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Inositol triphosphate (IP3) and cyclic ADP-ribose (cADPr) evoke a marked decrease in the free calcium concentration inside zymogen granules (ZG) of pancreatic acinar cells. Using confocal microscopy, it has been shown that IP3 and cADPr induce the rapid release Ca2+ from the granules. A novel high resolution method further showed that IP3-mediated Ca2+ release causes a spike in cytosolic Ca2+ generation outside the ZG.

Microscopic studies using fluorescent probes of the release and uptake of Ca2+ from cell nuclei in liver show that the Ca2+-sensitive fluorescent probe Fura 2 localizes mainly to the nuclear envelope, indicating that the inward and outward transport of Ca2+ from the nuclear envelope is responsible for the inositol triphosphate-dependent secretion of Ca2+. Fluorescent Ca2+ indicator-tagged dextrans display a uniform distribution in the nucleoplasm, while variations in external Ca2+ alter the nucleoplasmic Ca2+ level. Ca2+ released from the nuclear envelope enters nucleoplasm and diffuses out through nuclear pore complexes.

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. [ABSTRACT FROM AUTHOR]

• 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 IP 3 Rs 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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 &y& Elsevier]

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 Ca2+, 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 Ca2+ ion concentration, showing apical to basal progression, in acinar cells from 14 patients with unobstructed pancreata. The cytosolic Ca2+ ion concentration increases were followed by increases in mitochondrial adenosine triphosphate production and secretion. CCK-elicited Ca2+ signals and exocytosis were not inhibited by atropine (1 μmol/L) or tetrodotoxin (100 nmol/L), showing that CCK was unlikely to have acted via neurotransmitter release. CCK-elicited Ca2+ signals were inhibited reversibly by caffeine (5-20 mmol/L), indicating involvement of intracellular inositol trisphosphate receptor Ca2+ release channels. Acetylcholine (50 nmol/L) elicited similar Ca2+ signals. Conclusions: CCK at physiologic concentrations in the presence of atropine and tetrodotoxin elicits cytosolic Ca2+ 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. [Copyright &y& Elsevier]

Background & Aims: Previous studies of pancreatic acinar cells characterized the effects of Ca2+-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca2+, 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. Ca2+-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 &y& Elsevier]

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 Ca2+ 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 Ca2+ release sites in the apical region of the cell and the important Ca2+ sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca2+ signals in the apical region, large Ca2+ 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 Ca2+ transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria. [ABSTRACT FROM AUTHOR]

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 Ca2+-dependent and Ca2+-independent components of the Na+ concentration increase. The Ca2+-independent component was resolved in conditions when the cytosolic Ca2+ level was buffered with a high concentration of the calcium chelator 1,2-bis(oaminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca2+-dependent component of intracellular Na+ increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca2+ oscillations the recovery of cytosolic Na+ was much slower than the recovery of Ca2+, creating a possibility for the summation of Na+ transients. The bile-induced Ca2+-independent current was found to be carried primarily by Na+ and K+, with only small Ca2+ and Cl- contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 µM). 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. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of secreta- gogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mi- tochondrial membrane potential using the tetramethyl- rhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensi- tivity to the uncoupler carbonyl cyanide m-chlorophenyl- hydrazone, 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. [ABSTRACT FROM AUTHOR]

Investigates the characteristics of cytosolic Ca[sup 2+] signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. Examination of the mechanism of formation of localized Ca[sup 2+] signals.

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. [ABSTRACT FROM AUTHOR]

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 µg/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 (Galβ1,3GalNAc-α (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1- transfected, Ras-transformed, canine kidney epithelial-hike (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 IF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with anti-bodies against E-Selectin or CD44H, but not integrin-β1, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 Ca2+. Using two-photon plasma membrane permeabilization and measurement of [Ca2+] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca2+ release pathways. Inhibition of inositol trisphosphate receptors (lP3Rs), by caffeine and 2-APB, reduced Ca2+ 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 IP3Rs and RyRs abolished Ca2+ 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 Ca2+ liberation from both the ER and the apical acidic stores depends on both RyRs and IP3Rs. [ABSTRACT FROM AUTHOR]