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MUCOSAL BIOLOGY

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms

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

Submitted 10 July 2008 ; accepted in final form 2 April 2009

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR₂-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I_sc) responses to basolateral application of the selective PAR₂ activating peptide, SLIGRL-NH₂, were monitored as a measure of net electrogenic ion transport caused by PAR₂ activation. SLIGRL-NH₂ induced a transient I_sc response that was significantly reduced by inhibitors of PLC (U73122 [GenBank] ), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCβ and PLC following PAR₂ activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKC/βI (Gö6976), and PKC (rottlerin), but not PKC (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCβI, PKC, and PKC, but not PKC or PKC, in membrane fractions following PAR₂ activation. Pretreatment of the cells with U73122 [GenBank] , ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKC translocation. Immunoblots revealed that PAR₂ activation induced phosphorylation of both cRaf and ERK1/2 via PKC. Inhibition of PKCβI and PI3K had only a partial effect on this response. We conclude that basolateral PAR₂-induced chloride secretion involves activation of PKCβI and PKC via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

ion transport; proteases; signal transduction; epidermal growth factor receptor; cRaf