The role of endothelial nitric oxide synthase (eNOS) uncoupling in acute hyperglycemia – induced oxidative stress and vascular endothelial dysfunction by measuring blood nitric oxide and hydrogen peroxide in real-time

Philadelphia Campus

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased eNOS derived nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H₂O₂), are the major characteristics of vascular endothelial dysfunction. Furthermore, eNOS can change from coupled to an uncoupled status resulting in SO production instead of NO production. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction is unclear in vivo. In this study we hypothesized that acute hyperglycemia (200 mg/dL) would increase H₂O₂ and decrease NO release in blood relative to saline control. By contrast, 5,6,7,8-tetrahydrobiopterin (BH₄, an essential cofactor of coupled eNOS) (MW=241.247 g/mol, 6.5 mg/kg) or L-arginine (the substrate of coupled eNOS) (MW=210.66 g/mol, 600 mg/kg) would attenuate acute hyperglycemia-induced blood NO/H₂O₂ change. However, 7,8-dihydrobiopterin (BH₂, an oxidized form of BH₄ and serves as a cofactor for uncoupled eNOS) (MW=239.231 g/mol, 4 mg/kg) will exacerbate acute hyperglycemia-induced blood NO/H₂O₂ change. Blood NO or H₂O₂ levels were measured simultaneously using calibrated NO or H₂O₂ microsensors (100 µm WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug and converted into concentration based on the calibration curve. We found that acute hyperglycemia (200 mg/dL) significantly increased H₂O₂ (n=6) and reduced NO (n=6) blood levels compared to the saline group (n=7, p2 exacerbated hyperglycemia– induced increased H₂O₂ levels (n=7) and decreased NO levels (n=4) (p4 (n=6), significantly attenuated hyperglycemia– induced increased H₂O₂ levels and decreased NO levels (p2O₂ (n=5) and NO (n=6) blood levels as BH₄, showing significant reduction of blood H₂O₂ and enhancement of blood NO (p2O₂ and reduced NO blood levels. Uncoupled eNOS serves as a significant source mediating acute hyperglycemia-induced vascular dysfunction. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult.