Respiratory properties of mitochondrial fractions prepared from astrocytes after mitochondrial glutathione depletion and exposure to SIN-1
| Oxygen uptake rate (ng-atom 0·min−1·mg of protein−1) | ||||
|---|---|---|---|---|
| Conditions | State 3 respiration | State 4 respiration | Uncoupled respiration | ADP/0 |
| Respiratory substrate: pyruvate plus malate | ||||
| Untreated cells | 25.1 ± 4.1 | 8.9 ± 3.0 | 25.8 ± 2.0 | 2.4 ± 0.6 |
| Ethacrynic acid | 54.0 ± 5.9** | 19.0 ± 7.3* | 59.1 ± 6.0** | 2.0 ± 0.5 |
| Ethacrynic acid plus SIN-1 | 15.8 ± 0.5* | 17.9 ± 6.4 | ||
| Respiratory substrate: succinate | ||||
| Untreated cells | 43.6 ± 8.9 | 14.2 ± 3.7 | 48.5 ± 9.1 | 1.3 ± 0.3 |
| Ethacrynic acid | 42.1 ± 3.3 | 20.5 ± 6.7 | 47.3 ± 3.4 | 1.5 ± 0.2 |
| Ethacrynic acid plus SIN-1 | 24.2 ± 2.0* | 24.3 ± 5.7** | ||
Mitochondrial fractions were isolated from astrocytes after 24 hrs in HBSS. Treated cells were exposed to ethacrynic acid to deplete mitochondrial glutathione, and some preparations were further treated for 3 hr with 200 μm SIN-1. Respiratory properties were measured using an oxygen-sensitive electrode. State 3 respiration rate was determined in the presence of ADP plus respiratory substrates. State 4 respiration was measured following depletion of the ADP, and the uncoupled rate was determined after the addition of CCCP. Samples from cells treated with ethacrynic acid plus SIN-1 were poorly coupled, and state 4 values could not be reliably determined. Values are shown as mean ± SD (n = 3-5). Values significantly different from those for mitochondrial fractions from the untreated cells are indicated as follows: *p < 0.05, **p < 0.01 (one-way ANOVA with Student-Newman-Keuls test). All of the values for mitochondria from cells treated with ethacrynic were also significantly different from those for cells exposed to ethacrynic acid plus SIN-1 (p < 0.01).