer (MSM).The vessel lumen (L) is filled with nucleated red blood cells. Bar = 50 Fm. (B) A coronary artery from a spawning chinook salmon showing a severe arteriosclerotic lesion, which significantly occludes the vessel lumen (devoid of red blood cells). The main tissue component of the lesion is intimal vascular smooth muscle (ISM), which has a different fiber arrangement than the MSM. Bar = 50 pm.

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ANTHONY P. FARRELL AND DAVID R. JONES

(Farrell et al., 1986), and correlations are found between plasma low density lipoproteins and lesions (Eaton et al., 1984; Farrell et al., 1986).The possibility exists that lesions form because of mechanical stresses imposed on the coronary artery by unusually large expansions and contractions of the compliant bulbus arteriosus on which the coronary artery lies. Preliminary studies show no lesions in coronary arteries that lie on the conus arteriosus of elasmobranchs (A. P. Farrell, 1992). Van Citters and Watson (1968), for steelhead trout, and Maneche et al. (1973), for Atlantic salmon, concluded that lesions regress when individuals return to the sea for repeated spawning. This idea was challenged by Saunders and Farrell(1988), who reported that coronary lesions in Atlantic salmon continue to develop and parallel the resumption of growth after spawning.

111. CARDIAC PHYSIOLOGY A. Cardiac Cycle

The cardiac cycle refers to synchronized contractions and relaxations of the cardiac chambers (Randall, 1970). The cycle consists of isometric and isotonic contractions (systole), during which the ventricle is emptied, and a period of relaxation (diastole), during which the chamber is refilled. Diastasis is a period when there is no blood flow into or out of a cardiac chamber. The electrical events (waves ofmuscular depolarization and repolarization), recorded as the electrocardiogram (ECG) can be related to contractile events, as revealed by intracardiac pressure and ventral aortic flow measurements. When comparisons are made between fish species (Fig. 6), important differences emerge. This section considers general and species-specific aspects of the ECG, cardiac contractility, and cardiac work.

1. ELECTROCARDIOGW The ECG terminology is the same in fishes as in mammals. The P wave represents atrial contraction, the QRS complex, ventricular contraction, and the T wave, ventricular relaxation. However, additional Fig. 6. Electrocardiograms and blood pressure traces during the cardiac cycle of representative fishes. Trout heart, adapted from Randall (1970) and Farrell (1991~). Shark heart, adapted from Satchel1 (1971). Hagfish heart, adapted from Davie et al. (1987).

0

0

In

In N

1

0

O

-

3 N

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ANTHONY P. FARRELL AND DAVID R. JONES

waves are present in fish compared with mammalian ECGs. Contraction of conal cardiac muscle in elasmobranchs produces a B wave during the S-T interval (Fig. 6). Contraction of cardiac muscle in the sinus venosus appears as a V wave during the P-T interval and is prominent in A. anguilla and hagfishes. Given the anatomical factors presented previously (a paucity of valves to prevent backflow of blood into the veins and the absence or limited amount of cardiac muscle in many species), it seems unlikely that the sinus venosus functions effectively as a contractile chamber to propel venous blood into the atrium, even though such a role has been ascribed to it in the past. In the mammalian heart, coordination between the atrial and ventricular contractions is provided by specialized cardiac fibers, which first delay conduction to the ventricle (atrioventricular junctional fibers) and then speed up its propagation over the ventricle (fastconducting fibers). An atrioventricular delay, as indicated by the P-Q interval, is evident in fish ECGs. This delay is important in allowing the complete contraction of the atrium before ventricular contraction starts. Atrioventricular conduction in some fish can be seriously compromised at low temperatures (Peyraud-Waitzenegger et al., 1980). In perfused rainbow trout hearts, atrioventricular desynchrony can occur at 5 to 7°C (Bennion, 1968; Graham and Farrell, 1989), but is corrected with adrenergic stimulation (10 nmol adrenaline). This contrasts with the situation in A. anguilla at 8"C, in which stimulation of a-adrenoceptors with high concentrations of adrenaline (>5 pmol) actually causes atrioventricular desynchrony (Pennec and Peyraud,

1983). The propagation of the action potential from the atrioventricular node to the various parts of the ventricle is coordinated and synchronized despite a lack of histological evidence for fast-conducting fibers in fish ventricles (Satchell, 1991). Whether the various layers of fiber bundles or the different myocyte diameters in the ventricle are important in the spread of excitation is unknown. At the level of individual myocytes, current flow between cells is affected by the degree of folding and number of gap junctions in the intercalated discs. Davie et al. (1987)noted that teleosts have intercalated discs that are less convoluted and have fewer gap junctions than those of higher vertebrates. Gap junctions may be absent or few in number in hagfish myocytes.

2. CONTRACTILITY Cardiac contractility is a term that expresses the vigor of contraction or, more specifically, the change in developed force at a given resting fiber length (Berne and Levy, 1988).An increase in fiber length above

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resting increases the force of contraction (Frank-Starling mechanism), but it does not increase contractility. Measures of contractility in vitro include peak isometric tension and maximal shortening velocity at a fixed initial length. Measurement of contractility in vivo is less precise. The dP/dt during the isovolumic phase of the cardiac cycle (isometric contraction) and initial velocity of blood flow in the aorta are used as indirect indices. Davie et al. (1987) noted that dP/dt values for ventricular contractions in teleosts ranged from 370 mm Hg/sec to 480 mm Hg/sec, about five times slower than those of mammalian hearts. On the other hand, tunas have higher heart rates and ventral aortic pressures, and dP/dt values are much higher compared with other teleosts, falling into the mammalian range (Jones et al., 1992). In contrast, ventricular dP/dt for Myxine glutinosa and Eptatretus cirrhatus (approximately 22 mm Hg/sec) is more than 10 times slower than that for teleosts (Davie et al., 1987). Ventricular dP/dt in the leopard shark (Triakis semifasciata) is also slow (25 mm Hg/sec), increasing to only 36 mm Hg/sec during swimming (Lai et al., 1990a).The slow dP/dt in hagfish and elasmobranchs probably reflects low contractility and perhaps slow rates of electrical conduction between myocytes. Slow conduction rates would be expected in hagfish because of the lack of deeply penetrating T tubules in the SL and poor electrical couplings between myocytes (Davie et al., 1987). Neural (vagal and adrenergic nerve fibers), humoral, and local factors can increase (positive inotropy) and decrease (negative inotropy) cardiac contractility. Among the specific factors known to increase contractility of the atrium and/or ventricle in fishes are the following: increased temperature (Ask et al., 1981; Bailey and Driedzic, 1990), P-adrenergic stimulation (see Ask et aZ., 1981; Laurent et al., 1983; Nilsson, 1983; Farrell, 1984; Vornanen, 1989), the peptides arginine vasotocin and oxytocin (Chui and Lee, 1990), adenosine (Lennard and Huddart, 1989), prostacyclin (Acierno et aZ., 1990), and histamine (Temma et al., 1989). Negative inotropic effects on atrial or ventricular tissue occur with the following: hypoxia and acidosis (Gesser and Poupa, 1983; Farrell, 1984), acetylcholine (Randall, 1970; Holmgren, 1977; Cameron and Brown, 1981), a-adrenergic stimulation in some species (Tirri and Lehto, 1984), purinergic agents (Meghi and Burnstock, 1984), and adrenaline in combination with adenosine (Lennard and Huddart, 1989). Isolated atrial and ventricular tissues can have different sensitivities to pharmacological agents. For example, acetylcholine has negative inotropic effects on the Atlantic cod atrium but not on the ventricle, whereas the ventricle is more sensitive to adrenergic stimulation (Holmgren, 1977). This implies that vagal inner-

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ANTHONY P. FARRELL AND DAVID R. JONES

vation is confined to the atrial region. Interestingly, the affinity for P-adrenergic atrial stimulation is greater in Platichthys Jesus and Squalus acanthias (fish without adrenergic innervation) than in rainbow trout (Ask, 1983). Contractility, as measured by the force of contraction of cardiac muscle strips in vitro, is dependent on the duration of the active state (the period of contraction and relaxation) and its intensity (rate of contraction). The relationship between maximal isometric force and contraction frequency is documented for several species. In ventricular and atrial strips from hagfish (Myxine glutinosa) and a variety of teleost species, an increase in contraction frequency reduces the duration of the active state, decreases maximal isometric tension, and, at higher frequencies, the rate of contraction (Ask, 1983; Driedzic and Gesser, 1985; Vornanen, 1989). This inverse relationship between maximal isometric tension and contraction frequency for these teleosts and hagfish is referred to as a negative staircase effect. In other vertebrates, including ventricular tissue from mammals and several elasmobranch species (Driedzic and Gesser, 1988) and atrial tissue from skipjack tuna (Keen et al., 1992), there is a positive relationship between contraction frequency and maximal isometric tension at low frequencies, whereas at higher frequencies the relationship is again negative. Thus, the force-frequency relationship has an apex, i.e., a contraction frequency at which maximal isometric tension reaches a peak. In elasmobranchs, the apices occur at contraction frequencies (0.3-0.4 Hz) that correspond to in vivo heart rates. Similarly, the apex for skipjack tuna (1.4-1.6 Hz) also corresponds to in vivo heart rates. Moreover, skipjack atrial tissue can contract up to a maximal frequency of 3.4 Hz (Keen et aZ., 1992), a frequency that is well beyond those reported for other fish species. The relevance of these in vitro observations to the relationships between maximal isometric tension, contraction frequency, and the duration of the active state to the in vivo situation is further brought home by Vornanen’s observation that the duration of the action potential is very similar to the duration of contraction. In electrically paced cardiac strips in vitro, increasing temperature increases the rate of contraction, thereby increasing contractility (Ask, 1983).However, the duration ofthe active state is shorter, with increasing temperature through shorter contraction and relaxation times (Vornanen, 1989; Bailey and Driedzic, 1990), and this reduces maximal isometric tension. Nonetheless, because the active state is shorter, the muscle strips can contract at higher frequencies at high temperatures. An additional effect of temperature is revealed in spontaneously

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beating cardiac strips in vitro; the negative staircase effect is less pronounced at high than at low temperatures because the higher heart rates result in a shorter active state. Some of the changes in cardiac contractility associated with temperature acclimation were identified by Bailey and Driedzic (1990).Among these were a pronounced reduction in the duration of relaxation (e.g., yellow perch, Percaflavescens) and a shorter-duration contraction (e.g., smallmouth bass, Micropterus dolomieui, and yellow perch) in cold-acclimated compared with warmacclimated fish. The positive inotropic effect of P-adrenergic stimulation is the result of increases in both the rate and duration of contraction in crucian carp and rainbow trout (Vornanen, 1989).Again positive chronotropy also caused by P-adrenergic stimulation offsets, but does not overcome, the positive inotropic effects (Ask, 1983).In contrast, adrenaline increased maximal tension independent of frequency in atrial strips from skipjack tuna; the duration of contraction increased with no change in the rates of contraction and relaxation (Keen et al., 1992). Cardiac contractility in fishes is dependent on extracellular calcium concentrations, reflecting the overall importance of transsarcolemmal calcium movements in the availability of activator calcium for excitation-contraction coupling (see Chapter 6). In several species of elasmobranchs and teleosts, including skipjack tuna, maximal isometric force increases severalfold with increasing extracellular calcium (1-9 mM) (Driedzic and Gesser, 1985; Driedzic and Gesser, 1988; Keen et al., 1992). One consequence of the positive inotropic effect of extracellular calcium is to shift the relationship between maximal isometric tension and contraction frequency to the right (Driedzic and Gesser, 1988).Despite marked in vitro effects on contractility by extracellular calcium, in vivo effects may be quite limited because a reasonably good homeostatic mechanism maintains extracellular calcium levels above the threshold for major cardiac effects. An increase from 1 mM to 2 mM extracellular calcium substantially increased and pressure development in isolated perfused Atlantic cod hearts (Driedzic and Gesser, 1985). However, increases in extracellular calcium above the threshold level of around 1.5 mM resulted in very modest increases in maximal cardiac power output of in situ perfused heart preparations (Farrell et aZ., 1986a).

3. CARDIAC STROKE WORKAND POWER OUTPUT Pressure-volume loops are presented in Fig. 7 for rainbow trout and leopard shark at rest and during exercise. The area of the pressure-volume loop is proportional to the external work performed by

A. RAINBOW TROUT

VENTRICLE VOLUME (ml/kg body mass) B. LEOPARD SHARK

VENTRICLE VOLUME (ml/kg body mass)

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the ventricle. This is termed ventricular stroke work (units of joules). During exercise the ventricle clearly performs more work as a result of higher pressures and stroke volumes. However, usually stroke work is not obtained from pressure-volume loops but estimated as the product of stroke volume times mean driving pressure (ventral aortic pressure minus central venous blood pressure). If information on central venous blood pressure is absent, using a venous blood pressure of zero probably introduces an error of no more than 5% in the calculation. Myocardial power output is the product of stroke work times heart rate, or alternatively, the product of Q and ventral aortic pressure (units of watts). Stroke work and power output are often expressed per gram ventricular mass in fishes because ventricular mass per se is quite variable. Comparisons of power output, such as those presented in Table 11, are useful because power output measures the integrated performance of the heart (flow work and pressure work). A number of general points can be drawn from these data. The highest and lowest myocardial power output values are for tuna and hagfish, respectively, with the difference being over sixfold. With the exception of hagfish and a high value for Scyliorhinus canicula, most temperate water species have resting power output values in the range 0.9-1.8 mW/g ventricular mass a range which is similar to that for Antarctic species. Power output can increase two- to fourfold with exercise or adrenergic stimulation. The maximal power output observed for elasmobranchs and sluggish species of teleosts is about half that reported for the more active salmonids (3.2mW/g versus 6-7 mW/g). Maximal power output in rainbow trout is only 30-50% greater than the resting value for tuna. A threefold increase in the power output values reported for tuna in Table I1 would not be unexpected (i.e., a twofold increase in Q and a 50% increase in ventral aortic pressure). This would mean that maximal power output values for tuna are of the order of 15-18 mW/g as compared with 7.0 mW/g in rainbow trout, 3.3 mW/g in leopard shark, and 0.27 mWlg in Atlantic hagfish. The physiological and biochemical basis for this large range in power output per unit mass of ventricle warrants attention. The slow heart rates of Antarctic fish result in the highest values for

Fig. 7. Pressure-volume loops based on data from resting and exercising rainbow trout and leopard sharks. The area of the pressure-volume loops reflects cardiac work. With swimming, substantial increases in both stroke volume and blood pressure raise the work performed by the heart.

Cardiac Work and Power Output in Selected Fishes at Rest and while Swimming Stroke work (mJM

Temperate water fishes Myxine glutinosab Triakis semifasciata Scyliorhinus stelloris Scyliorhinus canicula Gadus morhua Ophiodon elongatus Hemitripterus americanus Anguilla australisb Anguilla australis Oncorhynchus mykiss Oncorhynchus kisutch Tropical fishes Katsuwonus pelamis Thunnus albacares Antarctic fishes Chaenocephalus aceratus Chaenocephalus aceratus Pseudochaenichthys georgianus Chionodraco hamatusc Pagothenia borkgreuinki Pagothenia bernacchii

Power output (mWk)

Rest

Exercise

Rest

Exercise

0.21 2.00 2.00 6.50 2.47 2.36 1.54 1.09 1.29 2.41 2.44

0.62 3.59 3.21

0.08 1.71 1.43 2.07 1.77 1.18 1.16 0.96 1.08 1.53 1.22

0.27 3.30 2.46

-

3.87 4.28 3.54 1.09 1.88 8.27 8.96

-

3.29 3.21 3.13 0.98 2.19 7.03 5.97

Temperature ("C)

11 14-24 19 14 10.5 10 10 16-17 16-20 11 5

2.23 3.46

4.70 5.60

26 26

4.02 6.60 5.32

0.98 1.80 0.72

0.5-2 1.2 0.5-2 0-2

-

5.58 4.69

4-6 6.0

-

1.05 0.82

-

1.6-3.4 2.00

-

0 0

Body mass (kg)

Source"

0.08 1.93 2.6 0.5-1.0 0.4-0.8 4.2 1.2 0.62 0.9-1.1 1.0 1.4 1-2 1-2 1.0 2.0 1.o 0.29-0.47 0.06

0.05

a (a)Axelsson et al. (1990) and Forster et 01. (1991);(b)Lai et al. (1989a,b, 1990a);(c)Piiper et al. (1977); (d) Short et al. (1977);(e)Axelsson and (f) Farrell(l982);(g) Axelsson et al. (1989)and Farrell et al. (1985);(h) Davie and Forster (1980);(i) Hipkins (1985);(i)Kiceniuk Nilsson (1986); (1) Bushnell(1988); (m) Hemmingsen and Douglas (1977);(n) Hemmingsen et al. (1972); (0) Tota et al. (1991); and Jones (1977);(k) Davis (1966); (p) Axelsson et al. (1992). Maximum values for postadrenaline infusion. Maximum values in perfused heat preparations. Where ventricle mass is not known 0.2glkg body and 0.08glkg body were assumed for elasmobranch and teleost species.

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resting stroke work (Table 11).Conversely, the fast heart rates in tuna result in a stroke work that is not so different from that of other fishes, even though power outputs in tuna are the highest reported. The impact of a large relative ventricular mass on overall cardiac performance can be assessed by expressing cardiac power output as a function of body mass rather than ventricular mass and comparing data for skipjack tuna and rainbow trout. The resting power output for skipjack tuna heart (-26 mW/kg body mass) is more than 10 times the resting power output for rainbow trout heart (-2 mW/kg body mass) (Farrell et al., 1992). Since a large ventricular mass increases total myocardial power output, a large ventricular mass will compensate for reduced contractility associated with cold temperatures. This sort of compensation may explain why cardiac hypertrophy is associated with cold-acclimation in fish and why Antarctic fishes have relatively large ventricles (Table I). 4. EFFICIENCY OF CARDIAC CONTRACTION

Efficiency is the quotient of external work divided by the total energy transformed and, like that of most mechanical and biological systems, the efficiency of the fish heart is low (Farrell et al., 1985; Farrell and Milligan, 1986). Hence, only a small proportion of cardiac 0 2 consumption appears as external work and, up to a point, functional or design strategies that improve external work performance will have relatively little impact on cardiac energetics. The rider, “up to apoint,” is included in the above statement because, theoretically, both mechanical and biological systems increase efficiency with increasing power output from idle or rest. Hence, it is possible for more external work to be performed with little or no reflection of this in increased cardiac 0 2 uptake. In this respect, Houlihan et al. (1988) report no increase in mechanical efficiency from the resting value of 21% for perfused rainbow trout hearts. In contrast, others have reported resting mechanical efficiency to be 11-15%, increasing to a maximum of 25% at higher power outputs (Farrell et al., 1985; Graham and Farrell, 1990). Really, it depends on where you start, because at subnormal power outputs, mechanical efficiency is greatly reduced (4% in sea raven; Driedzic et al., 1983). Hence, given that efficiency changes in fish hearts are small, then a general prediction is that more active fishes with larger hearts, higher Q’s and higher blood pressures also have a greater myocardial oxygen consumption per unit time ( V 0 2 ) . Furthermore, reasonably accurate predictions of myocardial V 0 2 will be possible from values of Q and ventral aortic pressure. The majority of myocardial V 0 2 is utilized for developing tension to

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ANTHONY P. FARRELL AND DAVID R. JONES

generate blood pressure, so anything that decreases cardiac tension (e.g., a fall in ventricular volume or blood pressure) or the time over which the heart is contracting (e.g., decrease in heart rate) will bring about an improvement in cardiac efficiency. As a consequence, if ventricular work is increased in mammals by raising mean aortic pressure (pressure work), at constant stroke volume and rate, then the increase in external work is paralleled by an increase in myocardial V02.Obviously, efficiency is unaltered. In contrast, if work is increased a like amount by increasing stroke volume alone (flow work), then myocardial VO, increases only slightly owing to a large increase in efficiency (Sarnoff, 1958a,b). However, in fish, both pressure and flow work increase myocardial V 0 2 by similar amounts for similar increases in external work (Farrell, 1984), so the increase in wall tension in enlarged fish ventricles must offset gains in efficiency due to increased external work, per se. As noted previously, external work is obtained as the area enclosed by the ventricular pressure-volume loop. More usually, this value is approximated as the product of mean aortic pressure and flow, which gives values within 20% of those obtained from pressure-volume loops. Using a colorific equivalent, power outputs can be converted to 0 2 cost, and comparing this value to measured values ofmyocardial VO, yields the efficiency of cardiac contraction. Unfortunately, making measurements of myocardial VO2 in intact fish is next to impossible owing to the variable development of the coronary system, lack of a coronary sinus, and difficulty in assessing 0 2 uptake from venous blood flowing through the heart. Consequently, studies with working perfused fish hearts have provided much of the basis for our understanding of myocardial VO2 in fish. Since myocardial VOZis directly proportional to cardiac power output under aerobic conditions (Fig. 8), then myocardial 002 appears to be similar in all hearts, being around 0.3 pl Oz/sec per mW of cardiac power output (Davie and Farrell, 1991a). Sea raven and Eptatretus cirrhatus hearts (type I) use 0.29 and 0.42 pl Oe/sec per mW, respectively (Farrell et al., 1985; Forster, 1991).Rainbow trout hearts (type 11)use 0.26-0.28 pl Oz/sec per mW (Houlihan et al., 1988; Graham and Farrell, 1990). Squalus acanthias hearts (type 111) use 0.30 pl Oz/sec per mW (Davie and Franklin 1992). Assuming a myocardial VO2 of0.3 pl/sec per mW for other species, it is possible to calculate the 0 2 cost of cardiac pumping in tuna and Antarctic hemoglobin-free fishes, two species with high Q values. Using a total resting V02value of 242 pl Oz/sec kg body mass for skipjack tuna (Bushnell and Brill, 1992) and a cardiac power output of

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HEART

I 1

2

3

CARDIAC POWER OUTPUT (mW)

Fig. 8. The relationship between cardiac Vo, and power output for perfused fish hearts. The data were obtained from the following sources: dogfish, Davie and Franklin (1992); hagfish, Forster (1991); sea raven, Farrell et al. (1985); rainbow trout, Graham and Farrell(l989).

4.7 mW/g ventricular mass (Table 11),the 4-g ventricle in a 1-kg skipjack tuna would develop 18.8 mW and consume 5.64 pl Oz/sec, representing 2.3%of total V02. For a 1-kg Chaenocephalus aceratus using a total VO2 of 5.8 pl Oz/sec (MacDonald et al., 1987) and a total cardiac power output of 3.0 mW (1.0 mW/g for a 3-g ventricle; Table 11), myocardial 602 is 1.2 PI Oelsec, which in contrast is 23% of resting metabolism. This high 0 2 cost of cardiac pumping may limit activity in C. aceratus, particularly since its oxygen supply must be obtained from the venous blood. Estimates of the 0 2 cost of cardiac pumping have been made for other species. Cameron (1975) estimated 0 2 cost of cardiac pumping as between 0.08 and4.0% oftotal VO2.Jones and Randall (1978) estimated myocardial V 0 2 in resting rainbow trout as 3.5% oftotal V02, increasing to 4.5% at maximal prolonged swimming speed (Ucrit).These early predictions are close to more recent values based on myocardial V 0 2 measurements. In sea raven, myocardial VOz is approximately 0.6%of

36

ANTHONY P. FARRELL AND DAVID R. JONES

total resting V 0 2 (Farrell et al., 1985). The 0 2 cost of cardiac pumping represents 4.6% of total V02 in rainbow trout, but decreases to 1.9% at Ucrit(Farrell and Steffensen, 1987a) and is unlikely to limit swimming performance in rainbow trout, as was suggested earlier (Jones, 1971). The reasons for this decrease in 0 2 cost are that the increase in Q provides around half of the increase in internal 0 2 convection and that myocardial efficiency increases. In E . cirrhatus, myocardial V02 for a resting power output of0.2 mW is approximately 3%oftotal resting 6 0 2 (2.77 uL Oz/s.kg body mass; Forster et al., 1992).Therefore, the 0 2 cost of cardiac pumping in a variety of temperate fishes ranges between 0.5% and 5.0%. It is also important to recognize that anaerobic metabolism can contribute significantly to cardiac work in certain fishes under hypoxic conditions. Since the capacity to generate adenosine triphosphate (ATP) anaerobically does not vary greatly between fish species (see Chapter 5), but power output can vary considerably between fish species, the relative contribution made by anaerobic metabolism to cardiac work is then dependent on the total cardiac power output (Farrell, 1991a). Forster (1991), after examining the myocardial V 0 2 and lactate release by hypoxic, perfused E . cirrhatus hearts, reported that most of the ATP requirement was met by anaerobic metabolism (see also Forster et al., 1991). Thus, hagfish, with an extremely low cardiac power output, have a low cardiac ATP demand and maintain resting cardiac function through glycolysis (Forster, 1989, 1991; Forster et al., 1991). In most other fish, glycolysis would appear to be inadequate to support the much higher power outputs (Farrell, 1991a). For example, anaerobic metabolism contributes at best 25% of the ATP requirement during hypoxic exposure in perfused sea raven hearts. Failure to account for anaerobic metabolism in calculating mechanical efficiency leads to erroneously high estimates, e.g., Farrell et al. (1985).

B. Control of Stroke Volume Cardiac output is the product of stroke volume and heart rate. A number of important differences exist between fish and mammalian hearts with respect to the control of stroke volume. Foremost, many fish can increase stroke volume to a much greater degree than heart rate (Farrell, 1991a; see also Section 111,D).Second, atrial contraction is the primary, if not sole determinant of ventricular filling. Third, atrial filling is achieved through uis-b-fronte and uis-b-tergo mechanisms. (Vis-h-fronte filling directly utilizes some of the energy of ven-

1. THE

HEART

37

tricular contraction, “force from in front,” whereas uis-a-tergo filling utilizes energy remaining in the venous circulation, “force from behind.”) Fourth, end-systolic volume of the ventricle is quite small. Because of these differences, it is important to fully appreciate the control of stroke volume in fish. The following describes in some detail the Frank-Starling mechanism, atrial filling, and other factors that affect stroke volume. I n mammalian hearts, atrial contraction provides around 25% of ventricular filling, the majority occurring directly from the veins, with a small proportion due to elastic recoil of the ventricle. In contrast, it is generally held that atrial contraction is the sole determinant of ventricular filling in fish (Randall, 1970; Johansen and Burggren, 1980).This being the case, the fish heart needs to be viewed as two pumps (atrium and ventricle) in series. The volume pumped by each chamber is set by the end-diastolic volume minus the end-systolic volume (Fig. 7 ) . End-diastolic volume of the atrium is determined by the time available for filling and the volume and distensibility of the atrium. With uis-u-tergo filling, central venous pressure and perhaps sinus contraction provide the energy for atrial filling. With uis-u-fmnte filling, the force of ventricular contraction will b e an additional factor because of the effect it has on the negative (suction) pressure within the pericardium. Atrial end-systolic volume is determined by (1) the FrankStarling mechanism, which will be described; (2) atrial contractility; ( 3 )the atrioventricular delay, which sets ventricular filling time; and (4) ventricular volume and distensibility. End-systolic volume of the ventricle is determined by the force of contraction, the diastolic output pressure in the aorta, and the timing of the closure of the ventricular outflow valves. Force of contraction is altered either through the Frank-Starling mechanism, or by factors that modulate contractility (see preceding sections). End-systolic volume of the ventricle is much lower in fish than in mammals. Normal contraction appears to almost completely empty the ventricle. Mean ejection fraction is estimated at 80% in the leopard shark (Lai et al., 1990a). In rainbow trout, the ejection fraction is normally near 100% (Franklin and Davie, 1992). Thus, ventricular end-diastolic volume approximates atrial stroke volume. Clearly, modulation of atrial contractility and the Frank-Starling mechanism are particularly important in determining ventricle filling and hence cardiac stroke volume. I n terms of the pressures developed, the atrium can be viewed as a preamplifier and the ventricle as a power amplifier. An intrinsic property of cardiac muscle is that end-systolic volume

38

ANTHONY P. FARRELL AND DAVID R. JONES

of the ventricle is normally independent of mean aortic output pressure. This is termed homeometric regulation. Homeometric regulation has been quantified in perfused fish hearts, by measuring the heart’s intrinsic ability to maintain resting stroke volume over a range for aortic output pressure (Farrell, 1984).As expected, the range is species specific and correlates well with in vivo ventral aortic pressures, i.e., hearts from more active fishes are capable of sustaining stroke volume at higher aortic pressures (Fig. 9A). However, at extremely high pressures homeometric regulation breaks down, the heart does not empty completely, end-systolic volume increases (Franklin and Davie, 1992), and stroke volume decreases (Fig. 9A). Factors that alter cardiac contractility also affect the maximal output pressure against which stroke volume can be maintained. The Antarctic fish Chionodraco is unusual in that even small increases in output pressure decrease stroke volume substantially in perfused heart preparations (Tota et al., 1991). An anatomical constraint on stroke volume is the size of the ventricular chamber. This anatomical constraint is presumably reflected in the maximal stroke volume. Maximal stroke volume varies between fish species in the general range ofO.7 to 1.4 ml/kg body mass (Fig. 9B). Such a small interspecific range is perhaps surprising because fish hearts develop different blood pressures, and the size of the ventricle relative to body mass vanes by more than 10-fold (Table I). Antarctic fishes are important exceptions in that hemoglobin-free and redblooded species have unusually high stroke volumes, ranging from 2 to 10 ml/kg (Holeton, 1970; Hemmingsen et al., 1972; Tota et al., 1991; Axelsson et al., 1992). Stroke volume is not entirely independent of heart rate (Farrell, 1984, 1985). In isolated hearts of rainbow trout, increasing cardiac pacing frequency within the physiological range reduced maximal stroke volume to such a degree that 0 did not increase at higher heart rates (Farrell et al., 1989).This observation can be interpreted as either cardiac filling time being compromised, or the force of contraction being reduced at high heart rates (an expression of the negative staircase effect). Furthermore, there seems to be an antagonistic rather than synergistic interaction between increasing inotropic and chronotropic forces in yellowfin tuna. An increase in stroke volume of 30%,caused by injecting phenylephrine into the circulation of restrained fish, was accompanied by a 50% increase in the length of the systolic ejection phase. In fact, in one tuna, stroke volume reached 1.2 ml/kg, but heart rate fell from 112 to 105 beats per minute (bpm) (Jones et al., 1992).

1.

.

,tuna

5 9

04-

y”- 0.3 O?

$Z

0.2

0c w

dogfish

trout

4

4

t’

-

+sea

hagfish I

raven

ocean pout

01I

39

-1 .o 0 1 .o 2.0 CARDIAC FILLING PRESSURE, (mrnHg)

-2.0

03

--

THE HEART

I

1

I

I

1

I

I

I

I

I

I

Fig. 9. (A) The effect of increased output pressure on the ability of fish hearts to maintain constant stroke volume (homeometric regulation). At resting Q levels, increases in output pressure have little effect on resting stroke volume over a physiological pressure range. The maximum pressure is species specific, being higher in active fish. (B) A general comparison of Starling curves obtained for perfused hearts from several teleost species. Note that, through uis-a-fronte filling, a large portion of the curve for rainbow trout is at subambient pressure. The positive inotropic effect of adrenaline is to shift the Starling curve for rainbow trout upward and to the left. From Farrell (1991a).

1. FRANK-STARLING MECHANISM With greater cardiac filling, the force of contraction must be greater to achieve complete cardiac emptying. According to the FrankStarling mechanism of the heart, “the energy of contraction is a function of the length of the muscle fibre.” Thus, increased end-diastolic volume results in a greater force of contraction and a greater stroke volume. This intrinsic property of muscle applies to the atrium and ventricle. Since an increase in cardiac filling pressure increases enddiastolic volume, the relationship between cardiac filling pressure and stroke volume is referred to as a Starling curve.

40

ANTHONY P. FARRELL AND DAVID R. JONES

The Starling curve has functional significance in fish because stroke volume varies appreciably. Figure 9B shows a series of Starling curves for perfused hearts from teleosts. Similar curves have been obtained with E. cirrhatus (Forster et al., 1991) and S . acanthias (Davie and Farrell, 1991b).These curves illustrate four important points. First, the in vitro range for stroke volume corresponds well with the in vivo range (see Table V). Second, the cardiac filling pressures in vitro are low, even subambient on occasions, and are comparable with central venous pressures in vivo. Third, only small changes in cardiac filling pressures (about 1-2 mm Hg) are needed to move along the Starling curve from resting to near maximal stroke volume. In this regard, perfused fish hearts are much more sensitive to filling pressure than perfused mammalian hearts, which often require filling pressures 10 times higher than those in fish to evoke maximal stroke volume. The greater sensitivity of the fish heart to filling pressure may reflect a combination of a highly distensible, thin-walled atrium and atrial contraction determining ventricular filling. Fourth, inotropic actions of neurohumoral factors create a family of Starling curves, making the heart more or less sensitive to filling pressure. For example, the positive inotropic effect of adrenaline increases the sensitivity of the rainbow trout heart to filling pressure, shifting the Starling curve to the left and upward (Fig. 9B). The negative inotropic action of acetylcholine probably shifts the Starling curve to the right and makes it flatter. What is not clear in terms of the regulation of stroke volume in vivo is whether venous filling pressure, cardiac contractility at a constant filling pressure, or some combination of both are utilized to adjust stroke volume. Increased performance due to the Frank-Starling mechanism causes a shift along a given curve, whereas altered contractility causes a shift to another curve. Blood pressure in the cardinal vein increased significantly with swimming in the leopard shark (Lai et al., 1990b). In rainbow trout, the increase in venous pressure at Ucrit (maximal prolonged swimming speed), though not statistically significant (1.9 mm Hg; Kiceniuk and Jones, 1977),corresponds to the upper arm of the Starling curve. Hence these data suggest that increased venous pressure, alone, may make an important contribution.

2. CARDIAC FILLING a. Atrial Filling. Atrial filling is achieved by one of two mechanisms: vis-a-tergo and vis-a-fronte. Vis-ci-tergo atrial filling utilizes potential and kinetic energy that either remains in the venous circulation, or is generated by sinus contraction. Intuitively, the large unvalved communications between the Cuverian ducts and the sinus

1. THE

HEART

41

venosus should render contraction of the sinus cardiac muscle, when present, relatively ineffective at filling the atrium. Therefore, vis-utergo atrial filling is determined primarily by the pressure gradient set by the central venous blood pressure. In contrast, vis-h-fronte filling uses some of the energy of ventricular contraction directly to distend the atrium and bring about atrial filling. This is achieved by subambient intrapericardial pressures, which create a negative atrial transmural pressure gradient. A rigid pericardial cavity is necessary to develop subambient intrapericardial pressures. The anatomical arrangements for a rigid pericardial cavity include a thick pericardial membrane (e.g., elasmobranchs) or a rigid musculoskeletal framework over which a relatively thin pericardial membrane is attached (e.g., active teleosts). An important consequence of vis-u-fronte filling is that central venous blood pressures will be subambient. The relative importance of the vis-a-fronte and uis-a-tergo atrial filling mechanisms in fish has been examined with in situ perfused hearts. What has emerged from these studies is that benthic teleosts such as the sea raven and ocean pout use only vis-a-tergo filling. Above-ambient filling pressures are required for normal and elevated stroke volumes (Farrell, 1984, 1985). In contrast, elasmobranchs and active teleosts employ both vis-u-fronte and vis-a-tergo filling, with the vis-u-tergo mechanism increasing in importance at high stroke volumes. For perfused hearts from rainbow trout (Fig. 8A), S . acanthias, yellowfin tuna (Farrell et a1., 1992), A. dieffenbachii (Franklin and Davie, 1991), and SerioZa grandis (P. S. Davie and C. E. Franklin, personal communication), the lower half of the range for stroke volume can be generated with subambient filling pressures (uis-ci-fronte filling); but maximal stroke volume requires positive filling pressures (vis-u-tergo filling). Subambient intrapericardial pressures are reported for rainbow trout (Farrell et al., 1988b), various elasmobranchs (Satchell, 1971; Shabetai et al., 1985), and tuna (Lai et aZ., 1987). Typically, intrapericardial pressure oscillates during the cardiac cycle with a sharp decrease in pressure during ventricular systole coincident with atrial filling (Fig. 10A). The observation that intrapericardial pressures increased when stroke volume increased in the leopard shark (Lai et al., 1989a) is also consistent with the idea of transition from vis-u-fronte to vis-a-tergo filling at higher stroke volumes. When the pericardium is either punctured or is absent in perfused hearts from rainbow trout, S . acanthias, and A. dieffenbachii, intrapericardial pressure remains at ambient pressure, and the Starling curve is right shifted (Fig. 10B); above-ambient filling pressures are

42

ANTHONY P. FARRELL AND DAVID R. JONES

pericardioperitoneal

C

I

L I

I r 1

D

t

bulbus arteriosus

Fig. 10. (A) Cardiovascular measurements from a perfused rainbow trout heart during low (equivalent to a resting in uiuo status) and high (elevated and diastolic output pressure) work conditions. The pressure in the sinus venosus (Psv) is below zero with the low-work condition, but above zero with the high-work condition. From A. P. Farrell, unpublished observations. (B) The effect of puncturing the pericardium on the Starling curve of a perfused rainbow trout heart. Normally, filling pressures that are below zero account for 50% of the Starling curve, indicating a uis-d-frontecardiac filling mechanism. When the pericardium is open, there is a right shift in the Starling curve, and filling pressures are above zero. Adapted with permission from Farrell et QZ. (1988b). (C) and (D)illustrate how certain fish modulate stroke volume even though they have a rigid pericardial cavity that facilitates uis-d-fronte filling. In each case the solid lines represent the end-diastolic volume when 0and stroke volume increase. (C) The situation in elasmobranchs. Atrial volume can be matched with ventricular volume, and, as ventricle

1. THE HEART

43

then needed for uis-u-tergo filling (Farrell et aZ., 1988b; Davie and Farrell, 1991b; Franklin and Davie, 1991). Satchel1 and Jones (1967) report that puncturing the pericardium had no effect on Q in Heterodontis portusjacksoni. Hence, the potential for wis-u-fronte filling exists, but whether this potential is realized, especially during activity, is unclear. For example, Lai et al. (1990a) suggest that atrial filling is uis-u-tergo in the leopard shark. They reported a triphasic filling pattern for the atrium, involving atrial relaxation, ventricular ejection, and sinus venosus contraction. Also cardinal sinus blood pressures were higher than pericardial pressures, which contrast with the typically subambient venous pressures recorded in other elasmobranchs (see Chapter 3, this volume). Furthermore, recent recordings from tuna show no evidence of large subambient intrapericardial pressures ( Jones et al., 1992). Potential advantages and disadvantages of uis-u-fronte atrial filling are worth considering. First, subambient venous pressures, which uis-u-fronte filling permits, will tend to keep the stressed venous blood volume (see Part B, Chapter 3) at a minimum (Farrell et al., 1988b). This may be particularly advantageous in fish with either fast circulation times (e.g., active teleosts), or capacious venous blood vessels (e.g., elasmobranchs). Second, higher aortic pressures may be possible with uis-d-fronte filling. Puncturing the pericardium to prevent uis-dfronte filling reduced the maximal pressure that could be developed by perfused rainbow trout and A. dieffenbachii hearts (Farrell et al., 198813; Franklin and Davie, 1991). However, the ventricle has to generate pressure against a small negative intrapericardial pressure, which must reduce efficiency. Third, ois-u-fronte filling may be more rapid than uis-b-tergo filling since filling time is set by the duration of isometric ventricular contraction, rather than by the longer diastolic period. This may permit higher heart rates (Farrell, 1991b). In terms of disadvantages, the rigid pericardial cavity needed for uis-u-fronte filling has a finite volume, which theoretically should limit any increase in stroke volume. The fact that teleosts and elasmobranchs switch to wis-u-tergo filling at high stroke volumes is undoubtedly a reflection of

end-diastolic volume and intrapericardial pressure increase, pericardial fluid is displaced from the pericardium into the peritoneum through the pericardio-peritonea1 canal. (D) The probable situation in trout. The volumes of the sinus venosus and atrium are larger than the volume of the ventricle and act as variable-volume reservoirs. When ventricular end-diastolic volume increases, the end-diastolic volumes of the atrium and sinus venosus decrease. From Farrell(1991a).

44

ANTHONY P. FARRELL AND DAVID R. JONES

this problem. Nonetheless, two- to threefold increases in stroke volume are common in fishes, and up to 50% of this change is possible with vis-d-fronte filling (Fig. 10B). There appear to be two solutions to the problem of a finite pericardial volume. Elasmobranchs have a pericardio-peritonea1 canal, which allows a controlled and rapid removal of fluid from the pericardial cavity to peritoneal cavity (Shabetai et al., 1985; Lai et aZ., 1989a).Thus, when stroke volume increases, the ventricle can occupy a greater proportion of the pericardial volume b y displacing pericardial fluid into the peritoneum (Fig. 1OC).Pericardial fluid is replaced at a slower rate by secretion. Teleosts do not have a pericardio-peritonea1 canal, and the near zero end-systolic volume of the ventricle exacerbates the problem (i.e.,increases in stroke volume cannot draw on an end-systolic reserve of the ventricle). To accommodate an increase in stroke volume within the rigid pericardial cavity, the mechanism suggested for rainbow trout is that the atrium and sinus act as variable-volume reservoirs; their end-diastolic volumes are larger under low than under high stroke volume conditions (Fig. 10D). For this to occur, maximal end-diastolic volume of the sinus and atrium must be larger than that of the ventricle (i.e., maximal stroke volume), and this is the case (Farrell, 1991a). Tuna could circumvent the problem of a rigid pericardial cavity by increasing heart rate rather than increasing stroke volume to any significant degree (see the following), although recent recordings in vivo suggest that stroke volume can change by a factor of two (Jones et al., 1992). Another problem with uis-u-frontefilling relates to the atrial and sinus venosus distensibility being quite similar, with the result that ventricular contraction would produce uis-u-fronte filling of the sinus venosus and would tend to short-circuit vis-d-fronte filling of the atrium. Tonic contraction of the cardiac muscle in the sinus venosus, or a sinus venosus with a low volume, as in elasmobranchs and tuna, could minimize this problem (Farrell, 1991a).

b. Ventricular FiZling. Evidence for atrial filling being the sole determinant of ventricular filling comes from blood pressure measurements, which show an unfavorable pressure gradient for filling directly from either the sinus venosus or the veins during atrial diastole, and from angiographic images, which show no filling of the ventricle before atrial contraction (See Randall, 1970; Satchel], 1971; Johansen and Burggren, 1980). However, Lai et al. (1990a),working with the leopard shark and using echo-Doppler and angiographic imaging techniques, challenged the concept of atrial contraction as the sole determinant of ventricular filling. They provided evidence for biphasic ventricular

1. THE

HEART

45

filling. Blood first entered the ventricle during early diastole, probably as a result of elastic recoil, and was followed by a period of diastasis. A second surge of flow accompanied atrial contraction. The velocitytime integrals were similar for these two filling phases, suggesting that the relative flow contributions were similar. C. Control of Heart Rate Heart rate is set by the intrinsic rhythm of the sinoatrial pacemaker in the absence of extrinsic modulation. At least four mechanisms modulate the pacemaker rhythm to some degree and set the in vivo heart rate. These are stretch of the pacemaker cells, cholinergic nerve fibers, adrenergic nerve fibers, and hormones (Randall, 1970). The interplay of these effector mechanisms is species specific and temperature dependent. As will be evident from the following, temperature directly affects the pacemaker rate, alters the responses to modulators, and produces acclimatory changes. Positive chronotropy and negative chronotropy refer to increases (tachycardia) and decreases (bradycardia) in heart rate, respectively.

1. INTRINSIC HEARTRATE It is very difficult to remove all extrinsic modulation of heart rate in vivo and measure intrinsic heart rate. However, based on an analysis of the various modulators (see the following), it is clear that the adrenergic and cholinergic controls are by far the most important. Thus, heart rate in vivo after P-adrenoceptor blockade (e.g., propranolol or sotalol) and muscarinic blockade (e.g., benzetimide or atropine) provides a good approximation of intrinsic heart rate. In fact there is good agreement between estimates of intrinsic heart rate obtained in this way and measurements of intrinsic heart rate in spontaneously beating isolated heart preparations. For example, a heart rate of 38 bpm in sea raven at 10°C after pharmacological blockade (Table 111)compares well with an intrinsic heart rate of 39 bpm for a working perfused heart preparation (Farrell et al., 1983). In view of this, the data base on heart rate after muscarinic and adrenergic blockade (Table 111) is used to provide general insights into the factors that might affect intrinsic heart rate. On the basis of the data presented in Table 111, intrinsic heart rate is lowest in cyclostomes, higher in elasmobranchs, and highest in certain teleosts. In fact, a sevenfold difference in heart rate exists between hagfish and tuna. Furthermore, active fish tend to have higher heart rates within a phylogenetic grouping. There is not enough information to determine whether intrinsic heart rate in fish scales with a

Table 111 Estimates of the Intrinsic Heart Rate and the Relative Levels of Cholinergic and Adrenergic Tone which Set the Resting Heart Rate in Various Fish Species

Species Cyclostomes Myxine glutinosa Eptatretus cirrhatus Elasmobranchs Squalus acanthias Squalus acanthias Teleosts Pagothenia borchgreoinki Pagothenia bernacchii Chionodraco hamatus Oncorhynchus kisutch Oncorhynchus tshawytscha Oncorhynchus mykiss Gadus morhua Hemitripterus americanus Hemile pidotus hemilepidotus Pollachius pollachius Labrus tnixtus Labrus berggylta Ciliata mustela Raniceps raninus Zoarces oiviparus Myoxocephalus scorpius Carassius auratus Macrozoarces americanus Anguilla dieffenbachii Seriola grandis Katsuwanus pelamis Thunnus albacares

Cholinergic tone (%)

Adrenergic tone (%)

-

50.8 86.1

-

-c 30.8

27.3

55.4

Ratio

Intrinsic“ heart rate (bpm)

Temp. (“C)

17 14

10-11 17-18

1.2

-

19 40

7-8 15

3.2

17.2

23

0-0.5

80.4

27.5

3.9

22

0-0.5

-

-

-

26

0.6

55.2

60.0

0.9

30

11-13

15.4

38.8

0.4

54

8-9

-

-

-

45

10

37.7 34.8

21.0 39.6

1.9 0.9

39 38

10- 11 10-12

17.4

19.9

0.9

47

8-10

19.7

33.2

0.6

38

11-12

14.2 33.9 14.5 12.5 18.9 11.5

14.7 15.4 29.6 28.8 67.1 25.6

1.o 2.2 0.5 0.4 0.3 0.4

52 48 55 28 39 42

11-12 11-12 11-12 11-12 11-12 11-12

66.0

22.0

0.3 -

57 56

20-25 10

-

-

-

41

15

-

-

-

-

-

80 138

19 25

-

-

-

123

25

-

-

-

-

-

~

~~

Source”

~

(continues)

1. THE

47

HEART

negative exponent of body mass. Dissimilar rates have been measured for closely related and similar sized (500 g) chinook salmon (Oncorhynchus tshawytscha) and rainbow trout, whereas heart rate in larger (3-6 kg) coho salmon (0.kitsutch) is lower than both of the former, indicating a potential for species as well as size-related differences. Intrinsic pacemaker rate varies directly with temperature (Table 111) as a result of a direct effect on the membrane permeability of pacemaker fibers (Randall, 1970). Moreover, the factor by which rates change for an increase in temperature of 10°C (Qlo) for intrinsic heart rate is 2.0 or greater, indicating conformer-type responses to acute temperature changes. For in vitro working perfused sea raven hearts, the Qlo is 2.0 (Graham and Farrell, 1985)and is 2.0-2.3 in nonworking, isolated goldfish hearts (Tsukuda et al., 1985). A similar Q l o of 2.0 is reported for P . bernacchi over a temperature range of 0”-5”C after P-adrenergic and muscarinic blockade (Axelsson et al., 1992). In fact, similar Q l o values of 2.1 for Scyliorhinus canicula (Butler and Taylor, 1975), 2.1 for 15°C- and 25°C-acclimated A. anguilla (Seibert, 1979), and 2.5-2.9 for 10°C- and 24°C-acclimated sole (SoEea vulgaris) (Sureau et al., 1989) are also reported after only muscarinic blockade. Likewise, the Qlo for heart rate is 2.1 for 6.5”C- and 15°C-acclimated rainbow trout after bilateral vagotomy (Priede, 1974). Temperature acclimation results in a compensatory change in pacemaker rate, so that cold-acclimated fish have a higher pacemaker rate than warm-acclimated fish at a given test temperature. For example, at the same test temperature pulsation rates in vitro (Tsukuda et al., 1985) and heart rates in vivo (after muscarinic blockade) are lower in warmacclimated compared with cold-acclimated Carassius auratus, Solea vulgaris and A. anguilla (Seibert, 1979; Sureau et al., 1989).As a result, a Q l o value derived from the heart rates at the two acclimation temperatures is typically less than 2.0 (Qlo = 1.46 for bilaterally vagotomized rainbow trout, Priede, 1974; Qlo = 1.73 for muscarinically blocked A.

Table In (Continued) Intrinsic heart rate was estimated in uioo after pharmacologic blockade with atropine and a B-adrenergic antagonist. These values are indicated by the accompanying values for cholinergic and adrenergic tone. Other values of intrinsic heart rate are from perfused heart preparations. (a) Axelsson et al. (1990);(b) Forster et al. (1991);(c) Holmgren et al. (1992); (d) Davie and Farrell(1991b);(e)Axelsson et al. (1992); (f) Tota et al. (1991); (8) Axelsson and Farrell (unpub.); (h)Thorarensen and Farrell (unpub.);(i) Farrell et al. (1991);(j) Axelsson (1988);(k) Axelsson et al. (1989);(1) Axelsson et al. (1992);(m)Axelsson et al. (1987);(n) Cameron et al. (1979a); (0)Farrell et al.(1983); (p) Davie et al.(1992); (9)Davie et al. (1992); (r) Farrell et a1. (1992). No vagal innervation of the heart.

48

ANTHONY P. FARRELL AND DAVID R. JONES

anguilla, Seibert, 1979; 910 = 1.64 for isolated goldfish hearts, Tsukuda et al., 1985; Q l o = 1.4-1.9 for perfused sea raven hearts, Graham and Farrell, 1985; 010= 1.4 for perfused rainbow trout hearts, Graham and Farrell, 1989).The mechanism underlying the adjustment of pacemaker rate in response to temperature acclimation is not understood.

2. RESTINGHEARTRATE Resting heart rate is rarely the intrinsic pacemaker rate and, at a given temperature, is determined primarily by a “push-pull” type modulation as set by the relative levels of adrenergic and cholinergic tone on the heart (Farrell, 1991a). Numerous studies show that atropine injections in resting fish increase heart rate, from as little as 10 to 50% in rainbow trout, Atlantic cod, and dogfish (Priede, 1974; Short et al., 1977;Taylor et al., 1977; Wood et al., 1979; Axelsson et al., 1987),to as much as 175%in A. anguilla (Seibert, 1979) and 300% in red-blooded Antarctic fish (Axelsson et al., 1992). All species examined so far, except for cyclostomes, have a resting cholinergic tone to the heart (Table 111). In addition, all species have a resting adrenergic tone as revealed by P-adrenergic antagonists. In some species, e.g., goldfish, cholinergic tone is greater than the adrenergic tone, resulting in slow heart rates, whereas in others, the adrenergic tone is greater than the cholinergic tone, resulting in higher heart rates, e.g., Zoarces viuiparus. Cholinergic tone and adrenergic tone are similar in some species, e.g., sea raven (Table 111). Thus, resting heart rate can be slower than, faster than, or the same as the intrinsic pacemaker frequency, depending on the relative contributions of adrenergic and cholinergic tone.

3. CHOLINERGIC CONTROL OF HEART RATE Cholinergic fibers carried in the vagus are responsible for lowering heart rate. Increased cholinergic tone is the primary mechanism for abrupt bradycardia, as is often observed during exposure to hypoxia, at the initiation of burst swimming, and with visual and olfactory stimuli. Vagotomy or atropine injections abolish bradycardia. It is likely that acetylcholine, released from the large number of cholinergic nerve terminals in the sinoatrial region, hyperpolarizes pacemaker cells and slows the pacemaker rate (Saito, 1973). However, the specific action of neurotransmitter release on pacemaker cells in fish probably needs reexamining, since Hirst et al. (1991) find that in amphibian and mammalian cardiac cells, acetylcholine applied as a pharmacological agent causes pacemaker hyperpolarization whereas acetylcholine released as a result of vagal stimulation does not.

1. THE

HEART

49

A cholinergic inhibitory tone exists in resting fish, so a reduction in the cholinergic tone (vagal release) causes tachycardia. Obviously, the greater the resting cholinergic tone, the greater the potential for tachycardia from vagal release. During swimming a twofold increase in heart rate is observed in P . borchgrevinki as a result of vagal release of resting cholinergic tone (Axelsson et al., 1992), whereas in rainbow trout and Scyliorhinus stellaris, vagal release produces a much smaller tachycardia during exercise (Priede, 1974; Piiper et al., 1977). The cholinergic mechanisms that control heart rate are affected by temperature acclimation. For example, the level of cholinergic inhibition of heart rate is greater in cold-acclimated compared with warmacclimated Scyliorhinus canicula and rainbow trout (Taylor et al., 1977; Wood et al., 1979).In contrast, the level of cholinergic inhibition of heart rate is lower in cold-acclimated A. anguilla (Siebert, 1979). Hydrostatic pressure also affects cholinergic control of heart rate. Heart rate in A. anguilla is elevated at a hydrostatic pressure of 101bar. In addition, unlike the bradycardia observed at 1bar hydrostatic pressure, tachycardia is normally observed with motor activity at a hydrostatic pressure of 101 bar, unless water temperature is >24.5"C, and then bradycardia occurs (Sebert and Barthelomy, 1985).These observations suggest a direct cardiac effect of hydrostatic pressure which is temperature dependent. Indeed, Gennser et al. (1990) report that twitch tension and frequency of atrial strips in vitro are both decreased by hyperbaric conditions at 8"-10"C, but twitch tension increases and frequency is unchanged with hyperbaric conditions at 16"-24"C.

4. ADRENERCICCONTROL The cardiac response to adrenergic stimulation depends to a large degree on the relative density of SL adrenoceptor subtypes, the sensitivity of the adrenoceptor, and the concentration and type of ligand (adrenaline and noradrenaline). The relative density of adrenoceptor subtypes affect the chronotropic response in a qualitative way, because a- and P-adrenoceptor subtypes, both of which are found in fish hearts, have opposite chronotropic effects; cardiac a-adrenoceptors mediate bradycardia, and P-adrenoceptors mediate tachycardia. The absolute density and sensitivity of the adrenoceptors, as well as the concentration and type of ligand, affect the chronotropic response in a quantitative way. For example, adrenaline and noradrenaline have different potencies for adrenoceptor subtypes and are coreleased in different ratios. Circulating levels of adrenaline and noradrenaline (nanomolar concentrations) are often similar (e.g., Butler et al., 1978; Nilsson, 1983; Ristori and Laurent, 1985; Butler, 1986; Primmett et al., 1986; Milligan

50

ANTHONY P. FARRELL AND DAVID R. JONES

et al., 1989),but with stress and disturbance, these levels increase 10to 100-fold (Nilsson, 1983; Butler, 1986). In cyclostomes, elasmobranchs, and dipnoans, either noradrenaline becomes the dominant hormone, or adrenaline and noradrenaline increase to similar levels. In contrast, in teleost fish, with the exception of Cyprinus carpio, adrenaline becomes the predominant circulating hormone. Adrenaline and noradrenaline are also coreleased from adrenergic nerve endings in fish hearts (in contrast to mammalian hearts), with adrenaline being the primary neurotransmitter (Cameron, 1979b; Pennec and Le Bras, 1984). The majority of studies indicate that adrenergic stimulation of fish hearts causes positive chronotropy (Nilsson, 1983; Laurent et al., 1983; Farrell, 1984; Butler and Metcalfe, 1988).Studies with in vitro cardiac muscle strips and perfused hearts suggest that positive chronotropy is mediated by j3-adrenoceptors, probably the j3z subtype (Ask et al., 1980,1981; Cameron and Brown, 1981; Farrell et al., 1986b; Temma et al., 1986; Davie et al., 1992). There are, however, a few species in which a-adrenoceptors mediate negative chronotropy, and these include s. acanthias (Capra and Satchell, 1977),carp (Laffont and Labat, 1966; Temma et al., 1989), perch (Tirri and Ripatti, 1982), and eel (Forster, 1976; Chan and Chow, 1976; Peyraud-Waitzenegger et al., 1980).In perch, a-adrenoceptor-mediated negative chronotropy dominates over a very weak j3-adrenoceptor-mediated positive chronotropy (Tirri and Ripatti, 1982). In C. carpio, however, a-adrenoceptormediated negative chronotropy is revealed only at high concentrations of noradrenaline, with positive chronotropy occurring at lower concentrations (Temma et al., 1989). Cardiac a-adrenoceptors are apparently not involved in adrenergically mediated chronotropy in either rainbow trout or Pleuronectes platessa (Farrell et al., 1986b; Temma et al., 1986). Thus, a- and P-adrenoceptor subtypes may both be found in fish hearts, but both receptor types are not necessarily found in a single species. An additional confounding factor, which is not yet fully understood, is the impact of seasonal changes in temperature on adrenoceptor function. In rainbow trout, for example, the number of cardiac aand P-adrenoceptors and adrenoceptor sensitivity, but apparently not the concentrations of circulating catecholamines, vary with acclimation temperature (Ask, 1983; Graham and Farrell, 1989; Milligan et al., 1989). Adrenergic control of heart rate is possible through three means, which can be grouped phylogenetically (Laurent et at., 1983; Nilsson, 1983):(1)endogenously, as in cyclostomes and dipnoans, which have chromaffin tissue within the heart (endogenous catecholamine stores);

1.

THE HEART

51

(2) exogenously, for all fish that have chromaffin tissue outside the heart (exogenous catecholamine stores), release of catecholamines being under sympathetic control in elasmobranchs and teleosts; and (3) neurally, as in the teleosts and holosteans that have adrenergic cardiac innervation. Bearing in mind these groupings, the relative importance of these three adrenergic control mechanisms will be examined. a. Endogenous Catecholamine Stores. Of the three adrenergic mechanisms, the role of cardiac catecholamine stores is the most poorly understood (see Laurent et al., 1983; Dashow and Epple, 1985). Nonetheless, based on the observations that in vivo injection of P-adrenergic antagonists reduces heart rate significantly in hagfish (Axelsson et al., 1990; Forster et al., 1992) and that depletion of catecholamine stores in hagfish with reserpine also depresses heart rate (Bloom e t al., 1961), a suggestion worth further study is whether endogenous catecholamines play a role in tonic cardiac stimulation in cyclostomes (Forster et al., 1991).

b. Circulating Catecholamines. The low levels of circulating catecholamines in resting fish are probably responsible for the excitatory adrenergic tone observed in fish, with the possible exception of cyclostomes (see above). Nilsson et al. (1976)first suggested that the adrenergic tone on the heart was a result of circulating catecholamines, after noting that the threshold concentration for adrenergic stimulation of isolated cardiac muscle strips was similar to the blood concentrations of catecholamines. Since then several studies have found that nanomolar concentrations of adrenaline and noradrenaline stimulate perfused teleost and elasmobranch hearts and make them less prone to deterioration with time (Graham and Farrell, 1989; Davie and Farrell, 1991). Tonic adrenergic stimulation in rainbow trout may be particularly important for maintaining atrioventricular conduction at low temperature (Graham and Farrell, 1989). Because the sensitivity of cardiac tissue to catecholamines matches the concentration range for circulating catecholamines, the 10- to 100fold increase in circulating catecholamine concentrations under stressful conditions should produce tachycardia. Thus, elevated levels of circulating catecholamines may be responsible for the tachycardia following burst swimming (Farrell, 1982). They may also oppose the vagally mediated bradycardia observed with exposure to hypoxia, and may prevent the negative chronotropy produced by extracellular acidosis.

52

ANTHONY P. FARRELL A N D DAVID R. JONES

During prolonged swimming, circulating catecholamine levels do not increase appreciably except at the maximal prolonged swimming speed (Ucrit),when there is a small component of burst swimming (Nilsson, 1983; Butler, 1986). The implication of this observation is that tachycardia observed during aerobic exercise is probably determined largely by vagal release rather than by additional adrenergic stimulation beyond the tonic level observed in resting fish. Even though circulating catecholamine levels increase at Ucrit,heart rate has already reached a plateau (Priede, 1974; Kiceniuk and Jones, 1977).An anomalous observation is that in bilaterally vagotomized rainbow trout heart rate increased by 8% and 83% at 6.5"C and 15"C, respectively, with exercise (Priede, 1974). The tachycardia after vagotomy could reflect an involvement of adrenergic innervation other than that contained in the vagal trunk, an abnormal increase in circulating catecholamines during prolonged swimming, or some other positive chronotropic mechanism.

c . Cardiac Adrenergic Znneruation. One of the major difficulties in separating the role of adrenergic nerves from that of circulating catecholamines is that catecholamine injections in d u o produce vasoconstriction and cross the blood-brain barrier, either of which can elicit cardiac reflexes that can mask any direct cardiac effect (PeyraudWaitzenegger et al., 1979; Wood and Shelton, 1980a,b; Jones and Milsom, 1982; Farrell, 1986). However, these roles have been separated in one study on the Atlantic cod (Axelsson and Nilsson, 1986). Blockade of adrenergic nerve activity with bretylium reduced heart rate in both resting and swimming fish by only 3 bpm (7%). Thus, chronotropic effects mediated by adrenergic nerves are minimal in Atlantic cod. Nonetheless, it would be worth studying fishes that have a more active lifestyle before making firm conclusions regarding the limited involvement of adrenergic nerves in terms of chronotropic effects. In this regard, it is interesting to note that in fishes lacking adrenergic cardiac innervation (e.g., elasmobranchs), tachycardia associated with swimming is usually quite small; a change of often less than 10 bpm in elasmobranchs (Lai et al., 1989a,b). 5. MECHANICAL STRETCH OF THE PACEMAKER CELLS Pacemaker cells are stretched with increased cardiac filling and mechanical stretch of the pacemaker cells increases the intrinsic heart rate. In the functionally aneural heart of hagfish, stretch of the pacemaker cells may be an important mechanism to increase heart rate (Jensen, 1960, 1961). Even so, normal heart rate in hagfish probably

1.

THE HEART

53

varies by no more than 10-20% (Forster et al., 1991,1992). Mechanical stretch of the pacemaker has even less physiological significance in terms of modulating heart rate in elasmobranchs and teleosts. Physiological increases in cardiac filling pressure of perfused hearts have little, if any, effect on the intrinsic pacemaker rate and, furthermore, the effect is to decrease rather than increase heart rate by 1to 2 bpm (Farrell, 1984; Farrell et al., 1986; Davie and Farrell, 1991a). Thus, the mechanism for mechanical modulation of heart rate has been superseded b y neurohumoral control of pacemaker rate in phylogenetically advanced fishes. The mechanical effects on heart rate, reported previously for lampreys, elasmobranchs, and teleosts ( Jensen, 1969,1970) were observed with isolated perfused hearts performing a subphysiological work load and beating either at rates slower than the sinoatrial pacemaker rate (8bpm and 24 bpm at 21°C in rainbow trout, and 12 bpm at 13°C in elasmobranchs), or not beating at all (lampreys). In view of these observations, it is possible that either depolarization rates of cardiac tissues other than the sinoatrial pacemaker are more sensitive to stretch than is the sinoatrial pacemaker, or the sinoatrial pacemaker can be kick-started by stretch. 6. OTHERFACTORS AFFECTINGHEARTRATE In A. japonica, arginine vasotocin, oxytocin, metosotocin, and isotocin, but not arginine vasopressin, increase heart rate in isolated atria (Chui and Lee, 1990).Both metosotocin and isotocin are more effective at 28°C. Moderate levels of hypoxia do not have a direct affect on pacemaker frequency, but severe hypoxia and anoxia can lead to cardiac failure. Heart rate is unchanged when the hearts from sea raven, rainbow trout, A. dieffenbachii, and S . acanthias are perfused with hypoxic saline (Farrell et al., 1985; Farrell et al., 1989; Davie and Farrell, 1991b; Davie et aZ., 1992). High extracellular calcium reduces intrinsic heart rate in sea raven and rainbow trout (Farrell et aZ., 1983; Farrell et al., 1986b).

7. MAXIMALHEARTRATE In mammals, an allometric relationship exists between heart rate and body mass so that resting and maximal heart rates are higher in smaller mammals. Presently there is no evidence for or against a similar allometric relationship in fishes. Instead, there is evidence to suggest that lower vertebrates, with the exception of tunas, have a range for heart rate which does not exceed 120 bpm (Farrell, 1991a). Examples of the highest heart rates reported for fish at high temperatures

54

ANTHONY P. FARRELL AND DAVID R. JONES

include 112 bpm for rainbow trout at 20°C (Wood et al., 1979), 120 bpm for spangled perch, Leipotherupon unicolor at 30°C (Gehrke and Fiedler, 1988), 30 bpm for C. carpio at 25°C (Moffit and Crawshaw, 1983), 56 bpm for A. japonica at 22°C (Chan, 1986), and 80 bpm for Seriola gradis at 2Q°C (€?S. Davie and C. E. Franklin, 1991, personal communication). Thus, if an allometric relationship for heart rate does exist for fish, it would have to be compressed into a range that has a maximal value of 120 bpm or lower, depending on the species and water temperature. Tuna have heart rates that exceed 120 bpm, the highest rates among lower vertebrates. Heart rates are 117-126 bpm in spinally blocked skipjack tuna (Bushnell, 1988; Bushnell et al., 1989; Bushnell and Brill, 1991) and range from 91 to 172 bpm in perfused hearts from skipjack tuna and yellowfin tuna (Farrell et al., 1992). Rates of 180 to 240 bpm have been measured in swimming tuna (Kanwisher et al., 1974). What might be the modifications that allow tuna hearts to beat faster? Possibilities include the following:

1. Membrane permeabilities in ion-pumping activities. The SL membrane events associated with the action potential (the sodium leak, channel opening and closing, and pumps that restore ionic status) obviously match a given heart rate, but whether any of these SL membrane events are unusual in tuna is unknown. Bailey and Driedzic (1990)noted that cold-acclimation in yellow perch was associated with a more rapid rate of relaxation, suggesting some modification of SL calcium ATPase to improve maximal contraction frequency. 2. Calcium diffusion during excitation-contraction (E-C) coupling. Since the majority of calcium that binds to troponin-C (activator calcium) is derived from the extracellular space and passes through L-type calcium channels during excitation (see Chapter 6) the rate of diffusion of extracellular calcium to and from the innermost troponin-C could set a limit for contraction frequency in fishes. Skeletal muscle (mammalian and fish) and mammalian cardiac muscle, all of which contract at high frequencies, have intracellular calcium stores in the SR, bringing most of the activator calcium nearer to the troponin C. In tuna, SR calcium is involved to a significant degree in E-C coupling (Keen et al., 1992). In contrast, hagfish, with exceptionally slow heart rates, appear to “protect” the extracellular calcium with an unusually thick glycocalyx, which surrounds the myocyte (Poupa et al., 1985).

1. THE

55

HEART

3. Conduction of electrical activity. Every myocardial fiber is activated with each heart beat, and so the anatomical factors affecting the transmission of the action potential may be different in tuna. 4. Oxygen supply. A high dP/dt in tunas coupled with a high heart rate increases not only the absolute myocardial 0 2 demand but also the rate at which 0 2 must diffuse through the myocardium. The presence of very high cardiac myoglobin concentrations, and the presence of and wider distribution pattern for the coronary circulation in tuna may mean that diffusion of 0 2 from blood to mitochondria is not limiting heart rate. In fact, Poupa and Brix (1984) note that cardiac capillary density, blood P50, and mitochondrial density are all high in fast-beating hearts.

D. Cardiac Output Cardiac output (Q) is the product of stroke volume and heart rate. Since the mechanisms controlling heart rate and stroke volume have been described, this section focuses on interspecific differences in 0 and changes in Q in response to environmental perturbations. The importance of in terms of internal oxygen convection is evident from a rearrangement of the Fick equation:

Q

=

VoJA - Vo2

where Vo, is the oxygen consumption and A-Voz is the difference in arterial-venous blood 0 2 content. Differences in Q account for a major proportion of interspecific differences in VO,. For example, there is a 75-fold difference between the routine VO,of Eptatretus cirrhatus and Katsuwonus pelamis, which is reflected in a 15-fold difference for Q and a five fold difference for A-V0, content (Farrell, 1991a; Table IV). Despite the obvious importance of Q in terms of 0 2 transport in fishes, in vivo measurements of Q are far from comprehensive. The available Q values, arranged according to measurement technique, species, and temperature, are presented in Table IV. In order to evaluate this information, it is essential to first appreciate the technical limitations of these Q measurements.

1. MEASUREMENT

Q can be measured by direct or indirect techniques. Of the indirect methods used for measuring Q, the Fick equation is the most common. In fact, two thirds of the 0 values presented in Table IV are based on the Fick principle. Unfortunately, Fick estimates in fish are unreliable

56

ANTHONY P. FARRELL AND DAVID R. JONES

because errors introduced by the method can substantially (20-40%) overestimate or underestimate Q (Metcalfe and Butler, 1982; Daxboeck et al., 1982; Hughes et al., 1982; Butler, 1988; PeyraudWaitzenegger and Soulier, 1989). However, in some cases, the sum of the errors can be such that the Fick principle may accurately estimate (Randall, 1985). Direct techniques most commonly use cuff-type or cannulating electromagnetic or Doppler-flow probes on the ventral aorta. In teleosts, the ventral aorta is often accessible, but limited space in active species favors the use of miniature Doppler probes over the more bulky electromagnetic probes. In elasmobranchs, posterior afferent branchial arteries form immediately after the conus arteriosus reaches the anterior pericardial wall. Therefore Q can be estimated only by using assumptions regarding flow distribution to the various gill arches because a flow probe must be placed on an unbranched segment of the ventral aorta between branchial arteries (Satchel1et al., 1970; Short et al., 1977; Lai et al., 1989b). Doppler and electromagnetic techniques have different technical merits. Electromagnetic flow probes can be calibrated accurately, but a zero level must be established regularly in vivo (because of the sizeable diastolic blood flow in the ventral aorta) by means of either a cuff-type occluder, if anatomy permits, or bradycardia (evoked by cholinergic antagonists or by frightening the fish). Therefore, the accuracy of the zero level is a major concern. In contrast, Doppler-flow probes have a reliable “built-in” zero, but calibration is less accurate even when extreme care is taken. Thus, Doppler-flow probes are particularly useful when measuring relative changes in Q. The recent development of ultrasonic flow probes may resolve some of these technical difficulties. An additional concern is the state of the animal, which is not always clearly defined in the literature. Stress, spontaneous activity, and blood loss all will tend to elevate resting 0. Also, opening the pericardium will be of concern given the importance of uis-u-frontecardiac filling in certain teleosts. Hence, the following description of some of the factors that influence 0is at best tentative.

2. BODYMASS Vo2,Q and end-diastolic volume scale to body mass in mammals with an exponent of 0.75-0.77, whereas heart rate scales with an exponent of -0.25 (see Schmidt-Nielsen, 1989). This dogma for endothermic mammals may not be true for ectothermic fish. For example, the exponent for the allometric relationship between resting metabolic rate and body mass in the catfish (Silurus meridionah) decreases with

57

1. THE HEART

increasing water temperature, being 0.94 at 10°C and 0.75 at 30°C (Xiaojun and Ruyung, 1990). The significance of this in terms of Q needs to be established. 3. ACTIVITY Resting Q varies by 15-fold among fish species ranging from hagfish to tuna (Table IV). Fish that display high levels of activity often have a higher resting Q than sluggish forms. For example, benthic fishes such as sea raven and lingcod (Ophiodon elongatus) have a resting near 10 ml/min/kg at 1O"C, whereas 17 mI/min/kg is reported for rainbow trout at similar temperatures. Tuna have the highest Q values among fishes. In fact, the 132 ml/min/kg at 26°C reported for spinally blocked skipjack tuna is about half that of resting Q values reported for similar sized mammals at 37°C. Most of the 15-fold range in between hagfish and tuna is accounted for by the seven fold difference in heart rate (Table 11). Fish exercise anaerobically (burst exercise) for short periods. Burst exercise is often accompanied by decreases in heart rate, Q, and blood pressure (Stevens et al., 1972; Farrell, 1982). The bradycardia may begin even before an exercise bout begins (Farrell, 1982). The depression of cardiac activity during burst exercise may be related to preventing hypertension as the skeletal muscle contracts violently and closes off peripheral blood flow. Recovery from burst exercise is characterized by increases in Q, heart rate, and ventral aortic blood pressure (pva).

With aerobic swimming, increases in Q and A-Vo, increase internal 0 2 convection by similar amounts (Kiceniuk and Jones, 1977; Jones and Randall, 1978; Farrell, 1991a). In response to prolonged swimming, Q increases by 47% in Atlantic cod, 64% in sea raven, 70% in Scyliorhinus stellaris, 70% in leopard shark, and threefold in rainbow trout (Table V). Brill and Bushnell (1991)estimate that tuna increase Q twofold. An anomalous observation is that 6, does not change from resting values in swimming eels (Table V). Information on maximal values for Q (Qmax)is available from work with in situ perfused heart preparations. The is determined by elevating heart rate with adrenaline in the perfusate and elevating stroke volume by mechanically increasing the filling pressure of the heart (e.g., Farrell et al., 1988b; Milligan and Farrell, 1991).These data suggest that the Qmaxfor in oitro perfused heart studies is similar to the highest value observed with exercise in tho; Omaxhas been measured at 50 to 70 ml/min/kg for rainbow trout and 25 ml/min/kg for sea raven (Farrell et al., 1983; Farrell et al., 1986b; Farrell et al., 1991),and

omax

Table IV Cardiac Output (Q) for Fishes at Rest

Q Species

Cyclostomes Eptatretus cirrhatus Myrine glutinosa Elasmobranchs Dasyastis sabina Raja rhina" Scyliorhinus canicula

Scyliorhinus stellaris Squalus acanthias

Squalus suckleyii Triakis semi$asciatac Antarctic teleosts Chaenocephalus aceratusc Chaenocephalus aceratus

(ml/min . kg)

Temp ("C)

Method" (source)'

15.8 8.7

17 10-11

d (1)

Q

Species

(ml/min . kg)

Temp "C

Pseudo pleuronectes americanus

15.5 23.1 41.8 12.2 8.7 11.5 9.3 16.6-25.7 18.8 9.1 6.2-10.2 10.4 10.9-11.3 12.8 18.3 20.7 36.5 24.6-34.2 36.6 15-30 19.8 38.7 62.5 18.3 18.6

5 10 15 8.5-10.5 8.5-10.5 15 15 18-20 21-24 15.5-18.5 16-20 16-20 16.6-17.0 21-23 9-11 15-16 24-25 25.1-25.7 4-8 6 12 18 8.5-8.9 8.5-8.9

d (2) Anguilla anguilla

47 2 1.2-23.3 19.2 23.2 39.8 43.7 32.1 22.3 52.5 24.8 25.5 26.7 21 32 33.1 61 20-30 119 66 94-104

21.5-24.2 10 7 12 17 15 14-16 16 18.5-19.3

-

6-7 9-10 14-24

0-1 1-2 1-2 0.5-2 1-2

i (3) d (4) i (5) i (6) i (7) i (8) i (9) i (10) i (11) i (12) i (13)

Anguilla australis

Anguilla australis schmidtii Anguilla japonica Cyprinus carpio

i

d(14) i (15) d(16) 1

d (17) 1

Oplegnathusfasciatus Salmo gairdneri

Method" (source)' i (30) 1 1

d (31) i

d (32) i

d,i (33) i(34) d (35) d(36) d(37) i (38)

i(39) i(40) i(34) i(41) i (42) i (34) i (43) i (44) i 1

i (45)

i (46)

Pseudochaenichth yes Pagothenia bernacchii Pagothenia borchgrevinki Temperate water teleosts Gadus morhua

Hemitripterus americanus Myoxocephalus scorpius Ophiodon elongatus Platichthys stellatus UI (0

50-87 29.6 17.6

0.5-2

17-26 29.1 17.3 19.2 10.8 14.6 18.8 27.7 10.9 11.2 5.9 45.1 93.5

9-10 10 10-11 10-12 7 10.5 10-12 15-18 10 10 13 9-11 19.5-20.0

0 0

Tinca tinca Tropical teleosts Katsuwonus pelamis Thunnus albacares Air-breathing fish Protopterus aethiopicus Electrophorus electricus Amphipnous cuchia Hoplerythrinus unitaeniutus

17.6 34 31.2-36.7 65-100 45.9 16 14-18

9.0-10.5 13-16 13-16

14-15 14.8-15.2 15-16 12-14

50-80 132.3 115

23-25

20 40-70 80 27.6-32.2

18 28-30 20-30 26-30

24-26 24-26

i (47) d (48) d (49) i (50) i (51) i (34) i (52) i (53) d (54) d

d (55) d i (56)

d (57)

Method of measurement: i, indirectly measured, d, directly measured. (1)Forster et al. (1992);(2)Axelsson et 01. (1990);(3)Cameron et al. (1971);(4) Satchel1et al. (1970); (5)Butler and Taylor (1975);(6)Tayloret al. (1977); (7)Short et al. (1979); (8)Baumgarten-Schumann and Piiper (1968); (9) Piiper et al. (1977); (10) Robin et al. (1964); (11)Robin et al. (1966);(12) Murdaugh et al. (1965);(13) Hanson and Johansen (1970);(14) Lai et al. (1989a);(15)Holeton (1970);(16) Hemmingsen et al. (1972); (17) Hemmingsen and Douglas (1977); (18)Axelsson et al. (1992); (19) Jones et al. (1974);(20) Peterssen and Nilsson (1980); (21) Axelsson and Nilsson (1986); (22) Axelsson (1988); (23) Farrell (1986);(24) Axelsson et al. (1989); (25) Goldstein et al. (1964); (26) Farrell(l981); (27) Farrell (1982); (28) Stevens et al. (1972);(29)Watters and Smith (1973); (30)Cech et al. (1976);(31)Hughes et al. (1982);(32) Peyraud-Waitzenegger and Soulier (1989); (33)Motais et al. (1969); (34)Itazawa (1970);(35)Hipkins et al. (1986);(36)Hipkins and Smith (1983);(37) Hipkins (1985); (38) Davie and Forster (1980); (39) Chan (1986); (40) Garey (1970); (41) Itazawa and Takeda (1978); (42) Takeda (1990); (43) Stevens and Randall (1967);(44)Barron et al. (1987);(45)Cameron and Davis (1970);(46)Davis and Cameron (1971);(47)Kiceniuk and Jones (1977);(48)Wood (1974); (49)Wood and Shelton (1980a,b);(50)Holeton and Randall (1967);(51)Neumann et ul. (1983);(52)Eddy (1974);(53)Stevens (1972);(54)Bushnell (1988);(55)Johansen et al. (1968);(56) Lomholt and Johansen (1976); (57)Farrell(l978). Assumptions made regarding distribution of blood flow.

60

ANTHONY P. FARRELL AND DAVID R. JONES

39 ml/min/kg in S. acanthias (Davie and Franklin, 1992). However, in

omax

A. dieffenbachii, (22 mllminlkg) is about twice that observed in vivo (Davie et al., 1992). Whereas interspecific differences in resting Q can be largely accounted for by differences in heart rate, changes in stroke volume are important in bringing about increased Q during aerobic exercise. When both stroke volume and heart rate have been measured during aerobic exercise, the relative contribution of stroke volume is usually similar to or greater than that of heart rate (Table V). For example, during prolonged swimming the percentage change in stroke volume versus heart rate is, respectively, 200% versus 50% in rainbow trout (Kiceniuk and Jones, 1977), 55-63% versus 7-15% in Scyliorhinus stellaris (Piiper et al., 1977), 33% versus 7% in leopard shark (Lai et al., 1989b), 26% versus 18% in the Atlantic cod (Axelsson and Nilsson, 1986),and 25% versus 31% in the sea raven (Axelsson et aZ., 1989).Farrell(1991a) notes that cardiac pumping in mammals, birds, reptiles, and amphibians is primarily frequency modulated and not volume modulated. Thus, the predominantly stroke volume-modulated increase in Q in response to exercise is different from other vertebrates. However, two notable exceptions to this trend exist among fishes; they are tunas and certain Antarctic fishes. The reported range for heart rate (90240 bpm) in tuna would adequately account for the predicted twofold increase in Q (Brill and Bushnell, 1991; Farrell, 1991). In the redblooded Antarctic notothenid (2.' borchgrevinki), swimming in a swimtunnel results in a twofold increase in heart rate with little change in stroke volume (Axelsson et al., 1992). Interestingly, these two fish live at opposite ends of the temperature spectrum for fishes. 4. TEMPERATURE Acute and chronic (or seasonal) changes in temperature are expected to affect Q. Temperature effects can be assessed using Q l o values with a Q l o value of around 2.0 indicating temperature conformity and a 9 1 0 value of 1.0 indicating complete temperature compensation. When subjected to an acute 5°C increase in temperature, Q l o values for resting Q were reported as 1.56,2.12 and 2.35 for 5°C-, lO"C-, and 1ti"C-acclimatedwinter flounder, respectively (Cech et aZ., 1976). In S. acanthias the Q l o values are 2.10 for both i> and heart rate for an acute temperature change from 7" to 17°C (Butler and Taylor, 1975). A Qlo value of 2.00 was reported with perfused hearts from the sea raven for the temperature range 3"-13°C (Graham and Farrell, 1985). In contrast, an acute increase in temperature from 0°-5"C pro-

Table V Effect of Swimming Activity on Cardiac Output (Q),Heart Rate, Stroke Volume, Ventral Aortic Blood Pressure (PJ, and Oxygen Uptake (VoJ" Stroke Heart rate volume P,, VO, (mllmin . kg) (beatdmin) (mllkg) (mm Hg) (ml Oz/min . kg)

Q

Species (mass)

Activity state

Anguilla australis schmidtii (0.62 kg) Gadus morhua (0.35-0.73 kg) Gadus morhua (0.4-0.8 kg) Hemitripterus americanus (0.67-1.40 kg) Salmo gairdneri (0.9-1.5 kg)

Rest 15 cm/sec Rest 2/3 bl sec-' Rest 213 bl sec-' Rest 30 cmlsec Rest 41-6370 Ucrit 70-78% U,,, 81-91% U,,it Maximum Rest 30 min postexercise Rest 0.27 bl . sec-' Rest 0.3-0.7 bl sec-' postexercise Rest 1bl * sec-'

Salmo gairdneri (1.4-1.7 kg) Scyliorhinus stellaris (2.8 kg) Triakis semifasciata (1.93 kg) Pagothenia borchgrevinki (0.064kg)

9

-

11.3 11.3 19.2 30.2 17.3 25.4 18.8 30.9 17.6 28.4 34.8 42.9 52.6 45.9 71.9 52.5 89.2 33.1 56.2 60.4 29.6 51.8

53.5 53.9 30.5 42.7 43.2 51.2 37.3 49.1 37.8 42.7 49.0 51.3 51.4

-

43 46 51.3 55.1 49.5 11.3 21.0

0.21 0.21 0.49 0.61 0.39 0.49 0.51 0.64 0.46 0.62 0.70 0.86 1.03

-

-

1.21 1.94 0.77 1.02 1.22 2.16 2.16

40 40 -

38 48 29 36 38 40 50 60

-

25 26

0.56 1.52 1.90 3.12 4.34 2.2 3.1 1.30 1.90

48

-

58 55 28 28

-

-

Method (temp.)

Source"

Indirect (17°C) Direct ( 11°C) Direct (10.5"C) Direct (10-12~C) Fick (9-10°C)

Fick ( 15T)

Fick (19°C) Direct (14-24°C) Direct (0-1°C)

Swimming speeds are presented as cm/sec, bodylengths per second (bl . sec-l), or a percentage of the maximum prolonged swimming speed (Ucrit). (a) Davie and Forster (1980); (b) Axelsson (1988); (c) Axelsson and Nilsson (1986); (d) Axelsson et al. (1989); (e) Kiceniuk and Jones (1977); (f) Neumann et al. (1983); (9) Piiper et al. (1977);(h) Lai et al. (1989a; 1990a);(i) Axelsson et al. (1992).

62

ANTHONY P. FARRELL AND DAVID R. JONES

duces only a 20% increase in Q in P . bernacchi (Axelsson et al., 1992). Following temperature acclimation of winter flounder, a Qlo value of 2.70 was reported for resting Q values over the temperature range 5"to 15°C (Cech et aE., 1976). Following temperature acclimation of rainbow trout Barron et al. (1987) report a similar Q l o of 2.6 for resting Q over the temperature range 6" to 18°C. In contrast, the Qmax for perfused hearts from temperature-acclimated rainbow trout had a Qlo of 1.36 over the temperature range 5" to 15°C (Graham and Farrell, 1989).This observation raises the possibility that resting Q and Qmax are affected differently with temperature acclimation.

5. ACIDOSIS Extracellular acidosis occurs in a number of situations including exposure to hypercapnic and acid environments, and after burst exercise. Under these conditions, extracellular pH can decrease by as much as 0.5 pH units (see Wood et al., 1977; Ruben and Bennett, 1981; Graham et al., 1982; Milligan and Wood, 1986).The effects of extracellular acidosis on isolated cardiac muscle strips and perfused hearts have been reviewed previously (Gesser and Poupa, 1983; Farrell, 1984; Gesser, 1985). In general, extracellular acidosis has negative chronotropic and inotropic effects, with some species being more acidosis sensitive than others. An extreme extracellular pH of6.8 to 7.1 reduces the force developed by ventricular muscle strips by as much as 55% in rainbow trout, perch, Atlantic cod, plaice, eel, and flounder (Gesser et al., 1982; Gesser and Poupa, 1983; Gesser, 1985). Perfused hearts exposed to pH 7.4, as is more usual in extracellular acidosis, show a small decrease (10-20%) in resting Q, primarily through negative chronotropy (Farrell, 1984). The negative inotropic effect of acidosis is better revealed at higher cardiac work loads. Figure 11A illustrates that at pH 7.4, Qmaxfor the perfused sea raven heart is reduced by more than 50% compared with that of controls at pH 7.9. Perfused Fig. 11. The effect of hypercapnic acidosis on cardiac performance in perfused sea raven (A) and rainbow trout (B) hearts. Curves for cardiac power output versus cardiac filling pressure are presented. Since mean output pressure and heart rate change only slightly with the increases in filling pressure, the changes for Q and stroke volume were quantitatively similar. The sea raven heart is clearly more sensitive than the rainbow trout heart to extracellular acidosis, as indicated by the greater percentage reduction in maximal power output. When physiological levels of adrenaline (0.1 p M ) are added to the perfusate during exposure to acidosis, maximal cardiac performance is improved beyond the control level in both species. In contrast, doubling the concentration of extracellular calcium in the perfusate restores cardiac performance only for rainbow trout hearts. Exposure to hypoxic perfusate has a similar negative inotropic action on sea raven hearts. Data adapted from Farrell(1985) and Farrell et al. (1986b).

A

zE

CT)

6 -

F” 5 3

a

I-

z

4-

U

0 a

3 -

0

5 n

2-

U

a 0

1-

Control

3

2

1

FILLING PRESSURE, mmHg

I

1

I

I

Control

1

2

3

FILLING PRESSURE, mmHg

64

ANTHONY P. FARRELL AND DAVID R. JONES

rainbow trout hearts are more tolerant to a similar acidotic challenge (Fig. 11B); Qmaxis higher and is reduced by only 15%.In the absence of in viuo data, a direct effect of extracellular acidosis in vivo is likely to be a reduction in maximum being more significant in acidosissensitive compared with acidosis-tolerant fishes. An important observation made with perfused hearts and isolated muscle strips is that the negative inotropic and chronotropic effects of extracellular acidosis can be ameliorated. In isolated muscle strips, contractility can be restored during acidosis with an increase in either extracellular calcium concentration or adrenergic stimulation (Gesser and Poupa, 1983; Gesser, 1985). Somewhat different results are obtained with perfused hearts (Fig. 11). Adrenergic stimulation of perfused sea raven and rainbow trout hearts during extracellular acidosis does improve cardiac performance beyond even the control level (Fig. 11). Further, only in rainbow trout (not sea raven) does increasing extracellular calcium have an ameliorative effect during extracellular acidosis. Thus, it seems likely that in uivo, increased levels of circulating catecholamines protect the heart against the negative inotropic and chronotropic effects of extracellular acidosis. The most likely explanation for the cardiac effects of extracellular acidosis is that it causes an intracellular acidosis in cardiac muscle, which results in a functional deficit of activator calcium for E-C coupling (Gesser and Poupa, 1983). This idea is supported by the in udtro effects on isolated muscle strips of increasing extracellular calcium. Furthermore, following adrenergic stimulation, there can be a greater influx of extracellular calcium into the myocardium since by activating cardiac P-adrenoceptors, the probability that SL calcium channels are in an open state is increased. [The possibility that adrenaline exerts its protective effect though intracellular pH regulation was discounted in perfused rainbow trout hearts (Milligan and Farrell, 1986).] In addition to extracellular calcium, release of calcium from intracellular stores is thought to be involved in the recovery of cardiac performance that occurs in acidosis-tolerant species during a 15- to 20-minute exposure to extracellular acidosis (Gesser, 1985). Sensitivity to acidosis is therefore set by factors that include the sensitivity of the myofilaments to calcium availability, the level of adrenergic stimulation, and the potential for calcium release from intracellular stores. A problem does exist with the activator calcium deficit hypothesis, and it relates to the fact that myocardial intracellular acidosis is not necessarily observed in vivo even though intracellular acidosis is observed in vitro during extracellular acidosis (Gesser and Jorgensen, 1982; Farrell and Milligan, 1986). For example, a marked extracellular

0,

1. THE HEART

65

acidosis is produced by strenuous activity in sea raven, rainbow trout, and starry flounder (Platichthys stel2aris). However, during the first 30 min of this extracellular acidosis, ventricular intracellular pH increased significantly in sea raven, was not significantly changed in rainbow trout, and decreased in starry flounder (Milligan and Wood, 1986; Wood and Milligan, 1987).Based on these limited data, the level to which myocardial intracellular acidosis occurs in vivo appears to be greater in the acidosis-tolerant species of fish as measured by in vitro approaches. 6. HYPOXIA During environmental hypoxia, many fish can maintain Q if levels are moderate (water Po, > 70 torr). Moderate to severe hypoxia (water P o , = 40-70 torr) causes reflex bradycardia, but in some fishes, Q is maintained because of a compensatory increase in stroke volume (e.g., rainbow trout, Holeton and Randall, 1967; Scyliorhinus canicula, Butler and Taylor, 1975) whereas in others, i> decreases even though stroke volume increases somewhat (e.g., lingcod, Farrell, 1982; A. japonica, Chan, 1986; A. anguilla, Peyraud-Waitzenegger and Soulier, 1989). Tuna are extremely hypoxia sensitive and, at a water P o , between 85 and 104 torr, hypoxic bradycardia develops, but there is no compensatory increase in stroke volume to maintain Q (Bushnell et al., 1990; Bushnell and Brill, 1991). Hagfishes are exceptional in terms of hypoxic tolerance. Myxine glutinosa maintain Q and heart rate under severely hypoxic (water P o , = 13-20 torr) conditions (Axelsson et al., 1990), whereas Eptatretus cirrhatus increase Q during hypoxia (water PO, = 40 torr) (Forster et al., 1992). The direct effect of hypoxia on cardiac tissue is negative inotropy (Gesser et al., 1982). Factors that probably influence the overall cardiac response to hypoxia, but have not been fully examined, include the following:

1. The anaerobic ATP generating potential of the heart relative to total cardiac power output (see preceding sections). 2. The intrinsic ability of the contractile machinery to tolerate hypoxia. The eel myocardium, for example, has a remarkable intrinsic tolerance to hypoxia (Gesser et al., 1982). In fact, the maximal capacity of perfused eel hearts is reduced by only 20% with a perfusate PO, of 11.5 torr (Davie et al., 1992); rainbow trout hearts fail at a perfusate PO, of 40 torr (Farrell et al., 1988). 3. The presence of a coronary circulation. Increased coronary blood flow during exposure to moderate to severe hypoxia may

66

ANTHONY P. FARRELL AND DAVID R. JONES

be important in maintaining oxygen delivery to the myocardium as partial pressure of oxygen in venous blood PVo,decreases (Farrell, 1984). In this way, Q could be maintained during hypoxic exposure. 4. Temperature and the normoxic level of cholinergic tone. At a high water temperature, hypoxia-induced bradycardia is greater in S. canicula (Butler and Taylor, 1975). This may reflect increased myocardial 0 2 demand and a lower normoxic level of cholinergic tone at higher temperatures. In P. barnacchi at VC, the normoxic level of cholinergic tone is near maximal, and bradycardia does not develop during hypoxia (water Po, = 40 torr). Since the heart is already under an unusually high resting cholinergic tone, a further increase associated with hypoxia may be difficult (Axelsson et al., 1992). 5. Involvement of higher cerebral centers. Despite the high hypoxic tolerance of the eel myocardium i n uitro, 0 is not maintained in uiuo at water Po, below 40 torr (Chan, 1986).Therefore, it would appear that eels down-regulate resting Q during hypoxic exposure. 6. Humoral factors. Hypoxia is often accompanied by extracellular acidosis. The effects of hypoxia and acidosis are additive in terms of negative inotropy (Gesser et al., 1982). However, circulating catecholamines increase during hypoxia to levels that should prevent or at least moderate this negative inotropy (Gesser et al., 1982). E. Myocardial 0 2 Supply, and the Threshold Venous Po, Since the majority of fish have a type I ventricle and spongiosa accounts for the major portion of type I1 ventricles, luminalO2 is the predominant myocardial 0 2 supply in fish. Coronary arteries supply 0 2 to the compacta and to some extent to the spongiosa in ventricle types I11 and IV. The extent to which luminal blood obviates the need for a coronary circulation and the extent to which the coronary circulation supplements the luminal 0 2 supply is not entirely clear (Davie and Farrell, 1991a). Luminal blood provides such a high volume of 0 2 relative to the myocardial 0 2 demand (because the entire Q passes through the heart) that a measurable decrease in the 0 2 content ofblood leaving the heart, compared with that entering, is unlikely (Jones and Randall, 1978; Farrell, 1984). This means that venous 0 2 content is unlikely to limit

1. THE

HEART

67

myocardial 0 2 delivery. Instead, luminal Pvo, which, to a large degree, sets the gradient for O2 diffusion across the spongiosa, might limit the luminal02 supply under certain conditions. Whether or not a threshold P,o, exists has not been rigorously tested, but a number of observations that bear on the issue were examined by Davie and Farrell (1991a). They suggested a threshold PVo, of around 10 torr; the exact value of which would vary somewhat according to the exact distribution pattern of the coronary circulation, the level of 0 2 demand as set by myocardial power output, the blood residence time in the heart as set by heart rate, the intracellular myoglobin concentration, and the hemoglobin 0 2 dissociation curve. For example, at Ucrit,when myocardial VO, is near maximal, the range of PVo2values is 9-16 torr (e.g., 16 torr in rainbow trout, Kiceniuk and Jones, 1977; 9 torr in leopard shark, Lai et al., 1990b). During hypoxia, when cardiac performance is lower, PVo2 is correspondingly lower at the point when Q decreases below a resting value (e.g., 7 torr in S. canicula, Short et al., 1977; 6-10 torr in rainbow trout, Holeton and Randall, 1967; Wood and Shelton, 1980b; 6 torr in A. dieffenbachii, Forster, 1985).As a means of experimentally lowering P,o, in a progressive fashion and to establish a threshold P,o, for rainbow trout, Steffensen and Farrell (1992) exposed coronary-ligated and sham-operated fish to stepwise decreases in the water PO, while they were swimming at 50% of Ucrlt.Ventral aortic blood pressure (Pva)was used as a measure of cardiac performance. The sham-operated fish swam until Pvo, was 8.6 torr and the coronary-ligated fish that had only a luminal myocardial 0 2 supply swam until Pvo,was 6.8 torr (Steffensen and Farrell, 1992). Furthermore, at a water PO, of 60 torr when Pvoz was 14.3 torr and 13.6 torr in sham-operated and coronary-ligated, respectively, P,, increased with the increasing levels of hypoxia in sham-operated fish, but decreased in coronary-ligated fish, suggesting earlier cardiac failure when there was no coronary circulation to supplement the luminalO2 supply. The problem of a Pvo, threshold is particularly acute in the hemoglobin-free Antarctic fishes. The myocardial Vo, of 1.2 p1 0 2 / set-kg body mass in Chaenocephalus aceratus requires a high 0 2 extraction from luminal plasma. Assuming a plasma 0 2 solubility of 0.056 pl Oz/ml torr at O'C, a Pvo, of 30 torr and of 100ml/min.kg body mass, venous plasma will supply 2.8 p1 Oz/sec.kg body mass. Myocardial 0 2 demand therefore uses 43%of the luminalO2 supply. Clearly, myocardial 0 2 supply in these fish will be jeopardized in situations when the luminalO2 supply is reduced, e.g., a decrease in either PVo2,or 0 or some combination. A coronary circulation either reduces or eliminates the reliance of

68

ANTHONY P. FARRELL AND DAVID R. JONES

the heart on the luminalO2 supply. In view of the possibility of a PVo, threshold, it is not surprising, therefore, that fish that encounter environmental hypoxia or are active swimmers (two situations in which Pvo,decreases) have a coronary circulation. Nevertheless, there is no clear estimate of the relative contributions made by the luminal and coronary 0 2 supplies to the 0 2 requirement of the heart. Indirect information has been obtained from in vivo studies in which the coronary circulation is interrupted, from coronary perfusion studies in vitro, and from theoretical estimates of 0 2 delivery via the coronaries. Three important points have emerged from studies with salmonids in which coronary blood flow has been stopped by surgical ablation or ligation of the main coronary artery (Daxboeck, 1982; Farrell and Steffensen, 1987b; Farrell et d., 1990a). First, the coronary circulation in rainbow trout and chinook salmon is not essential for short-term survival in captivity. Coronary-ligated fish can survive for several months. Second, the coronary artery can regrow around the ligation or ablation site. Angiogenesis is quite rapid, a matter of weeks, which presumably indicates a long-term survival value. Third, swimming is still possible after stopping the coronary 0 2 supply, but Ucritis reduced by 10-33% (Farrell and Steffensen, 1987b; Farrell et al., 1990a) provided there is no coronary regrowth (see Daxboeck, 1982). Four adaptations alleviate the problem of Pvo, threshold. 1. A coronary circulation. This well-oxygenated blood supply to the heart supplements the luminal 0 2 supply in all elasmobranchs, as well as active and hypoxia-tolerant teleosts. 2. High myocardial myoglobin concentration. Myoglobin facilitates 0 2 diffusion under hypoxic conditions in fish hearts and is found in high concentrations in very active and hypoxia-tolerant species. 3. Hypoxia (and acidosis) tolerance. The myocardia of some teleosts and elasmobranchs are particularly tolerant to hypoxia and the attendant acidosis produced by anaerobic metabolism. Hagfish hearts in vitro can function under anoxic conditions, and in oivo P,o, is 5 torr after exercise (Wells et al., 1986), or 2 torr during exposure to progressive hypoxia (Forster et al., 1992). 4. Slow heart rates. Slow heart rates in Antarctic fishes, for example, increase the residence time of blood in the lumen of the heart.

Of the above adaptations, the development of a coronary circulation is particularly interesting. Fish are, in effect, at the evolutionary inter-

1. THE

HEART

69

face in the development of the vertebrate coronary circulation. The coronary 0 2 supply in salmonids supplements rather than replaces the luminalO2 supply, and appears to be important for supporting normal pressure development by the ventricle. Experimental support for this suggestion is provided by the observation that when coronary-ligated trout swim at 50%of their critical swimming speed and are exposed to progressive hypoxia to systematically lower venous P02, they cannot develop normal ventral aortic pressures below a venous PO, of approximately 15 torr and, as a result, cardiac power output was reduced by an estimated 37% (Steffensen and Farrell, 1992). Similarly perfused hearts from rainbow trout, S. acanthias and A. dieffenbachii, cannot generate high output pressures when pumping hypoxic saline without coronary perfusion, but can maintain normal or elevated flow rates (Farrell et al., 1989; Davie and Farrell, 1991b; Davie et al., 1992). A higher cardiac performance is possible when the coronary circulation is perfused with either red blood cell suspensions under a condition of luminal hypoxia (Davie and Farrell, 1991; Davie et al., 1992) or with saline under aerobic conditions (Houlihan et al., 1988).These in vitro observations are consistent with the coronary 0 2 supply having a greater impact on homeometric regulation than on the Frank-Starling mechanism. Whereas it is unlikely that luminal 0 2 can supply sufficient 0 2 to meet the entire myocardial 0 2 demand in salmonids because of a diffusion limitation, it is equally unlikely that the coronary circulation normally satisfies the entire myocardial 0 2 demand. Based on a coronary flow of0.43 ml/min.kg body mass and a simultaneously measured myocardial power output of 4.7 mW in coho salmon, the coronary 0 2 supply would be 1.42 pl Oa/sec to the heart of a 1-kg fish, assuming an 0 2 content of 11 vols% (Axelsson and Farrell, 1992).This would represent approximately half the myocardial 0 2 requirement for this particular cardiac work load. Tuna may be different from salmonids in terms of requirement for coronary 0 2 supply. This conclusion is based on limited success perfusing tuna hearts using techniques previously successful with other fish. Resting cardiac power outputs could be achieved in perfused yellowfin tuna but not in perfused skipjack tuna hearts (Farrell et al., 1992). Furthermore, the hearts released large quantities of lactate into the perfusate even though luminal Po, exceeded 600 torr. The most likely conclusion to be drawn from these observations is that the myocardial Vo2of skipjack tuna is predominantly supplied by the arterial blood in the coronary circulation.

70

ANTHONY P. FARRELL AND DAVID R. JONES

E. Control of Coronary Blood Flow Cameron (1975) estimated coronary flow as 0.56 to 0.65% of Q for burbot (Lota Zota) and sucker (Catastornus catastornus) using microspheres. These values should be treated with some caution because microspheres injected into the dorsal aorta should not reach the coronary circulation in appreciable numbers. Axelsson and Farrell (1992) simultaneously measured coronary artery blood flow, dorsal aortic blood pressure, and Q in coho salmon (Fig. 12). Coronary flow was 0.20 ml/min.g ventricular mass (0.43ml/min.kg body mass), which represented 1.1% of 0. Coronary flow in two anaesthetized school sharks (Galeorhinus australis) was measured as 0.64 ml/min.g ventricular mass (0.37 ml/min.g body mass) (P. S. Davie and C. E. Franklin, 1992, personal communication). Pressure-flow relationships for the coronary circulation have been used to estimate coronary flow as 0.38 and 0.67 m1lmin.g ventricular mass in rainbow trout and skipjack tuna (Farrell, 1987; Farrell et al., 1992), which are 1.5%and 1.9%of resting Q, respectively. These flows are higher than in viuo measurements, perhaps because a large coronary vasodilatory reserve exists in uiuo and is lost with in vitro saline perfusion. At Ucritin rainbow trout, increases threefold, ventral aortic blood pressure increases by 50% (Kiceniuk and Jones, 1977),and mechanical efficiency of the heart doubles (Graham and Farrell, 1989),so myocar-

601............. ..","l.l"."..",

250-

3-

Q Pda

200-

F

-

8 2 -

-.-

-& X150-

c

r

E -

E

E

v

E

E100-

81-

5

U

45 -

E

.c

Y

ol

r

m

a" 30-

50 -

OJ

OJ

15-1

250 rns

(oh

Fig. 12. Simultaneous measurements of ventral aortic blood flow dorsal aortic blood pressure (Pa,)and coronary blood flow (qcor)in acoho salmon (4kg, 10°-lloC).The continuous but phasic nature of blood flow in the main coronary artery during one cardiac cycle is clearly ilhstrated. With permission, Axelsson and Farrell (1992).

1. THE HEART

71

dial Vozmust quadruple. To meet this increase in myocardial 0 2 demand, total coronary blood flow must also increase as much as fourfold ifthere is no change in 0 2 extraction. How a change in coronary flow of this magnitude is achieved is not entirely clear. Coronary vasoactivity and changes in dorsal aortic blood pressure affect coronary blood flow. Recent in vivo measurements of coronary blood flow support the idea of coronary vasoactivity altering coronary blood flow (Axelsson and Farrell, 1992). In coho salmon, coronary vascular resistance decreased when coronary flow increased by 114%with hypoxia, 60%with adrenaline injections, and 40%with isoproterenol injections. Axelsson and Farrell (1992) concluded that aadrenergic constriction and P-adrenergic dilatation, as well as increases in dorsal aortic pressure are involved in regulating coronary flow in coho salmon. Analysis of the pressure-flow relationships for rainbow trout coronaries suggests that the small increase in dorsal aortic blood pressure associated with sustained swimming would, by itself, increase total coronary blood flow by 50%(Farrell, 1987), which means that there must be a large coronary vasodilatory reserve. That is, under resting conditions there is a tonic vasoconstriction of the coronary circulation, which can be released to increase coronary flow. Satchel1 (1971) noted that the coronary arteries in fish, being derived from postbranchial arteries, have a blood pressure significantly less than the ventricular pressure. This may be a good reason why coronary blood flow in fish hearts is not so tightly linked to coronary blood pressure as in the mammalian heart, where coronary perfusion pressure closely follows pressure developed by the ventricle and therefore, its 0 2 requirement (Feigl, 1983). The mechanisms responsible for coronary vasoactivity have been studied in vitro at the arteriolar and arterial levels. The picture emerging is one of species diversity. Based on perfusion studies of the entire coronary circulation, a-adrenergic contraction dominates P-adrenergic relaxation in Atlantic salmon (Farrell and Graham, 1986), rainbow trout (Farrell, 1987), and marlin (Davie and Daxboeck, 1984). In the conger eel, 0-adrenergic relaxation predominates (Belaud and Peyraud, 1971). a-Adrenergic vasoconstruction is more pronounced in 15°C-than 5°Cacclimated rainbow trout (Farrell, 1987).Acetylcholine causes contractions in the coronary circulation of the conger eel (Belaud and Peyraud, 1971). Even though most circulatory control is normally exerted at the arteriolar level, there is significant vasoactivity at the arterial level in mammalian coronaries (Kalsner, 1982). The vasoactive responses of isolated coronary vascular rings from three species of fishes are summarized in Table VI, and some of the responses are different from

72

ANTHONY P. FARRELL AND DAVID R. JONES

Table VI A Summary of the Vasoactive Responses of Arterial Rings from the Main Coronary Artery in Fishes" ~~~~~

Agonist Acetylcholine Noradrenaline Adrenaline Isoproterenol Adenosine ATP ADP Histamine Bradykinin Serotonin Prostaglandin F% Nitroglycerine Nitroprusside Endothelin Thrombin

~

Rainbow troutb

Mako Skate" sharkd

+ -&+

-

+ +

+

+I+/-

0 0

X

(-1 ++ -

0

++ +

X

++ X X

X X

t +, very strong contractions; +, contractions; -, relaxations; 0, no response; x, not tested; ( ), (weak response); /, two responses observed at low/high concentrations; &, both responses observed but on different rings. Small et al. (1990). " Farrell and Davie (1992). Farrell and Davie (1991b)

those of the whole coronary circulation. P-Adrenergic relaxations predominate over a-adrenergic contractions, and postaglandin FzQproduces strong contractions in all three species. In contrast, the responses to purinergic agents and acetylcholine are species specific. Clearly, receptor subtypes, their relative density, and location in the coronary network are quite variable between fish species. For example, in salmonids the a-adrenoceptors dominate in arterioles, whereas P-adrenoceptors dominate in the main artery. The mechanism underlying the proposed vasodilatory reserve in fish has not been established, but the high sensitivity to and potent constrictor effects of endothelin and prostaglandin FzQwarrant further study as tonic vasoconstrictor agents. Physical factors also affect coronary flow. Contraction of the mammalian left ventricle can reverse coronary flow for up to 20% of the

73

1. THE HEART

cardiac cycle. The effects of vascular compression are less dramatic in fish hearts. There are beat-by-beat oscillations in coronary perfusion pressure in perfused rainbow trout and S. acanthias hearts, and alterations to distolic output pressure also affect coronary perfusion pressure (Farrell, 1987; Davie and Farrell, 1991b). I n uiuo blood flow in the main coronary artery of coho salmon is phasic but continuous (Fig. 12). The systolic rise in coronary flow lags behind ventral aortic flow and is almost coincident with the rise in dorsal aortic blood pressure. The lowest flow always just precedes the onset of ventral aortic flow (coincident with ventricular contraction), and a second rise in coronary flow occurs during the early phase of diastole. However, in the anaesthetized school shark, coronary blood flow in the main artery is briefly reversed, coincident with ventricular systole (P. S. Davie and C. E. Franklin, 1992, personal communication). This difference may be related to conal contraction and the fact that the coronary circulation goes to the spongiosa in sharks but not in rainbow trout.

ACKNOWLEDGMENTS Work by the authors was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the British Columbia and Yukon Heart Foundation, and the British Columbia Health Care Research Foundation. We are indebted to Joanne Harrington for her excellent typing skills in preparing the many drafts of this manuscript and to Alan Kolok for his comments on early drafts.

REFERENCES Acierno, R., Agnisola, C., Venzi, R., and Tota, B. (1990).Performance of the isolated and perfused working heart of the teleost Conger conger: Study of the inotropic effect of prostacyclin. J . Comp. Physiol. B . 160,365-371. Ask, J. A. (1983). Comparative aspects of adrenergic receptors in the hearts of lower vertebrates. Comp. Biochem. Physiol. 76A, 543-552. Ask, J.A., Stene-Larsen, G., and Helle, K. B. (1980).Atrial &-adrenoceptors in the trout. J. Comp. Physiol. 139,109-116. Ask, J . A., Stene-Larsen, G., and Helle, K. B. (1981). Temperature effects on the &adrenoceptors of the trout atrium. J. Comp. Physiol. 143, 161-168. Axelsson, M. (1988).The importance of nervous and humoral mechanisms in the control of cardiac performance in the Atlantic cod Gadus morhua at rest and during nonexhausting exercise. J. Erp. Biol. 137,287-303. Axelsson, M., and Farrell, A. P. (1992).Coronary blood flow in uiuo in the coho salmon (Oncorhynchus kisutch). Society for Exptal. Biol. Meeting. Abstract A12.1.

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133-148. Wells, R. M. G., Forster, M. E.,Davison, W., Taylor, H. H., Davie, P. S., and Satchel], G. H. (1986).Blood oxygen transport in the free-swimming hagfish, Eptatretus cirrhatus. J , Exp. Biol. 123,4343, Wood, C. M. (1974). A critical examination ofthe physical and adrenergic factors affecting blood flow through the gills of the rainbow trout.]. E r p . Biol. 60,241-265. Wood, C . M., and Milligan, C. L. (1987).Adrenergic analysis of extracellular and intracellular lactate and H + dynamics after strenuous exercise in the starry founder, Platichthys stellatus. Physiol. Zool. 60,69-81. Wood, C. M. and Perry, S. F. (1985).Respiratory, circulatory, and metabolic adjustments to exercise in fish. In “Circulation, Respiration, and Metabolism ” (R. Gilles, ed.), pp. 2-22.Springer-Verlag. Berlin, Germany. Wood, C. M., and Shelton, G . (1980a).Cardiovascular dynamics and adrenergic responses of the rainbow trout in oioo. ]. E x p . Biol. 87,247-270. Wood, C. M., and Shelton, G. (1980b). The reflex control of heart rate and cardiac output in the rainbow trout: Interactive influences of hypoxia, haemorrhage and systemic vasomotor tone.]. E x p . Biol. 87,271-284. Wood, C.M., McMahon, B. R., and McDonald, D. G . (1977).An analysis of changes in blood pH following exhausting activity in the starry flounder (Platichthys stellatus). J . EX^. B i d . 69,173-185. Wood, C. M., Pieprzak, P., and Trott, J. N. (1979).The influence of temperature and anaemia on the adrenergic and cholinergic mechanisms controlling heart rate in the rainbow trout. Can.J . Zool.57,2440-2447. Wright, C.M. (1984).Structure of the conus arteriosus and ventral aorta in the sea lamprey, Petromyzon murinus, and the atlantic hagfish, Myxine glutinosa: Microfibrils, a major component. Can.]. Zool. 62,2445-2456. Xiaojun, X.,and Ruyung, S. (1990).The bioenergetics of the southern catfish (Silurus merldionalis chen). I. Resting metabolic rate as a function of body weight and temperature. Physiol. 2001.63,1181-1195. Yamauchi, A. (1980).Fine structure of the fish heart. In “Hearts and Heartlike Organs” (C. H. Bourne, ed.), pp. 119-148.Academic Press, New York. Yamauchi, A., and Burnstock, G. (1968).Electron microscopic study on the innervation of the trout heart. J . Camp. Neurol. 132,567-588. Zummo, G. and Farina, F. (1989).Ultrastructure of the conus arteriosus of Scyliorhhus stellads. J . E x p . Zool. (Suppl.) 2,158-164.

2 THE ARTERIAL SYSTEM P . G. BUSHNELL and DAVID R. JONES Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

ANTHONY P . FARRELL Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada I. Introduction 11. Physical Factors Affecting Blood Flow 111. Roles of the Conus and Bulbus Arteriosus

A. Role of the Conus Arteriosus B. Role of the Bulbus Arteriosus IV. Pressure and Flow Relationships in the Ventral and Dorsd Aortas V. Blood Flow Distribution and Vascular Resistance A. Blood Flow Distribution at Rest B. Blood Flow during Exercise C. Blood Flow during Hypoxia VI. Heat-Exchange Retial Systems A. Anatomy of Heat-Exchange Retia B. Blood Flow in Heat-Exchange Retia References

I. INTRODUCTION The primary circulation of fishes follows the typical vertebrate pattern in that a heart forces blood into a ventral aorta, which then gives off paired vessels (afferent branchials), which arch upward between successive gill clefts and rejoin (efferentbranchials) to form the dorsal aorta, which is typically paired anteriorly and a single vessel posteriorly. The dorsal aorta distributes blood to the vascular beds in the tissues where exchange of metabolites, nutrients, and waste prod89 FISH PHYSIOLOGY, VOL. XIM

Copyright8 1992 by Academic Press, Inc.

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ucts occur. A unique feature of the piscine circulation is that the gas exchanger (gills) and systemic capillaries are perfused in series. Hence the piscine circulation is an arterial portal system in which the whole cardiac output (Q) flows through two successive vascular beds of comparable resistance, and each influences the perfusion and hemodynamics of the other (Langille et al., 1983).The nature and regulation of blood flow through the gills has been the subject of extensive review in the previous volume of this series (Volume XI), and further discussion would be repetitive and probably redundant. A further embellishment of the arterial system exists in those fish having arterial retia interspersed between the gills and some tissue vascular beds. The descriptor rete mirubile refers to a blood vessel that divides into many small vessels and then rejoins to form a single vessel (Fange, 1983).Retial systems can be found in many teleosts and elasmobranchs. In most cases arterial and venous retia are closely apposed and form countercurrent systems that concentrate gases (swim bladder retia or choroid retia in the eye), metabolites (lactate in the swim bladder rete), or heat (heat-exchanger retia). The contribution of retia to vascular resistance is small since the retial vessels are resistances in parallel, although in those fishes with retial exchangers protecting large areas of the body (i.e., muscle) changes in blood flow distribution subserving thermoregulation may have a major influence on circulatory dynamics. Retia associated with swim bladders are well studied, and reviews by Blaxter and Tytler (1978)and Fange (1983)provide an in-depth discussion. Further, the anatomy and physiology of cephalic retial systems have been reviewed extensively by Block and Carey (1985) and Block (1991), whereas anatomical descriptions of muscle and visceral retia date back 150 years and are summarized in a previous volume of this series (Volume VII) and in reviews by Carey (1982)and Satchell (1991).Consequently, our discussion will focus on the physiology of countercurrent retial systems that trap heat (heat exchange retial systems or heat exchangers), concentrating on contributions made in recent years. The primary circulation is remarkably constant in general form in cyclostomes, elasmobranchs, and teleosts. However, major modifications of the posterior branchial arteries occur in air-breathing fishes. These modifications have been reviewed by, for instance, Fishman et al. (1989)and Satchell (1975)and will not be dealt with in the present account. The primary circulation is, in many vascular regions, paralleled by a secondary system of arteries, capillaries, and veins which is discussed in Chapter 4 by Steffensen and Lomholt. The role of the conus and bulbus arteriosus, two structures that are found within the

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pericardium in all fishes except cyclostomes, is discussed in this chapter. Regardless of whether the origin of the conus or bulbus arteriosus is cardiac or arterial both of these structures have a marked influence on pressure-flow relationships in the arterial system and consequently, that influence is described here. In this chapter circulatory dynamics and regulation will be emphasized. Consequently, we will look at the “nature” of the fluid flowing in the vessels and effect of blood viscosity on cardiac energetics. The “windkessel” function of the major arteries is also of great importance because of influences on both cardiac work and central and peripheral blood flow. Arterial elasticity is the major contributor to the “windkessel” by storing blood in systole, when the walls are stretched by the blood pressure, and maintaining blood flow in diastole by their passive recoil. Hence, the arteries are more than just conduits for blood from the heart to the peripheral blood vessels. Control of flow distribution resides in the arterioles that terminate the arterial system and their role in regulating blood flow in response to exercise, hypoxia, and for thermoregulation is discussed in the final section.

11. PHYSICAL FACTORS AFFECTING

BLOOD FLOW Blood flow in major blood vessels of fishes will be laminar with turbulence occurring, if at all, only during peak systolic ejection in the ventral aorta of the largest animals (Langille et al. 1983).Hence if flow is laminar then the pressure gradient for steady flow can be obtained from Poiseuille’s Law F=

(Pi- P,)

P

r4

8LP

where F = flow (cm3 * S-’), Pi = pressure at the upstream point (dynes - cm-’), P , = pressure at the downstream point (dynes cm-’), L = distance between the two measuring points (cm), r = radius of the vessel (em), and p = viscosity (Poise). Hence, there will be a pressure drop whenever flow occurs, energy being dissipated in overcoming the “inner friction” or viscosity of the blood. Both pressure and flow in the major arteries are pulsatile, and due to inertia within the blood, the flow amplitude may no longer be related to the pressure gradient, so that Poiseuille’s Law will not apply.

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The deviation from Poiseuille’s Law and the extent of the phase lag is determined by a nondimensional constant a where

where T = radius of the vessel, p density (gm * crnp3), and f = frequency (Hz). If a is less than unity, then instantaneous flow rate will vary by less than 2%from that predicted by Poiseuille’s Law, and the phase difference between pressure gradient and flow will be less than 10%.Assuming viscosity to be constant, then the same a may arise from a low frequency in a wide tube or a high frequency in a narrow tube. The small heart and narrow blood vessels of fish, combined with low frequencies of contraction, mean that (Y will be small in the vast majority of species. This discussion highlights the importance of viscosity to the dynamics of blood flow in fishes. Blood viscosity is related to the fraction of red blood cells in the blood, so from Poiseuille’s Law [Eq. ( l ) ] , vascular resistance is also related to hematocrit (Hct). Furthermore, since the heart must generate sufficient blood pressure to overcome vascular resistance, blood pressure is in turn linked to Hct. The complex and, as yet, incompletely understood interrelationships between blood viscosity, Hct, cardiac performance, and 0 2 transport are represented globally by the optimal hematocrit concept. Blood viscosity increases with both increasing Hct and decreasing temperature (Fig. 1A and B). In addition, blood is a non-Newtonian fluid, that is, its viscosity is shear-rate dependent (Fig. 1C) and increases exponentially at low shear rates. consequently, blood viscosity will be highest in fishes that have high Hcts and low blood velocities and live in cold water. The lowest viscosities will be found in warmwater fish with low hematocrits and high flows. The viscosity of plasma is also shear-rate dependent (Fig. 1D) and, in some species, can contribute over 50% to the whole blood viscosity, even at high temperatures. Interestingly, in rainbow trout the viscosity of plasma is independent of shear rate (Fletcher and Haedrich, 1987). The shear-rate dependency of fish blood has been examined in a number of species, and it appears that it is less in active fishes (Graham and Fletcher, 1985; Fletcher and Haedrich, 1987; Wells and Baldwin, 1990). However, at very high shear rates these species differences are considerably reduced. It is important to note that the major site of vascular resistance is the arterioles, and the shear-rate value in arterioles will have the greatest influence on the overall vascular resistance. However, shear rate is inversely proportional to radius of the

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Fig. 1. The effect of hematocrit on viscosity at 0°C (A) and 15°C (B) at shear rates indicated to the immediate right of each line describing a viscosity-hematocrit relation, Average hematocrits and mean cellular hemoglobin concentrations, respectively, are as follows: 0% (plasma); 11.5%and 24.8; 22.3%and 20.6; 43.1%and 26.3; and 75.0%and 25.7. The effect of temperature (indicated in "C to the left of each line) and shear rate on the viscosity ofwhole blood (C)and plasma (D).For C and D, total protein concentration, 4.32 2 0.12 g * 100 ml-I; average Hb concentration, 4.93 f 0.18 g . 100 ml-', average hematocrit, 22.26 0.20%.Redrawn from Graham and Fletcher (1983).

*

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blood vessel, so shear rate in arterioles would be expected to be high (Graham and Fletcher, 1983). The optimal hematocrit concept recognizes that, in terms of cardiac work per unit of internal 0 2 convection, there is a trade-off between increasing Hct to increase the 02-carrying capacity of the blood and decreasing the Hct to reduce the work of the heart generating blood pressure. The upper limit for Hct may, therefore, be set by the speciesspecific maximal blood pressure that the heart can develop (see Chapter 3), and the exceptional increase in blood viscosity at Hct values around 50430%. Conversely, the lower limit for Hct may be set by the maximum Q of the heart. One would further expect these limits to be affected by temperature, not only because of the effect of Hct on blood viscosity, but also because the demand for internal 0 2 convection is temperature dependent. Wells and co-workers (Baldwin and Wells, 1990; Wells and Baldwin, 1990;.Wells and Weber, 1991) examined the optimal hematocrit concept in a number of fish species by measuring blood viscosity with a cone-plate viscometer and relating this to the 02-transport capacity (OTC) as calculated by OTC =

1.3 [Hb] El.

(3)

where [Hb] = hemoglobin concentration. Reasonably discreet peaks for the calculated optimal Hct were obtained and these ranged among various species from 20 to 40%. The range for calculated optimal hematocrit clearly encompasses measured Hct values of many teleosts and elasmobranchs. However, for individual species examined, there were discrepancies between the calculated optimal Hct and the measured Hct. For example, calculated optimal Hct values were much higher than Hct values actually measured in rainbow trout (Oncorhynchus my kiss) and a variety of tropical elasmobranchs. Furthermore, in tropical reef teleosts, measured Hct was greater than the calculated optimum in an active species, but measured values were lower than the calculated optimum of two less-active species (Wells and Baldwin, 1990). A number of reasons can be advanced for these equivocal findings in respect to the validity of the optimal hematocrit concept. On the one hand, the plasma may or may not make a significant contribution to measured .viscosity whereas, on the other hand, variations in erythrocyte size and rigidity of the red blood cell membranes may decrease or increase whole blood viscosity. Also, the concentration of hemoglobin

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packed into each blood cell will affect differentially oxygen transport capability even when Hcts are similar. Furthermore, red blood cells have functions other than 0 2 transport and the measured Hct is likely to reflect compromises with these functions. Finally, an optimal Hct for 0 2 transport may be more relevant to a fish's physiology when internal 0 2 convection is pushed near its limit, i.e., near maximum VOZ. For example, prolonged and especially burst swimming are accompanied by increased Hct as a result of splenic contraction releasing stored red blood cells. The most extreme example of this is observed in the red-blooded Antarctic fish, Pagothenia borchgrevinki, in which Hct almost doubles when they are stressed (Davison et al., 1988; Franklin et al., 1991).These fish live in frigid waters and have low Hct at rest, so there will be an impact of the increased Hct on blood viscosity. In view of these considerations, it is more likely that optimal Hct spans a broad range and attempts to define the optimal Hct should recognize the importance of many other factors. Antarctic fishes are an extreme example of how blood viscosity influences overall cardiovascular design. Blood viscosity is exceptionally high around O'C, and Antarctic fishes living exclusively at temperatures of -1.7" to 2.0"C have either fewer or no red blood cells compared with temperate teleost species (see MacDonald et al., 1987). This appears to be an effective strategy to reduce blood viscosity, since the viscosity of the blood of hemoglobin-free Chaenichthyids at 0°C (2.9-3.9 centipoise; Hemmingsen and Douglas, 1972; Wells et d., 1990) is comparable to that at much higher temperatures in temperate water species of fish. While low or zero Hcts reduce blood viscosity, they also reduce blood 0 2 carrying capacity. To compensate, both hemoglobin-free and red-blooded Antarctic fishes have a high Q for internal 0 2 convection. The immediate advantage of this strategy is not clear in terms of cardiac work because high Q values increase cardiac work, thereby negating some or all of the benefits accrued by reducing blood viscosity. Further, since most cardiac work is done in generating tension within the muscle and not as external work, then Laplacian relationships dictate that a large-volume, low-frequency pump will operate at a low mechanical efficiency. However, the low blood pressures of Antarctic fishes will tend to offset the effects of a large ventricular volume on wall tension. Perhaps the most important advantage of the low Hct and high Q strategy for internal 0 2 convection in Antarctic fishes is to minimize the shear-rate effect on blood viscosity (Wells et az., 1990). Considerable modifications of the cardiovascular system of Antarctic fishes are necessitated by having a high Q. To achieve high Q

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values, the ventricle is enlarged and accommodates an exceptional stroke volume (e.g., 10 ml kg-’ in Chionodraco hamatus; Tota et al., 1991). Because of the high resting stroke volume, large increases in stroke volume are unlikely so heart rate rather than stroke volume increases with swimming. The increase in heart rate during exercise is brought about by a marked reduction in the high resting vagal tonus (Axelsson et al., 1992). Further, stroke volume is particularly sensitive to increases in diastolic output pressure in Chionodraco hamatus (Tota et al., 1991), indicating a limited capacity for homeometric regulation. Antarctic fishes also have large capillaries which, combined with a low Hct, yields an unusually low vascular resistance (Axelsson et al., 1992). One disadvantage of low Hct blood is that venous 0 2 content will be low. Consequently, myocardial 0 2 demand will be a major proportion ofthe 0 2 normally contained in venous blood (see Chapter 3).The long residence time of blood in the heart as a result of slow heart rates will favor 0 2 extraction, whereas the low dP/dt due to low arterial blood pressures, will be advantageous in terms of myocardial 0 2 demand. However, low venous PO, in hemoglobin-free fishes may seriously limit myocardial 0 2 delivery and thereby limit exercise capability.

-

111. ROLES OF THE CONUS AND BULBUS ARTERIOSUS For efficient gas exchange, water flow over the gills and blood flow through the gills should be continuous. Continuous blood flow may be achieved by the elastic recoil of blood vessel walls stretched during systolic ejection. The best estimate of the extent of this “windkessel” function of the ventral aortic system is from flow measurements made on the main vessel either just outside the conus or bulbus or between pairs of branchial arteries. Recordings from these situations show major differences between flow patterns in cyclostomes and elasmobranchs on the one hand and teleosts on the other, although these flow patterns may not be truly indicative of gill blood flow. In the ventral aorta of elasmobranchs, flow recorded either just outside the pericardium or between the second and third pairs of branchial arteries, stops or even reverses at some stage during diastole (Fig. 2; Satchel1 and Jones, 1967; Butler and Taylor, 1975; Short et al., 1977; Metcalfe and Butler, 1982; Abel et al., 1987). In contrast, flow in the teleost ventral aorta is usually continuous during disatole, a testament to the extreme capacity and compliance of the bulbus arteriosus (Fig. 5; Johansen, 1962; Stevens et al., 1972; Farrell, 1981; Hipkins, 1985; Axelsson et al., 1989; Jones et al., 1992a).

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Fig. 2. Schematic representation of heart structure and pressure within the pericardium, atrium, ventricle, and conus, aligned with ventral aortic pressure and flow of the Port Jackson shark, Heteredontus portusjacksoni. Not all pressures were recorded simultaneously. Figure from Satchel1 (1971), with permission.

A. Role of the Conus Arteriosus At present, the role of the elasmobranch conus arteriosus is ohscure. Nevertheless, many aspects of its function are clear. There is no doubt that ventricular systole is prolonged by contraction of the conus. However, the conal contribution to flow is slight (Johansen et al., 1966) since its volume is so much smaller than that ofthe ventricle. Ideas that conal contraction can provide an extra, active addition of propulsive

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power to that released by the ventricle (Johansen, 1965) have never been substantiated. The contrary idea that the conus could “depulsate” or reduce the peak systolic pressure also seems a doubtfbl proposition. “Depulsation” results from the elasticity of the central arterial system and since the conus is contracting during ventricular ejection (March et al., 1962; Sudak, 1965), its walls will be stiff and the conal contribution to overall elasticity will be small. Conal contraction ensures the competency of the lower (proximal) tiers of conal valves. In the isolated, relaxed conus of Heterodontus portusjacksoni only the top (distal) valves remain competent against back flow. The middle or lower tiers are not large enough to bridge the relaxed conus and must, therefore, depend on conal contraction to reduce conal diameter and bring them into contact with one another (Satchell and Jones, 1967).Satchell and Jones (1967)envisage a “peristaltic” wave moving up the conus and closing each tier of valves in turn. Certainly, the conus contracts with a time delay with respect to the ventricle, which allows the conus to distend with the initial ventricular ejection so that outflow is not impeded. Nodal tissue is present at the ventricular-conal junction and the electrocardiogram (ECG) shows a delay between deflections caused by contraction of the ventricle and conus (Satchell, 1991).Furthermore, the conduction velocity of the conal depolarization wave is some 20 times slower than the ventricular wave of depolarization (TebCcis, 1967), slow enough in fact (24 cm - sec-’) to provide the required to proximal distal wave of contraction. Satchell and Jones (1967)envisaged the primary role of the conus as postponing valve closure until the nadir of pericardial negative pressure, caused by ventricular ejection, was passed, reducing ventral aortic backflow. However, in their traces, marked aortic backflow was associated with closure of the lower not the upper set of valves (Fig. 2). It seems strange that this backflow would not also passively shut the upper conal valves, which are competent in relaxed preparations. Furthermore, there seems no intrinsic reason why negative external pressures should have more effect on valve competency than positive internal pressures. In fact, Satchell and Jones (1967) were unable to control arterial and pericardial pressures independently, and the only conclusion that can be drawn from their experiments is that aortic backflow increases when transmural pressures increase. Finally, pericardial pressures in Horn sharks (Heterodontus fruncisci) are not always negative, indicating that further research on the outflow dynamics of the elasmobranch heart should prove worthwhile (Abel et ul., 1986).

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B. Role of the Bulbus Arteriosus Depulsation and prolongation of aortic flow during diastole are undoubted functions of the teleostean bulbus. The bulbus of rainbow trout, yellowfin tuna (Thunnus albacares), and carp (Cyprinus carpio) are extremely elastic, with those of the latter two species being exceptionally extensible over pressure ranges to which the vessels are subjected in uiuo (Fig. 3A; Licht and Harris, 1973; Priede, 1976; Jones et al., 199213). Licht and Harris (1973) report that the bulbus of carp is 30 times more distensible than the human aorta over a pressure range of 7.3 to 33 mm Hg. One of the reasons for the high distensibility may be A 100

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Fig. 3. (A) Quasi-static pressure-volume curves for the bulbus arteriosus of tuna (Jones e t al., 1992b), trout (Priede, 1976), and carp (Licht and Harris, 1973). Redrawn from published traces, scaling the ordinate (volume) to 100% at maximal volume. (B) Quasi-static pressure-volume curves for the bulbus arteriosus and dorsal and ventral aortas of tuna. Ordinate scaled to 100%at maximal volume.

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that teleost elastin has fewer cross-links than that of mammals, so it is more elastic (Serafini-Fracassini et aE., 1978). Further, the “higgledypiggledy” arrangement of elastic fibrils in the bulbus may allow the fibers to slide between one another, allowing for greater distension (Benjamin et aE., 1983, see also Chapter 3 of this volume). Finally, the elastin :collagen ratio usually gives a good indication of blood-vessel stiffness. The larger the ratio, the more elastic the tissue. In the trout bulbus this ratio is about 14 (Serafini-Fracassini et al., 1978),compared with 1.5 in the proximal mammalian aorta (McDonald, 1974),and 0.4in the frog aorta (Gibbons and Shadwick, 1991a). Hysteresis is the proportion of energy lost through viscous processes during inflation and deflation and is evidenced by a pressure difference occurring at similar volumes during the increasing and decreasing limbs of a volume cycle (Fig. 3A). On a pressure-volume loop, the area within the loop as a proportion of the area under the inflation part of the cycle, is a measure of hysteresis. For the carp, hysteresis is negligible, whereas for the rainbow trout, it is substantial (Fig. 3A). The reason for this difference is obscure. Vascular smooth muscle is the main contributor to hysteresis in the mammalian arterial wall (Dobrin, 1978), and it is plentiful in the bulbi of both carp and rainbow trout. Furthermore, smooth muscle cells in bulbi of both rainbow trout and carp are firmly joined to one another by desmosomes. Lack of hysteresis in carp probably results from insufficient conditioning of the vessel. The first 4 to 6 volume cycles do not give identical results, and this probably obscured the hysteresis since Licht and Harris (1973) averaged values of 51 volume cycles done on bulbi from six fish. Bulbi are round, swollen proximally, and tapering distally to meet the ventral aorta (Santer, 1985).The functional significance of differing bulbar shapes is unknown, although Priede (1976) has discussed some hydromechanical advantages and disadvantages. Basically the bulbus expands in preference to the ventral aorta and this seems to be achieved by the bulbus having a much larger diameter and different wall construction. Dead space within the expanded bulb is reduced by trabeculae in rainbow trout, yellowfin tuna (Thunnus albacaves), turbot (Scophthalmus maximum), and other less phylogenetically advanced teleosts. The trabeculae may be irregular and anastomosing (Santer, 1985) or may be regularly disposed into longitudinal and radial elements as in rainbow trout and tuna species (Priede, 1976; Jones et al., 1992b). The longitudinal and radial elements provide for strain equalization within the bulbar wall. When the bulbus is expanded there is far more strain on the inner than the outer bulbar wall,

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but the inner longitudinal elements do not follow the outer wall during expansion (see Chapter 3). The longitudinal elements arch outward and support the outer wall by means of the obliquely inserted radial elements (Fig. 4) (Priede, 1976).A smooth bulbar lumen represents the apogee of teleostean phylogenetic advancement implying more differentiation in strain-limiting wall structure than in that of the conus of more primitive forms. Cyclostomes with a smooth inner wall to the conus appear to present an exception. However, the conus in cyclostomes has features that suggest it is an isolated evolutionary experiment rather than a predecessor of the conus or bulbus in the elasmobranchs and teleosts. Collagen, which is relatively inexpansible, is usually confined to the pericardial layer of the wall and will check bulbar expansion. Hence, collagen and elastin form a two-phase system in the bulbus similar to their role in blood vessels. Further, although it is the pericar-

A

B

Fig. 4. Isometric projection diagrams of the bulbus arteriosus as reconstructed in the contracted (A) and expanded (B) states. 1, Anterior wall of pericardial cavity; 2, wall of ventricle; 3, ventriculo-bulbar valve; 4, adventitia composed of pericardial elements and epicardium; 5, longitudinal elements; 6, radial elements; 7, compact layer of media with predominantly circumferential fibers. Caption and figure from Priede (1976),with permission.

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dial confinement of the bulbus that allows expansion in the first place, ultimately the pericardial wall itself will limit bulbar enlargement. The discussion so far has been restricted to circumferential and not longitudinal enlargement of the bulbus. The attachment of the pericardium to the distal end of the bulbus would appear to constrain bulbar lengthening in vivo. However, there is nothing to stop an elongating bulbus from pushing the ventricle backward. Hence, it is no surprise that collagen fibers in the outer wall may be oriented to resist longitudinal much more than circumferential stretch. For instance, in bigeye tuna (Thunnus obesus),the outer wall is twice as extensible in the circumferential as in the longitudinal direction (Jones et al., 1992b). Concentration of the major portion of arterial distensibility just outside the heart is more effective in reducing peak systolic pressure than a similar compliance distributed throughout the arterial system (Campbell et al., 1981; Jones, 1991). On the other hand, diastolic pressures will be raised equally regardless of the location of the compliance. In other words, the heart “sees” all of the compliance added on the outflow tract, whereas compliance added at other locations is effectively hidden from the heart during systolic ejection. Hence, the bulbar “windkessel” not only creates steady flow in the aorta, and presumably enhances gas exchange at the gills, but also reduces peak systolic pressures (Fig. 5 ) .As the majority of cardiac 0 2 uptake is utilized for developing tension, generating the blood pressure, any reduction in cardiac tension will bring about an improvement in cardiac efficiency. Efficiency is the quotient of external work divided by total energy transformed and, like that of most biological systems, the efficiency of the fish heart is low (Farrell et al., 1985; Farrell and Milligan, 1986).Hence, a reduction in peak systolic pressure will have a marked impact on the efficiency of heart function, much more so than improving external work performance, since so little of cardiac oxygen consumption appears as external work. Nevertheless, compromise must remain an important feature of cardiovascular function. For instance, increases in input frequency to the “windkessel” will further reduce pulsatility around the mean pressure, and promote continuous flow, but energetic savings will be compromised by the increase in the proportion of time that the cardiac muscle is generating tension. Priede (1976) calculated that 25% of the stroke volume in a trout could be held in the bulbus. Pressure-volume loops suggest that this may be an underestimate. For instance, in 1- to 2-kg tuna, stroke volumes vary from 0.3 to 1.3 ml - kg-l (Bushnell and Jones, 1992) and the bulbus can contain up to 0.5 to 1ml * kg-’ at normal ventral aortic

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1000

Fig. 5. Pressures in the ventricle (VP), ventral (VAP) and dorsal (DAP) aortas and ventral aortic flow (FLOW) of yellowfin tuna (Thunnusalbacares). Superimposed from original traces in Jones et ul. (1992a).

blood pressures when examined using quasi-static pressure-volume loops (Fig. 3A and B; Jones et al., 1992b).However, in vivo, expansion of the bulbus will be limited owing to its position within the pericardium. Furthermore, there is a lot of smooth muscle in the bulbar wall, individual cells being tightly bound together by desmosomes. The muscle cells are innervated by autonomic nerves at their outer ends, not laterally, and low-resistance gap junctions connect the cells in a cablelike electrical syncytium (Watson and Cobb, 1979). Hence, contraction or relaxation of smooth muscle will considerably reduce the capacity and compliance of the bulbus. Acetylcholine causes contraction of spiral strips of trout bulbus (Klaverkamp and Dyer, 1974) and considerably stiffens the wall of the isolated bulbus and ventral aorta in dynamic pressure-volume tests (Farrell, 1979). On the other hand, adrenergic agents and hypoxic exposure cause an increase in compliance in dynamic pressure-volume tests (Farrell, 1979). In vivo, these changes would tend to increase depulsation by accentuating the reduction in systolic pressure. No ventral aortic back-flow is measured during valve closure in fish with a bulbus arteriosus (Fig. 5). It is possible that backflow occurs in the region of the ventriculobulbar valves but, because of the com-

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P. G . BUSHNELL ET AL. 50

-EE

30

W

a

2

Y 2

20 10

0

0

.5

1

1.5

2

2.5

TIME (sec)

Fig. 6. Superimposed traces of ventricular (VP) and ventral aortic (VAP)pressures in the carp (Cyprinus carpio) from original traces in Ngan et al. (1974).

pliance of the bulbus, backflow is not transmitted to the ventral aorta. During ventricular ejection, vortices may be set up between the valve cusp and the wall so that as ventricular output slows, the pressure difference between the vortex and decelerating blood is sufficient to close the ventriculobulbar valves. This idea, first attributed to Leonard0 da Vinci, seems to hold for mammals, although it is doubtful that this mechanism will apply in fish owing to much lower rates of acceleration and deceleration during ventricular ejection. A notable feature of high-fidelity pressure and flow recordings in teleosts (i.e., carp, Ngan et al., 1974; yellowfin tuna, Jones et al., 1992a) is that ventricular pressure generation rises throughout the ejection phase, and aortic and ventricular pressures part company, indicating valve closure, at or soon after peak pressure (Fig. 5 and 6). This is unusual in vertebrates, for valve closure usually occurs on the descending limb of the ventricular pressure profile. In fact, the situation in teleosts is suggestive of active processes being involved in valve closure. In some teleosts, there is a muscular ring of tissue at the junction between the bulbus and ventricle and its activation could bring about valve closure. The muscular ring may represent a vestige of the more phylogenetically ancient conus. Alternatively, it may be that the valve is actively held open during ejection and closes passively when the ventricular muscle relaxes. Sanchez-Quintana and Hurle (1987) have described the insertion of cardiac muscle fibers on the bulboventricular ring that could actively open the valve. These are superficial fibers in swordfish (Xiphias gladius) and deep fibers in

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105

Atlantic bluefin tuna (Thunnus thynnus). Nonpassive cardiac valving is a common feature among the vertebrates, and a careful hemodynamic analysis of the cardiac outflow tract in teleosts should be most rewarding.

IV. PRESSURE AND FLOW RELATIONSHIPS IN THE VENTRAL AND DORSAL AORTAS Blood vessel structure appears to be similar from cyclostomes to the most advanced teleosts. The wall consists ofthree layers or tunics. The luminal tunic (intima) is endothelial, and the endothelium may be drawn into long spindle-shaped folds that run parallel to the aortic axis (Leknes, 1985). The middle tunic (media) is largely smooth muscle with elastic fibers interspersed between the smooth muscle cells. The outer tunic (adventitia) is mostly collagen. The collagenous layer is believed to be much thicker in the dorsal than in the ventral aorta (Lander, 1964). In the ventral aorta the smooth muscle cells are often loosely arranged with respect to the long axis of the vessel. However, in some teleosts smooth muscle is more organized, and in trout, for example, it is arranged both circumferentially and longitudinally (SerafiniFracassini et al., 1978). Elastic fibrils are often arranged randomly although grouping into distinct laminae, resembling amorphous elastin of mammalian vessels, occurs in carp, eel, rainbow trout, and yellowfin and skipjack (Katsuwonus pelamis) tuna (Dornesco and Santa, 1963; Isokawa et al., 1988, 1990; Jones et al., 1992b). Large wall strain due to the continuous pounding of high blood pressures may be a prerequisite for laminae formation, which explains their absence in fishes with low blood pressures. However, no determinations have been made of either the requisite wall strain or the period over which strain must be exerted to promote laminae formation. Leknes (1986) reports that no laminae occur up to 21 days posthatching in the guppy (Poecilia reticulata), although some alignment of fibrils is observed. A unique feature of cyclostome blood vessels (and the cyclostome conus) is that the fibrillar network in the media does not consist of elastin (Wright, 1984; Isokawa et al., 1989). In lamprey, the ventral aorta has a thicker wall than the hagfish, although there is a much higher collagen content in the wall of the latter (Wright, 1984). Interestingly, the New Zealand hagfish (Eptatretus cirrhatus) has a paired

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P. G. BUSHNELL ET AL.

ventral aorta, whereas Myxine glutinosa has a single ventral aorta (Axelsson et al., 1990; Forster et al., 1992). In elasmobranchs, elasticity measurements indicate that the ventral aorta is some six times more distensible than the dorsal aorta (Lander, 1964). However, this does not appear to be the case in at least one teleost, the yellowfin tuna (Jones et al., 199213).Pressure-volume loops show that the compliance of the ventral or dorsal aortae are similar. In the ventral aorta, distensibility is high up to the physiological pressure range but above that range, the pressure-volume curve increases sharply as the wal1 becomes stiffer (Fig. 3B, Jones et al., 1992b). The shape of this curve is generally attributed to the interaction of elastin and collagen, in that the highly distensible portion is dominated by the contribution of elastin, whereas the stiff portion is predominantly the result of collagen fibers. In contrast, pressurevolume loops for the dorsal aorta are nearly linear (Fig. 3B) over the range of pressures experienced physiologically. Both loops show hysteresis, which is more marked for the dorsal than ventral aorta. Hence, viscous losses in each cycle are higher for the dorsal aorta. Pressure and flow relationships in arteries are usually analyzed using an analog (pressure = flow x resistance) of Ohm’s law for direct current electrical circuits. However, both pressure and flow are pulsatile, and hemodynamic analysis is better served by considering not only the mean values of pressure and flow but also the sine wave harmonics of the pulsatile waveforms. The harmonics are obtained from a Fourier analysis of the original pressure and flow waveforms (McDonald, 1974; Jones, 1991). The quotient of dividing oscillatory pressure by oscillatory flow is termed impedance. Further, it can be seen from traces presented in Fig. 5 (Jones et al., 1992a) that in the ventral aorta, flow leads pressure. Consequently, the phase angle between any pair of harmonics of pressure and flow of the same frequency will usually be negative. Impedance modulus and phase differences for pressures and flows in the ventral aorta of a yellowfin tuna are shown in Fig. 7 (Jones et al., 1992a).It is noteworthy that the shape of the modulus and phase curves is unchanged by a doubling of heart rate. It can be seen from the modulus curve that zero-frequency impedance is some 3 to 10 times greater than that for the first and succeeding harmonics. What this means from the fish’s point of view is that the energetic costs of pulsatile pumping are much lower than they would be if impedance remained at steady flow values. The phase curves in Fig. 7 show that flow leads pressure at all pulsatile harmonics, which is a characteristic feature of a “windkessel” system. During the past 20 years, electrical,

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THE ARTERIAL SYSTEM

3 K

a

0.24

I

0

2 T

0 T-22

0 T-15

1

2 3 FREQUENCY

4

5

Fig. 7. Input impedance modulus and phase of the ventral aortic circulation in a yellowfin tuna (Thunnus albacares). For T-22 heart rate was 120 min-' and for T-15 heart rate was 58 . min-'. Figure and caption from Jones et al. (1992a).

mechanical, and mathematical models have been used to supplement recordings of pressure and flow in fish to better understand and attempt to explain pressure and flow relationships in the ventral and dorsal aortae. Models, consisting of two sets of compliant and resistive elements coupled in series (Satchell, 1971; Jones et al., 1974) have been analyzed in great detail by Langille et al. (1983) and consequently will not be dealt with here. The analysis of vascular impedance shown in Fig. 7 was limited by only being taken to five harmonics but, even at heart rates in excess of 2 Hz, there are no signs of the oscillations in modulus and phase that are so apparent in analyses of mammalian circulations. Pulse wave velocities will be slow, although accelerating toward the gills owing to the increased stiffness of the distal ventral aorta. Nevertheless, it is unlikely that pulse transmission will occupy a large enough portion of the cardiac cycle (10-20%) for significant phase changes to occur be-

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Whdkessel

Reflection free

Transmission line

-4

-b

1

0.25

I

0.50

I

I

0.75

1.00

Lh Fig. 8. A generalized curve showing impedance amplitude as a function of frequency expressed as the ratio of the arterial tree length (L)to the fundamental pressure wavelength (A). A short arterial tree and/or a low heart rate shift the impedance spectrum to the left, making the system function as a “windkessel.” A very long arterial tree or a very high heart rate shifts the spectrum to the right, making it more like a reflection-free transmission line. Impedance modulus (Z) is normalized by dividing by peripheral resistance (R). Z, is the characteristic impedance. Figure and caption from Gibbons and Shadwick (1991b), by permission.

tween pressure and flow pulses at different arterial sites. These phase changes can lead to marked deviations between pulses expected in a “windkessel” and those actually recorded, and are caused by wave propagation effects. In mammals, wave-propagation effects cause amplification of the pressure pulse as it moves toward the periphery, distortion ofthe pulse profile, and the appearance of diastolic secondary waves. These features result from both geometric and elastic tapering of the arteries as the periphery is approached, as well as discrete reflections either from discontinuities in the arterial system or in the termina1 impedance (Taylor, 1964; McDonald, 1974; Gibbons and Shadwick, 1991b; Jones, 1991). However, reflection effects in mammals are more pronounced the higher the terminal resistance. The terminal resistance for the ventral aorta is the gill resistance, which is generally less than that of the systemic resistance. Consequently, a number of factors contribute to a lack of wave transmission effects in the ventral aortic circulation of teleosts, and it is entirely possible that circumstances may exist (i.e., an elongated ventral aorta, extremely high heart rate, low pulse wave velocity, and high gill resistance) to promote considerable deviation from the “windkessel” model.

2. THE

ARTERIAL SYSTEM

109

Pressure and flows in the dorsal aortic circulation are pulsatile, albeit with some loss of the high-frequency components of the waveform seen in the ventral aorta. Unfortunately, simultaneous recordings of pressure and flow have been made only in Atlantic cod (Gadus rnorhua)and yellowfin tuna (Jones et al., 1974; 1992a), although the numerous recordings of pressures made in the postbranchial circulation suggest that neither cod nor tuna are in any way unique. Dorsal aortic flow pulsatility as a function of mean flow, is about 2.5 to 4 times pressure pulsatility (as a function of mean pressure). If the dorsal aortic circulatory system were purely resistive, then pulse flow and pulse pressure would be equal percentages of their means. Hence, there is considerable compliance in the dorsal aortic system, which subserves a “windkessel” function. Even if the vessels of the dorsal aortic circulatory system are stiffer than those of the ventral, as seems to be the case in elasmobranchs (Lander, 1964), their considerable length means that compliance of this part of the circulation will be substantial. Analysis of electrical and mechanical models of the fish circulation by Langille et aE. (1983) shows that dorsal and ventral aortic pressure oscillations are inversely proportional to dorsal aortic compliance, whereas ventral aortic flow oscillations are directly proportional. However, the effect of increasing dorsal aortic compliance on ventral aortic pressure pulsatility is rather small, so energetic savings in terms of reduced cardiac work will not be that big (Jones et al., 1974).On the other hand, a large dorsal aortic compliance may have a deleterious effect on gas exchange. With a large dorsal aortic compliance, cardiac stroke flow rushes through the gills to charge the dorsal aortic capacitance, increasing flow oscillations not only in the ventral aorta but also at the gas-exchange surface of the gills. Since the dorsal aorta is long, a significant proportion of the cardiac cycle may be taken by the pulse wave in traveling along it. Hence, one of the basic tenets of the “windkessel,” that pressure changes generated by cardiac contraction occur simultaneously throughout the arterial system, will not be fulfilled. Thus, under certain conditions, wave transmission effects might be expected in the dorsal circulation. For instance, body length is related to body mass to the minus onethird power, whereas the correlation between heart rate and body mass in fish (if one exists) has not been determined. Only if heart frequency decreases in proportion to the change in aortic length will haemodynamics of large and small fish be the same. But, if there is no heart frequency :body mass relation, and if both large and small fish have similar heart rates, then wave transmission effects can be expected in the former but not in the latter. Also, heart rate may increase with

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increase in temperature and with exercise, so it is possible that wavetransmission effects will become more significant in fish exercising in warm waters. A major benefit of wave transmission phenomena is that the high terminal impedance, represented by the vascular beds, is essentially decoupled from the input to the arterial system. This represents a considerable energetic saving, which is totally realized when the ratio of the length (L)of the arterial tree to the fundamental pressure wavelength (A) is one-quarter (Fig. 8). A is the pulse wave velocity divided by the repetition (heart) rate. However, A is not fixed and varies directly with wall stiffness, which can be changed as a result of neural, humoral, and hormonal influences. Furthermore, an increase in blood pressure that occurs particularly during initial stages of exercise, will increase stretch of the arterial walls and make them stiffer. Nevertheless, the major determinant of the properties of the dorsal aortic circulation is LIA (Fig. 8; Gibbons and Shadwick, 1991b).A short dorsal arterial vessel or low heart rate will shift the curve left, making the system function as a “windkessel” (Fig. 8).A long vessel and high heart rate confer the benefits of wave transmission effects. However, in extremely long fish, these benefits may be lost as the system will function as a reflection-free transmission line (Fig. 8).

V. BLOOD-FLOW DISTRIBUTION AND VASCULAR RESISTANCE Blood-flow distribution is regulated by smooth muscle activity in the resistance vessels induced neurally, humorally, or locally. However, the basal vascular tone is set by myogenic activity within electrically coupled smooth muscle cells, contracting to counteract stretch caused by the blood pressure. In addition, contraction of smooth muscle cells can be modified by endothelial-derived substances (Vanhoutte et al., 1986; Miller and Vanhoutte, 1986). Hence blood flow distribution is affected not only against a background set by the basal vascular tone, but also modulation by endothelial-derived factors. In fish the branchial and systemic vascular beds represent the two major sites of resistance in the circulatory system. Approximately 30% (range 18-40%) of the resting total peripheral resistance (TPR)occurs in the gills, while the remaining 70% (range 60-82%) resides in the visceral and somatic vasculature (TableI). In the few species oftuna in which it has been examined, branchial resistance (R,) is significantly elevated (41-65% of TPR), presumably as a result of their much larger

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111

gill surface area. The innervation and control of the branchial vasculature has been reviewed in a previous volume (XA) in this series (Nilsson, 1984a)and elsewhere (Nilsson, 1983; 1986; Nilsson and Axelsson, 1987) and will not be considered here. Further, nonadrenergic and noncholinergic (NANC) neurotransmitters such as bombesin, neurotensin, and vasoactive intestinal peptide (Holmgren and Nilsson, 1983a,b)are considered in detail in Part B, Chapter 5. This section will emphasize adrenergic and cholinergic control and their effects on distribution of systemic blood flow. A. Blood Flow Distribution at Rest

Although little is known about the autonomic innervation of the systemic vasculature in cyclostomes, anatomical and histochemical evidence suggest that there is some adrenergic spinal innervation to the blood vessels in lampetroids (Leont’eva, 1966). In addition, infusion of adrenaline and acetylcholine into hagfish, Myxine glutinosa, produces a small reduction (10-30%) in systemic resistance (R,), whereas adenosine reduces R, by almost 50% (Axelssonet al., 1990). In spite of unusually low dorsal ( Pda) and ventral ( Pva) aortic pressures (Table I), TPR in M . glutinosa is similar to teleosts because of a correspondingly low 0. The autonomic innervation of the arterial system in elasmobranchs has not been extensively studied. Histochemical and in vitro studies have demonstrated the existence of adrenergic innervation of systemic arteries in Squalus acanthias and Scyliorhinus canicula (Nilsson et al., 1975). However, direct neurogenic control of R , has not been demonstrated. Instead, in vivo and in vitro experiments suggest that control of R, at rest is mainly via circulating catecholamines (Short et al., 1977; Butler et al., 1978; Opdyke et al., 1981, 1982). Injection of catecholamines in vivo produces a large a-adrenoceptor mediated systemic vasoconstriction, as well as a smaller p-adrenoceptor-mediated vasodilation (Kent and Pierce, 1978; Opdyke et al., 1982). Certainly, indirect neurogenic control of vascular resistance may exist, however, since ganglionic blockade of pre- and postsynaptic transmission has been shown to reduce the release of catecholamines from chromaffin cells in S. acanthias (Opdyke et al., 1983). Histochemical, in uitro, and in vivo evidence shows that the major systemic arteries and arterioles in teleosts receive widespread adrenergic innervation (Nilsson, 1983; 198413). While both a- and P-adrenoceptors are present, the a-adrenergic constrictor mechanism dominates the P-adrenergic dilator mechanism in the systemic vascu-

Table I Systemic and Vascular Resistance Calculated from Data Measured in Fish at Rest and during Exercise (cm . s - l ) or Hypoxia" Resistance

Q

Species

Mass (kg)

Myxine gbtinosa

.55-.91

Scyliorhinus stellaris S . canicula

2.8 0.75

Anguilla anguilla

0.51

A. australis

0.62

A. japonica

0.3-0.6

Gadus morhua

0.4-0.8

G . morhua

0.4-1.3

(ml-' . min-' Condition

W')

rest hypoxia-15 rest -24 cm . s-' rest h ypoxia-77 rest hypoxia-40 rest 15 cm . s-l 24 cm . s-l rest hypoxia-80 hypoxia-40 rest 26 cm * s-l rest

8.7 8.7 52.5 89.2 32.1 35.7 11.8 7.8 11.3 11.3 10.3 11.0 9.5 4.8 17.3 25.4 19.2

.

P,, Pda (mmHg)

7.8 9.4 25.2 25.5 38.0

32.0 37.9 32.3 38.6 39.0 46.8 24.2 23.3 14.3 36.8 46.5 36.8

5.8 6.8 18.5 17.5 29.0 24.0 25.0 17.6 23.5 23.6 21.8 16.9 17.4 9.7 24.0 30.0 23.3

Branch System Total (mmHg. ml-' . min-' . kg-') 0.23 0.30 0.13 0.09 0.28 0.22 1.12 1.88 1.34 1.36 2.42 0.66 0.62 0.97 0.74 0.65 0.70

0.66 0.78 0.35 0.20 0.90 0.67 2.17 2.26 2.08 2.09 2.11 1.54 1.83 2.05 1.39 1.18 1.21

0.90 1.08 0.48 0.29 1.18

0.90 3.29 4.14 3.41 3.45 4.53 2.20 2.45 3.02 2.12 1.83 1.91

Branch System (% of total) 26.0% 28.0% 26.6% 31.4% 23.7% 25.0% 34.0% 45.3% 39.1% 39.4% 53.4% 29.8% 25.2% 32.1% 34.7% 35.5% 36.7%

74.0% 72.wo 73.4% 68.6% 76.3% 75.0% 66.0% 54.7% 60.9% 60.6% 46.6% 70.2% 74.8% 67.9% 65.3% 64.5% 63.3%

Reference"

Hemitripterus americanus Ophiodon elongatus Oncorhynchus mykiss

0.mykiss Thunnus albacases

C

T. albacares T. alalunga Katsuwonus pelamis

h ypoxia-35 rest 30 cm . s-l 3.8-6.5 rest h y poxia-75 h ypoxia-35 0.9-1.5 rest 44 cm . s-l 63 cm . s-l 73 cm . s-l 0.1-0.7 rest 1.4 spinal block hypoxia-130 h y poxia-90 h ypoxia-50 anesthetized 1.0-2.0 7.8- 10.7 spinalectomy spinal block 1.6 hypoxia-130 hypoxia-90 h y poxia-50

0.67-1.4

19.2 18.8 30.9 11.2 9.9 7.7 17.6 28.4 34.8 42.9 36.7 115 115 115 74.1 61.9 29.4 132.3 132.3 105.3 75.0

47.3 28.5 35.3 38.0 39.2 33.8 38.8 40.2 48.7 52.2 31.2 89.7 89.7 89.7 89.7 84.4 84.2" 87.3 87.3 87.3 87.3

37.5 23.3 26.3 28.3 30.2 23.9 31.0 30.0 30.0 33.7 25.4 32.6 32.6 32.6 32.6 44.9 48.1 40.2 40.2 36.2 40.2

0.51 0.28 0.29 0.87 0.91 1.29 0.44 0.36 0.45 0.43 0.16 0.49 0.50 0.50 0.77 0.64 1.23 0.36 0.36 0.49 0.63

1.95 1.24 0.85 2.53 3.05 3.10 1.76 1.06 0.95 0.79 0.69 0.28 0.28 0.28 0.44 0.73 1.64 0.30 0.30 0.34 0.54

2.46 1.52 1.14 3.39 3.96 4.39 2.20 1.42 1.40 1.22 0.85 0.78 0.78 0.78 1.21 1.36 2.86 0.66 0.66 0.83 1.16

20.6% 18.4% 25.5% 25.5% 23.0% 29.3% 20.1% 25.4% 32.3% 35.1% 18.8% 63.7% 63.7% 63.7% 63.7% 46.8% 42.9% 54.0%

54.0% 58.5% 54.0%

79.4% 81.6% 74.5% 74.5% 77.0% 70.7% 79.9% 74.670 67.7% 64.6% 81.2% 36.3% 36.3% 36.3% 36.3% 53.2% 57.1% 46.0% 46.0% 41.5% 46.0%

9

10 11

12 13

14 15 13

PO2 given in mmHg. Central venous pressure was assumed to be zero in all conditions. Mean ventricular systolic pressure, (1)Axelsson et al., 1990; (2) Piiper et al., 1977; (3)Short et al., 1979; (4) Peyraud-Waitznegger and Soulier, 1989; (5)Davie and Forster, 1980; (6)Chan, 1986; (7)Axelsson and Nilsson, 1986;(8)Fritsche and Nilsson, 1989; (9)Axelsson et al., 1989; (10)Farrell, 1982; (11)Kiceniuk and Jones, 1977; (12)Wood and Shelton, 1980; (13)Bushnell and Brill, 1992;(14)Jones et al., 1992a;(15)Lai e t al., 1987.

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P. G . BUSHNELL ET AL.

lature (Wood, 1976; Farrell, 1981; Nilsson and Holmgren, 1985).Although a cholinergic innervation of major arteries in eel and trout was postulated by Kirby and Burnstock (1969),experimental evidence has not substantiated the idea of primary cholinergic vascular control in the systemic circuit (Nilsson, 1983).The relative importance of neural versus humoral control of resting R, has been a matter of some controversy (Wahlqvist and Nilsson, 1977; Smith, 1978). It is now generally agreed that in rainbow trout (Smith, 1978; Wood and Shelton, 1980), resting R, is maintained by a neurally mediated adrenergic tonus. This was also thought to be the case in Atlantic cod (Smith et al., 1985), but recent evidence suggests that the neural adrenergic influence on systemic vasculature in Atlantic cod is extremely low, verging on the nonexistent (Fritsche and Nilsson, 1990). In eels (Anguilla australis), adrenergic innervation of dorsal aorta, visceral arteries, and small arterioles has been demonstrated histochemically (Gannon, 1972), and both adrenaline and spinal stimulation increase R,, yet pharmacological blockade of a-adrenoceptors fails to reduce R,. Hence, it appears that in eels, unlike in rainbow trout, there is no resting adrenergic tonus to the systemic circulation (Hipkins et al., 1986). Due to the difficulty of measuring blood flow and pressure in visceral and somatic vascular beds, R, is usually considered a lumped parameter. A few studies, however, have attempted to study the distribution and control of blood flow to individual vascular beds within the systemic circuit using radioactive microspheres. In this technique, activity of tissue samples following injection of microspheres is taken as a measure of relative tissue perfusion and converted to absolute blood flow by multiplying by measured or assumed Q. Results indicate that blood flow distribution to muscle and visceral organs in albacore tuna, Thunnus alalunga (White et al., 1988), rainbow trout (Barron et al., 1987),and arctic grayling, Thymalus arcticus (Cameron, 1975) are similar (Fig. 9A). A high proportion of Q in all three species is distributed to the red (10-38%) and white (28-50%) swimming muscles, which agrees with earlier findings of flow distribution in the dogfish, S. acanthias (Kent et al., 1971,1973).Among the visceral organs, most of the blood flow in the tuna and grayling goes to the liver and kidney, whereas in the rainbow trout the kidney and pyloric caeca (not shown in Fig. 9A) receive the highest percentage of Q. Although the white muscle in all three species receives a large percentage of Q, the actual perfusion rate (ml min-' * g-l) is low when compared with red muscle, liver, and kidney (Fig. 9B). Recently, direct measurements of blood flow to the viscera in the

2.

115

THE ARTERIAL SYSTEM

B

--.-

'm

.-

0.6

I

8

I1

II

I

t

,

0.5

'C

-EE

$

0.4

0.3

Fig. 9. The percentage distribution of cardiac output (A) and absolute blood flow (B) to selected organs and tissues of albacore tuna, Thunnus alalunga (open bars) (White et al., 1988), arctic grayling, Thymalus arcticus (solid bars) (Cameron 1975),and rainbow trout, Oncorhynchus mykiss (striped bars) (Barron et al., 1988) as determined by the injection of radioactive microspheres.

sea raven, Hemitripterus americanus, have been made by placing electromagnetic flow cuffs around the celiac artery, a major branch of the celiaco-mesenteric artery, which provides blood to approximately 50% of the gut (Axelsson et al., 1989). Blood flow in the artery of unfed, resting fish was 2.9 ml * min-l(15% of Q) and doubled after feeding to reach approximately 30% of Q,There appeared to be a tonically active adrenergic innervation of the visceral vasculature since administration

116

P. G . BUSHNELL ET AL.

of phentolamine, an a-adrenoceptor blocking agent, resulted in a quadrupling of resting celiac artery blood flow. Although the exact site of visceral resistance could not be established, pressure and flow measurements demonstrated that in sea raven, at least, resistance changes did not occur upstream or proximal to the measuring site. This contradicts suggestions made for rainbow trout (Smith, 1978; Randall and Daxboeck, 1982) that the celiaco-mesenteric artery is a major control site for R,. Resistance changes, presumably at the arteriolar level of the gastrointestinal circuit, make an important contribution to R,, since changes in celiac blood flow are accompanied by opposite and proportional changes in dorsal aortic pressure (Axelsson et al., 1989). Doppler blood flow measurements in the celiac and mesenteric arteries of resting Atlantic cod showed that gastrointestinal (stomach, pyloric caeca, liver, and intestine) blood flow accounted of approximately 40% (7.5 ml - min-') of Q (Axelsson and Fritsche, 1991). Control of resistance to blood flow in celiac and mesenteric arteries in resting cod is unclear, however. Whereas portions of Axelsson and Fritsche's (1991) study could find no evidence for adrenergic tonus acting on the gut vessels, a second portion of the same study clearly demonstrated resting humoral and neuronal adrenergic tonus on the celiac artery. Postprandial blood flow to the gut increased to 52% of Q but did not change after the injection of the a-adrenoceptor blocker, phentolamine, which contrasts with that of the sea raven, in which blood flow increased substantially after phentolamine treatment. The difference may reflect the fact that resting adrenergic tonus is already reduced to elevate postprandial blood flow in the Atlantic cod.

B. Blood Flow during Exercise Brett (1964) divided swimming into three categories, burst, prolonged, and sustained, in order to reduce confusion that was associated with the many types of swimming behavior. Burst swimming is high-speed anaerobic exercise lasting < 20 sec, whereas sustained swimming is aerobic exercise lasting > 200 min. Between these two extremes is prolonged swimming, which may be composed of aerobic and anaerobic components. The description that follows will focus on cardiovascular events that occur during prolonged or sustained swimming. Cardiovascular responses to exercise ye surprisingly variable among fish species. In dogfish for instance, Q increases by 70% with little change in Pda and Pva.Thus R, falls by 43%while R, falls by 40% (Table I). In the short-finned eel (Anguilla australis), on the other

2. THE ARTERIAL SYSTEM

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hand, 0 and Pd, remain the same or decrease slightly (Table I), while P,, increases significantly. This is indicative of a substantial increase in R, and virtually no change in R, (Davie and Forster, 1980). Responses to exercise in the sea raven, cod, and rainbow trout are similar to each other, and one might suggest that they are more typical of the cardiovascular adjustments to exercise in teleosts. Increasing swimming speeds (Table I) generally cause an increase in Q, an increase in Pd, and P,,, and a decrease in R, (Kiceniuk and Jones, 1977; Randall and Daxboeck, 1982; Axelsson et al., 1989; Axelsson and Fritsche, 1991).The increase in P,, and Pda is biphasic in response to a step increase in swimming speed and consists of an initial increase in pressure, which peaks in 10-15 min, followed by a slow decline, which stabilizes at a value slightly higher than before the increase in swimming speed (Kiceniuk and Jones, 1977; Randall, 1982).In exercising cod, R, has been shown to be under adrenergic neuronal control since pharmacological blockade eliminates or reverses the changes in Pda and P,, seen in untreated fish (Axelsson and Nilsson, 1986). However, Axelsson and Fritsche (1991) found that a-adrenoceptor blockade following neuronal blockade further reduced Pd,, indicating either a possible role for circulating catecholamines in exercise or a compensatory release of circulating catecholamines in response to the pharmacological neural blockade. Although plasma levels of adrenaline and noradrenaline were thought to increase 8- to 26-fold in response to exercise (Nakano and Tomlinson, 1967), recent work has shown this to be a result of “stress” due to how the exercise was induced, rather than a direct result of exercise per se. When dogfish, trout, or cod are allowed to swim spontaneously or are induced to exercise without violence (i.e., no prodding, handling, or shocking) exercise levels of circulating catecholamines are only slightly higher (two to three times) or no different than resting values (Ristori and Laurent, 1985; Butler et al., 1986; Hughes et al., 1988). In fact, when proper care is taken, fish can be swum to exhaustion without raising plasma catecholamine levels at all (Butler et al., 1989). Therefore, although plasma catecholamine levels can be significantly elevated during repeated burst exercise, for instance, their role in controlling R, during normal increases in swimming speed is considered to be physiologically insignificant (Butler, 1986). The distribution of systemic blood flow is significantly altered in exercise when compared with that in rest. In exercising rainbow trout, estimates of blood flow using radioactive microspheres show that red muscle blood flow increases from 9% (rest) to 42% of Q (Randall and Daxboeck, 1982) and remains elevated for at least 2 hr postexercise

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(Neumann et al., 1983).The mechanism behind the dramatic increase in blood flow to the swimming muscles is still unclear, although vasodilation resulting from locally produced metabolites (COz, H+, K+, lactic acid) has been suggested (Randall and Daxboeck, 1982; Canty and Farrell, 1985). Redistribution of blood flow to swimming muscle comes at the expense of blood flow to visceral organs. In rainbow trout, for instance, blood flow to the liver, spleen, and intestine is curtailed when muscle flow is enhanced (Randall and Daxboeck, 1982). In exercising cod, blood flow in the celiac and mesenteric arteries was significantly reduced (29% and 36%, respectively) as a result of a 75% increase in vascular resistance of the celiac and 101% increase in the mesenteric circuit (Axelsson and Fritsche, 1991). Pharmacological studies indicated that resistance in the mesenteric artery was controlled by an adrenergic tonus that has nervous and humoral components, whereas no adrenergic control of the celiac artery could be demonstrated. In Atlantic cod, therefore, the blood vessels to the gut respond to adrenergic as well as nonadrenergic mechanisms. In this regard, it should be mentioned that the gut vasculature has a significant innervation with fibers that contain NANC neurotransmitters (Holmgren and Nilsson, 1983a,b). Although we do not know much about their role in controlling R, and blood flow during exercise, their potential contribution should not be ignored, C. Blood Flow during Hypoxia Few studies have made the necessary measurements required to properly evaluate changes in blood-flow distribution in response to environmental hypoxia. Nevertheless, when it has been studied, R, has been reported to increase ( Japanese eel, AnguiZZajaponica, Atlantic cod, lingcod), decrease (dogfish, yellowfin tuna) or remain unchanged (European eel, hagfish, skipjack tuna) (see Table I for references). The changes in R , are reflected in equally varied changes in Q, P,,, and Pda. Although some of the alterations in R, are simply the result of passive changes in vessel resistance (Farrell, 1982), other factors have been implicated. In Atlantic cod, for instance, Q does not change during hypoxia (water PO, = 30-40 mm Hg) while Pva and Pda increase (Fritsche and Nilsson, 1989,1990).Pharmacological blockade of adrenergic nerves significantly reduces hypoxia-induced hypertension in the ventral and dorsal aortas. Subsequent treatment with a-adrenoceptor blockade further reduces P,,, which led the authors to conclude that arterial hypertension in hypoxia was primarily a result of increased nervous adrenergic tone combined with a small, but signifi-

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cant, tonus resulting from circulating catecholamines (Fritsche and Nilsson, 1990). Although levels of circulating catecholamines do not increase substantially in exercise, levels of adrenaline and noradrenaline increase markedly in hypoxia. A number of studies with rainbow trout (Tetens and Christensen, 1987; Boutilier et al., 1988; Ristori and Laurent, 1989) have shown that while concentrations of both catecholamines increase 300-400% during deep hypoxia (water Poz = 20-40 mm Hg), noradrenaline becomes the predominant catecholamine, attaining levels as high as 20.5 nM - 1-' (Ristori and Laurent, 1989). However, in Atlantic cod subjected to 30 to 40 mm Hg hypoxia (Axelsson and Fritsche, 1991) both noradrenaline and adrenaline reach similar levels (30 nM 1-', noradrenaline; 50 nM - 1-' adrenaline). Although it is clear that plasma catecholamine levels rise in response to hypoxia, in uitro studies indicate that they do not appear to reach a level high enough to alter R,. In Atlantic cod, rainbow trout, and short-finned eel tail preparations, significant changes in R, occur only when catecholamine levels reach 100 to 300 nM * 1-' (Wood and Shelton, 1975; Wahlqvist, 1980; Davie, 1981). In contrast to the redistribution that occurs during exercise, distribution of blood flow to muscles and visceral organs in the arctic grayling remains unchanged when ambient oxygen is reduced to 55 mm Hg (Cameron, 1975). However, when Atlantic cod are exposed to hypoxia (water Po, = 30-40 mm Hg), mesenteric artery blood flow decreases 62% and celiac artery decreases 42%, in spite of a 32% increase in Q (Axelsson and Fritsche, 1991). The increases in vascular resistance in both arteries were eliminated by adrenergic nerve blockade with bretylium, and further reduced by application of the a-adrenoceptor blocker phentolamine. These results agree with earlier findings (Fritsche and Nilsson, 1989,1990)that vascular resistance in the Atlantic cod is controlled by both neuronal and humoral adrenergic mechanisms during hypoxia. It is obvious from the preceding discussion that our understanding of the distribution of systemic blood flow is extremely limited. Obviously there are marked species differences, not only in the control of resting blood pressure and resistance, but also in control of responses to hypoxia and exercise. Even within a species, responses to similar experimental conditions differ. In Atlantic cod, for example, exposure to hypoxia (water PO, = 30-40 mm Hg) results either in an increase in Q and a decrease in R, (Axelsson and Fritsche, 1991) or an increase in R,, while Q remains unchanged (Fritsche and Nilsson, 1989). Standardized procedures, a wider variety of species, and more sophisti-

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cated techniques will be required before we can begin to make the most basic generalizations about the regulation of blood flow in fish.

VI. HEAT-EXCHANGERETIAL SYSTEMS Endothermy, the ability to maintain elevated body temperature by trapping metabolic heat with vascular retia has evolved independently three times in the family Scombridae and once in the elasmobranch family Lamnidae (Block, 1991). In nonendothermic fish, heat produced by metabolism is lost to the surrounding water as blood flows through the gills. As a result, the largest steady-state excess temperature that can be maintained is about 0.5”C (Carey, 1982). Heatexchange retial systems effectively uncouple the heat-production and heat-loss pathways and allow body temperature to exceed ambient water temperature by 5” to 20°C (Carey and Teal, 1969a,b; Graham, 1973). Vascular heat exchangers are found in three locations: the swimming muscles, the viscera, and the cranial cavity (Table 11).Benefits of endothermy include niche expansion (Carey et al., 1971; Graham, 1975) and increases in muscle power output (Carey et d.,1971), digestion rate (Stevens and McLeese, 1984), metabolite and oxygen flux rate (Stevens and Carey, 1981),visual threshold (Block and Carey, 1985), and recovery from burst activity (Stevens and Neill, 1978). A. Anatomy of Heat-Exchange Retia

Vascular heat exchangers, consisting of small interdigitating arteries and veins, are associated with swimming muscles in all true tunas (Scombridae), lamnid sharks (Carey, 1982), and swordfish (Block, 1991) (Table 11).A single, large rete is located centrally in the hemal canal of tropical species of tuna. However, temperate and subtropical tuna, lamnid sharks, and swordfish lack a central rete. In the latter, a pair of well-developed lateral or cutaneous retia are present on the dorsal and ventral surface of the red swimming muscles (Fig. 10) (Stevens and Neill, 1978; Carey, 1982). The lateral heat exchanger’s effectiveness in limiting heat loss in cold water (7°C)is exemplified by the giant bluefin tuna in which core temperatures of large (200-400kg) tuna can be as high as 29°C (Carey and Teal, 1969b). Although both location and basic anatomy of heat-exchanging retia in lamnid sharks are similar to those of the bluefin tuna (Carey and Teal, 1969a,b), the retia in mako and great white shark (Cacharodon carchardas) do not

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Table I1 The Occurrence of Retial Heat-Exchange Systems in Billfish, Tuna, and Sharks Swimming muscle" Common name Billfish'*" Blue marlin Black marlin White marlin Striped marlin Long bill spearfish Short bill spearfish Sailfish Swordfishd TunacSe.f Bullet tuna Frigate tuna Kawakawa Little tunny Black skipjack Skipjack tuna Longtail tuna Blackfin tuna Yellowfin tuna Albacore Bigeye Atlantic bluefin tuna Pacific bluefin tuna Southern bluefin tuna Butterfly mackerel8 Sharks' Mako Long-finned mako Porbeagle Salmon shark White shark Big-eyed thresher Thresher

Scientific name

Cranial

Central

Lateral

Visceral

Makaira nigricans M.indica Tetrapterus albidus T. audax T. pjluegeri T. angustirostris lstiophorus platypterus Xiphias gladius

X X X X X X X X

0 0 0 0 0 0 0 X

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

Auxis rochei

? ? ? ?

X X X X X X X X X 0 0 0 0 0 0

X X X X X X X X X X X X X

0 0 0 0 0

0

0 0 0 X X X X X 0

0 0 0 0 0 0 ?

X X X X X X ?

X X X X X 0 X

A. thazard

Euthynnus afjnls E. allatteratus E. lineatus Katsuwonus pelamis Thunnus tonggol T. atlanticus T. albacares T. alalunga T. obesus T. thynnus thynnus T. t. orientalis T . maccoyii Gasterochisma melampus Isurus oxyrhinchus 1. paucus Lamna nausas L. ditropis Carcharodon carcharias Alopias superciliosus A. vulpinus

X, present; 0, absent, ?, unknown. Block (1986). Carey (1982). Carey (19~0). Collette (1978). f Sharp and Pirages (1978). 4 Block (pers. comm.). a

X X ? ?

X X X X X ?

X X X

x

X X X 0

X

0

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P. G . BUSHNELL ET AL.

Fig. 10. A schematic diagram of the blood supply to muscle of bluefin tuna. Four pairs of cutaneous vessels branch into many smaller vessels and form the counter-current heat exchange retia above and below the red swimming (dark) muscle. The white (light) muscle is supplied by bands of alternating arteries and veins that pass through the muscle from the segmental vessels, thus acting as small heat exchangers. Other nonheat-exchanging arteries and veins (not shown) run out from dorsal aorta and post cardinal vein, respectively. Redrawn from Carey (1973).

form a compact mass of tissue, but are dispersed throughout the red muscle (Carey and Teal, 196913). A second, smaller rete, formed by approximately 20 vessels that branch to form triads of two arterioles and one venule, provides blood to the white swimming muscles in both tuna and sharks, except in the mako and great white sharks. In the latter, ribbons of vessels, arising from the rete, course through the white muscle (Carey and Teal, 1969b). Among tuna species, only bluefin, albacore, and bigeye have a visceral rete (Table 11).It is well developed, and visceral temperature is as high as the temperature of the swimming muscles (Carey et al., 1971). The single rete is located on the dorsal side of the liver and consists of small veins that intermingle with small arteries branching from the celiomesenteric artery (Carey et al., 1971). Billfish do not have a visceral rete. In contrast, porbeagle, mako, and great white sharks have paired visceral or suprahepatic retia, which are located on the ventral and lateral surfaces of the esophagus (Burne, 1923, Carey et

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al., 1971). Unlike the red muscle retia, the suprahepatic rete in sharks consists of a spongelike meshwork of arteries, which fills the lumen of a venous sinus. Warm venous blood from the hepatic circulation fills the sinus bathing the arterial rete. The venous sinus drains into the Cuvierian ducts. Cranial retia that protect the eyes and brain from heat loss are found in tuna, lamnid sharks, and billfish. Brain and eye temperature in bluefin tuna swimming in 20°C water can be as high as 27°C (Linthicum and Carey, 1972), and despite large fluctuations in ambient water temperature of 7" to 23"C, brain and eye temperature change by only 6" to 7°C. In tuna and lamnid sharks the heat-exchange retia are paired structures located dorsal and anterior to the first efferent branchial arteries (Stevens and Fry, 1971; Linthicun and Carey, 1972). Brain and eye temperatures of lamnid sharks are often 5°C above ambient water temperature (Block and Carey, 1985). A pair of cranial heat-exchange retia, consisting of an arterial plexus passing through a venous sinus in the orbit, is found in the porbeagle shark (Burne, 1923), salmon shark (Larnna ditropis), shortfin mako (Zsurus oxyrhinchus), longfin mako (1. paucus), and great white shark (Block and Carey, 1985). Recent evidence (Wolf et al., 1988) suggests that in lamnid sharks, brain metabolism alone is not sufficient to account for the reported brain and eye temperatures. Heat may be imported to the brain via a "red muscle vein," which carries blood 0.3"to 4.5"C warmer than ambient water temperature from the red swimming muscle to the plexus of veins in the membrane covering the brain (Wolf et al., 1988). In this respect, the carotid retia located in the crania of billfish, swordfish, and butterfly mackerel are associated with thermogenic tissues derived from skeletal muscles attached to the eyeball (Block, 1986).Although the heater tissue has few myofibrils and is incapable of force generation, it has many mitochondria, which are used for heat generation (Block, 1986, 1987; Block and Franzini-Armstrong, 1988). The paired carotid retia at the base of the heater organs are formed by extensive branching of carotid arteries and veins. Carotid artery blood is warmed as it passes through the retia and is delivered to a fine network of capillaries on the ventral surface of the retina (Block, 1986). B. Blood Flow in Heat-Exchange Retia The heat exchanger's effectiveness in trapping heat is determined, in part, by three factors; the shunt fraction (how much blood bypasses the exchanger), the surface area of the rete available for heat exchange, and rate of blood flow through the rete (Graham, 1983).A measure of

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P. G . BUSHNELL ET AL.

effectiveness that is often used is heat exchanger efficiency (H.E.), defined mathematically as

H.E. = Txm

-

Txvb

1xm

(4)

where T,, = the difference between red muscle and ambient water temperature and Txvb = the difference between the temperature of venous blood after passing through the heat exchanger and ambient water temperature (Brill et al., 1992). A high- and fixed-efficiency rete can be detrimental as well as advantageous. During bouts of strenuous activity, for example, the inability to shed heat can lead to overheating (Sharp and Vlymen, 1978).Because core temperature rises as a hnction of size, as well as activity (Neill et al., 1976), the distribution of larger fish could be limited to colder waters (Barkley et al., 1978). Finally, since heat exchangers reduce heat gain as well as heat loss between the fish and the surrounding water, cool endothermic fish returning to warm surface water after a vertical migration will warm at a much slower rate than nonendothermic fish (Brill et al., 1992). The control of heatexchange efficiency through blood-flow changes, therefore, has physiological, as well as ecological implications. The extent of blood-flow rearrangement necessary to significantly alter body temperature depends on heat-exchanger efficiency. A hypothetical model of steady-state (heat production equals heat loss) muscle temperature in tuna with countercurrent heat exchangers (Brill et al., 1992) shows that excess muscle temperature [T,,,,,Eq. (4)l is related to heat-exchanger efficiency in a highly nonlinear manner (Fig. 11)

H.E. =

(Txm- 0.42)

(5)

1 xm

(Graham, 1983; Brill et al., 1992). At high efficiencies (>95%),a 3% reduction in efficiency is predicted to result in a 6°C decrease in body temperature. Calculations based on body temperature and metabolism estimate heat-exchanger efficiency in skipjack and albacore tuna to be between 95 and 98% (Neill et ul., 1976; Graham, 1983). Direct measurements, however, show efficiency in kawakawa (Brill et d.,1992) and skipjack tuna (Stevens and Neill, 1978) to be only 70%. Owing to the flatness of the efficiency-excess temperature curve below 80% efficiency, large changes in efficiency would be required to cause significant changes in body temperature.

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Fig. 11. Hypothetical steady state (heatproduction equals heat loss) muscle temperature of a tuna with a range of counter-current heat-exchanger efficiencies. Redrawn from Brill et al. (1992).

Anatomical studies have shown that in most fish, alternate routes exist that allow blood to reach organ systems (red swimming muscles, brain, eyes, or viscera) without passing through retia. In fish with lateral heat exchangers, for example, cool blood from the gills can be delivered to the swimming muscles via dorsal aortic branches that are not associated with retial systems (Graham, 1975; Graham and Dickson, 1981). In tuna and swordfish that have centrally located heat exchangers and poorly developed or nonexistent lateral heat exchangers, blood shunted laterally instead of centrally effectively alters the thermal conductivity of the fish (Dizon and Brill, 1979a; Carey, 1990). Alternatively, whole-body thermal conductivity can also be changed by modifying the perfusion of red muscle relative to white muscle because, although blood flow to the white muscle passes through a small rete of its own, it is not as large and well developed as those surrounding the red muscle. Shunts bypassing arterial and venous sides of the suprahepatic retia also exist. The celio-mesenteric, lieno-gastric, and spermatic arteries

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act as shunt vessels around the arterial side of suprahepatic retia (Carey et al., 1981).The venous side of the rete can be bypassed via the hepatic sinus, a pathway that returns blood directly to the heart. The internal carotid arteries act as shunt vessels around the carotid retia in tuna and lamnid sharks, although the internal carotids are quite small (Block and Carey, 1985). The red muscle vein in lamnid sharks may also be used to regulate brain temperature, since warm blood flowing to the brain through this vessel can be diverted away from the brain to the lateral cutaneous rete (Wolf et al., 1988). Although it has never been demonstrated experimentally, changes in the relative resistance of retial and shunt vessels would be an effective mechanism for altering heat-retaining efficiency of the rete. Carey et at. (1981), for example, estimated that if venous flow through the hepatic sinus shunt (around the suprahepatic rete) increased from 0 to 20%,heat-exchanger efficiency would fall from 96 to 77%, thereby reducing visceral temperature from 8" to 1.5"C above ambient. The effective surface area of the heat-exchange retia could also be altered by changing the resistance of vessels within the retia. Experimental and anatomical studies suggest that catecholamines may be important in altering resistance in and around the retial vascular beds. Although no direct innervation has been demonstrated, both Stevens et al. (1974) and Dickson (1988) have confirmed the existence of smooth muscle in the walls of arterial retia in skipjack and albacore tuna. Injecting circulating catecholamines into skipjack tuna dramatically reduces heat exchanger efficiency (Brill et al., 1992), but the mechanism of action is unknown. Heat-exchanger efficiency is inversely proportional to blood flow through the retial system (Mitchell and Meyers, 1968).Theoretically, it is possible that in heat exchangers operating at close to 100% efficiency, additional heat generated by exercise might be dissipated by increased flow through the rete. Carey and Teal (1969a) reported that violent struggling by captured bluefin tuna reduced, rather than increased, deep body temperature, yet laboratory experiments have failed to demonstrate any relationship between body temperature and exercise in skipjack tuna (Dizon and Brill, 1979a). To date, there is no direct evidence that blood flow through, or bypassing, any retia occurs in response to external or internal perturbations. However, there is at least an anatomical basis for believing that fish with heat-exchange retia may be able to control rates of heat loss to, or heat gain fiom, the environment (i.e., phyysiologically thermoregulate). Unfortunately, even indirect laboratory and field evidence to support the concept of physiological thermoregulation is sparse be-

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cause changes in body temperature are difficult to interpret. Alternative explanations for the observed alterations in body temperature (i.e., changes in activity, metabolism, body size, thermal inertia) must be eliminated before physiological thermoregulation can be cited as an explanation. The above notwithstanding, physiological thermoregulation has been demonstrated, indirectly, using body-temperature measurements, in free-swimming bluefin tuna (Carey and Lawson, 1973),bige y e tuna (Holland et al., 1992),and swordfish (Carey, 1990).However, in at least one case (Carey and Lawson, 1973),the authors’ conclusions were challenged (Neil1and Stevens, 1974)because mathematical models of passive thermal inertia accounted for most of the change in body temperature. Evidence of physiological thermoregulation is often based on measures of the animal’s whole body thermoconductivity (k),an empirically derived parameter in the non-steady-state heat-transfer equation [Eq. (6)] which applies to cases in which heat production and heat loss are not equal and body temperature is changing

dTbldt = ( k X (Tb - T,)) + To

(6) where dTbldt = the instantaneous rate of body temperature change, k = an empirically derived parameter describing whole body thermal conductivity, Tb and T, = body and ambient water temperature, respectively, and To = the temperature increase due to heat production of the animal (Brill et al., 1992).This equation is oAen used to mathematically describe heat transfer (gain or loss) with the environment occurring when fish make large and rapid movements up and down in the water column. Figure 12 presents body-temperature data from free-swimming fish equipped with ultrasonic temperature and depth transmitters. As expected, body temperature in the blue shark (Fig. 12A), an elasmobranch lacking heat exchangers associated with the swimming muscles, closely follows changes in ambient water temperature. The swordfish (Fig, 12B),on the other hand, has a small lateral heat exchanger and warms approximately 10 times more quickly than it cools (Carey, 1990). Although k was not calculated, the data suggest that swordfish are making circulatory adjustments in blood flow to swimming muscles and heat-exchange retia that increase whole-body thermal conductivity as they rise through the water column into warm water, and reduce thermal conductivity when they return to the colder depths (Carey, 1990). In contrast to the fairly long-term (hours) physiological thermal regulation demonstrated by the swordfish in Fig. 12B, field data from

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P. G. BUSHNELL ET AL.

A 20

a v

18

!P

2 16 E

e

14 12 10

B

0600

1200 Time of day

1800

5 T ~ " " : " " ' " " ' ' ~ ' " " " ' ' ~ 1200 1800 2400 0600 Time of day Fig. 12. Muscle temperature of a blue shark, Prionace glauca (A), and swordfish, Xiphias gladius (B), measured telemetrically. (A) Muscle temperature (heavy line) in the blue shark changes rapidly with water temperature (dotted line). The blue shark is a poikilotherm and equilibrates with water after an hour or so, as is seen at the beginning ofthe record. Redrawn from Carey, (1982).(B) Muscle temperature (heavy line) cools at a slower rate than ambient water temperature (dotted line) in the swordfish, which has a small lateral heat exchanger. The muscle rewarms very rapidly when the fish returns to warm water. The rate of warming is more than 10 times as fast as the rate of cooling. When the fish reentered cool water on the second day, it again cooled very slowly. Redrawn from Carey, (1990).

bigeye tuna (Fig. 13; Holland et al., 1992) suggest rapid, short-term (minutes) changes in whole-body thermal conductivity. The rate of warming is approximately 50 to 200 times faster than of cooling (Fig. 13).Although metabolic heat (To)is expected to increase warming and decrease cooling rates, [see Eq. (6)]its contribution to body temperature was calculated to be only 0.02"C* min-', and therefore, insignifi-

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25

--

20 --

t

5 a

z

I-

15

--

0800

0900

1000 TIME

1100

1200

1300

OF DAY

Fig. 13. Records of water temperature (water) and swimming muscle temperature (muscle) in a bigeye tuna (Thunnusobesus) obtained telemetrically. As for the swordfish (Fig. 12,B),tuna cool much more slowly than they warm. The rapid warming and slow cooling curves generated by computer modeling (model, dotted line) closely match the observed data. Whole-body thermal conductivity [k,Eq.(S)] during warming (high k) and cooling (low k) can change by over two orders of magnitude. Redrawn from Holland et al. (1992).

cant. Computer modeling of body temperature fluxes (Fig. 13), which utilized a variable whole-body thermal conductivity, fitted the observed data quite well, indicating that k could change by as much as two orders of magnitude. Whereas field data suggest that physiological thermoregulation occurs, there is little supporting evidence from controlled laboratory experiments. Dizon et al. (1978) and Dizon and Brill (1979a,b) studied thermoregulatory behavior when temperature and activity were closely regulated or monitored. Under these conditions, twofold to fourfold changes in whole-body thermal conductivity were recorded in swimming skipjack and yellowfin tuna. The pattern of change in core temperature of restrained albacore tuna subjected to acute temperature change suggest that they are also capable of physiological thermoregulation (Graham and Dickson, 1981). Most recently, Dewar et al. (1991) and Brill et al. (1992) have minimicked the temperature changes encountered by tuna swimming

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P. G . BUSHNELL ET AL.

up and down in the water column in a large swim tunnel. Tunas with thermocouples inserted into red swimming muscle were exposed to abrupt step changes in temperature (3"to 5°C) while swimming at a constant speed [i.e., To,Eq. (6) is constant]. Under these conditions, thermal conductance changed rapidly, varying inversely with ambient water temperature (Dewar et aE., 1991; Brill et al., 1992).Whole-body thermal conductivity also changed when tuna were held at a constant temperature and briefly forced to swim at higher speeds. In this case, an increase in metabolism was reflected by an increase in body temperature. The rate of body temperature increase at 25°C was seven to eight times higher than at 30"C,which suggests that at 30"C,the additional heat load resulting from increased metabolism is being dissipated, perhaps through control of heat-exchange efficiency (Dewar et az., 1991). Results presented in this discussion suggest that physiological thermoregulation occurs in endothermic fish. This, presumably, is a result of changes in the heat-exchange efficiency of the retial systems resulting from alterations in blood flow through or around the retia. Unfortunately¶there is no direct evidence for neuronal and humoral control of blood Bow in retia. Future research will make an important contribution toward our understanding of thermoregulation effected through changes in blood flow.

ACKNOWLEDGMENTS

Work by the authors was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the British Columbia and Yukon Heart and Stroke Foundation, and the British Columbia Health Care Research Foundation.

REFERENCES Abel, D. C., Graham, J. B., Lowell, W. R., and Shabetai, R. (1986). Elasmobranch pericardial function. 1. Pericardial pressures are not always negative. Fish Physiol. Biochem. 1 , 7 5 4 3 , Abel, D. C., Lowell, W. R., Graham, J. B., and Shabetai, R. (1987). Elasmobranch pericardial function. 2. The influence of pericardial pressure on cardiac stroke volume in horn sharks and blue sharks. Fish Physiol. Biochem. 4,5-14. Axelsson, M., and Fritsche, R. (1991). Effects of exercise, hypoxia, and feeding on the gastrointestinal blood flow in the atlantic cod Gadus morhua. J . Exp. Biol. 158, 181-196.

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322,191-198. Vanhoutte, P. M., Rubanyi, G. M., Miller, V. M., and Houston, D. S. (1986).Modulations of vascular smooth muscle contraction by the endothelium. Annu. Reu. Physiol. 48,

307-320. Wahlqvist, I. (1980).Effects of catecholamines on isolated systemic and brachial vascular beds of the cod, Gadus m0rhua.j. Comp. Physiol. 137(B),139-143. Wahlqvisf I., and Nilsson, S. (1977).The role of sympathetic fibres and circulating catecholamines in controlling the blood pressure and heart rate in the cod, Cadus morhua. Comp. Biochem. Physiol. 57(C),65-67. Watson, A. D., and Cobb, J. L. S.(1979).A comparative study on the innervation and the vascularization of the bulbus arteriosus in teleost fish. Cell Tissue Res. 196,337-346. Wells, R. M.G., and Baldwin, J, (1990).Oxygen transport potential in tropical reef fish with special reference to blood viscosity and hemat0crit.J. Erp. Mar. Biol. Ecol. 141,

131-143. Wells, R. M. G., and Weber, R. E. (1991).Is there an optimal hematocrit for rainbow trout Oncorhynchus mykiss (Walbaum)? An interpretation of recent data based on blood viscosity measurements. J . Fish. Biol. 38,s-65. Wells, R. M. G., MacDonald, J. A., and Diprisco, G. (1990).Thin-blooded Antarctic fishes: A rheological comparison of the hemoglobin-free icefish Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii. j . Fish. BioE. 36,595-609. White, F. C., Kelly, T., Kemper, D., Schumacker, P. T., Gallagher, K. R., and Laurs, R.M.

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(1988).Organ blood-flow hemodynamics and metabolism of the albacore tuna Thunnus alalunga (Bonnaterre). E x p . Biol. 47,161-169. Wolf, N. G., Swift, P. R., and Carey, F. G.(1988).Swimming muscle helps warm the brain of lamnid sharks. J . Comp. Physiol. B 157,709-715. Wood, C. M. (1976).Pharmacological properties of the adrenergic receptors regulating systemic vascular resistance in the rainbow trout. /. Comp. Physiol. ( B ) 107,211228. Wood, C. M., and Shelton, G.(1975).Physical and adrenergic factors affecting systemic vascular resistance in the rainbow trout: A comparison with branchial vascular resistance. J. E r p . Biol. 63,505-523. Wood, C . M., and Shelton, G. (1980).Cardiovascular dynamics and adrenergic responses of the rainbow trout in uiuo. J . E x p . Biol. 87,247-270. Wright, G . M. (1984). Structure of the conus arteriosus and ventral aorta in the sea lamprey, Petromyzon marinus, and the atlantic hagfish, Myxine glutinosa: Microfibrils, a major component. Can. J . 2001.62,2445-2456.

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3 THE VENOUS SYSTEM GEOFFREY H . SATCHELL Department of Physiology Otago Medical School Dunedin, New Zealand

I. Introduction 11. The Capillaries A. Interchanges across the Capillary Wall: The Starling Principle B. The Two Pathways through the Capillary Wall 111. The Structure of Fish Veins IV. Blood Pressure in Fish Veins V. The Capacitative Function of the Venous System VI. Valves in Fish Veins VII. The Veins of the Somatic System A. The Hemal Arch Pump B. Pumps Powered by Respiratory Muscles: The Branchial Pump C. The Renal Portal System and Its Branches VIII. The Veins of the Hepatic Portal System IX. The Secondary Veins of the Skin A. Venous Pumps of the Secondary System: Median Fin Pumps B. The Caudal Pump C. The Caudal Heart of the Carpet Shark D. The Caudal Heart of the Eel E. The Caudal Heart of the Hagfish F. The “Cardinal Heart” of the Hagfish X. Discussion and Conclusion References

I. INTRODUCTION The veins of fish differ in how directly they return blood to the heart, and they can be grouped into three subsystems in this regard. Blood from somatic muscles, such as those of the myotomes in the trunk and the jaw muscles in the head, is returned on each side, into a transverse vessel, the ductus cuvieri, which channels these flows di141 FISH PHYSIOLOGY,

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rectly into the sinus venosus of the heart. The anterior and posterior cardinal sinuses or cardinal veins (Fig. 1A) are examples. In the trunk the paired posterior cardinal veins run beneath or between the kidneys, and behind these, the caudal vein is an important highway back to the heart, for blood from the myotomes of the postabdominal trunk and tail. A second and clearly defined vascular bed is that of the hepatic portal system. The term portal draws attention to the fact that at its proximal end this system is recruited from the many capillaries of the absorptive part of the gut, and at its distal end it terminates among the

c cs

K.

L.R.P.V.

C.V.

R.L.C.V. 1.l.C.V

I!c:v.

Fig. 1. The venous system of fish. (A) The somatic and hepatic portal systems of the lingcod Ophiodon elongutus. (B) The cutaneous veins of the stargazer, Urunoscopus scaber. A.C.S., anterior cardinal sinus; C. V., caudal vein; D.C.V., dorsal cutaneous vein; Du.Cv., ductus cuvieri; E., esophagus; F.V., fin veins; H.P.V., hepatic portal vein; Ic.V., intercostal vein; I.J.S., inferior jugular sinus; In., intestine; K, kidney; L, liver; L.L.C.V., left lateral cutaneous vein; L.R.P.V., left renal portal vein; Md.V., mandibular vein; Mx.V., maxillary vein; P.C.S., posterior cardinal sinus; P.Mes.V., posterior mesenteric vein; R.L.C.V., right lateral cutaneous vein; S.V., sinus venosus; V., ventricle; V.C.V., ventral cutaneous vein. Redrawn after (A) Allen (1905);(B) Trois (1880).

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thousands of liver sinusoids; the blood within the hepatic portal system cannot flow directly into the heart. Within the liver, soluble nutrients such as glucose and amino acids pass into the hepatocytes where they are processed into glycogen and proteins. Blood that has passed through the sinusoids is collected into two to four short vessels, the hepatic veins (Fig. 1A). These open directly into the sinus vgnosus, thus bypassing the ductus cuvieri, which lie more laterally. The hepatic portal system is a separate circulation confined within the boundaries of the abdominal cavity, although, as we shall see, some teleosts have connections that can allow caudal vein blood to enter it. The third subsystem comprises veins that drain the skin ofthe body and fins, and of some internal surfaces such as the buccal and opercular cavities. It also includes certain veins draining the gills. The most visible are the four longitudinal vessels (Fig. lB), the dorsal, lateral, and ventral cutaneous veins, which run the length of the trunk beneath the dermis, and extend onto the tail. Blood in these veins has been derived from the capillaries of the secondary blood system; some of this blood is pumped back into the caudal vein by the caudal heart or caudal pump, but in elasmobranchs and some teleosts, there is also a rostra1 connection with the ductus cuvieri. These three systems differ, not only in the vascular beds they drain, the presence and distribution of valves within them, and the blood pressures they sustain, but also in the histology of their vessel walls (Fig. 3). Before we discuss these topics however, it is necessary to consider something of the structure and dynamics of the vascular beds from which blood enters the veins, i.e., the capillaries.

11. THE CAPILLARIES

The capillaries (Fig. 2), in fish as in higher vertebrates, are the smallest of all the blood vessels; they are the vessels of that part of the circulatory system specialized for diffusion and exchange of substances between the blood and the tissues. The overall architecture of the system facilitates this, because each arteriole gives rise to many capillaries, and the total cross-sectional area of the capillary bed increases several hundredfold. There is a corresponding reduction of the velocity of flow, and this provides a longer time for diffusional exchanges to proceed toward completion. Endothelial cells are much flattened (Fig. 2A); only at the nucleus are they appreciably thickened. Some fish such as the pond loach (Misgurnus fossilis), which live in

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09

@

E.C\.A.C.

CV.

B.H

E.C.V.C.

Fig. 2. The structure of capillaries. (A) A capillary from the intestine of the spiny dogfish, Squalus aconthias. (B) A capillary from the respiratory intestine of the pond loach Misgurnusfossilis. (C) A reconstruction of the membrane of a cerebral capillary of the Atlantic hagfish, Myxine glutinosa. (D)A wall of a capillary of the Port Jackson shark Heterodontus portusjacksoni showing desmosomes and intercellular clefts. (E)A wall of a venous capillary of a rete from the gas bladder of the common eel Angullla anguilla, showing pinocytotic vesicles. B.M., basement membrane; Cp., capillary; D., desmosome; E.C.A.C., endothelial cell of arterial capillary; E.C.N., endothelial cell nucleus; E.C.V.C., endothelial cell of venous capillary; En.C., endothelial cell; Ep.C., epithelial cell; Fn., fenestra; I.C., intercellular cleft; O.T.I., openings of tubular invaginations; P.V.,pinocytotic vesicles; T.I.Tubular invaginations. Redrawn after (A) Rhodin and Silversmith (1972);(B) Jasinski (1973);(C)Bundgaard (1987); (D)Casley-Smith and Mart (1970); (E) Stray-Pedersen and Nicolaysen (1975).

water that may, through the decay of organic materials, come to have a low oxygen tension, have their posterior intestine modified as an auxillary gas-exchange organ. A network of large capillaries exposes blood to a reservoir of air, gulped from the surface. The nuclei in their endothelial cells are located uniformly at their bases (Fig. 2B) where they do not increase the thickness of the gas-exchange surfaces, and both endothelial cell and intestinal epithelium are very thin (Jasinski, 1973). A capillary consists of a single layer of endothelial cells rolled up to form a tube 4-10 Fm in diameter and commonly 0.2-1 mm long. Underlying the endothelial layer is a basal membrane (Fig. 2A),which

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at high magnification is seen to consist of three layers. The middle layer has many 2- to 5-nm fibrils in it, and larger 10-nm elements occur in the outer layer (Bendayan et al., 1975). In the capillaries of the intestine, kidney, and pancreas of elasmobranch fish, the basal membrane is anchored into the surrounding tissue by strands that extend radially outward from it, and these may help the vessel to withstand subambient pressures (Casley-Smith and Mart, 1970). In some air-breathing fish, such as the climbing perch, Anabas testudineus, gas exchange occurs through islets of tissue lining the suprabranchial chambers, which can be ventilated. The capillaries of the respiratory islets have a unique structure in that each endothelid cell of the transverse channels has a tonguelike extension that points downstream and may serve to deflect red blood cells upward against the respiratory surface (Munshi et al., 1986). The tonguelike cells are rich in mitochondria and have a large area in contact with the plasma; the authors suggest that they may be involved in the metabolism of catecholamines. Certain endothelial cells lining small venous sinuses in the interlamellar network of rainbow trout have been shown to bind labeled norepinephrine (Nekvasil and Olson, 1985). Axial alignment of endothelial cells is characteristic of vessels with high mean linear-flow velocities, and is seen in the afferent gill respiratory pathways of many fish. Endothelial cells of the small vessels of the venous interlaminar network have an irregular orientation, suggesting that this is a low-pressure, low-flow pathway (Olson, 1981). The scanning electron microscope (EM) studies of the filamental central canal in the toadfish Torquiginer glaber (Cooke and Campbell, 1980) suggest that the endothelium is lacking altogether in places, and extracellular tissue fluid may flow directly into such vessels. Certain ultrastructural features have been supposed, by earlier authors, to increase permeability. Much of our knowledge of these features and of the dynamics of diffusional exchanges have been derived from a study of the retial “capillaries” of the fish swimbladder. This gas-filled buoyancy organ has in its walls, a gas gland and one or more retia mirabilia. The latter consist of closely packed arrays of arterial and venous vessels of capillary dimensions in which each venous vessel is surrounded by a ring of three to seven vessels. These so-called capillaries are of great length. In the swimbladder of the eel they may be 4 mm long, and the vessels leading to and from them are large enough to be canulated. They have been much used in functional studies. The endothelial cells of arterial capillaries are relatively tall, 2-4 pm high, whereas those of the venous capillaries are less so, 0.2-1.0 pm high. A thin layer of adventitial tissue separates

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their adjacent basement membranes (Stray-Pedersen and Nicolaysen,

1975). The endothelial cells of the cerebral capillaries of hagfish (Bundgaard, 1987) (Fig. 2C)contain many tubular invaginations of the surface membrane. Beneath these lie smooth-surfaced cisternae, many of them similar to the sarcoplasmic reticulum of smooth muscle cells. The reconstructions demonstrate clearly, however, that these invaginations are not the openings of transendothelial passages. Bundgaard (1987) suggests they may be sites of ingress of Ca2+, and the presence of smooth-surfaced cisternae nearby recalls a similar association in smooth muscle. Other investigations of the blood-brain barrier of the hagfish, a site that, as in higher vertebrates, is known to be impermeable to large molecules, show that radiolabeled polyethylene glycol (4000 daltons) and microperioxidases remain confined to the capillary lumen (Bundgaard and Cserr, 1981). Similar invaginations occur in teleost arterial retial capillaries (Bendayan et GI., 1975).When gold is coupled to albumin and localized on the capillary wall with albumin antisera (Bendayan, 1980),it can be seen to penetrate these intuckings, but it does not pass through the endothelial cell. Venous retial endothelial cells of the eel have fenestrations closed by a diaphragm (Fig. 2D); they are 20-80 nm wide, and the diaphragm is 5 nm thick. They tend to occur where the arteriovenous barrier is thinnest and to be arranged in groups of three to five. Fenestrations have been thought to facilitate transendothelial movement. Open fenestrations may be present in glandular organs and have been reported in the pineal gland of the pike (Owman and Rudeberg, 1970) and the gas gland of the perch (Jasinski and Kilarski, 1971). Prominent among the ultrastructural features of endothelial cells are the pinocytotic vesicles (Fig. 2E); they are abundant in the endothelial cells of certain dogfish tissues (Rhodin and Silversmith, 1972). These structures facilitate, it is believed, transport across the endothelial cell. Earlier studies suggested that vesicles are tucked into the cell by endocytosis, transported through the cell and extruded to the opposite side by exocytosis. Some workers claimed to have seen a front of labeled vesicles moving progressively across the endothelial cell membrane. Bundgaard (1987) reports that ultrathin serial sectioning shows many of these profiles to be sections through the tubular invaginations previously noted. Van Deurs et al. (1989)in a general review of endocytosis, summarizes the doubts that are now held as to the importance of pinocytosis and suggest that only a small percentage of vesicles are involved in transcellular movements at any one time.

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While pinocytotic vesicles and fenestrae may sometimes provide for a transcellular movement of substances, larger molecules can enter or leave the capillary through spaces between the ends of endothelial cells. In arterial retial capillaries of the eel, shrunk by perfusing them with hyperosmotic solutions, these junctions are seen (Fig. 2D) to be held together at many points by desmosomes (Rasio et al., 1977,1981). They possess a line a dense material in the intercellular space, and this provides a firm adhesion of the two membranes. Ultrathin (18 nm) serial sectioning has shown that tortuous pathways span the capillary wall between these points of fusion (Bundgaard, 1987) and, it is believed, provide a paracellular pathway into and out of the capillary. In elasmobranchs the junctions between the intercellular clefts of the cerebral endothelial cells allow horseradish peroxidase to pass through to the basement membrane (Brightman et al., 1970). It also must not be assumed that permeability of endothelial cells is the same along the length of a capillary. Cultured endothelial cells from the pulmonary veins of sheep (Vecchio et al., 1987) are more permeable than those from its pulmonary artery. A. Interchanges across the Capillary Wall: The Starling Principle

The movement of water across the capillary wall depends on a balance between three variables (Michel, 1988).At the arteriolar end of a capillary, the hydrostatic or blood pressure will be higher than at the venous end and will tend to force water through the capillary wall into the surrounding tissue spaces. This permeability to water differs from one capillary to another; it is a property of the capillary wall and is termed the hydraulic permeability, Lp. It is defined as the flow of water across a unit area of capillary wall per unit difference of hydrostatic pressure. This osmotic or hydrodynamic flow is independent of the movement of substances by diffusion. Substances such as the plasma proteins, in solution in the plasma, may have molecules too large to pass freely through the wall. They will, to a greater or lesser extent, exert a colloid osmotic pressure, COP, which acts to oppose L, and draws water back into the capillary. To such substances, the term diffusional permeability P d is appropriate. P d is a coefficient applied to a particular substance; it is defined as the mass transport of a substance per unit area, per unit of concentration difference under conditions when the flow through the wall is zero. Related to this is the reflection coefficient cr, which indicates the proportion of the substance that remains within the capillary, i.e., is reflected back into it. If, through

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features of molecular size, charge, or some property of the endothelial cells, the wall were completely impermeable to the substance, u would equal 1. If the substance passed through the wall without hindrance, u would equal 0, and the substance would exert no COP. The hydrostatic pressure at the arteriolar end of the capillary in mammals is some 40 mm Hg, which exceeds the COP of 25 mm Hg; thus fluid is forced through the capillary wall into the tissue spaces. The resistance of the capillary reduces the hydrostatic pressure to some 15 mm Hg, i.e., lower than the COP, so that at the venule end, fluid tends to flow back into the vessel. The fluid part of the blood that passes along such pathways may be 60%of the cardiac output. We owe to Starling (1866-1927) this hypothesis of fluid exchange across the capillary wall, and we may enquire what values of arteriolar and venule hydrostatic pressure and colloid osmotic pressure are appropriate to apply in fish. In mammals it is known that some 50% of the arterial pressure is lost across the arterioles, and a pressure at the arteriolar end of a capillary of a cod is thus likely to be around 13 mm Hg (Wahlqvist and Nilsson, 1977, 1981). The COP of cod blood is known to be 8 mm Hg (Hargens et al., 1974). A capillary pressure of 2 mm Hg at the venule end of the capillary may be estimated from the caudal vein pressures in other teleost fish such as the stary flounder (Platichthys stellatus), determined by Wood et al. (1979). All three pressures are very much lower than those in mammals, but they are in much the same proportion. That the capillary wall is partly permeable to the plasma proteins, and that u is less than 1, is suggested by the observations of Forster et al. (1989) on the hagfish Eptatretus cirrhatus. When its blood volume was assessed with "Cr-labeled red cells, the volume of distribution did not increase markedly after 4 hr. When it was assessed with radiolabeled human serum albumin, '251-HSA, the volume of distribution was 110-150 ml/kg-' after 2 hr, but this increased to 200 ml after 6 hr and to 285 ml after 20 hr. The data suggest that the label escaped steadily from the blood into the extracellular fluid. Casley-Smith and Casley-Smith (1975) report that hagfish capillaries are fenestrated and permeable to molecules the size of human serum albumin.

B. The Two Pathways through the Capillary Wall The study of these three coefficients, Lp, Pd, and CT, of capillaries in the mesenteries and muscles of higher vertebrates, confirms that there are two pathways through the capillary wall: a transcellular pathway

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through the endothelial cells, and a paracellular pathway that traverses the clefts between them. Such capillaries show a high value of (T for albumin, suggesting that most of it is held back in the capillary, and a value for L,, which is too high if it is assumed to traverse the same pathway as the protein. Water, it seems, has a pathway available to it that the albumin does not traverse. Stray-Pedersen and Steen (1975) reported high values of L,, for tritiated water in the retial capillaries of eel. Rasio et al. (1977)suggested that, in fish as in other vertebrates, the entire inner surface of the capillary wall is available for the passage of water, while larger molecules such as albumin can pass only through the intercellular clefts. Permeability to water and to larger molecules in eel retial capillaries is very much enhanced if they are perfused with solutions that lack the plasma proteins (Myhre and Steen, 1977). These are believed to organize the molecules of fibrous protein of the endothelial cell membrane into a more regulated lattice that reduces the number of larger interstices between the fibrous elements (Michael, 1988). Blood in the veins draining the somatic muscles is likely to have lost much of its oxygen; carbon dioxide and various tissue metabolites such as lactate may have been added. We may expect that the blood in the hepatic portal veins, and thus in the gut microcirculation, will be at a higher pressure, for an additional vascular bed, that of the liver sinusoids, intervenes between its downstream end and the vis-hfronte of the heart. It will in addition carry the nutrients such as glucose, amino acids, and fatty acids that entered it from the gut. The secondary blood system will be discussed in detail in Chapter 4,but we can note briefly here that the narrow, coiled, anastomotic vessels that link it to the gill arteries and dorsal aorta may have a higher resistance than the arterioles of the primary system, and pressure in its microcirculation may be quite low. Moreover, the vascular beds in which the secondary capillaries are most concentrated, i.e., those of the skin and gills, face into the environment, and the blood draining from these into the secondary veins may have altered concentrations of the respiratory gases and of ions and water. In mammals it is known that endothelial cells contain filaments of actin and myosin and can contract. Smirnov et al. (1989) have shown that this contraction is regulated by Ca" phosphatidylserinedependent protein kinase C and cyclic adenosine monophosphate (AMP). Endothelial cells undergo rapid and reversible changes in shape, and their actin bundles disappear when their adenylate cyclase is activated with forskolin. Endothelial cells have in their membranes stress-sensitive ion channels which are opened, it has been suggested,

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when excited by tensions generated by flow. This is accompanied by an inward K+-selective ion current, which hyperpolarizes the endothelial cell membrane (Olesen et al., 1988).In larger vessels, endothelial cells secrete EDRF (endothelium-derived relaxing factor), which can relax smooth muscle. The permeability of endothelial cells can change very rapidly, and it may be that such changes of flow activate the actidmyosin filaments that pull on the ends of the endothelial cells and increase the diameter of the passages within the clefts (Crone, 1986,1987).While we do not yet know that these mechanisms occur in fish capillaries, they direct us to the view that the endothelial cell layer is a dynamic and responsive tissue. 111. THE STRUCTURE OF FISH VEINS

The walls of veins in higher vertebrates commonly display the same three layers, intima, media, and adventitia, that occur in arteries. They differ from arteries in that overall, the wall is much thinner and the media has less smooth muscle and more collagen fibers in it. Such a vessel can be dilated to its full volume by a relatively small transmural pressure; its pressure-volume curve has an initial steeply rising segment and lies to the left of that of comparable arteries; hence, its volume approaches maximum with quite small transmural pressures. Some of the main veins of the hepatic portal system of elasmobranchs exhibit this structure, and Rhodin and Silversmith (1972) have described the wall of a large intestinal vein of the spiny dogfish Squalus acanthias. The thin media shows a continuous layer of smooth muscle. In the adventitia are many large bundles of collagen fibers, and this layer composes more than half of the wall thickness. Neuville (1901) reports that the intraintestinal vein, a vessel specifically draining the spiral valve of the absorptive intestine, has elastic and collagenous elements and smooth muscle fibers in its wall. In the hepatic portal veins of the carp (Cyprinus carpio), however, smooth muscle is entirely lacking (Amlacher, 1954), but there is a well-developed collagenous adventitia (Fig. 3B). The histology of the caudal vein and posterior cardinal vein have been little investigated. Allen (1908)figured the caudal vein of Lepistosteus osseus (Fig. 3C) and specifically comments that it consists only of an endothelium. A loosely organized connective tissue surrounds the vein and binds it to the wall of the hemal canal. His figures of the branchial veins of Polyodon (1907)show a fibrous wall in which are just a few smooth muscle cells. Dornesco and Santa (1963) briefly describe

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Fig. 3. The structure of veins. Transverse sections of (A) the hepatic vein; (B) the hepatic portal vein of the carp, Cyprinus carpio; (C) the caudal vein and hemal canal of the garpike, Lepisosteus osseus; (D) longitudinal section through the hepatic sphincter of the spiny dogfish; (E) transverse section of the hepatic sphincter of the big skate Raja binoculata. Ad, adventitia; C.T., connective tissue; En., endothelium; H. C., hemal canal; H.V., hepatic vein; S.M.C., smooth muscles cells; S.V., sinus venosus. Redrawn after (A,B) Amlacher (1954);(C) Allen (1908);(D,E) Johansen and Hanson (1967).

(Fig. 3A,B) the histology of the veins and of the carp and note that the caudal vein and the free wall of the posterior cardinal sinus are very thin, have an endothelial lining and an investment of collagenous fibers, among which are scattered a few smooth muscle cells and elastic fibers.

IV. BLOOD PRESSURE IN FISH VEINS The pressures given in Table I have been collected from the literature. They have inevitably been derived from rather few species of fish. Moreover, almost all ofthem have been obtained from resting fish. We might well expect that the operation of the hemal arch pump, to be described later in this chapter, and of various other venous pumps actuated by fin movement, would facilitate venous return and increase

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Table I Venous Pressures in Fish System Somatic veins (Subsystem 1) Port Jackson shark Posterior cardinal vein Spiny dogfish Posterior cardinal vein Carpet shark Caudal vein Starry flounder Caudal vein (Platichthys stellatus) Hepatic portal system (Subsystem 2) Spiny dogfish Hepatic portal vein (Squalus acanthias) Trout Ventral intestinal vein (Salrno gairdneri) Common cardinal vein Secondary veins (Subsystem 3) Port Jackson shark Lateral cutaneous vein

(Heterodontus portusjacksoni) Carpet shark (Cephaloscyllium isabella) Lingcod (Ophiodon elongatus)

Lateral cutaneous vein, caudal end Small veins in gills

Pressure (mm Hg)

Reference"

-2.0 f 1.2 -0.4 to +0.2 + 1.6 +2.1 0.13

*

+ 1.7 to + 1.9 +6.9 to +9.9 +1.4 0.3

*

Central end -3.1 to -1.7 Caudal end +0.6 to +1.2 -2 to -3

+4.6to +6.5

(3)

" (1)Satchell (1971);(2) Satchell and Weber (1987);(3)Farrell and Smith (1981);(4) Stevens and Randall (1967);(5)Wood et al. (1979);(6) Kiceniuk and Jones (1977).

central venous pressure. Farrell (1984) reports that central venous pressure in exercising trout rises by around 1 mm Hg, a change in conformity with the view that flow into the central veins and cardiac output increases. However, Kiceniuk and Jones (1977) noted that pressure in the right common cardinal vein of resting rainbow trout was 1.4 +- 0.3 mm Hg and increased by only an insignificant amount, to 1.8 mm Hg, when they were swum at critical velocity. No increase in venous pressure was obtained at intermediate speeds. Wood et al. (1979) in an investigation of the effects of anemia on starry flounder (Platichthys stellatus),reported that caudal vein pressure rose from 2.1 +- 0.13 mm Hg in normal fish to 3.0 ? 0.4 mm Hg in severely anemic fish. This presumably reflected the fact that severe anemia caused the cardiac output to double. Nevertheless, it seems clear that, apart from

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the hepatic portal system, venous pressures are very low and close to ambient pressure, and in some vessels are below ambient. V. THE CAPACITATIVE FUNCTION OF THE VENOUS SYSTEM

The heart is mechanically coupled to the blood vessels, and this coupling dictates that circulatory parameters can alter cardiac output, in addition to features of the heart itself, such as its contractility and its rate of beat. Active changes in the muscles of the vessel walls may effect shifts of blood into the central veins carrying blood to the heart. Our present understanding of this coupling is derived from mammalian studies such as those of Caldini et al. (1974) and Green and Jackman (1984). These authors and others have used models in which two compartments, i.e., vascular beds, with different capacitances and different compliances are in parallel and each is in series with the heart. The terms splanchnic compartment (s) and peripheral compartment (p) have been used; the latter includes all of the vessels other than those of the viscera. Capacitance, in this sense, defines the total contained volume of the vessels at a given transmural pressure within the pressure range of the species concerned. Their compliance is the ratio of change of volume caused by a change in this transmural distending pressure, i.e., AVIAP (Rothe, 1983). In mammals, blood flows from the veins into the right heart; its myocardium contracts more vigorously when stretched, and an increase in central venous pressure may thus cause an increase in cardiac output. As inflow from the veins occurs at rates proportional to the difference between their upstream driving pressure and the back pressure in the heart, such a rise in right heart pressure will also tend to diminish inflow (Caldini, 1974), a change that would offset the rise in cardiac output. It is this balance between opposing tendencies that makes the right heart, in mammals, the region where coupling between the heart and the blood vessels is primarily effected. The pressure within a venous compartment depends on the ratio between the volume of the veins when stressed and their compliances. In mammals the greatest vascular volume is contained in the small veins (Drees and Rothe, 1974).When the resistance of the arterioles of the splanchnic compartment increases, blood flow through the capillaries and veins is reduced, the transmural pressure in both falls, and the volume of blood contained in them diminishes. Blood leaves the

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splanchnic compartment, because of the passive elastic recoil of its walls, passes through the heart, and is redistributed to the peripheral compartment. The effectiveness of this transfer from one to the other depends on differences in the compliances of their vessel walls. It is likely that the time constant T, i.e., the product of the sum of the arteriolar and venous resistances and the capacitances, of the two circuits ( R A s + Rvs) x CSand ( R A P + Rvp) x Cp, will differ. It is known that in dogs the time constant in the splanchnic compartment, TS is three times that of the peripheral compartment, TP (Green, 1977). To what extent, we may ask, do such interpretations help us understand the capacitative functions of the venous system of fish? Adrenergic fibers are known to innervate the vascular smooth muscle of the gut of many teleosts (Nilsson, 1984). Smith (1978)and Wood and Shelton (1980) have shown that a neurally mediated vasoconstriction is normally present in trout and increases when they are exercised; these studies do not locate this vasoconstriction specifically in the splanchnic or peripheral vascular beds. However, when radiolabeled microspheres are injected into trout, their distribution shows that during exercise there is an increased flow of blood to red muscle and a decreased flow to the liver, spleen, and stomach. It thus seems likely that, in fish as in mammals, a neurally mediated, arteriolar reduction of flow to the gut leads to a fall in transmural pressure, and, by passive elastic recoil, a transfer of blood from the splanchnic to the peripheral vessels. The extent to which this can occur will depend on the magnitude of the difference between 7s and TP for a change in compartmental flow, Qv, is inversely proportional to the time constant, i.e., Qv = AV/r. If we assume that TS is three times TP, a reduction of flow by AV in the splanchnic compartment would diminish flow in it only by AV/3, but would increase flow in the peripheral compartment by AV/l. The increase in flow in the peripheral compartment would be three times as great as the decrease of flow in the splanchnic compartment. We do not as yet know what values to ascribe to TSand TP in any fish. The study by Thorarensen et al. (1991)of the vasculature of the gut of the chinook salmon shows that the ventral intestinal and dorsal intestinal veins form a complex interdigitating system of featherlike venules, and the hepatic portal vein within the liver is so extensively branched that castings of it come to have the general shape of the liver. The large volume of blood contained in such an extensive vascular bed suggests at least a potential for capacitative shifts. In fish an additional feature is evident. The part of the hepatic vein that lies outside the liver and carries blood through the pericardioperi-

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toneal diaphragm to the sinus venosus is exceedingly short. It is surrounded by a prominent sphincter of smooth muscle, the hepatic sphincter (Fig. 3D,E).Amlacher (1954)reports that in carp the hepatic vein has blocks of smooth muscle in the medial layer (Fig. 3A). The margins ofthe openings are partly tethered by the collagenous tissue of the pericardioperitoneal diaphragm, and the radial tension that this will provide must act to oppose the constrictor action of the sphincter. Johansen and Hanson (1967) report that in the spiny dogfish S. acanthias, 10 ,ug ml-l of adrenaline relaxes the sphincter if it has previously been caused to contract by acetylcholine in the same concentration. We know that in elasmobranchs catecholamines are liberated into the blood during exhaustive exercise (Butler et al., 1986). In mammals too, @ activation of hepatic vein smooth muscle causes it to relax (Green 1977). A rise in central venous pressure must diminish the gradient between the splanchnic compartment and the heart and diminish flow; relaxation of the hepatic sphincter by lowering resistance will allow blood to leave the splanchnic circulation despite this. Thus the outflow of the whole of the hepatic portal circulation into the heart has an additional regulatory mechanism at this, its most distal point. The hepatic sphincter appears to be strategically placed to provide a means of changing the conductance of the splanchnic veins independent of changes in the filling pressure of the heart. In the previous paragraphs stress has been laid on the fact that the changes in CS and Cp are passive changes effected primarily by the smooth muscle ofthe upstream arterioles. What, we may ask, is the role of the layer of innervated smooth muscle so commonly present in mammalian veins. In mammals it has, it seems, a specific part to play in the response to changes in blood volume. When dogs are rapidly hemorrhaged sufficient to reduce their blood pressure to 50 mm Hg, the lost volume is, within 5 min, compensated in roughly equal amounts by the passive elastic recoil outlined above, by venoconstriction and by transcapillary fluid shifts (Rothe, 1983). Most of the venoconstriction occurs in the 10-30 sec after a volume change (Rothe and Drees, 1976). Venoconstriction in mammals is also of importance in the capacitative adjustments that are necessary when the animal changes its position in the gravitational field. The paucity of smooth muscle in the peripheral veins of fish suggests that venoconstriction is of little importance, and such smooth muscle as does occur in the splanchnic veins is not known to be innervated. The veins of the peripheral compartment are for the most

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part reduced to simple conduits, the compliance of which cannot be altered. A change in the capacitance of these vessels must therefore be largely passive. Veins close to the heart are, in some fish, swollen to form sinuses that provide a volume reservoir called on during pump diastole, but the walls of such sinuses have little or no smooth muscle.

VI. VALVES IN FISH VEINS Blood has weight, and the long vertical columns of blood extending, for example, from the right atrium of humans down to the foot, will increase the pressure at the venule end of the capillaries and favor outflow from them. Capillaries can withstand much higher pressures than those to which they are normally subject, but an excess of outflow over inflow, leading to edema, will eventually occur if raised vascular pressure is prolonged in the absence of movement. In land vertebrates (Fig. 4A) this is largely prevented because valves extend across the lumen of the long veins of the limbs, and even small movements of the limb muscles adjacent to them compresses them and cause the valves distal to the compression to close, and the blood to be moved toward the heart. Hence the long columns of blood become segmented into a series of shorter lengths, and as long as there is any movement of muscles in the limb, the capillary beds in the extremities are spared the increased pressure. Such valves are termed parietal valves (Franklin, 1937),and they are found only in veins that can be subject to pressure from without. In the fetus they arise around the time that involuntary movements of the limb muscles commence; the valves of the heart have arisen much before this. Parietal valves are to be distinguished from ostial valves, which occur in the mouths of tributary veins at the point where they open into a main vein (Fig. 4). When movement occurs and muscles contract, their vascular beds are compressed by the activity; blood is expressed from them, past the ostial valve and into the longitudinal vein. Earlier writers reported that the veins of fish lack valves; they were wrong, but it is certainly true that parietal valves are seldom, if ever, present. In the longitudinal veins of all three subsystems (Fig. 4B), there are no valves with the ability to segment the column of blood, as it moves forward to the heart, into a series of separate lengths. The flow of blood in such veins may be pulsatile, owing to the fluctuations of cardiac suction, and the activity of auxillary pumps, but it does not follow the stop-start pattern of flow to be observed in the veins adjacent to the long bones of a human when walking. Ostial valves, in contrast, are

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common in fish veins. They are very evident at the junctions of the intercostal veins with the caudal vein and renal portal veins. They occur commonly in the various auxillary venous pumps shortly to be described. With this general introduction completed, it is time to look at particular features of the three subsystems of veins. VII. THE VEINS OF THE SOMATIC SYSTEM

Blood flowing from the serial array of myotomes that extends the length of the body and comprises 40-65% of its weight must be returned to the heart. A basic problem besets this return, for in all but a few fish that swim with their fins, forward motion is derived from the backward movement of sigmoid flexures that pass along the length of

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the trunk toward the tail. The backward moving wave of tissue pressure that this generates would tend to propel blood away from, rather than toward the heart. Forward flow is facilitated by the provision of the hemal arch pump, which exploits the pressures generated by the myotomal contractions. A. The Hemal Arch Pump

In that region of the spine that lies behind the abdominal cavity and which in many fish is between a third and a half of its length, ventral extensions of the vertebrae unite below the spine to enclose the hemal canal (Fig. 5). In this lies the caudal artery (an extension of the dorsal aorta), the caudal vein, and paired secondary veins. The caudal vein extends from the tip of the tail to the commencement of the abdominal cavity (Fig. lA), where it divides into the two renal portal veins. A neural and hemal artery arise from the aorta at each alternate vertebra, and a neural and a hemal vein unite to empty into the caudal vein beneath the vertebrae between the veins (Allen, 1908). The caudal

v. c.v Fig. 5. The hemal arch pump of the Port Jackson shark. (A) General view; the left and right sides portrayed as if at the same level; (B) detail of arterial valves; (C) detail of venous valves. A.V., arterial valves; C.A., caudal artery; C.V., caudal vein; D.C.V., dorsal cutaneous vein; Ic.A., intercostal artery; Ic.V., intercostal vein; L.C.V., lateral cutaneous vein; Nch., notochord; N.C., nerve cord; V.C.V., ventral cutaneous vein; V.V., venous valves. Redrawn after Satchel1 (1965, 1971).

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vein thus lies in the midline, a region in which it is least likely to be compressed by the contracting myotomes, and it is additionally sheltered by the bony or cartilaginous hemal arch. Blood from the capillaries of, and the small veins within and between the myotomes, enters the caudal vein via the so-called intercostal veins (ribs may or may not be present), and at their openings prominent ostial valves occur (Fig. 4C).The mechanism is such that no matter which region of the column of myotomes is contracted, or in what sequence, blood from it will be injected into the caudal vein in which it can only flow forward to the abdomen. In some elasmobranchs the mechanism is made additionally effective by the occurrence of valves in the openings of the intercostal arteries (Fig. 5A).These valves are directed so as to prevent blood from returning to the dorsal aorta when the vascular bed is compressed. In resting fish, deflections of the trunk (Fig. 6A) either to right or to left, causes pulselike elevations of caudal vein pressure. When the perfused postabdominal trunk of a Port Jackson shark was caused to undergo swimming movements b y electrical stimulation of its spinal cord (Satchell, 1965), the flow of blood from the caudal vein increased by 46% (Fig. 6B). When the blood enters the renal portal veins, the continued operation of the hemal arch pump propels it onward, through the capillary beds around the renal tubules into the posterior cardinal veins and

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Fig. 6. (A) Changes in vascular pressures (in mm Hg), caused by trunk flexion in the Port Jackson shark, evoked by touching either the left or the right side of the snout. Upper trace, pressure in dorsal aorta; middle trace, pressure in caudal vein; lower trace, movement of trunk. (B) Volume of perfusate flowing from the caudal vein in the isolated perfused post pelvic trunk of a Port Jackson shark. Upper trace, movement; lower three traces, flow A, before, B during, and C after 1 min of swimming movements evoked by electrically stimulating the spinal cord.

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forward via the ductus cuvieri to the heart. Within the abdomen, forward flow is minimally hindered by pressures generated by swimming, for the extent of lateral deflection of the trunk at this level is less. The sigmoid waves of movement increase in amplitude and decrease in wavelength as they move toward the tail, and the viscera protect and shelter the vessels. Moreover, the returning blood is, at this level, subject to the uis-ci-fronte of the heart. In some fish such as the shark Centrophoms calceus (Woodland, 1906) and the freshwater teleosts Perca, Luciperca, and all the salmonids (Jourdain, 1859; Einstmann, 1913), the renal portal veins pass straight through the kidney and are continuous with the cardinal veins. The hemal arch pump overcomes the haemodynamic problem of how to use a backward-moving wave of muscular compression to achieve a forward movement of blood. It is an example of a propulsor, i.e., a pump powered by muscles that primarily serve some other end, such as ventilation or locomotion. Propulsors contrast with hearts, which are pumps powered by their own intrinsic muscles. The hemal arch pump is probably an ancient mechanism; the hemal arch is an essential part of it, and arches are visible in the skeletons of such fossil sharks as Xenacanthus (late Devonian to Triassic), which extend back some 400 million years.

B. Pumps Powered by Respiratory Muscles: The Branchial Pump The return of blood from the head is not beset with the difficulties that arise owing to the remoteness of the vascular bed from the heart. In its great veins the uis-u-fronteof the heart is evident, for both the anterior cardinal sinus and the inferior jugular sinus, which drain the dorsal and ventral parts of the head respectively, open into the ductus cuvieri. Ostial valves guard these openings and the anterior cardinal sinus, lying directly above the gills, is well placed to exploit the ventilatory movements of the branchial skeleton. In elasmobranchs and teleosts, venous blood from the nutrient circulation of the gill lamellae and from the muscles that extend between the skeletal elements of the branchial arches, enters veins that pass up the gill bars, immediately beneath the skin of the pharynx, and open by valved openings into the floor of the anterior cardinal sinus. In a ventilating dogfish, the expulsive phase of each ventilation, when pressure in the orobranchial cavity rises, can be seen to cause blood to pass from these valved openings into the sinus. The contraction of the constrictor muscles also compresses the sinus and forces blood past the

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ostial valve at its junction with the ductus cuvieri. Cardiorespiratory synchrony, a reflex coordination of the heartbeat with ventilation (Satchell, 1960),may serve to link this outflow with the systolic peak of cardiac suction. Rowing (1981) has drawn attention to a rather different branchial pump in the eel and what appears to be a parietal valve along the length of the vein lying above its gills. The homology of this vessel is obscure; it is not the anterior cardinal vein, as in most teleosts, and has been termed the internal jugular vein by Mott (1950).It is in part a vein of the branchial secondary system, and the valve occurs at the junction of this with the primary venous system. Rowing (1981) suggests that this valve may protect the low-pressure secondary veins from the higher pressures developed in the external jugular vein, generated by coughing. C. The Renal Portal System and Its Branches The caudal vein, we have noted, divides into paired renal portal veins, which ramify in the substance ofthe kidney and supply capillary networks around the tubules; from these, blood is collected and enters the posterior cardinal veins (Audige, 1910). In the lingcod (Allen, 1905), the caudal vein gives rise, just above this division, to branches, the posterior intestinal vein and the bladder vein, which terminate in capillary networks in the mucosa of the posterior intestine and the bladder. Capillaries ramify around a specialized epithelium of cuboidal cells in the dorsal posterior wall of the bladder; these cells, it is believed, are concerned with the active reabsorption of sodium ions from the urine (Loretz and Bern, 1980).The posterior intestine, too, is the site of active sodium reabsorption. Blood from the bladder capillary network is ultimately returned to the posterior cardinal veins, and the vessels constitute a separate portal system, parallel with the renal portal system. The caudal vein receives the drainage from the neurohemal organ at the tip of the spinal cord, the urophysis, from which peptide hormones, the urotensins are released. Two of these, urotensins 1 and 2, have been sequenced and synthesized (Lederis, 1984),and are known (1)to inhibit and (2) to stimulate salt uptake from the isolated postintestine of 5%sea water-adapted Gillichthys (Loretz et al., 1983).The possibility exists that these vessels provide the pathway for urotensins to reach these epithelia. When, during swimming, the hemal arch pump is operating, pressures in the caudal vein are likely to exceed those in the hepatic portal system. Connections occur between the

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caudal vein and the bladder and posterior intestinal circulations in Scorpaenichthys, Sebastodes, and Hexagrammus (Allen, 1905). We have noted that in other genera there is a direct connection between the right renal portal vein and the right posterior cardinal vein; this forms a shunt vessel that bypasses the capillaries of the kidney tubules. The condition occurs in such freshwater genera as Perca, Salmo, Luciperca, Misgurnus, and in some marine forms, too, such as Cyclopterus and Gadus. In such genera the kidney tubules receive venous blood only from the segmental intercostal veins.

VIII. THE VEINS OF THE HEPATIC PORTAL SYSTEM We noted above that the hepatic portal system is localized entirely within the abdominal cavity, and its veins connect two microcirculations, those of the absorptive part of the gut and those of the liver sinusoids. The branches of the hepatic portal system are the only veins in fish in which the wall is sufficiently differentiated from the surrounding tissue to enable the vessel to be dissected free. In elasmobranchs their walls may have the usual three layers of veins of higher vertebrates. The veins originate from two quite separate capillary beds (Friedrich, 1956). One lies between the longitudinal and circular muscle of the gut; the other ramifies immediately beneath the mucosa. Both are supplied with arterialized blood by median arteries from the dorsal aorta. Blood within the first capillary bed presumably carries oxygen, nutrients, and the mediators that regulate the activity of the smooth muscle, to this tissue. The second is likely to be involved in the forward transport of dissolved nutrients derived from the food, for further processing in the liver. Maclean and Ash (1989) in a study of rainbow trout (wet weight, 1192 & 104 g), chronically cannulated in the hepatic portal vein, report that mean blood flow in the vein was 12 & 2.87 ml/min/kg-' in anesthetized fish. This is some 40% of the cardiac output (Wood and Shelton, 1980) of this species. Pressure in the hepatic portal vein is usually above ambient (Table I). In it, as in other parts of the circulation, the pressure at a particular site represents a point along a gradient of pressure extending from the base of the ventral aorta, through the circulation, to the entrance to the sinus venosus. It represents a particular combination of uis-cl-tergoand

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uis-d-fronte. In branches of the hepatic portal system, pressure is elevated by the resistance of the liver sinusoids. Nevertheless, we know that flow in the hepatic portal vein is powerfully influenced by the uis-d-fronte of the heart. Maclean and Ash (1989), in a Doppler-flow study of the flow pulse in the hepatic portal vein of rainbow trout show that the velocity of flow rises almost vertically at systole and declines steadily during diastole. The velocity profile changes cyclically in time with the heart beat and at no point has a level course. The number of major veins recognizable in the hepatic portal system is related to the degree of gut elaboration. The pond loach has a short straight intestine, and blood passes to the liver from it in a single ventral subintestinal vein. (Talikowska, 1962). In the barbel (Barbus fluuiatiZis) the intestine bends back on itself at the end of the abdominal cavity and passes rostrally toward the pharynx before bending again to pass caudally toward the vent. In this species the gut is drained by four major veins, which unite to form the common trunk of the hepatic portal vein (Koniar, 1947). Connections between the hepatic portal and somatic venous circulations have been reported in some teleosts. Thorarensen et al. (1991) note that in chinook salmon Oncorhynchus tshawytscha the ventral intestinal vein receives blood from the ventral body wall via seven veins. The digestion and absorption of food itself make demands on cardiac output, and in many genera, occurs most when the fish is less active. Certainly there are no features of the gut vasculature that appear to exploit pressures generated by swimming muscles. The gut and liver are slung centrally below the column of myotomes, in a region in which body flexure is minimal. The arteries to and the veins from the viscera are each single median vessels, not paired structures such as might harness energy from the waves of myotomal contraction that pass down the trunk. We know of no auxillary pumps, comparable to those described in the previous section, except that of the portal heart of the hagfish, and its investment of true cardiac muscle sets it apart from all others. The pressure developed by the heart of hagfish is lower than that of any elasmobranch or teleost. Dorsal aortic pressure in the Atlantic hagfish Myxine glutinosa is 6-7 mm Hg (Satchell, 1986); in the New Zealand hagfish Eptatretus cirrhatus, it is 10 mm Hg. The pressure in the branches of the hepatic portal system is only 1-2 mm Hg, and an auxillary pump, the portal heart, is located just before its entrance into the liver. This heart (Fig. 7A) is invested with true cardiac muscle, which resembles in its gross and fine structure that of the true, or branchial heart. The portal heart consists of a single chamber, with

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Fig. 7. The portal heart of hagfish. (A) The portal heart of Myrine glutinosa; (B) tracings of the sinus intestinalis in systole (solid line) and diastole (broken line); (C) simultaneous recordings of portal heart ECG (upper trace) and pressure in mm Hg (lower trace) of Eptutretus cirrhatus. Br.H.P.V., branches of hepatic portal vein; H.V., hepatic vein; I.V., inlet valves; O.V., outlet valves; P.H., portal heart; S.I., sinus intestinalis. Redrawn after (A,B) Fange et al. (1963);(C) Davie et ul. (1987).

valves at the openings of the single outflow and the two inflow veins. It generates its own electrocardiogram with a P and a T wave (Fig. 7C). It can be likened to an isolated atrium, and like the atria of many fish, it develops a pressure of 2-3 mm Hg. A wavelike contraction (Fig. 7B) can be seen to pass along the region of the hepatic portal vein proximal to the portal heart, termed the sinus intestinalis, and this region appears to have the role of a sinus venosus. The much larger electrocardiogram (ECG) of the true heart tends to interfere with records of the portal heart ECG, and this may explain the failure to report an electrical event associated with the contraction of the sinus intestinalis. The beat of the portal heart is not coordinated with that of the true heart. The provision of an auxillary pump powered by cardiac muscle represents an alternative solution to the problem of ensuring that the pump may need to be active even when the fish is at rest. The property of inherent rhythmicity, characteristic of cardiac muscle, substitutes for the provision of an independent rhythm generator located in the tip of the spinal cord, to be described later in this chapter.

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IX. THE SECONDARY VEINS O F THE SKIN In most fish the lateral cutaneous veins (Fig. lB, Fig. 5), are the longest veins in the body, for they extend, at the level of the lateral line, from the pectoral region onto the surface of the tail fin. The blood in them is derived from secondary capillary beds in the skin and scales. In many genera the blood is aspirated from them by a caudal pump or a caudal heart, and is propelled into the caudal vein. In elasmobranchs and some bony fish such as Pleuronectes and Arnia (Sappey, 1880; Hopkins, 1893), there is an additional communication with the ductus cuvieri rostrally at the level of the sinus venosus, either directly or via a short vessel, the subscapular vein. Blood from the skin is also collected into segmental subdermal vessels that link the lateral cutaneous veins with the dorsal and ventral cutaneous veins. These sometimes communicate rostrally with the sinus venosus, but the main outflow from them is into the caudal vein by one or another of the fin pumps, to be described shortly. In most elasmobranchs the caudal pump in the tail is active only when the fish swims, and at rest, the pressure profile along the length of the lateral cutaneous vein is dominated b y the rostra1 outflow and the uis-h-fronte of the heart. Pressure is subambient (Fig. 8A) and fluctuates in time with the heartbeat (Satchell, 1971).The amplitude of this fluctuation diminishes away from the heart. In the resting fish, venous pressure does not attain ambient until the level of the pelvic fins. There are no valves along the length of the cutaneous veins, but ostial valves occur where they enter the caudal pump in the tail, and rostrally, where they enter the subscapular vein. There is reason to believe that at rest these subscapular valves, which are subject to the fluctuating vis-h-fronte of the heart, open and close in time with its beat. When, at ventricular systole, blood leaves the heart, and pericardial pressure falls below that in the cutaneous vein, blood is aspirated from it. As diastole proceeds, pericardial pressure rises toward ambient, and these ostial valves close, entrapping a region of subambient pressure within the cutaneous system. The subambient venous pressure is maintained by cardiac suction, and if the vein is blocked behind the valves by the injection of warmed vaseline, the gradient of pressure along the vein is lost (Fig. 8B). The movement of these venous ostial valves is thus dictated by the heartbeat, and this is unique in the vertebrate classes (Birch et al., 1969). The opening and closing of venous valves in mammals is at the dictates of the skeletal or visceral muscles adjacent to them.

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Fig. 8. (A) Pressures along the length of the lateral cutaneous vein of a resting Port Jacksonshark. Ordinate, pressure in mm Hg. Abscissa, distance in cm from entrance to subscapular sinus. (B) Pressures observed when the rostra1 end of the vein was blocked. Redrawn after Birch et al. (1969).

A. Venous Pumps of the Secondary System: Median Fin Pumps In elasmobranchs, blood draining from the skin into the dorsal and ventral cutaneous veins can be pumped into the caudal and renal portal veins by small venous pumps (Fig. 9) associated with the median fins; their vascular anatomy was first described by Mayer (1885). In fish, forward movement of the body is imparted by the alternate contraction of the left and right myotomes; the tendency to roll is countered by right and left deflection of the median fins, effected by small radial muscles inserted onto the radial elements of the fin skeleton. The dorsal and ventral cutaneous veins divide around the median fins to form venae circulares (Fig. 9) into which the secondary veins of the fins open. From the caudal end of the loop vessel, paired veins pass to paired spherical vesicles located between the radial muscles and the fin skeleton. Each vesicle is valved at its entrance and exit, and their outflows join to form a single common vein, the vena profunda, which passes ventrally, around the spine, to enter the caudal vein or renal portal vein via one of the intercostal vessels. When the median fin is moved to the leA, the right chamber is stretched across and com-

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Fig. 9. The median fin pump. (A) The vessels surrounding the posterior median fin of the Port Jackson shark. (B) Aspiratory pulse of pressure in mm Hg, in the dorsal cutaneous vein (upper trace) and fin movement (lower trace). D.C.V.,dorsal cutaneous vein; C. V., caudal vein; L.C.V., lateral cutaneous vein; V.Circ., vena circularis; V.Prf., vena profunda; Vsc., vesicle with inlet and outlet valves. Redrawn after Birch et al. (1969).

pressed against the fin skeleton, while the left is decompressed within the larger space formed at the fin base. As the fin sweeps to the right, the left chamber is emptied and the right one fills. This basic mechanism underlies the operation of most of the caudal pumps and caudal hearts of fish. In the bony fish there is much variation in the way in which blood from the median fins finds its way into the caudal vein, and in Lepisosteus the vena circulares and vesicles are absent; however, the vessels passing down into the caudal vein are somewhat dilated at the base of the fin (Allen, 1908).

B. The Caudal Pump In the tail of most fish, the subepidermal net of vessels is concentrated to form a marginal vein at the junction of the fin with the more solid portion containing the spinal column and radial muscles. Figure 10 shows the veins of the tail of the Port Jackson shark Heterodontus portusjacksoni. Vessels derived from the extensions of the cutaneous veins onto the tail, and smaller veins draining the fin itself, pour their blood into the marginal vein. From it many small branches pass through the layer of radial muscles to enter the caudal sinus, a deeper vessel that runs between the muscles and the fin skeleton on each side. In elasmobranchs and the more primitive bony fish, the marginal vein

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Fig. 10. The caudal pump ofthe Port Jacksonshark. C.A., caudal artery;C.S., caudal sinus; C.V., caudal vein; D.C.V., dorsal cutaneous vein; L.C.V., lateral cutaneous vein; M.V., marginal vein; V.C.V., ventral cutaneous vein. Redrawn after Birch et al. (1969).

and caudal sinus bear many valves so orientated as to direct the blood entering from the tip and the base of the tail toward the mid-region, where most of the connections to the caudal vein occur. Radial muscles extend from the fin radials to a fascia of dense connective tissue beneath the skin, and as the fish swims, they contract alternately, left and right, and deflect the lower lobe of the tail. In so doing they alternately compress or relax the caudal sinus of the two sides in the manner outlined for the median fin pump; blood gathered from the periphery into the caudal sinus is expelled through valved connections into the caudal vein. The elongate caudal sinus spanning several tail segments intergrades in different genera, with the single chambered condition seen in many teleosts. The caudal fin pump, like the median fin pump, is thus a propulsor, powered by muscles primarily concerned with moving the fin.

C. The Caudal Heart of the Carpet Shark The location and interconnections of these three vessels, the marginal vein, the caudal sinus, and the caudal vein, differ from one species of fish to another, depending on the shape and size of the tail, but follow a common plan. In the resting carpet shark Cephaloscyllium isabella and the smooth hound Mustelus antarcticus, a muscular ripple can be seen to pass from the margin of the tail fin toward its base, in

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a line directly over the origin of the radial muscles. The tail remains quite stationary, and the muscles of the two sides are clearly contracting synchronously in a sequence directed toward the base of the tail. This has been confirmed by strain-gauge records and electromyography (Satchell and Weber, 1987).The arrangement of the three vessels (Fig. 11)is broadly like that of the Port Jackson shark except that the caudal sinus is tapered from the tip toward a swollen region at the base of the tail; all of its valves are directed toward the basal region, and only a single, or occasionally two connecting veins, on each side, join it to the caudal vein. Records of blood pressures in the lateral cutaneous vein, caudal sinus, and caudal vein (Fig. 12) suggest that the radial muscles pull the skin down toward the fin radials and subject the caudal sinus to a rolling wave of compression that forces blood past the valves and into the caudal vein. The pulse of pressure in the caudal sinus coincides with the rise in pressure in the caudal vein. Diastolic pressure in the caudal sinus is maintained subambient, and this enhances the pressure gradient across, and presumably flow from, the cutaneous circulation. The pressure gradient along the length of the lateral cutaneous vein, compared with that in a resting Port Jackson shark, is thus reversed, for the lowest pressures are at the caudal end. When the radial muscles are paralyzed with D-tubocurarine (Fig. 13), the pump fails and pressure in the caudal sinus rises toward that in the

Fig. 11. The caudal heart ofthe carpet shark, Cephaloscyllium isabella. Many ofthe radial muscles and smaller vessels have been omitted for the sake of clarity. C.A., caudal artery; Cn.V., connecting vessel; C.S., caudal sinus; C.V., caudal vein; D.C.V., dorsal cutaneous vein; L.C.V., lateral cutaneous vein; M.V., marginal vein; R.M., radial muscles; V.C.V., ventral cutaneous vein. Redrawn after Satchel1 and Weber (1987).

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Fig. 12. Pressures in the caudal vein, the lateral cutaneous vein, and the caudal sinus of a resting unanesthetized carpet shark. After Satchel1 (1991).

caudal vein; the increase in pressure at the downstream end of the cutaneous circulation must presumably lessen flow from it. The pump is unlike the branchial heart or the portal heart, in that it consists of many similar valved chambers in sequence, and blood enters laterally into each so that the volume of blood that is propelled is summed and thus increases along its length. When the fish is swimming, it presumably acts like that of the Port Jackson shark, and it must then be regarded as a propulsor. At rest the radial muscles serve only to power the pump, and it acts as a caudal heart; the distinction previously made between propulsors and hearts is not so complete as was thought. The significant advance achieved by the carpet shark lies in the spinal cord, for a separate motor center has evolved, controlling the caudal radial muscles, and able to act autonomously in the resting fish. In addition, the motoneurons activating the muscles of the two sides can be discharged synchronously so that the tail remains still and they serve solely to compress the caudal sinuses.

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Fig. 13. Records similar to those in Fig. 12 recorded at slower speed, following the injection of D-tubocurarine. The slight pulse remaining in the caudal vein, following paralysis of the caudal heart, is that of the true heart, directly communicated within the hemal canal from the dorsal aorta. After Satchel1 (1991).

D. The Caudal Heart of the Eel In the tail of the eel, as in many other teleosts, a tiny caudal heart (Fig. 14B,C), only a few mm long, is located below the last vertebrae. (Favaro, 1906).It is a median structure consisting ofjust two chambers; the right chamber, or “atrium” receives blood from the cutaneous veins and the small vessels of the fin, and propels it through a median opening between the fin radials, into the left chamber, the “ventricle.” The openings into the “atrium” and “ventricle” are valved and the “ventricle” has a valved opening into the caudal vein. In the tench Tinca tinca (Fig. 14B) the “atrium” is on the left and the “ventricle” is on the right (Przemyska-Smosarska, 1952). On each side, a delicate band of skeletal muscle runs from the last vertebra onto the radials below; right and left muscles contract alternately, compressing the chambers of the heart against the fin skeleton. Blood passes from the “atrium” to the “ventricle” and from the “ventricle” to the caudal vein. This alternate contraction of the muscles of the two sides does not cause the tail to move, for the muscles are too small. One beat of the caudal heart involves the compression, in

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@

@

t.V

c.v

0 c.v

t Fig. 14. Caudal hearts of (A) Myxine glutinow, (B) tench, Tinw tincu, (C) the common eel. Arrows indicate direction of blood flow. C.F.V., inflow from caudal fin veins; C.V., caudal vein; C.V.S., caudal vein sinus; Ds.C.V.,distal part of caudal vein; L.Ch., left chamber; L.C.V., inflow from lateral cutaneous veins; M.V., marginal vein; R.Ch., right chamber; R.Ds.C.V., right distal caudal vein; V.Sc.S., vein from subcutaneous sinus. Redrawn after (A) Satchel1 (1984), (B) Przemyska-Smosarska(1952).

sequence, of both sides, so that blood is delivered into the caudal vein only once in each cycle. In the holostean genera Lepdsosteus and Arnia, the left and right chambers are more elongate than those in the eel (Allen, 1908).Not all teleost fish have a caudal heart; it is absent in all the Acanthopterygi, a group that includes such spiny-finned fish as the wrasse, flounders, and perch. Motoneurons of the caudal heart center are located in the last two segments of the spinal cord; Mislin (1969) notes that the center has some independence, for when the eel is cooled, the caudal heart becomes inactive at 11"C,at which temperature the fish continues to ventilate slowly. Davie (1981)has recorded trains of action potentials in some afferent fibers in the cardiac nerves that return from the caudal heart muscles, suggesting that there is some regulation of the beat, although this has not, as yet, been investigated. Chan (1975)reports that urotensin II increases the rate of caudal heartbeat in the eel.

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Caudal heart and urophysis may be jointly involved in regulating flow from the cutaneous circulation.

E. The Caudal Heart of the Hagfish The hagfish, or Myxinoids, have many primitive features, and the venous system differs notably from that of teleosts and elasmobranchs in the presence of many blood-filled sinuses, the homology of which is uncertain. The dorsal, lateral, and ventral cutaneous veins are not present; beneath the skin is a subcutaneous sinus that extends from the snout almost to the tip of the tail and from the middorsal to the midventral line. Approximately a third of the blood volume is contained in it (Forster et al., 1989). At its caudal end a small caudal heart pumps blood from it into the caudal vein. It differs from that of a teleost in that the left and right chambers are quite separate and do not communicate across the midline (Fig. 14A). The caudal heart muscle forms a thin strap of fine fibers on each side, which insert, not onto the spine, but onto a knob of cartilage (Fig. 15) so arranged that the center part of the median fin skeleton can be swung left and right, free of the main fin skeleton (Satchell, 1984). Between the muscle and the fin skeleton, on each side, is a valved caudal heart chamber formed as a dilation in the course of the left and right divisions of the caudal vein. Blood enters the heart from the fin veins and from the subcutaneous sinus and is

R :V

M.Y.

Fig. 15. The caudal heart of Myrine glutinosa in side view. C.H.M., caudal heart muscle; C.V., caudal vein; Ds.C.V., distal caudal vein; K.M.P., knob on movable portion of median plate; L.C.H.C., left caudal heart chamber; M.N., motor nerve fibers; M.P., median plate; M.V.,marginal vein; N.C., nerve cord; R.V., radial veins; V.Sc.S., vein from subcutaneous sinus. Redrawn after Kampmeier (1969).

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pumped into the caudal vein. The existence of what is, in effect, a separate caudal heart on each side ensures that in each cycle, caudal vein pressure shows two elevations (Fig. 16), as the left and right chambers are successively emptied, rather than the single pulse seen in the carpet shark. The caudal heart muscle is more extensive than that in the eel, but the separation of a portion of the median fin cartilage from the remainder ensures that the tail fin does not move, despite the alternate contraction of the muscles. The caudal heart of the Atlantic hagfish Myrine glutinosa does not beat continuously. In aquaria, hagfish alternate periods of swimming with periods of rest, and the caudal heart becomes active shortly after the end of a swim (Fig. 17). It exhibits cycles of beating with brief periods of rest until swimming once more commences. It may be that the sinus system of the hagfish is the forerunner of the secondary blood system of the true fish. We can note that in both a portion of the blood volume is deployed in vascular spaces that are located peripherally beneath the skin. In both, the caudal heart, unequivocally part of the secondary blood system of teleosts, is powered by skeletal muscles innervated by motor neurons in the tip of the spinal cord that form an autonomous motor center. In both, their contained blood has a lower hematocrit than central venous blood because the entrances into the secondary system and sinus system have filtering devices that hold back red cells. We need to study the shifts of blood between it and the primary circulation resulting from exercise

Fig. 16. Pressure pulses in the caudal vein of Myxine caused by the caudal heart. Top, pressure trace; bottom, mechanogram from surface of caudal heart. Redrawn after Satchel1 (1984a).

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and hypoxia. We cannot, at present, explain why hagfish, often considered to be among the most primitive of living chordates, should have their trunk virtually enveloped in a bag of blood. The physiological role of the hagfish sinus system is quite obscure, and its further investigation presents a challenge (Satchell, 1991). Fish, in the course of their evolution, have progressed from longbodied creatures that swim in the anguilliform mode to short-bodied forms that swim in the carangiform mode. The return of blood from the microcirculation of the skin in long-bodied forms is likely to be less rapid, as the cutaneous vessels are yet further removed from the vis h fronte of the heart. The caudal heart appears to be strategically located so as to enhance flow from that part of the cutaneous circulation that is most remote from the branchial heart. Poiseuillean flow cannot occur against a pressure gradient, and pressures in the caudal vein, usually between 2 and 3 mm Hg, have to be sufficiently high to propel blood through the capillary beds surrounding the renal tubules. In longbodied fish at rest, cutaneous vein pressure may be below this level; the caudal heart lifts pressure sufficiently to enable flow to occur from the secondary to the primary circulation.

Time after first swimming period in mins

Fig. 17. Record of rate of caudal heart beat of Myxine following a period of swimming. Solid circles, 1"C,hollow circles, 14°C. The arrow indicates a second short period of swimming (5 min), which occurred in the course of the 14°C record. After Satchell (1991).

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F. The “Cardinal Heart” of the Hagfish Ventilation in the hagfish is very different from that of the true fish. The gill lamellae are arranged in meridional rows lining spherical sacs; these are encircled with bands of muscle that by their contraction, constrict them. In the roof of the pharynx, a velum, shaped like a matador’s clock, is waved up and down by the agency of four muscles and a series of cartilages (Johansen and Strahan, 1963). It propels water into the pharynx and along it to the various gill pouches. Between the hyoid plate and the olfactory organ (Fig. 18)in the roof of the pharynx is a blood sinus, the hypophysiovelar sinus (Cole, 1925; Brodal and Fange, 1963).Two of the muscles that take part in the complex movement of the velum are the veloquadratus and the velospinalis; they lie within the sinus and extend from parts of the velar skeleton to the hyoid plate (Cole, 1906-1907). When they contract they primarily

Fig. 18. The cardinal heart of myxinoids. (A)The veins and sinuses; (B) the skeleton and muscles. H.V.S., hypophysio-velar sinus; Hy.P., hyoid plate; I.V., inlet valves; L.V.B., lateral velar bar; Nc., notochord; S.A.C.V., swollen portion of anterior cardinal vein; Sc.A., subcutaneous anastomosis; Sc.S., subcutaneous sinus; Vq.M.1-3, veloquandratus muscle, divided into three portions; V.Sp.M., velospinalis muscle.

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move the velar skeleton but additionally move the hyoid plate up and down against the olfactory organ and compress and dilate the sinus. Small veins draining blood from the subcutaneous sinus have valved openings into the hypophysiovelar sinus; a larger valved vein provides an outlet from it into an expanded portion of the anterior cardinal vein. The structure has been termed the cardinal heart, and the name is firmly embedded in the literature. Simple inspection in the living hagfish shows, however, that the two pairs of muscles that activate the pump are primarily involved in ventilation; it is therefore a propulsor and not a heart. Nevertheless, as Cole (1925) has noted, there are resemblances between the cardinal and the caudal heart of the hagfish. Both pump blood from the subcutaneous sinus into the veins of the primary circulation, but they are located at its opposite ends. The muscles ofboth look alike in that they are thin, straplike, parallel arrays of delicate striped muscle fibers rich in myoglobin.

X. DISCUSSION AND CONCLUSION The capillaries constitute a diffusion and exchange system in which the pressure is required to be low to reduce filtration. The entry of blood into this segment of the circulation is regulated by the arterioles, which are innervated by the autonomic nervous system. Contraction of the abundant smooth muscle cells in their walls increases their resistance, decreases flow through them, and lowers transmural pressure in the capillary and venous segments of the downstream circulation. Blood issuing from the distal ends of capillary vessels will have lost some 80-90% of the pressure imparted to it by the heart. It is an evident fact that the pressure the heart is able to generate has increased through the phylogenetic series, and dorsal aortic pressures in fish are between a fifth and a half of those in mammals. The gradient of pressure between the capillary beds and the heart can be increased by cardiac suction. This has been enhanced by the stiffening of the pericardium caused by its adherence to skeletal elements in the pectoral girdle and branchial arches, but the magnitude of this suction inevitably decreases with distance from the heart. The vascular bed of the myotomal muscles extends the length of the trunk and much of it is located in the solid column of myotomes that extends beyond the end of the abdominal cavity. The hemal arch pump provides a means of increasing venous return from this at the very time, i.e., during swimming, that it is most needed. The array of ostial valves located along each side of the caudal vein are well placed to exploit that

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ancient feature of vertebrate structure, metameric segmentation. As waves of neuronal excitation pass down the spinal cord, and the serial array ofmyotomes successively contracts, so are their regional vascular beds compressed and emptied into the longitudinal vessels. Additional venous pumps are associated with the unpaired fins. In the tailfin, the skeletal muscles that originally served to move the fin lobe come to power the caudal heart, which is provided with an independent rhythmic motor center in the terminal part of the spinal cord. The caudal heart is specifically concerned with returning blood from the cutaneous circulation to the primary venous system. The caudal heart of the carpet shark maintains subambient pressures in the caudal sinus and the veins leading into it (Fig. 12,13).The hemal arch pump may generate subambient pressure in the tributaries of the intercostal veins. Those that lie in the septa between the myotomes of one side will be expanded as the myotomes of the opposite side contract. If blood is to flow along veins subject to subambient pressure, their walls must be stiffened or they will collapse. This stiffening also ensures that the vessel dilates of its own accord as the aspiratory or compressive pressures lessen; it also enables subambient pressures to be propagated further along their length. Veins represent that component of a circulatory system associated with the return ofblood to the heart. Fish live in a medium, water, with a specific gravity closely similar to that of their blood, and their veins are thus largely spared the effects of gravity. Their tissues and the vessels within them are everywhere buoyed up by the water that surrounds them. This contrasts with the conditions present in land vertebrates. The capillary beds in the foot of a standing human are subject to the pressure generated by the column of blood extending from the foot up to the level of the right atrium. This extra pressure, some 85-90 mm Hg, has the potential to disturb the Starling balance of fluid distribution across the capillary walls and cause edema. The veins of fish differ from those of terrestrial vertebrates in two important respects. They lack parietal valves. These first appear in the limb veins of the anuran amphibia (Suchard, 1907)and serve to protect dependent vascular beds from the high pressures generated by gravity. Fish, buoyed by the water around them, need no such provision. Ostial valves have a different function; they serve to prevent the backflow of blood from the major longitudinal veins into the vascular beds of the muscles. They are commonly present in the openings of segmental vessels into the longitudinal channels. They are probably part of a more ancient system related to the strongly metameric repetition of the myotomes along the length of the spine. Both parietal valves and an

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innervated layer of smooth muscle serve to combat the hemodynamic problems crated by gravity. The pressure at the junctions of the venous system with the heart must attain a level sufficient to cause an adequate flow of blood into it. In a terrestrial animal this pressure must be continuously adjusted as its body is moved about within the gravitational field. If the returning blood fails to achieve this critical level, the heart will be unable to fill. The problem appeared in the vertebrates only when the supporting buoyancy of the surrounding water was removed. The innervated layer of smooth muscle in the veins in mammals plays a part in the adjustments that match the blood volume to the capacitance of the vessel system. The problem is presumably less acute in creatures largely removed from gravitational stresses. The presence of parietal valves and of an innervated layer of smooth muscle may need to be added to the long list of structural changes that must have occurred in that great Devonian-Carboniferous transition: the emergence of the vertebrates from the seas onto the land.

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Mayer, P. (1885). Die unpaaren Flossen der Selachier. Mitt. Zool. Stats. Neapel 6, 217-285. Michel, C. C. (1988).Capillary permeability and how it may change. J. Physiol. (London) 404,l-29. Mislin, H. (1969). Zur Funktionsanalyse des lymphatischen Kaudalherzens beim Aal (Anguilla anguilla L.).Rev. Suisse Zool. 67,262-269. Mott, J. C. (1950). The gross anatomy of the blood vascular system of the eel Anguilla anguilla. Proc. Zool. SOC. Lond. 120,503-518. Munshi, J. S.D., Olsen, K. R., Ojha, J., and Ghosh, T. K. (1986).Morphology and vascular anatomy of the accessory respiratory organs of the air-breathing climbing perch, Anabas testudineus (Bloch).Amer. J . Anat. 176,321-331. Myhre, K. and Steen, J. B. (1977).The effect of plasma proteins on the capillary permeability in the rete mirabile of the eel (Anguilla uulgaris L.) Acta Physiol. Scand. 99, 98-104. Nekvasil, N. P., and Olson, K. R. (1985).Localization of 3H-norepinephrine binding sites in the trout gil1.J. E x p . Zool. 235,309-313. Neuville, H. (1901).A 1’Ctude de la vascularisation intestinal chez les cyclostomes et les selaciens. Ann. Sci. Naturelles. Zool. 13,l-116. Nilsson, S . (1984).Adrenergic control systems in fish. Mar. Biol. Lett. 5,127-146. Olesen, S. P., Clapham, D. E., and Davies, P. F. (1988). Haemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature (London) 331,168-170. Olson, K. R. (1981). Morphology and vascular anatomy of the gills of a primitive airbreathing fish, the bowfin (Amia calva). Cell Tissue Res. 218,499-517. Owman, C. H.and Rudeberg, C. (1970). Light fluorescence and electron microscopic studies on the pineal organ of the pike Esox lucius L. with special regard to 5hydroxytryptamine. Z. Zellforsch. 107,522-550. Przemyska-Smosarska,J, (1952).Serca limfatyczne ryb kostnoszkieletowych. Bull. Acad. Pol. Sci. Lettr. 77-85. Rasio, E. A., Bendayan, M., and Goresky, C. A. (1977). Diffusion permeability of an isolated rete mirabile. Circ.Res. 41,791-798. Rasio, E. A., Bendayan, M., and Goresky, C. A. (1981).The effect of hyperosmolality on the permeability and structure of the capillaries of the isolated rete mirabile of the eel. Circ. Res. 49,661-676. Rhodin, J. A. G., and Silversmith, C. (1972). Fine structure of elasmobranch arteries, capillaries and veins in the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 42A, 59-64. Rothe, C. F. (1983).Venous system: Physiology ofthe capacitance vessels. “Handbook of Physiology” Section 2, Vol. 3, Pt. 1 (J.T. Shepherd and F. M. Abboud, eds.), pp. 397-452. American Physiological Society, Bethesda, Maryland. Rothe, C. F., and Drees, J. A. (1976).Vascular capacitance and fluid shifts in dogs during prolonged hemorrhagic hypotension. Circ. Res. 38,347-356. Rowing, C. G. M. (1981). Interrelationships between arteries, veins and lymphatics in the head region of the eel, Anguilla anguilla L. Acta Zool. (Stockholm) 62,159-170. Sappey, P. C. (1880).“Etudes sur 1’AppareilMucipare et sur le System Lymphatique des Poissons.” V. A. Delahaye et Cie, Paris. Satchell, G. H..,(1960).The reflex co-ordination of heartbeat with respiration in the dogfish.]. E x p . Biol. 37,719-731. Satchell, G. H. (1965).Blood flow through the caudal vein of elasmobranch fish. Aust.]. Sci. 27,240-241.

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Satchell, G. H. (1971). “Circulation in Fishes.” Cambridge University Press, Cambridge, England. Satchell, G. H. (1984).On the caudal heart of Myxine (Myxinoidea: Cyclostomata). Acta Zool. (Stockholm)65,125-133. Satchell, G. H. (1986). Cardiac function in the hagfish, Myxine, Myxinoidea : Cyclostomata. Acta 2001.(Stockholm)67, 115-122. Satchell, G. H. (1991). “Physiology and Form of Fish Circulation.” Cambridge University Press. Cambridge, England. Satchell, G. H., and Weber, L. J. (1987).The caudal heart of the carpet shark, Cephaloscyllium isabella. Physiol. Zool. 60,692-698. Smirnov, V. N., Antonov, A. S., Antonova, C. N., Romanov, Y. A., Kabaeva, N. V., Tchertikhina, I. V., and Lukashev, M. E. (1989). Effects of forskolin and phorbolmyristate-acetate on cytoskeleton, extracellular matrix and protein phosphorylation in human endothelial cells. J . Mol. Cell Cardiol. 21,3-11. Smith, D. G. (1978). Neural regulation of blood pressure in rainbow trout (Salmo gairdnert) Can.J . Zool. 56,1678-1683. Stevens, E. D., and Randall, D. J. (1967).Changes of gas concentrations in blood and water during moderate swimming activity in rainbow trout. J. E x p . Blol. 46,329-337. Stray-Pedersen, S. and Nicolaysen, A. (1975).Qualitative and quantitative studies of the capillary structure in the rete mirabile of the eel, Anguilla vulgaris L. Acta Physiol. Scand. 94,339-357. Stray-Pedersen, S., and Steen, J. B. (1975). The capillary permeability of the rete mirabile of the eel, Anguilla vulgaris L. Acta Physiol. Scand. 94,401-422. Suchard, M. E. (1907).Sur les valvules des veins de la grenouille. C. R. SOC. Biol.(Paris) 62,452-453. Talikowska, H. (1962).Uklad krwionosny przewodu pokarmowego pikorza (Misgunus fossilis L.). Acta Biologica Cracoviensia Sr. Zool. 5,141-153. Thorarensen, H., McLean, E., Donaldson, E. M., and Farrell, A. P. (1991). The blood vasculature of the gastrointestinal tract in chinook, Oncorhynchus tshawytscha (Walbaum),and coho, 0. kisutch (Walbaum), salmon. J . Fish Biol. 38,525-531. Trois, E. F. (1880).Del sistema linfatico dei teleostei. Atti. 1st. Veneto. Sci.40,401-416. Van Deurs, B., Petersen, 0. W., OIsnes, S., and Sandvig, K. (1989). The ways of endocytosis. Intern. Rev. Cytol. 117,131-160. Vecchio, P. J.. Del., Siflinger-Birnboim, A., Shepard, J. M.,Bizois, R., Cooper, J. A., and Malik, A. B. (1987). Endothelial monolayer permeability to macromolecules. Fed. PTOC.46,2511-2515. Wahlqvist, I., and Nilsson, S. (1977). The role of sympathetic fibres and circulating catecholamines in controlling the blood pressure and heart rate in the cod, Gadus morhua. Comp. Biochem. Physiol. 57C, 65-67. Wahlqvist, I., and Nilsson, S. (1981).Sympathetic nervous control of the vasculature in the tail of the Atlantic cod, Gadus morhua.]. Comp. Physiol. (B) 144, 153-156. Wood, C. M., and Shelton, G. (1980).Cardiovascular dynamics and adrenergic responses of the rainbow trout in vivo. J . E r p . Biol.87,247-270. Wood, C. M., McMahon, B. R.,and McDonald, D. G. (1979).Respiratory, ventilatory and cardiovascular responses to experimental anaemia in the starry flounder, Platichthys stellatus. J. Exp. Biol. 82,139-162. Woodland, W .(1906).On the anatomy of Centrophorus calceus (crepidalbusBocage and Capello) Gunther. Proc. Zool. SOC. London 865-886.

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4 THE SECONDARY VASCULAR SYSTEM 1.F . STEFFENSEN Marine Biological Laboratory Copenhagen University DK-3000 Helsinger Denmark

J . P . LOMHOLT Department of Zoophysiology University of Aarhus DK-8000 Aarhus C Denmark 1. Introduction 11. Morphology of the Secondary System A. Teleosts

B. Cyclostomes C. Elasmobranchs 111. I n Vioo Microscopic Observations IV. Physiological Experiments A. Volume of the Secondary Vascular System B. Exchange between the Primary and Secondary Vascular Systems C. Determination of Plasma Skimming D. Pressure in the Secondary System V. Functional Aspects of the Secondary Vascular System VI. Evolution of Lymphatic Vessels References

I. INTRODUCTION The aim of this chapter is to discuss recent advances in our knowledge of that part of the vascular system in fish that has up to now been described under the heading “lymphatic vessels.” Until recently teleosts have been believed to possess a lymphatic vessel system of basically the same nature as that known to exist in 185 FISH PHYSIOLOGY, VOL. XIIA

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mammals (Bertin, 1958;Ruszniak, Foldi, and Szabo, 1967; Kampmeier, 1969;Prosser, 1973;Romer and Parsons, 1977;Casley-Smith, 1983).A mammalian-type system consists of terminal lymphatic vessels or networks of lymphatic capillaries originating in various tissues. These initial lymphatics coalesce to form larger and larger lymphatic trunks, which finally empty into the systemic venous system. In mammals a great deal is known about its role in returning tissue fluid to the venous system and thus contributing to the maintenance of normal tissue fluid balance. The importance of this is evident from the pathological conditions of edema seen if, for some reason, the normal flow of lymph has been obstructed (Foldi and Casley-Smith, 1983;Adair and Guyton, 1985). In contrast, cyclostomes and elasmobranchs were believed to have a less differentiated circulatory system with no clear distinction between lymphatic and venous vessels (Kampmeier, 1969; CasleySmith, 1983). The existence of a piscine lymphatic system was challenged when Vogel(1981a,b)and Vogel and Claviez (1981)first described what was termed the secondary vascular system. This system constitutes a separate, parallel circulatory system and includes the vessels hitherto described as lymphatics. It originates from systemic arteries, forms its own capillary networks, and returns to the systemic venous system. The history of the study of fish lymphatics is interesting. Among the many older anatomical papers, several describe observations not complying with the concept of a lymphatic system in the strict sense of the word. More than 100years ago Jourdain (1880)described circulation in the fins of small flatfish. Besides arteries and veins, he saw vessels along the fin rays containing no red cells but some white cells. Some of these vessels showed flow into the fin, and some showed flow returning from the fin. Accordingly he spoke of a circulation of lymph comparable to the circulation of blood. Mayer (1917)confirmed and extended these observations and was able to observe capillaries connecting “lymph arteries” and “lymph veins.” He was clearly aware that he was looking at something different from a blind-ending lymphatic system with flow in only one direction, and concluded that these vessels should not be called lymphatics. Instead he used the term “Weissadern” (white vessels). Wardle (1971)proposed that the observations of lymph circulation in the fins of flatfishes could be explained by different vessels along the fin rays being connected to different lymph sinuses with different pressures. In his study of the sinus system of lampreys, Tretjakoff (1926) concluded that they were not lymphatic but were connected to the

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arterial system. He also argued that certain vessels in elasmobranchs considered to be lymphatics should rather be compared to the Weissadern of Mayer (1917). Finally, Burne (1926, 1929) was very close to discovering the secondary vascular system (see Section 11,A). Although none of this work was conclusive, it seems, at least in retrospect, that it called for further investigations. Instead these authors were largely forgotten and through the work of Glaser (1933),Weidenreich et al. (1933), and Hoyer (1938), it was concluded that teleosts have a lymphatic system basically similar to that of mammals. It is difficult to avoid the impression that a preconception of the traditional concept of the mammalian lymphatic system prevented the realization of the different layout of this part of the circulatory system of fish (Vogel, 1981a). With this chapter we want to draw the attention of physiologists to the secondary circulatory system, with the result we hope, that it is not forgotten once more. The chapter will largely be confined to a treatment of the systemic part of the secondary circulation. The arteriovenous vascular circuit of the gills may be considered the branchial part of the secondary circulation (Vogel, 1978a). It has been studied much more intensively than the systemic secondary vascular system, and several reviews concerning morphological as well as physiological aspects are available (Laurent and Dunel, 1976; Laurent, 1982,1984). 11. MORPHOLOGY OF THE SECONDARY

VASCULAR SYSTEM

A. Teleosts The discovery of the secondary vascular system may be said to have begun with the elucidation of the structure of the nonrespiratory vascular pathway of the gill (Vogel et al., 1973; Laurent and Dunel, 1976; Laurent, 1984). Briefly, this pathway consists of small anastomoses branching mostly from the efferent vessels of the gill and leading to the central venous sinus of the gill filament and through branchial venous vessels to the general venous circulation (Randall, 1985).The central venous sinus was thus found not to be of a lymphatic nature as previously thought, but to be part of a vascular pathway parallel to the respiratory pathway. A somewhat similar pathway arranged in parallel to the normal system of arteries, capillaries, and veins was shown to be present throughout the body in several teIeosts as well as the holosteans Amia

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and Lepisosteus (Vogel, 1981a,b; Vogel and Claviez, 1981; Vogel, 1985a). The term secondary vascular system was proposed for these vessels and accordingly, the normal vascular system was termed the primary vascular system, The secondary system originates from tiny vessels with a diameter of about 10 to 14 pm branching from primary arteries. They are irregularly curled, often branching, and occur in clusters. The glomeruli vasali mentioned by Giacomini (1922) are undoubtedly identical to these vessels (Vogel, 1981a). The small vessels immediately merge to form secondary arteries and are hence termed arterioarterial anastomoses (Fig. 1).Around and particularly upstream to the opening of the arterioarterial anastomoses a cluster of microvilli covers the wall of the primary artery (Fig. 2). Arterioarterial anastomoses are found along the aorta and segmental arteries of the trunk and tail (Vogel, 1981a;Vogel and Claviez, 1981; Vogel, 1985a; Steffensen et al., 1986). From the work of Burne (1926, 1929)we can safely say that anastomoses occur along various arteries of the head region as well. The secondary arteries are normally smaller than the primary ones and run closely parallel to them. The system of “fine vessels” described by Burne (1926,1929) is clearly identical to secondary arteries. The smallest ramifications of the fine vessels (Fig. 3) are certainly identical to arterioarterial anastomoses. Since Burne’s injection material failed to pass through these, he could not prove the existence of an open communication with the arterial system. In section Burne (1929) observed that the walls of the secondary arteries were quite thick and resembled those of small primary arteries. The “satellite” vessels described by Mosse (1980) are also quite clearly identical to secondary arteries. The secondary arteries supply secondary capillary beds of their own. These beds are distributed to the skin of the body surface, the fins, the surfaces of mouth and pharynx, and to the peritoneum (Vogel, 1985a).According to Vogel there may be some doubt as to the presence of primary capillaries in the skin of trout, Onchorhynchus mykiss, and tilapia, Sarotherodon mossambica. In the flounder, Plucthithysflesus, both types of capillaries are present (Vogel, 1985, pers. comm.; Jakubowski, 1960). In the glass catfish, Kryptopterus bicirrhis, secondary capillaries are absent from the general body surface and are present only in the fin membranes (Fig. 4). Burne (1929)could not find the fine vessels in the intestinal tract. Vogel and Claviez (1981) also found secondary vessels to be absent from the intestinal tract as well as from the central nervous system. Later Vogel (1985a) said that secondary

Fig. 1. Scanning electron micrograph of a corrosion cast showing the arterioarterial anastomoses between the primary and secondary arteries in Oncorhynchus mykiss. The origins of the anastomoses are marked by arrowheads. From Vogel(1985a).

Fig. 2. Scanning electron micrograph of the wall of a segmental artery from the luminar side. An anastomosis with specialized endothelial cells is indicated by small m o w . Flow direction is indicated by large arrow. Tentacular microvilli are found around and especially upstream from the anastomoses. From Vogel(1981a).

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Fig. 3. Burne’s illustration ofthe “fine vessels” (black)surroundinga hypobranchial artery (white) in angler fish, Lophius piscatorus. From Burne (1926).

vessels may be present in the intestine, but no details were given. Based on the description of intestinal lymphatics by Glaser (1933) it may seem probable that they are present, but further study is required to clarify this. The secondary capillaries are in turn drained by secondary veins, which empty into the primary venous circulation. The secondary veins are often of a rather large caliber, thin walled, and may show an irregular outline (Fig. 5). The many descriptions of lymphatic vessels in the anatomical literature (Kampmeier, 1969; Wardle, 1971; Rowing, 1981)(Fig. 6) are in most cases venous vessels ofthe secondary system. The secondary veins drain into the primary venous system close to the

Fig. 4. Photograph of distal part of the anal fin containing only vessels of the secondary system in a live glass catfish, Kyptopterus biciwhis. Number legends in Fig. 8. Bar = 250 pm. From Steffensen et al. (1986).

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Fig. 5. Photograph of the ventral part of the trunk and the proximal part of the anal fin in a live glass catfish, Ktyptopterus bicirrhis. Number legends in Fig. 8. Bar = 250 pm. From Steffensen et al. (1986).

heart and in the tail, often via a caudal heart (Vogel, 1985b; Chapter 3 in this volume). The number and diversity of species in which the secondary system has been observed seem to be sufficiently large to justify the statement that the system is of general occurrence among the teleosts. In Fig. 7 a schematic diagram of the circulation of a teleost is shown. Many details of its distribution remain to be worked out, and most likely many differences among species will appear. At present the answer to the question whether teleosts do have lymphatics at all seems to be no (Vogel, 1981a; Vogel and Claviez, 1981; Vogel, 1985a). Until the status of the intestinal circulation has been definitely clarified, it may, however, be best to leave the question open.

B. Cyclostomes The anatomy of the circulatory system of the hagfishes has been studied for more than 150 years, and the older anatomical literature has been reviewed by Kampmeier (1969). A most conspicuous feature of hagfishes is the occurrence of large

Fig. 6. Generalized scheme of systemic lymphatics (white), veins (black), and arteries (grey) in teleosts. From Kampmeier (1969).

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Fig. 7. Vogel’s concept of the primary and secondary circulatory systems in a typical teleost. From Vogel(1985a).

subcutaneous and other sinuses, which have often been observed to contain variable amounts of red blood cells. Some authors were inclined to consider the occurrence of red cells in the sinuses as an artifact from the stress of bringing the animals up from great depth (Retzius, 1890; Jackson, 1901; Grodzinski, 1926).Others preferred to ascribe to the sinuses and “red lymphatics” a mixed venolymphatic function (Klinkowstrom, 1891; Cole, 1912, 1925; Allen, 1913). Cole (1912, 1925) described hollow papillae on the branchial and common carotid arteries, and believed that red blood cells could enter the system of sinuses by way of these “curious structures.” He also suspected that such connections would exist at other points in the arterial system. The return of sinus fluid to the venous system via accessory hearts is discussed in Chapter 3 in this volume. Grodzinski (1932) confirmed the presence of Cole’s vascular papillae but expressed doubt as to their continuity with the sinuses. The papillae were observed by Pohla et al. (1977),but they were not sure if they did form an open communication between the branchial artery and the peribranchial sinus. They did, however, find other communications

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between radial gill arteries and adjacent sinuses and also observed these sinuses to communicate with the peribranchial sinus. Elger (1987) presented a detailed description of the communications between the gill arteries and sinuses and concluded that the sinuses of the hagfish gill correspond well to the central venous sinus of the gills of other fishes. Johansen et al. (1962)found that sinus fluid and blood plasma were identical with respect to concentration of dissolved protein, and that sinus fluid showed a hematocrit that was variable but lower than that of blood. They concluded that the sinuses are not lymphatic spaces, but are perfused with blood, which undergoes plasma skimming as it enters the sinuses (Johansen, 1963). Plasma skimming occurs as a consequence of the tendency of cells to concentrate in the central stream of a vessel. A small sidebranch will drain off the relatively cell-free plasma flowing along the vessel wall, and thus show a lower hematocrit than that of the parent vessel (Krogh, 1930). Previously the circulation of a fraction of the blood through the sinuses and “red lymphatics” would have seemed to be just another among several unusual traits of the hagfishes. The arrangement clearly resembles that of the secondary circulation now believed to be a general characteristic of the teleosts. In the light of this, the occurrence of red cells in hagfish sinuses and “red lymphatics” may turn out to be less puzzling. Certain vessels of the hagfishes have never been observed to contain red cells and accordingly have been termed “white lymphatics” by Cole (1925).Johansen et al, (1962)measured protein concentration of fluid from white lymphatics and found the value to be less than half the value of blood plasma. They concluded that the white lymphatics are true lymphatics, i.e., vessels that carry an ultrafiltrate of plasma from the interstitial space back into the circulation. Various lymphatic vessels and sinuses have been described in lampreys (Kampmeier, 1969).As in hagfishes, the occurrence of red blood cells in parts of the system has raised doubts about their true lymphatic nature (Tretjakoff, 1926). The occurrence of red cells in these vessels suggests something in the manner of a secondary circulatory system, unless it is just an artifact, as held by Hoyer (1934). C. Elasmobranchs The anatomical literature reflects the same difficulties in defining lymphatic vessels as is the case in the Cyclostomes. According to Glaser (1933), the only vessels that can be said with certainty to be

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lymphatics are found in the intestine of Torpedo and related rays. Skates and sharks were stated not to have true lymphatics. Large subcutaneous vessels have been claimed to be lymphatics, but most authors have held them to be veins (Kampmeier, 1969). Mayer (1888)observed these subcutaneous vessels to contain variable amounts of red blood cells. This suggests that they may belong to a secondary circulation, a view held by Satchell in Chapter 3 in this volume. Arterioarterial anastomoses have, however, not been demonstrated in elasmobranchs. The nonrespiratory vessels of the elasmobranch gill are basically similar to those of teleosts (Cooke, 1980). If these vessels in the gill are accepted to be the branchial part of a secondary circulatory system, their occurrence suggests that elasmobranchs also have a secondary circulatory system. The similarity of the caudal hearts of elasmobranchs and teleosts lends further support to this suggestion, as discussed by Satchell in Chapter 3.

111. In Vivo MICROSCOPIC OBSERVATIONS The vascular corrosion casts demonstrated in teleosts (Fig. 1) the communication between primary and secondary arteries through the arterioarterial anastomoses (Vogel, 1981a,b; Vogel and Claviez, 1981). Filling the secondary system from the primary arteries was, however, technically difficult and required the use of vasodilatory drugs and sometimes the blocking of the primary microcirculation by the injection of microspheres (Vogel, 1985a). Furthermore, electron micrographs of sectioned anastomoses from gills showed them to be more or less completely closed (Vogel et al., 1974). These observations raised questions as to the state of the anastomoses in viuo. The secondary circulation has been observed in vivo in the transparent glass catfish, Kryptopterus bicirrhis (Steffensen et al., 1986). Unanesthetized fish were placed in a dish of water and observed under the microscope. In the ventral part of the tail, the entire secondary circulation could be observed from the arterioarterial anastomoses through secondary arteries to secondary capillaries in the membrane of the anal fin and finally to secondary veins. A semischematic representation of the secondary circulation in this part of the fish is given in Fig. 8. Groups of anastomoses occur along primary segmental arteries and give rise to one or two secondary arteries, which lead to the membranous part of the anal fin. Secondary arteries along both sides of the fin rays give rise to characteristic long, straight, secondary capillaries, which run across the fin membrane to join secondary veins along the

Fig. 8. Semidiagrammatic drawing of the vascular system of the ventral part of the trunk of the glass catfish, Kryptopterus bicirrhis, between the anus and the caudal fin. Redrawn from Steffensen et al. (1986). Number legends for figures 4, S, 8, and 9: 1, vertebra; 2, henial arch and ventral spinal process; 3, fin supporting bone (radial); 4, fin ray; 5, dorsal aorta; 6, caudal vein; 7 , longitudinal secondary venous collecting vessel along ventral aspect of spinal column (secondary subvertebral vein); 8, group of arterioarterial anastomoses; 9, primary segmental artery; 10, secondary segmental artery; 11, capillary of primary vessel system; 12, electroreceptor; 13, capillary of secondary vessel system; 14, primary vein; 15, secondary vein along fin ray; 16, secondary venous collecting vessel along base of anal fin; 17, distal margin of anal fin membrane.

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opposite fin rays (Fig. 4).Most of the secondary veins along the fin rays join a large secondary vein in the base of the fin (Fig. 5 ) , but some proceed centrally to join another, smaller secondary vein just ventral to the spinal column. The secondary capillaries are the only capillaries in the fin membrane, whereas the rest of the body surface is supplied exclusively by capillaries from the primary circulation. The observations on the glass catfish showed a very pronounced plasma skimming as blood passed through the anastomoses. Vogel and Claviez (1981)also found very few cells in the secondary vessels. They saw the plasma skimming as a result of a closing down of the secondary system, which they believed to be caused by stress. The observations on the glass catfish suggest that plasma skimming and thus a paucity of cells in the secondary vessels is the normal condition. The microscopical observations of course imposed a certain degree of stress, but the fish behaved normally immediately after return to the holding aquarium. Rather, if a fish was in a bad condition, showing reduced blood flow and signs of stasis in the primary microcirculation, flow through the secondary system would become so sluggish that cells would begin to settle and accumulate in the secondary vessels (Lomholt and Steffensen, 1984, unpublished observations). Two features of the anastomoses may contribute to the plasma skimming. One is the rather wide opening, which only gradually tapers into capillary dimensions as evidenced by the deformation of passing cells (Fig. 9).The larger diameter at the opening will result in a lower velocity, and this may augment the skimming of only the relatively cell-free layer of plasma along the vessel wall. Occasionally very small primary arteries are seen to branch off directly from large ones. In that case the diameter of the small vessel is constant from the very beginning, and no substantial plasma skimming is observed (Lomholt and Vang, 1990, unpublished). The other feature is a cluster of microvilli around and immediately upstream from the opening of the anastomoses (Fig. 2). Shortly upstream of an anastomosis, single blood cells are seen in the glass catfish to slow and eventually to pass through. This slowing is observed only rarely along other parts of the arterial wall. In a few cases a simple, unbranched anastomosis was observed with a cell stuck inside and thus blocking the flow. The slowing of cells

Fig. 9. Photographs from video recording showing a red cell at two different stages of passage through an arterioarterial anastomosis in the glass catfish, Kryptoptems bicirrhis. The line drawing is based on the original video *cording. Blood-flow direction is indicated by arrows. See Fig. 8 for number legends. From Steffensen et al. (1986).

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persisted, but they could not enter the anastomosis. This indicates that the slowing of the cells does not depend on a flow through the anastomosis (Lomholt and Vang, 1990, unpublished). Microvilli are not visible in vivo and have not yet been looked for with electron microscopy in the glass catfish. Since they have been demonstrated in several species of fish (Vogel, 1981a), it seems probable that the observed slowing of cells is somehow dependent on their presence in the glass catfish as well. Based on these observations, it appears that the role of the microvilli may not be an augmentation of plasma skimming, but rather may be to assure that at least some cells do enter the secondary system. White blood cells show a tendency to drop out of the axial stream of blood and move slowly along the vessel wall (Schmid-Schonbein et al., 1975).It is tempting to see the opening of the anastomosis as a system, which could favor the passage of white blood cells into the secondary vessels. At certain points in the venous part of the secondary circulation where velocity is sufficiently low, red and white cells can sometimes be distinguished in vivo. In fact, the ratio of white to red cells appears much higher in the secondary system than would be expected in full blood (Lomholt and Vang, 1990, unpublished). This observation is in accordance with Wardle (1971), who found roughly the same number of white cells in blood and “lymph.” He considered the small number of red cells also found in lymph as an artifact. Practically nothing is known about control of flow through the secondary system apart from the fact that it may be almost shut off as a result of preparatory procedures in connection with anatomical studies. Observations on the glass catfish have shown that flow may vary considerably between neighboring segments. Whether these variations reflect resistance changes in the anastomoses or in the peripheral secondary vessels is not known. At the capillary level local variations in flow have been observed. Figure 10 shows an example of how flow in two adjacent secondary capillaries of the anal fin membrane may change independently. These changes can be correlated with visible changes in the lumen of the arterial end of the capillaries. Observations of this sort may suggest that the secondary capillaries are equipped with a precapillary sphincter (Lomholt and Vang, 1990, unpublished), The secondary venous system is connected to the primary system anteriorly as well as in the tail via a caudal heart. The movements of the caudal heart may sometimes be observed, but details of its structure are not visible in vivo and have not been investigated anatomically in the glass catfish. As a consequence of the dual connection of the secondary venous system to the primary circulation, the direction of flow

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Number of cells pr. 10 sec.

60 I

A

u IL

B

14 12 10

8 6 4 2

0

1

2

3

4

Time, min Fig. 10. Spontaneous changes in flow, as judged from the number of cells passing, in two neighboring secondary capillaries (A, B) in the glass catfish, Kryptopterus bicirrhis. The flow changes independently in the two vessels, showing that they are capable of individual, independent changes in resistance. Lomholt and Vang (unpublished).

may b e seen to reverse in parts of the system from time to time. Normally the secondary flow from the anal fin is drained anteriorly via the large secondary vein at the base of the fin. Sometimes fluid from the posterior segments of the anal fin is drained toward the tail and returned via the caudal heart, together with flow from the caudal fin. Flow in the longitudinal secondary vein ventral to the spinal column is quite low compared to the vein at the base of the anal fin and often shows reversals.

202

J. F. STEFFENSEN AND J. P. LOMHOLT

IV. PHYSIOLOGICAL EXPERIMENTS A. Volume of the Secondary Vascular System

Blood volumes of fishes have typically been determined either by the dye dilution technique, or by radioactively labeling red blood cells or a plasma protein, i.e., albumin, and extrapolating to concentration of tracer at time zero (Conte et al., 1963; Bernier et al., 1981).There have been numerous determinations of the blood volume of fish and Mott (1957), Holmes and Donaldson (1970),and Satchel1(1971)give a selection of values derived from the earlier literature. Blood volume ranged from approximately 3 4 % of body weight. Many authors have, however, indicated that complete mixing of the injected dye with the venous blood is a very slow process. By allowing 4-5 hr to elapse before blood sampling, higher blood volume values are obtained, according to Smith and Bell (1964). Smith (1966) mentioned that blood volumes estimated from plasma samples taken 20,25, and 30 min after injection were 35% less than from a second estimate taken 1,2, and 3 hr after the dye injection. Nikinmaa et al. (1981) took four blood samples for estimation of blood volume at 40 min, 1hr, 1hr 30 min and 2 hr, but discarded the 40-min sample as it invariably failed to fall on the dilution line of the three other samples. Further, it has been found that depending on the type of tracer, different volumes of blood are obtained. Conte et aE. (1963) reported that the rate of loss of tracer from the vascular compartment, or primary circulatory system, was greatest for dye, less for human serum a l b ~ m i n - ~and ~ ~least I , for 'lcr-labeled red blood cells. It has been suggested that the capillary wall in teleosts is permeable to plasma proteins based on observations of Nichols (1987),Gingerich et al. (1987), and Duff et al. (1987).They found that blood volume based on plasma protein markers was 25-40% higher than values based on labeled red blood cells, and they concluded that some of the plasma protein had leaked from the blood vessels. From visual observations of the glass catfish (Steffensen et al., 1986), it was obvious that the volume of the secondary system was considerable compared to that of the primary system. The longitudinal collecting vein of the secondary system at the fin base is by far the largest vessel in this fish. These observations initiated a study of the blood volume of the primary and secondary system ofthe rainbow trout utilizing plasma protein and red blood cell markers simultaneously, based on the assumption that the red blood cells would stay in the primary system, while the plasma would equilibrate with the total

4. THE

203

SECONDARY VASCULAR SYSTEM

volume of the primary and secondary system (Steffensen et al., in prep.). The experiment confirmed that the red blood cells stayed within the primary system during a 24-hr period, and that the plasma proteins equilibrated between the primary and secondary systems (Fig. 11).The decay of marked plasma protein in the primary system was solved with a curve-fitting program assuming a twocompartmental recirculation between the primary and secondary system and a constant, concentration-independent, one-way Ieakage of plasma from the two systems (equal to the slope of the dotted line in Fig. 11). The volumes of the primary and primary plus secondary

Plasma

-2 350.-

131

,

RBC51 Cr

E

2 300c C

a

0

250-

5

10

15 TIME, hr

20

25

Fig. 11. Dilution curves of "Cr-labeled red blood cells and human serum albumin '%I injected simultaneously in the dorsal aorta, and sampled from the same vessel. From Steffensen et u1. (in preparation).

204

J. F. STEFFENSEN AND J. P. LOMHOLT

systems were 3.4and 8.3%of body weight, respectively (Steffensen et al., in preparation). This means that the volume of the secondary system is approximately 1.5 times that of the primary system. Most of the previous estimations of blood volume fall within this range of 3 4 % of body weight, and the variation can to a large extent be explained by the time allowed for equilibration and the duration of the sampling. The observations of Steffensen et aZ. (in prep.) agree with those of Nichols (1987), Gingerich et d. (1987), and Gingerich and Pityer (1989), in that the estimated plasma volume is larger than would be expected from the marked red blood cells. Nichols (1987),however, was not aware of the existence of a secondary system, whereas Gingerich and Pityer (1989)suggested that the expanded plasma volume was the result of either the distribution of erythrocyte-poor blood in the secondary circulation, or the result of extravascular exchange of plasma proteins. Gingerich and Pityer (1989)and Gingerich et al. (1990) suggested that owing to leakage of plasma protein markers to the extracellular space, their estimation of plasma volume may be too great. According to Steffensen et al. (in preparation), the leakage of plasma proteins from the primary system was found to be less than 1-2% per hour, and without importance to the volume determination of the primary and secondary circulation, since the curve-fitting program incorporated, calculated, and corrected for the leakage. Hence the expanded plasma volume can be ascribed solely to the volume of the secondary circulation.

B. Exchange between the Primary and Secondary Vascular Systems From the curve-fitting analysis of data to the two-compartmental wash-out of labeled plasma proteins from the primary to the secondary system in resting rainbow trout, used to estimate blood volume, the flow between the primary and secondary circulation was found to be 2.5 2 0.8 ml/hr for fish with an average weight of0.378 kg (See Table I). This is only approximately 0.3% of the total cardiac output at rest, found to be 13 ml/min for similar size fish (Randall and Daxboeck, 1982).Knowing the volume of the primary and secondary systems, the washout between the two systems can be calculated for different flows. Figure 12 illustrates the wash-out curve for the empirically measured flow of 2.5 ml/hr as well as calculated curves assuming blood flows of 5 and 1.25 ml/hr. The figure illustrates the effect of flow on equilibration time. The half-times for equilibration are indicated by lines.

4.

205

THE SECONDARY VASCULAR SYSTEM

Table I Blood Volume of the Primary and Secondary Circulatory Systems of Rainbow Trout, Oncorhynchus mykiss, Determined Simultaneously with 51Cr and lZ5I

Plasma volume of primary system, ml Plasma volume of primary

+ secondary systems

Mean

?

8.8

k

SD

4.7

27.1 k 7.2

Plasma volume of secondary system, ml

18.3

*

5.4

Plasma volume ratio secondary: primary

2.2

4

0.7

3.8 2 1.7

Volume of RBC in primary system, ml Volume of RBC

+ plasma in primary system, ml

3.4

Percent of body weight Volume of RBC

12.6 f 4.3

+ plasma in primary + secondary systems, ml

Percent of body weight

*

1.3

30.9 5 8.8 8.3

*

2.5

Blood volume ratio secondary: primary

1.5 4 0.4

Flow between primary & secondary systems ml/hr

2.5

2

0.8

Time for 50% wash out, hrs

1.8

*

0.7

Time for 95% wash out, hrs

7.8 2 3.0 378

Body weight, g

"N

= 7. Values from Steffensen et

k41

al. (in preparation).

From previous experiments aimed at determining blood volume, mixing was found to be slow, and a higher blood volume was obtained the longer the period from injection of dye to sampling (Smith and Bell, 1964; Smith, 1966; Nikinmaa et al., 1981; Nichols, 1987; Gingerich et al. 1987). Most of the differences in estimated blood volume may be explained by the effect of the time of sampling after injection of the marker, due to the slow equilibration between the primary and secondary systems, illustrated in Fig. 12. Solid bars in Fig. 13 show the period of sampling. The figure indicates how, in earlier experiments, a longer period of equilibration before sampling, and a longer duration of blood sampling, tend to result in a larger estimate of blood volume. Dotted lines in Fig. 13 show the estimate of the volume of the primary system and the primary plus secondary system, based on the twocompartmental model. The blood volumes resulting from the shortest sampling periods of earlier investigators are seen to correspond to the volume of the primary system calculated on the basis of the twocompartmental model. The highest values from the literature are lower

206

J. F. STEFFENSEN AND J. P. LOMHOLT WASH-OUT BETWEEN PRIM. & SEC. SYSTEMS Prim.Vol. 8.8 ml - Sec.Vol. 18.3 ml

5

e 2

2010-

Fig. 12. Theoretical wash-out curves for a 378-g rainbow trout with a volume of the primary system of 3.8% body weight, primary plus secondary system of 8.3% body weight, and flows between the two systems of 1.25,2.5, and 5 ml/hr. From Steffensen et al. (in preparation). Straight lines indicate half times at the respective flow rates.

than the value of the combined volume of the primary plus secondary systems resulting from the two-compartmental analysis. The reason for this is that the time allowed for equilibration by previous investigators was too short. From Fig. 11 and Fig. 12 it is evident that equilibration between the two systems lasts from 8 to 12 hr. In agreement with this, Forster et aZ. (1989) demonstrated that approximately 30% of the total blood volume of the hagfish, E ptatretus cirrhatus, was contained within the subcutaneous sinus, and that at least 8 hr was necessary to get thorough mixing of central and sinus blood. From in vim observations on the glass catfish (Steffensen et al., 1986), variations of hematocrit and flow in the secondary system were evident. The innervation of a sphincter in the efferent branchial artery in teleosts has been studied by Bailly and Dunel-Erb (1986) and Dunel-Erb and Bailly (1986). A similar system may be involved in control of blood flow and plasma skimming from the primary to the secondary system through the non-gill arterioarterial anastomoses, but no experiments have addressed this question. Variation in hematocrit in the primary system has been observed by several authors, and has been explained by either red blood cell swelling/shrinking (Nikinmaa, 1983), or release of erythrocytes from

4. THE SECONDARY VASCULAR SYSTEM

E0,

9-

I

8-

.-

207

JFS et al. (In Prep.): Vol of primary + secondary systems. .............

h

U

0

P $

5-6 >o

7-

sm

6-

mw

'Id 54-

~

- ni

8

........................... CO 3-Im

u

2-

U

3

.gi-90 st 91-89

JFS et al. (In Prep.): Vol. of prim

............

Q)

4-

a

.-E

1-

4-

8

0o

5

10

15

20

25

Fig. 13. The effect of the time allowed for equilibration after injection of marker, and of the period of sampling (indicated as horizontal bars), on the estimated blood volume from previous experiments, yi, Yamamoto and Itazawa (1989); co, Conte et al. (1963); ni, Nikinmaa et al. (1981); sm, Smith (1966); hd, Houston and DeWilde (1969); mw, Milligan and Wood (1982); st, Stevens (1968);gi-89, Gingerich and Piteyer (1989); gi-90, Gingerich et al. (1990). Redrawn from Steffensen et al. (in preparation). Further explanation in text.

the spleen, as well as water shifts out of the blood vessels (Yamamotoet al., 1980; Yamamoto and Itazawa, 1989). Yamamoto et al. (1980) reported that 35% of a hematocrit elevation in exercised carp, Cyprinus carpio, could be ascribed to water shifts out of the blood vessels. How large a proportion of this water shift can be ascribed to the movement of a plasma from the primary to the secondary system is not known, but the possibility should be considered. Changes in hematocrit may also be caused by changes in plasma skimming. The functional significance of the flow of plasma between the primary and secondary system is not known, but must depend on the time required to exchange plasma from one system to the other. From Steffensen et a2. (in preparation) the flow between the two systems was determined only during rest, but a possible increase during or after exercise will decrease the time for equilibration, and must not be neglected in acid-base status experiments, since the total plasma volume is approximately three times as large as the plasma volume in the primary system.

208

J. F. STEFFENSEN AND J. P. LOMHOLT

C. Determination of Plasma Skimming Observations by Iwama et al. (1990) on cannulated rainbow trout gave hematocrit values in the secondary system from 1.2 to 1.7%. Similarly, samples from the secondary vein along the lateral line showed hematocrits of 0.5 to 1%whether taken acutely from anesthetized fish or from a permanently implanted cannula (Lomholt and Steffensen, 1984, unpublished). These observations support the view that a low hematocrit in the secondary system is the normal condition and not the result of stress. In uiuo analysis of the partitioning of cardiac output in rainbow trout, based on blood sampled from the branchial (secondary) vein, dorsal aorta, and sinus venosus, showed that plasma skimming occurred in the gills, since hematocrit and hemoglobin concentration was only 3.5% and 1.04 g/100 ml, respectively, in the branchial vein (Ishimatsu et al., 1988). The systemic flow, estimated from hemoglobin concentration, was reported to account for 93% of the total cardiac output, and the remaining 7% of the cardiac output shunted through the central venous sinus (CVS). According to Vogel(1985a), the central filament arteries of Fromm (1974) are secondary arteries, connected to the systemic secondary vessels (Vogel, 1978a). Similarly, the central venous sinus of the gill filaments and the branchial venous vessels belong to the secondary system. This may involve problems in the calculation of the partitioning of the cardiac output if indeed low hematocrit blood from systemic secondary vessels is drained into the CVS and branchial veins. The result will be a dilution of the CVS fluid and hence an overestimation of plasma skimming in the gills. Further, Randall (1985) suggested that interstitial fluid presumably drains into the central venous sinus. If so, this will also cause an overestimation of plasma skimming. Ishimatsu et a!. (1988)found more red blood cells in the fluid from the branchial vein of fish undergoing surgery than in recovered, resting fish. This can either be owing to less plasma skimming in the gills, allowing more red blood cells to pass, or a decrease in a possible low hematocrit supply to the central venous sinus from the systemic secondary vessels.

D. Pressure in the Secondary System The pressure in the secondary system is not known, but it must be lower than that in the primary system. Farrell and Smith (1981) recorded pressures between 23 and 49 cm HzO in the primary filamental

4.

THE SECONDARY VASCULAR SYSTEM

209

arteries in the gills of anesthetized lingcod, Ophiodon elongatus. Two pressure measurements were substantially lower, approximately 6 and 9 cm HzO, respectively, and were believed to represent blood pressure in the “venolymphatic” system, that is, a branchial secondary vessel. Part of the fluid in the secondary system is pumped back into the primary system via the caudal heart to the caudal vein. The literature offers few references to venous blood pressures in fishes (see Satchell, Chapter 3 in this volume). The presence of a caudal heart suggests that the pressure in the secondary system afferent to the caudal heart is lower than that in the primary venous system. According to Wardle (1971), the pressure in the neural lymph duct, which is a secondary vein, is negative due to a pumping action generated by respiratory movements. V. FUNCTIONAL ASPECTS OF THE SECONDARY VASCULAR SYSTEM

Speculations about the possible functions of the secondary vascular system will depend on whether the plasma skimming resulting in a low hematocrit in the system is considered to be the normal state of affairs or whether it is an artifact resulting from stress. The difficulties in injecting the system led Vogel(1981a, 1985a)and Vogel and Claviez (1981) to propose that stress causes the secondary system to close down and that this closure is the cause for the few red cells found in the system. They thought that if stress could be avoided, the system would be more open, and a higher hematocrit should be expected. In contrast to this, the in uiuo observations (Steffensen et al., 1986) show that a high flow in the system does not result in a high hematocrit in the secondary vessels. In other words, a high flow appears to be fully compatible with a high degree of plasma skimming. Only in cases of near stasis in the secondary system is an accumulation of red cells observed. As a consequence of this, we believe that attention should be focused on possible nonrespiratory functions of the secondary vascular system. Wardle (1971) discussed the possibility that “lymph” to some extent functionally replaces blood with respect to the release of lactic acid from muscle. According to present knowledge, this is not possible, since neither lymphatic nor secondary capillaries have been found in muscle. Wardle (1971) observed the protein concentration of what he believed to be the neural lymph duct to be 80.4% of that in the blood

210

J. F. STEFFENSEN AND J. P. LOMHOLT

plasma. The neural lymph duct now must be considered to belong to the secondary vascular system. Why the protein concentration was not identical to that in the plasma is not known. If the difference in protein concentration is correct, it would indicate that the secondary system may functionally work as a lymphatic system. Our own unpublished observations on colloid osmotic pressure on plasma from the primary and secondary systems from rainbow trout show identical values, and thus do not agree with the observations of Wardle. Wardle (1971),even though he was aware of Burne’s work (1926,1929),had no doubt about the existence of the lymphatic system, but stated that “nearly all substances present or injected in the blood will be present in the lymph so that estimates of the total amounts of a circulating substance based on measurements of blood levels must also take into account the volume of circulating lymph” (p. 987). The respiratory gas exchange of the skin of fishes has been shown to take place directly with the surrounding water (Kirsch and Nonotte, 1977; Nonotte and Kirsch, 1978; Nonotte, 1981; Steffensen et al. 1981; Steffensen and Lomholt, 1985).An obvious implication is that secondary capillary networks in the skin may supply nutrients, particularly to the mucus-producing cells. There is evidence that the secondary circulation of the gill plays this role in relation to the salt-secreting chloride cells (Laurent, 1984). Based on the observation that secondary capillary networks are restricted to surfaces, that is, to potential sites of infection, Vogel (1981a) proposed that the secondary system may be of importance to the distribution of white blood cells. As mentioned in Section 111, there is evidence that the process of plasma skimming favors the entrance of white cells into the secondary system. If it is accepted that lymphatic vessels are not present, questions arise concerning two important functions known to be carried out by the mammalian lymphatic system. The first is the specialized function of the intestinal lymphatics in the transport of lipids, and the other is the general role of the lymphatic system in the maintenance of tissue fluid balance. Although the status of the secondary system in the intestine may not be definitely clarified, it appears certain that fish do not have vessels comparable to the lacteals of the intestinal villi of mammals. In mammals, absorbed lipid is incorporated into chylomicrons, which are transferred to the lacteals and transported from the intestine by lymphatic vessels. It has been claimed that in fish, lipids are transferred directly to the blood stream in the form of fatty acids (Robinson and Mead, 1973; Kayama and Ijima, 1976). Later investigators found that lipid particles much like chylomicrons are formed in fish (Sire et al.

4. THE

SECONDARY VASCULAR SYSTEM

211

1981). If this is so, the lipid particles must somehow be taken up and transported from the intestine by primary blood vessels. Vogel(1981a) considered the possibility that the secondary vascular system may fulfill functions in tissue fluid balance similar to those ascribed to the mammalian lymphatic system. In the light of our present knowledge of the restricted distribution of the secondary system to the surfaces of the fish, it seems improbable that it could play a general role in regulation of tissue fluid balance. Furthermore, the function of the mammalian lymphatics depends on the special valvelike structure of the blind-ending terminal lymphatics (Adair and Guyton, 1985). Knowing the serious consequences of malfunctioning lymphatic vessels in mammals (Johnston, 1985), the question arises how fish can function without this system. Part of the answer may rest with the lower blood pressure of fish compared to mammals (Hargens et a2. 1974). More generally, the answer may have to do with the fact that fish live surrounded by a medium of a density roughly equal to that of their own bodies. As a consequence of this, their vascular systems are not subjected to hydrostatic pressure gradients as are those of terrestrial animals. The dogfish, Mustelus canis, has been found to be susceptible to circulatory impairment caused by gravitational stress resulting from a head-up tilt out of water (Ogilvy and DuBois, 1982), In contrast the bluefish, Pornatornus saZtatrix, was found to tolerate gravitational stress quite well. This was seen as an adaptation to resist the impact of acceleration on the vascular system during fast swimming. The tolerance was shown to depend on an intact autonomic innervation of the vasculature (Ogilvy and DuBois, 1982; Ogilvy et al. 1989). Implications of the presence or absence of a lymphatic system were not discussed in this context. Whether the different mechanical conditions prevailing for aquatic versus terrestrial animals are reflected in tissue mechanics in such a way that only the terrestrial animals “need” the lymphatic function of draining the tissue spaces is not clear to the present authors, but may warrant consideration in the future. V. EVOLUTION OF LYMPHATIC VESSELS

Teleosts have been believed to be the first group of vertebrates in which a true lymphatic system came into existence (Kampmeier, 1969; Rusznyak et al., 1967). In cyclostomes and cartilaginous fish the distinction between lymphatics and veins was seen as being less clear-

212

J. F. STEFFENSEN AND J. P. LOMHOLT

cut. In these animals certain vessels have often been classified as “ venolymphatic,” a condition that was seen as primitive in the sense that the separation of the lymphatic system from the venous system was not yet fulfilled. The first deviation from this primitive condition was thought to be the occurrence of true lymphatics in the intestine of rays (Glaser, 1933).The distinction between red and white lymphatics in the hagfish (Cole, 1925) as well as the previously mentioned observations of Johansen et al. (1962) could be taken to indicate that certain vessels in the hagfish have the nature of true lymphatics. Until these observations have been reaffirmed, the existence of true lymphatics in these groups remains doubtful. With the demonstration of the secondary vascular system in teleosts and the failure to find vessels of a true lymphatic nature, a different picture emerges. Various pieces of evidence presented in Sections II,B and II,C point to the existence of some sort of a secondary vascular system in cyclostomes and elasmobranchs. There are, however, large gaps in our knowledge, and a reexamination of the vascular system of these groups with modern corrosion-casting technique is much needed. Laurent et al. (1978) described cisternae in the gills of African lungfish, Protopterus, which appear to be able to pump fluid from the interstitial space into branchial veins. Similar cisternae were found along veins of the body. Their similarity to small lymph pumps in amphibians was pointed out, but no information was given concerning their possible association with lymphatic vessels. Vogel(l988) reported true “tetrapod type” lymphatics to be present in the South American lungfish, Lepidosiren parudoxa, and in the Australian lungfish, Neocerutodus. It thus seems that true lymphatics appear for the first time in the evolutionary lineage leading to the earliest tetrapods, a lineage to which the lungfishes are closely related. The question naturally arises whether the secondary system may be seen as an evolutionary forerunner of a true lymphatic system. It is tempting to imagine an elimination of the arterial part of the system leaving the secondary capillaries and veins to form a lymphatic system, as suggested by Vogel(1981a). One immediate difficulty for this view is the distribution of the secondary system, which is confined to the various inner and outer body surfaces, whereas the lymphatics of the higher vertebrates are distributed to most organs of the body (Rusznyak et aZ., 1967; Johnston, 1985). The alternative possibility that a secondary vascular system is not to be considered a direct predecessor of the lymphatic system of the higher vertebrates appears to gain some support from embryological observations in mammals (Kampmeier,

4. THE SECONDARY VASCULAR SYSTEM

213

1960, 1969; Vogel, 1985a). If this is so, the idea of a continuous evolution of the lymphatic system from cyclostomes to mammals can no longer be upheld.

ACKNOWLEDGMENTS We are grateful for financial support from The Danish Natural Science Research Council, the Carlsberg Foundation, and the E. & K. Petersen Foundation.

REFERENCES Adair, T. H., and Guyton, A. C. (1985).Introduction to the lymphatic system. In “Experimental Biology of the Lymphatic Circulation” (M. G. Johnston, ed.) pp. 1-12. Elsevier, Amsterdam, Netherlands. Studies on the development ofthe venolymphatics in the tail region Allen, W. F.(1913). of Polistotrema (Bdellostoma)stouti. Q. J . Micr. Sci. 59,309-360. Bernier, D. R., Langan, J. K., and Wells, L. D. (1981). “Nuclear Medicine Technology and Techniques.” Mosby Company, London. Bailly, Y., and Dunel-Erb, S. (1986).The sphincter of the efferent artery in teleost gills. I. Structure and parasympathetic innervation. J . Morphol. 187,219-237. Bertin, L. (1958).Systeme lymphatique. In “Trait6 d e zoologie” (P. P. Grass6, ed.).Vol. 13 11, pp. 1444-1451. Masson et Cie, Paris. Booth, J. H. (1978).The distribution of blood flow in the gills offish: Application ofa new technique to rainbow trout (Salmo gairdneri).].E x p . Biol. 73, 119-129. Burne, R. H. (1926). A contribution to the anatomy of the ductless glands and lymphatic system of the angler fish (Lophius piscatorius).Phil. Trans. R . Soc. (London).B215, 1-57. Burne, R. H. (1929).A system of “fine” vessels associated with the lymphatics in the cod (Gadus morrhua). Phil. Trans. R. SOC. (London).B 217,235-366. Casley-Smith, J. R. (1983).The phylogeny of the lymphatic system. In “Lymphangiology” (M. Foldi and J. R. Casley-Smith, ed.). pp. 1-25. F. K. Schattauer Verlag, Stuttgart-New York. Cole, F. J. (1912).A monograph on the general morphology ofthe myxinoid fishes, based on a study of Myxine. Part IV. On some peculiarities of the afferent and efferent branchial arteries of Myxine. Trans. R . Soc. Edinburgh 48,215-230. Cole, F. J. (1925).A monograph on the general morphology of the myxinoid fishes based on a study on Myxine. Part VI. The morphology ofthe vascular system. Trans.R. SOC. Edinburgh 54,309-342. Conte, F. P., Wagner, H. H., and Harris, T. 0. (1963).Measurement of blood volume in the fish, (Salmo gairdneri). Am.J . Physiol. 205,533-540. Cooke, I. R.C. (1980). Functional aspects of the morphology and vascular anatomy ofthe gills of the endeavour dogfish, Centrophorus scalpratus. Zooniorphologie 94,167183.

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Duff, D. W., Fitzgerald, D., Kullman, D., Lipke, D. W., Ward, J., and Olsson, K. R. (1987). Blood volume and red cell space in tissues of the rainbow trout, Salmo gairdneri. Comp. Bfochem.Physiol. 87A, 393-398. Dunel-Erb, S., and Bailly, Y. (1986).The sphincter of the efferent filament artery in teleost gills. 11. Sympathetic innervation. J . Morphol. 187,239-246. Elger, M. (1987).The branchial circulation and the gill epithelia in the Atlantic hagfish, Myxine glutionosa L. Anat. Embryol. 175,489-504. Farrell, A. P., and Smith, D. J. (1981). Microvascular pressures in gill filaments of lingcod, Ophiodon elongatus.J. E x p . Biol. 216,341-344. Forster, M. E., Davison, W., Satchell, G. H., and Taylor, H. H. (1989).The subcutaneous sinus ofhagfish, Eptatretus cirrhatus, and its relation to the central circulating blood volume. Comp. Biochem. Physiol. 93A, 607-612. Fromm, P. 0. (1974).Circulation in trout gills: Presence of “blebs” in afferent filamental vessels.]. Fish. Res. Board. Can.. 31,1793-1796. Foldi, M., and Casley-Smith, J. R. (1983).“Lymphangiology.” F. K. Schattauer Verlag, Stuttgart-New York. Giacomini, E. (1922). Sopra ad alcune particulari disposizioni nel sistema arterioso (glomeruli vasali) e nel sistema linfatico dei teleostei. Rend. R. Accad. Sci. Bologna. N . S. 26,188-196. Gingerich, W. H., Pityer, R. A,, and Rach, J. J. (1987).Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Sulmo gairdneri). Comp. Biochem. Physiol. 87A, 251-256. Gingerich, W. H., and Pityer, R. A. (1989).Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with ILZ5bovine serum albumin and C?’ erythrocyte tracers. Fish Physiol. Biochem. 6,3947. Gingerich, W. H., Pityer, R. A., and Rach, J. J. (1990).Whole body and tissue blood volumes of two strains of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 97A, 615-620. Glaser, G. (1933).Beitrage zur Kenntnis des Lymphgefasssystems der Fische. 2. Anat. Entw. Gesch. 100,433-511. Grodzinski, Z . (1926).Uber das Blutgefksssystem von Myxine glutinosa. Bull. Znt. Acad. Pol. Sci. Lett., Cl. Sci. Math. N u t . B38, 123-157. Grodzinski, Z. (1932). Bemerkungen iiber das Lymphgefasssystem von Myxine glutinosa L. Bull. Znt. Acad. Pol. Sci. Lett., Cl. Sci. Math. Nat. B44,221-236. Hargens, A. R., Millard, R. W. & Johansen, K. (1974). High capillary permeability in fishes. Comp. Biochem. Physiol. 48A, 675-680. Holmes, W.N., and Donaldson, E. M. (1970).The body compartments and the distribution ofelectrolytes. In “Fish Physiology” (W. S. Hoar, and D. J. Randall, eds.),Vol. I, pp. 1-89. Academic Press, New York. Houston, A. H., and DeWilde, M. A. (1969).Environmental temperature and the body fluid system of the fresh-water teleost. 11. Hematology and blood volume of thermally acclimated brook trout. Salvelinus fontinalis. Comp. Biochem. Physiol. 28, 877-885. Hoyer, H. (1934). Das Lymphgefasssystem der Wirbeltiere vom Standpunkte der vergleichende Anatomie. Mem. Acad. Pol. Sci. (Cracow). A(l), 1-205. Hoyer, H. (1938). Das Lymphgefiisssystem. In “Bronns Klassen und Ordnungen des Tierreichs 6/1, Echte Fische, Teil2”. Akademische Verlagsgesellschaft, Leipzig. Ishimatsu, A., Iwama, G. K., and Heisler, N. (1988). In oivo analysis of partitioning of cardiac output between systemic and central venous sinus circuits in rainbow trout: A new approach using chronic cannulation of branchial vein. J. E x p . Biol. 137, 75-88.

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Iwama, G. K., Ishimatsu, A., and Heisler, N. (1990).The effect of environmental hypercapnia on the ionic and acid-base status ofthe fluid in the veno-lymphatic system of rainbow trout, Oncorhynchus mykiss. Abstract from an International Symposium “Mechanisms of Systemic Regulation in Lower Vertebrates: Respiration, Circulation, Ion Transfer and Metabolism.” Gottingen, Germany. Jackson, C. M. (1901).An investigation of the vascular system of Bdellostoma dombeyi.J . Cincinnati SOC. Nat. Hist. 20, 13. Jakubowski, M. (1960). The structure and vascularization ofthe skin ofthe leathern carp (Cyprinus carpio L. oar nuda) and flounder (Pleuronectesflesus luscus Pall.) Acta Biologia Cracoo. Zoologia 111, 139-162. Johansen, K. (1963). The cardiovascular system of Myxine glutinosa L.. In “The Biology of Myxine ” (A. Brodal and R. Fange, eds.), pp. 289-316. Universitetforlaget, Oslo, Norway. Johansen, K., Fange, R., and Johannessen, M. W. (1962).Relations between blood, sinus fluid and lymph in Myxine glutinosa L. Comp. Biochem. Physiol. 7,23-28. Johnston, M . G . (1985). “Experimental Biology of the Lymphatic Circulation.” Elsevier, Amsterdam, The Netherlands. Jourdain, S. (1880). Sur I’existence d’une circulation lymphatique chez les Pleuronectes. C . R. Acad. Sci. Paris 90,1430-1432. Kampmeier, 0. F. (1960). The development of the jugular lymphsacs in the light of vestigial, provisional, and definitive phases of morphogenesis. Am. J. Anat. 107, 153- 175. Kampmeier, 0. F. (1969).“Evolution and Comparative Morphology of the Lymphatic System.” Charles C Thomas, Springfield, Illinois. Kayama, M., and Iijima, N. (1976). Studies on lipid transport mechanisms in the fish. Bull. Jpn. SOC. Sci. Fish. 42,987-996. Kirsch, R., and Nonnotte, G. (1977). Cutaneous respiration in three freshwater teleosts. Respir. Physiol. 29,339-354. Klinkowstrom, A. (1891).Ueber die blutfuhrenden Lymphraume bei Myxine glutinosa. Biol. Forening. Forhandl (Stockholm)4. Krogh, A. (1930). “The Anatomy and Physiology of Capillaries.” Yale Univ. Press, Reprinted with new introduction and preface by E. M. Landis (1959) Hafner Publ. Co., New York. Laurent, P. (1982). Structure ofvertebrate gills. In “Gills” (D. F. Houlihan, J. C. Rankin, and T. J. Shuttleworth, eds.). pp. 25-43. SOC.Exp. Biol. Sem. Ser. 16. Laurent, P. (1984). Gill internal morphology. In “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. X, Part A, pp. 73-183. Academic Press, New York. Laurent, P., and Dunel, S. (1976).Functional organization of the teleost gill. I. Blood pathways. Acta 2001.(Stockh.) 57, 189-209. Laurent, P., Delaney, R. G.,and Fishman, A. P. (1978). The vasculature of the gills in the aquatic and aestivating lungfish (Protopterus aethiopicus).J . Morphol. 156, 173-208. Mayer, P. (1888). Ueber Eigentiimlichkeiten in den Kreislauforganen der Selachier. Mitt. 2001.Stat. Neapel. 8,307. Mayer, P. (1917). Uber die Lymphgefasse der Fische und ihre mutmassliche Rolle bei der Verdaung. Jena Z. Naturw. 55,125-174. Milligan, C. L., and Wood, C. M. (1982). Disturbances in haematology, fluid volume distribution and circulatory function associated with low environmental pH in the rainbow trout, Salmo gairdneri.]. E x p . Biol. 99,397-415. Mosse, P. R. L. (1980). Vascular anatomy of the lateral musculature of the flathead, Plattycephalus bassensis (Teleostei, Perciformes). Zoomorphologie 95, 133-148.

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Mott, J. C. (1957).The cardiovascular system. In “Physiology of Fishes” (M. E. Brown, ed.). Vol. I., pp. 81-108. Academic Press, New York. Nichols, D. J. (1987). Fluid volumes in rainbow trout, Salmo gairdneri: Application of compartmental analysis. Comp. Biochem. Physiol. 87A, 703-709. Nikinmaa, M. (1983).Adrenergic regulation of haemoglobin oxygen affinity in rainbow trout red cells. J. Comp. Physiol. 152,67-72. Nikinmaa, M.,Soivio, A., and Railo, E. (1981).Blood volume of Salmo gairdneri: Influence of ambient temperature. Comp. Biochem. Physiol. 69A, 767-769. Nonnotte, G . (1981).Cutaneous respiration in six freshwater teleosts. Comp. Biochem. Physiol. 70A, 541-543. Nonnotte, G . , and Kirsch, R. (1978).Cutaneous respiration in seven sea-water teleosts. Respir. Physiol. 35, 111-118. Ogilvy, C. S., and DuBois, A. B. (1982).Effect oftilting on blood pressure and interstitial fluid pressures of bluefish and smooth dogfish. Am. J. Physiol. 242,70-76. Ogilvy, C. S., Fox, S. H., and Dubois, A. B. (1989). Mechanisms of cardiovascular compensation for gravity in bluefish (Pomatomus saltatrir). Biol. Bull. 176, 176190. Pohla, H., Lametschwandtner, A., and Adam, H. (1977).Die vaskularisation der kiemen von Myrine glutinosa L. 2001.Scrlpta. 6,331-341. Prosser, C. L. (1973).Circulation of body fluids. In “Comparative Animal Physiology,” Vol. 2, pp. 822-856. W. B. Saunders, Philadelphia, Pennsylvania. Randall, D. J. (1985).Shunts in fish gills. In “Cardiovascular Shunts.” A. Benzon Symposium 21. K. Johansen, and W. W. Burggren, eds.). pp. 71-87. Munksgaard, Copenhagen, Denmark. Randall, D. J., and Daxboeck, C. (1982).Cardiovascular changes in the rainbow trout (Salmo gairdneri) during exercise. Can.J . 2001.60, 1135-1140. Retzius, G. (1890). Eine soganntes Caudalherz bei Myxine glutlnosa. Biol. Untersuchungen (Neue Folge), Stockholm 1,94-96. Robinson; J. S., and Mead, J. F.(1973).Lipid absorption and deposition in rainbow trout (Salmo gairdneri). Can.J . Biochem. 51,1050-1058. Romer, A. S., and Parson, T. S. (1977).“The Vertebrate Body.” W. B. Saunders, Philadelphia, Pennsylvania. Rowing, C. G. M. (1981). Interrelationships between arteries, veins and lymphatics in the head region of the eel, Anguilla anguilla L. Acta 2001.62, 159-170. Rusznyak, I., Foldi, M., and Szabo, G. (1967).“Lymphatics and Lymph Circulation.” Pergamon Press, London. Satchell, G. H. (1971). “Circulation in Fishes.” Cambridge University Press, Cambridge, Massachusetts. Satchell, G. H. (1992). The venous system. In “Fish Physiology” (W. S. Hoar, D. J. Randall, and S. F! Farrell, eds.), Vol. 12A,pp. 141-183. Academic Press, San Diego. Sire, M-F., Lutton, C., and Vernier, J-M. (1981).New views on intestinal absorption of‘ lipids in teleostean fishes: An ultrastructural and biochemical study in the rainbow trout. J . Lipid. Res. 22,81-94. Schmid-Schonbein, C. W., Fung, Y. C., and Zweifach, B. W. (1975). Vascular endothelium-leucocyte interaction: Sticking shear force in venules. Circ. Res. 36,173184. Smith, L. S. (1966). Blood volumes of three sa1monids.J. Fish. Res. Bd. Can. 23, 14391446. Smith, L. S., and Bell, G. R. (1964). A technique for prolonged blood sampling in free-swimming salmon. J . Fish. Res. Bd. Canada, 21,711-717.

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Steffensen, J. F., Lomholt, J. P., and Johansen, K. (1981). The relative importance ofskin oxygen uptake in the naturally buried plaice, Pleuronectes platessu, exposed to hypoxia. Respir. Physiol. 44,269-275. Steffensen, J. F., and Lomholt, J. P. (1985). Cutaneous oxygen uptake and its relation to skin blood perfusion and ambient salinity in the plaice, Pleuronectes platessa. Comp. Biochem. Physiol. 81A, 373-375. Steffensen, J. F., Lomholt, J. P., and Vogel, W. 0. P. (1986). In uivo observations on a specialized microvasculature, the primary and secondary vessels in fishes. Acta ZOOZ.67, 193-200. Steffensen, J. F., Heies, M., Randall, D. J., and Iwama, G. Blood volume of the primary and secondary vascular systems in rainbow trout, Oncorhynchus mykiss. (In preparation). Stevens, E. D. (1968). The effect ofexercise on the distribution ofblood to various organs in rainbow trout. Comp. Biochem. Physiol. 25,615-625. Tretjakoff, D. (1926). Die orbitalen Venensinusse der niederen Wirbeltiere. Gegenbaurs Morph.Jaht-b.56,402-445. Vogel, W. 0. P. (1978a). The origin of Fromm’s arteries in trout gills. Z. mikrosk. onot. Forsch. Leipzig 92,565-570. Vogel, W. 0. P. (1978b). Arterio-venous anastomoses in the afferent region of trout gill filaments (Salrno gairdneri R., Teleostei). Zoomorphologie 90,205-212. Vogel, W. 0. P. (1981a). Struktur und Organisationsprinzip im Gefasssystem der Knochenfische. Gegenb. Morph. Jahrb. 127,772-784. Vogel, W. 0. P. (l98lb). Das Lymphgefasssystem der Knochenfische-eine Fehlinterpretation? Verh.Anat. Ges. 75,733-735. Vogel, W. 0. P. (1985a). Systemic vascular anastomoses, primary and secondary vessels in fish, and the phylogeny of lymphatics In “Cardiovascular Shunts.” A. Benzon Symposium 21 (K. Johansen, and W. W. Burggren, eds.), pp. 143-159. Munksgaard, Copenhagen, Denmark. Vogel, W. 0. P. (1985b). The caudal heart of fish: Not a lymph heart. Actu Anat. 121, 41-45. Vogel, W. 0. P. (1988). Structure and evolution of lymphatics. Abstract # 509, Proc. 2nd lnt. Congr. Comp. Physiol. Biochem. Baton Rouge, Louisiana. Vogel, W. 0. P., and Claviez, M. (1981).Vascular specialization in fish, but no evidence for lymphatics. Z. Naturforsch. 36c, 490-492. Vogel, W. 0. P., Vogel, V., and Kremers, H. (1973).New aspects of the intrafilamental vascular system in gills of a euryhaline teleost, Tilapia mossambica. Z . Zellforsch. 144,573-583. Vogel, W. 0.P., Vogel, V., and Schlote, W. (1974). Ultrastructural study of arterio-venous anastomoses in gill filaments of Tilapia mossambica. Cell Tissue Res. 155,491-512. Wardle, C. S. (1971). New observations on the lymph system of the plaice Pleuronectes platessu and other teleosts. J . Mar. Biol. Assoc. U . K. 51,977-990. Weidenreich, F., Baurn, H., and Trautmann, A. (1933). Lymphgefasssystem. In “Handbuch der Vergleichende Anatomie der Wirbeltiere.” (L. Bolk, E. Goppert, E. Kallius, and W. Lubosch, eds.), Vol. 6. Urban und Schwartzenhurg, Berlin, Germany. Yamamoto, K., and Itazawa, Y. (1989). Erythrocyte supply from the spleen of exercised carp. Comp. Biochem. Physiol. 92A, 139-144. Yamamoto, K., Itazawa, Y., and Kobayashi, H. (1980). Supply of erythrocytes into the circulating blood from the spleen of exercised fish. Comp. Biochem. Physiol. 65A, 5-11.

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5 CARDIAC ENERGY METABOLISM WILLIAM R. DAZEDZZC Biology Department Mount Allison University Sackville, New Brunswick, Canada

I. Introduction 11. Energy Demand and Supply under Normoxic Conditions 111. Fuels of Aerobic Metabolism A. Blood Levels of Potential Metabolic Fuels B. Maximal Enzyme Activity Levels C. Respiration of Isolated Mitochondria D. 14C02Production from Labeled Fuels E. Support of Performance by Metabolic Fuels F. Summary IV. Metabolism in Epicardial and Endocardia1 Layers V. Metabolism under Dysoxic Conditions A. Heart Performance In Viuo B. Importance of Myoglobin C. Carbohydrate and High-Energy Phosphate Metabolism In Viuo D. Metabolism in Isolated Preparations E. Metabolic Control at the Enzyme Level F. Summary VI. Impact of Low Temperature on Metabolism A. Acute Temperature Transitions B. Chronic Low-Temperature Exposure in Nonpolar Teleosts C. Antarctic Teleosts References

I. INTRODUCTION

Hearts require a continuous supply of adenosine triphosphate (ATP)to maintain performance. Energy derived from the hydrolysis of ATP is utilized to support contractility directly and other processes such as ion balance. The synthesis of ATP from adenosine diphosphate (ADP) plus inorganic phosphate (Pi) is dependent on the energy liber219 FISH PHYSIOLOGY, VOL. XIIA

Copyright 0 1992 by Ac;ldernic Press, Inc. All rights of reproduction in any form reserved.

220

WILLIAM R . DRIEDZIC

ated from oxidation of metabolic fuels. Energy metabolism in fish hearts is particularly interesting since, in contrast to better studied mammalian hearts, ATP production is sustained under highly variable extracellular conditions both within individuals and across species. This chapter will focus on the general organization and subsequently on the impact of oxygen limitation and low temperature on energy metabolism. Each section is prefaced by a brief description of cardiac performance, since this is the physiological context in which energy metabolism must operate. The overall objective is to elucidate how heart metabolism, which is necessary to underwrite performance, is maintained under diverse conditions.

11. ENERGY DEMAND AND SUPPLY UNDER

NORMOXIC CONDITIONS Sustained heart performance requires an ongoing match between energy demand and supply. In this section the range of power output/g heart, which is the aspect of cardiac performance most directly related to energy metabolism, is summarized. Foremost, in v im power output for a number of selected species is presented. When not provided in the original literature, power output/g heart has been calculated from the mean aortic pressure, cardiac output, and gravitational acceleration as follows: power (mW/g) = mean pressure (cm HzO) X cardiac output (ml/min) x l/heart mass (g) x 980/60 x lod4. Thereafter, performance and metabolism in isolated preparations is described to gain insight into the aerobic versus anaerobic contributions to metabolism and the efficiency of energy transformation. In quiescent Atlantic hagfish (Myxine glutinosa), maintained in normoxic water at 1O"C, cardiac power development is about 0.15 mW/g heart (Axelsson et al., 1990). In resting Pacific hagfish (Eptatretus cirrhatus), ventral aortic pressure is slightly higher (Forster et d., 1988) than in Atlantic hagfish, and isolated, perfused hearts can achieve power outputs of 0.4mW/g (Forster, 1989). Heart power outputs under resting conditions and in normoxic water b y representative teleosts are as follows: short-finned eel (Anguilla australis), 0.9 mW/g heart at 18°C (Hipkins, 1985); Chaenocephalus aceratus, 1mW/g at 1°C (Holeton, 1970; Hemmingsen et al., 1972); sea raven (Hernitripterus americanus), 1.7 mW/g at 11°C (Axelssonet al., 1989); Atlantic cod (Gadus morhua), 1.8mW/g at 10°C

5. CARDIAC ENERGY

METABOLISM

221

(Axelsson and Nilsson, 1986); rainbow trout (Onchorhynchus mykiss), 1.5 mW/g at 10°C (Kiceniuk and Jones, 1977) and 2.5 mW/g at 15°C (Wood and Shelton, 1980a). During swimming, cardiac power output increased in cod, sea raven, and rainbow trout by factors of l.8,2.0, and 4.7, respectively, reaching a value of 7 mW/g in rainbow trout (Kiceniuk and Jones, 1977). Power outputs are probably higher in some species such as tuna, which may have very high cardiac outputs (see Chapter 1 this volume). Comprehensive measurements of cardiovascular parameters are available for only a few species of elasmobranchs. Under resting conditions and in normoxic water at 15"-20°C, the sedentary dogfish, Scyliorhinus canicula and Scyliorhinus stellaris, and the more active lemon shark (Triakis sem+sciata), have power outputs between 2 and 4 mW/g (Piiper et al., 1970; Taylor et al., 1977; Piiper et al., 1977;Lai et al., 1989; Lai et al., 1990).In lemon shark, swimming is associated with significant increases in cardiac output and pressure development (Lai et al., 1989; Lai et al., 1990) such that power output approaches 7 mW/g. In overview, there is considerable species variability in power development by fish hearts. Teleosts and elasmobranchs exhibit similar levels of cardiac power output/g heart and by extension, similar metabolic supplies. There appears to be about a two- to fivefold scope for increases in power output, which must be tracked by energy production mechanisms. Maximal in vivo power development by commonly studied species at 1Oo-20"C is about 7 mW/g, which is about the resting power development of heart from a 2.5-kg mammal operating at 37°C (Driedzic et al., 1987) and about one half of the maximal power output of fish white muscle performing oscillatory work cycles (Moon et al., 1991). Studies with perfused and isolated hearts add the additional information of simultaneous measurements of oxygen consumption and lactate production. In hagfish (Eptatretus cirrhatus), sea raven, ocean pout (Macrozoarces americanus),and rainbow trout hearts, the anaerobic contribution to energy metabolism is minimal when oxygen is available (Driedzic et al., 1983; Driedzic, 1983; Forster, 1991; Arthur et al., 1992).Isolated hearts generating close to resting levels of power have oxygen consumptions ranging between 0.27 and 1 pmolO2 8-l min-' at temperatures from 10 to 18°C (Table I). Apparent mechanical efficiency ranges from 11to 25% and increases in power output result in proportional increases in oxygen consumption. Considered together, these studies reveal that under non-oxygen-limiting conditions, fish hearts oxidize metabolic fuels aerobically to provide the energy necessary to rephosphorylate ADP.

Table I Power Output and Oxygen Consumption by Isolated Heart Preparations Reference

Species Cyclostome Epatatretus cirrhatus Teleosts Anguilla rostrata Carassius aratus Enophrys blson Esox niger Hemitripterus americanus Hemitripterus americanus Macrozoarces americanus Onchorynchus mykiss Onchorynchus mykiss

0.5

0.42

18

16

Forster (1991)

I

0.61 0.27 0.29 0.51 0.89 0.79 0.89 1 2.7 0.84 1.2

15 10 12 15 15 10 10 15 15 10 10

22

Bailey et al. (1991) Tsukuda et al. (1985) Nichols and Weber (1990) Bailey et al. (1991) Sephton et al. (1990) Farrell et al. (1985) Bailey and Driedzic (1986) Graham and Farrell(l990)

0.5 0.95 0.96 1

0.75 1

3 1 2

23 25 14 17

11 13 15 16 11

Houlihan et al. (1988)

Aerobic efficiency was calculated by dividing energy output by energy input calculated on the basis of 1 liter 0

2 =

20 kJ.

5. CARDIAC

ENERGY METABOLISM

223

111. FUELS OF AEROBIC METABOLISM The question of what fuels are utilized to support oxygen consumption is addressed in this section. The major potential fuels for vertebrate heart are generally considered to be glucose, lipid in the form of free fatty acids, lactate, and ketone bodies. Insight into which fuels are important is drawn from many approaches and different levels of physiological organization. A. Blood Levels of Potential Metabolic Fuels

Sustained aerobic performance is dependent on continuous delivery of metabolic fuel. Plasma concentrations are not necessarily directly related to use, but extremely low or undetectable levels of any particular metabolite must imply limited use, and high levels set the stage for potential utilization. Plasma glucose concentration typically ranges from 1 to 15 mM. Following stress-associated, intense activity, blood glucose levels frequently double in a number of teleost species (Black et al., 1960; Schwalme and Mackay, 1985; Milligan and Farrell, 1986). Under these conditions glucose utilization may be enhanced in concert with elevated levels of cardiac work. In many species glucose increases during hypoxic excursions and serves as an anaerobic fuel (for references and further discussion of this point, see Section V,C). Blood glucose remains relatively constant during starvation (Chavin and Young, 1970; Larsson and Lewander, 1973; Zammit and Newsholme, 1979; Bever et al., 1981). Plasma levels of free fatty acids occur in different concentrations among the major classes. In cyclostomes and elasmobranchs, concentrations are often around 0.1 mM (John et al., 1977; Larsson and Fange, 1977; Zammit and Newsholme, 1979; Fellows et al., 1980; Plisetskaya et al., 1983) although free fatty acids are below detectable limits in some elasmobranchs (Fellows et al., 1980).In teleosts, free fatty acid concentrations are typically between 0.5 and 1.5mM; however, some species have levels as low as 0.1-0.2 mM (Larsson and Fange, 1977; Zammit and Newsholme, 1979; Fellows et al., 1980; Black and Skinner, 1986; Sephton and Driedzic, 1991). Starvation has very little effect on blood levels of free Catty acids (Zammit and Newsholme, 1979; Black and Skinner, 1986). Further insight into the potential for free fatty acids to serve as fuel for cardiac metabolism is gained from knowledge of levels of fatty acid-binding proteins in plasma. Albumin or an

224

WILLIAM R. DRIEDZIC

albuminlike protein was detected in lamprey (Mordacia mordax) and a variety of teleosts but was absent in six species of elasmobranchs (Fellows and Hird, 1981).Free fatty acid and albumin levels imply that this fuel source is available to a limited degree to hearts of cyclostomes and elasmobranchs but to a much greater extent in teleosts. The level of plasma lactate in resting fish is usually 0.5-2.0 mM. Most fish exhibit elevated levels of plasma lactate following bouts of extreme exercise. In active animals the concentration typically reaches about 20 mM and may remain elevated for 12-24 hr (Bennett, 1978).In light of high concentrations of plasma lactate for prolonged periods, this metabolite must be considered a quantitatively important fuel for aerobic metabolism. The concentration of ketone bodies is typically less than 0.05 mM in plasma of fed fish. During prolonged starvation of dogfish (Scyliorhinus canicula; Elasmobranchii), plasma concentrations of 3hydroxybutyrate and acetoacetate increased from negligible levels to 1.9 and 0.4 mM, respectively. No alteration as a function of starvation was noted in sea bass (Dicentrarcus labrar; Teleostii) (Zammit and Newsholme, 1979). In overview, carbohydrates and fatty acids are available as energy sources for the teleost heart. In cyclostomes and elasmobranchs, plasma fatty acid levels are low, but glucose and, at least under starvation conditions, ketones are potential metabolic fuels.

B. Maximal Enzyme Activity Levels The maximal in uitro activity of enzymes expressed in tissue homogenates is often used to assess the organization of energy metabolism in viuo. There is a sound theoretical and experimentally supported basis for the use of enzymes that catalyze rate-limiting and nonequilibrium reactions as predictors of metabolic flux rates in uiuo (Newsholme and Crabtree, 1986). It is also generally accepted that the in vitro activity of enzymes that catalyze reactions maintained close to equilibrium in vivo provide at least qualitative information regarding the presence or absence of particular metabolic pathways. The enzymes most frequently measured and the rationale for their use will be outlined. Total activity of adenosine triphosphatase (ATPase) is a qualitative measure of the maximal capacity of a tissue to consume ATP. Oxygen consumption of perfused, isolated hearts and in uitro ATPase activity has been obtained for sea raven, pickerel, and eel (Sephton et al., 1990;

5. CARDIAC

225

ENERGY METABOLISM

Table I1 Calculated Supply and Utilization of ATP Equivalents in Fish Hearts"

MOz ATPase HK (aerobic) HK (anaerobic) CPT Carnitine palmitoleoyl CoA transferase

Sea raven

Pickerel

Eel

5.3 2.4 140 7.4 4.3 4.3

3.1 4.2 59 3.1 4.0 1.9

3.7 3.9 110 5.8 3.1 12.5

a Oxygen consumption was determined on perfused, isolated hearts generating power outputs of =1 mW/g and offered both glucose and palmitoleate as exogenous fuels. Enzyme activities are maximal levels assessed on crude homogenates. All measurements were made at 15°C. ATP supply from oxygen consumption is based on a P:O of3. ATP yield from HK is based on 38 ATPIglucose under fully aerobic conditions and 2 ATP/glucose under fully anaerobic conditions. Carnitine acyl CoA transferase activities were divided by two to account for the position of the enzyme on either side of the mitochondria1 membrane, and ATP yield was based on 130 ATP/fatty acid. Data are taken from Sephton et al. (1990) and Bailey et al. (1991).

Bailey et al., 1991)(Table 11). Isolated hearts were generating about 1 mWIg, so oxygen consumption was probably two- to threefold lower than the maximal in vivo level. On the other hand, enzyme activities were maximal in vitro levels. Given this interpretive constraint there is a reasonable match between ATPase activity (i.e., ATP demand) and predicted ATP yield (following conversion of oxygen consumption to ATP equivalents), suggesting that in vitro ATPase activity is a good indicator of cardiac ATP demand in this group of animals. Hexokinase (HK),phosphofructokinase (PFK),and pyruvate kinase (PK)are all requisite and potential regulatory enzymes for the degradation of blood-borne glucose. Hexokinase catalyzes the first step in the utilization of exogenously supplied glucose in which glucose is phosphorylated to glucose (G-6-P). In addition, PFK and PK are required for the breakdown of hexose units derived from glycogen, and PFK is a well-recognized rate-controlling enzyme, in the top segment of glycolysis, and catalyzes the conversion of fructose-6-phosphate (F-6-P) plus ATP to fructose-1,6-diphosphate (F-1,6-P) plus ADP. Pyruvate kinase occurs in the final segment of the reaction pathway and catalyzes the conversion of phosphoenolpyruvate (PEP) plus ADP to pyruvate plus ATP. Lactate dehydrogenase (LDH) catalyzes the conversion of lactate to pyruvate for further oxidation and lactate production from pyruvate during anaerobic metabolism.

226

WILLIAM R. DRIEDZIC

Carnitine palmitoyl (CPT) and 3-hydroxyacyl CoA dehydrogenase (HOAD) are both mitochondrial enzymes required for fatty acid metabolism. The oxidation of fatty acids is dependent on the sequential action of two mitochondrial membrane-bound carnitine fatty acyl coenzyme A (CoA) transferases that facilitate the movement of fatty acid derivatives across the mitochondrial membrane to the site of p-oxidation. As palmitate is the fatty acid most commonly investigated, the enzyme system is usually referred to as carnitine palmitoyl CoA transferase. CPT I converts palmitoyl CoA to palmitoyl carnitine, which is translocated across the inner mitochondrial membrane and subsequently reconverted to palmitoyl CoA by CPT I1 for entry into the p-oxidation spiral (Vary et al., 1981).The potential for the use of these enzymes as indicators of fatty acid metabolism in teleost hearts is evident from measurements of oxygen consumption and maximal enzyme activities (Table 11). The CPT or carnitine palmitoleoyl CoA transferase activities provide calculated ATP yields from fatty acid metabolism that are similar to predicted yields (given the assumptions of this approach) from oxygen consumption or demands from ATPase activity. This suggests that carnitine acyl CoA transferase activities are reasonable reflectors of maximal rates of fatty acid metabolism in fish hearts. One of the difficulties with carnitine acyl CoA transferase measurements is that at the physiological temperature of most fish, activity is at the lower limit of accurate quantitation with standard spectrophotometer assays. An alternative enzyme that is frequently used as a qualitative index of fatty acid oxidation is HOAD, a component of p-oxidation which catalyzes a near-equilibrium reaction. The advantage of HOAD over CPT as a mirror of fatty acid oxidation capability is the much greater activity of the former enzyme at physiological assay temperatures. A possible pitfall in the use of HOAD is that this enzyme is involved in the oxidation of fatty acids in peroxisomes, as well as in mitochondria, and measurements of total enzyme activity take into account both compartments. Peroxisomal fatty acid oxidation in fish hearts, though, appears to be minimal (Moyes et aE., 1990). Citrate synthesis (CS) catalyzes the first reaction of the citric acid cycle in which acetyl CoA is condensed with oxaloacetate to form citrate. Acetyl CoA may be derived from any of the potential aerobic metabolic fuels. Maximal in uitro activity of CS correlates with resting and by extension, maximal power development in heart from a wide spectrum of vertebrate animals when both parameters are expressed per g of tissue (Driedzic et al., 1987).Thus, in vitro activity of CS may be used as a marker of relative maximal carbon flux through the citric acid cycle. Cytochrome oxidase (Cyt Ox) catalyzes the final step in the

Table 111 Maximal in Vitro Enzyme Activities in Fish Hearts" Species Cyclostomes Myxine glutinosa

HK

PFK

1.7

2.6

PK

35.9

LDH CPT HOAD 114

0.06

8.3

CS

CYTOX

6.9

4.2

ATPase

5.3

Assay temp.("C)

15

Reference Hansen and Sidell(1983); Sidell ef al. (1987)

Eptatretus stouti Elasmobranchs i'otamotrygon magdalenae Raja erinacea Raja en'nacea

-

-

-

-

0.04

4.4

-

0.01 5.0 1.1

0.13 5.3 3.8

-

39

N.D.

15.2 46.7

119 67

N.D. N.D.

0.1 0.62 0.13

14.4 8.8 4.1

21.3

9.5

8.3

-

-

-

12.8

11.5 23.1

3.4 2.3 3.3

Squalus acanthias Squalus acanthias Nonpolar teleosts

4.4

10.4

66.7

251

N.D.

-

-

-

-

<.01

1.8 1.6

A nguill a rostra ta Anguilla rostrata

2.9 17.2 6.6 6.4

-

-

-

0.05

-

21.7 19.3 1.9

41.7

185

-

-

-

-

8.2

9.5

46.8

208

0.39 0.59

3.3

0.73

-

168

0.17

1.4

Carassius auratus Cyprinus carpio Cyprinus carpio Dicentrarchus labrax Esox niger

Esox niger Gadus morhua Gaidropsarus vulgaris

172

454 415

-

-

-

15

Moyes et al. (1990)

-

-

19.1

3.8

-

-

25 15 10

Singer and Ballantyne (1989) Sidell et al. (1987) Moon and Mommsen (1987)

15 15

Sidell et al. (1987) Moyes et al. (1990) Bailey et al. (1991) MacIntyre and Driedzic (1981) Duncan and Storey (1991)

Way Kleckner and Sidell (1985); Sidell et al. (1987) Bailey et al. (1991)

-

3.9

-

-

-

4.5

20.9

17.9

-

-

-

13.2

27.4

31.2

15 25 22 15 15 15

9.2

17.4

8.7

15

-

1.6

-

-

-

6.4

-

4.2

15

9.6

58.9

330

0.06 0.44

-

6.2

17.2

12.1

38.9

-

15

8.1

67.8

132

0.41

2.4

8.9

36.2

4.1

15

11

Sidell et al. (1987) Moyes et al. (1990) Sidell et al. (1987)

Hansen and Sidell (1983);Sidell et al. (1987) Sidell et al. (1987)

(continues)

Table I11 continued Species Hemitripterus americanus Hemitripterus americanus Hemitripterus americanus Lophius piscatonus Macrozoarces americanus Macrozoarces americanus Makaira nigricans Micopterns dolomieui Morone americanus Morone americanus Morone saratilis

Myorocephalus 0ctodecimspinosuP Onchorhynchus mykiss Onchorhynchus mykiss Onchorhynchus mykiss

HK

PFK

2.5

1.3

3.7

-

5.2 2.5

3.8 1.2

6.9 5.2 3.9 11.9 5.1 12 20.2

-

PK

LDH

37

155

-

-

18.2 36.3

112 128

-

-

104

708

CFT 0.03 0.16

HOAD

12.1

35.8

-

0.07

-

16.5

-

0.23 0.21

1.4 1.8

7.0 12.8

13.4 29.8

-

-

69.2

252

8.6

-

-

-

0.02

-

9.5 3.6

29.9 36.9

149

0.52 0.31

3.1 1.2

34

99 36.9

268

-

-

ATPase

Assay ternp.("C)

11.3

15

2.4 16.8

15

-

10

10

11.7

2.4

-

CYTOX

2.9

0.39 0.05 0.32

137

CS

0.28

14.2 26.2 13.6 5.2 4.8 8.2

-

-

27.2 7.2

32.2

-

25.1

39.3 7.4

12.7 17.5 8.1 35.9

15

15 25 20 15 20 15 10

22 25 15

Reference Driedzic and Stewart (1982) Sidell et al. (1987) Sephton et al. (1990) Sidell et al. (1987) Diredzic and Stewart (1982) Sidell et al. (1987) Suarez et al. (1986) Sephton and Driedzic (1991) Sidell et al. (1987) Sephton and Driedzic (1991) Sidell et al. (1987) Crockett and Sidell(l990)

Brooks and Storey (1988) Farrell et al. (1990) Moyes et al. (1990)

Perm jlaoescens Protopterus aethiopicus Salmo salar

-

-

8.8

-

-

0.02

-

7.9

-

17.4

23.2 17.9

-

101

-

0.38

34.8 16.4

19.2 32.4

20 25 15

Sephton and Driedzic (1991) Dunn et al. (1983) Ewart and Driedzic (1987)

0.8

13.1 13.2

16.5

15

Ewart and Driedzic (1987); Driedzic et al. (1987) MacIntyre and Driedzic (1981)

13.5

15.4

58

594 215

Salvelinus fontinalis

8.3

17.7

37.7

225

Salvelinus fontinalis

10.6

26.7

3.8

9.9

-

-

Scomber scombrus Tautoga onitis Zoarces viviparous

35.1

34.9 23.3 31.2

144

-

6.5

34

156 103

0.28 0.35

9.5 1.1 5.4

-

-

25

19.1 10.1

36.0

15

-

12.8

95.1

10 10

32.1 6

Sidell et al. (1987) Feller and Gerday (1987)

15.1 40 38.9

Crockett and Sidell (1990) Fitch (1989) Sidell et a / . (1987)

8.9 13.7

Feller and Gerday (1987) Crocket and Sidell (1990)

Sidell et al. (1987) Crockett and Sidell (1990) Driedzic et a / .(1985a)

Antarctic teleosts

Chaenocephalus aceratus

t.a WJ

Channichthys rhinoceratus Notothenia gibberifrons Notothenia neglecta Notothenia rossi Paranotothenia magellanica Trematomus newnesi

7 3.7

1.2

1.32 1.8 5 1.74 0.94

2.1 0.6 0.77 0.65 2.1

1

24.3 27

10.2

135 608

0.39 0.33

6.7

-

-

8.5

99 145 89

0.18

2.6 0.5

13.7

75 37

0.43 0.24

5.5

-

-

11.3

0.29

3.3

9.1

All values are processed as (pmol substrate converted to product) . g-' . min-'. N.D., not detected. The symbol reported.

- indicates that the enzyme level was not

230

WILLIAM R. DRIEDZIC

electron transport system, which involves the reduction of 0 2 . As such, this enzyme is often used as a qualitative measure of maximal oxygen consumption. The experimental approach of assessing maximal in uitro enzyme activities has been used many times in the study of fish heart metabolism. Table I11 presents the activity of key enzymes of energy metabolism from a variety of species. References have been selected that present data for a number of enzymes assayed under relatively similar analytical conditions. In this survey no attempt has been made to distinguish enzyme activities in endocardia1 versus epicardial tissue since, as is discussed in Section IV, differences occurring at this level are small relative to between-species variation. Foremost, the marker enzymes necessary for glucose catabolism are routinally detected in high activities in almost all species. The capacity to oxidize fatty acids, as evidenced by enzyme activities, is not uniform across the major classes. Two species of hagfish and numerous teleosts have detectable levels of CPT and HOAD that are prerequisite for fatty acid metabolism. The situation with respect to elasmobranchs seems quite clear, as there were undetectable or only marginally detectable levels of CPT in six different species in association with low levels of HOAD in two of three species (additional references, Zammit and Newsholme, 1979; Moyes et al., 1990). Further, insight can be gained by regression analysis of maximal enzyme activities found in the wide spectrum of animals presented in Table 111. In these analyses, each point represents a single species, and all data were obtained at assay temperatures within the normal thermal range of the animals. The data set contains information from animals as diverse as Antarctic teleosts operating at - 1.8"Cto African lungfish (Protopterus aethiopicecs)with body temperatures in excess of 25°C and were collected over a period of many years in a number of different laboratories. Regression analysis reveals a significant positive correlation between HK and CS activities (Fig. 1).Similar significant correlations exist for PFK versus CS (r = ,574; n = 26) and PFK versus Cyt Ox (r = .686; n = 25).This suggests that in fish hearts, as the demand for acetyl CoA per g heart is increased, there is a concomitant expansion in the use of glucose as a metabolic fuel. (The metabolic significance of the relationship between HK and ATPase is treated under Section V,E,3). Regressions of CPT versus ATPase and HOAD versus CS (with elasmobranchs excluded in both cases) yield significant linear correlations (Fig. 1).These relationships, as with those for glucose utilization, suggest that as energy demand per g heart increases across teleost species, there is an increase in capacity for fatty acid oxidation.

5. CARDIAC

0

231

ENERGY METABOLISM

10

20

ATPase (pmol g

30 - 1

40

0

m t i ')

10

20

30

40

ATPase (pmol g * 'mln' ')

' C

15

m 0

0

10

15

Y

=

' n

Y

o 0

10

20

30

cs (pmol g- ' miri

40

5 '0

10

20

30

I

D

cs (pmoi g- ' m i i '1

Fig. 1. Relationships between maximal in oitro enzyme activities in fish hearts. Information is taken from Table 111. Left panels include data from all species. Right panel includes data from cyclostomes and teleosts only. All enzyme activities are expressed at the assay temperatures, which fall within normal physiological ranges. Regression equations are as follows: HK = (.34)CS + 1.31, r = ,611,n = 31; HK = (.17)ATPase + 3.74, r = .550,n = 20; CFT = (.Olrl)ATPase + .093, r = .575,n = 18; HOAD = (.49)CS + .06,r = .634, n = 23.

The production of acetyl CoA from ketone bodies is dependent on the concerted action of 3-hydroxybutyrate dehydrogenase, succinylCoA ketotransferase, and acetoacyl-CoA thiolase. These enzymes have been detected in heart of six different species of elasmobranchs (Zammit and Newsholme, 1979; Moon and Mommsen, 1987; Singer and Ballantyne, 1989), implying a potential to utilize ketones as metabolic fuels. Activity of 3-hydroxybutyrate dehydrogenase is minimal in three species of teleosts; however, succinyl-CoA ketotransferase and acetoacyl-CoA thiolase occur at levels comparable to those in elasmobranchs (Zammit and Newsholme, 1979). C. Respiration of Isolated Mitochondria Mitochondria isolated from heart of the elasmobranch, Squalus acanthias, consume oxygen when pyruvate or ketone bodies are of-

232

WILLIAM R. DRIEDZIC

fered as metabolic fuels but not when long-chain fatty acids or carnitine derivatives are provided (Moyes et d., 1990). In contrast, mitochondria from two species of teleosts Channichtys rhinoceratus and Notothenia rossii exhibit vigorous metabolic rates fueled by palmitoyl CoA in the presence of carnitine (Feller et al., 1989). Respiration rate driven with 2-oxoglutarate was similar for mitochondria1 preparations from all three species. These studies, quite independent from the enzyme analysis previously discussed, suggest that fatty acids are a suitable metabolic fuel for teleost but not elasmobranch hearts. Moreover, elasmobranch heart has the potential to utilize ketone bodies.

D. 14C02Production from Labeled Fuels Ventricle sheets, perfused isolated hearts, or isolated myocytes of Atlantic hagfish (Sidell et al., 1984), brook trout (Saluelinusfontinalis) (Lanctin et al., 1980), sea raven (Sephton et al., 1990), rainbow trout (Milligan, 1991), and Notothenia gibbefifrons (Sidell et al., 1988)produced labeled 14C02from exogenous labeled glucose. It is not possible to calculate true glucose catabolic rates from these data since much of the added label exchanges with the intracellular pools of glycogen and lipid (Sephton et al., 1990) and thus potentially alters the true specific activity of acetyl CoA that enters the citric acid cycle. Moreover, some of these studies utilized uniformly labeled glucose, and there is no justification in the assumption, necessarily involved in the back calculation of metabolic rate, that complete combustion of glucose proceeds for each labeled I4CO2 evolved. Nevertheless, the isotope studies are important in establishing that glucose may be oxidized to C02 and are useful in revealing the competitive nature of metabolic fuels. In the case of Atlantic hagfish (Sidell et al., 1984), the inclusion of palmitate as a representative free fatty acid in the bathing medium did not decrease production of I4CO2from glucose. Thus, the inhibition of glucose metabolism by fatty acids, so prevalent in mammalian heart (Neely and Morgan, 1974), does not occur in hagfish. Isolated heart preparations of Atlantic hagfish (Sidell et al., 1984), Notothenia gibberifrons (Sidell et al., 1988), sea raven (Sephton et al., 1990), and white perch (Morone americanus) (Sephton and Driedzic, 1991)produced labeled 14C02 from exogenous labeled fatty acids. As with the glucose studies, it is not possible to calculate accurate oxidation rates of fatty acids from presented data since exogenously added label is incorporated into the intracellular lipid pool (Sephton et al., 1990). Mixing between the added label and the intracellular fatty acid

5. CARDIAC

ENERGY METABOLISM

233

pool would result in a dilution of the specific activity of the acetyl CoA pool and lead to underestimates of true rates of fatty acid oxidation. Once more, though, the labeling studies are useful in elucidating potential interactions of metabolic fuels. In Atlantic hagfish heart, inclusion of glucose in the bathing medium decreased the rate of release of labeled C02 from palmitate (Side11 et al., 1984), implying that glucose is utilized in preference to exogenous fatty acid. Homogenates, isolated myocytes, and perfused hearts of teleosts produced 14C02from exogenous labeled lactate, and apparent rates of fuel utilization generally increased as extracellular lactate was elevated (Bilinski and Jonas, 1972; Gemelli et al., 1980; Lanctin et al., 1980; Driedzic et al., 1985b; Milligan, 1991; Milligan and Farrell, 1991). In isolated rainbow trout hearts, apparent lactate oxidation at high (10 mM) but not low (1 mM) extracellular levels increased in association with elevated levels of power output (Milligan and Farrell, 1991).The inclusion of putative inhibitors of lactate transporters in the perfusion media substantially impaired 14C02 production from labeled lactate at high extracellular lactate concentrations suggesting that lactate uptake is carrier mediated (Milligan and Farrell, 1991).In isolated brook trout hearts, the inclusion of 2 mM lactate in the perfusion medium decreased the apparent oxidation of exogenous glucose by 10-fold (Lanctin et al., 1980), implying that lactate was preferentially utilized. As the concentration of extracellular lactate offered to perfused isolated sea raven and ocean pout (Macrozoarces americanus) hearts was increased from 2 to 10 mM, the oxidation of 14C-3 lactate was increased two- to fourfold, showing that exogenous lactate was being channeled into the citric acid cycle (Driedzic et aE., 1985b). When lactate and pyruvate were offered at the same concentrations, the decarboxylation of pyruvate was about fourfold greater than lactate. The last point implies that pyruvate production is a rate-limiting step in lactate oxidation. The primary determinants in the use of exogenous lactate are probably availability, transport, and the low activity of lactate oxidase at physiological pH (Gesser and Sundell, 1971; Driedzic and Stewart, 1982; Stewart and Driedzic, 1986; Milligan and Farrell, 1991) although the possibility that transport of nicotinamide adenine dinucleotide, reduced (NADH) into the mitochondria may be a critical factor cannot be ruled out (Hochachka et al., 1979).

E. Support of Performance by Metabolic Fuels Further insight into the efficacy of various metabolic fuels comes from studies of performance of isolated preparations. Ventricle sheets

234

WILLIAM R. DRIEDZIC

from hagfish maintained performance for up to 4 hr when provided with glucose or lactate as exogenous fuel but deteriorated when palmitate was the only exogenous energy source (Side11 et al., 1984). Perfused isolated sea raven hearts initially operating at close to resting levels of power output developed only 40% of initial power output following 30 min of treatment with Ringer's, without exogenous fuel and containing iodoacetic acid (IAA) to impair glycolysis (Fig. 2). The inclusion of postexercise levels (Milligan and Farrell, 1986) of lactate (0.5 mM) resulted in maintenance of 50% of initial power development, and 2.0 mM lactate increased this to 90% (Driedzic and Hart, 1984).The addition of 0.5 mM palmitate or 1.0mM palmitate resulted in maintenance of 60 and 100%,respectively, of initial power output (Driedzic and Hart, 1984). Acetoacetate (0.5 mM) increased performance to 75%of initial power output. Ventricle strips of Atlantic cod, eel (Anguilla anguilla), and plaice (Pleuronectes platessa), developing maximal isometric tension, failed when poisoned with IAA. The deterioration in function was ameliorated by the inclusion of lactate in the bathing medium (Gesser and Poupa, 1975).Farrell et al. (1988b) noted that in the absence of exogenous fuel, perfused rainbow trout hearts exhibited a gradual deterioration in maximal power output, but the addition of lactate to the medium resulted in maintenance of performance. Later studies did not support this contention, possibly due to the inclusion of adrenaline, which allowed achievement of maximal levels of performance even in the presence of IAA and blockers of lactate transport (Milligan and Farrell,

i Q)

P

60 40

40 20

s

l 200 S k0a t e5 1 0 1i 5 2 0 2 5 30 k

Sea raven

0

0

5

1 0 1 5 2 0 25 3 0

Tlme (mln)

s Tlme (mln)

Fig. 2. Percentage initial performance of perfused, isolated sea raven (Hemitripterus americanus; Teleostii) and little skate (Raja erinecea; Elasmobranchii) hearts treated with IAA (solid circles) plus additional fuels. Left panel: solid squares, 2.0 mM lactate; open squares, 1.0 mM palmitate. Right panel: open squares, 0.5 mM acetoacetate. Data taken from Driedzic and Hart (1984).

5. CARDIAC ENERGY METABOLISM

235

1991). The authors attribute this to adrenergic activation of lipolysis. This study is particularly insightful in that simultaneous measurements of power development and lactate oxidation were obtained. Assuming that most of the lactate was completely catabolized and hearts were working at efficiencies of 15-25%, it was calculated that lactate oxidation could supply all the required energy to support performance (Milligan and Farrell, 1991). Isolated skate (Raja erinacea) hearts perfused with media containing IAA and no exogenous fuel failed within 30 min; however, the inclusion of acetoacetate (0.5 mM) resulted in a 70% maintenance of performance (Fig. 2 ) (Driedzic and Hart, 1984).

F. Summary The use of glucose as a fuel for aerobic metabolism is well developed in all fish hearts examined to date with the possible exception of the freshwater elasmobranch, Potamotrygon magdalenae. This conclusion is based on the availability of the fuel, prerequisite enzymes necessary for glucose oxidation, the production of COz from extracellular glucose, and failure of isolated preparations treated with a glycolytic blocker. Positive correlations between predictors of glucose utilization (HK and PFK) and ATPase or the citric acid cycle (CS) suggest that as energy demand per g heart is increased across species, there is a concomitant expansion in the utilization of exogenous glucose. Lactate appears to be an excellent fuel of metabolism, especially for the teleost heart. This is supported by fuel availability, substantial activities of lactate dehydrogenase (LDH), production of 14C02from 14C-lactate,the maintenance of performance in the presence of glycolytic blockage, and the fact that lactate may be preferentially utilized even when glucose is available. The use of fatty acids as metabolic fuels is more diverse. Hagfish hearts appear to have a limited capability to utilize this fuel. They produced 14C02from labeled palmitate, but the apparent preference of glucose over fatty acids by hagfish heart is in marked contrast to the situation in mammalian hearts, which utilize fatty acids even when glucose is available (Neely and Morgan, 1974). Elasmobranch hearts do not utilize exogenous free fatty acids as metabolic fuels. For many species of elasmobranchs, plasma is devoid of fatty acids and albumin, hearts lack CPT, and isolated mitochondria do not respire when fatty acids or fatty acid derivatives are the only metabolic fuel. In contrast, fatty acids are an excellent metabolic fuel for teleost hearts. Free fatty acids are available in plasma, hearts have the necessary enzyme com-

236

WILLIAM R. DRIEDZIC

plement to utilize this fuel, fatty acids can support respiration of isolated mitochondria, isolated preparations generate 14C02from labeled palmitate, and palmitate can ameliorate contractile failure due to glycolytic blockage in perfused hearts. There is a linear increase in the activities of CPT and HOAD in association with energy demand across species. The use of ketones as metabolic fuels is well developed in elasmobranch hearts. Plasma levels of ketone bodies increase during prolonged starvation. Hearts have the enzyme complement necessary to oxidize ketones, and in isolated hearts, physiological levels of acetoacetate resulted in partial maintenance of performance under glycolytic blockage, which otherwise resulted in complete failure.

111. METABOLISM IN EPICARDIAL AND ENDOCARDIAL LAYERS In teleosts, the ventricle consists of a spongy endocardium which in some species is surrounded by varying degrees of a compact epicardium (see Chapter 1, this volume). The compact layer is enhanced in animals with high sustained swimming speeds and may account for as much as 35% (e.g., rainbow trout) to 70% (e.g., bigeye tuna, Thunnus obesus) of the total myocardium (Santer and Greer Walker, 1980). In Atlantic salmon (Salmo salar), the compact layer increases from 5% of the myocardium in young animals to 25%in adults (Poupa et al., 1974). In teleosts, the endocardia1 cells are supplied primarily by the venous blood from the intertrabecular spaces of the ventricular lumen; whereas, the epicardium is nourished by discrete coronary arteries providing oxygenated blood (Tota, 1989). The elasmobranch heart also consists of two layers, with the epicardium representing about 25% of the myocardium in numerous species (Santer and Greer Walker, 1980). In elasmobranchs, subepicardial arteries anastomose with the intertrabecular spaces of the spongy endocardium, thus providing this area with oxygenated blood as well (Tota, 1989) Maximal levels of enzymes of energy metabolism have been assessed in the compact epicardium and the spongy endocardium for a number of teleosts. The most complete scan to date was obtained on adult Atlantic salmon (Ewart and Driedzic, 1987). Hexokinase and LDH activities were significantly higher (1.5 times and 1.2 times, respectively) in the spongy than the compact layer, but CPT (1.2 times), HOAD (1.2times), and Cyt Ox (1.3times) activities were significantly higher in the compact than in the spongy layer. No differences

5.

CARDIAC ENERGY METABOLISM

237

were noted in PFK, PK, CS, or 3-oxoglutarate dehydrogenase. Similarly, in rainbow trout, HK activity was 1.4 times higher in the spongy than in the compact layer, and Cyt Ox activity was 1.4 times higher in the compact than in the spongy layer (summer fish only). No differences were noted in HOAD, CS, or LDH activities (Poupa et al., 1974; Farrell et al., 1990).The enzyme activity levels suggest a subtle difference in metabolic fuel utilization between the spongy and cortical layers of the salmonid heart. The cortical layer with its high level of oxygen delivery may be better poised for the aerobic oxidation of fatty acids; whereas, carbohydrate metabolism may be better developed in the spongy layer, which operates on venous blood. The HK activity was also higher in the spongy than in the compact layer of carp (1.4 times) (Bass et al., 1973) and Conger conger (2 times) but similar in the two layers of tuna (Thunnus thynnus) heart (Tota, 1983). In these species, CS was more active in the spongy than in the compact myocardium (carp, 2 times; Conger, 1.5times; tuna, 1.3times) and in carp, Cyt Ox was more active (1.4 times) in the spongy than in the compact layer (Kalous et al., 1989). These findings suggest an enhanced aerobic activity in the endocardium of carp, C. conger, and tuna, a situation quite different from that found in salmonids. There is evidence that mitochondria may differ in the compact and spongy layers. In shark (Alopia d p i n u s ) , swordfish (Xiphias gladius), and tuna (Thunnus thynnus) mitochondria isolated from endocardia exhibited higher rates of oxygen consumption with succinate as a substrate than mitochondria isolated from the epicardia (Tota, 1983). In tuna, the apparent K,(succinate) was lower for mitochondria from the spongy than from the compact layer (Greco et al., 1982) and mitochondria from the spongy layer had a higher cristae density than those from the compact layer (Basile et al., 1976).The functional significance of these findings is yet to be established. In summary, it is not possible to generate a comprehensive and uniform picture of metabolic differences between the endo- and epicardium. Overall the differences in metabolic characteristics between the compact and spongy layers seem small relative to differences in oxygen delivery. For instance, in trout swimming at maximum speeds, partial oxygen pressure (Poz) and oxygen content are about sevenfold higher in arterial than in venous blood perfusing the compact and spongy layers, respectively (Kiceniuk and Jones, 1977). Yet key metabolic enzymes differ by no more than a factor of 1.5 between the two layers. In Atlantic salmon, myoglobin levels are 2.3 times higher in the spongy than in the compact layer (Ewart and Driedzic, 1987). This would allow the endocardium to maintain aerobic metabolism at lower

238

WILLIAM R. DRIEDZIC

levels of extracellular Po2 than the epicardium (see Section V,B) and subsequently, obviate the necessity for major differences in the organization of energy metabolism.

IV. METABOLISM UNDER DYSOXIC

CONDITIONS A. Heart Performance In V i m

In Atlantic hagfish, power output is maintained in hypoxic water (PO, = 2 kPa for 15-35 min) under conditions where blood perfusing the heart must be extremely low (Axelsson et al., 1990). In teleosts, acute hypoxic exposure is often associated with a rapid reflex bradycardia. This has been demonstrated, for instance, in rainbow trout (Wood and Shelton, 1980b; Holeton and Randall, 1967) and Atlantic cod (Fritsche and Nilsson, 1989 and 1990).Heart rates are typically decreased to about 50% of control level at the limit of whole animal viability. In contrast, hypoxic bradycardia is weak or absent in other species (Saunders and Sutterlin, 1971; Cech et al., 1977; Berschick et al., 1987; Fritsche, 1990).Under hypoxia blood pressure increases in some species (Holeton and Randall, 1967; Saunders and Sutterlin, 1971; Wood and Shelton, 1980b; Fritsche and Nilsson, 1990) but decreases in others (Cech e t al., 1977; Farrell, 1982; Fritsche, 1990).Cardiac output has been measured in only a few cases. In rainbow trout and Atlantic cod, cardiac output is maintained under hypoxiadue to an increase in stroke volume (Wood and Shelton, 1980b; Fritsche and Nilsson, 1989),but in lingcod (Ophiodon elongatus),it decreases (Farrell, 1982). As a representative elasmobranch, dogfish exhibit a decrease in power output, during hypoxic exposure at elevated temperature, to about 60% of the level in normoxic water (Satchell, 1961; Piiper et al., 1970; Taylor et al., 1977). Variability in response is probably due to species differences, experimental approaches, and depth of hypoxia. But it appears that some species attempt to maintain cardiac performance prior to succumbing to oxygen limitation, whereas, in others, performance immediately declines under environmental hypoxia, and power development may be reduced to about 50% of normoxic levels. There is no evidence that fish hearts enter into prolonged states of very low power output (i.e., less than 10% of normal) in response to hypoxia. Cardiac “hypometabolism” as occurs in diving turtles (Kelly and Storey, 1988) does not seem to be a strategy invoked by fish.

5.

CARDIAC ENERGY METABOLISM

239

B. Importance of Myoglobin One strategy for coping with a problem of low extracellular oxygen levels is to extend the lower limit at which oxygen extraction from the extracellular space is possible. In some species this is achieved by enhanced levels of the intracellular protein myoglobin. It is well recognized that myoglobin facilitates diffusion of oxygen in mammalian skeletal muscle (Wittenberg and Wittenberg, 1989). A similar role is demonstrated in hearts of fish, which may face low PO, levels owing to either environmental hypoxia or intense exercise. The latter results in blood with a lower P o , reaching the endocardium, due to utilization by swimming muscles. For instance, in trout, ventral aortic PO, falls from 4.43 kP (3.17mM 02) at rest to 2.13 kPa (0.6 mM) during maximal activity (Kiceniuk and Jones, 1977). Myoglobin content in fish hearts is extremely species variable. Comprehensive listings of cardiac myoglobin levels in various species are provided elsewhere (Giovane et al., 1980; Driedzic, 1988),and the following summary only illustrates the extremes. Antarctic icefish that are devoid of hemoglobin have undetectable (Feller and Gerday, 1987; Sidell et al., 1987)or possibly very low levels of cardiac myoglobin (Douglas et al., 1985). Very low levels of myoglobin are not restricted to icefish, as some species of nonpolar teleosts, such as lumpfish (Cyclopterus lumpus), monkfish (Lophius piscatorius), and ocean pout (Macrozoarces americanus) have either zero or only marginally detectable myoglobin contents (Driedzic and Stewart, 1982; Sidell et al., 1987). Isolated, perfused hearts from these species appear white after blood washout (Driedzic, 1983). At the other extreme some species have hearts that are extremely rich in myoglobin, such as mackerel (Scomber scombrus) (332 nmol/g) (Sidell et at., 1987) and tuna (Thunnus thymus) (= 580 nmol/g) (Giovane et al., 1980). Fish that exhibit high levels of sustainable swimming invariably have high levels of cardiac myoglobin. Elevated levels are also found in species that are tolerant of environmental hypoxia such as carp (C. carpio) (488 nmol/g) (Sidell et al., 1987) and eel (Anguilla rostrata) (239 nmol/g) (Bailey et al., 1990). Low levels of myoglobin are found only in species that are relatively inactive. The oxygen-binding characteristics of fish myoglobin are well suited for function at physiological temperatures. At 20°C, the PSOfor myoglobin from buffalo sculpin (Enophrys bison) (0.15 kPa) and that for coho salmon(0nchorhynchus kisutch) (0.24kPa) heart are significantly higher (i.e., bind with a lower affinity) than myoglobin from rat (0.10 kPa) heart or sperm whale (0.06 kPa). Affinity increases with a

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WILLIAM R. DRIEDZIC

decrease in analysis temperature. If fish myoglobins bound oxygen with the same affinity as mammalian myoglobin, unloading would be restricted at physiological temperatures (Nichols and Weber, 1989). Myoglobin concentration across species does not correlate in any obvious fashion with activity of enzymes of energy metabolism. In three independent studies (Driedzic and Stewart, 1982; Feller and Gerday, 1987; Sidell et al., 1987) comparisons between myoglobinrich and myoglobin-poor hearts from ectotypically similar species showed no difference in maximal activities of key enzymes from carbohydrate metabolism, fatty acid metabolism, citric acid cycle, or the electron-transport system. In a study encompassing a wide spectrum of species with different behavioral characteristics, there was no correlation between heart myoglobin content and any of eight key enzymes of energy metabolism (Sidell et al., 1987). Regardless of the myoglobin content, fish hearts have an aerobic metabolic profile similar to redtype skeletal muscle and quite distinct from anaerobic white muscle (Driedzic and Stewart, 1982; Feller and Gerday, 1987; Fitch, 1989). Indirect evidence for myoglobin function under hypoxia was first obtained from performance studies of isolated, perfused hearts (Driedzic et al., 1982). Hearts from ocean pout (myoglobin content = 5 nmol/ g) and sea raven (myoglobin content = 75 nmol/g) were treated with IAA to block glycolysis and subjected to hypoxia (5kPa; 0.07 mM 0 2 ) . Ocean pout hearts failed more rapidly than sea raven hearts. The inclusion of hydroxylamine in the medium to convert myoglobin to a form incapable of binding oxygen resulted in more rapid failure of sea raven but not ocean pout hearts. These studies were subsequently extended to include oxygen-consumption measurements (Bailey and Driedzic, 1986). A stepwise change in input PO, from 21 kPa (0.3 mM 0 2 ) to 5 kPa (0.07 mM 0 2 ) resulted in a decrease in oxygen consumption by ocean pout but not sea raven hearts. The inclusion of hydroxylamine in the medium led to a decrease in oxygen consumption by sea raven hearts under both normoxic and hypoxic conditions. Hydroxylamine treatment had no effect on ocean pout hearts under normoxia and only a small effect under hypoxia. These experiments suffered from two potential problems. Foremost, when perfusion was from a fixed filling pressure, decreases in oxygen consumption were associated with decreases in cardiac output. In the perfused fish heart, this results in a decrease in bulk flow of oxygen and nutrients to the myocytes. Second, the possibilty of alternative effects of hydroxylamine at low but not at high levels of Po, were not ruled out. In later experiments, isolated hearts from eel, sea raven, and lumpfish were subjected to stepwise changes in input oxygen content from 21 to 1.3 kPa (Fig. 3)

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24 1

Input PO:! (kPa)

Fig. 3. Oxygen extraction by perfused isolated hearts at different input oxygen levels. Solid symbols, untreated hearts. Open symbols, hearts received 5 mM sodium nitrite to decrease content of functional myoglobin. Myoglobin content in eel and untreated sea raven hearts was 239 and 135nmol/g, respectively. Functional rnyoglobin was undetectable in treated sea raven and all lumpfish hearts. Data from Bailey et al. (1990).

(Bailey et al., 1990).Hearts were force-filled with perfusate at set flow rates, afterload, and frequency of contraction. This resulted in maintenance of stroke volume and stretch on the ventricle. Sea raven and lumpfish hearts were treated with sodium nitrite to block myoglobin function. Sodium nitrite treatment is completely reversible (Bailey and Driedzic, 1988)and does not impair respiration of isolated mitochondria even at oxygen levels approaching zero (Bailey et al., 1990). Myoglobin-rich hearts extracted a constant amount of oxygen until perfusate PO, fell below 10.6 kPa. At this point oxygen uptake began to decline, but these hearts consumed oxygen until input Po,was 1.3kPa. Naturally myoglobin-poor lumpfish hearts and nitrite-treated sea raven hearts were unable to maintain constant levels of oxygen consumption in the face of declining perfusate Pol and failed at an input PO, of 2 4.6 kPa. Nitrite treatment had no effect on lumpfish hearts. Half-maximal oxygen consumptions were attained by myoglobin rich hearts at lower input Po, than either untreated lumpfish hearts or nitrite-treated sea raven hearts. The impact of myoglobin in the support of oxygen consumption was evident at relatively high extracellular PO, (5.3-10.6 kPa) in this model system. Two experiments with fish hearts have failed to reveal a functional significance for myoglobin. The inactivation of myoglobin with phenylhydroxylamine had no effect on cardiac performance or oxygen consumption by isolated buffalo sculpin hearts even under hypoxic conditions (Nichols and Weber, 1990). Hearts were perfused in a fashion similar to that utilized in the study of sea raven already cited (Bailey and Driedzic, 1986.) In addition, oxygen consumption and myoglobin levels are comparable in buffalo sculpin and sea raven hearts, so the apparent lack of myoglobin function in buffalo sculpin

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heart is perplexing. It is possible that for any given species, myoglobin plays a critical role only in a narrow band of oxygen supply and energy demand. As such, it may be very difficult to hit on the set of conditions that illustrate the function of this protein. In an alternative approach, Zoarces viviparous were subjected to hypoxic conditions for 4 to 6 weeks, such that hematocrit and hemoglobin content of blood were significantly elevated. This treatment did not lead to alterations in myocardial myoglobin content (Driedzic et al., 1985a). In summary, most of the evidence supports the concept that the presence of myoglobin allows hearts of some fish to maintain oxygen consumption at lower levels of extracellular Po, than would otherwise be possible. This is similar to findings with mammalian heart that show the presence of myoglobin allows better maintenance of relaxation times (Braulin et al., 1986) and defense of high-energy phosphates (Taylor et al., 1986) under hypoxic conditions. The fish studies are important in a broad sense in that they were the first to show a direct impact of myoglobin on oxygen consumption in intact heart for any species. C. Carbohydrate and High-Energy Phosphate Metabolism In V i m Atlantic hagfish subjected to 20 hours of anoxia (water PO, < 0.3 kPa) showed a decrease in cardiac glycogen (22 to 0.95 pmoles glucose/g) in association with an increase in tissue lactate (0.4 to 11 pmol/g). Blood glucose doubled from 0.5 to 1pmol/ml. Glycolysis was essential to the maintenance of heart function. Animals, pharmacologically immobilized with gallamine triethiodide, maintained cardiac performance at control levels for up to 3 hr under anoxic conditions following treatment with cyanide or azide to impair oxidative phosphorylation; however, glycolytic blockage with IAA severely impaired performance even under normoxic conditions (Hansen and Sidell, 1983). Cardiac glycogen levels in teleosts typically ranges between 40 and 150 pmol/g (Merrick, 1954; Driedzic et al., 1978; Jpjrgensen and Mustafa, 1980a; Driedzic and Stewart, 1982; Dunn et al., 1983; Dunn and Hochachka, 1986); however, a value as low as 5 pmol/g has been reported for European eel (Anguilla anguilla) (van Waarde et al., 1983). During exposure to environmental hypoxia there is usually a decrease in glycogen (Merrick, 1954; Driedzic et al., 1978; Dunn et al., 1983; Dunn and Hochachka, 1986) with a concomitant increase in heart lactate (Driedzic et al., 1978; Jpjrgensen and Mustafa, 1980a;

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Dunn et aZ., 1983; Shoubridge and Hochachka, 1983; Dunn and Hochachka, 1986). Blood glucose increased under oxygen limitation in bluegill sunfish (Lepomis macrochirus) (Heath and Pritchard, 1965), goldfish (Walker and Johansen, 1977; Shoubridge and Hochachka, 1983), flounder (Platichtys Jesus) ( Jgrgensen and Mustafa, 1980a), European eel (van Waarde et al., 1983), and African lungfish (Protopterus aethiopicus) (Dunn et al., 1983), but no increase occurred in rainbow trout (Dunn and Hochachka, 1986) or cutthroat trout (Salmo clarki) (Heath and Pritchard, 1965). The use of exogenous glucose under anoxic conditions by heart was clearly shown in goldfish. Animals subjected to anoxia and injected with 14C-glucose showed labeled glucose, glycolytic intermediates, and lactate in the heart. The specific activity of tissue lactate exceeded that of blood lactate (Shoubridge and Hochachka, 1983). African lungfish (Dunn et al.,1983), European eel (van Waarde et d.,1983), and flounder (Jgrgensen and Mustafa, 1980b) maintained levels of ATP, ADP, and adenosine monophosphate (AMP) under conditions of environmental hypoxia, which led to either decreases in glycogen and/or increases in lactate concentration. The creatine phosphate (CP) levels were constant in flounder and lungfish heart as well ( Jgrgensen and Mustafa, 1980b; Dunn et al., 1983).In contrast, CP and ATP levels in trout decreased, while ADP and AMP concentrations remained the same under hypoxia (Dunn and Hochachka, 1986).With the exception of trout, it appears that high-energy phosphate levels are maintained rather well via anaerobic metabolism within the range of sustainable life. The in oivo studies reveal that glycogen is mobilized and lactate accumulates in heart during periods of oxygen limitation. The extent of alterations in glycogen and metabolite levels depends on the level and length of time under hypoxia. For the most part, high-energy phosphate levels are held fairly constant under reversible oxygen deprivation challenges. That is, glycolytic potential is sufficient to maintain bulk ATP at a level equivalent to ATP demand. D. Metabolism in Isolated Preparations Isolated preparations are useful in that they allow simultaneous assessment of performance and the calculation of rates of anaerobic metabolism, since both tissue and glucose/lactate concentrations in the bathing medium can be quantitated. The importance of anaerobic glycolysis to the maintenance of contractility was evident by a much accelerated failure of trout ventricle

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strips under anoxia with IAA in the media to block glycolysis relative to preparations with glycolysis intact (Hansen and Gesser, 1987). A series of experiments that assessed anaerobic metabolism in oxygen-deprived preparations, that ultimately failed, are especially informative about the role of high-energy phosphates. Perfused isolated hearts of sea raven, ocean pout (Turner and Driedzic, 1980), and smooth dogfish (Mustelus canis) (Driedzic, 1978) and ventricle strips of rainbow trout (Nielsen and Gesser, 1984) all entered into contractile failure under anoxia in association with an increase in lactate production. In each of these studies the preparations entered into contractile failure prior to a significant decrease in ATP levels (Fig. 4). When rainbow trout ventricle strips received an elevation in external Ca2+, twitch force development increased even under anoxia, in association with an increase in the rate of lactate production and no change in ATP levels (Nielsen and Gesser, 1984). Alterations in the,CP pool are more difficult to define. There was no decrease in CP content in the case of sea raven, ocean pout, or dogfish; however, levels at the initiation of the perfusion period were low, and it may be that CP had already discharged (Driedzic, 1978; Turner and Driedzic, 1980). CP levels decreased in rainbow trout ventricle strips (Nielsen and Gesser, 1984) under anoxia. The rate of release of lactate by rainbow trout ventricle strips, maintained in media of 1.25 mM Ca2+, was linear over the 60-min anoxic challenge. The rate of lactate production, taking into consideration both release and accumulation in the tissue, was equiva-

CP = 1.4 i 0.3 ATP I1.3 f 0.2

ATP

=

CP = 2.0 i 0.2 ATP = 2.6 f 0.4

0.1 1.2 f 0.2

40 Trout

040

t

CP = 2.1 f 0.9 ATP = 3.7 f 0.1

CP I 0.5 i 0.1 ATP 5 1.4 f 0.2

10 20 30 40 50 60

Time (mln)

0

20 4 0 60 80100120

Time (min)

Fig. 4. Performance of ventricle strips from rainbow trout and perfused, isolated hearts from dogfish. Solid squares, oxygenated medium. Open symbols, anoxic medium. The arrow in the trout experiment indicates an increase in Ca2+ from 1.25 to 5.0 mM. Concentrations of CP and ATP were determined at the 60- and 120-min points for trout and dogfish preparations, respectively. Values are expressed in pmol/g wet weight on the assumption of 85%wet weight. Data from Nielsen and Gesser (1984)and Driedzic (1978; published by The University of Chicago Press).

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lent to -0.25 pmol * g-' * min-'. Assuming linearity of lactate release for the perfusion period, comparable values for sea raven and ocean pout were 0.5 and 0.13 pmol * g-' min-'. These values are all well below the maximal potential of glycolysis suggested by in vitro enzyme activity levels (Table 111). It is probable that these isolated preparations failed for reasons other than a limitation of glycolysis and subsequent supply of ATP. Metabolism under hypoxic conditions within the range of tolerance has been studied less frequently. Perfusion of isolated hearts of hagfish ( E . cirrhatus) (Forster, 1991) and rainbow trout (Arthur et al., 1992) with hypoxic media results in a decline in sustainable performance and activation of lactate production. Hagfish hearts, perfused with media having an input P o , of 4 kPa release lactate at a rate of about 1.1 pmol 8-l * min-', with anaerobic metabolism accounting for 70% of the ATP production. Short-term, peak lactate production rate was 2.5 pmol g-' min-' at a power output of 0.3 mW/g (Forster, 1991). Perfused rainbow trout hearts can sustain subphysiological levels of power output under close to anoxic conditions. Lactate production is elevated 35-fold to a sustainable rate of 4.3 pmol * g-' * min-' at a power output of 0.5 mW/g (Arthur et al., 1992). In each of the cited studies, involving either perfused hearts or ventricle strips, extracellular glucose was available and is apparently a requirement. Perfused isolated hearts of the teleosts, Hoplias malabaricus and Hoplerythrinus unitaeniatus, when treated with cyanide failed in 23 and 3 min, respectively. Hearts receiving glucose maintained contractility for at least 90 min (Driedzic et al., 1978). Studies with isolated heart preparations corroborate in vivo findings in that they emphasize the importance of the activation of anaerobic metabolism under oxygen-limiting conditions with the use of exogenous glucose as a fuel. Perfused hearts can maintain low levels of power output under hypoxic conditions in concert with elevated rates of anaerobic metabolism. Heart preparations from teleosts and elasmobranchs, induced to perform resting levels of work under anoxia, invariably fail before total ATP pools are decreased.

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E. Metabolic Control at the Enzyme Level 1. MAXIMAL ENZYME ACTIVITY LEVELS AND ANAEROBIOSIS

The maximum in vitro activity levels of enzymes of cardiac energy metabolism are not obviously related to survival capabilities at either the whole-animal or isolated-tissue level (Table 111; Gesser and

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Poupa, 1974). There are some descriptive relationships, though, that warrant comment. A linear relationship exists between maximal PFK activitylresting work and capacity of in uitro preparations to maintain performance under anoxia in a sample size of three species (Side11and Driedzic, 1985). In seven species of marine fish there was a linear correlation between maximal in uitro activities of PKlCytOx versus the time under anoxia required to reduce initial level of force to 50% (Gesser and Poupa, 1974). These studies suggest modifications in enzyme activity levels across species in association with anoxic cardiac performance. However, energy metabolism, although essential, in itself may not be the limiting factor in maintaining functional integrity. Perhaps this is why it is so difficult to reveal direct relationships between anaerobic performance and enzyme capacity to sustain anaerobic ATP production.

2. GLYCOGEN PHOSPHORYLASE In goldfish subjected to anoxia for 24 hr at 7"C, the percentage of glycogen phosphorylase in the active a form decreased from 49 to 8%of the total. The total activity of glycogen phosphorylase did not change (Storey, 1987). This finding implies a decrease in the rate of glycogenolysis at a time when it is probable that glycogen reserves are exhausted (Merrick, 1954). 3. HEXOKINASE Hexokinase occurs in high activities in most fish hearts, and extracellular glucose is utilized as an energy source under hypoxia. In many species, there is a close 1: 1match between ATP demand predicted on the basis of ATPase activity, and potential anaerobic ATP yield from glucose based on the activity of HK (Tables I1 and 111).The calculation is based on the net production of two ATP per glucose utilized by the HK reaction, with the ultimate conversion of glucose to lactate. Furthermore, regression of HK activity against ATPase activity in a wide spectrum of species yields a significant linear equation (Fig. 1).Considered together, these findings suggest that as the total demand for ATP is increased across species, there is an increase in capacity to meet the demand via anaerobic metabolism of exogenous glucose. The importance of HK to the anaerobic fish heart is illustrated by lactate production rates. Maximal lactate production was 2.5 pmol g-' min-' at 18°C by perfused isolated hagfish (Eptatretus cirrhatus) hearts (Forster, 1991) and 4 pmol * 8-l * min-' at 16°C by hypoxic rainbow trout hearts, performing subphysiological levels of work (Arthur et al., 1992). Maximal in uitro HK activity in Atlantic

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-

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hagfish hearts is 1.7 pmol - g-' * min-' at 15°C (Hansen and Sidell, 1983). In rainbow trout, in uitro HK activity ranges from 12 pmol g-l * min-' at 22°C (Brooks and Storey, 1988) to 20 pmol * g-' - min-' at 25°C (Farrell et al., 1990). The Qlo for HK for a number of species is 2.6 (see Section V1,A). Once corrections for temperature are made, rates of lactate production fall within a factor of three- to fourfold the predicted maximal yield from HK activity. Assuming that lactate production rates could be driven even higher with elevations in imposed workload, the match between isolated enzyme activity and lactate formation suggests that the maximal in vitro activity of HK is closely related to maximal rates of lactate production in intact tissue. The HK activity is lower than that of PFK activity in both hagfish (Myxine glutinosa) and rainbow trout heart (Hansen and Sidell, 1983; Brooks and Storey, 1988). The cumulative data imply that HK may be a rate-limiting step in glycolysis. Proof ofthis contention awaits simultaneous measurements of glucose transport, use of exogenous glucose, glycogenolysis, and lactate production. The role of HK in the control of anaerobic glycolysis in the turtle heart is well documented (Reeves, 1966).

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4. PHOSPHOFRUCTOKINASE The key role of PFK in control of glycolysis is illustrated by crossover plots of metabolites. In this experimental approach, the concentrations of glycolytic intermediates are assessed before and after a transition period. Identification of control sites is made by expressing the tissue content of each intermediate, after the onset of anoxia/ hypoxia, as a percentage of the aerobic value. When glycolytic flux increases, there will be a relative depletion of the reactants and accumulation of the products of the rate-limiting reaction. The control sites are identified by crossovers in the sequence of glycolytic intermediates. Crossover points only identify control sites in the metabolic sequence and do not necessarily imply that such sites control the flux. For instance, HK could still control the overall glycolytic rate, even though PFK is maximally activated. Phosphofructokinase was activated during oxygen limitation as indicated by crossover analysis in intact Atlantic hagfish (Hansen and Sidell, 1983), African lungfish (Dunn et al., 1983) and goldfish (Shoubridge and Hochachka, 1983) (Fig. 5). In goldfish heart, PFK activation was still indicated after 60 hr of anoxia at 4°C (Shoubridge and Hochachka, 1983). The kinetics of PFK have been assessed on partially purified preparations from a number of species, including flounder, rainbow trout,

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06P FI.6P G3P PYR F6P DHAP PEP

G6P

F1,6P G3P PYR F6P DHAP PEP

Fig. 5. Crossover plots of glycolytic intermediates in heart following the transition from aerobic to anaerobic conditions. Atlantic hagfish were exposed to anoxic water for 20 hr. African lungfish, which are air breathers, were subjected to forced dives for 3 hr. Data from Hansen and Side11 (1983) and Dunn et al. (1983).

cod, and goldfish (Jensen and Gesser, 1980; Jplrgensen and Mustafa, 1980b; Jensen, 1981; Storey, 1987). Fish heart PFK, similar to that of other systems, is inhibited by low pH, high ATP, and citrate: activators include AMP, Pi, and fructose-2,6-diphosphate (F-2,6-Pz). There are no obvious differences between hypoxia tolerant and intolerant myocardia with respect to potential regulation by pH and the adenylates (Jensen and Gesser, 1980). Of the regulators, F-2,6-Pz may be especially important with a K , of about 0.16 pM for the goldfish enzyme. Following 24 hr of anoxia at TC,F-2,6-Pz levels in goldfish heart increased from 0.13 to 0.47nmol/g, Under anoxia, a rise from about the K , level to a saturating level would stimulate a large increase in PFK activity by increasing the affinity for F-6-P to a concentration closer to the physiological range of the substrate (Storey, 1987). Another method of glycolytic activation in muscle is considered to be increased binding of key enzymes to contractile fibers. Enhanced binding could help to localize ATP production by anaerobic glycolysis into close proximity with the myofibrils to support anaerobic muscle work. Following 24 hr of anoxia at 7"C, binding of PFK to subcellular fractions increased from 35 to 48%of the total activity in goldfish heart (Duncan and Storey, 1991). An alternative view on long-term regulation of PFK is offered by Rahman and Storey (1987). Goldfish were subjected to 24 hr of anoxia at 7°C. Under these conditions the maximal in vitro velocity of PFK was decreased in concert with an increase in K , (F-2,6-P2) and &(AMP). The authors argue that this could indicate a covalent modification of the enzyme via a dephosphorylation, which results in a less active PFK, and that this could result in a curtailment of glycolysis following long-term anoxia. However, crossover plots indicate an acti-

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vation of PFK even after 60 hr of anoxia at low temperature (Shoubridge and Hochachka, 1983), and heart performance studies do not support the concept of a substantial metabolic depression in hearts of teleost fish (see Section V,A). The significance of anoxia-induced alterations in kinetic constants for PFK remains to be elucidated. 5. PYRUVATE KINASE Flounder heart PK is inhibited by ATP and alanine ( JZrgensen and Mustafa, 1980b). Goldfish enzyme is activated by fructose-1,6diphosphate (F-1,6-Pz) and inhibited by alanine. Following 24 hr of anoxia at 7"C, there was a marked reduction in Iso(a1anine)from 12 to 3 mM. The significance of this is unknown.,,V and kinetic constants for PEP, ADP, and F-lY6-P2were unaltered (Rahman and Storey, 1988). Large increases in F-l,6-P2 under anoxia (Shoubridge and Hochachka, 1983) suggest stimulation of PK via feed-forward activation. Consistent with an activation of glycolysis at the level of PK is an increase in %PK bound to the particular fraction following a period of anoxia in goldfish (Duncan and Storey, 1991)and cross-over of metabolites at this site in Atlantic hagfish (Fig. 5). 6. LACTATE DEHYDROGENASE Although not considered a regulatory enzyme, LDH is frequently studied with respect to anaerobic potential. A single B4/H4 LDH homotetramer is expressed in Atlantic hagfish heart. The enzyme is inhibited by high concentrations of pyruvate; however, K,(pyr) is higher than values for Bq isozymes from a number of teleosts and endothermic vertebrates. The isozyme may be functionally similar to a A4/M4 skeletal muscle-type isozyme (Side11and Beland, 1980). The New Zealand hagfish also has a Bq LDH with a relatively high K,(pyr) (Baldwin et al., 1989). Teleost hearts usually exhibit one primary Bq LDH isozyme. In most cases, kinetics of purified enzyme exhibit a low Km(pyr) and inhibition at high pyruvate concentrations in the pyruvate-to-lactate direction (Lush et al., 1969; Gesser and Sundell, 1971; Sensabaugh and Kaplan, 1972; Lim et al., 1975; Place and Power, 1979; Baldwin et al., 1989). In other species, though, the heart isozyme resembles a skeletal muscle-type A4 LDH with a high Km(pyr)and lack of pyruvate inhibition in the pyruvate to lactate direction (Jeirgensen and Mustafa, 1980b; Driedzic et al., 198513; Stewart and Driedzic, 1986; Fitch, 1989). The extreme species variability among fish in maximal in vitro LDH activity and the ratio of activity at low versus high levels of pyruvate was apparent in a survey of 16 species (Gesser and Poupa, 1973).

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Regardless of the kinetic properties of LDH, there is much evidence to show that lactate may be either produced or utilized dependent on the specific set of conditions.

F. Summary Some fish species have elevated levels of myoglobin, which extend the lower limit of extracellular Po, at which oxygen extraction is possible. Transitions from aerobic to oxygen-limiting conditions are associated with an activation of glycogenolysis and glycolysis. Hagfish are able to maintain heart performance under hypoxia via anaerobic ATP production. This does not imply that glycolytic rates are exceptionally high in this species but only that ATP supply can meet demand. Cardiac power output decreases in teleosts and elasmobranchs under oxygen-limiting conditions. Under anoxia/hypoxia, glycogen and blood-borne glucose serve as metabolic fuels, and glycolytic flux remains elevated during the entire period. The use of exogenous glucose is consistent with high activities of HK in this group of animals. Glycolysis is activated at the level of PFK and at least in goldfish, F-2,6-P2 appears to be an important regulator, in concert with increased enzyme binding to contractile fibers. There may also be feed-forward activation at PK via increases in F-l,6-P2, and increased binding to contractile fibers may occur at this site as well. Production of ATP via anaerobic glycolysis keeps pace with ATP demand, and hearts fail for reasons other than whole-tissue ATP availability. That is, the metabolic potential is in place to meet anaerobic ATP demand. Alterations in CP levels with subsequent increases in Pi are still in question and require further investigation, since Pi directly impairs contractility in skinned muscle from rat ventricle (Kentish, 1986).

V. IMPACT OF LOW TEMPERATURE ON METABOLISM A. Acute Temperature Transitions 1. In Vivo AND In Vitro PERFORMANCE Acute decreases in temperature invariably result in decreases in resting heart performance in uiuo. Heart rate decreases with a QKIof about 2 (Farrell, 1984;Gehrke and Fielder, 1988),and there are reduc-

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25 1

tions in cardiac output (Stevens et al., 1972; Cech et al., 1976; Barron et al., 1987). The impact of acute temperature transitions on force development of electrically paced ventricle strips has been assessed in yellow perch (PercafEauescens),smallmouth bass (Micropterns dolornieui), pickerel (Esox niger), and American eel (Anguilla rostrata), and leads to the following generalizations (Bailey and Driedzic, 1990; Bailey et al., 1991).A decrease in temperature results in a lengthening of the times to peak tension and relaxation. In preparations contracting at low frequencies, temperature decreases within the physiological range have very little impact on ability to develop force and in some cases actually result in an increase in force development. At elevated contraction frequencies, however, force development is compromised by low temperature owing to either increases in resting tension or inability to follow imposed pacing regimes. These experiments imply that maximal levels of heart performance and hence ATP demand and supply are reduced as temperature is decreased. The impact of a decrease in temperature at low levels of work is not so clear. There may be some situations where heart power output is maintained because of an enhancement of force development, possibly associated with enhanced calcium delivery to the contractile apparatus (see Chapter 6, this volume). This implies that ATP production mechanisms keep pace with at least basal rates of demand over a range of temperatures. 2. METABOLISM IN ISOLATEDPREPARATIONS The rate of contraction of spontaneously beating hearts decreases with a decrease in temperature in goldfish (Tsukuda, 1990), perch (Percafluviatilis)(Bowler and Tirri, 1990),and sea raven (Graham and Farrell, 1985). Spontaneously beating goldfish hearts, immersed in Ringer's, have higher rates of contraction and oxygen consumption at 15°C than at 10°C (Tsukuda et al., 1985).Experiments conducted with electrically paced preparations negate the impact of temperature on the pace-maker cells and allow a more direct assessment of the metabolic support system. Perfused, sea raven (with glucose alone as a metabolic fuel), pickerel, and eel hearts receiving medium containing low levels of Ca2+, all showed lower rates of oxygen consumption in association with lower temperatures (lo" or 15°C to 5°C). Changes in overt power output were small but in the same direction as the alterations in oxygen consumption (Bailey and Driedzic, 1989; Bailey et al., 1991).An elevation in extracellular Ca2+resulted in a twofold increase in oxygen consumption by sea raven hearts but no change in pickerel and eel hearts. At least for sea raven it appears that neither oxygen nor

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fuel utilization was the limiting factor in performance at the lower calcium level. In these studies hearts were generating power outputs of 0.5-1 mW/g, levels close to in vivo resting conditions. The impact of acute temperature changes on maximal levels of performance and metabolism is yet to be assessed. Perfusion of sea raven hearts with alternative fuel sources has revealed a complex and yet-to-be-resolved role for fatty acids at low temperature (Sephton et al., 1990). Hearts receiving glucose alone as an exogenous fuel showed a decrease in power output and oxygen consumption at 5°C relative to those at 15°C. The inclusion of palmitoleate (a 16-carbon fatty acid with 1 double bond) in the medium enhanced both oxygen consumption and performance at 5°C only, such that there was no difference in these parameters between the two temperatures. The simplest explanation for the finding is that glucose metabolism is limited at 5"C, but fatty acid metabolism is functional and provides the necessary energy to support contractility. Attempts to directly test this hypothesis with radiolabeled fuels were unsuccessful as I4CO2 production from either ''C-6-glucose or '*C-1-palmitate yielded only about 2% of predicted rates on the basis of oxygen consumption. Label was incorporated into glycogen and lipid pools at similar rates at both 5" and 15°C. It is not known how carbon from glucose enters the lipid pool. It may be at the level of glycerol forming the backbone for triglycerides, in which case the possibility of impairment of glucose catabolism at some site in the final segment of glycolysis such as PK or pyruvate dehydrogenase cannot be ruled out. Alternatively if glucose was catabolized to acetyl CoA prior to entry into the lipid pool, an impaired use of exogenous glucose at low temperatures cannot be invoked as the explanation for the fatty acid-induced enhancement in oxygen consumption and power output. Enzyme studies suggest that fatty acid metabolism in sea raven heart should be curtailed at low temperature since the 910 for CPT is high (see the following). An impairment of fatty acid oxidation could potentially result in the elevation of intracellular free fatty acids. Fatty acids can function directly as modifiers of membrane channels in a number of tissues (Ordway et al., 1991). The possibility that fatty acids are inducing alterations in intracellular ion activity, which results in an enhancement of cardiac performance and oxygen consumption at low temperature, should be assessed. 3. In Vitro ENZYME ACTIVITIES The impact of acute temperature transitions within the physiological range on maximal in uitro enzyme activity has been determined for

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key enzymes of energy metabolism (Crockett and Sidell, 1990; Bailey et al., 1991). A decrease in assay temperature always results in a decrease in HK activity, which follows a Qlo of 2.63 2 0.29 (mean -+ S.E. for 7 species of teleosts). In longhorn sculpin (Myoxcephalus octodecimspinosus) and tautog (Tautoga onitis) the Qlo values for PFK, PK, and LDH were close to 2.5 (Crockett and Sidell, 1990). These findings suggest that the maximal rate of carbohydrate metabolism should be curtailed with a decrease in body temperature. Temperature transitions have a much lower impact on CS activity that follows a Q l o of 1.60 -+ .09 for eight species of teleosts. The response of CPT to altering temperature appears to be quite species specific. I n sea raven and smallmouth bass (Micropterus dolomieui) the Qlo values were very steep at 5.5 and 9.9, respectively (Sephton et al., 1990; Sephton and Driedzic, 1991). In longhorn sculpin and tautog, the Q l o was 2.3, but in four other species of teleosts, a change in assay temperature did not alter the activity of CPT. It should b e appreciated that the activity of this enzyme is extremely low in these animals, thus making quantitation difficult. The Qlo for HOAD was 3.1 and 9.3 for longhorn sculpin and tautog, respectively. The impact of temperature on aerobic fatty acid metabolism warrants further study. The available data suggest extreme species variability in maximal rates of fatty acid metabolism ranging from temperature insensitivity to decreases of about 10-fold for every 10°C decrease in temperature.

B. Chronic Low-Temperature Exposure in Nonpolar Teleosts Some teleosts such as carp show an enhancement of swimming performance following acclimation to low temperature (Rome et al., 1985). Such behavior, which must be dependent on cardiac performance, may allow for active foraging and/or escape from predators at winter temperatures. This section summarizes the evidence for adaptive responses in heart, which presumably underwrite additional demands of blood supply either during or following activity periods. It should be recognized that positive thermal acclimation at the wholeanimal level does not occur in all species as some, such as eels, become quite dormant at low temperatures. 1. INCREASES IN HEARTMASS One frequently observed response to low temperature is an increase in heart size. This occurs in carp (Goolish, 1987), goldfish (Tsukuda et al., 1985), sunfish (Lepornis cyaneZZus) (Kent et aZ., 1988),

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catfish (Zctalurus punctatus) (Kent et al., 1988), rainbow trout (Farrell et aZ., 1988a; Graham and Farrell, 1989), and smallmouth bass (Sephton and Driedzic, 1991). The increase in heart mass relative to body mass is typically in the range of 10 to 25% following an acclimation period of a few weeks to a temperature 10"-15"C lower. Cardiac growth does not result in changes in protein contentig heart weight (Farrell et al., 1988a; Kent et al., 1988).An increase in heart size following acclimation to low temperature need not always occur, as no changes were noted in white perch (Morone arnericanus) or yellow perch (Perca jlauescens) (Sephton and Driedzic, 1991), species that are active at low temperatures, Moreover, an increase in heart mass may occur in species that are quite sedentary at low temperature, such as smallmouth bass. Maximal performance levels were determined for in situ perfused trout hearts. Hearts from animals acclimated to 5°C generated about 60% of maximal power output/g as hearts from fish acclimated to 15°C when both were tested at their respective holding temperature (Graham and Farrell, 1989, 1996). The larger heart size of the coldacclimated animals contributed to enhanced stroke volumes at low temperatures, and consequently there was considerable overlap between the two groups in the maximal level of power output per heart. Acute effects were not assessed for either 5" or 15°C acclimated fish, so it is not known whether additional adaptational responses occurred. AND METABOLISM 2. In Vitro PERFORMANCE Positive thermal adaptation has been demonstrated in Perca, which remain active at winter temperatures. In P. Bauescens, acclimated to 20"C, the maximal rate of contraction of electrically paced ventricle strips was >48 b e a t s h i n at 20°C but only 30 beatslmin at 5°C. Moreover, at F C , twitch force development decreased substantially with an increase in contraction frequency. Following acclimation to 5"C, hearts could be paced at frequencies >48 beatdmin, and twitch force development was enhanced. The enhanced chronotropic responsiveness was associated with about a 25% decrease in time to relaxation (Bailey and Driedzic, 1990).Although acclimation to low temperature increases heart performance and energy demand, there were no changes in HK, CS, or carnitine acyl CoA transferase activities in .2' flauescens (Sephton and Driedzic, 1990). The impact of lowtemperature acclimation has also been studied in the congeneric P. fluviatilis (Bowler and Tirri, 1990). Atria from this species, acclimated to 5°C had higher rates of spontaneous contraction at low test temperatures than atria from animals acclimated to 19"C, and similarly, non-

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contracting isolated hearts had higher rates of oxygen consumption. Low-temperature acclimation resulted in an increase in the volume density of sacroplasmic reticulum but no alteration in mitochondrial volume density or myoglobin content. The situation with respect to thermal acclimation in Perca seems to be both clear and consistent. Low-temperature acclimation leads to alterations in ability to maintain higher rates of contraction and presumably cardiac output with no increase in heart mass. The increase in sarcoplasmic reticulum is possibly responsible for shorter relaxation times and subsequently higher maximal contraction frequencies. The metabolic machinery necessary to supply ATP is quite adequate and requires no further expansion as evident by constant mitochondrial volume density, constant activity of the mitochondrial marker enzymes, and myoglobin content. Goldfish hearts also exhibit enhanced performance following lowtemperature acclimation. Spontaneously contracting, isolated preparations from cold-acclimated goldfish had higher frequencies and/or amplitudes of contraction and higher rates of oxygen consumption per g than hearts from warm acclimated animals at low test temperatures (Tsukuda et al., 1985; Tsukuda and Liu, 1987; Tsukuda, 1990).At low test temperature glucose had a protective effect on performance of hearts isolated from warm-acclimated but not cold-acclimated goldfish suggestive of alterations in ability to utilize exogenous glucose as a function of thermal history (Tsukuda and Kihara, 1989). Adaptations in cardiac enzyme levels are evident in some species. Hearts from chain pickerel acclimated to 5" exhibited higher maximal i n vitro activitiedg of HK, CPT, CS, and Cyt Ox than hearts from animals acclimated to 25°C when assays were conducted at the common temperature of 15°C.Similarly animals sampled in winter had higher enzyme activities than those sampled in summer (Way Kleckner and Sidell, 1985). Carp showed an increase in Cyt Ox following acclimation to low temperature (Cai and Adelman, 1990).White perch hearts exhibited a 2- to 40-fold increase in activity of carnitine palmitoleoyl CoA transferase and carnitine oleoyl CoA transferase (oleate is a 18-carbon fatty acid with 1 site of unsaturation) following lowtemperature acclimation. This finding suggests an enhanced ability to oxidize unsaturated fatty acids. Increases in enzyme activities are taken to mean an increase in the concentration of enzyme as opposed to the expression of specific isozymes at different temperatures. The fish species in which there were increases in cardiac enzyme activities, as a function of cold-temperature acclimation, all show maintenance of whole-animal swimming performance at low temperatures. Positive thermal adaptation in cardiac performance and/or metabo-

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lism does not occur in all species. In situ performance of perfused sea raven hearts from summer and winter acclimation conditions was assessed (Graham and Farrell, 1985). Inherent heart rate of summer fish was higher at both 14" and 4°C test temperatures. There was no evidence for a positive thermal compensation in power output, as hearts from summer animals tested at 4°C exhibited marginally higher levels than hearts from winter animals tested at 4°C.These findings are consistent with the very low activity patterns that sea raven in captivity exhibit at low temperatures. Acclimation to low temperature did not result in changes in HK, CS, or CPT in white perch, yellow perch, or smallmouth bass (Sephton and Driedzic, 1990).

3.

SUMMARY

Some fish species exhibit positive thermal compensation in response to low-temperature acclimation. This is evident by increases in maximal rates of contraction and ability to maintain force development at elevated frequencies. An increase in heart mass is a frequent response to low temperature. Both increases in maximal contraction frequency and heart size could contribute to enhanced maximal cardiac output. In some species, metabolic potential as evidenced by enzyme levels is sufficient to meet energy demands over a range of temperatures. In other species, there is an increase in metabolic potential, following low temperature acclimation, owing to elevated levels of some enzymes of energy metabolism. Most frequently observed are increases in mitochondria1 enzymes necessary for aerobic fatty acid catabolism.

C. Antarctic Teleosts Fish living in the Antarctic have evolved at temperatures close to 0°C. Channichthyidiae have hearts three to five times larger than those of other nonpolar and Antarctic teleosts (Johnston and Harrison, 1987; Harrison et al., 1991).This, however, is not an adaptation to cold temperature alone, as other hemoglobin-containing Antarctic teleosts have heart sizes within the range typically encountered in nonpolar species (Holeton, 1970; Johnston et al., 1983; Feller and Gerday, 1987). Despite the low temperature, Chaenocephalus acerutus has a resting power output per g heart comparable to those of temperate zone species (see Section 11).Aspects of energy metabolism relevant to a lack of myoglobin in heart are discussed in Section V,B. Maximal in vitro activities of enzymes of energy metabolism have been assessed for five species of red-blooded Antarctic fish and two

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species of hemoglobin-free icefish (Feller and Gerday, 1987; Sidell et al., 1987; Fitch, 1989; Crockett and Sidell, 1990). Following adjustments for assay temperature activities (concentrations) of enzymes of carbohydrate metabolism (i.e., HK, PFK, PK), on the basis of a Q ~ of o 2.5, and CS, on the basis of a Qlo of 1.6, are similar in Antarctic fish and in other teleosts. Activities of CPT and HOAD, necessary for fatty acid catabolism, and Cyt Ox,an indicator of the electron-transport system capacity, are comparable to activities in nonpolar teleosts, despite large differences in assay temperatures (Table 111).This general contention is consistent with the comparison of enzyme activities in heart from two species on Antarctic fish with two temperate-zone species of similar body form and behavior. At a common assay temperature of 1"C, enzymes of aerobic fatty acid catabolism were always significantly higher in the Antarctic species (Crockett and Sidell, 1990). Overall the enzyme data suggest that there is an increase in the concentration of metabolic enzymes necessary for aerobic metabolism and that there may be a predilection for the oxidation of fatty acids as opposed to carbohydrates as metabolic fuels in hearts of Antarctic fish. Isolated heart preparations from N. gibberifrons preferentially oxidize unsaturated as opposed to saturated fatty acids. This view is based on the observations that l4CO2 release was 2.5-fold greater from oleate than from palmitate when both were offered at the same specific activity, and in mixed substrate studies, palmitate had no effect on oleate oxidation, but oleate inhibited palmitate oxidation (Sidell et al., 1988). In summary, information on Antarctic fishes is minimal. Metabolic adaptations allow at least resting levels of power output per g heart to be as high as that which occurs in species operating at much higher temperatures. There is a general increase in mitochondria1 enzyme levels and an enhancement of potential to oxidize fatty acids as metabolic fuels. In one species there is a demonstrated preference for unsaturated over saturated fatty acids. These responses are the same as those that occur in some species of temperature zone teleosts acclimated to cold temperature.

ACKNOWLEDGMENTS During the writing of this paper, WRD was the recipient of a Royal Society (U.K.)Natural Sciences and Engineering Research Council of Canada Exchange Fellowship, which was held at the Marine Biological Association, Plymouth, England. Appreciation is extended to the staff ofthe M.B.A. for their support. Research in WRD's laboratory is supported by N.S.E.R.C.and the New Brunswick Heart and Stroke Foundation.

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Harrison, P., Zummo, G., Farina, F., Tota, B., and Johnston, I. (1991).Gross anatomy, myoarchitecture, and ultrastructure of the heart ventricle in the hemoglobinless icefish, Chaenocephalus aceratus. Can.J . Zool.69,1339-1347. Heath, A. G., and Pritchard, A, W. (1965). Effects of severe hypoxia on carbohydrate energy stores and metabolism in two species of fresh-water fish. Physiol. Zool. 38, 325-334. Hemmingsen, E.A., Douglas, E. L., Johansen, K., and Millard, R. W. (1972).Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus. Comp. Biochem. Physiol. 43A, 1045-1051. Hipkins, S . F. (1985). Adrenergic responses of the cardiovascular system of the eel, Anguilla australis, In vivo.]. E x p . Zool.235,7-20. Hochachka, P. W., Storey, K. B., French, C. J., and Schneider, D. E. (1979).Hydrogen shuttles in air-versus water-breathing fishes. Comp. Biochem. Physiol. 63B, 45-56. Holeton, G. F. (1970).Oxygen uptake and circulation by a hemoglobinless Antarctic fish (Chaenocephalus aceratus Lonnberg) compared with three red-blooded Antarctic fish. Comp. Biochem. Physiol. 34,457-471. Holeton, G . F., and Randall, D. J. (1967).Changes in blood pressure in the rainbow trout during hypoxia. /. Exp. Biol. 46,297-305. Houlihan, D. F., Agnisola, C., Lyndon, A. R., Gray, C., and Hamilton, M. N. (1988). Protein synthesis in a fish heart: Responses to increased power 0utput.J. Exp. Biol. 137,565-587. Jensen, G. S. (1981). A comparison of some properties of myocardial phosphofmctokinase from the rainbow trout (Salmo gairdneri) and the diving turtle (Pseudemys scripta). Comp. Biochem. Physiol. 70B, 161-164. Jensen, G. S.,and Gesser, H. (1980).Properties of phosphofructokinase and tolerance to hypoxia in vertebrate myocardia. Comp. Biochem. Physiol. 67B, 175-178. John, T. M., Thomas, E., George, J. C., and Beamish, F. W. H. (1977).Effect ofvasotocin on plasma free fatty acid level in the migrating anadromous sea lamprey. Arch. Int. Physiol. Biochim. 85,865-870. Johnston, I. A., and Harrison, P. (1987).Morphometrics and ultrastructure of myocardial tissue in Notothenioid fishes. Fish Physiol. Biochem. 3,l-6. Johnston, I. A., Fitch, N.,Zummo, G., Wood, R. E., Harrison, P., and Tota, B. (1983). Morphometric and ultrastructural features of the ventricular myocardium of the haemoglobin-less icefish Chuenocephalus aceratus. Comp. Biochem. Physiol. 76A, 475-480. Jgirgensen, J. B., and Mustafa, T. (1980a).The effect of hypoxia on carbohydrate metabolism in flounder (PlatichthyefEesus L.)-I. Utilization of glycogen and accumulation of glycolytic end products in various tissues. Comp. Biochem. Physiol. 67B, 243248. Jgirgensen, J. B., and Mustafa, T. (198Ob).The effect of hypoxia on carbohydrate metabolism in flounder (Platichthysjesus L.)-11. High-energy phosphate compounds and the role of glycolytic and gluconeogenic enzymes. Comp. Biochem. Physiol. 67B, 249-256. Kalous, M., Rauchovh, H., Maresca, A., Prochbka, J., and Drahota, Z. (1989).Oxidative metabolism of the inner and outer ventricular layers of carp heart (Cyprinus carpio L.). Comp. Biochem. Physiol. 94B, 631-634. Kelly, D. A., and Storey, K. B. (1988). Organ-specific control of glycolysis in anoxic turtles. Am. J . Physiol. 255 (RegulatoryIntegrative Comp. Physiol. 24), R774-R779. Kent, J. M., Koban, M., and Prosser, C. L. (1988). Cold-acclimation-induced protein hypertrophy in channel catfish and green sunfish.]. Comp. Physiol. B . 158,185-198.

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Kentish, J. C. (1986).The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J. Physiol. 370,585-604. Kiceniuk, J. W., and Jones, D. R. (1977). The oxygen-transport system in trout (Salmo gairdneri) during sustained exercise. J . Exp. Biol. 69,247-260. Lai, N. C., Graham, J. B., Lowell, W. R., and Shabetai, R. (1989).Elevated pericardial pressure and cardiac output in the leopard shark Triakis semifmciata during exercise: The role of the pericardioperitoneal canal.]. E x p . Biol. 147,263-277. Lai, N. C., Graham, J. B., and Burnett, L. (1990).Blood respiratory properties and the effect of swimming on blood gas transport in the leopard shark Triakis semifasciata. J . Exp. Biol. 151,161-173. Lanctin, H. P., McMorran, L.E., and Driedzic, W. R. (1980).Rates of glucose and lactate oxidation by the perfused isolated trout (Saluelinus fontinalis) heart. Can.J . Zool. 58,1708-1711. Larsson, A,, and Fange, R. (1977). Cholesterol and free fatty acids (FFA) in the blood of marine fish. Comp. Biochem. Physiol. 57B, 191-196. Larsson, A.,and Lewander, K. (1973).Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A,367-374. Lim, S. T., Kay, R. M., and Bailey, G . S. (1975).Lactate dehydrogenase isozymes of salmonid fish. Evidence for unique and rapid functional divergence of duplicated Hq lactate dehydrogenases. J. Biol. Chem. 250,1790-1800. Lush, I. E., Cowey, C. B., and Knox, D. (1969).The lactate dehydrogenase isozymes of twelve species of flatfish (Heterosomata).J. E x p . Zool. 171,105-118. MacIntyre, A. B., and Driedzic, W. R. (1981).Activities of enzymes in cardiac energy metabolism. Can.J . Zool. 59,325-328. Merrick, A. W. (1954).Cardiac glycogen following fulminating anoxia. Am. J . Physiol. 176,83-85. Milligan, C. L. (1991).Adrenergic stimulation of substrate utilization by cardiac myocytes isolated from rainbow tr0ut.J. E x p . B i d . 159, 185-202. Milligan, C. L., and FarreI1, A. P. (1991).Lactate utilization by an in situ perfused trout heart: Effects of workload and blockers of lactate transport. J . E x p . Biol. 155,357373. Milligan, C. L.,and Farrell, T. P. (1986).Extracellularand intracellular acid-base status following strenuous activity in the sea raven (Hemitripterus americanus).J . Comp. Physiol. B . 156,583-590. Moon, T. W., Altringham, J. D., and Johnston, I. A. (1991).Energetics and power output of isolated fish fast muscle fibres performing oscillatory work. J. Exp. Biol. 158, 261-273. Moon, T.W.,and Mommsen, T. P. (1987).Enzymes of intermediary metabolism in tissues of the little skate, Raja erinacea.j . Exp. Zool. 244,9-15. Moyes, C. D., Buck, L.T., and Hochachka, P. W. (1990).Mitochondria] and peroxisomal fatty acid oxidation in elasmobranchs. Am. J. Physiol. 258 (Regulatory Zntegratiue Comp. Physiol. 27).R7564762. Neely, J. R.,and Morgan, H. E. (1974).Relationship between carbohydrate and lipid metabolism and the energy balance of the heart. Annu. Rev. Physiol. 36, 413459. Newsholme, E. A., and Crabtree, B. (1986).Maximum catalytic activity of some key enzymes in provision of physiologically useful information about metabolic fluxes. J. E X P .2001.239, 159-167. Nichols, J. W., and Weber, L. J. (1989).Comparative oxygen affinity of fish and mammalian myoglobins. J . Comp. Physiol. B 159,205-209.

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EXCITATION-CONTRACTION COUPLING I N THE TELEOST HEART GLEN F . TZBBITS A N D CHRZSTOPHER D . MOYES Cardiac Membrane Research Laboratory Kinesiology Simon Fraser University Burnaby, British Columbia, Canada

LEIF HOVE-MADSEN Department of Zoophysiology University of Aarhus 8000 Aarhus C., Denmark

I. Introduction 11. Myocardial Contraction

A. Contractile Proteins 8. Calcium Requirements of the Contractile Element 111. Regulation of Caz+ Delivery to Myofibrils A. Depolarization B. Transsarcolemmal Ca2+ influx C. Calcium Release from the Sarcoplasmic reticulum IV. Myocardial Relaxation A. Sarcolemma Ca2+ATPase B. Na+-Ca*+ exchange V. Conclusions References

I. INTRODUCTION Cardiac output, the product of heart rate and stroke volume, must be regulated in vertebrates to maintain appropriate blood flow to exercising muscle, brain, and other tissues under a wide variety of conditions. Stroke volume, in turn,is controlled primarily by the modulation of two important parameters: the preload or end-diastolic volume and myocardial contractility. This chapter will focus on the mechanisms of 267 FISH PHYSIOLOGY, VOL.XlIA

Copyright Q 1992 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

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excitation-contraction (E-C) coupling that serve to regulate contractility in the teleost heart. In the discussion it will be apparent that there is a paucity of information on the regulation of contractility at the cellular level in the fish heart, and therefore, it is necessary to draw inferences from what is known in the amphibian and mammalian heart. One of the intentions of this chapter is to stimulate discussion and research in these critical areas. A general scheme of E-C coupling is shown in Fig. 1,which serves to compare and contrast the model developed for mammals (Fig. 1A) with what is known in teleosts (Fig. 1B). The major difference in E-C coupling between poikilotherms and homeotherms relates to the source of Ca" that is delivered to the myofibrils. It has been known for more than a century (Ringer, 1883)that myocardial contraction has an absolute extracellular Ca2+ requirement. It has been shown that an influx of calcium across the sarcolemma (SL) plays a critical role in the regulation of contractility. This transsarcolemmal Ca2+influx has been proposed by Fabiato (1983) to trigger the release of a greater quantity of calcium from the sarcoplasmic reticulum (SR). Despite the fact that the magnitude of Ca2+ influx across the SL is variable between mammalian species (Bers, 1985),most studies indicate that is inadequate in magnitude to support contraction, and therefore Ca" released from the SR is the major source of Ca2+ for contraction in the hearts of all mammals examined (Wier, 1990). In the hearts of lower vertebrates, however, the situation appears to be quite different. For example, in the amphibian myocardium, it has Fig. 1. (A) Schema for excitation-contraction coupling in the mammalian heart. Contraction occurs due to a 5- to 20-fold rise in cytosolic [Ca2+]from a resting value of approximately 100 nM. The intensity of the arrows indicate relative roles of Ca2+ released from the SR (SO-SO%) and transsarcolemmal influx (20-40%). The SR Cap+ release occurs in response to Ca2+ influx through the Ca2+ channel or DHPR, which is obligatory for contraction. Relaxation occurs due to the lowering of [Cap+],back to 100 nM by the action of the SR Ca2+ pump and the SL Na+-Ca2+ exchanger. SL, sarcolemma; SR, sarcoplasmic reticulum. See (B). Proposed schema for excitation-contraction coupling in the teleost heart. The figure illustrates several differences in cardiac myocyte structure between fish and mammals, including differences of cell diameters, absence ofT-tubules, and sparsity of SR. It appears that the primary source ofcontractile Ca2+is from the transsarcolemmal influx through the DHPR. The dashed lines indicate a putative role for these sources of Cap+under certain physiological conditions in some teleost species. For example, Ca2+from the SR appears to be important for contraction when the temperature is relatively high (>2OoC)in salmonids and tuna. Reverse mode of the SL Na+-Ca2+ exchanger may also be a source of contractile Cap+in the teleost heart. Relaxation occurs by removal of Ca2+ from the cytosol, and we propose that this occurs primarily by the SL Na+-Ca2+ exchanger and, to a lesser degree, the SL and SR Cap+ pumps. See text for details.

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I 3Na

\

i

W

B

+

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Ca

I

Ca

SR

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been suggested (Klitzner and Morad, 1983; Morad et al., 1983)that SR Ca2' release is of no significance in contraction. Contraction in both the amphibian (Bers, 1985) and teleost (Driedzic and Gesser, 1988; Hove-Madsen and Gesser, 1989) ventricle appears to be relatively insensitive to ryanodine at concentrations demonstrated to block SR Ca2' release (Sutko and Kenyon, 1983). Given these findings, one would expect in the hearts of lower vertebrates that the magnitude of transsarcolemmal Ca2+transport to be sufficient to support contraction unless another organelle (e.g., mitochondria) is able to contribute to the beat-to-beat regulation of the intracellular concentration of Ca2+ [Ca2']i. Whereas it should be noted that this potential contribution of mitochondria has been ruled out in mammalian hearts, based on a variety of kinetic and pharmacological arguments (Fabiato, 1983), the validity of these arguments needs to be tested in teleosts. In this chapter, we will examine what is known about E-C coupling in the teleost heart and attempt to integrate it into a conceptual framework based on the literature of all species. It is unlikely that the scheme for mammals would serve teleosts equally well. Anatomical and ultrastructural distinctions between the hearts of mammals and teleosts (sparse SR, absence of T-tubules, often absent or rudimentary coronary circulation) underlie important physiological differences. The scope of cardiovascular function evident in fish (see Chapter 3 of this volume), and wide-ranging environmental regimes impose unique demands on the regulation of contractility and hence E-C coupling. Of these, temperature is perhaps the most dominant environmental factor. These challenges would be expected to differ in eurytherms (e.g., carp), low temperature-strict stenotherms (e.g., icefish), and endotherms (e.g., tuna). Furthermore, active teleosts, such as salmonids, face special challenges in maintaining significant cardiac outputs at temperatures that are cardioplegic to mammals. Thus, it is also our intention in this chapter to examine possible mechanisms by which the teleost heart is able to regulate contractility despite these different demands. 11. MYOCARDIAL CONTRACTION A. Contractile Proteins 1. ULTRASTRUCTURE OF THE CONTRACTILE ELEMENT The ultrastructure of the fish heart has been thoroughly reviewed recently by Santer (1985). Electron microscopy reveals the distinct pattern of banding of the contractile element observed in striated

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muscle of other species. A, I, and 2 bands are obvious, but the H zone varies with the degree of contraction. M lines, which play a role in maintaining myosin alignment, are not so well documented in fish heart. N lines, which are found on either side of the Z line, have been observed in a number of fish and, although the composition of these bands is not established, it has been suggested that they play a role in Ca2+ binding in the I band. As in mammalian striated muscle, six actin-containing thin filaments surround one myosin-containing thick filament in the hearts of several fish. The distance between thick filaments varies from 20 to 30 nm. There are no apparent phylogenic trends in the intracellular distribution of myofibrils. In some species, the myofibrils are found predominantly in the periphery of the cell; in other species they are evenly distributed. Morphometric analyses have indicated that the proportion of the cell taken up by contractile element appears to be higher in ventricle compared to atrium (e.g., Salmo trutta, 47% of ventricular intracellular volume is myofibril versus 37% of atrium; Yamauchi and Burnstock, 1968). The ventricular content of myofibrils may vary between species. At least in tuna (Thunnus alalunga), there are no differences in the myofibrillar content between the morphologically distinct compact and spongy ventricular layers, each 52-55%. 2. ACTOMYOSIN ATPASE

Myofibrillar proteins include those of the thick filament (mainly myosin) and thin filament (mainly actin, troponin, and tropomyosin). Native mammalian cardiac myosin is composed of two myosin heavy chains (HC) and four myosin light chains (LC).The two HC subunits of mammalian ventricle (a, 8)are combined into three possible dimers VI (acy),V2 (ap),and V3 (Pp), which can be distinguished by enzymic (ATPase) activity, rates of contraction, and electrophoretic mobility in nondissociating gels. The capacity of mammalian heart to express different isoforms of myosins provides plasticity in the cardiac response to changes in cardiovascular demands. Thus, long-term changes in ventricular function accompanying development, exercise training, or changes in metabolic demands (e.g., starvation, hormonal conditions) can be met by structural adaptations (Solar0 et al., 1989). Unlike the mammalian situation, only one native myosin isoform is detected in the ventricles of most teleosts (Karasinski, 1988; Martinez et al., 1991). Goldfish (Carassius auratus L.) ventricle demonstrates two isoforms (Karasinski, 1988). Although only one myosin isoform could be detected in carp (Cyprinus carpio) ventricle (Karasinski, 1988), myosin ATPase activity (pmol phosphate released/min/mg myosin) of the carp compact myocardium is about 50% higher than that in spongy myocar-

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dium (Bass et al., 1973). This suggests the presence of at least two isoforms differing in catalytic activity, which are electrophoretically indistinguishable using the techniques applied to date. Native myosins of atrium migrate as a single band in four cyprinids (Tinca tinca L., Rutilis rutilis L. Leuciscus leuciscus L., Gobio gobio L.; Karanski, 1988)and a salmonid (Salvelinus alpinus, L.; Martinez et al., 1991),but two bands in three other cyprinids (Cyprinus carpio L., Carussius auratus gibelio Bloch, Carussius carussius L.; Karanski, 1988).Native atrial myosin is distinct from that of ventricle in each species (Karanski, 1988; Martinez et al., 1991). Electrophoresis of myosin under denaturing conditions reveals subunit composition (HC, LC). Single HC subunits are detected in both ventricle and atrium of Arctic char, (Suluelinus alpinus) consistent with the expression of only one apparent native myosin (Martinez et aZ., 1991). Atrium and ventricle each possess two types of myosin light chains (LC1, LC2) (Karanski, 1988; Martinez et al., 1991). Each atrial isoform comigrates with its ventricular counterpart on twodimensional gel electrophoresis (Martinez et al., 1991). Changes in myofibrillar proteins occur in relation to acclimation temperature in skeletal muscle (see Guderley and Blier, 1988; Johnston et al., 1990). Johnston et al. (1975) demonstrated that goldfish skeletal myofibrillar ATPase activity is 2.8-fold higher in fish acclimated to 1°Cthan to 26°C. Also, marked differences in thermal stability were obvious, as indicated by inactivation at 37°C. Acclimationinduced changes in skeletal HC profiles have not been found using native protein analysis (Johnston et al., 1990). Peptide mapping of myosin proteins of skeletal muscle of Cyprinus carpio yield little (a-chymotrypsin treated HC subfragment-1; Hwang et al., 1991)or no difference (V8 protease or chymotrypsin treatment of HC; Johnston et al., 1990)) induced by acclimation to different temperatures. An increase in messenger RNA (mRNA)encoding for the fast migrating HC subunit was detected under similar acclimation regimens (Gerlach et al., 1990). Myofibrillar changes have not been examined in heart, where cold acclimation in some fish species leads to changes in heart size, heart rate, and mechanical efficiency (e.g., Graham and Farrell, 1990). In mammals, there is evidence for changes in cardiac myosin isoforms in relation to development (V3 to Vl), and after swimming training (see Solar0 et al., 1989).Again, there have been no comparable studies in fish. The thin filaments backbone is double-stranded F-actin, composed of G-actin monomers assembled into filaments. The energetics of the conformation change associated with assembly vary between fish species in a manner that suggests adaptation of the structure of the protein

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to both temperature and hydrostatic pressure (Swezey and Somero, 1982). Troponin (Tn) is composed of three proteins; TnC is the calcium-binding protein, TnI inhibits actin from binding to myosin heads, and TnT binds tropomyosin. Mammalian heart possesses only one isoform of TnC, shared by slow-twitch skeletal muscle but distinct from fast-twitch skeletal muscle TnC (Solaro et al., 1986).Changes in TnI isoforms occur in development of mammals. These differences help explain the differential effects of acidosis on Ca2' sensitivity of Tn from neonates and adult rats (see Solaro et al., 1989). Interspecies differences in mammalian and avian TnC and TnI are evident from sequence analyses (Collins, 1991; Murphy et aZ., 1991), but corresponding physiological differences have not been demonstrated. As many as five TnT isoforms have been identified in mammalian hearts. Although their functional roles are not well established, the different isoforms can affect ATPase activties (see Solaro et al., 1989). Intra- and interspecific differences in cardiac Tn-tropomyosin components have not been demonstrated in fish. Isoforms of cardiac myofibrillar proteins in fish have not been observed in most studies. The plasticity afforded by isoforms in mammals is important in the response of the heart to long-term changes in cardiovascular demand. The physiological effectors that lead to changes in isoform expression in mammals (starvation, exercise, basal metabolic rate, temperature adaptation) can be much more extreme in many fish species. Consequently, it is unlikely that diveristy of cardiac myofibrillar isoforms in fish is as limited as suggested by the studies performed to date. Perhaps the introduction of a wider range of techniques (e.g., mRNA hybridization; Gerlach et aZ., 1990) may reveal differences in contractile proteins not currently identified using conventional protein analysis.

B. Calcium Requirements of the Contractile Element A discussion of the beat-to-beat regulation of contractility demands a knowledge of the Ca2+ requirement of the myofibrils. Generation of force is predicated on Ca2+ binding to the single, low-affinity Ca2+specific site on TnC. The effective Ca2+ concentration required for contraction is dependent on the intracellular Ca2+ buffering capacity as well as the affinity of TnC for Ca2+. 1. CALCIUM BUFFERING It has been shown that the contractile element requires about 22 pmol of Ca2+/kgwet weight to generate 50% tension in the dog heart

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(Solar0 et al., 1974). Intracellular buffering of Ca2+ requires the total released to be severalfold higher (Fabiato, 1983). Ca2+ binds to TnC itself, but also to the SR, the inner face of the SL and to cytosolic molecules such as calmodulin. Fabiato (1983),based on calculations using appropriate concentrations and apparent association constants (K,) for several critical Ca2+-binding moieties, suggested that a substantially higher Ca" release (i.e., >50 pmollkg wet weight) is needed to achieve 50%maximal tension generation. Pierce et al. (1985)found an even higher value (
6.

EXCITATION-CONTRACTION COUPLING IN THE HEART

275

with frog (Rana pipiens) ventricle suggest myofibrils from poikilotherms exhibit some degree of thermal adaptation. Dropping temperature from 22” to 1°C decreases Ca2+ sensitivity to a similar degree in both frog and mammalian cardiac myofibrils, but the frog preparation has a higher Ca2+ affinity at each test temperature (Fig. 2). At physiological temperatures each preparation has similar Ca2’ affinity, analogous to the “conservation of Km” observed for metabolic enzymes (Somero, 1986).The inhibition of C, observed with decreasing temperature in mammalian cardiac myofibrils is much less pronounced in frog heart myofibrils (Fig. 2). As in heart, poikilotherm skeletal myofilaments are less sensitive to decreasing temperature than homeotherms myofilaments; skeletal myofilaments from temperate amphibians (Ranaesculenta, Bufo bufo)are less sensitive than those from tropical climates (Bufo marinus; see Stephenson and Williams, 1985). Comparable studies, to examine whether teleost cardiac myofibrils are temperature-adapted, have not been performed. Johnston (1979)reported that the differences in the thermal dependence of skeletal actomyosin ATPase in goldfish acclimated to 2 and 31°C were related to unidentified changes in Tn-tropomyosin. Mam-

100 h

#’

;

80-

0

60-

u

n

v

6

5

4

PCR

Fig. 2. Effects of temperature on Ca2+sensitivity of skinned cardiac myofibrils from rat and frog. Hill plots were drawn from mean K l l z values and Hill coefficients reported by Harrison and Bers (1990a).Tension is expressed as a percentage of maximal tension achieved at the highest temperature in each species. Calcium concentration is expressed as pCa (-log[Ca2+]).

276

GLEN F. TIBBITS ET AL.

malian studies point specifically to TnC as an important determinant of the thermal response of the heart contractile element. Cardiac myofilaments become less sensitive to Ca2+ at lower temperature, whereas fast-twitch skeletal myofilaments become more sensitive (Stephenson and Williams, 1981; Godt and Lindley, 1982). When cardiac myofilaments are depleted of their native TnC and reconstituted with skeletal TnC, the decrease in Ca2+sensitivity observed in native fibers at low temperature is greatly diminished (Harrison and Bers, 199Ob). Similarly, slow-twitch skeletal muscle fibers, which share the cardiac isoform of TnC, are less temperature sensitive than fast-twitch skeletal myofibrils (Stephenson and Williams, 1985). If poikilotherm myofilaments are temperature adapted, it may be reflected in the structure of their TnC. When Ca2+binds to TnC, changes in the orientation of TnC a-helices are thought to result in an increase in hydrophobic interactions between TnC and TnI (Herzberg et al., 1986). Thus, physical factors such as temperature and intracellular solutes, which affect the hydrophobicity of proteins (see Somero, 1981, 1986), have the potential to alter TnC structure, as well as the interactions between TnC and TnI that are critical in triggering contraction. Adaptation to lower temperature in bacterial enzymes, particularly in a-helical regions of the protein, involves a substitution of particular amino acids with less hydrophobic residues, conserving the degree of hydrophobicity of the protein at the optimal temperature of the organism (Menendez-Arias and Argos, 1989). When comparing skeletal TnC primary sequences of mammals and birds with that of frog, several amino acid residues that are highly conserved in homeotherm sequences are replaced with more hydrophobic residues in frog, the only poikilotherm sequenced to date (Collins, 1991). The influence of these changes on TnC structure and TnI-TnC interactions are not established. Since no poikilothermic vertebrate cardiac TnC has been sequenced, it is not known whether similar sequence differences occur in fish heart. Tissue acidosis is another factor known in mammalian cardiac myofilaments to profoundly affect Ca2+sensitivity. Decreasing pH from 7.0 to 6.5 (each at 22OC) decreases TnC affinity for Ca2+ severalfold. The effect of pH is enhanced when TnC is complexed to TnI, suggesting that TnC-TnI interactions may be involved in the inhibition by pH (Solar0 et al., 1986).Whereas tissue acidosis would occur in fish under the same conditions as in mammals (ischemia, environmental hypoxia, exercise-induced acidemia), there are also the effects of temperature on intracellular pH to consider. Poikilotherms adjust blood and tissue

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pH in response to temperature in a manner that keeps the relative alkalinity (OH-/H+) constant. This relationship between pH and temperature (-0.016 to -0.019 pHPC over physiological temperatures) is thought to be important in poikilotherm biochemistry, as it would keep constant the ionization state of a-imidizole groups of histidyl residues of proteins (alpha-stat hypothesis-see Reeves, 1972). Histidine is involved in active sites of many enzymes and is one of the few amino acids that possess side-chains with pK values close to physiological pH, such that changes in OH-/H+ could appreciably affect protonation state (see Somero 1981,1986). Mammalian cardiac and skeletal TnC are not likely to be affected directly in this manner by temperature and pH, as these proteins possess, at most, one histidyl residue, which is replaced in frog skeletal TnC (Collins, 1990), icefish (Champsocephalus gunnari; Feller and Gerday, 1989),pike (Esox lucius;MeCubbin et al., 1982),carp (Cyprinus carpio),and eel (Anguilla anguilla; Gerday et d., 1984), as well as each cardiac TnC sequenced to date (Collins, 1990). The effects of temperature/pH on TnC-TnI interactions and secondary structure of TnI have not been determined in any species. Cardiac TnI possesses several histidine residues throughout the molecule (Murphy et d.,1991) and in sites known to interact with TnC (residues 96-116, e.g., Leszyk et al., 1990). Regardless of the mechanism of the pH effect on Ca2' sensitivity, it has important implications in fish, which can experience extreme pHhemperatwe perturbations. Furthermore, fish intracellular pH changes much less in heart than in other tissues when temperature is varied (-0.003 to -0.0014 pH/"C; Heisler, 1979; Cameron, 19841, such that decreasing temperature would result in a relative acidification in this tissue, possibly enhancing the direct effects of hypothermia on Ca2+ sensitivity in vivo. It should be noted that in the mammalian experiments cited (Harrison and Bers, 1989, 1990a), pH was maintained at 7.0 across the temperature spectrum, which would also lead to a relative acidification with decreasing temperature. For a better understanding of the regulation of contractility in the teleost heart, a more detailed knowledge of the Ca" requirement of the teleost myofilaments and the effects of temperature/pH on Ca2+ sensitivity is required. Indeed, extending investigations to include fish species experiencing different thermal regimes (eurytherms, low and high temperature stenotherms) may help elucidate the mechanism underlying the temperature dependence of the contractile element in vertebrate heart.

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111. REGULATION OF Ca2+ DELIVERY TO MYOFIBRILS A. Depolarization

Electrophysiological measurements of action potentials (AP) and ionic currents have provided information about E-C coupling in hearts from a variety of species. In particular, information about the role of ionic currents in the E-C coupling in mammalian heart cells has increased tremendously since the introduction of the patch clamp technique to study isolated cardiac myocytes (Lee et al., 1979; Hume and Giles, 1983).The capability to measure the Ca2+-currentacross the SL and intracellular Ca2+transients simultaneously (Lipp et al., 1990; Spurgeon et al., 1990; Sipido and Wier, 1991) has lead to the development of quantitative models of cellular Ca2+ movements (Sipido and Weir, 1991). In lower vertebrates, the E-C coupling is well characterized only for amphibia (Hume and Giles, 1982, 1983; Morad et al. 1983; Gurney et al., 1989), and mathematical models based on empirical data have been described (Rasmusson et al., 1990a, b). 1. THEACTION POTENTIAL The action potential is composed of a complex of electrical currents across the SL and reflects changes in the sum of these currents after stimulation of the heart cell. The type and number of ionic and capacitative currents, which account for the AP, differ between types of heart tissue and between species. Fish heart pacemaker cells show characteristics similar to those reported for amphibian and mammalian heart pacemaker cells (Huang, 1973; Saito, 1973; Rasmusson et al. 1990a). Atrial cells in the teleost heart (golden carp) exhibit shorter action potentials and have a less pronounced plateau phase (Huang, 1973) than ventricular cells, which have the longest action potentials. The relative importance of SL flux as primary source of Ca2+ for contraction is suggested by the action potential duration (APD).At one extreme is frog, which depends almost solely on SL-mediated Ca2' entry (Fabiato and Fabiato, 1978; Morad and Cleemann, 1987). The frog heart atrial cell is characterized hylaloiig APD (723 2 106 msec SD at room temperature; Hume and Giles, 1982) and a pronounced plateau phase. The rat represents the other extreme, with a very short APD and practically no plateau phase (Shattock and Bers, 1987). Even in mamma1ian gpecies that demonstrate relatively long action potentials (e.g., rabbit, 372 r?: 14 msec at 23°C and pH 7.4; Shattock and Bers, 1987), the APD amounts to only half the value reported for frogs.

6. EXCITATION-CONTRACTION COUPLING IN THE

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279

As is the case for mammalian species, fish AP characteristics vary among species. Flounder (Pleuronectesflesus)ventricle demonstrates long APD with a pronounced plateau phase, resembling the situation in the frog, whereas rainbow trout (Oncorhynchus mykiss) ventricle exhibits a less pronounced plateau phase with APD less than 50% of that in flounder (Hoglund and Gesser, 1987). Time to peak tension (TPT) is a more commonly determined parameter and has been shown to correlate with APD (Rumberger and Reichel, 1972). Table 1, in which TPT and AP durations determined in a number of fish are compiled, highlights the significant variation found between species. The relationship of fish heart AP with contractile characteristics gives indirect evidence that SR is not an important source of Ca2+ for contraction in these species. Flounder ventricle demonstrates a long AP plateau phase and is not sensitive to agents that affect the ability of the SR to release Ca" (e.g., El-Sayed and Gesser, 1989).This is similar to the situation with frog atrium and ventricle, consistent with the notion that contraction is supported entirely by a transsarcolemmal source of Caz+. A similar conclusion was reached by Lennard and Huddart (1991)based on correlations between the AP and contractile properties in flounder ventricle. Also, Maylie et QZ. (1979)found parallels between the elasmobranch and frog ventricles; each species lacks postextrasystolic potentiation, and twitch force is highly sensitive to both [Ca2+], and APD. Conflicting conclusions arise from studies with rainbow trout ventricle, which has a shorter AP than does flounder, with a less pronounced plateau phase. Although changes in twitch force in the trout ventricle correlated to changes in APD under some conditions and showed little ryanodine sensitivity (Hove-Madsen and Gesser, 1989), potentiation of twitch force after a rest period is sensitive to agents that affect SR Ca" release (Hove-Madsen and Gesser, 1989; El-Sayed and Gesser, 1989). It should be noted that comparisons of mechanisms, and their importance in the E-C coupling in different species, in general are difficult because of differences in experimental conditions. In particular, comparison is difficult between homeothermic mammalian species with a physiological temperature of 37°C and pH 7.4 and poikilothermic fish species with a significantly lower physiological temperature range as well as pH 7.6-7.8 (Ruben and Bennett, 1981; Andreasen, 1985).For example, the APD in rabbit venticle is reported to be 372 2 14 msec at 23°C (Shattock and Bers, 1987), which is comparable to values reported for some teleost ventricles at 25°C or less (Huang, 1973; Hoglund and Gesser, 1987; Hove-Madsen and Gesser, 1989).

Table I Comparative Aspects of Action Potential Duration (APD) and Time-to-Peak Tension (TPT) in Various Poikilothermic Species" Frequency (bpm) Species Osteichtyhes Teleosts Lumpfish Mackerel Sea raven

Paced

Unpaced

108 108 96

TPT (msec

APD (msec)

400 490 560 39

Alewife Ocean pout Salmon Sculpin Goosefish Cod Rainbow trout

60 48 48 48 36

500 570

600 620 860 41

120

430

12* 36*

350

360 300

75 55 29 Yellow perch

48 48

Smallmouth bass

48 30

Golden carp atrium

600 700 500 900 105

197

Temp ("C)

[Ca2+] (mM)

15 15 15 10 15 15 15 15 15 10 15 15 15 20 12 5 20 5 20 5 26-28

1.00 1.00 1.00

-

1.00 1.00 1.00 1.00 1.00

-

1.25 1.25 1.25

-

1.00 1.OO 1.00 1.00 1.50

Reference

Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Farrell, 1984 Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Driedzic and Gesser, 1985 Farrell, 1984 Dybro and Gesser, 1986 Hoglund and Gesser, 1987 Hove-Madsen and Gesser, 1989 Wood et al., 1979 Wood et al., 1979 Wood et al., 1979 Bailey and Driedzic, 1990 Bailey and Driedzic, 1990 Bailey and Driedzic, 1990 Bailey and Driedzic, 1990 Huang, 1973

SA-pacemaker SA-pacemaker Flounder

26-28 22-25 15 15 10

1.50 1.80 1.25 1.25

Huang, 1973 Saito, 1973 Dybro and Gesser, 1986 Hoglund and Gesser, 1987 Cobb and Santer, 1973

24 28 51

15 25 17

-

Seibert, 1979 Seibert, 1979 Hughes et al., 1982

16 11 16

0.5 0.5 0.5-2

3.00 3.00

37 25 20

10 10 7 17 12 7

-

Driedzic and Gesser, 1988 Driedzic and Gesser, 1988 Maylie et al., 1979 Taylor et al., 1977 Taylor et al., 1977 Taylor et al., 1977

10

3.00

Driedzic and Gesser, 1988

15 20

1.25 2.50

Dybro and Gesser, 1986 Hume and Giles, 1982

145 25-45 580

76 12*

atrium Anguillifoms European eel

194 700 200

60

Australian eel Antarctic fish

NI

Notothenia gibberifrons Paracheannichtys charcocti Chaenochephalus aceratus Chrondrichthyes Elasmobranchs Little skate Spiny dogfish

Agnathans Hagfish Amphibians Frog atrium

36 48 60

150

30

66 30*

1020 723

-

-

5.00

-

Holeton, 1970 Holeton, 1970 Hemmingsen and Douglas, 1977

a Unless indicated otherwise the values refer to ventricular tissue. For paced preparations the value indicated represents the highest sustainable frequency unless shown with *.

282

GLEN F. TIBBITS ET AL.

However, at 37°C the APD in rabbit ventricle is 222 2 12 msec (Shattock and Bers, 1987),which is shorter than values reported for the fish heart at physiological temperatures. OF CARDIAC EXCITATION 2. NERVOUSREGULATION In the bullfrog heart, a mathematical model based on experimental data has been proposed to explain electrophysiological activity during E-C coupling in pacemaker and atrial cells (Rasmusson et al., 1990a, b). In contrast to the atrial cell, the pacemaker cell lacks a tetrodotoxinsensitive Na’ current, and a background inwardly rectifying K+ current. Thus, after repolarization of an AP of the pacemaker cell, the lack of the background K+ current causes a gradual depolarization of the resting potential, until the threshold for activation of the Ca” current is reached. This, in turn, elicits a new AP, and the cycle is repeated. The number of electrophysiological measurements performed in the fish heart are much more limited compared to those in frog, but measurements in the carp (Huang, 1973; Saito, 1973) showed that the sinoatrial pacemaker generates the heart rate and apparently controls ventricular frequency by having a self-excitation rate up to twice as fast as that of atrial or ventricular cells. The pacemaker cells show characteristics similar to those of the amphibian and mammalian heart with a maximal diastolic potential of -65 mV, 74 mV AP amplitude, 9 mV overshoot, and a 10-mV prepotential (Huang, 1973). I n the fish heart, the frequency of the pacemaker is regulated cholinergically through vagal stimulation. In some species, both excitatory and inhibitory effects of vagal stimulation are mediated cholinergically (Cobb and Santer, 1973; Laurent et aZ., 1983),whereas in others, inhibition is cholinergically and excitation adrenergically mediated (Gannon and Burnstock, 1968; Gannon, 1971; Huang, 1973). Furthermore, neither preload nor afterload appears to have any effect on heart rate in teleosts (Farrell et al., 1982; Farrell et al., 1983), whereas heart rate is strongly affected by temperature (Seibert, 1979; Wood et al., 1979; Lennard and Huddart, 1991). Regulation of heart rate in elasmobranchs and hagfish appears to differ from that in teleosts, as heart rate increases with preload, and isolated hearts appear to have an intact pacemaker (Jensen, 1961,1970).

3. TEMPERATURE In the teleost heart, temperature affects the frequency of the pacemaker cells (Seibert, 1979; Wood et aZ., 1979). In isolated ventricular strips, a lowering of temperature has also been reported to reduce the maximal stimulation frequency (Bailey and Driedzic, 1990). At low

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283

temperatures, the frequency of the pacemaker cells may, in fact, approach that of ventricular cells, which may cause changes in the regulation of the heart rate; i.e., regulation becomes dependent on ventricular factors such as pre- and afterload (Farrell, 1984). In mammalian and frog hearts, APD is increased with lowered temperature (Rumberger and Reichel, 1972; Shattock and Bers, 1987). This has also been reported for flounder ventricle where increasing temperature from 10" to 30°C reduces APD (Lennard and Huddart, 1991).Lowering of temperature has also been reported to prolong TPT (Ask et al., 1981; Bailey and Driedzic, 1990).In the trout heart, increasing the temperature by 10°C reduces TPT and increases the maximal rate of force development (dFldt,,,) and temperature, therefore, has little impact on the magnitude of twitch force. Furthermore, both twitch force and dFldt,, are potentiated after a rest period. This rest potentiation is strongly increased by an increase in temperature from 15"to 25"C, and can be abolished by ryanodine (Hove-Madsen, 1992). The influence of temperature on the AP and ionic currents would provide essential information about the E-C coupling in the fish heart.

4. HUMORAL FACTORS Myocardial acidosis, anoxia, hypoxia, and the level of circulating catecholamines are important factors affecting the E-C coupling. However, the bulk of research on the fish heart so far has been concerned with the effects on the contractility of the heart, and little information exists about the influence of these factors on electrical parameters. In some teleost species, adrenaline has been reported to have positive inotropic and chronotropic effects (Cobb and Santer, 1973; Wood et al., 1979; Ask et al., 1981; see Chapter 3). The increase in heart rate can be related to an increase in the self-excitation rate of the pacemaker cells by adrenaline, which is brought about by an increase in the slope of the diastolic depolarization of the pacemaker cell. (Huang, 1973). In contrast, adrenaline increases the duration and plateau phase of the AP in atrial and ventricular cells of the golden carp without any significant effect on beat frequency (Huang, 1973). The increased duration of the plateau phase in atrial and ventricuIar cells may reflect an increased Ca2+ flux across the SL, which in turn could account for a positive inotropic effect of adrenaline. Extracellular acidosis can induce profound intracellular acidification in the fish heart; however, the response of isolated ventricular strips to acidosis varies between teleost species. Most species exhibit an initial decrease in force development. In rainbow trout (Oncorhynchus mykiss), inhibition of force development persists (Gesser et

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GLEN F. TIBBITS ET AL.

al., 1982; Hoglund and Gesser, 1987), whereas acidosis-resistant species like flounder (Pleuronectesflesus)and eel show a secondary recovery of twitch force after 10-20 min (Gesser and Poupa, 1979,1981; Gesser et al., 1982). Hypercapnic acidosis initially increases APD for both trout and flounder; however, during extended exposures, only the flounder ventricle exhibits a secondary reduction in APD (Hoglund and Gesser, 1987). Hoglund and Gesser (1987) suggest that the secondary decrease in APD for the flounder heart could be the result of an increased [Ca2+Iiin this species, which is known to cause shortening of the APD in other species (Maylie et al., 1979; Noble, 1983). It has been suggested that the increase in [Ca2+]iis caused by release of Ca2+ from mitochondria (Gesser and Poupa, 1979). The relationship between pHi and pCa is extremely complex, because pHi affects the Ca2+ transporters as well as intracellular Ca2+ buffering. Furthermore, the relationship between pCa and force of contraction is pHi dependent, and both pCa and pHi are temperature dependent. Thus, further information about the effect of acidosis on contraction will be obtained only from experiments in which all of these variables are simultaneously monitored. B. Transsarcolemmal Ca2+ Influx

1. L-TYPECa2+ CHANNELS It has been recognized for some time that a variety of different classes of Ca2+ channels exist in nature (see Hagiwara and Byerley, 1981). Tsien (1983) has developed a system of nomenclature for Ca2+ channels based on their electrophysiological and pharmacological properties and subdivides voltage-dependent Ca2+channels into L, T, and N types. The mammalian ventricle is characterized by a relatively high density of Gtype channel only (Bean, 1989).The L-type channels are distinguished from others by long-lasting currents and sensitivity to a class of Ca2+ antagonists known as dihydropyridines (DHP). It is known that the teleost ventricle contains a high density of DHP receptors (DHPR) or L-type Ca2+ channels, but other types of voltagedependent Ca2+ channels have not been investigated. It has been shown in mammals that the L-type Ca2+ channel accounts for most, if not all, of Ca2+ influx across the SL with each excitation (Wier, 1990), and it has been suggested that in the heart of lower vertebrates, Ca2+ influx through the DHPR is the only route of Ca2+ entry for contraction. Thus, this channel is pivotal in the link between excitation and contraction in the mammalian and teleost heart alike and is worthy of detailed discussion.

6. EXCITATION-CONTRACTION

COUPLING IN THE HEART

285

a. Structure. The DHPR appears to be critical for contraction in striated muscles from all species examined to date including the teleost heart. Skeletal muscle T-tubule membranes from the rabbit have been found to be the most enriched source of DHPR. The purified DHPR from skeletal muscle T-tubule consists of five subunits (a1,( ~ 2 , p, 6, and y ) that exist in a stoichiometric relationship. Mammalian cardiac muscle contains less than 1% of DHPR density of skeletal muscle, and as a result, the purification of DHPR from cardiac muscle has proven to be more difficult. However, photoafhity-labeling experiments with Ca2+ channel ligands and immunostaining with antibodies against the a1 and a2 subunits of skeletal muscle DHPR have identified related polypeptides in partially pure cardiac DHPR preparations. The a1 subunit in mammalian cardiac and skeletal muscle, which has been shown to contain ligand-binding domains of DHP and other types of Ca2+ channel antagonists, can function as a voltage sensor and Ca2+ channel in E-C coupling. The a1 subunit from skeletal muscle has an M , of 170 kDa in sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE), whereas cDNA cloning studies indicate a M , of 212 kDa. The mammalian cardiac 01 subunit has an M, of 170-195 kDa in SDS-PAGE and 242 kDa calculated from amino acid data. Recently, we have shown that the rainbow trout (0. mykiss) cardiac muscle contains an a1 subunit that has an M, of 190 kDa, which is comparable to the mammalian heart a1 subunit and different from the mammalian skeletal muscle form (Murphy et al., 1992). In this regard, recent studies indicate that carp skeletal muscle contains an a1 subunit that is comparable in size to its mammalian counterpart and exhibits a high degree of homology (Grabner et al., 1991). The a2 subunit, which is glycosylated and the only major glycoprotein in the DHPR, has an M , 175 kDa in both skeletal and cardiac muscle of mammals. The a2 subunit is also distinguishable from other subunits of the DHPR in that it changes its apparent mobility in SDSPAGE to 150 kDa on reduction of disulfide bonds. In trout hearts, the a2 subunitlike polypeptide is larger, with an M,of 220 kDa that shifts to 150 kDa (Murphy et al., 1992). The 220/150 kDa polypeptide is the only glycoprotein present in the isolated DHPR preparation. Whereas the DHPR is retained by the lectin columns, which specifically bind glycoproteins, the a1 subunit could have been retained only through its association with the a2 subunit. These results indicate that in fish heart, the a2 subunit of the DHPR is different from that in the mammalian heart. Differences in a2 subunit between mammals and fish are also evident in skeletal muscle DHPR. In this regard, DHPR purified

286

GLEN F. TIBBITS ET AL.

from C. carpio skeletal muscle has indicated the presence of two a2 subunits of M , 235 and 220 kDa that shift mobility to 159 kDa under reducing conditions (Grabner et al., 1991). It is apparent that the rainbow trout cardiac DHPR contains only one a2 of M, 220, and its relationship to the skeletal form needs to be studied further. Whereas the exact role of the a2 subunit in Ca2+ channel function is unclear, it has been suggested that it may serve to stabilize the a1 subunit (Mikami et al., 1989) in the membrane. The functional significance of the differences in a2 subunits in fish are yet to be studied. It has been proposed that the mammalian skeletal muscle p subunit influences the activation and inactivation characteristics of the Ca2+ channels as well as increases the density of DHPR sites during expression. Whereas the DHPR preparation from trout heart contains polypeptides of M, 50-58 kDa and 30 kDa (Murphy et al., 1992), it is unclear whether these polypeptides are comparable to the p and y subunits of the Ca2' channel structure from skeletal muscle. The presence of p- and y-like subunits in mammalian cardiac and fish skeletal muscle remains to be established.

b. E lectrophysiology. The examination of the L-type Ca2+channel by patch-clamping techniques in isolated myocytes from bullfrog atrium shows that the Ca2+current (ZCJ density is in the lower end of the range of current densities found in mammalian species (McDonald et al., 1986) and agrees with the results for multicellular amphibian preparations (Reuter, 1983).Although current densities are similar, the 5-10 times higher surface-to-volume ratio in the frog heart myocytes results in a much higher capacity to deliver Ca2+via transsarcolemmal influx (see Tibbits et al., 1991). In agreement with this, a number of other experiments suggests that Ca2+flux across the SL is sufficient to support contraction entirely in the amphibian heart (Klitzner and Morad, 1983; Bers, 1985). Although Ca2+ current densities are not known in the fish heart, teleosts, like the amphibians, generally have small myocyte diameters (2-10 pm; Santer, 1985). Consequently, calcium flux across SL would also be expected to be relatively more important in these species, but no direct evidence of the importance or the magnitude of this has been provided. In the ventricles of rainbow trout (0.mykiss), however, a number of observations indirectly suggest that the transsarcolemmal Ca2+ influx is large relative to the cytosolic and myofilament requirement for Ca2+.These findings include the following:

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1. the presence of a substantial AP and twitch at 15 mM external potassium (a concentration that depolarizes the cell to a point where Na+ channels are expected to be largely inactivated); 2. the potential prominence of the Ca2+current in teleost ventricle is indicated by a severalfold higher binding of the DHP PN200110 to both crude homogenates and purified SL from the hearts of cold-adapted rainbow trout compared to mammalian species (Tibbits et d.,1990); and 3. the insensitivity of contraction to ryanodine, which blocks SR Ca2+ release. The latter is discussed in more detail in Section 111, C. These observations, however, require corroboration with more direct approaches before it can be concluded that the SL Ca2+influx is sufficiently large to support contraction in the teleost heart. Interestingly and potentially very important to the understanding of the regulation of contractility in the teleost heart, the L-type Ca2+ channel in myocytes from the mammalian heart has a strong temperature dependence. Generally, voltage-dependent channels have a QIO of between 1.3and 1.6 in describing the changes in current amplitude as a function of temperature (Hille, 1984).The dependence of Zca amplitude is described with a Q l o of almost 3 (Cavalie et al., 1985).This high value precludes a simple diffusion model. Unfortunately, little is known about the L-type Ca2+channel and Zcain the trout heart, but the strong temperature dependence observed in mammals would have a profound impact on the regulation of contractility in teleosts. A role for P-adrenergic agonists in augmenting Ica and regulating contractility in teleosts is suggested from studies on the in situ teleost heart (Farrell, 1984). The regulation of the L-type Ca2+ channel by P-adrenergic agonists through G,-protein and cyclic adenosine monophosphate (CAMP) is well documented in both mammals and amphibians (Reuter, 1983). It has been shown that CAMP-dependent protein kinases phosphorylate the a1 and p subunits of the DHPR, increasing the probability of channel opening (Tsien et al., 1986), thereby increasing Zc,. Alterations in the modulation of L-type Ca2+ channel by P-adrenergic agonists may constitute an important adaptive mechanism to cold in the teleost heart (Graham and Farrell, 1990). For a detailed understanding of E-C coupling in the fish heart, more information about both the structure and the function of the channel are required. Thus, the sequence of both the a1 and a2 subunits in the fish hearts would be extremely useful, as would more detailed electrophysiological analyses.

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2. Na+-Ca2+ EXCHANGE The role of Na+-Ca2+ exchange in the fish heart is described in detail in Section IV, as this transporter is a prime mechanism in myocardial relaxation in the fish heart (Tibbits et al., 1991). However, it is well documented the exchanger is bidirectional (Philipson, 1985). Although under normal conditions Na+-Ca2+ exchange transports 1Ca2+ out of the myocyte for 3Na+ transported into the cell, it is capable of transportation in the opposite direction under certain conditions. The direction of transport is determined by equilibrium potential membrane potential (Ec,), (ENa)and (Em)with decreases in E N a and Em and increases in EC, augmenting the potential for reverse mode operation of the exchanger. During a single action potential, EN^ remains essentially unchanged, while Em and Eca both decrease. Thus, whereas the Na+-Ca2+ exchanger may contribute to Ca2+ influx during excitation, this is difficult to predict without detailed knowledge of Eta, EN^, and Em. It has been suggested, however, that Ca2+ influx through Na+-Ca2+ exchange plays an important role in Ca2+-induced Ca2+release in mammalian cardiac muscle (Leblanc and Hume, 1990). These relationships need to be established in the teleost heart. C. Calcium Release from the Sarcoplasmic Reticulum Electron microscopic observations of a number of poikilothermic hearts, including those of the teleosts, demonstrate both a paucity and a lack of organizational complexity of SR in comparison to the mammalian heart. The SR accounts for approximately 0.5% of myocyte volume in fish and amphibians in comparison to about 7% found in the rat (Santer, 1985; Page and Niedergerke, 1972). Secondly, the T-tubular system, which plays a vital role in the propagation of the AP to the 'interior of the myocyte in the mammalian heart is absent, apparently, in the teleost heart (Santer and Cobb, 1972; Santer, 1974). Experiments with ryanodine and caffeine also suggest the SR to be of minor importance in the activation of the contractile element in the teleost heart. Ryanodine, added in concentrations known to block SR Ca2' release in the mammalian heart, does not induce a substantial reduction in contractile force in teleosts at physiological stimulation frequencies (above 0.2 Hz) and temperatures (10-15°C) in ventricular strips (Driedzic and Gesser, 1988; Hove-Madsen and Gesser, 1989) or the in situ heart (Keen and Farrell, unpublished observations). For reasons mentioned, the role of SR Ca2+ release in tension generation in the hearts of lower vertebrates is normally dismissed.

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This appears to be generally true in the hearts of lower vertebrates, but there are several caveats that need to be addressed. Niedergerke and Page (1981)suggested that catecholamines can induce SR Ca2' release in the amphibian heart via an a-mediated process. In teleosts also there are data that defy total dismissal of a role for SR Ca2' release in contraction. As discussed previously (Section III,A,l), ventricular strips of trout but not plaice demonstrate pronounced postrest potentiation after 5 min of quiescence at 15°C (El-Sayed and Gesser, 1989). In the mammalian heart, this phenomenon has been ascribed to increased calcium release from the SR (Bers, 1985). In agreement with this interpretation, ryanodine was shown to inhibit both the potentiation force and 45Ca2+uptake subsequent to a rest period in trout (El-Sayed and Gesser, 1989). More recently, it was demonstrated that contractility in atrial strips from skipjack tuna (Katsuwonus pelamis) had a pronounced ryanodine sensitivity at 25°C (Keen et al., 1992). Thus as shown in Fig. lB, under some conditions in certain teleost species, SR Ca2' release appears to play a role in contraction. In mammalian skeletal and heart muscle, ryanodine binding to the SR has been examined extensively (Pessah et al., 1985; Meissner, 1986) and the & of 8-10 nM agrees with the concentration needed for one half maximal reduction of contractile force (KI) in papillary muscles (Shattock and Bers, 1987). Furthermore, the &, the K I , and the maximal reduction of contraction by ryanodine have been shown to vary with temperature in mammalian muscle. Thus, it is possible that the failure to demonstrate effects of ryanodine in teleosts may be owing to the lower temperatures under which these experiments are generally conducted. In agreement with this, the rainbow trout heart exhibits a similar & for ryanodine binding at room temperature (Tibbits and Kashihara, unpublished observations) as observed in mammals and a K I that agrees with that found in mammals (Hove-Madsen, unpublished observations). It may be important, therefore, to examine SR calcium release in both teleost and mammalian species over a similar range of temperatures. We have hypothesized that the role of SR Ca2' release in contraction becomes progressively less with cooler temperatures (Tibbits et al., 1991). If the ryanodine-sensitive Ca2' release channel opens in trout SR in response to cold as it does in the mammalian heart, then this would render the SR ineffective under hypothermic conditions as there would not be adequate SR loading. As the temperature increases, however, the SR may be able to maintain Ca2' levels sufficient for a contribution to contraction. The significance of this hypothesis in species that experience different environmental conditions is obvious. This notion would also lead one to pre-

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dict that the SR plays a more prominent role in warm-water fish and would serve to increase both the speed of contraction and relaxation. Cooling the myocardium to about 0°C within a few seconds in mammals results in a contracture that is caused apparently by the temperature-dependent opening of the SR Ca2+ release channel mentioned, and a relative inability of the normal transport systems to reduce [Ca2+Iiin the cold. Thus, rapid cooling contractures (RCCs) have given valuable information about the calcium content of the SR. In the guinea pig heart, values of 85-235 pmol Ca2+per kg wet weight are estimated (Bers et al., 1989), indicating that SR calcium content is sufficient in magnitude to support a normal twitch in the mammalian heart (see Section II,B,l). Under similar conditions in the frog, only very small RCCs are observed, consistent with a minor role for SR Ca2+ release in frog ventricle. However, in both the amphibian and the teleost heart, assessment of the SR Ca2+ content by rapid cooling is complicated by the fact that other cellular Ca2+-regulating mechanisms, such as the Na+-Ca2+ exchanger, are still active at low temperatures (Bersohn et al., 1991; Tibbits et al., 1992),and no estimates of SR Ca2+ content are available for these species.

IV. MYOCARDIAL RELAXATION In both mammalian and teleost species, myocardial relaxation occurs by lowering cytosolic concentration of Ca2+ back to a diastolic level of about 100 nM. In theory, the lowering of free calcium could be brought about by Ca2+ transport proteins in 3 organelles: (1)the SR Ca2+ pump, (2) the SL Na+-Ca2+ exchanger and Ca2+ pump, and (3)mitochondria. In the mammalian heart, the SR Ca2+pump and the SL Na+-Ca2+ exchanger are the prime means of reducing cytosolic Ca2+ to induce maximal relaxation (Bridge et al., 1988; Bers and Bridge 1989). Na+Ca2+ exchange is the primary mechanism responsible for the efflux of Ca2+ across the SL. The best estimates at present suggest that under normal conditions, 60-80% of the calcium is removed by the SR and the remainder by Na+-Ca2+ exchange. It is not known what the role of the SL Ca2+ pump and mitochondria1 Ca2+uptake is (if any) in relaxation. It has been suggested that the SL Ca2+ pump with its relatively high affinity for Ca", yet low capacity for transport, contributes to the maintenance of low diastolic Ca2+ activity (Carafoli, 1987). The reduced SR content in fish heart relative to mammals necessi-

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tates fundamentally different relative contributions to Ca2+removal in myocardial relaxation. Indirect studies in teleost ventricular muscle suggest Na+-Ca2+ exchange to be important in reduction of [Ca2+Ii. Hove-Madsen and Gesser (1989) found that in trout ventricle, elevation of "a+],, which increases the efficacy of Ca2+ extrusion by Na+Ca2+ exchange, reduced both the positive inotropy of an increase in stimulation frequency from 0.2 to 1.0 Hz and the transient overshoot in twitch force after lowering stimulation frequency back from 1.0 to 0.2 Hz. Also, simultaneous measurements of twitch force and 45Ca efflux in plaice ventricle showed a concurrent increase in twitch force and 45Ca uptake, whereas 45Ca efflux decreased on a lowering of "a+], (Gesser and Mangor-Jensen, 1984). Furthermore, a Na+dependent Ca2+ extrusion in the fish heart has been examined in experiments with application of lowered "a+],, or nominally Na+free solutions. As has been found for most other species (Chapman, 1983), application of nominally Na+-free solutions causes a contracture, which gradually relaxes with time (Busselen and Carmeliet, 1973; Gesser and Mangor-Jensen, 1984). Measurements of 45Ca efflux in goldfish ventricles exposed to nominally Na+-free solutions showed an increase in 45Ca efflux with increasing "a+],, which was dependent on [K+l, (Busselen and Van Kerkhove, 1978; Busselen, 1981). The differences in Ca2+-removal transporters between mammals and fish hearts are illustrated by the differences in relaxation in response to decreased temperature, especially RCCs. At physiological temperatures, relaxation in the fish heart, as reflected by the time to 50% relaxation of a contraction, has been reported to be 290 and 300 msec at 15°C for rainbow trout (0.mykiss) and flounder (P. #ems), respectively (Dybro and Gesser, 1986) and 680 msec at 5°C for yellow perch ( P . ftauescens; Bailey and Driedzic, 1990). These values are longer than corresponding values for the mammalian heart at 37°C (50 msec for rat; Dybro and Gesser, 1986). At low temperatures, however, both the SR Ca2+ pump and the Na+-Ca2+ exchange are very inefficient in the mammalian heart. Relaxation from a RCC takes several tens of seconds (Bridge, 1986) in mammals, whereas rapid cooling of the trout heart from 25" to 1°C elicits a contraction that relaxes in Iess than 2 sec (Hove-Madsen, unpublished observations). It seems clear that mechanisms involved in the relaxation of the fish heart must exhibit characteristics that are distinct from those in mammals. In the lower vertebrate heart, the SR does not play a dominant role in the E-C coupling as discussed previously. The relatively large transsarcolemmal Ca2+ influx must be removed from the cytosol during relaxation either by Na+-Ca2+ exchange or the SL Ca2+ pump.

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1. SARCOLEMMA Ca" ATPASE It has been suggested that the SL Ca2+ ATPase is important for myocardial relaxation in lower vertebrates (Brommundt and Kavaler, 1985). Efflux of 45Ca2+in bullfrog ventricle was primarily ATP and not "a+], dependent, implying that the Ca2+pump and not the exchanger is important for efflux. Their interpretation was based on the analysis of a very slowly exchanging flux component ( t I 1 2 >50 min), which may be limited by an internal compartment rather than by SL Ca2+flux. Thus, the temporal resolution of efflux in this study precludes an accurate determination of the beat-to-beat Ca2+ efflux mechanisms. In the steady state, the rate of Ca2+ efflux should be comparable to that of Ca2+ influx, since over the cardiac cycle, the cell must extrude the same amount of Ca2+ that entered during activation. However, it is premature at this stage to dismiss the role of the cardiac SL Ca2+pump in myocardial relaxation in lower vertebrates, as has been done in mammals. For example, the in uitro characteristics of the SL Ca" pump including both the V, and apparent K, for Ca2', as well as the temperature dependence of these parameters are not known in the teleost heart. 2. Na+-Ca2+ EXCHANGE A prominent role for Na+-Ca2+ exchange in the mechanical relaxaton of the teleost heart is consistent with studies mentioned and the following observations in teleosts: (1)rainbow trout heart Na+-Ca2+ exchange specific activity at 21°C is comparable to the highest activities observed in mammals (Reeves and Philipson, 1989; Tibbits et al., 1992); (2) as previously mentioned, the myocyte surface to cytosolic volume ratio in teleost hearts appears to be several times higher than that in mammals (because of the substantially smaller myocyte diameter), thereby increasing the efficacy of the SL transport proteins in general; and (3)unlike in mammals, the Na+-Ca2+ exchanger in teleost is extremely active over the temperature range of 4-15°C which is physiological for these species (Tibbits et al., 1992). Several lines of evidence suggest a high degree of homology in the structure of Na+-Ca2+ exchanger in the teleost and mammalian hearts. Recently, the canine Na+-Ca2+ exchanger cDNA was cloned and sequenced (Nicoll et al., 1990).A cDNA probe from the canine cardiac Na+-Ca2+ exchanger hybridized to trout mRNA with a brand at -7 kb similar to that in mammals (Tibbits et al., 1992). This indicates that there is some homology between Na+-Ca2+ exchanger cDNAs from trout and that of mammals, and the transcripts are approximately the same size.

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Western blots of mammalian, amphibian, and trout SL run on SDSPAGE and reacted with polyclonal antibodies raised in rabbits to Na+-Ca2+ exchanger purified from dog heart have an identical pattern of banding, with prominent bands at 70,120, and 160 kDa (Tibbits et al., 1992).Whereas the pattern of banding observed in Western blots is identical in fish and mammals, the reaction is less pronounced in trout, as compared to dog. This is probably owing to a lower antigenicity of trout Na+-Ca2+ exchanger compared to dog, but is also possibly owing to lower exchanger density in the SL preparations used in these experiments. The K+ pNPPase activity in canine SL was approximately 2.5-fold higher compared to trout SL, and the fold purification was also comparably higher. Whereas the density of the Na+-Ca2+ exchanger in the mammalian heart has been estimated to be approximately 200500 pm-2, we are unable to make this determination at present in teleost. However, based on preliminary evidence, it appears that at least in rainbow trout the density is high. Furthermore, use of monoclonal antibodies with electron microscopy have localized the Na+Ca2+ exchanger in mammals to the T-tubules (Frank, Mottino, Reid, Molday, and Philipson, unpublished observations). Since in teleosts there is an absence of T-tubules, it is not known whether this protein is distributed homogeneously in the SL or whether it is localized. The deduced molecular mass of the canine Na+-Ca2+ exchanger from the cDNA sequence is 108 kDa (Nicoll et al., 1990). The 120-kDa band observed in Western blots may be the consequence of posttranslational modification such as glycosylation. Regardless, the native molecular mass of the Na+-Ca2+ exchanger appears to be similar in the trout and mammals, suggesting that a similar degree of posttranslational modification takes place. Trout myocardial Na+-Ca2+ exchange is stimulated by limited chymotypsin proteolysis is similar to that observed in mammals (Tibbits et al., 1992). The Na+-Ca2+ exchanger of both the higher and lower vertebrates can be distinguished from that ofthe invertebrate Artemia, which is insensitive to limited proteolysis. Despite this similarity of structure, pronounced differences between mammals and trout in the response of Na+-Ca2+ exchanger to temperature and pH are obvious. Bersohn et al. (1991) recently demonstrated that the amphibian heart Na+-Ca2+ exchange was less temperature dependent than that in mammals. The Arrhenius plot for Na+-Ca2+ exchange showed clear breaks at -22'C for both mammalian and amphibian hearts (Fig. 3). More recently, Tibbits et al. (1992) found that the teleost cardiac Na+-Ca2+ exchange shows even less temperature dependence than that of the frog and no obvious breakpoint in the Arrhenius plot. These temperature dependencies of

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1

1

v

Canine

0

Trout

1

3.2

1

1

3.3

1

I

3.4

I

I

3.5

I

I

*

3.0

Temp-' (1000/'K)

Fig. 3. Arrhenius plots of Na+-dependent Ca2' uptake into purified sarcolemmal vesicles from the hearts of mammals, amphibians, and teleosts. Data for mammalian and teleost heart Na+-Ca2+ exchange are taken from Tibbits et al. (1992) and those for the amphibian heart from Bersohn et al. (1991).

the teleost, amphibian, and mammalian Na+-Ca2+ exchange were maintained in asolectin vesicles in all species. Therefore, the species differences in temperature dependencies of Na+-Ca2+ exchange are apparently independent of possible differences in bilayer composition and must reflect differences in protein structure. In salmonids (Tibbits and Kashihara, 1991), amphibians, and mammals (Bersohn et al., 1991), the K,(Ca) of Na+-Ca2+ exchange is unaffected by temperature, and thus the observed effects of temperature are owing strictly to changes in V-. The species variation in temperature dependence is an important physiological difference because active cold-adapted fish maintain relatively high cardiac outputs at temperatures as low as 4°C (Graham

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and Farrell, 1990).If Na+-Ca2+ exchange is responsible for the majority of cytosolic Ca2+ removal with each beat, as has been suggested (Tibbits et al., 1991), then the temperature insensitivity of the fish Na+-Ca2+ exchanger represents an important evolutionary adaptation. Na+-Ca2+ exchange from crayfish skeletal muscle has a reasonably similar temperature profile to that for trout myocardial Na+-Ca2+ exchange. In both trout cardiac and crayfish skeletal muscle Na+-Ca2+ exchange, there is an obvious decline in activity when the membranes are maintained at >25"C for more than a brief period. Furthermore, we have determined that a component of the acclimation of the trout to warmer water is an increased stability of the Na+-Ca2+ exchanger at temperatures >25"C (Tibbits and Kashihara, 1991). Although this is above the upper lethal temperature of the species, it must be noted that these studies are conducted in uitt-o and this component of the acclimation may be important physiologically. Whether Na+-Ca2+ exchange in eurythermal fish, such as carp, which experience temperatures up to 30°C, is more amphibianlike or troutlike remains to be determined. In the warm-adapted trout, there were no other differences in myocardial Na+-Ca2+ exchange activity (KJCa), V,,, and temperature dependence over the range of T-21"C) compared to that in the cold-adapted trout. Not only is fish myocardial Na+-Ca2+ exchange relatively insensitive to temperature, but it also demonstrates a unique pH dependency. Surprisingly, alkaline pH does not stimulate trout Na+-Ca2+ exchange (Tibbits et al., 1991) as it does in mammalian hearts and invertebrate tissues (Philipson, 1985) such as squid giant axons and crayfish skeletal muscle. As with temperature, this stimulation is observed when the exchanger is reconstituted into asolectin in mammals, indicating that this property is intrinsic to the protein itself. The stimulation of the mammalian cardiac exchanger is the result of lowering the K,(Ca) of the exchanger at alkaline pH (Philipson et al., 1982). It was postulated that a histidine residue near a Ca2+ binding site on the exchanger becomes deprotonated at higher pH and changes the conformation of the Ca2+ binding site. The interactions between pH and temperature have not been explored for the teleost Na+-Ca2+ exchanger. Whereas a teleological explanation for the differences in the temperature/pH dependencies between poikilo- and homeotherm cardiac Na+-Ca2+ exchange is apparent, a mechanistic interpretation is not. Based on the temperature profiles of cardiac Na+-Ca2+ exchange activity, there appear to be at least three different classes (mammalian, amphibian, and teleost) of exchangers (Fig. 3) that presumably reflect differences in the primary structure of the protein. As discussed pre-

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viously, similar temperature-dependent species differences have been observed for the Ca2+ sensitivity of the cardiac contractile element (Harrison and Bers, 1990a). It should be noted, however, that trout K+pNPPase, a sarcolemmal protein, is highly temperature dependent, similar to that of the mammalian Na+-Ca2+ exchanger, and radically different from that from fish hearts (Tibbits, unpublished observations). In general, a high degree of similarity in the properties of the cardiac Na+-Ca2+ exchangers from mammals and teleosts is observed. These similarities include K,(Ca), stimulation by both valinomycin and limited chymotrypsin proteolysis, apparent molecular weight of the protein, and size of transcript. The most striking difference between the two species is the temperature dependence, which is probably caused by differences in the protein amino acid sequence. Because the mammalian Na+-Ca2+ exchanger antibodies cross-react with trout Na+-Ca2+ exchanger, and because mammalian Na+-Ca2+ exchanger cDNA hybridizes to trout Na+-Ca2+ exchanger mRNA, there is significant homology between the two species. A mechanistic interpretation of the different temperature profiles will require a more detailed knowledge of the structure of the myocardial Na+-Ca2+ exchangers. In summary, we are suggesting that like that of mammals, the Na+-Ca2+ exchanger is the prime means of Ca2' efflux across the sarcolemmal, and because of the limited role of SR Ca2+,the primary means of myocardial relaxation in teleosts. However, physiological studies designed to address the relative roles of the SR Ca2+ pump, Na+-Ca2+ exchanger, and SL Ca2+ pump in mechanical relaxation have not been performed in the fish heart.

V. CONCLUSIONS It has been suggested that the sparsity of the sarcoplasmic reticulum and the absence of T-tubules in the hearts of lower vertebrates has dictated that the myocyte diameters be small in order to maintain appropriate diffusional distances for Ca2+ to the myofilaments. Whereas the larger myocyte diameter that is observed in mammals offers certain advantages such as increased conduction velocity, it is not obvious what benefits it confers in terms of contractile performance. It could be argued that the more extensive SR found in the hearts of higher vertebrates allows for more rapid sequestration of cytosolic Ca2+.The latter is consistent with the higher rates of relaxation and the shorter cardiac cycles observed at maximal heart rates in

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mammals, although clearly this relationship is not a simple one attributable, in part, to differences in temperatures. Whether the more rudimentary E-C coupling mechanism in lower vertebrates limits the scope of cardiac performance in these species remains to be determined; however, a preliminary analysis would suggest that it does. The problems of cardiac functioning in the cold are numerous. It would appear that both the delivery and removal of Ca2+ from the myofilaments represent a challenge in teleosts because of the colder temperatures and larger diffusional distances associated with the lack of a developed SR. If the Qlo for the L-type channel in lower vertebrates proves to be 3 as it is in mammals, then this would have a crucial influence on the rate of Ca2+ delivery to the myofilaments. Furthermore, if the calcium requirement for contraction increases in the cold in the teleost heart as it does in mammals, then a greater delivery would be required. On the other hand, the duration of the Ca2+ transient may be increased under hypothermic conditions in teleosts, as it is in the mammalian heart, increasing the probability of troponin C occupation by Ca2+. In the frog heart, it was noted that although the K1/2 decreases with cold, as it does in higher vertebrates, the absolute Kl,2 was higher than the mammalian species and, at least on theoretical grounds, the Ca2+delivery could be less for a given temperature. From the study of Harrison and Bers (1990a), it can be determined that the K l / 2 for frog myofilaments at 1°C (-pCa 5 ) is similar to that of rats at 18°C. If the observation of a higher affmity of the amphibian myofilaments for Ca2+ is a general property of ectothermic vertebrates, then this has important ramifications for regulation of myocardial contractility in these species. Although myofilament Ca2+ loading may be enhanced, there may be problems associated with unloading; either the mechanisms for cytosolic Ca2+ removal are more efficacious or a reduced rate of relaxation is the consequence. There is evidence that some of the Ca2+ transport systems in the teleost are less sensitive to cold (e.g., Na+-Ca2+ exchange), but more study along these lines is required.

REFERENCES Andreasen, P. (1985). Free and total calcium concentrations in the blood of rainbow trout, Salmo gairdneri, during “stress” conditions.J . E x p . Blol. 118, 111-120. Ask, J. A,, Stene-Larsen, G. and Helle, K. B. (1981). Temperature effects on the &adrenoceptors of the trout atrium. J . Comp. Physiol. 143, 161-168.

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Bailey, J. R., and Driedzic, W. R. (1990). Enhanced maximum frequency and force development of fish hearts following temperature acclimation. J . Exp. Biol. 149, 239-254. Bass, A,,Ostadal, B., Pelouch, V., and Vitek, V. (1973).Differences in weight parameters, myosin-ATPase activity and the enzyme pattern of energy supplying metabolism between the compact and the spongious cardiac musculature of carp (Cyprinus carpio) and turtle (Testudo horsfieldi).PPugers Arch. 343,65-77. Bean, B. P. (1989).Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51,367-384. Bers, D. M. (1985). Ca influx and SR Ca release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248, H366-H381. Bers, D. M.,and Bridge, J. H. B. (1989). Relaxation of rabbit ventricular muscle by Na-Ca exchange and sarcoplasmic reticulum calcium pump. Circ. Res. 65,334-342. Bers, D. M., Christensen, D. M., and Nguyen, T.X. (1988).Can Ca entry via Na/Ca exchange directly activate cardiac muscle contraction? J. MoZ. Cell. Cardiol. 20, 405-4 14. Bers, D. M., Bridge, J. H. B., and Spitzer, K. W. (1989). Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes. J. Physiol.417, 537-553. Bersohn, M. M., Vemuri, R., Schuil, D. S., Weiss, R. S., and Pbilipson, K. D. (1991). Effect of temperature on Na+-Ca2+ exchange in sarcolemma from mammalian and amphibian. Biochim. Btophys. Acta 1062,19-23. Bridge, J. H. B. (1986).Relationship between the sarcoplasmic reticulum and sarcolemma1 calcium transport revealed by rapidly cooling rabbit ventricular musc1e.J. Gen. Physiol. 88,437-473. Bridge, J. H. B., Spitzer, K. W., and Ershler, P. R.(1988).Relaxation of isolated ventricular cardiomyocytes by a voltage-dependent process. Science 241,823-825. Brommundt, G., and Kavaler, F. (1985).Greater ATP dependence than sodium dependence of radioactive efflux in bullfrog ventricle. Am. J. Physiol. 249, C129-C139. Busselen, P. (1981). Effect of potassium depolarisation on sodium-dependent calcium efflux from goldfish heart ventricles and guinea-pig atria. J. Physiol. 327,309-324. Busselen, P., and Carmeliet, E. (1973). Protagonistic effects of Na and Ca on tension development in cardiac muscle at low extracellular Na concentrations. Nature, New Biol. 243,5749. Busselen, P., and Van Kerkhove, E. (1978).The effect of sodium, calcium and metabolic inhibitors on calcium efflux from goldfish heart ventric1es.J. Physiol. 282,263-283. Cameron, J. N. (1984).Acid-base status of fish at different temperatures. Am.J. Physiol. 246, R452-R459. Carafoli, E. (1987).Intracellular calcium homeostasis. Annu. Reo. Biochem. 56,395-433. Cavalie, A., McDonald, T. F.,Pelzer, D., and Trautwein, W. (1985). Temperatureinduced transitory and steady-state changes in the calcium current of guinea pig ventricular myocytes. Pjlugers Arch. 405,294-296. Chapman, R. A. (1983). Control of cardiac contractility at the cellular level. Am. J. Physiol. 245, H535-H552. Cobb, J. L. S.,and Santer, R. M. (1973).Electrophysiology ofcardiac function in teleosts: Cholinergically mediated inhibition and rebound excitation. J. Physiol. 230, 561573. Collins, J. H. (1991). Myosin light chains and troponin-C-Structural and evolutionary relationships revealed by amino acid sequence comparisons. J. Muscle Res. Cell Motility 12,3-25.

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AUTHOR INDEX Numbers in italics refer to pages on which the complete references are listed. A

B

Abbot, E., 20, 84 Abel, D. C., 86, 96, 98, 130 Ahrahamson, T., 84 Acierno, R.,27, 73,87,138 Adair, T. H., 186,211,213 Adam, H., 216 Adams-Ray, J., 74 Adelman, I. R., 255,259 Agnisola, C., 73, 80, 87, 138,262 Akaike, N., 301 Akamine, M., 87 Allen, W. F., 150, 158,161, 162, 166, 172, 179,213 Altringham, J. D., 263 Amlacher, E., 150, 155,179 Anantaraman, A., 82, 135 Andreasen, P., 279, 297,299 Andresen, P., 79 Antonov, A. S., 183 Antonova, G. N., 183 Aree, S . M.,79 Argos, P., 276, 302 Arthur, P. G., 221,245,246,258 Ash, R., 162, 163, 181 Ask, J. A., 27, 28, 29, 50, 51, 73, 283, 297 Audige, J., 161, 179 Axelsson, M., 5, 32, 38, 47, 48,49, 51, 53, 59, 60,62, 65, 66, 69, 71, 73, 74, 78, 80,96, 106, 111, 113,115, 116,117, 118, 119,130,132,133,136,220,221, 238,258

Baartz, G., 303 Bailey, G. S.,263 Bailey, J. R., 16,27,28,29,55,74,222,224, 227,239,240,241,251,253,254,258, 264,280,282,283,291,298 Bailly, Y.,206, 213, 214 Baldwin, J., 94, 131, 138, 249, 258 Ballantyne, J. S.,231,265 Balzer, P. J., 75,259 Barkley, R. A,, 124, 131 Barron, M. G., 59, 74, 114, 131,251, 258 Barthelemy, L., 49, 84 Barthelmey, L.,86 Basile, C., 74,237, 258 Bass, A., 5, 74, 237, 258, 272, 298 Baumgarten-Schumann, D., 59, 74 Baumgarten, D., 264 Baum, H., 217 Beamish, F. W. H., 262 Bean, B. P., 284,298,304 Beckett, J. S., 132 Beland, K. F., 249,264 Belaud, A., 71, 74 Bell, G. R., 202, 205,216 Bendayan, M., 145,179,182 Benditt, E. P., 23, 80 Benjamin, M., 17, 19, 74, 79, 100, 131 Bennett, A. F., 62,85,224,258,279,303 Bennion, G. R., 26, 74,86,138,265 Berne, R. M., 26, 74 Bern, H. A., 161,181 Bernier, D. R., 202, 213

305

306 Bers, D. M., 87,268,270,274,276, 277, 278,279,282,283,286,289,290,296, 297,298,300,303,304 Berschick, P., 238,258 Bersohn, M. M., 290,293,298,302 Bertin, L., 186,213 Bever, C. T., 135 Bever, K., 223,258 Bhargava, V., 82,86 Bilinski, E., 233, 259 Birch, M. P., 165, 166, 167, 168, 179 Bizois, R., 183 Black, D., 259 Black, E. C., 223,259 Black, W., 77 Blanchard, E. M., 303 Blaxter, J. H. S., 90, 131 Blier, P., 272,300 Block, B. A,, 90, 120, 123, 126, I31 Bloom, G., 51, 74,180 Booth, J. H., 213 Bourke, R. W., 75 Boutilier, R. G., 85, 119, 131 Bowler, K., 251, 254, 259 Bramieh, N. J , , 80 Brams, P., 79, 299 Branscombe, R., 260 Braulin, E. A., 242, 259 Braunwald, E.,85 Breisch, E. A., 14, 15, 74 Brett, J. R., 116,131 Bridge, J. H. B., 290,298 Bridges, C. W., 258 Bridges, D. W., 259 Bridges, R. W., 75 Briggs, F. N., 303 Brightman, M. W., 147,179 Brill, R. W., 2,34,54,60,62,65,74,75,76, 78,81,82,113,124,125,126,127,128, 129, 130,131,133,134,135,301 Brix, O., 55, 84 Brodal, A., 176,180 Brommundt, G., 292,298 Brooks, S. P. J., 228,247,259 Brown, A. M., 301 Brown, C. E., 83 Brown, S. E., 27, 50, 75 Buck, L. T., 263 Bullock, J., 136 Bundgaard, M., 144, 146, 147,180

AUTHOR INDEX

Burggren, W. W., 37,45,81 Burne, M. A,, 122,123,131 Burne, R. H., 187,188,210,213 Burnett, L., 82,263 Burnstock, G., 15, 19, 27, 79, 83, 88, 114, 135,271,282,299,304 Bushnell, P. G., 2, 32, 34, 54,60, 62, 65, 74, 75,81, 113, 131, 135 Busselen, P., 291, 298 Butler, P. J., 2, 48, 50, 52, 56, 59, 62, 65, 66, 75, 83, 87, 96, 111, 117, 132,136, 137,155,180,265,304 Byerley, L., 284, 300

C Cai, Y. J., 255, 259 Caldini, P., 153,180 Callewaert, G., 301 Cameron, J, N., 27, 35, 47, 50, 59, 70, 75, 76, 114, 119,132,298 Campbell, D. L., 302 Campbell, G., 145, 180 Campbell, K. B., 102, 132 Canty, A. A., 132 Capogrossi, M. A,, 303 Capra, M. F., 50, 75 Capra, M. F.,75,132 Carafoli, E., 290,298 Carey, F. G., 90,120, 122, 123, 125, 126, 127,131,132,135,138,139 Carey, P. W., 133 Carmeliet, E., 291, 298, 301 Carre, C. G., 179 Carroll, R. G., 136 Case, R. B., 85 Casida, J. E., 302 Casley-Smith,J. R., 144,145,148,180,186, 213,214 Cavalie, A., 287,298,302 Cech, J. J., 59, 62, 75 Cech, J. J., Jr., 238,251,259 Chancey, B., 78 Chan, D. K.0.,50,59,65,66,75,113,132, 172,180 Chang, R. K. C., 134,136 Chapman, D. J., 260 Chapman, R. A., 298 Charnet, P., 300

307

AUTHOR INDEX

Chavin, W., 223,259 Chenoweth, M., 258 Chiu, W-G., 259 Chow, P. H., 50,75 Christensen, D. M.,298 Christensen, N. J., 119,138 Christiansen, J. S., 301 Chui, K.W., 27, 54, 75 Cimini, V., 87 Clapham, D. E., 182 Clark, J. W., 302 Claviez, M., 188, 192, 196, 198, 209, 21 7 Cleeman, L., 278,302 Cobb, J. L.S., 19, 76, 88, 103, 281, 282, 283,288,298,303 Cockett, F. B., 157, 180 Cole, F. J., 176, 177, 180, 194, 195, 212, 213 Collette, B. B., 132 Collins, J. H., 273, 277,298,301 Conte, F. P., 202,213 Cooke, I. R. C., 145,180, 196,213 Cooper, J. A., 183 Cowey, C. B., 263 Cox, R. H., 132 Crabtree, B., 224,263 Crawshaw, L. I., 54,83 Crockett, E. L.,228,229,253,257,259,265 Crockford, T., 301 Crone, C., 150, 180 Cserr, H. F., 146, 180 D

Dashow, L., 51, 76 Davidson, W., 260 Davie, P. S., 2,5,8, 11, 17,20,21,26,27, 32,34,37,38,40,41,43,44,47,50,52, 53,54,59,60,65,66,67,69,70,71,76, 77,78,88,113, 117,119,132,164, 172, 180,261 Davies, P. F., 182 Davis, F. K., 84 Davis, J. C., 32, 59, 75, 76 Davison, B., 76 Davison, W., 12, 74, 75, 78, 88, 95, 131, 132,133,180,181,124,258,261,301 Daxboeck, C., 56,68,71, 76, 77, 116, 117, 118,137,216,265

DeAndres, A. V., 20, 76 Delaney, A. G., 215 Delcourt, R., 87 Dewar, H., 79,128, 130,131,133 DeWilde, M.A., 124 Dickson, K. A., 125, 126, 128,133,134 Diener, D. R., 79 Diprisco, G., 138 Dizon, A. E., 76, 125, 126, 128, 133,136 Dobrin, P. B., 100, 133 Dobson, G., 131 Dodd, H., 157,180 Donaldson, E. M.,183,202, 214 Dornauer, R. J., 80 Dornesco, G. T., 105,133, 150,180 Douglas, E. L., 32, 59, 80, 95, 134, 239, 260,262,281,300 Drahota, A., 262 Drees, J. A., 153, 155,180,182 Drewry, W. F., 83 Driedzic, W. R., 16, 27,28,29,33,55, 74, 76,77,78,131,133,221,222,223,226, 227,228,229,232,233,234,235,236, 237,239,240,241,242,244,245,246, 249,251,253,254,256,258,259,260, 263,264,265,270,280,281,282,283, 288,291,298,299 DuBois, A. B., 211, 216 Dubois, I., 299 Duff, D. W., 202,214 Duncan, J. A., 227,248,249,260 Dunel, S., 187, 215 Dunel-Erb, S., 206, 213,214 Dunn, A., 258 Dunn, F. J., 229,242,243, 247,260 Dunn, J. F., 242,243,260 Dybro, L., 280,281, 291,299 Dyer, D. C., 103,135 E

Eaton, R. P., 24, 77 Eddy, F. B., 77 Edwards, F. R., 80 Ehredstrom, F., 74 Ehrenstrom, F., 132 Einstmann, J. W., 160, 180 El-Sayed, M. F., 279,289,299 Elger, M.,194,214

308

AUTHOR INDEX

Fox, S . H., 216 Foxon, G. E. H., 20, 78 Francois, J. M., 299 Franklin, C. E., 34, 37, 38, 43, 44, 60, 70, 76, 78, 95, 132, 133 Franklin, D. L., 135 Franklin, K. J., 156, 181 Franzini-Armstrong, C., 123, 131 Freel, R. W., 181 F Freeman, H. C., 78 French, C., 259,262 Friedrich, G., 162, 181 Fabiato, A., 268, 270, 274, 278, 299 Friedrich, K., 299 Fabiato, F., 278, 299 Fritsche, R., 113, 114, 116, 117, 118, 119, Fanelli Sr., G. M., 79 130,133,238,261 Fange, R., 90, 133 Fromm, P. O., 214 Fange, R., 164, 176,180,125,223,263 Fry, F. E. J., 123, 138 Farina, F., 19, 88,261 Farrell, A. P., 2,5, 8, 11, 12, 14, 16,20,21, Fujii, K. I., 87, 265 23,24,26,27,29,32,33,34,35,37,40, Fung, Y. C., 216 4 1,43,44,45,47,48,51,52,53,54,56, 57,60,62,63,64,65,66,67,68,69,70, G 73,74,76,77,78,79,80,82,83,84,86, 96, 102, 103, 113, 114, 118,131, 132, 133,152,181,183,208,214,222,223, Gabrisa, S . , 137 228,233,234,235,237,238,250,251, Galante, R. G., 133 254,256,258,260,261,263,280,282, Gallagher, K. R., 138 Candy, J., 302 283,287,294,299,300,301,304 Cannon, B. J., 19, 79,133,282,299 Favaro, G., 171,181 Garbisa, S . 86 Feder, N., 179 Garcia-Garrido, L., 76 Feigl, E. O., 71, 78 Carey, W., 59, 79 Feller, G., 229, 232, 239, 240, 256, 257, Gehrke, P. C., 54, 79, 250, 261 260,277,299 Gemelli, L., 233, 261 Fellows, F. C. I., 224,260 Gennser, M., 50, 79 Fielder, D. R., 54, 79, 250, 261 George, J. C., 262 Field, J. M., 86,137 Gerday, Ch., 229, 239, 240,256, 257,260, Fields, J. H. A,, 259 277,299 Fishman, A. P., 133 Gergely, J., 301 Fitch, N., 81,229,240,257,260 Gerlach, G. F., 272,273,299 Fitzgerald, D., 214 Gesser, H., 27, 28, 29, 62, 63,64, 65, 66, Fleming, J. D., 301 76,79,81,84,260,261,262,263,233, Fletcher, G. L., 92, 94, 133, 134 234,244,245,246,247,248,249,264, Foldi, M., 186,214,216 270,279,280,281,283,284,288,291, Forster, M. E., 2,32,34,36,40,47,50,51, 299,300 53, 59, 65, 67, 68, 74, 76, 78, 88, 106, 113, 117,131,132,133,148,172,180, Ghosh, T. K., 182 181,206,214,220,221,222,245,246, Giacomini, 188,214 Gibbons, C. A., 100, 108, 110,134 258,260,261 Giles, W., 278, 300, 302 Forster, R. P., 79 Gillot, P. H., 87 Fournier, D., 134 Gingerich, W. H., 204,205,214 Fox, I. J., 259 Elson, P. F., 83 Emerson, L., 85 Emery, S. H., 2, 5, 12, 77 Epple, A., 51, 76 Ershler, P. R., 298 Evans, B. K., 80,134 Ewart, H. S., 229, 236,237,260

309

AUTHOR INDEX

Ginley, S. A., 132, 180 Giovane, A., 16, 79,239,261 Glaser, G., 187, 191, 195, 212, 214 Glasgow, J. S., 259 Glossman, H., 299 Godt, R. E., 276,299 Goldman, Y. E., 302 Goldspink, G.,74,258,264,299,301 Goldstein, L., 59, 79 Gooding, R. M., 131 Goodrich, E. S., 17, 79 Goolish, E. M., 12, 79, 253, 261 Gorbman, A,, 264 Goresky, C. A,, 182 Grabarek, Z., 301 Grabner, M.,285,286,299 Graham, J. B., 26,79,82,86,120,123, 124, 125,128,130,131,134,135,263 Graham, M. S., 12, 33, 34, 48, 51, 52, 62, 70,71,77,78,83,92,94,222,251,256, 260,261, 294, 300 Grant, R. T., 20, 79 Gray, C., 80, 262 Gray, W. R., 20, 85 Greco, G., 79,237, 261 Green, J. F., 153, 154, 155, 181 Greer Walker, M., 2, 5, 6, 17, 21, 79, 85, 236,264 Grieshaber, M. K., 258 Grodzinski, Z., 194,214 Grove, D. J., 84,136 Guderley, H., 80, 272,300 Guppy, M.,80,260 Gurney, A. M., 278,300 Guyton, A. C., 186, 211, 213 Gysi, J. R., 260 H

Haedrich, R. T., 92, 133 Hagiwara, S., 284, 300 Halpern, M. H., 20,80 Hamilton, M. N., 262 Hamilton, N, M., 80 Hammons, A. M., 78,260 Hansen, C. A., 227,242,247,261,265 Hansen, S. P., 261 Hansford, R. G., 303 Hanslip, A. R., 259

Hanson, D., 59,80,81,86,151,155,181 Hanyu, I., 136 Hargens, A. R., 148,181,211,214 Harrison, P., 81,256,261,262 Harrison, S . M., 274, 276, 277, 296, 297, 300 Harris, T. O., 213 Harris, W.S., 19,82,99, 100,135 Hart, T., 78,133,234,235,259 Hashimoto, K., 300 Hayton, W.L., 74, 131, 258 Heath, A. G., 243, 261 Heies, M., 217 Heisler, N., 83, 136, 124, 125, 277, 300 Helle, K. B., 13, 73, 80,84, 85,297 Hemmingsen, E. A., 32,38,59,80,95,134, 220,262,281,300 Herzberg, O., 276,300 Hess, P., 304 Hibya, T., 136 Hille, B., 287, 300 Hipkins, S. F., 32, 80, 96, 114, 134,220, 262 Hird, F. J. R., 260 Hirst, G. D. S., 49, 80 Hnath, J. G., 77 Hochachka, P. W., 14,80,233,242,243, 247,249,258,260,262,263,264,265 Hoeger, U., 131 Hoglund, L., 279, 280, 281, 284, 300 Holeton, G. F., 5,12,38,59,65,67,80,83, 136,220,238,256,, 262,281,300 Holland, K.,128,134 Holmes, K., 136 Holmes, W. N., 202,214 Holmgren, S., 27,47,80,82,111, 118,134, 136,301 Holt, B. D., 82 Hopkins, G. S., 165,181 Hougie, C., 83 Houlihan, D. F., 33,34, 69, 80,222, 262 House, E. W., 23,80,86 Houston, A. H., 214 Houston, D. S., 138 Hove-Madsen, L., 87,270, 279, 280, 283, 288,291,300,304 Hoyer, H., 187, 195,214 Huang, T. F., 279,280,281,282,283,300 Huddart, H., 27,82,83,279, 282,283, 301

310

AUTHOR INDEX

Hughes, G. M., 56, 59, 80, 117, 134, 281, 300 Hulbert, W. C., 80 Hume, J. R., 278, 281, 288, 300,301 Hunter, W. C., 132 Hurle, J. M., 2, 9, 85, 104, 137 Hwang, G. -C., 300

Jonsson, S., 84, 264 Jorgensen, E., 64, 79 Jflrgensen, J. B., 242,243, 248,249,262 Jourdain, M. S., 160, 181 Jourdain, S., 186, 215

I

Kabaeva, N. V., 183 Kalous, M., 237, 262 Kalsner, S., 71, 81 Kampmeier, 0. F., 173,186,181,191,192, 195,196,211,212,215 Kanwisher, J., 54, 81 Kanwisher, J. W., 132 Kao, R. L., 301 Kaplan, N. O., 249, 264 Karasinski, J., 271, 272, 301 Karpe, F., 79 Karttunen, P., 14, 82 Kashihara, H., 294, 295, 304 Katano, Y., 87 Kavaler, F., 292, 298 Kayama, M., 210,215 Kay, C. M., 301 Kay, R. M., 263 Keen, J. E., 28, 29, 55, 82, 258, 289, 301, 304 Keller, N. E., 136 Kelly, D. A., 238. 262 Kelly, T., 138 Kemper, D., 138 Kent, B., 111, 114,135 Kentish, J. C., 250, 262 Kent, J. M., 253,254,262 Kenyon, J. L., 270,304 Kesbeke, F., 265 Keyes, R. S., 86 Kiceniuk, J. W., 32, 41, 52, 59, 60, 67, 70, 82, 113, 117, 135, 152, 181, 221, 237, 239,262 Kihara, M., 255, 265 Kilarski, W., 146, 181 Kirby, S., 114, 135 Kirsh, R., 210, 215, 216 Kitazawa, T., 87 Kitoh, K., 8, 82 Klassen, G. A., 83 Klaverkamp, J. F., 103, 135

Ijima, N., 210, 215 Imoto, K., 302 Isaia, J., 83 Ishimatsu, A., 208, 214, 215 Isokawa, K., 20, 80, 81, 105, 134 Itazawa, Y., 59,81,207,217 Iwama, G. K., 208,214,215,217 J

Jackman, A. P., 153, 181 Jackson, C. M., 194,215 Jakubowski, M., 215 James, M. N. G., 300 Jasinski, A,, 144, 146,181 Jennings, R. B., 303 Jensen, D., 53,81,282,301 Jensen, G. S., 247,248, 262 Johannessen, M. W., 215 Johansen, J. A., 78,260 Johansen, K., 2, 11, 37, 45, 59, 80,81, 82, 86, 87, 96, 97, 98, 135, 151, 155, 181, 195,212,215,217,260,262 Johansen, P. H., 243,265 Johnson, D. W., 83 Johnston, I. A., 12, 15, 81, 256,261,262, 263,265,272,275,301 Johnston, M. G., 211,212,215 John, T. M., 223,262 Jonas, R. E. E., 259 Jones, D. R., 1, 27, 32, 35, 36, 38, 40, 44, 45, 52, 53, 59, 60, 66, 67, 70, 81, 82, 96, 99, 100, 102, 103, 104, 105, 106, 108, 109, 113, 117,131,135,152,181, 221,237,239,262 Jones, H. M., 74 Jones, L., 302 Jones, M. P., 43, 86, 96, 98, 107, 137

K

AUTHOR INDEX

Klemm, M. F.,80 Klinkowstrom, A., 194,215 Klitzner, T., 270,286,301 Knaus, H-G., 299 Knox, D. E., 86,263 Koban, M., 262 Kobayashi, H., 217 Koch, W. J., 299 Kondo, H., 87 Koniar, St., 163, 181 Krogh, A., 195,215 Kubasch, A., 23, 82 Kullman, D., 214 Kumar, P., 303

311

Leont’eva, G. R., 111,135 Leszyk, J., 277,301 Levy, M. N., 26, 74 Lewander, K., 223,263 Licht, J. H., 19, 82, 99, 100, 135 Lida, J., 299 Lim, S. T., 249,263 Lindley, B. D., 276,299 Lindstrom, L., 11, 12, 14,M Linthicum, D. S., 123,135 Lipke, D. W., 124 Lipp, P., 278, 301 Lishajko, F., 74 Liu, B., 87,255,265 Lomholt, J. P., 59, 82, 217 Lomsky, M.,84 L Longoni. S., 302 Loretz, C. A., 161,181 Labat, R., 50, 82 Loughna, P. T., 264 Laffont, J., 50, 82 Lowell, W. R., 79, 82, 130,135,263 Lagardere, J. P., 87 Lower, R. R., 135 Lai, N. C., 27,32,37,40,43,44,45,53,56,Lucas, R. V., 259 59, 60, 67, 79, 82, 113, 135,221,263 Lukashev, M. E., 183 Lakatta, E. G., 303 Lush, I. E.,249,263 Lametschwandtner, A., 216 Lutton, C., 216 Lanctin, H. P., 232,233, 263 Lykkeboe, G., 87 Lander, J., 105, 106, 109, 135 Lyndon, A. R., 80,262 Langer, G. A., 301,302 Langille, B. L., 19, 82, 90, 107, 109, 135 M Langille, B. W., 81 Langon, J. K., 123 Lansman, J. B., 304 MacDonald, C., 86 Laparra, J., 84 MacDonald, J. A., 35,83,95, 136,138 Larsson, A., 223, 263 MacIntyre, A. B., 227, 229,263 Laurent, P., 2,3, 12, 13,27,50,51,82,85, Mackay, W. C., 223,264 117, 119,137, 187,210,212,215,282, Maclean, E., 163,181,183 301 MacLeod, K. R.,78, 260,299 Laurs, R. M., 74, 82, 135, 139 Maetz, 83 Lawson, K., 81 Malik, A. B., 183 Lawson, K. D., 127,132 Malik, K. T. A., 299 Leblanc, N., 288,301 Mallet, M. D., 265 LeBras-Pennec, Y.,134 Maneche, H.C., 23, 24,83 LeBras, Y. M., 50, 84 Mangano, C., 77 Lederis, K., 161, 181 Mangor-Jensen, A., 291,299 Lee, K. S., 278, 301 March, H., 98, 135 Lee, Y. C., 27, 54, 75 Maresca, A., 79,84, 261,262 Lehto, H, 27,87 Martin, A., 303 Leknes, I. L., 19,82,105,135 Martinez, I., 271, 272,301 Lenfant, C., 81 Martino, G., 261 Lennard, R., 27,82,83,279, 282,283,301 Mart, P. E., 144, 145,180

312 Matthews, P. M., 265 Mayer, P., 166,182, 186, 187, 196,215 Maylie, J., 279, 281, 284, 301 May, M. M., 20,80 Mayr, W.,83 Mazeaud, M., 85 McConnell, T., 77 McCubbin, W.D., 277,301 McDonald, D. A., 108,136 McDonald, D. G., 88, 100, 106,183 McDonald, T. F. 286,298,302 McKenzie, J. E., 23, 83 McLean, E., 162, 181 McLean, R. M., 260 McLeese, J. M., 120,138 McMahon, B. R., 88,183 McMorran, L.E., 263 McNairn, G., 260 McWilliam, J. G., 83 Mead, J. F., 210,216 Meghi, P., 27,83 Meissner, G., 289, 302 Meltcalfe, J. D., 83 Menendez-Arias, I., 276,302 Merrick. A. W., 242, 263 Metcalfe, J. D., 2,50,56, 75,96,132,136,

AUTHOR INDEX

Morgan, H. E., 232,235,263 Mori, Y.,302 Mosse, P. R. L., 188,215 Motais, R., 83 Mott, J. C., 161, 182,202,216 Moult, J., 300 Moyes, C. D., 78,226,227,228,230,232, 260,263 Muir, B. S., 83 Munoz-Chapuli, R., 76 Munshi, J. S. D., 145, 182 Murdaugh, H. V., 59,83,85 Murphy, B. J., 273, 277, 285, 286,302 Mustafa, T., 242, 243, 248, 249,262 Myhre, K., 149, 182 Myklebust, R., 85 N

Nakano, T., 117,136 Nargeot, J. 300 Narumiya, S., 302 Neely, J, R., 232, 235,263,265 Neill, W.H., 120, 124, 127, 131,136, 138 Nekvasil, N. P., 145, 182 Neumann, P., 61, 83, 118, 136 180 Neuville, H., 150, 182 Meyer, M., 84,137,264 Newsholme, E. A.,223,224,230,231,263, Meyers, G. E., 126,136 Michael, C.C., 147, 149,182 266 Ngan, V., 104,136 Midtun, B,,15,83 Nguyen, T. X.,298 Mikami, A., 286,302 Nichols, J. W., 222, 240, 241,263 Millard, R, W., 80, 181,262 Nicolaysen, A., 144, 146, 183 Millen, J. E., 83, 85 Nicoll, D. A., 292, 293, 302 Miller, B. F., 85 Nicols, D. J., 204, 216 Miller, V. M., 110, 136, 138 Milligan, C. L., 33, 50, 51, 60, 62, 64,65, Niedergerke, R., 288, 289, 302 77,83,88,102,133,215,223,232,233, Nielsen, K. E., 244,263 Niidome, T.,302 234,235,263 Nikinmaa, M., 202,205, 206,216 Milsom, W.K., 53,81 Mislin, H., 172, 182 Nilius, B., 304 Mitchell, J. W., 126, 136 Nilsson, S., 2, 12,27,32,50,51,52,53,59, Modigh, M., 74,258 60,74, 78, 82, 83,84, 111, 113, 114, 118, 119,131,132,133,134,136,138, Moffitt, B. P., 54, 83 148, 154,182,183,221,238,258,261, Mommsen, T. P., 78,227,231,263 301 Montgomery, J. C., 83,136 Nioshimoto, A. Y ., 302 Moon, T.W.,221,227,231,263 Moore, J.F., 23,83 Noble, M. I. M., 284,302 Nonnotte, G., 210,215,216 Morad, M., 270,278,286,301,302 Noordergraaf, A,, 132 Morfin, R., 84

313

AUTHOR INDEX

Norman, D., 79,131 Nowycky, M. C., 304 Nunzi, M.G., 301

0 OConnor, E. F., 75 O’Donoghue, C. H., 20,84 Ochiai, Y.,300 Ofstad, R., 301 Ogilvy, C. S., 211, 216 Oguri, M., 8, 82 Ohta, T., 8 7 Oikawa, K., 301 Ojha, J., 182 Olesen, S. P., 150,182 Olsen, K. R., 182 Olsen, R. L., 301 Olsnes, S., 183 Olson, K. R., 145,182,214 Opdyke, D. F., 111,136 252,264 Ordway, R. W,, Ornhagen, H. C., 79 Ostadal, B., 21,84,258 Ostadal, B., 8, 74,298 Ostlund, E., 74, 180 Owman, C. H. 146,182

Philipson, K. D., 288,292,295,298,302, 304 Phleger, C. F., 259 Pieprzak, P., 88,304 Pierce, I. G., 274, 302 Pierce, M.,111, 135 Piiper, J., 32,49,59, 60,74,84, 113, 137, 221,238,264 Pirages, S. W., 137 Pityer, R. A., 204, 214 Place, A. R., 249,264 Plisetskaya, E., 223, 264 Pohla, H., 194, 216 Pott, L., 301 Poupa, O., 5, 8, 11, 12, 14, 16, 27, 55, 62, 63,64, 79,84,234,236,237,246,249, 261,264,284,299 Poupin, J.. 87 Powers, D. A,, 249,264 Priede, I. G., 16,17,48,49,52,85,99,100, 101,102,137 Primmett, D. R. N., 50,85 Pritchard, A. W., 243,261 Proch zka, J., 262 Prosser, C. L., 186,216,262 Przemyska-Smosarska, 171, 172, 182 Pye, J. M., 300 R

P Pack, A. I., 133 Page, S. G., 288,289,302 Parker, G . H., 20,84 Parker, T. J., 20, 84 Parson, T. S., 216 Peirce, E. C., 135 Pelouch, V., 74,258, 298 Pelzer, D., 298, 302 Pennec, J. P., 26, 50,84,87, 134 Permutt, S., 180 Perry, S. F., 2, 76,84, 88 Pessah, I. N., 289, 302 Petersen, 0. W., 183 Peterson, K. S., 260 Peterson, K., 59, 84 Peyraud-Waitzenneger, M.,26,50,53,56, 59,65,80,84,113,136,300 Peyraud, C., 26, 71, 74,80,84,300

Rach, J. J., 214 Rada, G. K., 265 Raffaeli, S., 303 Rahman, M. S.,248,249,264 Railo. E., 216 Randall, D. J., 1,24, 27, 35, 37, 45, 56, 59, 65,66, 67, 75, 76,80,81, 85,86 116, 117, 118,131,135,137,138, 152,183, 187, 208,216,217,238,262,265 Randazzo, V., 77 Rankin, J. C., 83 Rasio, E. A., 147, 149, 179,182 Rasmusson, R. L. 278, 302 Rauchov , H., 262 Reese, T. S., 179 Reeves, J. P., 277,292, 302,303 Reeves, R. B., 247,264 Regnier, M.,20, 79 Reibel, D. K., 265

314 Reichel, H., 279,283,303 Retzius, G.,194,216 Reuter, H.,286,287,303 Rhode, E.A., 132 Rhodin, J. A. G., 144,150,182 Riley, R. L.,180 Ringer, S. A., 268,303 Ripatti, R.,50,51,87 Ristori, M.T.,50,85,117, 119,137 Riben, M.,74 Robertson, A. C.,259 Robertson, 0.H.,21,23, 85 Robin, E.D.,59,83,85 Robinson, J. S., 210,216 Romanov, Y. A.,183 Rome, L.C.,253,264 Romer, A. S., 216 Ross, J. K.,135 Rothe, C.F., 153, 155,180,182 Rourke, A. W., 23,82 Rowell, D.M.,75,259 Rowing, C. G.M.,161,182,191,216 Rubanyi, G.M.,138 Ruben, J. A.,62,85,279,303 Rudeberg, C.,146,182 Rumberger, E.,279,283,303 Rusznyak, I,, 211,212,216 Ruyung, S , , 57,88

AUTHOR INDEX

172,173,174,175,179,180,181,182, 202,214,216,238,261,264 Saunders, R. L., 23,24, 78,86,238,264 Savina, A.,84 Scarborough, D.,74,131 Scheffauer, F.,299 Schiebler, T.H.,8,21,84,87 Schlote, W.,21 7 Schmid-Schbein, G.W.,200,216 Schmidt-Nielsen, K.,57,86 Schmidt, S. P., 23,86 Schneider, D.E.,262 Schuil, D.S., 298 Schumaker, P. T.,139 Schwartz, A., 299 Schwlame, K.,223,264 Scott, C.,260 Scott, D.L.,77,259,260 Scutt, A.,299 Sebert, P. H., 49,86 Seibert, H.,48,86,281,282,303 Senior, H.D., 16,86 Sensabaugh, G.R., Jr., 249,264 Sephton, D. H.,74,222,223,224,228,229, 232,252,253,254,256,258,264 Serafini-Fracassini, A,, 19,86, 100,105, 137 Sflinger-Birnboim, A., 183 Shabetai, R.,43,44,82,86,130,263 Shadwick, R. E.,100, 108,110,134 S Sharp, G.D.,124,137 Shattock, M.J,, 278,279,282,283,289,303 Saetersdal, T. S., 19,85 Shelton, G., 53,67,81,86,88,113, 114, Sage, E. H.,20,85 119,135,138,139,154,162,183,221, Saito, T.,49,85,281,282,303 238,265,266 Salvatore, G.,87 Shepard, J. M.,183 Sanchez-Quintana, D.,2,9,85,104,137 Shibata, E. F.,302 Sandborn, E. B., 179 Shimizu, T.,87 Sandvig, K., 183 Shiner, J. S., 303 Sans-Coma, V.,76 Short, S.,32,48,56,59,67,87,96,111,113, Santa, V., 105,133,150,180 137,265,304 Santer, R. M.,2,3,5,6,9, 12,13, 14,19, Shoubridge, E.A., 243,247,249,264 21,23,76, 79,85,LOO, 131,137,236, Sibert, J., 134 264,270,274,281,282,283,286,288,Sidell, B. D.,227,228,229,232,234,239, 240,246.247,249,253,255,257,259, 298,303 Sappey, P.C., 165,182 260,261,264,265,266 Siebert, H., 86 Sarnoff, S. J., 34,85 Satchell, G. H.,2,3,26,43,45,50,56,59, Silversmith, C.,144,150,182 71,86,88,90,96,98,107,137, 152, Sims, H.F.,302 158,159,161,163,165,169,170,171, Singer, J. J., 231,264

315

AUTHOR INDEX Singer, T. D., 227, 265 Sipido, K., 278, 303 Sire, M-F., 210,216 Skinner, E. R., 223,259 She-Goffart, C. M.,260 Sluse, F. E., 260 Small, S. 78 Small, S. A., 86 Smirnov, V. N., 149,183 Smith, D. G., 80,114,116,134,137,138, 154,183 Smith, D. J., 152,181,124 Smith, L. S., 59,88,202,205,208,216 Soivio, A., 216 Solaro, R. J.. 271,272,273, 274, 276, 303 Somero, G. N., 275, 276, 277, 303, 304 Sommer, J., 303 Sorensen, E., 85 Soulier, P., 56, 65, 80, 84, 113, 136 Sower, S. A., 264 Spina, J., 137 Spina, M., 86 Spitzer, K. W., 298 Spurgeon, H., 278,303 Stachell, G. H., 75, 76 Stainsby, W. N.,85 Staudinger, R., 299 Steen, J. B., 149,182,183 Steffensen, J. F.,36,67,68,69,77,78,86, 188,196,202,204,206,209,210,217, 260 Stephenson, D. G., 275,276,303 Stene-Larsen, G., 73,297 Stern, M. D., 303 Stevens, E. D., 57,59,82,86,96, 120, 123, 124, 126, 127,135,136,138, 152, 183, 217,251,265 Stewart, J. M., 228,239,240,242,259,260, 265 Storey, K. B., 80, 227,228, 238,246,247, 248,249,259,260,262,264,265 Stowe, B. D., 260 Stowe, D., 265 Strahan. R.,176,181 Straus, A. W., 302 Stray-Pedersen, S., 144, 146, 149, 183 Striessnig, J., 299 Stuart, R. J., 86, 137 Suarez, R. K., 78, 228, 260,265 Suchard, M. E., 178,183

Sudak, F. N., 98,138 Sullivan, L., 84,264 Sund-Laursen, J., 79, 299 Sundell, L. E., 233, 249, 261 Sundness, G., 81 Sunnerhagen, K. S., 82 Sureau, D., 48, 87 Sutko, J. L. 270, 304 Sutterlin, A. M., 238, 264 Swartz, R. E., 77 Swayze, C. R., 259 Swezey, R. R., 273,304 Swift, P. R., 139 Sykes, B. D., 301 Szabo, G., 186,216

T

Takagi, M., 80,81,134 Takeda, T., 59,81,87 Takeshima, H., 302 Talikowska, H., 163, 183 Talo, A., 303 Tanabe, T., 302 Tarr, B. D., 74,131,258 Taylor, D. J., 221, 242,265 Taylor, E., 281,304 48,49,59,62,65,66,75,87, Taylor, E.W., 96,132,137,238,265 Taylor, H. H., 88,132,181,214 Taylor, M. G., 108, 138 Tchertikhina, I. V., 183 Teal, J. M., 120, 122, 126, 132 TebBcis, A. K., 98, 138 Temma, K., 27, 50, 51, 87 Tetens, V., 119, 138 Thomas, E., 262 Thomas, M. J., 304 Thomas, S., 87 Thorarensen, H., 154,163,183 Tibbits, G. F., 78,82,87,260,286,287, 288,289,290,292,293,294,295,301, 302,304 Tirri, R., 14, 27, 50, 51, 82, 87, 251, 254, 259 Toda, Y.,80,81,134 Tomlinson, N., 117, 136 Tota, B., 2,5,6,8,12,20,21,32,38,73,74,

316

AUTHOR INDEX

Walker, T. I., 260 Walsh, J. V. Jr., 264 Ward, J., 214 Wardle, C. S., 186, 191,200,209,210,217 Watabe, S., 300 Waterhouse, A. L., 302 Watson, A. D., 19, 24, 88, 103, 138 Watson, C., 135 Watters, K. W., 59, 88 Way Kleckner, N. W., 255,266 Weber, L. J., 152, 169, 183,222, 240,241, 263 Weber, R. E., 94,138 Weeks, T. A., 301 Weidenreich, F., 187, 217 Weir, W. G., 304 Weiss, R. S., 298 v Welch Jr., G. H., 85 Wells, L. D., 213 Wells, R. M. G., 68,83,88,92,94,95,131, Van Citters, R. L., 24, 87, 135 136,138,261 Van den Thillart, G., 265 West, T. G., 78,260 Van Deurs, B., 146,183 Wexler, B. C., 85 Vanhoutte, P. M., 110,136,138 White, F., 74 Van Kerkhove, E., 291,298 White, F. C., 114, 139 Van Lenten, B. J., 80 Wier, W. G., 268,278, 284,303 Van Waarde, A., 242,243,265 Williams, D. A., 275, 276, 303 Vary, T. C., 226,265 Willmer, H., 84, 137,264 Vastesaeger, M. M., 23, 87 Wise, R. M., 303 Vecchio. P. J., 147, 183 Wittenberg, B. A., 239,266 Vemuri, R., 298 Wittenberg, J. B., 239,266 Venzi, R., 73 Witthames, P. R., 85 Vernier, J-M., 216 Witztum, K., 86 Vitek, V., 74, 258, 298 Wolf, N. G., 123, 126, 139 Vlymen, W. J. 111, 124, 137 Wood, C. M., 2,48,49, 53, 54, 59,62, 65, Voboril, Z., 21, 87 67,79,83,88,113, 114, 119,139,148, Vogel, V., 21 7 152, 154, 162,183,215,221,238,266, Vogel, W. 0. P., 186, 187, 188, 192, 196, 280,282,283,304 200,208,209,210,211,212,213,217 Wood, R. E., 81 Von Euler, U. S., 74 Wood, S., 78,133, 299 Vornanen, M., 27, 28,29,88 Woodhouse, S. P., 83 Woodland, W., 160, 183 W Worth, H., 84,137,264 Wright, G. M., 17, 20, 88, 105, 139 Waddell, J. A., 180 Wagner, H. H., 213 X Wahler, G. M., 259 Wahlqvist, I., 114, 119, 138, 148, 183 Xiaojun, X., 57, 88 Walker, R.M., 243,265

79, 81,84, 87, 96, 138, 236, 237, 258, 261,262,265 Trautmann, A., 217 Trautwein, W., 298, 302 Trentham, D. R., 302 Tretjakoff, D., 186, 195,217 Trois, E. F., 183 Trott, J. N., 88,304 Tsien. R. W., 284, 287, 304 Tsukuda, H., 12,48,87,222,251,253,255, 265 Tuana, B. S., 302 Turay, L., 299 Turner, J. D., 79,244,265 Tytler, P., 90, 131

317

AUTHOR INDEX Y

Yamamori, K., 136 Yamamoto, K., 207,217 Yamauchi, A., 3, 13, 15,88,271,304 Young, J. E., 223,259 Yuen, H. S. H., 76,133

z Zammit, V. A., 223,224,230,231,266 Zummo, G., 19, 81, 87, 88, 261 Zweifach, B. W., 216

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SYSTEMATIC INDEX Names listed are those used by the authors of the various chapters. No attempt has been made to provide the current nomenclature where taxonomic changes have occurred. Boldface letters refer to Parts A and B of Volume XII. A

Abramis brama, B, 104 Acanthgopagms, B, 17 Acipenser, A, 17, B, 29,38 A. brevirostris, B, 18, 29, 196 A. fluuescens, B, 140, 149, 168 A. montanus, B, 45 Albacore, A, see Thunnus alalunga Alewife, A, 280 Alopias A. superciliosus, A, 121 A. uulpinus, A, 121,237 Alosa sapidissima, A, 16, B, 203 Ambassis, B, 3 Amia, A, 17, 165, 172, 187, B, 32, 39, 279-282 A. caloa, B, 71, 149, 169, 196, 324, 399 Amiurus melas, B, 332 Amphipnous cuchia, A, 59, B, 85, 171 Anabas testudineus, A, 145, B, 85 Anarhichas lupis, B, 181, 186 Anchovy, see Engraulis encrasicolus Angler fish, see Lophius piscatorus Anguilla, B, 3,27, 107, 110,267 A. anguilla, A, 3,26,47-49,58,65, 112, 118,144,234,242,243,277, B, 6,13, 41, 140,154,159,192,200,202,204, 207,211,316,363 A. australis, A, 58, 112, 114, 116, 119, 220, B, 18, 34, 176 A. diffenbachii, A, 41-42,44,46,53,60, 67,69 A. japonica, A, 53,58,65, 112, 118, B,

69,169,198,200,202,207,211,216, 218,221,224,225 A. rostrata, A, 16,227,239,251, B, 7,11, 72,142,153,154,155,156,160,164, 166, 169, 174, 192, 197,198,207, 224,268,270,272 A. uulgaris, A, 3, 13 Aphanopus carbo, B, 7 Arapaima, B, 12 A. gigas, A, 14, B, 11 Artemia, A, 292-293 Arwana, see Osteoglossum Amazonian discus fish, see Symphysodon Atractosteus tn'stoechus, B, 169 Auxis A. rochei, A, 121 A. thazard, A, 121 B

Barb, see Barbus Barbus, B, 22 B. Juoiatilis, A, 163 Bass, B, 102 kelp, see Paralabrax largemouth, see Micropterus salmoides sea, see Dicentrarcus labrax smallmouth, A, 29,280, see also Microptems dolomieui striped, see Morone saratilis Bichir, Nile, see Polypterus senegalus Billfish, A, 122-123 Bitterling, see Rhodeus amams 319

320 Bluefish, 211, see also Pomatomus salatrix Boreogadus saida, B, 91 Bowfin, see Amla caloa Bream, see Abramis brama Buffalo fish, see Megastomatobus or Ictiobus Bufo B. bufo, A, 275 B. marinus, A, 275, B, 326-327 Bullhead brown, see lctalurus nebulosus Bull rout, A, see Myxocephalus scorpius Burbot, A, 70, see Lota lota

SYSTEMATIC INDEX

Channa punctatus, B, 159, 165, 171 Channichthyidiae, A, 256 Channichthys rhinoceratus, A, 229,232 Chanos chanos, B, 110 Char, Arctic, see Saloelinus alpinus Chelidonichthys kumu, A, 5 Chimaera, B, 3, 19, 37-40 C . monstrosa, A,4, B, 157,196,220,268 Chionodraco, A, 38 C. hamatus, A, 46,96 Cichlasoma, B, 11 Ciliata mustela, A, 46 Clarias, B, 35 C. batrachus, B, 41,85, 169,192 Clupea, B, 11 C C. harengus, A, 4, B, 4,8, 17,24-25 Clupidae, A, 11 Cod, A, 247, B, 71,228,276,316,327,349 Cacharodon carcharias, A, 4, 120-123 Atlantic, see Gadus morhua Caiman crocodylus, B, 324 Coelacanth, B, 21,39,43,98, 197, see also Callorhyncus millii, B, 142 Latimeria chalumnae Carassius Conger, B, 3 C. auratus, A, 46,47,227,246-249,253, C. conger, A, 237 255, 271, B, 17, 32, 59-60, 66, 81, Coryphaenoides rupestris, A, 5 149,358, 362,397 Cottus C. carassius, B, 78,268 C. bairdi, B, 210 Carcharhinus sp. ,B, 107 Carp, A, 3,12,50,100,105,151,155,207, C. gobio, B, 9 C. poectlopus, B, 9 253,255,278,282, B, 71,80, 104,290, Crassius, B, 258 395, 397, 416, see also Cyprinus C . auratus, A, 3,12,48,243,B,210,212 carpio C. crassius, A, 272 crucian, see Carassius carassius Crocodylus porosus, B, 324 grass, see Ctenopharyngodon idell Ctenopharyngodon idell, B, 64 Catastomus Culpea pallasii, B, 94 C . catastomus, A, 70 C. commersoni, B, 17-18,74,109, 150, Cycloptews, A, 162 C. lumpus, A, 239, B, 4,79-80,206,207 157, 165, 169 Cyprinus carpio, A, 3, 13, 50-51, 54, 58, C. macrocheilus, B, 211 99,150,227,239,271-272,277, B, 14, Catfish, A,57, B,4,35,401, see also Silurus 17-18,25-26,34,68,82,150,160,162, meridionalis 169,191,204,268,306,310,372,375, armored, see Pterygoplichthys 391 channel, see Ictalurus punctatus glass, see Kryptoptenrs bicirrhis Centrophorus calceus, A, 160 D Cephaloscyllium isabella, A, 168-170,174 Cetorhinus, B, 351 Dasyatis, B, 37-38 Chaenocephalus aceratus, A, 35,58,67, D. sabina, A, 58, B, 80 220,229,256 Dicentrarcus labrax, A, 224,227, B, 59-60, Chalinura profundicola, A, 17 103, 105 Champsocephalus gunnari, A, 277 Dogfish, A, 9, 19,48, 114, 116-118, 160, Chanda, B, 3

321

SYSTEMATIC INDEX

211,238, B, 26,71, 103,261,266,270, 275-276,280,347,355-357,360-361, 365-369,371-375,377-381,401,403, 408, 417, see also Scyliorhinus stellaris; Galeorhinus galeus, Squalus acanthius smooth, see Mustelus canis spiny, A, 281, B, 105,323,327 spotted, see Scyliorhinus canicula E

Ebchytraeus, B, 36 Eel, A, 20, 50, 63, 66, 105, 114, 145, 146, 147, 149, 161, 171,224-225,240, B, 66,71, 102, 185,209,219,279 Australian, A, 281 conger, A, 71 electric, see Electrophorus European, A, 3, 118, 144,281, B, 76 see also Anguilla anguilla North American, see Anguilla rostrata Japanese, A, 118, B, 70, see also Anguilla japonica short-finned, see Anguilla australis Eelpout, Antarctic, see Rhigophila dearborni Electrophorus, B , 12, 399 E . electricus, A, 59 Eleginus garcills, B, 91 Engraulis encrasicolus, A, 4 Enneacanthus obesus, B, 165 Enophrys bison, A, 239, 241, B, 171, 192 Entosphenus E. tridentatus, B, 11 E. japonicus, B, 12 Epinephelus striatus, B, 150, 171 Eptatretus, B, 25, 86-87 E . burgeri, B, 42, 205,220 E. cirrhatus, A, 3, 5, 27, 34, 36, 40, 46, 55, 58, 65, 105, 148, 163, 206, 220, 221,246, B, 168, 173, 186 E . stouti, A, 3, 227, B, 31, 82, 90, 140, 149, 168, 186, 194 Erpetoichthys, B, 358 Esor E . lucius, A, 277, B , 8, 17, 26, 45, 76 E . niger, A, 224-225,227,251

Euthynnus E . affinis, A, 121 E. allatteratus, A, 121 E . lineatus, A, 121 F Flounder, A, 63, 243, 247, 281, B, 71, 92, see also Pleuronectes jlesus starry, see Platichthys stellaris yellow tail, see Limanda feruginea Fundulus, B, 4 F . catenatus, B , 163 F. grandis, B, 150, 163 F. heteroclitus, B, 60,160,162,163,218, 221,230 F . oliuaceus, B, 163

G Gadus, A, 162 G. ogac, B, 91 G. merlangus, B, 6-7 G. morhua, A, 29,46,48,53,59-60,63, 109, 112, 114, 116-119,220, 227, 234, 238, B, 11, 13, 17, 70, 90-91, 102-103, 181, 186,226,228, 258-259,265,268,319,329-331, 358 Gaidropsarus vulgaris, A, 227 Galeocerdo cuuieri, A, 4 Galeorhinus G. australis, A, 70 G. galeus, A, 9 Gar, B, 13 longnose, see Lepisosteus osseus Garpike, see Lepisosteus osseus Gasterosteus, A, 17 G. aculeatus, B, 18,35, 162 Gastrochisma melampus, A, 121, 123 Gila atraria, B, 221, 224 Gillichthus mirabilis, B, 160 Gillichthys, A, 161 Ginglymostoma cirratum, B, 19, 30, 33, 34, 195,200 Glassfish, Asiatic, see Chanda Gnathonemus, B, 358 Gobio gobio, A, 272

322

SYSTEMATIC INDEX

Goldfish, see Carassius auratus Gonostomidae, B, 5 Goosefish, A, 280 Grayling, B, 8 arctic, see Thymalus arcticus Gourami, blue, see Trichogaster trichopterus Guppy, see Poecilia reticulata Gymnothorax funebris, B, 5, 150, 171

1. punctatus, A, 254, B, 16-17,29-30,

81, 98, 109, 150,153, 157, 165,358, 403 Ictiobus, B, 23 1. cyprinellus, B, 150, 169 lsurus 1. oxyrinchus, A, 4,9, 120, 122-123, B,

78,310 1. paucus, A, 121, 123

H

K

Hagfish, A, 17,26,28,31,53,68,111, 118, 146, 163, 173-177, 192, 195,212, 244-245,281-282, B,5,11,26,30-31, 35, 37,42-43, 90, 108, 139, see also Myxine cirrhatus Atlantic, see Myxine glutinosa New Zealand, see Eptatretus cirrhutus Pacific, see Eptatretus stouti Hemilepidotus hemilepidotus, A, 46 Hemitripterns amerlcanus, A, 5,46,59, 113, 115, 228, B, 176,372, 391 Herring, see Clupea harengus Pacific, see Culpea pallasii Heterodontus, B, 35, 38, 347 H . francisci, A, 98 H . japonicus. B, 206 H . portusjacksoni, A, 43,98, 144, 158-159,166-170 Heteropneustes fossilis, B,34,41,85, 169, 192 Hexagrammus, A, 162 Hippocampus hudsonia, B, 203 Hippoglossoides elassodon, B, 78 Hoplerythrinus unitaeniatus, A, 59, 245 Hoplias malabaricus, A, 245 Hydrolagus colliei, B, 140, 149, 168, 196, 315,322,351

Katsuwonus pelamis, A, 5, 11,21, 28, 35,

I

Icefish, A, 257,277 Antarctic, A, 239, B, 3 Ictalurus, B, 17,42 1. melas, B, 319 1. nebulosus, A, 3

46,54,55,59,69, 105 113, 118, 121 124, 126,289, B, 76,78 Killijish, see Fundulus heteroclitus Kryptoptems bicirrhis, A, 188, 196-201, 206

L

Labrus L. berggylta, A, 46, B, 13 L. mixtus, A, 46 Lamna ditropis, A, 121, 123 Lampetra, B, 12,31,40, 303, 344, 350 L. jluuiatilis, B, 4, 22, 26, 186, 194 L. japonica, B, 27, 33, 205,220 L. lamottenii, B, 79 L. planed, B, 140,149, 168,173 L. reissneri, B, 25 L. tridentata, 90 Lamprey, A, 12,17,53,105,186, B,71,75, 90, 104, 108,224,271, see also Mordacia mordax; Lampetra fluoiatilis Latimeria, B, 10,39,85,358 L. chalumnae, B, 21, 43, 98, 108, 197 Leipotherapon unicolor, A, 54 Lepidoseus, A, 17 Lepidosiren, B, 12, 36, 39 Lepidosiren paradoxa. A, 212, B, 196 Lepisosteus, A, 167,172,188, B, 32,39,42, 71, 399 L. oculatus, B, 418-419 L. osseus, A, 150, B, 13, 196 L. patostomum, B, 149, 168

323

SYSTEMATIC INDEX

L. platyrhincus, B, 27,41,307 L. productus, B, 13 L. spatula, B, 324 Lepomis L. cyanellus, A, 253 L. gibossus, B, 165, 397 L. macrochirus, A, 243 Leuclscus leuciscus. A, 272 Limanda L. feruginea, B, 91 L. limanda, B, 77,230 Lingcod, see Ophiodon elongatus Loach, see Misgurnus anguillicaudatus Lophius L. americanus, B, 207,210,325 L. litulon, B, 198 L. piscatorius, A, 191, 228, 239, B, 4 Lota lota, A, 70 Luciperca, A, 160, 162 Lumpfish, 240, 241, 280 Lungfish, A, 3, B, 5,45 African,see Protopterus acthiopicus Australian, see Neoceradotus forsteri South American, see Lepidosiren paradoxa Lutianus L. campechanus, B, 150, 171 L. griseus, B, 150, 171

M

Mackerel, A, 280, see also Scomber scombrus butterfly, see Gastrochisma melampus Spanish, see Scomberomorus maculatus Macrouridae, A, 17 Macrozoarces americanus, A, 16,41,46, 221,228,233,239-240,244,280, B, 92 Makaira M . indica, A, 121, B, 78 M . nigricans, A, 4, 121,228, B, 78 Marlin, A, 71 black, see Makaira indica blue, see Makaira nigricans striped, see Tetrupterus audax white, see Tetrapterus albidus Maurolicus miilleri, B, 5 Megastomtobus sp., B, 86

Microgadus tomcod, B, 91 Micropterus M . dolomieui, A, 29,228,251,253, 254, 256 M. salmoides, B, 71 Milkfish, see Chanos chanos Misgurnus, A, 162 M . anguillicaudatus. A, 3, 13, B, 17-18 M.fossilis, A, 143, 163 Monkfish, see Squalus squatina; Lophius piscatorius Mordacia mordax, A, 224, B , 97 Morone M . americanus, A, 228,232,254,255, 256 M . saxatilis, A, 228, B, 25, 29, 86 Mud skipper, see Periophthalmodon schlossen' Mugil cephalus, B, 101,224 Mullet, stripped, see Mugil cephalus Mumrnichog, see Fundulus heteroclitus Mustelus, B, 347 M . antarcticus, A, 168 M . canis, A, 211, B, 6, 11, 36, 182, 195 Mycteroperca tigris, B, 150, 171 Myliobatis, B, 37-38 M yoxocephalus M. awnaeus, B, 92 M. octodecimspinosus, A, 228,253, B, 207,210,212,224 M. scorpius, A, 3,46, 59, B, 91 Myxine, B, 12,344 M. cirrhatus, A, 28 M . glutinosa, A, 27,46,58,65, 106, 111, 113,144, 163, 172,173, 174,220, 227,232,233,238,242,247, B, 4,8, 10, 13,21-23,26-27,31,36,43,98, 140, 142, 149, 157, 162, 194,204, 220,303,310-311

N

Narke japonica, B, 220 Natropis cornutus, B, 210 Negaprion brevirostus, B, 85 Neoceradotus, A, 212, B, 11,26,29,39 N.forsteri, B, 21, 169, 196, 201,206 Neothunnus mamopterus, B, 199

324

SYSTEMATIC INDEX

Notothenia N. gibberifrons, A, 229,232,257,281 N. neglecta, A, 229 N . rossi, A, 229,232 Notothenid, Antarctic, A, 62,see also Pagothenia borchgreuinki U

Oncorhynchus, A, 23 0. gorbuscha, B , 94,182 0. keta, B, 64,67,182,198 0. kitsutch, A, 47,71,73,239, B,59,66,

P. borchgreoinki, A, 5,46,49,59,60,95,

B,174.363 Paracheannichtys charcocti, A, 281 Paralabrax sp. ,B,76 Paralichthys lethostigma, B,145,150,163 Paranotothenia rnagellanica, A, 229 Parophys uetulus, B, 84,159,164 Pelteobagrus fuluidraco, B, 221 Perca, A, 160,162,255,B,40 P. jlauescens, A, 29,229,251,254,256,

280,2,"Ql -

P.fEuolatilis, A, 254,B,18,104,148, 154,

155,159 P. perca, A, 14 68-69,72 Perch, A, 3,50,51,63, see also Perca perca 0.mykiss, A, 8,12,14,19-21,23,29,31, climbing, A, 145 33,35,37-38,40-41,43-44,4649, spangled, A, 54 52,54,60,62,63,69,71,73,94, white, see Morone americanus 99-100,102-103,105,113,114, yellow, see Perca jlauescens 116-119,162-163, 188,202-205, Periophthalmodon schlosseri, B, 70 208,210,221,228,232,234, Petromyzon, B, 12 236-238,243,245,254,280,283, P. marinus, B, 9,22,31,75,108, 140, 285-287,291,B, 6,8, 13,17,25, 149,168,173 27-29,31,33,37,59,66-67,69, 70-73,75,77,81,84,86-88,92-93,

96,109-111,136,141,150-151, 153-156,158-159,162,169-170, 189-190,203,207,221, 316,332, 358 0. nerka, B,81,98,170 0. tshawytscha, A, 47,163,B, 72,100, 182 Ophiodon elongatus, A, 57,59,113,118, 142,209,238, B,94 Oplegnathus fasciatus, A, 58 0p sa nus 0.beta, B, 78,85, 203,221,224,227 0. tau, B,85,197,200,203,207,210, 226,228-229 Oreochromis 0. alkalicus grahamin, B,85 0. niloticus, B, 34,84,311 Osmerus mordax, B, 92 Osteoglossum, B, 12 P

Paddlefish, see Polyodon

Pagothenia P. bernacchi, A, 5,46,48, 59,62, 66,B,

363

~~

Pickerel, see Esor niger Pike, see Esox lucius Pikeperch, see Stizotedion lucioperca Pipefish, see Syngnathus fuscus

Piraruca, see Arapaima Plaice, A, 3,63,see also Pleuronectes platessa Platichthys P.jlesus, A, 28,188,243,B,150,159,

206,207,310 P. stellaris, A, 65,148,B, 166 P. stellatus, A, 59,152,B, 72,78,150,

153-154,156,159,164 Pleuronectes, A, 165 P.fEesus, A, 279,284,291, B, 10,23, 74,

162,211-212,230-231

P.microcephalus, B, 197 P. platessa, A, 3,5,19,51,234, B,16-17, 22,24,26,29,35,156,159,181,186,

226,230 Poecilia reticulata, A, 105 Polistotrema stouti, see Eptatretus stouti Pollachius pollachius, A, 46 Polyodon, A, 150,B,23,31,38-39 P. spathula, B,18,24,34, 149,169 Polypterus, A, 17,B, 43 P. senegalus, B, 196 Pomatomus salatrix, A, 211,B,171,404

325

SYSTEMATIC INDEX

Pomolobus pseudoharengus, B, 203 Pond loach, see Misgurnus fossilis Potamotrygon, B, 86,107-108 P. circularis, B, 195 P. hystrix, B, 140, 142, 161 P. magdalenae, A, 227,235 Pout, ocean, see Macrozoarces amerlcanus Prionace, B, 86 P. glauca, A, 4, 9, 127, B, 4 Protopterus, B, 36, 399 P. acthiopicus, B,76, 87,206 P. aethiopicus, A, 3, 59, 212, 229, 230, 243, 247, B, 196 P . annectens, B, 21 Pseudochaenichthyes, A, 59 Pseudopleuronectes americanus, A, 58, B, 78,91, 155-156, 159, 174,210,212, 224,230, -231 Pseudoscarus guacamaia, B, 150, 171 Pterygoplichthys, B, 12 P . multiradiatus, B, 10 Pungitius, A, 17 R

Rabbit fish, see Chimaera Raja, B, 75, 324, 355 R . batis, B, 18 R. binoculata, A, 151, B, 140, 149, 168 R. clavala, B, 220 R. clavata, A, 4, B , 351, 355, 375 R . elanteria, B, 85, 355 R. erinacea, A, 227, B, 6, 152,163, 195-196,322,324 R. hyperborea, A, 4, B, 32 R. kenojei, B, 32 R. microocellata, B, 355 R . nasuta, B, 310 R. ocellata, B, 78, 148 R. radiata, B, 90,324-325 R. rhim,A,58, B, 140,149,168,323-325 Rana esculenta, A, 275 Rana pipiens, A, 275 Raniceps raninus, A, 46 Ratfish, see Hydrolagus colliei Rays, B, 3,37,40,356-3577 freshwater, see Potamotrygon thomback, see Raja clavata Torpdeo, A, 196 Reedfish, see Erpetoichthys

Rhigophila dearborni, B, 176 Rhinoptera bonasus, B, 195 Rhizoprionodon terraenovae, B, 195 Rhodeus amarus, B, 78 Rockfish, red, see Sebastodes reberrimus Rutilis rutllis, A, 272 S

Salmo, A, 162, B, 42 S. clarki, A, 243 S . fario, B, 72 S.gairdneri,A,%, B,6,17,27,30,268,310 S. salar, A, 8,23,71,229,236, B,65-66, 73,91-92,94,202,391 S . trutta, A, 3,8,271,274,B,12,58,154, 32 1 Salmon, A, 280, B, 69 Atlantic, see Salmo salar chinook, see Oncorhynchus tsha w ytscha chum, see Oncorhynchzls keta coho, see Oncorhynchus kitsutch Pacific, A, 21 sockeye, see Oncorhynchus nerka Salvelinus, B, 110 S . alpinus, A, 272, B, 105 S. fontinalis, A, 229,232-233, B, 13,16, 67,150,157,162,163,165,170,191 S. namaycush, B, 111, 171 Sand dab, see Limanda limanda; Spicara chryselis Sarotherodonmossambica,A,188,B,66,160 Scaphirhynchus platorynchus, B, 196 Scomber, B, 11 S . scomber, B, 86 S. scombrus, A, 4,229,239, B, 4 Scomberomorus maculatus, B, 13 Scombridae, A, 120 Scophthalmus maxima, A, 100 Scorpaena, B, 40 Scorpaenichthys, A, 162 Scorphaeichthys mamnoratus, B, 322 Sculpin, A, 280 buffalo, see Enophrys bison grubby, see Myoxocephalus awnaeus longhorn, see Myoxcephalus octodecimspinosus mottled, see Cottus bairdi shorthorn, see Myoxocephalus scorpius

326 Scyliorhinus S . africanus, B,85 S. canicula, A, 31,48,49,58,65,66,67, 111-112,221,224, B, 19,74-75,78, 82, 103,105, 140,195,196,200,

SYSTEMATIC INDEX

Smelt, see Osmerus mordax Solea oulgaris, A, 47 Sole, see Solea vulgaris lemon, see Parophrys vetulus; Pleuronectes micmcephalus Somniosus microcephalus, B,4,13,19,22 205-206,218,220,260,310,315, Spearfish 319,322,345,351 longbill, see Tetrapterus pjluegeri S . stellads, A, 19,49,57,58,112, B,322 Sea raven, A, 34,35,41,47,48,49,57,60, short bill, see Tetrapterus angustirostris Sphryna lewini, B, 195 65, 115-117,220,221,224-225,232, 233,210,241,244,251-252,256,280, Sphyraena barracuda, B, 150,171 B, 78,92,391, see also Hemitripterus Spicara chryselis, B.,77, 103 Squalus americanus S. acanthias, A, 4,28,34,40-41,42,46, Sebastodes, A, 162 50,53,58,60, 69,73, 111, 114, 144, S. reberrimus, B, 94 Selache maxima, B, 351 150,155,227,231, B, 4,77,94,140, 142, 149, 152, 157, 162, 168, 172, Seriola S . grandis, A, 41, 46, 54 190, 195-196,205,220,224-225, S. quinquerdiata, B, 171, 191 227, 265, 268, 270, 312,319, Shad, see Alosa sapidissima 321-322,324-325,327,351,393 Sharks, B, 3.37,40,45,205,228,230,417 S. squatina, A, 4 blue, see Prionace glauca S. suckleyii, A, 58, B, 315 bull, see Carcharhinus Squatina aculeata, B, 315, 322 bullhead, see Heterodontus Stickleback, see Gasterosteus aculeatus Stizotedion lucioperca, B, 14 carpet, see Cephaloscyllium isabella great white, see Cacharodon carcharias Sturgeon, B, 29,37,45 Greenland, see Somniosus shovelnose, see Scaphirhynchus mkcrocephalus platorynchus horn, see Heterodontus francisci Sucker, A, 70, see also Catastomus catastomus lamnid, A, 20, 120, 123, 126 lemon, A, 221 white, see Catastomus commersoni leopard, see Triakis semifasciata Sunfish, see Lepomis cyanellus longfin mako, see lsurus paucus bluegill, see Lepomis macrochirus pumpkinseed, see Lepomis gibossus mako, see lsurus oxyrinchus nurse, see Ginglymostoma cirratum Swordfish, see Xiphias gladius Symphysodon, B, 32 porbeagle, A, 121-123 Port Jackson, see Heterodontus Synbranchus marmoratus, B, 150, 165, 399 portusjacksoni salmon, see Lamna ditropis Syngnathus fuscus, B, 6 shortfin mako, see Isurus oxyrhinchus tiger, see Galeocerdo cuvieri Shorthorn, see Myoxocephalus scorpius T Silurus S. glands, B,45 S. glads, B, 358 Tautog, see Tautoga onitis S . meridionalis, A, 56 Tautoga onitis, A, 229,253 Siphonostoma typhle, B, 4 Tetaptusrus audas, B, 78 Tetrapterus Skate, A, 196, B, 18,80,325 arctic, see Raja hyperborea T. albidus, A, 121 T. angustirostris, A, 121 spiny rasp, see Raja kenojei

327

SYSTEMATIC INDEX

T. audar, A, 121 T. pfEuegeri,A, 121 Thresher, see Alopias vulpinus big-eyed, see Alopias superciliosus Thunnus T. alalunga, A, 9, 14, 113-114,271,274, B, 171 T. albacares, A,4,41,46,54,59,99-100, 103, 105-106,109, 113,118, 121, B, 78 T. atlanticus, A, 121 T. maccoyii, A, 121 T. obesus, A, 4, 8, 102, 121-122, 127-128,236 T. orientalis, A, 121 T. thynnus, A, 9, 11, 16, 105, 121, 237, 239,B,4, 13 T. tonggol, A, 121 Thymalus T. arcticus, A, 114, 119 T. thymallus, B, 8, 94 Tilapia, B, 16, 34, 185, see also Sarotherodon mossambica T. mossambica, B, 160, 162 T. nilotica. B, 155, 156, 159 Tinca, B, 42 T. tinca,A,59,171,272,B,9,17-18,104, 362 Toadfish, see Torquiginer glaber oyster, see Opsanus Torpedo marmorata, B,375 Torquiginer glaber, A, 145 Trematomus newnesi, A, 229 Triakis T. semifasciata, A, 27,29,31,37,45,58, 60,67,221 T. scyllia, B, 206, 220 Trichogaster trichopterus, B, 41 Trout, A, 154,292-295, B, 185, 187, 199, 208-209,215,217,228-229,259-261, 265,272,276,280,290,319,321,362, 364,373,392-393,395,405,413,415 brook, see Salvelinus fontinalis brown, see Salmo trutta cutthroat, see Salmo clarki lake, see Salvelinus namaycush rainbow, see Oncorhynchus mykiss Tuna, A, 20, 31, 38, 43,54, 55,57,61, 69, 121, 125, 126, 129, B, 186 albacore, A, 114,121-122,124,126,129

Atlantic bluefin, see Thunnus thynnus bigeye, see Thunnus obesus black skipjack, see Euthynnus lineatus blackfin, see Thunnus atlanticus bluefin, A, 4, 120, 122, 127 bullet, see Auxis rochei frigate, see Auxis thazard kawakawa, see Euthynnus affinis little tunny, see Euthynnus allatteratus longtail, see Thunnus tonggol Pacific bluefin, see Thunnus orientalis skipjack, see Katsuwonus pelamis southern bluefin, see Thunnus maccoyii yellowfin, see Thunnus albacares Turbot, A, 100, see Scophthalmus maximum U

Uranoscopus scaber, A, 142 Urolophus sp. , B, 86 V

Valencienellus tripunctatus, B, 5 Vinciguerria, B, 5

w Wels, see Silurus glanis Whiting, see Gadus merlangus Wolf fish, see Anarhichas lupis, B, 181 X

Xenacanthus, A, 160 Xiphias gladius, A, 9, 20, 104, 120, 122, 127

Z Zauo platypus, B, 221 Zebra danio, A, 3 Zebra fish, see Zebra danio Zoarces viviparous, A, 46, 49, 229, 242

This Page Intentionally Left Blank

SUBJECT INDEX Boldface A refers to entries in Volume XIIA; B refers to entries in Volume XIIB. Acronyms that occur in the text are indexed and identified by a cross reference. A

AADC, see Amino acid decarboxylase ACE, see Angiotensin converting enzyme Action potential (AP) duration of (APD), A, 278-282 in heart muscle, A, 278-282 Actomyosin ATPase, in myofibrils, A, 271-273 Adrenaline, B, 256-257, 264, see also Catecholamines Adenosine 5-triphosphate (ATP), catabolism of, B, 307 Afferent branchials, see Blood vessels Amino acid decarboxylase, B, 256-257 Anaerobiosis, and cardiac metabolism, A, 245-246 Anaphylaxis, B, 33-34 AChE, see Enzymes, in plasma ACTH, see Adrenocorticotropic hormone Adrenocorticotropic hormone, B, 62, 71 AFPIAFGP, see Antifreeze proteins Albumin spaces, in various tissues, B, 178 Alk-Pase, see Enzymes, in plasma Alkyldiacylglycerol, B, 98 Amino acids in blood, B, 80-83 essential (EAA), B, 80-81 tabulation of, B, 82-83 Ammonia, in fish blood, B, 84-85 Androgen, B, 63, 75 ANF (atrial natriuretic factor), B, 61, see also Atrial natriuretic peptides ANC I, ANG 11, see Angiotensins

Angiotensin converting enzyme (ACE), B, 193-212 Angiotensins, B, 193-212 ANP, see Atrial natriuretic peptides Antibody producing cells, B, 30-31 Antifreeze proteins, B, 91-92 AP, APD, see Action potential Arginine vasotocin plasma levels, B, 67 and renin secretion, B, 204 Arterial system, A, 89-139 pattern of, 89-91 Arterioarterial anastomoses, A, 188, 189, 196, 198 Arteriosclerotic lesions, see Coronary circulation, lesions in Atrial natriuretic peptides (ANP), B, 217-231 cardiac effects, B, 228-229 cardiovascular effects, B, 225-228 on chloride cells, B, 230 distribution of, B, 219-223 families of, B, 217 mechanism of action, B, 231 and osmoregulation, B, 225 and rectal gland, B, 219, 224, 230 renal effects, B, 229-230 salinity and, B, 221-224 structures of, B, 218 and volume expansion, B, 224-225 Atriopeptin, B, 61 Atrium anatomy, A, 3-5 filling of, A, 36, 40-44 pressure in, A, 97

329

330

SUBJECT INDEX

Autonomic innervation, of systemic vasculature, A, 111, 114, 115, 116 Auxillary body, B, 347, 348 AVT, see Arginine vasotocin

B

Baroreceptors, B, 401-402 Basophils, see Granulocytes Bile pigments, B, 79-80 Bilirubin, B, 80, 89 Biliverdin, B, 80 Blaschko pathway, B, 256-260 Blood, see also Blood chemistry chemical properties, B, 55-133 in different tissues, B, 174-179 plasma space (PS), B, 166 plasma volume, B, 190 red cell space (RCS), B, 166, 177-178 total volume (TBV), B, 166-174 venous capacitance, B, 188-190 volume and pressure, B, 186-188 Blood cells, B, 1-54, see also various cell types Blood chemistry die1 cycles in, B, 58-60, 68 sampling methods, B, 57-58 Blood flow catecholamines and, A, 117, 118, 119 to different organs, A, 114-116, 117 exercise and, A, 116-118 hypoxia and, A, 118-120 metabolites and, A, 118 physical factors and, A, 91-96 and viscosity, A, 92-96 Blood pressure blood volume and, B, 186-188 dorsal aortic, A, 70 and U S , B, 204-212 and ventilation, B, 380 ventral aortic, A, 61 Blood vessels afferent branchials, A, 89 dorsal aorta, A, 89 efferent branchials, A, 89 elasticity of, A, 91 pattern of, A, 89-91 rete mirabile, A, 90 ventral aorta, A, 89

Blood volume, see also Plasma volume determinants of, B, 179-191,204-212 regulation of, B, 166-193, 204 Blood-brain barrier, A, 146; B, 174,282, 283 BM, see Bombesin Bombesin, B, 314,321-324 Bovine serum albumin, B, 176 Bradykinin, B, 213, 214, 216 Brain, see Central nervous system Branchial innervation, B, 392 Branchial pump, A, 160-161 Branchial vasculature, B, 303, 305, 306, 331 innervation of, B, 313 BSA, see Bovine serum albumin Bulbus arteriosus distensibility, A, 100 hypoxia and, A, 103 pharmacological agents on, A, 103 pressure-volume for, A, 99 role of, A, 99-105 C

CA, see Catecholamines Caerulein, B, 330-331 Calcitonin, B, 63, 69-70 Calcium, see also Electrolytes Ca2+ ATPase in SL, A, 292 in cardiac contraction, A, 268-269, 273-277 delivery to myofibrils, A, 278-284 release from SR, A, 288-291 transsarcolemmal influx, A, 284-286 Capillaries, A, 143-150 colloid osmotic pressure (COP), A, 147-148 diffusional permeability, A, 147 fenestrated, A, 148 physiology of, A, 147-150 pinocytic vesicles in, A, 146-147 retial, A, 145, 146 structure of, A, 143-147 in suprabranchial chamber, A, 145 Carbonic anhydrase, B, 10, 14,277,284, 285 Cardiac contractility, A, 26-29, see also Heart calcium and, A, 29

SUBJECT INDEX

inotropic and chronotropic effects, A, 29 neural control, A, 27 pharmacological agents on, A, 26-29 Cardiac metabolism dysoxic conditions and, A, 238-250 energy demands and supply, A, 220-222,242-243 enzyme activity levels, A, 225-231 fuel of metabolism, A, 223-236 hypoxic conditions and, A, 245-246 in isolated preparations, A, 243-245 and temperature, A, 250-257 Cardiac morphometrics, tabulation, A, 4-5 Cardiac nerves, qrigin of, B, 306-307 Cardiac output (Q), A, 55-69, 90,95 acidosis and, A, 62-65 activity and, A, 57-62 body mass and, A, 56 calcium and, A, 64-65 defined, A, 36.55 exercise and, A, 112-113 hypoxia and, A, 65-68, 112-1 13, 118 measurement of, A, 55-56 temperature and, A, 60-62 to various organs, A, 114-116 Cardiac performance myoglobin and, A, 239-242 Cardiac stroke work, A, 29-33 Cardinal heart (myxinoids), A, 176-177 Cardiorespiratory interaction, B, 371-375 and synchrony, B, 375-381 Cardiovascular regulation and 5-HT, B, 301-302,311-317 and neuropeptides, B, 301-302, 317-331 and purines, B, 301-302,307-311 Cardiovascular system, see also Heart anatomy of, A, 1-24 physiology of, A, 24-73 Cardioventilatory control afferent input, B, 389426 central sensory areas of, B, 419-420 reflexes, B, 402 Carnitine palmitoyl (CPT), in cardiac metabolism, A, 225-230, 235, 236, 253,257 Catecholamines, B, 63,71-72,74-75 adrenaline, B, 63, 71 biological halftime, B, 272 biosynthesis, B, 256-260 and blood distribution, B, 283-284

331 blood volume and, B, 191 and cardiac output, A, 64, 66 and cardiac rate, A, 48-53 circulatory leveIs, B, 263-267 and CO, transport, B, 284-287 control of release, B, 269-272 degradation of, B, 260-263 diurnallseasonal effects, B, 287-290 exercise and, B, 271.279, 284 on gill ventilation, B, 279-283 hypoxia and, B, 288-290 ion movement, B, 283 metabolism of, B, 255-263 and neuropeptide Y, B, 326 noradrenaline, B, 63, 71 on 0, exchange, B, 276-283 physiological effects, B, 275-287 plasma clearance, B, 272-275 and renin secretion, B, 203-205,207 sources of, B, 267-269 stress and, B, 265467,271-273 uptake of, B, 274 on venous capacitance, B, 189 Carnitine, A, 232 Carotid labyrinth, B, 401, 402 Catechol-0-methyl transferase, B, 260-262 Caudal heart, A, 171-177 of carpet shark, A, 168-171 of eel, A, 171-173 of hagfish, A, 172-175 and secondary vascular system, A, 192, 200,209 Caudal urophysis, see Urophysis CCK, see Gastrin/Cholecystokinin Central nervous system cranial nerve nuclei of, B, 350-360 respiratory motor nuclei, B, 352-354 vagal motor column, B, 352-354,360 Central respiratory pattern generator, B, 366 Central rhythm generator, B, 374 Central venous sinus, A, 187, 208 Chemoreceptors, see ulso Nociceptors branchial 0, sensitivity, B, 405-410 carotid, B, 372 COslpH receptors, B, 414-416 effects of hypoxia, B, 413 glomus cells, B, 411-412 and heart rate, B, 371-372 O2 receptors, B, 404-414

332 pharmacological agents on, B, 413-414 receptors), B, 411-414 transduction (0, Cholesterol in blood, B, 98,99-101 migration and spawning, B, 100-101 ChromafXn cells, B, 63, 71,256,257,259, 265,268,269,347 in heart, A, 19,50-51 Chylomicrons, A, 210; B, 97,99 Circulation, see Arterial system and Venous system Citrate synthesis (CS), in heart metabolism, A, 226-230,235,237,254 Coagulation of blood, A, 35-36, 44 Colloid osmotic pressures, B, 180-185 plasma proteins and, B, 182-183 Compacta, see Ventricle Complement (C), B, 32-33 Compliance, A, 102-104,106; B, 189-191 COMT, see Catechol-0-methyl transferase Conus arteriosus ECG of, A, 98 pressure in, A, 97 role of, A, 96-98 Conus and bulbus arteriosus, description of, A, 16-20 Coronary circulation, A, 6, 9 anatomy of, A, 20-24 catecholamines and, A, 71-72 control of, A, 70-73 evolution of, A, 21 and hypoxia, A, 65 lesions in, A, 21-24 prostaglandin and, A, 72 purines and, A, 72 Cortisol, B, 63,70-71, 75 Cough reflex, B, 417 Counter current heat exchange, see Heat exchange systems Countercurrent retial systems, see Heat exchange systems CPG, see Central respiratory pattern generator CPK, see Enzymes, in plasma CP, see Creatine phosphate CPT, see Carnitine palmitoyl CreatineICreatinine, B, 86-87 Creatine phosphate, A, 243-244,250 CRG, see Central rhythm generator CS, see Citrate synthesis

SUBJECT INDEX CT, see Calcitonin Cushing reflex, B, 403-404 CVM, cardiac vagal motoneurons, B, 359 CVS, see Central venous sinus Cytochrome oxidase, in cardiac metabolism, A, 226,230,237,246,257 Cyt Ox, see Cytochrome oxidase D

DA, see Dopamine DBH, see Dopamine-P-hydroxylase Defense receptors, see Nociceptors DHPR, see Dihydropyridine receptors Dihydroxyphenylalanine (DOPA), B, 256-257 Dihydropyridine receptors (DHPR), in sarcolemma, A, 284-286 DOPA, see Dihydroxyphenylalanine Dopamine (DA), B, 256,257, see also Catecholamines Dopamine-F-hydroxylase, B, 258-260 Dorsal aorta pressure, A, 112-113 pressure-flow relations, A, 105-110 pressure-volume curves, A, 99,103,106 Dorsal vagal motoneuron, B, 354 Drinking, angiotensins and, B,210-212 DVN, see Dorsal vagal motoneuron E

EAA, see Amino acids E-C coupling, see Excitation-contraction coupling ECFV, see Water, extracellular ECG, see Electrocardiogram EDRF, see Endothelium-derived relaxing factor Efferent branchials, see Blood vessels Electrocardiogram (ECG), A, 24-26 Electrolytes in plasma, B, 106-113 and pollutants, B, 110-111 table of blood, B, 108-109 Endocardium, A, 6 , 8 metabolism of, A, 236-238 Endothelial cells, A, 143-147, see also Capillaries

333

SUBJECT INDEX

contractile filaments of, A, 149 secretion of, A, 150 structure of, A, 144 Endothelial factors, and vascular reactions, B, 331-332 Endothelium-derived relaxing factor (EDRF), A, 150; B, 307,311,317, 332 Endothermy, and heat exchangers, A, 120 Enteramine, see 5-Hydroxytryptamine Enterochromafin cells, B, 308,311, 312 Enzymes in cardiac metabolism, A, 224-231, 236-238,245-250,252-257 in plasma, B, 92-96 Eosinophils, see Granulocytes Epicardium, A, 6, 8 metabolism of, A, 236-238 Epigonal organ, B, 39 Erythroblasts, B, 8 Erythrocytes, see also Erythropoiesis cell membrane, B, 11-13 coagulation of, B, 35-36 gas transport by, B, 13-14 hemoglobin content, B, 4 immature forms, B, 8 metabolism of, B, 9 morphology, B, 3-8 nonnucleated, B, 5 nucleoside phosphates in, B, 11, 12 numbers of, B, 3-4 permeability of, B, 12-13 pH regulation, B, 278 physiology and biochemistry, B, 8-14 sedimentation rate, B, 34 Erythroplastids, B, 5 Erythropoiesis, B, 9, 39-42, see also Erythrocytes Erythropoietin, B, 41,43 Estrogen, B, 63,65,74 Ethanol, in blood, B, 78 Excitation-contraction coupling general scheme of, A, 268-270 humoral factors and, A, 282-284 temperature and, A, 282-283 Exercise and blood flow, A, 154-155 and blood volume, B, 191-192 Extracellular fiuids exercise and, B, 164 muscle fluid volumes, B, 157-160

photoperiod, pH, Pot, B, 164-165 salinity and, B, 161-163 stress and, B, 165-166 table of volumes, B, 149-156 volume regulation, B, 143-193 Extracellular space, B, 147

F Fatty acids free, B, 79 in heart metabolism, A, 223-236, 252, 257 Fick equation, A, 56 F i n pumps (venous), B, 166-168 Fluid compartments, B, 137 Frank-Starling mechanism, A, 37,39-40, 69; B, 344

G Galanin, B, 328-330 Gas gland, A, 145 Gastrin/Cholecystokinin (CCK), B, 330-331 Gastrin-releasing peptide, B, 321-324 GDH, see Enzymes, in plasma GFR, see Glomerular filtration rate GH, see Growth hormone Gills, vasomotor innervation, B, 348 GK, see Kallikrein, glandular Glomerular filtration rate, B, 195,205-206, 208,210 GLU, see Glucagon Glucagon, B, 63,72-73 Glucagon-like peptide, B, 63,72-73 Glucose, in cardiac metabolism, A, 223-236 GLP, see Glucagon-like peptide Glycogen, B, 76 in cardiac metabolism, A, 243,246 Glycogenolysis, A, 246, 250 Glycolysis, A, 250 Glucose, in blood, B, 76-77 Glycerol, B, 104-105 Gonadotropins (GtH), plasma levels, B, 61-65

334

SUBJECT INDEX

GOT, see Enzymes, in plasma GPT, see Enzymes, in plasma Granulocytes, B, 2, 16-22, 44 basophils, B, 18 eosinophils, B, 17, 21, 22 heterophils, B, 16, 17, 18 neutrophils, B, 16, 18 polymorphonuclears, B, 16 Growth hormone (GH), plasma levels, B, 65-66 GRP, see Gastrin-releasing peptide GtH, see Gonadotropin Gut hormones, B, 317-318

H

HBDH, see Enzymes, in plasma Hct, see Hematocrit Head kidney (Pronephros), B, 38,71,256 Heart, see also Cardiac contractility, Myocardial relaxation, Myocytes, M yofibrils adrenergic control, A, 49-52 anatomy, A, 2-24 atrial filling, A, 40-44 cardiac cycle, A, 24 cardiac filling, A, 40,41-45 cholinergic control, A, 48-49 chromaffin tissue in, A, 50-51 circulation (coronary), A, 20-24 efficiency of contraction, A, 33-36 electrical events, A, 24-26 enzymes of muscle, A, 224-231, 252-257 epicardium and endocarcium, A, 236-238 excitation-contraction coupling, A, 267-304 innervation, A, 12-13; B, 344-350 metabolism of, A, 219-266 morphometrics (tabled), A, 4-5 myocytes of, A, 13-16,26,27 myoglobin in, A, 16,55 nervous regulation, A, 282; B, 343-387 0,supply, A, 34-36, 66-73,222,225 pacemaker, A, 45,278,282; B,346,360 performance, A, 238 physiology of, A, 24-73

stroke work and volume, A, 29-33, 36-39 temperature and size effects, A, 253-254 valves of, A, 11 ventricular filling, A, 44-45 Heart rate, see also Heart body mass and, A, 53-54 calcium and, A, 54,55 control of, A, 48-52 exercise and, A, 52 hypoxia and, A, 51,53 intrinsic rate, A, 45-48 maximal rate, A, 53-55 modulation of, B, 371-375 nervous control, A, 45-53 neuropeptides and, A, 53 pharmacological agents on, A, 45-53 stretch effects, A, 52-53 Heat exchange systems, A, 90,120-130 anatomy of, A, 120-123 blood flow in, A, 123-130 diagram of, A, 122 efficiency of, A, 124-126 occurrence of, A, 120-123 Hemal arch pump, A, 158-160 Hematocrit, B,3-4,167,173,174,178,179, 191 and hemodynamics, A, 92-96 large vessel (LVH),B, 167 optimal, A, 92-94 in secondary vascular system, A, 208 Hemoglobin, types of, B, 10 Hemopoiesis, B, 39-42, 43 organs of, B, 37-38 in peripheral blood, B, 41-42 stimulation of, B, 41 Hemosiderin, B, 9, 39 Heparin, B, 22, 36 Hepatic portal system, A, 162-164 pressures in, A, 162, 163 Hepatic sphincter, A, 155 Hexokinase (HK), in cardiac metabolism, A, 225-231,235,237,246-247,250, 253 High energy phosphates, in heart muscle, A, 219,224 Hindbrain, see Central nervous system HK, see Hexokinase HOAD, see 3-Hydroxylacyl CoA dehydrogenase

SUBJECT INDEX

Homeometric regulation, A, 38, 39, 96 Hormones, see also various hormones molecular weights, B, 62-63 plasma levels, B,60-75 tabulation of, B, 62-63 HRVS, Heart rate variability signal, B, 364-365 5-HT, see 5-H ydroxytryptamine Hydraulic pressure (Pt), in determining blood volume, B, 182 3-Hydroxylacyl CoA dehydrogenase (HOAD), in cardiac metabolism, A, 226-230,236,237,253,257 5-Hydroxytryptamine, B, 302 on cardiovascular system, B, 315 in fish tissues, B, 311-317 source of, B, 311 Hypocalcin, see Stanniocalcin Hypophysiovelar sinus, A, 176-177 Hypoxia bradycardia and, A, 238 cardiac response to, B, 360-365 Hysteresis, A, 100, 106 I ICFV, see Water, intracellular Immune responses, B, 27,29-34 Immunoglobulins, B, 31, 32 Impedance (vascular), A, 106, 107, 108 Inflammation, B, 34-35 Insulin (INS), B, 63,72-73 Ions, in blood, see Electrolytes J

Jacob-Stewart cycle, B, 277 Juxtaglomerular cells (JG), B, 194-196 K

Kallidin, B, 213 Kallikrein glandular (GK), B, 214-215 inhibitor (PPAMCK),B, 215 Kallikrein-kinin system, B, 213-217

Ketones, B, 78-79 in cardiac metabolism, A, 224,236 11-Ketotestosterone (KT), B, 63, 64 Kinins, B, 213,214,216 KKS, see KaIlikrein-kinin system KT, see 11-Ketotestosterone L

Lactate in blood, B, 77-78 in cardiac metabolism, A, 233,235,243, 246 Lactate dehydrogenase (LDH), in cardiac metabolism, A, 223, 227-229, 235, 236,247,249-250 Lacteals, A, 210 LAP, see Enzymes, in plasma Lateral vagal motonucleus, B, 354 LDH, see Lactate dehydrogenase LDLIHDL, see Lipoproteins, in blood Leucocytes, B,2 biochemistry of, B, 26-36 blast cells, B, 26 classification of, B, 15-16 granulocytes, B, 16-22 homeostasis of, B, 26-27 lymphocytes and plasma cells, B, 23-24 macrophages, B, 24 mast cells, B, 22-23 monocytes, B, 24 physiology of, B, 26-36 spindle cells, B, 24 staining methods, B, 15 Leydig organ, B, 32,39 Lipids in blood, B, 96-105 total, B, 96 Lipoproteins, in blood, B, 97-99 L-type Caz+ channels, in sarcolemma, A, 284-286 LVH, see Hematocrit LVN, see Lateral vagal motonucleus Lymph pumps, A, 212 Lymphatics, A, 186,192,193,196, see also Secondary vascular system evolution of, A, 211-213 red, A, 194, 195,212 white, A, 195, 212

336

SUBJECT INDEX

Lymphatic system, see Secondary vascular system Lymphocytes, B, 23-24, see also Leucocytes antibody producing cells, B, 30 functions of, B, 29-34 infiltrations of, B, 38-39 killer cells (NK),B, 30 plasma cells and, B, 30 rosette complexes, B, 31 types of, B, 29-30,43 Lymphomyeloid tissue, B, 24, 25, 36-39 Lysozyme, B, 28

Myocytes, see olso Heart, A, 13-16,26 electrophysiology of, A, 286-287 M yofibrils calcium delivery to, A, 268-269, 278-284 contractile proteins, A, 270-273 troponin in, A, 273,276,277 ultrastructure of, A, 270-271 Myoglobin, A, 16,55,67,68,239-242,256 Myosins, A, 271-272 N

NaC-Ca2 exchange in myocyte activity, A, 288-296 temperature and pH on, A, 293-295 Nicotinamide adenine dinucleotide (NADH), A, 233 Nonadrenergic, noncholinergic transmitters (NANC),B, 302 NEFA, see Nonesterified fatty acids Neuropeptide Y, B, 324-326 Neuropeptides, B, 317-331 Neurotransmitters, in perivascular nerves (table), B, 314 Neutrophils (heterophils), see Granulocytes Nociceptors, B, 416-420 in air breathers, B, 418-419 chemical irritants, B, 417-418 mechanical trauma, B, 416-417 Nonesterified fatty acids (NEFA) in blood, B, 101-104 starvation and, B, 103 Nonprotein nitrogen, in blood, B, 80-87 Noradrenaline, B, 256, 257, 264, see also Catecholamines NPY, see Neuropeptide Y +

M

Macula densa, B, 194, 195 Magnesium, see Electrolytes MAO, see Monoamine oxidase Mast cells, B, 22-23, 33 Mechanoreceptors, see also Nociceptors and Proprioceptors in air breathing organs, B, 397-400 arterial, B, 401-402 of gill filaments, B, 391-393 of gill rakers, B, 393-395 and heart rate, B, 372,380 intracardiac, B, 402-403 intracranial, B, 403-404 orobranchial, B, 397 Median fin pumps, A, 166-167 Melanomacrophage centers, B, 9, 39 Melatonin (MLT), B, 62, 67-68 Metabolites, see also different substances plasma levels of, B, 76-80 Metanephrine (MN), B, 261-262 3-Methoxy-4-hydroxyphenyl glycol (MOPEG), B, 261,262 Microvilli, A, 198,200 Mitochondria, cardiac, A, 231-232, 237 MLT, see Melatonin MN, see Metanephrine Monoamine oxidase (MAO), B, 260-263 MOPEG, see 3-Methoxy-4-hydroxyphenyl glycol Muscle extracellular volume, B, 157-160 intracellular fluids, B, 161 Myocardial relaxation, A, 290-296

0 Obex, B, 356, 357, 359 Oncotic pressures, see Colloid osmotic pressures Opsonins, B, 31 Ornithine-urea cycle, B, 85 Osmolarity, B, 106-110 Osmotic fragility (red cells), B, 13

337

SUBJECT INDEX Oxygen myocardial supply, A, 67-73 transport capacity, A, 94 P

Pacemaker activity, see also Heart modulating, A, 45 neurons of, B, 374 stretch effect, A, 52 Pancreas, hormones of, B, 63, 72-73 Pancreatic peptide (PP), B, 72 PAS-positive granulocytes, B, 22-23 PEG, see Polyethylene glycol PEP, see Phosphoenolpyruvate Pericardium, A, 2 anatomy, A, 41, 43 and atrial filling, A, 36-37,40-41 pressures in, A, 41, 43-45, 97, 98 and venous return, A, 177 Peripheral resistance branchial, A, 110-114 total (TPR), A, 110, 112-113 PFK, see Phosphofructokinase Phagocytosis, B, 27-28 Phen ylethanolamine-N-methyl transferase (PNMT), B, 257-260 Phosphate, see Electrolytes Phosphoenolpyruvate (PEP), in cardiac metabolism, A, 225 Phosphofructokinase (PFK), in cardiac metabolism, A, 225-231,235,237, 247-249 Pinocytotic vesicles, A, 146-147 PK, see Pyruvate kinase Plasma cells, B, 24, 30, 32, 43, see also Leucocytes Plasma proteins, B, 87-96 albumin, B, 88-89 antifreeze, B, 91-92 capillary exchanges of, A, 147-148 hormone binding by, B, 90-91 immunoglobulins, 8, 89-90 Plasma renin activity (PRA), B, 200-203 Plasma skimming, A, 195, 198, 206, 207, 208,210 Plasma volume, B, 190 PNMT, see Phenylethanolamine-Nmethyl transferase

Poiseuille’s Law, A, 91-92 Polyethylene glycol, B, 145, 148 Polyvinylpyrrolidone, B, 176 Portal heart, A, 163-164 Potassium, see Electrolytes PP, see Pancreatic peptide, B, 72 PPAMCK, see Kallikrein, inhibitor PRA, see Plasma renin activity Pressure-volume loops, A, 29-31 PRL, see Prolactin Proerythrocytes, B, 8 Propulsor (venous blood), A, 160 Prolactin (PRL), B, 66 Proprioceptors, see also Mechanoreceptors in air breathers, B, 400 of gill arch, B, 395-396, 397 opercular, B, 397,398 Progestogen, B, 64,74 Purinergic nerves, B, 308-310, 311 Purines and cardiovascular control, B, 307-31 1 on heart, B, 309-310 and nerve transmission, B, 309 on vasculature, B, 310 PVP, see Polyvinylpyrrolidone Pyruvate kinase (PK), in cardiac metabolism, A, 225-229,237,249

Q QlO

of cardiac enzymes, A, 252-253,257 of heart rate, A, 47-48 in myocyte excitation, A, 287, 297 Q,see Cardiac output

R

Radioimmunoassays, B, 61 Ram ventilation, and heart rate, B, 373 Rapid cooling contracture, A, 290,291 RAS, see Renin-angioteisin system RCC, see Rapid cooling contracture Red blood cells, see Erythrocytes Reflection coefficient (m),B, 180-181 Renal portal system, A, 161-162

338

SUBJECT INDEX

Renal portal veins, A, 158,159,160, 161-162 Renin, B, 193, 195 Renin-angiotensin system, B,61,193-212 activating stimuli, B, 200-204 components of, B, 193-194 corticosteroid secretion and, B,212 effects of, B, 204-212 occurrence in fish, B, 194-199 Respiratory muscles, innervation of, B, 346 Retia mirabile, A, 90, 121-123, 145 Reticulo-endothelial system (RES),B, 25 Reticulocytes, B, 8 RIA, see Radioimmunoassays RVM, Respiratory vagal motoneurones, B, 366 S

Salinity, effects on body water, B,161-163 Sarcolemma (SL),of myofibrils, A, 268,270 Sarcoplasmic reticulum, A, 268, 270, 296 Secondary vascular system, A, 185-217 in Cyclostomes, A, 192-195 in Elasmobranchs, A, 195-196 evolution of, A, 211-213 exchanges with primary, A, 204-206 functions of, A, 209-211 morphology of, A, 187-196 pressures in, A, 208-209 in teleosts, A, 187-192 volume of, A, 202-205 Serotonin, see 5-Hydroxytryptamine Sex steroids, plasma levels of, B, 61-65, 74-75 Sexual maturation, and coronary lesions, A, 23 Shear rate,A, 92-93 Single nephrun filtration rate, B, 208 Sinus intestinalis, A, 164 Sinusoids, liver, A, 149 Sinus venoms, A, 2-3 SL, see Sarcolemma Smoltification, A, 8 hormone changes of, B, 66,67,69,71,73 lipids in, B, 104, 105 SNGFR, see Single nephron filtration rate Somatostatin (SST), B, 63, 72, 326-327

Spindle cells, B, 24.25-26 Spleen, B, 37 erythrocyte release, A, 95; B, 279 Spongiosa, see Ventricle Squalene, B, 98 SR, see Sarcoplasmic reticulum SR CaZt (sarcoplasmic release ofca"), see Calcium Stanniocalcin (STC), B, 63-73 Stannius corpuscles, renin-like activity of, B, 197-198 Starling curve, A, 39-43; B, 180 Starling principle, A, 147-148 STC, see Stanniocalcin Stress, A, 117; B, 75 in blood sampling, B, 57-58 control of, A, 36-45 electrolytes and, B, 111 fatty acids in, B, 104 glucose in, B, 76 hormone effects of, B, 67, 70-71,72 Stroke volume, A, 36-45,55,61,102 Subcutaneous sinus, A, 173-175; B, 173 Substance P, B, 327-329 Sulfate, see Electrolytes Suprarenal bodies, B, 347-348 Swimbladder, retial capillaries of, A, 145 Systemic resistance (Rs), see also Peripheral resistance autonomic innervation and, A, 111 exercise and, A, 112-113 hypoxia and, A, 112-113 T

T, see Testosterone T3, T4, see Thyroid hormone Tachykinins, B, 317,327,328 TBV, see Blood, total volume TBW, see Water, total body Teleocalcin, see Stanniocalcin Temperature, see also Qlo and blood volume, B, 191 body muscle, A, 128-129 cardiac metabolism and, A, 251-257 cardiac output and, A, 60, 62, 66 cardiac performance, A, 250-256 contraction rate, A, 28-29 contracture and, A, 290,291

SUBJECT INDEX

and ECG, A, 26 myocardial relaxation and, A, 293-296 and myofibrillar contraction, A, 274-276,282-283 and myofibrillar proteins, A, 272-273 on pacemaker, A, 45 on vagal tone, B, 360-365 and ventricular mass, A, 12 Testosterone (T), B, 64,74 TG, see Triglycerides Thebesian system, A, 21 Thennoregulation, A, 120, 126-130 Thrombocytes, B, 23,25-26,35-36 Thymus, B, 37,43 Thyroid hormones plasma levels of, B, 68-69, 75 thyroxine (TJ. B,62,68-69, 91 triiodothyronine (T3),B, 62, 68-69, 91 Time to peak tension (TPT), in muscle contraction, A, 279-282 TMAO, see Trimethylamine oxide Tn, TnC, TnI, see Myofibrils, troponin TMW, see Water, total muscle Toxicity (metals), and erythropoiesis, B,42 TPR, see Peripheral resistance TPT, see Time to peak tension Triglycerides, B, 104-105 Trimethylamine oxide, B, 86, 106, 139 Troponin, A, 273,276,277,297, see ulso M yofibrils T-tubules, A, 270, 288, 293, 296

U UI and UII, see Urotensins Urea, B, 85, 86, 106, 108-109, 139 Urophysis, A, 161, 173; B, 63 Urotensins, A, 161, 172; B, 63, 74

V

Vaccination, B, 34 Vagus nerve efferent activity (cardiac), B, 365-370 hindbrain nucleus, B, 350-360 vagal tone, B, 360-365

Valves ostial, A, 156, 157, 159, 160, 161, 177 parietal, A, 156, 176, 179 of veins, A, 156-157 Vanillymandelic acid (VMA), B, 261-262 Vascular compliance, B, 189-191 Vascular resistance, A, 92, see also Peripheral resistance Vascular tone, A, 110 Vasoactive intestinal peptide, B, 314, 318-321 Vasomotor nerves, origin of, B, 303-306 Veins capacitance of, A, 153-157, 188-191 compliance of wall, A, 154-156 hepatic portal, A, 162-164 intercostal, A, 159 renal portal, A, 158,161-162 of skin, A, 165-166 somatic system, A, 157-162 structure of, A, 150-151 valves of, A, 156-157 venous pressures, A, 151-153 venous pumps, A, 151,158-178 Venae circulares, A, 166, 167 Venous system, A, 141-183, see ulso Veins Ventilation cardiac rhythm and, B, 375-381 hypercapnia on, B, 415-416 rate of, B, 378 Ventral aorta, pressure in, A, 97, 99, 103, 106, 112-113 Ventricle anatomy, A, 5-12 fiber architecture, A, 10-11 pressure in, A, 97 relative mass, A, 11-12, 15 spongiosa and compacta, A, 2-11 types of,A, 7 Vis-a-fronte, A, 36-37,4044 Viscosity of blood, A, 92-96 hematocrit and, A, 92-93 temperature and, A, 92-93 Vis-8-tergo, A, 36-37, 40-44 Vitellogenin, B, 98-99 VLDL (very low density lipids), see Lipoproteins VIP, see Vasoactive intestinal peptide VMA, see Vanillylmandelic acid Volume regulation, B, 136-193

340

SUBJECT INDEX

W

Water extracellular (ECFV), B, 143-161 intracellular (ECFV), B, 141-143 metabolism, B, 138 salinity on ECFV, B, 161-163

total body (TBW), B, 137-141 total muscle (TMW), B, 162 WBH, see Hematocrit Weissadern, A, 186, 187 White blood cells, B, 14-36, see also Leucocytes Windkessel, A, 91, 102,106,108,109, 110