Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.
Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21224.
S P
SP
Sanders
J L
JL
Zweier
P
P
Kuppusamy
S J
SJ
Harrison
D J
DJ
Bassett
E W
EW
Gabrielson
J T
JT
Sylvester
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002478:Cells, Cultured; D003486:Cyanides / Q000494:pharmacology; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000098:blood supply*; D008833:Microcirculation; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*; D012402:Rotenone / Q000494:pharmacology; D012756:Sheep; D013113:Spin Labels
D003486:Cyanides; D003497:Cyclic N-Oxides; D005609:Free Radicals; D013113:Spin Labels; D012402:Rotenone; D000968:Antimycin A; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D010100:Oxygen