Adenosine triphosphate-evoked cytosolic calcium oscillations in human granulosa-luteal cells: Role of protein kinase C

ATP has been shown to modulate progesterone production in human granulosa-l uteal cells (hGLCs) in vitro. After binding to a G protein-coupled P2 purin ergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activates protein kinase C (PKC) and intracellular calcium [Ca2+](i) mobilization, respectively. In the pres ent study, we examined the potential cross-talk between the PKC and Ca2+ pa thway in ATP signal transduction. Specifically, the effect of PKC on regula ting ATP-evoked [Ca2+](i) oscillations were examined in hGLCs. Using micros pectrofluorimetry, [Ca2+](i) oscillations were detected in Fura-X loaded hG LCs in primary culture. The amplitudes of the ATP-triggered [Ca2+](i) oscil lations were reduced in a dose-dependent manner by pretreating the cells wi th various concentrations (1 nM to 10 muM) of the PKC activator, phorbol-12 -myristate-13-acetate (PMA). A 10 muM concentration of PMA completely suppr essed 10 muM ATP-induced oscillations. The inhibitory effect occurred even when PMA was given during the plateau phase of ATP evoked [Ca2+](i) oscilla tions, suggesting that extracellular calcium influx was inhibited. The role of PKC was further substantiated by the observation that, in the presence of a PKC inhibitor, bisindolylmaleimide I, ATP-induced [Ca2+](i) oscillatio ns were not completely suppressed by PMA. Furthermore, homologous desensiti zation of ATP-induced calcium oscillations was partially reversed by bisind olylmaleimide I, suggesting that activated PKC may be involved in the mecha nism of desensitization. These results demonstrate that PKC negatively regu lates the ATP-evoked [Ca2+](i) mobilization from both intracellular stores and extracellular influx in hGLCs and further support a modulatory role of ATP and P2 purinoceptor in ovarian steroidogenesis.