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

van der Merwe JQ, Moreau F, MacNaughton WK. Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms. Am J Physiol Gastrointest Liver Physiol 296: G1258-G1266, 2009. First published April 9, 2009; doi:10.1152/ajpgi.90425.2008.-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 PAR(2)-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 PAR(2) activating peptide, SLIGRL-NH2, were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH2 induced a transient I-sc response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLC beta and PLC gamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKC alpha/beta I (Go6976), and PKC delta (rottlerin), but not PKC zeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKC beta I, PKC delta, and PKC epsilon, but not PKC alpha or PKC zeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKC delta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKC delta. Inhibition of PKC beta I and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKC beta I and PKC delta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.