Nitrotyrosine generation via inducible nitric oxide synthase in vascular wall in focal ischemia-reperfusion.
Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037 +/- 0.040% (n = 8) and 0.064 +/- 0.035% (n = 6), respectively, being significantly higher than that in the sham operation group (n = 7) (P < 0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039 +/- 0.025%, n = 7), not in knockout or sham-operated mice (P < 0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.
Hirabayashi
H
H
Department of Neurology, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Takizawa
S
S
Fukuyama
N
N
Nakazawa
H
H
Shinohara
Y
Y
D000138:Acidosis / Q000378:metabolism; D000818:Animals; D001786:Blood Glucose; D001794:Blood Pressure; D002245:Carbon Dioxide / Q000097:blood; D002540:Cerebral Cortex / Q000098:blood supply / Q000378:metabolism; D002560:Cerebrovascular Circulation / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000201:enzymology*; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism; D002546:Ischemic Attack, Transient / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010100:Oxygen / Q000097:blood; D015427:Reperfusion Injury / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D001786:Blood Glucose; D009566:Nitrates; D002245:Carbon Dioxide; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D010100:Oxygen