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 H2O2 are produced, resulting i n brain oedema and changes in cerebral vascular permeability. We have found that H2O2 (100 mu M) induces a significant intracellular acidosi s 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-crossin g ferrous iron chelators, phenanthroline and deferoxamine, almost comp letely inhibited H2O2-induced intracellular acidosis, while the non-me mbrane-crossing iron chelator apo-transferrin had no effect. Furthermo re, the acidosis was completely inhibited by two potent membrane-cross ing . OH scavengers, N-(2-mercaptopropionyl)-glycine (N-MPG) and dimet hyl thiourea (DMTU). Since . OH can be produced during iron-catalysed H2O2 breakdown (Fenton reaction), ive have shown that a large reductio n in pH(1) in glial cells can result from the production of intracellu lar . OH via H2O2 oxidation. 3. We ha ve ruled out the possible involv ement of: (i) an increase in intracellular Ca2+ levels; and (ii) inhib ition of oxidative phosphorylation. 4. Our results suggest that . OH i nhibits glycolysis, leading to, ATP hydrolysis and intracellular acido sis. This conclusion is based on the following observations: (i) in gl ucose-free medium, or in the presence of iodoacetate or 2-deoxy-D-gluc ose, H2O2-induced acidosis is completely suppressed; (ii) H2O2 and iod oacetate both produce an increase in levels of intracellular free Mg2, an indicator of ATP breakdown; and (iii) direct measurement of intra cellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatme nt with 100 mu M H2O2. 5. Inhibition of the pH(1) regulators (i.e. the Na+-H+ exchange and possibly the Na+-HCO3--dependent pH(1) transporte rs) resulting from H2O2-induced intracellular ATP reduction may also b e involved in the H2O2-evoked intracellular acidosis in glial cells.