title: Adenosine triphosphate-evoked cytosolic calcium oscillations in human granulosa-luteal cells: role of protein kinase C.
abstract: ATP has been shown to modulate progesterone production in human granulosa-luteal cells (hGLCs) in vitro. After binding to a G protein-coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activates protein kinase C (PKC) and intracellular calcium [Ca(2+)](i) mobilization, respectively. In the present study, we examined the potential cross-talk between the PKC and Ca(2+) pathway in ATP signal transduction. Specifically, the effect of PKC on regulating ATP-evoked [Ca(2+)](i) oscillations were examined in hGLCs. Using microspectrofluorimetry, [Ca(2+)](i) oscillations were detected in Fura-2 loaded hGLCs in primary culture. The amplitudes of the ATP-triggered [Ca(2+)](i) oscillations were reduced in a dose-dependent manner by pretreating the cells with various concentrations (1 nM to 10 microM) of the PKC activator, phorbol-12-myristate-13-acetate (PMA). A 10 microM concentration of PMA completely suppressed 10 microM ATP-induced oscillations. The inhibitory effect occurred even when PMA was given during the plateau phase of ATP evoked [Ca(2+)](i) oscillations, 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 [Ca(2+)](i) oscillations were not completely suppressed by PMA. Furthermore, homologous desensitization of ATP-induced calcium oscillations was partially reversed by bisindolylmaleimide I, suggesting that activated PKC may be involved in the mechanism of desensitization. These results demonstrate that PKC negatively regulates the ATP-evoked [Ca(2+)](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.
authors.affiliation: Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5.
authors.forename: C J
authors.initials: CJ
authors.lastname: Tai
authors.forename: S K
authors.initials: SK
authors.lastname: Kang
authors.forename: P C
authors.initials: PC
authors.lastname: Leung
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000166:cytology / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009991:Oscillometry; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium