PubMed · 4891011
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I Katsushima. 1969. [Aldosterone].. https://pubmed.ncbi.nlm.nih.gov/4891011/
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To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) or hMR (hMR 711-984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone-hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1-944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711-984 region is sufficient for hsp90-MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand.
Studies from our laboratory have demonstrated rapid (<1 min) non-genomic activation of Na+-H+ exchange and potassium recycling by mineralocorticoids in human and rat colonic epithelium. It has previously been demonstrated that Na+-H+ exchange may be stimulated by protein kinase C (PKC) activation; therefore, we examined the effect of mineralocorticoids on PKC activity in rat colonic epithelium. Activation (after 15 min of incubation) of basal PKC activity was observed in cytosolic fractions of rat colonic epithelium by aldosterone, fludrocortisone, and deoxycorticosterone acetate. In all instances, PKC activation was inhibited by the PKC inhibitor bisindolylmaleimide (GF109203X). Hydrocortisone failed to activate PKC activity. Stimulation of basal intracellular free calcium [Ca2+]i was observed, in isolated rat colonic crypts, following aldosterone addition. This stimulatory effect was inhibited by the PKC inhibitor, chelerythrine chloride. Hydrocortisone failed to increase [Ca2+]i. These results indicate that intracellular signaling for aldosterone involves changes in [Ca2+]i via activation of PKC. Since the stimulation of PKC and increase in [Ca2+]i are apparent at normal circulating levels of aldosterone, our findings have major implications for the reassessment of mineralocorticoid effects on electrolyte homeostasis.