Rat ovarian granulosa cell as a site of endothelin reception and action: Attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway

Endothelins (ETs) are a family of vasoactive peptides that may be involved in granulosa cell (GC) luteinization or follicular maturation. However, the precise role of ET in ovarian physiology remains unknown. We have investigated whether the rat GC is a site of ET reception and have characterized the antigonadotropic effect of ET in cultured GC from immature rats. Two major ET binding species (52 and 30 kDa) were observed after cross- linking of GC membranes with radiolabeled ET-1, although the smaller protein may represent a degradative product. Unlabeled ET-1, ET-2, or ET-3 were equipotent in displacing radiolabeled ET-1 from these putative ET receptors, with EC(50s) of 0.3-0.7 x 10^-9 M. Similarly, ET-1, ET-2, and ET-3 were equipotent (EC(50s) of about 10^-10 to 10^-9 M) in inhibiting the FSH- supported accumulation of progesterone. ET-1 (10^-7 M) also inhibited (> 90%) FSH-supported estrogen accumulation. Maximum progesterone inhibition (> 90%) by ET-1 (10^-7 M) was achieved throughout the range of FSH doses and cell densities tested and by 48 h or 72 h of culture. ET-1 was not cytotoxic in the dose range tested. Forskolin-stimulated progesterone accumulation was similarly inhibited by ET-1, suggesting that ET-1 inhibits cAMP-mediated (e.g., FSH or forskolin-stimulated) progesterone accumulation. ET-1 inhibited (74%) the FSH-stimulated accumulation of cAMP suggesting that it acts at sites related to cAMP generation or degradation. In addition, ET-1 inhibited 8-bromo-cAMP-generated progesterone accumulation (60%), suggesting that it also acts at sites distal to cAMP generation. These studies demonstrate that the rat GC is a site of ET reception and action, and that ET inhibits cAMP- mediated steroidogenesis via interaction at sites related to cAMP generation and/or action.