title: EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells.
abstract: We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.
authors.affiliation: Inflammation Research Network, Department of Physiology, University of Calgary, Calgary, AB, Canada T2N 4N1.
authors.forename: Jacques Q
authors.initials: JQ
authors.lastname: van der Merwe
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
authors.forename: Wallace K
authors.initials: WK
authors.lastname: MacNaughton
mesh_terms: D001693:Biological Transport, Active; D001711:Biotransformation; D015153:Blotting, Western; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D066246:ErbB Receptors / Q000235:genetics / Q000502:physiology*; D016391:Genes, src / Q000502:physiology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D002712:Chlorides; D044464:Receptor, PAR-2; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011451:Prostaglandin-Endoperoxide Synthases; D066246:ErbB Receptors; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases