Mechanism of action of gonadotropin releasing hormone upon gonadotropin secretion: involvement of protein kinase C as revealed by staurosporine inhibition and enzyme depletion.
The role of protein kinase C (PKC) in the mechanism of action of gonadotropin-releasing hormone (GnRH) upon gonadotropin secretion is controversial and therefore was investigated in primary cultures of rat anterior pituitary cells. A relatively selective PKC inhibitor, staurosporine, inhibited both GnRH- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced luteinizing hormone (LH) release with half-maximal inhibition (IC50) of about 80 nM. Inhibition of GnRH action was not complete suggesting also a PKC-insensitive component in GnRH-induced gonadotropin release. Staurosporine had no effect on basal LH release, or on cellular LH content, neither did the drug interfere with the binding of [125I]iodo-[D-Ser(t-Bu)6]des-Gly10-GnRH N-ethylamide to its receptor in pituitary cells. When cultured pituitary cells were incubated with TPA (1 microM) for 24-48 h no measurable cellular PKC activity could be detected. The decrease in total PKC activity was accompanied by an increase in Ca2+, phosphatidylserine (PS), diacylglycerol (DG)-insensitive activity suggesting the release of a portion of the catalytic domain of PKC (M-kinase) by the phorbol ester treatment. TPA-induced LH release was nearly abolished in PKC-depleted cells and the response to GnRH was markedly reduced (40%). The stimulatory effect of the Ca2+ ionophore, ionomycin, was not impaired in PKC-depleted cells. Impaired responses to GnRH in PKC-depleted cells were only noticed at a later phase (2-4 h) of the exocytotic response of the neurohormone. The data strongly suggest a role for PKC during the second phase of GnRH-induced gonadotropin secretion.
Dan-Cohen
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Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Naor
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D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002064:Buserelin / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000378:metabolism*; D010718:Phosphatidylserines / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D004075:Diglycerides; D010718:Phosphatidylserines; D007987:Gonadotropin-Releasing Hormone; D015759:Ionomycin; D007986:Luteinizing Hormone; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002064:Buserelin; D002118:Calcium