title: PACAP activates calcium influx-dependent and -independent pathways to couple met-enkephalin secretion and biosynthesis in chromaffin cells.
abstract: Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) caused a dose-dependent increase in met-enkephalin secretion and increased production of met-enkephalin peptide and proenkephalin A (PEnk) mRNA in bovine chromaffin cells, at concentrations as low as 300 pM. PACAP-38 was less potent than PACAP-27, but had similar effects. Vasoactive intestinal polypeptide (VIP) (1-100 nM) was without appreciable effect on either enkephalin secretion or biosynthesis, implicating PACAP type I receptors in PACAP-stimulated enkephalin secretion and synthesis. PACAP type I receptors can activate adenylate cyclase and stimulate phospholipase C through heterotrimeric G protein interactions, leading to increased intracellular cyclic AMP (cAMP), inositol triphosphate (IP3)-mediated calcium mobilization, and calcium- and diacylglycerol (DAG)-mediated protein kinase C (PKC) activation. Enkephalin secretion evoked by 10-100 nM PACAP-27 was not inhibited by 1 microM (-)-202-791, an L-type specific dihydropyridine calcium channel blocker, but was inhibited 65-80% by the arylalkylamine calcium channel blocker D600. Forty mM potassium-evoked secretion was inhibited > 90% by both D600 and (-)-202-791, 25 microM forskolin-induced secretion was blocked < 50% by D600 and was unaffected by (-)-202-791, and 100 nM phorbol myristate acetate (PMA)-induced secretion was unaffected by either D600 or (-)-202-791. Enkephalin biosynthesis was increased by 10 nM PACAP-27, as measured by increased met-enkephalin pentapeptide content and PEnk A mRNA levels. PACAP-, forskolin-, and PMA-stimulated enkephalin synthesis were not blocked by D600 or (-)-202-791. Elevated potassium-induced enkephalin biosynthesis upregulation was completely blocked by either D600 or (-)-202-791 at the same concentrations. PACAP acting through type I PACAP receptors couples calcium influx-dependent enkephalin secretion and calcium influx-independent enkephalin biosynthesis in chromaffin cells. Restriction of the effects of enhanced calcium influx to stimulation of secretion, but not of biosynthesis, is unique to PACAP. By contrast, potassium-induced enkephalin biosynthesis upregulation is completely calcium influx dependent, specifically via calcium influx through L-type calcium channels. We propose that subpopulations of voltage-dependent calcium channels are differentially linked to intracellular signal transduction pathways that control neuropeptide gene expression and secretion in chromaffin cells.
authors.affiliation: Section on Molecular Neuroscience, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-4090, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Hahm
authors.forename: C M
authors.initials: CM
authors.lastname: Hsu
authors.forename: L E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D020746:Calcium Channels, L-Type; D020013:Calcium Signaling / Q000187:drug effects*; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects* / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D004744:Enkephalin, Methionine / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004745:Enkephalins / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D008297:Male; D008954:Models, Biological; D009479:Neuropeptides / Q000494:pharmacology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011188:Potassium / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics; D011863:Radioimmunoassay; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004745:Enkephalins; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011498:Protein Precursors; C029992:proenkephalin; D005576:Colforsin; D014660:Vasoactive Intestinal Peptide; D004744:Enkephalin, Methionine; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium