Regulation of alpha-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and alpha T3-1 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of alpha-subunit protein characteristic of the pituitary. The present study used primary pituitary cell cultures and the alpha T3-1 pituitary cell line to investigate how PACAP affects alpha-subunit mRNA transcripts. Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased alpha-subunit mRNA levels 1.5-fold, whereas GnRH more effectively (P<0.01) stimulated alpha-subunit protein release than did PACAP38 (3.2- vs. 2.0-fold). alpha-Subunit mRNA levels in alphaT3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h (P<0.05), and alpha-subunit protein secretion rose proportionately and in parallel with alpha-subunit mRNA levels. PACAP38 was a 100-fold more potent stimulator of alpha-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors. Type I receptor mRNA transcripts were identified by Northern analysis in alphaT3-1 cells. Depletion of PCK activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing alpha-subunit mRNA levels and alpha-subunit secretion. PACAP38, like 8Br-cAMP and forskolin, stimulated (P<0.05) luciferase (LUC) activity in alphaT3-1 cells transfected with a plasmid containing the first 846 of 180 base pairs of the 5'-flanking region of the human alpha-subunit gene linked upstream to a LUC reporter gene. Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change alpha-subunit mRNA half-life. These findings are consistent with the proposal that PACAP contributes to the high levels of alpha-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating alpha-subunit gene transcription in part by the cAMP/PKA pathway.