Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells.
OBJECTIVE
The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro.


METHODS
OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining.


RESULTS
Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)ornithine and nitro-L-arginine, and an NO scavenger, the Fe(2+)-N-methyl-D-glucamine dithiocarbamate complex.


CONCLUSIONS
OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.
Xu
J
J
Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.
He
L
L
Ahmed
S H
SH
Chen
S W
SW
Goldberg
M P
MP
Beckman
J S
JS
Hsu
C Y
CY
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001812:Blood-Brain Barrier / Q000502:physiology; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism*; D061945:Caspase Inhibitors; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D005609:Free Radicals / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D019335:Nitroarginine / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D013012:Sorbitol / Q000031:analogs & derivatives / Q000494:pharmacology; D013113:Spin Labels; D013859:Thiocarbamates / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000235:genetics
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002614:Chelating Agents; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D005609:Free Radicals; D012333:RNA, Messenger; D013113:Spin Labels; D013859:Thiocarbamates; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019335:Nitroarginine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D013012:Sorbitol; C041262:N-methyl-D-glucamine dithiocarbamate; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D005947:Glucose; D010100:Oxygen