Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca 2+ concentration inside pancreatic acinar cells ([Ca 2+ ] i ), due to excessive release of Ca 2+ stored inside the cells followed by Ca 2+ entry from the interstitial fluid. The sustained [Ca 2+ ] i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca 2+ release-activated Ca 2+ channels (CRAC) would prevent sustained elevation of [Ca 2+ ] i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca 2+ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca 2+ . Ca 2+ entry then occurred upon admission of Ca 2+ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca 2+ entry in a concentrationdependent manner within the range of 1 to 50 μM (IC 50 = 3.4 μM), but had little or no effect on the physiological Ca 2+ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca 2+ ] i , which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca 2+ ] i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.capacitative Ca 2+ entry | alcohol metabolite | pancreas | hepatocyte Ca 2+ entry | AR42JA cute pancreatitis is a human disease mostly caused by alcohol abuse or complications from biliary disease. In this disease, against which there is currently no effective therapy, digestive proenzymes are prematurely activated inside the acinar cells leading to autodigestion and necrosis (1-3). Intracellular Ca 2+ plays a critical role in the initiation of this disease process (2-4), but intracellular Ca 2+ also plays a critical role in the physiological regulation of the normal exocytotic secretion of the digestive proenzymes (5).The pancreatic acinar cells are capable of generating multiple patterns of cytosolic Ca 2+ signals depending on the type and concentration of the stimulating agent (5). The physiological Ca 2+ signals regulating secretion-evoked by the neurotransmitter acetylcholine (ACh) or the hormone cholecystokinin (CCK)-consist of repetitive short-lasting rises in the cytosolic Ca 2+ concentration ([Ca 2+ ] i ). These are mostly confined to the apical area, in which the secretory (zymogen) granules (ZGs) are concentrated, by a belt of perigranular mitochondria operating as a firewall against...

Key points Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions.In this study, we have investigated bradykinin‐elicited Ca2+ signal generation in normal mouse pancreatic lobules.We found complete separation of Ca2+ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca2+ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca2+ signals in PACs, never elicited Ca2+ signals in PSCs.The bradykinin‐elicited Ca2+ signals were due to initial Ca2+ release from inositol trisphosphate‐sensitive stores followed by Ca2+ entry through Ca2+ release‐activated channels (CRACs). The Ca2+ entry phase was effectively inhibited by a CRAC blocker.B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. AbstractNormal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca2+ signals. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The BK‐induced Ca2+ signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca2+ rise in PSCs was due to inositol trisphosphate receptor‐mediated release from internal stores, whereas the sustained phase depended on external Ca2+ entry through Ca2+ release‐activated Ca2+ (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca2+ signal generation in PSCs and this may be helpful in treating acute pancreatitis.

This review deals with the roles of calcium and ATP in the control of the normal functions of the different cell types in the exocrine pancreas as well as the roles of these molecules in the pathophysiology of Acute Pancreatitis. Repetitive rises in the local cytosolic calcium ion concentration in the apical part of the acinar cells do not only activate exocytosis but also, via an increase in the intra-mitochondrial calcium ion concentration, stimulate the ATP formation that is needed to fuel the energy-requiring secretion process. However, intracellular calcium overload, resulting in a global sustained elevation of the cytosolic calcium ion concentration, has the opposite effect of decreasing mitochondrial ATP production and this initiates processes that lead to the necrotic destruction of the cells. In the last few years it has become possible to image calcium signalling events simultaneously in acinar, stellate and immune cells in intact lobules of the exocrine pancreas. This has disclosed processes by which these cells interact with each other, particularly in relation to the initiation and development of Acute Pancreatitis. By unravelling the molecular mechanisms underlying this disease, several promising therapeutic intervention sites have been identified. This provides hope that we may soon be able to effectively treat this often fatal disease.

Rationale Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear. Objective To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering, and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart. Methods and Results Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with post-pause AP prolongation, afterdepolarizations and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction. Conclusions Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, AP prolongation and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.

Key points Ca2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared.Ca2+ signals evoked by K+‐induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca2+ signals in acinar but not in stellate cells.Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca2+ signals in response to membrane depolarization.The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol‐related acute pancreatitis, but they became sensitive to stimulation with trypsin.Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. AbstractPhysiological Ca2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca2+ signals in the peri‐acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization‐evoked Ca2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca2+ signals in response to membrane depolarization. The principal agent evoking Ca2+ signals in the stellate cells is bradykinin, but in experimental alcohol‐related acute pancreatitis, these cells become much less responsive to bradykinin and then acquire sensitivity to trypsin. Our new findings have implications for our understanding of the development of acute pancreatitis and we propose a scheme in which Ca2+ signals in stellate cells provide an amplification loop promoting acinar cell death. Initial release of the proteases kallikrein and trypsin from dying acinar cells can, via bradykinin generation and protease‐activated receptors, induce Ca2+ signals in stellate cells which can then, possibly via nitric oxide generation, damage more acinar cells and thereby cause additional release of proteases, generating a vicious circle.

Immune cells were identified in intact live mouse pancreatic lobules and their Ca2+ signals, evoked by various agents, characterised and compared with the simultaneously recorded Ca2+ signals in neighboring acinar and stellate cells. Immunochemistry in the live lobules indicated that the pancreatic immune cells most likely are macrophages. In the normal pancreas the density of these cells is very low, but induction of acute pancreatitis, by a combination of ethanol and fatty acids, markedly increased the number of the immune cells. The principal agent eliciting Ca2+ signals in the pancreatic immune cells was ATP, but these cells also frequently produced Ca2+ signals in response to acetylcholine and to high concentrations of bradykinin. Pharmacological studies, using specific purinergic agonists and antagonists, indicated that the ATP-elicited Ca2+ signals were mediated by both P2Y1 and P2Y13 receptors. The pancreatic immune cells were not electrically excitable and the Ca2+ signals generated by ATP were primarily due to release of Ca2+ from internal stores followed by store-operated Ca2+ entry through Ca2+ Release Activated Ca2+ channels. The ATP-induced intracellular Ca2+ liberation was dependent on both IP3 generation and IP3 receptors. We propose that the ATP-elicited Ca2+ signal generation in the pancreatic immune cells is likely to play an important role in the severe inflammatory response to the primary injury of the acinar cells that occurs in acute pancreatitis.

Acute pancreatitis (AP), a human disease in which the pancreas digests itself, has substantial mortality with no specific therapy. The major causes of AP are alcohol abuse and gallstone complications, but it also occurs as an important side effect of the standard asparaginase-based therapy for childhood acute lymphoblastic leukemia. Previous investigations into the mechanisms underlying pancreatic acinar cell death induced by alcohol metabolites, bile acids, or asparaginase indicated that loss of intracellular ATP generation is an important factor. We now report that, in isolated mouse pancreatic acinar cells or cell clusters, removal of extracellular glucose had little effect on this ATP loss, suggesting that glucose metabolism was severely inhibited under these conditions. Surprisingly, we show that replacing glucose with galactose prevented or markedly reduced the loss of ATP and any subsequent necrosis. Addition of pyruvate had a similar protective effect. We also studied the effect of galactose in vivo in mouse models of AP induced either by a combination of fatty acids and ethanol or asparaginase. In both cases, galactose markedly reduced acinar necrosis and inflammation. Based on these data, we suggest that galactose feeding may be used to protect against AP.

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