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Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway

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Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway
  Malonate induces cell death  via  mitochondrial potential collapseand delayed swelling through an ROS-dependent pathway 1 Francisco J. Fernandez-Gomez,  1 Maria F. Galindo,  1 Maria Go ´mez-La ´zaro,  2 Victor J. Yuste, 2 Joan X. Comella,  3 Norberto Aguirre & * ,1,4 Joaquı ´n Jorda ´n 1 Departamento de Ciencias Me´dicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Albacete, Spain; 2 Grup de Neurobiologia Molecular, Departmento de Ciencies Mediques Basiques, Universitat de Lleida, Spain; 3 Departamento de Farmacologı ´a, Facultad de Medicina, Universidad de Navarra, Pamplona, Spain and 4 Centro Regional de Investigaciones Biome ´dicas, Albacete, Spain 1  Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primarytarget, the mitochondrion. 2  Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome  c  (Cyt  c )release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H)stores in brain-isolated mitochondria. 3  Although, mitochondrial potential collapse was almost immediate after malonate addition,mitochondrial swelling was not evident before 15min of drug presence. This latter effect was blockedby cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4  Malonate added to SH-SY5Y cell cultures produced a marked loss of cell viability together withthe release of Cyt  c  and depletion of GSH and NAD(P)H concentrations. All these effects were notapparent in SH-SY5Y cells overexpressing Bcl-xL. 5  When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection affordedby Bcl-xL overexpression was not evident anymore. 6  Taken together, all these data suggest that malonate causes a rapid mitochondrial potentialcollapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacityand leads to mitochondrial swelling. Further permeability transition pore opening and the subsequentrelease of proapoptotic factors such as Cyt  c  could therefore be, at least in part, responsible formalonate-induced toxicity. British Journal of Pharmacology  (2005)  144,  528–537. doi:10.1038/sj.bjp.0706069Published online 17 January 2005 Keywords:  Bcl-xL; cytochrome  c ; mitochondria; malonate; neurodegeneration; ROS; glutathione; mitochondrial permeabilitytransition; cytotoxicity Abbreviations:  A 540 , absorbance at 540nm; BSO, buthionine sulfoximine; COX-IV, cytochrome  c  oxidase subunit IV; CsA,cyclosporin A; Cyt  c , cytochrome  c ; DMEM, Dulbecco’s modified Eagle’s medium; GSH, glutathione; LDH,lactate dehydrogenase;  DC m , mitochondrial potential; mBCl, monochlorobimane; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride; NAD(P)H, nicotinamide adenine dinucleotide coenzyme (NADH/NAD þ ) and itsderivatives (NADPH/NADP þ ); 3-NP, 3-nitorpropionic acid; 6-OHDA, 6-hydroxydopamine; PBS, phosphatebuffered saline; PTP, permeability transition pore; ROS, reactive oxygen species; RR, ruthenium red; TCA,trichloroacetic acid; TMRE, tetramethylrhodamine ethyl ester Introduction Mitochondria are involved in a number of important cellularfunctions, including essential pathways of intermediate metabo-lism, amino-acid biosynthesis, fatty acid oxidation and steroidmetabolism. Of key importance is the role of mitochondria inoxidative phosphorylation and apoptosis. Thus, mitochondriaare considered the headquarters in apoptosis pathways (for areview, see Susin  et al  ., 1998; Kroemer & Reed, 2000; Jordan et al  ., 2003). Many apoptotic stimuli cause either functional ormorphological mitochondrial alterations such as collapse of thetransmembranal potential or swelling. Hallmarks of thesemitochondrial alterations are increased free radical productionand release of cytochrome  c  (Cyt  c ) from mitochondria to thecytosol through the permeability transition pore (PTP) (Kroe-mer & Reed, 2000; Vila & Przedborski, 2003). The Bcl-2 familyof proteins, which is implicated in the regulation of apoptosis bymodulating PTP aperture, comprises members that have eitherantiapoptotic (such as Bcl-2 and Bcl-xL) or proapoptotic (suchas Bax and Bak) effects (Brenner  et al  ., 2000; Gross, 2001). Inthis sense, Bcl-2 or Bcl-xL overexpression has been shown toconfer protection to cells exposed to apoptotic stimuli includingstaurosporine (Yuste  et al  ., 2002), 6-hydroxydopamine(6-OHDA) (Galindo  et al  ., 2004), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride (MPTP) (Yang  et al  ., 1998) or 3-nitorpropio-nic acid (3-NP) (Bogdanov  et al  ., 1999). *Author for correspondence at: Facultad de Medicina, CentroRegional de Investigaciones Biome ´dicas, Universidad de Castilla-LaMancha, Avda. Almansa, s/n, 02006 Albacete, Spain;E-mail: joaquin.jordan@uclm.esPublished online 17 January 2005 British Journal of Pharmacology  (2005) 144,  528–537  &  2005 Nature Publishing Group All rights reserved 0007–1188/05  $  There is substantial evidence indicating that oxidative stressplays a role in the progression of a constellation of neurological disorders. Mitochondria have been proposed tobe the main source of reactive oxygen species (ROS) inneuronal cells. Cells contain antioxidant systems to block ROSoverproduction, including glutathione (GSH) and nicotina-mide adenine dinucleotide coenzyme (NADH/NAD þ ) and itsderivatives (NADPH/NADP þ ) (NAD(P)H), which play acrucial role as part of the primary cellular defence againstoxidative stress. It has been shown that GSH levels in thecerebrospinal fluid decline during aging (Cudkowicz  et al  .,1999). The involvement of GSH in the control of cell death inneurodegenerative diseases is striking and it has been suggestedthat GSH depletion might be an upstream biochemical event inneurodegeneration (Dexter  et al  ., 1989a,b). We and othershave also observed a GSH depletion after different apoptoticstimuli including 6-OHDA (Galindo  et al  ., 2004), veratridine(Jordan  et al  ., 2002), MPTP (Selley, 1998) or malonate(Ehrhart & Zeevalk, 2003).On the other hand, it is clear that impairment of mitochondrial energy metabolism is the key pathogenic factorin a number of neurodegenerative disorders (see review bySchon & Manfredi, 2003). Accordingly, toxins that affectmitochondria are being used as pharmacological tools tomimic several of these diseases. Among others, 3-NP, MPTP,rotenone and malonate are well-established mitochondrialcomplex inhibitors frequently used to investigate the keycellular pathways that provoke neurodegeneration in Parkin-son’s or Huntington’s diseases (Browne & Beal, 2002).Malonate has been shown to cause dose-dependent neuro-toxicity both ‘ in vivo ’ and ‘ in vitro ’ by inhibition of succinatedehydrogenase and depletion of striatal ATP (Beal  et al  ., 1993;Greene & Greenamyre, 1995; Stokes  et al  ., 2001, VanWesterlaak  et al  ., 2001) resulting in neuronal depolarizationand secondary excitotoxicity (Henshaw  et al  ., 1994; Greene &Greenamyre, 1996). In a recent study by Schulz  et al  . (1998), itwas suggested that malonate toxicity involves neurons dyingnot only by secondary excitotoxicity but also by delayedcaspase activation and apoptosis (Schulz  et al  ., 1998). Thus,the exact mechanism by which malonate induces toxicityremains unclear.The aim of this study was to analyse the role played bymitochondria in the mechanisms underlying malonate-inducedcell death. We used isolated mitochondrial preparations tostudy the effect of malonate on this organelle. We presentevidence showing that malonate induces mitochondrial poten-tial collapse and depletion of mitochondrial antioxidantdefence which leads to mitochondrial swelling and releaseof proapoptotic proteins including Cyt  c . The role of theantiapoptotic protein Bcl-xL in these processes is alsoaddressed. Methods Mitochondrial isolation Mitochondria were isolated from the brains of adult Sprague– Dawley rats. All the procedures followed in the present workwere in compliance with the European Community CouncilDirective of 24 November 1986 (86/609/EEC) and wereapproved by the Ethical Committee of the University of Castilla-La Mancha. To exclude that the observed effects weredue to contaminating synaptosomes, we isolated brainmitochondria using a Percoll gradient as previously described(Sims, 1990). Rats were killed by decapitation, forebrains wererapidly removed, chopped and homogenized in ice-coldisolation buffer (225m M  mannitol, 25m M  sucrose, 10m M Hepes, 1m M  K 2 EDTA, pH 7.4 at 4 1 C). The homogenatewas centrifuged at 1330   g  for 3min, and the pellet obtainedwas resuspended and recentrifuged at 1330   g  for 3min. Thepooled supernatants were centrifuged at 21,300   g  for 10min.The pellet was resuspended in 15% Percoll and layered onpreformed gradients (40 and 23%). The Percoll gradients werethen centrifuged at 31,700   g  for 10min. The mitochondrialfraction located at the interface of the lower two layers wasremoved, diluted with isolation buffer and centrifuged at16,700   g  for 10min. The mitocondrial pellet was resuspendedin solution III (215m M  mannitol, 71m M  sucrose, 10m M succinate and 10m M  HEPES, pH 7.4) and kept on ice foranalysis. Permeability transition pore activity Permeability transition pore opening was assayed spectro-photometrically as previously described (Kristal  et al  ., 2000).Specifically, mitochondria were suspended to reach a proteinconcentration of 1mgml  1 in 200 m l of solution containing125m M  KCl, 20m M  Hepes, 2m M  KH 2 PO 4 , I  m M  EGTA, 1m M MgCl 2 , 5m M  malate and 5m M  glutamate with the pH adjustedto 7.08 with KOH. Changes in absorbance at 540nm ( A 540 ),indicating mitochondrial swelling due to PTP opening, weredetermined, after addition of different compounds, using amicroplate reader (BioRad, Hercules, CA, U.S.A.). Initial  A 540 values were D 0.8, and minor differences in the loading of thewells were compensated by representing the data as thefraction of the initial absorbance determination remaining ata given time. Mitochondrial protein concentrations werequantified spectrophotometrically (Micro BCA Protein Re-agent Kit), with bovine serum albumin used as standard. Assay for NAD(P)H levels Levels of NAD(P)H were determined using autofluorescenceas previously described (Rover Jr  et al  ., 1998). The chemicalstability of nicotinamide adenine dinucleotide coenzyme(NADH/NAD þ ) and its derivatives (NADPH/NADP þ ) wereinvestigated by using changes in the UV-visible absorptionspectra of these compounds. We therefore refer to NAD(P)H,indicating the signal derived from either NADH or NADPH,or both. NAD(P)H fluorescence in intact mitochondria(1mgml  1 at 25 1 C) was measured fluorimetrically usingexcitation and emission wavelengths set at 340nm (slit 3nm)and 460nm (slit 5nm), respectively, in a Perkin–Elmer LS50Bluminescence-spectrophotometer using a quartz cell with a1cm optical path as previously described (Rover Jr  et al  .,1998). On the day of the experiment, culture medium wasremoved, cells were washed three times with 1ml of ice-coldphosphate-buffered saline (PBS), scraped into 500 m l 0.2%Triton X-100, centrifuged (800   g , 4 1 C, 5min). Fluorescencewas determined in 300 m l of the cell extract. F.J. Fernandez-Gomez  et al  Malonate induces delayed mitochondrial swelling  529 British Journal of Pharmacology vol 144 (4)  Measurement of GSH  Levels of GSH were determined by using monochlorobimane(mBCl) fluorescence. GSH is specifically conjugated withmBCl to form a fluorescent bimane-GSH adduct, in a reactioncatalysed by glutathione  S  -transferases (Shrieve  et al  ., 1988).The concentration of the bimane-GSH adduct increases duringthe initial 10–12min period of this reaction with first-orderkinetics, before levelling off (Young  et al  ., 1994). Fluorescencelevels at 15min were used as an indication of intracellular GSHcontent, as it has been described previously (Shrieve  et al  .,1988). Culture medium was removed and cells were washedthree times with 1ml PBS (37 1 C) and incubated for 30min at37 1 C in 1ml fresh PBS containing 80 m M  mBCl. Afterincubation, cells were washed twice with ice-cold PBS andscraped in PBS, centrifuged and 300 m l of the extract was usedfor GSH determination. Fluorescence was measured at anexcitation wavelength of 340nm and emission wavelength of 460nm. Protein content was determined by the bicinchoninicacid method. Measurement of cell viability SH-SY5Y cultures were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 2m M L -glutamine,penicillin (20Uml  1 ), streptomycin (5 m gml  1 ), and 15%(volvol  1 ) heat-inactivated fetal calf serum (GIBCO, Gaithers-burg, MD, U.S.A.) as reported previously by Galindo  et al  .(2004). Cells were grown in a humidified cell incubator at 37 1 Cunder a 5% CO 2  atmosphere. For viability experiments, cellswere plated at a density of 4  10 4 cellscm  2 and allowed toattach overnight. Cell viability after malonate additions wasassessed by measurement of lactate dehydrogenase activityaccording to the protocol provided by the manufacturer(Promega). Briefly, the reaction mixture was added toconditioned media and removed from 24-well plate aftercentrifugation at 250   g  for 10min. Absorbance of samples at490nm was measured in a microplate reader (BioRad,Hercules, CA, U.S.A.) after 30min of incubation at roomtemperature. Cyt  c  determinations Immunoblot analysis was performed on extramitochondrialextracts and cytosolic proteins. Briefly, isolated mitochon-drial suspensions were treated with resuspension (control),malonate 1 and 10m M ) for 30min and then centrifuged at15,000r.p.m. for 15min. The supernatants, extramitochon-drial fractions, were precipitated by trichloroacetic acid(TCA) (10%, 4 1 C overnight) and centrifuged (15,000   g ;15min). Pellets were resuspended in 40 m l of loading bufferand boiled for 15min. For culture experiments, cytosolicextracts from control and malonate-treated cultures wereobtained as previously described (Maestre  et al  ., 2003). Cellswere washed once with PBS and collected by centrifugation(2000   g ; 5min). The cell pellet was resuspended in 200 m lof extraction buffer containing (m M ): sucrose 250, Tris–HCl50, EGTA 1, EDTA 1, DTT 1, PMSF 0.1, pH 7.4. Cells werehomogenized in a Teflon-glass homogenizer (five strokes)and, after 15min on ice, the suspension was centrifuged(15,000   g ; 15min). Supernatants, that is, cytosolic fractions,were removed and stored at   80 1 C until analysed by gelelectrophoresis. TCA-samples or 15 m g of cytosolic proteinsfrom control and treated conditions were loaded onto thesame 15% SDS–polyacrylamide gel, separated and trans-ferred to a PVDF membrane, that was incubated with anti-Cyt  c  (1:1000 dilution of rabbit polyclonal IgG, Santa CruzBiotechnology Inc.). The signal was detected using anenhanced chemiluminescence detection kit (Amersham ECLRPN 2106 Kit). Analysis of cytosolic Cyt  c  from SH-SY5Y-Neo  versus  Bcl-xL overexpressing cells were performed on thesame gel and lot for comparative purposes. cytochrome c  oxidase subunit IV (COX-IV) protein levels were usedas mitochondrial protein loading controls by using withantiCOX-IV (BD Biosciences). Analysis of DNA fragmentation The nuclear morphology of cells was studied by using the cell-permeable DNA dye Hoechst 33342. SH-SY5Y cells werecultured on poly- D -lysine-coated glass coverslips. Cells treatedwith malonate (100m M , 24h) were washed twice with PBS,stained with 5 m gml  1 Hoechst 33342 for 5min, and rinsedonce with PBS. Cells were observed under an UV illuminationmicroscope (excitation/emission 350/460nm). Cells withhomogeneously stained nuclei were considered to be viable,whereas the presence of chromatin condensation and/orfragmentation was indicative of apoptosis. Living andapoptotical cells were counted on adjacent fields of eachcoverslips totalling  B 300–450 cells. The percentage of apoptotical cells was determined on three or four coverslipsfor each condition and normalized to parallel controls. Eachindependent coverslip was treated as a single observation, andat least three coverslips were used in each experiment. Theaverage relative percent apoptotical from at least threeseparate cultures was determined. Statistical analysis Statistically significant differences between groups weredetermined by ANOVA followed by a Newman–Keuls  posthoc  analysis. The level of statistical significance was set at P o 0.05. Materials Most chemicals, including malonic acid, cyclosporin A (CsA),vitamin E, catalase, trichloroacetic acid and ruthenium red(RR) were obtained from Sigma (St Louis, MO, U.S.A.).Monochlorobimane, tetramethylrhodamine ethyl ester(TMRE) and Hoechst 33342 were purchased from MolecularProbes, Inc., (Eugene, OR, U.S.A.). The Micro BCA ProteinReagent Kit and Lactate dehydrogenase activity kit camefrom Pierce (Rockford, IL, U.S.A.) and from Promega(Madison, WI, U.S.A.) respectively. Monoclonal anti-Cyt c  was purchased from R&D system (Minneapolis, MN,U.S.A.) and anti-COX-IV from BD Biosciences (Heidelberg,Germany). 530  F.J. Fernandez-Gomez  et al  Malonate induces delayed mitochondrial swelling British Journal of Pharmacology vol 144 (4)  Results Malonate induces a delayed mitochondrial swellingand Cyt  c  release Mitochondria are key players in triggering apoptosis signallingpathways that lead to cell death (Susin  et al  ., 1998). In the firstset of experiments, we investigated whether malonate wouldinduce mitochondrial swelling in isolated mitochondria bymonitoring 540nm absorbance ( A 540 ) decrease, and its effectwas compared to that produced by a control stimuli, CaCl 2 . Asshown in Figure 1 (top panel), malonate induced mitochon-drial swelling. The mitochondria suspension did not respondto malonate as quickly as it did after CaCl 2  additions. Whilemitochondria started to swell right after Ca 2 þ addition,malonate did not significantly modify mitochondrial suspen-sion absorbance at times earlier than 20min. By 10min,mitochondrial suspension absorbance dropped around 5%of control values in malonate-treated mitochondria ( P 4 0.05, n ¼ 6), while Ca 2 þ -induced decrease was around 20% at thistime point ( P o 0.01,  n ¼ 6) (Figure 1a). However, oncemalonate initiated mitochondrial swelling, mitochondrialsuspension absorbance started to drop resulting in similarvalues to those achieved after Ca 2 þ additions at the end of theexperiments (Figure 1b). Consistent with the well-establishedconsequence of mitochondrial swelling, release of Cyt  c  wasevident by 30min after malonate addition (1 and 10m M )(Figure 1c).Next, we were interested in gaining insight into themechanisms underlying malonate-induced mitochondrialswelling. To address this question, we performed a series of experiments using malonate (1m M ) in combination with drugsknown to act through different mechanisms of action. Thepresence of CsA (10 m M ) blocked malonate-induced changes inabsorbance, suggesting the involvement of the PTP (Figure 2).As PTP opening is regulated by the external inhibitory Mg 2 þ -binding site (Bernardi  et al  ., 1993), we added Mg 2 þ at a finalconcentration of 2m M . As expected, Mg 2 þ also abolishedmalonate-induced swelling, confirming the role of PTP. Asshown in Figure 2, blockade of Ca 2 þ influx, in the presence of the mitochondrial Ca 2 þ uptake blocker RR, also inhibitedmalonate-induced mitochondrial swelling. Finally, the role of ROS in malonate-induced mitochondrial swelling was assessedby using the broad lipophylic antioxidant vitamin E, thescavenger enzyme catalase (10UmL  1 ) or GSH (2.5m M ).Pretreatment with these antioxidant agents for 1h caused a 1010Malonate (mM) 0 500 1000 1500 2000 25000. 2 Drug    A    5   4   0    (   N  o  r  m  a   l   i  z  e   d   )   A    5   4   0    (   N  o  r  m  a   l   i  z  e   d   )   A    5   4   0    (   N  o  r  m  a   l   i  z  e   d   ) Time (s)0 1 10 50 100*[Malonate] (mM)CaCl 2 0 1 10 50 100[Malonate] (mM)CaCl 2 *********** —15 kDaCytc abc Figure 1  Malonate induces a delayed mitochondrial swelling.Mitochondrial swelling was followed by measuring changes in  A 540 in mitochondria suspensions. Malonate mediates a loss in absor-bance in isolated mitochondria suspension. Different amounts of malonate, in a final volume of 25 m l, were added at 5min as noted bythe arrow. Final malonate concentrations are expressed at the end of traces. Trace control represents no added drug, and 25 m l of bufferwere added to control for dilution effects. Dashed line representCaCl 2  (75 m M ) addition. Data represent mean values obtained fromone experiment performed in triplicate. Histograms representmeans 7 s.e.m. of changes in absorbance at 600 (a) and 2400s (b)after drug additions from five experiment performed by triplicate.* P o 0.05; *** P o 0.001. (c) Malonate induces Cyt  c  release.Mitochondria were incubated during 30min with resuspension(control) or malonate (1,10m M ) and then centrifuged for 15min at15,000r.p.m. at 4 1 C. Protein precipitated overnight with 10% TCAis shown. Pellets were subjected to polyacrylamide gel electrophor-esis and immunoblot analysis using an antibody that recognizesCyt  c . F.J. Fernandez-Gomez  et al  Malonate induces delayed mitochondrial swelling  531 British Journal of Pharmacology vol 144 (4)  slight but significant blockade of malonate-induced swelling(Figure 2). Malonate induces mitochondrial potential collapse In some apoptotic models, changes in mitochondrial potential( DC m ) take place. To address the plausible effect of malonateon  DC m , we monitored the release of TMRE, a cationicmembrane-permeant fluorescent probe, from preloaded mito-chondria. Under these conditions, total fluorescence of mitochondrial suspension will increase if the organellesdepolarise. Mitochondria responded to malonate releasingTMRE in a concentration-dependent manner, indicating thatmalonate additions result in  DC m  collapse. As shown inFigure 3, the time required for malonate to induce  DC m collapse was shorter than that required to induce mitochon-drial swelling. Malonate disrupts mitochondrial redox state GSH and NAD(P)H belong to the antioxidant systems used bycells to prevent ROS damage. In the next set of experiments,we analysed whether malonate could compromise these twoROS scavenger systems. To address this issue, we treatedmitochondrial suspensions with different concentrations of malonate (1–100m M ) and 15min later, GSH and NAD(P)Hlevels were determined. Our results show that malonatedepletes these two antioxidant agents in a concentration-dependent manner (Figure 4), suggesting that it causes enoughROS to overwhelm mitochondrial antioxidant capacity. Bcl-xL overexpression blocks malonate-induced cell death Bcl-xL is an antiapototic protein located in the mitochondriathat has been shown to block cell death under severalparadigms, including those mediated by ROS (Vander Heiden et al  ., 1997). In the next set of experiments, we used theneuroblastoma cell line SH-SY5Y to investigate the role of thisantiapoptotic protein in malonate-induced toxicity. Cellcultures were either stably transfected with DNA containingthe open reading frame of Bcl-xL subcloned into pcDNA3(SH-SY5Y/Bcl-xL) or with empty/pcDNA3 (SH-SY5Y/Neo)(Yuste  et al  ., 2002). We used the lactate dehydrogenase (LDH)cell viability assay method to analyse the effects of malonate(0.1–100m M ) on SH-SY5Y cell viability. The lower concentra-tions of malonate tested, up to 10m M , did not compromise cellviability, while higher concentrations (50–100m M ) resulted ingross morphological changes (data not shown). Staining of these cells with the DNA-binding dye, Hoechst 33342, showedchromatin condensation and fragmentation as comparedto control cells (100m M , about 40%, Figure 5b). Theoverexpression of Bcl-xL protein protected SH-SY5Y cells 0 500 1000 1500 2000 25000. EGSHRR    A    5   4   0    (   N  o  r  m  a   l   i  z  e   d   ) 1 mM MalonateTime (s) C    o   n    t    r    o   l     C    s   A    R    R    M      g    C    a    t    a    l     a    s   e    V     i     t     E     G    S     H      0.6************    A    5   4   0    (   N  o  r  m  a   l   i  z  e   d   ) ab Figure 2  Role of calcium and ROS in malonate-induced mito-chondrial swelling. Malonate addition (1m M /500 m g protein; arrow)induces mitochondrial swelling in isolated mitochondria in an PTP-sensitive manner. Mitochondrial suspensions were incubated during15min with Cyclosporin A (CsA, 10 m M ), Magnesium (Mg, 2m M ),Ruthenium Red (RR, 5 m M ) or 1h with Vitamin E (Vit E, 50 m M ),Catalase (Cat, 10UmL  1 ) and Glutathione (GSH, 2.5m M ) prior toaddition of malonate. Control nontreated mitochondria are alsoshown. Lines represent mean values of one experiment performed intriplicate. Histogram panels represent mean values 7 s.e.m. of normalised  A 540  at 2200s from at least five different mitochondrialpreparations. * P o 0.05; *** P o 0.001 Students unpaired  t -test. 0 100 200 300 400 500 600100200300800900MitMalonate15010Control100    T   M   R   E   (   A .   F .   U .   ) Time (s) Figure 3  Malonate induces mitochondrial potential collapse. Mi-tochondrial membrane potential was measured by using TMRE.Addition of mitochondria (Mit, 500protein m g) caused a decrease influorescence intensity due to TMRE uptake. Malonate (1–100m M )was added at 240s. Final malonate concentrations are expressed atthe end of traces. Data are expressed as mean values obtained fromone experiment performed in triplicate. Similar data were found inat least four different experiments. 532  F.J. Fernandez-Gomez  et al  Malonate induces delayed mitochondrial swelling British Journal of Pharmacology vol 144 (4)
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