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• W2093485207 abstract "In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect ∼40%, IC50, 12 μm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60–70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 μm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased ΔΨ, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks. In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect ∼40%, IC50, 12 μm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60–70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 μm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased ΔΨ, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks. It is generally believed that the mitochondrial electron transfer chain is one of the major cellular generators of reactive oxygen species (ROS), 1The abbreviations used are: ROSreactive oxygen speciesHRPhorseradish peroxidaseMOPS4-morpholinepropanesulfonic acidGRglutathione reductaseGPXglutathione peroxidaseFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneRRruthenium red.1The abbreviations used are: ROSreactive oxygen speciesHRPhorseradish peroxidaseMOPS4-morpholinepropanesulfonic acidGRglutathione reductaseGPXglutathione peroxidaseFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneRRruthenium red. which include superoxide (O2·¯), H2O2, and the hydroxyl free radical OH· (1Loschen G. Flohe L. Chance B. FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (444) Google Scholar, 2Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1207) Google Scholar, 3Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4780) Google Scholar). The mitochondrial production of ROS is supposed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (4Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2885) Google Scholar, 5Jenner P. Trends Neurosci. 2001; 24: 245-247Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Furthermore, evidence has been presented recently that diabetic complications may be secondary to hyperglycemia-induced generation of ROS by mitochondria (6Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (6928) Google Scholar). It was found that some electrons leak out from accumulating unstable intermediates of the respiratory chain, performing a partial reduction of molecular oxygen, generating O2·¯, which is in turn rapidly dismutated by Mn-superoxide dismutase to H2O2 (7Loschen G. Azzi A. Flohe L. FEBS Lett. 1974; 42: 68-72Crossref PubMed Scopus (498) Google Scholar, 8Boveris A. Cadenas E. FEBS Lett. 1975; 54: 311-314Crossref PubMed Scopus (329) Google Scholar). There is, however, some controversy as to whether mitochondria are an important source of ROS under physiological and pathological conditions (9Forman H.J. Azzi A. FASEB J. 1997; 11: 374-375Crossref PubMed Scopus (109) Google Scholar, 10Staniek K. Nohl H. Biochim. Biophys. Acta. 2000; 1460: 268-275Crossref PubMed Scopus (133) Google Scholar). H2O2 production has been ascribed to Complex I and Complex III of the respiratory chain, being induced in deenergized mitochondria by succinate in the presence of antimycin or by NAD-linked substrates in the presence of rotenone (11Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1349) Google Scholar, 12Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1061) Google Scholar, 13Zoccarato F. Cavallini L. Deana R. Alexandre A. Biochem. Biophys. Res. Comm. 1988; 154: 727-734Crossref PubMed Scopus (43) Google Scholar, 14St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Some studies were conducted more recently in coupled mitochondria in the absence of respiration inhibitors. In these, it was shown that H2O2 is produced during controlled (state 4) succinate oxidation (succinate feeds electrons to Complex II, which in turn reduces Complex III) and that such production is abolished by decreasing Δψ (i.e. during ADP-stimulated respiration, state 3), and oddly, also in the presence of the Complex I inhibitor rotenone (15Cino M. Del Maestro R.F. Arch. Biochem. Biophys. 1989; 269: 623-638Crossref PubMed Scopus (125) Google Scholar, 16Hansford R.G. Hogue B.A. Mildaziene V. J. Bioenerg. Biomembr. 1997; 29: 89-95Crossref PubMed Scopus (394) Google Scholar, 17Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1367) Google Scholar, 18Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (958) Google Scholar, 19Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar, 20Votyakova T.V. Reynolds I.J. J. Neurochem. 2001; 79: 266-277Crossref PubMed Scopus (507) Google Scholar). These results are a strong evidence that succinate-supported H2O2 production in coupled mitochondria does not derive from Complex III (as with antimycin) but rather from energy-dependent reverse electron transfer to Complex I and autooxidation of some carrier therein, either iron-sulfur centers (21Herrero A. Barja G. Mech. Ageing Dev. 1997; 98: 95-111Crossref PubMed Scopus (175) Google Scholar, 22Genova M.L. Ventura B. Giuliano G. Bovina C. Formiggini G. Parenti-Castelli G. Lenaz G. FEBS Lett. 2001; 505: 364-368Crossref PubMed Scopus (259) Google Scholar, 23Lenaz G. Bovina C. D'Aurelio M. Fato R. Formiggini G. Genova M.L. Giuliano G. Pich M.M. Paolucci U. Castelli G.P. Ventura B. Ann. N. Y. Acad. Sci. 2002; 959: 199-213Crossref PubMed Scopus (328) Google Scholar) or the active site flavin (18Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (958) Google Scholar) whose reduction is prevented by rotenone when the electrons originate from succinate. Some experiments were reported using NAD-dependent substrates, and it appeared that little or no H2O2 was produced in these conditions during state 4 respiration (19Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar, 20Votyakova T.V. Reynolds I.J. J. Neurochem. 2001; 79: 266-277Crossref PubMed Scopus (507) Google Scholar).In this study, we analyze a previously undescribed aspect of H2O2 handling by mitochondria, namely their ability to remove exogenously supplied H2O2. We demonstrate that H2O2 is actively removed by respiring mitochondria and identify a physiologically significant inhibitory control exerted by Ca2+ on such removal. We also report on the properties and controls of mitochondrial production of H2O2 and figure out the balance between H2O2-removing and H2O2-producing processes in non-inhibited respiring mitochondria. The possibility that mitochondria may be engaged in active removal of H2O2 has been suggested some years ago (24Sandri G. Panfili E. Ernster L. Biochim. Biophys. Acta. 1990; 1035: 300-305Crossref PubMed Scopus (76) Google Scholar).EXPERIMENTAL PROCEDURESReagents—Scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one), bovine serum albumin (essentially fatty acid free), the Ca2+ indicator Fura-2, pentapotassium salt, and glutathione reductase (EC 1.6.4.2) were supplied from Sigma. Peroxidase from horseradish (HRP) (grade I, EC 1.11.1.7) was from Roche Applied Science. The Ca2+ fluorescent dye Calcium-Green-5N was from Molecular Probes. All other reagents were of analytical grade.Preparation of Rat Brain Mitochondria—The cerebral cortices of two 6–7-week-old rats were rapidly removed into 20 ml if ice-cold isolation medium (320 mm sucrose, 5 mm HEPES, 0.5 mm EDTA, 0.05 mm EGTA, pH 7.3) and homogenized. The homogenate was centrifuged at 900 × g for 5 min at 4 °C. The supernatant was centrifuged at 8500 × g for 10 min, and the resulting pellet was resuspended in 1 ml of isolation medium. This was layered on a discontinuous gradient consisting of 4 ml of 6% Ficoll, 1.5 ml of 9% Ficoll, and 4 ml of 12% Ficoll (all prepared in isolation medium) and centrifuged at 75,000 × g for 30 min. The myelin, synaptosomal, and free mitochondrial fractions formed respectively above the 6% layer, as a doublet within the 9% layer and as a pellet. The pellet was resuspended in 250 mm sucrose, 10 mm K-HEPES, pH 7.2, and centrifuged at 8500 × g for 15 min before being resuspended in this last medium to 10–20 mg of protein/ml by the Gornall protein assay. The mitochondria were well coupled as judged by the increment of the oxygen consumption rate upon the addition of ADP (respiratory control ratio), which was between 3.5 and 7.0 with glutamate/malate as substrates. Oxygen consumption was monitored with a Clark-type oxygen electrode.Standard Incubation Procedure—Mitochondria (0.4–0.6 mg/ml) were incubated at 30 °C in a medium containing 125 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgCl2, 500 μg/ml defatted bovine serum albumin, 20 mm MOPS (pH 7.2, adjusted with KOH).Hydrogen Peroxide Measurements—Mitochondrial H2O2 removal and H2O2 release were assessed by the oxidation of scopoletin by horseradish peroxidase in the presence of H2O2 (1Loschen G. Flohe L. Chance B. FEBS Lett. 1971; 18: 261-264Crossref PubMed Scopus (444) Google Scholar, 25Boveris A. Nartino E. Stoppani A.O.M. Anal. Biochem. 1977; 80: 145-158Crossref PubMed Scopus (105) Google Scholar). Scopoletin fluorescence was monitored at excitation and emission wavelengths of 365 and 450 nm, respectively, on a Shimadzu RL-5000 spectrofluorometer in a stirred cuvette thermostatted at 30 °C. For measurements of H2O2 consumption, H2O2 was added to respiring mitochondria generally at 3 min of incubation, and residual H2O2 was determined by the addition of scopoletin (5 μm) followed 20 s later by HRP (15 μg/ml, 3.75 units). The interval between scopoletin and HRP was necessary to allow stabilization of the fluorescence signal after scopoletin. In the absence of HRP, the scopoletin fluorescence remained unmodified for over a minute. HRP promoted an immediate fluorescence decrease, which was proportional to H2O2. After reaching equilibrium, each trace was calibrated with standard H2O2 additions. The H2O2 calibration scale is slightly non-linear, and the overall fluorescence decrease in each trace was compared with a calibration scale obtained by the addition of known amounts of H2O2 to mitochondria that were treated with HRP plus scopoletin. The addition of HRP to a scopoletin containing medium in the absence of H2O2 gave a small non-specific fluorescence variation, which was subtracted from each curve to obtain the correct measurements. The zero time H2O2 concentration was determined by adding H2O2 to mitochondria that were already treated with HRP and scopoletin. Alternatively, H2O2 was supplemented to uncoupled scopoletin-treated mitochondria in the presence of malonate, and HRP was added few s later. No fluorescence decrease was detected in the absence of HRP.For measurements of H2O2 production, mitochondria were supplemented with scopoletin and HRP at 2 min of incubation followed by the addition of substrates and inhibitors as detailed in the figures. At the end of the experiment, each trace was calibrated with standard H2O2.Measurement of Mitochondrial NAD(P)H—The mitochondrial NADP reduction was monitored by recording its relative fluorescence intensity at 348 nm (excitation) and 464 nm (emission) with a Shimadzu RL-5000 spectrofluorometer in a stirred cuvette thermostatted at 30 °C. Mitochondria were suspended at 0.4 mg/ml in sucrose-containing medium (250 mm mannitol, 75 mm sucrose, 5 mm MgCl2, 2.5 mm KH2PO4, 500 μg/ml defatted bovine serum albumin, 5 mm MOPS, pH 7.3, adjusted with KOH). Substrates were added at 90 s of incubation. Since the baseline fluorescence trace without substrates exhibited a decline with time, which was also more accentuated in the absence of EGTA, the fluorescence traces reported in Fig. 7 represent the difference fluorescence between the trace with substrate and a parallel trace (with or without EGTA) without substrateGlutathione Reductase (GR) and Glutathione Peroxidase (GPX) Activities—For measurements of GR and GPX activities, mitochondrial pellets were resuspended in a small volume of H2O and subjected to 2–3 cycles of freeze-thawing. Aliquots of disrupted mitochondria (∼0.9 mg) were suspended in 1.6 ml of incubation medium (pH 7.35). NADPH was supplemented at 125 μm. To monitor the combined GR and GPX activities, NADPH oxidation, measured at 345 nm (excitation) and 450 nm (emission), was initiated by the addition of GSH (1 mm) and H2O2 (as detailed in the figures) (Scheme 1).Scheme 1GR activity was monitored as detailed in the legend for Fig. 8. GPX activity was measured by following NADPH oxidation with GSH and H2O2 and the further presence of commercial dialyzed GR (0.4 μg/1.6 ml:0.4 units/1.6 ml).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Other Assays—The mitochondrial glutathione was determined in mitochondrial pellets after incubation, by the method of Tietze et al. (26Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5502) Google Scholar) as modified by Xia et al. (27Xia Y. Hill K.E. Burk R.F. J. Nutr. 1985; 115: 733-742Crossref PubMed Scopus (66) Google Scholar) as in Ref. 28Zoccarato F. Deana R. Cavallini L. Alexandre A. Eur. J. Biochem. 1989; 180: 473-478Crossref PubMed Scopus (19) Google Scholar. Free Ca2+ concentrations in the incubation medium were calculated using the Ca2+-sensitive fluorescent dye fura-2, pentapotassium salt (with excitation and emission wavelengths set at 340 and 505 nm, respectively; KD 320 nm) and the fluorescent dye Calcium-Green-5N (with excitation and emission wavelengths set at 505 and 535 nm, respectively; KD 14 μm). The use of the low affinity Ca2+ indicator Calcium-Green-5N and the high affinity Ca2+ indicator fura-2 (and pentapotassium salt) can give an indication of the magnitude of free Ca2+ concentration in the incubation medium. Δψ was estimated using fluorescence quenching of the cationic dye safranine O, which is accumulated and quenched inside energized mitochondria (29Akerman K.E.O. Wikstrom M.K.F. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (665) Google Scholar). The excitation wavelength was 495, and the emission wavelength was 586 nm, and the dye concentration used was 2 μm.RESULTSIn the first group of experiments, we investigated the fate of exogenous H2O2, supplied in small amounts to brain mitochondria during controlled (state 4) or ADP-stimulated (state 3) respiration. As shown in Fig. 1, exogenous H2O2 was removed slowly in the absence of respiratory substrates. In these experiments, malonate, which by inhibiting succinate dehydrogenase limits the cycling of endogenous substrates, and an uncoupler to minimize NADPH generation via energy-dependent transhydrogenase, were generally also included (Fig. 1). This background removal may depend on contaminating catalase or the non-specific action of heme proteins. When controlled (state 4) respiration was activated with glutamate/malate, which feed electrons to Complex I (NADH dehydrogenase) of the respiratory chain, the rate of exogenous H2O2 removal was substantially increased. In a series of experiments, the net removal rate was 6.4 ± 0.6 nmol × min–1 × mg–1, mean ± S.E., n = 7. Coupled respiration leads to extensive NADP reduction via energy-dependent transhydrogenase; in turn, NADPH promotes GSSG reduction via GR, and the GSH thus formed activates the GPX-mediated H2O2 removal. The GR/GPX system is considered to account for most of the mitochondrial peroxidase activity. These results show that respiring mitochondria are net removers, rather than producers, of H2O2 (see Scheme 2).Fig. 1Respiration-supported removal of H2O2. Mitochondria (0.85 mg/1.6 ml) were incubated in standard incubation medium containing EGTA (375 μm). H2O2 (8 nmol) was added at 2 min. Residual H2O2 was determined at selected times as detailed under “Experimental Procedures.” Further additions are as follows: malonate (0.3 mm) and FCCP (3 μm) (○); glutamate (2 mm) and malate (2 mm) (•); succinate (2 mm)(▵); succinate followed by rotenone (1.5 μm) added 20 s prior to H2O2 (□); succinate, glutamate, and malate (▪); succinate followed by ruthenium red (1 μm) added 20 s prior to H2O2 (▴). Data points from a single experiment are representative of at least seven independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 2Succinate (succ) oxidation (1) generates Δψ, which drives reverse electron flow from NAD to NADH at Complex I (2) and NADH-supported NADP reduction via energy dependent transhydrogenase (3). Glutamate plus malate (glut/mal) generate NADH, whose oxidation also generates Δψ and NADPH. NADPH promotes GSSG reduction via GR (4); in, turn GSH promotes H2O2 removal via GPX (5). Reactions 4 and 5 are Ca2+-inhibited. Succinate-induced reverse electron flow at Complex I is accompanied by intramitochondrial generation of O2·¯. O2·¯ production is inhibited by rotenone, Ca2+, RR, glutamate plus malate, and decreased Δψ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next studied H2O2 removal using succinate as the substrate. Succinate feeds electrons to CoQ of the inner mitochondrial membrane, via Complex II. Also, with succinate, NAD(P) is highly reduced, due to reverse electron transfer through Complex I to NAD followed by NADP reduction via energy-dependent transhydrogenase (Refs. 16Hansford R.G. Hogue B.A. Mildaziene V. J. Bioenerg. Biomembr. 1997; 29: 89-95Crossref PubMed Scopus (394) Google Scholar and 18Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (958) Google Scholar and see below). Despite the similarity of the redox state of the NADP pool with succinate and glutamate/malate, the removal of exogenous H2O2 was ∼50% slower with succinate (6.4 ± 0.6 (with glutamate/malate) versus 3.3 ± 0.4 (with succinate) nmol of H2O2 removed/mg × min, mean ± S.E., n = 5). However, if rotenone was supplied immediately prior to H2O2 during succinate oxidation, the succinate-supported H2O2 removal increased, to a level comparable with that with glutamate/malate (Fig. 1). Thus, manipulating the activity of Complex I, which lies uphill of the entry point of electrons from succinate, affects the activity of mitochondrial H2O2 removal. This result is reminiscent of the finding that succinate-supported H2O2 production is inhibited by rotenone (15Cino M. Del Maestro R.F. Arch. Biochem. Biophys. 1989; 269: 623-638Crossref PubMed Scopus (125) Google Scholar, 16Hansford R.G. Hogue B.A. Mildaziene V. J. Bioenerg. Biomembr. 1997; 29: 89-95Crossref PubMed Scopus (394) Google Scholar, 17Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1367) Google Scholar, 18Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (958) Google Scholar, 19Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar, 20Votyakova T.V. Reynolds I.J. J. Neurochem. 2001; 79: 266-277Crossref PubMed Scopus (507) Google Scholar).When the experiments were performed under state 3 conditions, i.e. in the presence of ADP, the net glutamate/malate-supported peroxide removal was slightly slower than in state 4 (5.3 ± 0.5 nmol × min–1 × mg–1, mean ± S.E., n = 5). The succinate-supported removal in state 3 was just about the same as in state 4. In the presence of the uncoupler FCCP, H2O2 removal was strongly decreased. Some residual activity was, however, still clearly evident with glutamate/malate (Table I).Table IH2O2 removal by respiring mitochondria in different metabolic conditionsH2O2 removalnmol/mg × minMalonate and FCCP0.3 ± 0.2Glutamate/malate and EGTA6.7 ± 0.6Glutamate/malate, EGTA, and ADP (1.5 mm)5.6 ± 0.5Glutamate/malate, EGTA, and FCCP2.6 ± 0.6Glutamate/malate1.4 ± 0.4Glutamate/malate, EGTA; ruthenium red at 45 s, CaCl2 at 1 min6.5 ± 0.5Succinate, EGTA3.2 ± 0.4Succinate, EGTA, and ADP3.5 ± 0.4Succinate, EGTA, and FCCP0.9 ± 0.4Succinate, EGTA, and rotenone5.4 ± 0.5Succinate, EGTA, and glutamate/malate4.8 ± 0.5Succinate1.2 ± 0.4 Open table in a new tab Ca2+and Arsenite Inhibit the Glutamate-Malate-supported H2O2 Removal—The experiments of Fig. 1 were performed in the presence of EGTA. Surprisingly, omitting EGTA from the incubation medium reduced by about 80% the glutamate/malate-supported net rate of measured exogenous H2O2 removal (Fig. 2). Such inhibition was due to Ca2+ contamination of the incubation medium (∼6 μm). In fact, a similar level of inhibition was observed also in EGTA-containing media supplied with enough Ca2+ to increase its free concentration to the low μm range, thus ruling out an effect of EGTA per se or of some other contaminating ion. Furthermore, Ca2+ influx into the mitochondria was required for inhibition to occur since the Ca2+ inhibition of exogenous H2O2 removal was prevented by ruthenium red (RR), an inhibitor of the Ca2+ uniporter, supplemented in the presence of EGTA and prior to Ca2+ addition. As will be reported below, the Ca2+ effect depends on the Ca2+ inhibition of the GR/GPX system and represents a novel unanticipated control of the mitochondrial handling of ROS.Fig. 2Ca2+ and arsenite inhibit the respiration-supported removal of H2O2. Mitochondria were incubated in standard incubation medium. Further additions are as follows: malonate and FCCP (○); glutamate and malate (•); glutamate/malate, EGTA, and CaCl2 (375 μm)(▴); glutamate/malate and EGTA followed by ruthenium red at 45 s and CaCl2 at 1 min(▵); glutamate/malate, EGTA, and arsenite (50 μm) (□); glutamate/malate, arsenite, EGTA, and CaCl2 (▪). Data points from a single experiment are representative of at least five independent experiments. Inset, dose-response effect of the arsenite inhibition of glutamate/malate-supported H2O2 removal. EGTA is present. The data ± S.E. are collected from three preparations, with at least four determinations per each arsenite concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The glutamate/malate-supported removal of exogenous H2O2 in the presence of EGTA was partially inhibited by the vicinal thiol reagent arsenite (Fig. 2). The arsenite-sensitive component represents about 40% of the overall peroxidase activity. The half-maximal inhibitory concentration of arsenite was ∼12 μm (Fig. 2, inset). Arsenite was without effect on the rate of glutamate/malate oxidation. The highest inhibition (over 90%) of mitochondria-associated peroxidase activity was obtained when Ca2+ and arsenite were present together (Fig. 2). An overall picture of the H2O2 removal activity and of some relevant controls is reported in Table I.Succinate-supported H2O2 Production and Its Controls—We investigated the possibility that the different rates of exogenous H2O2 removal with glutamate/malate and succinate in the presence of EGTA as described above could be related to different rates of H2O2 production by the mitochondria with different substrates and in different metabolic situations, leading to a competition between external and internal H2O2 for the mitochondrial peroxidase(s). Peroxide production in coupled mitochondria was studied in the presence of HRP and scopoletin, providing a trap system that removes H2O2 immediately upon reaching the extramitochondrial space. In the absence of a suitable trap, no H2O2 accumulation took place. This may be expected in view of the new finding that mitochondria are net removers of H2O2. As shown in Fig. 3A (trace a), coupled mitochondria respiring on succinate in the presence of EGTA released H2O2. On the contrary, H2O2 release with glutamate/malate was hardly detectable (trace d). The succinate-dependent H2O2 was removed by rotenone and during state 3 respiration, which decreases Δψ (traces f and e) as already described by others. Furthermore, H2O2 release was prevented if EGTA was omitted (or in the presence of EGTA and enough Ca2+ to reach a low μm free Ca2+ concentration) (trace c). Such Ca2+ inhibition occurred without affecting ΔΨ, as monitored following safranine fluorescence (Fig. 4), and appears to represent a direct action of Ca2+ on the site of superoxide production. Also RR inhibited H2O2 release, in the presence of EGTA, apparently via a direct interaction with the superoxide production site at Complex I (Fig. 3A, trace b). Furthermore, H2O2 release was strongly depressed by glutamate/malate, probably because they decrease reverse electron flow from succinate (trace g). The latter effect increased with decreasing succinate concentration, Fig. 3C.Fig. 3H2O2 production by respiring mitochondria. Mitochondria (0.7 mg/1.6 ml) were incubated in standard medium. Scopoletin (scop), HRP, and substrates (subs) were added as indicated. A, succinate (2 mm) and EGTA (trace a); succinate and EGTA, RR added at the arrow (trace b); succinate, no EGTA (trace c); glutamate/malate, EGTA (trace d); succinate, EGTA, and ADP (trace e); succinate, EGTA, and rotenone (trace f); succinate, glutamate/malate, EGTA (trace g). B, same as panel A, with arsenite (50 μm). C, succinate (0.6 mm) EGTA (trace a); succinate, EGTA, and glutamate/malate (trace b). D, same as panel C, with arsenite. Traces are representative of duplicate traces from at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The mitochondrial membrane potential in the absence and presence of EGTA. Mitochondria (0.7 mg/1.6 ml) were treated with 3 μm safranin (S) as indicated and supplemented where indicated (subs) with glutamate/malate (traces a and b) or succinate (traces c and d) in the absence (traces a and c) and presence (traces b and d) of EGTA. FCCP (F) was 3 μm. Typical traces are reported, representative of duplicate experiments from two independent preparations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is noticeab" @default.
• W2093485207 created "2016-06-24" @default.
• W2093485207 creator A5046180256 @default.
• W2093485207 creator A5048853036 @default.
• W2093485207 creator A5090075910 @default.
• W2093485207 date "2004-02-01" @default.
• W2093485207 modified "2023-10-16" @default.
• W2093485207 title "Respiration-dependent Removal of Exogenous H2O2 in Brain Mitochondria" @default.
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