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1 Physiology and Biophysics, University of Calgary, Calgary, Canada
2 Pharmacology & Therapeutics, and Medicine, University of Calgary, Calgary, Canada
3 Department of Physiology & Biophysics, University of Calgary, Calgary, Canada
* To whom correspondence should be addressed. E-mail: wmacnaug{at}ucalgary.ca.
We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR2) stimulates chloride secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR2 activating peptides SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2. Short circuit current was used as a measure of net electrogenic ion transport. Basolateral but not apical application of SLIGRL-NH2 or 2-furoyl-LIGRLO-NH2 caused a concentration-dependent change in short circuit current that was significantly reduced in chloride-free buffer and by the intracellular calcium blockers, thapsigargin and BAPTA-AM, but not by the calcium channel blocker, verapamil. Inhibitors of PKA (H-89), CFTR (glybenclamide) and calcium-dependent chloride channels (niflumic acid) also significantly reduced PAR2-stimulated chloride transport. PAR2 activation was associated with increases in cAMP and intracellular calcium. Immunoblot analysis revealed increases in phosphorylation of EGF receptor (EGFr)-tyrosine-kinase, Src, Pyk2, cRaf and ERK1/2 in response to PAR2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFr (PD153035), MEK (PD98059 or U0126) and Src (PP1) inhibited SLIGRL-NH2-induced increases in short circuit current. Inhibition of Src, but not of matrix metalloproteinases, reduced EGFr phosphorylation. Reduced EGFr phosphorylation paralleled the reduction in PAR2-stimulated short circuit current. We conclude that activation of basal, but not apical, PAR2 induces epithelial chloride secretion via cAMP- and calcium-dependent mechanisms. The secretory effect involves EGF receptor transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.
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