DETERMINATION OF THE MECHANISM OF FREE-RADICAL GENERATION IN HUMAN AORTIC ENDOTHELIAL-CELLS EXPOSED TO ANOXIA AND REOXYGENATION

Endothelial cell-derived oxygen free radicals are important mediators of postischemic injury; however, the mechanisms that trigger this radi cal generation are not known, and it is not known if this process can occur in human cells and tissues. The enzyme xanthine oxidase can be a n important source of radical generation; however, it has been reporte d that this enzyme may not be present in human endothelium. To determi ne the presence and mechanisms of radical generation in human vascular endothelial cells subjected to anoxia and reoxygenation, electron par amagnetic resonance measurements were performed on cultured human aort ic endothelial cells using the spin trap 5,5-dimethyl-1-pyrroline N-ox ide (DMPO). These measurements were correlated with cellular injury, x anthine oxidase activity, and alterations in cellular nucleotides. Upo n reoxygenation after 60 min of anoxia, large DMPO-OH (a(N) = a(H) = 1 4.9 G) and smaller DMPO-R (a(N) = 15.8 G, a(H) = 22.8 G) signals were seen. Superoxide dismutase totally quenched this radical generation. T he ferric iron chelator deferoxamine prevented cell death and totally quenched the DMPO-R signal with a 40% decrease in the DMPO-OH signal. Xanthine oxidase was shown to be present in these cells and to be the primary source of free radicals. While the concentration of this enzym e did not change after anoxia, the concentration of its substrate, hyp oxanthine, markedly increased, resulting in increased free radical gen eration upon reoxygenation. Thus, reoxygenated human vascular endothel ial cells generate superoxide free radicals, which further react with iron to form the reactive hydroxyl radical, which in turn causes cell death. Xanthine oxidase was the primary source of radical generation w ith this process triggered by the breakdown of ATP to the substrate hy poxanthine during anoxia.