Oxidative stress and S-nitrosylation of proteins in cells.
The effect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte-derived Jurkat and L929 fibroblast human cell lines using a constant concentration of nitric oxide (1.5 microM) released by the nitric oxide donor DETA-NO (0.5 mM). Nitric oxide inhibited immediately the respiration of the cells acting reversibly at complex IV. With time, the inhibition became progressively persistent, i.e. not reversed by trapping of nitric oxide with oxyhaemoglobin, and was preceded by a decrease in the concentration of the intracellular reduced glutathione. This persistent effect of nitric oxide on respiration was due to inhibition of complex I activity which could be reversed by addition of reduced glutathione or by cold light, suggesting that it was due to S-nitrosylation of thiols necessary for the activity of the enzyme. The activity of other enzymes also known to be susceptible to inhibition by S-nitrosylation, i.e. glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase, was progressively decreased by exposure to nitric oxide with a similar time course to that observed for the inhibition of complex I. Furthermore, inhibition of these enzymes only occurred when the concentrations of reduced glutathione had previously fallen and could be prevented by increasing the intracellular concentrations of reduced glutathione. Our results suggest that S-nitrosylation of different enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.
Beltrán
B
B
The Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WCIE 6BT.
Orsi
A
A
Clementi
E
E
Moncada
S
S
D002460:Cell Line; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; D005347:Fibroblasts; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D010101:Oxygen Consumption / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014226:Triazenes / Q000494:pharmacology
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011506:Proteins; D017382:Reactive Oxygen Species; D014226:Triazenes; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009247:NADH, NADPH Oxidoreductases; D005980:Glutathione Reductase; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; D005978:Glutathione