Mechanism of oxidative stress-induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells

1. Following ischaemic reperfusion, large amounts of superoxide anion (·O₂ ^-), hydroxyl radical (·OH) and H₂O₂ are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H₂O₂ (100 μM) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 ± 0.04 pH units) and C₆ glioma cells (0.33 ± 0.07 pH units). 2. Two membrane-crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H₂O₂-induced intracellular acidosis, while the non-membrane-crossing iron chelator apo-transferrin had no effect. Furthermore, the acidosis was completely inhibited by two potent membrane-crossing ·OH scavengers, N-(2-mercaptopropionyl)-glycine (N-MPG) and dimethyl thiourea (DMTU). Since ·OH can be produced during iron-catalysed H₂O₂ breakdown (Fenton reaction), we have shown that a large reduction in pH(i) in glial cells can result from the production of intracellular ·OH via H₂O₂ oxidation. 3. We have ruled out the possible involvement of: (i) an increase in intracellular Ca²⁺ levels; and (ii) inhibition of oxidative phosphorylation. 4. Our results suggest that ·OH inhibits glycolysis, leading to ATP hydrolysis and intracellular acidosis. This conclusion is based on the following observations: (i) in glucose-free medium, or in the presence of iodoacetate or 2-deoxy-D-glucose, H₂O₂-incluced acidosis is completely suppressed; (ii) H₂O₂ and iodoacetate both produce an increase in levels of intracellular free Mg²⁺, an indicator of ATP breakdown; and (iii) direct measurement of intracellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatment with 100 μM H₂O₂. 5. Inhibition of the pH(i) regulators (i.e. the Na⁺-H⁺ exchange and possibly the Na⁺-HCO₃ ^- -dependent pH(i) transporters) resulting from H₂O₂-induced intracellular ATP reduction may also be involved in the H₂O₂-evoked intracellular acidosis in glial cells.