We examined how pulsatile stimulation with adenylate cyclase-activating polypeptide 1 (ADCYAP1) affected gonadotrophs. In static culture, gonadotropin-releasing hormone (GnRH) stimulated transcription of all the gonadotropin subunits. In contrast, ADCYAP1 increased common alpha-glycoprotein subunit gene (Cga) promoter activity but failed to increase luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoters. Messenger RNAs for Lhb and Fshb were slightly but significantly increased by ADCYAP1 stimulation. The results of cotreatment of the cells with GnRH and ADCYAP1 was not different from the effects of GnRH alone on Lhb and Fshb transcriptional activities as well as on mRNA expressions. To determine the effect of pulsatile ADCYAP1 stimulation on gonadotropin subunit gene expression, perifused LbetaT2 cells were stimulated either at high frequency (5-min ADCYAP1 pulse every 30 min) or at low frequency (5-min ADCYAP1 pulse every 120 min). High-frequency ADCYAP1 pulses preferentially increased Lhb gene expression 2.29-fold +/- 0.15-fold, and low frequency pulses resulted in a 1.55-fold +/- 0.16-fold increase. Fshb gene expression was increased 1.87-fold +/- 0.3-fold by high-frequency ADCYAP1 pulses and 4.3-fold +/- 0.29-fold by low-frequency pulses. These results were similar to the frequency-specific effects of pulsatile GnRH. Follistatin (Fst) gene expression was specifically increased by high-frequency GnRH pulses. High-frequency ADCYAP1 pulses increased Fst to a larger extent (4.7-fold +/- 0.57-fold) than did low-frequency pulse (2.72-fold +/- 1.09-fold). ADCYAP1 receptor gene (Adcyap1r) expression was increased significantly following pulsatile GnRH regardless of pulse frequency. Low-frequency ADCYAP1 pulses, however, increased Adcyap1r expression (16.49-fold +/- 8.41-fold) to a larger extent than high frequency pulses did. In addition, high-frequency ADCYAP1 pulses specifically increased Gnrhr (GnRH receptor) expression by 4.38-fold +/- 0.81-fold; however, low-frequency pulses did not result in an increase. These results suggest that ADCYAP1, like GnRH, specifically regulates Lhb and Fshb subunit gene in a pulse frequency-specific manner. This regulation may involve alteration in numbers of GnRH and ADCYAP1 receptors as well as FST expression.

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs-and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3 0 ,5 0 -cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.cyclic adenosine monophosphate, hypothalamic hormones, kinases, neuroendocrinology, pituitary hormones

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