Fish Physiology: Part B : The Cardiovascular System

FISH PHYSIOLOGY VOLUME X I I , Part B The Cardiovascular System

CONTRIBUTORS MARK L. BURLESON RAGNAR FANGE SUSANNE HOLMGREN D. G. McDONALD C . L. MILLIGAN WILLIAM K. MILSOM

STEFAN NILSSON KENNETH R. OLSON S. F. PERRY D . J. RANDALL NEAL J. SMATRESK E. W. TAYLOR

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

D. J. R A N D A L L DEPARTMENT OF ZOOLOGY THE UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, BRITISH COLUMBIA, CANADA

A. P. F A R R E L L DEPARTMENT OF BIOLOGICAL SCIENCES SIMON FRASER UNIVERSITY BURNABY, BRITISH COLUMBIA, CANADA

VOLUME X I I , Part B The Cardiovascular System

A C A D E M I C PRESS, INC. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London

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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, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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Academic Press Limited 24-28 Oval Road, London NWl 7DX

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. 11. Conte, Frank P., date. 111. Title. QL639.1.H6 597.01 76-84233 ISBN 0-12-350436-8 (v. 12B) CIP PRINTED IN THE UNITED STATES OF AMERICA 92 93 94

95 96 91

MV

9

8 1 6 5 4 3 2

1

CONTENTS CONTENTS OF PARTA

ix

CONTRIBUTORS

xi ...

Xlll

PREFACE

xvii

OF OTHERVOLUMES CONTENTS

Fish Blood Cells Ragnar Fange

1.

I. Introduction

11. Red Cells: Morphology 111. Red Cells: Physiology and Biochemistry IV. White Cells: Morphology V. White Cells: Physiology and Biochemistry VI. Lymphomyeloid Tissues VII. Hemopoiesis VIII. Future Research References

2.

2 3 8 14 26 36 39 42 46

Chemical Properties of the Blood D. G. McDonald and C . L. Milligan

I. Introduction 11. Hormones: Teleosts 111. Hormones: Cyclostomes and Chrondricthyes IV. Metabolites V. Nonprotein Nitrogenous Compounds VI. Plasma Proteins VII. Lipids VIII. Electrolytes References V

56 60 74 76 80 87 96 106 113

vi

CONTENTS

Blood and Extracellular Fluid Volume Regulation: Role of the Renin-Angiotensin System, Kallikrein-Kinin System, and Atrial Natriuretic Peptides Kenneth R. Olson

3.

I. Introduction 11. Fluid Compartments

111. IV. V. VI.

Renin-Angiotensin System Kallikrein-Kinin System Atrial Natriuretic Peptides Summary References

136 136 193 213 217 23 1 232

4. Catecholamines D. J. Randall and S. F . Perry I. Catecholamine Metabolism 11. Control of Blood Catecholamine Levels 111. Actions of Circulating Catecholamines IV. Factors Influencing Actions of Catecholamines References

5.

Cardiovascular Control by Purines, 5-Hydroxytryptamine, and Neuropeptides Stefan Nilsson and Susanne Holmgren

I. Introduction 11. Origin of Vasomotor and Cardiac Nerves 111. Purines IV. 5-Hydroxytryptamine (Serotonin) V. Neuropeptides VI. Endothelial Factors References

6.

255 263 275 287 290

30 1 303 307 311 317 33 1 333

Nervous Control of the Heart and Cardiorespiratory Interactions E . W. Taylor

I. Introduction 11. Innervation of the Heart 111. The Central Location of Vagal Preganglionic Neurones

343 344 3.50

CONTENTS

IV. Control of the Heart and Branchial Circulation V. Cardiorespiratory Interactions VI. Cardiorespiratory Synchrony References

7.

vii 360 37 1 375 38 1

Afferent Inputs Associated with Cardioventilatory Control in Fish Mark L. Burleson, Neal J . Smatresk, and William K . Milsom

I. Introduction 11. Mechanoreceptors 111. Chemoreceptors IV. Nociceptors V. Central Projections of Sensory Neurons References

390 390 404 416 419 420

AUTHORINDEX

427

SYSTEMATIC INDEX

453

SUBJECTINDEX

463

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CONTENTS OF PART A The Heart Anthony P . Farrell and David R . Jones The Arterial System P . G . Busnell, David R.Jones, and Anthony P . Farrell The Venous System Geoffrey H. Satchel1 The Secondary Vascular System J. F . Steffensen andJ. P. Lornholt Cardiac Energy Metabolism William R. Driedzic Excitation-Contraction Coupling in the Teleost Heart Glen F . Tibbits, Christopher D . Moyes, and Leif Hove-Madsen Author Index-Systematic

Index-Subject

ix

Index

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

Mark L. Burleson (390), Department of Biology, University of Texas at Arlington, Arlington, Texas 76019 Ragnar Fange (2),Department of Zoophysiology, University of Goteborg, S-40031, Goteborg, Sweden Susanne Holmgren (301),Department of Zoophysiology, University of Goteborg, S-40031, Goteborg, Sweden D. G. McDonald (56), Department of Biological Sciences, McMaster University, Hamilton, Ontario, Canada L8S 4 K 1

C. L. Milligan (56), Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 William K. Milsom (390), Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A9 Stefan Nilsson (301), Department of Zoophysiology, University of Goteborg, S-40031, Goteborg, Sweden Kenneth R. Olson (136), South Bend Center f o r Medical Education, lndiana University School of Medicine, University of Notre Dame, Notre Dame, Indiana 46556 S. F. Perry (255), Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K 1 N 6N5

D. J. Randall (255), Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A9 Neal J . Smatresk (390),Department of Biology, University of Texas at Arlington, Arlington, Texas 76019

E . W. Taylor (343), Department of Zoology and, Comparative Physiology, University of Birmingham, Birmingham B15 2TT, United Kingdom Xi

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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 circuxiii

xiv

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 control 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.

xv

PREFACE

Cardiovascular regulation centers around control of the heart’s activity, modulation of central blood pressure, and alterations to vascular resistance to effect regional control ofblood 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, 5B, 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-SYSTEMATICINDEX-SUBJECTINDEX

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

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 . Chester Jones, 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 111 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 Wolf and M . C . Quimby Chromatophores and Pigments Ryozo Fujii Bioluminescence J . A. C. Nicol Poisons and Venoms Findlay E. Russell AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume IV Anatomy and Physiology of the Central Nervous System Jerald J . 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 R i g s Gas Exchange in Fish D . J . Randall The Regulation of Breathing G . Shelton Air Breathing in Fishes Kjell Johansen The Swim Bladder as a Hydrostatic Organ Johan B . Steen Hydrostatic Pressure Malcolm S . Gordon Immunology of Fish John E . Cushing

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

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

xix

xx

CONTENTS OF OTHER VOLUMES

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

CONTEXTS 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 JohnJ . 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 AUTHOR

INDEX-SYSTEMATICINDEX-SUBJECTINDEX

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

xxi

xxii

CONTENTS OF OTHER VOLUMES

Cytogenetics J. R. Gold Population Genetics Fred W. Allendorfand Fred M . Utter Hormonal Enhancement of Growth Edward M . Donaldson, Ulf H . M . Fagerlund, David A. Higgs, a n d J . R . McBride Environmental Factors and Growth 1.R. Brett Growth Rates and Models W. E . Ricker AUTHOR INDEX-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.]. van Oordt and]. Peute Teleost Gonadotropins: Isolation, Biochemistry, and Function David R . Idler and T . Bun N g The Functional Morphology of Teleost Gonads Yoshitaka Nagahama The Gonadal Steroids A . Fostier, B.]alabert, R. Billard, 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 V a n Der Kraak

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

CONTENTS OF OTHER VOLUMES

xxiii

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

xxiv

CONTENTS OF OTHER VOLUME!

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 lsaia Branchial Ion Movements in Teleosts: The Role of Respiratory and Chloride Cells P. Payan,]. 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 1.A. Zadunaisky Hormonal Control of Water Movement across the Gills J . C . Rankin and Liana Bolis 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 PeterJ. Rombough

CONTENTS OF OTHER VOLUMES

xxv

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]. 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, and Julian Lombardi First Metamorphosis John H . Youson Factors Controlling Meristic Variation C. C. Lindsey The Physiology of Smoking Salmonids W. S. Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes David L. G. Noakes andlean-Guy].Godin

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

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1 FISH BLOOD CELLS RAGNAR FANGE Department of Zoophysiology University of Goteborg Goteborg, Sweden

I. Introduction 11. Red Cells: Morphology A. Hematocrit B. Shape and Size C. Cytoplasmic Structures D. Immature and Senescent Cells 111. Red Cells: Physiology and Biochemistry A. Erythrocyte Homeostasis B. Metabolism C. Proteins, Phosphates, and Nitrogen Metabolites D. Membrane Properties E. Osmotic Fragility F. Gas Transport IV. White Cells: Morphology A. Occurrence B. Staining Methods, Classification C. Granulocytes D. Mast Cells, PAS-Positive Granulocytes E. Lymphocytes, Plasma Cells F. Monocytes and Macrophages G. Thrombocytes or Spindle Cells H. Blast Cells V. White Cells: Physiology and Biochemistry A. Leucocyte Homeostasis B. Phagocytosis C. Granulocytic Defense Mechanisms D. Lymphocytic Functions, Immune Responses E. Pathology, Inflammation F. Thrombocytes and Blood Coagulation VI. Lymphomyeloid Tissues A. Thymus B. Spleen C. Kidney 1 FISH PHYSIOLOGY, VOL. XlIB

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

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RAGNAR FANGE

D. Lymphocytic Infiltrations E. Granulo(cyto)poieticTissues F. Melanomacrophage Centers VII. Hemopoiesis A. Stem Cells B. Tissue Microenvironment C. Factors Stimulating Hemopoiesis D. Erythropoiesis in the Peripheral Blood E. Toxic Effects on Erythropoiesis VIII. Future Research A. Gaps in Knowledge B. Hemopoiesis C. Lymphocyte Functions D. Blood Coagulation E. Granulocytic Function F. Electron Microscopy C. Immune System of Long-Lived Fishes H. Microcirculation of Hemopoietic Tissues I. Cell Interactions References

I. INTRODUCTION

Aquatic vertebrates originated hundreds of millions of years ago and evolved in different directions (Jarvik, 1980; Bjerring, 1985). In spite of systematic diversity (Nelson, 1984) all fishes possess two main types of blood cells, erythrocytes (red cells) and leucocytes (white cells), a property shared by the land-living vertebrates, which are derived from early fishlike ancestors. Detailed studies, however, reveal considerable variations in the structure and function of the blood cells between different groups of fishes. Drzewina’s (1911) thorough description of fish granulocytes was based on her own microscopic studies of68 species, but she and Jordan (1938), and Grodzinski and Hoyer (1938) in their article on comparative hematology, refer to many previous authors. A bibliography on fish hematology was assembled by Hawkins and Mawdesley-Thomas (1972). Rowley et al. (1988) have put together an extensive work on fish blood cells illustrated by numerous electron microscopic photos. Comparative hematology is discussed by Andrew (1965)and Ratcliffe and Millar (1988).Yasutake and Wales (1983),in an atlas of the microscopic anatomy of salmonids, include a chapter on blood cells. Ivanova (1983) has illustrated the microscopic morphology of different stages of fish blood cells, and Golovina and Trombitsky (1989) have treated some hematological parameters of freshwater fishes. Methods in fish hema-

1. FISH BLOOD

CELLS

3

tology are described by Blaxhall and Daisley (1973) and Houston (1990). Ellis (1977) has reviewed works on fish leucocytes dealing with morphology, staining characteristics, physiology, biochemistry, immunology, and the relationships of mast cells and eosinophils. Hine et al. (1987) describe enzyme cytochemistry of leucocytes of different species of fishes. Nikinmaa (1990)has published an excellent monograph on functional aspects of vertebrate erythrocytes that includes material on fish red blood cells. Articles or books on fish immunology are written by Cushing (1970), Anderson (1974), Corbel (197~4,Marchalonis (1977), Ingram (1980),Manning and Tatner (1985), and Ellis (1989). Cooper (1983), in a textbook on general immunology, also treats fishes. Litman et al. (1990) discuss the function of immunoglobulin genes in lower vertebrates. 11. RED CELLS: MORPHOLOGY

A. Hematocrit The concentration of erythrocytes in the blood can b e expressed as hematocrit or as the number of red cells per volume blood. The hematocrit values of fish blood range from almost 0 to more than 50% in actively swimming, surface-feeding species (Table I). I n most teleosts the hematocrit is between 20 and 40%, but some members ofthe family Chaenichthyidae (Antarctic icefishes) have colorless blood containing extremely few erythrocytes, which are fragile and lack hemoglobin. In Nototheniidae, another group of Antarctic fishes, the blood contains 0.38-1.2 x lo6 erythrocytes per pl, less than normal values for teleosts (Hureau, 1966; Barber et al., 1981; D’Avino et al., 1990). Average number of red blood cells in a number of marine teleosts of Puerto Rico was 2.3-5.3 x 106/pl (Saunders, 1966a).Low amounts of erythrocytes have been noted in stomiiforms and alepocephalids (Hine et al., 1987). Leptocephalus larvae (Anguilla, Conger) may lack erythrocytes. No data on the erythrocyte number of the Asiatic glassfish, Chanda (Ambassis) (Nelson, 1984), or other transparent fishes seem to be available. Elasmobranchs in general show lower erythrocyte number in the blood than teleosts (0.1-0.4 x 106/p1:Grodzinski and Hoyer, 1938). This may partly be related to the large size of elasmobranch red cells. However, in the North Atlantic region chondrichthyans (sharks, rays) and holocephalans (Chimaera) show not only lower hematocrits but also lower hemoglobin values than the teleosts (Fange, 1978).

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RAGNAR F A N G E

Table I Data on Erythrocytes and Hemoglobin Concentrations in Fish Blood"

(70)

Erythrocyte NO.^ (103/4)

(g%)

Erythrocyte lengthb (ym)

19.1 (5)

0.12-0.19 (8)

4.1 (5)

15-35 (6)

16.3 (5) 20 (5)

0.06-0.07 (8) 0.24 (4) -

3.2 (8) 3.2 (5)

2U4) 31-38 (1)

25 (2)

0.76 (2)

17.2 (5) 19.3 (5)

0.97-1.30 (8) 1.09 (4)

52.4 (3) 52.5 (5) 51.2 (5)

2.15 (3) 3.87 (4) -

Hemoglobin Hematocrit Fish C yclostomes Myxine glutinosa Elasmobranchs Squalus acanthias

Somniosus microcephalus Prionace glauca Teleosts, slow swimmers Lophius piscatorius Cyclopterus lumpus Teleosts, fast swimmers Thunnus thynnus Scomber scombrus Clupea harengus

10.0 (2)

3.2 (5) 3.3 (5) 15.4 (3) 15.2 (5) 14.0 (5)

15.7 (2) 13-15 (8) 10-12 (1) 13.1 (3) 12.0 (7) 12.0 (7) 9.0-14 (9)

Only a few species and groups have been included. Examples from other fishes may be found in the text. Numbers in parentheses: (1)own estimation; (2) Glazova (1977);(3) CutiCrrez (1967); (4) Kisch (1951);(5) Larsson et al. (1976);(6) Mattisson and Fange (1977);(7) Wilkins and Clarke (1974);(8)Wintrobe (1933);(9) Sherburne (1973). (I

The hematocrit, hemoglobin concentration, and other hematological parameters are highly sensitive to physiological changes, for instance those occurring due to stress (Soivio and Oikari, 1976). Contraction of splenic vessels may force stored red cells into the general circulation (Fange and Nilsson, 1985).

B. Shape and Size Mature fish red cells usually are oval and disk-shaped with a compact nucleus. The erythrocytes of the lamprey, Lampetra fluuiatilis, are biconcave with an almost circular outline, rather similar to mammalian erythrocytes (Potter et al., 1982). Round disc-shaped red cells occur in some teleost species (Siphonostoma typhle: Undritz, 1963). The average red cell size differs between systematic groups of fishes. Teleost red cells usually measure between about 8 and 15 pm. Shrivastava and Griffith (1974) recognized a tendency for brackish

1. FISH

5

BLOOD CELLS

water species of Fundulus to have slightly smaller blood cells than freshwater species. Glazova (1977) noted that the erythrocytes are slightly smaller in active species than in nonactive. Probably small dimensions facilitate physiological exchanges by a favorable cellular surface/volume ratio. Erythrocytes larger than normal for teleosts are found in certain deep sea forms: Alepocephalidae, Halosauridae, Stomiiformes (Hine et al., 1987), and Saunders (1966a) observed large erythrocytes (17 pm) in a muraenid teleost, Gymnothorax funebris. Exceptionally large red cells, about 40 pm in length, occur in dipnoans (lungfishes) (Parker, 1892). The dipnoan cell sizes almost approach those of some urodelan amphibians (Amphiuma,Necturus). Elasmobranchs, holocephalans (chimaeroid fishes), and myxinoids (hagfishes) also possess relatively large blood cells (Wintrobe, 1933). The physiological consequences of the enormous cell sizes in lungfishes and amphibians are not well understood. Nonnucleated red cells (erythroplastids, hemoglobin packets) in marine deep water teleosts of the family Gonostomidae (Maurolicus mulleri, Valencienellus tripunctatus, and Vinciguerria sp.) have dimensions of 5.5 x 2.5 pm. The presence of nonnucleated red cells is associated with unusually small dimensions of blood vessels (2 pm

0

A

.*...:.. C

B

n D

F I

10 Am Fig. 1. Camera lucida drawings at the same scale of cells in MGG-stained blood smears of a teleost, Gadus morhua (cod) (A, B, C), and a lungfish (Protopterus aethiopicus (D, E, F). A and D, erythrocytes; B and E, polymorphomucleated granulocytes (B, neutrophil; E, eosinophil); C and F, lymphocytes. The lungfish cells are several times larger than the corresponding teleost cells.

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internally) (Hansen and Wingstrand, 1960; Hine et al., 1987). The sizes of blood and other cells are correlated to the content of DNA (Ohno and Atkin, 1966; Pedersen, 1971; Hinegardner, 1976). In a general sense the amount of nuclear DNA increases with biological complexity. However, within a systematic group of animals, such as fishes, variations of the amount of nuclear DNA depend mainly on the amount of repeated DNA (Schmidtke et al., 1978) and the polyploidy level. Erythrocyte nuclear measurements have been used to distinguish between diploid and triploid individuals of fish species (Wolters et al., 1982). The diploid cellular DNA content in teleosts is around 2 pg, in elasmobranchs 5.6-18.6 pg, and values as high as 248 pg are noted in dipnoans (Hinegardner, 1976). The average size of the red blood cells seems to vary in parallel with this series (Table I: erythrocyte length). C. Cytoplasmic Structures Ultrastructurally the cytoplasm of mature fish red cells has few organelles other than pinocytotic vacuoles (Fig. 2A,B) and microtubules (marginal bands). Single small mitochondria may be present (Keen et al., 1989). Kreutzmann and Jonas (1978) describe a Golgi complex and a so-called segregation apparatus in adult red cells of the eel (Anguilla anguilla) and the rainbow trout (Oncorhynchus mykiss). The segregation apparatus appears to be associated with the hemoglobin formation (Keen et al., 1989). Cell elongation during maturation of red cells in rainbow trout (Salmo gairdneri) is correlated with the appearance of a marginal band system (Keen et al., 1989). The peripheral bundles of microtubules presumably protect the shape of the erythrocytes and hinder deformation of red cells during passage through capillaries ( Joseph-Silverstein and Cohen, 1984). The number of microtubules in teleost red cells varies with species from 6-10 to 27 (Kreutzmann and Jonas, 1978).The microtubules connect with other types of filaments of the cytoskeleton (Nikinmaa, 1990). Centrioles participate in the biogenesis of the marginal bands as concluded from ultrastructural studies of the erythrocytes of the skate, Raja erinacea (Cohen, 1986). Microtubules of the marginal bands of the red cells of the smooth dogfish (Mustelus canis) have been isolated and examined after lysis of the cells with detergents in the presence of protease inhibitors (Sanchez and Cohen, 1988). Intracellular crystallization of hemoglobin commonly is observed in teleostean red cells (Yoffey, 1929: whiting, Gadus merlangus; Dawson, 1932: the pipefish, Syngnathus fuscus; Kisch, 1949: the eel,

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7

Fig. 2. Erythrocytes. (.4)Cyclostome: Myxine glutinosa. The cytoplasm contains pinocytotic vacuoles (rhopheocytosis), canaliculi, and minute granules. (B) Teleost: Gadus rnorhua (blood cells inside a vessel). The cytoplasm more homogenous and free from organelles. Electron micrographs. Bar = 1 pm.

Anguilla rostrata; Hansen and Wingstrand, 1960: stomatids and myctophids; and Mattisson, pers. comm.: Aphanopus carbo). It is con-

sidered as a postmortal phenomenon (Hansen and Wingstrand, 1960), but according to Thomas (1971) the hemoglobin already in the erythrocytes of the living normal whiting (Gadus merlangus) may exist in a paracrystalline state as bundles of parallel tubules or filaments. The tendency to form hemoglobin crystals in the red cells of certain teleosts may be a phenomenon somewhat similar to the propensity for crystal formation inside red cells of human beings suffering from sickle cell disease.

8

RAGNAR F A N G E

Virus particles or sporozoan parasites often occur in fish blood cells. Occasionally it may be hard to decide if inclusions represent parasites or organelles (Cenini, 1984; Rodger et al., 1991). D. Immature and Senescent Cells

The blood of just-hatched larva of the rainbow trout (Oncorhynchus mykiss) contains round disc-shaped larval erythrocytes with special embryonic hemoglobin (Iuchi, 1973). Bielek (1975) described similar primary or larval erythrocytes in the pike, Esox lucius, and the grayling, Thymallus thymallus. In adult teleosts, the blood normally contains a certain percentage of immature (juvenile) red cells (proerythrocytes, reticulocytes) (Dawson, 1933, Hardig, 1978, Boomker, 1980; Keen et al., 1989, Houston, 1990). Sherburne (1973) found between 6 and 38% immature erythrocytes (average 21%) in the herring (Clupea harengus). Immature red cells differ from the mature ones by a more circular outline, by presence of mitochondria, polyribosomes and other organelles, and by less hemoglobin (Lane and Tharp, 1980). When maturing they lose organelles, get loaded with hemoglobin, and elongate and develop marginal bands (Keen et al., 1989). Hemoglobin-free erythroblasts with a basophilic cytoplasm and a large nucleus are abundant in elasmobranch embryos (Saunders, 1966b) and in the blood of the hagfish, Myxine glutinosa (Mattisson and Fange, 1977). Small numbers of erythroblasts normally occur in the circulation of adult elasmobranchs, teleosts and dipnoans (Saunders 1966a; Boomker, 1980; see Section on Blast Cells). With senescent red cells a part of the circulating erythrocytes are aged or effete. “Nuclear shadows” in blood smears may consist of disintegrated red cells (Weinberg et al., 1972). Seven and one-half percent of senescent red cells occur in the blood of the rainbow trout (Keen et al., 1989). 111. RED CELLS: PHYSIOLOGY

AND BIOCHEMISTRY A. Erythrocyte Homeostasis

The hematocrit values are relatively constant within species but vary between species. The amount of erythrocytes (evaluated as hematocrit or erythrocyte count) and the total hemoglobin concentration

1.

FISH BLOOD CELLS

9

in the blood vary in accordance to life habits. Fast swimming species of fishes on average have more erythrocytes, larger hematocrits, and more hemoglobin than less mobile forms (Table I). A hemoglobin content of more than 20 g/100 ml is found in tropical scombrids (Klawe et al., 1963).Low oxygen in the environment stimulates erythropoiesis. Thus values for hematocrit and hemoglobin increase in freshwater fishes (Cottus poecilopus and C . gobio),which were transferred to water with low oxygen concentration (Starmach, 1970).Seasonal variations in the hematocrits of the winter flounder, Pseudopleuronectes americanus, may depend on nutritional or hormonal factors (Bridges et al., 1976). However, under normal conditions fishes usually are able to keep the concentration of red cells in the blood relatively constant. Such a red cell homeostasis results from a dynamic equilibrium between new formation (erythropoiesis) and destruction of erythrocytes. New erythrocytes are continuously entering the circulation, and effete erythrocytes are destroyed at the same rate. Destruction and elimination of aged or damaged erythrocytes are brought about by macrophages in the kidney and the spleen. Whereas the anucleate mammalian red cells have a life span of about 120 days, the less differentiated fish red cells presumably live somewhat longer. Hevesy et al. (1964), by isotope labeling, estimated a life span of more than 150 days for erythrocytes of a teleost fish, the tench (Tinca tinca). Youson (1971) has observed destruction of red blood cells in the kidney of the sea lamprey (Petromyxon marinus). Binding of antibodies to aged red cells or exposure of fish to pollutants may increase the rate of destruction of red cells in salmonids (Nikinmaa, 1990). The hemoglobin from decomposed erythrocytes is transformed into bile pigments and iron. The iron, stored as ferritin or hemosiderin, may be reused in erythropoiesis. Main iron stores are found in lymphomyeloid tissues and in the liver. In the tench (Tinca tinca) the spleen contains 10-15 times more iron per gram of tissue than the liver (Dijk et al., 1975). Probably the iron in the spleen occurs largely in melanomacrophage centers (Walker and Fromm, 1976; Agius, 1985). B. Metabolism

The metabolism of vertebrate red cells generates energy for the maintenance of cell shape and for transport of substances across the cell membrane (Nikinmaa, 1990). The sodium pump and phosphorylation processes consume about 50% of the total energy. Fish erythrocytes are metabolically more active than mammalian erythrocytes and, like unripe mammalian red cells or reticulocytes, consume oxygen by

10

RAGNAR FANGE

respiration (Bushnell et al., 1985). In mammals a large part of the erythrocytic energy production is based on anaerobic glycolysis and lactate formation. A fraction of glucose is metabolized along the pentose phosphate pathway producing reduced nicotiamide adenine dinucleotide phosphate (NADPH), reduced glutathione and sulfhydryl groups being needed for the detoxification of free radicals. The importance of the pentose phosphate pathway in fish erythrocytes is not known. Although glycolytic enzymes are present, glycolysis may be limited by low permeability to glucose of the red cells in some fish species (Bolis et al., 1971; Bachand and Leray, 1975). C. Proteins, Phosphates, and Nitrogen Metabolites The hemoglobins of vertebrates with the exceptions of cyclostomes (hagfishes and lampreys) are tetrameric, built of four peptide chains, each with one heme and a molecular weight of about 17,000. Monomeric and dimeric hemoglobins are found in the cyclostome red cells. The hemoglobin of the coelacanth, Latimeria, shows features of fish as well as tetrapod relationships. Sequence analyses of the hemoglobin indicate that Latimeria is more closely related to tetrapods than are dipnoans (Weber et al., 1973; Gorr et al., 1991). Fish embryos contain special kinds of hemoglobin, which are electrophoretically different from adult hemoglobins (Iuchi, 1973). Multiple hemoglobin systems in many fishes may be important in physiological adaptations to variable environments. Pterygoplichthys multiradiatus, a catfish of the Amazon basin, has multiple erythrocytic hemoglobin. It is a facultative air breather adapted for periods with low oxygen availability (Val et al., 1990). But even fishes living in constant oceanic environment often possess many forms of hemoglobin. In addition to hemoglobin, erythrocytes contain other proteins such as glykolytic metabolic enzymes, but mitochondria1 enzymes of the aerobic cell metabolism are poorly represented. Vislie (1978) found lysosomal enzymes such as p-N-acetylglucosaminidase in mature erythrocytes of the flounder (Platichthys Jesus). The enzyme carbonic anhydrase occurs in red cells of all vertebrates, but the activity in the blood of the flounder (P. f l e w s ) is low (Mashiter and Morgan, 1975). It catalyzes the reversible hydration/ dehydration of carbon dioxide and functions in the transport of carbon dioxide. Carbonic anhydrase from red cells of hagfish (Myxine glutinosa) has been investigated by Carlsson et al. (1980), who found its chemical properties to resemble those in other vertebrates. Superoxide dismutase has been isolated from the red cells of salmonids (Scott and Harrington, 1990).

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Nucleoside triphosphates are physiologically important in fish red cells because they function as allosteric modifiers controlling the oxygen affinity of the hemoglobin. In many fish species adenosine triphosphate (ATP) is the main compound acting in this way (Clupea: Everaarts, 1978; Scomber: Bartlett, 1982; Cichlasomu: Gillen and Riggs, 1971), but the erythrocytes of some species possess both ATP and quanosine triphosphate (GTP) (smooth dogfish, Mustelus canis, and the American eel, Anguilla rostrata: Peterson and Poluhowich, 1976; Bartlett, 1982; the Australian lungfish, Neocerutodus: Isaacks and Kim, 1984). Quanosine triphosphate is more efficient than ATP in lowering the oxygen affinity in certain species. Other phosphates present in the red cells of some air breathing species are inositolpentaphosphate in Arapaima gigas, inositol-diphosphate in the South American lungfish (Lepidosiren), and 2,3-diphosphoglycerate (DPG) in Pterygoplichthys sp. (Isaaks and Kim, 1984).A considerable amount of DPG is found in the red cells of the lamprey (Entosphenus tridentat u s ) but the erythrocytes of hagfish (Eptatretus) contain varying amounts of ATP (Bartlett, 1982). In fishes a major part of nitrogenous excretory products are excreted as ammonia via the gills. Experiments on the carp show that ammonia is transported in the blood in about equal amounts by red cells and by plasma (Ogata and Murai, 1988). D. Membrane Properties Red cell membranes consist of a bimolecular lipid layer associated with carbohydrate-containing antigens intermingled with proteins, among which are contractile proteins and enzymes. Spectrin, actin, and other proteins form an intricate membrane skeleton interacting with hemoglobin, membrane transport proteins, and tubulin of the marginal bands. Spectrin and actin occur in the red cell membrane of elasmobranchs (Cohen e t ul., 1982), but in cyclostomes spectrin is lacking (Ellory e t ul., 1987; Nikinmaa, 1990). A few studies have been made in order to find blood group systems in fish (Cushing, 1970). Studies of erythrocyte antigens in cod, Gadus morhua (Moller, 1967), and in the American eel, Anguilla rostrata (Sindermann and Krantz, 1968), are of genetic interest. The American eel shows complex isoagglutinin-isoantigen systems. A number of cod blood group antigens were discovered by using antiserum from rabbits. Somewhat less than half of the erythrocyte membrane consists of lipids. The membrane lipids composition have been investigated in a few species. Phospholipids make up about 80% of the total lipids.

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RAGNAR FANGE

Polyunsaturated fatty acids constitute a high percentage of the fatty acids (Bolis and Fange, 1979). The function of the membrane lipid components is influenced by environmental factors such as diet and temperature (Bly and Clem, 1988). Low environmental temperature induces increased degree of unsaturation in fish lipids (Cowey and Sargent, 1977; see the review by Nikinmaa, 1990). The red cell membrane contains transport proteins for both anions and cations. The exchange of cations may be brought about by a sodium pump. Little is known about molecules responsible for cation transport across the erythrocyte membrane in fishes. Chloride ions pass extremely slowly through the red cell membrane of the hagfish, Myxine, and in lampreys (Larnpetra, Petrornyzon) the red cells are impermeable to bicarbonate. This makes exchange between chloride and bicarbonate difficult, and cyclostome erythrocytes seem incapable of intracellular buffering during transport of carbon dioxide (Nikinmaa, 1990). The erythrocytes from the Japanese lamprey, Entosphenus japonicus, show 50 or 100 times higher adenosine triphosphatase (ATPase) activity than mammalian erythrocytes. The ATPase is activated by Mg and Ca ions, but not by Na and K ions (Asai et al., 1976). Many vertebrate erythrocyte membranes possess P-adrenergic receptors for catecholamines. Under influence of catecholamines adenylate cyclase is activated catalyzing the conversion of ATP to cyclic adenosine monophosphate (CAMP). However, according to Tufts and Randall (1988) the erythrocytes of elasmobranchs and cyclostomes, in contrast to those of other vertebrates, are not influenced by catecholamines, and adrenergic receptors may not occur in the erythrocyte membranes of cyclostomes (Nikinmaa, 1990). Erythrocytes are frequently used to investigate the permeability of organic substances through cell membranes. The observations may be based on hematocrit estimations, hemolysis experiments ( Jacobs, 1931), or tracer technique. Relatively few studies concern fish erythrocytes. The permeability of fish red cells to glucose and other carbohydrates varies considerably between species. The red cells of armored catfish (Pterygoplichthys)and brown trout (Salmo trutta) are impermeable to glucose, those of arawana (Osteoglossum)slightly permeable, and those of the electric eel (Electrophorms)and lungfish (Lepidosiren) show the greatest permeability (Bolis et aZ., 1971; Kim and Isaacks, 1978). In a series of South American fish (lungfish, Lepidosiren; electric eel, Electrophorus; arawana, Osteoglossum; armored catfish, Pterygoplichthys; piraruca, Arapaima) the red cells proved to be permeable to urea in decreasing order (Kim and Isaacks, 1978).The

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effects of urea on red cells do not seem to have been specifically studied in those fish that use urea retention for their osmotic balance (elasmobranchs, holocephalans, the coelacanth, Latimeria). E. Osmotic Fragility Hemolysis is caused by the addition of distilled water or hypotonic sodium chloride solutions to blood. The osmotic fragility of the erythrocytes differs between species. The erythrocytes of euryhaline species, such as the rainbow trout (Oncorhynchus mykiss) (Hughes et al., 1986a,b)and gars (Lepisosteus osseus and L. productus), show higher tolerance against hypotonic salt solutions than those of exclusively marine teleosts (Ezell et al., 1969). Pitombeira et al. (1971) studied osmotic fragility in tuna (Thynnus thynnus) and Spanish mackerel (Scomberomorus maculatus) before and after spawning. The osmotic fragility of the red cells may be expressed as a percentage of NaCl causing 50% hemolysis (time and temperature standardized). Erythrocytes of the brook trout (Saluelinusfontinalis) show an osmotic fragility of 0.30-0.40% NaCl (Christensen et al., 1978: O"C,60 min). In Esox Zucius a value of 0.32% NaCl is measured (Mulcahy, 1970). The erythrocytes of the hagfish (Myxine glutinosa) seem more resistant against hypotonic salt solutions than those from an elasmobranch. The erythrocytes from the hagfish and marine teleosts (Labrus berggylta, Gadus morhua) hemolyze in 0.07-0.1 M NaCl solution, while those of the greenland shark (Somniosus microcephalus) hemolyze at sodium chloride concentration of 0.13-0.21. M (Fange, 1985). Taurine and amino acids are important in the intracellular osmotic volume regulation of the erythrocytes of teleosts and elasmobranchs (Fugelli, 1967; Goldstein and Boyd, 1978; Bedford, 1983). Studies on amphibian blood show that the capacity of red cells to resist osmotic swelling is affected by the cell metabolism. Increased metabolism, resulting in higher ATP levels, increases the resistance against hypotonic NaCl solutions (Goniakowska-Witalinska, 1974).

F. Gas Transport The oxygen binding capacity of the blood is dependent on the concentration and properties of the hemoglobin containing red cells. Fish in different ecological environments need different functioning hemoglobins. The oxygen binding capacity of the hemoglobin molecules varies with the chemical structure of the protein moiety of the molecule and with the intraerythrocytic content of certain substances

14

RAGNAH FdNGE

such as phosphates, which modulate the properties ofthe hemoglobin molecule. The oxygen binding properties of the blood are controlled by changes in the physicochemical environment of the erythrocytes. The Bohr and Root effects on fish hemoglobins, the effects of urea on the oxygen affinity in shark blood, and temperature effects on the blood gas binding properties are discussed by Nikinmaa (1990). The oxygen affinity of the blood of water breathing fishes generally is higher than in air-breathing fishes due to different intrinsic properties of the hemoglobins (Johansen and Lenfant, 1972; Johansen et al., 1978). The efficiency of the transport of carbon dioxide in vertebrate circulation depends on a rapid exchange of chloride and bicarbonate through the erythrocyte cell membrane (Deuticke, 1970). The enzyme carbonic anhydrase facilitates the anion exchange through the erythrocytic membrane. However, in fish the anion permeability of the red cells varies. In certain teleosts (carp, Cyprinus carpio; pikeperch, Stizotedion lucioperca) anion exchange of the red cells is slow, and in cyclostomes-due to nonexistent intracellular buffering of the blood (Nikinmaa, 199O)-the capacity for carbon dioxide excretion probably is lower than in any other vertebrates.

IV. WHITE CELLS: MORPHOLOGY A. Occurrence

Except for a few hours during which they are transported b y the blood, vertebrate leucocytes function outside the circulation (Tavassoli and Yoffey, 1988). In mammals there are about 60 times more mature granulocytes and perhaps 400 times more lymphocytes in the tissues than inside the blood vessels (Antonioli, 1961). In fishes large amounts of leucocytes, in all phases of development, occur in specific lymphomyeloid tissues and organs and infiltrate the skin, mucosal membranes, and connective tissue areas all over the organism (Drzewina, 1905; Kanesada, 1956; Fange, 1984, 1987). Fish blood is remarkably rich in leucocytes (Parker, 1892; Drzewina, 1911;Wintrobe, 1933; Reznikoff and Reznikoff, 1934; Weinberg et d., 1972). In teleosts and elasmobranchs the blood contains 15-135 x lo3 respectively 22-57 x lo3 white cells per p1 (Kisch, 1951), as compared with about 7 x lo3 leucocytes per p1 in human blood. The high values may be explained by nucleated thrombocytes being counted as leucocytes.

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B. Staining Methods, Classification

Since Ehrlich (1879) leucocytes are classified according to their affinities to acid and basic dyes. Application of differential staining methods (May-Griinwald-Giemsa, Romanowsky, Pappenheim, Wright, etc.) on fish blood has resulted in a nomenclature based on human hematology (Jakowska, 1956; Cenini, 1984; Rowley et al., 1988). Thus fish leucocytes may be divided into granulocytes (neutrophil, eosinophil, and basophil) and nongranulocytes (lymphocytes, monocytes, and thrombocytes). The thrombocytes are often regarded as forming their own cell line. Blast cells, early undifferentiated blood cells with a large nucleus and basophilic cytoplasm, may be included in the nongranulate fraction of circulating leucocytes. Stem cells are in part identical with blast cells. However, sometimes differential staining methods seem to work less well on fish blood cells than on mammalian cells. When possible microscopy studies of fixed and stained smears of fish blood should be complemented with observations on living cells, either illuminated by phase contrast, dark field, polarized or ordinary light, or supravitally stained with neutral red or fluorescent dyes like acridine orange. Cytoplasmic particles containing hydrolytic enzymes, such as lysosomes, take up stains by a vital process (Koehring, 1930; Allison, 1968). As an example shark granulocytes, probably due to the lysosomal nature of the cytoplasmic granules, accumulate supravital stains very rapidly (Fange, 1968). Phagocytosis may be demonstrated by injections of latex particles or yeast cell membranes (zymosan). Density gradient centrifugations with Percoll or Ficoll-Paque are used to separate types of fish leucocytes (Braun-Nesje et al., 1981; Fujii, 1981; Savage, 1983; Blaxhall and Sheard, 1985; Mainwaring and Rowley, 1985a,b; Fange, 1987; Suzuki, 1988; Plytycz et al., 1989). Leucocyte types can also be separated by means of their different adherence to glass (Mainwaring and Rowley, 1985a,b) and by flow cytometry (Ellsaesser et al., 1985). Electron microscopic, cytochemical, and immunological methods are increasingly used to study fish blood cells. Difficulties are involved in overbridging the gap between light microscopic and electron microscopic observations. Fish hematology is especially complicated because of differences between systematic groups. Combinations of many methods are needed if classification of fish blood cells shall improve its present provisional state.

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C. Granulocytes The majority of granulocytes are mobile, phagocytically active cells. The cytoplasm contains lysosomal granules, vacuoles, mitochondria, and other organelles or particles. Contractile vacuoles occur in elasmobranch granulocytes (Fey, 196613).The properties of the granulocytes vary extremely, especially between systematic groups of fishes, and Rowley et al. (1988, p. 41) rightly remark that “no other leucocyte type has caused as much confusion in the fish literature.” 1. TELEOSTS In teleosts, granulocytes constitute 4.5-18% or more of the leucocytes in the blood (Duthie, 1939; Watson et al., 1963; Wardle, 1971; Hines and Spira, 1973). They measure about 9-12 pm in diameter in blood smears, less in the living state, and resemble mammalian granulocytes in appearance. Ellis et al. (1976) and Ellsaesser et al. (1985) distinguish only one type of granulocyte in the plaice (Pleuronectes platessa) and the channel catfish (lctalurus punctatus) respectively. Other authors describe several categories: (a) neutrophils (heterophils), (b) eosinophils, and (c) basophils. a . Neutrophils (Heterophils).The predominating, sometimes only existing, type of granulocyte in teleost blood is named neutrophil due to its similarity with neutrophils of human blood. Neutrophils constitute 5.9% of the total leucocytes in the brook trout, Salvelinus fontinalis (Christensen et al., 1978). They are phagocytic in most species (Phromsuthirak, 1977). Neutrophils is the usual denomination (Catton, 1951; Weinreb, 1963; Watson et al., 1963, Kelknyi and Nkmeth, 1969; Wardle, 1971; Javaid and Lone, 1973; Lester and Desser, 1975; Ellis, 1977; Cannon et al., 1980; Bielek, 1981; Garavini et al., 1981; Roubal, 1986). But many synonymous terms are used, such as heterophils (Fey, 1966b; Barber and Westermann, 1978), “fine” or “specific” leucocytes, or “polymorphonuclears.” In many teleosts, for instance salmonids and cyprinids, the neutrophils possess polymorph (segmented or multilobed) nuclei (Fey, 1966b; Rowley et al., 1988),but in other species the nuclei are round or bilobed (the eel, Anguilla anguilla: Fey, 1966b; the plaice, Pleuronectes platessa: Ellis, 1976; Tilapia sp.: Ezzat et al., 1974; and the channel catfish, lctalurus punctatus: Elssaesser et al., 1985). The cytoplasm of neutrophils contains numerous fine granules (Phromsuthirak, 1977). These stain faintly red, pink, or violet in blood smears (Catton, 1951; Gardner and Yevish, 1969; Ezzat et al.,

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1974; Lester and Desser, 1975; Ellis, 1977; Hightower et al., 1984; Roubal, 1986), or azurophil (Haider, 1967). But often the granules are unstained by ordinary staining methods or need extra long staining time (Durand, 1950; Catton, 1951). Finn and Nielsen (1971)observed granulocytes in the rainbow trout, Oncorhynchus mykiss, that did not stain. Granules of the granulocytes in the cod, Gadus morhua, are unstained by Giemsa but show intense peroxidase response and are visible with phase contrast or dark field (Fange and Koskinen, 1984, unpublished). The teleost neutrophil granules generally are peroxidase positive and show acid phosphate reactions and affinity to Sudan black B (Cannon et al., 1980 and Garavini et al., 1981: Ictnlurus; Bielek, 1981: Cyprinus carpio, Tinca tinca, Salmo gairdneri; Hine et al., 1987: various species). But peroxidase negative neutrophils occur in certain species of eels (Hine et al., 1987). Neutrophils ofthe plaice, Pleuronectes platessa, are stained with PAS indicating presence of glycogen or other polysaccharides (Ellis, 1976). However, Barber et al. (1981) describe a special type of PAS-positive granulocytes in freshwater fish. In the main cytochemical responses of teleost neutrophils resemble those of similarly named mammalian cells. In the electron microscope the neutrophils show Golgi apparatus, mitochondria, ribosomes, endoplasmic reticulum, vacuoles, glycogen particles, and specific granules (Weinreb, 1963, Cenini, 1984, and Fujiinaki and Isoda, 1990: cyprinids; Bielek, 1980: salmonids and cyprinids; Ferguson, 1976: plaice, Pleuronectes platessa; Savage, 1983: Esox lucius; Cannon et al., 1980: channel catfish, Ictalurus punctatus; Lester and Desser, 1975: white sucker, Catastomus commersoni; Ishizeki et al., 1984: loach, Misgurnus sp.). The granules are round or elongate with dimensions of 0.1-0.5 pm. The interior of the granules is either homogenous or contains fibrillary or rodlike inclusions (Ferguson, 1976). Subpopulations of granules may exist. Small and large granules in heterophils of the pike (Esox lucius) structurally resemble primary and secondary granules of mammalian neutrophils (Savage, 1983). Fujimaki and Isoda (1990) distinguish three kinds of granules in the neutrophils of the goldfish, Carassius auratus.

b. Eosinophils. Eosinophils contain cytoplasmic granules that stain by acid stains. In most teleosts they are scarce or lacking in the circulation (Clupea harengus: Sherburne, 1973; Acanthopagrus: Roubal, 1986), but in labrids and a few other groups they are relatively abundant (Drzewina, 1911).Unique eosinophils containing one large

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RAGNAR FANGE

granule (2.5-2.8 pm) occur in Misgurnus anguillicaudatus (Ishizeki et al., 1984). Teleosts lacking eosinophils in the blood may possess such cells in the tissues and the peritoneum. The abundance of eosinophils in the intestinal mucosa of many teleosts led Jordan and Speidel (1924) to assume that eosinophils are responsible for immunity against bacteria. In the stickleback, Gasterosteus aculeatus, eosinophils are a rare component among other leucocytes infiltrating the skin (Phromsuthirak, 1977). Lester and Daniels (1976) found eosinophils in histological sections of inflammatory tissue of the white sucker (Catastomus commersoni) affected by parasites. The sections were stained with hematoxylin-eosin. However, Romanowsky stained blood smears showed no eosinophils. Eosinophilic granule cells in peritoneal exudates of the eel, Anguilla australis, give peroxidase positive reaction (Hine and Wain, 1989). Electron microscopic studies show that teleost eosinophil granules, in contrary to those of mammals, do not possess crystalline inclusions. c . Basophils. The granules of basophils stain by basic dyes. These cells scarcely occur in the blood of some teleosts such as carp (Cyprinus carpio), tench (Tinca tinca), and perch (Perca fluuiatilis) (Fey, l966b; Haider, 1968).

2. CHONDROSTEANS The blood of chondrosteans (sturgeons, paddlefish) contains neutrophils (heterophils) and eosinophils (Good et al., 1966; Kelenyi, 1972; Ivanova, 1983; Fange, 1986b). Neutrophils with 1-5 lobed nuclei predominate in the sturgeon, Acipenser brevirostris. The neutrophi1 granules are of two kinds, large, elongate, and uniformly electron dense, and small with a fibrillary interior. Eosinophils, also with lobed nuclei, are scarce. The eosinophils of sturgeons possess large, round or oval homogenous granules, which are peroxidase-positive (Hine and Wain, 1988b).Tissue infiltration with eosinophils is observed in paddlefish, Polyodon spathuZa, infected by nematode larvae (Miyazaki et ul., 1988). 3. ELASMOBRANCHS The elasmobranch blood is exceptionally rich in granulocytes with distinct granules, predominantly showing various grades of eosinophilia. Already in 1846 Wharton Jones (p. 63) described such a cell in the blood of the skate (Raja batis) as “composed of an agglomerulation of granules surrounded by a cell-membrane. The granules are clear and strongly refract the light.” There is little agreement on the number

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of existing types of elsasmobranch granulocytes. Some investigators distinguish two types (Drzewina, 1911; Jordan, 1938; Fange, 1968; Johansson-Sjobeck and Stevens, 1976). Pica et al. (1983)and Saunders (1966a,b) describe eosinophils and heterophils but state that in some species heterophils are replaced by neutrophils. Other authors find three types of elasmobranch granulocytes (Fey, 1966a; Stokes and Firkin, 1971). In the Greenland shark, Somniosus microcephalus, the blood contains: (a) cells with large, intensely eosinophilic granules, (b) cells with small, weakly eosinophilic granules, and (c) cells containing round granules of high density, which do not stain by Giemsa (Fange, 1987). The “heavy granulocytes” may correspond to the type I1 cells of Morrow and Pulsford (1980) or G3 cells of the type I1 cells of Morrow and Pulsford (1980) or G3 cells of Parish et al. (1986)found in the blood of the dogfish (Scyliorhinus canicula) by electron microscopy (see Section IV,D). Electron microscopic studies confirm and extend results from light microscopic observations (Fig. 3A,B). Two types of granulocytes are distinguished by their ultrastructure in nurse shark, Ginglymostoma cirriitum (Hyder et al., 1983), and in rays (Torpedo spp.: Pica et al., 1983; Raja spp.: Mainwaring and Rowley, 1985a). But in the blood of the dogfish, Scyliorhinus canicula, investigators found a large diversity of granulocyte ultrastructure (Morrow and Pulsford, 1980; Mainwaring and Rowley, 1985a; Parish et al., 1986). The most abundant granulocytes have relatively large (0.8 pm in diameter) membrane bound eosinophil granules; in addition three or four less common types are found. Electron microscopic and cytochemical observations on a series of other elasmobranchs (Hine and Wain, 1987a,b,c; Hine et al., 1987) show granulocyte populations difficult to put into categories corresponding to those of other vertebrates. At present there is no generally accepted nomenclature for elasmobranch granulocytes. The cytoplasmic granules are mostly eosinophilic and have different dimensions and structure. A scarce type of granulocytes contains high density granules, which do not stain by Giemsa. The granulocytes of rays and sharks as a rule are peroxidase negative but contain acid phosphatase and esterases (Hine et al., 1987). 4. HOLOCEPIIALANS Microscopic and ultrastructural studies of the blood of ratfishes such as Chimaera rnonstrosa show two types of granulocytes, one with fine, faintly red granules, the other with coarse, brightly red granules. In the adults the fine granulocytes predominate (Fange and Sundell, 1968; Mattisson et al., 1990). Obvious species differences exist. The

20

KAGNAR FANGE

Fig. 3. Leucocytes. Elasmobranch: Raja radiata. (A) Granulocyte (from Leydig organ). Electron-dense granules with rod-shaped inclusions are grouped around a Golgi apparatus ( G ) .Part of the nucleus (N) has been sectioned. (B) Granulocyte containing large, round, electron-dense granules. The cytoplasm is highly vacuolated. (C) Small lymphocyte. The nucleus is surrounded by a narrow rim of nongranulated cytoplasm. Electron micrographs. Bar = 1 wm.

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21

holocephalan granulocytes are peroxidase negative but contain acid phosphatase and esterases (Hine and Wain, 1988a,b).

5. DIPNOANS Dipnoan white blood cells are exceptionally abundant and prominent. The leucocytes of the Australian lungfish, Neoceratodus forsteri, measure 25 pm in diameter (probably measured in histological sections: Ward, 1969). According to Parker (1892)the largest leucocytes of the African lungfish, Protopterus annectens, may exceed the erythrocytes (length 40-46 pm) in size. The proportion of leucocytes to erythrocytes in the blood was about 1: 3, but probably erythroblasts were counted as leucocytes. In the blood of Neoceratodus, about 16% of the cells of the erythrocytic line are in the blast stage (Ward, 1969; Hine et al., 1990a,b). Parker (1892) found that lungfish leucocytes may continue amoeboid movements for hours under the cover glass and regarded the cells as “admirably adapted for examination in the living condition” (p. 169). Further studies have shown that the white blood cell pattern of lungfishes is relatively complex (Protopterus: Jordan, 1938; DeLaney et al., 1976; Neoceratodus: Ward, 1969; Hine et al., 1990a,b). Heterophils, or small eosinophils, which constitute 69% of the leucocytes (Ward, 1969), have find eosinophilic granules. Large eosinophils have an ovoid or bilobed nucleus and coarse intensely eosinophilic granules with a rodlike internal structure. Neutrophils possess azurophilic granules and segmented (polymorph) nuclei. As in chondrosteans (Hine and Wain, 1988b) eosinophil granules are peroxidase-positive, while heterophil and neutrophil granules contain no peroxidase. 6. COELACANTH Granulocytes constitute the majority of leucocytes in the blood of Latimeria chalumnae. Most common are neutrophils (pseudoeosinophils) with large granules and eosinophils. However, the blood cells of the coelacanth have not been much investigated (Millot et al., 1978; Locket, 1980). 7. CYCLOSTOMES Granulocytes constitute about 50% of the total leucocytes in the blood of the hagfish, Myxine glutinosa. Only one granulocyte type is recognized. The nuclei are polymorph with one to three segments. The granules resemble ultrastructurally the primary or azurophil granules of mammalian neutrophils (Mattisson and Fange, 1977) but are peroxidase-negative ( Johansson, 1973). The hagfish granulocytes are

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phagocytically active. Very large and active phagocytic granulocytes occur in the peritoneal cavity together with nongranulated macrophages (Fange and Gidholm, 1968; Mattisson and Fange, 1977). The granulocytes of the lamprey (Lampetra fluzjiatilis) resemble those of the hagfish but are smaller. As in the hagfish, they constitute about 50% of the leucocytes and have nonsegmented or 2-3 lobed nuclei. The granules are heterogenous in size (0.07-0.40 pm) and peroxidase negative (Page and Rowley, 1983). The granulocytes of the sea lamprey (Petromyzon marinus) resemble teleostean neutrophils ultrastructurally (Potter et al., 1982). They are phagocytically active and are opsonized by antiserum (Page and Rowley, 1984; Fujii, 1982). Blood eosinophils are found in the ammocoetes stage (Potter et al., 1982). D. Mast Cells, PAS-Positive Granulocytes

Mast cells are supposed to belong to the same cell line as the blood basophils. The cytoplasmic granules are basophilic and stain metachromatically with toluidin blue. The granules contain sulfated polysaccharides (heparin), which explains metachromacia, and substances such as histamine, which are released during anaphylaxia. The mast cells are found in mammalian connective tissue, especially around blood vessels, but it has been debated if similar cells occur in fish (Ellis, 1977). In teleosts basophilic (or eosinophilic) granulated cells form a stratum granulosum in the stomach and intestine (Drzewina, 1911; Bolton, 1933; Bielek, 1975).The cells have been regarded as mast cells but are not metachromatic and do not react to the histamine liberator 48/80 (Arvy, 1955; Weinreb and Bilstad, 1955). Also to some extent resembling mast cells are PAS-positive granular leucocytes (PAS-GL) in certain freshwater fishes (Barber and Westermann, 1975, 1978), and eosinophilic granular cells (EGC) in the plaice Pleuronectes platessa (Ellis, 1977). T h e PAS-GL are considered as “forerunners to mast cells.” The granules contain nonsulfated, neutral polysaccharides, which may be experimentally sulfated to produce a metachromatic reaction (Barber and Westermann, 1978). Davina et uZ. (1980) describe a PAS-positive granulocyte in the intestine of a barb (Barbus); the nucleus is eccentric, the granules do not stain with Giemsa but are intensively red with PAS. The “heavy granulocytes” that constitute a minor fraction of the granulocytes in the blood of the Greenland shark, Soinniosus microcephalus, also possess granules that do not stain by Giemsa. Due to the density of the granules the heavy granulocytes can be isolated by centrifugation. The granules may contain polysac-

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charides, but PAS staining was not tried (Fange, 1987). Heavy granulocytes of teleosts (blood of puffers, and pronephros of carp: Suzuki, 1986, 1988), “secretory granulocytes” of the buffalofish, Ictiobus, and the paddlefish, Polyodon ( Jordan, 1938), and “finely reticular cells” (Hines and Spira, 1973) show similarities to mast cells. But it is doubtful if any mast cell-like cells in fishes represent true mast cells; they usually lack metachromacia, and there is no direct evidence that they contain histamine and heparin (Ellis, 1982). E . Lymphocytes and Plasma Cells 1. LYMPHOCYTES Lymphocytes may be defined either morphologically or functionally (Ellis, 1977). Lymphocytes in the morphological sense are relatively small cells with a round or oval nucleus (Fig. 2C). The cytoplasm is nongranulated or contains few minute granules and usually stains blue in routinely stained smears. Teleost lymphocytes measure between 4.5 and 8.2 pm (Ellis, 1977),but some authors distinguish small, medium, and large lymphocytes. Remarkably large lymphocytes are found in lungfishes and certain elasmobranchs that possess large cells generally. Lymphocytes are mobile but usually nonphagocytic. Ultrastructurally the cytoplasm shows mitochondria, rough and smooth endoplasmic reticulum, ribosomes, and a Golgi system. They constitute from 50 to 80% of the leucocytes, but sometimes thrombocytes are reported to be more frequent than lymphocytes in fish blood. Vertebrate lymphocytes are supposed to be the predominating cells of lymph, but all kinds of leucocytes, not exclusively lymphocytes, are found in the supraspinal fluid of the plaice (Pleuronectes flesus) (Wardle, 1971). However, so-called lymph collected from fishes is claimed to originate from plasma skimming and does not correspond to the lymph of mammalian lymph vessels (Vogel and Claviez, 1981). There are few if any morphological criteria that can be used to distinguish lymphocytes from other types of nongranulated cells such as circulating stem cells, blast cells, monocytes, or thrombocytes. Phase-contrast microscopy, vital and fluorescence staining, and electron microscopy may solve part of the problem, but experimental methods, preferably immunological, are needed. The matter is discussed by Ellis (1977) and Rowley et al. (1988). In practice all nongranulated white blood cells, not identified morphologically as thrombocytes, may be provisionally described as lymphocytes or “lymphocyte-like” cells. Lymphocytes of hagfish ( M y x i n e glutinosa) cannot be morphologically distinguished from early stages of erythrocytes, the “lymphoid

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hemoblasts” of Jordan (1938) and Good et al. (1966), or from varieties of spindle cells (Mattisson and Fange, 1977). In fact lymphocytes and spindle cells in Myxine may belong to the same cell line. Destruction of microtubules by incubation with vinblastine (1pg/ml) or colchicine causes spindle cells to transform into round lymphocyte-like cells (Fange et al., 1974).

CELLS 2. PLASMA Plasma cells have a basophil, usually nongranulated cytoplasm, and an eccentric spherical nucleus. They occur in connective tissue, rarely in the blood, and may originate from blast transformed antigenactivated lymphocytes (B cells). In the electron microscope the cytoplasm exhibits numerous ribosomes in a rough endoplasmic reticulum that forms flat or irregular cisternae. Downey (1911) studied plasma cells in the renal lymphomyeloid tissue of the paddlefish, Polyodon spathula, before the immunological function of these cells was known. The plasma cells in the paddlefish resemble mammalian plasma cells except in nuclear structure. Good et al. (1966) observed large numbers of plasma cells in the spleen and the pericardial lymphomyeloid tissue of paddlefish immunized against Brucella microorganisms. T h e cells were identified by their ultrastructure. Plasma cells in lymphomyeloid tissues of elasmobranchs, teleosts, lampreys, and hagfishes have been identified electron microscopically (Fujii, 1982; Zapata et al., 1984).The plasma cells are considered as the main producers of immunoglobulins, but lymphocytes and other cells probably also produce antibodies (see Section V,D).

F. Monocytes and Macrophages Monocytes are mobile, phagocytic cells, usually slightly larger than other leucocytes. They have a vacuolated, weakly basophilic cytoplasm, which lacks distinct granules. The nucleus is generally oval or kidney shaped. Monocytes constitute only a minute fraction of the blood leucocytes in fishes and may be lacking in some species. Sherburne (1973) found no monocytes in the blood of the herring, Clupea harengus, but Ellis et al. (1976) noted 0.1-0.2% monocytes among the blood leucocytes in the plaice, Pleuronectes platessa. The plaice monocytes increased in number after injection of carbon particles. Macrophages, or phagocytically active cells in tissues and body cavities, are supposed to belong to the same cell line as monocytes, but occasionally macrophages may be related to granulocytes and contain cytoplasmic granules or may be derived from connective tissue cells.

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Tissue bound macrophages are often collectively termed the reticuloendothelial system (RES) (Mc Cumber et al., 1982; Page and Rowley, 1984). In teleosts, such as the rainbow trout (Oncorhynchus mykiss), macrophages are especially abundant in the spleen and the renal lymphomyeloid tissue (“pronephros”; Bielek, 1980) but they also occur in other tissues, for instance the olfactory mucosa (migrating Baltic salmonids: Bertmar, 1980). Macrophages that ingest India ink and sheep red cells are described from the pronephros of the carp, Cyprinus carpio (Smith et al., 1970).Weak phagocytic responses were observed when killed bacteria (Bacillus cereus) were injected into the peritoneum of a teleost, the striped bass (Morone saxatilis).The weakness of the reaction might have been due to absence of opsonizing effects because no immunization had taken place (Bodammer, 1986). Melanomacrophages are pigment containing cells that form nodule-like accumulations in lymphomyeloid tissues (Agius, 1985). Yamaguchi et al. (1979) found macrophages in the cavernous bodies at the base of the gill filaments of lampreys (Larnpetrareissneri). These phagocytic cells are considerably larger, about 50 pm in diameter, than the blood granulocytes. The cavernous bodies constitute unique filtration organs, the extensive phagocytic capabilities of which may keep the blood free from infections more efficiently than the circulating granulocytes (Page and Rowley, 1984). Granulated and nongranulated macrophages in the peritoneal cavity of hagfishes ingest intraperitoneally injected heat-killed yeast cells (Myxine: Fange and Gidholm, 1968; Mattisson and Fange, 1977). Thoenes and Hildemann (1970) used thioglycolate to activate the peritoneal macrophages in the California hagfish, Eptatretus. G. Thrombocytes or Spindle Cells Thrombocytes, or spindle cells, usually are oval or spindle shaped. Contrary to the mammalian platelets (or thrombocytes), they are nucleated and occasionally consist of almost naked nuclei (Ellis, 1977). Teleostean thrombocytes look like hemoglobin-free, slightly deformed erythrocytes or are difficult to distinguish by light microscopy from lymphocytes. The thrombocytes constitute up to 80.2% of the white cells in the herring (Clupea harengus) (Sherburne, 1973) but only 0-7% in other teleost species (Boyar, 1962). The large variation in thrombocyte numbers probably reflects difficulties in identifying the thrombocytes and the tendency of these cells to aggregate, adhere to surfaces, and disappear from the blood samples. In stained smears the cytoplasm of thrombocytes is nongranulated, grayish blue, or unstained. Ultrastructurally it shows mitochondria, ribosomes, glycogen

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granules, and bundles of microtubules (Ferguson, 1976: plaice, Pleuronectes platessa; Savage, 1983: pike, Esox Zucius). It contains vacuoles or vesicles, sometimes arranged like a string of pearls and connected with the surface (Bielek, 1979; Zapata and Carrato, 1980; Hyder et al., 1983; Cenini, 1984: Cyprinus carpio). Granules similar to a-granules of mammalian platelets occur in dogfish thrombocytes (Cannon et al., 1980). Lamellated inclusion bodies, probably phospholipids, are observed in lungfish thrombocytes (Neoceratodus, Tanaka and Saito, 1981). Dogfish thrombocytes show cytochemical responses for PAS, p-glucuronidase, and aliesterase (D’Ippolito et al., 1985). In the lamprey (Larnpetra fluviatilis) thrombocytes constitute 27.4% of the leucocytes (Page and Rowley, 1983). Spindle shaped cells constitute about 50% of the leucocytes in the blood of hagfish (Myxine). They contain mitochondria and microtubules. The spindle cells of Myxine may easily transform into round lymphocyte-like cells (Fange et al., 1974; Mattisson and Fange, 1977; see Section on Lymphocytes).

H. Blast Cells Blast cells represent early stages of red or white blood cells that arise by transformation of hemopoietic stem cells. Blast cells have increased nuclear volume, and the cytoplasm is intensely basophilic due to numerous ribosomes. Blast cells have the capacity to divide and form new cells. Rapid synthesis of DNA and RNA is indicated by intense cellular uptake of [3H]thyn~idineand [3H]~ridine(Fange and Edstrom, 1973), but few experimental studies have been made on blast cells in fish blood. Although blast cells are regularly present in small numbers in fish circulation (Saunders, 1966b; Rubashev, 1969; Boomker, 1980; Hine et al., 1990a,b),they are ignored by many investigators, probably due to difficulties in distinguishing them from lymphocytes and monocytes in microscopic studies. In teleosts the blast cells, to a great extent, may remain in the hemopoietic tissues, but in the blood of the cyclostome, hagfish (Myxine glutinosa), erythroblasts, and leucocytoblasts are abundant (Mattisson and Fange, 1977). V. WHITE CELLS: PHYSIOLOGY A N D BIOCHEMISTRY

A. Leucocyte Homeostasis

The frequency of leucocytes in the blood of fishes is influenced by physiological conditions. The number of leucocytes is affected by

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hormones in similar ways as in other vertebrates. In the rainbow trout (Salmo gairdneri), cortisone and adrenocorticotropic hormone (ACTH) produce lymphopenia and thrombocytopenia, whereas experimental injections of small amounts of turpentine cause inflammation and increase of granulocytes (Weinreb, 1958). The amount of circulating white blood cells increases with infection and parasitic infestation. Drzewina (1911)found high leucocyte counts in the blood of eel (Anguilla)infected with trypanosomes, and leucocytosis was noted by Murad and Kustafa (1988) in the catfish, Heteropneustes fossilis, parasitized by metacercariae. In higher vertebrates the granulocytes are relatively short-lived and must be replaced by continuous granulocytopoiesis. Conditions in fishes are unknown, but the granulocytes probably have a short life span in fishes too.

B. Phagocytosis Phagocytically active cells in fish blood are mainly neutrophil (heterophil) granulocytes and monocytes (mononuclear phagocytes). Macrophages are the main cells responsible for phagocytosis in the peritoneum, spleen, kidney, liver, gills, and other tissues. Usually the granulocytes are the most efficient blood phagocytes, but in a holostean, the gar (Lepisosteus platyrhincus) monocytes dominate (McKinney et al., 1977). The phagocytes destroy ingested microorganisms by chemical mechanisms. Deficiency of phagocytic killing mechanisms may explain why microorganisms and parasites cause disease in fishes and other animals (MacArthur and Fletcher, 1985).Phagocytic uptake of antigens is an important step in the initiation of humoral immune responses. Special antigen-trapping or antigen-presenting cell systems may be distinguished. The phagocytically very active cells in the blood and peritoneal cavity of hagfish, Myxine glutinosa, attack foreign material such as heat-killed yeast cells without previous immunization (Fange and Gidholm, 1968).Phagocytically active polymorphonuclear granulocytes isolated from the blood of the lamprey, Lampetrajaponica, by the Ficoll-Paque technique were studied by electron microscope (Fujii, 1981). In the lamprey antigenic material (sheep red blood cells) was more easily ingested by phagocytes if it was bound to specific lamprey immunoglobulins. Braun-Nesje et al. (1981) isolated and cultivated macrophages from the pronephros of the rainbow trout (Oncorhynchus mykiss). Ferguson (1984) examined the kidney of rainbow trout in the electron microscope after inoculation with killed bacteria. The bacteria were phagocytosed by macrophages associated with the endothelium of the

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renal portal veins. The venous renal portal system is a large area for antigen trapping. Datta Munshi et al. (1990) in cytochemical studies have investigated damage on fish macrophages caused by heavy metals. 1. NERVEAND HORMONE EFFECTS

Phagocytes are affected by nerve transmitters. a-adrenergic and cholinergic receptor agonists enhance the ability of stimulated pronephric macrophages and granulocytes to produce reactive oxygen species. Adrenaline and P-adrenergic agonists suppress the chemiluminescent response of pronephric phagocytes from the rainbow trout, Oncorhynchus mykiss (Flory and Bayne, 1991). Hydrocortisone depresses the activity (chemiluminescense response) of striped bass phagocytes (Stave and Roberson, 1985)indicating that phagocytes may be controlled by stress hormones. C. Granulocyte Defense Mechanisms Phagocytically active granulocytes destroy bacteria and fungal parasites by using enzymes localized in the cytoplasmic granules. Several antibacterial peptides are isolated from mammalian neutrophils (Cline, 1981; Lehrer and Ganz, 1990). Similar peptides are present in fish granulocytes. 1. HYDROLYTIC ENZYMES The bacteriolytic enzyme lysozyme (N-acetylmuramylhydrolase) is active against gram-positive bacteria. It occurs in granulocytes and peritoneal macrophages of the teleost plaice (Pleuronectes platessa) (Murray and Flechter, 1976) and may occur in leucocytes of other species of teleosts. High activities of lysozyme, chitinase, and other glycosidases are found in granulopoietic tissues of elasmobranchs and other lymphomyeloid tissues of fishes (Fkinge et al., 1980). Cytochemical observations confirm that fish granulocytes generally are rich sources of hydrolytic enzymes (Hine et al., 1987). Leucocytic enzymes besides those mentioned are acid and alkaline phosphatase, sulfatases, and various esterases. The molecular aspects of fish leucocytic enzymes and their biological importance are virtually unknown, but one might feel tempted to assume that several of the enzymes may play a role in defense against parasites and microbes, for example by dissolving cell walls of phagocytically ingested microbes. Other enzymes may act in cellular production of chemotactic substances or other biologically active products.

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2. OXYGEN-DEPENDENT BACTERICIDAL SYSTEMS Peroxidase (myeloperoxidase: MPO) is a component of mammalian granulocytes and is located in primary or azurophil granules. Peroxidase is demonstrated cytochemically in the neutrophils of most teleosts but is lacking in the leucocytes of certain teleost families (Johansson, 1973; Hine et al., 1987; Hine and Wain, 1 9 8 8 ~ )In . the Australian lungfish, Neoceratodus, and the sturgeon, Acipenser brevirostrum, eosinophils but not neutrophils show peroxidase reaction (Hine and Wain, 1988b; Hine et al., 1990a,b). The enzyme is poorly represented in elasmobranch granulocytes, and hagfishes lack leucocytic peroxidase (Johansson, 1973). Leucocytic peroxidase is supposed to function in a bacteria killing system of phagocytic granulocytes. During phagocytosis the cells show an intense increase of oxygen consumption, a “respiratory burst.” This results in production of superoxide anion ( 0 2 - ) , hydrogen peroxide (HZOZ),and hydroxyl radicals (OH‘).Under catalytic influence of peroxidase hydrogen peroxide reacts with chloride ions producing antibacterial substances. The respiratory burst can be shown by the substance luminol, which produces light in the presence of oxygen superoxide. Chemiluminescent phagocytes, probably neutrophils, occur in the blood and the pronephros of teleosts: striped bass, Morone saxatilis (Stave et al., 1984); plaice, Pleuronectes platessa (Nash et al., 1987); rainbow trout, Oncorhynchus mykiss (Plytycz et al., 1989); and the channel catfish, Lctalurus punctatus (Dexiang and Ainsworth, 1991).Apparently teleosts possess similar oxygen-dependent, bacteria killing leucocytic mechanisms as those found in mammals. D. Lymphocyte Functions, Immune Responses Vertebrate lymphocytes were long regarded as mature cells, end products of hemopoietic cell differentiating processes. However, they have the capacity to develop and undergo blast transformation when appropriately stimulated. Then the nucleus enlarges and the amount of ribosomes increases in the cytoplasm. Stimulating substances are lectins or mitogens usually of plant origin. Phyto-haemagglutinin (PHA) stimulates T lymphocytes to enlarge and divide, and lipopolysaccharide (LPS) stimulates B cells. Formation of plasma cells may be regarded as a blast transformation of lymphocytes (B cells) when stimulated by foreign antigens (Cooper, 1983). 1. LYMPHOCYTIC HETEROGENEITY Immunocompetent lymphocytes constitute the basis of immune

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reactions. Fishes, like all other vertebrates, show both cell mediated and humoral immune responses and may possess functional equivalents to T and B cells of higher vertebrates. Teleostean lymphocytes respond to mitogens such as PHA, concanavalin A (Con A), and LPS, which are considered as specific to mammalian subclasses of lymphocytes. However, subpopulations of fish lymphocytes may not necessarily be analogous to the mammalian T and B lymphocytes (Warr and Simon, 1983; Caspi et aZ., 1984). Gilbertson et al. (1986), by flow cytometry of hagfish (Eptatretus) peripheral blood, separated a fraction of granulocytes and macrophages from a fraction of small cells, mainly lymphocytes. Heterogeneity of the hagfish leucocytes was noted using monoclonal antibodies against cell surface antigens. Small leucocytes reacted in mixed lymphocyte tests as T cells. 2. NONSPECI~~C CYTOTOXIC LYMPHOCYTES Nonspecific cytotoxic cells or natural killer cells (NK cells) are thought to play a role in natural immunity of higher vertebrates. They are cytolytic for cultured tumor cell lines and virus infected cells. Natural killer cells are also produced in lymphomyeloid tissues of freshwater teleosts. Thus the channel catfish (Ictalurus punctatus) is protected against the protozoan disease ichthyophthiriasis by the combined effects of nonspecific cytotoxic cells and a humoral immune response. Immobilization of the protozoans by immunoglobulins facilitates adherence of NK cells to the parasites (Hinuma et al., 1980; Graves et al., 1985; Greenlee et al., 1991). Cells in the blood of the nurse shark, Ginglymostoma cirratum, react unspecifically cytotoxic to red cells of other species after exposure to the mitogens PHA, Con A, or LPS (McKinney et al., 1977; Mc Cumber et al., 1982; Haynes and McKinney, 1991). 3. HUMORAL IMMUNE RESPONSE Antibody producing cells are assumed to derive from lymphocytes (B cells), which are influenced by antigens to transform into blast-like cells, the plasma cells. Antigen presenting macrophages cooperate in the process. However, it is not clear if plasma cells are the only producers of immunoglobulins in fish. Antibody forming cells have been demonstrated in the anterior kidney (pronephros) and the spleen of immunized teleosts by the Jerne hemolytic plaque assay (Anderson et al., 1979; Smith et al., 1967, 1970), and by rosette test (immunocytoadherence test) (Chiller et al., 1969a,b).According to Chiller et al. (1969a) most of the rosette forming cells of the pronephros and spleen of the teleost, the rainbow trout (Salmo gairdneri), have the appearance of small, medium, or large

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lymphocytes. More seldom they are plasma cells. Plasma cells are lymphocyte-like cells involved in especially intense synthesis of immunoglobulins, but other kinds of leucocytes may produce antibodies too. Plasma cells have been demonstrated by light and electron microscopy in several main groups of fishes, including cyclostomes (Zapata et al., 1984). In cyclostomes such as hagfish (Myxine, Eptatretus) and lampreys (Lampetra),the amount of immunoglobulins produced is estimated at about 1/50ofthat found in mammals. In elasmobranchs up to 60% of the plasma proteins consist of immunoglobulins, in a chondrostean, the paddlefish (Polyodon)about 40% (Litman et al., 1990; Legler et al., 1971). The immunoglobulins of cyclostomes have properties that are intermediate between immunoglobulins of vertebrates and invertebrate lectins. The immune responses of lampreys are weak and slow acting (Corbel, 1975; Marchalonis, 1977; Fujii et al., 1979). In the Pacific hagfish, Eptatretus stoutii, about half of the blood leucocytes show surface immunoglobulins as revealed by radioimmunoassay (Raison and Hildemann, 1984).

4. RADIATION SENSITIVITY Lymphocytes are considered to be extraordinarily sensitive to irradiation. However, high doses of irradiation (1200, 5000, or 10,000 R) caused little change in leucocyte counts in the hagfish (Myxine glutinosa), while the white blood cells of the sea lamprey, Petromyzon marinus, proved very susceptible. The reason for the unusual resistancy of Myxine leucocytes is not known (Finstad et al., 1969).

5. OPSONINS Several observations indicate that phagocytosis in fishes is accelerated by the presence of antibodies. Michel et al. (1990) reported opsonizing properties of natural antibodies of rainbow trout (Oncorhynchus mykiss). Scott et al. (1985) showed an opsonin effect on peripheral blood phagocytes from channel catfish. 6. ROSETTE (IMMUNOADHERENCE)

Chiller et al. (1969a,b) showed that lymphocytes and macrophages from the spleen and kidney of rainbow trout immunized against sheep red cells form rosette complexes with the sheep cells. SYNTHESIS 7. TEMPERATURE EFFECTSON ANTIBODY It has been known for a long time that temperature affects synthesis of antibody in fishes. Although production of antibodies takes place at

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low temperature, it takes a longer time, and it increases if the temperature goes up (Harris, 1973). Probably antibody production is greater and biologically more important in species living in warm tropical water than in fish living in cold water. 8. IMMUNOGLOBULIN MOLECULES Teleosts, holosteans ( A d a ,Lepisosteus),and polypterids have tetrameric immunoglobulins of the IgM type with a molecular weight of about 600,000 (Warr, 1983; Acton et al., 1971; Bradshaw et al., 1971). Elasmobranchs possess pentameric and monomeric immunoglobulins that resemble the mammalian immunoglobulin (Ig)type IgM. The pentameric IgM has a molecular weight of about 900,000 (Bradshaw et al., 1971; Acton et al., 1971; Corbel, 1975). Dipnoans (lungfishes) (Corbel, 1975; Marchalonis, 1977)possess Igs oftwo classes, analogous to IgM and igG types in mammals. Among elasmobranchs, the spiny rasp skate, Raja kenojei, and the Aleutian skate, Bathyraja aleutica, possess two kinds of Igs, a high molecular weight Ig analogous to mammalian IgM and a low molecular weight Ig. Two distinct populations of Ig-forming plasma cells were found in the spleen and the intestinal mucosa. In the Leydig organ, the epigonal organ, and the liver plasma cells were less frequent (Tomonaga et al., 1985, 1986; Kobayashi et al., 1985). Immunoglobulins do not occur only in blood plasma and mucus secretions but are also associated with cell membranes of lymphocytes acting as antigen-specific receptors. Immunoglobulins from splenic lymphocytes of the goldfish, Carassius auratus, differ from the Ig of' the plasma (Warr and Marchalonis, 1977). 9. SECRETORY ANTIBODIES Secretory Igs are found in skin mucus of teleosts and holosteans (Corbel, 1975; Ourth, 1980). The mucous antibodies are of the IgM type as those of the plasma but further molecular properties are not known. In the Amazonian discus fish, Symphysodon, young fishes feed from their parent's epidermal mucus, probably acquiring immunity in this way. The cells producing secretory antibodies may be leucocytes infiltrating the skin (Hildemann, 1962). 10. COMPLEMENT (C) The reaction between antibodies and antigen-carrying foreign cells stimulate proteins of the complement system in the plasma to start a cascade of enzyme reactions that damage and ultimately destroy cells covered by an antibody. In mammals the C system consists of nine

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components. The C factors have been studied in some fish groups (Marchalonis, 1977). Hemolytic Cs, extremely labile to freezing, are found in the blood of elasmobranchs (Legler and Evans, 1967). Jensen et al. (1981) investigated the C system of the nurse shark, Ginglymostoma cirratum. It consists of only six components, but in a mammalian-like way it forms holes in the stroma of antibodysensitized sheep erythrocytes. Teleost complement is species specific (bluegills, salmonids: Smith et d., 1967, Chiller et d., 1969b). Complement-like activity is detected in lampreys (Lampetra japonica). However, information on structure and function of C systems in lower vertebrates are fragmentary (Fujii and Murakawa, 1981). The C factors are supposed to be produced by liver cells and by leucocytes. 11. “NATURALANTIBODIES” Fish blood plasma contains nonspecific lectin-like proteins or glycoproteins, different from immunoglobulins, which provide some protection against infections. These substances may act together with the immune mechanisms (Ingram, 1980). ON IMMUNE PROCESSES 12. NERVEINFLUENCE The teleost spleen has a rich adrenergic innervation, which may affect the antibody producing cell system. According to Flory and Bayne (1991), damage of the adrenergic nerves increases secretion of antibodies. In the rainbow trout, Oncorhynchus mykiss, a-adrenergic and cholinergic agents increase the number of antibody secreting cells while P-adrenergic substances have the opposite effect.

13. CHEMICAL FACTORS INFLUENCING IMMUNE PROCESSES Chemical pollutants in the environment are thought to depress fish immune mechanisms leading to an increase of infections or tumors (Vos et al., 1989). Production of stress hormones such as corticosteroids may also unfavorably affect immune functions (Grimm, 1985).

14. ANAPHYLAXIS Anaphylactic reactions are thought to be caused by substances that are released from mast cell granules, when Igs of IgE type react with specific antigens at the cell surface. A few observations indicate that anaphylaciic-like hypersensitivity reactions exist in fishes (Goven et al., 1980; Ellis, 1982). Cells responsible could be PAS-positive granulocytes or EGC, but they lack histamine and Igs of the IgE type are unknown in fishes. The question of anaphylactoid responses in fishes is unsolved (Ellis, 1977, 1982).

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15. VACCINATION Several methods of vaccination against infections of fish in aquaculture have been developed. Intraperitoneal injection of vaccines is superior to other methods in experiments but may also be used on a commercial scale. Other methods build on adding the vaccines to the food or to the water (Horne et al., 1984; Ellis, 1988).

E. Pathology ZnAammation 1. ERYTHROCYTE SEDIMENTATION RATE

Estimation of erythrocyte sedimentation rate (ESR) is an unspecific method used in human medicine to detect infections. The method has been tried in hematological studies on salmonids but probably is of little practical value (Blaxhall and Daisley, 1973). Schumacher e t al. (1956) reported increased ESR in brook trout infected by furunculosis, and Murad and Mustafa (1988) found a similar response in catfish (Heteropneustes fossilis) parasitized by metacercariae and also reported low hematocrit and increased white cell counts in the diseased fishes. 2. INFLAMMATION Pathological processes caused by infections or parasites are relatively rarely examined in fishes, but leucocytes are known to be engaged. The role of lymphocytes in inflammatory processes in fishes is described b y Ellis (1976). In mirror carp (Cyprinus carpio L.) infection with lchthyophthirius causes a rise in the percentage of neutrophils and granuloblasts and a temporary drop in lymphocytes in the blood (Wines and Spira, 1973). Appearance of immature neutrophils with intensely basophilic cytoplasm and reduced numbers of granules are seen in inflammatory conditions in the eel, A n g u i h australis (Hine and Wain, 1988a). Suzuki (1986) obtained peritoneal exudate cells b y injection of liquid paraffin into tilapia (Oreochromis niloticus) and carp (Cyprinus curpio). Eosinophils appeared later in the exudate and were less phagocytic than macrophages-monocytes and neutrophils. Hyder Smith e t al. (1989) found that in the nurse shark (Ginglymostoma cirrhatum) granulocytes and mononucleate macrophages, like mammalian neutrophils and monocytes-macrophages, react chemotactically to endotoxin-activated rat serum. Infection of paddlefish, Polyodon spathula, with larval nematodes causes ulcers of the gastric mucosa with accompanying infiltration with eosinophils. The lymphomyeloid tissue of'the epicardium reacts with

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extensive proliferation of macrophages (Miyazaki et al., 1988). Phromsuthirak (1977) followed leucocytic responses at healing of an incision in the skin of Gasterosteus aculeatus. Macrophages, neutrophils, eosinophils, and lymphocytes accumulated in the skin. Leucocytes migrated into the skin from the blood. The number of neutrophils reached a peak after 1 day, that of macrophages after 3 days.

F. Thrombocytes and Blood Coagulation In cyclostomes and elasmobranchs blood clotting is relatively slow, in teleosts and lungfishes very fast (Doolittle and Surgenor, 1962; Ward, 1969). Addition of seawater accelerates blood clotting in hagfish ( M y x i n e )(Fange and Gidholm, 1973) and in an elasmobranch (Heterodontus) (Stokes and Firkin, 1971). In mammals hemostasis (i,e., stoppage of bleeding from damaged vessels) results from interaction of vasoconstriction and processes taking place in the blood. The latter involve (a) aggregation of platelets leading to plug formation and ( b )blood coagulation. Hemostatic responses of fishes have not been much investigated, but evidences are that similar mechanisms are at work as in other vertebrates. Vertebrate blood coagulation results from a cascade of reactions producing an enzyme, thrombin, that splits fibrinogen into insoluble fibrin and peptides. Two pathways or systems are distinguished. The extrinsic system is initiated by tissue factors, the intrinsic by factors from platelets or other blood cells. The nucleated thrombocytes of fish blood participate in the coagulation process in the conversion of prothrombin to thrombin and in clot retraction (Doolittle and Surgenor, 1962; Belamarich et al., 1962; Fey, l966a; Gardner and Yecish, 1969; Stokes and Firkin, 1971; Rowley et al., 1988),but detailed knowledge on the roles played b y thrombocytes and other cells in fish blood coagulation is lacking. 1. ACTIVATIONOF THROMBOCYTES Stobbe (1963) observed in phase-contrast microscope that the remarkably large thrombocytes of the salamander, Amphiuma, have a tendency to aggregate and undergo “viscous metamorphosis.” Similar observations by Wardle (1971) on thrombocytes in lymph from the supraneural duct of the plaice (Pleuronectesplatessa) show that, in the absence of anticoagulants, thrombocytes send out filaments that attach to the glass, after which radiating fibrin threads link the thrombocytes together. Boomker (1980)briefly reports that reactive stages of thrombocytes, similar to those in avian blood, are observed in the catfish, Clarias. Phase-contrast microscopy of coagulating blood of the hagfish

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(Myxine glutinosa) blood show disintegrating leucocytes to form centers of clot retraction. The exact morphological type of leucocytes initiating the process could not be identified but probably spindle cells or lymphocyte-like cells were involved (Fange and Gidholm, 1973; Mattisson and Fange, 1977). Small amounts of ADP cause mammalian platelets to aggregate, whereas serotonin (5-HT) has a similar effect on the nucleated thrombocytes in avian blood (Stiller et al., 1975). The factors that induce aggregation of thrombocytes in fish blood are unknown. Belamarich et al. (1968) found no aggregating effect of adenosine diphosphate (ADP) on thrombocytes in any nonmammals. and contrary to mammalian platelets the thrombocytes of dogfish (Mustelus canis) do not accumulate or produce serotonin (Belamarich et al., 1962).

2. LAMELLAR INCLUSION BODIES Inclusion bodies, probably consisting of phospholipid membranes, occur in thrombocytes of lungfishes (Lepidosiren, Protopterus: Tanaka and Saito, 1981). In annelid coelomocytes somewhat similar inclusions form myelin figures during a clotting-like process (Enchytraeus: Fange, 1951). In mammalian platelets lamellar bodies are visualized by the use of tannic acid (Baker et al., 1982). The “viscous metamorphosis” of activated platelets and thrombocytes may be explained by myelin figures developing from intracellular phospholipid particles. The “platelet factor 3” functioning in mammalian blood coagulation probably is a phospholipid. 3. ANTICOAGULANTS Anticoagulants used in hematological studies on fishes are EDTA, citrates, oxalates, and heparin. Smit and Hattingh (1980) found heparin to be the most suitable anticoagulant in experiments on freshwater fishes. The anticoagulatory effect of heparin is assumed to be caused by activation of antithrombin, a protein that inhibits coagulation enzymes. Curiously, Jordan (1983) was unable to isolate antithrombin from the blood of various fish species. It seems as if in fishes the plasma contains heparin dependent coagulation inhibitory factors with properties different from those of the antithrombins of other vertebrates. VI. LYMPHOMYELOID TISSUES

Lymphocytes and other blood cells originiate from and are stored in so-called lymphomyeloid tissues. These do not always form distinct

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organs but may consist of temporary accumulations of proliferating blood cells. A continuous migration of lymphocytes unites the different structures into a “lymphomyeloid complex” (Yoffey and Courtice, 1970; Yoffey, 1985). Although lymph nodes and hemopoietic bone marrow are lacking, fishes have a rich array of lymphomyeloid structures. The thymus and the spleen belong to the lymphomyeloid complex of tissues. In total, the lymphomyeloid tissues of fishes compose 0.5-1.5% of the body weight (Fange, 1987), or about half the percentual weight of analogous tissues in mammals. A. Thymus

A thymus exists in all fishes except cyclostomes, but diffuse accumulations of lymphocytes in the gill region of larval lampreys are supposed to represent a “protothymus.” In holocephalans and chondrosteans the thymus is well developed, lobated, and organized in cortex and medulla as in mammals, but no Hassall’s corpuscles are found (Fange and Sundell, 1968; Fange, 1986b). The teleost thymus has an intraepithelial position inside the epithelium of the gill chamber (rainbow trout, Oncorhynchus mykiss: Chilmonczyk, 1985). B. Spleen

A spleen is present in all fishes, but in cyclostomes it is represented by lymphomyeloid aggregations in the typhlosole (lamprey) and the intestinal submucosa (hagfish). The structures are unlike real spleens (hagfish: Tomonaga et al., 1973; Fujii, 1982)but they may serve similar functions. In elasmobranchs, teleosts, and chondrosteans (sturgeons) the spleen consists of red and white pulp, although the boundary between these is diffuse. In elasmobranchs (sharks, rays) the lymphoid white pulp areas are more distinct than in teleosts, and in the white pulp of the spleen of rays (Dasyatis, Myliobatis) follicle-like structures occur (Tomonaga et al., 1986). However, it may be difficult to distinguish between lymphoid and erythropoietic centers. In elasmobranchs as in holocephalans (rabbit fish, Chimaera) and a series of teleost species, the spleen is the primary hemopoietic organ (Fange and Nilsson, 1985). It is also an important immune organ containing plasma cells and phagocytes and has a blood filtering, antigencapturing function.

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C. Kidney

The kidney of most fishes contains lymphomyloid tissue, but holocephalans (chimaeroids, rabbit fishes) and many elasmobranchs are exceptions. The pronephros or head kidney, the anterior part of the kidney, is a complex tissue found in many teleosts. It contains lymphomyeloid, renal and endocrine components richly supplied with blood from arteries and caudal portal veins and innervated from the sympathetic ganglionic chain. It serves as the analog of bone marrow, lymph nodes, and, in part, the adrenal gland of higher vertebrates. It is the main hemopoietic organ in many teleosts producing erythrocytes, granulocytes, lymphocytes, macrophages, thrombocytes, and plasma cells, and it is a main source of antibodies. Both granulopoietic and erythropoietic areas can be identified histologically in the pronephros of teleosts. Smith et al. (1970)describe the histology of the pronephros of the carp as resembling the subcortical region of mammalian lymph nodes, while Zapata (1981) emphasizes the similarity between teleostean pronephros and mammalian red bone marrow. In some teleosts lymphomyeloid tissue extends into areas of the ordinary kidney, the mesonephros. Like the spleen the lymphomyeloid structures of the kidney are rich in macrophages and are supposed to have a blood filtering, antigen-capturing function.

D. Lymphocytic Infiltrations Lymphocytes occur in all lymphomyeloid tissues, and these may contain follicle-like accumulations of lymphocytes, although real follicles or germinal centers, such as found in avian and mammalian lymphoid tissues, probably never occur. Lymphocytes and other types of leucocytes infiltrate mucosal and other membranes all over the organisms. They occur regularly in the intestinal mucosa and submucosa giving rise to voluminous accumulations in the spiral intestinal valve of elasmobranchs (bullhead shark, Heterodontus: Tomonaga et al., 1985; rays, Dasyatis, Myliobatis: Tomonaga et al., 1986) and chondrosteans (sturgeons, and paddlefish, Polyodon: Weisel, 1973). Rich development of intestinal lymphoid tissue as in the paddlefish may be related to the presence of parasites. In the massive lymphoid accumulations in the spiral valve of rays, follicle-like structures have been observed. Lymph node-like cell masses in the pericardium of the heart of sturgeons (Acipenser) and paddlefish (Polyodon) contain large numbers of lymphocytes that migrate through the endothelium of venous sinuses (Fange, 1986a). Interactions between immunocompe-

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tent lymphocytes and endothelial cells may have physiological implications (Baldwin, 1982).

E. Granulo(cyt0)poietic Tissues A whitish, mainly granulopoietic bone marrow-like tissue is found in the esophagus (Leydig organ), the gonads (epigonal organ), and occasionally in the kidney of elasmobranchs. Similar tissue, analogous to the red bone marrow of terrestrial vertebrates but producing leucocytes only, forms a “cartilage marrow” in recesses and canals of the skeleton of holocephalans (Chimaera)(Fange, 1987; Mattisson et al., 1990). Granulopoiesis also takes place in the meninges of chondrosteans (sturgeon, Acipenser, paddlefish, Polyodon) and holosteans (Amia,Lepisosteus). In lungfish (Lepidosiren,Neoceratodus) granulocyte producing lymphomyeloid tissue is distributed around the kidney, gonads, spleen, and pancreas, but histological investigations are impeded by the abundance of large pigmented cells (Bargmann, 1934; Rafn and Wingstrand, 1981). Extensive lymphomyeloid masses are found in the viscera of the coelacanth, Latimeria chalumnae, but complete anatomical data are missing (Millot et al., 1978; Locket, 1980).

F. Melanomacrophage Centers Accumulations of macrophages and pigment into melanomacrophage centers are found in lymphomyeloid structures in many fishes, especially in the spleen. According to one theory these centers are primitive analogues of the germinal centers or secondary follicles in avian and mammalian lymphoid tissues. However, there are major differences between melanomacrophage centers and germinal centers (Agius, 1985), and their importance is unclear. They are involved in the metabolism and store iron as hemosiderin, but they may have other functions as well. VII. HEMOPOIESIS

Hemopoiesis is the production of cells and fluid of the blood, but usually the term is restricted to cells. A. Stem Cells According to current views (Cline and Golde, 1979; Hoffbrand and Pettit, 1980) vertebrate blood cells arise from pluripotent stem cells in

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hemopoietic tissues. The concept of stem cells in mammalian hematology is founded on experiments in which cells from nonirradiated animals form colonies in the spleen when injected into irradiated animals (Weiss, 1981). Such experiments have not been made on fishes. Present knowledge about piscine stem cells is founded on analogy with mammalian hemopoiesis and indirect morphological evidence. Yoffey (1985)considers hemopoietic stem cells (mammalian) to be identical with transitional cells. These are lymphocyte-like with a basophilic cytoplasm, a leptochromatic nucleus, and a high tendency to incorporate tritiated thymidine. Attempts have been made to find and characterize hemopoietic stem cells in fishes. The descriptions by Jordan and Speidel (1924) and Jordan (1938) of “lymphoid hemoblasts” or “hemocytoblasts” indicate that these function as stem cells, but the conception of lymphoid hemoblasts is criticized by Ellis (1977). Zapata (1981) in an electron microscopic study found cells in the teleost kidney similar to transitional cells of mammalian bone marrow.

B. Tissue Microenvironment Comparative studies led Jordan (1933) to assume that erythropoiesis occurs in tissues with a sluggish or stagnant sinusoidal venous circulation, because blood with a high carbon dioxide concentration was needed for the synthesis of hemoglobin. In fishes the portal circulation of the kidney and the sinusoidal venous blood flow of the splenic red pulp seem to favor erythropoiesis. Thus in most teleosts (bony fish), in chondrosteans (sturgeons, paddlefish), and holosteans (bow fin, gar) erythropoiesis occurs in the kidney. And in other teleosts (Tautoga and Stenotomus: Jordan and Speidel, 1924; Perca: Catton, 1951; Scorpaena: Fey, 1965), in elasmobranchs (sharks, rays), and in holocephalans (rabbit fish: Chimaera) the spleen is the main erythropoietic organ. In larval lamprey (Lampetra) the typhlosole (primitive spleen), together with the kidney, is the major site of hemopoiesis (Fujii, 1982). According to Jordan (1933)erythrocytes and thrombocytes (spindle cells) form inside and granulocytes form outside the blood vascular system. The formation of leucocytes needs a sparse blood supply. Granulocytes differentiate within sparsely vascularized mesenchymal connective tissue. In accordance with these theories the granulopoietic tissues of holocephalans and elasmobranchs receive only sparse arterial supply. However, the tissues are associated with prominent venous sinuosities (Stahl, 1967; Fange, 1986a). T h e granulopoietic

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areas of the teleost pronephros would seem to have a rich blood supply, which does not fit well with the theories given. It is of interest to examine in greater detail the microcirculation and microenvironment of hemopoietic tissues in fish. Accumulation and proliferation of lymphocytes seem to occur at anatomical sites where fluids are filtered such as mucosal membranes, renal tissue, and the pericardium (chondrosteans: sturgeons, paddlefish). C. Factors Stimulating Hemopoiesis The stem cells grow, multiply, and develop into different lines of blood cells if adequately influenced. Stimulatory factors may include hormones and microenvironmental factors (suitable concentrations of oxygen, carbon dioxide, nutritional substances, metabolites). The developing blood cells seem to possess receptors for hormones such as erythropoietin, which stimulate differentiation into specific cell lines. However, whether or not a specific “leucocytopoietin” exists is an open question. Certain observations indicate that erythropoietin mechanisms exist in fishes. Anemia after bleeding or phenylhydrazine-induced hemolysis stimulates new formation of red cells and hemoglobin (Cameron and Wohlschlag, 1969; Smith et al., 1971). A few days after withdrawal of blood the number of erythroblasts and proerythrocytes increased to 55% of the total number of red cells in the blood of the eel (Anguilla anguilla) (Kreutzmann, 1976a), and in the gar (Lepisosteus platyrhincus) erythroblasts increased to 26%. In the latter case, hemopoiesis was stimulated by anemia but not by hypoxia (McLeod et al., 1978). Blood plasma from experimentally anemic fishes stimulates erythropoiesis and hemoglobin formation in the tropical fish Blue Gourami (Trichogaster trichopterus) (Zanjani et al., 1969; Yu et al., 1971), and mammalian urinary erythropoietin, although in high doses, increases erythropoiesis in the Blue Gourami (Zanjani et al., 1969)and in the tropical teleosts, Clarias batrachus and Heteropneustes fossilis (Pradhan et al., 1989). But fish erythropoietins have not been isolated and analyzed.

D. Erythropoiesis in the Peripheral Blood The late phase of fish erythropoiesis, including hemoglobin synthesis, occurs in the circulating blood. The earliest red cells in the blood are erythroblasts, spherical cells with a cytoplasm, which is rich in RNA and stains intensely blue with Giemsa. The erythroblasts possess receptors for transferrin, the iron-transporting protein of the blood

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(Fletcher and Huehns, 1968). Uptake of iron into immature red cells goes on in the blood (Tinca:Hevesy et al., 1964; Lepisosteus: McLeod et al., 1978). The immature cells continue to synthesize hemoglobin until synthesis ends, and they lose RNA and transform into adult erythrocytes. The blood of hagfish (Myxine)is the site of both the early proliferative and the late differentiating phase of erythropoiesis. It may be regarded as a fluid red bone marrow. It contains mitotically dividing erythroblasts (Mattisson and Fange, 1977). These show strong nuclear uptake of tritiated thymidine, indicating synthesis of DNA, while spindle cells show intense nuclear uptake of uridine (Fange and Edstrom, 1973). Undifferentiated spindle cells or lymphocytes may b e stem cells in the sense of Jordan’s (1938) lymphoid hemoblast theory. The spindle cells easily transform into lymphocytes (Fange et al., 1974). When stimulated by the mitogen PHA spindle cells of Myxine blood transform into erythroblast-like cells (Fange and Zapata, 1985). Several observations, although preliminary, indicate that spindle cells and lymphocytes of hagfish are undifferentiated cells with a considerable capacity of growth and differentiation. Concerning late hemopoiesis Tomonaga et al. (1973) have shown by autoradiography that in hagfish (Eptatratus burgeri) incorporation of iron into erythroblasts does not take place in the hemopoietic tissues but probably in the blood. A transferrin in the blood plasma, similar to that of other vertebrates (Aasa, 1973), probably supplies iron.

E. Toxic Effects on Erythropoiesis Chronic exposure of fish to sublethal concentrations of cadmium impedes erythropoiesis and hemoglobin formation ( JohanssonSjobeck and Larsson, 1978: Carassius; Houston and Keen, 1984). Lead, absorbed from very low concentrations in the surrounding water, inhibits the enzyme S-aminolevulinic acid dehydratase, which is involved in hemoglobin synthesis ( Johansson-Sjobeck and Larsson, 1979). Copper significantly changes hemoglobin values in the blood of freshwater fish (McKim et al., 1970; Christensen et al., 1972: Salmo, I c t a lurus).

VIII. FUTURE RESEARCH A. Gaps in Knowledge The hematology of teleostean fishes is better known than that of most other fish groups, but many dark points remain. Variations in

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terminology have created minor confusions, which ought to be overcome. Much work is required to reach a better understanding of the function and the hemopoietic origin of fish blood cells. The concept of cellular migration streams (Yoffey, 1985) may be useful in investigations on leucocytic function. The blood cells of phylogenetically important fishes are rarely studied. Although some aspects of the blood of the coelacanth, Latimeria chalumnae, are known (Weber et al., 1973; Gorr et al., 1991),the blood cytomorphology of our closest relative among fishes living now (Gorr et al., 1991) is practically unknown. Also the blood cells of dipnoans (lungfishes), holosteans (gars, bowfin), chondrosteans (sturgeons, paddlefish), and brachiopterygians (bichirs, Polypterus) have been insufficiently investigated. B. Hemopoiesis

In elasmobranchs, erythrocytes and granulocytes are produced in different tissues, whereas in teleosts both kinds of blood cells originate in the same tissue, usually the head kidney (pronephros). The factors that lead to accumulation of multipotent stem cells in certain tissues and to differentiation of these cells into different lines of blood cells are poorly known. Investigations are needed on the importance of endocrine factors (erythropoietin) and of tissue hypoxia (or hypercapnia?) for red cell formation.

C. Lymphocyte Functions Few vertebrate cells are more important than the lymphocytes. Hagfishes (myxinoids) seem central in understanding the evolution of lymphocyte functions. The conspicuous lymphocytes/spindle cells of Myxine glutinosa may play roles in both hemopoiesis and immune functions. Responses of myxinoid lymphocytes to various mitogens are important to investigate. Vertebrate lymphocytes generally are very sensitive to irradiation. The remarkably low susceptibility of hagfish lymphocytes (Finstad et al., 1969) is still waiting for explanation. Plasma cells are supposedly derived from lymphocytes (B cells) that have been activated by immunization processes. Plasma cells are found in most major groups of fishes. It is not known if cells, identified as plasma cells on morphological criteria, are the only source of Ig in fishes. Investigations on functional categories of fish lymphocytes are in a preliminary stage. A thymus probably exists in all fishes except cyclostomes, but it is not clear if it functions in maturation of T lymphocytes

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as in mammals. The existence of cytotoxic lymphocytes, NK cells, and similar cells in fishes has to be further investigated. D. Blood Coagulation

The importance of activation of thrombocytes (spindle cells) or other types of cells during fish blood coagulation is poorly understood. The number and chemical nature of the coagulation factors are imperfectly known, and the clotting mechanisms may differ between systematic groups of fishes. Facts are few and need to be completed.

E. Granulocytic Function The abundant and prominent granulated white blood cells of elasmobranchs and dipnoans may have functions that are still unknown. Probably, together with lymphocytes and macrophages, they participate in the defense against parasites and microbes. The great diversity of granulocyte types may indicate a variety of functions. Enzymes released from granulocytes could influence growth or repair in the tissues, blood coagulation, and so on. In spite of some histochemical work having been done, the composition and function of substances, which may be released from the leucocytic granules, are practically unknown. The blood and the granulocyte-producing lymphomyeloid tissues of large sharks may be rich sources for investigations of the properties of leucocytic granules. In this connection it is well to remember that sharks, according to some popular views, are remarkably free from cancer diseases. However, scarce tumors, probably benign, have been observed (Harshbarger, 1981). Certain types of teleost granulocytes may kill ingested bacteria by mechanisms similar to those of mammalian neutrophils. It is not known if the eosinophils of elasmobranchs and labrids (and some other teleosts) resemble the mammalian eosinophils in showing activities directed against parasites. The “heavy granulocytes” and other odd types of fish granulocytes require further investigations.

F. Electron Microscopy The results of electron microscopical studies need to be better correlated with those obtained by light microscopy. Probably electron microscopy, in combination with experimental methods, will be of use to improve the complicated classification of fish granulocytes.

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G. Immune System of Long-Lived Fishes

Large fishes with a long life presumably have evolved more efficient immune mechanisms than small short-lived forms. Long-lived species are found among chondrosteans (sturgeons), elasmobranchs (sharks), dipnoans (lungfishes), and certain teleosts (pike, Esox Zucius; the wels, Silurus glanis). Certain chondrosteans such as the American white sturgeon, Acipenser montanus, get sexually mature at around 20 years of age and may live more than 80 years (Gahlbreath, 1979). During their long, life sturgeons must resist influences from bacteria, viruses, parasites, and cancerogenic agents. However, few works have been done on the hematology and immunology of sturgeons.

H. Microcirculation of Hemopoietic Tissues The distribution of lymphomyeloid tissues at different anatomical sites of the fish organism is not only a comparative anatomical problem. The fact that erythropoiesis predominates in some tissues and lymphopoiesis or granulopoiesis in others may partly be due to local differences in circulation. Studies of the tissue microenvironment of proliferating and differentiating stem cells is an important research task.

I. Cell Interactions In red bone marrow erythroblasts agglutinate around macrophages forming erythroblastic islands (Bernard, 1991), nurse cells and lymphocytes interact in the thymus (Wekerle and Ketelsen, 1980), and something goes on between lymphocytes and capillary endothelial walls, perhaps production of substances causing lymphocyte accumulation (Baldwin 111,1982).These examples are from mammals. Studies of analogous phenomena in fishes may enlighten general biological problems.

ACKNOWLEDGMENTS I thank A. Mattisson for electron micrographs and for reading the manuscript, Inger Holmqvist for technical work, and D. I. Randall for constructive advice.

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Morrow, W. J. W., and Pulsford, A. (1980).J.Fish Biol. 17,461-475. Mulcahy, M. F. (1970).J.Fish B i d . 1,333-338. Murad, A., and Mustafa, S. (1988).J.Fish Dis. 11,365-368. Murray, C. K., and Fletcher, T. C. (1976).J.Fish Biol 9,329-334. Nash, K. A., Fletcher, T. C., and Thomson, A. W. (1987). Comp. Biochem. Physiol. B 86, 31-36. Nelson, J. S. (1984). “Fishes of the World,” 2nd ed. John Wiley & Sons, New York. Nikinmaa, M. (1990). “Vertebrate Red Blood Cells.” (Zoophysiology 28) SpringerVerlag, Berlin-Heidelberg. Ogata, H., and Murai, T. (1988).]. Fish Biol. 33,471-479. Ohno, S., and Atkin, N. B. (1966).Chromosorna 18,455-466. Ourth, D . D. (1980). Deo. Comp. Zmmunol. 4,65-74. Page, M., and Rowley, A. F. (1983).A cytochemical, light and electron microscopical study of the leucocytes of the adult river lamprey, Lampetrafluviatilis (L. Gray). J. Fish Biol. 22,502-517. Page, M., and Rowley, A. F. (1984).J.Fish Dis. 7,339-353. Parish, N., Wrathmell, A., Hart, S., and Harris, J. E. (1986).J.Fish Biol. 28,545-561. Parker, W. N. (1892). Trans. Roy. Zrish Acad. (Dublin)30, Part 111, 109-130. Pedersen, R. A. (1971).J.E x p . Zool. 177,65-78. Peterson, A. J., and Poluhowich, J. J. (1976). Comp. Biochern. Physiol. A 55,351-354. Phromsuthirak, P. (1977).J. Fish Biol. 11,193-206. Pica, A., Grimaldi, M. C., and Della Corte, F. (1983). Monitore Zoo/ Ztal ( N . S . ) 17, 353-374. Pitombeira, M. S., Barrets Gomes, F. V., and Martins, J. M. (1971). Mar. B i d . (Berlin)9, 250-252. Plytycz, B., Flory, C. M., Galvan, I., and Bayne, C. J. (1989).Dec. Comp. Zmmunol. 13, 217-224. Potter, I. C., Percy, L. R., Barber, D. L., and Macey, D. J. (1982). In “The Biology of Lampreys” (M. W. Hardisty, and I. C. Potter, eds.), Vol. 4A, pp. 233-292. Academic Press, London. Pradhan, A. K., Saini, S. K., Biswas, J., and Pati, A. K. (1989).Gen. Comp. Endocrinol. 76, 382-389. Rafn, S., and Wingstrand, K. G. (1981). Zool. Scripta 10,223-239. Raison, R. L., and Hildemann, W. H. (1984).Dev. Comp. Immunol. 8,99-108. Ratcliffe, N. A., and Millar, D. A. (1988).In “Vertebrate Blood Cells” (A. F. Rowley and N. A. Ratcliffe, eds.), pp. 1-17. Cambridge University Press, Cambridge. Reznikoff, P., and Reznikoff, D. G. (1934). Biol. Bull. 66, 115-123. Rodger, H. D., Deinan, E. M., Murphy, T. M., and Lunder, T. (1991).Bull. Eur. Ass. Fish Pathol. 11, 108-111. Roubal, F. R. (1986).J . Fish Biol. 28,573-593. Rowley, A. F., Hunt, T. C., Page, M., and Mainwaring, G. (1988).In “Vertebrate Blood Cells” (A. F. Rowley, and N. A. Ratcliffe, eds.), pp. 19-127. Cambridge University Press, Cambridge. Rubashev, S. I. (1969). Trydy Borodin. Biol. S t . (Proc. Biol. Station, Borodino) 9(1), 61-69; (Fisheries Res. Bd. Canada. Translation Series No. 1312). Sanchez, I., and Cohen, W. D. (1988).B i d . Bull. 175,302. Saunders, D. C. (1966a).Trans. Amer. Microsc. Soc. 85,427-449. Saunders, D. C. (1966b).Elasmobranch blood cells. Copeia (2), 348-351. Savage, A. G. (1983).J . Morphol. 178,187-206. Schmidke, J., Schmitt, E., and Engel, W. (1978).Comp. Biochem. Physiol. B 64,117-120.

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Schumacher, R. E., Hamilton, C. H., and Longtin, E. J. (1956). Progr. Fish Cult. 18, 147- 148. Scott, A. L., Rogers, W. A,, and Klesius, P. H. (1985).Deu. Comp. Immunol. 9,241-250. Scott, E. M., and Harrington, J. P. (1990). Comp. Biochem. Physiol. B 95,91-93. Sherburne, S . W. (1973).Fishery Bull. 71, 1011-1017. Shrivastava, A. K., and Griffith, R. W. (1974). Copeia (l),136-141. Sindermann, C. J., and Krantz, G. E. (1968). Chesapeake Science 9,94-98. Smit, G . L., and Hattingh, J. (1980).J . Fish B i d . 17,337-341. Smith, A. M., Potter, M., and Merchant, E. B. (1967).J . Immunol. 99,876-882. Smith, A. M., Wivel, N. A., and Potter, M. (1970).Anat. Rec. 167,351-356. Smith, C. E., McLain, L. R., and Zaugg, W. S. (1971). Toxicol. Appl. Pharmacol. 20, 73-81. Soivio, A., and Oikari, A. (1976).J.Fish B i d . 8,397-411. Stahl, B. (1967). Bull. Mus. Comp. Zool. Hart;ard 135, 141-213. Starmach, J . (1970).Acta Biol. Cracooiensia Series: Zoologia 13,243-249. Stave, J . W., and Roberson, B. S. (1985). Det;. Comp. Immunol. 9 , 7 7 4 4 . Stave, J. W., Roberson, B. S., and Hetrick, F. M. (1984).J.Fish Biol. 25, 197-206. Stiller, R. A., Belamarich, F. A,, and Shepro, D. (1975).A m . J . Physiol. 229,206-210. Stobbe, H. (1963). Folia Haematol. 80,452-462. Stokes, E. E., and Firkin, B. G. (1971).Brit.J.Haematol. 20,427-435. Suzuki, K. (1986).J . Fish Biol. 29,349-364. Suzuki, Y. (1986). Bull.Jap. Soc. Sci. Fish. 52,1895-1899. Suzuki, Y. (1988). Bull. Jap. Soc. Sci. Fish. 54, 1257. Tanaka, Y., and Saito, Y. (1981).J.Electron Microsc. 30,253-273. Tavassoli, M., and Yoffey, J. M. (1988).“Bone Marrow. Structure and Function.” Alan R. Liss, Inc., New York. Thoenes, G. H., and Hildemann, W. H. (1970). In “Developmental Aspects ofAntibody Formation and Structure” (Sterz et al., eds.), Vol. 2, pp. 711-726. Czechoslovak Akad. Sci./Academic Press, Prague, New York. Thomas, N. W. (1971).J.Cell Sci. 8,407-412. Tomonaga, S., Hirikane, T., Shinohara, H., and Awaya, K. (1973).2002.Mag. (Tokyo)82, 215-217. Tomonaga, S., Kobayashi, K., Hagiwara, K., Sasaki, K., and Sezaki, K. (1985).Det;. Comp. Immunol. 9,617-626. Tomonaga, S., Kobayashi, K., Hagiwara, K., Yamaguchi, K., and Awaya, K. (1986). 2001. Science (lapan) 3,453-458. Tufts, B. L., and Randall, D. J. (1988). Can.1.Zoo/. 67,235-238. Undritz, E. (1963). The Physician’s Panorama (Sandoz) Febr., 4-5. Val, A. L., D e Almeida-Val, V. M., and Affonso, E. G. (1990). Comp. Biochem. Physiol. B 97,435-440. Vendrely, R. (1955).In “The Nucleic Acids” (E. Chargaff, and J. N. Davidson, eds.), Vol. 2, pp. 155-180. Academic Press, New York. Vislie, T. (1978). Comp. Biochem. Physiol. B 60, 35-40. Vogel, V. 0. P., and Claviez, M. (1981).Z. Naturforsch. 36,490-492. Vos, J . , Van Loveren, H., Wester, P., and Vethaak, D. (1989).TZPS 10,289-292. Walker, R. L., and Fromm, P. 0. (1976).Comp. Biochem. Physiol. A 55,311-318. Ward, J . W. (1969). Copeia (3), 633-635. Wardle, C. S. (1971).J.Mar. Biol. Ass. U . K . 51,977-990. Warr, G. W. (1983). Comp. Biochem. Physiol. B 76,507-514. Warr, G. W., and Marchalonis, J. J. (1977). Deo. Comp. Immunol. 1, 15-22.

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2 CHEMICAL PROPERTIES OF THE BLOOD D. G. MCDONALD Department of Biology McMaster University Hamilton, Ontario, Canada

C . L. MZLLZGAN Department of Zoology University of Western Ontario London, Ontario, Canada

I. Introduction A. Effects of Sampling Method on Blood Chemistry B. Die1 Cycles in Blood Chemistry 11. Hormones: Teleosts A. Gonadotropins and Sex Steroids B. Growth Hormone C. Prolactin D. Arginine Vasotocin E. Melatonin F. Thyroid Hormones G. Calcitonin H. Cortisol I. Catecholamines J. Pancreatic Hormones K. Stanniocalcin L. Urotensins 111. Hormones: Cyclostomes and Chrondricthyes IV. Metabolites A. Glucose B. Lactate C. Ethanol D. Ketone Bodies E. Bile Pigments V. Nonprotein Nitrogenous Compounds A. Amino Acids B. Ammonia C. Urea and Uric Acid 55 FISH PHYSIOLOGY, VOL. X l l B

56

D. G . MCDONALD AND C. L. MILLIGAN

D. Trimethylamine Oxide E. Creatine and Creatinine VI. Plasma Proteins A. Total Plasma Protein B. Albumin C. Immunoglobulins D. Hormone-Binding Proteins E. Antifreeze Proteins F. Plasma Enzymes VII. Lipids A. Total Lipids B. Lipoproteins C. Cholesterol D. Nonesterified Fatty Acids E. Triglycerides VIII. Electrolytes A. Na+, C1-, and Osmolarity B. Calcium C. Magnesium D. Potassium E. Phosphate and Sulfate References

I. INTRODUCTION In this Chapter our objective is to compile normal values for such blood variables as hormones, metabolites, proteins, lipids, and electrolytes as well as variations resulting from such factors as temperature, hypoxia, exercise, salinity, feeding, development, and reproductive state. To make this review comprehensive is essentially an impossible task as there are over 25,000 species of fish in 6 major groups (cyclostomes, chrondricthyes, dipnoans, chondrosts, holosts, and teleosts) and there is a considerable bias toward species that are readily available and of economic importance. Most data are for salmonids, and there is a considerable bias toward species that are readily available and of economic importance. Most data are for salmonids, al., 1988; Bergheim et al., 1990). Thus, we have not attempted to compile all available data but rather report the “best” or “most representative” available value for each variable, some estimate of its maximum range in the healthy organism, and the condition(s) under which the maxima are reached. To this end, we have used our judgment as to what are representative values. Furthermore, we have tended to emphasize those areas of blood chemistry that have previously been neglected. Consequently, our emphasis is on plasma levels of hormones and certain key metabolites, while

2.

CHEMICAL PROPERTIES OF THE BLOOD

57

our treatment of such topics as electrolytes and acid-base chemistry is highly selective because this subject has already been comprehensively reviewed (see, in particular, Chapters by Holmes and Donaldson in Volume 1and Heisler in Volume XA). Irrespective of the endogenous or exogenous factor of interest, attention must first be paid to two factors that have profound influence on blood chemistry: the method employed to obtain the blood sample for analysis and the time of day the samples are collected. A. Effects of Sampling Method on Blood Chemistry It is now well established that blood chemistry is extremely sensitive to sampling procedure (e.g., Railo et al., 1985; Laidley and Leatherland, 1988a; Houston, 1990). Two approaches to blood sampling are in routine use: acute sampling of stunned or anesthetized fish via either cardiac or caudal puncture, or caudal severence; and chronic sampling via an indwelling catheter, usually implanted in the dorsal aorta. Acute sampling procedures have often been successful in obtaining resting or routine levels of blood variables but potentially alter blood chemistry more than chronic procedures. The trauma associated with capture, handling, and sampling can activate physiological stress responses that will have an effect on blood chemistry. This can become compounded if the sampling duration on each fish is greater than about 30 sec and if the sampling protocol involves serial removal of individual fish from the same tank (Laidley and Leatherland, 1988a). The initial physiological effect of primary importance is the mobilization of catecholamines, particularly adrenaline. Adrenaline will cause mobilization of red cells from the spleen and red cell swelling (Nikinmaa and Heustis, 1984) thus elevating hematocrit. It will also increase blood pressure and gill blood flow, which will, in effect, increase electrolyte permeability of the gills and can quickly cause a depression in plasma Na+ and C1- in freshwater teleosts (Gonzalez and McDonald, 1992) or a net gain in seawater teleosts (Boutilier et al., 1984; Wells et al., 1986). If the agitation of the remaining fish is prolonged beyond 5 min or so, then significant elevation in white muscle lactate levels will occur. This will cause acid-base disturbances, elevation in plasma lactate, and a shift of fluid to the intracellular compartment, concentrating most plasma constituents. While the amount of the disturbance can vary from species to species and with sampling methodology, it is generally agreed that repetitive sampling by acute methodology is inappropriate because of the long-term effects of the stress on a single sample. For example, Pickering et al. (1982) found in brown trout, Salmo trutta,

58

D. G. MCDONALD A N D C. L. MILLIGAN

that a minimum of 2 wk was required for complete recovery of all blood parameters from a 2-min bout of handling stress. Chronic indwelling catheters are the method of choice for obtaining resting levels of blood parameters (in particular for catecholamines), but they are also not entirely free from stress. A certain amount of blood loss is inevitable during surgery, and these losses will increase with repetitive blood sampling and with procedures required to keep the catheters clear and working. Hematocrits are typically below average as a result. Although some splenic compensation for blood loss can be expected (Pearson and Stevens, 1991),the reduction in blood volume will produce changes in physiological state such as renin release and activation of angiotensin I1 (Bailey and Randall, 1981). The procedure of anesthesia and surgery is stressful in itself and, at least in salmonids, up to 4 days is required for plasma electrolytes, metabolites, and acid-base status to return to normal (Heisler, 1984). Also, chronic catheterization is impractical for animals much smaller than 100 g. The effects of anesthesia on blood chemistry have been studied extensively; see Laidley and Leatherland (1988a), Iwama et al. (1989), and Summerfelt and Smith (1990) for discussions. B. Die1 Cycles in Blood Chemistry The levels of many plasma constituents exhibit daily (diel) variations that can modify and complicate any analysis of the influence of environmental factors. These diel fluctuations are often considered endogenous. This designation, however, requires that they be shown to free-run under constant conditions, and this has not been demonstrated in most instances. Hence their designation as diel cycles is more appropriate (Laidley and Leatherland, 198813). Daily fluctuations in plasma cortisol concentration are one of the most studied and reproducible of the cyclic phenomena (Peter et al., 1978; Spieler, 1979; Bry, 1982; Pickering and Pottinger, 1983; Thorpe et al., 1987; Laidley and Leatherland, 1988b; Planas et al., 1990). This has led to the suggestion that plasma cortisol might be one of the endogenous variables to which other endocrine and metabolic rhythms are tied (Meier, 1984). Entraining stimuli include the day/ night cycle and time of feeding. The cortisol peak typically occurs just before the onset of light and precedes an increase in locomotory activity (Spieler, 1979) although there is a wide variation in the frequency, amplitude, and phasing of such rhythms (Pickering and Pottinger, 1983).

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CHEMICAL PROPERTIES OF THE BLOOD

59

In fishes, other hormones reported to show diel cycling include prolactin (Spieler, 1979), growth hormone (Bates et al., 1989), thyroid hormones (White and Henderson, 1977; Spieler and Noeske, 1979; Cook and Eales, 1987), gonadotropin (Hontela, 1984; Zohar and Billard, 1984), gonadal steroids (Lamba et al., 1983; Zohar and Billard, 1984), insulin (Gutikrrez et al., 1984), and melatonin (Gern et al., 1978). It is significant, however, that in many studies, diel endocrine rhythms have been looked for but not found. Two reasons can be suggested for their apparent absence. In some instances the rhythms may be present but masked by the method of blood sampling. In those studies where blood has been sampled acutely and each animal sampled only once, rhythmicity would not be detected unless the population was synchronized. In other studies where animals are repetitively sampled by means of indwelling catheters, the rhythms may be masked by the effects of the surgery and serial sampling on the physiology and behavior of the fish. In other instances, however, the absence of the rhythms may be real. For example, the intensity of the cortisol rhythm fluctuates with the season and may disappear in winter months under short photoperiod conditions (Rance et aZ., 1982) and may also fluctuate with the age and sex of fish (Peter et al., 1978).Also, Marchant and Peter (1986) were unable to find a reproducible daily rhythm in circulating growth hormone in goldfish (Carassius auratus) at any time of the year even though Bates et al. (1989), using a similar methodology, found a pronounced nocturnal peak in growth hormone in coho salmon (Oncorhynchus kisutch). The frequent appearance of diel fluctuations in many of the circulating hormones are often, not surprisingly, accompanied by fluctuations in other plasma constituents. For example, daily fluctuations in plasma glucose and plasma lipids in goldfish appear related to cortisol and thyroid hormone fluctuations (Delahunty et al., 1978). Similarly, in sea bass (Dicentrarchus Zabrax) there were significant and inverse rhythms in glucose and insulin with glucose peaking during the day and insulin peaks during the dark period (Gutikrrez et al., 1984). Although these fluctuations were tied to feeding times, their continuation during a fast of 7 days indicates an endogenous rhythm. Diel fluctuations in plasma protein in rainbow trout (Oncorhynchus mykiss) were significantly correlated with fluctuations in thyroid hormone levels (Laidley and Leatherland, 1988b). Diel fluctuations in plasma electrolytes (Na+,Ca2+,Mg2+,K+ ) have also been reported (Toews and Hickman, 1969; Houston and Koss, 1982; Carillo et al., 1986; Laidley and Leatherland, 1988b; Peterson

60

D. G . MCDONALD AND C. L. MlLLIGAN

and Gilmore, 1988) although, to this point, they have not been consistently linked to hormonal fluctuations (Kiihn et al., 1986; Laidley and Leatherland, 1988b). Daily temperature fluctuations appear to be an important entraining stimulus for electrolyte fluctuations (Toews and Hickman, 1969; Houston and Koss, 1982), but the rhythms persist in a constant temperature environment indicating their circadian nature (Houston and Koss, 1982). A recent report suggests that electrolyte rhythms are more pronounced in euryhaline species inhabiting estuarine environments than in stenohaline freshwater species (Peterson and Gilmore, 1988). Interpreting daily endogenous variations in plasma constituents is further complicated by the presence of additional cycles with longer period lengths. These include cycles related to the tides, phases of the moon (i.e., lunar cycles), and seasonal variations in photoperiod and/or temperature, particularly in temperate zone animals. The latter variations have prominent effects on growth rate and on reproductive status, and thus it is not surprising to find significant seasonal fluctuations in virtually all plasma constituents. For example, lunar cycles have been reported for thyroid hormones and some related metabolites such as plasma glucose and triglycerides in salmonids (Grau et al., 1981; Hopkins and Sadler, 1987; Farbridge and Leatherland, 1987) and for gonadal steroids in the semilunar spawning mummichog, Fundulus heteroclitus (Taylor, 1984). Other examples include seasonal changes in growth hormone in goldfish related to changes in day length (Marchant and Peter, 1986), in cortisol in trout and sea bass related to water temperature (Pickering and Pottinger, 1983; Thorpe et al., 1987; Planas et al., 199O), in gonadotropins and gonadal steroids related to either water temperature or photoperiod (Peter, 1981; Crim, 1982; Zohar and Billard, 1984), and in electrolytes associated with vitellogenesis (e.g., Carillo et al., 1986). In the following text we have avoided reporting data where the effects of sampling may, in our judgment, have been prominent. As for the circadian fluctuations, we report the amplitude wherever possible. However, it is common practice to sample fish at the same time of the day so as to minimize contributions from die1 fluctuations. 11. HORMONES: TELEOSTS

There has been a considerable increase in information concerning the plasma levels of hormones in fishes over the last decade associated in large part with improvements in measurement technologies. Com-

2.

CHEMICAL PROPERTIES OF THE BLOOD

61

mercial kits developed for mammalian plasma have been exploited, particularly in those instances where the hormones are identical between fish and mammals (e.g., steroid hormones, thyroid hormones, catecholamines, melatonin). There have also been a number of sensitive homologous radioimmunoassays (RIAs) developed for various fish hormones, although such assays apply to relatively few species, almost exclusively teleosts, and mostly salmonids and cyprinids. This discussion is restricted mainly to the hormone measurements that have been validated for a particular fish species. Table I summarizes the teleost hormones reviewed here. Hormones of cyclostomes and elasmobranchs are summarized briefly later. Also, there are a number of real or putative hormones that we have chosen to leave out because only limited information is available. Fish endocrinology is a rapidly changing field and rather than report information that may soon be out of date we have restricted ourselves to those hormones for which there is already extensive literature. The group not covered includes the hypothalamic releasing hormones, some of the trophic hormones (adrenocorticotropic hormone [ACTH], thyroid stimulating hormone), melanocyte stimulating hormone, other neuropeptides, and the gut peptides (e.g., cholecystokinin and gastrin). We have also not covered the renin-angiotensin system (RAS) or atrial natriuretic factor (ANF or atriopeptin) as these are comprehensively reviewed by Olson (see Chapter 3). There is no universal standard for reporting plasma hormone concentrations at the present time so we have adopted the common practice of reporting nanogram or picogram quantities for most hormones except for the catecholamines, which are reported in nanomolar quantities. Table I lists molecular weights (where known) for teleost hormones. A. Gonadotropins and Sex Steroids Studies on gonadotropins (GtH) have been largely confined to salmonid and cyprinid species (see Billard et al., 1978; Peter, 1981; Idler and Ng, 1983; Hontela, 1984; Zohar and Billard, 1984; Donaldson, 1990 for reviews); species for which there are homologous RIAs (see Table I for references). The sex steroids have been examined in a much wider range of species (see Fostier et al., 1983 in Volume IXA for an extensive review), but comments here are largely confined to salmonids and cyprinids. There has been a considerable controversy over whether there are one or two GtHs in teleosts. Although initially it was believed that

Table I Partial Survey of Hormones of Teleost Fishes Glandular tissue Pituitary Pars distalis

Hormone

Nature

MW

Measurement

Gonadotropin(s) (GtH)

Glycoprotein

Growth hormone (GH)

Protein

27 K

RIAs for carp', Pacific salmon', 3, cod4

Prolactin (Prl)

Protein

25 K

RIAs for tilapia', Pacific salmon2, eel3

Adrenocorticotropic hormone (ACTH) Thyrotropic hormone VSH) Melanocyte stimulating hormone (aMSH) Arginine vasotocin (AVT) Melatonin (MLT)

Peptide

45 K

Mammalian RIA validated for salmonids In oioo bioassay

Triiodothyrouine (T,) Thyroxine (T4)

36-56 K

RIAs for salmonids',2. 3 ; cyrinids4, 5 , ', eel7, catfish*

Q,

E3

Pars intermedia Pars nervosa Pineal Thyroid

Glycoprotein

-30 K

Peptide

13 aas

Octapeptide

1050

Indoleamine

232

RIA

Iodiuated tyrosine derivatives

651 777

KIA

Mammalian RIA validated for salmonids RIA

References 1. Crim et ul., 1973; 2. Breton and Billard, 1977; 3. Suziiki et al., 1988; 4. Breton et al., 1971; 5. Crim et al., 1976; 6. Hontela and Peter, 1978; 7 . Dufour et al., 1983; 8. Goos et al., 1986 1. Cook et al., 1983; 2 . Wagner and McKeown, 1986; 3. Bolton et al., 1986; 4. RandWeaver et al., 1989 1. Nicoll et d.,1981; 2. Hirano et al., 1985; 3. Suzuki and Hirano, 1991 Sumpter and Donaldson, 1986

Swanson et al., 1989 Rodrignes and Sumpter, 1984 Holder et al., 1982; Hontela and Lederis, 1985 Gern et al., 1978; Kezuka et al., 1988 Commercial kits available

Ultimobrancial bodies Interrenal tissue Chromaffin tissue

Pancreas

Q,

W

Corpuscles of Stannius Gonads Ovary Testes

Caudal urophysis

Calcitonin (CT)

Peptide

Cortisol Adrenalin (-4)

C21 steroid Catecholamine

362.5 183.2

Noradrenaline (NA) Insulin (INS)

Catecholamine Peptide

169.2 5784

Glucagori (GLU)

Peptide

3508

Glucagon-like peptide (GLP) Somatostatin (SST-25) Stanniocalcin (STC, formerly hypocalcini teleocalcin)

Peptide Peptide Glycoprotein

Estrogen (17pestradiol) Androgens (11-keto testosterone) Urotensin I (UI) Urotensin I1 (UII)

C19 steroid C19 steroid

41 aas 12 aas

3432

RIAs for salmon’ and eel” RIA HPLC’, fluorimetric2 or radio-enzymatic methods”

1. Deftos et al., 1974; 2. Orimo et al., 1977 Commercial kits available 1. Woodward, 1982; 2. Nakano and Tomlinson, 1967, 3. Peuler and Johnson, 1977

31 aas

Very similar to inanimalian glucagon’, RIA for salmon2 RIA for salmon

1. Plisetskaya et al., 1986a; 2. Gutierrez et al., 1984; 3. Thorpe and Ince, 1976 1. Gutierrez et al., 1986; 2. Plisetskaya et al., 1989 Plisetskaya et al., 1989

25 aas 52 K

RIA for salmon ELISA for salmon

Plisetskaya et al., 198613 Mayer-Gostan et al., 1992

RIA RIA

Commercial kits available Commercial kits available

RIA for white sucker RIA for white sucker

Suess et al., 1986 Kobayashi et al., 1986

272 302

4864 1351

RIAs for chum salmon’, bonito (tuna)’, cod”

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D. G . MCDONALD AND C. L. MILLIGAN

there was only one GtH (see review by Peter and Crim, 1979), Idler and N g (1979) demonstrated the presence of two GtHs, one rich in carbohydrate (Con A-I1 or maturational GtH) and one low in carbohydrate content (vitellogenic GtH). Further research has provided evidence that the two active gonadotropins in teleosts are, in fact, both glycoproteins (GtH I MW 50,000, GtH 11, MW 36,000 in chum salmon [Oncorhynchus keta]; Suzuki et al., 1988; 56,000 and 53,000 in the grass carp [Ctenopharyngodon idell]; Yu and Shen, 1989). GtH I is mainly secreted during early gonadal development, whereas GtH I1 is secreted at the time of spermiation and ovulation (Kawauchi et al., 1989). GtH I1 is comparable to the previously isolated maturational GtH and is biochemically similar to mammalian leutinizing hormone (Kawauchi et al., 1989) whereas GtH I (similar to mammalian follicle stimulating hormone) is newly identified and, therefore, its plasma concentrations are not widely reported (Yu and Shen, 1989). T h e principal circulating sex steroids in male teleosts are the androgens, 11-keto-testosterone (KT) and testosterone (T) (in order of importance, Zohar and Billard, 1984; Barry et al., 1990), while in female teleosts it is 17p estradiol (Ez).Final maturation in males and females in many teleosts is brought about by the progestogen, 17a, 200dihydroxyprogesterone (P) (Donaldson, 1990). Significant circulating levels of T are also found in females because it is the immediate precursor to Ez. In sexually immature animals the plasma levels of gonadotropins and sex steroids are either very low or undetectable; less than 1 ng ml-' for GtHs, 0.2-0.3 ng ml-' for the sex steroids. Sexual maturation is associated not only with an increase in the average plasma concentrations of gonadotropins and sex steroids but also with an increase in the frequency and amplitude of daily fluctuations in plasma levels (Hontela, 1984; Zohar and Billard, 1984). Timing of gonadrotropin release and steroidogenesis in temperate zone teleosts is primarily controlled by variations in temperature and photoperiod (Peter, 1981;Crim, 1982). Salmonids, spawning mainly in autumn and winter, are cued primarily by decreasing day length whereas cyprinids, spawning in spring and summer are more dependent on increasing temperature (Billard et d.,1978). In all fish, plasma GtH levels (GtH 11) increase first gradually during the major part of gonad development in both sexes (i.e., vitellogenesis and spermatogenesis) and then sharply around the time of oocyte maturation and ovulation and before the start of spermiation. In salmonids the concentration of maturation GtH is elevated for several days before, during, and after ovulation whereas in cyprinids plasma GtH changes tend to be restricted to a brief surge associated with final

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maturation and ovulation (Stacey et al., 1979; Donaldson, 1990). In salmonids there are considerable interspecific and strain differences in GtH levels. Domesticated species show less of an elevation than natural spawning wild strains (Billard et al., 1978), and species that spawn only once have substantially higher GtH levels during this period than repeat spawners (Suzuki et al., 1988).The maximum daily average level may be as little as 10 ng ml-' in domesticated species and up to 10 times higher in some other salmonid species allowed to spawn naturally (Zohar and Billard, 1984; Dye et al., 1986). In cyprinids the GtH peaks range from 30 to 160 ng ml-' with no major differences apparent between males and females (Billard et al., 1978; Stacey et al., 1979; Hontela and Peter, 1983; Barry et al., 1990). Considering the cyclic nature of plasma GtH and the effects of such exogenous factors as water temperature on GtH levels (Peter, 1981), this amount of variation in GtH levels among studies is not surprising. In female teleosts plasma Ez and T gradually increase during vitellogenesis, followed, in many cases, by a decrease in Ez before and during oocyte maturation. There is also a temporary increase in T before P increases, which induces oocyte maturation. In males, KT and T rise during spermatogenesis with KT reaching a maximum just prior to the start of spermiation. The concentrations of both androgens decrease during spermiation, whereas P shows an important rise related to its putative role in regulation of spermiation and control of male spawning behavior (see Barry et al., 1990 for a discussion). However, P is not the final maturation inducing steroid in all teleosts, and absolute plasma steroid concentrations vary dramatically among families. For example, salmonids tend to have high steroid concentrations and cyprinids low concentrations (Donaldson, 1990). In male salmonids, maximum values for the androgens approach 45 ng ml-' (Billard et al., 1978; Zohar and Billard, 1984) while in females the peak estrogen levels are around 30 ng ml-' (Dickhoff et al., 1989) and progestogen levels are very high; 300-600 ng ml-' at the peak in the preovulatory period (e.g., Scott et al., 1982; Dye et al., 1986). In male cyprinids androgen levels are lower than in male salmonids. For example, peak T and KT levels were 12 and 25 ng ml-', respectively, in the male cyprinids (Barry et al., 1990) whereas in female cyprinids E and P levels rarely exceed 10 ng ml-' (Peter et al., 1984; Venkatesh et al., 1989). B. Growth Hormone Growth hormone (GH) is a protein hormone molecular weight (MW) of Atlantic salmon [Salmo salar] GH is 23,000; Skibelli et al.,

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1990) released by GH-specific cells in the rostra1 pars distalis (RPD) of the pituitary. Homologous RIAs have now been developed for carp, salmon, and cod (see Table I for references). Detection limits vary from <1 to 3 ng m1-l. Growth hormone levels in resting fish in freshwater can be as low as 5 ng ml-'. However, levels can increase from this baseline with starvation (6-fold elevation after 30 days starvation in rainbow trout, Barrett and McKeown, 1988), exercise (&fold elevation after 24 h at 1.5 body lengths sec-'; Barrett and McKeown, 1988), seawater transfer (2- to 3-fold elevation after 2 days in seawater (SW); rainbow trout, Sakamoto et al., 1990; coho salmon, Sweeting and McKeown, 1987), smoltification @-fold elevation during parr-smolt transformation in Atlantic salmon reared in freshwater; Prunet et al., 1989); rearing at a higher temperature (&fold higher in rainbow trout at 16 versus 5°C; Barrett and McKeown, 1989), or at different times of the day (8-fold difference in juvenile coho salmon between daytime minimum and midnight maximum; Bates et al., 1989). There is also a consistent relationship between GH levels and the age (size) of the fish (B. A. McKeown, personal communication) with small fish invariably showing much higher GH circulating levels. In addition to daily variations, there are variations associated with changing season and daylength. In goldfish, the seasonal peak in plasma GH levels occurs in spring to late summer where levels of GH are about 2-fold higher than seasonal mimima in November (Marchant and Peter, 1986). C . Prolactin

Prolactin (PRL) is a protein hormone (MW of chum salmon PRL -23,000; Kawauchi et al., 1983) released by prolactin specific cells in the RPD of the pituitary. It is very similar in structure to GH from which it is thought to have arisen by gene duplication (Hirano et al., 1987). Homologous RIAs are available for a few species, all euryhaline teleosts (tilapia, salmon, and eel; see Table I for references). All measurements point clearly to high PRL levels in freshwater fish and low levels in seawater fish. In all seawater fish, PRL levels are at or below detection limits of the RIAs (0.1-1.0 ng ml-l) and rise on transfer to fresh water; to 5-15 ng ml-' in salmon and eels (Hirano, 1986;Ogasawara et al., 1989;Fargher and McKeown, 1990; Suzuki and Hirano, 1991)and to 60 ng ml-' in tilapia (Sarotherodonmossambicus; Nicoll et al., 1981).The higher PRL levels in tilapia may be related to the more critical dependence of this species on PRL for freshwater survival. Salmon and eels survive in fresh water without prolactin (i.e., after hypophysectomy) while tilapia do not (Hirano, 1986; Bern, 1990).

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The major stimulus to prolactin release appears to be a reduction of plasma osmolarity, and/or specific ions such as Ca2+, or both (see Nishioka et al., 1988 for detailed review). In SW-adapted fish, depression of plasma Ca2+ stimulates an increase in PRL levels in plasma; for example, injection of EGTA into SW adapted coho salmon lowers plasma Ca2+ and causes PRL levels to double (Fargher and McKeown, 1989). The latter reflects the now well-established hypercalcemic action of PRL in teleosts (Clark, 1983; Flik et al., 1989). Nonetheless, PRL levels can change independently of changes in plasma osmolarity or electrolytes. Prolactin levels rise in response to stress in either fresh water or seawater (Avella et al., 1991) and decline with smoltification in fresh water (Prunet et al., 1989; Younget al., 1989).The high variation in plasma PRL among individual fish (all collected at similar times of the day) suggests pulsatile release of the hormone (Nicoll e t al., 1981).There also is some evidence of a marked difference between the sexes in PRL release. Plasma PRL levels rose about 4-fold higher in mature female chum salmon compared to males when both were transferred from seawater to fresh water (Hirano et al., 1985).

D. Arginine Vasotocin Arginine vasotocin (AVT) from the pars nervosa circulates in the blood of teleosts. It has cardiovascular effects although its status as a hormone and its role in osmoregulation are uncertain (Sawyer, 1987). It has been measured in teleosts by RIA, but appears to be present in body fluids at or near the limit of sensitivity of the assay, 5-10 pg ml-' (Holder et id.,1982; Hontela and Lederis, 1985). Perrot et al. (1986), however, reported somewhat higher levels of AVT in rainbow trout and flounder in fresh water, 88 and 192 pg ml-', respectively, and significantly lower levels in seawater, 35 and 82 pg ml-', thus suggesting a role for AVT in freshwater adaptation. While unable to measure plasma AVT levels in freshwater brook trout (Saluelinus fontinalis),Hontela et al. (1991) showed an increase in pituitary AVT levels in acid stressed animals. Acid stress produces ion losses in fresh water similar to those experienced by euryhaline teleosts acutely transferred from seawater to fresh water, suggesting a role for AVT in hyperosmotic regulation.

E, Melatonin Melatonin (MLT) is an indole-amine secreted by the pineal gland. A RIA based on a rabbit antiserum to a melatonin-BSA (bovine serum

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albumin) conjugate has been validated for use on teleost plasma (Gern et al., 1978; Kezuka et al., 1988)and has detection limits of about 50 pg ml-' (Kezuka et al., 1988).The most prominent feature of plasma MLT in teleosts is marked day-night fluctuations. In common carp (Cyprinus carpio),MLT levels were high during darkness (220-540 pg ml-') and low (23-104 pg ml-l) in the light phase (Kezuka et al., 1988). I n rainbow trout the daily fluctuations were similar but the amplitude was less; 81 t 19 pg ml-' in the light and 153 +- 16 pg ml-' in the dark (Gern et al., 1978). Studies indicate that MLT secretion is light sensitive as well as having a circadian or endogenous rhythm (Iigo et al., 1991). Other than strictly day/night fluctuations, plasma MLT levels increase with seawater adaptation in salmonids (Gern et al., 1984) and cyclic MLT levels in plasma have been implicated in sexual maturation by altering daily cycles in plasma GtH concentrations (Hontela, 1984). F. Thyroid Hormones The thyroid hormones are thyroxine (T4) and triiodothyronine (TJ). While both hormones circulate in the blood, T4 is the primary, if not the only, substance released by the thyroid gland, at least in teleosts (Eales and MacLatchy, 1989), while T3 is mainly formed by deiodination of T4 in peripheral tissues. Triiodothyronine is regarded as the active hormone and T4 a prohormone; nuclear receptors have a much higher binding affinity for T3 than T4 (at least fourfold higher; Eales, 1985). Triiodothyronine and Tq levels are readily measured in plasma with commercial kits that have detection limits (-0.1 ng ml-') appropriate for measuring routine minimum plasma levels in fish (0.1-1.0 ng ml-'). It has become fairly common practice to measure both substances simultaneously in plasma, T4 levels because they are more sensitive to activation of the hypothalamic-pituitary-thyroid axis and thus to environmental influences (Leatherland, 1988), and Tx levels because this is the biologically active molecule. The interpretation of the meaning of changing plasma hormone levels is, however, particularly difficult in the case of the thyroid hormones. For example, a decrease in plasma T4 levels could reflect a decrease in secretion by the thyroid gland or an increase in conversion of T4 to T3 in peripheral tissues. Since T3 levels are a function not only of T4 secretion rates but also of the rate of extrathyroidal deiodination, there is no universally established relationship between plasma T3 and T4 levels. For example, in brook trout, plasma T3 levels are usually consistently higher than T4 levels (White and Henderson, 1977), while in coho salmon the

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reverse is true (Dickhoff and Darling, 1983), and in still others, the levels of the two are similar, Most of the circulating TBand T4 are bound to plasma proteins (Eales and Shostak, 1985) leaving less than 1% of the total hormones in the free or diffusible form. Variations in plasma protein concentration can then explain some of the fluctuations in plasma thyroid hormone levels (Laidley and Leatherland, 1988a). In general, thyroid hormones are elevated when fish are under conditions favorable for somatic growth (after food intake or treatment with androgens or growth hormone) and lowered when conditions are unfavorable (starvation, stress, or high estradiol levels associated with vitellogenesis) (Dickhoff and Darling, 1983; Eales and MacLatchy, 1989). In addition, thyroid hormones can show diurnal (increasing during daylight hours) and annual variations (White and Henderson, 1977) and are elevated during larval development, smoltification (parrsmolt transformation), and during reproductive maturation (Specker, 1988; Dickhoff and Darling, 1983). These latter elevations indicate an important role in preparing animals to exploit new habitats; a switch to active feeding in the case of larvae and increased adaptability to seawater in the case of smolts. Environmental factors such as daylength, temperature, lunar cycle events, pH, salinity, and nutrition are all implicated in stimulating thyroid activity (Grau, 1988). For salmonids at least, the elevation of thyroid hormones during smoltification is apparently the largest observed. Peak levels around 10-15 ng ml-' are usually reported for salmonids smolts (Prunet et al., 1989; Young et al., 1989), but levels as high as 40 ng ml-' (T4)have been reported in coho salmon (Dickhoff and Darling, 1983). In other circumstances the elevation in thyroid hormones is less, with an increase from 1 to 4 ng ml-I being fairly typical (e.g., due to die1 variation [rainbow trout, Cook and Eales, 19871 and in response to feeding [Leatherland and Hilton, 1988; Himick and Eales, 19901). G. Calcitonin Calcitonin (CT) is a peptide hormone (MW of salmon C T is 3452) produced by the ultimobranchial bodies. Calcitonin function in fishes is reviewed in Copp (1969) in Volume 11, Clark (1983);and Pang and Pang (1986). Homologous RIAs have been developed for salmon and for Japanese eel (Anguilla japonica; see Table I for references) with detection limits <1 ng ml-'. Although CT is present in large amounts in the gland and can be greatly elevated in the circulation, it does not appear to play a promi-

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nent role, at least directly, in Ca2+ regulation (Clark, 1983). This is in contrast to tetrapods where CTs role is to correct excessive calcium levels, although in this context it is antihypercalcemic rather than hypocalcemic (Talmage et al., 1980). Hypercalcemia in coho salmon, brought about by either netting and confinement stress in freshwater or b y seawater transfer, does not elevate plasma C T levels (Bjornsson et al., 1989), nor do C T levels change in Atlantic cod (Gadus morhua) exposed to Ca2+-enriched (100 mM) seawater (Bjornsson and Deftos, 1985).In contrast, hypercalcemia in mud skipper (Periophthalmodon schlosseri) induced by air exposure could be corrected by daily CT injections (Fenwick and Lam, 1988). Fish have very precise plasma Ca2+ homeostasis and most, if not all, of the major changes in plasma C T levels and in plasma [Ca"] are associated with gonadal maturation. I n rainbow trout C T levels are around 1 ng ml-' in immature males and females but rise to 23 and 47 ng ml-', respectively, with sexual maturation (Fouchereau-Peron et al., 1990). Female Japanese eels exhibit a similar increase (from 1 to 40 ng ml-l) with sexual maturation (Yamauchi et al., 1978). Accompanying maturation in females are major increases in plasma Ca2+ associated with vitellogenesis. However, the Ca2+ increase, by itself, is not responsible for the elevation in CT (Yamauchi et al., 1978; Bjornsson et al., 1986).Rather, the rise in C T in both sexes is probably most closely associated with the rise in plasma concentrations of the gonadal steroids, GtH, or both. However, it is still possible that C T may play a role in Ca2+ homeostasis by protecting the skeleton during periods of the increased Ca2+ demand of oogenesis (Bjornsson et al., 1986).

H. Cortisol Cortisol is the main (380%) circulating corticosteroid hormone in teleosts (Donaldson, 1981). It is probably the most frequently measured hormone in fishes. Commercial kits are available and are valid for measuring teleost cortisol levels, providing the protein levels of standards and unknowns are similar. Detection limits are 3-8 ng ml-' for commercial kits, but improvements in assay procedures have permitted detection limits of 0.5-2 ng ml-' (Bry, 1982; Pickering and Pottinger, 1983).Resting levels as low as 5 ng ml-' have been reported for salmonids by Pickering and co-workers, although studies on other teleosts (Peter et al., 1978; Lamba et al., 1983; Venkatesh et aZ., 1989) report minimum levels that are higher (10-50 ng ml-'). The most usual reason for an increase in plasma cortisol levels is stress. Indeed,

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cortisol measurements are the method of choice for quantifying stress in fishes (Donaldson, 1981). Stressful circumstances include handling, close confinement, transport or other physical disturbances, and rapid changes in water quality (e.g., pH, salinity, or temperature). The response to stress is mediated by ACTH and can be very rapid. For example, in salmonids exposed to handling stress, ACTH levels were significantly elevated in 2 min and cortisol levels within 10 min of the start of the stress (Sumpter et al., 1986). For a given stress there are marked interspecific differences in the amount of cortisol elevation (Davis and Parker, 1983,1986).The greatest elevations appear to be in salmonids (Davis and Parker, 1986), although the response is greater in wild than in cultured (i.e., domesticated) salmonids (Woodward and Strange, 1987) and the increase is larger and faster at higher temperatures (Sumpter et al., 1985). Generally speaking, there appears to be an approximate correlation between metabolic scope and cortisol elevation in response to stress. In metabolically active species such as salmonids and cyprinids, cortisol elevations as high as 400600 ng ml-' have been reported, while species of intermediate activity such as largemouth bass (Micropterus salmoides) have levels around 200 ng ml-' and inactive species such as the holosts, gar (Lepisosteus sp.), and bowfin (Amia calua) have peaks around 5060 ng ml-' (Davis and Parker, 1986). Other than strictly stressful conditions, there are other circumstances under which cortisol rises. These include die1 cycles (see earlier discussion), smoltification in anadromous salmonids (Young et aZ., 1989), feeding (Bry, 1982; Pickering and Pottinger, 1983), and sexual maturation (Pickering and Christie, 1981).

I. Catecholamines The main circulating catecholamines (CAs) in teleosts are adrenaline (A) and noradrenaline (NA), which originate predominantly from the chromaffin tissue, mostly in the head kidney and postcardinal veins. Plasma levels have been extensively measured but only in a few species (e.g., rainbow trout, eel, cod, carp, flounder, dogfish, and lamprey; see summary tables in Mazeaud and Mazeaud, 1981; Milligan and Wood, 1987; Tang and Boutilier, 1988; Perry et al., 1989; Thomas and Perry, 1991; Randall and Perry, 1992). Measurement techniques include fluorimetric, radioenzymatic, and HPLC methods, with the latter being the most popular. Detection limits are 51 nM. Resting levels of A and NA are usually similar and are typically less than 5 nM but such low levels are achievable only by blood sampling

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via indwelling catheters. Acute sampling methods (see earlier discussion) cause rapid and massive elevation of CA levels in plasma, at least in salmonids. The degree of elevation depends on the method of immobilizing the animal and the rapidity of sampling; however, plasma levels of A reach 700 nM in rainbow trout stunned by a blow to the head, and blood collected by caudal severence, all within 30 sec (McDonald and Goldstein, 1992). Even higher levels (>1000 nM) have been reported for Pacific salmon (Oncorhynchus tshawytscha) after struggling in air, handling, and hauling in the net (Mazeaud and Mazeaud, 1981). Treatments that cause CA elevation in cannulated animals are reviewed in Randall and Perry (Chapter 4)and include hypoxia, hypercapnea, air exposure, exhaustive or violent exercise, metabolic acidosis, and anemia. Violent, enforced exercise and deep hypoxia appear to be the most effective means to elevate CAs with levels in cannulated animals reaching as high as >200 and >100 nM respectively for A and NA in rainbow trout after enforced exercise (Butler et al., 1986), and A > 400 nM in rainbow trout during exposure to severe hypoxia (PO, < 20 mmHg = 3 KPa; Thomas and Perry, 1992). In salmonids, the elevation in A is typically greater than NA reflecting the higher A content of the chromaffin tissue. Although only a limited number of teleosts have been examined there does appear to be marked species differences in CA mobilization that may in part be related to the activity level of the species. For example, Milligan and Wood (1987) showed that A levels were marginally elevated in starry flounder (Platichthys stellatus) after enforced exhaustive exercise, whereas a similar treatment in rainbow trout produced a 10-to 100-fold elevation in A. Similarly, deep hypoxia in the American eel (Anguilla rostrata) produced A levels of only about 10 nM while a comparable treatment produced 400 nM in the rainbow trout and in Salmo fario (Thomas and Perry, 1991). In addition to marked species differences there are seasonal and water temperature related variations in catecholamine release (Mazeaud and Mazeaud, 1981; Randall and Perry, Chapter 4).

J. Pancreatic Hormones

The pancreas of teleosts contain the following peptide hormones (listed in order of decreasing content in coho salmon endocrine pancreas): insulin (INS), somatostatin-25, glucagon-like peptide (GLP), glucagon (GLU), somatostatin-14, and pancreatic peptide (PP) (see Plisetskaya, 1989, 1990 for reviews). Homologous RIAs have been developed for INS, GLU, GLP, and somatostatin-25 in coho salmon

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and for INS in cod and tuna (see Table I for references). Although structurally similar to mammalian INS, fish INS are very different immunologically, hence mammalian RIAs are unsuitable (Plisetskaya, 1989). In contrast, fish GLU can be measured with mammalian RIAs (Gutikrrez et aZ., 1986). Routine INS levels in regularly fed teleosts range from 5 to 20 ng ml-' with no consistent interspecific differences (Gutikrrez et aZ., 1984; Moon et aZ., 1989; Dickhoff et al., 1989; Hemre et al., 1990). With prolonged starvation (3-6 wk) INS drops to very low levels (0.2-1.0 ng ml-'; Moon et al., 1989; Hemre et al., 1990). Feeding typically increases INS secretion. Amino acids are the most powerful stimulators of INS secretion in fish with arginine being the most potent of the amino acids tested (Plisetskaya, 1989).Nonetheless, high carbohydrate diets are more effective at stimulating INS release than low carbohydrate diets (Hilton et aZ., 1987; Hemre et al., 1990). In one extreme case, INS levels as high as 48 ng ml-I were reported in rainbow trout 3 h after a high carbohydrate meal (Hilton et al., 1987). Insulin levels also increase with the parr-smolt transition in salmonids (Dickhoff et al., 1989; Plisetskaya, 1990), decrease during spawning and with a drop in water temperature, and show seasonal fluctuations (GutiCrrez et al., 1987a). Starvation may be responsible for some of the drops in INS levels. Glucagon and GLP levels fluctuate to a smaller extent than INS. For example, both are in the range of 0.5-2.0 ng ml-l in coho salmon and rainbow trout (Plisetskaya, 1990).Glucagon and GLP levels decrease with starvation (Gutikrrez et al., 1990; Moon et al., 1989; sea bass, rainbow trout) but the ratio of GLU (and GLP) to INS increases; this change is consistent with activation of gluconeogenesis (Moon et al., 1989).

K. Stanniocalcin Stanniocalcin (STC, formerly called hypocalcin or teleocalcin, Flik et al., 1990) is a glycoprotein hormone released from the Stannius corpuscles (CS, unique to teleost and holostean fish) and is the predominant hypocalcemic hormone in fish. Removal of the glands results in prolonged hypercalcemia, particularly in seawater fish. A homologous enzyme-linked immunosorbent assay (ELISA) has been developed for Atlantic salmon STC (MW 54,000; Mayer-Gostan et al., 1992) that has detection limits of 0.2 ng ml-'. In Atlantic salmon, STC levels were 40 and 150 ng ml-' in freshwater- and seawater-adapted forms, respectively. A survey of STC levels in 11 marine teleost species yielded values of 76-186 ng ml-'.

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L. Urotensins Urotensin I (UI) and urotensin II (UII) are neuropeptide hormones released from the caudal urophysis in teleosts. Urotensin I is a homologue of mammalian corticotropin releasing factor (CRF) and has a molecular weight of 4864 while UII is a partial homologue of mammalian somatostatin and is a 12-amino-acid peptide of MW 1351. Homologous RIAs have been developed for urotensins I and I1 extracted from white suckers, Catastomus commersoni (Suess et al., 1986; Kobayashi et al., 1986).Blood levels are low (74 and 55 pg ml-’, respectively) and difficult to measure (Hontela et al., 1989). Nevertheless, measurements of urotensins in urophysial tissue indicate a role in osmoregulation. Tissue levels increase in brook trout in response to low p H exposure (Hontela et al., 1989) and increase in flounder, Platichthys flesus, while adapting to seawater (Arnold-Reed et al., 1991). 111. HORMONES: CYCLOSTOMES

AND CHRONDRICTHYES At the present time neither the presence or absence nor the identity of most of the peptide and protein hormones of cyclostomes and chron-

dricthyes is known and no homologous RIAs have, therefore, been developed. However, there has been some limited use of heterologous RIAs (e.g., insulin levels in spotted dogfish [Scyliorhinus canicula; Gutierrez et al., 19881). Consequently, the following discussion is confined to nonprotein hormones: steroids, thyroid hormones, and catecholamines. In cyclostomes the steroidogenic tissues have not been localized (Gorbman, 1989) and the steroid hormones not precisely identified. A variety of steroids have been detected as circulating in the plasma (Katz et al., 1982; Kime and Larsen, 1987) but the active sex steroids are thought to be hydroxylated derivatives of T and Ez in males and females respectively (Larsen, 1990). In chondrichthyes, the sex steroids are similar to those of teleosts. In females, the important gonadal steroids are Ez, T, and progesterone. In immature animals steroid levels are 51 ng ml-’. Ez and T are the principal steroids during the preovulatory phase of the cycle, while P is elevated approximately 24-48 h prior to ovulation. Peak levels of sex steroids vary depending on the steroid and the species but most peaks fall within the range of 10-40 ng ml-’ (Dodd, 1983; Callard and Klosterman, 1988). I n males, there are less data available but, like

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teleosts, T is apparently the main sex steroid. Testosterone levels are around 10 ng ml-' in mature male spotted dogfish (Dobson and Dodd, 1977) but very high levels have been reported in skate, Raja sp. (>2000 ng ml-') associated with very high levels ofa plasma testosterone binding protein (Callard, 1988). Cortisol is riot the steroid stress hormone in either cyclostomes or chrondrichthyes. In the sea lamprey, Petromyzon marinus, androstenedione may be the cortisol analog. It increases from 4 to 14 ng ml-' with agitation stress (Katz et al., 1982). In elasmobranchs the main interrenal steroid is 1a-hydroxycorticosterone, but its precise physiological function remains unclear, although mineralocorticoid rather than glucocorticoid activity has been reported. Resting levels in spotted dogfish are around 4 ng ml-' and increase three- to fourfold when salinity is reduced (Balment et al., 1987). In general, it appears that the corticosteroid stress responses in cyclostomes and chrondricthyes are small relative to those of teleosts. The physiological role of thyroid hormones in cyclostomes is not precisely known (Plisetskaya et al., 1983; Specker, 1988), and the thyroid does not appear to be under pituitary control (Dickhoff and Darling, 1983). Most of the studies have been done on lampreys. Thyroid hormone levels are particularly high in larvae (ammocoetes), Tq is 70 ng ml-' and T3 is 16 ng ml-' (Wright and Youson, 1977) but decrease (50-90%) at the onset of metamorphosis to adults. The levels in ammocoetes are as much as 10 times higher than those of most other vertebrates but levels in juveniles are within the normal vertebrate range (Youson, 1988). Larval and metamorphosing forms also have a much higher liver T3 binding capacity than adults (Lintlop and Youson, 1983). The circulating catecholamines in cyclostomes and chrondrichthyes are identical to those of teleosts (Dashow et al., 1982). Resting levels in lampreys, measured on cannulated animals, are substantially higher than those measured in resting teleosts but the response to stress has a much lower amplitude. Adrenaline and noradrenaline were 20 and 8 nM, respectively, and rose only 3- and %fold, respectively, after 5 min of agitation stress (Dashow et id.,1982). In the spotted dogfish resting catecholamine levels were similar to those of the lamprey (A and NA were 6 and 14 nM, respectively) but the response to agitation stress (repeated burst swimming for 2-3 min) was much larger (16-and y-fold, respectively; Butler et al., 1986).Nonetheless, in comparison to active teleost species, adrenaline mobilization is still relatively small. In rainbow trout, treated comparably, A and NA elevations were 150- and g-fold, respectively (Butler et al., 1986).

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D. G. MCDONALD AND C . L. MILLIGAN

IV. METABOLITES A. Glucose

Glucose is one of the most frequently measured blood metabolites and is, perhaps, the most variable. Typically, whole blood rather than plasma levels are reported. Free glucose in the red cells is quite low (<1mM) and small variations in hematocrit have little impact on whole blood glucose levels. Resting blood glucose levels range from lows of 0.2 mM in the African lungfish (Proptopterus acthiopicus; Dunn et al., 1983) to highs of 15 mM in tuna (Katsuwonus pelamis; Arthur et al., 1991).Interspecific variation in whole blood glucose levels has been attributed to differences in activity levels of fish: More sluggish, benthic species tend to have lower blood glucose levels than d o more active, pelagic species (Umminger, 1977). Glucose levels also vary among individuals within a species. In the extreme, blood [glucose] ranged from 0.3 to 8.8 mM in 26 individual kelp bass (Paralabrax sp., Bever et al., 1977) caught from the wild and held in the laboratory. Some of this individual variation can be attributed to differences in size, age, and the nutritional and reproductive states of the fish (Fletcher, 1985). Blood glucose levels are often cited as being a sensitive physiological indicator of stress in fish (Wedermeyer and McLeay, 1981). In general, stress (e.g., hypoxia, crowding, handling, forced exercise, disease, angling, captivity) causes a relative hyperglycemia of variable extent and duration. The severity of the stress in large part dictates the degree and duration of the hyperglycemic response. For example, in pike (Esox lucius),angling stress resulted in a threefold increase (from 2 to 6.2 mM) in blood glucose, whereas mild chasing was without effect (Schwalme and McKay, 1985). The source of the elevated blood glucose is hepatic glycogen, which is mobilized in response to catecholaminergic stimulation (Mazeaud and Mazeaud, 1981). The intra- and interspecific variability in glycemic response to stress may reflect differences in catecholamine secretion (Mazeaud and Mazeaud, 1981),hepatic glycogen reserves, or both. The latter may explain why, in some instances, chronic stress results in hypoglycemia (Fletcher, 1985).To further complicate matters, in some species (e.g., the European eel, Anguilla anguilla; Sokolowska and Bieniarz, 1981) the time of day the stress is applied influences the glycemic response. Nutritional status also influences blood glucose levels. All species examined show a postprandial hyperglycemia, the duration and magnitude of which is dependent on the dietary carbohydrate level (Hilton

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77

and Atkinson, 1982; Fletcher, 1985; Suarez and Mommsen, 1987; Moon, 1988). The effect of starvation on blood glucose levels is species- and time-dependent. Mild hypoglycemia (i.e., blood glucose rarely drops by more than 25-30% of resting levels) is observed after 6 weeks starvation in rainbow trout (Moon et al., 1989) and sea bass (Zammit and Newsholme, 1979; Gutikrrez et al., 1990)and after 22 but not 7 days starvation in the sand dab (Limanda limanda; Fletcher, 1985). Several species show blood glucose homeostasis with prolonged starvation; after 150 days starvation, blood glucose was unaltered in Squalus acanthias (deRoos et al., 1985), the spotted dogfish (Zammit and Newsholme, 1979), and the kelp bass (Bever et al., 1977). Perhaps the record for tolerance to food deprivation belongs to the American eel; after 36 months of starvation, blood glucose levels remained constant at about 9 mM (Cornish and Moon, 1985). The mechanism for the maintenance of blood glucose during prolonged starvation is not clear. Metabolic depression, resulting in a decrease in glucose utilization, may be involved (Cornish and Moon, 1985) or, alternatively, gluconeogenesis from amino acids and fatty acids (Suarez and Mommsen, 1987) may be activated. In those species that show seasonal variations in blood glucose levels, it is associated with the reproductive cycle. In rainbow trout (Miller et al., 1983), the spotted dogfish (Gutikrrez et al., 1988), and Spicara chryselis (FernBndez and Planas, 1980), the lowest blood glucose levels (3.9, 0.5, and 2.7 mM, respectively) were associated with peak gonadal development and spawning, whereas the highest levels (11.7, 1.0, and 8.3 mM, respectively) were associated with postspawning feeding activity. Although blood glucose levels vary considerably among species (Fletcher, 1985),within a species, and over time within an individual (Bever et al., 1977),severe hypoglycemia (i.e., >50% decline in blood glucose) does not occur. Suarez and Mommsen (1987) argue that because glucose turnover is directly related to blood [glucose], maintenance of a critical blood glucose level, albeit over a wide range, is important in ensuring that glucose turnover continues in the face of starvation. This strategy appears to be important in maintaining fuel to the nervous system, particularly in species that experience prolonged starvation as part of their life cycle.

B. Lactate Lactate is the major end product of anaerobic metabolism in vertebrates and is routinely found in low levels, <1 mM, in fish blood. Its

D. G. MCDONALD AND C . L. hlILLIGAN

78

accumulation in the blood is usually indicative of oxygen limitation. Blood lactate levels increase in response to environmental hypoxia (low inspired P o , ) , internal hypoxemia (e.g., anemia, gill dysfunction), or strenuous activity. The elevation of blood lactate in response to environmental hypoxia is directly related to the degree of hypoxia: the lower the Po,, the higher the blood lactate (Boutilier et al., 1988).The P o , at which anaerobic metabolism is activated and lactate appears in the blood, the critical P o , , varies among species (Boutilier et al., 1988). In general, the more active the species, the higher the critical Po,. Similarly, the degree to which lactate accumulates in the blood following a bout of strenuous activity (e.g., enforced swimming, angling) is species specific and appears to be correlated with life-style and habitat. In active, pelagic species, including pike, salmonids, tunas ( K . pelamis, Thunnus albacares),marlins (Tetaptusrus audas, Makaira nigricans, M. indica), and a number of sharks (Zsurus oxyrhinchus, S . canicula; see Wood and Perry [1985] for a review; Schwalme and McKay l19851; Wells et al. [1986]), blood lactate levels are often in the range of 15-20 mM following a bout of activity. In contrast, in benthic, more sluggish species, including flatfish (e.g., P . stellatus, Hippoglossoides elassodon, Pseudopleuronectes americanus), toadfish (Opsanus beta), sea raven (Hemitripterus americanus), and skates (Raja ocellata) (see Wood and Perry [1985] for a review; Walsh [1989]), strenuous activity rarely raises blood lactate levels in excess of 1-2 mM. Though well-documented, the reasons for the differences in postexercise blood lactate levels between active, pelagic, and benthic, sluggish species are unknown. C. Ethanol

While lactate is considered the main anaerobic end product in vertebrates, there are a few fish species in which ethanol has been found to accumulate in significant amounts under anaerobic conditions. Goldfish (Shoubridge and Hochachka, 1980; van den Thillart and Verbeek, 1982), crucian carp (Carassius carassius; Johnston and Bernard, 1983), and bitterling (Rhodeus amarus; Wissing and Zebe, 1988) produce ethanol only in response to severe (Po, < 1.33 kPa, 1 mmHg = 0.133 kPa) prolonged hypoxia or anoxia and can reach levels of 2-4 mM in the blood (versus resting levels of <1 mM; Shoubridge and Hochachka, 1980; Johnston and Bernard, 1983). D. Ketone Bodies

Ketone bodies, acetoacetate and P-hydroxybutyrate, are produced

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79

primarily in the liver from nonesterified fatty acids. The abundance of ketone bodies in elasniobranch blood and their virtual absence in that of teleosts may be linked to the absence of free fatty acids in elasmobranch plasma (see Section VII). Elasmobranchs may rely upon ketone bodies generated in liver as a fuel substitute for fatty acids (Zammit and Newsholme, 1979). Elasmobranch blood contains both P-hydroxybutyrate and acetoacetate, although the former predominates (0.089.6 mM versus 0.03-055 mM; Zammit and Newsholme, 1979; deRoos et al., 1985; Gutikrrez et al., 1988). Teleost blood, however, contains only acetoacetate and at much lower levels (0.05-0.1 mM; Zammit and Newsholme, 1979; Fernandez et al., 1989).Plasma ketone levels are influenced by both starvation and reproductive status. In elasmobranchs, plasma levels of both acetoacetate and P-hydroxybutyrate increase in response to long-term starvation (weeks), although the latter increases to a much greater extent (2O-foldversus 6-fold increase; Zammit and Newsholme, 1979; deRoos et al., 1985). In teleosts, starvation was without effect on plasma acetoacetate levels (Zammit and Newsholme, 1979; FernBndez et al., 1989). In both elasmobranchs and teleosts, the highest plasma levels of ketones (8-9 mM and 0.10.5 mM, respectively) were observed in prespawning individuals, during the period of gonadal development ( F e r n h d e z and Planas, 1980; Gutierrez et al., 1987a; Fernandez et al., 1989). E. Bile Pigments

Biliverdin and bilirubin are common bile pigments formed in the liver from the breakdown of heme and other porphyrine proteins. In most fish, they are stored in the gall bladder and are removed from the body when food moves along the gut. Thus, in most species, the plasma levels ofthese pigments are quite low (e.g., 1-5 p M ; Wells et al., 1986). The lamprey (Lampteru lamottenii), however, is a notable exception. Upon metamorphosis from the ammocoete to adult form, the liver undergoes extensive reorganization; the bile ducts degenerate and the gall bladder is lost (Sidon and Youson, 1983). Bile pigments have not been detected in the plasma of ammocoetes, but both bilirubin (-20 p M ) and biliverdin (-2 p M ) are found in juvenile and adult upstream migrant fish (Makos and Youson, 1987). A number of cottids and anguillids have blue-green, lavender, and purple colored plasma (Yamaguchi, 1973; Low and Bada, 1974; Ellis and Poluhowich, 1981; Makos and Youson, 1987; Eng and Youson, 1991). Also, the plasma of female lumpfish (CycZopterus Zumpus) is a deep blue-green whereas that of males is reddish in color (Mudge and Davenport, 1986).The blue-green coloration is due to the presence of

80

D. G. MCDONALD AND C. L. MILLIGAN

biliverdin and to a lesser extent, bilirubin. It is not clear why these pigments accumulate in the plasma and why they are not toxic since biliverdin levels in these species are in excess of 100 p M ; well above the levels considered toxic in mammals (25 pM;Low and Bada, 1974; Mudge and Davenport, 1986). The purpose (if any) of the serum pigmentation is obscure, although Mudge and Davenport (1986) have suggested that plasma pigmentation may be involved in sexual signalling in the lumpfish. V. NONPROTEIN NITROGENOUS COMPOUNDS A. Amino Acids Amino acids are transported within erythrocytes and the plasma, although the relative importance of each pool is a matter of some debate (Dabrowski, 1982; Murai and Ogata, 1990; van der Boon et al., 1991). The distribution of free amino acids between erythrocytes and plasma differs among species (see Table 11), and there are no obvious trends. Furthermore, in some instances, the response of red cell free amino acids is independent of that of the plasma. For example, in rainbow trout after exhaustive exercise, some free amino acids in red cells decrease whereas other amino acids in the plasma increase (S. Eros and L. Milligan, unpublished observations). Although the data are sparse, it appears that plasma amino acid levels are higher in marine elasmobranchs than in either marine or freshwater teleosts or marine agnathids (Boyd et aZ., 1977).This is most likely a consequence of the high extracellular osmotic pressure maintained by organic osmolytes in elasmobranchs. When skate and stingray (Dasyatis sabina), euryhaline elasmobranchs, are moved into a dilute environment (15%)there is a decrease in the level of several amino acids, particularly taurine, in the red cells (Boyd et aZ., 1977). There is, however, no effect of salinity changes on plasma amino acid levels, further illustrating the independence of free amino acids in red cells and plasma. Fish show minimal dietary requirements for certain amino acids. The essential amino acids requirements have been determined for only a few species, but these show little interspecific variation. Whole blood and plasma levels of essential amino acids (EAA) are mainly a function of dietary protein intake and are positively correlated with their levels in the diet (e.g., carp, Dabrowski, 1982; Murai and Ogata,

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CHEMICAL PROPERTIES OF THE BLOOD

81

1990; trout, Walton and Wilson, 1986). Nonessential amino acids, in contrast, are independent of dietary levels (Plakas et al., 1980;Walton and Wilson, 1986). Although the plasma levels of EAA are similar among species (at least in the few examined),the abundance of nonessential amino acids in the plasma varies considerably. For example, in the common carp (Dabrowski, 1982) and tilapia (Yamada et al., 1982) plasma is abundant in proline and threonine, whereas in sockeye salmon (0. nerka; Mommsen et al., 1980) and rainbow trout (Nose, 1972; Kaushik and Luquet, 1977), glycine, alanine, and lysine are most abundant, and serine and lysine are abundant in channel catfish (Zctalurus punctatus; Wilson et al., 1985). By and large, the majority of studies on plasma amino acids in fish have examined the influence of food deprivation and various diets, with the goal being to define EAA requirements. In all species examined, the highest whole blood and plasma amino acid levels are seen within hours after a meal. The response of plasma amino acid levels to fasting is complex; dependent on, among other things, species and duration of starvation. In general, plasma levels of the EAA tend to decline at the start of starvation (i.e., within days), and subsequently increase to prestarvation levels, while there is little change in the level of nonessential amino acids (Timoshina and Shabalina, 1972; Blasco et al., 1991). A striking example of the preservation of plasma amino acid levels during prolonged starvation is seen in sockeye salmon. The long-term starvation associated with spawning migration caused only very modest changes in plasma amino acid levels (Mommsen et al., 1980), despite significant muscle proteolysis. Some amino acids that are poor metabolic substrates (e.g., lysine and valine) tend to accumulate in the plasma as a result of muscle proteolysis. Few studies have examined the influence of seasonality or photoperiod on circulating levels of free amino acids. To our knowledge, the influence of photoperiod on circulating amino acid levels has been assessed in only two species: goldfish (Carrillo et al., 1980) and sea bass (Carrillo et al., 1982). The story emerging is complex: Only some amino acids show a circadian rhythm and, at least in the goldfish, the rhythmicity is dependent on duration of the light phase. For example, plasma serine showed a circadian rhythm when fish were exposed to short day lengths (9L : 13D) and natural day lengths (1OL : 14D) but no rhythmicity was observed in fish exposed to long day lengths (15L : 9D) (Carrillo et al., 1980). The explanation for this is not at all clear but is undoubtedly linked to the complex hormonal changes accompanying changes in day length.

Table I1 Whole Blood, Plasma, and Erythrocyte Amino Acid Levels in an Agnathid, Elasmobranch, and Teleost Species QI

M

Pacific hagfish Eptatretus stoutin

Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phen ylalanine Threonine

Erythrocyte (mM cell water)

3.36 1.22 3.18 8.76 4.00 2.72 4.73 7.58

Dogfish Scyliorhinus caniculab

Plasma (mM)

Whole blood'/ (mM)

0.265 0.014 0.059 0.159 0.352 0.053 0.075 0.113

0.20 0.11 0.32 0.52 1.01 0.07 0.14 0.24

Common carp C yprinus carpio"

Plasma (mM)

Whole blood' (mM)

0.31 0.08 0.28 0.39 0.57 0.12 0.11 0.07

0.256* 0.169* 0.103* 0.183* 0.451* 0.162* 0.103* 0.702*

Plasma (mM)

Calculated erythrocyte' (mM whole blood)

Measured erythrocytef (mM whole blood)

0.138* 0.092* 0.068* 0.127* 0.214* 0.069* 0.076* 0.304*

0.047* 0.042* 0.035* 0.056* 0.052* 0.093* 0.027* 0.350*

0.088* 0.040*

0.017* 0.035* 0.067* 0.065* 0.013* 0.328*

Valine Cystine Tyrosine Alan i n e Aspartate Glutamate Glutamine Glycine Proline Serine Taurine ?-Amino butyric acid (GABA) a-Amino-n-butyrate

6.27 4.16 8.99 1.63 3.00 6.93 3.72 16.8 4.15 N 1.09 2.23

0.106 0.058 0.041 0.049 0.064 0.108 0.115 0.097 0.092 ND 0.020 0.11

1.03

-

0.11 1.25 0.22 0.22 0.22 3.66 0.56 0.36 7.44

0.43 0.10 0.32 0.04 0.03 0.12 0.19 0.36 0.15 0.23

0.161* 0.042 0.068 0.418 0.134 0.101 0.602 0.572 0.236

* Essential amino acid for this species. 0

CJ

Fincham et al. (1990). GutiCrrez et al., (198713). Samples taken in November, 24 h after feeding. Dabrowski (1982)Samples taken 72 h after feeding. Measured in deproteinized extracts of whole blood. Hematocrits not given. Plasma [amino acid] was subtracted from whole blood [amino acid]. f Erythrocytes were washed twice with 0.65% Ringer solution prior to deproteinizing red cells. g Not detectable.

0.112* 0.021 0.054 0.218 0.134 0.053 0.315 0.246 0.115

0.048* 0.014 0.157 0.153 0.109 0.264 0.167 0.115 -

0.033* 0.034 0.008 0.264 0.125 0.105 0.332 0.349 0.135

84

D. G. MCDONALD AND C. L. MILLIGAN

B. Ammonia Because of the high pk of ammonia (-9.5; Cameron and Heisler, 1983) relative to the physiological p H of fish plasma (-7.6-8.0; see Heisler, 1984 for a review of fish acid-base physiology), >99% of ammonia is present in the ionized form, NH4+. Ammonia, as NH3 and NH4+,is the primary transport vehicle for nitrogen in plasma of most fish, and the unionized form, NH3, can be extremely toxic if allowed to accumulate in the body. Under aerobic conditions most ammonia is produced in the liver, but under anoxic conditions, liver production is reduced and muscle proteolysis becomes the main source of ammonia (van Waarde, 1983). During exhaustive exercise, adenylate deamination in the muscle becomes a major source of ammonia (Dobson and Hochachka, 1987), although its quantitative importance to total ammonia production depends on the activity level of the animal; increasing with increasing workload (Driedzic and Hochachka, 1976). Plasma total [ammonia] is variable and dependent, among other things, on the site of blood sampling. In tilapia, Oreochromis nilotica, plasma ammonia levels in blood sampled via caudal puncture (prehepatic blood) were greater than that in plasma obtained via cardiac puncture (posthepatic blood), indicating a significant hepatic ammonia uptake (Wood et al., 1989). Similarly in rainbow trout, the plasma total [ammonia] in blood drawn from a dorsal aorta catheter (posthepatic) was about 30%ofthe plasma total [ammonia] in the blood sampled by caudal puncture (prehepatic blood; Wood et al., 1989), although the stress associated with caudal sampling may have contributed to the higher ammonia levels in the caudal blood. In blood drawn from the ventral aorta (pregill), the plasma [ammonia] is 1.5-2.0 times that in dorsal aortic (postgill) blood (Cameron and Heisler, 1983; Wright and Wood, 1985); the difference representing ammonia excreted at the gills. In most fish, plasma total [ammonia] ranges from 0.1 to 0.8 mM (Watts and Watts, 1974; Wright and Wood, 1985; Perry and Vermette, 1987; Perlman and Goldstein, 1988; MacKenzie and Randall, 1990); fed fish typically have higher levels than do starved ones. Plasma [ammonia] increases in response to exhaustive exercise (e.g., Turner et al., 1983a,b),air exposure (van Waarde, 1983; Walsh et al., 1990), increases in temperature and water [ammonia] (Thurston et al., 1984),and exposure to alkaline (pH -9:5; Wright and Wood, 1985) and acidic (pH -4.5; McDonald, 1983a; H6be et al., 1984) environments (see Randall and Wright, 1987, for a review). Interestingly, in the lemon sole (Parophrys uetulus), hypercapnia results in a reduction in plasma ammo-

2. CHEMICAL

PROPERTIES OF THE BLOOD

85

nia which may be due, in part, to a COZ-mediated suppression of metabolism (Wright et al., 1988).

C. Urea and Uric Acid Elasmobranchs, holocephalans (chimaeras), and the coelocanth (Latimeria),whose milieu interieur are isotonic or slightly hypertonic to seawater, are distinguished by the high levels (250-400 mM) of urea in their plasma. The source of this urea, which serves as the animal’s most abundant organic osmolyte, is de novo synthesis by the ornithineurea cycle in the liver. In some euryhaline elasmobranchs (e.g., Scyliorhinus africanus; Haywood, 1973; Raja elanteria; Watts and Watts, 1974; Perlman and Goldstein, 1988),plasma urea levels decline when animals are placed in dilute seawater and, conversely, increase when fish are exposed to concentrated seawater. In others (e.g., lemon shark, Negaprion brevirostus; Watts and Watts, 1974), plasma [urea] is constant in the face of changing salinities. A reduction in food intake over a period of several weeks can reduce plasma [urea] in elasmobranchs and, hence, their ability to hyperosmoregulate. At any given salinity, plasma [urea] was consistently greater in well-fed sharks ( S . africanus fed twice weekly; Haywood, 1973) than in poorly fed (once a month) sharks. Plasma [urea] increased almost immediately on refeeding. The dependence of plasma [urea] on food availability may explain, to some extent, the variation in reported plasma urea levels for marine elasmobranchs. Urea is also present in the plasma of marine and freshwater teleosts, although at much lower levels (1-10 mM), and plays an insignificant role in osmoregulation. In most teleosts, urea is derived from the degradation of purines via uric acid, the hydrolysis of arginine, or both. In some species, e.g., Gulf and oyster toadfish (0.beta, 0. tau; Walsh et al., 1990); tilapia [Oreochromis alkalicus grahami; Wood et al., 1989) and several air-breathing fish from the Indian subcontinent (Heteropneustes fossilis, Clarias batrachus, Anabas testuidneus, Amphipnous cuchia; Saha and Ratha, 1989), urea is synthesized via the ornithine-urea cycle. Plasma urea levels in these ureogenic teleosts, even when air exposed, are generally not much greater than those in ammoniotelic species. In the marine, ureogenic 0. beta, plasma [urea] is about 9-10 mM (Walsh et al., 1990) compared to 4-5 mM in the freshwater ammoniotelic rainbow trout (Wood et al., 1989). Unlike air breathing teleosts, urea in the African lungfish builds up to high levels during estivation (200 mM after 13 months). When aquatic, lungfish

86

D. G. MCDONALD AND C. L. MILLIGAN

plasma [urea] (1-7 mM) is not unlike that in teleosts (DeLaney et al., 1977). For a complete discussion of the evolutionary and physiological significance of urea synthesis in fish, see Mommsen and Walsh (1989, 1991). Uric acid is formed by the degradation of purine nucleotides and protein catabolism via purines, primarily in the liver and white muscle. Uric acid is generally converted to urea for excretion, so blood levels are typically low. In rainbow trout, plasma uric acid ranges from 40 to 100 pM (Hille, 1982). Plasma uric acid is not commonly measured, so there is little data available on phylogenetic trends, effects of starvation, stress, or seasonal variations.

D. Trimethylamine Oxide Trimethylamine oxide [ (CH3)3N-O; TMAO], like urea, occurs in high concentrations in marine elasmobranchs (22-120 mM; Griffith, 1981)and the coelocanth (Griffith et al., 1974) and contributes substantially to plasma osmotic pressure. Unlike urea, however, plasma TMAO levels are quite variable both among and within species (Griffith, 1981).Some of this variability might be due to differences in diet, since diet is thought to be the principle source of TMAO (Watts and Watts, 1974). Levels of TMAO in the freshwater elasmobranch, Potamotrygon sp., are considerably lower (often <1 mM) than in their marine counterparts. Trimethylamine oxide in holocephalans are also quite low, averaging 5 mM (Griffith et al., 1974). Trimethylamine oxide is normally considered negligible in the blood of teleosts (<1 mM; Griffith, 1981); however, several marine teleosts are reported to have substantial plasma TMAO levels. In several shallow water marine teleosts (e.g., Scomber scomber, Morone saxatilus, 0.tau; Griffith, 1981), plasma TMAO levels range from 1 to 38 m M . In deeper water fish, which are often moribund when sampled, plasma TMAO levels are considerably higher (often 60-90 mM). E. Creatine and Creatinine

Creatine is an amino acid that is an end product of the metabolism of glycine, arginine, and methionine and is found primarily in the white muscle. Creatine is a precursor for the high energy phosphate, phosphocreatine. The source of creatine in fish is unclear. The necessary enzymes for de novo synthesis of creatine have been found in carp, but are absent, or undetectable, in the Pacific hagfish (Eptatertus),a shark (Prionace sp.), ray (Urolophus sp.), and a teleost, the buffalo fish (Mega-

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CHEMICAL PROPERTIES OF THE BLOOD

87

stomatobus SP.) (Van Pilsum et al., 1972). Creatine is present at fairly high concentrations in seawater (-0.1 mM), but only Eptatertus is known to obtain creatine via absorption from the water (Van Pilsum et al., 1972). In other fish, it has been concluded that creatine is supplied in sufficient quantities in the diet (Danulat and Hochachka, 1989). Creatine in the blood, therefore, is in a dynamic state, representing a balance between that absorbed via the gut and that released from muscle. Whole blood creatine levels average 270 pM in starry flounder (Danulat and Hochachka, 1989) and are unaffected b y 6 wk of starvation. Whole blood creatine in trout averages 1 mM and is about half the level found in extracts of red cells (2.5 mM; Danulat, unpublished, cited in Danulat and Hochachka, 1989).Danulat and Hochachka (1989) have suggested that the differences between creatine levels in flounder and trout may reflect differences in swimming activity or nutritional status. Creatinine is formed by spontaneous (i.e., nonenzymatic) cyclization of creatine. Its levels in plasma are typically low, 10-80 pM (Sandnes et al., 1988), and appear to be unaffected by stress (Wells et al., 1986) or, in the African lungfish, estivation (DeLaney et al., 1977). Creatinine is not metabolized further and is excreted by the kidneys.

VI. PLASMA PROTEINS The plasma proteins in fish have not been studied in detail in any species, and other than electrophoretic mobility patterns there is little information about many of the plasma proteins. The nomenclature for fish plasma proteins is adopted from that used for mammals, which is based on molecular weight and pattern of electrophoretic mobility. Among the plasma proteins identified, most fish plasma contains albumin-like proteins, a number of &"-binding proteins (e.g., ceruloplasmin, vitellogenin), blood clotting proteins (e.g., fibrinogen and prothrombin), metal-binding proteins (e.g., transferrin), immunoglobulins, lipoproteins (e.g., high density lipoproteins [HDL], low density lipoproteins [LDL]; see Section VII,B), and hormone-binding proteins (Ts-binding protein; steroid-binding proteins). In Arctic and Antarctic species, there are specialized antifreeze proteins and glycoproteins. Aside from albumin or para-albumin, there is very little information on the phylogenetic distribution of these proteins. Most of the information about these proteins is descriptive (e.g., molecular weights, binding characteristics, electrophoretic mobility), with very little information available as to the amounts in plasma. For a thorough discussion of the evolution of plasma proteins, see Doolittle (1984, 1987).

D. G . MCDONALD AND C. L. MILLIGAN

88 A. Total Plasma Protein

Total plasma protein can be determined relatively simply from the refractive index of plasma. Since proteins form approximately 80% of the plasma solutes, it is possible to use the measurement of refractive index to show changes in total plasma [protein] (Gowenlock, 1988). There are, however, differences between total protein estimated by colorimetric techniques (e.g., Lowry method, Biuret reaction; see Gowenlock, 1988 for a detailed discussion of these methods) and by refractometry. Alexander and Ingram (1980) and more recently Hunn and Greer (1990) reported that the plasma [protein] estimated by the colorimetric methods was lower than that estimated by refractometry. Nonetheless, there were good correlations between refractometry estimates and colorimetric estimates. This led Alexander and Ingram (1980)and Hunn and Greer (1990)to suggest that, because of its ease of use and small sample volume required, refractometry is the method of choice for estimating plasma [protein], providing the refractometer measurements are calibrated with a colorimetric method of analysis. The total plasma [protein] in fish ranges from 2 to 8 g dl-' (Fletcher, 1975; Fellows et al., 1980; Miller et al., 1983; Sandnes et al., 1988; Hunn and Greer, 1990) and appears to be fairly constant within and among species. Total plasma [protein] is altered mainly by changes in plasma volume; an increase is caused by a shift of fluid from the plasma to the intracellular compartment, and a decrease can be caused by hydration of the plasma. Fluid shifts out of the plasma are caused by an osmotic imbalance between the extracellular and intracellular compartments, and any stress that induces such an imbalance can lead to increases in plasma [protein] (see Olson, Chapter 3, for a discussion on the regulation of fluid volume). For example, total plasma [protein] increases in rainbow trout in response to strenuous exercise (Milligan and Wood, 1986) and exposure to low environmental pH (Milligan and Wood, 1982). Reductions in plasma [protein] are less common, and are generally associated with prolonged starvation (Love, 1980) and severe stress (Stevens, 1968). Several studies have examined seasonal variations in total plasma [protein] with often conflicting results. In rainbow trout, Haider (1970) reported peak plasma levels in midwinter, whereas Schlotfeldt (1975) found maximal levels at the end of the summer.

B. Albumin Albumin has a molecular mass of about 68-70 kDa. It reversibly

2.

CHEMICAL PROPERTIES OF THE BLOOD

89

binds with fatty acids, bilirubin, and many other substances and serves as a major transport protein in the blood. In fish, albumin-like proteins have been identified in lamprey, hagfish, and several teleost species. There is, however, some question as to the presence of albumin in elasmobranchs (Fellows et al., 1980; Fellows and Hird, 1981, 1982). The bromocresol green method (see Gowenlock, 1988 for details) is routinely used to measure plasma albumin. In teleosts, plasma albumin concentration ranges from 1.0 to 2.4 g dl-' and constitutes 25 to 50% of the total protein (Fellows et al., 1980; Miller et al., 1983; Sandnes et al., 1988).Albumin levels tend to be low in the lamprey, representing less than 15% of the total protein (Fellows and Hird, 1981). Although the presence of albumin in elasmobranchs has been questioned (see following discussion), plasma albumin has been detected by the bromocresol green method in several elasmobranchs but at very low levels: 0.5-0.7 g dl-l (Fellows et al., 1980).When albumin levels are low, the bromocresol green method tends to overestimate [albumin] because of the binding of bromocresol green to lipoproteins and transferrin (Gowenlock, 1988). The presence of albumin in fish plasma has been confirmed on the basis of electrophoretic mobility, fatty acid-binding properties, and molecular mass (Fellows and Hird, 1981; Davidson et al., 1988). Agnathid, elasmobranch, and teleost plasma all contain a protein with a molecular mass similar to that of human albumin (68-70 kDa; Fellows and Hird, 1981; Davidson et al., 1988).In the agnathids and teleosts, this protein reversibly binds fatty acids (palmitate and oelate; Fellows and Hird, 1981; Davidson e t al., 1988) and bilirubin (Fellows and Hird, 1982). In elasmobranchs, however, this albumin-like protein does not bind fatty acids but does bind bilirubin (Fellows and Hird, 1982). The significance of the absence of a fatty acid-binding protein in elasmobranchs is not clear but is consistent with the low levels of plasma free fatty acids found in these species.

C. Immunoglobulins Next to albumin, the immunoglobulins are the most abundant plasma proteins, representing anywhere from 2 to 15%of total protein (Kobayashi et al., 1982; Olsen and J@rgesen, 1986; Haversein et al., 1988; Klesius, 1990). Immunoglobulin levels are quite variable in fish (44-650 mg ml-') and tend to be higher in wild-caught fish (Klesius, 1990). These higher levels in fish from natural environments most likely reflect exposure to many different antigens. In channel catfish (Klesius, 1990)and rainbow trout (Olsen and Jqjrgensen, 1986), plasma

D. G. MCDONALD A N D C. L. MILLIGAN

90

immunoglobulin levels were independent of temperature. Immunoglobulin levels significantly increased with increasing size and age of catfish (Klesius, 1990),which is similar to the pattern found for immunoglobulins from several other species, including humans.

D. Hormone-Binding Proteins T h e steroid and thyroid hormones are transported in the plasma bound to proteins. It has been argued that during blood transit through the capillary, rapid dissociation of the hormone from a large proteinbound reserve helps keep the free hormone level low and thereby facilitates tissue uptake (Ekins, 1986). Steroid hormones bind to three classes of proteins: 1. Albumin binds most steroids with low affinity and very high

capacity. 2. Corticosteroid-binding globulin binds glucocorticosteroids and progesterone with high affinity but binds aldosterone and sex steroids with low affinity. 3 . Sex hormone-binding globulin binds sex steroids with a specificity and affinity that is species specific (Wingfield, 1988). Two steroid-binding globulins have been demonstrated with electrophoretic techniques in adult sea lamprey (Boffa et al., 1972); one preferentially binds progesterone and the other has a high affinity for estradiol. However, these proteins also show a high cross-reactivity with testosterone and corticosterone, suggesting a low specificity for steroid binding. In contrast, only very low affinity and nonspecific binding of sex steroids is observed in the blood of the Pacific hagfish and the anadromous lamprey (L. tridentata) (Wingfield, 1988),which may be linked to generally low levels of steroids found in agnathan blood. Elasmobranchs appear to have only a single protein system that binds steroids with low specificity but high affinity. Although this protein binds both cortisol and corticosteroid, the major corticosteroid in elasmobranchs, la-hydroxycorticosterone, is bound with low affinity (Martin, 1975).Skate (Raja rudiata) plasma possesses a protein that binds sex steroids with a rather high affinity but low capacity (Wingfield, 1988). I n teleosts, steroid hormones appear to bind to a single protein system showing specificity and high affinity for CIS and C19 steroids (e.g., testosterone, estrogen) and low affinity for Czl steroids (e.g., progesterone, corticosteroids). In Atlantic salmon and Atlantic cod a

2.

CHEMICAL PROPERTIES OF THE BLOOD

91

second protein has been found that binds cortisol with a very high affinity (Freeman and Idler, 1971). In general, the steroid hormone-binding proteins in fish tend to have a higher affinity and higher capacity than those in mammals. Wingfield (1988) has argued that this may explain how fish are able to maintain the very high blood steroid levels typical of most fish. Less is known about the thyroid-binding hormones in fish. It is known that only a small portion of T3 and T4 (<3%) is present in the circulation in the free form (Cyr and Eales, 1989). I n brook trout (Faulkner and Eales, 1973)and rainbow trout, T3 appears to bind to an albumin-like protein with high affinity. Thyroxine appears to bind to vitellogenin in several cyprinid species (MacKenzie et al., 1987) but not in trout. In both salmonids and cyprinids, the level of thyroid hormone binding protein increases in response to estradiol administration (MacKenzie et al., 1987; Cyr and Eales, 1989).

E. Antifreeze Proteins A number of marine teleosts inhabiting polar and subpolar regions produce antifreeze proteins (AFP) or antifreeze glycoproteins (AFGP). These proteins act in a noncolligative fashion; they lower the freezing point of plasma without affecting the melting point. This property of antifreeze proteins is exploited and used to estimate AFP activity, which is expressed in terms of thermal hysteresis: The difference between the melting and freezing points. The greater the difference, the greater the antifreeze protein activity (DeVries, 1983; Fletcher et al., 1984). Antifreeze glycoproteins are found in the Antarctic Nototheniidae and in a number of cod species (e.g., Gadus ogac, G. morhua, Bareogadus saida, Eleginus garcills, Micragadus tomcod; DeVries, 1983) from the Arctic and North Atlantic. The AFGP are well conserved and consist of a repeating structure of alanine-alanine-threonine with a disaccharide attachment to the threonine side chain. It is estimated that AFGP constitutes about 3.5% of the total plasma protein in nototheniids (see DeVries, 1983 for a complete review). Antifreeze proteins have been characterized from a number of species found in the North Atlantic and three distinct types are recognized on the basis of amino acid composition and secondary structure: AFP I, AFP 11, and A F P 111. Unlike AFGP, AFP contain no sugar moieties. Antifreeze protein I are a-helical and rich in alanine and have been found in winter (Pseudopleuronectesamricanus)and yellow tail flounder (Limanda feruginea) and in shorthorn (Myaxocephalus scorpius) and

D. G . MCDONALD AND C. L. MILLIGAN

92

grubby sculpins ( M . awnaeus) (Davies et al., 1988).Antifreeze protein I1 is alanine poor, half-cysteine rich, and has been found in sea raven (Hemitripterus americanus), smelt (Osmerus mordax), and Atlantic herring (Clupea harengus harengus) (Davies et al., 1988; Ewart and Fletcher, 1990).Antifreeze protein 111is different from the other two in that no amino acid predominates and, to date, it has been found only in ocean pout (Macrozoarces americanus) (Davies et al., 1988). There is considerable species variation in the regulation of expression of AFP and AFGP. Winter flounder and shorthorn sculpin produce AFP immediately prior to environmental freezing, whereas Atlantic cod produce AFGP only in response to freezing (Davies et al., 1988). Ocean pout and the Antarctic nototheniids have high levels of AFP and AFGP, respectively, year round (DeVries, 1983; Davies et al., 1988). From studies with winter flounder, it appears that the reduction in day length is the primary environmental cue responsible for initiation of AFP synthesis. Water temperature appears to be an important cue for the clearance of AFP from the plasma in spring (see Davies et al., 1988 for a review). F. Plasma Enzymes

Plasma enzymes are broadly classified into two categories: (a)plasma specific enzymes, having function and purpose in the blood;

and (b) plasma nonspecific enzymes that are derived from moribund cells (Gowenlock, 1988).Measurement of the activities of the plasma nonspecific enzymes has diagnostic potential in fish toxicology and pathology because enzyme activities can often be related to cell damage in specific organs. For example, the liver is rich in glutamicoxalacetic transaminase (GOT) and glutamic-pyruvate transaminase (GPT), and changes in plasma levels of these enzymes may be indicative of liver dysfunction. In trout treated with carbon tetrachloride (CC14), a known hepatotoxicant, plasma GOT and GPT levels increased significantly (Racicot et al., 1975; see Table 111). A few studies have attempted to use plasma enzyme values for the assessment of general fish health and these are summarized in Table 111. However, there are few data available for fish other than rainbow trout and, more importantly, information about the impact of nonpathological factors (e.g., salinity, temperature, reproductive state) is lacking. Furthermore, when available the effects are often complex. For example, in trout, acclimation temperature has a complex effect on the activities of plasma creatine phosphokinase (CPK) and alkaline phosphatase (AlkPase). Creatine phosphokinase activity in trout plasma

Table I11 The Activity of Nonspecific Plasma Enzymes in Different Fish Species Enzyme activity

u1-'

~

LDH"

Tissue specificity:

Muscle Liver Kidney Heart Red blood cells

~-

GPT

AlkPase

CPK

GDH

HBDH

AchE

LAP

Heart

Liver

Liver Kidney Red blood cells Small intestine

Muscle Heart

Liver Kidney

Liver

Red blood cells Nerve tissue

Small intestine

244 157 171 192 267 125 275 310

7.2 4.9 8.0 7.8 17.9 7.0 11.5 15.2

138 68 136 191 111 72 140 122

68.3 45.7 40 49 38 35 55 60

373 230 198 185 249 50 200 225

196 347 167

26.6 47 12.9

GOT

Species Rainbow trout Oncorhynchus mykiss Fedb Starvedb Fresh waterb 10% salinityb 20% salinityb 3.5"C" 10°C" 15°C" Electroshockedd Seinedd Angledd Caught by dip net? Controle CC14 injectione Aeromonas infection; early stage"

1111 730 610 49 1 579 150 825 910 204 222 213 198 86 1 1793 1560

4.2 3.6 3.6

365 1,032 382 1,620 630 920 790 2,940

0.6 1.2 16.2

66 66 74 88 62 50 51 80 10.2 10.8 11.3 11.9

continues

Table 111 continued Enzyme activity UI - 1 LDH"

Tissue specificity:

GPT

AlkPase

CPK

GDH

HBDH

AchE

LAP

Heart

Liver

Liver Kidney Red blood cells Small intestine

Muscle Heart

Liver Kidney

Liver

Ked blood cells Nerve tissue

Small intestine

2468

450

70.7

10,147

32.4

4952 882

187 170

85 51

278

6

Muscle Liver Kidney Heart Red blood cells

GOT

Species

Aeromonas infection; late stage" Caudal puncture' $ Cardiac puncturef Atlantic salmong Salmo salar Pink salmon" Oncorrhynchus gorbuscha Nonspawning Prespawning Spawning Pacific herring'' Culpea pallasii Lingcod" Ophiodon elongutus Red rock fish" Sebastodes

reberrimus Dogfish"

7.2

2,940 678

33

216 50

853

518 2427 2463

309 500 797

1,091 2,936

965 3387 3421

3358

1778

2,948

3228

107

28

141

18

389

0

12

Squolus acarithias Grayling' Thymallus thymallus Male: prespawning Male: spawning Male: postspawning Female: prespawning Female: spawning Female: postspawning

149

128

243 702 756 248 526 455

0

111

18.6 78 27 17 62 29

5,767 5,064 2,802 5,130 6,228 15,600

60 195 94

178 236 164

'I Total IJDH activity is reported. LDH i s a ubiquitous enzyme and most vcrtcbrates show five isozymes, which can be broadly1 classified into three functional groups: heart, spleen, red cell; gill, kidney and liver; white muscle. These groups have distinctive biochemical characteristics that can be exploited to determine the contribution of various tissues to circulating LDH activity (for details, see Gaudet et al., 1975). Sauer and Haider (1979). Fish (100-150 g) held at 15°C; fed group were fed 2 g fish-' day-'; food was withheld from starved group for 20 days. Blood collected via cardiac puncture. Fish held at 10 and 20%0salinity for 20 days. ED Sauer and Haider (1977). Fish (180-222 g) held at various temperatures for 20 days and fed daily. Blood collected via cardiac puncture. Bouck et al. (1978). Fish held at 12°C. Blood collected from anesthetized fish via cardiac puncture. Angled fish struggled for 1 min prior to sampling. Fish were electroshocked with 75 V until stunned. Seined fish were netted 2 at a time. Dip netted fish were netted individually, ' Racicot et a / . (1975). Fish (150-300 g) held at 15°C. Blood sampled via caudal puncture. CC14, 0.4 mg/kg administered in mineral oil via intraperitoneal injection. Aermonas infection: Early stage, fish were starting to lose scales and white spots appeared in caudal region; late stage: dorsal and ventral sides of the peduncle were badly affected. f Gaudet et al. (1975). Fish (100-250 g) held at 15°C. Sandnes et a1. (1988). Fish (1-1.8 kg) held at ambient temperature (2-8°C) at a salinity of 32%. Blood collected from anesthetized fish from the ductus cuvieri. Marquez (1976). Prespawning fish collected by angling. Spawning and nonspawning fish collected by netting. Lingcod, red rock fish, and dogfish were collected via hook and line. Fish placed in holding tanks 12-24 h prior to blood collection via caudal puncture. Hlavovi (1989). Fish (220-350 g) were captured via electroshocking, and blood was collected 2-4 min after capture via cardiac puncture. Abbreviations: LDH: lactate dehydrogenase (E.C. 1.1.1.27)GOT: glutamic-oxalacetic transaminase also known as aspartate aminotransferase (E.C. 2.6.1.1); GPT: glutamic-pyruvate transaminase, also known as alanine aminotransferase (E.C. 2.6.1.2); AIkP: alkaline phosphatase (E.C. 3.1.3.1); CPK: creatine phosphokinase (E.C. 2.7.3.2); GDH: glutamate dehydrogenase (E.C. 1.4.1.3); HBDH: a-hydroxybutyric dehydrogenase (E.C. 1.1.1.30);AchE: acetylcholinesterase (E.C. 3.1.1.7); LAP: leucine amino peptidase (E.C. 3.4.11.1) AlkP Activity expressed in phosphatase unit: Activity which liberates 1pmol ofp-nitrophenol per 30 min at 25°C. AchE activity expressed in Happaport units: Activity that hydrolyzes 1pmol of acetylcholine in 39 min at 25°C. All other enzyme activities expressed in international units: 1 U = 1pmol of substrate used per minute at 25°C.

96

D. G. MCDONALD AND C. L. MILLIGAN

increases when acclimation temperature is increased from 3.5" to 10°C but then declines at 15°C. Alkaline phosphatase activity decreases when acclimation temperature is increased from 6"to 19°C (Table 111). There are also methodological considerations that can influence the interpretation of plasma enzyme activities. In rainbow trout, the method of blood sampling influences the activity of at least two plasma-nonspecific enzymes: lactate dehydrogenase (LDH) and CPK. These enzymes, which are abundant in skeletal muscle, are greater in blood sampled via caudal puncture than via cardiac puncture (Table 111).When catheterization is not possible, Gaudet et aZ. (1975) recommend withdrawing blood via cardiac puncture for enzyme analysis. In addition, the methods commercially available for measurement of plasma enzymes have been developed for mammalian plasma. For some enzymes (e.g., LDH), mammalian assay systems are applicable to fish plasma (D'Apollonia and Anderson, 1980).However, for analysis of enzymes of ammonia metabolism, GTP, GOT, and glutamate dehydrogenase (GDH) assay kits designed for analysis of mammalian plasma may not be directly applicable. D' Apollonia and Anderson (1980) have indicated that unlike the mammalian system, GDH interferes with the transaminase assays in rainbow trout and if the level of GDH activity is high, this may lead to overestimation of GPT and GOT activity. Furthermore, the higher ammonia levels found in fish plasma relative to mammals can lead to higher transaminase activities. This is particularly important when attempting to interpret changes in plasma GOT and GPT activity: An increase in activity may be indicative of liver dysfunction or reflect changes in plasma [ammonia] (D'Apollonia and Anderson, 1980). VII. LIPIDS

A. Total Lipids Lipids are a heterogeneous class of compounds that are grouped together by virtue of their solubility in organic solvents (e.g., chloroform, acetone, ether) and their relative insolubility in water. The lipids include fatty acids and some of their esters (e.g., wax esters), cholesterol and its esters, triglycerides, and phospholipids (Sheridan, 1989). In fish, triglycerides and phospholipids are the most abundant lipid classes. Total plasma lipid levels in most species range from 1200 to 3000 mg dl-' (Dindo and MacGregor 111,1981; Freemont et d.,1981;

2.

CHEMICAL PROPERTIES O F THE BLOOD

97

Sheridan, 1989) and are influenced by many factors, including diet, stress, and reproductive state. In general, the lowest plasma lipid levels are associated with spawning activity and the highest are observed within hours after a meal (Sheridan, 1989). Stress in the form of exhaustive exercise can also deplete plasma lipids; immediately following 5 min of forced swimming, plasma lipids in rainbow trout declined from 3400 to 1000 mg dl-' (Girard and Milligan, 1992).

B. Lipoproteins Plasma lipoproteins facilitate the transport of the otherwise insoluble lipids in plasma from sites of storage (e.g., liver and muscle) to sites of utilization (Henderson and Torcher, 1987). Lipoproteins are thought to consist of a core of the more hydrophobic lipids (e.g., triglycerides, cholesterol esters) surrounded by phospholipids, free cholesterol, and proteins (Darnel1 et al., 1990). Lipoproteins in fish are classified based on their densities according to the system devised for mammals: chylomicrons, very low density lipoproteins (VLDL), LDL, and HDL. The lipid composition of each density class of plasma lipoproteins is comparable to those in mammals; chylomicrons, when found, are rich in triglycerides and are involved in the absorption of lipids from the gut; VLDL is rich in triglycerides; LDL is rich in cholesterol; and HDL is rich in cholesterol and phospholipids (McKay et al., 1985). For a comprehensive review of the subject of plasma lipoproteins in fish and a summary of plasma levels for numerous fish species, see Babin and Vernier (1989). 1. VLDL, LDL, AND HDL

Although plasma lipoprotein levels vary both within and among species and are affected by such things as nutritional and reproductive status, a few generalizations can be made. Relative to mammals, fish, with the exception of elasmobranchs and garpike, are considered hyperlipidemic. The distribution of lipids within each class of lipoprotein shows a strong phylogenetic trend. a. Agnathids. In the agnathids, no one lipoprotein predominates; VLDL, LDL, and HDL are present in more or less equal amounts. In the lamprey (Mordacia rnordax), plasma levels of VLDL, LDL, and HDL during a spawning migration are 426, 664, and 507 mg dl-', respectively (Fellows and McLean, 1982). The LDL fraction is rich in cholesterol, while HDL is associated mainly with phospholipids.

13. G. MCDONALD AND C. L. MILLIGAN

98

While the plasma lipoprotein levels are similar in the Atlantic hagfish (Myxine glutinosa), they contain very little cholesterol; rather, all classes of lipoproteins are rich in triglycerides and phospholipids (Mills and Taylaur, 1973).

b. Chondrichthyes. Very low density lipoproteins and LDL are the predominant lipoproteins in elasmobranchs, with levels ranging from 196 to 415 mg dl-' and 120 to 230 mg dl-', respectively, while HDL levels are quite low; 20-40 mg dl-' (Babin and Vernier, 1989). The lipoproteins of elasmobranchs are distinct from those of other fish in that they are high in squalene and alkyldiacylglycerols. These two constituents are poorly metabolized, have low densities, and tend to concentrate in the liver, suggesting that they play a role in hydrostatic equilibrium in fish lacking a swimbladder (Sargent, 1976). c. Osteichthyes. High density lipoproteins tend to dominate the lipoprotein classes in the bony fish, accounting for as much as 50% of the total lipoprotein. These levels are not only variable among species but also within species, affected primarily by nutritional and reproductive status. Most of the available data are for the salmonids. High density lipoprotein levels range from 238 mg dl-' in juvenile sockeye salmon to as high as 3300 mg dl-' in prespawning pink salmon (Babin and Vernier, 1989). The high HDL levels are associated with high cholesterol levels. Very low density lipoprotein and LDL levels in plasma tend to be lower (167-650 mg dl-' and 225-1189 mg dl-'; Babin and Vernier, 1989) than HDL and are very low or absent from the plasma of spawning salmonids. Also, prolonged starvation (8 weeks) in rainbow trout (Black and Skinner, 1986)and channel catfish (McKay et al., 1985) results in approximately 60-70% and 40-50% reductions in plasma levels of VLDL and LDL, respectively, but HDL levels were unaffected. Although the datum is limited to a single observation on plasma obtained from a moribund specimen, the coelacanth (Latimeria chaZumnae) appears to differ from other bony fish. In coelocanth plasma, VLDL predominates (1105 mg dl-l) with LDL and HDL levels considerably lower (194 and 127 mg dl-', respectively; Mills and Taylaur, 1973). Also, like the hagfish, the coelocanth lipoproteins are cholesterol poor. 2 . VITELLOCENIN

One further class of lipoproteins, vitellogenin, is found in large

2.

CHEMICAL PROPERTIES OF THE BLOOD

99

quantities in mature oviparous female fish (Dye et al., 1986; Henderson and Torcher, 1987). Vitellogenin is a higher density lipoprotein than H D L and contains approximately 80% protein and 20% lipid, most of which is phospholipid (Henderson and Torcher, 1987).Vitellogenesis has been most extensively studied in the salmonids, but similar trends are observed in other species. Although vitellogenin is found in both males and females, the plasma level in females is orders of magnitude greater than in males. For example, in male rainbow trout (mature and immature), plasma vitellogenin ranges from 0.3 to 0.8 p g dl-', whereas plasma vitellogenin ranges from 115 fig dl-' in immature females to as high as 5000 mg dl-' in ovulating females (Scott and Sumpter, 1983; Copeland et al., 1986), representing >50% of the total plasma protein. After ovulation, vitellogenin levels drop to around 50-100 mg dl-' (Scott and Sumpter, 1983).

3. CHYLOMICRONS Chylomicrons are microscopically visible, triglyceride-rich lipoprotein particles formed in the intestinal mucosa following the absorption of' dietary fatty acids. As they circulate in the blood, triglycerides are stripped off by the action of lipoprotein lipases present in plasma and several tissues. The remaining protein particles are thought to be metabolized in the liver (Sheridan, 1988). Chylomicron particles have been observed in plasma from a number of elasmobranchs (Mills et al., 1977) and teleosts (Sheridan, 1988). I n rainbow trout, the particles appear as early as 2-4 h after feeding and are about 80% triglyceride, with minor amounts of cholesterol, cholesterol esters, and phospholipids (Sheridan, 1988). The size of the particles, which ranges from 1000 to 6500 A, is dependent on the lipid content of the diet; the higher the lipid content, the larger the chylomicron particle size (Sheridan, 1988). C. Cholesterol Most fish, with the exception of elasmobranchs and garpike, are hypercholesterolemic relative to mammals. In the agnathids, approximately 50% of the cholesterol is carried by LDL, whereas in elasmobranchs and teleosts, 60-90% of the cholesterol is carried by HDL (Babin and Vernier, 1989). 1. ACNATHIDS

Plasma cholesterol levels in agnathids are high (e.g., 400 mg dl-I in the Atlantic hagfish), though only about 50% of it is associated with

100

D. G. MCDONALD AND C. L. MILLIGAN

lipoproteins (Larsson and Fange, 1977). As with most fish, the highest cholesterol levels are associated with spawning and the lowest seen in food-deprived animals (Babin and Vernier, 1989). 2. ELASMOBF~ANCHS Elasmobranchs have substantially lower plasma cholesterol levels than do agnathids or teleosts; typical values range from 86 to 200 mg dl-' (Larsson and Fange, 1977; Babin and Vernier, 1989; Garcia-Garrido et al., 1990). Plasma cholesterol levels vary during sexual maturation, and at least in the spotted dogfish (Garcia-Garrido et al., 1990), there are pronounced sex differences. Females had consistently lower levels of plasma cholesterol (82 mg dl-') than males (103rng dl-') and, in fact, the lowest were seen in egg-carrying females (Garcia-Garrido et al., 1990). The highest cholesterol levels (112 mg dl-') were seen in males during spermatogenesis. There appeared to be no effect of 3 months of starvation on cholesterol levels in the spotted dogfish (Garcia-Garrido et al., 1990). 3. TELEOSTS

Plasma cholesterol levels show considerable intra- as well as interspecific variability. For example, serum cholesterol in Pacific salmon (Oncorhynchus tshawytscha) varies from 300 to 1470 mg dl-' (Robertson et al., 1961) and in the Atlantic cod, values range from 399 to 1598 mg dl-' (Larsson and Fange, 1977). These extreme individual variations are no doubt linked to differences in diet, activity, and sexual development. Generally, the highest plasma cholesterol levels tend to be seen in both male and female prespawning fish and appear to be linked to the time when fish are actively feeding. Upon spawning, plasma cholesterol tends to drop, though more so in females than in males. In female migrating Pacific salmon, plasma cholesterol reaches highs of 635 mg dl-', but in spawning fish, cholesterol levels drop precipitously, to 126 mg dl-' (Robertson et al., 1961).Although this trend is observed in most species examined, the decline in plasma cholesterol is more severe in fish that undergo an active migration. For example, following a 1000-km upstream migration, plasma cholesterol levels in sockeye salmon dropped from 570 to 294 mg dl-' in males and from 585 to 202 mg dl-' in females (Idler and Tsujuki, 1958). In contrast, in Atlantic salmon captured at sea and transported to the spawning site, plasma cholesterol levels did not drop with spawning, but remained at prespawning levels, 576 mg dl-' (Farrell and Munt, 1983). Furthermore, in Lake Erie coho salmon that undergo only short migrations, plasma cholesterol is 355 mg dl-' in spawning fish com-

2.

CHEMICAL PROPERTIES OF THE BLOOD

101

pared to 540 mg dl-l in prespawning fish (Leatherland and Sonstegard, 1981). Although not as extensively studied, nonsalmonid teleosts tend to show the same general trends: In prespawning sea bass, plasma cholesterol levels peak at about 900 mg dl-' and fall to about 350 mg dl-' in spawning fish (FernBndez et al., 1989). Similarly in plaice (White et al., 1986) and the stripped mullet (Mugil cephalus; Dindo and MacGregor III,1981), peak plasma cholesterol levels (225 and 300 mg dl-', respectively) were seen in the summer months when fish were actively feeding and lowest levels (150 mg d1-l) were seen when fish were spawning. Whether or not the changes in plasma cholesterol associated with spawning are a direct consequence of the physiological changes associated with spawning per se is not clear. Many species cease feeding while spawning, and it may be starvation that reduces plasma cholesterol levels. Few studies have systematically examined the effect of starvation on plasma cholesterol. Farrell and Munt (1983) reported that plasma cholesterol levels declined only slightly in immature Atlantic salmon starved for 6 months (454 mg dl-' in starved, immature females versus 572 mg dl-' in fed, mature females). However, in salmon that had spawned and were starved for 11 months, plasma cholesterol levels were substantially reduced (243 mg d1-l; Farrell and Munt [ 19831). These observations suggest that spawning and the activity associated with it may be the more important determinant of plasma cholesterol levels.

D. Nonesterified Fatty Acids Nonesterified fatty acids (NEFA) are the most metabolically active form of lipid in the blood and are indicative of the extent to which fish rely on lipid as a fuel. Thus, measurement of plasma levels of NEFA in response to environmental and physiological changes yields information about fuel use. A problem that became evident when surveying the literature is that a variety of methodologies have been used to measure plasma [NEFA]; colorimetric (Larsson and Fange, 1977; Zammit and Newsholme, 1979; Moon, 1983; Black and Love, 1986), enzymatic (Santulli et al., 1988), gas-liquid chromatographic (Fellows et al., 1980),and gas chromatographic (Singer et aZ., 1990; Gong and Farrell, 1990; Singer and Ballantyne, 1991). Singer et al. (1990) suggested that the colorimetric, enzymatic, and earlier gas-liquid chromatographic methods may underestimate plasma [NEFA] in some cases b y as much

102

D. G. MCDONALD AND C. L. MILLIGAN

as 30%. The colorimetric method tends to miss the longer chain (18-22 carbon) NEFA because they are easily oxidized and antioxidants were not routinely used. The longer chain NEFA can constitute as much as 30% of the total [NEFA] (Singer et al., 1990). Similarly the enzymatic assay tends to underestimate the longer chain NEFA because the assay is designed primarily to measure palmitic acid (16 carbon chain) in human plasma. In some chromatographic methods, the plasma is extracted and, as with most extraction procedures, there is a tendency to lose sample and unless internal standards are added to the plasma, the loss cannot be quantified. Comparisons between studies employing different methodologies are quite difficult and make not only interspecific comparisons nearly impossible but even intraspecific comparisons difficult. For example, in Atlantic cod, [NEFA] ranges from 0.20 mM (Black and Love, 1986), as determined by one colorimetric method (described by Duncombe, 1963), to 1.28 mM (Larsson and Fange, 1977) using a different method (described by Laurel1 and Tibbling, 1967). Thus, there is no advantage to be gained in citing “typical” values; values reported range from 0.10 to 2.5 mM. For a comprehensive tabulation of plasma [NEFA] in a variety of fish, see Plisetskaya (1980). Despite these methodological problems, plasma [NEFA] determined using the same methodology can be compared and, in doing so, some trends become evident. The lowest [NEFA] are consistently observed in elasmobranchs and the highest levels in cod (Gadus sp.) (Zammit and Newsholm, 1977; Larsson and Fange, 1979; Fellows et d.,1980; Black and Love, 1986).Typically, codfish plasma [NEFA] is two to three times that of other fish and elasmobranch plasma [NEFA] is one-tenth that seen in teleosts and cyclostomes. It is not clear why the cod have such high plasma [NEFA]. However, the striking difference in plasma [NEFA] between elasmobranchs and teleosts may be a direct consequence of the lack of a serum fatty acid-binding protein in the former (see Section VI1,B). Since the solubility of fatty acids in plasma is quite low (pmolar range), only very low levels would be expected in the absence of a fatty acid-binding protein. Many fish species undergo periods of starvation as part of their natural life cycles; some in association with migration and spawning (e.g., salmonids, eels) and others in association with seasonal low temperatures (e.g., eels, bass). Consequently, the response of fish plasma [NEFA] to starvation has received wide attention, and the variability in response is tremendous. An important variable in determining the effect of starvation on plasma [NEFA] is the duration of food deprivation. In the American eel, plasma [NEFA] was unaffected

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by 95 days of starvation (Larsson and Lewander, 1973), whereas after 6 months [NEFA] nearly tripled (Moon, 1983). In sea bass, plasma [NEFAI increased 65% after 40 days of starvation (0.9 to 1.5 mM), but declined after 150 clays of starvation (0.76-0.44 mM; Zammit and Newsholme, 1977). In other species, the response of plasma [NEFA] levels to starvation is more consistent. For example, in the spotted dogfish starved for 40 and 150 days, plasma [NEFA] decreased by about 30% (0.13-0.09 mM; Zammit and Newsholme, 1979) and after 85 days of starvation plasma [NEFA] in the Atlantic cod declined from 8.8 to 2.8 mM and to a low of 0.8 mM after 154 days of food deprivation (Black and Love, 1986). Not only are there extreme variations among species in the response of plasma [NEFA] to starvation but within species as well. For example, rainbow trout starved for 1-4 months show either a steady increase in plasma [NEFA] (Love, 1980)or no change at all (Robinson and Mead, 1973; Black and Skinner, 1986).Also, in some experiments, long-term starvation ( 145 days) raised plasma [NEFA] in the European eel from 0.38 to 0.54 mM, but in other experiments, there was no effect (Larsson and Lewander, 1973; Dave et al., 1976). The interspecific variations of the response of plasma [NEFA] to starvation may be related to the site of lipid storage. In teleosts with extrahepatic lipid stores (the so-called "fatty fish," that is, salmonids, eels), starvation results in a rise in plasma [NEFA] (e.g., Zammit and Newsholme, 1979), as stored triglycerides are mobilized for hepatic metabolism. However, in those species where the liver is the major storage site for lipids (e.g., cod), plasma [NEFA] tend to fall with starvation since triglycerides are already in the liver and their products need not enter the blood stream (Black and Love, 1986; Garcia-Garrido et al., 1990). Plasma [NEFA] levels are also affected by spawning and migration. In all species examined to date, plasnia [NEFA] consistently increases during spawning. For example, in plaice, the peak plasma [NEFA] (0.5-0.6 mM; White et ul., 1986)was seen at spawning and during the period of increased feeding postspawning. Similar trends are seen in the sand dab (Fletcher, 1985), Spicara chryselis ( F e r n h d e z and Planas, 1980), the sea bass (FernAndez et al., 1989), spotted dogfish (Garcia-Garrido et al., 1990), and salmonids (Plisetskaya, 1980). The peak in plasma [NEFA] associated with spawning is associated with gametogenesis, especially vitellogenesis, and reflects the mobilization of lipid reserves required for gondal development (Fletcher, 1985). The increased activity associated with spawning and migration may also be a contributing factor.

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D. G. MCDONALD AND C. L. MILLIGAN

The changes in plasma [NEFA] associated with maturation are not well documented and are limited to observations in salmonids. Generally, smoltification results in a reduction in plasma [NEFA] (by about 50%; Sheridan, 1989; Gong and Farrell, 1990) and a change in plasma [NEFA] composition. The change in NEFA composition accompanying smoltification may be explained by differences in diet, water temperature, and salinity, although the physiological significance remains obscure (see Sheridan, 1989 for a review). Although the response of plasma [NEFA] to stress has not received systematic study, it is apparent that the response is quite variable, again making generalizations difficult. Stress is often associated with hyperglycemia, which, in mammals, results in an inhibition of lipid mobilization and a reduction in plasma [NEFA]. However, this is not necessarily so in fish. In extensive experiments with common carp, Mazeaud and co-workers showed that stress both increased and decreased plasma [NEFA], independent of changes in blood glucose (Mazeaud and Mazeaud, 1981). In response to hypoxia and handling stress, plasma [NEFA] increased in rainbow trout but not until 4-5 h after the stress (Mazeaud and Mazeaud, 1981). Tench (Tinca tinca) show a biphasic response to stress: Plasma [NEFA] was elevated immediately following the stress and later declined to below prestress levels (see Plisetskaya, 1980). Plisetskaya grouped fish into two very broad categories based on the response of plasma [NEFA] to hypoxic stress: (a) those that respond to stress by increasing both plasma [glucose] and [NEFA] (e.g., lamprey, trout); and (b)those that show hyperglycemia and a lowering of plasma [NEFA] (e.g., carp, pike, perch (PercaJuviatilis),bream (Abramis brama).However, given the inconsistencies in the type of stress and the influence of such variables as starvation, sexual maturation, and even time of the day the fish are stressed (Mazeaud and Mazeaud, 1981) on the stress response, such generalizations are premature. E. Triglycerides Triglycerides (TG) are the primary storage form of lipid in most fish species and are readily mobilized in response to physiological demand. Triglycerides released from storage sites (e.g., liver, adipocytes) are transported in the plasma in association with VLDL and LDL. Fatty acids are released from TG by the action of extracellular lipases; the fatty acids are taken up by the tissue, and the glycerol backbone is transported back to the liver via the plasma.

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105

As is the case with the other lipids, the level of plasma TG varies throughout the life cycle of many fish and is affected by such factors as sexual maturation, smoltification (in the salmonids), spawning, and nutritional status. In the salmonids, smoltification generally results in a depletion of body lipids, reflected in a reduction in plasma TG levels from 1100 mg dl-' in parr to 700 mg dl-' in smolts (Dannevig and Norum, 1982; Sheridan, 1989). In all teleost species examined, the highest levels of plasma TG are associated with the period of peak feeding in preparation for spawning. In feeding prespawning plaice, channel catfish, and Arctic char (Saluelinus alpinus), plasma TG levels peaked at 100, 600, and 750 mg dl-', respectively (Dannevig and Norum, 1983; McKay et al., 1985; White et al., 1986). During the period of spawning, plasma TG levels generally decline by about 20-50%. The extent of the decline appears to be related to the level of activity during spawning: The more active fish (e.g., Arctic char) experience a greater decline in TG levels (McKay et al., 1985; Sheridan, 1989). Similarly, starvation results in a loss of TG from the plasma: The longer the period of starvation, the greater the decline in TG levels. In sea bass starved for 40 days, plasma TG levels dropped from 560 to 280 mg dl-' and after 150 days, TG levels fell to about 70 mg dl-' (Zammit and Newsholme, 1979). Similarly, in Arctic char that have ceased feeding due to low temperature, plasma TG levels fell by about 60% to 200 mg dl-' after 60 days (Dannevig and Norum, 1983). The decrease in plasma TG levels is accompanied by an increase in free glycerol. In sea bass, free glycerol doubled in spawning fish, increasing from 2.76 to 5.5 mg dl-' (White et ul., 1986). Similarly, after 100 days of starvation, glycerol increased from 0.18 to 6.7 mg dl-' (Zammit and Newsholme, 1979).There was, however, no further increase after 150 days of starvation. Generally, elasmobranchs tend to have lower plasma T G levels than do teleosts and are less dependent on the physiological and nutritional state of the animal. In spotted and spiny dogfish, plasma TG levels ranged from 30 to 120 mg dl-' (Zammit and Newsholme, 1979; Garcia-Garrido et al., 1990) and, at least in the spotted dogfish, were unaffected by 100-150 days of starvation (Zammit and Newsholme, 1979) or sexual maturation (Garcia-Garrido et al., 1990). This difference may be explained b y the fact that elasmobranchs do not mobilize lipid reserves as triglycerides or NEFA, but rather as ketone bodies, which is supported by the absence of detectable free glycerol in the plasma (Zammit and Newsholme, 1979).

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D. G. MCDONALD AND C. L. MILLIGAN

VIII. ELECTROLYTES The major trends in electrolyte/osmolyte composition in fish from different groups is well known and only the highlights will be reviewed here. For more detailed information the comprehensive review by Holmes and Donaldson (1969) in Volume I11 should be consulted. A. Na+, C1-, and Osmolarity Na+ and C1- are the major ions in the blood of all fishes. Na+ concentration typically exceeds [Cl-] in all but the hagfishes (Table IV) where the Na :C1 ratio is 0.95 reflecting the similarity of hagfish plasma to seawater (seawater Na: C1 ratio = 0.86). In lampreys and marine elasmobranchs the Na: C1 ratio is 1.04 while it averages 1.1 in most teleosts (both marine and fresh water). The one major exception is the freshwater anguillids that have unusually low plasma [Cl-] and where the ratio averages 1.55 (cf. Farrell and Lutz, 1975). Furthermore, in all but the chondrichthyes and the coelocanths, NaCl contributes over 75% of the osmolarity of the plasma. In marine cartilaginous fishes, NaCl makes u p only about 50% of the total osmolarity. Nonprotein nitrogen compounds, mostly urea, secondarily TMAO, make up most of the balance. Plasma Na+ and C1- measurements are much more common than measurements of plasma osmolarity, and the electrolyte measurements are typically more reliable because of the simplicity and standardization of the techniques. Hence, we have used the equation OsmNaCl= (Na+ + C1-) * 0.91 (the NaCl osmotic activity coefficient; Robertson, 1989) instead of osmolarity in a survey of the electrolyte composition (degree of dilution of body fluids) among several groups (see Table IV). AS Lutz (1975) has argued, there are broad trends in the osmolarity and electrolyte composition of fish plasma that reflect the evolutionary and environmental history of each group. The basic notion is that, with the exception of the hagfish, all groups of fishes originated in fresh water (or brackish water), 400 million years ago in the major radiation that occurred in the Ordivician and Silurian periods. In the evoiutionary ferment of this period, selection pressures, Lutz argues, would have strongly favored dilution of body fluids to reduce the cost of osmoregulation. Hence, the lowest electrolyte levels are found in the lungfishes (OsmN,cl = 172) and the African polypterids (OsmN a C l = 172) that have had a long and continuous history of life in fresh water. Other groups (elasmobranchs, coelocanths, lampreys, chon-

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drosts, holosts, and teleosts) have at some point in their evolutionary history reinvaded seawater, and some representatives of all groups except coelocanths later returned to fresh water. Ion levels in the fresh water representatives of these groups are correspondingly higher than in the lungfishes and reflect, approximately, the duration in evolutionary time spent in freshwater habitats. Among the freshwater elasmobranchs, the euryhaline species (e.g., Lake Nicaragua shark, bull shark; Carcharhinus sp.), which are quite recent emigrants to fresh water (McFarland et al., 1979), have the highest OsmNaCl of all freshwater fish (381) and still retain urea at levels about 50%of that of their marine counterparts (137 versus 374 mM; Table IV). I n contrast, the freshwater rays, Potarnotrygon sp., which are stenohaline and have been in fresh water for at least 15 million years (McFarland et al., 1979), have a much lower OsmNaCl (281), while urea (0.7 mM) makes a negligible contribution to plasma osmolarity. The OsmNaClof the remaining freshwater fish species fall within the range of 195-252 (lampreys [201], chondrosts [2161, holosts [2521, and teleosts [ 195-2523). Freshwater teleosts exhibit almost the full range of OsmNaCl in freshwater fishes but the trends within this group are not as readily apparent. OsmNaCl in stenohaline freshwater species such as the white sucker (195)are low relative to the euryhaline salmonids (252),but other euryhaline species such as eel (Anguilla sp.) are also low (229). Among marine teleosts, a clear distinction can be drawn between euryhaline and stenohaline forms. In stenohaline species (average of 21 species) OsmNaCl averaged 346, while in euryhaline species (average of 16 species) it was significantly lower, averaging 311 (Table IV). Plasma Naf and C1- levels are sensitive to a wide variety of environmental and endogenous influences. In addition to circadian fluctuations described earlier, these influences can be divided into three categories: (a) changes in [NaCI] related to a change in the osmotic gradient across the gills (i.e., due to a change in external salinity), (b) changes brought about by effects of external pollutants on gill function, and (c) stress-related changes. 1. EFFECTS OF SALINITY CHANGE

The emphasis in research has been on euryhaline species (salmon, trout, killifish, eels, flounders, and mullet; Evans, 1984), particularly on salmonids and on the transfer from fresh water to seawater (see Evans, 1984 in Vol XB for a review of euryhalinity; Hoar, 1976,1988 in Vol XIB for reviews on smoltification in salmonids). The adaptability of salmonids to seawater is both species and age dependent and is also

Table IV Summary of Plasma Electrolytes and Urea" in Fish"

OSM Na+ C1-

NaCl

OSM

K+

Seawater Hagfishes

458 499

535 524

903 930

1070 1069

10.5 9.5

9.8 5.4

42.6 18.2

0 6.5

29.1 5.2

Lamprey (Petromyzon marinus) Marine elasmobranchs Coelacanth (Latimeria chalumnae) Stenohaline marine teleosts Euryhaline marine teleosts Freshwater elasmobranch sp. Potamotrygon sp. Lamprey (freshwater adults)

159 153 266 259 197 187 20 1 174 178 164 22 1 189 152 158 117 104

284 476 349 346 311 381 281 201

331 991 932 425 527 295 256

5.4 8.4 5.8 8.9 7.3 7.0 3.1 4

3.5 5.6 4.9 3.4 3.2 3.9 1.4 1.9

7.0 2.8 5.3 4.7

-

4.0 5.1 5.8

4.4 2.3 4.8 4.2

106 130 102 147

172 216 172 252

238 -

2.5 3.3 2.2 2.6

1.8 2.0 2.2 2.8

Ca2+ Mg2+ PO4 SO4

Urea

Comments

References

L 0

E

Dipnoi (lungfish) Chondrostei Pol ypteridae Holostei

83 108 87 131

-

-

-

_

-

1.5 4.5 1.2

3.8 5

0.6 -

1.2 1.9 1.3 1.5

1.8 1.5 1.5 1.9

0.9 0.6 0.6 1.4

-

6 analyses 0 3.3 Eptatretus, Myxine, Polis t remata 9 species 374 1 living specimen 377 8.3 21 species - 15 species 137 3 species 0.7 P . marinus and L. Juuiatilis 0.7 5 species - 2 species 2 species 0.5 2 species

1 1 2-4 1,5-7 8 1,6 1

1,9 1, 10, 11 1,3, 12-14 1, 11, 15

15 15 1

Teleosts Salmonidae (Oncorhynchus mykiss) Cyprinidae Anguillidae Catostomidae (Catastomus commersoni) Ictaluridae (Ictaluris punctatus)

147

122

246

-

3.7

2.5

1.3

2.6

2.8

133 151

118 110

228 229

274 293

3.3 3.0

1.8 2.9

1.5 1.7

0.4 1.5

-

-

-

120

94

195

245

2.6

2.1

0.8

-

1.0

-

20-22

142

110

229

259

3.1

-

-

-

-

-

23

1.0 17 studies

Goldfish and carp A. rostrata and A. anguilla

16,22 17-18 1, 13, 19

(’All measurements are in mM. Except where noted values are means of 3-21 measurements with sources indicated. The osmolarity contributed by NaCl (OsrnNacl)was calculated as the sum of Na+ and C1- multiplied by 0.91 (After Robertson, 1989). 1. Holmes and Donaldson (1969).2. Mathers and Beamish (1974).3. Beamish et al. (1978).4. Beamish (1980).5. Wells et al. (1986).6. Griffith (1981).7. Robertson (1989).8. Griffith et al. (1974).9. Thorson et al. (1973). 10. Griffith et al. (1973). 11. Mangum et al. (1978). 12. Pickering and Morris (1970).13. Robertson (1984).14. Tufts and Boutilier (1989).15. Urist et al. (1972). 16. Hille (1982).17. Houston and Koss (1982).18. Fuchs 0 and Albers (1988). 19. Farrell and Lutz (1975).20. Wilkes et al. (1981).21. H6be et al. (1983).22. Hdbe et al. (1987).23. Cameron (1980). (D

110

D. G. MCDONALD AND C. L. hlILLIGAN

dependent, in some species, on there being preparatory physiological changes in fresh water prior to seaward migration (smoltification). The latter is particularly prominent in those species that migrate to sea only once (e.g., all Oncorhynchus sp. except 0.mykiss) (Hoar, 1988). Upon abrupt transfer of Oncorhynchus sp. to seawater there are transient increases in plasma Na+ and C1-, not normally exceeding 20-40 mM in the successfully adapting animal; the peak is reached within 12 h of transfer and thereafter plasma levels decline to stabilize at or near freshwater values, usually by 24-36 h (Sweeting and McKeown, 1987; Yada et al., 1991). The corresponding return to fresh water for Oncorhynchus sp. is accompanied by a transient depression of plasma electrolytes followed by a more gradual recovery, but still within 7 days (e.g., Ogasawara et al., 1989). In most other teleosts, including salmonids that have a lower seawater tolerance (e.g., Salvelinus sp.; Finstad et al., 1989) and other teleosts with similar or higher seawater tolerance (e.g., milkfish, Chanos chanos; Ferraris et al., 1988) the response to salinity transfer is usually more exaggerated. Plasma Na+ and C1levels take a much longer time to stabilize after salinity transfer (up to 2 weeks) and typically stabilize at levels substantially higher in seawater than in fresh water. For example, in eels (Anguilla sp.), seawater adapted forms have plasma Na+ and C1- levels 50-60 mM higher than freshwater forms (Table IV). 2. EFFECTS OF POLLUTANTS

A variety of environmental contaminants in fresh water and seawater are toxic to fish, at least in part, because they disrupt gill function. The primary effect of many of these contaminants is the impairment of gill iono-regulation and, therefore, disruption of plasma electrolyte balance (see McDonald et al., 1989 for review). Some toxins, such as Cd and Zn, disrupt gill Ca2+ fluxes but the majority (e.g., acidic pH, Cu, Al, Hg) act principally by the disruption of NaCl fluxes. This disruption develops in part through inhibition of NaCl transport, but mostly through increasing the NaCl permeability of the gills (McDonald, 1983b; McDonald et al., 1989). Substantial depression of plasma [NaCl] can result in freshwater fish (or elevation in marine fish; Bouquegneau and Gilles, 1980) even with sublethal exposures. The response of freshwater fish is characterized by an initially rapid depression in plasma NaCl, which can be as much as 30%,and is usually complete within 24 h (in sublethal exposures) followed by a more gradual recovery where fish may either regain original ion levels or, more commonly, reach a new steady state with continued depression of plasma [NaCl]. For example, Audet et al. (1988) showed in rainbow

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CHEMICAL PROPERTIES OF THE BLOOD

111

trout surviving and feeding through 84 days at p H 4.8 that NaCl levels were still about 20 mM lower than in neutral p H controls.

3. EFFECTS OF STRESS Stress has profound effects on plasma electrolytes, particularly Na+ and C1-. A variety of acutely stressful procedures such as angling or capture, enforced exhaustive exercise, and blood sampling trauma provoke catecholamine and cortisol release and niuscle lactacidosis. Over the time frame of a very few minutes, the latter increases intracellular osmolarity, provoking a shift in fluid from the extracellular space and elevating plasma [NaCll. For example, in rainbow trout, 5-6 min of enforced exercise produced a 10-20 mM increase in plasma Na+ and C1-, and both remained significantly elevated for 24 h (Graham et al., 1982; Holeton et al., 1983). If the stress is prolonged over the longer term, >0.5 h, then the impact of chronic catecholamine elevation on branchial and renal ion and electrolyte flows will become more apparent. For freshwater teleosts this will mean a net loss of NaCl and for marine teleosts a net gain. For example, 8 h of crowding stress in lake trout, Salvelinus namaycush, led to a 25-30 mM depression in Naf and C1+ in the first 4 h, little change over the subsequent 4 h and, when crowding stress was removed, over 24 h was required for recovery (McDonald and Robinson, 1992). B. Calcium In freshwater fishes total plasma Ca2+ levels are quite uniform, falling within the narrow range of 2-3 mM, except for the euryhaline elasmobranchs where plasma Ca2+ is 3.9 mM (Table IV). In marine fishes, Ca2+ levels are higher (ranging from -3 to 2 5 mM in coelocanths, elasmobranchs, and myxinoids, Table IV) but are still one half or less seawater values (-10 mM). [Ca"] is apparently very tightly regulated in both freshwater and marine fishes. For example, a variety of normal stresses produced either no effect on plasma [Ca"] in rainbow trout (hypoxia, hypercapnia, sustained exercise) or caused a slight increase (exhaustive exercise; Andreason, 1985). A much more extreme treatment, elevating [Ca2+]from 10 to 100 mM in seawater, led to only a 40% increase in plasma [Ca"] in the Atlantic cod (Bjornsson and Deftos, 1985). Furthermore, circadian fluctuations in plasma [Ca2' 1 appear to be negligible (Houston and Koss, 1982; Kuhn et al., 1986; Laidley and Leatherland, 1988b). A fraction of total plasma calcium is always protein bound. The bound fraction in freshwater teleosts ranges from 30 to 48% in males

112

D. G. MCDONALD AND C. L. MILLIGAN

and nongravid females (Bjornsson and Haux, 1985). Marine fishes are less well studied but the bound fraction in the cod was 22% (Bjornsson and Deftos, 1985). Any change in plasma protein will effect total plasma [Ca" I although, generally speaking, the free [Ca2+I tends to remain constant. The largest change in plasma protein (>threefold increase) is that associated with vitellogenesis, where total Ca2+ levels reach 5 mM in normally ovulating rainbow trout, but can be as high as 9 mM with estrogen injection (Bornsson and Haux, 1985; Bjornsson et al., 1986). However, the ionic [Ca2+] remained constant at about 1.4 mM. C . Magnesium

Plasma M 2 + levels are lower than Ca2+ levels in all freshwater fishes (except Potamotrygon sp.) and in the marine elasmobranchs (Table IV). In the other marine fish, Mg2+ levels exceed Ca2+ levels with the highest M 2 + levels being found in the myxinoids (18 mM) although this is still less than half that of seawater (43 mM). Mg2+ is less well studied than Ca2+ with only a few studies, all on freshwater teleosts. However, the present evidence (van der Velden et al., 1989) suggests that Mg2+ is as tightly regulated as Ca2+, even though the mechanisms of Mg2+ regulation are unknown. Like Ca2+, Mg2' is bound by plasma proteins; about 25% of the total plasma Mg2+ in rainbow trout (Bjornsson and Haux, 1985). Since Mg2+ levels in erythrocytes are 210-fold higher than plasma levels (Houston, 1985),hemolysis can contribute significantly to erroneous plasma values.

D. Potassium Plasma levels are 2-4 mM in freshwater fish and 5-10 mM in marine fish (Table IV). Less than 2% of the total body K+ is contained in extracellular fluids (Eddy, 1985). Consequently, plasma K+ levels are relatively unaffected by any treatments that increase gill electrolyte permeability because any branchial influx or efflux would be readily buffered by transfers to and from the intracellular compartment. However, treatments that produce an intracellular acidosis, such as strenuous exercise, will cause an outward leak of K+ from muscle cells. For example, in rainbow trout, 6 min of exhaustive exercise led to a progressive increase in plasma [K+ 1, reaching a peak twice preexercise levels at 4 h postexercise and still significantly elevated at 8 h (Graham et al., 1982). Similar increases in plasma [K)] are seen in marine teleosts following strenuous exercise (e.g., Boutilier et al.,

2.

CHEMICAL PROPERTIES OF THE BLOOD

113

1984; Wells et al., 1986). There is also the potential for a substantial error in estimates of plasma K+ levels if hemolysis occurs because of high K+ levels in erythrocytes (90-130 mM).

E. Phosphate and Sulfate Data for both of these anions are sparse relative to other plasma ions. Available data (Table IV) indicate that the levels of phosphate are usually higher than sulfate and that both are higher in marine fish compared to freshwater fish. A fraction of phosphate is protein bound, about 17% in freshwater rainbow trout (Bjornsson and Haux, 1985),but the amount bound increases with increasing plasma protein levels so the free phosphate fraction remains unchanged (Bjornsson and Haux, 1985).Little, however, is known of the regulation of either ion nor of the factors that affect plasma concentrations (c.f. Bjornsson and Haux, 1985; Hille, 1982; HGbe, 1987).

ACKNOWLEDGMENTS We gratefully acknowledge the information and advice contributed by our colleagues, in particular, C . Audet, J . Ballantyne, A. P. Farrell, G. Flik, J. Gutibrrez, A. H. Houston, B. A. McKeown, T. W. Moon, M. Nikinmaa, R. E. Peter, E. M. Plisetskaya, and C . M. Wood. Any errors, however, are ours.

REFERENCES Alexander, J. B., and Ingram, G. A. (1980).Acomparison offive ofthe methods commonly used to measure protein concentrations in fish sera.]. Fish B i d . 16, 115-122. Andreason, P. (1985). Free and total calcium concentrations in the blood of rainbow trout, Salmo gairdneri, during ‘stress’ conditions. J. E x p . B i d . 118, 111-120. Arnold-Reed, D. E., Balment, R. J., McCrohan, C. R., and Hackney, C . M. (1991). The caudal neurosecretory system of Platichthys flesus: General morphology and responses to altered salinity. Comp. Biochern. Physiol. 99A, 137-143. Arthur, P. G., West, T. G., Brill, R. W., Schulte, P. M., and Hochachka, P. W. (1992). Recovery metabolism of tuna white muscle: Rapid and parallel changes of lactate and phosphocreatine after exercise. Can.J . Zool. (in press.) Audet, C., Munger, R. S., and Wood, C . M. (1988). Long-term sublethal acid exposure in rainbow trout (Salmo gairdneri) in soft water: Effects on ion exchanges and blood chemistry. C u n J . Fish. Ayuat. Sci. 45, 1387-1398.

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3 B L O O D AND EXTRACELLULAR F L U I D VOLUME REGULATION: R O L E OF THE RENINANGIOTENSIN SYSTEM, KALLIKREIN-KININ SYSTEM, AND ATRIAL NATRIURETIC P E P T I D E S KENNETH R . OLSON Indiana University School of Medicine, South Bend Center University of Notre Dame Notre Dame, Indiana

1. Introduction 11. Fluid Compartments A. Total Body Water B. Intracellular Water C. Extracellular Water D. Blood Volume 111. Renin-Angiotensin System A. Components of the Renin-Angiotensin System B. Occurrence and Distribution in Fish C. Stimulus for Activation of the Renin-Angiotensin System D. Effects of Angiotensins IV. Kallikrein-Kinin System A. Components of the Kallikrein-Kinin System B. Occurrence and Distribution in Fish C. Effects of Kinins V. Atrial Natriuretic Peptides A. Structure of Natriuretic Peptides B. Distribution in Fish C. Physiological Significance of Natriuretic Peptides VI. Summary References

135 FISH PHYSIOLOGY, VOL. XIIB

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

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I, INTRODUCTION Osmoregulation has long been a fascinating subject for biologists and in fish it has probably been more extensively studied than any other physiological or biochemical process. Volume regulation, on the other hand, even though it is inexorably interwoven with osmoregulatory activities, has been less frequently examined and is still poorly understood. Why volume regulation has received so little attention is not clear. Certainly technical difficulty in measuring and monitoring the various fluid compartments has been a contributory factor. Perhaps more importantly, however, is the fact that volume regulation in most vertebrates is poorly understood. The intent of this chapter is to provide a conceptual basis for volume regulation in fish and to evaluate the role of the renin-angiotensin system (RAS), kallikrein-kinin system (KKS), and atrial natriuretic peptides (ANP) in this process. Admittedly there is considerable bias in this approach because many of the concepts regarding fluid compartments and volume regulation have been developed in mammalian models and may not be directly applicable to fish. Water is not actively transported and, therefore, movement of fluid between compartments or within subcompartmental spaces is governed by hydraulic and osmotic forces across compartmental boundaries and the permeability barriers between them. Volume regulatory processes can be considered at various levels from the intact fish to single cells and even within subcellular compartments. Events that affect one compartment invariably impact on all others and initiate a cascade of physiological changes and responses. The emphasis of this chapter is on extracellular volume regulation. Because water accounts for around 95% of the mass of the mobile extracellular compartment, the assumption is made that extracellular volume equals extracellular water. Furthermore, the importance of intracellular fluids in regulating extracellular volume is assumed to be minimal because individual cells attempt to maintain constancy of their internal milieu. Therefore, while intracellular water can provide a substantial fluid reservoir it appears to react to, rather than control, the extracellular fluid environment. 11. FLUID COMPARTMENTS

Organization of the various fluid compartments has been described by Holmes and Donaldson (1969) and is briefly outlined on the following page. Only three compartments (*) can be measured directly.

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The Basic Fluid Compartments

I. Total body water* A. Intracellular fluid B. Extracellular fluid* 1. Interstitial fluid 2. Intravascular fluid* a. Unstressed volume b. Stressed volume 3. Lymph 4. Transcellular fluid The lymphatic system is discussed elsewhere in this volume. Transcellular fluids have been described by Holmes and Donaldson (1969) and will not be considered further. A. Total Body Water Total body water (TBW) is the amount of water in an intact fish. Total body water is most accurately determined by desiccating the carcass to a constant weight in an oven, although the terminal nature of this process may be of limited value in some experiments. Methods that employ indicator dilution techniques (Isaia and Masoni, 1976; Kitzman et al., 1990), neutron activation analysis (Talbot et al., 1986), and freeze drying (Cameron, 1980)have also been used. Values for TBW are usually reported as a fraction of total body weight (bwt) (i.e., g or ml HzO/100 g bwt). Some authors (cf. Assem and Hanke, 1979; Bittner and Lang, 1980; Loretz, 1979a,b) prefer to express body water as a fraction of dry cell solids (dcs) (g HzO/g dcs). Water expressed as a fraction of total tissue weight will be less sensitive to a change in either water or solids content, whereas slight changes in tissue solids may overemphasize the importance of the g HzO/g dcs value. With regard to TBW, neither calculation method seems to have a particular advantage, although Shearer (1984) has shown in rainbow trout, Oncorhynchus mykiss, that water weight per total tissue (wet) weight is a better indicator of elemental status than dry weight concentration. Presumably there is more variability in the dry weight calculation due to individual differences in body fat. To be consistent with the majority of values in the literature, volumes and spaces originally presented as g HzO/g dcs have been converted to ml/kg total weight using the formula: ml HzO/kg bwt

=

[g HzO/g dcs/(l+g HzO/g dcs)] . (1000/1.05), (1)

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where 1.05 is the assumed density of extracellular fluid. Values reported in the literature as g/100 ml body weight have also been converted to ml/kg by multiplying by 10/1.05. Total body water reflects the net water balance between a fish and its environment and is determined by osmotic and hydraulic components. Osmotic forces are affected by active and passive exchange of electrolytes across the gills, skin, gastrointestinal and renal epithelia, and nonionic osmolytes generated through metabolic activities. Net water balance is also affected by mechanical compliance of the body (total body fluid volume divided by total body fluid pressure). Total body compliance determines the relationship between total fluid volume and average internal hydraulic pressure. Whole body compliance has not been measured in fish. The entropic and homeostatic movements of water and osmolytes across fish epithelia are summarized for typical freshwater (FW) and saltwater (SW) teleosts in Fig. 1. All freshwater fish counter a volume expanding environment by actively accumulating ambient and dietary FRESH WATER

SALT WATER

/-mm--. H 2 0 NaCl

Fig. 1. Salt and water metabolism in freshwater (FW) and saltwater (SW) fish. In FW there is a net accumulation of water and loss of salt that is compensated by active accumulation of ions by the gills and through dietary sources. Dilute urine excreted by the kidneys eliminates the water load while conserving electrolytes. Passive water and salt fluxes are reversed in SW, and here fish drink SW to restore volume. Monovalent ions, ingested by drinking and through the diet, are excreted by the gills, divalent ions are excreted by the kidneys in a minimum of urine. Intracellular volume, indicated by box, is regulated by adjusting intracellular osmolytes (ions, organics, or both). Coiled spring on dorsum suggests net whole body compliance conditions, expansion in FW and contraction in SW.

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BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

139

sodium and chloride across the gills and gut, respectively. The kidney in these fish rids the body of excess water with a minimum solute loss. Renal mechanisms, although not completely understood, appear to derive much of their efficacy toward pressure and volume regulation through pressure natriuresisldiuresis responses (Nishimura, 1985a), similar in principle to those described in mammals (Guyton, 1990; Guyton et al., 1990; Roman, 1986). Not all saltwater fish are hypoosmotic to their environment. Marine myxinoids (hagfishes) are osmoconformers; that is, they are very close to being isosmotic with seawater and extracellular electrolytes almost mirror the environment. However, most studies have shown that these fish are in fact around 2-10% hyperosmotic to their environment (Alt et aZ., 1981; Cholette et al., 1970; Robertson, 1974, 1976, 1986). One might assume that total body volume regulation in myxinoids is subjected to the same constraints and responses as freshwater fish, albeit at a reduced scale. Marine elasmobranchs maintain body electrolytes somewhat higher than teleosts but well below that of the environment. However, elasmobranchs are also slightly hyperosmotic (1-10%) to seawater due to internal generation and retention of organic osmolytes, most notably urea and trimethylamine oxide (Holmes and Donaldson, 1969; Robertson, 1975, 1989). Thus, similar to myxine, elasmobranchs osmoregulate in a slightly hypotonic environment. Saltwater teleosts are hypoosmotic to their environment and respond to volume depletion by drinking seawater and excreting the monovalent ions across the gill. Divalent cations are excreted by the kidney in minimal solvent. It may well be that marine teleosts and osmoregulating marine lampreys are the only fish capable of living in a substantially dehydrating environment.' Representative water contents of whole fish and skeletal muscle, and the effect of environmental salinity are shown in Tables I and 11. Some variation in the data can be attributed to interspecific differences in dry weight composition due to scales (Thorson, 1961), bone (Cameron, 1985), and fat (Shearer, 1984; Thorson, 1961).There is also a 10-15% decrease in TBW with age (Denton and Yuosef, 1976). However, as previously observed (Thorson, 1958, 1959, 1961), the most notable features of total water volume, or any of the fluid volumes for that matter, are the remarkable constancy between species and the minimal effect of environmental salinity. Lungfish and other species may encounter transient dehydration but restoration of

full physiological function in these fish is not achieved without external hydration in a hypoosmotic environment.

140

KENNETH R. OLSON

Table I Total Body Water in Fish Species Cyclostomes Myxine glutinosa Eptatretus stoutii Petromyzon marinus Lampetra planeri Chondrichthyes Hydrolagus colliei Squalus acanthias Raja binoculata Raja rhina Squalus acanthias Scyliorhinus canicula Potamotrygon hystrix Osteichthyes Chondrosteans AcipenserfEuvescens Polyodon spathula Holosteans Lepiosteus patostomum Amia calaa Teleosteans lctiobus cyprinellus Notropus cornutus Cyprinus carpio lctalurus punctatus lctalurus punctatus Catostomus commersoni Catostomus commersoni Fundulus grandis Enneacunthus obesus Channa punctatus Lepomis gibbosus Perca fluuescens Pseudoscarus guacamuia Anguilla anguilla Epinephelus striatus Gymnothorax funebris Sphyraena barracuda Sarotherodon mossaiizbicu5 Mycteroperca tigris Lutianus griseus Lutianus campechanus Platichthys stellatus

Environment'

Valueb (ml/kg)

Reference

sw sw sw sw

689 746 720 760'

Robertson (1976) McCarthy and Conte (1966) Thorson (1959) Bull and Morris (1967)

sw sw sw

Thorson (1958)

FW

680 683 787 78 1 712 730 793

FW FW

692 705

Thorson (1961)

FW FW

635 710

Thorson (1961)

FW FW FW FW FW

672 758 680 653 664 531d 709 775 737 737 668 768 758 696 708" 683 607 672 742 677 689 676 784

Thorson (1961) Freda and McDonald (1988) Thorson (1961) Cameron (1980) Kitzman et al. (1990)

SW

sw

FW FW FW FW FW FW FW

sw sw sw SW

sw sw sw sw sw sw

Robertson (1975) Robertson (1989) Bittner and Lang (1980)

Thorson (1961) Hdbe (1987) Spence et a1. (1977) Gonzalez and Dunson (1987) Sinha and Munshi (1979) Gonzalez and Dunson (1987) Freda and McDonald (1988) Thorson (1961) Isaia and Masoni (1976) Thorson (1961)

Loretz (1979b) Thorson (1961) Thorson (1961) Milligan and Wood (1987b) (continues)

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

141

Table I (Continued) Species

Salmo trutta Salmo salar Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss

Environmenta FW SW FW

sw

Oncorhynchus mykiss

FW FW FW FW FW FW SW FW SW FW

Oncorhynchus mykiss

FW

Oncorhynchus mykiss Fundulus olivaceus

FW FW 50% SW FW 40% sw

Oncorhynchus mykiss

Fundulus catenatus

sw sw

Valueb (mllkg) 735 720 694f,r 752" 7 14 731 740 773 8 10 773 736 770 759 782 780 737 739 731 711 751 734 748

Reference Madsen (1990) Talbot et al. (1986) Freda and McDonald (1988) Milligan and Wood (1982) Hdbe (1987) Madsen (1990) Schiffman and Fromm (1959) Eddy and Bath (1979) Finstad et a / . (1988) Bath and Eddy (1979a) Jackson (1981) Munger et al. (1991) Duff and Fleming (1972a) Duff and Fleming (1972b)

Environment-salt water (SW) or fresh water (FW). Ml/kg body weight, determined by dessication unless otherwise indicated. Ammocoete. Antipyrine space. 3 H z 0 space. f Parr. R Determined by neutron activation. a

B. Intracellular Water Control of intracellular fluid volume (ICFV) and composition enables solvation and movement of appropriate molecules upon which cellular functions are achieved. When confronted with hyper- or hypotonic environments, most cells behave initially as near-perfect osmometers. However, in time these cells restore part, or all, of the volume perturbation by adjusting intracellular osmolytes, thereby producing a regulatory volume increase or decrease, respectively. Both intracellular ions (Assem and Hanke, 1979; Lang et al., 1990; Schmidt-Nielsen,

KENNETH R. OLSON

142 Table I1 Total Water in Fish Muscle ~~

~~

~

Species C yclostomes Myxine glutinosa Myxine glutinosa Chondrichthyes Potamotrygon hystrix Squalus acanthias Callorhyncus millii Osteichthyes Anguilla rostrata Catostomus commersoni Saloelinus fontinalis

Sarotherodon mossambicus Perca fluviatilis Salvelinus alpinus Platichthys stellatus Anguilla anguilla

Environment"

Reference

SW SW

74 1 683

Robertson (1986) Cholette et al. (1970)

FW SW SW

799 782 792

Bittner and Lang (1980) Bedford (1983) Bedford (1983)

FW FW FW

736 79 1 758 (751-768)

FW FW SW' SWd SW FW

77 1 764 782 783 804 746f 737f 742" 734" 788 792 783 (768-804)

Walsh and Moon (1982) Gras et al. (1971) Houston et al. (1971) Houston et al. (1969) Nichols et al. (1985) Assem and Hanke (1981) Lutz (1972) Finstad et al. (1989)

sw

FW Brecortiu tyranicus

sw sw

Cyprinus carpi0

FW FW

sw Oncorhynchus naykiss

FW SW

Salnio salar

sw sw FW

Sulmo salar

Valueb (ml/kg)

sw

FW

749" 769 (728-817) 744 (706-802)

753" 644 610 773 779

Milligan and Wood (l987b) Chan et al. (1967)

Engel et al. (1987)

Abo Hegab and Hanke (1982) Cupta and Hanke (1982) Abo Hegab and Hanke (1982) Eddy and Bath (1979) Bath and Eddy (19794 Leray et al. (1981) Cras et al. (1971) Murphy and Houston (1977) hlearow and Houston (1980) H6be (1987) Houston and Mearow (1979) hlilligan and Wood (1982) Munger et al. (1991) Parry (1961) Talbot and Potts (1989)

(continues)

3.

B L O O D A N D EXTRACELLULAR F L U I D V O L U M E REGULATION

143

Table I1 (Continued) Species

Environment"

Salmo trutta

FW

Gasterosteus aculeatus

FW SW FW SW FW SW FW SW FW SW

Fundulus heteroclitus Gillichthys mirabilis Tilapia mossambica Pleuronectes jiesus

sw

Valueb (ml/kg) 742 735 781 764 755 736 79 1 785 769 753 (744-763) 773 768

Reference Madsen (1990) Lange and Fugelli (1965) Schmidt-Nielsen (1977) Loretz (1979,) Assem and Hanke (1979) Lange and Fugelli (1965)

Environment-salt water (SW) or fresh water (FW) ml/kg tissue wet weight. 7 glliter NaCI. 15 glliter NaCl. Smolt. fyellow eel. Silver eel.

1975, 1977) and organic solutes (Abo Hegab and Hanke, 1983; Assem and Hanke, 1983; Ballantyne et al., 1987; Goldstein and Kleinzeller, 1987; King and Goldstein, 1983; Kleinzeller and Ziyadeh, 1990; McConnell and Goldstein, 1990) may be regulated. Fluid movement in and out of the intracellular compartment appears to be an obligatory response to changes in intracellular and extracellular osmolarity, and it is probable that the regulatory responses of individual cells are designed to protect intracellular volume rather than to control extracellular fluids. The assumption is made, then, that although fluid shifts between intracellular and extracellular compartments may be substantial, neurohumoral regulation of fluid volume is achieved principally, if not entirely, by adjustments in the volume and composition of extracellular fluids. C. Extracellular Water Extracellular water is the sum of interstitial, plasma, lymph, and transcellular fluids. Regulation of extracellular fluid volume (ECFV)

144

KENNETH R. OLSON

and composition is necessary to achieve a stable, or at least manageable, environment around the cells and to serve as an avenue of molecular commerce between cells and tissues. Extracellular fluid is interposed between the intracellular compartment and the environment and is thereby directly subjugated to hydraulic and osmotic forces at both the cellular level in the tissues and across the various body epithelia. If, as indicated in the previous section, transcellular water movement is dictated initially by extracellular osmolarity, then extracellular fluid volume is largely governed by transepithelial exchange processes. Tissues responsible for these activities include the gill, skin, gastrointestinal tract, and kidney. Extracellular fluid volumes have been estimated from either GibbsDonnan distribution of endogenous ions (cf. Holmes and Donaldson, 1969) or with indicator dilution methods. Because of a somewhat incongruous distribution of the various ions or indicators, it is difficult to determine the absolute extracellular volume, and the distribution space of the specific extracellular volume marker is cited. Sodium spaces probably provide the least reliable estimate of extracellular fluid volume (Houston and Mearow, 1979; Lutz, 1972; Wood and Randall, 1971)and will not be described further. In many fish, plasma ion concentrations can probably be substituted for extracellular concentrations because of the relatively limited Gibbs-Donnan effect due to high protein permeability of fish capillaries (Houston and Mearow, 1979). Indicator dilution methods are based on the relationship: where V1 is the unknown volume, V, and Cz are the volume and concentration of the indicator prior to dilution in the unknown volume (Vz * Cs is the amount of indicator injected), and C1 is the concentration of the indicator after it has been evenly distributed within the compartment. An ideal indicator is (a) physiologically and biochemically innocuous, (b)not metabolized or modified in any way that might affect its distribution or detection, (c)completely mixed and homogeneously distributed within the compartment in question, (d) confined to the compartment, and (e)precisely quantified (Feldschuh, 1990).The time required for adequate distribution of an indicator in the test volume must also be compatible with the experimental protocol. Much of the rationale for selection of suitable indicators for determining volumes in fish has been based on the behavior of these indicators in mammalian systems, a tenuous and perhaps hazardous supposition. In addition, indicators must be introduced into the space in

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

145

question and that, in turn, may impose some degree of stress on the fish and thereby affect volume homeostasis (Houston et al., 1971). The methods employed to measure extracellular space in fish are provided in the following paragraphs. An excellent summary of the early literature has been provided by Holmes and Donaldson (1969). Over the past 20 years the extracellular space markers most commonly used in fish have been inulin, mannitol, sucrose, 4000 molecular weight polyethylene glycol (PEG), [35S]sulfate, [32Plphosphate, glofil ( 1251-iothalamate),51Cr-ethylene diamine tetraacetate (EDTA), 1251-albumin,or radionuclides of sodium or chloride. Two methods of administering the indicator, constant infusion or single injection, may be employed. In the former, the indicator is infused until compartmental concentrations reach steady state. The infusion is then stopped, and the excreted indicator is collected until compartmental concentrations fall to zero, which may take weeks (Hickman, 1972).The impracticality of this method has limited its usefulness, and the single injection procedure has been the method of choice. Hickman (1972) compared two methods, kinetic and net retention, to determine extracellular space following a single inulin injection into the flounder, Paralichthys lethostigma. With the kinetic method, blood samples are taken at timed intervals after the indicator is injected and the activity of the indicator in plasma is then plotted as a function of time on a semilogarithmic paper (Fig. 2). Typically, the resultant curve has a minimum of two components, a fast component due to distribution of the indicator in the vascular and interstitial spaces and a slower component as the indicator is gradually removed from the extracellular compartment. Extrapolation of the slow component back to the injection time provides the theoretical “instantaneous” concentration of indicator in the extracellular space. This concentration is then used in the dilution equation (Equation 2) to solve for the indicator space (Hickman, 1972). Nichols (1987) has shown that, in trout, any one of several three-compartment models provides a better description of inulin kinetics than either one- or two-compartment models, and a three-compartment mammillary model provides the best estimate of inulin kinetics. Presumably this model accounts for differential accessibility of inulin into extravascular spaces of both highly and poorly vascularized tissues; the latter taking as long as 10.5 h to reach equilibrium in trout (Nichols, 1987) and 8 h in eels (Chester Jones et al., 1969). The net retention method requires quantification of the indicator lost from the compartment during the mixing period. In these experiments it is assumed that the ‘lost’ indicator is excreted into the urine,

146

KENNETH H. OLSON

10

20

30

40

50

60

HOURS AFTERINJECTION

Fig. 2. Kinetic method. The decrease in plasma radioactivity (composite curve) following injection of [3H]methoxy inulin into southern flounder, P . lethostigrna, has two components, A and B. The fast component (A) represents inulin equilibration with the interstitial fluid; curve B primarily indicates inulin excretion from the body. The intercept resulting from extrapolation of curve B to time 0 (C,) is equivalent to inulin dilution in the extracellular fluid space and is substituted for C1 in Equation 2 (see text). [Redrawn from Hickman (1972),with permission.]

and/or environment and can thus be accurately measured. The amount of indicator excreted (AE) is then subtracted from the total amount injected and Equation 2 becomes:

Only a single blood sample is necessary once the indicator has equilibrated throughout the compartment in question (Fig. 3 ) .This is advantageous in minimizing the volume disturbance to the fish but requires preliminary information on the time required for indicator equilibration, a point often overlooked in these studies. The net retention method is also insensitive to internal removal (e.g., cellular binding or uptake) of indicator from the test compartment and can result in overestimation of the space. Extracellular space of individual tissues is often calculated as the ratio of the concentrations of indicator in tissue relative to plasma at the time of tissue collection. This method optimistically assumes that the concentration of indicator in interstitial fluids temporally mirrors plasma concentrations.

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

a-

147

Freshwater

14.9

15.3

15.9

_------ -

$1 Marine

20-

0

10

20

I 3J

HOURS AFTER INJECTION Fig. 3. Net retention method. Extracellular fluid space determined in FW and SW southern flounder, P . lethostigma, at 12, 18, and 24 h after injection of [3H]methoxy inulin using the net retention method. Arrows indicate time when composite plasma inulin disappearance curve (see Fig. 2) begins an exponential decline. Dots on curve prior to 10 h indicate calculated extracellular space prior to inulin equilibrium and underestimate the apparent space. [From Hickman (1972), with permission.]

Probably none of the indicators used to date give an exact representation of extracellular space. At least 8-12 h are necessary for inulin to be sufficiently equilibrated with the extracellular fluids (Chester Jones et al., 1969; Hickman, 1972; Munger et al., 1991; Nichols, 1987), and studies over shorter intervals may underestimate distribution volumes. Thorson observed that, after 4-5 h circulation, the volume distribution of saccharides in an elasmobranch (Thorson, 1958) and teleost (Thorson, 1961) was inversely proportional to the indicator's molecular weight, and he suggested that this was due to the relative diffusivity of these molecules in water. Certainly a 5-h equilibration period would seem to limit inulin penetration into certain compartments and thus underestimate inulin spaces. However, if equilibration periods become prolonged there is a risk of tissue binding or metabolism. Although neither [3Hl nor [l4C1inulin are metabolized after 146 h in the circulation (Hickman, 1972), the 3H-labeled compounds often produce larger extracellular spaces, even during 10- to

148

KENNETH R . OLSON

24-h equilibration periods (Hickman et al., 1972; Munger et al., 1991; Schmidt-Nielsen et al., 1972). When pairs of commonly used indicators, PEG, inulin, and mannitol, are injected, [ 14C]PEG generally provides the most conservative estimate of whole body and tissue extracellular volumes. Inulin, especially the tritiated form, is accumulated by kidney tissue (Beyenbach and Kirschner, 1976; Hickman et al., 1972; Schmidt-Nielsen et al., 1972) and [14C]inulin may be removed by liver (Munger et al., 1991). Schmidt-Nielsen et al. (1972) also observed elevated [3H]inulin spaces in muscle, gut, and liver after a 24-h equilibration period while Munger et al. (1991) found [ 14C]inulin spaces lower in red muscle than [3H]PEG spaces after 13h. Mannitol spaces are consistently greater than either PEG or inulin in most tissues, especially after 6 h (Cameron, 1980; Munger et al., 1991). Mannitol appears to be accumulated by gill (Munger et al., 1991),liver (Milligan and Wood, 1986a,b, 1987a,b; Munger et al., 1991), and heart (Munger et al., 1991). Mannitol (Munger et al., 1991), or other small saccharides such as sucrose (Fenstermacher and Patlak, 1977), may more readily penetrate the blood-brain barrier and therefore be preferable to other indicators in this tissue. However, Wood et al. (1990) have shown that even after 12 h in the circulation, mannitol penetration into the cerebrospinal fluid space (CSF) of the skate, Raja ocellata, is only around 26% of that predicted that equilibrium between CSF and plasma. In some tissues, PEG spaces may compare favorably with spaces calculated by Gibbs-Donnan methods. Houston and Mearow (1979) used an indicator dilution method to compare the distribution space of [14C]PEG to Na+, C1-, and C1-/Kf spaces in tissues of rainbow trout. Twelve hours after injection, ['*C]PEG spaces were similar to C1- and Cl-/K+ spaces in epaxial and cardiac muscle and gut; however, [14C]PEG appeared to provide a more realistic estimate than ion spaces in spleen and brain. [14C]PEGspaces in liver were only slightly higher than those determined by other methods (Houston and Mearow, 1979).A number of investigators (H6be and McMahon, 1988; Mearow and Houston, 1980; Munger et al., 1991; Sinha and Munshi, 1980) have found that Cl-/K+ spaces are satisfactory in skeletal muscle, whereas Munger et al. (1991) found Cl-/K+ underestimated white muscle space. Chloride and inulin spaces are virtually identical in perch, Perca fluviatilis, muscle, whereas C1- space is 60 and 90% greater in liver and gut, respectively (Lutz, 1972). However, compared to inulin, Gibbs-Donnan methods may be less applicable when fish are adapted to different salinities (Eddy and Bath, 1979). Tables 111, IV, and V summarize extracellular fluid spaces for whole

Table 111 Whole Body Extracellular Fluid Volumes in Fish Env"

Cyclostomes Lampetra planeri

Myxine glutinosa Eptatretus stouti Petromyzon marinus Chondrichthyes H ydrolagus colliei Squalus acanthias

Squalus acanthias Raja binoculata Raja rhina Osteichthyes Chondrosteans Acipenserfluvescens Polydon spathula Holosteans Amia calva Lepiosteus patostomum Teleosteans Carassius auratus Catastomus commerso ni

Space'' (nil/kg)

Indicator [ ''C]Inulin [ "CC]Sucrose

Method"

Comments

Reference

244 223 285 255 254 228

c1[ "C]Inulin [ '"CIInulin Sucrosed Inulind Inulind Ramnosed Sucrosed [ 3H]Inulin Inulind Inulind

K (4-5 K (4-5 K (4-5 K (4-5

sw

101 121 145 202 122 126 112

K (4-5 h) K (4-5 h)

Benyajati and Yokota (1989) Thorson (1958) Thorson (1958)

FW FW

191 149

Sucrose" Sucrosed

K (3-5 h) K (3-5 h)

Thorson (1961) Thorson (1961)

FW FW

180 130

Sucrosed Sucrosed

K (3-5 h) K (3-5 h)

Thorson (1961) Thorson (1961)

FW FW

114 228

C1CI-/K+

G-D G-D

Houston (1962) H6be (1987)

FW

SW

sw sw SW Sw

sw SW

Inj (1 h) Inj (1h) G-D Inj (14-18 h) K (104 h ) K (40-235 min) h) h) h) h)

Ammocoete

Bull and Morris (1967) Cholette et al. (1970) McCarthy and Conte (1966) Thorson (1959) Thorson (1958) Thorson (1958)

(continues)

Table 111 (Continued) Env“

g O

Catastomus commersoni Cyprinus carpio lctiobus cyprinellus Salzjelinus fontinalis

FW FW FW FW

S ynbranchus

FW

marmoratus Pseudoscarus guacamaia

sw

Fundulus grundis Mycteroperca tigris Gymnothoraxfunebris Epinephelus striatus Sphyraena barracuda Lutianus campechanus Lutianus griseus Platich thyesflesus Platichthys stellatus

sw sw sw sw sw sw sw sw sw

lctalurus punctatus

FW

lctalurus punctatus Oncorhynchus mykiss

FW

Oncorhynchus mykiss

sw

sw

sw

Space’’ ( m k )

Indicator

Method’

Comments

Reference

116 148 126 118 112 163

Sucrosed Sucrosed Sucrosed [I4C]InuIin [ ‘‘C]Inulin [ 3H]Inulin

K (3-5 h) K (3-5 h) K (3-5 h) K (4 h) K (4h) K (12 h)

Thorson (1961) Thorson (1961) Thorson (1961) Nichols et a1. (1985)

158 137 109 236 119 151 138 1.51 133 133 308 255 259 319 166 178 183 181 263 231 262 280

Sucrosed Raffinosed Inulind [ ’‘C]Inulin Sucrose“ Sucrosed Sucrosed Sucrosed Sucrosed Sucrosed

K (3-5 h) K (3-5 h) K (3-5 h) Inj (24 h) K (3-5 h) K (3-5 h) K (3-5 h) K (3-5 h) K (3-5 h) K (3-5 h) Inj (75 min) NR (12 h) NR (12 h) NR (36 h) K (?I K (9 K (4h) K (50 h) K (50 h) K (50 h) K (50 h) G-D

Thorson (1961)

36~1-

F3H]Mannitol l3H1Mannitol [ 3H]Mannitol [3H]Inulin [3H]Mannitol SCN”.“ [ I4C]PEG [:’H]Inulin [ 3H]Inulin ’2”-glofil

c1-

Heisler (1982)

Spence et al. (1977) Thorson (1961) Thorson (1961) Thorson (1961) Thorson (1961) Thorson (1961) Thorson (1961) Macfarlane and Maetz (1975) Milligan and Wood (l987a)

Cameron (1980) Kitzman et al. (1990) Beyenback and Kirschner (1976) different fish 10 days

Bath and Eddy (19794

Oncorhynchus mykiss

FU' SW FW

Oncorhynchus mykiss Oncorhynchus mykiss

FW FW FW

Oncorhynchus mykiss

FW

Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss Channa punctatus

FW FW FW FW FW

Paralichthys lethostigma

SW

FW

460 151 157 124 138 273 127 287 193 278 201 216 216 280 172 241 101 196 156 166 178 186 145 142 146 151

c1-

C-D G-D c1G-D c1G-D c1G-D [ 14C]Mannitol K (12 h) [ ''C]Inulin Inj (6 h) [3H]Mannitol Inj (6 h) [ 14C]Inulin Inj (13h) [ 'HIMannitol Inj (13h) [ 14C]Inulin Inj (24 h) CI-/K+ G-D c1K c1G-D [ ''C]Inulin K (24 h) ['HIMannitol NR (12 h) Inulind Inj (30 min) c1G-D [metho~y-~H]InulinK (50 h) [meth~xy-~H]InulinNR (12 h) [rnetho~y-~H]Inulin NR (18 h) [metho~y-~H]InulinNR (24 h) [metho~y-~H]InulinK (50 h) [metho~y-~H]InulinNR (12 h) [metho~y-~H]InulinNR (18 h) [metho~y-~H]InulinNR (24 h)

c1-

Transfer-100 h <40g BWT 70g BWT

Houston (1959)

Milligan and Wood (1982) Munger et al. (1991)

Eddy and Bath (1979

3 Pool model

Habe (1987) Bath and Eddy (197%) Nichols (1987) Milligan and Wood (1986a) Sinha and Munshi (1980) Hickman (1972)

Environment-salt water (SW) or fresh water (FW). In mllkg body weight. Kinetic method (K), net retention method (NR), Gibbs-Donnan method (C-D), space at time after injection (inj), equilibration times in parentheses. Unlabeled. '' SCN, sodium thiocyanate. a

Table IV Tissue Extracellular Fluid Volume: Nonskeletal Muscle Env“ Elasmobranchs Brain Raja erinacea

Squalus acanthias

SW SW SW

Space (ml/kg)

Indicator

Method

Comments

Telencephalon Medulla Telencephalon Medulla Spinal cord

Cserr et a / . (1983)

259 243 40 105 29 71 47 121

C1-

[3H]Sucrose [ 14C]Inulin [“HISucrose [14C]Inulin [3H]Sucrose

G-D G-D Inj (20 h) Inj (20 h) Inj (20 h) Inj (20 h) Inj (20 h) Inj (20 h)

c1[ 14C]Inulin

Reference

Fenstermacher and Patlak (1977)

Gut Squalus acanthias

SW

242

[14C]PEG

Inj (12 h)

Assume tissue 80% H20

Schmidt-Nielson et al. (1972)

Kidney Squalus acanthias

SW

29 1

[14C]PEG

Inj (12 h)

Assume tissue 80% HzO

Schmidt-Nielson et al. (1972)

Liver Squalus acanthias

Sw

208

[ l4C1PEG

Inj (12 h)

Assume tissue 80% HzO

Schmidt-Nielson et al. (1972)

Rectal Gland Squalus acanthias

SW

182

[ 14C]PEG

Inj (12 h)

Assume tissue 80% H20

Schmidt-Nielson et a / . (1972)

Spleen S o u a h ucunthius

SW

212

[‘%]PEG

Inj (12 h)

Assume tissue 80% H20

Schmidt-Nielson et a1. (1972)

Teleosts Bone Ictalurus punctutus Brain Ictalurus punctatus Oncorhynchus mykiss

FW

190 117

FW FW

['HH]Inulin [3H]Inulin

Inj (24 h) Inj (24 h)

[3H]Inulin

Cl-/K+ CI-,Cl-/K+

Inj (24 h) Inj (12 h) G-D G-D G-D

[14C]Inulin 13H]Mannitol [3H]PEG [3H]Mannitol [14C]Mannitol ['4C]Inulin [3H]Mannitol

Inj (6 h) Inj (6 h) Inj (13 h) Inj (13 h) Inj (13h) Inj (13h) NR (12 h)

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

FW

Platichthys stellatus

SW

68 63 317 345 277 405 25 67 30 101 125 35 224

FW FW

238 296

[3H]Inulin [3H]Inulin

Inj (24 h) Inj (8 h)

Oncorhynchus mykiss

FW

240

[3HlMannitol

Oncorhynchus mykiss

FW

80

[3H]lnulin

In vitro (20 min) In vitro

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

FW

190 210 215 290 22 1

[I4C]PEG c1CI-/K+ CI-,CI-/K+

Inj (12 h) G-D G-D G-D

Heart Ictalurus punctatus Anguilla rostrata

[ 14C]PEG

c1-

Skull Vertebrae

Cameron (198.5) Cameron (1985) Houston and Mearow (1979)

Summer Winter

Mearow and Houston (1980) Munger et a1. (1991)

Wood and Milligan (1987) Cameron (1985) Walsh and Moon (1982) Farrell and Milligan (1986) Nielsen and Gesser (1984) Houston and Mearow (1979) Summer Winter

Mearow and Houston (1980)

(continues)

Table IV (Continued) Space

Env"

r

(mlk)

Indicator

Method

Comments

Munger et al. (1991)

FW

148 279 170 364 285 180 164

[ ''C]Inulin ['HIMannitol ['HIPEG [3H]Mannitol [ 14C]Mannitol ['4C]Inulin Inulin"

Salmo truttu

FW

166

[ L4C]Inulin

Inj (6 h) Inj (6 h) Inj (13h) Inj (13 h) Inj (13h) Inj (13h) I n uitro (1h) Inj (24 h)

Platichthys stellatus

sw

224

['HIMannitol

NR (12 h)

Gill Oncorhynchus mykiss

FW

[3H]PEG ['HIMannitol [ 14C]Mannitol CI-/K+ C1-

Inj (13 h) Inj (13 h) Inj (13h ) G-D G-D

Munger et ul. (1991)

sw

218 440 344 424 474

FW

130

"CR-EDTA

Inj (27 h)

Babiker et a1. (1979)

FW

420 44s 472 277 405 306

[ 14C]PEG

Houston and Mearow (1979)

CI-IK' CI-,CI-/K'

Inj (12 h) G-D G-D G-D

Inulin'

Inj (24 h)

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

Hansen and Gesser (1987) Fugelli and Vislie (1980) Wood and Milligan (1987)

cn

I&

Reference

Anguilla unguillu

Gonad Tilapia nilotica Gut Oncorhynchus mykiss

Oncorhynchus mykiss

FW

Perca Juljiatilis

FW

c1-

Isaia and Masoni (1976)

Summer Winter Intestine

Mearow and Houston (1980) Lutz (1972)

Tilupia nilotica Pseudopleuronectes umericanus Anguillu rostrata Kidney Tilapia nilotica Pseudopleuronectes americanus

Pseudopleuronectes americanus Anguilla rostrata I-

%

Liver Anguilla rostrata

51CR-EDTA

Inj (27 h)

SW

58 94 200

[ 14C]PEG

Inj (24 h)

FW SW

171 186

['4C]PEC [l4C1PEG

Inj (24 h)

FW SW

51CR-EDTA [ 14C]PEG [3H]PEG [ I4C]Inulin ['H]Inulin [ 14C]PEG

Inj (27 h) In vitro

SW

208 229 358 367 349 357

FW SW

492 635

[ I4C]PEG

Inj (24 h)

FW

256

[14C]Inulin(or

Inj (24 h)

Intestine Hepatopancreas Assume tissue 75% HzO

Assume tissue 75% HzO

FW

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

FW

Perca fluviatilis

FW

322 295 310 323 327 140 821 195 145 862 790 220

c1CI-IK' CI-/K+ CI-/K+ [ 14C]Inulin i3H] Mannitol [ ''C]Inulin [3H]PEG [3H]Mannitol [ 14C]Mannitol Inulin

Schmidt-Nielson et al. (1972) Schmidt-Nielson et al. (1972)

Inj (8 h)

Walsh and Moon (1982)

Inj (12 h) G-D G-D G-D G-D Inj (6 h) Inj (6 h) Inj (13 h) Inj (13 h) Inj (13 h) Inj (13h) Inj (24 h)

Houston and Mearow (1979)

"Oncorhynchus mykiss

Schmidt-Nielson et a1. (1972) Schmidt-Nielson et al. (1972) Babiker et al. (1979) Hickman et al. (1972)

[14C]PEG

4C]Methoxy inulin) [14C]PEG

Babiker et al. (1979)

18 h light 6 h light

Murphy and Houston (1977) Munger et al. (1991)

Lutz (1972) (continues)

Table IV (Continued) Env"

u1 Q)

Space (ml/kd

Indicator

Method

"CR-EDTA [3H]Mannitol [ ''C]Inulin [ 14C]PEG

Inj (27 h) NR (12 h) (12 h) Inj (24 h)

Comments

Reference

Tilapia nilotica Platichthys stellatus

FW SW

Pseudopleuronectes americanus

SW

101 -571* 170 229

Anguilla rostratu

FW SW

186 171

[ "CIPEG [ 14C]PEC

SW

607

'311-albumin

Inj (50 h)

Wardle (1971)

FW

234 306 336 157

[l4C1PEG C1Cl-/K+ "'CR-EDTA

Inj (12 h) G-D G-D Inj (27 h)

Houston and Mearow (1979)

Skin Pleuronectes platessa Spleen Oncorhynchus mykiss

Tilapia nilotica

FW

" See Table I11 for abbreviations

' Unlabeled.

*Unsuitable 14C = 3H Assume tissues 75% H 2 0

Babiker et a1. (1979) Milligan and Wood (1987b) Schmidt-Nielson et al. (1972)

Babiker et al. (1979)

Table V Muscle Extracellular Fluid Volume Env" Cyclostomes Myxine glutinosa Myxine glutinosa

I-

%

SW

sw

Myxine glutinosa

sw

Myxine glutinosa

SW

Space Wkg)

128 194 128 278 188 155 136

Elasmobranchs Chimaera monstrosa Squalus acanthias

SW

Squalus acanthias

sw

210 120 177 50

FW

47

FW

55 156 70 73 52

Teleosts Catastomus commersoni lctalurus punctatus Salvelinus fontinalis Saluelinus fontinalis Salvelinus fontinalis

sw

FW FW FW

Indicator Inulinb

Method

[I4C]Inulin [I4C]Inulin [14C]PEG

Inj (24 h) G-D Inj (24 h) G-D Inj (14-18 h) Inj (14-18 h) Inj (24 h)

Inulin" Inulin"

In vitro Inj (14-22 h)

[14C]PEG

G-D Inj (12 h)

c1-

Inulinh

c1-

c1-

Comments

Robertson (1976) Robertson (1986) Parietal tongue Assume tissue 80% HzO

c1c1[ '*C] Inulin

Cholette et al. (1970) Schmidt-Nielson et d . (1972) Robertson (1976) Robertson (1975)

Assume tissue 80% H 2 0

CI-/K+ [3H]Inulin [3H]Inulin

Reference

Schmidt-Nielson et al. (1972) H6be (1987)

K (24 h) K (24 h) G-D G-D K (12 h)

White Red

Epiaxial

Cameron (1985) Houston et al. (1971) Houston et al. (1969) Nichols et al. (1985) -

(continues)

Table V (Continued) Env"

Oncorhynchus mykiss

Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss

FW

FW FW FW Summer Winter

Oncorhynchus mykiss

FW

Space (ml/kd

44 41 37 37 38 32 75 66 63 73 53 52 33 60 53 44 50 45 54 85 74 45 64 23 76 126 92 140

Indicator [14C]PEG C1CI-/K+ [14C]PEG

c1-

Cl-IK+ [ 14C]PEG

c1-

CI-/K+ [I4C]Mannitol CI-/K+ CI-,CI-IK'

['4C]Inulin [3H]PEG [3H]Mannitol ['4CC]Mannitol [ ''C]Inulin [3H]Ma~initol CI-/K+ [ 14C]I n uli n [3H]Mannitol [ ''C]Inulin [3H]PEG

Method Inj (12 h) G-D G-D Inj (12 h) G-D G-D Inj (12 h) G-D G-D K (12 h)

G-D

Inj (13 h) Inj (13 h) Inj (13 h) Inj (13 h)

Inj (6 h) Inj (6 h) G-D Inj (6 h) Inj (6 h) Inj (13 h) Inj (13 h)

Comments Postopercular

Reference Houston and Mearow (1979)

Subdorsal

Caudal Epiaxial Postopercular Subdorsal Caudal Postopercular Subdorsal Caudal White Muscle

Red Muscle

Milligan and Wood (1982) Hdhe (1987) Mearow and Houston (1980)

Munger et d . (1991)

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

FW

Oncorhynchus mykiss

FW

Oncorhynchus mykiss Perca Juvia tilis Tilapia nilotica Channa punctatus

FW FW FW

Platichthys stellatus Platichthys stellatus Pleuronectes platessa Parophrys vetulus Pla tichthys Jesus

sw . sw

Pseudopleuronectes americanus Anguilla anguilla

FU'

SW

sw sw sw sw

FW

sw

131 145 48 46 73 105 222 224 82 74 51 157 99 71 120 85 4070 100 170 213 50 46 100 63 150 147 229 102 185

c1-

Inj (13 h) Inj (13 h) G-D G-D Inj (24 h) G-D In vitro In vitro Inj (12 h) Inj (24 h) Inj (27 h) G-D

[3HlMannitol [3H]Mannitol 1311-albumin [3HlMannitol [35S1SO4 [35SI SO4 [ 14C]PEG

NR (12 h) NR (12 h) Inj (50 h) Inj (12 h) Inj (8 h) Inj (8 h) Inj (24 h)

[35S]S04

Inj (58 h)

[3H]Mannitol [ 14C]Mannitol CI-/K+ CI-/K+ [ ''C]Inulin Cl-/K+ 32P04 Inulin [3H]Mannitol huhb 51Cr-EDTA

[35S1SO4

Inj (58 h)

18 h light 6 h light

Murphy and Houston (1977) Eddy and Bath (1979)

Tissue slices

Gras et al. (1971)

Hi gonad wt' Low gonad wt" Hi gonad wtC

White 6°C 16°C Assume tissue 75% HzO Parietal (y) Tongue (Y) Parietal (s) Tongue (s) Parietal (y) Tongue (Y) Parietal (s) Toneue(s)

Milligan and Wood (1986b) Lutz (1972) Babiker et ( 1 1 . (1979) Sinha and Munshi (1979)

Milligan and Wood (1987b) Wood and Milligan (1987) Wardle (1971) Wright et ( 1 1 . (1988) Maetz and Evans (1972) Schmidt-Nielson et a1 (1972) Chan et a / . (1967) y = yellow s =

silver

(continues)

Table V (Continued) Env"

Anguilla rostrata Anguilla rostrata Cyprinus carpio

FW

[3H]Inulin [3H]Inulin [I4C]PEG [ I4C]PEG [ '4C]Inulin [ ''C]Inulin [ '*C]Inulin [ ''C]Inulin ['4C]PEG [ 14C]PEG CI[ ''C]Inulin CI [ ''C]Inulin ['*C]Inulin

SW

65

FW SW

59 60 70

FW

sw

FW

sw

FW

Fundulus heteroclitus

FW

Gillichthys mirabilis

FW

sw

sw

sw a

See Table I11 for abbreviations

" Unlabeled.

' Mature fish.

Indicator

72 149 50 79 97 176 159 72 54 58 70 40 92 52 49

FW

C yprinus carpio

Sarotherodon mossambicus Sarotherodon mossambicus Tilapia mossambica

Space (ml/kg)

Method Inj (8 h) Inj (8 h)

Comments White Red

Inj ( 3 h) 15 gll NaCl Inj ( 3 h) Inj (6 h) Inj (6 h)

Reference Walsh and Moon (1982)

Abo Hegab and Hanke (1982) Gupta and Hanke (1982) Schmidt-Nielsen (1977) Loretz (1979a)

Inj (8 h) Inj (8 h) Inj (3 h)

Assem and Hanke (1981)

[ ''C]Inulin

Inj (24 h)

Loretz (1979b)

[ ''C]Inulin ['4C]Innlin ['*C]Inulin

Inj ( 3 h)

Assem and Hanke (1979) 27 g/l NaCl 35 g/l NaCl

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

161

fish, skeletal muscle, and tissues other than skeletal muscle, respectively. It becomes readily apparent that the variability due to different methods is often as great, if not greater, than actual differences between fish. Comparatively little work has been done on nonteleostean fish since the elegant surveys by Thorson (1958,1959,1961). However, even these studies should be repeated using other indicators and longer circulation times because many spaces reported by Thorson, especially for teleosts, are somewhat lower than those determined since then (see Table 111). Based on sucrose spaces, Holmes and Donaldson (1959) concluded that extracellular space is lower in the more advanced fish (teleosts). As is evident from the variability in these measurements (Table 111),further work is required to substantiate this hypothesis. Frequently, tissue extracellular fluid spaces are measured not for their intrinsic properties, but incidental to analyses of intracellular composition. This is especially true for teleost muscle (see Tables IV and V). Because teleost muscle tissue is around 50% of the total body weight (Gingerich et al., 1987; Heisler, 1982; although Stevens, 1968 reports 66%), it is clear that muscle intracellular fluid and solutes constitute a major fraction of the total body composition. It is also tempting to think that muscle extracellular fluid is a major component of the total extracellular volume although this is probably not the case. Most estimates of muscle extracellular space (Table V) are around 50-100 ml/kg tissue weight, which is only around 30-40% of the extracellular spaces reported for most other tissues or for the whole body (Tables 111 and IV). Thus muscle may contain as little as 20-30% of the total extracellular fluid and, therefore, may not be the most quantitative index of total intercompartmental fluid movements. 1. EFFECTS OF

SALINITY ON

ECFV

Few factors pose a greater immediate threat to fish volume regulation than the osmolarity of the environment. Extracellular fluids appear to be more sensitive than intracellular fluids to an initial change in transepithelial osmolarity gradients. When ECFV is evaluated in the context of the change in TBW, both organismal and cellular regulatory systems can be appreciated. Studies such as these, although limited to date, are clearly necessary to understand the homeostatic mechanisms that accompany life in these diverse environments, and they may well provide the key to an appreciation of volume regulation in vertebrates in general. Water is lost from intracellular stores by transfer of FW fish to media of higher salinity. This has been observed in the elasmobranch Potamotrygon hystrin: (Bittner and Lang, 1980), the stenohaline FW tele-

162

KENNETH R . OLSON

ost Cyprinus carpio (Abo Hegab and Hanke, 1982),and the euryhaline teleosts Tilapia mossambica (Asseni and Hanke, 1979, 1981), Pleuronectes flesus (Lange and Fugelli, 1965), Gasterosteus aculeatus (Lange and Fugelli, 1965), Fundulus heteroclitus (Schmidt-Nielsen, 1977), Oncorhynchus mykiss (Eddy and Bath, 1979; Finstad et al., 1988), Salvelinus fontinalis (Nichols et al., 1985),and Eugerres plumieri (Plaza-Yglesias et al., 1988). Intracellular water and ambient salinity are inversely related in stenohaline Myrine glutinosa (Cholette et al., 1970) over the range of salinity tolerance (600-1540 mOsm/liter). An increase in extracellular osmolarity accompanying transfer of Cyprinus carpio to hypertonic mannitol (Gupta and Hanke, 1982) or hemorrhage in Oncorhynchus mykiss (Duff and Olson, 1989) also extracts water from the intracellular compartment. Conversely, events that increase intracellular osmolarity (e.g., exercise) draw extracellular water into the cells (Milligan and Wood, 1986a,b, 1987a,b; Wright et al., 1988). An osmoconformer such as the hagfish, Myxine glutinosa, is also a volume conformer. Cholette et al. (1970) have shown that within the range of salinity tolerance of myxine (600-1500 mOsm) muscle ECFV and environmental osmolarity (ENVosm)are directly related by the relationship: ECFV

=

0.013(ENVoS,)

+ 4.87,

(4)

while total muscle water (TMW) and ENVosm are inversely related according to TMW

=

-0.017(ENVoSm)+ 90.1.

(5)

Thus, as ambient salinity increases from 600 to 1500 mOsm, muscle ECFV increases from 130 to 225 g/kg while TMW decreases from 780 to 640 g/kg. Although this study (Cholette et al., 1970) is limited to muscle, it can be tentatively concluded that as ambient salinity increases, there is a net loss of water from the fish and a concomitant shift of fluid from the intracellular to the extracellular compartment, the latter as a result of the increased extracellular osmolarity. Extracellular fluid volume in the dogfish shark, Squalus acanthias, is dramatically affected by ambient salinity; transfer from 100% SW to 90 or 110% SW changes ECFV from 122 ml/kg to 220 or 82 ml/kg, respectively (Benyajati and Yokota, 1989; Yokota and Benyajati, 1988). Progressive increases in salinity from -0 to 460 mOsm/liter do not affect TBW or muscle or liver water in the FW stingray (Bittner and Lang, 1980), while 525 mOsm/liter decreases both total and muscle water. Cserr et al. (1983) have shown that during transient salinity

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

163

perturbations, C1- space, but not non-C1- space, is regulated in the skate, Raja erinacea, telencephalon, whereas all spaces in the medulla are unregulated. Holmes and Donaldson (1969) concluded, primarily from Thorson’s survey of three FW and seven SW teleosts (Thorson, 1961), that SW teleosts have a greater extracellular volume whereas blood volume and TBW are the same. Examination of reported values (Tables III-V) clearly illustrates that the variability in ECFV, which may be largely methodological, obscures definitive confirmation of this hypothesis. However, there does appear to be a decrease in muscle and TBW and an increase in ECFV as teleosts are adapted to progressively higher salinities, although exceptions to this trend are not uncommon. Few studies have used indicator dilution methods to compare whole body ECFV in fish adapted to FW or SW. In the flounder, Paralichthys lethostigma, ECFV appears to decrease after FW adaptation (Hickman, 1972), although in this study only four fish, one each adapted to FW for 4 days, 1 week, 2 week, and 3 months were examined. In rainbow trout, SW adaptation (3-4 weeks) slightly decreases ECFV (Kellogg et al., 1990; M. D. Kellogg and K. R. Olson, unpublished). Short-term (48 h) transfer of brook trout, Salvelinusfontinalis, to SW does not significantly affect ECFV (Nichols et al., 1985). These few studies do not permit a generalized hypothesis regarding the affects of salinity on ECFV and clearly this is an open area for further investigation. A number of studies have reported the effects of salinity on ECFV in individual tissues, and they provide some idea of fluid economy in different environments. Salinity adaptation increases ECFV of muscle tissue in Anguilla, Fundulus, Gillichthys, and Tilapia, although these effects are often not substantial (Table V). Muscle ECFV also increases in stenohaline Carpio when adapted to 380-400 mOsm NaC1, their upper limit of salinity tolerance (Abo Hegab and Hanke, 1982). Gut and kidney ECFV are greater in SW Anguilla, whereas liver ECFV is greater in the FW fish (Table IV). However, salinity adaptation does not affect muscle ECFV in the killifish, Fundulus heteroclitus, (Schmidt-Nielsen, 1977) and, in fact, transfer from FW to 40-50% SW increased TBW in Fundulus catenatus and F . olivaceus (Duff and Fleming, 1972a,b). Ten days after SW transfer, TBW of 13-g trout also returns to FW values (Bath and Eddy, 1979b), whereas eel TBW is 5% less in SW after 3 weeks (Sokabe et al., 1973). Inulin spaces and TBW in F . grandis do not temporally correspond to circadian oscillations in the chloride balance (Spence et al., 1977). Generally, in teleosts, as in cyclostomes, when ambient salinity increases tissue and whole body ECFV, it does so at the expense of intracellular volume.

164

KENNETH R . OLSON

2. OTHERFACTORS AFFECTING ECFV There is a well-known allometric reduction in ECFV with fish age or size (Holmes and Donaldson, 1969). These are not considered to be under physiological regulation in the context of this chapter. a . Exercise. In an early study, Stevens (1968) reported that severe exercise is associated with an increase in body weight (1.5g) in 224-g trout. Later, Milligan and Wood (1986a,b; 1987a,b) found that severe exercise does not affect tissue or TBW in either FW trout or SW starry flounder, Platichthys stellatus. However, exercise decreases total and muscle ECFV ( [3H]mannitol space; -20% in trout and 28% in flounder) indicative of fluid influx into the intracellular compartment, presumably due to increased intracellular osmolarity (Milligan and Wood, 1986a,b, 1987a,b). Fluid spaces in both flounder and trout brain, and probably heart and liver as well, are unaffected by exercise (Milligan and Wood, 198613; Wood and Milligan, 1987). Extracellular fluid volume in both fish recovers in 4-8 h. Similar muscle-specific increases in ECFV have been observed to accompany exercise in the lemon sole, Parophrys vetulus (Wright et al., 1988).

b. Environment: Temperature, Photoperiod, p H , and POS. In eels, Anguilla rostrata, an increase in ambient temperature from 5 to 20°C has no affect on total tissue water or inulin spaces in heart, liver, or either red or white muscle (Walsh and Moon, 1982). Total tissue water and C1- or Cl-/K+ spaces (Na+ space in liver) in trout tissues are variously affected by seasonal acclimation and ambient temperatures between 4 and 20°C (Mearow and Houston, 1980). Muscle water is greater in summer than winter acclimated fish and is not appreciably affected b y ambient temperature. Muscle ion spaces increase with increasing temperature but are not affected by season. Cardiac water is slightly greater in winter, and it is independent of temperature in both summer and winter fish, whereas ion spaces are greater in summer and increase with temperature in both acclimated groups. Gut water and spaces behave somewhat similarly to muscle, although total water decreases as temperature increases in summer fish. Liver water is greater in summer fish and ion spaces increase slightly with temperature in winter fish. Total water in the brain is unaffected by season or temperature, whereas C1- space increases with temperature. Murphy and Houston (1977) have shown an interactive effect between temperature (2 versus 1SOC) and photoperiod (6 versus 18 h light) on C1-/Kt spaces and total tissue water in muscle and liver of rainbow trout. Low

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

165

light increases total water and decreases Cl-/K+ space in both tissues at 2°C; at 18"C, photoperiod has no effect on either fluid compartment. High temperature increases muscle water in 6-h light-exposed fish, increases liver water at both photoperiods, and increases liver Cl-/K+ space in 6-h light-exposed fish. Low temperature (1°C) also disrupts TBW regulation in trout transferred from FW to SW (Finstad et al., 1988), and seasonal effects on TBW responses to salinity have been observed (Finstad et al., 1989). In Channa punctatus muscle C1- space is correlated with the reproductive cycle, doubling in October when gonad weight increases (Sinha and Munshi, 1980). Total body water also increases in spawning FW trout (Parry, 1961). Fish exposed to acidic water lose extracellular ions and may also experience volume disturbances. While exposure of trout to acid water (pH 4.0-4.5) for 1-3 days does not affect TBW, both ECFV ( L3HImannitol or Cl-/K+ space) and plasma volume decrease, indicative of a fluid shift into the intracellular compartment (Habe, 1987; Milligan and Wood, 1982). Similar responses are found in the white sucker, Catastomus commersoni (Habe and McMahon, 1988), although in these fish total water in muscle (but not TBW) also increases (Habe, 1987).Total body water in acid tolerant sunfish, Enneacanthus obesus, is unaffected by acid exposure, whereas it increases in the less acid tolerant Lepomis gibbosus; ECFV was not measured in these fish (Gonzalez and Dunson, 1987). Despite the pronounced effects of acidic medium on extracellular and intracellular fluid volume, related acid-base disturbances appear to be relatively innocuous. Environmental hypercapnia has no affect on ECFV ( [3H]mannitol) or total water of catfish, Zctalurus punctatus, white or red skeletal muscle, heart, or bone (Cameron, 1985), or of sole, Parophrys vetulus, white muscle, heart, and brain (Wright et al., 1988). Adaptation of the air breathing teleost, Synbranchus marmoratus, to nitrogen-gassed (hypoxic) water for 4-5.5 days decreases [3H]inulin space from 163 to 149 ml/kg (Heisler, 1982), although it is not known if the response is due to hypoxia per se, or the attendant acidosis. An acidosis effect is more likely because iodoacetate, cyanide, or anoxia have no effect on total water or inulin space in isolated ventricular strips from trout (Nielsen and Gesser, 1984; Hansen and Gesser, 1987).

c . Other Stress. Surgical stress, but not anesthesia per se, transiently (<6 h) decreases muscle C1- space in Salvelinus fontinalis but does not affect TMW (Houston et al., 1971). However, dorsal aortic cannulation depresses muscle C1- space and increases TMW for up to 72 h (Houston et al., 1969).Twelve months of starvation does not affect

166

KENNETH R . OLSON

liver water content in the eel, A. rostrata (Moon, 1983). Catecholamines, secreted by fish in response to a variety of stressors, may also affect fluid volume. Although P-adrenergic blockade has no effect on ECFV ( [3H]mannitol) or total water content in starry flounder muscle, heart, and brain (Wood and Milligan, 1987), 5 x M epinephrine increases mannitol space in the in situ perfused heart without affecting total water, indicating a loss in ICFV (Farrell and Milligan, 1986). A similar increase in inulin or 32P04 space at the expense of intracellular water has been observed by Gras et al. (1971) in l-mm-thick skeletal muscle tissue slices. This effect can only be completely inhibited by combined a and /3 blockade. Catecholamine-induced cellular dehydration of tissues contrasts with the well-known @mediated hydration of red blood cells. The mechanisms of catecholamine action at the tissue level are not known.

D. Blood Volume Total blood volume is influenced by the sum of factors that govern the volume occupied by red cells and the plasma volume. It is assumed in this chapter that regulation of total blood volume is achieved primarily, if not exclusively, through adjustments in plasma volume, as it is in mammals (Manning and Guyton, 1982). In this context, blood volume is regulated by factors that affect ECFV (see Section II,C) and b y factors that affect fluid movement between intravascular and interstitial compartments. 1. WHOLEFISH

In the last 20 years nearly all blood volume measurements on fish have employed indicator dilution methods. Apart from the requirement that the indicator must be confined to the vascular compartment, these methods are identical to those already described for measuring ECFV. The major constraints, loss of indicator from the compartment and incomplete equilibration, apply here as well. In practice, both kinetic and single-sample net retention techniques are used. Net retention methods are simplified because indicators sufficiently large enough to be restricted from the interstitial compartment are also poorly filtered by the kidney. However, some indicators, especially those diluted in the plasma, may distribute to extravascular compartments. Usually only one space, red cell (RCS) or plasma (PS), is measured in a single experiment. Total blood volume (TBV) is then corrected for the fractional volume of cells (hematocrit, Hct) or plasma by the relationship:

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

TBV

=

RCS/Hct, or TBV

=

PS/(1-Hct).

167 67)

Correction for plasma trapped between cells is often ignored, which may result in under estimation of TBV calculated from plasma spaces by several percentage points if whole blood is counted. Hematocrits for the above calculations are usually measured on blood taken from a large vessel. This may bias TBV if large vessel hematocrit (LVH) is not identical to the whole body hematocrit (WBH).That the latter two are dissimilar has been known in mammals for some time where the ratio WBH/LVH, commonly called Fcell ratio, is around 0.91 (Albert, 1971). Fcel1 ratios calculated from experiments on fish in which red cell and plasma volumes have been determined simultaneously are considerably lower than 0.91. In hagfish, the Fcell calculated from the data of Forster et al. (1989)on 20 h distribution of 1251-humanserum albumin, 51Cr red cells, and ventral aorta hematocrit is 0.54. Substitution of subcutaneous sinus hematocrit (Forster et al., 1989) results in a Fcell of 0.74, still well below mammalian values. Fcell values in trout, determined from 4 h circulation of '251-bovine serum albumin, 51Cr red cell spaces, and dorsal hematocrit, are similarly low, around 0.78 (Gingerich and Pityer, 1989). The comparatively low Fceil values reported in fish could be due to a proportionally greater plasma volume in fish tissues, a red cell inaccessible plasma space (i.e., secondary circulation; see Part A, Chapter 4), or an artifact due to loss of plasma indicator from the vascular compartment (see following discussion). It becomes apparent in species where multiple indicators have been employed that larger TBV are associated with PS indicators such as Evans blue dye (T1824) or radioiodinated albumins than with radiolabeled red cells (Conte et al., 1963; Duff and Olson, 1989; Forster et al., 1989; Gingerich and Pityer, 1989;Gingerish et al., 1990; Huggel et al., 1969; McCarthy and Conte, 1966; Wardle, 1971; summarized in Table VI). Conte et al. (1963)also observed that TI824 is lost from the circulation at a faster rate than 1311-human serum albumin. If T1824 is allowed to bind with plasma proteins prior to injection into coho salmon, Oncorhynchus kisutch, less dye is lost from the circulation 6 h after injection and the estimated blood volume (45 ml/kg) is nearly 30% lower than when unbound dye is injected (62 ml/kg; Smith, 1966). However, this is still around 30% higher than blood volumes reported in other salmonids using tagged red cell methods and equivalent circulation times (Table VI). This indicates that dye and protein together are lost from the primary circulation because once Ti824 is bound to fish plasma it remains bound for days (Nichols, 1987; Smith, 1966; Takei, 1988). No dye is excreted in trout urine or bile for at least 24 h after

Table VI Blood Volume ~

Species

Environment

Blood volume (mllkg)

Method"

~

Circulation time

Reference

Cyclostomes Eptatretus stouti

sw

206

Eptatretus stouti

sw

169

Eptatretus cirrhatus

sw 16°C

177

Inj (24 h)

Forster et al. (1989)

FW FW

85 80

K (30 min)

Thorson (1959) Bull and Morris (1967)

sw

sw

52 68 80 72 66 79

K (40 min) K (40 min) K (40 rnin) K (40 min) K (2 h) K (15 min) 20% Hct assumed

Thorson (1958) Thorson (1958) Thorson (1958) Thorson (1958) Opdyke et al. (1975) Solomon et al. (1985b)

FW FW FW

37 30 38

K (40 min) K (40 min) K (40 min)

Thorson (1961) Thorson (1961) Thorson (1961)

K (104 h)

McCarthy and Conte (1966)

McCarthy in Holmes and Donaldson (1969)

I-

Q1 QI

Petromyzon marinus Lampetra planeri (ammocoete) Elasmobranchs Hydrolagus colliei Squalus acanthias Raja binoculata Raja rhinus Squalus acanthias Squalus acanthias

Osteichthyes Acipenser fluvescens Polyodon spathula Lepiosteus patostomum

SW

sw sw SW

A tractosteus tristoechus Clarias batrachus Amia calva Neoceratodus forsteri Anguilla japonica Anguilla rostrata

Catastomus commersoni C yprinus carpio Cyprinus carpio

c

g

FW

31

FW FW FW FW

47 34 49

sw

FW (5 wk) FW FW FW

+ Hct

Inj (60 min)

Siret et al. (1976)

Inj (30 min) K (40 min) K (?) K ( 1 h) K (50 min)

Pandey et al. (1978) Thorson (1961) Sawyer et al. (1976) Takei (1988) Nishimura et al. (1976)

22

K (40 min)

Thorson (1961)

K (40 min) K (15 min) K (15miti)

Thorson (1961) Avtalion et al. (1974)

Inj (30 min)

Pandey et al. (1976)

35 28 29

”Cr-RBC

Heteromeustes fossilis Heteropneustes fossilis Zctiobus cyprinellus Oncorhynchus mykiss Oncorhynchus mykiss

FW

30 53 53 53 13-20

FW

14

Inj (30 min)

Pandey (1977)

FW FW FW

28 23 35 33 28

K (40 min) Inj (10 min) K (30-90 min)

Thorson (1961) Schiffman and Fromm (1959) Conte et al. (1963)

Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss

FW FW FW

K (3h) K (45 min)

Smith (1966) Nikinmaa et al. (1981) Huggel et al. (1969)

Oncorhynchus mykiss

FW

24 35 35 44 56

K (6 h)

Milligan and Wood (1982) (continues)

Table VI (continued)

Species I

Environment

Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus mykiss

F W 6°C FW 12°C FW 18°C FW FW FW

Oncorhynchus mykiss

FW

Oncorhynchus m y k i s s

I

0

(Wythville strain)

Blood volume (mlikg) 34 33 36 34 32 60 35 51 4s

51Cr-RBC+ Hct 'lCr-RBC + Hct 51Cr-RBC+ Hct lZ5I-A+ Hct "Cr-RBC + Hct 12'I-A + Hct "Cr-RBC + "'I-A

53 46 41 69 62 45 54

51Cr-RBC + '"I-A "Cr-RBC + 12sI-A Ti824 + Hct Tie24 + Hct Ti824 + Hct "bound" T1H24 Ti824 + Hct

41

Oncorhynchus mykiss Oncorhynchus mykiss Oncorhynchus m y k i s s Oncorhynchus kisutch

FW FW FW SW SW

Oncorhynchus nerka

FW

(Wythville) (Kamloops)

Circulation time

Method"

Reference

K (P)

Barron et ul. (1987)

Inj (4h) K (4 h) Inj (4 h)

D u f f e t al. (1987) Gingerich et al. (1987) Duff and Olson (1989) Gingerich and Pityer (1989)

K (4 h)

Gingerich et a/. (1990)

K (24 h ) K (56 h) K (56 h)

Nichols (1987) Smith (1966) Smith (1966)

K (56 h )

Smith (1966)

Salvelinus fontinalis Salvelinus fontinalis

c. 4 L

Sahelinus namaycush Amphipnous cuchia Channa punctatus Pseudoscarus guacamaia Enophrys bison Epinephelus striatus Gymnothorax funebris Lutianus campechanus Lutianus griseus Mycteroperca tigris Sphyraena barracuda Seriola quinquerdiata Pomatomus saltatrix Thunnus alalunga

FW FW SW (48h) FW FW FW SW

55 45 44 43 31 30 36

Ti824 + Hct Inulin + Hct

K (5 h) K (4 h)

Houston and DeWilde (1969) Nichols et al. (1985)

'lCr-RBC + Hct Ti824 + Hct Ti824 + Hct Ti824 + Hct

Inj (15 min) Inj (30 min) K (30 min) K (40 min)

Hoffert (1966) Munshi et al. (1975) Sinha and Munshi (1981) Thorson (1961)

SW

sw sw

71 26 22

+ Hct Ti824 + Hct Ti824 + Hct

K ( 3 h) K (40 min) K (40 min)

Sleet and Weber (l983a) Thorson (1961) Thorson (1961)

sw

22

Ti824

+ Hct

K (40 min)

Thorson (1961)

sw sw sw

20 33 28 47 43 132

K (40 min) K (40 min) K (40 min) K (45 h) Inj (10 min) K (30 min)

Thorson (1961) Thorson (1961) Thorson (1961) Yamamoto et al. (1980) Ogilvy et al. (1988) Laurs et al. (1978)

SW

sw sw

Ti824

Ti824 + Hct Tim4 + Hct Ti824 + Hct Ti824 + Hct Ti824 + Hct 1311-A + Hct

Abbreviations: A, albumin; Dil, red cell dilution; RBC, red blood cell; Hct, hematocrit. Other abbreviations as in Table 111. Blood volume expressed as ml/kg body weight.

172

KENNETH R. OLSON

injection (Nichols, 1987). Tagged red cells are also removed from the circulation by the spleen (Gingerich et al., 1987; Duff et al., 1987), which may result in overestimation of blood volume by this method as well. It is likely that plasma-borne indicators, such as T1824 and albumin, are lost from the vascular compartment in addition to slowly equilibrating with the secondary circulation. Gingerich and Pityer (1989) point out that there is no direct correlation between the amount of secondary vascularization of tissues and the albumidred cell space. Tissue Fcell ( F c e l l T ,the ratio of '251-albumin and 'lCr red cell calculated hematocrit within a tissue to LVH) is high in a tissue with an extensive secondary circulation, such as gill (0.96), yet low in muscle (0.44) which lacks a secondary circulation. The opposite would be expected if the secondary circulation was a significant sink for 12'I-albumin. Furthermore, turnover of plasma in the gill secondary circulation is quite rapid (-20 min; Olson, 1984; Olson et al., 1989) and inconsistent with the rate of protein efflux observed in volume determinations. Nichols (1987) found that in trout, TI824 was distributed to two anatomically and kinetically distinct compartments that he attributed to plasma and interstitial lymph. Blood volume calculated from the plasma compartment and hematocrit was 41 ml/kg; the interstitial-lymph compartment was 41-48 ml/kg (Nichols, 1987).The volume of the interstitial compartment measured by Nichols (1987) is considerably greater than that predicted from anatomical studies of the secondary circulation (Vogel, 1985a,b; Vogel and Claviez, 1981) but less than that determined with other indicators (i.e., inulin, T1824, etc.). Extravasation of the albumin is further supported by several reports that suggest some fish capillaries are relatively permeable to proteins (see following discussion). Optimal mixing times for blood volume indicators have not been established and often there appears to be a trade-off between adequate mixing throughout the vascular compartment and loss of indicator from the vasculature. Nichols (1987) reported that T1824 mixes with trout plasma in 2 min and then slowly exchanges with the extravascular compartment at the rate of 1% per minute. Adequate mixing of T1824 in the shark, Squalus acanthias, takes 20-40 min, yet 30% of the dye leaves the plasma every hour (Opdyke et al., 1975). In these instances, short equilibration times for plasma indicators may provide better estimates of intravascular volume as they minimize extravasation, whereas longer mixing times may be more desirable from a practical standpoint, especially for kinetic methods (see Table VI). Volume markers such as T1824 may not be suitable for use in fish in either

3.

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instance. Blood volume calculated from 51Cr red cell distribution is less sensitive to mixing time; in trout there is a slight but insignificant increase in calculated blood volume for 4 h after injection of 51Cr red cells (Duff and Olson, 1989; Duff et al., 1987). However, tagged red cells are not rapidly distributed to all tissues and at least 4 h is necessary to ensure adequate mixing within individual tissues (Duff et al., 1987). Longer circulation times (24)may be preferable for studies of organ blood volume (Duff and Olson, 1989; Duff et al., 1987; Gingerich and Pityer, 1989; Gingerich et al., 1987, 1990). Several alternative methods have also been used to measure blood volume. In some instances where indicator methods are not possible, such as the small Lampetra planeri ammocoete, blood volume may b e estimated from the total body hemoglobin (Bull and Morris, 1967). Avtalion er al. (1974) estimated blood volume from the change in hematocrit after hemorrhage. In this method, an initial hematocrit (Hctl) is determined, a volume of blood (a) between 20 and 50% of the estimated blood volume is removed, and 2 h later, when blood and extravascular fluids are reestablished, a second hematocrit (Hctz) is determined. Blood volume (BV) is then calculated from the relationship: BV = a * Hctl/(Hctl-Hctz).

(8)

Blood volumes determined with this method agree with those obtained by TI824 or 1311-albumindilution methods (Table VI). However, the imposition of hemorrhage stress may not be desirable in many instances, and the error incurred by splenic contraction or depletion of extracellular fluid volume needs to be examined. Blood volumes for representative fish are shown in Table VI. The largest volumes are found in the hagfish followed by lampreys and skates. In the hagfish, Eptatretus cirrhatus, 30% of the blood volume is located in a subcutaneous sinus that extends over most of the body (Forster et al., 1989). Hematocrit is lower in the subcutaneous sinus than in the remaining vasculature (4.3 versus 13.5),and the time necessary for equilibrium between the two compartments is in excess of 8 h (Forster et al., 1989).The blood volume accounts for around 50%of the extracellular space in Myxinoids. Brain blood volume in Petromyzon marinus is also higher than that of other vertebrates (Heisey, 1968). Blood volumes or spaces of individual organs or tissues have not been systematically measured with indicator dilution methods in either agnatha or chondrichthyes. Osteichthyes collectively have the lowest blood volumes of any vertebrate (Table VI). The tunas appear to be an exception (BV = 132 ml/kg), perhaps due to their extensive heat-exchanging rate (Laurs et

174

KENNETH R . OLSON

al., 1978). However, the results of the latter study (Laurs et ul., 1978) should be confirmed because Brill (unpublished observations) measured blood volumes around 50 ml/kg in tunas using idocyanine green. In their survey of the literature, Holmes and Donaldson (1969) concluded that blood volumes of SW and FW teleosts are virtually identical. This information, summarized in Table VI, does little to refute this hypothesis, although, as is the case with extracellular fluids, the variation attributable to methodology may preclude accurate comparison. Surprisingly, BV has rarely been measured in euryhaline fish adapted to different salinities. Nishimura et al. (1976) adapted SW eels, A. rostrata, to FW and found no change in BV (1251-albuminhematocrit) after 1, 2, and 5 weeks in FW (28 versus 29, 28, and 29 ml/kg, respectively); hematocrit was similarly unaffected. Similar findings have been observed in rainbow trout adapted to SW for 3 weeks or longer (Kellogg and Olson, unpublished). Thus blood volume appears to be well maintained in both hydrating and dehydrating environments. 2. TISSUEBLOODVOLUME AND SPACES Tissue fluid compartments have seldom been determined in fish, although they have been perhaps most extensively studied in salmonids. Vascular space and calculated blood volumes for a variety of salmonid tissues are given in Table VII. Kidney vascular volumes are the largest followed by gill and liver; muscle volume per unit tissue weight is around 10-15% of that found in the kidney. Tissue blood volumes determined with labeled red cells are consistently lower than volumes calculated from 1251-albumin spaces except in gill and brain (Duff and Olson, 1989; Gingerich and Pityer, 1989; Gingerich et al., 1987, 1990) and sometimes kidney (Duff and Olson, 1989; Gingerich and Pityer, 1989). The blood-brain barrier appears to effectively exclude albumin from the interstitial compartment in trout and probably all fish. An excellent review of the phylogenetic development of the blood-brain barrier is provided by Cserr and Bundgaard (1984). In agnatha (both hagfish and lamprey), holocephalans and teleosts, brain vascular endothelium restricts macromolecular movement, whereas glial end feet provide this barrier function in elasmobranchs (Cserr and Bungaard, 1984). Hydrostatic pressures are greater in gills than in any other tissue, and a tight endothelial and pillar cell barrier undoubtedly serves to minimize fluid filtration. Anatomical studies have corroborated the barrier capability of gills. In winter flounder, Pseudopleuronectes americanus, and Antarctic cod, Pagothenia borchgrevinki, gill endothelium and pillar cells prevent protein extravasation (Boyd et al.,

Table VII Estimated Tissue Blood Volumes, "Cr Red Cell, and lZ5I-Albumin Spaces in Salmonids Blood volume

Brain Eye Fat Gill Heart (atrium) Heart (ventricle) Large intestine Small intestine Anterior kidney Posterior kidney Liver Red muscle White muscle Pseudobranch Pyloric cecae Spleen Stannius corpuscles Stomach Swimbladder

Mean

Range

59 66 37 175 625 125 104 70 32 1 367 150 24 8 332 56 1830 285 34 120

18-100 47-110 33-40 97-315

-

47-250 65-175 23-120 157-509 252-525 85-240 17-38 4-12

-

43-79 34-6250 19-50 16-183

51Cr RBC space* (Ref)

Mean

19 15 6 48 125 19 10 8 158 62 29 4 1 66 5 765 57 4 22

* Values in ml/kg tissue weight. Oncorhynchus mykiss (Duff and Olson, 1989),30 min. Oncorhynchus mykiss (Duff et al., 1987),4 h. Oncorhynchus mykiss Wythville (Gingerich and Pityer, 1989), 4 h. Oncorhynchus mykiss (Gingerich et al., 1987),Hct=37 (4 h). Oncorhynchus mykiss Kamloops (Gingerich et ul., 1990),4 h. foncorhynchus mykiss Wythville (Gingerich et al., 1990),4 h. Oncorhynchus mykiss (M. D. Kellogg and K. R. Olson, unpublished), 8 h. I, Oncorhynchus mykiss (Stevens, 1968), Hct=27 (4 h). Saloelinus namaycush (Hoffert, 1966), Hct= 19.8 (15 min). a

Range

4-34 9-22 3-9 32-63 7-50 0-35 3-24 31-777 42-105 17-48 3-8 1-2 4-5 7-1410 2- 10 3-45

'"I-plasma (Ref)

Mean

39 38 24 113

-

68 64 46 260 252 110 16 6 42 151 22 138

Range

14-85 35-45 17-31 71-155 19-181 11-108 10-78 197-434 144-475 78-162 9-22 3-9

-

13-75 140-161

-

6-39

-

space* (Ref)

176

KENNETH R. OLSON

1980) and eight different vital dyes, including T1824, are effectively excluded from trout gill extravascular spaces (Davie and Daxboeck, 1982). Neither cationized nor native ferritin bind to or cross gill endothelial or pillar cell barriers of winter flounder, Antarctic cod, sea raven, Hemitripterus americanus, or Antarctic eelpout, Rhigophila dearborni (Boyd et al., 1990). In contrast, the rete mirabile of the eel swimbladder is quite permeable to ferritin and dextran (and to some extent albumin) but is somewhat more restrictive to charged macromolecules (Rasio and Goresky, 1985). There are conflicting reports on fluid exudation and the importance of colloid in preventing branchial edema in perfused gill preparations. Bornancin et al. (1985) reported that in a perfused trout head preparation the gills become edematous when perfused with saline and that edema could be prevented by addition of 4 g/liter bovine serum albumin (BSA) and 4 g/liter gelatin to the perfusate. Similar observations are reported in the eel, Anguilla australis. Profound edema is observed in eel gills perfused with saline, 143% polyvinylpyrrolidone (PVP; 30,000-40,000 molecular weight; MWT) or 2-3% dextran (70,000 MWT), but not when gills are perfused with mammalian blood or plasma (Ellis and Smith, 1983). Addition of 0.2% BSA to the PVP or dextran perfusate does not prevent edema formation (Ellis and Smith, 1983). Other studies indicate that perfused trout gills, in both whole head (Perry et al., 1984) and isolated holobranch preparations (Olson, 1984), are resistant to edema formation, even in the complete absence of colloid in the perfusate. Factors other than colloid impermeability may be involved in preventing fluid extravasation in the trout gill. The volume of blood in the central circulation (i.e., conducting arteries and veins and cardiac chambers) is not known. Gingerich et al. (1990) compared the TBV, determined from combined 'lCr red cells and 12'1-albumin spaces, to the sum of blood volumes in 13 tissues (also determined from red cell and plasma spaces) and found that even though the tissues account for 60% of the total body weight, the combined tissue blood volume is only 27-43% of the total volume. Gingerich et aZ. (1990) suggest that either a portion of the unaccounted volume is in tissues not examined (head and fins) or in the central vessels and heart. Table VIII extends this analysis using the organ weights provided by Gingerich et al. (1990) and the average tissue red cell or plasma spaces from Table VII. Table VIII shows that although nearly 90% of the total body weight is accounted for, the total red cell space of the tissues examined is only 4.44 ml per 883 g body weight or 5 ml/kg. This is only half of the "Cr red cell distribution volume measured in the whole fish (approximately 11 ml/kg; Duff et al., 1987; Duff and Olson, 1989; Hoffert, 1966; Gingerich and Pityer, 1989; Gingerich et

3.

BLOOD AND EXTRACELLULAR FLUID VOLUME REGULATION

177

al., 1987, 1990). Presumably a significant fraction of the volume that cannot be accounted for represents blood trapped in the heart and conducting vessels and blood lost from large vessels in the tissues when the tissues are removed. Thus the large vessel, or “central” blood volume in salmonids and perhaps many teleosts, represents a sizable reservoir that may be accessible under appropriate conditions. It should also be noted that similar calculations based on ‘251-albumin spaces cannot be made at this time because they grossly overestimate the TBV. This is due largely to the lack of information on skin albumin space. The only estimate available of skin albumin space is on the plaice (607 pllg; Wardle, 1971). This value produces unusually high skin and fin plasma spaces when used in the calculations for salmonids (45 and 17 ml/kg fish weight, respectively) and results in calculated blood volumes for whole fish in excess of 120 ml/kg. As Satchel1 (Chapter 3, Part A) notes, skin and fins are well endowed with a secondary circulation and an accurate estimation of skin and fin blood volumes is needed to estimate central blood volume. Table VIII also shows the estimated TBV and Hct calculated from red cell and albumin spaces in each tissue. Red cell space in white muscle accounts for less than 15% of the total red cell space in salmonids (19% if the head is included). This is consistent with the relatively low total extracellular volume found in this tissue (see earlier). Interestingly, 51Cr red cell spaces in trout muscle remain constant between 12 and 240 min after injection of tagged cells indicating little, if any, vasomotor activity (Duff et al., 1987). Splanchnic tissues, including swimbladder but excluding spleen, account for 8% of the total red cell space. Because many of the larger vessels, especially veins, are also located on or near these tissues, it is likely, a considerable fraction of the TBV is located in the splanchnic region. Kidney contains around 10% of the red cell space and gills around 25%. Splenic tissue, which readily accumulates radiolabeled red cells (Duff et al., 1987; Duff and Olson, 1989; Hoffert, 1966; Gingerich and Pityer, 1989; Gingerich et al., 1987, 199O),is undoubtedly a source for red cells during emergency situations (Pearson and Stevens, 1991; Yamamoto, 1987; Yamamoto and Itazawa, 1989; Yamamoto et al., 1980,1983) but is probably similar to the small intestine, on a per weight basis, in its ability to contribute to fluid volume. ‘251-albumin space in the spleen (120 pl/kg fish) is probably more representative of the actual vascular volume; this equates to a red cell space of -50 pllkg fish. Blood distribution in mammals, as a percent of TBV, is: muscle, 17%; splanchnic, 33%; kidney, 1%; and pulmonary, 10% (Greenway and Lautt, 1986). Thus fish have proportionally more blood in the respiratory organs (gills) and kidney and less in the splanchnic, although the latter is probably

178

KENNETH R . OLSON

Table VIII Total 51CrRed Cell and '251-Albumin Spaces in Salmonid Tissues

Bone Brain Eye Fat Fins Gill Head Heart Large intestine Small intestine Anterior kidney Posterior kidney Liver Ked muscle White muscle Pseudobranch Pyloric cecae Skin Spleen Stannius corpuscles Stomach Swimbladder Total a

Tissue weight" (g/kg fish)

"'Cr red cell spaceh (&kg fish)

""I-albumin space" (pllkg fish)

Total blood volume

99.7 1.2 7.1 8.9 28.6 20.0 90.2 1.4 3.9 2.7 2.4 5.7 9.7 34.3 467.0 0.1 10.1 74.8 0.8 0.01 13.3 1.8 883.71

128' 23 103 53 172* 1000 234' 13 46 23 167 311 193 132 600 7 45 449" 642 1 53 39 4434

631' 47 270 211 17,630* 3,089 997' 95 249 125 625 1,435 1,064 543 2,958 18 424 45,404" 120 13" 293 197' 76,168

759 70 373 264 17,532 4,089 1,231 108 295 148 792 1,746 1,257 675 3,558 25 469 45,853

(PI)

-

14 346 236

Calculated hematocrit 17 33 28 20 1 24 19 12 16 16 21 18 15 20 17 28 10 1 7 15 17

Tissue weight from Gingerich et al. (1990)for Oncorhynchus mykiss, Wythville Strain.

" Spaces averaged from Table VII. Estimated from muscle.

* Estimated from skin.

Estimated from red and white muscle. Estimated from gill. From Wardle, 1971, red cell space = 6 pl/g, albumin space Estimated from kidney. ' Estimated from liver.

f

=

607 p l / g .

artificially low. The proportion of blood volume in muscle is similar in both vertebrates. The calculated Hct (Table VIII) for tissues other than brain, eye, gill, and pseudobranch is lower than the average large vessel hematocrit (28%)from which these data were obtained (Duff et al., 1987; Duff and Olson, 1989; Hoffert, 1966; Gingerich and Pityer, 1989; Gingerich et al., 1987, 1990). Low Hcts in many tissues are probably due to at

3.

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179

least two factors: albumin extravasation and the fact that these samples may be biased toward capillary Hcts if blood is lost from large vessels during sampling. Capillary Hcts in mammals may be 10% or less (Johnson, 1971; Klitzman and Duling, 1979). This issue cannot be resolved until a suitable indicator of plasma space is identified.

3. DETERMINANTS OF BLOODVOLUME The primary determinant of blood volume is the physical dimensions of the vascular “container.” Within this constraint, blood volume is regulated by factors that affect ECFV and by factors that affect change between intravascular and interstitial fluid compartments. Relatively little is known about these parameters in fish and, of necessity, the general principles of blood volume regulation in mammals are offered as a convenient, albiet preliminary, starting point. Figure 4 summarizes these variables; details are provided in Sections a-c.

a. Extravascular Variables of Blood Volume Regulation. These are factors that affect water movement between a fish and its environment or between intracellular and extracellular compartments. Extra-

Cutaneous

+ Gill

I..................

Lymph? Fig. 4. Factors that govern vascular and interstitial fluid volume. See Sections 3,a,b for details.

180

KENNETH R. OLSON

vascular variables are determinants of ECFV and thereby affect both intravascular and interstitial compartments. Intracellular osmolyte generation further affects fluid balance between intracellular compartments as described earlier. Regulation of extravascular variables by the RAS, KKS, and ANP will be described in later sections.

b. Intravascular-lnterstitial Variables of Blood Volume Regulation. These are factors that govern fluid exchange between intravascular and interstitial compartments. Intravascular-interstitial variables can be further divided into primary and secondary determinants. The two primary determinants of blood volume are the so-called Starling forces and return of filtered solute and protein via extravascular (e.g., lymphatic) pathways. Starling forces directly affect fluid balance across the capillary and venule and are summarized b y the equation (Taylor, 1981; Taylor and Granger, 19842): where JV is the net volume of fluid flow across the capillary wall per unit time; Kf,, is the capillary filtration coefficient in volume per mmHg pressure (usually also per unit tissue weight); P , and P, are hydraulic pressures in capillary and tissue (interstitial) spaces, respectively; (T is the reflection coefficient; and rr, and rr, are colloid osmotic (oncotic) pressures in capillary and interstitial spaces, respectively. By convention, a positive value indicates either a pressure gradient or flow from the capillary to the interstitium. In mammals, moderate increases in Jv produce corresponding increases in lymph flow without significant effects on interstitial fluid volume. Further increases inJv, beyond the capacity of the lymphatics, result in tissue edema, exudation of fluid into body cavities, or both. The importance, or even existence, of fish lymphatics in return of ultrafiltrate and protein to the vasculature is not known. The secondary determinants of Starling forces are factors that affect Kf,,, CT, P,, Pt, rrc, and rrt. Kt;, and (T have not been measured in fish and although factors that might affect them are unknown, one can assume that they are similar to those found in mammals. A summary of the secondary determinants is provided in the following paragraphs. Kf,, is the permeability surface area product and is therefore an index of both the hydraulic conductivity of the exchange vessel and the Other models for fluid filtration have been proposed to better explain transcapillary fluid movement (Bert et al., 1988; Bassingthwaight and Goresky, 1984; Reed et al., 1989). These are not considered in the present discussion.

3.

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181

exchange surface area. Hydraulic conductivity is directly related to the number and radius of filtering pores and inversely related to barrier thickness and viscosity of the filtering fluid (Taylor, 1981). Kf,,may vary b y as much as 10-fold between tissues. Local or remote vasoactive stimulants may affect Kf,, by altering barrier permeability or surface area in addition to their effects on hydraulic pressure. The reflection coefficient (a)is an index of the effectiveness of the wall of the exchange vessel in acting as a barrier to colloid (usually plasma proteins). In its simplest form a is defined by the equation: a = l-[col~I/[col,],

(10)

where [ColJ and [Col,] are the concentrations of colloid (usually protein) in lymph and plasma, respectively. a approaches 1in capillaries that are impermeable to protein and becomes 0 when the protein barrier is absent. It may also be affected by hormones (i.e., it is reduced in frogs by atrial natriuretic peptide) (Huxley et aZ., 1987). Although a h a s not been directly measured in fish, protein concentration in the subcutaneous sinus and peritoneal fluid of cod, Gadus morhua, and flounder, Pleuronectes platessa, and in peritoneal fluid from the wolf fish, Anarhichas Zupis, is similar to that found in plasma (Hargens et al., 1974; Turner, 1937). This suggests that a may approach 0 in some fish tissues. A low (+ would offset the predicted low P , in these tissues thereby stabilizing Starling forces. It is important to note, however, that the fluid sampled by Hargens et al. (1974) and Turner (1937) is derived from tissues with the highest apparent albumin permeability (Table VIII) and may not be representative of all capillary beds. Tissues that appear leaky to '251-albumin(Table VIII) may have lower (T values, whereas a in gill, brain, and eye may, in fact, approach 1 (see earlier). a may also approach 0 in venular endothelium of carp orbital tissue (Suzuki and Hibiya, 1981b)or fins (Suzuki and Hibiya, 1981a)during inflammatory reactions. The anatomy of elasmobranch systemic capillaries (Rhodin, 1972) is suggestive of a leaky endothelium and very low a; this has been corroborated by physiological studies (see later). Capillary hydrostatic pressure (P,) is determined by arterial and venous pressure ( P a and P,, respectively) as discussed elsewhere in this volume. At constant Pa and P,, P , will still increase or decrease if the ratio of precapillary to postcapillary resistance ( R J R , ) decreases or increases, respectively. Pulse pressure, often ignored in determining filtration gradients, may also be important. Davie and Daxboeck (1982) calculated that half of the total fluid flux moves back and forth across the gill endothelium with each heart beat.

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KENNETH R . OLSON

Tissue (interstitial) hydraulic pressure (Pt)opposes capillary filtration. Interstitial compliance (C,) determines the relationship between interstitial volume (V,) and pressure according to the compliance relationship:

Ct

=

AVtIAPt,

(11)

where AV, and AP, denote the change in interstitial volume and pressure, respectively. Pt is subatmospheric in many mammalian tissues (- 1 to -6 mmHg; Guyton, 1991). Under normal circumstances Ct is quite low in mammals, which prevents accumulation of interstitial fluid. However, if filtration exceeds reabsorption and lymph removal capabilities, slight accumulation of interstitial fluid greatly increases Ct and substantial amounts of fluid will accumulate without a corresponding increase in Pt (Aukland, 1984; Bert and Pearce, 1984; Bert et al., 1988; Reed et al., 1989; Wiig and Reed, 1987). Measurements of P, in fish are limited to one elasmobranch, the smooth dogfish shark, Mustelus canis, and five teleosts: bluefish, Pomatomis saltatrix; pink salmon, Oncorhynchus gorbuscha; chum salmon, 0. keta; chinook salmon, 0. tschawytscha; and coho salmon, 0. kisutch. All values were obtained with the cotton wick method (Hargens and Perez, 1975; Ogilvy and DuBois, 1982). Average P, in anterior and posterior muscle of seven unanesthetized smooth dogfish is 1.5 and 2.1 mmHg, respectively, although pressures as low as -1.5 and - 1.0 mmHg, respectively, were observed in these tissues in one fish. In anesthetized bluefish, anterior and posterior muscle Pt are 0.3mmHg; in one control, -0.7 and -0.2 mmHg were recorded. Headup tilting (30")in air, which places a hydrostatic load on the posterior region, does not affect anterior Pt in the smooth dogfish but greatly increases posterior Pt (to 10.3 mmHg). Head-up tilt slightly increases anterior P, in the bluefish and increases posterior P, (to 1.0 mmHg). Considerable edema and extravasation accompany head-up tilt in the smooth dogfish, whereas neither are observed in bluefish. Subcutaneous and peritoneal fluid pressures in all four species of Pacific salmon are negative (--1 to -2 mmHg) when these fish are in seawater; however, upon their spawning migration into FW, fluid pressures become positive (- 1 to 2 mmHg) and the fish develop profound edema (Hargens and Perez, 1975). Plasma protein concentration is the primary determinate of rr, and both are low in most fish. The approximate correlation between rr, and protein concentration in fish plasma has been provided by Burton (1988): rr, =

0 . 0 3 4 ~+ 0.0073c2,

(12)

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where c is protein concentration in g/liter and reis in mmHg. Plasma protein concentrations in most fish are between 20 and 50 g/liter. The predicted r,,calculated from Eq. 12, is between 3.5 and 20 mmHg. These pressures, and those determined by direct measurement of rC, may overestimate physiological r,because both methods assume that, as is the case in mammals, plasma albumins are a major component of rC. Although albumins are found in all fish (Doolittle, 1984,1987), they probably account for less than 20% of the total plasma protein and, in subteleostean species, albumin-like molecules may exist as large molecular weight (150-360 kDa) monomers or dimers (Davidson et al., 1988; Elger et al., 1987, 1988; Fellows and Hird, 1981; Filosa et al., 1982,1986; Hilmy et al., 1978; Logan and Morris, 1981; Nagano et al., 1976; Perrier and Perrier, 1978; Perrier et al., 1977; Yanagisawa and Hashimoto, 1984). Two problems arise in calculating rC from plasma protein concentrations. First, mammalian-size albumins (-60 kDa) may not be retained by fish capillaries and hence they will be osmotically ineffective. Second, large molecular weight proteins (the major constituents of fish plasma) are less osmotically active on a per weight basis. Errors are undoubtedly made in direct measurement of r, (Table IX) as well, because the molecular cut-off of membranes used in most oncometers is 10-30 kDa, and this is probably physiologically inappropriate for fish. This becomes apparent in the study is reported in excess of of polar fish by Hargens (1972) in which rTT, 100 mmHg (Table IX). Resolution of this issue awaits characterization of the permselectivity of fish capillaries and identification of the relative proportion of physiologically important colloid to total protein. There has been some attempt to correlate elevated plasma protein concentration in salmonids with salinity adaptation (Alexander, 1977) or smoltification (Bradley and Rourke, 1984, 1988). Table IX also suggests that rcis slightly greater in SW species. However, these relationships have not been corroborated b y protein measurements in eel (Robertson, 1984), Tilapia (Gupta and Hanke, 1982), or trout (Elger et d.,1988),nor by measurement of rein FW and SW eels (Keys and Hill, 1972). rTT, drops from 13-14 mmHg to 7-11 mmHg during the anadromous spawning migration of Pacific salmon, although this is probably a consequence of the associated degenerative processes observed in these species (Hargens and Perez, 1975). Doolittle (1984,1987) claims that, in view of the variability in size and concentration of proteins in different fish in the same salinity, a significant, salinity-dependent, adaptive effect of protein in volume homeostasis seems doubtful. Additional measurements of r, and intravascularlinterstitial compartment volumes during salinity adaptation are required to resolve this issue.

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KENNETH R. OLSON

Table IX Plasma Oncotic Pressure Species Cyclostomes Eptatretus stouti Eptatretus stouti Lampetra jluviatilis

Elasmobranchs Raja erinacea Carcharias taurus Mustelus canis Dasybatus marinus Raja fullonica Chimaera monstrosa Galeus vulgaris Squalus acanthias Pristiurus catulus Raja oxyrhynchus Etmopteris spinax Osteichthyes Teleosteans Anguilla japonica Carassius carassius C yprinus carpio Tinca vulgaris Oncorhynchus mykiss Oncorhynchus gorbuscha Oncorhynchus keta Oncorhynchus tschawytscha Oncorhynchus kisutch Esox lucis Tantoga onitis Sauda savda Anarhichas lupus Opsanus tau Molua molva Gadus morhua

Environment

sw sw

Pressure (mmHg)

FW

7.7 10.5 8.9 8.6 7.8 9.3 10.7 8.5 9.0 4.1 8.8

sw sw sw sw sw sw sw sw sw sw sw

2.7 3.4 3.0 2.7 2.4 2.0 2.0 2.0 1.9 1.6 1.3

FW FW FW FW FW

14.3 5.1 5.2 7.7 3.6 13.3 14.6 14.9 13.9 6.6 9.5 17.2 11.3 7.7 10.3 8.2

20% sw 30% SW 40% SW 50% SW FW female FW male

sw sw sw

SW FW

sw SW

sw sw sw SW

Reference Riegel (1978) Riegel (1986) McVicar and Rankin (1983)

Immature Maturity 2-week adaption Kakiuchi et al. (1981)

Turner (1937)

Kakiuchi et al. (1981) Keys and Hill (1972) Duff and Olson (1989) Hargens and Perez (1975)

Keys and Hill (1972) Turner (1937)

Turner (1937) Hargens et al. (1974) (continues)

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Table I X (Continued) Species

Gadus morhuu Gadus pollachius Gadus cirens Eleginus gracilis Myxocephalus scorpioides Brosmius bromsome Lophius piscatoris Belone acus Echene.s naucrates Cyclopterus lumpus Prionotus carolinus Prionotus strigatus Scomber scombrus Pseudopleuronectes americanus Pleuronectes platessa Pleuronectes platessa Anguilla vulgaris

Environment

sw sw sw sw 5°C SW -1.8"C SW -1.4"C

sw sw SW

sw SW

sw sw sw sw SW FW SW FW

sw

FW

Chaenocephalus aceratus Pseudochaenichthys georgiaiius Notothenia corriceps Notothenia gibberifrons

sw sw

SW 2°C sw -1.8"c sw 2°C sw -1.8"C

Pressure (mmHg)

8.6 6.9 3.0 129.0 158.0 94.0 10.3 3.1 12.8 15.9 6.0 7.8 7.5 14.6 8.9 9.0 9.1 20.7& 18.8" 10.3' 8.:3' 21.0 4.0 59.0 75.0 52.0 73.0

Reference Turner (1937)

Hargens (1972)

Turner (1937)

Hargens et al. (1974) Keys and Hill (1972)

Hargens (1972)

Abbreviations as in Table 111. Starved 7 days. Starved 60 days.

There does not appear to be a significant correlation between protein concentration or rC(Table IX) and blood pressure or phylogeny, although available data are limited. F, is as high, or higher, in osmoconforming hagfish and osmoregulating lamprey as it is in teleosts, even though the hagfish arterial blood pressure is only -30% that of a teleost (Riegel, 1986; Satchell, 1986). Riegel(1978,1986)found rC was from 1.4 to 2 times greater than dorsal aortic pressure (7.7 versus 3.9 mmHg) in the Pacific hagfish, Eptatretus stouti, which raises ques-

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KENNETH R. OLSON

tions regarding the relationship of Starling forces to filtration even in the kidney. This apparent imbalance may contribute to the disproportionately high blood volume in hagfish, which may be 50-60% of the total extracellular fluid. However, it should be pointed out that considerably higher arterial pressures (10.8 mmHg) have been recorded in Eptatretus cirrhatus (Forster et al., 1988), and these are more in line with the plasma oncotic pressures described by Riegel (1978; 1986). Interestingly, injection of even small amounts of saline into hagfish will raise blood pressure for several hours (Satchell, 1986), whereas this has only a minimal transient effect in elasmobranchs (Opdyke et al., 1975) and teleosts (Chester Jones et al., 1966; Duff and Olson, 1986; Hipkins et al., 1986; Lee and Malvin, 1987). rc in lamprey is surprisingly high (8-10 mmHg), especially in young fish (Table IX), considering that plasma protein in the river lamprey, Larnpetra fluuiatilis, is only 38.5 g/liter (Robertson, 1984). r, is lower in elasmobranchs than teleosts, and this correlates with slightly (20-30%) lower blood pressure in the former (Satchell, 1991). In teleosts, there is also some correlation between arterial pressure and rC,both of which are notably elevated in tuna (White, 1988;Table IX), although pressure and T,have not been simultaneously measured in individual fish or species. rcin eels (14-20 mmHg) is considerably greater than would be predicted by dorsal aortic pressure (-2030 mmHg, Hipkins et al., 1986), but it is consistent with the measured protein concentrations (74 and 71 g/liter in FW and SW, respectively; Robertson, 1984). The reason for comparatively high r, and protein concentration in eels is unknown. Little information is available regarding rt in fish. Turner (1937) collected peritoneal fluid from the wolf fish, Anarhichas lupis, and observed that rt was 7.2 mmHg compared to 9.2 mmHg in serum. Similar findings have been reported by Hargens et al. (1974). Subcutaneous, peritoneal and plasma protein concentrations in cod, Gadus morhua, are 56, 51, and 57 g/liter, respectively; in flounder, Pleuronectes platessa, they are 56, 57, and 57 g/liter, respectively (Hargens et al., 1974). I n these tissues, rt virtually offsets rc and, assuming Pt is negative or near atmospheric (see previous discussion), it is difficult to envisage how intravascular fluid can be retained unless P , is exceptionally low (which is doubtful) or lymph return is high. The presence of additional factors or forces may be necessary to adequately explain transcapillary fluid balance in these vertebrates. The relationship among arterial blood pressure, blood volume, and capillary fluid balance can be examined when blood pressure is manually controlled through an external reservoir connected to the dorsal

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aorta. In trout, a decrease in arterial pressure by 15 mmHg produces an initial rapid loss of blood (-10 ml/kg) due to vascular decompression, followed by slow, sustained loss of an additional 12 ml/kg fluid from interstitial and intracellular compartments (Duff and Olson, 1989). Both 51Crred cell and 1251-albuminspaces contract to the same volume (16 ml/kg) after the hypotension. If arterial pressure is raised in two consecutive 5 mmHg steps over 240 min, fish accumulate fluid. The extent of volume expansion depends on the composition of fluid entering the fish from the reservoir (Fig. 5; Table X). Neither Ringer nor Ringer-containing albumin are retained within the red cell (i.e., vascular compartment) even though 7r of the latter is hyperoncotic to trout plasma. On the other hand, steelhead trout plasma is partially retained, doubling the vascular space and resulting in a lower rate of fluid accumulation. Vascular compliance, computed from the sum of the rapid fluid intake volume for steelhead trout plasma, is around 1 ml/ mmHg-kg-'. Compliance of the Ringer and albumin distribution volumes is in excess of 3 ml/mmHg.kg-'. These experiments support the idea that 7rC in fish is determined by proteins larger than mammalian-

''

i

0 0

40

80

120

160

200

240

TIME ( m i d

Fig. 5. Cumulative fluid intake by trout connected via the dorsal aorta to reservoirs containing Ringer ( 0 ;N = 12).5% bovine serum albumin ( 0 ;N = 5), or steelhead plasma (0; N = 13).Reservoir height was maintained at a height equivalent to resting arterial pressure for 3 h then raised at time 0 min to produce a 5-mmHg increase in pressure. At 120 min (small arrow) reservoirs were raised an additional 5 mmHg.

188

KENNETH R . OLSON

Table X Effect of Increased Arterial Pressure on Calculated Blood Volume of Trout

a

Raised pressure ~

Blood volumeb '251-albumin 51Cr red cells Ratiod

Control

Ringer'

59.6 (k2.3) 31.7 (A3.0) 1.9 (N=14)

140.3* (A24.7) 42.8 (55.7) 3.3* (N=12)

5% Albumin" 129.5*

Steelhead' plasma 122.7*

(k48.0)

( A 12.7)

28.5 ("3.5) 4.5* (N=5)

61.7* (29.1) 2.0 ( N = 13)

Pressure was increased above resting in two 5-mmHg increments of 120 min each. '251-albumin and 51Cr red cell spaces were determined after a 30-min circulation (270 min after initial pressure elevation). Blood volume (ml/kg) determined from albumin space/(l-hematocrit) or red cell space/hematocrit; mean SE. Reservoir fluid. Ratio of albumidred cell blood volume. * Significantly ( P 5 0.05) greater than respective control.

size albumins and perhaps even albumin-size molecules in trout plasma are of little significance in transcapillary fluid balance.

c. Venous capacitance. Total blood volume (i.e., vascular capacitance) is the sum of the unstressed and stressed vascular volumes (Rothe, 1983a,b). The unstressed volume is the volume of blood remaining in the vasculature when mean circulatory filling pressure is zero. Mean circulatory filling pressure is the blood pressure at any point in the vasculature when cardiac output is zero, and after pressure has equilibrated throughout the vasculature (Guyton et al., 1973). Mean circulatory filling pressure is probably the major long-term determinant of capillary hydrostatic pressure. The unstressed volume is physiologically inert, and its removal does not affect cardiovascular function even though the unstressed volume may be 60-75% of the TBV (Greenway and Lautt, 1986; Rothe, 1983a,b). The stressed vascular volume is dependent on blood pressure according to the compliance relationship: where C\I is vascular compliance, AVV is the change in vascular volume, and APV is the change in vascular transmural (intraluminal-

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extraluminal) pressure. Pv equates to mean circulatory filling pressure. In mammals, systemic compliance and capacitance are largely attributable to the venous system because 70-75% of the systemic blood volume is located in the veins (Guyton, 1991; Hainsworth, 1986) and venous compliance is around 25 times greater than arterial compliance (Guyton, 1991). Sympathetic activation in response to exercise, hemorrhage, or other factors decreases unstressed volume in mammals by increasing venous tone without substantially affecting compliance (Greenway and Lautt, 1986; Rothe, 1983a, 1986). This shifts blood to the stressed volume and, if other reflexes are prevented, will increase venous pressure (and therefore capillary hydraulic pressure) according to the compliance relationship. Thus the unstressed volume, because of its physical size and the potential for vasoactive regulation, is an important blood reservoir that is potentially affected by a variety of vasoactive stimuli. Even changes in arterial resistance affect venous capacitance through their effects on intravenous pressure; a decrease in arterial pressure decreases venous pressure, which, at constant compliance, decreases venous volume (Rothe, 1986). Nearly 70% of the TBV mobilized by sympathetic stimulation in mammals is supplied by the splanchnic circulation (Greenway and Lautt, 1986), which also has the major portion of the total body compliance (Hainsworth, 1986). On the other hand, atrial natriuretic peptide decreases mean circulatory filling pressure and decreases blood volume; unstressed volume also decreases due to passive vascular recoil and possibly venoconstriction (Trippodo et al., 1986).Venous compliance is not affected by inhibiting angiotensin I1 (ANG 11) formation with the converting enzyme inhibitor, captopril (Ogilvie, 1985). Compliance characteristics of isolated rainbow trout vessels are remarkably similar to their mammalian counterparts (Conklin and Olson, 1990). Compliance of unstimulated anterior cardinal veins (5-8.5 mm long) from 300- to 600-g fish is 2.1 k 1.0 pl/mmHg; some 21 times greater than 4- to 7-mm-long efferent branchial arteries (0.10 t 0.03 pl/mmHg). Epinephrine, norepinephrine (both 10-lo lo-‘ M ) , or ANG I1 (lo-” M ) do not affect venous compliance, whereas epinephrine increases, norepinephrine decreases, and ANG I1 does not affect arterial compliance. Because trout anterior cardinal veins contract in response to catecholamines (Olson et al., 1991),it is possible that the major effects of adrenergic stimulation of trout veins are on the unstressed volume. The efficacy of vasoconstrictors on other venous reservoirs, especially those with little obvious smooth muscle (Satchell, 1991; also see Satchell, Part A, Chapter 3 ) , remains to be determined.

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KENNETH R. OLSON

Vascular compliance has not been measured in cytostomes, and only one attempt has been made to measure it in elasmobranchs. Opdyke et al. (1975) monitored blood pressure in recently killed dogfish sharks during consecutive 5- to 10-ml injections of saline or 100,000-200,000 MWT dextran. Compliance calculated from their data at 1 min after the first injection is -0.6 ml/mmHg.kg-'. Curiously, blood pressure during subsequent injections decreases, indicating large increases in compliance perhaps caused by stress relaxation (Opdyke et al., 1975) or LaPlace effects (final compliance after six injections is 6.3 ml/mmHg-kg-l). Injection of saline, urea, or dextran increases plasma volume by as much as 65% yet has no effect on blood pressure (Opdyke et al., 1975). Mean circulatory filling pressure, vascular compliance, and unstressed volume have been measured in freshly killed FW and SW adapted trout after incremental hemorrhage or volume expansion with trout plasma from -60 to 160% of their estimated blood volume (Fig. 6; M. D. Kellogg and K. R. Olson, unpublished). With this method, mean circulatory filling pressure for FW ( n = 8) or SW ( n = 5) trout is 2.8 5 0.6 and 4.2 0.6 mmHg, respectively; unstressed volume is 54 -+ 5 and 30 -+ 5% of estimated blood volume, respectively; and vascular compliance is 5.5 +- 0.6 and 6.9 0.9 ml/mmHg.kg-'. As shown in Fig. 6, compliance increases greatly with volume expansion, as is the case in mammals. Opdyke et al. (1975) estimated mean circulatory filling pressure of 5.7 mmHg in the shark, Squalus acanthias, by measuring blood pressure in freshly killed fish. Slightly lower values (3.8 5 0.4 mmHg, n =

*

*

-

2

0

2 4 mmHg

6

8

Fig. 6. Vascular compliance curves for 5ix SW trout. Blood pressure is measured in freshly killed trout during incremental hemorrhage or volume expansion between 60 and 160%of estimated blood volume. Mean circulatory filling pressure can be estimated from the compliance curve at 100%(i.e.,control, volume [horizontal line]). Unstressed volume is the blood volume at 0 mmHg pressure (vertical line). The slope of the curve is equivalent to compliance.

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5) have been determined in anesthetized trout during temporary cardiac arrest (M. D. Kellogg and K. R. Olson, unpublished). The latter agree with filling pressures determined from compliance curves above (Fig. 6). 4. FACTORS THATCAUSEREDISTRIBUTION OF FLUID BETWEEN PLASMA AND INTERSTITIAL COMPARTMENTS Exhaustive exercise is perhaps the best known effector of apparent fluid efflux from the vascular compartment of fish (Milligan and Wood, 1987a,b; Randall and Daxboeck, 1982; Stevens, 1968; Pearson and Stevens, 1991; Yamamoto, 1987, 1988; Yamamoto and Itazawa, 1989; Yamamoto et al., 1980, 1985). Hemoconcentration, interpreted from an increase in Hct, may be produced by the addition of red cells to the circulation through splenic discharge (see earlier discussion), by catecholamine-induced red cell swelling (Milligan and Wood, 1987a; Pearson and Stevens, 1991; Primmett et al., 1986),as well as by loss of vascular fluid to the interstitium. In perhaps the most thorough series of experiments to date, Yamamoto and colleagues (Yamamoto, 1987, 1988; Yamamoto and Itazawa, 1989; Yamamoto et al., 1980, 1985) determined that 35% of the increase in Hct in exercising SW yellowtail, Seriola quinqueradiata, and 20-40% in exercising FW carp, Cyprinus carpio, was due to fluid loss from the vascular compartment (a net decrease in plasma volume of 18 and -lo%, respectively). Pearson and Stevens (1991) estimate that in trout, 50% of the increase in Hct associated with strenuous exercise or air exposure is due to loss of plasma from the circulation. This is equivalent to a 3-ml/kg reduction, or around 13% of the plasma volume, and is similar to the findings of Yamamoto's group. The mechanisms involved in capillary fluid efflux during exercise are complex and probably include changes in P, and Kf,, as well as branchial and renal responses. Catecholamines could contribute somewhat to an exercise hypovolemia. In the perfused eel tail, catecholamines decrease vascular volume by -2% and increase interstitial (or possibly secondary circulation) volume by -3% (Davie, 1982). Acid exposure also decreases trout blood volume by -20% (Milligan and Wood, 1982). It is not known if an exercise induced acidosis could have similar effects. Temperature acclimation of FW trout between 6" and 18°C does not affect blood volume (Barron et al., 1987; Nikinmaa et al., 1981). However, 2°C adapted brook trout, Salvelinus fontinalis, have a 20% lower blood volume then 20°C adapted fish. A slight decrease in blood volume at lower temperatures is also apparent in the air breathing singi

192

KENNETH R. OLSON

catfish, Heteropneustes fossilis (Pandey et al., 1976). L-Thyroxine and progesterone decrease and hydrocortisone increases blood volume in the walking catfish, Clarias batrachus (Pandey et al., 1978). Anesthesia decreases TI824 space in trout, presumably due to impaired mixing of indicator (Smith, 1966). Conversely, hyperosmotic hemodilution following dorsal aortic cannulation increases blood volume in the SW buffalo sculpin, Enophrys bison (Sleet and Weber, 1983a,b). The relationships between blood pressure and blood volume and the effects of environmental salinity on these cardiovascular parameters have received surprisingly little attention. Blood pressure of Squalus acanthias adapted to 70% seawater for 24 h is nearly double that of seawater sharks (Solomon et al., 1988). The mechanisms mediating the hypertension are unknown, although they might include stress induced catecholaminergic responses that would increase peripheral vascular resistance and an increase in cardiac output resulting from increased venous return. Increased venous return could be the result of intra- and extravascular factors that increase vascular filling pressure, including general hydration of the fish. Both SW adapted eels, A. anguilla (Chan et al., 1967; Chester Jones et al., 1966, 1969), and rainbow trout (Olson and Duff, 1992) have significantly lower blood pressures than their FW counterparts. Because blood volume does not decrease when either eels, A. rostrata, or trout are transferred to SW (Kellogg et al., 1990; Nishimura et al., 1976), it is apparent that a dehydration-induced hypovolemia cannot be the cause of hypotension in these fish. Clearly other, perhaps multiple, effectors of pressure and volume are involved. It is also possible that under certain circumstances there is a trade-off between pressure and volume regulation, osmoregulation, or both such that one variable is conserved at the expense of the others. The RAS and ANP systems deserve special consideration because of their well-known involvement as antidrop and antirise effectors of pressure and volume in mammals and because of their ubiquity in a variety of fish. The KKS also impacts on fluid balance in mammals and may have similar activities in fish. Although there is considerable information regarding the effects of the RAS and ANP on blood pressure, renal function, and to some extent osmoregulation in fish, there is virtually nothing known about the involvement of these two systems, or of the KKS, in volume regulation. For that matter, there is no known mechanism for detecting either ECFV or TBW in mammals (Cowley and Roman, 1989).The following sections describe how the RAS, KKS,

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and ANP may affect these parameters and contribute to volume homeostasis.

111. RENIN-ANGIOTENSIN SYSTEM A. Components of the Renin-Angiotensin System The renin-angiotensin system is an enzyme-activated and peptidemediated effector of extracellular electrolyte and fluid balance in many vertebrates. The RAS has been extensively examined in mammals. Historically this has formed the basis for comparative studies in fish, and a number of analogies between osteichthean and mammalian systems have been established. In nonosteichthean fish these analogies are less evident, and it has been argued that these fish lack a RAS. However, it is not yet clear if nonosteichthean fish actually lack a complete RAS or if the criteria are too stringent or specific for a primordial system to be identified. The mammalian RAS has been described in a number of reviews (Dzau et al., 1988; Hall and Brands, 1992) and can be summarized as follows. Renin, a highly specific aspartyl proteinase, hydrolyzes the decapeptide angiotensin I (ANG I) from the crz-globulin, angiotensinogen. A dipeptide is then hydrolyzed from the carboxy terminal end of ANG I, principally by angiotensin converting enzyme (ACE; E.C. 3.4.15.1), thereby producing ANG 11. In some instances the amino terminal residue is also hydrolyzed and the heptapeptide des-Asp1ANG 11, or angiotensin I11 (ANG 111),is formed. Angiotensins may be inactivated by a variety of peptidases. ANG I is for the most part biologically inactive, whereas ANG I1 has potent vascular, renal, adrenal, and other effects. Angiotensin 111 is less vasoactive than ANG I1 but may be equipotent in stimulating aldosterone secretion; ANG III-like homologs have not been reported in fish. Messenger RNAs for angiotensinogen and renin have been identified in kidney, brain, adrenal, heart, vascular, and other tissues, suggesting a local RAS is operative. Renin may be circulated in the plasma (as much as 90% in the inactive prorenin form) or bound to membranes, and it has been proposed that in mammals the RAS has autocrine, paracrine, and endocrine functions. A number of comprehensive reviews have described the RAS in fish (Henderson et al., 1980, 1985; Malvin, 1984; Nishimura, 1980a,b, 1985a,b, 1987; Nishimura and Ogawa, 1973; Sokabe and Ogawa, 1974;

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KENNETH R. OLSON

Taylor, 1977; Wilson, 1984a,b).These provide an excellent summary of the initial morphological and physiological evidence regarding the phylogenetic development of the RAS. B. Occurrence and Distribution in Fish A complete juxtaglomerular apparatus is absent in all fish, being limited in teleosts by the lack of a true macula densa (Sokabe, 1974; Sokabe and Ogawa, 1974; Nishimura, 1980a). Criteria for the presence or absence of a RAS in nonmammalian vertebrates has been defined by Sokabe et al. (1969) and Nishimura et al. (1973; summarized by Nishimura, 1985b)as (a) presence in the kidney of granulated cells morphologically and histochemically resembling juxtaglomerular ( JG) cells; (b) time dependent production of an angiotensin, or angiotensin-like, pressor substance on incubation of homologous plasma (substrate, i.e., angiotensinogen) with kidney extract (renin source) in the presence of angiotensinase inhibitors, both plasma and renal extract being heat labile; and (c)formed product must resemble angiotensin in its pressor response in the rat, be resistant to a-adrenergic blockade in the rat bioassay, dialyzable, heat stable, susceptible to a-chymotrypsin digestion, and adsorbed onto Dowex 50W-X2 resin. The major disadvantages of these criteria are the assumptions that angiotensin-like peptides are pressor in all animals and the dependence on mammalian angiotensin receptors to recognize heterologous peptides. Undoubtedly, application of modern molecular biology techniques will greatly clarify this issue. 1. CYCLOSTOMES

Based on the above criteria there is little evidence to support the existence of a complete RAS in cyclostomes. Cyclostomes lack granulated JG cells, and angiotensin-like pressor activity has not been generated from cyclostome plasma by either homologous or heterologous kidney extracts (Nishimura, 1980a, 198513).Renal renin activity has not been demonstrated in cyclostomes (Nishimura, 198%) and, although a pressor substance can be generated by Lampetra jluviatilis kidney (Henderson et al., 1980),this is not unequivocally attributable to renin activity (Nishimura, 198513).However, ANG I1 has pressor activity in M . glutinosa (see later), suggesting some attributes of a receptor activation process and ACE-like activity has been found in Pacific hagfish, Eptatretus stouti, plasma and liver (Lipke and Olson, 1988).Thus some aspects ofthe RAS are present in cyclostomes. How they function and what their contribution might be to volume homeostasis remain to

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be determined. Because blood pressure appears to be the primary, if not sole determinant of glomerular filtration rate (GFR) and urine output, and hence fluid volume, in hagfish (Alt et al., 1981), it may be that some volume regulatory mechanisms such as the RAS have an extrarenal origin. The phylogenetic development of an association between “juxtaglomerular” cells, initially with distributing renal vessels in teleosts and ending with the glomerular circulation in mammals (Sokabe, 1974; Sokabe and Ogawa, 1974; Sokabe et al., 1969), suggests the evolutionary refinement of an initially systemic homeostatic system for service in intrarenal regulation. 2. CHONDRICHTHYES a. Elasmobranchs. Anatomical studies of a variety of marine sharks, rays, and the FW stingray, Potamotrygon circularis, have failed to reveal granulated JG or specialized macula densa cells, even though the distal nephron returns to the glomerulus as it does in mammals (see Nishimura, 1985b for summary). This, plus the inability to demonstrate angiotensin formation or renin activity in elasmobranch tissues (Nishimura et al., l970,1985b), led Nishimura (1985b)to conclude that a RAS was also absent in elasmobranchs. However, Lacy and Reale (1990) observed a complete JG apparatus, including granulated afferent arteriolar smooth muscle cells, close apposition of the distal tubule with glomerular arteries and some specialization of macula densa-like cells, and glomerular mesangial cells, in four marine elasmobranchs: Squalus acanthias, Mustelus canis, Raja erinacea, and Rhinoptera bonasus. Lacy and Reale (1990) postulated that the granules they observed with electron microscopy may be a different form of renin and thus undetectable using other histochemical methods. Granulated peripolar cells are also found at the junction between the parietal and visceral epithelia in the glomerulus of five species of elasmobranchs: Raja erinacea, Mustelus canis, Rhizoprionodon terraenovae, Sphryna lewini, and Rhinopetra bonasus (Lacy and Reale, 1989). These cells, never before observed in fish, have been postulated to be involved in osmoregulation via kallikrein-kinin or renin-angiotensin mechanisms, although they are not immunoreactive with mammalian antibodies to kallikrein or renin (Lacy and Reale, 1989). Other evidence, albeit limited, supports a RAS in elasmobranchs. Renin-like activity has been reported in kidney extracts from Scyliorhinus canicula (Henderson et al., 1981) and, in a preliminary report, immunoreactivity with antibodies to ANG I1 [Val5] has been found in the plasma, anterior kidney, rectal gland, hypothalamus, brainstem, and pituitary of the nurse shark, Ginglymostoma cirratum (Galli and

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Kiang, 1990).However, Hazon et al. (1989)could not detect any immunoreactive ANG I1 (irANG 11) in S. canicula using antibodies against ANG I1 [Ile5]. Abundant ACE-like activity is found in a variety of tissues, especially brain, gill, heart, kidney, and rectal gland from the little skate, Raja erinacea, and dogfish shark, Squalus acanthias (Lipke and Olson, 1988). The ability of angiotensin to produce various physiological responses in elasmobranchs (see following discussion) suggests that the effector end of a RAS loop is also present.

b. Holocephaluns. Based on current criteria, the holocephalans appear to have a complete RAS. Granulated JG cells have been reported in Hydrolagus colliei and Chimaera monstrosa (Nishimura et al., 1973; Oguri, 1978). Modest renin-like activity is evident in H . colliei kidneys (Nishimura et al., 1973). 3. OSTEICHTHYES In general, osteichthyes exhibit most characteristics of a complete RAS. In the few fish where RAS-like attributes are missing, one might logically question the sensitivity, or selectivity, of the assay or the experimental conditions. u. Subteleosts. A RAS is probably present in chondrosteans, although information is somewhat fragmentary. Granulated JG cells are not present in chondrosteans (Nishimura et al., 1973), however, there is evidence for renin in shortnose sturgeon, Acipenser brevirostris, and for renin and angiotensinogen in Nile bichir, Polypterus senegalus (Nishimura et al., 1973). In holosteans, renin activity and plasma angiotensinogen are found in both the longnose gar, Lepisosteus osseus, and bowfin, Amia calva; however, only L. osseus have granulated JG cells (Nishimura et al., 1973; Youson and Butler, 1988). Renin activity, angiotensinogen, and granulated JG cells are found in two dipnoians, the African and South American lungfish, Protopterus aethiopicus and Lepidosiren paradoxa (Nishimura et al., 1973); plasma renin activity is also found in the Australian lungfish, Neoceratodus forsteri, (Blair-West et al., 1977). Angiotensin converting enzyme-like activity is present in chondrostean (shovelnose sturgeon, Scaphirhynchus platorynchus), holostean (A. calva), and dipnoan ( P . aethiopicus) tissues and is often concentrated in gill, gut, kidney, heart, and accessory respiratory organs (Lipke and Olson, 1988; Olson et al., 1987).Juxtaglomerular cells are found in the larger renal arteries of the coelacanth, Latimera chalumnae, but are rare in arterioles and absent in afferent arterioles and mesangial tissue (Lagios, 1974).

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b. Teleosts. Granulated cells have been observed in the renal arteries and afferent arterioles of a wide variety of teleosts (Nishimura, 1980b, 1985b; Sokabe, 1974). Granulated cells are often more numerous in the terminal arterioles, decrease in number near the glomerulus, and are absent in postglomerular vessels (Christensen et al., 1989; Sokabe, 1974). They also appear to be innervated with adrenergic nerves (Elger et al., 1984), although monoamine specific fluorescence is absent in renal vessels of the toadfish, Opsanus tau (Madey et al., 1984). Antibodies to human, rat, and mouse renins do not react with salmon tissues (Christensen et al., 1989), although human, and to a lesser extent murine, renin antibodies react with epithelioid cells in renal arteriolar networks of the aglomerular lemon sole, Pleuronectes microcephalus (Christensen et al., 1987). Receptor-type binding of '251-ANG I1 [Ala', Val5] to glomeruli of FW trout has also been observed (Brown et al., 199Ob). Stannius corpuscles contain renin-like activity (Chester Jones and Henderson, 1965; Chester Jones et al., 1966; Ogawa and Sokabe, 1982; Pang et al., 1981a,b; Sokabe, 1968; Sokabe et al., 1970)and irANG I1 (Yamada and Kobayashi, 1987).The amount of renin in Stannius corpuscles is less than 1% of that found in the kidney (Sokabe et al., 1970), and it is probable that corpuscular renin-angiotensin has a paracrine function. However, Stannius corpuscles may contribute to the regulation of systemic blood pressure or volume as stanniectomy in the eel lowers blood pressure by 33% (Chester Jones et al., 1966). To date, neither angiotensinogen nor renin from fish has been purified and sequenced. Angiotensins from several teleosts have been sequenced and synthesized, and their structures are compared to a mammalian angiotensin in Table XI. In this chapter ANG I and ANG I1 denote the human sequence and residue substitutions are indicated in brackets. There is considerable homology among all peptides with variations occurring only at positions 1 , 5 , and 9. Some of the variation in the amino terminal residue of Anguilla rostrata (and perhaps other fish as well) may be due to a plasma L-asparaginase amidohydrolase capable of converting asparaginyl angiotensins to aspartyl angiotensins (Khosla et al., 1985). Val5 is common to all fish, whereas the residue in position 9 is species specific. Plasma and renal renin and plasma angiotensin levels in fish appear to be generally lower than levels in mammals (Nolly and Fasciolo, 1972; see also Nishimura, 1985b). Angiotensin converting enzyme-like activity is found in tissues of nearly all teleosts, although enzymatic activity is generally lower in teleosts than in other vertebrates, including subteleostean species

Table XI Fish Angiotensins Formed by Incubation of Homologous Plasma with Renal or Stannius Corpuscle Extracts ANG I ANG I1 Species

1

2

3

4

5

6

7

8

9

10

Arg

Val

Tyr

Ile

His

Pro

Phe

His

Leu

Reference

Human, rat, dog, pig, horse, rabbit, guinea pig

Asp

Akagietal. (1982)

Lophius litulon (aglomerular goosefish; renal or SC*)

Asn

Val

His

Hayashi et al. (1978) Hasegawa et al. (1984)

Oncorhynchus keta (chum salmon; renal or SC)

Asn Asp**

Val Val

Asn Asn

Takemoto e t al. (1983)

Anguilla rostrata (American eel; renal)

Asn Asp**

Val Val

GlY GlY

Khosla et al. (1985)

Anguilla japonica (Japanese eel; renal)

Asp** Asn

Val Val

GlY GlY

Hasegawa et al. (1983)

Abbreviations: SC, Stannius corpuscles. *, minor ANG in SC not identified. **, may be due to in eitro conversion from Asn (Khosla et a / . , 1985). Multiple sequences within a species are listed in descending order of' prevalence.

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(Lipke and Olson, 1988; Olson et al., 1987; Polanco et al., 1990). Trout ACE is similar to mammalian pulmonary ACE with respect to K , and chloride dependence; however, trout ACE has a higher p H optimum and is active over a greater range of pH, presumably reflecting the tendency of fish toward higher plasma alkalinity (Lipke et al., 1987). Teleost gills frequently have higher ACE activity than other tissues (Galardy et al., 1984; Lipke and Olson, 1988; Olson et al., 1987; Polanco et al., 1990).In gills, ACE is predominant in vascular endothelium and pillar cells of secondary lamellae (Olson et al., 1989). Because the entire cardiac output must first traverse the lamellae before entering the systemic circulation, the gills are in an ideal position to condition arterial blood; in fact, the isolated perfused gill converts over 60% ofANG I to ANG I1 in a single pass (Olson et al., 1986).ACE activity in nonrespiratory tissues may also contribute to circulating ANG I1 or may indicate the presence of a local RAS. Kohama et al. (1988)purified a novel ACE inhibitor from muscle of the tuna, Neothunnus macropterus, the sequence being: Pro-Thr-HisIle-Lys-Trp-Gly-Asp. The physiological significance of this octapeptide is not known, although its presence could account for the unusually low ACE activity in tuna tissues (Lipke and Olson, 1988)and the relative insensitivity of tuna blood pressure to '4CE inhibitors (R. W. Brill and K. R. Olson, unpublished). In contrast, vascular ACE activity is quite prevalent in trout tissues. Equal doses of ANG I [Asn', Val5, Asng] or ANG I1 [Asn', Val5] produce an identical increase in resistance during single-pass perfusion of either skeletal musclekidney or splanchnic circulations, whereas in the presence of an ACE inhibitor, ANG I is ineffective (K. R. Olson and R. Ferlic, unpublished). Angiotensins are inactivated b y peptidases, which are abundant in most tissues including kidney, Stannius corpuscles (Chester Jones et al., 1966),and the extensive alamellar vasculature in the core of the gill filament (Olson et al., 1986). Angiotensin metabolites may be selectively removed by the liver and secreted into the bile (Olson et al., 1986), although the catabolic process has not been examined in detail. The half-time (tllz) for circulating ANG I1 in trout, estimated by a variety of methods, is 3-7 min (Kellogg and Olson, 1990). In fact, based on the rate of ANG I1 infusion necessary to maintain blood pressure in ACE inhibited trout, the rate of ANG I1 inactivation in vivo appears to be limited as much by tissue perfusion as it is by peptidase activity (Kellogg and Olson, 1990). Thus the RAS in fish appears to be a rapid effector of blood pressure.

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KENNETH R. OLSON

C. Stimulus for Activation of the RAS Hypotension, hypovolemia, and, to a lesser extent, osmotic perturbation are the best known activators of systemic RAS responses in fish although there are considerable species and experimentally induced variations. It is inherently difficult, if not impossible, to separate these stimuli, especially at the level of the intact animal, and, therefore, much of the afferent limb of RAS control remains to be described. Studies in elasmobranchs indicate that hypotension, hypovolemia, or both stimulate a RAS-like response. Angiotensin converting enzyme inhibitors generally have little effect on blood pressure in resting sharks (Opdyke and Holcombe, 1976; Hazon et al., 1989),which can be interpreted as lack of evidence for a RAS. However, moderate hypotension produced by the smooth muscle relaxant papaverine in the dogfish, Scylorhinus canicula, makes captopril an effective hypotensive drug, presumably by blocking the now active RAS (Hazon et al., 1989). Furthermore, a 30% hemorrhage increases immunoreactive ANG II [Val5]in plasma, hypothalamus, brainstem, and pituitary of the nurse shark, Ginglymostoma cirratum; plasma irANG I1 [Val5] is also increased in G . cirratum after removal of the rectal gland or adaptation to 50% seawater for 7 days (Galli and Kiang, 1990). Hemorrhage, or other perturbations that produce hypotension or hypovolemia, usually activates the teleost RAS. Consecutive l-ml hemorrhages increase drinking and plasma ANG I1 in FW eels, Anguilla japonica (Kobayashi et al., 1989).Transfer of eels to SW after the first hemorrhage, which presumably exacerbates the hypovolemia, enhances drinking, and further increases plasma ANG I1 (Kobayashi et al., 1980).In the aglomerular toadfish, Opsanus tau, either a decrease in blood pressure (produced by captopril, isoproterenol, or papaverine) or a combined decrease in blood pressure and volume produced by repeated small volume (-5% of blood volume) or rapid, large volume hemorrhage increases plasma renin activity (PRA; Fig. 7; Madey et al., 1984; Nakamura and Nishimura, in: Nishimura and Bailey, 1982; Nishimura and Madey, 1989; Nishimura et al., 1979). Bailey and Randall (1981) have also observed a positive correlation between the amount of hemorrhage and PRA in intact trout and between renin release and perfusion pressure in the perfused kidney. However, Henderson et al. (1985)found that neither 15 nor 30%hemorrhage affected irANG I1 in the eel, A . anguilla, and they hypothesized that plasma sodium concentration, but not plasma volume, is the primary stimulus for activation of the RAS in fish. It should be noted that Henderson et al. (1985) measured ANG I1 30 min after hemorrhage, which might

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I

201

blood pressure -0

D

--4

0

P

--8 73

--I2

- -16 --20

; v1 v,

c XJ m

-

(5) (10) (20) (40) (60) BIOODLOSS ml/kg ( % e s t blood vol)

Fig. 7. Relationship between plasma renin activity (PRA) and blood pressure after graded hemorrhage in the unanesthetized toadfish, Opsunus tau. Mean *SE, N = 8;+, P<0.05; ++, P
provided sufficient time for pressure and volume restoration (Duff and Olson, 1989). Volume expansion would be expected to produce an inactivation of the RAS. Expansion of Australian lungfish, Neoceratodus forsteri, with isoosmotic NaCl (0.6% at 28 ml/h.kg-l for 5 h) decreases PRA by 50% (Blair-West et al., 1977), consistent with the predicted response. However, infusion of hypoosmotic saline (0.3%)at the same rate increases PRA to 140% above control (Blair-West et al., 1977). In both experiments dorsal aortic pressure increases by -2 mmHg while plasma sodium decreased during 0.3%saline infusion (Blair-West et al., 1977). The mechanism for the elevation in PRA in these fish remains to be determined. Transfer of fish between hypo- and hyperosmotic environments has been used to examine the effects of ion, volume, or pressure perturbation on the RAS. While ionic changes are readily quantified, volume and pressure measurements have rarely been attempted in the same experiment, and it is difficult to evaluate cause and effect relationships

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KENNETH R . OLSON

between cardiovascular parameters and the RAS. If one assumes that the general effect of a hyperosmotic environment is to increase extracellular ions, increase (or not change) extracellular and blood volume, and decrease blood pressure and intracellular water (see earlier sections), then three parameters-plasma ions, blood pressure, and cellular dehydration-would be predicted to have the greatest stimulatory effect on the RAS. Correlative relationships between plasma and tissue renin activity or angiotensin concentration may be further confounded by different responses in systemic versus local RAS or by concomitant alteration in turnover rates. Further experiments will need to be carefully designed to separate these variables. Known relationships between salinity transfer and the RAS are summarized in the following paragraphs, and it is evident that species differences contribute additional sources of variation. The effects of salinity on the RAS have been most extensively studied in eels. Henderson et al. (1976) found that PRA of eels, Anguilla anguilla, transferred to SW slowly increases over 3-5 days to levels more than twice as great as FW fish and is maintained thereafter at this level. Plasma sodium and osmolarity are also greater in SW fish (Henderson et al., 1976). Transfer of SW fish to FW rapidly (4-24 h) lowers eel PRA (Henderson et al., 1976). Plasma irANG I1 in SW adapted eels, A. anguilla, is nearly 20 times greater than that of eels adapted to distilled water; values for FW and 50% seawater are between these extremes (Henderson et d., 1985). Similar qualitative responses have been reported by Nishimura et al., (1976) and Okawara et al. (1987). In A. rostrata,transfer from SW to FW (3days to 5 weeks) decreases PRA, angiotensinogen, renal renin activity (RRA), and plasma sodium, but does not affect blood volume; RRA returns to SW level after 2-5 weeks (Nishimura et al., 1976). Because no correlation between PRA and either plasma sodium or cortisol is apparent in either eels or toadfish, Nishimura et al. (1976) hypothesized that PRA may be correlated with blood pressure. In A. japonica, plasma irANG I1 does not change during the first 2 h after transfer to SW even though drinking initially increases, whereas over the next 3 days a further increase in drinking is accompanied by an increase in plasma irANG I1 (Okawara et al., 1987). Captopril prevents the delayed increase in drinking but does not affect the early response (Okawara et al., 1987). Conversely, PRA has been shown to only transiently increase in A. japonica transferred from FW to 30, 50, or 100% SW or air before returning to FW levels (Sokabe et al., 1973; Takei et al., 1988). Effects of salinity transfer on the salmonid RAS are less clear. Granulated epithelioid cells in renal arteries of Atlantic salmon, Salmo

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203

salar, decrease in number between l-year FW and 2-year SW fish, which the authors suggested might be indicative of reduced renin secretion b y FW fish (Christensenet al., 1989; although see following). Salinity adaptation of rainbow trout reduces glomerular size, flattens podocytes, and increases pedicel density, often occluding the filtration pores (Brown et al., 1983; Gray and Brown, 1987). Angiotensin I1 [Asn', Val5] infusion for 45 min into FW trout, or added to trout glomeruli in oitro, produces similar effects except that glomerular diameter is unaffected (Brown et al., 1990a; Gray and Brown, 1987). However, PRA in trout adapted to SW for 10 days is not different from FW fish (1.65versus 1.19 ng ANG I e q / m l / l 6 h; Brown et al., 1980). In nonsalmonids, RRA is usually inversely related to environmental salinity, whereas the effects on PRA are less obvious, perhaps suggesting that the predominant effect of the RAS is on kidney function. Renal renin activity is elevated when aglomerular (Opsanus beta and Hippocampus hudsonia) or glomerular euryhaline (Alosa sapidissmia and Pornolobus pseudoharengus) teleosts are adapted to dilute media (Capelli et al., 1970). PRA does not change when Tilapia mossambica are transferred from SW to FW for from 1to 7 days (Malvin and Vander, 1967), although RRA decreases after transfer from FW to SW (Sokabe et al., 1968,1973). Plasmarenin activity does not change when aglomerular toadfish, Opsanus tau, are transferred from 50 to 5% SW (Nishimura et al., 1976). In other fish, RRA is also higher in FW species (Capelli et al., 1970; Mizogami et al., 1968). Other factors that affect fish RAS have received but little attention. In trout, unionized ammonia increases ir-RRA in a log-dose related fashion (Arillo et al., 1981); this may, in turn, be the cause of the hypertension (Smart, 1978) and diuresis (Lloyd and Orr, 1969) associated with ammonia toxicity. Unlike the situation in mammals, renin secretion in fish does not appear to be regulated by adrenergic mechanisms. The renin response of the perfused trout kidney to reduced perfusion pressure is not affected by prior a, p, or combined a + p adrenergic blockade (although basal secretion appeared to be decreased and increased by a and p blockade, respectively; Bailey and Randall, 1981). A similar insensitivity of renin secretory mechanisms to the fi adrenergic agonist or antagonist, isoproterenol or propranolol, respectively or to chemical sympathectomy is found in intact toadfish, Opsanus tau (Madey et al., 1984; Nishimura and Madey, 1989). Renin secretion by the toadfish kidney also appears to be independent of prostanoid regulators (Madey et al., 1984). Renin secretion from superfused toadfish kidney slices is inhibited by polarizing concentrations of K+ (50 mM) or the

204

KENNETH H. OLSON

calcium channel agonist Bay K 8644 and is restored by the calcium channel antagonist nifedipine (Nishimura and Madey, 1989). Furthermore, renin secretion by toadfish kidney slices does not appear to be regulated by /3 adrenergic, cholinergic, cyclic adenosine monophosphate (CAMP),or cyclic guanosine monophosphate (cGMP) mediated systems (Nishimura and Madey, 1989). Reflex inhibition of the RAS in FW eels, A. anguilla, may be mediated in part through arginine vasotocin (AVT) as exogenous AVT reduced PRA activity by 60% (Henderson et al., 1985).However, in SW eels AVT effects are less evident, perhaps due to the elevated plasma AVT levels in these fish or other factors (Henderson et al., 1985). Feedback regulation of the RAS by other hormonal signals has not been critically examined. Little is known regarding control of ACE activity in fish tissues. Polanco et a2. (1990) found that ACE activity in carp, C yprinus carpio, gill, but not kidney, was reduced by 50%after 1week in hypoxic water. However, in these experiments hypoxia was produced by raising water temperature from 10"to 25"C, and it is not clear if the response was due to hypoxia per se or to temperature. In obligate air breathing fish the dependence on branchial ACE is reduced commensurate with a reduction in gill vascularity, and in these species there is a corresponding increase in ACE activity in the accessory respiratory organs, presumably reflecting their increased vascularity (Olson et al., 1987). D. Effects of Angiotensins

T h e RAS appears to serve primarily as an antidrop effector of blood pressure, fluid volume, or both in fish. Angiotensin-mediated pressor responses have been demonstrated in virtually all species examined. Dipsogenic and antidiuretic effects attributable to the € U S have also been reported in select species suggesting the importance of this system in volume homeostasis. Pressor doses of exogenous angiotensin may produce hypovolemia by increasing fluid filtration across glomerular and other systemic capillaries. Perhaps this is a physiological response, but until more evidence is available it can be assumed to be a pharmacological artifact. Surprisingly, the effects of the RAS on vascular or extracellular fluid volume have not been directly examined. 1. CYCLOSTOMES Information on RAS actions in cyclostomes is limited. Angiotensin 11 is pressor in hagfish, Myxine glutinosa, but its effects appear to b e mediated exclusively by catecholamines as angiotensin responses are

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205

abolished by prior a-adrenergic blockade (Carroll and Opdyke, 1982). Drinking cannot be induced in either the marine hagfish, Eptatretus burgeri, or FW arctic lamprey, Lampetra japonica japonica, by intraperitoneal injection of high (50-100 mg/kg) doses of ANG I1 [Am', Val5] (Kobayashi et al., 1983). 2. CHONDRICHTHYES a. Cardiovascu1ar.A variety of angiotensins including ANG I and 11,ANG I [Asp', Val5, Serg], ANG I1 [Asn', Val5], and ANG I11 produce phentolamine sensitive pressor responses in Squalus acanthias (Carroll, 1981; Carroll and Opdyke, 1982; Khosla et d.,1983; Opdyke and Holcombe, 1976), and ANG I1 [Asp', Ile'] is pressor in Scyliorhinus canicula (Hazon et al., 1989). Angiotensin I1 [Asn', Val'] (5 pg/kg) injection increases plasma epinephrine and norepinephrine in S. acanthias by -10-fold (Opdyke et al., 1981), and this probably accounts for the entire pressor effect. Angiotensin converting enzyme inhibitors SQ 20881 or captopril inhibit pressor responses to any form of ANG I, but do not affect resting blood pressure in either S. acanthias or S. canicula (Hazon et al., 1989; Khosla et al., 1983; Opdyke and Holcombe, 1976), suggesting that the RAS is not important in resting blood pressure regulation in elasmobranchs. However, in S. canicula, captopril is hypotensive if administered after the smooth muscle relaxant papaverine (Hazon et al., 1989), indicating that a RAS-like response may be activated under certain circumstances. Classical mammalian angiotensin antagonists, ANG I or I1 [Ile'], ANG I1 [Sar', Ile'] or ANG I1 [Sar', Thr8] do not block S. acanthias receptors but are often pressor as well (Khosla et al., 1983).Angiotensin I1 does not affect cardiac contractility in S. acanthias (Opdyke et al., 1982). In vitro studies confirm the dependence of the angiotensin response on catecholamines. ANG I or I1 has no effect on vascular resistance in the perfused gut (Opdyke and Holcombe, 1978) or on systemic or gill resistance of whole-body perfused dogfish, S. acanthias (Opdyke et al., 1982). Topical application of ANG I1 [Asn', Val5] to mesenteric circulation in zjivo or ANG I1 [Asn', Val5] added to celiac artery or anterior intestinal vein strips in vitro is likewise ineffective. However, if chromaffin tissue is added to the medium in which arterial strips are incubated, then ANG I1 [Am', Val5] stimulates catecholamine release and contracts the muscle (Carroll, 1981).

b. Renal. Much of the control of GFR in sharks appears to be regulated by catecholamines. Epinephrine, norepinephrine, and phenylephrine increase GFR and urine formation, whereas both vari-

206

KENNETH R. OLSON

ables are inhibited by isoproterenol and phenoxybenzamine (Yokota and Benyajati, 1986).Subpressor doses of ANG I1 (18-36 ng/min.kg-') do not affect GFR, urine flow, or sodium excretion in elasmobranchs (Churchill et al., 1985a). c. Drinking. Drinking is not stimulated by ANG I1 [Asn', Val5] in the banded dogfish, Triakis scyllia, or bull-head shark, Heterodontus japonicus (Kobayashi et al., 1983). Studies by Hazon et al. (1989) indicate that some angiotensin-dependentdipsogenic effectors may be present in elasmobranchs. ANG I1 [Asp', Ile5] increases drinking more than 2-fold in S. canicula (Hazon et al., 1989).Captopril has no effect on drinking in otherwise untreated S. canicula (Hazon et al., 1989). However, papaverine stimulates drinking in S. canicula by 30-fold, and this dipsogenic response can then be 80% inhibited by captopril (Hazon et al., 1989).These studies also suggest that a RAS may function in elasmobranchs under certain circumstances.

d . Other Effects. Infusion of homologous renal extracts (0.3 mg/ h.kg-') or ANG I1 (36 pglhakg-') increases the secretion rate and plasma levels of la-hydroxycorticosterone in Scyliorhius canicula but does not affect plasma osmolarity or sodium (Hazon and Henderson, 1985). However, ANG I1 [Asp', Ile5] decreases plasma osmolarity and sodium in this species (Hazon et al., 1989). Effects of angiotensins have not been reported in holocephalans. 4. OSTEICHTHYES T h e effects of angiotensins in chondrosteans or holosteans have not been reported. a. Dipnoians. ANG I1 [Val5] is pressor in both African and Australian lungfish, Protopterus aethiopicus and Neoceratodus forsteri, respectively, but produces slight to negligible diuresis and natriuresis; heart rate does not change (Blair-West et al., 1977; Sawyer, 1970; Sawyer et al., 1976).Infusion of 0.2-4 pg/h.kg-' ANG I1 [Val5] into N . forsteri for 2-4 h has no effect on plasma aldosterone or deoxycorticosterone (Blair-West et al., 1977).

b. Teleosteans. ( i ) Cardiovascular. Exogenous angiotensins produce pressor responses in all teleosts thus far examined including SW lumpfish, Cyclopterus lumpus (ANG 11; Carroll and Opdyke, 1982), 50% SW flounder, Platichthyes Jesus (ANG I1 [Asp', Val']; Perrott and Bal-

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ment, 1990), and both the aglomerular toadfish, Opsanus tau (ANG I1 [Asn', Val5]; Zucker and Nishimura, 1981), and goosefish, Lophis americanus (Churchill et al., 1979). Angiotensins are also pressor in FW eels, Anguilla rostrata (ANG I [Val5, Ser'], partially purified eel ANG I, ANG I1 [Am', Val5]; Nishimura and Sawyer, 1976; Nishimura et al., 1978), A . japonica (ANG I, 11; Hirano and Hasegawa, 1984) intact, hypophysectomized, or stanniectomized A. anguilla (ANG I1 [Asp', Val5]; Henderson et al., 1976; ANG I1 [Val5]; Chester Jones et al., 1966),and in rainbow trout, Oncorhynchus mykiss (ANG I1 [Asn', Val5]; Gray and Brown, 1985; ANG I [Asn-l, Val', Asn'l, ANG I1 [Asn', Val']; H. Xu and K. H. Olson, unpublished). Angiotensin I11 is pressor in A.japonica but it has only one-tenth of the activity of either ANG I or I1 (Hirano and Hasegawa, 1984). Angiotensin converting enzyme hydrolysis of decapeptide angiotensins to the octapeptide is necessary for pressor activity (Hirano and Hasegawa, 1984; Madey et al., 1984; Nishimura et al., 1978). The hypotensive effect of ACE inhibition in SW toadfish, Opsanus tau (Madey et al., 1984; Nakamura and Nishimura, in: Nishimura and Bailey, 1982), and FW rainbow trout (Galardy et al., 1984; Lipke and Olson, 1990) indicates that the RAS is active in blood pressure regulation in these fish even under resting conditions. In other species such as SW or FW flounder, Platichthyes Jesus, ACE inhibition has no effect on resting blood pressure (Balment and Carrick, 1985), but ACE inhibition prevents the gradual recovery of pressure following papaverine injection into 50% SW flounder (Perrott and Balment, 1990). Curiously, infusion of captopril into FW (40 pg/min.kg-'- or SW (160 pg/min-kg-')-adapted trout does not affect blood pressure (Kenyon et al., 1985)even though the total dose infused is greater than that which produces hypotension in the same species after a single injection (1mg/kg, Galardy et al., 1984; Lipke and Olson, 1990). Unlike elasmobranchs, only a fraction of the pressor effects of angiotensins are mediated by catecholamine release in teleosts even though angiotensin may increase circulating catecholamines two- to threefold (Carroll and Opdyke, 1982). a-Adrenergic blockade reduces ANG I1 effects by 10% in lumpfish, Cyclopterus lumpus (Carroll and Opdyke, 1982), and by 30-40% in A . rostrata (Nishimura et al., 1978), although reserpine produces 70% inhibition in eels (Nishimura et al., 1978). The in vitro vascular effects of angiotensins are consistent with their in vivo actions. Ventral aortic strips from longhorn sculpin, Myoxocephalus octodecirnspinosus, are directly stimulated by ANG I1 [Asn', Val'] independent of a-adrenergic inhibition with phentolamine (Carroll, 1981). Topical application of ANG I1 [Asn', Val'] also

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contracts sculpin mesenteric circulation (Carroll, 1981). Although major arteries in trout, such as ventral aorta and celiacomesenteric artery, are not contracted by mammalian or salmonid angiotensins, these peptides are vasoconstrictory in perfused systemic tissues (Olson and Meisheri, 1989; K. R. Olson, Ferlic and T. Kne, unpublished) and in perfused gills of both trout (K. R. Olson, unpublished) and eels (Fenwick and So, 1981). Classical angiotensin antagonists, such as ANG I1 [Sar', Thr'l, [Sar', Ile'l, and [Sar', Val5, Ala'], are ineffective in fish in vivo and in vitro, and, in fact, they often have slight agonistic properties (Carroll, 1981; Churchill et al., 1985b; Khosla et al., 1983; Nishimura et al., 1978). Lack of suitable angiotensin antagonists has severely limited further examination of the RAS in fish cardiovascular function. ( i i ) Renal. In glomerular fish the renal effects of exogenous angiotensins are often obscured by a concomitant systemic hypertension and it is difficult to identify specific intrarenal RAS actions. In the eel, Anguilla rostrata, ANG I1 [Val5], and partially purified eel ANG I, increased GFR and urine formation and, at high doses (100 ng/ minakg-'), decreased fractional sodium reabsorption (Nishimura aiid Sawyer, 1976). These responses were only seen after pressor doses of angiotensin, suggesting a primary extrarenal effect. In order to stabilize blood pressure, Brown et aZ. (1978, 1980) continuously infused pressor doses of norepinephrine into anesthetized trout. In these experiments Brown et al. (1978, 1980) found that FW trout have more filtering nephrons, but the single nephron filtration rate (SNGFR) is lower in FW than SW trout; the percent filtering, nonfiltering, aiid nonperfused nephrons in FW fish is 45,40, and 13%, respectively, and in SW fish it is 5,40, and 51%, respectively. After infusion of ANG I1 (Asn', Val5] in FW fish the percent filtering and nonfiltering nephrons is 9 and 46%, respectively, and in SW trout it is 6 and 12%, respectively. The single nephron filtration rate is unaffected. Thus ANG I1 in FW fish appears to reduce GFR b y reducing the number of filtering nephrons to the nonperfused type. In SW trout, ANG I1 [Asn', Val5] reduces the SNGFR to FW levels without affecting the number of filtering nephrons while nonfiltering nephrons are converted to the nonperfused type (Brown et al., 1980). Because ANG I1 reduced GFR and urine formation by 50% in FW fish (GFR from 140 to 61 and urine formation from 76 to 30; all in pllmin-kg-'), ANG I1 appears to exert a direct intrarenal antidiuretic effect (Brown et al., 1980). Much higher infusion rates of ANG I1 (600 versus 150 ng/min-kg-') are needed in SW than FW fish for an equivalent reduction in urine formation, suggesting that endogenous angiotensins are already elevated in the

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former (Brown et al., 1980). In subsequent experiments, ANG I1 [Asn', Val'] effects were examined in anesthetized FW trout without concomitant norepinephrine infusion, and these experiments confirm the antidiuretic nature ofangiotensin (Gray and Brown, 1985). In these experiments (Gray and Brown, 1985),ANG I1 [Asn', Val5] infusion increased blood pressure and heart rate, caused an initial antidiuresis, reduced GFR, and slightly decreased free water clearance (CH,O); all parameters returned to control levels over 45 min of continuous ANG I1 infusion (CH,O slightly increased) and remained at control even after cessation of ANG I1 infusion (Gray and Brown, 1985).Tubular maximum for glucose (TMG) was reduced by ANG I1 and did not fully recover until after ANG infusion stopped; ANG had no effect on fractional sodium excretion but during the recovery in urine flow, in the latter stages of infusion, fractional osmolyte excretion increased (Gray and Brown, 1985). The only partial recovery of TMG, despite complete recovery of urine formation rate and GFR, was taken to indicate that there are still fewer filtering nephrons in the later stages of ANG infusion but that GFR in the remaining nephrons is now greater, perhaps due to the hypertension. Angiotensin 11 [Asn', Val5] infusion (45 min) into FW trout, or added to glomeruli in vitro, flattens podocytes and obliterates pedicels, consistent with an antidiuretic effect in vivo (Gray and Brown, 1987). Angiotensin converting enzyme inhibition provides further evidence of an endogenous renal RAS in fish. Captopril infusion at a nonhypotensive rate into FW or SW trout increases urine flow rate, GFR, and TMG, indicating an increase in the number of functioning tubules (Kenyon et al., 1985).Captopril also increases urinary electrolyte concentration, fractional sodium excretion, solute concentration, and free water excretion in FW fish (Kenyon et al., 1985). In SW fish, captopril decreases urinary sodium concentration, but because urine formation rate increases, sodium excretion rate does not change (Kenyon et al., 1985). In both FW and SW trout, urine formation rate and GFR are linearly correlated; because this is not affected by captopril, changes in urine production are not attributable to increase in tubular water reabsorption (Kenyon et al., 1985). The ability of captopril to increase GFR in SW trout to FW levels indicates that angiotensin may be an important factor in regulating renal function in SW adaptation of trout (Kenyon et al., 1985). In eels this effect is less clear. Captopril injected into eels prior to abrupt seawater transfer greatly increases the cortisol response (from a 2 x increase without captopril to 10x with captopril), enhances the fall in plasma potassium, but does not affect the increase in plasma sodium (Kenyon et al., 1985). Furthermore,

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captopril injected into either FW or SW adapted eels does not affect plasma sodium or potassium or cortisol in SW eels and only transiently decreases cortisol in FW fish (Kenyon et al., 1985). Aglomerular fish have been used to examine angiotensin responses in the absence of GFR-mediated effects with mixed results. In goosefish, Lophius americanus, ANG I1 increases urine flow rate, urinary "a+], and urinary Na+ and K+ excretion but has no effect on Mg2+ or Ca2+excretion, and it has been proposed that pharmacological doses of ANG I1 inhibit Na+ and K+ reabsorption in the distal segment (Churchill e t al., 1979). However, in later experiments, saralasin ANG I1 [Sar', Val5, Alas] appeared to decrease urine formation and excretion of K+, Mg2+, and Ca2' indicating an inhibition of the effect of physiological levels of ANG I1 on divalent ion secretion (Churchill et al., 1985b). In view of the ineffectiveness of angiotensin antagonists on cardiovascular responses (see previous discussion) and on 1251-ANGI1 [Ala', Val5] binding to glomeruli of FW trout (Brown et al., 1990b), it remains to be determined if saralasin actually inhibited goosefish renal angiotensin mechanisms or was itself agonistic. In contrast, ANG I1 [Am', Val5] has no effect on urine flow or electrolyte excretion in the toadfish, Opsanus tau, even at pressor doses (Zucker and Nishimura, 1981). (iii) Drinking. Kobayashi et al. (1983) observed a dipsogenic response to ANG I1 [Asnl, Val'] in 10 of 19 FW and 6 of 14 SW teleosts and hypothesized that the ability of angiotensin to stimulate drinking in FW fish is correlated with an estuarine habitat or an ability to survive hypersaline water; in SW fish angiotensin mediated drinking is commonly associated with fish inhabiting tide pools or brackish water. Angiotensin I1 responses occurred irrespective of whether the fish were euryhaline or stenohaline (Koyabashi et al., 1983).Kobayashi et al. (1983) also proposed that angiotensins do not usually affect fish inhabiting solely FW or SW. High doses (10-50 pg/kg) of ANG I1 [Asn', Val'] frequently inhibited drinking (Kobayashi et al., 1983), perhaps due to secondary hypertensive effects. Beasley et al. (1986) found that drinking is not stimulated by angiotensins in fish that inhabit only FW, such as goldfish, Crassius auratus, common shiner, Natropis cornutus, and mottled sculpin, Cottus bairdi, whereas in fish that inhabit only SW (e.g., winter flounder, Pseudopleuronectes americanus, and longhorn sculpin, Myoxocephalus octodecemspinosus), ANG I1 increases drinking two- threefold. Additional species need to be examined to further define these relationships. Interestingly, there are some apparent intraspecies differences; ANG I1 [Val5] stimulates

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drinking in a hypersaline tolerant Japanese strain of Carassius auratus (Kobayashi et al., 1983; Okawara and Kobayashi, 1988) but not in hypersaline intolerant C . auratus raised in North America (Beasley et al., 1986). Angiotensin effects on drinking are usually most pronounced in euryhaline fish adapted to seawater. In euryhaline flounder, Platichthyes Pesus, SW adaptation increases drinking, plasma osmolarity, and [Cl-1; ANG I1 [Asp', Val5] infusion, even at hypertensive doses, is a greater stimulus for drinking in SW fish (Balment and Carrick, 1985; Carrick and Balment, 1983). In FW flounders, papaverine lowers blood pressure, plasma osmolarity, and [Cl-] yet increases drinking (Balment and Carrick, 1985). Captopril alone has no affect on drinking in FW fish but prevents the dipsogenic response to papaverine and decreases drinking in SW fish (Balment and Carrick, 1985; Carrick and Balment, 1983). Hypertonic saline (10%) infusion plus papaverine and captopril further increases drinking (Balment and Carrick, 1985). These studies demonstrate the involvement of the RAS in drinking and indicate that hypovolemia may be a potent stimulus for RAS activation during cellular hydration. The fact that cellular dehydration plus hypovolemia produce an augmented response suggests that these effectors act through independent mechanisms. Volume or pressure dependent drinking, and multiple dipsogenic foci have also been shown in Anguilla japonica (Hirano and Hasegawa, 1984; Hirano et al., 1978; Okawara et al., 1987; Takei et al., 1988),and some of these may be independent of systemic angiotensin. Volume expansion and concomitant cellular dehydration produced by infusion of 7 or 14% saline or 65% sucrose increases plasma irANG 11, yet decreases drinking in esophageal cannulated FW or 113 SW eels (Takei et al., 1988). In FW or SW eels, Anguilla japonica, intraarterial ANG I1 [Asn', Val5] stimulates drinking; however, intracerebroventricular ANG I1 [Asn', Val5] is a more potent dipsogen than intraarterial injection (Takei et al., 1979). Removal of the telencephalon, diencephalon, and part of the mesencephalon does not affect ANG II-stimulated drinking, but drinking is inhibited by vagotomy, implicating the medulla as a dipsogenic center (Hirano et al., 1978; Takei et al., 1979). Angiotensin I11 does not have a dipsogenic function in eels (Hirano and Hasegawa, 1984). Okawara et al. (1987) found a transient plasma irANG II-independent increase in drinking in A . japonica during the first 2 h after transfer to SW, whereas over the next 3 days a further increase in drinking was accompanied by an increase in plasma irANG 11. Captopril prevented the delayed increase in drinking at 3

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and 10 days after transfer but did not affect the early response (Okawara et al., 1987), suggestive of multiple dipsogenic mechanisms. Angiotensin converting enzyme inhibitors consistently inhibit ANG I-stimulated drinking in fish (Carrick and Balment, 1983; Hirano and Hasegawa, 1984; Okawara and Kobayashi, 1988). Both ACE inhibition and saralasin decrease drinking by SW killifish, Fundulus heteroclitus (Malvin et al., 1980), and flounder, Platichthysflesus (Carrick and Balment, 1983), whereas neither affect drinking by control or hemorrhaged SW winter flounder, Pseudopleuronectes americanus, or by longhorn sculpin, M. octodecemspinosus (Beasley et al., 1986). Okaware and Kobayashi (1988) found that in goldfish, C . auratus, low doses of captopril (0.4 and 4.0 pg/fish, -0.1-1 mg/kg) stimulated drinking, whereas higher doses were ineffective. The authors (Okawara and Koyabashi, 1988) proposed that during low dose captopril treatment ANG I levels increased, and then as captopril was metabolized proportionally more ANG I1 was formed, thereby stimulating drinking. ( i v ) Other effects. An ability of the RAS to stimulate corticosteroid production in fish has received attention because of the well-known stimulatory response in mammals and because of a somewhat temporal correlation between the RAS and cortisol during salinity adaptation. The results to date are equivocal. With in vitro preparations using interrenal tissue or isolated cells, angiotensins may (Hanke, 1990) or may not (Decourt and Lahlou, 1987; Hanke, 1990; Takahashi et al., 1985; Vetter and Hanke, 1985) stimulate corticosteroid release. However, ANG I, ANG I [Asn', Val5, Asn'l, and ANG I1 [Asp', Ile5] stimulate ACTH release from dispersed goldfish anterior pituitary cells independent of ovine corticotropin releasing factor or urotensin I (Weld and Fryer, 1987, 1988). Angiotensin may also act indirectly on trout interrenal tissue in vitro by interacting synergistically with other secretagogues (Decourt and Lahlou, 1987). The in vivo response is also species specific. Angiotensin I1 [Asp', Val5] injection increases plasma cortisol in 50% SW flounder, Platichthysflesus,as does ANG I1 [Asp', Val5] and renal extracts in intact or hypophysectomized eels, Anguilla anguilla (Henderson et al., 1976). However, SW adaptation only transiently (24-48 h) increases plasma cortisol in eels, whereas PRA increases slowly and steadily over 3-5 days (Henderson et al., 1976) and ACE inhibition does not diminish the cortisol response (Kenyon et al., 1985). Furthermore, immunoreactive plasma aldosterone in largescale suckers, Catostomus macrocheilus, is not affected by injection of homologous renin extracts or ANG I1 but does increase in response to hemorrhage (Reinking, 1983).

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IV. KALLIKREIN-KININ SYSTEM A. Components of the Kallikrein-IGnin System

The kallikrein-kinin system is the physiological enantiomer of the RAS (Fig. 8). In its simplest form, the enzyme kallikrein cleaves a biologically active peptide (kinin) from an inactive precursor (kininogen). Kinins are inactivated by a variety of peptidases, most notably ACE. (The latter was originally thought to be a separate enzyme, kininase 11.) In mammals, the KKS is divided into plasma and glandular forms based on the location of kallikrein and kininogen (Nustad et al., 1980; Schachter, 1980). T h e plasma KKS consists of a circulating inactive “prekallikrein” and kininogen, a high molecular weight kinintransporting a-globulin. Upon activation of kallikrein by Hageman factor, or other stimuli, the nonapeptide bradykinin is cleaved from kininogen. Glandular KKS is found in the pancreas, salivary glands, and kidney. Glandular kallikrein cleaves the pentapeptide lysylbradykinin (kallidin) from a low molecular weight tissue kininogen, although it can also form kinins from plasma kininogens (Rabito et al., 1972; Claeson et al., 1978). The physiological significance of the KKS in mammals is unclear. The KKS has been shown to have potent effects on renal salt and water

.-+I

RAS Angiotensinogen

KKS

--r-’l-... Kininogens

Kallidin (dve)

Angiote&I

Bradyldnin

C i v e )

1

-4

Angiotensin II

(active)

AngiOtensin

converting Enzyme

+-

(Kininase II)

1

Metabolites (inactive)

Fig. 8. Activation steps in the renin-angiotensin (RAS) and kallikrein-kinin (KKS) systems. See text for details.

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balance in the kidney (Margolius, 1984; Scicli and Carretero, 1986). Kinins are among the most potent vasodilators known, and many of their functions are undoubtedly related to this vasorelaxant activity. Kinins have potent effects on capillary permeability and are often involved in inflammatory responses; in pathological situations, they may produce considerable fluid loss from the vasculature (Margolius, 1989; Nustad et al., 1980; Sander and Huggens, 1972; Schacter, 1980). A complete KKS has also been identified in the brain, and kinins appear to be involved in the central control of the cardiovascular system (Diz, 1985).

B. Occurrence and Distribution in Fish Evidence for a complete KKS in fish should include the presence of (a) a kinin precursor; (b) kallikrein-like enzymatic activity; (c) kinin receptors, which when stimulated mediate a physiological response;

and (d) a mechanism for inactivating kinins. Kinin-like substances were identified in neural tissue from carp as early as 1961 (Inouye et al., 1961). However, subsequent reports, indicating that the KKS is absent in anamniotic vertebrates (Rabito et al., 1972; Seki et al., 1973), did much to quell enthusiasm for further study. Surprisingly, the demonstration of the ability of mammalian glandular kallikrein to produce a kinin-like substance from rainbow trout plasma (Dunn and Perks, 1975) did little to promote further investigation. In an examination of the phylogenetic expression of ACE, Lipke and Olson (1988) observed that this enzyme is found in virtually all vertebrate classes and thereby may predate the supposed origin of the RAS. They (Lipke and Olson, 1988) hypothesized that perhaps ACE was originally important in other, non-RAS, proteolytic functions. Because the Michaelis-Menten constant (K,) for bradykinin is lower than for ANG I (8 x M versus 3.3 x lop5M , respectively; Ryan, 1983), the KKS seemed to be a possible candidate. This formed the basis for the reexamination of the KKS in the rainbow trout (see next section) and, although these studies are far from definitive, they provide a general framework for future investigations. 1. EVIDENCE FOR KININOGEN IN FISH

In vivo and in vitro studies indicate that a biologically active product can be generated from trout plasma by porcine glandular kallikrein (GK; Dunn and Perks, 1975; Lipke and Olson, 1990; Lipke et al., 1990a). Injection of GK into intact trout pretreated with the ACE inhibitor captopril increases dorsal aortic blood pressure, whereas in

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untreated trout, GK has no effect (Lipke and Olson, 1990). This indicates that the pressor substance generated by GK is rapidly inactivated by endogenous ACE, consistent with the kininolytic function of the latter. Incubation of trout plasma for 1 h with GK likewise generates a product that is pressor in intact trout (Lipke et al., 1980a). This product, termed T60K (trout plasma incubated 60 min with kallikrein) is a heat stable, low molecular weight (<10,000 kDa), peptide (Lipke et al., 1990a). T60K is either very potent or trout plasma contains substantial quantities of precursor, because as little as 10-50 p l of activated plasma has pressor activity when injected into 300-g trout or rats (Lipke et al., 1990a). It is not clear whether T60K is a kinin- or angiotensin-like molecule as it has properties of both peptides yet it is not identical to either. T60K is similar to angiotensin in that it is pressor in both trout and rats and that the response in trout is sensitive a-adrenergic blockade, whereas, in rats, the effect is blocked by the angiotensin receptor agonist saralasin [Sar', Alas] ANG 11. T60K also competes with mam1990a). malian ANG I1 in homogenized rat adrenal tissue (Lipke e t d., T60K is dissimilar to angiotensin in that (a) it appears to be inactivated by ACE in uiuo, (b)it contracts isolated trout arteries that are refractory to angiotensions, (c)the time course for contraction of rabbit arteries is much longer for T60K than ANG 11, and (d) T60K is not detected in a radioimmunoassay that is 70 and 100% cross reactive to trout ANG I and ANG 11, respectively. T60K is similar to bradykinin in that both are pressor in trout and both are inactivated by the arterioarterial pathway of the isolated, perfused gill (Lipke et al., 1990a,b). Dissimilarities between T60K and bradykinin include the pressor effect of T60K in rats and sensitivity of the T60K, but not bradykinin, pressor response to a-adrenoceptor blockade in trout.

2. EVIDENCE FOR KALLIKREIN The limited information available suggests that kallikrein is present in trout tissues, although definitive studies have yet to be performed. Results from two spectrophotometric assays, based on the hydrolytic activity of kallikrein on synthetic substrate, indicate that kallikrein-like activity is present in trout gill and kidney, but not in liver, muscle, or plasma (Lipke and Olson, 1990). However, these results could not be confirmed with a third method that employed a different substrate (Lipke and Olson, 1990). Further examination of gill and kidney tissue, in the presence of a specific mammalian kallikrein inhibitor, Phe-Phe-Arg-chloromethylketone (PPAMCK), showed that a fraction of the gill estrolytic activity was indeed due to a

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kallikrein-like enzyme (Lipke and Olson, 1992). PPAMCK did not affect kidney estrolytic activity (Lipke and Olson, 1992). Other kallikreinlike enzymes, undetectable by these methods, may also be present in fish as, even in mammals, there are a large number of genes that encode kallikrein (MacDonald et al., 1988).

3. KININ METABOLISM Angiotensin converting enzyme, present in many fish tissues, especially gills, may be important in kinin inactivation, although other peptidase are undoubtedly involved as well. In the mammalian lung, ACE is located in the lumenal surface of the pulmonary endothelium, and there it efficiently (-80%) inactivates kinins and effectively prevents these vasodilators from entering the systemic circulation. Inactivation of bradykinin by the gill is less efficient (-20-40%) and appears to involve incorporation of the peptide into the endothelial/ pillar cell prior to metabolism (Lipke et al., 1990b). Because kinins are not hypotensive in fish (see later), there is apparently no immediate need for their rapid inactivation. The comparatively low rate of kinin inactivation by the gill may indicate that circulating kinins have systemic functions in these vertebrates. Interestingly, as noted above, gills and lungs are similar in their ability to activate angiotensins (Olson et al., 1986).

C. Effects of Kinins Unlike the potent depressor action of kinins in mammals, bradykinin and other kinin analogs are pressors in rainbow trout (Lipke et al., 1990b),and bradykinin is a pressor in the eel, Anguilla japonica (Chan and Chow, 1976). In trout, bradykinin also fails to relax endotheliumintact arterial and venous rings or to lower resistance in perfused organs (Olson and Villa, 1991). Similar results are obtained with acetylcholine. These results are taken to indicate that trout, and perhaps other fish as well, lack nonprostanoid endothelium derived relaxing factors (EDRF; Olson and Villa, 1991). The pressor effects of bradykinin in vivo are unaffected by a-adrenoceptor blockade and are therefore unlike angiotensin in this regard (Lipke et al., 199Ob). Identification of the physiological function(s) of the KKS awaits further characterization of this system in fish tissues and the development of suitable antagonists to block endogenous KKS activity. The

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apparently high level of circulating kallikrein substrate in trout plasma (Lipke and Olson, 1990; Lipke et al., 1990a) suggests a systemic function of the KKS (see previous discussion). Yet injection of mammalian kallikrein inhibitor, PPAMCK, has no overt effects on trout blood pressure in vivo (Lipke and Olson, 1992) and a circulating kallikrein has not been detected, both of which argue against the KKS as a tonic pressor system. Certainly, local regulation (or remote regulation through a central nervous system KKS) is possible and the effects ofthe KKS on salt and water balance, which have yet to be measured, could exert a powerful, but less immediately obvious, role in cardiovascular homeostasis. V. ATRIAL NATRIURETIC PEPTIDES In the early 1 9 8 0 d~e~Bold and co-workers demonstrated that peptides with diverse biological activity were synthesized and released by mammalian cardiac tissues (de Bold et al., 1981; d e Bold, 1982). Since then the genetic code and biochemical processing from the 151 or 152 amino acid (human or rat) preprohormone through 126-128 residue prohormone to an active 28-residue peptide has been well characterized, as have a number of other important peptide variants (see reviews: Brenner et al., 1990; Needleman et al., 1989; summaries edited by Needleman, 1988; Samson and Quirion, 1990). Three families of naturetic peptides (NP), A-, B-, and C-type (ANP, BNP, and CNP, respectively), have been identified so far, and more are possible. These peptides were originally named after the tissues from which they were first isolated (e.g., atrium [ANP] and brain [BNP]). Most peptides have since been found in multiple sites and, in fact, may be more prevalent in tissues other than those in which they were first identified. In spite of the intense activity focused on NP, and the variety of pharmacological responses examined, there is as yet little consensus regarding their physiological role (cf. Blaine, 1990; Goetz, 1990). In fish the situation is exacerbated by the great physiological diversity of these vertebrates, a limited appreciation of their homeostatic attributes, and, until recently, the lack of native peptides with which to study them. In spite of these limitations a growing amount of evidence indicates that these peptides are ubiquitous among fish, and they have somewhat stereotypic effects. Here too, however, the challenge remains to delineate their physiological function.

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A. Structure of Natriuretic Peptides Figure 9 compares the amino acid sequence of rat ANP and fish NPs. By convention, the lowercase letter preceding the peptide family indicates the animal from which the peptide was isolated: h, human; p, porcine; r, rat; e, eel, Anguilla japonica; k, killifish, Fundulus heteroclitus; and s, shark, Scyliorhinus canicula. The obvious confusion generated by this practice will, undoubtedly, limit its value in the future as more species are examined. Sequence homology of eANP to rat, fowl, and frog ANP is 59, 52, and 46%, respectively (Takei, et al., 1989); eCNP is 95 and 86% homologous to kCNP and pCNP, respectively (Takei, 1990a). sCNP is 82% homologous to rCNP (Suzuki et al., 1991). Residues between the cysteine disulfide bridge of all peptide families are highly conserved, this is especially true for fish. Three NP receptors have been identified in mammals. The ANPRA and ANPR-B (also known collectively as B-ANP receptors, B denoting "biologically active") is a 130-kDa transmembrane protein. ANPRA receptors are stimulated by ANP and to a lesser extent by BNP; CNP is the most efficacious agonist of ANPR-B receptors (Koller et al., 1991). The intracellular segments of both ANPR-A and ANPR-B receptors contains a guanylate cyclase tail through which physiological activity is conferred (Brenner et al., 1990; Needleman et al., 1989). The ANPR-C receptor (also known as the C-ANP receptor; C denoting "clearance," but confusing relative to the active CNP peptide) is a -60-kDa protein that lacks the guanylate cyclase tail and has been proposed to clear NPs from the circulation and thus provide a non-

Fig. 9. Amino acid sequence for atrial natriuretic peptide family. Diagram shows full sequence for rat ANP plus substitutions for shark CNP ((sCNP)),killifish CNP [kCNP], eel CNP {eCNP}, and eel ANP (eANP). * denotes amino acid deletion; + denotes amino acid insertion. References: a, Suzuki et al. (1991);b, Price et al. (1990); C, Takei et al. (1990a);d, Takei et al. (1989); e, Brenner et al. (1990).

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proteolytic pathway for NP inactivation (Maack et al., 1987). The ring heptapeptide residues 10-16 in Fig. 9 are essential for ANPR-C receptor binding. ANPR-C receptors are otherwise relatively insensitive to residue deletion or substitution (Bovy, 1990). On the other hand, ANPR-A and ANPR-B receptors are quite sensitive to the structural integrity of the peptide; minor substitutions variously affect vasodilator or natriuretic responses in different tissues (Bovy, 1990). Substitutions in the eANP undoubtedly account for the 110-fold increase in potency of this peptide compared to mammalian peptides when injected into eels (Takei et al., 1989) and the decrease in potency of eANP in trout (Olson and Duff, 1992),toadfish (Evans, 1991),and quail or rat tissues (Takei et al., 1989,199Oa,b).sCNP is a considerably more potent agonist of rectal gland secretion than ANP-related peptides or nonshark CNPs, and the amino acid at position 4 on sCNP appears functionally important (Solomon et al., 1992). A concomitant interspecific selectivity of NP receptors is also implied by these studies. Natriuretic peptides can be removed from the circulation by ANPRC receptors prior to proteolysis or by receptor independent proteolysis through vascular and tissue peptidases. One of the most significant enzymes in this regard is the neutral endopeptidase E C 24.11 ( E C 3.4.24.11; Erdos and Skidgel, 1989). One, or both, ofthese inactivation mechanisms appear operative in trout. Simultaneous injection of a competitive ANPR-C receptor inhibitor (SC-46542) and an inhibitor of neutral endopeptidase (phosphoramidon) into trout produces diuresis, saliuresis, and decreases pulse pressure, consistent with elevation of endogenous NP (Duff and Olson, 1992). 1251-ratANP(lZ5I-rANP)extraction by the perfused gill is also reduced by ANPR-C receptor inhibition (Olson and Duff, submitted). These results indicate that the mechanisms of NP inactivation in fish are qualitatively similar to those found in mammals. The observation that eANP effects last longer than hANP in eels, quail, and rats (Takei et al., 1989) may indicate quantitative differences in peptide inactivation mechanisms.

B. Distribution in Fish Atrial and often ventricular cardiocytes of virtually all fish examined contain -0.2- to 0.4-pm-diameter dense core granules, somewhat smaller in size and fewer in number, but otherwise similar to ANP secretory granules found in mammalian cardiocytes (Chapeau et al., 1985; Hirohama et al., 1988;Reinecke et al., 1985,1987a,b; Solomon et al., 1985a; Uemura et al., 1990; Westenfelder et al., 198813).Additional evidence for ANP distribution in fish has been obtained largely

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KENNETH R. OLSON

through immunohistochemical and radioimmunoassay techniques using antibodies to mammalian ANP, and through bioassay of fish heart extracts. To date, natriuretic peptides have been isolated from only one elasmobranch and two teleosts (Fig. 9; Price et al., 1990; Suzuki et al., 1991;Takei et al., 1989,1990a). A summary ofANP levels in fish tissues and plasma is provided in Table XII. 1. CYCLOSTOMES Immunoreactive ANP (ir-ANP) granules are distributed throughout atrial tissue of Myxine glutinosa (Reinecke et al., 1987a) and atrial subepicardium of Eptatretus burgeri (Uemura et al., 1990). Immunoreactive ANP is rare in M . glutinosa and absent in E. burgeri ventricles but very prevalent in the portal vein heart of M . glutinosa (Reinecke et al., 1987a). Immunoreactive ANP has also been demonstrated in various regions of M . glutinosa brain, including primordium hippocampi, pars ventralis thalami, hypothalamus, medulla, and spinal cord (mainly somato motor tracts; Reinecke et al., 1987a). lZ5I-rANP binding has been identified in myxine glomerular arterioles, capsular epithelia, neck cells, and smooth muscle of the archinephric duct as well as aortic endothelium and smooth muscle (Kloas et al., 1988). Myxine heart extracts also relax mammalian arteries (Reinecke et al., 1987b). Although granules are present in lamprey, Lampetra japonica, hearts, they do not bind antibody directed against hANP (Uemura et al., 1990). 2. ELASMOBRANCHS Immunoreactive ANP has been demonstrated in hearts of Chimera monstrosa, Squalus acanthias, Scyliorhinus canicula, Raja clavala, and Triakis scyllia (Hirohama et al., 1988; Reinecke et al., 1987b; Uemura et al., 1990)but not in the ray, Narkejaponica (Uemura et al., 1990). Plasma concentrations of ir-ANP in chondrichthyes are equivalent to those found in osteichthyes (Table XII). A high-molecularweight (115 residue) C-type (presumably prohormone) has been isolated from the heart of the shark, Scyliorhinus canicula (Suzuki et al., 1991). The putatively active sCNP fragment is shown in Fig. 9. This study (Suzuki et al., 1991) is noteworthy in two regards; it demonstrates that the peptide ring is highly conserved phylogenetically, and it is also the first instance in which a CNP has been isolated from tissue other than brain. 3 . OSTEICHTHYES

Immunohistochemical and immunoassay techniques have demonstrated ir-ANP in hearts of nearly all teleosts thus far examined (Galli et

3.

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22 1

al., 1988; Chapeau et al., 1985; Hirohama et al., 1988; Kim et al., 1989; Reinecke et al., 1985, 198713; Uemura et al., 1990); only rarely is ir-ANP absent (Chapeau et al., 1985; Hirohama et al., 1988).Ventricular concentrations of ANP are usually lower than those in the atria, although in some species such as Zauo platypus and Pelteobagrus fulvidraco ventricular ir-ANP concentrations are similar (Kim et al., 1989), and in Oncorhynchus mykiss and Opsanus beta ventricular extracts have equal or greater potency in bioassay experiments (Duff and Olson, 1986; Evans et al., 1989). Considering the relative mass of the two cardiac chambers, it is probable that the ventricle contributes significantly to circulating ANP in some fish. Similar ir-ANP secretory rates by primary cultures of Gila atraria atria and ventricles, 3.9 and 2.8 ng/105 cells, respectively (Baranowski and Westenfelder, 1989), support this contention. Natriuretic peptides have been purified from eel, Anguilla japonica, heart (eANP; Takei et al., 1989) and brain (eBNP, Takei et al., 1990a) and killifish, Fundulus heteroclitus, brain (kBNP; Price et al., 1990).Truncation of both eBNP and kBNP at the carboxy terminus of the ring (Fig. 9) indicates that these peptides are C- rather than B-type, and eCNP and kCNP notation will be followed in the present discussion.

C. Physiological Significance of Natriuretic Peptides Inferences regarding the physiological significance of NP in fish are based on two types of experiments: (a) those in which circulating and tissue NP concentrations (more appropriately ANP because most antibodies used to date are directed against this peptide) are correlated with physiological perturbation, and (b)those that examine the physiological responses to NP administration. Many of these maneuvers directly or indirectly affect ion and volume regulatory processes and the associative link between NPs and volume regulation in fish is probably more than coincidental. 1. FACTORS AFFECTINGPLASMA OR TISSUE NP (ANP) a. Salinity. Depending on species, environmental salinity may have either no effect on, or it may be directly correlated with, ANP synthesis and secretion. In an elasmobranch and several teleosts there is no apparent correlation between salinity and ANP. Abrupt transfer or 24-h adaptation of the dogfish shark, Squalus acanthias, to 70%

Table XI1 Distribution of ANP-like Immiinoactivity (IA) in Fish Tissue and Plasma Species

a.t

Environment

Tissue

IA

Antibody

Reference

Cyclostomes Myxine glutinosa Eptatretus burgeri

sw sw

Plasma* Heart a/v Plasma*

187 0.3610.01 38.7

hANP hANP

Evans et al. (1989) Uemura e t nl. (1990)

Chondrichthyes Narke juponicu

SW

Uemura et al. (1990)

SW

0.3310.01 21.5 0.0210.01 2.7 208 129 60

hANP

Triakis scyllia

Heart alv Plasma* Heart alv Plasma* Plasma* P 1asm a * Plasma* Plasma*

154 127 0.081ND 5.210.04 45.7 44.612.6 90.5 197

Dosyatis sabina Squalus acanthias Squalus acanthias Osteichthyes Anguilla rostrata

SW SW SW

Anguilla japonicu Anguilla japonica

SW FW FW FW

Cyprinus carpio

FW

Gila atruria

FW anesth

Oncorhynchus mykiss

Awake Pithed FW 1%NaCI FW

Heart a h Heart a1v Plasma Heart aiv Plasma Plasma

Plasma Plasma

218 207 146 347 184

hANP Evans et al. (1989)

hANP hANP hANP

Epstein et al. (1988)

hANP

Epstein et al. (1989)

AP 111 hANP hANP hANP hANP hANP

Kim et al. (1989) Uemura et al. (1990)

hANP

Westenfelder e t al. (198813)

Westenfelder et ul. (1988a)

1

Oncorhynchus mykiss

FW

Levomis macrochirus

FW

Channa maculata

FW

Pelteobagrus fuluidraco Zauo platypus Mugil cephalus

FW FW

M yoxocephalus octodecimspinosus Pseudo pleuronectes americanus Opsanus beta

>

(1990)

Heart a/v Plasma* Heart a/v Plasma*

0.3510.01 8.9 0.23/0.05 31.0

hANP hANP hANP hANP

0.29/0.03 70.3 0.1610.21 0.2110.47 33 16 5 102

hANP hANP AP 111 AP I11 1-28 ANP

Galli et al. (1988)

50% SW, 7d FW,7d SW

Heart a h Plasma* Heart a h Heart a/v Plasma Plasma Plasma P 1asm a *

hANP

Evans et al. (1989)

SW

Plasma*

sw

Plasma Brain Heart Plasma Brain Heart Heart a/v P 1asm a * Heart a/v Plasma* Heart a/v Plasma* Heart aiv Plasma* Heart a/v Plasma*

sw

5% sw Conger myriaster

SW

Oplegnathus fasciatus

SW

Pagrus major

SW

Trachurusjaponicus

SW

Hexagrammos otakii

SW

30 61 47 49 23 20 29 47.3/7.03 69.1 0.53/0.02 45.5 0.05/0.01 11.3 0.04/0.01 11.1 0.08/0.01 13.2

Uemura et

(11.

Kim et al. (1989)

hANP Epstein et al. (1989)

hANP hANP hANP hANP hANP hANP hANP hANP hANP hANP

Uemura et al. (1990)

Abbreviations: a, atrium; v, ventricle; APIII, atriopeptin 111 (1-26); hANP, human (1-28ANP); *, extracted plasma. Plasma concentrations in pg/ml; tissue concentrations in ng/g.

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KENNETH R. OLSON

seawater does not change plasma ir-ANP but enhances the renal response to exogenous ANP (Solomon et al., 1988). Long-term adaptation or rapid transfer of eels, Anguilla rostrata, or rainbow trout between FW and SW does not affect plasma ir-ANP (Duff and Olson, 1990a; Epstein et al., 1989; Y. Takei, unpublished observation). Similarly, Uemura et al. (1990) did not find a significant difference in plasma ir-ANP when they examined five species of FW and five species of SW teleosts. However, it should be noted that caution must b e exercised when interpreting “no response” results until these studies are confirmed with assays using antibodies directed against homologous ANP. This is underscored by the findings of Epstein et al. (1988) in which only one of four commercial radioimmunoassay kits (two anti-rANP and two anti-hANP) indicated a change in plasma ANP in volume expanded sharks. In other teleosts there is a direct correlation between salinity and ANP secretion (Table XII). Evans et al. (1989) observed that 1 week after transfer from seawater to dilute media (-200 mOsm), circulating ir-ANP is reduced by 90% in longhorn sculpin, Myoxocephalus octodecimspinosus, and winter flounder, Pseudopleuronectes americanus. Plasma ir-ANP is also lower in toadfish, Opsanus beta, mullet, Mugil cephalus, and catfish adapted to dilute media; mullet heart and brain ir-ANP concentrations decline as well (Galli et al., 1988). In Gila atraria, a fish with somewhat limited osmoregulatory capability, an increase in medium osmolarity from 106 to 491 mOsmol/kg increases plasma ir-ANP from 213 to 691 pg/ml and increases plasma osmolarity from 272 to 486 mOsmol/kg; plasma sodium is highly correlated with ANP (Westenfelder et al., 1988b). Uemura et al. (1990) observed that cytochemical immunoreactivity in cardiocytes is greater in FW than SW teleosts and claimed (as an unpublished observation) that FW to SW transfer stimulates ANP synthesis in eel, Anguilla japonica.

b. Volume Expansion. The effect of volume expansion on ANP secretion by fish has received scant attention despite the fact that an increase in central blood volume is thought to be the primary stimulus for ANP secretion in mammals. In the dogfish shark, S . acanthias, volume expansion with shark Ringer increases rectal gland volume excretion and nearly doubles plasma ir-ANP (Epstein et al., 1988). Because the shark rectal gland is involved in regulating intravascular volume rather than osmoregulation (Solomon et al., 1985b), and volume expansion (but not salinity transfer) is a stimulus for ANP secretion, a strong case can be made for ANP as a volume regulating hormone in elasmobranchs.

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The effects of volume expansion on ANP secretion in teleosts are not clear. In a preliminary report Duff and Olson (1990a) found that trout plasma ir-ANP is unaffected by volume expansion with saline, albuminated saline, or steelhead plasma. Whether trout are unresponsive to volume expansion or whether the antibody used (hANP) is insensitive to trout NPs is not known. However, Westenfelder et al. (1988a,b) reported that trout ANP is immunologically similar to hANP. Thus, the study by Duff and Olson (1990a) suggests that trout lack ANP-mediated volume regulatory responses. Additional work with trout and other teleosts is clearly needed to resolve this issue. Volume, electrolyte load, or both could stimulate salinity-mediated ANP responses. There is clearly a correlative relationship between plasma ir-ANP and plasma electrolytes, suggestive of ANP’s role in osmoregulation (summarized by Evans, 1990). Whether the ANP response is directed toward ionic or volume imbalance is unknown. Evans (1990) argued against volume stimulation as a factor because of the hypovolemic condition of marine teleosts. However, as previously indicated in this chapter, SW teleosts may not be hypovolemic relative to their FW counterparts. An even better case can be made for ANP as a volume regulating hormone in hagfish and elasmobranchs as both fish are slightly hyperosmotic to their environment and they may continually have to deal with volume expansion. If this is the case then the lineage of ANP and volume regulation is indeed ancient and persistent, perhaps explaining the conservatism between structure and function of this peptide in vertebrates.

2. PHYSIOLOGICAL EFFECTS a . Fluid Compartments. Continuous infusion of trout with 300 ng/kg min-’ rANP [Ile26](versus an equal rate of saline infusion) for 8 h lowers blood volume (51Cr red cell method) from 29.8 2 1.9 to 22.9 0.97 ml/kg and ECFV (“Co-EDTA method) from 244 t 11 to 156 2 10 ml/kg (D. W. Duff and K. R. Olson, unpublished). The reduction in extracellular fluid volume (36%) is greater than the reduction in blood volume (23%), indicative of reabsorption of interstitial fluid to maintain plasma volume. There have not been any other reports of ANP on fluid compartments in fish.

*

b. Cardiovascular Effects in Vivo. Hypotension is the most common response to ANP injection into intact fish. Atriopeptin I1 (AP 11)is depressor in Squalus acanthias (Benyajati and Yokota, 1990; Solomon

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KENNETH R. OLSON

et al., 1988).In eels, Anguilla japonica, a-rANP, a-hANP, a-hANP(5,25), eANP and BNP, and eel heart extracts all lower arterial pressure (Takei et al., 1989, 1990a,b). Similar responses are found in cod, Gadus morhua, to a-rANP (Acierno et al., 1991) and toadfish, Opsanus tau, to homologous heart extracts (Lee and Malvin, 1987). hANP (10 pg/kg) produces only a modest drop in blood pressure (1-2 mmHg) in the flounder, Pleuronectes platessa, although the hypotension persists for several hours (Arnold-Reed et al., 1991). Atriopeptin 111(AP 111)may lack cardiovascular effects in fish even though it is less truncated than the depressor AP I1 (AP 11 lacks the C-terminal tyrosine of AP 111).In both shark (Solomon et al., 1988)and toadfish (Lee and Malvin, 1987),AP 111has no effect on blood pressure even though substantial effects on renal function are observed in both species. It is not clear if this insensitivity to AP I11 is a general feature of piscine cardiovascular systems. Human cardiodilantin 1-16 (the 1-16 N-terminal residues of pro-ANP) is also without effect in cod (Acierno et al., 1991). Intraarterial injection of ANP into trout may produce a biphasic pressor-depressor response (Duff and Olson, 1986), no response (Eddy et al., 1990), or a depressor response (Olson and Duff, 1992). The variability of this response is probably attributable to the rate of ANP administration. Injection of 10 pglkg of either rANP[IleZ6] or eANP as a single bolus produces a pressor-depressor response (Duff and Olson, 1986,1990b). If injected over 10 min, 10 pg/kg a-hANP has no effect on blood pressure in either control trout or trout fed a high salt diet (Eddy et al., 1990). Constant infusion of rANP[Ilez61 (18 pg/ hekg-') produces a steady decline in blood pressure (Olson and Duff, 1992).The reason for this variability is not known, although it probably is related to the different plasma concentrations of the peptide following injection and the resultant titers of ANP at different receptor/ effector sites. The implications from these studies are that there are multiple foci for ANP action in fish and that perhaps some of these are dependent on extravascular sources of the peptide for their activation. The ability of a-adrenergic antagonists to inhibit the rANP[Ile26]mediated pressor response in trout (Duff and Olson, 1990b; Olson and Duff, 1992) suggests that the sympathetic nervous system is involved. Both the nonspecific a-receptor antagonist, phenoxybenzamine, and the specific al-receptor antagonist, prazosine, prevent the pressor response; in fact, the former unmasks an ANP-mediated drop in arterial pressure (Duff and Olson, 1990b; Olson and Duff, 1992). Both antagonists prevent ANP diuresis and saliuresis.

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c. Cardiovascular Effects in Vitro:Perfused Tissues. Studies of the effects of ANP on perfused tissues are limited to three species. ANP vasodilates tissues precontracted with either carbachol, such as the isolated head of Opsanus beta (Evans et al., 1989), or with epinephrine, such as gill, muscle-kidney, and splanchnic bed of trout, 0. mykiss (Olson and Meisheri, 1989). Tissues not precontracted, such as trout muscle-kidney and splanchnic (Olson and Meisheri, 1989) and Squalus acanthias rectal gland (Solomon et al., 1985a), do not respond to ANP. ANP-mediated vasodilation of the trout gill occurs even if the gill vessels are not precontracted (Olson and Meisheri, 1989) and favors perfusion of the arterioarteriolar (lamellar) pathway. The effective ANP concentration producing half maximal response (ECJO)in the perfused toadfish head is 3 x lO-'M (Evans et al., 1989). ECsos have not been reported for other tissues. This ECso is similar to those reported for isolated vessels which indicates that the microcirculation of the head is neither more or less sensitive to ANP than are large vessels.

d . Cardiovascular Effects in Vitro: Isolated Vessels. Atrial peptides relax all isolated fish vessels examined to date including ventral aortas from agnatha, elasmobranchs, and teleosts (Fig. 10).Relaxation

I

I

Fig. 10. Dose response curves for ANP-mediated relaxation ofhagfish, M . glutinosa, ventral aorta (B; Evans, 1991), shark, S. acanthias, ventral aorta ( 0 ; Evans, 1991), and trout, 0. mykiss, celiacomesenteric artery (X;Olson and Meisheri, 1989). Approximate concentrations for half-maximal response (EC,) are hagfish, 4 x lo-”; shark, 7 x lo-’; and trout, 1.5 x lo-’.

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KENNETH R. OLSON

can be demonstrated in unstimulated vessels (Evans et al., 1989;Olson and Meisheri, 1989)and in vessels stimulated with a variety of agonists such as carbachol (Solomon et al., 1985a),acetylcholine, arginine vasopressin, epinephrine, norepinephrine, serotonin, and a thromboxane A2 agonist (Olson and Meisheri, 1989). With relatively few exceptions the ECsos for relaxation by mammalian ANPs are in the 1-10 nanomolar range; these concentrations are two orders of magnitude greater than plasma ir-ANP levels. However, the potency of homologous ANP may be considerably greater, as it is in the eel in vivo (Takei et al., 1989, 1990a) and perfused shark rectal gland (Solomon et al., 1992), and more in line with circulating titers. Nearly all isolated vessel studies have used rings from ventral aortas and in some species (e.g., dogfish shark and trout) these vessels are refractory to common agonists, such as catecholamines (Olson and Villa, 1991; Solomon et al., 1985a). Trout celiacomesenteric and epibranchial arteries demonstrate sensitivity to other agonists including catecholamines and these vessels are relaxed by ANP independent of agonist type (Olson and Meisheri, 1989).Additional studies on other postgill vessels and vessels from a variety of species are needed to better interpret the systemic effects of these peptides. ANP also relaxes trout anterior cardinal veins (Olson et al., 1991). Thus, some of the depressor response to ANP in vivo could be mediated through changes in vascular filling pressure and venous return. Evans et al. (1989) found that ventral aorta rings from toadfish adapted to 5% seawater are more sensitive to rat (101-126) ANP than rings from seawater adapted toadfish (EC50 of 3 x lo-'' and 4 X lo-' M , respectively). Evans et al. (1989) proposed that this is likely due to an up-regulation of ANP receptors secondary to the fall in circulating ANP titers. The physiological significance of this response is unclear as ANP sensitivity of the perfused toadfish head does not change with salinity adaptation (Evans et al., 1989). e. Cardiac Effects. In vivo, rANP increases heart rate in cod, Gadus morhua, and trout (Acierno et al., 1991; Olson and Duff, 1992), whereas neither heart extract nor AP I11 affects heart rate in Opsanus tau (Lee and Malvin, 1987). hANP has no effect on blood pressure but profoundly reduces pulse pressure when slowly (10 min) injected into normal trout but not fish fed a high salt diet (Eddy et al., 1990),an effect attributed to an ANP-mediated decrease in gill vascular resistance. Continuous infusion of rANP [IleZ6]into trout also reduces pulse pressure; however, this appears to be associated with an increase in heart rate; both effects precede, and appear to be temporally dissociated

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from, the depressor response (Olson and Duff, 1992). ANP-induced tachycardia is partially inhibited by propranolol in trout (Olson and Duff, 1992) and by atropine in cod (Acierno et al., 1991) suggesting a reflexogenic response. Neither eANP nor rANP affect heart rate or cardiac power output in the denervated, spontaneously rhythmic, in situ perfused trout heart (J. Keen, H. Thoranensen, A. P. Farrell, and K. R. Olson, unpublished observation). Thus the changes in pulse pressure and heart rate observed in fish in vivo probably result from extracardiac events.

f . Renal Effects. The effects of ANP on renal function in cyclostomes have not been examined. In the elasmobranch, Squalus acanthias, AP I1 and A P I11 reduce GFR, urine flow, and sodium, chloride, and osmolar excretion b y 40-50% (Benyajati and Yokota, 1990; Solomon et al., 1988).Potassium excretion is reduced by AP I1 but not AP I11 (Benyajati and Yokota, 1990). The renal response appears to b e mediated primarily through changes in GFR because fractional electrolyte excretion is unaffected. Renal responses are also independent of systemic cardiovascular effects; inhibition of renal function temporally lags behind, and persists several hours after, the depressor effect of AP I1 and occurs without a concomitant cardiovascular response to AP I11 (Benyajati and Yokota, 1990). If sharks are placed in 70% SW, urine formation rate increases nearly six times and chloride excretion triples (Solomon et al., 1988); in 90% SW, GFR and urine formation increases nearly twofold (Benyajati and Yokota, 1988). Under these circumstances AP I1 or I11 increases urine formation and GFR and has a synergistic stimulatory effect with norepinephrine (Benyajati and Yokota, 1989; Solomon et al., 1988; Yokota and Benyajati, 1988). ANP-mediated diuresis and saliuresis are found in teleosts with (trout) or without (toadfish, Opsanus tau) glomeruli. In trout, rANP [Ilez61increases urine formation and electrolyte excretion irrespective of a hypertensive (Duff and Olson, 1986) or hypotensive (Olson and Duff, 1992) cardiovascular response. rANP [Ile2"] increases urinary excretion of sodium and chloride three to four times, whereas urine formation and potassium excretion are doubled (Duff and Olson, 1986). This suggests that both GFR and fractional excretion of sodium and chloride contribute to the response. Homologous heart extracts and AP I11 increase urine formation and sodium excretion in the aglomerular toadfish but do not affect potassium or divalent cation excretion (Lee and Malvin, 1987), indicative of a direct stimulatory effect of ANPs on renal tubular cells, although local changes in renal perfusion cannot be

230

KENNETH R. OLSON

excluded. A potassium-sparing effect of heart extracts and eANP or BNP is also observed when fish extracts or peptides are injected into mammals (Takei et al., 1989,1990a; Westenfelder et al., 198813);in fact, high doses of eel peptides may be antikaliuretic (Takei et al., 1989, 1990a). g. ANP Effects on Other Zonoregulatory Organs. Although information is quite limited, there is some evidence to indicate that ANP affects several nonrenal volume or osmoregulatory organs. ANP acts as a hypovolemic hormone in the shark rectal gland. Synthetic ANP and shark heart extracts increase rectal gland chloride and volume excretion (Solomon et al., 1985a) by releasing vasoactive intestinal peptide within the gland (Silva et al., 1987). Shark CNP is a potent stimulus for M rectal gland secretion; during continuous perfusion as little as sCNP increases secretion over threefold (Solomon et al., 1992). Unlike ANP and BNP, sCNP appears to have a direct effect on glandular secretion as it alone will bind to rectal gland membranes and stimulate guanylate cyclase (Solomon et aZ., 1992). AP I1 stimulates short-circuit current and conductance by the chloride cell-rich opercular epithelium of either FW- or SW-adapted killifish, Fundulus heteroclitus (Scheide and Zadunaisky, 1988). The AP 11 effect is independent of neural activity or P-adrenergic stimulation. AP I11 does not affect salt transport by the opercular epithelium of the SW adapted winter flounder, Pseudopleuronectes arnericanus (O’Grady et al., 1985). In contrast to the apparent stimulatory effects of ANPs on renal, rectal gland, and opercular epithelial ion transport, A P I and I11 inhibit salt transport by the intestine of SW winter flounder, P . americanus (O’Grady, 1989;O’Grady et al., 1985).The intestine is more sensitive to AP I11 than API; ECsos of 7 x lO-’M and 7 x lops M , respectively (O’Grady et al., 1985). The efficacy of AP I11 is similar to ECSOSreported for fish vessels (see earlier discussion). The physiological significance of ANP effects on opercular and gut epithelia are not known, although these studies suggest that NPs decrease salt intake and promote salt excretion. In uliuo, a single injection of hANP into either FW trout or trout on a high salt diet has no effect on ionoregulation or branchial sodium exchange (Eddy et al., 1990). However, hANP increases sodium efflux when it is injected into SW flounder, Platichthys jlesus, dab, Limanda limanda, and plaice, Pleuronectes platessa (Arnold-Reed et al., 1991). Furthermore, hANP increased plasma cortisol in flounder (Arnold-Reed et al., 1991) indicating that NPs may have both direct short-term and indirect long-term effects on osmoregulatory organs. The effects of long-term ANP administration on osmoregulatory processes remain to be determined.

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h. Mechanisms ojANP Action: Interaction with Other Endocrine Systems. The relationship of atrial peptides to other endocrine systems in fish, especially those with known osmoregulatory functions, is just beginning to be evaluated. ANP stimulates release of l-ahydroxycorticosterone by perifused elasmobranch interrenal tissue (O’Tool et al., 1990). I n teleosts, hANP increases plasma cortisol in SW flounder, Platichthys flesus, and stimulates cortisol release by perifused interrenal tissue from SW but not FW trout (Amold-Reed and Balment, 1991; Arnold-Reed et al., 1991). These stimulatory effects of ANP are opposite to the well-known inhibitory effect of ANP on aldosterone release in mammals but support the general concept of an interactive mechanism of action of ANP on other endocrine systems. It is likely that many of the physiological effects of ANP and related peptides are mediated through similar indirect processes. i. Subcellular Mechanisms. Relatively little is known about the subcellular mechanism(s) of action of atrial peptides in fish tissues, however, in most instances they appear similar to those found in mammals. Olson and Meisheri (1989)have shown that rANP [Hez6]inhibits norepinephrine stimulated contractions of celiacomesenteric arteries in Ca”-free medium. Thus, as is the case in mammals, the peptide acts at a level central to receptor activated release of intracellular calcium. The effects of NP on guanylate cyclase activity in all fish tissues examined to date are consistent with those observed in mammals. Incubation of trout arteries with rANP [Ilez6]increases tissue levels of cGMP (D. W. Lipke and K. R. Olson, unpublished observation) and hANP increases cGMP in gills of SW, but not FW rainbow trout (Balment and Lahlou, 1987). AP 111 also increases cGMP in the winter flounder, P . americanus, intestine (O’Grady, 1989; O’Grady et al., 1985). Thus it would appear that the basic intracellular response of fish vessels to ANP is similar to that found in mammals.

V. SUMMARY Fluid volume, fluid hydraulic pressure and osmolarity are intimately related; in fact they are probably different attributes of a more general physiological phenomenon. Although osmoregulation and blood pressure regulation have been intensively examined in fish, fluid compartments and their control have received surprisingly little attention. Blood and extracellular fluid volume are governed by a variety of physical and physiological processes. Factors that affect

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water and salt movement across the gills, skin, gut, and kidney determine overall fluid balance. Fluid distribution between intravascular and extravascular compartments is further subjugated to hydraulic, osmotic, and mechanical forces in the microcirculation. The remarkable constancy of compartmental volumes, even in the face of adverse circumstances, emphasizes that homeostatic reflexes, especially in bony fish, are highly developed and efficient. Appreciation of volume regulatory mechanisms in fish has suffered from a lack of information on the nature and disposition of the fluid compartments themselves, the relative importance of various factors that affect intercompartmental fluid movements, and the regulatory processes involved. T h e afferent limb of volume regulatory reflexes in fish, or for that matter vertebrates in general, has not been characterized. Efferent control of appropriate effectors of fluid balance is under investigation. Two systems, the renin-angiotensin system and the atrial natriuretic peptides, have been shown to affect a variety of vascular and extravascular effectors and these systems may play an important role in volume homeostasis. The kallikrein-kinin system may also prove to have important systemic as well as intraorgan cardiovascular functions. Undoubtedly, other central and peripheral systems are also involved. Of all vertebrates, fish are probably the most versatile and manipulative model, from both phylogenetic and physiological perspectives, in which fluid compartments and volume regulation can be examined. I hope this chapter provides a convenient starting point for such studies.

ACKNOWLEDGMENTS This chapter is dedicated to the colleagues and students that I have had the privilege to work with and learn from. I would like to express my sincere appreciation to Connie Gordon and Kathy Drajus for their excellent secretarial and librarian help and to Dixie Kullman for her technical assistance. Unpublished experiments were supported by National Science Foundation Grants DCB-8616028, DCB-9004245, and DCB-9105247.

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uretic peptide decreases circulatory capacitance in areflexic rats. Circulation Research 59,291-296. Turner, A. H. (1937). Serum protein measurements in the lower vertebrates. 11. In marine teleosts and elasmobranchs. Biol. Bull. 73,511. Uemura, H., Naruse, M., Hirohama, T., Nakamura, S., Kasuya, Y.,and Aoto, T. (1990). Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy, immunohistochemistry and radioimmunoassay. Cell Tissue Res. 260,235-247. Vetter, S., and Hanke, W. (1985). A study comparing in vitro adrenal cortex responses of fish and amphibia. Acta Endocrinol. (Copenhagen) 108,157-161. Vislie, T., and Fugelli, K. (1975). Cell volume regulation in flounder (Platichthyspesus) heart muscle accompanying an alteration in plasma osmolality. Comp. Biochem. Physiol. [A] 52,415-418. Vogel, W. 0. (1985a). Systemic vascular anastomoses, primary and secondary vessels in fish, and the phylogeny of lymphatics. In “Cardiovascular shunts, Alfred Benzon Symposium 21” (K. Johansen and W. W. Burggren, eds.), pp 143-159. Munksgaard, Copenhagen. Vogel, W. 0. (1985b). The caudal heart of fishes: Not a lymph heart. Acta Anat. 121, 41-45. Vogel, W. O., and Claviez, M. (1981).Vascular specialization in fish, but no evidence for lymphatics. Z. Naturforsch. 36c, 490-492. Walsh, P. J., and Moon, T. W. (1982). The influence of temperature on extracellular and intracellular pH in the American eel, Anguilla rostrata (LeSueur). Respir. Physiol. 50,129-140. Wardle, C. S. (1971). New observations on the lymph system of the plaice Pleuronectes platessa and other teleosts. J. Mar. Biol. Assn. U . K . 51,977-990. Warner, M. C., and Williams, R. W. (1977). Comparison between serum values of pond and intensive raceway cultured channel catfish lctalurus punctatus (Rafinesque). J . Fish Biol. 11,385-391. Weld, M. M., and Fryer, J. N. (1987). Stimulation by angiotensins I and I1 of ACTH release from goldfish pituitary cell columns. Gen. Comp. Endocrinol. 68, 19-27. Weld, M. M., and Fryer, J. N. (1988). Angiotensin I1 stimulation ofteleost adrenocorticotropic hormone release: Interactions with urotensin I and corticotropin-releasing factor. Gen. Comp. Endocrinol. 69,335-340. Wells, R. M. G., and Forster, M. E. (1989). Dependence ofblood viscosity on haematocrit and shear rate in a primitive vertebrate. J . E r p . Biol. 145,483-487. Westenfelder, C., Baranowski, R. L., Shiozawa, D. K., and Kablitz, C. (1988a). Atrial natriuretic factor (ANF) in two teleost fish Gila atraria (GA) and Salmo gairdneri (SG). Proc. Am. Soc. Neph. 142A. [Abstract] Westenfelder, C., Birch, F. M., Baranowski, R. L., Rosenfeld, M. J., Shiozawa, D. K., and Kablitz, C. (198813).Atrial natriuretic factor and salt adaptation in the teleost fish Gila atraria. Am. J . Physiol. 255, Fl281-F1286. White, F. C. (1988). Organ blood flow haemodynamics and metabolism of‘the albacore tuna Thunnus alalunga (Bonnaterre).E x p . Biol. 47,161-169. Wiig, H., and Reed, R. K. (1987). Volume-pressure relationship (compliance)of interstitium in dog skin and muscle. Am. J . Physiol. 253, H291-H298. Wilkes, P. R. H., and Mchlahon, B. R. (1986). Responses of a stenohaline freshwater teleost (Catostomus commersoni) to hypersaline exposure 11. Transepithelial flux of sodium, chloride and ‘acidic equivalents.’]. E z p . Biol. 121,95-113. Wilkes, P. R. H., Walker, R. L., McDonald, D. G., and Wood, C. M. (1981). Respiratory,

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4 CATECHOLAMINES D. J . RANDALL Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

S. F . PERRY Department of Biology University of Ottawa Ottawa, Ontario, Canada

I. Catecholamine Metabolism A. Catecholamine Biosynthesis B. Catecholamine Degradation 11. Control of Blood Catecholamine Levels A. Circulating Levels during “Rest” and “Stress” B. Origin of Plasma Catecholamines C. Control of Catecholamine Release D. Fate of Plasma Catecholamines 111. Actions of Circulating Catecholamines A. Introduction B. Catecholamines and Gill Diffusing Capacity C. Catecholamines and Blood Oxygen Capacity D. Catecholamines and Gill Ventilation E. Catecholamines and Ion Movements F. Catecholamines and Blood Flow and Distribution G. Carbon Dioxide Transport H. General Conclusions Concerning the Action of Circulating Catecholamines IV. Factors Influencing Actions of Catecholamines References

I. CATECHOLAMINE METABOLISM The biosynthesis and metabolic degradation of catecholamines in fish, with few exceptions, appears to be essentially identical to the metabolism of catecholamines in other vertebrate groups. There is, 255 FlSH PHYSIOLOGY, VOL. XIIB

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

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D. J. RANDALL A N D S. F. PEKKY

however, relatively little information on the metabolic pathways in fish, and the data available are based on studies of very few species. The topic of catecholamine metabolism in fish was reviewed by Nilsson (1983). A. Catecholamine Biosynthesis

Catecholamines are synthesized in both nonneural chromaffin cells and adrenergic neurons by identical processes. A single biosynthetic pathway is responsible for the production of the three catecholamines, dopamine, noradrenaline, and adrenaline. This series of enzymatic reactions, known as the “Blaschko pathway” (Blaschko, 1939; Holtz, 1939), is summarized in Fig. 1. The first step is the hydroxylation of tyrosine to dihydroxphenylalanine (DOPA) by tyrosine hydroxylase (TH).This conversion, which occurs in the cytoplasm, is believed to be the rate-limiting process in the synthesis of dopamine and noradrenaline. The activity of T H is rapidly controlled, in part, via negative feedback from the end products dopamine and noradrenaline (e.g., Spector et al., 1967). Thus, increases in catecholamine release will cause an immediate increase in the formation of DOPA. In addition, T H may be subject to long-term modulation in which additional enzyme molecules are induced slowly during chronic hypersecretion of catecholamines (e.g., Lewander et al., 1977). For these reasons, catecholamine stores are rarely depleted in chromaffin tissue even during periods of intense release into the circulation. Molecular oxygen is a co-factor in the hydroxylation reaction catalyzed by TH, thus the availability of oxygen may also limit catecholamine synthesis. Indeed, it was demonstrated that long-term exposure of crucian carp (Carassius) to anoxia markedly reduced the catecholamine content of head kidney chromaffin tissue (Nilsson, 1989, 1990). Upon return to normoxia after 17 days of anoxia (Nilsson, 1990), the noradrenaline levels recovered only very slowly indicating that chronic lack of oxygen may damage the catecholamine synthesizing system within the chromaffin tissue. The pH and temperature optima of T H (see Table I) in chromaffin tissue of Atlantic cod (Gadus morhua) are 6.0 and 3Oo-35”C, respectively (Jonsson and Nilsson, 1983). The decarboxylation of DOPA to yield dopamine (DA) is catalyzed b y the cytoplasmic enzyme aromatic L-amino acid decarboxylase (AADC). Although the activity of this enzyme has not been directly measured in fish, its presence in Atlantic cod is clearly indicated by the increased levels of DA in a variety of peripheral tissues following injections of DOPA (Johansson and Henning, 1981). In mammals,

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257

Fig. 1. The biosynthesis of catecholamines within a chromaffin cell by the “Blaschko pathway.” Enzyme abbreviations: TH, tyrosine hydroxylase; AADC, aromatic L-amino acid decarboxylase; DBH, dopamine-P-hydroxylase; P N M T , phenylethanolamine-N-methyltransferase.

AADC is distributed ubiquitously in both neural and nonneural tissue and has a broad substrate specificity. Aromatic L-amino acid decarboxylase is available in sufficient quantities so that DOPA does not accumulate to any great extent, and AADC has never been shown to be rate-limiting in the production of catecholamines. The dopamine thus formed is taken up into storage vesicles and is either stored as such for

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D. J. RANDALL A N D S. F. PERRY

Table I pH and Temperature Optima ofthe Blaschko Pathway Enzymes TH, DBH, and PNMT in Chromaffin Tissue of Atlantic Cod (Gadas rnorhua) and Dogfish (Squalus acanthius) Enzyme

PH optimum

Temperature optimum

TH

6.0

30-35°C

C. morhzra

DBH

5.4

27°C

G. morhua

DBH

5.4-6.2

24.5-32°C

S. acanthius

Jonsson and Nilsson (1983) Jonsson and Nilsson (1979) Jonsson (1982)

PNMT PNMT

7.9 -

37°C 30°C

S. ucanthias C. morhua

Abrahamsson (1979) Abrahamsson (1979)

~

~

_

_

_

Species

_

_

_

Reference

_

_

_

~

~

~

Abbreviations: DBH, dopamine-p-hydroxylase; PNMT, phenylethano1amine-Nmethyl transferase; TH, tyrosine hydroxylase.

later release (e.g., dopaminergic neurons) or further metabolized (e.g., adrenergic neurons, chromaffin cells). The conversion of dopamine to L-noradrenaline occurs within storage vesicles by a stereospecific hydroxylation by the enzyme dopamine-P-hydroxylase (DBH),which is a copper containing tetrameric glycoprotein that requires ascorbate and molecular oxygen as cofactors. This requirement of oxygen for noradrenaline synthesis may also partially explain the drastic reductions in chromaffin tissue catecholamine content in chronically anoxic carp (Nilsson, 1990; see earlier). A portion of the vesicular DBH is bound to the vesicular matrix while another portion is soluble and released with other vesicular contents (catecholamines, adenosine triphosphate (ATP), chromogranin A; see Nilsson, 1983)on neural or other appropriate stimulation. The pH and temperature optima for DBH are shown in Table I. The pH optima of 5.4 and 5.4-6.2 for DBH from chromaffin tissue of Atlantic cod (Jonsson and Nilsson, 1979) and dogfish (Jonsson, 1982), respectively, may be physiologically significant because the internal pH of isolated bovine chromaffin cell storage vesicles has been estimated to be 5.5 (Johnson and Scarpa, 1976). The temperature optima for cod and dogfish DBH appear somewhat lower than values reported for the mammalian enzyme (Nilsson, 1983). The final step in the biosynthesis of adrenaline is the N methylation of noradrenaline catalyzed by the enzyme phenylethanolamine-N-methyl transferase (PNMT) in the cytoplasm. It is unclear how the noradrenaline formed within storage vesicles is made avail-

4. CATECHOLAMINES

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able to the cytoplasmic enzyme or how the adrenaline synthesized is repackaged into vesicles. In mammals, the enzyme is specific for phenylethylamines with the highest affinity for L-noradrenaline suggesting that this catecholamine is the natural substrate. In fish, unlike mammals, adrenaline is synthesized in both adrenergic neurons and chromaffin cells, and thus both catecholamines can act as neurotransmitters. Phenylethanolarnine-N-methyltransferase activity has been reported in a variety of fish species including rainbow trout (Mazeaud, 1972), Atlantic cod (Abrahamsson and Nilsson, 1976; Abrahamsson, 1980), and dogfish (Abrahamsson, 1979). A comparison of the optimal catalytic activities of TH, DBH, and PNMT from cod chromaffin tissue (cardinal vein) or coeliac ganglia (PNMT, Abrahamsson and Nilsson, 1976; DBH, Jonsson and Nilsson, 1979; TH, Jonsson and Nilsson, 1983) indicates that the rate-limiting step in the biosynthesis of adrenaline is the PNMT-catalyzed methylation of noradrenaline because the activity of PNMT is so much lower than either T H or DBH. This view is further supported by the observation of accumulated [3H]noradrenaline in Atlantic cod after injection of [3H]tyrosine (Jonsson and Nilsson, 1983). For these reasons, it is likely that the ratio of adrenaline/noradrenaline in any catecholaminesynthesizing tissue will be determined, in part, by the activity of PNMT. In Atlantic cod, adrenaline is the predominant catecholamine in chromaffin tissue and most adrenergically innervated organs with the exception of the swim bladder and urinary bladder (Abrahamsson and Nilsson, 1976). This is perhaps surprising given the relatively low activities of PNMT and presumably indicates a low turnover rate of adrenaline. Although adrenaline may be formed from noradrenaline produced locally in both neural and nonneural tissue, an additional route may involve uptake of circulating noradrenaline and subsequent methylation. The importance of this pathway for adrenaline synthesis will depend on the extent of blood flow to the particular organ. The synthesis of catecholamines in some vertebrate groups is under hormonal control specifically by the stimulatory actions of adrenocortical steroids on DBH and PNMT (Nilsson, 1983). In fish, little is known about the endocrine control of catecholamine biosynthesis although it would appear that in rainbow trout, PNMT is not modified by corticosteroids (Mazeaud, 1972; Nilsson, 1983; Jonsson et al., 1983). On the other hand, administration of cortisol was shown to significantly increase the activity of chromaffin tissue DBH in rainbow trout (Jonsson et al., 1983), presumably by reducing the extent of enzyme degradation.

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D. J. RANDALL AND S. F. PERRY

The biosynthesis of catecholamines can be blocked at several steps by specific enzyme inhibitors (see Nilsson, 1983). The most widely used compounds are similar to L-tyrosine in structure and are used to inhibit TH. Enzyme inhibitors have been used only rarely and with limited success in fish. Metcalfe and Butler (1989) administered a-methyl-p-tyrosine, an inhibitor of T H (Spector et al., 1965), to dogfish (Scyliorhinus canicula) for 5 days in an attempt to prevent the increase in circulating levels of catecholamines on exposure to environmental hypoxia. This protocol was effective in lowering both the “resting” and hypoxic levels of circulating catecholamines. However, the hypoxic fish still released physiologically significant quantities of catecholamines (see Table I in Metcalfe and Butler, 1989) into the circulation so it is unclear whether or not this protocol can adequately assess the importance of elevated catecholamines during such environmental stresses. Furthermore, as discussed by Metcalfe and Butler (1989),chronic treatment of dogfish with a-methyl-p-tyrosine substantially altered oxygen consumption and blood oxygen status because of specific effects on oxidative metabolism at the cellular level. Similar usage of a-methyl-p-tyrosine in rainbow trout does not impair the release of catecholamines during acute external hypoxia or physical disturbance (S. F. Perry and R. Kinkead, unpublished observations). The methylation of noradrenaline to adrenaline can be blocked by SK&F 64139, a specific inhibitor of PNMT (Pendleton et al., 1976). This compound has been used in Atlantic cod to effectively deplete sympathetic neurons of adrenaline (Abrahamsson, 1980).Owing to the apparent low rate of adrenaline turnover, several days of treatment are required to significantly deplete stores of adrenaline. This protocol, although effective in reducing the adrenaline content of catecholamine-synthesizing tissues, is not surprisingly associated with significant increases in noradrenaline content (Abrahamsson, 1980). An alternate technique to deplete chromaffin tissue or adrenergic neurons of endogenous catecholamines is to present a-methylated precursors (e.g., a-methyltyrosine, a-methyldopa, a-methyldopamine) that are recognized as substrates by the Blaschko pathway enzymes and metabolized to inactive (or less active) “false transmitters” (see Nilsson, 1983). B. Catecholamine Degradation

The two predominant enzymes involved in the catabolism of catecholamines are monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT), and these produce inactive deaminated and 0-

4.

CATECHOLAMINES

26 1

methylated catabolites, respectively. The mitochondrial enzyme, MAO, is particularly important in neuronal degradation whereas COMT may play a more important role in the catabolism of circulating catecholamines because it is largely extraneural. The relative importance of deamination versus O-methylation for the degradation of circulating catecholamines in fish is probably related to species (Mazeaud and Mazeaud, 1973; Ungell and Nilsson, 1979, 1983; Nekvasil and Olson, 1986a,b; Colletti and Olson, 1988). In Atlantic cod (Ungell and Nilsson, 1979), the major urinary catabolite of adrenaline is metanephrine (MN) suggesting that O-methylation is the predominant route of degradation. In dogfish (Mazeaud and Mazeaud, 1973; Ungell and Nilsson, 1983), the urinary catabolites of adrenaline are MN, vanillylmandelic acid (VMA), and 3-methoxy-4-hydroxyphenylglycol (MOPEG),indicating that both O-methylation and deamination are involved. In rainbow trout (Nekvasil and Olson, 1986a; Colletti and Olson, 1988),deamination by MA0 appears to be the more important route of catecholamine inactivation. The relative importance of the two enzymes cannot be determined with certainty because at least two catabolites (VMA and MOPEG; see Fig. 2) are produced by the combined effects of O-methylation/deamination (see Nekvasil and Olson, 1986a). In trout (Nekvasil and Olson, 1986a) and cod (Ungell and Nilsson, 1979), O-methylated derivatives are the first to appear following injections of exogenous catecholamines. These results are consistent with the established view (Kopin, 1960) that COMT acts before deamination. The temporal differences in the appearance of O-methylated and deaminated catabolites may reflect the mitochondrial and cytoplasmic locations of MA0 and COMT, respectively. A variety of tissues exhibit MA0 and COMT activities (Edwards et al., 1986; Nekvasil and Olson, 1986b; Colletti and Olson, 1988)including gill, liver, and kidney. The relative importance of any particular organ to the overall metabolic degradation of circulating catecholamines depends on the enzymatic activities, percentage of blood flow, relative mass, and the ability to extract catecholamines from the plasma. Based on the few available data, the gill is probably the dominant site of plasma catecholamine degradation (Nekvasil and Olson, 1986a,b; Colletti and Olson, 1988). The gill receives 100% of the cardiac output, possesses both deaminating and O-methylating activities, and forms an extensive cell-plasma interface through which catecholamines can be extracted. There are more catabolites released into the arteriovenous (AV) circulation of the gill compared to the arterioarterial circulation (Colletti and Olson, 1988)indicating that cells per-

-Hq=+

av

NMl HOO 3

t VMlA Z

-

h

-

" q9 - - -

HOO

OSm

P -

Fig. 2. Metabolic dcgradittion of' adrenaline arid Iioratireiialiue by dearnination (MAO. monoamine oxidase) and O-methylation (COMT, catechol-0-methyl transfel-ase). Abbreviations: AD, adrenalirte: DOMA, 3,4-dihydroxymandelic acid; IIOPEC, 3,4-dihydroxyphenylglycol; MN, metanephrine; NA, n o r a d r e d i n e ; N M , noi-mctanephrine; MOPEG, 3 - 1 ~ 1 ~ ~ l i ~ ~ x y - 4 - 1 i ~glycol; ~ ~ ~ xVMA, y ~ l 1vanill ~ 1 1 ylmandelic v~ acid.

4. CATECHOLAMINES

263

fused by the AV circulation are the major sites of catecholamine degradation. Since chloride cells are most abundant on the gill filamental epithelia (bathed by the AV circulation), these cells may be especially important in this regard. Noradrenaline is the preferred substrate for catabolism in both gill tissue homogenates (Nekvasil and Olson, l986b) and perfused gill preparations (Colletti and Olson, 1988). The oxidative deamination of catecholamines requires molecular oxygen. Thus, the oxygen status of the blood, in vivo or in vitro, will affect the rate of catecholamine catabolism. Exposure of blood in vitro to hyperoxia is a convenient technique to rapidly deplete endogenous catecholamines. It is possible that hypoxic conditions may attenuate the rate of degradation and in this way prolong the physiological effects of circulating catecholamines. The deamination of catecholamines by M A 0 can be blocked by a class of compounds termed M A 0 inhibitors; these include pargyline and nialamide (see Nilsson, 1983).Monoamine oxidase inhibitors have been used in vitro to impede catecholamine degradation in whole blood suspensions (Milligan and Wood, 1987), although the benefit is questionable owing to the low activity of M A 0 in blood.

11. CONTROL OF BLOOD CATECHOLAMINE LEVELS A. Circulating Levels during “Rest” and “Stress” The levels of plasma catecholamines under resting conditions and after a variety of imposed stresses have been summarized previously (Nilsson, 1983; Tetens et al., 1988; Hart et al., 1989; Perry et al., 1989; Thomas and Perry, 1991; McDonald and Milligan, 1992; see also Fig. 3).In unanesthetized cannulated fish, the concentrations of adrenaline or noradrenaline are usually between 1and 5 nmol.liter-l. The absolute values and the ratios of adrenalinelnoradrenaline at rest vary among and between species with no obvious pattern emerging. There is a slight predominance of adrenaline in teleosts whereas noradrenaline appears to be the dominant catecholamine in elasmobranchs. In general, however, any one species does not appear markedly different from any other. The eel may be an exception among all fish in that it has unusually low levels of plasma catecholamines (Le Bras, 1982; Epple et al., 1982; Epple and Nibbio, 1985; Hyde and Perry, 1990). The circulating levels of catecholamines rise during or immediately following a variety of physical and environmental disturbances

264

-

vn

D. J. RANDALL AND S. F. PERRY

400 350

1B =

0Pre-hypoxic Hypoxic

/

n "

--

I

=

-

(4) (5) (6) (7) ( 8 ) (9) (10) (11) (12) (13) Fig. 3. Changes in the plasma levels of (A) adrenaline and ( B ) noradrenaline in fish during environmental hypoxia. The data were obtained from the following studies corresponding to the numbers in parentheses in the lower panel. 1.Thomas et (11. (1991); 2 . Metcalfe and Butler (1989); 3. Wright et a / . (1989); 4.Aota et a / . (1990); 5 . Boutilier et al. (1988);6. Ristori and Laurent (1989); 7. Kinkead et al. (1991); 8. Thomas et a / . (1991); 9. Perry et al. (1991b); 10. Fievet et al. (1987); 11. Tetens and Christensen (1987); 12. Fritsche and Nilsson (1990); 13. Fievet et a1. (1990). See text for further details. (1)

(2)

(3)

4.CATECHOLAMINES

265

including external hypoxia (Fritsche and Nilsson, 1990; Perry et al., 1991b; Thomas et al., 1991; Metcalfe and Butler, 1989; Aota et al., 1990; Boutilier et al., 1988; Fievet et al., 1990; Ristori and Laurent, 1989; Wright et al., 1989; Butler et al., 1979; Fievet et al., 1987; Kinkead et al., 1991; Tetens and Christensen, 1987), hypercapnia (Hyde and Perry, 1990; Perry et al., 1989; Perry et al., 1987), air exposure (Fuchs and Albers, 1988; Nilsson et al., 1976),exhaustive or “violent” exercise (Wood et al., 1990; Ristori and Laurent, 1985; Primmet et al., 1986; Axelsson and Nilsson, 1986; Butler et al., 1986; Milligan and Wood, 1987; Tang and Boutilier, 1988; Wood, 1991; Opdyke et al., 1982; Nakano and Tomlinson, 1967), metabolic acidosis (Boutilier et al., 1986; Aota et al., 1990), anemia (Iwama et al., 1987; Perry et al., 1989), and exposure to “softwater” (Perry et al., 1988a).There is considerable disparity among the various studies that have evaluated plasma catecholamine levels during stress owing, in part, to species and methodological differences. Adrenaline usually is the predominant catecholamine during stress. Although it is expected that the relative quantities of adrenaline and noradrenaline in the plasma should reflect, at least partially, the storage levels in the chromaffin cells of the head kidney and elsewhere, this is only partly supported by the available data. In Atlantic cod, the ratio of stored catecholamine within the head kidney is 86% adrenaline to 14% noradrenaline (Abrahamsson and Nilsson, 1976), while in rainbow trout the levels of each catecholamine in the chromaffin cells of the anterior kidney are approximately equal (Nakano and Tomlinson, 1967). The ratio of circulating adrenaline/ noradrenaline in trout and cod during hypoxia (e.g., cod, Fritsche and Nilsson, 1990; Perry et al., 1991; trout, Fievet et al., 1990; Thomas et al., 1991) or exhaustive exercise (e.g., trout, Primmett et al., 1986; Wood et al., 1991; cod, Butler et al., 1989)do not appear to be different. On the other hand, the chromaffin tissue of the axillary bodies of dogfish (Squalus acanthias) contains predominantly noradrenaline (83% noradrenaline/l7% adrenaline) (Abrahamsson, 1979). This may explain the obvious predominance of noradrenaline in the circulation of dogfish during hypoxia (Metcalfe and Butler, 1989; Butler et al., 1978; Butler et al., 1979) or after exhaustive exercise (Opdyke et al., 1982) or intravascular K+ injection (Opdyke et al., 1983). Several other factors may potentially influence the levels of circulating catecholamines during stress including the rate of metabolic degradation (Nekvasil and Olson, 1986a,b; Colletti and Olson, 1988), accumulation by neural and extraneural tissues (Nekvasil and Olson, 1986a; Busacker and Chavin, 1977; Nekvasil and Olson, 1985; Ungell, 1985a,b), and overflow from adrenergic neurons. Furthermore, dis-

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crepancies among the various studies may be related to the site of blood withdrawal. Hathaway and Epple (1989) demonstrated regional differences in the levels of plasma catecholamines with the highest levels occurring near the presumed major site of release, the head kidney. Since the gill is believed to be an important location of catecholamine removal from the blood (Nekvasil and Olson, 1986a,b), preversus postbranchial blood samples might be expected to vary considerably. However, the only two studies to measure catecholamine levels in pre- and postbranchial blood (eel, Hathaway and Epple, 1989; cod, Kinkead et al., 1991)demonstrated essentially equivalent values. Finally, the measured levels in the blood may be affected by the speed with which the stress is imposed and the nature of the sampling protocol. Perry et d.(1991b) demonstrated two different patterns of catecholamine release in Atlantic cod during hypoxia depending on how rapidly the final water Po, (Pwo,) was attained. A gradual reduction of PWO,caused significant elevations of plasma adrenaline levels without affecting noradrenaline levels while a more rapid induction of hypoxia caused similar increases in the levels of both catecholamines. During progressive stress, one catecholamine may be more important initially while the other appears later. For example, during progressive hypoxia, noradrenaline appearance in the plasma may precede adrenaline appearance by several minutes (Fritsche and Nilsson, 1990). In contrast, dogfish preferentially release adrenaline into the blood during brief stress, but if the stress persists, noradrenaline becomes the more important plasma catecholamine (Abrahamsson, 1979). In other instances, adrenaline and noradrenaline levels may initially increase in the plasma at equal rates but noradrenaline may subsequently stabilize while adrenaline levels continue to rise (Thomas et al., 1991). It would appear that the chromaffin cells release variable quantities of adrenaline and noradrenaline depending on the nature and duration of the particular stimulus. Because of the rapid clearance of catecholamine from the blood by the combined effects of tissue accumulation and metabolism (see following discussion), peak levels normally occur within several minutes after cessation of the stress (e.g., Tang and Boutilier, 1988; Wood et al., 1990). Less severe or prolonged stresses may cause delayed increases in plasma catecholamine levels (Thomas et al., 1991).Catecholamine levels may return to control values despite the continuation of the stress that originally initiated the release (e.g., Perry et al., 1987). It is informative that plasma catecholamine levels remain essentially unchanged during a variety of physical and/or environmental disturbances that are usually classified as “stressful” including moder-

4.

CATECHOLAMINES

267

ate or mild hypoxia (Boutilier et al., 1988; Ristori and Laurent, 1989; Kinkead and Perry, 1990) and sustained aerobic exercise (Ristori and Laurent, 1985; Butler et al., 1986; Hughes et al., 1988; see also review by Wood, 1991). It is becoming increasingly clear that plasma catecholamine levels do not rise substantially until the degree of stress becomes very severe. Only at such times can circulating catecholamines play a significant role in the initiation of adaptive responses. B. Origin of Plasma Catecholamines The two potential sources of circulating catecholamines are “overflow” from adrenergic nerve terminals and release from chromaffin cells. Few studies have directly assessed the importance of adrenergic neural overflow. It has been argued (Wahlqvist and Nilsson, 1980; Butler et al., 1989) that the inability to totally prevent the rise in circulating catecholamines during stress after bilateral sectioning of the nerves to the chromaffin tissue may reflect a minor component of adrenergic neural overflow. On the other hand, Perry et al. (1991b) concluded that “overflow” from peripheral adrenergic nerve terminals did not significantly contribute to the elevation of circulating catecholamines in hypoxic cod since pretreatment with bretylium (an inhibitor of catecholamine release from adrenergic nerve terminals) did not alter the pattern of catecholamine release. Although it is unlikely that “overflow” of catecholamines significantly contributes to systemic changes in the circulating levels, it is conceivable that local levels may change in the region of densely adrenergically innervated organs or tissues. Pennec and Le Bras (1984) showed that the perfused eel (Anguilla) heart spontaneously releases catecholamines stored within adrenergic neurons into the perfusion fluid. This “overflow” of neuronal catecholamines was enhanced by stimulation of the vagus nerve and significantly affected cardiac function. The relative proportions of catecholamines released b y the eel heart closely matches the storage levels (noradrenaline > adrenaline > dopamine). Significant quantities of neuronal catecholamines also “overflow” from the perfused cod spleen after electrical stimulation (Nilsson and Holmgren, 1976; Ehrenstrom and Ungell, 1990). The predominant catecholamine released by the cod spleen is adrenaline in accordance with the higher concentration of this catecholamine stored within this tissue (Abrahamsson and Nilsson, 1976). The physiological significance of catecholamine “overflow” by the spleen has yet to be elucidated. Overflow from other adrenergically innervated organs may also contribute slightly to circulating levels or cause regional increases. In teleosts, adrenaline is the

D. J. RANDALL AND S. F. PERRY

268

dominant catecholamine in most adrenergic neurons (Abrahamsson and Nilsson, 1976; Nilsson, 1983)while in elasmobranchs noradrenaline appears to be more important (Abrahamsson, 1979). By far, the most important source of circulating catecholamines is the chromaffin tissue (Nakano and Tomlinson, 1967; Abrahamsson and Nilsson, 1976; Abrahamsson, 1979; Nilsson, 1983; Hathaway and Epple, 1989). I n teleosts, the chromaffin tissue is contained primarily within the anterior or head kidney often in association with the walls of the posterior cardinal veins (Nilsson, 1983). Table 11 summarizes the levels of adrenaline and noradrenaline in the chromaffin tissue of various teleost and elasmobranch species. It would appear that the absolute levels of the catecholamine stored within the chromaffin tissue-containing organs vary considerably among species as does the ratio adrenaline/noradrenaline. It is unclear to what extent methodological differences have contributed to these variable patterns. Furthermore, the values presented in Table I1 are expressed in terms of tissue weight. Since the proportion of chromaffin cells varies in the particular tissues, little can be inferred about the absolute values in the chromaffin tissue itself. I n elasmobranchs, the axillary bodies are the major sites of chromaffin tissue and unlike in teleosts, noradrenaline Table 11 Catecholamine Levels in the Chromaffin Tissue of Teleosts and Elasmobranchs* Reference Teleost S. gairdneri Nakano and Tomlinson (1967) A . rostrata

Hathaway and Epple (1989) G. morhua Abrahamsson and Nilsson (1976) C . carassius Nilsson (1990) C . carpio Stabrovskii (1968) Elasmobranch S. acanthias Abrahamsson (1979) C . monstrosa Pettersson and Nilson (1979)

Adrenaline

Noradrenaline

Site

4.7

4.5

84.8

42.4

PCB

38.2

14.3

PCV

Head kidney

0.05

0.16

Kidney

0.05

0.84

Head kidney

445

2139

Axillary body

3780

9390

Axillary body

* All values are given as pg g-' tissue

4.

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CATECHOLAMINES

always is the prevalent catecholamine (Abrahamsson, 1979; Nilsson, 1983; see also Table 11). In cyclostomes and dipnoans, the heart is an important site of chromaffin tissue (Nilsson, 1983).Depletion of cardiac catecholamine stores reduces heart rate in hagfish but the exact role of these catecholamine stores in cardiac function is not clear (see Part A, Chapter 1).

C. Control of Catecholamine Release The chromaffin tissue of teleosts and elasmobranchs is innervated by sympathetic preganglionic nerve fibers (Gannon and Campbell, 1972; Nilsson, 1976; Nilsson et al., 1976; Hathaway et al., 1989). These fibers are evidently cholinergic because electrical stimulation (Nilsson et al., 1976; Abrahamsson, 1979; Wahlqvist, 1981) or application of acetylcholine (Ach) to simulate nerve activity (Nilsson et al., 1976; Perry et al., 1991b) causes the release of both adrenaline and noradrenaline from in situ perfused preparations; the cholinergic ganglionic blocker hexamethonium abolishes the release of catecholamines after nerve stimulation. The ratio of adrenaline/noradrenaline released from perfused chromaffin tissue may deviate markedly from the predicted ratio based on the chromaffin tissue catecholamine content (Abrahamsson, 1979; Perry et al., 1991b;see also Fig. 4). Moreover, the

0

1

2

3

4

5

6

7

8

9

1011121314

Time (min) Fig. 4. Catecholamine overflow in an in situ, saline-perfused head kidney preparation of Atlantic cod (Gadus rnorhua) during a control period and immediately after administration of 10-6M acetylcholine. Noradrenaline overflow is represented by the open boxes, and adrenaline overflow is represented by the shaded boxes. * indicates a statistically significant difference compared to the corresponding catecholamine overflow value immediately before addition of acetylcholine (6 min); t indicates a statistically significant difference compared to the overflow value of other catecholamines. [From Perry et al. (1991b).]

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D. J. RANDALL AND S. F. PERRY

proportions of catecholamines released may vary in relation to the nature and duration of the stimulus. For example, Nilsson et al. (1976) demonstrated variable adrenaline/noradrenaline overflow ratios in a perfused head kidney preparation of Atlantic cod depending on whether the preparation was stimulated electrically or with Ach. Similarly, Abrahamsson (1979)reported that brief (1min) electrical stimulation caused preferential adrenaline overflow in a perfused dogfish preparation although noradrenaline clearly is the dominant circulating catecholamine in elasmobranch plasma during sustained stress. These results suggest that adrenaline and noradrenaline may be stored in different chromaffin cell types, as demonstrated in amphibians (Coupland, 1971). Furthermore, these cell types may respond differently to potential catecholamine-releasing stimuli. The importance of neural stimulation ofthe chromaffin tissue in the mobilization of circulating catecholamines during stress has been evaluated by bilateral sectioning of spinal nerves 1-4 innervating the head kidney. This procedure was shown to significantly, although not entirely, decrease the increase of plasma catecholamines during air exposure (Wahlqvist and Nilsson, 1980) or after exhaustive exercise (Butler et al., 1989). Although it is obvious that neural stimulation of chromaffin tissue and consequent release of Ach contributes to the elevation of plasma catecholamines during periods of stress, the intermediary pathways are unknown. A variety of chemoreceptors exist in fish that monitor the chemical or physical properties of the internal, external, or both environments. These receptors have been implicated primarily in the control of cardiovascular and ventilatory functions, although it is also conceivable that they (or other similar receptors) may play a role in the control of catecholamine release. If so, the sensory thresholds to elicit release must b e considerably different than those eliciting cardiovascular and ventilatory effects because the latter responses commence with slight changes in external or internal chemistry, or both whereas catecholamine release only begins under conditions of extreme stress. The inability of bilateral denervation to completely prevent catecholamine release into the circulation or to substantially alter adrenaline mobilization indicates that other factors are involved in the control of catecholamine release from chromaffin tissue. This contention is further supported by several experimental observations. First, the catecholamine-induced (catecholaminotropic) release of catecholamines from chromaffin tissue of American eels (Anguilla rostrata) is unaffected by removal of the preganglionic innervation (Hathaway et al., 1989; Hathaway and Epple, 1989). Second, dogfish (Squalus acanthias) liberate catecholamines into the circulation in response to

4. CATECHOLAMINES

27 1

intravascular injections of potassium even after ganglionic blockade using hexamethonium (Opdyke et al., 1983). This observation supports the idea that increases in plasma K+ arising from skeletal muscle may be an important secondary stimulus contributing to the release and maintenance of plasma catecholamine after exhaustive exercise in elasmobranchs (Opdyke et al., 1982).Third, the noninnervated cardiovascular chromaffin cells of the sea lamprey release catecholamines in response to carbon dioxide (Dashow and Epple, 1985). Fourth, the demonstration by Perry et al. (199lb) of a local direct stimulatory effect of blood hypoxemia on adrenaline release from the chromaffin tissue in an in situ, blood perfused head kidney preparation of Atlantic cod. This observation of a specific modulatory effect of blood oxygen on the release of catecholamine from the chromaffin tissue is consistent with the general consensus that lowering of blood oxygen content is the dominant factor initiating the release of catecholamines and also consistent with the view that the principal effects of elevated circulating catecholamine levels are related to enhancement of blood oxygen transport (see following discussion). It was suggested (Perry et al., 1991b) that the release of either stored catecholamine (adrenaline or noradrenaline) from the chromaffin tissue could be controlled independently as a function of the inflowing (local) plasma levels of that particular catecholamine (Perry et al., l99lb). The basis of this “negative feedback” phenomenon is that catecholamine “overflow” from the chromaffin tissue into the circulation is the net result of two opposing processes; clearance of inflowing catecholamines and release of sequestered catecholamines. Clearance, in turn, is the summated effect of reuptake, metabolism, and tissue binding. Thus, in the event of a prolonged catecholamine-releasing stimulus, catecholamine net “overflow” from chromaffin tissue into the circulation will stop when the inflowing plasma levels rise to a point when clearance exceeds release. This mechanism is probably one of several means (see later) by which plasma catecholamines are prevented from rising to unnecessarily high levels and, in addition, allows independent control of the plasma levels of each catecholamine. A common feature shared by the various stresses in which catecholamine levels are elevated is a requirement for enhanced oxygen transport (see Table I in Thomas and Perry, 1991). This additional requirement may arise from the increased metabolic demands associated with exercise or reductions in blood oxygen content caused by environmental hypoxia or internal acidosis. Since blood acid-base status (specifically red blood cell acid-base status) and oxygen content are interrelated, it is difficult to distinguish the potential independent effects of

272

D. J. RANDALL AND S. F. PERRY

acidosis and hypoxemia on promoting the release of catecholamines from chromaffin tissue. There is no evidence, however, that acidosis per se is a direct stimulus for catecholamine release or that it is a prerequisite for catecholamine release during hypoxia. The release of catecholamines during periods of internal acidosis, at least in rainbow trout, is related to the lowering of blood oxygen content by the effects of H + on hemoglobin-oxygen binding (Perry et al., 1989) and can be prevented by exposing fish to hyperoxic water (Perry et al., 1989; Aota et al., 1990).Acidotic conditions may augment catecholamine release during hypoxemia because the extent of the reduction in oxygen content required to elicit catecholamine release is much larger during anemia than during an acidosis. It is likely that the lowering of the blood oxygen content (or a closely related variable), rather than a reduction of PO, per se, is the proximate stimulus for catecholamine mobilization since blood PO, is often unchanged or even elevated when catecholamines are released during several stresses including anemia (Iwama et al., 1987; Perry et al., 1989), hypercapnia (Perry et al., 1987), and exhaustive exercise (Wood et al., 1990).Moreover, results of studies suggest that the principal “zone” of catecholamine release corresponds to the area of maximal capacitance on the oxygen dissociation curve (Thomas et al., 1992; S. F. Perry and S. D. Reid, unpublished observations). It would appear that both inter- and intraspecific differences in the pattern of catecholamine release during hypoxia simply reflect intrinsic differences in the properties of hemoglobin-oxygen binding. For example, rainbow trout and American eel both release catecholamines during exposure to environmental hypoxia but with widely different arterial P o , thresholds (Fig. 5 ) corresponding to nearly identical arterial oxygen content thresholds. In each case, the release of catecholamines is initiated at arterial P o , values roughly equivalent to the hemoglobin P50 value as determined from in vivo oxygen dissociation curves (S. F. Perry and S. D. Reid, unpublished observations; Fig. 5). D. Fate of Plasma Catecholamines

Catecholamines released into the circulation are rapidly cleared from the plasma by the combined effects of tissue accumulation/ binding and metabolic degradation (see previous discussion). In rainbow trout, the biological half-time of an injected dose of adrenaline or noradrenaline is less than 10 min (Nekvasil and Olson, 1986a). Indeed, by 10 min after a bolus injection of catecholamine in trout, only 10% of the injected dose is physiologically active (Nekvasil and Olson, 1986a).

273

4. CATECHOLAMINES

1.4

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60

80

100

120

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0.6 0.4

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20

40

60

80

100

120

146

PaO, (torr)

Fig. 5. The relationships between arterial Po, (Pa0,)and plasma adrenaline levels during acute external hypoxia in (A) rainbow trout (Salmo gairdneri) and (B) American eel (Anguilla rostrata).In each case, in oioo hemoglobin oxygen dissociation curves are shown. The shaded areas represent the zones of adrenaline release. For each species, the release of adrenaline commences at 50-60% Hb-02 saturation. Note the expanded scale ofthe adrenaline axis for A. rostrata.[From S. F. Perryand S. Reid (unpublished).]

Only a few studies have attempted to quantify the half-time of catecholamines in the circulation of fish (Mazeaud, 1972; Ungell and Nilsson, 1979), but they appear to be in general agreement with the detailed study of Nekvasil and Olson (1986a). The relatively short residence time of biologically active catecholamines in the circulation

274

D. J. RANDALL AND S . F. PERRY

corresponds with the brief physiological effects of catecholamines following single injections of exogenous catecholamines or sudden releases of endogenous catecholamines. The accumulation of catecholamines into tissues occurs by two processes termed neuronal (type 1)or extraneuronal (type 2 ) uptake (see Nilsson, 1983).Neuronal uptake refers to the absorption of catecholamines by adrenergic nerve terminals after which the amines are either metabolized or repackaged within the storage vesicles for subsequent rerelease. The neuronal uptake mechanism can be blocked by cocaine or desmethylimipramine (Nilsson, 1983).Extraneuronal uptake refers to accumulation by nonneural tissues for subsequent metabolic degradation. Extraneuronal uptake can be blocked by either metanephrine or corticosterone (Nilsson, 1983). The importance of any particular tissue in the accumulation of circulating catecholamines is determined by several factors including its relative mass, blood flow, complement of catabolic enzymes, uptake affinity, endogenous catecholamine levels, and density of adrenergic neurons (neuronal uptake only). Thus, skeletal muscle, although relatively inefficient at accumulating catecholamines (Ungell, 1985; Nekvasil and Olson, 1986a), may nevertheless play an important role in inactivating circulating catecholamines owing to its enormous mass (Nekvasil and Olson, 1986a). Conversely, the highly efficient chromaffin tissue may be relatively unimportant because of its small size. With the exception of the brain, all tissues that have been examined accumulate both catecholamines to varying degrees (Busacker and Chavin, 1977; Ungell, 1985a,b; Nekvasil and Olson, 1986a). The brain apparently does not accumulate adrenaline (Busacker and Chavin, 1977; Nekvasil and Olson, 1986a) and has an extremely low affinity for noradrenaline accumulation (Nekvasil and Olson, 1986a). In general, tissues preferentially accumulate noradrenaline regardless of the nature of the uptake mechanism (neuronal versus extraneuronal; Busacker and Chavin, 1977; Ungell, 1985a,b; Nekvasil and Olson, 1985, 1986a,b). The gill tissue is presented with enormous levels of inflowing catecholamines owing to its anatomical location. It is probably not surprising, therefore, that the gill possesses highly efficient “neuronallike” and extraneuronal uptake mechanisms (Colletti and Olson, 1988).Although the gill is innervated by adrenergic nerves (Donald, 1984), these apparently are not the sites of neuronal-like accumulation since autoradiographic studies (Nekvasil and Olson, 1985) have revealed preferential accumulation/binding of noradrenaline by the noninnervated pillar cells. A considerably greater quantity of catechol-

4.CATECHOLAMINES

275

amines are extracted and metabolized as blood flows through the AV, relative to the arterio-arterial circulation. Since the relative proportion of the cardiac output flowing through the AV circulation of the gill is, in part, controlled by circulating catecholamines, a model has been proposed (Nekvasil and Olson, 1986b) in which inactivation of plasma catecholamines is partially controlled by the resistance of the AV pathway. In this scheme, the release of catecholamines will reduce blood flow through the AV circuit (see Nilsson, 1984) and the rate of catecholamine inactivation, and hence it will assist in maintaining high circulating systemic levels during periods of stress.

111. ACTIONS OF CIRCULATING CATECHOLAMINES A. Introduction Changing levels of circulating catecholamines have numerous physiological effects, both direct and indirect, all of which lead to either increases in, or maintenance of, energy turnover and oxygen supply under adverse conditions such as extreme hypoxia or acidosis. The metabolic effects have been studied extensively in fish and injected catecholamines, acting via p-adrenoreceptors, have been shown to increase plasma glucose levels in trout (Wright et al., 1989) activating liver glycogenolysis, gluconeogenesis, or both and inhibiting glycolysis. Catecholamine injections had no effect on either muscle or liver glycogen levels, and glycogen depletion seen during hypercapnia is related to factors other than adrenergic activation of liver glycogen phosphorylase (Perry et al., 1988).Hypercapnia (Perry et al., 1988) and hypoxia (Wright et al., 1989) depress glycogen phosphorylase activity but increase pyruvate kinase activity leading to a reduction in glucose levels. The effect of catecholamines, in both cases, was to reverse this trend and maintain glucose availability. Catecholamines also help to sustain oxidative metabolism in trout red blood cells during acidotic states resulting from exhaustive exercise. This is achieved in part by enhanced erythrocyte lactate oxidation (Wood et al., 1990), an effect only seen under acidic conditions. Catecholamines were without effect on this aspect of erythrocyte metabolism at normal blood pH (Wood et al., 1990). Treatment of dogfish with a-methyl-tyrosine reduced circulating levels of catecholamines and oxygen uptake in resting fish (Metcalfe and Butler, 1989). They concluded that the reduction in oxygen con-

276

D. J . RANDALL A N D S. F. PERRY

sumption was due to a direct effect of the drug on cellular metabolism, having observed that a-methyl-tyrosine depresses oxygen uptake in isolated hepatocytes. It is also reported, however, that heart function in vitro is dependent on low levels of catecholamines in the perfusate (see Part A, Chapter l ) , so it is possible that the reduction in circulating catecholamines following a-methyl-tyrosine treatment also contributed to the decrease in oxygen uptake. Catecholamines have a marked effect on oxygen delivery to the tissues in teleosts, modulating changes in gill diffusing capacity, increases in erythrocyte number, volume and intracellular pH, and changes in blood flow (see Part A, Chapter 2) and breathing. In elasmobranchs infusion of catecholamines caused an increase in breathing in quiet dogfish (Randall and Taylor, 1991) but inhibition of the rise in circulating catecholamines associated with hypoxia, by treatment with a-methyl-tyrosine, did not have any marked effect oxygen transfer or breathing rate in dogfish exposed to moderate hypoxia (Metcalfe and Butler, 1989). B. Catecholamines and Gill Diffusing Capacity

Increases in gill diffusing capacity probably result from elevations in circulating catecholamines (Randall and Daxboeck, 1984) that occur during hypoxia or following exhaustive exercise. An increase in circulating catecholamines raises dorsal aortic blood pressure, which, in turn, increases the width of the gill blood sheet and results in a more even distribution of blood throughout the lamellae (Farrell et al., 1980). In addition, there is probably an increase in the number of lamellae perfused (Randall and Daxboeck, 1984), and it has been suggested that adrenaline increases the permeability of the gill epithelium to oxygen and other nonelectrolytes (Isaia et al., 1978). All of these factors will increase the gill diffusing capacity. There is no direct evidence for a role of catecholamines in augmentation of gill diffusing capacity in intact animals, but Pettersson (1983)and Perry et al. (1985) demonstrated that adrenaline enhanced gill oxygen diffusing capacity in saline perfused cod and trout heads, respectively. C. Catecholamines and Blood Oxygen Capacity Catecholamines have a marked effect on blood oxygen capacity via a stimulation of Na+/H+ exchange across the erythrocyte membrane,

and the subsequent elevation of intracellular p H (Nikinmaa, 1990; Motais et al., 1990) and via increases in hematocrit due to the splenic release of red blood cells into the circulation (Nilsson, 1983).

4. CATECHOLAMINES

277

Many teleosts have a blood with a marked Root shift: That is, a reduction in blood p H results in a decrease in hemoglobin oxygen binding capacity. This is important for oxygen transfer into the swimbladder as acidosis in the gas gland causes the release of oxygen from hemoglobin for diffusion into the bladder. If the acidosis was general, however, one might expect blood oxygen capacity to be reduced and oxygen delivery to other tissues impaired. This does not occur as the release of catecholamines into the blood maintains erythrocytic p H in the face of an acidosis, and no Root shift is observed (Tufts and Randall, 1989). This increase in erythrocytic pH is caused by a P-adrenergic activation of Na+/H+transfer across the red blood cell membrane that results in a disequilibrium of the proton gradient across the erythrocyte membrane, raising intracellular p H (Nikinmaa, 1990).Adrenergic stimulation of trout erythrocytes increases nucleotide triphosphate (NTP)use, probably as a result of increased adenosine triphosphatase (ATPase) activity and production of cyclic adenosine monophosphate (CAMP) (Ferguson and Boutilier, 1989). Nucleotide triphosphate levels do not fall under aerobic conditions because oxygen uptake and NTP production is increased as well, to match use. Under anoxic conditions, however, NTP levels fall following adrenergic stimulation, presumably production no longer keeps pace with use (Ferguson and Boutilier, 1989). Decreases in erythrocytic levels of NTP during hypoxia, however, are not simply a consequence of adrenergic stimulation because ATP levels can fall during hypoxia in the absence of any change in circulating catecholamines (Val, 1991). The erythrocytic membrane is relatively impermeable to protons and so acid is transferred between the plasma and the red blood cell via the Jacobs-Stewart cycle. The increase in red blood cell pH is not immediately short-circuited by the Jacobs-Stewart cycle, because the plasma carbon dioxide hydration/ dehydration reaction is uncatalyzed and the rate of proton transfer is much faster than the plasma bicarbonate dehydration reaction velocity (Forster and Steen, 1969; Motais et al., 1990; Nikinmaa et al., 1990). This means that, unlike the situation in mammals, the capillary endothelium in fish cannot contain carbonic anhydrase activity because if it did the catecholamine-stimulated rise in erythrocytic p H would be short-circuited and blood oxygen-carrying capacity would be reduced. Thus, all carbon dioxide transfer must occur through the red blood cell, as shown by Perry et al. (1982), and there must be a tight coupling of oxygen uptake and carbon dioxide excretion. Hemoglobin oxygenation supplies the protons for bicarbonate dehydration and, therefore, carbon dioxide excretion. Hemoglobin oxygenation not only supplies protons for bicarbonate dehydration but also for Na+/H+ exchange

278

D. J. RANDALL AND S. F. PERRY

A

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Fig. 6. (A) The regulation of erythrocytic pH during a metabolic acidosis, following adrenergic stimulation. The increased Na+/H+ transfer transports protons out of the erythrocyte at a greater rate than the Jacobs-Stewart cycle replaces them, owing to the absence of carbonic anhydrase (CA) in the plasma. Hb, hemoglobin.

4.

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(Motais et al., 1990) (Fig. 6). Thus adrenergic stimulation of the sodium/proton antiporter may limit proton availability for bicarbonate dehydration. Carbon dioxide excretion is maintained, however, even though erythrocytic sodium/proton exchange is stimulated by catecholamines (Steffensen et al., 1987). Catecholamines cause the release of erythrocytes from the spleen into the blood stream. The splenic contraction causing this release is mediated by stimulation of a-adrenoreceptors (Nilsson and Grove, 1974), first appears at low levels of catecholamines, and is dosedependent in hypercapnic trout (Perry and Kinkead, 1989).Catecholamines are probably involved in the recruitment of red blood cells from the spleen during hypoxia (Yamamoto et al., 1985) and following exhaustive exercise (Nikinmaa et al., 1984; Primmett et al., 1986). Perry and Kinkead (1989) showed that splenic contraction was the dominant response causing arterial blood oxygen content to increase during hypercapnic induced elevations in circulating catecholamines. During exhaustive exercise, however, reductions in plasma volume, due to fluid shifts between blood and muscle, probably also contribute to the increase in blood oxygen content. Thus, oxygen content of arterial blood is maintained or even elevated under a variety of adverse conditions due to a rise in both the number of circulating red blood cells and erythrocytic p H caused by the rise in circulating catecholamines.

D. Catecholamines and Gill Ventilation Catecholamines infused into the dorsal aorta of eels caused a hyperventilation in the summer animals but a hypoventilation in winter eels (Peyreaud-Waitzeneggar, 1979). Hyperventilation was blocked by the P-adrenergic antagonist propranolol whereas the hypoventilatory responses were inhibited by a-adrenergic blockers. Infusion into trout resulted in a reduction in the breathing rate but an increase in breathing amplitude and gill water flow (S. Aota, unpublished observations). In Amia, there was an increase in both rate and amplitude of breathing following catecholamine infusion (McKenzie et al., 1991a). (B) Respiratory exchange ratio (R. E.) (i) prior to and following a burst swim and (ii) prior to and following adrenaline infusion in fish at rest. [Modified from Steffenson et al. (1987).] Bars show SD ( n = 6). Asterisks indicate values significantly different from resting value ( P 5 0.025).(iii) Maintenance of COz excretion during adrenergic stimulation of the erythrocyte. Protons essential for HCOS- dehydration are released from the hemoglobin on oxygenation. [From Randall and Brauner (1991).]

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In dogfish catecholamine infusion resulted in a marked increase in breathing rate but only in those fish with a low initial rate (Randall and Taylor, 1991). If the dogfish were agitated and had a high initial breathing rate, there was no effect of catecholamines infusion on breathing rate. Catecholamine infusion into trout has been reported to depress gill ventilation in normoxic (Kinkead and Perry, 1990; Playle et al., 1990.), hypoxic, and hypercapnic trout (Kinkead and Perry, 1991). Kinkead et al. (1991) found that neither a-nor P-adrenoreceptor agonists significantly impaired the hyperventilatory responses of cod to hypoxia, despite an increase in circulating catechokmine levels. Thus, there is evidence that catecholamine infusion has an effect on breathing in fish, causing either an increase or a decrease in rate depending on the species, the time of year, and the physiological state of the animal. In some instances, for example in hypoxic cod (Kinkead et al., 1991), elevated catecholamines may have little or no effect on gill ventilation. The question remains: Is there a physiological role for catecholamines in the control of ventilation (see Randall and Taylor, 1992; Perry et al., 1992)?Catecholamine levels in the blood increase following anemia, hypoxia, and exhaustive exercise, and all these conditions are associated with increases in ventilation. Changes in ventilation in intact fish during moderate hypoxia (trout, Kinkead and Perry, 1990; cod, Kinkead et al., 1991) and hyperoxia (trout, Kinkead and Perry, 1990),however, were not due to changes in circulating catecholamines but rather to gill oxygen chemoreceptor activity (Burleson and Smatresk, 1990; Burleson, 1991). These chemoreceptors can be stimulated by NaCN to increase breathing (Burleson and Smatresk, 1990). Denervation of the gills of Amia resulted in a reduced ventilatory response to hypoxia and the effects of NaCN are obliterated. The reduced response in the denervated fish is similar to that seen following catecholamine infusion in the normoxic fish. Hypoxia in Amia causes an increase in circulating catecholamines and could account for the response to hypoxia following denervation of the peripheral oxygen chemoreceptors. Thus, although the hyperventilatory response to hypoxia in fish is largely driven by chemoreceptor stimulation due to oxygen lack, there may be some additional or even supplementary action of catecholamines in extreme hypoxia, when chemoreceptor output has been reported to decline. It has been shown that propranolo1 does not effect the initial increase in breathing but does impair the sustained hyperventilatory response associated with hypercapnia in trout (R. Kinkead and S. F. Perry, unpublished observations), hence catecholamines may play some role in prolonged ventilatory responses

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to hypercapnia. Catecholamines may stimulate breathing during exhaustive exercise and during anemia, but this has yet to be demonstrated. If there is a role for circulating catecholamines in the control of breathing how could they have an effect? In mammals, P-adrenergic stimulation of the carotid body of rabbits (Milsom and Sadig, 1983), lambs (Jansen et al., 1986),and cats (Mulligan et al., 1986) results in an increase in ventilation. Denervation of the carotid body of lambs inhibits the ventilatory response to catecholamines. Catecholamines have no effect on the carotid body activity but stimulate ventilation in goats (Hudgel et al., 1986). Denervation of the carotid and aortic bodies of cats does not inhibit the increase in ventilation following catecholamine infusion (Eldridge and Gill-Kumar, 1980). The increase in ventilation could not be explained by the associated brain acidosis caused by catecholamines (Eldridge et al., 1985).Thus it was concluded that there was a central stimulation of breathing by catecholamines in some mammals. I n others, peripheral stimulation of chemoreceptors by catecholamines was of greater importance. In the lamb the blood brain barrier appears to be complete and there is no central effect of circulating catecholamines, hence only peripheral mechanisms can respond to changes in circulating catecholamines. In cats perhaps circulating catecholamines can cross the blood brain barrier and have a central, as well as peripheral, action. The situation in fish is much less clear and is based on only a few studies. Catecholamines have no direct effect on activity from trout gill oxygen chemoreceptors (Burleson, 1991). Amia with denervated gill chemoreceptors show no response to NaCN either peripherally (McKenzie et al., 1991b) or centrally (Hedrick et d.,1991)but still retain a ventilatory response to catecholamines. This rather fragmentary and sparse evidence indicates that catecholamines in fish do not stimulate ventilation via action on peripheral chemoreceptors. Catecholamines have a marked effect on the circulation and changes in breathing could be secondary to changes in blood flow, pressure, or both. S. Aota (unpublished observations), however, found that infusion of low doses of catecholamines (3-5 nM) resulted in a rise in blood pressure but no change in breathing, indicating that the two responses were separate. At moderate (physiological) doses of catecholamines there was a hyperventilation, as well as an increase in blood pressure, whereas at high levels there was no effect on ventilation but a very marked increase in blood pressure. Kinkead et al. (1991) observed no change in breathing associated with modulations of blood pressure during hypoxia and concluded that adrenergically medi-

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ated changes in blood pressure had no effect on breathing in the hypoxic cod. Hedrick et al. (1991)were unable to stimulate breathing by altering the pH, oxygen, or carbon dioxide content of the extradural fluid of Amia brains. They could find no evidence for central pH/C02 or oxygen receptors. This is despite the fact that many investigators have observed correlations between blood pH and ventilation (Janssen and Randall, 1975). In many fish, however, acid infusion is associated with a rise in circulating catecholamines. If hyperoxic fish are made acidotic, catecholamines are not released and ventilation is in fact reduced, presumably due to the rise in blood oxygen content. Aota et al. (1990) concluded that the increase in ventilation following acid infusion was due to the action of catecholamines but changes in blood oxygen content could also be involved. These experiments did not differentiate between the effects of catecholamines and hypoxemia. Circulating noradrenaline can cross the blood brain barrier in fish (Peyreaud-Waitzeneggar et al., 1980; Nekvasil and Olsen, 1986a) and stimulate fictive ventilation when injected into the caudal vein or directly into the fourth ventricle of the dogfish brain (Fig. 7) (Randall and Taylor, 1991). Injections of noradrenaline close to the cell body of a respiratory motorneurone in the dogfish medulla caused an increase in its firing rate, which occurs within 3-5 sec of the injection (Randall and Taylor, 1991). This was in contrast to much longer laten-

Adr

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I

, , I

I

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Fig. 7. Effect of injection of adrenaline into the fourth ventricle of a dogfish (male Squalus ucunthias, 1260) upon bursting efferent activity recorded from the central cut end of the third branchial branch ofthe vagus. Injection of20 pl of lo-* mol . I-' adrenaline (Adr), after a delay of about 100 sec, induced slow bursts of increased amplitude, which progressively increased in rate. Repeated injections of adrenaline together with propranolol (AdrProp) abolished the stimulatory response, and on the second injection may have unmasked an a-adrenergic inhibition of efferent respiratory activity. [From Randall and Taylor (1991).]

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cies following infusion into the blood or the fourth ventricle of the brain, presumably because noradrenaline must first cross the blood brain barrier before it can act on these central respiratory sites. The short latency also indicates that the site of action is in the region of the respiratory motoneurons. This central effect of catecholamines can be blocked by infusion of propranolol into the blood, indicating that not only is the effect via P-adrenoreceptors but that the site is accessible from the blood. Thus, it seems possible that there is a central respiratory site sensitive to catecholamines from the blood that may be involved in regulating gill ventilation in fish. The importance of this mechanism has been debated, with Randall and Taylor (1992) arguing for and Perry et al. arguing (1992) against physiological relevance.

E. Catecholamines and Ion Movements McDonald and Rogano (1986) showed that during a 2-h period of epinephrine infusion, NaCl efflux was initially elevated by about 6fold above resting levels, declined to resting levels about 15 min after the onset of infusion, and remained at that level for the rest of the infusion period. Vermette and Perry (1987), also based on infusion studies, concluded that catecholamines were involved in anion and cation transfer across fish gills. Despite the fact that catecholamines are involved in ion transfer across the gills, Vermette and Perry (1988) could find no positive evidence that catecholamines played a role in pH regulation during hypercapnic exposure in trout nor could Perry et al. (1988a) find any evidence for a role for catecholamines in calcium balance in fish exposed to low calcium water. Catecholamines may be involved in the retention of lactate in muscle following exhaustive exercise in plaice (Wardle, 1978), but this has not been confirmed in other fish (Wood and Milligan, 1987; Tang et al., 1989). Catecholamines have a marked effect on erythrocytic pH due to P-adrenoreceptor activation of NaIH exchange, as discussed earlier (see Nikinmaa, 1990; Motais et al., 1990; for further discussion and review). F. Catecholamines and Blood Flow and Distribution The role of circulating catecholamines on the cardiovascular system has been reviewed by Nilsson (1983, 1984), and some aspects are

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discussed in Part A, Chapter 1by Farrell and Jones. Adrenergic control of the heart is largely neural, and it is only during stresses such as exhaustive exercise (Primmett et al., 1986)or severe hypoxia (Boutilier et al., 1988) that circulating levels may increase enough to have an effect on the heart (Axelsson and Nilsson, 1986; Butler et al., 1989). A neural innervation of the heart is present in most teleosts but absent in elasmobranchs (Nilsson, 1983; Nilsson and Holmgren, Chapter 5). Adrenergic stimulation generally elicits positive inotropic and chronotropic effects on the heart, but under some conditions, in some species, negative chronotropic effects have been observed (see Farrell and Jones, Part A, for further discussion). There is some adrenergic tone to the teleost heart, but this appears to be less important than cholinergic tone during both rest and exercise (Axelsson and Nilsson, 1986; Axelsson, 1988). The vasculature of teleosts, unlike that of elasmobranchs, is usually well innervated by adrenergic nerves. Catecholamine infusion usually evokes a rise in blood pressure and a vasoconstriction mediated by a-adrenoreceptors. In addition, there is a pool of P-adrenoreceptors that cause a vasodilation when stimulated (Nilsson, 1983). Vascular resistance in the systemic circuit is modulated by adrenergic tone; circulating catecholamines have an effect only under very adverse conditions (Wood and Shelton, 1975; Smith et al., 1985; Axelsson, 1988). Exercise results in an increase in adrenergic tone to the heart and the rise in blood pressure is modulated by neurally mediated a-adrenergic vasoconstriction in the systemic circuit but a vasodilation in the branchial circuit (Jones and Randall, 1978). Gut blood flow increases following feeding but decreases during exercise (Axelsson et al., 1989).These changes in gut blood flow are modulated, at least in part, by adrenergic fibers innervating a-adrenoreceptors that cause vasoconstriction. G. Carbon Dioxide Transport

The pattern of carbon dioxide transport in blood and the mechanisms of its excretion across the gill epithelium have been reviewed (Perry, 1986; Perry and Wood, 1989; Perry and Laurent, 1990; Randall, 1990). As blood arrives at the gills, plasma HC03- is rapidly dehydrated by carbonic anhydrase within red blood cells to form molecular COZ. The rate-limiting step in the overall conversion of plasma HC03to COZ is probably the entry of HC03- into the red blood cell (Perry et al., 1982) in exchange for C1- via the band 3 anionic exchanger (Romano and Passow, 1984). The COe thus formed diffuses from the red

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cell into the plasma and across the gill lamellar epithelium into the water according to existing COz partial pressure gradients. Relatively few studies have addressed the potential impact of elevated circulating catecholamines on COZ excretion or blood COZ transport. According to theory, however, activation of the red blood cell Na+/H+exchanger by catecholamines is expected to impair branchial carbon dioxide excretion and markedly affect blood COz transport. A transient inhibition of COz excretion is predicted for the following reasons. First, the passive entry of HCO3- into the red cell must be reduced owing to a rise in the intracellular levels of HC03-- associated with the abrupt alkalinization of the cell interior (see previous discussion). This, in turn, would slow the rate of formation of excretory COz. Second, the PC02 gradient between the plasma and red blood cell must be temporarily reversed owing to a reduction of intracellular PCOZ (see Thomas and Perry, 1991).At such times, the red blood cell is excluded from any role in net C02 excretion. Third, the dehydration of HC03- to COZ within the red blood cell must be temporarily “rate-limited” by the availability of H owing to the enormous consumption of H + by the Na+/H+ exchanger. The extent of this reduction of bicarbonate dehydration will depend on the relative rates of Nat/H+ exchange and the catalyzed bicarbonate dehydration reaction during the early phases of oxygenation and subsequently on the relative rates of Na+/H+transfer and Cl-/HC03- exchange. There would appear to be some adrenergically mediated expulsion of the protons liberated from the hemoglobin into the plasma because there is a large, but slow, increase in blood PaCOz after exhaustive exercise (Perry and Wood, 1989)that is reduced by infusion of carbonic anhydrase into the blood. The notion of adrenergic inhibition of COz excretion remains controversial with considerable empirical evidence both for and against the hypothesis as originally proposed by Wood and Perry (1985). Direct measurements of COz excretion in vivo (Steffensen et al., 1987; Playle et al., 1990) or in vitro (Tufts et al., 1988) failed to provide evidence in favor of adrenergic impairment. On the other hand, in vitro studies (Perry et al., 1991a; Wood and Perry, 1991)clearly demonstrate an inhibitory effect of catecholamines on red blood cell COz excretion and establish that the underlying mechanism is related to adrenergic activation of the Na+/H+ exchanger. A difference between the in vitro and in vivo studies is the absence of proton production as a result of hemoglobin oxygenation that occurs in vivo simultaneously with bicarbonate dehydration. This probably explains the differences between the in vitro and in vivo results. Elevation of circulating catecholamines markedly affects blood car+

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bon dioxide transport by the concerted actions of ( a )inhibition of COZ excretion and ( b )titration of plasma HC03- by Hf extruded by the red blood cell Na+/H+ exchanger. Both of these effects contribute to an elevation in blood Pco, during intravascular infusion of exogenous, or release of endogenous, catecholamines (Perry and Vermette, 1987; Vermette and Perry, 1988; Perry and Thomas, 1991) that cannot be otherwise explained by ventilatory adjustments (see Fig. 8). It has 2.6 2.4 2.2

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PaO, (tom) Fig. 8. P , O , - P , ~ o , diagrams obtained from continuous recordings of arterial blood respiratory status of rainbow trout using an extracorporeal loop. Injections of exogenous adrenaline during normoxia and hypoxia (inset) or the release of endogenous catecholamines during hypoxia cause pronounced deviations from the predicted P,~,-P,~o, relationship shown in (A) and reproduced in (B). In each case, the elevation of circulating catecholamines is associated with a rise of arterial PC02 without an appropriate accompanying change in arterial Po,. These data indicate that the origin of the adrenergic respiratory acidosis is unrelated to ventilatory or other branchial adjustments but likely reflects activation of red blood cell Na+/H+exchange. See text for further details. [Data from S. F. Perry and S. Thomas (unpublished observations).]

4. CATECHOLAMINES

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been suggested (e.g. Wood and Perry, 1985; Perry and Wood, 1989) that the rise in arterial blood PCO, associated with the recovery from exhaustive exercise in fish may partially result from the effects of catecholamines on blood COZ transport and excretion. The functional significance of the catecholamine-induced effects on arterial blood COZ transport is unknown although it has been proposed (Perry and Wood, 1989) that the associated respiratory acidosis may assist in the stimulation of ventilation during recovery from exhaustive exercise.

H. General Conclusions Concerning the Action of Circulating Catecholamines Catecholamines may be involved in regulating gill water flow, gill diffusing capacity, blood oxygen content, and blood flow and distribution, as well as energy turnover. The metabolic effects appear to be to maintain function in the face of adverse conditions rather than to increase metabolism. The same can be said to be true for both cardiac function (see Part A, Chapter 1)and oxygen transfer. The evidence is implied rather than direct, increasing catecholamine levels have a marked effect on each ofthese factors. In a study on dogfish in which an increase in circulating catecholamines during hypoxia was inhibited, no marked effect on oxygen transfer was observed (Metcalfe and Butler, 1989). Randall et al. (1987), however, observed that fish swim as well after exhaustive exercise as before, even though they are acidotic, whereas fish in acidic water do not swim as well as in neutral water (Ye and Randall, 1991). Fish in acid water become acidotic but do not release catecholamines, whereas catecholamines are released after exhaustive exercise and the fish can maintain oxygen delivery to the tissues. IV. FACTORS INFLUENCING ACTIONS OF CATECHOLAMINES Fish exhibit large intra- and interspecific differences in both the nature of catecholamine mobilization in response to stress and the responsiveness of target tissues to the catecholamines. These differences are related to diurnal and seasonal cycles, interactive effects of other hormones, acclimation history, and the prevailing internal respiratory/acid-base status. The quantities of catecholamines stored within tissues and circulating in the plasma vary diurnally and seasonally both under resting

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conditions and in response to stress. At rest, diurnal or daily fluctuations (Boehkle et al., 1967; Le Bras, 1984; Ehrenstrom and Johansson, 1987)are probably more important than seasonal variations (Van Dijk and Wood, 1988; Milligan et al., 1989; Temma et al., 1990). Indeed, there is little agreement on the influence of season on resting plasma catecholamine levels; for example, the unusually high values in rainbow trout plasma reported by Van Dijk and Wood (1988) were attributed to “winter” acclimation yet Milligan et al. (1989)were unable to demonstrate any effects of seasonality on resting levels in trout. Similarly, the effects of season on stress-induced release of catecholamines are poorly understood. Milligan et al. (1989) observed an obvious reduction in the circulating levels of noradrenaline yet an increase in the levels of adrenaline associated with exhaustive exercise in winter acclimated rainbow trout. On the other hand, Van Dijk and Wood (1988) measured extremely high values of both catecholamines after exhaustive exercise that, again, were attributed to winter acclimation. Seasonality may influence the pattern of catecholamine release in response to environmental disturbances owing to temperature related effects on metabolism and blood oxygen transport because the internal oxygen status (or a closely related variable) is a key factor controlling catecholamine release (see previous discussion). Thus, the water PO, threshold for catecholamine release during hypoxia is expected to be directly proportional to temperature from the combined effects of increased metabolic rate and decreased affinity of hemoglobin-oxygen binding. This idea has not been tested directly, although a comparison of two studies on hypoxic trout (Pwo, = 60 torr) performed at 12°C (Thomas et al., 1991) or 4°C (S. D. Reid and S. F. Perry, unpublished observations) reveals that significantly greater quantities of catecholamines were released at the warmer water temperature (see Fig. 9). Diurnal, seasonal, or long-term elevations of plasma catecholamines resulting from chronic stress may significantly affect the adrenergic responsiveness of target tissues by desensitization or down regulation” of adrenoreceptors (Lefkowitz et al., 1990) although this has yet to be clearly demonstrated in fish. An often cited example of seasonal effects on adrenergic function is the variable responsiveness of the teleost red blood cell Na+/H+ exchanger to catecholamines (Nikinmaa and Jensen, 1986; Van Dijk and Wood, 1988;Cossins and Kilbey, 1989).Although not always obvious (Tetens et al., 1988; Milligan et al., 1989),it would appear that the red blood cells of winter-acclimated fish are considerably less responsive to adrenergic stimulation than summer-acclimated fish. The reasons for these seasonal differences remain uncertain but may involve changes “

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Time (h) Fig. 9. The effects of long-term (48 h) exposure to moderate environmental hypoxia (Pwo2 = 60 torr, open symbols) or normoxia (closed symbols) on plasma catecholamine (adrenaline and noradrenaline) levels in rainbow trout acclimated to (A) winter conditions (temperature = 12°C) or (B) summer conditions (temperature = 4°C). All values shown are means 2 1 SEM. [Data from Thomas et al. (1991)and S. G . Reid and S. F. Perry (unpublished observations).]

in cell surface P-adrenoceptor numbers, affinities, or both (Marttila and Nikinmaa, 1988). Changes in the surface population of red blood cell P-adrenoceptors may also partially explain the enhanced adrenergic responsiveness during acute hypoxia (Motais et al., 1987; Marttila and Nikinmaa,

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1988; Fuchs and Albers, 1988; Salama and Nikinmaa, 1990; Reid and Perry, 1991). In both trout (Reid and Perry, 1991) and carp (Marttila and Nikinmaa, 1988),short-term exposure of red blood cells to severe hypoxia facilitates the recruitment of internal cytomplasmic p-adrenoceptors (Reid et al., 1991) to the cell surface where they become functionally coupled to adenylate cyclase. Interestingly, chronic exposure of fish to hypoxia appears to desensitize red blood cell adrenergic responsiveness (Thomas et al., 1991) and is perhaps related to down regulation of P-adrenoceptors. The sensitivity of teleost red blood cells to catecholamines is also related to the extracellular acid-base status with increasing sensitivity at lower p H values (Nikinmaa et al., 1987; Borgese et al., 1987; Cossins and Kilbey, 1989). There is also a marked influence of season on the cardiovascular (e.g. Part et al., 1982) and ventilatory responses (Peyreaud-Waitzenegger et al., 1980) to catecholamines. Specifically, in “summer”acclimated fish, P-adrenoceptor mediated responses are dominant while in winter-acclimated fish, a-adrenoceptor mediated responses appear to be relatively more important. The reason(s) for such seasonal switches in adrenoceptor dominance is unknown but may involve alterations in the proportions of adrenoceptor subtypes present in particular target tissues. The heart from a cold-acclimated rainbow trout shows a greater sensitivity to adrenaline than that from a warmacclimated fish. This increase in sensitivity is probably due to the twofold increase in P-receptors in the sarcolemma with no change in the total P-receptor population in hearts from cold-acclimated fish (Keen, 1992). Little is known about the interactive effects of noncatecholamine hormones on adrenergic function. The glucocorticoid, cortisol, however, has been shown to enhance the responsiveness of trout red blood cells (Reid and Perry, 1991) and hepatocytes (Reid et al., 1991)to catecholamines specifically by increasing the abundance of cell surface P-adrenoceptors. It is possible that chronic stress and the associated elevation of plasma cortisol levels may increase the ability of fish to physiologically adapt to any subsequent acute stresses, at least with respect to the adrenergic stress responses. REFERENCES Abrahamsson, T. (1979). Phenylethanolamine-N-methyltransferase (PNMT) activity and catecholamine storage and release from chromaffin tissue of the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. C 64, 169-172.

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Abrahamsson, T. (1980). The effect of SK and F 64139, an inhibitor of' phenylethanolamine-N-methyl transferase (PNMT), on adrenaline and noradrenaline content in sympathetic neurons of the cod Gadus morhua. Comp. Biochem. Physiol. C 67, 49-54. Abrahamsson, T., and Nilsson, S. (1976). Phenylethanolamine-N-methyltransferase (PNMT) activity and catecholamine content in chromaffin tissue and sympathetic neurons in the cod, Gadus morhua. Acta Physiol. Scand. 96,94-99. Aota, S., Holmgren, K. D., Gallaugher, P., and Randall, D. J . (1990).A possible role for catecholamines in the ventilatory responses associated with internal acidosis or external hypoxia in rainbow trout Oncorhynchus mykiss. J . E x p . Biol. 151,57-70. 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 nonexhaustive exercise. J . E x p . Biol. 137,287-303. Axelsson, M., and Nilsson, S. (1986). Blood pressure control during exercise in the Atlantic cod, Gadus morhua. J. E x p . Biol. 126,225-236. Axelsson, M., Driedzic, W. R., Farrell, A. P., and Nilsson, S. (1989).Regulation ofcardiac output and gut blood flow in the sea raven, Hemitripterus americanus. Fish Physiol. Biochem. 6,315-326. Blaschko, H. (1939). The specific action ofL-DOPA decarboxylase.]. Physiol. (Lond.) 96, 50-51. Boehkle, K. W., Tiemeier, 0. W., and Elephteriou, B. E. (1967). Diurnal rhythm in plasma epinephrine and norepinephrine in the channel catfish (Ictalurus punctatus). Gen. Comp. Endocrinol. 8, 189-192. Borgese, F., Garcia-Romeu, F., and Motais, R. (1987). Ion movements and volume changes induced by catecholamines in erythrocytes of rainbow trout: Effect of pH. J. Physiol. (Lond.)382, 145-157. Boutilier, R. G., Iwama, G. K., and Randall, D. J . (1986).The promotion ofcatecholamine release in rainbow trout, Salmo gairdneri, by acute acidosis: Interaction between red cell pH and haemoglobin oxygen carrying-capacity.]. E x p . Biol. 123,145-157. Boutilier, R. G., Dobson, G. P., Hoeger, U., and Randall, D. J . (1988).Acute exposure to graded levels of hypoxia in rainbow trout (Salmo gairdneri): Metabolic and respiratory adaptations. Respir. Physiol. 71,69-82. Burleson, M. L., and Smatresk, N. J. (1990). Effects of sectioning cranial nerves IX and X on cardiovascular and ventilatory reflex responses to hypoxia and NaCN in channel catfish. J. E x p . Biol. 154,407-420. Burleson, M. L. (1991). Oxygen-sensitive chemoreceptors and cardiovascular and ventilatory control in rainbow trout. Ph.D. Thesis, University of British Columbia. Busacker, G. P., and Chavin, W. (1977). Uptake, distribution, and turnover of catecholamine radiolabel in the goldfish, Carassius auratus L. Can. J . Zool. 55, 16561670. Butler, P. J., Axelsson, XI., Ehrenstrom, F., Metcalfe, J. D., and Nilsson, S. (1989). Circulating catecholamines and swimming performance in the Atlantic cod, Gadus morhua. J . E x p . Biol. 141,377-387. Butler, P. J . , Metcalfe, J. D.,and Ginley, S . A. (1986).Plasma catecholamines in the lesser spotted dogfish and rainbow trout at rest and during different levels of exercise. /. E x p . Biol. 123,409-421. Butler, P. J . , Taylor, E. W., Capra, M. F., and Davison, W. (1978).The effect of hypoxia on the levels of circulating catecholamines in the dogfish Scyliohinus canicula. J . Comp. Physiol. 127,325-330. Butler, P. J., Taylor, E. W., and Davison, W. (1979). The effect of long term, moderate hypoxia on acid-base balance, plasma catecholamines and possible anaerobic end

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Tufts. B. L., Ferguson, R. A., and Boutilier, R. G. (1988).In tjiuo and in uitro effects of adrenergic stimulation on chloride/bicarbonate exchange in rainbow trout erythrocytes./. E x p . Biol. 140,301-312. Ungell, A. L. (1985a).Accumulation of intra-arterially administered [3Hladrenaline and [3H]noradrenaline in various tissue of the Atlantic cod, Gadus morhua. C o m p . Biochem. Physiol. C 81,25-28. Ungell, A. L. (198513).Uptake of 3H-adrenaline and 14C-noradrenaline into neuronal and extraneuronal tissue compartments in the perfused gas gland of the swimbladder of the Atlantic cod, Gadus m0rhua.J.Comp. Physiol. B 155,479-485. Ungell, A. L., and Nilsson, S. (1979). Metabolic degradation of I3H]-adrenaline in the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. C 64, 137-141. Ungell, A. L., and Nilsson, S. (1983). Catabolism and excretion of [3H]adrenaline in the spiny dogfish, Squalus acanthias. C o m p . Biochem. Physiol. C 74,319-322. Val, A. L. (1991). Adaptations of fishes to extreme conditions in freshwaters. In “The Vertebrate Gas Transport Cascade: Adaptations to Environment and Mode of Life (E. Bicudo, ed.). (In press.) Van Dijk, P. L. M., and Wood, C. M. (1988). The effect ofp-adrenergic blockade on the recovery process after strenuous exercise in the rainbow trout, Salmo gairdneri Richardson. J . Fish Biol. 32,557-570. Vermette, M. G., and Perry, S. F. (1987). The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo Gairdneri. J. E x p . Biol. 128,255-267. Vermette, M. G., and Perry, S. F. (1988). Effects of prolonged epinephrine infusion on blood respiratory and acid-base states in the rainbow trout: Alpha and beta effects. Fish. Physiol. Biochem. 4(4), 189-202. Wahlqvist, I. (1981). Branchial vascular effects of catecholamines released from the head kidney of the Atlantic cod, Gadus morhua. Molec. Physiol. 1,235-241. Wahlqvist, I., and Nilsson. S. (1980).Adrenergic control of the cardio-vascular system of the Atlantic cod, Gadus morhua, during “stress.”/. C o m p . Physiol. B 137, 145-150. Wardle, C. S. (1978). Non-release of lactic acid from anaerobic swimming muscle of plaice, Pleuronectes platessa L.: A stress reaction./. E x p . Biol. 77, 141-155. Wood, C. M. (1991). Acid-base and ion balance, metabolism, and their interactions after exhaustive exercise in fish./. E x p . Biol. 160,285-308. Wood, C. M., and Milligan, C. L. (1987).Adrenergic analysis of extracellular and intracellular lactate and H + dynamics after strenuous exercise in the starry flounder, Platichthys stellatus. Physiol. 2001.60,69-81. Wood, C. M., and Perry, S. F. (1985).Respiratory, circulatory and metabolic adjustments to exercise in fish. I n “Circulation, Respiration and Metabolism” (R. Gilles, ed.), pp. 1-22. Springer-Verlag, Berlin, Heidelberg. Wood, C. M., and Perry, S. F. (1991). A new in uitro assay for C 0 2 excretion by trout red blood cells: Effect of catecho1amines.J. E x p . Biol. 157, 349-366. 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 x p . Biol. 63,505-523. Wood, C. M., Walsh, P. J., Thomas, S., and Perry, S. F. (1990). Control of red blood cell metabolism in rainbow trout (Oncorhynchus mykiss) after exhaustive exercise. /. E x p . Biol. 154,491-507. Wright, P. A., Perry, S. F., and Moon, T. W. (1989).Regulation ofhepatic gluconeogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia./. E x p . Biol. 147, 169-188. ”

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Yamamoto, K., Itazawa, Y., and Kobayashi, H. (1985). Direct observation offish spleen by an abdominal window method and its application to exercised and hypoxic yellowtail. J a p .J . Icth. 31,427-433. Ye, X., and Randall, D. J. (1991). The effect of water pH on swimming performance in rainbow trout (Salmo gairdneri, Richardson). Fish. Physiol. Biochem. 9, 15-21.

5 CARDIOVASCULAR CONTROL BY PURINES, 5HYDROXYTRYPTAMINE, AND NEUROPEPTIDES STEFAN N I L S S O N A N D S U S A N N E H O L M G R E N Department of Zoophysiology University of Goteborg Giitehorg, Sweden

I. Introduction 11. Origin of Vasomotor and Cardiac Nerves A. Vasomotor Nerves B. Cardiac Innervation 111. Purines A. Purine Derivatives B. Purinergic Nerves C. Purine Actions on the Heart D. Purine Actions on the Vasculature IV. 5-Hydroxytryptamine (Serotonin) A. 5-HT in Cyclostomes B. 5-HT in Elasmohranchs C. 5-HT in Teleosts V. Neuropeptides A. Vasoactive Intestinal Polypeptide 13. Bombesin C. Neuropeptide Y D. Somatostatin E. Substance P F. Galanin G. Gastrin/Cholecystokinin V l . Endothelial Factors References

I. INTRODUCTION Although cardiovascular ph rsiology in nonmammalian rertebrates and its relation to respiration is a major area of comparative physiology, the systems that control the cardiovascular functions have been given 301 FISH PHYSIOLOGY, VOL XIIB

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modest attention. Knowledge of vasomotor control in fish is still fragmentary: Apart from studies on certain peptides with major functions in cardiovascular control in mammals (notably angiotensin and atrial natriuretic peptide; see Chapter 3), research on cardiovascular control systems has been largely restricted to adrenergic vasomotor control (via adrenergic neurons and circulating catecholamines; see also other chapters in this volume) and the double antagonistic cholinergic (inhibitory) and adrenergic (excitatory) innervation of the teleost heart. Only recently has data started to accumulate, which demonstrate the involvement in cardiovascular control of a number of neuropeptides, and also amines (5-hydroxytryptamine, serotonin) and certain purine derivatives (adenosine and its nucleotides). With the exception of the branchial innervation in some teleosts, vasomotor nerves in fish appear to be derived solely from the spinal autonomic (“sympathetic”) division of the autonomic nervous system. These fibers were previously regarded as solely adrenergic (i.e., releasing adrenaline, and/or noradrenaline as the neurotransmitter). Increasing evidence does, however, suggest that vasomotor control in many organs, especially the gut, may also depend on several types of spinal autonomic neurons that release nonadrenergic, noncholinergic (NANC) transmitters (alone or co-released with catecholamines or acetylcholine; see later). The concept of neurons releasing a transmitter substance other than the classical (adrenaline/noradrenaline or acetylcholine) arose in the 1960s through work on the guinea pig gut by Greeff et al. (1962) and Burnstock et al. (1963,1964). The nature of these NANC neurons is, in many cases, still not at all clear although a number of candidates, notably the neuropeptides (sometimes also known as “brain-gut peptides” or “regulatory peptides”), have been proposed as putative neurotransmitters, also in the cardiovascular system (Burnstock and Griffith, 1988). General descriptions of the autonomic innervation patterns in fish and other vertebrates are given by Nicol(1952), Burnstock (1969), and Nilsson (1983). In this chapter, we give a brief description of the origin of vasomotor and cardiac nerves in fish outlining the “classical” adrenergic and cholinergic pathways. The status of knowledge regarding purines, 5-hydroxytryptamine (serotonin), and neuropeptides in the cardiovascular system of fish is discussed. However, the available information is still fragmentary, and parallels need to be drawn with the situation in mammals where the functions of NANC neurotransmitters and their relations to the adrenergic and cholinergic systems are better understood.

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11. ORIGIN OF VASOMOTOR AND CARDIAC NERVES The autonomic nervous system has a major function in the control of the cardiovascular system in all vertebrates, with the possible exception of some cyclostomes (myxinoids) that have a poorly developed autonomic nervous system. Nerve fibers from the cranial autonomic division (“parasympathetic nerves”) reach the heart in the paired vagus nerve, and fibers from the sympathetic chains (spinal autonomic nervous system or sympathetic nervous system) either run in separate nerves to the heart (for instance, the nervi acceleruntes of mammals) or join the vagi and run as a “vago-sympathetic trunk” to the heart. Spinal autonomic (sympathetic) fibers run to the viscera in the splanchnic nerves, which consist of postganglionic neurons in fish, or join the spinal nerves and run to somatic vascular beds. A detailed account of the structure and function of the autonomic nervous system of fishes can be found in Nilsson (1983). A brief summary of the origins of the vasomotor and cardiac nerves is offered in Fig. 1. A. Vasomotor Nerves

1. CYCLOSTOMES Knowledge of the structure of the autonomic nervous system of cyclostomes is fragmentary, and differentiation between autonomic and sensory neurons is hard to make. Visceral branches from the ventral spinal nerves in the hagfish, Myxine glutinosu, probably carry vasomotor fibers (Nicol, 1952; Fange et al., 1963; Campbell, 1970). In the lamprey, Lampetra sp., vasomotor fibers that may be regarded as sympathetic leave the central nervous system (CNS) in both the dorsal and ventral spinal nerves (Tretjakoff, 1927; Nicol, 1952). Histochemical evidence suggests that these fibers are adrenergic (Leont’eva, 1966; Govyrin, 1977). There is no evidence for vasomotor innervation of the branchial vasculature of cyclostomes. 2. ELASMOBRANCHS The paravertebral autonomic ganglia in elasmobranchs are arranged segmentally, but longitudinal connections are irregular, which means that true sympathetic chains of the type found in teleosts and tetrapods are absent. The most anterior sympathetic ganglia on each side form, together with masses of chromaffin cells, the axillary bodies. These contain large quantities of catecholamines, mainly in the chro-

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Gyclostome

Elasmobranch

I I I

@ artery

I

KI9

vein heart

Dipnoan

artery

artery

vein heart

Teleost

heart

artery

heart

Fig. 1. Simplified diagrammatic representation of the origins of'the vasomotor and cardiac nerves in fish from four different groups. Preganglionic neurons are shown as solid lines, while the postganglionic neurons are shown as broken lines. Chromaffin tissue occurs in the (large) veins of all four groups and in cyclostomes and dipnoans also within the heart and possibly in some arteries (such as the intercostal arteries of Protopterus). The vagal innervation of the heart is inhibitory in all vertebrates except cyclostomes (lampetroids) and may be lacking (myxinoids). Note that the sympathetic chains in teleosts continue into the head, bearing ganglia that send gray rami communicantes into the cranial nerves. cc, chromaffin cells; grckpl, gray rami communicantes or splanchnic nerves; sc, sympathetic chains; sg, sympathetic ganglion; wrc, white rami communicantes, X, vagus nerve.

maffin tissue, which can be released to the blood within the posterior cardinal sinuses (Nicol, 1952; Young, 1933; Nilsson, 1983; Nilsson and Holmgren, 1988). The elasmobranch paravertebral ganglia are connected to the spinal nerves via white rami communicantes. Recurrent gray rami are absent in elasmobranchs, and vasomotor fibers to the somatic vasculature, if such an innervation does indeed exist, must take other pathways. Splanchnic nerves, carrying vasomotor (and other) fibers to the

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viscera, are plentiful in the elasmobranchs, and there is thus a rich autonomic innervation of spinal autonomic (sympathetic) origin. Early anatomical reports suggested an innervation of the branchial vasculature of elasmobranchs via the cranial nerves (Nicol, 1952). However, later examination has shown that effects on gill blood flow observed during stimulation of the branchial nerves can be attributed to contractions of skeletal muscle of the gill arch (Metcalfe and Butler, 1984). Spinal autonomic pathways do not enter the head in elasmobranchs, and there is, therefore, no evidence for an autonomic innervation of the branchial vasculature in these fish (Nilsson, 1983). 3. TELEOSTS AND OTHERACTINOPTERYGIANS In teleosts and other actinopterygians, there are well-developed sympathetic chains and, at least in teleosts and some ganoids, these chains continue into the head bearing ganglia in connection with the cranial nerves. Both white and gray rami communicantes are present, and fibers of spinal autonomic (sympathetic) origin thus run in the spinal nerves to the systemic blood vessels. Although there are no white rami connecting the sympathetic chains to the cranial nerves, autonomic fibers from the spinal segments of the sympathetic chains can run forward in the chains and enter the cranial nerves via gray rami communicantes. A spinal autonomic (sympathetic) vasomotor innervation in those parts of the body that are innervated by the cranial nerves (or rather cranio-sympathetic nerve trunks) is thus possible (Nicol, 1952; Nilsson, 1983). In addition to the vasomotor control exerted by fibers that originate in the sympathetic chains, there is good evidence for a cranial (parasympathetic) innervation of the branchial vasculature (see extensive reviews by Laurent (1984)) and Nilsson (1984)). Histochemical and electron microscopical studies of the innervation of the branchial vasculature of teleosts has revealed the presence of cholinergic-type nerve profiles, that also show strong acetylcholinesterase activity, in the sphincter at the base of the efferent filamental arteries (Bailly and Dunel-Erb, 1986) (Fig. 2). These findings are in concert with physiological studies that demonstrate a cholinergic constriction of the arterioarterial vascular pathway of the gills (Smith, 1977, 1978; Pettersson and Nilsson, 1979; Nilsson and Pettersson, 1981). Formaldehyde-induced fluorescence histochemistry (FalckHillarp technique) and electron microscopy have been used to show the distribution of adrenergic nerves in the gills of several teleost species (Donald, 1984, 1987; Dunel-Erb and Bailly, 1986; Nilsson,

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EFA

AFA

Fig. 2. Simplified and generalized diagram showing the distribution of cholinergic (ACh: solid line), serotonergic (5-HT-immunoreactive:dotldash lines), and adrenergic (Adr, formaldehyde induced fluorescence: dashed lines) fibers in the teleost gill. Serotonergic fibers running in the branchial nerve innervate the sphincter at the base of the efferent filamental artery, the bases of the efferent filamental arterioles and the central venous sinus. The occurrence ofadrenergic nerve terminals varies with species. In many species, an innervation of the afferent filamental arteries, afferent lamellar arterioles, and the central venous sinus and nutritive vasculature has been observed, and in some species there are also occasional fibers to the efferent lamellar arterioles. ABA, EBA, afferent and efferent branchial artery; AFA, EFA, afferent and efferent filamental artery; ALa, ELa, afferent and efferent lamellar arteriole; BV, branchial vein; CVS, central venous sinus; FC, filamental cartilage; Sph, sphincter at the base of the efferent filamental artery. [Figure based primarily on histochemical data from Bailly et al. (1989)and Donald (1984, 1987).]

1986; Dunel-Erb et al., 1989). An innervation of the branchial arteries b y fluorescent nerve fibers was observed in the carp (Cyprinus carpio) only, while an innervation of the afferent filamental artery and lamellar arterioles appears to be a general feature of the teleost gill. In addition, there are generally dense adrenergic plexuses in the nutritive vasculature and central venous sinus of the gill filament. B. Cardiac Innervation

With the probable exception of the myxinoids, all vertebrates possess a vagal innervation of the heart. This innervation is excitatory in lampetroids, possibly due to release of local stores of catecholamines

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(adrenaline and noradrenaline), although evidence for an extrinsic innervation of the endogenous catecholamine storing cells is wanting (Caravita and Coscia, 1966; Beringer and Hadek, 1973). In all other vertebrates studied, the vagal innervation is inhibitory. For an in-depth account of the vagal innervation and control of the fish heart, see Chapter 6 in this volume. Other reviews on fish cardiac innervation and control are those by Nilsson (1983), Laurent et al. (1983), and Farrell (1984). The presence of spinal autonomic (sympathetic) pathways to the heart has not been confirmed either in cyclostomes or in elasmobranchs, and anatomical evidence for the existence of such an innervation in dipnoans is ambiguous. In teleosts (with the exception of pleuronectids where they seem to be absent) and the holostean (Lepisosteus platyrhincus),however, there is a well-established cardiac innervation by spinal autonomic fibers that reach the heart chiefly in the vagosympathetic trunks, but that may also run along the first pair of spinal nerves or enter the heart along the coronary arteries (Gannon and Burnstock, 1969; Holmgren, 1977; Nilsson, 1983; Donald and Campbell, 1982). These fibers are excitatory and were originally demonstrated as adrenergic neurons using histochemical and ultrastructural techniques (Govyrin and Leont'eva, 1965; Yamauchi and Burnstock, 1968). 111. PURINES

A. Purine Derivatives

Adenosine 5'-triphosphate (ATP) and its derivatives occur ubiquitously in living cells and serve major functions in the life of the cells. Adenosine 5'-triphosphate is catabolized by the action of various adenosine triphosphatase (ATPases), and the formed adenosine diphosphate (ADP) and adenosine monophosphate (AMP) can be further degraded by 5'-nucleotidase to adenosine and finally deaminated to inosine (Burnstock, 1972). From mammalian systems it is known that ATP from cells of the vascular endothelium may be released in response to disturbances of the endothelium (such as hypoxia) and affect the vascular smooth muscle. This occurs either directly, with ATP activating Pz-purinoceptors that cause contraction of the vessel, or via release of endothelium derived relaxing factor(s) (EDRF) that inhibit the musculature of the media (Fig. 3) (Mione et al., 1990).

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STEFAN NILSSON AND SUSANNE HOLMGREN

Adventitia I

Media I

Endothelium Vascular I lumen

Nerve terminals

Fig. 3. Hypothetical diagram, based mainly on observations from mammals, showing possible relationships in the mechanisms involving various transmitters and factors that control the tension ofthe blood vessel wall. Nerve fibers (vasomotor nerves) running as a plexus at the adventitio-medial border release transmitters, neuromodulators, or both that may affect the smooth muscle of the media directly or control (via autoregulation) the release of neurotransmitters. Catecholamines (CA) from adrenergic nerves or in the form of circulating (humoral) catecholamines normally constrict arteries by acting on a-adrenoceptors, although inhibitory P-adrenoceptors may be dominating in certain vascular beds (e.g., the branchial vasculature of teleosts). Whether released as a cotransmitter from adrenergic nerves, as a transmitter in its own right from purinergic nerves (if such exist in fish), or as ATP or its metabolites from endothelial cells, ATP may act directly on vascular purinoceptors (PI or P2-receptors) of the media, or by stimulating release of endothelium-derived relaxing factor (EDRF) from endothelial cells. It should be emphasized, however, that the presence of an EDRF in fish blood vessels has yet to be confirmed. 5-Hydroxytryptamine (serotonin) is released either as a neurotransmitter from serotonergic vasomotor nerves, from enterochromaffin cells in the gut, or from endothelial cells of the type demonstrated in fish (see Fig. 4). As in the case of ATP, 5 - H T may also exert some of its (inhibitory) effects via the release of EDRF. A number of neuropeptides, exemplified by substance P (SP) and vasoactive intestinal polypeptide (VIP), may also act in a vasoregulatory system directly from nerves or carried in the blood stream, and may also act via factors from the endothelium (EDRF).

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The effects of purine compounds show certain patterns, which have made a classification of the receptors into PI- and Pe-purinoceptors useful. The PI-purinoceptors are more sensitive to adenosine than to ATP, can be blocked by methylxanthines such as caffeine and theophylline, and affect the levels of cyclic adenosine 3’,5‘monophosphate (CAMP). The Pz-purinoceptors are more sensitive to ATP than to adenosine. Quinidine and 2,2’-pyridylisatogen act as antagonists, and the effect is not mediated via the adenylate cyclase system (Burnstock, 1976, 1978). B. Purinergic Nerves One of the first hypotheses regarding the nature of the NANC transmitters was the “purinergic nerve hypothesis,” originally conceived by Burnstock and collaborators (1972, 1975). This hypothesis suggests that ATP or a related purine nucleotide acts as a transmitter of autonomic nerves. Most of the evidence in favor of this hypothesis derives from studies of mammalian systems, notably the gut innervation, although purinergic transmission in other systems, including the cardiovascular system, has also been suggested (Burnstock and Griffith, 1988). It is well known and accepted that ATP is stored and released with other neurotransmitters (e.g., catecholamines), and one function of nervously released ATP (or ATP-like substances) may be that of a neuromodulator that affects synaptic transmission of the “true” transmitter (Burnstock, 1990; Mione et al., 1990). Practically nothing is known about the possible existence of purinergic nerves in the fish cardiovascular system. Thus, the control exerted by purine compounds will be summarized disregarding their origin (nervous, endothelial, metabolic, etc.). C. Purine Actions on the Heart

During periods of hypoxia, ATP resynthesis may be inhibited in some tissues and the concentration of adenosine can increase. Although several studies of the fish heart suggest a close control of the ATP levels [which can be sustained by linkage to creatine phosphate (JGrgensen and Mustafa, 1980; Nielsen and Gesser, 1984; Koke and Anderson, 1986)1, levels of the dephosphorylated adenosine compounds may increase, and the physiological effects of these can be of significance in cardiac control during hypoxia. The pattern of effects of adenosine compounds on the fish heart is, however, not uniform. A positive inotropic response to adenosine occurs in the heart ofthe

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flounder, Platichthys flesus. The effect was antagonized by the PIpurinoceptor antagonist caffeine (Lennard and Huddart, 1989). In the carp (Cyprinus carpio), adenosine and adenine nucleotides produced negative inotropic and chronotropic effects that could be blocked by theophylline, again demonstrating purinoceptors of the PI-variety (Cohen et al., 1981; Rotmensch et al., 1981). Studies of the rainbow trout (Salmo gairdneri = Oncorhynchus mykiss) heart by Meghji and Burnstock (1984a) showed negative inotropic (and positive chronotropic) effects of adenosine and ATP on the atrium: The effects were, however, insensitive to a PI-antagonist, and it was concluded that the rainbow trout heart purinoceptor differs from the type found in other vertebrates (Meghji and Burnstock, 1984a). Studies by the same authors on the heart of the dogfish, Scyliorhinus canicula, show the presence of PI-purinoceptors, mediating negative inotropic and chronotropic effects in the atrium, while the ventricle was largely insensitive to the adenosine compounds (Meghji and Burnstock, 198413). D. Purine Actions on the Vasculature Theophylline-sensitive contraction of coronary artery ring preparations caused by adenosine, ATP, and ADP have been demonstrated in rainbow and steelhead trout (Small et al., 1990; Small and Farrell, 1990), and also in the skate, Raja nasutu, at low concentrations while higher concentrations produced relaxation (Farrell and Davie, 1991b). In contrast, vascular rings from coronary arteries of the maco shark, Zsurus oxyrinchus, relaxed in response to adenosine and ADP (in high concentration). This effect was also inhibited by theophylline (Farrell and Davie, 1991a). The effect of adenosine compounds on other systemic vessels is similarly variable. Adenosine infusion was without effect on the isolated perfused trunk preparation of the rainbow trout (Colin et al., 1979), while Wood (1977) demonstrated systemic vasoconstriction in response to ATP injected in vivo. Adenosine dilates the branchial vasculature in the hagfish, Myxine glutinosa (Axelsson et al., 1990), and several studies have demonstrated effects of adenosine compounds on the branchial vasculature in teleosts. A marked arterioarterial branchial vasoconstriction occurs in the isolated-perfused head preparation of the rainbow trout, and adenosine infusion in vivo also markedly increased the branchial vascular resistance. Isolated gill arches with the filaments removed showed a higher sensitivity in the efferent than in the afferent vessels,

5. PURINES,

5-HT, AND NEUROPEPTIDES

31 1

and the authors implicated the sphincter at the base of the efferent filamental artery (see Fig. 2) in the contractile response (Ristori and Laurent, 1977; Colin et al., 1979). Similarly, adenosine produced theophylline-sensitive vasoconstriction in the isolated perfused gills of Oreochromis niloticus, and in these experiments a vasodilator effect of ATP was also observed (Okafor and Oduleye, 1986). From a thorough pharmacological analysis of the relative activities of a number of adenosine analogues, Colin and Leray (1981) concluded the presence of specific vascular purinoceptors of the gill vasculature. It would thus seem that the branchial vasculature of teleost fish possesses PI-purinoceptors responsible for arterioarterial vasoconstriction in response to adenosine. IV. 5-HYDROXYTRYPTAMINE (SEROTONIN)

5-Hydroxytryptamine (5-HT, serotonin, enteramin), originally described as a vasoactive factor present in blood serum (hence the name serotonin) (Rapport et al., 1948; Erspamer, 1954), emanates from several sources, notably enterochromaffin cells of the gut, but also from serotonergic” nerves. 5-HT fulfills the criteria for a neurotransmitter in mammals (Griffith, 1988) and it is reasonable to believe that the histochemical findings of 5-HT nerves indicate a role for 5-HT as a transmitter also in nonmammalian species. The species variations in the source of 5-HT, and whether or not the neuronally located 5-HT is involved in cardiovascular control in fish, are discussed later. In addition to the possible effects of 5-HT as a neurotransmitter or hormone from enterochromaffin cells, 5-HT stored in and released from specialized endothelial cells may affect blood vessels directly or via stimulation of the release of EDRF (Figs. 2 and 4). Tables I, 11, and I11 summarize the known distribution and effects of 5-HT in fish. “

A. 5-HT in Cyclostomes

In cyclostomes (Atlantic hagfish, Myxine glutinosa, and Pacific hagfish, Polistotrema [Eptatretus]stouti) the effects of 5-HT on the cardiovascular system may be indirect, due to release of catecholamines. Injections of 5-HT caused a small increase in dorsal aortic blood pressure and heart rate and perfusion of 5-HT with constant flow rate through the branchial system or systemic vessels to the gut, in situ, produced both increases and decreases in vascular resistance. The

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Fig. 4. Electron micrograph of endothelial cells (E)lining the vascular lumen (L) of a swimbladder artery of the eel (Anguilla anguilla). Smooth muscle cells of the media (M) are also seen. Note the darker 5-HT immunoreactive cell. [Courtesy of K. Lundin, unpublished.]

responses mimicked, but were much weaker than, the responses to the same concentrations of adrenaline or noradrenaline and were blocked by adrenergic antagonists but not by the serotonergic antagonist methysergide (Reite, 1969). The formaldehyde-induced fluorescence histochemical technique (Falck-Hillarp technique), autoradiography studies of the uptake of tritiated 5-HT, and immunohistochemistry performed on the gut of different species of cyclostomes have demonstrated the presence of 5-HT-containing nerve fibers in the gut (Honma, 1970; Baumgarten et al., 1973; Sakharov and Salimova, 1980; S. Nilsson and S. Holmgren, unpublished results). However, these fibers show no particular relation to the vessels of the gut, and no 5-HT-containing (enterochromaffin) cells of the mucosa (which could possibly provide a “hormonal source” of 5-HT) have been observed in either species (El-Salhy et al., 1985; S. Nilsson and S. Holmgren, unpublished studies). It is, therefore, possible that the effects of 5-HT observed by Reite (1969) are merely pharmacological and of little physiological significance. B. 5-HT in Elasmobranchs

In Squalus acanthias there are numerous endocrine cells showing 5-HT-like immunoreactivity (IR) in the gut mucosa, but there is only a sparse innervation of the muscular layers of the gut, and no 5-HT fibers

Table I Summary of Anatomical Data Concerning the Innervation of the Branchial Vasculature in Teleost Fish Species Adrenergic neurons C yprinus carpio Platycephalus bassensis P . caeuruleopunctatus Tetractenos glaber Anguilla australis Gadopsis marmoratus Salmo trutta Salmo gairdneri Gadus morhua

Cholinergic neurons Perca fluviatilis Gadus morhua Serotonergic neurons/cells Salmo gairdneri

BA

+ ~

-

-

+ -

-

AFA

ALA

Lam

ELA

EFA

Sph

+ + + + + + + +

CVS

+

Nut

+

Donald (1'387) Donald (1987) Donald (1987) Donald (1987) Donald (1987) Donald (1987) Donald (1984) Donald (1984); Dunel-Erh and Bailly (1986) Nilsson (1986)

+

Bailly and Dunel-Erb (1986) Bailly and Dunel-Erb (1986)

+ + -

t

+

+

+

Reference

Abbreviations: AFA, EFA, afferent and efferent filamental artery; ALA, ELA, afferent and efferent filamental arterioles; BA, branchial artery; CVS, central venous sinus; Lam, (secondary) lamellae; Nut, nutritive vasculature; Sph, sphincter at the base of the efferent filamental artery.

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Table I1 Putative Neurotransmitters in Perivascular Nerves of Fish, Revealed by Immunohistochemistry Tissue Gut/c.a,ni.a

Transmitter

Species

Reference

CGRP GAL N PY SOM

Lampetrapuviatilis Raja erinacea Scyliorhinus canicula Squatina aculeata Squalus acanthias Lampetra fluviutilis G a d u s morlzua Raja erinucea, R. radiata Squalus acanthias

SP VIP

Gadus morhua Squalus acanthias

S. Holmgren, unpublished Bjenning e t al. (1991) Tagliafierro e t al. (1988) Tagliafierro e t al. (1988) Bjenning e t al. (1990) S . Holmgren, unpublished S. Holmgren, unpublished Bjenning e t al. (1989) Holmgren and Nilsson (1983a) Jensen and Holmgren (1991) Holmgren and Nilsson (1983,) Holmgren and Nilsson (198313) Lundin and Holmgren (1984); Jensen and Holmgren (1985); S. Holmgren, unpublished Holmgren et al. (1982)

BM

Lepisosteus platyrhincus Gadus morhua

Gills

Coronary artery

GiCCK 5-HT NPY VIP BM

N PY Swimbladder

5-HT VIP

Salmo gairdneri Gadus morhua Salmo gairdneri Raja erinacea, R . rudiata Gadus morhua Raja erinacea Salmo guirdneri

Raja erinacea, R. radiata Anguilla anguilla

S. Holmgren, unpublished Bailly e t al. (1989) Bjenning et al. (1989) S. Holmgren, unpublished Bjenning et al. (1991) Bjenning and Holmgren (1989) Bjenning et ul. (1989) Lundin and Holmgren (1989); K. Lundin, unpublished Lundin and Holmgren (1984)

Gallbladder

VIP

Gudus niorhua Gudus morhua

Aldman and Holmgren (1987)

Gonads

VIP

Gudus morhua

Uematsu e t al. (1989)

Urinary bladder

VIP

Brain Ducts of Cuvier

BM VIP

Gadus morhua Scyliorhinus G a d u s morhua

Vallarino e t a / . (1990) S. Holmgren, unpublished

Lundin and Holmgren (1986)

Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); BM, bombesin; c.a, coeliac artery; CGRP, calcitonin gene related peptide; GAL, galanin; GICCK, gastrin/cholecystokinin; m a . , mesenteric artery; NPY, neuropeptide Y; SOM, somatostatin; SP, substance P; VIP, vasoactive intestinal polypeptide.

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Table 111 Effects of 5-HT on the Cardiovascular System in Fish Effect Increased blood pressure

Organ!tissue Dorsal aorta

Species

Reference

Anguilla

Reite (1969) Reite (1969) Reite (1969) Reite (1969) Reite (1969) R. Fritsche, unpublished Reite (1969)

Decreased blood pressure Tachycardia

Dorsal aorta

Five teleost species

Reite (1969)

Heart

Myxine glutinosa Polistotrema stouti

Reite (1969) Reite (1969)

Increased pressure

Ventricle

Salmo gairdneri

Increased vascular resistance

Gills

Myxine, Polistotrema Squalus acanthias Anguilla anguilla Zoarces uiuiparus Labrus berggyl tu Gadus morhua Salmo gairdneri Myxine, Polistotrema Salmo gairdneri

Reite (1969) Reite (1969) Ostlund and Fange (1962) Ostlund and Fange (1962) Ostlund and Fange (1962) Ostlund and Fagne (1962) Ostlund and Fagne (1962) Katchen et a / . (1976) Reite (1969)

Ventral aorta

Relaxation

Systemic vascul. Coronaiy vascul.

Myxine glutinosa Polistotrema stouti Squalus suckley Hydrolagus collei Gadus morhua

Small et a / . (1990)

innervating visceral vessels have been observed (Holmgren and Nilsson, 1983a; El-Salhy et al., 1985). However, in Scyliorhinus canicula and Squatina aculeata fibers surround gut vessels (Tagliafierro et al., 1988). This may indicate true species differences but may also depend on different sensitivity of the antisera used. Using the formaldehyde-induced fluorescence method, Bailly (1983) failed to show a serotonergic innervation of the gill filaments of the dogfish, Scyliorhinus canicula. However, this method is not as sensitive as immunochemical methods and does not, therefore, completely rule out the possible existence of branchial serotonergic nerves. I n uivo experiments in dogfish (Squalus suckley) and ratfish ( H y drolagus collei) showed a small increase in ventral aortic pressure after 5-HT injection, while the pressure in the dorsal aorta was unaffected. In skates, 5-HT produced no response (Reite, 1969).The indications of an effect on the gill vasculature is in agreement with the findings in teleosts (see later) and could point to a similar control function of 5-HT

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on the gills, although histochemical evidence for serotonergic nerves in elasmobranch gills is still wanting.

C. 5-HT in Teleosts The most conspicuous action of 5-HT on the cardiovascular system in teleosts is a marked constriction of the branchial vasculature. Injections of 5-HT caused a decrease in dorsal aortic pressure in five teleost species studied, while the ventral aortic pressure, measured in cod and eel (anesthetized), and the intraventricular systolic pressure in rainbow trout (anesthetized)increased rapidly (Reite, 1969). Direct effects on systemic vessels were small, while perfused branchial vessels from cod and eel showed a marked constriction that could be blocked by the specific serotonergic antagonist methysergide (Ostlund and Fange, 1962; Reite, 1969; Katchen et aZ., 1976). Similar results were obtained in a preliminary study of unrestrained cod in our laboratory. Injection of 5-HT caused an increase in ventral aortic pressure and heart rate, while dorsal aortic pressure was reduced and lost its pulsatile nature, indicating a constriction of the branchial vessels (R. Fritsche, personal communication). In the rainbow trout, injection of 5-HT produced the same response as exposure to acidified water or infusion of HCl (i.e., an increased frequency and magnitude of opercular movements, a drop in arterial oxygen tension (P,o,), a rise in arterial COZ tension (PLco2),and a decrease in arterial pH) while the heart rate remained unchanged. The responses to both acidification and 5-HT were antagonized by methysergide. The results were interpreted as a mechanism for redistributing blood resulting in a decrease in gas exchange during acidification (Thomas et al., 1979). Careful anatomical and histological studies of the gills from rainbow trout made by Dunel-Erb and co-workers have shown the presence of 5-HT in at least three types of cells: neurons (and nerve fibers), polymorphous granular cells (PGCs), and neuroepithelial cells (NECs) (Dunel-Erb et aZ., 1982; 1989; Bailly et al., 1989). The neurons are of vagal origin, they innervate the proximal part of the efferent filament artery (including the sphincter) and extend to the efferent lamellar arterioles and central venous sinus (see Fig. 2). The fibers were observed impinging on the vascular smooth muscle, which supports the theory that 5-HT is involved in branchial vasomotor control. In view of the constrictor effects on gills obtained in physiological experiments (see earlier), it may be hypothesized that 5-HT, released from nerves or other cells within the gills, cause a constriction of the

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efferent arteries, thereby increasing lamellar recruitment during conditions such as hypoxia, exercise, or stress. In mammals, 5-HT has been demonstrated in endothelial cells of certain blood vessels (Lincoln et al., 1990), and 5-HT has been shown to produce vasodilatation by stimulating a release of EDRF from the epithelium of coronary vessels (Cocks and Angus, 1983)(see also Fig. 3 ) .In the eel, Anguilla anguilla, 5-HT-immunoreactive cells with long varicose processes, possibly paracrine in nature, are present in the mucosa of the pneumatic duct of the swimbladder (Lundin and Holmgren, 1989). At the ultrastructural level, it was observed that these cells were confined to the vascular endothelium of small vessels supplying the pneumatic duct (Lundin, 1991; Fig. 4). It is possible that these 5-HT-immunoreactive cells are involved in the control of the blood flow through this resorptive part of the swimbladder, thereby affecting the rate of resorption of gas from the swimbladder. Whether or not this mechanism involves an EDRF in fish, and whether or not 5-HT has a dilatory effect on the swimbladder vessels remains to be elucidated. 5-HT has weak inhibitory effects on isolated coronary arteries from the rainbow trout, but in this case, it is unlikely that the response is mediated by EDRF, because most of the endothelium was removed during preparation of the artery rings (Small et al., 1990). V. NEUROPEPTIDES

Neuropeptides are bioactive peptides of about 4-40 amino acids present in autonomic neurons as well as in neurons of the CNS. Identical or closely related peptides (“gut hormones”) occur in endocrine cells of the gut and pancreas. Variations in the amino acid sequences between animal species are common. The number of amino acids substituted, their position, and the types of substitution indicate two things: (a) the evolutionary relationship between different animal groups, and (b) the importance of different parts of the molecule in the bioactivity of the peptide. Related peptides form families such as the tachykinins, the vasoactive intestinal polypeptide/peptide histidine isoleucin-like (VIP/PHI) peptides, and the gastrin/cholecystokinin-like(gastrin/CCK-like)peptides. Several members of a peptide family often occur in one animal species, and may have either similar or clearly separate functions. Closely related peptides from different species may be given the same name with a species prefix (e.g., porcine VIP, human VIP, and cod VIP).

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Research on neuropeptides and gut hormones has evolved dramatically over the last two decades, and accumulating evidence shows the involvement of these compounds in cardiovascular control (Burnstock and Griffith, 1988). In the following account, only those peptides have been included that have been implicated in the control of the fish cardiovascular system (Tables 11, IV-VII). A. Vasoactive Intestinal Polypeptide The 28-amino-acid peptide vasoactive intestinal polypeptide (VIP) holds a prominent position in mammalian cardiovascular physiology as a neuropeptide early recognized to possess vasoactive properties. In mammals, cerebral arteries are densely innervated by VIP-containing fibers, while the density of the innervation of peripheral systemic vessels and vascular beds varies; blood vessels of the gastrointestinal tract, the respiratory tract, and the urogenital tract are densely innervated, while systemic, coronary, and blood vessels of the liver, the

Table I V Effects of VIP on the Cardiovascular System in Fish Effect

Orgadtissue

Species

Reference

Perfused intestine

lctalurus melas

Holder et al. (1983)

Perf. swimbladder

Gadus morhua

Perf. rectal gland

Squalus acanthias

Perfused gills A.mes. in uiuo A.coe1 in uioo Ventral aorta in oioo Overall systemic

Salmo trutta Gadus morhua Cadus morhua Gadus morhua

Lundin and Holmgren (1984) Solomon et al. (1984) Thorndyke et al. (1989) Bolis et al. (1984) Jensen et al. (1991) Jensen et al. (1991) Jensen et al. (1991)

Coeliac vasculature

Squalus acanthias

Increased stroke volume

Heart

Gadus morhua

Tachycardia

Heart

Squalus acanthias

Decreased vascular resistance

Increased vascular resistance

Squalus acanthias

S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished Jensen et al. (1991) S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished

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spleen, the pancreas, and the kidney generally show a sparse innervation (Edvinsson and Uddman, 1988). Also in fish, an increasing number of studies point to the involvement of one or several VIP-like peptides in the cardiovascular control (Tables I1 and IV). Immunohistochemical studies suggest that major systemic arteries are more densely innervated than the peripheral vascular beds. In the spiny dogfish, Squalus acanthias, a moderately dense plexus of fibers was found in the walls of the coeliac and mesenteric arteries (Holmgren and Nilsson, 1983a). In the cod, Gadus morhua, there is a dense perivascular nerve plexus in the adventitiomedial border of the mesenteric artery and its branch to the swimbladder (swimbladder artery) (Lundin and Holmgren, 1984). Welldeveloped perivascular plexuses were observed along the small mesenterial branches of the coeliac and mesenteric arteries, and in the branches on the surface of the gut wall (S. Holmgren unpublished; Fig. 5 ) . The VIP innervation of small arteries and veins running within the walls of the swimbladder, the urinary bladder, the gonads, and the gallbladder appears to be sparse (Lundin and Holmgren, 1984, 1986, 1989; Aldman and Holmgren, 1987; Uematsu et al., 1989).Similarly, the density of perivascular VIP fibers innervating vessels intrinsic to the gut wall was low in investigated fish species, such as the holostean, Lepisosteus platyrhiricus (Holmgren and Nilsson, 1983b), the cod (Jensen and Holmgren, 1985), and the rainbow trout (Holmgren et al., 1982). In a study of the myenteric plexus and muscular layers of 18 elasmobranch and teleost species, it was concluded that VIPimmunoreactive fibers follow and surround vessels of these gut layers to some extent only (Bjenning and Holmgren, 1988). VIP extracts from rainbow trout and catfish gut, like porcine VIP, induced a vasodilation in a perfused intestinal loop of the catfish, Zctalurus melas (Holder et aZ., 1983).This is to our knowledge the only published study using native fish VIP on the fish cardiovascular system. Instead, most studies of the presence and function of VIP in fish have been performed using mammalian VIP or antibodies raised against mammalian VIP. Sequence analyses of VIP from the dogfish, Scyliorhinus canicula, and the cod, Gadus morhua, showed that the 28 amino acid sequence of dogfish and cod VIP varies in five positions only from porcine VIP (Dimaline and Thorndyke, 1986; Dimaline et al., 1987; Thwaites et aZ., 1989). Furthermore, the elasmobranch VIP has full affinity for mammalian pancreatic VIP receptors (Dimaline et al., 1987). It is, therefore, reason to believe that available immunohistochemical data give a good indication of the distribution of nerve

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Fig. 5. Immunohistochemistry of blood vessels from cod (Gadus rnorhua) showing VIP-immunoreactive nerve fibers in the wall of the ducts of Cuvier (A), and transverse section (B) and whole mount preparation (C) of small arteries on the surface of the gut. (D)and (E)show NPY and bombesin immunoreactive fibers, respectively, in the wall of small arteries entering the stomach wall.

fibers containing VIP-like material in fish. Most studies in fish using mammalian VIP give results that agree with the effects of VIP on the mammalian cardiovascular system, but it must be kept in mind that the differences in amino acid sequence between mammalian and fish VIP could give the mammalian VIP antagonistic, rather than agonistic, properties when tested in fish. Generally, porcine VIP appears to be vasodilator in teleosts, as in mammals. In the cod, VIP caused a long-lasting decrease in vascular resistance during perfusion of the isolated gas gland and swimbladder, probably due to vasodilation of the vascular beds fed by the mesenteric and swimbladder arteries, which possess VIP-immunoreactive perivascular nerves (Lundin and Holmgren, 1984). The arterioarterial flow through isolated gill arches from the brown

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trout, Salmo trutta, was dose dependently increased by VIP (Bolis et al., 1984). However, in the cod only a few, usually single, varicose fibers were observed running along the gill vessels (S. Holmgren, unpublished), and injection of VIP in vivo produced no effects on blood pressure, flow, or vascular resistance that may be attributed to an effect on the gills (Jensen et al., 1991). It is premature at this stage to speculate whether or not these differences between the rainbow trout and the cod studies are due to the different experimental approaches or to true species differences. Injections of porcine VIP into the cardiovascular system of unanesthetized cod in vivo caused an increase in the gut blood flow, due to an increase in flow in the coeliac and mesenteric arteries and in the ventral aorta. The increased cardiac output was caused by an increase in stroke volume, while the heart rate was largely unaffected. Surprisingly, the increase in cardiac output appeared to be the only reason for the increase in flow in the mesenteric artery. In the coeliac artery, on the other hand, a decrease in vascular resistance caused by vasodilation further increased the flow (Jensen et al., 1991). The reasons for this discrepancy between the two vascular beds are uncertain, but preliminary immunohistochemical studies show a more dense VIP innervation of the coeliac artery than the mesenteric artery, indicating that this vessel normally is more influenced by a VIP-like peptide than the mesenteric artery. In the elasmobranch Squalus acanthias, a different effect of porcine VIP has been obtained. Injections into unrestrained fish in vivo caused increased total vascular resistance including the coeliac artery vascular bed. The blood flow to the gut was consequently reduced. The dorsal aortic blood pressure was slightly increased (S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished). Whether this reflects true differences between elasmobranchs and teleosts, differences between individual species, or depends on the difference between porcine VIP and the native Squalus VIP remains to be elucidated. It is notable, however, that in the rectal gland of Squalus, VIP caused the same effect as that described in mammalian exocrine glands: a vasodilation, combined with an increase in glandular secretion (Solomon et al., 1984; Thorndyke et al., 1989).

B. Bombesin It is not yet clear exactly which of the approximately 20 bombesinlike peptides known today are present in fish. Most studies indicate the presence of several related peptides, and the presence of both longer, gastrin-releasing peptide-like (GRP) forms and shorter,

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bombesin-like forms, possibly with species differences, have been concluded. Thus, Conlon et al. (1987) found that bombesin-like material extracted from the intestine of the common dogfish, Scyliorhinus canicula, occurs in large and small forms; the long, partly sequenced 25 amino acid form shows a clear homology with mammalian and chicken GRP, and it has been argued that authentic bombesin is not present in this species. On the other hand, one of the two bornbesin/ GRP-related peptides present in the gut of Scyliorhinus stellaris appears closely related to bombesin (Cimini et al., 1985). Several forms of bombesin-like material were obtained from the cod gut, one of which shows similar properties to bombesin (Holmgren and Jonsson, 1988), and bombesin-like material isolated from the intestine of the ratfish, Hydrolagus colliei, was equipotent with synthetic amphibian bombesin in stimulating amylase secretion; the effect being blocked by a specific bombesin antagonist (Thorndyke et al., 1990). Binding sites for bombesin have been demonstrated in the stomach of the teleost Scorphaeichthys marmoratus (Vigna and Thorndyke, 1989), and there are several studies in elasmobranchs and teleosts that show effects of exogenous bombesin on gut smooth muscle (Lundin et d., 1984; Holmgren, 1983;Jensen and Holmgren, 1985; Holmgren and Jonsson, 1988; Thorndyke and Holmgren, 1990), on gastric acid secretion (Holstein and Humphrey, 1980), and on behavior (Kavaliers and Hawkins, 1981; Beach et al., 1988). Less attention has been paid to the possible bombesin innervation of the cardiovascular system in fish (Table 11), but a moderately dense to dense plexus of perivascular nerves showing bombesin-like immunoreactivity has been demonstrated in systemic vessels to the gut in the elasmobranchs, Squalus acanthias and Raja erinacea; the distribution of the fibers to the adventitiomedial border implies an involvement in the control of the vascular smooth muscle (Bjenning et d., 1990, 1991). A sparse innervation has been found in the heart and coronary vessels of the little skate, Raja erinacea (Bjenning et al., 1991). A colocalization of bombesin-like peptide(s) with 5-HT in perivascular nerves of the gut was suggested after immunohistochemical studies in the elasmobranchs Scyliorhinus canicula and Squatina aculeata (Tagliafierro et al., 1988), but the physiological significance of this has not been looked into further. In the brain of Scyliorhinus, bombesin immunoreactive fibers innervate vascular structures of the median eminence and may be involved in hypophysiotropic actions similar to the situation in mammals (Vallarino et al., 1990). In the rainbow trout, immunoreactive fibers surround ganglion cells in the sinoatrial region

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and innervate the myocardium (Bjenning and Holmgren, 1989).This is compatible with the view of a regulatory function on the heart activity. Physiological experiments (Table V) further suggest a role of bombesin-like peptides in the cardiovascular control of fish. The flow through the vascularly perfused stomach of the spiny dogfish shows several phases of increase and decrease in resistance, possibly reflecting activation of several mechanisms (Bjenning et al., 1990). In uuiuo, bombesin causes an major increase in somatic vascular resistance, which causes a shunting of blood into the coeliac artery, although the resistance in the coeliac artery vascular bed is slightly increased (S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished). A small, but significant bradycardia is obtained in the spiny dogfish in uiuo; this occurs after a significant increase in dorsal aortic pressure and may possibly demonstrate the presence of a reflex bradycardia (S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished). However, a negative chronotropic effect (and a negative inotropic effect) of bombesin is obtained in the isolated perfused heart of the rainbow trout (C. Bjenning and S. Holmgren, unpublished), suggesting a direct effect on the heart tissues at least in this species. Coronary vessels from the longnose skate, Raja rhina, contract in response to bombesin (Bjenning et al., 1991). Injections of bombesin in the cod in viuo failed to produce an effect on blood pressure, heart performance, or flow to the gut (M. Axelsson

Table V Effects of Bombesin on the Cardiovascular System in Fish Effect

Organ/tissue

Species

Reference

ncreasedldecreased vascular resistance mcreased vascular resistance

Perfused stomach

Squalus acanthias

Bjenning et (11. (1990)

Overall systemic and perfused tail

Squalus acanthias

Bradycardia

Perfused heart

Oncorhynchus mykiss

I n aiuo

Squalus acanthias

S. Holmgren, M. Axelsson, and A. P. Farrell, unpublishe Bjenning and Holmgren (1989) S. Holmgren, hl. Axelsson, and A. P. Farrell, unpublishe

Decreased stroke force

Perfused heart

Oncorhynchus mykiss

Zontraction

Coronary vasculature

Raja rhina

Bjenning and Holmgren (1989) Bjenning et al. (1991)

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STEFAN NILSSON AND SUSANNE H O L M G R E N

and S. Holmgren, unpublished). Blood vessels of the cod appear to be sparsely innervated by bombesin fibers: bundles of bombesinimmunoreactive fibers run along arteries on the surface of the gut wall, but very few fibers seem to innervate the vessels themselves (S. Holmgren, unpublished). T h e results available give little support for a general theory on the effect of bombesin-like peptides on fish circulation. The immunohistochemical data suggest a variation in innervation between species and between cardiovascular beds. It is possible that bombesin, in some species such as Squalus acanthias, is involved in the redistribution of blood between different vascular beds. This has been observed in the crocodiles, Caiman crocodylus and Crocodylus porosus, where the flow to the gut increased and the flow to the lung decreased due to changes in vascular resistance after bombesin administration (Holmgren et al., 1989).In mammals, nerve fibers showing bornbesin/ GRP-like immunoreactivity supply the brain vasculature and lung vessels, but no vasomotor effects could be demonstrated in isolated pial arteries (Uddman et al., 1983,1984), and it has been suggested that the nerves are sensory or that the major function of bombesin/GRP in the perivascular nerves is that of a modulator. Indeed, in the rainbow trout and cod, bombesin potentiates the effect of acetylcholine on gut wall smooth muscle (Thorndyke and Holmgren, 1990). C. Neuropeptide Y

Immunohistochemical neuropeptide Y (NPY) studies in fish suggest a striking species variation in the cardiovascular innervation. Thus a comparative study of three species of elasmobranchs, Squalus acanthias, Raja erinacea, and Raja radiata, and of two teleosts, the cod and the rainbow trout, revealed NPY-like immunoreactivity in the two Raja species only, although in all these species, as well as in several other teleost and Raja species, the same antisera reveal immunoreactive fibers innervating the gut smooth muscle (Bjenning and Holmgren, 1988; Burkhardt-Holm and Holmgren, 1989; Bjenning et al., 1989). It is, however, clearly premature to conclude that skates are unique among fish in the possession of cardiovascular NPY-containing fibers. The amino acid sequences of NPY isolated from the fish pancreas show good homology with mammalian NPY, especially in the Cterminal region. Interestingly, the primary structure has been strongly conserved among skate (Raja rhina), ganoids (Lepisosteus spatula, Amia calua), and salmon and eel NPY, and (in the C-terminal region)

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between these species and mammals, while the variation among teleost appears much larger (Andrews et al., 1985; Conlon et al., 1986a, 1991;Kimmel et al., 1986; Pollock et al., 1987). No neuronally derived NPY from fish has been sequenced to date, but immunoreactive material from the brain of the anglerfish, Lophius americanus, shows even closer similarity to mammalian NPY than fish pancreatic NPY (aPY) in elution and radioimmunoassay studies (Andrews et a/., 1985; Noe et al., 1986, 1989). There is as yet no information on the characteristics of NPY-like peptides in cardiovascular nerves from fish, or whether or not different forms are expressed depending on which organ is innervated. In skates, the perivascular fibers form a plexus in the adventitiomedial border. This plexus is especially dense in systemic vessels to the gut; less dense in the conus arteriosus, the coronary vessels, the gill arteries, the dorsal aorta, vessels within the gut wall, and the portal vein; and sparse in the ducts of Cuvier. In the heart, the sinus venosus and the atrium receive a sparse innervation, while the ventricle is moderately innervated in Raja erinacea but devoid of fibers in Raja radiata (Bjenning et al., 1989). Neuropeptide Y often (but not always) coexists with adrenaline/ noradrenaline in perivascular nerves in mammals, amphibians, and reptiles (Gibbins et al., 1988; Morris, 1989). In Raja radiata, adrenergic nerves innervate gut arteries and arterioles and to some extent coronary vessels, while the heart, larger arteries, and veins are devoid of such nerves (Bjenning et al., 1989). The distribution thus agrees with part of the NPY-immunoreactive nerves, making coexistence a possibility. In Squalus acanthias in vivo, cardiac output and coeliac artery Bow increased in response to NPY; both the overall systemic vascular resistance and the vascular resistance of the coeliac vascular bed decrease, which suggests an inhibitory effect on the vascular smooth muscle (Table VI). The response is probably independent of adrenergic mechanisms, since injected adrenaline or noradrenaline increase total systemic and coeliac vascular resistance (S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished). himunohistochemical studies have failed to demonstrate cardiovascular nerves containing NPY in Squalus acanthias (see earlier). However, NPY-like peptides are common in the fish pancreas (see earlier), and a humoral action of these peptides on the cardiovascular system is possible. Experiments performed on isolated coronary arteries from the longnose skate, Raja rhina, show that NPY only occasionally produces an effect of its own on these vessels, while the amplitude of the response

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Table VI Effects of Neuropeptide Y (NPY) on the Cardiovascular System in Fish ~

Effect Decreased vascular resistance

Organ/tissue Overall systemic

Coeliac vasculature

Tachycardia

Heart

Potentiates Coronary vasculature noradrenaline induced contraction

Species

Reference

Squalus acanthias S . Holmgren, M. Axelsson, and A. P. Farrell, unpublished Squalus acanthias S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished

Squalus acanthias S . Holmgren, M. Axelsson and A. P. Farrell, unpublished Raja rhina

C. Bjenning and A. P. Farrell, unpublished

to noradrenaline was significantly enhanced in the presence of NPY (C. Bjenning and A. P. Farrell, unpublished). This has also been seen in isolated canine coronary arteries, while vascular resistance in the coronary vascular bed of the whole perfused heart increased by NPY alone, suggesting a different effect in the more peripheral parts of the vessels (Macho et al., 1989).The potentiating effect of NPY on adrenergic vasoconstriction has been reported from several studies in mammals (see Polak and Bloom, 1988), and NPY coexists with catecholamines in sympathetic perivascular neurons of most vertebrate species examined, including the toad, Bufo marinus (Morris et a,?.,1986; Morris, 1989). Taken together, these results point to some interesting features: 1. It is evident that the interaction between NPY and catecholamines occurs early in the vertebrate lineage. 2. The interaction of NPY with catecholamines appears independent of the effect of NPY alone, which may vary between tissues and species. 3. Regional differences in NPY-mechanisms may occur along the vascular tree.

D. Somatostatin Somatostatins of various lengths are widely distributed in vertebrate tissues: as a neuropeptide in the CNS and in endocrine cells in the gut and pancreas. Somatostatin also occurs in peripheral nerves,

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but the presence in cardiovascular nerves is not widespread (Dahlstrom et al., 1988; Morris, 1989; Plisetskaya, 1989). In general, somatostatin has inhibitory effects on secretory events. In the toad, Bufo marinus, somatostatin is present in vagal postganglionic neurons and has inhibitory effects on the heart, supporting the cholinergic effects (Campbell et al., 1982). Dense plexuses of nerve fibers that show somatostatin-like immunoreactivity are present in the walls of the coeliac and mesenteric arteries of the spiny dogfish, Squalus acanthias (Holmgren and Nilsson, 1983a). In the cod in vivo, somatostatin had inconsistent and usually weak effects on heart rate and on flow in the ventral aorta, the coeliac artery and the mesenteric artery, and on dorsal aortic pressure. The ventral aortic pressure increased in 50% of the tested fish (S. Holmgren and M. Axelsson, unpublished). E. Substance P Substance P was the first peptide to be recognized of the large tachykinin family with the common C-terminal sequence Phe-X-GlyLeu-Met-NHZ (Euler and Gaddum, 1931). Tachykinins were demonstrated in fish at a relatively early stage (Euler and Ostlund, 1956), and a number of fish tachykinins have been sequenced since (Conlon et al., 1986b, 1990; Conlon and Thim, 1988). The teleost gut is densely innervated by substance P-immunoreactive fibers (Jensen, 1989), but in the cod the innervation of the cardiovascular system is sparse with only few blood vessels in the gut wall innervated by substance P-immunoreactive fibers. However, endocrine cells containing tachykinin-like peptides are common in most fish species investigated, and, in addition, transmitter overflow during nerve activity may affect the vasculature of the stomach ( Jensen, 1989; Jensen and Holmgren, 1991). Numerous mammalian studies have demonstrated a vasodilator effect of substance P, and studies in the cod and the spiny dogfish demonstrate a similar situation (Jensen et al., 1991; S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished; Table VII). However, in contrast to the effects of substance P in mammals, heart rate remains unaffected in these two fish species. In Squalus acanthias, substance P reduced the overall systemic vascular resistance; the effect was particularly evident in the vascular bed of the coeliac artery. Both cardiac output and coeliac artery blood flow increased, while blood pressure decreased slightly (S. Holmgren and M. Axelsson, unpublished). In vivo injections of substance P in the cod decreased the vascular

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Table VII Effects of Substance P on the Cardiovascular System in Fish Effect Decreased vascular resistance

Orgadtissue

Species

Reference

Overall systemic

Squalus ucanthius

Coeliac vasculature

Squalus acanthias

Coeliac vasculature Mesenteric vasculature

Gudus morhua Gadus morhua

S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished S. Holmgren, M. Axelsson, and A. P. Farrell, unpublished Jensen et al. (1991) Jensen et al. (1991)

resistance of the coeliac and mesenteric arteries with very small effects on dorsal or ventral aortic blood pressures (Fig. 6). The flow in the coeliac artery reached a single peak, and then declined to its basic value, while the flow in the mesenteric artery described a three-phasic pattern. After an initial increase in flow, the flow decreased to its initial value, or below, and then increased again. Blockade with atropine abolished the phase of flow decrease, leaving an increase in flow roughly agreeing in time with the three phases prior to blockade. The transient flow decrease occurred even during continuous exposure to substance P and after vagotomy and was not caused by mechanical obstruction of the vessel walls due to contractile activity in the stomach. It thus appears that substance P somehow triggers a local cholinergically mediated vasoconstrictor reflex in the vascular bed perfused by the mesenteric artery, but the physiological significance of this remains to be elucidated (Jensen et al., 1991). Ingestion of food leads to an increase in blood flow to the gut (mammals, Fara, 1984; crocodiles, M. Axelsson, unpublished; teleost fish, Axelsson et al., 1989), and in the dog there is a postprandial increase in plasma levels of a substance P-like peptide, suggesting an involvement of a tachykinin in the postprandial hyperemia. Whether or not this is also the case in fish remains to be elucidated. F. Galanin Galanin, a 29-amino acid neuropeptide, was first isolated and described by Tatemoto et al. (1983), and its actions in mammals have mostly been related to pancreatic functions (Plisetskaya, 1989).

5.

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PSTOM

k Pa

‘1

FMA kHz

‘] A 0

FCA kHz

0

k Pa

PVA k h

‘1 ‘1

--

0

HR bpm

20 A Substance P

A

A

Atropine

Substance P

2 rnin

Fig. 6. Effects of substance P injection in wioo in the cod (Gadus rnorhua) on the intraluminal pressure in the stomach (PSTOM), mesenteric artery blood flow ( F i t * ) , coeliac artery blood flow (FCA),dorsal and ventral aortic blood pressure ( P D A and PvA, respectively), and heart rate (HR). Note triphasic response in FMA,which is changed to a simple flow increase after atropine treatment.

However, studies in mammals (e.g., Kummer, 1987; Morris et al., 1992),toads (Morris et al., 1989),and lizards (Gibbins et al., 1989)show the presence of galanin in perivascular, sympathetic nerves, often in co-existence with NPY. In the cod, Gadus morhua, galanin-like immunoreactivity is present in perivascular nerves innervating arterial branches on the gut surface (Fig. 7), and galanin stimulates strip preparations of the coeliac and mesenteric arteries by a direct action on the smooth muscle (P. Karila and S. Holmgren, unpublished results; Fig. 7). Little is known of the effects of galanin in the cardiovascular system of vertebrates other than mammals, but the results in the cod agree with the direct effects of galanin on smooth muscle of the gut reported in the rat (Ekblad et al., 1985).

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A * mN0

1 4 t

100 t

300 10 min

Galanin (nM) 10

Fig. 7. Contractile effect of galanin on isolated mesenteric artery strip preparation from cod (Gadus morhua) (A), and galanin-immunoreactive nerve fibers running along a small artery at the surface of the stomach (B) and forming a perivascular plexus around a branching visceral artery (C).

G. Gastrin/Cholecystokinin

Although there has been much discussion of the exact identity of the gastrin/CCK-like peptide(s) present in fish, there is to date no sequence analyses made of a native fish gastrin or CCK. With the support of circumstantial evidence, evolutionary theories have been put forward suggesting that caerulein is the most primitive variant, first appearing in fish (Larsson and Rehfeld, 1977)or that ancestral gastrin/ CCK resembles CCK rather than gastrin (Crim and Vigna, 1983; Vigna, 1985). Whichever the case, it is clear from radioimmunoassay studies combined with Sephadex gel filtration and ion-exchange fractionation that multiple forms of gastrin/CCK exist in extracts from the fish gut (Aldman et al., 1989; Jonsson, 1989). Gastrin/CCK has mainly been associated with digestive events, and the few investigations dealing with their effects on the cardiovascular system in mammals have mainly focused in the control of the gastrointestinal blood flow. In our preliminary experiments with the cod (Gadus morhua) in vivo, there appears to be a reduction of gut blood flow after injection of sulfated CCK8 and caerulein ( J . Gunnarsson, unpublished, Fig. 8). There was also a dramatic increase in ventral aortic blood pressure in response to these peptides, without a

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331

pVA

Fig. 8. Effect of sulfated caerulein (CAER-S, 1 pmollkg) on heart rate (fH, beats/ min), ventral aortic blood pressure (Pv,, kPa), mesenteric and coeliac artery blood flow (F,,, and Fcoel, respectively, both shown as uncorrected AkHz doppler shift) in the cod (Gadus rnorhua). Note marked increase in ventral aortic blood pressure, concomitant with the (probably barostatic reflexogenic) decrease in heart rate and reduction in gut blood flow. [Courtesy of J . Gunnarsson, unpublished.]

significant effect on the dorsal aortic pressure ( J . Jensen, M. Axelsson, and S. Holmgren, unpublished). In addition, CCK8-S caused a marked vasoconstriction of the branchial vasculature in the isolated-perfused head preparation from the cod (Fig. 9; L. Sundin, unpublished). This suggests an effect of the gastrin/CCK-like peptides on the gill vasculature, a conclusion that was supported by immunohistochemical findings of CCK-immunoreactive nerve fibers along gill blood vessels in the cod (S. Holmgren, unpublished).

VI. ENDOTHELIAL FACTORS There is an increasing number of studies in mammals that indicate that neurotransmitters may act both directly on the vascular smooth muscle cells or indirectly by the release of endothelial factors. It is

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1

8 7 pi

kPa

Fig. 9. Effect of a bolus injection of sulfated CCK-8 (0.5 nmol) on inflow pressure

(P,;kPa), efferent arterial outflow (Fa;dropsimin), and inferior jugular vein outflow (F”; dropslmin) in the gill apparatus from the cod (Gadus rnorhua) perfused at constant flow from a pulsatile pump. Note marked vasoconstriction, reflected as both an increase in inflow counter-pressure and a reduction in both flows. [Courtesy of L. Sundin, unpublished.]

important to bear in mind that the effect produced by a certain amine or peptide may be quite different depending on the route of administration of the substance during an experiment (Cocks and Angus, 1983; D’Orleans-Juste et al., 1985; Daly and Hieble, 1987; Burnstock, 1988). In mammals, the endothelium-related compounds include EDRF, later identified as nitric oxide (NO) (Ignarro et al., 1986; Palmer et al., 1987; Mione et al., 1990; Burnstock, 1990).Whether or not EDRF also occurs in fish is not clear, but nitroglycerine (which is broken down to nitrous oxide) causes relaxation of coronary artery rings (Small et al., 1990). A second substance that has major effects on fish blood vessels is endothelin. Endothelin-1, a 21-amino acid peptide that was originally isolated from porcine endothelial cells, contracts blood vessels from several vertebrates including the catfish (Arniurus rnelas) and rainbow trout (Oncorhynchus mykiss) (Poder et al., 1991; Olson et al., 1991). Olson et al. (1991) also observed a transient decrease in dorsal aortic blood pressure after injection of 500 ngekg-’ endothlin-1 into rainbow trout, while higher doses (1500ngskg-’) produced a triphasic (pressor/ depressor/pressor) response. They concluded that the rainbow trout vasculature “is exquisitely sensitive” to endothelin-1 and suggested that the physiological expression of the peptide has been highly conserved during the course of vertebrate evolution.

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ACKNOWLEDGMENTS Our own work on cardiovascular control in fish is currently supported by the Swedish Natural Science Research Council and the Swedish Forestry and Agriculture Research Council. We thank Kersti Lundin, Michael Axelsson, Jorgen Jensen, Regina Fritsche, Jonas Gunnarsson, and Lena Sundin for letting us use and quote their, as yet, unpublished material.

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Katchen, M. S., Olson, K. R., and Wayne, C. (1976).Effects ofhistamine and serotonin on isolated perfused gill of rainbow trout (Salmo gairdneri). Fed. Proc. 35,528. Kavaliers, M., and Hawkins, M. F. (1981).Bombesin alters behavioral thermoregulation in fish. Life Sci. 28, 1361-1364. Kimmel, J. R., Plisetskaya, E. M., Pollock, H. G., Hamilton, J . W., Rouse, J. B., Ebner, K. E., and Rawitch, A. B. (1986). Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y. Biochem. Biophys. Res. Commun. 141, 1084- 1091. Koke, J. R., and Anderson, D. R. (1986).Changes in metabolite levels and morphology of teleost ventricular myocytes due to hypoxia ischemia and metabolic inhibitors. Cytobios 45,97-108. Kummer, W. (1987). Galanin- and neuropeptide Y-like immunoreactivities coexist in paravertebral sympathetic neurons of the cat. Neurosci. Lett. 78,127-131. Larsson, L.-I., and Rehfeld, J. F. (1977).Evidence for a common evolutionary origin of gastrin and cholecystokinin. Nature 269,335-338. Laurent, P. (1984).Gill internal morphology. In “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. X, pp. 73-183. Academic Press, Orlando. Laurent, P., Holmgren, S., and Nilsson, S. (1983). Nervous and humoral control of the fish heart: Structure and function. Comp. Biochem. Physiol. 76A, 525-542. Lennard, R., and Huddart, H. (1989). Purinergic modulation of cardiac activity in the flounder during hypoxic stress. J . Comp. Physiol. 159, 105-114. Leont’eva, G. R. (1966). Distribution of catecholamines in blood vessel walls of cyclostomes, fishes, amphibians and reptiles. J . Evol. Biochem. Physiol. 2,31-36. Lincoln, J., Loesch, A., and Burnstock, G. (1990). Localization of vasopressin, serotonin and angiotensin-I1 in endothelial cells ofthe renal and mesenteric arteries ofthe rat. Cell Tissue Res. 259,341-344. Lundin, K. (1991). “The Teleost Swimbladder: A Study of the Non-adrenergic, Noncholinergic Innervation.” Ph.D. Thesis, Department of Zoophysiology, University of Goteborg. Lundin, K., and Holmgren, S. (1984).Vasointestinal polypeptide-like immunoreactivity and effects of VIP in the swimbladder of the cod, Gadus morhua. J . Comp. Physiol. 154B, 627-633. Lundin, K., and Holmgren, S. (1986). Non-adrenergic, non-cholinergic innervation of the urinary bladder of the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. 84C, 315-323. Lundin, K., and Holmgren, S. (1989). The occurrence and distribution of peptide- or 5-HT-containing nerves in the swimbladder of four different species of teleosts, (Gadus morhua, Ctenolabrus rupestris, Anguilla anguilla, Salmo gairdneri). Cell Tissue Res. 257,641-647. Lundin, K., Holmgren, S., and Nilsson, S. (1984). Peptidergic functions in the dogfish rectum. Acta Physiol. Scand. 121,46A. Macho, P., Perez, R., Huidobro-Toro, J. P., and Domenech, R. J. (1989).Neuropeptide Y (NPY): A coronary vasoconstrictor and potentiator of catecholamine-induced coronary constriction. Eur. J . Pharmacol. 167,67-74. Meghji, P., and Burnstock, G. (1984a).Actions of some autonomic agents on the heart of the trout (Salmo gairdneri) with emphasis on the effects of adenyl compounds. Comp. Biochem. Physiol. 78C, 69-75. Meghji, P., and Burnstock, G. (1984b). The effect of adenyl compounds on the heart of the dogfish, Scyliorhinus canicula. Comp. Biochem. Physiol. 77C, 295-300. Metcalfe, J. D., and Butler, P. J. (1984).On the nervous regulation ofgill blood flow in the dogfish Scyliorhinus canicula. J . E x p . Biol. 113,253-267.

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Mione, M. C., Ralevic, V., and Burnstock, G. (1990). Peptides and vasomotor mechanisms. Pharmacol. Ther. 46,429-468. Morris, J. (1989).The cardiovascular system. In “The Comparative Physiology of Regulatory Peptides” (S. Holmgren, ed.), pp, 273-307. Chapman and Hall, London. Morris, J. L., Gibbins, I. L., Campbell, G., Murphy, R., Furness, J. B., and Costa, M. (1986). Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons. Cell Tissue Res. 243, 171-184. Morris, J. L., Gibbins, I. G., and Holmgren, S. (1992).Galanin is more common than NPY in vascular sympathetic neurons ofAustralian marsupials. Regul. Pept. 37, 101-109. Morris, J. L., Gibbins, I. L., and Osborne, P. B. (1989). Galanin-like immunoreactivity in sympathetic and parasympathetic neurons of the toad Bufo marinus. Neurosci. Lett. 102, 142-148. Nicol, J. A. C. (1952). Autonomic nervous systems in lower chordates. Biol. Reo. 27, 1-49. Nielsen, K. E., and Gesser, H. (1984).Energy metabolism and intracellular pH in trout heart muscle under anoxia and different calcium ii concentrations.]. Comp. Physiol. 154,523-528. Nilsson, S. (1983). “Autonomic Nerve Function in the Vertebrates.” Springer-Verlag, Berlin, Heidelberg, New York. Nilsson, S. (1984). Innervation and pharmacology of the gills. I n “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. XA, pp. 185-227. Academic Press, Orlando. Nilsson, S. (1986).Control of gill blood flow. In “Fish Physiology: Recent Advances” (S. Nilsson and S. Holmgren, eds.), pp. 86-101. Croom Helm, Dover, New Hampshire. Nilsson, S., and Holmgren, S. (1988).The autonomic nervous system of elasmobranchs: Structure and function. In “Physiology of Elasmobranch Fishes.” (T. J. Shuttleworth, ed.), pp. 143-169. Springer-Verlag, Berlin. Nilsson, S., and Pettersson, K. (1981). Sympathetic nervous control of blood flow in the gills of the Atlantic cod, Gadus morhua.]. Comp. Physiol. 144, 157-163. Noe, B. D., McDonald, J. K., Greiner, F., Wood, J. G. and Andrews, P. C. (1986). Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY. Peptides 7,147-154. Noe, B. D., Milgram, S. L., Balasubramaniam, A,, Andrews, P. C., Calka, J., and McDonald, J. K. (1989). Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus). Cell Tissue Res. 257,303-:311. Okafor, M . C. J., and Oduleye, S. 0.(1986). Hemodynamic effects ofpurinergic receptor stimulation in isolated fish Oreochromis niloticus gills. Actu Physiol. Hung. 67, 45-52, Olson, K. R., Duff, D. W., Farrell, A. P., Keen, J , , Kellogg, M. D., Kullman, D., and Villa, J . (1991). Cardiovascular effects of endothelin in trout. A m . J .Physiol. 260, H1214H1223. Ostlund, E., and Fange, R. (1962).Vasodilation by adrenaline and noradrenaline, and the effects of some other substances on perfused fish gills. Comp. Biochem. Physiol. 5 , 307-309. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987).Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327,524-526. Pettersson, K., and Nilsson, S. (1979).Nervous control of the branchial vascular resistance of the Atlantic cod, Gadus morhua.]. Comp. Physiol. 129, 179-183. Plisetskaya, E. M. (1989). Pancreatic peptides. In “The Comparative Physiology of Regulatory Peptides” (S. Holmgren, ed.), pp. 174-202. Chapman and Hall, London.

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6 NERVOUS CONTROL OF THE HEART AND CARDIORESPIRATORY INTERACTIONS E . W. TAYLOR School of' Biological Sciences The University of Birmingham Birmingham, United Kingdom

I. Introduction 11. Innervation of the Heart A. Cyclostomes B. Elasmobranchs C. Teleosts 111. The Central Location of Vagal Preganglionic Neurons A. Cyclostomes B. Elasmobranchs C. Teleosts IV. Control of the Heart and Branchial Circulation A. Vagal Tone on the Heart B. Efferent Activity Recorded from Cardiac Vagi C. Central Origin of Efferent Activity in Cardiac Vagi V. Cardiorespiratory Interactions A. Reflex Modulation of Heart Rate B. Central Interactions Modulating Heart Rate VI. Cardiorespiratory Synchrony References

I. INTRODUCTION The fish heart is composed of typical vertebrate cardiac muscle fibers. Contraction is initiated by a propagated muscle action potential that originates from a myogenic pacemaker and generates a characteristic electrocardiogram (ECG) wave form (Randall, 1968; Satchell, 1991).Its functioning is influenced by intrinsic mechanisms, such as 343 FISH PHYSIOLOGY, VOL. XIIB

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the relationship between the force of contraction and stretch applied to the muscle fibers, which is identical to the Frank-Starling relationship described in mammals (Johansen, 1971). Thus, the increase in diastolic filling time that accompanies cardiac slowing, because it results in increased atrial volume, causes an increase in cardiac stroke volume. Short et al. (1977) concluded that maintenance of cardiac output in the dogfish during a hypoxia-induced bradycardia was wholly attributable to the Frank-Starling relationship. Heart rate operates under the influence of nervous and hormonal controls in order that it may respond to changes in supp:y or demand with respect to oxygen or metabolites. This chapter considers the efferent arm of the reflex nervous control of the fish heart. The afferent, sensory arm is reviewed by Burleson et al. in Chapter 7 of this volume. Anatomical and pharmacological evidence suggests that efferent nervous control of the heart in elasmobranchs is due solely to an inhibitory parasympathetic input supplied by the cardiac vagus. Although the teleost heart receives an excitatory sympathetic supply, parasympathetic control predominates, and the heart in both groups operates under varying levels of inhibitory vagal tone. Consequently, this chapter concentrates on efferent vagal innervation of the heart and considers the central projections of the cardiac vagi together with associated branchial and visceral branches of the vagus. A detailed description of the neuranatomy of the vagal motor column is related to recordings of efferent activity in branches of the vagus. Changes in heart rate with temperature and oxygen partial pressure, together with cardiorespiratory interactions, are described with emphasis on their neurophysiological bases, including the roles of central interactions and peripheral chemoreceptors and mechanoreceptors in determining the efferent output from cardiac vagal motoneurons in the brainstem. 11. INNERVATION OF THE HEART

A. Cyclostomes This group of vertebrates is composed of the myxinoids (e.g., Myxine, the hagfish) and the petromyzonts (e.g., Lampetra, the lamprey). The heart of myxinoids is aneural, that is, it is not innervated by the vagus or the sympathetic nervous system (Green, 1902; Carlson, 1904); whereas the heart of the lamprey (although similarly devoid of a sympathetic supply) is innervated by the vagus (Ransom and Thompson, 1886; Augustinsson et al., 1956). The cardiac fibers leave the thin,

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nonmyelinized epibranchial trunk of the vagus and run to the median jugular vein (Fange, 1972). The main effect of vagal stimulation in petromyzonts is an acceleration of the heart with an accompanying decrease in the force of contraction (Falck et al., 1966).Acetycholine induces an acceleration of the heart, a response unique among vetebrates. Nicotinic cholinoceptor agonists, such as nicotine, have the same effect (Augustinsson et al., 1956; Falck et al., 1966).The excitatory effect ofvagal stimulation or nicotinic agonists can be blocked by nicotinic cholinoceptor antagonists such as tubocurarine and hexamethonium (Augustinsson et al., 1956; Falck et al., 1966; Lukomskayo and Michelson, 1972).The heart in cyclostomes contains large quantities of adrenaline and noradrenaline, stored in chromaffin-type cells, that may be released to maintain normal cardiac function (Nilsson and Axelsson, 1987). Adrenaline, noradrenaline, isoprenaline, and tyramine stimulate the petromyzont heart, although the effects are less pronounced than that of acetylcholine. These effects are blocked by propranolol, suggesting involvement of P-adrenoceptors on the heart as in the higher vertebrates (Augustinsson et al., 1956; Falck et al., 1966; Nayler and Howells, 1965). B. Elasmobranchs The elasmobranchs are phylogenetically the earliest group of vertebrates in which a well-developed autonomic nervous system with clearly differentiated parasympathetic and sympathetic components has been described (Nicol, 1952). They are also the earliest group known to have an inhibitory vagal innervation of the heart. In the dogfish, Scyliorhinus canicula, the vagus nerve divides to form, at its proximal end, branchial branches 1, 2, 3, and 4 that contain skeletomotor fibers innervating the intrinsic respiratory muscles of gill arches 2, 3, 4, and 5, respectively, as well as sensory fibers to the gill arches and walls of the pharynx (Fig. 1).The first gill arch is innervated by the glossopharyngeal (IXth cranial) nerve. Other respiratory muscles operating around the jaws and pharyngeal skeleton are innervated by branches of cranial nerves V and VII (Fig. 1).The vagus also sends, on each side of the fish, two branches to the heart: the branchial cardiac branch, which arises from the fourth branchial branch (Norris and Hughes, 1920), and the visceral cardiac branch, which arises from the visceral branch of the vagus (Marshall and Hurst, 1905). These branches were redescribed by Lutz (1930c), Taylor et al., (1977), Barrett and Taylor (1985a), and Withington-Wray et al. (1986) and are

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Vmand

VII spiracle

brain

IX gill cleft X br. 1 X br. 2 X br.

post. lat. lin. ant. card. sin.

3

4

X br. c. duct. Cuv. Xvisc. c.

heart pericard. visc. X

Fig. 1. Schematic diagram of a dorsal view of the left side of the head of the dogfish to display the course of the cranial nerves innervating respiratory muscles and the heart. These constitute the mandibular branch of the trigeminal, Vth nerve (V mand.); the spiracular branches of the facial, VIIth nerve; the glossopharyngeal, IXth; and the vagus Xth nerves. The vagus divides to form four branchial branches (X br.1-4), which innervate the intrinsic respiratory muscles in the gill arches on either side of gill clefts 2-5. The first gill cleft is innervated by IX. Both IX and X are located on the wall of the anterior cardinal sinus (ant. card. sin.). The vagus, X also supplies two branches to the heart: the branchial cardiac (X br. c), which branches from the fourth branchial branch, and visceral cardiac (X visc. c), which branches from the visceral branch (visc. X), supplying the esophagus and foregut. Both cardiac branches enter the ductus Cuveri (duct. Cuv.) and run toward the heart where they form a dense plexus on the sinus venosus and atria. (pericard., pericardium; post. lat. lin., posterior lateral line nerve).

illustrated in Fig. 1. The two pairs of cardiac nerves pass down the ductus Cuveri and then break up into an interwoven plexus on the sinus venosus, terminating at the junction with the atrium (Young, 1933).The sinoatrial node is thought to be the site of the pacemaker in elasmobranch fishes (Rybak and Cortok, 1956; Satchell, 1971). The remainder of the vagus is termed the visceral branch, and this innervates the anterior part of the gut down to the pylorus and the anterior part of the spiral intestine (Young, 1933). Stimulation of the vagus nerve, as well as application ofacetycholine, has an inhibitory effect on heart rate (Short et al., 1977). The effects are antagonized b y atropine,

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implying that the effect is mediated by muscarinic cholinoceptors as in the higher vertebrates (Lutz, 1930a-c; Johansen et al., 1966; Butler and Taylor, 1971; Capra and Satchell, 1977; Taylor et al., 1977). Injection of atropine into intact fish abolished a reflex bradycardia in response to hypoxia (Butler and Taylor, 1971; Taylor et al., 1977) and hyperoxia (Barrett and Taylor, 1984a; and see Fig. 9). The sympathetic system of elasmobranchs consists of an irregular series of ganglia, lying dorsal to the posterior cardinal sinus and extending back above the kidneys (Young, 1950). These paravertebral ganglia are arranged approximately segmentally, except in the most anterior part where there is a concentration of associated neurosecretory tissue forming the auxillary bodies (see later). The segmentally arranged ganglia are irregularly connected longitudinally and with the contralateral paravertebral ganglia, but there are no distinct sympathetic chains of the type found in higher vertebrate groups. A peculiarity of the sympathetic system of elasmobranchs is that it does not extend into the head. This condition is unique among vertebrates, but it is not clear whether it is primary or the result of a secondary loss (Young, 1950). Gannon et al. (1972) described a sparse adrenergic innervation of the sinus venosus in Heterodontus, and it is conceivable that sympathetic fibers reach the elasmobranch heart and may influence heart rate, but the pathway is disputed. With rare exceptions (e.g., Mustelus, Pick, l970), contributions to the vagi or direct cardiac nerves from paravertebral ganglia have not been traced anatomically (Izquierdo, 1930; Lutz, 1930a-c; Young, 1933; Pick, 1970; Short et al., 1977). However, Izquierdo (1930) described a branch from the suprarenal bodies (see following discussion) that joined the vagi at the ductus Cuveri and found that electrical stimulation of this region increased sinoatrial conduction velocity in atropinized preparations. Short et al. (1977) found that vagal stimulation in atropinized dogfish had no effect on heart rate. However, our stimulation sites may have excluded a sympathetic nerve joining the vagi close to the heart. In turn, Izquierdo’s methods did not exclude an effect on the heart due to release of catecholamines from the auxillary body (see later). Thus, the existence of a cardiac sympathetic innervation in elasmobranchs is not proven, and there is no direct evidence for it having a functional role (Randall, 1970; Nilsson and Axelsson, 1987). Aggregates of chromaffin tissue, the “suprarenal bodies,” are juxtaposed to the paravertebral ganglia in elasmobranchs. They represent the homologue of the more discrete adrenal medulla of mammals, birds, and reptiles and contain high concentrations of catecholamines,

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predominantly noradrenaline, that are released into the circulation by activity in sympathetic preganglionic fibers. The most anterior and largest suprarenal body, the auxillary body, lies in the dorsal wall of the posterior cardinal sinus from where its products will b e aspirated directly into the heart (Johansen, 1971).The reported effects of adrenaline and noradrenaline on the elasmobranch heart are variable (Nilsson, 1983) but a positive chronotropic and large inotropic effect, mediated by a P-adrenoreceptor mechanism, has been described for the isolated heart (Capra and Satchell, 1977), and some degree of cardioregulation may be exercised by catecholamines released from the auxillary bodies. Circulating levels of catecholamines are relatively high in elasmobranchs and increase during hypoxia (Butler et al., 1978) so that it seems possible that they may exert tonic control over the cardiovascular system (Short et al., 1977); compensating for lack of sympathetic innervation of the heart and branchial circulation. An additional adrenergic influence on the heart may be exerted by specialized catecholamine storing endothelial cells in the sinus venosus and atrium. These cells are innervated by cholinergic vagal fibers (Saetersdal et al., 1975; Pettersson and Nilsson, 1979). There is no evidence for any vasomotor innervation of elasmobranch gills (Nilsson, 1983; Metcalfe and Butler, 1984b), but an intrinsic vasoconstriction during deep hypoxia (Satchell, 1962) may be released by a rise in circulating catecholamines (Butler et al., 1978), and it is possible that these vasomotor effects on the branchial vasculature involve variations in the relative proportion of total blood flow directed through the parallel arterioarterial and arteriovenous routes or changes in the patterns of perfusion of the gill lamellae (Nilsson, 1983). C. Teleosts

In teleost fish the vagus innervates the gills, the heart, and the viscera (pharynx, esophagus, stomach, and swimbladder) (Campbell, 1970). The teleost heart is innervated via a branch of the visceral vagus (Nicol, 1952; Randall, 1970; Johansen, 1971). The cardiac branches of the vagi follow the ductus Cuveri to the sinus venoms and atrium but vagal fibers may not reach the ventricle. Vagal ganglia lie close to the sinoatrial border and appear to consist solely of nonadrenergic cell bodies (Laurent, 1962; Gannon and Burnstock, 1969; Santer and Cobb, 1972; Santer, 1972; Holmgren, 1977,1981). The vagus is cardioinhibitory as in all vertebrates, with the exception of the cyclostomes. As in elasmobranchs, this inhibitory affect is due to the release of acetycholine affecting muscarinic cholinoceptors associated with the car-

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diac pacemaker and atrial musculature (Young, 1936; Randall, 1966; Randall and Stevens, 1967; Gannon and Burnstock, 1969; Holmgren, 1977,1981; Cameron, 1979).Atropine or hyoscine block a reflex bradycardia in intact fish (Stevens et al., 1972; Priede, 1974; Nilsson and Axelsson, 1987), the inhibition of rate following vagal stimulation (Randall, 1966; Holmgren, 1977) and the negative inotropic effects of acetylcholine on isolated perfused atria (e.g., Donald and Cambell, 1982).Acetylcholine was without effect on the contractility of isolated ventricles from the cod (Holmgren, 1977) and seven other teleost species (Donald and Cambell, 1982).Although the negative inotropic influence of the vagi does not reach the ventricle, cardiac output is greatly affected by the inotropic control of the atrium, which directly regulates the filling of the ventricle (Jones and Randall, 1978; Johanson and Burggren, 1980). Historically, sympathetic cardioaccelatory innervation was generally assumed to be lacking in teleosts (Randall, 1968). However, an adrenergic innervation of the heart via a mixed vagosympathetic trunk as well as separate sympathetic postganglionic fibers has since been described in several teleost species (Gannon and Burnstock, 1969; Holmgren, 1977; Cameron, 1979; Donald and Campbell, 1982). As a whole the teleosts may be considered phylogenetically at the first group of vertebrates in which there is both sympathetic and parasympathetic control of the heart, with innervation similar to that found in tetrapods (Laurent et al., 1983; Nilsson and Axelsson, 1987). An adrenergic tonus has been demonstrated on the heart of the cod (Gadus), but the relative importance of the neuronal and humoral adrenergic control of the heart remains uncertain (Nilsson, 1983). The positive chronotropic and inotropic effects on the teleost heart, produced by adrenergic agonists and adrenergic nerves, are mediated via P-adrenoceptor mechanisms associated with the pacemaker and the myocardial cells (Randall and Stevens, 1967; Gannon and Burnstock, 1969; Holmgren, 1977; Wahlqvist and Nilsson, 1977; Cameron and Brown, 1981).Adrenergic control may be important during exercise as a rapid cardioacceleration induced by enforced swimming in the goldfish was abolished by propranolol (Cameron, 1979), and Priede (1974) found that increases in heart rate associated with swimming in rainbow trout continued after bilateral vagotomy. In contrast to elasmobranchs where the branchial branches are solely skeletomotor (Metcalfe and Butler, 1984b), the branchial branches of the vagus (going to the gills) have both a vasomotor and skeletomotor function (Pettersson and Nilsson, 1979). There are sympathetic ganglia associated with cranial nerves IX and X in teleosts and

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the branchial nerves are mixed vago/glossopharyngeosympathetic trunks (Nilsson, 1983). Stimulation of these nerves may produce a cholinergically mediated constriction of the arterioarterial pathway in the gills whereas stimulation of the adrenergic fibers favors blood flow through this respiratory route rather than the arteriovenous route. Despite the clear demonstration of mixed autonomic innervation of the heart and branchial vasculature in teleosts, it remains probable that much of the functional control of gill perfusion is exercised via circulating catecholamines (Nilsson, 1983; Part A, Chapter 1).

111. THE CENTRAL LOCATION OF VAGAL PREGANGLIONIC NEURONS A. Cyclostomes The central nervous system of the cyclostomes may represent a prototype of the vertebrate brain (Ariens Kappers, 1929, 1947). The hindbrain is identical in superficial appearance to that of the rest of the vertebrates, with vagal rootlets leaving on either side to innervate the viscera. No study has been made of the topographical representation of vagally innervated structures within the vagal motor column of cyclostomes. In Lampetra two separate divisions of the vagal motor column have been identified using normal staining techniques: a rostral and a caudal motor nucleus of X (Niewenhuys, 1972). The caudal motor nucleus of X, which cannot be delineated from the spinal visceromotor cells, is thought to represent a splanchnic center, and the rostral nucleus is considered to be branchiomotor in nature (i.e., to innervate the branchial pouches) (Addens, 1933). The location of the caudal motor nucleus in cyclostomes, which centers around obex, is similar to the region of the dorsal vagal motor nucleus (DVN) in the cat (Bennett et al., 1981)and to the nucleus motorius nervi vagi medialis (Xmm) in the dogfish (Barrett and Taylor, l985b) in which the cell bodies contributing axons to the cardiac vagi are found.

B. Elasmobranchs T h e gross location of the vagal motor column in the hindbrain has been described in a number of elasmobranchs, although almost nothing was known of the topographical origin of vagal preganglionic fibers in elasmobranchs (Smeets et al., 1983). Classic neuranatomical techniques were used to describe a continuous column of large cell bodies

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of preganglionic neurons constituting motor nuclei of the IXth and Xth cranial nerves in a number of elasmobranchs, namely Selache maxima (Black, 1917), Squalus acanthias and Scyliorhinus canicula (Smeets and Niewenhuys, 1976), and Hydrolagus collei and Raja clavata (Smeets et al., 1983). In the shark Cetorhinus and in the Holocephali, Addens (1933) divided the vagal motor nucleus into separate rostral and caudal parts and suggested that the rostral portion subserves either a visceromotor or branchiomotor function, whereas the caudal portion represents a general visceromotor or splanchnic center. In Squalus this vagal part of the visceromotor column was designated the nucleus motorius nervi wagi medialis (Xmm) (Smeets and Niewenhuys, 1976). An area lateral to the caudal part of the visceromotor column contained a distinct aggregation of large bipolar and triangular cells and Smeets and Niewenhuys (1976) considered this to represent part of the motor nucleus of X and accordingly named it the nucleus motorius nervi vagi lateralis (Xml). The Xmm and Xml, by virtue of their locations, may be the homologues of the mammalian DVN and the nucleus ambiguus (NA), respectively (Smeets and Niewenhuys, 1976; Barrett et al., 1983). In this account they will be referred to as the DVN and LVN signifying dorsal and lateral vagal motonuclei. Retrograde intraaxonal transport of horseradish peroxidase (HRP) to identify vagal preganglionic neurons (Withington-Wray et al., 1986; Levings, 1990) showed that the vagal motor column in the dogfish, Scyliorhinus canicula, extends over 5 mm in the hindbrain (2.1 mm caudal to 2.9 mm rostral to obex, Figs. 2, 3, and 4),which agrees well with the extent described by Smeets and Niewenhuys (1976) for fish of similar size. Caudal to obex there appeared at first to be two distinct groups of vagal motoneurons, the majority found dorsomedially, and a smaller ventromedial group, both close to the lateral edge of the fourth ventricle (Withington-Wray et al., 1986).The ventromedial group were continuous with cells in the spino-occipital motor nucleus (Black, 1917), and almost certainly constituted a forward extension of this nucleus-contributing axons to the hypobranchial nerve that innervates the ventral muscles of the gill region (Levings, 1990; Levings and Taylor, 1988).The majority of vagal motoneurons caudal of obex contribute axons to the visceral branch of the vagus. The other branch of the vagus whose motoneurons were found caudal of obex was the visceral cardiac branch (Fig. 3). Visceral cardiac motoneurons were found in the dorsomedial division of the vagal motor column. Rostra1 of obex the medial vagal motoneurons were found clustered close to the ventrolateral edge of the fourth ventricle (Fig. 5) in the visceromotor

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;mi

Fig. 2. Schematic diagram of a dorsal view of the hindbrain and anterior spinal cord of the dogfish showing the distribution of the neuron cell bodies supplying efferent axons to cranial nerves innervating respiratory muscles and the heart; namely, the vagus, Xth; glossopharyngeal, IXth; spiracular branches of the facial, VIIth; and the adductor mandibulae branches of the trigeminal, Vth nerves. The individual motor nuclei are represented as hatched areas; the area of overlap between adjacent motor nuclei is indicated in black. 1, Adductor mandibulae motor nucleus (V); 2, Facial motor nucleus (VII); 3, Glossopharyngeal motor nucleus (IX); 4, Dorsal motor nucleus of the vagus (X); 5, Lateral motor nucleus of the vagus (X); 6, Occipital nerve (XI); 7, Vagus nerve (X); 8, Glossopharyngeal nerve (IX); 9, Octavus nerve (VIII); 10, Branches of the facial nerve (VII); 11, Branches of the facial and trigeminal nerves (VII and V); aur, cerebellar auricle; smi, sulcus medianus inferior; siv, sulcus intermedius ventralis; slH, sulcus limitans of His; oli, inferior olive; R, rhomboid fossa; 0, obex. [Redrawn from Levings (1990).]

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Fig. 3. The topographical organization of the vagal motor column and respiratory motor nuclei of the Vth, VIIth, IXth, and Xth cranial nerves in the dogfish to show their sequential rostro-caudal distribution on either side of obex. The lines indicate, from the top down, the rostro-caudal extent of pools of motoneurons supplying the mandibular branch of the Vth cranial nerve; the facial branch of the VIIth cranial nerve; the whole vagus (X), the glossopharyngeal (IXth) cranial nerve; and the vagus (Xth) cranial nerve, separated into its constituent branches (the first three branchial branches, X Br 1-3; the branchial cardiac, X Br C; and visceral cardiac, X visc. C, branches, and the visceral branch, visc. X). [Redrawn from Taylor (1989).]

IJl

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Fig. 4. Rostro-caudal distribution of vagal preganglionic motoneurons with respect to obex in the medulla of the dogfish, Scyliorhinus canicula. The majority of labeled motoneurons are located medially in the DVN. A small number (8%)are located ventrolaterally and supply axons solely to the branchial cardiac branch of the vagus innervating the heart. Medial cells supplying this nerve are indicated by the unshaded portion of the upper histogram. Motoneurons supplying axons to branchial branches 1, 2, and 3 (Br 1-Brlll) of the vagus,innervating gill arches 2, 3, and 4,occupy the rostral part of the vagal motor column, while the more caudal motoneurons supply axons sequentially to the heart, esophagus, and stomach. [Redrawn from Taylor and Elliott (1989).]

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Fig. 5. The location of preganglionic motoneurons identified in the brainstem of elasmobranch fish, following retrograde transport of HRP along identified branches of the Xth, vagus, and IXth glossopharyngeal cranial nerves. Cell bodies of preganglionic motoneurons, their axons and dendrites, as well as afferent sensory projections are stained with dark reaction product. (A) T.S. of medulla from dogfish, Scyliorhinus canicula, taken at obex to show preganglionic vagal motoneurons in the dorsal motonucleus (DVN) close to the 4th ventricle and in a scattered ventrolateral location (the LVN) (scale bar 200 pm). (B) A schematic diagram of a T.S. at obex to show the relationship between vagal motoneurons and other structures in the brainstem. (C) T.S. of dogfish medulla taken 0.4 mm rostral of obex to show the location of cardiac vagal motoneurons that are distributed both in the DVN close to the 4th ventricle and are scattered ventrolaterally right to the lateral edge of the brainstem in the LVN (scale bar 100 pm). (D) T.S. of medulla from the ray Raia claoata, 1.2 mm rostral of obex following application of HRP to the right glossopharyngeal nerve. Sensory neurons are labeled in the visceral sensory nucleus of IX (IX sn) together with their axons that course through the solitary tract (ts). The motor nucleus of IX is located medially in the visceral motor column surrounding Steida’s fasiculus (fst) (scale bar 200 pm). (ax, axons; ax. X, axons of vagus nerve; DVN, dorsal vagal motonucleus; F1, nucleus funiculi lateralis; flm, fasiculus longitudinalis medialis; fst, fasiculus medianus of Steida; IXsn, glossopharyngeal sensory nucleus; LVN, lateral vagal motonucleus; Oh, Oliva inferior; rdv, radix descendens nervi trigemini; siv, sulcus intermedius ventralis; slH, sulcus limitans of His; smi, sulcus medianus inferior; ts, nucleus tractus solitarius; 4 Vent, fourth ventricle; Xr, vagal rootlet; Xsn, vagal sensory nucleus.)

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column. The majority of vagal motoneurons were found in this rostromedial division of the vagal motor column (i.e., the DVN), which contributed axons to the branchial cardiac branch and to the visceral branch in its most caudal one-third, and to the branchial branches of the vagus in its rostra1 two-thirds (Fig. 3 ) . A clearly distinguishable ventrolaterally scattered group of cells was identified that had a rostrocaudal extent of approximately 1 mm, rostrally from obex (Figs. 2,4, and 5). This population of motoneurons contributed axons solely to the branchial cardiac branch of the vagus (Barrett et al., 1983;Barrett and Taylor, 1985b) and composed 8% ofthe total population of vagal motoneurons. The cells in this lateral division supply 60% of the efferent axons running in the branchial cardiac nerve, with the other 40% supplied by cells in the rostromedial division (Fig. 4).When the medial cells contributing efferent axons to the heart via the visceral cardiac branches are taken into account, then the lateral cells supply 45% of vagal efferent output to the heart (Withington-Wray et al., 1987). Thus branchial cardiac motoneurons are found rostromedially and solely compose the lateral division ofthe vagal motor column (Fig. 4). It is thought that these two locations of cardiac vagal preganglionic neurons give rise to the two different types of efferent activity recorded from the cardiac vagi of the dogfish (Taylor and Butler, 1982; Barrett and Taylor, 1985a,c); this point is examined in the following discussion. An as yet unpublished study using retrograde transport of HRP along identified branches of the vagus to identify the detailed topography of the vagal motor column in two species of rays Raja clavata and R . microocellata (Levings, 1990) supported our results for the dogfish. T h e majority of vagal preganglionic neuron cell bodies were located as a “classic DVN” forming a continuous longitudinal column in the ipsilateral hindbrain. In the midportion of the vagal motor nucleus, labeled neurons were observed to be located both in the DVN and scattered in a separate ventrolateral location, called the LVN that extended rostro-caudally for approximately 2 mm (Fig. 6). Barry (1987) described the vagal motor nucleus of Raja eglanteria as being located as separate dorsal and ventral nuclei. Examination of his data revealed a typical DVN (which he termed Xmd) and a separate group of neurons, which were scattered ventrolaterally, almost to the edge of the hindbrain (termed Xmv). The location of these scattered neurons is very similar to that of neurons sited in the LVN of the Raja species examined by Levings (1990). However, the location of neurons sited caudally in his Xmv closely resembles that of hypobranchial motoneurons, and it is probable that they arise from confusion between vagal and

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A Whole vagus DVN LVN Branchial 1 Branchial 2 Branchial 3 Branchial 4 Cardiac DVN LVN Visceral I

-4

I

I

I

I

1

I

-1 OBEX *1 +2 -2 -3 Distribution with respect to obex (mm)

I

+3

Fig. 6 . The rostro-caudal distribution, with respect to obex, of preganglionic motoneurons in the vagal motor nucleus of the ray Raja clauata. (A) The distribution of vagal motoneurons found following application of HRP to the whole vagal trunk. Dorsal vagal motonucleus, DVN; lateral vagal motonucleus, LVN. (B) The topography of the vagal motor nucleus. The distribution of vagal motoneurons supplying efferent axons to the four branchial branches, the cardiac branch (supplied by the DVN and LVN) and the visceral branches of the vagus nerve. [Redrawn from Levings (1990).]

hypobranchial branches, similar to that experienced by WithingtonWray et al. (1986)working on the dogfish. Application of HRP to the individual branches of the vagus revealed that the cells of origin of the branchial nerves were serially represented in the DVN in the rostral and midportion of the vagal motor nucleus (Figs. 6 and 7). A similar sequential topographical representation of the cells of origin of the individual branchial branches of the vagus in the DVN was described in the dogfish (Fig. 3 ) and can be identified in Fig. 4.Additional neuranatomical study of branches ofthe Vth and VIIth cranial nerves to respiratory muscles in dogfish and rays (Levings, 1990) revealed that their efferent cell bodies were located ipsilaterally in the brainstem rostral of the vagal motor column, contributing to the sequential rostro-caudal distribution of discrete motor nuclei innervating the respiratory apparatus (Figs. 2 and 3 ) . The rays possess only one pair of cardiac vagi. Cardiac vagal motoneurons (CVM) were found in the DVN and LVN, and their distribution overlapped that of the respiratory vagal motoneurons supplying axons to the fourth branchial branch of the vagus (Fig. 6). Thus the cardiac nerves in the rays would, on neuranatomical criteria, ap-

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Br. 2

r 60 -

40 -

. I -

20 -

0

0 z

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0 caudal

rostra1

Distribution with respect to obex (mm) Fig. 7. The rostro-caudal distribution, with respect to obex, of respiratory vagal motoneurons supplying axons to branchial branches 2 , 3 , and 4 of the ray Raja clawata. They show a sequential rostro-caudal distribution with some overlap of cell bodies innervating neighboring gill clefts. [Redrawn from Levings (1990).]

pear to be homologous to the branchial cardiac branches of the dogfish. Labeled visceral vagal motoneurons were located in the caudal portion of the vagal motor nucleus, in the ipsilateral DVN (Fig. 6). These results agree with our findings in the dogfish. In summary, the vagal motor column of elasmobranchs consists of distinct divisions. Caudal to obex it contains dorsomedial and possibly ventromedial divisions, though the latter distribution is contentious. Rostra1 of obex there is a single rostro-medial division and a short lateral division that contains about 8% of the vagal preganglionic neurons. This scattered lateral group supplies axons solely to the heart. There is a sequential topographic representation of the vagus nerve in the vagal motor column with neurons supplying the structures distal to the hindbrain (gastrointestinal tract) located caudally, those supplying the cardiac nerves located in the midportion of the column, and those supplying the proximal structures (gill arches) located most rostrally in a sequential topography, with anterior gill arches provided by more rostrally located cell bodies. This sequential topography is extended rostrally by the motor nuclei of the IXth, VIIth, and Vth cranial nerves supplying efferent innervation to the first gill cleft and to the respiratory muscles of the spiracles and jaws, respectively (Figs. 1,2, and 3 ) . In some fish anterograde transport of HRP filled the sensory projections of the cranial nerves. This revealed darkly stained axons connecting to a diffuse area of small neurons and dendrites in the visceral sensory area, dorsal of the motonuclei but in the same rostrocaudal location as the motor projection for each nerve (Fig. 5 ) .

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C. Teleosts Initial topological studies of the brain in the Crossopterygian fish Latimeria (Kremers and Niewenhuys, 1979), Gnathonemus (Szabo and Libouban, 1979), and the reedfish Erpetoichthys (Niewenhuys and Oey, 1983) described a single vagal motor nucleus, and it seemed that the equivalent of an LVN was absent from the hindbrain of teleosts. Subsequent work on the channel catfish, Zctalurus punctatus (Kanwal and Caprio, 1987), revealed that all of the IX and Xth efferent roots originated from cell bodies located in a continuous longitudinal motor column, bordering the fourth ventricle, along the ventromedial portion of the medulla, terminating caudally at obex. As in Silurus glaris (Black, 1917)the vagal motor column (DVN) terminated rostrally before the appearance of the VII facial motor nucleus. A study using HRP transport and immunocytochemistry on the goldfish, Carassius auratus (Morita and Finger, 1987), revealed that a lateral subnucleus of the vagal complex, possibly equivalent to the mammalian nucleus ambiguus, provided axons to respiratory muscles. The cardiac and visceral motoneurons were located medially, rostral of obex. We have applied HRP to the whole vagus nerve and to selected branches of the vagus in two species of teleost, the cod (Gadus morhua) and the trout (Oncorhyncus mykiss).The results obtained in these two species of teleost were essentially similar to each other (Withington-Wray et al., 1987).We found that, for example in the cod, vagal preganglionic neurons were located over a distance of 4 mm in the ipsilateral hindbrain from 1.8 caudal to 2.2 mm rostral to obex. Labeling the entire vagus also showed that approximately 11%of the neurons were located ventrolaterally, while all the others were found in a dorsomedial location (the DVN) clustered close to the edge of the fourth ventricle. The lateral group of vagal motoneurons, constituting the LVN, were concentrated into a more discrete group or nucleus compared with the scattered distribution of elasmobranchs. When HRP was applied to the cardiac branch of the vagus labeled neurons (CVM) were found at the caudal extent of the LVN as well as in the DVN. The application of HRP to one of the branchial branches of the vagus also labeled cells in the LVN as well as the DVN, this time with the lateral cells located in a more rostral group (Fig. 8). Although the majority of cardiomotor neurons were located medially and a small number of respiratory vagal motoneurons were ventrolaterally located, there is a clear difference between the distribution we describe for cod and trout and that described in the goldfish by Morita and Finger (1987) that must be resolved by further study.

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medial

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Fig. 8. The rostro-caudal distribution of respiratory and cardiac vagal motoneurons in the cod, Gadus morhua. Motoneurons supplyingaxons to the heart (CVM) on the right side ofthe fish had their cell bodies predominately in a medial location (the DVN) with a small proportion in a discrete lateral nucleus (the LVN). Respiratory vagal motoneurons (RVM) supplying axons to the third branchial branch on the left side ofthe fish (L branch 3 ) were similarly located in the DVN and in smaller numbers in the LVN. These RVM were located rostrally of the CVM indicating a sequential topography of vagal motoneurons. [Unpublished data from E. W. Taylor, D. J. Withington-Wray, and J. D. Metcalfe.]

The identification of lateral cardiac vagal preganglionic neurons in the cod and trout is similar to our findings in elasmobranchs. In contrast, however, some branchial motoneurons are also located in the lateral division in teleosts, whereas they are confined to a medial location in elasmobranchs. This may reflect the observation that the branchial branches of the vagus serve both a vasomotor and skeletomotor function in teleosts but only a skeletomotor function in elasmobranchs (see earlier). We suggest that the dual function ofthe branchial branches in teleosts may be reflected in the dual origin of the branchial efferent axons fibers in the brain and that the medial neurons may give rise to the skeletomotor axons whereas the lateral neurons may give rise to the vasomotor axons (Withington-Wray et al., 1987). This hypothesis remains to be tested. A larger percentage of vagal preganglionic neurons is located in a lateral division in teleosts (11%)when compared to elasmobranchs (8%),with the increment associated with the supply of axons ofboth branchial and cardiac branches of the vagus. In teleosts there is also a sequential topographic representation of the vagus within the vagal motor column that is evident in Fig. 8. The most rostral neurons supply axons to the most proximal organs (gills),

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and the caudal neurons innervate the viscera (Kanwal and Caprio, 1987).The cardiac neurons are located in the middle of the vagal motor column. Morita and Finger (1987) described a sequential viscerotopy in the lateral motor column of the goldfish. So in both classes of fish in which the topographical layout of the vagus has been studied there is a sequential representation of the target organs in the vagal motor column. IV. CONTROL OF THE HEART AND BRANCHIAL CIRCULATION A. Vagal Tone on the Heart

As described previously, the heart in all fish except cyclostomes is supplied with inhibitory parasympathetic innervation via the vagus nerve. The inhibitory effect is mediated via muscarinic cholinoreceptors associated with the pacemaker and atrial myocardium (Holmgren, 1977). The heart in vertebrates typically operates under a degree of inhibitory vagal tone that varies with physiological state and environmental conditions. Lutz (1930~) found that bilateral vagotomy caused an increase of about 60%in heart rate in the dogfish. Butler and Taylor (1971) established a direct relationship between vagal tone and normoxic heart rate in lightly restrained, unanesthesized dogfish, with the increase in rate following injection of the muscarinic cholinergic blocker atropine inversely proportional to the normal rate in individual fish. Heart rate in dogfish restrained in a standard set of experimental conditions at 15"-17°C varied directly with Po,. Hypoxia induced a reflex bradycardia (Butler and Taylor, 1971; Taylor et al., 1977), a normoxic vagal tone was released by exposure to moderate hyperoxia, and extreme hyperoxia induced a secondary reflex bradycardia, possibly resulting from stimulation of venous receptors (Barrett and Taylor, 1984a). All of these affects were abolished by atropine (Fig. 9). In addition, cholinergic vagal tone, assessed as the proportional change in heart rate following atropinization, increased with an increasing temperature of acclimation (Taylor et al., 1977; Fig. 10). These data indicate that the level of vagal tone on the heart in dogfish varies with temperature and oxygen partial pressure and may be determined peripherally by graded stimulation of P o , receptors, with large variations in this inhibitory tone causing reflex bradycardia or tachycardia. However, the relationship between heart rate and oxygen availability is complex in dogfish. Butler and Taylor (1971) found that a rapid

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.-

'C

E m

4,02(mmHg)

Fig. 9. Mean ventilation rate (VR) and heart rate in normal (HR) and atropinized (HR atr) dogfish at a range of oxygen partial pressures ranging between 30 (hypoxia) and 650 mmHg (extreme hyperoxia). Hypoxia caused a bradycardia but no change in VR. Hyperoxia caused a progressive decrease in VR and an initial increase in HR above the rate in normoxia, followed by a progressive bradycardia. HR atr did not vary with Po2, indicating that these changes in HR were reflex resulting from oxygen dependent variations in vagal tone. The initial increase in hyperoxic HR was the result of a reduction in vagal tone with HR approaching the HR atr and also VR indicating that cardiorespiratory synchrony may arise when cardiac vagal tone is low. [Data from Barrett and Taylor (1984b) and Taylor and Barrett (1985).]

reduction in inspired oxygen levels (within 1 min) caused a transient intense bradycardia. Heart rate then recovered to a stable rate the same as that measured in fish gradually exposed (e.g., over 30 min) to the same hypoxic P o , (Fig. 11). The increase in heart rate on recovery from hypoxia also anticipated complete reaeration of the water, so that rapid fluctuations in Po, caused changes in heart rate that related to the direction and rate of change of P o , as well as to the level of hypoxia (Fig. 12).The efferent arm for all these recorded changes in heart rate was the cardiac vagi, because the complete hypoxic response was abolished by atropine or cardiac vagotomy (Butler and Taylor, 1971; Taylor et al., 1977, see Figs. 9 and 11).Thus, variations in the degree of cholinergic vagal tonus on the heart seems to serve as the predominant mode of nervous cardioregulation in elasmobranchs (Taylor et al., 1977; Barrett and Taylor, 1984a; Taylor, 1985, 1989). In teleosts the heart receives both a cholinergic vagal supply and an adrenergic sympathetic supply. Available data on the extent of vagal tone on the teleost heart give a wide range of values revealing species differences, and the effects of different environmental or experimental conditions. Injection of atropine into the pericardial cavity of normoxic

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A

$ 1 ,

c

5

disturbed

I

I

I

,

10

15

20

25

temperature "C

Fig. 10. Mean ventilation rate (VR) and heart rate in normal (HR) and atropinized (HR atr) dogfish at a range oftemperatures between 7" and 23°C. Fish at 7", 12", and 17°C were restrained in an experimental chamber. Fish at 23°C were cannulated then released into a large tank of seawater where they were unrestrained and routinely active. Under these latter conditions they typically became inactive (settled) and heart rate and ventilation became synchronous. When they swam spontaneously (active)heart rate fell and ventilation increased, and when they were physically disturbed this trend was increased until ventilation rate was double heart rate. These disturbed rates at 23°C had a straight line relationship with the rates of restrained animals at lower temperature. Atropinization raised heart rate at all temperatures but was most effective at high temperatures, indicating that vagal tone on the heart of disturbed or restrained dogfish increased with temperature. [Data from Taylor et al. (1977) and Taylor (1985).]

tench, Tinca tinca, caused a 15-35% increase in heart rate at 11"-15"C but was without effect on the goldfish (Randall, 1966). However, Cameron (1979) found that goldfish at 25°C had a calculated parasympathetic tone (released by atropine) of 66% of intrinsic heart rate while sympathetic tone was 22% and a rapid cardioacceleration induced by enforced swimming was abolished by propranolol. Axelsson (1988) used injections of atropine and sotalol to estimate the cholinergic and adrenergic influences on the heart of the cod. At rest cholinergic and adrenergic tonus were 38 and 21%, respectively. After a period of exercise cholinergic tonus decreased to 15% and adrenergic tonus increased to 28% suggesting that variations in cholinergic tone largely determined heart rate. In the trout vagal tone on the heart, although higher than in the dogfish at all temperatures, decreased at higher temperatures, but the cardioacceleration induced by adrenaline injection into atropinized fish increased with temperature (Wood et al.,

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363

vagotomized (d)

N S 1

N H

N T S

N T S l

Fig. 11. Time related changes in mean heart rate (2SEM) in restrained, unanesthetized dogfish ( S . canicula) during exposure to hypoxia. (a) Exposure to gradual hypoxia with ambient Poz reduced from normoxic levels down to 30 mmHg over about 30 min caused a moderate bradycardia. (b) Rapid reduction of Poz to 30 mmHg within 1 min caused a transient, more intense bradycardia followed by recovery over 3 min to the same rate as measured during gradual hypoxia. (c)A similar but more marked response to rapid hypoxia was followed by injection of atropine, which released a normoxic vagal tone on the heart, signified by the hatched area on A. (d) Following branchial cardiac vagotomy there was an increase in normoxic heart rate, resulting from a reduction in vagal tone as signified by the reduced area of hatching on A, and the transient intense bradycardia was virtually abolished. (N, normoxic rate; H, rate during gradual hypoxia; T, transient rate during rapid hypoxia; S, stable rate during rapid hypoxia; A, rate following injection of atropine.) [Redrawn from Butler and Taylor (1971) and Taylor et al. (1977).]

1979). An inhibitory vagal tonus was significantly greater in wannacclimated than in cold-acclimated eels, Anguilla anguilla, and blocking vagal function with benzetimide reduced a nearly complete temperture compensation (Seibert, 1979). These data indicate that adaptation of heart rate to temperature in the eel was largely mediated by the parasympathetic nervous system. Further evidence for temperature related changes in heart rate being determined centrally is provided by work on antarctic fishes. Resting heart rate in Pagothenia benacchii and P . borchgrevinki at 0°C was about 11beats min-' with steady rates as low as 3-6 beats min-' recorded from some P . benacchii. However, the intrinsic rate following vagal and sympathetic blockade was about 23 beats min-l (Axelsson et al., 1992). The low normal rate in P . benacchii was due to an 80% inhibitory vagal tone, released by atropine, antagonized by a 28% excitatory adrenergic tone, released by propranolol. Pagothenia borchgrevinki exhibited a 55% vagal tone and

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Fig. 12. Continuous record showing the effects of rapid hypoxia and partial recovery on blood pressure and heart rate in the unanesthetized dogfish. The traces are, from top downwards: ambient Pop; blood pressure in the ventral aorta; blood pressure in the dorsal aorta (mmHg). The values of Pop (mmHg) and heart rate (beatdmin-') at specific times are given above the P o p trace. Heart rate falls to a very low level at the onset of rapid hypoxia then recovers to a stable rate, characteristic of the Pop.When P o p is raised heart rate shows a transient overshoot then again adapts to a rate typical of the Pol. As a consequence heart rate at any Po2 can be markedly different, dependent upon the rate and direction of change (e.g., at a Popof approx 45 mmHg heart rate varied from 28 to 19 beats/min-' and at 17 mmHg from 4.5 to 7.5 beatdmin-'). [Taken from Butler and Taylor (1971).]

an excitatory adrenergic tone of only 3%. The intrinsic heart rate at 5°C rose to 32 beats min -l, which is similar to the trout at the same temperature. Thus, despite their increased cardiac volume the intrinsic pacemaker frequency in these antarctic fishes may be similar to that of temperate species, with the low normal resting rates attributable to high levels of vagal tone, imposed from the central nervous system (CNS). Recordings of heart rate often show short-term variation in beat-tobeat intervals that can be plotted as a heart rate variability signal (HRVS). It has been suggested that analysis of the HRVS in mammals can be used as a quantitative means of investigating control of cardiac function (McDonald, 1980) and that, in particular, HRVS may represent fluctuations in vagal tone in the heart. An HRVS can be recognized in the ECGs recorded by telemetry from free swimming trout at a range of temperatures (de Vera and Priede, 1991). The power spectra of the HRVS showed two major peaks, corresponding to two main periodic components in the signal. At 5" and lWC, the power of the lowfrequency peak was significantly greater than that of the highfrequency one; but at 15°C this was reversed with both spectral peaks

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at higher frequencies. Mammals typically exhibit a range of HRVS spectral peaks, and it has been shown that the high- and midfrequency peaks are caused by parasympathetic activity whereas the lowfrequency peak includes the sympathetic component (Akselrod et al., 1981). The implications of these recordings of HRVS in the trout for our understanding of the control of the heart in teleost fish were considered by de Vera and Priede (1991). They stated that cholinergic inhibitory pathways show feedback approximately twice as fast as adrenergic stimulatory pathways. We know that tonic inhibitory control of the heart exerted by the vagus varies with chemoreceptor and mechanoreceptor stimulation and that it includes efferent components that show respiration related activity. Consequently, a predominance of vagal control could result in high frequency components in the HRVS. Adrenergic control relates to influences on the peripheral circulation, possibly due to circulating catecholamines, which are likely to have a longer latency. The two pathways appear to vary in importance according to temperature, with the cholinergic pathway (high frequency) dominating at high temperature. This is at variance with the earlier suggestion by Priede (1974) and Wood et al. (1979) that the cholinergically mediated parasympathetic inhibitory tonus dominates at low temperatures, and also contradicts the data from antarctic fishes, but agrees with our observations on dogfish (Taylor et al., 1977). Clearly, the degree of tonic control of heart rate varies in complex ways, both in the short-term and with long-term factors such as acclimation temperature. A systematic study of variation in vagal and sympathetic tone on the hearts of fishes at a range of temperatures and activity levels may clarify the present contradictory data. B. Efferent Activity Recorded from Cardiac Vagi The existence of a varying level of inhibitory vagal tone on the heart of the experimentally restrained dogfish prompted an investigation of vagal output to the heart. Experiments involving transection and electrical stimulation of the two pairs of cardiac vagi in the dogfish revealed that the branchial cardiac branches are more effective in cardioinhibition than the visceral cardiac branches (Taylor et al., 1977; Short et al., 1977), accounting for the majority of normoxic vagal tone and the reflex bradycardia during hypoxia (Taylor, 1985). Branchial cardiac vagotomy markedly reduced normoxic vagal tone and abolished a transient intense bradycardia during rapid hypoxia (Butler and Taylor,

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1971; Fig. 11).Recordings from a branchial cardiac branch contained high levels of spontaneous efferent activity, separable into two types of units. Some, typically smaller, units fired sporadically and increased their firing rate during hypoxia (Taylor and Butler, 1982; see Fig. 13). These nonbursting units seem to play the major role in initiating the reflex hypoxic bradycardia and may also determine the overall level of vagal tone on the heart (Taylor, 1985). Other, larger units fired in rhythmical bursts that were synchronous with ventilatory movements (Taylor and Butler, 1982; see Figs. 13 and 18).Recordings from the less-effective visceral cardiac branches consisted of bursting units alone. These bursts were often not synchronous with ventilation and their motor function is as yet unclear (Barrett and Taylor, 1984a); their primary function may be sensory (Short et al., 1977). The bursts recorded from branchial cardiac branches continue in decerebrate, paralyzed dogfish and are synchronous with efferent activity in branchial branches of the vagus that innervate respiratory muscles (Fig. 13).This evidence alone suggests that the bursting activity originates in the CNS through some interaction, either direct or indirect, with the central respiratory pattern generator (CPG). Randall (1966) recorded bursting activity from the cardiac vagus of the tench that he concluded could either originate peripherally from stimulation of receptors on the gills or in the blood stream dorsal to the gills (i.e., efferent vessels) or may result from connections between the “vagal and respiratory centers” in the medulla. The temporal relationships of respiration-related activity recorded from cranial nerves innervating the respiratory muscles and the heart are described in Section VI. C. Central Origin of Efferent Activity in Cardiac Vagi As has been previously described the branchial cardiac branch of the vagus in dogfish is unique in having some of its CVM located as a ventrolateral group of neurons (the LVN) that supply axons solely to this branch of the vagus, with the remainder located, together with respiratory vagal motoneurons (RVM) in the DVN (Barrett and Taylor, 1985b).Extracellular recordings from CVM identified in the hindbrain of decerebrate, paralyzed dogfish by antidromic stimulation of a branchial cardiac branch revealed that neurons located in the DVN were spontaneously active, firing in rhythmical bursts that contributed to the bursts recorded from the intact nerve (Fig. 14). Neurons located in the LVN were either spontaneously active, firing regularly or sporadically but never rhythmically, or were silent (Barrett and Taylor,

6. CONTROL OF HEART AND CARDIORESPIRATORY INTERACTIONS

A

branchial cardiac nerve

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hypoxia

2s

I

branchial

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Fig. 13. (A) Efferent activity recorded from the left branchial cardiac branch of the vagus of a decerebrate dogfish in normoxia ( P o , 150 mmHg) and hypoxia ( P o 245 mmHg). The smaller, sporadically active units markedly increased the firing rate during hypoxia, while the larger, rhythmically bursting units were less active. [Data from Taylor and Butler (1982).] (B) Simultaneous recordings of efferent activity in the third branchial branch (branchial) and the branchial cardiac branch (cardiac) of the left vagus from a decerebrate, paralyzed dogfish. The persistence of rhythmic bursting activity in both nerves indicates that it is generated in the CNS and not by stimulation of mechanoreceptors on the gill arches. Sporadically active units that are present in recordings from the cardiac nerve are less evident in the branchial nerve. [Data from Barrett and Taylor (1985a).]

l984b; 1 9 8 5 ~ )It. seems that the two types of efferent activity described in the branchial cardiac branches of the vagus have separate origins in the CNS, which may indicate a separation of function. All of the spontaneously active CVM from both divisions and some of the silent CVM fired in response to mechanical stimulation of a gill arch (Fig. 14). A study in our laboratory (M. Young, unpublished observations) revealed that electrial stimulation of the central cut end of a branchial branch of the vagus in the decerebrate, curarized dogfish could entrain efferent activity recorded from the branchial cardiac branch (Fig. 15). These data indicate that activity in CVM could be entrained to ventilatory movements in the spontaneously breathing fish.

368

E. W. TAYLOR medial cardiac motoneuron

cardiac nerve

u

1 mm

B

stim

1 sec

lateral cardiac motoneuron

cardiac nerve

-

u

1 mm

1 sec

lateral cardiac motoneuron

C cardiac nerve U

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Fig. 14. Central recordings from cardiac vagal motoneurons, identified by antidromic stimulation of the branchial cardiac nerves, in different locations in the medulla of the dogfish, Scyliorhinus canicula, together with recordings of spontaneous efferent activity in the cardiac nerve: (A) A rhythmically bursting unit, identified in the DVN just rostra1 to obex, that contributed action potentials to the regular bursts of activity in the nerve and responded to mechanical stimulation of the gill arches (stim); (B) a regularly firing unit identified in a ventrolateral location that contributed action potentials within and outside the bursts of activity recorded from the nerve; (C) another ventrolateral cell that was not spontaneously active but responded to mechanical stimulation. The location of the tip of the recording electrode is indicated by the filled circle on the diagrammatic TS of the medulla. [Data from Barrett and Taylor (1985c).]

The central origin of the respiration-related bursting activity in the medial group of CVM is of particular interest because respiratory modulation of CVM has been observed in mammals. The sensitivity of mammalian CVM to inputs from arterial baroreceptors and chemoreceptors is reduced during inspiration and, when their excitability

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CONTROL OF HEART AND CARDIORESPIRATORY INTERACTIONS Br. card. (rec)

49 m1n-l

Br. 2 (stim)

39 min-'

369

1 1 1 1 1 1 1 1 1

t Br card (recl

49 rnin-'

Br. 2 (stirn)

62 min-'

2 sec

111111111111

t

stim. on (100 sec-l)

Fig. 15. The effect of electrical stimulation of the central cut end of a branchial branch of the vagus on efferent activity in the ipsilateral branchial cardiac nerve. Spontaneous activity in the cardiac branch consisted of regular bursting units firing at a rate of 49 burstslmin-' plus relatively low levels of background activity in smaller units. Central stimulation of the branchial branch with bursts of stimuli at 100/sec-' entrained the bursts in the cardiac nerve to either a slower rhythm of 39 bursts/min-' or a faster rhythm of 62 burstdmin-'. During periods of stimulation the background activity was also increased. [Unpublished data from M. Young, P. J. Butler, and E. W. Taylor.]

is raised experimentally, they fire in the postinspiratory and expiratory phases of ventilation and are silent during inspiration, when the phrenic nerve is firing (Spyer, 1982; Jordan and Spyer, 1987). This modulation, which is the central origin of respiratory sinus arrhythmia in the mammal, is thought to arise from direct, inhibitory synaptic contact between collaterals from RVM and CVM in the ventrolateral NA. Direct connections between bursting CVM and RVM are possible in the dogfish hindbrain as both are located in the medial division of the vagal motor column (DVN) with an overlapping rostrocaudal distribution (Barrett and Taylor 1985b; Fig. 3 ) . As the bursts recorded from branchial and branchial cardiac vagal branches are synchronous (Fig. 13)the influence of RVM on CVM is likely to be excitatory rather than inhibitory, and it is equally possible that a direct drive from the CPG operates on the RVM and the CVM. In addition, the activity in CVM and RVM may be modulated by afferent input from gill mechanoreceptors, stimulated by ventilatory movements, and by chemoreceptors in various locations (see later). The possible connections to and from the CPG, RVM, and CVM are summarized diagrammatically in Fig. 16.

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GILL ARCHES

HEART

Fig. 16. Diagram of the possible afferent and efferent connections of preganglionic vagal motoneurons in the hindbrain of the dogfish that control and coordinate gill ventilation and heart rate. There are several established connections in the nervous control of ventilation; (1)the respiratory central pattern generator neurons (CPG) show endogenous bursting activity that drives respiratory motoneurons (RVM); (2) the RVM innervate the intrinsic muscles in the gill arches; ( 3 )the activity ofthe C P G is modulated by feedback from mechanoreceptors and possibly chemoreceptors located on or near the gills and innervated by vagal sensory neurons (RVS). Heart rate is controlled by inhibitory input from the vagus nerve that receives axons from cardiac vagal motoneurons (CVM), which are topographically and functionally separable into: (4)a ventrolateral group, some of which fire continuously and may be responsible for reflex changes in heart rate (e.g., hypoxic bradycardia) and for the varying level ofvagal tone on the heart, and ( 5 ) a medial group, which burst rhythmically and may cause the heart to beat in phase with ventilation. Other more speculative connections may determine the activity in the CVM: (6) collaterals from neighboring RVM may have an excitatory effect on bursting medial CVM (or release a tonic inhibition); (7)the CPG may connect directly to medial CVM; (8)stimulation of receptors on the gill arches may directly modify activity in medial and some ventrolateral CVM; (9)stimulation of receptors in the cardiovascular system close to the heart innervated by vagal sensory neurons (CVS) may affect vagal outflow to the heart. This diagram is highly schematic and ignores the existence and possible roles of interneurons and inputs from and to higher centers in the CNS. A, efferent termination; A,afferent termination; S, sinus venosus; A, atrium; V, ventricle.

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V. CARDIORESPIRATORY INTERACTIONS A. Reflex Modulation of Heart Rate 1. CHEMORECEPTORS The aquatic environment often varies markedly in oxygen content, and this is reflected immediately as an internal change, across the countercurrent on the gills. The physiological responses to hypoxia typically include an increase in ventilation rate and a reduction in heart rate that has been characterized as a reflex bradycardia with the vagus as the efferent limb (Taylor, 1985). The complex cardiac chronotropic responses to hypoxia in the dogfish were described in Section IV,A. Study of the hypoxic responses of fish, combined with nerve transection, stimulation experiments, and external and internal injection of cyanide have eliminated the brain as a site for central chemoreception but identified a number of putative reflexogenic sites around the gills and vasculature of fish. These data are reviewed by Burleson et al. in Chapter 7. The best evidence suggests that receptors on the gills, sensitive to oxygen partial pressure, mediate the reflex hypoxic bradycardia as well as ventilatory responses and that vascular receptors, responding to oxygen supply (i.e., content and rate of delivery), mediate reflex changes in ventilation (Randall, 1982; Taylor, 1985). Evidence indicates a role in the reflex cardiac responses in the dogfish for venous oxygen content receptors (Barrett and Taylor, 1984a).Thus fish may possess receptors monitoring P o , levels on both the afferent and efferent sites of the countercurrent exchanger potentially enabling them to match the relative flow rates of water and blood over the gill lamellae in order to optimize respiratory gas exchange, saturating the blood with oxygen while minimizing the energy cost of ventilation and perfusion (Taylor, 1985). From our standpoint, it is of interest that many of the putative reflexogenic areas are innervated by branches of the cranial nerves innervating respiratory muscles, including X, IX, VII, and V (e.g., Butler et al., 1977), SO that the afferent and efferent arms ofthe reflex responses to hypoxia have the same peripheral routes and neighboring central projections described earlier (Figs. 1,2,3, and 5 ) and in Chapter 7. Central stimulation of branchial branches of the vagus in the dogfish, as well as entraining efferent bursting activity in the branchial cardiac, also caused increased activity in nonbursting units (Fig. 15). Activity in these nonbursting units may determine heart rate with reference to ambient PO, (Taylor and Butler, 1982) so that central

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stimulation of a branchial nerve appears to stimulate both chemoreceptor and mechanoreceptor afferents. Stimulation of carotid chemoreceptors can activate higher centers such as the defense areas in mammals, to evoke the defense or visceral alerting response that may culminate in fight or flight (Marshall, 1987). Although some fish show behavioral avoidance reactions to hypoxia ( Jones, 1952), the unrestrained dogfish responds to environmental hypoxia with a reduction in activity, which remains suppressed throughout the hypoxic period despite an increase in circulating catecholamines (Metcalfe and Butler, 1984a). This would seem to be the opposite of a defense or alerting response and is reflected in the apparent absence or suppression of a flight syndrome in dogfish, which often fails to respond overtly to physical disturbance, though they typically exhibit a reflex bradycardia (Taylor, 1989). 2. MECHANORECEPTORS The respiratory muscles in fish contain length and tension receptors in common with other vertebrate muscles, and the gill arches bear a number of mechanoreceptors with various functional characteristics. Satchel1 and Way (1962) characterized mechanoreceptors on the branchial processes of the dogfish and Sutterlin and Saunders (1969) described receptors on the gill filaments and gill rakers of the sea raven, Hemitripterus americanus. Using a single-unit recording from vagal epibranchial ganglia in spontaneously breathing carp Cyprinzis carpio, de Graaf et al. (1987) and de Graaf and Ballintijn (1987) described slowly adapting position receptors on the gill arches and phasic displacement receptors on the gill filaments and rakers of the trout. Some of these mechanoreceptors are stimulated by the ventilatory movements of the gill arches and filaments, and afferent information, reaching the brain in the IXth and Xth cranial nerves, is known to influence the respiratory rhythm, with fictive breathing rate slowing in teleosts and increasing in elasmobranchs following transection of the branchial nerves or paralysis of the ventilatory muscles (Johansen, 1971; Barrett and Taylor, 1985a; Ballintijn, 1987). The importance of these observations for control of the heart is that the activity recorded from CVM was increased by mechanical stimulation of the gill arches in the curarized dogfish (Fig. 14). Efferent respiratory bursts recorded from the branchial branches of the dogfish were entrained to electrical stimulation of the central cut-end of a neighboring branchial branch (M. Young, unpublished observations). Consequently, normal breathing movements in the intact fish may indirectly affect cardiac vagal outflow and subsequently heart rate (cf. Fig. 16).Stimuli such as

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hypoxia, which increase ventilation rate, amplitude, or both, could result in an increase in mechanoreceptor input and in a consequent reduction in heart rate, both by central interactions between RVM and CVM and by this reflex route, so that the typical hypoxic bradycardia, although it clearly represents a direct response to environmental hypoxia, may partly arise as a consequence of a ventilatory response. This is reminiscent of, but opposite in kind to, the cardiovascular responses to hypoxia in mammals where heart rate in the intact animal increases during hypoxia, due to stimulation of lung stretch receptors (Daly and Scott, 1962). It is probably stimulation of branchial mechanoreceptors by increasing rates of water flow that triggers ram ventilation in swimming fish (Johansen, 1971). Onset of ram ventilation in the trout is accompanied by an increase in heart rate ( J . Steffensen, personal communication; E. W. Taylor and D. J. Randall, unpublished observations) (Fig. 17), and if this can be attributed to a reduction in cardiac vagal tone then it may result either from cessation of phasic stimulation of branchial mechanoreceptors or from the reduction in efferent outflow from RVM with an associated reduction in activity in the bursting CVM.

-ram

ventilation(1)

-(2)

,

,

active ventilation ( 2 )

-

ram ventilation (3)

I

10 x c

Fig. 17. Recordings of ventilation (measured with EMG electrodes which may have recorded movement artifact) and heart rate (as an ECG) from rainbow trout swimming in a continuous flow respirometer. When the speed of water flow increased above a threshold level, the fish switched from active to ram ventilation. Heart rate was elevated during ram ventilation, relative to active ventilation (top traces). When the fish switched to ram ventilation, heart rate increased immediately as active ventilation ceased (lower traces). Unpublished data from J. F. Steffensen.

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B. Central Interactions Modulating Heart Rate The respiration-related activity recorded from the cardiac vagi of the dogfish (Taylor and Butler, 1982), because it continues in the paralyzed fish (Barrett and Taylor, 1985a),must be generated by direct or indirect central interactions between the respiratory CPG and CVM. Consequently, a brief consideration of the central generator of the respiratory rhythm in fish is appropriate for this chapter. Bilaterally symmetrical areas in the medulla of fish and mammals show rhythmic neuronal activity in phase with various parts of the ventilatory cycle. In fish, these areas are located in the ventral medulla and in or around the motor nuclei of the trigeminal, facial, glossopharyngeal, and vagal nerves (Shelton et al., 1986, Ballintijn, 1987, for reviews). In mammals, there are two main areas of such activity, the ventral respiratory group and the dorsal respiratory group. These supply premotor neurons to areas in the spinal cord that innervate the muscles of the chest and diaphragm (Euler, 1986, for review). Rhythmic respiration-related activity continues in isolated goldfish and cat medullas (Adrian and Buytendijk, 1931; Euler and Soderberg, 1952), indicating that the rhythmicity is inherent to neural structures within the medulla. Evidence from isolated mammalian brainstem preparations indicates that the rhythm is generated by pacemaker neurons composing the central rhythm generator (CRG), which then stimulate a neural network, the CPG, which coordinates the various muscular activities of ventilation (Feldman and Smith, 1989). The site of the CRG is unidentified. It is unknown whether or not fish have a similar neuranatomical arrangement, although attempts to localize the CRG in lampreys suggest that it lies within the motonuclei in or near the caudal trigeminal nucleus (Russell, 1986). Ballintijn (1987) suggested that the CPG in teleosts may be located diffusely throughout the reticular formation, although our work on dogfish suggests that it may be intrinsic to the rostrocaudally distributed respiratory motoneurons. Respiratory motor or preganglionic neurons in fish are located in the motor nuclei composing the trigeminal Vth, facial VIIth, glossopharyngeal IXth, and vagal Xth motor nuclei, which together drive the respiratory muscles (Figs. 2 and 3 ) . These motor nuclei are interconnected and each receives an afferent projection from the descending trigeminal nucleus and has efferent and afferent projections to and from the reticular formation. The intermediate facial nucleus, which receives vagal afferents from the gill arches that innervate a range of tonically and physically active mechanoreceptors (de Graaf and Ballintijn, 1987), projects to the motor nuclei (Ballintijn et al., 1983). Finally,

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areas in the midbrain such as the mesencephalic tegmentum have efferent and afferent connections with the reticular formation (Ballintijn et al., 1979; Juch and Luiten, 1981). Central recordings from the medulla oblongata of the carp (Ballintijn and Alink, 1977) suggested that adjacent neurons have different firing patterns. They identified the target muscle of individual motoneurons b y simultaneous recordings of neuronal activity and electromyograms (EMGs) from the respiratory muscles. However, we know from the neuranatomical studies already described that the cell bodies of the neurons supplying efferent axons to the nerves that innervate the respiratory muscles are located in motor nuclei that are distributed in a sequential series in the brainstem (Figs. 2 , 3 , 6 ,and 7). Recordings of efferent activity from the central cut ends of the nerves innervating the respiratory muscles of the dogfish, Scyliorhinus canicula (Barrett and Taylor, 1985a), and the ray, Raia clavata (E. W. Taylor and J. J. Levings, unpublished data), have revealed that the branches of the Vth, VIIth, IXth, and Xth fire sequentially in the order of the sequential rostrocaudal distribution of their motonuclei in the brainstem (Fig. 18). The resultant coordinated contractions of the appropriate respiratory muscles may relate to their original segmental arrangement before cephalization-an arrangement that is retained in the hindbrain of the fish in the sequential topographical arrangement of the motor nuclei, including the subdivisions of the vagal motonucleus. The phase relationship, with these respiratory bursts, of the bursting units recorded from the branchial cardiac branches of the vagus in the dogfish seems complex. They fire virtually synchronously with activity in the branchial branches of the vagus but careful examination of recordings revealed that the onset of each burst in the cardiac branch may anticipate the branchial burst. This would seem to preclude a direct link between RVM and CVM, with the former driving the later, and renews our speculation over central and peripheral interactions summarized in Fig. 16.

VI. CARDIORE SPIRATORY SYNCHRONY

A link between heart beat and ventilation in fish was first noted in 1895 by Schoenlein (cited by Satchell, 1960) who described 1:l synchrony in Torpedo marmorata. This original observation triggered numerous investigations of the occurrence and mechanisms underlying cardiorespiratory synchrony in fish. The supposed functional sig-

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E. W. TAYLOR

A

1s

c I

ul

I I I I I --t---

Vent

I I

Fig. 18. Sequential bursting activity in the respiratory and cardiac branches of cranial nerves in the dogfish. (A) Regular bursting activity recorded from the posttrematic glossopharyngeal IXth (IX Gloss) and the mandibular Vth (V Mand) cranial nerves in a spontaneously ventilating fish. The start of a burst of activity in the mandibular Vth nerve preceded the start of a burst in the glossopharyngeal nerve by 152 2 6 ms. (B) An example of efferent activity recorded from the mandibular (V), glossopharyngeal (IX), the first branchial branch of the vagus nerve (Br. I), and the branchial cardiac branch of the vagus nerve (Br. Card.), from the left side of the same fish, together with ventilatory movements (Vent) recorded from a gill septum. The taped recordings were passed through an integrator acting as a spike counter (time constant 0.1 sec) and the approximate spiking rate is indicated by the vertical bar. The vertical solid line drawn through all the traces indicates the onset of a contraction of the first gill septum; the dashed line indicates the start of a burst of activity in the mandibular nerve, which preceded that in all other nerves. The bursts in the glossopharyngeal preceded those in the branchial branch of the vagus, which occurred virtually simultaneously with bursts in the other branchial branches of the vagus. The onset of the bursts in the branchial cardiac branch typically preceded those in the adjacent third branchial branch and recordings from this nerve include sporadic activity between the bursts that is absent from the respiratory nerves. [Reformated from Barrett and Taylor (19854.1

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nificance of cardiorespiratory synchrony relates to the importance of matching relative flow rates of water and blood over the countercurrent at the gill lamellae. Although virtually continuous, both water and blood flow over the lamellae are markedly pulsatile (see Fig. 20). Recordings of differential blood pressure and gill opacity in the dogfish revealed a brief period of rapid blood flow through the lamellae early in each cardiac cycle (Satchell, 1960),and as the ECG tended to occur at or near the mouth-opening phase of the ventilatory cycle this could result in coincidence of the periods of maximum flow rate of blood and water during each cardiac cycle (Shelton and Randall, 1962; Shelton, 1970). The improvement in gill perfusion and consequent oxygen transfer resulting from changes in transmural pressure and intralamellar blood flow, described by Farrell et al. (1980), may be further improved by synchronization of the pressure pulses associated with ventilation and perfusion. Cardiorespiratory synchrony may, by a combination of these effects, increase the relative efficiency of respiratory gas exchange (i.e., maximum exchange for minimum work). However, ventilation rate is usually faster than heart rate in experimental dogfish so that if one ventilatory cycle coincides appropriately with heart beat then the second or third in a sequence will occur in a wholly inappropriate phase of the cardiac cycle (Shelton, 1970). In the restrained dogfish, ventilation rate was approximately twice heart rate and these showed a drifting relationship (Taylor and Butler, 1971; Taylor, 1985). Hughes (1972) explored evidence for phase coupling between ventilation and heart beat in dogfish released into a fish box that included a movement restrictor. Sophisticated analysis using event correlograms revealed that in some cases the heart tended to beat in a particular phase of the ventilatory cycle for short periods. Use of polar coordinates revealed some significant coupling at varied phase angles between the two rhythms with individual fish varying in both the degree of coupling and the phase angle, during a period of observation. The absence of synchrony, or even consistent close coupling, as opposed to a drifting phase relationship, was most often attributable to changes in heart rate, which was more variable than ventilation rate (Taylor and Butler, 1971; Hughes, 1972; Taylor, 1985). This may be reliably interpreted in the dogfish as variations in cardiac vagal tone, possibly exerted by changes in the rate of firing of the nonbursting units recorded from the branchial cardiac nerves. Activity in these units is high in the restrained dogfish, when cardiorespiratory synchrony is absent (Taylor and Butler, 1971, 1982). As noted earlier, activity in the nonbursting units increases during hypoxia when a

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reflex bradycardia is elicited (Taylor and Butler, 1982; Fig. 14).A decrease in vagal tone on the heart, such as that recorded during exposure to moderately hyperoxic water (Fig. 9),caused heart rate to rise toward ventilation rate (Barrett and Taylor, 1984a),suggesting that when vagal tone was relatively low a 1:1 synchrony could occur. When cannulated dogfish were allowed to settle in large tanks of running, aerated seawater at 23°Cthey showed 1:1 synchrony between heart beat and ventilation for long periods (Taylor, 1985).This relationship was abolished by atropine (Fig. 19)confirming the role of the vagus in the maintenance of synchrony and providing a hypothetical role for the bursting units recorded from the cardiac vagi. Whenever the fish was spontaneously active or disturbed the relationship broke down due to a reflex bradycardia and acceleration of ventilation (Fig. 19) so that the 2:l relationship between ventilation and heart rate characteristic of the experimentally restrained animal was reestab-

A VR

76

normal

atropinlzed

78

76

04

76

------r-,

lnactlve

. move

I

I

r

,

I

I

1

I

I

,

1

,

awlmmlng

Fig. 19. (A) Ventilation rate (VR), measured as orobranchial water pressure and heart rate (HR), measured as ventral aortic blood pressure (beats/min-'), recorded from an unrestrained dogfish enclosed in a large tank of running seawater at 23°C. When the animal was stationary, resting on the bottom of the tank (normal), the two rates were identical, and there were clear signs of maintained synchrony. Atropinization (atropinized) caused an increase in heart rate and loss of synchrony. (B) When the normal, inactive animal moved (move) and then spontaneously commenced swimming, it showed a bradycardia and then an increase in ventilation rate so that ventilation became considerably faster than heart beat, a condition previously observed in disturbed or restrained animals. [Data from Taylor (1985).]

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lished (Fig. 10). Thus, it is possible that the elusiveness of data supporting the proposed existence of cardiorespiratory synchrony in dogfish was due to experimental procedures that increase vagal tone on the heart, exerted by the nonbursting units, and masked the more subtle control exerted by the bursting units recorded from the cardiac vagi. These data from elasmobranchs suggest that cardiorespiratory synchrony, when present, is due to central interactions generating respiration-related activity in CVM located in the DVN, which are then effective in determining synchronous heart beating when overall cardiac vagal tone is relatively low in normoxic or hyperoxic fish. Work on teleosts has stressed the importance of inputs from peripheral receptors in the genesis of cardiorespiratory synchrony. Randall (1966) recorded efferent nervous activity from the cardiac branch of the vagus in the tench that was synchronized with the mouth-opening phase of the breathing cycle. It was suggested that this activity maintains synchrony between heart beat and breathing movements and that both a hypoxic bradycardia and synchrony were mediated by reflex pathways. Randall and Smith (1967) described the development of exact synchrony between breathing and heart beat in the trout during progressive hypoxia. In normoxia heart rate was faster than ventilation; hypoxia caused an increase in ventilation rate and a reflex bradycardia resulting in 1:1 synchronization of the two rhythms. Both the bradycardia and synchrony were abolished by atropine. In addition, Randall and Smith (1967) were able to demonstrate 1:l synchronization of hypoxic heart rate with pulsatile forced ventilation, which was clearly generated by reflex pathways because the spontaneous breathing efforts of the intubated fish were out of phase with imposed changes in water velocity and were without effect on heart beat. Thus, we are left with an apparent conflict of evidence on the generation of cardiorespiratory synchrony, which in elasmobranchs may be centrally generated in inactive, normoxic, or hyperoxic fish when cardiac vagal tone is low; while in teleosts it appears during hypoxia and is generated reflexly by increased vagal tone. The differences between these two groups of fish may be real and it is of interest that branchial denervation increases fictive ventilation rate in elasmobranchs but decreases it in teleosts. However, it is as likely that further experimentation will establish that both central and peripheral mechanisms are important in each group. Activity recorded centrally from CVM in the dogfish was increased by mechanical stimulation of the gill arches (Fig. 14), and central stimulation of a branchial branch entrained efferent activity in the branchial cardiac branch (Fig. 15).Thus, respiration-related efferent

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cardiac vagal activity may be generated both centrally and peripherally, and this is likely to be the case in teleosts. Experimentally restrained dogfish show no hypoxic ventilatory response (Butler and Taylor, 1971) and no evidence of maintained cardiorespiratory synchrony (Taylor and Butler, 1971; Hughes, 1972). However, unrestrained fish show reduced normoxic ventilation rates, synchronous with heart beat, as described previously, and also exhibit a ventilatory response to hypoxia (Metcalfe and Butler, 1984a). When cod were cannulated and released into large holding tanks of normoxic seawater they showed periods of 1:1synchrony ( Jones et al., 1974). The importance of these observations is that they measured dorsal aortic blood flow that was markedly pulsatile (Fig. 20), confirming a role for cardiorespiratory synchrony in the generation of concurrent flow patterns of ventilation and perfusion over the gills. Thus, both unrestrained dogfish and cod can show synchrony, and as our understanding of the underlying mechanisms increases, it seems likely that elasmobranchs and teleosts will share common characteristics with respect to the generation and potential physiological advantages of cardiorespiratory synchrony. What emerges from our understanding is that a potent mechanism for the generation of cardiorespiratory synchrony in fish exists in the form of the bursting units present in recordings of efferent activity in the cardiac vagi, whether generated by central interactions or reflexly b y stimulation of branchial mechanoreceptors. Interestingly, in the

Dorsal Aortic Pressure (mm Hg)

30

Dorsal Aortic

Flow (ml/min)

0

-

Buccal Cavity +ve Pressure +vet

,

,

50 seconds

5 seconds Time

Fig. 20. Dorsal aortic blood pressure and flow recorded together with water pressures in the buccal cavity of a cod, Gadus morhua. Synchrony (1:l) is observable between heart beat and ventilation with markedly pulsatile blood flow measured in the dorsal aorta. [Data from Jones et al., (1974).1

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dog, stimulation of the vagus nerves toward the heart with brief bursts of stimuli, similar to those recorded from efferent cardiac vagal fibers, caused heart rate to synchronize with the stimulus, beating once for each vagal stimulus burst over a wide frequency range (Levy and Martin, 1984).Similar entrainment with the bursts of activity recorded from the cardiac vagi could explain the 1 : l synchrony observed in “settled” normoxic dogfish and in hypoxic trout.

ACKNOWLEDGMENTS I am very grateful to Marilyn Nicholl for her patient and good humored processing of the manuscript and to Pauline Hill for her skill in drawing the figures. The contributions of a succession of co-workers to the development ofthe ideas in this chapter are apparent in the reference list, but I wish to acknowlege in particular Jenny Levings and Mike Young for their permission to use as yet unpublished data. Much of our work has been supported by the Science and Engineering Research Council.

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7 AFFERENT INPUTS ASSOCIATED WITH CARDIOVENTILATORY CONTROL IN FISH MARK L. BURLESON ANV NEAL J . SMATRESK Department of Biology University of Texas, Arlington Arlington, Texas

WlLLlAM K . MILSOM Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

I. Introduction 11. Mechanoreceptors

A. Mechanoreceptors Associated with Respiratory Passages B. Mechanoreceptors Associated with the Cardiovascular System 111. Chemoreceptors A. 02-Sensitive Chemoreceptors B. C02/pH-Sensitive Chemoreceptors IV. Nociceptors A. Mechanoreceptors B. Chemoreceptors C. Nociceptors in Air-Breathing Fish? V. Central Projections of Sensory Neurons References

389 FISH PHYSIOLOGY, VOL. XIIH

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I. INTRODUCTION

T h e basic respiratory rhythm and rhythmic contractions of the heart result from the actions of endogenous rhythm generators and do not require afferent feedback for their initiation or maintenance in fish (see Smatresk, 1990b for review). Modulation of the cardiorespiratory system to meet environmental and metabolic demands, however, requires afferent feedback from a host of sensory receptors. In addition to regulating the frequency and stroke volume of ventilatory and cardiac pumps, sensory feedback is needed from entero- and exteroreceptors to match ventilation to perfusion in gas exchange organs and to match flow to demand in systemic vascular beds. Despite the large body of work describing cardiorespiratory responses to various environmental and metabolic challenges (discussed in Chapter 6), remarkably little is known about the receptors and afferent pathways mediating these responses in fishes. The focus of this chapter will be to discuss the receptor physiology and afferent pathways for the three major groups of receptors believed to contribute to cardiorespiratory control, including mechanoreceptors monitoring physical events in the respiratory passages or cardiovascular system, chemoreceptors monitoring gas tensions and acid-base balance, and the more poorly defined “defense” receptors that may act to override normal cardiorespiratory control during exposure to hazardous environmental conditions. Given the balance of existing data, discussion of the control of ventilation will predominate over discussion of cardiovascular control. Given the degree of cardioventilatory synchrony seen in fish, however (reviewed in Chapter 6), there are good grounds to believe that the same receptors found to be instrumental in ventilatory control will also feature heavily in cardiovascular control. 11. MECHANORECEPTORS A. Mechanoreceptors Associated with Respiratory Passages

Mechanoreceptors sensitive to displacement of pharynx, pharyngeal pads, gill arches, gill rakers and filaments, and air-breathing organs (lungs) have been identified in fishes. As in other vertebrates, these mechanoreceptors appear to be simple free nerve endings located in connective tissue or muscle (Ballintijn and Bamford, 1975). Consequently, it is thought that the locations of these mechanorecep-

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tors primarily determine their sensory modalities and response characteristics. All of these receptors have been implicated in the control of the respiratory pattern and cardiorespiratory coupling in fish although the specific reflex roles have not been worked out, as yet, for any receptor group. 1. GILLMECHANORECEPTORS

a. Gill Filament Mechanoreceptors. Recordings of single unit afferent information from vagal epibranchial ganglia in spontaneously breathing carp (Cyprinus carpio) (de Graff et al., 1987; de Graaf and Ballintijn, 1987) demonstrated rapidly adapting bursts of activity in response to filament displacement or tactile stimulation of the gill lamellae. The tactile stimulation of lamellae suggested that each receptive field was limited to a single filament. Lateral or vertical displacement of several adjacent filaments could stimulate activity in a single afferent fiber, however, presumably due to mechanical interactions between adjoining gill filaments. The afferent pathway for these receptors was the external pre- and post-trematic branches of the branchial nerves (Fig. l),with each epibranchial ganglion appearing to innervate a whole gill slit (i.e., the posterior demibranch of one arch and the anterior demibranch of the next arch). These rapidly adapting mechanoreceptors were not normally activated during the respiratory cycle in this study using lightly curarized fish, and thus the authors argued that it was unlikely that they would normally b e involved in respiratory or cardiovascular control. It was more likely, they felt, that these receptors would be stimulated during feeding, coughing, or in the presence of gill parasites or particulate matter in the water. Stimulation of these receptors elicited filament adduction, which should lower gill resistance, and thus they suggested that these receptors might facilitate cough or expulsion reflexes, or help clear the gills of debris. Foreign matter that could obstruct water flow would reflexly lower gill resistance and increase water flow velocity across the lamellae (de Graaf et al., 1987). Mechanoreceptor discharge activated by gill filament displacement has also been recorded from the branchial nerves of isolated sea raven (Hemitripterus americanus) and salmon (Salmo salar) gills (Sutterlin and Saunders, 1969). Whether these were post- or pretrematic nerves was not addressed in these studies. The sea raven filament receptors were rapidly adapting, like carp filament mechanoreceptors, although several adjacent filaments appeared to compose each receptive field in this species. Similar activity has subsequently also been recorded from the post-trematic branch of the glossopharyngeal nerve of isolated

392

BURLESON ET AL. POSTERIOR

EBAL 4

PST TREM BR X 2 EBAL 3

E X PRET BR X 2 IN PRET BR X 2 EBAL 2

D PH BR X 2

EBAL 1

-

ANTERIOR

2mm

Fig. 1. Diagram showing branchial innervation in carp. D PH BR X 2 , dorsal pharyngeal branch of the second branchial branch of the vagus; EBAL 1-4,external branchial arch levator muscles; E M BR X2, epibranchial motor branch of the second branchial branch of the vagus; EX PRET BR X2, external pretrematic branch of the second branchial branch of the vagus; EXO, exoccipital bone; GG, glossopharyngeal ganglion; IBAL 1-2, internal branchial arch levator muscles; IN PRET BR X 2 , internal pretrematic branch of the second branchial branch of the vagus; PRO, prootic bone; PST TREM BR X2,posttrematic branch of the second branchial branch of the vagus; VG 1-4, vagal ganglia. (Reproduced from de Graaf, 1990 by permission, copyright 0 Wiley-Liss, a division of John Wiley and Sons. Inc.)

perfused rainbow trout (Oncorhynchus mykiss) first gill arches (Burleson and Milsom, 1992a). In addition to rapidly adapting mechanoreceptors, however, Burleson and Milsom (1992)also described slowly adapting gill filament mechanoreceptors (Fig. 2). Because these studies were done on isolated perfused gills, it was not possible to tell

7.

ENG

393

AFFERENT INPUTS

Deflection

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Fig. 2. Traces showing the responses of slowly (SAR) and rapidly (RAR) adapting receptors to filament displacement in an isolated, perfused first gill arch from a rainbow trout (Onchorhynchus m y k i s s ) . Lower trace shows the discharge (ENG) of a single fiber of each type while the top trace shows the instantaneous discharge rate (impulses/ second). The middle trace is a time marker (in seconds) and the bars below the traces indicate the period during which the gill filament was displaced. (From Burleson and Milsom, 1992a).

whether or not these receptors were active during normal breathing movements. Gill filament adduction occurs during normal ventilation in many fishes (Pazstor and Kleerekoper, 1962; Ballintijn, 1984), however, Sutterlin and Saunders (1969) felt that this filament adduction during normal ventilatory movements should elicit regular ventilatory related afferent discharge from the rapidly adapting receptors in sea ravens and salmon. Since the slowly adapting gill filament mechanoreceptors in trout may be tonically active, they are even more likely to provide phasic afferent feedback during normal breathing. Thus, at present, the reflex role of gill filament mechanoreceptors remains unknown. T o determine whether these receptors simply help protect respiratory surfaces by eliciting coughing or expulsion reflexes, or whether they are also regularly activated by normal breathing movements and thus contribute to respiratory control on a breath by breath basis will require further description of their discharge characteristics and reflex roles in normally breathing fishes.

b. Gill Raker Mechanoreceptors. The most thorough description of gill raker mechanoreceptors comes from a study by Satchel1 and Way (1962) who characterized the afferent discharge in the post-trematic branch of the vagus, elicited by deflection of the branchial processes in dogfish sharks (Squalus acanthias). They identified a population of rapidly adapting receptors that responded to very small displacements with receptive fields confined to a single process. More commonly, however, they found medium to high threshold, slowly adapting re-

394

BUHLESON ET AL.

ceptors that often had levels of tonic discharge that were correlated to stimulus strength. These receptors also had a phasic component to their response that increased as the rate of stimulus onset increased (Fig. 3 ) . Although their study was done on isolated gills, Satchell and Way (1962) suggested that the tonically active, slowly adapting gill

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Fig. 3. Responses of mechanoreceptors in the branchial processes (rakers) ofdogfish (Squalus acanthias) gills to deflection. (A) Effect of stimulus amplitude on discharge frequency. (B) Effect of the rate of stimulus onset on discharge frequency. (Reproduced from Satchell and Way, 1962 by permission, 0 The Company of Biologists, Ltd.)

7 . AFFERENT

INPUTS

395

process receptors should be activated by normal respiratory movements. In contrast to these studies, de Graaf et al. (1987) only recorded rapidly adapting bursts of activity from epibranchial ganglia of the internal pretrematic branch of the vagus (Fig. 1) during mechanical stimulation of gill rakers in carp. They could not record any activity during normal breathing. Thus in carp, they argued, these receptors did not modulate normal breathing movements. Because gill raker stimulation elicited filament adduction, de Graaf et al. (1987) suggested that these receptors were likely to be involved in coughing and expulsion reflexes or branchial responses during feeding. Gill raker mechanoreceptors have also been identified in the posttrematic branch of the glossopharyngeal nerve in trout (Burleson and Milsom, 1992a)and in unidentified branchial nerves of sea raven (Sutterlin and Saunders, 1969), but the discharge characteristics for these receptors have not been described in detail. Given species differences in the presence of slowly adapting receptors and the lack of in vivo recordings from receptors in species other than carp, as with the gill filament mechanoreceptors, it remains unclear to what extent gill raker mechanoreceptors are active during normal ventilation in fishes and the extent to which they participate in cardiorespiratory control.

c. Gill Arch Proprioceptors. In addition to mechanoreceptors located in the gill filaments and rakers, de Graaf and Ballintijn (1987) described mechanoreceptors located in the cartilaginous strip between the epibranchial and ceratobranchial elements activated by displacement of the gill arch of carp. These receptors were innervated only by pretrematic branches of the vagi (Fig. 1; de Graaf, 1990) and responded to gill arch adduction by decreasing discharge and to abduction by increasing discharge. Unlike carp gill filament or raker mechanoreceptors, these receptors were tonically activated by gill arch displacement and were activated during normal breathing (Fig. 4).Burleson and Milsom (1992a) have also identified gill arch proprioceptors, with discharge characteristics similar to those described for carp, innervated by the post-trematic branch of the glossopharyngeal nerve in isolated perfused trout gills. Based on discharge characteristics, d e Graaf and Ballintijn (1987b) described the receptors in carp as proprioceptive and argued that of all the receptors found in gills, these receptors were most likely to contribute to control of breathing and cardiorespiratory synchronization.

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Fig. 4. Gill arch proprioceptive activity recorded during ventilation in a carp. Panel Al shows an interval plot ofproprioceptor activity arising from the second gill arch during normal breathing while A2 illustrates the simultaneous movement (MVT) of the second gill arch (GA 2 ) . The symbols above the recordings in both traces indicate coughs. Panel B, shows an interval plot of proprioceptive activity arising from the same arch during intermittent ventilation with the corresponding gill arch movement shown in Bz. (Reproduced from de Graaf'and Ballintijn, 1987 by permission, 0 Elsevier Science Publishers.)

7 . AFFERENT

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397

2. OTHER MECHANORECEPTORS ASSOCIATED WITH THE RESPIRATORY PASSAGES a . Orobranchial Mechunoreceptors. In addition to the gill mechanoreceptors already described, it is likely that mechanoreceptors lining the orobranchial cavity or associated with the skeletal or muscular system of the buccal opercular pump provide afferent feedback for cardiorespiratory modulation. These receptors have not been systematically investigated, however, and despite knowledge of reflex responses to manipulation of the orobranchial cavity (Satchell, 1959),our understanding of them is confined to a few studies. Roberts and Rowel1 (1988)have described mechanoreceptors in the opercula of goldfish and pumpkinseed sunfish (Lepomis gibbosus) that overlie the jaw muscles. These receptors are fine nerve endings with varicosities in the hypodermis, innervated by the infraorbital nerve, a branch of cranial nerve V (Fig. 5A). They respond to abduction of the mandible with a burst of activity, and thereby provide afferent information about the rate and extent of buccal expansion produced by the adductor mandibulae muscle complex. These receptors are active during normal ventilation (Fig. SB) and, although the role of afferent information provided by these receptors is not known, they could be involved in cardioventilatory reflexes. In central recording studies, Ballintijn and Roberts (1976)have also identified medullary neurons that were activated by jaw or operculum abduction, thus providing evidence for jaw or opercular proprioception. Ballintijn and Bamford (1975) also provided evidence for muscle stretch receptors and tendon organ receptors in carp associated with respiratory muscles. Receptors located in tendons responded to muscle contraction during normal respiration by firing during the contraction phase of ventilation. The muscle stretch receptors responded to changes in muscle length and were activated twice during the normal ventilatory cycle, during both contraction and relaxation (Ballintijn and Bamford, 1975). While details on the reflex roles of these receptors are not well understood, they appear to be involved primarily in load matching (i.e., matching the performance of the respiratory muscles to the demands placed on the ventilatory pump). The afferent pathways for these muscle proprioceptors appear to be cranial nerves V and VII (Nilsson, 1984).

b. Air-Breathing Organ Mechanoreceptors. There is tremendous diversity in the structures used for aerial gas exchange in fishes, including the orobranchial cavity, specialized regions of the pharyngeal

398

BURLESON ET AL.

B

Fig. 5. (A) Camera-lucida drawing ofan opercular proprioceptor field formed by two nerves in a pumpkinseed sunfish (Lepomis gibbosus). Scale bar = 0.5 mm. (B) Cheek propriceptor afferent activity (upper trace) recorded from the infraorbital nerve (a branch of cranial nerve V) during ventilation in a goldfish (Curussius uurutus). Lower trace represents mandibular movement, abduction is down (Horizontal bar = 100 msec). (Reproduced from Roberts and Rowell, 1988 by permission, 0 National Research Council.)

cavity, gills, gut, and swimbladders. It seems likely that the types of mechanoreceptors already described for the gills and orobranchial cavity would also be adequate for providing proprioceptive feedback for the control of air breathing, or cardiorespiratory modulation, when air breathing was confined to the mouth, gills, or pharynx. Afferent activity arising from these receptors correlated to inflation or deflation

7.

399

A F F E R E N T INPUTS

of these areas during air breathing, however, has not yet been recorded, although reflex tachycardia following inflation of the buccal cavity in Synbranchus inarmoratus (Johansen, 1966; Roberts and Graham, 1985) and the electric eel (Johansen et al., 1968) provide evidence for volume mechanoreception in teleost air breathers (Roberts and Graham, 1985). When air breathing involves swimbladder “lungs,” however, proprioceptive feedback for the control of air breathing and cardiorespiratory modulation would be expected to arise from these structures and, indeed, mechanoreceptors arising from swimbladder lungs have been described in detail in lungfish (Protopterus, DeLaney et al., 1983), bowfin (Amia calva, Milsom and Jones, 1985), and gar (Lepisosteus, Smatresk and Azizi, 1987). These receptors are all innervated by a branch of the ramus intestinalis of the vagus and include slowly and rapidly adapting receptors (Fig. 6). The slowly adapting receptors display both dynamic and tonic changes in activity on inflation or deflation (Fig. 6), indicating that they are able to provide information about the rate of change, as well as the absolute change in the volume of the air-breathing organ. The rapidly adapting receptors respond to inflation, deflation, or both. Both groups of receptors in lungfish and gar are inhibited by 6-10% inspired CO2 (Fig. 7) (DeLaney et al., 1983; Smatresk and Azizi, 1987). Although the sum of the evidence suggests that mechanoreceptors

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Fig. 6. Representative nerve activity o f a slowly adapting receptor (SAR) and a rapidly adapting receptor (RAR) in the air-breathing organ (ABO) of a spotted gar (Lepisosteus oculatus) in response to ABO inflation (between arrows). Time marker applies to both traces. (From Smatresk and Azizi, 1987by permission, 0The American Physiological Society.)

400

BURLESON E T AL. 15r

Pressure, cm H,O

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0

Activity, imp * secl

10% CO,

-

Activity, imp sec-1

+ air

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Fig. 7. Representative traces showing the inhibitory effect of 10% COZ on the activity of a slowly adapting receptor during ramp inflation and sudden deflation of the air-breathing organ of a spotted gar. The upper trace in each pair is the pressure measured inside the air-breathing organ while the lower trace is the instantaneous discharge frequency of the receptor. (From Smatresk and Azizi, 1987 by permission, 0The American Physiological Society.)

associated with the respiratory passages are instrumental in the regulation of breathing pattern and cardiorespiratory synchronization in fish, the details of these processes are far from clear. Studies of the cardiorespiratory responses to stimulation of proprioceptors remain difficult to interpret due to differences in species, anesthetics, and experimental protocols used in different studies (Azizi and Smatresk, 1986; Azizi, 1989; Milsom, 1990; Taylor, Chapter 6 this volume). Much remains to be done to elaborate the specific cardiorespiratory responses to physiological stimulation of each of these receptor groups.

7.

A F F E R E N T INPUTS

401

B. Mechanoreceptors Associated with the Cardiovascular System 1. ARTERIAL MECHANORECEPTORS Mechanoreceptors located in the walls of blood vessels are generally termed baroreceptors. They are responsive to the mechanical distortion of the blood vessel caused by changes in blood pressure. Lutz and Wyman (1932a,b) demonstrated that sudden increases in perfusion pressure to the gill arches and electrical stimulation of the branchial nerves (cranial nerves IX and X) in dogfish caused a reflexive decrease in heart rate. They attributed the observed bradycardia to stimulation of baroreceptive areas, although it should be noted that the resultant bradycardia could also have been due to stimulation of chemoreceptor or nociceptor afferent fibers in these nerves (see e.g. Burleson and Smatresk, 1990b; Satchell, 1978). Subsequently, however, Irving et al. (1935) were able to record afferent nerve activity from these nerves and confirm the presence of receptors that increased neural activity in response to increased blood pressure. In dogfish (Irving et al., 1935) a bursting pattern of activity, synchronous with systole, was observed but was abolished by reducing blood pressure by hemorrhage. Increasing blood pressure by injecting adrenaline changed the bursting pattern into a continuous pattern of discharge. By statically increasing ventral aorta blood pressure to produce step changes of 10 mmHg in the gills, Irving et al. (1935) observed a burst of baroreceptor activity followed by a rapid decrease in discharge frequency to zero, thus indicating that these were rapidly adapting receptors. Branchial baroreceptors have since been demonstrated directly and indirectly in several species of teleost fish (Mott, 1951; Laurent, 1967; Ristori, 1970; Ristori and Dessaux, 1970; Burleson and Milsom, 1992a) (Fig. 8).Although the precise location of piscine baroreceptors remains unknown, histological studies suggest that two likely baroreceptive regions within the gills are located at the junction of the afferent branchial artery and efferent filamental arteries (Boyd, 1936; DeKock, 1963).Baroreceptive activity has been observed in the posttrematic branches of cranial nerves IX and X arising from all branchial nerves. Baroreceptive areas other than the gills have not been identified in fish. In catfish (order Siluriformes), however, there are carotid labyrinths associated with the carotid arteries (Srivastava and Singh, 1980; Olson et al., 1981).The location and morphology of these labyrinths are similar to those of reptiles and amphibia, and it has been suggested

402

BURLESON ET AL.

ENG

Fig. 8. Afferent discharge recorded from a neuron responding to changes in perfusion pressure in the isolated, perfused first gill arch o f a rainbow trout. The upper trace depicts the mean discharge frequency (impulses/second) of the single fiber whose raw discharge (ENG) is shown in the third trace. The second trace is a time marker (in seconds) while the bottom trace illustrates the change in gill perfusion pressure measured in the afferent branchial artery. (From Burleson and Milsom, 1992a).

(Olson et al., 1981).Although the innervation of the piscine carotid labyrinth is not known, the branchial nerve section that denervates the gills does not denervate the carotid labyrinths. Such denervation, however, abolishes 0 2 chemoreflexes in channel catfish (Zctalurus punctatus) suggesting that the carotid labyrinths may be baroreceptive rather than chemoreceptive loci. Despite its intriguing morphology, however, to date there is no physiological evidence to support a baroreceptive function for the carotid labyrinth in catfish. 2. INTRACARDIAC MECHANORECEPTORS

There is direct and indirect evidence that indicates that some fishes may possess intracardiac receptors homologous to mammalian atrial and ventricular stretch receptors. Very few studies, however, have examined cardioventilatory reflexes arising from receptors located in the heart of fishes.

7.

AFFERENT INPUTS

403

Innervation of the heart of cyclostomes is poor. Cardiac innervation is absent in some myxinoids although lampetroids possess a cardiac branch of the vagus (see Laurent et al., 1983for review). To date, there are no data available to say whether or not cyclostomes possess intracardiac receptors. In elasmobranchs, however, reflex studies indicate that there are sensory receptors in the heart that mediate cardioventilatory reflexes. Electrical and mechanical stimulation of the heart and central cut ends of cardiac vagi in dogfish inhibit heart rate and ventilation (Lutz, 1930). Elasmobranchs have two pair ofvagal cardiac nerves, one pair arising from the visceral branches of the vagus and the other pair arising from the post-trematic branchial branches of the vagi (Marshall and Hurst, 1905; Norris and Hughes, 1920). Both pairs of nerves contain afferent and efferent pathways, but nerve stimulation studies indicate that the branchial cardiac vagi are primarily efferent, playing an important role in cardiac inhibition, whereas the visceral cardiac vagi appear to be primarily sensory (Short et al., 1977). In teleost fishes, the heart is innervated by one pair of cardiac branches of the vagus nerves. Innervation of fish hearts has been extensively reviewed by Laurent et al. (1983). Briefly, three types of afferent nerve fibers have been histologically identified in the cardiac vagi of teleosts (Laurent, 1962; Kumar, 1979): atrial nonmyelinated endings from ribbon-like fibers, subendocardial arborescent endings within the ventricle, mainly located in the atrioventricular funnel, and a subepicardial sensory plexus. The nerve endings of the latter are unencapsulated and sometimes form coiled or bulb-like structures (Kumar, 1979). The adequate stimulus of the atrial receptors appears to be the active contraction of the atrium, and the activity of these receptors is influenced by the degree of atrial filling (Laurent, 1962) (Fig. 9). It has been suggested that activity arising from the subendocardial arborescent receptors is a function of ventricular pressure and that activity in the subepicardial sensory plexus is synchronous with the isometric phase of ventricular contraction and is influenced by the degree of ventricular filling (Laurent, 1962).The role that these cardiac stretch receptors play in cardiovascular control remains unclear but they may contribute to the cardiac-ventilatory coupling (Randall, 1982; Smatresk, 1986) observed in some fishes. 3. INTRACRANIAL MECHANORECEPTORS (CUSHING REFLEX) The Cushing response is a reflexive increase in blood pressure and heart rate in response to increased intracranial pressure (Cushing, 1901).The Cushing response has been demonstrated in bluefish (Po-

404 Groups

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Fig. 9. Effect of increasing atrial filling on cardiac afferent receptor activity in catfish. Recordings were multiunit recordings and three groups refer to (A) all activity occurring in association with the P wave of the EKG; (B) all acitivity occurring in association with the P-Q interval in the EKG, and (C) all activity occurring in association with the QRS complex of the EKG. Note the increase in discharge associated with atrial contraction with increasing atrial filling (Goups A and B) but not that associated with ventricular contraction (Group C). (From Laurent, 1962 by permission, 0 Biological Structures and Morphogenesis.)

matomus sultatrix) (Fox et al., 1990). This reflex may maintain brain blood flow during periods of high intracranial pressure. For example, during fast swimming, compression of the vertebral column raises intracranial pressure and could restrict central blood flow if this reflex was not present. DuBois et d.(1990),however, suggest that central hypoxia, caused by ischemia, rather than elevated cranial pressure, may be the stimulus for the Cushing response in bluefish since presaturating the fish with 0 2 delays the onset of this response. 111. CHEMORECEPTORS A. 02-Sensitive Chemoreceptors Matching oxygen uptake to metabolic demands requires that animals sense and respond to respiratory and blood oxygen concentrations. In fishes, the need for fine control over branchial gas exchange is exacerbated b y the low 0 2 capacitance of water and the tremendous

7.

AFFERENT INPUTS

405

spatial and temporal variability in oxygen availability in the aquatic environment. A large body of work documents the cardiorespiratory responses of fishes to hypoxia, hypoxemia, and elevated metabolic demands (see Smatresk, 199Ob; Taylor, Chapter 6, this volume, for reviews), but we have begun to understand the receptors and afferent pathways mediating these responses. Lacking direct histological identification of oxygen-sensitive chemoreceptors, a variety of reflexogenic areas have been tested as putative oxygen-sensitive loci using a variety of indirect techniques. Based on reflex and nerve section studies, the brain (Bamford, 1974; Jones, 1983), gills (Powers and Clark, 1942), pseudobranch (Laurent and Rouzeau, 1972), venous vasculature (Barrett and Taylor, 1984),arterial vasculature (Randall, 1982), and afferent branchial vasculature (Smatresk et al., 1986) have all been proposed to be chemoreceptive sites. Some studies provide fairly strong evidence that the oxygen-sensitive chemoreceptors are located predominantly, if not exclusively, in the gills of most Actinopterygian fishes and are innervated by cranial nerves VII, IX, and X (Burleson and Smatresk, l99Ob; McKenzie et al., 1991; Smatresk, 1987). In elasmobranchs, their innervation is more diffuse and includes cranial nerve V, suggesting that other sites in the orobranchial cavity may also be involved (Butler et al., 1977). It should be noted, however, that this evidence only removes the brain from the list of putative chemoreceptor sites. 1. BRANCHIAL CHEMORECEPTORS a . Receptor Discharge Characteristics. The only direct evidence for the presence of 02-sensitive chemoreceptors in fish comes from three studies involving the branchial areas in teleosts (specifically, the pseudobranch and fist gill arch). In an early series of studies, Laurent (1967,1969) and Laurent and Rouzeau (1969,1972)demonstrated low sensitivity, low voltage amplitude changes in recordings of multiunit afferent activity from pseudobranchs of the rainbow trout, in vitro, during hypoxia. Subsequently, Milsom and Brill (1986)recorded afferent activity from single receptors in the isolated first gill arch of yellowfin tuna. These receptors increased their discharge in response to decreasing perfusion rate, decreasing perfusion Po2, and, in most fibers, decreasing external Po,. Fibers responding to environmental hypoxia exhibited an exponential increase in discharge to decreasing external PO, with a sensitivity similar to that exhibited by mammalian carotid body chemoreceptors (Fig. 10). In a study on isolated first gill arches of rainbow trout, Burleson and Milsom (1992a) obtained similar results with the exception that trout receptors were much less sensitive to changes in gill perfusion flow.

406

BURLESON ET A L .

1

trouf cot c.6. tuna

, 20

60

100 ’ 1LO

Po2 (tow) Fig. 10. Composite diagram comparing the O2 response curves of oxygen chemoreceptors in the gills of trout and tuna as well as in the carotid and aortic bodies of the cat (modified from Milsom and Brill, 1986 and data from Burleson, 1991 by permission, 0 Elsevier Science Publishers.)

Both of the latter studies noted afferent activity arising from fibers that responded only, or preferentially, to external 0 2 stimulus levels, and others that responded only, or preferentially, to internal 0 2 stimulus levels (Milsom and Brill, 1986; Burleson, 1991; Burleson and Milsom, 1992a) (Fig. 11).These observations support results from reflex studies that suggest the presence of a population of internally oriented 0 2 chemoreceptors that elicit only ventilatory responses (Randall and Smith, 1967; Cameron and Wohlschlag, 1969; Cameron and Davis, 1970; Holeton, 1971; Wood et al., 1979; Smith and Jones, 1978; Smatresk et al., 1986; Burleson and Smatresk, 1990a; McKenzie et al., 1991; Burleson, 1991; Burleson and Milsom, 1992a) and a separate set of externally oriented receptors that elicit both ventilatory and cardiovascular reflex effects (Randall and Smith, 1967; Saunders and Sutterlin, 1971; Smith and Jones, 1978; Smatresk et al., 1986; Burleson and Smatresk, 1990a; McKenzie et al., 1991; Burleson, 1991; Burleson and Milsom, 1992a).

b. Receptor Location. Cells have been identified in the gill filaments of fish resembling the chemoreceptor cells in mammals. These candidate chemoreceptor cells display monoamine fluorescence using

7.

407

AFFERENT INPUTS saline Po2 =

76

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Aperfu;,Io;

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Fig. 11. ENG ofactivity from oxygen chemoreceptors in the first gill arch of skipjack tuna illustrating the effect of interrupting perfusion on afferent activity. The fiber depicted in panel A is not sensitive to changes in the P o 2 of the bathing solution but does respond to changes in perfusion. The fiber illustrated in panel B does respond to changes in the Poz of the bathing solution but is not very responsive to changes in perfusion. (Reproduced from Milsoni and Brill, 1986 by permission, 0 Elsevier Science Publishers.)

408

BURLESON ET AL.

the Falck-Hillarp technique (Figs. 12A and B) (Donald, 1984, 1987; Dunel-Erb et al., 1982; Laurent, 1984), as do the carotid body chemoreceptors in mammals (Niema and Ojala, 1964). Furthermore, under light and electron microscopy, they resemble mammalian Type I (glomus) chemoreceptor cells (Fig. 12C). They are located in the primary gill epithelia between the inhalant water and blood flow pathways (Figs. 12A and B) (Dunel-Erb et al., 1982). Unfortunately, the location and similarity of these cells to mammalian carotid body chemoreceptors are not strong enough evidence to convincingly conclude that these are the cells that give rise to the electrophysiological data previously reported. Other cellular structures like noradrenergic fibers or taste cells may potentially display Falck fluorescence (Donald, 1987; Roper, 1989) and, although chemoreceptors in this position would probably respond well to aquatic hypoxia, they would be unlikely to provide distinct responses to hypoxemia. Based on NaCN localization studies, Smatresk et al. (1986)suggested that the internally oriented chemoreceptors respond to the mixed venous blood somewhere between the ventral aorta and the afferent filamental artery. Barrett and Taylor (1984) also suggested that the responses to hypoxemia in dogfish were monitored by venous side receptors. No chemoreceptor-like cells, however, have been found histologically in such a site as yet. Thus, at present there is a dichotomy between the electrophysiological and reflex response data suggesting there are two distinct receptor populations at functionally different sites within the gill and the morphological data that demonstrate the existence of putative chemoreceptor cells but with a uniform distribution. Fig. 12. (A) Fluoresence micrograph of a cross-section of a catfish (1. punctutus) gill filament (efferent side). Note the fluoresent neuroepithelial cells (NEC) lying midway between the edge of the primary epithelium (PEP) and the efferent filamental artery (EFA). (B) Midsagittal section through a catfish gill filament showing several neuroepithelial cells (NEC) displaying faint green fluoresence (indicating the presence of catecholamines within the cell) lying between the efferent lamellar arterioles (ELA)and the edge of the primary epithelium (PEP) facing the water flow. A group of cells containing serotonin (5-HT CELLS), as indicated by their intense yellow fluoresence, are also commonly found scattered through the primary epithelium and interlamellar space. [Fluoresence micrographs prepared using the Falck-Hillarp technique by P. Anderson, J. Butler, and N. Smatresk.] (C) Transmission electronmicrograph o f a NEC lying on the basal lamina (bl) of a trout (S. gairdneri) gill filament, showing its similarity to mammalian carotid body chemoreceptor glomus (Type I) cells. A nerve fiber (nf) is shown in an indentation in the NEC cell. [From Dunel-Erh et ul. (1982).](D) Transmission electron micrograph of a NEC separated from a vesiculated nerve fiber lying in the basal lamina (bl) that overlies a smooth muscle fiber (smf).(From Dunel-Erb et al., 1982 by permission, 0 The American Physiological Society.)

7 . AFFERENT

INPUTS

409

410

BURLESON E T A L

Fig. 12-Continued

7.

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411

b. Mechanisms of 02-Sensitive Chemotransduction. The mechanism of' 0 2 chemotransduction in fish chemoreceptors has not been studied. Given the similarities in afferent discharge recorded from mammalian carotid bodies and fish gills in response to hypoxia, however, it seems likely that the 02-sensitive chemoreceptors in fishes and mammals utilize similar transduction mechanisms. Furthermore, sodium cyanide, which blocks electron transport and mitochondrial oxidative phosphorylation, stimulates 02-sensitive chemoreceptor activity in all vertebrates in which they have been found (Eclancher and Dejours, 1975; Bouverot, 1978; Ishii et al., 1985a,b; Smatresk, 1986; Smatresk et al., 1986; Burleson and Smatresk, 1990; Burleson and Milsom, 1992a) suggesting that the 08-sensitive chemoreceptors of all vertebrates may be similar. Further elucidation of the similarities between chemoreceptor transduction mechanisms in vertebrates will require unequivocal identification of the chemoreceptor cells and primary afferent projections but, for now, the possibility that 0 2 chemotransduction is similar in all groups will allow us to explore data relevant to this topic collected from various species. The application of the whole cell patch clamp technique to the study of carotid body glomus cells has begun to reveal the cellular basis for the transduction of a hypoxic stimulus to afferent discharge in mammalian chemoreceptors (Lopez-Barneo et al., 1988; Biscoe and Duchen, 1990). While many of the details of chemotransduction are not resolved, two hypotheses have arisen from this research. Studies by L6pez-Barneo et al. (1988)and L6pez-L6pez et al. (1989) suggest that hypoxia directly acts to reduce K+ conductance, thereby depolarizing the cell, which in turn should promote neurotransmitter release. On the other hand, Biscoe and Duchen (1990) found that hypoxia promotes the release of Ca2+ from mitochondrial stores and suggest that this release is the cellular event ultimately leading to neurotransmitter release. The second major step in the process of chemotransduction is believed to be the release of a neurotransmitter from the chemoreceptor, the glomus, or type I cell, and the initiation of action potentials in the primary afferent neuron. Unfortunately, elucidating this step has turned out to be very problematic. The synaptic arrangement between type I cells and their primary afferent neurons is complex and involves reciprocal synapses (Fig. 13). Furthermore, a large number of neuromodulators appear to be present in the chemoreceptor complex that have multiple interactions. This is a problem that is exacerbated by the fact that the presence, or relative efficacy of different neuromodulators, appears to be both species specific and dependent on the anesthetic

412

BURLESON E T AL.

Sustentacuhr cell

Efferent nerve

Glomus d l

Efferent synapse

Afferent nerve ending

I

Afferent synapse

Fig. 13. Diagrammatic representation of the presumed chemoreceptive unit in vertebrates, composed of glomus cells, sustentacular cells, and both afferent and efferent nerve endings. The calyx type afferent nerve ending makes reciprocal synaptic contact (afferent and efferent synapses) with the glomus cell. Different efferent nerves make synaptic contact with afferent nerves, glomus cells, and capillaries. (From Jones and Milsom, 1982 by permission, 0 The Company of Biologists, Ltd.)

used in in vivo preparations. The arrangement of gustatory chemoreceptor cells and their primary afferent neurons is strikingly similar to that seen in the mammalian glomus cell complex (for review see Roper, 1989). Understanding the significance of' the reciprocal synaptic arrangement and the interplay of neurotransmitters and neuromodulators has been equally confusing in the taste system. It has been argued that this synaptic arrangement appears to be primitive, however, and reciprocal innervation between the chemoreceptor cells and primary afferent neurons is critical for the viability of the chemoreceptor cells (Fidone and Gonzalez, 1986; Roper, 1989). While it is not known which neurotransmitter(s) activates the primary afferent neuron in the fish chemoreceptor complex, Burleson and Milsom (1992b) have assessed the afferent response of trout branchial chemoreceptors to a variety of pharmacological agents (Table I). In the isolated perfused gill arch preparation used in these studies, afferent

7.

413

AFFERENT INPUTS

Table I Summary of Effects of Hypoxia and Different Pharmacological Agents on Afferent Neural Activity in Glossophaqngeal Slips Arising from the First Gill Arch in Rainbow Trout

Agent"

Site/Dose

Hypoxia

External

Hypoxia

Internal

NaCN

External 1000 pg/ml Internal 25 pg 100 nmol 100 nmol 100 nmol 100 nmol 5-1000 nmol 500 nmol 100 nmol 500 nmol 100 nmol

NaCN ACH NIC MUSC ATRO NOREPI EPI IS0 PROP DOP 5-HT

100 nmol

Effect Stimulates some cells preferentially, others respond only to internal hypoxia Stimulates some cells preferentially, others respond only to external hypoxia Strong stimulation Strong stimulation Strong stimulation Strong stimulation Weak stimulation Inhibits effects of ACH, NIC, and MUSC but not hypoxia No effecth

No effect No effect Partially inhibits effects of NaCN and hypoxia Weak stimulation followed by weak inhibition Weak stimulation followed by weak inhibition

a ACH, acetylcholine; NIC, nicotine; MUSC, muscarine; ATRO, atropine; NOREPI, norepinephrine; EPI, epinephrine; ISO, isoproterenol; PROP, propranolol; DOP, dopamine; 5-HT, 5 hydroxy-tryptamine (serotonin). Mild effect at 1000 nmol on 1 of 17 receptors.

activity was fairly insensitive to changes in perfusate flow and thus the effects of neurochemicals on neural discharge were probably direct effects and not secondary to changes in vascular perfusion. In these studies the cholinergic agonists acetylcholine and nicotine promptly stimulated afferent activity. Muscarine stimulated only a small, slow increase in afferent activity, suggesting that it exerted its effects primarily on oxygen delivery to the chemoreceptor by altering vascular tone, rather than directly on the chemoreceptor (Burleson, 1991; Burleson and Milsom, 1992b). A dosage of atropine sufficient to block the effects of muscarine, nicotine, and acetylcholine slightly attenuated, but did not block, the effects of NaCN. Thus, although there is strong cholinergic (nicotinic) modulation of chemoreceptor activity in trout

414

BURLESON ET A L .

(as seen for cats, see Fidone and Gonzalez, 1986 for review), this appears to be a neuromodulatory mechanism rather than a part of the chemotransduction process. Adrenergic agonists have little or no effect on afferent discharge from 02-sensitive chemoreceptors in the trout gill, but propranolol, an adrenergic antagonist, attenuates the afferent responses to hypoxia and sodium cyanide (Burleson and Milsom, 1990).The lack ofa response to adrenergic agonists suggests that these effects may be due to proprano101’s membrane stabilizing (anesthetic) properties rather than to P-adrenergic blockade per se. It has been suggested that elevated circulating catecholamines during hypoxia or following exercise are important for cardiorespiratory modulation in trout (Aota et al., 1990), although this point is debated (Kinkead and Perry, 1990). In either case, it is clear from the studies of Burleson and Milsom (1990) that these putative effects are not likely to arise from catecholaminergic stimulation of branchial chemoreceptor activity. Dopamine, the dominant catecholamine found in mammalian carotid body chemoreceptors, may stimulate or inhibit chemoafferent activity in mammals (Fidone and Gonzalez, 1986). In fishes, it stimulated a small burst of activity followed by inhibition of afferent activity. This pattern of response has been observed previously in cats and appears to be dependent on the dose and interval between injections (Okajima and Nishi, 1981). Serotonin, another common biogenic amine, also caused a brief burst of increased activity followed by inhibition in trout branchial chemoreceptors (Burleson, 1991; Burleson and Milsom, 1992b). In fishes, serotonin appears to be the major monoamine found in gill neuroepithelial cells and aquatic hypoxia alters its concentrations in these cells (Dunel-Erb et al., 1982). The responses recorded from chemoreceptor afferent neurons would suggest, however, that serotonin and dopamine are likely to be neuromodulators, at best, rather than a part of the transduction mechanism. B. COz/pH-Sensitive Chemoreceptor The ability to sense and respond to changes in COZ/pH represents an important stage in the evolution of terrestriality and also is an important component of the responses of fishes to environmental hypercapnia, acidosis, and exercise. The anatomical location, afferent pathways, and discharge characteristics of the receptors that mediate cardioventilatory reflex responses to arterial acidosis in fishes, however, remain unknown. The sensitivity to hypercapnia and acidosis in terrestrial vertebrates arises from the combined responses of

7.

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415

peripheral and central chemoreceptors (see Smatresk, 1990b for review). It is likely that responses to hypercapnic acidosis in fishes are also mediated by peripheral, central, or both chemoreceptors, but there have been no concerted efforts to identify whether the characterized branchial chemoreceptors of fishes are C02/pH sensitive, and there is no compelling evidence for central CO2/pH chemosensitivity in fishes (Graham et al., 1990; Wood et al., 1990; Hedrick et al., 1991). Because of the high ratio of C 0 ~ / 0 2solubility in water, fish are typically “hyperventilated” with respect to C02, and C 0 z tensions are normally quite low in fishes, typically ranging from 2 to 8 torr. Respiratory regulation of p H is, therefore, limited even though aquatic hypercapnia can elicit significant cardiorespiratory responses (see Shelton et al., 1986 for review). The effects of hypercapnic acidosis on cardioventilatory reflexes have been attributed to both indirect and direct effects. Smith and Jones (1982) found that changes in ventilation during hypercapnic acidosis in trout were better correlated to the concomitant reduction of arterial 0 2 content via Root or Bohr effects than to arterial PcO2 or p H per se. Thus, a major portion of the reflex responses to hypercapnia appear to arise indirectly, when branchial 02-sensitive chemoreceptors are stimulated by reduced 0 2 content. That ventilation is reduced in fishes exposed to aquatic hyperoxia, despite a significant respiratory acidosis, is generally taken as further evidence for the lack of a significant direct effect of hypercapnia on ventilation (Dejours, 1981; Shelton et al., 1986). Several studies, however, have demonstrated significant respiratory responses to the acidosis accompanying hypercapnia or exercise, which is independent of arterial 0 2 content (Hesiler et al., 1988; Shipman, 1989; Wood et al., 1990).Some studies suggest that acidosis may elicit the release of catecholamines, which in turn stimulate ventilation (Boutilier et al., 1986; Aota et al., 1990; Taylor and Randall, 1990). Other studies have found that the release of catecholamines has little effect on ventilation and that endogenous catecholamine release is not due to hypercapnia, but instead is correlated to low blood 0 2 content (Perry et al., 1989). Regardless of the role of catecholamines on ventilation, there is now ample evidence that acidosis provokes a modest stimulation of ventilation that is independent of arterial 0 2 content or endogenous catecholamines. The cardiovascular effects of hypercapnic acidosis, however, have largely been ignored. Given that systemic arterial chemoreceptors in all other animals examined respond to both O2 and COz (Smatresk, 1990b), it seems probable that the branchial chemoreceptors of fishes mediate reflex responses to hypercapnic acidosis as well as to hypoxemia. The re-

416

BURLESON ET AL.

sponses are likely to be weak at the relatively low temperatures and low PcOz in blood or water, but it could be predicted that at elevated temperatures the responses to Pco, and acidosis should be more pronounced. The respiratory responses to COe at different temperatures have not, however, been systematically investigated in fishes. It is interesting to consider whether the respiratory centers of fishes may respond directly to acidosis or if there are distinct central chemoreceptors as there are in terrestrial vertebrates. Superfusion of the brain with acidotic solutions elicits a modest ventilatory stimulation in lamprey brains while superfusion with bicarbonate solutions elicits a modest hypoventilation in both lamprey and carp brains (Hughes and Shelton, 1962; Rovainen, 1977), but later studies found no significant effects of intracisternal or extradural fluid acidosis (Graham et al., 1990; Wood et al., 1990; Hedrick et al., 1991).Taste or olfactory receptors, sensitive to COz, have been identified in carp (Yoshii et al. 1979), trout (Yamashita et al., 1989), gar (Smatresk, 1990a), and catfish (J. Caprio and N. J. Smatresk, unpublished observations), but there is no indication that stimulation of these receptors leads to an increase in ventilation. In fact, as discussed in the next section on nociceptors, stimulation of olfactory receptors by hypercapnia is more likely to lead to inhibition of ventilation in gar and other air-breathing vertebrates (Smatresk, 1988; Smatresk, 1990a). IV. NOCICEPTORS The obvious behavioral responses of fish to a wide range of potentially harmful substances provide ample evidence that they possess nociceptors, but few studies have described the location, afferent discharge patterns in response to specific stimulants, or specific cardiorespiratory responses of these “defense” receptors. The major responses to irritating or noxious stimuli are (a)avoidance, (b)coughing or expulsion reflexes to remove large particles, and (c) inhibited exchange to limit uptake of noxious solutes. The variety of responses suggests that several modalities, including mechanical and chemical stimulation, are involved in eliciting these reactions. A. Mechanoreceptors

Responses to mechanical trauma or stimulation of the gills, by such things as silt, particulate matter, or parasites on the gill curtain, most likely arise from stimulation of gill raker or filament mechanorecep-

7.

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417

tors. This function of these receptors was described in some detail earlier in Section II,A. They appear to elicit filament adduction, which should lower gill resistance and facilitate expulsion (de Graaf et al., 1987; de Graaf and Ballintijn, 1987). In addition, mechanical stimulation of the pharyngeal wall also evokes cough reflexes (Satchell and Maddalena, 1972). In the case of mechanical stimulation of the respiratory passages, Satchell and Maddalena (1972) observed a parabranchial cough that served to clear the gills. Although such expulsion reflexes will obviously disrupt the normal breathing rhythm, their effects on cardiac rhythms and on overall respiratory and cardiovascular control (rhythm generation, pattern generation, resetting of rhythm, and cardiorespiratory synchrony) have not been examined in any detail. Nociceptors with characteristics similar to juxtapulmonary receptors ( J receptors; Paintal, 1955) in mammalian lungs have been demonstrated in the gills of dogfish (Satchell, 1978; Poole and Satchell, 1979). These receptors are innervated by both pre- and post-trematic branches of the vagus nerve and respond to topical or injected phenyldiguanide and 5-hydroxytryptamine (agents that specifically stimulate c-fiber irritant receptors), and mechanical stimulation (Poole and Satchell, 1979). The receptive field for these nociceptors appears to be similar to that of gill mechanoreceptors, and based on the tactile responses described for these receptors (Poole and Satchell, 1979), it seems most likely that they are simply rapidly adapting gill filament mechanoreceptors rather than a distinct subclass of chemosensory receptors. In mammals they are believed to be stimulated normally by the tension generated with pulmonary edema. Injection of phenyldiguanide into conscious dogfish elicits shallow breathing, bradycardia, and hypotension (Satchell, 1978). The net effect is a reduction in gill perfusion pressure that would reduce the production of gill ultrafiltrate, a result consistent with their suggested role as the sensory component of a mechanoreceptive reflex acting to limit gill capillary fluid loss. B. Chemoreceptors Chemical irritants, like ammonia or dilute acid, appear to elicit an orobranchial cough in sharks, which serves to expel water from the buccal cavity and may limit contact between noxious solutes and the respiratory exchange surfaces (Satchell and Maddalena, 1972). This response to chemical irritants appears to arise from receptors associated with the gills, nares, and less well-defined portions of the orobranchial cavity.

418

BURLESON ET A L .

In brown bullhead, on the other hand, exposure to food stimulates respiration and heart rate (Sawyer and Heath, 1988). Although not a noxious stimulus, this indicates that activation of gustatory receptors with more favorable stimuli also modulates cardiorespiratory activities but in a very different manner. This suggests that there is substantial integration of olfactory and gustatory afferent information with medullary cardiorespiratory control centers. C. Nociceptors in Air-Breathing Fish?

In water-breathing fishes stimulation of nociceptors as described by Poole and Satchel1 (1979) may help to minimize exposure or uptake of potentially harmful solutes. Because of the high demand for convective transport in fish that breathe only water, however, ventilation and blood flow responses can only be momentarily compromised. In the gar, Lepisosteus oculatus, an air-breathing fish, stimulation of receptors in the nares elicits nonadapting branchial apnea in response to a wide variety of irritants and ionic solutes (Fig. 14). Under these conditions the animal simply switches to lung ventilation. Sectioning the terminal and olfactory nerves abolishes the response (Smatresk, 1990a). The specific receptor type mediating this response has not been identified, but olfactory and gustatory receptors sensitive to COZ and acidic solutions have been identified in carp (Konishi et aZ., 1969), eels (Yoshii et aZ., 1979), trout (Yamashita et al., 1989), and catfish barbels (N. J. Smatresk and J. Caprio, unpublished observations). These results suggest that receptors that are equivalent to those medi-

cm h r AH . 2 0 30

1

20 L

NaCN off

NaCN on

1 min

Fig. 14. Traces of' ventral aortic pressure (PvA)and the pressure in the branchial cavity (Pt,) (reflecting heart rate and breathing rate respectively) illustrating reflex apnea in gar in response to stimulation of nociceptors in the nares. (Reproduced from Smatresk, 1988 by permission, 0 National Research Council Canada.)

7.

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419

upper airway defense reflexes found in terrestrial vertebrates (Ballam, 1984, 1985) exist even in fishes. In addition to protecting respiratory structures from potentially harmful solutes, the ability to limit branchial exchange during exposure to the salty or brackish water in estuaries or bayous may help gar to limit ionic uptake, and thus “behaviorally” osmoregulate (Smatresk and Cameron, 1982a,b; Smatresk, 1990a).

V. CENTRAL PROJECTIONS OF SENSORY NEURONS Data regarding central sensory areas controlling cardioventilatory control in fishes are sparse. Almost all of the sensory information involved in cardiorespiratory control, however, appears to be conveyed in cranial nerves, and there is some general information concerning central afferent projections of cranial nerves. The afferent component of a cranial nerve may be composed of one or more of four types of afferent fibers as described by Nieuwenhuys and Pouwels (1983). These are: (a) general somatic afferent fibers from receptors in the skin, skeletal muscle, joints, or ligaments; (b) general visceral afferent fibers from receptors in internal organs and vasculature; (c) special somatic afferent fibers from the specialized sense organs (i.e., vision, hearing, lateral line); and (d) special visceral afferents from visceral sense organs (i.e., taste and olfaction). Inputs from several, if not all, of these types of fibers may be involved in cardiorespiratory control as discussed previously. The organization of the brain stem of actinopterygian fishes has been extensively reviewed by Nieuwenhuys and Pouwels (1983) who state that on entering the medulla, the various classes of fibers that compose a cranial nerve separate and travel to specific zones. Within the medulla of fishes, the sites of termination for afferent sensory fibers are located dorsally and laterally above the sulcus limitans of His (Fig. 15). In contrast, the zones of motor efferents are located ventral and lateral to the sulcus limitans of His. The sensory zone is divided into two regions with the somatic sensory zone above and the visceral sensory zone below the sulcus intermedius dorsalis. Other than these general delimitations, however, the partitioning of the central sensory regions in the brain stem of fishes has not been extensively described. Consequently, the central projections of the sensory afferents contributing to cardioventilatory control, described in this chapter, have not been identified. Ultimately, of course, they must converge either directly, or via inter-

420

-

BUHLESOK ET AL.

=

%

T - somatic motor zone visceral motor zone visceral sensory zone somatic sensory zone

Fig. 15. Diagram showing the ventricular sulci and sensory zones in the hindbrain of longnose gar (Lepisosteus osseus). oli, Oliva inferior; sid, sulcus intermedius dorsalis; siv, sulcus intermedius ventralis; slH, sulcus limitans of His; vem, nucleus vestibularis magnocellularis; Vm, nucleus motorius nervi trigemini; VI, nucleus motorius nervi abducentis; VIIm, nucleus motorius nervi facialis; IXm, nucleus motorius nervi glossopharyngei; Xm, nucleus motorius nervi vagi. (Reproduced from Nieuwenhuys and Pouwels, 1983 by permission, 0 T h e University of Michigan Press.)

neurons, on the motor output pools involved in the reflexes described in Chapter 6. The anatomical and physiological interactions that occur between the afferent inputs and efferent projections, however, remain an area in need of examination.

REFERENCES Aota, S., Holmgren, K. D., Gallaugher, P., and Randall, D. J. (1990).A possible role for catecholamines in the ventilatory responses associated with internal acidosis or external hypoxia in rainbow trout Oncorhynchus mykiss. J. E x p . Biol. 151,57-70. Azizi, S. Q . (1989).“The Role of Air-Breathing Organ Mechanoreceptors in Gar, Lepisosteus oculatus and Lepisosteus osseus. MSc. Thesis, University of Texas, Arlington. Azizi, S. Q., and Smatresk, N. J. (1986).Relationships between vagal efferent activity and pressure in the air-breathing organ of gar. Physiologist 29, 177. [Abstract] Ballam, G. 0. (1984).Ventilatory response to inspired COZ in the lizard, Tupinarnbis nigropunctatus. Comp. Biochem. Physiol. A. 78,757-762. Ballam, G. 0. (1985). Breathing response of the tegu lizard to 1-4% COz in the mouth and nose or inspired into the lungs. Respir. Physiol. 62,375-386.

7 . AFFERENT

INPUTS

42 1

Ballintijn, C. M. (1984).The respiratory function of gill filament muscles in the carp. Respir. Physiol. 60,59-74. Ballintijn, C. M., and Bamford, 0. S.(1975). Proprioceptive motor control in fish respirati0n.J. E x p . Biol. 62,99-114. Ballintijn, C. M., and Roberts, J . L. (1976). Neural control and proprioceptive load matching in reflex respiratory movements of fishes. Fed. Proc. 35,1983-1991. Bamford, 0. S . (1974).Oxygen reception in the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 48,69-76. Barrett, D. J., and Taylor, E. W. (1984).Changes in heart rate during progressive hypoxia in the dogfish, Scyliorhinus canicula L.: Evidence for a venous oxygen receptor. Comp. Biochem. Physiol. 78,697-703. Biscoe, T. J., and Duchen, M. R. (1990).Monitoring Poz by the carotid chemoreceptor. N I P S 5,229-233. Boutilier, R. G., Heming, T. A., and Iwama, G. K. (1986).Acute extracellular acidosis promotes catecholamine release in rainbow trout (Salmo gairdneri): Interactions between red cell pH and Hb-02 carrying capacity. 1.E x p . Biol. 123, 145-157. Bouverot, P. (1978).Control ofbreathing in birds compared with mammals. Physiol. Reo. 58,604-655. Boyd. J . D. (1936). Nerve supply to the branchial arch arteries of vertebrates. J . Anat. Lond. 71,157-158. Burleson, M. L. (1988).Sensory receptors in the first gill arch of rainbow trout. Bull. Can. Soc. Zool. 20,34. [Abstract] Burleson, M. L. (1991). “Oxygen-Sensitive Chemoreceptors and Cardiovascular and Ventilatory Control in Rainbow Trout.” Ph.D. Thesis, University of British Columbia, Vancouver, Canada. Burleson, M. L., and Milsom, W. K. (1990).Propranolol inhibits oxygen sensitive chemoreceptor activity in trout gills. A m . J . Physiol. 258, R1089-R1091. Burleson, M. L., and Milsom, W. K. (1992a). Sensory receptors in the first gill arch of rainbow trout. (Submitted.) Burleson, M. L., and Milsom, W. K. (1992b). Effects of neurochemicals on Oz-sensitive chemoreceptor afferent activity in rainbow trout. (Submitted.) Burleson, M. L., and Smatresk, N. J. (l990a). Evidence for two oxygen-sensitive chemoreceptor loci in channel catfish. lctalurus punctatus. Physiol. Zool. 63,208-221. Burleson, M. L., and Smatresk, N. J. (1990b). Effects of sectioning cranial nerves IX and X on cardiovascular and ventilatory reflex responses to hypoxia and NaCN in channel catfish. J . E x p . B i d . 154,407-420. Butler, P. J., Taylor, E . W., and Short, S. (1977).The effect of sectioning cranial nerves V, VII, IX and X on the cardiac response of the dogfish, Scyliorhinus canicula, to environmental hypoxia. J . E x p . Biol. 69, 233-245. Cameron, J. N., and Davis, J. C. (1970).Gas exchange in rainbow trout (Salmo gairdneri) with varying blood oxygen capacity.J. Fish. Res. Board Can. 27, 1069-1085. Cameron, J. N., and Wohlschlag, D. E. (1969).Respiratory response to experimentally induced anaemia in the pinfish (Lagodon rhombiodes).J . E x p . Biol. 50,307-317. Cushing, H. (1901). Concerning a definite regulatory mechanism of the vaso-motor center which controls blood pressure during cerebral compression. Bull. Johns Hopkins Hosp. 12,290-292. d e Graaf, P. J. F. (1990). Innervation pattern of the gill arches and gills of the carp (Cyprinus carpio)./. Morphol. 206,71-78. de Graaf, P. J. F., and Ballintijn, C. M. (1987).Mechanoreceptor activity in the gills ofthe carp. 11. Gill arch proprioceptors. Respir. Physiol. 69, 183-194.

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de Graaf, P. J. F., Ballintijn, C. M., and Maes, F. W. (1987). Mechanoreceptor activity in the gills of the carp. I. Gill filament and gill raker mechanoreceptors. Respir. Physiol. 69,173-182. Dejours, P. (1981). “Principles of Comparative Respiratory Physiology,” 2nd ed. ElsevierINorth-Holland Biomedical Press, New York. DeKock, L. L. (1963). A histological study of the head region of two salmonids with special reference to pressor- and chemo-receptors. Acta Anat. 55,39-50. DeLaney, R. G., Laurent, P., Galante, R., Pack, A,, and Fishman, A. P. (1983). Pulmonary mechanoreceptors in the dipnoi lungfish Protopterus and Lepidosiren. A m . J. Physiol. 244, R418-R428. Donald, J. (1984). Adrenergic innervation of the gills of brown and rainbow trout, Salmo trutta and S. gairdneri. J. Morphol. 182,307-316. Donald, J. (1987). Comparative study of the adrenergic innervation of the teleost gill. J. Morphol. 193,63-73. DuBois, A. B., Fox, S. H., and Ogilvy, C. S. (1990). Preoxygenation delays onset of the Cushing response in a bluefish. (Abstr.) The Physiologist 33, A113. Dunel-Erb, S., Bailly, Y. S., and Laurent, P. (1982). Neuroepithelial cells in fish gill primary lamellae. J. Appl. Physiol. 53, R1324-R1353. Eclancher, B., and Dejours, P. (1975). ContrBle de la respiration chez les poissons tekostkens: Existence de chemorecepteurs physiologiquement analogues aux chemorecepteurs des vertebres supCrieurs. C . R. Acad. Sci. Der. D . 280,451-453. Fidone, S. J., and Gonzalez, C. (1986).Initiation and control of chemoreceptor activity in the carotid body. In “Handbook of Physiology, The Respiratory System” (A. P. Fishman, N. S. Cherniack, J. S. Widdicombe, and S. R. Geiger, eds.), Sec. 3, Vol. 11, part. 1, pp. 247-312. American Physiology Society, Bethesda, MD. Fox, S. H., Ogilvy, C. S., and DuBois, A. B. (1990). Search for the biological stimulus of the Cushingresponse in bluefish (Pomatomussaltatrix).(Abstr.)Biol. Bull. 179,233. Graham, M. S., Turner, J. D., and Wood, C. M. (1990). Control of ventilation in the hypercapnic skate Raja ocellata: I. Blood and extradural fluid. Respir. Physiol. 80, 259-277. Hedrick, M. S., Burleson, M. L., Jones, D. R., and Milsom, W. K. (1991). An examination of central chemosensitivity in an air-breathing fish (Amia caloa).J . E x p . Biol. 155, 165- 174. Heisler, N., Towes, D. P., and Holeton, G. F. (1988). Regulation of ventilation and acid-base status in the elasmobranch Scyliorhinus stellaris during hyperoxia induced hypercapnia. Respir. Physiol. 71,227-246. Holeton, G . F. (1971). Oxygen uptake and transport by the rainbow trout during exposure to carbon monoxide. J. Exp. Biol. 54,239-254. Hughes, G. M., and Shelton, G. (1962). Respiratory mechanisms and their nervous control in fish. Ado. C o m p . Physiol. Biochem. 1,275-364. Irving, L., Solandt, D. Y., and Solandt, 0. M . (1935). Nerve impulses from branchial pressure receptors in the dogfish. J . Physiol. Lond. 84, 187-190. Ishii, K., lshii, K., and Kusakabe, T. (1985a). Electrophysiological aspects ofreflexogenic area in the chelonian, Geoclemmys reeoesii. Respir. Physiol. 5 9 , 4 5 5 4 . Ishii, K., lshii, K., and Kusakabe, T. (1985b).Chemo- and baroreceptor innervation ofthe aortic trunk of the toad Bufo vulgaris. Respir. Physiol. 60,365-375. Johansen, K. (1966). Air breathing in the telost Symbrunchus mannoratus. Cornp. Biochem. Physiol. 18,383-395. Johansen, K., Lenfant, C . ,Schmidt-Nielsen, K., and Petersen, J. A. (1968). Gas exchange and control of breathing in the electric eel, Electrophorus electricus, Z . Vgl. Physiol. 61.137-163.

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423

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7.

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Rovainen, C. M. (1977). Neural control of ventilation in the lamprey. Fed. Proc. 36, 2386-2389. Satchel], G. H. (1959).Respiratory reflexes in the dogfish. J . Exp. Biol. 36, 62-71. Satchel], G. H. (1978). The J reflex in fish. In “Respiratory Adaptations, Capillary Exchange and Reflex Mechanisms” (A. S. Paintal and P. Gill-Kumar, eds.), pp. 432-441. Vallabhbhai Patel Chest Institute, University of Delhi, Delhi. Satchel], G. H., and Maddalena, D. J. (1972). The cough or expulsion reflex in the Port Jackson shark, Heterodontus portusjacksoni. C o m p . Biochem. Physiol. 41A, 49-62. Satchel], G. H., and Way, H. K. (1962). Pharyngeal proprioceptors in the dogfish Squalus acanthias L. J . E x p . Biol. 39,243-250. Saunders, R. L., and Sutterlin, A. M. (1971).Cardiac and respiratory responses to hypoxia in the sea raven, Hemipterus americanus, an investigation of possible control mechanisms. J . Fish. Res. Bd. Can. 28,491-503. Sawyer, P. L., and Heath, A. G. (1988). Cardiac, ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide. Fish Physiol. Biochem. 4,203-219. Shelton, G., Jones, D. R., and Milsom, W. K. (1986). Control ofbreathing in ectothermic vertebrates. In “Handbook of Physiology-The Respiratory System,” pp. 857-909. American Physiological Society, Bethesda, Maryland. Shipman, B. N. (1989). “Patterns of Ventilation and Acid-Base Recovery following Exhausting Activity in the Air-Breathing Fish Lepisosteus oculatus.” M.Sc. Thesis, University of Texas, Arlington. Short, S., Butler, P. J., and Taylor, E. W. (1977). The relative importance of nervous, humoral and intrinsic mechanisms in the regulation of heart rate and stroke volume i n the dogfish Scyliorhinus canicula. J . E x p . Biol. 70,77-92. Sniatresk, N. J. (1986). Ventilatory and cardiac reflex responses to hypoxia and NaCN in Lepisosteus osseus, and air-breathing fish. Physiol. Zool. 59,385-397. Smatresk, N. J. (1987). Vagal afferent control over water and air-breathing patterns in Lesisosteus oculatus, on air-breathing fish. A m . Zool. 27, 11A. [Abstract] Smatresk, N. J. (1988).Control of the respiratory mode in air-breathing fishes. Can. J . Zoo/. 66, 144-151. Smatresk, N. J. (199Oa).Respiratory defense reflexes in an air-breathing fish. Lepisosteus oculatus. A m . Zool. 30,67A. [Abstract] Smatresk, N. J. (1990b).Chemoreceptor modulation of endogenous respiratory rhythms in vertebrates. A m . J . Physiol. 259, R887-R897. Smatresk, N. J., and Azizi, S. Q. (1987). Characteristics of lung mechanoreceptors in spotted gar, Lepisosteus oculatus. A m J . Physiol. 252, R1066-R1072. Smatresk, N. J., and Cameron, J. N. (1982a). Respiration and acid-base physiology of the spotted gar, a bimodal breather. I. Normal values and the response to severe hypoxia. J . E x p . Biol. 96,263-280. Smatresk, N. J., and Cameron, J. N. (1982b). Respiration and acid-base physiology ofthe spotted gar, a bimodal breather. 111. Response to a transfer from fresh water to 50% sea water, and control of ventilation. J . Exp. B i d . 96,295-306. Smatresk, N. J., Burleson, M. L., and Azizi, S. Q. (1986).Chemoreflexive responses to hypoxia and NaCN in longnose gar: Evidence for two chemoreceptive loci. A m . J . Physol. 251, R116-Rl25. Smith, F. M., and Jones, D. R. (1978). Localization of receptors causing hypoxic bradycardia in trout (Salmo gairdneri). Can.J . Zool. 56, 1260-1265. Smith, F. M., and Jones, D. R. (1982). The effect of changes in blood oxygen-carrying capacity on ventilation volume in the rainbow trout (Salmo gairdneri).J . Exp. Biol. 97.325-334.

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Srivastava, C. B. L., and Singh, M. (1980). Occurrence of carotid labyrinth in the catfish group of teleost fishes. Experientia 36,651-653. Sutterlin, A. M., and Saunders, R. L. (1969).Proprioceptors in the gills ofteleosts. Can.]. ZOO/..47,1209-1212. Taylor, E. W., and Randall, D. J. (1990). Control ofventilation in fish. In “Fish Physiology, Fish Toxicology and Fish Management,” pp. 146-156. U.S. EPA/600/9-90/011. 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 . E x p . Biol. 82, 139-162. Wood, C. M., Turner, J. D., Munger, R. S., and Graham, M. S. (1990). Control of ventilation in the hypercapnic skate Raja ocelata: 11. Cerebrospinal fluid and intracellular pH in the brain and other tissues. Respir. Physiol. 80,279-298. Yamashita, S., Evans, R. E., and Hara, T. J. (1989). Specificity of the gustatory chemoreceptors for COZ and H + in rainbow trout (Oncorhynchus mykiss).Can]. Fish. Aquatic Sci. 46, 1730-1734. Yoshii, K., Kashiwayanagi, M., Kurihara, K., and Kobatake, Y. (1979). High sensitivity of the eel palatine receptors to carbon dioxide. Comp. Biochem. Physiol. 66,327-330.

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

Aasa, R., 42, 46 Aasjord, P. M., 121 Abe, K., 123 Abo Hegab, S. A,, 142, 143, 160, 162, 163, 232,233 Abrahamsson, T., 259, 260, 265. 266, 267, 268, 269, 270,290,291,296 Acierno, R., 226, 228, 229, 233 Acton, R. T., 32, 46, 51 Addens, J. L., 350, 351,381 Addink, A. D. F., 131 Adrian, E. D., 374, 381 Agapito, M. T., 248 Agius, C., 9, 25, 39, 46 Ahlman, H., 335 Ahmad, M., 247 Aida, K., 114, 123 Aihara, M., 247 Ainsworth, A. J., 29, 47 Akiyanam, T., 126 Akselrod, S., 365, 381 Albers, C., 109, 120, 265, 290, 292 Albert, S. N., 167, 233 Aldman, G., 314, 319, 330,333,337 Alexander, J. B., 88,113, 185, 233 Alink, G. M., 375, 382 Allison, A., 15, 46 Allonso, E. G., 53 Almeida, F. A., 244 Almendras, J. M., 119 Alt, J . M., 139, 233 Andersen, O., 129 Anderson, D. P., 3, 30, 46 Anderson, D. R., 309, 338

Anderson, P. D., 96, 116 Ando, K., 252 Andreason, P., 111, 113 Andrew, W., 2, 46 Andrews, P. C., 325, 333, 339 Angus, J . A., 317,332, 334 Anthony, J., 51 Antonioli, A,, 46 Antonsson, B., 47 Aota, S., 265, 291, 294, 414, 415, 420 Aoto, T., 241, 252 Aponte, G. E., 235 Applegate, S., 131 Arai, S., 126 Arai, T., 242 Ariens Kappers, C. U., 350,381 Arillo, A., 203, 233 Arlot-Bonnemains, Y., 120 Armour, K. J., 247 Arnesen, A. M., 119,238 Arnold-Reed, D. E., 74,113,226,230,231, 233 Arthur, P. G., 76,113 Amy, L., 46 Asai, H., 12, 46 Askensten, U., 249 Assem, H., 137, 141, 142, 143, 160, 162, 233 Atkin, J., 234 Atkin, N. B., 6, 52 Atkinson, J. L., 77, 121 Audet, C., 110, 113 Aughton, P., 115 Augustinsson, A., 344, 345, 381 Aukland, K., 182, 233 Aurell, L., 236

427

428

AUTHOR I N D E X

Avella, M.,67, 114 Avranieas, S., 51 Avtalion, R. R., 47, 169, 173, 233 Awaya, K., 53, 54 Axelson, M., 233, 265, 284, 291, 310, 328, 333,337,345, 347,349,362, 363,381, 385 Azizi, S. Q., 399, 400, 420, 42.5 B

Babiker, M. M., 154, 155, 156, 159, 233 Babin, P. J., 97, 98, 99, 100, 114 Bachand, L., 10,46 Bada, J. L., 80, 124 Badawi, H. K., 241 Bailey, J. R., 58, 114, 200, 203, 207, 233, 24 6 Bailly, Y., 305,313,314,315,316,333,335, 336 Bailly, Y. S., 422 Baker, B. I., 128 Baker, N. J., 36, 46 Balasubramaniam, A,, 339 Baldwin, 111, W. M., 39, 45, 46 Ballam, G. O., 419, 420 Ballantyne, J. S., 101, 129, 143, 233 Ballerman, B. J., 235 Ballintijn, C. M., 372, 374, 375, 381, 382, 383,390,391,393,395,397,417,421, 422 Balment, H., 75, 113, 114, 127, 207, 211, 212,231,233,235,240,248 Bamford, 0. S., 390, 397, 405, 421 Barajas, L., 244 Baranowski, R. L., 221, 233, 253 Barber, D. L., 3, 16, 17, 22, 46, 52 Barger, A. C., 381 Bargmann, W., 39, 46 Baroncelli, V., 46 Barret, B. A., 66, 114 Barrets Gomes, F. V., 52 Barrett, D. J., 345, 347, 350, 351, 355 360, 360,361,366,369,371,372,374,375, 378,382,386,421 Barrett, I., 51 Barrington, J., 50 Barron, M. G., 170, 191, 233 Barry, M. A., 355, 382

Barry, T. P., 64, 65, 114 Bartelt, D., 47 Barthelemy, L., 297 Bartlett, G. R., 11, 46 Barun-Nesje, R., 46 Bassingthwaighte, J. B., 180, 234 Bates, D. J., 59, 114 Bath, R. N., 141, 142, 148, 151, 159, 162, 163, 234,237 Baumgarten, H. G., 312, 333 Bayne, C. J., 28, 33, 49, 52 Beach, M. A., 322, 333 Beamish, F. W. H., 109,114, 125 Beardsley, A. M., 334 Beasley, D., 210, 211, 212, 234, 236 Bedford, J. J., 13, 46, 142, 234 Belamarch, F. A,, 36, 46,53 Belaud, A., 340 Belle, N. D. L., 118 Benfey, T. J., 130 Bennett, G. L., 243 Bennett, J. A., 350,382 Bennett, J. C., 46 Bennett, M., 334 Bennion, G. R., 386 Benyajati, S., 149, 162,206, 225,229,234, 254 Bergheim, A., 56, 114 Beringer, T., 307, 333 Bern, H. A., 66, 114, 120, 122, 126, 133 Bernard, J., 45, 46 Bernard, L. M., 78, 123 Bernard, M., 130 Bernard, M. G., 117 Bert, J. L., 180, 182, 234, 248 Bertheussen, K., 46 Bertmar, G., 25, 46 Betzler, D., 248, 249 Bever, K., 76, 77, 114 Beyenbach, K. W., 148, 150, 234 Bielek, E., 8, 16, 17, 22, 25, 26, 46 Bieniarz, K., 76, 129 Billard, R., 59,60, 61, 62,64,65,114,115, 119,133 Bilstad, N. M., 22, 54 Binia, A., 248 Birch, F. M., 253 Birt, T. P., 117, 236 Birt, V. L., 117, 236 Biscoe, T. J., 411, 421

429

AUTHOR INDEX

Bisgard, G. E., 293 Biswas, J., 52 Bitner, A., 137, 140, 142, 161, 162, 234 Bjenning, C., 314,319,322,323,324,325, 333,334,335 Bjerring, H. C., 2, 46 Bjorklund, A,, 333 Bjornsson, B. T.,70,111,112,113,114,133 Black, D., 101,102,103,115,351,358,382 Blaine, E. H., 217, 234, 245 Blair-West. J. R., 196, 200, 206, 250 Blaschko, H., 256, 291 Blasco, J., 81, 115, 121 Blaxhall, P. C., 3, 15, 34, 46 Bloom, S. R., 326,334,340 Bly, J. E., 12, 46 Bodarnmer, J. E., 25,46 Boehkle, K. W., 288, 291 Boeuf, G., 50,128 Boffa, G. A,, 115 Bohle, A., 236 Bol, J . F., 54 Bolis, L., 10, 12, 46, 318, 321, 334 Bolton, J. P., 62, 115, 122, 128 Bolton, L. L., 22, 46 Bonini, E., 340 Boornker, J., 8, 26, 35, 46 Booth, C. E., 248, 296 Borelli, B., 49 Borenstein, B., 236 Borgese, F., 290,291,295 Bouck, G. R., 95,115 Boudon, M., 120 Bouquegneau, J. M., 110, 115 Boustead, N. C., 50 Boutilier, R. G., 57, 71, 78, 109, 112, 115, 130,131,248,265,266,267,277,284, 291,292,293,297,298,299,415,421 Bouverot, P., 411, 421 Bovy, P. R., 219, 234 Bowen, B. D., 234,248 Box, B. E., 251 Boyar, H. C., 46 Boyd, J. D., 401, 421 Boyd, R. B., 174, 175, 234 Boyd, T. A., 13,49,80,115 Brace, R. A., 248 Bradbury, M. W. B., 236 Bradley, T. M., 185, 234 Bradshaw, C. M., 32,46

Brands, M. W., 193, 240 Brauner, C., 297 Braun-Nesje, R., 15, 27, 46 Brenner, B. M., 217, 218, 235 Breton, B., 114, 115, 119 Breton, R., 62, 115, 119 Bridges, D. W., 9, 47 Brill, R. W., 1 1 3, 405, 406, 423 Brinn, J. E., 293 Brown, J. A., 197, 203,207, 208, 209, 210, 235, 239,292 Brown, J. J., 235 Brown, R. A., 49 Brown, S. E., 349, 382 Brown, W. D., 238 Brungs, W. A., 47 Bry, C., 58, 71,115 Budde, R. B., Jr., 424 Buijs, R. M., 122 Bull, J. M., 140, 149, 168, 173, 235 Bullock, J., 296 Bumpus, F. M., 242,246 Bundgaard, M., 174, 236 Burggren, W., 349, 383 Burkhardt-Holm, P., 324, 334 Burleson, M. L., 280, 281, 291, 293, 294, 392, 395, 401,405, 406, 411,412, 413, 414,421,422,423,425 Burnstock, G. 302,307,309,310,318,332, 334,336,338,339,341,348,349,383 Burt, D. W., 237 Burton, R. F., 235 Burzawa-Gerard, E., 115, 120 Busacker, G. P., 265, 274,291 Bushnell, P. G., 10, 47 Butler, D. G., 196, 254 Butler, P. J., 72,75,115,260,265,267,270, 275,276,284,287,291,294,305,338, 347, 348, 349, 355, 360, 361, 365, 366, 371,372,374,377,378,380,382,384, 385, 386,387,405,421,425 Buytendijk, F. J. J., 374, 381 Byrns, R. E., 337

C Cade, J. T., 125 Cagen, L. M., 244 Cairns, J., 125

430 Cairns, M. A,, 115 Calka, J., 339 Callard, G. V., 115 Callard, I. P., 74, 75, 115 Callejas, J., 250 Cameron, J. N., 41, 47, 84, 109, 115, 137, 139, 140, 148, 150, 153, 157, 165,235, 406, 419,421,425 Cameron, J. S., 349, 362,382 Campbell, G., 283,303,307,327,334,335, 339, 348, 382 Campbell, G. D., 269,292,349,383 Canals, P., 127 Cannon, A. M., 47 Cannon, M. S., 16, 17, 26, 47 Cantin, M., 235 Capelli, J. P., 203, 235 Capra, M. F., 291, 347, 348, 382 Caprio, J., 358, 360, 384 Caravita, S., 307, 334 Carlson, A. J., 344, 383 Carlsson, U., 10, 47 Carmena, A. O., 250 Caroff, J., 292 Caron, M. G., 294 Carragher, J. C., 128 Carrato, A,, 26, 54 Carretero, 0. A., 214, 250 Carrick, S., 127, 207, 211, 212, 233, 235 Carrillo, M., 60, 81, 116, 119, 120, 127 Carrol, R. G., 205,206,207,208,235,247, 296 Caruso, C., 47 Caspi, R. R., 30, 47 Castillo, G. A., 247 Catton, W. T., 16, 17, 40, 47 Cayer, M. L., SO Cech, J. J., 295 Cech, J. J., Jr., 47 Cenini, P., 8, 15, 17, 26,47 Chagny, F., 120 Chambers, V. C., 47 Chan, D. K. O., 142, 159, 192, 216,235 Chapeau, C., 219,221,235 Chapman, M. J., 126 Chavin, W., 265, 274, 291 Chenoweth, M., 114 Cherniak, V., 293 Chernow, B., 293 Chester-Jones, I., 145, 147, 186, 192, 197, 199,207,235

AUTHOR INDEX

Chevalier, G., 122 Chiller, J. M., 30, 31, 33, 47 Chilmonczyk, S., 37,47 Chipouras, E., 238 Cho, K. W., 242 Cholette, C., 139, 142, 149, 157, 162, 236 Choubey, B. J., 247 Chow, P. H., 216,235 Chrisman, C. L., 54 Christensen, G. M., 13, 16, 42, 47, 51 Christensen, J. A., 197, 203, 236 Christensen, N. J., 265, 298 Christian, A. R., 115 Christie, P., 127 Chung-Ja, C., 115 Churchill, M. C., 236 Churchill, P. C., 206, 207, 208, 210, 236 Cimini, V., 322, 334 Claeson, G., 213, 236 Clark, B., 238 Clark, N. B., 67, 69, 70, 116 Clark, R. T., 405, 424 Clark, S., 236 Clarke, F. D., 4, 54 Claviez, M., 23, 53, 252 Clem, L. W., 12, 46, 47, 48 Cline, M. J., 28, 39, 47 Cobb, J. L. S., 348, 386, Cocks, T. M., 317, 332, 334 Coghlan, J. P., 234 Cohen, R. J., 381 Cohen, S., 310, 334, 340 Cohen, W. D., 6, 11,47,51,52 Cole, F. E., 252 Coleman, T. G., 240 Colin, D. A,, 310, 311, 335 Colletti, A. E., 261, 263, 274, 292 Collie, N. L., 122 Comfort, D., 241 Conklin, D. J., 189, 236 Conlon, J. M., 322, 325, 327, 335 Conte, F. P., 140, 167, 168, 169, 236, 244 Cook, A. F., 62, 116, 127, 130 Cook, J. E., 247 Cook, R. F., 59, 69, 116 Cooper, E. L., 3, 29,47 Copeland, P. A., 99, 116 Copp, D. H., 117 Copp, H., 69,116 Corbel, M. J., 3, 31, 47 Corneillie, S., 124

431

AUTHOR INDEX

Cornish, I., 77, 116 Cortok, H., 346,385 Coscia, L., 307, 334 Cossins, A. R., 288, 290, 292 Costa, M., 334, 336, 339 Coston-Clements, L., 238 Coupland, R. E., 270, 292 Courtice, F. C., 37, 54 Coviello, A., 242 Cowan, C. M., 131 Cowey, C. B., 12,47 Cowley, A., 192,236 Crim, J. W., 330,335 Crim, L. W., 60, 62, 64, 116, 127 Croft, M., 116 Crosby, S., 292, 383 Cserr, H. F., 152, 162, 174, 236 Cuchens, M. A,, 48,51 Curatolo, A,, 129 Cushing, H., 403, 421 Cushing, J . E., 3, 11, 47 Cyr, D. G., 91, 116 D

Dabrowski, K., 80, 81, 116 Dahlstrom, A., 327, 335 Daisley, K. W., 3, 34, 46 Daly, de B. M., 373, 383 Daly, R. N., 332, 335 Dalziel, T. R. K., 125 D’Amelio, V., 129 Daniels, B. A,, 18, 51 Dannevig, B. H., 105,116 Danulat, E., 87, 116, 132 D’Apollonia, S., 96, 116 Darling, D. S., 69, 75, 117 Darnell, J., 97, 116 Dashow, I., 75, 123 Dashow, L., 271,292 Datta Munshi, J. S., 28, 47, 171, 236 Dave, G., 103, 117 Davenport, J., 79, 80,126 Davidson, W. S., 89, 117, 183, 236 Davie, P. S., 132, 176, 181, 191, 236, 239, 296, 310,336 Davies, P. L., 92, 117 D’Avino, R., 3, 47 Davis, J . C., 406, 421 Davis, K. B., 71, 117

Davison, W., 118,239, 291, 381,382 Dawe, D. L., 49 Dawson, A. B., 6, 8, 47 Daxboeck, C., 176,181,191,236,248,276, 296,297 De, C., 46 de Goeij, J. J . M., 131 Deacon, C. F., 335 De Almeida-Val, V. M., 53 deAndres, A. V., 120 deBold, A. J., 217, 236 Decourt, C., 212,237,247 Deftos, L. J., 63, 70, 111, 112, 114, 117 de Graaf, P. J. F., 372, 374, 383,391, 395, 417, 421,422 Deinan, E. M., 52 Dejours, P., 411, 415, 422 DeKock, L. L., 401,422 Delahunty, R. G., 59, 117 DeLaney, R. G., 21,47,85,87,117,237, 399, 422 Della Corte, F., 47, 52 Denton, D. A., 234 Denton, J. E., 139, 237 deRoos, C. C., 77, 117 deRoos, R., 77, 79, 117 Deshimaru, O., 127 Dessaux, G., 401, 424 Desser, S. S., 16, 17, 51 Deste, L., 341 Deuticke, B., 14, 47 de Vera, L., 364, 365,383 devlaming, V., 117 DeVries, A. L., 91, 92, 1 1 7, 234 DeWilde, M. A,, 171,241 Dexiang, C., 29, 47 Dick, L., 238 Dickhoff, W. W., 65, 69, 73, 75, 117, 120, 128,130 Dietz, T. H., 251 Dijk, J. P., 9, 47 Dimaline, R., 319, 335, 337, 340 Dindo, J. J., 96, 101, 117 Dion, S., 335 D’Ippolito, S., 26, 47 Dixon, J. E., 333 Dixon, 0. W., 46 Diz, D. I., 214, 237 Doble, K. E., 248 Dobson, G., 115 Dobson, G. P., 84,117,291

432

AUTHOR INDEX

Dobson, S., 75, 117 Dodd, J. M, 74,75,117 Dodgen, C. L., 48 Dogterom, J., 122 Domenech, R. J., 338 Donald, J., 274, 292, 305, 307, 313, 335, 349,383,408,422 Donald, J. A,, 305, 313, 335 Donaldson, E. M., 61, 62, 64, 65, 70, 71, 106,109,116,117,118,122,130,144, 145, 161, 163, 164, 168, 174,241 Doolittle, R. F., 35, 46, 47, 87, 118, 183, 185,237 D’Orleans-Juste, P., 332, 335 Dorsey, D., 251 Downey, H., 24, 47 Driedzic, W. R., 84, 117, 291,333 Drzewina, A,, 2, 14, 17, 19, 22, 27, 48 Dubey, A., 251 DuBois, A., 182, 246 DuBois, A. B., 404, 422 Duchen, M . R., 411,421 Ducret, C. G., 242 Duff, D. W., 141, 162, 163, 167, 170,172, 173, 174, 175, 176, 177, 178, 186, 187, 192,219,221,224,225,226,228,229, 237,242,247,339 Dufour, S., 62, 118 Duling, B. R., 179, 243 Duncombe, W. G., 118 Dunel-Erb, S., 305,306,313,316,333,335, 336,408, 414,422 Dunn, A,, 114 Dunn, B. M.,248 Dunn, J. I?., 76, 118 Dunn, R. S., 214, 237 Dunson, W. A., 140, 165, 239 Dupree, H. K., 51 Durand, J., 17, 48 Duthie, E. S., 16, 48 Dye, H. M., 65,118,130 Dzau, V. J., 193, 237 E

Eales, J. G., 59, 68, 69, 91, 116, 118, 119, 121 East, B. W., 252 Eastman, J . T., 234

Ebner, K., 338 Ebner, K. E., 340 Eclancher, B., 411, 422 Eddy, E. B., 112, 118 Eddy, F. B., 141, 142, 151, 159, 162, 163, 226,228,230,234,237 Edstrom, A,, 26, 42, 48 Edvinsson, L., 319, 336, 341 Edwards, D., 261, 292 Ehrenstrom, F., 288,291,292 Ehrlich, P., 15, 48 Eido, G., 233 Eiger, S., 244 Eijk, H. G. van, 47 Eisenbach, G. M., 233 Ekblad, E., 329, 336 Ekins, R., 90, 118 El-Doniiaty, N. A,, 241 Eldridge, F. L., 281, 292 Elephteriou, B. E., 291 Elger, B., 185, 237 Elger, E., 183, 237 Elger, M., 197, 237 Elliot, C. J. H., 387 Ellis, A. E., 3, 16, 17,22,23,24,25,33,34, 40,48 Ellis, A. G., 176, 237 Ellis, M. J., 118 Ellory, J. C., 11, 48 Ellsaesser, C. F., 15, 16, 48 El-Salhy, M., 312, 315, 336 Endersen, C., 121 Eng, F., 118 Engel, D. W., 142, 238 Engel, W., 52 Enger, P. S., 336 Epple, A., 116,123,263,266,268,270271, 292,293 Epstein, F. H., 224, 238, 250, 251, 340 Erdos, E. G., 219, 238,244,250 Eriksson, B.-M., 298 Erspamer, V., 311,336 Euler, von, C., 374, 383 Euler, von U. S., 327, 336 Eurell, T. E., 47 Evan, A. P., 247 Evans, B. K., 241 Evans, D. H., 107,118, 159,219,221,224, 225, 227, 228,238,239,244,248 Evans, D. L., 49

AUTHOR INDEX

Evans, E. E., 33,51 Evans, R. E., 426 Everaarts, J., 11, 48 Ewart, K. V., 92, 118 Ewing, D., 297 Ezell, A. A., 13, 48 Ezzat. A. A,, 16, 48

433

Fievet, B., 265, 292 Filosa, M. F., 183, 238 Fincham, D. A., 119 Finger, T. E., 358,359, 385 Finn, J. P., 17, 48 Finstad, B., 110, 119, 141, 142, 162, 165, 238 Finstad, J., 31, 43, 48, 49 Firkin, B. G., 19, 35, 53 F Fishman, A. P., 47, 117, 237, 422 Fitzgerald, D., 237 Fleming, W. R., 141, 163, 237 Fagerlund, U. H. M., 118 Fletcher, D., 76, 77, 103, 119 Falck, B., 345, 383 Falkmer, S., 249, 335 Fletcher, G. L., 91,92,117,118,119,238, Fange, R., 3,4, 8, 12, 14, 15, 17, 18, 19 21, 239 22,23,24,25,26,27,28,35,36,38,39, Fletcher, J., 48 Fletcher, T. C., 27, 28, 42, 51, 52, 88, 132 40, 42, 46, 48, 51, 54, 100, 101, 102, 124,238,242,303, 31.5, 336,339,345, Flik, G., 67, 73, 119, 125, 131, 297 381,383 Flint, K. B., 424 Fara, J. W., 328, 336 Flint, P. F., 129 Faraldi, G., 340 Flory, C . M., 28, 33,49,52 Farbridge, K. J., 60, 118 Flugge, G., 243 Folmar, L. C., 120 Farghaly, A. M., 48 Fargher, R. C., 66, 118 Fontaine, Y. A., 118 Farina, L., 340 Forlin, L., 114 Farrell, A. P., 100, 101, 104, 106, 109 118, Forssmann, W. G., 248,249 119,120, 153, 166,238,247,276,291, Forster, G. R., 126 292,295,307,310,333,334,336,339, Forster, M. E., 167, 168, 173, 186,239 253, 340, 377, 381,383 381 Fasciolo, J. C., 197, 246 Forster, R., 115 Fasulo, S., 340 Forster, R. E., 277, 292 Faulkner, N. W., 119 Foster, G. D., 126 Feeney, R. E., 238 Fostier, A,, 61, 114, 119 Feldman, A. P., 374, 383 Fouchereau-Peron, M., 70, 120 Feldschuh, J., 144, 238 Fox, S. H., 404, 422 Fellows, F. C. I., 88,89,97, 101,119, 183, Franklin, D. L., 384 238 Freda, J., 140, 141, 239 Fenstermacher, J. D., 148, 152, 238 Freeman, H. C., 91,120 Fenwick, J. C., 70, 119, 208, 238 Freeman, R. H., 242 Ferguson, H. W., 17, 26, 27, 48 Freemont, L., 96,120 Ferguson, R. A., 277, 292,299 Friberger, P., 236 Fernandez, J., 77, 79, 101, 103, 115, 119, Friche, H., 49 120, 127 Fritsche, R., 265, 266, 292, 296, 294, 297 Ferraris, R. P., 110, 119 Frohlich, E. D., 252 Ferrige, A. G., 339 Fromm, P. O . , 9, 53, 141, 169, 250 Festa, E., 50 Fryer, J. N., 253 Fey, F., 16, 18, 19, 40, 48 Fuchs, D. A., 109,120,265, 290, 292 Fiandt, J. T., 47 Fuchs, E., 243 Fidone, S. J., 412, 414, 422 Fugelli, K., 13,49, 143, 154, 162,239,243, Field, M., 246 252

434

AUTHOR INDEX

Fujii, T., 15, 22, 24, 27, 31, 33, 37, 40, 49 Fujimaki, Y., 17, 49 Furness, J. B., 334, 336, 339 Furukawa, K., 114, 123

G Cabrielsen, A. E., 49

Gaddum, J. H., 327,336 Gagnon, A,, 236 Galante, R., 422 Galardy, R., 199, 207, 239 Galbreath, J., 45, 49 Gallaugher, P., 127, 291,297,420, 424 Galli, S. M., 195, 200, 224, 239, 248 Gallwitz, B., 335 Galvan, I., 52 Gannon, B. J., 269,292,307,336,347,348, 349, 383 Ganz, T., 28, 51 Garavini, C., 16, 17, 49 Garcia, R., 235 Garcia-Garrido, L., 100, 103, 105, 120 Garcia-Romeu, F., 291, 295 Gardner, G. R., 16, 49 Gatlin, D. M. 111, 132 Gaudet, M., 95, 96, 120, 128 Gautvik, K. M., 129 Geffard, M., 333 Genest, J., 235 Germain, P., 236 Gem, W., 59, 62, 68, 120 Gerst, J. W., 120 Gesse, J. M., 121 Gesser, H., 153, 154, 165, 240, 245, 309, 339 Gessner, G., 296 Ghosh, T. K., 247 Gibbins, I. L., 325, 329, 334, 336, 339 Gibson, A. P., 234 Gidholm, L., 22, 25, 27, 35, 36, 48 Gilbertson, P., 30, 49 Gillen, R. G., 11, 49 Gilles, R., 110, 115 Gill-Kumar, P., 281, 292 Gilmore, R. G., 60, 127 Gingerich, W. H., 161, 167, 170, 172, 173, 174, 175, 176, 177, 178,239 Ginley, S. A., 115, 291

Giordano-Lanza, G., 334 Glazova, T. N., 4, 5, 49 Goeddel, D. V., 243 Goetz, K. L., 217, 239 Golde, D. W., 39, 47 Goldstein, L., 13,49,84,85,115,120,127, 143,239,242,244 Golovina, N. A., 2, 49 Gong, B., 101, 104,120 Goniakowska-Witalinska, 13, 49 Gonzales, R., 51 Gonzalez, C., 412, 414, 422, 423 Gonzalez, R. J., 57, 120, 140, 165, 239 Good, R. A,, 18, 24,48,49 Goos, H. J. T., 62, 120 Gorbman, A., 74, 120, 128 Gordon, A. S., 54 Gordon, D., 381 Gordon, R., 298 Goresky, C. A., 176, 180, 234,248 Gorlin, A,, 233 Gorr, T., 10, 43, 49 Goswami, S. V., 124 Coven, B. A,, 33,49 Govyrin, V. A., 303, 307, 336 Gowenlock, A. H., 88, 89, 92, 120 Gozzelino, M. T., 120 Graham, J., 399,424 Graham, M. S., 111,112,120,253,295,415, 416,422,426 Granger, D. N., 180, 252 Grant, B. F., 120 Gras, J., 120, 142, 159, 239, 248 Gratzek, J. B., 49 Grau, E. G., 60, 69, 120 Craves, S. S., 30, 49 Gray, C. J., 203, 207, 209235, 239 Greef, K., 302, 336 Green, A. A., 340 Green, C. W., 344,383 Green, J. M., 117, 236 Greenlee, A. R., 30, 49 Greenwald, J. E., 245 Greenway, C. V., 177, 188, 189,239 Greer, I. E., 88, 123 Greger, R., 250 Greiner, F., 339 Grierson, C., 237 Griffith, R. W., 4, 53, 86, 109, 121 Griffith, S. C., 309, 311, 318, 334, 336

435

AUTHOR INDEX Grimaldi, M. C., 47, 52 Grimin, A. S., 33, 49 Grodzinski, H., 2, 3, 49 Grove, D. J., 279, 295, 296 Grubb, S. A., 130 Gudefin, Y., 120,239 Guern6, J. M., 122 Gunning, M. E., 235 Gunter, G., 251 Guppy, M., 118 Gupta, 0. P., 142, 160, 162, 185, 240 Gustavsson, S., 236 GutiCrrez, J., 59, 63, 73, 74, 77, 79, 115, 119, 121,127 GutiCrrez, M., 4, 49 Gutowska, J., 235 Gutwirth, E., 233 Guyton, A. C., 139,166,182,188,189,240, 244

H Hackney, C. M., 113 Hadek, R., 307,333 Haedrich, R. T., 239 Hagiwara, K., 53 Haider, G., 17, 18, 49, 88. 95, 121, 129 Haines, H. G., 51 Hainsworth, R., 189, 240 HQkanson, R., 336 Hall, J. E., 193, 240 Hall, S. J . , 46 Hall, T. R., 292 Hamilton, C. H., 53 Hamilton, J . W., 338, 340 Hamilton, R., 1 1 7, 237 Hanke, W., 137, 141, 142, 143, 160, 162 163, 185, 212, 240,232,233,252 Hansen, S. P., 154, 165, 240 Hansen, V. K., 6, 7, 49 Hanssen, R. G. J. M., 119 Hansson, T., 293 Hanyn, I., 114 Hanyu, I., 123 Hara, A., 117, 124 Hara, T. J., 426 Hardig, J., 8, 49 Hargens, A. R., 181,182,183,184,185 186, 240

Harrington, J. P., 10, 53 Harris, J. E., 32, 49, 52 Harris, T. O., 236 Harshbarger, J. C., 44, 49 Hart, B. B., 263, 293 Hart, S., 52 Hartvigsen, R. E., 49 Hasegawa, S., 122,123,207,211,212,241 Hasegawa, Y., 242 Hashimoto, K., 183, 254 Haswell, M. S., 125 Hata, M., 50 Hathaway, C. B., 266, 268, 269, 270,293 Hattingh, J., 2, 53, 250 Hausdorff, W. P., 294 Haussinger, D., 243 Haux, C., 112, 113,114 Haversein, L. S., 89, 121 Hawke, D., 333 Hawkins, M. F., 322, 338 Hawkins, R. I., 49 Haya, K., 119 Haynes, L., 30, 49 Hayton, W. L. 233 Haywood, G. P., 85,121,293 Hazon, N., 114, 196, 200, 205, 206,233, 237, 240,247,251 Heath, A. G . , 418, 425 Hedrick, M. S., 281,282,293,415,416,422 Heiser, J. B., 125 Heisey, S. R., 173, 240 Heisler, N.,58,84,115,121,122,150,161, 240,415,422 Helle, K. B., 385 Heming, T. A., 293,421 Hemre, G-I., 73, 121 Henderson, I. W., 193, 194, 195, 196,200, 202, 204, 206, 207, 212,235, 240, 242, 247, 335 Henderson, N. E., 59, 68, 69, 97, 99,132 Hendricks, A. C., 125 Henning, M., 256, 293 Hentschel, H., 237 Herrera, E., 116 Herrera, F. C., 248 Hetrick, F. M., 53 Hettler, W. F., 238 Heustis, W. H., 57, 126 Hevesy, G., 9 , 4 2 , 4 9 Hew, C. L., 117

436 Hibiya, T., 181, 251 Hickman, C. P., 59, 60, 131 Hickman,C. P., Jr., 145,147,148,151,155, 163, 241 Hidaka, M., 423 Hieble, J. P., 332, 335 Hightower, J. A., 17, 49 Hildemann, W. H., 25, 31, 32, 49, 52, 53 Hille, S., 56, 86, 113, 121 Hill, R. M., 185, 242 Hilmy, A. M., 183,241 Hilton, J. W., 69, 73, 77, 121, 124 Himick, B. A., 69,121 Hinds, K., 51 Hinegardner, R., 6, 50 Hine, P. M., 3, 5, 6 , 17, 18, 19, 21, 26, 28, 29, 49, 50 Hines, R. S., 16, 23, 34, 50 Hinuma, A., 30, 50 Hipkins, S. F., 186, 241 Hirai, H., 124 Hirano, T., 62, 66, 67, 115, 122, 123, 126, 129,130,133,207,211, 212,241,251 H i d , F. J. R., 89, 119, 183, 238 Hirikane, T., 53 Hirohama, T., 219, 220, 221, 252 Hlavov , V., 95, I22 Hoar, W. S., 107, 110, 122 HBbe, H., 109,113,122,131,141,142,148, 150, 157, 159, 165,241 Hochachka, P. W., 78, 84, 87, 113, 116,117,118, 126,129 Hodgkins, H. O., 47 Hoeger, U., 115,291 Hoffbrand, A. V., 39, 50 Hoffert, J. R., 171, 176, 177, 178, 241 Holcombe, R., 200, 205, 247 Holder, F. C., 62, 67, 122, 318, 319, 337 Holeton, G. F., 111, 122, 406, 422 Holey, J. H., 242 Holman, M., 334 Holmes, K., 296 Holmes, W. N., 106, 109, 122, 136, 139, 144, 145, 161, 163, 164, 168, 174,241 Holmgren, K. D., 291, 420 Holmgren, S., 267,296,304,307,314,315, 317,318, 319, 320,322, 323, 324, 327, 333, 334,336,337,338,339,340,341, 348, 349, 360,383,384,423 Holstein, B., 322, 337

AUTHOR INDEX

Holtz, P., 256, 293 Honma, S., 312, 337 Hontela, A., 59,61, 62,64,67,68,74,122, 127 Hopkins, C. L., 60, 123 Horimoto, M., 242 Horne, M. T., 34, 50 Hosaka, K., 245 Hoss, D. E., 238 Houston, A. H., 2,8,42.50,51,59,60,109, 111, 112, 123, 142, 144, 145, 148, 149, 151, 153, 154, 155, 156, 157, 158, 159, 164, 165, 166, 171,241,242,244 Howells, J. E., 345, 385 Hoyer, H., 2, 3, 49 Huber, W. G . , 242 Huddart, H., 310, 338 Hudgel, D. W., 281,293 Huehns, E. R., 42,48 Huggel, H. J., 169, 242 Huggins, C. G., 214, 249 Hughes, G. M., 13,50,242,267,293,377, 380,383, 416,422 Hughes, K., 240 Hughes, S. P., 345, 385, 403, 424 Huidobro-Toro, J. P., 338 Humphrey, C. S., 322, 337 Hunn, J. B., 88, 123 Hunt, E. P., 47, 51 Hunt, T. C., 52 Hureau, J. C., 3, 50 Hurst, C. H., 345, 384, 403, 423 Huxley, V. H., 181, 242 Hyde, D. A,, 263, 265,293 Hyder, S. L., 26, 50 Hyder Smith, S., 34, 50 I

Ichinohe, K., 243 Idler, D. R., 61, 91, 120, 123, 127 Ignarro, L. J., 332, 337 Ihara, K., 54 Iida, S., 50 Iigo, M., 68, 123 Imai, M., 246 Ince, B. W., 63, 130 Ingram, G . A., 3, 33, 50, 88, 113 Inouye, A., 214,242

437

AUTHOR INDEX

Iorio, R. J., 50 Irving, L., 401, 422 Isaacks, R. E., 11, 12, 50, 51 Isaia, J., 137, 140, 154, 234, 242, 276, 293 Ishii, K., 411, 422 Ishii, S., 122 Ishizeki, K., 17, 18, 50 Isoda, M., 17, 49 Itatsu, N., 243 Itazawa, Y., 177, 191, 254. 300 Itoh, S., 123 Iuchi, I., 8, 10, 50 Ivanova, N. T., 2, 18,50 Iwama, G. K., 58, 123,265, 272,291, 293, 421 Iwata, M., 122, 298 Izquierdo, J., 347, 383

Johnston, I. A., 78, 123 Jonas, L., 6, 51 Jones, C. E., 240 Jones, D. R., 284,293,294,349,380,384, 386,399,405,406,415,422,423,425 Jones, I. C., 235 Jones, J. R. E., 372, 384 Jonsson, A.-C.,256,258,259,293,322,330, 333,334,337 Jordan, D., 369, 384 Jordan, H. E., 2, 18, 19,21, 23, 24, 36, 40, 42,50 Jordan, R. E., 50 Jgirgensen, J. B., 309,337 Jgirgensen, P. E., 89, 126 Joseph-Silverstein, J., 51 Jotisankasa, V., 240 Juch, P. J. W., 375, 382, 384

J

Jackson, A. J., 141, 242 Jackson, B. A., 235 Jackson, L. L., 54 Jacobs, M. H., 12, 50 Jaeger, J., 47 Jakowska, P., 15, 50 Jalabert, B., 114, 119 Janssen, A. H., 281, 282, 293 Jarvik, E., 2, 50 Javaid, M. Y., 16, 50 Jazul, A. P., 119 Jensen, J., 314,318,319,321,327,328,333, 337 Jensen, J. A., 33, 50 Jenson, F. B., 288,295 Joh, T. H., 294 Johansen, K., 14,50,54,125,240,242,344, 347,348, 349,372, 373, 383,384,399, 422 Johansen, L., 246 Johansson, M. L., 21, 29, 50 Johansson, P., 256, 288,292,293 Johansson-Sjobeck, M. L.: 19, 42, 48, 50, 51, 117 Johnels, A,, 381 Johnels, A. G., 336 Johnson, G. A., 63,128 Johnson, P. C., 179, 242 Johnson, R. G., 258,293

K

Kablitz, C., 253 Kah, O., 335 Kaiser, C., 296 Kajii, T., 51 Kakiuchi, Y., 242 Kanamori, A., 130 Kanesada, A., 14, 51 Kangawa, K., 243 Kanje, M., 48 Kann, G., 115 Kanwal, J. S., 358, 360, 384 Kao, M. H., 119 Kaplan, K., 46 Karakida, T., 247 Karlsson, G., 236 Kashiwayanagi, M., 426 Kasperat, H., 336 Kasuya, Y., 252 Kataoka, K., 242 Katayama, T., 127, 133 Katchen, M. S., 315, 316, 338 Katz, S. A., 244 Katz, Y., 74, 75, 123 Kaushik, S., 81, 123 Kavaliers, M., 322, 338 Kawauchi, H., 64,66,115,122,123,130, 251

438 Keen, J., 339 Keen, J. E., 6, 8, 42, 50, 51, 247 Keen, K., 290, 294 Kehati-Da, T., 47 KelCnyi, G., 16, 18, 51 Keller, N. E., 247, 296 Kellog, M. D., 163, 192, 199,242,247,339 Kenyon, C. J., 209,210,212,242 Ketelsen, U. P., 45, 54 Keys, A., 185,242 Kezuka, H., 62,68,123 Khosla, M. C., 197, 205, 208, 242 Kiang, K., 196, 200, 239 Kibota, J., 251 Kidd, C., 382 Kiessling, A., 296 Kikuchi, Y., 50, 242 Kilbey, V. K., 288, 290, 292 Kiley, C. W., 54 Kiley, J. P., 292 Kim, H. D., 11, 12, 50, 51 Kim, S. H., 221,242 Kime, D. E., 74,123 Kimmel, J. R., 325, 340 Kimura, B., 239 King, E., 131 King, P. A., 143, 242 Kinkead, R., 127, 265, 266, 267, 279, 280, 281,294,296,297,298,414,423,424 Kinter, W. B., 241 Kirsch, R., 335 Kirschner, L. B., 148, 150,234 Kisch, B., 4, 6, 14, 51 Kitzman, J. V., 137, 140, 150, 242 Kjellstrom, B., 47 Klawe, B. M., 51 Klawe, W. L., 9, 51 Kleerekoper, H. J., 393, 424 Klesius, P. H., 53, 89, 90, 123 Klitzman, B., 179, 243 Kloas, W., 220, 243 Klosterman, L., 74, 115 Kniaz, D., 250 KO, D., 122,124,130 Kobatake, Y., 426 Kobayashi, H., 197,200,205,206,210 211, 212,241,243,246,247,251,253,254, 300 Kobayashi, K., 32, 51, 53, 89, 124 Kobayashi, Y., 63, 74, 124

AUTHOR INDEX

Koehring, V., 15,51 Kohama, Y., 199, 243 Koh, G. Y., 242 Koke, J . R., 309, 338 Kokubu, F., 51 Kolar, Z., 131 Koller, K. J., 218, 243 Kondo, H., 298 Konishi, J. I., 418, 423 Kopin, I. J., 261, 294 Koss, T. F., 59, 60, 109, 111, 123 Koyama, T., 242 Krantz, G. E., 11, 53 Kremers, J. W. P. M., 358, 384 Kressin, N. A,, 293 Kreutzmann, H. L., 6, 41, 51 Kroglund, F., 114 Kubota, J., 115, 122 Kuhn, E. R., 60, 111,124 Kullman, D., 237, 247, 339 Kullman, D. E., 242 Kumagai, K., 50 Kumar, S., 403, 423 Kummer, W., 329,338 Kunwar, 247 Kurihara, K., 426 Kusakabe, T., 422 Kustafa, S., 27, 52

L

Labedz, T. 119 Lachenmayer, L., 333 Lacy, E. R., 195,243 Lagerwerf, A. J., 47 Lagios, M. D., 197, 243 Lahiri, S., 117, 237, 295 Lahlou, B., 212,231, 233,237, 247 Laidley, C. W., 57, 58, 59, 60, 111, 124 Lake, C. R., 293 Lam, T. J., 70, 119, 131 Lamba, V. J., 70, 124 Lambersten, G., 121 Lance, V., 340 Lane, H. C., 8,51,242 Lang, F., 137, 141, 243 Lang, S., 140, 142, 161, 162, 234 Lange, F., 143, 162,243

439

AUTHOR INDEX

Langford, G., 47 Langille, B. L., 384 Larsen, L. O., 74, 123, 124 Larsson, A., 4, 42, 50, 51, 100, 101, 102, 103, 1 1 7, 124 Larsson, L.-I., 330, 338 Lass, Y., 334, 340 Latif, A. B., 382 Laufer, M., 248 Laurell, S., 102, 124 Laurent, D. J., 248 Laurent, P., 265, 267, 284. 296, 298, 305, 307,311,333,336,337,338,340, 348, 349,384,401, 403, 405,422,423 Laurs, R. M., 171, 173, 174,243 Laut, W. W., 177, 188, 189, 239 Lawrence, J., 130 Lear, S., 250 Leatherland, J. F., 57, 58, 59, 68, 69, 111, 118,121,124 Le Bras, Y. M., 263, 267, 288, 294, 296 Le Bras-Pennec, Y., 293 Lederis, K., 62, 67, 122, 124, 130 Lee, C. M., 335 Lee, J., 186, 226, 228, 229, 243 Lee, R. F., 125 Lee, T. D., 248 Leeuw, R. D., 120 Lefkowitz, R. J., 288, 294 Leger, C., 120 Legler, D. W., 31, 33, 51 Lehrer, R. I., 28, 51 Leijsne, B., 47 Lenfant, C., 14, 50,422 Lennard, R., 310, 338 Leont’eva, G. R., 303, 307, 336,338 Leray, C., 10, 46, 120, 128, 142, 243, 311, 335 Lester, R. J. G., 16, 17, 18, SO, 51 Lever, A. F., 236 Levings, J . J., 351, 355, 356, 384 Levy, M. N., 381,384 Lewander, K., 103, 117,124 Lewander, T., 256,294 Lewicki, J. A., 244 Lewis, D. H., 47 Libey, G. S., 54 Libouban, S., 358, 386 Lidman, U., 117 Lie, O., 121, 129

Lin, R. J., 114, 133 Lincoln, J., 317, 338 Lind, J., 48 Lintlop, S. P., 75, 124 Lipke, D., 247 Lipke, D. W., 194,196,199,207,214,215, 216,217,237,243,247 Litman, G. W., 3, 31, 51 Liu, V. K. Y., 247 Lloyd, R., 203, 244 Lobb, C. J., 48 Locket, N. A,, 21, 39, 51 Lockner, D., 49 Loesch, A., 338 Logan, A. G., 183,244 Lone, K. P., 16, 50 Longtin, E. J., 53 Lopez, J., 423 Lopez-Barneo, J., 411, 423 Lbpez-Lbpez, J., 411,423 Loretz, C. A., 137, 140, 143, 160, 244 Love, R. M., 88, 101, 102, 103, 115, 124 Low, P. S., 80, 124 Lowe, D. G., 243 Luedtke, R. J., 131 Luiten, P. G. M., 375, 382, 384 Lukomskaya, N. J., 345,384 Luly, P., 46 Lundblad, 48 Lunde, L. G., 246 Lunder, T., 52 Lundgren, O., 335 Lundin, 314, 317, 318, 319, 320, 322, 338 Lundqvist, M., 336 Luquet, P., 81, 123 Lutz, B. R., 345, 347, 360, 384, 401, 403, 423 Lutz, P. L., 106, 109, 118, 124, 142, 148, 154, 155, 159,244 Lykkeboe, G., 298

M

Maack, T., 219,244 MacArthur, J. I., 27, 51 Macdonald, C., 340 MacDonald, R. J., 216, 244 Macey, D. J., 52

440 MacGregor, R., 111, 96, 101, 117 Macho, P., 326, 338 MacKenzie, D. J., 84, 124 MacKenzie, D. S., 91, 125 Mackie, K., 236 MacLatchy, D. L., 68, 69, 118 MacPhee, A. A., 252 Maddalena, D. J., 417, 425 Madden, J. A., 241,242 Madey, M. A., 197,200,203,204,207,244, 246 Madsen, S. S., 141, 143, 244 Maes, F. W., 422 Maetz. J., 150, 159, 244, 293 Mahadevappa, V. G., 129 Mainwaring, G., 15, 19, 51, 52 Makos, B. K., 79, 125 Malone, S., 297 Malvin, R. L., 186, 193,212,226,228,229, 234,236,243,244 Mandolfino, M., 334 Mangum, C. P., 50, 109,125 Manning, M. J., 3, 51 Manning, R. D., Jr., 166, 240, 244 Marchalonis, J. J., 3, 31, 32, 33, 51 Marchant, T. A., 59, 60, 66, 125 Margolius, H. S., 214, 244 Marino, D., 334 Marquez, E. D., 95,125 Marshall, A. H., 345, 384, 403, 423 Marshall, J. M., 372, 384 Martelli, P., 49 Martin, B., 90, 115, 125 Martin, J. F., 242 Martinex, I., 50 Martins, J. M., 52 Marttila, 0. N. T., 289,290,294 Mashiter, K. E., 10, 51 Masoni, A., 137, 140, 154,234,242 Mata, M. I., 248 Mathers, J. S., 109, 125 Matsuda, H., 423 Matsumoto, S., 243, 295 Matsuo, H., 243 Mattisson, A., 4 , 7 , 8 , 19,21,22,24,25,26, 36, 39, 42, 51, 54 Matuo, M., 126 Maubras, L., 120 Mawdesley-Thomas, L. E., 2, 49 Maxime, V., 298

AUTHOR INDEX

Mayer-Gostan, N., 63, 73, 125 Mazeaud, F.,71,72,76,104,125,261,294 Mazeaud, M., 248,293,297 Mazeaud, M. M., 71,72,76, 104,125,259, 261, 273,294 McCarthy, J. E., 140, 149, 167, 168, 244 McConnell, F. M., 143, 244 McConway, M. G., 131 McCrohan, C. R., 113 McCumber, L. J . , 25, 30,49, 51 McDonald, A. H., 364, 384 McDonald, D. G., 57, 110, 120, 125, 132, 140, 141,239,253,263,283,294,296, 298,426 McDonald, J. K., 339 McDonald, T. J., 340 McEnroe, G. A., 244 McEnroe, M., 295 McFarland, W. N., 107, 125 Mcfarlane, N. A. A,, 150, 244 McGeer, J. C., 123 McGregor, G. P., 334 McGregor, K. H., 295 Mclntyre, R. H., 132 McKay, A. H., 125 McKay, M. C., 97, 98,125 McKay, W. C., 76, 78, 105,129 McKeever, A., 240,242 McKenzie, D. J., 279, 281, 294, 405, 406, 423 McKeown, B. A., 62,66,67, 110,114,118, 130,132 McKim, J., 42, 51 McKim, J. M., 47 McKinney, E. C., 27, 30, 49, 51 McLain, L. R., 53 McLean, R. M., 97,119 McLeay, D. J., 132 McLeod, T. V., 41,42,51 McMahon, B. R., 122, 148, 165, 241,253, 426 McMahon, R. F., 236 McMaster, D., 124 McNabb, R. A,, 241 McVean, A,, 333 McVicar, A. J., 244 McWilliam, P. N., 382 Mead, J. F., 103, 128 Mearow, K. M., 142, 144, 148, 153, 154, 155, 156, 158, 164,241,244

AUTHOR INDEX

Mecklenburg, Cv., 383 Meghji, P., 310, 338 Meier, A. H., 58, 125, 251 Meiniel, A., 122 Meisheri, K. D., 208, 227, 228, 231, 243, 24 7 Mense, D., 297 Merchant, E. B., 53 Metcalfe. J. D., 115, 260, 265, 275, 276, 287,291,294,305,338,348,349,372, 380,384,385 Meyer, D. S., 236 Meyer, R. K., 116 Meyn, E. L., 131 Michel, C., 31, 51 Michelson, M. J., 345, 384 Michener, M. L., 245 Mikeler, E., 236 Miles, H. M., 242 Miles, M. S., 131 Milgram, S. L., 339 Milhaud, G., 120 Milhorn, D. E., 292 Millar, D. A,, 2, 52 Millard, R. W., 240 Miller, N. W., 48 Miller, W. R., 56, 77, 88, 89, 125 Milligan, C. L., 71, 72, 88. 125, 126, 140, 141, 142, 148, 150, 151, 153, 154, 156, 158, 159, 162, 164, 165, 166, 169, 191, 238,245,253,263,265,283,288,294, 29,5 299 Millot, J . , 21, 39, 51 Mills, G. L., 98, 99, 126 Milne, R. W., 235 Milsoin, M. K., 392, 405, 406, 423 Milsoin, W. K.,281,293,295,386,395,399, 400, 401,405, 406, 411, 412, 413, 414, 421,422,423,425 Mimura, T., 243 Minamino, N., 243 Mione, M. C., 307, 309, 332,339 Mishra, N., 47, 236 Miwa, I., 250 Miyazaki, T., 18, 35, 51 Mizogami, S., 245, 251 Mizrahi, J., 335 Modica, A,, 129 Moghimzade, E., 341 Moitra, A,, 247

44 1 Mokashi, A., 295 Mollenhauer, H. H., 47 Moller, D., 11, 51 Mommsen, T. P., 77,81,86,126,128,130, 132,297 Moncada, S., 339 Montoro, R., 116 Moody, E. J., 236 Moon, T. W., 73, 77, 101, 116, 126, 142, 155, 160, 164,233,245,252,297,299, 335 Morgan, A. K., 132 Morgan, M. R., 10, 51 Morita, Y., 358, 360, 385 Morris, J., 325, 326, 327, 339 Morris, J. L., 326, 329, 334, 336 Morris, R., 109, 127, 140, 149, 168, 173, 183,235,244 Morrison, D. C., 243 Morrow, W. J. W., 19, 52 Mosley, W., 235, 240 Motais, R., 276, 277, 279, 283, 289, 291, 292,295 Mott, J. C., 401, 423 Moukhtar, M. S., 120 Moyes, C. D., 233 Mudge, S. M., 79, 80, 126 Muir, J. S., 131 Mulchay, M. F., 52 Mulligan, E., 281, 295 Munger, R. S., 113, 141, 142, 147, 148, 151, 153, 154, 155,245,253,297,426 Muhoz-Chapuli, R., 120 Munroe, A. L., 48 Munshi, J. S. D., 140, 148, 151, 159, 165, 247,250 Munt, B., 100, 101,119 Mura, T., 11, 52 Murad, A., 27, 34, 52 Murai, T., 80, 126 Murakawa, S., 33,49 Murphy, P., 142, 155, 159, 164, 245 Murphy, R., 334, 339 Murphy, T. M., 52 Murray, C. K., 28, 52 Mustafa, S., 34, 52 Mustafa, T., 309, 337 Mutt, V., 340 Myhrberg, H., 383 Mykleburst, R., 385

442

AUTHOR INDEX

N

Nagahama, Y., 123, 130 Nagano, H. 183,245 Naito, N., 123 Nakagawa, H., 49 Nakai, Y., 123 Nakajima, K., 251,252 Nakajima, T., 250 Nakamura, H., 244 Nakamura, S., 241, 252 Nakano, T., 63,126, 265,268,295 Nardini, V., 334 Narkates, A. J., 247 Naruse, M., 252 Nasea, S. S. T., 247 Nash, K. A., 29, 52 Nash, N. T., 246 Navarro, I., 121 Nawa, T., 50 Nayler, W. G., 345, 385 Needleman, P., 217, 218, 245 Neelissen, J. A. M., 119 Negri, L., 341 Nehls, M., 249 Nekvasil, N. P., 261, 263, 265, 266, 272, 273,274,275,282,295 Nelson, J. S., 2, 3, 51, 241 Nemeth, A., 16, 51 Nemhauser, I., 47 Ness, S., 121 Neumann, P., 122 Newcomb, E. W., 111, 241 Newsholme, E. A., 77, 79, 101, 102, 103, 105,133 Ng, T. B., 61, 123 Nibbio, B., 116, 263, 292 Nichols, D. J., 142, 145, 147, 150, 151, 157, 163, 167, 170, 171, 172,245 Nicol, J. A. C., 302,303,304,305,339,345, 348,385 Nicoll, C. S., 62, 66. 67, 126 Niedermeier, W., 46 Nielsen, A. M., 293 Nielsen, K. E., 153, 165, 245, 309, 339 Nielsen, N. O., 17, 48 Niema, J., 408, 424 Nieuwenhuys, R., 350,351,358,384,385, 386, 419. 424 Nigrelli, R. F., 54, 246

Nikinmaa, M., 3,6,9,11, 12, 14,47,52,57, 128, 169, 191,245,276,277,279, 283, 288,289,290, 294,295,298 Nilssen, K. J., 119, 238 Nilsson, G. E., 295 Nilsson, S., 4, 37, 48, 233, 256, 258, 259, 260,261,263,265,266,267, 268, 269, 270,273,274,275,276,279,283, 284, 291,292,293,294,295,296,297,298, 299,302,303,304,307,313,314, 315, 319,327,333,335,337,338,339,341, 345, 347, 348, 349, 350,384,385,387, 397,423,424 Nishi, K., 414, 424 Nishimura, H., 139,169,174,192,193194, 195, 196, 197,200,202,203,204,207, 208,210,242,244,245,246,251,254 Nishioka, R. S., 67, 120, 122, 126 Nobin, A., 333 Noe, B. D., 325, 339 Noeske, T. A, 59, 130 Nolly, H. L., 197, 246 Norimatsu, H., 130 Norris, H. W., 345, 385, 403, 424 Norton, V. M., 246 Norum, K. R., 105, 116 Nose, T., 81, 126 Nozaki, M., 123,130 Nussenzveig, D., 244 Nustad, K., 213, 214, 246

0 Obenauf, S. D., 50 Oddie, C. J., 234 Oduleye, S. O., 247, 311, 339 Oey, P. L., 358, 385 Ogasawara, T., 66, 110, 122, 126, 129 Ogata, H., 11, 52, 80, 126 Ogawa, M., 193, 195, 197,246,251 Ogilivie, R. I., 246 Ogilvy, C. S., 171, 182, 246, 422 O’Grady, S. M., 230, 231, 246 Oguri, M., 196, 240, 245, 246, 251 O’Harte, F., 335 Ohono, S., 6, 52 Ohta, T., 298 Ohyama, T., 126 Oide, H., 251

443

AUTHOR INDEX

Oikar, A., 47 Oikari, A,, 4, 53 Ojala, K., 408, 424 Ojka, J., 47 Oka, € I . , 243 Okabe, hl., 243 Okada, M., 131 Okafor, M. C. J., 311, 339 Okajima, Y., 414, 424 Okawara, Y., 202, 210, 211, 212, 246, 247 Okubo, J., 251 Olcese, J., 117 Oliver, J. A., 235, 240, 242 Oliver, J. R., 334 Ollevier, F., 116, 124 Olsen, N . J., 89, 126 Olson, K. R., 162, 167, 172, 173, 174, 175, 176, 178, 186, 187, 189, 192, 194, 196, 199,204,207,208,214,215,216,217, 219, 221, 224,225, 226, 227, 228, 229, 231,236,237,239,242,243,247,261, 263, 265,266, 272,273, 274, 275, 282, 292, 295, 332, 338, 33.9, 401, 402, 424 Oparil, S.,243, 247 Opdyke, D. F., 172, 186, 190, 200, 205, 206, 207, 235,242, 247, 265, 271, 296 Orice. G . C., 247 Orimo, H., 63, 126, 133 Om, L. D., 203, 244 Orstavik, T. B., 246 Osborne, P. B., 339 Ostlund, E., 315, 316, 327, 336, 339, 381 Oswald, W., 336 Otani, M., 126 O’Toole, L., 335 O’Toole, L. B., 231, 240 Ottolenghi, T., 128 Ottonello, I., 341 Ourth, D. D., 32, 52 Owens, D. W., 120 Owman, C., 341 Ozawa, M., 243 Ozon. R., 115 P

Page, I. H., 340 Page, M., 22, 25, 26, 52 Paintal, A. S., 417, 424

Palmer, R. M. J., 332,339 Pandey, B. N., 192, 247 Pandey, P. K., 169, 236, 247 Pang, P. K. T.,69,121,125,127,197,247, 248 Pang, R. K., 69, 127,247 Paquette, T. L., 128 Parish, N., 19, 52 Parker, N. C., 71, 117 Parker, W. N., 5, 14, 21, 52 Parry, G., 142, 165, 248 Part, P., 290, 296 Parten, B., 248 Passow, H., 284, 298 Pasztor, V. M., 393, 424 Pati, A. K., 52 Patlak, C. S., 148, 152, 238 Paulencu, C. R., 127 Pawluk, M. P., 123 Payne, J. A., 238 Pearce, R. H., 182, 234 Pearson, M. P., 58,127, 177, 191, 248 Pedersen, R. A., 6, 52 Pedro, D. N., 47 Pendelton, R. G., 260, 296 Pennec, J.-P., 267, 293 Pennec, Y., 298 Percy, L. R., 52 Peres, G., 248 Perez, J., 121 Perez, M., 182, 184, 185, 240 Perez, R., 338 Perks, A. M., 214, 237 Perlman, D., 84, 84, 127 Perlmutter, A., 54 Perrier, C., 183, 239, 248 Perrier, H. 120, 183, 239, 248 Perrott, M., 240 Perrott, M. N., 67, 114,127, 206, 207, 248 Persson, H., 383 Perry, S. F., 71, 72, 78, 84, 127, 128, 130, 132, 176,248,263,265,266,267,269, 271,272,275,276,277,279,280,283, 284,285,286,287,290,293,294,296, 297, 298,299, 414, 415, 423, 424 Peter, R. E., 58, 59, 60, 61, 62, 64, 65, 66, 70,116,122,125,127,130 Petersen, J. A., 422 Peterson, A. J., 11, 52 Peterson, M. S., 59, 60, 127

444 Pettersson, K., 276,297,339,348,349,385 Pettey, C. L., 50 Pettit, J. E., 39, 50 Peuler, J. D., 63, 128 Pevet, P., 122 Peyreaud, C., 297,340 Peyreaud-Waitzenegger, M., 279,282, 290,297 Phillips, M. I., 239 Phromsuthirak, P., 16, 35, 52 Pica, A., 19, 47, 52 Pick, J., 347, 385 Pickering, A. D., 58, 60, 70, 71, 109, 127, 130 Pickford, G. E., 120 Pieprzak, P., 387 Pierce, J. V., 246 Pietra, G. G., 234 Pitombeira, M. S., 52 Pityer, R. A., 167, 170, 172, 173, 174, 175, 176, 177, 178,239 Plakas, S. M., 127 Planas, J., 58, 60, 77, 103, 119, 121, 127 Playle, R. C., 280, 285, 297 Plaza-Yglesias, M., 162, 248 Plisetskaya, E. M., 63, 72,73,75,102, 103, 104,117,121,126,127,128,327,328, 338,339 Plytycz, B., 15, 29, 52 Poder, T. C., 332,340 Podhasky, P., 239 Poe, W. E., 132 Poeschl, B. A,, 47 Polak, J. M., 326, 334, 340 Polanco, M. J., 199, 204, 248 Pollara, B., 49 Pollatz, M., 122 Pollock, H. G., 128, 325, 338, 340 Poluhowich, J. J., 11, 52, 118 Poole, C. A., 417, 418, 424 Pope, J. A., 132 Potter, I. C., 4,22, 52 Potter, M., 53 Pottinger, T. G., 58,60, 70, 71,127,130 Potts, J. T. Jr., 117 Potts, W. T. W., 142,252 Pough, F. H., 125 Poulin, P., 124 Pouwels, E., 419, 424 Power, G. G., 248

AUTHOR I N D E X

Powers, E. B., 405, 424 Prack, M., 117 Pradhan, A. K., 41, 52 Pratt, R. E., 237 Preston, T., 252 Price, D. A,, 220, 221, 248 Priede, I. G., 349, 364, 365, 383, 385 Primmett, D. R. N., 191,248,265,279,284, 297 Protter, A., 251 Prunet, P., 66, 67, 69, 114, 122, 128, 133 Pulsford, A., 19, 52 Q

Quinn, J., 245 Quirion, R., 217, 249 R

Rabito, S. F., 213, 214, 248 Rach, J. J., 239 Racicot, J-G., 92, 95, 120, 128 Rafn, S., 39, 52 Railo, E., 57, 128, 245 Raison, R. L., 49, 52 Ralevic, V., 339 Ralph, C. L., 120 Rampe, D., 340 Rance, T. A., 59,128 Rand-Weaver, M., 62,128 Randall, D. J., 12, 53, 58, 71, 72, 84, 115, 124,127,128, 144, 191,200,203,248, 253,276,277,280,282,283,284,287, 291,292,293,294,295,296,297,298, 300, 343, 347, 348, 349, 362, 366, 371, 377, 379,383, 384,385,386, 403, 406, 415,420,423,424,426 Rankin, J. C., 233, 235, 244, 334 Ransom, W. B., 344,385 Rao, M. C., 246 Rapport, M. M., 311,340 Rasio, E. A., 176, 248 Rasmussen, J . B., 122 Ratcliffe, N. A., 2, 52 Ratha, 8.K., 85, 128 Rausch, A., 247 Rawitch, A. B., 338, 340

445

AUTHOR INDEX

Reader, J. P., 125 Reale, E., 195, 243 Recio, J. M. 248 Reed, R. K., 180, 182, 234, 248, 253 Reeve, J. R., 340 Regoli, D., 335 Rehfeld, J. F., 330, 338 Reid, C., 46 Reid, S., 290, 297 Reid, S. D., 245 Reinecke, M., 219,220, 221,248,249 Reinking, L. N., 212, 249 Reis, D. J., 294 Reite, 0. B., 312, 315, 316, 340 Renda, T., 341 Renfro, J. L., 250 Reznikoff, D. G., 14. 52 Reznikoff, P., 14,52 Rhodin, J . A,, 181, 249 Richter, C. J. J., 120 Riddell, J. H., 340 Riegel, J . A., 186, 249 Riete, 0. B., 238 Riggs, A., 11, 49 Ring, O., 296 Ristori, M. T., 265,267,298,311,337 340, 401,424 Ristow, S. S., 49 Rivier, J., 124 Roberson, B. S., 28, 46, 53 Roberts, B. L., 382,386,387 Roberts, J., 399,424 Roberts, J. L., 397,421, 424 Roberts, M. G., 333 Roberts, R. J., 48 Robertson, J. D., 100, 106, 109,128, 139, 140, 142, 157, 186, 249 Robertson, J. I. S., 236 Robineau, D., 51 Robinson, J. S., 103, 128 Rodger, H. D., 8, 52 Rodriques, K. T., 62, 128 Rogano, M. S., 283,294 Rogers, W. A,, 53 Rogers, W. T., 51 Rokaeus, A., 340 Roman, R. J., 139, 192, 236,249 Romano, L., 284,298 Romer, A. S., 424 Roper, S. D., 408, 412,424

Rosenfeld, M. J., 253 Rosengren, E., 333 Rosseland, B. O., 114 Rossi, G. G., 340 Rothe, C. F., 188, 189, 249 Rotmensch, H. H., 310,334,340 Roubal, F. R., 16, 17, 52 Rourke, A. W., 185,234 Rouse, J. B., 128,338,340 Rouzeau, J. D., 405,423 Rovainen, C. M., 416,424 Rowell, D. M., 397, 424 Rowley, A. F., 2, 15, 16, 19,22,23,25,26, 35, 51, 52 Roy, P. K., 247 Roy, Y., 122 Rubashev, S. I., 26, 52 Rubinstein, R., 334, 340 Ruhs, H., 237 Russel, T. R., 51 Russell, D. F., 374, 385 Russo, R. C., 131 Ryan, J. W., 214,247,249 Rybak, B., 346,385 Ryu, H., 242

S Sadig, T., 281, 295 Sadler, W. A,, 60, 123 Saetersdal, T., 348, 385 Saha, N., 85,128 Saini, S. K., 52 Saito, Y., 26, 36, 53 Sakakibara, S., 251, 252 Sakakura, Y., 50 Sakamato, T., 66, 129 Sakharov, D. A., 312,340 Salama, A., 290, 298 Salimova, N. B., 312,340 Sameshima, M., 133 Samson, W. K., 217,249 Sanchez, I., 6, 52 Sander, G. E., 214,249 Sandnes, K., 56, 87, 88, 89, 95, 129 Santer, R. M., 348, 386 Santos, A. J . G., 114 Santulli, A., 101, 129 Saper, C. B., 245

446 Sargent, J. R., 12, 47, 98, 129 Sargent, P. A,, 238 Sarot, D. A,, 54 Sasaki, K., 53 Satchel], G. H., 186, 189, 239, 343, 346, 347, 348,372,375,377,382,383,386, 393,394,397,401,417,418,424,425 Sauer, D. M., 95,129 Saunders, D. C., 3, 5, 8, 19, 26, 52 Saunders, R. L., 372, 386, 391, 393, 395, 406,425,426 Savage, A. G., 15, 17, 26, 52 Sawyer, M. K., 250 Sawyer, P. L., 418,425 Sawyer, W. H., 67,129,169,206,207,208, 234,246,250 Scarborough, R. M., 244 Scarpa, A., 258, 293 Schachter, M., 213,214, 250 Scheide, J. I., 230, 250 Scheuring, F., 295 Schiff, D., 244 Schiffman, R. H., 169,250 Schiller, P. W., 235 Schinina, M. E., 47 Schlotfeldt, H. J., 88, 129 Schmidke, J., 6, 52 Schmidt, W. E., 335 Schmidt-Nielsen, B., 141, 143, 148, 152, 155, 156, 157, 159, 160, 162, 163, 250 Schmidt-Nielsen, K., 422 Schmitt, E., 52 Schreck, C. B., 114,117 Schroeder, M. D., 122 Schulte, P. M., 113 Schumacher, R. E., 34, 53 Schwalme, K., 78, 129 Scicli, A. G., 214, 250 Scoggins, B. A., 234 Scott, A. L., 31, 53 Scott, A. P., 65, 99, 129 Scott, E. M., 10,53 Scott, M. J., 373, 383 Segovia, R., 248 Seibert, H., 363, 386 Seki, T., 214, 250 Seljelid, R., 46 Semmens, K. J., 51 Sessler, F. M., 244

AUTHOR INDEX

Seul, K. H., 242 Sezaki, K., 53 Shabalina, A. A,, 81, 131 Shabana, M. B., 48 Shahrabani, R., 47 Shakoumakos, C., 131 Shannon, D. C., 381 Sheard, P. R. 15, 46 Shearer, K. D., 139, 250 Shechmeister, I. L., 54 Sheldrick, E. L., 129 Shelton, E., 46 Shelton, G., 115, 374, 377, 384, 386, 415, 416, 422,425 Shen, S. T., 64, 133, 299 Shepro, D., 53 Sherburne, S. W., 4, 8, 17, 24, 25, 53 Sheridan, M. A., 96, 97, 99, 104, 105, 128, 129 Sheth, S., 251 Shier, D. N., 234,236,244 Shinohara, H., 53 Shipman, B. N., 415, 425 Shiozawa, D. K., 253 Shively, J. E., 333 Short, S., 344,346,347,348,365,366,386, 387,403,421,425 Shostak, S., 118 Shoubridge, E. A,, 78, 129 Shrivastava, A. K., 4, 53 Shub, C., 47 Shukuya, R., 245 Shuttleworth, T. J., 335 Sidon, E. W., 79, 129 Siegel, C. D., 54 Sigel, M. M., 46,49, 51 Silberberg, S. D., 340 Silva, M., 238 Silva, P., 230, 238, 250, 251, 340 Simon, R. C., 30,53 Simpson, K. L., 133 Simpson, P. A., 250 Sindermann, G. J., 11, 53 Singer, T. D., 101, 102, 129 Singh, M., 401, 425 Singh, N. K., 47 Singh, 0. N., 247 Sinha, N. D. P., 140, 148, 151, 159, 165, 171,250 Sinnenberg, H., 236

AUTHOR INDEX

Siret, J. R., 168, 250 Sjoerdsma, A., 298 Skibelli, V., 65, 129 Skidgel, R. A,, 219, 238 Skinner, E. R., 103, 115 Sleet, R. B., 192, 250 Sletten, K., 49 Slettengren, K., 48 Small, P. A., Jr., 47 Small, S. A., 310, 315, 317, 332, 340 Smart, G. R., 203,250 Smatresk, N. J., 280, 291, 390, 399, 400, 401,403,405,406, 408,411,415,416, 418,419,420,421,425 Smeets, W. J. A. J., 350, 351, 386 Smit, G. L., 36, 53, 250 Smith, A. M., 25, 30, 33, 38, 53 Smith, C. E., 41, 53, 131 Smith, D. G., 176,237,241,284,298,305, 340 Smith, D. S., 50 Smith, F. M., 406, 415, 425 Smith, G., 234 Smith, J. C., 374, 379, 383,385, 406, 424 Smith, L. S., 58, 130, 167. 169, 170, 171, 192, 250 Smith, M. A. K., 125 Smith, N. F., 237 Smith, S. B., 51 So, J. N., 208, 242 So, Y. P., 238 Sobin, S. S., 292, 383 Soderberg, U., 374, 383 Soivio, A., 4, 53, 128, 245 Sokabe, H., 193, 194, 195, 197,202,203, 245, 246,247,248,251 Sokolowska, W. P., 76, 129 Sokowska, M., 127 Solandt, D. Y., 422 Solandt, 0. M., 422 Solomon, R., 318, 321, 340 Solomon, R. J., 168,192,219,224,225,226, 227,228,229,230,238,250,251 Sonstegard, R. A,, 124 Sorensen, E., 385 Specker, J. L., 69,130 Spector, S., 256, 298 Speidel, C. C., 18, 40, 50 Spence, B., 140, 150, 163, 251

447 Spieler, R. E., 58, 59, 130 Spira, D. T., 16, 23, 34, 50 Spryer, K. M., 386 Spyer, K., 369, 384 Srivastava, A. K., 121 Srivastava, C. B. L., 401, 425 Stabrovskii, E. M., 298 Stacey, N. E., 65,130 Stahl, B., 40, 53 Stanford, G. G., 293 Starmach, J., 9, 53 Staurnes, M., 238 Stave, J. W., 28, 29, 53 Steele, A. M. C., 51 Steen, J. B., 277, 292 Steffensen, J. F., 279, 285, 295, 298 Stephens, G. C., 131 Stevens, E. D., 58, 88, 127, 130, 164, 177, 191,248,251,349,385,386 Stevens, J. D., 50 Stiller, R. A., 36, 53 Stobbe, H., 35, 53 Stockmann, P. T., 245 Stoff, J. S., 250, 340 Stokes, E. E., 19, 35, 53 Stolte, H., 233, 237,243 Strachan, P. D., 114 Strange, R. J., 71, 132 Suarez, R. K., 77, 130 Suess, U., 63, 74, 130 Sullivan, C. V., 117 Sulya, L. L., 48, 251 Summerfelt, R. C., 58, 130 Sumpter, J. P., 62, 71, 99, 116, 118, 128, 129,130 Sundararajm, B. I., 124 Sundby, A., 121 Sundell, G., 19, 37, 48 Sundell, K., 337 Sundler, F., 336, 341 Surgenor, D. M., 3 5 , 4 6 , 4 7 Sutterlin, A. M., 372, 386, 391, 393, 395, 406,425,426 Suzuki, K., 62, 64, 65, 66, 130 Suzuki, M., 244 Suzuki, R., 218,219, 220,251 Suzuki, Y., 15, 23, 34, 53, 181, 251 Swanson, P., 62,117, 123, 130 Sweeting, R. M., 66, 110, 130 Szabo, T., 358, 386

448

AUTHOR INDEX

Thomas, N. W., 53 Thomas, P., 125 Thomas, R. L., 243 Tachibana, T., 50 Thomas, S., 71,72,130,263,266,271,272, Tagawa, M., 126 Tagliafierro, G., 314, 315, 322, 340 285,286,288,290,292,296,298,299, Takada, K., 122 316, 340 Thompson, D’Arcy W., 344,385 Takagi, H., 46 Takahashi, A., 115,122,123,212,251,252 Thompson, V. W., 234 Takahashi, H., 133 Thomson, A. W., 52 Thorndyke, M., 249 Takahashi, K., 133 Thorndyke, M. C., 317,319,321,322,324, Takano, K., 124,133 Takao, T., 252 333,335,340,341 Takei, Y., 167,169,202,211,218,219,220, Thorpe, A., 63,130 221,224,226,228,230,241,243,251, Thorpe, J. E., 58, 60, 131 252 Thorson, T., 139, 140, 147, 149, 150, 161, 163, 168, 169, 171, 252 Talbot, C., 137, 141, 142, 252 Thorson, T. B., 109,120,131 Talmage, R. V., 70, 130 Tamaki, H., 252 Thurston, R. V., 84, 131 Tanaka, Y., 26,36,53,127,133 Thwaites, D. T., 319, 335, 340 Tibbling, G., 102, 124 Tan, C. H., 131 Tiemeier, 0. W., 291 Tang, Y., 71,130,265, 283,298 Timoshina, L. A., 81,131 Tarr, B. D., 233 Toews, D. P., 59, 60, 131 Tatemoto, K., 328, 340 Tolunay, H. E., 245 Tatner, M. F., 3, 50, 51 Tomlinson, K., 63, 126 Taugner, R., 236 Tomlinson, N., 265, 268,295 Tavassoli, M., 14, 53 Tomonaga, S., 32, 37, 38, 42, 51, 53, 54 Taylaur, C. E., 98, 126 Torcher, D. R., 97, 99, 121 Taylor, A. A., 247, 252 Taylor, A. E., 180, 181, 252 Tota, B., 233 Towes, D. P., 422 Taylor, D., 131 Taylor, E. W., 276,280,282,283,291,297, Towle, D. W., 125 345, 347, 350, 351, 355, 360, 361,365, Toyota, M., 423 366,369,371,372,374,375,377,378, Trauger, R. J., 51 380,382,384,386,387,408, 415,421, Tree, M., 236 Tremml, P. G., 246 425,426 Tretjakoff, D., 303,341 Taylor, H. H., 239 Trippodo, N. C., 189,252 Taylor, M., 238, 251, 340 Trombitsky, I. D., 2,49 Taylor, M. H., 60, 130 Taylor, S. M., 235 Trott, J. N., 387 Temma, K., 288,298 Truscott, B., 127 Tsujioka, T., 242 Teramoto, T., 243 Terlou, M., 120 Tsunoda, S., 46 Teshima, K., 51 Tsuyuki, H., 123 Tucker, V. L., 242 Tetens, V., 263, 265, 288,298 Tufts, B. L., 12,53,109,131,277,285,295, Tharp, T. P., 8, 51 298,299 Thibault, G., 235 Turner, A. H., 181, 186, 252 Thim, L., 327,335 Turner, J. D., 84,120,131,253,422, 426 Thoenes, G. H., 25,53 426 Thomas, E., 114 T

449

AUTHOR INDEX

U

Ubel, F. A,, 381 Uddnian, R., 319, 324, 336, 341 Udenfriend, S., 298 Uematsu, K., 314, 319, 341 Uemura, H., 219,220, 221, 241,243,252 Ulevitch, R., 243 Umminger, B. L., 76, 120, 131 Undritz, E., 4, 53 Ungell, A. L., 261,265, 273,274,292,299 Urena, J., 423 Urist, M. R., 109, 131 Utida, S., 251 Uva, B., 233 Uyeno, S., 131 V

Vaillant, C., 337 Val, A. L., 10, 53, 277, 299 Vallarino, M., 233, 314, 322, 341 Van Breeman, E. D., 241 Van Citters, R. L., 384 Van Coillie, R., 122 van den Thillart, G., 78, 131 van den Thillart, G. E. E. J., 131 Vander, A. J., 244 van der Boon, J., 80,131 van der Velden, J. A., 112, 131 VanderWeil, C. J., 130 Van Dijk, P. L. M., 288,299 Van Loveren, H., 53 Van Noorden, S., 334 van Pilsum, J. F., 86, 87, 131 van Waarde, A., 84, 131 Vatne, D. F., 114 Vendrely, R., 53 Venkatesh, B., 65, 70, 131 Verbeek, R., 78, 131 Verbost, P. M., 297 Verburg, K. M., 242 Veress, A. T., 236 Verhagen, M. A. W. H., 131 Vermette, M. G., 84,127,283, 286, 296, 297,299 Vernier, J. M., 97, 98, 99, 100, 114 Vethaak, D., 53

Vetter, S., 252 Vigna, S. R., 322, 330, 335, 340, 341 Villa, J., 228, 247, 339 Villena, A., 54 Vincent, B., 337 Vislie, T., 10, 53, 154, 239, 252 Vivien-Roels, B., 122 Vodienik, M. J., 117 Vogel, V. 0. P., 23, 53 Vogel, W. H., 292 Vogel, W. O., 252 Volkl, H., 243 Vos, J., 33, 53

w Waagbo, R., 129 Wagner, G. F., 62,132 Wagner, H. H., 236 Wahlestedt, C., 336 Wahlqvist, I., 237,269, 270,293,298,299, 349,387 Wain, J. M., 18, 19, 21, 29, 34, 49, 50 Wales, J. H., 2, 54 Walker, R. L., 9, 53, 122, 132, 253 Walker, T. J., 119 Walker, T. K., 116 Walsh, J., 127 Walsh, P. J., 78, 84, 85, 86, 126, 132, 142, 155, 160, 164,252,296,297,298,299 Walton, M. J., 81, 132 Walvig, F., 233 Ward, J., 237 Ward, J. W., 21, 35, 53 Ward, P. D., 50 Ward, R. L., 53 Wardle, C . S., 16,35,53,156,1.59,167,253, 283,299 Warner, J., 125 Warner, M. C., 253 Warr, G. W., 30, 32, 53 Watanabe, T. X., 251,252 Watson, D. E., 131 Watson, L. J., 16, 54 Watts, D. C., 84,85,132 Watts, E. G., 117 Watts, R. L., 84, 85, 132 Way, H. K., 372, 386, 393, 394, 425

450 Wayne, C., 338 Weber, L. J., 192, 250 Weber, R. E., 10, 43, 50,54 Webley, G., 128 Wedemeyer, G. A., 132 Weil, C., 114 Weinberg, S. R., 8, 14, 54 Weingarten. K. E., 238 Weinheimer, P. F., 46, 51 Weinreb, E. L., 16, 17, 22, 27, 54 Weisbart, M., 245 Weisel, G. F., 38, 54 Weiser, R. S., 47 Weiss, L., 40, 54 Wekerle, H., 45, 54 Weld, M. M., 253 Wells, R. M. G., 57, 78, 79, 87, 109, 113, 132,239,253 Welsh, M. G., 49 Wendelaar Bonga, S. E., 131 Wenderlaar Bonga, S., 119 Werner, C. S., 117 Werner, H., 117 Wesson, L. G., Jr., 235 West, T. G . , 113 Westenfelder, C., 219, 221, 225, 230, 233, 253 Wester, P., 53 Westermann, J. E. M., 16, 22, 46 Wharton Jones, T., 18, 54 Whatley, D. S., 49 White, A., 101, 103, 105, 132 White, €5. A., 59, 68, 69, 132 White, F. C., 186, 253 White, M. G., 46 Whitmore, D. H., Jr., 236 Wiig, H., 182, 253 Wilander, E., 336 Wilde, D. W., 247 Wilkes, P. R. H., 109, 122,132, 253 Wilkins, N. P., 4, 54 Williams, R. W., 253 Wilson, J. X., 253 Wilson, R. P., 81, 132 Wilson, S. W., 116, 126 Winchenne, J. J., 115 Wingfield, J . C., 90, 91, 132 Wingstrand, K. G., 6, 7, 39, 49, 52 Wintrobe, M. M., 4, 5, 14, 54 Wissing, J., 78, 132

AUTHOR INDEX

Withington-Wray, D. J., 345,351,355,356, 387 Wive], N. A., 53 Wohlschlag, D. E., 41,47,406, 421 Wojdani, A., 233 Wolomyk, M. W., 48, 119 Wolters, W. R., 6, 54 Wood, C. M., 71, 72, 78, 84, 85, 88, 113, 120,122,125,126,131,132, 140, 141, 142, 144, 148, 150, 151, 153, 154, 156, 158,159,162, 164, 165,166, 169, 191, 245,253,263, 265,266,267,272,275, 283,284,285, 287, 288294,296,297, 298,299,337,341,362,365,387,406, 415,416,422,426 Wood, J. G., 339 Wood, S. C., 54 Woods, R. J., 242 Woodward, J. J., 63, 71, 132 Wotherspoon, J., 49 Wrathmell, A,, 52 Wright, P. A., 75,84,85,128,132,133,159, 162, 164, 165,253,275,299 Wyman, L. C., 401,423 Y

Yada, T., 133 Yamada, C., 197,253 Yamada, S., 81, I33 Yamaguchi, K., 25, 53, 54,133, 247, 251 Yamamoto, K., 171,177,191,253,254,279, 300 Yamashita, S., 416, 418, 426 Yamauchi, A., 307,341 Yamauchi, H., 70,133 Yan, L., 117 Yanagisawa, T., 183,254 Yang, M. C. M., 248 Yasutake, W. T., 2, 54 Ye, X., 287, 300 Yevish, P. P., 16,49 Yoffey, J. M., 6, 14, 37, 40, 43, 53, 54 Yokota, S. D., 162,206,225,229,234,254 Yoshii, K., 416,418, 426 Young, G., 67,69,71,114,128,133 Young, J., 335, 340 Young, J. D., 48 Young, J. Z., 304,341,346, 347, 349,387

45 1

AUTHOR INDEX

Yousef, M. K., 139,237 Youson, J . H . , 9 , 5 4 , 7 5 , 7 9 , 1 1 8 , 1 2 5 , 1 2 9 , 132,133, 196,238,335 Yu, J. Y. L., 64, 133 Yu, M. L., 41, 54 Yunnis, A. A., 51

Z

Zaccone, G., 340 Zadunaisky, J. A., 230, 250

Zammit, V. A., 77, 79, 101, 102, 103, 105, 133 Zanjani, E. D., 41, 54 Zanny, S., 116, 121 Zanny, Z., 119 Zapata, A., 24,26,31,38,40,42,48,51,54 Zaugg, W. S., 53 Zebe, E., 78,132 Zeidel, M. L., 235 Zigler, M. G., 293 Ziyadeh, F. N., 242 Zohar, Y., 59, 60, 61, 64. 65, 119, 133 Zucker, A., 210,246,254

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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 Acanthgopagrus, B, 17 Acipenser, A, 17, B, 29, 38 A . breuirostris, 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 Amiu, 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. dqfenbachii, A, 41-42,44,46,53,60,

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 tristoechus, B , 169 Auxis A . rochei, A, 121 A . thazard, A, 121

67,69

A . japonica, A, 53, 58, 65, 11%118, B, 453

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

SYSTEMATIC INDEX

Bluefish, 211, see also Pomatomus salatrix Boreogadus saida, B, 91 Bowfin, see Amia calva 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 c; Cacharodon carcharias, A, 4, 120-123 Caiman crocodylus, B, 324 Callorhyncus millii, B, 142 Carassius C . auratus,A,46,47,227,246-249,253, 255, 271, B, 17, 32, 59-60, 66, 81, 149, 358,362, 397 C . carassius, B , 78, 268 Carcharhinus s p . , B, 107 Carp, A, 3, 12,50, 100, 105, 151,155,207, 253,255,278,282, B, 71,80, 104,290, 395, 397, 416, see also Cyprinus carpio crucian, see Carassius carassius grass, see Ctenopharyngodon idell Catastomus C . catastomus, A, 70 C . commersoni, B, 17-18, 74, 109, 150, 157, 165, 169 C . macrocheilus, B, 211 Catfish, A, 57, B, 4,35,401, see also Silurus meridionalis armored, see Pterygoplichthys channel, see Zctalurus punctatus glass, see Kryptopterus bicirrhis Centrophorus calceus, A, 160 Cephaloscyllium isabella, A, 168-170,174 Cetorhinus, B, 351 Chaenocephalus aceratus, A, 35,58, 67, 220,229, 256 Chalinura profundicola, A, 17 Champsocephalus gunnari, A, 277 Chanda, B, 3

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 . harengus, A, 4, B , 4,8,17, 24-25 Clupidae, A, 11 Cod, A, 247, B, 71,228,276,316,327,349 Atlantic, see Gadus morhua Coelacanth, B, 21,39,43,98, 197, see also Latimeria chalumnae Conger, B, 3 C . conger, A, 237 Coryphaenoides rupestris, A, 5 Cottus C . bairdi, B, 210 C . gobio, B, 9 C . poecilopus, B, 9 Crassius, B , 258 C . auratus, A, 3, 12,48,243,B, 210,212 C . crassius, A, 272 Crocodylus porosus, B, 324 Ctenopharyngodon idell, B, 64 Culpea pallasii, B, 94 Cyclopterus, A, 162 C . lumpus, A, 239, B, 4,79-80,206,207 Cyprinus carpio, A, 3, 13, 50-51, 54, 58, 99,150,227,239,271-272,277, B, 14, 17-18,25-26,34,68,82,150,160,162, 169, 191,204,268, 306, 310,372, 375, 39 1 D

Dasyatis, B, 37-38 D. sabina, A, 58, B, 80 Dicentrarcus labrar, A, 224,227,B , 59-60, 103, 105 Dogfish, A, 9, 19, 48, 114, 116-118, 160,

455

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 Anguzlla 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 Esox 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 Pleuronectesflesus 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 . olivaceus, 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 cuvieri, 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

456

SYSTEMATIC INDEX

Goldfish, see Carassius auratus Gonostomidae, B, 5 Goosefish, A, 280 Grayling, B, 8 arctic, see Thymalus arcticus Gourami, blue, see Trichogaster trichop terus 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 lctiobus, 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,

Katsuwonus pelamis, A, 5, 11, 21, 28, 35, 46, 54,55, 59,69, 105 113, 118, 121 124, 126,289, B, 76,78 Killijish, see Fundulus heteroclitus Kryptopterus bicirrhis, A, 188, 196-201, 206

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 cirrhatus Pacific, see Eptatretus stouti Hemilepidotus hemilepidotus, A, 46 Hemitripterus americanus, 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 Heragrammus, 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

I Icefish, A, 257,277 Antarctic, A, 239, B, 3

lctalurus, B , 17, 42 1. melas, B, 319 I . nebulosus, A, 3

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. jluviatilis, B, 4, 22, 26, 186, 194 L. japonica, B, 27, 33, 205, 220 L. lamottenii, B, 79 L. planeri, 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 jluviatilis 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

457

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 Leuciscus 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 Scomher 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 Tetrapterus audax white, see Tetrapterus albidus Maurolicus mulleri, B, 5 Megastomatobus 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 schlosseri Mugil cephalus, B, 101, 224 Mullet, stripped, see Mugil cephalus Mummichog, 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 Myoxocephalus 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 macropterus, B, 199

SYSTEMATIC INDEX

458 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

0 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, 68-69,72 0.mykiss, &8,12,14,19-21,23,29,31, 33,35,37-38,40-41,43-44,46-49, 52, 54, 60,62,63, 69, 71, 73, 94, 99-100, 102-103, 105, 113, 114, 116-119,162-163, 188,202-205, 208, 210, 221, 228, 232, 234, 236-238,243,245,254,280,283, 285-287, 291, B, 6, 8, 13, 17, 25, 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 Opsanus 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

P . borchgreuinki, A, 5,46,49,59,62,95, B, 174,363 Paracheannichtys charcocti, A, 281 Paralabrax sp. ,B, 76 Paralichthys lethostigma, B, 145, 150,163 Paranotothenia magellanica, A, 229 Parophrys uetulus, B, 84, 159, 164 Pelteobagrus fulvidraco, B , 221 Perca, A, 160, 162, 255, B, 40 P. jlauescens, A, 29,229,251, 254,256, 280,291 P.jluuiatilis, A, 254, B, 18,104,148,154, 155, 159 P. perca, A, 14 Perch, A, 3,50,51,63, see also Perca perca climbing, A, 145 spangled, A, 54 white, see Morone americanus yellow, see Perca flauescens Periophthalmodon schlosseri, B , 70 Petromyzon, B, 12 P. marinus, B, 9, 22, 31, 75, 108, 140, 149, 168, 173 Pickerel, see Esor niger Pike, see Esor lucius Pikeperch, see Stizotedion lucioperca Pipefish, see Syngnathus fuscus Piraruca, see Arapaima Plaice, A, 3, 63, see also Pleuronectes platessa Platichthys P.flesus, 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.jlesus, 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 reticdata, 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 salatrir, A, 211, B , 171, 404

459

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 americanus 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 Pseiidochaenichthyes, A, 59 Pseiidopleuronectes 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. rhina,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 thornback, see Raja claoata 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 rutilis, A, 272

S Salmo, A, 162, B, 42 S. clarki, A, 243 S . fario, B, 72 S . gairdneri,A,58, B,6,17,27,30,268,310 S . salar, A, 8,23,71,229,236, B, 65-66, 73, 9.1-92, 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 tshaw ytscha chum, see Oncorhynchus 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 marmoratus, 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

460 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, 205-206,218,220,260,310,315, 319,322, 345,351 S . stellaris, A, 19,49,57,58, 112, B, 322 Sea raven, A, 34, 35, 41, 47,48,49, 57, 60, 65, 115-117,220,221,224-225,232, 233,240,241,244,251-252,256,280, B, 78,92, 391, see also Hemitripterus americanus Sebastodes, A, 162 S. reberrimus, B, 94 Selache maxima, B, 351 Seriola S . grandis, A, 41, 46, 54 S . quinquerdiata, B, 171, 191 Shad, see Alosa sapidissima Sharks, B, 3. 37,40,45,205,228,230,417 blue, see Frionace glauca bull, see Carcharhinus bullhead, see Heterodontus carpet, see Cephaloscyllium isabella great white, see Cacharodon carcharias Greenland, see Somniosus microcephalus horn, see Heterodontus francisci lamnid, A, 20, 120, 123, 126 lemon, A, 221 leopard, see Triakis semifasciata longfin mako, see Isurus paucus mako, see Isurus oryrinchus nurse, see Ginglymostoma cirratum porbeagle, A, 121-123 Port Jackson, see Heterodontus portusjacksoni salmon, see Lamna ditropis shortfin mako, see Isurus oxyrhinchus tiger, see Galeocerdo cuvieri Shorthorn, see Myoxocephalus scorpius Silurus S. glanis, B, 45 S . glaris, B, 358 S . meridionalis, A, 56 Siphonostoma typhle, B, 4 Skate, A, 196, B, 18, 80, 325 arctic, see Raja hyperborea spiny rasp, see Raja kenojei

SYSTEMATIC INDEX

Smelt, see Osmerus mordax Solea vulgaris, A, 47 Sole, see Solea vulgaris lemon, see Farophrys vetulus; Fleuronectes microcephalus Somniosus microcephalus, B, 4, 13, 19,22 Spearfish longbill, see Tetrapterus pfluegeri short bill, see Tetrapterus angustirostris Sphryna lewini, B, 195 Sphyraena barracuda, B, 150, 171 Spicara chryselis, B., 77, 103 Squalus S . acanthias, A, 4,28,34,40-41,42,46, 50, 53, 58, 60,69,73, 111, 114, 144, 150, 155,227,231, B, 4, 77, 94, 140, 142, 149, 152, 157, 162, 168, 172, 190, 195-196, 205, 220, 224-225, 227, 265, 268, 270, 312, 319, 321-322, 324-325,327,351, 393 S . squatina, A, 4 S . suckleyii, A, 58, B, 315 Squatina aculeata, B, 315, 322 Stickleback, see Gasterosteus aculeatus Stizotedion lucioperca, B , 14 Sturgeon, B, 29, 37, 45 shovelnose, see Scaphirhynchus platorynchus Sucker, A, 70, see also Catastomus catastomus white, see Catastomus commersoni Sunfish, see Lepomis cyanellus bluegill, see Lepomis macrochirus pumpkinseed, see Lepomis gibossus Swordfish, see Xiphias gladius Symphysodon, B, 32 Synbranchus mannoratus, B, 150, 165, 399 Syngnathus fuscus, B , 6

T

Tautog, see Tautoga onitis Tautoga onitis, A, 229, 253 Tetaptusrus audas, B, 78 Tetrapterus T. albidus, A, 121 T . angustirostris, A, 121

46 1

SYSTEMATIC INDEX

T . audax, A, 121 T . ppuegeri, A, 121 Thresher, see Alopias uulpinus 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 blackfin, see Thunnus atlanticus black skipjack, see Euthynnus lineatus bluefin, A, 4, 120, 122, 127 bullet, see Auxis rochei frigate, see Auxis thazard kawakawa, see Euthynnus af&nis 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 ANG 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

463

464

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 RAS, 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 Bulhus 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 Catecholaniines 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, 112-113, 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, 389-426 central sensory areas of, B, 419-420 reflexes, B, 402 Carnitine palmitoyl (CPT), in cardiac metabolism, A, 225-230, 235, 236,53, 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

465 blood volume and, B, 191 and cardiac output, A, 64, 66 and cardiac rate, A, 48-53 circulatory levels, B, 263-267 and COz transport, B, 284-287 control of release, B, 269-272 degradation of, B, 260-263 diurnaUseasona1 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 2 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, 265-267, 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 also Nociceptors branchial O2 sensitivity, B, 405-410 carotid, B, 372 C02/pH receptors, B, 414-416 effects of hypoxia, B, 413 glomus cells, B, 411-412 and heart rate, B, 371-372 0 2 receptors, B, 404-414

466 pharmacological agents on, B, 413-414 transduction ( 0 2 receptors), B, 411-414 Cholesterol in blood, B, 98,99-101 migration and spawning, B, 100-101 Chromaffin 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 CreatinelCreatinine, 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-P-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

467

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 Enterochromaffin 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 ulso 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 fluids exercise and, B, 164 muscle fluid volumes, B, 157-160

photoperiod, pH, POZ, 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 Fin 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 GLP, see Glucagon-like peptide GLU, see Glucagon Gliicagon, B, 63, 72-73 Glucose, in cardiac metabolism, A, 223-236 Glucagon-like peptide, B, 63, 72-73 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

468

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

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

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 Hematocrit,B,3-4,167,173,174,178,179, atrial filling, A, 40-44 191 cardiac cycle, A, 24 and hemodynamics, A, 92-96 cardiac filling, A, 40,41-45 large vessel (LVH), B, 167 cholinergic control, A, 48-49 optimal, A, 92-94 chromaffin tissue in, A, 50-51 in secondary vascular system, A, 208 circulation (coronary), A, 20-24 Hemoglobin, types of, B, 10 efficiency of contraction, A, 33-36 Hemopoiesis, B, 39-42,43 electrical events, A, 24-26 organs of, B, 37-38 enzymes of muscle, A, 224-231, in peripheral blood, B, 41-42 252-257 stimulation of, B, 41 epicardium and endocarcium, A, Hemosiderin, B, 9,39 Heparin, B, 22,36 236-238 excitation-contraction coupling, A, Hepatic portal system, A, 162-164 267-304 pressures in, A, 162,163 innervation, A, 12-13;B, 344-350 Hepatic sphincter, A, 155 metabolism of, A, 219-266 Hexokinase (HK), in cardiac metabolism, morphometrics (tabled), A, 4-5 A, 225-231,235,237,246-247,250, myocytes of, A, 13-16,26,27 253 myoglobin in, A, 16,55 High energy phosphates, in heart muscle, nervous regulation, A, 282;B, 343-387 A, 219,224 0, supply, A, 34-36,66-73,222,225 Hindbrain, see Central nervous system pacemaker, A, 45,278,282; B, 346,360 HK, see Hexokinase performance, A, 238 HOAD, see 3-Hydroxylacyl CoA physiology of, A, 24-73 dehydrogenase

469

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-Hydroxytryptamine 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 (JC;), 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 Kallikrein-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 LDL/HDL, 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 Ca2+ 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

470

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 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

Myocytes, see also Heart, A, 13-16, 26 electrophysiology of, A, 286-287 Myofibrils 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

Na+- 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 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

471

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, 110-1 14 total (TPR), A, 110, 112-113 PFK, see Phosphofructokinase Phagocytosis, B, 27-28 Phenylethanolamine-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, B, 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 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, 307-311 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 Qio

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 (u),B, 180-181 Renal portal system, A, 161-162

472

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),ofmyofibrils, 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 venosus, 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 SRCa2+(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 (RJ, see also Peripheral resistance autonomic innervation and, A, 111 exercise and, A, 112-113 hypoxia and, A, 112-113 T

T, see Testosterone T,, T,, 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

473

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 Thermoregulation, A, 120, 126-130 Thronibocytes, 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 triiodothyronirie (TJ, 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 also 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, 318-32 1 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 also 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, 40-44 Viscosity of blood, A, 92-96 hematocrit and, A, 92-93 temperature and, A, 92-93 Vis-k-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

474

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