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Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês

10.1074/jbc.m701589200

1083-351X

Mauro Salvi, Valentina Battaglia, Anna Maria Brunati, Nicoletta La Rocca, Elena Tibaldi, Paola Pietrangeli, Lucia Marcocci, Bruno Mondovı̀, Carlo Rossi, Antonio Toninello,

Metabolomics and Mass Spectrometry Studies

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue. Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue. Many human diseases, including cancer and other pathologies associated with aging, such as arteriosclerosis and cataracts, are related to mitochondrial dysfunctions provoked by reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; ATZ, aminotriazole; Gpx, glutathione peroxidase; NEM, N-ethylmaleimide; MAO, monoamine oxidase; MPT, mitochondrial permeability transition; PMCA, plasma membrane Ca2+-ATPase; RLM, rat liver mitochondria; Tbh, tert-butylhydroperoxide; TNF-α, tumor necrosis factor-α; TPP+, tetraphenylphosphonium. 2The abbreviations used are: ROS, reactive oxygen species; ATZ, aminotriazole; Gpx, glutathione peroxidase; NEM, N-ethylmaleimide; MAO, monoamine oxidase; MPT, mitochondrial permeability transition; PMCA, plasma membrane Ca2+-ATPase; RLM, rat liver mitochondria; Tbh, tert-butylhydroperoxide; TNF-α, tumor necrosis factor-α; TPP+, tetraphenylphosphonium. (1Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7202) Google Scholar). In this regard, the so-called free radical theory of aging has been proposed (2Afanas'ev I.B. Biogerontology. 2005; 6: 283-290Crossref PubMed Scopus (37) Google Scholar). ROS are highly reactive and may be extremely toxic in biological systems, as they attack a variety of molecules, including proteins, polyunsaturated lipids, and nucleic acid (3Tappel A.L. Fed. Proc. 1973; 32: 1870-1874PubMed Google Scholar), causing the cell to die by apoptosis or necrosis. In physiological conditions, 1-2% of molecular oxygen consumption during mitochondrial respiration undergoes incomplete reduction by single electrons to form superoxide anion ( O2ċ¯) at the level of NADH-ubiquinone reductase (complex I) and ubiquinol-cytochrome c reductase (complex III). These two segments of the respiratory chain generate the superoxide radical by autoxidation of reduced flavin and by transferring an electron from reduced ubisemiquinone to molecular oxygen, respectively (4Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar). Superoxide is rapidly converted to hydrogen peroxide by mitochondrial superoxide dismutase, which then produces the highly reactive hydroxyl radical (OH·) by interacting with transition metal ions (Fe2+) of the respiratory complexes (Fenton reaction), unless H2O2 is removed by the action of glutathione peroxidase (Gpx) or catalase (see below).In physiological conditions, the primary defense against superoxide anion and hydrogen peroxide in mitochondria not containing catalase is performed by the concerted action of the above mentioned superoxide dismutase and Gpx. However, despite the activity of these enzymes, significant amounts of H2O2 can diffuse out from mitochondria to cytosol, where detoxification occurs through cytosol Gpx or by peroxisome catalase. In pathological conditions, e.g. during inflammation, hyperoxia (5Freeman B.A. Crapo J.D. Lab. Investig. 1982; 47: 412-426PubMed Google Scholar), or chemotherapy (6Davies K.J. Doroshow J.H. J. Biol. Chem. 1986; 261: 3060-3067Abstract Full Text PDF PubMed Google Scholar), i.e. in the presence of large amounts of ROS, catalase becomes the most important scavenger of H2O2 in the cytosol. These conditions may be even more serious in mitochondria, as also observed in "in vitro" investigations with the organelle treated with monoamines (7Marcocci L. De Marchi U. Salvi M. Micella Z.G. Nocera S. Agostinelli E. Mondovi B. Toninello A. J. Membr. Biol. 2002; 188: 23-31Crossref PubMed Scopus (32) Google Scholar), flavones (8Hodnick W.F. Milosavljevic E.B. Nelson J.H. Pardini R.S. Biochem. Pharmacol. 1998; 37: 2607-2611Crossref Scopus (188) Google Scholar), isoflavones (9Salvi M. Brunati A.M. Clari G. Toninello A. Biochim. Biophys. Acta. 2002; 1556: 187-196Crossref PubMed Scopus (84) Google Scholar), and cyclic triterpenes (10Salvi M. Fiore C. Armanini D. Toninello A. Biochem. Pharmacol. 2003; 66: 2375-2379Crossref PubMed Scopus (63) Google Scholar, 11Fiore C. Salvi M. Palermo M. Sinigaglia G. Armanini D. Toninello A. Biochim. Biophys. Acta. 2004; 1658: 195-201Crossref PubMed Scopus (61) Google Scholar). By interacting with transition metals of the respiratory chain, these compounds produce H2O2 and the highly toxic hydroxyl radical. These species, besides causing peroxidation of the phospholipid bilayer, with consequent severe damage to membrane integrity (12Kowaltowski A.J. Vercesi A.E. Free Radic. Biol. Med. 1999; 26: 463-471Crossref PubMed Scopus (699) Google Scholar), can also induce mitochondrial permeability transition (9Salvi M. Brunati A.M. Clari G. Toninello A. Biochim. Biophys. Acta. 2002; 1556: 187-196Crossref PubMed Scopus (84) Google Scholar, 10Salvi M. Fiore C. Armanini D. Toninello A. Biochem. Pharmacol. 2003; 66: 2375-2379Crossref PubMed Scopus (63) Google Scholar, 11Fiore C. Salvi M. Palermo M. Sinigaglia G. Armanini D. Toninello A. Biochim. Biophys. Acta. 2004; 1658: 195-201Crossref PubMed Scopus (61) Google Scholar, 13Salvi M. Fiore C. Battaglia V. Palermo M. Armanini D. Toninello A. Endocrinology. 2005; 146: 2306-2312Crossref PubMed Scopus (30) Google Scholar, 14Battaglia V. Salvi M. Toninello A. J. Biol. Chem. 2005; 280: 33864-33872Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) with release of cytochrome c and activation of the pro-apoptotic cascade. The presence of catalase is of great importance, as its scavenging of H2O2 protects these organelles against the above damaging effects (15Bai J. Cederbaum A.I. Biol. Signals Recept. 2001; 10: 189-199Crossref PubMed Scopus (109) Google Scholar). The problem of the presence of catalase in mitochondria was examined many years ago by Neubert et al. (16Neubert D. Wojtczak A.B. Lehninger A.L. Proc. Natl. Acad. Sci. U. S. A. 1962; 48: 1651-1658Crossref PubMed Scopus (69) Google Scholar), but so far the presence of this enzyme has been clearly demonstrated only in rat heart mitochondria (17Radi R. Turrens J.F. Chang L.Y. Bush K.M. Crapo J.D. Freeman B.A. J. Biol. Chem. 1991; 266: 22028-22034Abstract Full Text PDF PubMed Google Scholar). More recently, it has been demonstrated that yeast catalase A, which contains two peroxisomal targeting signals, can also enter mitochondria, although the enzyme lacks a classical mitochondrial import sequence (18Petrova V.Y. Drescher D. Kujumdzieva A.V. Schmitt M.J. Biochem. J. 2004; 380: 393-400Crossref PubMed Scopus (95) Google Scholar). The key role that a possible mitochondrial catalase may play in oxidant defense has been demonstrated by several research groups, by experimentally targeting catalase to mitochondria. In these studies, the introduction of catalase into mitochondria provides better protection than cytosol expression against H2O2-induced injury (19Arita Y. Harkness S.H. Kazzaz J.A. Koo H.C. Joseph A. Melendez J.A. Davis J.M. Chander A. Li Y. Am. J. Physiol. 2005; 290 (-L986): L978Google Scholar), oxidative damage, and mitochondrial DNA deletion, and in extending the murine life span (20Schriner S.E. Linford N.J. Martin G.M. Treuting P. Ogburn C.E. Emond M. Coskun P.E. Ladiges W. Wolf N. Van Remmen H. Wallace D.C. Rabinovitch P.S. Science. 2005; 308: 1909-1911Crossref PubMed Scopus (1343) Google Scholar). In light of the importance of these results and considering the general opinion that catalase is present only in heart mitochondria (15Bai J. Cederbaum A.I. Biol. Signals Recept. 2001; 10: 189-199Crossref PubMed Scopus (109) Google Scholar, 17Radi R. Turrens J.F. Chang L.Y. Bush K.M. Crapo J.D. Freeman B.A. J. Biol. Chem. 1991; 266: 22028-22034Abstract Full Text PDF PubMed Google Scholar), the aim of this study is to ascertain if catalase is constitutively present in the mitochondria of liver, a multifunctional organ, responsible for vital functions ranging from control of the endocrine system to bile secretion or vesicular trafficking, and if it is part of the liver defense system against H2O2.EXPERIMENTAL PROCEDURESChemicals—Monoclonal anti-catalase antibody was purchased from Sigma; anti-flavoprotein of succinate ubiquinone reductase (complex II) monoclonal antibody was from Molecular Probes; anti-Bcl-2 antibody was from Santa Cruz Biotechnology; anti-PMCA was from Upstate; anti-Golgi 58K antibody was from Sigma, and anti-calreticulin was from Upstate. All other reagents were of the highest purity commercially available.Mitochondrial Isolation and Purification—Rat liver was homogenized in isolation medium (250 mm sucrose, 5 mm Hepes, 0.5 mm EGTA, pH 7.4) and subjected to centrifugation (900 × g) for 5 min. The supernatant was centrifuged at 12,000 × g for 10 min to precipitate crude mitochondrial pellets. The pellets were resuspended in isolation medium plus 1 mm ATP and layered on top of a discontinuous gradient of Ficoll diluted in isolation medium, composed of 2-ml layers of 16 (w/v), 14, and 12% Ficoll and a 3-ml layer of 7% Ficoll. After centrifugation for 30 min at 75,000 × g, mitochondrial pellets were suspended in isolation medium and centrifuged again for 10 min at 12,000 × g. The resulting pellets were suspended in isolation medium without EGTA, and their protein content was measured by the biuret method, with bovine serum albumin as a standard. The absence of other contaminating subcellular compartments in our mitochondrial preparations has been demonstrated in previous studies (21Salvi M. Brunati A.M. Bordin L. La Rocca N. Clari G. Toninello A. Biochim. Biophys. Acta. 2002; 1589: 181-195Crossref PubMed Scopus (93) Google Scholar). In addition, electron microscopy demonstrated the complete absence of contaminating membrane fragments or peroxisomes in the preparations (data not shown). It should be emphasized that the experiments with isolated rat liver mitochondria (RLM) have been performed with organelles highly purified by means of the above mentioned Ficoll gradient. The mitochondrial fraction obtained by this gradient does not exhibit any activity of urate oxidase and NADPH-cytochrome c reductase, peroxisomal and microsomal markers, respectively (data not shown).Mitoplasts were obtained by hypotonic shock of mitochondria (1:10 dilution in deionized water for 5 min on ice) followed by centrifugation (10 min at 10,000 × g) to precipitate mitoplasts. Outer membrane and intermembrane space proteins were collected in the supernatant. MAO activity was assayed spectrophotometrically in the two fractions by monitoring the oxidation of benzylamine to benzaldehyde at 250 nm (ɛ = 12,500 m-1 cm-1).Subcellular Fractionation—Rat liver (250 mg) was homogenized in 1 ml of isolation medium (250 mm sucrose, 5 mm Hepes, pH 7.4) and subjected to centrifugation for 10 min at 900 × g (nuclei fraction, pellet I). The supernatant was then centrifuged for 1 h at 100,000 × g to separate cytosol from the post-nuclear particulate fraction (pellet II). The two pellets were resuspended in 1 ml of isolation medium.Subcellular fractionation of the post-nuclear particulate fraction was performed using Optiprep™ (Accurate Chemical and Scientific Corp.). A discontinuous gradient was prepared using 30, 25, 20, 15, and 10% Optiprep™ dissolved in 50 mm Tris/HCl, pH 7.5, containing protease inhibitor mixture. The particulate fraction, resuspended in 200 μl of the above described isotonic buffer, was overlaid onto the discontinuous gradient and centrifuged at 100,000 × g for 3h at 4 °C. The gradient was removed in 15 equal fractions collected from the top of the gradient. Fractions 13-15 were collected and subjected to a new discontinuous gradient (35, 30, 25, and 20 Optiprep™ dissolved in 50 mm Tris/HCl, pH 7.5, containing protease inhibitor mixture) (as described in Refs. 22Joly E. Bendayan M. Roduit R. Saha A.K. Ruderman N.B. Prentki M. FEBS Lett. 2005; 579: 6581-6586Crossref PubMed Scopus (20) Google Scholar, 23Van Veldhoven P.P. Baumgart E. Mannaerts G.P. Anal. Biochem. 1996; 237: 17-23Crossref PubMed Scopus (52) Google Scholar) and centrifuged at 100,000 × g for 3h at 4 °C. 50 μl of each fraction were analyzed by Western blotting. Transferred to nitrocellulose membranes, proteins were incubated with the indicated antibody, followed by the appropriate second biotinylated antibody, and developed by means of an enhanced chemiluminescent detection system (ECL, Amersham Biosciences). Densitometric analysis of the anti-catalase spots was performed by Image Station 440 (Eastman Kodak Co.). It should be emphasized that the fractions containing RLM (6Davies K.J. Doroshow J.H. J. Biol. Chem. 1986; 261: 3060-3067Abstract Full Text PDF PubMed Google Scholar, 7Marcocci L. De Marchi U. Salvi M. Micella Z.G. Nocera S. Agostinelli E. Mondovi B. Toninello A. J. Membr. Biol. 2002; 188: 23-31Crossref PubMed Scopus (32) Google Scholar, 8Hodnick W.F. Milosavljevic E.B. Nelson J.H. Pardini R.S. Biochem. Pharmacol. 1998; 37: 2607-2611Crossref Scopus (188) Google Scholar), of the second Optiprep™ gradient, also do not exhibit any activity by urate oxidase and NADPH-cytochrome c reductase, as occurs with the mitochondrial fraction obtained with the Ficoll gradient (see above) (data not shown).Standard Incubation Procedures—Mitochondria (1 mg of protein/ml) were incubated in a water-jacketed cell at 20 °C. The standard medium contained 200 mm sucrose, 10 mm Hepes, pH 7.4, 5 mm succinate, and 1.25 μm rotenone. Variations and/or other additions are given with the individual experiments presented.Determination of Mitochondrial Functions—Membrane potential (Δψ) was calculated on the basis of movement of the lipid-soluble cation tetraphenylphosphonium through the inner membrane, measured on a tetraphenylphosphonium-specific electrode (24Kamo N. Muratsugu M. Hongoh R. Kobatake Y. J. Membr. Biol. 1979; 49: 105-121Crossref PubMed Scopus (886) Google Scholar). Matrix volume was determined as reported previously (7Marcocci L. De Marchi U. Salvi M. Micella Z.G. Nocera S. Agostinelli E. Mondovi B. Toninello A. J. Membr. Biol. 2002; 188: 23-31Crossref PubMed Scopus (32) Google Scholar).Mitochondrial swelling was determined by measuring the apparent absorbance change of mitochondrial suspensions at 540 nm on a Kontron Uvikon model 922 spectrophotometer equipped with thermostatic control.Oxygen uptake was measured by a Clark electrode. The redox state of endogenous pyridine nucleotides was followed fluorometrically in an Aminco-Bowman 4-8202 spectrofluorometer with excitation at 354 nm and emission at 462 nm. Respiratory control index and P/O ratio were calculated as reported previously (where P/O is the ratio of the number of ATP molecules formed to the number of oxygen molecules reduced by electron transport) (14Battaglia V. Salvi M. Toninello A. J. Biol. Chem. 2005; 280: 33864-33872Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Determination of oxidation of glutathione oxidation of glutathione was performed as described by Tietze (25Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5505) Google Scholar).Determination of Glutathione Peroxidase (Gpx) Activity—Gpx activity was determined by the method of Paglia and Valentine (26Paglia D.E. Valentine W.N. J. Lab. Clin. Med. 1967; 70: 158-169PubMed Google Scholar) in the presence of catalase inhibitors.Determination of Urate Oxidase—Urate oxidase was determined by measuring the decrease in absorbance at 290 nm resulting from the oxidation of uric acid to allantoin. One unit oxidizes 1 μmol of uric acid per min at 25 °C in 0.1 m sodium borate buffer, pH 8.5, containing 0.1 mm uric acid (27Angelini R. Rea G. Federico R. D'Ovidio R. Plant Sci. 1996; 119: 103-113Crossref Scopus (23) Google Scholar).Determination of NADPH-Cytochrome c Reductase—NADPH-cytochrome c reductase activity was assayed as described previously (28Sottocasa G.L. Kuylenstierna B. Ernster L. Bergstrand A. J. Cell Biol. 1967; 32: 415-438Crossref PubMed Scopus (1812) Google Scholar).Assay of H2O2 Concentration—H2O2 was added to a mitochondria suspension (final concentration 0.80 mg/ml protein) in a buffer containing 10 mm Hepes, pH 7.4, with or without 15 mm ATZ, and incubated for 20 min at room temperature. The residual level of H2O2 in the suspension was then evaluated as reported by Angelini et al. (27Angelini R. Rea G. Federico R. D'Ovidio R. Plant Sci. 1996; 119: 103-113Crossref Scopus (23) Google Scholar) by adding a volume of a solution containing 4 mm 3,5-dichloro-2-hydroxybenzenesulfonic acid, 2mm 4-aminoantipyrine, and 30 units/ml horseradish peroxidase and incubating for 15 min. The absorbance was then measured at 515 nm against a reference containing mitochondria suspension alone.Proteinase K Treatment—Purified rat liver mitochondria was treated with 50 ng/ml proteinase K in isolation medium without EGTA (see below) with or without 0.5% Triton X-100 at room temperature for 30 min. The reaction was stopped by the addition of protease inhibitor mixture and then analyzed by Western blotting with antibodies to catalase, Bcl-2, and the flavoprotein of succinate ubiquinone reductase (complex II).Immunogold Labeling—Isolated RLM or rat liver tissue samples were fixed for 2h in 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 m phosphate buffer, pH 7.2, post-fixed for 1 h in 1% osmium tetroxide in the same buffer, dehydrated in ethanol, and embedded in London Resin White. Ultrathin sections picked up on gold grids were de-osmicated with sodium metaperiodate, washed with 0.01 m phosphate-buffered saline, pH 7.2, incubated for 20 min on 1% bovine serum albumin in phosphate-buffered saline, and treated with primary monoclonal antibody against catalase. After washing with phosphate-buffered saline, sections were incubated with colloidal gold (15 nm) conjugated with goat-mouse antibody. Sections were then stained with uranyl acetate followed by lead citrate and examined under the electron microscope. A control experiment was performed by eliminating the incubation of sections with the primary antibody.RESULTSThe main problem in identifying constitutive catalase in RLM and evidencing its physiological significance on mitochondrial function is to have a mitochondrial preparation completely free of contaminant peroxisomes and other organelles. This condition was achieved by ultracentrifugation of isolated RLM on a Ficoll discontinuous gradient, as described under "Experimental Procedures." Instead, to evaluate the distribution of catalase among differing intracellular organelles, post-nuclear particulate fractions were separated by ultracentrifugation on an Optiprep™ discontinuous gradient, as also described under "Experimental Procedures."As the transit of mitochondria through a concentration gradient may cause some structural and functional damage, it is of primary importance to verify their integrity. The experiment (Fig. 1, panel A, inset) allows calculation of both the respiratory control index (=6) and phosphorylative capacity (ADP/O = 1.9; where ADP/O indicates mol of ADP added/mol of oxygen consumed during phosphorylation). These parameters, together the ΔΨ value of 170 mV (Fig. 1, panel B), demonstrate that the organelles did not undergo any damage during purification and that they maintain optimal respiration-phosphorylation coupling.In the presence of insufficient defense systems, oxidative stress, induced in mitochondria by ROS action, is responsible for a number of damaging effects, including membrane lipid alterations, enzyme inactivation, mutations, and mtDNA strand breaks. Indeed, at high Ca2+ concentrations, ROS action induces or amplifies the phenomenon of the mitochondrial permeability transition (MPT) (for reviews see Refs. 29Kim J.S. He L. Lemasters J.J. Biochem. Biophys. Res. Commun. 2003; 304: 463-470Crossref PubMed Scopus (630) Google Scholar, 30Toninello A. Salvi M. Mondovi B. Curr. Med. Chem. 2004; 11: 2349-2374Crossref PubMed Scopus (70) Google Scholar). The occurrence of the MPT is mainly characterized by colloidosmotic swelling of the mitochondrial matrix and collapse of membrane potential (ΔΨ). The results shown in Fig. 1 reveal some effects on RLM by tert-butylhydroperoxide (Tbh). When treated with 100 μm Tbh, RLM incubated in standard medium, supplemented with 50 μm Ca2+, exhibit an apparent absorbance decrease of the suspension of about 1 unit (Fig. 1, panel A). This event is accompanied by a parallel and complete drop in ΔΨ (Fig. 1, panel B). The observation that cyclosporin A prevents both these effects (data not shown) indicates that the peroxide, as also observed by other authors (e.g. Ref. 33Nieminen A.L. Byrne A.M. Herman B. Lemasters J.J. Am. J. Physiol. 1997; 272 (-C1294): C1286Crossref PubMed Google Scholar), induces the opening of the MPT pore. Mitochondrial swelling and ΔΨ drop are also concomitant with almost complete oxidation of glutathione (Fig. 1, panel C) and oxidation of most of the pyridine nucleotides (panel D). All these events indicate that pore opening is closely related to oxidative stress in which the glutathione peroxidase/glutathione reductase (Gpx/Gpr) system is involved. Control determinations of Gpx activity in these RLM preparations gave a mean value of 424 ± 8.5 milli-units, in agreement with recent determinations by other authors (31Schild L. Plumeyer F. Reiser G. FEBS J. 2005; 272: 5844-5852Crossref PubMed Scopus (9) Google Scholar).When Tbh is substituted with the same 100 μm concentration of either hydrogen peroxide or the monoamine tyramine, the oxidation of which by mitochondrial monoamine oxidase (MAO) also generates hydrogen peroxide, none of the above mitochondrial alterations can be observed. Only at 200 μm H2O2 is very low oxidation of glutathione (Fig. 1, panel C) induced, and higher oxidation of pyridine nucleotides (Fig. 1, panel D), accompanied by reduced opening of the MPT pore (Fig. 1, panel A). Note that pyridine nucleotide oxidation is blocked after 5 min.Fig. 2, panel A, shows that the addition of antimycin A to RLM, respiring in state 4 conditions, results in respiration block, quickly followed by a reversal of the oxygen uptake trace, indicative of O2 generation (trace a). This reverse trend, as a result of O2 production, is further augmented when 50 μm H2O2 is added to the incubation (Fig. 2, trace c). The generation of O2 upon addition of antimycin A is completely inhibited by the catalase inhibitors aminotriazole (ATZ) or KCN (Fig. 2, trace b). Indeed, in the presence of the inhibitors, the respiration block because of antimycin A addition is not followed by a significant reversal of the O2 trace after subsequent addition of H2O2 (Fig. 2, trace d).FIGURE 2Oxygen generation from endogenous or added H2O2 in isolated RLM. Panel A, effect of treatment with antimycin A and ATZ or KCN (both in presence of antimycin A). RLM were incubated in standard medium, as described under "Experimental Procedures." When present in medium, ATZ and KCN were 10 μM and 200 μm, respectively. 1 μm antimycin A (Ant. A) and 50 μm H2O2 were added (see arrows). Trace a, antimycin A; trace b, ATZ or KCN plus antimycin A; trace c, antimycin A plus H2O2; trace d, ATZ or KCN plus antimycin and plus H2O2. Assays were performed three times with comparable results. Panel B, effect of exogenous H2O2. RLM were incubated in standard medium, deprived of succinate, in same conditions as in panel A. 50 μm H2O2 were added where indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Prompt addition of H2O2 to RLM at the beginning of incubation, in the absence of an energizing substrate to avoid any consumption of O2 and production of H2O2, yields molecular oxygen in a 2:1 stoichiometry, i.e. 50 nmol of H2O2 generates 25 nmol of O2, (Fig. 2, panel B).Fig. 3 shows that RLM completely consume exogenously added hydrogen peroxide by a first-order reaction, with t½ = 20 s. The rate of H2O2 consumption is not sensitive to the presence of respiratory substrates, succinate/rotenone or glutamate/malate, nor to the alkylating reagent N-ethylmaleimide (NEM) in concentrations known to deplete mitochondrial glutathione (Fig. 3). Instead, KCN exhibits significant inhibition with a t½ = 7 min 30 s (Fig. 3). In this regard, it should be noted that pure bovine liver catalase is inhibited 95% by 200 μm KCN (results not shown). The inset of Fig. 3 shows the calculation of the first-order rate constant, k0, of H2O2 consumption, which has a value of 0.0346 s-1 (r = 0.994).FIGURE 3Hydrogen peroxide consumption by isolated RLM. RLM (0.5 mg/ml) were incubated in standard medium, in conditions described under "Experimental Procedures," with 1 mm H2O2 (curve a). When added to medium, 200 μm KCN (curve b), 1 mm NEM (curve c), succinate and 1.25 μm rotenone (curve d), and 5 mm glutamate and 5 mm malate (curve d) were present. Background H2O2 consumption in absence of RLM is shown in curve e. Data are means of four determinations. Inset, calculation of rate constant of catalase, k0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The observed inefficacy in inducing serious oxidative stress and MPT induction by H2O2 from external sources (directly added or generated by MAO activity) (Fig. 1) and the results of experiments on O2 generation and H2O2 consumption (Figs. 2 and 3) may be explained in several ways. Considering the results of Fig. 1 perhaps added H2O2 or generated by tyramine oxidation is not sufficiently transported into the matrix and is unable to induce the MPT. However, the important role of cardiolipin in favoring the diffusion of hydrogen peroxide in liposomes has already been reported (32Mathai J.C. Sitaramam V. J. Biol. Chem. 1994; 269: 17784-17793Abstract Full Text PDF PubMed Google Scholar). The effect of cardiolipin is because of the induction of stretch sensitivity in membranes made up of binary mixtures of lipids, resulting in the formation of the large voids responsible for H2O2 diffusion