Role of glutathione redox cycle in TNF-alpha-mediated endothelial cell dysfunction

Modulation of the glutathione redox cycle may influence tumor necrosis factor-alpha (TNF)-mediated disturbances of endothelial integrity. To test this hypothesis, normal endothelial cells or cells with either increased or decreased glutathione levels were exposed to 100 ng (500 U) TNF/ml. Increased glutathione levels were achieved by exposure to 0.2 mM N-acetyl-L-cysteine (NAC) and decreased glutathione levels by exposure to 25 microM buthionine sulfoximine (BSO). Several components of the glutathione redox cycle as well as markers of endothelial integrity, such as cytoplasmic free calcium and transendothelial albumin transfer, were measured in the treated cells. Exposure to TNF for 3 and 6 h decreased total glutathione levels, which was followed by an increase at later time points. Moreover, treatment with TNF resulted in an increase in the ratio of oxidized to reduced glutathione, intracellular free calcium, albumin transfer across endothelial monolayers and lipid hydroperoxides. However, an increase in lipid hydroperoxides was seen only when endothelial cell cultures were supplemented with iron. BSO treatment increased susceptibility of endothelial cells to TNF-mediated metabolic disturbances. On the other hand, NAC partially protected against TNF-induced injury to endothelial monolayers. Our results demonstrate the important role of the glutathione redox cycle in TNF-mediated disturbances of the vascular endothelium and indicate that modulation of glutathione levels may potentiate the injurious effects of this inflammatory cytokine.