Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 by adrenocorticotropin in cultured bovine adrenocortical cells

The long-term action of adrenocorticotropin (ACTH) to stimulate side chain cleavage of cholesterol has been studied utilizing confluent monolayer cultures of adult bovine adrenal cortex cells maintained for periods of time up to 72 h in the absence or presence of ACTH (10^-6 M). Following incorporation of [³⁵S]methionine into cellular protein at various times after ACTH addition, cholesterol side chain cleavage cytochrome P-450 (P-450(scc)) was immunoisolated and quantitated by autoradiography following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under these conditions, the radiolabel was incorporated into a protein identical in size with purified bovine cytochrome P-450(scc). The rate of incorporation was increased 9-fold over control rates 36 h following initiation of ACTH treatment and subsequently declined. Total cellular RNA was extracted from cells maintained for various times in the absence or presence of ACTH (10^-6 M) and translated in a rabbit reticulocyte lysate translation system. Radiolabel was incorporated into a protein approximately 5500 daltons large than native cytochrome P-450(scc) isolated from bovine adrenocortical mitochondria. The cell-free synthesis of this precursor was increased 6-fold over the control level 36 h following initiation of ACTH treatment and subsequently declined. The synthesis of pregnenolone by cells treated with ACTH also reached a maximum at 36 h when compared to untreated cells and subsequently declined. On the basis of these results it is proposed that ACTH stimulates the rate of synthesis of cytochrome P-450(scc) by means of an increase in the translatability of RNA, presumably resulting from an increase in the transcription of specific mRNA sequences. Once incorporated into the mitochondrion, newly synthesized cytochrome P-450(scc) catalyzes the conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone biosynthesis.