Antigonadotropic action of adenosine triphosphate in human granulosa-luteal cells: Involvement of protein kinase c alpha

The presence of P2U purinoceptor in human granulosa-luteal cells (hGLCs) in dicates a potential role of ATP in regulating ovarian function. In this stu dy an inhibitory effect of ATP on hCG-induced cAMP production was observed. Extracellular ATP has been shown to activate protein kinase C (PKC) after binding to a purinoceptor. To understand the role of PKC in mediating ATP a ction, hCG-stimulated cAMP level was examined in the presence of the PKC ac tivator, 1 mu mol/L phorbol 12-myristate 13-acetate (PMA), or the PKC inhib itor, 1 mu mol/L staurosporin or 1 mu mol/L bisindolylmaleimide I. PMA, lik e 10 mu mol/L ATP, significantly reduced hCG-evoked cAMP production. In add ition, the inhibitory effect of ATP was reversed by staurosporin and bisind olylmaleimide I. To further investigate the involvement of PKC isoforms in mediating the inhibitory effect of ATP, the presence of PKC isoforms in cul tured hGLCs was examined by Western blot using monoclonal antibodies agains t specific isoforms. Translocation of PKC isoforms from cytosolic fraction to membrane fraction was studied to identify the active PKC isozymes subseq uent to ATP treatment. The change in PKC isoform in PKC-depleted cells (ach ieved by exposure to PIMA for 18 h) was also examined. Our results demonstr ated the presence of PKC alpha, -delta, -iota, and -lambda isoforms in hGLC s and the translocation of PKCa subsequent to ATP treatment. In PKC-deplete d cells the PKCa level was reduced, and no significant effect of ATP on hCG -stimulated cAMP production was observed. To our knowledge, this is the fir st demonstration of PKC isoforms in hGLCs and the involvement of activated PKC in mediating the antigonadotropic effect of extracellular ATP. Taken to gether, these results further support a role of this neurotransmitter in re gulating human ovarian function.