Specific inhibition of the last steps of aldosterone biosynthesis by 18-vinylprogesterone in bovine adrenocortical cells

Abstract

18-Vinylprogesterone (18-VP), designed for mechanism-based specific inhibition of the last steps of the aldosterone biosynthesis, was used to characterize the mechanism of the 11- and 18-hydroxylase activities of bovine cytochrome P450 11 β . In the present work, its action was studied by observations on a primary culture of bovine adrenocortical cells. First, we investigated the effects of 18-VP on the different enzymatic steps of the biosynthesis of cortisol and aldosterone. The production of cortisol, baseline or hormone-stimulated (ACTH or AII), was inhibited by 18-VP in a dose-dependent manner with a maximal inhibition at 5 μM. Supply of different exogenous substrates to support steroidogenesis revealed an inhibition of the last step of cortisol or corticosterone biosynthesis. We then used specific blockers to measure individual activities and conclude that 11β-hydroxylation was the only enzymatic activity affected. Aldosterone, as well as 18-hydroxycorticosterone, was also measured following addition of corticosterone. The 18-hydroxylation of corticosterone was inhibited by 18-VP, with 50% inhibition occurring at 0.04 μM compared with the 50% inhibition value of 0.3 μM obtained for 11-hydroxylation. Surprisingly, 18-ethynyl-progesterone (18-EP), which has a structure very similar to 18-VP, only weakly inhibits 11β-hydroxylation. The inhibition of aldosterone formation was also much lower with 18-EP than with 18-VP. These studies demonstrate that 18-VP inhibits only the later steps of aldosterone biosynthesis and more specifically 18- than 11-hydroxylation activity.