Effect of the ulcerogenic agents ethanol, acetylsalicylic acid and taurocholate on actin cytoskeleto

PO Box 2345, Beijing 100023, China置d/ Gastroenterol 2005; 11(26)wig@wigner.comELSEVIER@2005 The WG Press and Elsevier Inc. All rights I·9B4 SIC RESERCH·Effect of the ulcerogenic agents ethanol, acetylsalicylic acid andtaurocholate on actin cytoskeleton and cell motility in culturedrat gastric mucosal cellsSiamak Bidel, Harri Mustonen, Giti Khalighi-Sikaroudi, Eero Lehtonen Pauli Puolakkainen Tuula Kiviluoto Eero KivilaaksoSiamak Bidel, Harri Mustonen, Giti Khalighi-SikaroudiIn addition ethanol and aSa caused degradation of actinLehtonen, Pauli Puolakkainen tuula Kiviluoto, Eero Kivcytoskeleton Oxidative stress seems to underlie ethanolDepartment of Surgery, Helsinki University Central Hebut not aSa or taurocholate, induced cytoskeletal damageHaartmaninkatu 8, Helsinki, FinlandSupported by the Grants from Antti and Jenny Wihuri Foundation 2005 The w3G Press and Elsevier Inc. All rights reservedand Research Foundation of Helsinki University Central HospitalTYH4228Co-first-authors: Harri MustonenKey words: Gastric mucosa; Ethyl alcohol; Taurocholate;Correspondence to: Harri Mustonen DSc, Helsinki University Aspirin; ActinCentral Hospital, Department of Surgery, Biomedicum HelsinkiHaartmaninkatu 8. Helsinki 00290Bidel S, Mustonen H, Khalighi-Sikaroudi G, Lehtonen E,Finland. harri mustonen @helsinki. fiPuolakkainen P, Kiviluoto T, Kivilaakso E. Effect of theTelephone:+3589471-7829Fax:+3589471-74675ulcerogenic agents ethanol, acetylsalicylic acid andReceived: 2004-09-01 Accepted: 2004-10-07taurocholate on actin cytoskeleton and cell motility incultured rat gastric mucosal cells. World J Gastroenterol2005;11(26):4032-4039http://www.wjgnet.com/1007-9327/11/4032.aspAbstractAIM: To assess the effects of ulcerogenic agents on actincytoskeleton and cell motility and the contribution ofoxidative stressINTRODUCTIONMETHODS: Rat gastric mucosal cell monolayers wereThe gastric mucosa is frequently exposed to differentor without allopurinol( 2 mmol/L), for 15 min to ethanol aspirin and taurocholate. These agents have been extensively10-150 mL/L), ASA(1-20 mmol/L)or taurocholateds, including biochemistry, mor( 1-20 mmol/L), then the cells were processed for actinlogy, electrophysiology, tissue permeability, etc but theand vinculin staining. Cell migration after wounding wascellular mechanisms of injury are still poorly definedalso measuredIn digestive epithelia actin cytoskeleton is involved, forexample, in organization of cytoplasm, maintenance of cellRESULTS: Exposure to 10 my/L ethanol caused divergence shape, generation and maintenance of epithelial polarityof zonula adherens-associated actin bundles of adjacent in migration. The cytoplasm of epithelial cell is spatiallyells and decreased rate of migration. These actions were and temporally organized by microfilaments, microtubulesopposed by xanthine oxidase inhibitor allopurinol Exposure and intermediate filaments, which form a lattice-liketo 50 mL/L ethanol induced degradation and divergence intracellular meshwork!2.of zonula adherens- associated vinculin from adjacent cells,The actin cytoskeleton is highly conserved in eukaryoticwhich was, again, partially reverted by allopurinol. With cells, including more than 70 categorized types of actin-1 mmol/L ASA actin filaments became shorter and thicker. binding proteins. The human actin family includes a-B,However, higher concentrations(10, 20 mmol/L)of ASA and Y-actin, which share most of their amino acid sequencreturned microfilaments thinner and longer, and decreased The actin filaments are formed by polymerization of actinrate of migration. Zonula adherens-associated actin bundleswere moderately distorted with 10 mmol/L ASA and withmonomers. The polymeric actins are assembled into10 mmol/L taurocholate. Exposure to taurocholate provokedfilamentous network, which is regulated by actin-bindingchanges resembling those of ASA. Taurocholate 5-20 mmol/L proteins. These proteins regulate, for example, polymcrization,decreased the rate of migration dose dependently. The crosseffects of aSa and taurocholate were not prevented by中国煤化工 LallopurinolCNMH Gf actin filaments/#.Theactinlieu wgcuicr by various actin crossCONCLUSION: All ulcerogenic agents decreased the rate linking proteins to form complex three-dimensional structuresof migration dose dependently and induced divergence of The filaments are extensively branched near the plasmazonula adherens-associated actin bundles of adjacent cells. membrane, focal adhesions, and adherens and tight junctionsBidelS et al. Ulcerogenic agents and gastric F-actin4033Arp2/3 complex is involved in this branchingthe cytoskeleton. We further studied the possible role ofDifferent components of the cytoskeleton are tightly reactive oxygen species in epithelial injury by inhibitinginvolved in cell motility Actin based cell motility has several endogenous intracellular generation of superoxide radicalsimportant tasks in epithelial cell functions, such as in secretory from purines with a xanthine oxidase inhibitor, allopurinolvesicle movement and cell migration during wound healing 1The cell migration starts with lamellipodia extension and MATERIALS AND METHODSof the cell body and detachment of the tail. Rho guanosine Immortal rat gastric mucosal cells(RGM-1, Riken Cell Bank,triphosphatases have a key role in regulating different phases Riken, Japan) I were cultured on coverslips to confluentof migration. Racl seems to be involved in initiation of monolayers at 37 C in humidified atmosphere containingmigration and in stimulation of lamellipodium extension", 50 mL/L COz in air. The medium used was an equalwhile Rho seems to contribute mainly to cell body contraction. mixture of Ham's F12 and Dulbeccos's MEM supplementedEpithelial cells have specialized mechanisms to enable with inactivated 200 mL/L fetal bovine serum, 100 U/mLcell-cell and cell-extracellular matrix adhesions. These penicillin, 100 ug/mL streptomycin and 0. 125 ug/mLconnections are formed by transmembrane proteins, which amphotericinbind to extracellular matrix or adjacent cells with theirThe cells were exposed for 15 min to the followingextracellular domains, while the intracellular domains bind ulcerogenic agents: ethanol (10-150 mL/L), acetylsalicylicto intracellular cytoskeleton via cytoplasmic adaptor proteins, acid (ASA, 1-20 mmol/L)and taurocholate(1-20 mmol/L)many of which interact with the actin cytoskeleton o, at 37C. In allopurinol experiments the samples were treatedIntegrins mediate cellular adhesion to the extracellular matrix. with 2 mmol/L allopurinol 24 h before and during theThey are linked to the actin cytoskeleton in focal adhesion exposure of the ulcerogenic agent. Thereafter the cells werecomplexes, which include a multitude of proteins, such as fixed with 35 mL/L paraformaldehyde in PBS for 15-20 minvinculin, talin, Paxillin and calpain. These focal adhesions at room temperature and stained as described beloware involved in regulation of the cell migration andActin filaments and focal adhesion plaques were stainedproliferation. The classical E-cadherins mediate Ca2+ with phallotoxins conjugated to Alexa 488 fluorophoredependent cell-cell adhesion through their cxtracellular Ca2* Molecular Probes, A-12379). After fixation the cells werebinding repeats, while their cytoplasmic part binds to the washed three times for 5-10 min with PBS at roomactin cytoskeleton via B-and a-catenin. This complex is temperature, permeabilized with 1 mL/L Triton-X, 1 mL/Lassociated with a variety of other proteins, including vinculin, bovine serum albumin in PBS and washed again threea-actinin, and paxillin Zonula adherens is an E-cadherin times for 5-10 min in PBS. Thereafter, the samples werebased belt-like adherent junction just below the tight junctions incubated for 30-60 min in PBS with 80 mL/L bovine serumencircling the epithelial cclls adhered to each other. Along albumin and washed three times for 5-10 min with PBSthe cytoplasmic side of the zonula adherens runs a contractile and incubated overnight with the mouse monoclonal anti-bundle of actin filaments, which are associated to zonula vinculin(10 ug/mL, with 10 mL/L bovine serum albumin)adherens with a set of intracellular proteins, such as a-catenin, The samples were washed three times for 5-10 min withvinculin,-actinin and plakoglobin. Vinculin is a large proteinPBS and were incubated with secondary antibody goatwith multiple binding sitcs to different adhesion related anti-mouse Alexa 568(10 ug/mL)and Alexa 488 phalloidinproteins, including actin, a-actinin, talin, paxillin, VASP, (1: 1 000) with 1 mL/L bovine serum albumin in PBS forponsin, vincxin and PKC. 2. Vinculin is thought to mediate 30 min at room temperature. Thereafter, the samples werethe linkage between actin cytoskeleton and cadherins or washed three times for 5-10 min with PBS and mountedintegrins in cell-cell or cell-extracellular matrix adhesions. with DABCO containing mounting media. ConfocalRecently vinculin has been shown to transiently associate fluorescence microscopy was performed with a Leica TCSwith arp2/3 complex in the nascent focal complexes in the SP2 confocal microscope and normal fluorescenceleading edgell), possibly linking cellular protrusion and microscopy with an Olympus IX50 microscopedhesion to extracellular matrix. Ethanol is known to causeoxidative stress in rat hepatocytes and in gastric mucosal Cell migration assaycells14, 151. Oxidative stress can cause various effects in the RGm cells were cultured to confluency on plastic cultureell, such as oxidation and fragmentation of cellular proteins, dishes as described above. In allopurinol scrics the confluentperoxidation of membrane lipids, fragmentation of DNA monolayers were treated with allopurinol after and 24 hand mitochondrial damage. Among the very early effects before wounding. Artificial round-shaped wounds wereof oxidative stress might be disruption of the cytoskeleton 6l. created in the cell monolayer with a silicon tip, where afterThe aim of the present study was to characterize the control (water)or ulcerogenic agents were added. The migrationeffects of different ulcerogenic agents on actin cytoskeleton, distance of the cells at the wound edge was measured duringcell adhesion molecules and cell migration, and to investigate the nethe possible role of oxidative stress in the cellular injury free3H中国煤化工 ring the change in cellinduced by them. This was accomplished by confocal and chanCNMHGand 6 h, as describednormal fluorescence imaging of actin and vinculin during previouslyloyexposure of the epithelial cells to the ulcerogenic agentsWe also measured the rate of cell migration during exposure Statistical analysisto the ulcerogenic agents to assess the functional state of The results are expressed as mean+95% confidence intervals4034ISSN 1007-9327 CN 14-1219/r World J Gastroenterol July 14, 2005 Volume 11 Number 26Student's unpaired /-test was used for statistical analysis of cells could not be distinguished from each other. Exposurehe raw data. P values less than 0.05 were considered as to 10 mL/L ethanol caused actin belts of adjacent cells to divergestatistically significant.from each other and the gap between them was widened withincreasing cthanol concentrations(20-50 mL/L, Figure 2)RESULTSTreatment with allopurinol (2 mmol/L), which acts asEthanolinhibitor of xanthine oxidase and inhibits productionIn control cells, actin filaments were thin, showing branched intracellular reactive oxidative species, prevented completelyactin networks and zonula adherens-associated actin bundles the divergence of actin belts in cells exposed to 10 mL/Lnear the cell-cell contact sites(Figures 1 and 2). Exposure cthanol and partially in cells exposed to 20 mL/L ethanol,to 10 mL/L(vol/vo) ethanol for 15 min(Figure 1)did not but not in cells exposed to 50 mL/L ethanoL.cause any apparent changes in actin cytoskeleton. IncreasingIn control cultures. vinculin was visible at the focalthe ethanol concentration to 50 mL/L caused moderate adhesions as well as in the zonula adherens between thedegradation and irregularity in the actin filament organization. adjacent cells. The zonula adherens-associated vinculin ofThese changes became more prominent with 100 mL/L adjacent cells could not be distinguished. Exposure toethanol, displaying almost complete degradation of the 10-20 mL/L ethanol did not change the distribution ofactin filaments. Exposure to 150 mL/L ethanol for 15 min vinculin, but 50 mL/L ethanol exposure caused degradationprovoked detachment of the cells from each other and divergence of zonula adherens-associated vinculin(Figure 1). The damage of the actin filaments was severe, staining. Allopurinol treatment moderately attenuated thisand in some microscopic fields only shadows of cells and change(Figure 3)nuclei remained visibleExposure to 10 mL/L ethanol decreased the rate ofIn control cultures, the zonula adherens-associated actin migration during the first six hours after wounding(27+3bundles were normally seen as continuous belt-like accumu- and 17+2 um/h for control and cthanol experiments,lations between the cells, and the actin belts of adjacent respectively, P<0.001, n=20, Figure 4). This decrease in中国煤化工CNMHGFigure 1 Effects of different doses of ethanol (ETOH), acetylsalicylic acid(AS/and taurocholate(TC)on actin filaments of RGM rat gastric surtace epithelial cellsBidel s et a/ Ulcerogenic agents and gastric F-actin4035Figure 2 Effects of different doses of ethanol (ETOH), acetylsalicylic acid(asa) in RGM cells and their modulation by allopurinol (2 mmoWL-)treatment. Arrowstaurocholate(TC)on the zonula adherens-associated belt-like actin bundles indicate perturbations in the actin beit organization.migration was partially abolished by allopurinol pretreatment was中国煤化工wih30mL/ L ethanol(17+2 and 23+2 um/h for ethanol and ethanol+allopurinol andexperiments, respectively; P=0.025, n= 20). AllopurinolEYHCNMHG-I7+13 um/h withof the wound, possiblytreatment alone had no effect on the rate of migration due to a direct toxic effect of ethanol and /or to overall cell27+3 and 27+2 um/h for control and allopurinol experiments, shrinkage in the monolayer caused by ethanol. At theserespectively). The migration distances are shown in Figure 4. higher concentrations of ethanol (30-50 mL/L) allopurinolWith higher ethanol concentrations the rate of migration did not prevent the cthanol promoted decrease in migration4036ISSN 1007-9327 CN 14-1219/R World J Gastroenterol July 14, 2005 Volume 11 Number 26Figure 3 Effects of different doses of ethanol (ETOH), acetylsalicylic acid (ASA) by allopurinol (2 mmol/L)treatment. Arrows indicate perturbations in vinculinand taurocholate(TC)on vinculin distribution in RGM cells and their modulation organizationAcetylsalicylic acid (ASA)Exposure to 1-10 mmol/L ASA had no effect on vinculinAfter 15 min exposure to 1 mmol/L ASA, the actin filaments distribution( Figure 3). Likewise, exposure to 1-5 mmol/Lwere contracted and became shorter and thicker, but noASA had no effect on the rate of migration, but exposuresigns of cell detachment from each other were visible. However, to 10 mmol/L ASA retarded migration after wounding fromwith higher concentrations of ASA(10 and 20 mmol/L) 16.8:+0.5 to 13.7+1.2 um/hthe microfilaments became thinner and longer again, butfailed to resume their original size and shape. Increasing TaurocholateASA concentration to 20 mmol/L detached the cells partially Following exposure to low concentrations of taurocholatecom each other, which was manifested also as a divergence (1of the zonula adherens-associated actin bundles of adjacent becH中国煤化工 Is. These effso degradaton oCN MH Easing concentrationslatt, taurocholate causedExposure to 1-5 mmol/L. ASA had no effect on the similar changes as was seen with low concentrations ofoula adherens-associated belt-like actin bundles. Exposure ASA, but the alterations were of lesser degree. Exposure toto 10 mmol/L ASA moderately distorted these actin belts and 1-5 mmol/L taurocholate did not cause any change in thethis was not prevented with allopurinol treatment(Figure 2). zonula adherens-associated actin bundies, but 10 mmol/LBidel S et al. Ulcerogenic agents and gastric F-actin4037there was an extensive distortion of the actin cytoskeletalControlnetwork, and even stronger ethanol (150 mL/L) causedsevere damage to the cells with complete degradation of allt-10 mL/L ETOHactin filament0804010 mL/L ETOHThe zonula adhcrens-associated belt-like actin bundlesof adjacent cells were diverged from each other alreadyduring exposure to a rather low(10 mL/L)concentrationof ethanol. This divergence was prevented with 2 mmol/L0allopurinol treatment. Allopurinol is a cell membranepermeable xanthine oxidase inhibitor, which blocks endogenousTime(h)intracellular enzymatic generation of superoxide radicalsfrom purines. This suggests that superoxide radicals arealipurre 4 Effect of ethanol on cellular migratinvolved in ethanol induced divergence of the actin beltsourinol treatment. Ethanol significantly reduced the migrated distanc. Ethanol also perturbed the distribution of the zonula adherensAlopurinol partially abolished the ethanol promoted inhibition of cell migrationjunction-associated protein, vinculin, but only after exposureto 50 mL/L ethanol. Further, this change was moderatelytaurocholate showed a moderate perturbation of these belts, opposed by allopurinol treatment. The damage in zonulawhich was not prevented by allopurinol treatment(Figure 2). adherens-associated vinculin became apparent only withExposure to taurocholate(1-10 mmol/L) had no effect onhigher ethanol concentrations than was needed for changesvinculin distribution(Figure 3)in zonula adherens-associated actin bundles, which suggestsExposure to 1 mmol/L taurocholate had no effect onthat the changes in the actin belts might precede the zonulathe rate of migration after wounding, but 5-20 mmol/Iadherens junction damage. Allopurinol also opposed thetaurocholate retarded migration dose dependentlyethanol provoked decrease in cell migration after artificial(migration speed 16.8*0.6, 14.5+0.8(P=0.01), 12.7+0.4 monolayer wounding, a further indication of the involvement(P<0.001)and 1.6+0.8(P<0.001)um/h for 0 mmol/l damageof superoxide radicals in ethanol induced cytoskeletal(control, 5, 10 and 20 mmol/L taurocholate, respectively;Pvalues are given in comparison with control, #=6)The above findings suggest that oxidative stress mightunderlie the observed ethanol induced changes in actincytoskeleton. Epithelial cells exposed to ethanol increaseDISCUSSIONsuperoxide production 2l and superoxide dismutase activityThe gastric mucosa is normally exposed to a large number is decreased in rat stomach exposed to pure ethanol. Also,of ulcerogenic compounds, including ethanol, ASA(aspirin) oxidative stress and mitochondrial damage precede deathand bile salts. The effects of these agents on cell cytoskeleton in gastric mucosal cells exposed to ethanols. The numberhave not been thoroughly studied. In the healing of superficial of propidium iodide positive cells, indicating loss of cellgastric mucosal lesions, intact actin cytoskeleton is essential viability, was increased following exposure to 50 mL/Lfor cell migration and survival. The normal wound healing ethanol and ethanol caused mitochondrial cell membraneprocess in gastric mucosa is initiated by epithelial restitution depolarization and mitochondrial permeability transitionwhereby the surviving epithclial cells around wound edge indicating mitochondrial dysfunction 5migrate over the injured area to cover it with a flattenedIt has been suggested, that NSAIDs, including ASA,neo-epithelium. The migrating cells form lamellipodia and damage gastrointestinal tract both by direct local effectsvinculin, RhoA and Rac are strongly expressed along the and by systemic inhibition of prostaglandin synthesiswound edge. Ulcerogenic agents can profoundly modulate Davenport first demonstrated that ASa diffuses into thethis healing process.gastric mucosa as an undissociated molecule leading toEthanol promotes narrowing of lamellipodia as well as disruption of the gastric mucosal barrier and backdiffusiontards cell migration and inhibits cellular proliferation after of luminal acid into the mucosa, which, in turn, leadounding in rabbit gastric mucosa. 2. Exposure to 50- presumably via generation of an inflammatory cascade,to100 mL/L ethanol causes extensive disorganization and break-down of the mucosal tissue/. Although prostaglandinsragmentation of the actin cytoskeleton leading to its contribute to cell migration in several cell types 28 29),additioncomplete collapse in an intestinal epithelial cell line2. We of exogenous prostaglandins or inhibition of endogenoushave previously shown that 50 mL/L luminal ethanol causes prostaglandin synthesis had no effect on gastric epithelialthe opening of basolateral potassium channcls leading to cell migration e On the other hand, NSAIDs may directlycell volume shrinkage 24. In the present work we demonstrated retard cell migration and decrease actin staining in gastithat the same concentration of ethanol affects also various epithelial cell (RGMi)monolayer ycomponents of the cytoskeleton. The changes in the structureThe present resnlts with ASa are well in accordanceand arrangement of the cytoskeleton are in line with cell with中国煤化工 auses major cell shapeshrinkage and the consequent increase in the gaps between deforthe cells. Since an intact cytoskeleton is of utmost importance TheCNMHG(unpublished results)Probably contributes tofor the epithelial integrity and function, these minor changes cell-cell detachment, which was shown to occur at higher ASAmay be the first manifestations of the cthanol-induced damage concentrations. ASA decreased migration at 10 mmol/Lto the epithelial cclls. At 100 mL/L ethanol concentration, concentration and degradation of actin filaments was visibleISSN 1007-9327 CN 14-1219/R World J Gastroenterol July 14, 2005 Volume 11 Number 26at 20 mmol/L ASA concentration. Zonula adherens-2 Seksek O, Biwersi J, Verkman AS Translational diffusion ofssociated actin bundles were moderately damaged withmacromolecule-sized solutes in cytoplasm and nucleus. Ceil10 mmol/L ASA and this was not affected by allopurinol 3 Alberts b, johnson A, Lewis ), Raff M, Roberts K, Walter PMolecular Biology of the Cell. 4th ed. New York: Garlandinduced divergence of these actin bundlesScience 2002To our knowledge there is very little information about 4 Ayscough KR In vivo functions of actin-binding proteinsthe actions of ASa on cellular actin cytoskeleton in theCurr Opin Cell Bio! 1998; 10: 102-111gastric epithelium. Electrophysiological measurements haveu NO, Zhou X, Toivola DM, Omary MB. The cytoskeletonshown that there is a slight initial increase in transepithelialof digestive epithelia in health and disease. Am/ Physiol 1999;277:G1108G1137resistancefollowing exposure to 10 mmol/L ASA (at pH 6 Mitchison TL, Cramer LP. Actin-based cell motility and cell3), which is, however, due to increase in apical cell membranelocomotion Cell 1996: 84: 371-379resistance. However, during more prolonged exposure to 7 Weber I, Gerisch G, Heizer C,Murphy J,Badelt K,StockASA the transepithelial resistance decreased, which mighSchwartz JM, Faix J Cytokinesis mediated through the rebe a consequence of tight junction disruption, widening ofruitment of cortexillins into the cleavage furrow. EMBO Jintercellular space and/or loosening of cell-cell adhesions)The changes in the zonula adherens-associated actin bundles 8 Ridley A]. Rho GTPases and cell migration. / Cell Sci 2001114:2713-2722observed in the present study might contribute to the 9 Pollard TD, Blanchoin L, Mullins RD Molecular mechanismspreviously observed decrease in transepithelial resistance.controlling actin filament dynamics in nonmuscle cells. AnnuRev Biophys Biomol Struct 2000; 29: 545-576In earlier studies bile salts have been shown to retard 10 Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura Swound restitution and repair in cultured rabbit gastricTsukita S, Tsukita S Occludin: a novel integral membraneepithelial cells 4. In the present study, taurocholate causedtein localizing at tight junctions. Cell Biol 1993: 123similar effects in cell migration reducing it dose dependently17771788in concentrations of 5 mmol/L and higher. The mechanism 11 Critchley DR Focal adhesions-the cytoskeletal connectionf taurocholate induced decrease in migration speed is stillCurr Opin Cell biol 2000: 12: 133-13912 Pokutta S, Weis WI. The cytoplasmic face of cell contactunclear. but bile salts are known to increase intracellularsites. Curr Opin Struct Bio! 2002; 12: 255-262calcium levels,34I and this might have an cffect on cellular 13 DeMali KA, Barlow CA, Burridge K Recruitment of the Arp2migration3 complex to vinculin: coupling membrane protrusion toTaurocholate is known to interact with cell membranesatrix adhesion. / Cell Biol 2002: 159: 881-891and as a lipophilic compound it may diffuse into the cell mem- 14 Higuchi H, Adachi M, Miura S, Gores G), Ishii H. The mito-branes increasing its permeability to extracellular agentsondrial permeability transition contributes to acute ethaTaurocholate may also act as a detergent dissolving cell34:320-328membrane phospholipids eventually breaking up the cell 15 Hirokawa M, Miura S, Yoshida H, Kurose I, Shigematsu T,membranes. Apical cell membrane resistance was decreasedignificantly with 10 mmol/L taurocholate in NecturusKato S, Ishii H Oxidative stress and mitochondrial damageecedes gastric mucosal cell death induced by ethanolantrum, suggesting that cell membranes may be the mainadministration. Alcohol Clin Exp Res 1998: 22: 111$-114starget in taurocholate induced cellular damage. Zonula 16 Bellomo G, Mirabelli F Oxidative stress and cytoskeletaladherens associated actin bundles were moderately damagedIterations. Ann N y Acad Sci 1992; 663: 97-109with 10 mmol/L taurocholate, but this was not affected by 17 Kobayashi I, Kawano S, Tsuji S, Matsui H, Nakama A,Sawaoka H, Masuda E, Takei Y, Nagano K, Fusamoto H,allopurinol treatment suggesting, again, a mechanismOhno T Fukutomi H, Kamada T, RGM1, a cell line deriveddifferent from ethanol induced damage. Previously, it has beenfrom normal gastric mucosa of rat In Vitro Cell Dev Biol Animshown that bile salts induce depolarization of mitochondrial1996;32:259-261membrane potential, which might be due to disturbed 18 Ranta-Knuuttila T, Kiviluoto T, Mustonen H,Puolakkainenoxidative phosphorylation in mitochondria ". In the presentP, Watanabe S, Sato N, Kivilaakso E Migration of primatudy, the possible oxidative damage was not prevented withcultured rabbit gastric epithelial cells requires intact proteikinase C and Ca2+/calmodulin activity. Dig Dis Sci 2002; 47:allopurinol exposure, suggesting that xanthine oxidase is not1008-1014involved in taurocholatc-induced cellular injury19 Watanabe S, Hirose M, Yasuda T, Miyazaki A, Sato N RoleIn conclusion, our results show that deformations ofof actin and calmodulin in migration and proliferation ofthe actin cytoskeleton are involved in the epithelial cellabbit gastric mucosal cells in culture. 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