PROTEIN-KINASE-C ACTIVATION AND POSITIVE AND NEGATIVE AGONIST REGULATION OF 3', 5'-CYCLIC ADENOSINE-MONOPHOSPHATE LEVELS IN CULTURED RAT SERTOLI CELLS

The present study examines the effects of 12-O-tetradecanoylphorbol-13 -acetate (TPA) on agonist-regulated 3', 5'-cyclic adenosine monophosph ate (cAMP) formation and cAMP-mediated effects in cultured Sertoli cel ls from immature rats. Concentration-dependent stimulation of cAMP lev els by follicle-stimulating hormone (FSH) was inhibited dramatically b y the coaddition of 100 nmol/l TPA, which exerted a similar inhibition of glucagon- and isoproterenol-stimulated cAMP production. These resu lts show that protein kinase C (PKC) activation by TPA attenuates G(s) -protein-mediated agonist activation of cAMP production. (-)-N6(R)-Phe nylisopropyladenosine (L-PIA), an A1-adenosine receptor agonist, inhib ited cAMP stimulation by FSH in a concentration-dependent manner. When L-PIA was added in increasing concentrations simultaneously with 100 nmol/l TPA, the L-PIA still inhibited FSH-stimulated cAMP production i n a concentration-dependent manner. In the presence of TPA, the half-i nhibitory concentration (IC50) for L-PIA inhibition of cAMP formation was reduced by more than one order of magnitude, indicating that PKC a ctivation by TPA increases the sensitivity of Sertoli cells to G(i)-pr otein-mediated agonist inhibition of cAMP production. The inhibitory e ffects of TPA on FSH-stimulated cAMP production were still observed wh en cAMP phosphodiesterase activity was inhibited by 1 mmol/l methyliso butylxanthine or when the activity of G(alphai)-protein was eliminated by pretreatment with 100 mug/l pertussis toxin. Taken together, the r esults indicate that PKC activation inhibits agonist-dependent stimula tion of cAMP production by phosphorylation of components common to all the activating agonists used, and not via stimulation of G(i)-protein activity or degradation of cAMP by cAMP phosphodiesterase activity. T he increased sensitivity to L-PIA inhibition of cAMP formation induced by TPA may simply be a result of the reduced activity of the agonist- receptor/G(s)-protein/C complex.