Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH4

We hypothesized that transient high-glucose concentration interferes with mediation by nitric oxide (NO) of flow-induced dilation (FID) of arterioles due to enhanced production of superoxide. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles (similar to130 mum) after transient high-glucose treatment (tHG; incubation with 30 mM glucose for 1 h), FID was reduced (maximum: control, 38 +/- 4%; after tFIG, 17 +/- 3%) which was not further diminished by the NO synthase (NOS) inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME; 18 +/- 2%). Correspondingly, an enhanced polyethylene-glycol-SOD (PEG-SOD)-sensitive superoxide production was detected after tHG in carotid arteries by dihydroethydine (DHE) staining. Presence of PEG-SOD during tHG prevented the reduction of FID (41 +/- 3%), which Could be inhibited by L-NAME (20 +/- 4%). Administration of PEG-SOD after tHG did not prevent the reduction of FID (22 +/- 3%). Sepiapterin, a precursor of the NO synthase cofactor tetrahydrobiopterin (BH4), administered during tFIG did not prevent the reduction of FID (maximum, 15 +/- 5%); however, it restored FID when administered after tHG (32 +/- 4%). Furthermore, inhibition of either glycolysis by 2-deoxyglucose or mitochondrial complex II by 2-thenoyltrifluoroacetone reduced the tHG-induced DHE-detectable enhanced superoxide production in carotid arteries and prevented FID reduction in arterioles (39 +/- 5 and 35 +/- 2%). Collectively, these findings suggest that in skeletal muscle arterioles, a transient elevation Of glucose via its increased metabolism, elicits enhanced production of superoxide, which decreases the bioavailability of NO and the level of the NOS cofactor BH4. resulting in a reduction of FID mediated by NO.