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

Ke Li Tsai, Seu Mei Wang, Ching Chow Chen, Tsorng Harn Fong, Mei Lin Wu

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69 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)161-174
Number of pages14
JournalJournal of Physiology
Volume502
Issue number1
DOIs
Publication statusPublished - Jul 1 1997
Externally publishedYes

ASJC Scopus subject areas

  • Physiology

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