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).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, PAR-2 by trypsin and tryptase, and PAR-4 by both thrombin and trypsin. Trypsin and tryptase cleave within the extracellular NH 2 terminus of PAR-2 (in humans, at the arrow N-SKGR2SLIGRL-C) to yield a tethered ligand (N-SLIGRL-C) that activates the cleaved receptor. In various tissues PAR-2 couples with PLC to hydrolyze PIP 2 into IP 3 and DAG. IP 3 in turn increases [Ca 2ϩ ] i , whereas DAG activates PKC (2-4).PAR-2 is highly expressed in several tissues including pancreas, kidney, intestine, liver, and heart (3) where it mediates both physiologic and pathologic functions (2, 5, 6). PAR-2 function in the pancreas is of particular interest because its activator, trypsin, is a digestive enzyme produced by pancreatic acina. In pancreatic ducts, which channel digestive enzymes from the acina into the duodenum and further secrete fluid and electrolytes into the lumen, we demonstrated that PAR-2 mediates the activation of Ca 2ϩ -actived K ϩ and Cl Ϫ channels (7). In addition, we also demonstrated that exocytosis from PDEC is induced by [Ca 2ϩ ] i increases and by activation of protein kinases A and C (8, 9).We now report that PAR-2 activation stimulates PLC-mediated hydrolysis of PIP 2 into IP 3 and DAG, increasing [Ca 2ϩ ] i via Ca 2ϩ mobilization from intracellular stores and Ca 2ϩ influx through store-operated Ca 2ϩ channels (SOC) and activating PKC. Each of these signals promotes exocytosis and mucin secretion. EXPERIMENTAL PROCEDURESCell 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 * 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.
Cardiac glycosides are known to inhibit cation transport and the activity of Na+-K+-dependent ATPase in most tissues (1). In oxygenated in vitro frog gastric mucosa Davenport (2) reported that ouabain inhibited active transport of H+ and Na+, and Cooperstein (3) observed inhibition of H+ and C1-transport with strophanthidin. In rat gastric mucosa Sernka and Hogben (4) found inhibition of active H+ and C1transport by ouabain.The purposes of our study were (a) to explore the effects of ouabain on active transpolt and the electrical properties of an in vivo canine stomach preparation and (b) to compare these findings with flux measurements in an in vitro canine stomach preparation. This comparison suggests that simpler measurements in the in vivo preparation permit prediction of findings in the in vitro.Materials and Methods. In vivo preparation. Six fasted mongrel dogs ( 13-22 kg) of either sex were anesthetized with intravenous chloralose and ethyl carbamate ( 1 ml/kg of a solution containing 9.25 g chloralose and 92.5 g ethyl carbamate in 150 ml normal saline). Our in vivo chambered stomach preparation has been described previously (5).The chamber divided the stomach flap into two sides. Each side was bathed with 10 ml of isotonic saline maintained at 37". The effluent from one side was collected and titrated (Radiometer autoburette) at 15 min interyals to determine the gastric acid output. The other side of the chamber was connected to calomel electrodes and Ag-AgC1 electrodes to determine transmural potential difference (PD) and current ( I ) necessary to reduce the PD to zero, respec-Recipient of Research Scientist Development Award 1 KO2 MH70463-0 1. tively . These measurements were determined with a Shanbour voltage-clamp system (6). The relative resistance (R) was then calculated as the ratio of PD to I . The pH of this side of the chamber was kept above 2.5 by constantly flushing the mucosa with normal saline at 3437".Histamine base (1.2-2.0 pg/kg min) was infused through a femoral vein for 90 min. Then, with continuous histamine infusion, ouabain (Lilly) was injected as a bolus dose (50 pg/kg) into the other femoral vein. Throughout both control and postouabain periods, measurements of PD, I , and acid output were obtained at intervals noted above. Only those animals which achieved an acid secretory rate above 60 pEq/ 15 min were used in the study.In vitro preparation.
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