Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling. Free Radic Biol Med 2013 Oct;63:143-50
Date
05/28/2013Pubmed ID
23707606Pubmed Central ID
PMC3748942DOI
10.1016/j.freeradbiomed.2013.04.035Scopus ID
2-s2.0-84884840975 (requires institutional sign-in at Scopus site) 19 CitationsAbstract
Tetrahydrobiopterin (BH₄) is required for NO synthesis and inhibition of superoxide release from endothelial NO synthase. Clinical trials using BH₄ to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH₄. One of the oxidation products of BH₄, 7,8-dihydrobiopterin (7,8-BH₂), is recycled back to BH₄ by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH₄ treatment is lacking. To characterize this reaction, we applied a novel multielectrode coulometric HPLC method that enabled the direct quantification of 7,8-BH₂ and BH₄, which is not possible with fluorescence-based methodologies. We found that basal untreated BH₄ and 7,8-BH₂ concentrations in human endothelial cells (ECs) are lower than in bovine and murine endothelioma cells. Treatment of human ECs with BH₄ transiently increased intracellular BH₄ while accumulating the more stable 7,8-BH₂. This was different from bovine or murine ECs, which resulted in preferential BH₄ increase. Using BH₄ diastereomers, 6S-BH₄ and 6R-BH₄, the narrow contribution of enzymatic DHFR recycling to total intracellular BH₄ was demonstrated. Reduction of 7,8-BH₂ to BH₄ occurs at very slow rates in cells and needs supraphysiological levels of 7,8-BH₂, indicating this reaction is kinetically limited. Activity assays verified that human DHFR has very low affinity for 7,8-BH₂ (DHF<Km>7,8-BH₂) and folic acid inhibits 7,8-BH₂ recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies, which may be further aggravated by folate supplements.
Author List
Whitsett J, Rangel Filho A, Sethumadhavan S, Celinska J, Widlansky M, Vasquez-Vivar JAuthors
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of WisconsinMichael E. Widlansky MD Associate Director, Professor in the Medicine department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AnimalsCattle
Cells, Cultured
Endothelial Cells
Endothelium, Vascular
Humans
Mice
Nitric Oxide
Nitric Oxide Synthase
Oxidation-Reduction
Superoxides
Tetrahydrofolate Dehydrogenase
Vascular Diseases