The Role of Endothelial Nitric Oxide Synthase (eNOS) Coupling Status During Acute Hyperglycemia as Determined by Real-time Measurements of Blood Nitric Oxide And Hydrogen Peroxide in Rat

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased endothelial-derived nitric oxide (NO) bioavailability and increased concentrations of reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H202), are the major characteristics of vascular endothelial dysfunction. Normally, vascular endothelial function depends on NO production from coupled endothelial NO synthase (eNOS) in the presence of 5,6,7,8- tetrahydrobiopterin (BH4). By contrast, 7,8-dihydrobiopterin (BH2, Oxidized form of BH4) and/or lack ofL-arginine (coupled eNOS substrate) causes eNOS uncoupling to produce SO, which can be quickly converted to H202. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction in vivo is unclear. In this study; we hypothesized that acute hyperglycemia (200 mg/dL) would increase H202 and decrease NO release in blood relative to saline control. By contrast, BH4 or L-arginine would attenuate the acute hyperglycemia-induced blood NO and H20 2 changes. However, BH2 will exacerbate the acute hyperglycemia-induced blood NO and H202levels. To test the hypothesis, blood NO or H20 2 levels were measured simultaneously using calibrated NO or H20 2 micro sensors ( 100 )lm; WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces from microsensors were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug (BH4,BH2, or L-arginine) and converted into a concentration based on a calibration curve. Acute hyperglycemia (200 mg/dL by i.v. 20% D-glucose) significantly increased H20 2 (n=6) and reduced NO (n=6) blood levels in comparison to the saline group (n=7, p\u3c0.05). BH2 (MW=239.23 g/mol, 4mg/kg) exacerbated hyperglycemia,- induced increased H20 2 levels (n=7) and decreased NO levels (n=6) (p\u3c0.05). By contrast, BH4 (n=6, MW=314.20 glmol, 6.5mglkg), significantly reduced blood H20 2 levels and increased blood NO levels during acute hyperglycemia compared to saline control (p\u3c0.05). Moreover, L-arginine (MW=21 0.66 glmol, 600mglkg) had similar effects on H20 2 (n=5) and NO (n=6) blood levels as BH4, showing significant reduction of blood H202 and enhancement ofblood NO compared to saline control (p\u3c0.05). In summary, uncoupled eNOS serves as a significant mechanism mediating acute hyperglycemiainduced vascular dysfunction and oxidative stress. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult