Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) 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 Ca(2+) 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 cytochrome c 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 Ca(2+)-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 Ca(2+) levels induce apoptosis.

[1]  C. Richter,et al.  Nitric oxide synthase activity in mitochondria , 1997, FEBS letters.

[2]  S. Moncada,et al.  Nitric oxide: physiology, pathophysiology, and pharmacology. , 1991, Pharmacological reviews.

[3]  M. Mattson,et al.  Calcium and reactive oxygen species mediate staurosporine‐induced mitochondrial dysfunction and apoptosis in PC12 cells , 1998, Journal of neuroscience research.

[4]  W. Koppenol,et al.  Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis. , 1997, Chemical research in toxicology.

[5]  L. Colom,et al.  Cell death induced by β-amyloid 1–40 in MES 23.5 hybrid clone: the role of nitric oxide and NMDA-gated channel activation leading to apoptosis , 1995, Brain Research.

[6]  T. Dawson,et al.  Manganese Superoxide Dismutase Protects nNOS Neurons from NMDA and Nitric Oxide-Mediated Neurotoxicity , 1998, The Journal of Neuroscience.

[7]  M. Beal,et al.  Increased nitrotyrosine immunoreactivity in substantia nigra neurons in MPTP treated baboons is blocked by inhibition of neuronal nitric oxide synthase , 1999, Brain Research.

[8]  B. Corkey,et al.  Assay of citric acid cycle intermediates and related compounds--update with tissue metabolite levels and intracellular distribution. , 1979, Methods in enzymology.

[9]  B. Brüne,et al.  Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis* , 1997, The Journal of Biological Chemistry.

[10]  E. Carafoli,et al.  Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria. , 1980, The Journal of biological chemistry.

[11]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[12]  P. Nicotera,et al.  Caspase-Mediated Apoptosis in Neuronal Excitotoxicity Triggered by Nitric Oxide , 1997, Molecular medicine.

[13]  C. Richter,et al.  Mitochondria, nitric oxide, and peroxynitrite. , 1999, Methods in enzymology.

[14]  C. Giulivi,et al.  Production of Nitric Oxide by Mitochondria* , 1998, The Journal of Biological Chemistry.

[15]  M. Mattson,et al.  Pivotal Role of Mitochondrial Calcium Uptake in Neural Cell Apoptosis and Necrosis , 1999, Journal of neurochemistry.

[16]  B. Freeman,et al.  Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. , 1991, Archives of biochemistry and biophysics.

[17]  Dean P. Jones,et al.  Superoxide in Apoptosis , 1998, The Journal of Biological Chemistry.

[18]  Jung-Ae Kim,et al.  Intracellular Ca2+ release mediates ursolic acid–induced apoptosis in human leukemic HL‐60 cells , 1997, International journal of cancer.

[19]  B. Spur,et al.  Peroxynitrite-induced Apoptosis in HL-60 Cells (*) , 1995, The Journal of Biological Chemistry.

[20]  J. B. Hutchins,et al.  Mitochondrial Manganese Superoxide Dismutase Prevents Neural Apoptosis and Reduces Ischemic Brain Injury: Suppression of Peroxynitrite Production, Lipid Peroxidation, and Mitochondrial Dysfunction , 1998, The Journal of Neuroscience.

[21]  S. Orrenius,et al.  The role of calcium in the regulation of apoptosis. , 1996, Journal of leukocyte biology.

[22]  G. Kroemer,et al.  Role of the Mitochondrial Permeability Transition Pore in Apoptosis , 1997, Bioscience reports.

[23]  S. Barnes,et al.  Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. , 1994, The Journal of biological chemistry.

[24]  L. Barbeito,et al.  Nitric Oxide and Superoxide Contribute to Motor Neuron Apoptosis Induced by Trophic Factor Deprivation , 1998, The Journal of Neuroscience.

[25]  C. Richter,et al.  Photosensitization of isolated rat liver mitochondria by tetra(m-hydroxyphenyl)chlorin. , 1997, Archives of biochemistry and biophysics.

[26]  John Calvin Reed,et al.  Nitric oxide induced poly(ADP‐ribose) polymerase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl‐2 , 1996, FEBS letters.

[27]  S. Rocha,et al.  Ceramide Induces Cytochrome c Release from Isolated Mitochondria , 1999, The Journal of Biological Chemistry.

[28]  M. Theus,et al.  Cyclosporine A inhibits mitochondrial pyridine nucleotide hydrolysis and calcium release. , 1990, Biochemical pharmacology.

[29]  J. Albrecht,et al.  l-Arginine uptake in rat cerebral mitochondria , 1998, Neurochemistry International.

[30]  Ian J. Reynolds,et al.  Glutamate-induced neuron death requires mitochondrial calcium uptake , 1998, Nature Neuroscience.

[31]  Á. Almeida,et al.  Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion , 1998, Brain Research.

[32]  J. Martinou,et al.  The Release of Cytochrome c from Mitochondria during Apoptosis of NGF-deprived Sympathetic Neurons Is a Reversible Event , 1999, The Journal of cell biology.

[33]  S Moncada,et al.  Nitric oxide synthases in mammals. , 1994, The Biochemical journal.

[34]  M. Mattson,et al.  HIV-1 Protein Tat Induces Apoptosis of Hippocampal Neurons by a Mechanism Involving Caspase Activation, Calcium Overload, and Oxidative Stress , 1998, Experimental Neurology.