Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochrome c Release from Isolated Mitochondria

Abstract

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca2+ dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca2+ by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochromec release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca2+-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca2+ levels induce apoptosis. Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca2+ dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca2+ by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochromec release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca2+-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca2+ levels induce apoptosis. Nitric oxide (NO) 1The abbreviations used are:NOnitric oxideNOSnitric-oxide synthasemtNOSmitochondrial nitric-oxide synthaseΔΨmitochondrial transmembrane potentialONOO−peroxynitriteSODsuperoxide dismutaseLPOlipid peroxidationl-NMMAN ω-monomethyl-l-arginine1400WN-(3-(aminomethyl)benzyl) acetamidineMnTBAPmanganese (III) tetrakis (4-benzoic acid) porphyrinBH4tetrahydrobiopterinCSAcyclosporin ARRruthenium red is a molecule of prime importance in biology. The most-cited and best understood physiological target for NO is the heme-containing protein, soluble guanylyl cyclase (1Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). However, at physiological concentrations, NO also binds to another hemoprotein, cytochrome oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, and thereby controls cellular functions via reversible inhibition of respiration (reviewed in Ref. 2Ghafourifar P. Richter C. Wagner E. From Symbiosis to Eukaryotism-Endocytobiology VII. Geneva University Press, 1999: 503-516Google Scholar). NO is synthesized by members of the NO synthase family (NOS, EC 1.14.13.39; reviewed in Ref. 3Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2507) Google Scholar). In 1997, we reported for the first time on the presence of a constitutively expressed and continuously active NOS in mitochondria (mtNOS), its localization in the inner mitochondrial membrane, its Ca2+ dependence, and that the enzyme exerts substantial control over mitochondrial respiration and mitochondrial transmembrane potential (ΔΨ) (4Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (535) Google Scholar). Soon thereafter the presence of mtNOS and its localization were confirmed, and the enzyme was enriched and shown to cross-react with antibodies directed against inducible NOS (5Giulivi C. Poderoso J.J. Boveris A. J. Biol. Chem. 1998; 273: 11038-11043Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). nitric oxide nitric-oxide synthase mitochondrial nitric-oxide synthase mitochondrial transmembrane potential peroxynitrite superoxide dismutase lipid peroxidation N ω-monomethyl-l-arginine N-(3-(aminomethyl)benzyl) acetamidine manganese (III) tetrakis (4-benzoic acid) porphyrin tetrahydrobiopterin cyclosporin A ruthenium red Apoptosis, also called programmed cell death, is an evolutionary conserved phenomenon which regulates normal cellular turnover. Mitochondria are essential for at least certain forms of apoptosis (reviewed in Ref. 6Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). For example, cytochrome c, a mitochondrial protein that is part of the respiratory chain, triggers apoptosis once it is dislocated from the organelle. It is now well accepted that many factors drive cells into apoptotic deathvia mitochondrial cytochrome c release. NO reacts with O2− to produce the powerful oxidizing agent, peroxynitrite (ONOO−). Recently many studies focused on the role of NO and ONOO− in apoptosis (7Lin K.T. Xue J.Y. Nomen M. Spur B. Wong P.Y.K. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 8Lin K.T. Xue J.Y. Lin M.C. Spokas E.G. Sun F.F. Wong P.Y.K. Am. J. Physiol. 1998; 274: C855-C860Crossref PubMed Google Scholar, 9Szabo C. Cuzzocrea S. Zingarelli B. O'Connor M. Salzman A.L. J. Clin. Invest. 1997; 100: 723-735Crossref PubMed Scopus (356) Google Scholar, 10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar). Their exact source(s) and the mechanism(s) are, however, not yet fully elucidated. An increase in cytosolic Ca2+ level caused by, e.g. glutamate receptor stimulation, is apoptogenic (reviewed in Ref. 11McConkey D.J. Orrenius S. Biochem. Biophys. Res. Commun. 1997; 239: 357-366Crossref PubMed Scopus (383) Google Scholar). Many recent reports show that mitochondrial Ca2+ uptake is an essential step in Ca2+-induced apoptosis (12Stout A. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar, 14Kruman I.I. Mattson M.P. J. Neurochem. 1999; 72: 529-540Crossref PubMed Scopus (294) Google Scholar). This kind of programmed cell death is accompanied by increased NOS activity (12Stout A. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar) and prevented by mitochondrial superoxide dismutase (SOD), MnSOD (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 15Gonzalez-Zulueta M. Ensz L.M. Mukhina G. Lebovitz R.M. Zwacka R.M. Engelhardt J.F. Oberley L.W. Dawson V.L. Dawson T.M. J. Neurosci. 1998; 18: 2040-2055Crossref PubMed Google Scholar), or by the ONOO− scavenger, urate (16Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (347) Google Scholar). Mitochondria produce NO in a Ca2+-dependent manner (4Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (535) Google Scholar) and are a rich source of O2−. Therefore, intramitochondrial Ca2+-dependent ONOO− formation seems likely. Here we show that upon Ca2+ uptake by isolated mitochondria, mtNOS is stimulated and cytochrome c is released in a Bcl-2-sensitive manner. We provide evidence that the observed cytochrome c release is due to intramitochondrial ONOO− formation because it is prevented by NOS inhibitors, an SOD mimic, and an ONOO−scavenger. We suggest that Ca2+-induced apoptosis is at least partly mediated via mtNOS. N ω-monomethyl-l-arginine (l-NMMA), N-(3-(aminomethyl)benzyl) acetamidine (1400W), and manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) were obtained from Alexis Biochemicals (Läufelfingen, Switzerland). Horse heart cytochrome c andl-[2,3-3H]arginine (36.8 Ci/mmol) were from Sigma, mouse monoclonal cytochrome c antibody from RDI (Flanders, NJ), anti-mouse Ig, horseradish peroxidase from Amersham Pharmacia Biotech (Dübendorf, Switzerland), 6-His-human Bcl-2 from Novartis (Basel, Switzerland), andl-[ureido-14C]citrulline (58.8 mCi/mmol) from NEN Life Science Products. Isolation of rat liver mitochondria was performed by differential centrifugation as described (17Lötscher H.R. Winterhalter K.H. Carafoli E. Richter C. J. Biol. Chem. 1980; 255: 9325-9330Abstract Full Text PDF PubMed Google Scholar). The protein content of mitochondria and the mitochondrial supernatants were determined by the Biuret method with bovine serum albumin as standard. Freshly isolated mitochondria (50 mg of protein/ml) were incubated at 4 °C in 0.1m HEPES buffer, pH 7.1, containing protease inhibitors: aprotinin, pepstatin A, phenylmethanesulfonyl fluoride, and leupeptin (2 μg/ml each). NOS inhibitors, l-NMMA (10 mm) or 1400W (100 μm), 6-His-human Bcl-2 (8 pmol/mg of mitochondrial protein) (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), SOD mimic, MnTBAP (10 μm) or peroxynitrite scavenger, urate (100 μm) (16Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (347) Google Scholar) were also present in the incubation buffer as indicated. After 10 min of incubation, 100 μmCa2+, 100 μml-arginine, and 12 μm tetrahydrobiopterin (BH4; from a 1.2 mm stock solution prepared instantaneously before the experiment) (4Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (535) Google Scholar) were added, and the mitochondrial suspensions were incubated for 5 min at room temperature. Mitochondrial respiration was then supported by 0.8 mm K+-succinate in the presence of 5 μm rotenone for 10 min at room temperature. Samples were spun at 12,000 × g for 10 min at 4 °C, and the resulting supernatants were spun at 100,000 ×g for 15 min at 4 °C. The supernatants of the second centrifugation were used for the detection of cytochrome cby Western blot analysis as described (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Cyclosporin A (CSA; 1 μm) or mitochondrial Ca2+ uptake blocker ruthenium red (RR; 8 μm) were added before Ca2+. Mitochondrial de-energization was performed by addition of 50 nm antimycin A to block complex III and 1.7 μg of oligomycin/mg of mitochondrial protein to block ATPase before succinate (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) or by omitting the respiratory substrate. Samples were prepared as described above, except that mitochondria were incubated at 2 mg/ml and in the absence of protease inhibitors. Mitochondrial LPO was determined by thiobarbituric acid assay as described (19Klein S.D. Walt H. Richter C. Arch. Biochem. Biophys. 1997; 348: 313-319Crossref PubMed Scopus (25) Google Scholar). Samples were prepared as described above except that mitochondria were incubated at 10 mg/ml and in the absence of protease inhibitors. mtNOS activity was determined by measurement of the conversion ofl-[3H]arginine tol-[3H]citrulline as described (20Richter C. Schweizer M. Ghafourifar P. Methods Enzymol. 1999; 301: 381-393Crossref PubMed Scopus (30) Google Scholar) and is expressed as cpm/mg of mitochondrial protein. Mitochondria (1 mg/ml) were incubated in 0.1 m HEPES, pH 7.1, at room temperature. Oxygen consumption was measured under continuous stirring with a Clarke-type electrode, as described (4Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (535) Google Scholar). Mitochondrial transmembrane potential (ΔΨ) was measured in an Aminco DW-2A spectrophotometer at 511–533 nm in the presence of 10 μmsafranin as described (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). mtNOS stimulation induces cytochrome c release from isolated mitochondria in a manner that is prevented by two NOS inhibitors, l-NMMA and 1400W (Fig.1 A). This panel also shows that Bcl-2 prevents mtNOS-induced cytochrome c release, which indicates that the release is not because of a general mitochondrial damage followed by a nonspecific protein release, but it is a specific phenomenon relevant to apoptosis. A considerable number of recent reports show that endogenously formed NO induces Bcl-2-sensitive apoptosis (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar, 15Gonzalez-Zulueta M. Ensz L.M. Mukhina G. Lebovitz R.M. Zwacka R.M. Engelhardt J.F. Oberley L.W. Dawson V.L. Dawson T.M. J. Neurosci. 1998; 18: 2040-2055Crossref PubMed Google Scholar, 21Estevez A.G. Spear N. Manuel S.M. Radi R. Henderson C.E. Barbeito L. Beckman J.S. J. Neurosci. 1998; 18: 923-931Crossref PubMed Google Scholar, 22Brüne B. Götz C. Meßmer U.K. Sandau K. Hirvonen M. Lapetina E.G. J. Biol. Chem. 1997; 272: 7253-7258Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 23Messmer U.K. Reimer D.M. Reed J.C. Brune B. FEBS Lett. 1996; 384: 162-166Crossref PubMed Scopus (104) Google Scholar, 24Ferrante R.J. Hantraye P. Brouillet E. Beal M.F. Brain Res. 1999; 823: 177-182Crossref PubMed Scopus (75) Google Scholar), which is accompanied by mitochondrial dysfunction (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar), increased LPO (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar) and ONOO− formation (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 24Ferrante R.J. Hantraye P. Brouillet E. Beal M.F. Brain Res. 1999; 823: 177-182Crossref PubMed Scopus (75) Google Scholar, 25Leist M. Volbracht C. Kuhnle S. Fava E. Ferrando-May E. Nicotera P. Mol. Med. 1997; 3: 750-764Crossref PubMed Google Scholar). It is also well demonstrated that increased cytosolic Ca2+-induced apoptosis requires mitochondrial Ca2+ uptake (12Stout A. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar, 14Kruman I.I. Mattson M.P. J. Neurochem. 1999; 72: 529-540Crossref PubMed Scopus (294) Google Scholar), is paralleled by increased NOS activity (12Stout A. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google Scholar, 13Almeida A. Heales S.J.R. Bolanos J.P. Medina J.M. Brain. Res. 1998; 790: 209-216Crossref PubMed Scopus (130) Google Scholar, 26Le W.D. Colom L.V. Xie W.J. Smith R.G. Alexianu M. Appel S.H. Brain Res. 1995; 686: 49-60Crossref PubMed Scopus (141) Google Scholar), and is prevented by lowering the mitochondrial O2− level by MnSOD (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 15Gonzalez-Zulueta M. Ensz L.M. Mukhina G. Lebovitz R.M. Zwacka R.M. Engelhardt J.F. Oberley L.W. Dawson V.L. Dawson T.M. J. Neurosci. 1998; 18: 2040-2055Crossref PubMed Google Scholar) or by scavenging ONOO− with urate (16Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (347) Google Scholar). The reaction of NO and O2− with the rate constant of 1.9 × 1010m−1 s−1 (27Kissner R. Nauser T. Bugnon P. Lye P.G. Koppenol W.H. Chem. Res. Toxicol. 1997; 10: 1285-1292Crossref PubMed Scopus (567) Google Scholar) is one of the fastest reactions known in biology. Mitochondria produce NO in a Ca2+-dependent fashion (4Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (535) Google Scholar), and they are well known sources of O2− radicals. Intramitochondrial Ca2+-dependent ONOO− formation is, therefore, very likely. Fig.1 A shows that mtNOS-induced cytochromec release is prevented by the SOD mimic, MnTBAP, as well as by the ONOO− scavenger, urate. This finding strongly suggests that ONOO− is indeed formed within mitochondria and that it releases mitochondrial cytochrome c. This may explain the mechanism by which an elevated Ca2+ level induces apoptosis in a manner that requires mitochondrial Ca2+ uptake and is prevented by inhibiting NOS activity, lowering O2− level, and scavenging ONOO−. It is known that ONOO− induces LPO (10Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 28Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2050) Google Scholar, 29Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar). Fig.2 shows that, upon mtNOS stimulation, LPO is increased in a manner that is sensitive to l-NMMA, Bcl-2, MnTBAP, and urate. This figure also shows that exogenously added cytochrome c prevents increased LPO. From the elegant study by Cai and Jones (30Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar) it is known that cytochrome c release is the cause and not the consequence of reactive oxygen species formation in mitochondria. We have recently confirmed this finding by showing that the decreased mitochondrial O2 consumption, ΔΨ and Ca2+ retention, consequent to ceramide-induced mitochondrial cytochrome c loss, are recovered by addition of exogenous cytochrome c (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Very recently, it has also been reported that both the shape and the volume alterations of mitochondria because of cytochrome c loss are reversible (31Martinou I. Desagher S. Eskes R. B. E. S. J.C. J. Biol. 1999; PubMed Scopus Google Scholar). release of cytochrome c from its within the mitochondrial respiratory III and complex III and therefore, for O2− by Bcl-2 of cytochrome c release the for to from complex III to and from to complex the of for the formation of one of the two of ONOO−. In the reported above, and were to mitochondria. 1 shows that Ca2+ is to cytochrome c release in and Bcl-2 sensitive manner and shows that the of Ca2+ is It is not that Ca2+ is for mtNOS-induced cytochrome c release, because to in mitochondria in Intramitochondrial M. J. Neurochem. 1998; PubMed Scopus Google Scholar) and Methods Enzymol. PubMed Scopus Google Scholar) are in the and are of mitochondrial respiratory and therefore, present in mitochondria I. Smith P. M. and Press, Scholar). is also evidence for the presence of H. K. J. Biochem. PubMed Scopus Google Scholar) and O. A. T. H. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, T. H. A. O. Biochem. Google Scholar) in mitochondria. that the observed cytochrome c release requires the uptake of Ca2+ into the mitochondria, we used specific mitochondrial release and uptake 1 shows that of Ca2+ within mitochondria by a known to block the specific mitochondrial C. M. J. Biochem. Pharmacol. PubMed Scopus Google Scholar), mtNOS-induced cytochrome in an manner. is also reported to a of the nonspecific the inner mitochondrial membrane, the mitochondrial permeability transition which is to the for many of apoptosis cytochrome c release (reviewed in Ref. T. I. G. 1997; PubMed Scopus Google Scholar). the release of cytochrome by mtNOS stimulation 1 we conclude that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability of mitochondrial Ca2+ uptake by prevents Ca2+-induced cytochrome c release 1 This finding is with reports by that prevents apoptosis by elevated cytosolic Ca2+ levels (12Stout A. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google I.I. Mattson M.P. J. Neurochem. 1999; 72: 529-540Crossref PubMed Scopus (294) Google Scholar, H.C. J. 1997; PubMed Scopus Google Scholar, I.I. A. Mattson M.P. 1998; PubMed Scopus Google Scholar). mitochondria were by antimycin A or by omitting succinate and not mtNOS activity was decreased and cytochromec release was prevented 1 This finding also recent reports (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, I. Desagher S. Eskes R. B. E. S. J.C. J. Biol. 1999; PubMed Scopus Google Scholar) that cytochrome c is not from the mitochondrial inner because of a in mitochondrial transmembrane e.g. upon uptake of Ca2+. the mechanism by which Bcl-2 prevents mtNOS-induced cytochrome c release, we measured mitochondrial Ca2+ uptake III as the (18Ghafourifar P. Klein S.D. Schucht O. Schenk U. Rocha S. Pruschy M. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) and observed that Bcl-2 not mitochondrial Ca2+ uptake we measured mtNOS activity and observed that Bcl-2 not it A). shows that Ca2+-induced decreased mitochondrial is prevented by l-NMMA, but not by shows that uptake of Ca2+ by mitochondria causes a in ΔΨ that is prevented by l-NMMA but not that decreased O2 consumption and ΔΨ by mitochondrial Ca2+ uptake is because of NO formation by and not cytochrome c release, and that by Bcl-2 of mtNOS-induced cytochrome c release is not because of a decreased mtNOS In mitochondrial de-energization by antimycin A prevents mtNOS-induced cytochromec release 1 because of a in mtNOS activity A). show that uptake of Ca2+ by mitochondria followed by mtNOS stimulation causes mitochondrial cytochrome c release and increased and provide evidence that are mediated via intramitochondrial ONOO− We propose that mtNOS a role in apoptosis by elevated cytosolic Ca2+ We for in the and for We Novartis for Bcl-2, and K. H. for and