Mineralocorticoid Receptors and Hormones: Fishingfor Answers

In tetrapod vertebrates, aldosterone is the main mineralo-corticoid hormone, playing a key role in the regulation ofsodium transport across epithelia. Metabolic functions areregulated by the glucocorticoid hormones cortisol and/orcorticosterone. The mineralocorticoid and glucocorticoidhormones are produced by the adrenal cortex and exert theireffects through separate, well-characterized receptors be-longing to the superfamily of steroid hormone receptors thatact as ligand-dependent transcription factors (1). By contrast,most fish appear to lack aldosterone; neither attempts tomeasure the hormone itself (2) nor attempts to find an en-zyme with significant aldosterone synthesizing activity havebeen successful (3, 4). Cortisol is the principal corticosteroidproduced by the interrenal tissue, the piscine equivalent ofthe adrenal cortex, and contributes to the regulation of saltand water balance as well as metabolism (5). Yet despite theapparent absence of a selective mineralocorticoid hormone,fish possess, as Sturm et al. report in this issue of Endocri-nology (6), mineralocorticoid receptors (MRs). Their workreopens the debate about whether fish possess a mineralo-corticoid hormone, while at the same time adding to a recentbut growing body of evidence supporting the presence ofmultiple corticosteroid receptors in teleost fish (7–9).

Sturm et al. (6) cloned two MR isoforms from rainbow troutand examined their activation by various corticosteroids. Asexpected, cortisol was a potent agonist of the trout MRs,exhibiting EC50 values of 0.5–1.1 nm. This sensitivity to cor-tisol is similar to or greater than that of the trout glucocor-ticoid receptors (GRs), which exhibit EC50 values of 0.7–46nm (9). A similar comparison has been made for only oneother fish species, a cichlid, and here again the MR appearsto be more sensitive to cortisol than the GRs (7). In thisregard, the fish MRs parallel their mammalian counterparts,which are considered to be high-affinity cortisol receptors.Indeed, the high affinity of the mammalian MR for cortisolcoupled with the approximately 100-fold higher circulatinglevels of glucocorticoids than aldosterone would result incontinuous activation of MRs by cortisol were it not for thepresence of a cellular sentinel that excludes cortisol fromsome MR-expressing cells. In the mammalian kidney, forexample, MRs are coexpressed with the enzyme 11�-hydrox-ysteroid dehydrogenase type 2 (11�-HSD) which inactivatescortisol but not aldosterone, conferring selective activationby the mineralocorticoid hormone (10). Whether a similarprotective mechanism exists in fish remains to be deter-mined, but fish do at least possess the necessary enzyme (11)

and a variety of tissues exhibit both MR and 11�-HSD geneexpression (6, 7, 11).

If selective mineralocorticoid activation of the fish MR isin fact possible, what is the compound with selectivemineralocorticoid action in fish? Sturm et al. (6) surveyed anumber of corticosteroids and found that, in addition toaldosterone, 11-deoxycorticosterone was the most potent ac-tivator of the trout MRs; EC50 values for aldosterone and11-deoxycorticosterone were about 10 times lower than thosefor cortisol. Like aldosterone, 11-deoxycorticosterone is aselective MR agonist: it does not activate the trout GR (9).Unlike the situation for aldosterone, however, the capacity tosynthesize 11-deoxycorticosterone is present in fish (Fig. 1;Ref. 12) and, although data are sparse, the compound itselfappears to be present in fish blood at levels that are not onlymeasurable but are comparable to the concentrations re-quired to activate the trout MR (13). In suggesting the po-tential for 11-deoxycorticosterone to function as a fish min-eralocorticoid hormone, Sturm et al. (6) have presented anintriguing challenge to the research community. To establish11-deoxycorticosterone as a physiologically significant fishMR ligand in vivo will require that changes in circulating11-deoxycorticosterone levels occur in response to a relevantdisturbance, and that physiological responses appropriatefor the correction of that disturbance be initiated by 11-deoxycorticosterone administration. However, the physio-

Abbreviations: GR, Glucocorticoid receptor; 11�-HSD, 11�-hydrox-ysteroid dehydrogenase type 2; MR, mineralocorticoid receptor.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

FIG. 1. Schematic of the probable pathways involved in cortisol and11-deoxycorticosterone biosynthesis in fish. Solid black arrows indi-cate that the enzyme required for that step has been cloned in fish anddisplays appropriate activity (3, 12); unfilled arrows indicate steps forwhich confirmation of enzyme activity is required. The gray arrowindicates that the enzyme required for that step has been cloned infish but did not display the appropriate activity (12).

0013-7227/05/$15.00/0 Endocrinology 146(1):44–46Printed in U.S.A. Copyright © 2005 by The Endocrine Society

doi: 10.1210/en.2004-1390

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logical consequences of MR activation in fish have yet to beidentified.

In mammals, MRs in the kidney are activated by aldoste-rone for the regulation of sodium retention, and hence saltand water balance (14). Aldosterone administration into fish,by contrast, appears to promote salt loss (15). Although thiseffect is the opposite of that in mammals, it does suggest thatMRs play a role in electrolyte balance in fish. In fish, regu-lation of hydromineral balance relies on the integrated re-sponses of the kidney and gills, unlike the situation in mam-mals, in which the kidneys predominate. Attempts todistinguish between kidney and gill responses suggestedthat mineralocorticoid administration promoted renal saltretention but branchial salt loss in rainbow trout—and, co-incidentally, deoxycorticosterone acetate was the MR agonistused in these experiments (16). The direction of net sodiummovement in fish depends on the salinity of the environment.Regardless of whether freshwater or marine environmentsare inhabited, the body fluids of teleost fish are maintainedat an osmotic concentration that is about one third seawaterstrength. Freshwater fish counter the resultant water gainand ion loss by the copious production of dilute urine andby active salt uptake across the gills. Marine teleosts drink seawater to replace water losses and actively excrete the result-ant salt load via the gills and kidney. Fish that move betweenthese two environments (euryhaline species) arguably pro-vide the most useful models for examining the hormonalcontrol of salt and water balance.

Studies of such euryhaline species have revealed a keyregulatory role for cortisol (5, 17). Cortisol levels increasewhen euryhaline fish are transferred from fresh water to seawater, and cortisol treatment improves salinity tolerance, inpart by increasing the activity of a key element of the gillsalt-secreting mechanism, Na�-K�-ATPase. However, par-allel increases in salt uptake and the prevalence of ion trans-porting cells in the gills occur after cortisol treatment infreshwater fish. Additionally, transient increases in cortisollevels accompany transfer to more dilute environments.Thus, cortisol appears to be a piscine switch hitter, promotingsalt secretion in sea water but salt uptake in fresh water (17).Interestingly, at least some of these mineralocorticoid actionsof cortisol are mediated by GRs. Gill GR gene expression (18,19) and numbers increase with seawater acclimation (20),and these changes translate into greater stimulation of Na�-K�-ATPase activity by cortisol (17). Also, the intestinal ab-sorption of imbibed water that accompanies the transitionfrom fresh water to sea water in developing salmon wasinhibited by GR blockade (21). To what extent MRs are reg-ulated by salinity changes remains to be determined, as dothe osmoregulatory responses that are linked to MR activa-tion. Indeed, unraveling the roles of GRs and MRs in themaintenance of salt and water balance in fish will requirepatience and ingenuity, given the suite of corticosteroid re-ceptors that has been identified in two teleost fish (6–9). Thisprocess must also take into consideration possible mecha-nisms, such as 11�-HSD, that may regulate access to the tworeceptor types. Finally, in light of the findings of Sturm et al.(6), a clear need exists to measure 11-deoxycorticosteronelevels during osmoregulatory disturbances and to readdress

the question of whether fish possess a selective mineralo-corticoid hormone.

Fish MRs are broadly distributed beyond the tissues (suchas gill; Fig. 2) that are important in salt and water balance.Interestingly, of the 12 rainbow trout tissues examined bySturm et al. (6), MR mRNA levels were highest in brain. Brainwas also the only cichlid tissue examined that displayedgreater MR than GR gene expression (7). In mammals, MRsare found in the brain in the absence of 11�-HSD expression.Without the protective effect of 11�-HSD, brain MRs prob-ably function as high-affinity cortisol receptors, but identi-fying the physiological roles of brain MRs vs. GRs is anongoing challenge (22). Thus, investigating the physiologicalfunctions of fish MRs in nontraditional tissues is yet anotheritem to add to the growing “to-do” list. In short order,corticosteroid signaling in fish has expanded from a onehormone-one receptor system to a system encompassingmultiple receptor types (6–9) and, given the findings ofSturm et al. (6), potentially multiple hormones. Additionalfishing for answers will clearly be required to fully elucidatethe complexity of this corticosteroid signaling system.

FIG. 2. Immunofluorescence localization of mineralocorticoid (MR)protein in the gills of rainbow trout (Oncorhynchus mykiss). A com-mercially available goat polyclonal antibody raised against the N-terminal region of the human MR together with an antigoat fluores-cein isothiocyanate-conjugated secondary antibody were used to labeltrout gill sections (A). Nuclei were visualized using 4�,6�-diamidino-2-phenylindole and are shown in overlay in blue. The specificity of theimmunoreactivity is indicated by the absence of immunostaining insections incubated without the primary antibody (B), or incubatedwith primary antibody in the presence of excess peptide against whichthe antibody was raised (C).[Figure courtesy of M. Bell, Departmentof Biology, University of Ottawa, Ottawa, Ontario, Canada.]

Gilmour • News & Views Endocrinology, January 2005, 146(1):44–46 45

Kathleen M. GilmourDepartment of BiologyUniversity of OttawaOttawa, Ontario, Canada K1N 6N5

Acknowledgments

Received October 21, 2004. Accepted October 26, 2004.Address all correspondence and requests for reprints to: K. M. Gil-

mour, Department of Biology, University of Ottawa, 150 Louis Pasteur,Ottawa, Ontario, Canada K1N 6N5. E-mail: katie.gilmour@science.uottawa.ca.

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46 Endocrinology, January 2005, 146(1):44–46 Gilmour • News & Views