Inhibition of bovine adrenocortical steroidogenesis by benzodiazepines: a direct effect on microsomal hydroxylation or an inhibition of calcium uptake?

Abstract

We have previously reported that benzodiazepines inhibit microsomal steroid hydroxylases. We have now studied their effects at much lower drug concentrations and have also addressed the suggestion that benzodiazepines alter cellular calcium metabolism. We investigated the in-vitro effects of midazolam on microsomal steroid hydroxylation by measuring basal and ACTH-stimulated cortisol and 17 alpha-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3.4 mumol midazolam/l while ACTH-stimulated production required 13.6 mumol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13.6 mumol midazolam/l for basal and 6.8 mumol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6.8 mumol/l and additional effects on 17 alpha-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 mumol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 mumol/l). Both midazolam and diazepam inhibited K(+)-stimulated aldosterone synthesis, with IC50 values of 1.2 mumol/l and 0.8 mumol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11 beta-hydroxyprogesterone as substrate, the KI for the inhibition of aldosterone synthesis by midazolam was 54 mumol/l. Potassium stimulates aldosterone biosynthesis at least partly by changing intracellular free calcium levels. To investigate possible antagonistic effects of benzodiazepines on calcium metabolism, we measured 45Ca uptake in the presence of midazolam.(ABSTRACT TRUNCATED AT 250 WORDS)