In previous studies, the EC50s of trypsin and AP for the rat PAR-2 were, respectively, 25 nM and 17 M; for the human PAR-2, the corresponding EC50s were 2.3 nM and 18 M (8). trypsin and AP stimulated a short-circuit current from your basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical ClC and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on doggie pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is usually prematurely activated, PAR-2Cmediated ductal secretion may promote clearance of toxins and debris. Introduction Proteinase-activated receptor-2 (PAR-2) is the second member of the new family of G proteinCcoupled receptors that are activated by proteolysis rather than binding to a soluble ligand (examined in ref. 1). PAR-1, PAR-3, and PAR-4 are receptors for thrombin (2C5); PAR-2 is usually a receptor for pancreatic trypsin and mast cell tryptase (6, 7). Trypsin and tryptase cleave within the extracellular NH2-terminus of PAR-2 at SKGRSLIGRL, yielding a tethered ligand (SLIGRL) that binds to and activates the cleaved receptor. Synthetic peptides corresponding to this tethered ligand domain name selectively activate PAR-2 without proteolysis. They are thus useful reagents for studying receptor function without the use of proteases, which may cleave other proteins. The gene encoding PAR-2 has been cloned in humans, and PAR-2 has been found to be highly expressed in the pancreas and kidney as well as intestine, liver, prostate, heart, lung, and trachea (8). High pancreatic expression is supported by abundant PAR-2 expression in several cell lines derived from pancreatic acinar and duct cells. However, although the tissue distribution of PAR-2 has been examined, its precise cellular localization, ligands, and physiological function are unknown for most tissues. The very high level of PAR-2 expression in the pancreas is usually intriguing, as trypsin, the protease that cleaves and triggers PAR-2 with highest potency and efficacy, is usually synthesized and secreted by pancreatic acinar cells. Although trypsin is usually traditionally considered as a digestive enzyme, we have recently reported (9) that physiological concentrations of trypsin in the intestinal lumen cleave and activate PAR-2 at the apical membrane of enterocytes, suggesting that trypsin also functions as a signaling molecule that specifically targets cells through PAR-2. It is therefore possible that 20(S)-NotoginsenosideR2 trypsin also activates PAR-2 in the pancreas and thereby regulates pancreatic function. However, trypsin is mostly secreted as its inactive zymogen precursor, trypsinogen, which is usually inactive until it is cleaved by enterokinase in the intestinal lumen. Although small amounts of active trypsin are created within the pancreas under normal circumstances, trypsin is usually prematurely autoactivated within 20(S)-NotoginsenosideR2 the inflamed pancreas and is believed to contribute to pancreatitis (10). Indeed, the genetic defects of hereditary pancreatitis are amino acid mutations of trypsin that render it resistant to degradation following premature autoactivation (11, 12). Therefore, trypsin may cleave and activate PAR-2 within the inflamed pancreas. A role for PAR-2 in inflammation is also supported by the finding that tryptase, a prominent component of secretory granules of most subsets of human mast cells that is released upon degranulation, also activates PAR-2 (7, 13). Tryptase may also trigger PAR-2 in the pancreas during inflammation, when mast cells are present (Nguyen, T.D., developed methods to isolate and culture doggie PDEC that are nontransformed, well-differentiated, and polarized, and which retain many of the characteristics of PDEC, such as mucin secretion (14) and the presence Dnmt1 of cAMP- and Ca2+-activated ClC channels (15), and Ca2+-activated K+ channels (16). They are thus ideally suited for detailed examination of the regulation of ion channels by specific receptors (17, 18). In the present investigation, 20(S)-NotoginsenosideR2 we examined the hypothesis that trypsin regulates PDEC through PAR-2. Our aims were to ([ln (8C13 cells for each measurement). Immunostaining. Pancreas from an adult dog was fixed in 4% paraformaldehyde in 100 mM PBS for 48C72 h at 4C and placed in 25% sucrose in PBS for 24 h at 4C. Specimens were either (assessments. Results Iodide efflux studies. Trypsin activation of ion channels of PDEC was first evaluated. Trypsin stimulated 125IC efflux in a concentration-dependent manner, with efflux peak rate coefficients of 0.268 0.25/min (peak increase above baseline: 0.085/min), 0.620 0.051/min (peak increase: 0.436/min), and 0.615 0.089/min (peak increase: 0.456/min) observed, respectively, 105, 45, and 30 seconds after the addition of 0.1, 1, and 10 M trypsin (Fig. ?(Fig.113). (3). (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl) ester 4,4-diisothiocyanatostilbene-2,2-disulfonic acid; 0.001 compared with control for both inhibitors) (Fig. ?(Fig.223), 1 min after its addition. This effect was inhibited by 100 nM charybdotoxin, an inhibitor of Ca2+-activated K+ channels, to a peak efflux rate coefficient of 0.081 0.007/min (peak increase: 0.059/min, 0.001 compared with.Bar, 50 m in and and and em f /em ) and when the primary antibodies were replaced with an unrelated rabbit IgG (not shown), both indicating specificity. chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on doggie pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is usually prematurely activated, PAR-2Cmediated ductal secretion may promote clearance of toxins and debris. Introduction Proteinase-activated receptor-2 (PAR-2) is the second member of the new family of G proteinCcoupled receptors that are activated by proteolysis rather than binding to a soluble ligand (examined in ref. 1). PAR-1, PAR-3, and PAR-4 are receptors for thrombin (2C5); PAR-2 is usually a receptor for pancreatic trypsin and mast cell tryptase (6, 7). Trypsin and tryptase cleave within the extracellular NH2-terminus of PAR-2 at SKGRSLIGRL, yielding a tethered ligand (SLIGRL) that binds to and activates the cleaved receptor. Synthetic peptides corresponding to this tethered ligand domain name selectively activate PAR-2 without proteolysis. They are thus useful reagents for studying receptor function without the use of proteases, which may cleave other proteins. The gene encoding PAR-2 has been cloned in humans, and PAR-2 has been found to be highly expressed in the pancreas and kidney as well as intestine, liver, prostate, heart, lung, and trachea (8). High pancreatic expression is supported by abundant PAR-2 expression in several cell lines derived from pancreatic acinar and duct cells. However, although the tissue distribution of PAR-2 has been examined, its precise cellular localization, ligands, and physiological function are unknown for most tissues. The very high level of PAR-2 expression in the pancreas is usually intriguing, as trypsin, the protease that cleaves and triggers PAR-2 with highest potency and efficacy, is usually synthesized and secreted by pancreatic acinar cells. Although trypsin is usually traditionally considered as a digestive enzyme, we have recently reported (9) that physiological concentrations of trypsin in the intestinal lumen cleave and activate PAR-2 at the apical membrane of enterocytes, suggesting that trypsin also functions as a signaling molecule that specifically targets cells through PAR-2. It is therefore possible that trypsin also activates PAR-2 in the pancreas and thereby regulates pancreatic function. However, trypsin is mostly secreted as its inactive zymogen precursor, trypsinogen, which is usually inactive until it is cleaved by enterokinase in the intestinal lumen. Although small amounts of active trypsin are created within the pancreas under normal circumstances, trypsin is usually prematurely autoactivated within the inflamed pancreas and is believed to contribute to pancreatitis (10). Indeed, the genetic defects of hereditary pancreatitis are amino acid mutations of trypsin that render it resistant to degradation following premature autoactivation (11, 12). Therefore, trypsin may cleave and activate PAR-2 within the inflamed pancreas. A role for PAR-2 in inflammation is also supported by the finding that tryptase, a prominent component of secretory granules of most subsets of human mast cells that is released upon degranulation, also activates PAR-2 (7, 13). Tryptase may also trigger PAR-2 in the pancreas during inflammation, when mast cells are present (Nguyen, T.D., developed methods to isolate and culture doggie PDEC that are nontransformed, well-differentiated, and polarized, and which retain many of the characteristics of PDEC, such as mucin secretion (14) and the presence of cAMP- and Ca2+-activated ClC channels (15), and Ca2+-activated K+ channels (16). They are thus ideally suited for detailed examination of the regulation of ion channels by specific receptors (17, 18). In the present investigation, we examined the hypothesis that trypsin regulates PDEC through PAR-2. Our aims were to ([ln (8C13 cells for each measurement). Immunostaining. Pancreas from an adult dog was fixed in 4% paraformaldehyde in 100 mM PBS for 48C72 h at 4C and placed in 25% sucrose in PBS for 24 h at 4C. Specimens were either (assessments. Results Iodide efflux studies. Trypsin activation of ion channels of PDEC was first evaluated. Trypsin stimulated 125IC efflux in a.