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 LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) 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 PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 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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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 LbetaT2 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 LbetaT2 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.