Protease-activated Receptor-2 Increases Exocytosis via Multiple Signal Transduction Pathways in Pancreatic Duct Epithelial Cells

摘要

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH2 terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PHPLC-δ1-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cγ (PKCγ)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca²⁺ stores and a subsequent Ca²⁺ influx through store-operated Ca²⁺ channels cause a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]i), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca²⁺]i in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 μm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 μm trypsin at 37 °C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca²⁺]i and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.