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Cardiovascular-Renal Mechanisms in Health and Disease

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

¹Department of Pharmacology, Weill Medical College of Cornell University, New York; and ²Renal Research Institute, Division of Nephrology, New York Medical College, Valhalla, New York

Submitted 16 July 2007 ; accepted in final form 4 January 2008

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 BH₄ insufficiency, but rather results from a diminished ratio of BH₄ vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH₂). In support of this hypothesis, [³H]BH₄ binding studies revealed that BH₄ and BH₂ bind eNOS with equal affinity (K_d 80 nM) and BH₂ can rapidly and efficiently replace BH₄ in preformed eNOS-BH₄ complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH₂ levels increased from undetectable to 40% of total biopterin. This BH₂ 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, BH₄ supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH₄:BH₂ and not absolute intracellular levels of BH₄. Reciprocally, superoxide production was found to negatively correlate with intracellular BH₄:BH₂. Hyperglycemia-associated BH₄ 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 BH₄:BH₂, rather than BH₄ depletion per se, as the molecular trigger for NO insufficiency in diabetes.

nitric oxide; diabetes; endothelial dysfunction