Effect of medium pH on glutathione redox cycle in cultured human umbilical vein endothelial cells

Abstract

Impairments of the glutathione redox cycle in cultured endothelial cells under acidic pH conditions were measured. Glutathione-dependent H 2O 2-degrading activities decreased by 20% ( P < .01) at pH 6 and by 51% ( P < .01) at pH 4 compared with activities at pH 7.4 1 hour after a change with fresh medium. Intracellular reduced glutathione (GSH) content increased by 85% ( P < .01) following the change with pH 7.4 medium. Such increases in GSH content were impaired after exposure to acidic medium. After exposure to 500 μmol/L H 2O 2, intracellular GSH content decreased by 61% compared with the level obtained in the absence of H 2O 2 at pH 7.4 ( P < .01). Compared with the level at pH 7.4, the H 2O 2-induced decrease in intracellular GSH content was 32% lower ( P < .01) at pH 6 and did not change at all at pH 4. After exposure to 500 μmol/L H 2O 2, the intracellular oxidized glutathione (GSSG) content increased by 160% at pH 7.4 ( P < .01), 370% at pH 6 ( P < .01), and 90% at pH 4 compared with treatment without H 2O 2, respectively. After exposure to 500 μmol/L H 2O 2, the release of GSSG from cells at pH 6 decreased by 38% compared with the value found at pH 7.4 ( P < .05), and the release at pH 4 completely disappeared. Both glutathione peroxidase (GPO) and glutathione reductase activities decreased as a function of a decrease in pH from 7.4 to 4. The results (1) indicate that the glutathione redox cycle in cultured endothelial cells exposed to acidic conditions is profoundly impaired because of a decrease in GSH content, GPO and glutathione reductase activities, and GSSG transport out of the cells; and (2) suggest that impairment of active oxygen degradation under acidic conditions may induce endothelial cell dysfunction.