Mechanisms underlying endothelin's inhibition of FSH-stimulated progesterone production by ovarian granulosa cells.

In previous studies in porcine granulosa cell cultures, endothelin-1 (ET-1) was shown to inhibit FSH-stimulated cAMP and progesterone accumulation, and to increase inositol phosphate formation and cytosolic calcium ion concentration. The latter results suggest an action of ET-1 via the activation of phospholipase C. Here we have investigated the following experimental questions. (1) Does ET-1 activate PKC in ovarian cells? (2) Does the cellular mechanism(s) whereby ET-1 interferes with the steroidogenic action of FSH in granulosa cells involve an impairment of cAMP generation or action? And (3) how does the site(s) of the inhibitory effect(s) of ET-1 and TPA on FSH-stimulated progesterone accumulation in cultured granulosa cells compare? In the present investigation, ET-1 (1 microM) induced rapid cytosol-to-membrane translocation of [3H]phorbol 12,13-dibutyrate binding sites, indicating protein kinase C (PKC) activation. At 24 or 48 h, ET-1 inhibited FSH-, but not forskolin (1 microM)-induced, cAMP accumulation. Cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) messenger RNA (mRNA) accumulation was stimulated by FSH, 8-bromo-cAMP (8Br-cAMP, 0.5 mM) and forskolin. ET-1 significantly inhibited this effect of FSH, but not the effects of 8Br-cAMP and forskolin. Progesterone production decreased commensurately with this inhibitory action of ET-1 on the FSH-stimulated accumulation P450scc mRNA. The PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), suppressed steroidogenesis stimulated by forskolin and 8Br-cAMP as well as FSH. In conclusion, ET-1 inhibited FSH-stimulated cAMP accumulation, P450scc expression, and progesterone production in porcine granulosa cell cultures. The data are compatible with pre-adenylate cyclase site of action. Although ET-1 activated PKC, TPA, unlike ET-1, seems to inhibit steroidogenesis by interfering with cAMP action.