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PAR2-INDUCED EPITHELIAL CHLORIDE SECRETION INVOLVES PHOSPHOLIPASE C (PLC) AND PROTEIN KINASE C (PKC) ISOFORM-SPECIFIC SIGNALING
J van der Merwe, WK MacNaughton
Inflammation Research Network, University of Calgary, Calgary, Alberta
BACKGROUND: Proteinase-activated receptor (PAR)2 is activated by trypsin-like serine proteinases and selective synthetic peptides. It has been implicated in intestinal inflammation, but its role in the regulation of intestinal mucosal function remains unclear. We have previously shown that PAR2-induced chloride secretion involves both calcium and cAMP signaling, and Src-dependent EGFr transactivation leading to subsequent ERK1/2 activation in a cell culture model (SCBNs). We have further studied the stimulus-secretion coupling mechanisms of PAR2 activation on epithelial chloride transport in SCBNs, focusing on specific PLC and PKC isoforms.
METHODS: 1. SCBN monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short circuit current (Isc) was monitored as a measure of net electrogenic ion transport. The roles of specific PLC and PKC isoforms in PAR2-induced chloride secretion were determined by the use of selective PLC (U73122, ET-18 and D609) and PKC inhibitors (GFX, Gö6976, rottlerin, PKCzeta pseudosubstrate inhibitor). 2. Immunoblot analysis was conducted for both PLCbeta and PLCgamma following PAR2 activation by the activating peptide, SLIGRL-NH2 (50 µM), for 5 minutes. Immunoblots for total amounts of each protein were conducted as controls.
RESULTS: 1. Pretreatment with U71322, D609 or ET-18 resulted in a significant decrease in PAR2-induced ion secretion, suggesting a role for both phosphatidylcholine-specific PLC and phosphoinositol-specific PLC in the Isc response. Furthermore, PAR2 stimulation by SLIGRL-NH2 resulted in a significant increase in both PLCbeta and PLCgamma phosphorylation. Pretreatment of the cells with GFX, rottlerin and Gö6976, but not the PKCzeta pseudosubstrate inhibitor, resulted in a significant reduction in the chloride secretory response to PAR2 activation by SLIGRL-NH2, thereby suggesting a role for PKCdelta, PKCalpha and/ or PKCbeta1 in PAR2 mediated chloride secretion.
CONCLUSIONS: PAR2 activation induces epithelial chloride secretion that is mediated by both PC-PLC and PI-PLC, and involves increases in PLCbeta/gamma phosphorylation. Furthermore PKCdelta, PKCalpha and/or PKCbeta1, but not PKCzeta, are important in PAR2-induced epithelial chloride secretion.