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Gonadotropin-Releasing Hormone Couples to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway through Novel Protein Kinase C and - in LßT2 Gonadotrope Cells

Unité Mixte de Recherche Centre National de la Recherche Scientifique 7079 (S.L., G.G., J.-N.L., R.C., J.C.-T.), Physiologie and Physiopathologie, Université Pierre and Marie Curie-Paris 6, 75252 Paris, France; Department of Pharmacology and Experimental Therapeutics (V.S.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and Biomedical Research Center for Signal Transduction Networks (J.-W.S.), Inha University Incheon, 402-751 Incheon, Korea

Address all correspondence and requests for reprints to: Prof. J. Cohen-Tannoudji, UMR CNRS 7079 Université Pierre and Marie Curie-Paris 6, Case 256, 4 Place Jussieu, 75252 Paris cedex 05, France. E-mail: joelle.cohen-tannoudji{at}snv.jussieu.fr.

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LßT2 gonadotrope cells (maximal increase, 2.5-fold; EC₅₀, 0.30 nM), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca²⁺ independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKC, -, and - by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKC or - but not PKC impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKC and - are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LßT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

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