cortisol en peces

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Reviews in Fish Biology and Fisheries 9: 211268, 1999. 1999Kluwer Academic Publishers. Printed in the Netherlands.

211

Cortisol in teleosts: dynamics, mechanisms of action, and metabolicregulation

Thomas P. Mommsen1

, Mathilakath M. Vijayan2

& Thomas W. Moon3

1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada, V8W 3P6;2Department of Biology, University of Waterloo, Waterloo, Ontario, Canada;3Department of Biology, University

of Ottawa, Ottawa, Ontario, Canada (E-mail: [email protected])

Accepted 8 April 1999

Contents

Abstract page 212

Abbreviations 212Introduction 213Dynamics of cortisol 214

BiosynthesisSecretionPlasm-binding proteinsMetabolic clearanceUptake and catabolism

Mechanism of action of cortisol 222Tissue cortisol receptors

Mammalian corticosteroid receptorsFish corticosteroid receptors

Receptor compartmentationGene regulationGlucocorticoid receptor regulationNongenomic actions

Metabolic regulation 230CarbohydratesProtein and amino acids

Protein turnoverAmino acid metabolism

Ammonia outputEnzymes

Glutamine synthetaseAminotransferasesArginase

Other effectsMetyrapone, RU 486 and dexamethasone

MetyraponeRU 486Dexamethasone

LipidsInteractions with other hormones 250Physiological role of cortisol 252

Glucose regulation during stressToxicant exposureOsmoregulationGrowth and reproduction

Future directions 256Summary 257Acknowledgements 257References 257


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Key words: cortisol biosynthesis, gluconeogenesis, hormone responsive elements, metabolic regulation,physiology, steroid receptor

Abstract

Cortisol is the principal corticosteriod in teleost fishes and its plasma concentrations rise dramatically duringstress. The relationship between this cortisol increase and its metabolic consequences are subject to extensivedebate. Much of this debate arises from the different responses of the many species used, the diversity ofapproaches to manipulate cortisol levels, and the sampling techniques and duration. Given the extreme differencesin experimental approach, it is not surprising that inconsistencies exist within the literature. This review attemptsto delineate common themes on the physiological and metabolic roles of cortisol in teleost fishes and to suggestnew approaches that might overcome some of the inconsistencies on the role of this multifaceted hormone.We detail the dynamics of cortisol, especially the exogenous and endogenous factors modulating production,clearance and tissue availability of the hormone. We focus on the mechanisms of action, the biochemical andphysiological impact, and the interaction with other hormones so as to provide a conceptual framework for cortisolunder resting and/or stressed states. Interpretation of interactions between cortisol and other glucoregulatoryhormones is hampered by the absence of adequate hormone quantification, resulting in correlative rather than

causal relationships.The use of mammalian paradigms to explain the teleost situation is generally inappropriate. The absence of aunique mineralocorticoid and likely minor importance of glucose in fishes means that cortisol serves both gluc-ocorticoid and mineralocorticoid roles; the unusual structure of the fish glucocorticoid receptor may be a directconsequence of this duality. Cortisol affects the metabolism of carbohydrates, protein and lipid. Generally cortisolis hyperglycaemic, primarily as a result of increases in hepatic gluconeogenesis initiated as a result of peripheralproteolysis. The increased plasma fatty acid levels during hypercortisolaemia may assist to fuel the enhancedmetabolic rates noted for a number of fish species. Cortisol is an essential component of the stress response infish, but also plays a significant role in osmoregulation, growth and reproduction. Interactions between cortisoland toxicants may be the key to the physiology of this hormone, although cortisols many important housekeepingfunctions must not be ignored. Combining molecular approaches with isolated cell systems and the whole fish willlead to an improved understandingof the many faces of this complex hormone in an evolutionaryand environmentalframework.

Abbreviations:ACTH adrenocorticotrophic hormone; ANP atrial natriuretic peptide; CBG corticosteriod-binding globulins; CR corticosteroid receptor; CRE cAMP-responsive element; CYP1A1 cytochrome P4501A1; DBD DNA-binding domain; Dex dexamethasone; ELISA enzyme-linked immunosorbent assay;EROD 7-ethoxyresorufin-O-deethylase; F1,6BPase fructose 1,6-bisphosphatase; FFA unesterified fattyacids; FW fresh water; G6PDH glucose 6-phosphate dehydrogenase; GDH glutamate dehydrogenase;GK glycerokinase; GNSase glutamine synthetase; GPase glycogen phosphorylase; GR glucocorticoidreceptor; GRE glucocorticoid-responsive element; GSase glycogen synthase; HOAD hydroxyacyl-coenzymeA dehydrogenase; HPI hypothalamic-pituitary-interrenal (axis etc.); HRE hormone-responsive elements; HSD 3-hydroxysteroid dehydrogenase; HSP heat shock protein (hsp 90 etc.); MCR metabolic clearance rate;MR mineralocorticoid receptor; MSH melanocyte stimulating hormone;-NF -naphthoflavone; NLS nuclear localization signals; PEPCK phosphoenolpyruvate carboxykinase; POMC pro-opiomelanocortin;PR

progesterone receptor; SERPINS serine proteinase inhibitors and substrates; SW sea water; TA triamcinoloneacetonide; TAT tyrosine aminotransferase; TCBP 3,3 ,4,4-tetrachlorobiphenyl.


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Introduction

The past decade has seen a flood of informationon the metabolic and physiological effects of stressin fish and several recent reviews have summarizedthese findings (Barton and Iwama, 1991; Gamperl

et al., 1994; Pickering and Pottinger, 1995; Wende-laar Bonga, 1997; Iwama et al., 1998). One ofthe most commonly measured indicators of stressin fish is the concentration of the major circulatingcorticosteroid, cortisol (hydrocortisone). The reasonsfor this abundance of data on plasma cortisol aremanifold, but, as often, grounded in experimentalaccessibility: (1) cortisol can be measured easilyand accurately using commercially available radioim-munoassay (RIA; Gamperl et al., 1994) or enzyme-linked immunosorbent assay kits (ELISA; Barry et al.,1993; Boesgaard et al., 1993); (2) it is possible toobtain unstressed levels of plasma cortisol by propersampling procedure, including anaesthesia (Laidleyand Leatherland, 1988; Iwama et al., 1989); and (3)plasma cortisol levels tend to increase with exposureto stressors (see earlier reviews).

However, there are inconsistencies and confusionin the literature regarding the action of cortisol dur-ing stress in fish. Much of the confusion probablyarises owing to differences among species (Vijayanand Moon, 1994), methods employed to raise cortisollevels (Gamperl et al., 1994), sampling procedures(Laidley and Leatherland, 1988; Iwama et al., 1989),seasonal and diel changes (Bry, 1982; Rance et al.,

1982; Pickering and Pottinger, 1983; Nichols andWeisbart, 1984; Thorpe et al., 1987), photoperiod(Audet et al., 1986), nutritional conditions (Vijayanet al., 1993a; Reddy et al., 1995) and sexual matur-ity of the fish (Pickering et al., 1987). One of theunderlying assumptions in several studies is that anelevated plasma concentration of cortisol during stressis deleterious to the fish, although direct causeeffectrelationships attributed to cortisol have yet to be estab-lished (Barton and Iwama, 1991).

Nevertheless, there is increasing evidence suggest-ing that cortisol directly and/or indirectly plays an

important role in intermediary metabolism (Vijayanet al., 1994b, 1996a, 1997a), ionic and osmoticregulation (McCormick, 1995) and immune function(reviewed by Wendelaar Bonga, 1997), all of whichargues for an adaptive role for cortisol during stress infish.

The majority of studies tends to correlate thephysiological changes associated during stress with

cortisol, based primarily on the circulating level ofthe steroid. However, plasma concentration of thesteroid is determined to a large extent by the pro-duction and plasma clearance of the hormone, i.e. asum of dynamic processes, all of which can regulatethe physiological response to cortisol. Indeed, Foster

and Moon (1986) and Vijayan and co-workers (1991)showed that cortisol-induced changes in tissue meta-bolism occur even in the absence of increased plasmacortisol levels.

The objective of this review is to synthesize themany metabolic and physiological roles of cortisolreported in fish and to put them into an evolutionaryand environmental framework. In this, we chose tofocus on the teleost fishes, and to address some of theproblems associated with the interpretation of cortisoleffects based on plasma cortisol concentration. Wewill restrict our review to the processes involved incortisol dynamics and its physiological and metabolic

actions, and will attempt to stay clear of the stressresponse in fish, especially because several reviewson this topic exist already (see above). Nevertheless,when appropriate, we will discuss the implications ofsome of the metabolic and physiological actions ofcortisol in the context of stress in fish.

Owing to the nature of the hormone and the typeof literature available for fish, the current review has anumber of limitations.

1. Information on fish systems is relatively sparse.2. The literature is generally biased towards the use

of cortisol as an indicator of stress; useful or not,

this has overshadowed its obvious importance inother physiological processes such as metabolism.3. The isolated nature of many observations and the

differing experimental protocols employed makeit nearly impossible to derive a coherent picturefor more than the smallest corners of cortisolsdomains.

4. Quantification of actual concentration of the hor-mone (free or physiologically active) is clouded bythe question of the importance and role of cortisol-binding proteins, and whether plasma hormoneconcentration is an adequate indicator of hor-mone effect, compared with estimates of hormoneturnover or clearance.

5. Because correlation does not equal causation, it isoften impossible to identify cortisol as a causativeagent in particular experiments.

6. The absence of specific mineralocorticoids infish and their partial functional replacement withcortisol introduces an interesting dualism for


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cortisol, which applies only to the fishes, withsome startling evolutionary implications.

7. Dexamethasone, an excellent model analogue forcortisol, is often used to inhibit the hypothalamicpituitaryinterrenal (HPI) axis, thus indirectlyactivating and inhibiting competing routes of

cortisol action.8. Fish studies are lagging behind mammalianstudiesin the availability of antibodies, probes, cDNA lib-raries and, moreover, owing to the variability andevolutionary distance of fishes, the usefulness ofmammalian probes is limited.

9. Smoltification is unique to some salmonids andhas no equal in the mammals; yet, its potentialto serve as an interesting model for nonstandardactions of cortisol and to exemplify the breadth ofits workings still needs to be developed fully.

10. Finally, nongenomic actions of cortisol have beennoticed in fishes, yet researchers have been con-tent merely to describe the associated phenomenawithout developing their observations into chal-lenging the dogma for general steroid hormoneaction.

Basically by default, therefore, the usefulness ofobservations as model systems has been limited andwe would like to make the case that researchers work-ing with fishes finally go beyond the descriptive stageand approach their quarry more in mechanistic terms.

Intrinsically, the structure of our review there-fore follows simple, albeit frustrating lines. Oftenwe must set the stage using mammalian models and

concepts. We describe similar observations for thefishes, always happy to point out the uniqueness andusefulness of fish models, before stating, rather lac-onically and repetitively, that more work needs to bedone on the fish systems. Yet, what is patently obvi-ous is that fish are highly underutilized models, notonly from an evolutionary point of view, although at least for developmental biology zebrafish (Daniorerio, Cyprinidae) have started to fill an obvious void.Unfortunately, for our specific task, zebrafish aretoo small to deliver data on plasma cortisol dynam-ics, metabolic clearance rates, amino acid utilization,

control of glycogen synthesis or similar physiolo-gical parameters. Until appropriate molecular probesbecome available, larger fish species must be used asmodels.

Cortisol is a multifaceted hormone, not only chem-ically, but especially physiologically and metabolic-ally. First, it is lipid soluble, yet, because of thepresence of binding proteins in plasma, its physiolo-

gically effective concentration may differ substantiallyfrom what chemical analysis reveals. Second, it mayexert its action by different modes of action: oneof them rapid, nongenomic; the other, the genomicroute, slower and generally longer lasting, and exper-imentally more accessible. Third, cortisol is always

present in vertebrates, even under unstressed con-ditions, playing housekeeping roles; yet its actionstend only to be apparent and appreciated when itsconcentration and actions go well beyond housekeep-ing range. Finally, the hormone does not work in avacuum, devoid of other hormones, but rather it inter-acts at many levels, with many other hormones, settingup a bewildering array of multilevel, multihormone,multitarget interactions. As expected, the reduction-ist approach may lead to interesting insights about theminutiae, but does little to help develop the big pic-ture. With all these limitations in mind, we will tryto compose a coherent picture of what and why and

how our quarry controls key processes in our finnedfriends.

Dynamics of cortisol

Despite the interest in plasma cortisol measurementas an indicator of stress, few studies have actuallymeasured the kinetics of cortisol in fish. Plasmacortisol concentrations reflect the net effect of pro-duction and plasma clearance of the hormone. Highor low plasma cortisol concentrations may not be agood indicator of tissue responses to cortisol stim-ulation. During chronic stress, plasma cortisol fallsback to the resting levels, even though the fish maystill be responding to the stressor (Vijayan and Leath-erland, 1990). The clearance from the plasma isdependent upon binding proteins, target tissue recept-ors, tissue uptake and catabolism of cortisol. Thusthe animals physiological response to the hormonemay be modulated by the above-mentioned processes.Factors affecting any of these parameters will in turnmodify the cortisol response in the animal. Therefore,a good understanding of the regulatory factors that canmodulate plasma cortisol concentration is paramount

to obtaining some perspective on plasma concentra-tion and the physiological responses associated withcortisol.

Biosynthesis

Fish do not possess a discrete adrenal gland as inmammals, and the steroidogenic cells called inter-


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Figure 1. Biosynthesis of cortisol in teleost fishes. The shaded arearepresents the mitochondrial compartment, whereas those reactionsoccurring in the nonshaded area occur within the cytosolic compart-ment. Abbreviations: 3-HSD, 3-hydroxysteroid dehydrogenase;P450s, various forms of cytochrome P450.

renal cells are distributed in the head-kidney region,mostly along the posterior cardinal veins and theirbranches. These steroidogenic cells lie in close prox-imity to the chromaffin cells (which secrete cat-echolamines), raising the possibility of a paracrinecontrol in the release of these hormones (Reid etal., 1996). The morphology of the interrenals andthe biosynthesis of corticosteriods by these cells havebeen reviewed previously) Idler and Truscott, 1972;Butler, 1973; Sandor et al., 1984). Briefly, the bio-synthesis of cortisol in fish is similar to that in mam-mals and involves the microsomal enzymatic path-

ways, including 21-hydroxylation (P450c21), 17-hydroxylation (P450c17), and 3-hydroxy steroiddehydrogenation(3-HSD, Figure 1). In addition, fishpossess the mitochondrial inner membrane monooxy-genase enzymes, such as the cholesterol side-chaincleavage enzyme (cytochrome P450scc, desmolase)and the 11-hydroxylase that catalyses the 11-hydroxylation of deoxycortisol/deoxycorticosterone

(cytochrome P450c11) (Lehoux et al., 1972). Recentstudies have also identified an adrenodoxin-like pep-tide in the interrenal cells of a teleost the Asiansea bass (Lates calcarifer, Centropomidae) (Sampath-Kumar et al., 1996); this peptide in mammals func-tions as a shuttle between adrenodoxin reductase and

cytochrome P450scc/P45011 in the mitochondrialsteroidogenic electron-transport chain. The presenceof adrenodoxin-like peptide immunoreactivity in fishinterrenals hints at the possibility of distinct type I andtype II steroid-hydroxylating enzyme systems (cyto-chrome P450s), as in mammals. As recently reportedfor mammals, control of steroidogenesis may lie out-side of the reactions mentioned above, and the keylimiting factor may be the transport of cholesterol tothe outer mitochondrial membrane and its subsequenttranslocation across the aqueous intermembrane spaceof the mitochondria to the inner membrane (Stoccoand Clark, 1996).

Cortisol is present in eggs and larvae of a numberof species, including Japanese flounder (Papalichthysolivaceus, Bothidae) (De Jesus et al., 1991), chumsalmon (Oncorhynchus Keta, De Jesus and Hirano,1992; Hwang et al., 1992), Mozambique tilapia (Oreo-chromis massambicus, Hwang and Wu, 1993), rain-bow trout (Oncorhynchus mykiss, Salmonidae), Asiansea bass (Sampath-Kumar et al., 1997a) and com-mon carp (Cyprinus carpio, Cyprinidae) (Stouthart etal., 1988a). Cortisol in unfertilized eggs appears tobe largely of maternal origin (Feist et al., 1990) andis likely to have been transferred into the growing

oocyte adventitiously via vitellogenin, similar to otherlipophilic hormones such as thyroxine (Specker andSullivan, 1995). Endogenous production of cortisol isfirst detectable 36 h after fertilization in the commoncarp (Stouthart et al., 1998a), but is believed to startafter hatching in flounder (De Jesus et al., 1991), chumsalmon (Hwang et al., 1992), tilapia (Hwang and Wu,1993), rainbow trout (Barry et al., 1995b) and Asiansea bass (Sampath-Kumar et al., 1997a). While theexisting database is limited, it hints at species differ-ences in the ontogeny of steroidogenic cell function.Certainly the regulating mechanisms need to be elu-cidated as does a functional analysis of the presenceand the production of cortisol in early ontogeny.

Although cortisol is the major circulating adreno-cortical steroid in fish, several studies have shown thatstress also increases the concentrations of cortisonein teleost plasma (Weisbart and McGowan, 1984;Patio et al., 1987; Pottinger et al., 1992). In vitro,cortisone can be produced by the head kidney (Idler


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and Truscott, 1972; Butler, 1973), but the major-ity of plasma cortisone is due to 11-oxidation ofcortisol in other tissues (Patio et al., 1985). Forinstance, incubation of 3H-cortisol with liver slicesof rainbow trout, perch (Perca fluviatilis, Percidae)or pike (Esox lucius, Esocidae) confirms that cortisol

is rapidly converted into cortisone (Kime, 1978).In mammals, the reciprocal conversion of cortisoneto cortisol occurs readily and is catalysed by 11-hydroxysteroid dehydrogenase; however, this inter-conversion has not been clearly established in fish(Donaldson and Fagerlund, 1972). Because the poten-tial roles of cortisone in fish remain to be identified,this review will focus on the major corticosteroid,cortisol. To our knowledge, very few studies havedirectly examined the environmental regulation ofcortisol biosynthesis in fish. This may be an import-ant area for future research, especially in light of theendocrine-disruption action of some environmental

contaminants. Indeed, chemicals inducing cytochromeP450s may compete for the same substrates in thesteroidogenic pathway, thereby altering the corticost-eroid biosynthetic capacity. In birds, the adrenotoxicenvironmental pollutant 3-methylsulphonyl-2,2-bis-(4-chlorphenyl)-1, 1-dichloroethene (MeSO2-DDE, ametabolite of the insecticide DDT) decreased plasmacorticosterone (Jnsson et al., 1994). This decreaseis mediated by the bioactivation of MeSO2DDE byadrenal cytochrome P450c11, a key mitochondrialenzyme involved in cortisol biosynthesis. The out-come is an inhibition of steroid hydroxylase affecting

the final steps of corticosteronebiosynthesis (Figure 1)(Lund and Lund, 1995).

Secretion

The secretion of cortisol is under the control ofthe hypothalamuspituitaryinterrenal axis (HPI axis;reviewed by Donaldson, 1981). Adrenocorticotrophichormone (ACTH) released from the anterior pituitarygland is the main secretagogue for cortisol (Table 1).The control of ACTH secretion has been reviewedby Lederis and colleagues (Fryer and Lederis, 1986;

Lederis et al., 1993) and suffice it to say that sev-eral factors, including hormones, stress (Sumpter etal., 1986; Balm et al., 1994) and negative feed-back of cortisol at the level of the hypothalamus andpituitary may modulate ACTH secretion in fish, andconsequently cortisol production.

Most studies on cortisol secretion employed head-kidney preparations containing the majority of the

interrenal tissue, either in static or superfusion systems(Table 1). The basal unstimulated release of cortisolis higher from interrenal tissue obtained from stressedfish than from unstressed fish. Stressors, includinghigh stocking density (Patio et al., 1986; Vijayan andLeatherland, 1990), confinement (Balm and Pottinger,

1995) and toxicants (Brown, 1993), result in higherbasal release of cortisol from the interrenal tissue.However, -naphthoflavone (-NF), a potent inducerof cytochrome P4501A1, failed to elicit any increasein basal cortisol release in implanted fish (Wilson etal., 1998), suggesting that the type of contaminant andthe duration of exposure may be important factors inthe cortisol release process. Also, the circulating levelsof ACTH, which are modulated by stress, may play animportant role in the regulation of the HPI axis (Balmand Pottinger, 1995). It is hypothesized that the higherbasal unstimulated release of cortisol in stressed fishis due to chronic stimulation of the interrenal cells by

the higher concentration of circulating ACTH (Balmand Pottinger, 1995) a stimulation that is retainedfor some time in the isolated interrenals even in theabsence of ACTH.

Interrenal tissue from stressed fish is less sensitiveto ACTH-stimulation than tissue from unstressed fish.For example, interrenals from brook charr (Salvelinus

fontinalis, Salmonidae) held at high stocking densityhad decreased sensitivity to ACTH-induced cortisolproduction (Vijayan and Leatherland, 1990). Thisstudy used a static system, so it is possible thatthe build-up of cortisol in the incubation medium in

itself may have affected the release of cortisol. Highlevels of cortisol in the medium have been shownto influence interrenal sensitivity in vitro, via anultrashort-loop feedback mechanism (Bradford et al.,1992). A recent study using a superfusion system,which overcomes cortisol build-up, confirmed thatprior confinement stress resulted in decreased sensit-ivity to ACTH in rainbow trout interrenals (Balm andPottinger, 1995). Together these results suggest thateither ACTH receptor downregulation and/or desens-itization associated with the higher ACTH stimulationin vivomay be responsible for the decreased sensit-ivity to ACTH in vitro. Very little is known aboutACTH receptor dynamics in fish interrenal cells andit is likely that ACTH receptor regulation may beplaying an underappreciated role in cortisol produc-tion associated with stress. For instance, organochlor-ine insecticide or -NF completely abolished the invitroACTH-induced cortisol production in fish inter-renal tissue (Ilan and Yaron, 1980a; Wilson et al.,


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Table 1. Cortisol secretagogues other than adrenocorticotrophin (ACTH) in teleostean fishes

Secretagogue Comments Reference

-MSH Also acts via ACTH Lamers et al. (1992)

Thyroxine Sensitizes response to ACTH in presmolts Young (1986)Thyroxine Desensitizes response to ACTH in postsmolts Young and Lin (1988)Angiotensin II Direct and synergistic action with ACTH Decourt and Lahlou (1987),

Arnold-Reed and Balment (1994)Growth hormone Young (1988)Catecholamines In vivo, but notin vitro White and Fletcher (1985),

Schreck et al., (1989)

N-terminal of POMC Slight potentiation of ACTH effects Takahashi et al. (1985)ANP Arnold-Reed and Balment (1991)

Urotensin I Direct and potentiating effects with ACTH Arnold-Reed and Balment (1994)Urotensin II Arnold-Reed and Balment (1994)Prostaglandin E1 Gupta et al. (1985),

Wales and Gaunt (1986)17, 20-DHPO Barry et al. (1997)17, 20-DHPO Barry et al. (1997)11-Ketotestosterone No effect (rainbow trout) Barry et al. (1997)

11-Ketotestosterone Suppression (coho salmon) Young et al. (1996)Salmonid gonadotrophins Schreck et al. (1989)

Dibutyryl-3 ,5-cyclic AMP Ilan and Yaron (1980a,b),Patio et al. (1986)

Forskolin Patio et al. (1986)Calcium Decourt and Lahlou (1986)Increased osmolarity Physiological role questionable Decourt and Lahlou (1986),

Kniehl et al. (1987),Patio et al. (1988)

DHPO, dihydroxy-4-pregnen-3-one; other abbreviations may be found in the list on page 212.Extracellular calcium is essential for ACTH action (Decourt and Lahlou, 1986).

1998). Furthermore, ACTH-induced cortisol produc-tion could be suppressed by treating interrenal tissuewith organochlorines in vitro(Ilan and Yaron, 1980a),implying that the changes in interrenal responsive-ness are mediated directly by the toxicants at thelevel of the interrenal cells and not by the circulatingconcentrations of other hormones such as cortisol orACTH. Interestingly, when cAMP was substituted forACTH in the above study, the suppressive effect ofo,p-DDD on cortisol output was prevented (Ilan andYaron, 1983), raising the possibility that the effectmay be at the level of receptor desensitization more

so than receptor downregulationper se. As far as weare aware, studies addressing ACTH receptor dynam-ics and/or the regulation of the signal-transductionpathways in fish interrenal cells do not exist. Unfor-tunately, obstacles in this area include the lack of adistinct adrenal cortex in fish and the difficulty in isol-ating interrenal cells forin vitroreceptor characteriza-

tion. Any study using a whole head-kidney preparationwill, of course, be influenced by the surrounding cells,including the chromaffin cells. Because Reid and co-workers (Reid et al., 1996) showed that ACTH inducescatecholamine release in fish and epinephrine stim-ulates cortisol release in mammals (Mokuda et al.,1992), the possibility of paracrine influence on cortisolproduction seems reasonable.

Although ACTH is the primary secretagoguefor cortisol, several other hormones can indirectlymodify cortisol secretion from the interrenal tissue(Table 1). Some of these hormones may modulate

the ACTH-induced steroidogenesis. The N-terminalpeptide of salmon pro-opiocortin, for instance, exertsa weak potentiating effect on ACTH-induced inter-renal function in rainbow trout (Takahashi et al.,1985), while angiotensin II and the urophysial pep-tides, urotensins I and II, stimulate cortisol secre-tion in rainbow trout interrenals both in fresh water


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(FW) and in sea water (SW) (Arnold-Reed and Bal-ment, 1994), with angiotensin II acting synergistic-ally with ACTH (Decourt and Lahlou, 1987). Thepossibility that urotensin I enhances the steroido-genic action of ACTH in SW could contribute tothe adaptive changes in osmoregulatory mechanisms

necessary for SW acclimation (see Osmoregulation,below). Atrial natriuretic factor (ANF) may alsoassist in the SW acclimation process by increasingACTH-induced corticosteroidogenic capacity in rain-bow trout acclimated to SW, but not to FW (Arnold-Reed and Balment, 1991). In addition to these peptidehormones, 11-ketotestosterone (11-KT) suppressesACTH-induced cortisol production in rainbow troutinterrenal cells (Young et al., 1996). These resultswere further corroborated in vivo in rainbow troutand brown trout (Salmo trutta, Salmonidae) given11-KT implants and subjected to confinement stress,suggesting that gonadal steroids also play a role in

the regulation of the cortisol-release process (Pot-tinger et al., 1996; Young et al., 1996) and thus willexert a significant effect on the pituitary interrenalaxis.

Some of the hormones above and several otherhormones have been directly implicated in controllingthe interrenal function of fish (Table 1), includingprostaglandin PGE1, but not PGF2 , -melanocytestimulating hormone (-MSH), and salmon gonado-trophins. These effects clearly imply a complex envir-onmental endocrine interaction in the control ofinterrenal function in fish. Very little, however, is

known about the significance of these pituitary andextra-pituitary hormones on cortisol production. Onehypothesis put forward recently was that these pituit-ary and extra-pituitary hormones may take on promin-ence in the absence of ACTH stimulation in order tomaintain plasma cortisol concentration (Wilson et al.,1998). Indeed, plasma cortisol concentration is main-tained in -NF fish despite the abolition of interrenalsensitivity to ACTH in vitro, suggesting an augmen-ted role for other hormones in the functioning ofthe interrenal tissue, or a direct impact of environ-mental factors. The lack of response or muted cortisolresponse to stress in contaminant-exposed fish, how-ever, clearly implies an important role for ACTHin the stress-induced plasma cortisol levels (Wilsonet al., 1998). Finally, an increase in extracellularosmotic pressure will result in a sharp, but short-lived, increase in the rate of cortisol excretion fromsuperfused trout interrenal tissue (Decourt and Lahlou,1986). Activation of adenylyl cyclase and cAMP are

implicated in the steroidogenic actions described, withextracellular calcium playing an ancillary role in theprocess.

Similar to the synthesis of cortisol, the HPI axisis functional immediately after hatching in numer-ous species, including tilapia (Hwang et al., 1992;

Hwang and Wu, 1993), milkfish (Hwang et al., 1992),Asian sea bass (Sampath-Kumar et al., 1997a), yellowbream, Japanese flounder (De Jesus et al., 1991) andrainbow trout (Pottinger and Mosuwe, 1994; Barryet al., 1995b). A recent study presented evidence fora functional pituitary-interrenal axis prior to hatchingin carp (Stouthart et al., 1998a), while cultured rain-bow trout interrenal cells produced significant levelsof cortisol in response to ACTH at the time of hatching(Barry et al., 1995a). Carp eggs generated ACTH and-MSH endogenously 24 h after fertilization. Also,ACTH and -MSH immunoreactivity was present inthe pituitary of chum salmon two weeks prior to hatch-

ing, with the staining intensity increasing graduallyduring embryonic life and markedly after hatching(Naito et al., 1984; Saga et al., 1993). While a cortisolhypo-responsive period as in mammals was sugges-ted for the rainbow trout (Barry et al., 1995b), nosuch reduced HPI activation was evident in develop-ing carp (Stouthart et al., 1998a), leaving the questionopenwhether different experimentalprotocols or onto-genetic species differences account for these opposingobservations.

The environmental modulation of interrenal func-tion during development has been poorly studied.

Stouthart and co-workers (Stouthart et al., 1998a)raised the possibility that rearing temperature foreggs and larvae may influence the induction of thecortisol response. Further, it appears that xenobiot-ics can affect the HPI function during development,because exposure of carp embryos to PCB 126 res-ults in an increase in whole-body ACTH, -MSHand cortisol levels (Stouthart et al., 1998b). The sig-nificance of elevated cortisol during development isnot clear. Some larval fish (1 day after hatching)increase body cortisol content when transferred to20% SW, and cortisol-fed larvae were able to sur-vive better when transferred from FW to SW (Hwangand Wu, 1993), arguing for a role for cortisol in theSW, but not the FW, acclimation process. Researchis certainly warranted in a number of areas: first,to assess the environmental control of the HPI axis;second, to probe the role of elevated (or decreased)cortisol concentrations on development and growth;and third, to analyse the effect of maternal exposure


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to stress (and resultant high cortisol levels) on larvaldevelopment.

Plasma-binding proteins

Mammalian plasma contains steroid-binding glo-

bulins, a group of glycoproteins that bind steroidsselectively and with higher affinities than plasma albu-min or other plasma proteins. The structure of thecorticosteroid-binding globulin (CBG) is unrelated tosex-steroid-binding globulin or to any other steroid-binding protein, receptor or enzyme, but is a mem-ber of the serine proteinase inhibitors and substrates(SERPINS) superfamily (Hammond, 1995; Seralini,1996). The idea that CBG is a protease inhibitor orsubstrate fits well with its ability to regulate the plasmadistribution and bioavailability of cortisol at its site ofaction. Only free cortisol constitutes the physiologic-ally active form and in mammals 9095% of plasmacortisol is bound to CBG (Fleshner et al., 1995); thusthe overwhelming fraction of cortisol neither reachesthe target tissues nor binds to target receptors. Becauseit harbours the bulk of total plasma cortisol, smallalterations in the concentration in CBG will producelarge changes in the levels of free (biologically act-ive) cortisol. The presence of specific CBG-bindingsites on steroid target cells adds a further local levelof control over cortisol concentrations, with poten-tial interactions between CBG and specific proteinases(Hammond, 1995). Plasma CBG levels increase whencortisol levels rise, as in chronic stress (Flesher et

al., 1995) or at a site of inflammation (Hammond,1995; Garrel, 1996). Dietary manipulations, such asfeeding low-fat diets, will increase plasma CBG alongwith free cortisol (Garrel, 1996), providing clear evid-ence that mammalian CBGs modulate levels of plasmacortisol.

The situation in fishes appears to differ dramat-ically. Idler and Truscott (1972) found little evid-ence for a plasma CBG-like protein in a variety offish species. Using equilibrium dialysis (indicationof total binding), the steriod bound was found tobe 4050% of the total steroid, but using gel filt-

ration, less than 5% was bound. On the one hand,binding of cortisol in fish plasma is one hundredthof what would be expected in the presence of trueCBGs. On the other hand, it is about tenfold greaterthan the binding for human serum albumin, and fishplasma albumin concentrations fall into the samerange as those of mammals (Maillou and Nimmo,1993). Studies in the ensuing quarter century have

not changed the view regarding the absence of aCBG in fish, perhaps because very few studies haveactually addressed this issue in fish. Nichols and Weis-bart (1985) reported that total cortisol plasma binding(equilibriumdialysis) decreased when Atlantic salmon(Salmo salar, Salmonidae) were transferred from FW

to SW, but this was paralleled by a general decrease inplasma protein. Later studies from the same laborat-ory (Chakraborti et al., 1987) failed to find bindingin brook trout plasma by a dilution method. Pot-tinger (1990) was unable to identify specific plasmacortisol binding in rainbow trout, although othersshowed that mature females had higher plasma bind-ing of cortisol than mature males or immature trout(Caldwell et al., 1991). This latter study used acentrifugal ultrafiltration isodialysis method, and rep-resents the only study to identify the presence of aCBG-like protein in fish blood. One wonders whetherthe presence of vitellogenin that binds a number of

lipophilic substances adventitiously may have skewedthese results for the maturing female trout. Addi-tional studies using competition analysis are neededto resolve this issue, and even though plasma sex-steroid-binding proteins exist in fish (Foucher et al.,1991), further studies on CBG proteins are usefulif only to show that general plasma proteins suf-fice for limited binding and transport of cortisol infish.

Metabolic clearance

The volume of blood cleared completely and irrevers-ibly of hormone per unit time is known as the meta-bolic clearance rate (MCR). The MCR for cortisolindicates the availability of the hormone for tissuefunction. The clearance of cortisol from the plasmarepresents the net effect of tissue capacity for cortisoluptake and catabolism. As shown above, turnoverand biological actions in mammals are determinedto a large extent by the dynamics of steroid-bindingproteins in the plasma. In the absence of specificcorticosteroid-binding proteins (CBG) in fishes, otherregulatory sites can be expected. The MCR of cortisol

ranges between 45 mL kg

1

h

1

(American eel,Anguilla rostrata, Anguillidae) and 270 mL kg1 h1

(mature sockeye salmon, Oncorhynchus nerka, Sal-monidae), values that are considerably lower than theMCR of 449 mL kg1 h1 measured for cortisonein the sea raven (Hemitripterus americanus, Cottidae)(Table 2). Many environmental factors will modifythe clearance of cortisol, including stress, ambient


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salinity, maturity, and nutritional state. Some of theseeffects are collated in Table 2, but it should be kept inmind that the different methods constant infusion orbolus injection used for the determination of MCRmay have influenced the actual value of MCR. Nev-ertheless, statements about changes within concurrent

treatments should hold. Independent of experimentalprocedures, it is quite obvious that fish plasma cortisolconcentrations are labile and may be modulated by theclearance of hormone from the circulation. Becausemany of the factors listed in Table 2 alter the plasmaclearance of cortisol, these treatments in turn willexert significant effects on the plasma concentrationof the hormone and its bioavailability. We believe thatan understanding of the factors modulating cortisoldynamics is essential to appreciate the physiologicalconsequences of raised or lowered plasma cortisolconcentrations.

Uptake and catabolism

The clearance of cortisol is essentially maintained bytissue uptake and catabolism. Because of the lipophilicnature of cortisol, its entry into cells is thought tooccur by passive diffusion. However, recent studieshave provided evidence of carrier-mediated uptake ofsteroid hormone in the rat liver (Allera and Wildt,1992). In fish, only a few studies have examinedthe uptake of cortisol into cells. In isolated rain-bow trout hepatocytes, evidence was presented fora low-affinity carrier protein located in the cellu-

lar membrane (Porth-Nibelle and Lahlou, 1981), aconclusion confirmed in recent studies on the samespecies (Vijayan et al., 1997b). The attenuation inthe uptake rate of 3H-cortisol (40%) by trout hep-atocytes in the presence of excess unlabelled cortisol,over and above the expected decline in specific activ-ity, led us to postulate a distinct uptake mechanism forcortisol (Vijayan et al., 1997b). These data raise thepossibility of a saturable membrane component thatmight play a role in cortisol uptake, similar to theprocess observed in rat liver cells (Allera and Wildt,1992).

Once inside the cell, the steroid is bound to areceptor and activated or it is metabolized and thusinactivated. Hormone bound to the receptor will even-tually be released from the hormonereceptor complexand will subsequently also be subject to processing.The major steroid-metabolizing enzymes in rat livermicrosomes are reductases, oxidoreductases and cyto-chrome P-450 dependent hydroxylases. The presence

of most of these enzymes has been confirmed forfish. In rainbow trout, for instance, intra-arterialinjection of 3H-cortisol resulted in the accumula-tion of metabolites mostly as water-soluble conjug-ates of polar derivatives of cortisol (Truscott, 1979).The steroid moieties of the conjugates, released by

-glucuronidase and aryl sulphatase, were identifiedin order of their quantitative importance as tetrahy-drocortisone, 20-cortolone, tetrahydrocortisol, 5-dihydrocortisone, cortisol and cortisone. The enzymesinvolved are 4-ene-5-reductase, 3-hydroxysteroiddehydrogenase, 20-hydroxysteroid dehydrogenase,and 11-hydroxysteroid dehydrogenase. In contrastto the situation in mammalian liver, no evidence wasfound for 5-reduction or oxidative cleavage of theside-chain. In vivo, the major biliary conjugated ster-oids in rainbow trout 4 h after stress were tetrahydro-cortisone, tetrahydrocortisol, cortisone, cortisol and-cortolone in that order of appearance (Pottinger et

al., 1992).Liver is the key target organ for cortisol disposal

with the hepato-biliary system as the main route forcortisol clearance (Idler and Truscott, 1972; Reddinget al., 1984; Vijayan and Leatherland, 1990; Vijayanand Moon, 1994; Wilson et al., 1998). Renal andbranchial routes play subordinate roles in steroid elim-ination (Idler and Truscott, 1972; Cravedi et al., 1993).Very little is known about factors regulating cortisoluptake and metabolism. As cytochrome P450s are keyplayers in steroid metabolism and some of these P450genes are also triggered by xenobiotics, it is likely that

contaminants modulate cortisol metabolism. Vijayanand co-workers (Vijayan et al., 1997b) explored thispossibility by exposing rainbow trout to the PCB con-gener, 3,3,4,4-tetrachlorobiphenyl (TCBP), a potentinducer of CYP1A, then examined cortisol uptakeand catabolism in isolated hepatocytes. Fish exposedto TCBP had significantly higher rates of hepatocyte3H-cortisol uptake, concomitant with increased meta-bolism of cortisol, with tetrahydrocortisone as themajor metabolite (Vijayan et al., 1997b). This studyprovided the first evidence that contaminants can sig-nificantly alter cortisol clearance. The mechanism(s)involved in this process is not clear, although oneof the hypotheses is that TCBP influences membranefluidity, thereby enhancing cortisol uptake rates. Arecent study using -NF, another potent cytochromeP450 inducer, showed no effect on cortisol uptakerate or membrane fluidity. This result suggests thatthe effect on cortisol uptake rate may be contaminantspecific, but may also be dependent upon the dura-


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Table 2. Factors affecting metabolic clearance rates (MCR) for cortisol in teleostean fishes

Species MCR Changes / comments / references(mL h1 kg1)

Sockeye salmon (Donaldson and Fagerlund, 1968, 1972)(Oncorhynchus nerka) 54 Three fold increase in MCR with maturation in immature males

83 Three fold increase in MCR with maturation in immature females

Sea raven (Owen and Idler, 1972)(Hemitripterus americanus) 127 Cortisol constant infusion

449 Cortisone constant infusion

(Vijayan and Moon, 1994)89102 No effect of confinement on cortisol MCR666 6-Week food deprivation increases cortisol MCR

6-Week food deprivation decreases plasma cortisol

American eel (Butler, 1973)

(Anguilla rostrata) 45 SW adapted, single injection

European eel (Leloup-Hatey, 1974, 1976; Henderson et al., 1976)(Anguilla anguilla) 28.4 (SW) Higher in SW than FW, plasma cortisol unaltered

20.6 (FW) MCR higher in hypophysectomized eels, together with decreased plasma cortisoland decreased cortisol synthesisMCR doubled after stanniectomy together with increased cortisol synthesis;plasma cortisol unaltered

Coho salmon (Redding et al., 1984; Patio et al., 1985)(Oncorhynchus kisutch) 5358 (FW) MCR higher in SW than FW fish

84 (SW) Plasma cortisol higher in SW than FW fish

Chronic but not acute stress increases MCR in SW and FWSeasonal increases in MCR correlate with gill Na+/K+-ATPase (smoltification)

No correlation of MCR with plasma cortisol

Brook trout (brook charr) (Nichols et al., 1985; Vijayan and Leatherland, 1990, 1992)(Salvelinus fontinalis) 236 (FW) FW to SW transfer increases cortisol titres

227 (SW) FW to SW transfer increases cortisol synthesis, but not MCR105118 No effect of high stocking density on cortisol MCR

No effect of RU486 on cortisol MCR

Rainbow trout (Brown et al., 1986, 1989)(Oncorhynchus mykiss) 176213 FW to SW transfer increases MCR, and lowers CBP

260 Total plasma cortisol unchanged

No difference in MCR between fish held at pH 5.0 and pH 7.7Increased MCR if exposed to aluminium at low pH

(Wilson et al., 1998)7696 No effect of -naphthoflavone on MCR

Decreased interrenal sensitivity to ACTH with-naphthoflavone


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tion of exposure and the metabolic state of the animal(Wilson et al., 1998). The regulation of cytochromeP450s and steroid biosynthesis and metabolism will bea very important area of research, especially with theemerging use of fish as indicator species for endocrinedisrupters in the aquatic environment.

Mechanism of action of cortisol

Corticosteroid hormones are hydrophobic moleculesthat travel bound to plasma proteins and act as tran-scription factors by binding to specific tissue DNAsequences. To accomplish this action, corticosteroidreceptors (CRs) must be expressed, or a particulartissue cannot be said to be a target tissue for thatparticular hormone. The dogma regarding corticost-eroid hormone action in general, and glucocorticoidaction in particular, is that receptors are localized

in the cytosolic and nuclear fractions only, as mem-brane permeability does not restrict cellular corticost-eroid movements. This dogma has been challenged bymammalian studies, but work on fish lags well behind.

Tissue cortisol receptors

There have been many recent reviews on corticoster-oid receptors (CRs) in mammalian systems (Evans,1988; Burnstein and Cidlowski, 1989; Fuller, 1991;Gehring, 1993; Bamberger et al., 1996; Beato et al.,1996; Guiochon-Mantel et al., 1996; Wehling, 1997)and it is not the object of this review to exhaustively

present these studies. However, some background isnecessary to provide a framework for the literatureconcerned with fishes.

Mammalian corticosteroid receptors

Two cytosolic CRs have been demonstrated in mam-malian tissues, termed the glucocorticoid receptor(GR, type II corticosteroid) and the mineralocortic-oid receptor (MR, type I corticosteroid). Classicallya type I receptor has a high affinity for the miner-alocorticoid aldosterone, while the type II receptorhas a high affinity for dexamethasone, but low affin-

ity for aldosterone (Knoebl et al., 1996). Both GRand MR are members of the steroid/thyroid hor-mone/retinoic acid receptor superfamily, the membersof which are phosphoproteins and ligand-dependenttranscription factors (Burnstein and Cidlowski, 1989,1992; Fuller, 1991; Schmidt and Meyer, 1994). Thesereceptors share a high degree of sequence homo-logy in the three linearly arranged domains making

up this class of receptors. Each domain has a spe-cific role based primarily on studies using site-directedmutagenesis. The central domain contains the DNA-binding domain (DBD) with a sequence predictinga zinc finger structure with two loops stabilizing thezinc ion. It is this highly conserved DBD region that

binds to hormone-responsive elements (HRE) calledglucocorticoid-responsive elements (GRE) activatingspecific regions of nuclear DNA. The carboxyl ter-minal contains the ligand-binding site and shows ahigh degree of homology between species, but lessbetween receptor types within the family. The aminoterminal is the least conserved region between spe-cies, it is of variable length between receptor typesand even for the same receptor in different species,it is thought to be the site of receptor regulation andit contains consensus phosphorylation sequences. TheGRs of mammals are between 85 and 100 kDa in mass,from 775 to 800 amino acids in length and differ sig-

nificantly from the MRs of the same species (Fuller,1991; Gehring 1993).

The composition of the GR in vivohas been moredifficult to establish. It is clear that two molecules ofhsp90 (heat shock protein) are absolutely required forGR folding and ligand binding (Pratt, 1993; Gehring,1993; Bohen, 1995). Hsp90 binds at the carboxyl ter-minal and maintains the GR in an inactivated state;hsp90 mutants may destabilize the GR aporeceptorand modify GR signalling (Bohen, 1995). The pres-ence of hsp70 and hsp56 in the aporeceptor is lesswell accepted and possibly varies between species,

although recent literature tends to support a role forboth in DNA-binding, transcriptional activities andreceptor trafficking (Diehl and Schmidt, 1993; Czaret al., 1995). Therefore, the functional GR is com-posed of one receptor polypeptide, two molecules ofhsp90, and one subunit of about 50 kDa, for a totalmolecular mass of about 330 kDa (Bohen, 1995).There is also evidence that alternate splicing of thepre-mRNA can result in two human GRs, termed GRand GR, which differ only in the replacement ofthe last 50 amino acids of the C-terminal region witha unique 15 amino acid sequence preventing GRfrom binding glucocorticoid hormones (Bamberger etal., 1996). The presence of this altered form hassignificant mechanistic impact.

The binding of ligands to the GR results in receptoractivation (transformation) and a significant changeto the ligandreceptor complex allowing the move-ment of the complex to the nucleus and DNA binding.Although localization of receptor in the nucleus in


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Table 3. Kinetic characteristics of ligand binding to fish tissue glucocortoicoid receptors

Organ/tissue Ligand Kd(nM) Bmax References

and species (fmol per mg protein)

Liver

Rainbow trout 3H-TA 36.0 78.0 Vijayan et al. (1997b)

(Oncorhynchus mykiss)

3

H-TA 2.75 61.1 Vijayan et al. (1993a)3H-Dex 16.9 87.8 Lee and Struve (1992)3H-Dex 16.0 82.3 Lee et al. (1992)

Potency: dexamethasone > TA > cortisol>>corticosterone3H-Cortisol 5.1 197 Pottinger (1990)

Potency: dexamethasone = cortisol > RU486>>corticosterone

Brook trout 3H-Cortisol 5.6 167 Chakraborti and Weisbart (1987)(Salvelinus poutinualis) 3H-TA 0.6 171

Potency: dexamethasone > TA > cortisol>>corticosterone

Mozambique Tilapia 3H-Cortisol 66 100 Kloas et al. (1998)(Oreochromis mossambicus) 3H-Dex 9 121

Red tilapia 3H-Dex 11.9 Chen et al. (1997)

(Oreochromis mossambicus O. niloticus)

Common carp 3H-Cortisol 31 91 Kloas et al. (1998)(Cyprinus carpio) 3H-Dex 36 160

Potency: dexamethasone > TA cortisol > corticosterone > aldosterone; both tilapia and carp

Gill

Rainbow trout 3H-TA 1.4 271 Sandor et al. (1984)

Coho salmon 3H-TA 0.5 60 Shrimpton et al. (1995)(Oncorhynchus kisutch) 3H-TA 1.0 45 Shrimpton and Randall (1994)

Brook trout 3H-Cortisol 3.2 224 Chakraborti et al. (1987)Potency: dexamethasone > TA > 11-deoxycortisol>>cortisol > corticosterone >>cortisone

American eel 3-TA 2.8 188 Chakraborti et al. (1987)

(Anguilla rostrata)Potency: TA > dexamethasone > cortisol > 11-deoxycortisol > 21- deoxycortisol

Mozambique tilapia 3H-Cortisol 32 59 Kloas et al. (1998)3H-Dex 7 125

Common carp 3H-Cortisol 31 112 Kloas et al. (1998)3H-Dex 27 122

Brain

Chinook salmon 3H-Cortisol 4.5 25.4 Knoebl et al. (1996)(Oncorhynchus tshawtscha) 3H-TA 0.85 22.4

Potency: TA > dexamethasone > cortisol >>RU486>>corticosterone >>17-oestradiolRainbow trout hypothalamus 3H-Dex 12.5 Lee et al. (1992)

Potency: dexamethasone = TA>>>corticosterone

Rainbow trout hypothalamus 3H-Dex 1.22 296 Allison and Omeljaniuk (1998)Potency: dexamethasone > cortisol > corticosterone > TA = 11- deoxycortisol >>aldosterone >>17-oestradiol

Red tilapia 3H-Dex 9.6 66 Chen et al. (1977)Potency: dexamethasone > cortisol > 11-deoxycortisol >>17,20-dihydroxy-4-pregnen-3-one


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Table 3. Continued

Organ/tissue Ligand Kd(nM) Bmax Referencesand species (fmol per mg protein)

Intestine

Brook trout 3H-Cortisol 4.5 26.6 Chakraborti et al. (1987)

Intestinal mucosa

American eel 3H-TA 2.3 900 Dibattista et al. (1983)Potency: TA > dexamethasone > cortisol > 11-deoxycortisol

Kidney

Common carp 3H-Cortisol 58 51 Kloas et al. (1998)3H-Dex 70 82

Muscle

Brook trout 3H-Cortisol 2.8 8.3 Chakraborti et al. (1987)

Leukocytes

Chinook salmon 3H-Cortisol 2.2 41.4 Maule and Schreck (1991)3H-TA 0.38 37.8

Potency: TA > cortisol > 17-hydroxyprogesterone > cortisone > aldosteroneCommon carp, PBL 3H-Dex 3.8 490 Weyts et al. (1998)

Potency: TA > dexamethasone >>cortisol > cortisone

Erythrocytes

Rainbow trout 3H-Cortisol 4.7 0.33 Pottinger and Brierley (1997)Potency: cortisol = dexamethasone>>11-ketotestosterone >>17-oestradiol = cortisone

Not done; Dex, dexamethasone; TA, triamcinolone.Peripheral blood leukocytes.Value in binding sites per cell.

some systems has not been demonstrated (Akner etal., 1995), presumably the ligandGRhsp complexmoves to the nucleus to allow for DNA binding (Bam-berger et al., 1996; Guiochon-Mantel et al., 1996). Thehsp complex appears to be essential for this transport(Pratt, 1993) and specifically hsp56 (Czar et al., 1995)using a protein-transport system (Akner et al., 1995).The binding of the activated receptor to GREs willinitiate transcription.

Fish corticosteroid receptors

In contrast to the above, little is known concerningCR structure in fish. Fish are thought to have onlyone CR type, unlike mammals which contain distinctmineralocorticoid and glucocorticoid (MR and GR)receptors. This one receptor is termed a GR, consistentwith the lack of a significant amount of a unique min-eralocorticoid hormone in fish (Ducouret et al., 1995).

Northern blot analysis and other molecular techniquesindicate that GR mRNAs are expressed in a large num-ber of rainbow trout tissues, including liver, kidney,gill, intestine, skeletal muscle and brain (Ducouret etal., 1995; Teitsma et al., 1997, 1998). These methodssupplement earlier studies identifying CRs using com-petitive binding assays for different ligands, in tissuessuch as gills, liver, brain, intestinal mucosa, leuko-cytes, and erythrocytes of a number of fish species(Table 3).

The structure of the GR, its association with hspsor the existence of multiple forms has yet to beestablished. The rainbow trout GR (rtGR) is 758amino acids in length (similar to human GR, hGR)and has high sequence homology with hGR; thisincludes 90% homology within the DBD and 70%in the glucocorticoid-binding domain. However, thertGR contains an expanded region within the zinc


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finger sequence (Ducouret et al., 1995). This nineamino acid insert is located between the two zincfinger sequences, extending this region, while retain-ing high homologies within the zinc finger sequenceitself (Takeo et al., 1996; Tagawa et al., 1997). Theinsert is encoded by a unique exon not found in the

human gene. This modified gene is expressed in alltissues assayed in the trout, although testes expressboth this extended form and a form that lacks thenine amino acid insert, making this latter form moresimilar to other vertebrate GRs (Takeo et al., 1996;Tujague et al., 1998b). The functional significance ofthis insert is not clear. Using cotransfection assays inCHO cells, Tujague and co-workers (Tujague et al.,1998b)noticed that the insert does not affect transcrip-tional efficiency, but it may affect constitutive activitybecause cells with the insert had twofold higher levelsof GR expression than those without. Certainly thisdifference in sequence may affect other functions of

rtGR and may possibly be involved in the dual func-tioning of this CR in trout (Ducouret et al., 1995;Tujague et al., 1998a,b).

There is no report of hsp association with anyfish GR, but given the conserved nature of hsps ininvertebrate and vertebrate species, it is likely thata GRhsp complex exists in fish species as well.Evidence in support of the presence of a GRhsp com-plex is that the cytosol 3H-cortisol binding activityof brook trout liver and gill have molecular massesof 319 kDa (Chakraborti and Weisbart, 1987) and326 kDa (Chakraborti et al., 1987), respectively, while

American eel gill (Sandor et al., 1984) and intest-inal mucosa (Dibattista et al., 1983) are approximately335 and 230 kDa, respectively. Therefore, their func-tional molecular masses differ slightly from that ofthe complexed heteromeric mass of330 kDa repor-ted for mammalian GR (Gehring, 1993). Given thatthe number of amino acids and the predicted molecu-lar mass of GR in trout (Ducouret et al., 1995) andhuman (Gehring, 1993) are similar (85100 kDa), thisadditional mass of some 130 to 250 kDa is likelyattributable to hsps or related proteins.

Whether all CRs are GRs or type II receptors in fishtissues remains to be established. Although Ducouretand co-workers (Ducouret et al., 1995) reported onlyone type of CR in trout tissues with strong homolo-gies to hGR, their specific molecular approach a priorifavoured identification of only GR, not MR. Althoughdata in Table 3 would support the existence of only asingle GR type, we feel these data are not entirely con-clusive. Recepor densities (Bmax) differ for the various

tissue types, dissociation constants (Kd) are within the0.2 to 40 nM range and potency orders always favourthe synthetic steroids dexamethasone or triamcinoloneacetonide. In the few studies employing aldosterone(Sander et al., 1984; Knoebl et al., 1996; Allison andOmeljaniuk, 1998; Kloas et al., 1998), binding was

ineffectual compared with the classic glucocorticoidsor their analogues, providing further evidence that thereceptors present in fish tissues group with type II(GR) and not type I (MR) receptors. Hence, unlikemany mammalian tissues that express MRs and GRs,fish tissues may have only a single class of receptors.Additional pharmacological studies using aldosteroneare needed to validate the presence of a single receptortype in all fish tissues. It should also be noted thatthe species listed on Table 3 are primarily salmonids,and there is evidence for species differences withinmammalian CRs (Fuller, 1991; Burnstein and Cid-lowski, 1992; Gehring, 1993). Chen and colleagues

(Chen et al., 1997) using the red tilapia (Oreochromismossambicus niloticus) found only a single CR inall tissues examined. In addition, if as detailed below,fish skeletal muscle is an important target tissue forcortisol, the muscle GR needs to be examined, asthe only evidence that such a receptor exists is foundin the molecular studies of Ducouret and co-workers(Ducouret et al., 1995; Ducouret, 1996).

The brain represents a key site of glucocortic-oid and mineralocorticoid function in mammals (Joelsand De Kloet, 1995). With the availability of thertGR mRNA, experiments have now been undertaken

to establish expression and distribution of GRs inthe brain of trout. Previous studies demonstrated thepresence of GRs in fish brain (Table 3), but onlythe study of Allison and Omeljaniuk (1998) showedspecific binding to a part of the brain (hypothal-amus). Teitsma and colleagues (Teitsma et al., 1997)used rtGR mRNA and Northern blotting and in situhybridization to study brain expression and distribu-tion of GRs. Intensive hybridization to the preopticnucleus and the nucleus lateralis tuberis, the mainhypophysiotrophic regions in fish, occurred, lendingsupport to the basic physiological feedback mechan-isms reported in many fish species. These studies arekey to a better understanding of the effects and theinteractions of glucocorticoids within fish tissues.

Receptor compartmentation

The subcellular distribution of GRs is controversialin mammals (Akner et al., 1995; Guiochon-Mantel


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et al., 1996; Htun et al., 1996) and few studieshave examined this in fish (cf. Knobel et al., 1996).Evidence supports the nuclear localization of steroidreceptors in the presence or absence of steroid for allsteroid receptors, except the GRs. Translocation of areceptor protein to the nucleus requires the presence

of nuclear localization signals (NLS) and recognitionof these by binding proteins within the nuclear porecomplex. Studies have identified these NLS to specificregions between the DBD and the hormone-bindingregions, and if mutated, these receptors are localizedtothe cytosol until hormone activated when they move tothe nucleus (Guiochon-Mantel et al., 1996). Althoughthe mammalian GR has a homologous NLS sequencealigning with that of the progesterone receptor (PR)that is localized to the nucleus (Guiochon-Mantel etal., 1996), Picard and Yamamoto (1987) identified aregion within the carboxyl terminal or ligand-bindingsite that was more important. It is believed that upon

GR transformation by ligand the homologous NLSis critical for translocation. Certainly the binding ofhsp90, which is cytosolic, could protect these trans-locating sites, potentially restricting the GR to thecytosol, unlike other steroid receptors. The import-ance of hsps to the native structure of GR and itstranslocation to the nucleus has been reviewed extens-ively (Gehring, 1993; Pratt, 1993; Akner et al., 1995),but conflicting views of GR localization continue tobe reported (Brink et al., 1992; Akner et al., 1995)although the recent visualization of a rat GR bound toa green fluorescent dye should assist our understand-

ing of this issue (Htun et al., 1996). The prevailingview is that the exact composition of the GRhspcomplex determines the predominant direction of itsmovement (Bamberger et al., 1996).

Working with fresh tissues, Weisbart and col-leagues have separated and characterized trout cytoso-lic and nuclear GRs by using a low-osmotic-strengthhomogenization medium and differential centrifuga-tion. Brook trout liver cytosolic GR has a lower disso-ciation constant (Kd5.6 vs. 30 nM) and a lower max-imum binding capacity (Bmax167 vs. 858 fmol per mgprotein) than the nuclear GR using 3H-cortisol as a lig-and (Chakraborti and Weisbart, 1987) as does the gillGR (Kd3.2 vs 50 nM; Bmax224 vs 425 fmol per mgprotein; Chakraborti et al., 1987). Similar differencesexist between cytosol and nuclear GRs of rainbowtrout gill (McLeese et al., 1994), and values for fishGRs fall into comparable ranges to those reported forrats (Kd1.2 vs. 65 nMand Bmax40 vs. 1.1 fmol per mgprotein using3H-dexamethasone; Audouin-Chevallier

et al., 1995). The differences between nuclear andcytoplasmic binding constants and Bmaxindicate thatthe cytosolic GR has a higher affinity than the nuclearGR, and in fact Pottinger (1990) has suggested that thecharacteristics of the nuclear GRs would be expectedfor a nonreceptor-binding protein.

The physiological state of the fish can alter thecompartmental abundance of the GR receptor. Inbrook trout gill, for instance, the number of nuc-lear GRs is increased while that of cytosolic GRsdecreased after SW transfer (Weisbart et al., 1987).Because plasma cortisol levels were not correlatedto cytosolic GRs, but nuclear cortisol levels were tonuclear GRs, the authors suggested that the nuclearGRs were important to SW tolerance in this species.It is interesting to note that the complex of hsp90and GR is necessary to display a high-affinity GR(Simons and Pratt, 1995); as the hsp90 dissociatesfrom the GR within the nucleus, this low-affinity GR

reported by Weisbart and co-workers may in fact rep-resent a GR devoid of hsp90. Other studies with fishhave failed to quantify nuclear vs. cytosolic GRs, orhave found that nuclear GRs represented a very smallcomponent of the total GR pool, or that the poolsremained unchanged after ligand activation (Lee et al.,1992; Pottinger et al., 1994; Shrimpton et al., 1995).This discrepancy between the results of Weisbart andcolleagues and others may represent a simple method-ological difference; if not, the fish system may be quitedifferent from that in mammals and may be an appro-priate model system to probe the dilemma of cytosol

vs. nuclear localization for GRs. It should be noted,however, that most studies on fish report data for totalGR binding and do not distinguish between cytosolicand nuclear GRs.

Gene regulation

The GR has been considered to be a direct signal-transduction system, in that it binds hormone andin its transformed state moves to the nucleus whereit affects the transcription of specific genes (Pratt,1993; Jewell et al., 1995). Although this view is

slowly changing, the principal role of the GR remainsas a transacting transcription factor. The evidenceis that once ligand-bound GR is in the nucleus, itbinds through its DNA-binding domain to one or morehormone-response elements, termed glucocorticoid-response elements (GREs), located in the 5-region ofthe promoter of glucocorticoid-sensitive genes. Bam-berger et al. (1996) used the term type 1 mechanism


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to describe this GR action. The GRE lacks specificityas it can be bound by a variety of steroid receptorsincluding those for progesterone, androgen and miner-alocorticoids (Beato et al., 1996). GR binds as a dimerand the interaction of the zinc fingers of the DBDand the GRE with the two trans-activation domains

on GR are probably responsible for the specificity ofgene transcription (Evans, 1988; Fuller, 1991; Beatoet al., 1996). In addition, a series of interactionswith other transcription factors called co-activatorsare thought to increase the efficiency of transcriptionby RNA polymerase II. The list of co-activators isincreasing rapidly, but already includes TFII andsteroid receptor activator 1 (SRC-1) (Bamberger etal., 1996) and possibly the cAMP response-element-binding protein (CREB) (Smith et al., 1996). GREsand oestrogen-responsive elements are closely related.For instance, by making one or two base substitutions(Klock et al., 1987) to this oestrogen-response ele-

ment, it is possible to confer a glucocorticoid responseto a conserved palindromic sequence in the 5-flankingregion present in fish, frog and chicken vitellogeningenes.

A second (type 2) mechanism of GR action inhib-its rather than activates transcription (Bamberger etal., 1996). The promoter lacks a specific GRE, butGR binds to transcription factors such as Jun and Fosfamily proteins and blocks their stimulatory actions ongenes including those of the immune system.

There is no comparable information on any fishspecies or tissue. Glucocorticoids have been shown to

modify metabolism and activities of specific enzymesas well as growth, implying changes in transcrip-tion. Given that the GREs are promiscuous, that thetrout GR protein is altered at the zinc finger sequence(Ducouret et al., 1995), and that fish are devoid ofan unique mineralocorticoid, the fish GR-transcriptionsystem could be important to assist our understand-ing of the complex evolution of this steroid-receptorfamily. This area could also be important in terms ofthe possible relationship between the responses of fishto toxicants such as dioxins, and the stress responseinitiated by glucocorticoids. Recently, Celander andco-workers (Celander et al., 1996) reported that dexa-methasone potentiates the actions of aryl hydrocar-bon receptor inducers on both cytochrome P4501A(CYP1A) activities and protein, but not on CYP3A.This result suggests a specific interaction betweeninducer elements and a GRE on the CYP1A gene, andnot only a widespread location of GREs in the fishgenome, but also that the fish system is not unlike that

seen for mammals. For instance, a GRE-like motifwas localized on the rainbow trout prolactin gene(tPRL) promoter (Argenton et al., 1996). Activation oftPRLapparently requires the simultaneous occupancyof both the GRE site and a second site for an addi-tional transcription factor (GHF1). During vertebrate

evolution, the conservation of GHF1 appears criticalfor PRL, but what seems to differ are the modulatorsof this particular site. Studies in this area may dis-cover some very fascinating evolutionary differencesand may explain the absence of a mineralocorticoid inteleosts.

Glucocorticoid receptor regulation

The regulation of GR activity has been reviewed forthe mammals (Gehring, 1993; Schmidt and Meyer,1994; Bamberger et al., 1996). Clearly, the sensit-ivity of a particular tissue to corticosteroids will beestablished by the type and level of receptor expressedand will also be affected by either down-regulationor up-regulation of expression. Arriza and co-workers(Arriza et al., 1988) have also suggested, for mam-malian tissues that coexpress GR and MR, that the MRmay function as a high-affinity GR. GR, in turn, mayonly be activated at much higher circulating concen-trations of corticosteroids. This selectivity mechanismis in addition to the well-known enzymatic conversionof cortisol to cortisone by 11-hydroxycorticosteroiddehydrogenase, permitting aldosterone binding to MRrather than cortisol (Fuller, 1991). Autoregulation of

GRs is dependent upon the tissue or cell type, devel-opmental stage and the prior corticosteroid history ofthe animal/tissue. Homologous down-regulation hasbeen shown to occur by decreasing the rate of GR genetranscription (thus reducing GR mRNA and protein),destabilization of GR mRNA (post-transcriptional)and reduction in the half-life of GR protein (post-translational, including phosphorylation). In eachcase, it is clear that either the glucocorticoid or the GRinteracts with specific sequences on the GR mRNA orcDNA. The mechanism of homologous up-regulationis not understood, but is known in both normal and

adrenalectomized mammals. Heterologous regulationby other steroid hormones, and in particular the sexsteroids, by regulating levels of GR mRNA, has alsobeen reported. The cloning of the human GR pro-moter (Nobukuni et al., 1995) should assist in ourunderstanding of these regulatory features; certainlythe existence of a large number of sites for transcrip-tion factors means that many components could alter


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GR mRNA levels. Recent studies also indicate theexistence of endogenous, low-molecular-weight mod-ulators (a phosphoglyceride) of corticosteroid recept-ors (GR and MR) in mammalian liver cytosol (Bodineand Litwack, 1995), but how these modulators arerelated to GR regulation is at the moment unclear.

It is clear, however, that many other components ofthe glucocorticoid signal cascade system are modu-lated by a host of factors which in themselves aremodulated (Bamberger et al., 1996). This complexitywill increase as additional components of the systembecome better defined. GR is a phosphoprotein, andseven phosphorylation sites have been found in themouse receptor, all but one in the amino terminaldomain (Kuiper and Brinkmann, 1994). Although therole of phosphorylation is clear for some of the sexsteroid receptors (e.g., oestrogen), no such evidenceis available for GR. In fact, some feel (Bamberger etal., 1996) that phosphorylation has no impact on GR

activity, but may be more important to establish cel-lular localization. This is an obvious area for furtherstudies.

Again, studies with fish systems have shown GRautoregulation, but the precise mechanism(s) is com-monly not understood. Generally, an inverse correl-ation is noted between plasma cortisol level and thenumber of GRs in fish tissues. This has been clearlydemonstrated in liver by both confinement stress,which increases plasma cortisol, and injection orimplants containing either cortisol or dexamethasone(Pottinger, 1990). No such correlation was noted

in trout injected with 3,3

,4,4

-tetrachlorobiphenyl(TCBP), even though plasma cortisol values increasedby 3.5 fold (Vijayan et al., 1997b). Other studiesfound a significant linear correlation in trout betweenapproximately 1 and 30 ng mL1 cortisol and Bmaxvalues in control and cortisol-dosed fish, without alter-ations in the Kd (Pottinger, 1990; Lee et al., 1992).Similarly, trout brain GR numbers were inverselyrelated to plasma cortisol (Lee et al., 1992), witha unique increase in Kd. Carp fed a single cortisol-containing meal had increased plasma concentrationsof the hormone for one day (Weyts et al., 1998).This increase was associated with a 50% decrease incortisol binding to peripheral blood leukocytes by 3 hthat persisted for more than 4 days even though plasmacortisol had returned to baseline. The observation thataddition of linoleate (C18:2) to trout liver cytosolincreased the Kd, but did not affect the Bmaxfor 3H-dexamethasone binding, was interpreted as indicativeof changes in GR conformation, but the precise mech-

anism is unknown (Lee and Struve, 1992; Lee et al.,1992). Unsaturated fatty acids inhibit hepatic cytosol3H-dexamethasone binding in a mammal by a mixed,noncompetitive manner (Sumida, 1995), without alter-ing GR mRNA, suggesting that the fatty acids maybe acting indirectly through a protein kinase system.

Because fish fatty acid composition differs from thatof endothermic mammals and can be altered experi-mentally by thermal adaptation (Hazel, 1993) and bydietary manipulation (Labbe et al., 1995), fatty acidcontrol over fish GRs could be an intriguing area forfurther research.

The situation with gill GR binding is complic-ated by developmental stage, especially smoltific-ation in salmonids. As noted elsewhere in thisreview, smoltification is associated with increasedplasma cortisol levels, increased gill Na+/K+-ATPaseactivities, chloride cell proliferation and enhancedSW tolerance. Generally, numbers of gill GRs are

positively related to the ability of a salmonid forhypo-osmoregulation, i.e. to undergo smoltification(Shrimpton et al., 1994). However, a number of stud-ies have reported that both acute and chronic elevationof cortisol decreased the maximum binding capacityof gill GRs in salmon and trout (Maule and Schreck,1991; Shrimpton and Randall, 1994; Shrimpton et al.,1994; McLeese et al., 1994). It is this decreased sens-itivity to circulating cortisol that is thought to interferewith smoltification in stressed salmonids (Shrimptonand Randall, 1994). In a related experiment, injectionof the somatotrophic (mammalian) placental lactogen

or (piscine) growth hormone increased both the num-ber of gill GRs and gill Na+/K+-ATPase activitieswithout consistent impact upon plasma cortisol levelsin coho salmon (Shrimpton et al., 1995). These resultswere interpreted as indicating that the simultaneousrelease of somatotrophins and cortisol during smoli-tification may ameliorate the direct effects of cortisolon decreasing gill GRs. Recently Tagawa et al. (1997)also found increased intensity of GR mRNA in gillsfrom tilapia raised in FW compared with fish raised infull-strength SW.

In no case has there been a mechanisticexplanationfor down-regulation of any fish tissue GR, althoughas mRNA probes and antibodies become available formore GRs, mechanistic answers will not be far behind.Sea water transfer in brook trout was associated withincreased numbers of gill nuclear GRs (Weisbart etal., 1987), suggesting this translocation was critical,but no other study has confirmed this observation. Thestudies using somatotrophins are intriguing (Shrimp-


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ton et al., 1995), but the authors indicate that theeffects of growth hormone are additive; as a result, up-regulation, in this case heterologous autoregulation,is not an issue. The fact that cortisol has permissiveeffects on fish metabolism, as in mammals, mayimplicate autoregulation of fish tissue GRs by other

hormones. Studies examining the GR promoter in thefish system need to be undertaken to achieve a bet-ter understanding of which factors may regulate fishGRs. These molecular technologies have already beenused successfully for other fish promoters, for exampleprolactin in the context of glucocorticoid activation(Argenton et al., 1996) and insulin (Argenton et al.,1997), and should be readily transferable to the studyof the GR.

Fasting in rainbow trout resulted in increasedamounts of cortisol specifically bound to erythrocytes(Pottinger and Brierley, 1997). Few similar studieshave been undertaken, although for tilapia, red blood

cells have been shown to express higher levels of GRmRNA than any other tissue examined (Tagawa et al.,1997). This study points to a lack of information onhow stressors affect the density of GRs in fish speciesand should provide new avenues for future studies onthe control of fish GRs. Now that antibodies have beenproduced for the rtGR (Tujague et al., 1998a), we arecloser to more appropriate models for assessing GRexpression under a variety of conditions.

Nongenomic actions

Increasing evidence suggests that steroid hormones,including glucocorticoids, exert nongenomic effects.For the mammals, this area has been reviewed(Wehling, 1997) and the list of steroid hormones andtissues involved is extensive. Nongenomic effects ofsteroid hormones are rapid (seconds to a few minutes).For instance, the effects of dexamethasone on glyco-gen metabolism peak within 515 min of exposureto this analogue (Baqu et al., 1996). The effects arealso insensitive to inhibitors of transcription or proteinsynthesis, actinomycin D and cycloheximide, and gen-erally involve membrane actions and changes in intra-

cellular calcium ([Ca

2+

]i). The best-studied cases arealdosterone and progesterone actions in mammaliankidneys and sperm/oocytes, respectively.

The case for nongenomic actions of glucocor-ticoids is less well established, but, again, sup-porting evidence is accumulating. Radioligand (3H-dexamethasone) and immunofluorescence studies ledGrote and colleagues (Grote et al., 1993) to sug-

gest GR distribution to both cytosol and membranecompartments of rat liver cells. Others were ableto distinguish between cytosol and membrane 3H-dexamethasone binding by rate of association, differ-ential competition with glucocorticoid agonists, andsensitivity to the thiol reactive agent arsenite, again in

rat liver (Wright and Paine, 1995). Other studies havebeen summarized by Wehling (1997).Comparable phenomena of rapid action have been

described for cortisol and dexamethasone in fish. Ina tilapia pituitary perifusion preparation, for instance,the stimulation of prolactin release by a hyposmoticmedium was blocked by cortisol (200 nM) and becamesignificant compared with controls by 20 min (Bor-ski et al., 1991). In addition, the increase in cAMPand Ca2+ in these cells to hyposmotic medium couldalso be blocked by cortisol within 15 min, imply-ing that cortisol can have a rapid effect and thatthis effect is mediated through pathways that modify

cAMP and Ca2+ levels. Similarly, dexamethasoneexerted a short-term tropic effect on a goldfishmelano-cytoma cell line. Within 20 min of application of theanalogue, cells flatten out and undergo extension andbroadening of dendrites. The effect was specific forglucocorticoid, as it was blocked by the corticoster-oid antagonist RU486. More importantly, however,the reaction to dexamethasone remained unaffected bycycloheximide or actinomycin D (Shih et al., 1990),clearly indicating that the effect was nongenomic andindependent of transcription and protein synthesis. Asin the case of prolactin release (Borski et al., 1991),

Ca2+

seems to play an important role in the rapidchanges in melanocytoma cell morphology (Shih andLo, 1993). The observation that3H-cortisol binds spe-cifically, albeit to a limited extent at around 8% oftotal binding, to trout erythrocyte membranes (Pot-tinger and Brierley, 1997) provides another indicationthat nonstandard mechanisms for corticosteroid actiondo occur in fish. It remains to be seen how widespreadthese phenomena are and whether they have meta-bolic consequences. Metabolic studies using cortisolin fish generally preclude the identification of suchshort-term, possibly nongenomic, actions of this hor-mone. Even so-called acutein vitrohepatocyte stud-ies (Renaud and Moon, 1980; Foster and Moon, 1986)sample after 1 to 2 h, i.e. well past the time of changesnoted in the tilapia pituitary or goldfish melanocyt-oma cells, and those particular hepatocyte studies didnot use inhibitors of protein synthesis or transcrip-tion. Therefore, careful short-term studies need to beundertaken, possibly using the hepatocyte system to


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include the possibility of identifying surface receptorsfor cortisol or its agonists in fish cells.

Metabolic regulation

The treatment of fish with cortisol is even more variedthan the number of species examined. Apart from theexpected in vivoversusin vitroapproaches, treatmentshave been done with suspected agonists (cortisol,dexamethasone, triamcinolone), and different modesof application(single or repeated injection, at differingsites, oral application, various oil deposits or osmoticminipumps). These varied approaches are exacerbatedby a bewildering array of treatment times, rangingfrom minutes to weeks. Other routes of inquiry haveemployed crowding, exhaustive exercise or similarstress situations with the aim of increasing endogen-ously produced cortisol concentrations. While this isnot the place to discuss the relative merits of thesedifferent approaches, it must be kept in mind that theexperimental design is likely to strongly influence theoutcome of a particular experiment (Gamperl et al.,1994). As outlined in the following, this considerationis nowhere more obvious than in a discussion of gluc-ocorticoid action on carbohydrate, protein and aminoacid metabolism. Furthermore, biological variationbetween species will have a bearing on experimentaloutcomes, rendering the derivation of common themesfor cortisol action a difficult task, especially for proteinmetabolism, which is a priori not easily accessible

experimentally.A clear differentiation must be made between

short-term, acute effects and long-term treatmentswith cortisol. Under acute stress situations, plasmacortisol concentrations tend to shoot up within aminute to hour time frame, followed by a gradualdecrease to pretreatment levels within a day or so,depending upon subsequent maintenance conditions.Hence, under longer-term stressful situations, the fishseem to adapt to the stress and plasma cortisol fluctu-ates within the range considered normal for a partic-ular species. An acute stress can be simulated experi-

mentally with a single injection of cortisol (generallya stress itself, thus releasing endogenous cortisol; seebelow), although the time course of an augmentedcortisol titre tends to be longer than under acute stresssituations. In contrast, long-term exposure to exogen-ous cortisol with oil or similar deposits usually resultsin chronically elevated plasma cortisol concentrations.Independent of macro- or micro-control over plasma

cortisol concentrations, it is clear that carbohydrates,protein turnover, amino acid dynamics and lipids areconsistently identified as important control points forthis hormone.

Carbohydrates

Glucocorticoids modulate hepatic glucose metabol-ism in vivo in mammals (Goldstein et al., 1992,1993) and in isolated hepatocytes in vitro (Jones etal., 1993), generally stimulating gluconeogenesis andincreasing liver glycogen content. Two key mechan-isms have been identified by which glucocorticoidsstimulate gluconegoenesis. The first is induction ofphosphoenolpyruvate carboxykinase (PEPCK) activ-ity by increased transcription (Granner et al., 1986)of PEPCK mRNA by sixfold, especially in the pres-ence of glucagon, together with a fourfold increasein the stability of its mRNA. This stabilization isa process dependent on a glucocorticoid-responsiveelement (GRE) in the 3-noncoding region of the PEP-CKmRNA (Heinrichs et al., 1994). At the same time,glucocorticoids destabilize the mRNAs of other hep-atic proteins, including those of interleukin 1 and3-hydroxymethylglutaryl-CoA reductase. The secondmechanism is a stimulatory effect on hepatic precursorsupply, thereby sustaining gluconeogenesis in vivo(Fujiwara et al., 1996). Stimulation of glycogen syn-thesis is thought to be due to the hormone-dependentactivation of glycogen synthase by increasing glyco-gen synthase phosphatase activity (Vanstapel et al.,

1982), although glucocorticoid stimulation of glyco-gen synthesis in mammalian liver is not supportedby all studies. At various times, the absence of gly-cogen synthesis (Hems and Whitton, 1980), reducedglycogen content and activated glycogenolysis (Baquet al., 1996) have been reported. In isolated hep-atocytes, glucocorticoid did not stimulate glycogensynthase, even though glycogen synthase phosphatasewas activated (Laloux et al., 1983). In recent studieswith rat hepatocyte primary culture, dexamethasoneincreased both glycogen synthase and glycogen phos-phorylase activities without changing their mRNA

content, suggesting that the model glucocorticoid con-trols the activity of these enzymes by modifying theirphosphorylation state rather than by affecting geneexpression (Baqu et al., 1996). Also, the rapid activ-ation of glycogen phosphorylase with dexamethasone,peaking between 5 and 15 min after dexamethasoneadministration, clearly supports possible nongenomiceffects of glucocorticoid on glycogen metabolism.


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Table 4. Changes in blood glucose and liver glycogen with exposure to corticosteroids in teleost fish

Change Species References

Plasma glycaemia

Increase American eel (Anguilla rostrata) Butler (1968)Japanese eel (Anguilla japonica) Inui and Yokote (1975), Chan and Woo (1978)European eel (Anguilla anguilla) Lidman et al. (1979)

Roach (Rutilus rutilus, Cyprinidae) Mller and Hanke (1974)Gulf toadfish (Opsanus beta, Batrachoididae) Mommsen et al. (1992)Sea raven (Hemitripterus americanus) Vijayan et al. (1997a)Mozambique tilapia (Oreochromis mossambicus) Vijayan et al. (1997a)Killifish (Fundulus heteroclitus, Cyprinodontidae) Leach and Taylor (1982)Coho salmon (Oncorhynchus kisutch) Vijayan and Leatherland (1989)Cutthroat trout (Oncorhynchus clarki) Morgan and Iwana (1996)Rainbow trout (Oncorhynchus mykiss) de la Higuera and Cardenas (1986), Barton et al. (1987)

Decrease American eel Foster and Moon (1986)

Brook charr (Salvelinus fontinalis) Vijayan et al. (1991)Unaltered Channel catfish (Ictalurus punctatus, Ictaluridae) Davis et al. (1985)

Brook charr Tam et al. (1988)

Rainbow trout Leatherland (1987), Andersen et al. (1991), Vijayan et al. (1994a)

Liver glycogen

Increase American eel Butler (1968)Japanese eel Inui and Yokote (1975), Chan and Woo (1978)

European eel Lidman et al. (1979)Killifish Leach and Taylor (1982)Mozambique tilapa Swallow and Fleming (1970)Brook charr Whiting and Wiggs (19

cortisol en peces

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