ACTIONS OF ENDOTHELIN-1 ON SWINE OVARIAN (GRANULOSA) CELLS

We have investigated the regulatory actions of endothelin-I (ET-1) on inositol phosphate accumulation, cytosolic free Ca2+ ion concentrations ([Ca2+]i), and basal and FSH-stimulated progesterone and cAMP accumulation by swine granulosa cells in serum-free cultures. ET-1 induced a rapid stimulation of phosphoinositide hydrolysis in populations of granulosa cells, as inferred by the rapid appearance of soluble inositol polyphosphates in response to ET-1 exposure. At the single cell level, fura-2 videomicroscopy was used to measure [Ca2+]i in individual granulosa cells. We observed cell-cell variability in the threshold concentration of ET-1 required to induce a rise in [Ca2+]i. More than 75% of granulosa cells responded to maximal doses of ET-1. The following parameters of [Ca2+]i were influenced by ET-1 concentration: percentage of responding cells, lag time for the onset of response, amplitude, and kinetics of the response. Two types of ET-1-mediated [Ca2+]i rises were observed. One type exhibited rapid Ca2+ kinetics, reaching at least a 2-fold increase above basal (spike phase) within 1-10 sec and returning to a new steady state (plateau phase) 2 min after onset. The other mode of response had slower [Ca2+]i kinetics, in which 50 sec or more were required to double [Ca2+]i, which remained at this level throughout the observation period (2.5 min). These responses to ET-1 were specific and were not initiated by vasopressin or tumor necrosis factor-a. In cell population studies using monolayer cultures of swine granulosa cells, ET-1 inhibited FSH-stimulated accumulation of progesterone and cAMP. The ET-1-mediated inhibition of FSH-stimulated accumulation of progesterone required at least 4 h of ET-1 exposure. The ET-1-mediated inhibition of both the FSH-stimulated accumulation of progesterone and cAMP after 24-h incubation was mimicked by an activator of protein kinase-C, phorbol 12-myristate 13-acetate, but not by an inactive phorbol. These observations in either single cells or populations of swine ovarian (granulosa) cells are consistent with a possible regulatory role of an ET-1-activated intracellular signaling pathway involving inositol phosphates, [Ca2+]i, and protein kinase-C in the mammalian granulosa cell.