High glucose-induced tyrosine nitration in endothelial cells: Role of eNOS uncoupling and aldose reductase activation

PURPOSE. Analyses in diabetic rats have shown that breakdown of the blood-retina barrier is associated with increased formation of peroxynitrite, nitric oxide, and lipid peroxidation. The permeability increase is blocked by treatments that also prevent the increases in reactive oxygen species, suggesting their causal role in vascular dysfunction. The purpose of this study was to determine the specific effects of high glucose and high osmolarity on the formation of nitrotyrosine, nitric oxide, and superoxide anion in retinal vascular endothelial cells and to evaluate the metabolic pathways involved. METHODS. Cultured retinal endothelial cells were maintained for 5 days in media with different concentrations of glucose or osmotic control reagents and tested for effects on protein tyrosine nitration and nitric oxide synthase (NOS) expression, using immunoblot techniques. NOS activity was determined by assays for nitrite formation and conversion of arginine to citrulline. Superoxide anion formation was assayed by hydroethidine staining. RESULTS. Increased concentrations of glucose or 3-methyl-o-glucose stimulated formation of nitric oxide (NO) and superoxide induced protein nitration on tyrosine and increased expression and activity of endothelial nitric oxide synthase (eNOS). The effects of glucose were more potent: Inhibiting NOS or aldose reductase (AR), scavenging superoxide or peroxynitrite, or supplementing the NOS substrate L-arginine or cofactor tetrahydrobiopterin blocked the formation of reactive oxygen species and prevented protein tyrosine nitration. CONCLUSIONS. Increases in glucose levels and osmotic stress similar to those in diabetic patients increase the formation of nitrotyrosine in retinal endothelial cells because of their actions increasing NOS activity and causing superoxide formation due to eNOS uncoupling and AR activation.