A novel mechanism of action of corticotropin releasing factor in rat Leydig cells

We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.