Reactive oxygen species generated by mitochondrial injury in human brain microvessel endothelial cells.
Generation of reactive oxygen species (ROS) and their detrimental effects on the brain after transient ischemia are widely recognized. We studied ROS production from mitochondria in human brain microvessel endothelial cells (HBEC) under chemical hypoxia. HBEC in confluent conditions were incubated for 30 min with 10 microM 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein (DCF) diacetate, which was hydrolyzed and trapped inside the cells. ROS were measured with a fluorescent microscope, a CCD camera and an image analyzing system. Injury to mitochondrial respiratory chain was induced either with rotenone (an inhibitor of mitochondrial complex I) or with m-chlorocarbonyl cyanide phenylhydrazone (CCCP) (an uncoupler of ATP synthetase). Shortly after application of 10 microM rotenone, fluorescent intensity started to increase and the gradual increase continued for 10 min. Similarly, CCCP (10, 50 and 100 microM) dose-dependently increased the fluorescent intensity (p<0.01). Edaravone, a free radical scavenger widely used for treatment of cerebral infarction in Japan, at 100 microM successfully suppressed this ROS production (p<0.05). These data show that chemical hypoxia with normal concentration of oxygen in the medium induced free radicals generation in HBEC. Importance of endothelial mitochondria as a source of free radicals after reperfusion is suggested.
Itoh
Yoshiaki
Y
Department of Neurology, Keio University School of Medicine, Tokyo 160-8582, Japan. yoshiito@sc.itc.keio.ac.jp
Takaoka
Rie
R
Ohira
Masayuki
M
Abe
Takato
T
Tanahashi
Norio
N
Suzuki
Norihiro
N
D000983:Antipyrine / Q000031:analogs & derivatives / Q000494:pharmacology; D001921:Brain / Q000098:blood supply; D002545:Brain Ischemia / Q000188:drug therapy; D002560:Cerebrovascular Circulation; D000077553:Edaravone; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy; D008833:Microcirculation / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D000077553:Edaravone; D000983:Antipyrine