Abstract
In order to determine the impairment to glutathione redox cycle in cultured endothelial cells (ECs) under acidic conditions, we measured glutathione-dependent degradation of hydrogen peroxide (H2O2), H2O2-induced changes in cytosolic glutathione contents (GSH, GSSG), GSH and GSSG transport rates from ECs, and both glutathione peroxidase and reductase activities in an acidic pH medium. 1) Glutathione-dependent H2O2 degradation activities decreased by 19% at pH 6, and 52% at pH 4 as compared with those of pH 7.4 (p<0.01), respectively. 2) The basal GSH content of ECs under the acidic conditions fell by 17% at pH 6, and by 53% at pH 4 as compared with that of pH 7.4. The decrease in basal GSH content under acidic pH (pH 4) could be reversed by the change in pH of the medium pH 7.4 after 30 minute-acidic pH condition. After an exposure of 500μM H2O2, intracellular GSH content fell by 61% of the control level at pH 7.4 (p<0.01). The H2O2-induced decrease in GSH level under acidic condition was impaired at pH 6 by 50% (p<0.01) of the value of pH 7.4, and there was no response at pH 4. 3) Intracellular GSSG content increased to 2.6 times the control level at pH 7.4 (p<0.01), 4.7 times the control level at pH 6 (p<0.01), and 1.9 times the control level at pH 4 after exposure to 500μM H2O2. The H2O2-induced increase in GSSG level at pH 6 was 2.6 times greater than the value at pH 7.4 (p<0.001). 4) After exposure to 500μM H2O2, the release of GSSG from the cells at pH 6 decreased by 38% as compared with the value at pH 7.4 (p<0.05) and the release at pH 4 completely disappeared. 5) Both glutathione peroxidase and glutathione reductase activities progressively decreased as the pH of the medium decreased from pH 7.4 to pH 4.
These results indicate that glutathione redox cycle in cultured endothelial cell was profoundly impaired by way of the decreases in both glutathione peroxidase and reductase activities and GSSG transport from the cells under the acidic conditions. The impairment of active oxygen degradation under the acidic conditions may induce endothelial cell dysfunction.
