The localization of DMPO spin adducts of OH in endothelial cells exposed to hydrogen peroxide.
Examination by electron spin resonance (ESR) spectroscopy revealed the localization of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) spin adducts of hydroxyl radicals (.OH) produced by bovine endothelial cells exposed to hydrogen peroxide. Addition of 10 mM chromium oxalate, a line-broadening agent, to the reaction mixture virtually abolished the signal of DMPO-OH spin adducts. Moreover, the spin adducts were recovered in the filtrated fraction of the cell suspension. We, therefore, concluded that the location of DMPO-OH due to .OH radicals produced by endothelial cells was extracellular. Contrastingly, the site of formation of DMPO-OH was confirmed to be intracellular by the effect of Desferal, an iron chelator, and the effect of poly(ethylene glycol), an extracellular scavenger of OH radicals, as previously reported. The DMPO-OH adducts in the cell suspension mixture were degraded by a cyanide sensitive pathway and they were apparently more unstable than in the extracellular fraction. The initial amount of DMPO-OH adducts formed in endothelial cells could potentially be monitored by the DMPO-OH signals in the extracellular reaction mixture better than those in the cell suspension mixture.
Kaneko
M
M
Biophysics Division, National Cancer Center Research Institute, Tokyo, Japan.
Kodama
M
M
Inoue
F
F
D000818:Animals; D000931:Antidotes / Q000494:pharmacology; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D003497:Cyclic N-Oxides / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000008:administration & dosage / Q000633:toxicity*; D007502:Iron Chelating Agents / Q000494:pharmacology; D010070:Oxalates / Q000633:toxicity; D016877:Oxidants / Q000008:administration & dosage / Q000633:toxicity*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013113:Spin Labels
D000931:Antidotes; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D007502:Iron Chelating Agents; D010070:Oxalates; D016877:Oxidants; D013113:Spin Labels; D011092:Polyethylene Glycols; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D003676:Deferoxamine