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J. Biol. Chem., Vol. 283, Issue 27, 18711-18720, July 4, 2008

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

Mean-Hwan Kim¹, Bo-Hwa Choi, Seung-Ryoung Jung, Thomas J. Sernka^¶, Seunghwan Kim, Kyong-Tai Kim, Bertil Hille^||, Toan D. Nguyen^¶**, and Duk-Su Koh^||2

From the Departments of Physics and Life Science, POSTECH, Pohang 790-784, Republic of Korea and ^¶Veterans Affairs Puget Sound Health Care System and Departments of ^||Physiology and Biophysics and ^**Medicine, University of Washington, Seattle, Washington 98195

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, PH_PLC-1-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase C (PKC)-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.

Received for publication, February 29, 2008 , and in revised form, April 30, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM083913 and DK55885. This work was also supported by the R&D Medical Research Office of the Department of Veterans Affairs (Merit Review) and by Korea Science and Engineering Foundation Grant R01-2002-000-00285). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Brain Korea 21 and the Korea Research Foundation Grant KRF-2006-612-C00011.

² To whom correspondence should be addressed: Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Health Sciences Bldg., Seattle, WA 98195-7290. Tel: 206-543-6661; Fax: 206-685-0619; E-mail: koh{at}u.washington.edu.

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