Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway

Proteinase-activated receptor 1 (PAR-1) is activated by thrombin and induces chloride secretion by intestinal epithelial cells. To elucidate further the mechanisms whereby PAR -1 stimulates secretion, monolayers of SCBN intestinal epithelial cells were studied in modified Ussing chambers. Short circuit current responses were determined after basolateral application of thrombin and the PAR-1 -activating peptide, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-Citrulline-Tyr (Cit-NH2) in the presence or absence of a variety of signal transduction and cyclo-oxygenase (COX) pathway inhibitors. Increased kinase activity was monitored by immunoprecipitation and Western blot analysis of target phosphoproteins. The PAR 1-induced chloride secretory response was significantly attenuated by inhibitors of the EGF receptor tyrosine kinase, Src-kinase, MEK1/2, as well as by inhibitors of cytosolic phospholipase (cPL) A(2), COX-1 and COX-2. PAR-1-induced activation of cPLA(2), as shown by Western blot of phosphoserine residues, was blocked in cells treated with the MEK inhibitor U0126, indicating that the MEK-ERK1/2 MAP kinase pathway mediated PAR-1-induced cPLA2 phosphorylation. Our data show that PAR 1-induced chloride secretion in SCBN cells involves Src, EGF receptor trans-activation, activation of a MAPK pathway, phosphorylation of cPLA2, COX activity, but not PGF(2alpha) or PGE(2). These findings may be of clinical importance in inflammatory diseases of the intestine where secretory dysfunction is evident and thrombin levels are elevated.