Abstract
Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) 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, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-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 Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](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 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees 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 [Ca(2+)](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.
Highlights
Of the four protease-activated receptors (PARs),3 G-protein-coupled receptors activated by proteolysis [1, 2], PAR-1 and PAR-3 are activated by thrombin, Protease-activated receptor-2 (PAR-2) by trypsin and tryptase, and PAR-4 by both thrombin and trypsin
In this report we define the different signaling pathways activated by PAR-2 and investigated how they interact to stimulate the complex function of exocytosis
Trypsin-induced PAR-2 Signaling—In different tissues PAR-2 effects are typically mediated through PLC-mediated generation of IP3 and DAG [3, 4]; induction of a cAMP increase has been suggested (16 –18)
Summary
Cell Culture—Canine PDEC, originally derived from the accessory pancreatic duct of a dog, were cultured on Vitrogencoated Transwell inserts above a confluent feeder layer of human gall bladder myofibroblasts, as previously obtained and. Measurement of PIP2 and DAG—Plasma membrane PIP2 and DAG were monitored using PHPLC-␦1-EGFP (GFP-PH) and PKC␥-C1-YFP (YFP-C1), the fluorescent translocation probes, respectively For these experiments, PDEC on glass coverslips were transfected with cDNA Measurement of [Ca2ϩ]i—For single cell experiments, PDEC were loaded with 2 M membrane-permeant Ca2ϩ-sensitive dye Indo-1 AM for 30 min. Fura-2 fluorescence was excited at 340 and 380 nm and detected at 510 nm In this imaging system only changes in [Ca2ϩ]i could be determined through the ratio of fluorescence at 340 and 380 nm, as no cell-free region was present for background determination and correction. Significant differences (p Յ 0.05 (*)) between the means of two groups were determined by Student’s two-tailed, unpaired t test
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