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.
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 Ca2+ from intracellular stores, followed by excessive entry of Ca2+ from the interstitial fluid. However, Ca2+ 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 Ca2+ 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 Ca2+ release‐activated Ca2+ currents in pancreatic acinar cells offers remarkable protection against Ca2+ 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 (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.
Abstract
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.
•Ca2+ overload and ATP depletion are the main pathological processes in AP.•The CRAC channel is the most attractive therapeutic target to reduce excessive Ca2+ entry.•Modulation of IP3Rs and RyRs can ...efficiently inhibit pathological Ca2+ release.•Energy supplementation can effectively reduce ATP depletion in AP.
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 (Ca2+ plateau) and significantly reduced intracellular energy (ATP depletion). These hallmarks are interdependent, i.e., Ca2+ plateau increase energy demand for its clearance while energy production is greatly affected by the pathology. Result of long standing Ca2+ 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 Ca2+ overload or reduction of ATP depletion. This review will summarise these approaches, including recent developments of potential therapies for AP.
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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.
The antiapoptotic protein Bcl-2 1, 2 plays important roles in Ca2+ signaling 3 by influencing inositol triphosphate receptors and regulating Ca2+-induced Ca2+ release 4–6. Here we investigated ...whether Bcl-2 affects Ca2+ extrusion in pancreatic acinar cells. We specifically blocked the Ca2+ pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca2+ concentration after a period of enhanced Ca2+ entry. Because external Ca2+ was removed and endoplasmic reticulum Ca2+ pumps were blocked, Ca2+ extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca2+ level much faster than control cells. The enhanced Ca2+ extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na+/Ca2+ exchange activity, because removal of external Na+ did not influence the Ca2+ extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca2+ extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca2+ extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca2+ pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca2+ extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.
► Bcl-2 reduces Ca2+ extrusion through PMCA in control cells as compared to Bcl-2 KO ► Bcl-2 reduces Ca2+ extrusion though PMCA in AR42J cells overexpressing Bcl-2 ► Loss of Bcl-2 reduces Ca2+ overload, decreases necrosis, and promotes apoptosis
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.
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 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.
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.
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