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061 PAR2-INDUCED EPITHELIAL CHLORIDE SECRETION INVOLVES BOTH PROTEIN KINASE C AND CYCLOOXYGENASE ACTIVITIES JQ van der Merwe, WK MacNaughton Intestinal epithelial proteinase-activated receptor-2 (PAR2) is activated by trypsin-like serine proteinases to stimulate intestinal epithelial chloride secretion, but the cellular mechanism is unclear. We have previously shown that PAR2-induced chloride secretion involves Src-dependent EGF receptor transactivation leading to subsequent ERK1/2 activation in a cell culture model (SCBN). In the present study, we investigated the role of specific protein kinase (PK) C isoforms and cyclooxygenases (COX-1, COX-2) in epithelial chloride transport stimulated by PAR2 activation in the SCBN epithelial cell line.
Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta
METHODS: SCBN monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short circuit current (Isc) was used to measure net electrogenic ion transport, primarily chloride secretion, stimulated by the PAR2-activating peptide, SLIGRL-NH2 (50 µM). First, the role of PKC was determined by pretreating the cells with inhibitors of different PKC isoforms (GFX, Go6976, rottlerin, PKC5 pseudosubstrate inhibitor). The effect of PKC inhibition on PAR2-induced ERK1/2 MAP kinase activation was determined by immunoblot. Second, the role of arachidonic acid metabolites in the response to PAR2 activation was examined using specific inhibitors of phospholipase A2 (AACOCF3), COX-1 and COX-2 (indomethacin, celecoxib and SC560).
RESULTS: 1. Pretreatment of the cells with GFX, rottlerin and Go6976, but not the PK5 pseudosubstrate inhibitor, resulted in a significant reduction in the Isc response to PAR2 activation by SLIGRL-NH2, thereby suggesting a role for PKCdelta, PKCalpha and/or PKCbeta1 in PAR2-mediated chloride secretion. Furthermore inhibition of PKCdelta, but not PKCalpha or PKCbeta1, prevented ERK1/2 phosphorylation, suggesting that PAR2-induced ERK1/2 phosphorylation is dependent on PKCdelta. Thus, PKCalpha and/or PKCbeta1 mediate PAR2-induced chloride secretion independently of ERK1/2 activation. 2. Inhibition of PLA2, COX-1 and COX-2 resulted in a significant reduction PAR2-induced chloride secretion.
CONCLUSIONS: The ERK1/2-dependent component of PAR2-induced epithelial chloride secretion is mediated by PKCdelta, whereas the ERK1/2-independent component is mediated by PKCalpha and/or PKCbeta1. PAR2-activation also stimulates PLA2 to release arachidonic acid which is metabolized by COX-1 and COX-2 to produce a secretory prostanoid.