A V Tepikin

University of Liverpool, Liverpool, England, United Kingdom

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Publications (151)812.42 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca2+ concentration, which is responsible for the damage and eventual death of pancreatic acinar cells. Vacuolisation is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as "initiating" organelles in the development of the cell injury. In this study we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis ((bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)). We found that the inhibitor of STIM/Orai channels GSK-7975A effectively suppressed both Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry induced by thapsigargin (inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ pumps) and observed strong inhibition of thapsigargin-induced vacuole formation by GSK-7975A. Finally we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation.
    The Biochemical journal. 11/2014;
  • Pancreatology 06/2014; 14(3):S10. · 2.04 Impact Factor
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    ABSTRACT: Aims: The aim of this study was to develop a fluorescent ROS probe which is preferentially localised in cellular membranes and displays a strong change in fluorescence upon oxidation. We also aimed to test the performance of this probe for detecting pathophysiologically relevant ROS responses in isolated cells. Results: We introduced a novel lipophilic ROS probe dihydrorhodamine B octadecyl ester (H2RB-C18). We then applied the new probe to characterise the ROS changes triggered by inducers of acute pancreatitis (AP) in pancreatic acinar cells. We resolved ROS changes produced by L-ornithine, L-arginine, cholecystokinin-8 (CCK-8), acetylcholine (ACh), taurolithocholic acid 3-sulfate (TLC-S), palmitoleic acid ethyl ester (POAEE) and the bacterial toxin pyocyanin. Particularly prominent ROS responses were induced by pyocyanin and L-ornithine. These ROS responses were accompanied by changes in cytosolic Ca2+concentration ([Ca2+]i), mitochondrial membrane potential (ΔΨ) and NAD(P)H concentration. Innovation: The study describes a novel sensitive lipophilic ROS probe. The probe is particularly suitable for detecting ROS in near-membrane regions and therefore for reporting the ROS environment of plasma membrane channels and pumps. Conclusions: In our experimental conditions the novel probe was more sensitive than CM-H2DCF and H2R123 and allowed us to resolve ROS responses to secretagogues, pyocyanin and L-ornithine. Changes in the fluorescence of the new probe were particularly prominent in the peripheral plasma membrane - associated regions. Our findings suggest that the new probe will be a useful tool in studies of the contribution of ROS to the pathophysiology of exocrine pancreas and other organs/tissues.
    Antioxidants & Redox Signaling 03/2014; · 8.20 Impact Factor
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    ABSTRACT: This brief review discusses recent advances in studies of mitochondrial Ca(2+) signaling and considers how the relationships between mitochondria and Ca(2+) responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been "How to win ATP and influence Ca(2+) signaling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells". But "brevity is a virtue" and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Among these mechanisms are mitochondrial regulation of Ca(2+) entry and the role of mitochondria in the formation of localized Ca(2+) responses. The roles of Ca(2+) signaling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed.
    Cell calcium 02/2014; · 4.29 Impact Factor
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    ABSTRACT: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Intracellular calcium ([Ca(2+)]C), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)]C rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.
    Gut 10/2013; · 10.73 Impact Factor
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    ABSTRACT: We demonstrated that increasing intracellular cAMP concentrations results in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation are mediated by PKA, whilst EPAC potentiates migration.
    Biochimica et Biophysica Acta 06/2013; · 4.66 Impact Factor
  • Pancreatology 03/2013; 13(1):e9. · 2.04 Impact Factor
  • Pancreatology 03/2013; 13(1):e12. · 2.04 Impact Factor
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    ABSTRACT: We demonstrated three novel forms of dynamic behaviour of junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) in migrating cancer cells: saltatory formation, long distance sliding and dissolution. The individual ER-PM junctions formed near the leading edge of migrating cells (usually within 0.5µm of polymerised actin and close to focal adhesions) and appeared suddenly without sliding from the interior of the cell. The long distance sliding and dissolution of ER-PM junctions accompanied the tail withdrawal.
    Biochemical Journal 01/2013; · 4.65 Impact Factor
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    ABSTRACT: The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.
    Current biology: CB 06/2012; 22(13):1241-6. · 10.99 Impact Factor
  • Svetlana Voronina, Alexei Tepikin
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    ABSTRACT: The remarkable recent discoveries of the proteins mediating mitochondrial Ca(2+) transport (reviewed in this issue) provide an exciting opportunity to utilise this new knowledge to improve our fundamental understanding of relationships between Ca(2+) signalling and bioenergetics and, importantly, to improve the understanding of diseases in which Ca(2+) toxicity and mitochondrial malfunction play a crucial role. Ca(2+) is an important activator of exocrine secretion, a regulator of the bioenergetics of exocrine cells and a contributor to exocrine cell damage. Exocrine secretory cells, exocrine tissues and diseases affecting exocrine glands (like Sjögren's syndrome and acute pancreatitis) will, therefore, provide worthy research areas for the application of this new knowledge of the Ca(2+) transport mechanisms in mitochondria.
    Cell calcium 05/2012; 52(1):86-92. · 4.29 Impact Factor
  • Geng Tian, Alexei V Tepikin, Anders Tengholm, Erik Gylfe
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    ABSTRACT: The events leading to the activation of store-operated Ca(2+) entry (SOCE) involve Ca(2+) depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca(2+) channel protein Orai1 to activate Ca(2+) influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing β-cells and glucagon-secreting α-cells within intact mouse and human pancreatic islets. ER Ca(2+) depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca(2+) store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in α- than in β-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in α-cells and retranslocation in β-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.
    Journal of Biological Chemistry 02/2012; 287(13):9862-72. · 4.65 Impact Factor
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    ABSTRACT: Pancreatic acinar cells exhibit a remarkable polarization of Ca2+ release and Ca2+ influx mechanisms. In the present brief review, we discuss the localization of channels responsible for Ca2+ release [mainly IP3 (inositol 1,4,5-trisphosphate) receptors] and proteins responsible for SOCE (store-operated Ca2+ entry). We also place these Ca2+-transporting mechanisms on the map of cellular organelles in pancreatic acinar cells, and discuss the physiological implications of the cellular geography of Ca2+ signalling. Finally, we highlight some unresolved questions stemming from recent observations of co-localization and co-immunoprecipitation of IP3 receptors with Orai channels in the apical (secretory) region of pancreatic acinar cells.
    Biochemical Society Transactions 02/2012; 40(1):108-11. · 2.59 Impact Factor
  • Dingsdale H, Haynes L, Lur G, Tepikin AV
    Store-operated Ca2+ entry (SOCE) pathways, Edited by K. Groschner, W.F. Graier, Romanin C., 01/2012: chapter The role of ER and ER junctions with the plasma membrane in the regulation of SOCE; Springer.
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    ABSTRACT: Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca²⁺ entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP₃Rs (InsP₃ receptors). The co-localization between Orai1 and IP₃Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca²⁺ store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca²⁺ store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP₃Rs and a basolateral pool that interacts with STIM1 following the Ca²⁺ store depletion. Experiments on IP₃R knockout animals demonstrated that the apical Orai1 localization does not require IP₃Rs and that IP₃Rs are not necessary for the activation of SOCE. However, the InsP₃-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP₃Rs could have an inhibitory effect on this Ca²⁺ entry mechanism.
    Biochemical Journal 06/2011; 436(2):231-9. · 4.65 Impact Factor
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    ABSTRACT: Pancreatic acinar cells possess a very large Ca(2+) store in the endoplasmic reticulum, but also have extensive acidic Ca(2+) stores. Whereas the endoplasmic reticulum is principally located in the baso-lateral part of the cells, although with extensions into the granular area, the acidic stores are exclusively present in the apical part. The two types of stores can be differentiated pharmacologically because the endoplasmic reticulum accumulates Ca(2+) via SERCA pumps, whereas the acidic pools require functional vacuolar H(+) pumps in order to maintain a high intra-organellar Ca(2+) concentration. The human disease acute pancreatitis is initiated by trypsinogen activation in the apical pole and this is mostly due to either complications arising from gall bladder stones or excessive alcohol consumption. Attention has therefore been focussed on assessing the acute effects of bile acids as well as alcohol metabolites. The evidence accumulated so far indicates that bile acids and fatty acid ethyl esters - the non-oxidative products of alcohol and fatty acids - exert their pathological effects primarily by excessive Ca(2+) release from the acidic stores. This occurs by opening of the very same release channels that are also responsible for normal stimulus-secretion coupling, namely inositol trisphosphate and ryanodine receptors. The inositol trisphosphate receptors are of particular importance and the results of gene deletion experiments indicate that the fatty acid ethyl esters mainly utilize sub-types 2 and 3.
    Cell calcium 03/2011; 50(2):193-9. · 4.29 Impact Factor
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    ABSTRACT: Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.
    Proceedings of the National Academy of Sciences 03/2011; 108(14):5873-8. · 9.81 Impact Factor
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    ABSTRACT: 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. 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). 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. 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.
    Gastroenterology 02/2011; 140(7):2116-25. · 12.82 Impact Factor
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    ABSTRACT: Short title: Orai1 co-localises with IP 3 receptors Abbreviations: PAC, pancreatic acinar cells; ER, endoplasmic reticulum; SOCE, store-operated Ca 2+ entry; STIM, stromal interaction molecule; IP 3 R, IP 3 receptors; ACh, acetylcholine; CCK, cholecystokinin; [Ca 2+ ]c , cytosolic Ca 2+ concentration; ZO1, zona occludens 1; TG thapsigargin. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
    Biochemical Journal 01/2011; 436(2):231-239. · 4.65 Impact Factor
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    ABSTRACT: SOCCs (store-operated Ca(2+) channels) are highly selective ion channels that are activated upon release of Ca(2+) from intracellular stores to regulate a multitude of diverse cellular functions. It was reported previously that Golli-BG21, a member of the MBP (myelin basic protein) family of proteins, regulates SOCE (store-operated Ca(2+) entry) in T-cells and oligodendrocyte precursor cells, but the underlying mechanism for this regulation is unknown. In the present study we have discovered that Golli can directly interact with the ER (endoplasmic reticulum) Ca(2+)-sensing protein STIM1 (stromal interaction molecule 1). Golli interacts with the C-terminal domain of STIM1 in both in vitro and in vivo binding assays and this interaction may be modulated by the intracellular Ca(2+) concentration. Golli also co-localizes with full-length STIM1 and Orai1 complexes in HeLa cells following Ca(2+) store depletion. Overexpression of Golli reduces SOCE in HeLa cells, but this inhibition is overcome by overexpressing STIM1. We therefore suggest that Golli binds to STIM1-Orai1 complexes to negatively regulate the activity of SOCCs.
    Biochemical Journal 09/2010; 430(3):453-60. · 4.65 Impact Factor

Publication Stats

5k Citations
812.42 Total Impact Points

Institutions

  • 1992–2014
    • University of Liverpool
      • • Department of Cellular and Molecular Physiology
      • • School of Biological Sciences
      Liverpool, England, United Kingdom
  • 2012
    • Cardiff University
      • School of Biosciences
      Cardiff, WLS, United Kingdom
  • 2008–2010
    • Bogomolets Institute of Physiology NASU
      • Department of General Physiology of Nervous System
      Kievo, Kyiv City, Ukraine
  • 2002
    • Sungkyunkwan University
      • Department of Physiology
      Sŏul, Seoul, South Korea
  • 1991–1993
    • Ukrainian Academy of Agrarian Sciences
      Kievo, Kyiv City, Ukraine