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 17α-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3·4 μmol midazolam/1 while ACTH-stimulated production required 13·6 μmol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13·6 μmol midazolam/l for basal and 6·8 μmol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6·8 μmol/l and additonal effects on 17α-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 μmol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 μmol/l).
Both midazolam and diazepam inhibited K+-stimulated aldosterone synthesis, with IC50 values of 1·2 μmol/l and 0·8 μmol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11β-hydroxyprogesterone as substrate, the KI for the inhibition of aldosterone synthesis by midazolam was 54 μmol/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. Both basal (P < 0·01) and K+-stimulated 45Ca uptake (P < 0·05) were inhibited by the drug although the effects of K+ were not completely abolished. Comparison of the dose-dependent effects of midazolam on basal 45Ca uptake in cell suspensions prepared from different areas of the adrenal cortex indicated that zona glomerulosa cells are more sensitive to midazolam.
We confirm that benzodiazepines at low concentrations have a direct effect on microsomal steroid hydroxylase enzymes in vitro and postulate that the greater sensitivity to benzodiazepines of K+-stimulated aldosterone synthesis, when compared with either ACTH-stimulated cortisol synthesis or conversion of 21-deoxycortisol to cortisol, may be explained by additional effects of these drugs on plasma membrane calcium transport.