Abstract
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, [
3
H]BH
4
binding studies revealed that BH
4
and BH
2
bind eNOS with equal affinity (
K
d
≈ 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 undetectable to 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.