PACAP ACTIVATES CALCIUM-INFLUX-DEPENDENT AND CALCIUM-INFLUX-INDEPENDENT PATHWAYS TO COUPLE MET-ENKEPHALIN SECRETION AND BIOSYNTHESIS IN CHROMAFFIN CELLS

Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) cause d a dose-dependent increase in met-enkephalin secretion and increased production of met-enkephalin peptide and proenkephalin A (PEnk) mRNA i n bovine chromaffin cells, at concentrations as low as 300 pM. PACAP-3 8 was less potent than PACAP-27, but had similar effects. Vasoactive i ntestinal 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. P ACAP type I receptors can activate adenylate cyclase and stimulate pho spholipase 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 evok ed by 10-100 nM PACAP-27 was not inhibited by 1 mu M (-)-202-791, an L -type specific dihydropyridine calcium channel blocker, but was inhibi ted 65-80% by the arylalkylamine calcium channel blocker D600. Forty m M potassium-evoked secretion was inhibited >90% by both D600 and (-)-2 02-791, 25 mu M forskolin-induced secretion was blocked <50% by D600 a nd 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 level s. PACAP-, forskolin-, and PMA-stimulated enkephalin synthesis were no t blocked by D600 or (-)-202-791. Elevated potassium-induced enkephali n biosynthesis upregulation was completely blocked by either D600 or ( -)-202-791. at the same concentrations. PACAP acting through type I PA CAP receptors couples calcium influx-dependent enkephalin secretion an d calcium influx-independent enkephalin biosynthesis in chromaffin cel ls. Restriction of the effects of enhanced calcium influx to stimulati on of secretion, but not of biosynthesis, is unique to PACAP. By contr ast, potassium-induced enkephalin biosynthesis upregulation is complet ely calcium influx dependent, specifically via calcium influx through L-type calcium channels. We propose that subpopulations of voltage-dep endent calcium channels are differentially linked to intracellular sig nal transduction pathways that control neuropeptide gene expression an d secretion in chromaffin cells.