Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17α-hydroxylase/c17-20 lyase in mouse leydig cell cultures: Relation to steroid production

Abstract

The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17α-hydroxylase/C17-20 lyase (P-45017α) was investi-gated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-45017α decreased to less than 5% by day 4 and was unde-tectable between days 7 and 11. In contrast, the amount of immunoreactive P-450scc remained rela-tively constant throughout the same period. Treat-ment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-45017α with relatively little effect on the amount of P-450scc The rate of de novo synthesis of each of the P-450 enzymes was studied by deter-mining [35S]methionine incorporation into newly syn-thesized protein. In the absence of cAMP, de novo synthesis of P-45017α ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-45017α on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly iso-lated Leydig cells. The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-45017α ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture. This treatment has little effect on de novo synthesis of P-450scc. The capacity of Leydig cells for steroid production was determined by in-cubating cultures for 2 h with 1 mM 8-bromo-cAMP and measuring the amount of pregnenolone, proges-terone, androstenedione, and testosterone pro-duced. Total steroid production declined somewhat on day 2 and remained relatively unchanged during the remaining 9 days of culture in the presence or absence of chronic cAMP treatment. However, the pattern of steroid production changed markedly. In the absence of cAMP treatment, testosterone pro-duction decreased while progesterone production increased. Treatment of cultures with cAMP re-versed this pattern of steroid production. These studies demonstrate that cAMP is essential for the synthesis of P-45017α. P-450scc, in contrast, is con-stitutively synthesized. Decreases in acute cAMP-stimulated testosterone production in Leydig cell cultures, maintained in the absence of cAMP treat-ment, are not due to a reduced capacity for steroid production but reflect decreases in the amount of P-45017α. © 1987 by The Endocrine Society.