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Reactive oxygen metabolites have an important role in ischemia-reperfusion injury. One of the sources of reactive oxygen metabolites is xanthine oxidase, which is present in several tissues but is also released into the circulation after ischemia. We studied the effect of several potentially protective compounds on adenine nucleotide depletion induced by extracellular xanthine oxidase and hypoxanthine, in concentrations relevant to human pathophysiology. In umbilical vein endothelial cells prelabeled with 14C-adenine, cellular adenine nucleotides retained 64 +/- 9% of the initial radioactivity over a 4-h incubation with culture medium (controls), whereas in the presence of xanthine oxidase (80 mU/mL) and hypoxanthine (100 microM), only 3 +/- 4% of radioactivity remained in cellular nucleotides, the rest appearing in catabolic products in the medium. Glutathione and 3-aminobenzamide, an inhibitor of poly-ADP-ribose polymerase, partly prevented the nucleotide depletion (adenine nucleotide radioactivity 15 +/- 6% to 33 +/- 13% of total), but scavengers of the hydroxyl radical, dimethylthiourea and DMSO, as well as vitamins E and C, were without effect. Superoxide dismutase prevented the leakage of nucleotides into the culture medium but not intracellular nucleotide catabolism, whereas the latter process was decreased by catalase, consistent with predominant effects of superoxide and hydrogen peroxide at the cell membrane and interior, respectively.

Nucleotide Depletion Due to Reactive Oxygen Metabolites in Endothelial Cells: Effects of Antioxidants and 3-Aminobenzamide