SPECIFIC-INHIBITION OF THE LAST STEPS OF ALDOSTERONE BIOSYNTHESIS BY 18-VINYLPROGESTERONE IN BOVINE ADRENOCORTICAL-CELLS

18-Vinylprogesterone (18-VP), designed for mechanism-based specific in hibition of the last steps of the aldosterone biosynthesis, was used t o characterize the mechanism of the 11- and 18-hydroxylase activities of bovine cytochrome P450(11 beta). In the present work, its action wa s studied by observations on a primary culture of bovine adrenocortica l cells. First, we investigated the effects of 18-VP on the different enzymatic steps of the biosynthesis of cortisol and aldosterone. The p roduction of cortisol, baseline or hormone-stimulated (ACTH or AII), w as inhibited by 18-VP in a dose-dependent manner with a maximal inhibi tion at 5 mu M. Supply of different exogenous substrates to support st eroidogenesis revealed an inhibition of the last step of cortisol or c orticosterone biosynthesis. We then used specific blockers to measure individual activities and conclude that 11 beta-hydroxylation was the only enzymatic activity affected. Aldosterone, as well as 18-hydroxyco rticosterone, was also measured following addition of corticosterone. The 18-hydroxylation of corticosterone was inhibited by 18-VP, with 50 % inhibition occurring at 0.04 mu M compared with the 50% inhibition v alue of 0.3 mu M obtained for 11-hydroxylation. Surprisingly, 18-ethyn yl-progesterone (18-EP), which has a structure very similar to 18-VP, only weakly inhibits 11 beta-hydroxylation. The inhibition of aldoster one formation was also much lower with 18-EP than with 18-VP. These st udies demonstrate that 18-VP inhibits only the later steps of aldoster one biosynthesis and more specifically 18- than 11-hydroxylation activ ity. (C) 1996 Elsevier Science Ltd.