The splanchnic nerve, innervating the adrenal medulla, releases a variety of neurotransmitters that stimulate genes involved in catecholamine biosynthesis. In particular, cholinergic agonists have been shown to induce phenylethanolamine N-methyltransferase (PNMT) gene expression through activation of both nicotinic and muscarinic receptors in vivo and in vitro. By contrast, the role of peptidergic neurotransmitters in adrenal medullary PNMT gene expression remains unclear. Using transient transfection assays, we demonstrate that rat PNMT promoter-luciferase reporter gene constructs are markedly activated by 10 nM PACAP when expressed in PC12 cells. PACAP appears to mediate its effects primarily through PAC1 receptors and, subsequently, cAMP-protein kinase A (PKA) and extracellular Ca(2+) signaling mechanisms. Activation of these signal transduction pathways markedly increases nuclear levels of the immediately early gene transcription factor Egr-1 and the developmental factor AP2. A slight decrease in Sp1 expression may also occur, whereas MAZ and glucocorticoid receptor expression remains unaltered. Although PACAP stimulates rapid changes in transcription factor expression and PNMT promoter activity, its effects are long lasting. PNMT promoter induction continues to rise and is sustained for > or=48 hours. By contrast, while muscarine, nicotine, or carbachol (100 micro M) also evoke rapid increases in rat PNMT promoter activity, peak activity is observed at 6 hours, followed by a decline and restoration to basal levels by 24 hours. Cholinergic activation of the PNMT promoter also seems to involve the cAMP-PKA signaling mechanism. However, the magnitude of stimulation and antagonist blockade with H-89 or the polypeptide inhibitor PKI suggests that the extent of activation is much less than that with PACAP.