Inhibition of mitochondrial respiration by endogenous nitric oxide: a critical step in Fas signaling

Abstract

We have found that activation of human adult T cell leukemia (Jurkat) cells with anti-Fas Ab leads, in a concentration-dependent manner, to an early burst of production of nitric oxide (NO), which inhibits cell respiration. This results in mitochondrial hyperpolarization, dependent on the hydrolysis of glycolytic ATP by the F1F(o)-ATPase acting in reverse mode. During this early phase of activation, there is a transient release of superoxide anion. All these processes can be prevented by an inhibitor of NO synthase. Approximately 2 h after stimulation with anti-Fas Ab, a distinct second phase can be detected. This comprises a concentration-dependent collapse in mitochondrial membrane potential, a second wave of free radical production, and activation of caspase-8 leading to apoptosis. This second phase is abolished by an inhibitor of caspase activation. In contrast, inhibition of NO synthesis leads to an enhancement and acceleration of these latter processes, suggesting that the early NO-dependent phase represents a protective mechanism. The significance of the two phases in relation to cell survival and death remains to be studied.

Figure 1

Anti-Fas Ab-induced generation of intracellular NO. (a) Anti-Fas Ab (10–100 ng/ml) induced a concentration-dependent shift in fluorescence of DAF-2/DA. Measurements made 40 min after addition of the Ab. (b) Time dependence of the production of NO in response to anti-Fas Ab (10 ng/ml). (c) l-NAME (500 μM) alone had no effect on the DAF-2/DA fluorescent signal but caused a significant shift in the response to anti-Fas Ab (10 ng/ml) toward the baseline value. Measurements made 1.5 h after addition of Ab. Results shown are representative or the mean ± SEM of five experiments.

Figure 2

Inhibition of mitochondrial respiration after administration of anti-Fas Ab. Anti-Fas Ab (10–1,000 ng/ml) caused a time-and concentration-dependent inhibition of mitochondrial respiration (see Materials and Methods), which was prevented by treatment with l-NAME (500 μM). n = 3; *, P < 0.05.

Figure 3

Effect of anti-Fas Ab on Δψ_m. Anti-Fas Ab (10 and 100 ng/ml) caused an initial hyperpolarization of the mitochondrial membrane, followed by a depolarization, the onset of each of which occurred earlier at the higher concentration. n = 4; *, P < 0.05.

Figure 4

Anti-Fas Ab-induced generation of ROS. After treatment with anti-Fas Ab (10 ng/ml), there was an initial burst of ROS reaching a maximum at 1 h; this was followed, after a delay of several hours, by a prolonged generation of ROS. The early phase of ROS production was inhibited by l-NAME (500 μM) but the second phase was enhanced. In contrast, zVADfmk (50 μM) had no effect on the early phase of ROS generation but inhibited the second phase. n = 5; *, P < 0.05.

Figure 5

Effect of anti-Fas Ab on cell viability determined in cells treated with annexin V and PI. (a) Anti-Fas Ab caused a time- and concentration-dependent fall in cell viability. (b) Treatment with anti-Fas Ab led to apoptosis (cells annexin V positive, PI negative). (c) Treatment with anti-Fas Ab led to necrosis (cells annexin V positive, PI positive). (d) l-NAME (500 μM) alone had no effect on cell viability but significantly enhanced cell mortality 3 h after treatment with anti-Fas Ab (10 ng/ml). n = 5; *, P < 0.05.