Research Article Open Access

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

Srinidi Mohan1, Harsh Patel1, Jorge Bolinaga1, Nathania Soekamto1, Lum Achu1 and Kefali Teklemariam1
  • 1 University of New England, United States

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.

American Journal of Biochemistry and Biotechnology
Volume 8 No. 2, 2012, 54-62

DOI: https://doi.org/10.3844/ajbbsp.2012.54.62

Submitted On: 24 March 2012 Published On: 22 June 2012

How to Cite: Mohan, S., Patel, H., Bolinaga, J., Soekamto, N., Achu, L. & Teklemariam, K. (2012). Dihydrobiopterin (BH2): Key Determinant in Influencing Arginine Mediated Endothelial Tolerance and Dysfunction. American Journal of Biochemistry and Biotechnology, 8(2), 54-62. https://doi.org/10.3844/ajbbsp.2012.54.62

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Keywords

  • Tetrahydrobiopterin
  • dihydrobiopterin
  • eNOS
  • arginine
  • super-oxide