Background & AimsSustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.MethodsMouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.ResultsGSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.ConclusionsCytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

The intracellular activation of trypsinogen, which is both pH-and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokininelicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-Larginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ؎ 0.1, and the calcium ion concentration was 37 ؎ 11 M. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within Ϸ100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.Ca 2ϩ ͉ endocytosis ͉ pancreatitis ͉ trypsin

The effect of the natural bile acid, taurolithocholic acid 3‐sulfate (TLC‐S), on calcium signalling in pancreatic acinar cells has been investigated. TLC‐S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC‐S in a calcium‐free external solution. TLC‐S‐induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC‐S application, displayed vectorial apical‐to‐basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC‐S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid‐induced cellular damage.

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Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca 2؉ toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca 2؉ . Using two-photon plasma membrane permeabilization and measurement of [Ca 2؉ ] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca 2؉ release pathways. Inhibition of inositol trisphosphate receptors (IP 3 Rs), by caffeine and 2-APB, reduced Ca 2؉ release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP 3 Rs and RyRs abolished Ca 2؉ release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca 2؉ liberation from both the ER and the apical acidic stores depends on both RyRs and IP 3 Rs.Bile acids, hydrophobic derivatives of cholesterol, have been suggested as a possible cause of acute pancreatitis (1). Many patients with acute pancreatitis have gallstones, blocking the ampulla of Vater, which may allow reflux of bile into the pancreatic duct system, leading to inflammation. It has been proposed that bile acids can trigger acute pancreatitis (2-4), and they have also been postulated to be tumor promoters (5), although the mechanism of action is not clear (6). Bile salts are known to induce severe experimental pancreatitis (7-9). Recent research points mainly toward abnormal calcium signaling as a possible cause of acute pancreatitis (10 -13) while casting doubt on the originally proposed ionophore-like mechanism of action of bile acids (14,15). Previous work has shown that application of bile acids can cause an increase in the levels of cytosolic [Ca 2ϩ ] in both hepatocytes (16,17) and pancreatic acinar cells (10). Specifically, bile acids activate calcium entry into the cell and cause depletion of internal calcium stores (12). Other effects not linked to calcium signaling (18) have also been observed, including an increase in the intracellular Na ϩ concentration (19) and depolarization of the inner mitochondrial membrane (20, 21).Ca 2ϩ signaling has been extensively studied in pancreatic acinar cells (22,23) and their organelles (24 -26). They are highly polarized, with distinct basal and apical poles. The secretory granules are confined to the apical region (27), which is surrounded by a perigranular Golgi apparatus and a mitochondrial belt (28, 29). All Ca 2ϩ signals start in the apical pole, and those elicited by low agonist concentrations are most...