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. 1998 Jun 23;95(13):7631-6.
doi: 10.1073/pnas.95.13.7631.

Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

E Clementi  1 , G C Brown, M Feelisch, S Moncada
Affiliations

Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

E Clementi et al. Proc Natl Acad Sci U S A. .

Abstract

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

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Figures

Figure 1
Figure 1
Effects of exposure to DETA-NO on cell respiration in J774 cells. Cells were incubated with different concentrations of DETA-NO (0.03 mM, triangles; 0.1 mM, circles; 0.5 mM, squares) and oxygen consumption was measured in samples taken at the indicated time points: before (open symbols) or after (filled symbols) addition of Hb (8 μM; n = 5). Oxygen consumption in DETA-NO-treated cells, here and in the subsequent figures, is reported as percentage of that observed in samples treated in the same way but without DETA-NO (controls). The rate of oxygen consumption in control cells was 14.3 ± 0.78 μM min−1 (n = 5) and did not vary significantly throughout the experiment.
Figure 2
Figure 2
Role of ONOO− and superoxide in the irreversible inhibition of respiration induced by DETA-NO. (A) J774 cells were treated with DETA-NO (0.5 mM) alone (squares) or in combination with the ONOO− scavenger methionine (20 mM, circles) or the superoxide dismutase mimetic MnTBAP (0.3 mM, triangles), and oxygen consumption was measured before (open symbols) or after (filled symbols) addition of Hb (8 μM, n = 4). (B) Cells of the J774 mutant C3C clone were treated with DETA-NO (0.5 mM) and oxygen consumption was measured before (open symbols) or after (filled symbols) addition of Hb (8 μM). The rate of oxygen consumption in control C3C cells was 11.8 ± 1.41 μM min−1 ( n = 3).
Figure 3
Figure 3
Spontaneous and DTT-induced recovery of respiratory function in J774 cells after long-term exposure to NO. (A) Cells were treated with DETA-NO (0.5 mM) for 6 h in the incubation medium under sterile conditions, then washed, resuspended in culture medium, and maintained in the cell incubator. Oxygen consumption by cell samples was determined at the indicated time points (n = 3). (B) Cells were treated with DETA-NO (0.5 mM) for 6 h in the incubation medium. Increasing concentrations of DTT were added 5 min before addition of Hb (8 μM), after which oxygen consumption was measured (n = 6).
Figure 4
Figure 4
Mechanism of inhibition of respiration at complex I by DETA-NO. (A) Changes in GSH concentration were measured in control cells (triangles) or in cells treated with DETA-NO (0.1 or 0.5 mM, circles and squares, respectively) after an 18-h incubation in culture medium in the absence (filled symbols) or presence (open symbols) of l-BSO (0.3 mM). Results shown are representative of two experiments. (B) J774 cells were incubated for 18 h in the culture medium in the absence (squares) or presence of l-BSO (0.3 mM, diamonds) and then treated with DETA-NO (0.5 mM). Oxygen consumption was measured before (open symbols) or after (filled symbols) addition of Hb (8 μM). After 5 h of incubation, DETA-NO was removed by washing and the cells were incubated with GSH–methylester (1 mM; filled symbols, Lower) or exposed to a high intensity light (filled symbols, Upper; n = 3).
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