Search

Competing Interests: J.G. holds the position of Editorial Board Member for Function and is blinded from reviewing or making decisions for the manuscript.

Exocrine pancreas has been the field of many successful studies in pancreatic physiology and pathology. However, related disease - acute pancreatitis (AP) is still takes it toll with more than 100,000 related deaths worldwide per year. In spite of significant scientific progress and several human trials currently running for AP, there is still no specific treatment in the clinic. Studies of the mechanism of initiation of AP have identified two crucial conditions: sustained elevations of cytoplasmic calcium concentration (Ca 2+ plateau) and significantly reduced intracellular energy (ATP depletion). These hallmarks are interdependent, i.e., Ca 2+ plateau increase energy demand for its clearance while energy production is greatly affected by the pathology. Result of long standing Ca 2+ plateau is destabilisation of the secretory granules and premature activation of the digestive enzymes leading to necrotic cell death. Main attempts so far to break the vicious circle of cell death have been concentrated on reduction of Ca 2+ overload or reduction of ATP depletion. This review will summarise these approaches, including recent developments of potential therapies for AP., Competing Interests: Declaration of Competing Interest The authors of the manuscript entitled “The role of Ca(2+) signalling in the pathology of exocrine pancreas” declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca 2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca 2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca 2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca 2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca 2+ -handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca 2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca 2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis., (© 2022. The Author(s).)

The S protein subunit 1 (S1) of SARS-CoV-2 is known to be responsible for the binding of the virus to host cell receptors, but the initial intracellular signalling steps following receptor activation of cells in the exocrine pancreas are unknown. Using an intact live mouse pancreatic lobule preparation, we observed that S1 elicited Ca 2+ signals in stellate cells and macrophages, but not in the dominant acinar cells. The Ca 2+ signals occurred mostly in the form of repetitive Ca 2+ spikes. The probability of observing Ca 2+ signals depended on the S1 concentration. The threshold was close to 70 nM, whereas at 600 nM, all cells responded. The SARS-Cov-2 nucleocapsid protein did not elicit any Ca 2+ signals in any of the three cell types tested. The S1-induced Ca 2+ signals in stellate cells started much faster (122 ± 37s) than those in macrophages (468 ± 68s). Furthermore, the interleukin-18 binding protein (IL-18BP) abolished the responses in macrophages without affecting the Ca 2+ signals in stellate cells. The S1-elicited Ca 2+ signals were completely dependent on the presence of external Ca 2+ and were abolished by a selective inhibitor (CM4620) of Orai1 Ca 2+ Release Activated Ca 2+ channels. SARS-CoV-2 may contribute to acute pancreatitis, an often fatal inflammatory human disease. The S1-elicited Ca 2+ signals we have observed in the pancreatic stellate cells and endogenous macrophages may play an important part in the development of the inflammatory process., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

This review deals with the roles of calcium ions 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 not only activate exocytosis but also, via an increase in the intramitochondrial 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 necrosis. In the last few years it has become possible to image calcium signaling 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 unraveling 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.

Immune cells were identified in intact live mouse pancreatic lobules and their Ca 2+ signals, evoked by various agents, characterized and compared with the simultaneously recorded Ca 2+ 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 (AP), by a combination of ethanol and fatty acids, markedly increased the number of the immune cells. The principal agent eliciting Ca 2+ signals in the pancreatic immune cells was ATP, but these cells also frequently produced Ca 2+ signals in response to acetylcholine and to high concentrations of bradykinin. Pharmacological studies, using specific purinergic agonists and antagonists, indicated that the ATP-elicited Ca 2+ signals were mediated by both P2Y1 and P2Y13 receptors. The pancreatic immune cells were not electrically excitable and the Ca 2+ signals generated by ATP were primarily due to release of Ca 2+ from internal stores followed by store-operated Ca 2+ entry through Ca 2+ release-activated Ca 2+ channels. The ATP-induced intracellular Ca 2+ liberation was dependent on both IP 3 generation and IP 3 receptors. We propose that the ATP-elicited Ca 2+ 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 AP., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

Background and Purpose: Many cancer cells depend on anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins for their survival. Bcl-2 antagonism through Bcl-2 homology 3 (BH3) mimetics has emerged as a novel anti-cancer therapy. ABT-199 (Venetoclax), a recently developed BH3 mimetic that selectively inhibits Bcl-2, was introduced into the clinic for treatment of relapsed chronic lymphocytic leukaemia. Early generations of Bcl-2 inhibitors evoked sustained Ca 2+ responses in pancreatic acinar cells (PACs) inducing cell death. Therefore, BH3 mimetics could potentially be toxic for the pancreas when used to treat cancer. Although ABT-199 was shown to kill Bcl-2-dependent cancer cells without affecting intracellular Ca 2+ signalling, its effects on PACs have not yet been determined. Hence, it is essential and timely to assess whether this recently approved anti-leukaemic drug might potentially have pancreatotoxic effects., Experimental Approach: Single-cell Ca 2+ measurements and cell death analysis were performed on isolated mouse PACs., Key Results: Inhibition of Bcl-2 via ABT-199 did not elicit intracellular Ca 2+ signalling on its own or potentiate Ca 2+ signalling induced by physiological/pathophysiological stimuli in PACs. Although ABT-199 did not affect cell death in PACs, under conditions that killed ABT-199-sensitive cancer cells, cytosolic Ca 2+ extrusion was slightly enhanced in the presence of ABT-199. In contrast, inhibition of Bcl-xL potentiated pathophysiological Ca 2+ responses in PACs, without exacerbating cell death., Conclusion and Implications: Our results demonstrate that apart from having a modest effect on cytosolic Ca 2+ extrusion, ABT-199 does not substantially alter intracellular Ca 2+ homeostasis in normal PACs and should be safe for the pancreas during cancer treatment., Linked Articles: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc., (© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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.

Key Points: Ca 2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Ca 2+ signals evoked by K + -induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca 2+ signals in acinar but not in stellate cells. Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca 2+ 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 Ca 2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation., Abstract: Physiological Ca 2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca 2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca 2+ signals in the peri-acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca 2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization-evoked Ca 2+ 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 Ca 2+ signals in response to membrane depolarization. The principal agent evoking Ca 2+ 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 Ca 2+ 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 Ca 2+ 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., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Biliary acute pancreatitis (AP) is a serious condition, which currently has no specific treatment. Taurolithocholic acid 3-sulfate (TLC-S) is one of the most potent bile acids causing cytosolic Ca 2+ overload in pancreatic acinar cells (PACs), which results in premature activation of digestive enzymes and necrosis, hallmarks of AP. The inositol 1,4,5-trisphosphate receptor (IP 3 R) and the ryanodine receptor (RyR) play major roles in intracellular Ca 2+ signaling. Inhibition of these endoplasmic reticulum-located channels suppresses TLC-S-induced Ca 2+ release and necrosis, decreasing the severity of AP. Anti-apoptotic B-cell lymphoma (Bcl)-2-family members, such as Bcl-2 and Bcl-X L , have emerged as important modulators of IP 3 Rs and RyRs. These proteins contain four Bcl-2 homology (BH) domains of which the N-terminal BH4 domain exerts critical roles in regulating intracellular Ca 2+ release channels. The BH4 domain of Bcl-2, but not of Bcl-X L , binds to and inhibits IP 3 Rs, whereas both BH4 domains inhibit RyRs. Although clear cytoprotective effects have been reported for these BH4 domains, it remains unclear whether they are capable of inhibiting pathological Ca 2+ -overload, associated with AP. Here we demonstrate in PACs that the BH4 domains of Bcl-2 and Bcl-X L inhibit RyR activity in response to the physiological agonist cholecystokinin. In addition, these BH4 domains inhibit pathophysiological TLC-S-induced Ca 2+ overload in PACs via RyR inhibition, which in turn protects these cells from TLC-S-induced necrosis. This study shows for the first time the therapeutic potential of BH4 domain function by inhibiting pathological RyR-mediated Ca 2+ release and necrosis, events that trigger AP., Competing Interests: The authors declare that they have no conflict of interest.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2' and HA14-1, induce Ca 2+ release from intracellular stores followed by a sustained elevation of the cytosolic Ca 2+ concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca 2+ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax -/- cells, Ca 2+ loss from the ER was reduced compared to WT cells. The Ca 2+ -like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca 2+ -binding proteins, significantly reduced excessive Ca 2+ signals and necrosis caused by two BH3 mimetics: BH3I-2' and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca 2+ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca 2+ leak in bax -/- cells as well as the fact that BH3 mimetics trigger substantial Ca 2+ signals by liberating Bax, indicate that Bax may regulate Ca 2+ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca 2+ -binding sites by CALPs can be used to ameliorate excessive Ca 2+ signals caused by BH3 mimetics and shift necrotic death towards apoptosis.

Store-operated Ca 2+ entry (SOCE), an important Ca 2+ signaling pathway in non-excitable cells, regulates a variety of cellular functions. To study its physiological role, pharmacological tools, like 2-aminoethyl diphenylborinate (2-APB), are used to impact SOCE. 2-APB is one of the best characterized SOCE inhibitors. However, 2-APB also activates SOCE at lower concentrations, while it inhibits inositol 1,4,5-trisphosphate receptors (IP 3 Rs), sarco/endoplasmic reticulum Ca 2+ -ATPases (SERCAs) and other ion channels, like TRP channels. Because of this, 2-APB analogues that inhibit SOCE more potently and more selectively compared to 2-APB have been developed. The recently developed DPB162-AE is such a structural diphenylborinate isomer of 2-APB that selectively inhibits SOCE currents by blocking the functional coupling between STIM1 and Orai1. However, we observed an adverse effect of DPB162-AE on the ER Ca 2+ -store content at concentrations required for complete SOCE inhibition. DPB162-AE increased the cytosolic Ca 2+ levels by reducing the ER Ca 2+ store in cell lines as well as in primary cells. DPB162-AE did not affect SERCA activity, but provoked a Ca 2+ leak from the ER, even after application of the SERCA inhibitor thapsigargin. IP 3 Rs partly contributed to the DPB162-AE-induced Ca 2+ leak, since pharmacologically and genetically inhibiting IP 3 R function reduced, but not completely blocked, the effects of DPB162-AE on the ER store content. Our results indicate that, in some conditions, the SOCE inhibitor DPB162-AE can reduce the ER Ca 2+ -store content by inducing a Ca 2+ -leak pathway at concentrations needed for adequate SOCE inhibition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Key Points: Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas. Bile acids are known to induce Ca 2+ 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 Ca 2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3-sulfate primarily affects acinar cells. Ca 2+ signals and necrosis are strongly dependent on extracellular Ca 2+ 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., Abstract: Acute 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca 2+ 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 Ca 2+ and significantly reduced in the absence of Na + , showing that bile-dependent cell death was a downstream event of Ca 2+ 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., (© 2016 The Authors The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca(2+) signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca(2+) elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca(2+) signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5-10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca(2+) release followed by Ca(2+) entry and also substantially reduced Ca(2+) extrusion because of decreased intracellular ATP levels. The toxic Ca(2+) signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca(2+) signals and necrosis. We tested the effects of inhibiting the Ca(2+) release-activated Ca(2+) entry by the Ca(2+) channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca(2+) entry and also protected effectively against the development of necrosis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'., (© 2016 The Authors.)

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 Ca(2+) signals. Only stimuli that evoke Ca(2+) 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 Ca(2+)-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 Ca(2+) and NO signalling pathways in cell-cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies., (© 2016 The Authors.)

Hepatic and pancreatic stellate cells may or may not be regarded as stem cells, but they are capable of remarkable transformations. There is less information about stellate cells in the pancreas than in the liver, where they were discovered much earlier and therefore have been studied longer and more intensively than in the pancreas. Most of the work on pancreatic stellate cells has been carried out in studies on cell cultures, but in this review we focus attention on Ca(2+) signalling in stellate cells in their real pancreatic environment. We review current knowledge on patho-physiologically relevant Ca(2+) signalling events and their underlying mechanisms. We focus on the effects of bradykinin in the initial stages of acute pancreatitis, an often fatal disease in which the pancreas digests itself and its surroundings. Ca(2+) signals, elicited in the stellate cells by the action of bradykinin, may have a negative effect on the outcome of the acute disease process and promote the development of chronic pancreatitis. The bradykinin-elicited Ca(2+) signals can be inhibited by blockade of type 2 receptors and also by blockade of Ca(2+)-release activated Ca(2+) channels. The potential benefits of such pharmacological inhibition for the treatment of pancreatitis are reviewed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 Ca(2+) signal generation in normal mouse pancreatic lobules. We found complete separation of Ca(2+) signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca(2+) signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca(2+) signals in PACs, never elicited Ca(2+) signals in PSCs. The bradykinin-elicited Ca(2+) signals were due to initial Ca(2+) release from inositol trisphosphate-sensitive stores followed by Ca(2+) entry through Ca(2+) release-activated channels (CRACs). The Ca(2+) 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., Abstract: Normal 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 Ca(2+) signals. We have compared Ca(2+) signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca(2+) 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 Ca(2+) signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca(2+) signals in PSCs. The BK-induced Ca(2+) signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca(2+) rise in PSCs was due to inositol trisphosphate receptor-mediated release from internal stores, whereas the sustained phase depended on external Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca(2+) signal generation in PSCs and this may be helpful in treating acute pancreatitis., (© 2015 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Intracellular Ca(2+) release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca(2+) release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca(2+) release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca(2+) release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca(2+) release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca(2+) release by 86% (64% vs. 86%, p<0.0002). In RyR3-KO, NAADP-evoked Ca(2+) release reduced by ∼50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca(2+) liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca(2+) release from the all stores is RyR1>TPC2>RyR3>TPC1>>RyR2. However, when assessing NAADP-induced Ca(2+) release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca(2+) liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acute pancreatitis is a human disease in which the pancreatic pro-enzymes, packaged into the zymogen granules of acinar cells, become activated and cause autodigestion. The main causes of pancreatitis are alcohol abuse and biliary disease. A considerable body of evidence indicates that the primary event initiating the disease process is the excessive release of Ca(2+) from intracellular stores, followed by excessive entry of Ca(2+) from the interstitial fluid. However, Ca(2+) release and subsequent entry are also precisely the processes that control the physiological secretion of digestive enzymes in response to stimulation via the vagal nerve or the hormone cholecystokinin. The spatial and temporal Ca(2+) signal patterns in physiology and pathology, as well as the contributions from different organelles in the different situations, are therefore critical issues. There has recently been significant progress in our understanding of both physiological stimulus-secretion coupling and the pathophysiology of acute pancreatitis. Very recently, a promising potential therapeutic development has occurred with the demonstration that the blockade of Ca(2+) release-activated Ca(2+) currents in pancreatic acinar cells offers remarkable protection against Ca(2+) overload, intracellular protease activation and necrosis evoked by a combination of alcohol and fatty acids, which is a major trigger of acute pancreatitis.

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 concentration-dependent manner within the range of 1 to 50 μM (IC50 = 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.

The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

As a primary energy producer, mitochondria play a fundamental role in pancreatic exocrine physiology and pathology. The most frequent aetiology of acute pancreatitis is either gallstones or heavy alcohol consumption. Repeated episodes of acute pancreatitis can result in the development of chronic pancreatitis and increase the lifetime risk of pancreatic cancer 100-fold. Pancreatic cancer is one of the most common causes of cancer mortality with only about 3-4 % of patients surviving beyond 5 years. It has been shown that acute pancreatitis involves Ca²⁺ overload and overproduction of reactive oxygen species in pancreatic acinar cells. Both factors significantly affect mitochondria and lead to cell death. The pathogenesis of inflammation in acute and chronic pancreatitis is tightly linked to the induction of necrosis and apoptosis. There is currently no specific therapy for pancreatitis, but recent findings of an endogenous protective mechanism against Ca²⁺ overload--and particularly the potential to boost this protection--bring hope of new therapeutic approaches.

Background & Aims: Oxidative stress is implicated in the pathogenesis of pancreatitis, but clinical trials of antioxidants have produced conflicting results. We examined the role of intracellular reactive oxygen species (ROS) in pancreatic acinar cell injury., Methods: Freshly isolated murine and human pancreatic acinar cells were studied using confocal microscopy to measure changes in intracellular and mitochondrial ROS concentrations ([ROS]I and [ROS]M), cytosolic and mitochondrial calcium concentrations ([Ca2+]C and [Ca2+]M), reduced nicotinamide adenine dinucleotide phosphate levels, and death pathways in response to taurolithocholate acid sulfate (TLC-S) or the oxidant menadione. Ca2+-activated Cl- currents were measured using whole-cell patch clamp, with or without adenosine triphosphate (ATP)., Results: TLC-S induced prolonged increases in [Ca2+]C and [Ca2+]M, which led to dose-dependent increases in [ROS]I and [ROS]M, impaired production of ATP, apoptosis, and necrosis. Inhibition of the antioxidant reduced nicotinamide adenine dinucleotide phosphate quinine oxidoreductase by 2,4-dimethoxy-2-methylnaphthalene potentiated the increases in [ROS]I and apoptosis but reduced necrosis, whereas the antioxidant N-acetyl-L-cysteine reduced [ROS]I and apoptosis but increased necrosis. Inhibition of mitochondrial ROS production prevented apoptosis but did not alter necrosis; autophagy had no detectable role. Patched ATP prevented sustained increases in [Ca2+]C and necrosis., Conclusions: Increases in [ROS]M and [ROS]I during bile acid injury of pancreatic acinar cells promote apoptosis but not necrosis. These results indicate that alternative strategies to antioxidants are required for oxidative stress in acute pancreatitis., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

The exocrine pancreas synthesizes all the enzymes needed for intestinal breakdown of proteins, fats, and carbohydrates in our diet. Unfortunately, the proteases needed for the digestion of the meat we eat can, if inappropriately activated inside the acinar cells, also digest the pancreas itself as well as the surrounding tissues, which is what happens in the sometimes fatal human disease acute pancreatitis. The disease is currently untreatable, but significant progress has recently been made in understanding the fundamental processes initiating the pathological changes underlying pancreatic autodigestion. It is now clear that intracellular trypsin activation-a crucial step in pathogenesis-is due to excessive release of Ca(2+) from intracellular stores, principally via two types of inositol trisphosphate receptor. The unexpected recent discovery of an intrinsic protective mechanism caused by intracellular calmodulin and, specifically, the finding that this protective effect can be boosted by a membrane-permeable Ca(2+)-like peptide are promising.

Background & Aims: Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria., Methods: ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases., Results: Inhibition of oxidative phosphorylation produced a substantial decrease in cytosolic ATP comparable to that induced by inhibition of glycolysis. Cholecystokinin-8 (CCK) increased cytosolic ATP in spite of accelerating ATP consumption. Acetylcholine, caerulein, and bombesin had similar effect. A bile acid, taurolithocholic acid 3-sulfate (TLC-S); a fatty acid, palmitoleic acid (POA); and palmitoleic acid ethyl ester (POAEE) reduced cytosolic ATP. The ATP decrease in response to these substances was observed in cells with intact or inhibited oxidative phosphorylation. TLC-S, POA, and POAEE reduced mitochondrial ATP, whereas physiological CCK increased mitochondrial ATP. Supramaximal CCK produced a biphasic response composed of a small initial decline followed by a stronger increase., Conclusions: Both glycolysis and oxidative phosphorylation make substantial contributions to ATP production in acinar cells. Ca(2+)-releasing secretagogues increased ATP level in the cytosol and mitochondria of intact isolated cells. TLC-S, POA, and POAEE reduced cytosolic and mitochondrial ATP. When cells rely on nonoxidative ATP production, secretagogues as well as TLC-S, POA, and POAEE all diminish cytosolic ATP levels., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Store-operated Ca(2+) entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels. In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP(3)) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca(2+) loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex, and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca(2+) release and Ca(2+) reloading) and that any potential interference between Ca(2+) release and Ca(2+) influx is minimized by the spatial separation of the two processes.

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 mum 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.

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca(2+). This excessive Ca(2+) liberation induces Ca(2+)-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca(2+) release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca(2+) concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca(2+) release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca(2+) release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP(3)Rs) of types 2 and 3. POAEE evoked very little Ca(2+) release and trypsin activation when IP(3)Rs of both types 2 and 3 were knocked out. Antibodies against IP(3)Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca(2+) release and trypsin activation. We conclude that Ca(2+) release through IP(3)Rs of types 2 and 3 in the acid granular Ca(2+) store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta-structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1-ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca(2+) influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1-ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P(2) depletion.

Background & Aims: Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as on rodent pancreatic acinar cells. We tested whether CCK (CCK-8 and human CCK-58) can act directly on human pancreatic acinar cells., Methods: Human acinar cells were freshly isolated from pancreatic transection line samples, loaded with Fluo4-AM or quinacrine, and examined for Ca(2+), metabolic and secretory responses to CCK-8, human CCK-58, or acetylcholine with confocal microscopy., Results: CCK-8 and human CCK-58 at physiologic concentrations (1-20 pmol/L) elicited rapid, robust, oscillatory increases of the cytosolic Ca(2+) ion concentration, showing apical to basal progression, in acinar cells from 14 patients with unobstructed pancreata. The cytosolic Ca(2+) ion concentration increases were followed by increases in mitochondrial adenosine triphosphate production and secretion. CCK-elicited Ca(2+) signals and exocytosis were not inhibited by atropine (1 mumol/L) or tetrodotoxin (100 nmol/L), showing that CCK was unlikely to have acted via neurotransmitter release. CCK-elicited Ca(2+) signals were inhibited reversibly by caffeine (5-20 mmol/L), indicating involvement of intracellular inositol trisphosphate receptor Ca(2+) release channels. Acetylcholine (50 nmol/L) elicited similar Ca(2+) signals., Conclusions: CCK at physiologic concentrations in the presence of atropine and tetrodotoxin elicits cytosolic Ca(2+) signaling, activates mitochondrial function, and stimulates enzyme secretion in isolated human pancreatic acinar cells. We conclude that CCK acts directly on acinar cells in the human pancreas.

Here, we describe novel mechanisms limiting a toxic cytosolic Ca(2+) rise during adenosine 5'-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca(2+) signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca(2+) extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca(2+) influx. Alternative inhibition of Ca(2+) extrusion by carboxyeosin had a much smaller effect on Ca(2+) influx. The coordinated metabolic inhibition of Ca(2+) influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca(2+) oscillations, which was due to the inhibition of Ca(2+) release from internal stores. This could be particularly important for limiting Ca(2+) toxicity during periods of hypoxia. In contrast, metabolic control of Ca(2+) influx and Ca(2+) release from internal stores spectacularly failed to prevent large toxic Ca(2+) responses induced by bile acids-activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca(2+) release from internal stores nor Ca(2+) influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms.

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

Cell-death programs executed in the pancreas under pathological conditions remain largely undetermined, although the severity of experimental pancreatitis has been found to depend on the ratio of apoptosis to necrosis. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10, or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half-maximum activation (t(0.5)) of caspase-9 occurred within approximately 2 min and was identified at or in close proximity to mitochondria, whereas t(0.5) for caspase-8 occurred within approximately 26 min of menadione application and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (preincubation with Gly-Phe beta-naphthylamide, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsin L or D. Using pepstatin A-BODIPY FL conjugate, we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway that is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled.

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 cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine 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 microM. 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 approximately 100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.

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.

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.

Inositol trisphosphate and cyclic ADP-ribose release Ca2+ from the endoplasmic reticulum via inositol trisphosphate and ryanodine receptors, respectively. By contrast, nicotinic acid adenine dinucleotide phosphate may activate a novel Ca2+ channel in an acid compartment. We show, in two-photon permeabilized pancreatic acinar cells, that the three messengers tested could each release Ca2+ from the endoplasmic reticulum and also from an acid store in the granular region. The nicotinic acid adenine dinucleotide phosphate action on both types of store, like that of cyclic ADP-ribose but unlike inositol trisphosphate, depended on operational ryanodine receptors, since it was blocked by ryanodine or ruthenium red. The acid Ca2+ store in the granular region did not have Golgi or lysosomal characteristics and might therefore be associated with the secretory granules. The endoplasmic reticulum is predominantly basal, but thin extensions penetrate into the granular area and cytosolic Ca2+ signals probably initiate at sites where endoplasmic reticulum elements and granules come close together.

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.

In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.

Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.