NUCLEOTIDE DEPLETION DUE TO REACTIVE OXYGEN METABOLITES IN ENDOTHELIAL-CELLS - EFFECTS OF ANTIOXIDANTS AND 3-AMINOBENZAMIDE

Reactive oxygen metabolites have an important role in ischemia-reperfu sion injury. One of the sources of reactive oxygen metabolites is xant hine 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 indu ced by extracellular xanthine oxidase and hypoxanthine, in concentrati ons relevant to human pathophysiology. In umbilical vein endothelial c ells prelabeled with C-14-adenine, cellular adenine nucleotides retain ed 64 +/- 9% of the initial radioactivity over a 4-h incubation with c ulture medium (controls), whereas in the presence of xanthine oxidase (80 mU/mL) and hypoxanthine (100 mu M), only 3 +/- 4% of radioactivity remained in cellular nucleotides, the rest appearing in catabolic pro ducts 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 pr evented the leakage of nucleotides into the culture medium but not int racellular nucleotide catabolism, whereas the latter process was decre ased by catalase, consistent with predominant effects of superoxide an d hydrogen peroxide at the cell membrane and interior, respectively.