The Effects of Hormones, Cholera Toxin, and iVa-Tosyl-LLysine Chloromethyl Ketone on Adenylate Cyclase in Viable Granulosa Cells*

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Abstract
Porcine small (1*2 mm) follicle granulosa cells before [day 0 (d.O)] or after 6 days in suspension culture with 10 ng/ml FSH [day 6 (d.6)] were incubated at 37 C for up to 3 h in medium containing 1 mM methylisobutylxanthine in the absence or presence of FSH, hCG, cholera toxin (CT), epinephrine, and/ or Afa-tosyl-L-lysine chloromethyl ketone (TLCK). The cellular cAMP content and basal cAMP production were approximately 10 times higher in d.6 than in d.O cells. d.O granulosa cells responded maximally to FSH and less to hCG, CT, and epinephrine, whereas d.6 cells responded maximally to hCG and CT, less to epinephrine, and least to FSH. CT responsiveness increased with hCG responsiveness. In neither cell did the stimulators have additive effects. These findings suggest that either CTbinding components are missing from d.O cells or hCG and CT activate adenylate cyclase via a system or component distinct from that via which FSH activates cyclase. TLCK, a watersoluble, synthetic, low molecular weight inhibitor of serine proteases, potentiated FSH-, hCG-, CT-, and epinephrine-activated cAMP production in both d.O and d.6 cells. At higher concentrations, TLCK blocked cAMP production. Preincubation of d.6 cells with 100 μM (potentiating dose) TLCK did not alter its potentiating effect on hCG-activated cAMP production; if TLCK was added 1 h after hCG, it was only 74% as effective as when it was added with hCG. Preincubation of d.6 cells with 1 mM (inhibitory dose) TLCK did not alter its inhibitory effect; however, if 1 mM TLCK was added 1 h after hCG, it had a potentiating rather than an inhibitory effect on hCG-activated cAMP production. The evidence indicates that TLCK affects one or more components of the intact granulosa cell, probably not the receptor or the phosphodiesterase and possibly of cytoplasmic origin.