A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

Abstract

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11β-hydroxysteroid dehydroxgenase (11β-HSD1 or 11β-HSD2) enzymatic activity and so are without apparent “protective” enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6β-hydroxylase that transforms corticosterone to 6β-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3α,5β-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11β-HSD1) and 11-dehydrocorticosterone and 11β-hydroxy-3α,5β-tetrahydroprogesterone (inhibitors of 11β-HSD2) and carbenoxalone, which inhibits both 11β-HSD1 and 11β-HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current ( I sc)] in A6 cells. All of these 11β-HSD inhibitory substances induced a significant increment in corticosterone-induced I sc, which was detectable within 2 h. However, none of these agents caused an increase in I sc when incubated by themselves with A6 cells. In all cases, the additional I sc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original I scelicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6β-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6β-hydroxylase inhibition and the MR-mediated increase in I scin the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6β-hydroxylase and also significantly enhanced corticosterone-induced I sc at 2 h. These experiments indicate that the enhanced MR-mediated I sc in A6 cells may be related to inhibition of 6β-hydroxylase activity in these cells and that this 6β-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).