MEASUREMENT AND CHARACTERIZATION OF FREE-RADICAL GENERATION IN REOXYGENATED HUMAN ENDOTHELIAL-CELLS

The endothelial cell is thought to be an important site of free radica l generation in ischemic tissues. It has been demonstrated that endoth elial cells from several species generate a burst of free radical gene ration upon reoxygenation; however, it has been suggested that human e ndothelial cells are not similarly capable of generating free radicals on reoxygenation. In view of the central importance of revascularizat ion with accompanying reoxygenation in the clinical treatment of tissu e ischemia/ infarction, we have performed studies to determine the pre sence, mechanism, and kinetics of free radical generation in human end othelial cells. Therefore, we subjected cultured human umbilical vein endothelial cells to anoxia followed by reoxygenation. Cell suspension s of 10(7) cells/ml were subjected to varying periods of anoxia and re oxygenation. On reoxygenation with addition of a 50 mM concentration o f the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), after 90 min of anoxia an electron paramagnetic resonance (EPR) signal was observed consisting of 2 components: a quartet 1:2:2:1 DMPO-OH signal, a(N) = a(H) = 14.9 G, and a six-peaked DMPO-R signal, a(N) = 15.6 G a(H) = 22 .9 G, whereas cells in air gave no signal. The observed signal was que nched by superoxide dismutase (SOD) or catalase. Deferoxamine decrease d the measured radical signals by 40%. Cyclooxygenase blockers did not decrease radical generation, but the xanthine oxidase blocker oxypuri nol did decrease radical generation by 60%. Both the magnitude of radi cal generation and the cellular xanthine oxidase activity were measure d to be 4- to 5-fold lower than that seen in identical preparations of bovine aortic endothelial cells, further suggesting that the presence and concentration of xanthine oxidase has a major influence on the ma gnitude of the radical generation. Thus human endothelial cells can ge nerate oxygen free radicals when subjected to anoxia and reoxygenation . Both xanthine oxidase and redox cycling of chelatable iron are impor tant mechanisms of this free radical generation.