title: Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures.
abstract: 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.
mesh_terms: D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000305:Adrenal Cortex Hormones / Q000096:biosynthesis*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000735:Androstenedione / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D003345:Corticosterone / Q000096:biosynthesis; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006854:Hydrocortisone / Q000096:biosynthesis; D007700:Kinetics; D013250:Steroid Hydroxylases / Q000378:metabolism
chemical_list: D000305:Adrenal Cortex Hormones; D000735:Androstenedione; D000324:Adrenocorticotropic Hormone; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D013250:Steroid Hydroxylases; D002786:Cholesterol Side-Chain Cleavage Enzyme; D003345:Corticosterone; D006854:Hydrocortisone