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

Research output: Contribution to journalArticle

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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

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Acidosis
Glioma
Astrocytes
Oxidative Stress
Hydroxyl Radical
Adenosine Triphosphate
Iodoacetates
Iron
Chelating Agents
Neuroglia
Lactic Acid
Thiourea
Deferoxamine
Phenanthrolines
Membranes
Oxidative Phosphorylation
Brain Edema
Deoxyglucose
Capillary Permeability
Glycolysis

ASJC Scopus subject areas

  • Physiology

Cite this

Mechanism of oxidative stress-induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells. / Tsai, Ke Li; Wang, Seu Mei; Chen, Ching Chow; Fong, Tsorng Harn; Wu, Mei Lin.

In: Journal of Physiology, Vol. 502, No. 1, 01.07.1997, p. 161-174.

Research output: Contribution to journalArticle

Tsai, Ke Li ; Wang, Seu Mei ; Chen, Ching Chow ; Fong, Tsorng Harn ; Wu, Mei Lin. / Mechanism of oxidative stress-induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells. In: Journal of Physiology. 1997 ; Vol. 502, No. 1. pp. 161-174.
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N2 - 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.

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