title: Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling.
abstract: 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.
authors.affiliation: Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Whitsett
authors.forename: Artur
authors.initials: A
authors.lastname: Rangel Filho
authors.forename: Savitha
authors.initials: S
authors.lastname: Sethumadhavan
authors.forename: Joanna
authors.initials: J
authors.lastname: Celinska
authors.forename: Michael
authors.initials: M
authors.lastname: Widlansky
authors.forename: Jeannette
authors.initials: J
authors.lastname: Vasquez-Vivar
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014652:Vascular Diseases / Q000201:enzymology / Q000378:metabolism / Q000473:pathology
chemical_list: D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; D019001:Nitric Oxide Synthase; D013762:Tetrahydrofolate Dehydrogenase; C003402:sapropterin
keywords: Dihydrobiopterin; Dihydrofolate; Endothelial drug metabolism; Free radicals; Methotrexate; Nitric oxide synthase