The teleost fish are thought to lack the mineralocorticoid hormone aldosterone but possess mineralocorticoid receptor (MR) homologs. Here we describe the characterization of two rainbow trout (Oncorhynchus mykiss) MRs, called rtMRa and rtMRb. The open reading frame of rtMRa cDNA encoded a protein of 1041 amino acids. The rtMRb predicted protein sequence is similar, differing in only 10 amino acids in the nonconserved A/B domain and lacking a three-amino acid insertion between the two zinc fingers of the C domain. Expression of rtMR mRNA (sum of both forms), measured in juvenile trout by real-time RT-PCR, shows that the transcripts are ubiquitous. Expression was significantly higher in brain than the other tissues studied (eye, trunk kidney, head kidney, gut, gills, liver, spleen, ovary, heart, white muscle, skin). Hormonal stimulation of receptor transactivation activity was studied in COS-7 cells transiently cotransfected with receptor cDNA and a mouse mammary tumor virus-luciferase reporter. The mineralocorticoids 11-deoxycorticosterone and aldosterone were more potent enhancers of rtMRa transcriptional activity (EC50 = 1.6 +/- 0.5 x 10(-10) and 1.1 +/- 0.4 x 10(-10) M, respectively) than the glucocorticoids cortisol and 11-deoxycortisol (EC50 = 1.1 +/- 0.3 x 10(-9) and 3.7 +/- 1.9 x 10(-9) M, respectively). A similar response was observed in transactivation assays with rtMRb. These results are discussed in the view of reported circulating levels of corticosteroids in trout.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7·3 kb transcript was observed in various tissues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K d =5·05±0·45 nM; GR2 K d =3·04±0·79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10 −6 M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC 50 =0·72±0·87 nM) compared with rtGR1 (cortisol EC 50 =46±12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Aldosterone regulates renal sodium reabsorption through binding to the mineralocorticoid receptor (MR). Because the glucocorticoid receptor (GR) is expressed together with the MR in aldosterone target cells, glucocorticoid hormones bound to GR may also intervene to modulate physiological functions in these cells. In addition, each steroid can bind both receptors, and the MR has equal affinity for aldosterone and glucocorticoid hormones. Several cellular and molecular mechanisms intervene to allow specific aldosterone regulatory effects, despite the large prevalence of glucocorticoid hormones in the plasma. They include the local metabolism of the glucocorticoid hormones into inactive derivatives by the enzyme 11beta-hydroxysteroid dehydrogenase; the intrinsic properties of the MR that discriminate between ligands through differential contacts; the possibility of forming homo- or heterodimers between MR and GR, leading to differential transactivation properties; and the interactions of MR and GR with other regulatory transcription factors. The relative contribution of each of these successive mechanisms may vary among aldosterone target cells (epithelial vs. nonepithelial) and according to the hormonal context. All these phenomena allow fine tuning of cellular functions depending on the degree of cooperation between corticosteroid hormones and other factors (hormonal or tissue specific). Such interactions may be altered in pathophysiological situations.

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