Interactions of protein kinase C with receptor- and non-receptor-mediated cyclic AMP generation in swine granulosa cells

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The swine granulosa cell provides a model to study the interactions between the cAMP and calcium-lipid-dependent signaling pathways. To this end, porcine granulosa cells were incubated in monolayer culture for 1-4 days in the presence of FSH (200 ng/ml), forskolin (85 μM), or cholera toxin (3 μg/ml) with or without an activator of protein kinase C, the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) (30 ng/ml). TPA had little effect on basal cAMP generation (1-4 days) or on follicle-stimulating hormone (FSH)-stimulated cAMP formation during the first 24 h. Phorbol ester did inhibit cAMP formation on day 2 (by ~ 25%), on day 3 (by ~ 70%) and on day 4 (by > 80%). Forskolin-mediated cAMP generation was inhibited (33-56%) on days 1-4, respectively. TPA suppressed dose-dependent FSH (3-300 ng/ml)-stimulated cAMP production on day 2, virtually abolished FSH-provoked cAMP formation on day 4 and inhibited dose-dependent forskolin-stimulated cAMP production on both days. TPA had no effect on the half-maximally effective dose, ED₅₀, of FSH-stimulated cAMP production but did decrease the ED₅₀ of forskolin and the maximal stimulatory effect of FSH and forskolin on days 2 and 4. Similar effects were observed with the synthetic diacylglycerols DOG (1,2-dioctanoylglycerol) and OAG (1-oleoyl-2-acetylglycerol). The TPA effect was limited to the mammalian adenylate cyclase as it had no effect on bacterially derived adenylate cyclase from Bordetella pertussis. In contrast, although phorbol ester treatment inhibited the dose-dependent (30-1000 ng/ml) stimulation of cAMP accumulation by cholera toxin on day 2, unexpectedly, the action of TPA on day 4 was reversed in the presence of cholera toxin becoming stimulatory instead of inhibitory. Pretreatment of granulosa cells with pertussis toxin (100 ng/ml for 18 h) did not override the inhibitory action of TPA on either receptor- or non-receptor-mediated cAMP accumulation after 2 or 4 days of incubation. We conclude that the protein kinase C effector pathway is effectively coupled in an inhibitory fashion to both receptor-mediated and non-receptor-mediated activation of adenylate cyclase in porcine ovarian cells. Moreover, the present observations are consistent with the hypothesis that this inhibitory coupling is enacted at least in part at the level of the catalytic subunit of adenylate cyclase.