@article {10.3844/ajbbsp.2012.54.62,
article_type = {journal},
title = {Dihydrobiopterin (BH2): Key Determinant in Influencing Arginine Mediated Endothelial Tolerance and Dysfunction},
author = {Mohan, Srinidi and Patel, Harsh and Bolinaga, Jorge and Soekamto, Nathania and Achu, Lum and Teklemariam, Kefali},
volume = {8},
number = {2},
year = {2012},
month = {Jun},
pages = {54-62},
doi = {10.3844/ajbbsp.2012.54.62},
url = {https://thescipub.com/abstract/ajbbsp.2012.54.62},
abstract = {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.},
journal = {American Journal of Biochemistry and Biotechnology},
publisher = {Science Publications}
}