Regulation of the content and phosphorylation of RII by adenosine 3',5'-monophosphate, follicle-stimulating hormone, and estradiol in cultured granulosa cells

The mechanisms by which FSH and cAMP induce receptors for LH (RLH) and increase progesterone (P) production in estradiol (E)-primed ovarian granulosa cells remain unclear, but may involve increases in the regulatory subunit of cAMP-dependent protein kinase II (RII) and the phosphorylation of specific cellular proteins. To examine the relationship of these events, primary cultures of granulosa cells (10(6) cells/ml) from E-treated (1.5 mg/day for 3 days) immature female rats were incubated with 10 nM E with or without FSH (25 ng/ml) for 0-120 h. The cytosolic content of RII was analyzed by four techniques: 1) immunoblotting using an antibody to bovine heart RII; 2) photoaffinity labeling with [32P]8-azido-cAMP; 3) phosphorylation with [gamma-32P]ATP with or without 2 microM cAMP or with the catalytic subunit of cAMP-dependent protein kinase; and 4) phosphorylation of intact cells with [32P] orthophosphate. All approaches revealed a time-dependent 5- to 6-fold increase in RII content in granulosa cells cultured for 48 h with E and FSH compared to that in cells treated with E alone. The content of RI, the regulatory subunit of protein kinase type I, remained low throughout the culture period regardless of hormone treatment. Granulosa cells were also cultured with E (10 nM) and 8-bromo-cAMP (8-Br-cAMP; 0.25-3 mM) or forskolin (0.5-100 microM), agents that increase intracellular cAMP, for 48 or 72 h. The cytosolic content and phosphorylation of RII were increased by culturing granulosa cells in E and 8-Br-cAMP (1 mM) or forskolin (50 microM) for 48 h. The increase in RII was associated with a FSH-mediated increase in the content and phosphorylation of other cAMP-dependent phosphoproteins. The increases in RII and cAMP-dependent phosphoproteins were associated with specific alterations in granulosa cell function: a FSH-mediated rise in 1) RLH [59.3 +/- 7.4 cpm/micrograms DNA (without FSH) to 1171.5 +/- 157 cpm/micrograms DNA (with FSH]) and 2) P accumulation in the medium [0.05 +/- 0.03 ng/ml (without FSH) to 25.3 +/- 4.6 ng/ml (with FSH]) at 48 h. A dose-dependent increase in the RLH and P accumulation in the medium was observed at 48 h of culture with E and 8-Br-cAMP or E and forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)