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1
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Benchikh I, Ziani K, Benalia A, Djebbar AA, Argoub H, Khaled MB. Thirty-day oral exposure to acetamiprid induces biochemical and histological alterations in rat pancreas: protective effects of carnosine supplementation. Toxicol Mech Methods 2025; 35:329-339. [PMID: 39627014 DOI: 10.1080/15376516.2024.2435350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2024] [Revised: 11/21/2024] [Accepted: 11/24/2024] [Indexed: 01/03/2025]
Abstract
Acetamiprid is a neonicotinoid insecticide used against various insect pests. Serious concerns are emerging regarding their adverse effects on non-target organisms and organs. This study aimed to investigate the mechanistic toxic effect of oral administration of acetamiprid at 21.7 and 43.4 mg/kg body weight on the histological structure and pancreatic function of male Wistar rats and the potential effect of carnosine in mitigating this toxicity for 30 consecutive days. Thirty-six animals were divided into six groups: the control group received distilled water, the second group received 200 mg/kg body weight of carnosine, two groups received 21.7 and 43.4 mg/kg of acetamiprid, and two groups received 21.7 and 43.4 mg/kg + 200 kg/kg body weight of acetamiprid and carnosine, respectively. Acetamiprid caused a significant decrease in body weight (p < 0.001), pancreatic somatic index (p < 0.001), and amylase level (p ≤ 0.0001) and increased lipase level (p ≤ 0.0001), blood glucose level (p ≤ 0.0001), histological scores (p ≤ 0.01), and malondialdehyde level (0.01
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Affiliation(s)
- Imen Benchikh
- Laboratory of Applied Hydrology and Environment, Faculty of Science and Technology, University of Ain Temouchent, Ain Témouchent, Algeria
- Laboratoire de Nutrition, Pathologie, Agro-Biotechnologie et Santé (NuPABS), Department of biology, Faculty of Natural and Life sciences, Djillali Liabès University, Sidi Bel Abbès, Algeria
| | - Kaddour Ziani
- Laboratory of Biotoxicology, Pharmacognosy and Biological Valorization of Plants, Department of Biology, University of Saida, Dr. Taher Moulay, Saida, Algeria
| | - Abdelkrim Benalia
- Laboratory of Environment and Health Research (LRES), Faculty of Medicine, Djillali Liabès University, Sidi Bel Abbès, Algeria
| | - Ahmed Abdelhammid Djebbar
- Laboratory of Environment and Health Research (LRES), Department of Biology, Faculty of Natural Sciences and Life, Djillali Liabès University, Sidi Bel Abbès, Algeria
| | - Hayat Argoub
- Service of Anatomy & Pathology, University Hospital Center Hassani Adbelkader, Sidi Bel Abbès, Algeria
| | - Méghit Boumediène Khaled
- Laboratoire de Nutrition, Pathologie, Agro-Biotechnologie et Santé (NuPABS), Department of biology, Faculty of Natural and Life sciences, Djillali Liabès University, Sidi Bel Abbès, Algeria
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2
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Petersen OH. Watching Living Cells in Action in the Exocrine Pancreas: The Palade Prize Lecture. FUNCTION 2022; 4:zqac061. [PMID: 36606242 PMCID: PMC9809903 DOI: 10.1093/function/zqac061] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Revised: 12/04/2022] [Accepted: 12/05/2022] [Indexed: 12/13/2022] Open
Abstract
George Palade's pioneering electron microscopical studies of the pancreatic acinar cell revealed the intracellular secretory pathway from the rough endoplasmic reticulum at the base of the cell to the zymogen granules in the apical region. Palade also described for the first time the final stage of exocytotic enzyme secretion into the acinar lumen. The contemporary studies of the mechanism by which secretion is acutely controlled, and how the pancreas is destroyed in the disease acute pancreatitis, rely on monitoring molecular events in the various identified pancreatic cell types in the living pancreas. These studies have been carried out with the help of high-resolution fluorescence recordings, often in conjunction with patch clamp current measurements. In such studies we have gained much detailed information about the regulatory events in the exocrine pancreas in health as well as disease, and new therapeutic opportunities have been revealed.
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Affiliation(s)
- Ole H Petersen
- School of Biosciences, Sir Martin Evans Building, Cardiff University, Wales, CF10 3AX, UK
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3
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Delgado AAA, Sethio D, Munar I, Aviyente V, Kraka E. Local vibrational mode analysis of ion-solvent and solvent-solvent interactions for hydrated Ca 2+ clusters. J Chem Phys 2020; 153:224303. [PMID: 33317306 DOI: 10.1063/5.0034765] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hydrated calcium ion clusters have received considerable attention due to their essential role in biological processes such as bone development, hormone regulation, blood coagulation, and neuronal signaling. To better understand the biological role of the cation, the interactions between the Ca2+ ions and water molecules have been frequently investigated. However, a quantitative measure for the intrinsic Ca-O (ion-solvent) and intermolecular hydrogen bond (solvent-solvent) interactions has been missing so far. Here, we report a topological electron density analysis and a natural population analysis to analyze the nature of these interactions for a set of 14 hydrated calcium clusters via local mode stretching force constants obtained at the ωB97X-D/6-311++G(d,p) level of theory. The results revealed that the strength of inner Ca-O interactions for Ca(H2O)n 2+ (n = 1-8) clusters correlates with the electron density. The application of a second hydration shell to Ca(H2O)n 2+ (n = 6-8) clusters resulted in stronger Ca-O interactions where a larger electron charge transfer between lp(O) of the first hydration shell and the lower valence of Ca prevailed. The strength of the intermolecular hydrogen bonds, formed between the first and second hydration shells, became stronger when the charge transfers between hydrogen bond (HB) donors and HB acceptors were enhanced. From the local mode stretching force constants of implicitly and explicitly solvated Ca2+, we found the six-coordinated cluster to possess the strongest stabilizations, and these results prove that the intrinsic bond strength measures for Ca-O and hydrogen bond interactions form new effective tools to predict the coordination number for the hydrated calcium ion clusters.
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Affiliation(s)
- Alexis A A Delgado
- Department of Chemistry, Computational and Theoretical Chemistry Group (CATCO), Southern Methodist University, 3215 Daniel Avenue, Dallas, Texas 75275-0314, USA
| | - Daniel Sethio
- Department of Chemistry - BMC, Uppsala University, Husargatan 3, 75237 Uppsala, Sweden
| | - Ipek Munar
- Department of Chemistry, Boǧaziçi University, Bebek 34342, Istanbul, Turkey
| | - Viktorya Aviyente
- Department of Chemistry, Boǧaziçi University, Bebek 34342, Istanbul, Turkey
| | - Elfi Kraka
- Department of Chemistry, Computational and Theoretical Chemistry Group (CATCO), Southern Methodist University, 3215 Daniel Avenue, Dallas, Texas 75275-0314, USA
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4
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He Z, Feng G, Yang B, Yang L, Liu CW, Xu HG, Xu XL, Zheng WJ, Gao YQ. Molecular dynamics simulation, ab initio calculation, and size-selected anion photoelectron spectroscopy study of initial hydration processes of calcium chloride. J Chem Phys 2018; 148:222839. [DOI: 10.1063/1.5024279] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Affiliation(s)
- Zhili He
- Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
| | - Gang Feng
- Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
- School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, China
| | - Bin Yang
- Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lijiang Yang
- Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
| | - Cheng-Wen Liu
- Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
- Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA
| | - Hong-Guang Xu
- Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xi-Ling Xu
- Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Wei-Jun Zheng
- Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yi Qin Gao
- Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
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5
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Kohagen M, Mason PE, Jungwirth P. Accurate Description of Calcium Solvation in Concentrated Aqueous Solutions. J Phys Chem B 2014; 118:7902-9. [DOI: 10.1021/jp5005693] [Citation(s) in RCA: 104] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Miriam Kohagen
- Institute of Organic Chemistry
and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo
nam. 2, 16610 Prague
6, Czech Republic
| | - Philip E. Mason
- Institute of Organic Chemistry
and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo
nam. 2, 16610 Prague
6, Czech Republic
| | - Pavel Jungwirth
- Institute of Organic Chemistry
and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo
nam. 2, 16610 Prague
6, Czech Republic
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6
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Orabi AI, Muili KA, Javed TA, Jin S, Jayaraman T, Lund FE, Husain SZ. Cluster of differentiation 38 (CD38) mediates bile acid-induced acinar cell injury and pancreatitis through cyclic ADP-ribose and intracellular calcium release. J Biol Chem 2013; 288:27128-27137. [PMID: 23940051 DOI: 10.1074/jbc.m113.494534] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Aberrant Ca(2+) signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca(2+) signals due to bile acid exposure is the intracellular Ca(2+) channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca(2+) signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38(-/-)). Cytosolic Ca(2+) signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μM). To focus on intracellular Ca(2+) release and to specifically exclude Ca(2+) influx, cells were perifused in Ca(2+)-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mM) or the cADPR antagonist 8-Br-cADPR (30 μM) abrogated TLCS-induced Ca(2+) signals and cell injury. TLCS-induced Ca(2+) release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca(2+) signaling.
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Affiliation(s)
| | | | | | | | - Thottala Jayaraman
- Departments of Internal Medicine, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15224
| | - Frances E Lund
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35213
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7
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Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy. Proc Natl Acad Sci U S A 2013; 110:13186-91. [PMID: 23878235 DOI: 10.1073/pnas.1300910110] [Citation(s) in RCA: 152] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
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.
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8
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Mitochondrial function and malfunction in the pathophysiology of pancreatitis. Pflugers Arch 2012; 464:89-99. [PMID: 22653502 DOI: 10.1007/s00424-012-1117-8] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2012] [Revised: 05/11/2012] [Accepted: 05/15/2012] [Indexed: 12/13/2022]
Abstract
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.
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9
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Muili KA, Ahmad M, Orabi AI, Mahmood SM, Shah AU, Molkentin JD, Husain SZ. Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury. Am J Physiol Gastrointest Liver Physiol 2012; 302:G898-905. [PMID: 22323127 PMCID: PMC3355562 DOI: 10.1152/ajpgi.00545.2011] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2011] [Accepted: 01/31/2012] [Indexed: 02/07/2023]
Abstract
Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.
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Affiliation(s)
- Kamaldeen A Muili
- Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania, USA
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10
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Gerasimenko OV, Petersen OH, Gerasimenko JV. Role of intracellular acid Ca(2+) stores in pathological pancreatic protease activation. Expert Rev Gastroenterol Hepatol 2012; 6:129-31. [PMID: 22375516 DOI: 10.1586/egh.12.5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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11
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Kar P, Nelson C, Parekh AB. Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels. J Biol Chem 2011; 286:14795-803. [PMID: 21325277 DOI: 10.1074/jbc.m111.220582] [Citation(s) in RCA: 105] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
NFATs are a family of Ca(2+)-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of numerous distinct cell types and organ systems. Here, we visualize NFAT1 movement in and out of the nucleus in response to transient activation of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in nonexcitable cells. We show that NFAT migration is exquisitely sensitive to Ca(2+) microdomains near open CRAC channels. Another Ca(2+)-permeable ion channel (TRPC3) was ineffective in driving NFAT1 to the nucleus. NFAT1 movement is temporally dissociated from the time course of the Ca(2+) signal and remains within the nucleus for 10 times longer than the duration of the trigger Ca(2+) signal. Kinetic analyses of each step linking CRAC channel activation to NFAT1 nuclear residency reveals that the rate-limiting step is transcription factor exit from the nucleus. The slow deactivation of NFAT provides a mechanism whereby Ca(2+)-dependent responses can be sustained despite the termination of the initial Ca(2+) signal and helps explain how gene expression in nonexcitable cells can continue after the primary stimulus has been removed.
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Affiliation(s)
- Pulak Kar
- Department of Physiology, Oxford University, Sherrington Building, Parks Road, Oxford OX1 3PT, United Kingdom
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12
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Parekh AB. Decoding cytosolic Ca2+ oscillations. Trends Biochem Sci 2010; 36:78-87. [PMID: 20810284 DOI: 10.1016/j.tibs.2010.07.013] [Citation(s) in RCA: 176] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2010] [Revised: 07/28/2010] [Accepted: 07/29/2010] [Indexed: 12/19/2022]
Abstract
A rise in cytosolic Ca(2+) concentration is used as a universal signalling mechanism to control biological processes as diverse as exocytosis, contraction, cell growth and cell death. Ca(2+) signals are often presented to cells in the form of Ca(2+) oscillations, with signalling information encoded in both amplitude and frequency of the Ca(2+) spikes. Recent studies have revealed that the sub-cellular spatial profile of the Ca(2+) oscillation is also important in activating cellular responses, thereby suggesting a new mechanism for extracting information from the ubiquitous Ca(2+) oscillation.
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Affiliation(s)
- Anant B Parekh
- Department of Physiology, Anatomy and Genetics, Sherrington Building, Parks Road, Oxford OX1 3PT, UK.
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13
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Baumgartner HK, Gerasimenko JV, Thorne C, Ferdek P, Pozzan T, Tepikin AV, Petersen OH, Sutton R, Watson AJM, Gerasimenko OV. Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening. J Biol Chem 2009; 284:20796-803. [PMID: 19515844 PMCID: PMC2742844 DOI: 10.1074/jbc.m109.025353] [Citation(s) in RCA: 211] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2009] [Indexed: 01/16/2023] Open
Abstract
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.
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Affiliation(s)
- Heidi K. Baumgartner
- From the Physiological Laboratory, School of Biomedical Sciences
- the Division of Gastroenterology, School of Clinical Sciences, and
| | | | | | - Pawel Ferdek
- From the Physiological Laboratory, School of Biomedical Sciences
| | - Tullio Pozzan
- the Department of Biomedical Sciences and CNR Institute of Neurosciences, University of Padua, Viale G Colombo 3, 35121 Padua, Italy
| | | | - Ole H. Petersen
- From the Physiological Laboratory, School of Biomedical Sciences
| | - Robert Sutton
- the Division of Surgery and Oncology, School of Cancer Studies, Liverpool University, Liverpool L69 3BX, United Kingdom and
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14
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Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors. Proc Natl Acad Sci U S A 2009; 106:10758-63. [PMID: 19528657 DOI: 10.1073/pnas.0904818106] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
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.
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15
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Criddle DN, McLaughlin E, Murphy JA, Petersen OH, Sutton R. The pancreas misled: signals to pancreatitis. Pancreatology 2007; 7:436-46. [PMID: 17898533 DOI: 10.1159/000108960] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/25/2007] [Indexed: 12/11/2022]
Abstract
Acute pancreatitis is an increasingly common and sometimes severe disease for which there is little specific therapy. Chronic pancreatitis is a common and grossly debilitating sequel that is largely irreversible, whatever treatment is adopted. In the face of these burdens, the absence of specific treatments is a spur to research. The acinar cell is the primary target of injury from alcohol metabolites, bile, hyperlipidaemia, hyperstimulation and other causes. These induce abnormal, prolonged, global, cytosolic calcium signals, the prevention of which also prevents premature digestive enzyme activation, cytokine expression, vacuole formation and acinar cell necrosis. Such agents increase calcium entry through the plasma membrane and/or increase calcium release from intracellular stores, shown to result from effects on calcium channels and calcium pumps, or their energy supply. A multitude of signalling mechanisms are activated, diverted or disrupted, including secretory mechanisms, lysosomal regulators, inflammatory mediators, cell survival and cell death pathways, together with or separately from calcium. While recent discoveries have increased insight and suggest prophylaxis or treatment targets, more work is required to define the mechanisms and interactions of cell signalling pathways in the pathogenesis of pancreatitis.
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Affiliation(s)
- David N Criddle
- MRC Group, Physiological Laboratory, University of Liverpool, Liverpool, UK
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16
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Jin SW, Zhang L, Lian QQ, Yao SL, Wu P, Zhou XY, Xiong W, Ye DY. Close functional coupling between Ca2+ release-activated Ca2+ channels and reactive oxygen species production in murine macrophages. Mediators Inflamm 2007; 2006:36192. [PMID: 17392583 PMCID: PMC1775034 DOI: 10.1155/mi/2006/36192] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Aim. To investigate the role of Ca2+ release-activated Ca2+ (CRAC) channels in the ROS production in macrophages. Methods. The intracellular [Ca2+]i was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. Results. Both LPS and thapsigargin induced an increase in intracellular [Ca2+]i, either in the presence or absence of extracellular Ca2+ in murine macrophages. The Ca2+ signal was sustained in the presence of external Ca2+ and only initiated a mild and transient rise in the absence of external Ca2+. CRAC channel inhibitor 2-APB completely suppressed the Ca2+ entry signal evoked by thapsigargin, and suppressed approximately 93% of the Ca2+ entry signal evoked by LPS. The increase in intracellular [Ca2+]i was associated with increased ROS production, which was completely abolished in the absence of extracellular Ca2+ or in the presence of CRAC channel inhibitors 2-APB and Gd3+. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses. Conclusions. These findings indicate that the LPS-induced intracellular [Ca2+]i increase depends on the Ca2+ entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages.
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Affiliation(s)
- Sheng-Wei Jin
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430022, China
- Department of Anesthesiology, Second Affiliated Hospital, Wenzhou Medical College, Wenzhou 325027, China
- *Sheng-Wei Jin:
| | - Li Zhang
- Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Qin-Quan Lian
- Department of Anesthesiology, Second Affiliated Hospital, Wenzhou Medical College, Wenzhou 325027, China
| | - Shang-Long Yao
- Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430022, China
| | - Ping Wu
- Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Xiao-Yan Zhou
- Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Wei Xiong
- Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Du-Yun Ye
- Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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17
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Yang K, Ding YX, Chin WC. K+-induced ion-exchanges trigger trypsin activation in pancreas acinar zymogen granules. Arch Biochem Biophys 2007; 459:256-63. [PMID: 17270141 DOI: 10.1016/j.abb.2006.12.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2006] [Revised: 12/01/2006] [Accepted: 12/03/2006] [Indexed: 11/30/2022]
Abstract
Trypsin premature activation has been thought to be a key event in the initiation phase of acute pancreatitis. Here we test a hypothesis that a sustained increase of cytosolic Ca(2+) concentration ([Ca(2+)](C)) can trigger K(+) influx into pancreas acinar zymogen granules (ZGs) via a Ca(2+)-activated K(+) channel (K(Ca)), and this influx of K(+) then mobilizes bound-Ca(2+) by K(+)/Ca(2+) ion-exchange to increase free Ca(2+) concentration in the ZGs ([Ca(2+)](G)) and release bound-H(+) by K(+)/H(+) ion-exchange to decrease the pH in ZGs (pH(G)). Both the increase of [Ca(2+)](G) and the decrease of pH(G) will facilitate trypsinogen autoactivation and stabilize active trypsin inside ZGs that could lead to acute pancreatitis. The experimental results are consistent with our hypothesis, suggesting that K(+) induced ion-exchanges play a critical role in the initiation of trypsin premature activation in ZGs.
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Affiliation(s)
- Kai Yang
- Department of Biological Sciences, Florida State University, Tallahassee, FL 32310, USA
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18
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Ding YX, Yang K, Chin WC. Ethanol augments elevated-[Ca2+]C induced trypsin activation in pancreatic acinar zymogen granules. Biochem Biophys Res Commun 2006; 350:593-7. [PMID: 17026963 DOI: 10.1016/j.bbrc.2006.09.086] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2006] [Accepted: 09/19/2006] [Indexed: 11/17/2022]
Abstract
It has been long recognized that significant percentage of patients with acute pancreatitis often presents with a history of excessive alcohol consumption; however, the patho-physiological effect of ethanol on acute pancreatitis remains poorly understood. Abnormally elevated cytosolic Ca2+ ([Ca2+]C) has been found to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation. Here, we present the effects of ethanol to sensitize zymogen granules (ZGs) of pancreas acinar cells to elevated [Ca2+]C leading to zymogen premature activation that could result in acute pancreatitis. The pH fluctuations ([pH]G), Ca2+ concentration ([Ca2+]G), and premature trypsin activation inside the ZGs were monitored directly with specific fluorescence indicators. Our results showed that ethanol could act directly on ZGs and cause ZGs more receptive to elevated [Ca2+]C that could induce premature activation of zymogen (trypsin). This alcohol-induced effect is concentration dependent and strongly influenced by the surrounding [Ca2+]C. The K+ channels on ZGs membranes are required in the sensitization process. Our observations provide a mechanistic understanding of the role of ethanol in the initiation phase of alcoholic pancreatitis.
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Affiliation(s)
- Yong-Xue Ding
- College of Engineering, Florida State University, Tallahassee, FL 32310, USA
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19
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Abstract
Whereas alcohol itself, even in high concentrations, has little effect on the functional performance of isolated pancreatic acinar cells, non-oxidative metabolites (fatty acid ethyl esters [FAEE] and fatty acids [FA]) can cause Ca(2+)-dependent necrosis. The mechanism of action of FAEE has been investigated using a combination of patch clamp whole-cell current recording and Ca(2+) imaging. At low stimulation intensities, FAEE evoke repetitive short-lasting cytosolic Ca(2+) spikes, which are inhibited by caffeine, used as an inositol trisphosphate receptor antagonist. With more intense stimulation, sustained elevations of the cytosolic Ca(2+) concentration are observed, which can be prevented by pharmacological inhibition of the conversion of FAEE to FA. It is therefore the FA and not the FAEE that cause necrosis. The effect of FA cannot be blocked by inositol trisphosphate receptor antagonists. Fatty acids elicit a marked reduction in the cytosolic adenosine triphosphate (ATP) level. The patch clamp experiments show that the toxic sustained Ca(2+) signal generation induced by FA can be prevented by adding ATP to the cell interior. The toxic alcohol effects are principally due to FAEE produced under non-oxidative conditions and their subsequent conversion to FA. The FA-induced necrosis is Ca(2+)-dependent. The destructive sustained Ca(2+) signals are due to inhibition of mitochondrial function with failure of ATP generation.
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Affiliation(s)
- David N Criddle
- MRC Group, Physiological Laboratory, University of Liverpool, Liverpool, UK
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20
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Abstract
In non-excitable cells such as T lymphocytes, hepatocytes, mast cells, endothelia and epithelia, the major pathway for calcium [Ca2+] entry is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, however, neither the gating mechanism nor the downstream targets of these channels has been clear established. Here, I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.
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Affiliation(s)
- Wei-chiao Chang
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
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21
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Abstract
In electrically nonexcitable cells, Ca2+influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca2+entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca2+stores activates Ca2+influx (store-operated Ca2+entry, or capacitative Ca2+entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca2+release-activated Ca2+current, ICRAC. Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for ICRAC-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca2+content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca2+sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca2+entry. Recent work has revealed a central role for mitochondria in the regulation of ICRAC, and this is particularly prominent under physiological conditions. ICRACtherefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of ICRACand other store-operated Ca2+currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca2+entry pathway.
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Affiliation(s)
- Anant B Parekh
- Department of Physiology, University of Oxford, United Kingdom.
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22
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Zhou ZG, Chen YQ, Liu XB, Hu WM, Tian BL, Chen HQ. Changes of cytosolic [Ca 2+]i in neutrophils in pancreatic microcirculation of rats with caerulein-induced acute pancreatitis under fluid shear stress. World J Gastroenterol 2004; 10:3185-7. [PMID: 15457570 PMCID: PMC4611268 DOI: 10.3748/wjg.v10.i21.3185] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the fluid shear stress induced changes of [Ca2+]i in neutrophils in pancreatic microcirculation of experimental acute pancreatitis (AP).
METHODS: Wistar rats (n = 36) were randomized into three groups. A model of AP was established by subcutaneous injection of caerulein. Low-shear 30 viscometer was used to provide steady fluid shear stress on separated neutrophils. The mean fluorescent intensity tested by flow cytometry was used as the indication of [Ca2+]i quantity.
RESULTS: Under steady shear, cytosolic [Ca2+]i showed biphasic changes. The shear rate changed from low to high, [Ca2+]i in different groups decreased slightly and then increased gradually to a high level (P < 0.05). A close correlation was observed between the cytosolic [Ca2+]i level and the alteration of fluid shear stress in regional microcirculation of AP.
CONCLUSION: The increase of [Ca2+]i is highly related to the activation of neutrophils, which contributes to neutrophil adhesion to endothelium in the early phase of AP. The effect of fluid shear stress on [Ca2+]i may play a crucial role in pancreatic microcirculatory failure of AP.
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Affiliation(s)
- Zong-Guang Zhou
- Department of General Surgery and Institute of Gastroenteric Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.
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23
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Criddle DN, Raraty MGT, Neoptolemos JP, Tepikin AV, Petersen OH, Sutton R. Ethanol toxicity in pancreatic acinar cells: mediation by nonoxidative fatty acid metabolites. Proc Natl Acad Sci U S A 2004; 101:10738-43. [PMID: 15247419 PMCID: PMC490004 DOI: 10.1073/pnas.0403431101] [Citation(s) in RCA: 161] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2004] [Indexed: 12/22/2022] Open
Abstract
Ethanol causes pancreatic damage by an unknown mechanism. Previously, we demonstrated that a sustained rise of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes pancreatic acinar cell injury. Here we have investigated the effects of ethanol and its metabolites on Ca(2+) signaling in pancreatic acinar cells. Most cells exposed to ethanol (up to 850 mM) showed little or no increase in [Ca(2+)](i) (and never at concentrations <50 mM). During sustained exposure to 850 mM ethanol, acetylcholine (ACh) evoked a normal [Ca(2+)](i) elevation and following ACh removal there was a normal and rapid recovery to a low resting level. The oxidative metabolite acetaldehyde (up to 5 mM) had no effect, whereas the nonoxidative unsaturated metabolite palmitoleic acid ethyl ester (10-100 microM, added on top of 850 mM ethanol) induced sustained, concentration-dependent increases in [Ca(2+)](i) that were acutely dependent on external Ca(2+) and caused cell death. These actions were shared by the unsaturated metabolite arachidonic acid ethyl ester, the saturated equivalents palmitic and arachidic acid ethyl esters, and the fatty acid palmitoleic acid. In the absence of external Ca(2+), releasing all Ca(2+) from the endoplasmic reticulum by ACh (10 microM) or the specific Ca(2+) pump inhibitor thapsigargin (2 microM) prevented such Ca(2+) signal generation. We conclude that nonoxidative fatty acid metabolites, rather than ethanol itself, are responsible for the marked elevations of [Ca(2+)](i) that mediate toxicity in the pancreatic acinar cell and that these compounds act primarily by releasing Ca(2+) from the endoplasmic reticulum.
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Affiliation(s)
- David N Criddle
- Physiological Laboratory, Medical Research Council Secretory Control Research Group, University of Liverpool, Liverpool L69 3BX, United Kingdom
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24
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Sutton R, Criddle D, Raraty MGT, Tepikin A, Neoptolemos JP, Petersen OH. Signal transduction, calcium and acute pancreatitis. Pancreatology 2003; 3:497-505. [PMID: 14673201 DOI: 10.1159/000075581] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Evidence consistently suggests that the earliest changes of acute pancreatitis are intracellular, the hallmark of which is premature intracellular activation of digestive zymogens, accompanied by disruption of normal signal transduction and secretion. Principal components of physiological signal transduction include secretagogue-induced activation of G-protein-linked receptors, followed by generation of inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose. In response, calcium is released from endoplasmic reticulum terminals within the apical, granular pole of the cell, where calcium signals are usually contained by perigranular mitochondria, in turn responding by increased metabolism. When all three intracellular messengers are administered together, even at threshold concentrations, dramatic potentiation results in sustained, global, cytosolic calcium elevation. Prolonged, global elevation of cytosolic calcium is also induced by hyperstimulation, bile salts, alcohol and fatty acid ethyl esters, and depends on continued calcium entry into the cell. Such abnormal calcium signals induce intracellular activation of digestive enzymes, and of nuclear factor kappaB, as well as the morphological changes of acute pancreatitis. Depletion of endoplasmic reticulum calcium and mitochondrial membrane potential may contribute to further cell injury. This review outlines current understanding of signal transduction in the pancreas, and its application to the pathophysiology of acute pancreatitis.
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Affiliation(s)
- Robert Sutton
- Department of Surgery, University of Liverpool, 5th Floor UCD Block, Royal Liverpool University Hospital, Daulby Street, Liverpool L69 3GA, UK.
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25
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Abstract
A calcium signal is a sudden increase in concentration of calcium ions (Ca2+) in the cytosol. Such signals are crucial for the control of many important functions of the body. In the brain, for example, Ca2+ signals are responsible for memory, in muscle cells they switch on contraction, whereas in gland cells they are responsible for regulation of secretion. In many cases Ca2+ signals can control several different processes in the same cell. As an example, we shall deal with one particular cell type, namely the pancreatic acinar cell, which is responsible for the secretion of the enzymes essential for the digestion of food. In this cell, Ca2+ signals do not only control the normal enzyme secretion, but also regulate growth (cell division) and programmed cell death (apoptosis). Until recently, it was a mystery how the same type of signal could regulate such diverse functions in one and the same cell. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited.
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Affiliation(s)
- O H Petersen
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Liverpool, UK
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26
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Abstract
Studies of Ca2+ transport pathways in exocrine gland cells have been useful, chiefly because of the polarized nature of the secretory epithelial cells. In pancreatic acinar cells, for example, Ca2+ reloading of empty intracellular stores can occur solely via Ca2+ entry through the basal part of the plasma membrane. On the other hand, the principal site for intracellular Ca2+ release-with the highest concentration of inositol 1,4,5-trisphosphate (IP(3)) receptors-is in the apical secretory pole close to the apical plasma membrane. This apical part of the plasma membrane contains the highest density of Ca2+ pumps and is therefore the principal site for Ca2+ extrusion. On the basis of the known properties of Ca2+ entry and exit pathways in exocrine gland cells, the mechanisms controlling Ca2+ exit and entry are discussed in relation to recent direct information about Ca2+ transport into and out of the endoplasmic reticulum (ER) and the mitochondria in these cells.
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Affiliation(s)
- Ole H Petersen
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK.
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27
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Parekh AB. Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane. J Physiol 2003; 547:333-48. [PMID: 12576497 PMCID: PMC2342659 DOI: 10.1113/jphysiol.2002.034140] [Citation(s) in RCA: 149] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store-dependent and is required for controlling a host of Ca2+-dependent processes ranging from exocytosis to cell growth and proliferation. In many cell types, store-operated Ca2+ entry is manifest as a non-voltage-gated Ca2+ current called ICRAC (Ca2+ release-activated Ca2+ current). Just how store emptying activates CRAC channels remains unclear, and some of our recent experiments that address this issue will be described. No less important from a physiological perspective is the weak Ca2+ buffer paradox: whereas macroscopic (whole cell) ICRAC can be measured routinely in the presence of strong intracellular Ca2+ buffer, the current is generally not detectable under physiological conditions of weak buffering following store emptying with the second messenger InsP3. In this review, I describe some of our experiments aimed at understanding just why InsP3 is ineffective under these conditions and which lead us to conclude that respiring mitochondria are essential for the activation of ICRAC in weak intracellular Ca2+ buffer. Mitochondrial Ca2+ uptake also increases the dynamic range over which InsP3 functions as the second messenger that controls Ca2+ influx. Finally, we find that Ca2+-dependent slow inactivation of Ca2+ influx, a widespread but poorly understood phenomenon that helps shape the profile of an intracellular Ca2+ signal, is regulated by mitochondrial Ca2+ buffering. Thus, by enabling macroscopic store-operated Ca2+ current to activate and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca2+ influx. Store-operated Ca2+ entry reflects therefore a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.
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Affiliation(s)
- Anant B Parekh
- Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
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28
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Bakowski D, Parekh AB. Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes. Cell Calcium 2002; 32:379-91. [PMID: 12543097 DOI: 10.1016/s0143416002001914] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward. Like all Ca(2+)-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na(+) currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I(CRAC) in divalent-free external solution, notably omission of Mg(2+)/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg(2+)-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is approximately 200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.
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Affiliation(s)
- D Bakowski
- Department of Physiology, University of Oxford, Parks Road, OX1 3PT, Oxford, UK
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29
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Weber CK, Adler G. From acinar cell damage to systemic inflammatory response: current concepts in pancreatitis. Pancreatology 2002; 1:356-62. [PMID: 12120214 DOI: 10.1159/000055834] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Acute pancreatitis represents a local inflammatory disorder with severe systemic consequences. Significant progress in understanding the pathophysiology of acute pancreatitis has been achieved in recent years. However, there is no clear concept about initialization and propagation of the disease both in experimental models and in humans. Furthermore, reliable strategies to evaluate prognosis and perform therapy are still missing. The review focuses on mechanisms originating from acinar cells leading to a systemic inflammatory response in experimental pancreatitis.
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Affiliation(s)
- C K Weber
- Department of Medicine I, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany
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30
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Gerasimenko JV, Gerasimenko OV, Palejwala A, Tepikin AV, Petersen OH, Watson AJM. Menadione-induced apoptosis: roles of cytosolic Ca2+elevations and the mitochondrial permeability transition pore. J Cell Sci 2002; 115:485-97. [PMID: 11861756 DOI: 10.1242/jcs.115.3.485] [Citation(s) in RCA: 120] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In normal pancreatic acinar cells, the oxidant menadione evokes repetitive cytosolic Ca2+ spikes, partial mitochondrial depolarisation,cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca2+ spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca2+ spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca2+ uptake, which permits rapid spread of Ca2+ throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca2+ rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca2+buffering power by BAPTA prevents cytosolic Ca2+ spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca2+ and induction of the permeability transition pore are required for initiation of apoptosis.
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Affiliation(s)
- Julia V Gerasimenko
- Medical Research Council Secretory Control Research Group, Physiological Laboratory, University of Liverpool L69 3BX, UK
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31
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Nilius B, Prenen J, Vennekens R, Hoenderop JG, Bindels RJ, Droogmans G. Pharmacological modulation of monovalent cation currents through the epithelial Ca2+ channel ECaC1. Br J Pharmacol 2001; 134:453-62. [PMID: 11588099 PMCID: PMC1572972 DOI: 10.1038/sj.bjp.0704272] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
1. The recent identification of the epithelial Ca(2+) channel, ECaC1, represents a major step forward in our knowledge of renal Ca(2+) handling. ECaC1 constitutes the rate-limiting apical Ca(2+) entry mechanism of active, transcellular Ca(2+) reabsorption. This unique highly selective Ca(2+) channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR). 2. We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca(2+) dependent inactivation of ECaC1. 3. Several modulators were tested, including inorganic cations, putative store-operated Ca(2+) entry (SOC) blockers, the vanilloid receptor (VR-1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red. 4. Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC(50) values of 111 nM and 1.3 microM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective. 5. The divalent cation current block profile for ECaC1 is Pb(2+)=Cu(2+) >Zn(2+) >Co(2+) >Fe(2+) with IC(50) values between 1 and approximately 10 microM. 6. In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca(2+) homeostasis could benefit from such developments.
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Affiliation(s)
- B Nilius
- Department of Physiology, Campus Gasthuisberg, KU Leuven, Belgium.
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32
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Park MK, Lomax RB, Tepikin AV, Petersen OH. Local uncaging of caged Ca(2+) reveals distribution of Ca(2+)-activated Cl(-) channels in pancreatic acinar cells. Proc Natl Acad Sci U S A 2001; 98:10948-53. [PMID: 11535807 PMCID: PMC58579 DOI: 10.1073/pnas.181353798] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
In exocrine acinar cells, Ca(2+)-activated Cl(-) channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl(-) uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca(2+), was used to reveal the Cl(-) channel distribution in mouse pancreatic acinar cells, where approximately 90% of the current activated by Ca(2+) in response to acetylcholine was carried by Cl(-). When caged Ca(2+) in the cytosol was uncaged locally in the apical pole, the Cl(-) current was activated, whereas local Ca(2+) uncaging in the basal or lateral areas of the cell had no effect. Even when Ca(2+) was uncaged along the whole inner surface of the basolateral membrane, no Cl(-) current was elicited. There was little current deactivation at a high cytosolic Ca(2+) concentration ([Ca(2+)](c)), but at a low [Ca(2+)](c) there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca(2+)-activated Cl(-) channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca(2+)](c) and membrane potential. Ca(2+)-activated Cl(-) channels do not mediate Cl(-) uptake at the base, but acetylcholine-elicited local [Ca(2+)](c) spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.
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Affiliation(s)
- M K Park
- Medical Research Council Secretory Control Research Group, Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom
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Petersen OH, Tepikin A, Park MK. The endoplasmic reticulum: one continuous or several separate Ca(2+) stores? Trends Neurosci 2001; 24:271-6. [PMID: 11311379 DOI: 10.1016/s0166-2236(00)01787-2] [Citation(s) in RCA: 123] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The Ca2+ store and sink in the endoplasmic reticulum (ER) is important for Ca2+ signal integration and for conveyance of information in spatial and temporal domains. Textbooks regard the ER as one continuous network, but biochemical and biophysical studies revealed apparently discrete ER Ca2+ stores. Recent direct studies of ER lumenal Ca2+ movements show that this organelle system is one continuous Ca2+ store, which can function as a Ca2+ tunnel. The concept of a fully connected ER network is entirely compatible with evidence indicating that the distribution of Ca2+ -release channels in the ER membrane is discontinuous with clustering in certain localities.
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Affiliation(s)
- O H Petersen
- The MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Liverpool, UK, L69 3BX.
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Park MK, Ashby MC, Erdemli G, Petersen OH, Tepikin AV. Perinuclear, perigranular and sub-plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport. EMBO J 2001; 20:1863-74. [PMID: 11296220 PMCID: PMC125431 DOI: 10.1093/emboj/20.8.1863] [Citation(s) in RCA: 242] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
We have identified three distinct groups of mitochondria in normal living pancreatic acinar cells, located (i) in the peripheral basolateral region close to the plasma membrane, (ii) around the nucleus and (iii) in the periphery of the granular region separating the granules from the basolateral area. Three-dimensional reconstruction of confocal slices showed that the perigranular mitochondria form a barrier surrounding the whole of the granular region. Cytosolic Ca(2+) oscillations initiated in the granular area triggered mitochondrial Ca(2+) uptake mainly in the perigranular area. The most intensive uptake occurred in the mitochondria close to the apical plasma membrane. Store-operated Ca(2+) influx through the basolateral membrane caused preferential Ca(2+) uptake into sub-plasmalemmal mitochondria. The perinuclear mitochondria were activated specifically by local uncaging of Ca(2+) in the nucleus. These mitochondria could isolate nuclear and cytosolic Ca(2+) signalling. Photobleaching experiments indicated that different groups of mitochondria were not luminally connected. The three mitochondrial groups are activated independently by specific spatiotemporal patterns of cytosolic Ca(2+) signals and can therefore participate in the local regulation of Ca(2+) homeostasis and energy supply.
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Affiliation(s)
- Myoung Kyu Park
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, UK Present address: Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea Corresponding author e-mail:
| | - Michael C. Ashby
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, UK Present address: Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea Corresponding author e-mail:
| | - Gul Erdemli
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, UK Present address: Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea Corresponding author e-mail:
| | - Ole H. Petersen
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, UK Present address: Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea Corresponding author e-mail:
| | - Alexei V. Tepikin
- MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, UK Present address: Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea Corresponding author e-mail:
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