SIGNAL-TRANSDUCTION IN AVIAN GRANULOSA-CELLS - EFFECTS OF PROTEIN-KINASE-C INHIBITORS

We evaluated the effects of two protein kinase C (PKC) inhibitors, sta urosporine (ST) and H-7, on LH-activated phospholipase C and adenylate cyclase activity by measuring the production of inositol phosphates ( IF) and cAMP in freshly dispersed granulosa cells from mature preovula tory follicles of laying hens. ST and H-7 dose-dependently potentiated LH-stimulated IP generation, whereas a protein kinase A (PKA) inhibit or (H-8) had no effect. The PKC activator, phorbol ester TPA (50 nM), significantly inhibited LH-stimulated IP production, which was complet ely prevented by ST. Both ST and H-7, while having no effect on basal cAMP levels, significantly and dose-dependently potentiated LH-stimula ted, but not forskolin-stimulated cAMP production. However, progestero ne production in response to LH, forskolin, and 8-Br-cAMP was inhibite d in granulosa cells preincubated for 30 min with H-7 or ST. H-7 and S T had no effect on 25-hydroxycholesterol- and pregnenolone-supported p rogesterone production. These results support a negative feedback role for PKC in LH-initiated signal transduction in avian granulosa cells. PKC blockade removes the inhibitory effect on LH-stimulated phospholi pase C and adenylate cyclase activity. The inhibitory effect of H-7 an d ST on progesterone synthesis could be attributed to inhibition of PK A and/or steps proximal to cholesterol side-chain cleavage. (C) 1994 A cademic Press, Inc.