Protease-activated receptor-2 increases exocytosis via multiple signal transduction pathways in pancreatic duct epithelial cells.
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.
Kim
Mean-Hwan
MH
Department of Physics, POSTECH, Pohang 790-784, Republic of Korea.
Choi
Bo-Hwa
BH
Jung
Seung-Ryoung
SR
Sernka
Thomas J
TJ
Kim
Seunghwan
S
Kim
Kyong-Tai
KT
Hille
Bertil
B
Nguyen
Toan D
TD
Koh
Duk-Su
DS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004195:Disease Models, Animal; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005089:Exocytosis / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007473:Ion Channels; D009077:Mucins / Q000378:metabolism; D010183:Pancreatic Ducts / Q000378:metabolism* / Q000473:pathology; D010195:Pancreatitis / Q000378:metabolism / Q000473:pathology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D015220:Calcium Channels; D007473:Ion Channels; D009077:Mucins; D019269:Phosphatidylinositol 4,5-Diphosphate; D044464:Receptor, PAR-2; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; D014357:Trypsin; D002118:Calcium