Table Of ContentPreface
The idea of editing a series of volumes on The Biochemistry and Molecular Bi-
ology of Fishes was born out of the present-day lack of a forum for state-of-the-art
review articles in this rapidly expanding field of research. On the one hand, researchers
and students in this area always find themselves combing the literature on general
(rat-dominated) biochemistry before discovering short and usually incomplete and
disappointing coverage of the situation in the piscine setting. On the other hand, the
rapidly expanding volume and quality of the primary literature in fish biochemistry
and molecular biology supply convincing evidence for a maturing field. This discipline
is no longer the younger sibling of rat or human biochemistry but has recently led to
a number of major conceptual breakthroughs; for this reason, and because its activ-
ity domain is sometimes nonoverlapping with 'mainstream' biochemistry, the field is
certainly ripe and ready for a review series of its own.
Comparative biochemistry and molecular biology and comparative physiology as
disciplines by definition use organisms as a special kind of experimental parameter
for probing general mechanisms and principles of function. In theory this approach is
relatively blind to phylogenetic boundaries, but in practise the realities of funding and
availability of experimental material greatly narrow the field of play. As a result, two
phylogenetic groups - the insects and the fishes - have over the last several decades
provided the bulk of the experimental data base in these disciplines. Interestingly,
although comparative biochemistry in many ways grew out of comparative physiol-
ogy, the growth and development of these two activities in the insect field have to
major extent proceeded along independent paths. By contrast, the comparative phys-
iology and biochemistry of fishes have not been so independent of one another and
the tendency has been for the former to envelope the latter. We believe that the cur-
rent conceptual developments in the fields as well as the simple logistics of dealing
with massive data bases make this the right time for the reality of independence to
match the perception of independence, which we feel is another important rationale
for this review series.
Our goal is to provide researchers and students with a pertinent information source
from theoretical and experimental angles. To be useful to students, theoreticians,
and experimentalists alike, contributing authors are urged to emphasize concepts
as well as to relate experimental results to the biology of the animals, to point out
controversial issues, and to delineate as much as is possible directions for future
research.
Peter .W Hochachka
Thomas E Mommsen
Vancouver and Victoria, B.C.
Contributors
Vera Maria E Almeida-Val, Instituto Nacional ed sasiuqseP ad ,ainOzamA ,suanaM
AM .96( ,)380 Brasil retpahC( )3
Istvfin Benedeczky, Department of ,ygolooZ A. J6zsef ,ytisrevinU ,degezS yragnuH
retpahC( )61
Nicholas J. Bernier, Department of Biology, ytisrevinU of ,awattO 03 Marie ,eiruC
,awattO ,oiratnO Canada K1N 5N6 retpahC( )81
Mark .L Burleson, Section of evitarapmoC ,ygoloisyhP Department of ,ygoloiB -revinU
ytis of ,saxeT ,notgnilrA Box ,89491 ,notgnilrA saxeT ,91067 ASU retpahC( )1
James .J Childress, Department of lacigoloiB secneicS and Marine Science ,etutitsnI
ytisrevinU of ,ainrofilaC Santa ,arabraB ainrofilaC ,60139 ASU retpahC( )9
Andrew R. Cossins, Environmental ygoloisyhP hcraeseR ,puorG Department of Envi-
ronmental and yranoitulovE ,ygoloiB ytisrevinU of ,loopreviL PO Box ,741 Liverpool
L69 3BX, KU retpahC( )6
Eva Danulat, datlucaF ed ,saicneiC Depto. ed ,aifargonaecO UNDECIMAR, m(tsirT
ajavraN ,4761 00211 ,oedivetnoM yaugurU retpahC( )21
Gert Flik, Department of Animal ,ygoloisyhP Faculty of Science, dlevioonreoT ,1
NL-6525 ED ,negemjiN ehT sdnalrehteN retpahC( )31
Allen Gibbs, Department of ygolocE and yranoitulovE ,ygoloiB ytisrevinU of -rofilaC
,ain ,enivrI ,enivrI ainrofilaC ,71729 ASU retpahC( )01
Peter .W Hochachka, Department of ,ygolooZ ytisrevinU of hsitirB ,aibmuloC -uocnaV
,rev British ,aibmuloC Canada V6T 2A9 retpahC( )3
Christopher .J Kennedy, Department of lacigoloiB ,secneicS Simon resarF ,ytisrevinU
,ybanruB British ,aibmuloC ,adanaC A5V 6S1 retpahC( )51
Stephen .C Land, ygoloiB ,tnemtrapeD sdooW Hole cihpargonaecO ,noitutitsnI sdooW
,eloH sttesuhcassaM ,34520 ASU retpahC( )81
Timothy .S Moerland, Department of lacigoloiB ecneicS ,751-B adirolF State -revinU
,ytis ,eessahallaT adirolF ,0503-60323 ASU retpahC( )4
Thomas E Murray, egelloC of ,ycamrahP nogerO State ,ytisrevinU ,sillavroC nogerO
,13379 ASU (Chapter )8
iiiv srotubirtnoC
Jfinos Nemcs6k, Department of ,yrtsimehcoiB A. J6zsef ,ytisrevinU Szeged, Hungary
retpahC( )61
G6ran E. Nilsson, Department of Limnology, Uppsala ,ytisrevinU Box 560, S-752 36
,alasppU Sweden (Chapter 2)
A. D. Pickering, The Institute of retawhserF ,ygolocE Windermere Laboratory, Amble-
side, Cumbria LA22 ,PLO UK (Chapter )71
.T G. Pottinger, The Institute of retawhserF ,ygolocE Windermere Laboratory, Amble-
side, Cumbria LA22 ,PLO UK (Chapter )71
Scott D. Reid, Department of ,ygoloiB Okanagan ytisrevinU ,egelloC Kelowna, British
Columbia, Canada V1V 1V7 (Chapter )11 ......
Lawrence .C Rome, Department of ,ygoloiB ytisrevinU of Pennsylvania, ,aihpledalihP
Pennsylvania 19104, ASU (Chapter 5)
Pablo J. Schwarzbaum, Instituto ed Quimica y Fisicoquimica Biol6gicas (UBA-
CONICET). Facultad de Farmacia y Bioquimica, Junin 956, 1113 Buenos Aires,
Argentina (Chapter )6
Joseph E Siebenaller, Department of Zoology and ,ygoloisyhP Louisiana State -revinU
,ytis Baton Rouge, Louisiana ,30807 ASU (Chapter )8
Erik .V Thuesen, Department of Biological Sciences and Marine Science Institute,
ytisrevinU of ,ainrofilaC Santa Barbara, California 93106, USA (Chapter )9
Hiroshi Ueda Toya Lake Station for Environmental Biology, Faculty of ,seirehsiF
Hokkaido ,ytisrevinU Abuta, Hokkaido ,75-940 Japan (Chapter )41
Pieter M. Verbost, Department of Animal ,ygoloisyhP Faculty of Science, Toernooiveld
,1 NL-6525 ED Nijmegen, ehT Netherlands (Chapter )31
Cheryl Watson, Department of Medicine, Division of ,ygoloidraC ytisrevinU of -yraM
land, 22 .S Greene St., Baltimore, MD 21201, USA (Chapter )7
Wolfgang Wieser, Institute of Zoology, ytisrevinU of Innsbruck, essartsrekinhceT ,52
A-6020 Innsbruck, Austria (Chapter )6
Kohei Yamauchi Department of ,ygoloiB Faculty of ,seirehsiF Hokkaido ,ytisrevinU
Hakodate, Hokkaido 041, Japan (Chapter )41
Abbreviations
17a,20/~-diOHprog GABA y-Aminobutyric acid
17a,20fl-dihydroxy-4-pregnen-3- GDH Glutamate dehydrogenase
one GH Growth hormone
2,4,5-T 2,4,5-Trichlorophenoxyacetic acid iG Inhibitory guanine nucleotide
2,4-D Phenylacetic acid binding protein
2,4-DNP 2,4-Dinitrophenol GnRH Gonadotropin releasing hormone
5-HT 5-Hydroxytryptamine (serotonin) GRP Glucose regulated protein
AChE Acetylcholinesterase GS Glutamine synthetase
ACTH Adrenocorticotropic hormone sG Stimulatory guanine nucleotide
ADA Adenosine deaminase binding protein
ADH Alcohol dehydrogenase GSH Glutathione (reduced form)
AEC Adenylate energy charge GST Glutathione S-transferase
AFP Antifreeze protein GTH Gonadotropin
AHH Aryl hydrocarbon hydroxylase GTPyS 5'-O-(3-thiotriphosphate)-GTP
ALA-D d-Amino levulinic acid dehydratase HCB Hexochlorobenzene
AlaAT Alanine aminotransferase HPI Hypothalamic-pituitary-interrenal
APUD Amine precursor uptake and HSP Heat shock protein
decarboxylation (cells) IAA Iodoacetic acid
AspAT Aspartate aminotransferase IGF-I Insulin-like growth factor- 1
BaP Benzoapyrene LAN Leucine aminonaphthylamidase
BiP Immunoglobulin binding protein LDH L-lactate dehydrogenase
BNF /~-naphthoflavone MCH Melanocyte concentrating
CHA Cyclohexyladenosine hormone
CNS Central nervous system MCHC Mean cell haemoglobin
CPA Cyclopentyladenosine concentration
CRF Corticotropin releasing factor MD Metidathion
CS Citrate synthase MDH Malate dehydrogenase (NAD)
CT Calcitonin MDO Minimum depth of occurrence
D Diffusion coefficient MFO Mixed-function oxidase
DDA 2,2-Bis(p-chlorophenyl)-acetic acid ML Muscle length
DDD 1,1-Dichloro-2,2-bis 3-MP 3-Mercaptopropionic acid
(p-chlorophenyl)ethane HSM-to Melanocyte stimulating hormone
DDT siB-2 (p-methylthiophenyl)- -1,1,1 MTAL Medullary thick ascending limb
trichloroethane or NANA N-Acetyl-neuraminic acid
sib-2 (p-chlorophenyl)- -1,1,1 NECA 5'-N-Ethylcarboxamidoadenosine
trichloroethane NMR Nuclear magnetic resonance
DOM Dissolved organic matter NPP Nucleus preopticus
E2 loidartse-3/71 periventricularis
eEF eukaryotic elongation factor OLC Ouabain-like compound
EH Epoxide hydrolase OUC Ornithine-urea cycle
elF eukaryotic initiation factor HAP cilcycyloP aromatic hydrocarbon
EMG Electromyogram BP Phenobarbital
EPO Erythropoietin PC Phosphatidylcholine
EROD Ethoxyresorufin O-deethylase PCB Polychlorinated biphenyls
ESR Electron spin resonance PFK Phosphofructokinase
FAH Fatty acid ot-hydroxylase iP Inorganic phosphate
FCCP Carbonylcyanide p-trifluoro- PK Pyruvate kinase
methoxyphenylhydrazone; PKA Protein kinase A
FFA free fatty acid(s) PKC Protein kinase C
xii Abbreviations
PQ Paraquat T3 Tri-iodothyronine
PRL Prolactin T4 Thyroxine (tetraiodothyronine)
PTH Parathormone TAT Tyrosine aminotransferase
PTX Pertussis toxin TCDD rteT-8,7,3,2 achlorodibenzo-p-
Q Coenzyme Q dioxin
R-PIA N6-(R)-phenylisopropyladenosine TFM 3-Trifluoromethyl-4-nitrophenol
.L.S Sarcomere length Tn Troponin
SL Somatolactin TPCK lynehp-lysoT
SR Sarcoplamic reticulum TRH Thyrotropin releasing hormone
STC Stanniocalcin TSH Thyroid stimulating hormone
STX Stanniectomy UDPGT UDP-glucuronosyltransferase
akhcahcoH dna Mommsen ,).sde( yrtsimehcoiB dna ralucelom ygoloib of ,sehsif .lov 5
(cid:14)9 5991 Elsevier Science .V.B All rights reserved.
CHAPTER 1
Oxygen :ytilibaliava Sensory systems
MARK L. BURLESON
Section of Comparative Physiology, Department of Biology, University of ,saxeT Arlington, Box
19498, Arlington, TX 76019, USA
I. Introduction
II. Chemoreceptor localization
.1 Morphology
III. Discharge characteristics
.VI Oxygen transduction mechanisms
.V Pharmacology of oxygen sensitive chemoreceptors
.1 Metabolic poisons
.2 Catecholamines
.3 Acetylcholine
.4 Serotonin
VI. Conclusions
VII. References
I. Introduction
In order to maintain oxidative metabolism active animals must be able to sense
and respond to changes in environmental 02 availability and metabolic 02 demand.
This si especially true for aquatic animals such as fish because they are subject to
extreme spatial and temporal changes in ambient or environmental 02 tensions.
In addition, energetic activities (i.e. migration, pursuing prey, fleeing predators)
require that fishes regulate the delivery of 02 internally to metabolically active
tissues. This review focuses on chemoreceptors that are sensitive to 02 and alter the
performance of fishes cardiovascular and ventilatory systems to maintain oxidative
metabolism in the face of decreasing environmental 02 availability, compromised
02 uptake ability and/or increased metabolic demand.
The effects of hypoxia, hypoxemia and elevated metabolism on various aspects
of fish physiology have been studied extensively (see ref. 66 for review). Regardless
of the response or reflex elicited, virtually all fish species studied to date are
sensitive to hypoxia and/or hypoxemia. Because cardiovascular and ventilatory
variables have been technically easier to measure than the receptors mediating
these responses, most of what we know about 02 chemoreception in fish (i.e. loci
and afferent pathways) si based primarily on their reflex contribution to cardio-
ventilatory variables. The most noticeable effects of 02 receptor stimulation are
decreased heart rate (hypoxic bradycardia), increased blood pressure and increased
2 M.L. Burleson
ventilatory frequency and amplitude. These responses are generally regarded as the
typical 'chemoreflex' responses of a water-breathing fish to hypoxia. In practice,
however, depending upon the species, if it is an air-breather, and physical condition
of the fish (i.e. anesthetic, stress, temperature, etc.), the responses may vary
considerably.
Most experimental evidence indicates that cardiovascular and ventilatory func-
tions are predominately driven by 02 in aquatic animals. Carbon dioxide (CO2) also
appears to have direct effects on cardioventilatory reflexes in teleost fishes 66'71 and
elasmobranchs ~3,72 but these effects are more variable and less intense than the
responses to 02. The fact that cardioventilatory reflexes are primarily driven by 02
in aquatic respiring animals can be explained, in part, by the physico-chemical prop-
erties of 02 and CO2 in water. CO2 is approximately 30 times more soluble in water
than 02, therefore the convection requirement for 02 uptake (i.e. the volume of
ventilation required to extract a given amount of 02) in a water-breathing animal is
much greater (approximately 01 to 30 times higher) than in air-breathing animals .81
As a result of this hyperventilation with respect to CO2 elimination, arterial CO2
levels are normally very low (1-5 torr Pco2) in fishes as compared with terrestrial
vertebrates (30-40 torr Pco2). Thus, cardiovascular and ventilatory performance in
fishes, in contrast to most air-breathing vertebrates, is driven predominately by the
requirements for 02 uptake rather than CO2 elimination or acid-base regulation.
It has been shown that some of the effects of hypercapnia on cardioventilatory
reflexes in trout are due to hypoxemia and not a direct CO2 response per se 17 (Fig.
1). Hypercapnic hypoxemia is caused by reductions of blood 02 content due to
500 - RAINBOW TROUT
A 9.~,,,. Hypoxia
C 400 -
E
17~ ",,T,~.o
i n p o c r e pi" ya,H,,,,,
,. 300 - ,,'
Anaemia
0
~
200 " 14.6"~, ,' Normoxia
33.~",~ Hyperoxic
' .' hypercapnia
I00 i , i l i
4 5 6 7 8 9
Arterial blood oxygen content (Vols.%)
Fig. .1 Ventilatory volume (ml/min) versus arterial blood 02 content in rainbow trout. Numbers by
each point are the blood 02 tensions (in KPa) at each condition. Hypercapnia causes an increase in
ventilation indirectly by decreasing blood 02 content but there also appears to be a direct effect of CO2
on ventilation (from Smith and Jones, 1982) 07 .
Oxygen availability: Sensory systems 3
Bohr and Root shifts in hemoglobin 02 affinity and carrying capacity. Thus, 02
receptor stimulation may mediate a large portion of the cardio-ventilatory reflexes
to hypercapnic hypoxemia in rainbow trout 17 (Fig. .)1 At the present time, however,
it is not known if the branchial O2-sensitive chemoreceptors in fishes are directly
sensitive to CO2 as are the aortic and carotid body 02 receptors in mammals ~4 or
if CO2-sensitive gustatory chemoreceptors in the palate 67,57,93 or barbels (Smatresk
and Caprio, unpublished observations) mediate cardio-ventilatory responses to
hypercapnia.
Remarkably few studies have focused directly on O2-sensitive chemoreceptors in
teleost fishes and even fewer in elasmobranchs. As a result, comparisons between
different groups of fish, between fish and other vertebrates and generalizations
about the mechanisms of 02 chemoreception tend to be working hypotheses rather
than absolute fact. A major goal of this chapter is to point out gaps in our knowledge
in order to stimulate further research into the mechanisms of 02 chemoreception in
fishes and other lower vertebrates.
II. Chemoreceptor localization
Various anatomical locations have been proposed for the 02 receptors that mediate
the cardio-ventilatory responses to hypoxia (see 21 for review). Recent nerve
section and nerve recording studies, however, indicate that in teleost fishes these
receptors are located primarily in the gills and are innervated by branches of cranial
nerves IX (glossopharyngeal) and X (vagus). Other putative loci include the venous
vasculature ,4 arterial vasculature ~6 afferent branchial vasculature 86 and indefinite
areas of the orobranchial cavity .31 There ,si however, no convincing experimental
evidence for central (brain) chemoreception in fishes .92,72
Nerve section studies have localized 02 chemoreceptive loci and identified
afferent pathways in several different species of fishes. The 02 receptors that
mediate hypoxic bradycardia in rainbow trout suhcnyhrocnO( mykiss), coho salmon
suhcnyhrocnO( kisutch) and Atlantic cod (Gadus morhua) appear to be restricted
to the first gill arch and are innervated by branchial branches of cranial nerves
IX and X .07'96'62'71 In the dogfish sunihroilycS( ,)alucinac the receptors controlling
bradycardia appear to be widespread throughout the orobranchial cavity and are
innervated by cranial nerve V (facial) as well as IX and X .31
The receptors that mediate ventilatory responses to hypoxia appear to be dis-
tributed throughout all of the gill arches. All nerves to the gills (branchial branches
of cranial nerves IX and X) and pseudobranch must be sectioned to abolish ventila-
tory responses to hypoxia and cyanide (a potent 02 receptor stimulant) in channel
catfish surulatcI( )sutatcnup ,9 bowfin (Amia )avlac ,84 and longnose gar suetsosipeL('
oss eus ) 67.
Reflex studies indicate that there are at least two functional groups of 02-
sensitive chemoreceptors in the gills of fishes (Fig. .)2 Aquatic (external) hypoxia or
cyanide stimulates both an increase in ventilation and bradycardia in longnose gar ,86
channel catfish 8 and bowfin .84 Internal hypoxia (hypoxemia) or cyanide (during
4 M.L. Burleson
Fig. .2 Representative recordings showing the cardio-ventilatory responses of channel catfish to 500/zg
sodium cyanide (CN-) injected into the ventilatory water flow (A), 50/zg injected into the dorsal aorta
(DA) (B), and 50/zg injected into the ventral aorta (VA) (C). Stimulation of external receptors elicits
both cardiac (bradycardia) and ventilatory reflexes whereas stimulation of internal receptors only elicits
ventilatory reflexes (from Burleson and Smatresk, 1990) .8
aquatic normoxia) stimulates ventilation, but has no significant effect on heart
rate. Thus, one group of 02 receptors appears to monitor the external (aquatic)
environment and mediates hypoxic bradycardia and ventilatory stimulation. Another
group of chemoreceptors is apparently only sensitive to changes in internal (blood
or tissue) 02 levels and mediates ventilatory reflexes only .37'07'26'84'23'51'41~8
Neural recordings have confirmed the presence of internal and externally ori-
ented chemoreceptors in the gills of yellowfin tuna (Thunnus )seracabla ~5 and
rainbow trout (Oncorhynchus mykiss) .11'01 Some branchial 02 receptors are sen-
sitive to either external or internal changes in 02 and cyanide while others are
sensitive to both ~5'11 It si not known if the ventilatory and cardiac reflexes to
hypoxia/hypoxemia are mediated by separate chemoreceptor groups or if stimula-
tion of one group, external ones for example, mediates both cardiac and ventilatory
reflexes.
negyxO :ytilibaliava yrosneS smetsys 5
An appealing explanation of this arrangement of two receptor groups is because
there is a need to monitor both external and internal environments. External
O2 receptors probably initiate rapid cardiovascular and ventilatory adjustments to
hypoxic water before 2O delivery to metabolically active tissue is compromised.
This is supported by studies that show that the response time of fish to external
hypoxia and/or cyanide is very short, 5-7 sec (refs. 3 and 8) and that despite the
fact that aquatic oxygen tensions decline during aquatic hypoxia, O2 uptake can be
maintained down to a relatively low 2oP in most fishes .66 Internal receptors, on the
other hand, respond preferentially to hypoxemia and can do so in the absence of
aquatic hypoxia. Stimulation of internally oriented 2O receptors by hypoxemia, or
cyanide mediates a hyperventilatory response but does not significantly affect heart
rate. The ability to monitor internal 2O demand during external normoxia may play
an important role in the responses to hypermetabolic conditions such as exercise.
Aquatic hypoxia is a common occurrence, especially in fresh water, and may be
due to a variety of biological and physical factors (see ref. 81 for review). Hypoxemia
in the absence of any changes in aquatic 2O availability also appears to be routine
and can severely compromise 2O uptake. Anemic hypoxemia can develop as a
result of blood loss due to injury or parasitism and may be a frequent affliction in
wild fish .37 Chemical pollutants, particularly phenol compounds, are hemolytic and
have the potential to cause anemia in certain populations of fishes .51 A situation
not uncommon in nature and a significant problem in aquaculture occurs when
excess nitrite (NO2) and nitrate (NO 3), formed through natural processes, cause a
condition in fish known commonly as 'brown blood disease'. High levels of nitrite
in water are able to enter the fish's blood and combine with hemoglobin to form
methemoglobin, turning it a brown color and effectively blocking the ability of
hemoglobin to bind with O2. Nitrite poisoning has been shown to significantly
reduce the blood 2O content in carp (Cyprinus carpio) and cause ventilation to
increase .73 Thus, two 2O receptor groups enable fishes to respond differently to the
dissimilar conditions of aquatic hypoxia and hypoxemia in order to maintain 2O
uptake.
.1 Morphology
Vertebrate 02 chemoreception is associated with glomus or Type I cells. These cells,
together with Type II cells and nerve endings make up the putative 'chemoreceptive
unit' (Fig. 3). Most studies support the hypothesis that Type I cells are the primary
O2-transducing cell (see ref. 24 for review). Type I cells have been identified in
amphibians, reptiles, birds and mammals (see ref. 38 for review) and although the
reflexogenic regions and afferent neural pathways for the responses of teleost fishes
to hypoxia has been identified, the precise location and identification of Oz-sensitive
receptor (i.e. Type I) cells within the gills and/or branchial vasculature is, a matter
of conjecture.
While the specific 2O sensory cells in the gill have not been positively identified,
one would expect that branchial 2O receptors would share anatomical characteristics
with other vertebrate 2O sensory cells. Type I cells are similar in structure and
