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...

Abstract-Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly reduced LPS-mediated secretion of tumor necrosis factor-␣, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPSstimulated macrophages treated with the S1P1 receptor-specific agonist SEW2871. Pharmacological antagonism of the S1P1 receptor on macrophages using the S1P1-specific antagonist VPC44116 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow-derived macrophages from S1P2-deficient mice revealed that the S1P2 receptor did not play a pivotal role in this process. Thus, activation of the S1P1 receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S1P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S1P1 receptor activation on macrophages. Analysis of a 1.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor B, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor B-mediated induction of inducible NO synthase. These findings demonstrate an important role for S1P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S1P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S1P1 receptor. (Circ Res. 2008;102:950-958.)Key Words: macrophage Ⅲ sphingosine-1-phosphate Ⅲ arginase I Ⅲ iNOS Ⅲ NFB Ⅲ inflammation A therosclerosis is a chronic inflammatory disease. 1 Monocytes/macrophages play key roles in the initiation and progression of atherosclerosis, and can alter their phenotype in response to changes in the local cytokine environment. 2 Macrophages can be distinctly activated to either a classically activated, or M1, phenotype by proinflammatory molecules such as interferon-␥ and lipopolysaccharide (LPS) or to an "alternatively activated," or M2, phenotype by Th-2 antiinflammatory cytokines such as interleukin (IL)-4. 3 Macrophages can also display a M2b activated phenotype when stimulated by immune complexes and LPS. 4 The M1 and M2 phenotypes are specifically distinguished by the cytokines produced and by the way in which arginine is processed, whereas the M2b phenotype is mainly distinguished by high IL-10 and low IL-12 levels. In classically activated macrophages, Th-1 cytokines trigger the induction of inducible NO synt...

We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

Objective-Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. Methods and Results-On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT Յ23), medium (moderately active, GTϭ24 to 28), and long (least active, GT Ն29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J 2 , hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1␤, interleukin-6, and soluble intercellular adhesion molecule-1. Conclusion-The

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

Pancreatic stellate cells, normally quiescent, are capable of remarkable transition into their activated myofibroblast-like phenotype. It is now commonly accepted that these cells play a pivotal role in the desmoplastic reaction present in severe pancreatic disorders. In recent years, enormous scientific effort has been devoted to understanding their roles in pancreatic cancer, which continues to remain one of the most deadly diseases. Therefore, it is not surprising that considerably less attention has been given to studying physiological functions of pancreatic stellate cells. Here, we review recent advances not only in the field of pancreatic stellate cell pathophysiology but also emphasise their roles in physiological processes.

Key points Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. AbstractAcute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na+‐dependent bile acid uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca2+ and significantly reduced in the absence of Na+, showing that bile‐dependent cell death was a downstream event of Ca2+ signals. Finally, combined application of TLC‐S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC‐S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells.

The mammalian diffuse stellate cell system comprises retinoid-storing cells capable of remarkable transformations from a quiescent to an activated myofibroblast-like phenotype. Activated pancreatic stellate cells (PSCs) attract attention owing to the pivotal role they play in development of tissue fibrosis in chronic pancreatitis and pancreatic cancer. However, little is known about the actual role of PSCs in the normal pancreas. These enigmatic cells have recently been shown to respond to physiological stimuli in a manner that is markedly different from their neighbouring pancreatic acinar cells (PACs). Here, we demonstrate the capacity of PSCs to generate nitric oxide (NO), a free radical messenger mediating, for example, inflammation and vasodilatation. We show that production of cytosolic NO in PSCs is unambiguously related to cytosolic Ca2+ signals. Only stimuli that evoke Ca2+ signals in the PSCs elicit consequent NO generation. We provide fresh evidence for the striking difference between signalling pathways in PSCs and adjacent PACs, because PSCs, in contrast to PACs, generate substantial Ca2+-mediated and NOS-dependent NO signals. We also show that inhibition of NO generation protects both PSCs and PACs from necrosis. Our results highlight the interplay between Ca2+ and NO signalling pathways in cell–cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies.