Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2, spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 μM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (ρ−) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

[1]  G. Brown,et al.  Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. , 2001, Biochimica et biophysica acta.

[2]  S Moncada,et al.  The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Freeman,et al.  Concentration-dependent Effects of Nitric Oxide on Mitochondrial Permeability Transition and Cytochrome cRelease* , 2000, The Journal of Biological Chemistry.

[4]  A. Mathur,et al.  Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. , 2000, Cardiovascular research.

[5]  J. Howl,et al.  Critical Evaluation of ECV304 as a Human Endothelial Cell Model Defined by Genetic Analysis and Functional Responses: A Comparison with the Human Bladder Cancer Derived Epithelial Cell Line T24/83 , 2000, Laboratory Investigation.

[6]  E. Cadenas,et al.  The Regulation of Mitochondrial Oxygen Uptake by Redox Reactions Involving Nitric Oxide and Ubiquinol* , 1999, The Journal of Biological Chemistry.

[7]  J. Lancaster,et al.  Cellular antioxidant and pro-oxidant actions of nitric oxide. , 1999, Free radical biology & medicine.

[8]  C. Richter,et al.  Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation. , 1999, The Journal of biological chemistry.

[9]  P. Ping,et al.  The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Erkki Ruoslahti,et al.  Anti-cancer activity of targeted pro-apoptotic peptides , 1999, Nature Medicine.

[11]  T. Hughes,et al.  Trafficking of Endothelial Nitric-oxide Synthase in Living Cells , 1999, The Journal of Biological Chemistry.

[12]  F. Kiessling,et al.  Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics , 1999, Cell and Tissue Research.

[13]  T. Billiar,et al.  Nitric Oxide Suppresses Apoptosis via Interrupting Caspase Activation and Mitochondrial Dysfunction in Cultured Hepatocytes* , 1999, The Journal of Biological Chemistry.

[14]  E. Clementi,et al.  On the mechanism by which vascular endothelial cells regulate their oxygen consumption. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Moskowitz,et al.  The Cerebral Metabolic Consequences of Nitric Oxide Synthase Deficiency: Glucose Utilization in Endothelial and Neuronal Nitric Oxide Synthase Null Mice , 1999, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[16]  R. Radi,et al.  Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite. , 1998, Archives of biochemistry and biophysics.

[17]  A. Nègre-Salvayre,et al.  Apoptosis and Activation of the Sphingomyelin-Ceramide Pathway Induced by Oxidized Low Density Lipoproteins Are Not Causally Related in ECV-304 Endothelial Cells* , 1998, The Journal of Biological Chemistry.

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

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

[20]  A. Zeiher,et al.  Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[21]  A. Bowie,et al.  Lipid Peroxidation Is Involved in the Activation of NF-κB by Tumor Necrosis Factor but Not Interleukin-1 in the Human Endothelial Cell Line ECV304 , 1997, The Journal of Biological Chemistry.

[22]  D. Kipnis,et al.  Interleukin-1 Reduces the Glycolytic Utilization of Glucose by Pancreatic Islets and Reduces Glucokinase mRNA Content and Protein Synthesis by a Nitric Oxide-dependent Mechanism* , 1997, The Journal of Biological Chemistry.

[23]  I. Zachary,et al.  Vascular Endothelial Growth Factor Stimulates Tyrosine Phosphorylation and Recruitment to New Focal Adhesions of Focal Adhesion Kinase and Paxillin in Endothelial Cells* , 1997, The Journal of Biological Chemistry.

[24]  T. Pozzan,et al.  A Role for Calcium Influx in the Regulation of Mitochondrial Calcium in Endothelial Cells (*) , 1996, The Journal of Biological Chemistry.

[25]  M. King,et al.  Isolation of human cell lines lacking mitochondrial DNA. , 1996, Methods in enzymology.

[26]  T. Hintze,et al.  Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. , 1995, Circulation.

[27]  J. Lancaster,et al.  Nitrogen oxide‐induced autoprotection in isolated rat hepatocytes , 1995, FEBS letters.

[28]  Richard Graham Knowles,et al.  Endotoxin causes reciprocal changes in hepatic nitric oxide synthesis, gluconeogenesis, and flux through phosphoenolpyruvate carboxykinase. , 1994, Biochemical and biophysical research communications.

[29]  James B. Mitchell,et al.  Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Brüne,et al.  Nitric oxide-induced S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation. , 1992, The Journal of biological chemistry.

[31]  M. King,et al.  Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. , 1989, Science.

[32]  D. W. Thomas.  Handbook of Methods for Oxygen Radical Research , 1988, Journal of Pediatric Gastroenterology and Nutrition.

[33]  D. Hinshaw,et al.  Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. , 1988, The Journal of biological chemistry.