P450scc-dependent cholesterol metabolism in rat adrenal mitochondria is inhibited by low concentrations of matrix...

Pregnenolone formation at P450scc in isolated rat adrenal mitochondria is determined equally by the amount of ACTH-stimulated reactive inner membrane cholesterol and by the matrix generation of NADPH. Evidence is presented here that both are sensitive to an increase in matrix Ca2+ produced by low levels of external Ca2+ (0.4-4 microM). Cholesterol availability to P450scc and intramitochondrial NADPH are shown to be highly interdependent. The proportion of mitochondrial cholesterol which is readily available to P450scc in the intact mitochondria increases as conditions become more favorable for NADPH generation. Preincubation of mitochondria without reductant causes substantial decreases in cholesterol metabolism when weaker reducing conditions are used without effect on NADPH generation as evidenced by unchanged metabolism of 20 alpha-hydroxycholesterol at P450scc or DOC at P450(11) beta. Increased Ca2+free not only increases this sensitivity to preincubation but also inhibits reductant transfer from succinate and isocitrate to both P450scc and P450(11) beta. Succinate activity was inhibited much more than that of isocitrate. These actions of Ca2+, which quantitatively explain the stimulatory characteristics of EGTA in standard media, were reversible providing exposure was short (< or = 3 min). Ruthenium red (inhibits Ca2+ uptake antiporter) is as effective as EGTA in preventing all these effects of Ca2+ while cyclosporin A (prevents opening of Ca(2+)-activated inner membrane channels) is partially effective. Ca2+ entry into the matrix is, therefore a necessary step prior to inhibition of cholesterol metabolism by several mechanisms, including the consequences of changes in inner membrane permeability.