Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

• Published in: Free radical biology & medicine Vol. 63; pp. 143 - 150
• Main Authors: Whitsett, Jennifer, Rangel Filho, Artur, Sethumadhavan, Savitha, Celinska, Joanna, Widlansky, Michael, Vasquez-Vivar, Jeannette
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2013
• Subjects: Animals; Biopterins - analogs & derivatives; Biopterins - metabolism; Cattle; Cells, Cultured; clinical trials; diastereomers; Dihydrobiopterin; Dihydrofolate; dihydrofolate reductase; endothelial cells; Endothelial Cells - enzymology; Endothelial Cells - metabolism; Endothelial drug metabolism; Endothelium, Vascular - enzymology; Endothelium, Vascular - metabolism; folic acid; Free radicals; high performance liquid chromatography; Humans; Methotrexate; Mice; nitric oxide; Nitric Oxide - metabolism; Nitric oxide synthase; Nitric Oxide Synthase - metabolism; oxidation; Oxidation-Reduction; Superoxides - metabolism; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - metabolism; Vascular Diseases - enzymology; Vascular Diseases - metabolism; Vascular Diseases - pathology; Dihydrofolate; Endothelial drug metabolism; Nitric oxide synthase; Free radicals; Methotrexate; Dihydrobiopterin
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2013.04.035

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 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. [Display omitted] •Clinical trials using BH4 to treat endothelial dysfunction have reported mixed results.•BH4 uptake and recycling in human endothelial cells was examined.•BH4 uptake is fast but transient, leading to accumulation of high 7,8-BH2 levels.•DHFR-dependent reduction of 7,8-BH2 to BH4 is exceedingly slow in human cells.