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

Abstract

5,6,7,8-Tetrahydrobiopterin (BH₄) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH₄, 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 BH₄insufficiency, but rather results from a diminished ratio of BH₄vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH₂). In support of this hypothesis, [³H]BH₄binding studies revealed that BH₄and BH₂bind eNOS with equal affinity ( K_d≈ 80 nM) and BH₂can rapidly and efficiently replace BH₄in preformed eNOS-BH₄complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH₂levels increased from undetectable to 40% of total biopterin. This BH₂accumulation 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, BH₄supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH₄:BH₂and not absolute intracellular levels of BH₄. Reciprocally, superoxide production was found to negatively correlate with intracellular BH₄:BH₂. Hyperglycemia-associated BH₄oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH₄:BH₂, rather than BH₄depletion per se, as the molecular trigger for NO insufficiency in diabetes.