Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction.
We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.
Liscovitch
M
M
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Amsterdam
A
A
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D005260:Female; D020404:Glycerophospholipids; D006107:Granulosa Cells / Q000201:enzymology*; D007700:Kinetics; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010906:Pituitary Hormone-Releasing Hormones / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D015398:Signal Transduction; D013268:Stimulation, Chemical
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010906:Pituitary Hormone-Releasing Hormones; C051521:phosphatidylethanol; D011433:Propranolol; D011493:Protein Kinase C; D010740:Phospholipases; D010739:Phospholipase D