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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Prostaglandin E₂ Derived from Cyclooxygenases 1 and 2 Mediates Intestinal Epithelial Ion Transport Stimulated by the Activation of Protease-Activated Receptor 2

Inflammation Research Network and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada

Proteinase-activated receptor (PAR)₂ is activated by trypsin-like serine proteinases and has been implicated in intestinal inflammation. However, its role in the regulation of intestinal mucosal function remains unclear. Using the intestinal epithelial cell line, SCBN, we have studied the stimulus-secretion coupling mechanisms of PAR₂-induced epithelial chloride transport, focusing on cyclooxygenase (COX)-1 and COX-2 activities and prostaglandin (PG) E₂ secretion. SCBN monolayers were grown on Snapwell supports, mounted in modified Ussing chambers, and exposed to the activating peptide, SLIGRL-NH₂ (50 µM), to activate PAR₂. Pretreatment with inhibitors of cytosolic PLA₂ (cPLA₂) (AACOCF3, arachidonyltrifluoromethyl ketone), COX-1 [SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2 (celecoxib) resulted in a significant concentration-dependent attenuation of PAR₂-induced changes in short-circuit current. Immunoblot analysis showed a PAR₂-induced increase in cPLA₂ phosphorylation that was blocked by the mitogen-activated protein kinase kinase inhibitor, PD98059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C₁₆H₁₃NO₃], and the pan-protein kinase C inhibitor, GFX (bisindolylmaleimide). PAR₂ stimulation also resulted in a large increase in the production of PGE₂ as determined by enzyme-linked immunosorbent assay and was also blocked by PD98059 and GFX. Immunofluorescence and immunoblot analysis determined that EP2 and EP4 are expressed at the basolateral membrane of SCBN cells. Through the use of selective inhibitors (EP2, AH6809 [6-isopropoxy-9-oxoxanthene-2-carboxylic acid]; EP4, GW627368X [N-{2[4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl] acetyl}benzene sulphonamide]), it was found that both EP2 and EP4 were involved in mediating the PAR₂-induced chloride secretory response. We conclude that basolateral PAR₂ activation induces epithelial chloride secretion that is mediated by cPLA₂, COX-1, COX-2, and the subsequent release of PGE₂. The production of PGE₂ results in an autocrine secretory response that is dependent on basolateral EP2 and EP4 receptors.

Address correspondence to: Dr. Wallace MacNaughton, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1. E-mail: wmacnaug{at}ucalgary.ca