1 The eect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte-derived Jurkat and L929 ®broblast human cell lines using a constant concentration of nitric oxide (1.5 mM) released by the nitric oxide donor DETA-NO (0.5 mM). 2 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 eect 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. 3 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. 4 Our results suggest that S-nitrosylation of dierent enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.

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Nitric oxide (NO) plays a key role in many physiological and pathophysiological events, including the control of cell respiration. Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of NO by cells. We have exposed the murine macrophage cell line J774 to high concentrations of NO, such as are generated in some pathological conditions, and determined their effect on oxygen consumption. We observed a persistent inhibition of respiration which was due to a redox-dependent, progressive inhibition of complex I activity. No other enzyme of the respiratory chain was inhibited in this way. At the same time, we detected a paradoxical removal of oxygen by the extracellular medium. This removal was due to a chemical interaction between dissolved oxygen and NO-related species released from cells exposed to NO. A similar removal of oxygen by the cell supernatant also occurred following activation of cells with cytokines and bacterial products. Thus, the amounts of NO generated during pathological conditions may contribute to tissue hypoxia both by inhibiting cell respiration and by promoting removal of oxygen from the extracellular medium.