Mechanism of oxidative stress‐induced intracellular acidosis in rat cerebellar astrocytes and C 6 glioma cells

1 Following ischaemic reperfusion, large amounts of superoxide anion (.O 2 − ), hydroxyl radical (.OH) and H 2 O 2 are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H 2 O 2 (100 μm) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 ± 0.04 pH units) and C 6 glioma cells (0.33 ± 0.07 pH units). 2 Two membrane‐crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H 2 O 2 ‐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)‐grycine ( N ‐MPG) and dimethyl thiourea (DMTU). Since .OH can be produced during iron‐catalysed H 2 O 2 breakdown (Fenton reaction), we have shown that a large reduction in pH 1 in glial cells can result from the production of intracellular .OH via H 2 O 2 oxidation. 3 We have ruled out the possible involvement of: (i) an increase in intracellular Ca 2+ 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, H 2 O 2 ‐induced acidosis is completely suppressed; (ii) H 2 O 2 and iodoacetate both produce an increase in levels of intracellular free Mg 2+ , 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 μ m H 2 O 2 . 5 Inhibition of the pH 1 regulators (i.e. the Na + –H + exchange and possibly the Na + –HCO 3 − –dependent pH 1 transporters) resulting from H 2 O 2 ‐induced intracellular ATP reduction may also be involved in the H 2 O 2 ‐evoked intracellular acidosis in glial cells.