Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Secretoneurin Release and Secretogranin II Gene Transcription in Bovine Adrenochromaffin Cells through Multiple Signaling Pathways and Increased Binding of Pre-Existing Activator Protein-1-Like Transcription Factors
Résumé
Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by pro-teolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC 50 ϳ 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroen-docrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl 2 equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator for-skolin or cell-depolarization by high K ϩ were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to transacting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein -1-related proteins. These data indicate that SN biosynthe-sis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.