title: Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells.
abstract: Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED(50)=4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl(2), the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl(2) and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.
authors.affiliation: INSERM U413, European Institute for Peptide Research IFRMP 23, Laboratory of Cellular and Molecular Neuroendocrinology, University of Rouen, 76821 Mont-Saint-Aignan, France.
authors.forename: Johann
authors.initials: J
authors.lastname: Guillemot
authors.forename: Djida
authors.initials: D
authors.lastname: Aït-Ali
authors.forename: Valérie
authors.initials: V
authors.lastname: Turquier
authors.forename: Maité
authors.initials: M
authors.lastname: Montero-Hadjadje
authors.forename: Alain
authors.initials: A
authors.lastname: Fournier
authors.forename: Hubert
authors.initials: H
authors.lastname: Vaudry
authors.forename: Youssef
authors.initials: Y
authors.lastname: Anouar
authors.forename: Laurent
authors.initials: L
authors.lastname: Yon
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000502:physiology*; D011863:Radioimmunoassay; D053381:Secretogranin II / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C557436:EM66 peptide; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D053381:Secretogranin II; D020928:Mitogen-Activated Protein Kinases