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Gonadotropin-Releasing Hormone Inhibits Pituitary Adenylyl Cyclase-Activating Polypeptide Coupling to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway in LβT2 Gonadotrope Cells through Novel Protein Kinase C Isoforms and Phosphorylation of Pituitary Adenylyl Cyclase-Activating Polypeptide Type I Receptor

Unité Mixte de Recherche-Centre National de la Recherche Scientifique 7079 Physiologie et Physiopathologie (S.L., G.G., V.S., R.C., J.C.-T.), Université Pierre et Marie Curie-Paris 6, 75252 Paris, France; Université Paris Diderot-Paris 7 (V.S., J.C.-T.), 75205 Paris, France; and Molecular Neuropharmacology (N.S., A.B.), Graduate School of Pharmaceutical Sciences, Osaka University, 565-0871 Osaka, Japan

Address all correspondence and requests for reprints to: Pr J. Cohen-Tannoudji, Unité Mixte de Recherche-Centre National de la Recherche Scientifique 7079 Physiologie and Physiopathologie, 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.

Gonadotrope cells are primarily regulated by GnRH but are also targets of the pituitary adenylyl cyclase-activating polypeptide (PACAP). Although it has been reported that reciprocal interactions between both neuropeptides contribute to regulation of gonadotrope function, the underlying mechanisms remain poorly understood. In this study, we reevaluated PACAP coupling to the cAMP pathway in LβT2 gonadotrope cells and analyzed GnRH effect on PACAP signaling. We established that PACAP₃₈ markedly increases intracellular cAMP levels (EC₅₀ of 4.7 ± 1.3 nM) through the PACAP type 1 receptor (PAC1-R), as evidenced by pharmacological and RT-PCR studies. Interestingly, although GnRH couples to cAMP pathway in LβT2 cells, the effects of both neuropeptides were not synergistic. Instead, the GnRH agonist (GnRHa) triptorelin rapidly and strongly inhibited (70% inhibition as early as 5 min) PACAP₃₈-induced cAMP production. Inhibition was calcium independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindoylmaleimide, indicating that GnRHa inhibitory action relies on PKC. Selective down-regulation of both conventional and novel PKC prevented a GnRHa effect, whereas pharmacological inhibition of conventional PKC only was ineffective, strongly suggesting the involvement of novel PKC isoforms. GnRHa did not inhibit forskolin- or cholera toxin-stimulated cAMP accumulation, suggesting that PAC1-R is the predominant target of GnRH. Accordingly, we demonstrated for the first time that GnRH increases PAC1-R phosphorylation through PKC, providing a potential molecular mechanism which may account for GnRH inhibitory effect.

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