Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS

摘要

5,6,7,8-Tetrahydrobiopterin (BH_4) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH_4, in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH_4 insufficiency, but rather results from a diminished ratio of BH_4 vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin(BH_2). In support of this hypothesis, [~3H]BH_4 binding studies revealed that BH_4 and BH_2 bind eNOS with equal affinity (K_d approx 80 nM) and BH_2 can rapidly and efficiently replace BH_4 in preformed eNOS-BH_4 complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH_2 levels increased from undetectableto 40% of total biopterin. This BH_2 accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH_4 supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH_4: BH_2 and not absolute intracellular levels of BH_4. Reciprocally, superoxide production was found to negatively correlate with intracellular BH_4:BH_2. Hyperglycemia-associated BH_4 oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH_4:BH_2, rather than BH_4 depletion per se, as the molecular trigger for NO insufficiency in diabetes.