Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells

Background and Purpose-The cerebral endothelial cells (ECs) are a primary t arget of hypoxic or ischemic brain insults. EC damage may contribute to pos tischemic secondary injury. Massive production of NO after inducible NO syn thase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression af ter oxygen-glucose deprivation (OGD) in vitro. Methods OGD in bovine cerebral ECs in culture was induced by deleting gluco se in the medium and by incubating the cells in a temperature-controlled an aerobic chamber. The extent of cell death was assessed by trypan blue exclu sion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling we re used to examine DNA fragmentation. The expression of iNOS mRNA and prote in was detected by reverse transcription-polymerase chain reaction and West ern blotting, respectively. Nitrotyrosine expression was confirmed with Wes tern blot analysis and immunostaining. Results-Bovine cerebral EC death was dependent on the duration of OGD and s howed selected biochemical, morphological, and pharmacological features sug gestive of apoptosis. OGD also induced the expression of iNOS mRNA and prot ein in bovine cerebral ECs. Increased expression of nitrotyrosine, the prod uct formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reductio n of cell death by NO synthase inhibitors, including L-N-G-(1-iminoethyl)or nithine and nitro-L-arginine, and an NO scavenger, the Fe2+-N-methyl-D-gluc amine dithiocarbamate complex. Conclusions-OGD-induced bovine cerebral EC death involves an apoptotic proc ess. Induction of iNOS with subsequent peroxynitrite formation may contribu te to bovine cerebral EC death caused by OGD.