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

Abstract

Background and Purpose: 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 transferasemediated 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)omithine and nitro-L-arginine, and an NO scavenger, the Fe2+-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.