Cortisol resistance in the degu (Octodon degus).

Cortisol resistance has also been reported in the degu, Octodon degus, a New World hystricomorph endemic to central Chile. The degu is used as a model for studies of stress and diurnal rhythms, parental behaviour and female masculinization. Another New World hystricomorph, the guinea pig, also exhibits glucocorticoid resistance, a result of amino acid sequences that differ from other mammalian glucocorticoid receptors (GR). Mutations in the ligand-binding domain of the human GR have been identified in familial or sporadic generalised cortisol resistance as have variants in the guinea pig. To address the possibility that the high levels of cortisol observed in the degu are a result of the same or similar sequence variations observed in the guinea pig GR, we have cloned, expressed and characterised the ligand-binding domain (LBD) of the degu GR. Somewhat unexpectedly, neither the amino acids nor the region involved in the resistance observed in the guinea pig GR are relevant in the degu GR. The relative resistance to cortisol observed in the degu GR is conferred by the substitution of two isoleucine residues, which are highly conserved in the GR across species, with a valine doublet. These amino acids lie in the region between helices 5 and 6 of the GR LBD, a region known to be important in determining the affinity of ligand-binding in steroid receptors.

Characterization of the zebrafish (Danio rerio) mineralocorticoid receptor.

Comparison between evolutionarily distant receptors can provide critical insights into both structure and function. Sequence comparison between the mineralocorticoid receptors (MR) of the zebrafish (zMR) and human (hMR) reveals a high degree of sequence conservation in the major functional domains. We isolated a zMR cDNA to contrast the transcriptional response to a range of ligands and to establish whether a teleost MR exhibits the amino/carboxyl-terminal interaction (N/C-interaction) previously reported for the hMR. Aldosterone, deoxycorticosterone (DOC) and cortisol induced zMR transcriptional activity with similar efficacy to that observed with the hMR. The hMR antagonist, spironolactone, acted as an agonist with the zMR. The zMR exhibited an N/C-interaction in response to aldosterone but, in contrast to the hMR, cortisol and DOC predominantly stimulated the interaction in the zMR. Conservation of the N/C-interaction between evolutionarily distant MR provides evidence of functional significance.

A direct effect of aldosterone on endothelin-1 gene expression in vivo.

Aldosterone regulates sodium reabsorption in epithelial tissues such as the kidney and colon, via a pathway involving the activation of intracellular mineralocorticoid receptors (MR), induction of specific target genes, and a subsequent increase in sodium channel activity. Characterized aldosterone target genes in epithelia include the serum and glucocorticoid-regulated kinase 1 and the corticosteroid hormone-induced factor. Endothelin-1 (ET-1) is a potent vasoconstrictor that alters both sodium transport and hydrogen ion secretion in the kidney. Recent studies in a mouse medullary collecting duct cell line and rat A-10 smooth muscle cells have demonstrated an acute response of ET-1 gene expression to aldosterone. In the present study, we have investigated the ET-1 gene in vivo as a potential direct aldosterone-regulated target gene in the kidney and colon. Adrenalectomized rats given a single dose of aldosterone were found to have a 2-fold increase in ET-1 mRNA levels in the kidney and colon after 1 h. No significant changes in mRNA levels were detected for the related isoforms ET-2 or ET-3. Cotreatment with aldosterone and potassium canrenoate, a MR antagonist, blocked induction of ET-1 mRNA, suggesting that induction was mediated via the MR. In a time course study, ET-1 mRNA levels were induced rapidly by aldosterone, with levels of ET-1 mRNA maximally increased 2- and 2.5-fold after 1 h in the kidney and colon, respectively. These results suggest that ET-1 is a direct aldosterone gene target in the kidney and colon and may play an important role in aldosterone-regulated ion homeostasis.

Mammalian K-ras2 is a corticosteroid-induced gene in vivo.

Aldosterone acts via the mineralocorticoid receptor to regulate gene expression. A number of aldosterone-induced genes have been characterized in the distal colon and/or the distal nephron. Using the Xenopus kidney-derived A6 cell line, the K-ras transcript of the K-ras gene was identified as aldosterone induced, with a role in epithelial sodium transport. This study sought to establish whether K-ras expression is also increased in mammalian epithelia in vivo in response to aldosterone. RNA was extracted from the kidney and distal colon of rats treated with aldosterone or dexamethasone. Northern blot analysis and real-time RT-PCR were performed using probes and primers specific for the K-rasA isoform and for total K-ras. The expression of both total K-ras and of the A isoform is induced in the distal colon by aldosterone and by dexamethasone. Given the relative abundances of the two isoforms, this would appear to indicate induction of both isoforms. The time course of the response is consistent with a primary transcriptional response. In contrast to the documented up-regulation in the amphibian kidney, we did not observe regulation by corticosteroids in the kidney. However, regulation in a subpopulation of cells cannot be excluded.

Mineralocorticoid receptor binding, structure and function.

The isolation of aldosterone 50 years ago was a critical first step in elucidating the mechanism by which corticosteroids regulate electrolyte homeostasis. The broad principles of this mechanism involving an intracellular receptor acting on specific genes to induce the expression/repression of aldosterone-induced proteins (AIP) were established 30 years ago. The cloning of the mineralocorticoid receptor (MR) has enabled studies of the subcellular mechanisms of aldosterone action, including the molecular dissection of structure-function relationships in the receptor. We have exploited the close structural and functional similarity of the MR with the glucocorticoid receptor to identify the regions in the MR that confer ligand-binding specificity. The critical region is located, not as might be expected in the ligand-binding pocket but rather on the surface of the molecule. These studies have been extended to an analysis of the interactions between the N-terminal and ligand-binding domains of the MR. In the last decade, AIP have been identified; the regulation of the genes encoding these AIP are discussed.

Dissecting mineralocorticoid receptor structure and function.

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.