Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures.

The early events of steroidogenesis, following adrenocorticotropin (ACTH) stimulation, were investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide, even though cytochrome P-450scc only increases after 12 h. Mitochondrial free cholesterol levels increased during the same time period (16.5 to 25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. It is concluded that early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial free cholesterol. The maximum rate of cholesterol transport to mitochondria in aminoglutethimide-blocked cells (8.6 micrograms/mg of protein/h) was consistent with both the maximum rate of mitochondrial cholesterol SCC and cellular steroidogenesis (6.0 micrograms of pregnenolone/mg/h and 5.5 micrograms of steroid/mg of mitochondria/h, respectively). Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial free cholesterol. The distribution of steroid products fell into three distinct phases during a 24-h period following ACTH stimulation: an initial increase in SCC activity (0-4 h), elevation of androstenedione in place of corticosterone (4-12 h), and then in place of cortisol (12-24 h). The changes from 4 to 24 h result from a progressive stimulation by ACTH of 17 alpha-hydroxylase activity (but not 21-hydroxylase or C17:20 lyase activities) that is maintained even when stimulation of total steroidogenesis has stopped.