Dihydrobiopterin (BH₂): Key Determinant in Influencing Arginine Mediated Endothelial Tolerance and Dysfunction

The redox-sensitive tetrahydrobiopterin (BH₄) is an essential cofactor that is required by endothelial Nitric Oxide Synthase (eNOS) for L-arginine (ARG) mediated Nitric Oxide (NO) generation. Oxidation of BH₄ causes cofactor insufficiency and uncoupling of eNOS, resulting in product switching from NO to O₂^⋅– 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 versus its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH₂). Human Umbilical Vein Endothelial Cells (HUVEC) were incubated for 2 h in Locke’s buffer with 100 µM ARG with or without other agents for 2 h (acute) or in medium for 7 days and challenged in buffer for 2 h (chronic). eNOS activity was determined by cellular accumulation of nitrite/nitrate and its expression was measured using ELISA method. Dihydroethidium fluorescence technique was used to measure O₂^⋅– accumulation. For binding studies, cell extracts were quantified for levels of BH₄, BH₂, quinonoid isoform of BH₂ (qBH₂) and biopterin using a modified HPLC method. [³H]BH₄ binding studies revealed BH₄ and BH₂ bind eNOS with equal affinity and BH₂ can efficiently replace BH₄ in preformed eNOS-BH₄ complexes. While the total pterin pool of HUVEC was unaffected by chronic (7 days) exposure to ARG, BH₂ levels increased from undetectable to 40% of total pterin. This BH₂ accumulation was associated with diminished NO activity and accelerated O₂^⋅– production. Reciprocally, O₂^⋅– production was found to negatively correlate with intracellular ratio of BH₄-to-BH₂. Our findings implicate intracellular BH₄-to-BH₂ ratio, not simply BH₄ amount, as a critical in vitro determinant of eNOS product formation during continuous ARG supplementation. Accordingly, diminished ratio of BH₄-to-BH₂ is likely to be the fundamental molecular link between oxidative stress and endothelial dysfunction during ARG mediated tolerance development.