The endothelial cell is thought to be an important site of
free radical generation in ischemic tissues. It has been demonstrated that endothelial cells from several species generate a burst of
free radical generation upon reoxygenation; however, it has been suggested that human endothelial cells are not similarly capable of generating
free radicals on reoxygenation. In view of the central importance of revascularization with accompanying reoxygenation in the clinical treatment of tissue
ischemia/
infarction, we have performed studies to determine the presence, mechanism, and kinetics of
free radical generation in human endothelial cells. Therefore, we subjected cultured human umbilical vein endothelial cells to
anoxia followed by reoxygenation. Cell
suspensions of 10(7) cells/ml were subjected to varying periods of
anoxia and reoxygenation. On reoxygenation with addition of a 50 mM concentration of 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, aN = aH = 14.9 G, and a six-peaked DMPO-R signal, aN = 15.6 G aH = 22.9 G, whereas cells in air gave no signal. The observed signal was quenched by
superoxide dismutase (SOD) or
catalase.
Deferoxamine decreased the measured radical signals by 40%.
Cyclooxygenase blockers did not decrease radical generation, but the
xanthine oxidase blocker
oxypurinol did decrease radical generation by 60%.