Dihydrobiopterin (BH2): Key Determinant in Influencing Arginine Mediated Endothelial Tolerance and Dysfunction
Srinidi Mohan, Harsh Patel, Jorge Bolinaga, Nathania Soekamto, Lum Achu and Kefali Teklemariam
DOI : 10.3844/ajbbsp.2012.54.62
American Journal of Biochemistry and Biotechnology
Volume 8, Issue 2
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.
© 2012 Srinidi Mohan, Harsh Patel, Jorge Bolinaga, Nathania Soekamto, Lum Achu and Kefali Teklemariam. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.