Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-α

Abstract

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O 2 − and H 2O 2, in endothelial cells. Then we examined the influence of tumor necrosis factor-α and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O 2 − and H 2O 2. We determined a basal intracellular H 2O 2 concentration of 32.8 ± 10.7 p m in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 ± 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 ± 3.79 mU/ mg protein. Only 0.48 ± 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H 2O 2 release was 75 ± 12 pmol · min −1 · mg cell protein −1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 ± 0.75 μU/mg with 47.1 ± 11.7% in the oxidase form. Intracellular purine levels of 1.19 ±1.04 nmol hypoxanthine/mg protein, 0.13 ± 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/ oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 μg/ml lipopolysaccharide (LPS) for 1–12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H 2O 2 in the direct endothelial toxicity of TNF and LPS.