Article Figures & Data
Figures
- Fig. 1.
Inhibition of aconitase by H2O2. Nerve terminals were incubated in the absence (control) or presence of different concentrations of H2O2. Aconitase activity was measured after incubation with H2O2 for 5 min. In the control samples the activity of aconitase was 86.4±3.4 nmol · min−1 · mg−1protein taken as 100%. Enzyme activities are expressed as percentage of control. Data are the average ± SEM of eight determinations made in four independent experiments. *Significant compared with the control, p < 0.05.
- Fig. 2.
Inhibition of α-ketoglutarate dehydrogenase by H2O2. Nerve terminals were incubated in the absence (control) or presence of different concentrations of H2O2 for 5 or 10 min (a), or with 100 and 500 μmH2O2, respectively, for different lengths of time (b). In control samples the activity of α-KGDH was 14.2 ± 1.2 nmol · min−1 · mg−1taken as 100%. Data are the average ± SEM of eight determinations made in three independent experiments. *Significant compared with the control, p < 0.05.
- Fig. 3.
Decrease in the NAD(P)H level by H2O2. Fluorescence of NAD(P)H was monitored in synaptosomes (0.5 mg/ml protein) incubated in standard medium in the presence or absence of H2O2. Five minutes after application of H2O2, rotenone (2 μm) was added. To calculate ΔNAD(P)H, the fluorescence measured 10 sec before addition of rotenone was subtracted from that obtained 100 sec after rotenone application. ΔNAD(P)H representing the effect of rotenone in the absence of H2O2(inset, trace a) was taken as control (100%). The rotenone-induced NAD(P)H signals obtained in the presence of 100 or 500 μm H2O2 are shown (% of control) as a function of time; 100% represents 1.18 ± 0.04 nmol NAD(P)H, calibrated with added amounts of NADPH. Results are mean ± SEM of five determinations from three independent experiments. *Significant compared with the control, p < 0.05.
- Fig. 4.
Decrease in the NAD(P)H level by H2O2 in the absence or presence of BCNU, inhibitor of glutathione reductase. Synaptosomes (6 mg/ml) were incubated in the presence (b) or absence (a) of BCNU for 30 min at 37°C, then cooled to 0°C. Aliquots (1 mg protein) were incubated in standard medium (0.5 mg/ml), and NAD(P)H fluorescence was measured as described for Figure3, in the presence of different concentrations of H2O2 for 5 min. Results are expressed as mean ± SEM of three independent experiments; 100% represents 1.17 ± 0.06 nmol NAD(P)H. Inset shows the activity of glutathione reductase measured after incubation with different concentrations of BCNU for 30 min. The activity of glutathione reductase in control samples (100%) was 28 ± 0.56 nmol · min−1 · mg−1protein. Data are mean ± SEM of five determinations,p < 0.05.
- Fig. 5.
Relationship between inhibition of aconitase or α-KGDH and decrease in the rotenone-induced NAD(P)H fluorescence. Decreases in the rotenone-induced NAD(P)H signal (data in Fig. 4,curve b) are shown as a function of percentage inhibition of aconitase (derived from Fig. 1) or α-KGDH (from Fig.2a) as measured after incubation with H2O2 for 5 min. H2O2concentrations (in micromoles) are indicated inbrackets. We have shown in separate control experiments (data not shown) that BCNU at 200 μm concentration has no effect on the activities of aconitase or α-KGDH, nor does it influence the effect of H2O2 on these enzymes. Data are average of five [for NAD(P)H measurement] or eight (for enzyme assays) determinations ± SEM.
- Fig. 6.
Reactions in the Krebs cycle influenced by low or high concentrations of H2O2. In the presence of low concentrations of H2O2, (a) when aconitase is completely inactivated but α-KGDH is still functional, glutamate becomes a key metabolite driving a segment of the Krebs cycle (thick arrows) and NADH production is maintained. When α-KGDH is also partially inhibited (b) in the presence of higher concentrations of H2O2 (≥100 μm), NADH generation becomes limited, resulting in an impaired respiratory capacity.
Tables
- Table 1.
The effect of H2O2 on the activity of citrate synthase, succinate dehydrogenase, and malate dehydrogenase
Activity (% of control) 100 μm H2O2 500 μm H2O2 5 min 10 min 5 min 10 min Citrate synthase ND ND 98.2 ± 1.8 98.3 ± 1.2 Succinate dehydrogenase 88 ± 1.121-a 76 ± 2.91-a 74 ± 4.61-a 70.8 ± 3.71-a Malate dehydrogenase 101.2 ± 1.8 104 ± 1.8 97.4 ± 2.3 105 ± 4.1 Nerve terminals were incubated with H2O2as indicated, then enzyme activities were determined in assay media containing Triton X-100 to permeabilize the plasma membrane as described in Materials and Methods. Results are expressed as percentage activity of the corresponding controls measured without H2O2 treatment. The following activities were taken as 100%: (1) citrate synthase, 778 ± 70 nmol · min−1 · mg−1 protein (n = 4); (2) succinate dehydrogenase, 30 ± 0.69 nmol · min−1 · mg−1 protein (n = 4); and (3) malate dehydrogenase, 12 ± 0.26 μmol · min−1 · mg−1 protein (n = 4). Results are the average of four independent determinations ± SEM. ND, Not determined.
↵F1-a Significantly different from the corresponding control.
- Table 2.
Comparison of the effect of H2O2 on the rotenone-induced NAD(P)H signal in the presence or absence of glucose
ΔNAD(P)H nmol/mg protein Control 25 μmH2O2 100 μmH2O2 500 μmH2O2 + Glucose 1.054 ± 0.14 0.840 ± 0.13* 0.544 ± 0.08* 0.518 ± 0.07* − Glucose 1.285 ± 0.17 0.850 ± 0.17* 0.567 ± 0.09* 0.418 ± 0.06* Synaptosomes were incubated with H2O2 for 5 min, then rotenone (2 μm) was added, and the increase in the NAD(P)H signal [ΔNAD(P)H] was measured as shown in Figure 3,inset. Data are mean ± SEM of three independent experiments. Values obtained in samples containing H2O2 are significantly different (
↵* ) from the corresponding controls. Differences between values obtained at a given H2O2 concentration in glucose-containing or glucose-free medium are not statistically different.
NAD+/NADH (pmol/mg protein) NADP+/NADPH (pmol/mg protein) 0 min 5 min 10 min 0 min 5 min 10 min Control 3381 ± 174 3509 ± 370 3357 ± 570 359 ± 55 387 ± 42 351 ± 62 100 μmH2O2 ND 3199 ± 225 3277 ± 381 ND 433 ± 86 403 ± 62 500 μmH2O2 ND 3016 ± 410 2852 ± 425 ND 380 ± 49 349 ± 54 Synaptosomes (0.5 mg/ml protein) were incubated in standard medium with or without H2O2 as indicated, and the content of pyridine nucleotides was measured as described in Materials and Methods. Results are given as mean ± SEM of four independent experiments. ND, Not determined. The content of pyridine nucleotides in H2O2-treated samples is not significantly different from the corresponding controls.
Glutamate (nmol/mg protein) 0 min 10 min 20 min Control 36.1 ± 3.2 34.5 ± 2.4 35.9 ± 2.2 H2O2 (5 μm) ND 34.5 ± 3 33.6 ± 5.1 H2O2 (10 μm) ND 29.3 ± 2.1 28.3 ± 1.74-a H2O2 (50 μm) 36.2 ± 1.8 24.4 ± 1.44-a 18 ± 1.54-a H2O2 (500 μm) 31.6 ± 4.3 20.6 ± 1.94-a 18.2 ± 1.84-a − Glucose 30.9 ± 3.7 25.1 ± 5.84-a 16.9 ± 0.54-a Synaptosomes were incubated in the presence or absence of H2O2 or in glucose-free medium as indicated. After different lengths of time, aliquots were taken, and glutamate content was measured as described in Materials and Methods. Each point represents a mean ± SEM of four experiments performed in duplicate.
↵F4-a Significantly different from the corresponding controls (p < 0.005).
In this issue
